A single mutation in the PB1-F2 of H5N1 (HK/97) and 1918 influenza A viruses contributes to increased virulence

Abstract

The proapoptotic PB1-F2 protein of influenza A viruses has been shown to contribute to pathogenesis in the mouse model. Expression of full-length PB1-F2 increases the pathogenesis of the influenza A virus, causing weight loss, slower viral clearance, and increased viral titers in the lungs. After comparing viruses from the Hong Kong 1997 H5N1 outbreak, one amino acid change (N66S) was found in the PB1-F2 sequence at position 66 that correlated with pathogenicity. This same amino acid change (N66S) was also found in the PB1-F2 protein of the 1918 pandemic A/Brevig Mission/18 virus. Two isogenic recombinant chimeric viruses were created with an influenza A/WSN/33 virus background containing the PB1 segment from the HK/156/97: WH and WH N66S. In mice infected with WH N66S virus there was increased pathogenicity as measured by weight loss and decreased survival, and a 100-fold increase in virus replication when compared to mice infected with the WH virus. The 1918 pandemic strain A/Brevig Mission/18 was reconstructed with a pathogenicity-reducing mutation in PB1-F2 (S66N). The resultant 1918 S66N virus was attenuated in mice having a 3-log lower 50% lethal dose and caused less morbidity and mortality in mice than the wild-type virus. Viral lung titers were also decreased in 1918 S66N-infected mice compared with wild-type 1918 virus-infected mice. In addition, both viruses with an S at position 66 (WH N66S and wt 1918) induced elevated levels of cytokines in the lungs of infected mice. Together, these data show that a single amino acid substitution in PB1-F2 can result in increased viral pathogenicity and could be one of the factors contributing to the high lethality seen with the 1918 pandemic virus.

Figure 1. Alignment and Location of N66S Mutation in the PB1-F2 Protein

Alignment of human isolates of influenza A viruses from a H5N1 Hong Kong outbreak and from the 1918 H1N1 pandemic. Viruses in red are of the high-pathogenic phenotype, those in purple are of intermediate pathogenicity, and those in blue are of low pathogenicity in mice. Yellow indicates C-terminal region with α-helical structure, and green indicates the minimal mitochondrial targeting sequence.

Figure 2. In Vitro Growth Curve of Recombinant Viruses

(A) MDCK cells were inoculated at an MOI of 0.1 and 0.001, and virus growth of WH and WH N66S was assessed at the time points indicated. The figure is representative of three similar experiments. (B) MDCK cells were inoculated at an MOI of .01 and .001, and virus growth of r1918 and r1918 S66N was assessed at the time points indicated.

Figure 3. Contribution of PB1-F2 N66S Mutation to Pathogenicity of Recombinant Virus

(A) Mice were inoculated with 1 × 10⁴ PFU of virus or PBS, and their weights were recorded every day after infection. (B) Virus titers from lung homogenates were measured from mice infected with WH or WH N66S virus at days 1, 2, 3, 5, 7, and 8 after inoculation. Error bars represent 1 standard deviation.

Figure 4. The Effect of the S66N Mutation on Pathogenicity in Mice

(A) Eight mice were inoculated with recombinant wt 1918 virus at 10², 10³, 10⁴, 10⁵, or 10⁶ PFU, and their weights were measured every day. Average weights are represented in the graph. (B) Eight mice were inoculated with recombinant 1918 S66N virus at 10²,10³, 10⁴, 10⁵, or 10⁶ PFU, and their weights were measured every other day. Average weights are represented in the graph. (C) Effect of S66N mutation on mouse lung titers in r1918 virus. Virus titers from lung homogenates were measured from 3 mice infected per time point with r1918 or r1918 S66N virus at days 1, 2, 3, 6, 7, and 8 after inoculation. Error bars represent 1 standard deviation.

Figure 5. Cytokine Levels in Lungs of Infected Mice

(A) IFN-γ enzyme-linked immunosorbent assay was performed on lung homogenates with four mice per time point for each virus and is represented here in ng/ml. *p < .05. (B) TNF-α enzyme-linked immunosorbent assay was performed on lung homogenates with four mice per time point for each virus represented here in ng/ml. *p < .05. (C) Lung homogenates collected on day 4 after inoculation were measured for the levels of IL-1β, IFN-γ, and TNF-α using the Bioplex Protein Array system. All error bars represent 1 standard deviation.