Evolutionary pattern of the H 3 haemagglutinin of equine influenza viruses: multiple evolutionary lineages and frozen replication.

The nucleotide and deduced amino acid sequences of the haemagglutinin genes coding for the HA 1 domain of H3N8 equine influenza viruses isolated over wide regions of the world were analyzed in detail to determine their evolutionary relationships. We have constructed a phylogenetic model tree by the neighbour-joining method using nucleotide sequences of 15 haemagglutinin genes, including those of five viruses determined in the present study. This gene tree revealed the existence of two major evolutionary pathways during a twenty five-year period between 1963 to 1988, and each pathway appeared to consist of two distinct lineages of haemagglutinin genes. Furthermore, our analysis of nucleotide sequences showed that two distinct lineages of equine H3N8 viruses were involved in an equine influenza outbreak during the period of December 1971-January 1972 in Japan. The number of nucleotide changes between strains was proportional to the length of time (in years) between their isolation except for three of the HA genes. However, there are three exceptional strains isolated in 1971, 1987, and 1988, respectively. The haemagglutinin gene in these strains showed a small number of nucleotide substitutions after they branched off around 1963, suggesting an example of frozen replication. Although the estimated rate (0.0094/site/year) of synonymous (silent) substitutions of the haemagglutinin gene of equine H3N8 viruses was nearly the same as that of human H 1 and H 3 haemagglutinin genes, the rate of nonsynonymous (amino-acid changing) substitutions of the former equine virus gene was estimated to be 0.00041/site/year--that is about 5 times lower than that estimated for the human H 3 haemagglutinin gene. The present study is the first demonstration that multiple evolutionary lineages of equine H3N8 influenza virus circulated since 1963.