Development of DNA and Subunit Vaccines for Spring Viremia of Carp Virus and Koi Herpes Virus

NON-TECHNICAL SUMMARY: Spring viremia of carp virus and koi herpes virus are pathogens that affect fish in the family Cyprinidae, which includes carp produced for human consumption and ornamental koi. Both viruses have been associated with substantial economic losses to both industries. Vaccines will be developed for spring viremia of carp virus and koi herpes virus, and tested for safety and efficacy.

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APPROACH: An SVCV strain isolated during the 2002 outbreak in NC will be propagated in the monolayers of epithelioma papillosum cyprni (EPC) cells as described (Goodwin 2002). Koi herpes virus isolated from clinically ill koi will be grown in Koi Fin cell-line (KF-1). Cells will be infected with SVCV or KHV and purified. After the appearance of cytopathic effect (CPE), cells will be harvested, pelleted, and the cell free supernatant will be collected. After resuspension and repelleting, additional processing and transfection, protein expression in the cells will be confirmed by Western blotting at 3 day post-transfection.. The clones expressing the protein of the right size will then be used as a DNA vaccine vector against SVCV and KHV, respectively. BAC-TO-BAC baculovirus expression system (Invitrogen) will be used to generate recombinant baculoviruses and is based on site-specific transposition of an expression cassette into a baculovirus shuttle vector (bacmid) that is propagated in E. coli. We will use the donor plasmid pFastBac HTC, which consists of a baculovirus polyhedrin promoter (Polh) and cloning sites that facilitate cloning expression of the desired genes. The transfer vector also carries a 6x histidine tag downstream of the promoter that will enable purification of the expressed protein. Viral RNA from SVCV and KHV will be purified and target proteins will be generated as described above. The purified cDNA will then be cloned into pFastBacHTC vector after double digestion with Nco and Xho II sites. The resulting donor plasmid will be then propagated in E. coli DH5+/- cells and transposed into E. coli DH10Bac cells to generate recombinant bacmid as described by manufacturer. Recombinant bacmid DNA will be isolated from the above cultures and transfected into Spodoptera frugiperda (Sf-9) cells using CellFectin reagent (Invitrogen). The cells will be monitored for CPE. Once the CPE is noticed, protein expression will be confirmed by Western blotting technique. Upon confirmation of protein expression, the proteins will be purified using nickel ion chromatography columns (Ni-NTA) and will be used either as a subunit vaccine or diagnostic reagent for development of a pond-side test. Vaccines will be tested for safety and efficacy in a series of controlled clinical trials.
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PROGRESS: 2005/11 TO 2008/11 <BR>
OUTPUTS: Carp, members of the fish family Cyprinidae, are the most common finfish species grown by aquaculturists. They are an important food-fish, and represent more than 40% of the total international aquaculture finfish harvest. Ornamental carp, or koi, are common carp (Cyprinus carpio) bred to enhance and maintain natural color features for display aquaria. The global economic value of the ornamental carp industry approaches three billion dollars. Carp and other members of the family Cyprinidae, are susceptible to two viral diseases, spring viremia of carp, and koi herpes virus disease that are associated with production loses and mortality in cultured carp and wild populations. Both diseases have a profound impact on the economics of carp culture. Spring viremia of carp virus (SVCV), a rhabdovirus, has caused significant economic losses to European aquaculture enterprises. Since April 2002, however, SVC has also been reported in the United States, leading to the depopulation of one of the largest koi (ornamental carp) hatcheries in the country. The virus poses a major threat to the entire carp industry, as well as free ranging cyprinid species. Koi herpes virus (KHV), a herpesvirus, is a DNA virus that causes mass mortality in common and ornamental carp. The virus was first isolated in 1998 from koi in the United States and Israel and has since spread to Germany, England, Italy, Netherlands, Israel, and Indonesia. These studies were conducted to work toward the development of potential vaccines for preventing infection and minimizing losses associated with these diseases. Assay techniques developed during the course of these studies have also enhanced our ability to detect both viruses. Purified stocks of SVCV isolated during the 2002 outbreak in North Carolina were propagated on monolayers of carp cells, and quantified. Constructs for two molecular approaches to vaccine development, the use of a DNA plasmid, and a baculovirus expression system for inducing an immune response to the SVCV G gene were developed. Human cytomegalovirus is traditionally used to develop constructs for DNA plasmid-based vaccines. To avoid concerns about potential use of CMV as a promoter, a fish promoter was used for construct development. For KHV, the target was the KHV envelope protein. To conduct the studies a unique biosecurity facility was renovated with state funds to accommodate clinical trials focused on the pathogenesis of fish diseases. Spring viremia of carp virus, is considered a foreign animal disease. Rapid recognition of both SVCV and KHV are essential to efforts to control its introduction and spread. Real-time polymerase chain reaction assays were developed for both SVCV and KHV. Culture methods and polymerase chain reaction-based assays have been used previously to diagnosis the infection. Both approaches require substantial technical expertise. To improve the rapid detection of SVCV, and reduce the cost of initial screening for the virus, a loop-mediated amplification (LAMP) assay was developed for detecting the virus. The assay proved to rapid, and inexpensive and offers great promise as a tool for the timely field detection of SVCV virus. <BR> PARTICIPANTS: Jay Levine, principal investigator Raghunath Shivappa, post-doctoral student Shannon Kozlowicz, graduate student Tony Szempruch, technician Maria Serano, technician Contributors: James Guy, North Carolina State University Mac Law, North Carolina State University Michael Stoskopf, North Carolina State University Laboratory of Marine Biotechnology, University of Miyazaki, Japan Savan, R, Kono, T, Sakai, M, E. Western Fisheries Research Center, US Geological Survey Emmenegger, Kurath, G, <BR> TARGET AUDIENCES: Target audiences for the project included aquaculture disease researchers, fisheries biologists, commercial food-fish aquaculturists, commercial ornamental carp aquaculturists, and ornamental carp hobbyists, and related trade-groups and associations. The facilities developed with state funds to support these studies have the value-added benefit of providing a biosecure facilities-related resource for investigators conducting clinical trials with fish pathogens. <BR> PROJECT MODIFICATIONS: The primary modification to the project was in the time-line for the studies. State supported facilities renovation was repeatedly delayed, and necessitated no-cost extensions for the project <BR> <BR>

IMPACT: 2005/11 TO 2008/11<BR>
Aquaculture is a global industry and the most rapidly growing segment of world livestock production. Aquaculture species represent an increasingly important proportion of total seafood sales, with an annual value exceeding $70 billion. Carp, fishfish in the family Cyprinidae, are the most commonly raised foodfish. Koi, ornamental carp, are common carp, bred and raised for hobbyists and display aquaria. Their production and sales support a global industry with an economic value exceeding three billion dollars. Carp and other cyprinids are susceptible to two viral pathogens, spring viremia of carp virus (SVCV), and koi herpes virus (KHV) associated with production loses and mortality in cultured carp and wild populations. Vaccines are needed to prevent their potential introduction into aquaculture farms, and inexpensive assays are needed for their rapid detection. Vaccine constructs for potential use in vaccine production were developed for both SVCV and KHV. Constructs for a potential DNA-plasmid-based vaccine were developed targeting a glycoprotein of SVCV and an envelope protein of KHV. A baculo-virus expression vector system was also used to develop additional constructs to both viruses. On-going efforts focus on the further development and evaluation of these constructs for potential vaccine production. Real-time polymerase chain reaction assays were developed for both SVCV and KHV. In addition, a novel assay using loop-mediated isothermal amplification of DNA was developed to detect SVCV, and provide an inexpensive, rapid alternative for SVCV virus field detection.