Sequence variation of the glycoprotein gene identifies three distinct lineages within field isolates of viral haemorrhagic septicaemia virus, a fish rhabdovirus.

To evaluate the genetic diversity of viral haemorrhagic septicaemia virus (VHSV), the sequence of the glycoprotein genes (G) of 11 North American and European isolates were determined. Comparison with the G protein of representative members of the family Rhabdoviridae suggested that VHSV was a different virus species from infectious haemorrhagic necrosis virus (IHNV) and Hirame rhabdovirus (HIRRV). At a higher taxonomic level, VHSV, IHNV and HIRRV formed a group which was genetically closest to the genus Lyssavirus. Compared with each other, the G genes of VHSV displayed a dissimilar overall genetic diversity which correlated with differences in geographical origin. The multiple sequence alignment of the complete G protein, showed that the divergent positions were not uniformly distributed along the sequence. A central region (amino acid position 245-300) accumulated substitutions and appeared to be highly variable. The genetic heterogeneity within a single isolate was high, with an apparent internal mutation frequency of 1.2 x 10(-3) per nucleotide site, attesting the quasispecies nature of the viral population. The phylogeny separated VHSV strains according to the major geographical area of isolation: genotype I for continental Europe, genotype II for the British Isles, and genotype III for North America. Isolates from continental Europe exhibited the highest genetic variability, with sub-groups correlated partially with the serological classification. Neither neutralizing polyclonal sera, nor monoclonal antibodies, were able to discriminate between the genotypes. The overall structure of the phylogenetic tree suggests that VHSV genetic diversity and evolution fit within the model of random change and positive selection operating on quasispecies.

The glycoprotein of viral hemorrhagic septicemia virus (VHSV): antigenicity and role in virulence.

In order to study the antigenic structure of the G protein of VHSV, we produced several anti-G monoclonal antibodies (MAbs) and used 4 neutralizing MAbs (NMAbs) to select resistant (MAR) mutants. Each MAR mutant was confronted with the 4 NMAbs in a neutralization test, and also with our panel of MAbs in surface plasmon resonance (SPR) analysis to determine the extent of their relatedness. Determination of the sequence of the entire G gene of representative MAR mutants allowed us to map the mutations responsible for the resistant phenotypes. We identified several locations on the G protein sequence, which represent, most probably, critical positions within the binding sites of the neutralizing MAbs. In addition, the MAR mutants selected with a cross-reactive MAb exhibited a reduced pathogenicity for fish. This indicated that the regions bearing the point mutations selected with MAb C10 were probably involved in the determination of the virulent phenotype.

Genetic diversity and phylogenetic classification of viral hemorrhagic septicemia virus (VHSV).

The present study was undertaken to determine the genetic diversity of viral hemorrhagic septicemia virus (VHSV) and to gain insight into the molecular epidemiology of this fish rhabdovirus. The sequences of the nonstructural (NV) protein and the transmembrane (G) protein of sequential North American and European isolates of VHSV were determined and used to compute phylogenetic trees. According to the percentage of nucleotide or amino acid similarities, North American and European isolates formed 2 clearly distant genetic groups. While North American isolates clustered into a highly homogeneous genetic group, European isolates exhibited a higher genetic variability. Subgrouping based on this variability could be correlated with both the geographic origin and the serological classification.