Preliminary Investigation Of Norovirus Infection In Housed Monkeys

Human norovirus(huNoVs)is the main cause of acute nonbacterial gastroenteritis.In recent years the incidence and death rate are increasing persistently.All age groups were sensitive to huNoVs infection.Although the majority of people infected with huNoVs can be self-healing,huNoVs infection still pose a serious threat to children and immunocompromised population.Factors that make huNoVs outbreak seriously include:fast molecular evolution of huNoVs,sustained infection after detoxification,low dose of infection and transmission diversity.Therefore,the rapid diagnosis and timely treatment after infection are very important for controlling huNoVs infection.Effective vaccines are considered not only to greatly reduce the threat to public health,but also to improve the efficiency of the national economy and the quality of life.However,development of vaccines against huNoVs is very difficult due to lack of a effective culture system in vitro and animal model for future vaccine evaluation.The aims of this study is to investigate NoVs infections in the housed monkeys and lay a solid foundation for further establishment of non-primate animal model of NoVs infection in the future.To do this,five parts of studies were performed as follows:1)We analyzed the epidemic situation of noroviruses in some certain population of monkeys and patients in a certain Hospital by RT-PCR method,detection the norovirus epidemic situation of monkeys in the promise of the for the late animal model select appropriate animals and virus strains,the detection norovirus epidemic situation of the population for selected human noro virus strains to infection animal experiments.2)NoVs-positive samples were selected for the amplification of whole genomes,through the whole gene comparison we can understand the evolution and variation of Norovirus.3)The P domain and variant protein of one GII.17 NoV were cloned into the pGEX-4T-1 vector,following the optimization of sequences according to the bias of E.coli expression system.The recombinant constructs were expressed in the prokaryotic expression system.The antiserums were prepared via immunization of mice with the purified P domain or its variant protein.And the antigenicity of proteins was analyzed by Western Blot and ELISA,prepare for test samples of animals infected by norovirus.4)Next we infected Rhesus Macaques orally with GII.17 NoVs identified from extraction of monkey feces in this study,followed by monitoring the clinical symptoms and viral shedding in 40 consecutive days,which was a preliminary exploration for the construction of Norovirus infection animal model.5)Saliva from these housed monkeys were typed using ELSIA,these data will be contribute to the study of the mechanism of Norovirus infection.6)Finally,we tried to establish a 3D culture system of the intestinal epithelial cells of rhesus monkey,lay the foundation for the establishment of Norovirus conventional culture system and vaccine neutralization evaluation.Through the above experiments,we have obtained the following results:(1)We analyzed 30 fecal samples from monkeys and 50 from patients using RT-PCR.The results indicated that positive rates of NoVs in monkeys and in patients were 28.57%and 37.63%respectively.(2)One complete genomic sequence of GII.17 virus was obtained from monkey fecal samples by RT-PCR amplification,which was submitted to Genebank with gene ID KX356908.In addition,three complete genome sequences of the NoVs GII.4 strain from patients were amplified at the same time.(3)The sequencing results showed P domain and its variant proteins were successfully cloned into the prokaryotic expression vector.SDS-PAGE analysis indicated that P proteins were expressed in both supernatant and inclusion body of prokaryotic cells after ultrasonication,and the variant was expressed mainly in the supernatant.Western Blot and ELISA showed the purified P domain and its variants protein have good antigenicity.The antibody titer reached 1:32 000 by ELISA.(4)The challenging experiment showed that no clinical symptom was observed in the challenged monkeys,but obvious viral shedding was recorded after challenging.ELISA further showed the titer of serum antibody against P protein was detected over time,but prechallenged sera from the two monkeys also showed high titer of NoVs specific antibody.(5)In rhesus macaques,the distribution of salivary phenotype A,B,AB,H was 15.79%,68.42%,14.04%,and 1.75%.The orthogonal experiment showed that phenotypes in saliva and blood were matched.Saliva binding assay showed that no differences between NoVs P-RGD and P domain variants protein.Further experiments are necessary for confirming this results.(6)The intestinal crypt epithelium of the colon was successfully isolated via the EDTA method.The growth of intestinal crypt was observed in the matrix via the three-dimensional culture.However,the histological morphology and epithelial marker need to be further characterized via HE staining and immunofluorecence analysis.Conclusions:In present study,we investigated the prevalence of NoVs in the housed monkeys and a certain hospital,showing 28.57%and 34.69%positive samples.Four complete genomic sequences of NoVs were amplified from fecal samples.We successfully expressed and purified the antigenic P domain and its variant protein of NoVs.The identified NoVs could reinfect rhesus macaques,leading to viral shedding and induction of antibody against NoVs.Finally,we successful isolated and cultured macaque intestinal epithelial cells via 3D culture system.The results in this study lay a foundation for the establishment of in vitro culture system and non-human Primate infection model of NoVs.