广东省一株新型肠道病毒EV-B83型的全基因组序列进化分析及重组分析

本研究对2001年广东省急性弛缓性麻痹(Acute flaccid paralysis,AFP)病例中分离到的一株肠道病毒AFP341_GD-CHN_2001株进行了高通量测序后,获取该全基因组序列,经鉴定该毒株为EV-B83。为了解该广东省首株EV-B83分离株的全基因组特征及其重组特点,本研究对AFP341_GD-CHN_2001毒株通过最大似然法构建系统进化树,利用bootscanning分析该毒株与其它型别肠道病毒的重组位点,最后根据该重组位点分区段构建系统进化树,进一步验证重组分析结果。全长VP1序列构建的分子进化树表明,AFP341_GD-CHN_2001毒株与EV-B83原型株及柬埔寨SEP025_KH_2012分离株形成一簇;初步构建P1、P2、P3编码区序列系统进化树,提示该分离株在其分子进化中可能存在基因重组,而bootscanning分析表明非结构蛋白编码区是其发生基因重组的区域。根据bootscanning分析的断裂点对该毒株的全基因组序列分成3段构建系统进化树,该系统进化分析结果进一步明确该分离株于2B、2C、3D非结构编码区存在型间重组,与AFP341_GD-CHN_2001发生重组的毒株为CV-A9分离株NSW-V20_AU_2008、CV-B2分离株NSW-V53_AU_2010以及CV-B3分离株SSM_JL-CHN_2006。当前GenBank数据库中仅有来自美国以及中国云南的两株EV-B83全基因组序列,缺乏对该基因型的分子进化特征描述,本研究中的AFP341_GD-CHN_2001毒株为广东省首株EV-B83分离株,扩展了EV-B83数据信息,为今后EV-B83的研究提供了基础性资料。     

中国大陆地区埃可病毒25型全基因组进化与重组分析

埃可病毒25型（Echovirus 25,E25）属于肠道病毒B组（Enterovirus B, EV-B），其在临床上可引起包括手足口病（Hand, foot, and mouth disease, HFMD），无菌性脑膜炎（Aseptic meningitis, AM），急性弛缓性麻痹（Acute flaccid paralysis, AFP）等在内的多种疾病。本文从全国HFMD监测网络选取了2009-2018年的18株E25毒株进行全基因组测序，将所得全长序列及GenBank上目前已发表的E25全长序列，以及EV-B组其它原型株全长序列分别构建全基因组和各编码区的亲缘进化树，并依据不同进化分支选取具有代表性的两株E25毒株进行重组分析。结果显示，中国大陆地区E25在P1区具有更高的亲缘关系，而P2和P3区建树中各出现了两个分支，且在每个分支上，与多个EV-B组的原型株具有较高的核苷酸同源性，提示E25可能存在多种重组模式。此外所选的两株毒株均与EV-B的其它血清型流行株在P2及P3不同区域发生重组，且重组的模式不相同，因此中国大陆地区的E25至少存在两种重组模式，并且致病力可能与...     

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。     

我国新分离ECHO30病毒VP1序列分析

测定了引起2003年苏北地区无菌性脑膜炎暴发流行的病因病毒FDJS03分离株的VP1基因序列,并与国外同型流行毒株做比较,以了解本流行株的分子生物学特点及遗传变异规律。随机选取FDJS03分离毒株中的4株,用肠道病毒、VP1序列的特异性引物008／011进行RT-PCR,扩增产物经凝胶纯化后测序。将序列输入GenBank,用BLAgr程序进行核苷酸和氨基酸序列比对;选取32株不同地区不同年代的ECHO30分离毒株,在PHYLIP3．573C和TREE-PUZZLE5．0中构建进化树,比较它们完整VP1序列(876nt)的进化关系。核苷酸和氨基酸同源性比较结果证明：4株分离病毒均为ECHO30。进化树分析显示：本次FDJS03分离株与欧美20世纪70、80年代流行株亲缘关系最近,但自成一簇,与国外毒株仍然有地区差别。ECHO30的VP1基因进化有时间效应,但存在地区差异。本次流行的病原可能是单一基因型的ECHO30病毒。     

柯萨奇病毒A组2型江苏分离株(JS16-80)全基因组序列分析

柯萨奇病毒A组2型(CV-A2)是引起手足口病和疱疹性咽峡炎暴发流行的重要病原体。本文选取中国大陆流行的D基因型代表毒株JS16-80/JS/CHN/2016(简写JS16-80)进行全基因组序列测定和分析,了解其基因特征,并进行重组和进化分析。结果提示,JS16-80在非结构蛋白P2和P3多区段均有出现重组现象,并且多个A组肠道病毒血清型参与了非结构蛋白的重组。本研究可帮助了解CV-A2的全基因组特征和重组特点,进一步为CV-A2重组对其进化和潜在致病力的影响提供基础数据。     

2009年云南省边境地区健康儿童肠道病毒测序定型和ECHO7、ECHO13病毒的基因特征分析

了解2009年缅甸入境健康儿童和云南省口岸地区健康儿童肠道病毒(enterovirus,EV)带毒情况并对埃柯病毒7型(ECHO7)和埃柯病毒13型(ECHO13)的基因特征进行了描述。采集9个边境口岸小于15岁的健康儿童粪便标本271份,进行病毒分离和基因测序定型。271份便标本中共检测到EV30株(带毒率为11.1%),其中脊髓灰质炎病毒(poliovirus,PV)6株(阳性率2.8%),均为疫苗株,未发现脊灰野病毒;检测到非脊灰病毒(NPV)24株(阳性率8.9%)。经VP1区核苷酸序列测定,24株NPV全部为人类肠道病毒B组(HEV-B,6个血清型),其中13株为埃柯病毒7型(echovirus 7,ECHO7,占54.17%),5株为ECHO13(占20.83%)。未分离到HEV-A组,HEV-C组和HEV-D组病毒。2009年缅甸入境健康儿童和云南省口岸地区健康儿童中肠道病毒携带率较高,且均为HEV-B组病毒,其中主要型别为ECHO7和ECHO13,这两种病毒存在基因多样性特点(即存在不同的基因型)。     

引起甘肃省一起病毒性脑炎疫情的ECHO30全基因组序列分析

为了解引起2015年甘肃省酒泉市瓜州县病毒性脑炎疫情的ECHO30的基因特点,分析其重组变异和进化规律,对从患者脑脊液标本中分离到的3株ECHO30进行全基因序列测定和分析,采用Simplot3.5.1软件进行重组分析,并利用MEGA6.0软件构建系统进化树。结果表明,本次疫情中分离到的ECHO30与E30/SD/2010/CHN株的相似度最高(92%~99%)。重组分析表明甘肃ECHO30株在非结构蛋白区P2区和P3区出现了多处重组现象。由于对当地流行的肠道病毒谱不清楚,虽然发现此次流行的病毒存在重组,但是对重组片段的来源尚不明确,因此需要加强对甘肃省流行肠道病毒的本底调查工作和遗传背景的研究工作。     

云南省2006～2010年非脊灰肠道病毒分子流行病学特征分析

为了解云南省非脊髓灰质炎(脊灰)肠道病毒(NPEV)的基因型分布及分子进化特征,对2006～2010年间从急性迟缓性麻痹(AFP)病例中分离到的105株NPEVs进行VP1区部分核苷酸扩增和序列测定。所获得的云南地方株基因序列与各基因型原型株进行核苷酸与氨基酸同源性比较,并与GenBank中选取的代表株构建基因进化关系树。结果分析显示:105株NPEVs分别属于HEV-A、HEV-B、HEV-C,其中HEV-A 18株(7个血清型)所占比例为17.1%;HEV-B 77株(22个血清型)所占比例为73.3%,表明云南省AFP病例中流行的NPEV还是以HEV-B为主;HEV-C 10株(4个血清型)所占比例为9.5%;没有分离到HEV-D组肠道病毒;基因进化树中各种血清型病毒与对应原型株及代表株聚集一起,除CA2、EV90和EV76外,云南地方株与原型株位于不同分支。相同型别的毒株在5年的流行过程中变异程度亦不同,亲缘关系远近不一,表明这些病毒在云南省存在不同的传播链。     

湖南省2009～2014年分离埃可病毒6型的基因特征分析

为了解中国埃可病毒6型(Echovirus type 6,ECHO6)的基因特征。通过对2009~2014年从湖南省手足口病病例监测中分离到的6株ECHO6病毒部分VP1序列测定,并与来自中国5个省和世界范围的138株ECHO6病毒进行序列对比和构建系统进化树。中国ECHO6病毒形成2个彼此独立的进化分支,分支1和分支2。湖南省ECHO6病毒均聚集在2c亚分支中,可能具有共同的进化来源。分支2病毒在我国分布较广且流行时间较长,可能是我国的优势流行株。以核苷酸差异30%为分组依据,可将ECHO6病毒划分为4个基因组A~D,以核苷酸差异在15%~30%之间分组,将D基因组再划分为10个基因亚组,D1~D10。湖南省ECHO6病毒均属于D7基因亚型。研究结果显示,2009~2014年期间,湖南省存在着具有共同进化来源的ECHO6病毒流行。中国至少存在4个不同进化分支的ECHO6病毒流行。     

龙岩市一起暴发性脑炎疫情分离的埃可病毒6型基因特征分析

对2010年福建省龙岩市长汀县埃可病毒6型(ECHO6)肠道病毒引起的暴发性脑炎,进行病原学分析,为该病的防控提供有价值的信息。对病例脑脊液标本进行荧光RT-PCR、病毒分离(RD细胞)、中和鉴定进行病原确认,再经PCR法对其中4株毒株进行VP1片段或全基因组核苷酸序列测定,用DNAStar软件中的MegAlign等软件进行同源性分析,Mega 4.0软件进行进化树分析。经病原学检测方法确认为ECHO6型肠道病毒,VP1片段核苷酸序列分析显示为C2亚型。全基因组序列共7 407个核苷酸,与其他ECHO病毒和CVB(Coxsackie virus B)病毒基因组构成相似,同源性为78.5%~87.3%。此次暴发性脑炎是由肠道病毒ECHO6C2亚型引起,病毒株全基因组序列长度与标准株(U16283)相近,同源性达80.4%。     

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.     

Analysis of molecular variation in porcine reproductive and respiratory syndrome virus in China between 2007 and 2012

In the present study, 89 porcine reproductive and respiratory syndrome virus (PRRSV) isolates in China during 2007 to 2012 were randomly selected from the GenBank genetic sequence database. Evolutionary characteristics of these isolates were analyzed based on the sequences of non-structural protein 2 (Nsp2) and glycoprotein 5 (GP5). The genetic variations of the isolates were also compared with six representative strains. The results showed that a high degree of genetic diversity exists among the PRRSV population in China. Highly pathogenic PRRSV isolates, with a discontinuous deletion of a 30 amino acid residue in the Nsp2 region, remained the most dominant virus throughout 2007–2012 in China. Owing to the extensive use of representative vaccine strains, natural recombination events occurred between strains. Three isolates — HH08, DY, and YN-2011 — were more closely related to vaccine strains than the other isolates. Both YN-2011 and DY were the evolutionary products of recombination events between strains SP and CH-1R. The results of the present study provide useful information for the epidemiology of PRRSV as well as for vaccine development.     

The association of recombination events in the founding and emergence of subgenogroup evolutionary lineages of human enterovirus 71

Enterovirus 71 (EV71) is responsible for frequent large-scale outbreaks of hand, foot, and mouth disease worldwide and represent a major etiological agent of severe, sometimes fatal neurological disease. EV71 variants have been classified into three genogroups (GgA, GgB, and GgC), and the latter two are further subdivided into subgenogroups B1 to B5 and C1 to C5. To investigate the dual roles of recombination and evolution in the epidemiology and transmission of EV71 worldwide, we performed a large-scale genetic analysis of isolates (n = 308) collected from 19 countries worldwide over a 40-year period. A series of recombination events occurred over this period, which have been identified through incongruities in sequence grouping between the VP1 and 3Dpol regions. Eleven 3Dpol clades were identified, each specific to EV71 and associated with specific subgenogroups but interspersed phylogenetically with clades of coxsackievirus A16 and other EV species A serotypes. The likelihood of recombination increased with VP1 sequence divergence; mean half-lives for EV71 recombinant forms (RFs) of 6 and 9 years for GgB and GgC overlapped with those observed for the EV-B serotypes, echovirus 9 (E9), E30, and E11, respectively (1.3 to 9.8 years). Furthermore, within genogroups, sporadic recombination events occurred, such as the linkage of two B4 variants to RF-W instead of RF-A and of two C4 variants to RF-H. Intriguingly, recombination events occurred as a founding event of most subgenogroups immediately preceding their lineage expansion and global emergence. The possibility that recombination contributed to their subsequent spread through improved fitness requires further biological and immunological characterization.     

Prevalence of Nonpolio Enteroviruses in the Sewage of Guangzhou City,China, from 2009 to 2012

The human-pathogenic viruses in urban sewage have been extensively monitored to obtain information on circulating viruses in human communities. Enteroviruses (EVs) excreted by patients who present with diverse clinical syndromes can remain infectious in the environment for several weeks, and limited data on circulating environmental EVs are available. A 4-year (2009 to 2012) surveillance study was conducted to detect nonpolio enteroviruses (NPEVs) in the urban sewage of Guangzhou city, China. After the viruses in the sewage samples were concentrated and isolated, molecular identification was used to detect and type the NPEVs. During the 4-year study, 17 different NPEV serotypes were identified in the sewage of Guangzhou city. The most common serotypes were echovirus 11 (ECHO11), ECHO6, ECHO7, and ECHO12 and coxsackie group B viruses 5 (CVB5) and CVB3. The predominant serotypes were influenced by spatial and temporal factors and differed each year. CVB5 was commonly detected in 2009 and 2010 but was rarely isolated in 2011 and 2012. In contrast, CVB3 was not observed in 2009 and 2010 but was increasingly detected in 2011 and 2012. Our study provides an overview of the serotype distribution and circulation patterns of NPEVs in the sewage of Guangzhou, China. In the absence of a systematic EV disease surveillance system, the detection and characterization of sewage-borne NPEVs will help us better understand the changes in EV disease trends and the epidemic background of circulating EVs, which could help interpret the EV trends and warn of future outbreaks in this area.     

一起传染病暴发中肠道病毒血清型鉴定和ECHO30基因特征分析

2003年5～9月,山东省泰安市发生了由肠道病毒(Enterovirus,EV)感染所致的传染病暴发,临床症状以手足口病(HFMD)为主,同时有心肌炎和无菌性脑膜炎等中枢神经系统症状患者也占较大比例。131份病人(粪便、咽拭子、脑脊液)标本中共分离到EV62株,其中ECHO1939株,EV716株,ECHO304株,其它肠道病毒13株。4株ECHO30病毒中的2株分离自2个患者的粪便标本,但用WHO肠道组合血清中和试验未能定出型别。另外2株分离自同一患者的粪便和脑脊液标本。病原学分析表明,ECHO30是引起该患者无菌性脑膜炎的病原。抗E—CHO30标准株的血清中和这4株病毒的滴度低于标准株5～20倍。VP1区全基因序列测定和同源性比较分析表明,4株ECHO30分离株病毒核苷酸同源性在98．0％～98．5％,氨基酸同源性在98．9％～99．3％,提示这4株病毒来源于同一传播链,2003年5～9月ECHO30在该地区可能有局部流行。系统进化树分析表明,ECHO30病毒可以划分为6个基因型,其中基因型1～5为GenBank中已发表的ECHO30分离株,山东分离株与其它5个基因型成员核苷酸差异分别在9．4％～24．4％,在进化树上形成了较独立的分支,是一个新基因型,将其划分为第6基因型。     

Genetic relationship of diarrheagenic Escherichia coli pathotypes among the enteropathogenic Escherichia coli O serogroup

The genetic relationship among the Escherichia coli pathotypes was investigated. We used random amplified polymorphic DNA (RAPD) data for constructing a dendrogram of 73 strains of diarrheagenic E. coli. A phylogenetic tree encompassing 15 serotypes from different pathotypes was constructed using multilocus sequence typing data. Phylogram clusters were used for validating RAPD data on the clonality of enteropathogenic E. coli (EPEC) O serogroup strains. Both analyses showed very similar topologies, characterized by the presence of two major groups: group A includes EPEC H6 and H34 strains and group B contains the other EPEC strains plus all serotypes belonging to atypical EPEC, enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC). These results confirm the existence of two evolutionary divergent groups in EPEC: one is genetically and serologically very homogeneous whereas the other harbors EPEC and non-EPEC serotypes. The same situation was found for EAEC and EHEC.     

Evidence of recombination among enteroviruses.

Human enteroviruses consist of more than 60 serotypes, reflecting a wide range of evolutionary divergence. They have been genetically classified into four clusters on the basis of sequence homology in the coding region of the single-stranded RNA genome. To explore further the genetic relationships between human enteroviruses and to characterize the evolutionary mechanisms responsible for variation, previously sequenced genomes were subjected to detailed comparison. Bootstrap and genetic similarity analyses were used to systematically scan the alignments of complete genomic sequences. Bootstrap analysis provided evidence from an early recombination event at the junction of the 5' noncoding and coding regions of the progenitors of the current clusters. Analysis within the genetic clusters indicated that enterovirus prototype strains include intraspecies recombinants. Recombination breakpoints were detected in all genomic regions except the capsid protein coding region. Our results suggest that recombination is a significant and relatively frequent mechanism in the evolution of enterovirus genomes.     

Comparative Genomic Analysis Shows That Avian Pathogenic Escherichia coli Isolate IMT5155 (O2:K1:H5; ST Complex 95, ST140) Shares Close Relationship with ST95 APEC O1:K1 and Human ExPEC O18:K1 Strains

Avian pathogenic E. coli and human extraintestinal pathogenic E. coli serotypes O1, O2 and O18 strains isolated from different hosts are generally located in phylogroup B2 and ST complex 95, and they share similar genetic characteristics and pathogenicity, with no or minimal host specificity. They are popular objects for the study of ExPEC genetic characteristics and pathogenesis in recent years. Here, we investigated the evolution and genetic blueprint of APEC pathotype by performing phylogenetic and comparative genome analysis of avian pathogenic E. coli strain IMT5155 (O2:K1:H5; ST complex 95, ST140) with other E. coli pathotypes. Phylogeny analyses indicated that IMT5155 has closest evolutionary relationship with APEC O1, IHE3034, and UTI89. Comparative genomic analysis showed that IMT5155 and APEC O1 shared significant genetic overlap/similarities with human ExPEC dominant O18:K1 strains (IHE3034 and UTI89). Furthermore, the unique PAI I₅₁₅₅ (GI-12) was identified and found to be conserved in APEC O2 serotype isolates. GI-7 and GI-16 encoding two typical T6SSs in IMT5155 might be useful markers for the identification of ExPEC dominant serotypes (O1, O2, and O18) strains. IMT5155 contained a ColV plasmid p1ColV₅₁₅₅, which defined the APEC pathotype. The distribution analysis of 10 sequenced ExPEC pan-genome virulence factors among 47 sequenced E. coli strains provided meaningful information for B2 APEC/ExPEC-specific virulence factors, including several adhesins, invasins, toxins, iron acquisition systems, and so on. The pathogenicity tests of IMT5155 and other APEC O1:K1 and O2:K1 serotypes strains (isolated in China) through four animal models showed that they were highly virulent for avian colisepticemia and able to cause septicemia and meningitis in neonatal rats, suggesting zoonotic potential of these APEC O1:K1 and O2:K1 isolates.