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Caracterización Molecular Del Virus Culex Flavivirus Y Estudios De Co- Infección Con El Virus West Nile Con Otros Flavivirus Aislados En Mosquitos Del Género Culex

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Caracterización Molecular Del Virus Culex Flavivirus Y Estudios De Co- Infección Con El Virus West Nile Con Otros Flavivirus Aislados En Mosquitos Del Género Culex Tesis Doctoral Caracterización molecular del virus Culex flavivirus y estudios de co- infección con el virus West Nile con otros flavivirus aislados en mosquitos del género Culex Goenaga, Silvina 2016-09-05 Este documento forma parte de la colección de tesis doctorales y de maestría de la Biblioteca Central Dr. Luis Federico Leloir, disponible en digital.bl.fcen.uba.ar. Su utilización debe ser acompañada por la cita bibliográfica con reconocimiento de la fuente. This document is part of the doctoral theses collection of the Central Library Dr. Luis Federico Leloir, available in digital.bl.fcen.uba.ar. It should be used accompanied by the corresponding citation acknowledging the source. Cita tipo APA: Goenaga, Silvina. (2016-09-05). Caracterización molecular del virus Culex flavivirus y estudios de co-infección con el virus West Nile con otros flavivirus aislados en mosquitos del género Culex. Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Cita tipo Chicago: Goenaga, Silvina. "Caracterización molecular del virus Culex flavivirus y estudios de co- infección con el virus West Nile con otros flavivirus aislados en mosquitos del género Culex". Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. 2016-09-05. Dirección: Biblioteca Central Dr. Luis F. Leloir, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires. Contacto: [email protected] Intendente Güiraldes 2160 - C1428EGA - Tel. (++54 +11) 4789-9293 UNIVERSIDAD DE BUENOS AIRES FACULTAD DE CIENCIAS EXACTAS Y NATURALES DEPARTAMENTO DE QUÍMICA BIOLÓGICA “Caracterización molecular del virus Culex flavivirus y estudios de co-infección con el virus West Nile con otros flavivirus aislados en mosquitos del género Culex” Tesis presentada para optar al título de Doctor de la Universidad de Buenos Aires en el área Química Biológica SILVINA GOENAGA Director de Tesis: Dr. Oscar Daniel Salomón Director Asistente: Dra. Silvana del Carmen Levis Consejero de Estudio: Dra. Laura Alché Lugar de trabajo Instituto Nacional de Enfermedades Virales Humanas “Dr. Julio I. Maiztegui”. Pergamino, Buenos Aires. Buenos Aires, 2016 Fecha de defensa: 5 de septiembre de 2016 i “Caracterización molecular del virus Culex flavivirus y estudios de co-infección con el virus West Nile con otros flavivirus aislados en mosquitos del género Culex” Resumen Las enfermedades transmitidas por vectores representan un importante problema de salud pública, la dinámica de tales enfermedades es compleja, ya que diversos factores pueden contribuir a la emergencia de brotes. En esta Tesis doctoral, inicialmente se analizó la presencia de flavivirus en poblaciones de mosquitos de distintas regiones de la Argentina. Luego, se caracterizó una cepa de Culex flavivirus (CxFV), un flavivirus específico de insectos, y se desarrollaron ensayos in vitro e in vivo para evaluar el fenómeno exclusión por superinfección entre CxFV y el virus Nhumirin (NHUV) de reciente descubrimiento, con el virus West Nile (WNV). Los resultados demostraron una alta prevalencia de CxFV en distintas especies de mosquitos del género Culex, y además, señalan la presencia de dos cepas de flavivirus nuevos, aún no descriptos, lo cual demuestra la diversidad de flavivirus que están asociados a poblaciones de mosquitos en la naturaleza. A su vez, se demostró la incapacidad de CxFV para replicar en ratón lactante y en cultivos celulares de vertebrados. Los ensayos de co-infección in vitro entre CxFV y WNV mostraron un efecto inhibitorio por parte de CxFV, sólo cuando la multiplicidad de infección fue mayor para CxFV que para WNV. Se observó, también en ensayos in vitro, un efecto inhibitorio en la replicación de dos cepas de WNV por parte de NHUV. Los ensayos in vivo en Culex pipiens y Culex quinquefasciatus, señalaron una disminución en la tasa de transmisión de WNV en ejemplares Culex quinquefasciatus previamente infectados con NHUV. Como conclusión se remarca la interacción que existe entre flavivirus y se sugiere que la misma podría interferir en la transmisión de flavivirus patógenos como WNV en la naturaleza. Este trabajo provee información básica para estudios futuros de la dinámica de algunas enfermedades transmitidas por vectores en mosquitos y para la exploración de estrategias potenciales de control. Palabaras claves: Argentina, mosquitos, flavivirus, exclusión por superinfección, virus West Nile, virus Culex flavivirus. 1 “Molecular characterization of Culex flavivirus and co-infection studies with West Nile virus and other flaviviruses isolated from Culex mosquitoes” Summary Vector-borne diseases remain a major public health concern as new diseases are emerging and previously controlled diseases are now resurging. Many factors contribute to the spatial and temporal dynamics of disease outbreaks. In this Thesis we analyzed flavivirus infection in mosquitoes from different regions of Argentina and a molecular characterization of one strain of CxFV was done. To explore potential interactions between flaviviruses, in vitro and in vivo studies were performed. Superinfection exclusion assays were performed between WNV and Nhumirin (NHUV), a new flavivirus isolated in Brazil, more closely genetically related to Mosquito borne flaviviruses than insect-specific flaviviruses, and between WNV and CxFV. Results show a high prevalence of CxFV in different species of Culex genera mosquitoes. Besides, two new flavivirus strains were isolated from mosquitoes which indicate the diversity of flaviviruses that could be found in mosquitos’ population in nature. CxFV was unable to replicate in vertebrate cells and in suckling mouse brain. Co-infection in vitro assays between WNV and CxFV evidenced an inhibitory effect on WNV only when multiplicity of infection was high. An inhibitory effect was determinate by NHUV in the replication of two strains of WNV, in two cell lines of Aedes albopictus. In vivo assays performed in Culex pipiens and Culex quinquefasciatus mosquitoes, showed a 40% reduction in WNV transmissibility in mosquitoes co- infected with NHUV when compared with the WNV control group. As conclusions, it is highlights the potential interaction between flaviviruses and suggest that it could be interfering in the transmission of medical importance flaviviruses in nature. Moreover, these data indicated the potential modulatory effect of certain pre-existing flaviviruses on the capacity for establishment of WNV superinfection in mosquitoes and highlights a potential method for blocking mosquito infection as a public health. 2 Los resultados de este trabajo han sido y serán publicados en los siguientes artículos con referato: Goenaga, S., Kenney, J. L., Duggal, N. K., Delorey, M., Ebel, G. D., Zhang, B.,Levis S.C., ENria D.A., and Brault, A. C. (2015). Potential for Co-Infection of a Mosquito-Specific Flavivirus, Nhumirim Virus, to Block West Nile Virus Transmission in Mosquitoes. Viruses, 7(11), 5801–5812. http://doi.org/10.3390/v7112911 Goenaga, S., Fabbri, C.M., García, J.B., Rondán, J.C., Gardenal, N., Calderón, G.E., Enria,D.A., Levis, S.M., (2014). New strains of Culex flavivirus isolated in Argentina. J. Med. Entomol. 51 (4), 900–906. Goenaga, S., Romo H., Levis S., Enria D., and Brault A. Characterization of a novel flavivirus isolated in mosquitoes from Argentina (Manuscrito). Goenaga S., Enria D. and Levis S. Superinfection exclusion studies between West Nile virus and Culex flavivirus strains from Argentina (Manuscrito). Los resultados de este trabajo de Tesis han sido publicados en las siguientes reuniones científicas internacionales: Goenaga S.; Enria D. and Levis S. Co-infection studies between West Nile virus and Culex flavivirus determine an inhibitory effect on West Nile virus infection. 2016. 65th Annual Meeting of American Society of Tropical Medicine and Hygiene. Atlanta, Georgia USA. Goenaga S., Kenney J.L., Duggal N., Levis S., Enria D. and Brault A.C . Potential for co-infection of novel mosquito-specific flaviviruses to block human flaviviral disease agent infection and/or transmission in mosquitoes. 2015. 64th Annual Meeting of American Society of Tropical Medicine and Hygiene. Philadelphia, Pennsylvania USA. Goenaga S., Kenney J.L., Duggal N., Levis S., Enria D. and Brault A.C. Inhibición de la replicación del virus West Nile por infección previa con otro flavivirus en cultivos celulares. 2015. XI Congreso Argentino de Virología y II Congreso Latinoamericano de Virología.Buenos Aires, Argentina. Los resultados de este trabajo de Tesis han sido publicados en las siguientes reuniones científicas nacionales: Fabbri C.; Morales, M.A.; Luppo V.C.; Cappato Berger F.; Salanitro B.; Manrique M.; Fierro L.; Goenaga, S.; Enría, D.A. and Levis, S. 2011. Brote de Encefalitis de San Luis en la Provincia de San Juan, Argentina.X Congreso Argentino de Virología Buenos Aires, Argentina. Goenaga, S.; García, J.; Morales, A.; Levis, S.; Enria, D. y Calderón, G. Detección de RNA de Flavivirus en poblaciones de mosquitos capturados en las provincias de Corrientes y Chaco. 2010. XIII Simposio Internacional sobre Control Epidemiológico de Enfermedades Trasmitidas por Vectores. Buenos Aires.Argentina. 3 Agradecimientos En primer lugar les agradezco a mis directores Daniel y Silvana, por confiar en mí, por sus enseñanzas, sus consejos, por tanta paciencia y tiempo dedicado a esta Tesis. A Delia y Silvana, por todo el apoyo que me han brindado desde mis inicios en el INEVH. Desde el primer momento sentí un apoyo incondicional y una gran valoración de mi trabajo por parte de ustedes, siempre fueron un estímulo y ejemplo para mí. Gracias por su empuje, por valorar mi capacidad y por confiar en mí. Mi formación se debe, en gran parte, a ustedes. A todo el personal del INEVH por su paciencia, por su ayuda y compañerismo, y por brindar todo su conocimiento. Sobretodo quiero agradecer a Maria Esther y Laura que colaboraron con los cultivos de pollo y a Marcelo por su ayuda en la edición de esta Tesis. Particularmente, quiero agradecer a todo el personal del Departamento de Investigación del INEVH, sin todos ustedes no hubiera podido culminar esta etapa. Gracias por hacerme reír todos los días, por hacer cada día divertido y por escuchar mis ideas, por más locas que sonaran.
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