Characterization of the Non-Structural Gene of Influenza A Virus H1N1 in Iran

The non-structural (NS) genomic segment of influenza A virus expresses two proteins (NS1 and NS2) responsible for virulence and pathogenicity. Here, we characterize the NS gene of H1N1 influenza viruses isolated from Iranian patients during the 2015 and 2017 outbreaks.

Influenza A positive specimens with high viral load were selected for virus amplification on MDCK (Madin-Darby canine kidney) cells to obtain sufficient viral RNA for RT-PCR. The NS segments were amplified and sequenced from randomly selected samples. Genetic characterization, phylogenetic, and protein modeling analyses were carried out using Bioedit, MEGA7 software, and Muster, Modweb, Modfold, Tm-align web servers. Reference sequences from other geographic regions were available on GenBank.

Phylogenetic analysis on the NS gene of A/H1N1 isolates revealed that all Iranian isolates, except the A/Tehran/SMO08/2015, were close to strains from Continental countries of Asia and North America, with 99% identity. Meanwhile the mentioned exception was clustered with some strains from Iran, Kazakhstan, Myanmar, Turkmenistan, and Jordan. Overall six substitutions were observed through the deduced Iranian NS1 and NS2 amino acid sequences. One of the mutations (the E96D mutation from the isolate A/Tehran/A7106/2017) was first observed in the present project. Prediction of three-dimensional structure of the Iranian NS1 and NS2 proteins in comparison with counterpart references available in PDB showed a slight deviation in the functional domains. However, the high similarity of the Iranian NS gene with those from other countries indicate no significant changes in the molecular features of the NS genes.

The severity of the symptoms does not seem to be caused by the observed mutations. Molecular analysis of other alleles is needed to explain the high pathogenic feature of the 2015 isolates.