Establishment Of Tree Shrew Animal Model Infected By EB Virus From Nasopharyngeal Carcinoma Patients

Objective:As a kind of tumor virus,Epstein-Barr virus(EBV)plays critical role in the generation of carcinomas,including Burkett's lymphoma,nasopharyngeal carcinoma(NPC),lung cancer,stomach tumor and so on.However,researches about the relationship between EBV and NPC is not going well,since the lack of effective cell and animal models.Although it has been proved that EBV spreads widely in crowed and some EBV infectious animal models has been built,the precise role of EBV in the development of NPC is still uncertain.Therefore,it is critical to establish a kind of efficacious animal model infected by EBV from NPC patients.In order to achieve the objective,we designed an experiment including five parts,which are performed as follows.Methods:1.The investigation of EBV DNA copy number and EBV particles in nasopharyngeal carcinoma patients:The nasopharyngeal biopsy tissues were collected from 10 NPC patients,whose serum EBVCA-IgA and EBNA-IgA were positive identified by Clinical Laboratory of The First Affiliated Hospital of Guangxi Medical University.There were 20ml anticoagulant peripheral blood and 10ml non-anticoagulant peripheral blood been collected.Tissues were brokenanddilutedinto2mlsuspension.Bothanticoagulantand non-anticoagulant blood were used to extract mononuclear cell(PBMC)and serum,which were also concentrated into 2ml.There were 200?l liquids from these three samples were used for extraction of DNA.The copy number of EBV DNA in each sample was determined by fluorescence quantitative PCR.At the same time,transmission electron microscope(TEM)was applied to explore the existence of intact EBV viral particles,then labeled the virus particles with EBV gp340/220 envelope protein antibody and colloidal gold particles and observed them by transmission electron microscopy.2.The exploration of EBV DNA copy number for the effective infection of Tree Shrews:The NPC tissue suspensions were diluted into 2×10~6,2×10~4,2×10~2,2×10~0 copies/?l according to the copy number of EBV DNA in the first part.Each group was inoculated with 5 tree shrews by nasal spray.Blood samples were collected from tree shrews in certain time(the 1~stt week,the 2~ndd week,the3~rdd week,the 1~stt month,the 2~ndd month).Following a 2-month observation,after EBV changed into latent infection,20mg/Kg of cyclophosphamidum was injected into enterocoelic for immunosuppressant.And then,3-week continued observation to survey EBV-relative gene fragments and the level of EBVCA-IgG in serum for the confirmation of infection effect.3.Establishment of tree shrew animal model infected by EBV in a long term and the analysis of infection stages of EBV in vivo:There were 10 tree shrews randomly assigned to the experimental group and 3 tree shrews in control group,without gender consideration.The experimental group tree shrews were infected with EBV virus suspension from NPC tissue by nasal spray,while the same volume of saline in the control group.EBV DNA copy number in peripheral blood PBMC of all tree shrew was monitored regularly by RT-PCR detection.Further,enzyme-linked immunosorbent assay(ELISA)was used for testing EBVCA-IgG level in serum.The expression of EBERs in organs of tree shrews was detected by ISH.Analysis of different infection stages of EBV in tree shrews:RT-PCR detection was applied to evaluate the expression of EBV-relative gene fragments,including BARF1,BHRF1,LMP1 and EBNA1,since they present for the lytic infection and latent infection stages in the lifetime of EBV.4.BARF1 gene sequencing:Methods including RT-PCR and cDNA gene sequencing were used to detect the BARF1 gene and to compare with the sequence results of Human herpes virus(HHV-4),nasopharyngeal carcinoma(HHNPC1-9),lymphoma(B95-8)and gastric cancer(Akata-GC1)strains in the published literature,for finding the virus genotypes of the tree shrews infected.5.Preliminary study on tumor genesis of EBV infection induced by cyclophosphamide:By using cyclophosphamide to disturb body immunity,tree shrews were fad continually after one week of cyclophosphamide application.All of the animals were sacrificed after 3 months,and part of their nasopharynx,spleen,liver,lung and stomach tissues were embedded in paraffin wax.Then,tissues were sliced into 4um which were stained by HE and Immunochemistry marking by EBNA1,LMP1,EA and BZLF1 monoclonal antibodies.These slices were also strained by hybridization in situ for detecting the expression of EBERs.Finally,they were observed by light microscope.Results:1.There is the highest EBV DNA copy number in NPC tissue,and EBV particles could be observed under TEM and immune TEM.Through qPCR detection of EBV DNA copy number in NPC tissue,PBMC and serum,there were the highest copy number of EBV DNA in NPC tissue,which was(14.3±5.41)×10~7 copies/DNA,while only(1.58±1.04)×10~4 and(11.6±2.96)×10~2 copies/DNA in PBMC and serum,respectively.Besides,under TEM,herpesvirus like granules were observed,which could be labed by EBV gp340/220 envelope protein specific antibody and marked by colloidal gold particles(diameter=8nm).2.In this experiment,the critical value of EBV DNA copy number for tree shrews infected by EBV was 2×10~2 copies.Through the RT-PCR detection of the expression of genes,like BamHI-W,BZLF1,BARF1,BHRF1,LMP1,EBNA1 in PBMC of tree shrews,the ten tree shrews in group A and group B were clearly infected with EBV.And EBV-relative gene fragments expressed in the first week,while that of C group expressed later.Meanwhile,there were strong expression of EBV-relative gene fragments in group A and group B,but some weak expression in group C.Interestingly,there were two tree shrews infected by EBV,but both of their EBV genes showed a weak expression.After infected two months,their immunity were suppressed by cyclophosphamide,which actived EBV in vivo,showing a high expression of BamHI-W.By contrast,even suppressed the immunity,tree shrews in D group never showed any expression of EBV-relate genes.In addition,the level of EBVCA-IgG increased after infected with EBV suspenant in group A and group B.However,the two tree shrews in D group did not show any upward in the level of EBVCA-IgG.Therefore,we argue that the critical value of EBV DNA copy number for tree shrews infection is 2×10~2 copies,and the best effects of EBV infection in vivo is the B group,which was infected with 2×10~4 copies.We also confirm that the use of immunosuppressive agent can induce EBV change from latent infection to lytic infection.3.Tree shrews could be infected by EBV from NPC patients'tumor tissues for establishing a long time infection,and the time of lytic infection occurred irregularly.During the 10-month observation,tree shrews that lived longer than four weeks were detected the expression of EBV relative gene fragments through RT-PCR.The expression of BARF1,BHRF1,LMP1 and EBNA1showed a significantly increase,which proved that all tree shrews were infected by EBV.With the change of copy number of EBV DNA in tree shrews,the level of EBVCA-IgG also changed obviously.Meanwhile,EBNA1,EBNA2 and LMP1 expression in nasopharyngeal tissue of tree shrew could be detected by immunohistochemical method.The expression of EBNA1 and EBNA2 proteins in spleen and/or liver was detected by Western blot.ISH was efficient method for detecting EBERs in spleen,liver,lung and stomach tissues of tree shrews.Therefore,the results suggest that EBV might exist in tissues of nasopharynx,spleen,liver,lung and stomach.Then,cDNA from dead tree shrews'spleen,liver,lung and stomach were examined by RT-PCR.The expression of BamHI-W and BZLF1 were tested in some tissues.During EBV lifetime in vivo,EBVCA-IgG level showed an obvious high titer comparing with control group?Interestingly,according to the analysis of EBV infection stage based on RT-PCR results,the expression of lytic genes(BARF1 and BHRF1)and latent genes(LMP1 and EBNA1)were detected in tree shrews,which suggested that there were latent infection and lytic infection in these tree shrews.4.The results of gene sequencing:By analyzing the RT-PCR results of tree shrews infected for a long time in Chapter 3,it was found that the early gene BARF1 of EBV expressed the best in the whole observation population and observation cycles.Compared with EBNA1,LMP1 and BHRF1 gene fragments,BARF1 might be the best one to represent infection status of EBV in vivo.Therefore,BARF1was chosen for gene sequencing.The result of gene sequencing showed that BARF1 gene sequences could be compared with the subtype strains of EBV from NCBI GENE bank,which indirectly confirmed that tree shrews in experimental group were infected by EBV.Additionally,according to phylogenetic tree analysis,it was found that B1,B3,B6 and B8were infected by HHV-4 subtype.The subset of EBV strain infected in B5,B7and B9 was close to subtypes out of standard types presenting in this article.HNNPC1 EBV subtype might be the strain which infected in B2 and B10.5.Preliminary study on tumor genesis of tree shrew model induced by EBV infection and cyclophosphamide:Through the application of cyclophosphamide,there were 6 tree shrews from experimental group showing inflammatory changes in the nasopharyngeal HE staining section.Although some lymphocytes and plasma cells were found,the abnormal proliferation or carcinogenesis of nasopharyngeal epithelial cells has been observed.Cyclophosphamide could promote the expression of EBERs in spleen and liver,but had no effect on the expression of ebers in lung and stomach.In addition,no positive expression of EBERs was found in the nasopharyngeal lymphatic follicles of tree shrews.The expressions of EBNA1,LMP1,EA and BZLF1in nasopharynx,spleen,liver,lung and stomach of tree shrews were observed by immunohistochemistry.It proved that whether cyclophosphamide was applied or not,latent and lytic proteins of EBV could present at the same time.It also indicated that once been infected with EBV,there could be both latent infection and lytic infection.This might provides a new point of view for further exploring the existence of EB virus in the organism.Conclusion:Tree shrews could be infected by EBV from patients with nasopharyngeal carcinoma.It was indirectly confirmed that there were infectious EBV granules in the tissues of patients with NPC.Further,the minimum copy number of EBV DNA in tree shrews infected with EBV was 2×10~2 copies.And,when tree shrews received EBV DNA copy number was 2×10~4 copies,the infection effects in group B was the best comparing with other groups in this experiment.Besides,in NPC patients'tumor tissues,peripheral blood mononuclear cells and serum,where the EBV derived from,could abstract infectious EB virus to form tree shrew animal model.With 10-month of observation,the reaction of tree shrews to EB virus were clearly reflected in the animal model,which were constructed successfully by us.For the importance of animal model in both science and nature,we argue that the establishment of tree shrew animal model infected with EBV will provide precious accordance for further research about the anti-virus medicines and vaccines,and for the exploration of relationship between EBV and the generation and development of NPC.Gene sequencing was used to type EBV,which laid a foundation for further study of EBV subtype in nasopharyngeal carcinoma in Guangxi.Through the exploration of the method of inducing tumor by immunosuppressant,although no malignant changes such as abnormal tissue structure or abnormal nucleus were observed,the experimental results confirmed that immunosuppressant could promote the latent presence of EB virus in the spleen of tree shrews.B cells infected by EB virus can migrate to liver with blood flow,and finally to nasopharynx,lung and stomach to cause inflammatory lesions.These findings may provide a basis for the further study of the infection characteristics of EB virus and the tumorigenic mechanism of EB virus.