我国柯萨奇病毒A组6型VP1区基因分子流行病学特征

  目的  分析我国2010―2018年柯萨奇病毒A组6型（Coxsackievirus A6,CV-A6）毒株VP1区基因流行和进化规律,为手足口病的防治提供科学依据。  方法  从GenBank获得2010―2018年我国CV-A6病毒全长VP1区核苷酸序列,用MEGA V7.0和Interactive Tree of Life V5（iTOLV5）软件构建进化树,用DNAstar V8.1.3软件对核苷酸同源性进行分析。  结果  880条序列来源的CV-A6毒株以D3亚型为主,占毒株总数的95.45%,不同基因型核苷酸同源性为80.8%~86.0%,同基因型内核苷酸同源性为88.1%~100.0%。D2亚型VP1区相对稳定,无整体氨基酸变异情况;而D3毒株VP1区发生多点氨基酸替换,其中A5T、T283A、N137S、V242I、A30V、I174V氨基酸替换循环出现,这种进化模式与越南地区相似,但与日本地区却不同。VP1区5T、30A、137N、242V比例逐年增加,可能与CV-A6引起我国手足口的轻症和重症病例比例上升有关。  结论  具有VP1区遗传多样性的CV-A6 D3株的出现可能是CV-A6在反复流行的一个重要因素,这有助于解释我国CV-A6的流行情况和流行特点。     

潍坊市9例重症与轻症手足口病患者肠道病毒71型5′非编码区、VP1基因特征分析

目的 探讨重症手足口病肠道病毒71型(enterovirus 71,EV71)5′非编码区(untranslated region, UTR)、VP1区是否存在潜在的毒力位点。 方法 分离培养不同临床症状手足口病患者粪便标本中EV71,提取病毒总RNA,逆转录PCR扩增5′-UTR、VP1区后测定基因序列,对两个区域核苷酸序列及VP1区编码氨基酸序列进行比对和同源性分析,构建VP1区及不同临床症状EV71病毒5′- UTR基因进化树。 结果 本实验共获得9株EV71毒株(5株来源于重症病例,4株来源于轻症),5′-UTR、VP1区核苷酸同源性均为94.5%~ 99.7%,VP1区氨基酸同源性为97.3%~99.9%。9株EV71毒株5′-UTR共有67个核苷酸突变位点,与4株轻症EV71毒株相比,5株重症EV71毒株VP1区编码氨基酸有2处发生替换(S283T、A289T),5′-UTR共有37个核苷酸位点发生突变。VP1区基因进化树显示9个EV71毒株均属于C4a基因亚型,并且两组不同临床症状毒株处于同一较小分支中。5′-UTR核苷酸序列的系统进化树显示临床表现不同的EV71株呈交错分布,相同临床表现不单独聚类。 结论 9株EV71毒株均属于C4a基因亚型,5′-UTR核苷酸突变和VP1区氨基酸替换可能影响病毒毒力,对EV71病毒的全基因组序列特征分析及与宿主之间的相互作用机制进行研究非常必要。     

中国柯萨奇病毒A组6型分离株全基因序列特征分析

  目的  分析中国2007-2019年柯萨奇病毒A组6型(coxsackievirus A6,CVA6)毒株遗传进化规律,以期为疾病预防及疫苗研发提供理论依据。  方法  收集中国截至2021年3月1日Genbank数据库中454株CVA6毒株全基因序列,分析毒株分离年份、地区信息,构建系统发育树揭示进化规律并进行基因重组分析。  结果  地区信息显示,毒株主要分布于中国沿海及南方地区,时间信息表明,自2013年起,CVA6毒株报告数量逐渐增加。系统进化结果表明,454株CVA6毒株分10个分支,A分支中3株分离自江西省的毒株与CVA6原型毒株Gdula(AY421764)亲缘关系极为接近,核酸序列相似度近乎100%,J分支是中国流行最广泛的毒株群,包含了中国较多省份如山东省、广东省等。基因重组分析显示,基因重组现象较为普遍,大部分CVA6代表毒株在P2区和P3区与其他肠道病毒A组毒株之间存在基因重组现象。  结论  2007-2019年CVA6毒株在中国广泛传播,毒株间基因重组现象普遍存在。     

手足口病相关柯萨奇病毒A组8型全基因组序列特征分析

目的 研究中国2013-2018年手足口病病例分离的柯萨奇病毒A组8型（CV-A8）全基因组序列特征及对全基因组各编码区进行遗传进化分析。方法 对我国不同地区手足口病患者分离的11株CV-A8的全基因组序列,采用Sequencher 5.0、MEGA 7.0等软件对获取的全基因组序列进行比对和遗传进化分析。结果 序列比对显示11株CV-A8基因组长度在7 393~7 400 bp之间,与原型株比较,在编码区无碱基插入或缺失,在非编码区存在个别碱基的插入或缺失。11株CV-A8毒株VP1区核苷酸和氨基酸同源性分别为78.3%~98.6%和92.6%~99.7%;与CV-A8原型株的核苷酸和氨基酸序列同源性分别为78.3%~98.2%和92.6%~99.7%。对CV-A8的VP1区序列进行了系统发育分析,可将CV-A8分为5个基因型：A、B、C、D和E,本研究11株CV-A8分属于C（1株）、D（2株）、E（8株）3个基因型。11株CV-A8毒株全基因组序列核苷酸和氨基酸同源性分别为81.3%%~98.8%和95.9%~99.5%,P2区进化树图显示,本研究的8株E基因型CV-A8和CV-A4、CV-A14和CV-A16原型株进化距离最近,P3区进化树图显示,本研究8株E基因型CV-A8和CV-A5、CV-A16、CV-A14和CV-A4进化距离最近。结论 本研究中11株CV-A8其衣壳区呈现基因多样性,非衣壳区呈现重组多样性,提示CV-A8正在经历变异动态变化;CV-A8有可能成为手足口病的重要病原体,因此需要进一步加强监测CV-A8。     

我国部分省市手足口病EV71 VP1基因特征分析

摘要:目的　分析我国部分省市手足口病EV71 VP1基因特征。方法　对从NCBI 上获得我国部分省市和本实验室来源的VP1 基因序列99株进行分析,采用MEGA软件构建系统进化树,计算相同或不同基因型及基因亚型毒株的核苷酸与氨基酸的相似性。结果　研究序列中C4亚型占96.0%,A亚型（3.0%）和B5亚型（1.0%）较少;A亚型毒株氨基酸变异为非同义突变;部分毒株发生氨基酸突变位点与病毒致病力相关;南方株相对北方株基因序列变异较大,东部株相对中部和西部株基因序列变异较大。结论　我国EV71以C4亚型为主;南方株较北方株、东部株较中西部株基因序列变异较大可能与我国气候、交通和人口流动有关;我国A亚型毒株非同义突变及2011年分离自浙江的KF358275突变位点增多是否会引起新的流行需加强后续监测。     

2009—2019年上海市闵行区手足口病病原谱及EV-A71和CV-A16病毒分离株VP1区基因特征分析

目的 了解2009—2019年上海市闵行区手足口病的病原谱,分析肠道病毒71型(enterovirus 71,EV-A71)和柯萨奇病毒A组16型(coxsackievirus A16,CV-A16)VP1区基因特征,为手足口病的综合防治提供科学依据。 方法 对2009—2019年闵行区手足口病监测点送检的标本应用实时荧光定量PCR进行病原学检测,分析病原学特征。EV-A71和CV-A16病毒分离株进行VP1区核苷酸序列测定,分析其同源性并构建系统进化树。 结果 2009—2019年共收集到5 364例手足口病病例标本,病原学检测阳性检出率为86.74%(4 653/5 364),其中EV-A71检出率为40.12%(2 152/5 364)、CV-A16为16.61%(891/5 364)、CV-A6为20.86%(1 119/5 364)、CV-A10为1.49%(80/5 364)。2009—2014年主要呈EV-A71和CV-A16共同流行趋势,2015—2019年主要以CV-A6和CV-A16共同流行为主。EV-A71分离株均为C4a型,VP1区核苷酸序列同源性为92.1%~99.3%、氨基酸序列同源性为98.1%~100.0%; CV-A16分离株均为B1基因型,存在B1b和B1a基因亚型的共同流行,VP1区核苷酸序列同源性为87.2%~99.6%、氨基酸序列同源性为97.9%~100.0%。 结论 2009—2019年上海市闵行区手足口病呈现EV-A71、CV-A16和CV-A6共同流行态势,不同年份的优势毒株呈现动态变化;而EV-A71流行株属于C4a亚型、CV-A16流行株存在B1b和B1a基因亚型的共同流行,与国内大部分地区的流行株基因亚型一致。     

四川省德阳市柯萨奇病毒A组4型基因特征分析

目的分析四川省德阳市柯萨奇病毒A组4型(coxsackievirus A4,CV-A4)的基因特征。方法对2014-2015年德阳市手足口病咽拭子标本进行病毒分离和核苷酸测序,VP1区序列与世界各地的CV-A4参考株序列构建进化树并分析基因特征。结果德阳市5株CV-A4分离株之间,VP1区核苷酸序列一致性91.5%~96.8%(推导氨基酸序列一致性97.7%~99.3%);同CV-A4原型株(High Point)比较,核苷酸序列一致性≥84.0%(推导氨基酸序列一致性≥97.0%);进化树分析显示,德阳市CV-A4分离株均为G1B基因亚型,分别与云南、上海和浙江分离株有较近的亲缘关系。G1B基因亚型内的平均核苷酸遗传距离为10.4%。结论德阳市CV-A4为G1B基因亚型,为中国大陆的优势基因亚型;VP1区核苷酸变异较大,形成不同的进化分支并在德阳市循环传播。     

河南省甲型H1N1流感病毒神经氨酸酶基因进化分析

目的分析河南省甲型H1N1流感病毒神经氨酸酶(NA)基因的变异情况,了解其变异现状及特点。方法对35株甲型H1N1流感病毒毒株提取病毒总RNA,设计引物运用RT-PCR技术扩增编码NA蛋白的基因序列,测序分析核酸序列并用MEGA4.1软件构建进化树,用BLAST进行比对分析。结果分离株NA基因316位G/A和742位A/G(N端106位V/I和248位N/D)发生变异,2个突变位点同时存在;同时,通过BLAST分析,发现我国多个省份和亚洲其它多个地区病毒株NA基因存在945位A/G和1 338位T/C突变,进化树分析发现,这两个位点的突变可能来自于中国猪-人之间相互感染。结论甲型H1N1流感病毒神经氨酸酶(NA)基因在中国内地传播期间个别核酸位点发生了突变,其氨基酸抗原区有一位点发生变异(106 V/I)。     

Genome-wide population structure inferences of human coxsackievirus-A; insights the genotypes diversity and evolution

Coxsackievirus-A (CV-A) is a causative agent of Hand Foot Mouth Disease (HFMD) worldwide. It belongs to the Human Enterovirus genus of the family Picornaviridae. The genomics data availability of CV-A samples, isolated from human host across different continental regions, provide an excellent opportunity to study its genetic composition, diversity, and evolutionary events. The complete genome sequences of 424 CV-A isolates were analyzed through a model-based population genetic approach implemented in the STRUCTURE program. Twelve genetically distinct sub-populations were identified for CV-A isolates with a marked Fst distinction of 0.76991 (P-value = 0.00000). Besides, genetically admixed strains were characterized in the G-Id, G-IIIb clusters constituted by the CV-A12 and CV-A6 enterovirus serotypes. The serotypes depicted inter/intra-genotype recombination and episodic positive selection signatures in the structural and non-structural protein-coding regions. The observed genetic composition of CV-A samples was also deduced by the phylogenetic tree analyses, where a uniform genetic structure was inferred for most of the CV-A genotypes. However, the CV-A6 serotype samples genetically stratified into three sub-populations that may lead to the emergence of new lineages in future. These informations may implicate in planning the effective strategies to combat the coxsackievirus-A-mediated infection.     

Molecular evolution and epidemiology of echovirus 6 in Finland

Echovirus 6 (E-6) (family Picornaviridae, genus Enterovirus) is one of the most commonly detected enteroviruses worldwide. The aim of this study was to determine molecular evolutionary and epidemiologic patterns of E-6. A complete genome of one E-6 strain and the partial VP1 coding regions of 169 strains were sequenced and analyzed along with sequences retrieved from the GenBank. The complete genome sequence analysis suggested complex recombination history for the Finnish E-6 strain. In VP1 region, the phylogenetic analysis suggested three major clusters that were further divided to several subclusters. The evolution of VP1 coding region was dominated by negative selection suggesting that the phylogeny of E-6 VP1 gene is predominantly a result of synonymous substitutions (i.e. neutral genetic drift). The partial VP1 sequence analysis suggested wide geographical distribution for some E-6 lineages. In Finland, multiple different E-6 lineages have circulated at the same time.     

Identification of a novel Aichivirus D in sheep

A novel kobuvirus was found in diarrheal fecal samples of Tibetan sheep using a viral metagenomics approach, and a full kobuvirus genome was successfully obtained by RT-PCR from a diarrheal fecal sample. The full genomic sequence was 8485 nucleotides (nt) in length with a standard picornavirus genome organization. The novel genome shares 62.9% and 77.8% nt homology with Aichivirus D1 genotype strain 1-22-KoV, and Aichivirus D2 genotype strain 2-44-KoV, respectively. According to the species classification criteria of the International Committee on Taxonomy of Viruses (ICTV), the new kobuvirus belongs to Aichivirus species D. Interestingly, compared with 2 known Aichivirus D genotype strains, the novel Aichivirus D has unique amino acid substitutions in the 5'untranslated region (-UTR), VP0, VP3, and VP1, with a recombination event in the 2C region.These characteristics make the novel Aichivirus D cluster into an independent branch in the phylogenetic tree, suggesting that strain may represent a novel genotype in Aichivirus D. Moreover, the novel Aichivirus D was detected in 9.2% (18/195) of the sheep diarrheal fecal samples from 4 farms in 3 counties of the Qinghai Tibet Plateau in China. In addition, full-length VP0, VP3, and VP1 genes were successfully obtained from 12 samples from 4 farms, and phylogenetic analysis based on these genes revealed a unique evolutionary pattern for this novel Aichivirus D strain.This study identified a novel Aichivirus D that is circulating in sheep in Qinghai Tibet Plateau in China and these findings provide a better understanding of the epidemiologic and genetic evolution of kobuviruses.     

Molecular detection and genomic characteristics of bovine kobuvirus from dairy calves in China

In this study, 96 diarrheic and 77 non-diarrheic fecal samples from dairy calves were collected from 14 dairy farms in 4 provinces to investigate the molecular prevalence and genomic characteristics of Bovine Kobuvirus (BKoV) in China. The results showed that the BKoV positive rate for the diarrheic feces (35.42%) was significantly higher than that for the non-diarrheic feces (11.69%, p < 0.001). Interestingly, three potential novel VP1 lineages were identified from 15 complete VP1 sequences, and a unique triple nucleotide insertion which can result in an aa insertion, was first observed in the 11/12 VP0 fragments with 660 bp long in this study, compared with known BKoV VP0 sequences. Moreover, the first Chinese BKoV genome was successfully obtained from a diarrheic fecal sample, named CHZ/CHINA. The open reading frame (ORF) of the genome from strain CHZ/China shares 87.4%–88.3% nucleotide (nt) and 93.7%–96.4% amino acid (aa) identity, compared with the three known genomes of BKoV. Interestingly, phylogenetic tree based on aa sequences of these genomes showed that CHZ/CHINA was clustered into an independent branch, suggesting the strain may represent a novel BKoV strain. The findings contribute to better understanding the molecular characteristics and evolution of BKoV.