Temperature-sensitive mutants of influenza A virus. Transfer of the twots-1 A 2ts lesions present in the Udorn/72-ts-1 A 2 donor virus to the influenza A/Alaska/6/77 (H 3 N 2) wild type virus

Abstract

The Udorn/72-ts-1 A 2 temperature-sensitive influenza A virus has a 37° C shutoff temperature and ats mutation on the genes coding for the P1 and P3 proteins. Thists donor virus was produced with the expectation that the transfer of its twots genes would regularly and predictably attenuate each new variant of influenza A virus. It had previously been mated with the A/Victoria/75 (H 3 N 2) virus and five Vic/75-ts-1 A 2 recombinants were isolated that had bothts-1 A 2ts genes andin vitro andin vivo genetic and biological properties similar to their Udorn/72-ts-1 A 2 parent. The present study was designed to determine if the acquisition of the twots-1 A 2ts genes would also confer a specific level of attenuation on the influenza A/Alaska/6/77 (H 3 N 2) wild type virus. Fifteen recombinant Alaska/77-ts-1 A 2 viruses were isolated and characterized genetically for the number and location ofts mutations. These clones were also studied for their level of replication and genetic stability in hamsters. Four recombinants possessed both of thets-1 A 2 mutations and had a 37° C shutoff temperature for plaque formation. Two recombinants possessed only ats P1 gene and had either a 38° C or 39° C shutoff temperature. The remaining nine clones had thets P3 gene and a shutoff temperature of 37° C, 38° C or 39° C. Each of the four 37° C shutoff temperature recombinants that possessed bothts P1 and P3 genes were restricted at least 10,000-fold in replication in the hamster's lung and approximately 100-fold in the nasal turbinates compared to the level of replication of wild type virus in these sites. All isolates from these animals retained thets phenotype. The level of replicationin vivo of thets P1 and P3 segregants was related to their shutoff temperature of plaque formationin vitro, e.g. the 38° Cts P3 segregant was less restricted in replication in the lungs than a 37° Cts P3 segregant. All isolates from animals infected with thets P3 segregants werets, whereas a low level of genetic instability was detected for one of thets P1 segregants. Since ten independentts-1 A 2 recombinants (one Udorn/72, 5 Victoria/75 and 4 Alaska/77) with bothts-1 A 2 mutations exhibited the same genetic and biologic properties, it is likely that thesets P1 and P3 genes were the prime determinants of attenuation and could effect a similar level of attenuation in other influenza A viruses within the H 3 N 2 subtype.