Susceptibility of Pallid Sturgeon to viral hemorrhagic septicemia virus genotype IVb.

OBJECTIVE: Viral hemorrhagic septicemia virus (VHSV) is an aquatic rhabdovirus causing severe disease in freshwater and saltwater fish species. The susceptibility of endangered Pallid Sturgeon Scaphirhynchus albus to VHSV genotype IVb (VHSV-IVb) infection was investigated. METHODS: An in vitro assessment using two Pallid Sturgeon cell lines derived from skin and spleen tissue and in vivo evaluation of juvenile Pallid Sturgeon after exposure to VHSV-IVb were performed. RESULT: Plaque assay and RT-PCR results confirmed VHSV-IVb replication in Pallid Sturgeon cell lines. Sturgeon were also susceptible to VHSV-IVb infection after immersion and injection exposures during laboratory experiments. However, after widespread mortality occurred in all treatment groups, including negative control fish, it was determined that the Pallid Sturgeon stock fish were infected with Missouri River sturgeon iridovirus (MRSIV) prior to experimental challenge. Nevertheless, mortalities were equal or higher among VHSV-exposed fish than among negative controls (MRSIV infected), and histopathological assessments indicated reduced hematopoietic cells in spleen and kidney tissues and hemorrhage in the gastrointestinal organs only in fish from the VHSV treatment. CONCLUSION: These results indicate that Pallid Sturgeon is a susceptible host for VHSV-IVb, but the degree of pathogenicity was confounded by the underlying MRSIV infection. Research comparing susceptibility of specific pathogen-free and MRSIV-infected fish to VHSV-IVb is needed to accurately assess the vulnerability of Pallid Sturgeon to VHSV-IVb.

Effects of Stocking Density on Stress Response and Susceptibility to Infectious Hematopoietic Necrosis Virus in Rainbow Trout.

The goals of this study were to examine the effect of stocking density on the stress response and disease susceptibility in juvenile rainbow trout (Oncorhynchus mykiss). Fish were sorted into one of 2 stocking densities (high density "HD", 20-40 kg/m³) or (low density, "LD", 4-8 kg/m³) and 3 stress indices (cortisol levels in serum and water, and neutrophil: lymphocyte (N:L) ratios from blood smears) were measured at multiple time points over 21 d. Serum cortisol was significantly increased at 1 h in LD samples and at 14 d in HD samples. Water cortisol concentrations were significantly higher in LD tanks as compared with HD tanks on day 14. N:L ratios were significantly higher in HD tanks on day 14 as compared with LD tanks and with baseline. The effect of stocking density on mortality after exposure to infectious hematopoietic necrosis virus (IHNV) was compared between fish held in HD or LD conditions, with or without prior acclimation to the different density conditions. No significant differences in survival were found between HD and LD treatments or between acclimated and nonacclimated treatments. Cumulative results indicate that 1) 1 to 4 gram rainbow trout did not generally demonstrate significant differences in stress indices at the density conditions tested over a 21-d period, 2) independent differences were found in 3 stress indices at day 14 after sorting into LD and HD holding conditions; and 3) LD and HD stocking densities did not have a significant effect on mortality due to IHNV.

Consequences of Piscine orthoreovirus genotype 1 (PRV-1) infections in Chinook salmon (Oncorhynchus tshawytscha), coho salmon (O. kisutch) and rainbow trout (O. mykiss).

Piscine orthoreovirus genotype 1 (PRV-1) is the causative agent of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar L.). The virus has also been found in Pacific salmonids in western North America, raising concerns about the risk to native salmon and trout. Here, we report the results of laboratory challenges using juvenile Chinook salmon, coho salmon and rainbow trout injected with tissue homogenates from Atlantic salmon testing positive for PRV-1 or with control material. Fish were sampled at intervals to assess viral RNA transcript levels, haematocrit, erythrocytic inclusions and histopathology. While PRV-1 replicated in all species, there was negligible mortality in any group. We observed a few erythrocytic inclusion bodies in fish from the PRV-1-infected groups. At a few time points, haematocrits were significantly lower in the PRV-1-infected groups relative to controls, but in no case was anaemia noted. The most common histopathological finding was mild, focal myocarditis in both the non-infected controls and PRV-1-infected fish. All cardiac lesions were judged mild, and none were consistent with those of HSMI. Together, these results suggest all three species are susceptible to PRV-1 infection, but in no case did infection cause notable disease in these experiments.

Effect of growth rate on transcriptomic responses to immune stimulation in wild-type, domesticated, and GH-transgenic coho salmon.

BACKGROUND: Transcriptomic responses to immune stimulation were investigated in coho salmon (Oncorhynchus kisutch) with distinct growth phenotypes. Wild-type fish were contrasted to strains with accelerated growth arising either from selective breeding (i.e. domestication) or genetic modification. Such distinct routes to accelerated growth may have unique implications for relationships and/or trade-offs between growth and immune function. RESULTS: RNA-Seq was performed on liver and head kidney in four 'growth response groups' injected with polyinosinic-polycytidylic acid (Poly I:C; viral mimic), peptidoglycan (PGN; bacterial mimic) or PBS (control). These groups were: 1) 'W': wild-type, 2) 'TF': growth hormone (GH) transgenic salmon with ~ 3-fold higher growth-rate than W, 3) 'TR': GH transgenic fish ration restricted to possess a growth-rate equal to W, and 4) 'D': domesticated non-transgenic fish showing growth-rate intermediate to W and TF. D and TF showed a higher similarity in transcriptomic response compared to W and TR. Several immune genes showed constitutive expression differences among growth response groups, including perforin 1 and C-C motif chemokine 19-like. Among the affected immune pathways, most were up-regulated by Poly I:C and PGN. In response to PGN, the c-type lectin receptor signalling pathway responded uniquely in TF and TR. In response to stimulation with both immune mimics, TR responded more strongly than other groups. Further, group-specific pathway responses to PGN stimulation included NOD-like receptor signalling in W and platelet activation in TR. TF consistently showed the most attenuated immune response relative to W, and more DEGs were apparent in TR than TF and D relative to W, suggesting that a non-satiating ration coupled with elevated circulating GH levels may cause TR to possess enhanced immune capabilities. Alternatively, TF and D salmon are prevented from acquiring the same level of immune response as TR due to direction of energy to high overall somatic growth. Further study of the effects of ration restriction in growth-modified fishes is warranted. CONCLUSIONS: These findings improve our understanding of the pleiotropic effects of growth modification on the immunological responses of fish, revealing unique immune pathway responses depending on the mechanism of growth acceleration and nutritional availability.

Complete Genome Sequences of the Index Isolates of Two Genotypes of Pacific Salmon Paramyxovirus.

We report here the genome sequences of two index strains of Pacific salmon paramyxovirus isolated in 1982 and 1983 from adult salmon in Oregon. The isolates are most closely related to Atlantic salmon paramyxovirus, the type species of the genus Aquaparamyxovirus, but are sufficiently distinct to be considered two genotypes of a novel species.

Development and characterization of two cell lines from gills of Atlantic salmon.

Gill disease in Atlantic salmon, Salmo salar L., causes big losses in the salmon farming industry. Until now, tools to cultivate microorganisms causing gill disease and models to study the gill responses have been lacking. Here we describe the establishment and characterization of two cell lines from the gills of Atlantic salmon. Atlantic salmon gill cell ASG-10 consisted of cells staining for cytokeratin and e-cadherin and with desmosomes as seen by transmission electron microscopy suggesting the cells to be of epithelial origin. These structures were not seen in ASG-13. The cell lines have been maintained for almost 30 passages and both cell lines are fully susceptible to infection by infectious hematopoietic necrosis virus (IHNV), viral hemorrhagic septicemia virus (VHSV), infectious pancreatic necrosis virus (IPNV), Atlantic salmon reovirus TS (TSRV) and Pacific salmon paramyxovirus (PSPV). While infectious salmon anemia virus (ISAV) did not cause visible CPE, immunofluorescent staining revealed a sub-fraction of cells in both the ASG-10 and ASG-13 lines may be permissive to infection. ASG-10 is able to proliferate and migrate to close scratches in the monolayer within seven days in vitro contrary to ASG-13, which does not appear to do have the same proliferative and migratory ability. These cell lines will be useful in studies of gill diseases in Atlantic salmon and may represent an important contribution for alternatives to experimental animals and studies of epithelial-mesenchymal cell biology.

Isolation and characterization of the fall Chinook aquareovirus.

BACKGROUND: Salmon are paramount to the economy, ecology, and history of the Pacific Northwest. Viruses constitute one of the major threats to salmon health and well-being, with more than twenty known virus species that infect salmon. Here, we describe the isolation and characterization of the fall Chinook aquareovirus, a divergent member of the species Aquareovirus B within the family Reoviridae. METHODS: The virus was first found in 2014 as part of a routine adult broodstock screening program in which kidney and spleen tissue samples from healthy-appearing, adult fall Chinook salmon (Oncorhynchus tshawytscha) returning to a hatchery in Washington State produced cytopathic effects when inoculated onto a Chinook salmon embryo cell line (CHSE-214). The virus was not able to be confirmed by an RT-PCR assay using existing aquareovirus pan-species primers, and instead was identified by metagenomic next-generation sequencing. Metagenomic next-generation sequencing was used to recover the full genome and completed using 3' RACE. RESULTS: The genome of the fall Chinook aquareovirus contains 11 segments of double-stranded RNA totaling 23.3 kb, with each segment flanked by the canonical sequence termini found in the aquareoviruses. Sequence comparisons and a phylogenetic analysis revealed a nucleotide identity of 63.2% in the VP7 gene with the Green River Chinook virus, placing the new isolate in the species Aquareovirus B. A qRT-PCR assay was developed targeting the VP2, which showed rapid growth of the isolate during the initial 5 days in culture using CHSE-214 cells. CONCLUSIONS: This sequence represents the first complete genome of an Aquareovirus B species. Future studies will be required to understand the potential pathogenicity and epidemiology of the fall Chinook aquareovirus.

Pufferfish mortality associated with novel polar marine toxins in Hawaii.

Fish die-offs are important signals in tropical marine ecosystems. In 2010, a mass mortality of pufferfish in Hawaii (USA) was dominated by Arothron hispidus showing aberrant neurological behaviors. Using pathology, toxinology, and field surveys, we implicated a series of novel, polar, marine toxins as a likely cause of this mass mortality. Our findings are striking in that (1) a marine toxin was associated with a kill of a fish species that is itself toxic; (2) we provide a plausible mechanism to explain clinical signs of affected fish; and (3) this epizootic likely depleted puffer populations. Whilst our data are compelling, we did not synthesize the toxin de novo, and we were unable to categorically prove that the polar toxins caused mortality or that they were metabolites of an undefined parent compound. However, our approach does provide a template for marine fish kill investigations associated with marine toxins and inherent limitations of existing methods. Our study also highlights the need for more rapid and cost-effective tools to identify new marine toxins, particularly small, highly polar molecules.

Analytical validation of a reverse transcriptase droplet digital PCR (RT-ddPCR) for quantitative detection of infectious hematopoietic necrosis virus.

Infectious hematopoietic necrosis virus (IHNV) is an important pathogen of salmonid fishes. A validated universal reverse transcriptase quantitative PCR (RT-qPCR) assay that can quantify levels of IHNV in fish tissues has been previously reported. In the present study, we adapted the published set of IHNV primers and probe for use in a reverse-transcriptase droplet digital PCR (RT-ddPCR) assay for quantification of the virus in fish tissue samples. The RT-ddPCR and RT-qPCR assays detected 13 phylogenetically diverse IHNV strains, but neither assay produced detectable amplification when RNA from other fish viruses was used. The RT-ddPCR assay had a limit of detection (LOD) equating to 2.2 plaque forming units (PFU)/µl while the LOD for the RT-qPCR was 0.2 PFU/µl. Good agreement (69.4-100%) between assays was observed when used to detect IHNV RNA in cell culture supernatant and tissues from IHNV infected rainbow trout (Oncorhynchus mykiss) and arctic char (Salvelinus alpinus). Estimates of RNA copy number produced by the two assays were significantly correlated but the RT-qPCR consistently produced higher estimates than the RT-ddPCR. The analytical properties of the N gene RT-ddPCR test indicated that this method may be useful to assess IHNV RNA copy number for research and diagnostic purposes. Future work is needed to establish the within and between laboratory diagnostic performance of the RT-ddPCR assay.

Molecular characterization of a novel orthomyxovirus from rainbow and steelhead trout (Oncorhynchus mykiss).

A novel virus, rainbow trout orthomyxovirus (RbtOV), was isolated in 1997 and again in 2000 from commercially-reared rainbow trout (Oncorhynchus mykiss) in Idaho, USA. The virus grew optimally in the CHSE-214 cell line at 15°C producing a diffuse cytopathic effect; however, juvenile rainbow trout exposed to cell culture-grown virus showed no mortality or gross pathology. Electron microscopy of preparations from infected cell cultures revealed the presence of typical orthomyxovirus particles. The complete genome of RbtOV is comprised of eight linear segments of single-stranded, negative-sense RNA having highly conserved 5' and 3'-terminal nucleotide sequences. Another virus isolated in 2014 from steelhead trout (also O. mykiss) in Wisconsin, USA, and designated SttOV was found to have eight genome segments with high amino acid sequence identities (89-99%) to the corresponding genes of RbtOV, suggesting these new viruses are isolates of the same virus species and may be more widespread than currently realized. The new isolates had the same genome segment order and the closest pairwise amino acid sequence identities of 16-42% with Infectious salmon anemia virus (ISAV), the type species and currently only member of the genus Isavirus in the family Orthomyxoviridae. However, pairwise comparisons of the predicted amino acid sequences of the 10 RbtOV and SttOV proteins with orthologs from representatives of the established orthomyxoviral genera and a phylogenetic analysis using the PB1 protein showed that while RbtOV and SttOV clustered most closely with ISAV, they diverged sufficiently to merit consideration as representatives of a novel genus. A set of PCR primers was designed using conserved regions of the PB1 gene to produce amplicons that may be sequenced for identification of similar fish orthomyxoviruses in the future.

Identification of the major capsid protein of erythrocytic necrosis virus (ENV) and development of quantitative real-time PCR assays for quantification of ENV DNA.

Viral erythrocytic necrosis (VEN) is a disease of marine and anadromous fish that is caused by the erythrocytic necrosis virus (ENV), which was recently identified as a novel member of family Iridoviridae by next-generation sequencing. Phylogenetic analysis of the ENV DNA polymerase grouped ENV with other erythrocytic iridoviruses from snakes and lizards. In the present study, we identified the gene encoding the ENV major capsid protein (MCP) and developed a quantitative real-time PCR (qPCR) assay targeting this gene. Phylogenetic analysis of the MCP gene sequence supported the conclusion that ENV does not group with any of the currently described iridovirus genera. Because there is no information regarding genetic variation of the MCP gene across the reported host and geographic range for ENV, we also developed a second qPCR assay for a more conserved ATPase-like gene region. The MCP and ATPase qPCR assays demonstrated good analytical and diagnostic sensitivity and specificity based on samples from laboratory challenges of Pacific herring Clupea pallasii The qPCR assays had similar diagnostic sensitivity and specificity as light microscopy of stained blood smears for the presence of intraerythrocytic inclusion bodies. However, the qPCR assays may detect viral DNA early in infection prior to the formation of inclusion bodies. Both qPCR assays appear suitable for viral surveillance or as a confirmatory test for ENV in Pacific herring from the Salish Sea.

Potential drivers of virulence evolution in aquaculture.

Infectious diseases are economically detrimental to aquaculture, and with continued expansion and intensification of aquaculture, the importance of managing infectious diseases will likely increase in the future. Here, we use evolution of virulence theory, along with examples, to identify aquaculture practices that might lead to the evolution of increased pathogen virulence. We identify eight practices common in aquaculture that theory predicts may favor evolution toward higher pathogen virulence. Four are related to intensive aquaculture operations, and four others are related specifically to infectious disease control. Our intention is to make aquaculture managers aware of these risks, such that with increased vigilance, they might be able to detect and prevent the emergence and spread of increasingly troublesome pathogen strains in the future.

Characterization of a Novel Hepadnavirus in the White Sucker (Catostomus commersonii) from the Great Lakes Region of the United States.

UNLABELLED: The white sucker Catostomus commersonii is a freshwater teleost often utilized as a resident sentinel. Here, we sequenced the full genome of a hepatitis B-like virus that infects white suckers from the Great Lakes Region of the United States. Dideoxy sequencing confirmed that the white sucker hepatitis B virus (WSHBV) has a circular genome (3,542 bp) with the prototypical codon organization of hepadnaviruses. Electron microscopy demonstrated that complete virions of approximately 40 nm were present in the plasma of infected fish. Compared to avi- and orthohepadnaviruses, sequence conservation of the core, polymerase, and surface proteins was low and ranged from 16 to 27% at the amino acid level. An X protein homologue common to the orthohepadnaviruses was not present. The WSHBV genome included an atypical, presumptively noncoding region absent in previously described hepadnaviruses. Phylogenetic analyses confirmed WSHBV as distinct from previously documented hepadnaviruses. The level of divergence in protein sequences between WSHBV and other hepadnaviruses and the identification of an HBV-like sequence in an African cichlid provide evidence that a novel genus of the family Hepadnaviridae may need to be established that includes these hepatitis B-like viruses in fishes. Viral transcription was observed in 9.5% (16 of 169) of white suckers evaluated. The prevalence of hepatic tumors in these fish was 4.9%, and only 2.4% of fish were positive for both virus and hepatic tumors. These results are not sufficient to draw inferences regarding the association of WSHBV and carcinogenesis in white sucker. IMPORTANCE: We report the first full-length genome of a hepadnavirus from fishes. Phylogenetic analysis of this genome indicates divergence from genomes of previously described hepadnaviruses from mammalian and avian hosts and supports the creation of a novel genus. The discovery of this novel virus may better our understanding of the evolutionary history of hepatitis B-like viruses of other hosts. In fishes, knowledge of this virus may provide insight regarding possible risk factors associated with hepatic neoplasia in the white sucker. This may also offer another model system for mechanistic research.

Molecular identification of erythrocytic necrosis virus (ENV) from the blood of Pacific herring (Clupea pallasii).

Viral erythrocytic necrosis (VEN) is a condition affecting the red blood cells of more than 20 species of marine and anadromous fishes in the North Atlantic and North Pacific Oceans. Among populations of Pacific herring (Clupea pallasii) on the west coast of North America the disease causes anemia and elevated mortality in periodic epizootics. Presently, VEN is diagnosed by observation of typical cytoplasmic inclusion bodies in stained blood smears from infected fish. The causative agent, erythrocytic necrosis virus (ENV), is unculturable and a presumed iridovirus by electron microscopy. In vivo amplification of the virus in pathogen-free laboratory stocks of Pacific herring with subsequent virus concentration, purification, DNA extraction, and high-throughput sequencing were used to obtain genomic ENV sequences. Fragments with the highest sequence identity to the family Iridoviridae were used to design four sets of ENV-specific polymerase chain reaction (PCR) primers. Testing of blood and tissue samples from experimentally and wild infected Pacific herring as well as DNA extracted from other amphibian and piscine iridoviruses verified the assays were specific to ENV with a limit of detection of 0.0003 ng. Preliminary phylogenetic analyses of a 1448 bp fragment of the putative DNA polymerase gene supported inclusion of ENV in a proposed sixth genus of the family Iridoviridae that contains other erythrocytic viruses from ectothermic hosts. This study provides the first molecular evidence of ENV's inclusion within the Iridoviridae family and offers conventional PCR assays as a means of rapidly surveying the ENV-status of wild and propagated Pacific herring stocks.

Genetic variation in bacterial kidney disease (BKD) susceptibility in Lake Michigan Chinook Salmon and its progenitor population from the Puget Sound.

Mass mortality events in wild fish due to infectious diseases are troubling, especially given the potential for long-term, population-level consequences. Evolutionary theory predicts that populations with sufficient genetic variation will adapt in response to pathogen pressure. Chinook Salmon Oncorhynchus tshawytscha were introduced into Lake Michigan in the late 1960s from a Washington State hatchery population. In the late 1980s, collapse of the forage base and nutritional stress in Lake Michigan were thought to contribute to die-offs of Chinook Salmon due to bacterial kidney disease (BKD). Previously, we demonstrated that Lake Michigan Chinook Salmon from a Wisconsin hatchery have greater survival following BKD challenge relative to their progenitor population. Here, we evaluated whether the phenotypic divergence of these populations in BKD susceptibility was due to selection rather than genetic drift. Comparison of the overall magnitude of quantitative trait to neutral marker divergence between the populations suggested selection had occurred but a direct test of quantitative trait divergence was not significant, preventing the rejection of the null hypothesis of differentiation through genetic drift. Estimates of phenotypic variation (VP ), additive genetic variation (VA ) and narrow-sense heritability (h (2)) were consistently higher in the Wisconsin relative to the Washington population. If selection had acted on the Wisconsin population there was no evidence of a concomitant loss of genetic variation in BKD susceptibility. The Renibacterium salmoninarum exposures were conducted at both 14°C and 9°C; the warmer temperature accelerated time to death in both populations and there was no evidence of phenotypic plasticity or a genotype-by-environment (G × E) interaction. High h (2) estimates for BKD susceptibility in the Wisconsin population, combined with a lack of phenotypic plasticity, predicts that future adaptive gains in BKD resistance are still possible and that these adaptive gains would be stable under the temperature range evaluated here.

Isolation and molecular characterization of a novel picornavirus from baitfish in the USA.

During both regulatory and routine surveillance sampling of baitfish from the states of Illinois, Minnesota, Montana, and Wisconsin, USA, isolates (n = 20) of a previously unknown picornavirus were obtained from kidney/spleen or entire viscera of fathead minnows (Pimephales promelas) and brassy minnows (Hybognathus hankinsoni). Following the appearance of a diffuse cytopathic effect, examination of cell culture supernatant by negative contrast electron microscopy revealed the presence of small, round virus particles (∼ 30-32 nm), with picornavirus-like morphology. Amplification and sequence analysis of viral RNA identified the agent as a novel member of the Picornaviridae family, tentatively named fathead minnow picornavirus (FHMPV). The full FHMPV genome consisted of 7834 nucleotides. Phylogenetic analysis based on 491 amino acid residues of the 3D gene showed 98.6% to 100% identity among the 20 isolates of FHMPV compared in this study while only 49.5% identity with its nearest neighbor, the bluegill picornavirus (BGPV) isolated from bluegill (Lepomis macrochirus). Based on complete polyprotein analysis, the FHMPV shared 58% (P1), 33% (P2) and 43% (P3) amino acid identities with BGPV and shared less than 40% amino acid identity with all other picornaviruses. Hence, we propose the creation of a new genus (Piscevirus) within the Picornaviridae family. The impact of FHMPV on the health of fish populations is unknown at present.

Universal reverse-transcriptase real-time PCR for infectious hematopoietic necrosis virus (IHNV).

Infectious hematopoietic necrosis virus (IHNV) is an acute pathogen of salmonid fishes in North America, Europe and Asia and is reportable to the World Organization for Animal Health (OIE). Phylogenetic analysis has identified 5 major virus genogroups of IHNV worldwide, designated U, M, L, E and J; multiple subtypes also exist within those genogroups. Here, we report the development and validation of a universal IHNV reverse-transcriptase real-time PCR (RT-rPCR) assay targeting the IHNV nucleocapsid (N) gene. Properties of diagnostic sensitivity (DSe) and specificity (DSp) were defined using laboratory-challenged steelhead trout Oncorhynchus mykiss, and the new assay was compared to the OIE-accepted conventional PCR test and virus isolation in cell culture. The IHNV N gene RT-rPCR had 100% DSp and DSe and a higher estimated diagnostic odds ratio (DOR) than virus culture or conventional PCR. The RT-rPCR assay was highly repeatable within a laboratory and highly reproducible between laboratories. Field testing of the assay was conducted on a random sample of juvenile steelhead collected from a hatchery raceway experiencing an IHN epizootic. The RT-rPCR detected a greater number of positive samples than cell culture and there was 40% agreement between the 2 tests. Overall, the RT-rPCR assay was highly sensitive, specific, repeatable and reproducible and is suitable for use in a diagnostic setting.

Cutthroat trout virus as a surrogate in vitro infection model for testing inhibitors of hepatitis E virus replication.

Hepatitis E virus (HEV) is one of the most important causes of acute hepatitis worldwide. Although most infections are self-limiting, mortality is particularly high in pregnant women. Chronic infections can occur in transplant and other immune-compromised patients. Successful treatment of chronic hepatitis E has been reported with ribavirin and pegylated interferon-alpha, however severe side effects were observed. We employed the cutthroat trout virus (CTV), a non-pathogenic fish virus with remarkable similarities to HEV, as a potential surrogate for HEV and established an antiviral assay against this virus using the Chinook salmon embryo (CHSE-214) cell line. Ribavirin and the respective trout interferon were found to efficiently inhibit CTV replication. Other known broad-spectrum inhibitors of RNA virus replication such as the nucleoside analog 2'-C-methylcytidine resulted only in a moderate antiviral activity. In its natural fish host, CTV levels largely fluctuate during the reproductive cycle with the virus detected mainly during spawning. We wondered whether this aspect of CTV infection may serve as a surrogate model for the peculiar pathogenesis of HEV in pregnant women. To that end the effect of three sex steroids on in vitro CTV replication was evaluated. Whereas progesterone resulted in marked inhibition of virus replication, testosterone and 17ß-estradiol stimulated viral growth. Our data thus indicate that CTV may serve as a surrogate model for HEV, both for antiviral experiments and studies on the replication biology of the Hepeviridae.

Kinetics of viral load and erythrocytic inclusion body formation in Pacific herring artificially infected with erythrocytic necrosis virus.

Viral erythrocytic necrosis (VEN) is a condition that affects marine and anadromous fish species, including herrings and salmonids, in the Atlantic and Pacific oceans. Infection is frequently associated with severe anemia and causes episodic mortality among wild and hatchery fish when accompanied by additional stressors; VEN can be presumptively diagnosed by (1) light microscopic identification of a single characteristic-a round, magenta-colored, 0.8-µm-diameter inclusion body (IB) within the cytoplasm of erythrocytes and their precursors on Giemsa-stained blood films; or (2) observation (via transmission electron microscopy [TEM]) of the causative iridovirus, erythrocytic necrosis virus (ENV), within erythrocytes or their precursors. To better understand the kinetics of VEN, specific-pathogen-free Pacific herring Clupea pallasii were infected with ENV by intraperitoneal injection. At 1, 4, 7, 10, 14, 21, and 28 d postexposure, samples of blood, spleen, and kidney were collected and assessed (1) via light microscopy for the number of intracytoplasmic IBs in blood smears and (2) via TEM for the number of virions within erythrocytes. The mean prevalence of intracytoplasmic IBs in the blood cells increased from 0% at 0-4 d postexposure to 94% at 28 d postexposure. Viral load within circulating red blood cells peaked at 7 d postexposure, fell slightly, and then reached a plateau. However, blood cells observed within the kidney and spleen tissues demonstrated high levels of ENV between 14 and 28 d postexposure. The results indicate that the viral load within erythrocytes does not correlate well with IB prevalence and that the virus can persist in infected fish for more than 28 d.