Epstein-Barr virus (EBV) subtype in EBV related oral squamous cell carcinoma in Okinawa, a subtropical island in southern Japan, compared with Kitakyushu and Kumamoto in mainland Japan

Abstract

Aim: In Okinawa, a subtropical island located between the East China Sea and Pacific Ocean, 2000 km south of mainland Japan, the incidence of oral squamous cell carcinoma is 1.5 times higher than that seen in mainland Japan, and a large number of these patients have been reported to be infected with the Epstein-Barr virus (EBV). This study aimed to gain a better understanding of the pathogenesis of this malignancy in this area by carrying out genomic analysis of EBV. Methods: Fifty four patients with oral squamous cell carcinoma reported from 1997 to 1999 in Okinawa were compared with 21 and 20 patients from Kitakyushu and Kumamoto in Kyushu, mainland Japan, respectively. Diagnosis was confirmed by conventional histological examination of paraffin wax sections. EBV was detected by non-isotopic in situ hybridisation (NISH) and the polymerase chain reaction (PCR) (Bam HI-F, EBV nuclear antigen 2 (EBNA2), and latent membrane protein 1 (LMP-1) regions). Sequence analysis of the PCR products was also carried out. Results: In Okinawa, 25 patients were found to be infected with EBV type A by analysing the 3′ sequence divergence of the EBNA2 genes. Six patients were positive for EBV type B, and eight for both type A and B. Therefore, type A virus infection was demonstrated in 33 of 54 patients, and type B in 14 of 54. In total, 39 of 54 patients were infected with EBV. However, the "f" variant was shown in only one patient, who was also infected with type A virus. In contrast, 97.0% of EBV type A infected patients showed a 30 bp deletion of the LMP-1 gene, but those infected with EBV type B did not. Sequence analysis of the type A virus EBNA2 gene revealed slight variations of the sequence (mutations)-48991 G→T and 48998 C→A-in 18 of 33 cases compared with the B95-8 strain, and in 14 cases, in addition to these, a further mutation of 48917 T→C was demonstrated; in the single remaining case, only one mutation at 49137 A→G was detected. The mutations at 48991 (G→T), and 49137 (A→G) are associated with amino acid changes Arg→Met and Thr→Ala, respectively. In contrast, no mutation was seen in the EBNA2 DNA from the 14 cases of type B virus when compared with that of the Jijoye strain. In Kitakyushu and Kumamoto, only 10 of 41 patients (six in Kitakyushu and four in Kumamoto) were infected with EBV. Among them, nine patients were infected with type A virus, and only one patient from Kitakyushu was infected with type B virus. The 48991 G→T and 48998 C→A mutations of the EBNA2 region were demonstrated in type A virus, but the 48917 T→C and 49137 A→G mutations were not when compared with the B95-8 strain. In the case of type B virus no mutation was noted. A 30 bp deletion was found in these nine cases of type A, but not in type B. The sequence analysis of EBV type A in Okinawa, Kitakyushu, and Kumamoto showed slight variations when compared with B95-8, but EBV type B LMP-1 did not when compared with the Jijoye strains. Conclusion: In Okinawa, EBV infection was frequently demonstrated in oral squamous cell carcinoma (p < 0.001)....

Distribution of Epstein–Barr virus genotypes in throat washings, sera, peripheral blood lymphocytes and in EBV positive tumor biopsies from Slovenian patients with nasopharyngeal carcinoma

“…DOI 10.1002/jmv patients with NPC were infected by genotype A. The only exceptions are the patients from Alaska and sporadic patients from China, where genotype B predominates [Abdel-Hamid et al, 1992;Chen et al, 1992;Choi et al, 1993;Bouzid et al, 1994;Wu et al, 1996;Higa et al, 2002]. The high incidence of genotype A in the patients with NPC may be related to its transforming ability as determined in cell culture [Rensburg et al, 2000].…”

Primary Central Nervous System Lymphoma Expressing the Human Neurotropic Polyomavirus, JC Virus, Genome

“…Of note, only five samples were negative for all JCV DNA sequences, whereas the eight samples that contained VP1 DNA also contained sequences corresponding to both T antigen and Agnogene. In addition, PCR followed by Southern blot hybridization performed with primers specific for the LMP-1 gene, as described previously, demonstrated the presence of EBV in 16 of 23 samples (70%) (9,22). All samples were negative for SV40 and BKV T-antigen sequences as determined by Southern blot hybridization with specific probes performed on blots containing sequences amplified with the Pep primers.…”

“…DNA was extracted from several 10-m sections from the tissue samples by using the QIAamp tissue kit according to the manufacturer's instructions (Qiagen, Valencia, Calif.). PCR amplification was performed on DNA extracted from the tumors with four individual sets of primers: Pep1 and Pep2, which amplify sequences (nucleotides [nt] 4255 to 4274 and nt 4408 to 4427, respectively) in the N-terminal regions of the T antigen of the polyomaviruses JCV, BK virus (BKV), and simian virus 40 (SV40); VP2 and VP3, which amplify regions of the JCV VP1 capsid protein (nt 1828 to 1848 and nt 2019 to 2039, respectively, of JCV); Agno1 and Agno2, which amplify sequences within the coding region of the JCV Agnogene (nt 279 to 298 and nt 438 to 458, respectively); and LMP1 and LMP2, which amplify portions of the EBV LMP1 gene (nt 168190 to 168209 and nt 168350 to 168331) (9,22). Amplification was carried out on 500 ng of template DNA with Failsafe Taq polymerase in Failsafe Buffer B (Epicenter) in a total volume of 50 l in the presence of 0.5 nM concentrations of the primers.…”

Epstein-Barr Virus Genotypes and Strains in Central Nervous System Demyelinating Disease and Epstein-Barr Virus-Related Illnesses in Australia

“…EBV genotype characterisation is important as epidemiological studies have shown that genotypes are geographically linked and can be associated with specific disease syndromes [12,28]. In this study, a real-time PCR method coupled with DNA sequence analysis was developed to identify EBV genotypes (A and B) and strains circulating in individuals with and without a first clinical diagnosis of CNS demyelination and EBV-related diseases in Australia.…”

“…Notwithstanding this general geographical variation, there is more localised regional variation in relation to specific diseases. For example, in Okinawa, genotype B is ten times more common in oral squamous cell carcinoma than on mainland Japan [12]. In Australia, EBV genotypes have been characterised in patients with MS [13], Hodgkin’s disease [14], HIV infection [15] and in healthy individuals [16].…”