Preliminary Study On Molecular Epidemiology Of Norovirus And Its Relationship With Intestinal Microflora In Zhenjiang

Objective:Noroviruses（Noroviruses,NoVs）are common pathogens in food borne diseases and public health emergencies in Zhenjiang,which can easily lead to outbreaks in the population.In order to clarify the molecular epidemiological characteristics of NoV in Zhenjiang and its relationship with intestinal flora,this study from molecular biology,bioinformatics,genomics and other aspects systematically analyzed the molecular epidemiological characteristics,evolution and recombination of NoV in this region and the effects on intestinal microflora.Methods:1.In order to analyze the prevalence of different genotypes of NoV in Zhenjiang,this study used a combination of real-time fluorescent quantitative PCR（q RT-PCR）and reverse transcription PCR（RT-PCR）.Collected stool samples of food borne diseases with diarrhea and conventional public health emergency cases during 2015.1-2016.12 in Zhenjiang city,q RT-PCR was performed using NoV GI/GII double PCR kit,then primers were designed based on NoV capsid protein of genome to implement RT-PCR and sequencing for genetic typing.2.Amplified RNA-dependent RNA polymerase（Rd Rp）region and sequenced,combined with the results of the Capsid region,retrieved by Gen Bank for similarity search.The phylogenetic tree was constructed using Mega 6.06 software Neighbor-joining（N-J）based on nucleotide to carry out phylogenetic analysis.At the same time,the systematic evolution and recombination of NoV in Zhenjiang area were analyzed by using RDP 4.0software for Rd Rp region and Capsid region sequences.3.Extracting the total DNA of samples,and specific primers were designed to amplify the 16 S v4-v5 region,after amplification,the amplified fragments were obtained,plused connector,using Hiseq 2500 sequencing platform to get longer sequence by splicing.The 16 S r RNA gene was used to carry out the population,abundance and community structure and other diversity analysis to clarify the relationship between NoV infection and intestinal microflora.Results:1.Infection status: 2015-2016,a total collection of feces and anal swab samples of1605,NoV positive rate was 5.30%（85/1605）,the detection rate of GI group was 0.87%（14/1605）,GII group was 4.55%（73/1605）,and 2 samples infected GI/II group at the same time,there was statistical significance between GI group and GII group detection rate difference（P< 0.05）.There was no significant difference in NoV detection rate of male and female（P> 0.05）.NoV’s popular peak was mainly concentrated in November to April of the next year,with the highest detection rate in Dantu District.Children aged 0 to 10 years old in this region and older people over 60 years old were mainly infected with NoV GII group,both GI and GII group can affect other age groups.The positive rate of GII group was the highest among young adults aged 10 to 20 years,up to 17.80%,and the differences were statistically significant（P< 0.05）compared with other age groups.There was no significant difference in GI group infection.2.NoV genotype: NoV GII.17 genotype accounted for 63.89% of the highest proportion in 2015,which were statistically significant differences with other genotypes,other genotypes were GI.3,GII.3,GII.4,and GII.13.In 2016,the proportion of GII.4Sydney₂012 was highest,up to 35%;other genotypes were GI.3,GI.4,GI.5,GI.6,GI.7,GI.8,GII.2,GII.3,GII.14,and GII.17.3.Evolution and recombination: Phylogenetic analysis results further confirmed that there were 31 strains of GII.17 genotype sequences in Zhenjiang area,3 of which were in the same evolutionary branch with GII.17 sequences in 1978-2002 years,the other 28 strains were GII.17 genotype variants,which were homologous with GII.17 previous,but there was a certain variation.There also found 2 recombination events,GII.P12_GII.3genotypes of ZJ086/2016/CHN and ZJ096/2016/CHN strains were U02030/Toronto/CA/GII.3 and X86557/Lordsdale/UK/GII.4 recombinant.GII.P16_GII.2genotype of ZJ152/2016/CHN strain was obtained from GII.P2_GII.2 genotype of ZJ124/2016/CHN strain and GII.P16_GII.13 genotype of ZJ105/2015/CHN strain recombination,the recombination site was NoV recombination hotspots: ORF1-ORF2 junction area.4.The relationship with intestinal microflora: The Operational Taxonomic Units of people infected with NoV were higher than that of the normal population.In the level of phylum,the number of proteobacteria in NoV infected patients was significantly reduced than normal population,Firmicutes and Bacteroidetes increased significantly;in the level of genus,Escherichia,Pseudomonas were significantly reduced,Streptococcus increased,Bacteroides increased significantly,while incertae sedis significantly increased.Intestinalbacteria had statistically significant were as much as 43 species between NoV infection and normal population,including Pseudomonas,Microbacterium,Rhodococcus etc.significantly decreased in NoV infection population,Bacteroides,Veillonella spp.,Leptotrichia,anaerobic bacteria and other increased significantly,and found that NoV infection often accompanied by a significant increase in a variety of bacteria.Conclusion:1.NoV was a common pathogen of food borne diseases and public health emergencies in Zhenjiang.The prevalence of NoV in this area had the characteristics of time,region and population,the NoV epidemic peak was mainly from November to April,with the highest detection rate in Dantu District,and the infection was mainly NoV GII genogroup.The positive rate of GII genogroup was highest in 10 to 20 years old,and there was no significant age difference in GI genogroup.2.There were a variety of NoV genotypes in Zhenjiang region,and were in the dynamic changes,the main prevalent genotypes were GII.17 and GII.4,which GII.17 genotype was a new variant and GII.4 genotype was GII.4 Sydney₂012.3.There were 2 recombination events of NoV in this region,namely GII.P12_GII.3genotype was GII.3 recombined with GII.4 genotype;GII.P16_GII.2 genotype was GII.P2_GII.2 recombined with GII.P16_GII.13 genotype.4.High-throughput sequencing based on 16 S r RNA gene found that NoV infection was associated with changes in intestinal bacterial communities.