Isolation of intertypic recombinants of Epstein-Barr virus from T-cell-immunocompromised individuals.

All wild-type isolates of Epstein-Barr virus (EBV) analyzed to date for allelic polymorphisms of the nuclear antigen EBNA2 gene (in the BamHI YH region of the genome) and of the EBNA3A,-3B, -3C genes (tandemly arranged in the BamHI E region) have proved either uniformly type 1 or uniformly type 2 at all four loci. The absence of detectable intertypic recombination in the wild probably reflects the rarity with which individual carriers, and certainly individual target cells, become coinfected with both virus types. Studying a group of human immunodeficiency virus-positive T-cell-immunocompromised patients known to be at enhanced risk of multiple EBV infections, we have isolated intertypic EBV recombinants from 2 of 40 patients analyzed. These recombinants, whose in vitro transforming capacity appeared at least equal to that of type 1 strains, carried a type 1 EBNA2 allele and type 2 EBNA3A,-3B, and -3C alleles. This was clearly demonstrable at the DNA level by PCR amplification using type-specific primer-probe combinations and was confirmed at the protein level (for EBNA2 and EBNA3C) by immunoblotting with type-specific antibodies. In one patient, the recombinant appeared to be the predominant strain, being the virus most commonly rescued by in vitro transformation both from the blood and from the throat washings on two separate occasions 20 months apart. A regular type 1 virus strain was also present in this individual, but this was not related to the recombinant since the two viruses carried type 1 EBNA2 genes with different patterns of variance from the B95.8 prototype sequence. In the other patient, recombinants were isolated on one occasion from the blood and on a separate occasion, 21 months later, from the throat; these recombinants were almost certainly related, being identical at several genomic polymorphisms and differing only in one facet of the "EBNAprint," the size of the EBNA1 protein. Three different type 1 viruses were also isolated from this patient, two of which carried EBNA2 genes with the same pattern of sequence variation from B95.8 as the recombinant; however, since this is a fairly common pattern of variance, the relationship of these viruses to the recombinant remains an open question. We infer that intertypic recombinants of EBV are not uncommon in HIV-positive T-cell-immunocompromised patients, that they arise in such individuals as a consequence of their increased frequency of mixed-type infections, and that they will prove capable of efficient transmission in the human population.     

Novel intertypic recombinants of epstein-barr virus in the chinese population

Among 34 Epstein-Barr virus isolates from nonimmunocompromised Chinese donors, we identified three intertypic recombinants with type 1 sequences at the EBNA2 locus and type 2 sequences at some or all of the EBNA3A, -3B, and -3C loci. These appear to have arisen from independent, evolutionarily recent recombination events; such events may be commoner in nonimmunocompromised populations than hitherto imagined.     

Epstein-Barr virus types 1 and 2 differ in their EBNA-3A, EBNA-3B, and EBNA-3C genes.

The two Epstein-Barr virus (EBV) types, EBV-1 and EBV-2, are known to differ in their EBNA-2 genes, which are 64 and 53% identical in their nucleotide and predicted amino acid sequences, respectively. Restriction endonuclease maps and serologic analyses detect few other differences between EBV-1 and EBV-2 except in the EBNA-3 gene family. We determined the DNA sequence of the AG876 EBV-2 EBNA-3 coding region and have compared it with known B95-8 EBV-1 EBNA-3 sequences to delineate the extent of divergence between EBV-1 and EBV-2 isolates in their EBNA-3 genes. The B95-8 and AG876 EBV isolates had nucleotide and amino acid identity levels of 90 and 84%, 88 and 80%, and 81 and 72% for the EBNA-3A, -3B, and -3C genes, respectively. In contrast, nucleotide sequence identity in the noncoding DNA adjacent to the B95-8 and AG876 EBNA-3 open reading frames was 96%. We used the polymerase chain reaction to demonstrate that five additional EBV-1 isolates and six additional EBV-2 isolates have the type-specific differences in their EBNA-3 genes predicted from the B95-8 or AG876 sequences. Thus, EBV-1 and EBV-2 are two distinct wild-type EBV strains that have significantly diverged at four genetic loci and have maintained type-characteristic differences at each locus. The delineation of these sequence differences between EBV-1 and EBV-2 is essential to ongoing molecular dissection of the biologic properties of EBV and of the human immune response to EBV infection. The application of these data to the delineation of epitopes recognized in the EBV-immune T-cell response is also discussed.     

Distinction between Epstein-Barr virus type A (EBNA 2A) and type B (EBNA 2B) isolates extends to the EBNA 3 family of nuclear proteins.

The Epstein-Barr virus (EBV) nuclear antigens EBNA 3a, 3b, and 3c have recently been mapped to adjacent reading frames in the BamHI L and E fragments of the B95.8 EBV genome. We studied by immunoblotting the expression of the family of EBNA 3 proteins in a panel of 20 EBV-transformed lymphoblastoid cell lines (LCLs) carrying either type A (EBNA 2A-encoding) or type B (EBNA 2B-encoding) virus isolates. Certain human sera from donors naturally infected with type A isolates detected the EBNA 3a, 3b, and 3c proteins in all type A virus-transformed LCLs (with a single exception in which EBNA 3b was not detected) but detected only EBNA 3a in LCLs carrying type B isolates. These results were confirmed with human and murine antibodies with specific reactivity against sequences of the type A EBNA 3a, 3b, or 3c expressed in bacterial fusion proteins. Conversely, selected human sera from donors naturally infected with type B strains of EBV identified the EBNA 3a encoded by both types of isolates plus two novel EBNAs present only in type B, and not in type A, virus-transformed LCLs; these novel proteins appear to be the type B homologs of EBNA 3b and 3c. The distinction between type A and type B EBV isolates therefore extends beyond the EBNA 2 gene to the EBNA 3 family of proteins. This has important implications with respect to the evolutionary origin of these two EBV types and also places in a new light recent studies which identified differences between type A and type B transformants in terms of growth phenotype (A. B. Rickinson, L. S. Young, and M. Rowe, J. Virol. 61:1310-1317, 1987) and of detection by EBV-specific cytotoxic T cells (D. J. Moss, I. S. Misko, S. R. Burrows, K. Burman, R. McCarthy, and T. B. Sculley, Nature [London] 331:719-721, 1988).     

Epstein-Barr virus recombinants from BC-1 and BC-2 can immortalize human primary B lymphocytes with different levels of efficiency and in the absence of coinfection by Kaposi's sarcoma-associated herpesvirus

Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are human gammaherpesviruses associated with numerous malignancies. Primary effusion lymphoma or body cavity-based lymphoma is a distinct clinicopathological entity that, in the majority of cases, manifests coinfection with KSHV and EBV. In previous analyses, we have characterized the EBV in the BC-1 and BC-2 cell lines as potential intertypic recombinants of the EBV types 1 and 2. In order to examine the infectious and transforming capacities of KSHV and the intertypic EBV recombinants from the BC-1 and BC-2 cell lines, viral replication was induced in these cell lines and fresh human primary B lymphocytes were infected with progeny virus. The transformed clones were analyzed by PCR and Western blotting. All analyzed clones were infected with the intertypic progeny EBV but had no detectable signal for progeny KSHV. Additionally, primary B lymphocytes incubated with viral supernatant containing KSHV alone showed an unsustained initial proliferation, but prolonged growth or immortalization of these cells in vitro was not observed. We also show that the EBV recombinants from BC-1 were less efficient than the EBV recombinants from BC-2 in the ability to maintain the transformed phenotype of the infected human B lymphocytes. From these findings, we conclude that the BC-1 and BC-2 intertypic EBV recombinants can immortalize human primary B lymphocytes, albeit at different levels of efficiency. However, the KSHV induced from BC-1 and BC-2 alone cannot transform primary B cells, nor can it coinfect EBV-positive B lymphocytes under our experimental conditions with B lymphocytes from EBV-seropositive individuals. These results are distinct from those in one previous report and suggest a possible requirement for other factors to establish coinfection with both viral agents.     

Epstein-Barr virus recombinants from overlapping cosmid fragments.

Five overlapping type 1 Epstein-Barr virus (EBV) DNA fragments constituting a complete replication- and transformation-competent genome were cloned into cosmids and transfected together into P3HR-1 cells, along with a plasmid encoding the Z immediate-early activator of EBV replication. P3HR-1 cells harbor a type 2 EBV which is unable to transform primary B lymphocytes because of a deletion of DNA encoding EBNA LP and EBNA 2, but the P3HR-1 EBV can provide replication functions in trans and can recombine with the transfected cosmids. EBV recombinants which have the type 1 EBNA LP and 2 genes from the transfected EcoRI-A cosmid DNA were selectively and clonally recovered by exploiting the unique ability of the recombinants to transform primary B lymphocytes into lymphoblastoid cell lines. PCR and immunoblot analyses for seven distinguishing markers of the type 1 transfected DNAs identified cell lines infected with EBV recombinants which had incorporated EBV DNA fragments beyond the transformation marker-rescuing EcoRI-A fragment. Approximately 10% of the transforming virus recombinants had markers mapping at 7, 46 to 52, 93 to 100, 108 to 110, 122, and 152 kbp from the 172-kbp transfected genome. These recombinants probably result from recombination among the transfected cosmid-cloned EBV DNA fragments. The one recombinant virus examined in detail by Southern blot analysis has all the polymorphisms characteristic of the transfected type 1 cosmid DNA and none characteristic of the type 2 P3HR-1 EBV DNA. This recombinant was wild type in primary B-lymphocyte infection, growth transformation, and lytic replication. Overall, the type 1 EBNA 3A gene was incorporated into 26% of the transformation marker-rescued recombinants, a frequency which was considerably higher than that observed in previous experiments with two-cosmid EBV DNA cotransfections into P3HR-1 cells (B. Tomkinson and E. Kieff, J. Virol. 66:780-789, 1992). Of the recombinants which had incorporated the marker-rescuing cosmid DNA fragment and the fragment encoding the type 1 EBNA 3A gene, most had incorporated markers from at least two other transfected cosmid DNA fragments, indicating a propensity for multiple homologous recombinations. The frequency of incorporation of the nonselected transfected type 1 EBNA 3C gene, which is near the end of two of the transfected cosmids, was 26% overall, versus 3% in previous experiments using transfections with two EBV DNA cosmids. In contrast, the frequency of incorporation of a 12-kb EBV DNA deletion which was near the end of two of the transfected cosmids was only 13%.(ABSTRACT TRUNCATED AT 400 WORDS)     

Latent gene sequencing reveals familial relationships among Chinese Epstein-Barr virus strains and evidence for positive selection of A11 epitope changes

Epstein-Barr virus (EBV) strains from the highly HLA-A11-positive Chinese population are predominantly type 1 and show a variety of sequence changes (relative to the contemporary Caucasian prototype strain B95.8) in the nuclear antigen EBNA3B sequences encoding two immunodominant HLA-A11 epitopes, here called IVT and AVF. This has been interpreted by some as evidence of immune selection and by others as random genetic drift. To study epitope variation in a broader genomic context, we sequenced the whole of EBNA3B and parts of the EBNA2, 3A, and 3C genes from each of 31 Chinese EBV isolates. At each locus, type 1 viruses showed <2% nucleotide divergence from the B95.8 prototype while type 2 sequences remained even closer to the contemporary African prototype Ag876. However, type 1 isolates could clearly be divided into families based on linked patterns of sequence divergence from B95.8 across all four EBNA loci. Different patterns of IVT and AVF variation were associated with the different type 1 families, and there was additional epitope diversity within families. When the EBNA3 gene sequences of type 1 Chinese strains were subject to computer-based analysis, particular codons within the A11-epitope-coding region were among the few identified as being under positive or diversifying selection pressure. From these results, and the observation that mutant epitopes are consistently nonimmunogenic in vivo, we conclude that the immune selection hypothesis remains viable and worthy of further investigation.     

Second-site homologous recombination in Epstein-Barr virus: insertion of type 1 EBNA 3 genes in place of type 2 has no effect on in vitro infection.

This study was undertaken to develop a general strategy for the introduction of mutations into specific sites in the Epstein-Barr virus (EBV) genome. Previous approaches were limited by the need for physical linkage of the transfected EBV DNA fragment to a positive selection marker. In our experiments, a positive selection marker was introduced into one site in the EBV genome and a distant, nonlinked, marker was introduced into another site. Each marker was on a large EBV DNA fragment and was inserted into the genome by transfection into cells carrying a resident EBV genome. The resident EBV genome was simultaneously induced to replicate by using a cotransfected expression plasmid for the EBV immediate-early transactivator, Z (J. Countryman, H. Jenson, R. Seibl, H. Wolf, and G. Miller, J. Virol. 61:3672-3679, 1987; G. Miller, M. Rabson, and L. Heston, J. Virol. 50:174-182, 1984). Eleven percent of the resultant EBV genomes which incorporated the positive selection marker also incorporated the nonlinked marker. Both markers uniformly targeted the homologous EBV genome site. In this way novel EBV recombinants were constructed in which the EBV type 1 EBNA 3A, EBV type 1 EBNA 3A and 3B, or EBV type 1 EBNA 3A, 3B, and 3C genes were introduced into a largely type 2 EBV genome, replacing the corresponding type 2 gene(s). No difference was observed in primary B-lymphocyte growth transformation, in latent EBV gene expression, or in spontaneous lytic EBV gene expression. These new recombinants should be useful for ongoing analyses of the type specificity of the immune response.     

Reducing the complexity of the transforming Epstein-Barr virus genome to 64 kilobase pairs.

Transformation-competent, replication-defective Epstein-Barr virus (EBV) recombinants which are deleted for 18 kbp of DNA encoding the largest EBNA intron and for 58 kbp of DNA between the EBNA1 and LMP1 genes were constructed. These recombinants were made by transfecting three overlapping cosmid-cloned EBV DNA fragments into cells infected with a lytic replication-competent but transformation-defective EBV (P3HR-1 strain) and were identified by clonal transformation of primary B lymphocytes into lymphoblastoid cell lines. One-third of the lymphoblastoid cell lines were infected with recombinants which had both deletions and carried the EBNA2 and EBNA3 genes from the transfected EBV DNA and therefore are composed mostly or entirely from the transfected EBV DNA fragments. The deleted DNA is absent from cells infected with most of these recombinants, as demonstrated by Southern blot and sensitive PCR analyses for eight different sites within the deleted regions. Cell growth and EBNA, LMP, and BZLF1 gene expression in lymphoblastoid cell lines infected with these recombinants are similar to those in cells infected with wild-type EBV recombinants. Together with previous data, these experiments reduce the complexity of the EBV DNA necessary for transformation of primary B lymphocytes to 64 kbp. The approach should be useful for molecular genetic analyses of transforming EBV genes or for the insertion of heterologous fragments into transforming EBV genomes.     

Influence of the Epstein-Barr virus nuclear antigen EBNA 2 on the growth phenotype of virus-transformed B cells.

Epstein-Barr virus (EBV) isolates show sequence divergence in the BamHI YH region of the genome which encodes the nuclear antigen EBNA 2, a protein thought to be involved in the initiation of virus-induced B-cell transformation; type A isolates (such as B95-8 EBV) encode a 82- to 87-kilodalton EBNA 2A protein, whereas type B isolates (such as AG876 EBV) encode an antigenically distinct 75-kilodalton EBNA 2B protein. In the present work 12 type A isolates and 8 type B isolates have been compared for their ability to transform resting human B cells in vitro into permanent lymphoblastoid cell lines. Although the kinetics of initial focus formation was not markedly dependent upon the EBNA 2 type of the transforming virus, on subsequent passage type A virus-transformed cells (type A transformants) yielded cell lines much more readily than did type B transformants. Direct comparison between the two types of transformant revealed clear differences in several aspects of growth phenotype. Compared with type A transformants, cell lines established with type B virus isolates consistently displayed an unusual growth pattern with poor survival of individual cells shed from lymphoblastoid clumps, a lower growth rate and a greater sensitivity to seeding at limiting dilutions, and a significantly lower saturation density that could not be corrected by supplementation of the medium with culture supernatant containing B-cell growth factors. This is the first direct evidence that, in EBV-transformed B-cell lines, the EBNA 2 protein plays a continuing role in determining the cellular growth phenotype.     

Frequency of multiple Epstein-Barr virus infections in T-cell-immunocompromised individuals.

The Epstein-Barr virus (EBV) carrier state is characterized by latent infection of the general B-cell pool and by chronic virus replication at oropharyngeal sites. In Caucasian populations, most healthy carriers seem to harbor one dominant transforming virus strain, usually of type I rather than type 2, which persists over time and is detectable both in the blood and in the throat. This finding implies that once the virus carrier state is established, both viral reservoirs are largely if not completely protected from infection with additional strains. However, it is not known which facets of the immune response offer that protection. Here we address this question by a detailed study of EBV carriage in patients T-cell immunocompromised as a result of chronic human immunodeficiency virus (HIV) infection. Resident EBV strains were rescued from blood and from throat washings by using an in vitro transformation assay which aims to minimize bias toward faster-growing transformants; in this way, a mean of 16 independent isolations were made from each of 35 HIV-positive (predominantly male homosexual) patients. These virus isolates were characterized first at the DNA level by PCR amplification across type-specific polymorphisms in the EBNA2 and EBNA3C genes and across the 30-bp deletion and 33-bp repeat loci in the LMP1 gene and then at the protein level by immunoblotting for the strain-specific "EBNAprint" of EBNA1, -2, and -3C molecular weights. By these criteria, 18 of 35 patients harbored only one detectable EBV strain, usually of type 1, as do healthy carriers. However, the other 17 patients showed clear evidence of multiple infection with different EBV strains. In eight cases these strains were of the same type, again usually type 1, and were more often found coresident in throat washings than in the blood. By contrast, a further nine patients gave evidence of coinfection with type 1 and type 2 strains, and in these cases both virus types were detectable in the blood as well as in the throat. Immunological assays on these HIV-positive patients as a group showed a marked impairment of T-cell responses, reflected in reduced levels of EBV-specific cytotoxic T-cell memory, but an elevation of humoral responses, reflected in raised antibody titers to the EBV envelope glycoprotein gp340 and by the maintenance of virus neutralizing antibodies in serum. We infer that selective impairment of the T-cell system predisposes the host to infection with additional exogenously transmitted EBV strains.     

Polymerase chain reaction genotyping of Epstein-Barr virus in scraping samples of the tongue lateral border in HIV-1 seropositive patients

The Epstein-Barr virus (EBV) is the etiological agent of oral hairy leukoplakia (OHL), an oral lesion with important diagnostic and prognostic value in acquired immunodeficiency disease syndrome. The two EBV genotypes, EBV-1 and EBV-2, can be distinguished by divergent gene sequences encoding the EBNA-2, 3A, 3B, and 3C proteins. The purpose of this study was to identify the EBV genotype prevalent in 53 samples of scrapings from the lateral border of the tongue of HIV-1 seropositive patients, with and without OHL, and to correlate the genotypes with presence of clinical or subclinical OHL with the clinic data collected. EBV-1 and EBV-2 were identified through PCR and Nested-PCR based on sequence differences of the EBNA-2 gene. EBV-1 was identified in the 31 samples (15 without OHL, 7 with clinical OHL and 9 with subclinical OHL), EBV-2 in 12 samples (10 without OHL, 1 with clinical and 1 subclinical OHL), and a mixed infection in 10 samples (2 without OHL, 3 with clinical and 5 with subclinical OHL). The presence of EBV-1 was higher in women, but a significant statistical result relating one the EBV genotypes to the development of OHL was not found. We conclude that the oral epithelium in HIV-1 seropositive patients can be infected by EBV-1, EBV-2 or by a mixed viral population.     

EBV increases phosphoinositide kinase activities in human B cells.

To determine whether EBV affects phosphoinositide kinase activities of human B cells, we compared the activities between EBV- and EBV+ human B cell lymphoma lines. The two types of human B cells contained both phosphatidylinositol (PtdIns) 4-kinase and phosphatidylinositol 4-phosphate (PtdIns(4)P) kinase activities irrespective of the presence of EBV. However, both activities were increased in EBV+ cells compared to EBV- cells. The increases were associated with neither altered Km values for substrates nor altered elution profiles in DEAE-cellulose chromatography. Furthermore, expression of a latent EBV protein, EBV nuclear Ag1 (EBNA1) in BHK cells by the transfection of EBNA1 DNA was accompanied by increased PtdIns 4-kinase and PtdIns(4)P kinase activities. These increases also were not associated with altered Km values for substrates. However, phospholipase C activity was altered in neither EBV+ cells nor in EBNA1-expressing cells. These results indicate that EBV selectively increases the two phosphoinositide kinase activities in human B cells, although the viral gene product has no intrinsic phosphoinositide kinase activity. PtdIns 4-kinase and PtdIns(4)P kinase cooperatively synthesize PtdIns 4,5-bisphosphate, the major source of 1,2-diacylglycerol and inositol 1,4,5-triphosphate, the two second messengers in transducing signals for cell activation. Such increase therefore may play a role in EBV-induced human B cell activation.     

Cytotoxic T-lymphocyte responses to a polymorphic Epstein-Barr virus epitope identify healthy carriers with coresident viral strains

Cytotoxic T-lymphocyte (CTL) responses to Epstein-Barr virus (EBV) tend to focus on a few immunodominant viral epitopes; where these epitope sequences are polymorphic between EBV strains, host CTL specificities should reflect the identity of the resident strain. In studying responses in HLA-B27-positive virus carriers, we identified 2 of 15 individuals who had strong CTL memory to the pan-B27 epitope RRIYDLIEL (RRIY) from nuclear antigen EBNA3C but whose endogenous EBV strain, isolated in vitro, encoded a variant sequence RKIYDLIEL (RKIY) which did not form stable complexes with B27 molecules and which was poorly recognized by RRIY-specific CTLs. To check if such individuals were also carrying an epitope-positive strain (either related to or distinct from the in vitro isolate), we screened DNA from freshly isolated peripheral blood mononuclear cells for amplifiable virus sequences across the EBNA3C epitope, across a different region of EBNA3C with type 1-type 2 sequence divergence, and across a polymorphic region of EBNA1. This showed that one of the unexplained RRIY responders carried two distinct type 1 strains, one with an RKIY and one with an RRIY epitope sequence. The other responder carried an RKIY-positive type 1 strain and a type 2 virus whose epitope sequence of RRIFDLIEL was antigenically cross-reactive with RRIY. Of 15 EBV-seropositive donors analyzed by such assays, 12 appeared to be carrying a single virus strain, one was coinfected with distinct type 1 strains, and two were carrying both type 1 and type 2 viruses. This implies that a small but significant percentage of healthy virus carriers harbor multiple, perhaps sequentially acquired, EBV strains.     

Epstein-Barr Virus Nuclear Antigen 1 Sequences in Endemic and Sporadic Burkitt’s Lymphoma Reflect Virus Strains Prevalent in Different Geographic Areas

The Epstein-Barr virus (EBV) nuclear antigen EBNA1 is the only viral protein detectably expressed in virus genome-positive Burkitt’s lymphoma (BL); recent work has suggested that viral strains with particular EBNA1 sequence changes are preferentially associated with this tumor and that, within a patient, the tumor-associated variant may have arisen de novo as a rare mutant of the dominant preexisting EBV strain (K. Bhatia, A. Raj, M. J. Gutierrez, J. G. Judde, G. Spangler, H. Venkatesh, and I. T. Magrath, Oncogene 13:177–181, 1996). In the present work we first study 12 BL patients and show that the virus strain in the tumor is identical in EBNA1 sequence and that it is matched at several other polymorphic loci to the dominant strain rescued in vitro from the patient’s normal circulating B cells. We then analyze BL-associated virus strains from three different geographic areas (East Africa, Europe, and New Guinea) alongside virus isolates from geographically matched control donors by using sequence changes in two separate regions of the EBNA1 gene (N-terminal codons 1 to 60 and C-terminal codons 460 to 510) to identify the EBNA1 subtype of each virus. Different geographic areas displayed different spectra of EBNA1 subtypes, with only limited overlap between them; even type 2 virus strains, which tended to be more homogeneous than their type 1 counterparts, showed geographic differences at the EBNA1 locus. Most importantly, within any one area the EBNA1 subtypes associated with BL were also found to be prevalent in the general population. We therefore find no evidence that Burkitt lymphomagenesis involves a selection for EBV strains with particular EBNA1 sequence changes.     

Phylogenetic comparison of Epstein-Barr virus genomes

Technologies used for genome analysis and whole genome sequencing are useful for us to understand genomic characterization and divergence. The Epstein-Barr virus (EBV) is an oncogenic virus that causes diverse diseases such as Burkitt’s lymphoma (BL), nasopharyngeal carcinoma (NPC), Hodgkin’s lymphoma (HL), and gastric carcinoma (GC). EBV genomes found in these diseases can be classified either by phases of EBV latency (type-I, -II, and -III latency) or types of EBNA2 sequence difference (type-I EBV, type-II EBV or EBV-1, EBV-2). EBV from EBV-transformed lymphoblastoid cell line (LCL) establishes type-III latency, EBV from NPC establishes type-II latency, and EBV from GC establishes type-I latency. However, other important factors play key roles in classifying numerous EBV strains because EBV genomes are highly diverse and not phylogenetically related to types of EBV-associated diseases. Herein, we first reviewed previous studies to describe molecular characteristics of EBV genomes. Then, using comparative and phylogenetic analyses, we phylogenetically analyzed molecular variations of EBV genomes and proteins. The review of previous studies and our phylogenetic analysis showed that EBV genomes and proteins were highly diverse regardless of types of EBV-associated diseases. Other factors should be considered in determining EBV taxonomy. This review will be helpful to understand complicated phylogenetic relationships of EBV genomes.     

Natural genetic exchanges between vaccine and wild poliovirus strains in humans

In a previous study of poliovirus vaccine-derived strains isolated from patients with vaccine-associated paralytic poliomyelitis (VAPP) (9, 11), we reported that a high proportion (over 50%) of viruses had a recombinant genome. Most were intertypic vaccine/vaccine recombinants. However, some had restriction fragment length polymorphism (RFLP) profiles different from those of poliovirus vaccine strains. We demonstrate here that five such recombinants, of 88 VAPP strains examined, carried sequences of wild (nonvaccine) origin. To identify the parental wild donor of these sequences, we used RFLP profiles and nucleotide sequencing to look for similarity in the 3D polymerase-coding region of 61 wild, cocirculating poliovirus isolates (43 type 1, 16 type 2, and 2 type 3 isolates). In only one case was the donor identified, and it was a wild type 1 poliovirus. For the other four vaccine/wild recombinants, the wild parent could not be identified. The possibility that the wild sequences were of a non-poliovirus-enterovirus origin could not be excluded. Another vaccine/wild recombinant, isolated in Belarus from a VAPP case, indicated that the poliovirus vaccine/wild recombination is not an isolated phenomenon. We also found wild polioviruses (2 of 15) carrying vaccine-derived sequences in the 3' moiety of their genome. All these results suggest that genetic exchanges with wild poliovirus and perhaps with nonpoliovirus enteroviruses, are also a natural means of evolution for poliovirus vaccine strains.