Naturally occurring NS gene variants in an avian influenza virus isolate

The A/Turkey/Wisconsin/68 (H5N9) isolate of avian influenza (AI) consists of two virus populations which have different NS genes and differ in their biological responses in chicken embryos. They were classified as being either rapidly embryo-lethal (REL) or slowly embryo-lethal (SEL), (Avian Dis., 33 (1989) 695-706). In this study, sequence analysis identified only two nucleotide differences between the two NS genes, creating single amino acid differences in both the NS1 and the NS2 protein. The difference in the NS1 protein appears to be neutral, while the differences in the NS2 places a phenylalanine at position 48. This amino acid has not been previously demonstrated at this position in an NS2 sequence and its presence results in a distinct hydrophobic shift in the region. The sequence specifying the phenylalanine also creates an EcoRI site in the cDNA of the REL NS gene. Analysis of several clones showed that this site appears to co-segregate with the REL characteristic. Molecular differences between the two NS gene variants were reflected by differences in the kinetics of early protein synthesis in infected cells. In particular, the NS2 protein is in higher concentration (relative to the NS1) in SEL-infected cells than in REL-infected cells. No differences were detectable, however, in the rates of viral replication, either in cell culture or in embryos. Also, the REL or SEL rate was established early during infection of the embryo and could not be competed out by the other variant population 3 h after inoculation. Thus, these two natural NS gene variants appear to specify early differences which influence the time of death of an infected embryo but the differences do not appear to influence virus replication.