| Research background and significanceNoroviruses(NoVs)is the main etiological agent causing viral gastroenteritis in all ages.NoVs infection has caused huge economic losses worldwide,leading to serious public health problems.Therefore,it is of great significance to develop an effective NoVs vaccine or antiviral drugs for the prevention and treatment of NoVs gastroenteritis.However,since the discovery of NoVs,due to the lack of mature cell culture system and appropriate animal infection model,the research on the pathogenesis of NoVs has been slow.At present,there is still a lack of effective vaccines and antiviral drugs for NoVs gastroenteritis.After more than ten years of research,it has been confirmed that human histo-blood group antigens(HBGAs)on the intestinal mucosa are the receptors of NoVs.At present,the binding/blocking model of NoVs and HBGAs receptor as an alternative to the neutralization experimental model has been widely used in the neutralization of antibodies and the evaluation of the effects of antiviral drugs.Based on the homology of the amino acid sequence of the main capsid protein VP1,NoVs can be divided into different genomes.Each genome can be divided into different genotypes,and the same genotype can be divided into multiple gene clusters and variant strains.At present,NoVs can be divided into at least ten genogroup(GI-GX),among which the two GI and GII genogroups that mainly infect human are composed of at least 9 and 27 different genotypes respectively.Due to the high diversity of NoVs genes and antigens,it has brought huge challenges to vaccine development strategies.In order to formulate a scientific and reasonable vaccine plan,in 2016,Parra et al.divided the NoVs genotypes into larger related clusters by in-depth analysis of the genetic relationship and natural development pattern of the virus strains,and proposed a new model called "immunetype".Among them,GI includes A-C and GII includes D-L.However,there is still a lack of research on the specific protection of antibodies produced by the body after infection with NoVs and the cross-protection of different genotypes.Fully clarifying the antigenic correlation between the different genotypes of NoVs will provide a scientific basis for the immunotyping theory of NoVs,and will have great guiding significance for the development strategy of NoVs vaccines.Theory and practice have proved that traditional Chinese medicine has broad application prospects and good research value in the treatment of viral diarrhea.In the early stage of the research group,through the literature screening and analysis of the effective prescriptions of traditional Chinese medicine for antiviral gastroenteritis,more than 50 main core Chinese medicines were counted,and the HBGAs receptor was used as the target to block the binding of NoVs and HBGAs receptors for traditional Chinese medicine.The screening of the active ingredients found that Chinese medicine gallnut and pomegranate peel of astringent intestines and antidiarrheal can significantly block the binding of the epidemic strain GII.4/VA387 NoV to the HBGAs receptor.However,whether it has a blocking effect on a variety of NoVs epidemic strains remains to be further evaluated.Based on this,this study selects pomegranate peel and their active ingredients tannic acid as the research object to explore its blocking effect on a variety of GⅡ/GⅠ.NoVs epidemic strains,and provides a new scientific basis for the use of traditional Chinese medicine "astringent intestines to relieve diarrhea" in the treatment of No Vs gastroenteritis.Research methods1.The recombinant plasmids of GⅡ.NoVs genogroup,GⅡ.2,GⅡ.4/VA387,GⅡ.6,GⅡ.12,GⅡ.17,GⅡ.21 and the common endemic strains GⅠ.2,GⅠ.3 and GⅠ.9 in GⅠ.NoVs genogroup were constructed by molecular biology methods.Then,the Escherichia coli prokaryotic system was used to express the P particles of the above GⅡ and GⅠ.NoVs genotypes.Mice were immunized with a variety of successfully expressed NoVs P particles to prepare mouse NoVs serum.2.Based on HBGAs binding experiment in vitro,a variety of expressed GⅡ/GⅠNoVs P particles were combined with the salivary phenotypes of HBGAs identified.Thus,the binding characteristics of various NoVs strains with different HBGAs receptors were analyzed to further clarify the important role of HBGAs in the epidemic of NoVs of different genotypes.3.The three outbreaks of GⅡ.6,GⅡ.17 and GⅠ.3.NoV acute phase serum,acuteconvalescent phase paired serum collected in the early stage of the research group and immunized mouse NoVs serum were used.Through antibody blocking experiments,the role of antibody levels in the outbreak of NoVs and the cross-protection of antibodies in the GⅡ genogroup and between GⅡ and GⅠgenogroup were explored to analyze the characterization of antigenic relatedness of various NoVs strains.4.The blocking experiment model of NoVs-HBGAs in vitro was used to explore the blocking effect of pomegranate peel and active ingredient tannin on the epidemic strain of GⅡ/GⅠ.NoVs,and provide further scientific basis for the,astringent intestines and antidiarrheal" Chinese medicine to resist NoVs gastroenteritis.The results of the study1.Successfully constructed the recombinant expression plasmids of nine different NoVs strains and expressed nine kinds of GⅡ/GⅠ NoVs P particles(GⅡ.2,GⅡ.4/VA387,GⅡ.6,GⅡ.12,GⅡ.17,GⅡ.21,GⅠ.2,GⅠ.3 and GⅠ.9)with the prokaryotic expression system at a concentration of 0.1～0.7 μg/μl,which can meet the research needs of HBGAs receptor binding/blocking effect and The nine expressed NoVs P particles were immunized to mice respectively,and the NoVs mouse serum of the corresponding genotype was successfully prepared.2.Different binding modes of GⅡ/GⅠ.NoVs genogroups and HBGAs receptor.In GⅡ.NoVs,the epidemic strain GⅡ.6 can bind to all secretory HBGAs,GⅡ.2 only has a low level of binding B secretory type,GⅡ.12 has a low level of recognition secretory type and does not bind to non-secretory saliva,GⅡ.21 not only recognizes all secretor types of A,B,AB,and O,but also has a good ability to bind to the saliva phenotype of non-secreted HBGAs.All the three strains of GⅠ could bind secretory and non-secretory to different degrees,and the binding degree of type B to GⅠ was relatively low,suggesting that individuals with type B HBGA might be protected from the infection of GⅠ.NoVs.3.Three outbreaks of GⅡ.6,GⅡ.17 and GⅠ.3 NoV serum antibody levels showed the antibody blocking titers of infected persons were low,with GMT±GSD of 18±2,18±2 and 25±1 respectively,and the serum antibody titers(18±2)of GⅡ.6 outbreak infection were significantly lower than Non-infected(66±2)(P<0.001).At the same time,the horizontal comparison of the antibody level of the GⅡ/GⅠ epidemic strain in the sera of the outbreaks showed the antibody titer of outbreak GⅡ.6 NoV strain(18±2)was significantly lower than that of heterotypic GⅡ.4(1681±5,P<0.001),GⅡ.17(59±3,P<0.001)in serum from an outbreak of GⅡ.6.The antibody titer of outbreak GⅡ.17 NoV(18±2)was significantly lower than that of heterotypic GⅡ.4(2832±5,P<0.001),GⅡ.6(200±3,P<0.001)and GⅠ.9(54±2,P<0.001)in the serum of GⅡ.17 NoV outbreak.In the serum of GⅠ.3 outbreak,the titer of outbreak GⅠ.3 NoV antibody(25±1)was significantly lower than that of heterotype GⅡ.4(3652±4,P<0.001),GⅡ.6(118±3,P<0.001)and GⅡ.17(122±3,P<0.001).In addition,this study further lonGⅠtudinally compared the levels of antibodies against epidemic strains GⅡ.6,GⅡ.17 and GⅠ.3 in the acute phase of the three outbreaks.The results showed that the antibody-blocking titer(18±2)of serum of GⅡ.6 outbreak against the homologous GⅡ.6 NoV was significantly lower than that of serum of GⅡ.17 and GⅠ.3,which were 200±3(P<0.001)and 118±3(P<0.001),respectively.Similarly,the level of antibody against homologous GⅡ.17 NoV in serum of GⅡ.17 outbreak(18±2)was significantly lower than that in serum of GⅡ.6 and GⅠ.3 outbreaks,which were 59±3(P<0.001)and 122±3(P<0.001),respectively.The results suggest that low-level serum antibody blocking titers are related to the outbreak of No Vs.4.Characterization of antigenic relatedness among GⅡ/GⅠ.NoVs genotypes,the results of antibody blocking of GⅡ/GⅠ NoVs P particles from the paired-sera of three NoVs outbreaks showed that in the sera of the GⅡ.6 NoV outbreak,there were 11 paired-sera with antibody blocking titers of the homologous GⅡ.6 NoV in the recovery period increased by more than 4 times than that in the acute period,the 11 paired-sera against GⅡ.17 NoV also have a 4-64-fold increase,GⅡ.21 NoV antibody blocking level during the recovery period also showed the same increase level as GⅡ.17.There is only one pair of paired sera for GⅡ.2,GⅡ.4,GⅡ.12 and 3 GⅠ strains with a 4-fold increase or no cross-blocking reaction in GⅡ.6 outbreak serum.In the sera of the GⅡ.17 outbreak,6 pairs of sera against homologous GⅡ.17 NoV have antibody blocking titers increased≥4 times in the recovery phase compared with the acute phase.GⅡ.21 NoV has 5 pairs of serum antibody titer increased by 4-16 times,GⅡ.6 NoV has 4 pairs of serum increased 4-8 times.For GⅡ.2,GⅡ.4,GⅡ.12 and 3 GI strains,there is only one pair or no cross-blocking effect with GⅡ.17 outbreak serum.In the sera of the GI.3 outbreak,this experiment randomly selected 6 pairs of paired sera with a blocking titer of ≥4-fold increase against the homologous GI.3 NoV,among them,GG14 is the co-infected serum of GⅡ.17 and GI.3 NoVs,the serum has a 16-fold increase in GⅡ.17 and GⅡ.21 NoV and a 4-fold increase in GⅡ.6.for GⅡ.2,GⅡ.4,GⅡ.12 NoV has no cross-blocking effect,the remaining 5 pairs of GI.3 outbreak sera have no cross-blocking effect on GⅡ genotype.Therefore,this part of the research shows that NoV serum after infection produces the strongest immune protection against the homologous strain,and there is no cross-protection between GI and GⅡ genogroups,among the GⅡ.NoVs,there is no cross-protection between GⅡ.2,GⅡ.4,GⅡ.12 and GⅡ.6 or GⅡ.17 NoV,which also belong to different immunotypes(belong to immunotypes E,immunotypes G,immunotypes D and immunotypes H or immunotypes J respectively).GⅡ.17 and GⅡ.21 NoV has a cross-blocking effect,which belongs to the same immunotype J.GⅡ.17 and GⅡ.6 NoV also have a partial cross-blocking effect,but they belong to different immunotypes(immunotype J and immunotype H).The results of antibody blocking in the serum of NoVs mice of different genotypes showed that No Vs mouse serum has the strongest blocking ability against the homologous NoVs strain,and the antibody titer is between 200～3200.In the GⅡ.17 mice serum,the blocking titer of antibodies to the heterotypic GⅡ.21 NoV reached 400.In the GⅡ.21 mice serum,the blocking titer of antibodies to the heterotypic GⅡ.17 NoV was 200,and the blocking titers of cross-antibodies of the other NoVs mouse sera against the heterotypic NoVs strains are mostly between 12.5～50.Therefore,this part of the research also shows that:NoVs serum after infection produces the strongest immune protection against the homologous NoVs strain and there is no cross-blocking effect between the GI and GⅡ genomes.Among the GⅡ.NoV,GⅡ.17 and GⅡ.21 NoV have cross-blocking effects,which belong to the same immunotype J,while the other genotypes have no cross-blocking effect,and they also belong to different immunotypes.In addition,it was found that there are some differences in the results of GⅡ.17 and GⅡ.6 NoV between mouse serum and outbreak serum,which may be related to the past infection history of the population.5.Study on the blocking effect of pomegranate peel and effective ingredient tannic acid on NoVs-HBGAs receptor binding show that the water extract of pomegranate peel can significantly block the binding of NoV P particles of GⅡepidemic strains GⅡ.4,GⅡ.6,GⅡ.17 and GI epidemic strains GI.2,GI.3,GI.9 to HBGAs receptors,and as the concentration of the water extract of pomegranate peel increases,its blocking ability also increases.In addition,its blocking rate for each strain is relatively stable,reaching the minimum concentration required to block the combination of multiple NoVs strains is about 62.5 p.g/ml.Tannic acid also has a significant blocking effect on GⅡ/GI No Vs epidemic strains,and its minimum concentration required to block the binding of multiple NoVs strains is about 0.12～0.23μmol/ml.The research conclusion1.GⅡ/GI.NoVs have different HBGAs binding characteristics.The non-secreted type of GⅡ.NoVs can protect the body from most GⅡ.NoVs infection.While GI.NoVs infection has nothing to do with secretion status,type B HBGAs can protect individuals from most GI.NoVs infections.HBGAs are an important factor in determining binding specificity and transmission risk.2.The low-level NoVs antibody blocking titre is an important mechanism for the outbreak of NoVs in susceptible populations;the immune serum after NoVs infection produces the strongest immune protection against the homologous strain and there is no cross-protection between the GⅠ and GⅡ genogroups.In the GⅡgenogroup,It has a cross-protective effect on genotypes belong to the same immunotype,while there is almost no cross-protection effect on genotypes belong to different immunotypes.However,the past exposure history or infection history of the population can affect the body’s immune response to different NoVs strains.3.Pomegranate peel and the active ingredient tannic acid can significantly block the binding of a variety of GⅡ/GⅠ.NoVs epidemic strains to HBGAs receptors.The broad-spectrum blocking effect on the binding of HBGAs receptors may be the main mechanism of action of the traditional Chinese medicine "astringent intestines and antidiarrheal" in the treatment of viral gastroenteritis. |
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