Synergistic HA and NS mutations enhanced the virulence of a mouse-adapted H1N1 influenza A virus

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H1N1 reassortants between the swine Eurasian avian-like (EA) and H1N1 2009 pandemic (H1N1 pdm/09) viruses have been circulating stably in pig populations for more than ten years, and they may have contributed to increased human infections. Whether these H1N1 viruses acquire adaptive mutations to increase their pathogenicity towards a new host is unknown. To address this problem, mouse-adapted (MA) variants of swine-origin EA H1N1 influenza virus isolated from dogs (A/canine/Guangxi/LZ57/2015[LZ57-MA]) were generated by serial lung-to-lung passages in BALB/c mice. These exhibited greater virulence and replication capability than the wild-type virus (LZ57-WT). Of the six adaptive mutations, two were mapped to the ribonucleoprotein (RNP) complex (PB2-E578D and PA-T97I), two to hemagglutinin (HA-N198D and HA-A227E) and two to the non-structural protein 1 (NS1) and nuclear export protein (NS1-A53D and NEP-R42K, respectively). Reverse genetic substitution of the viral genes and mutation experiments demonstrated that the mutations in PA-T97I could enhance the polymerase activity, but a significant downregulation of activity was seen with PB2-E578D, which was consistent with a decrease in virulence. However, HA and NS, which are genes that act synergistically, were found to be determinants of virulence in mice. The reassortant viruses bearing HA mutations (N198D and A227E) were acquired during adaptation enhanced early-stage viral replication in mammalian cells. The single-point mutations in the NS genes had limited effects on virulence. Furthermore, a combination of HA (N198D and A227E) with NS(A53D) in the rLZ57-WT backbone resulted in efficient replication and a significant increase in virulence. The results suggest that these substitutions could compensate for the polymerase function and contribute to enhanced virulence, which highlights a major role for mutations in the HA and NS genes.