| [Background]Norovirus(NoV)belongs to family Caliciviridae and genera norovirus with a 27～40 nm viral particle.Its genome is composed of a linear,positive-sense RNA that is 7.5～7.7 kb in length,and consists of three open reading frams(ORFs).The 5’ and 3,in its genome are Untranslated region(UTR),and the 3’ end has the poly(A)tail.ORF1 encodes a multimeric protein,and ORF2 and ORF3 encode the main and minor structural proteins(VP1 and VP2),respectively.VP1 subunit has a shell(S)and a protruding(P)domain consisting of a middle P1 and a distal P2 subdomain that is an insertion in the virus sequence.The P1 subdomain has been implicated in the antigenicity of noroviruses,while the P2 subdomain has been suggested to bind to cellular receptors of these viruses.Based on the sequence difference in the capsid of VP1,NoV can be divided into 7 genogroups(GⅠ-GⅦ)and more than 40 genotypes.Of these,GⅠ,GⅡ and GIV can infect humans,and GⅢ,GⅤ,GⅥ and GⅦ can infect bovine,murine,feline and canine.NoV is highly contagious and mainly transmitted by the fecal-oral route.Humans may be infected by NoV when contacted with cases,contaminated food and water or other environments.NoV infections present the symptom of acute gastroenteritis(AGE),such as diarrhea,vomitting,nausea,abdominal pain,headache,fever,chills and muscle aches.Although NoV infection is self-limiting,it can cause severe results or death in the infant,the elderly and immunocompromised patients.In addition,the human immune system drives selection and antigenic change in NoV,resulting in replacement of circulating dominant viruses every 2-3 years,with new variants able to re-infect hosts immune to earlier viruses.NoV is the significant viral agent in sporadic and outbreak events of AGE,and lead to heavy disease and economic burden in many countries.NoV infection is also common and outbreak events caused by NoV has been reported in many provinces and cities in our country.However,AGE is included in the C class infectious diseases and laboratory network for NoV is lacking in China,which result in that epidemiological data and evolutionary information on NoV is limited in our country.In this study,we selected 4 sentinel hospitals based on the foodborne diseases active surveillance network and conducted molecular epidemiology surveillance for NoV,to obtain the distribution of NoV genotypes in cases with AGE in Shandong province and analyze evolutionary characteristics of NoV.[Objectives](1)To obtain the positive rate and epidemiological characteristics of NoV in cases with foodborne AGE in Shandong province.(2)To investigate the distribution of NoV genotypes in cases with foodborne AGE and evolutionary characteristics of NoV in Shandong.[Methods](1)Sampling:From January to December of 2016,a total of 671 fecal samples were collected in 4 sentinel hospitals(Jinan Children’s Hospital,Yuhuangding Hospital,Laizhou People’s Hospital and Linyi People’s Hospital).Demographic and epidemiological data were also collected.(2)Samples processing and RNA extraction:All stool samples were converted to 10%～20%(w/v)suspensions in phosphate-buffered saline,which were used to extract viral RNA.(3)Detection of viruses and sequencing:Real time qRT-PCR was used to detect NoV GⅠ,NoV GⅡ,rotavirus,human astrovirus,sapovirus and enteric adenovirus.Conventional RT-PCR was used to amplify partial capsid sequences of VP1.After agarose gel analysis,the positive product was sequenced.(4)Sequences analysis:Multiple sequence alignment and homologous comparison was performed by BioEdit 7.0.5.3 between NoV sequences obtained in this study and reference sequences downloaded from GenBank database.Phylogenetic tree was constructed by MEGA 7 according to Neighbor-Joining method and Kimura 2-parameter model.(5)Statistic analysis:Statistical analysis was carried out with chi-square test by SPSS 20.0.All tests were two-tailed and considered significant when the P-value was<0.05.[Results](1)After detected by real time RT-PCR,a total of 70 fecal samples were positive for NoV with the detection rate of 10.43%.Of these,six fecal samples were positive for NoV GⅠ with the proportion of 8.57%,and 64 fecal samples were positive for NoV GⅡ with the proportion of 91.43%.Co-infection between GI and GII was not observed.The detection rates of rotavirus,human astrovirus,sapovirus and enteric adenovirus were 5.81%,2.24%,1.79%and 12.37%,respectively.(2)NoV was detected in both male and female cases with the detection rates of 10.12%and 10.73%(x2=0.016,p=0.898).The detection rates of NoV GⅠ in male and female cases were 0.87%and 0.95%,respectively(x2=0.000,p=1.000).The detection rates of NoV GⅡ in male and female cases were 9.25%and 9.78%,respectively(x2=0.054,p=0.816).(3)NoV was detected in all age groups,and two highest detection rates were observed in the children ≤5 years(14.22%)and cases 16～30 years(12.50%).The difference of detection rates in different age groups was not significant(X2=10.69,p=0.058).In addition,NoV GI was only detected in the cases 16～45 years,and NoV GⅡ was detected in all age groups.(4)NoV was detected in every month.The detection rate in February was the highest(14.49%)and in March was the lowest(5.13%).The number of cases and the detection rates were higher in cold months and lower in other months.(5)All cases positive for NoV presented with watery stool,and the high frequency of diarrhea was 10 episodes/day.Besides diarrhea,the common clinical manifestations were abdominal pain(44.93%,31/69),vomiting(44.93%,31/69)and fever(5.80%,4/69).The differences in incidence of vomiting and fever in cases positive and negative for NoV were not significant.(6)After sequenced,obtained sequences belonged to 6 genotypes(GⅠ.3,GⅠ.5,GⅡ.3,GⅡ.4,GⅡ.6 and GⅡ.17).There were 4 GⅠ.3 sequences and only 1 GⅠ.5 sequence,respectively.In all NoV genotypes,GⅡ.17(53.49%,23/43)and GⅡ.4(32.56%,14/43)were two most common genotype.Phylogenetic analysis revealed that all GⅡ.17 and GⅡ.4 sequences in this study belonged to Kawasaki 2014 variant and Sydney 2012 variant,respectively.[Conclusions](1)NoV was the significant viral agent responsible for foodborne AGE in Jinan,Linyi and Yantai,and NoV GⅡ was more prevalent than GⅠ.(2)NoV showed the high incidence in cold months,but no significant differences were observed in different genders and age groups.(3)Watery stool,abdominal pain and vomiting were the most common clinical manifestations in the cases positive for NoV,and water stool was the most important feature.(4)Multiple genotyped co-circulated in the study area,and GⅠ.3,GⅡ.17 and GⅡ.4 were the dominant GⅠ and GⅡ genotypes,respectively.All GⅡ.17 and GⅡ.4 sequences in this study belonged to Kawasaki 2014 variant and Sydney 2012 variant,respectively. |
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