The sequence of the latent membrane protein 1 (LMP-1) gene was analysed in Epstein-Barr virus (EBV) isolates from specific regions representing both type 1 and type 2 EBV. A predominant strain marked by an XhoI restriction enzyme polymorphism (REP) within the LMP-1 gene has been identified in type 1 EBV in nasopharyngeal carcinoma (NPC) from Southern China. This polymorphism was also present in type 2 EBV in NPC from Alaska. In this study, the sequence of the LMP-1 gene was determined in these samples representing type 1 and type 2 EBV and was compared with the prototype lymphoid strains. Consistent nucleotide variation in the amino terminus of LMP-1 was identified in strains marked by the XhoI REP. These changes were present in both EBV type 1 and type 2 strains. Three types of sequence variation were detected in the carboxy terminus of LMP-1. The LMP-1 sequences differed in the number of an 11 amino acid repeat element. In the prototype EBV type 1 (B95-8) sequence and in the type 1 Raji and type 2 HR-1 strains, the third repeat element contained an insertion of 5 amino acids that were also the first five unique amino acids after the last partial repeat element. The third variation was a deletion of amino acids 343 to 352 of the B95-8 LMP-1. This deletion was detected in the type 1 Chinese EBV strains, but was not detected in the type 2 Alaskan strains although the Chinese and Alaskan strains have nearly identical amino acid changes at the amino terminus. Numerous other amino acid changes were detected in the carboxy terminus which did not cosegregate with either EBV type, amino acid changes in the amino terminus, or specific geographic regions. These data indicate that EBV strains can be distinguished by sequence differences within LMP-1 and that unlike the divergence between type 1 and type 2 EBV in Epstein-Barr nuclear antigen sequences, different EBV types are nearly identical in LMP-1 sequence.

The aged immune system is characterized by clonal expansions of CD8+ T cells of which a substantial portion are directed against Epstein-Barr virus (EBV) and cytomegalovirus (CMV). It is unknown if these expansions represent increased viral reactivation or simply reflect an accumulation over time. We investigated herpesvirus reactivation in young and old subjects co-infected with CMV and EBV. Using molecular and serological techniques, we found significant increases in both the frequency and magnitude of EBV and CMV reactivation in elderly subjects. CMV DNA was frequently detected in the urine of elderly subjects; EBV load in peripheral blood was also significantly increased. Notably, EBV DNA in plasma was detected in a majority of the elderly subjects which was supported by frequent transcription of late structural genes. Furthermore, CD8+ T cells specific for EBV structural antigens were detected in samples from the elderly. Samples from our younger control group were negative for EBV DNA in plasma, CMV DNA in urine, expression of structural transcripts, and lacked CD8+ T cells specific for EBV structural antigens. These findings indicate that the aged immune system is no longer able to control EBV and CMV reactivation that could now be characterized as chronic instead of latent.

The phylogeny and evolution of Epstein-Barr virus (EBV) genetic variation are poorly understood. EBV latent membrane protein-1 (LMP-1) gene sequences are especially heterogeneous and may be useful as a tool for EBV genotype identification. Therefore, LMP-1 sequences obtained directly from EBV-infected human tissues were examined by PCR amplification and cloning. EBV genotypes were defined as "strains" from among 22 identified LMP-1 sequence patterns. Three molecular mechanisms were identified by which genetic diversity arises in the LMP-1 gene: point mutation, sequence deletion or duplication, and homologous recombination. The rate of LMP-1 gene evolution was found to be accelerated by coinfection with multiple EBV strains. The results of this study refine our understanding of LMP-1 sequence variation and enable accurate discrimination between independent EBV infection events and the consequence of intrahost EBV evolution. Thus, this LMP-1 sequence-based approach to EBV molecular epidemiology will facilitate the study of intrahost EBV infection, coinfection, and persistence.

We employed a newly developed genotyping technique with direct representational detection of LMP-1 gene sequences to study the molecular epidemiology of Epstein-Barr virus (EBV) infection in healthy individuals. Infections with up to five different EBV genotypes were found in two of nine individuals studied. These results support the hypothesis that multiple EBV infections of healthy individuals are common. The implications for the development of an EBV vaccine are discussed.Multiple Epstein-Barr virus (EBV) infections are common among immunocompromised individuals (21,29,31,39,41,43,44,46), but the origin of the multiple EBV strains remains a mystery. Multiple EBV strains could accumulate as superinfections in individuals who have lost previous protective immunity to EBV. Alternatively, they could represent the reactivation of latent EBV strains that were acquired prior to the onset of immunodeficiency. The reported prevalence of multiple EBV infections in healthy individuals ranges broadly between 0 and 100% (Table 1) (4,7,13,16,19,20,23,30,32,35,38,45; M. L. Lung and R. S. Chang, Letter, J. Infect. Dis. 162:994-995, 1990), but differences among these studies in the molecular detection and definition of an EBV strain confound the interpretation of their results.Molecular epidemiologic studies requiring EBV isolation by B-lymphocyte transformation (16,19,23,45; Lung and Chang, letter) suffer from selection bias toward transformation-competent EBV isolates (10,27,33). PCR amplification directly detects the EBV genome and avoids transformation selection bias, but the genetic definition of an EBV strain has been inconsistent across studies. Restriction fragment length polymorphisms detect either point mutations within restriction enzyme cleavage sites or variations of large repetitive regions within genome fragments (19,23,35; Lung and Chang, letter). Similarly, size variation in EBNA proteins ("EBNotype" or "EBNAprint") (16, 45) and size variation in specific gene PCR products (LMP-1, BZLF1, EBNA-6) (13, 35) reflect variations in repetitive and other genome sequences. However, many EBV genome sequences are susceptible to intrastrain homologous and nonhomologous recombination during productive replication and the number of repeat units present may vary in different isolates of the same EBV strain (12,38,(41)(42)(43). Studies examining the major sequence divergence between EBV types 1 and 2 have reported EBV coinfection rates ranging from 0 to 53% (4,13,20,34,35,45). However, EBV types 1 and 2 can both be further subdivided into different strains (1,24,41) and only three studies to date have utilized EBV gene nucleotide sequence variation to define EBV strains in healthy individuals (7,30,38).EBV genotyping assay. A consistent approach is needed for the definition and nomenclature of EBV genomes. It is impractical or even impossible to physically isolate (culture) and fully characterize the EBV genome(s) in clinical infections. A reasonable goal would be to identify an EBV genetic marker that represents the broad...