TESIS Gamino Rodríguez PDF

Infeccin por flavivirus en aves cinegticas en Espaa:
distribucin del virus y alteraciones estructurales

en los tejidos del hospedador
Memoria presentada por

Virginia Gamino Rodrguez
para optar al grado de Doctor
por la Universidad de Castilla-La Mancha
Bajo la direccin de la Doctora

Ursula Hfle Hansen
Ciudad Real, 2013
GRUPO DE SANIDAD Y BIOTECNOLOGA

INSTITUTO DE INVESTIGACIN EN RECURSOS CINEGTICOS
(CSIC-UCLM-JCCM)
DEPARTAMENTO DE CIENCIA Y TECONOLOGA AGROFORESTAL
Y GENTICA
UNIVERSIDAD DE CASTILLA-LA MANCHA
La Dra. Ursula Hfle Hansen hace constar que la tesis titulada Infeccin por
flavivirus en aves cinegticas en Espaa: distribucin del virus y alteraciones
estructurales en los tejidos del hospedador realizada bajo su direccin por Virginia
Gamino Rodrguez en el Instituto de Investigacin en Recursos Cinegticos, rene
los requisitos necesarios para su defensa y aprobacin y, por tanto, para optar al
grado de Doctor.
VB de la Directora
Dra. Ursula Hfle Hansen
Este trabajo de tesis doctoral se realiz gracias a los siguientes proyectos:

Proyecto AG2008-02504GAN, Ministerio de Ciencia e Innovacin
Proyecto PAC08-0296-7771, Junta de Comunidades de Castilla-La Mancha
Proyecto FAU2008-00006, Instituto Nacional de Investigacin y Tecnologa Agraria y
Alimentaria
ndice
NDICE
ESTRUCTURA DE LA TESIS ............................................................................... 1
INTRODUCCIN .................................................................................................... 3
Antecedentes ......................................................................................................... 5
El gnero Flavivirus ............................................................................................... 6
Flavivirus en Europa y Espaa ........................................................................... 10
Patogenia y desarrollo de encefalitis en la infeccin por flavivirus transmitidos
por mosquitos ..................................................................................................... 26
La perdiz roja y su relacin con los flavivirus ...................................................... 37
Uso de aves como modelo experimental de la infeccin por flavivirus ................. 38
Bibliografa .......................................................................................................... 39
OBJETIVOS ......................................................................................................... 61
CAPTULO 1. Revisin sobre la patologa y el tropismo tisular del virus
West Nile en aves infectadas de forma natural ................................................. 65
Resumen ............................................................................................................. 67
Abstract............................................................................................................... 68
Introduction ........................................................................................................ 69
Etiology ............................................................................................................... 69
Eco-epidemiology ................................................................................................ 71
Pathogenesis in birds .......................................................................................... 74
Pathology of natural WNV infection in birds ........................................................ 80
Discussion and conclusion .................................................................................. 91
Acknowledgements .............................................................................................. 94
References ........................................................................................................... 95
CAPTULO 2. Patogenia de la infeccin experimental con dos aislados

mediterrneos del virus West Nile en perdiz roja ...................................... 105
Resumen ........................................................................................................... 107
Abstract............................................................................................................. 108
Introduction ...................................................................................................... 109
Material and methods ....................................................................................... 110
Results .............................................................................................................. 114
Discussion......................................................................................................... 125
Acknowledgements ............................................................................................ 130
References ......................................................................................................... 131
Tesis doctoral
CAPTULO 3. Infeccin experimental de una especie gallincea

euromediterrnea con una cepa norteamericana del virus West Nile ........ 137
Resumen ........................................................................................................... 139
Abstract............................................................................................................. 140
Introduction ...................................................................................................... 141
Results .............................................................................................................. 146
Discussion......................................................................................................... 156
References ......................................................................................................... 161
CAPTULO 4. Infeccin natural por el virus Bagaza en aves cinegticas

en el sur de Espaa .................................................................................. 167
Resumen ........................................................................................................... 169
Abstract............................................................................................................. 170
Introduction ...................................................................................................... 171
Results .............................................................................................................. 175
Discussion......................................................................................................... 183
References ......................................................................................................... 187
CAPTULO 5. Hallazgos patolgicos en ojos de aves gallinceas infectadas

por flavivirus ............................................................................................ 191
Resumen ........................................................................................................... 193
Abstract............................................................................................................. 194
Introduction ...................................................................................................... 195
Results .............................................................................................................. 196
Discussion......................................................................................................... 200
References ......................................................................................................... 202
CAPTULO 6. Deteccin del virus Usutu en zorzales migratorios en

Espaa .................................................................................................... 203
Resumen ........................................................................................................... 205
Abstract............................................................................................................. 205
Introduction ...................................................................................................... 206
The study .......................................................................................................... 206
ii
ndice
Conclusions ...................................................................................................... 208

References ......................................................................................................... 209
SNTESIS ................................................................................................. 211

Bibliografa ........................................................................................................ 222
CONCLUSIONES ...................................................................................... 225
iii
Lista de abreviaturas
LISTA DE ABREVIATURAS
ARN
cido ribonucleico
BLAST
Basic Local Alignment Search Tool
bp
Base pairs
CD3/79
Cluster of differentiation 3/79
Ct
Cycle threshold
DNA
Deoxyribonucleic acid
ELISA
Enzyme-linked immunosorbent assay
GFAP
Glial fibrillary acidic protein
hr
Hour
IgG
Immunoglobulin G
kb
Kilobase
min
Minute
mL
Milliliter
mM
Millimolar
NK cells
Natural killer cells
No./Nos.
Number/Numbers
PCR
Polymerase chain reaction
PFU
Plaque-forming unit
pmol
Picomol
RCA
Ricinus communis agglutinin
RNA
Ribonucleic acid
RT-PCR
Reverse transcription-polymerase chain reaction
sec
Second
sp./spp.
Species
TNF
Tumoral necrosis factor
Units
UFP
Unidades formadoras de placa
Microgram
Microliter
Micrometer
Estructura de la tesis
ESTRUCTURA DE LA TESIS
La presente tesis doctoral se encuentra organizada de acuerdo al orden habitual
con el que se estructura un trabajo cientfico. La primera parte consiste en una
Introduccin en la que se realiza una sntesis sobre la informacin disponible
relacionada con los flavivirus transmitidos por mosquitos, centrndose en aquellos
que se encuentran circulando en Europa y, ms concretamente, en Espaa, y que
producen mortalidad en aves. A continuacin se enumeran los Objetivos generales
y especficos que se han planteado para realizar ste trabajo y que sern abordados
en cada uno de los seis captulos que componen esta tesis.
En el Captulo 1 se hace una revisin detallada sobre la informacin
disponible de la patogenia y los cambios morfolgicos asociados a la infeccin por el
virus West Nile (WNV) en aves. En los Captulos 2 y 3 se describen las lesiones y la
distribucin del virus durante el curso de la infeccin experimental de la perdiz roja
(Alectoris rufa) con dos aislados mediterrneos y un aislado norteamericano,
respectivamente, de WNV. En el Captulo 4 se describen los cambios morfolgicos y
la distribucin del virus en tejidos de perdiz roja, faisn comn (Phasianus
colchicus) y paloma torcaz (Columba palumbus) infectados de forma natural por el
virus Bagaza (BAGV). En el Captulo 5 se realiza una breve descripcin de las
lesiones y la distribucin del antgeno viral en ojos de aves gallinceas cinegticas
infectadas con WNV y BAGV. Finalmente, el Captulo 6 describe el primer caso de
mortalidad asociado a la infeccin por el virus Usutu en aves en Espaa. Todos
estos captulos estn redactados en ingls y corresponden a artculos cientficos en
diferente estado de publicacin.
Por ltimo, en el apartado de Sntesis se realiza una discusin general sobre
los hallazgos ms importantes obtenidos en cada uno de los trabajos que componen
la tesis doctoral, para terminar con las Conclusiones generales obtenidas de los
mismos.
1
Introduccin
Introduccin
ANTECEDENTES
Debido a la situacin estratgica que presenta Espaa en relacin al paso de aves
migratorias entre Europa y frica, su elevada riqueza en humedales (que sirven
como reas de invernada y nidificacin de muchas aves), su proximidad a frica
(considerada el origen de numerosos flavivirus), y las temperaturas relativamente
suaves que se suelen registrar durante gran parte del ao, fundamentalmente en el
rea mediterrnea (que permiten una amplia actividad de vectores capaces de
transmitir flavivirus), existen las condiciones ptimas para la introduccin de un
flavivirus y el establecimiento de una circulacin local y persistencia del mismo. A
pesar de que la circulacin de flavivirus en Espaa se detect hace ms de
cincuenta aos, ha sido en la ltima dcada cuando la actividad de estos virus ha
incrementado, provocando casos de enfermedad y mortalidad en humanos, caballos
y aves. Una situacin similar se est dando no slo en el resto de Europa, sino
tambin en Norteamrica, especialmente en relacin a uno de los flavivirus ms
distribuidos del mundo, considerado patgeno para las aves silvestres, el virus West
Nile (WNV). La emergencia o re-emergencia de la infeccin por un flavivirus est
asociada a factores no slo relacionados con el propio virus, sino tambin con el
ambiente, los vectores que los transmiten y los hospedadores a los que infectan. La
combinacin de estos factores hace que estos virus no resulten igualmente
patgenos en todas las reas del mundo y que no todos los hospedadores se vean
igualmente afectados por la infeccin. En la presente tesis doctoral se han realizado
una serie de estudios destinados a mejorar el conocimiento sobre las causas de la
emergencia o re-emergencia de la infeccin por estos virus en aves, as como de las
diferencias interespecie en la evolucin y las consecuencias de la infeccin.
Tesis doctoral
EL GNERO FLAVIVIRUS
Etiologa
El gnero Flavivirus, de la familia Flaviviridae, comprende aproximadamente 70
virus encuadrados en doce complejos antignicos (Heinz et al., 2000).
Los flavivirus son pequeos (50nm de dimetro) virus de morfologa
redondeada conformados por una nucleocpside de simetra polidrica rodeada por
una envoltura lipdica (Lindenbach y Rice, 2003) (Figura 1a). Su genoma consiste
en una cadena simple de ARN de polaridad positiva de 11kb de longitud que
configura tres protenas estructurales (cpside (C), premembrana/membrana
(prM/M) y envoltura (E)) y siete no estructurales (NS1, NS2A, NS2B, NS3, NS4A,
NS4B y NS5) (Brinton, 2002; Lindenbach y Rice, 2003) (Figura 1b). La protena E es
la ms importante de las protenas de la superficie de la partcula vrica,
participando en la unin a los receptores celulares y la fusin con la membrana
celular. Esta protena constituye el principal determinante antignico del virus,
siendo,
fundamentalmente
el
dominio
III,
el
objetivo
de
los
anticuerpos
neutralizantes del hospedador (Lindenbach y Rice, 2003; Martn-Acebes y Saiz,

2012). Las protenas no estructurales poseen diferentes funciones que, sobre todo,
son importantes en la replicacin vrica, pero tambin ejercen efectos inhibidores
sobre la respuesta inmune del hospedador (Brinton, 2002; Lindenbach y Rice,
2003; Martn-Acebes y Saiz, 2012). La protena NS5 es la de mayor tamao y la ms
conservada (Lindenbach y Rice, 2003) y su secuencia se utiliza para estudios
filogenticos (Kuno et al., 1998; Gaunt et al., 2001).
Introduccin
Figura
1.
a)
Morfologa
de
los
flavivirus
(Ilustracin
extrada
de
http://flavivirus.wordpress.com/general-flavivirus-info/). b) Organizacin del genoma y
funciones de las protenas virales (Ilustracin adaptada de Fernndez-Garca et al., 2009).
Clasificacin
Los flavivirus pueden ser clasificados en base a su secuencia nucleotdica,
antigenicidad, patogenicidad, distribucin geogrfica o forma de transmisin
(Calisher et al., 1989; Kuno et al., 1998; Gould y Solomon, 2008) (Figura 2). En
base
la
forma
de
transmisin,
hasta
el
ao
2009
se
contabilizaron
aproximadamente 54 arbovirus (39 flavivirus transmitidos por mosquitos y 15 por

garrapatas) y 17 flavivirus sin vector conocido (Mackenzie y Williams, 2009). Dentro
de los flavivirus transmitidos por mosquitos a su vez se pueden distinguir dos
grupos, el de los flavivirus transmitidos por Aedes spp. y el de los transmitidos
por Culex spp. (Gaunt et al., 2001; Kuno y Chang, 2005; Gould y Solomon, 2008)
(Figura 2). Sin embargo, todas estas clasificaciones no son absolutas, pudiendo
existir variaciones y discrepancias.
Tesis doctoral
Figura 2. rbol filogentico del gnero Flavivirus (Familia Flaviviridae). Asociacin en grupos
en base a su hospedador invertebrado, hospedador vertebrado y distribucin geogrfica
(Ilustracin extrada de Gould y Solomon, 2008).
Epidemiologa
Existen evidencias de que los flavivirus se originaron en el Viejo Mundo (Gaunt et
al., 2001; Gould et al., 2003), desde donde se distribuyeron a todos los continentes
excepto la Antrtida (Mackenzie y Williams, 2009). De esta forma, ha sido sugerida
Introduccin
la existencia de un ancestro africano para todos los virus que forman parte del
complejo antignico Encefalitis Japonesa (Mackenzie y Williams, 2009).
La circulacin de los flavivirus transmitidos por garrapatas y por mosquitos
Aedes spp., a excepcin del virus del dengue y de la fiebre amarilla, se restringe al
Viejo Continente, mientras que los transmitidos por mosquitos Culex spp. circulan
tanto en el Viejo como en el Nuevo Continente (Gaunt et al., 2001; Gould et al.,
2003) (Figura 2).
La mayora de los flavivirus transmitidos por mosquitos se caracterizan por
ser virus emergentes, es decir, tienden a establecerse en nuevas reas con relativa
facilidad y frecuencia. Esta propiedad se debe sobre todo a su epidemiologa
multifactorial, en la que influyen factores relacionados con el virus, el hospedador,
el vector o el ambiente (Monath, 1993; Kilpatrick et al., 2008; Pfeffer y Dobler,
2010; Jimnez-Clavero, 2012). Algunos ejemplos de flavivirus emergentes son el
virus Usutu (USUV) y su emergencia en Centroeuropa (Weissenbck et al., 2013),
WNV y su emergencia en Norteamrica (Lanciotti et al., 1999) y el virus Bagaza
(BAGV) y su emergencia en el sur de Europa (Agero et al., 2011).
Ecologa
Los flavivirus transmitidos por mosquitos pueden infectar a una gran variedad de
especies de mosquitos y hospedadores vertebrados. Algunos han sido asociados a
importantes mortalidades en humanos (virus del dengue y de la fiebre amarilla) y
otros, aparentemente, no resultan patgenos ni para el hombre ni para otros
vertebrados (virus Cacipacore) (Weissenbck et al., 2010).
La capacidad para producir enfermedad en humanos es considerada muy
importante, pero tambin los efectos econmicos y/o ecolgicos derivados de la
infeccin en animales.
Tesis doctoral
En general, los flavivirus transmitidos por Aedes spp. se mantienen en un

ciclo en el que participan mosquitos y primates (incluido el hombre), asocindose a
casos de enfermedad hemorrgica (Gaunt et al., 2001) (Figura 2). Sin embargo, los
flavivirus transmitidos por Culex spp., donde se encuadran los pertenecientes a los
complejos antignicos Encefalitis Japonesa y Ntaya, suelen mantenerse en ciclos
enzoticos que implican, sobre todo, a mosquitos ornitfagos y aves (Figura 2).
stos ltimos generalmente circulan de forma silenciosa, pero bajo ciertas
circunstancias pueden dar lugar a brotes en sus hospedadores naturales o ser
transmitidos a hospedadores accidentales, como son los mamferos, y entre ellos el
hombre, dando lugar a epizootias o epidemias (Monath, 1993; Gaunt et al., 2001).
Generalmente, los mosquitos que producen este tipo de transmisin no son los
mismos que mantienen el ciclo enzotico y estos hospedadores accidentales, si bien
pueden sufrir casos de encefalitis grave, son considerados como fondos de saco,
ya que no desarrollan una viremia suficiente que permita la transmisin del
patgeno a mosquitos (Gaunt et al., 2001; Mackenzie y Williams, 2009).
FLAVIVIRUS EN EUROPA Y ESPAA

Los flavivirus transmitidos por mosquitos que circulan en Europa y que
recientemente han provocado brotes en sus hospedadores naturales son WNV,
USUV y BAGV. WNV y USUV adems son capaces de producir enfermedad en
humanos y WNV ha dado lugar a episodios de mortalidad en caballos.
Por otro lado, anualmente se producen casos de enfermedad en humanos
asociados a infecciones por flavivirus adquiridos en los trpicos, como son por
ejemplo el virus del dengue, de la fiebre amarilla, de la encefalitis japonesa y el
virus Chikungunya (Hublek, 2008a; Reiter, 2010; Zeller, 2012).
Dada la situacin estratgica de Espaa en relacin al paso de aves
migratorias entre Europa y frica, y la importancia de nuestros humedales como
10
Introduccin
reas de invernada y nidificacin de muchas de estas aves, en nuestro pas existe

un riesgo elevado de aparicin de brotes asociados a la infeccin por virus
introducidos por aves migratorias. La circulacin de flavivirus en nuestro pas ha
sido demostrada por medio de serologa desde los aos 60, sobre todo en la zona
mediterrnea (Filipe y Andrade, 1990; Figuerola et al., 2007a; Bueno-Mar y
Jimnez-Peydr, 2010). La circulacin de WNV haba sido silenciosa hasta hace
relativamente poco tiempo. Sin embargo, en los ltimos seis aos se han producido,
por primera vez, casos de mortalidad en aves asociados a la infeccin por WNV,
BAGV y USUV (Jimnez-Clavero et al., 2008; Agero et al., 2011; Hfle et al., 2013).
Virus West Nile

El virus West Nile es uno de los flavivirus ms ampliamente distribuidos del
mundo, con una gran importancia tanto en sanidad animal como en salud pblica.
WNV fue aislado por primera vez de la sangre de una mujer con un proceso
febril en Uganda en 1937 (Smithburn et al., 1940) y desde ah pas a ser detectado
en numerosas partes del mundo, donde emergieron nuevos genotipos (Petersen,
2009). Los aislados de WNV se han clasificado filogenticamente en 7 linajes, siendo
los linajes 1 y 2 los ms importantes (Mackenzie y Williams, 2009) (Figura 3). El
linaje 1 contiene las cepas consideradas ms patgenas, que estn ampliamente
distribuidas por Europa, Amrica, Oriente Medio, India, frica y Australia (Lanciotti
et al., 2002). El linaje 2 estaba conformado por cepas que slo circulaban en el sur
de frica y Madagascar pero recientemente ha sido detectado en Europa, donde ha
dado lugar a casos de encefalitis grave en humanos, aves y caballos (Bakonyi et al.,
2006; Bagnarelli et al., 2011; Kutasi et al., 2011; Papa et al., 2011). Antes de la
dcada de los 90, a pesar de que WNV era uno de los flavivirus ms distribuidos en
humanos, aves y mosquitos de frica, Oriente Medio y el suroeste de Europa, slo
ocasionalmente daba lugar a infecciones que generalmente eran subclnicas o poco
11
Tesis doctoral
importantes y que afectaban sobre todo a humanos y caballos (Hublek y Halouzka,

1999; Murgue et al., 2002). Sin embargo, a partir de los aos 90 la frecuencia y
gravedad de las infecciones en humanos increment, as como el nmero de casos
en otros vertebrados, incluyendo animales de compaa, de granja y silvestres (van
der Meulen et al., 2005). En el ao 1999 WNV fue introducido en Norteamrica
(Lanciotti et al., 1999; Nash et al., 2001; Ludwig et al., 2002), donde en el ao 2002
emergi un nuevo genotipo (WN02) con capacidad para diseminarse de forma ms
eficiente, que desplaz al anterior (Davis et al., 2005). Desde entonces han ido
emergiendo nuevos genotipos que han dado lugar a miles de casos de encefalitis en
humanos, caballos y aves (CDC; McMullen et al., 2011).
12
Introduccin
Figura 3. rbol filogentico de WNV (Ilustracin extrada de Vzquez et al., 2011).
WNV pertenece al complejo antignico Encefalitis Japonesa y, por tanto, su

reservorio natural son las aves silvestres (McLean et al., 2001). Las aves migratorias
13
Tesis doctoral
juegan un papel importante en la dispersin local y a larga distancia del virus

(Malkinson et al., 2002a,b; Peterson et al., 2003), habindose demostrado que stas
presentan ttulos de anticuerpos frente al virus ms altos que las aves residentes
(Lpez et al., 2008; Dusek et al., 2009; Valiakos et al., 2012). Existen especies de
anfibios, reptiles y mamferos, entre ellos el hombre, que pueden infectarse y sufrir
encefalitis, sin embargo, en la mayor parte de los casos no desarrollan suficiente
viremia como para ser consideradas especies reservorio (Kostiukov et al., 1985,
1986; Rodhain et al., 1985; Turell et al., 2000; Sardelis et al., 2001; Xiao et al.,
2001; Komar et al., 2003a; Klenk et al., 2004; Root, 2012).
WNV ha sido aislado de numerosas especies de mosquitos, pero slo aquellos
en los que se replica y llega hasta las glndulas salivares para poder ser
transmitido, son vectores competentes (Hublek, 2008a). En Europa, los principales
vectores del virus son Culex pipiens, Cx. modestus y Coquillettidia richiardii
(Hublek y Halouzka, 1999).
Otras vas de transmisin de WNV en aves son el consumo de presas
infectadas o agua contaminada y el contacto con individuos infectados (Komar et
al., 2003b; Nemeth et al., 2006a) (Figura 4). Adems, en humanos el virus se puede
transmitir mediante trasplante de rganos, transfusiones de sangre, consumo de
leche materna, a travs de la placenta y en accidentes de laboratorio (CDC
2002a,b,c; Iwamoto et al., 2003; Pealer et al., 2003).
Contacto
Oral
Figura 4. Ciclo de transmisin de WNV (Elaboracin propia).
14
Introduccin
En reas endmicas, la infeccin en aves suele tener lugar en primavera y a

principios de verano, apareciendo los picos de mortalidad desde mitad del verano
hasta principios de otoo. Los casos en humanos y en caballos, si existen, suelen
producirse algunas semanas despus del inicio de la mortalidad en aves (Phalen y
Dahlhausen, 2004).
La susceptibilidad de las aves a la infeccin vara en funcin de la especie,
por lo que no todas llegan a sufrir enfermedad (Komar et al., 2003b). En Europa,
WNV ha sido detectado en numerosas aves residentes y migratorias, acuticas y
terrestres, pero los casos de mortalidad han sido
espordicos, afectando
fundamentalmente a aves rapaces (Bakonyi et al., 2006; Jimnez-Clavero et al.,

2008; Wodak et al., 2011). Por el contrario, en Norteamrica existen en torno a 300
especies de aves susceptibles (CDC) y la tasa de mortalidad en muchas de ellas es
elevada.
Algunas
poblaciones
como
la
de
cuervos
americanos
(Corvus
brachyrhynchos) han sufrido un grave declive debido a la mortalidad provocada por

este virus (LaDeau et al., 2007; Foppa et al., 2011).
En humanos, slo un 20% de las infecciones son de carcter clnico y, de
stas, menos del 1% son neuroinvasivas. La mayor parte de los casos clnicos se
producen en individuos de edad avanzada o inmunocomprometidos y la mortalidad
se mantiene en torno al 10% (Hayes et al., 2006; Sejvar, 2007). En caballos, slo un
10% de individuos desarrolla enfermedad neurolgica, con una mortalidad que
puede llegar al 50% (Castillo-Olivares y Wood, 2004; Garca-Bocanegra et al.,
2011a).
WNV en Espaa
La presencia de anticuerpos frente a WNV fue descrita por primera vez en
humanos y micromamferos entre los aos 60 y 80 (Filipe y Andrade, 1990) (Tabla
1). Posteriormente, se han seguido detectando anticuerpos en humanos, aves y
caballos, fundamentalmente en la zona mediterrnea (Tabla 1).
15
Tesis doctoral
La mayora de los estudios realizados, sobre todo en aves, se centran en el

suroeste de Espaa debido a que es un rea considerada de alto riesgo como
consecuencia de la existencia de zonas de cra de aves migratorias y poblaciones
abundantes de aves silvestres y vectores competentes, as como por su proximidad
a frica, donde WNV es endmico (Garca-Bocanegra et al., 2011b, 2012a,b). La
circulacin de WNV en esta zona es tan importante que en el ao 2006 se detect
en un pool de Cx. pipiens un posible nuevo linaje del virus (Vzquez et al., 2010)
(Tabla 1).
Los estudios serolgicos realizados en aves adems indican que existe
transmisin vertical de anticuerpos (Figuerola et al., 2007a), que hay circulacin
local de WNV en el sur de Espaa (Figuerola et al., 2007b) y que la seroprevalencia
est relacionada con la actividad migratoria (Lpez et al., 2008) y con el tamao
corporal y la ecologa del ave (Figuerola et al., 2008).
El primer caso de enfermedad asociada a la infeccin por WNV se produjo en
humanos en el ao 2004 (Kaptoul et al., 2007) (Tabla 1). En 2007 tuvo lugar el
primer y nico caso de encefalitis y mortalidad en aves y WNV fue aislado de
muestras de dos guilas reales (Aquila chrysaetos) y un guila perdicera (Aquila
fasciata) (Jimnez-Clavero et al., 2008) (Tabla 1). La cepa aislada mostr una gran
homologa gentica con la aislada en Italia en 1998 (Sotelo et al., 2009) y una muy
similar a sta fue detectada y aislada de mosquitos Cx. perexiguus un ao despus
en la cuenca del Guadalquivir (Vzquez et al., 2011) (Tabla 1). En el ao 2010 se
produjo el primer caso de encefalitis causado por WNV en caballos y, coincidiendo
con ste, dos casos de encefalitis en humanos en el suroeste del pas (GarcaBocanegra et al., 2011a; Jess-De La Calle et al., 2012) (Tabla 1). La cepa que
caus este brote mostr mayor homologa gentica con las aisladas en Italia en
2008 y 2009 que con las aisladas en Espaa en 2007 y 2008 (Sotelo et al., 2011a).
16
Introduccin
En el ao 2011 y 2012 tuvieron lugar nuevos casos de enfermedad en caballos en la

misma zona (RASVE, 2013) (Tabla 1).
Tabla 1. Deteccin cronolgica de la circulacin de WNV y otros flavivirus en Espaa.
Elaboracin propia.
Ao de
muestreo
Especie
1960-1970
Humanos
1973
Humanos
Humanos
1978-1979
Micromamferos
Finales de los
70
-
rea de
muestreo
Tipo de deteccin
Referencia bibliogrfica
Galicia, Castilla y
Len, Asturias
Comunidad
Valenciana
Andaluca
Anticuerpos
Gonzlez y Filipe, 1977
Anticuerpos
Sanchs-Bayarri, 1974
Anticuerpos
Lozano, 1980
Anticuerpos
Chastel et al., 1980
Humanos
Aragn,
Extremadura,
Andaluca
Catalua
Anticuerpos
Lozano y Filipe, 1998
Humanos
Andaluca
Anticuerpos
Pujol et al., 2004
2001
Humanos
Catalua
Anticuerpos
Bofill et al., 2006
2001-2005
guila imperial ibrica

(Aquila adalberti)
Aves
Castilla-La
Mancha
Andaluca
Anticuerpos, genoma
y antgeno viral
Anticuerpos
Hfle et al., 2008
Andaluca
Anticuerpos
2003?
Focha comn (Fulica

atra)
Humanos
Andaluca
Anticuerpos
2003-2011
Jabal (Sus scrofa)
Centro y sur de
Espaa
Anticuerpos
2004
Humanos
Catalua
2004-2006
Aves
Andaluca
Anticuerpos, caso
clnico
Anticuerpos
2005-2008
Caballos
Andaluca
Anticuerpos
2006
Halcn de Eleonora
(Falco eleonorae)
Mosquitos (Cx. pipiens)
Islas Canarias
Anticuerpos
Andaluca
Vzquez et al., 2010
2006-2008
Ciervo (Cervus
elaphus)
Zorro (Vulpes vulpes)
Anticuerpos
2006-2009
Aves
Centro y sur de
Espaa
Centro y suroeste
de Espaa
Andaluca
Genoma viral y
aislamiento
Anticuerpos
2007
guila real
guila perdicera
Urraca (Pica pica)
Castilla-La
Mancha
Gutirrez-Guzmn et al.,
2012
Garca-Bocanegra et al.,
2011b
Jimnez-Clavero et al.,
2008
2008
Mosquitos
Andaluca
2009-2010
Cerdo ibrico
2010
Humanos
Centro y suroeste
de Espaa
Andaluca
2003-2005
2003-2006
2006
2006
Anticuerpos
Anticuerpos, genoma
viral y aislamiento,
caso clnico y
mortalidad
Genoma viral y
aislamiento
Anticuerpos
Anticuerpos, caso
clnico
Figuerola et al., 2007a,

2008
Figuerola et al., 2007b
Bernabeu-Wittel et al.,
2007
Boadella et al., 2012;
2012
Kaptoul et al., 2007
Lpez et al., 2008, 2011
Jimnez-Clavero et al.,
2007, 2010
Gangoso et al., 2010
Boadella et al., 2012
Vzquez et al., 2011

2012
2011a; Jess-De La Calle
et al., 2012
17
Tesis doctoral
Tabla 1. Deteccin cronolgica de la circulacin de WNV y otros flavivirus en Espaa (cont).

Elaboracin propia.
Ao de
muestreo
Especie
rea de
muestreo
Tipo de deteccin
2010
Mulas y burros
Andaluca
Anticuerpos
2010-2012
Caballos
Andaluca
2011-2012
Perdiz roja (Alectoris rufa)

Faisn comn
(Phasianus colchicus)
Andaluca
Anticuerpos y
genoma viral, caso
clnico y mortalidad
Anticuerpos
2012c
2011a, 2012a,d; RASVE,
2013
Llorente et al., 2013
Estudio de la infeccin por WNV en aves

La infeccin por WNV en aves ha sido fundamentalmente
estudiada en
Norteamrica, puesto que es donde ms casos de mortalidad se han producido.

Existen varios trabajos describiendo las lesiones macroscpicas y microscpicas
asociadas a la infeccin natural, fundamentalmente en crvidos y en rapaces, pero
tambin numerosos trabajos experimentales en los que se han descrito la patogenia
de la infeccin as como la distribucin temporal de WNV en los diferentes tejidos.
En
Europa,
existen menos
trabajos
de esta
naturaleza,
los
que
hay
fundamentalemente se refieren a aves rapaces diurnas (Bakonyi et al., 2006; Hfle

et al., 2008; Wodak et al., 2011; Busquets et al., 2012; Ziegler et al., 2012; Dridi et
al., 2013).
En el captulo 1 de esta tesis profundizaremos en los datos existentes sobre
la dinmica de la infeccin por WNV en aves.
Virus Usutu
El virus Usutu fue aislado por primera vez de mosquitos Culex neavei en Sudfrica
en 1959 (Woodall, 1964) y de otros mosquitos y aves en el continente africano en
las dcadas posteriores (Williams et al., 1964; Adam y Digoutte, 2005). En el ao
1996, el virus lleg a Europa produciendo mortalidad en mirlo comn (Turdus
merula) en Italia (Weissenbck et al., 2013). A partir de ese momento, se sospecha
18
Introduccin
que se estableci una circulacin local entre aves y mosquitos del centro de Europa,
dando lugar a brotes posteriores que han afectado exclusivamente a aves
(Weissenbck et al., 2002, 2003; Bakonyi et al., 2007; Manarolla et al., 2010; Savini
et al., 2011; Steinmetz et al., 2011; Becker et al., 2012) (Tabla 2). La homologa
gentica de las cepas que circulan en el centro de Europa es muy elevada (Chvala et
al., 2007; Steinmetz et al., 2011; Peletto et al., 2012), pero en la Cuenca
Mediterrnea circulan adems otras cepas que probablemente hayan sido
introducidas posteriormente desde frica (Busquets et al., 2008; Savini et al., 2011;
Vzquez et al., 2011; Peletto et al., 2012).
Al igual que WNV, USUV pertenece al complejo antignico Encefalitis
Japonesa, por lo que su reservorio natural son las aves. En Europa, los principales
vectores de USUV pertenecen al gnero Culex (Weissenbck et al., 2007; Tamba et
al., 2011).
Las aves del orden Passeriformes, fundamentalmente el mirlo comn, y las
del orden Strigiformes son las ms susceptibles a la infeccin (Weissenbck et al.,
2002; Bakonyi et al., 2007). Sin embargo, la circulacin del virus ha sido detectada
mediante PCR, aislamiento vrico y serologa en varias especies de aves residentes y
migratorias en diferentes pases europeos (Tabla 2).
19
Tesis doctoral
Tabla 2. Deteccin de USUV en muestras de aves en diferentes pases de Europa. Elaboracin

propia.
Pas
Ao
Tipo de deteccin
Alemania
2000-2005
2011
2000-2006
Anticuerpos
Genoma y antgeno viral, aislamiento
Genoma y antgeno viral
2005-2006
2001-2002
2004
1996
2005
2006-2008
2007
2008-2009
2010
2006
Genoma y antgeno viral, aislamiento

Anticuerpos
Anticuerpos
Anticuerpos
Anticuerpos
Anticuerpos, genoma viral,
aislamiento viral
Genoma viral
Anticuerpos
Linke et al., 2007

Becker et al., 2012
Weissenbck et al., 2002, 2003; Chvala
et al., 2004, 2007; Meister et al., 2008
Bakonyi et al., 2007
Buckley et al., 2003
Buckley et al., 2006
Weissenbck et al., 2013
Rizzoli et al., 2007
Manarolla et al., 2010
Lelli et al., 2008
Savini et al., 2011; Tamba et al., 2011
2005
2011-2012
2006-2007
2009
Anticuerpos
Genoma viral, aislamiento viral
Anticuerpos, genoma y antgeno viral
Austria
Hungra
Inglaterra
Italia
Polonia
Repblica Checa
Suiza
Calzolari et al., 2012

Hublek, 2008b
Hublek, 2008c
Hublek, 2012
Steinmetz et al., 2007, 2011
Steinmetz et al., 2011
La susceptibilidad de los humanos a la infeccin parece ser baja en

comparacin a la que existe frente a otros flavivirus como WNV, sin embargo, USUV
fue aislado de un paciente con fiebre y erupciones en frica (Williams et al., 1964;
Adam y Digoutte, 2005). Adems, en Europa ha sido detectado por PCR en un
paciente en Austria y en pacientes inmunodeprimidos con un proceso neurolgico
en Italia (Weissenbck et al., 2007; Cavrini et al., 2009, 2011; Pecorari et al., 2009),
y por PCR y serologa en donantes de sangre asintomticos en Italia y Alemania
(Gaibani et al., 2010, 2012; Allering et al., 2012).
Se desconoce la susceptibilidad de otros vertebrados a la infeccin, pero en
Italia y Serbia se han detectado anticuerpos frente a USUV en caballos (Lupolovic et
al., 2011; Savini et al., 2011).
USUV en Espaa
La informacin relativa a la circulacin de USUV en Espaa es muy escasa. El virus
se detect por primera vez en un pool de mosquitos Cx. pipiens en Catalua en el
ao 2006 (Busquets et al., 2008) y en un pool de Cx. perexiguus en el sur de
20
Introduccin
Espaa en el ao 2009, mostrando ambas cepas una gran homologa gentica

(Vzquez et al., 2011). En un trabajo realizado en 2013 se ha demostrado la
presencia de anticuerpos frente al virus en una perdiz roja en el sur de Espaa
(Llorente et al., 2013).
Estudio de la infeccin por USUV en aves
Los hallazgos clnicos y los cambios morfolgicos asociados a la infeccin natural
por USUV en aves han sido descritos fundamentalmente en mirlo comn y en
algunas especies de rapaces nocturnas. Los signos clnicos que se pueden observar
son depresin, apata, anorexia, erizamiento de las plumas, incapacidad para volar,
incoordinacin y convulsiones, y muerte rpida, aproximadamente a los tres das
del comienzo de la signologa (Manarolla et al., 2010; Steinmetz et al., 2011; Becker
et al., 2012). Las lesiones macroscpicas sobre todo se detectan en hgado y en bazo
y microscpicamente lo ms caracterstico es la presencia de encefalitis y necrosis e
infiltrados inflamatorios multiorgnicos (Tabla 3).
Existen muy pocos estudios experimentales por lo que se tienen pocos datos
sobre la patogenia de la infeccin por USUV en aves. En un experimento realizado
con pollos de gallina domstica (Gallus domesticus) y en otro realizado con ganso
domstico (Anser anser domesticus) los animales no mostraron signos clnicos y las
lesiones macroscpicas y microscpicas fueron moderadas (Chvala et al., 2005,
2006). En ninguno de los casos se consigui demostrar la presencia del antgeno
viral mediante inmunohistoqumica (IHQ). Se detect material gentico del virus en
tejidos desde el da 2-3 post inoculacin (pi) hasta el da 14-16, viremia en muy
pocos individuos entre los das 5 y 7, y desarrollo anticuerpos el da 10 pi. Ninguna
de las dos especies, por tanto, puede ser considerada como un hospedador
competente o altamente susceptible a la infeccin por USUV. Los resultados
obtenidos en pollos de gallina son muy similares a los descritos en la infeccin
experimental con WNV (Senne et al., 2000). No es as en el caso de ganso
21
Tesis doctoral
domstico, en el que la infeccin experimental con WNV dio lugar a una encefalitis
fatal, demostrando que esta especie es ms susceptible a la infeccin por WNV que
por USUV (Swayne et al., 2001).
22
Weissenbck et al., 2003; Chvala et

al., 2004; Manarolla et al., 2010;
Steinmetz et al., 2011
Clulas glomerulares y epiteliales

tubulares del rin
Fibrocitos y clulas musculares de la

cpsula del bazo, macrfagos y
clulas dendrticas
Clulas inflamatorias
intravasculares, clulas del ovario
Hepatitis, necrosis coagulativa

heptica y hemorragias
Nefritis y degeneracin de clulas

epiteliales tubulares
Necrosis coagulativa esplnica,

hiperplasia folicular y de la pulpa
roja
Enteritis seromucosa
Hepatomegalia, congestin heptica y

focos hepticos amarillentos
Nefromegalia
Esplenomegalia
No descritas
Sistema gastrointestinal
Sistema endocrino
Sistema urinario
Sistema inmunitario
Otros
Leucocitolisis
Weissenbck et al., 2002, 2003;

Chvala et al., 2004; Bakonyi et al.,
2007; Manarolla et al., 2010;
Steinmetz et al., 2011; Becker et al.,
2012; Calzolari et al., 2012
Clulas de Kupffer en hgado
Proventriculitis y abscesos en criptas

intestinales

al., 2004; Bakonyi et al., 2007;
Manarolla et al., 2010; Steinmetz et
al., 2011; Weissenbck et al., 2013

2012

Manarolla et al., 2010
Ganglios autnomos, clulas

glandulares y de la tnica muscular
de proventrculo y molleja, y clulas
epiteliales de las criptas y tnica
muscular de intestino
Miocarditis y miocitolisis
No descritas
Sistema cardiovascular

Manarolla et al., 2010; Steinmetz et
al., 2011; Becker et al., 2012
Miofibrillas y clulas intersticiales del

corazn, clulas musculares y
endoteliales de la pared de grandes
vasos
Neumona y edema
Congestin, edema y hemorragia

pulmonar
Sistema respiratorio

2012; Weissenbck et al., 2013
al., 2004; Manarolla et al., 2010;
2012
Neuronas, clulas de la gla y clulas

inflamatorias
Neuronofagia, gliosis, manguitos

perivasculares, satelitosis,
degeneracin y necrosis neuronal y
edema
Hiperemia en meninges y
parnquima cerebral
Sistema nervioso central
REFERENCIA BIBLIOGRFICA
Clulas parenquimatosas del pulmn
ANTGENO VIRAL
LESIONES MICROSCPICAS
LESIONES MACROSCPICAS
SISTEMA ORGNICO
Tabla 3. Infeccin natural por USUV en aves. Lesiones macroscpicas, microscpicas y distribucin del antgeno viral (IHQ) en los diferentes sistemas
orgnicos del hospedador. Elaboracin propia.
Introduccin
23
Tesis doctoral
Virus Bagaza
El virus Bagaza es un flavivirus relativamente desconocido. Fue aislado por primera
vez de mosquitos del gnero Culex en la Repblica Centroafricana en 1966
(Digoutte, 1978) y despus detectado en mosquitos en otros pases de frica y la
India (Traore-Lamizana et al., 1994; Diallo et al., 2005; Bondre et al., 2009).
BAGV pertenece al grupo antignico Ntaya (Calisher et al., 1989), donde se
encuentran otros flavivirus como el de la meningoencefalitis del pavo (ITMV), que es
considerado sinnimo de BAGV (Kuno et al., 1998). ITMV fue descrito por primera
vez en Israel en 1960 (Komarov y Kalmar, 1960) y es un virus de alta patogenicidad
para aves domsticas, fundamentalmente pavos (Meleagris gallopavo), causando un
proceso neurolgico muy similar al encontrado en la infeccin por otros flavivirus
(Komarov y Kalmar, 1960; Barnard et al., 1980). Este flavivirus slo ha sido
detectado en Oriente Medio y el sur de frica (Kuno et al., 1998) y aparentemente
no es patgeno para humanos (Guy y Malkinson, 2008).
Se desconoce cul es el reservorio de BAGV. Algunos estudios han indicado
que son las aves y que se transmite fundamentalmente por mosquitos del gnero
Culex, al igual que WNV y USUV (Gaunt et al, 2001). Hasta hace relativamente poco
se desconoca la suceptibilidad de las aves a la infeccin por BAGV, pero al ser
genticamente muy similar a ITMV se especulaba que podra causar enfermedad en
el hospedador aviar (Kuno y Chang, 2007).
La suceptibilidad de los humanos est poco caracterizada, sin embargo, en el
ao 1996 se detectaron anticuerpos frente al virus en pacientes con encefalitis en la
India (Bondre et al., 2009) y Woolhouse y cols. (2006) identificaron a BAGV como
un patgeno emergente o re-emergente capaz de producir enfermedad en humanos.
BAGV en Espaa
El primer caso de mortalidad asociado a la infeccin por BAGV en aves se produjo
en el suroeste de Espaa en el verano de 2010 (Agero et al., 2011). BAGV afect
24
Introduccin
fundamentalmente a perdiz roja (Alectoris rufa) y faisn comn (Phasianus

colchicus), con una mayor mortalidad en la primera (Agero et al., 2011; GarcaBocanegra et al., 2012b).
El modo en el que BAGV lleg a Espaa se desconoce, sin embargo, su
secuencia gentica mostr una gran homologa con la cepa aislada en frica y en
menor medida con la detectada en la India (Agero et al., 2011), por lo que se
baraja la posibilidad de su introduccin por aves migratorias desde frica (Agero
et al., 2011; Garca-Bocanegra et al., 2012b). La elevada morbilidad de la infeccin
pudo estar relacionada con la abundancia de vectores competentes y la alta
densidad de aves en la zona (Garca-Bocanegra et al., 2011a, 2012b).
Estudio de la infeccin por BAGV en aves
Existen muy pocos trabajos en los que se describan las lesiones asociadas a la
infeccin por BAGV en aves. En general, los signos clnicos y las lesiones
macroscpicas y microscpicas encontradas son similares a los provocados por la
infeccin por ITMV (Komarov y Kalmar, 1960; Barnard et al., 1980). Las aves
infectadas en el brote de Espaa mostraron debilidad, postracin, incoordinacin,
prdida de peso y diarrea blanquecina (Agero et al., 2011; Garca-Bocanegra et al.,
2012b). Las lesiones macroscpicas principales fueron caquexia y congestin
generalizada y dentro de las microscpicas lo ms caracterstico fue la presencia de
meningoencefalitis y neuritis (Garca-Bocanegra et al., 2012b).
En el captulo 4 de esta tesis se darn ms datos sobre la infeccin natural
por este virus en aves.
25
Tesis doctoral
PATOGENIA Y DESARROLLO DE ENCEFALITIS EN LA INFECCIN POR

FLAVIVIRUS TRANSMITIDOS POR MOSQUITOS
La patogenia de la infeccin tras la inoculacin de un flavivirus por un mosquito
infectado en un hospedador vertebrado suele seguir el mismo esquema (Figura 5).
En mamferos, una vez que el virus es inoculado en la piel, y tras una posible
replicacin en los queratinocitos (Lim PY et al., 2011), ste es transportado por
clulas dendrticas o clulas de Langerhans desde la zona de inoculacin hacia los
linfonodos regionales (Johnston et al., 2000) (Figura 5). Aqu, se produce la
replicacin inicial del flavivirus (McMinn et al., 1996) ante la cual el hospedador
desarrolla una respuesta inmune inmediata en la que intervienen, entre otros,
mediadores como el interfern (IFN) tipo I y clulas como los macrfagos (Chambers
y Diamond, 2003; Arjona y Fikrig, 2009; Lim SM et al., 2011) (Figura 5). Diversos
trabajos han indicado que la produccin de IFN por las clulas dendrticas de la piel
es la primera lnea de defensa (Libraty et al., 2001) y que los macrfagos son clulas
diana de algunos flavivirus, jugando un papel importante en la patogenia de la
enfermedad (Steele et al., 1999; Chambers y Diamond, 2003; Garca-Tapia et al.,
2006). Desde los linfonodos regionales el virus pasa a la circulacin sangunea, a
travs de la cual llega a diferentes rganos, establecindose una infeccin sistmica
y producindose una segunda replicacin vrica (Figura 5). Los flavivirus muestran
un amplio tropismo celular y tisular pudiendo infectar clulas epiteliales,
parenquimatosas, endoteliales y musculares, as como fibroblastos (Chambers y
Diamond, 2003; Weingartl et al., 2004). Ante la infeccin sistmica, el hospedador
desarrolla una respuesta inmune en la que intervienen, adems de otros efectores,
linfocitos B y T que reaccionan frente a diferentes determinantes antignicos del
virus, como la protenas E y NS1, y que tratan de frenar la replicacin vrica
(Chambers y Diamond, 2003; Samuel y Diamond, 2006) (Figura 5). La informacin
sobre la fase inicial de la patogenia en aves es muy limitada, pero se sospecha que
26
Introduccin
tras una amplificacin local en la zona de inoculacin se produce una rpida

viremia e infeccin sistmica (Nemeth et al., 2011, para ms detalles ver captulo 1).
Los flavivirus han desarrollado numerosos mecanismos que les permiten evadir la
respuesta inmune del hospedador (Ye et al., 2013), por lo que en funcin del nivel
de replicacin perifrica y la efectividad de la respuesta inmune y, por tanto, del
nivel de viremia, stos pueden llegar a alcanzar el sistema nervioso central (SNC)
dando lugar a encefalitis (Albrecht, 1968; McMinn et al., 1996; Diamond et al.,
2003; Chambers y Diamond, 2003; Samuel y Diamond, 2006; Lim SM et al., 2011)
(Figura 5).
27
Tesis doctoral
Figura 5. Patogenia de la infeccin por un flavivirus transmitido por mosquitos en un

hospedador mamfero (Ilustracin modificada de Suthar et al., 2013).
28
Introduccin
En el caso de WNV, la entrada va oral o inhalatoria han sido sugeridas en

humanos y han sido demostradas en animales infectados de forma natural y
experimental (Nir et al., 1959, 1965; Odelola y Oduye, 1977; Garmendia et al.,
2000; Kuno, 2001; CDC 2002a, 2003; Komar et al., 2003b; Miller et al., 2003;
Austgen et al., 2004; Klenk et al., 2004). Sin embargo, debido al desconocimiento
de la importancia epidemiolgica de estos tipos de transmisin, existen muy pocos
trabajos en los que se haya estudiado la patogenia. En un experimento realizado en
el ao 1965, la exposicin de ratones a aerosoles contaminados con WNV provoc la
entrada del virus al cerebro a travs de los bulbos olfatorios sin necesidad de
viremia previa, as como la replicacin inicial del virus en pulmn y diseminacin
posterior a sangre, bazo y otros tejidos (Nir et al., 1965). Por otro lado, la infeccin
experimental por va oral en ratones permiti detectar el virus en cerebro dos das
antes que en sangre, por lo que, de nuevo, se asumi que el virus accedi al SNC a
travs de los bulbos olfatorios sin necesidad de una replicacin perifrica previa
(Odelola y Oduye, 1977). En general, tanto en aves como en otros hospedadores, la
infeccin por va oral provoca un retraso en la aparicin de viremia, aunque sta
suele alcanzar los mismos niveles que en el caso de la transmisin por la picadura
de un mosquito, las lesiones suelen ser ms moderadas y en muchos casos los
individuos infectados no muestran signos clnicos ni existe mortalidad (Komar et
al., 2003b; Klenk et al., 2004; Sbrana et al., 2005; Nemeth et al., 2006a,b;
Steinman et al., 2006).
La patogenia de la infeccin por un flavivirus est influenciada por factores
relacionados con el virus y el hospedador. Existen numerosos determinantes
genticos del virus que modulan su interaccin con el hospedador y una simple
mutacin puede dar lugar a un cambio en su virulencia, ya sea por una
modificacin en la capacidad de replicacin, en la susceptibilidad a la respuesta
inmune del hospedador, o en la sensibilidad a la temperatura corporal del mismo
29
Tesis doctoral
(Brinton et al., 2002; Kinney et al., 2006; Ye et al., 2013). En animales infectados
experimentalmente se ha demostrado que la existencia de cambios en las protenas
virales como la protena E, la NS3 o la NS4b pueden modificar la virulencia de un
flavivirus (Beasley et al., 2005; Wicker et al., 2006; Brault et al., 2007). As por
ejemplo, la glicosilacin de la protena E de WNV incrementa la replicacin
perifrica en pollos infectados de forma experimental y finalmente su virulencia
(Murata et al., 2010; Totani et al., 2011) y la sustitucin del aminocido treonina
por prolina en la posicin 249 de la protena NS3 incrementa la virulencia de WNV
en la infeccin experimental de cuervos americanos (Brault et al., 2004, 2007). El
estado inmunitario del hospedador es el factor ms importante del que depende la
susceptibilidad a la infeccin (Solomon y Winter, 2004). La capacidad de respuesta
inmune est a su vez condicionada por numerosos factores como la existencia de
inmunidad previa frente al virus o a flavivirus relacionados antignicamente (Tesh
et al., 2002; Fang y Reisen, 2006), la presencia de enfermedades concomitantes
(Murray et al., 2006) y la edad (Eldadah et al., 1967). De la edad del hospedador
depende la expresin de receptores celulares a los que se pueda unir el virus o de
factores intracelulares que participan en la patogenia de la infeccin (Chambers y
Diamond, 2003). Numerosos autores han indicado que la susceptibilidad a la
infeccin tambin tiene componentes hereditarios (Chambers y Diamond, 2003;
Bigham et al., 2011; Clark et al., 2012).
En funcin de la combinacin de estos factores la infeccin por un flavivirus
puede desencadenar tres cuadros clnicos diferentes (Monath, 1986):
a) Infeccin inaparente, con una viremia transitoria, replicacin perifrica
limitada y ausencia de neuroinvasin.
b) Encefalitis subclnica, con una viremia moderada, establecimiento tardo de
infeccin en el SNC y eliminacin del virus con un desarrollo mnimo de
lesiones.
30
Introduccin
c) Encefalitis severa, con una viremia temprana y de gran magnitud, invasin

del SNC y desarrollo de lesiones severas.
Desarrollo de encefalitis asociada a la infeccin por un flavivirus

La mayora de los trabajos publicados describen el desarrollo de encefalitis asociada
a la infeccin por flavivirus en roedores y humanos, y fundamentalmente se centran
en la infeccin por WNV (revisin en McMinn, 1997; Chambers y Diamond, 2003;
Lim SM et al., 2011; Cho y Diamond, 2012; Clark et al., 2012).
La habilidad de un flavivirus para provocar un proceso neurolgico depende
de su neuroinvasividad, es decir, la capacidad para replicarse perifricamente
alcanzando suficiente viremia para invadir el SNC, y de su neurovirulencia, o
capacidad para iniciar una infeccin citoptica en el SNC y provocar encefalitis
(Monath, 1986; McMinn, 1997). La mayora de las infecciones del SNC se producen
por va hematgena (Chambers y Diamond, 2003; Turtle et al., 2012), pero debido a
las caractersticas estructurales de la barrera hematoenceflica (BHE) (Abbott et al.,
2006, 2010) (Figura 6), el paso del virus a travs de la misma requiere mecanismos
especiales como pueden ser:
a) Infeccin y replicacin del virus en las clulas endoteliales de la BHE (Liou et
al., 1998; Verma et al., 2009).
b) Disrupcin de la BHE por medio de factores que alteren su permeabilidad (en
ratones, la produccin perifrica de TNF- en respuesta a la infeccin
experimental con WNV altera las uniones estrechas entre las clulas
endoteliales de la BHE permitiendo el paso de partculas vricas y clulas
inflamatorias (Wang et al., 2004)).
31
Tesis doctoral
Figura 6. Estructura de la BHE. La BHE es una barrera fsica conformada por clulas
endoteliales conectadas por uniones estrechas que hacen que sta acte como una barrera
selectiva, forzando el paso trascelular de la mayora de molculas. Rodeando a las clulas
endoteliales se sitan pericitos y una lmina basal (BL1). El parnquima cerebral a su vez est
rodeado por otra lmina basal (BL2) tras la cual se sita la capa limitante de gla, formada por un
entramado constituido por las porciones finales de los astrocitos y de algunas clulas de la
microgla. En algunos vasos sanguneos existe un espacio perivascular (Virchow-Robin) entre BL1
y BL2 (Ilustracin extrada de Abbot et al., 2010).
Existen otros mecanismos de acceso al SNC como son la difusin pasiva del
virus a travs de las clulas endoteliales del plexo coroideo (Kramer-Hmmerle et
al., 2005), la infeccin de neuronas olfatorias tras la invasin del epitelio olfatorio
(McMinn, 1997), el transporte axonal retrgrado desde neuronas motoras infectadas
perifricamente (Samuel et al., 2007a) o el transporte por medio de clulas
inflamatorias infectadas, proceso que se conoce con el nombre de caballo de Troya
(Garca-Tapia et al., 2006). El mecanismo de neuroinvasin depende de la
virulencia del virus y de la va de entrada del mismo en el hospedador (Beasley et
al., 2002).
Las principales clulas diana de los flavivirus en el SNC son las neuronas
(Desai et al., 1995; Shieh et al., 2000; Steele et al., 2000; Xiao et al., 2001;
Shrestha et al., 2003), pudiendo producir su muerte por necrosis o por apoptosis
(Xiao et al., 2001; Shrestha et al., 2003; Weissenbck et al., 2004; Samuel et al.,
2007b). El predominio de una u otra forma de muerte celular ha sido relacionado
con la virulencia y la dosis infectiva del virus en ratones y cultivos celulares (Xiao et
32
Introduccin
al., 2001; Chu y Ng, 2003). En ocasiones, las neuronas que estn muriendo como
consecuencia de la infeccin liberan citoquinas txicas que daan otras neuronas
que no estn infectadas (Shrestha et al., 2003; Kumar et al., 2010). Los flavivirus
adems pueden infectar clulas de la gla in vivo e in vitro y aunque las
consecuencias de este tropismo estn menos estudiadas, algunos trabajos in vitro
sugieren su papel en el mantenimiento de la infeccin (Jordan et al., 2000; Steele et
al., 2000; Diniz et al., 2006; Thongtan et al., 2010).
En el momento en el que el virus infecta las neuronas se inicia la respuesta
inmune del hospedador (Cho y Diamond, 2012). El SNC dispone de una serie de
componentes celulares que le permiten responder a estmulos de forma inmediata:
a) Clulas de la microgla: consideradas como los macrfagos residentes del
SNC, con capacidad para fagocitar y presentar antgenos a las clulas T y
producir citoquinas (Gehrmann et al., 1995; Kettenmann et al., 2011).
b) Astrocitos: su funcin bsica es el mantenimiento de la homeostasis para
una correcta actividad neuronal (Montgomery, 1994; Magistretti y Ransom,
2002). Su papel en la inflamacin es menos importante que el de la microgla
pero pueden actuar como clulas fagocitarias y presentadoras de antgeno,
as como liberar molculas que intervienen en el proceso inflamatorio
(Montgomery, 1994).
c) Otras clulas: en mamferos se ha visto que existen otras poblaciones
celulares en menor proporcin como son macrfagos y clulas dendrticas
perivasculares, coroideas y menngeas, adems de linfocitos T, B y monocitos
en el lquido cefalorraqudeo (LCR) (Ousman y Kubes, 2012; Ransohoff y
Engelhardt, 2012).
Por tanto, la primera lnea de defensa del SNC frente a la infeccin por un
flavivirus es la activacin de la microgla y los astrocitos, la expresin de molculas
de adhesin, y la produccin de mediadores de la inflamacin que terminar en el
33
Tesis doctoral
reclutamiento de clulas inflamatorias desde la periferia (Chambers y Diamond,

2003; Garca-Tapia et al., 2007; Rezai-Zadeh et al., 2009; Cho y Diamond, 2012;
Ousman
Kubes,
2012).
El
SNC
es
considerado
como
un
lugar
inmunoprivilegiado, entre otras razones debido a que las caractersticas de la BHE

hacen que el acceso de clulas inflamatorias perifricas sea mucho menor que en el
caso de otros tejidos (Muldoon et al., 2013). No obstante, existen tres vas por las
que estas clulas pueden acceder al SNC (Ransohoff et al., 2003):
1. De la sangre al LCR a travs del plexo coroideo, considerndose una va de
entrada en condiciones fisiolgicas (Figura 7).
2. De la sangre al espacio subaracnoideo y de ah al espacio perivascular de
Virchow-Robin (Figura 8).
3. De la sangre directamente al espacio perivascular intraparenquimatoso. En
este caso requieren atravesar la BHE y la lmina basal endotelial, por lo que
slo se va a producir en situaciones patolgicas en las que se altere su
permeabilidad (Wang et al., 2004).
Figura 7. Entrada de leucocitos en el LCR.

Las clulas inflamatorias se unen a citoquinas
expresadas en el endotelio fenestrado y entran
por diapdesis en el estroma del plexo
coroideo. Una vez aqu los leucocitos se
pueden unir a otras citoquinas expresadas por
las clulas epiteliales del plexo coroideo y
migrar hacia el LCR (Ilustracin extrada de
Ransohoff et al., 2003).
34
Introduccin
Figura 8. Entrada de leucocitos en el espacio subaracnoideo. La sangre llega desde las ramas
finales de la arteria cartida interna al espacio subaracnoideo. Cuando entran en el parnquima
del cerebro, los vasos estn rodeados inicialmente por un espacio perivascular (Virchow-Robin)
que a su vez conecta con el espacio subaracnoideo (Ilustracin extrada de Ransohoff y
Engelhardt, 2012).
En la encefalitis asociada a la infeccin por un flavivirus participan sobre

todo linfocitos T CD4+ y CD8+ pero tambin linfocitos B, neutrfilos y monocitos
(Sampson et al., 2001; Kelley et al., 2003; Brhin et al., 2008; Rezai-Zadeh et al.,
2009). Estas clulas suelen ser retenidas en el espacio perivascular, donde se
preparan para reaccionar frente al virus, atravesando posteriormente la capa
limitante de gla para penetrar en el parnquima nervioso (Bechmann et al., 2007).
La magnitud y el tipo de respuesta inflamatoria en el SNC depende de la
virulencia del virus, la va de infeccin y el estado inmunitario del hospedador
(Chambers y Diamond, 2003). En ocasiones la respuesta es tan intensa que puede
tener efectos perjudiciales sobre la integridad y la funcionalidad de los componentes
del SNC (bystander injury) (Shrestha et al., 2003; Brhin et al., 2008; Wee Yong et
al., 2010; Zhang et al., 2010). La importancia del papel que juega en el desarrollo
de enfermedad neurolgica la muerte neuronal por accin directa del virus o
secundaria a la inflamacin no est demasiado clara (Turtle et al., 2012). En
ratones se ha visto que los linfocitos T CD8+ y las clulas productoras de
anticuerpos son importantes para la recuperacin de la encefalitis, pudiendo
35
Tesis doctoral
permanecer en el SNC durante varias semanas (Wang et al., 2003; Stewart et al.,
2011).
El virus puede persistir en el SNC de humanos, aves, roedores y otros
mamferos infectados de forma natural o experimental durante varias semanas o
meses, dando lugar o no a secuelas neurolgicas (Pogodina et al., 1983; Ravi et al.,
1993; Xiao et al., 2001; Penn et al., 2006; Nemeth et al., 2009a,c; Appler et al.,
2010; Sadek et al., 2010; Wheeler et al., 2012).
Existen diversos factores vricos que van a modular la neuroinvasividad y la
neurovirulencia. La protena E es considerada el determinante antignico ms
importante, ya que es la que interacciona con los receptores celulares, pero existen
regiones no estructurales y no codificantes en el genoma viral que tambin ejercen
un efecto modulador (Monath y Heinz, 1996; Clark et al., 2012). La glicosilacin de
la protena E de WNV ha sido asociada a un incremento en la neuroinvasividad en
ratones (Shirato et al., 2004; Beasley et al., 2005), en parte porque los virus con
gran habilidad para replicarse perifricamente en el hospedador presentan una
mayor capacidad neuroinvasiva (Huang y Wong, 1963; Albrecht, 1968; Shirato et
al., 2004). Existen adems diferentes factores genticos en humanos, aves y
roedores que han sido relacionados con la susceptibilidad a sufrir encefalitis en la
infeccin por estos virus (Monath, 1986; Sangster et al., 1994; Chambers y
Diamond, 2003; Clark et al., 2012; Tag-El-Din-Hassan, 2012).
A pesar de los numerosos estudios experimentales realizados en aves, la
informacin sobre el mecanismo de entrada de los flavivirus en el SNC as como los
mediadores y componentes celulares que intervienen en el desarrollo de encefalitis y
muerte neuronal es muy escasa. La BHE en aves es considerada similar a la de
mamferos (Stewart y Wiley, 1981). La presencia de vasculitis, hemorragias y
manguitos perivasculares en algunas especies podra relacionarse con la llegada del
virus va hematgena. De igual forma, la deteccin del antgeno viral en clulas
36
Introduccin
endoteliales del SNC podra indicar una infeccin y replicacin del virus en las
mismas (Wnschmann et al., 2004; Lopes et al., 2007; Nemeth et al., 2011),
aunque la llegada por medio de clulas del sistema inmune infectadas no se
descarta, ya que en numerosas ocasiones se detecta el antgeno viral en stas antes
que en ninguna otra clula del SNC (Steele et al., 2000; Weingartl et al., 2004;
Gibbs et al., 2005).
Las clulas inflamatorias que intervienen en el desarrollo de encefalitis
asociada a la infeccin por un flavivirus en aves tampoco estn caracterizadas, sin
embargo, es probable que sean muy similares a las encontradas en mamferos. En
la encefalitis asociada a la infeccin por otros virus neurotrpicos, adems de
participar astrocitos y clulas de la microgla, se produce una infiltracin de
linfocitos T y B y macrfagos (Ecco et al., 2011; Brjer et al., 2012).
LA PERDIZ ROJA Y SU RELACIN CON LOS FLAVIVIRUS

La perdiz roja es una ave galliforme mediterrnea cuya poblacin se distribuye de
forma natural en el noroeste de Italia, centro y sur de Francia y la Pennsula
Ibrica, y que ha sido introducida en el sur de Inglaterra y las islas Canarias
(Blanco-Aguiar et al., 2003). Ms del 50% de dicha poblacin se encuentra en la
Pennsula Ibrica (Aebischer y Lucio, 1997), dnde presenta un gran valor
socioeconmico y ecolgico, ya que es una de las especies de caza menor ms
importante (Bernabeu, 2000) y un recurso trfico de una gran variedad de especies,
alguna de ellas amenazada como el guila imperial ibrica (Caldern, 1975; BlancoAguiar, 2007). La perdiz roja ha sufrido un declive en el 95% de sus reas de
distribucin (Aebischer y Potts, 1994; Aebischer y Lucio, 1997). La prdida del
hbitat, la introduccin de especies alctonas y la sobreexplotacin de recursos
parecen
estar
relacionadas
con
este
hecho
(Blanco-Aguiar,
2007).
Como
consecuencia de la elevada demanda cinegtica y el declive de sus poblaciones, esta

37
Tesis doctoral
especie es producida en granjas cinegticas de manera intensiva y al aire libre

(Aebischer y Potts, 1994), y las altas densidades que se alcanzan en estas
instalaciones pueden favorecer o incrementar el riesgo de transmisin o aparicin
de enfermedades (Blanco-Aguiar, 2007). Esto, unido al hecho del bajo o nulo
control sanitario de las repoblaciones (Gortzar, 1998), puede dar lugar a graves
problemas sanitarios, no slo para esta especie sino para muchas otras que estn
presentes en su entorno o que tengan relaciones ecolgicas con la misma.
En general las aves galliformes son consideradas poco susceptibles a la
infeccin por WNV (Guy y Malkinson, 2008; van der Meulen et al., 2005). La
mayora no muestra signos clnicos ni muere a causa de la enfermedad, si bien
desarrolla viremia y produce anticuerpos (Senne et al., 2000; Swayne et al., 2000;
Langevin et al., 2001; Komar et al., 2003b). Sin embargo, en Norteamrica se
produjeron sendos brotes de elevada mortalidad en granjas de perdiz chukar
(Alectoris chukar) y faisanes imperiales (Lophophorus impeyanus) (Wnschmann y
Ziegler, 2006) y la perdiz roja demostr ser susceptible a la infeccin experimental
con cepas mediterrneas de WNV, mostrando signos clnicos y mortalidad (Sotelo et
al., 2011b). Por otro lado, y como se ha mencionado anteriormente, tanto la perdiz
roja como el faisn comn son susceptibles a la infeccin natural por BAGV,
sucumbiendo incluso a la enfermedad, pero se desconoce su susceptibilidad a la
infeccin por USUV.
USO DE AVES COMO MODELO EXPERIMENTAL DE LA INFECCIN POR

FLAVIVIRUS
Como ya ha sido mencionado anteriormente, en el estudio experimental de
enfermedades infecciosas que afectan a humanos, entre ellas la infeccin por
flavivirus, se emplean fundamentalmente roedores. Estos modelos experimentales,
aunque con ciertas diferencias, presentan un sistema inmune similar al humano,
38
Introduccin
permitiendo emular de forma ms o menos fiable la patogenia de enfermedades que

afectan al hombre (Buer y Balling, 2003).
En relacin a los flavivirus, los ratones y hmsteres han sido ampliamente
utilizados para estudios de virulencia y tropismo viral, identificacin de factores
vricos y del hospedador que intervienen en la patogenia, as como en tests de
eficacia y seguridad de vacunas y tratamientos teraputicos (Clark et al., 2012). Sin
embargo, cuando se desea realizar estudios relacionados con la patogenia de la
infeccin en aves, reservorio natural de la mayora de los flavivirus transmitidos por
mosquitos, la utilidad de los roedores disminuye. Ciertas aves galliformes,
fundamentalmente los pollos de gallina domstica, han sido utilizadas para
estudios experimentales desde hace ms de 100 aos ya que, al igual que los
roedores, son fciles de manejar y suponen un bajo coste de adquisicin,
mantenimiento y alimentacin (Davis, 2003). La perdiz roja adems rene las
caractersticas de ser una especie autctona, distribuida en los ambientes
mediterrneos donde pueden persistir y amplificarse diferentes flavivirus, ser
fcilmente manejable en cautividad y tener un gran potencial como especie
centinela debido a la amplia distribucin de granjas cinegticas en las zonas de
inters. Por ello, los trabajos experimentales incluidos en esta tesis doctoral se han
desarrollado en esta especie.
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59
Objetivos
Objetivos
Como se ha expuesto anteriormente, en las ltimas dcadas estn incrementado de

forma importante los casos de mortalidad en aves silvestres asociados a la infeccin
por virus del gnero Flavivirus tanto en Norteamrica como en Europa, siendo
Espaa uno de los pases en los que se han registrado mortalidades en los ltimos
aos. Sin embargo, aunque se barajan numerosas posibilidades, las causas que
han llevado a esta situacin continan siendo objeto de estudio. Por otro lado,
aunque WNV es un flavivirus considerado como patgeno importante para las aves,
se ha visto que la infeccin no afecta a todas las especies por igual y que,
aparentemente, las consecuencias de dicha infeccin son menores para las aves
nativas europeas que para las que habitan en Norteamrica. A pesar de esto,
apenas existen trabajos en los que se estudie la patogenia o las lesiones asociadas a
la infeccin por WNV en aves endmicas de Europa. Finalmente, y aunque la
infeccin por WNV se caracteriza por dar lugar a un proceso neurolgico, no existen
trabajos que determinen los componentes celulares inflamatorios que intervienen
en el desarrollo de la encefalitis en aves. Por lo tanto, los objetivos de esta tesis
doctoral son:
Objetivo general
Estudiar los cambios morfolgicos y el tropismo celular y tisular del virus en aves
infectadas por flavivirus.
Objetivos especficos
1) Evaluar la informacin disponible sobre la patogenia y las lesiones asociadas
a la infeccin por WNV en aves para determinar los mecanismos patognicos
de los que deriva la variacin interespecie en la presentacin clnica de la
enfermedad y la mortalidad asociada (Captulo 1).
63
Tesis doctoral
2) Describir la patogenia de la infeccin experimental con tres cepas diferentes

de WNV en perdiz roja. Determinar los componentes celulares inflamatorios
que intervienen en el desarrollo de la encefalitis as como su evolucin
durante el curso de la infeccin (Captulos 2 y 3).
3) Estudiar las diferencias en la patogenia, severidad de lesiones y nivel de

replicacin viral en la infeccin experimental con tres cepas diferentes de
WNV en perdiz roja. Determinar la existencia de diferencias en la
susceptibilidad, patogenia y lesiones asociadas a la infeccin experimental de
un ave endmica europea con WNV respecto a lo descrito en aves de
Norteamrica (Captulos 2 y 3).
4) Describir las lesiones y la distribucin tisular del virus en la infeccin

natural de perdiz roja, faisn comn y paloma torcaz por BAGV. Determinar
las existencia de diferencias interespecie en relacin a estos aspectos
(Captulo 4).
5) Caracterizar las lesiones y la distribucin del antgeno viral en secciones

oculares de aves galliformes cinegticas infectadas con WNV y BAGV.
Determinar la va de entrada de WNV al ojo y los hallazgos microscpicos que
puedan explicar la ceguera observada en perdices rojas infectadas por BAGV
(Captulo 5).
6) Caracterizar el primer caso de infeccin por USUV en aves en Espaa.

Determinar el origen de la cepa infectante y describir brevemente las lesiones
asociadas a la infeccin (Captulo 6).
64
Captulo 1
Revisin sobre la patologa y el tropismo
tisular del virus West Nile en aves
infectadas de forma natural
Gamino V, Hfle U. Pathology and tissue tropism of natural West Nile

virus infection in birds: a review. Veterinary Research 2013, 44(1):39.
doi: 10.1186/1297-9716-44-39.
Captulo 1
RESUMEN
El virus West Nile (WNV) es un flavivirus transmitido por artrpodos distribuido
mundialmente y capaz de infectar a una gran variedad de hospedadores
vertebrados, siendo las aves su reservorio natural. Aunque este virus haba sido
considerado un patgeno de escasa importancia para las aves, desde la dcada de
los 90, y especialmente tras ser introducido en el continente norteamericano en
1999, miles de aves han muerto como consecuencia de la infeccin. En la presente
revisin se realiza una sntesis sobre la patogenia y la patologa asociadas a la
infeccin por WNV en aves, resaltando las diferencias en la distribucin y severidad
de lesiones y la presencia de antgeno viral entre rdenes y familias de aves. A pesar
de la diferencias encontradas entre especies en la susceptibilidad a la infeccin, en
un ave infectada por WNV existen lesiones y antgeno viral en la mayora de los
rganos. Las diferencias encontradas en cuanto a la distribucin y severidad de
lesiones y la presencia de antgeno viral estn relacionadas, en parte, con el curso
de la infeccin, segn sea sta no progresiva, aguda o crnica. Es muy probable que
la patogenia se vea modulada por la combinacin de numerosas variables del
hospedador, factores ambientales y factores intrnsecos del virus como la virulencia
y la patogenicidad de la cepa infectante.
67
Tesis doctoral
ABSTRACT
West Nile virus (WNV) is a globally distributed arthropod-borne flavivirus capable of
infecting a wide variety of vertebrates, with birds as its natural reservoir. Although
it had been considered a pathogen of little importance for birds, from the 1990s,
and especially after its introduction in the North American continent in 1999,
thousands of birds have succumbed to West Nile infection. This review summarizes
the pathogenesis and pathology of WNV infection in birds highlighting differences in
lesion and antigen distribution and severity among bird orders and families. Despite
significant species differences in susceptibility to infection, WNV associated lesions
and viral antigen are present in the majority of organs of infected birds. The nonprogressive, acute or more prolonged course of the disease accounts for part of the
differences in lesion and viral antigen distribution and lesion severity. Most likely a
combination of host variables and environmental factors in addition to the intrinsic
virulence and pathogenicity of the infecting WNV strain influence the pathogenesis
of the infection.
68
Captulo 1
INTRODUCTION
West Nile virus (WNV) is an arthropod-borne virus of the genus Flavivirus capable of
infecting a wide variety of vertebrates, with birds as its natural reservoir (McLean
and Ubico, 2007). It was first isolated in Uganda in 1937 from a woman with a
febrile process (Smithburn et al., 1940). During the 1960s WNV was one of the
most widely distributed flaviviruses in humans, birds and mosquitoes in Africa, the
Middle East and south-western Europe, where it was considered a pathogen of little
importance, causing subclinical infection or sporadic self-limiting outbreaks in
horses and humans (Murgue et al., 2002; Dauphin et al., 2004). From the 1990s
the frequency and severity of human infections as well as the number of cases in
other vertebrates including companion, farm and wild animals has increased (van
der Meulen et al., 2005). In Europe, WNV causes disease in horses and humans,
and recently sporadic mortality events have been reported primarily in birds of prey
(Bakonyi et al., 2006; Hfle et al., 2008; Jimnez-Clavero et al., 2008). In contrast,
after its introduction in North America in 1999, where it caused one of the most
important outbreaks in New York, thousands of birds, horses and humans have
died of the disease (CDCa; LaDeau et al., 2007). Nowadays it is one of the most
widely distributed flaviviruses in the world and an important public and animal
health and conservation concern.
ETIOLOGY
West Nile virus belongs to the genus Flavivirus of the family Flaviviridae. It
has been serologically classified within the Japanese encephalitis antigenic group.
The viral positive sense single-stranded RNA genome encodes a polyprotein which is
translated into different structural (envelope protein E, membrane precursor
protein prM and capsid protein C) and non-structural (NS1, NS2a, NS2b, NS3,
NS4a, NS4b, and NS5) proteins that play an important role in the viral host range,
69
Tesis doctoral
tissue tropism, viral replication and assembly, and host immune system
stimulation (Deubel et al., 2001; Brinton, 2002; Lindenbach and Rice, 2003). Based
on phylogenetic analysis, WNV strains have been grouped into seven genetic
lineages (Mackenzie and Williams, 2009) but there are two major lineages, lineage 1
and lineage 2. Lineage 1 is widespread and contains isolates from Europe, America,
the Middle East, India, Africa and Australia (Lanciotti et al., 2002; Charrel et al.,
2003). Lineage 2 had originally only been isolated in Southern Africa and
Madagascar but has recently also been detected in Europe (Bakonyi et al., 2006;
Papa et al., 2011a; Valiakos et al., 2011; Savini et al., 2012). In general lineage 1
viruses were considered to be more virulent than lineage 2 viruses, however it has
been demonstrated that both lineages contain neuroinvasive phenotypes and some
of the recent outbreaks in Europe, that also affect birds, have been caused by the
latter (Bakonyi et al., 2006; Wodak et al., 2011; Savini et al., 2012) (Table 1).
Increased virulence of WNV strains has been associated to genetic and aminoacidic
changes in structural and non-structural proteins (Charrel et al., 2003; Sotelo et
al., 2009; McMullen et al., 2013). Substitution to a proline in position 249 of the
non-structural protein NS3 has been associated with increased virulence in WNV
lineage 1 strains in American crows (Corvus brachyrhynchos) (Brault et al., 2007)
and the same substitution has been detected in recent lineage 2 strains causing
important outbreaks in Europe (Papa et al., 2011b).
70
Captulo 1
Table 1. Bird species found dead or with symptoms of encephalitis in Europe in which the
presence of WNV has been evidenced by real time RT-PCR or by virus isolation.
Year
Country
Order
Family
Species
20012004
Spain
Falconiformes
Accipitridae
2003
Hungary
Anseriformes
Anatidae
2004
Hungary
Falconiformes
Accipitridae
France
Passeriformes
Corvidae
Spanish imperial
eagle
(Aquila adalberti)
Domestic goose
(Anser anser
domesticus)
Goshawk
(Accipiter gentilis)
Common magpie
(Pica pica)
House sparrow
(Passer
domesticus)
Goshawk
Passeridae
2005
Hungary
Falconiformes
Accipitridae
2007
Spain
Falconiformes
Accipitridae
2008
Austria
Falconiformes
Accipitridae
Sparrow hawk
(Accipiter nisus)
Golden eagle
(Aquila chrysaetos)
Bonellis eagle
(Aquila fasciata)
Sparrow hawk
Lineage
1
(Hfle et al., 2008)
(Glvits et al., 2005;

Bakonyi et al., 2006)
(Bakonyi et al., 2006;

Erdlyi et al., 2007)
(Jourdain et al.,
2007b)
(Bakonyi et al., 2006;

Erdlyi et al., 2007)
(Jimnez-Clavero et
al., 2008)
(DEFRA; Wodak et al.,

2011)
Goshawk
Falconidae
Italy
Psittaciformes
Strigopidae
Charadriiformes
Laridae
Columbiformes
Columbidae
Passeriformes
Corvidae
Suliformes
Phalacrocoracidae
2009
Austria
Falconiformes
Accipitridae
2011
Italy
Columbiformes
Columbidae
2012
Italy
Falconiformes
Accipitridae
Gyrfalcon
(Falco rusticolus)
Kea
(Nestor notabilis)
Herring gull
(Larus argentatus)
Pigeon
(Columba livia)
Common magpie
Carrion crow
(Corvus corone)
Eurasian jay
(Garrulus
glandarius)
Great cormorant
(Phalacrocorax
carbo)
Goshawk
Collared dove
(Streptopelia
decaocto)
Goshawk
Reference
(DEFRA)
1
(Monaco et al., 2010)
(Wodak et al., 2011)
(Savini et al., 2012)
(Savini et al., 2013)
ECO-EPIDEMIOLOGY
The virus is maintained in an enzootic ornitophilic-mosquito-bird cycle (Hublek
and Halouzka, 1999). Migratory birds are considered to play an important role in
the local and long distance viral dispersal (Malkinson and Banet, 2002; Peterson et
71
Tesis doctoral
al., 2003; Rappole et al., 2006; Jourdain et al., 2007a). The most susceptible
species to infection belong to the family Corvidae of the order Passeriformes such as
the American crow, the blue jay (Cyanocitta cristata), the black-billed magpie (Pica
hudsonia) or the fish crow (Corvus ossifragus) (Komar et al., 2003; LaDeau et al.,
2007). Some species of this order such as the American robin (Turdus migratorius)
or the house sparrow (Passer domesticus) are also considered the main reservoir of
WNV in urban areas of North America and Europe (Langevin et al., 2005; Kilpatrick
et al., 2006). Other vertebrates susceptible to infection include amphibians, reptiles
and mammals, among which the virus can be especially pathogenic for equids and
humans. However, most of them are considered accidental or dead-end hosts as
they rarely develop sufficient viremia to infect feeding mosquitoes (van der Meulen
et al., 2005; Kramer et al., 2008).
Although WNV transmission in birds occurs primarily by mosquitoes of the
genus
Culex,
transmission
through
infected
prey
or
contaminated
water
consumption and horizontal contact transmission have also been documented

(Langevin et al., 2001; Swayne et al., 2001; Banet-Noach et al., 2003; Komar et al.,
2003; Nemeth et al., 2006a). The virus is maintained in a silent mosquito-bird cycle
in natural habitats (rural cycle) but can cause outbreaks in humans and horses
when it is introduced in humanized habitats where mosquitoes with a wider host
range are present, establishing an urban cycle (Jimnez-Clavero, 2012). In
endemic areas wild bird infection usually starts in spring and early summer,
mortality peaks from midsummer to early fall, and human and horse cases take
place a few weeks after the onset of bird mortalities (Phalen and Dahlhausen,
2004).
Before the 1990s WNV had been isolated in different parts of Europe, Asia
and Africa but was rarely associated with clinical disease. Although some studies
conducted in Africa in the 1950s underlined the role of birds as amplifying hosts
72
Captulo 1
and mortality was reported in host competence studies, especially in hooded crows
(Corvus corone sardonius) (Work et al., 1955), prior to 1997 WNV was considered
low-pathogenic for birds (Zeller and Schuffenecker, 2004). In 1998 a large outbreak
took place in Israel with high mortality in birds, particularly in geese (Anser anser
domesticus), and WNV was isolated from the brain of dead free-living storks (Ciconia
ciconia) (Malkinson et al., 2002). Currently, WNV is considered endemic in Europe
(Jimnez-Clavero, 2012). Although viral circulation has been reported in many bird
species, cases of encephalitis or mortality due to viral infection in wild birds have
been sporadic (Zeller and Schuffenecker, 2004) and observed mainly in raptors
(Table 1). In North America WNV was detected for the first time in 1999 in New York
(Lanciotti et al., 1999) and since then it has caused thousands of bird deaths being
detected in at least 326 bird species (CDCb) (Table 2). WNV became endemic within
ten years of its introduction in this continent (De Filette et al., 2012). The
epidemiological behavior in North America, with an intense viral circulation, differs
from the epidemiological behavior in other parts of the world including Europe, and
the reasons remain largely unknown (Jimnez-Clavero, 2012). Viral phenotype, host
heterogeneity in the area, vector abundance, feeding activity or host preference, and
host susceptibility have been identified as possible modulating factors (Ostfeld and
Keesing, 2000; Brinton, 2001, 2002; Ezenwa et al., 2006; Hamer et al., 2009;
Jimnez-Clavero, 2012). Host susceptibility to infection has been associated to
geographic range, mating and breeding systems, body size, migratory behavior and
to co-evolution with the virus or antigenically-related flaviviruses (Gancz et al.,
2004; Fang and Reisen, 2006; Reisen and Hahn, 2007; Figuerola et al., 2008; Kwan
et al., 2012).
73
Tesis doctoral
Table 2. Bird orders and families from North America in which mortality due to WNV infection
has been demonstrated.
ORDER
FAMILY
Anseriformes
Anatidae
Apodiformes
Apodidae, Trochilidae
Caprimulgiformes
Caprimulgidae
Casuariiformes
Dromaiidae
Charadriiformes
Charadriidae, Laridae
Ciconiiformes
Ardeidae, Cathartidae, Ciconiidae, Threskiornithidae
Columbiformes
Columbidae
Coraciiformes
Alcedinidae, Bucerotidae
Cuculiformes
Cuculidae
Falconiformes
Accipitridae, Falconidae
Galliformes
Numididae, Odontophoridae, Phasianidae
Gaviiformes
Gaviidae
Gruiformes
Aramidae, Gruidae, Rallidae
Musophagiformes
Musophagidae
Passeriformes
Bombycillidae, Cardinalidae, Cinclidae, Corvidae, Emberizidae, Estrildidae,

Fringillidae, Icteridae, Laniidae, Mimidae, Paridae, Parulidae, Passeridae,
Sittadae, Sturnidae, Thraupidae, Troglodytidae, Turdidae, Vireonidae
Pelecaniformes
Pelecanidae, Phalacrocoracidae
Phoenicopteriformes
Phoenicopteridae
Piciformes
Picidae
Podicipediformes
Podicipedidae
Psittaciformes
Aratingidae, Cacatuidae, Psittacidae
Sphenisciformes
Spheniscidae
Strigiformes
Strigidae, Tytonidae
Struthioniformes
Struthionidae
Tinamiformes
Tinamidae
PATHOGENESIS IN BIRDS
Most information about the pathogenesis of WNV infection is derived from
experimental studies done in mammals, mainly rodents.
In mammals, after mosquito blood-feeding, WNV replicates in the skin and is
transported by Langerhans dendritic cells to draining lymph nodes. There the virus
replicates and primary viremia and peripheral organ dissemination take place
(Samuel and Diamond, 2006). On the contrary, the exact mechanism and sites of
74
Captulo 1
WNV replication in avian hosts are still not well understood. It is supposed that,
like in mammals, it replicates locally at the inoculation site and is rapidly
distributed to all organ systems (Nemeth et al., 2011). However, it has been
demonstrated that the virus can be detected in the blood as early as 30-45 min
after the mosquito feeding period, suggesting that in birds local replication is not
necessary for the primary viremia (Reisen et al., 2007). In general, WNV can be
isolated from the blood of infected birds at one day post-infection (dpi) (one day
later in less susceptible species such as chicken or turkeys or in case of oral
infection) (Senne et al., 2000; Komar et al., 2003; Weingartl et al., 2004). Viremia
can peak as early as 2-3 dpi in geese and some Passeriformes such as crows and
jays, or 4-6 dpi in raptors, owls and chicken (Senne et al., 2000; Swayne et al.,
2001; Weingartl et al., 2004; Nemeth et al., 2006b; Ziegler et al., 2013). Mean peak
viremia is higher in those birds that finally succumb to the disease (Langevin et al.,
2005). WNV can be detected in the blood until day 6-7 pi in geese, passerines and
owls and up to 10 dpi in raptors and turkeys (Swayne et al., 2000; Banet-Noach et
al., 2003; Nemeth et al., 2006b; Lapointe et al., 2009; Ziegler et al., 2013).
Little is known about innate avian host defenses against WNV infection. In
mammals this includes IFN production, complement activation, phagocytosis and
cytotoxicity, with the participation of macrophages, neutrophils, NK cells and T
cells (Samuel and Diamond, 2006; Suthar et al., 2013).
WNV infects all major organ systems and a wide variety of individual cell
types. The targeting of cells of the mononuclear phagocytic system may play an
important role in the pathogenesis in infected birds, as the virus can replicate
within these cells and disseminate to a wide variety of tissues (Steele et al., 2000;
Weingartl et al., 2004). As early as 1 dpi WNV can be detected in the spleen of crows
and one day later it is widely distributed. In this species the highest viral titer in
tissues can be reached on day 4 pi, decreasing from day 5 pi (Weingartl et al.,
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Tesis doctoral
2004). On the contrary, in falcons and owls maximum viral titer in tissues can be
delayed until day 7-8 pi, decreasing from day 9 pi, and while in falcons the virus
remains detectable on day 14 pi (Busquets et al., 2012), it can be totally cleared
from most tissues of owls (Nemeth et al., 2006a,b). Flaviviruses have evolved
different strategies that modulate the host immune response (Ye et al., 2013) and
depending on the effectiveness of this response and, therefore, viremia level, WNV
can reach the central nervous system (CNS). While WNV can be detected in the
brain of crows as early as 2 dpi, in owls it may not be present before 5 dpi
(Weingartl et al., 2004; Nemeth et al., 2006b). In the former, maximum viral titer
can be reached on day 4 pi while this may not take place in the latter until day 8 pi
or until 14 dpi in falcons (Weingartl et al., 2004; Nemeth et al., 2006b; Busquets et
al., 2012). The mechanisms by which WNV crosses the blood-brain barrier and
reaches the CNS remain unknown. Some authors have suggested that TNF-
mediated change in endothelial cell permeability may facilitate its entry, at least in
mammals (Wang et al., 2004). Other mechanisms proposed are the infection of or
passive transport through the endothelium or epithelial cells of the choroid plexus
(McMinn, 1997), direct axonal retrograde transport from olfactory or motor neurons
(Monath et al., 1983; Samuel et al., 2007) or transport by infected immune cells
(Garca-Tapia et al., 2006). In birds, WNV detection in endothelial cells by
immunohistochemistry (IHC) and the presence of perivascular inflammatory
infiltrates (Wnschmann et al., 2004a; Lopes et al., 2007) may indicate that WNV
reaches the CNS via the blood stream and infects endothelial cells, although viral
transport through infected immune cells may also be probable (Weingartl et al.,
2004).
The development of clinical disease is caused by the invasion of the CNS
and/or other major organs such as the liver, spleen, kidney and heart (Steele et al.,
2000). In most cases, clinical signs appear approximately on 5 dpi in experimentally
76
Captulo 1
WNV infected birds, but may be absent both in experimental and natural infections
(Fitzgerald et al., 2003; Komar et al., 2003; Bertelsen et al., 2004; Nemeth et al.,
2006a; Wnschmann and Ziegler, 2006; Nemeth et al., 2011; Sotelo et al., 2011).
Unspecific clinical signs include depression, anorexia, dehydration and ruffled
feathers. In more than 60% of infections convulsions are present, approximately in
30% appear ataxia, abnormal head posture and head movements, and in up to 20%
tremors, uncoordinated flight, paresis and disorientation are observed (Steele et al.,
2000; Fitzgerald et al., 2003; D'Agostino and Isaza, 2004; Wnschmann et al.,
2004b; Joyner et al., 2006; Saito et al., 2007; Palmieri et al., 2011). The
development of impaired vision and blindness is common in raptors and owls
(Wnschmann et al., 2004b, 2005; Gancz et al., 2006). Long-term sequelaes have
been detected in long-lived birds such as raptors, in which relapses of neurologic
signs, feather pulp abnormalities and abnormal molt can persist up to 4 years
(Nemeth et al., 2009a), and may have a negative impact on the longevity of these
species (Nemeth et al., 2006a, 2009a).
Systemic infection of the host also activates the adaptive immune response
that includes B and T cell activation and antibody production (Samuel and
Diamond, 2006; Suthar et al., 2013). In most bird species seroconversion can first
be detected between 4-6 dpi (Weingartl et al., 2004; Sotelo et al., 2011) and
neutralizing antibodies, at least in some species, persist for longer than a year and
can be transferred from adult females to their progeny (Gibbs et al., 2005a; Hahn et
al., 2006; Wilcox et al., 2007; Nemeth et al., 2009b). In mice it has been indicated
that CD8+ and CD4+ T lymphocytes combined with antibodies and chemokines are
essential for viral clearance from the CNS and peripheral organs (Lobigs et al.,
2009). In birds it has been demonstrated that the virus can persist in different
organs such as the spleen, kidney, eye, brain or the skin (Komar et al., 2003;
Reisen et al., 2006; Nemeth et al., 2009c; Wheeler et al., 2012a,b). Wheeler et al.
77
Tesis doctoral
(2012a) demonstrated that viral persistence in naturally infected Passeriformes can

last for 4 months extending to up to 6 months in experimentally infected birds.
Viral persistence consequences for the avian host are still not clear (Wheeler et al.,
2012a) but it can play an important role in viral overwintering and mosquito
infection in case of host immune defenses impairment and viremia recrudescence
(Semenov et al., 1973; Reisen et al., 2006; Wheeler et al., 2012c).
In case of host defense failure, death often occurs within 24 hours or two to
four days after the onset of clinical signs in experimentally or naturally infected
birds, respectively (Komar et al., 2003; Glvits et al., 2005; Wnschmann and
Ziegler, 2006; Erdlyi et al., 2007). Sometimes death is not directly related to WNV
associated tissue lesions but to concurrent disease such as trauma or bacterial,
fungal or parasitic infections (Steele et al., 2000; Wnschmann et al., 2005; Nemeth
et al., 2006a; Saito et al., 2007; Hfle et al., 2008; Nemeth et al., 2009a).
The outcome of WNV infection and development of lesions and disease in
individual birds depend mainly on viral and host factors. Glycosylation in the E
protein of WNV has been demonstrated to increase peripheral viremia and virulence
for birds and neuroinvasiveness in rodents (Shirato et al., 2004; Murata et al.,
2010). Brault et al. (2007) experimentally demonstrated that a T249P NS3
substitution in the WNV NY99 strain increased virulence for American crows.
However, this substitution appears not to have the same effect for some other North
American bird species and an experimental study has shown that this substitution
alone in European WNV strains does not necessarily lead to an increased virulence
for susceptible European birds (Sotelo et al., 2011). Papa et al. (2011b) related the
H249P NS3 substitution to the increased virulence of the lineage 2 WNV strain that
caused the outbreak in humans in Greece in 2010 (Papa et al., 2010), but the role
of this substitution in the virulence for birds is not clear, as lineage 2 strains that
have caused mortalities in birds do not show this change (Papa et al., 2011b; Savini
78
Captulo 1
et al., 2012, 2013). Therefore, the presence of other genetic markers of virulence
could be implicated (Botha et al., 2008; McMullen et al., 2013). The existence of
natural infections of goshawks (Accipiter gentilis) by either WNV lineage 1 and 2
strains allows comparison of the associated pathology. In both cases viral cellular
and tissue tropism is similar but lesion localization and nature slightly differ. Thus
for example splenic lymphoid depletion, nephritis and hepatitis present in lineage 2
infected goshawks are not described in those infected by lineage 1 which in change
have hepatic and myocardial necrosis (Wnschmann et al., 2005; Erdlyi et al.,
2007; Wodak et al., 2011). Another determinant factor is the ability of the WNV
strain to replicate at the high corporal temperature of the avian host, as has been
demonstrated for the WNV NY99 genotype (Kinney et al., 2006). Moreover, viral
inoculation dose should not be forgotten, although dose-dependent pathological
differences are not always evidenced in experimental infections (Ziegler et al., 2013).
Finally, infection route is other viral factor that can influence WNV pathogenicity.
Some authors have indicated that in orally infected birds, tissue lesions are less
severe and the incidence of clinical signs or mortality is lower than in those
subcutaneously infected (Komar et al., 2003; Nemeth et al., 2006a).
Bird species is an important host factor that influences the pathogenesis of
WNV infection, as has been demonstrated by numerous authors (Steele et al., 2000;
Komar et al., 2003; Nemeth et al., 2006a; Wnschmann and Ziegler, 2006; Lopes et
al., 2007). Also, the immune status of the host plays an essential role in the
pathogenesis, as it determines the capacity of the host to clear the virus (Samuel
and Diamond, 2006). It can be modulated by the presence of previous immunity
against the virus or cross immunity against antigenically-related flaviviruses, the
presence of concurrent diseases, the influence of hormonal factors and stress, as
well as age (Eldadah et al., 1967; Langevin et al., 2001; Fang and Reisen, 2006;
Wnschmann and Ziegler, 2006; Nemeth and Bowen, 2007; Nemeth et al., 2008;
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Tesis doctoral
Shirafuji et al., 2009). Age not only determines the maturity of the immune system
but also the expression of cellular receptors that play an important role on viral
entry and intracellular factors that participate in the pathogenesis of the disease
(Chambers and Diamond, 2003). Finally, it has been demonstrated in avian and
rodent models that genetic factors influence host susceptibility to flaviviral
infections (Sangster et al., 1994; Tag-El-Din-Hassan et al., 2012).
PATHOLOGY OF NATURAL WNV INFECTION IN BIRDS

The pathology of natural WNV infection in birds has been documented in more than
57 species of at least 13 families and 10 orders.
Because of the wide cellular and tissue tropism of WNV, there are no
pathognomonic macroscopic lesions. Bird species with low susceptibility to WNV
infection such as chicken may have no observable macroscopic lesions at necropsy
(Senne et al., 2000) and those highly susceptible such as crows or jays will die
rapidly after a short incubation period and may have few acute or no observable
lesions (Bertelsen et al., 2004; Wnschmann et al., 2004a). In contrast, birds that
survive longer will have more pronounced macroscopic lesions, that may be chronic
in the case of individuals that survive days to months (Wnschmann et al., 2005;
Lopes et al., 2007). Emaciation, dehydration, multiorgan hemorrhages, petechiae
and congestion are the most characteristic macroscopic changes, but also
splenomegaly, hepatomegaly, myocardial pallor and pale mottling in the liver,
spleen or kidney. Cerebral atrophy and malacia can be observed in raptors (Steele
et al., 2000; Wnschmann et al., 2005; Nemeth et al., 2006a; Wnschmann and
Ziegler, 2006; Lopes et al., 2007; Palmieri et al., 2011).
In the vast majority of bird families, microscopic lesions due to WNV infection
are predominantly found in the CNS, heart, kidney, spleen and liver (Table 3).
Pathological changes can be the result of the direct effect of the virus or secondary
80
Captulo 1
to the host inflammatory response (Chambers and Diamond, 2003; Pauli et al.,
2007; Lim et al., 2011). Lymphoplasmacytic and histiocytic infiltrates, cellular
degeneration and necrosis, and hemorrhages are the main microscopic findings.
However, there are differences in antigen abundance and distribution and in
microscopic lesion severity and distribution depending on the species infected or
the period between infection and death of the analyzed individual (McLean and
Ubico, 2007) (Table 3 and figure 1). Highly susceptible species such as crows and
jays usually have large amounts of virus in the blood and WNV antigen is widely
distributed in major organs. Microscopic changes in these cases are acute with
minimal inflammatory reaction and can be absent in the CNS (Weingartl et al.,
2004; Wnschmann et al., 2004a; Gibbs et al., 2005b). In contrast, birds that
survive longer, such as raptors and owls, may develop chronic lesions that can be
also found in the CNS (Wnschmann et al., 2005; Nemeth et al., 2006a).
120
Percentage of tissues
100
n=15
n=14
n=4 n=9
n=2
n=18
n=18
n=10
n=10
n=13
n=15
n=7
n=7
n=10
n=19 n=19 n=19
n=19
80
60
n=10
n=10
Lesion
Antigen
40
20
Bird order
Figure 1. Lesion extension and antigen distribution among examined tissues in WNV infected
bird orders as reported in the reviewed literature. Each column represents the percentage of
tissues collected (n) in which lesions or antigen is present.
81
Tesis doctoral
Central nervous system. CNS lesions can appear on 8-9 dpi in falcons and owls,
increasing their severity during the course of infection, and can be maintained
chronically (Nemeth et al., 2006a,b; Busquets et al., 2012). In chicken, the
appearance of lesions can be delayed until 21 dpi (Senne et al., 2000).
Lymphoplasmacytic and histiocytic meningoencephalitis characterized by gliosis,
perivascular cuffing and glial nodules is the main microscopic finding. This
inflammation is usually present in the molecular layer of the cerebellum and in
some cases also in the cerebrum, mesencephalon and medulla oblongata. Neuronal
necrosis and degeneration are usually found in raptors and owls (Nemeth et al.,
2006a; Ellis et al., 2007). Vasculitis is detected in red-tailed hawks (Buteo
jamaicensis), yellow-billed magpies (Pica nuttalli) and house sparrows (Nemeth et
al., 2006a; Ernest et al., 2010; O'Brien et al., 2010) and hemorrhages are observed
in guanay cormorants (Phalacrocorax bougainvillea), turkeys and owls (Steele et al.,
2000; Gancz et al., 2006; Zhang et al., 2006) (Table 3). WNV antigen is usually
detected by IHC in the CNS. Cells showing antigen labeling include neurons
(including Purkinje cells) and glial cells as well as other inflammatory cells and
vascular endothelial cells.
Eye. The eye is not routinely microscopically studied due to the difficulty of
obtaining sections suitable for interpretation. However, it has been more closely
examined in raptors and owls, probably because of the importance of vision in these
species. Moderate to marked lymphoplasmacytic and histiocytic inflammatory
infiltrates, disarray of the retinal pigmented epithelial cell layer and retinal cell
necrosis and mineralization have been described in naturally WNV infected hawks
(Pauli et al., 2007). Birds of the order Psittaciformes and Strigiformes only show
inflammatory infiltrates that are mainly detected in the conjunctiva, iris, choroid,
pecten and retina (Gancz et al., 2006; Stockman et al., 2010) (Table 3). WNV
antigen is detected in different cells of the retina and in inflammatory infiltrating
82
Captulo 1
cells. Although Wnschmann et al. (2004a) described the presence of WNV antigen
in endothelial cells of the pecten and choroid in the eye of infected American crows,
they did not describe associated lesions in this species. In contrast, WNV antigen
presence has not been documented in the eye of Psittaciformes (Stockman et al.,
2010).
Peripheral nervous system. WNV related lesions in the peripheral nervous system
have only been described in raptors and owls (Ellis et al., 2007). Mononuclear or
mixed inflammation is usually present in the sciatic nerve and in the myenteric,
proventricular and ventricular ganglia (Table 3). Neuronal positivity can be detected
by IHC.
Heart. Myocardial lesions may not be detected in falcons until 14 dpi, but appear
earlier in owls (8 dpi) and crows (6 dpi) (Weingartl et al., 2004; Nemeth et al.,
2006b; Busquets et al., 2012). Lymphoplasmacytic and histiocytic myocarditis with
myocardial necrosis, degeneration, mineralization or fibrosis and hemorrhages are
the main microscopic findings. Vasculitis can be seen in blue jays and American
kestrels (Falco sparverius) (Gibbs et al., 2005b; Nemeth et al., 2006a) (Table 3).
Cardiomyocytes, inflammatory and endothelial cells, and smooth muscle cells of
arteries usually show immunostaining. Although the heart is usually affected in
WNV infected birds, Anseriformes rarely have lesions in this tissue, even though
viral antigen is demonstrated by IHC (Steele et al., 2000).
Spleen and other lymphoid organs. Splenic lesions can appear as early as 2 dpi in
jays and crows, increasing in severity as infection progresses (Weingartl et al., 2004)
but in falcons they may not be prominent until 7 dpi (Busquets et al., 2012). In
this organ, lymphoid necrosis or apoptosis with fibrin deposition and hemosiderosis
are
consistently
observed.
Hemorrhages
are
described
in
ducks,
guanay
cormorants, and black-crowned night heron (Nycticorax nycticorax) (Steele et al.,

2000) and lymphoid depletion in goshawks and owls (Gancz et al., 2004; Erdlyi et
83
Tesis doctoral
al., 2007) (Table 3). Viral antigen is detected in dendritic cells/macrophages and
smooth muscular cells of arterioles. Necrosis can be found in bursal and thymic
lymphoid cells (Ellis et al., 2007; Himsworth et al., 2009) but these tissues have
been only analyzed in some naturally WNV exposed birds, most probably because
they are only present in juvenile birds (Table 3).
Liver. Similarly to the spleen, hepatic lesions can be found in crows as early as 3
dpi and increase in severity during the progress of the infection (Weingartl et al.,
2004). In owls and falcons on day 5 pi these lesions are mild changing to severe by
day 14 pi (Nemeth et al., 2006b; Busquets et al., 2012). Lymphoplasmacytic and
histiocytic hepatitis as well as coagulative hepatocyte necrosis are the most
frequent lesions. Hemorrhages in goshawks and blue jays (Gibbs et al., 2005b;
Erdlyi et al., 2007), biliary duct hyperplasia in northern bald eagles (Haliaeetus
leucocephalus alascanus) and Chilean flamingos (Phoenicopterus chilensis) (Steele et
al., 2000), and vasculitis in American kestrels can also be found (Nemeth et al.,
2006a) (Table 3). Birds that show hemosiderosis in the spleen usually also have it
in the liver (Zhang et al., 2006; Ellis et al., 2007; Ernest et al., 2010) (Table 3). WNV
antigen is mainly seen in hepatocytes and Kupffer cells. Anseriformes do not show
lesions (Glvits et al., 2005) but WNV antigen is detected by IHC.
Kidney. Renal lesions can be moderate on 6 dpi in crows and 9 dpi in owls but
may not become apparent until 14 dpi in falcons (Nemeth et al., 2006b; 2011;
Busquets et al., 2012). Lymphoplasmacytic and histiocytic interstitial nephritis and
tubular epithelial cell degeneration and/or necrosis are detected in most WNV
infected birds. Glomerular cell degeneration and/or necrosis are described in birds
of the order Psittaciformes and Strigiformes (Gancz et al., 2006; Palmieri et al.,
2011), hemorrhages in ducks (Himsworth et al., 2009) and vasculitis in American
kestrels (Nemeth et al., 2006b) (Table 3). IHC demonstrates WNV antigen in tubular
84
Captulo 1
and collecting duct epithelial cells, glomerular cells, infiltrating inflammatory cells,
interstitial fibroblasts and, less frequently, in endothelial cells.
Lung. WNV antigen labeling and associated lesions are not usually described in the
lung or if present they are mild. Lymphohistiocytic inflammatory infiltrates have
occasionally been described and interstitial edema and necrosis can also be found.
In naturally WNV infected Anseriformes and Psittaciformes pulmonary lesions have
not been documented (Himsworth et al., 2009; Stockman et al., 2010) (Table 3).
Viral antigen is detected mainly in inflammatory cells although it can also be found
in epithelial cells of the air capillaries.
Gastrointestinal tract. Lesions in the gastrointestinal tract are mainly described in
the proventriculus, ventriculus and intestines with enterocyte and intestinal crypt
cell necrosis, lymphoplasmacytic and histiocytic infiltrates, and proventricular
gland and ventricular hemorrhages as main microscopic findings. In owls, but even
less so in goshawks, lesions in the gastrointestinal tract are rarely found or if
present changes are mild (Fitzgerald et al., 2003; Erdlyi et al., 2007) (Table 3).
Viral antigen is usually detected in enterocytes and intestinal crypt cells,
inflammatory cells and smooth muscle cells of the lamina muscularis.
Endocrine system. Lymphoplasmacytic and histiocytic pancreatitis and acinar cell
necrosis are present in most species (Table 3). WNV antigen is detected in exocrine
cells. Adrenalitis has been described in different bird species but adrenal gland cell
necrosis has only been documented in Psittaciformes (Palmieri et al., 2011) (Table
3). Viral antigen is present in both cortical and medullary cells. Thyroid gland has
rarely been analyzed in the available studies but follicular cell necrosis has been
described in ducks (Himsworth et al., 2009) and thyroiditis in goshawks
(Wnschmann et al., 2005) (Table 3). Viral antigen in follicular cells has only been
described in owls and goshawks (Wnschmann et al., 2005; Gancz et al., 2006;
Erdlyi et al., 2007).
85
Tesis doctoral
Gonads. WNV associated lesions in gonads have only been documented in owls in
which authors described oophoritis, epididimitis and necrosis of granulosa cells,
oocytes and seminiferous tubular cells (Gancz et al., 2006) (Table 3). Viral antigen
has been detected in different species in oocytes, theca and granulosa cells, stromal
cells, seminiferous tubule cells and infiltrating inflammatory cells.
Skeletal muscle. In the skeletal muscle WNV infection can produce myositis,
myofibril degeneration, necrosis and fibrosis (Table 3). IHC shows viral antigen in
myofibers but this is not consistently observed (Stockman et al., 2010).
Skin.
Lymphocytic
dermatitis
has
only
been
described
in
WNV
infected
Psittaciformes (Palmieri et al., 2011) (Table 3). Viral antigen is detected in

keratinocytes of the basal layer of the epidermis, fibrocytes and macrophages of the
dermis and subcutis, interstitial cells, and epithelial cells of the feather pulp.
Although in raptors and owls skin lesions are not described, viral antigen is usually
detected (Wnschmann et al., 2005).
Bone marrow. Cellular necrosis in American crows (Wnschmann et al., 2004a)
and hypercellularity in turkeys (Zhang et al., 2006) are the only lesions that have
been described in the bone marrow (Table 3). WNV antigen can be detected in
hematopoietic cells.
Despite all the cited differences among bird orders, families or even species, lesions
and viral antigen are described in the majority of organs in WNV infected birds
(Figure 1).
86
ANAT
FAMILY
+
+
+
+
Neuronal vacuolization
Meningeal inflammatory infiltrates
Gliosis
Perivascular cuffs
Glial nodules
Vasculitis
Hemorrhage
Inflammatory infiltrates
+
+
+
Myofibril necrosis
Myocytolisis-mineralization
Vasculitis
Hemorrhage
HEART
ND
NT
PERIPHERAL NERVOUS SYSTEM
NT
Retinal cell necrosis
EYE
Neuronal necrosis-degeneration
SPINAL CORD
Neuronal necrosis-degeneration
BRAIN
TISSUE/Lesion
ANSE
ORDER*
ND
NT
NT
ND
ND
LARI
CHAR
ND
NT
NT
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ARDE
CICO
ACCI
NT
NT
NT
ND
ND
FALC
FALC
ND
NT
NT
ND
ND
PHAS
GALL
Table 3. Distribution of specific lesions in tissues of naturally WNV infected birds.
ND
ND
ND
ND
CORV
NT
NT
NT
NT
NT
LANI
PASS
NT
NT
NT
ND
ND
PASS
ND
NT
NT
ND
ND
PHAL
PELE
ND
NT
NT
ND
ND
PHOE
PHOE
ND
ND
ND
PSIT
PSIT
STRI
STRI
Captulo 1
87
ANAT
FAMILY
+
+
Fibrin deposition
Lymphoid cell depletion
Hemosiderosis
Hemorrhage
Bursal lymphoid cell atrophy-apoptosis
Thymic lymphoid cell necrosis
Kupffer cell necrosis
Vasculitis
Biliary duct hyperplasia
Hemosiderosis
Hemorrhage
Vasculitis
+
Glomerular cell necrosis
Hemorrhage
Tubular epithelial cell necrosis
KIDNEY
Hepatocyte necrosis
LIVER
Bursal epithelial cell atrophy-apoptosis
OTHER LYMPHOID ORGANS
Lymphoid cell necrosis/apoptosis
SPLEEN
TISSUE/Lesion
ANSE
ORDER*
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
NA
NA
NA
ND
ND
ND
ND
ND
LARI
CHAR
ND
ND
ND
ND
ND
ND
ND
NA
NA
NA
ARDE
CICO
NA
ACCI
NA
NA
NA
FALC
FALC
NA
NA
NA
PHAS
GALL
Table 3. Distribution of specific lesions in tissues of naturally WNV infected birds (cont).
NA
NA
NA
CORV
ND
ND
ND
ND
ND
NA
NA
NA
LANI
PASS
NA
PASS
NA
NA
NA
PHAL
PELE
NA
NA
NA
ND
ND
ND
ND
ND
PHOE
PHOE
ND
ND
PSIT
PSIT
STRI
STRI

Tesis doctoral
88
ANAT
FAMILY
Vasculitis
Edema
Intestinal crypt cell necrosis
Hemorrhage
ND
ND
+
Pancreatic inflammatory infiltrates
Adrenal gland cell necrosis
Adrenal inflammatory infiltrates
Thyroid gland cell necrosis
Thyroid inflammatory infiltrates
ND
ND
ND
Myofibril degeneration-necrosis
SKELETAL MUSCLE
ND
Cellular necrosis
GONADS
Pancreatic acinar cell necrosis
ENDOCRINE SYSTEM
Enterocyte necrosis
GASTROINTESTINAL TRACT
Cellular necrosis
LUNG
TISSUE/Lesion
ANSE
ORDER*
NA
NA
ND
ND
NA
NA
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
LARI
CHAR
NA
NA
ND
ND
NA
NA
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ARDE
CICO
ACCI
NA
NA
NA
NA
ND
ND
FALC
FALC
ND
ND
NA
NA
ND
ND
PHAS
GALL
ND
ND
NA
NA
NA
NA
CORV
NA
NA
ND
ND
NA
NA
NA
NA
LANI
PASS
NA
NA
NA
NA
NA
NA
NA
NA
ND
ND
ND
ND
ND
ND
PASS
NA
NA
ND
ND
NA
NA
ND
ND
ND
ND
ND
ND
PHAL
PELE
NA
NA
ND
ND
NA
NA
ND
ND
ND
ND
ND
ND
PHOE
PHOE
ND
ND
PSIT
PSIT
ND
ND
STRI
STRI
Captulo 1
89
NA
Hypercellularity
NA
NA
NA
NA
LARI
CHAR
NA
NA
NA
NA
ARDE
CICO
ND
ND
ND
ACCI
NA
NA
NA
FALC
FALC
NA
PHAS
GALL
NA
ND
CORV
NA
NA
ND
NA
LANI
PASS
NA
NA
NA
NA
PASS
NA
NA
NA
NA
PHAL
PELE
NA
NA
NA
NA
PHOE
PHOE
ND
ND
PSIT
PSIT
*ANSE = Anseriformes; CHAR = Charadriiformes; CICO = Ciconiiformes; FALC = Falconiformes; GALL = Galliformes; PASS = Passeriformes; PELE = Pelecaniformes; PHOE =
Phoenicopteriformes; PSIT = Psittaciformes; STRI = Sitrigiformes.
ANAT = Anatidae; LARI = Laridae; ARDE = Ardeidae; ACCI = Accipitridae; FALC = Falconidae; PHAS = Phasianidae; CORV= Corvidae; LANI = Laniidae; PASS = Passeridae; PHAL =
Phalacrocoracidae; PHOE = Phoenicopteridae; PSIT = Psittacidae; STRI = Strigidae.
ND: No described lesion. Tissues that were taken during necropsy but for which there are no lesions described.
NA: Tissue sample no analyzed.
+ Lesion present. Lesion described by at least one author for the tissue.
Lesion absent. Lesion stated as absent or not specifically described by any author for the tissue.
NA
NA
Cellular necrosis
BONE MARROW
SKIN
Fibrosis
SKELETAL MUSCLE (cont)

ND
ANAT
FAMILY
TISSUE/Lesion
ANSE
ORDER*
ND
ND
STRI
STRI

Tesis doctoral
90
Captulo 1
DISCUSSION AND CONCLUSION

West Nile virus, a pathogen considered of little importance for birds before the
1990s (Zeller and Schuffenecker, 2004), is nowadays one of the most widely
distributed arboviruses in the world that causes thousands of bird deaths, with a
locally significant impact on populations of native species in North America (LaDeau
et al., 2007; Foppa et al., 2011).
In this work we have reviewed the pathogenesis of WNV infection in
experimentally infected birds and the main pathologic findings that have been
described in natural infections. The information that has been reviewed indicates
on one hand that in most infected birds the virus first appears in the spleen, after
which it spreads rapidly to other organs such as the kidney, lung, heart and liver,
reaching later the CNS. However, tissue distribution takes place earlier in bird
species highly susceptible to infection such as Passeriformes and later in less
susceptible species such as Falconiformes, Galliformes or Strigiformes. On the
other hand, the reviewed information indicates that most infected birds that show
clinical disease and some of those that die without previous clinical manifestations
have macroscopic and/or microscopic lesions, but that there may be differences in
their distribution and severity between species even within the same family.
Accordingly, considerable differences exist among species in expression of clinical
disease that are not always clearly related to the severity and distribution of lesions.
Thus for example, nervous clinical signs do not always correlate with pathological
findings in the brain such as neuronal necrosis.
Acute lesions such as encephalitis and hemorrhages in different tissues are
consistently found in the vast majority of the described bird families. Hemorrhagic
fever associated to WNV infection has mainly been described in humans (Nash et
al., 2001; Paddock et al., 2006) in which hemorrhages are thought to be related to
direct or indirect microvascular damage (Paddock et al., 2006). Thus similarly, the
91
Tesis doctoral
vasculitis and endothelial WNV antigen found in some bird species could explain
the presence of blood extravasation. A study on human pathogenic flaviviruses has
associated sequence signatures in the envelope protein of the virus with the
primary syndrome that they produce (encephalitis or hemorrhagic disease) (Barker
et al., 2009), but envelope protein sequence is not available for most WNV strains
that infected the birds from which lesion descriptions exist. One lesion that is not
consistently found in every bird family or even in different species within families is
hemosiderosis in the spleen and liver. Avian hepatic hemosiderosis has been
observed in relation to iron overload in captive wild forest birds but is also
frequently associated to hemolytic processes in acute infectious disease (Cork et al.,
1995). Although Passeriformes develop necrosis and mild inflammation in the heart,
spleen, liver or kidney, they show only mild encephalitic lesions and neuronal
necrosis is absent (Steele et al., 2000; Wnschmann et al., 2004a; O'Brien et al.,
2010). This may be related to the higher susceptibility of this order to WNV
infection, leading to rapid viral distribution and host death that does not allow
development of encephalic lesions (Gibbs et al., 2005b). The absence of encephalitis
in
juvenile
chukar
partridges
(Alectoris
chukar)
and
Impeyan
pheasants
(Lophophorus impeyanus) (Wnschmann and Ziegler, 2006) could have the same
explanation. In contrast, neuronal degeneration, necrosis and phagocytosis are well
documented in Strigiformes and Falconiformes, potentially because the course of
the disease in these species is more prolonged. The detection of WNV antigen or
genome by IHC or real time RT-PCR in a particular tissue, even when there are no
observable macroscopic or microscopic lesions, or the apparent absence of viral
antigen immunolabeling although microscopic related lesions are present, may have
the same underlying cause (Gibbs et al., 2005b; Gancz et al., 2006). Thus, lesion
description and viral antigen detection should be considered together in order to
enable us to understand the pathogenesis of WNV infection in a particular host.
92
Captulo 1
Many pathologic differences are related to the time post-infection the animal
died which, in fact, is related to its susceptibility to infection and immune capacity
at the time the virus is inoculated, as well as the infection route (oral vs. mosquito),
the infective dose and the virulence of the infecting virus. Looking for example at
the description of Lopes et al. (2007), species specific factors are evident as in this
case the infecting virus, route and dose were probably very similar. Based upon the
evaluated information, apparently there are species in which the individuals
respond early and strongly to the infection clearing the virus rapidly, suffering thus
only mild microscopic lesions and causing only few individuals to succumb to the
disease (Busquets et al., 2012). On the contrary, other species potentially mount a
weak immune response of late onset that leads to high tissue viral loads and death
due to the severity of tissue lesions during the first general viremia (Lapointe et al.,
2009; Nemeth et al., 2011). Finally, there is a third group of species which
maintains low levels of viral replication that leads to chronic infection which can
finally produce host death or long-term sequelaes (Nemeth et al., 2006a).
However, we should consider that the literature about the pathology of
natural WNV infection in birds is limited to a number of species that may not
necessarily reflect the complete host spectrum of species affected by natural
disease. Thus, the information is unbalanced, meaning that the absence of lesions
in certain tissues in any particular bird species could be related to the limited
number of studies available. The reason why most studies about the pathology of
WNV infection have been carried out in raptors and owls may be related to the
higher visibility of dead or clinically affected animals of these species in the field.
Furthermore, these animals are frequently maintained in facilities such as zoos or
rehabilitation centers where they are observed daily and where necropsies of fresh
carcasses can be performed in case of death. Additionally, studies in corvids in the
US are probably driven by the magnitude of mortalities in these species and their
93
Tesis doctoral
visibility for the public as they are relatively abundant in urban and suburban
areas. The lack of information about WNV infection pathology in other species could
be related to the lack of fresh carcasses that enable a detailed pathological
description.
Certain bird species considered resistant to the disease in the field such as
the red-legged partridge (Alectoris rufa) develop lesions and succumbed to the
disease in experimental studies (Sotelo et al., 2011). Therefore, it is important to
consider that lesion and viral antigen distribution between experimentally and
naturally
infected bird
species
is
probably
different,
and
that numerous
contributing factors in the field are generally not reproduced under experimental
conditions.
In conclusion, differences in pathology of WNV infection in different bird
species are most probably related to a combination of host and environmental
factors in addition to the intrinsic virulence and pathogenicity of the infecting
strain. The non-progressive, acute or more prolonged course of the disease
accounts for part of the differences in the distribution of lesion and viral antigen
and in the severity of lesions. Finally, although experimental infections cannot
completely reproduce field situations, they can increase our understanding of
pathologic pathways and help in the identification of key factors that influence the
outcome of an infection in a given host.
ACKNOWLEDGEMENTS
This review is part of the work supported by grant AG2008-02504GAN funded by
the Spanish Ministry for Science and Innovation. V. Gamino (323/09) is a research
fellow supported by the regional government of Castilla La Mancha (JCCM). We
acknowledge F. Ruiz-Fons for critically reviewing an early draft of the paper.
94
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104
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Patogenia de la infeccin experimental con
dos cepas mediterrneas del virus West Nile
en perdiz roja
Gamino V, Prez-Ramrez E, Gutirrez-Guzmn AV, Sotelo E, JimnezClavero MA, Hfle U. Pathogenesis of two Mediterranean West Nile
virus strains in experimentally infected red-legged partridges. En
preparacin.
Captulo 2
RESUMEN
La re-emergencia del virus West Nile (WNV) en Europa y la cuenca mediterrnea en
las ltimas dos dcadas se atribuye a cambios aminoacdicos que se han producido
en las cepas del virus con el consecuente incremento en su virulencia para
humanos, caballos y aves. En el presente trabajo, estudiamos las diferencias en la
patogenia de la infeccin experimental de perdices rojas (Alectoris rufa), de siete
semanas de edad, con dos cepas mediterrneas de WNV, Morocco/2003 (MO03) y
Spain/2007 (SP07). El objetivo fue determinar las causas del diferente curso de la
infeccin y la diferente mortalidad encontrados en un trabajo previo. Adems,
estudiamos la dinmica de activacin y llegada de clulas inflamatorias al cerebro y
cerebelo de las perdices infectadas. En ambas infecciones, el da 6 post inoculacin
(dpi) fue cuando la carga viral fue mayor, el antgeno viral estuvo ms ampliamente
distribuido y fue ms abundante, y las lesiones microscpicas fueron ms severas.
Los rganos ms afectados fueron el corazn, hgado y bazo. En el cerebro y
cerebelo, la microgla fue la poblacin inflamatoria ms prevalente y las clulas T
CD3+ infiltraron este tejido el 6 dpi. Comparando ambas infecciones, se observaron
relativamente pocas diferencias en la carga viral, distribucin del virus y naturaleza
y severidad de las lesiones; sin embargo, en las perdices infectadas con MO03 las
lesiones microscpicas en el cerebro y cerebelo aparecieron de forma ms aguda y
fueron ms severas, lo que dio lugar a una encefalitis ms marcada. Considerando
nuestros resultados, el mayor impacto de la infeccin por MO03 para las perdices
rojas est probablemente relacionado con marcadores especficos que hacen que
esta cepa sea ms neurovirulenta.
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ABSTRACT
West Nile virus (WNV) re-emergence in Europe and the Mediterranean basin in the
last two decades is thought to be related to amino acid substitutions in the virus
strains with a subsequent increase in their virulence for humans, horses and birds.
In the present work, we studied differences in the pathogenesis of the experimental
infection of 7-week-old red-legged partridges (Alectoris rufa) with two different
Mediterranean WNV strains, Morocco/2003 (MO03) and Spain/2007 (SP07). The
objective was to elucidate the reasons of the different infection course and mortality
observed in a previous work. Additionally, we studied the dynamics of inflammatory
cell activation and recruitment into the brain of the infected partridges. In both
infections, day 6 post-inoculation (dpi) was the day when viral load was higher,
virus antigen was more widespread and abundant, and microscopic lesions were
more severe. The most affected organs were the heart, liver and spleen. In the brain,
the more prevalent inflammatory cell population was microglia and CD3+ T cells
infiltrated this tissue on 6 dpi. Comparing both infections, few differences were
observed in viral load, virus distribution and lesion nature and severity; however, in
MO03 infected partridges microscopic lesions in the brain were more acute and
severe, which led to a more marked encephalitis. Considering our results, the
higher impact of MO03 infection on red-legged partridges is probably related to
specific markers of this WNV strain that make it more neurovirulent.
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INTRODUCTION
West Nile virus (WNV) is an arthropod-borne flavivirus whose natural cycle involves
bird hosts and mosquito vectors, with horses and humans as accidental or deadend hosts (Kramer et al., 2008). Nowadays, it is considered one of the most widely
distributed arboviruses in the world, causing thousands of human, equine and bird
encephalitis cases and mortalities, both in the Old and New World (CDC; ECDC). In
the Mediterranean basin, WNV activity has increased in the last two decades, and it
has been associated to several outbreaks affecting mainly humans and horses
(Murgue et al., 2001; Sotelo et al., 2011a). In this area, with the exception of the
outbreak reported in migrating storks (Ciconia ciconia) and geese (Anser anser
domesticus) in Israel between 1998-1999 (Malkinson et al., 2002), there have only
been sporadic cases of avian mortality (Jourdain et al., 2007; Jimnez-Clavero et
al., 2008; Monaco et al., 2010; Savini et al., 2012, 2013). WNV re-emergence is
thought to be associated mainly to genetic factors of the virus and/or
environmental factors (Sotelo et al., 2011a). The variability of amino acid sequence
of Mediterranean WNV isolates between 1998 and 2003 was low. However, recent
isolates show higher frequency of amino acid substitutions that can lead to
phenotypic changes (Sotelo et al., 2009; 2011a), as demonstrated in field and in
several experimental studies (Beasley et al., 2005; Davis et al., 2005; Wicker et al.,
2006). In birds, genetic differences in WNV strains have been shown to modify the
degree of peripheral virus replication and its virulence (Brault et al., 2004; Langevin
et al., 2005; Totani et al., 2011).
In a recent experimental study we demonstrated that the red-legged partridge
(Alectoris rufa), an endemic Mediterranean gallinaceous bird species (Blanco-Aguiar
et al., 2003), is susceptible to the experimental infection with two Western
Mediterranean WNV strains, Morocco/2003 (MO03) and Spain/2007 (SP07) (Sotelo
et al., 2011b). However, virulence of both strains was markedly different; with 70%
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mortality in MO03 infected partridges as compared to 30% mortality in SP07

infected birds. In that study, it was indicated that one or a combination of the 13
amino acid differences detected between the two WNV strains were probably
determining differences in their virulence. Nevertheless, in the aforementioned
study, detailed pathogenesis of the infection by each strain that led to differences in
the onset of clinical signs and mortality was not addressed.
For this reason, in the present study, we compare the dynamics of virus
appearance and distribution as well as of microscopic lesion distribution and
severity in different tissues during the course of the experimental infection of redlegged partridges with these two Mediterranean WNV strains. Additionally, and
considering the importance of the central nervous system (CNS) infection in the
pathogenesis of WNV disease, we study the dynamics of immune cell activation and
recruitment into the brain of the partridges. To our knowledge, this has not been
previously documented in WNV infected birds.
MATERIAL AND METHODS

Animals
Recently hatched red-legged partridge chicks were obtained from a commercial
breeder and raised in the experimental farm of the Instituto de Investigacin en
Recursos Cinegticos (IREC) until they were six weeks of age. Prior to the
experimental infection, they were tested by ELISA for the presence of WNV
antibodies (ID Screen West Nile Competition, IdVet, Montpellier, France) and by
real time RT-PCR for detection of WNV genome in cloacal and oral swabs, in order
to ensure they were not previously infected by the virus (Sotelo et al., 2011b).
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Captulo 2
Viruses
In this study we used two different strains of WNV that had been isolated in the
Mediterranean basin; Morocco/2003 (MO03), isolated from a horse and cultivated
in Vero cells, and Spain/2007 (SP07), isolated from a golden eagle (Aquila
chrysaetos) and cultivated in BSR cells (clone of BHK-21 cell line). Details on the
inoculates are given in Sotelo et al. (2011b).
Experimental infection
Partridges were transported to the biosafety level 3 (BSL-3) facilities in the Centro
de Investigacin en Sanidad Animal (CISA). After five days for acclimatization, two
groups of ten 7-week-old red-legged partridges were subcutaneously inoculated in
the cervical region with 104 PFU/individual of either WNV SP07 or MO03 diluted in
up to 0.1 mL Dulbecco's Minimum Essential Medium (DMEM) (supplemented with
2 mM L-glutamine, 100 U/mL penicillin and 100 g/mL streptomycin). A control
group was sham-inoculated with an equivalent volume of DMEM and placed in a
separate cage. Inoculated partridges were observed daily for clinical signs or death
and two birds of each group were euthanized by intravenous injection of
embutramide (T61 , Intervet Schering-Plough, Madrid, Spain) on days 3 (Nos. 14), 6 (Nos. 5-8) and 14 (Nos. 9-11) post-inoculation (dpi).
The experiment was performed following biosafety animal welfare and ethical
rules applicable in the EU. Food and water were provided ad libitum throughout the
experiment.
Sample collection
Detailed necropsies were performed on euthanized individuals. Samples of brain,
heart, lung, liver, spleen, kidney, thymus, bursa of Fabricius and feather pulp were
collected into sterile polypropylene tubes filled with 1 mL of Hanks' balanced
solution (10% glycerol, 200 U/mL penicillin, 200 g/mL strepctomycin, 100 U/mL
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Tesis doctoral
polymixin B sulphate, 250 g/mL gentamicin and 50 U/mL nystatin) and stored at
-70 C until analysis. In addition, samples of brain, oral mucosa, thymus, heart,
trachea, lung, liver, spleen, kidney, small and large intestine, pancreas, cecal
tonsils, bursa of Fabricius, pectoral muscle and skin with feather follicles were fixed
in 10% neutral buffered formalin.
Virus genome detection

RNA was extracted from tissue samples after homogenization and tested by real
time RT-PCR for the presence of WNV genome as described in Sotelo et al. (2011b).
Histopathology
Formalin-fixed tissue samples were trimmed, embedded in paraffin and processed
to obtain hematoxylin and eosin stained sections. These were independently
examined by two different investigators (UH and VG) to determine the presence of
WNV associated lesions. When lesions were present, these were graded according to
their distribution (focal, multifocal or diffuse) and severity (mild, moderate or
marked).
Immunohistochemistry
Tissue sections were also mounted on Vectabond reagent (Vector Laboratories,
Inc., Burlingame, CA) pretreated slides. Immunohistochemical detection of WNV
antigen was performed using a rabbit polyclonal antibody (BioReliance, Product 81015, Rockville, MD) following the protocol described previously (Gamino et al.,
2012). We also characterized the inflammatory cell population in the cerebrum and
cerebellum. For that purpose, we used primary antibodies, reagents and protocols
detailed in table 1. In all cases endogenous peroxidase activity was inhibited with a
peroxidase blocking reagent (Dako EnVision+System-HRP (AEC), DakoCytomation,
Carpinteria, CA) (CD3, CD79, GFAP) or with 3% H202 diluted in methanol (RCA),
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Captulo 2
rinses were performed using 0.1% Tris-buffered saline/Tween20 (TBS 0.05 M, pH

7.5), non-specific primary antibody labeling was blocked with 2% albumin from
bovine serum (BSA) (Sigma-Aldrich Chemie, Steinheim, Germany) diluted in 0.1%
TBS/Tween20, and sections were counterstained with Mayer's hematoxylin.
In the immunohistochemistry (IHC) for WNV antigen detection, tissue
sections of experimentally infected red-legged partridges, in which presence of WNV
had been confirmed by real time RT-PCR, served as positive controls. Negative
controls included several sections with substitution of the primary antibody by 2%
BSA-0.1% TBS/Tween20 and negative rabbit antibody (BioReliance, Product 81
015), as well as tissue sections of a red-legged partridge negative for WNV infection.
In the IHC for T and B cells, positive controls included sections of spleen and
bursa of Fabricius of red-legged partridges. Negative controls included substitution
of the primary antibody by 2% BSA-0.1% TBS/Tween20 and a brain section of a
red-legged partridge that tested negative for WNV presence by real time RT-PCR. A
brain section of a non-infected partridge of the same age served as reference for
RCA and GFAP.
We graded virus antigen staining according to its distribution and
abundance. Moreover, we characterized the distribution of inflammatory cells in the
brain and analyzed changes in their relative abundance during the course of
infection.
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Table 1. Detail of reagents and protocols used in the IHC for inflammatory cell characterization
in the CNS of experimentally WNV infected red-legged partridges.
Cell
population/
marker
Antibody
reference*
Pretreatment**
Primary
antibody
dilution
Primary
antibody
incubation
Secondary
antibody
Detection
system
Microgliamacrophages
Lectin RCA-1
biotinylated
Citrate buffermicrowave heat

(22 min)
1:600
45 min RT
Goat antirabbit IgG
ABC-DAB
Astrocytes
Polyclonal rabbit
anti-GFAP
Proteinase K
(7 min RT)
1:500
4C ON
T cells
Polyclonal rabbit
anti-human CD3
1:500
4C ON
AEC+
substrate
chromogen
B cells
Monoclonal rabbit
anti-human
CD79a
Labelled
polymer-HRP
anti-rabbit
1:10
45 min RT
Citrate buffermicrowave heat

(22 min)
*Primary antibody products: RCA-1 product No. B-1085 (Vector Laboratories); GFAP product No. Z0334
(DakoCytomation, Glostrup, Denmark); CD3 product No. A0452 (DakoCytomation); CD79a product No. RM-9118
(Thermo Fisher Scientific, Runcorn, UK).
**Proteinase K (DakoCytomation); RT: room temperature (22-25 C).
Antibodies were diluted in 2% BSA-0.1% TBS/Tween20.
ON: overnight.
Goat anti-rabbit IgG (Vector Laboratories) was diluted 1:200 in 0.1% TBS/Tween20 and applied for 1 hr at RT;
Labelled polymer-HRP anti-rabbit (Dako EnVision+System-HRP (AEC), DakoCytomation) was applied according to
manufacturer's recommendation.
Avidin-biotinylated enzyme complex (ABC system, Vector Laboratories) was applied for 30 min according to
manufacturer's recommendation and 3,3-diaminobenzidine tetrahydrochloride (DAB, Vector Laboratories) was
applied for 30 sec according to manufacturer's recommendations. AEC+substrate chromogen (Dako
EnVision+System-HRP (AEC), DakoCytomation) was applied for 15 min (CD3, CD79) and 3 min (GFAP) according
to manufacturer's recommendation.
RESULTS
Macroscopic findings
Macroscopic lesions were observed in the necropsy of all euthanized animals from 3
dpi on. The most affected organs were the heart, spleen, liver and kidneys, with no
remarkable difference between SP07 and MO03 inoculated partridges. Main
macroscopic lesions were pale myocardium and pale hepatic, splenic and renal
parenchyma, which, in some cases, also showed diffuse petechiae. On 14 dpi, one
SP07 inoculated bird (No. 10) had congestion in the kidney and spleen that also
was observed in the heart and the lung of one MO03 infected partridge (No. 11).
Partridge No. 11 showed cerebral vessel injection that was also observed in one
SP07 infected bird (No. 9).
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Captulo 2

In the WNV real time RT-PCR, all tissue samples collected in the necropsy of
inoculated animals tested positive from 3 dpi on, with lower Ct values (i.e. higher
viral loads) on 6dpi (Table 2). Ct values were similar between MO03 and SP07
infected partridges or slightly lower in some tissues of the MO03 infected group
(Table 2). On 3 dpi, the spleen followed by the kidney and heart were the organs
with highest viral load (Table 2). On 6 dpi, the highest viral load was detected in the
heart but also in the spleen and kidney (Table 2). On this dpi, the feather pulp was
markedly positive, especially in bird No. 8, and the brain showed the lowest Ct
values (Table 2). On 14 dpi, viral loads were low in most cases, and several tissues
were negative in the SP07 infected partridge (No. 10) (Table 2). Control animals
tested negative for WNV RNA throughout the experiment.
Table 2. Detection of WNV genome by real time RT-PCR (Ct values) in tissues of experimentally
infected red-legged partridges.
3 dpi
SP07
6 dpi
MO03
SP07
14 dpi
MO03
SP07
MO03
TISSUE
No.1
No. 2
No. 3
No. 4
No. 5
No. 6
No. 7
No. 8
No. 9
No. 10 No. 11
Brain
30.3
32.8
31.4
33.9
25.2
26.5
25.2
24.8
NA
No Ct
37.6
Heart
28.7
28.5
25.4
24.8
19.6
20.3
23.7
18.8
NA
38.0
33.8
Lung
NA
30.9
31.0
30.1
26.3
25.7
29.2
24.2
NA
No Ct
38.8
Liver
NA
30.2
29.6
30.9
28.7
25.7
30.8
28.2
NA
No Ct
No Ct
21.8
NA
36.1
34.0
20.9
NA
37.5
36.1
Spleen
Kidney
25.3
26.3
23.7
27.5
26.5
24.5
24.4
26.0
29.7
23.0
22.7
22.4
24.6
23.7
Thymus
27.6
29.3
31.9
23.5
30.2
23.4
33.0
NA
NA
No Ct
29.0
Bursa of Fabricius
NA*
32.0
32.8
34.2
27.9
24.8
31.5
29.8
NA
No Ct
31.3
No Ct
31.4
27.4
31.3
18.2
20.8
22.8
16.9
NA
34.6
33.7
Feather pulp
*NA: tissue sample no analyzed.

No Ct: tissue sample with Ct 40 (considered negative).
Histopathology
Histologic lesions appeared as early as 3 dpi, but were more severe and widespread
on 6 dpi (Table 3). The most affected tissues in both groups were the heart, spleen,
liver and skin with feather follicles (Figures 1-4). The brain, lung and kidney were
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moderately affected and the oral mucosa, pancreas, cecal tonsils, thymus and
intestines showed, in most cases, mild lesions (Table 3). There were no microscopic
lesions in the trachea and pectoral muscle.
Main
microscopic
findings
were
the
presence
of
lymphoplasmacytic,
histiocytic and/or granulocytic inflammatory infiltrates, cellular necrosis and/or

degeneration, and hemosiderosis in the spleen and liver (Table 3 and figures 1-4). In
the brain, the most consistently observed lesion was endothelial cell swelling
(Figure 5), both in the cerebrum and cerebellum, but also mild neuronal necrosis
(affecting also Purkinje cells) diffuse gliosis or glial nodules, and rare perivascular
cuffing (Table 3 and figures 5 and 6). All these lesions were observed in both groups
but were slightly more severe in some tissues of MO03 infected partridges,
especially in the brain (Table 3).
On 3 dpi, the main microscopic finding was the presence of widespread
moderate to marked inflammatory infiltrates, although mild cardiac myofiber
degeneration was also observed (Table 3). In the brain, endothelial cell swelling was
the only detected lesion, mainly in MO03 infected partridges (Table 3). On 6 dpi,
microscopic lesions increased in severity, with the appearance of cellular necrosis
in many tissues, which was especially severe in the liver in both groups (Table 3
and figures 1 and 3). One MO03 infected partridge (No. 8) showed multifocal
macrophages with foamy and distended cytoplasm in the spleen (Figure 2). On this
dpi, mild to moderate neuronal necrosis and gliosis appeared in the brain of MO03
infected partridges (Table 3 and figures 5 and 6). On 14 dpi, microscopic lesions
were still widespread, with moderate to marked severity in the heart and spleen
that was milder in the liver (Table 3). On this dpi, renal tubular epithelial cell
necrosis appeared and both SP07 and M003 infected birds showed mild to
moderate lesions in the brain (Table 3).
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Captulo 2
Incidental findings included the presence of granulomas in the liver and

pancreas of birds euthanized on 3 and 6 dpi and coccidian oocysts in the
enterocytes of the large intestine. These parasites were especially numerous in
individuals euthanized on 6 dpi and in partridges infected with MO03. Thus, one
MO03 infected partridge (No. 8) showed an associated severe necrosis of the
intestinal mucosa.
Table 3. Microscopic lesions and IHC staining gradation on different days post-inoculation (dpi)
in tissues of experimentally WNV infected red-legged partridges.
3 dpi
6 dpi
SP07
MO03
14 dpi
SP07
MO03
SP07
MO03
No. 1
No. 2
No. 3
No. 4
No. 5
No. 6
No. 7
No. 8
No. 9
No. 10
No. 11
Neuronal necrosis
Gliosis
++
Perivascular cuffing
Endothelial cell swelling
++
++
++
++
Immunohistochemical staining
Purkinje cell necrosis
++
NA*
Gliosis
++
NA
++
NA
++
++
+++
++
++
NA
++
NA
Cerebrum
Cerebellum
Heart
Myofiber necrosis-degeneration
+++
++
+++
+++
++
++
Inflammatory infiltrate
++
++
+++
+++
+++
+++
++
+++
++
++
+++
BALT necrosis
+++
++
+++
+++
+++
+++
+++
Lung
Liver
Hepatocyte necrosis
+++
+++
++
++
++
++
++
++
+++
++
Hemosiderosis
++
++
++
Lymphoid cell necrosis
++
++
+++
+++
++
++
+++
+++
++
+++
Eosinophilic material deposits
+++
+++
++
++
++
++
Hemosiderosis
+++
+++
+++
++
Spleen
Granulocytic infiltrate
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Table 3. Microscopic lesions and IHC staining gradation on different days post-inoculation (dpi)
in tissues of experimentally WNV infected red-legged partridges (cont).
3 dpi
6 dpi
SP07
No. 1
MO03
No. 2
No. 3
14 dpi
SP07
MO03
SP07
MO03
No. 4
No. 5
No. 6
No. 7
No. 8
No. 9
No. 10
No. 11
Kidney
Tubular epithelial cell necrosis
+++
+++
+++
++
++
++
++
NA
NA
++
NA
++
NA
Acinar cell necrosis
NA
NA
NA
NA
NA
NA
++
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
++
NA
NA
NA
++
++
++
NA
NA
NA
NA
NA
Duodenum
Large intestine
Pancreas
Cecal tonsils
Bursa of Fabricius
Skin + feather follicle

Inflammatory infiltrate skin
NA
+++
NA
NA
NA
++
Inflammatory infiltrate feather

pulp
+++
NA
+++
NA
NA
NA
NA
+++
NA
+++
NA
NA
NA
Tissue lesions were graded according to their distribution and severity: , no lesion; +, focal and mild or moderate /
multifocal and mild; ++, focal and marked / multifocal and moderate / diffuse and mild; +++, multifocal and marked /
diffuse and moderate or marked.
WNV antigen immunostaining was graded according to its distribution and percentage of stained cells: , negative
staining; , focal single cells; +, focal or multifocal and < 20% cells stained; ++, multifocal or diffuse and 20-80% cells
stained; +++, multifocal or diffuse and > 80% cells stained.
*NA: tissue sample no analyzed.
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Figure 1. Heart; partridge inoculated with SP07 No. 6, 6 dpi. Diffuse and marked necrosis and
degeneration of cardiac myofibers, and infiltration of lymphoplasmacytic and histiocytic cells. HE,
x100. Inset: detail of necrotic cardiac myofibers which show degeneration, fragmentation and
accumulation of hyaline material in the cytoplasm. Infiltration of mononuclear inflammatory cells is
also observed. HE, x400.
Figure 2. Spleen; partridge inoculated with MO03 No. 8, 6 dpi. Distension of the cytoplasm of
splenic macrophages by a foamy material (black arrows) or a red-brown pigment (white arrows) that
displace the nuclei. Mild infiltration of granulocytes (arrowheads). HE, x400.
Figure 3. Liver; partridge inoculated with SP07 No. 6, 6 dpi. Multifocal to coalescing marked
necrosis of hepatocytes near the central vein. HE, x40. Inset: detail of the necrotic hepatocytes which
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Tesis doctoral
show picnosis and lysis of the nuclei and degeneration of the cytoplasm. Mild infiltration of
lymphoplasmacytic and histiocytic cells is also observed. HE, x400.
Figure 4. Feather follicle; partridge inoculated with MO03 No. 11, 14 dpi. Diffuse and moderate
infiltration of lymphoplasmacytic, histiocytic and granulocytic cells in the feather pulp. HE, x200.
Inset: detail of an inflammatory nodule composed by lymphocytes, histiocytes and granulocytes. HE,
x400.
Figure 5. Cerebrum; partridge inoculated with MO03 No. 7, 6 dpi. Focal necrosis of neurons,
degeneration of the neuropil and infiltration of lymphocytic and glial cells. HE, x400. Inset: swelling of
the nuclei of endothelial cells (arrowheads). HE, x400.
Figure 6. Cerebellum; partridge inoculated with MO03 No. 7, 6 dpi. Focal necrosis of Purkinje cells
(arrow) and glial cells (arrowheads), and a focus of gliosis and degeneration in the molecular layer. HE,
x400.
Virus antigen
Virus antigen was detected by IHC in several tissues from 3 dpi on, mainly in
inflammatory cells but also in different parenchymal and epithelial cells.
Immunopositivity was mild to moderate in most cases and WNV antigen was more
widespread on 6 dpi and in the tissues of MO03 infected partridges (Table 3).
On 3 dpi, virus antigen was detected in macrophages in the spleen (Figure 7)
and in inflammatory cells and myofibers of the heart. In MO03 infected partridges,
there was also mild immunostaining in tubular epithelial cells of the kidney, single
acinar cells of the pancreas and a small group of inflammatory cells in the cecal
tonsils. On 6 dpi, WNV antigen was present in inflammatory cells in the lung, heart,
spleen, kidney and pancreas. It was also detected in cardiac myofibers (Figure 8),
glomerular mesangial cells (Figure 9) and tubular epithelial cells of the kidney,
acinar cells of the pancreas and in enterocytes of the crypts and myofibers of the
lamina muscularis of the large intestine. In MO03 infected birds, virus antigen was
also present in some vascular walls in the spleen, hepatocytes and Kupffer cells of
the liver, and in one vascular wall and myofibers of the lamina muscularis of the
duodenum. On 14 dpi, IHC for WNV antigen detection was negative in every tissue.
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Inflammatory cells in the brain

Microglia activation and macrophage infiltration
In both groups there was an early reaction of microglial cells, but differences in
their relative abundance and morphology were detected during the course of the
infection and between SP07 and MO03 infected birds.
In the cerebrum, microglia activation and macrophage infiltration were more
prominent in the peripheral pallium and especially evident near the lateral
ventricle. On 3 dpi, few cells with no more than two thin cellular processes were
observed, but there was also mild presence of amoeboid cells, characterized by a
larger soma and short thick cellular processes, and rounded cells, corresponding
both to activated microglia and macrophages. In one MO03 infected bird (No. 3), few
neurons and vessels were surrounded by amoeboid cells. On 6 dpi, relative
abundance of stained microglia, mainly with an amoeboid morphology, and
macrophages increased both in SP07 and MO03 inoculated birds. Many of these
cells were grouped surrounding neurons and vessels, especially in MO03 infected
birds. On this dpi, SP07 infected partridges also showed numerous ramified cells
composed by more than three thin cellular processes. On 14 dpi, ramified cells were
scarce but amoeboid cells and macrophages still remained. Reactive microglial cells
were slightly more abundant in SP07 infected birds, especially in case No. 10.
In the cerebellum, amoeboid microglial cells and macrophages were
especially abundant in the molecular layer, mainly from 6 dpi on (Figure 10). These
cells were diffusely distributed or forming nodules that in many cases were
surrounding Purkinje cells. On 6 dpi, microglia activation was also moderate in the
granular layer and white matter, especially in MO03 infected partridges. Similarly
to the cerebrum, cellular activation was lower on 14 dpi.
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Astrocyte activation
GFAP+ cells were detected from 3 dpi on, with only mild changes in their
distribution and relative abundance during the infection course and between
infected groups. In comparison to a non-infected partridge, there were mild
differences in staining distribution but none in astrocyte morphology or staining
intensity.
In the cerebrum, all inoculated animals showed positive staining in
astrocytes with long cellular processes located in the subpial zone, in astrocytes
with medium cellular processes surrounding some vessel walls, and in star-shaped
astrocytes in the pallium near the lateral ventricle (hippocampus) (Figure 11). Mild
astrocytosis (i.e. increased number of astrocytes) was detected in the lamina
medularis dorsalis in MO03 infected birds.
In the cerebellum, there was mild astrocytosis in the granular layer, slightly
more marked on 6 dpi and in MO03 infected birds. In the MO03 infected group,
especially in one bird euthanized on 6 dpi (No. 7), some GFAP+ fibers invade the
molecular layer among Purkinje cells. In this group, there was also a moderate
astrocytosis in the white matter.
T cell infiltration
Although scarce CD3+ T cells were detected in the vascular lumen of some vessels
in MO03 infected partridges on 3 dpi, it was on 6 dpi when these cells were more
numerous, especially in MO03 infected birds.
In the cerebrum, T cell infiltration was more marked in the peripheral
pallium. These cells were mainly diffusely distributed, but also formed part of
perivascular infiltrates and of inflammatory foci/nodules associated, in some cases,
with neuronal necrosis. T cells were also detected within the vascular lumen. On 6
dpi, partridge No. 8 (MO03) showed many T cells forming part of large inflammatory
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Captulo 2
nodules. On 14 dpi, cellular infiltration decreased, more drastically so in the MO03

infected group.
In the cerebellum, most T cells were distributed in the molecular layer, in
some cases forming part of inflammatory nodules surrounding Purkinje cells
(Figure 12). There was also moderate infiltration in the granular layer and mild
infiltration in the white matter. On 6 dpi, this infiltration was much more
prominent in MO03 infected birds, especially in case No. 7, decreasing markedly in
both groups on 14 dpi.
Few T cells were detected in meningeal vessels, only in MO03 infected
partridges. In some cases, brain zones of T cell infiltration corresponded to brain
zones of microglia activation and/or macrophage infiltration.
B cell infiltration
B cells were not stained in the brain of the red-legged partridges on any dpi.
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Figure 7. Spleen; partridge inoculated with SP07 No. 1, 3 dpi. WNV antigen in the cytoplasm of
splenic macrophages. IHC for WNV with Mayer's hematoxylin counterstain, x400.
Figure 8. Heart; partridge inoculated with SP07 No. 6, 6 dpi. WNV antigen in the cytoplasm of
cardiac myofibers. IHC for WNV with Mayer's hematoxylin counterstain, x100. Inset: detail of WNV
antigen in the cytoplasm of cardiac myofibers. IHC for WNV with Mayer's hematoxylin counterstain,
x400.
Figure 9. Kidney; partridge inoculated with MO03 No. 8, 6 dpi. WNV antigen in the cytoplasm of
glomerular mesangial cells. IHC for WNV with Mayer's hematoxylin counterstain, x400.
Figure 10. Cerebellum; partridge inoculated with MO03 No. 8, 6 dpi. RCA-1 positive staining in
the cytoplasm of phagocytic foamy macrophages in the molecular layer. IHC for microglia
activation/macrophages with Mayer's hematoxylin counterstain, x400.
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Figure 11. Cerebrum; partridge inoculated with MO03 No. 8, 6 dpi. GFAP positive staining in
astrocytes diffusely located in the subpial zone, surrounding blood vessels and diffusely distributed
within the parenchyma near the lateral ventricle (LV). IHC for astrocytes with Mayer's hematoxylin
counterstain, x100. Inset: detail of stained astrocytes with medium cellular processes surrounding a
blood vessel and star-shaped astrocytes within the parenchyma. IHC for astrocyte activation with
Mayer's hematoxylin counterstain, x400.
Figure 12. Cerebellum; partridge inoculated with MO03 No. 7, 6 dpi. CD3 positive staining in T
cells located near the Purkinje cell layer. IHC for T cells with Mayer's hematoxylin counterstain, x400.
DISCUSSION
In this work, we studied differences in the pathogenesis of the experimental
infection of red-legged partridges with two different Mediterranean WNV strains, in
order to elucidate the different infection course and mortality observed in a previous
work (Sotelo et al., 2011b).
In both groups, WNV genome and antigen as well as macroscopic and
microscopic lesions were detected as early as 3 dpi. On this dpi, the most affected
tissues were the heart, liver, spleen and kidney and virus antigen was detected
mainly in inflammatory cells, suggesting that these cells play an important role in
WNV infection pathogenesis in red-legged partridges. However, day 6 pi should be
considered critical in the course of the infection, since at that time viral load in
tissues was highest, virus antigen was more widespread and abundant, and
microscopic lesions were more severe. On this dpi, most birds suffered encephalitis
and virus antigen was detected both in inflammatory cells and parenchymal and
epithelial cells of several tissues. On 14 dpi, tissue lesions were still observed,
however, viral loads were low or absent and detectable virus had been cleared from
many tissues, suggesting that, at that time, partridges had eliminated most
infecting virus. Nevertheless, potential virus maintenance in the tissues where viral
load was relatively low should be considered, as persistent infection has been
demonstrated in experimentally and naturally WNV infected birds (Reisen et al.,
2006; Wheeler et al., 2012).
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Tesis doctoral
Microscopic lesions found in this work were similar to those described in

other WNV infected gallinaceous birds (Steele et al., 2000; Wnschmann and
Ziegler, 2006; Zhang et al., 2006) but, while we found endothelial cell swelling,
gliosis and neuronal necrosis in the brain, CNS lesions were not described in
chukar partridges (Alectoris chukar) naturally infected with the virus (Wnschmann
and Ziegler, 2006). The presence of marked inflammatory infiltrates in the feather
pulp and skin from 3 dpi onwards was probably associated to the high viral load
found, but we were not able to demonstrate virus antigen by IHC. Nevertheless,
WNV has been detected in the skin and feather follicles in naturally and
experimentally infected birds (Wnschmann et al., 2005; Gancz et al., 2006;
Shirafuji et al., 2008), and feather-picking has been suggested as a potential way of
horizontal WNV transmission (Banet-Noach et al., 2003). The severe increase in the
number of intestinal coccidian oocysts from 3 to 6 dpi, only in inoculated animals,
suggests that immunosuppression associated to WNV infection exacerbated this
secondary parasitation. In fact, secondary pathological processes in WNV infected
birds have been documented on numerous occasions (Saito et al., 2007; Hfle et al.,
2008; Nemeth et al., 2009).
In general, viral loads obtained in the real time RT-PCR correlated well with
virus antigen detected by IHC and microscopic lesions, with some exceptions such
as the brain. Despite the fact that this tissue showed microscopic lesion and tested
positive for WNV genome, virus antigen was not detected by IHC. Different
sensitivity of PCR and IHC could be the reason (Steele et al., 2000). However, it
would be possible that the mild to moderate neuronal necrosis found in our study
was more related to the local inflammatory response than to viral infection and
replication within neurons and other parenchymal cells, especially on 14 dpi.
Once WNV reaches the CNS, it rapidly infects neurons, but also glial and
endothelial cells (Steele et al., 2000; Fitzgerald et al., 2003; Erdlyi et al., 2007;
126
Captulo 2
Lopes et al., 2007). This activates the CNS immune response that, at least in
mammals, includes microglia and astrocyte activation and proliferation, chemokine
and cytokine release, and leukocyte recruitment (Samuel and Diamond, 2006;
Brhin et al., 2008; Suthar et al., 2013). In our study, the most prevalent
inflammatory cell population was microglia, mainly on 6 dpi, when these cells
change their morphology to an activated amoeboid form. Microglial cells are one of
the resident immune effectors of the CNS (Kettenmann et al., 2011) and are
considered essential in the anti-flavivirus response in the CNS (Maximova et al.,
2009). However, in case viral infection cannot be controlled, prolonged activation of
these cells can have detrimental effects, contributing to neuron damage (Brhin et
al., 2008). In fact, in our study, mild to moderate neuronal necrosis was detected on
6 and 14 dpi, days on which the reaction of these immune effectors was more
intense. We also detected RCA-1 lectin+ round shaped cells that, in many cases,
corresponded to macrophages, which were especially numerous on 6 and 14 dpi.
The other resident immune effector of the CNS, astrocytes, played a limited role in
the encephalitis of the red-legged partridges, as we only found mild differences in
their distribution or abundance during the course of the infection and compared to
a non-infected partridge of the same age. The presence of GFAP+ fibers among
Purkinje cells in the molecular layer of the cerebellum, mainly in one MO03 infected
partridge, potentially corresponded to Bergmann glia (Kalman et al., 1993).
Astrocytes constitute 50% of the glial cell population in the brain and are a
structural support of the CNS (Montgomery, 1994). In case of CNS damage, their
role as phagocyte and antigen-presenting cell is less important than that of the
microglia (Montgomery, 1994). Astrocytes also participate in WNV-associated
encephalitis in humans (Kelley et al., 2003) and are able to reduce WNV-induced
neuropathology in mice (Hussmann et al., 2013). Some authors have indicated that
these cells can react later than the microglia and that their maximum activity can
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Tesis doctoral
occur 14 days after CNS injury (Montgomery, 1994). Therefore, it is possible that
astrocytosis and/or astrogliosis would have been detected later if our experimental
study had carried on. In response to activation of microglia and astrocytes, among
other stimuli, CD3+ T cell infiltrated the brain, especially on 6 dpi and mainly the
cerebral pallium and molecular layer of the cerebellum. T cells act destroying virusinfected cells and producing cytokines that increase immune cell recruitment and
stimulate other immune effectors (Dorries, 2001). In WNV infection, these cells are
essential for virus clearance and for recovery from the disease (Shrestha and
Diamond, 2004; Sitati and Diamond, 2006), both in humans and rodent models
(Sampson and Armbrustmacher, 2001; Brhin et al., 2008). However, it has been
indicated that a robust response of these inflammatory cells can also have
detrimental effects (Wang et al., 2003). The failure to detect B cells in the CNS of
the partridges may be related to primary antibody malfunction rather than to a true
absence of B cell infiltration. Nevertheless, B cell infiltration in WNV-induced
encephalitis is much lower than that of T cells, at least in rodents and humans
(Kelley et al., 2003; Wang et al., 2003). These cells are usually detected in
perivascular inflammatory infiltrates, so the low number of positive cells detected in
this compartment in our study could be related to the absence of B cell staining.
Despite the fact that similar infection dynamics was found in experimentally
WNV SP07 and MO03 infected partridges, some differences were noticeable in viral
loads, virus antigen distribution and microscopic lesions severity. However, the
most striking differences were found in the CNS, where microscopic lesions and
encephalitis were more acute and severe in MO03 infected partridges.
WNV infection pathogenesis depends mainly on viral and host factors, which
determine the level of viral replication and, therefore, the severity of the infection. In
our work, the experimentally infected partridges were maintained under similar
conditions and were challenged at the same age, so immunologic status should be
128
Captulo 2
very similar between groups. For this reason, we think that the aforementioned
differences were most probably related to factors related to the WNV strain. Genetic
changes that lead to amino acidic substitutions can modify virulence of WNV
strains, in fact substitution of threonine by proline in position 249 of the NS3
protein has been identified as a WNV virulence marker in American crows (Corvus
brachyrhynchos) (Brault et al., 2007). Although this amino acid substitution is
present in the SP07 strain, in our experimental study this did not lead to an
increased virulence, as had already been indicated (Sotelo et al., 2011b).
Considering that SP07 and MO03 differ in 13 amino acid positions (Sotelo et al.,
2009a), it is probable that other markers are determining the virulence of these two
Mediterranean WNV strains.
According to our results, in red-legged partridges, the different virulence of
the MO03 and SP07 WNV strains appears to be driven by differences in the
pathogenesis of the CNS infection, as differences in pathological findings and virus
replication in other tissues were not enough to explain the higher mortality induced
by the Moroccan strain. Pathogenesis of WNV infection in the CNS depends mainly
on the capacity of the virus strain to enter the CNS (neuroinvasiveness) and
produce lesions (neurovirulence) (Chambers and Diamond, 2003). Some authors
have indicated that, once a certain viremia level is reached, the ability of a WNV
strain to enter the CNS is much more important than its own neurovirulence
(Brault et al., 2004; Beasley et al., 2005). Although mean peak viremia titer was
higher in the group inoculated with MO03, there were no statistically significant
differences (Sotelo et al., 2011b), and, in both groups, WNV was present in the
brain as early as 3 dpi. Moreover, on 6 dpi, despite viral load being very similar
between infected groups, a more intense inflammatory reaction and more severe
microscopic lesions were observed in MO03 infected partridges. For these reasons,
we suspect that, in this study, differences in neurovirulence between the strains
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Tesis doctoral
were probably more important than their neuroinvasiveness and it is probable that
a combination of the higher neurovirulence of MO03 with the more exacerbated
inflammatory reaction resulted in a more acute and severe CNS pathology.
Nevertheless, the limited number of birds analyzed in the study forces us to be
cautious with the interpretation of the results obtained.
In conclusion, experimental WNV infection of red-legged partridges with two
different Western Mediterranean strains resulted in a similar pathogenesis with
slightly higher viral loads, higher distribution of virus antigen and a higher severity
of lesions in MO03 infected individuals. The more acute and severe lesions in the
brain of MO03 infected partridges in combination with an apparent similar viral
load imply that the higher impact of MO03 infection on this species may be related
to a higher neurovirulence of this strain. Further studies should enable us to
elucidate the specific markers that are determining this higher neurovirulence.
ACKNOWLEDGEMENTS
We thank the personnel of the experimental farm of the University of Castilla-La
Mancha La Galiana for their effort in this study. We would like to acknowledge the
Junta de Comunidades de Castilla-La Mancha (JCCM) for their support. This study
is a contribution of the epidemiological network of rehabilitation centers in the
community of Castilla-La Mancha, and the REVISA network, and has been
supported by the project PAC08-0296-7771 (JCCM), and from INIA-MARM funds
(INIA CC08-020). V. Gamino (323/09) is a research fellow supported by the regional
government of Castilla La Mancha (JCCM) and Elisa Perez Ramirez is a fellow
from the National Research Council (CSIC).
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Captulo 2
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Infeccin experimental de una especie
gallincea euromediterrnea con una cepa
norteamericana del virus West Nile
Gamino V, Escribano-Romero E, Blzquez AB, Gutirrez-Guzmn AV,

Saiz JC, Hfle U. Experimental infection of a Euro-Mediterranean
gallinaceous bird species with a North American West Nile virus
strain. En preparacin.
Captulo 3
RESUMEN
Mientras que la infeccin por el virus West Nile (WNV) da lugar a la muerte de miles
de aves en Norteamrica, la mortalidad de aves europeas es de carcter espordico
y afecta principalmente a las aves rapaces. El diferente comportamiento ecoepidemiolgico y la diferente virulencia de WNV entre estos dos escenarios y para
las diferentes especies de aves han sido relacionados, entre otras cosas, con la cepa
circulante del virus y la susceptibilidad del hospedador a la infeccin. En el
presente estudio inoculamos nueve perdices rojas (Alectoris rufa), de nueve
semanas de edad, con 107 UFP de una cepa norteamericana de WNV (NY99) con la
finalidad de determinar la susceptibilidad de una especie de ave euromediterrnea
endmica a esta cepa. Adems, estudiamos la dinmica de activacin y
reclutamiento de clulas inflamatorias en el sistema nervioso central (SNC) de las
aves eutanasiadas. La perdiz roja demostr ser susceptible a la infeccin con WNV
NY99, desarrollando lesiones y sucumbiendo a la enfermedad. Los tejidos ms
afectados fueron el corazn y el SNC. En este ltimo, las clulas de la microgla, los
astrocitos y las clulas T participaron en la encefalitis causada por WNV. A pesar de
la alta dosis de inoculacin utilizada, la replicacin del virus en tejidos fue mnima
y, en los individuos eutanasiados, no se detectaron lesiones severas, ms
probablemente relacionadas con la respuesta inflamatoria del hospedador, hasta el
da 10 post inoculacin, demostrando que esta cepa del virus es relativamente poco
virulenta para la perdiz roja.
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ABSTRACT
While West Nile virus (WNV) infection produces thousands of bird deaths in
Northern America, European bird mortalities are rather sporadic and mainly
reported in raptors. The different eco-epidemiological behavior and virulence of
WNV between these two scenarios and among bird species have been related,
among others, to the circulating virus strain and host susceptibility to the infection.
In the present study we inoculated nine 9-week-old red-legged partridges (Alectoris
rufa) with 107 PFU of a North American WNV strain (NY99) in order to determine the
susceptibility of an endemic Euro-Mediterranean bird species to this strain.
Moreover, we studied the dynamics of inflammatory cell activation and recruitment
into the central nervous system (CNS) of euthanized birds. The red-legged partridge
proved to be susceptible to WNV NY99 infection, developing lesions and
succumbing to the disease. The most affected tissues were the heart and the CNS.
In the latter, microglial cells, astrocytes and T cells participated in the encephalitis
caused by WNV. Despite the high viral dose inoculated, viral replication in tissues
was minimal and severe lesions, most probably related to host inflammatory
response, were not detected in euthanized birds until day 10 post-inoculation,
demonstrating that this virus strain is of relatively low virulence for the red-legged
partridge.
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INTRODUCTION
West Nile virus (WNV) is a globally distributed mosquito-borne flavivirus whose
main reservoir are birds (Kramer et al., 2008). Before the 90's this arbovirus was
considered a pathogen of limited importance for birds (Zeller and Schuffenecker,
2004), but nowadays this scenario has changed, although with differences in the
eco-epidemiological behavior and virulence of the virus between the New and the
Old World and among bird species. While in Northern America WNV infection leads
to numerous outbreaks with thousands of bird deaths (CDC), European bird
mortalities are rather sporadic and mainly reported in raptors (Erdlyi et al., 2007;
Jimnez-Clavero et al., 2008; Wodak et al., 2011). Many of these differences are
related to the probability of the host to be infected by the virus and to susceptibility
to suffer disease once infected. On one hand, infection probability is influenced by
factors such as host geographic range, mating and breeding system, migratory
behavior and body size (Gancz et al., 2004; Reisen and Hahn, 2007). On the other
hand, infection pathogenesis is modulated by virulence determinants of the virus
strain (Beasley et al., 2004; Brault et al., 2007) and host capacity to fight the
infection (Gancz et al., 2004), which depends on the presence of previous immunity
against the virus or antigenically-related flaviviruses (Fang and Reisen, 2006;
Nemeth et al., 2008; Kwan et al., 2012), genetic factors (Tag-El-Din-Hassan et al.,
2012), age (Eldadah et al., 1967; Shirafuji et al., 2009) and the presence of
concurrent disease (Hfle et al., 2008) among others.
Susceptibility to both New and Old World
WNV strains has been
experimentally studied in bird species in Europe (Sotelo et al., 2011; Busquets et

al., 2012; Dridi et al., 2013; Ziegler et al., 2013). European hybrid falcons have
proven to be susceptible to different New York/1999 strains (Busquets et al., 2012;
Ziegler et al., 2013) and an endemic Mediterranean gallinaceous bird species, the
red-legged partridge (Alectoris rufa), developed disease and mortality up to 70%
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after the experimental infection with two different Mediterranean WNV strains
(Sotelo et al., 2011). Based upon these results, the aim of the present study was to
describe the dynamics of virus appearance and distribution, as well as of
macroscopic and microscopic lesion distribution and severity in different tissues
during the course of the experimental infection of red-legged partridges with a WNV
New York/1999 strain. We also studied the dynamics of inflammatory cell
activation and recruitment into the brain of the WNV infected partridges, as this
information is available for murine models experimentally infected with similar
WNV strains, but not for birds.

Animals
Recently hatched red-legged partridge chicks were raised in the experimental farm
of the Instituto de Investigacin en Recursos Cinegticos (IREC) until they were
eight weeks of age. In order to confirm that these birds had not been previously
infected by WNV, prior to the experimental infection they were tested by ELISA (ID
Screen West Nile Competition, IdVet, Montpellier, France) and by real time RTPCR (in cloacal and oropharyngeal swabs) as described previously (Crdoba et al.,
2007).
Virus
In this study we used a cell culture-passaged WNV New York/1999 (NY99) strain
(Crdoba et al., 2007; Martn-Acebes and Saiz, 2011).
Experimental infection
Partridges were transported to our biosafety level 3 (BSL-3) facilities in the Instituto
Nacional de Investigacin y Tecnologa Agraria y Alimentaria (INIA), where they were
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Captulo 3
given five days for acclimatization. Nine 9-week-old partridges were subcutaneously
inoculated in the cervical region with 107 PFU/individual of the WNV NY99 strain
diluted in up to 0.1 mL Dulbecco's Minimum Essential Medium (DMEM)
(supplemented with 2 mM L-glutamine, 100 U/mL penicillin and 100 g/mL
streptomycin). Animals were observed daily for clinical signs or death and were
euthanized by intravenous injection of embutramide (T61 , Intervet ScheringPlough, Madrid, Spain) on days 3 (No. 2), 7 (No. 4), 10 (Nos. 6 and 7) and 14 (Nos. 8
and 9) post-inoculation (dpi).
All animals were handled in strict accordance with the guidelines of the
European Community 86/609/CEE and the protocols were approved by the
Committee on Ethics of animal experimentation of the INIA (permit number 2010015). Food and water were provided ad libitum throughout the experiment.
Sample collection
Detailed necropsies were performed on dead (Nos. 1, 3 and 5) and euthanized (Nos.
2, 4, 6-9) individuals. Samples of brain, heart, liver, spleen and kidney were
collected into sterile polypropylene tubes and stored at -70 C until analysis.
Additional samples of brain, oral mucosa, thymus, trachea, lung, heart, liver,
spleen, kidney, adrenal gland, testicle, ovary, small and large intestine, pancreas,
cecal tonsils, bursa of Fabricius, spinal cord, pectoral muscle and skin with feather
follicles were fixed in 10% neutral buffered formalin.

WNV RNA was extracted using a commercial Kit (Speedtools RNA virus extraction
kit, Biotools B&M Labs S.A, Madrid, Spain) and quantified by real time RT-PCR as
described previously (Crdoba et al., 2007). For RNA quantification a standard
curve was generated with previously titrated WNV (10 610-1 PFU) and samples were
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Tesis doctoral
considered negative when Ct35, equivalent to 102 PFU/sample (Blzquez and Saiz,
2010).
Histopathology
Formalin-fixed tissue samples were trimmed, embedded in paraffin and processed
to obtain 4 m sections that were stained with hematoxylin and eosin. These were
independently examined by two different investigators (UH and VG) to determine
the presence of WNV associated lesions. When lesions were present, these were
graded according to their distribution (focal, multifocal or diffuse) and severity
(mild, moderate or marked).
Tissue sections were also mounted on Vectabond reagent (Vector Laboratories,
Inc.,
Burlingame,
CA)
pretreated
slides
for
immunohistochemistry
(IHC).
Immunohistochemical detection of WNV antigen was performed using a rabbit

polyclonal antibody (BioReliance, Product 81-015, Rockville, MD) applying the same
protocol
used
previously
(Gamino
et
al.,
2012).
For
inflammatory
cell
characterization in the cerebrum, cerebellum, optic lobe and pons and medulla
oblongata, we used different antibodies, reagents and protocols detailed in table 1.
Endogenous peroxidase activity was inhibited with a peroxidase blocking reagent
(Dako EnVision+System-HRP (AEC), DakoCytomation, Carpinteria, CA) (CD3,
CD79, GFAP) or with 3% H202 diluted in methanol (RCA), rinses were done using
0.1% Tris-buffered saline/Tween20 (TBS 0.05 M, pH 7.5), non-specific primary
antibody labeling was blocked with 2% albumin from bovine serum (BSA) (SigmaAldrich Chemie, Steinheim, Germany) diluted in 0.1% TBS/Tween20, and sections
were counterstained with Mayer's hematoxylin.
In the IHC for WNV antigen detection, tissue sections of experimentally
infected red-legged partridges in which presence of WNV had been confirmed by real
144
Captulo 3
time RT-PCR served as positive controls. Negative controls included substitution of

the primary antibody by 2% BSA-0.1% TBS/Tween20 and a negative rabbit
antibody (BioReliance, Product 81015), as well as tissue sections of a red-legged
partridge negative in the WNV real time RT-PCR. Spleen and bursa of Fabricius
sections of red-legged partridges served as positive controls in the IHC for T and B
cells. In this case, negative controls included substitution of the primary antibody
by 2% BSA-0.1% TBS/Tween20 and a brain section of a red-legged partridge
negative in the WNV real time RT-PCR. A brain section of a non-infected partridge of
the same age served as reference for RCA and GFAP.
Virus antigen staining was graded according to its distribution and
abundance. We also determined the distribution of inflammatory cells within the
brain parenchyma and analyzed changes in their relative abundance.
Table 1. Detail of reagents and protocols used in the IHC for inflammatory
characterization in the CNS of experimentally WNV NY99 infected red-legged partridges.
cell
Cell
population/
marker
Antibody
reference*
Pretreatment**
Primary
antibody
dilution
Primary
antibody
incubation
Secondary
antibody
Detection
system
Microgliamacrophages
Lectin RCA-1
biotinylated
Citrate buffer
Microwave heat
(22 min)
1:600
45 min RT
Goat antirabbit IgG
ABC-DAB
Astrocytes
Polyclonal rabbit
anti-GFAP
Proteinase K
(7 min RT)
1:500
4C ON
T cells
Polyclonal rabbit
anti-human CD3
1:500
4C ON
Labelled
polymer-HRP
anti-rabbit
AEC+
substrate
chromogen
B cells
Monoclonal rabbit
anti-human CD79a
1:10
45 min RT
Citrate buffer
Microwave heat
(22 min)
*Primary antibody products: RCA-1 product No. B-1085 (Vector Laboratories); GFAP product No. Z0334
(DakoCytomation, Glostrup, Denmark); CD3 product No. A0452 (DakoCytomation); CD79a product No. RM-9118
(Thermo Fisher Scientific, Runcorn, UK).
**Proteinase K (DakoCytomation); RT: room temperature (22-25 C).
Antibodies were diluted in 2% BSA-0.1% TBS/Tween20.
ON: overnight.
Goat anti-rabbit IgG (Vector Laboratories) was diluted 1:200 in 0.1% TBS/Tween20 and applied for 1 hr at RT;
Labelled polymer-HRP anti-rabbit (Dako EnVision+System-HRP (AEC), DakoCytomation) was applied according to
manufacturer's recommendation.
Avidin-biotinylated enzyme complex (ABC system, Vector Laboratories) was applied for 30 min according to
manufacturer's recommendation and 3,3-diaminobenzidine tetrahydrochloride (DAB, Vector Laboratories) was
applied for 30 sec according to manufacturer's recommendations. AEC+substrate chromogen (Dako
EnVision+System-HRP (AEC), DakoCytomation) was applied for 15 min (CD3, CD79) and 3 min (GFAP) according
to manufacturer's recommendations.
145
Tesis doctoral
RESULTS
Morbidity and mortality
Clinical signs were observed in the three partridges that died on days 2, 7 and 8 pi
(mortality of 45.5%). These birds showed depression, ruffled feathers and
recumbence, dying 24 hours after the onset of clinical signs.
Macroscopic findings
Macroscopic lesions compatible with WNV infection were detected in every
necropsied partridge, with minimal differences between dead and euthanized birds,
with the exception of the emaciation found only in fatally infected birds. Lesions
were more widespread between days 7 and 10 pi and the most affected organs were
the heart, liver, spleen and kidney. Pallor of the myocardium (3/9), hepatic (3/9),
splenic (3/9) and renal parenchyma (3/9), splenomegaly (2/9) and hepatomegaly
(2/9), as well as multifocal yellowish or reddish areas in the liver (2/9) were the
main macroscopic findings.
Additional macroscopic lesions, probably non-related to WNV infection, such
as dilation of intestinal loops (7/9) and injection of serosa vessels (4/9) (mainly in
the
duodenum and
cecum), and
distension
of the
crop
with a whitish
pseudomembrane covering the mucosa (2/9) were observed in some animals.

All tissue samples were negative for the presence of WNV genome in the real time
RT-PCR, with the exception of the heart of the bird found dead on 7 dpi (No. 3) and
the brain of one of the birds euthanized on 10 dpi (No. 6).
146
Captulo 3
Histopathology
Microscopic changes were observed early in the course of the infection, but while
these were severe in the bird found dead on 7 dpi, in euthanized partridges a
similar severity was not observed until 10 dpi (Table 2).
The most markedly affected tissues were the brain, spinal cord and heart.
While in fatally infected animals the spleen, liver, kidney, large intestine, pancreas
and bursa of Fabricius showed moderate lesions, in euthanized birds these tissues
were mildly affected or lesions were absent (Table 2). No microscopic changes were
detected in the trachea, cecal tonsils, testicles, ovaries, adrenal gland, and skin and
feather follicles.
Main
microscopic
findings
were
the
presence
of
lymphoplasmacytic,
histiocytic and/or granulocytic inflammatory infiltrates, cellular necrosis and

degeneration and hemosiderosis in the spleen and liver (Table 2 and figures 1-4).
While fatally infected animals showed moderate to marked lymphoid cell depletion
and necrosis in lymphoid organs, this was rarely observed in the euthanized ones.
The CNS mainly showed gliosis, perivascular cuffing and endothelial cell swelling,
but also neuronal necrosis and phagocytosis (Table 2 and figures 5 and 6). The bird
euthanized on 3 dpi only showed endothelial cell swelling, and moderate to marked
lesions detected in the CNS of birds found dead on 7 and 8 dpi, were not observed
until 10 dpi in euthanized animals.
As incidental findings, one fatally infected (No. 3) and five euthanized (Nos. 2
and 6-9) partridges showed moderate to marked hyperplasia, necrosis and
desquamation of the mucosa of the crop, associated to inflammatory infiltrates. In
these individuals pseudohyphae and blastospores of Candida sp. were confirmed in
keratin debris by staining with periodic-acid Schiff (PAS). Moreover, partridges
found dead on 7 and 8 dpi (Nos. 3 and 5) had mild to moderate amounts of
147
Tesis doctoral
coccidian oocysts in the cecum, and those euthanized on 7 and 14 dpi (Nos. 4, 8
and 9) showed pancreatic granulomas.
Table 3. Microscopic lesions and antigen staining gradation on different days post-inoculation
(dpi) in tissues of experimentally WNV NY99 infected red-legged partridges.
2dpi
3dpi
7dpi
7dpi
8dpi
10dpi
No. 1
No. 2
No. 3
No. 4
No. 5
No. 6
Neuronal necrosis
Neuronal phagocytosis
++
Gliosis
+++
++

Immunohistochemical
staining**
Cerebellum
++
Purkinje cell phagocytosis
Gliosis
14dpi
No. 7
No. 8
No. 9
++
++
++
++
++
++
++
++
+++
+++
++
++
++
++
+++
++
++
Neuronal necrosis
++
Gliosis
++
++
++
++
++
Neuronal necrosis
++
Gliosis
++
++
++
++
++
++
++
++
++
++
Neuronal necrosis
++
++
++
Gliosis
++
++
++
++
++
+++
++
Myofiber necrosis-degeneration
+++
+++
++
++
++
+++
++
+++
++
++
++
++
++
+++
++
Cerebrum*
Optic lobe
Pons + medulla oblongata
Spinal cord
Heart
Lung
148
Captulo 3
Table 3. Microscopic lesions and antigen staining gradation on different days post-inoculation
(dpi) in tissues of experimentally WNV NY99 infected red-legged partridges (cont).
2dpi
3dpi
7dpi
7dpi
8dpi
No. 1
No. 2
No. 3
No. 4
No. 5
No. 6
10dpi
No. 7
No. 8
14dpi
No. 9
Hepatocyte necrosis
+++
Hemosiderosis
++
Follicular cell necrosis
Lymphoid depletion
+++
+++
Hemosiderosis
++
+++
+++
++
++
Eosinophilic material deposits
+++
+++
Epithelial cell necrosis
++
++
Epithelial cell degeneration
++
++
Glandular cell necrosis
+++
++
++
+++
+++
++
++
Bursa of Fabricius
Epithelia/lymphoid cell
necrosis
Lymphoid depletion
+++
++
NA
Lymphoid depletion
NA
++
+++
NA
++
NA
Liver
Spleen
Kidney
Proventriculus
Gizzard
Duodenum
Large intestine
Pancreas
Thymus
Partridges found dead

Euthanized partridges
*Tissue lesions were graded according to their distribution and severity: , no lesion; +, focal and mild or moderate /
multifocal and mild; ++, focal and marked / multifocal and moderate / diffuse and mild; +++, multifocal and marked /
diffuse and moderate or marked.
**WNV antigen immunostaining was graded according to its distribution and percentage of stained cells: , negative
staining; , focal single cells; +, focal or multifocal and < 20% cells stained; ++, multifocal or diffuse and 20-80% cells
stained; +++, multifocal or diffuse and > 80% cells stained.
NA: tissue sample no analyzed.
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Tesis doctoral
Figure 1. Heart, partridge found dead No. 5, 8 dpi. Focal and marked necrosis and degeneration of
cardiac myofibers, and infiltration of lymphoplasmacytic and histiocytic cells. Necrotic myofibers show
lysis of the nuclei, and fragmentation and accumulation of hyaline material in the cytoplasm. HE,
x400.
Figure 2. Liver, partridge found dead No. 3, 7 dpi. Diffuse and marked necrosis of hepatocytes, and
mild infiltration of lymphoplasmacytic and histiocytic cells. Necrotic hepatocytes show lysis and
picnosis of the nuclei, and degeneration of the cytoplasm. HE, x400.
Figure 3. Spleen, partridge found dead No. 6, 10 dpi. Distension of the cytoplasm of macrophages
by a red-brow pigment (arrows) and moderate infiltration of granulocytes (arrowheads). HE, x400.
Figure 4. Pancreas, partridge found dead No. 3, 7 dpi. Multifocal and marked necrosis of acinar
cells. Necrotic cells show lysis and picnosis of the nuclei and degeneration of the cytoplasm. HE, x400.
150
Captulo 3
Figure 5. Cerebrum, partridge found dead No. 5, 8 dpi. Diffuse and moderate gliosis with
perivascular infiltration of lymphoplasmacytic and histiocytic cells and satellitosis (arrowheads). HE,
x200.
Figure 6. Cerebellum, euthanized partridge No. 7, 10 dpi. Focal necrosis of Purkinje cells and focal
and moderate gliosis and degeneration in the molecular layer. HE, x400.
Virus antigen
Although the amount of WNV antigen detected by IHC was scarce, it was
widespread, especially on 8 and 10 dpi (Table 2).
Similar cell types were stained in euthanized and fatally infected birds. WNV
antigen was detected in cardiac myofibers (Figure 7), glomerular mensangial and
tubular epithelial cells of the kidney, acinar cells of the pancreas, and rare vascular
walls in the spleen and inflammatory cells in the lamina muscularis of the gizzard.
In fatally infected birds, WNV antigen was also observed in hepatocytes and Kupffer
cells (Figure 8), and in proventricular gland epithelial cells. By contrast, in
euthanized partridges, it was detected in splenic macrophages and in vascular walls
and muscular fibers of the lamina muscularis of the large intestine. In the CNS, the
partridge euthanized on 7 dpi had only one intravascular inflammatory cell stained
in the cerebrum and the two birds euthanized on 10 dpi showed positive staining
within the cytoplasm of one Purkinje cell, several neuronal axons and in very few
glial cells in the cerebellum (Figure 9). On 14 dpi, virus antigen was only detected in
bird No. 8, in one inflammatory cell in the spinal cord and one cardiac myofiber.
Inflammatory cells in the brain
Inflammatory cells that participated in the encephalitis associated to WNV infection
were only analyzed in euthanized partridges.
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Tesis doctoral
Microglia activation/macrophage infiltration

Microglial cells reacted as early as 3 dpi, but there were changes in their
morphology and abundance during the course of the infection and among brain
regions.
On 3 dpi, most microglial cells displayed a poorly ramified morphology,
although some amoeboid cells, characterized by a large soma and short thick
cellular processes, were observed. On 7 dpi, most cells changed to an amoeboid
morphology, and rounded cells, which corresponded both to activated microglia and
infiltrating macrophages, were also observed. All these cells were especially
numerous on 10 dpi, diffusely distributed or forming nodules or foci that, in some
cases, surrounded dying neurons and blood vessels. On 14 dpi, microglia remained
very active. Some macrophages were also detected in the vascular lumen of
meningeal vessels.
In the cerebrum, on 3 dpi, ramified cells were diffusely distributed but, from
7 dpi on, these were more numerous and intensely stained in the peripheral
pallium. Nevertheless, from 7 dpi on, amoeboid and rounded cells were
predominant and especially active in the parenchyma near the lateral ventricle,
where some cells were also detected near ependymal cells.
In the cerebellum, activated microglial cells were mainly detected in the
molecular layer. In this layer, on 10 dpi, and especially in bird No. 7, most cells
were grouped forming numerous nodules, many of them surrounding necrotic
Purkinje cells (Figure 10). Moderate activation was also observed in the white
matter that, in change, was mild in the granular layer.
In the optic lobe, activated microglial cells were detected in every layer, but
microglia nodules were mainly located in the stratum griseum et fibrosum
superficiale. These cells were especially numerous in bird No. 6 (10 dpi).
152
Captulo 3
The pons and medulla oblongata were the brain regions that showed more
marked microglia activation. As early as 3 dpi, ramified microglial cells were
diffusely distributed, but amoeboid and rounded cells were also abundant. On 10
dpi, and also on 14 dpi, there were multiple microglia nodules, also located in some
neuronal nuclei.
Astrocyte activation
GFAP+ cells were detected from 3 dpi on. Mild changes in cellular morphology,
abundance and distribution compared to a non-infected partridge were noticed, but
none in staining intensity. On 14 dpi, astrocyte activation was slightly higher.
In the cerebrum, GFAP+ cells with long cellular processes were stained in the
subpial region and many vessels were surrounded by astrocytes with medium
cellular processes, mainly located in the peripheral parenchyma. Mild astrocytosis
was detected in the peripheral pallium, especially near the lateral ventricle,
characterized by star-shaped cells with short or medium cellular processes.
In the cerebellum, the granular layer showed scarce stained astrocytes,
mainly located near the Purkinje cell layer (Bergmann glia) (Figure 11). GFAP+ cells
were also scarce in the white matter and absent in the molecular layer.
In the optic lobe, numerous astrocytes were stained in the stratum opticum,
some of them surrounding blood vessels. On 14 dpi, mild astrocytosis was detected
in the stratum album centrale, and the ependymal cells surrounding the ventricle
were GFAP+.
In the pons and medulla oblongata, astrocytes with long cellular processes
were stained in the dorsal and ventral subpial zone and many vessels were
surrounded by GFAP+ cells. On 3 and 7 dpi, mild astrocytosis was detected in the
medulla oblongata that, in change, was detected on 10 dpi in the pons. On 14 dpi,
moderate diffuse astrocytosis was observed, especially in bird No. 8.
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Tesis doctoral
T cell infiltration
CD3+ T cells were detected early, but changes in their distribution and abundance
during the course of the infection and differences among brain regions were
observed.
On 3 and 7 dpi, CD3+ T cells were mainly observed in the vascular lumen of
meningeal and parenchymal vessels, and only scarce cells infiltrated the
interstitium of the cerebrum, cerebellum and pons and medulla oblongata. It was
on 10 dpi when T cells were especially numerous in the brain. On this dpi, these
cells were mainly diffusely distributed or forming part of inflammatory nodules or
foci, but were also observed in the perivascular space and vascular lumen. On 14
dpi, while the cellular infiltration decreased markedly in the cerebellum and optic
lobe, numerous T cells were still detected in the cerebrum and pons and medulla
oblongata.
In the cerebrum, T cells were diffusely distributed in the parenchyma.
Numerous T cells were detected in inflammatory nodules, inflammatory foci and in
some perivascular cuffs, especially in bird No. 7 (10 dpi).
In the cerebellum, the molecular layer was markedly infiltrated on 10 dpi,
showing numerous cells diffusely distributed and forming part of inflammatory
nodules, many of them surrounding Purkinje cells, which in most cases showed
necrosis. At this time, moderate infiltration was also detected in the granular layer
and white matter, especially in bird No. 7.
The optic lobe was the brain region in which less T cell infiltration was
observed. Although T cells were detected in every layer, these were slightly more
numerous in the stratum opticum and stratum griseum et fibrosus superficiale
and, to a lesser extent, in the stratum griseum and album centrale.
154
Captulo 3
The pons and medulla oblongata were the brain regions most markedly
infiltrated by T cells, either diffusely distributed or forming part of inflammatory
nodules (Figure 12), both on 10 and 14 dpi.
B cell infiltration
CD79a+ cells were not detected in any brain region on any dpi.
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Tesis doctoral
Figure 7. Heart; partridge found dead No. 5, 8 dpi. WNV antigen in the cytoplasm of cardiac
myofibers. IHC for WNV with Mayer's hematoxylin counterstain, x400.
Figure 8. Liver; partridge found dead No. 3, 7 dpi. WNV antigen in the cytoplasm of hepatocytes
and Kupffer cells. IHC for WNV with Mayer's hematoxylin counterstain, x400.
Figure 9. Cerebellum; euthanized partridge No. 6, 10 dpi. WNV antigen in the cytoplasm and axons
of Purkinje cells. IHC for WNV with Mayer's hematoxylin counterstain, x400.
Figure 10. Cerebellum; euthanized partridge No. 7, 10 dpi. RCA-1 positive staining in the
cytoplasm of activated microglia/macrophages forming nodules in the molecular layer. IHC for
microglia activation/macrophages with Mayer's hematoxylin counterstain, x400.
Figure 11. Cerebellum; euthanized partridge No. 8, 14 dpi. GFAP positive staining in cell processes
of astrocytes diffusely distributed in the granular layer near the Purkinje cell layer (Bergmann glia).
IHC for astrocyte activation with Mayer's hematoxylin counterstain, x100.
Figure 12. Medulla oblongata; euthanized partridge No. 7, 10 dpi. CD3 positive staining in the
cytoplasm of T cells forming an inflammatory nodule in the neuropil. IHC for T cells with Mayer's
hematoxylin counterstain, x400.
DISCUSSION
In this work we have demonstrated that the juvenile red-legged partridge, an
endemic Euro-Mediterranean gallinaceous bird species, is susceptible to the
experimental infection with a North American WNV strain, developing clinical signs,
macroscopic and microscopic lesions, and succumbing to the disease. To our
knowledge, this is the first experimental infection of a European gallinaceous bird
species with a North American WNV strain and one of the few experimental studies
that have been done with a European endemic bird species.
Gallinaceous birds have been traditionally considered little susceptible to
WNV infection (Komar et al., 2003). However, sporadic cases of West Nile disease
and mortality have been described in Northern America (Steele et al., 2000; Zhang
et al., 2006), where an outbreak among chukar partridges (Alectoris chukar), a bird
species closely-related to the red-legged partridge, occurred (Wnschmann and
Ziegler, 2006). Although experimental infections in gallinaceous birds have
demonstrated their little susceptibility to WNV infection (Swayne et al., 2000), the
red-legged partridge proved to be susceptible to two Mediterranean WNV strains in
a previous experimental infection, suffering moderate (30%) to high (70%) mortality,
developing high viremia levels and producing high titers of neutralizing antibodies
(Sotelo et al., 2011). While no WNV associated mortality event has been recorded in
156
Captulo 3
gallinaceous birds in Europe, the red-legged partridge and the ring-necked

pheasant (Phasianus colchicus) have found to be susceptible to natural infection by
a closely-related flavivirus, Bagaza virus (Gamino et al., 2012).
In the present work, we only detected WNV genome by real time RT-PCR in
the heart of one partridge that succumbed to the infection on 7 dpi and in the brain
of one animal euthanized on 10 dpi. This could be related to poor virus replication
and a rapid virus clearance by the host, which matches with the scarce virus
antigen detected by IHC. However, most infected partridges developed multiorgan
macroscopic and
microscopic
lesions,
probably
more
related
to
the
host
inflammatory response than to virus replication. These lesions, as expected, were

more severe and widespread in fatally infected birds and as infection progressed.
The most severely affected tissues were the CNS and the heart, as it had been
previously described in Gyr-Saker hybrid falcons inoculated with the same WNV
strain (Busquets et al., 2012).
One partridge died as early as 2 dpi; however, this bird only showed mild
microscopic changes and we could not detect WNV by real time RT-PCR or IHC.
Considering that the partridge euthanized on 3 dpi had only mild acute lesions and
WNV antigen was scarcely present in different tissues, and that even highly
susceptible species such as crows or jays do not usually succumb to WNV infection
until 4-5 dpi (Weingartl et al., 2004; Nemeth et al., 2011), we cannot rule out other
underlying causes.
Incidental findings such as the presence of coccidian oocysts and Candida
sp. in some individuals were probably enhanced by the impairment of host defenses
by the viral infection, as it had been previously described in natural WNV infections
(Hfle et al., 2008).
In the CNS of euthanized birds, although resident immune cells (i.e.
microglia and astrocytes) showed mild reaction as early as 3 dpi, it was on 10 dpi
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Tesis doctoral
when the inflammatory reaction was more marked, virus genome was detected by
real time RT-PCR, virus antigen was detected by IHC, and moderate to marked
microscopic lesions were observed. A similar sequence of virus antigen appearance,
development of lesions, and inflammatory reaction in the CNS has been described
in rodent models experimentally infected with WNV NY99 strains (Brhin et al.,
2008) and in red-legged partridges experimentally infected with Mediterranean WNV
strains (the authors, under revision); however, in both cases, this took place earlier
in the course of the infection.
The more prevalent inflammatory cell population on 10 dpi was microglia.
These resident immune cells act as CNS macrophages, phagocytizing damaged cells
and releasing immune mediators (Gehrmann et al., 1995), and are essential in the
anti-flavivus response (Brhin et al., 2008; Maximova et al., 2009). The other
resident cells, astrocytes, showed mild changes during the course of the infection
and, as expected, were more active on 14 dpi. These cells also participate in the
encephalitis associated to WNV infection in humans and mice (Kelley et al., 2003;
Hussmann et al., 2013). Microglia and astrocyte activation, together with other
stimuli, resulted in CD3+ T cell recruitment to the CNS, which infiltrated the
parenchyma from 7 dpi onwards. These immune cells are able to destroy cells
infected by the virus and are essential for CNS recovering from WNV infection, both
in humans and rodents (Sampson and Armbrustmacher, 2001; Brhin et al., 2008).
All these inflammatory cells were especially active in the pons and medulla
oblongata. This could be related to the fact that, as in humans (Sampson et al.,
2000) and horses (Cantile et al., 2001), the brain stem is the primary target for
WNV. In fact, infected birds usually show microscopic lesions in this region (Steele
et al., 2000; Gancz et al., 2006; Zhang et al., 2006). The absence of CD79a stained
cells was probably related to malfunction of the primary antibody rather than the
real absence of B cell infiltration. Nevertheless, studies done in humans and
158
Captulo 3
rodents have indicated that B cells play a limited role in the encephalitis associated
to WNV infection and are mainly detected in the perivascular space (Kelley et al.,
2003; Wang et al., 2003). Despite the important role of inflammatory cells in the
recovery of the CNS from WNV infection, it has been demonstrated that an
exacerbated and prolonged activation or recruitment of these cells can have
detrimental effects, contributing to neuron damage (Wang et al., 2003; Brhin et al.,
2008). It is probable that, in our study, CNS lesions were more related to host
response than to virus replication within cells, as virus genome was only detected in
one animal euthanized on 10 dpi and virus antigen was scarcely present only in the
cerebellum of the two individuals euthanized on the same dpi.
Lesions observed in red-legged partridges were similar to those described in
birds naturally and experimentally infected with WNV (Steele et al., 2000; Nemeth
et al., 2006; Ziegler et al., 2013). Although similar moderate to marked microscopic
lesions have been described in the CNS of some naturally infected gallinaceous
birds (Steele et al., 2000; Zhang et al., 2006), these are not always present both in
natural and experimental infections (Senne et al., 2000; Swayne et al., 2000;
Wnschmann and Ziegler, 2006). However, differences in infection conditions such
as the specific virus strain and inoculation dose, and the age, species and number
of birds infected, could be determining differences in pathological findings. In fact,
considering the poor virus replication in tissues and the relatively low mortality
found
in our study,
despite
the
high virus inoculation dose
used
(107
PFU/individual), it might be possible that, if we had given a lower dose, viral

replication, development of lesions and clinical disease might not have taken place
or, at least, might have been milder.
If we compare the infection pathogenesis and pathogenicity observed in the
present study with the results of the experimental infection of the same species
with Mediterranean WNV strains, differences are evident, mainly compared to
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Tesis doctoral
Morocco/2003 (the authors, under revision). In the present study, virus replication
in tissues was much lower and microscopic lesions in euthanized partridges were
milder and appeared later in the course of the infection (the authors, under
revision). Although birds inoculated with the Mediterranean isolates were two weeks
younger, and host susceptibility to infection is strongly related to this factor
(Wnschmann and Ziegler, 2006; Shirafuji et al., 2009), these were inoculated with
104 PFU, which is three logs lower than the inoculation dose of the present study.
From our point of view, this implies that WNV NY99 is less virulent for the redlegged partridge than Morocco/2003 and Spain/2007. However, the low number of
birds used in both studies and the different experimental conditions, prevents us
from drawing clear conclusions.
Summarizing, in the present study we have demonstrated that an endemic
Euro-Mediterranean gallinaceous bird species is susceptible to a North American
WNV strain, developing lesions, such as myocarditis and encephalitis, clinical
signs, and succumbing to the disease. Considering our results, together with the
fact that some European birds have suffered mortalities due to WNV infection
(Bakonyi et al., 2006; Monaco et al., 2010; Wodak et al., 2011; Bakonyi et al.,
2013), the low consequences of WNV infection for birds in Europe, and especially in
the Mediterranean basin, where outbreaks of West Nile fever have mainly affected
humans and equids (Murgue et al., 2001; Monaco et al., 2010; Garca-Bocanegra et
al., 2011), apparently are not related with a low susceptibility of European and
Mediterranean birds to WNV infection and disease. Other factors such as the
presence of previous immunity in the host, host and vector abundance in the area,
the degree of biodiversity or difficulties to identify WNV infection as the cause of
bird mortalities should be considered among others.
160
Captulo 3
ACKNOWLEDGEMENTS
This study was supported by grants AG2008-02504GAN and SAF-2008-04232
funded by the Spanish Ministry for Science and Innovation, grants FAU2008 000600-00 and RTA2011-0036 funded by the Spanish Institute for Agricultural and
Alimentary Investigation and Technology (INIA), and The Network of Animal Disease
Infectiology and Research Facilities, NADIR-UE-228394, funded by the E.U. V.
Gamino (323/09) is a research fellow supported by the regional government of
Castilla La Mancha (JCCM). We are grateful to the personnel of the experimental
farm of the University of Castilla La Mancha, La Galiana, for their help during
the breeding of partridges.
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Infeccin natural por el virus Bagaza en
aves cinegticas en el sur de Espaa
Gamino V, Gutirrez-Guzmn AV, Fernndez-de-Mera IG, Ortz JA,

Durn-Martn M, de la Fuente J, Gortzar C, Hfle U. Natural Bagaza
virus infection in game birds in southern Spain. Veterinary Research
2012, 43(1): 64. doi: 10.1186/1297-9716-43-65.
Captulo 4
RESUMEN
A finales del verano de 2010 el virus Bagaza (BAGV), un flavivirus transmitido por
mosquitos que nunca haba sido detectado previamente en Europa, caus un brote
de elevada mortalidad en perdiz roja (Alectoris rufa) y faisn comn (Phasianus
colchicus). En el presente trabajo se estudiaron los hallazgos clnicos y la
distribucin de lesiones y del antgeno viral en aves cinegticas infectadas de forma
natural con BAGV, con el fin de explicar el impacto aparentemente ms severo en la
poblacin de perdiz roja. En ambas especies de galliformes y, en menor medida, en
paloma torcaz (Columba palumbus), la infeccin dio lugar a signos nerviosos.
Adems, en las perdices sta caus una hemosiderosis severa en el hgado y en el
bazo que, por el contrario, no estuvo presente en los faisanes y fue menos evidente
en las palomas. Mientras que el antgeno viral fue detectado en el endotelio vascular
en diversos rganos de las perdices y en el bazo de las palomas, en faisanes slo fue
encontrado en neuronas y clulas de la gla en cerebro. Estos hallazgos indican la
existencia de un tropismo endotelial por parte de BAGV y un proceso hemoltico
severo en perdiz roja que se sum al proceso neurolgico encontrado en las tres
especies.
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ABSTRACT
In late summer 2010 a mosquito-borne flavivirus not previously reported in Europe
called Bagaza virus (BAGV) caused high mortality in red-legged partridges (Alectoris
rufa) and ring-necked pheasants (Phasianus colchicus). We studied clinical findings,
lesions and viral antigen distribution in naturally BAGV infected game birds in
order to understand the apparently higher impact on red-legged partridges. The
disease induced neurologic signs in the two galliform species and, to a lesser extent,
in common wood pigeons (Columba palumbus). In red-legged partridges infection by
BAGV caused severe haemosiderosis in the liver and spleen that was absent in
pheasants and less evident in common wood pigeons. Also, BAGV antigen was
present in vascular endothelium in multiple organs in red-legged partridges, and in
the spleen in common wood pigeons, while in ring-necked pheasants it was only
detected in neurons and glial cells in the brain. These findings indicate tropism of
BAGV for endothelial cells and a severe haemolytic process in red-legged partridges
in addition to the central nervous lesions that were found in all three species.
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INTRODUCTION
In late summer 2010 an extremely high mortality was observed among game birds
in southern Spain, especially in red-legged partridges (Alectoris rufa), but also in
ring-necked pheasants (Phasianus colchicus), that was determined to be due to a
flavivirus not previously reported in Europe, Bagaza virus (BAGV) (Agero et al.,
2011). BAGV is a relatively unknown member of the Ntaya group of the genus
Flavivirus that was first isolated in the Central African Republic in 1966 from a pool
of Culex spp. mosquitoes (Digoutte, 1978).
Although a recent study has examined the epidemiology of the outbreak more
closely (Garca-Bocanegra et al., 2012), information on the pathogenesis of BAGV is
very limited. Thus, in the present study we review the main clinical findings, lesions
and viral antigen distribution in wild birds naturally infected with BAGV. Moreover,
we compare these features among BAGV-infected red-legged partridges, ring-necked
pheasants and common wood pigeons (Columba palumbus), in order to understand
the pathogenesis of the disease and the reason for the extreme impact of the
disease in red-legged partridges in comparison to other affected species.

Study area
The study area is located on the Mediterranean coast, in south-western Spain
(between 36 20 N and 5 48 O). The climate is Mediterranean, with wet winters
and dry summers. The habitat is a mosaic of intensively managed crops, primarily
rice and vegetables, and open woodland (dehesa) with cork oaks (Quercus suber),
and stone pines (Pinus pinea).
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Postmortem examination and sampling

Recently dead or moribund red-legged partridges (n = 6), ring-necked pheasants
(n = 5) and common wood pigeons (n = 2) were collected in the area during three
consecutive days. Oral and cloacal swabs were taken and stored in viral transport
medium (Hanks' balanced salt solution containing 10% glycerol, 200 U/mL
penicillin, 200 g/mL streptomycin, 100 U/mL polymixin B sulphate, 250 g/mL
gentamicin and 50 U/mL nystatin (Munster et al., 2007)) and frozen immediately in
liquid nitrogen.
Detailed necropsies were carried out, and a complete set of tissues was taken
(brain, oral mucosa, pectoral muscle, trachea, lung, heart, liver, spleen, pancreas,
duodenum, caecal tonsils, kidney, bursa of Fabricius, thymus and skin with feather
follicles)
and
fixed
in
10%
neutral
buffered
formalin
for
histopathologic
examination. Additionally, samples of heart, kidney, brain and spleen were also
collected into sterile containers and frozen immediately in liquid nitrogen.
Real time RT-PCR and sequence analysis

Swabs and frozen tissue samples were processed upon arrival at the laboratory.
Nucleic acid (RNA) was extracted using High Pure RNA Tissue Kit (Roche
Diagnostics, Barcelona, Spain), and analyzed by real time RT-PCR for the presence
of West Nile Virus (WNV) and flavivirus genome in general. For WNV detection, a
TaqMan MGB multiplex real time RT-PCR using a commercial kit (QuantiTEC
ProbeW RT-PCR, Qiagen, Madrid, Spain) was applied as described before (JimnezClavero et al., 2006). Flavivirus detection was carried out as described previously by
generic SYBR Green (Qiagen, Madrid, Spain) real time RT-PCR (Moureau et al.,
2007).
A generic nested RT-PCR previously described (Snchez-Seco et al., 2005)
was used to confirm flavivirus detection. Reactions were performed using the Access
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RT-PCR System (Promega, Madison, WI, USA) in an automated DNA thermal cycler
model TC-512 (Techne, Cambridge, UK). Briefly, 5 L (around 10 g) of nucleic acid
preparation was added to 45 L of a RT-PCR mix containing 2 mM MgSO4, 0.2 mM
of dNTP, 1 AMV/Tfl 5 reaction buffer, 5 U Tfl DNA polymerase, 5 U AMV reverse
transcriptase and 40 pmols of each primer (Flavi1+ and Flavi1-) and amplified using
an initial incubation at 38 C for 45 min followed by 94 C for 2 min, and 40 cycles
of 94 C for 30 sec, 47 C for 1 min, and 68 C for 1 min and 15 sec. The nested
PCR reaction was carried out in a final volume of 50 L, with similar concentrations
as in the first reaction, using primers Flavi2+ and Flavi2-, and 1 L of the product
of the first amplification. The mix was subjected to 94 C for 2 min, followed by 40
PCR cycles with similar conditions to those used in the primary generic RT-PCR.
Subsequently, 8 L of each PCR product was subjected to electrophoresis on
a 2% agarose gel to check the size of amplified fragments by comparison to a DNA
molecular weight marker (1 kb Plus DNA Ladder, Promega, Madison, WI, USA).
The DNA bands from the nested and the first generic amplification were
resin-purified (Wizard, Promega Madison, WI, USA) and cloned into pGEM-T
(Promega, Madison, WI, USA). At least four independent clones were sequenced
from both ends for each positive sample (Secugen SL, Madrid, Spain). Sequence
similarity search was performed using BLAST (BLAST).
Histopathology and immunohistochemistry

Formalin-fixed tissues were trimmed, embedded in paraffin, sectioned at 4 m and
processed to obtain haematoxylin-eosin stained sections. These were examined
independently by two different investigators (VG and UH).
Furthermore, the avidin-biotin-peroxidase complex (ABC) method was used
on paraffin-embedded tissue sections for immunohistochemical demonstration of
flavivirus antigen. For this purpose, a rabbit polyclonal antibody against WNV that
has been shown to cross-react with other flaviviruses (Steele et al., 2000), was used
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(BioReliance, Product 81015, Rockville, Maryland, USA). After deparaffinization

and hydration of the sections, they were incubated with 3% H2O2 diluted in
methanol for 30 min at room temperature (2025C) in order to block endogenous
peroxidase activity. After rinsing with water, the sections were treated with
proteinase K (Diagnostic BioSystems, Pleasanton, California) for 15 min at room
temperature. They were then rinsed with water, followed by three 5 min rinses in
0.1% Tris-buffered saline/Tween20 (TBS 0.05 M, pH 7.5), and incubated 1 hr at
room temperature with 2% albumin from bovine serum (Sigma-Aldrich Chemie,
Steinheim, Germany) diluted in 0.1% TBS/Tween20. The primary antibody was
applied overnight at 4C at a dilution of 1:1000 in 2% albumin-0.1% TBS/Tween20.
After three 5 min rinses in 0.1% TBS/Tween20, sections were incubated with a
biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, California, USA)
diluted 1:200 in 0.1% TBS/Tween20 for 1 hr at room temperature. After three 5
min rinses in 0.1% TBS/Tween20 an avidin-biotinylated enzyme complex (ABC
system, Vector laboratories) was applied for 30 min at room temperature according
to manufacturer's recommendations. For development, sections were incubated for
1 min with 3,3-diaminobenzidine tetrahydrochloride according to manufacturer's
recommendations (Vector Laboratories). Sections were counterstained with Mayer's
haematoxylin. Negative controls included substitution of the primary antibody by
2% albumin from bovine serum diluted in 0.1% TBS/Tween20 and negative rabbit
antibody (BioReliance, Product 81015). Tissue sections of experimentally infected
red-legged partridges in which presence of WNV had been confirmed by real time
RT-PCR served as positive controls.
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RESULTS
Morbidity and mortality
The outbreak affected mostly gallinaceous game birds and caused nervous signs
and high mortality. Apparently the primary species affected by the outbreak were
red-legged partridges and ring-necked pheasants. Carcasses and individuals
displaying clinical signs of apparent blindness, ataxia and lack of coordination
belonged primarily to these two species, although a low number of affected
Columbiformes (common wood pigeon) had also been observed. Mortality or nervous
signs in other avian species such as corvids or birds of prey were not detected.
Real time RT-PCR and sequence analysis

All samples tested negative for genomes of WNV lineage 1 and 2 by real time RTPCR. Flavivirus genome was detected by real time RT-PCR in all tested individuals
in at least one of the samples analyzed (swabs and tissues, Table 1). A positive
signal was obtained most frequently in the brain (67%) and oral swabs (67%) of
partridges, and in the kidney (80%), and brain (100%) of pheasants. In one of the
wood pigeons sampled, all tested tissues were positive for flavivirus, while only the
kidney of the other one was positive and oral and cloacal swabs were negative in
both individuals (Table 1).
Only one partridge brain sample yielded a DNA product sufficient for cloning
and sequencing in the primary conventional generic nested flavivirus RT-PCR. In
the sequence similarity search performed by using BLAST, the analyzed sequence
of 1048 bp showed 92% sequence identity to previously reported BAGV strain
DakAr B209 [GenBank: AY632545.2]. In the case of the nested PCR, the 104 bp
amplicon was sequenced from 8 different samples; 3 brain samples and 2 oral
swabs from partridges, and 3 brain samples from pheasants, showing 97-98%
sequence identity to the previously described BAGV (Table 1).
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Table 1. Detection of flavivirus genome in BAGV infected game birds. Detection of flavivirus
genome by rt RT-PCR (Ct values) and by sequencing from conventional PCR in different tissues and
swabs.
Partridge
Wood
pigeon
Pheasant
Sample
No.1
No.2
No.3
No.4
No.5
No.6
No.1
No.2
No.3
No.4
No.5
No.1
No.2
19
30
23*
19*
29
31
Clocacal swab
29
31
Brain
22*
22*
16*
30
26*
23*
24*
30
30
30
Heart
32
23
23
27
31
Spleen
30
30
27
Kidney
29
30
28
29
27
29
29
30
Oral swab
*Sequenced 104 bp amplicon from conventional PCR.

Sequenced 1048 bp amplicon from conventional PCR.
Samples that did not show amplification.
Gross and microscopic lesions

The distribution, nature and severity of lesions varied considerably between species.
Two partridges and two pheasants were emaciated. The most striking macroscopic
lesions in partridges included pallor of the pectoral muscle (4/6), pancreas (4/6)
and myocardium (3/6) and injection of encephalic (3/6) and epicardial (4/6)
vessels. Besides thickening of the pericardium (2/5) or pallor of the myocardium
(2/5), no significant gross lesions were observed in pheasants. Equally, no
significant macroscopic lesions other than encephalic vessel injection (2/2) were
found in wood pigeons.
In all cases, the most prominent microscopic lesions were congestion,
necrosis and mononuclear inflammatory infiltrates consisting of lymphoid cells,
plasma cells and histiocytes, although to a different degree in individuals of the
three species (Table 2).
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Captulo 4
The most affected systems were the central nervous system and the spleen.
Lesions in the brain were characterized by congestion, gliosis, neuronal necrosis
and phagocytosis, perivascular cuffing, capillary endothelial cell swelling and
Purkinje cell necrosis and disappearance. The perivascular infiltrates and glial
nodules were constituted by lymphocytes, plasma cells and histiocytes and were
mainly present in the gray matter of the cerebrum, the brain stem and molecular
layer of the cerebellum (Table 2 and figures 1-4). In the spleen, lymphoid cell
depletion, necrotic foci of lymphoid cells, thickened capsule, and multifocal
granulocytic infiltrates were evident. Other organs affected included the kidney,
heart and liver, where necrosis and lymphoplasmacytic and histiocytic infiltrates
were the most important lesions (Table 2 and figures 5 and 6).
While partridges showed severe haemosiderosis in the spleen and liver, this
was absent in pheasants and less evident in wood pigeons (Table 2 and figures 7
and 8). All examined partridges and, to a lesser extent wood pigeons, had Kupffer
cells and hepatocytes in the liver and macrophages in the spleen heavily laden with
brown pigment. Using Perls' stain, this brown pigment was shown to contain iron,
indicating that in fact, it corresponds to haemosiderin (Figures 9 and 10). In
pheasants no iron/haemosiderin was evidenced in the liver or spleen using this
technique.
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Table 2. Microscopic lesions in BAGV infected game birds.

Table 2. Microscopic lesions in BAGV infected game birds.
Partridge
Lesion
Wood
pigeon
Pheasant
No.1
No.2
No.3
No.4
No.5
No.6
No.1
No.2
No.3
No.4
No.5
No.1
No.2
Neuronal necrosis
NA
Neuronophagia
NA
Satellitosis
NA
Gliosis
NA
Glial necrosis
NA
NA
NA
NA
Purkinje cell disappearance
NA
Gliosis
NA
NA
NA
Neuronal necrosis
NA
NA
Gliosis
NA
NA
Cerebrum
Cerebellum
Optic lobe
Glial necrosis
NA
NA
NA
NA
NA
NA
Miofibrilar necrosis
degeneration
NA
NA
Septal epithelial cells

necrosis
Capsular thickening
NA
NA
NA
NA
NA
NA
Haemosiderosis
NA
NA
Granulocytic infiltration
NA
NA
Vascular wall thickening
NA
NA
NA
NA
Hepatocellular necrosis
Haemosiderosis
Tubular epith. cell degen.
Heart
Lung
Spleen
Liver
Kidney
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Captulo 4
Table 2. Microscopic lesions in BAGV infected game birds (cont).

Partridge
Lesion
Wood
pigeon
Pheasant
No.1
No.2
No.3
No.4
No.5
No.6
No.1
No.2
No.3
No.4
No.5
No.1
No.2
NA
NA
NA
NA
NA
NA
NA
NA
NA
Large intestine
Epithelial cell necrosis
Pancreas
Skin
+ Presence of lesion.
Absence of lesion.
NA: tissue sample no analyzed.
Figure 1. Cerebrum; ring-necked pheasant. Diffuse necrosis of neurons and multifocal perivascular
infiltration of lymphoplasmacytic cells. HE, 100.
Figure 2. Cerebellum; ring-necked pheasant. Necrosis and disappearance of Purkinje cells, and mild
infiltration of lymphoplasmacytic and histiocytic cells in the molecular layer. HE, 200.
Figure 3. Cerebrum; red-legged partridge. Diffuse necrosis of neurons and diffuse and moderate
infiltration of lymphoplasmacytic and histiocytic cells. Necrotic neurons show picnotic nuclei and
retracted cytoplasm. HE, 200.
Figure 4. Cerebellum; red-legged partridge. Disappearance of Purkinje cells, which are replaced by
glial cells (arrowhead), and endothelial cell swelling in the white matter. HE, 100. Inset: detail of the
endothelial cell swelling. HE, x400.
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Figure 5. Heart; red-legged partridge. Multifocal and moderate necrosis of cardiac myofibers and
infiltration of lymphoplasmacytic and histiocytic cells. Necrotic myofibers show fragmentation and
accumulation of hyaline material in the cytoplasm. HE, 100.
Figure 6. Kidney; red-legged partridge. Multifocal and moderate interstitial infiltration of
lymphoplasmacytic and histiocytic cells. HE, 100.
Figures 7. Spleen; red-legged partridge. Diffuse brown pigment in the cytoplasm of splenic
macrophages. HE 100.
Figure 8. Liver; red-legged partridge. Multifocal brown pigment in the cytoplasm of hepatocytes and
Kupffer cells. HE, 100.
Figure 9. Spleen; red-legged partridge. Blue staining in the cytoplasm of splenic macrophages. Perls'
stain, 100.
Figure 10. Liver; red-legged partridge. Blue staining in the cytoplasm of hepatocytes and Kupffer
cells. Perls' stain, 100
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Captulo 4
Using immunohistochemistry, BAGV antigen was detected in numerous

organs of the partridges, especially in capillary endothelial cells, while in the wood
pigeon it was only detected in the spleen and in the pheasants in the central
Percentage of individuals with positive

immunostaining
nervous system (Figure 11).

n=6
100
90
n=6
n=6
80
70
60
50
40
n=5
n=2
n=6
30
20
Red-legged partridge
n=6
n=6
n=6
n=5
Ring-necked pheasant
n=5
Common wood pigeon
10
0
Tissue
Figure 11. Distribution of positive flavivirus (presumptive BAGV) immunostaining in naturally
BAGV infected red-legged partridges, ring-necked pheasants and common wood pigeons. Each
column represents the percentage of individuals that showed positive immunostaining in each organ
and n is the number of individuals in which each tissue was tested.
In pheasants, only the cytoplasm of neurons and glial cells of the thalamus
and optic lobe, and Purkinje cells of the cerebellum were found to contain BAGV
antigen (Figures 12 and 13). In partridges, endothelial cells of capillaries of the
cerebrum, cerebellum, spleen, heart, kidney, pectoral muscle, caeca and adrenal
gland contained BAGV antigen in their cytoplasm. Other cells that tested positive
were neurons and glial cells of the cerebrum, Purkinje cells of the cerebellum,
cardiomyocytes of the heart and endothelial cells of the glomerular mesangium in
the kidney (Figures 14-17). One of the wood pigeons had BAGV antigen in capillary
endothelial cells in the spleen. As swabs and tissues tested by real time RT-PCR
were negative for WNV, and the only flavivirus identified was BAGV, we assumed
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Tesis doctoral
that positive labeling in immunohistochemistry preparations indicated presence of

BAGV antigen.
Figure 12. Cerebrum; ring-necked pheasant. BAGV antigen in the cytoplasm of neurons. IHC using
a cross-reactive WNV antibody with Mayer's haematoxylin counterstain, x100.
Figure 13. Cerebellum; ring-necked pheasant. BAGV labeling in the cytoplasm of Purkinje cells. IHC
using a cross-reactive WNV antibody with Mayer's haematoxylin counterstain, x200. Inset: detail of
the immunostaining. IHC using a cross-reactive WNV antibody with Mayer's haematoxylin
counterstain, x400.
Figure 14. Cerebrum; red-legged partridge. BAGV labeling in the cytoplasm of neurons and capillary
endothelial cells. IHC using a cross-reactive WNV antibody with Mayer's haematoxylin counterstain,
x200.
182
Captulo 4
Figure 15. Cerebellum; red-legged partridge. BAGV antigen in capillary endothelial cells and in the
cytoplasm of Purkinje cells. IHC using a cross-reactive WNV antibody with Mayer's haematoxylin
counterstain, x100. Inset: detail of the immunostaining. IHC using a cross-reactive WNV antibody with
Mayer's haematoxylin counterstain, x400.
Figure 16. Heart; red-legged partridge. BAGV antigen in capillary endothelial cells. IHC using a
cross-reactive WNV antibody with Mayer's haematoxylin counterstain, x400.
Figure 17. Kidney; red-legged partridge. BAGV antigen in capillary endothelial cells of the
glomerular mesangium. IHC using a cross-reactive WNV antibody with Mayer's haematoxylin
counterstain, x400.
Incidental findings included avian tuberculosis, anthracosis and coccidian

oocysts. Two of the pheasants and one of the wood pigeons had lesions compatible
with avian tuberculosis, in which presence of acid resistant bacilli was confirmed by
Ziehl-Neelsen stain. All of the partridges and one pheasant had brown pigment and
crystalline structures within the cytoplasm of peribronchial macrophages in the
lungs, compatible with anthracosis. Finally, two of the partridges showed less than
five coccidian oocysts/cross sections in the mucosal epithelium of the large
intestine.
DISCUSSION
The BAGV outbreak in 2010 in Spain is the first documented occasion in which this
virus has caused disease and mortality in birds, with a strikingly most severe effect
on a game bird species, the red-legged partridge (Agero et al., 2011). Recently,
authors reporting on the parallel outbreak of WNV in horses in the same region
made the hot summer and high mosquito abundance responsible for both
outbreaks (Garca-Bocanegra et al., 2011). While several cases of WN fever in horses
were notified, no new mortality events among game birds were detected during
2011.
Pathology due to BAGV infection had not been previously described in any
species, as the presence of neutralizing antibodies against BAGV in persons with
acute encephalitis in India, could not link the infection clearly to disease symptoms
(Bondre et al., 2009). However, based on sequence analysis, BAGV has been shown
183
Tesis doctoral
to be synonymous to Israel turkey meningoencephalitis virus (ITMV) (Kuno et al.,

1998), which causes a disease characterized by nonpurulent meningoencephalitis
with lymphocytic perivascular infiltrates and focal myocardial necrosis in turkeys in
Israel and South Africa, and is controlled by vaccination with live attenuated
vaccines (Komarov and Kalmar, 1960; Ianconescu et al., 1975; Barnard et al.,
1980). The close genetic relationship between the two viruses may mean that BAGV
is similarly pathogenic to at least some bird species (Garca-Bocanegra et al., 2012).
A local hunting estate in the area of the outbreak that conducts direct
transect counts on game birds prior to and after the hunting season, reported
reduction of red-legged partridge numbers by 86% and in ring-necked pheasants by
29%. Due to the high mortality in red-legged partridges, only ring-necked pheasants
were hunted in winter 2010/2011. Hunting bag data from the same hunting estate,
showed a reduction in the female/male ratio with respect to previous years (2.4
females/male as opposed to 4 females/male), suggesting that female pheasants
were more affected than males.
The degree to which the impact of BAGV in red-legged partridge populations
is higher than in ring-necked pheasants and other birds is evidenced by differences
in mortality and in the incidence of concomitant disease observed. In all examined
partridges BAGV infection appeared to be the primary cause of death (2/6) or
disease (4/6), while two of five pheasants and one of the two wood pigeons had
severe advanced lesions of concurrent chronic disease (avian tuberculosis). The
reason of the higher reduction in numbers in female pheasants, as opposed to
males is unclear. With view to a potential higher impact of BAGV, disease due to
ITMV has also been reported to be more severe and frequent in female than in male
turkeys (Barnard et al., 1980). However, other effects such as higher susceptibility
of female pheasants to predation, other diseases, or toxic substances employed in
agriculture in the area cannot be ruled out.
184
Captulo 4
Differences in the pathogenicity of BAGV for the three species could partly be
explained by the differences observed in the distribution and severity of lesions,
distribution of viral antigen, and the severe haemosiderosis in partridges, that was
moderate in wood pigeons and absent in pheasants. In partridges, BAGV apparently
has a wide tropism, targeting different cell types, but especially capillary endothelial
cells. In pheasants, neurotropism appears to be somewhat more important while in
pigeons only endothelial cells of splenic capillaries seemed to contain BAGV
antigen.
One of the most well studied flaviviruses that is known to cause disease in
birds is WNV, of which information is available on pathology in naturally infected
humans, horses and birds as well as experimental avian and mouse models
(Sampson et al., 2000; Steele et al., 2000; Cantile et al., 2001; Samuel and
Diamond, 2006). WNV is known to vary greatly in its virulence in avian species
although the mechanism and reasons are still poorly understood (Steele et al.,
2000; Wnschmann et al., 2005; Brault, 2009). As an example, both endothelial
and neural tropism has been described in native North American avian species after
natural WNV infection (Wnschmann et al., 2004a,b; Lopes et al., 2007). Also, in
humans and mouse models WNV has been shown to have a diverse cell tropism,
leading to a variety of lesions and clinical manifestations (Hayes et al., 2005;
Samuel and Diamond, 2006).
The severe haemosiderosis in red-legged partridges that is the most striking
difference to lesions due to BAGV in ring-necked pheasants has been described
previously in birds naturally infected by WNV. Hepatic haemosiderosis is most well
known as associated to iron overload in captive wild forest birds but is also
frequently associated to haemolytic processes in infectious disease (Cork et al.,
1995). Haemosiderosis and haemorrhage in relation to WNV has been described
previously
in
the
spleen and
liver of naturally
infected
North American
185
Tesis doctoral
Passeriformes such as the blue jay (Cyanocitta cristata) and the house sparrow
(Passer domesticus) as well as raptors and owls from the North American continent
(Gibbs et al., 2005; Ellis et al., 2007; O'Brien et al., 2010). It has also been
described in wild turkeys (Meleagris gallopavo) (Zhang et al., 2006), but in an
outbreak in farmed chukar partridges (Alectoris chukar) and Impeyan pheasants
(Lophophorus impeyanus), only erythrocytophagocytosis was reported in the spleen,
while haemosiderosis was not observed (Wnschmann and Ziegler, 2006). With view
to WNV-associated haemolysis in other species, a few cases of WNV-associated
haemorrhagic fever in humans have been described (Paddock et al., 2006). A recent
study has associated sequence signatures in the envelope protein of human
pathogenic flaviviruses with the primary syndrome that they produce (encephalitis
or haemorrhagic disease) (Barker et al., 2009). These authors speculated that the
electrostatic charge differences caused by the presence of either glycosilated
asparagine (Asn, haemorrhagic viruses) or Aspartic acid (Asp, encephalitic viruses)
at position 67 of the domain II of the envelope protein could be responsible for the
phenotype and related disease syndrome. It would be of interest to further
characterize the outbreak-related virus, in order to study these and other features.
In conclusion, BAGV is more pathogenic for red-legged partridges than for
ring-necked pheasants and common wood pigeons, and causes a severe haemolytic
process in this species. Further (experimental) studies will be necessary to
determine the factors that trigger BAGV susceptibility and pathogenesis of the
infection in red-legged partridges.
ACKNOWLEDGEMENTS
This study was supported by grant AG2008-02504GAN funded by the Spanish
Ministry for Science and Innovation. A.V. Gutirrez-Guzmn (PAC08-0296-7771)
and V. Gamino (323/09) are research fellows supported by the regional government
186
Captulo 4
of Castilla La Mancha (JCCM) and I.G. Fernndez-de-Mera was funded by MCINN

Program Juan de la Cierva, JCI-2009-04518, Spain. We acknowledge the help of
game wardens with sample and data collection in the field, and the assistance of M.
Pizarro with the photography of microscopic lesions.
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Barker WC, Mazumder R, Vasudevan S, Sagripanti J-L, Wu CH: Sequence
signatures in envelope protein may determine whether flaviviruses
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Barnard BJ, Buys SB, Du Preez JH, Greyling SP, Venter HJ: Turkey meningoencephalitis in South Africa. Onderstepoort J Vet Res 1980, 47(2):89-94.
Bondre VP, Sapkal GN, Yergolkar PN, Fulmali PV, Sankararaman V, Ayachit VM,
Mishra AC, Gore MM: Genetic characterization of Bagaza virus (BAGV)
isolated in India and evidence of anti-BAGV antibodies in sera collected
from encephalitis patients. J Gen Virol 2009, 90(Pt 11):2644-2649.
Brault AC: Changing patterns of West Nile virus transmission: altered vector
competence and host susceptibility. Vet Res 2009, 40(2):43. doi:
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Cantile C, Del Piero F, Di Guardo G, Arispici M: Pathologic and
immunohistochemical findings in naturally occuring West Nile virus
infection in horses. Vet Pathol 2001, 38(4):414-421.
Cork SC, Alley MR, Stockdale PH: A quantitative assessment of haemosiderosis
in wild and captive birds using image analysis. Avian Pathol 1995, 24(2):239254.
Digoutte JP: Bagaza (BAG) strain: Dak Ar B 209. Am J Trop Med Hyg 1978,
27:376-377.
Ellis AE, Mead DG, Allison AB, Stallknecht DE, Howerth EW: Pathology and
epidemiology of natural West Nile viral infection of raptors in Georgia. J
Wildl Dis 2007, 43(2):214-223.
M, Fernndez-Molera V, Arenas A: West Nile fever outbreak in horses and
humans, Spain, 2010. Emerg Infect Dis 2011, 17(12):2397-2399.
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Garca-Bocanegra I, Zorrilla I, Rodrguez E, Rayas E, Camacho L, Redondo I,

Gmez-Guillamn F: Monitoring of the Bagaza Virus Epidemic in Wild Bird
Species in Spain, 2010. Transbound Emerg Dis 2012, 60(2):120-126.
Gibbs SEJ, Hoffman DM, Stark LM, Marlenee NL, Blitvich BJ, Beaty BJ,
Stallknecht DE: Persistence of antibodies to West Nile virus in naturally
infected rock pigeons (Columba livia). Clin Diagn Lab Immunol 2005,
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Hayes EB, Sejvar JJ, Zaki SR, Lanciotti RS, Bode AV, Campbell GL: Virology,
pathology, and clinical manifestations of West Nile virus disease. Emerg
Infect Dis 2005, 11(8):1174-1179.
Ianconescu M, Hornstein K, Samberg Y, Aharonovici A, Merdinger M: Development
of a new vaccine against turkey meningo-encephalitis using a virus
passaged through the Japanese quail (Coturnix coturnix japonica). Avian
Pathol 1975, 4(2):119-131.
Jimnez-Clavero MA, Aguero M, Rojo G, Gomez-Tejedor C: A new fluorogenic realtime RT-PCR assay for detection of lineage 1 and lineage 2 West Nile
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Komarov A, Kalmar E: A hitherto undescribed disease-turkey meningoencephalitis. Vet Rec 1960, 72:257-261.
Kuno G, Chang GJ, Tsuchiya KR, Karabatsos N, Cropp CB: Phylogeny of the
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Lopes H, Redig P, Glaser A, Armien A, Wunschmann A: Clinical findings, lesions,
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Moureau G, Temmam S, Gonzalez JP, Charrel RN, Grard G, de Lamballerie X: A
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Hallazgos patolgicos en ojos de
gallinceas infectadas por flavivirus
aves
Gamino V, Escribano-Romero E, Gutirrez-Guzmn AV, Blzquez AB,

Saiz JC, Hfle U. Oculopathologic findings in flavivirus infected
gallinaceous birds. Veterinary Pathology. En revisin.
Captulo 5
RESUMEN
En el presente trabajo estudiamos el curso de aparicin de lesiones y antgeno viral
en las estructuras oculares de nueve perdices rojas (Alectoris rufa), de nueve
semanas de edad, infectadas de forma experimental con el virus West Nile (WNV).
De forma adicional, se estudiaron las lesiones y la distribucin del antgeno viral en
los ojos de seis perdices rojas y tres faisanes comunes (Phasianus colchicus)
infectados de forma natural con el virus Bagaza (BAGV), con el fin de explicar la
aparente ceguera observada. La rpida aparicin de lesiones microscpicas y
antgeno viral en el ojo de las perdices infectadas experimentalmente con WNV,
incluso antes que en el sistema nervioso central, fue indicativo de la llegada del
virus al ojo va hematgena. Las perdices infectadas con BAGV presentaron una
mayor reaccin inflamatoria y una distribucin ms amplia del antgeno viral en la
retina en comparacion con los faisanes y las perdices infectadas experimentalmente
con WNV. Esto podra explicar el mayor efecto de la infeccin sobre la visin en las
primeras. Nuestros resultados indican que, en aves gallinceas de caza, la
replicacin de un flavivirus y el desarrollo de lesiones en estructuras oculares
varan en funcin del virus y la especie infectada.
193
Tesis doctoral
ABSTRACT
Using eye samples of nine 9-week-old experimentally West Nile virus (WNV) infected
red-legged partridges (Alectoris rufa), time course of lesions and WNV antigen
appearance in ocular structures was examined. Additionally, eye samples of six redlegged partridges and three ring-necked pheasants (Phasianus colchicus) naturally
infected with Bagaza virus (BAGV) were used to study lesions and flavivirus antigen
distribution in relation to apparent blindness. The rapid onset of microscopic
lesions and early presence of viral antigen in the eye of experimentally WNV infected
partridges,
prior
to
the
central
nervous
system
involvement,
suggested
hematogenous spread of the virus into the eye. BAGV infected partridges had a
more pronunced inflammatory reaction and more widespread flavivirus antigen
distribution in the retina compared to pheasants and experimentally fatally WNV
infected partridges, which could explain the more prominent effect on the vision.
Our results suggest that flavivirus replication and development of lesions in ocular
structures of gallinaceous game birds vary with the specific virus and host species
involved.
194
Captulo 5
INTRODUCTION
Flaviviruses are small single-stranded RNA viruses with a virtually worldwide
distribution. West Nile virus (WNV), a pathogen of importance for birds, has a wide
tissue tropism, and the eye is one of the target organs in infected avian hosts.
Vision impairment and ocular lesions have been described, mainly in raptors
(Wnschmann et al., 2004, 2005; Pauli et al., 2007), but little is known about the
mechanisms by which this virus reaches the eye and produces ocular lesions (Pauli
et al., 2007).
Another flavivirus, Bagaza virus (BAGV), recently caused an outbreak among
game birds in south-western Spain, producing neurologic signs in ring-necked
pheasants (Phasianus colchicus) and, more severely, in red-legged partridges
(Alectoris rufa), which also showed apparent blindness (Gamino et al., 2012).

For the present study, we collected eye samples of nine 9-week-old red-legged
partridges (Nos. 1 to 9) subcutaneously inoculated with 107 PFU of a cell culture
passaged New York/1999 (NY99) WNV strain, and euthanized on days 3 (No. 2), 7
(No. 4), 10 (Nos. 6 and 7) and 14 (Nos. 8 and 9) post-inoculation (dpi), or found
dead during the experiment presumably due to WNV infection (Nos. 1, 3 and 5).
These samples were examined in order to follow the appearance and distribution of
lesions and antigen during the course of the infection.
Moreover, we collected eye samples of six sexually mature red-legged
partridges (Nos. 10 to 15) and three ring-necked pheasants (Nos. 16 to 18) that died
or were euthanized in a moribund state during the recent BAGV outbreak in Spain
(Gamino et al., 2012). These samples were examined in order to compare
microscopic lesions and flavivirus antigen distribution between the two avian
195
Tesis doctoral
species, with the aim of amplifying our recent description of species specific tissue
tropism of this virus and investigating blindness observed in the affected partridges.
Hematoxylin
and
eosin
stain
(HE)
and
viral
antigen
detection
by
immunohistochemistry (IHC) were performed. For the IHC, a rabbit polyclonal

antibody against WNV that has been shown to cross-react with other flaviviruses
(BioReliance, Product 81015, Rockville, MD), was used applying the protocol
described previously (Gamino et al., 2012), but using 3-Amino-9-ethylcarbazole
(AEC) as visualizing method. These sections were examined under a light
microscope by two different investigators (VG and UH). For comparative purposes
between species (BAGV) or among different dpi (WNV), the distribution and
abundance of the viral antigen were subjectively scored as absent, mild (focal or
multifocal, <10% cells stained), moderate (multifocal, 10-50% cells stained) or
marked (diffuse, >50% cells stained).
RESULTS
WNV and BAGV infection was confirmed in all experimentally and naturally infected
birds by real time RT-PCR and sequencing as previously described (Crdoba et al.,
2007; Gamino et al., 2012)
Unlike BAGV infected partridges, none of the experimentally WNV infected
birds showed visual impairment.
Experimentally
WNV
infected
partridges
that
were
euthanized
had
intravascular lymphoplasmacytic, histiocytic and, less frequently, granulocytic

inflammatory cells in the choroid, pecten and ciliary body on various dpi (Table 1).
Mild inflammation in the connective tissue of the sclera (7 dpi, No. 4) and ciliary
body (7 and 14 dpi, Nos. 4 and 9, respectively) and endothelial cell swelling in the
choroid (10 dpi, No. 7), periocular muscle and pecten (14 dpi, Nos. 8 and 9) were
also detected (Table 1 and figure 1). Compared to euthanized birds, fatally WNV
196
Captulo 5
infected
partridges
showed
mononuclear
inflammatory
cells
and
muscular
degeneration in the iris on days 2 and 7 pi (Nos. 1 and 3, respectively) and mild
optic neuritis on 7 dpi (No. 3) (Table 1). In both euthanized and fatally infected
partridges, WNV antigen was only detected in the rods and cones processes. The
immunostaining was mild and appeared earlier in euthanized birds (Table 1 and
figure 2).
Table 1. Lesion and antigen distribution in the eyes of experimentally WNV infected red-legged
partridges, collected between days 2-14 post-inoculation (dpi).
2 dpi
(No. 1)
Tissue
3 dpi
(No. 2)*
7 dpi
(No. 3)
7 dpi
(No. 4)*
8 dpi
(No. 5)
10 dpi
(Nos.
6 and 7)*
14 dpi
(Nos.
8 and 9)*
HE
IHC
HE
IHC
HE
IHC
HE
IHC
HE
IHC
HE
IHC
HE
IHC
Periocular muscle
+d
+d
+d
Conjunctiva
NA
NA
NA
NA
+a,b
+a,b
Ciliary body
+a,c
+a,c
+c
+a
Choroid
+c
+a,c
+c,d
+d
+c
Pecten
+c
+c
+c,d
+c,d
Optic nerve
+a
NA
NA
Retina
++
Iris
Partridges found dead.

* Euthanized partridges.
Lesion evaluation in HE stained sections: () lesion absence; (+) lesion presence:(a) inflammatory cell infiltration;
(b) myofiber degeneration; (c) intravascular inflammatory cells; (d) endothelial cell swelling.
Immunostaining scores: () negative; (+) mild; (++) moderate; (+++) marked.
NA: no analyzed.
In BAGV infected partridges, the presence of mononuclear or mixed

intravascular and infiltrating inflammatory cells was marked. These were widely
distributed in every ocular structure, including the optic nerve. Additionally,
myofibril degeneration (Nos. 11-14) and endothelial cell swelling (Nos. 13 and 14)
were detected, but only in the periocular muscle. In pheasants, inflammatory
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Tesis doctoral
infiltrates were mild, with the exception of the pecten and the optic nerve (Figures 3
and 4), and absent in the periocular muscle. Endothelial cell swelling was observed
in the choroid and pecten of case No. 16. In both species, BAGV antigen was
detected in different cell layers of the retina, scarcer in pheasants, with differences
in staining distribution and abundance (Figures 5-7).
198
Captulo 5
Figure 1. Pecten; experimentally WNV infected partridge No. 8, 14 dpi. Nuclear swelling in
capillary endothelial cells. HE, x400.
Figure 2. Retina; experimentally WNV infected partridge No. 6, 10 dpi. WNV antigen in
photoreceptor processes. IHC for WNV with Mayer's hematoxylin counterstain, x400.
Figure 3. Pecten; naturally BAGV infected pheasant No. 16. Diffuse and marked infiltration of
lymphoplasmacytic, histiocytic and granulocytic cells which is expanding the pecten. HE, x40. Inset:
detail of the presence of numerous intravascular and perivascular inflammatory cells. HE, x400.
Figure 4. Optic nerve; naturally BAGV infected pheasant No. 16. Diffuse and moderate gliosis. HE,
x400.
Figure 5. Retina; naturally BAGV infected partridge No. 13. BAGV antigen in photoreceptor
processes and within vertical cell processes in the inner nuclear layer. IHC using cross-reactive WNV
antibody with Mayer's hematoxylin counterstain, x400.
Figure 6. Retina; naturally BAGV infected partridge No. 15. BAGV antigen in photoreceptor
processes, within cell cytoplasm and vertical cell processes in the inner nuclear layer, and in cell
199
Tesis doctoral
cytoplasm in the ganglion cell layer. IHC using cross-reactive WNV antibody with Mayer's hematoxylin
counterstain, x400.
100%
Pa
Ph
Pa
Ph
Pa
Ph
Pa
Ph
90%
Percentage of individuals
80%
70%
Marked
60%
Moderate
Mild
50%
Absent
40%
30%
20%
10%
0%
Rods and
cones
Outer nuclear
Inner nuclear
Ganglion cell
Retinal layer
Figure 7. Immunostaining scores of BAGV antigen in different layers of the retina of red-legged
partridges (Pa) and ring-necked pheasants (Ph) naturally infected with the virus.
DISCUSSION
In the present study, we observed similar lesions in the eyes of experimentally WNV
infected partridges to those described in naturally infected raptors and owls
(Wnschmann et al., 2004, 2005; Gancz et al., 2006). Ocular infection of
flaviviruses is thought to occur either hematogenously during viremia or by
extension from the central nervous system (CNS) to the retina via the optic nerves
(Pauli et al., 2007). Studying eye samples of the experimentally WNV infected
partridges, microscopic lesions appeared very early in the course of the infection
and, in euthanized animals, WNV antigen was detected as early as 3 dpi.
Considering that WNV RNA, viral antigen and microscopic lesions were detected
later in the CNS of the same birds (7-10 dpi, data not shown), we assumed that
WNV distribution into the eye occurred hematogenously.
200
Captulo 5
In this study, we also found differences in the inflammatory response and

antigen abundance in the eyes of BAGV infected partridges and pheasants. The
mild inflammatory reaction and the scarce BAGV antigen presence in the retina of
pheasants could be related to the apparently lower virulence of this virus in this
species (Agero et al., 2011; Gamino et al., 2012). On the contrary, the higher
abundance and wider distribution of BAGV antigen in the retina of infected
partridges accompanied by a more severe inflammatory reaction may be the cause
of the more prominent effect of BAGV on visual capacity in this species, which
matches clinical observation of apparent blindness that was absent in the
experimental WNV infection. Nevertheless, pathological differences could be also
related to the experimental conditions in the WNV infection, the age of the infected
birds or the virus.
In conclusion, virus replication and development of lesions in ocular
structures of flavivirus infected gallinaceous game birds vary with the virus and
host species involved. Especially, retinal involvement can influence the development
of impaired vision, as evidenced here in the case of BAGV infected red-legged
partridges. Vision impairment could have an important impact on the survival of
individuals in the field that adds to primary mortality from the disease.
ACKNOWLEDGEMENTS
This study was supported by grants AG2008-02504GAN and SAF-2008-04232
funded by the Spanish Ministry for Science and Innovation, grants FAU2008 000600-00 and RTA2011-0036 funded by the Spanish Institute for Agricultural and
Alimentary Investigation and Technology (INIA), and The Network of Animal Disease
Infectiology and Research Facilities, NADIR-UE-228394, funded by the E.U. We are
grateful to F. Ruiz-Fons for assistance with figure 7 and to J. Rodriguez-Ramos for
initial critical discussion of the interpretation of ocular histopathology.
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Tesis doctoral
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Redig P. Pathologic findings in red-tailed hawks (Buteo jamaicensis) and
Cooper's hawks (Accipiter cooperi) naturally infected with West Nile virus.
Avian Dis 2004, 48(3):570-580.
Redig P. Pathologic and immunohistochemical findings in goshawks
202
Captulo 6
Deteccin del virus Usutu
migratorios en Espaa
en
zorzales
Hfle U, Gamino V, Fernndez-de-Mera IG, Mangold AJ, Ortz JA, de la

Fuente J. Usutu Virus in Migratory Song Thrushes, Spain. Emerging
Infectious Diseases 2013, 19(7):1173-1175. doi: 10.3201/eid1907.130199.
Captulo 6
RESUMEN
En noviembre del ao 2012 se detect el virus Usutu (USUV) en zorzales comunes
(Turdus philomelos), afectados por un proceso de encefalitis aguda, que se
encontraban invernando en el sur de Espaa. El carcter no residente de esta
especie en el sur de nuestro pas y la secuencia de la cepa viral detectada
proporcionan evidencias de la introduccin de USUV por aves migratorias
procedentes del norte de Europa y una posible recrudescencia de una infeccin
persistente.
ABSTRACT
We detected Usutu virus (USUV) in migratory wintering song thrushes (Turdus
philomelos) with acute encephalitis in southern Spain in November 2012. The nonresident nature of this species in southern Spain and sequence data provides
evidence of USUV introduction by northern European migrants and a possible
recrudescence of a persistent infection.
205
Tesis doctoral
INTRODUCTION
Usutu virus (USUV), a member of the Japanese Encephalitis antigenic group, was
first detected in 1959 in mosquitoes in South Africa (Woodall, 1964), and emerged
in 1996 in blackbirds (Turdus merula) in Italy (Weissenbck et al., 2013). Recent
cases of USUV infection in asymptomatic human blood donors (Allering et al., 2012)
and severe disease in immunocompromised patients (Pecorari et al., 2009) have
evidenced its zoonotic potential.
Epidemiology and molecular phylogeny of USUV isolated in Italy, Austria,
Hungary, Switzerland and Germany suggest establishment of stable endemic
mosquito bird cycles in Europe (Chvala et al., 2007; Becker et al., 2012). Where
active vector surveillance programs exist, USUV is detected in mosquitoes prior to
bird mortality and human cases. USUV similar to African strains was detected in
Spanish mosquitoes in 2006 and 2009 (Busquets et al., 2008; Vzquez et al., 2011).
THE STUDY
In November 2012, two live song thrushes (Turdus philomelos) with neurological
signs were recovered from a mortality of approximately ten birds in a hunting estate
in southern Spain. After death, a full detailed necropsy was conducted and samples
were collected for virus detection and histopathology. Total RNA was extracted from
oral and cloacal swabs, serum from a cardiac blood clot, and heart, kidney, spleen
and brain samples using High Pure RNA Tissue Kit (Roche Diagnostics, Barcelona,
Spain) and analyzed by generic flavivirus SYBR Green real time RT-PCR (Qiagen,
Madrid, Spain) and by a generic conventional nested flavivirus RT-PCR (Gamino et
al., 2012). The product of the first generic PCR (1,048 bp) was resin purified, cloned
into pGEM-T (Promega, WI, USA) and sequenced. The obtained sequence was
compared to European and African USUV sequences available in GenBank using
BLAST (BLAST). In addition to histopathology, a polyclonal primary rabbit antibody
206
Captulo 6
directed against WNV, with proven cross-reactivity to other flaviviruses, was used
for viral antigen detection by immunohistochemistry (Gamino et al., 2012).
The thrushes were an adult male and female in poor body condition that had
greenish urate soiled feathers around the cloaca. Subcutaneous or visceral fat
deposits were absent and the pectoral muscle was partially atrophied, more severely
in the male. Both birds showed severe generalised congestion.
The serum, brain and pool of cloacal and oropharyngeal swabs of both birds
yielded a strongly positive signal in the generic flavivirus real time RT-PCR.
Sequencing of the PCR product obtained in the generic flavivirus RT-PCR (GenBank
accession number KC437386) showed a 96-97% homology to published USUV
sequences. Nucleotide sequence analysis revealed a higher homology to Northern
European strains (97% to BH65/11-02-03 [HE599647] and Meise H, Germany
[JQ219843]; Budapest, Hungary [EF206350]; Italy 2009 [JF266698]; and Vienna
2001, Austria [AY453411]) than to USUV isolated in South Africa (96% to SAAR 1776 [AY453412]), a finding also supported by phylogenetic analysis of USUV
strains with similar results for Maximum Likelihood and Neighbor-Joining methods
(Figure 1A, data not shown).
Histologically,
both
birds
had
severe
encephalitis
characterized
by
congestion, neuronal and Purkinje cell necrosis, gliosis, satellitosis, neuronophagia,

endothelial cell swelling and vasculitis. Other lesions included multiorgan
congestion, necrosis of renal tubular epithelium and moderate hemosiderosis in the
liver and spleen. Intravascular cross sections of microfilariaes were detected in
pulmonary capillaries. Virus antigen was detected in neurons in the cerebral
hemispheres and brain stem, and in glial cells throughout the brain (Figure 1B).
Rare Purkinje cells and neurons in peripheral ganglia (e.g. of the gizzard) as well as
cardiac myofibers and renal tubular epithelial cells were positive.
207
Tesis doctoral
Figure 1. Usutu virus in migratory song thrushes in Spain. (A) Phylogenetic analysis of European
and African USUV. The evolutionary history was inferred using the Neighbor-Joining method. The
optimal tree with the sum of branch length = 1.18014408 is shown. The percentage of replicate trees
in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to
the branches. The tree is drawn to scale, with branch lengths in the same units as those of the
evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed
using the Maximum Composite Likelihood method and are in the units of the number of base
substitutions per site. The analysis involved 10 nucleotide sequences. All ambiguous positions were
removed for each sequence pair. There were a total of 929 positions in the final dataset. Evolutionary
analyses were conducted in MEGA5. USUV are identified by their GenBank accession numbers and a
capital letter indicating the country of origin (A = Austria, G = Germany, I = Italy, H = Hungary, S =
Spain, SA = South Africa); the sequences of the last two branches correspond to outgroup viruses used
to root the tree (M = Murray Valley encephalitis virus [GenBank AF161266], J = Japanese encephalitis
virus [GenBank HM596272]). The song thrush strain from Spain 2012 is highlighted with a black
triangle. (B) Immunohistochemical staining with cross-reacting antibody showing USUV antigen
labeling in a Purkinje cell of the cerebellum of a song thrush that died from encephalitis. Original
magnification x400.
CONCLUSIONS
The
molecular
genetic
analysis,
histopathology
and
immunohistochemistry
confirmed encephalitis due to USUV in two migratory wintering song thrushes

collected in a small mortality event in southern Spain. Based upon phylogenetic
analysis, the USUV infecting the diseased birds was more similar to USUV from
Austria, Hungary and Germany, than to the USUV isolated from mosquitoes in
Spain (Busquets et al., 2008; Vzquez et al., 2011) or in the African continent. This
result together with the fact that song thrushes are a non-resident migratory
wintering bird species in southern Spain (MAGRAMA) provides circumstantial
evidence of USUV introduction in Spain during bird migration from northern
Europe. Persistence of USUV in song thrushes and recrudescence of the infection
during southward migration with concomitant filarial infection, together with the
208
Captulo 6
presence of USUV in a local endemic cycle from previous introductions, high vector
abundance and high viral loads in infectious mosquitoes are possible scenarios for
this outbreak.
Our data implies that introduction of USUV (and potentially other
flaviviruses such as WNV lineage 2, not yet detected in Spain) from northern
Europe in addition to local endemicity and introduction from Africa occurs, and
that zoonotic European USUV strains may be co-circulating with strains of African
origin. At this time it is not clear if the Spanish/African lineage differs in virulence
for humans from the European/African lineage. However, virus introduction by
northern migrants in combination with locally favourable conditions for vector
populations implies a risk for virus amplification and transmission and disease
outbreaks in humans and horses outside the currently established mosquitotrapping period (MayOctober) of targeted flavivirus surveillance programs.
ACKNOWLEDGEMENTS
This research was supported by European Union grants 278976 from the following
programs of the EMIDA ERANET (Coordination of European Research on Emerging
and Major Infectious Diseases of Livestock; www.emida-era.net): ANTIGONE
(Anticipating the Global Onset of Novel Epidemics) and APHAEA (Harmonised
Approaches in Monitoring Wildlife Population Health).
REFERENCES
Allering L, Jost H, Emmerich P, Gunther S, Lattwein E, Schmidt M, Seifried E,
Sambri V, Hourfar K, Schmidt-Chanasit J. Detection of Usutu virus infection
in a healthy blood donor from south-west Germany, 2012. Euro Surveill
2012, 17(50).
Basic Local Alignment Search Tool (BLAST) [http://blast.ncbi.nlm.nih.gov/]
Becker N, Jost H, Ziegler U, Eiden M, Hoper D, Emmerich P, Fichet-Calvet E,
Ehichioya DU, Czajka C, Gabriel M, Hoffmann B, Beer M, Tenner-Racz K, Racz
209
Tesis doctoral
P, Gunther S, Wink M, Bosch S, Konrad A, Pfeffer M, Groschup MH, SchmidtChanasit J. Epizootic emergence of Usutu virus in wild and captive birds in
Germany. PLoS One 2012, 7(2):e32604.
14(5):861-863.
Chvala S, Bakonyi T, Bukovsky C, Meister T, Brugger K, Rubel F, Nowotny N,
Weissenbock H. Monitoring of Usutu virus activity and spread by using
dead bird surveillance in Austria, 2003-2005. Vet Microbiol 2007, 122(34):237-245.
Gamino V, Gutirrez-Guzmn AV, Fernndez-de-Mera IG, Ortiz JA, Durn-Martn
Pecorari M, Longo G, Gennari W, Grottola A, Sabbatini A, Tagliazucchi S, Savini G,
Monaco F, Simone M, Lelli R, Rumpianesi F. First human case of Usutu virus
neuroinvasive infection, Italy, August-September 2009. Euro Surveill 2009,
14(50).
Spanish Ministry of Agriculture, Food and Environment (MAGRAMA): Atlas de las
aves
reproductoras
de
Espaa
[http://www.magrama.gob.es/es/
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inventario-nacional-de-biodiversidad/index2010-11-11_20.52.58.7490.aspx]
Figuerola J, Tenorio A. West Nile and Usutu viruses in mosquitoes in Spain,
2008-2009. Am J Trop Med Hyg 2011, 85(1):178-181.
Weissenbck H, Bakonyi T, Rossi G, Mani P, Nowotny N. Usutu Virus, Italy, 1996.
Emerg Infect Dis 2013, 19(2):274-277.
Woodall JP. The viruses isolated from arthropods at the East African Virus
Research Institute in the 26 years ending December 1963. Proc E Afr Acad
1964, 2.
210
Sntesis
Sntesis
Los tres flavivirus que han sido objeto de estudio de la presente tesis doctoral, y que
circulan en Espaa produciendo mortalidad en aves, son agentes patgenos
emergentes en cuya epidemiologa influyen numerosos factores relacionados con el
virus, el hospedador, el ambiente y el vector. La OIE considera como una
enfermedad emergente a toda aquella enfermedad infecciosa que cumpla uno o
varios de los siguientes requisitos:
1) Una infeccin que aparece como consecuencia de la evolucin o de un
cambio en un agente patgeno existente.
2) Un
patgeno
una
enfermedad
no
descrita
previamente
que
es
diagnosticada por primera vez.

3) Una infeccin conocida que se distribuye hacia una nueva zona geogrfica o
una nueva poblacin hospedadora.
El ejemplo ms claro del primer tipo de enfermedad emergente en nuestro
pas es la infeccin por WNV. A pesar de que su circulacin en Espaa se conoce
desde hace medio siglo, no haba dado lugar a casos de enfermedad y mortalidad
hasta
la
ltima
dcada.
Este
flavivirus
ha
sido
ampliamente
estudiado,
fundamentalmente desde su introduccin en Norteamrica en el ao 1999, dada las

graves consecuencias que esto supuso, y que sigue suponiendo, no slo para aves
sino tambin para quidos y humanos. Despus de su introduccin en el Nuevo
Continente, este virus ha re-emergido como patgeno para humanos, quidos y
aves en el Viejo Continente. No obstante, como ya se ha indicado previamente en
esta tesis, mientras que WNV ha causado miles de muertes de aves en
Norteamrica,
los
brotes
detectados
en
Europa
son
muy
escasos
fundamentalmente parecen afectar a aves rapaces. Adems, mientras que algunas

especies como los crvidos han demostrado ser muy susceptibles a la infeccin,
otras, como las encuadradas en el orden Galliformes, si bien pueden infectarse,
raramente desarrollan enfermedad y sucumben a la misma. Ante esta situacin, se
213
Tesis doctoral
han realizado numerosos trabajos que tratan de explicar el porqu de la reemergencia de este flavivirus, el porqu de las diferencias en su comportamiento
eco-epidemiolgico entre Europa y el continente norteamericano y el porqu de las
diferencias en las consecuencias de la infeccin para las diferentes especies de
aves.
En relacin a
esto,
puesto
que
existen todava
pocos
estudios
experimentales realizados en aves, se plantearon los tres primeros captulos de esta

tesis doctoral.
El Captulo 1 se centr, fundamentalmente, en realizar una revisin de la
informacin existente sobre la patogenia, lesiones y tropismo celular y tisular en la
infeccin por WNV en aves, resaltando las diferencias encontradas entre rdenes y
familias que pudiesen explicar el hecho de que en unas especies la infeccin sea
ms grave que en otras. Con los datos recopilados se comprob que, efectivamente,
existen diferencias entre especies, incluso dentro de la misma familia, en relacin a
la abundancia y distribucin del antgeno viral as como en la severidad y
distribucin de las lesiones macroscpicas y microscpicas. Estas diferencias estn
relacionadas, fundamentalmente, con el momento post infeccin en el que el
hospedador muere, lo que a su vez est determinado por la susceptibilidad del
mismo a la infeccin. De esta forma, existen especies en las que la respuesta a la
infeccin es dbil, y en muchos casos tarda, permitiendo una elevada replicacin
del virus en los tejidos que provoca la muerte rpida del hospedador. En este grupo
se podran encuadrar algunas aves del orden Passeriformes, como los cuervos o los
arrendajos, que presentan lesiones de carcter agudo, sobre todo de tipo
inflamatorio, en algunos casos ausentes en el SNC, ya que estos individuos pueden
morir incluso antes de que el virus llegue a este tejido. Existe un segundo grupo de
hospedadores capaz de mantener una replicacin viral baja, que da lugar a una
infeccin crnica, la cual finalmente puede llevar a la muerte del individuo o al
desarrollo de secuelas a largo plazo. En este grupo podran encuadrarse las aves
214
Sntesis
rapaces, en las que es caracterstica la presencia de lesiones crnicas que tambin

son detectadas en el SNC. Finalmente, existe un tercer grupo de hospedadores que
responde a la infeccin de forma efectiva, en muchos casos de forma temprana,
eliminando el virus rpidamente, y en el que slo algunos individuos mueren como
consecuencia de la enfermedad. En este ltimo grupo se encuadraran algunas aves
del orden Galliformes, en las que se detectan lesiones de carcter moderado, en
muchos casos ms relacionadas con la respuesta inflamatoria del hospedador que
con la replicacin activa del virus.
No obstante, aunque la especie tiene cierto valor predictivo, el desarrollo de
lesiones, enfermedad y muerte asociado a la infeccin por WNV est modulado por
muchos otros factores relacionados, sobre todo, con el virus y el hospedador y que,
en muchos casos, hacen difcil predecir el desenlace de la infeccin. Como ya se ha
indicado anteriormente, se ha visto que una simple modificacin gentica y
aminoacdica en el virus puede generar un cambio en su virulencia, ya sea por una
mayor capacidad para replicarse o por una mayor habilidad para evadir la
respuesta inmune del hospedador (Brault et al., 2004; Ye et al., 2013). De este
modo, la emergencia o re-emergencia de la virulencia de WNV se ha relacionado en
gran parte con este factor (Brault et al., 2007; Sotelo et al., 2011a). Por otro lado,
cualquier factor que modifique la capacidad de respuesta del hospedador frente al
virus, como puede ser la edad, la existencia de enfermedades concurrentes, o la
presencia
de
inmunidad
previa
frente
al
flavivirus
infectante
otros
antignicamente relacionados, influye en la evolucin de la infeccin (Eldadah et

al., 1967; Fang y Reisen, 2006; Hfle et al., 2008).
Para determinar cmo las variaciones genticas y aminoacdicas de WNV
pueden modificar su virulencia para las aves, y si existen diferencias en la
susceptibilidad a la infeccin entre aves de Norteamrica y Europa, en los captulos
2 y 3 estudiamos la patogenia de la infeccin experimental de un ave mediterrnea
215
Tesis doctoral
endmica con tres cepas de WNV pertenecientes al linaje 1. En el Captulo 2, se

infectaron perdices rojas con dos cepas mediterrneas del virus, MO03 y SP07. El
objetivo fundamental fue estudiar las causas que pudiesen explicar el diferente
curso de la infeccin y la distinta mortalidad provocada por ambas cepas descritos
en un trabajo previo (Sotelo et al., 2011b). Se encontraron diferencias entre las dos
cepas en relacin a la distribucin del virus y nivel de replicacin viral en tejidos,
as como en la severidad de las lesiones, pero stas fueron consideradas
insuficientes como para determinar la mayor mortalidad causada por MO03. La
diferente patogenia de la infeccin en el SNC, donde MO03 dio lugar a una
aparicin de lesiones ms severas de forma ms temprana y a una reaccin
inflamatoria ms marcada, podra explicar la distinta mortalidad. Las lesiones que
WNV
provoca
en
el
SNC
estn
relacionadas
con
la
neuroinvasividad
neurovirulencia de la cepa. En nuestro trabajo la neuroinvasividad de ambas cepas

fue muy similar, puesto que el nivel de viremia, la llegada al SNC y el nivel de
replicacin viral en este tejido fueron muy similares. Por ello, determinamos que fue
la diferente neurovirulencia la que dio lugar a la diferente gravedad de la infeccin.
Sin embargo, an se desconocen los determinantes especficos que hacen que
MO03 resulte ms neurovirulenta. En el Captulo 3, se realiz un estudio similar,
pero en este caso las perdices fueron infectadas con una cepa norteamericana de
WNV, NY99. El objetivo de este trabajo fue determinar si esta especie es tambin
susceptible a la infeccin con esta cepa. Aunque las perdices desarrollaron lesiones
y murieron como consecuencia de la enfermedad, la replicacin del virus en los
tejidos fue mnima, a pesar de la alta dosis de inoculacin (107 UFP) que se utiliz.
As mismo, al comparar la patogenia de la infeccin en los individuos eutanasiados
con la observada en el estudio anterior, se demostr que las lesiones fueron menos
severas y que aparecieron de forma ms tarda en el curso de la infeccin. El hecho
de que las perdices infectadas con NY99 tuviesen dos semanas ms de edad podra
216
Sntesis
estar influyendo en estas diferencias; sin embargo, es ms probable que la causa

est asociada a una menor virulencia de la cepa NY99 de WNV.
Dados los resultados de estos dos trabajos, se demuestra, por un lado, que la
perdiz roja es susceptible a la infeccin por WNV, desarrollando lesiones similares a
las descritas en otras aves (Steele et al., 2000), que incluso llegan a producir la
aparicin de signos clnicos y mortalidad. Por otro lado, que las tres cepas de WNV
estudiadas
presentan
una
diferente
virulencia
para
esta
especie,
siendo
especialmente elevada la de MO03. Sin embargo, se desconoce qu determinantes

genticos o aminoacdicos (Sotelo et al., 2009) estn marcando esta diferente
virulencia, al no haber podido relacionarla con la sustitucin de treonina por
prolina en la posicin 249 de la protena NS3, considerada como un marcador de
virulencia para cuervos americanos (Brault et al., 2007). De cualquier forma,
considerando nuestros resultados, las diferencias a nivel gentico o protenico
podran explicar, al menos en parte, la emergencia o re-emergencia de la virulencia
de este flavirus para las aves y para otros hospedadores. No obstante, la
epidemiologa de WNV es muy compleja, y existen otros factores que podran estar
contribuyendo a una mayor circulacin del virus como, por ejemplo, el cambio
climtico (Jimnez-Clavero et al., 2012). Adems, es posible que el incremento de la
sensibilizacin de la poblacin sobre la importancia que tiene la infeccin por este
virus para las aves y, sobre todo, para los humanos, y la mejora de los planes de
vigilancia estn contribuyendo a la deteccin de casos que antes pasaban
desapercibidos (Burt et al., 2002).
Aunque ambos estudios son experimentales y han sido realizados con un
bajo nmero de animales, y se desconoce cmo transcurrira la infeccin en
condiciones naturales, se podra decir que al menos esta ave endmica europea es
moderadamente susceptible a la infeccin por WNV y que en Europa circulan cepas
que han demostrado ser virulentas para sta y otras especies de aves, tanto de
217
Tesis doctoral
forma experimental como natural (Jimnez-Clavero et al., 2008; Bakonyi et al.,

2013; Dridi et al., 2013). Por lo tanto, las menores consecuencias de la infeccin
por WNV para las aves europeas no parecen estar relacionadas ni con la baja
virulencia de las cepas circulantes ni con la baja susceptibilidad de las especies de
aves a la infeccin. Es probable que aquellos factores que modulan la efectividad de
la transmisin del virus o la probabilidad de infeccin del hospedador, como
pueden ser la abundancia de vectores, su actividad y la preferencia de hospedador,
o la abundancia de hospedadores reservorio y la biodiversidad (Ezenwa et al., 2006;
Brault, 2009; Hamer et al., 2011), estn jugando un papel mucho ms importante.
No obstante, el constante contacto de las aves del Viejo Continente con WNV, o con
flavivirus antignicamente relacionados, que hace que stas desarrollen inmunidad,
tambin podra explicar la baja incidencia de la enfermedad (Reiter, 2010).
La enfermedad asociada a la infeccin natural por BAGV encontrada en aves
cinegticas del sur de Espaa, constituye un ejemplo del segundo tipo de
enfermedad emergente previamente citado, ya que este flavivirus nunca haba sido
asociado a ningn caso de enfermedad o mortalidad. En el Captulo 4 se realiz,
por primera vez, una descripcin detallada de los hallazgos microscpicos y de la
distribucin del antgeno viral en aves infectadas de forma natural por este virus,
tratando fundamentalmente de determinar el porqu de su mayor virulencia para la
perdiz roja en comparacin con el faisn comn y la paloma torcaz. Los hallazgos
clnicos y las lesiones encontradas fueron muy similares a lo que se observa en la
infeccin por otros flavivirus, entre ellos WNV, detectndose una encefalitis
marcada en las tres especies. El mayor impacto de esta infeccin en la perdiz roja
se relacion con una distribucin tisular del virus mucho ms amplia y, sobre todo,
con el grave proceso hemoltico que provoc exclusivamente en esta especie. Este
estudio nos sirvi para corroborar que no todas las especies se ven igualmente
afectadas por la infeccin por un flavivirus, y podra constituir un ejemplo de lo que
218
Sntesis
ocurre cuando un nuevo virus es introducido en una poblacin hospedadora que

nunca antes haba contactado con el mismo. Sin embargo, se desconoce desde
cundo llevaba circulando BAGV en nuestro pas. El hecho de que fuese un brote
muy localizado, afectando a pocas especies, y que no se detectasen mortalidades
posteriores, pudo deberse a que fue un caso puntual favorecido por las condiciones
climatolgicas de ese ao y la elevada abundancia de vectores, as como la alta
densidad de aves en la zona. Es posible que la presencia de inmunidad cruzada
frente a otros flavivirus y el desarrollo de inmunidad frente BAGV (Llorente et al.,
2013) hayan evitado la aparicin de nuevas mortalidades.
A pesar de que, como se ha indicado en el apartado anterior, los signos
clnicos y las lesiones fueron muy similares en la infeccin por BAGV y WNV,
consideramos interesante la deteccin de ceguera slo en las perdices infectadas
por BAGV. Esto nos llev a plantear el estudio que constituye el Captulo 5, en el
que se determin que la ceguera de las perdices infectadas por BAGV est
relacionada con una inflamacin ocular ms severa y una mayor replicacin del
virus en la retina. Estos hallazgos son similares a los que se obtienen si se
comparan los encontrados en rapaces diurnas y nocturnas infectadas por WNV. En
las primeras se observa ceguera o visin alterada como consecuencia de la
presencia de infiltrados inflamatorios oculares extensos y una grave afectacin de la
retina asociada la replicacin del virus. Por el contrario, en las rapaces nocturnas, a
pesar de presentar una inflamacin moderada, la retina rara vez se ve afectada,
haciendo que sea poco frecuente la deteccin de signos relacionados con la
infeccin ocular (Wnschmann et al., 2005; Lopes et al., 2007; Pauli et al., 2007).
Por otro lado, nuestro estudio nos permiti determinar que la entrada de WNV
NY99 en el ojo de la perdiz roja se produce por va hematgena durante la viremia
primaria.
219
Tesis doctoral
La infeccin por USUV podra encuadrarse en el tercer tipo de enfermedad

emergente citado al principio de este captulo de sntesis, puesto que ya se conoca
su virulencia para las aves, sobre todo de la familia Turdidae, y, aunque el virus
haba sido detectado en mosquitos (Busquets et al., 2008; Vzquez et al., 2011), el
Captulo 6 constituye el primer trabajo en el que se describe la infeccin por USUV
en aves en nuestro pas. Los dos zorzales infectados mostraron lesiones muy
similares a las descritas en aves de Centroeuropa infectadas por el mismo virus
(Chvala et al., 2004). El hecho de que el zorzal comn sea un ave migratoria que
pasa los inviernos en Espaa (MAGRAMA) y que la cepa detectada mostrara mayor
homologa con las circulantes en Centroeuropa que con las detectadas previamente
en Espaa y frica, parece indicar que, probablemente, estas aves vinieron
infectadas y que el estrs asociado a la migracin recrudeci la infeccin,
provocando la muerte de las mismas. No obstante, es posible que esta cepa ya
estuviese circulando en Espaa y los zorzales se infectasen en nuestro pas. Puesto
que no se ha detectado enfermedad o mortalidad en otras aves de la zona, es difcil
determinar qu consecuencias tiene la presencia de esta cepa.
Los hallazgos microscpicos incidentales de los trabajos anteriores indican
una frecuente asociacin de la infeccin por estos flavivirus con la presencia de
otros patgenos. En unos casos estos patgenos aprovecharn la inmunosupresin
provocada por el flavivirus para multiplicarse, contribuyendo al deterioro del estado
de salud del hospedador, y en otros sern la causa de la inmunosupresin del
hospedador, haciendo que finalmente sucumba a la infeccin por el flavivirus. De
esta forma, los coccidios detectados en el intestino grueso y la candidiasis
observada en el buche de las perdices objeto de estudio de los captulos 2 y 3,
probablemente
vieron
favorecida
su
proliferacin
por
la
inmunosupresin
provocada por la infeccin con WNV. Por el contrario, la presencia de tuberculosis

crnica en algunos de los faisanes y las palomas infectadas por BAGV posiblemente
220
Sntesis
contribuy a la susceptibilidad de estos hospedadores a la infeccin por el

flavivirus.
En conjunto, en esta tesis hemos demostrado, por un lado, que en Espaa
circulan flavivirus capaces de infectar a aves cinegticas, dando lugar a la aparicin
de lesiones que, en algunos casos, puede llegar a producir la muerte del
hospedador. Sin embargo, tambin hemos visto que su eco-epidemiologa es
extremadamente compleja, y que la susceptibilidad del hospedador o la virulencia
del virus slo determinan una parte de la misma. Debido a que los tres flavivirus
comparten hospedadores susceptibles y vectores, que se ha demostrado que
circulan de forma silenciosa en nuestro pas (Llorente et al., 2013) y que al menos
WNV y USUV son zoonticos, existe un riesgo de aparicin de epizootias o
epidemias, sin embargo, su prediccin resulta muy complicada. Dada la
importancia socioeconmica que tienen algunas de estas aves, fundamentalmente
la perdiz roja, la infeccin por flavivirus podra tener consecuencias graves en caso
de
provocar grandes
mortalidades.
Adems,
considerando
que
estas
aves
constituyen el recurso trfico de otras especies (algunas de ellas amenazadas), que

estos virus se replican en tejidos y que la transmisin por ingestin de tejidos
infectados ha sido demostrada, al menos para WNV (Komar et al., 2003), la
infeccin por flavivirus tambin podra tener consecuencias ecolgicas graves.
Por otro lado, la perdiz roja ha demostrado ser un buen modelo experimental
de la infeccin por WNV, permitiendo ampliar los conocimientos sobre la patogenia
de la infeccin por este flavivirus en aves.
Considerando los resultados obtenidos en esta tesis, sera interesante
plantear futuros estudios que nos permitiesen, por un lado, determinar los factores
que hacen que la cepa MO03 de WNV sea ms virulenta para la perdiz roja y, por
otro lado, determinar la patogenia de la infeccin por BAGV en aves, para poder
221
Tesis doctoral
esclarecer, entre otras cosas, las causas de la severa hemlisis detectada en la

perdiz roja y su diferente virulencia para distintas especies de aves. Por ltimo,
consideramos interesante determinar si la cepa de USUV ya estaba circulando en
Espaa o, en caso de haber sido introducida por los zorzales, dnde se infectaron y
si la cepa sigue circulando en nuestro pas.
BIBLIOGRAFA
Bakonyi T, Ferenczi E, Erdlyi K, Kutasi O, Csorgo T, Seidel B, Weissenbck H,
Brugger K, Ban E, Nowotny N. Explosive spread of a neuroinvasive lineage 2
West Nile virus in Central Europe, 2008/2009. Vet Microbiol 2013. pii:
S0378-1135(13)00164-8. doi: 10.1016/j.vetmic.2013.03.005.
Brault AC, Langevin SA, Bowen RA, Panella NA, Biggerstaff BJ, Miller BR, Komar N:
Dis 2004, 10(12):2161-2168.
Holmes EC, Powers AM, Miller BR: A single positively selected West Nile
2007, 39(9):1162-1166.
Brault AC: Changing patterns of West Nile virus transmission: altered vector
competence and host susceptibility. Vet Res 2009, 40(2):43. doi:
10.1051/vetres/2009026.
Burt FJ, Grobbelaar AA, Leman PA, Anthony FS, Gibson GV, Swanepoel R:
Phylogenetic relationships of southern African West Nile virus isolates.
Emerg Infect Dis 2002, 8(8):820-826.
14(5):861-863.
Chvala S, Kolodziejek J, Nowotny N, Weissenbock H. Pathology and viral
distribution in fatal Usutu virus infections of birds from the 2001 and
2002 outbreaks in Austria. J Comp Pathol 2004, 131(2-3):176-185.
Dridi M, Vangeluwe D, Lecollinet S, van den Berg T, Lambrecht B. Experimental
infection of Carrion crows (Corvus corone) with two European West Nile
virus (WNV) strains. Vet Microbiol 2013. pii: S0378-1135(13)00061-8. doi:
10.1016/j.vetmic.2012.12.043.
Eldadah AH, Nathanson N, Sarsitis R: Pathogenesis of West Nile Virus
222
Sntesis
Ezenwa VO, Godsey MS, King RJ, Guptill SC: Avian diversity and West Nile virus:
testing associations between biodiversity and infectious disease risk. Proc
Biol Sci 2006, 273(1582):109-117.
Fang Y, Reisen WK: Previous infection with West Nile or St. Louis encephalitis
Trop Med Hyg 2006, 75(3):480-485.
Hamer GL, Chaves LF, Anderson TK, Kitron UD, Brawn JD, Ruiz MO, Loss SR,
Walker ED, Goldberg TL. Fine-scale variation in vector host use and force of
infection drive localized patterns of West Nile virus transmission. PLoS One
2011, 6(8):e23767. doi: 10.1371/journal.pone.0023767.
Fuente J, Gortzar C: West Nile virus in the endangered Spanish imperial
Jimnez-Clavero MA: Animal viral diseases and global change: bluetongue and
West Nile fever as paradigms. Front Genet 2012, 3:105. doi:
10.3389/fgene.2012.00105.
Llorente F, Prez-Ramrez E, Fernndez-Pinero J, Soriguer R, Figuerola J, JimnezClavero MA. Flaviviruses in Game Birds, Southern Spain, 20112012. Emerg
Infect Dis 2013, 19(6):1024-1026. doi: 10.3201/eid 1906.130122.
Lopes H, Redig P, Glaser A, Armien A, Wnschmann A: Clinical findings, lesions,
Dis 2007, 51(1):140-145.
Ministerio de Agricultura, Alimentacin y Medio Ambiente (MAGRAMA): Atlas de las
aves
reproductoras
de
Espaa
[http://www.magrama.gob.es/es/
biodiversidad/temas/inventarios-nacionales/inventario-especies-terrestres/
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Organizacin Mundial de Sanidad Animal (OIE) (2012): Cdigo Sanitario para los
Animales Terrestres (2012) [http://www.oie.int/es/normas-internacionales/
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Pauli AM, Cruz-Martinez LA, Ponder JB, Redig PT, Glaser AL, Klauss G, Schoster
JV, Wunschmann A: Ophthalmologic and oculopathologic findings in redtailed hawks and Cooper's hawks with naturally acquired West Nile virus
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Tesis doctoral
Reiter P: West Nile virus in Europe: understanding the present to gauge the
future. Euro Surveill 2010, 15(10):19508.
P, Tenorio A, Jimnez-Clavero MA: Phylogenetic relationships of Western
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42. doi: 10.1186/1297-9716-42-11.
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2008-2009. Am J Trop Med Hyg 2011, 85(1):178-181.
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Vaccine 2013, 31(3):461-471.
224
Conclusiones
Conclusiones
1. Aunque la mayora de las aves infectadas por WNV desarrollan lesiones

macroscpicas y/o microscpicas, existen diferencias interespecie en la
naturaleza, severidad o distribucin de las mismas relacionadas con el
momento post infeccin en el que el animal muere, lo que est determinado por
la susceptibilidad del mismo a la infeccin.
2. La
perdiz roja
es
susceptible a
la
infeccin experimental
con WNV,
desarrollando lesiones muy similares a las encontradas en otras aves infectadas

por el virus.
3. La perdiz roja constituye un modelo experimental apropiado para estudiar la

patogenia de la infeccin por WNV en aves.
4. La infeccin experimental de la perdiz roja con WNV provoca una encefalitis en

la que participan componentes celulares inflamatorios similares a los descritos
en roedores y humanos infectados por el mismo virus.
5. Las diferencias genticas y aminoacdicas entre diferentes cepas de WNV dan

lugar a variaciones en la patogenia de la infeccin experimental de la perdiz roja
que se traducen en una diferente patogenicidad.
6. El diferente comportamiento eco-epidemiolgico de WNV entre Norteamrica y

Europa no parece estar asociado a la menor susceptibilidad de las aves
europeas a la infeccin ni a la menor virulencia de las cepas circulantes en
Europa.
227
Tesis doctoral
7. BAGV es un flavivirus emergente capaz de producir enfermedad en la perdiz

roja, el faisn comn y la paloma torcaz, siendo ms virulento para la primera,
al mostrar un tropismo tisular ms amplio y generar un proceso hemoltico
grave.
8. La infeccin por BAGV causa ceguera en la perdiz roja como consecuencia del
desarrollo de lesiones inflamatorias oculares severas y la intensa replicacin del
virus en la retina.
9. La llegada de la cepa NY99 de WNV al ojo de perdices rojas infectadas de forma

experimental ocurre va hematgena.
10. La cepa de USUV que produjo el evento de mortalidad en zorzal comn en

Espaa probablemente fue introducida por los propios zorzales desde el centro
de Europa. Esta cepa dio lugar a una enfermedad similar a la encontrada en
otras aves infectadas por el mismo virus, que posiblemente se vio favorecida por
el estrs causado por la migracin.
228

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Infeccin por flavivirus en aves cinegticas en Espaa:

distribucin del virus y alteraciones estructurales

Memoria presentada por

Bajo la direccin de la Doctora

Ciudad Real, 2013

GRUPO DE SANIDAD Y BIOTECNOLOGA

Dra. Ursula Hfle Hansen

Este trabajo de tesis doctoral se realiz gracias a los siguientes proyectos:

CAPTULO 2. Patogenia de la infeccin experimental con dos aislados

Virginia Gamino Rodrguez

CAPTULO 3. Infeccin experimental de una especie gallincea

CAPTULO 4. Infeccin natural por el virus Bagaza en aves cinegticas

CAPTULO 5. Hallazgos patolgicos en ojos de aves gallinceas infectadas

CAPTULO 6. Deteccin del virus Usutu en zorzales migratorios en

Conclusions ...................................................................................................... 208

SNTESIS ................................................................................................. 211

CONCLUSIONES ...................................................................................... 225

Basic Local Alignment Search Tool

Cluster of differentiation 3/79

Enzyme-linked immunosorbent assay

Glial fibrillary acidic protein

Natural killer cells

Polymerase chain reaction

Ricinus communis agglutinin

Reverse transcription-polymerase chain reaction

Tumoral necrosis factor

Unidades formadoras de placa

Virginia Gamino Rodrguez

neutralizantes del hospedador (Lindenbach y Rice, 2003; Martn-Acebes y Saiz,

aproximadamente 54 arbovirus (39 flavivirus transmitidos por mosquitos y 15 por

Virginia Gamino Rodrguez

Virginia Gamino Rodrguez

En general, los flavivirus transmitidos por Aedes spp. se mantienen en un

FLAVIVIRUS EN EUROPA Y ESPAA

reas de invernada y nidificacin de muchas de estas aves, en nuestro pas existe

Virus West Nile

Virginia Gamino Rodrguez

importantes y que afectaban sobre todo a humanos y caballos (Hublek y Halouzka,

Figura 3. rbol filogentico de WNV (Ilustracin extrada de Vzquez et al., 2011).

WNV pertenece al complejo antignico Encefalitis Japonesa y, por tanto, su

Virginia Gamino Rodrguez

juegan un papel importante en la dispersin local y a larga distancia del virus

Figura 4. Ciclo de transmisin de WNV (Elaboracin propia).

En reas endmicas, la infeccin en aves suele tener lugar en primavera y a

fundamentalmente a aves rapaces (Bakonyi et al., 2006; Jimnez-Clavero et al.,

brachyrhynchos) han sufrido un grave declive debido a la mortalidad provocada por

Virginia Gamino Rodrguez

La mayora de los estudios realizados, sobre todo en aves, se centran en el

En el ao 2011 y 2012 tuvieron lugar nuevos casos de enfermedad en caballos en la

Gonzlez y Filipe, 1977

Chastel et al., 1980

Lozano y Filipe, 1998

Pujol et al., 2004

Bofill et al., 2006

guila imperial ibrica

Hfle et al., 2008

Focha comn (Fulica

Jabal (Sus scrofa)

Vzquez et al., 2010

Figuerola et al., 2007a,

Boadella et al., 2012

Vzquez et al., 2011

Virginia Gamino Rodrguez