Sequencing and Phylogenetic Analysis of NS Gene of Two Isolates of H9N2 Influenza Virus and The Growth Characteristics of the Virus on A549 Cells

Avian influenza virus of H9N2 subtype has become panzootic during the last two decades in Asia. In addition to causing disease in poultry, this virus has been identified as the reason for several human diseases in Asia. The present study aimed to investigate the phylogenetic relationship between NS gene of two viruses (SS2\2008 and SS8\2011) and other Iranian isolated viruses, and we characterized the growth ability of two viruses in human A549 cell. Among the H9N2 viruses isolated from 2008 to 2011, two isolated were placed in different positions in the phylogenetic tree. The whole NS gene fragments of two virus isolates were amplified using reverse transcription-polymerase chain reaction (RT-PCR) method; the fragment of each isolate was cloned and then sequenced. Possible changes in the NS gene sequence of isolates were compared with subtypes registered in Gene Bank Database and their growth abilities in human A549 cells were evaluated. GC3s value and replacement rate of Ka/Ks value in NS gene locus were compared. The initial phylogenetic process revealed that the NS gene of Iranian isolates had a homology of 96.4% belonging to clade Y439 and Allele A. These viruses, based on the time of isolation, were divided into two subgroups of viruses isolated from poultry during the years 1998-2004 and viruses isolated during a time curse of the years 2006-2011. GC3s value varied between 0.371-0.392 and the Ka/Ks value NS gene locus was 0.38 and 0.42 in subgroups 1 and 2, respectively. Processing of NS amino acid sequence not only exhibited that these two isolates contained KSLR sequence in their PDZ domain and but also it revealed that multiple simultaneous changes had been occurred. The viruses titers of two isolates, based on plaque assay, were not statistically significant. These data demonstrate that two Iranian H9N2 isolates are affected by some genetic drifts.