Mei Ying Liu

Genomic analysis of Epstein-Barr virus in nasal and peripheral T-cell lymphoma : A comparison with nasopharyngeal carcinoma in an endemic area

The Epstein‐Barr virus (EBV) is prevalent in nasal and peripheral T‐cell lymphoma (NPTL) in Taiwan, where nasopharyngeal carcinoma (NPC) is endemic. In order to understand the pathogenesis of these two malignancies in this endemic area, genomic analysis of EBV in NPTL with comparison to NPC is important. We investigated the EBV subtype (types A and B), BamH‐I “f” variant, and the Xho‐I site mutant of the latent membrane protein‐1 (LMP‐1) gene in 19 cases of EBV‐associated NPTL and in 30 cases of NPC. EBV DNA from three patients with infectious mononucleosis (IM) was simultaneously studied as representative of normal healthy carriers. Similar to NPC and IM, the EBV in NPTL was found to belong to the type A strain in the majority (18 of 19) of cases by analyzing the 3′ divergence of EBNA‐2 genes. The extra restriction enzyme site in the BamHI‐F region (“f” variant) of EBV DNA was frequently (15 of 30) demonstrated in NPC, but only rarely (1 of 19) was it detected in NPTL and IM (0 of 3). The Xho‐I site mutant of the LMP‐1 gene previously characterized in Chinese NPC also prevailed in NPTL and IM with an identical nucleotide sequence. No correlation exists between the EBV subtype and its variants. In conclusion, type A EBV is prevalent in Taiwanese NPTL, a finding much distinct from the dominance of type B virus in nonendemic European patients. The EBV genomes in NPTL are closely similar to those in IM or normal healthy carriers, but are distinct from NPC for the infrequency of the “f” variant. The prevalence of the LMP‐1 mutant in this endemic region suggests that this EBV strain may confer a growth advantage role in the pathogenesis of these EBV‐associated diseases. The rarity of the “f” variant in NPTL and its high frequency in NPC may explain the differential tumorigenesis of different EBV strains.

Expression of the Epstein-Barr virus BHRF1 gene, a homologue of Bcl-2, in nasopharyngeal carcinoma tissue

Epstein‐Barr virus (EBV) infection is associated closely with the pathogenesis of nasopharyngeal carcinoma (NPC). The EBV gene product, BHRF1, has been demonstrated in vitro and is structurally and functionally similar to the oncogene bcl‐2, that is able to protect cells from programmed cell death. To determine whether the BHRF1 gene is expressed in vivo, BHRF1 mRNA or protein were sought in tissues from NPC and non‐NPC patients. BHRF1 transcripts were specifically detected in the NPC tumours (32 out of 44, 72.7%) rather than the non‐NPC tissues (0 out of 25) by reverse transcription, polymerase chain reaction and Southern hybridization. Other EBV genes, such as the lytic gene BZLF1 and latent genes EBNA1 and LMP2A, were also investigated. BZLF1 transcripts also were found specifically in NPC tumours (33 out of 44, 75%). EBNA1 was expressed in 79.5% of NPC, and 28% of non‐ NPC, tissues and LMP2A was expressed in 70.5% of NPC, and 88% of non‐NPC, tissues. BHRF1 protein was detected by immunohistochemistry in 4 metastatic NPC, of 36 NPC tissue sections available. The BHRF1 protein was distributed in both the nucleus and cytoplasm of the neoplastic epithelial cells. IgG antibody against the BHRF1 protein was detected in 6 of 17 (35.3%) NPC plasma, but the protein and IgG were both absent from the non‐NPC controls. BHRF1 DNA sequences were determined for 11 NPC and 3 non‐NPC samples. No sequence was specific for the EBV isolates from NPC tissue. Amino acids 79 and 88 always appeared in the same form, however, for every tested isolate and both were valine or leucine. This particular characteristic was not present in the B95‐8 strain or in the corresponding regions of homologues, Bcl‐2 and Bcl‐Xl, and was regarded as unique to Oriental EBV strains. J. Med. Virol. 61:241–250, 2000.

Familial Tendency and Risk of Nasopharyngeal Carcinoma in Taiwan: Effects of Covariates on Risk

In the present study, the authors compared the long-term risk of nasopharyngeal carcinoma (NPC) of male participants in an NPC multiplex family cohort with that of controls in a community cohort in Taiwan after adjustment for anti-Epstein-Barr virus (EBV) seromarkers and cigarette smoking. A total of 43 incident NPC cases were identified from the 1,019 males in the NPC multiplex family cohort and the 9,622 males in the community cohort, for a total of 8,061 person-years and 185,587 person-years, respectively. The adjusted hazard ratio was 6.8 (95% confidence interval (CI): 2.3, 20.1) for the multiplex family cohort compared with the community cohort. In the evaluation of anti-EBV viral capsid antigen immunoglobulin A and anti-EBV deoxyribonuclease, the adjusted hazard ratios were 2.8 (95% CI: 1.3, 6.0) and 15.1 (95% CI: 4.2, 54.1) for those positive for 1 EBV seromarker and positive for both seromarkers, respectively, compared with those negative for both EBV seromarkers. The adjusted hazard ratio was 31.0 (95% CI: 9.7, 98.7) for participants who reported a family history of NPC and who were anti-EBV-seropositive compared with individuals without such a history who were anti-EBV-seronegative. The findings suggest that both family history of NPC and anti-EBV seropositivity are important determinants of subsequent NPC risk and that the effect of family history on NPC risk cannot be fully explained by mediation through EBV serologic responses.

Molecular subtyping of human T-lymphotropic virus type I (HTLV-I) by a nested polymerase chain reaction-restriction fragment length polymorphism analysis of the envelope gene: Two distinct lineages of HTLV-I in Taiwan

The major type of human T‐lymphotropic virus type I (HTLV‐I), generally referred to as the cosmopolitan type, has been grouped into three subtypes (A, B, and C) by phylogenetic analysis of the long terminal repeat sequences of the viral genome. Twelve subtype‐specific nucleotide variations have been deduced by comparison between the envelope (env) sequences of 16 HTLV‐I strains with defined subtypes and 9 Taiwanese HTLV‐I strains. To gain further insights into the molecular epidemiology of HTLV‐I and the origin of this virus in Taiwan, a rapid method of identification for the cosmopolitan subtypes was developed by using a nested polymerase chain reaction (PCR) and subsequent restriction fragment length polymorphism (RFLP) studies using the two subtype B‐specific and four subtype C‐specific nucleotides located within the positions 5708 to 6320 of the env gene. The nested PCR‐RFLP method was used to subtype HTLV‐I from four virus‐positive cell lines derived from 1 Japanese and 3 North American patients, as well as 41 blood‐unrelated Taiwan Chinese. The sequences of PCR products were determined and the six positions of subtype‐specific nucleotide variations were examined. The sequence data completely supported the subtyping data via the nested PCR‐RFLP method. The results demonstrated that, as is the case in Japan, at least two distinct cosmopolitan subtypes (A and B) of HTLV‐I were present in Taiwan, but the more prevalent subtype in Taiwan is A in contrast to subtype B in Japan. Furthermore, rapid subtyping could facilitate molecular epidemiological studies of HTLV‐I infection and linkage between HTLV‐I subtypes and virus‐associated diseases. J Med Virol 51:25–31, 1997.

Use of bacterially-expressed antigen for detection of antibodies to the EBV-specific deoxyribonuclease in sera from patients with nasopharyngeal carcinoma

A cDNA clone, BG9, corresponding to the open reading frame BGLF5 of Epstein-Barr virus (EBV) DNase was inserted into an E. coli expression vector, pET3a, to generate a recombinant plasmid, pDNase 5. High level of expression of a DNase activity was detected in the E. coli transformed with pDNase 5 following induction with IPTG. The enzyme activity was purified using DEAE-cellulose, phosphocellulose and DNA-cellulose column chromatography. The purified protein appeared to be nearly homogeneous in SDS-PAGE using Coomassie blue staining. The requirement for divalent cations and optimum pH as well as inhibitory concentrations of ionic strength and polyamines for the purified enzyme activity were determined and seemed to be very similar to those of the enzyme activity purified from an EBV producing lymphoblastoid cell line. Using the purified enzyme as an antigen and anti-IgA as the secondary antibody, 82% (64/78) and 91% (71/78) of sera from patients with nasopharyngeal carcinoma (NPC) were shown to be positive by dot immunobinding assay and ELISA, respectively. The results suggest that purified E. coli expressed EBV DNase may be useful for preparing specific test for large scale screening of patients with NPC.

Lowered risk of nasopharyngeal carcinoma and intake of plant vitamin, fresh fish, green tea and coffee: a case-control study in Taiwan.

Background A case-control study was conducted to evaluate the role of adult diet on nasopharyngeal carcinoma (NPC) in Taiwan. Methods A total of 375 incident NPC cases and 327 controls matched to the cases on sex, age, and residence were recruited between July 1991 and December 1994. A structured questionnaire inquiring complete dietary history, socio-demographic characteristics, and other potential confounding factors was used in the personal interview. Unconditional logistic regression analysis was used to estimate multivariate-adjusted odds ratio (ORadj) with 95% confidence interval (CI) after accounting for known risk factors. Results Fresh fish (ORadj, 0.56; 95% CI, 0.38–0.83 for the highest vs. lowest tertile of intake), green tea (ORadj, 0.61; 95% CI, 0.40–0.91 for drinking ≥1 times/week vs. never) and coffee (ORadj, 0.56; 95% CI, 0.37–0.85 for drinking ≥0.5 times/week vs. never) were inversely associated with the NPC risk. No association with NPC risk was observed for the intake of meats, salted fish, fresh vegetables, fruits and milk. Intake of vitamin A from plant sources was associated with a decreased NPC risk (ORadj, 0.62; 95% CI, 0.41–0.94 for the highest vs. lowest tertile). Conclusion The study findings suggest that certain adult dietary patterns might protect against the development of NPC.

Characterization of Monoclonal Antibody to the Epstein-Barr Virus BHRF1 Protein, a Homologue of Bcl-2

A monoclonal antibody (MAb), designated 3E8, was produced against the Epstein-Barr virus BHRF1 which is a viral homologue of the anti-apoptotic protein Bcl-2. The MAb recognized the BHRF1 protein in extracts from EBV-containing cell lines after activation and EBV-negative cell lines transfected by the BHRF1 gene. Epitope mapping by Western blot analysis revealed that the antibody bound region encompassing amino acid residues 28-33 of the BHRF1. In addition to immunoblotting, the MAb could be applied widely in detection of the BHRF1 in many assays, including immunofluorescence assay, immunohistochemistry, enzyme-linked immunosorbent assay and immunoprecipitation. Most of all, when used in immunoprecipitation experiments, the MAb 3E8 showed a better effect than the existing anti-BHRF1 MAbs since radioactive isotopes were not required to intensify signals of its target antigen. Based on its great use in a variety of immunological reactions, it is a powerful tool to elucidate the biological functions of BHRF1.

Correlates of anti-EBV EBNA1 IgA positivity among unaffected relatives from nasopharyngeal carcinoma multiplex families

Background:To determine whether non-viral nasopharyngeal carcinoma (NPC) risk factors might be associated with (and mediated through) Epstein–Barr virus (EBV) serological responses linked to NPC risk, we evaluated predictors of risk of anti-EBNA1 IgA seropositivity and other markers among unaffected relatives from a large NPC family study in Taiwan.Methods:Multivariate logistic regression conditioned on family was used to examine the associations between sociodemographic, dietary, lifestyle, and occupational variables and risk of anti-EBV EBNA1 IgA positivity, anti-VCA IgA, and anti-DNase positivity.Results:Among 2393 unaffected relatives from 319 multiplex families, 1180 (49.3%) were anti-EBV EBNA1 IgA seropositive. None of the associations with anti-EBNA1 IgA were statistically significant, except for being 31–50 years of age (vs <30, adjusted ORs 0.51–0.57). For one or more EBV serological markers, there were suggestive associations for older age, GuangDong firm salted fish, betel use, current alcohol use, and male gender.Conclusion:Overall, we found little evidence to suggest that non-viral NPC risk factors significantly alter EBV serological patterns, suggesting that non-viral NPC risk factors act through pathways independent of EBV serological responses.