Progress 12/01/00 to 11/30/03
Outputs
The main goal of this project was to determine the role of viral proteins in the virulence and persistence in infectious pancreatic necrosis virus (IPNV). The objectives of this project were to: 1) clone and sequence the genomic segments of a virulent and an attenuated IPNV strains; 2) construct chimeric cDNA clones of a tissue-culture adapted and virulent IPNV strains; 3) produce chimeric viruses using the reverse genetics system and evaluate the characteristics of these viruses in fish. To determine the molecular basis of virulence in IPNV, fry of Atlantic salmon (Salmo salar L.) were challenged with nine different field strains causing either high mortality and severe pathological changes or low mortality and no lesions. Complete nucleotide sequences of segment A of all these strains, as well as segment B of three selected strains were determined. All viruses tested had a unique genome sequence. Only minor differences were noted in the genes encoding VP1, VP3 and
VP4 proteins, whereas, a majority of changes were observed in the gene encoding VP2 protein. Some degree of variation was found in the small open reading frame (ORF), which encodes a 15-kDa non-structural (NS) protein, also known as VP5. One of the strains lacked the initiation codon for this protein, whereas, the other four could encode a truncated version of the NS protein. Additional data obtained by sequencing of the NS and VP2 genes directly from diseased fish demonstrated changes in the VP2 gene after two passages in cell culture, which could possibly be associated with attenuation. Comparison of the deduced amino acid sequences of the NS and VP2 genes reveal that all the virulent strains possess a 12-kDa coding NS gene and have residues Thr, Ala, Thr/Ala, and Tyr/His at positions 217, 221, 247 and 500 of the VP2 gene, respectively - the motifs identified to be involved in virulence of IPNV. To identify the putative amino acids involved in the virulence and cell tropism of IPNV,
we constructed chimeric cDNA clones of segment A and B between the attenuated (Sp103) and the virulent strain, rNVI15. Using the cRNA-based reverse genetics system developed for IPNV, we generated several chimeric viruses after transfection in Chinook salmon embryo (CHSE) cells. Three recovered viruses that encode different forms of VP5 protein had similar replication kinetics in cell culture, and were virulent in vivo studies. Substitution of residues at position 217 and 247 in the VP2 region of the virulent strain with the corresponding residues of the attenuated strain yielded a virus that had similar replication kinetics as the attenuated strain, suggesting that these residues are the important markers for virus adaptation and attenuation in vitro. Work is underway to evaluate the characteristics of the recovered viruses in Atlantic salmon to pinpoint the specific residues involved in cell adaptation and attenuation of IPNV.
Impacts
The results from these studies will aid in the development of a subunit or a live attenuated IPNV vaccine, which would be beneficial for the aquaculture industry.
Publications
- Song, H., Santi, N., and Vakharia, V.N. (2003) Molecular determinant of cell adaptation in infectious pancreatic necrosis virus. 22nd Annual Meeting of American Society for Virology, July 12-16, Davis, CA.
- Santi, N. Vakharia, V.N., and Evensen, O. (2004) Identification of putative motifs involved in the virulence of infectious pancreatic necrosis virus. Virology (in press)
- Shivappa, R.B., McAllister, P.E., Edwards, G.E., Santi, N., Evensen, O., and Vakharia, V.N. (2004) Development of a subunit vaccine for infectious pancreatic necrosis virus using a baculovirus insect/larvae system. Dev. Biol. Stand. (in press)
- Shivappa, R.B., Song, H., Aas-Eng, A., Evensen, O, and Vakharia, V.N. (2004) Molecular characterization of serotype Sp strains of infectious pancreatic necrosis exhibiting difference in virulence. Dis. Aqua. Org. (submitted)
- Song, H., Santi, N., Jagus, R., Evensen, O., and Vakharia, V.N. (2004) Characterization of the nonstructural proteins of infectious pancreatic necrosis virus using reverse genetics. Virology (submitted)
|
Progress 01/01/02 to 12/31/02
Outputs
The genomic segments of the virulent and attenuated IPNV strains have been completely cloned and sequenced. Comparison of the deduced amino sequences of the virulent NVI15 strain with tissue culture adapted attenuated strain (ALV103) show only six amino acid differences in the structural protein VP2 and two in the polymerase protein. These findings allowed us to identify the major determinants of virulence in IPNV. Full-length cDNA clones of the virulent NVI15 strain have been constructed, and additional clones have been prepared, which contains site-specific mutations of selected residues in VP2. In another study, we have cloned the genes of IPNV in a baculovirus expression system, which results in the synthesis of IPNV structural proteins in insect larvae that form artificial, virus-like particles. These virus-like particles have the external structure of the real IPNV, but it is devoid of the viral nucleic acid. Atlantic salmon that received a single dose of the
injectable, recombinant subunit vaccine evoked a protective response against IPNV challenge and exhibited significantly lower cumulative mortality rate (39%) than the control fish (77%). In addition, rainbow trout fry vaccinated by immersion also showed a lower cumulative mortality rate (8%) than the control fish (14%).
Impacts
The results of this study will greatly aid in the development of recombinant vaccines for IPNV and will minimize losses should virulent strains of IPNV emerge in the United States.
Publications
- Song, H., and Vakharia, V.N. 2002. Analysis of VP5 protein from three isolates of infectious pancreatic necrosis virus. 21st Annual Meeting of American Society for Virology, July 20-24, Lexington, KY.
- Santi, N., Vakharia, V.N., and Evensen, O. 2002. Sequence analysis of the A-segment of various infectious pancreatic necrosis strain with different virulence characteristics. 5th International symposium on viruses of lower vertebrates: comparative virology of amphibians, reptiles and fish, August 27-30, Seattle, WA.
- Evensen, O., Santi, N., and Vakharia, V.N. 2002. Genetic changes in infectious pancreatic necrosis due to cell culture adaptation. 4th International Symposium on Aquatic Animal Health, September 1-5, 2002, New Orleans, LA.
- Shivappa, R.B., McAllister, P.E., and Vakharia, V.N. 2002. A subunit vaccine for infectious pancreatic necrosis virus (IPNV) using a baculovirus insect/larvae system. 4th International Symposium on Aquatic Animal Health, September 1-5, 2002, New Orleans, LA.
|
Progress 01/01/01 to 12/31/01
Outputs
Infectious pancreatic necrosis virus (IPNV), a pathogen of major economic importance to the aquaculture industry, causes a highly contagious and destructive disease of juvenile rainbow trout as well as Atlantic salmon. Highly virulent strains of IPNV can cause greater than 90% mortality in hatchery stocks less than four months old and the surviving fish can become lifelong carriers of IPNV. The overall goal of this project is to determine which gene(s) of IPNV are responsible for causing mortality in fish. The objectives of this project are to: 1) clone and sequence the genomic segments of a virulent and an attenuated IPNV strains; 2) construct chimeric cDNA clones of a virulent and tissue-culture adapted IPNV strain; 3) produce chimeric viruses using reverse genetics and evaluate the characteristics of these viruses in fish. To this end, we have completed objective 1 and part of objective 2. The genomic segments of the virulent and attenuated IPNV strains have been
completely cloned and sequenced. Comparison of the deduced amino sequences of the virulent ALV122 strain with tissue-culture adapted attenuated strain (ALV122-10) show only two amino acid differences in the structural protein VP2, and none in the VP3, VP4 or VP1 (polymerase) proteins. Several cDNA clones of IPNV have been constructed including, full-length clones of the virulent ALV122 strain. Work is in progress to generate chimeric cDNA clones of the virulent and attenuated IPNV strains.
Impacts
The results of this study will greatly aid in the development of recombinant vaccines for IPNV and will minimize losses should virulent strains of IPNV emerge in the United States.
Publications
- Shivappa, R., and Vakharia, V.N. (2001). Identification of amino acid changes involved in the attenuation of highly virulent infectious pancreatic necrosis virus. 20th Annual Meeting of American Society for Virology, July 21-25, Madison, WI.
- Vakharia, V.N. (2001). Molecular basis of virulence in infectious pancreatic necrosis virus. International Symposium on fish virus pathogenesis and disease control, August 27-31, Taipei, Taiwan.
|
|