Hemorrhagic Septicemia Virus Genotype IVb Research Articles

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

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. 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. 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. 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.

Production of a monoclonal antibody against of muskellunge (Esox masquinongy) IgM heavy chain and its use in development of an indirect ELISA for titrating circulating antibodies against VHSV-IVB

This study reports the development of a monoclonal antibody (designated 3B10) against the muskellunge (Esox masquinongy) IgM. The 3B10 monoclonal antibody (mAb) belongs to the IgG3 kappa isotype. Western blotting demonstrated that 3B10 mAb reacted primarily to muskellunge IgM heavy chain. 3B10 also reacted strongly with the IgM heavy chain of other esocids, including the northern pike (Esox lucius), tiger muskellunge (E. masquinongy x E. lucius), and, to a much lesser extent, the chain pickerel (E. niger). The 3B10 mAb did not bind to IgM from 10 other fish species resident in the Great Lakes basin. Using the 3B10 mAb, it was possible to determine the muskellunge Ig ability to bind to antigens. Using trinitrophenyl hapten conjugated to keyhole limpet hemocyanin (TNP-KLH) as the eliciting antigen, muskellunge Ig subclasses exhibited a range of affinities with log aK values 5.56–6.25 that is considered intermediate compared to other fish species. 3B10 mAb was used to develop and evaluate an indirect ELISA for the detection and quantitation of circulating antibodies against the viral hemorrhagic septicemia virus genotype IVb (VHSV-IVb). Using the newly optimized assay, anti-VHSV-IVb antibodies were detected in sera of VHSV-IVb vaccinated muskellunge as well as from those of wild muskellunge sampled from an endemic waterbody. In addition to its use in immunoassays, the developed 3B10 mAb will enable future investigation aiming at deciphering immune mechanism of this important fish species to pathogens.

Potential Reservoirs and Risk Factors for VHSV IVb in an Enzootic System: Budd Lake, Michigan.

Viral hemorrhagic septicemia virus genotype IVb (VHSV IVb) has caused major, sporadic fish die-offs in the Laurentian Great Lakes region of North America since 2005. Presently, factors affecting VHSV IVb persistence in enzootic systems are not well understood. Even with annual surveillance, the virus can go undetected for several years after an outbreak before again re-emerging, which suggests that the virus is maintained in the system either below detectable levels or in untested reservoirs. The aim of this study was to identify potential reservoirs of VHSV IVb in Budd Lake, Michigan; VHSV IVb was first detected in Budd Lake in 2007 but remained undetected until 2011. Additionally, we explored the susceptibility of naive fish introduced into a water body enzootic for VHSV IVb by stocking age-0 Largemouth Bass Micropterus salmoides at varying densities into enclosures in the lake. The virus was not detected among samples of the fishes Notropis spp. and Lepomis spp., cylindrical papershell mussels Anodontoides ferussacianus, leeches (subclass Hirudinea), sediment, or water. However, the virus was successfully isolated from amphipods (family Hyalellidae) and Largemouth Bass held in the enclosures. Our finding of VHSV IVb in Hyalellidae amphipods in combination with other research that has detected the virus in Diporeia spp., a large benthic amphipod important as a food resource to Great Lake fishes, suggests that benthic macroinvertebrates are a reservoir for VHSV IVb in infected systems. If there are environmental reservoirs for VHSV IVb in infected systems, they are likely unevenly distributed. Findings of this study add to our understanding of the seemingly complex ecology of this deadly and economically detrimental virus. Received February 22, 2016; accepted October 16, 2016.

Does Herd Immunity Exist in Aquatic Animals?

Viral hemorrhagic septicemia virus genotype IVb (VHSV-IVb) is presently found throughout the Laurentian Great Lakes region of North America. We recently developed a DNA vaccine preparation containing the VHSV-IVb glycoprotein (G) gene with a cytomegalovirus (CMV) promoter that proved highly efficacious in protecting muskellunge (Esox masquinongy) and three salmonid species. This study was conducted to determine whether cohabitation of VHSV-IVb immunized fishes could confer protection to non-vaccinated (i.e., naïve) fishes upon challenge. The experimental layout consisted of multiple flow-through tanks where viral exposure was achieved via shedding from VHSV-IVb experimentally infected muskellunge housed in a tank supplying water to other tanks. The mean cumulative mortality of naïve muskellunge averaged across eight trials (i.e., replicates) was significantly lower when co-occurring with immunized muskellunge than when naïve muskellunge were housed alone (36.5% when co-occurring with vaccinated muskellunge versus 80.2% when housed alone), indicating a possible protective effect based on cohabitation with vaccinated individuals. Additionally, vaccinated muskellunge when co-occurring with naïve muskellunge had significantly greater anti-VHSV antibody levels compared to vaccinated muskellunge housed alone suggesting that heightened anti-VHSV antibodies are a result of cohabitation with susceptible individuals. This finding could contribute to the considerably lower viable VHSV-IVb concentrations we detected in surviving naive muskellunge when housed with vaccinated muskellunge. Our research provides initial evidence of the occurrence of herd immunity against fish pathogens.

Goldfish Carassius auratus susceptibility to viral hemorrhagic septicemia virus genotype IVb depends on exposure route.

We assessed the susceptibility of goldfish Carassius auratus to infection by genotype IVb of the viral hemorrhagic septicemia virus. Goldfish were infected by intraperitoneal injections of 106 plaque-forming units (pfu) fish-1, single bath exposure of 105 pfu ml-1 for 24 h, or consumption of 0.4 g of commercial fish feed soaked in 107 pfu per 8 fish. The mortality rate of intraperitoneal-infected goldfish was 10 to 32%, although the virus was detected by quantitative RT-PCR in 77% (65/84) of the survivors at the end of the 42 d trial, suggesting a carrier state. Severe gross lesions were observed in many of the moribund and dead goldfish such as hemorrhaging in the skin, fin, liver, kidney, brain, intestine, and eye as well as abdominal distension, bilateral exophthalmia, and splenomegaly. There was minimal morbidity or mortality in the immersion, feeding, or control groups.

Direct and Indirect Evidence Suggests Continuous Presence of Viral Hemorrhagic Septicemia Virus (Genotype IVb) in Budd Lake, Michigan: Management Implications

AbstractIn May 2007, viral hemorrhagic septicemia virus genotype IVb (VHSV IVb) was isolated from several fish species in Budd Lake, a 71‐ha inland lake in central Michigan. Because of the virulence and pathogenicity of VHSV IVb, follow‐up testing was conducted between 2007 and 2010. This testing found no further evidence of VHSV IVb presence. Similar patterns (i.e., initial mortality events followed by no evidence of additional infections) have been observed in other waterbodies, which leads to questions regarding the implications that these patterns could have for fisheries management (e.g., could stocking naive fish lead to additional epizootic occurrences?). This study was conducted to determine whether VHSV IVb was still present in Budd Lake by intensively sampling across multiple seasons, locations, and size‐groups of Muskellunge Esox masquinongy, Northern Pike Esox lucius, and Largemouth Bass Micropterus salmoides. Individuals were tested for both active VHSV IVb infection and antibody production. We found that VHSV IVb was still present in Budd Lake, as active infections were detected from samples in spring, summer, and fall 2011, with highest infection prevalence in spring 2011. Spring 2011 infection prevalence was 17% for esocids and 33% for Largemouth Bass. Between 74% and 80% of collected esocids produced antibodies, depending on the sampling season. Antibody production in Largemouth Bass ranged from 12% in spring and fall 2011 to 20% in spring 2012. Based on these results, we recommend sustained vigilance in the management of VHSV IVb–positive systems to limit the risk of virus spread as it is perhaps questionable whether systems that have experienced an outbreak will ever be virus‐free.Received September 16, 2014; accepted February 7, 2015

Detection of viral hemorrhagic septicemia virus-IVb antibodies in sera of muskellunge Esox masquinongy using competitive ELISA

A competitive enzyme-linked immunosorbent assay (cELISA) was developed for the detection of antibodies to viral hemorrhagic septicemia virus genotype IVb (VHSV-IVb) in fish sera. Assay conditions were standardized using known negative and positive muskellunge Esox masquinongy. A positive-negative threshold of 14.6% inhibition was established based on analysis of sera of 60 muskellunge with no previous exposure to VHSV-IVb. The cELISA was then used to investigate immune responses of wild muskellunge sampled from 5 water bodies in Michigan and Wisconsin, USA, between 2005 and 2012. Antibodies were detected in fish from Lake St. Clair, Michigan, and Lower Fox River/Green Bay, Wisconsin. Both water systems were considered enzootic for VHSV-IVb. Additionally, antibodies were detected in muskellunge from Thornapple Lake, a Michigan inland lake previously considered negative for VHSV-IVb based on virus isolation methods. Muskellunge populations from Lake Hudson, Michigan, and Butternut Lake, Wisconsin, lacked evidence of an immune response to VHSV-IVb. When results of the cELISA were compared to the 50% plaque neutralization test for several groups of fish, there was 78.4% agreement between the tests for antibody presence. The cELISA is a rapid and efficient test for the detection of binding antibodies to VHSV-IVb and will be a useful non-lethal tool for monitoring the spread of this serious pathogen.

Modeling the secondary spread of viral hemorrhagic septicemia virus (VHSV) by commercial shipping in the Laurentian Great Lakes

Researchers have only begun to study the role of shipping in the spread of invasive species in the Laurentian Great Lakes despite a well-documented history of introductions in these lakes due to ballast water release. Here, we determine whether ballast water discharge was a likely vector of spread of the fish disease, viral hemorrhagic septicemia virus genotype IVb (VHSV-IVb), throughout the Great Lakes and St. Lawrence Seaway. Three models were developed to assess whether the spread of VHSV was due to (1) chance (random model), or (2) ballast water discharge (location model), and whether (3) increased propagule pressure, as measured by the number of visitations by ships carrying ballast water from VHSV infected areas, increased the likelihood of a discharge location becoming infected with VHSV (propagule pressure model). The third model was also used to assess the probable point of initial introduction of VHSV. Presence and absence accuracies and weighted Cohen’s kappa were calculated to determine which models best predicted observed presences and absences of VHSV. Location models explain the patterns of VHSV detections better than random models, and inclusion of “propagule pressure” often improved model fit; however, the relationship is weak likely because of a long lag time between introduction and detection, a high rate of false negatives in reporting, and the possible contribution of other vectors of spread. Montreal was also identified as the more likely introduction site of VHSV, rather than Lake St. Clair, the site where the virus was first detected.

Experimental Transmission of VHSV Genotype IVb by Predation

Preliminary surveillance of wild baitfish during the 2006 viral hemorrhagic septicemia virus genotype IVb (VHSV IVb) outbreaks indicated Emerald Shiners Notropis atherinoides and Bluntnose Minnow Pimephales notatus were infected with high levels of VHSV without showing clinical signs of disease. The movement and use of baitfish was recognized as the most probable vector for the introduction of VHSV to inland waters, such as Conesus Lake and Skaneateles Lake in New York, Budd Lake in Michigan, and Little Lake Butte des Morts and Lake Winnebago in Wisconsin. While numerous government agencies implemented restrictions to stop the movement of potentially infected baitfish into new waters and prevent the spread of VHSV IVb, until now, studies to investigate whether these initial introductions were by an oral route of infection have not occurred. Our studies identified infected Fathead Minnow Pimephales promelas as suitable vectors for transmitting VHSV IVb when fed to Tiger Muskellunge ( ♂ Northern Pike Esox lucius × ♀ Muskellunge Esox masquinongy) during laboratory trials. Six of 16 Tiger Muskellunge were infected with VHSV IVb after consumption of infected Fathead Minnows when assayed with quantitative reverse transcriptase polymerase chain reaction and viral isolation in cell culture. Weekly sampling of water and feces from these Tiger Muskellunge individually reared showed intermittent shedding of VHSV IVb. Those exposed to similarly VHSV IVb-inoculated fathead minnows by cohabitation only became infected in 1 case out of 16. A similar trial of 12 Tiger Muskellunge fed Round Goby Neogobius melanostomus that survived a VHSV IVb immersion challenge did not result in infection. Overall, our findings imply that consumption of infected wild baitfish may be a risk factor for introduction of VHSV.

Persistence of viral RNA in fish infected with VHSV‐IVb at 15 °C and then moved to warmer temperatures after the onset of disease

Smallmouth bass, Micropterus dolomieu Lacepède, bluegill, Lepomis macrochirus Rafinesque (coppernose strain), koi carp, Cyprinus carpio L., and channel catfish Ictalurus punctatus (Rafinesque), were infected by intraperitoneal injection with viral haemorrhagic septicaemia virus genotype IVb (VHSV-IVb) at 15 °C. When clinical signs of disease developed, one-third of the fish was moved to 20°C and one-third to 25°C. Mortality in challenged fish at all three temperatures ranged from 25 to 45% in smallmouth bass and from 70 to 90% in bluegill. No koi carp or channel catfish died during the study. Viral copy numbers detected by quantitative real-time reverse transcriptase PCR (qrt-RTPCR) in fish dying at 20 and 25°C decreased over time. In survivors of the challenge, viral copy numbers were higher in the more susceptible species (smallmouth bass and bluegill) than in the more VHSV-IVb disease-resistant species (koi carp and channel catfish). In fish surviving 28days post-infection, prevalence of infection was 66-100% depending on species and temperature, and VHSV-IVb was detected at 10(3) -10(5) copies μg(-1) host RNA. Our results show that qrt-RTPCR is a useful tool to investigate fish kills even 28days after temperatures are elevated above those known to be permissive for VHSV replication.

Mortality and Carrier Status of Bluegills Exposed to Viral Hemorrhagic Septicemia Virus Genotype IVb at Different Temperatures

The emergence of the viral hemorrhagic septicemia virus (VHSV) genotype IVb (VHSV-IVb) in the Great Lakes of North America has led to concern that the virus might spread to natural fisheries and aquaculture in the southern USA. We exposed bluegills Lepomis macrochirus to VHSV-IVb by intraperitoneal injection at six temperatures from 10 degrees C to 30 degrees C and followed the disease course by quantitative real-time reverse transcriptase polymerase chain reaction (qrt-RT-PCR). Mortality of injected fish was 90% at 10 degrees C, 35% at 14 degrees C, and 10% at 18 degrees C; no mortality attributable to VHSV was observed at temperatures of 22-30 degrees C. In survivors tested at 21 d postchallenge, viral copies and prevalence determined by qrt-RT-PCR were inversely related to temperature, and VHSV-IVb could not be detected in fish held at temperatures above 22 degrees C. Similar results were obtained for bluegills that were exposed by cohabitation with the intraperitoneally injected fish. Acclimation of the fish to 12 degrees C after 21 d at higher temperatures did not appear to cause a re-emergence of the virus. Based on our findings, the temperature range of VHSV-IVb appears to be the same as published values for VHSV genotype I, which has an optimum of 9-12 degrees C and an upper limit of 18-20 degrees C.

Experimental studies confirm the wide host range of the Great Lakes viral haemorrhagic septicaemia virus genotype IVb

Experimental studies confirm the wide host range of the Great Lakes viral haemorrhagic septicaemia virus genotype IVb