Evolution and mammalian adaptation of H3 and H10 subtype avian influenza viruses in wild birds in Yancheng Wetland of China

Wild birds play a critical role in avian influenza virus (AIV) ecology and some outbreaks of avian influenza in human originate from wild birds, suggesting that epidemiological surveillance and interspecies-transmission analysis of AIVs in wild birds are critical. Since 2019, we have performed sampling in Yancheng Wetland along the East Asian-Australasian Flyway. Totally, 2054 fecal swabs were collected and one H3N8, two H3N1, one H10N8, and three H10N1 were isolated. Three H3 gene of AIVs we isolated belonged to Eurasian lineage, but the four H10 gene clustered into North American lineage. What's more, the H3 and the foreign H10 gene had generated novel reassortants in Yancheng wetland. Receptor binding assay indicated that nearly all strains, except D369/H10N1, presented a dual receptor-binding profile and bound to avian-type receptor preferentially. In animal experiment, all isolates could infect mice without prior adaptation and induce histopathological changes in mice lungs, moreover, all H3 subtype AIVs obviously triggered weight loss of mice. In addition to lung and turbinate, D322/H3N1, D338/H3N8, D211/H10N8 and D266/H10N1 could spread to brain and kidney or liver or spleen, showing a wider range of tissue tropism. Multiple mutants associated with mammalian adaptation were also detected in all isolates according to molecular analysis. These findings revealed that H3 and H10 AIVs circulating in wild birds in Yancheng Wetland underwent complex reassortment and increased mammalian adaptation, which highlighted the necessity to monitor the diverse reassortment of AIVs in wild birds and evaluate the risks of H3 and H10 viruses to human health.