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.

DNA Vaccination Partially Protects Muskellunge against Viral Hemorrhagic Septicemia Virus (VHSV-IVb).

A DNA vaccine containing the glycoprotein (G) gene of the North American viral hemorrhagic septicemia virus (VHSV) genotype IVb was developed to evaluate the immune response of fish following vaccination and evaluate its efficacy in protecting a susceptible species, the Muskellunge Esox masquinongy, against VHSV-IVb challenge. Seven weeks (539 degree-days) following vaccination with 10 µg of either pVHSivb-G or a control plasmid, Muskellunge were challenged by immersion with 105 plaque-forming units (pfu)/mL of VHSV-IVb. Fish vaccinated with pVHSivb-G had a relative percent survival (RPS) of 45%. Vaccinated fish also had significantly lower mean viral titers in tissues (4.2 × 102 pfu/g) and viral prevalence (4%) than fish receiving the plasmid control vaccine (3.3 × 105 pfu/g; 82%). Neutralizing antibodies were detected 28 d (308 degree-days) postchallenge (11 weeks postvaccination) in 100% of Muskellunge vaccinated with pVHSivb-G compared with only 12% of plasmid-control-vaccinated Muskellunge, suggesting robust induction of a secondary, adaptive immune response. In addition, pVHSivb-G-vaccinated Rainbow Trout Oncorhynchus mykiss challenged 7 d (100 degree-days) postvaccination with the heterologous novirhabdovirus, infectious hematopoietic necrosis virus (IHNV), experienced an RPS of 61%, compared to control fish, suggesting induction of an early and transient nonspecific antiviral immune response. This study provides an important starting point for VHSV-IVb vaccine development and useful information about the antiviral immune response elicited by DNA vaccination in a nondomesticated fish species. Received May 1, 2016; accepted September 1, 2016.

A DNA vaccine encoding the viral hemorrhagic septicemia virus genotype IVb glycoprotein confers protection in muskellunge (Esox masquinongy), rainbow trout (Oncorhynchus mykiss), brown trout (Salmo trutta), and lake trout (Salvelinus namaycush).

BACKGROUND: The viral hemorrhagic septicemia virus (VHSV) is one of the most serious fish pathogens. In 2003, a novel sublineage (genotype IVb) of this deadly virus emerged in the Great Lakes basin causing serious fish kills. We have previously demonstrated that a DNA plasmid (pcDNA), containing a cytomegalovirus (CMV) promoter and the viral hemorrhagic septicemia virus (VHSV) genotype IVb glycoprotein (G) gene insert (designated pVHSivb-G) confers moderate protection in muskellunge (Esox masquinongy), a highly susceptible species upon challenge. In order to achieve optimal protection, we investigated a number of factors including the incubation time [i.e. the number of degree days (° days)] before challenge, and viral challenge dose and route. Additionally, we tested if pVHSivb-G provides protection against VHSV-IVb to less susceptible salmonids such as rainbow trout (Oncorhynchus mykiss), brown trout (Salmo trutta) and lake trout (Salvelinus namaycush). RESULTS: An increase in the period lapsed between vaccination and challenge to 1880° days resulted in 95% relative percent protection (RPS) in muskellunge following a single administration of the pVHSivb-G plasmid and viral challenge. An RPS of 100% for muskellunge was achieved with a longer incubation period (2400° days) and in conjunction with a booster dose of the plasmid. The pVHSivb-G vaccine also elicited significant protection in all three salmonid species, reaching 100% RPS in lake trout following an incubation period of 1001° days prior to viral challenge. Vaccination with pVHSivb-G was also associated with the development of significant levels of circulating VHSV-binding antibodies in muskellunge as measured by indirect ELISA, which reached peak levels 6-7 weeks post-vaccination. Viral shedding in vaccinated survivors was minimal and of transient nature. CONCLUSIONS: The study shows that the pVHSivb-G plasmid can elicit a protective response against the wild virus strain in a range of species important in recreational and commercial Great Lakes fisheries.

Isolation of the Fathead Minnow Nidovirus from Muskellunge Experiencing Lingering Mortality.

In 2011, the Fathead Minnow nidovirus (FHMNV; Genus Bafinivirus, Family Coronaviridae, Order Nidovirales) was isolated from pond-raised juvenile Muskellunge Esox masquinongy suffering from lingering mortality at the Wild Rose Hatchery in Wild Rose, Wisconsin. Moribund Muskellunge exhibited tubular necrosis in the kidneys as well as multifocal coalescing necrotizing hepatitis. The FHMNV was also isolated from apparently healthy juvenile Muskellunge at the Wolf Lake State Fish Hatchery in Mattawan, Michigan. The identity of the two syncytia-forming viruses (designated MUS-WR and MUS-WL from Wild Rose Hatchery and Wolf Lake State Fish Hatchery, respectively) as strains of FHMNV was determined based on multiple-gene sequencing and phylogenetic analyses. The pathogenicity of the MUS-WL FHMNV strain was determined by experimentally infecting naive juvenile Muskellunge through intraperitoneal injection with two viral concentrations (63 and 6.3 × 10(3) TCID50/fish). Both doses resulted in 100% mortality in experimentally infected fish, which exhibited severely pale gills and petechial hemorrhaging in eyes, fins, and skin. Histopathological alterations in experimentally infected fish were observed mainly in the hematopoietic tissues in the form of focal areas of necrosis. Phylogenetic analysis of concatenated partial spike glycoprotein and helicase gene sequences revealed differences between the MUS-WL FHMNV, MUS-WR FHMNV, and two other FHMNV originally isolated from moribund Fathead Minnows Pimephales promelas including the index FHMNV strain (GU002364). Based on a partial helicase gene sequence, a reverse transcriptase PCR assay was developed that is specific to FHMNV. These results give evidence that the risks posed to Muskellunge by FHMNV should be taken seriously. Received May 1, 2015; accepted February 8, 2016.

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.

Spread of the emerging viral hemorrhagic septicemia virus strain, genotype IVb, in Michigan, USA.

In 2003, viral hemorrhagic septicemia virus (VHSV) emerged in the Laurentian Great Lakes causing serious losses in a number of ecologically and recreationally important fish species. Within six years, despite concerted managerial preventive measures, the virus spread into the five Great Lakes and to a number of inland waterbodies. In response to this emerging threat, cooperative efforts between the Michigan Department of Natural Resources (MI DNR), the Michigan State University Aquatic Animal Health Laboratory (MSU-AAHL), and the United States Department of Agriculture-Animal and Plant Health Inspection Services (USDA-APHIS) were focused on performing a series of general and VHSV-targeted surveillances to determine the extent of virus trafficking in the State of Michigan. Herein we describe six years (2005-2010) of testing, covering hundreds of sites throughout Michigan's Upper and Lower Peninsulas. A total of 96,228 fish representing 73 species were checked for lesions suggestive of VHSV and their internal organs tested for the presence of VHSV using susceptible cell lines. Of the 1,823 cases tested, 30 cases from 19 fish species tested positive for VHSV by tissue culture and were confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). Gene sequence analyses of all VHSV isolates retrieved in Michigan demonstrated that they belong to the emerging sublineage "b" of the North American VHSV genotype IV. These findings underscore the complexity of VHSV ecology in the Great Lakes basin and the critical need for rigorous legislation and regulatory guidelines in order to reduce the virus spread within and outside of the Laurentian Great Lakes watershed.

Heterogeneity in levels of serum neutralizing antibodies against viral hemorrhagic septicemia virus genotype IVB among fish species in Lake St. Clair, Michigan, USA.

The presence of neutralizing antibodies against viral hemorrhagic septicemia virus (VHSV-IVb) was investigated in sera of 13 fish species collected from Lake St. Clair, Michigan, USA, a VHSV-endemic water body. We tested 297 sera collected May 2004-June of 2010, using a complement-dependent 50% plaque neutralization test (50% PNT). Neutralizing antibodies were detected in 23% (67/297) of the samples. The highest overall antibody prevalence (85%, 34/40) and mean positive antibody titer (12,113 ± 11,699 SD) were detected in muskellunge (Esox masquinongy). Antibodies were also detected in 50% (15/30) of sampled northern pike (E. lucius), 25% (15/61) of freshwater drum (Aplodinotus grunniens), and 7% (3/41) of smallmouth bass (Micropterus dolomieu). All sera from channel catfish (Ictalurus punctatus), lake sturgeon (Acipenser fulvescens), quillback (Carpiodes cyprinus), rock bass (Ambloplites rupestris), shorthead redhorse (Moxostoma macrolepidotum), silver redhorse (M. anisurum), walleye (Sander vitreus), white perch (Morone americana), and yellow perch (Perca flavescens) were negative. Antibodies in one or more fish species were detected in all sampling years (2004, 2006, 2007, 2009, and 2010), whereas in parallel sampling periods, VHS virus was detected only in 2006 and 2009. Our results suggest the continued presence of VHSV-IVb in the Lake St. Clair ecosystem, and underscore the importance of assessing immune responses of fish populations to determine prior virus exposure.