Anti-rotavirus Pharmacodynamics Evaluation Of P-VP8~*IgY And The Preliminary Exploration On The Small Animal Model Of Norovirus

Norovirus(NoV)and rotavirus(RV)are the leading pathogens of viral diarrhoea worldwide,Causing serious social and public health problems.While there are no specific anti-NoV drugs or vaccines,no specific drug can prevent and treat NoV and RV infection at the same time.In 2005,Jiang et al successfully expressed Novovirus P particles by E.coli expression system.P particles had the immunogenicity similarity to NoV virus-like particles(virus-like particles,VLPs).Also the binding affinity of HBGAs receptor are similar to that of VLPs.P particles can be expressed stably and efficiently in Escherichia coli expression system,and have the characteristics of low cost and short cycle.P particles can be widely used in pathogenesis and antigen variation of NoV.molecular evolution and second generation subunit vaccine candidate.Norovirus P protein has an octahedral structure,which each surface contains three ring structures(loop1,loop2,loop3)that exposed to P protein surface,is suitable for Insertion of exogenous epitopes,.Also there is no influence on the formation and yield of P particles.Each surface of P protein can be inserted into an antigen epitope,exposeing a foreign fragment to the outermost.It can be used as an ideal antigen presentation platform that greatly enhances the immunogenicity of the antigen.Our research group inserted the VP8*gene fragment of rotavirus coat stinging protein into the surface ring region loop2 of norovirus P particles.Stable expression of chimeric P-VP8*protein in Escherichia coli.Preparation and purification of specific P-VP8*IgY from Sailing chicken by Immunization.Studies confirm that P-VP8*IgY can effectively block the binding of NoV to HBGAs receptor,It also inhibited the infection of MA104 cells with RV Wa strain.These results indicate that the P-VP8*IgY may be a promising bivalent drug against both norovirus and rotavirus.The purpose of this study was to evaluate the protective effect of P-VP8*IgY against rotavirus in vivo by using rotavirus infection animal model,the methods and results were as follows:48 neonatal SPF rats were randomly divided into Control group,Infection group,Prevention treatment group,low dose treatment group,middle dose treatment group and high dose treatment group(8 in each group).Establishing the RV infected sucking mice animal model by inoculom with 107PFU ZTR-68 strain RV(G1P[8]type)cultured in vitro.The low,medium and high dose groups were given different concentrations of P-VP8*IgY(3.3mg kg-1,10mg kg-1,30mg kg-1)for 3 consecutive days,while the prevention group was given a certain amount of P-VP8*IgY(20mg kg-1)2 days before the drug attack.The symptoms of physical signs and diarrhea were observed after inoculation,the rotavirus colloidal gold kit was used to detect the stool sample rotavirus antigen.virus-shedding of stool sample was tested using ELISA.RV DNA gene copies of small intestine were tseted by RT-qPCR and the RV protein expression level were tested by Western Blot.Pathological changes were observed by HE stain and small intestine RV expression were tested by IHC.Results:6-day-old suckling mouse presented yellow dilute water stool after inoculation with a dose of Human RV of 107PFU,which indicated that the model of RV infection in suckling mouse was successful.3 days after treatment,compared with IG,the degree of diarrhea in MDTG and HDTGwas significantly alleviated,the diarrhea score(1.75±0.49,1.50±0.27,respectively)was significantly lower than that in IG group(3.37 ±0.18)(P<0.05),the content of RV antigen in feces(60.0±26.2,40.0±18.5,respectively)was significantly lower than that in IG(130.0±41.4)(P<0.05),the amount of RV DNA gene copies and the expression of RV-VP6 protein in small intestine has a significant reduction(P<0.05),chorionic vacuolar changes were alleviated by HE stain.IHC showed a significant decrease in the expression of RV antigen.While there was no significant difference between PTG and LDTG group and IG group.Conclusion:P-VP8'IgY antibodies could protect the intestinal villi and alleviate diarrhea induced by human rotavirus through neutralizing the RV antigen from fecal and small intestinal tissues in the suckling mouse disease model,and provide protection against rotavirus infection.The antigenicity of norovirus P particles is much larger than that of VP8*protein,and P-VP8*IgY has a good anti-norovirus activity in theory.Previously,it was confirmed that P-VP8*IgY could block the binding of norovirus to HBGAs receptor by HBGAs receptor replacement neutralization test in vitro.Therefore,it is necessary to evaluate the effect of P-VP8*IgY on Noroviruses in vivo.Due to the lack of effective small animal models of norovirus,the second part of this study aims to establish the animal model of tree shrew virus-infected diseases for the first time.the methods and results were as follows:Fifteen 6-month-old tree shrews were randomly divided into three groups:A?B and C group with 5 trees hrews in each group.To neutralize stomach acids prior toinoculation,tree shrews received 500ul of 100 mM sodium bicarbonate orally.Each tree shrew in group A and B received one oral dose of 1 ml of fecal suspension containing 2× 105viral RNA copiesHuNoVs G?.4 2010 strain and Sydney strain separately.The symptoms of physical signs and diarrhea were observed for 7 days after HuNoVs challenge.HuNoVs RNA was detected by RT-PCR for stool samples collected every 24h,and then the tree shrews were sacrificed to collect the small intestinal tissue.HE staining was used to observe the pathological changes.At the same time,the HuNoVs antigen were determined by immunohistochemistry(IHC),the saliva HBGA of tree shrew was analyzed by ELISA.Results:No obvious infection symptoms and diarrhea were observed in the tree shrews after HuNoVs challenge.No HuNoVs RNA was detected in the stool specimens,and no HuNoVs antigen was detected in the small intestine.The pathological changes were not obvious.The HBGA antigen in the saliva of tree shrews was all negative.Conclusion:The unsusceptible to HuNoVs in tree shrews might be related to noexpression of HBGA and common breeding conditions.The small animal model of HuNoVs remains to be further explored.Conclusion:1.P-VP8 IgY antibodies could protect the intestinal villi and alleviate diarrhea induced by human rotavirus through neutralizing the RV antigen from fecal and small intestinal tissues in the suckling mouse disease model,and provide protection against rotavirus infection,It provides a basis for the further development of P_VP8*IgY,also provides a new direction for the modern application of traditional Chinese medicine EGG YOLK..2.The unsusceptible to HuNoVs in tree shrews might be related to noexpression of HBGA and common breeding conditions.It provides a new direction for the establishment of norovirus animal model with stable genetic components of HBGAs in the future.