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-1A2 temperature-sensitive influenza A virus has a 37 degrees C shutoff temperature and a ts mutation on the genes coding for the P1 and P3 proteins. This ts donor virus was produced with the expectation that the transfer of its two ts genes would regularly and predictably attenuate each new variant of influenza A virus. It had previously been mated with the A/Victoria/75 (H3N2) virus and five Vic/75-ts-1A2 rcombinants were isolated that had both ts-1A2 ts genes and in vitro and in vivo genetic and biological properties similar to their Udorn/72-ts-1A2 parent. The present study was designed to determine if the acquisition of the two ts-1A2 ts genes would also confer a specific level of attenuation on the influenza A/Alaska/6/77 (H3N2) wild type virus. Fifteen recombinant Alaska/77-ts-1A2 viruses were isolated and characterized genetically for the number and location of ts mutations. These clones were also studied for their level of replication and genetic stability in hamsters. Four recombinants possessed both of the ts-1A2 mutations and had a 37 degrees C shutoff temperature for plaque formation. Two recombinants possessed only a ts P1 gene and had either a 38 degrees C or 39 degrees C shutoff temperature. The remaining nine clones had the ts P3 gene and a shutoff temperature of 37 degrees C, 38 degrees C or 39 degrees C. Each of the four 37 degrees C shutoff temperature recombinants that possessed both ts 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 the ts phenotype. The level of replication in vivo of the ts P1 and P3 segregants was related to their shutoff temperature of plaque formation in vitro, e.g. the 38 degrees C ts P3 segregant was less restricted in replication in the lungs than a 37 degrees C ts P3 segregant. All isolates from animals infected with the ts P3 segregants were ts, whereas a low level of genetic instability was detected for one of the ts P1 segregants. Since ten independent ts-1A2 recombinants (one Udorn/72, 5 Victoria/75 and 4 Alaska/77) with both ts-1A2 mutations exhibited the same genetic and biologic properties, it is likely that these ts 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 H3N2 subtype.

Temperature-sensitive mutants of influenza A virus: Evaluation of the Alaska/77-ts-1 A 2 temperature-sensitive recombinant virus in seronegative adult volunteers

“…This virus has • a) 37 ° C shutoff temperature for plaque formation (i. e., 100-fold or greater reduction of plaque formation at 37 ° C compared to that at 34 ° C), b) ts mutations on the genes coding for the P 1 and P 3 proteins, and e) the A/Alaska/77 hemagglutinin and neuraminidase glycoproteins (8). The isolation and cloning of the Alaska/6/77 wild type virus have been described (8). Importantly, the Alaska/77 wild type virus used to infect volunteers was a cloned population that also served as the wild type parent in the production of Alaska/77-ts-lA2 clone 190 recombinant.…”

“…The production of the A/Alaska/6/77-ts-1 A 2 recombinant, clone 190, was described previously (8). This virus has • a) 37 ° C shutoff temperature for plaque formation (i. e., 100-fold or greater reduction of plaque formation at 37 ° C compared to that at 34 ° C), b) ts mutations on the genes coding for the P 1 and P 3 proteins, and e) the A/Alaska/77 hemagglutinin and neuraminidase glycoproteins (8).…”

“…The Vic/75-ts-lA2 recombinant was administered to adult volunteers who lacked detectable serum a n t i b o d y to the hemagglutinin and neuraminidase antigens and was found to be satisfactorily a t t e n u a t e d and immunogenic (7). Subsequently, an Alaska/77-ts-1A2 recombinant was produced and was found to share the same genetic and biologic properties as its Udorn/72-ts-1A 2 parent (8). This Alaska/77-t s -l A 2 recombinant was evaluated for its a t t e n u a t i o n and antigenicity in serum hemagglutination-inhibiting (HAI) a n t i b o d y negative (titer of ~1 : 8 ) a d u l t volunteers in order to obtain more information concerning the regularity with which the two ts genes of the Udorn/72-ts-1 A 2 donor virus satisfactorily a t t e n u a t e new epidemic viruses within the I t 3 N 2 subtype.…”