The Prevalence Of Sapovirus In China And The Study Of Different P Protein Of NoV GⅡ.17 Variant Binding To Saliva

Part One The analysis of Sapovirus infection in Childrenunder 5 years old with Diarrhea in Eight provinces ofChina, 2012-2014Objective: To investigate the molecular epidemiological characteristics of sapovirus in children under 5 years old with diarrhea in eight provinces of China from 2012 to 2014, know the predominant genotype of sapovirus in recent years, and lay the foundation for the study of evolutionary mechanism of sapovirus in China.Methods: From January 2012 to December 2014, a total of 3175 stool specimens and epidemiological data were collected from children under 5 years old with diarrhea. Sapovirus was detected by one step reverse transcription polymerase chain reaction(RT-PCR). All sapovirus strains were sequenced and the phylogenetic analysis was performed.Results: Sapovirus was detected in 22 of 3175 specimens(0.7%). The positive rates of age groups <6 moths, 6-12 months, 12-24 months were 0.53%, 0.70% and 0.52%, but the positive rate was 2.63% in the age group 4-5. Phylogenetic analysis showed that the predominant genotypes were GI.1(8) and GII.1(7), and GI.2, GII.3, GII.4, GII.5, GIV were also detected.Conclusion: Sapovirus was one of the pathogens causing acute gastroenteritis in some areas of China, and GI.1 and GII.1 were the predominant genotypes.Part Two The Characteristics of Different P protein of Norovirus GII.17 variant Binding to SalivaObjective: To analyze the characteristics of different P protein of norovirus GII.17 variant binding to its receptor histo-blood group antigens(HBGAs), explore the reason that norovirus GII.17 new variant got prevalent in the world, and provide the basis for research of vaccine, evolution and prevalent mechanism.Methods: 1. In 2014, we collected 183 saliva samples from an outbreak caused by a new variant of norovirus GII.17 in Huadu District, Guangzhou, China. The HBGA phenotypes of A, B, Lea, Leb, Lex, Ley, and H1 antigens of the saliva samples were determined by enzyme immunoassays(EIAs) using the corresponding monoclonal antibodies against individual HBGAs. 2. We downloaded complete genome sequence of different GII.17 strains(41621, KW323 and CS-E1) from Genbank, constructed corresponding p GEX4T-1 cloning vector based on their P region sequences. 3. Through corresponding p GEX4T-1 cloning vector, we expressed and purified P protein of different GII.17 strains. 4. We injected rabbits with P proteins of different GII.17 strains, and produced corresponding polyclonal antibodies. 5. We analyzed the characteristics of P protein binding with different saliva by EIAs.Results: 1. The HBGA phenotypes of different saliva showed: The positive rates of B, Lex, Ley antigen were 34.48%, 17.24%, 94.83% within the case group, and 17.91%, 37.31%, 76.12 % within the control group respectively, the differences were statistically significant. 2. We successfully constructed the cloning vectors of three GII.17 strains(41621, KW323, CS-E1) cloning vector, expressed corresponding P proteins, and produced corresponding polyclonal antibodies. 3. The mean OD450 of 41621, KW323, CS-E1 P proteins binding to saliva were 1.637, 0.9 and 0.142, the numbers of positive responses were 166,152 and 40 respectively, and there were significant differences each other.Conclusion: 1.For the infection of norovirus GII.17 new variant, Ley and B antigen is susceptible antigens, Lex antigen is protective antigen. 2. Compared with the old strain CS-E1, norovirus GII.17 new variants(41621, KW323) had new antigenic characteristics, and had stronger binding signals with the receptor HBGAs. These two changes helped GII.17 new variants escape from herd immunity, and lead to outbreak of acute gastroenteritis.