Five distinct Epstein-Barr virus (EBV)-deterinned nuclear antigens (EBNA-1 to were recently identified. Antibody responses to these antigens could conceivably differ, and thus prove of serodiagnostic value, in EBVassociated disease processes. As a rst step, murine or human cell lines transfected with appropriate EBV DNA fragments and stably expressing either EBNA-1 or EBNA-2 were used to determine the frequency and time of emergence of antibodies to these two antigens in the course of acute and chronic infectious mononucleosis (IM) and to assess their titers in so-called chronic active EBV infections. Following IM, pntibodies to EBNA-2 arose first and, after reaching peak titers, declined again in time to lower persistent or even nondetectable levels. Antibodies to EBNA-1 emerged several weeks or months after anti-EBNA-2 and gradually attained the titers at which they persisted indefinitely. The ratios between the anti-EBNA-1 and anti-EBNA-2 titers therefore were generally well below 1.0 during the first 6-12 months after IM and turned to well above 1.0 during the second year. In clear cases of chronic IM, the inversion of this ratio was delayed or prevented. In the less well-defined chronic EBV infections, low ratios were observed in only some of the patients. Because many of these illnesses were not ushered in by a proven IM and often showed EBV-specific antibody profiles within the normally expected range, a causal role of the virus in these cases remains doubtful.

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The pathogenesis of spontaneous abortion is complex, presumably involving the interaction of several genetic and environmental factors. The methylenetetrahydrofolate reductase (MTHFR) gene C677T and A1298C polymorphisms are commonly associated with defects in folate dependent homocysteine metabolism and have been implicated as risk factors for recurrent embryo loss in early pregnancy. In the present study we have determined the prevalence of combined MTHFR C677T and A1298C polymorphisms in DNA samples from spontaneously aborted embryos (foetal death between sixth and twentieth week after conception) and adult controls using solid-phase minisequencing technique. There was a significant odds ratio of 14.2 (95% CI 1.78-113) in spontaneously aborted embryos comparing the prevalence of one or more 677T and 1298C alleles vs the wild type combined genotype (677CC/1298AA), indicating that the MTHFR polymorphisms may have a major impact on foetal survival. Combined 677CT/1298CC, 677TT/1298AC or 677TT/1298CC genotypes, which contain three or four mutant alleles, were not detected in any of the groups, suggesting complete linkage disequilibrium between the two polymorphisms. The present finding of high prevalence of mutated MTHFR genotypes in spontaneously aborted embryos emphasises the potential protective role of periconceptional folic acid supplementation.

The association of Epstein-Barr virus (EBV) with nasopharyngeal carcinoma (NPC) has been known for some time, but the precise role of EBV in this cancer is poorly understood, due partly to the lack of an in vitro system for studying NPC cells and the effect of EBV on epithelial cells. Biopsies of NPC tumours have revealed expression of the EBV latent membrane protein (LMP) in 65% of cases, suggesting that in at least some NPC tumours LMP may contribute to cell transformation. Here we address the question of the effect of LMP expression on epithelial cells. Transfection of an immortalized, non-tumorigenic keratinocyte cell line (RHEK-1) with the LMP gene causes a striking morphological transformation: the originally flat, polygonal colonies change to bundles of spindle-shaped cells that form multilayer foci, and cytokeratin expression is down-regulated. Our results suggest that LMP expression may be an important causal factor in the development of NPC.

Previous studies suggest that the EpsteinBarr virus nuclear antigen EBNA2 participates in the regulation of the expression of the viral latent membrane protein (LMP). We have used reporter plasmids containing DNA fragments of the 5' flanking region of the LMP gene in cotransfection experiments to analyze the effect of EBNA2 on the activity of the LMP promoter. The results show that the LMP promoter is controlled by positive and negative transcription elements in a DNA fragment that contains the LMP transcription initiation site and 634 base pairs of upstream sequences. The promoter is activated by EBNA2. The region between position -54 and +40 relative to the mRNA cap site contains a positive transcription element that is constitutively active in DG75 cells and independent of EBNA2. The -106 to -54 region contains a negative regulatory element that prevents adjacent positive elements from functioning in the absence of EBNA2. Regulatory sequences between -324 and -144 participate in maintaining a high level of transcription of the LMP promoter after induction with EBNA2. The regulatory elements in the -634 to -54 promoter region have the characteristics of an inducible enhancer, including orientation independence and ability to regulate a heterologous promoter. In this context it might be relevant that transfected EBNA2 could induce the expression of the activation antigen CD23 on the surface of an EBV-negative Burkitt lymphoma (BL) cell line (6). The CD23 antigen is related, to the receptor for a B-cell growth factor (7) and has also been suggested to act as an autocrine growth factor for B cells after shedding from the cell surface (8). Epstein-Barr virus (EBVEBNA2 is a phosphorylated polypeptide with DNAbinding properties, which is encoded by the BYRF1 reading frame of the BamHI WYH region of the EBV genome (reviewed in ref. 1). The protein (the A subtype) has a rather unusual primary structure containing an almost continuous sequence of 40 proline residues, an arginine-and glycine-rich positively charged region, and a negatively charged Cterminal sequence. The overall proline content of the 487-amino acid long polypeptide is 29%. The recent demonstration of a new class of transcriptional activators with a proline-rich domain might provide some clues concerning the action of EBNA2 (9).It is thus conceivable that EBNA2 modulates the cell phenotype by influencing the expression of cellular genes. The effect might be direct, as suggested by the induction of CD23 in EBNA2-transfected cells (6), or indirect and mediated by way of other EBV genes, whose expression is in turn controlled by EBNA2. Evidence for the latter mechanism has been provided recently. The B95-8 virus strain, but not P3HR-1, has been shown to be able to induce expression of LMP in EBV-negative BL cell lines (10). Transfection of the EBNA2 gene into P3HR-1 virus-converted B-lymphoma cell lines induced the expression of LMP and a dramatic change in growth phenotype toward a LCL-like pattern (11). Some, but not all, of these changes could b...