青海省67份轮状病毒结构蛋白VP7基因分型和分布情况

目的 研究2016-2017年青海地区腹泻患者轮状病毒基因分型及流行病学分布。方法 收集门诊及住院部腹泻的粪便标本238份,采用实时荧光PCR法对轮状病毒A组进行检测,阳性标本进行VP7基因扩增和测序。结果 238份粪便标本中,通过实时荧光PCR 检测到轮状病毒A组阳性67份,阳性率为28.15%（67/238）;对67份轮状病毒A阳性标本进行VP7蛋白检测和测序,测序后得到29份核苷酸序列,用Clustral X Bootstap NJ Tree软件构件进化树,分析发现2016年3月-2017年12月青海轮状病毒以G9P8型为主,共26株,占89.66% (26/29),G2P4型2株(2/28),G3P8型1株(1/28), 轮状病毒腹泻发病高发季节为9-12月,其中以12月份检出最高,占总数的61.19%（41/67）。病人以成人为主,成人和5岁以下儿童比例为1.73[DK]∶1。结论 2016-2017年青海地区轮状病毒以流行病毒株G9P8型为主。     

pMD18-T作为A组轮状病毒VP7基因克隆载体的研究

目的:利用RT-PCR法从A组轮状病毒扩增基因片段VP7,再将产物重组于大肠杆菌pMD18-T载体,探讨pMD18-T作为A组轮状病毒VP7基因克隆载体的应用。方法:提取A组轮状病毒总RNA,通过RT-PCR扩增,获得目的基因片段VP7,进行分离纯化和回收,最后把纯化的VP7基因与pMD18-T载体进行连接,进行质粒抽提与鉴定。结果:成功将VP7基因连接到pMD18-T载体,转化感受态菌(DH5α),通过蓝白斑筛选得到DNA阳性重组子(载体+VP7基因片段),经DNA测序鉴定正确。结论:成构建功A组轮状病毒外壳蛋白VP7基因克隆载体,为进一步获得大量VP7基因以及研究和开发轮状病毒基因工程疫苗奠定基础。     

武汉市儿童医院婴幼儿腹泻轮状病毒的VP4型别分析

目的研究武汉市儿童医院腹泻门诊A组轮状病毒VP4基因的分子流行病学特征. 方法利用聚丙烯酰胺凝胶电泳,将检测出的A组轮状病毒阳性样利用多重RT-PCR技术对VP4基因进行分型研究. 结果武汉地区793份腹泻患儿粪便样本经检测轮状病毒阳性257例,阳性率为32.4%.其中P [8]型232例(90.3%),P [4]型3例(1.2%),P [8]与P [4]混合感染15例(5.8%),尚有7例(2.7%)未能分出型别.对检测结果按采样时间、年龄和性别分布分别进行了分析. 结论武汉地区A组轮状病毒以P[8]型为主要流行基因型,患儿以6月至1岁为主,男女性别差异不大,武汉地区婴幼儿腹泻A组轮状病毒VP4基因分型研究将为轮状病毒疫苗的研制提供基础.     

人轮状病毒结构蛋白VP7基因毕赤酵母表达质粒的构建

[目的]构建人轮状病毒结构蛋白VP7基因重组酵母表达质粒VP7-pPICZαA. [方法]从VP7-pET32a质粒中PCR扩增轮状病毒VP7基因,EcoR I、Xba,I双酶切VP7基因产物和酵母表达载体pPICZαA,T4 DNA连接酶连接目的基因片段和pPICZαA,转化到大肠杆菌Top10中,Zeocin筛选转化子并进行PCR、酶切和测序鉴定. [结果]阳性克隆菌经酶切和PCR和测序鉴定,结果与预期相符,目的基因正确插入pPICZαA中. [结论]成功构建轮状病毒结构蛋白VP7基因重组酵母表达质粒VP7-pPICZαA,为轮状病毒结构蛋白VP7基因的酵母表达奠定了重要的实验基础.     

宁波地区新生儿轮状病毒VP7基因序列分析

目的研究宁波地区新生儿轮状病毒流行株VP7基因的分子生物学特点以及遗传变异规律。方法通过聚丙烯酰胺凝胶电泳(PAGE)检测新生儿病毒性腹泻便样中的轮状病毒核酸;选择特殊电泳带型样品通过逆转录聚合酶链反应(RT-PCR)扩增VP7全基因并测序;测序结果利用DNAstar和Blast软件进行比对。结果通过PAGE发现11份样品中有9份阳性样品,其中带型为4-2-3-2的有7份,带型为3-2-2-2和4-2-1-2的各1份。3种带型各选一株进行VP7基因的扩增并测序,测序结果经分析发现,06NB3和06NB9与G1型标准株Wa的核苷酸同源性为84.4%和91.5%,氨基酸同源性分别为87.4%和94.8%,06NB2与G3型标准株YO的核苷酸同源性为95.3%,氨基酸的同源性为96.6%。结论宁波地区在新生儿中有A组轮状病毒流行,其电泳带型以典型的4-2-3-2为主,也可见特殊带型。06NB3和06NB9的VP7基因片段属G1型,06NB2则属于G3型。06NB3与国内外近几年流行的G1型毒株基因序列有较大差异,06NB9和06NB2则差异较小。     

北京地区2007-2008年G9型A组人轮状病毒VP7和VP4基因分析

目的 了解北京地区2007-2008年检测到的G9型A组人轮状病毒外壳蛋白VP7和VP4的基因特征.方法 选取经过轮状病毒核酸杂交方法检测为G9型轮状病毒的12份儿童腹泻患儿的粪便标本,应用针对VP7全长基因的特异引物对进行RT-PCR扩增,对所获得的VP7全长基因进行克隆和测序,将所获得的序列与GenBank中的G9型原型病毒株和近期流行株的VP7基因进行序列和种系进化分析;经巢式PCR对G9型的VP4进行P基因分型.结果 12株G9型轮状病毒经VP7基因的序列比较分析得到确认.P基因分型结果显示北京地区近年来存在G9P[8]和G9P[6]型两种组合的轮状病毒感染.序列和种系进化分析发现北京G9型株VP7基因与世界范围内近期流行的G9型株一样都属于进化分支Ⅲ,彼此间的核苷酸和氨基酸同源性较高,而与国内最早报道的G9型T203进化关系较远,且北京G9P[8]和G9P[6]型株分别与国内近期报道的新疆G9P[8]和G9P[6]型株及相应的武汉G9型株VP7基因,在氨基酸位点上存在一些共同的氨基酸残基取代.结论 北京地区近年存在G9P[8]和G9P[6]两种不同基因组合的G9型轮状病毒感染,需要进一步加强对G9型轮状病毒的分子流行病学监测.     

昆明地区22株人轮状病毒VP7及NSP4基因分析

目的研究昆明地区轮状病毒（RV）不同VP7血清型及NSP4基因变异与腹泻的流行及症状严重程度的关系。方法对2002年和2003年分离于昆明地区的RV，用PCR分型法对四种主要的VP7血清型进行分型，并对从150份RV腹泻标本VP7血清型为G1、G3、（34型RV株中挑出的14份一般腹泻株和8份重症腹泻株的NSP4基因序列进行了分析，与来自GenBank Database的4株人RV（Wa、KUN、AU-1、Hochi）和3株动物RV（EW、OSU、SA11）以及中国不同地区流行株NSP4的基因变异情况进行了比较。结果2002年昆明地区RV流行株以G1型为主，2003年RV腹泻株以G3型为主；昆明地区RV流行株间的氨基酸同源性高达98．9％～99．4％，22株流行株全都属于Wa组；NSP4变异与地域及VP7血清型无关；NSP4基因变异与RV腹泻临床症状严重程度不相关（P〉0．05）。结论不同年份相同季节不同地域流行的RVVP7血清型变异较大，而NSP4基因的相对保守性及其免疫原性使其有可能成为发展疫苗的候选基因。     

重庆地区婴幼儿轮状病毒腹泻VP7型别分析

目的：研究重庆地区1998－2000年度秋冬季婴幼儿轮状病毒腹泻分子流行病学,方法：采用逆转录－聚合酶链反应（RT－PCR）扩增婴幼儿腹泻便样中的编码轮状病毒VP7蛋白的全基因片段（1062bp),再用巢式－聚合酶链反应（net-PCR)对扩增得到的VP7基因进行分型,同时利用核苷酸序列分析方法进行分型。结果：在1998－1999年度130例婴幼儿腹泻便样中VP7基因阳性者50例（38．46％）,其中G1型占88％（44／50）,G3型占8％（4／50）,混合型占4％（2／50）,均为G1＋G3型,而1999－2000年度轮状病毒流行季节采集的112 标本中VP7基因扩增阳性者38例（33．93％）,其中G3型占78．95％（30／38）,G1型占13．16％（5／38）,混合型占7．89％（3／38）,均为G1＋G3型,苷酸序列分型结果与PCR分型结果一致。结论：重庆地区 1998－1999年度轮状病毒流行季节中流行的轮状病毒以G1型为主,而1999－2000年度轮状病毒流行季节中G3型为主,在连续两年的监测中出现轮状病毒血清型的转变。     

人轮状病毒外衣壳糖蛋白VP7基因毕赤酵母重组表达系统的构建

[目的]构建人轮状病毒糖蛋白VP7基因毕赤酵母表达系统.[方法]构建好的毕赤酵母重组表达质粒VP7-pPICZαA经Sac Ⅰ线性化后应用电转化法转化毕赤酵母菌株GS115感受态中,Zeocin平板筛选,PCR鉴定转化子,MDH和MMH平板鉴定Mut表型.[结果]线性化的重组质粒VP7-pPICZα成功转化进入毕赤酵母感受态中,PCR鉴定结果与预期相符,表型确定为Mut+.[结论]成功构建人轮状病毒耱蛋白VP7基因的毕赤酵母表达系统     

辽宁省首次检出G9型A组轮状病毒

目的了解辽宁省A组轮状病毒的感染情况,为轮状病毒的预防控制提供科学依据。方法采集辽宁省沈阳、大连、丹东、阜新4个市门诊及住院疑似病毒性腹泻患者粪便标本135份,采用酶联免疫吸附试验(ELISA)检测轮状病毒抗原,阳性标本用逆转录-聚合酶链反应(RT-PCR)扩增A组轮状病毒VP7基因和VP4基因,RT-PCR产物进行核苷酸碱基序列的测定和比对,并构建VP7基因遗传进化树。结果 135份粪便标本中,共检测出A组轮状病毒阳性标本21份;A组轮状病毒G基因分型:G9型15株,G3型2株,G2型1株,G1型1株,未分型2株;P基因型分型:P[8]型20株,P[4]型1株;G/P基因型组合以G9P[8]为主共15株,G3P[8]2株,G1P[8]1株,G2P[4]1株,G/P[8]2株。结论辽宁省首次检出G9型A组轮状病毒,主要流行G/P基因型组合为G9P[8]。     

Epidemiology and phylogenetic analysis of VP7 and VP4 genes of rotaviruses circulating in Rawalpindi,Pakistan during 2010

Human group A rotaviruses (RVAs) possess a large genetic diversity and new RVA strains and G/P genotype combinations are been identified frequently. Only a few studies reporting the distribution and co-circulation of RVA G and P genotypes are available for Pakistan. This hospital based study showed a RVA prevalence rate of 23.8%, which is similar to RVA detection rates estimated in other Eastern Mediterranean countries. During 2010, the following RVA strains were found to co-circulate: G1P[8] and G2P[4] (both 24.3%), G1P[6] (12.1%), G9P[8] (10.8%), G9P[6] (5.4%), G12P[6] (6.7%), G6P[1] (2.7%) and mixed infections (6.7%). Sequence analyses of selected G1, G2, G9 and G12 RVA strains revealed a close evolutionary relationship with typical human RVA strains. Sequence identities among the Pakistani VP7 RVA genes encoding G1, G2, G9 and G12 ranged between 91.5–98.7%, 99.6–98.9%, 97.7–99.5% and 99.2–99.9%, respectively. Analysis of the VP4 genes revealed co-prevalence of distinct lineages of the P[8] genotype. P[6] and P[4] showed a close relationship with typical human RVA strains detected in several Asian countries. The two G6P[1] RVA strains were closely related to typical bovine RVA strains, suggesting one or multiple interspecies transmission events. Our data provide important baseline data on the burden of RVA disease and genotype distribution in Rawalpindi, Pakistan, which is important with respect to vaccine introduction in national immunization programs.     

Genetic diversity of G1P[8] rotavirus VP7 and VP81 antigens in Finland over a 20-year period: No evidence for selection pressure by universal mass vaccination with RotaTeq® vaccine

Two live-attenuated oral vaccines (Rotarix™ and Rotateq®) against rotavirus gastroenteritis were licensed in 2006 and have been introduced into National Immunization Programs (NIPs) of several countries. Large scale use of rotavirus vaccines might cause antigenic pressure on circulating rotavirus types or lead to selection of new rotaviruses thus decreasing vaccine efficacy.We examined the nucleotide and amino acid sequences of the surface proteins VP7 and VP4 (cleaved to VP8¹ and VP5¹) of a total of 108 G1P[8] rotavirus strains collected over a 20-year period from 1992, including the years 2006–2009 when rotavirus vaccine (mainly Rotarix™) was available, and the years 2009–2012 after implementation of RotaTeq® vaccine into the NIP of Finland.In G1 VP7 no changes at amino acid level were observed. In VP8¹ periodical fluctuation of the sublineage over the study period was found with multiple changes both at nucleotide and amino acid levels. Most amino acid changes were in the dominant antigenic epitopes of VP8¹. A change in VP8¹ sublineage occurred between 2008 and 2009, with a temporal correlation to the use of Rotarix™ up to 30% coverage in the period. In contrast, no antigenic changes in the VP8¹ protein appeared to be correlated to the exclusive use of RotaTeq® vaccine after 2009.Nevertheless, long-term surveillance of antigenic changes in VP4 and also VP7 proteins in wild-type rotavirus strains is warranted in countries with large scale use of the currently licensed live oral rotavirus vaccines.     

Genotypic linkages of VP4, VP6, VP7, NSP4, NSP5 genes of rotaviruses circulating among children with acute gastroenteritis in Thailand

Rotavirus is the main cause of acute viral gastroenteritis in infants and young children worldwide. Surveillance of group A rotavirus has been conducted in Chiang Mai, Thailand since 1987 up to 2004 and those studies revealed that group A rotavirus was responsible for about 20-61% of diarrheal diseases in hospitalized cases. In this study, we reported the continuing surveillance of group A rotavirus in 2005 and found that group A rotavirus was detected in 43 out of 147 (29.3%) stool samples. Five different G and P genotype combinations were detected, G1P[8] (27 strains), G2P[4] (12 strains), G9P[8] (2 strains), G3P[8] (1 strain), and G3P[10] (1 strain). In addition, analysis of their genotypic linkages of G (VP7), P (VP4), I (VP6), E (NSP4), and H (NSP5) genotypes demonstrated that the rotaviruses circulating in Chiang Mai, Thailand carried 3 unique linkage patterns. The G1P[8], G3P[8], and G9P[8] strains carried their VP6, NSP4, NSP5 genotypes of I1, E1, H1, respectively. The G2P[4] strains were linked with I2, E2, H2 genotypes, while an uncommon G3P[10] genotype carried unique genotypes of I8, E3 and H6. These findings provide the overall picture of genotypic linkage data of rotavirus strains circulating in Chiang Mai, Thailand.     

G1P[8] Rotavirus in children with severe diarrhea in the post-vaccine introduction era in Brazil: Evidence of reassortments and structural modifications of the antigenic VP7 and VP4 regions

Worldwide rotaviruses A (RVA) are responsible for approximately 215,000 deaths annually among children aged <5 years. RVA G1P[8] remains associated with >50% of gastroenteritis cases in this age group. The aim of this study was to assess the genetic variability of G1P[8] strains detected in children with severe diarrhea in Belém, Pará, Brazil, during the post-rotavirus vaccine introduction era. Phylogenetic analysis clustered the VP4 and VP7 genes of 40 samples selected between 2009 and 2011 into lineages found to be different from the Rotarix® vaccine strain. A detailed investigation of their complete genotype constellations identified 2 reassortant viruses (5%), resulting from reassortments between the genogroups Wa-like and DS-1-like (G1-P[8]-I1-R2-C1-M1-A1-N1-T2-E1-H1) and Wa-like and AU-1-like (G1-P[8]-I1-R3-C1-M1-A1-N1-T1-E1-H1) genotype constellations. A comparison of the amino acid residues presents in the antigenic epitopes of VP7 and VP4, showed differences in the electrostatic charges distribution, between wild type Brazilian strains and the Rotarix® and RotaTeq® vaccine strains. These findings reflect the structural analyses of the antigenic regions of VP7 and VP4 of the RVA G1P[8] in children with gastroenteritis in Northern Brazil raising the hypothesis that structural modifications at these sites over time may account for the emergence of new strains that could possibly pose a challenge to current vaccines.     

Identification of a multi-reassortant G12P[9] rotavirus with novel VP1, VP2, VP3 and NSP2 genotypes in a child with acute gastroenteritis

The G12 rotavirus genotype is globally emerging to cause severe gastroenteritis in children. Common G12 rotaviruses have either a Wa-like or DS-1-like genome constellation, while some G12 strains may have unusual genome composition. In this study, we determined the full-genome sequence of a G12P[9] strain (ME848/12) detected in a child hospitalized with acute gastroenteritis in Italy in 2012. Strain ME848/12 showed a complex genetic constellation (G12-P[9]-I17-R12-C12-M11-A12-N12-T7-E6-H2), likely derived from multiple reassortment events, with the VP1, VP2, VP3 and NSP2 genes being established as novel genotypes R12, C12, M11 and N12, respectively. Gathering sequence data on human and animal rotaviruses is important to trace the complex evolutionary history of atypical RVAs.     

Sequence and phylogenetic analyses of human rotavirus strains: Comparison of VP7 and VP81 antigenic epitopes between Tunisian and vaccine strains before national rotavirus vaccine introduction

Group A rotaviruses (RVA) are the leading cause of severe gastroenteritis in infants and young children worldwide. Due to their epidemiological complexity, it is important to compare the genetic characteristics of vaccine strains with the RVA strains circulating before the introduction of the vaccine in the Tunisian immunization program. In the present study, the nucleotide sequences of VP7 and VP8¹ (n = 31), the main targets for neutralizing antibodies, were determined. Comparison of antigenic epitopes of 11 G1P[8], 12 G2P[4], 4 G3P[8], 2 G4P[8], 1 G6P[9] and 1 G12P[8] RVA strains circulating in Tunisia from 2006 to 2011 with the RVA strains present in licensed vaccines showed that multiple amino acid differences existed in or near putative neutralizing domains of VP7 and VP8¹. The Tunisian G3 RVA strains were found to possess a potential extra N-linked glycosylation site. The Tunisian G4 RVA were closely related to the G4 vaccine strain in RotaTeq, belonging to the same lineage, but the alignment of their VP7 amino acids revealed an insertion of an asparagine residue at position 76 which is close to a glycosylation site (aa 69–71). Despite several differences detected between Tunisian and vaccine strains, which may affect binding of neutralizing antibodies, both vaccines are known to protect against the vast majority of the circulating genotypes, providing an indication of the high vaccine efficiency that can be expected in a future rotavirus immunization program.     

Comparative genomic analysis of genogroup 1 (Wa-like) rotaviruses circulating in the USA, 2006–2009

Group A rotaviruses (RVA) are double stranded RNA viruses that are a significant cause of acute pediatric gastroenteritis. Beginning in 2006 and 2008, respectively, two vaccines, Rotarix™ and RotaTeq®, have been approved for use in the USA for prevention of RVA disease. The effects of possible vaccine pressure on currently circulating strains in the USA and their genome constellations are still under investigation. In this study we report 33 complete RVA genomes (ORF regions) collected in multiple cities across USA during 2006–2009, including 8 collected from children with verified receipt of 3 doses of rotavirus vaccine. The strains included 16 G1P[8], 10 G3P[8], and 7 G9P[8]. All 33 strains had a Wa like backbone with the consensus genotype constellation of G(1/3/9)-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. From maximum likelihood based phylogenetic analyses, we identified 3–7 allelic constellations grouped mostly by respective G types, suggesting a possible allelic segregation based on the VP7 gene of RVA, primarily for the G3 and G9 strains. The vaccine failure strains showed similar grouping for all genes in G9 strains and most genes of G3 strains suggesting that these constellations were necessary to evade vaccine-derived immune protection. Substitutions in the antigenic region of VP7 and VP4 genes were also observed for the vaccine failure strains which could possibly explain how these strains escape vaccine induced immune response. This study helps elucidate how RVA strains are currently evolving in the population post vaccine introduction and supports the need for continued RVA surveillance.