Preliminary Research For TCM Syndrome Differentiation Based On An Outbreak Gastroenteritis Associated GI.3 Norovirus And Study On The Cross-Antigenicity

Norovirus is one of the main pathogens to cause acute gastroenteritis worldwide with serious public health problems and economic losses.GII.4 is a globally prevalent strain with an update every 1-3 years,accounting for 85%of all NoV infections,while other genotypes of NoV are commonly sporadic.Due to the large number of NoV genotypes,the development of NoV vaccines is still a huge challenge.So far,there is no specific drugs and vaccines for NoV.In order to develop a broad-spectrum NoV vaccine,Parra classified immunotypes of GI and GII NoV according to the amino acid differences of capsid protein,which provided theoretical support for the development of a broad-spectrum NoV vaccine.But still need further experimental verification.It has a long history for traditional Chinese medicine to treat viral diarrhea.However,it is mainly focused on the research of rotavirus gastroenteritis.Since NoV has not been included in routine testing and reporting projects,the rule of traditional Chinese medicine differentiation and treatment of norovirus gastroenteritis has not been reported and clinical research was needed.In this study,we successfully carried out an outbreak caused by GI.3 NoV and conducted TCM syndrome differentiation based on outbreak population.The cross-antigenicity of different GI NoV was developed.1.The epidemiological investigation of an outbreak associated with GI.3 NoVIn July 2018,an outbreak with gastroenteritis occurred in a warehouse in Guigang,Guangxi.The research group organized an epidemiological field investigation to collect clinical symptoms,anal swabs and paired serum samples from the outbreak population.Through laboratory NoV nucleic acid testing and epidemiological risk factor analysis,it was confirmed that the outbreak was associtated with GI.3 NoV.An in-depth investigation revealed that this was a water-borne outbreak.The cases outside and inside the camp area were drinking tap water from epidemic-stricken area.Because the chlorine production plant of the waterworks had been out of repair for 2 months,the effective chlorine content in the water was low,only 0.06mg/mL(national water quality sanitation standard is 0.3-0.5mg/mL),which cannot achieve an appriciated disinfection effect.The results show that the outbreak linked with GI NoV cannot be ignored,and it is particularly important to strengthen water-related health management.2.A preliminary study on syndrome differentiation of NoV fulminant gastroenteritis.Based on the clinical symptoms of patients who were infected with NoV,the syndrome differentiation of Traditional Chinese Medicine was performed,mainly considered as the pattern of cold-damp exuberanting the spleen.Some of them were defined as the pattern of damp-heat brewing in the spleen.According to the infection and clinical characteristics of NoV gastroenteritis,it belongs to the category of"diarrhea" and "plague " of traditional Chinese medicine.Judge in view of the above,it is suggested that externally contracted epidemic toxin and spleen vacuity with dampness are the main etiopathogenesis,and the disease location mainly include spleen,stomach and intestine.Spleen movement and dampness formation should be the main treatment method.The clinical features of NoV infection was discussed and a preliminary clinical medication plan was proposed.The results of our exploration provide preliminary data for syndrome differentiation of NoV gastroenteritis by traditional Chinese medicine,and further clinical observation and efficacy evaluation are needed.3.Cross antigenicity study among defferent GI NoVRepresentative strain of GI.2(accession number KF306212),GI.3(fecal specimen from outbreak)and GI.9 NoV(provide by CDC of Guangdong Province)recombinant plasmids were constructed using molecular biology methods.Three kinds of GI NoV P protein were expressed through the prokaryotic expression system,which were selected to prepare immunized serum of mice and guinea pig.Phylogenic tress was constructed using software as MEGA7.0 based on the P domain sequences.The results showed that the nucleotide similarity between GI.3 and GI.2 was 58.7%,while the nucleotide similarity between GI.3 and GI.9 was 64.5%,indicating that a closer relationship was existed between GI.3 and GI.9 NoV.Based on the NoV-HBGA binding assays,the results show that GI.2 and GI.3 P protein have the best binding ability to type of A saliva,while the GI.9 P protein can bind to all type of secreted A,B,O and non-secreted HBGA,showing a binding pattern of O(0.86)>A(0.76)>N(0.56)>B(0.47),with the strongest combination to type of O saliva.Cross-antigenicity studies of GI.2,GI.9 and GI.3 were carried out using the immune sera of mouse and guinea pig.The results showed that the mouse sera induced by GI.2,GI.3 and GI.9 P protein has a high antibody titer tested by their own protein(≥204800),and the cross-reactivity with the other two genotypes also has a high antibody titer(≥ 51200).The three immunized mice sera had a high blockade titer tested by their own protein(BT50≥800),and the immunized mice sera of GI.3 and GI.9 NoV had a higher blockade titer tested by each other’s P protein(BT50≥ 100)than GI.2 P protein(BT50=25).There was no blockade titer that GI.3 and GI.9 P protein was blocked by immunized mice sera of GI.2 NoV(BT50≤12.5).It is preliminary proved that the cross-antigenicity and cross-protection of GI.3 and GI.9 are better than GI.2.Based on the NoV-HBGA blockade assays,the cross-protection studies on GI.3,GI.2 and GI.9 antibodies were conducted using the acute and convalescent phase sera collected during the outbreak.The results showed that there were 16 and 15 pairs of GI.3 convalescent sera against GI.3 and GI.9 NoV showed seroconversion for antibody levels(4-64 times),while only 13 pairs of GI.3 convalescent sera against GI.2 NoV showed seroconversion(4-32 times).Antibody titers of GI.3 convalescent sera against different genotypes were compared by Kruskal-Wallis test(x2= 6.7,df=2,P=0.036),the difference was statistically significant(P<0.05).Compared with GI.3 acute sera,the blockade titer of 15 pairs of GI.3 convalescent sera against their own HBGA receptors have 4-64 times increases(seroconversion,BT50≤3200),while the blockade titer of 11 pairs of GI.3 convalescent sera against GI.9 NoV-HBGA receptor have 4-32 times increases(BT50≤800),but only blockade titer of 5 pairs of GI.3 convalescent sera against GI.2 NoV-HBGA receptors have 4-8 time increases(BT50≤400).Blocking titers of GI.3 convalescent sera for different genotypes were compared by Kruskal-Wallis test(X2=20.3,df=2,P<0.05),indicating that the cross-antigenicity and antibody cross-protection ability between GI.3 and GI.9 are more excellent than GI.2.Our study provides a preliminary data for strategy of NoV vaccine.Main Conclusion1.it was confirmed that GI NoV can cause acute gastroenteritis outbreak through an epidemiological investigation of an outbreak associated with GI.3 NoV.In-depth investigation found that the effective chlorine content in the tap water from onset was relatively low,only 0.06mg/mL(national water quality sanitation standard is 0.3-0.5mg/mL),which cannot achieve an appreciated disinfection effect.Initially judgement points that water-borne pollution should be the main cause of the epidemic and relevant health management was needed to improve.2.According to the TCM syndrome analysis of NoV fulminant gastroenteritis,it is clarified that the TCM pathogenesis of NoV fulminant gastroenteritis is externally contracted epidemic toxin and spleen vacuity and dampness,and the disease location mainly include spleen,stomach and intestine.Spleen movement and dampness formation should be the main treatment method.3.The P protein were expressed successfully.A closer relationship between GI.3 and GI.9 NoV than GI.2 was observed based on the phylogenic trees.GI.2 and GI.3 P protein have the best binding profile with type of A saliva,while the GI.9 P protein can bind to all type of secreted A,B,O and non-secreted HBGA,with the strongest combination with type of O saliva.The cross-antigenicity and cross-protection between GI.3 and GI.9 are higher than GI.2.The experimental results initially confirmed that GI.3 and GI.9 belong to the same immunotype(type C),and GI.2 belongs to another immunotype(type B).