2022-2024年盐城市甲型H3N2流感病毒HA1和NA基因分子特征

Molecular characteristics of HA1 and NA genes of influenza A(H3N2)virus in Yancheng city from 2022 to 2024

目的研究2022-2024年盐城市分离的甲型H3N2流感病毒血凝素(hemagglutinin,HA)和神经氨酸酶(neuraminidase,NA)分子进化特征。方法收集盐城市2022年4月至2024年3月间的流感哨点监测医院以及各县市区流感暴发点采集的流感样病例标本,Rt-qPCR进行核酸检测,阳性样本进行病毒分离。分离的甲型H3N2毒株,采用一步RT-PCR方法扩增HA1和NA基因并测序,采用相关生物信息学软件分析基因核苷酸、氨基酸位点变异及进化特征。结果2022年4月至2024年3月间共采集5020份样本,流感病毒核酸阳性检出率为18.59%(933/5020)。2022年4月至2024年3月两个监测季冬春季流感峰明显,其中,仅2022年4月-2023年3月监测季夏季流感峰明显,甲型H3N2亚型流感病毒是两个监测季的优势流行株。遗传进化树显示:2022-2024年盐城市32株甲型H3N2毒株HA1和NA基因在各分支的聚类关系基本一致,2023-2024年24株分离株的HA1基因和NA基因与2022-2024年北半球疫苗株A/Darwin/9/2021(H3N2)聚类位于3C.2a1b.2a.2a.3a.1进化谱系;2022年8株分离株与2021-2022年北半球疫苗株A/Cambodia/e0826360/2020(H3N2)聚类位于3C.2a1b.2a.1a进化谱系。6株分离株(A/JSTH/11735/2023、A/JSTH/11788/2023、A/JSTH/11974/2023、A/JSYD/353/2023、A/JSYD/354/2023、A/JSTH/138/2023)均发生F79L、N122D、P239S、K276E氨基酸位点的变异,既存在于散发株又存在于暴发株。盐城市甲型H3N2毒株HA1基因编码区抗原表位、受体结合位点、糖基化位点发生了一定程度变异,基因层面上分析,2023-2024年24株分离株与2022-2024年北半球疫苗株A/Darwin/9/2021免疫原性匹配性较好,2022年8株分离株与2021-2022年北半球疫苗株A/Cambodia/e0826360/2020免疫原性匹配性较好。盐城市分离株未发生NA蛋白酶活性位点以及耐药位点的变化。结论2022-2024年盐城市流行的甲型H3N2毒株HA1和NA基因正逐渐发生抗原性漂移,增加推荐疫苗株的不匹配度,造成流感的暴发流行。2022年盐城市分离株相对于疫苗株A/Darwin/9/2021(H3N2)已发生实质性抗原漂移。需要对甲型H3N2流感病毒进行动态监测及时发现变异株。

ObjectiveTo analyze the molecular evolutional characteristics of the hemagglutinin and neuraminidase genes of influenza A(H3N2)viruses isolated in Yancheng from 2022 to 2024.MethodsThe throat swab specimens of influenza-like illness(ILI)from sentinel surveillance hospital and outbreak sites were detected using the method of real time Rt-qPCR.The influenza A(H3N2)viruses were isolated using MDCK cells culture method from April 2022 to Marh 2024.The strains isolated from 2022 to 2024 were selected randomly and their sequences of the HA1 and NA genes were amplified through one step RT-PCR method and the PCR products were sequenced.The nucleotide and amino acid site variations and evolutionary characteristics of the genes were analyzed using relevant bioinformatics software.The mutations of genes and nucleic acid locus were analyzed and the evolutional trees were generated using bioinformatics software.ResultsA total of 5020 samples were collected between April 2022 and March 2024,the positive detection rate of influenza virus nucleic acid was 18.59%(933/5020).The winter and spring influenza peaks were obvious in the two monitoring seasons from April 2022 to March 2024.Among them,the summer influenza peak was obvious in the monitoring season from April 2022 to March 2023,and the H3N2 subtype influenza virus was the dominant epidemic strain in the two monitoring seasons.Genetic evolution tree displayed:the clustering relationships of the respective branches of HA1 and NA genes of 32 strains isolated in Yancheng were basically the same.The HA1 and NA genes of 24 strains isolated from 2023-2024 in Yancheng and the 2022-2024 Northern Hemisphere vaccine strain A/Darwin/9/2021(H3N2)were located in the 3C.2a1b2a.2a.3a.1 evolutionary lineage,while the 8 strains isolated in the 2022 in Yancheng and the 2021-2022 Northern Hemisphere vaccine strain A/Cambodia/e0826360/2020(H3N2)were located in the 3C.2a1b.2a.1a evolutionary lineage.The 6 strains(A/JSTH/11735/2023,A/JSTH/11788/2023,A/JSTH/11974/2023,A/JSYD/353/2023,A/JSYD/354/2023,A/JSTH/138/2023)all exhibited variations in the F79L,N122D,P239S,and K276E amino acid sites,which were present in both sporadic and outbreak strains.Because the strains of the antigen epitopes,receptor binding sites and glycosylation sites in the HA1 genes had a certain degree of variations in Yancheng in the 2022-2024 year,the immunogenicity matching between the 24 strains isolated in the 2023-2024 and the Northern Hemisphere vaccine strain A/Darwin/9/2021 was good,while the immunogenicity matching between the 8 strains isolated in the 2022 and the Northern Hemisphere vaccine strain A/Cambodia/e0826360/2022 was good;32 strains isolated from 2022 to 2024 had no mutations in catalytic residues and drug resistant sites of NA genes.ConclusionThese result indicated that the HA1 and NA genes of influenza A/H3N2 viruses circulated in Yancheng city from 2022 to 2024 are changed gradually.The accumulation of these mutations would result in antigenic drift of influenza A(H3N2)viruses and increase the mismatching of the recommended vaccine strain.Compared with the vaccine strain A/Darwin/9/2021(H3N2),the strains isolated in the 2022 had substantially result in antigenic drift on the whole.The influenza A(H3N2)viruses surveillance should be strengthened to find the new mutant of virus in time.