Characterization Of Epstein-Barr Virus Type1Nuclear Antigen3C Sequence Patterns Of Nasopharyngeal And Gastric Carcinomas In Northern China

Objective Epstein-Barr virus (EBV) is a human herpesvirus associated with numerous human cancers, including Burkitt’s lymphoma (BL), Hodgkin’s disease (HD), nasopharyngeal carcinoma (NPC), gastric carcinoma (GC), and breast carcinoma. In previous studies, over90%of the human population had EBV infection. It is said that the distribution of EBV associated cancers has the character of regional. This raises the possibility that particular EBV strains may contribute to the development of specific EBV-associated malignancies. In this study, EBV positive NPC, GC and throat washing samples of healthy donor (TW) in northern China were chosen for gene polymorphism study and analysis in order to elustrate the association of EBV gene polymorphism with the happeness of NPC and GC.Mehods①polymerase chain reaction (PCR) and DNA sequencing were chosen for the EBNA3C polymorphism study in EBV positive NPC, GC and TW. After sequencing, the sequences were compared with EBV prototype B95-8by DNAStar5.0.②All sequences were aligned and categorized by their consensus changes. One sequence was chosen to represent the samples which have identical sequences. Alignments between representative sequences were performed using MegAlign in DNA Star software (DNASTAR, Inc, version5.0), and the phylogenetic tree was drawn using Clastal W method.③To analyze the variations of EBNA3C and to investigate the association between the gene variations with function influence.④The distribution of EBV EBNA3C subtypes in EBV-associated gastric carcinoma (EBVaGCs), NPCs and TWs from healthy donors were compared and analyzed statistically by Fisher exact test. We also compared our results with previous studies in order to find the variations that have the character of northern China.Results EBNA3C gene was successfully amplified from103samples including26EBVaGCs,50NPCs and27TWs. Based on the phylogenetic tree, all isolates can be divided into3subtypes, named3C-5,3C-6and3C-12respectively according to the common mutations in each pattern.①3C-6sequences were found in most isolates, including17/26(65.4%) EBVaGCs,25/50(50.0%) NPCs and14/27(51.9%) TWs. Six consensus nucleotide sequence changes were identified from this subtype compared with prototype B95-8, including655(Gly→Ser),677(Thr→Met),683(Ala→Val),687 (Cys→Thr),701(Glu→Gln) and246(gta→gtg).②3C-12subtype was the second most subtype that detected in8/26(30.8%) EBVaGCs,11/50(22.0%) NPCs and6/27(22.2%) TWs. There were12common mutations, including AA208(gcc→gct),213(Gln-His),336(Glu→Asp),337(cac→cat),AA348(Ile→Leu), AA367(gaa→gag),370(agt→agc),414(aag→aaa),467(ccg→cct),656(Arg→Gly),687(Cys→Thr),701(Glu→Gln).③3C-5subtype was detected in1/26(3.8%) EBVaGCs,14/50(28.0%) NPCs and7/27(25.9%) TWs. There were5common sequence changes for this subtype, including AA348(Ile→Leu), AA367(gaa→gag),370(agt→agc),687(Cys→Thr),701(Glu→Gln).④Variations of epitope. Four of the8epitopes had AA changes in partial isolates, including QNG (213Gln→His), LRG (254Gln→His), KEH (336Glu→Asp), FRK (348Ile→Leu), the others are strictly conserved.⑤The distribution of EBNA3C subtypes among EBVaGCs, NPCs and healthy donors was not significantly different (P=0.17).Conclusion①3C-6is the predominant subtype (54.4%,56/103) of EBNA3C in Northern China, the mutations between our study with Southern China and Japan were similar.②The distribution of EBNA3C subtypes among EBVaGCs, NPCs and healthy donors was not significantly different, suggesting that EBNA3C polymorphism exhibits geographical differences but no differences in EBVaGCs and NPCs compared with healthy controls.③In our study we identified a new subtype named3C-5(21.4%,22/103) which has not been reported previous, and may serve as a specific subtype for Northern China.④Four of the8epitopes that sequenced occurred AA changes (AA213,254,336,348), the others are strictly conserved. These epitope changes could block either correct endogenous processing of the antigen, major histocompatibility complex (MHC) binding, and/or optimal recognition of the peptide-MHC complex by T-cell receptors.