Molecular changes in A/Chicken/Pennsylvania/83 (H5N2) influenza virus associated with acquisition of virulence

One of the unresolved questions concerning the acquisition of virulence by the A/Chicken/Pennsylvania/83 (H5N2) influenza virus is which gene segments other than the hemagglutinin (HA) showed changes that were relevant. To answer this question, reassortants were made possessing the hemagglutinin gene of the virulent virus and the seven other genes from the avirulent parent. Since both the virulent and avirulent H5N2 strains are antigenically almost indistinguishable, it was necessary to transfer the genes of interest to a "carrier" virus before the appropriate reassortment could be selected. The gene compositions of the reassortants was established by a combination of sequence analysis and migration on polyacrylamide gels. These analyses established that the avirulent influenza virus present in April 1983 possessed seven of the eight gene segments necessary for virulence; mutation(s) in the HA gene were required for acquisition of virulence. Other viruses such as A/Seal/Mass/1/80 (H7N7) could provide the other genes necessary for virulence. Two changes in the HA have been associated with the acquisition of virulence; these are at amino acid residues 23 and 78 (H3 numbering) (Y. Kawaoka and R.G. Webster, Virology, 146, 130-137 (1985]. Isolation of an amantadine-resistant avirulent revertant virus provided the opportunity to determine which of the two amino acid changes in HA is critical. Sequence analysis of the revertant virus revealed amino acid changes at residues 23 in HA1 and 40 in HA2 (H3 numbering). The change at residue 23 of HA1 is probably associated with reversion to avirulence, of cleavability of HA, and inability to plaque in tissue culture without trypsin; while the change at residue 40 of HA2 may be associated with the amantadine-resistant phenotype. These studies establish that a single critical point mutation in the hemagglutinin gene of the avirulent A/Chicken/Pennsylvania/1/83 (H5N2) was probably all that was required to produce the highly virulent Chicken/Pennsylvania virus; the avirulent virus already possessed the other genes necessary for virulence.