柯萨奇病毒A组9型的基因分型方法的建立

血清型别鉴定及基因分型分析是开展肠道病毒（Enterovirus,EV）分子进化特征研究的重要内容。目前为止,国内外对柯萨奇病毒A组9型（Coxsackievirus A9,CVA9）的研究主要集中在衣壳蛋白区的细胞受体结合位点及其基因特性分析,而基于全长VP1序列的基因型划分结果尚未明确。本研究依托国家手足口病监测网络,对2010-2019年全国31个省级行政区（省、自治区、直辖市）上送的18 238份手足口病样本中分离出的24株CVA9进行全长VP1区序列测定,并与GenBank中所有全长VP1区序列一起进行基因型划分研究。测序结果显示24株CVA9分离株VP1全长为906bp,编码302个氨基酸,与CVA9原型株（Griggs）核苷酸和氨基酸相似性分别为80.5%～97.6%和92.3%～99.6%。结合系统进化树和同一血清型内不同基因型的核苷酸差异界值为15%～25%,将全球CVA9划分为A-H八个基因型。进化树显示B、C和D基因型在病毒进化过程中已消失,而E、F和G基因型呈现共循环的趋势,其中G基因型包含了亚洲、北美洲、大洋洲和欧洲等9个国家的毒株,是CVA9的优势基因型。大...     

全球EV-A71的基因型分布

肠道病毒A组71型(EV-A71)是引起手足口病暴发流行的主要病原体,也是除脊髓灰质炎病毒以外最为重要的人肠道病毒病原体。截止2017年2月,全球报道的EV-A71共包括7个基因型(A~G)和14个基因亚型,其中B基因型和C基因型是目前全球流行的优势基因型,目前已鉴定B基因型有8个基因亚型(B0~B7),C基因型有6个基因亚型(C1~C6)。C4基因亚型中C4a进化分支是引起中国大陆近年手足口病大规模暴发流行的主要病原之一,也是引起手足口病重症和死亡的绝对优势病原体。本文对全球流行的EV-A71基因(亚)型的地理和年代分布、新基因型的发现进行了系统的归纳分析,为中国检测和监测引起HFMD流行的EV-A71基因型或亚型的动态变化提供基础数据。     

云南省4株埃可病毒11型VP1基因特征分析

埃可病毒11型(Echovirus 11,ECHO11)属于肠道病毒B组,是最常见的人类肠道病毒(Human enterovirus,HEV)之一,常引起儿童无菌性脑膜炎、脑炎和急性迟缓性麻痹等疾病。为了对云南省手足口病(Hand,foot,and mouth disease,HFMD)监测中分离到的4株ECHO11毒株的VP1基因进行序列分析,并为云南地区ECHO11的进化及流行趋势等研究提供基础数据,本研究对收集到的HFMD病例标本进行病毒分离培养和鉴定,对鉴定为ECHO11的毒株VP1基因测定其完整序列,与GenBank上其他参考株的VP1区序列构建遗传进化树进行基因分析。结果显示,4株ECHO11型毒株分属A1和D5两个基因亚型,分别与各自基因亚型参考株的同源性为最高。D5基因亚型ECHO11分离株在进化树上聚集为3簇,提示存在多个传播链,其D5-1簇的VP1区氨基酸在多个位点上与同基因亚型的其他簇参考株存在特异突变。本研究揭示,云南地区存在两种不同基因亚型的ECHO11流行情况,特别是D5亚型的出现,提示我国周边流行的ECHO11基因型已进入中国大陆,应密切关注其流行变异情况,为今后制定由ECHO11引起的HFMD的公共卫生策略提供依据。     

2018-2021年广东汕头手足口病病原谱及CVA6/CVA10基因特征分析

本研究旨在通过对手足口病（Hand, foot and mouth disease,HFMD）病例的病原学标本进行肠道病毒分型鉴定,和对CVA6和CVA10开展VP1基因全长序列分析,探究广东省汕头市HFMD病原谱及CVA6和CVA10基因进化特征,为当地HFMD肠道病毒监测及预警提供一定的技术支撑。将汕头市病原监测哨点医院2018-2021年的HFMD样本进行RT-PCR肠道病毒病原学分型,筛选出CVA6和CVA10样本进行VP1区全长基因扩增和测序,并通过系统进化分析、同源性和突变位点分析了解其遗传进化特征。结果显示,2018-2021年共监测到HFMD阳性样本706份,其主要病原体为CVA6和CVA10。CVA6和CVA10流行株分别属于D3和C2亚型。CVA6样本VP1核苷酸和氨基酸的相似性分别为91.37%～100.00%和92.79%～100.00%,与2010年的中国台湾株高度同源（GenBank:JQ946055.1）。CVA10样本VP1核苷酸和氨基酸的相似性分别为92.28%～100.00%和96.98%～100.00%,与2014年的广东株高度同源（GenBank...     

肠道病毒ECHO13中国分离株的基因特征

为研究ECHO13病毒中国分离株的分子特征及其与世界其它分离株之间的基因关系,对1998年、2000年从中国福建省分离到2株ECHO13病毒进行基因序列对比分析.2株病毒分别命名为Fujian98-1和Fujian00-1,用逆转录-聚合酶链反应(RT-PCR)扩增出VP1蛋白编码基因全长861个核苷酸片段并进行序列测定,将2株ECHO13病毒的VP1序列与所有已发表的ECHO13病毒VP1基因全长进行同源性比较.结果显示,福建分离株之间核苷酸同源性为79.6%,氨基酸同源性为93.4%;与遗传距离最近的法国CF1089-91(AJ537604)毒株的核苷酸同源性分别为80%和88%,与代表株Del Carmen的核苷酸同源性分别为75.8%和77.9%.通过VP1基因分析,福建2株病毒均属于ECHO13病毒,与血清中和试验鉴定结果一致.下载所有已发表的ECHO13病毒VP1序列并构建进化树,发现福建2株病毒分属不同的分枝,提示这2株病毒来自不同的病毒传播链.进一步分析发现,整个ECHO13病毒可划分为3个不同的基因型:A、B和C基因型.福建Fujian98-1和Fujian00-1分别被划分在基因型B和C中,各基因型之间的核苷酸差异均大于20%.为验证该分型方法,将26株来自不同国家和时间的ECHO13病毒和2株福建分离ECHO13病毒部分VP1基因序列进行对比分析,建立进化树.结果显示,所有ECHO13病毒被分在A、B和C3个基因型中,而2株福建分离病毒仍然被分在B和C基因型中.除了B基因型1株病毒以外,所有3个基因型之间的核苷酸差异均大于15%,与VP1全长分型结果基本一致,说明部分VP1序列的基因分析也能用于对ECHO13病毒进行规律和分子流行病学的研究中.该研究首次报道了ECHO13病毒中国分离株的VP1蛋白基因全长序列,并推荐按VP1基因全长核苷酸差异≥20%作为划分基因型的标准,将已知的ECHO13病毒划分为A、B和C3个基因型.同时也可用病毒VP1基因5′端部分序列替代VP1全长序列来划分基因型.     

柯萨奇病毒A9型山东地方株的进化遗传学特征

柯萨奇病毒A9型(Coxsackievirus A9,CVA9)是常见的人类肠道病毒血清型,其感染可引起无菌性脑膜炎、脑炎等疾病.为探索其进化遗传学特征,本研究对山东省1991-2018年CVA9分离株的VP1完整编码区进行了序列测定,并与GenBank中获得的全球序列一并进行系统发生学和进化遗传学分析.结果 显示全球CVA9可分为Ⅰ-Ⅻ 12个基因型,优势基因型为Ⅶ,包括山东株在内的所有中国分离株均属于该基因型.进化遗传学研究显示,CVA9 VP1区序列的每年每个碱基的平均进化速率约为6.25×10-3,共同祖先起源于1919年.CVA9 VP1区的基因多样性在2003年以前无明显变化,2003年以后出现明显波动.本研究为我们了解CVA9的流行趋势和遗传变异提供了数据,对CVA9相关疾病的防控和诊断试剂的研发具有重要的现实意义.     

2010年云南省1株ECHO-9病毒全基因序列的遗传特性分析

为了解云南省手足口病患者标本中分离到的一株ECHO-9病毒基因组特征,对2010年云南省ECHO-9病毒分离株MSH-KM812-2010全基因组序列测序,并与GenBank中其它ECHO-9病毒株基因组序列比对和分析。MSH-KM812-2010基因组长为7 424bp,编码2 203个氨基酸,其结构基因区与其它ECHO-9病毒核苷酸和氨基酸的同源性高于其它型别肠道病毒;而在非结构区,与其它肠道病毒血清型的同源性高于ECHO-9病毒株。VP1系统进化分析显示ECHO-9病毒株可形成A、B和C三个进化分支,MSH-KM812-2010株以及其它中国分离株属于C簇。三个分支之间核苷酸序列的差异大于15.0%可将ECHO-9病毒分为三个基因型。通过重组检测软件3(RDP3)和基本局部比对搜索工具(BLAST)比对分析,发现在非结构区可能存在重组。本文首次对我国分离的ECHO-9病毒全基因组序列的测定和分析,对了解ECHO-9病毒遗传特性具有重要意义。     

鼻病毒A21基因型的划分及全基因组特征分析

人鼻病毒(Human rhinovirus,HRV)是导致儿童急性呼吸道感染的主要病毒之一。最近的研究报道发现HRV-A21可导致危重症急性呼吸道感染。本研究将2013～2015年河北省发热呼吸道症候群哨点监测病例中获得的一株HRV-A21病毒标本(本课题组之前的研究已获得VP4/2区序列),结合从GenBank数据库下载的全球98株HRV-A21代表株,基于VP4/2区构建亲缘性关系树。本研究首次将HRV-A21划分为A、B、C、D、E五个基因型,其中E基因型进一步划分为E1、E2和E3亚型,并发现E1、E2和E3亚型在年代分布上呈现出一定的时间进化趋势。我国流行的HRV-A21以C和E基因型为主。基于VP1区划分基因型的结果与VP4/2区相似。本研究进而对这一株HRV-A21进行了全基因组序列测定,并结合GenBank数据库下载的HRV-A21全基因组序列,对HRV-A21进行基因亲缘性关系分析、氨基酸变异和糖基化位点分析。结果显示VP2区、VP3区、VP1区和3D区都属于高变区,但变异程度VP1区>VP2区>VP3区>3D区,同时发现了保守的14个N-糖基化位点...     

2009-2019年中国大陆柯萨奇病毒A14型基因特征分析

柯萨奇病毒A14型属于肠道病毒A组，可引起手足口病和无菌性脑膜炎等疾病，但目前全球对CVA14的分子流行病学研究较少，尚无对CVA14基于全长VP1区进行明确分型的相关研究。因得到中国手足口病监测网络的技术支持，本研究获得2009-2019年在中国大陆分离到的15株CVA14，应用RT-PCR对其全长VP1区进行扩增、测序和分析，并与GenBank中下载的22条CVA14全长VP1序列共同构建系统发育树。结果显示，2009-2019年中国大陆分离的15株CVA14与原型株G-14的核苷酸与氨基酸相似性分别为81.8%～82.9%与95.6%～96.9%,15株CVA14之间的核苷酸与氨基酸相似性分别为91.7%～99.7%与98.3%～100%。中国大陆所有的22条CVA14序列之间的核苷酸与氨基酸相似性分别为91.7%～100.0%与98.3%～100.0%。中国大陆所有的CVA14与国外8条CVA14序列之间的核苷酸与氨基酸相似性分别为80.7%～86.0%与94.9%～97.9%。基于全长VP1系统发育树，全球CVA14可划分为A-G七个基因型，目前全球范围内流行的CVA14的基...     

2014年陕西省柯萨奇病毒A10型相关手足口病的流行特征和其VP1区基因特征研究

了解陕西省手足口病(Hand,foot and mouth disease,HFMD)的致病病原体柯萨奇病毒A10型(CV-A10)的流行特征及VP1区基因特征。对2014年收集的HFMD病例标本,通过荧光定量PCR检测确定肠道病毒型别,对CV-A10引起的HFMD流行特征进行描述性分析。使用RD细胞进行病毒分离,RT-PCR扩增CV-A10的VP1区基因片段并进行序列测定,使用Meg Align软件进行核苷酸及氨基酸的同源性分析,并使用MEGA5.0软件构建系统进化树。2014年CV-A10是陕西HFMD病原谱中的第三大病原,占其他肠道病毒的57.71%,13例重症HFMD病例的致病病原体鉴定为CV-A10,占重症病例的9.03%。CV-A10感染HFMD病例以≤3岁年龄组儿童为主(83.07%),男女性别比为1.15∶1。发病时间主要集中在4～7月。实验室分离出101株CV-A10,覆盖全省10市(区)。完成测序的18株CV-A10核苷酸和氨基酸同源性分别为94.0%～100.0%和97.3%～100.0%,与A型原型株的核苷酸和氨基酸同源性分别为76.2%～77.5%和91.9%～93.0%,与近年来河北、湖南和河南地区流行株具有较高的同源性。系统进化显示陕西CV-A10分离株属于C基因型。CV-A10是2014年陕西HFMD的优势病原,能引起重症HFMD,本次分离到的CV-A10毒株均属于C基因型。     

Molecular Epidemiology of Echovirus 18 Circulating in Mainland China from 2015 to 2016

Echovirus 18(E18), a serotype of Enterovirus B(EV-B) species, is an important pathogen in aseptic meningitis. E18 had rarely been detected in mainland China, but became the predominant pathogen associated with viral encephalitis(VE) and meningitis in Hebei province for the first time in 2015. To investigate the molecular epidemiology and genetic characteristics of E18 in mainland China, sixteen E18 strains from patient throat swabs with hand, foot, and mouth disease(HFMD) in six provinces in China collected between 2015 and 2016, and four E18 strains isolated from 18 patient cerebrospinal fluid specimens with VE in Hebei Province in 2015 were obtained and sequenced. Combined with the sequences from the GenBank database, we performed an extensive genetic analysis. Phylogenetic analysis of VP1 gene sequences revealed that all E18 strains from mainland China after 2015 belonged to subgenotype C2. There were no obvious specific differences in phylogenetic and variation analyses of E18 genome sequences between HFMD and VE/meningitis strains. Potential multiple recombination may have occurred in the 50-untranslated region and in the P2 and P3 nonstructural protein-encoding regions of E18 strains from China. The current E18 strains were potential multiplerecombinant viruses. Overall, these findings supported that E18 caused HFMD, VE, and meningitis, although there were no significant associations between clinical features and viral genomic characteristics.     

Prevalence of Multiple Enteroviruses Associated with Hand,Foot, and Mouth Disease in Shijiazhuang City,Hebei Province,China: Outbreaks of Coxsackieviruses A10 and B3

Hand, foot, and mouth disease (HFMD) has been one of the most common infectious diseases in Shijiazhuang City, as is the situation in China overall. In the National HFMD surveillance system, the pathogen detection was focused on EV-A71 and CVA16, and therefore, information on the other EVs is very limited. In order to identify the circulating EV serotypes in the HFMD outbreaks in Shijiazhuang City during 2010–2012, 4045 patients presented with HFMD were recruited in the study, and clinical samples were investigated. Typing of EV serotypes was performed using the molecular typing methods, and phylogenetic analyses based on entire VP1 sequences of human enterovirus 71 (EV-A71), coxsackievirus A16 (CVA16), CVA10 and CVB3 was performed. The results revealed that EV-A71 and CVA16 were the 2 most important pathogens but the circulating trends of the 2 viruses showed a shift, the spread of EV-A71 became increasingly weak, whereas the spread of CVA16 became increasingly stronger. CVA10 and CVB3 were the third and fourth most prevalent pathogens, respectively. Co-infection of two viruses at the same time was not found in these samples. Based on entire VP1 region sequences, the phylogenetic analysis revealed that C4a subgenotype EV-A71, B1a and B1b subgenotype CVA16 continued to evolve. The CVA10 strains were assigned to 4 genotypes (A–D), whereas the CVB3 strains were assigned to 5 genotypes (A–E), with clear geographical and temporal-specific distributions. The Shijiazhuang CVA10 sequences belonged to 4 epidemic lineages within genotype C, whereas the Shijiazhuang CVB3 sequences belonged to 2 epidemic lineages within genotype E, which may have the same origins as the strains reported in other part of China. CVA10 and CVB3, 2 pathogens that were previously infrequently detected, were identified as pathogens causing the HFMD outbreaks. This study underscores the need for detailed laboratory-based surveillances of HFMD in mainland China.     

Genomic surveillance of coxsackievirus A10 reveals genetic features and recent appearance of genogroup D in Shanghai,China, 2016-2020

Coxsackievirus A10 (CVA10) is one of the major causative agents of hand, foot and mouth disease (HFMD). To investigate the epidemiological characteristics as well as genetic features of CVA10 currently circulating in Shanghai, China, we collected a total of 9,952 sporadic HFMD cases from January 2016 to December 2020. In the past five years, CVA10 was the fourth prevalent causatives associated with HFMD in Shanghai and the overall positive rate was 2.78%. The annual distribution experienced significant fluctuations over the past five years. In addition to entire VP1 sequencing, complete genome sequencing and recombination analysis of CVA10 isolates in Shanghai were further performed. A total of 64 near complete genomes and 11 entire VP1 sequences in this study combined with reference sequences publicly available were integrated into phylogenetic analysis. The CVA10 sequences in this study mainly belonged to genogroup C and presented 91%-100% nucleotide identity with other Chinese isolates based on VP1 region. For the first time, our study reported the appearance of CVA10 genogroup D in Chinese mainland, which had led to large-scale outbreaks in Europe previously. The recombination analysis showed the recombination break point located between 5,100 nt and 6,700 nt, which suggesting intertypic recombination with CVA16 genogroup D. To conclusion, CVA10 genogroup C was the predominant genogroup in Shanghai during 2016-2020. CVA10 recombinant genogroup D was firstly reported in circulating in Chinese mainland. Continuous surveillance is needed to better understand the evolution relationships and transmission pathways of CVA10 to help to guide disease control and prevention.     

Population Dynamics and Genetic Diversity of C4 Strains of Human Enterovirus 71 in Mainland China, 1998–2010

Background

Since 1997, several countries within the Asian Pacific region have been affected by one or more massive outbreaks of Hand Foot and Mouth Disease (HFMD). Virus typing experiments revealed that these outbreaks were caused by strains of human enterovirus 71 (EV71) belonging to several different, recently emerged subgenogroups. In mainland China, a different situation was observed. The first outbreak, localized in Shangdong Province, was reported in 2007, and was followed by a wide-spread outbreak in mainland China in 2008. Since then, numbers of reported HFMD cases have been persistently high.

Methodology/Principal Findings

To gain insight in the epidemiological behavior of EV71 in China, we studied genetic diversity and EV71 population dynamics to address whether the increase in number of reported EV71 infections reflects a real increase in viral spread or is just the result of increased awareness and surveillance. We used systematically collected VP1 gene sequences of 257 EV71 strains collected in Guangdong province from 2008 to 2010 as part of HFMD surveillance activities, and supplemented them with 305 GenBank EV71 reference stains collected in China from 1998 to 2010. All isolates from Guangdong Province belonged to subgenogroup C4. Viral population dynamics indicated that the increased reporting of HFMD in China since 2007 reflects a real increase in viral spread and continued replacement of viral lineages through time. Amino acid sequence comparisons revealed substitution of amino acid in residues 22, 145 and 289 through time regularly with the VP1 gene of EV71 strains isolated in mainland China from 1998 to 2010.

Conclusions

EV71 strains isolated in mainland China mainly belonged to subgenogroup C4. There was exponential growth of the EV71 virus population in 2007 and 2008. There was amino acid substitution through time regularly with the VP1 gene which possibly increased viral spread and/or ability of the virus to circulate persistently among the Chinese population.     

The persistent circulation of enterovirus 71 in People's Republic of China: causing emerging nationwide epidemics since 2008

Emerging epidemics of hand-foot-and-mouth disease (HFMD) associated with enterovirus 71 (EV71) has become a serious concern in mainland China. It caused 126 and 353 fatalities in 2008 and 2009, respectively. The epidemiologic and pathogenic data of the outbreak collected from national laboratory network and notifiable disease surveillance system. To understand the virological evolution of this emerging outbreak, 326 VP1 gene sequences of EV71 detected in China from 1987 to 2009 were collected for genetic analyses. Evidence from both traditional and molecular epidemiology confirmed that the recent HFMD outbreak was an emerging one caused by EV71 of subgenotype C4. This emerging HFMD outbreak is associated with EV71 of subgenotype C4, circulating persistently in mainland China since 1998, but not attributed to the importation of new genotype. Originating from 1992, subgenotype C4 has been the predominant genotype since 1998 in mainland China, with an evolutionary rate of 4.6∼4.8×10⁻³ nucleotide substitutions/site/year. The phylogenetic analysis revealed that the majority of the virus during this epidemic was the most recent descendant of subgenotype C4 (clade C4a). It suggests that the evolution might be one of the potential reasons for this native virus to cause the emerging outbreak in China. However, strong negative selective pressure on VP1 protein of EV71 suggested that immune escape might not be the evolving strategy of EV71, predicting a light future for vaccine development. Nonetheless, long-term antigenic and genetic surveillance is still necessary for further understanding.     

肠道病毒71型安徽、河南株的分离与VP1序列进化分析

旨在研究手足口病患者中肠道病毒71型分离株的病毒基因型特征。采集手足口病患者的粪便标本,进行病毒分离和逆转录-聚合酶链式反应(RT-PCR)特异性扩增进行鉴定,同时选取其中9株EV71分离株,对其抗原决定簇部位VP1区进行核酸序列测定,并参考EV71 A、B、C各基因型的参考株和以往中国EV71的分离株进行同源分析和构建系统发生树。结果显示,所分析的9株病毒株均为C4亚型,3株安徽株H7、H8和H9的VP1序列相似度很高(≥98.8%,其中H7、H9的相似度为100.0%),4株河南株H3、H4、H5和H6相似度较高(≥98.4%,H3、H4和H5≥99.6%,其中H3、H4的相似度为100.0%),它们同河南株H1、H5的相似度也较高(≥97.2%),河南株H2虽然与其他河南株具有较高的序列相似度,但进化分析表明,其与安徽株同源性较高。结果表明,安徽株H7、H8和H9株变异速率明显加快,这可能导致了手足口病在安徽省的率先爆发与大流行,河南株H2最初可能由安徽传入河南。     

Genetic analysis of the VP1 region of human enterovirus 71 strains isolated in Fuyang,China, during 2008

Enterovirus 71 (EV71) is a common cause of Hand, foot, and mouth disease (HFMD) and may also cause severe neurological diseases, such as encephalitis and poliomyelitis-like paralysis. To examine the genetic diversity of EV71, we determined and analyzed the complete VP1 sequences (891 nucleotides) from nine EV71 strains isolated in Fuyang, China. We found that nine EV71 strains isolated were over 98% homologous at the nucleotide level and 93%-100% homologous to members of the C4 subgenogroup. At the amino acid level, these Fuyang strains were 99% -100% homologous to one another, 97%-100% homologous to members of the C4 subgenogroup, and the histidine(H) at amino acid position 22 was conserved among the Fuyang strains. The results indicate that Fuyang isolates belong to genotype C4, and an H at position 22 appears to be a marker for the Fuyang strains.