Epstein-barr Virus-associated Gastric Virus Gene Polymorphism

Objective:In order to elucidate the association of various Epstein-Barr Virus(EBV) polymorphisms with diseases or regions,it is important to investigate the distribution of EBV polymorphisms in EBV associated diseases and comparable healthy controls from various regions all over the world.However,molecular epidemiological studies to map the distribution of these subtypes in Epstein-Barr virus associated gastric carcinoma(EBVaGC) are still limited,especially regarding EBVaGC in China, despite China being one of the countries with a high incidence of gastric carcinoma. The purpose of this study was to characterize viral gene polymorphosims at the four genotyping sites and several latent or lytic genes by analyzing specific EBV polymorphic variants in EBVaGC cases and healthy controls from nasopharyngeal carcinoma(NPC) nonendemic area of China.EBV positive specimens from Shandong province were screened and the EBVaGC was characterized by the clinic data.The EBVaGCs were examined for type 1/2 EBV and the virus polymorphism at Bam HI F and Bam HI W1/I1 boundary regions and Xho I restriction site in latent membrane protein(LMP) 1 gene.The sequence variations of LMP1 and 2A,together with the virus lytic infection genes,BHRF1 and BARF1 were also determined in Shandong EBVaGC in the present study.Local throat washings(TWs) from healthy adults were explored as controls to analyze the relationship between EBV gene polymorphism and EBVaGC.Methods 362 fresh gastric carcinoma and 639 formalin-fixation and paraffin-embedding(FFPE) gastric carcinoma tissues were collected from Shandong province and tested for EBV genome by polymerase chain reaction(PCR)-Southern analysis.EBV-encoded small RNA1(EBER1) of the PCR positive specimens was detected by in situ hybridization(ISH) to confirm EBVaGC.EBV positive TWs were sceened from 260 blood donors by PCR-Southern analysis.PCR-RFLP,nested-PCR and DNA sequencing were explored to determine the virus genotypes in EBV positive specimens,including type 1/2 EBV and the virus polymorphisms at Bam HI F and Bam HI W1/I1 boundary regions and Xho I restriction site in LMP1 gene.Besides,the gene polymorphisms of LMP1,LMP2A,BHRF1 and BARF1 were also determined. The virus genotypes and sequences were compared among the groups,B95-8 strain as a reference,and analyzed together with the published EBV variation data as well,in order to investigate the association between virus gene polymorphisms and EBVaGC.Results①There were 63 EBV positive samples in 1001 gastric carcinomas(6.29%) and 60 in 260 TWs(23.08%).②The statistical analysis showed that age,location of cancer and pathological differentiation were different between EBV-negative gastric carcinomas(EBVnGC) and EBVaGC.EBVaGCs have a higher frequency in＜60 population(9.21%) than in≥60 population(3.96%)(x~2 =11.581,P=0.0007),a higher frequency in cardia and fundus(7.28%) than in pyloric antrum(3.99%)(x~2 =4.684,P=0.030),and a higher frequency in lowly differentiated adenocarcinoma than in other pathological types(x~2 =19.797,P=0.0005).Gastric stump cancer showed a high EBV infection rate of 33.3%.However,sex,clinical stages and lymph node metastasis were not different between the two groups.③The rates(%) of EBV type 1/2,type F/f,type I/i and LMP1 Xho I(+)/(-) polymorphism in EBVaGC are 92.2/7.8,92.3/7.7,13.5/86.5 and 10.2/89.8, respectively.The corresponding data from TWs are 82.8/17.2,94.7/5.3,43.9/56.1 and 47.4/52.6,respectively.The type i and LMP1 Xho I(-) were detected with much higher rates in EBVaGCs than in TWs,while the distributions of type 1/2 and type F/f between EBVaGC and TWs are not significantly different.Among those specimens with determinable genotypes through all the polymorphisms,the distribution rate of type \IXho I(-) in EBVaGC(77.6%) was significantly higher than in TWs(52.6%)(x~2 =7.115,P=0.008).④Among the available LMP1 sequences of 23 EBVaGCs,19 were perfect China 1 strain.This strain also accounts 18 of the 29 available healthy TWs,with no significantly distribution difference between the two groups(x~2=2.64,P=0.1064). Besides,the TWs showed several strains other than China 1,including 6 Med-,1 China 2,1 NC,and other 3 isolates showed mutant types beyond all the determined strains.Nineteen of 23 EBVaGCs and 22 of 29 TWs had a consensus 30bp deletion to the AA 343-352 of B95-8 strain(x~2=0.062,P=O.803).The GC isolates had significantly higher mutant rate at AA326(E→Q) than the TWs(x~2=23.284, PO.0001).⑤The LMP2A showed a sequence polymorphism with 30 AA mutations to the prototype B95-8 strain,resulting in alterations to 9 CTL epitopes,and 4 of those epitope variations were reported for the first time.Four mutations were shared by all the specimens.LMP2A exonl showed an inconsistent pattern of polymorphism to exon 2-8.Exon 1 sequence showed no significantly different ditribution of the main variation types among the three groups,while the main variation Group 1 of exon 2-8 showed significantly different distribution among groups.The important motifs in exon 1 were strictly conserved in all specimens.⑥The BARF1 and BHRF1 genes were both highly conserved in all the specimens, with no significant difference between the GC isolates and healthy TWs iaolates.The few changes of BHRF1 protein were predominantly clustered near the amino terminus of the BHRF1 protein outside conserved domains identified in the Bcl-2 homologues.Conclusion①EBV infection is involved in the tumorigenesis of 6.29%gastric carcinoma.EBV infection is related to age,pathological type and location of cancer.②Our work suggested that the EBV strain i/Xho I(-) was associated with Shandong EBVaGC.③China 1 strain,which has several characteristic sequence changes in LMP1 gene, was for the first time to be reported as the prevalence strain in both EBVaGC and healthy adults of Shandong province.EBVaGC isolates have significantly higher mutant rate at AA326(E→Q) than the healthy controls.④The whole LMP2A sequence was for the first time analyzed and grouped. Furthermore,the variation groups were associated with region or disease.The EBV isolates in Shandong province have some polymorphism in LMP2A sequence.Some of the mutations are region-restricted,especially the variation types in exon 1 and the four shared mutations which are characteristic to Chinese EBV isolates.However,the main type,Group 1,in exon 2-8 is thought to be associated with EBVaGC.⑤Both virus lytic infection genes,BARF1 and BHRF1,are highly conserved in all the specimens,indicating the critic role of these genes in remaning the virus foundamental biological activity.