Study Of Epidemiological Characteristics Of Astrovirus And Interaction Of G?.3 Norovirus With The Receptor In China,2015

Part One Study of molecular epidemiology of Astrovirus in children under 5 years old with diarrhea in seventeen provinces of China,2015Objective: To investigate the molecular epidemiological characteristics of astrovirus in children with diarrhea under 5 years of age from seventeen provinces of China,2015.To definite the main epidemic strains in China 2015 and provide scientific basis for the prevention and control of astrovirus diarrhea.Method: A total of 4672 specimens from patients with gastroenteritis under 5 years of age and relevant clinical data were collected from January to December in China,2015.The astroviruses were detected in specimens by RT-PCR and partial astrovirus positive RT-PCR products were sequenced and analyzed for phylogenetics.The epidemiological characteristics of the astrovirus was analized by the method of descriptive epidemiology.Results: The total positive detection rate of astrovirus was 3.40%.The astrovirus positive rate is higest in age group 0~2.The season peak occurred in January.The high detection rate of astrovirus occurred in Western and northern area.Of the 54 astrovirus positive strains which was selected,32 strains were successful for sequencing.Thirty-one strains were classical HAst V-1astrovirus wihile one strain was classical HAst V-5 astrovirus.Conclusions: Astrovirus is one of the important pathogens of diarrhea in children under five years of age in China and Classic HAst V-1 astrovirus was the predominant strain.Part Two The specific interaction of the epidemic G?.3 Norovirus strain with the receptor in China,2015Objective: To analyze the binding patterns of the epidemic G?.3 norovirus strain in China2015 with the receptor histo-blood group antigens(HBGAs)and explore the reason of the outbreaks.To provide important scientific basis for the prevention and treatment of G?.3norovirus diarrhea.Methods: 1.One positive stool specimen was selected from samples of the epidemic G?.3norovirus strain collected in Guangdong of China,2015.The RNA of this specimen was extracted and then RT-PCR reaction was used to obtain the gene sequence of the ORF2 region.2.The p GEX-4T1-ORF2 and p GEX-4T1-PP plasmids were constructed respectively based on the gene sequences of ORF2 region and P region.3.Through the constructed plasmids,we expressed and purified the VP1 protein and P protein of the G?.3 norovirus respectively.The VP1 protein and P protein were validated by Western Blot and the concentrations of the target proteins were measured.4.The interaction of the VP1 and P protein with HBGAs were analyzed by the ELISA test.Results: The plasmids of p GEX-4T1-ORF2 and p GEX-4T1-PP were constructed successfully.Through the corresponding plasmids,we expressed and purified the VP1 and P protein of the G?.3 norovirus validated by Western Blot.The concentrations were 0.54mg/ml and0.29mg/ml.The results of saliva binding tests showed that the average OD450 values of P protein of G?.4 norovirus 97-11 with A,B,O secretion and O non-secretory saliva were 0.3957,2.7384,0.3173 and 0.1171 respectively.The average OD450 values of VP1 protein with A,B,O secretion and O non-secretory saliva were all lower,the highest value was 0.2231,and other values were lower than the positive threshold value.The highest average OD450 value of P protein with A,B,O secretion and O non-secretory saliva was 0.9036,the lowest is 0.0987.At the same protein concentration,the binding ability of P protein with HBGAs was stronger than that of VP1(t=3.496,P<0.05).In addition,the P protein were found to bind to A,B,AB and O secretion Lewis-positive saliva.No binding was observed for the type O non-secretory saliva and Lewis-negative A,B,AB and O secretion saliva.There was no significant difference between the different types of HBGAs(c2=4.641,P>0.05).Conclusions: Compared to G?.4 norovirus,the binding ability of this epidemic G?.3norovirus strain in China 2015 with the receptor HBGAs was weak.At the same proteinconcentration,the binding ability of P protein with HBGAs was stronger than that of VP1.Furthermore,the G?.3 norovirus strain were found to bind to A,B,AB and O secretion saliva on different degrees and no binding was observed for the O non-secretory saliva.The Lewis antigen was susceptible antigen.