天津地区儿童轮状病毒腹泻的分子流行病学研究

目的了解天津地区婴幼儿轮状病毒感染腹泻情况及其型别特点。方法收集天津市儿童医院2008年8月到2009年7月腹泻患儿的粪便标本,先采用A组胶体金法快速检测RV抗原,将检测阳性的标本进行病毒RNA提取,RT—PCR扩增病毒VP7编码基因,并将部分PCR产物阳性标本进行测序,测序结果递交GentBand数据库进行BLAST搜索,及作同源性分析。结果1156份标本经胶体金法检测RV阳性305例,阳性率26．38％。阳性患儿中5岁以下占97％,5岁以上占3％;。轮状病毒有明显的季节特征,季节高峰从9月逐渐升高至次年2月为高发季节（18．4％～47．6％）,三月逐渐回落。其中1月份轮状病毒检出率最高,其次为10月、11月、2月。轮状病毒检出率最低的是6、7、8月。检测结果显示天津地区的优势株仍然是Gl型（72．7％）,其次是G3型（27．2％）。结论轮状病毒是天津地区婴幼儿腹泻的重要病原,5岁以下儿童为最主要的易感人群,流行基因型以G1为主。     

改进探针标记法斑点杂交在轮状病毒VP7分型中的应用

目的建立一种更加简便的A组人轮状病毒（HRV）核酸斑点杂交VP7分型方法,以便用于对HRV流行情况进行调查。方法在HRVVP7编码基因各G基因型问高度变异而型内高度保守区域设计分型探针,在该区域的两侧相对保守区域设计一对通用引物,利用PCR分别将地高辛标记HRV5种常见型别（G1～4,G9型）的DNA探针,建立基于VP7的斑点杂交方法;选取经抗原检测和聚丙烯酰胺凝胶电泳（PAGE）检测均为HRV阳性的2006至2008年住院腹泻患儿粪便标本200份,RT—PCR扩增VP7全基因,并对扩增阳性产物应用斑点杂交方法进行G型别分析。结果建立的斑点杂交方法在5种型别探针间无交叉反应,各型探针的检测灵敏度可达到10Pg。200份PAGE阳性标本中162份RT—RCR扩增VP7基因阳性,斑点杂交显示G1型41例（25．3％）,G2型2例（1．2％）,G3型63例（38．9％）,G9型35例（21．6％）,混合感染19例（11．7％）,杂交未分出型2例（1．2％）,未检测到G4型HRV。结论本研究所建立的斑点杂交方法敏感度和特异度强,适合在HRV大规模分子流行病学调查时应用。通过该方法的初步应用,发现北京地区婴幼儿HRV除了常见的G1、G2和G3型外,还有G9型感染。     

一起由水污染引起的儿童及成人A组轮状病毒腹泻暴发

目的 确定2006年11月广西大新县大范围腹泻暴发性流行的病原及其分子生物学特点.方法进行流行病学个案调查和粪便标本的实验室检测,用ELISA试剂盒进行A组轮状病毒的检测,对检测阳性标本进行轮状病毒分型并对其中4份检测阳性轮状病毒的vfy7全基因扩增.结果 采集的64份腹泻患者粪便标本中共有30份检测为轮状病毒阳性,总阳性率为46.9%,此次暴发的流行病学和临床特征也符合轮状病毒腹泻特征,RT-PCR进行G/P分型显示为G1P[8]型轮状病毒,VP7氨基酸显示第68位氨基酸改变.结论 G1P[8]型A组轮状病毒是此次腹泻暴发的病原.值得注意的是,此次A组轮状病毒暴发累及儿童和成人.     

2011年北京市5岁以下婴幼儿A组轮状病毒腹泻监测

目的了解北京市5岁以下婴幼儿轮状病毒腹泻患者的流行特点。方法2011年1月至12月在北京市3家医院肠道门诊收集输液两次以上5岁以下腹泻患者病例604例,采集粪便标本,应用酶联免疫吸附试验检测轮状病毒抗原,阳性标本用半巢式反转录PCR进行基因分型。结果604例标本中96例轮状病毒抗原阳性,阳性率为15．89％。秋冬季为A组轮状病毒腹泻的发病高峰,其中11月份A组轮状病毒阳性率最高（55．56％,30／54）。轮状病毒腹泻主要发生于3—23月龄患者（93．76％,90／96）。对轮状病毒抗原阳性标本进行G／P分型,G分型以G3＋G9型混合感染占第一位（37．50％,36／96）,其它型别主要有G3型（28．12％,27／96）,G1型（11．46％,11／96）,G9型（9．38％,9／96）,G2型（8．33％,8／96）。P分型以P8型为主要优势株（86．46％,83／604）,其次为P4型（10．42％,10／96）。最常见的G／P基因型组合为G3＋G9／[P8]（36．46％,35／96）,其次为G3／[P8]（26．04％,25／96）,GI／[P8]（11．46％,11／96）,G9／[P8]（9．38％,9／96）,C2／[P4]（8．33％,8／96）。结论A组轮状病毒是北京市婴幼儿腹泻的主要病原之一,2011年主要流行株基因型组合为G3＋G9／P[8]。     

兰州地区5岁以下婴幼儿病毒性腹泻的分子流行病学研究

目的调查兰州地区5岁以下婴幼儿病毒性腹泻的流行情况,了解四种主要腹泻病毒在儿童中的分布情况。方法采集2009年7月至2010年6月兰州大学第一医院儿科5岁以下腹泻患儿粪便标本290份及儿童保健中心健康婴幼儿正常粪便标本114份,采用酶联免疫吸附试验（ELISA）检测轮状病毒抗原,采用巢式聚合酶链反应对轮状病毒阳性标本进行分型;采用反转录．聚合酶链反应（RT—PCR）检测杯状病毒和星状病毒,聚合酶链反应（PCR）检测腺病毒。结果290份腹泻标本中四种病毒的阳性率分别为：轮状病毒39．31％,杯状病毒11．38％,腺病毒10．69％,星状病毒4．83％;对114份轮状病毒阳性标本G、P分型,G3型及P[8]型为优势株;114份正常标本轮状病毒检出率为0,杯状病毒检出7例,星状病毒检出1例,腺病毒检出5例。结论病毒性病原在兰州地区婴幼儿腹泻中占有重要地位,长期系统的监测具有重要意义。     

河南省2012—2015年儿童A组轮状病毒病原学监测及基因型别分析CSCD

目的分析2012-2015年河南省5岁以下腹泻儿童A组轮状病毒的感染状况及病原学特征,为A组轮状病毒感染的监测、防控及爆发病例的调查及疫苗研发提供基线数据和方法学参考。方法采集河南省两个监测哨点医院5岁以下儿童腹泻病例的粪便样本1 433份。双抗体夹心法检测A组轮状病毒,阳性样本抽提病毒RNA,两步巢式多重RT-PCR进行G/P基因分型。结果1 433份腹泻样本共检出A组轮状病毒482份,总阳性率33.6%;轮状病毒检出率的季节性特征显著,存在秋季（9-11月份）和春季（3-5月份）两个较为显著的高峰。A组轮状病毒G分型以G1、G2、G3、G9为主;P分型以P[4]、P[8]为主;型别组合以G9P[8]、G2P[4]、G3P[8]、G1P[8]为主;还存在混合感染型别。阳性病例中,男女性别比1.4∶1;以4-12个月龄的婴幼儿为主;农村感染率高于城市;临床症状以轻中度腹泻为主,部分病例存在发热、呕吐、脱水等现象。结论河南省5岁以下腹泻患儿中存在较高的A组轮状病毒感染率,以秋季和春季为主,且存在混合感染病例;病原体可分为多种基因型别,G9P[8]为优势型别;大部分感染病例以轻症为主。     

人A组G2P[4]型轮状病毒的分离培养和鉴定

目的:对我国轮状病毒A组G2P[4]流行株进行分离培养和鉴定。方法:采用轮状病毒抗原胶体金法筛查56份病毒性腹泻患儿粪便标本,对阳性样本VP7和VP4基因测序确定其G和P基因型别,并接种至MA-104细胞,持续传代。运用SDS-PAGE电泳、实时荧光定量PCR、间接免疫荧光、嗜斑实验和电镜等技术进行鉴定。结果:检出轮状...     

河北省卢龙地区2008-2009年度轮状病毒流行病学研究

目的 了解河北省轮状病毒腹泻的流行情况.方法 2008年9月至2009年8月收集卢龙县儿童医院及妇幼保健院5岁以下腹泻住院患儿标本共426份,采用酶联免疫吸附试验（ELISA）检测轮状病毒抗原,阳性标本用反转录-聚合酶链反应（RT-PCR）进行基因分型.结果 426份标本中轮状病毒检出率为47.42%（202/426）.对202份轮状病毒阳性标本进行G/P分型,G3型是主要优势株,占57.9%（117/202）,其次为混合G型感染（16.3%）,G9型（14.9%）,G1型（7.9%）,G4型（1%）,G2型（0.5%）,P基因型最常见的为P[8]占58.4%,其次为P型混合感染占28.7%,P[4]（6.9%）,P[9]（1%）,最常见的G/P组合为G3P[8].结论 轮状病毒是卢龙地区儿童腹泻住院的主要病原,G3P[8]为主要流行株,混合型在该地区多发,且G9血清型在该地区已经成为仅次于G3的第二大优势株.     

成都地区婴幼儿病毒性腹泻分子流行病学研究

目的通过分子流行病学研究成都地区婴幼儿病毒性腹泻的病原学特点,掌握本地区病原分布特征,为疫苗研制和疫情控制提供科学依据。方法采用酶联免疫吸附试验（ELISA）及逆转录聚合酶链反应（RT—PCR）对成都地区2006—2008年度376例婴幼儿腹泻粪便标本进行轮状病毒（RV）、杯状病毒（HuCV）、星状病毒（AstV）及肠道腺病毒（Adv）检测。结果RV的检出率为37．76％（142／376）,其中42例进行G分型,45例进行P分型,G分型以G3型为主21例（50％）,其次为G2和G1型,P分型以P[8]型为主21例,其次是P[4]型19例。RV感染主要为6～23月的婴幼儿,发病高峰在10—12月份（75．8％）。RT—PCR法检出HuCV、AstV及Adv的检出率分别为15．85％、1．64％及2．04％。结论RV是成都地区婴幼儿病毒性腹泻的主要病原,其流行的主要血清型为G3型、P[8]、P[4]型。除RV外,HuCV也是重要的病原。     

小儿急性感染性胃肠炎轮状病毒感染的病原学研究

目的 了解我国广大地区小儿腹泻中轮状病毒感染的情况。方法 从我国有代表性地区收集到1968例腹泻患儿粪便标本、148例无腹泻儿、135例正常新生儿的同样标本,以及36例成年腹泻患者和37例无腹泻成人粪便标本,采用聚丙烯酰胺凝胶电泳（PAGE）查标本中轮状病毒RNA,部分标本同时用ELISA法查轮状病毒抗原,以及电镜检查病毒,部分标本还进行常规细菌培养。所有腹泻患者均符合WHO诊断标准。结果 从1968例腹泻患儿标本中804例查出轮状病毒RNA,病毒RNA阳性率为40．9％。36例成年腹泻患者中1例轮状病毒RNA阳性,其余无腹泻者均未查出轮状病毒RNA阳性。在1968例患儿便标本中,1493例用ELISA法查出33．5％轮状病毒阳性。在804例轮状病毒RNA阳性中,A组轮状病毒阳性占99．6％,C组占0．4％。A组轮状病毒中RNA长型占65．8％,RNA短型占33．3％,其他占0．8％。轮状病毒RNA长、短型中又可各分为4个主要电泳型（4232、4222、3232、3222）,病毒基因变化集中在第2、3及7－9片段上。病毒感染有明显季节性,秋冬季感染多,春夏季感染少。从患儿中检出病毒以－1岁、－2岁为高,病毒感染主要集中在2岁以内,尤其是6个月至2岁。在不同地区、城市、年份、轮状病毒感染率不一,可能与当地气候条件等因素有关。此外,还比较不同检测方法对病毒检出率的影响。结论 通过本调查研究表明,我国广大地区小儿腹泻中轮状病毒感染是主要病原,多发于秋冬季和2岁以内的婴幼儿。     

2013年11月北京丰台区轮状病毒腹泻流行的实验室分析

目的 了解2013年11月北京市丰台区腹泻中轮状病毒和诺如病毒发病率升高的流行情况及其基因特征 方法 2013年11月在哨点医院门诊随机采集47份腹泻患者便标本、30份环境标本,使用real-time RT-PCR进行轮状病毒（rotavirus）和诺如病毒（norovirus）的筛查;并对轮状病毒阳性标本使用RT-PCR方法扩增VP4和VP7基因,扩增产物进行序列测定.使用Blast、BioEdit及Mega4.0等软件进行序列比对及基因进化分析.结果 47份粪便标本中,37份为轮状病毒检出率为78.7％（37/470,诺如病毒阳性率为14.9％（7/47）,轮状病毒和诺如病毒混合感染率为10.6％ （5/47）;30份环境标本中,轮状病毒检出率为23.3％ （7/30）,未检出诺如病毒.核酸序列比对及进化分析表明此次流行的轮状病毒为G9P[8]a型,与2010-2012年北京地区感染儿童的G9株高度同源.结论 2013年11月北京市丰台区其他感染性腹泻标本中以轮状病毒检出为主,其基因型别为G9P[8]a型,检出率远高于北京市往年水平,提示应加强对轮状病毒进行监测.     

Distribution of human group a rotavirus VP7 and VP4 types circulating in Seoul,Korea between 1998 and 2000

Three hundred forty-eight fecal specimens collected from young children with acute diarrhea in Seoul, Korea between January 1998 and February 2000 were examined for G and P types. Of these, 205 samples (59%) were confirmed as group A rotavirus by ELISA for the detection of VP6 antigen. Confirmed rotavirus isolates were characterized using G serotyping ELISA and RT-PCR methodologies for G and P genotyping of the outer capsid proteins VP7 and VP4, respectively. Serotyping of the outer capsid protein, VP7, revealed G4 as the dominant circulating serotype (41%) followed by G1 (28%) and quite a high incidence of mixed infection (14%). Genotyping of the VP4 protein was carried out on 55 of the rotavirus isolates with the dominant type being P[8] (46%). Of interest were a number of unusual G and P type combinations detected in Korea for the first time, especially the P[4] genotype associated with non-G2 serotypes. There were also a number of P[6] isolates identified including one G2P[6] isolate.     

Changing pattern of human group A rotaviruses: emergence of G12 as an important pathogen among children in eastern India.

BACKGROUND: Rotavirus genotypes, G1-G4 and G9 are associated with childhood diarrhoea throughout the world. In our previous study, we detected G1, G2, G4 and three G12 strains from Kolkata, India. OBJECTIVES: To study the prevalence of G- and P-genotypes of rotaviruses associated with dehydrating diarrhoea in children admitted to two leading hospitals in eastern India. STUDY DESIGN: An active surveillance was conducted for elucidation of rotavirus infection in two leading hospitals in Kolkata, West Bengal and Berhampur (GM), Orissa, India, separated by 603km from January 2003 to April 2005. The rotaviruses were detected by RNA electrophoresis in polyacrylamide gels. G- and P-typing of the positive samples were accomplished by amplifying VP7 and VP4 genes by RT-PCR and genotyped by seminested multiplex PCR methods. Sequencing, sequence analysis and phylogenetic analysis of VP7 genes of G12 strains were carried out to understand the variations between the strains isolated from different parts of the world. RESULTS: The genotypic distribution varied remarkably from our earlier study period (1998-2001) with G1 (53.8%) being the most predominant strain followed by G2 (22.5%), G12 (17.1%), G9 (2.1%) and not a single G3 or G4 isolate was detected separately. 35.2% samples exhibited mixed P-types followed by P[4] (31.7%), P[8] (21.8%) and P[6] (9.8%). The phylogenetic analysis of G12 strains revealed that the G12 strains detected from different parts of the world clustered into three different lineages. Though VP7 sequences of G12 strains isolated from Kolkata and Berhampur are conserved, their P-types were different. CONCLUSION: During this study period we reported emergence of G12 strains as an important pathogen among children in eastern India, thus necessitating its inclusion in future polyvalent vaccine to control rotavirus diarrhoea.     

G[P] type profiles of group A human rotavirus and their distribution in Nizhni Novgorod and Dzerzhinsk in 1997-2005

RT-PCR was used to determine G (VP7) and P (VP4) genotypes of group A rotavirus found in 2454 children with diarrhea in 1997-2005. Eight G[P] combinations, including G1-4 serotypes and P[4, 6, 8, 9] genotypes, were identified. The P[8] genotype was presented by the subtypes P[8]-1 and P[8]-2. In 1997-2005, the spectrum and distribution of G[P] types were as follows: G1P[8]-1 (10%), G1P[8]-2 (67%), G1P[6] (3%), G2P[4] (9%), G3P[8]-2 (5%), G3P[6] (1%), G3P[9] (1%), G4P[8]-2 (3%), G9 (0%), G?P[?] (1%). Five G[P] types of rotavirus were identified in the 2004-2005 season. The G2P[4] type was prevalent (37%), the GI P[8]-2 type was detected in 25% of cases; G3P[8]-2 in 22%; G4P[8]-2 in 12%; G3P in 2%), G[?]P[?] in 2%. The P[8]-1 subgemotype was not found. The spectrum of the G1P[8] types of rotavirus and their distribution should be taken into account while developing a vaccine prophylaxis program against rotavirus gastroenteritis in Russia.     

Analysis of homotypic and heterotypic serum immune responses to rotavirus proteins following primary rotavirus infection by using the radioimmunoprecipitation technique.

Three sequential serum samples collected from each of 20 young children with a naturally acquired primary rotavirus infection were assayed by the radioimmunoprecipitation technique for immunoglobulin G antibodies to rotavirus structural and nonstructural proteins of the four major human rotavirus serotypes G1, P1A; G2, P1B; G3, P2; and G4, P2. Fourteen children were infected with a serotype G1 rotavirus strain and six children were infected with a serotype G4 rotavirus strain. Sera were collected from each child in the acute and convalescent periods postinfection and also approximately 4 months later. Serum immune responses to rotavirus core antigens VP2 and VP3, to the major inner capsid antigen VP6, to nonstructural proteins NS35, NS28, and NS26, and to the outer capsid neutralization antigen VP4 of all test strains were detected in the majority of patients. These responses do not appear to be influenced by the G type or P type of the rotavirus strain used in the reactions. Homologous responses to the main neutralization antigen VP7 were detected in 93% of patients with serotype G1 infections and 50% of patients with serotype G4 infections. Heterologous VP7 responses were less frequently detected and were restricted to G1, G3, and G4 serotype rotavirus strains. No responses to VP7 of the serotype G2 rotavirus strain were detected in any patients. Heterotypic immune responses to the neutralization antigens, at least following serotype G1 and G4 infections, therefore appear to consist primarily of responses to VP4 rather than to VP7.