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Universidad Austral De Chile Facultad De Ciencias

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Universidad Austral De Chile Facultad De Ciencias UNIVERSIDAD AUSTRAL DE CHILE FACULTAD DE CIENCIAS VETERINARIAS INSTITUTO DE PATOLOGÍA ANIMAL CARACTERIZACIÓN MOLECULAR DE VIRUS ADN EN CORMORÁN YECO (Phalacrocorax brasilianus) Memoria de Título presentada como parte de los requisitos para optar al TÍTULO DE MÉDICO VETERINARIO NAOMI ARIYAMA TAKENAKA VALDIVIA – CHILE 2017 PROFESOR PATROCINANTE ___________________________________ Claudio Marcelo Verdugo Reyes PROFESORES INFORMANTES ___________________________________ Ángelo Ociel Espinoza Cabrera ___________________________________ Francisco Javier Morera Galleguillos FECHA DE APROBACIÓN: 29 de Junio de 2017 ÍNDICE Capítulos Página 1. RESUMEN………………………………………………………………. 1 2. SUMMARY………………………………………………………………. 2 3. INTRODUCCIÓN………………………………………………………. 3 4. MATERIAL Y MÉTODOS……………...………………………………. 9 5. RESULTADOS…………………………...……………………………… 12 6. DISCUSIÓN……………………………………………………………... 17 7. REFERENCIAS...………………………………………………………... 21 8. ANEXOS………………………………………………………………… 24 9. AGRADECIMIENTOS…..……………………………………………… 29 1 1. RESUMEN1 El estudio de las relaciones genéticas entre hospedero-patógeno ha ampliado el entendimiento de los mecanismos de adaptación mutua entre especies antagonistas. Dichas adaptaciones conllevan procesos coevolutivos y de especialización de patógenos con sus respectivos hospederos. Dentro de los patógenos en aves que pueden ser utilizados como modelo para el estudio de evolución molecular de los virus ADN de doble hebra, están las familias Adenoviridae y Herpesviridae. El objetivo de este trabajo fue identificar y caracterizar molecularmente virus ADN de doble hebra infectando al cormorán yeco o neotropical, Phaacrocorax brasilianus. Se analizaron tórulas cloacales y traqueales de 100 individuos silvestres provenientes de Arica, Chillán, Los Ángeles y Valdivia, de las que se extrajo el material genético viral y se detectaron mediante partidores universales virus de las familias Adenoviridae y Herpesviridae amplificando el gen de la proteína hexona y de la ADN polimerasa, respectivamente. Se encontraron ocho muestras positivas a adenovirus y cuatro positivas a herpesvirus que, luego de secuenciar los respectivos productos de PCR, revelaron una posible nueva especie de adenovirus de cormorán neotropical dentro del género Aviadenovirus, mientras que el herpesvirus de cormorán neotropical está relacionado genéticamente con el virus de la laringotraqueitis infecciosa (GaHV-1) del género Iltovirus. Los adenovirus y herpesvirus tienden a infectar un rango de hospederos restringido y a tener baja patogenicidad, indicando procesos coevolutivos. En este trabajo se evaluó a P. brasilianus como modelo de estudio de relaciones hospedero-patógeno, evidenciando que el adenovirus encontrado posiblemente ha coevolucionado con P. brasilianus. Sin embargo, debido a la cercanía genética del herpesvirus de cormorán con virus que infectan aves de producción, sugiere una reciente transmisión de este herpesvirus desde aves domésticas a aves silvestres. Es importante el monitoreo de patógenos que afectan la fauna silvestre, debido a su rol como reservorio de enfermedades que afectan el ecosistema y la salud animal, de animales domésticos y silvestres. Por lo que requieren más estudios de ambos virus detectados, en términos de prevalencia, virulencia, patogénesis y efectos en el sistema inmune del hospedero P. brasilianus, así como de su origen, distribución y rango de hospederos. Palabras clave: adenovirus, herpesvirus, filogenética, cormorán. Fuente de financiamiento: Proyecto FONDECYT 11130305 2 2. SUMMARY1 MOLECULAR CHARACTERIZATION OF DNA VIRUSES FROM THE NEOTROPIC CORMORANT (Phalacrocorax brasilianus) The study of the genetic interactions of host-pathogens has allowed a better understanding of the mechanisms of mutual adaptation between antagonistic species. Such adaptations involve coevolutionary processes and pathogens specialization to their respective hosts. Among the pathogens that infect birds and can be used as a model for studying the molecular evolution of double-stranded DNA viruses are the families Adenoviridae and Herpesviridae. The objective of this study was to identify and molecularly characterize double-stranded DNA viruses infecting the Neotropic cormorant or Yeco, Phalacrocorax brasilianus. Cloacal and tracheal swabs were obtained of 100 wild individuals from Arica, Chillán, Los Ángeles and Valdivia. The viral genetic material was extracted to detect viruses of the families Adenoviridae and Herpesviridae by using universal primers targeting the hexon and DNA polymerase gene, respectively. Eight samples were positive to adenovirus, whereas four samples were positive to herpesvirus. Sequences revealed a possible new species of cormorant adenovirus within the genus Aviadenovirus; whereas the herpesviruses detected were genetically related to the infectious laryngotracheitis virus (GaHV-1) of the genus Iltovirus. Adenoviruses and herpesviruses tend to infect a restricted host range with a low pathogenicity, indicating potential coevolutionary processes. In this work, P. brasilianus was evaluated as a model of host-pathogen interactions, evidencing that the adenovirus found has possibly coevolved with P. brasilianus. However, because the cormorant herpesviruses are highly related to poultry viruses, a recent transmission from poultry to wild birds is suggested. It is important the surveillance of pathogens that affect wildlife, due to their role as a reservoir of diseases that may affect the ecosystem and animal health, in domestic and wild animals. Therefore, more studies of both viruses detected, in terms of prevalence, virulence, pathogenesis and effects on the immune system of the host P. brasilianus, as well as their origin, distribution and range of hosts are required. Key words: adenovirus, herpesvirus, phylogenetics, cormorant. Fuente de financiamiento: Proyecto FONDECYT 11130305 3 3. INTRODUCCIÓN Las interacciones hospedero - patógeno han sido objeto de estudio debido a su importancia en el entendimiento de los diversos mecanismos recíprocos de adaptación y persistencia. Algunos ejemplos de estas interacciones entre antagonistas son la infectividad del patógeno y la resistencia del hospedero, las vías de ingreso de patógenos y los mecanismos de defensa de los hospederos, la capacidad del patógeno para evadir o suprimir el sistema inmune y la habilidad del hospedero de montar una respuesta inmune y eliminar una infección (Woolhouse y col 2002). El proceso recíproco de cambios genéticos adaptativos entre dos o más especies que interactúan se denomina coevolución. Otra definición más descriptiva de coevolución es la evolución que implica una serie de cambios recíprocos en dos o más poblaciones que no se reproducen entre sí, los cuales tienen una estrecha relación ecológica y actúan como agentes de la selección natural una respecto de la otra (Thompson 1994). A nivel de patógeno-hospedero, la coevolución es de particular interés para el área biomédica ya que puede ser estudiada en términos de caracteres fenotípicos pareados (ej. infectividad- resistencia), en términos de moléculas que interactúan entre el hospedero y el patógeno (ej. Receptores de las células T y antígenos bacterianos) o directamente en términos de genes o secuencias de nucleótidos (ej. Genes del MHC del hospedero y de proteínas de superficie virales) (Woolhouse y col 2002). Es más, estudios experimentales demuestran que una consecuencia de dichos mecanismos coevolutivos entre hospederos y patógenos es la diversificación genética, contribuyendo al mantenimiento de la diversidad biológica (Brockhurst y Koskella 2013). Una evidencia del proceso coevolutivo es el fenómeno de adaptación local, donde la compatibilidad, que en el sistema hospedero-patógeno podría ser la infectividad, entre poblaciones simpátricas es más alta que en poblaciones alopátricas (Woolhouse y col 2002). Esta adaptación se produce incluso en ausencia de una respuesta de evasión concreta del hospedero (ej. en casos de patógenos con baja virulencia) ya que el patógeno se adapta a las características específicas de éste, disminuyendo su rango de hospederos potenciales (Antonovics y col 2013), es decir, aumentando su especificidad por determinados hospederos. Los virus ADN de doble hebra presentan tasas de mutación bajas (ej. Herpes simple tipo 1 con 1.8 x 10–8 mutaciones por nucleótidos por replicación genómica (mut/nt/rep)) debido a que poseen una ADN polimerasa de alta fidelidad en comparación a virus ADN de hebra simple o virus ARN (ej. el fago ႷX174 de ADN de hebra simple presenta 7.4 x 10-6 mut/nt/rep y el fago Qβ1 de ARN 1.5 x 10–3 mut/nt/rep). Además, los virus ADN presentan genomas largos (ej. hasta 1,260 kilo pares de bases (kpb) en Megavirus chilensis), que junto a las bajas tasas de mutación producen que los cambios evolutivos sean más lentos que en los virus ARN y ADN de hebra simple, manteniendo señales filogenéticas por más tiempo que otros virus (Duffy y col 2008). Estas bajas tasas de mutación se asemejan a las que se estiman en los hospederos (ej. En humanos de 1.3 a 2.7 x 10–8 mut/nt/generación (Nachman y Crowell, 2000)) posibilitando la codivergencia y el proceso coevolutivo entre estos virus y sus hospederos. 4 Dentro de los virus ADN de doble hebra, se encuentran las familias Adenoviridae y Herpesviridae, se caracterizan por tener un rango de hospederos restringido y baja patogenicidad al infectar, sugiriendo procesos de adaptación local a hospederos específicos con los que han coevolucionado por prolongados periodos de tiempo (Harrach y col 2011, Pellet y col 2011).
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