一株柯萨奇病毒B组5型分离株V1641/YN/CHN/2016的全基因序列分析

对2016年云南省昆明市手足口病相关的柯萨奇病毒B组5型分离株V1641/YN/CHN/2016（简称V1641）全基因组进行测序,并分析其分子变异和进化特点。设计针对V1641的引物,提取病毒RNA,RT-PCR扩增和测序,拼接获得的全基因组序列。利用MEGA7.0.26、Geneious9.1.4和SimPlot3.5.1软件分析全基因序列。V1641毒株基因组全长7 392nt,5’-UTR和3’-UTR分别长744nt和90nt,编码区长6 558nt,与其他CVB5相比未见核苷酸的插入和缺失,编码一个2 185aa的多聚蛋白。CVB5流行株大体上分为两个基因组,中国大陆流行株同原型株Faulkner一起分布于GenogroupⅠ分支,外国流行株大多分布于GenogroupⅡ分支。在GenBank中,V1641与KY303900-417/JS-CHN-2013最为同源,相似性为97.86%。V1641与其他中国大陆流行株的全基因组序列的核苷酸和氨基酸相似性分别为85.1%～97.8%和97.1%～99.6%。V1641在P1、P2和P3区段均与EV-B不同血清型的原型株聚类,经...     

广东省2020年首次报道E基因型柯萨奇病毒B组3型的鉴定及基因特征分析

柯萨奇病毒B组3型(Coxsackievirus B3,CVB3)是肠道病毒中流行较为广泛的血清型之一,在全球多个国家和地区曾报道儿童急性心肌炎、无菌性脑膜炎、手足口病等的暴发流行,严重威胁儿童健康和公共卫生安全。本研究对广东省2020年手足口病患者标本中分离到的8株CVB3进行基因特征和进化分析,结果显示分离到的8株CVB3 VP1区核苷酸序列相似性为98.2%~99.5%,与原型株Nancy的核苷酸序列相似性在77.9%~78.5%之间,与其他CVB3中国大陆流行株的核苷酸序列相似性为79.6%~82.2%。8株CVB3均为E基因型,为广东省首次报道。8株CVB3分离株在进化树上聚集,提示病毒发生了局部传播。全基因组序列分析提示2株广东CVB3分离株在非结构蛋白区有重组现象的发生。本研究为E基因型CVB3在我国的流行传播和疾病防控提供基础资料。     

辽宁省首株柯萨奇病毒B组5型全基因组序列分析

研究引起辽宁地区手足口病的柯萨奇病毒B组5型（coxsackievirus B5,CV-B5）基因组特征。对2018年从辽宁省688份肠道病毒核酸阳性的标本中分离到的1株CV-B5进行高通量测序，并对其全基因组进行遗传进化分析。结果表明，CV-B5辽宁分离株与国内流行株的全基因组核苷酸序列同源性为78.5%～97%，氨基酸序列同源性为75.3%～96.7%。基于全基因组的进化分析将CV-B5流行株分为A～D四个基因型，辽宁分离株属于D基因型。通过重组分析发现其在P3区的3D区段发生重组。首次在辽宁地区手足口病患儿中分离出CV-B5，辽宁省分离株（LN2018-23-21/CHN/2018）可能为重组株。     

柯萨奇病毒A组16型河南省分离株基因组序列分析

为了解河南省手足口病患者标本中分离柯萨奇A组的16型(CoxAl6)病毒基因组特征,对2010年采集的手足口病患者临床标本406份进行RT-PCR扩增和病毒分离鉴定;通过10对引物分段扩增和拼接CoxAl6分离株基因组序列,利用生物信息学软件对序列分析,构建序列遗传发育树。测序获得河南省CoxAl6分离株HN1162/HN/CHN/2010基因组全长序列7 411bp,5′非编码区(5′UTR)、P1、P2、P3、3′非编码区(3′UTR)区域核苷酸序列与GenBank公布的其它分离株相似性分别为87.0%～97.9%、77.0%～95.4%、80.3%～96.9%、77.9%～96.2%、80.5%～100%;VP1区核苷酸相似性为91.4%～96.4%,氨基酸相似性为99.3%～99.7%;遗传发育树分析表明与我国深圳、广州、福建分离株处于同一分支。河南省手足口病患者标本CoxAl6病毒分离株属于C2基因亚型/B-2基因亚型,对加强该病毒变异检测,预防控制手足口病疫情具有重要意义。     

天津市致病毒性脑炎柯萨奇病毒B组5型全基因组序列分析

肠道病毒是我国病毒性脑炎(Viral encephalitis,VE)的主要病原体。本文研究对4株引起VE的天津柯萨奇病毒B组5型(Coxsackievirus B5,CV-B5)分离株进行Illumina MiniSeq高通量测序,并对其全基因组特征、进化及重组特点进行分析。结果提示,4株CV-B5天津分离株的全基因组核苷酸和氨基酸序列同源性分别为84.5%~100.0%和98.1%~100.0%,与国内流行株的全基因组核苷酸序列同源性为83.2%~96.5%,氨基酸序列同源性为96.4%~99.4%。基于全基因组的系统进化分析将CV-B5流行株分为A-D四个基因型,其中天津与国内流行株均属于C基因型。C基因型进一步分为3个进化分支,而天津分离株处在两个不同的分支上。基于基因组各区段序列的系统进化与SimPlot重组分析结果显示,天津分离株15-39N、15-41N与埃可病毒30型(Echovirus 30,E-30)原型株在P3区3B、3C、3D区域均检测到重组信号。本研究有助于了解CV-B5的全基因组特点和重组规律,为相关疾病的防控提供依据。     

柯萨奇病毒B组3型基因组剪切片段的序列分析

柯萨奇病毒B组(Coxsackievirus B,CVB)感染细胞时其基因组RNA存在不稳定现象,但产生机制尚不清楚。本研究将柯萨奇病毒B组3型(CVB3)感染细胞后,利用5′ cDNA末端快速扩增技术(5′ rapid amplification of cDNA ends,5′ RACE)扩增并克隆细胞内CVB3基因组片段,并对每条序列及其5′端的二级结构进行分析。结果获得的20条CVB3基因组片段,长度为 2 067～5 547 bp,片段断端主要分布于2Apro和2C编码区。RNAfold分析显示,这些片段多数在5′断点端形成二级茎-环结构。本研究显示,CVB在宿主细胞感染时可形成大量不完整基因组RNA片段,这些片段可在5′断点端形成局部双链结构,提示片段不是随机产生,可能是RNA酶剪切产物。此发现有助于理解CVB基因组不稳定的机制。     

柯萨奇病毒B5的VP1区域基因特征分析及蛋白二级结构预测

柯萨奇病毒B组5型(Coxsackievirus B5,CVB5)感染人体后可引起一系列疾病,包括上呼吸道感染、腹泻、手足口病(Hand,foot,and mouth disease,HFMD)、病毒性脑炎、无菌性脑膜炎、胰腺炎、弛缓性麻痹、扩张性心肌炎以及糖尿病等症状。为了解云南省手足口病和疱疹性咽峡炎患者中CVB5的分子特征,本研究从2018-2019年昆明市儿童医院收集的HFMD和疱疹性咽峡炎(Herpangina,HA)患者粪便中分离得到9株CVB5,基于CVB5的VP1区的系统进化树以及蛋白二级结构的预测来分析分离株的基因序列特征以及与CVB5原型株Faulkner之间的差异,并对CVB5的分子流行病学特征进行分析。结果显示,2018-2019年分离株均属于E基因簇,与Faulkner株相比,所有分离株的VP1区域的氨基酸出现共同的10个氨基酸突变且增加了1个蛋白连接位点。本研究初步探索了肠道病毒CVB5进化过程中突变导致患者症状的差异以及揭示CVB5的全球流行趋势。     

河北省石家庄市手足口病流行中健康人肠道病毒带毒情况及病原分析

为了解河北省石家庄市健康儿童中肠道病毒带毒情况及血清型构成,对2010~2012年石家庄市的3个县区1 223例0~6岁的健康儿童及406名监护人(健康成人)采集咽拭子和粪便标本进行核酸检测鉴定肠道病毒血清型,并进行分子流行病学分析,为肠道病毒感染相关疾病的防控和治疗提供基础资料。1 223名健康儿童的标本检测结果中,肠道病毒(Enterovirus,EV)阳性的283例,阳性率为23.14%(283/1 223)。其中,以非肠道病毒71型非柯萨奇病毒A组16型的EV(Other-Enterovirus,other-EVs)阳性为主,占EV阳性的62%(175/283),而引起手足口病常见的EV病原体肠道病毒71型(Enterovirus A71,EV71)和柯萨奇病毒A组16型(Coxsackievirus A16,CVA16)阳性率较低,分别占19%(55/283)和18%(51/283);将结果为other-EVs阳性的175名健康儿童的标本全部进行病毒分离鉴定,共分离到25株病毒,分离率为14.29%(25/175),其中柯萨奇病毒B组5型(Coxsackievirus B5,CVB5)分离率最高,为40%(10/25)、柯萨奇病毒B组13型(Coxsackievirus B3,CVB3)分离率为24%(6/25)、埃可病毒21型(Enteric cytopathic human orphan virus 21,ECHO21)分离率为16%(4/25)、其他EV株分离率为20%(5/25)。提示,应加强对CVB3和CVB5等other-EVs的监测。     

柯萨奇B组病毒致病性的分子生物学研究

柯萨奇B组病毒（coxsackievirus group B,CVB）是微小RNA病毒科肠道病毒属成员,病毒性心肌炎的病例中大约有20％-25％是由柯萨奇B组病毒引起。CVB的致病机制十分复杂,病毒基因组以及病毒蛋白均在病毒致病过程中发挥重要作用。因此,对柯萨奇B组病毒的基因组、结构蛋白以及某些非结构蛋白与靶细胞内分子间相互作用生物信息的认识是阐述该病分子机制的基础。     

Full Genome Sequence of a Novel Coxsackievirus B5 Strain Isolated from Neurological Hand,Foot, and Mouth Disease Patients in China

Coxsackievirus B5 (CVB5) belongs to the human enterovirus B species within the family Picornaviridae. We report the complete genome sequence of a novel CVB5 strain, CVB5/SD/09, that is associated with neurological hand, foot, and mouth disease in China. The complete genome consists of 7,399 nucleotides, excluding the 3′ poly(A) tail, and has an open reading frame that maps between nucleotide positions 744 and 7301 and encodes a 2,185-amino-acid polyprotein. Phylogenetic analysis based on different genome region regions reveals that CVB5/SD/09 belongs to a novel CVB5 lineage, and similarity plotting and bootscanning analysis based on the whole genome of CVB5 in the present study and those available in GenBank indicate that the genome of CVB5/SD/09 has a mosaic-like structure, suggesting that recombination between different CVB5 strains may occur.     

Structure of the 5' nontranslated region of the coxsackievirus b3 genome: Chemical modification and comparative sequence analysis

Coxsackievirus B3 (CVB3) is a picornavirus which causes myocarditis and pancreatitis and may play a role in type I diabetes. The viral genome is a single 7,400-nucleotide polyadenylated RNA encoding 11 proteins in a single open reading frame. The 5' end of the viral genome contains a highly structured nontranslated region (5'NTR) which folds to form an internal ribosome entry site (IRES) as well as structures responsible for genome replication, both of which are critical for virulence. A structural model of the CVB3 5'NTR, generated primarily by comparative sequence analysis and energy minimization, shows seven domains (I to VII). While this model provides a preliminary basis for structural analysis, the model lacks comprehensive experimental validation. Here we provide experimental evidence from chemical modification analysis to determine the structure of the CVB3 5'NTR. Chemical probing results show that the theoretical model for the CVB3 5'NTR is largely, but not completely, supported experimentally. In combination with our chemical probing data, we have used the RNASTRUCTURE algorithm and sequence comparison of 105 enterovirus sequences to provide evidence for novel secondary and tertiary interactions. A comprehensive examination of secondary structure is discussed, along with new evidence for tertiary interactions. These include a loop E motif in domain III and a long-range pairing interaction that links domain II to domain V. The results of our work provide mechanistic insight into key functional elements in the cloverleaf and IRES, thereby establishing a base of structural information from which to interpret experiments with CVB3 and other picornaviruses.     

Genomic determinants of cardiovirulence in coxsackievirus B3 clinical isolates: localization to the 5' nontranslated region

Coxsackievirus B3 (CVB3) infections can cause myocarditis in humans and are implicated in the pathogenesis of dilated cardiomyopathy. The natural genetic determinants of cardiovirulence for CVB3 have not been identified, although using strains engineered in the laboratory, cardiovirulence determinants have been identified in the CVB3 5' nontranslated region (5'NTR) and capsid. The myocarditic phenotypes of two CVB3 clinical isolates were determined using an established murine model of inflammatory heart disease. The 5'NTRs and capsid proteins of the noncardiovirulent CVB3/CO strain and cardiovirulent CVB3/AS strain were examined to determine their influence on the cardiovirulence phenotype. Six intratypic chimeric viruses were constructed in which 5'NTR and capsid sequences of the infectious cDNA copy of the cardiovirulent CVB3/20 genome were replaced by homologous sequences from CVB3/CO or CVB3/AS. Chimeric strains were tested for cardiovirulence by inoculation of C3H/HeJ mice. Sections of hearts removed at 10 days postinoculation were examined for evidence of myocarditis by light microscopy and assayed for the presence of virus. Replacement of the CVB3/20 capsid coding region by that from the homologous region of CVB3/CO resulted in no change in the cardiovirulent CVB3/20 phenotype, with virus recoverable from the heart at 10 days postinoculation. However, recombinant virus containing the CVB3/CO 5'NTR alone or the 5'NTR and capsid sequences together were not myocarditic, and infectious virus was not recovered from the myocardium. Chimeric viruses containing the CVB3/AS 5'NTR alone, capsid sequence alone, or both together preserved the myocarditic phenotype. These data support the 5'NTR as the primary site in the determination of the natural cardiovirulence phenotype of CVB3.     

Complete genome sequence of a recombinant coxsackievirus b4 from a patient with a fatal case of hand, foot, and mouth disease in guangxi, china

The coxsackievirus B4 (CVB4) belongs to human enterovirus B species within the family Picornaviridae. Here we report a novel complete genome sequence of a recombinant CVB4 strain, CVB4/GX/10, which was isolated from a patient with a fatal case of hand, foot, and mouth disease in China. The complete genome consists of 7,293 nucleotides, excluding the 3' poly(A) tail, and has an open reading frame that maps between nucleotide positions 742 and 7293 and encodes a 2,183-amino-acid polyprotein. Phylogenetic analysis based on different genome regions reveals that CVB4/GX/10 is closest to a CVB4 strain, EPIHFMD-CLOSE CONTACT-16, in the 5' half (VP4～2B) of the genome, although it is closer to a Chinese CVB5 strain, CVB5/Henan/2010, in the 3' half (2C～3D) of the genome. Furthermore, similar bootscan analysis based on the whole genomes demonstrates that recombination has possibly occurred within the 2C domain and that CVB4/GX/10 is a possible progeny of intertypic recombination of the CVB4 strain EPIHFMD-CLOSE CONTACT-16 and CVB5/Henan/2010 that occurred during their cocirculation and evolution, which is a relatively common phenomenon in enteroviruses.     

SiRNA抑制柯萨奇B3病毒的复制和表达

目的 研究观察体外合成siRNA对培养HELA细胞中柯萨奇B3病毒（Coxsackievirus B3,CVB3）的影响。方法根据siRNA靶序列设计原则,针对编码CVB3病毒聚合酶、VP1蛋白和5’非编码区基因组,特异性地体外合成三对siRNA,同时合成一对与CVB基因组序列无关的阴性对照siRNA。利用脂质体转染进入Hela细胞,用CVB3感染培养HELA细胞,观察转染后HELA细胞病变;采用RT-PCR技术检测感染CVB3各组的病毒RNA;用免疫荧光技术检测各组CVB3蛋白的表达;并用培养细胞上清液再感染HELA细胞观察病毒滴度。结果针对CVB3病毒聚合酶的siR-NA能有效的抑制病毒的复制和CVB3蛋白的表达,并能抑制病毒的再感染;而针对VP1蛋白和5’非编码区的siRNA能部分抑制病毒的复制和CVB3蛋白的表达。结论我们设计合成针对编码CVB3病毒聚合酶基因组的siRNA能有效抑制CVB3病毒复制和表达。     

Nucleotide sequence of the 5'nontranslated and virion polypeptides regions of coxsackievirus B6.

The nucleotide sequence of coxsackievirus B6 (CVB6) has been determined, and the nucleotides encoding the 5' nontranslated region (5' NTR) and virion polypeptides (VP4, 2, 3 and 1) were compared with other serotype CVBs. An Unweighted Pair-Group Method Analysis (UPGMA) of phylogenetic trees indicated that the 5' NTR of CVB6 locates on an independent branch from the other CVBs. The tree based on the amino acid sequences showed that CVB6 has close correlation with CVB4 in the VP4 and VP2 regions, with CVB1 and CVB5 in the VP3 region, and with CVB5 in the VP1 region. Amino acid sequences of variable regions within the VP2, VP3, and VP1 of CVB6 were unique among CVBs. Thus, by comparison of the nucleotide and amino acid sequences of these variable regions, CVB6 can be easily distinguished from other serotypes. In addition, serine, instead of glycine, was found to locate at the amino-terminus of the VP1 region of CVB6, indicating that CVB6 has a unique cleavage site (i.e., glutamine/serine instead of glutamine/glycine) for proteinase 3C of Picornaviridae.