| H9N2 subtype avian influenza virus(AIV)was first isolated in chicken flocks in mainland China in 1994.Since then,the H9N2 subtype AIV has spread widely from the south to the north and from the east to the west with the Pearl River Delta as the first transmission center and the Yangtze Delta as the second transmission center.Endemic epidemics have formed in most areas of our country.H9N2 avian influenza virus often co-infects poultry with other pathogens which causing egg production decline even death,especially in intensive farms with a higher incidence and a faster spread speed,leading to a huge economic loss in the poultry industry.H9N2 subtype AIV can also break through the host barrier through gene mutation and recombination,achieve cross-host transmission to infect humans and other mammals which can pose a certain threat to public health safety in some certain.Therefore,it is of great significance to study the genetic evolution,molecular evolution and biological characteristics of H9N2 subtype AIV in China from 2014 to 2019.We analyzed the genetic evolution of 306 H9N2 subtype AIVs in China from 2014 to2019 and defined the genotype according to the phylogenetic combination of each individual.Then the rate of nucleic acid substitution of the dominant genotype G57 and its positive selection pressure were calculated.Finally,four laboratory isolates were selected to study their pathogenicity in 4-week-old SPF chickens and 4-week-old SPF-grade BALB / c mice.In our analysis,we observed that the HA genes of all viruses evolved from viruses of the BJ / 94-like lineage and were assigned to 5 different sub-branches,which are clade 2(1)and clade 4(2),clade 5(1),clade 12(2),and clade 15(300).We found 300 strains clustered in clade 15,and only 6 strains clustered in other branches.According to Liu Jinhua’s research,it can be inferred that since 2013,clade 15 replaced other branches and became the main popular branch nationwide.The NA base is assigned to 4 different sub-branches,which are clade 0(1),clade 1(7),clade 2(296),and clade 3(2).Previous studies have shown that all NA stems belonging to the clade 2 strain possess a three amino-acid deletion in the NA stalk region,which was not present in the isolates in clade 1.It is worth noting that a human strain belonging to clade 2 did not have this deletion in this study.The results of internal genes showed that 99% of the PB2 genes were clustered on the clade 8 branch;The three internal genes of PB1,PA,and NP of other strains originated from SF / 98-like Sublines except for A / duck / Wuhan / WHYF05 / 2014;99% of the M genes were gathered in clade 3,which originated from the G1-like lineage;99% of the NS genes were located in clades 7,which is evolved from the BJ / 94-like virus.Based on the combination of phylogenetic origins of 8 genes,we found a total of 10 genotypes,of which 7 newly discovered were named G119,G120,G121,G122,G123,G124,G125.Among them,genotypes G57,G68,G118,and G121 have appeared for many years in succession which are called major genotypes.Genotypes G119,G120,G122,G123,G124,and G125 only appeared once in the same year,which are called transient genotypes.Among them,G57 genotype is the absolute dominant genotype,accounting for 94% of all strains.Therefore,according to the appearance and development of the G57 genotype H9N2 subtype AIV in China,we divided it into two periods:2007-2012 and 2013-2019,and calculated the nucleic acid replacement rate and positive selection pressure of HA and NA gene fragments in different periods respectively.The results show that the evolution rates of HA and NA in the period 2013-2019 are higher than in the period 2007-2012.Positive selection pressure is also higher than the latter.Viral pathogenicity test results show that none of the four H9N2 AIV strains can kill SPF chickens and BALB / c mice,but the virus can replicate in their multiple organs,and the main replication site in BALB / c mice is the lung And turbinate,the main sites of replication in SPF chickens are the lungs and trachea.All isolates did not have multiple consecutive basic amino acids near the cleavage site of HA protein,and all showed the sequence characteristics of low pathogenic avian influenza virus.There are mutations in some receptor binding sites of HA protein,especially at position 234 of the HA gene,98%of the strains are mutated from Q to L,which has the characteristic of binding to sialic acidα-2,6 galactosidase receptor,indicating that in recent years Most of the popular H9N2 already has the ability to infect mammals. |
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