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.