Continuous exposure to infectious pancreatic necrosis virus during early life stages of rainbow trout, Oncorhynchus mykiss (Walbaum).

Rainbow trout, Oncorhynchus mykiss (Walbaum), were exposed continuously to infectious pancreatic necrosis virus (IPNV) at 0, 10(1), 10(3) or 10(5) plaque forming units (pfu) L(-1) of water to estimate the effects of chronic IPNV exposure on early life stages. Fish density averaged 35 fish L(-1) (low density) or 140 fish L(-1) (high density), and the tank flow rate was 250 mL(-1) min. Virus exposure began at 6 days before hatch and continued until fish were 44 days old. Cumulative per cent mortality, analysis of survival and hazard functions, and discrete-time event analysis were used to explore the patterns of survival and mortality. In eggs and fish exposed to IPNV, mortality significantly greater than in the 0 pfu L(-1) exposure did not occur until IPNV concentration was 10(5) pfu L(-1) at low fish density and 10(3) pfu IPNV L(-1) at high fish density. These results suggest that in the natural aquatic environment, where rainbow trout densities are likely to be considerably lower than in this study, mortality resulting from infection with IPNV will very likely not occur when ambient concentrations of virus are < or =10(3) pfu IPNV L(-1). In aquaculture rearing units, trout density is likely to be as high or higher than the densities used in this study. Therefore, continuous inputs of virus at concentrations greater than 10(1) pfu L(-1) may result in IPN epidemics in aquaculture facilities.

Development of a subunit vaccine for infectious pancreatic necrosis virus using a baculovirus insect/larvae system.

Various attempts to develop a vaccine against infectious pancreatic necrosis virus (IPNV) have not yielded consistent results. Thus, at present, no commercial vaccine is available that can be used with confidence to immunize fry of salmon and trout. We generated a cDNA clone of the large genome segment A of an IPNV Sp strain and expressed all structural protein genes in insect cells and larvae using a baculovirus expression system. Green fluorescent protein was also coexpressed as a reporter molecule. High yields of IPNV proteins were obtained and the structural proteins self assembled to form virus-like particles (VLPs). We tested the immunogenicity of the putative VLP antigen in immersion vaccine experiments (two concentrations) in rainbow trout (Oncorhynchus mykiss) fry, and by intraperitoneal immunisation of Atlantic salmon (Salmo salar) pre-smolts using an oil adjuvant formulation. Rainbow trout were challenged by immersion using either the Sp or the VR-299 strain of IPNV two or three weeks post-vaccination, while Atlantic salmon were bath challenged with Sp strain after two months, after parr-smolt transformation. In the rainbow trout fry challenged two weeks post-immunization, cumulative mortality rates three weeks post challenge were 14 % in the fry that had received the highest dose versus 8 % in the control groups. No indication of protection was seen in repeated trials using a lower dose of antigen and challenge three weeks post-immunisation. The cumulative mortality rate of intraperitoneally immunised Atlantic salmon post-smolts four weeks post challenge was lower (56%) than in the control fish (77%), showing a dose-response pattern.

Experimental infectious pancreatic necrosis infections: propagative or point-source epidemic?

Experimentally initiated epidemics of infectious pancreatic necrosis in rainbow-trout fry were analyzed using a modification of the standard mathematical model for a simple propagative epidemic. Contrary to expectations, the value of the transmission parameter (beta) was inversely related to initial density of susceptible hosts. This anomaly can be explained if we assume that the experimental epidemics were point-source rather than propagative epidemics. The implications of this conclusion for modeling experimental and natural epidemics are discussed.

Detection and identification of aquatic birnaviruses by PCR assay.

A reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed for the detection and identification of aquatic birnaviruses. The four sets of primers (PrA, PrB, PrC, and PrD) that we used are specific for regions of cDNA coded by genome segment A of aquatic birnaviruses. PrA identifies a large fragment (1,180 bp) within the pVP2-coding region, and PrB identifies a 524-bp fragment within the sequence amplified by PrA. Primer set PrC frames a genome fragment (339 bp) within the NS-VP3-coding region, and PrD identifies a 174-bp sequence within the fragment identified by PrC. PrB and PrD amplified cDNAs from all nine recognized serotypes of aquatic birnavirus serogroup A as well as the N1 isolate that may represent a 10th serotype. These results indicate that these three primer sequences are highly conserved and can be used in PCR assays for group identification of these viruses. PrA routinely produced amplification products from eight serotypes but exhibited variable results with one serotype, and primer PrC identified 6 of the 11 virus isolates tested. The qualitative sensitivity of the RT-PCR assay was evaluated by comparison of the results with those of cell culture isolation assays. With the exception of one sample, the RT-PCR assay with primer PrD was as accurate as cell culture isolation for detecting virus in kidney and spleen tissues from naturally infected, asymptomatic carrier fish. These results indicate that the RT-PCR assay can be a rapid and reliable substitute for cell culture methods for the detection of aquatic birnaviruses.

Immunoblot assay: a rapid and sensitive method for identification of salmonid fish viruses.

An immunoblot assay was used to identify the viruses of infectious pancreatic necrosis, infectious hematopoietic necrosis, and viral hemorrhagic septicemia. Viral antigen in infected cell culture supernatant was adsorbed onto nitrocellulose membrane or Whatman 541 filter paper and detected by enzyme-linked immunosorbent assay techniques. The immunoblot assay took less than 4 hr to perform and required no special instrumentation. Assays using cell culture supernatant fluids showed immunoblot sensitivity was 10(5) - 10(6) PFU/ml. Assay sensitivity, determined using purified virus, is 0.85-4.0 ng of viral antigen. The immunoblot assay was used to detect and identify virus in cell culture fluids.

Effect of exogenous corticosteroids on circulating virus and neutralizing antibodies in striped bass (Morone saxatilis) infected with infectious pancreatic necrosis virus.

Corticosteroids have been reported to induce immunosuppression in fish exposed to many types of bacterial antigens. We document a similar phenomenon in fish exposed to infectious pancreatic necrosis virus (IPNV). Fingerling striped bass that were injected with the steroid triamcinolone acetonide (100 mg/kg body weight) 24 hours before receiving intraperitoneal inoculation with IPNV became viremic 3 days post inoculation (dpi) and virus was still detected in the buffy coat cells 14 dpi. In contrast, viremia could not be detected after 7 dpi in fish that received virus but not steroids. Circulating virus neutralizing antibodies were first detected in steroid treated fish at 10 dpi compared to 7 dpi for the virus injected fish and titers were consistently lower in the steroid group. Steroid treatment of chronic IPNV-carriers did not induce detectable viremia nor alter circulating antibody levels in chronic IPNV-carriers. None of the striped bass demonstrated clinical signs of viral disease.

Viral diseases of fish: first report of carp pox in golden ide (Leuciscus idus) in North America.

Carp pox, a putative viral disease exotic to North America, occurred in golden ide 1 yr after the fish were imported into the United States from the Federal Republic of Germany. The raised, white, plaque-like lesions, which occurred on about 5% of the fish, healed spontaneously and caused no mortality. Electron micrographs showed herpesvirus-like particles associated with lesion specimens; however, no infectious viruses were detected in tests with seven warmwater fish cell lines.

Virion RNA polymerases of two salmonid rhabdoviruses.

RNA-dependent RNA polymerases were found to be associated with two salmonid rhabdoviruses: infectious hematopoietic necrosis (IHN) virus and the virus of hemorrhagic septicemia (VHS). The protein composition of these rhabdoviruses closely resembles that of rabies virus rather than that of vesicular stomatitis virus (McAllister and Wagner, 1975). The optimal temperature for in vitro transcription was found to be approximately 18 degrees C for IHN virus and approximately 15 degrees for VHS,, closely approximating optimal temperatures for growth of these viruses in salmonid cells. Unlike vesicular stomatitis virus, manganese ion (1 mM) could be used as a divalent cation substitute for magnesium ion (5 mM). The in vitro transcription products of IHN and VHS viruses hybridized completely to the homologous genome but not at all to the heterologous genome.

Comparative membrane microviscosity of fish and mammalian rhabdoviruses studied by fluorescence depolarization.

The microviscosity of the hydrophobic region of the membrane of infectious hematopoietic necrosis virus was determined using fluorescence depolarization analysis of the probe 1,6-diphenyl-1,3,5-hexatriene and was found to be much lower at 37 C than that of another rhabdovirus, vesicular stomatitis virus. However, the microviscosity of this fish virus at 18 C, the temperature at which it was grown, corresponded to the microviscosity of vesicular stomatitis virus at 37 C. Data obtained with the fish virus host cell (chinook salmon embryo cells) grown at 18 C suggest that its membranes have a lower microviscosity than either L-929 or BHK-21 cells (the vesicular stomatitis virus host cells) grown at 37 C.

Differential inhibition of host protein synthesis in L cells infected with RNA - temperature-sensitive mutants of vesicular stomatitis virus.

The response of mouse L cells to infection with wild-type (wt) and temperature-sensitive (ts) mutants of vesicular stomatitis virus was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to delineate the synthesis of host cell and viral proteins. Experiments utilized transcriptase mutants of complementation group I (ts114 and ts13), a group IV mutant (ts44) that is restricted in total RNA synthesis (RNA-1) but not in primary transcription, and a group II mutant (ts52) variably restricted in RNA synthesis (RNA +/-). L cells infected with ts mutants at permissive temperature exhibited the wt response of progressive inhibition of host cell protein synthesis accompanied by accumulation of all five viral proteins. Mutant ts44 (IV) also switched off cell protein synthesis at restrictive temperature and accumulated all five viral proteins, but with disproportionate ratios of N and G proteins. At restrictive temperature, cells infected with group I ts mutants failed to accumulate any viral protein and did not exhibit significant reduction in host cell protein synthesis. These data suggest that vesicular stomatitis virus inhibits cell protein synthesis at a stage of viral infection after transcription and possibly translation but preceding replication of progeny viral RNA.

Structural proteins of two salmonid rhabdoviruses.

Purified infectious hematopoietic necrosis (IHN) virus and the virus of haemorrhagic septicaemia (VHS) (Egtved virus) each contain five structural proteins which were designated L, G, N, M-1, and M-2. The IHN viral polypeptides have molecular weights estimated to be 157,000, 72,000, 40,000, 25,000 and 20,000, respectively, whereas those of VHS viral polypeptides are estimated to be 157,000 74,000, 41,000, 21,500, and 19,000, respectively. The carbohydrate composition of the glycoprotein (G) was confirmed by demonstrating selective incorporation of [3H]glucosamine into the designated G protein of both viruses. Phosphoproteins were identified by incorporation of [32P]orthophosphate into the N and M-1 proteins of IHN virus and into the N protein of VHS virus. The glycoprotein of each virus was selectively solubilized by treatment with Triton X-100 in low salt buffer, whereas the M-1, and M-2 proteins along with the G protein were solubilized by Ttition X-100 in 0.43 M NaCl. The protein composition of the salmonid rhabdoviruses resembles that of the rabies virus group more closely than the vesicular stomatitis virus group.