Multiple Epstein-Barr Virus Strains Research Articles

Nasal natural killer/T-cell lymphoma and its association with type “i”/XhoI loss strain Epstein–Barr virus in Chile

Background: Nasal T/natural killer (NK)-cell lymphoma is an aggressive type of non-Hodking’s lymphoma associated with Epstein–Barr virus (EBV) and striking geographical variations worldwide. Aim: To characterise nasal NK/T-cell lymphoma associated...

A Prospective Follow‐Up of Epstein‐Barr VirusLMP1Genotypes in Saliva and Blood during Infectious Mononucleosis

To monitor multiple Epstein-Barr virus (EBV) infections during the early and convalescent stages of infectious mononucleosis (IM), a cloning and sequencing study of the LMP1 gene was conducted in saliva and peripheral blood mononuclear cells (PBMCs) from 23 patients with IM at day 0 (D0) and day 180 (D180) after the onset of the disease. Multiple EBV strains were detected in 9 (39%) of the patients during follow-up, with 7 of 9 cases detected as early as D0. Six of the nine patients harbored the same dominant strain in saliva and PBMCs during follow-up, with a trend toward a restriction of the number of EBV strains in saliva but not in PBMCs at D180. Furthermore, transmission of a minor strain was observed between partners in a heterosexual couple. There was no correlation between multiple infections and EBV DNA load in either compartment.

Coinfection by Multiple Strains of Epstein-Barr Virus in Infectious Mononucleosis in Immunocompetent Patients

Objective--A molecular study of Epstein-Barr virus (EBV)-induced infectious mononucleosis (IM) was performed. The presence of a 30-bp deletion on the latent membrane protein-1 (LMP-1) oncogene from EBV in Caucasian IM patients was evaluated. Material and Methods--Peripheral blood mononuclear cells were obtained from 27 IM patients and 18 adenoids, 28 tonsils and 16 EBV-related reactive lymphadenitis specimens were used as controls. DNA isolation, EBV polymerase chain reaction (PCR) and LMP-1 oncogene PCR analysis were performed. Results--The 30-bp deletion on LMP-1 was identified in 29.6% of IM patients, but was always seen in conjunction with full-length LMP-1. Although the LMP-1-deleted strain seemed to be more prevalent in IM (29.6%) and EBV-related reactive lymphadenitis (37.5%) than in adenoid (0%) or tonsil specimens (21.4%), these differences were not significant (p>0.05;χ2). Thus, a 30-bp deletion on LMP-1 was present in almost a third of Caucasian IM patients. Conclusion--The finding of coinfections in IM patients confirms that primary infection by more than one EBV strain is possible.

Compartmentalization and transmission of multiple epstein-barr virus strains in asymptomatic carriers.

Infection with the Epstein-Barr virus (EBV) is often subclinical in the presence of a healthy immune response; thus, asymptomatic infection is largely uncharacterized. This study analyzed the nature of EBV infection in 20 asymptomatic immunocompetent hosts over time through the identification of EBV strain variants in the peripheral blood and oral cavity. A heteroduplex tracking assay specific for the EBV gene LMP1 precisely identified the presence of multiple EBV strains in each subject. The strains present in the peripheral blood and oral cavity were often completely discordant, indicating the existence of distinct infections, and the strains present and their relative abundance changed considerably between time points. The possible transmission of strains between the oral cavity and peripheral blood compartments could be tracked within subjects, suggesting that reactivation in the oral cavity and subsequent reinfection of B lymphocytes that reenter the periphery contribute to the maintenance of persistence. In addition, distinct virus strains persisted in the oral cavity over many time points, suggesting an important role for epithelial cells in the maintenance of persistence. Asymptomatic individuals without tonsillar tissue, which is believed to be an important source of virus for the oral cavity, also exhibited multiple strains and a cyclic pattern of transmission between compartments. This study revealed that the majority of patients with infectious mononucleosis were infected with multiple strains of EBV that were also compartmentalized, suggesting that primary infection involves the transmission of multiple strains. Both the primary and carrier states of infection with EBV are more complex than previously thought.

Evidence for trafficking of Epstein-Barr virus strains between hairy leukoplakia and peripheral blood lymphocytes.

Hairy leukoplakia (HL), an epithelial lesion found on the side of the tongue in immunocompromised individuals, is characterized by high-level replication of Epstein-Barr virus (EBV) and multiple EBV strains. The source of these strains and their relationship to peripheral blood lymphocyte (PBL) strains has not previously been characterized. Using matched pairs of HL scrapings and PBL from 16 HIV-positive men, variation in EBV strain identity was characterized by detection of a 30 nucleotide deletion of the EBV latent membrane protein (LMP)-1 gene, variation in the LMP-1 repeat region and typing for Epstein-Barr nuclear antigen (EBNA)-2. Multiple EBV strains were found in both the HL and PBL specimens, but 13 of 16 (81%) patients showed evidence of strain identity for at least one strain and analysis of two patients suggested that EBV strains from HL could infect the PBL. Our data are consistent with active trafficking of EBV between these two compartments.

Coinfection of multiple strains of Epstein-Barr virus in immunocompetent normal individuals: reassessment of the viral carrier state

This study reassesses the occurrence of Epstein-Barr virus (EBV) diversity and coinfection versus dominance of a single viral strain within immunocompetent normal carriers. Polymerase chain reaction analysis of several different polymorphic loci of the EBV genome was performed on collections of peripheral blood mononuclear cells and multiple lymphoid and epithelial tissues of the same individuals. Autopsy specimens from 15 individuals who died of causes unrelated to EBV infection served as normal viral carriers. Unexpectedly, coinfection of multiple distinct strains of EBV of the same type (usually type 1) and less frequently of both types 1 and 2 was found to be very high within individual viral carriers. These data indicate that coinfection with multiple EBV strains is much more prevalent in normal carriers than previously appreciated, which in turn has direct implications on EBV persistence, host–viral interaction and pathogenesis.

Characterization of Intertypic Recombinants of the Epstein–Barr Virus from the Body-Cavity-Based Lymphomas Cell Lines BC-1 and BC-2

Epstein–Barr Virus (EBV) can infect and transform human B-lymphocytes and has been associated with numerous human malignancies. Two distinct types of EBV have been described, EBV-1 and EBV-2. Whereas type 1 is known to be most widespread throughout the healthy adult population, type 2 EBV has been shown to be significantly present in certain T-cell immunocompromised patients. Some evidence also suggests that such immune impairment promotes coinfection with multiple strains of EBV and fosters the development of intertypic recombinant viruses. In this work, we have analyzed two established body-cavity-based lymphoma or primary effusion lymphoma cell lines, BC-1 and BC-2, for the presence of intertypic EBV recombinants. Using PCR primers to amplify across several markers in the genome, we have typed the BC-1 and BC-2 EBV at these loci. Immunoblot analysis of the EBNA1 protein expressed by these cell lines also suggests a change in EBV typing at this locus in these genomes. Additionally, we have analyzed the expression patterns of the latent EBNA proteins from these viruses and performed Southern blot analysis of the BamHI- and EcoRI-digested genomes to detect variations occurring from type I and II genomes. On the basis of these data, we suggest that the genomes of EBV in BC-1 and BC-2 are intertypic recombinants of type 1 and type 2 EBV genomes. This work corroborates other reports that intertypic EBV recombinants occur in the immunocompromised population. It is likely that intertypic recombination is a mechanism by which novel variants of EBV emerge having selective advantages over a strictly type 1 or type 2 strain.

Analysis of Epstein-Barr virus (EBV) nuclear antigen 1 subtypes in EBV-associated lymphomas from Brazil and the United Kingdom.

EBNA-1 is the only viral protein consistently expressed in all cells latently infected by Epstein-Barr virus (EBV). There is a high frequency of sequence variation within functionally important domains of EBNA-1, with five subtypes identified. Individuals may be infected with multiple EBV strains (classified according to EBNA-1 subtype), but Burkitt's lymphoma (BL) tumours carry a single subtype and exhibit some subtype preference. Subtype variation has also been related to geographical location. In the present study EBNA-1 polymorphisms were examined in a series of haematological malignancies from two distinct geographical regions, Brazil and the United Kingdom. Nucleotide sequence analysis of the carboxy-terminal region of EBNA-1 in 34 cases revealed six distinct sequences, some of which are novel. A new subtype, named V-Ala, was identified. EBNA-1 subtype in tumours differed markedly according to geographical location. In contrast to previous studies, we found evidence of EBNA-1 sequence variation within individual BL tumour samples.

The molecular epidemiology and evolution of Epstein-Barr virus: sequence variation and genetic recombination in the latent membrane protein-1 gene.

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.

Transcription of Epstein–Barr Virus Latent Cycle Genes in Oral Hairy Leukoplakia

The hairy leukoplakia lesion (HLP) is a unique example of a permissive infection with Epstein–Barr virus (EBV) in the tongue epithelium. HLP contains abundant replicating viral DNA and may be coinfected with multiple EBV strains. In this study, characterization of viral gene transcription within HLP biopsy specimens revealed that several genes, usually expressed in latently infected lymphocytes, are also transcribed in the HLP lesion. TheBamHI W and C promoters, (Wp and Cp) are consistently active in the HLP lesion, resulting in transcription and processing of mRNAs that encode the Epstein–Barr nuclear antigens (EBNAs) EBNA-LP, EBNA1, EBNA2, EBNA3B, and EBNA3C. The EBNA2 protein has been shown to activate expression of the EBV receptor, CD21. In HLP, CD21 transcription is also detected, usually in samples that contain transcripts for EBNA2. Transcripts encoding the LMP1 gene, the LMP2 gene, and rightward transcripts from theBamHI A fragment of the EBV genome are also detected in HLP. These gene products are invariably expressed in latently infected lymphocytes. This pattern of transcription suggests that genes characteristic of latent infection are also expressed in HLP. The activation of Wp and expression of EBNA2 and CD21 may contribute to the unique ability of the HLP lesion to permit superinfection and viral replication of multiple EBV strains.

Epstein-Barr virus BMRF-2 and BDLF-3 expression in hairy leukoplakia.

Hairy leukoplakia (HL) is a lesion found on the side of the tongue of immunocompromised individuals, including those with human immunodeficiency virus (HIV) infection. The lesion has unique histopathologic features and is characterised by high-level Epstein-Barr virus (EBV) replication, multiple EBV strains, and extensive inter- and intra-strain recombination. Expression of EBV genes spanning the entire viral life cycle from latency-associated genes to late, replicative genes has been detected in the lesion. HL thus provides a unique opportunity to study EBV expression in oral epithelium, and to study expression of novel EBV genes. We therefore constructed a cDNA library from an HL biopsy and detected expression of two genes not previously described in vivo: BMRF-2 and BDLF-3. Sequence analysis of the cDNAs revealed few amino acid changes from the B95-8 sequence. Expression of both genes was localized to the lower prickle cell layer of the tongue epithelium. BMRF-2 protein expression was primarily detected in the cell nuclei of the upper prickle cell layer. BDLF-3 protein expression was observed in the peri-nuclear space and Golgi compartment. The function of these proteins is currently under investigation.

Immunohistochemical detection of placental ECM molecules in paraffin sections after optimization of tissue pretreatment

Epstein–Barr Virus (EBV) can infect and transform human B-lymphocytes and has been associated with numerous human malignancies. Two distinct types of EBV have been described, EBV-1 and EBV-2. Whereas type 1 is known to be most widespread throughout the healthy adult population, type 2 EBV has been shown to be significantly present in certain T-cell immunocompromised patients. Some evidence also suggests that such immune impairment promotes coinfection with multiple strains of EBV and fosters the development of intertypic recombinant viruses. In this work, we have analyzed two established body-cavity-based lymphoma or primary effusion lymphoma cell lines, BC-1 and BC-2, for the presence of intertypic EBV recombinants. Using PCR primers to amplify across several markers in the genome, we have typed the BC-1 and BC-2 EBV at these loci. Immunoblot analysis of the EBNA1 protein expressed by these cell lines also suggests a change in EBV typing at this locus in these genomes. Additionally, we have analyzed the expression patterns of the latent EBNA proteins from these viruses and performed Southern blot analysis of the BamHI- and EcoRI-digested genomes to detect variations occurring from type I and II genomes. On the basis of these data, we suggest that the genomes of EBV in BC-1 and BC-2 are intertypic recombinants of type 1 and type 2 EBV genomes. This work corroborates other reports that intertypic EBV recombinants occur in the immunocompromised population. It is likely that intertypic recombination is a mechanism by which novel variants of EBV emerge having selective advantages over a strictly type 1 or type 2 strain.

Detection of multiple Epstein-Barr viral strains in allogeneic bone marrow transplant recipients.

We have previously shown in 3 allogeneic bone-marrow transplant (BMT) recipients that complete replacement of recipient marrow was associated with the elimination of the pretransplant Epstein-Barr virus (EBV) strain of the recipient. To study the kinetics of EBV elimination and reinfection in more detail, we have performed a longitudinal study of BMT recipients combining serology, virus isolation from mouthwashes and peripheral blood, and EBV strain characterization. Oropharyngeal EBV excretion was found to persist after the cytoreductive therapy prior to BMT, whereas EBV-carrying cells in the blood were detected only after 5 weeks following BMT. During the first month post-BMT, 2 different EBV strains could be isolated from sequential mouth-washes of 3 patients. The initial strains were found to persist up to 7, 21, and 29 days post-BMT, whereas the subsequent strains appeared at 21, 42, and 34 days post-BMT, respectively. Thus, the original EBV strain may persist only for a limited time after BMT, and the oropharyngeal epithelium may be reinfected by a new EBV strain from the blood within 3 weeks. With respect to the coexistence of multiple EBV strains, 2 patterns were evident. From the day 62 mouthwash of 1 patient, 1 Type A and 1 Type B strain were isolated. From the day 180 mouthwash of a second patient, a dominant Type A strain was recovered, together with 6 "variant" strains that differed from each other by only a single EBNA protein (EBNA 1). This pattern may be explained by viral recombinations during replication, which may form the basis for the vast polymorphism of EBV observed in unrelated individuals.

Detection of Epstein-Barr Virus with Molecular Hybridization Techniques

The cord-blood transformation assay remains the standard method for detecting Epstein-Barr virus (EBV) in secretions. However, newer methods are much faster and more sensitive, although most are still regarded as research procedures. The most useful of these are Southern blot hybridization, particularly the variation that employs terminal genomic probe analysis; in situ cytohybridization; and polymerase chain reaction analyses. Use of these methods alone or in combination should disclose the infected cell type, whether the infection is productive or latent, and the presence of multiple strains of EBV. Such information may help establish whether EBV is a causal agent in chronic fatigue syndrome.