GII.P7/GII.14型诺如病毒暴发的分子特征分析

目的 了解江苏地区3起诺如病毒(Norovirus,NoV)暴发疫情中病毒感染情况,并对其分子流行病学特征进行初步研究.方法 收集暴发疫情中患者的肛拭子标本53份,采用实时荧光RT-PCR定性检测NoV,阳性标本利用普通RT-PCR对其聚合酶区(RNA-dependent RNA polymerase,RdRp)及衣壳蛋白区(VP1)片段进行扩增和测序分析.结果 3起NoV暴发疫情中,阳性检出率为56.6%(30/53).对30株阳性标本序列进行分析,其RdRp区与GII.P7的同源性最高(93.86%),VP1区与GII.14的同源性较高(97.13%).初步判断为GII.P7/GII.14重组毒株,SimPlot分析可能的重组位点在核苷酸5009位.对其氨基酸序列进行分析发现在RdRp区和VP1区均出现部分氨基酸突变.结论 3起NoV暴发疫情均由同一重组毒株引起,提示GII.P7/GII.14重组株传染性较强,有可能再次引起暴发,应加强监测防控.     

我国沈阳一起GI.6型札如病毒引起的胃肠炎暴发的分子病原学特征研究

目的明确我国沈阳一起急性胃肠炎暴发的病原学及其基因进化特征。方法采集2021年11月沈阳某高校暴发疫情中的学生病例肛拭子和环境涂抹标本共计57份, 采用实时荧光RT-PCR进行常规腹泻病毒基因检测, 并对札如病毒核酸阳性标本采用传统RT-PCR方法扩增、序列测定、拼接和遗传进化分析。结果实时荧光RT-PCR检测确定26份标本为札如病毒核酸阳性, 成功扩增15份标本的衣壳蛋白基因片段。遗传进化分析表明, 15个阳性标本札如病毒分子分型均为GI.6基因型, 各标本序列间同源性为99.99%, 与2005日本的LC380411毒株亲缘较近为98.84%。结论引起本次暴发的病原札如病毒在东北地区较少检出, 其基因型为GI.6亚型引起暴发全球罕见报道。     

引起河南省一起病毒性脑炎暴发的柯萨奇病毒B5的鉴定及其序列分析

目的 鉴定2011年引起河南省漯河地区一起病毒性脑炎(VE)暴发的病原体,并对分离的柯萨奇病毒B5(CVB5)进行基因特征分析.方法 采集漯河地区病毒性脑炎暴发期间29例患者的5份咽拭子、21份粪便和14份脑脊液,采用实时荧光RT-PCR方法分别检测总肠道病毒(PE)、EV71及CA16病毒核酸.所有标本均进行病毒分离培养,对其中5例患者的2份粪便和3份脑脊液的阳性分离产物进行VP1和5′-UTR区核苷酸序列测定及亲缘进化分析.结果 实时荧光RT-PCR结果显示,所有临床标本EV71和CA16病毒核酸检测均为阴性,咽拭子、粪便和脑脊液的PE核酸检测阳性率分别为60.0％(3/5)、61.9％(13/21)和85.7％ (12/14).咽拭子、粪便和脑脊液病毒分离率分别为20.0％(1/5)、25.0％(5/21)和29.0％ (4/14).VP1和5′-UTR区核苷酸序列BLAST分析及分子分型结果显示为CVB5,对其中5株分离毒株进行VP1全长基因分析显示彼此之间核苷酸同源性为97.9％ ～ 99.5％.与其同源性最近的毒株是2010年引起长春手足口病暴发的CVB5分离株CC10/10/Changchun,核苷酸同源性为97.1％～98.1％.与2010及2012年河南省平顶山地区的脑炎分离株COXB5/Henan/2010和03001N/HN/CHN/2011/CB5的同源性分别为89.0％～89.6％和91.8％～92.5％.VP1区遗传进化树显示此次分离株为基因型D,且与长春株成一簇.结论 导致此次病毒性脑炎暴发的病原体为肠道病毒CVB5,优势基因型的变迁可能与此次暴发相关.     

贵阳地区GⅡ.4型诺如病毒变异株的初步研究

目的 了解贵阳地区GⅡ.4型诺如病毒变异株的组成及其点突变.方法 监测2010年6-11月于贵阳地区哨点医院就诊的急性胃肠炎病例,收集粪便标本,采用荧光定量逆转录-聚合酶链反应(real-time RT-PCR)初步鉴定,随机选取部分GⅡ型诺如病毒阳性标本,采用一步法RT-PCR对其VP1基因区克隆及测序,基因序列与国内外流行的GⅡ.4型诺如病毒进行系统进化及氨基酸位点的突变分析.结果 检测标本426份,有267份(62.68％)GⅡ型诺如病毒阳性,测序获得了9份GⅡ.4型诺如病毒VP1基因组序列,贵州省2010年流行的GⅡ.4型诺如病毒包括2个变异株(GⅡ.42008a和GⅡ.4 2008b新型变异株),亚型组闻的VP1基因的同源性为95.90％ ～ 96.72％,亚型组内同源性为99.45％ ～ 100％,有2个氨基酸位点易发生突变.结论 贵阳地区2010年夏秋季急性胃肠炎以诺如病毒GⅡ型为主,并且变异株具有多样性.     

一起不明原因感染性腹泻疫情的病原学诊断

目的 明确一起不明原因感染性腹泻疫情的病原体及其基因型别.方法 通过流行病学调查的方法,查明罹患率.对28例疑似病例的28份样本(粪便22份,呕吐物3份,肛拭子3份)采用RT-PCR方法进行诺如病毒核酸检测,并对5份阳性标本的核酸进行序列测定及遗传进化分析.结果 在调查的5694人中,发病160例,罹患率为2.81%.2007年3月7-9日为发病高峰.RT-PCR检测28份样本中,确定14份为阳性.对其中的5株毒株的核酸序列测定及序列遗传进化分析确定为GⅡ/4型诺如病毒,并与2006年诺如病毒流行株2006b同源性最高,为97.9%.结论 本次感染性腹泻疫情由诺如病毒引起.     

分型引物RT-PCR法在杯状病毒分子流行病学研究中的应用

目的:应用分型引物RT-PCR法确定五个地区病毒性腹泻粪便标本中杯状病毒流行型别及感染情况。方法:对五个地区收集的<5岁病毒性腹泻粪便标本应用针对病毒壳体区域设计的四对引物(GI-SKF/GI-SKR、COG2F/G2-SKR、SLV5317/SLV5749、PreCAP1/82b)进行反转录-聚合酶链反应(RT-PCR)扩增诺如病毒GI和GII组、扎如、星状病毒(Astrovirus),扩增产物通过1·5%琼脂糖凝胶电泳鉴定。选取部分阳性标本测序研究型别流行特点。结果:本实验从663例标本中共检出单独感染诺如病毒、扎如病毒和星状病毒阳性标本数分别为84、4、11株,同时还发现3株混合感染标本。选取24株诺如病毒阳性标本测序分析发现19株为GII/4型,2株为GII/3型,1株为GII/1型,1株为GII/13型,1株为GII/15型,4株扎如病毒中2株为SGI/1型,1株为SGII/3型,1株为SGII/1型。结论:研究表明杯状病毒是我国婴幼儿腹泻重要病原体,诺如病毒流行株为GII/4型毒株,同时存在其它型别的散在流行;福建、兰州检测到不同型别扎如病毒证明我国存在多种型别的扎如病毒感染。     

无菌性脑膜炎暴发中柯萨奇B3病毒的基因特征分析

目的 明确2008年山东省临沂市郯城县无菌性脑膜炎暴发的病原,对分离到的柯萨奇B3病毒的基因特征、遗传变异规律及进化来源进行分析.方法 采集暴发病例的粪便、脑脊液标本,应用RD、Hep-2细胞进行病毒分离;阳性分离物分别采用中和试验、逆转录-聚合酶链式反应(RT-PCR)和核苷酸序列测定方法进行定型,最后与GenBank中检索到的其他CVB3毒株进行同源性分析,并构建VP1完整编码区亲缘进化树研究其遗传进化规律.结果 共采集22份粪便和120份脑脊液标本,分离到病毒35株,经鉴定34株为CVB3,1株为ECH030.34株CVB3分离株VP1区部分序列(381 bp)核苷酸同源性为90.5%～100%,氨基酸同源性为98.4%～100%;与Nancy原型株的核苷酸同源性为79.5%～81.6%,氨基酸同源性为98.4%～99.2%.012/2008TC/SD/CHN和177/2008TC/SD/CHN与安徽2008年分离的Fuyang19株同源性最高,核苷酸同源性分别为98.2%和91.0%,氨基酸同源性分别为99.2%和98.9%;进化树分析显示,中国大陆分离株独处一支,且呈单源性发生关系.结论 此次脑膜炎暴发的病原是CVB3.它与其他的中国分离株同源性高,与国外分离到的CVB3具有不同的基因特征.     

广东省暴发性胃肠炎中诺如病毒的分子流行病学特点分析

目的研究广东省暴发性胃肠炎中诺如病毒的分子流行病学特点。方法对2003年至2004年的13起急性胃肠炎患者的粪便和肛拭子标本,采用针对诺如病毒的特异引物和逆转录-聚合酶链反应(RT-PCR)技术进行检测,再经核苷酸序列测定分析病毒的基因型,同时收集患者相关流行病学资料。结果13起暴发性胃肠炎中有8起是由诺如病毒引起的,时间主要集中在每年的10月至12月,基因分析显示这些病毒都属基因Ⅱ组,但分属3个亚型,6株病毒属GⅡ-4型。结论诺如病毒是广东省暴发性非细菌性胃肠炎的重要病原体之一,具有分布广、流行时间集中在秋冬季的特点。引起暴发的病毒主要为基因Ⅱ组,在现阶段存在优势毒株。     

深圳市儿童秋冬季腹泻诺如病毒检测及基因型分析

目的 了解深圳市儿童秋冬季腹泻中诺如病毒（Nov）的感染状况,并对阳性株进行基因型分析.方法 收集深圳地区多家医院2009年9月至2010年1月腹泻患儿粪便标本307份,通过逆转录-聚合酶链反应（RT-PCR）方法进行诺如病毒核酸扩增,部分阳性标本的PCR产物经纯化、测序,结合GenBank参考株相应核酸序列构建系统进化树并进行基因型分析,采用SimPlot3.5.1对重组株进行分析和鉴定.结果 307份粪便标本中诺如病毒核酸阳性38例,阳性率为12.4％,以6～24个月龄婴幼儿感染为主,占全部病例数的81.6％（31 /38）,感染的发病高峰为10、11月份,占全部病例数的73.7％ （28/38）;26份测序标本中25株属于GⅡ.4型2006b变异体,1株为GⅡ.12/GⅡ.3型重组株.结论 诺如病毒是深圳市儿童秋冬季腹泻的重要病原体,优势流行株为GⅡ.4型2006b变异体,深圳首次检出的诺如病毒重组株为GⅡ.12/GⅡ.3型.     

我国新分离盖塔病毒的部分基因组分子特征研究

目的 通过分子生物学方法研究我国山东省2008年分离的盖塔病毒(DY0824)基因组分子生物学特征.方法 应用逆转录聚合酶链反应(RT-PCR)扩增出结构区基因和3' UTR片段,连接到载体中进行序列测定,然后用Clustal X1.83和MegaAlign软件对测定的核苷酸和推测的氨基酸序列进行比较分析,用Mega4软件绘制系统发生树.结果 DY0824病毒株衣壳蛋白由804个核苷酸组成,编码268个氨基酸,与国内外其他分离株的核苷酸同源性为95.4%～99.9%,氨基酸同源性为97.4%～100%.E2蛋白全长1266个核苷酸,编码422个氨基酸,与其他株的核苷酸同源性为94.8%～99.5%,氨基酸同源性为97.6%～100%.该病毒3' UTR由401个核苷酸组成,存在3个重复序列.结论 山东省新分离盖塔病毒衣壳蛋白和E蛋白基国与该病毒原型分离株相比分别存在7个和10个氨基酸差异位点,病毒3'UTR区域存在多个核苷酸差异位点.     

Molecular epidemiology of norovirus gastroenteritis outbreaks in North Carolina, United States: 1995-2000

Noroviruses (NoVs) are the most common cause of acute non-bacterial gastroenteritis outbreaks in the US. We investigated 16 gastroenteritis outbreaks in North Carolina (NC), from 1995 to 2000, to further characterize the epidemiology of NoV using RT-PCR on stool and ELISA on sera. NoV were identified in 14 outbreaks by RT-PCR. Sequence analyses of the amplicons indicated the outbreak strains belonged to the following clusters: five GII/4, three GI/3, one GI/4, one GII/2, one GII/5, one GII/7, and one GII/13 (prototype strain). We detected NoV in stool samples from one outbreak but could not determine its specific cluster within the GII genogroup based on polymerase sequence analysis. The five GII/4 strains were classified as the "95/96 US common strain" and occurred throughout the 5-year period. In contrast to national trends, the majority (86%) of NoV outbreaks identified in North Carolina were foodborne. Of the 12 food-related NoV outbreaks, we were able to document transmission by food handlers in two outbreaks. Person-to-person transmission from primary cases was suggested in three outbreaks. Our results indicate that NoVs are important agents of viral gastroenteritis outbreaks in NC.     

Tracking the transmission routes of genogroup II noroviruses in suspected food‐borne or environmental outbreaks of gastroenteritis through sequence analysis of the P2 domain

The aim of this study was to apply sequence analysis of a hyper variable region of the norovirus (NoV) genome in order to identify point source outbreaks associated with suspect food or water. The hyper‐variable region of the gene encoding the P2 domain was chosen as small differences in sequence are likely to indicate virus from different sources whereas identical sequence may reveal transmission routes and the source of contamination. Strains with 100% similarity were considered as originating from a common source, whereas, strains with one or more mutations in the hyper variable region sequenced were regarded as representing unrelated transmission events. This study was able to identify a point source outbreak of a dominant strain, GII‐4, on a cruise ship but also of a less common strain, GII‐2, between two schools. Also identical GII‐3 strains were demonstrated in food handlers amongst the same outbreak; however epidemiologically related outbreaks showed different GII‐3 strains indicating multiple sources of contamination. J. Med. Virol. 81:1298–1304, 2009. © 2009 Wiley‐Liss, Inc.     

Two gastroenteritis outbreaks caused by sapovirus in Shenzhen,China

Human sapoviruses (SaVs) are a common cause of the acute gastroenteritis epidemic worldwide, and SaV outbreaks and infections have become more frequent in recent years. Since the end of December, 2015 to December, 2016, 2 gastroenteritis outbreaks occurred in 2 kindergarten classes (outbreaks A and B) in the Baoan district, Shenzhen, China. Feces and swabs were collected for laboratory tests of causative agents, and no bacterial pathogens were detected. Both the outbreaks were positive for SaV with a detection rate of 75% of symptomatic cases (6/8) in outbreak A and 71% (10/14) in outbreak B. For outbreak B, 1 of 3 asymptomatic teachers was detected to be SaV positive. The genome of some of the strains in this study was obtained, and the strains from outbreak A were genotyped into GII.3, while those from outbreak B were genotyped into GI.2, according to the phylogenetic analysis of the polymerase and the capsid region. No recombination was identified in either the GII.3 or GI.2 strain. GI.2 strains experience chronological variation, and the GI.2 strain in this study belonged to the most recent variant, which was observed from 2008 to 2016. The variation mainly occurred in the P domain from 1990 to 2016. Meanwhile, GII.3 strains shared greater similarity (>98.2%) at the amino acid identities; from 1999 to 2015, only 5 of them were in the predicted P domain. Our results suggest that it is necessary to conduct routine SaV surveillance and molecular characterization to further understand the SaV epidemic.     

Molecular epidemiology of norovirus gastroenteritis outbreaks in New Zealand from 2002-2009

Noroviruses are the most common cause of acute non-bacterial gastroenteritis outbreaks worldwide, including New Zealand. New Zealand has a population of 4.4 million, which allows for centralized outbreak surveillance and a Norovirus Reference Laboratory, which facilitates efficient diagnosis, surveillance, and tracking of norovirus outbreaks. Norovirus outbreak strains are identified, sequenced, and compared with international reference strains. Between January 2002 and December 2009, 1,206 laboratory-confirmed norovirus outbreaks were recorded. The predominant outbreak settings were healthcare institutions for the elderly and acute care patients. Other outbreak settings included catering establishments, cruise ships, homes, community events, school camps, child-related settings, and consumption of contaminated shellfish. Of the 1,206 outbreaks, 105 (8.7%) were caused by norovirus genogroup I (GI) strains, 1,085 (89.9%) were caused by genogroup II (GII) strains, and both GI and GII strains were detected in 9 (0.8%) outbreaks. The genogroup was not identified in 7 (0.6%) outbreaks. A range of norovirus genotypes, including GI genotypes 1-6, GII genotypes 2-8, and GII.12, were associated with these outbreaks. The predominant genotype was GII.4, which was identified in 825 (68.4%) outbreaks. Norovirus GII.4 variant strains, including 2002 (Farmington Hills), 2004 (Hunter), 2006a (Laurens, Yerseke), 2006b (Minerva), and 2010 (New Orleans) implicated in overseas outbreaks also occurred in New Zealand, providing evidence of global spread.     

Epidemiological study of human calicivirus infection in children with gastroenteritis in Lanzhou from 2001 to 2007

Stool specimens were collected from 1,195 young children with acute diarrhea in Lanzhou, China, from 2001 to 2007. RT-PCR was used to detect human calicivirus (HucV). One hundred seventeen specimens were found positive for HucV. The infection rate was noticeably higher during 2006–2007 compared to the other years studied. Ninety-six specimens were sequenced to determine the genotypes of HucV. Eighty-six were norovirus and 10 were sapovirus, while GII/4 was the predominant strain of NV, followed by GII/3. The subtype of NV GII/4 changed from the Farmington Hills strain to the Bristol strain, and then to the Hunter strain and variant 2006b strain, over time. Variant 2006b has become the major epidemic strain in Lanzhou and should be closely monitored in the coming years.     

2014年北京市某校连续三起诺如病毒疫情调查分析

目的 调查北京市某校连续三起诺如病毒疫情,为减少同类事件提供经验.方法 应用描述性分析方法,对三起疫情的调查结果进行分析.结果 2014年,北京市某小学2013级新生中连续发生三起诺如病毒疫情,罹患率分别为13.5％、10.56％及9.0％.随疫情起数增加,患儿就诊率下降(趋势x2=34.112,P＜0.001),且就诊患儿均未被诊断为法定报告传染病.造成三起疫情的诺如病毒基因型分别为GII.7型、GII.6型和GII.17型.结论 诺如病毒极易在集体单位内传播,扎实做好日常消毒和疫情报送工作,是预防及处置诺如疫情的关键措施.