Association Between RV Infection And HBGA Based On "Different Diseases With Same Syndrome"

Viral gastroenteritis, also known as viral diarrhea, is a group of acute diarrheal diseases caused by a variety of viruses. Most common causes of viral diarrhea are rotavirus and norovirus. They are responsible for more than 60% of viral infection for diarrhea. Although pathogens varies in gene and structure, they show similar clinical manifestations including nausea, vomiting, abdominal pain, diarrhea, watery stools or loose stools, and accompanied with fever and other symptoms sometimes.As is known, RV and NoV are two different pathogens in modern medicine. Since the infection of them both have same symptoms and signs such as vomiting, diarrhea, fever. RV and NoV gastroenteritis belong to "diarrhea" in traditional Chinese medicine, and the key pathogenesis lies in deficiency in spleen and dampness excessive. Syndrome (zheng) is a pathological generalization of a disease at a certain stage in the course of its development. It reveals the nature of disease and the whole responding of individual to the disease. Since RV and NoV gastroenteritis have the same (or similar) syndrome, we wonder whether they have the same material basis for the syndrome in "same syndrome in different diseases". Analysising human body from the molecular composition, the nature of syndrome in TCM is a class of proteins and peptides functioning as messenger. Specific receptor on the surface of cells is the key factor for virus infection to infect human. So we propose the hypothesis that the material basis of syndrome in "same syndrome in different diseases for RV and NoV infection may be similar receptor or co-receptor, that is the two viruses have the same receptor.It has been conformed that human histo-blood group antigens (HBGAs) on mucosal epithelia of the digestive tracts is the receptor for NoV afer decade of study by volunteers attack test, molecular epidemiology and experiment of molecular biology. HBGAs are complex carbohydrates distributed abundantly on mucosal epithelia of the respiratory, genitourinary and digestive tracts. They are also present as free oligosaccharides in biologic fluids, such as saliva, intestinal content, milk and blood. Mucosal epithelia of intestine express ABH and Lewis antigens. Red blood cells express secretors and non-secretors in accordance with the ABO blood type. The finding that HBGA is receptor of NoV is the milestone for NoV research. It has helped our understanding of virus/host interactions, the evolutionary of virus, epidemiology, and host immune selection of NoVs. The research for RV receptors is mainly focus on functional protein of cells including toll-like receptors, integrin family, ganglioside, heat shock cognate protein (Hsc70). Studies have shown they play a role in RV attachment. However, the relationship between HBGA and RV infection remains to be confirmed further.Based on study RV infection may associated with HBGA, we speculated that the material basis of syndrome in "same syndrome in different diseases" for RV and NoV infection may be similar receptor or co-receptor. Thus, to find the relationship between RV infection and HBGA, we collected stool specimens and paired saliva from young children sick with diarrhea in clinic, utilizing pupils in school as healthy control. Meanwhile, we express the capsid protein of RV VP8*, establish the binding assay of RV-VP8* and saliva HBGA to confirmed the association between RV infection and HBGA again in vitro.1. Epidemiological study of rotavirus infection and HGBAFecal samples and paired saliva samples were collected from children who were diagnosed with acute gastroenteritis in Nanfang Hospital pediatric clinic, saliva of healthy control were collected from pupils in a primary school located in Guangzhou. Trizol reagent is used for viral RNA extraction of stool samples, and then RT-PCR was performed to amplification RV-VP4 region. Further genotyping PCR were carried for RVpositive samples. The HBGA phenotypes of paired saliva were identified by ELISA. Testing monoclonal antibody include A, B, H, Lewis a, Lewis b, Lewis x and Lewis y. individuals of Lea-b+ and/or Lex-y+ are classified into secretor, and Lea+b+ and/or Le x+y+ partial secretor, both of them belong to secretors; Lea+b- and/or Lex+y- are non-secretors. The results show that 266 stool specimens and paried saliva were collected in Nanfang Hopital, among which 75 RV positive cases were found. The HBGA distribution of the 75 cases was as followed: the cases of type A antigens positive were 22, and B were 17, O were 33, AB were 3 parts. There were 63 cases with Lea-b+/Lex-y+, and 12 with Lea+b+ /Lex+y+, none with Lea+b-/Lex+y-.the 75 RV infected children were all belong to secretors. For 191 RV negative cases from Nanfang Hopital, despite of 40 cases of NoV positive, there were 151 cases in total. There were 47 cases of A antigen positive,44 of B,50 of O,10 of AB. Lea-b+/Lex-y+ were found in 132 cases, and Lea+b+ /Lex+y+ in 9 cases, Lea+b-/Lex+y- in 10 cases. This means there were 141 cases of secretor and 10 cases of non-secretor in RV-negative patients. No significant difference was detected in blood type between RV positive and RV negative cases according to Chi square test(x2= 3.047, P= 0.384> 0.05), indicating that there is no correlation between RV infection and ABO blood. Statistical significance was found in Lewis phenotypes between the two groups by Fisher’s exact test (χ2= 10.659, P= 0.004<0.05), showed that RV infection is closely related with the individuals’Lewis phenotype. And significantly difference in secretor status between the two groups were fpond by the Fisher’s exact test(P= 0.033<0.05),HBGA analysis of 159 cases of healthy control showed that there were 35 cases of A antigen positive,39 of B,59 of O,6 of AB (ABO blood type were indentified for 139 cases). Lea"b+/Lex"y+were found in 134 cases, and Lea+b+/Lex+y+in 5 cases, Lea+b"/Lex+y’in 20 cases. This means there were 139 (87.4%) cases of secretor and 20(12.6%) cases of non-secretor in healthy control.No significant difference was detected in blood type between RV positive and healthy control cases according to Chi square test (%2= 1.546, P= 0.672> 0.05), Statistical significance was found in Lewis phenotypes between the two groups by Fisher’s exact test (P= 0.000<0.05), also in secretor status (P= 0.00K0.05). These results suggest that RV infection is closely related with the individuals’HBGA phenotype but not with ABO blood type. RVs infect HBGA secretors but they do not infect non-secretors.2. Binding assay of rotavirus VP8* protein and saliva HBGA in vitro3 cases of rotavirus P [4] (GZ705) and P [8] (GZ023) positive samples were repectively selected from the very beginning, then one-step RT-PCR to amplify VP4 region, and followed by re-fidelity enzymatic for VP8* amplification. The target gene VP8* fragments were cloned into pGEX-4T-1 using the Sail and BamHI endonuclease sites. The GST-VP8* fusion protein was expressed in Escherichia coli strain BL21 (DE3) with 0.4 mM IPTG at 22℃. The fusion protein was purified using GST resign (7 seabiotech, China) according to the manufacturer’s protocol. Finally, P[8] stain GZ023 and P[4] stain GZ705 was successfully expressed.The binding assay of rotavirus VP8* protein and saliva HBGA were carried out by ELISA. We used 84 cases of saliva sample (22 cases for A,17 cases for B, O cases for 33,3 cases for AB,9 cases for non-secretor). According to the gradient experimental results of GST-VP8* (1.7-10μg/ml), we choose 10μg/ml of RV P [8], P [4] GST-VP8* protein for bingding assay. The results showed that:P [8] GST-VP8* bind to HBGA secretor. The mean OD values were as follow:A= 1.082, B= 0.486, AB= 0.590, O= 1.151. Binding ability is O> A> AB> B, while P [8] GST-VP8* can not bind non-secretor. Similar to P [8] GST-VP8*, P [4] GST-VP8* binding HBGA secretor. The mean OD values were as follow:A=0.348, B= 0.157, AB= 0.186, O= 0.334, Binding ability is A> O> AB> B, P [4] GST-VP8* can not bind non-secretor, too. The binding ability of RV P [8] GST-VP8* protein is much better than the P [4] GST-VP8* protein. This result further confirmed that RV infection is related to the bingding of its capsid protein to HBGA secretor, and may explain the reason why RV P [8] and P[4] can be major epidemic strain.Based on the findings above, we firstly confirmed RV infection is significantly associated with HBGA secretor from clinical population in China, and established binding assay of RV GST-VP8*/HBGA. We find that both P[8] and P[4] can recognize HBGA secretors but not HBGA non-secretors. The result has preliminarily proved our hypothesis that the material basis of syndrome in "same syndrome in different diseases" for RV and NoV infection may be related to HBGA. It provides an example for research on nature of syndrome (different diseases with same syndrome) in TCM, and point out the direction of HBGA as RV receptor.