Phylogenetic Analysis And Pathogenicity Of H6 Subtype Avian Influenza Viruses Isolated From Waterfowl In Southern China During 2011 To 2017

H6 subtype avian influenza virus(AIV)is one of low pathogenic AIV,which does not cause clinical symptoms,so it has not attracted enough attention all over the world.H6subtype avian influenza viruses spread widely in birds and poultry in many regions of China.The prevalence and transmission of H6 subtype AIV in domestic poultry has increased over the last two decades,and the H6 subtype AIV has gradually become one of the common subtypes of avian influenza virus in poultry and waterfowl in southern China.However,there is no systematic study on the evolution and pathogenicity of H6 avian influenza viruses isolated in mice and chickens.A total of eight kinds of H6 subtype AIVs were isolated from Southern China before,including H6N1,H6N2,H6N4,H6N5,H6N6,H6N7,H6N8 and H6N9 subtypes.The isolation of H6N2 and H6N6 was much higher than that of other H6 subtype viruses.From2011 to 2017,22 H6 subtype AIV strains isolated from duck and geese in live poultry markets.Therefore,the ducks and geese are the primary hosts of H6 subtype AIV,the H6N2 and H6H6 are the main subtype.The HA gene evolution and variation of H6 subtype AIV from china were analyzed by bioinformatics technology.The HA gene of H6N2 AIV showed a gradual evolution in chronological order.The epidemic areas of H6N2 AIV were mainly concentrated in Hubei,Hunan,Zhejiang,Guangdong and Guangxi,which nearby the Yangtze River Basin,and some HA genes were similar to the H6 viruses from chickens,pigeons and pigs reported in China.In contrast,H6N6 was reported relatively late and the first H6N6 strain was isolated from Guangdong ducks in 2001.As shown in Fig.2-5,Clade ? and clade ? of the phylogenetic tree of HA were mainly composed of H6N6 subtype AIVs from chicken and duck from 2006 to 2017.That means the most of H6N6 AIVs have a similar phylogenetic characterization and share a common origin.It's also shown that H6N6 subtype AIV in waterfowls,ducks and geese tended to transit to chickens.Beside,the variation of HA of H6N2 and H6N6 subtypes were mainly reflected in 2002 to 2014,and showed the trend of accumulation of amino acids at the same site,compared with the recently isolated strains,some amino acids sites had completely changed.Among them,the mutation of 156aa,157aa,160aa and 186aa sites caused the receptor binding site region changed,which affected the binding ability with sialic acid receptor of host,and was related to the antigenic drift of HA protein of H6 subtype virus.The average nucleotide evolution rate(substitution/site/year)of HA1 and HA2 of H6N2 and H6N6 AIVs were calculated by using the software of best v1.10.4,and the time of the nearest common ancestor(TMRCA)were calculated and the evolutionary dynamic curve(BSP)were constructed.It's shown that the nucleotide variation rate of HA2 gene of H6N6 viruses were higher than that of HA1,while amino acid entropy and selection pressure analysis showed that the amino acids at HA2 remained relatively stable,the frequency of amino acid variation at all sites was lower than that of HA1.Therefore,we can infer that the mutations in the HA2 region are all non synonymous mutations,which have no effect on the antigenic specificity of HA.The evolutionary dynamic curve showed that the prevalence of H6N2 and H6N6 subtype AIVs increased gradually,especially after2006.To explore the genetic composition and dominant genotypes of H6 subtype AIVs prevalent in Southern China in recent years.The whole genome of 22 H6 subtype strains isolated from waterfowl in southern China were sequenced and analyzed.According to the nucleotide homology of H6 subtype AIVs,the eight genes of 22 H6 AIVs belonged to Eurasia genetic lineage.Most of H6 subtype isolates had high homology among PB2,PA and M genes.As shown in Table 3-24,Except the GD/F1473 virus,NS gene of all H6isolates were similar with the H5N1 virus(A/duck/Hong Kong/140/1998/(H5N1))from duck in Hong Kong.Based on the molecular biological characteristics analysis of 22strains,all H6 viruses are low pathogenic AIV.None of the amino acid residues involved in the recognition of human-type receptors was found in all 22 isolates,indicating that all the viruses prefer avian-like(?-2,3-sialic acid)receptor.All 22 H6 isolates can be divided into9 genotypes,as shown in Figure 3-11.Seven H6N2 strains can be divided into three genotypes,while 15 H6N6 AIVs have been divided into six genotypes.In addition to 2013,new genotypes appeared every year from 2011 to 2017,among which B602 genotype had the largest number of isolates.To determine the pathogenicity of the 22 H6 isolates in different hosts,mice and chicken were chosen as mammal model and poultry models respectively.Each of eight4-week-old BALB/c mice were anesthetized and inoculated intranasally with 10^6.0 EID50 in30.0?l PBS of each virus of the 22 H6 isolates.Eight control mice were inoculated intranasally with 30.0?l of PBS.All of the mice used for observation survived till 14 dpi.None of H6N2 viruses,except GD/1268,caused significant body weight loss(P<0.05)in mice compared with the control group(Fig.4-3).As for H6N6 viruses,only HN/A729 and ZJ/B1994 caused significant growth retard in mice,while the clinical signs caused by other viruses were not obvious.All of the H6H2 isolates were able to replicate efficiently in mouse lung and nasal turbinate without prior adaptation.The mean virus titers were ranged from 10^3.4 to 10^6.0 EID50/ml in the lungs and from 10^1.8 to 10^4.8EID50/ml in the nasal turbinates(Table 4-2).Specially,two H6N2 viruses were found in the spleen of one out of three inoculated mice,and one of those viruses GD/E3503 caused systemic infection in one inoculated mouse.In addition,the virus was recovered from liver and brain.The GD/1268and GD/1127 viruses of A201 genotype were detectable in the lungs,nasal turbinates and hearts consistently,while the 4 isolates of A202 genotype showed diversity in the tissue tropisms(Table 4-2).The H6N6 viruses,except ZJ/B2039 and JS/G91,replicated in the lungs of inoculated mice.ZJ/B2039 has not been detected in any of the other tested tissues,and lower titers of JS/G91 wered etected only in the nasal turbinate of one out of three mice,suggesting the limited replication of those 2 viruses in mice,especially the H6N2subtype virus,posed the biological security threat to mammals.The pathogenicity of 5 H6 viruses from different genotypes,was evaluated in SFP chickens.Each of six 4-week-old SPF chickens were inoculated with 10^6.0 EID50/bird of each virus in a volume of 100.0?l,respectively.The DF-1,DEF,MDCK and A549 cells were infected with 5 H6 subtype AIV strains at a MOI of 0.01,respectively.The results showed that the 5 strains of H6 subtype AIV had strong replication level in mammalian cells(Fig.4-14).The GD/1127 and GD/E3503 viruses were detected in the trachea of one inoculated chicken,respectively.FJ/D34806 was detected in the duodenum of one inoculated chicken,while HN/A729 and ZJ/B2028 were not detected in any tested tissues of 3 inoculated chickens at 3dpi.Only GD/E3503 was detected in the oropharyngeal swab from one chicken at 4 dpi.GD/E3503/H6N2 and HN/A729 were detected in the cloacal swab from one chicken at 2 dpi and 6 dpi,respectively.None of 3 chickens inoculated with HN/A729 showed sero-positive to specific H6 antibody,and only some chickens inoculated with the other four viruses were sero-converted.One contact chicken in the GD/E3503 group was seroconverted at 14 dpi,while no specific H6 antibody was detected in other contact chickens(Table 4-7).The results suggested the replication and transmission of H6 viruses with different genotypes were limited in chickens.Thus,the H6 AIVs have obtained potential infection ability to mammalian host through the multiple reassortment between aquatic and mammalian viruses.Our study contributed to the current understanding of evolution and pathogenicity of prevalent H6N2and H6N6 viruses isolated in Southern China.These findings emphasize the need for regular surveillance of H6 AIVs and extensive characterization of H6 isolates to better understand genetic changes and their implications.