长沙市GI.6[P11]重组型诺如病毒引起的暴发疫情全基因组序列特征分析

目的 对长沙市2017年5月一起诺如病毒暴发疫情中的病毒基因型别进行鉴定,并对病毒全基因组序列特征进行分析。 方法 采集疫情中4份病例粪便标本和2份饮用井水标本,提取病毒核酸后实时荧光逆转录聚合酶链反应进行诺如病毒GI和GII型检测,逆转录聚合酶链反应扩增病毒开放阅读框(open reading frame,ORF),测定ORF1与ORF2连接区域序列并进行基因型别鉴定,应用二代测序技术对阳性标本进行全基因组序列测定和分析。 结果 采集的6份标本中有2份病例标本和1份井水标本诺如病毒GI型阳性,基于核酸序列的进化树分析发现ORF1区域序列位于GI.P11型别分支,ORF2区域序列位于GI.6型别分支。3份阳性标本毒株序列之间的同源性为99.8%~100.0%。全基因组序列毒株编号为Hu/CSNV0501/2017,基因组编码区全长为7 602 bp。Simplot软件分析重组位点位于ORF1区域序列约5 341 bp。 结论 本次疫情的传染源为井水污染传播,引起此次胃肠炎疫情的病原体为诺如病毒GI.6[P11]重组型。应加强对长沙市诺如病毒少见重组亚型的监测,为重组毒株的遗传进化和疫情防控提供参考。     

2018年连云港市一起GII型诺如病毒暴发疫情调查

目的了解一起暴发疫情的致病因子、感染来源、传播途径和危险因素,为控制疫情提供依据。方法通过搜索病例,开展个案调查,采集病例的呕吐物和肛拭子进行病毒学检测,同时采集校内直饮水及杯具拭擦物进行细菌学检测,通过疫情时间和空间分析,推断暴发原因。结果该疫情共发现病例29例,不同班级罹患率差异有统计学意义(P<0.05),病例间性别罹患率和年龄罹患率差异均无统计学意义(P值均>0.05)。结论此次暴发疫情是由诺如病毒GII型感染引起的,主要以接触传播和病毒气溶胶传播为主。     

重组诺如病毒GII 4型衣壳蛋白的表达、纯化及鉴定

目的通过基因重组方式获取高浓度重组诺如病毒GⅡ4型衣壳蛋白。方法将诺如病毒GII 4型衣壳蛋白基因片段插入pHT A载体中,测序鉴定正确后,将重组质粒转化到MAX Efficiency~ DH10Bac~(TM)感受态细胞,表达目的蛋白。重组蛋白用Ni-NTA His蛋白亲和柱纯化,用诺如病毒检测试剂盒进行鉴定。结果 SDSPAGE结果表明成功表达了分子量约为60 kD的重组蛋白,经诺如病毒检测试剂盒鉴定,表达产物为重组诺如病毒衣壳蛋白,并且具有较好的免疫原性。结论构建了诺如病毒GII 4型衣壳蛋白表达载体,并获得重组诺如病毒GII 4型衣壳蛋白。     

一起急性胃肠炎疫情中重组诺如病毒基因特征分析

目的 明确成都市2018年5月某建筑工地发生的一起急性胃肠炎疫情中的诺如病毒的基因型别,并对其遗传进化和分子流行病学特征进行分析研究。方法 对实时荧光定量PCR检测结果为阳性的标本采用常规RT - PCR扩增其RNA依赖的RNA聚合酶区和衣壳蛋白区并测序,与国内外参考株进行序列比对和构建进化树等基因特征分析。结果 序列比对和进化树分析结果显示:此次疫情中检出的诺如病毒核苷酸同源性为100%;其RNA依赖的RNA聚合酶区与北京株处于同一分支,同源性95.4%;而衣壳蛋白区与莫斯科株同源性最高,为96.4%。结合SimPlot对其进行分析,重组可能位点在213 bp处,定位在ORF1/2交叠区。结论 引起本次急性胃肠炎疫情的重组诺如病毒基因型为GI.P4 - GI.5,这是成都市首次检出诺如病毒GI群重组株。     

苏州市14起诺如病毒胃肠炎暴发疫情流行特征分析

目的了解苏州市诺如病毒胃肠炎暴发疫情的流行病学特征。方法对2010-2014年发生的14起诺如病毒胃肠炎暴发疫情进行描述性流行病学分析,采用聚合酶链反应对病例肛拭子、粪便、呕吐物、水、环境涂抹标本进行核酸检测。结果 2011-2014年,苏州市共报告14起诺如病毒胃肠炎暴发疫情,罹患率波动范围为0.89%~21.18%。14起疫情中12起(85.71%)发生于托幼机构和中小学校,医院、农村各1起。全年均有发生,但以寒冷季节发生较多。仅1起查明与二次供水受到污染有关,13起未证实暴发原因。核酸检测结果12起由诺如病毒GII基因组引起,2起由GI基因组引起。结论诺如病毒已成为苏州市急性胃肠炎暴发疫情的重要病原,托幼机构和学校是高发场所,GII基因组是主要基因组。在暴发疫情处理中,应加强传染源和传播途径的调查。     

南京市某小学一起GII.P7 - GII.6型诺如病毒引起的急性胃肠炎暴发调查

目的 查明致病因子、传播方式、危险因素和感染来源,控制疫情蔓延。方法 搜索2017年2月15日 - 22日期间,某小学学生及教职员工中出现呕吐（≥2 次/24 h）或腹泻（≥3次/24 h同时伴有性状改变）之一者,开展个案调查;采集病例肛拭子标本,通过RT - PCR检测诺如病毒核酸;收集该校环境卫生情况、日常消毒以及供水、供餐等信息;开展病例对照研究分析发病的危险因素。结果 罹患率为7.8%（46/587）;病例主要表现为呕吐（87.0%）、腹泻（71.7%）、恶心（65.2%）;流行曲线提示为同源暴露后继之以接触传播,六（5）班罹患率（60.9%）远高于其他班级,有显著的班级聚集性（P＜0.05）;性别罹患率、学生与教职工罹患率都无统计学差异（P＞0.05）;便后不洗手（OR = 3.9,95%CI = 1.8～8.5）、关系好的同学中有人发病（OR = 3.8,95%CI = 1.8～7.9）会增加发病的风险;100%（10/10）病例肛拭子标本中检出GII.P7 - GII.6型诺如病毒核酸。结论 此次暴发是一起由GII.P7 - GII.6型诺如病毒引起的急性胃肠炎暴发,以接触传播为主,不排除气溶胶传播的可能。     

一起诺如病毒感染性腹泻暴发流行病学分析

目的了解某中学诺如病毒感染性腹泻暴发的特点和流行原因,探索暴发疫情调查处理的经验。方法按照病例定义开展病例搜索,采用描述性流行病学和回顾性队列研究方法进行分析,样本采用RT-PCR方法检测诺如病毒核酸。结果2011年2月14日至2月19日,该校共发生诺如病毒感染性腹泻478例,罹患率为6.72%(478/7 113);2月16日为发病高峰;病例主要集中在初三和高二两个年级;单独在第一食堂就餐师生的罹患率为4.09%(46/1 124),单独在第二食堂就餐师生的罹患率为4.86%(61/1 256),单独在两个食堂就餐师生的罹患率差异无统计学意义;采集患者肛拭子15份,诺如病毒RT-PCR检测阳性8份。结论这是一起由诺如病毒感染所致的感染性腹泻暴发疫情,供水系统受到一过性污染是本起疫情的主要传播途径。     

RT-PCR法检测诺如病毒结果分析

目的通过比较2种检测诺如病毒（NV）的方法,为临床实验室病毒检测提供参考依据。方法用荧光定量RT-PCR和ELISA法对泉州地区多起流行性急性胃肠炎事件中病人标本进行NV检测,观察方法的敏感性和特异性。结果荧光定量RT-PCR法阳性检出率（86.5%）高于ELISA法（43.7%）;以ELISA法为标准,荧光定量RT-PCR法的敏感性为94.5%,特异性为19.7%,两者的Kappa值=0.128,相关系数r=0.207,一致性较差。荧光定量RT-PCR法敏感性较高。结论荧光定量RT-PCR法敏感性较高,可用于应急快速检NV,但其特异性不高。     

GGⅠ诺如病毒致胃肠炎饮水型暴发的调查

诺如病毒是引起人类急性胃肠炎的重要病原体,其感染基因型包括GG Ⅰ、GG Ⅱ和GGⅣ[1].调查显示我国人群诺如病毒感染十分普遍[2],尽管如此,国内报道的诺如病毒基因型多为GGⅡ[3-5],而GGⅠ特别是多区域人群暴发的研究较少涉及.2008年3月杭州市某区发生一起GG Ⅰ诺如病毒的暴发疫情,调查结果如下.     

登革3型病毒07CHLS001株全基因组序列测定和分析

目的对登革3型病毒浙江分离株07CHLS001进行了全基因组序列测定和分析，为探讨其基因组特征和来源提供依据。方法根据登革3型病毒98TW407株设计22对引物，利用RT-PCR法扩增出07CHLS001株基因组不同区段的eDNA片段，通过序列测定和拼接获得其全基因组序列。结果07CHLS001株基因组全长10707 nt，包含一个开放读码框（95-10 267nt），编码3390个氨基酸。它与登革3型病毒ThD3-1687-98株、98TW407株和80-2株的全基因组序列核苷酸相似性依次为98．7％、98．5％和94．6％。prM／M-E核苷酸序列系统发生树分析表明，07CHLS001与来自泰国的5个株系形成了一个Bootstrap支持率为90％的分枝。结论07CHLS001属于登革3型病毒的亚型Ⅱ。07CHLS001株全序列的测定，对进一步研究该株系的特性有十分重要的意义。     

Emergence of GII.e as a major ORF 1 norovirus genotype and its associated ORF 2 GII.4 variant forms

The noroviruses are a major cause of outbreaks of gastroenteritis. The norovirus genotype “GII.e”, identified by ORF (Open Reading Frame) 1 nucleotide sequencing, appears to be an obligatory recombinant, in that no unique GII.e ORF 2 genotype has been identified. In 2012 GII.e norovirus became the predominant ORF 1 genotype in norovirus outbreaks in Victoria, Australia, and the current study documents changes in the ORF 1 region of GII.e norovirus since it first emerged in 2008, as well as in the ORF 2 genotypes associated with GII.e norovirus. GII.e norovirus underwent significant genetic change in ORF 1 between 2010 and 2012 and this genetic change corresponded to a significant increase in the prevalence of the virus. Nucleotide sequencing of the ORF 2 region of GII.e specimens showed that in 2008–2009, all the ORF 2 sequences corresponded to the GII.4 (2007) variant, in 2010 all the ORF 2 sequences corresponded to the GII.4 (2012-like) variant and in 2012 all the ORF 2 sequences corresponded to the GII.4 (2012) variant, the GII.4 (2012-like) variant, or the GII.4 (2009-like) variant. The evidence indicated that the development of the 2012 GII.e epidemic strains was due to evolutionary change rather than a novel recombination event. The results also support the notion that ORF 1 is critical in determining the virulence of a norovirus strain.     

Recombinant norovirus GII.g/GII.12 gastroenteritis in children

Recombinant GII.g/GII.12 norovirus (NoV) strains emerged in 2008 in Australia and subsequently have been associated with gastroenteritis outbreaks worldwide. In the winter season 2009-2010 GII.12 strains caused 16% of the NoV outbreaks in the United States. During 2009-2010 we also identified GII.g/GII.12 strains during surveillance of sporadic cases of gastroenteritis in Italian children. Severity scores were calculated for the GII.g/GII.12 NoV infections using the Vesikari scale and in two out of three paediatric cases they exceeded the median value calculated for concomitant GII.4 infections. Upon sequence analysis, the Italian strains were found to be recombinant viruses and displayed different patterns of nucleotide polymorphisms. Phylodynamic analysis with other GII.g/GII.12 recombinants showed a high rate of evolution, comparable to the rates observed for GII.4 viruses. The mechanisms leading to worldwide emergence of GII.12 NoV strains in 2008-2010 are not clear. Monitoring of GII.12 NoV circulation is necessary to understand these mechanisms of evolution.     

Phylogenetic analyses of Norovirus strains detected in Uruguay reveal the circulation of the novel GII.P7/GII.6 recombinant variant

Noroviruses (NoV) are one of the major etiological agent of acute gastroenteritis (AGE) outbreaks worldwide. Distinct NoV genotypes have been associated with different transmission patterns and disease severity in humans. Therefore, it is important to identify genetically different NoV genotypes circulating in a particular region. However, genotyping has become a challenge due to recombination events occurring mainly nearby ORF1/ORF2 junction of NoV genome, leading to distinct genotypes with polymerase and capsid regions derived from parenteral strains. Taking this into account, ORF1/ORF2 sequences were obtained from NoV strains collected from patients with AGE in Uruguay. This study reveals in silico evidences of recombination events taking place in four out of six strains analyzed for which its polymerase gene and its capsid region correspond to GII.P7 and to GII.6 genotype, respectively. These results also reveal the circulation of a GII.P7/GII.6 recombinant variant in the natural populations of NoV strains in the northwestern region of Uruguay. As far as we know this is the first report about the circulation of a NoV GII.P7/GII.6 recombinant variant in the Americas.     

Detection and analysis of recombination in GII.4 norovirus strains causing gastroenteritis outbreaks in Alberta

Recombination is an important mechanism generating genetic diversity in norovirus (NoV) that occurs commonly at the NoV polymerase-capsid (ORF1/2) junction. The genotyping method based on partial ORF2 sequences currently used to characterize circulating NoV strains in gastroenteritis outbreaks in Alberta cannot detect such recombination events and provides only limited information on NoV genetic evolution. The objective of this study was to determine whether any NoV GII.4 strains causing outbreaks in Alberta are recombinants. Twenty stool samples collected during outbreaks occurring between July 2004 and January 2012 were selected to include the GII.4 variants Farmington Hills 2002, Hunter 2004, Yerseke 2006a, Den Haag 2006b, Apeldoorn 2007, New Orleans 2009, and Sydney 2012 based on previous NoV ORF2-genotyping results. Near full-length NoV genome sequences were obtained, aligned with reference sequences from GenBank and analyzed with RDPv4.13. Two sequences corresponding to Apeldoorn 2007, and Sydney 2012 were identified as recombinants with breakpoints near the ORF1/2 junction and putative parental strains as previously reported. We also identified, for the first time, a non-recombinant sequence resembling the ORF2–3 parent of the recombinant cluster Sydney 2012 responsible for the most recent pandemic. Our results confirmed the presence of recombinant NoV GII.4 strains in Alberta, and highlight the importance of including additional genomic regions in surveillance studies to trace the evolution of pandemic NoV GII.4 strains.     

Genome characterization of a GII.6 norovirus strain identified in China

Noroviruses (NoVs) are the primary cause of non-bacterial acute gastroenteritis worldwide. Most NoV infections are caused by GII.4, but GII.6 is also an important genotype with a long-term persistence in human populations. In this study, the complete genome sequence of a NoV strain GZ2010-L96 isolated in China was identified and analyzed phylogenetically. The viral genome comprised 7550 nucleotides, and its phylogenetic analysis revealed that the strain belonged to GII.6 genotype. All reported GII.6 NoV capsid protein sequences were also collected for comparative analysis, and GZ2010-L96 was clustered into GII.6-b with other 8 strains. Meanwhile, it was found that 53 spots on viral capsid showed subcluster specificity according to multiple alignments. Moreover, homologous modeling of GZ2010-L96 based on comparison with GII.4 VA387 strain showed a different antigen distribution pattern. In summary, the genome of the GII.6 strain GZ2010-L96 detected in China was extensively characterized, and phylogenetic analyses of GII.6 NoVs based on the capsid proteins may reveal a different evolution process from the predominant genotype GII.4.     

Novel intergenotype human norovirus recombinant GII.16/GII.3 in Bangladesh

Noroviruses (NoVs) are one of the major etiological agents of acute gastroenteritis in all age groups. In this study, we identified an intergenotype NoV recombinant strain in the fecal specimens of two male infants with acute diarrhea in Bangladesh. Phylogenetic analysis showed that the identified strains were recombinant NoV strains with a GII.3 capsid and a GII.16 polymerase gene. The recombination breakpoint was located in the ORF1/ORF2 overlap region. To the best of our knowledge this is the first report of a NoV recombinant GII.16/GII.3 strain worldwide.     

长沙地区诺如病毒基因型分析

目的确定长沙地区引起急性胃肠炎诺如病毒的类型。方法从128例腹泻患者粪便中,提取RNA。将RNA进行逆转录、扩增、测序,并通过构建基因进化树进行分析导致长沙地区患者感染的诺如病毒的主要种类。结果63个样品中含有诺如病毒,分别为GII-3、GII-4、GII-12三种类型,其中GII-4为主要病原体。结论在长沙地区,诺如病毒GII-4是引起病毒性胃肠炎的的主要致病株。GII-4基因进化快速,已成为全球范围内导致急性胃肠炎的最常见病毒株。     

2012-2014年广东省哨点医院诺如病毒GII.4 Sydney变异株流行状况及暴发疫情特征分析

目的：分析2012年1月至2014年6月广东省哨点医院诺如病毒GII.4 Sydney变异株流行状况,以及由诺如病毒GII.4 Sydney变异株感染引起的暴发疫情特征。方法2012年1月至2014年6月在广东省选取22家医院的门诊科室为监测哨点,将采集的腹泻病例粪便样本（共10750份）送至市级CDC,进行病毒核酸提取及诺如病毒核酸检测,所有阳性样本递送至广东省CDC,并按照随机数字法抽取了855份进行诺如病毒基因分型,共成功测序样本690份。采用χ2检验比较不同年龄组、不同时期内腹泻病例诺如病毒感染情况。通过广东省突发公共卫生事件管理信息系统,收集2012年1月至2014年6月广东省13起由诺如病毒GII.4 Sydney变异株感染引起的社区暴发疫情数据,进行社区流行病学分析。结果2012年8月首次检出诺如病毒GII.4 Sydney变异株,2012年11月检出比例为13/15。2012年11月至2013年1月（T1时期）,各月份中腹泻病例诺如病毒感染阳性率分别为23.8%（100/421）、15.9%（61/383）、19.2%（95/495）,2013年11月至2014年1月（T2时期）,各月份中腹泻病例诺如病毒感染阳性率分别为17.0%（90/529）、8.7%（37/426）、11.2%（46/409）,均低于T1时期（χ2值分别为6.65、9.93、10.74,P值分别为0.010、0.002、0.001）。T1时期内≥15、<15岁组腹泻病例诺如病毒感染阳性率分别为26.3%（143/543）、14.9%（113/756）（χ2=25.90,P<0.001）;T2时期≥15、<15岁组阳性率分别为10.1%（52/516）、14.3%（121/848）（χ2=5.09,P=0.024）。13起暴发疫情中,食源性传播占10/13。结论广东省2012年8月首次检出诺如病毒GII.4 Sydney变异株,2012年11月起呈现社区流行,流行1年后,强度降低;由诺如病毒GII.4 Sydney变异株引起的暴发疫情主要以食源性传播为主。     

Molecular epidemiology of norovirus associated with gastroenteritis and emergence of norovirus GII.4 variant 2012 in Japanese pediatric patients

In late 2012, an outbreak of acute gastroenteritis due to norovirus variant Sydney_2012 occurred and have been reported from many counties. In this study, we described surveillance study of the incidence of norovirus infections among Japanese pediatric patients in association with gastroenteritis and investigated the antigenic change of the new variant Sydney_2012 circulated in Japanese populations. A total of 2381 fecal specimens collected from children with acute gastroenteritis in Hokkaido, Tokyo, Shizuoka, Kyoto, Osaka, and Saga from 2009 to 2013 were examined for norovirus and further analyzed molecularly. A high proportion (39.3%) of norovirus positive samples and several genotypes were detected. Norovirus GII.4 dominated over other genotypes (71.4%). The Den_Haag_2006b (43.2%) was detected as the predominant variant and co-circulated with New_Orleans_2009 (17.8%) until March 2012. Subsequently, they were displaced by Sydney_2012. The Sydney_2012 variant has been responsible for the majority of norovirus infections in 2012–2013 (85.7%). Although Sydney_2012 variant has a common ancestor with New_Orleans_2009 variant, analysis of P2 sub-domain showed a high level of diversity in comparison with other variants in four amino acid changes at the antigenic sites. The change in particular residue 393 of new variant may affect HBGA recognition. Analysis of noroviruses circulating in the past 4 years revealed a change of predominant variant of norovirus GII.4 in each epidemic season. The change of amino acid in putative epitopes may have led the virus escape from the existing herd immunity and explain the increase of new variant outbreaks.