Progress 11/15/05 to 11/14/08
Outputs
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. 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, 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. 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
Impacts
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.
Publications
- Shivappa, R.B., Savan, R, Kono, T, Sakai, M, E. Emmenegger, Kurath, G, and Levine, J.F. 2008. Detection of spring viremia of carp virus (SVCV) by loop-mediated isothermal amplification (LAMP) in koi carp, Cyprinus carpio L. Journal of Fish Diseases 31:249-258.
- Shivappa Rb, Savan R, Masahiro S, Levine Jf. Detection Of Spring Viremia Of Carp Virus By Loop Mediated Isothermal Amplification (Lamp). International Symposium on Aquatic Animal Health in San Francisco, November 2006
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Progress 11/15/06 to 11/14/07
Outputs
OUTPUTS: Outbreaks of infection with the rhabdovirus, spring viremia of carp virus (SVCV), have been associated with significant economic losses to aquaculture facilities in Europe, the middle East, and Asia. The fish pathogen was also recently recognized in the US. The disease affects cyprinid fish, which include carp, the most frequently cultured finfish, and ornamental carp, koi, which support a large international pet fish industry. Infected fish develop clinical signs easily confused with other fish diseases, and mortality can be high. A second disease affecting cyprinids, koi herpes virus (KHV), also contributes to substantial mortality. 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 initiated to explore the use of molecular technologies for the development of candidate antigenic targets for potential vaccines for both viruses. Two different
approaches for potential vaccine production are being pursued, a DNA plasmid-based vaccine and an insect baculovirus expression vector based vaccine. The glycoprotein (G) gene of SVCV, and the envelope protein gene of KHV are our antigenic targets. To generate DNA vaccine based on the CMV promoter for SVCV, viral RNA was extracted from SVCV infected cells and real-time polymerase chain reaction designed primers were used to amplify the G gene. The resulting cDNA was then cloned into pcDNA3.1(+) expression vector. A similar strategy was used for KHV viral DNA. Vaccines developed based on DNA plasmid techniques generally use a mammalian cytomegalovirus (CMV) promoter. Use of a human virus as a promotor, can complicate efforts to have a DNA-based vaccine approved. As an alternative, a fish promoter, carp beta-actin (provided by Dr. Marta Gomez-Chiarri), was used for sublconing the glycoprotein and envelope protein genes of SVCV and KHV. All plasmids have been confirmed and verified for
orientation by restriction digestion and nucleotide sequencing and are ready to be tested in fish. Constructs for sub-unit vaccine testing have been developed with a BAC-TO-BAC baculovirus expression system. Recombinant baculoviruses expressed SVCV G and KHV envelope proteins were generated with a baculovirus polyhedrin promoter (Polh) and restriction sites that facilitate directional cloning and expression of the desired genes. The genes were cloned into a pFastBacHTC donar plasmid propagated in E. coli DH5 cells and transposed into E. coli DH10Bac cells. The resulting recombinant bacmid DNA was purified. Recombinant baculoviruses will be generated using bacmid DNA for testing the antigenicity of the vaccine constructs. Renovations to a facility designed to conduct initial trials with the vaccine constructs are nearing completion. We have also been using the products of these studies to refine diagnostic tests for identifying fish infected with these viruses. A novel,
loop-mediated isothermal amplification system was used to develop an assay for detecting spring viremia of carp virus. The assay is a viable option for the development of a field diagnostic test for detecting the virus.
PARTICIPANTS: The project provided post-doctoral training for Ragunath Shivappa, and additional training opportunities for three graduate students, Shannon Kozlowicz, Otis Miller and Michael Bercz.
TARGET AUDIENCES: Nothing significant to report during this reporting period.
PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The fish diseases associated with spring viremia of carp virus and koi herpes virus have both contributed to substantial economic loses to carp aquaculture enterprises worldwide. Koi herpes virus is now endemic in the US, and spring viremia of carp virus has been identified in several outbreaks. Common carp are the most commonly cultured food-fish on the planet, and ornamental common carp, koi, and related product industries are a multi-billion dollar global industry. Introduction of these viruses into aquaculture facilities poses a potential significant economic loss. These studies provide an initial step toward the development of vaccines that could be used to prevent infection with these viruses or reduce morbidity and mortality after infection. Antigenic targets generated during the implementation of these studies also offer the opportunity to develop alternative assays for rapid spring viremia of carp virus and koi herpes virus detection; a key component of
efforts to prevent the introduction and spread of these viruses between aquaculture facilities.
Publications
- No publications reported this period
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Progress 11/15/05 to 11/15/06
Outputs
Spring viremia of carp virus (SVCV), a rhabdovirus, is associated with a severe disease in cyprinid fish, which has caused significant economic losses to European aquaculture enterprises. Koi herpes virus (KHV), a herpesvirus, is a DNA virus that causes mass mortality in common & ornamental carp. Virus was first isolated in 1998 from koi in the US and Israel and has since spread to Germany, England, Italy, Netherlands, Israel, and Indonesia. Proposed study was initiated with an overall objective of developing DNA vaccines for SVCV based on glycoprotein (G) gene and KHV based on an envelope protein gene. In addition, we have cloned these genes into baculovirus expression vectors that could be potentially expressed and used as a subunit vaccine and/or a reagent for a enzyme linked immuno-sorbent assay (ELISA) based pond-side diagnostic test for detection of SVCV and KHV. For DNA vaccines, though we had originally proposed to use the standard mammalian cytomegalovirus
(CMV) promoter, we have also generated plasmids based on a fish promoter, carp beta-actin. Use of fish promoter for developing DNA vaccines for fish pathogens will not only be effective, but also safe since DNA integration will not be a concern. To generate DNA vaccine based on the CMV promoter for SVCV, viral RNA was extracted from cell free SVCV infected EPC cells and subjected to RT-PCR with primers designed to amplify the G gene. The resulting cDNA was then cloned into pcDNA3.1(+) expression vector. A similar strategy was used for KHV using the viral DNA that was subjected to PCR. For DNA vaccines based on fish promoter, a plasmid carrying carp beta-actin promoter (provided by Dr. Marta Gomez-Chiarri, University of Rhode Island) was used for sublconing the glycoprotein and envelope protein genes of SVCV and KHV respectively. All these plasmids have been confirmed and verified for orientation by restriction digestion & nucleotide sequencing and are ready to be tested in fish. For
subunit vaccine, BAC-TO-BAC baculovirus expression system has been used. Objective of this strategy is to generate recombinant baculoviruses expressing SVCV G and KHV envelope proteins. We have used the donor plasmid pFastBacHTC, which consists of a baculovirus polyhedrin promoter (Polh) and restriction sites that facilitates directional cloning and 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 DNA from KHV were purified and respective genes were amplified as described above. The genes were then cloned into pFastBacHTC. The resulting donor plasmid was then propagated in E. coli DH5 cells and transposed into E. coli DH10Bac cells and the resulting recombinant bacmid DNA was purified. Recombinant baculoviruses will be generated using bacmid DNA when we are ready to test the vaccines. We are currently renovating at the CVM. Upon completion, the
facility will be used as an aquatic animal pathogen laboratory. Vaccines and diagnostic reagents that have resulted from this project will be tested in this facility. Renovations are slated for completing by April.
Impacts
Spring viremia of carp virus and KHV have caused devastating losses to the carp industry worldwide. While in the United States the losses have been less dramatic, the potential to spread rapidly throughout the hobby and trade industries as well as to wild fish is ever present. Research on medical treatments may help to decrease the mortality associated with these viral pathogens. However, prevention is essential to curtail the spread to populations of common and koi carp. Because the number and value of koi kept as pets continue to increase, it is essential that fish medicine enter the realms of traditional small and large animal medicine. Research aimed at developing vaccines and testing their safety and efficacy is crucial to the realization of this goal. The proposed vaccines would have the potential to: 1) curtail the spread of SVCV and KHV to ornamental koi kept as pets; 2) prevent the euthanasia of infected pet koi, and 3) prevent the potential introduction of
these pathogens into surface waters and wild fish populations.
Publications
- No publications reported this period
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