Genes coding for polymerase proteins are essential for attenuation of the cold-adapted A/Leningrad/134/17/57 (H2N2) influenza virus

Abstract

Background: The A/Leningrad/134/17/57 (H2N2) (Len/17) cold-adapted (ca) master strain used as a donor of attenuation for preparing live attenuated influenza vaccine was derived from the A/Leningrad/134/57 wild-type virus (Len/wt) after 17 passages in eggs at 25 °C. In contrast to Len/wt, the Len/17 virus is sensitive to high (40 °C) temperature ( ts), is able to grow at low (25 °C) temperature ( ca), and is attenuated ( att) for humans. Len/17 is different from Len/wt by eight coding mutations in internal genes PB2 (V-478-L), PB1 (K-265-N, V-591-I), PA (L-28-P, V-341-L), M1 (I-15-V), M2 (A-86-T) and NS2 (M-100-I). However, the role of individual mutant genes in the manifestation of the ts, ca, and att phenotypes was not established. To evaluate this, single gene reassortants (SGRs) with one mutated gene in the background of the Len/wt genome were studied. Methods: Since heterogeneity (A- or T-86 in the M2 gene) was noted in Len/17, the ts and ca clone 28 of this donor (Len/17-cl28) that has A-86 in M2 was used to prepare the SGRs. Preservation of mutations in SGRs was monitored by PCR-restriction (RFLP) analysis. Virus replication in ferret lungs was observed to evaluate the att phenotype of SGRs. Results: PB2- and PB1-SGRs each manifested the ts phenotype, and PB2-SGR demonstrated the ca phenotype as well. PA-SGR was ca, but not ts. Mutations in the M1 and NS2 genes did not affect the ts or ca phenotypes of corresponding SGRs. Ferrets infected with Len/17-cl28, Len/wt and PB2-, PB1- or PA-SGRs demonstrated comparable titers in nasal turbinates on the third day, while infectious virus was found only in the lungs of ferrets infected with Len/wt and PA-SGR. Conclusion: These data suggest that mutations in the PB2 (V-478-L) and PB1 (K-265-N, V-591-I) genes play a critical role in the attenuation of the ca Len/17 vaccine donor strain.