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Using Cell Lines to Study Factors Affecting Transmission of Fish Viruses

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Using cell lines to study factors affecting transmission of fish viruses by Phuc Hoang Pham A thesis presented to the University of Waterloo in fulfillment of the thesis requirement for the degree of Doctor of Philosophy in Biology Waterloo, Ontario, Canada, 2014 ©Phuc Hoang Pham 2014 AUTHOR'S DECLARATION I hereby declare that I am the sole author of this thesis. This is a true copy of the thesis, including any required final revisions, as accepted by my examiners. I understand that my thesis may be made electronically available to the public. ii ABSTRACT Factors that can influence the transmission of aquatic viruses in fish production facilities and natural environment are the immune defense of host species, the ability of viruses to infect host cells, and the environmental persistence of viruses. In this thesis, fish cell lines were used to study different aspects of these factors. Five viruses were used in this study: viral hemorrhagic septicemia virus (VHSV) from the Rhabdoviridae family; chum salmon reovirus (CSV) from the Reoviridae family; infectious pancreatic necrosis virus (IPNV) from the Birnaviridae family; and grouper iridovirus (GIV) and frog virus-3 (FV3) from the Iridoviridae family. The first factor affecting the transmission of fish viruses examined in this thesis is the immune defense of host species. In this work, infections of marine VHSV-IVa and freshwater VHSV-IVb were studied in two rainbow trout cell lines, RTgill-W1 from the gill epithelium, and RTS11 from spleen macrophages. RTgill-W1 produced infectious progeny of both VHSV-IVa and -IVb. However, VHSV-IVa was more infectious than IVb toward RTgill-W1: IVa caused cytopathic effects (CPE) at a lower viral titre, elicited CPE earlier, and yielded higher titres. By contrast, no CPE and no increase in viral titre were observed in RTS11 cultures infected with either genotype. Yet in RTS11 all six VHSV genes were expressed and antiviral genes, Mx2 and Mx3, were up regulated by VHSV-IVb and -IVa. However, replication appeared to terminate at the translational stage as viral N protein, presumably the most abundant of the VHSV proteins, was not detected in either infected RTS11 cultures. In RTgill-W1, Mx2 and Mx3 were up regulated to similar levels by both viral genotypes, while VHSV-IVa induced higher levels of IFN1, IFN2 and LGP2A than VHSV-IVb. The second part of the thesis examined the ability of two Ranaviruses, GIV and FV3, to infect non-host fish cells. This is referred to as cellular tropism and is one of many host-virus interaction events required to established successful infection in new organisms. Grouper iridovirus (GIV), belonging to the Ranavirus genus of the Iridoviridae family, was demonstrated to differentially express viral genes and induce apoptosis in three non-host fish cell lines rainbow trout monocyte/macrophage (RTS11), Chinook salmon embryon (CHSE-214) and fathead minnow Epithelioma papulosum cyprinii (EPC). These cells were challenged with GIV and virus entry into all three cell lines was confirmed by the expression of viral immediate early genes. The expression of the late major capsid protein gene was detected in CHSE-214 and EPC, iii but not in RTS11, suggesting an earlier termination in the viral replication cycle in RTS11. Approximately 12 h after infection with GIV, cell death was prominent in all three non-host cell lines. Death was later confirmed to be apoptosis by the presence of chromosomal DNA fragmentation and phosphatidylserine externalization. To determine whether apoptosis was protein related or gene expression related, the three cell lines were infected with heat-inactivated GIV and UV-treated GIV (GIVUV). The heat inactivation abolished apoptosis in all three cell lines, but each cell line responded differently to GIVUV. Relative to GIV, GIVUV caused no apoptosis in CHSE-214, decreased apoptosis in RTS11, and increased apoptosis in EPC. These results suggest that early GIV gene expression was needed for apoptosis in CHSE-214 but impeded apoptosis in EPC. At the cellular level, only EPC was a permissive host as EPC was the only cell line of the three capable of producing a moderate increase in virus titre. The three non- host cell lines present a good system for potentially identifying different components of GIV- induced apoptotic pathways in future studies. Rainbow trout are not highly susceptible to frog virus 3 (FV3) induced diseases, and had been suggested to be a potential carrier for the virus. To determine which rainbow trout cell types are permissive for FV3 and act as a potential source for virus replication in vivo, the ability of rainbow trout cell lines from gonads (RTG-2), skin (RTHDF), liver (RTL-W1), gills (RTgill- W1), intestine (RTgut-GC) and spleen (RTS11), and primary leukocyte cultures from peripheral blood (PBL) and head kidney (HKL) to support FV3 infection was examined. RTG-2 supported a moderate level of FV3 replication while viral replication in RTL-W1 was minimal. The rest of the cell lines did not support viral replication but all succumbed to the infection and were killed by FV3. Lymphocyte-like cells from PBL and HKL were not killed by FV3 while macrophage- like cells were. Most of the cell lines died by an apoptosis-independent mechanism, presumably necrosis, while the monocyte/macrophage cell line, RTS11, died by an apoptosis-dependent mechanism. In addition, neoplastic macrophage-like human U937 cell line, and T lymphocyte- like PEER cell line were also infected with FV3 to compare their response to that of rainbow trout immune cells. U937 cells were killed by FV3 in an apoptosis-dependent manner; however, PEER T cells did not die from FV3 infection, a result similar to the lymphocyte-like fraction of rainbow trout PBL and HKL. In summary, most rainbow trout cell lines do not support significant FV3 replication; furthermore, cells of the lymphocyte origin appeared refractory to iv FV3 induced cell death while those of macrophage origin underwent apoptosis as a response to FV3. The last factor affecting the transmission of aquatic viruses examined in this thesis is the persistence of viruses in the aquatic environment. Virus persistence is influenced by natural environmental factors such as temperature, pH, desiccation and salinity, but the often unexplored anthropogenic factors can play a role. Therefore, the focus of this section was on the effect of one particular anthropogenic substance, Corexit 9500, on the infectivity of aquatic viruses with different physical characteristics. The effect of Corexit 9500, a dispersant used to clean up oil spills, on invertebrates, lower vertebrates, birds and human health have been examined but there is a significant lack of study on the effect of this dispersant on aquatic viruses. In this study, the effect of Corexit 9500 on four aquatic viruses of different structural composition was examined. Corexit 9500 reduced the titre of the enveloped viral hemorrhagic septicemia virus (VHSV) at all concentrations (10% to 0.001%) examined. The titre of frog virus 3 (FV3), a virus with both enveloped and non-enveloped virions, was only reduced at the high Corexit 9500 concentrations (10% to 0.1%). Corexit 9500 was unable to reduce the titre of non-enveloped infectious pancreatic necrosis virus (IPNV), but enhanced the titre of chum salmon reovirus (CSV) by 2-4 logs. With the ability to inactivate enveloped viruses and possibly enhance some non-enveloped viruses, Corexit 9500 has the potential to alter the aquatic virosphere. v ACKNOWLEDGEMENTS To my family, I would like to thank you for your care and support throughout the years. Life has not been easy but it has definitely improved. To Dr. Niels Bols, I have many things to say thank you for. First and most important, thank you for your trust during one of the most challenging times in my life and allowing me to return to your lab those many years ago. I still have not forgotten. Second, thank you for being a great supervisor and mentor and for providing a comfortable environment for a young person to grow and mature. Finally, thank you for giving me the freedom to pursue my dream of travelling and seeing the world. Together with the rest of my experiences in your lab, my PhD has been very memorable. To my advisory committee, Drs. Christine Dupont, John Lumsden and Brian Dixon, thank you for being apart of my advisory committee and for offering your knowledge and advice. A special thanks to Dr. Christine Dupont for advising me through both my MSc and PhD! To all the members of the Bols lab with whom I have had the privilege to work with since 2006, I hope you all forgive me for not naming you all. Thank you for all the time you have shared with me, be it daily engagments or special events! I wish you the best on your future pursuits! vi DEDICATION This thesis is dedicated to my grandparents, parents and sister. vii TABLE OF CONTENTS AUTHOR'S DECLARATION ...................................................................................................................... ii ABSTRACT ................................................................................................................................................. iii ACKNOWLEDGEMENTS ......................................................................................................................... vi DEDICATION ............................................................................................................................................ vii LIST OF FIGURES ...................................................................................................................................
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    FACULTAD DE VETERINARIA GRADO DE VETERINARIA Curso 2015/16 Asignatura: MICROBIOLOGÍA E INMUNOLOGÍA DENOMINACIÓN DE LA ASIGNATURA Denominación: MICROBIOLOGÍA E INMUNOLOGÍA Código: 101463 Plan de estudios: GRADO DE VETERINARIA Curso: 2 Denominación del módulo al que pertenece: FORMACIÓN BÁSICA COMÚN Materia: MICROBIOLOGÍA E INMUNOLOGÍA Carácter: BASICA Duración: ANUAL Créditos ECTS: 12 Horas de trabajo presencial: 120 Porcentaje de presencialidad: 40% Horas de trabajo no presencial: 180 Plataforma virtual: UCO MOODLE DATOS DEL PROFESORADO __ Nombre: GARRIDO JIMENEZ, MARIA ROSARIO (Coordinador) Centro: Veterinaria Departamento: SANIDAD ANIMAL área: SANIDAD ANIMAL Ubicación del despacho: Edificio Sanidad Animal 3ª Planta E-Mail: [email protected] Teléfono: 957218718 _ Nombre: SERRANO DE BURGOS, ELENA (Coordinador) Centro: Veterinaria Departamento: SANIDAD ANIMAL área: SANIDAD ANIMAL Ubicación del despacho: Edificio Sanidad Animal 3ª Planta E-Mail: [email protected] Teléfono: 957218718 _ Nombre: HUERTA LORENZO, MARIA BELEN Centro: Veterianaria Departamento: SANIDAD ANIMAL área: SANIDAD ANIMAL Ubicación del despacho: Edificio Sanidad Animal 2ª Planta E-Mail: [email protected] Teléfono: 957212635 _ DATOS ESPECÍFICOS DE LA ASIGNATURA REQUISITOS Y RECOMENDACIONES Requisitos previos establecidos en el plan de estudios Ninguno Recomendaciones 1/11 MICROBIOLOGÍA E INMUNOLOGÍA Curso 2015/16 Se recomienda haber cursado las asignaturas de Biología Molecular Animal y Vegetal, Bioquímica, Citología e Histología y Anatomía Sistemática. COMPETENCIAS CE23 Estudio de los microorganismos que afectan a los animales y de aquellos que tengan una aplicación industrial, biotecnológica o ecológica. CE24 Bases y aplicaciones técnicas de la respuesta inmune. OBJETIVOS Los siguientes objetivos recogen las recomendaciones de la OIE para la formación del veterinario: 1. Abordar el concepto actual de Microbiología e Inmunología, la trascendencia de su evolución histórica y las líneas de interés o investigación futuras.
  • ICTV Code Assigned: 2011.001Ag Officers)

    ICTV Code Assigned: 2011.001Ag Officers)

    This form should be used for all taxonomic proposals. Please complete all those modules that are applicable (and then delete the unwanted sections). For guidance, see the notes written in blue and the separate document “Help with completing a taxonomic proposal” Please try to keep related proposals within a single document; you can copy the modules to create more than one genus within a new family, for example. MODULE 1: TITLE, AUTHORS, etc (to be completed by ICTV Code assigned: 2011.001aG officers) Short title: Change existing virus species names to non-Latinized binomials (e.g. 6 new species in the genus Zetavirus) Modules attached 1 2 3 4 5 (modules 1 and 9 are required) 6 7 8 9 Author(s) with e-mail address(es) of the proposer: Van Regenmortel Marc, [email protected] Burke Donald, [email protected] Calisher Charles, [email protected] Dietzgen Ralf, [email protected] Fauquet Claude, [email protected] Ghabrial Said, [email protected] Jahrling Peter, [email protected] Johnson Karl, [email protected] Holbrook Michael, [email protected] Horzinek Marian, [email protected] Keil Guenther, [email protected] Kuhn Jens, [email protected] Mahy Brian, [email protected] Martelli Giovanni, [email protected] Pringle Craig, [email protected] Rybicki Ed, [email protected] Skern Tim, [email protected] Tesh Robert, [email protected] Wahl-Jensen Victoria, [email protected] Walker Peter, [email protected] Weaver Scott, [email protected] List the ICTV study group(s) that have seen this proposal: A list of study groups and contacts is provided at http://www.ictvonline.org/subcommittees.asp .