Detection And Genetic Characterization Of Avian Influenza A (H5n6) Virus Clade 2.3.4.4 In Isolates From House Crow And Poultry In Bangladesh, 2017

Background: Bangladesh has experienced repeated house crow (Corvus splendens) mortality events due to highly pathogenic avian influenza (HPAI) viruses since 2011. We investigated one such event in Dhaka in 2017 to detect and molecular characterize the virus in crows and poultry from nearby live poultry markets (LPMs). Methods and materials: We collected cloacal and oropharyngeal swab samples from dead and moribund crows (N = 166) and environmental samples (N = 149) from 19 nearby LPMs. Samples were tested by qRT-PCR for influenza A (M gene) and H5/H7/H9/N1/N6. H5 positive samples were inoculated into embryonated eggs for virus culture. We performed conventional RT-PCR and Sanger sequencing of the isolated viruses and used MEGA7 for phylogenetic analysis. Results: 91.6% (n = 152; 95%Confidence Interval (CI): 86.3–95.3) of crows were positive with influenza A/H5 including 77.6% for H5N1 (n = 118), 2% for H5N6 (n = 3) and 20.4% H5Nx (n = 31). Of the 149 LPM samples, 46.3% (n = 69; 95%CI: 38.1–54.7) were positive for influenza A/H5 among which 85.5% for H5N1, 5.8% for H5N6 and 9% for H5Nx. We successfully isolated 2 H5N6; 1 from chicken and 1 from crow. Here we describe molecular characteristics of H5N6 only. Phylogeny of the HA gene showed 99.0% nucleotide similarity with the H5N8 virus belonging to clade 2.3.4.4B, isolated from Muscovy duck in Congo, 2017. NA gene showed 98.4% similarity with that of H4N6, isolated from duck in Mongolia, 2015. The PB1 and PA gene had high nucleotide identity, 98.0% and 99.0% respectively, with an H3N1 isolated from duck in Bangladesh, 2017. NP, M and NS genes of the two H5N6 isolates are closely related to H5N8. The isolates were identified as HPAIV based on multiple basic amino acids at the HA proteolytic cleavage site. Molecular markers in the HA protein suggested predicted binding to human receptor α2,6 linkage and increased pandemic potential. Conclusion: This investigation detected and characterized a novel H5N6 reassortant in wild birds for the first time and highlighted the potential for the exchange of HPAI between poultry and wild birds. Continued surveillance of both poultry and wild birds is needed for early detection of novel viral introductions, to trace the transmission route and to reduce public health risks.