Identification and molecular characterization of H9N2 viruses carrying multiple mammalian adaptation markers in resident birds in central-western wetlands in India

We report here a targeted risk-based study to investigate the presence of influenza A viruses at the migratory-wild-domestic bird interface across the major wetlands of central India's Maharashtra state during the winter migration season. The H9N2 viruses have been isolated and confirmed in 3.86% (33/854) of the fecal samples of resident birds. To investigate the genetic pools of H9N2 circulating in resident birds, we sequenced two isolates of H9N2 from distant wetlands. Sequence and phylogenetic analyses have shown that these viruses are triple reassortants, with HA, NA, NP, and M genes belonging to G1 sub-lineage (A/quail/Hong Kong/G1/1997), PB2, PB1, and NS genes originating from the prototype Eurasian lineage (A/mallard/France/090360/2009) and PA gene deriving from Y439/Korean-like (A/duck/Hong Kong/Y439/97) sub-lineage. It was confirmed not only that four of their gene segments had a high genetic association with the zoonotic H9N2 virus, A/Human/India/TCM2581/2019, but also that they had many molecular markers associated with mammalian adaptation and enhanced virulence in mammals including the unique multiple basic amino acids, KSKR↓GLF at the HA cleavage site, and analog N-and O-glycosylation patterns on HA with that of the zoonotic H9N2 virus. Furthermore, future experiments would be to characterize these isolates biologically to address the public health concern. Importantly, due to the identification of these viruses at a strategic geographical location in India (a major stop-over point in the Central Asian flyway), these novel viruses also pose a possible threat to be exported to other regions via migratory/resident birds. Consequently, systematic investigation and active monitoring are a prerequisite for identifying and preventing the spread of viruses of zoonotic potential by enforcing strict biosecurity measures.