Genetic evolution and pathogenicity analysis of a canine-origin H9N2 subtype influenza virus

【Objective】To understand the characteristics of molecular variation of canine-origin H9N2 subtype influenza virus and its pathogenicity in mice. It would provide the scientific evidence for the cross-species transmission of aviancanine.【Method】An H9N2 subtype influenza virus was isolated from stray dogs,eight of its gene segments were amplified by RT-PCR for BLAST homology comparison,and then genetic evolution analysis was conducted based on 22 reference strains at home and abroad,and pathogenicity of the isolated strains was evaluated by mouse nasal drip infection test.【Result】From collected 393 samples of dog nose swabs,one subtype H9N2 influenza virus strain was isolated,named A/canine/Guangxi/LZ11/2018(abbreviated as LZ11). Genetic evolution analysis showed that LZ11 was a triple-ressortant strain,which contained HA,NA and NS genes from BJ/94-like lineage,PB2 and M genes from G1-like lineage,PB1,PA and NP genes from F/98-like lineage,whose genetic composition belonged to S genotype circulating widely in China recently. Sequence analysis showed that there was a cleavage site(PSRSSR↓GL) in the HA protein at position 324-331 of LZ11 isolate,which was consistent with the characteristics of low pathogenic influenza virus. HA protein had ²²⁶ Q→L mutation,which preferentially bound human alpha-2,6 sialic acid receptors. There were no mutations in ¹¹⁹ E,¹⁵¹ D,²⁷⁶ E,²⁹² R and ²⁹⁴ N in NA protein,while there was an ³¹ S→N mutation occurred in M2 protein,indicating that the LZ11 isolate was sensitive to neuraminidase inhibitors but resistant to amantadine drugs. Multiple amino acid site mutations related to mammalian adaptation were found in polymerase proteins,including ²⁹² I→V in PB2 protein,³⁶⁸ I→V in PB1 protein,and ⁴⁰⁹ S→N and ³⁵⁶ K→R in PA protein. The results showed that although this isolate did not cause any typical clinical symptoms in mice,it could replicate effectively in the lungs and turbinates,that viral titer could reach 3.82 and 5.98 lg PFU/mL respectively.【Conclusion】The isolated canine-origin H9N2 subtype influenza virus belongs to to S genotype circulating widely in China recently,whose eight gene segments belong to three lineages,including BJ/94-like,G1-like and F/98-like lineage.It contains several adaptive amino acid mutations in mammals and can replicate well in the organs of mice with the molecular characteristics to infect mammals. Due to the closer relationship between human and pet dogs,the surveillance and prevention and control for canine-origin H9N2 subtype influenza virus should be strengthened in future.