Evolution Mechanism Of Rotavirus And Antiviral Effect Of Pomegranate Rind Based On VP8~*-HBGAs

As a common,widespread virus in the world,Rotavirus(RV)consists of 11 double-stranded RNA fragments with different molecular weights,which belongs to the reovirus family.According to the gene structure specificity,RVs can be divided into seven groups A-G.The serotype of the virus is determined by the outer shell protein VP4 and VP7 encoded by gene fragments 4 and 9,whihc can be divided into type P and type G.RV can cause viral gastroenteritis(as viral infection diarrhea)in humans and animals.RVA mainly causes diarrhea in infants,while RVB mainly infects adults.RVC mainly prevalent in pigs,and the group D-G are mainly associated with animal diseases.Around 200,000 children die of viral diarrhea caused by RV each year in the world.Background:viral receptors are the key initial steps to mediate viral infection of host cells,and the research of RV receptors has always been the hot topic in the field of RV.Previous studies have shown that some animal RVs can recognize Sialic acid(SA)as a receptor,but later it has been found that most animal RVs and human RVs are Sialic acid-insensitive.Other studies have shown that Heat shock cognate70(Hsc70),ganglioside,integrin family and toll-like receptors are associated with RV infection.Human histo-blood group antigens(HBGAs)is a non-sia glycoconjugates widely distributed on the surface of mucosal epithelial cells in respiratory tract,digestive tract,urogenital tract,and it can also exist in saliva,intestinal fluid,milk,blood and other body fluids in the form of free oligosaccharides.HBGAs is formed by gradually adding different monosaccharides to the precursor sugar chain under the catalysis of glycosyltransferase.HBGAs can be expressed as ABH and Lewis antigens on the intestinal epithelial cells,which corresponds to the secretory(A,B,O blood types)and non-secretory types on the red blood cells.Through nearly 10 years of volunteer challenge assay,molecular epidemiology and molecular biology experiment,our research group confirmed that HBGAs,which distributed on the intestinal mucosa,is the receptors of the Norovirus(NoV).Through the expression of the RV spike protein VP8*and the VP8*-HBGA interaction study,we confirmed that the RV infection was related to HBGAs for the first time.Three main P genotypes of RVs(P[?]:P[4],P[6],P[8])can combine type 1 H antigen and Leb.Subsequently,multiple combination patterns between different RVs genotypes and HBGAs were found:P[?](P[9],P[14]and P[25])can identify type A HBGAs antigen,while P[IV](P[11])mainly identified type 2 HBGA precursor LacNAc.Therefore,we inferred that HBGAs may play a selective regulatory role in the evolution and epidemic law of RVs,that is,HBGAs affects the evolution process of RVs.Further in-depth study of the interaction between RVs and HBGAs will help to understand the host adaptation characteristics of RVs and the evolution and epidemic law of the virus,and formulate reasonable anti-RVs vaccine strategies and develop antiviral drug.The theory and practice of traditional Chinese Medicine(TCM)have proved that TCM treatment of viral diarrhea has broad application prospect and good development value.Through the study on the screening of active components of TCM by targeting the binding of Norovirus(NoV)to HBGAs receptors,our research group found that gallnut and pomegranate rind of "astringent intestinal antidiarrhea" could significantly block the binding of NoV capsid protein to HBGA receptors.The model based on the RVs VP8*-HBGAs receptor binding/blocking can be theoretically used to screen anti-RVs drugs in vitro.However,whether pomegranate rind and other TCMs,which have the ability to block the binding of NoV capsid protein to HBGA receptors,can also block the binding of RVs to HBGA receptors remains to be further evaluated.Methods:Based on previous research of the combination of RVA P[?],P[?]and P[?]strains with HBGAs,RVA P[?]genome and RVC strain are taken as the research object.VP4 sequences of RVA/RVC are retrieved and downloaded from Genebank,and VP8*sequence(aa46-231)of VP4 is edited and intercepted using Laser Gene/Edit sequence software.Through sequence alignment and genetic evolutionary tree construction,the type strain genome and genetic classification are confirmed.Cloning and express of RV GST-VP8*proteins representative strains(RVA:P[3],P[5],P[6],P[7],P[15],P[16],P[23],P[28],and RVC:RVC/bovine BAM7272.1,RVC/human BAB83827.1)and based on the VP8*-HBGAs interaction model,glycan array screening and saliva HBGAs combined with ELISA test were used to determine the types of different carbohydrate antigens of the combination HBGAs with RVA P[?]and RVC.Based on the previous work,the correlation and rule between HBGAs binding spectrum of RVs and their host species were analyzed and summarized,and the regulatory role and evolutionary mechanism of HBGAs on the host range of RVs were clarified.Based on the established RV VP8*-HBGA binding model and taken TCM as intervention object,the feasibility of RV VP8*-HBGA combination/blocking role model as a resistance to the RV in vitro drug screening is evaluated,and the pomegranate peel water extraction liquid block the RV VP8*combining HBGA(A/B/O)is evaluated.The antiviral effect of pomegranate peel is further verified by the integration of virus plaque assay experiment method,CPE observation method and immunofluorescence at cellular level.Plaque assay methods:culture MA104 cells in a 6-well(or 12-well)culture plate;incubate the 2-fold gradient diluted pomegranate peel water and RV Wa virus(50-100pfu)at 37? for 1h;infect MA104 cells for 1h and then discard the cells;cover the semisolid cell culture medium with a low melting point of 0.6%agarose;culture at 37? for 4 days;fix with 4%formaldehyde solution;stain with 0.1%crystal violet solution.Through the observation of the number of plaque in the control group and the drug treatment group,the inhibition rate of plaque(IR)was calculated.Cytopathic CPE was used to observe cell morphology under an inverted microscope,and the replication level of RV Wa virus in MA104 cells was observed by immunofluorescence.Results:1.48 RVA genetic trees were divided into P[?]-P[?]genomes and 38 P genotypes.P[?]genome contains the most 25 P genotypes.P[36],P[37]and P[38]are the three new P genotypes.P[36]and P[37]belong to the P[?]genome,while P[38]belongs to the P[V]genome.The 32 genetic trees of RVC have converged into several branches,which include the pig,cow,dog and human genomes.The VP8*evolutionary tree of RVC and RVA is structurally separated from each other.2.The VP8*expression plasmids of 11 RV strains(RVA:P[3]?P[5]?P[6]?P[7]?P[15]?P[16]?P[23]?P[28]and RVC:RVC/bovine BAM7272.1,RVC/human BAB83827.1)were successfully constructed,and the expression of RV GST-VP8*protein was identified by SDS electrophoresis,and the protein size was consistent with the expected value.3.Ten expressed RV GST-VP8*fusion proteins(diluted to 5ug/ml and 50ug/ml,respectively)were used as probes to react with a sugar chip containing about 600 glycans.The activity of the specific RV GST-VP8*protein interacting with the glycosyl group is expressed by the relative fluorescence unit RFU.Fluorescent anti-GST-labeled RV monoclonal antibody was used as the detection antibody,and fluorescence was measured and quantified by microarray scanner.The results showed that:P[3],P[5],P[7],P[28],P[16]/EDIM,P[16]/EMcN and the bovine strain in the RVA[?]genome could identify the carbohydrate structure containing galal-3galb1-4glc.Among them,P[3],P[16]/EDIM,P[16]/EMcN and niuyuan RVC strains showed the strongest binding capacity with luumgal sugars,ranking first in the spectrum of binding capacity of sugars,while P[5],P[6],P[7]and P[28]showed relatively weak binding capacity with luumgal sugars.P[28]can combine with the sugars in the structure of Fucl-2galb1-3glcnac(in the first place in the binding ability map with P[28])in addition to the binding with lugogal,which proves that P[28]can recognize type 1 HBGAs-H antigen.P[3],P[7],P[15],P[23]could bind a carbohydrate containing the structure of neu5aca2-3galb1-4glcnac(including sialic acid carbohydrate structure),proving that P[3],P[7],P[15],P[23]could recognize sialic acid receptors.The human RVC strain was able to recognize sugars bound to GalNAc?1-3(Fuc?1-2)Galb1-3GlcNAc,that is,to recognize HBGAs-A binding antigen.Ten RV GST-VP8*fusion proteins were used in the binding experiments based on saliva and mucins VP8*HBG A on animals.The results showed that anthropogenic RVC and human type A saliva had a clear combination pattern of the RV(30%),while animals and human saliva combining ability is generally low(0-8%).The human/pork/beef bovine strains RVs and sticky proteins of group C can be combined,which prompted that there has existence population isolation barrier of the host range between different species and human.4.According to the specific binding effects of RV GST-VP8*and carbohydrate and through the RVs genetic evolutionary tree analysis combined with aGal,the results showed that:P[3],P[16],RVC/cow and P[5]have the strong combination ability with ?Gal and their the host species are monkey,rat and cow respectively,which belong to a relatively lower creatures.P[28]has a weaker combination ability with the aGal and its host is mainly the human.The strong and the weak combination ability with the aGal of these four strains corresponds to the high and low evolution of the infected host level.Conservative analysis of the sequences of RVA and RVC binding to type A HBGAs showed that the binding sites of RVA/RVC binding to type A antigen in the two genomes were highly conserved in each genome,but there were differences in the binding sites of A antigen between the two groups(RVA:101R,187S,188Y,189Y,190L,191T.RVC:109R,110A,152G,205P,206R,207S).5.After three times of regular water decoction,the obtained water solution was concentrated in a constant temperature water bath at 75?.The initial dose of 1mg/ml water agent was prepared.After 0.22um pore membrane filtration,it was used for antiviral effect evaluation.The results showed that the water extract of pomegranate rind could significantly block the binding of human RVC VP8*protein to type A,B and O saliva,and the concentration of BT50 was 1.2ug/ml.The antiviral effect of pomegranate rind water extract was further verified at the cellular level,and the results showed that:lOug/ml pomegranate rind water extract has obvious direct virus killing effect and antiviral effect.In other words,10ug/ml water extract from pomegranate rind can significantly inhibit cytopaemia(CPE)and viral plaque formation caused by RVs.It has been observed by immunofluorescence assay that the replication level of RV Wa virus interfered by pomegranate rind water extract in MA104 cells is significantly reduced.Conclusion:1.Through analyzing and comparing the binding types and associations of different genome RVA and RVC,the results showed that epidemiology of different genotypes of RVs matched their HBGA spectral,which illustrated that the seclectio effects of HBGAs receptors can regulate the evolution of RVs and reservoir types of infection.Therefore,it will help us understand RVs host adaptability and popular rule,make the new strategy of RVs vaccine and look for the development of new anti RVs drugs.2.RV VP8*-HBGAs receptor binding/blocking model is a new and effective model for in vitro screening of anti-RV drugs.The water extract of pomegranate rind could block the binding of RV VP8*to HBGAs(A/B/O)receptor obviously,which proved to have A good antiviral effect at the cellular level.Blocking receptor binding may be an important mechanism of "astringent intestinal antidiarrhea" of TCMs such as pomegranate rind.