Redesigned Duplex RT-qPCR for the Detection of GI and GII Human Noroviruses

Human noroviruses(HuNoVs)are major foodborne pathogens that cause nonbacterial acute gastroenteritis worldwide.As the tissue-culture system for HuNoVs is not mature enough for routine detection of the virus,detection is mainly dependent on molecular approaches such as reverse transcription polymerase chain reaction(RT-PCR)and reverse transcription quantitative real-time polymerase chain reaction(RTqPCR).The widely used primers and probes for RT-qPCR were established in the early 2000s.As HuNoVs are highly variant viruses,viral genome mutations result in previously designed primers and/or probes that were perfectly matched working less efficiently over time.In this study,a new duplex RT-qPCR(ND-RT-qPCR)was designed for the detection of genogroup Ⅰ(GⅠ)and genogroup Ⅱ(GⅡ)HuNoVs based on an analysis of viral sequences added in the database after 2010.Using long transcribed viral RNAs,the results demonstrate that the sensitivity of ND-RT-qPCR is as low as one genomic copy for both GⅠ and GⅡ HuNoVs.The performance of ND-RT-qPCR was further evaluated by a comparison with the commonly used Kageyama primer/probe sets for RT-qPCR(Kageyama RT-qPCR)for 23 HuNoV-positive clinical samples.All five GⅠ samples were registered as positive by ND-RT-qPCR,whereas only two samples were registered as positive by Kageyama RT-qPCR.All 18 GⅡ samples were registered as positive by ND-RT-qPCR,while 17 samples were registered as positive by Kageyama RT-qPCR.The sensitivity reflected by the quantification cycle(Cq)value was lower in ND-RT-qPCR than in Kageyama RT-qPCR.Our data suggest that ND-RT-qPCR could be a good fit for the detection of current strains of HuNoVs.

The complete genomic sequence analysis of human norovirus NVgz01 strain in Guangzhou

The aim of this study is to explore the genomic molecular organization and genogroup of human nomvirus from infected infants in Guangzhou of China. Primers were designed according to the genomic sequence of norovims in the GenBank, and the nomvirus genome was amplified by RT-PCR. The PCR- products were cloned into T vector and sequenced, and the genomic nucleotide sequences were analyzed with the programs CLUSTAL W/X, DNASTAR and RAT （Recombination Analysis Tool）. The NVgz01 strain genome is 7558 bp in length and encodes three open reading frames （GenBank accession No. is DQ369797）. The genomic sequences of NVgz01 were compared with those of nomvirus in GenBank, which revealed that the homology with genogroup Ⅱ ranges between 76%-90%, and genogroup Ⅰ between 43%-44%. The ORF1 region shared 94% and 88% identity with Mc37 and Famiington strains, respectively; the capsid region （ORF2） shared 65% and 94% identity with Mc37 and Farmington strains, respectively. Phylogenetic trees were reconstructed by the neighbor-joining method. Comparative complete sequence analysis of the NVgz01 with reported human norovirus genomic sequences revealed that this isolate belongs to genogroup Ⅱ . The ORF1 and ORF2 regions shared different identity with Mc37 and Fannington strains, suggesting NVgz01 could be a recombinant virus.

A large outbreak of acute gastroenteritis caused by the human norovirus GII.17 strain at a university in Henan Province,China

Background:Human noroviruses are a major cause of viral gastroenteritis and are the main etiological agents of acute gastroenteritis outbreaks.An increasing number of outbreaks and sporadic cases of norovirus have been reported in China in recent years.There was a large acute gastroenteritis outbreak at a university in Henan Province,China in the past five years.We want to identify the source,transmission routes of the outbreak by epidemiological investigation and laboratory testing in order to provide the effective control measures.Methods:The clinical cases were investigated,and analysed by descriptive epidemiological methods according to factors such as time,department,grade and so on.Samples were collected from clinical cases,healthy persons,the environment,water,and food at the university.These samples were tested for potential bacteria and viruses.The samples that tested positive for norovirus were selected for whole genome sequencing and the sequences were then analysed.Results:From 4 March to 3 April 2015,a total of 753 acute diarrhoea cases were reported at the university;the attack rate was 3.29%.The epidemic curve showed two peaks,with the main peak occurring between 10 and 20 March,accounting for 85.26%of reported cases.The rates of norovirus detection in samples from confirmed cases,people without symptoms,and environmental samples were 32.72%,17.39%,and 9.17%,respectively.The phylogenetic analysis showed that the norovirus belonged to the genotype GII.17.Conclusions:This is the largest and most severe outbreak caused by genotype GII.17 norovirus in recent years in China.The GII.17 viruses displayed high epidemic activity and have become a dominant strain in China since the winter of 2014,having replaced the previously dominant GII.4 Sydney 2012 strain.

Case study:May human norovirus infection be associated with premature delivery?

Human norovirus(HuNoV)is the leading cause of acute gastroenteritis.The varying severity of chronic infection in patients with underlying immune deficiencies poses additional burdens on public health.However,the potential effects of HuNoV infection during pregnancy,a specific immune perturbed state,have been rarely reported.Recently,four cases of HuNoV-infected patients in the late stages of pregnancy were admitted to the Guangzhou Women and Children's Medical Center,and premature rupture of membranes as primary adverse outcome was observed in these cases.Samples of fetal accessory tissue were collected from two of these cases at delivery to explore the potential pathogenesis.Pathological analysis showed placental malperfusion in both maternal and fetal vascular,while a decrease in vessels was not observed in villi of placenta.There was obvious pathological change in the chorion of fetal membrane,accompanied by a tendency of Th-1 immune bias.Notably,aggregation of M2 macrophages was observed in the chorion of the fetal membrane,potentially recruited for tissue repair.Next-generation sequencing showed minimal changes in immune pathways within placenta tissue.A gene panel associated with immunosuppression was identified in the fetal membrane of HuNoV-infected women compared to those of normal parturient.Taken together,this study provides clues for the association between the HuNoV and premature delivery,which requires the attention of the clinicians.

某高校不同地域学员诺如病毒抗体阳性水平的调查研究

目的拟初步了解NVs感染在我国是否普遍存在,以丰富NVs感染的流行病学信息。方法利用酶联免疫吸附试验检测不同省份学员血清中的诺如病毒rNV、rMX和r387特异性IgG抗体水平。结果来自于22个省、直辖市、自治区的学员血清中均检出rNV、rMX和r387IgG抗体。不同地域学员rNV、rMX和r387 IgG抗体阳性率,仅rMX抗体阳性率在不同地域间有显著性差异。结论我国在广泛的空间存在NVs的感染,NVs抗体阳性率与不同地区的社会经济及生活卫生条件有关,不同的基因型的NVs可能在我国的分布有所不同。

基于受体捕获的人源诺如病毒检测方法的优化及应用

在原位捕获反转录实时定量PCR法(In situ capture real-time quantitative reverse transcription polymerase chain reaction,ISC-RT-qPCR)建立的基础上,本研究旨在优化该新型感染性HuNoVs的检测方法,并将其应用于市售贝类样品中HuNoVs的检测。本研究以2份HuNoVs临床腹泻样本3010(GI组)和3009(GII组)对ISC-RT-qPCR中猪胃粘膜提取物(porcine gastric mucin,PGM)包被浓度、待测样本孵育时间和孵育pH进行了优化;最后,对随机采集的40份市售贝类样品(文蛤、花蛤、海蛏、扇贝和鲍鱼)进行感染性HuNoVs的检测。结果表明,ISC-RT-qPCR最佳试验参数分别为:PGM包被浓度为1.0 mg/mL、待测样本孵育时间为30min、孵育pH(GI组,pH=3.0;GII组,pH=7.0);40份市售贝类样品中,贝类中HuNoVs总检出率为22.5%,多重检出率为5.0%,GI组和GII组HuNoVs的检出率分别为17.5%和10.0%;其中,市售文蛤的GI组和GII组HuNoVs检出率均为最高。因此,ISC-RT-qPCR是一种可用于市售贝类样品中感染性HuNoVs的检测方法,为食品中HuNoVs的检测与监测提供了新选择。

我国贝类中人源诺如病毒检出状况的荟萃分析

人源诺如病毒(Human Noroviruses,HuNoVs)是引发食品安全事件的重要病原微生物。贝类为滤食性动物,是HuNoVs污染传播的重要媒介。本研究搜集了我国贝类污染调查的横断面研究文献,综合评价了贝类中HuNoVs的污染现状。通过检索中国知网、维普、万方、中国生物医学文献数据库、PubMed和EMbase数据库,从所获得的600篇关于贝类污染HuNoVs相关文献中筛选纳入37篇。采用Stata 14.0软件进行荟萃分析,结果显示,我国贝类中不同基因型HuNoVs的混合检出率达15%(95%CI:11%~18%)。亚组分析显示,GⅡ基因群检出率(11%)高于GⅠ基因群(4%);地理位置对贝类中病毒污染水平影响显著(P<0.01),华南地区、华北地区、华东地区的检出率分别达到19%、17%和11%,而东北和西北地区则分别为4%和9%;此外,季节差异明显,其中,冬季的病毒检出率最高(25%),而夏季仅为10%,春季、秋季则分别为16%和12%;不同品种贝类的病毒污染同样存在差异,其中,牡蛎(Ostreidae)(16%)、贻贝(Mytilusedulis)(10%)和蛤(Mactridae)(9%)中病毒检出率居前三。综上所述,我国贝类中HuNoVs污染较为普遍,地区、季节、贝类品种等因素均对病毒污染存在显著影响。本研究结果有助于综合掌握我国贝类中食源性病毒污染现状,为精准防控食源性HuNoVs传播提供研判依据,促进贝类产业的高质量发展。

高表达H2型人类组织血型抗原HEK-293T细胞系的构建及其应用

目的通过在人胚肾-293T(human embryo kidney-293T,HEK-293T)细胞中过表达岩藻糖转移酶2(fucosyltransferases 2,FUT2)基因,进而催化诺如病毒受体人类组织血型抗原(histo-blood group antigen,HBGA)的形成,构建介导诺如病毒和受体发生相互作用的细胞系。方法构建FUT2慢病毒质粒,采用Lipofectamine 2000将FUT2慢病毒质粒及pMD.2G和psPAX2包装质粒共转染HEK-293T细胞,收获慢病毒颗粒。慢病毒颗粒感染对数生长期的HEK-293T细胞,嘌呤霉素加压筛选获得HEK-293T/FUT2细胞系。PCR鉴定FUT2基因是否整合到基因组中,RT-qPCR检测HEK-293T/FUT2细胞FUT2 mRNA转录水平,流式细胞术分析HEK-293T/FUT2细胞HBGAs的表达,间接免疫荧光法分析HEK-293T/FUT2细胞与诺如病毒病毒样颗粒(virus-like particle,VLP)的结合活性。结果成功构建了序列正确的慢病毒质粒PLVX-IRES-Puro-FUT2,并获得了慢病毒颗粒。慢病毒颗粒感染HEK-293T细胞后,通过嘌呤霉素加压筛选获得了HEK-293T/FUT2细胞系,PCR验证显示,FUT2基因成功整合到HEK-293T细胞基因组中。HEK-293T/FUT2细胞FUT2 mRNA水平提高了330倍。99.9%的HEK-293T/FUT2细胞表达H2型人类组织血型抗原。GI.1重组诺如病毒VLP可以与HEK-293T/FUT2细胞产生很好的结合反应,EC50为2.007μg/mL。结论建立了H2型人类组织血型抗原稳定高表达的HEK-293T细胞系,并基于该细胞系初步建立了GI.1型重组诺如病毒VLP结合活性评价方法。

GII.12型诺如病毒与受体HBGAs结合模式分析

为阐明GII.12型诺如病毒与组织血型抗原(Histo-blood group antigens,HBGAs)受体结合模式,本研究首先合成GII.12型诺如病毒毒株Pune株(GenBank登录号:EU921353)P区基因序列,并在原核系统中表达P蛋白,利用快速液相色谱分析鉴定P粒子的形成,将P粒子免疫小鼠,应用HBGAs表型明确的唾液样本分析P粒子的HBGAs结合模式。通过唾液结合分析,GII.12型诺如病毒Pune株与B型、AB型唾液结合较高,与A型、O型分泌型及O型非分泌型唾液结合较低,表明GII.12型诺如病毒与B抗原亲和性更高,这与先前GII.12型诺如病毒晶体结构的研究一致。这些研究提示,B型、AB型个体相对于其他血型个体,可能对GII.12型诺如病毒更具易感性。GII.12型诺如病毒与B抗原有更高的亲和性,为GII.12型诺如病毒的预防和控制提供了科学基础。

GⅡ.23基因型诺如病毒P蛋白的寡糖结合特征

诺如病毒(Noroviruses,NoVs)是导致人急性胃肠炎的最重要病原体之一,也是引起食源性疾病暴发的首要病原体。组织血型抗原(Histo-blood groups antigens,HBGAs)是NoVs的受体或宿主易感因子。已有研究表明HBGAs与NoVs的感染和流行高度相关。GⅡ.23是最近报道的NoVs新基因型。为了研究GⅡ.23与HBGAs的结合特征,表达纯化GⅡ.23基因型的P蛋白之后,通过唾液和寡糖结合实验研究其与HBGAs的结合特性,并通过同源结构模拟探索GⅡ.23 P蛋白与糖抗原潜在的对接分子机制,与已经解析的GⅡ.10的P蛋白与岩藻糖的复合物结构进行重叠。结果发现,GⅡ.23 P蛋白可以与B型唾液结合,但不结合A、O^+和O^-非分泌型唾液;P蛋白与H双糖抗原发生结合;分子模拟显示GⅡ.23 P蛋白具有与岩藻糖环结合的类似特征。本研究首次揭示了GⅡ.23 P蛋白与HBGAs受体的结合特征,为深入探索GⅡ.23基因型NoVs的进化、感染以及流行的具体机制提供了基础资料。