| The diarrhea caused by acute gastrointestinal is still a common disease in children especially under3years old，and the viral diarrheaoccupies an important position. Norovirus, sapovirus, adenovirus and astrovirus induced diarrhea are also common except rotavirus induced diarrhea. Researching on the the law of morbility, clinical manifestations and the characteristic of epidemiology about these four viral induced diarrhea has great importance to the diagnosis、prevention and treatment of the disease，to the vaccine development. Through our three section research, it had been clarified about the molecular epidemiology and rules of NoV, SaV, EadV and AstV in Chongqing area, whichenriched the epidemiological data of children with diarrhea in Chongqing, provided scientific data and basis for the prevention and controlof diarrhea.1.Epidemiological study of NoV,SaV,EAdV and AstVMethods:Five hundred and Sixty Fecal specimens were collected fromout-patient children with acute diarrhea at the Children’s Hospital ofChongqing Medical University between Dec.2011and Nov.2012. Nucleotides segments of norovirus,sapovirus, Enteric Adenovirus andastrovirus were detected by RT-PCR. Phylogenetic tree was built aftergenotyping.Results：560fecal specimens were collected, the male to female ratiowas1.43:1, the children ranging in age from22days to12.5years. RT-PCRdetection showed the positive rate of norovirus was21.25%(119/560), thepositive rate of sapovirus was2.68%(15/560), the positive rate of EntericAdenovirus was5.17%(29/560), the positive rate of astrovirus was1.25%(7/560). Genotyping study showed norovirus GII/42006b as the dominantstrain, and the new variant strain GII.4Sydney was also found, followed byGII.61.25%(7/560), GII.30.71%(4/560), GII.20.36%(2/560), GII.70.36%(2/560), GII.120.54%(3/560), GII.140.36%(2/560), GI.10.18%(1/560), GI.30.54%(3/560). Sapovirus with GI.1as the dominantstrain1.43%(8/560), followed by GII.10.54%(3/560), GI.20.18%(1/560),GII.20.18%(1/560), GIV.10.36%(2/560). Enteric Adenovirus strains fortype G41, accounts for3.21%(18/560), the addition of G310.71%(4/560),G3and G12each strain0.18(1/560), G70.54%(3/560), G20.36%(2/560).Astrovirus with HastV-1is the dominant strain0.71%(4/560),followed byHastV-40.36%(2/560),HastV-20.18%(1/560).The study found that the8cases of mixed infection. The infection peak was in autumn and winter, nosex difference.Conclusion: Among these four kinds of viral diarrhea in Chongqing area, norovirus diarrhea was prominent, GII.4as the dominant strain, butthe new variant strain GII.4Sydney has occurred. Followed by EntericAdenovirus infection, with41. sapovirus infection with GI.1, astrovirus isdominated by HAstV-1. Viral diarrhea in which peaked in autumn andwinter, no gender difference between male and female incidence.2.Analysis of norovirus recombinationMethods:28Fecal specimensins were extracted from119positivesamples of norovirus randomly, amplification of virus ORFs1capsidprotein coding sequences in RT-PCR, and sequenced to genotyping. Andthen genotyping based on RdRp, the two type of inconsistent results weresuspicious of recombinant virus strains. Amplification of the RdRp to thepartial sequences of VP1region of the long fragment, after sequencing therecombinant analysis with software Simplot and RDP was executed to findpossible recombination events and sites. Mixed infections were excluded asinterference.Results: Among the28specimens extracted, two samples weredifferent between the first and the second genotyping. CQ242wasclassified GII.3genotype based on RdRp, and then classified GII.12genotype based on VP1; Likewise,CQ426was classified GII.4and GII.6.The two specimens were long fragment amplification base on primer pairJY12Y and G2SKR, CQ426sequencing failed, unable to determine.CQ242sequencing success, identified as the recombinant strain, recombinant type is GII.3/GII.12.Conclusion: in the Chongqing area, noroviruses recombinant typeis GII.3/GII.12, which belongs to intergenotype recombination. Due tothe recombination leads to the virulence of the virus, changes in the physicochemical properties of virulence、infection、Physical and chemical properties.It must pay close attention to large-scale outbreak of norovirus diarrhea.3. Bioinformation analysis of Norovirus capsid proteinMethod: It was download the capsid protein amino acid sequence ofAGS41450virus strains GII.42006b in Chongqing from the NCBI Proteindatabase. The Swiss Institute of bioinformatics expert protein analysissystem (Expert Protein Analysis System, ExPASy), analysis ofphysicochemical properties of protein primary structure, composed ofseconfary structure, and establish the three-dimensional model results byhomology protein modeling, prediction of B cell epitopes of the protein.Results: Norovirus capsid protein GII.4of2006b by540aa residues,the total molecular weight of58991.5, its isoelectric point is5.53,negatively charged residues47,35positively charged residues. Proteinhalf-life of about30hours, unstable coefficient was45.3, fat coefficient is78.15, the total average hydrophilicity of-0.195, extremely weakhydrophilic protein. The main transmembrane segment is107-124aa residues,1-106position of the part is the membrane,125-540membraneouter segment. Homology modeling model shows531-540aa residuesprominent extended to the outside, and the strongest antigenicity. B cellepitope prediction results also showed that the ten aa residues are B cellepitope sites.531-540aa the corresponding nucleotide sequences and thewhole NoV sequence alignment,6677-6707nucleotide position in allsequences, just in the coding sequence of the P region of the capsid protein.Conclusion: norovirus antigenic sites GII.42006b virus capsid proteinVP1is the531-540aa, the nucleotide sequences corresponding to6677-6707, homology modeling of protein three-dimensional structurerevealed that the aa residue protrude to the external, consistent with NoV Pdomain under electron microscope. |
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