PB2 and Hemagglutinin Mutations Are Major Determinants of Host Range and Virulence in Mouse-Adapted Influenza A Virus

Abstract

Serial mouse lung passage of a human influenza A virus, A/Hong Kong/1/68 (H3N2) (HK-wt), produced a mouse-adapted variant, MA, with nine mutations that was >10 3.8 -fold more virulent. In this study, we demonstrate that MA mutations of the PB2 (D701N) and hemagglutinin (HA) (G218W in HA1 and T156N in HA2) genes were the most adaptive genetic determinants for increased growth and virulence in the mouse model. Recombinant viruses expressing each of the mutated MA genome segments on the HK-wt backbone showed significantly increased disease severity, whereas only the mouse-adapted PB2 gene increased virulence, as determined by the 50% lethal dose ([LD 50 ] >101.4 -fold). The converse comparisons of recombinant MA viruses expressing each of the HK-wt genome segments showed the greatest decrease in virulence due to the HA gene (10 2 -fold), with lesser decreases due to the M1, NS1, NA, and PB1 genes (10 0.3 -to 10 0.8 -fold), and undetectable effects on the LD 50 for the PB2 and NP genes. The HK PB2 gene did, however, attenuate MA infection, as measured by weight loss and time to death. Replication of adaptive mutations in vivo and in vitro showed both viral gene backbone and host range effects. Minigenome transcription assays showed that PB1 and PB2 mutations increased polymerase activity and that the PB2 D701N mutation was comparable in effect to the mammalian adaptive PB2 E627K mutation. Our results demonstrate that host range and virulence are controlled by multiple genes, with major roles for mutations in PB2 and HA.

Fecal Influenza in Mammals: Selection of Novel Variants

“…This result is consistent with the findings of others (49,50). Fecal isolates were obtained from pooled samples from each group of mice, and lung isolates were obtained from individual mice.…”

“…The adaptive genetic variations were mainly detected in the PB2 and HA genes of the fecal and lung isolates, which is consistent with the findings of other laboratories, in terms of influenza virus adaptation and virulence in mice (49). Moreover, these mutations were generally maintained in the fecal isolates after the second passage, and additional mutations in different gene segments were acquired, especially on later days postinfection.…”

“…Similarly, the adaptation of human H3N2 virus (A/Hong Kong/1/68) requires 20 serial passages in the lung to observe adaptive mutations in the CD-1 mouse model (49). Increased pathogenicity has been observed with mouseadapted avian H9N2 following eight lung-to-lung passages (56).…”

Identification of RNA Helicase A as a Cellular Factor That Interacts with Influenza A Virus NS1 Protein and Its Role in the Virus Life Cycle

“…To each well, 500 l of the diluted A549 cells was added to obtain a final siRNA concentration of 40 nM in a final volume of 600 l. Cells were incubated at 37°C for 12 h; the medium was then replaced by OptiMEM. At 48 h after siRNA transfection, the cells were further transfected with phPolI-LUC-NP, which contains a firefly luciferase open reading frame flanked by noncoding regions of NP under the control of human RNA polymerase I protein (36), together with the plasmids pcDNA3-PB2, pcDNA3-PB1, pcDNA3-PA, and pcDNA-NP, which encode PB2, PB1, PA, and NP, respectively, and Renilla luciferase expression plasmid pTK-rLuc, using Lipofectamine LTX with Plus reagents per the manufacturer's protocol (Invitrogen). At 24 h posttransfection, the luciferase activity was measured using a DualLuciferase Reporter Assay kit (Promega) according to the manufacturer's protocol.…”

Influenza A Virus Acquires Enhanced Pathogenicity and Transmissibility after Serial Passages in Swine

“…Mice have been used extensively for investigating pathogenic mechanisms and host range determinants (10,11). Guinea pigs and ferrets support IAV transmission and have been used as models for IAV adaptation studies (12)(13)(14)(15)(16).…”