Poultry trading behaviours in Vietnamese live bird markets as risk factors for avian influenza infection in chickens

Sealy, Joshua E.; Fournie, Guillaume; Trang, Pham Hong; Dang, Nguyen Hoang; Sadeyen, Jean-Remy; Thanh, To Long; van Doorn, H. Rogier; Bryant, Juliet E.; Iqbal, Munir

To ensure we could obtain sequencing data from our samples, we employed next-generation sequencing (NGS) on PCR products generated from viral RNA taken directly from pooled swab samples (Passage 0) and from infected allantoic fluid (Passage 1). Embryonated hens' eggs were inoculated with VTM from pooled swabs which had a Ct < 27 for H9 or any Ct for H5 and "unknown" subtype-positive samples. Allantoic fluid was harvested after 48 hr of incubation and confirmed for influenza virus A by haemagglutination (HA) assay. Viral RNA was extracted from allantoic fluid as above. In total, 50 samples meeting the above criteria were passaged in eggs, and this yielded 34 samples positive for HA activity. Passage 0 and Passage 1 samples were both subjected to NGS, and where possible, sequencing data for Passage 0 were used in phylogenetic analysis. Sequencing data were generated for a total of 33 viruses (31 H9N2 and 2 H5N6).