| H9N2 subtype avian influenza virus（AIV）belongs to low pathogenic avian influenza virus（LPAIV）,which is the most widely prevalent subtype of avian influenza virus in Chinese poultry.It is also the most widespread and destructive AIV in the world.It usually causes respiratory and egg loss symptoms in poultry,as well as other serious respiratory disease secondary infection,which is a great harm to the poultry industry.Therefore,it is of great significance to strengthen surveillance,prevention and control of H9N2 subtype AIV to provide early warning of potential epidemics.In order to understand the molecular characteristics and pathogenic mechanism of H9N2 subtype AIV in China,the genetic evolution and key amino acid sites of Hemagglutinin（HA）and Neuraminidase（NA）of six strains of H9N2 subtype AIV isolated in the laboratory were analyzed.The results showed that the six strains belong to Eurasian lineage,HA and NA belonged to Y280/G9-like lineage,and were G57genotype,which were consistent with the current epidemic trend of H9N2 subtype AIV.four strains had Q226L mutation,two strains had A/T190V mutation,and HA of six strains all had increased or decreased potential glycosylation sites.The 62～64 sites were absent in the NA stalk of all the six strains.In conclusion,the six strains are low pathogenic avian influenza viruses,and both HA and NA contain mammalian adaptive mutations that increase the risk of cross-species transmission.The pathogenicity of six H9N2 subtype AIV strains in MDCK cells and mice showed that all six strains were pathogenic to MDCK cells,with titers ranging from10-5.3to 10-7.7/m L.The growth curves of the six strains peaked at 36～60 h,and the titer of the virus amplified in MDCK cells was higher than that amplified in chicken embryos.Pathogenicity tests in mice showed that the six strains slowed weight gain in mice,but did not kill them.Virus titers were detected in mouse lung tissue 3 and 5 days after infection.Pathological results of mouse lung tissue showed that inflammatory cell infiltration and destruction of alveolar wall were observed in the lung tissue of the 3-day infection group,but this phenomenon was alleviated or even disappeared on the 5-day infection,indicating that the six strains had low pathogenicity to mice.In order to further study the mechanism of molecular variation of the virus,we constructed a reverse genetic operating system of one H9N2 subtype AIV by homologous recombination method,and identified the rescue virus by observing the cytopathic changes of MDCK and sequencing the viral genome.The sequencing results showed that the saved strain was consistent with the original strain,which proved that the virus had been rescued.The six strains in this study are consistent with the current epidemic trend of H9N2 subtype AIV,contain mammalian adaptive mutations,and have strong replication ability in cells.They show low pathogenicity to mice,and may have the risk of crossing the species barrier to infect mammals.This study analyzed the molecular genetic evolution and the possibility of cross-species transmission,providing a theoretical basis for evaluating the potential threat of H9N2 subtype AIV to public health.The reverse genetic system provides conditions for the subsequent modification of the viral genome and helps to clarify the cross-species transmission mechanism and pathogenic mechanism of AIV. |
