Genotype Analysis Of Norovirus Infected Among Children In Fuzhou And Preliminary Establishment Of Capture ELISA For Genogroup ? Norovirus Detection

Objectives: To explore the genotype of noroviruses infected among hospitalized children with acute diarrhea under 5 years of age in Fuzhou area between 2009 and 2016 applying dual-gene sequencing,and establish capture ELISA preliminarily for genogroup II noroviruses detection.Methods:?1?With systematic sampling method,a total of 118 fecal specimens were selected randomly from the noroviruses nucleic acid positive specimens with real-time RT-PCR screening which collected from hospitalized children under 5 years age with acute diarrhea from Fuzhou Municipal Children Hospital and Pingtan County Hospital during 2009-2016.The VP1 and partial Rd Rp genes of noroviruses were amplified and sequenced.Based on viral sequences obtained,the genotypes/subgenotypes,revolution,potential recombination and dynamic change trend of noroviruses was revealed by online genotyping,phylogenetic and intratypic recombination analysis.?2?A representative strain of Sydney₂012 variant of GII.4 noroviruses was chosen and its encode sequence of protruding?P?domain of VP1 structural protein was amplified,cloned and expressed in vitro using E.coli prokaryotic expression system.The recombinant P protein,in inclusion body form,was purified crudely and dissolved subsequently in urea solution and refolded by dialysis against PBS.Furthermore,the forming of virus like particles?VLPs?was attempted.The immunoreactivities of soluble recombinant P antigens were determined by ELISA methods,and the morphology of VLPs was observed by transmission electronic microscope.?3?BALB/c mice of sixweek-old were immunized with soluble antigen-active recombinant P protein.Conventional methods for hybridoma antibody preparation including cellular hybrid,limited dilution subcloning and indirect ELISA screen were performed for production the specific mouse hybridoma cells against recombinant P protein.The ascite were prepared through i.p.inoculation of the specific hybridoma cells into mice,and specific monoclonal antibodies?m Abs?purified through protein G column chromography.Horseradish peroxidase?HRP?labeled and unlabeled mouse m Abs were applied for establishment of capture ELISA for genogroup II noroviruses detection.In comparison with real-time RT-PCR and imported commercial reagents,the sensitivity,specifitivity and consistency of capture ELISA established in this study were evaluated.Results:?1?From 118 noroviruses positive samples,the 93 sequences of entire VP1 and partial Rd Rp were obtained,and all of them belonged to genogroup II noroviruses.Of them,41 were 2006 b variant of GII.P4_GII.4 genotypes,31 were Sydney₂012 varaint of GII.Pe_GII.4 genotypes,and 13 were genotypes of GII.P12_GII.3.The viruses belonging to New Orleans 2009 variant of GII.P4_GII.4 genotypes and GII.P17_GII.17 genotypes were 2 each,and the 2006 b variant of GI.Pa_GII.4 genotype,GII.P7_GII.7,GII.P7_GII.6 and GII.P21_GII.3 genotype was only one each.?2?By using E.coli expression system,the recombinant P protein of a chosen Sydney₂012 strain of GII.4 noroviruses expressed in form of inclusion body in vitro successfully.The inclusion body of recombinant P protein can be effectively dissolved into 4 mol/L urea solution,and the soluble protein refolded by dialysis against PBS showed relative strong immunoreactivities when tested by ELISA.Furthermore,the recombinant P proteins can self-assemble into VLPs with diameter of 10-20 nm when dialyzed against appropriate solution.?3?BALB/c mice were immunized with soluble antigen active recombinant P protein,and two monoclonal hybridoma cells,212-1 and 46-2,which secreted stably antibodies against recombinant P protein obtained.The titers of the two purified m Abs were up to 1:102400?212-1,Ig G class?and 1:16000?46-2,Ig M class?respectively.Capture ELISA method for genogroup II noroviruses detection was established,using HRP labeled and unlabeled m Ab 212-1.The sensitivity and specificity of the capture ELISA,compared with real-time RT-PCR,were 50% and 100% respectively shown on detection of 32 noroviruses nucleic acid positive and 30 negative fecal specimens,and the consistency was 79.03% when compared with commercial kits.Conclusions:?1?The dual-gene genotyping revealed diversity of noroviruses among hospitalized children under 5 years of age with acute diarrhea in Fuzhou area.2006 b variant of GII.P4_GII.4 genotype and Sydney₂012 variant of GII.Pe_GII.4 genotype predominated in period of 2009-2012 and 2013-2016,respectively.?2?The antigen active recombinant P protein of genogroup noroviruses was expressed in vitro successfully,and the capture ELISA for genogroup II noroviruses detection was established preliminarily in this study,which could provide foundations for further development of high quality commercial reagents for convenient use in field.