| Avian influenza is caused by avian influenza virus,which manifests as respiratory diseases to systemic sepsis in birds.Among them,H3 subtype AIV can not only infect birds,causing economic losses to the development of poultry industry,but also has the risk of infecting mammals,threatening public health security.The interlaced water network and developed traffic in East China are the necessary places for many migratory birds to migrate.At the same time,the live poultry market model makes it easy for influenza viruses to evolve and recombine here and spread to poultry and other migratory bird populations.H3 subtype AIV has no obvious clinical symptoms after infecting poultry.When it is co-infected with other subtype influenza viruses,gene recombination may occur to generate new epidemic strains.At present,there are relatively few systematic studies on the genetic evolution,pathogenicity and transmission characteristics of H3 subtype AIV in China.Here,7 representative H3 subtype AIVs isolated from Eastern China during 2014 to 2021 were selected according to different regions,hosts,and period.The genetic evolution was analyzed,and biological characteristics and receptor binding characteristics of the isolates were studied.Finally,the pathogenicity and transmission characteristics of H3 subtype AIV in chickens and mammals were studied,which will provide references for making the prevention and control strategy of H3 subtype AIV.1 Epidemiology and genetic evolution of H3 subtype AIVs isolated from East China during 2014 to 2021During 2014 to 2021,among 27630 samples collected by our laboratory in East China,total 29 strains(0.1%)of H3 subtype AIVs were isolated.In detail,6 strains(0.03%)of H3 subtype AIVs were isolated from 19730 samples of chickens,and 4 strains(0.14%)of H3 subtype AIVs were isolated from 2800 samples of gooses.No H3 subtype AIV was isolated from 1750 pigeon samples,and 19 strains(0.57%)of H3 subtype AIVs were isolated from 3350 duck samples.The H3 subtype AIV was selected from the above samples according to different isolation regions,hosts and time,and was purified and identified with the H3 subtype AIV isolated from a swan disease material.A total of 7 strains of H3 subtype were successfully isolated from MDCK cells after three rounds of purification,including 5 H3N2,1 H3N3 and 1 H3N6 subtype AIVs.The hosts included swan,duck and chicken.One H3N2 strain from swan was from pathological tissue,and the rest were from live poultry market.The whole genome of 7 strains of H3 subtype AIVs were sequenced and analyzed and found that the cleavage mode of HA protein of the 7 strains was 340PEKQTR? GLF348and there was only one basic amino acid,in line with the characteristics of low pathogenic AIV,and the 226Q and 228G positions are more inclined to bind to avian sialic acid receptors.The isolates had T160A mutation of HA,I333T mutation of PB2,H436Y mutation of PB1,A100V,R356K,and N409S mutation of PA,M105V mutation of NP,and N209D mutation of M,which could increase the pathogenicity to animals or increase the polymerase activity of the virus.The genes of seven strains,including PB2,PB1,PA,HA,NP,NA,M,and NS,all belonged to the Eurasian branch.The HA fragment of W23910 strain had the highest homology with a canine strain(97.2%),and the NA and M fragment of J84610 strain had the highest homology with a mink strain(99.7%and 99.6%),implying that these two strains may have been cross-species recombination.Moreover,various fragments of H3 subtype AIVs also frequently recombined with H1,H4,H5,H6,H7,H9,H10,H11,and other subtypes.2 Biological characteristics and pathogenicity evaluation of H3 subtype AIVsThe biological characteristics of 7 H3 subtype AIVs,including HA titers,the mean death time(MDT),median tissue culture infective dose(TCID50),50%embryo infective dose(EID50),growth curve,and receptor binding levels were evaluated.The HA titer of the 7 strains ranged from 7 to 9 log2 and did not cause death of chicken embryos within 96 h.The EID50 was between 106.35-107.78 EID50/mL and the virus replicated well on CEF cells with 104.39-106.72 TCID50/mL.Among the 7 strains,except JY020416,all the other strains had poor replication on A549 cells,and the highest TCID50 titer did not exceed 104 TCID50/mL.Except J84610 strains,the other 6 strains replicated well on MDCK and CEF cells,and the replication peaks of 4D1-1 and LY were higher than other strains on CEF and MDCK cells.The results of goose erythrocyte assay and solid phase ELISA of the 7 H3 isolates showed that J84610 had only the avian ?-2,3 sialic acid receptor binding properties,while the other 6 isolates had both ?-2,3 and ?-2,6 sialic acid receptor binding properties.The pathogenicity of 7 H3 isolates in chickens,mice,and guinea pigs was also evaluated.The pathogenicity in SPF chickens showed that 4D1-1 strain was detected only in lung,W7-4 and LY strain was detected only in trachea,901084 strain was detected only in trachea and ileum,W23910 strain was detected only in ileum,and other strains were not detected in the above organs after 3 days of infection.The results of pathogenicity in mice showed that none of the strains killed mice,but JY020416,4D11,and LY strains caused a weight loss of more than 6%,and the highest weight loss of 4D1-1 strain was 10.5%.The weight loss was mainly in the first 3 days,and then gradually recovered.On day 3 and 5,W7-4,JY020416,4D1-1,and LY strains can be detected in lung and nasal cavity of mice.In addition,W7-4 and JY020416 strains can also be detected in brain of mice.W23910 strain was detected only in lung of mice on day 5,while J84610 and 901084 strains were not detected in nasal cavity,lung,ileum and brain.The results of pathogenicity in guinea pigs showed that LY can only replicate in nasal cavity of guinea pigs.The above results showed that although the abovementioned H3 subtype AIVs only replicated at low doses in some organs of chickens,5 strains could replicate in mouse lungs.3 Evaluation of transmission characteristics of H3 subtype AIVsHere,the contact transmission between chickens and cross-species contact transmission between chickens and guinea pigs were studied.In this experiment,3 SPF chickens without maternal antibody at 21 days of age were inoculated with nasal drops of H3 subtype AIVs(106 EID50/100 ?L).Three unchallenged chickens and guinea pigs were placed in the same cage as the contact group.On day 1,3,5,7,9,and 11,oropharyngeal and cloacal swabs of the challenged chickens were collected.On day 2,4,6,8,10,and 12,oropharyngeal and cloacal swabs of the contact chickens and nasal washing of guinea pigs were also collected to detect virus shedding.On day 21,serum was collected for detecting the serum transformation.We found that the five challenge groups,including W7-4,JY020416,W23910,901084,and LY,appeared the contact transmission among chickens.Except JY020416 strain,contact transmission from chicken to guinea pig occurred in the other six attack strain groups.On day 21,seropositive transformation was observed in all challenged groups with HI titer ranging from 4 to 8 log2.In the exposed groups,serum positive transformation was observed in all 6 groups except J84610 group,but the HI titer was lower than that in challenged group.In the contact groups,except JY020416 and 4D1-1 group,the other 5 groups,including W7-4,J84610,W23910,901084,and LY,had seropositive transformation.Furthermore,LY strain was used to perform the contact transmission and aerosol transmission assay among guinea pigs.The results showed that virus shedding was detected in all guinea pigs in challenge group,direct contact group,and aerosol group,and serum was positive both on day 14 and 21.These results indicated that the above H3 subtype AIVs was easily transmitted by contact between chickens and had the ability of cross-species contact transmission to guinea pigs,and LY strain had the ability of contact transmission and aerosol transmission in guinea pigs. |
PDF Full Text Request