Biological Characterization Of H3N2 (from 2009 To 2014) And H5 (from 2015 To 2016) Avian Influenza Viruses In China

The H5 subtype avian influenza virus(AIV)has been stably established in nature and gained high pathogenicity to its natural hosts through continual evolution,variation and adaptation.Its prevalence not only caused serious harm for the poultry industry,but also posed potential threat to human public health.H3N2 influenza virus,known as having a broad host range from wild birds and poultry to various mammalian species,such as pigs,dogs,cats,ferrets and so on.Moreover,H3N2 is one of the most directly pathogen of human seasonal influenza.In recent years,active avian influenza virus surveillance indicated that H3N2 subtype AIV was one of the highest isolation rate in waterfowls.Moreover,this subtype virus has been prevalent in most areas of China,and the number of isolated has a tendency to increase year by year.In order to understand the epidemics and evaluate the potential risk to mammalian species posed by the two subtypes,we analyzed the phylogenetic relationships,antigenicity,receptor binding properties,replication in mice(66 H5 AIVs isolated from 2015 to 2016 and 26 H3N2 AIVs isolated from 2009 to 2014),replication and transmission in guinea pigs.We isolated AIV with various HA and NA combinations,including H5N1,H5N2,H5N3,H5N6,and H5N8 avian influenza viruses(AIVs).The H5N6 was the dominant subtype among 66 viruses.The phylogenetic analysis showed that 66 H5 viruses formed 5 genotypes,which named A,B,C,D and E.The results of genotypes demonstrated that the H5 AIVs circulating in China currently displayed distinct diversity,and internal genes have undergone extensive reassortment with H1,H5,H7,H9,H12,H3 and H6 low pathogenic AIVs isolated from wild birds and poultry,respectively.In addition,the internal genes of some strains had a close relationship with human H5N6/H7N9 and swine-origin H3N2 viruses.The phylogenetic analysis of 26 H3N2 viruses indicated that they were divided into a complex of 26 genotypes,and showed distinct diversity.The HA genes of 26 H3N2 AIVs were markedly distinct from equine-?human-and swine-origin H3 influenza viruses.Furthermore,there were extensive reassortment of internal genes between H3N2 viruses and different kinds of low pathogenic,such as H1N2?H1N9?H2N3?H6N6 and H10N5 AIVs which isolated from wild birds.In addition,some genes which had a relatively independent evolution were most closely related to the genes of highly pathogenic H5N1 and H5N2 AIVs and even swine-origin H4N8 influenza viruses.The results of antigenic analysis showed that most of clade 2.3.4.4 viruses reacted well with antiserum generated against Re-8 vaccine starin,and a small number of viruses did not react well with it.Moreover,there was an obvious distinction of reaction between antiserum generated against Re-6 vaccine strain and clade 2.3.2.1 viruses.Besides,antigenicity analysis demonstrated that H3N2 viruses circulating in nature have undergone obvious antigenic drift,and showed significantly difference between human and swine-origin H3N2 influenza viruses.In mice study,we found that 66 viruses could efficiently replicate in respiratory system of mice without prior adaptation,and selective replication in brain,spleen and kidney in mice.The virulence of 66 viruses in mice various from each other: genotype A showed medium and high pathogenicity to mice;genotype B and C showed low and medium pathogenicity to mice;genotype D showed low,medium and high pathogenicity to mice and genotype E displayed high pathogenicity to mice.Moreover,most of the H3N2 AIVs could efficiently replicate in the nasal turbinate and lungs of infected mice without pre-adaptation.The mice after inoculation did not show obvious clinical symptoms,with no death,indicating the viruses displayed low pathogenic to mice.We conducted receptor binding specificity analysis for 10 viruses which possessed substitutions in receptor binding site and near the receptor binding site of HA protein.The results indicated that all viruses had a strong ?-2,3 receptor binding properties,but they bound to ?-2,6 receptor with no,mild,medium and strong affinity.In addition,10 viruses tested in our study could bind to ?-2,6-linked sialic acids,although their affinity for ?-2,3-linked sialic acids was higher.We selected 5 viruses that could efficiently replicate in mouse upper respiratory and have gained bind to ?-2,6-linked sialic acids to investigate their replication and transmission among guinea pigs.The replication in guinea pigs showed that all of the viruses selected could replicate in respiratory system of guinea pigs.The transmission study indicated that 5 viruses tested could efficiently transmit among guinea pigs by direct contact.In addition,two viruses could transmit among guinea pigs by respiratory droplet with medium level,and one virus could efficiently transmit among guinea pigs by respiratory droplet.In conclusion,H5 AIVs circulating in China had various combination and genomic displayed diversity.Antigenic differences between newly H5 viruses and vaccine strains were observed.Moreover,the H5 AIVs in our study could bind to human-type receptor and posed the potential risk to infect humans.In addition,H3N2 AIVs circulating in China posed potential risk to infect and transmit among humans.