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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.

Antibodies to Epstein-Barr-virus-specific DNase in children under 15 years old from Taipei, Taiwan were determined by an enzyme neutralization test. Serum was taken as positive for the anti-DNase antibody when 1 ml of serum could neutralizeor = 2 units of the DNase activity. Among the 1,292 sera tested, 5.6% of the children were shown to have anti-EBV DNase antibodies, a result similar to that for adults. Maternal antibodies against the EBV DNase were found to decrease very rapidly 2 months after the child's birth, and new antibody appeared upon natural EBV infection.

Two cDNA clones covering the N- and C-terminal portions of the EBV BXLFI open reading frame were selected from a cDNA library derived from P3HR1 cells. The two clones were ligated, the N-terminal untranslated region truncated, and the product inserted into an E. coli expression vector, pET3CP°. The fusion protein was expressed under control of the T7 phage phi 10 gene promoter and shown to possess thymidine kinase activity. The protein was then used as an antigen to detect antibody reactivities in serum samples of nasopharyngeal carcinoma patients and healthy blood donors. Using a 1 :400 dilution of serum samples in Western blot analyses, it was possible to differentiate the reactivities of serum IgA of NPC patients and healthy donors. The prevalence of positive reactivity to EBV TK in NPC was around 84%. The test was compared to others used for early diagnosis of NPC and was able to detect some patients who were negative in those tests. © 1992 Wiley-Liss, Inc.

Serum samples from 154 patients with nasopharyngeal carcinoma (NPC), 374 with other cancers, 1,000 normal controls from Government Employees' Clinic Center (GECC), and 3,642 individuals of various ethnic-dialect groups living in high-risk areas for NPC were collected and the concentration of antibodies to Epstein-Barr virus (EBV)-specific DNase activity was determined. Taking a serum sample where 1 ml will neutralize two or more units of the DNase activity as positive, 2-4 units as low level, 4-6 units as medium level, and more than 6 units as a high level of antibody, 90.3% of the NPC patients contained significant amounts of antibodies to EBV-specific DNase activity and most of those had high levels of the antibody. In contrast, only 11% of sera from patients with cancers other than NPC contained antibodies to EBV-specific DNase activity, and high levels were very rare (2.1%). The difference in positive rates between these two groups is highly significant according to the chi 2 test (P less than 0.001). The positive rate of this antibody in the control group (GECC) was 5.3% with 0.0%, 0.8%, and 4.5% having high, medium, and low levels of antibodies, respectively. Again, the difference in positive rates between the GECC group and the NPC group is statistically significant (P less than 0.001). Taken separately, the positive rates of anti-EBV DNase activity in the three high-risk groups were 11.7%, 13.0%, and 13.1%. No significant difference in age distribution for the levels of this antibody was observed in the control GECC group or the three high-risk groups. However, the positive rates of the three high-risk groups are more than twice those of the GECC group (11.7% approximately 13.1% vs 5.3%). This ratio coincides with the ratio of the probability of developing NPC in high-risk groups compared to that of the GECC group (also more than two times). The significance of this coincidence is discussed.