Phylogenetic and Mutational Analysis of Neuraminidase of Avian Influenza A (H9N2) Virus of B2-Sub Lineage, Isolated from Commercial Poultry Pakistan

Avian influenza A virus subtype H9N2 has been in circulation since last two decades in poultry industry of Pakistan. This virus is causing huge economic loss every year due to poor weight gain, drop in egg production and mortality of birds since first reported in 1998. Influenza viral surface protein neuraminidase enhances both virus replication and its release from host cells. In this study we isolated nineteen H9N2 viruses from infected birds of various farms from Punjab, Pakistan. Neuraminidase gene of these viruses was amplified using RT-PCR and sequenced to perform mutational analysis. Phylogenetic analysis revealed that B2 sub-lineage is endemic in the country as all isolated H9N2 strains belongs to this clade of G-1 lineage. Two isolated H9N2 strains A/Chick/Pak/303/2018 and A/Chick/Pak/436/2019 contain six glycosylation sites instead of seven as found in their ancestral strains. A comparative analysis of neuraminidase amino-acids sequences of B2 sub-lineage with Y-280 lineage and B1 sub-lineage demonstrated that there were no human related substitutions, insertion or deletions in hem-absorbing site and loop of H9N2 isolates. Two unique substitutions, Gln39Arg and Lys47Glu have been noted in NA stalk domain of all isolates of this study which might be indirectly enhancing the virulence of virus. These findings demonstrated that new mutations are emerging in B2 sub-lineage and there is need for constant surveillance of evolving genome of H9N2 virus prevailing in the country to combat the future challenges of avian influenza out breaks in Pakistan.