| Objective:Human norovirus(Hu NV)is are the leading cause of acute gastroenteritis,there have been several outbreaks of norovirus infectious diarrhea in many countries,It brings huge economic burden to public health.The detection and epidemiological study of norovirus are of great significance for the study of vaccines and prevention control of diseases.According to the report,research about norovirus is lacking in yunnan province.Therefore,the purpose of this study was to investigate the gene polymorphism,genotype distribution and relationship between clinical symptoms and genotype of the endemic strain of norovirus of the outbreak during November 2018 to January 2019 in Kunming,Yunan,China.A standard quality plasmid and its detection system for detecting copy number of norovirus G? virus in fecal samples have been successfully established,which will aid in epidemic prevention,epidemiology studies and vaccine development.Whole-Genome Sequences provided the genetic characteristics,potential sources and evolution of the virus genome information that might aid in epidemic prevention,epidemiology studies and vaccine development.Methods:(1)During October 2018 to February 2019,the 237 diarrhea samples of children younger than 10 years were collected in Children's Hospital of Kunming,including80 fecal samples from hospitalized children,157 fecal samples from outpatientchildren.there were 145 males and 92 females,with a sex ratio of 1.6:1.Fecal samples were detected by RT-PCR,then PCR products were sequenced in theTsing Ke Biotech Co of kunming,genotypes of all sequences were confirmed by using blast search and the clinical symptoms of all diarrhea cases were recorded.(2)NV G? standard plasmid was accomplished that the highly conserved NV G? gene sequence(500 bp)was cloned into p UC57 vector to construct,then the plasmid was verified by PCR.The corresponding standard curve equation was obtained that the standard curve of Ct value and copy number of norovirus G? was drawn according to real-time fluorescence quantitative PCR amplification curve.Then,4 positive samples were selected in the part from 84 NV positive samples were obtained in the above part and were tested and validated by the established norovirus G? detection system.(3)In the chapter,6 stool samples of norovirus infection positive of G? group were selected.whole-genome sequence of NV G? were downloaded in the NCBI database to designed primers,then 3 Fecal samples were detected by RT-PCR,PCR products were sequenced in the Tsing Ke Biotech Co of kunming,then genotypes of all sequences were confirmed by using blast search.Finally the Bio Edit sequence Alignment Editor was employed to splice sequences of complete gene and the whole genome sequences have been deposited to the NCBI Gen Bank database.The aim of this part was to explore the genetic characteristics,potential sources and evolution of the virus.Results:(1)In 237 stool samples of the study,two genogroups of Hu NV were detected in84(35.44%)samples of cases.There were 47 male and 37 female in NV positive cases.The GI genogroups contained 1 genotype,and the G? contained 11genotypes.G?.4 was still the dominant pandemic strain,then The recombinant strain G?.Pe-G?.4 and G?.3 were increased in G? genogroup infection.Experiment data show that 12-23 months(45.71%,32/70)was the age range with the highest NV infection rate in age group distribution,and genotype and corresponding clinical symptoms were statistically analyzed,and the analysis results showed that fever,vomiting,diarrhea>3 days were found associated significantly with NV infections.Statistical analysis of the ratio of male and female with NV infection positive showed that there was no statistical significance between NV infection positive and gender.(2)PCR proved that the standard plasmid for detection of copy number of NV G? was constructed correctly.Based on amplification curve of real-time fluorescence quantitative PCR acquired standard curve equation and the standard curve equation as follow:Y=-3.972X+39.03,R~2=0.9907.The viral copy numbers in four faeces samples were 30443.45,9468.40,53176.69,4493.12 copies/?L respectively.(3)The whole genomes of 3 positive samples were successfully sequenced through G? primer.The genome of G?.4 variant strain has a total length of 7545nucleotides and were made up of ORF1 of 5100 nucleotides,ORF2 of 1623nucleotides and ORF3 of 807 nucleotides.Phylogenetic tree analysis of ORF1,ORF2and ORF3 showed that the 2017 jinan strain(MG214988)of the genotype G?.4 were the highest similarity with whole genomes of the study.Conclusion:(1)G?.4 was still the dominant pandemic strain,then The recombinant strain G?.Pe-G?.4 and G?.3 were increased in G? genogroup infection.(2)Experiment data show that 12-23 months(45.71%,32/70)was the age range with the highest NV infection rate in age group distribution.(3)Fever,vomiting,diarrhea>3 days were found associated significantly with NV infections.(4)there was no statistical significance between NV infection positive and gender.(5)Based on amplification curve of real-time fluorescence quantitative PCR acquired standard curve equation.(6)The whole genomes of 2 positive samples were successfully sequenced through G? primer.(7)Phylogenetic tree analysis of ORF1,ORF2 and ORF3 showed that the 2017jinan strain(MG214988)of the genotype G?.4 were the highest similarity with whole genomes of the study. |
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