诺如病毒新重组株GⅡ.P16/GⅡ.2引起福建省2016年冬季病毒性胃肠炎暴发

目的 本文旨在鉴定福建省2016年冬季导致病毒性胃肠炎暴发的病原体,并对病原体进行分子特征研究.方法 对疫情暴发地上送的福建省2016年胃肠炎暴发急性病例标本,采用荧光PCR初步判定病原体,常规RT-PCR检测诺如病毒RNA聚合酶和衣壳蛋白基因片段,并进行序列测定和分子特征分析.结果 在3起暴发疫情中18份标本经荧光PCR检测均为诺如病毒核酸阳性,其中15份标本RT-PCR检测判为GⅡ型诺如病毒,9份标本成功测序.分析测序结果,证实引起本轮疫情为诺如病毒新重组株,该重组株有别于本地散发流行及全球暴发流行的优势基因型GⅡ.4.毒株RNA聚合酶核苷酸序列与2016年日本的GⅡ.4悉尼变异株Kawasaki194毒株的同源性最高,达98％,为GⅡ.P16亚型;衣壳蛋白核苷酸序列与2008年比利时的IPH2161-08VG06毒株同源性最高(97.7％～98.8％),为GⅡ.2亚型.结论 这是福建省首次报道诺如病毒重组株GⅡ.P16/ GⅡ.2的检出,并引起病毒性胃肠炎暴发.     

一起由诺如病毒和肠道腺病毒混合感染引起的急性胃肠炎聚集性疫情的病原鉴定及进化分析

目的?对一起儿童急性胃肠炎聚集性疫情的病原进行鉴定、分型及进化分析。 方法?收集本次疫情患儿粪便标本，提取核酸，采用实时荧光PCR初步进行病原鉴定。通过反转录-聚合酶链反应对诺如病毒阳性标本的聚合酶区和衣壳VP1区部分基因序列进行扩增；通过PCR对腺病毒阳性标本的六邻体区部分基因序列进行扩增。对扩增产物进行测序，利用生物信息学软件及网站对病毒进行型别鉴定及进化分析。结果?共收集到3例患儿粪便标本，诺如病毒和腺病毒均为阳性。3株诺如病毒毒株均为诺如病毒GII.P12-GII.3型，其聚合酶区部分基因序列（206 bp）与2015─2018年我国GII.P12-GII.3型诺如病毒参考序列的同源性为98%，且位于同一进化树分支上；3株腺病毒毒株均为腺病毒F亚属41型（F41型），其六邻体区部分基因序列（420 bp）与2015─2019年我国F41型腺病毒参考序列的同源性为100%，且位于同一进化树分支上。 结论?本次儿童急性胃肠炎聚集性疫情是由GII.P12-GII.3型诺如病毒和F41型腺病毒混合感染引起，毒株序列分别与近年来我国流行的GII.P12-GII.3型诺如病毒及F41型腺病毒序列高度同源。本研究为混合感染急性胃肠炎疫情病原的快速鉴定及溯源分析提供了参考。     

一起诺如病毒GⅡ.17型引起的急性胃肠炎病原学诊断及基因特征分析

目的对长沙市2014年12月某厂区暴发的一起急性胃肠炎疫情进行病原学诊断及对其致病原进一步的基因分型研究。方法共采集6例病人肛拭子标本和可疑水源标本4份,提取核酸后,诺如病毒GI/GII型real-time RT-PCR试剂盒检测;阳性标本普通RT-PCR扩增VP1基因片段,产物测序后BLAST比对确定其型别,并构建进化树分析其进化关系。结果 10份标本real-time RT-PCR扩增结果显示为诺如病毒GII型,VP1区片段RT-PCR扩增后产物测序,BLAST比对发现与2014年香港诺如病毒株GII/Hu/HKG/2014/GII.17/CUHK-NS-463同源性最高,达99%,证实为诺如病毒GII.17型;构建系统进化树分析显示本次疫情分离的毒株与日本、香港、台湾等亚洲地区的毒株亲缘关系更为接近,而与美国、法国等地区的毒株亲缘关系则较远。结论诺如病毒是引起此次急性胃肠炎疫情的病原体,且引起暴发的病毒株属于GII.17型。     

一起急性腹泻疫情中检出的GI.8型诺如病毒的序列测定和分子特征分析

目的对湖州地区一起急性腹泻疫情中检出的GI.8型诺如病毒进行序列测定和分子特征分析。方法根据GI型诺如病毒保守序列自行设计5对引物,用一步法RT-PCR分段扩增GI.8型诺如病毒CHN/Huzhou/N10的全基因组序列,采用生物信息学相关软件对序列进行整理和分析。结果 CHN/Huzhou/N10全长7 740bp,编码区包括3个开放读码框架(ORF1~ORF3),分别长5 400bp(5~5 404nt)、1 632bp(5 388~7 019nt)和642bp(7 019~7 660nt)。RdRp区,VP1区和VP2区的系统进化分析显示CHN/Huzhou/N10属于GI.8基因型。衣壳蛋白区的氨基酸序列分析表明,与原型株Boxer/2001/US相比,CHN/Huzhou/N10VP1区的氨基酸序列共有16个变异位点,12个位于P2区。其中aa347T→S,aa397T→E这2个变异位点分别位于HBGA受体结合袋位点Ⅱ和Ⅲ内。结论本文获得了我国GI.8型诺如病毒CHN/Huzhou/N10的全基因组序列,相关序列信息可用于病毒的遗传进化研究、快速诊断试剂的研发以及疫苗的设计。     

诺如病毒GⅡ.4型流行株基因序列分析和流行病学研究

目的研究诺如病毒的基因特征和变化规律,预测诺如病毒的进化方向及流行趋势,为预防诺如病毒病的暴发打下良好基础。方法采用RNA提取试剂Trizol提取诺如病毒毒株RNA,采用试剂盒TaKaRa Ex TAP进行PCR扩增,利用Bioedit软件对基因序列进行拼接,然后对基因序列分析。将PCR扩增出的P区域克隆到表达载体,构建原核表达质粒。在大肠埃希菌中表达P蛋白,重组蛋白经SDS-PAGE凝胶分离检测。以ABO血型健康人唾液为受体研究P粒子与唾液HBGA结合能力,检测方法采用间接ELISA法。结果实验用诺如病毒毒株与GⅡ.4流行株在RdRp区的核苷酸序列同源性达到94%以上,其中US95/96株94%,Farmington Hills株94.2%,Hunter株94.7%,Den_Haag₂006b株95.8%,Osaka₂007株96.5%,New_Orleans₂009株97.2%,Sydney₂012株98.4%。实验株与GⅡ.4流行株在衣壳蛋白区的核苷酸序列同源性达到94%以上,其中US95/96株94.3%,Farmington Hills株94.1%,Hunter株95.2%,Den_Haag₂006b株96.3%,Osaka₂007株96.8%,New_Orleans₂009株97.8%,Sydney₂012株98.6%;氨基酸同源性达到96%以上,其中US95/96株为96.1%,Farmington Hills株96.3%,Hunter株97.1%,Den_Haag₂006b株96.8%,Osaka₂007株97.3%,New_Orleans₂009株97.8%,Sydney₂012株98.9%。实验株在RdRp区核糖核苷酸序列的与New_orleans2009和Sydney₂012同源性较高,实验株中P2区氨基酸位点发生多点位定向突变,如P2区第95位由天冬酰胺（N）突变为组氨酸（H）。重组蛋白经SDS-PAGE凝胶分离检测产物大小为35ku的目的蛋白表达。检测P粒子与唾液HBGA结合能力（A450值）血型A为3.81⁵.12;血型B为3.12⁴.05;血型O为2.85³.51。结论诺如病毒在遗传上具有多样性,变异快的特点,因而很难有疫苗对它长期安全有效。通过对GII.4型基因序列的研究,有助于寻找其关键位点变化规律和流行株进化机理,有助于预测诺如病毒的进化方向及流行趋势。     

贝类中GI、GII诺如病毒快速检测与分群方法的建立

目的 建立贝类中GI、GII诺如病毒快速检测与分群方法。 方法 对比分析6株主要流行的GI、GII诺如病毒及参考诺如病毒的基因序列,筛选保守区域引物,优化建立SYBR Green I荧光定量检测与熔解曲线快速分群方法,并对实际样品检测验证。结果 引物P289/290可同时检测GI、GII诺如病毒,SYBR Green I荧光定量检测方法在病毒浓度10³~10⁹ copies 之间呈现良好的线性关系(R²=0.993,P＜0.01),熔解曲线Tm值可区分GI和GII不同基因群的诺如病毒(F=7 507.60,P＜0.05 )。120份贝类样品阳性检出率5.83%,其中阳性结果测序鉴定与快速分群方法结论一致。 结论 该方法成本低、检测快速、分群准确,具有良好重复性,适用于贝类中GI、GII诺如病毒的快速检测与分群。     

我国GII.4型诺如病毒Sydney株P颗粒的表达及其组织血型抗原结合特征

目的表达我国新发现GII.4型诺如病毒优势流行株—Sydney株的P颗粒,并探究其结合人类组织血型抗原(HBGAs)受体特征及变化规律。方法扩增SH 2012_05株中的P区域序列,构建进化树分析其氨基酸序列,并在原核表达系统中表达P颗粒。目的重组蛋白经Western blot确定其特异性,并采用ELISA法检测P颗粒结合HBGAs的能力及方式。结果 SH 2012_05毒株P区域与Sydney/2012原型株同源性为99.1%,为GII.4/Sydney基因簇。SDS-PAGE电泳和Western blot分析验证SH 2012_05株P颗粒具有特异性。其结合HBGAs受体方式与以往流行的基因簇一致,即与A、B、O、AB型分泌型唾液均能结合,其中与B型抗原亲和力最高。结论成功表达了我国新发现GII.4型诺如病毒Sydney株的P颗粒,明确了Sydney株能结合分泌型HBGAs受体的特征。本研究为诺如病毒疫苗株选择提供了技术储备,这将为GII.4型诺如病毒的预防和控制提供科学基础。     

2018—2021年长沙市诺如病毒暴发疫情流行特征分析

目的 了解2018年1月至2021年3月长沙市诺如病毒(Norovirus, NoV)暴发疫情的流行病学和进化特征。方法 采集2018年1月至2021年3月急性胃肠炎暴发疫情临床病例标本463份，采用实时荧光RT-PCR法进行诺如病毒GI和GII基因群的鉴定；对阳性标本采用特异引物RT-PCR扩增，所得扩增产物测序后进行进化分析。结果 2018年1月至2021年3月共报告诺如病毒疫情64起，采集标本463份，NoV检出阳性率为57.67%,男性检出率为60.16%,女性检出率为54.71%;12～18岁年龄组检出率最高，达74.24%。每年冬春季(10月至次年3月)为流行高峰季节；有64.06%(41/64)的疫情发生在托幼机构。64起暴发疫情中由GII基因群NoV引起的占82.81%(53/64),由GI基因群NoV引起的占7.81%(5/64),混合基因型占9.38%(6/64)。GI基因群NoV的具体基因型别有GI.2[P2]、GI.3[P13]、GI.5[P4]、GI.6[P11];测序分型成功的GII基因群NoV具体基因型别有GII.1[P16]、GII.2[P16]、GII...     

福建省2015年本地登革热病例的病原学特征

目的 分析2015年福建省本地登革热病例部分登革病毒流行株的基因组序列,调查相关病毒株之间的遗传联系,为福建省登革热防控提供病原学证据。方法 采集急性期患者血清,实时荧光RT-PCR检测登革病毒RNA,阳性者分离病毒,扩增病毒基因组并测序,以病毒全长编码区序列进行种系发生分析。结果 2015年福建省先后出现登革1型(DENV1)和登革2型病毒(DENV2)引起的本地暴发疫情,共发生4起聚集性病例,报告41例。分离到DENV1毒株9株,DENV2毒株8株。种系发生分析显示,9株DENV1高度相似,同属于基因1型(G1),提示其共同的输入来源可能为斯里兰卡;而8株DENV2病毒虽然同属于大都市基因型,却分为差异较大的两簇,表明莆田与福州两地疫情相关毒株的遗传关联度较低,可能分别来自马来西亚和印度。结论 2015年共出现4起聚集性本地登革热疫情,部分患者病毒分离株的病原学特征表明,福建省2015年本地登革热暴发疫情存在多个输入来源。     

重组酶聚合酶侧流层析技术检测新冠病毒核酸快速诊断方-法的初步建立

目的 建立重组酶聚合酶扩增侧流层析技术(RPA-LFD)检测新冠病毒(SARS-CoV-2)核酸快速诊断方-法,为基层医院和边远地区防控新冠病毒疫情提供技术支持。方法 根据新冠病毒N基因核苷酸序列中保守序列,通过生物信息学软件分析、设计和筛选最佳的重组酶聚合酶扩增引物及侧流层试纸探针,优化重组酶聚合酶扩增反应条件以及检验建立方-法的灵敏性和特异性。结果 利用RPA-LFD法在37 ℃下反应15 min可检测到每个反应最低100 fg含量的新冠病毒,并与流感病毒、副流感病毒、鼻病毒和腺病毒没有交叉反应,因此据此可以建立灵敏性高、特异性强和可操作性强的新冠病毒快速诊断方-法。结论 通过RPA-LFD技术建立的新冠病毒快速诊断方-法可获得较为理想的灵敏度、特异性,为进一步研制新冠病毒核酸快速诊断试剂盒奠定了基础。     

Epidemics of gastroenteritis during 2006 were associated with the spread of norovirus GII.4 variants 2006a and 2006b.

BACKGROUND: Acute gastroenteritis is commonly associated with norovirus genogroup II (GII) infection. Norovirus GII has 17 classified genotypes (GII.1-GII.17), but only 1 norovirus genotype (GII.4) is associated with global epidemics of gastroenteritis. In 2006, an increase in global norovirus activity was observed. METHODS: During the period from December 2005 through August 2006, a total of 231 fecal samples were obtained from patients with acute gastroenteritis from Australia and New Zealand. Norovirus RNA was amplified and sequenced to determine norovirus genotype and relatedness to known epidemic norovirus GII.4 variants. RESULTS: Two GII.4 variants, designated 2006a and 2006b, were identified in 61.8% and 11.3%, respectively, of the 186 cases investigated. Norovirus 2006a and 2006b have also been implicated as the predominant causes of norovirus-associated gastroenteritis across Europe in 2006. CONCLUSIONS: The global increase in norovirus-associated gastroenteritis in 2006 was linked to the emergence of 2 novel GII.4 variants, 2006a and 2006b.     

Virological and Epidemiological Features of Norovirus Infections in Brazil, 2017–2018

Noroviruses are considered an important cause of acute gastroenteritis (AGE) across all age groups. Here, we investigated the incidence of norovirus, genotypes circulation, and norovirus shedding in AGE stool samples from outpatients in Brazil. During a two-year period, 1546 AGE stool samples from ten Brazilian states were analyzed by RT-qPCR to detect and quantify GI and GII noroviruses. Positive samples were genotyped by dual sequencing using the ORF1/2 junction region. Overall, we detected norovirus in 32.1% of samples, with a massive predominance of GII viruses (89.1%). We also observed a significant difference between the median viral load of norovirus GI (3.4×10⁵ GC/g of stool) and GII (1.9×10⁷ GC/g). The most affected age group was children aged between 6 and 24 m old, and norovirus infection was detected throughout the year without marked seasonality. Phylogenetic analysis of partial RdRp and VP1 regions identified six and 11 genotype combinations of GI and GII, respectively. GII.4 Sydney[P16] was by far the predominant genotype (47.6%), followed by GII.2[P16], GII.4 Sydney[P31], and GII.6[P7]. We detected, for the first time in Brazil, the intergenogroup recombinant genotype GIX.1[GII.P15]. Our study contributes to the knowledge of norovirus genotypes circulation at the national level, reinforcing the importance of molecular surveillance programs for future vaccine designs.     

Norovirus Epidemiology and Genetic Diversity in Leipzig,Germany during 2013–2017

Globally and in all age groups, noroviruses are a main cause of gastroenteritis. To assess their local epidemiology and genetic diversity, stool samples of 7509 inpatients with gastrointestinal complaints from all age groups were analyzed. After detection of norovirus genogroup I and II RNA by real-time RT-PCR, viral capsids were genotyped by partial nucleic acid sequencing. In the case of GII.2 strains, polymerase genotypes were also assessed. Between October 2013 and September 2017, presence of norovirus RNA was shown in 611 samples (8.1%), of which 610 (99.8%) were typed successfully. Norovirus positivity rate was higher in patients aged below five years (14.8%) than in older patients (5.7%). Among the 611 norovirus positive samples, GII.4 (56.6%) strains prevailed, followed by GII.6 (11.3%), GII.3 (11.0%) and GII.2 (9.5%). The most common genogroup I (GGI) genotype was GI.3 (3.6%). In addition, rare genotypes such as GII.13, GII.14 and GII.26 were detected. Interestingly, GII.3 infections were most common in children under the age of five years. Assessment of polymerase genotypes in GII.2 viruses showed a shift from P2 to P16, with higher diversity in P2 sequences. The varying distribution of norovirus genotypes depending on season, age and setting of infection highlights the importance of frequent genotyping as a basis for vaccine development and needful adjustments.     

Virus Etiology,Diversity and Clinical Characteristics in South African Children Hospitalised with Gastroenteritis

Background: Viral gastroenteritis remains a major cause of hospitalisation in young children. This study aimed to determine the distribution and diversity of enteric viruses in children ≤5 years, hospitalised with gastroenteritis at Kalafong Provincial Tertiary Hospital, Pretoria, South Africa, between July 2016 and December 2017. Methods: Stool specimens (n = 205) were screened for norovirus GI and GII, rotavirus, sapovirus, astrovirus and adenovirus by multiplex RT-PCR. HIV exposure and FUT2 secretor status were evaluated. Secretor status was determined by FUT2 genotyping. Results: At least one gastroenteritis virus was detected in 47% (96/205) of children. Rotavirus predominated (46/205), followed by norovirus (32/205), adenovirus (15/205), sapovirus (9/205) and astrovirus (3/205). Norovirus genotypes GI.3, GII.2, GII.3, GII.4, GII.7, GII.12, GII.21, and rotavirus strains G1P[8], G2P[4], G2P[6], G3P[4], G3P[8], G8P[4], G8P[6], G9P[6], G9P[8] and sapovirus genotypes GI.1, GI.2, GII.1, GII.4, GII.8 were detected; norovirus GII.4[P31] and rotavirus G3P[4] predominated. Asymptomatic norovirus infection (GI.3, GI.7, GII.4, GII.6, GII.13) was detected in 22% of 46 six-week follow up stools. HIV exposure (30%) was not associated with more frequent or severe viral gastroenteritis hospitalisations compared to unexposed children. Rotavirus preferentially infected secretor children (p = 0.143) and norovirus infected 78% secretors and 22% non-secretors. Conclusion: Rotavirus was still the leading cause of gastroenteritis hospitalisations, but norovirus caused more severe symptoms.     

Epidemiological and Genetic Characterization of Norovirus Outbreaks That Occurred in Catalonia,Spain, 2017–2019

Molecular characterization of human norovirus (HuNoV) genotypes enhances the understanding of viral features and illustrates distinctive evolutionary patterns. The aim of our study was to describe the prevalence of the genetic diversity and the epidemiology of the genotypes involved in HuNoV outbreaks in Catalonia (Spain) between 2017 and 2019. A total of 100 HuNoV outbreaks were notified with the predominance of GII (70%), followed by GI (27%) and mixed GI/GII (3%). Seasonality was observed for GII outbreaks only. The most prevalent genotypes identified were GII.4[P31] Sydney 2012, GII.4[P16] Sydney 2012 and GII.2[P16]. As compared to person-to-person (P/P) transmitted outbreaks, foodborne outbreaks showed significantly higher attack rates and lower duration. The average attack rate was higher in youth hostel/campgrounds compared to nursing homes. Only genotypes GI.4[P4], GII.2[P16], GII.4[P16], GII.4[P31] and GII.17[P17] were consistently detected every year, and only abundance of GII.2[P16] showed a negative trend over time. GII.4 Sydney 2012 outbreaks were significantly associated to nursing homes, while GII.2[P16] and GI.3[P3] were most frequently identified in youth hostel/campgrounds. The average attack rate was significantly higher when comparing GII.2[P16] vs. GI.4[P4], GII.2[P16] vs. GII.4[P31] Sydney 2012, and GII.6[P7] vs. GII.4[P31] Sydney 2012. No correlations were found between genotype and outbreak duration or age of affected individuals.     

Norovirus Genogroup II Epidemics and the Potential Effect of Climate Change on Norovirus Transmission in Taiwan

The activity of norovirus varies from season to season, and the effect of climate change on the incidence of norovirus outbreaks is a widely recognized yet poorly understood phenomenon. Investigation of the possible association between climatic factors and the incidence of norovirus is key to a better understanding of the epidemiology of norovirus and early prediction of norovirus outbreaks. In this study, clinical stool samples from acute gastroenteritis outbreaks were collected from January 2015 to June 2019 in Taiwan. Data analysis from our study indicated that more than half of the cases were reported in the winter and spring seasons, including those caused by norovirus of genotypes GII (genogroup II).2, GII.3, GII.6, and GII.17, and 45.1% of the patients who tested positive for norovirus were infected by the GII.4 norovirus in autumn. However, GII.6 norovirus accounted for a higher proportion of the cases reported in summer than any other strain. Temperature is a crucial factor influencing patterns of epidemic outbreaks caused by distinct genotypes of norovirus. The results of this study may help experts predict and issue early public warnings of norovirus transmission and understand the effect of climate change on norovirus outbreaks caused by different genotypes and occurring in different locations.     

Detection of Norovirus in Saliva Samples from Acute Gastroenteritis Cases and Asymptomatic Subjects: Association with Age and Higher Shedding in Stool

Norovirus infections are a leading cause of acute gastroenteritis outbreaks worldwide and across all age groups, with two main genogroups (GI and GII) infecting humans. The aim of our study was to investigate the occurrence of norovirus in saliva samples from individuals involved in outbreaks of acute gastroenteritis in closed and semiclosed institutions, and its relationship with the virus strain, virus shedding in stool, the occurrence of symptoms, age, and the secretor status of the individual. Epidemiological and clinical information was gathered from norovirus outbreaks occurring in Catalonia, Spain during 2017–2018, and stool and saliva samples were collected from affected and exposed resident individuals and workers. A total of 347 saliva specimens from 25 outbreaks were analyzed. Further, 84% of individuals also provided a paired stool sample. For GII infections, norovirus was detected in 17.9% of saliva samples from symptomatic cases and 5.2% of asymptomatic individuals. Positivity in saliva occurred in both secretors and nonsecretors. None of the individuals infected by norovirus GI was positive for the virus in saliva. Saliva positivity did not correlate with any of the studied symptoms but did correlate with age ≥ 65 years old. Individuals who were positive in saliva showed higher levels of virus shedding in stool. Mean viral load in positive saliva was 3.16 ± 1.08 log₁₀ genome copies/mL, and the predominance of encapsidated genomes was confirmed by propidium monoazide (PMA)xx-viability RTqPCR assay. The detection of norovirus in saliva raises the possibility of oral-to-oral norovirus transmission during the symptomatic phase and, although to a lesser extent, even in cases of asymptomatic infections.