Institute of Aquaculture, University of Stirling, Stirling FK9 4LA, UK1
Author for correspondence: K. Riji John. Present address: Fisheries College and Research Institute, Tuticorin, 628 008 India. Fax +91 461 323187. e-mail rijijohn{at}vsnl.com
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() |
---|
![]() |
Main text |
---|
![]() ![]() ![]() ![]() |
---|
Stock BSNV used in this study was prepared by inoculating BSN cell culture supernatant onto bluegill (Lepomis macrochirus) fibroblast (BF-2) cells (Wolf et al., 1966 ). Once the CPE was complete, culture supernatant was clarified at 1500 g for 15 min and stored at -70 °C in aliquots. Catfish reovirus (CRV; Amend et al., 1984
), provided by R. P. Hedrick (School of Veterinary Medicine, University of California, USA), sandgoby virus (SGV; Hedrick et al., 1986
), provided by J. L. Fryer (Oregon State University, USA), IPNV reference strains WB and TV-1, provided by P. F. Dixon (CEFAS, Weymouth, UK), and IPNV Sp and IPNV Ab were also used in the present study. BSNV was propagated and assayed in striped snakehead (SSN-1) cells (Frerichs et al., 1991
) or in BF-2 cells. The brown bullhead (BB) cell line (Wolf & Quimby, 1969
) was used to grow CRV. For biochemical characterization, IPNV Sp and SGV were grown in chinook salmon (Oncorhynchus tshawytscha) embryo (CHSE-214) cells and for serological studies all the IPNV strains and BSNV were grown and assayed in BF-2 cells. The cell lines were maintained in Eagle's minimum essential medium (EMEM) or L-15 medium with 10% foetal bovine serum and after virus inoculation the serum content was reduced to 5%. Cells inoculated with viruses other than IPNV strains were incubated at 25 °C; IPNV-inoculated cells were incubated at 20 °C. Viruses grown in the various cell lines were purified after concentrating the virus in the clarified cell culture supernatant by PEG precipitation according to the method of Mahy & Kangro (1996)
. The virus from pelleted cell debris was repeatedly extracted by treating with trifluorotrichloroethane (Sigma) after freezethawing the pellet. The resulting aqueous phases from PEG precipitation and trifluorotrichloroethane treatment were layered on to a discontinuous CsCl gradient and the virus was banded isopycnically by centrifuging at 130000 g for 17 h. The virus bands were collected by puncturing the centrifuge tubes with syringe and needle, repelleted at 100000 g for 90 min and stored at -20 °C after resuspending in TNE (0·01 M TrisHCl, pH 7·5, 0·1 M NaCl, 1 mM EDTA).
BSNV grew well in SSN-1 cells at 2530 °C and in BF-2 at 25 °C but was unable to multiply in CHSE-214, EPC (Epithelioma papillosum cyprini), RTG-2 (rainbow trout gonad) or FHM (fathead minnow) cells. The CPE in SSN-1 differed from that of IPNV and was characterized by the aggregation of granular refractile cells forming a mesh-like appearance in the monolayer. Within 4872 h, the CPE spread across the entire cell sheet followed by detachment and cell lysis. Haemagglutination studies revealed that BSNV lacked the ability to haemagglutinate human O-type red blood cells at room temperature (25 °C).
The presence of a double-stranded RNA genome was demonstrated by the lack of inhibition of virus replication in SSN-1 cells growing in a culture medium containing 50 µg/ml 5-iodo-2'-deoxyuridine and the presence of yellowish-green cytoplasmic inclusions after acridine orange staining of 15 h virus-infected cell cultures (Rovozzo & Burke, 1973 ). The absence of a lipid-containing envelope was confirmed by resistance of infectivity to chloroform treatment (Feldman & Wang, 1961
). The virus was resistant to heat treatment at 56 °C for 2 h and was also stable at acid (3·0) and alkaline (9·0) pH for 30 min (Rovozzo & Burke, 1973
). Isopycnic centrifugation of pelleted virus at 130000 g for 17 h in a 2040% discontinuous CsCl gradient and assay of recovered fractions in SSN-1 cells established a buoyant density of 1·33 g/ml for infective particles. Transmission electron micrographs of ultrathin sections of an infected SSN-1 cell culture fixed in glutaraldehyde, post-fixed in 1% osmium tetroxide and stained in uranyl acetatelead citrate showed intracytoplasmic inclusions similar to viroplasms and scattered, fully formed virions. The virions were also sometimes found accumulated in the intercellular spaces (Fig. 1
a). Preparations of the virus negatively stained with 2% phosphotungstic acid revealed the presence of icosahedral, non-enveloped, single-shelled particles with a mean diameter of 57±1·6 nm (n=32) (Fig. 1b)
.
|
|
Serological comparison of BSNV and IPNV was performed by reciprocal ß cross-neutralization of BSNV and four classical strains of IPNV (WB, Sp, Ab and TV-1) by the method of Okamoto et al. (1983) with minor modifications. Antisera against two of the IPNV reference strains WB and TV-1 were provided by P. F. Dixon (CEFAS, Weymouth, UK). Antisera were serially five-fold diluted in microtitre plates (40 µl per well) and mixed with ~100 TCID50 (40 µl) virus. The virusantiserum suspension was incubated at room temperature (24 °C) for 60 min with frequent mixing on a plate shaker (Titretek, Flow Labs). BF-2 single-cell suspension (100 µl) was added to each well and the plates were incubated at the temperatures given above and observed for 1014 days for the development of CPE. Neutralizing antibody titre (ND50) of antiserum was expressed as the highest antiserum dilution protecting 50% of the inoculated cultures, as calculated by the SpearmanKärber method (Kärber, 1931
). Serological relationships (1/r) between the IPNV strains and BSNV, calculated from the formula r=
(r1xr2), where r1 and r2 are the titre ratios (Archetti & Horsfall, 1950
), are shown in Table 1
.
|
The type species of the genus Aquabirnavirus, IPNV, currently has 10 serotypes identified in two serogroups, A and B, which show some kind of cross-reactivity within each serogroup but not between the two serogroups (Hill & Way, 1996 ). The present study, involving three classical serotypes of serogroup A and the sole serotype of serogroup B, revealed that BSNV did not have any cross-reaction with strains of either serogroup. SGV, isolated from an ulcerated sandgoby in Thailand, is another tropical birnavirus reported to be significantly different from the classical IPNV strains (Hedrick et al., 1986
). This isolate, however, which showed some degree of cross-reaction with IPNV Ab and Sp, has later been recognized as a mixture of IPNV Ab and Sp (Hill & Way, 1996
).
Apart from serological distinctness, the differences in the molecular masses and migration patterns of capsid proteins and viral RNAs also distinguish BSNV from other reported aquabirnavirus strains. Phenotypically, the virus was different from all the IPNV strains, as it was unable to multiply in CHSE-214, RTG-2, EPC or FHM cell lines (Macdonald & Gower, 1981 ). Although BSNV was identified as belonging to the genus Aquabirnavirus, biochemical, biological and serological characteristics demarcated it from reported strains of the type species of the genus, IPNV. Since the virus cannot be compared with any of the existing IPNV strains and also since the virus was serologically distinct from the two reported serogroups of IPNV, BSNV could be classified currently as third serogroup of IPNV (serogroup C) in the genus Aquabirnavirus. Further studies would, however, be required to clearly identify whether the virus really forms a new serogroup or a new species altogether under the genus Aquabirnavirus. The name blotched snakehead virus is therefore proposed for this agent.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() |
---|
Archetti, I. & Horsfall, F. L.Jr (1950). Persistent antigenic variation of influenza A viruses after incomplete neutralization in ovo with heterologous immune serum. Journal of Experimental Medicine 92, 441-462.[Medline]
Burleson, F. G., Chambers, T. M. & Wiedbrauk, D. L. (1992). Virology: A Laboratory Manual, pp. 186246. London: Academic Press.
Chen, M. M., Kou, G. H. & Chen, S. N. (1993). Establishment and characterization of a cell line persistently infected with infectious pancreatic necrosis virus (IPNV). Bulletin of the Institute of Zoology, Academia Sinica 32, 265-272.
Dobos, P. (1996). The molecular biology of infectious pancreatic necrosis virus (IPNV). Annual Review of Fish Diseases 5, 25-54.
Dobos, P., Hallett, R., Kells, D. T. C., Sorensen, O. & Rowe, D. (1977). Biophysical studies of infectious pancreatic necrosis virus. Journal of Virology 22, 150-159.[Medline]
Dobos, P., Hill, B. J., Hallett, R., Kells, D. T. C., Becht, H. & Teninges, D. (1979). Biophysical and biochemical characterization of five animal viruses with bisegmented double-stranded RNA genomes. Journal of Virology 32, 593-605.[Medline]
Dobos, P., Nagy, E. & Duncan, R. (1991). Birnaviridae. In Viruses of Invertebrates, pp. 301-314. Edited by E. Kurstak. New York: Marcel Dekker.
Dobos, P., Berthiaume, L., Leong, J. A., Kibenge, F. S. B., Muller, H. & Nicholson, B. L. (1995). Family Birnaviridae. In Virus Taxonomy. Sixth Report of the International Committee on Taxonomy of Viruses, pp. 240-244. Edited by F. A. Murphy, C. M. Fauquet, D. H. L. Bishop, S. A. Ghabrial, A. W. Jarvis, G. P. Martelli, M. A. Mayo & M. D. Summers. Vienna & New York: Springer-Verlag.
Duncan, R. & Dobos, P. (1986). The nucleotide sequence of infectious pancreatic necrosis virus (IPNV) dsRNA segment A reveals one large ORF encoding a precursor polyprotein. Nucleic Acids Research 14, 5934.[Medline]
Feldman, H. & Wang, S. (1961). Sensitivity of various viruses to chloroform. Proceedings of the Society for Experimental Biology and Medicine 106, 736-738.
Frerichs, G. N., Morgan, D., Hart, D., Skerrow, C., Roberts, R. J. & Onions, D. E. (1991). Spontaneously productive C-type retrovirus infection of fish cell lines. Journal of General Virology 72, 2537-2539.[Abstract]
Hedrick, R. P., Eaton, W. D., Fryer, J. L., Groberg, W. G.Jr & Boonyaratpalin, S. (1986). Characteristics of a birnavirus isolated from cultured sandgoby, Oxyeleotris marmoratus. Diseases of Aquatic Organisms 1, 219-225.
Hill, B. J. & Way, K. (1996). Serological classification of infectious pancreatic necrosis (IPN) virus and other aquatic birnaviruses. Annual Review of Fish Diseases 5, 55-77.
Kärber, G. (1931). Beitrag zur kollektiven behandlung pharmakologischer Reihenversuche. Archives of Experimental Pathology and Pharmacology 162, 480-483.
Laemmli, U. K. (1970). Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680-685.[Medline]
Macdonald, R. D. & Gower, D. A. (1981). Genomic and phenotypic divergence among three serotypes of aquatic birnaviruses (infectious pancreatic necrosis virus). Virology 114, 187-195.[Medline]
Magyar, G. & Dobos, P. (1994). Expression of infectious pancreatic necrosis virus polyprotein and VP1 in insect cells and the detection of the polyprotein in purified virus. Virology 198, 437-445.[Medline]
Mahy, B. W. J. & Kangro, H. O. (1996). Virology Methods Manual. London: Academic Press.
Okamoto, N., Sano, T., Hedrick, R. P. & Fryer, J. L. (1983). Antigenic relationships of selected strains of infectious pancreatic necrosis virus and European eel virus. Journal of Fish Diseases 6, 19-25.
Roberts, R. J., Frerichs, G. N., Tonguthai, K. & Chinabut, S. (1994). Epizootic ulcerative syndrome of farmed and wild fishes. In Recent Advances in Aquaculture, pp. 207-239. Edited by J. F. Muir & R. J. Roberts. Oxford: Blackwell Science.
Rovozzo, G. C. & Burke, C. N. (1973). A Manual of Basic Virological Techniques, pp. 126163. Englewood Cliffs, NJ: Prentice Hall.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning. A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Wolf, K. & Quimby, M. C. (1969). Fish cell line and tissue culture. In Fish Physiology, pp. 253-305. Edited by W. S. Hoar & D. J. Randall. New York: Academic Press.
Wolf, K., Gravell, M. & Malsberger, R. G. (1966). Lymphocystis virus: isolation and propagation in centrarchid fish cell lines. Science 151, 1004-1005.[Medline]
Received 17 February 1999;
accepted 28 April 1999.