Infectious salmon anemia virus (ISAV) is classified in the genusIsavirus of the family Orthomyxoviridae. Although virulence variation of ISAV can be demonstrated experimentally in fish, virus strain identification is ambiguous because the correlates of pathogenicity and/or antigenicity of ISAV are not well defined. Thirteen ISAV isolates characterized for their ability to kill fish were used to search for markers of virulence on the virus surface glycoprotein genes; haemagglutinin-esterase (HE) and fusion (F) protein genes. A single amino acid change N 164 D in the putative globular head of the HE protein, and a deletion/insertion of ¡13 aa with the presence of a specific motif 352 FNT 354 in the highly polymorphic region spanning residues 337 V to M 372 in the HE protein stalk, in combination with a specific motif 265 YP 266 very close to the trypsin-cleavage site 267 RA/G 268 of the precursor F 0 protein were correlated with reduced cytopathogenicity and reduced virulence for Atlantic salmon. Phylogenetic analysis suggests that the original ancestral ISAV was virulent. The virulence of the North American genotype has not changed much, whereas the European genotype evolved into two genogroups, the real-European genogroup that is still virulent and the European-in-North America genogroup, which is of lower virulence. A novel phylogenetic software program, BACKTRACK, estimated that the North American and European genotypes diverged between 1879 and 1891, whereas the European-in-North America genogroup diverged from the real-European genogroup between 1976 and 1988. This direction of evolution supports insertion of specific motifs in the HE protein, resulting in ISAV attenuation.
The larger genome segment, segment A, of infectious bursal disease virus (IBDV) encodes VP2, VP3 and VP4 as a precursor polyprotein. The viral protease, VP4, is responsible for self-processing of the polyprotein, however, there are additional secondary precursor products such as VPX whose further processing has not been defined. Expression of IBDV cDNAs in vitro with rabbit reticulocyte lysates in a coupled transcription-translation system and in the Sindbis virus expression system (with BHK-21 and Vero cell cultures) were used to study processing of the polyprotein. In both expression systems, we identified three main gene products with molecular masses of 48, 34, and 30.5 kDa corresponding to VPX, VP3, and VP4, respectively, as found in IBDV-infected Vero cell cultures, although the amount of each product was variable. A translational time course of the polyprotein gene and analyses of products specified by various sub-clones of the full-length cDNA were used to distinguish primary processing products of translation from secondary products generated by proteolytic processing during in vitro coupled transcription-translation expression. The VPX, VP3 and VP4, which are the primary processing products, first appeared after 20 min of incubation and their production was maximum by 75 min of the coupled transcription-translation reaction. Cycloheximide chases demonstrated that there is no secondary processing of VPX (or VP3 and VP4). Thus VP2, the major capsid protein in virions, was not detected either in translation products of rabbit reticulocyte lysates or in lysates of Sindbis virus recombinant-infected cell cultures indicating the absence of secondary processing of VPX to VP2 during foreign expression of the segment A cDNA. We conclude that VPX maturation to VP2 does not involve cellular proteases.
Background: Infectious salmon anaemia (ISA) virus (ISAV), an important pathogen of fish that causes disease accompanied by high mortality in marine-farmed Atlantic salmon, is the only species in the genus Isavirus, one of the five genera of the Orthomyxoviridae family. The Isavirus genome consists of eight single-stranded RNA species, and the virions have two surface glycoproteins; haemagglutinin-esterase (HE) protein encoded on segment 6 and fusion (F) protein encoded on segment 5. Based on the initial demonstration of two 5'-coterminal mRNA transcripts by RT-PCR, ISAV genomic segment 7 was suggested to share a similar coding strategy with segment 7 of influenza A virus, encoding two proteins. However, there appears to be confusion as to the protein sizes predicted from the two open reading frames (ORFs) of ISAV segment 7 which has in turn led to confusion of the predicted protein functions. The primary goal of the present work was to clone and express these two ORFs in order to assess whether the predicted protein sizes match those of the expressed proteins so as to clarify the coding assignments, and thereby identify any additional structural proteins of ISAV.
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