No description available.

No description available.

No description available.

Recently, viral hemorrhagic septicemia virus (VHSV IVb) was associated with several walleye (Sander vitreus) mortality events in the Great Lakes. To examine the effects of route, strain-variation and temperature, walleye were experimentally infected with VHSV IVb using intraperitoneal (i.p.)-injection (102-108 pfu/fish) and immersion (w.; 1.4 x 107 virions mL-1). Walleye were relatively resistant to experimental infection with VHSV IVb, regardless of route or water temperature. High cumulative mortality (64-100%) and severe gross lesions associated with VHSV-IVb infection were only evident in fish i.p.-injected with 108 pfu at 12°C, which had mild to moderate, multifocal necrosis of several tissues including the gill and heart. There were significant differences in mortality between four walleye strains following i.p.-infection. Viral antigen was found in both i.p. and w.-exposed walleye using immunohistochemistry, mostly within the gill and skin epithelium of w.-exposed fish. VHSV IVb was detected in walleye tissues from 6-21 d post-infection using RT-qPCR. / Great Lakes Fisheries Commission and NSERC

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, 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 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.

No description available.

No description available.

Methods of studying the interaction between fish viruses with inanimate surfaces were developed and used to explore several variables. Viral hemorrhagic septicemia virus (VHSV) was used as the model virus. The EPC cell line, which is now known to be from Fathead Minnow, was used to detect the virus through the development of cytopathic effect (CPE); this allowed virus levels to be titrated and expressed as tissue culture infectious dose (TCID50). The labour and tedium of scoring hundreds of wells for CPE was overcome through the use of the fluorescent indicator dye, Alamar Blue, which is reduced by living cells and not by dead cells to yield a fluorescent product that can be measured as relative fluorescence units (RFUs) with a fluorescent microwell plate reader. Microsoft Excel 2007 was used to compare RFU values of wells and to create a scoring template in the computer program that allowed for easy summation of the total number of wells with infectious virus. With this system and as well as with conventional scoring, surface-virus interactions were studied in the following general way. Surfaces were incubated with a solution (L-15 with 2% fetal bovine serum or FBS) of virus, rinsed, and then incubated under various conditions, either wet or dry, before being evaluated for infectious virus. The transfer of viruses through their elution from surfaces is termed elution transfer (ET) and was investigated in two ways: agitated elution and static elution. Agitated elution was done through the repeated action of pipetting up and down on either glass or plastic surfaces with different eluting solutions. The best eluting solution was 2% FBS/L-15 and the worst was tissue culture grade water. Regardless of the eluting solution, no infectious virus could be removed by agitated elution from glass Petri dishes. Static elution was demonstrated through a two-compartment culture system linked by 3.0 m pores. L-15 with 2% FBS eluted VHSV from the surface of the top chamber to infect cells in the bottom chamber and from the surface of the bottom chamber to infect cells in the top chamber. The ability of different objects to carry infectious VHSV to a new culture vessel was investigated in a protocol termed object-associated transfer (OAT). The objects were incubated with VHSV, rinsed, and then incubated wet or dry for various periods before being transferred to EPC cultures. After up to ten days of wet incubation, pieces of glass, fishing line, plastic water bottle, and pop can were able to transfer infectious virus. In contrast, when the same objects were incubated dry, they were able to transfer VHSV after only one day of drying. Fishing hooks kept wet for a day were able to transfer VHSV but dry hooks had no capacity to transfer infectious virus. A third experimental protocol was used to detect infectious viruses on surfaces and involves the surface to cell transfer (SCT) of viruses. For this protocol, EPC cells were plated directly onto plastic or glass surfaces that previously had been exposed to virus, rinsed, and incubated dry or wet at various temperatures for up to 15 days. After 15 days being kept dry at 4 °C, infectious VHSV was still found to be present on both glass and plastic surfaces. At 14 °C and room temperature, the virus was found to survive longer on plastic than on glass, and at 26 °C both surfaces retained infectious VHSV for only one day of being dry. Survival time on plastic surfaces at different temperatures was compared for wet and dry incubation. VHSV kept on plastic surface in a dry state was more susceptible to temperature inactivation, with inactivation of the virus being detected clearly after 1 day 37 °C, 10 days at 26 °C, and 15 days at room temperature.

Methods of studying the interaction between fish viruses with inanimate surfaces were developed and used to explore several variables. Viral hemorrhagic septicemia virus (VHSV) was used as the model virus. The EPC cell line, which is now known to be from Fathead Minnow, was used to detect the virus through the development of cytopathic effect (CPE); this allowed virus levels to be titrated and expressed as tissue culture infectious dose (TCID50). The labour and tedium of scoring hundreds of wells for CPE was overcome through the use of the fluorescent indicator dye, Alamar Blue, which is reduced by living cells and not by dead cells to yield a fluorescent product that can be measured as relative fluorescence units (RFUs) with a fluorescent microwell plate reader. Microsoft Excel 2007 was used to compare RFU values of wells and to create a scoring template in the computer program that allowed for easy summation of the total number of wells with infectious virus. With this system and as well as with conventional scoring, surface-virus interactions were studied in the following general way. Surfaces were incubated with a solution (L-15 with 2% fetal bovine serum or FBS) of virus, rinsed, and then incubated under various conditions, either wet or dry, before being evaluated for infectious virus. The transfer of viruses through their elution from surfaces is termed elution transfer (ET) and was investigated in two ways: agitated elution and static elution. Agitated elution was done through the repeated action of pipetting up and down on either glass or plastic surfaces with different eluting solutions. The best eluting solution was 2% FBS/L-15 and the worst was tissue culture grade water. Regardless of the eluting solution, no infectious virus could be removed by agitated elution from glass Petri dishes. Static elution was demonstrated through a two-compartment culture system linked by 3.0 m pores. L-15 with 2% FBS eluted VHSV from the surface of the top chamber to infect cells in the bottom chamber and from the surface of the bottom chamber to infect cells in the top chamber. The ability of different objects to carry infectious VHSV to a new culture vessel was investigated in a protocol termed object-associated transfer (OAT). The objects were incubated with VHSV, rinsed, and then incubated wet or dry for various periods before being transferred to EPC cultures. After up to ten days of wet incubation, pieces of glass, fishing line, plastic water bottle, and pop can were able to transfer infectious virus. In contrast, when the same objects were incubated dry, they were able to transfer VHSV after only one day of drying. Fishing hooks kept wet for a day were able to transfer VHSV but dry hooks had no capacity to transfer infectious virus. A third experimental protocol was used to detect infectious viruses on surfaces and involves the surface to cell transfer (SCT) of viruses. For this protocol, EPC cells were plated directly onto plastic or glass surfaces that previously had been exposed to virus, rinsed, and incubated dry or wet at various temperatures for up to 15 days. After 15 days being kept dry at 4 °C, infectious VHSV was still found to be present on both glass and plastic surfaces. At 14 °C and room temperature, the virus was found to survive longer on plastic than on glass, and at 26 °C both surfaces retained infectious VHSV for only one day of being dry. Survival time on plastic surfaces at different temperatures was compared for wet and dry incubation. VHSV kept on plastic surface in a dry state was more susceptible to temperature inactivation, with inactivation of the virus being detected clearly after 1 day 37 °C, 10 days at 26 °C, and 15 days at room temperature.

No description available.