广东省2008-2015年诺如病毒感染暴发的危险因素分析

目的 研究2008-2015年广东省诺如病毒感染暴发疫情的危险因素,为诺如病毒感染的预防控制工作提供参考依据。方法 通过"突发公共卫生事件报告管理信息系统"收集2008年1月1日至2015年12月31日广东省报告的诺如病毒感染暴发疫情资料,并进行流行病学分析。采用RT-PCR 方法对2012-2015年73起诺如病毒感染暴发疫情的372份阳性标本进行基因测序亚型分析。结果 2008-2015年广东省共报告96起诺如病毒感染暴发疫情,其中2013-2015年共报告80起（占83.3%,80/96）。暴发地点在学校的占全部疫情的85.4%（82/96）;传播途径为食源性传播占40.6%（39/96）,接触传播占24.0%（23/96）,水源性传播占7.3%（8/96）。基因测序亚型分析显示, 2012-2013年的暴发主要由GⅡ.4/Sydney2012型诺如病毒感染引起（占30.0%,6/20）,2014-2015年的暴发主要由GⅡ.17型诺如病毒感染引起（占62.3%,33/53）。结论 食源性和接触传播及新出现的2种诺如病毒变异株GⅡ.4/Sydney2012变异株和GⅡ.17是引起广东省诺如病毒感染暴发的主要原因。     

牡蛎养殖区人群诺如病毒感染监测对暴发的预警作用

目的了解牡蛎养殖区人群诺如病毒感染病例的流行特征,研究病例监测对暴发的预警作用。方法 2014年1—12月,采用多阶段抽样方法从牡蛎养殖区选取部分社区人群进行入户调查,回顾其过去4周内急性胃肠炎的发病情况。采用实时RT-PCR与半巢式PCR相结合的方法检测诺如病毒,测序并绘制系统发育树。结果调查共发现75例急性胃肠炎病例,年发病率为0.10次/人年;诺如病毒阳性率为20.0%(15/75);系统发育分析显示,诺如病毒优势流行株为GII.4 Sydney 2012;发现新型GII.17变异株,检出时间为2014年3月。结论 GII.4 Sydney 2012仍是引起当地诺如病毒感染急性胃肠炎的主要流行株,在牡蛎养殖区哨点医院诺如病毒感染病例监测中发现的GII.17变异株对2014年冬季的暴发流行具有早期预警作用。     

广东省2012年病毒性腹泻病原学特征分析

目的系统掌握广东省病毒性腹泻的病原学特征及变化趋势,为病毒性腹泻防治工作提供参考依据。方法在广东省选择广州、深圳、肇庆等11个地级市23家哨点医院,每家哨点医院每周至少收集≤5岁和〉5岁门诊就诊腹泻病例粪便样本各3份进行轮状病毒和诺如病毒检测。结果2012年广东省11个哨点监测市23家哨点医院共采集门诊腹泻病例粪便样本4644份,轮状病毒和诺如病毒阳性检出率分别为13．94％（645／4627）和22．14％（1028／4644）。轮状病毒≤5岁年龄组阳性率为18．60％（445／2393）,〉5岁年龄组阳性率为8．95％（200／2234）,差异有统计学意义（P〈0．01）。诺如病毒≤5岁年龄组阳性率为21．88％（524／2395）,〉5岁年龄组阳性率为22．41％（504／2249）,差别无统计学意义（P〉0．05）。轮状病毒阳性检出率最高的月份是1月,为41．50％（105／253）;诺如病毒阳性检出率最高的月份是10月,为40．15％（218／543）。从1028份诺如病毒阳性样本中随机抽取226份进行序列测定,结果显示诺如病毒基因型GII型203份、GI型23份。203份GIL型中,GIL4型172份（其中GIL 4Sydney2012变异株87份、GIL 42006b变异株78份、GIL42010变异株4份、未分型3份）。结论2012年广东省病毒性腹泻的优势病毒是诺如病毒,而诺如病毒占优势与出现新的诺如病毒GIL4Sydney2012变异株有关。     

2013—2017年中山市20起诺如病毒暴发疫情的分子流行病学特征分析

目的 分析2013—2017年间中山市诺如病毒暴发疫情的分子流行病学特征,为中山市诺如病毒感染疫情防控提供分子生物学实验依据。 方法 收集2013—2017年间中山市20起诺如病毒引起的感染性腹泻暴发疫情病例标本,采用 RT-PCR方法测定诺如病毒VP1基因全序列,并对序列进行系统发育分析。 结果 20起诺如病毒引起的感染性腹泻暴发主要发生在幼儿园及小学,占疫情总数的80.0%(16/20)。获得了77株病毒的VP1基因全序列, 20起疫情分别由GII.4 Sydney_2012(25.0%, 5/20),GII.3(25.0%, 5/20),GII.17(25.0%, 5/20),GII.2(15.0%, 3/20),GII.21(5.0%, 1/20),及GII.6(5.0%,1/20)亚型引起,5起暴发中GII.4 Sydney_2012分为两个不同分支。 结论 GII.17型为中山市新发现流行毒株,2016年底发现的两起GII.4 Sydney_2012区别于以往出现GII.4 Sydney_2012,可能为新亚型的重组毒株(GII.P16-GII.4 Sydney_2012)。VP1基因序列分析可作为有效的诺如病毒分型工具, RdRp基因分析应作为重要补充以确定毒株的重组变异情况。     

安徽省诺如病毒G II.4/Sydney 2012变异株分子特征分析

目的了解安徽省某高校一起急性胃肠炎暴发疫情的病原体基因分型和分子特征。方法采用实时荧光定量PCR法(Real-time PCR)对6份粪便/肛拭子标本检测诺如病毒核酸,检出的阳性标本经传统RT-PCR扩增衣壳蛋白区(ORF2)部分序列、基因测序和型别鉴定,利用Clustal X 2.0和Mega 6.0软件对测定序列进行核苷酸同源性比对和构建基因进化和亲缘性关系树。结果检出4份标本诺如病毒核酸阳性,阳性率为66.67%(4/6);基因序列比对和进化亲缘性关系显示:诺如病毒基因型为GII.4型;2毒株序列之间核苷酸同源性为100%;与2014~2015年上海株、香港株核酸同源性最高,均为100%;与2012澳大利亚Sydney株(JX459908)同源性为99.6%;与上海和香港两地区毒株亲缘性关系最近,聚为独立一簇,同属GII.4/Sydney 2012分支。结论引发该起急性胃肠炎暴发的病原体为GII.4型诺如病毒,且属于GII.4 Sydney 2012变异株。     

广州市2017年某高校诺如病毒GⅡ.4 Sydney 2012变异株感染暴发调查

目的 分析2017年广州市GⅡ.4 Sydney 2012变异株引起的诺如病毒感染暴发疫情的流行特征和分子生物学特征,为诺如病毒感染疫情的防控提供依据。方法 设计调查表收集病例发病资料和疫情数据,采集现症病例和厨工的肛拭子以及环境标本进行病原学检测,阳性标本进行基因测序和同源性分析。采用病例对照研究方法分析疫情的相关危险因素。结果 2017年9月17-21日,广州市大学城某高校累计发生诺如病毒感染病例226例,包括223例在校学生和3例厨工,学生罹患率为0.73%（223/30 711）;宿舍A区学生罹患率最高（1.73%,164/9 459）,无院系和班级聚集性;主要危险因素为18-20日在A区食堂就餐（OR=10.75,95% CI：5.56~20.79）,发病风险最高的是18日晚餐,另一危险因素是同宿舍有病例（OR=3.65,95% CI：1.92~6.94）;诺如病毒核酸阳性的厨工全部来自A区食堂,阳性率为26.67%（12/45）,病毒基因测序结果为GⅡ.4 Sydney 2012变异株,与学生病例的病毒基因序列相似度为100%。结论 本次疫情是继2013年之后诺如病毒GⅡ.4 Sydney 2012变异株再次在学生人群引起的较大规模暴发疫情,由食源性传播的可能性较大,同时不排除接触传播。     

2016年新会区学校诺如病毒感染聚集性疫情流行病学特征分析

目的分析2016年新会区诺如病毒感染聚集性疫情的流行病学特征,为校园内诺如病毒防控工作提供科学依据。方法收集2016年新会区校园内发生的诺如病毒感染聚集性疫情的调查资料进行分析。结果 2016年共发生12起诺如病毒感染聚集性疫情,病例122例,暴发率为1.14%,病毒分型为诺如病毒GII型。临床症状主要为呕吐、腹痛、腹泻等。疫情多发生在小学和托幼机构。疫情主要集中于冬春季节,以11月、12月为主;城乡和年级之间的差异没有统计学意义。结论托幼机构和学校是诺如病毒感染聚集性疫情暴发防控的重点,应加强有关健康宣传教育,加强预防控制措施,减少诺如病毒感染聚集性疫情的发生。     

浙江省2006-2007年暴发性胃肠炎中诺如病毒的分子流行病学分析

目的 分析浙江省2006-2007年暴发性病毒性胃肠炎中诺如病毒感染的分子流行病学特征.方法 收集监测期间暴发性病毒性胃肠炎疫情患者的粪便标本及相关流行病学资料.采用RT-PCR及荧光定量RT-PCR检测诺如病毒.随机选掸部分阳性标本扩增部分多聚酶区和衣壳蛋白片段,进行序列测定,结合诺如病毒I(GI)、Ⅱ(GⅡ)基因型参考株进行进化分析.结果 诺如病毒感染暴发性胃肠炎共5起,发生时间均集中于9-12月,送检标本63份,诺如病毒检测阳性45份.序列分析结果显示,浙江省诺如病毒序列与诺如病毒GⅡ/4型参考株同源性最高,其中与北京2006年、荷兰2006年诺如病毒GⅡ/4型变异株最为接近,核苷酸同源性分别为99.7%和98.5%～99.0%.诺如病毒与北京、荷兰流行的GⅡ/4型变异株处于同一分支.结论 诺如病毒是浙江省病毒性腹泻暴发疫情的重要病原体,流行时间集中于秋季,其流行株为GⅡ/4型变异病毒株.     

广州地区病毒性腹泻的病原分布和病毒基因分型调查

摘要:目的　初步了解广州地区儿童和成人病毒性腹泻的病原学构成和分布情况及相关病毒的分子特征。方法　收集2012年11月-2013年5月于珠江医院就诊的腹泻患者的粪便标本,采用免疫层析法检测轮状病毒和腺病毒抗原,实时荧光PCR检测诺如病毒GI/GII,上述检测阳性标本进行病毒基因测序分型。结果　290例患者中,3种常见病毒的检出率分别为21.4%、15.5%和1.7%;男性患者病毒阳性率显著高于女性患者（χ2＝0.017,P＜0.05）;≤5岁患者轮状病毒阳性率最高,为28.49%（χ2＝0.017,P＜0.05）,＞5岁患者诺如病毒阳性率最高,为21.62%;轮状病毒性腹泻秋冬季节高发;轮状病毒G/P分型以G1P8、G9P8、G2P4和G3P8为主,占92.16%,诺如病毒共检测出8种基因型,以GII.4型为主,占53.33%;病毒合并其他常见腹泻病原体感染占病毒阳性标本的15.89%。结论　轮状病毒和诺如病毒分别是广州地区婴幼儿和成人病毒性腹泻的主要病原体,本地区流行的轮状病毒和诺如病毒优势株分别是G1P8和GII.4,诺如病毒基因型多样性更丰富;混合感染常见,需引起重视。     

诺如病毒GⅡ．4型Sydney 2012变异株与GⅡ．3型混合感染事件调查

目的 调查某大学一起急性胃肠炎暴发事件的感染来源和流行病学特征。方法 开展病例搜索,用描述性流行病学方法分析;用RT PCR法检测诺如病毒核酸,进行基因测序和核酸分型。结果 事件共发生感染性腹泻疑似病例473例,罹患率7.7%,确诊97例（20.5%）;临床症状以腹泻（100%）和呕吐（67%）为主,病例分布无聚集性,无性别差异;采集病例和厨工肛拭子、食堂剩余食物、环境涂抹样和生活饮用水样本共53份,细菌分离培养阴性,肛拭子诺如病毒RT PCR阳性率60.0%（9/15）,经基因测序分型5份为诺如病毒GⅡ.4型Sydney 2012变异株,4份为GⅡ.3型。结论 该起事件由诺如病毒GⅡ.4型Sydney 2012变异株和GⅡ.3型混合感染所致,传播途径可能为食源性传播和接触传播,卫生监管部门应加强对集体单位食堂的监管。     

中国7个地区诺如病毒G II．4／Sydney 2012变异株分析

目的　分析2012--2013年冬季中国地区诺如病毒新流行株GII．4／Sydney 2012的衣壳蛋白区核苷酸与氨基酸变异特点。方法　对已获得的22份2012--2013年冬季北京地区GⅡ．4／Sydney2012株进行全衣壳蛋白区基因扩增。从GenBank检索中国其他地区的GⅡ．4／Sydney 2012株衣壳蛋白区核苷酸序列。对获得的GII．4／Sydney 2012株的衣壳蛋白区核苷酸及氨基酸序列与往年GⅡ．4流行株进行系谱分析。结果　获得中国7个地区（北京、上海、江苏、湖州、荆州、香港和台湾）的GⅡ．4／Sydney 2012株资料共38份（至少包含完整VPl核苷酸序列）。GH．4／Sydney 2012株之间VPl核苷酸差异为0．1％～3．3％,氨基酸差异为0～3．1％。系谱分析发现GⅡ．4／Sydney2012株与Apeldoorn 2008和NewOrleans 2009起自共同的分支。中国和悉尼GH．4／Sydney 2012代表株的A、D、E抗原表位氨基酸序列组合有两种：TSRN-GTT-SNT和TSRN-STT-SNT。结论　G II．4／Sydney 2012株已在中国多个地区出现,各地区病毒株同源性较高。G H．4／Sydney 2012株的抗原表位氨基酸组合发生明显改变。     

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.     

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.     

Identification of a novel codon in the norovirus GII.4 polymerase region which acts as a marker of major GII.4 norovirus epidemics

Noroviruses are considered the most common cause of outbreaks of non-bacterial gastroenteritis in humans and the GII.4 genotype the most common norovirus genotype. Previous studies have shown that two adjacent codons acted as markers of the severity of GII.4 norovirus outbreak epidemics. In this study, a further such codon was identified at nucleotide position 4670–4672 relative to the norovirus strain Lordsdale virus (GenBank accession no. X86557). Taken together, the data indicate these epidemic marker sites occur, on average, about once in 30 amino acids (aa) in the polymerase region. None of the variant forms associated with the three codons resulted in an aa change. The three codons were not associated with the active sites of the polymerase gene. It is possible changes in these marker sites may influence norovirus virulence by altering the timing of co-translational folding in the norovirus genome.     

Genome characterization and temporal evolution analysis of a non-epidemic norovirus variant GII.8

Noroviruses are the primary cause of non-bacterial acute gastroenteritis worldwide, and GII.8 belongs to a non-epidemic genotype with a limited understanding currently. In this study, we assembled the first GII.8 norovirus genome from China and clarified the temporal evolutionary process of this non-epidemic variant. Using the “4+1+1” application strategy with newly designed primer sets, the genome of one GII.8 strain GZ2017-L601 from China was firstly sequenced that comprised 7476 nucleotides. The homology of the new genome and the previous only GII.8 genome reached 93.8% identity at the nucleotide level, but only 10, 6, 7 amino acid mutations occurred in three ORFs. When compared the new strain with other GII reference strains, p22 and P2 were calculated as the variable encoding regions, and NTPase, VPg, 3CL, RdRp and S were shown as the conserved ones. We then reconstructed the evolutionary process of the GII.8 genotype using other available sequences in GenBank. Based on the partial N/C region, all GII.8 strains could be subdivided chronologically into four clusters, which spans 1967–1994, 1997–2005, 2003–2009, and 2007–2017, respectively. Moreover, differences of capsid P proteins between GII.8 strains and the epidemic GII.4 strain VA387 were also compared. There existed 147/310 distinct amino acid sites in the alignment, including two insertion and three deletion mutations. Distribution of antigen epitopes of two GII.8 variants was comparable, but the numbers of antigenic sites of GII.8 strains were less than that of VA387. In summary, the first GII.8 genome from China was assembled and extensively characterized, and a time-order evolutionary process of this genotype was identified with a static pattern of antigenic variations.     

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.     

Occurrence of Norovirus GII.4 Sydney Variant-related Outbreaks in Korea

Human noroviruses are major causative agents of food and waterborne outbreaks of nonbacterial acute gastroenteritis. In this study, we report the epidemiological features of three outbreak cases of norovirus in Korea, and we describe the clinical symptoms and distribution of the causative genotypes. The incidence rates of the three outbreaks were 16.24% (326/2,007), 4.1% (27/656), and 16.8% (36/214), respectively. The patients in these three outbreaks were affected by acute gastroenteritis. These schools were provided unheated food from the same manufacturing company. Two genotypes (GII.3 and GII.4) of the norovirus were detected in these cases. Among them, major causative strains of GII.4 (Hu-jeju-47-2007KR-like) were identified in patients, food handlers, and groundwater from the manufacturing company of the unheated food. In the GII.4 (Hu-jeju-47-2007KR-like) strain of the norovirus, the nucleotide sequences were identical and identified as the GII.4 Sydney variant. Our data suggests that the combined epidemiological and laboratory results were closely related, and the causative pathogen was the GII.4 Sydney variant strain from contaminated groundwater.     

Pediatric norovirus GII.4 infections in Nicaragua, 1999–2015

ObjectivesInvestigate clinical and epidemiological factors of pediatric GII.4 norovirus infections in children with acute gastroenteritis (AGE) in Nicaragua between 1999 and 2015.MethodsWe retrospectively analyzed laboratory and epidemiologic data from 1,790 children ≤ 7 years with AGE from 6 hospitals in Nicaragua (n = 538), and 3 community clinics (n = 919) and households (n = 333) in León, between 1999 and 2015. Moreover, asymptomatic children from community clinics (n = 162) and households (n = 105) were enrolled. Norovirus was detected by real-time PCR and genotyped by sequencing the N-terminal and shell region of the capsid gene.ResultsNorovirus was found in 19% (n = 338) and 12% (n = 32) of children with and without AGE, respectively. In total, 20 genotypes including a tentatively new genotype were detected. Among children with AGE, the most common genotypes were GII.4 (53%), GII.14 (7%), GII.3 (6%) and GI.3 (6%). In contrast, only one (1.4%) GII.4 was found in asymptomatic children. The prevalence of GII.4 infections was significantly higher in children between 7 and 12 months of age. The prevalence of GII.4 was lowest in households (38%), followed by community clinics (50%) and hospitals (75%). Several different GII.4 variants were detected and their emergence followed the global temporal trend.ConclusionsOverall our study found the predominance of pediatric GII.4 norovirus infections in Nicaragua mostly occurring in children between 7 and 12 months of age, implicating GII.4 as the main norovirus vaccine target.     

Characterization of novel intragenotype recombination events among norovirus pandemic GII.4 variants

Recently, there has been an increase in the number of children hospitalized due to norovirus infection in Brazil. This is due both to the occurrence of more severe norovirus-related gastroenteritis cases after the introduction of the rotavirus vaccine and an increase in the tools for the detection of the disease. This pathogen is transmitted by the fecal-oral route, and the illness is characterized by diarrhea, vomiting, nausea and abdominal cramps. The genome of the virus is organized into three open reading frames showing strong mutation rates. Additionally, homologous recombination events, which can increase the virulence of the virus and lead to genotyping mistakes in molecular epidemiological studies, frequently occur. The purpose of this study was to describe two recombination events among different GII.4 variants that infected children who were hospitalized for severe acute gastroenteritis during distinct periods of time in Belém, Brazil. The recombination among the variants US95_96/Kaiso_2003 and Den Haag_2006b/Yerseke_2006a were observed in May 2003 and February 2009, respectively. In both cases, the association between the dominant variant at that point in time and another that was circulating at a low frequency in the population of Belém was demonstrated. Interestingly, the position of the breakpoint of the recombination event in the genome was the polymerase gene and was located at the nucleotide positions 4.834 and 5.002, which is an unusual location for the occurrence of recombination as other studies have previously reported the junction region as a breakpoint. In this study, both recombinant variant strains were related to severe cases of diarrhea that lead to hospitalization, demonstrating the viral evolution of GII.4 in response to selective pressures, which ultimately lead to the emergence of novel viral types in the pediatric population. The cases discussed here reinforce the need for continuous norovirus surveillance. To our knowledge, these two GII.4 variant recombinations have not yet been previously described.