Comparison of Pathogenicity between PR8F and Different Mutants on the NS1 Locus in Mice

The aim of this study was to explore the influence of mutation of different non-structural (NS) 1 amino-acid residues on the pathogenicity of influenza viruses and the function of NS1 virulence-related sites on the pathogenesis of influenza viruses. We analyzed segments of the NS1 protein gene and key sites related to virulence of influenza viruses based on a literature review. Fragments of the NS1 gene were cloned from the HIN1 subtype PR8F (non-mutated) and preserved by our research team with encoding sequence site-specific mutagenesis at aa42, aa8l, and aa149. Via a reverse genetics system, we rescued the mutant strains PR8F-42, PR8F-81, and PR8F-149, which were inoculated into chick embryos and could replicate stably after five passages. Efficiency of viral replication was measured by testing hemagglutination titers. BALB/c mice were inoculated With mutated or non-mutated PR8F (10(6) TCIDO(50)/100 μl for each mouse), respectively. The typical clinical manifestations (weight change and survival) were recorded. Autopsies, as well as observations of the pathologic features and pulmonary-tissue slices of mice that died after inoculation, were done. RNA of mouse lungs was extracted, and the residual quantity of virus in lungs was detected by quantitative polymerase chain reaction (qPCR). Results showed that mutation of NS1 at aa42 from Ser to Pro did not change the pathogenicity of PR8F in mice, but a low pathogenicity of PR8F occurred after mutation of NS1 at aa8l and aa149. The present study lays the foundation for further investigations of the function of NS1 pathogenicity-related sites in the pathogenesis of influenza viruses.