Phylogenetic analysis of influenza C virus nonstructural (NS) protein genes and identification of the NS2 protein

The nucleotide sequences of RNA segment 7 (nonstructural protein gene; NS) were compared among 34 influenza C virus strains isolated between 1947 and 1992. The results showed that all the NS genes analysed had the potential to encode NS1 and NS2 proteins of 246 and 182 amino acids, respectively. The deduced amino acid sequence of the previously unidentified NS2 was fairly well conserved, although it was more divergent than the NS1 protein sequence. Moreover, immunoprecipitation experiments with rabbit immune serum against a glutathione S-transferase fusion protein containing the C-terminal region of the 182 amino acid NS2 protein revealed synthesis of a protein with an apparent molecular mass of approximately 22 kDa in infected cells. A phylogenetic analysis showed that the 34 NS genes were split into two distinct groups, A and B. Comparison of the phylogenetic positions of the individual isolates in the NS gene tree with those in the haemagglutinin-esterase (HE) gene tree suggested that most of the influenza C viruses currently circulating in Japan, irrespective of their HE gene lineage, had acquired group B NS genes through reassortment events that presumably occurred either in the 1970s or in the early 1980s.