Molecular Epidemiology Of Norovirus Based On The Sentinel Hospitals In Three Cities Located In Shandong Province

BackgroundNoroviruses（NoV）found in human are the leading cause of acute gastroenteritis（AGE）.The clinical symptoms of NoV-related AGE include acute onset of nausea,vomiting,headaches,chills,abdominal cramps,and loose or watery diarrhoea.Pandemic forms of NoV cause substantial morbidity,mortality and economic costs across the globe each year.Using previously described 2×standard deviation（SD）criteria to group sequences into separate clusters,the number of genogroups can be divided into 10（GⅠ-GⅩ）.NoV can infect humans（GⅠ,GⅡ,GⅣ,GⅧ,and GⅨ）.GⅡ is the most predominant NoV genogroup,with GⅡ.4,GⅡ.17 and GⅡ.2 being the dominant genotypes.Studies have found that NoV-positive rate are various between diverse population,time and regional.The epidemic characteristics and genetic evolution mechanism of NoV are still research hotspots in this field.AGE causes considerable disease burden in Shandong province and NoV is the leading cause of gastroenteritis epidemics and a common cause of sporadic gastroenteritis.But there is a lack of understanding of NoVs’ epidemic characteristics,genotype distribution and related genetic evolution laws in parts of Shandong.Therefore,this study conducted case studies and laboratory tests on AGE patients treated in sentinel hospitals located in three cities in Shandong Province from June 2016 to May 2018.The epidemiological characteristics of patients with NoV-related AGE in some cities in Shandong Province were analyzed.The NoV sequences were analyzed by bioinformatics to elaborate the genetic evolution law of the whole genome of NoV.The results of this study revealed the epidemiological characteristics of patients with NoV-related AGE in some areas of Shandong Province,and also provided a molecular basis for studying the genetic evolution mechanism of NoV dominant genotypes,which has provided a theoretical basis for the prevention and control of NoV.Objective（1）The pathogenic spectrum of foodborne virus-related AGE in three cities in Shandong was mastered,for the purpose of providing statistics which can support for the formulation of AGE targeted control measures in some parts of Shandong Province.（2）Comprehend the epidemiological characteristics of NoV and the laws of genetic evolution.The aim of these is to eliminate NoV infection in a timely and effective manner,and control the public health risks and socio-economic burden caused by norovirus in time.（3）Master the distribution of NoV genotypes in foodborne AGE patients,and further study the genetic evolution of dominant genotypes.The specific aims are to inform targeted vaccine development for dominant genotypes.Methods（1）Case investigation of AGE patients:2,169 research objects were selected from sentinel hospitals in Jinan,Linyi,and Yantai between June 2016 and August 2018.All cases were defined as foodborne diarrhea.Epidemiological information,including gender,age,and location of patient’s onset,was collected.An Excel database was established and statistical analysis was conducted by SPSS 23.0 software.（2）Laboratory examination:Fecal specimens were collected from 2,169 patients.The real-time fluorescence quantitative PCR was used to detect common enteroviruses including NoV GI/GII,SaV,HaV,RoV and EAdV.For NoV amplification,the reverse transcription-polymerase chain reaction（RT-PCR）was carried out.（3）Sequencing and genetic diversity analysis of NoV:Stratified sampling was carried out according to genotype.Fecal samples from 37 patients were collected and sequenced by next-generation sequencing technology.Genotyping was identified using NoV online typing tool and Blast platform.Mafft and Bioedit software were used for alignment,homology comparison and amino acid mutation site analysis of NoV sequences.Raxml software was used to construct phylogenetic trees.Results（1）The positive test results:703 of the 2169 samples were tested positive for one or more viruses,with an overall positive rate of 32.41%.A total of 99 cases were associated with co-infection of multiple enteroviruses,including 92 co-infection with two viruses.RoV and AdV mixed with other viral infections were the most common situation,and RoV and AdV combined with other viral infections were 53 cases.A total of 211 NoV-positive cases were detected,with an overall positivity rate of 9.73%（21 1/2169）.（2）The epidemiological results:Overall,most cases（65）were found in the spring with the highest NoV-positive rate（13.24%）.On the contrary,the lowest rate was observed in the autumn（37/857,4.32%）with a statistically significance（χ2=88.891,P<0.001）.The highest prevalence（119/809,14.71%）was observed in Jinan,followed by 7.73%in Linyi.The patients from Yantai had the lowest positive rate（42/713,5.89%）and with a statistically significant difference（χ2=37.764,P<0.001）.There were statistical differences in the positive rate of NoV among various age groups（χ2=16.454,P<0.050）,the highest positive rate of NoV in the 16～30 age group（63/454,13.88%）,and the lowest in the>60 age group（14/244,5.74%）.There were statistical differences in the positive rate of NoV among the population of different occupations（χ2=18.310,P<0.050）,the highest positive rate among students（29/210,13.94%）,and the lowest positive rate among farmers（20/368,5.46%）.（3）The results of analysis of clinical manifestations:The difference between diarrhea symptoms in NoV-positive and negative patients was statistically significant（χ2=17.045,P<0.001）,and the incidence of diarrheal symptoms in NoV-positive patients was higher than that in negative patients.There was no statistically significant difference in the incidence of vomiting,abdominal pain,and fever between NoV-positive and negative patients.（4）Genotyping results:A total of 165 NoV genotypes were successfully genotyped in this study.According to 2×standard deviation（SD）criteria,a total of 12 recombinant genotypes were detected,of which GⅡ.2[P16]was the main recombinant genotype.A total of 11 VP1 genotypes were detected,including GⅡ.2,GⅡ.17,GⅡ.3 and GⅡ.4.A total of 11 RdRp genotypes were detected,among which GⅡ.P16,GⅡ.Pe and GⅡ.P12 were the most common.（5）The results of phylogenetic analysis showed that the GⅡ.4 sequences obtained in this study were all GⅡ.4 Sydney₂014 variant,the ORF2 of GⅡ.2[P16]were all Reemerge GⅡ.2[P16]variant,and the other sequences obtained were also the most prevalent variant of this genotype in the world.The GⅡ.P16 sequences were all located in the Reemerge GⅡ.P16 variant.The analysis of the mutation site showed that the RdRp mutation site（1548）in the sequencing GⅡ.2[P16]sequence was different from that of the Reemerge GⅡ.P16 variant which is currently prevalent in the world.Conclusion（1）In parts of Shandong Province,foodborne diarrhea can be caused by different viruses.NoV is one of the main pathogens of foodborne diarrhea in parts of Shandong Province.NoV infection varies by season,region and population,suggesting that early prevention and control warning should be implemented in summer and autumn,and health education should be done to high-risk populations.Vomiting is significant in determining the presence or absence of NoV infection and may be helpful for early identification of NoV.（2）Multiple NoV genotypes co-circulated in Shandong Province.GⅡ.2,GⅡ.4 and GⅡ.3 were common genotypes,circulating in the population.But there were differences in the prevalence of variant genotypes in diverse time,regions and age groups.The endemic strains of each genotype in Shandong Province were consistent with the dominant variant of this genotype in the world,suggesting that preventive vaccination should be carried out in high-risk populations as soon as possible according to the global NoV epidemic situation.（3）In GⅡ.4 and GⅡ.2 sequences that sequenced in this study,there were multiple mutation sites in VP1 region,which may be related to immune escape of NoV.The RdRp mutation site（1548）in the GII.2[P16]that we sequenced was different from that of the Reemerge GⅡ.P16 subtype currently prevalent in the world.