Molecular analysis of thets lesion residing on RNA1 of thecaA/AA/6/60 influenza virus vaccine.

Abstract

Predictors of the temperature sensitive (ts) phenotype exhibited by cold adapted (ca) A/AA/6/60-H2N2 influenza virus have been identified as two nucleotide changes on segment one of the RNA genome. These changes are associated with attenuation. Two modes whereby the ts phenomenon attributed to RNA1 of caA/AA/6/60 is lost have been characterized. The first deals with extragenic suppression and the second is attributed to the two sequence changes on RNA1. A reassortant of caA/AA/6/60 and A/Dunedin/4/73-H3N2, CR13 clone 5-3, was shown to be 5/3 in gene profile and ts+ as a result of extragenic suppression of RNA1. Although its RNA1 has the same nucleotide sequence as that of caA/AA/6/60 it has a ts+ phenotype. Through a series of backcross experiments and gene profile analysis of the resultant clones, the extrasuppressor genes were determined to be RNA2 coding for PB1 and/or RNA4 coding for HA. Sequence differences on RNA1 are related to the emergence of the ts+ phenotype of A/AA/6/60. RNA1 of seven different passages of wtA/AA/6/60, ranging from a ts virus two egg passages from the clinical isolate to a ts+ virus with a passage history of 51 tissue culture and egg passages at 35$\sp\circ$C have been sequenced. By correlating the appearance of the ts+ phenotype with the appearance of two mutations common to all ts+ RNA1's, nucleotides #141 and #821 were identified as predictors of the ts+ phenotype. Both nucleotide changes contribute to RNA folding configuration changes while the change at base #821 predicts an amino acid change from asparagine in the ts+ viruses to serine in the ts viruses. Possible implications for RNA and protein function are discussed. Viral virulence in ferrets indicates that there is no correlation between the phenotype of ts and virulence, but there is an association between the two bases predicting ts and attenuation. Viruses which are ts+ because of extragenic suppression of RNA1 are attenuated in ferrets whereas viruses which are ts+ because of sequence changes on RNA1 are virulent in ferrets and can replicate in ferret lungs. These two bases may be used to help predict the safety of a newly reassorted live influenza vaccine.