| H9N2 avian influenza virus(AIV)belongs to low pathogenic avian influenza virus(LPAIV),it can cause pathogenic synergy once mixed infection with other pathogens,which results in serious disease in infected chickens.Nowadays,the prevalence of H9N2 subtype AIV is very common in domestic chickens.Even in immunized chickens with high antibody titer,chickens are often found to be infected,which can lead to clinical morbidity seriously.The reason may be related to the variation of the strains which contain certain differences of antigenicity compared with the vaccine strains.Therefore,systematic epidemiological monitoring to clarify the genetic evolution of epidemic strains is of great significance for guiding the screening of vaccine strains and improving the effectiveness of clinical immune prevention.In this study,we tracked and monitored the infection and morbidity of H9N2 subtype AIV in large-scale chicken farms in Jiangsu province and surrounding areas during the year of 2016 to 2018,and isolated and identified the pathogens from the diseased chickens.Meanwhile,the analysis of systematic genetic evolution and antigenicity difference of isolated strains will provide reference for prevention and control of chicken infection with H9 subtype AIV in this region.1 Isolation,identification and phylogenetic analysis of H9N2 subtype AIVs53 strains of H9N2 subtype AIV were isolated and identified during the monitoring periods.All strains were subjected to hemagglutinin(HA)gene sequencing and genetic evolution analysis.According to the constructed phylogenetic tree,all strains belonged to the h9.4.2 lineage represented by A/chicken/Beijing/1/1994.They were more concentrated in the phylogenetic tree,which belonged to h9.4.2.5 lineage,and could be divided into different evolutionary branches.All the amino acid sequences of HA protein cleavage sites deduced from HA gene sequence were RSSR,which was consistent with the typical characteristics of LPAIV cleavage site sequence.The HA protein of the most strains were mutated at position 203 amino acid where changed from T to I/V,resulting in deletion of the 200th glycosylation site,mutation at position 297 where changed from P to S,thus inserting a potential NCS glycosylation site at position 295.The appearance of new glycosylation sites or the deletion of the original glycosylation site may affect viral virulence and antigenicity.Most of the strains changed from Q to L at position 234,which has been shown to increase the possibility of viral infection in mammals.2 Research on antigenic difference of H9N2 subtype AIV from different evolutionary branchesIn the h9.4.2.5 lineage branch selected 7 different strains:Ck/JS/YZ0418/2016,Ck/JS/TZ0218/2017,Ck/JS/YZ0725/2017,Ck/JS/YZ-HJ0510/2017,Ck/JS/YZ-GL0916/2016,Ck/JS/YZ-GY 1024/2017 and Ck/JS/YZ-GY0822/2017,in order to further determine whether there was a difference in antigenicity between the strains on different evolutionary branches under the same lineage.According to the phylogenetic tree drawn,the seven strains were performed of the comparative analysis of antigenic differences.In the first experiment,160 1-day-old healthy Hy-line Brown female chickens were randomly divided into 8 groups(n=20).Seven groups of chickens were immunized with the above inactivated vaccines at 7 and 35 days of age,respectively.Each chicken was intramuscularly injected with 0.5 mL of vaccine,and another group was non-vaccination group.Serum was collected at 30-day-old,60-day-old,100-day-old,and 127-day-old,and the antibody titer was measured by HI test using hemagglutination antigen prepared from the Ck/JS/NT0428/2018.The results showed that all of the 7 strains had good immunogenicity,but there was a significant difference in the titer of HI antibody measured by the same antigen,which reflected that there may be certain antigenic differences between each other.In the second experiment,according to the results of cross-immunity test,four strains with significant differences on different branches were selected(Ck/J S/YZ0418/2016,Ck/JS/TZ0218/2017,Ck/JS/YZ-GY 1024/2017 and Ck/JS/YZ-GY0822/2017)to conduct cross-challenge test.50 seven-day-old SPF chickens were randomly divided into 5 groups(n=10),the first 4 groups were immunized with four oil emulsion inactivated vaccines,the last group was non-vaccination group.21 days post immunization(dpi),five groups were challenged with the Ck/JS/NT0428/2018 strain by eye-nose drop at dose of 108.2 EID50/bird.The clinical symptoms were observed daily.The results showed that chickens in immunization group had no clinical symptom,and some chickens in the control group had a loss of appetite.Throat swabs and cloaca swabs of each group were collected on 3 days post challenge(dpc)?6 dpc?9 dpc,which were used for virus shedding quantitation by real-time PCR.The results showed that the four vaccines could significantly reduce the level of virus shedding,but there were significant differences among the groups.The group vaccinated with homologous vaccine corresponding to the same evolutionary branch of the challenge virus has the best protective effect.In conclusion,the results of this study showed that all strains belonged to the h9.4.2.5 lineage,but the antigenic differences of the virus strains in different evolutionary branches are obvious. |
PDF Full Text Request