Fatal infection of a novel canine/human reassortant H3N2 influenza A virus in the zoo-housed golden monkeys

Influenza A virus (IAV) is one of the most challenging pathogens that threaten human and animal health. In June 2022, seven golden monkeys (Rhinopithecus roxellana) developed flu-like symptoms in succession at a zoo in Jiangsu Province, China, and two died of respiratory distress. Histopathological and immunostaining results supported a diagnosis of pulmonary infection with influenza virus. An H3N2 canine/human reassortment virus, designated A/golden monkey/Jiangsu/1/2022 (Gm-1), was isolated from the lungs of deceased animals. Sequencing analysis revealed that except for PB1 gene that was 99% similar to that from human H1N1 virus (A/WSN/1933, WSN), the other 7 segments of Gm-1 were entirely derived from previously circulating canine H3N2 virus (A/canine/Jiangsu/06/2010, JS06). To determine whether the human-origin PB1 segment conferred a virulence advantage to Gm-1, we reconstructed this reassortant event using reverse genetics and generated two reassortment viruses, rGm-1 and rJS06. Experimental infection of mice revealed that the rGm-1 virus induced more severe pathological damage along with higher levels of proinflammatory cytokines in the lungs, suggesting that replacement of the PB1 segment of JS06 by PB1 of WSN enhanced viral virulence. Furthermore, we identified a unique cytotoxic motif (68I, 69L and 70V) in PB1-F2 protein that was present in Gm-1 but absent in JS06, potentially enhancing viral virulence. This is the first report confirming IAV infection in golden monkeys. Our finding highlight the the importance of enhanced biosecurity surveillance for this endangered species.