Epstein-Barr virus WZhet DNA can induce lytic replication in epithelial cells in vitro, although WZhet is not detectable in many human tissues in vivo.

OBJECTIVE: WZhet is a rearranged and partially deleted form of the Epstein-Barr virus (EBV) genome in which the BamH1W region becomes juxtaposed with and activates BZLF1, resulting in constitutive viral replication. We tested whether WZhet induces viral replication in epithelial cells, and we studied its prevalence in a wide range of lesional tissues arising in vivo. METHODS: A quantitative real-time PCR assay targeting EBV WZhet DNA was developed to measure this recombinant form of the EBV genome. RESULTS: WZhet DNA was undetectable in any of 324 plasma or paraffin-embedded tissue samples from patients with EBV-associated and EBV-negative disorders. These included specimens from patients with Hodgkin or non-Hodgkin lymphoma, post-transplant lymphoproliferation, nasopharyngeal or gastric adenocarcinoma, and infectious mononucleosis. However, WZhet DNA was detected in vitro in EBV-infected AGS gastric cancer cells. Additionally, transient transfection of infected AGS gastric cancer cells showed that viral replication could be induced by a WZhet plasmid. CONCLUSION: This is the first evidence that WZhet induces the EBV lytic cycle in an epithelial cell line. Our negative findings in natural settings suggest that WZhet is a defective viral product that thrives in the absence of a host immune system but is rarely present in vivo.

High levels of Epstein-Barr virus DNA in latently infected gastric adenocarcinoma.

Gastric adenocarcinoma is the second leading cause of cancer death worldwide. Epstein-Barr virus (EBV) is present in the malignant cells of approximately 10% of cases. It is unclear whether EBV is being missed in some gastric adenocarcinomas due to insensitive test methods or partial EBV genome loss. In this study, we screened 113 gastric adenocarcinomas from low- and high-incidence regions (United States and Central America) for the presence of EBV using a battery quantitative real-time PCR (Q-PCR) assays targeting disparate segments of the EBV genome (BamH1W, EBNA1, LMP1, LMP2, BZLF1, EBER1) and histochemical stains targeting EBV-encoded RNA (EBER), the latent proteins LMP1 and LMP2, and the lytic proteins BMRF1 and BZLF1. EBV DNA was detected by Q-PCR in 48/75 United States cancers (64%) and in 38/38 Central American cancers (100%), which was a significant difference. EBER was localized to malignant epithelial cells in 8/48 (17%) United States and 3/38 (8%) Central American cancers. Viral loads were considerably higher for EBER-positive vs EBER-negative cancers (mean 162 986 vs 62 EBV DNA copies per 100,000 cells). A viral load of 2000 copies per 100,000 cells is recommended as the threshold distinguishing EBER-positive from EBER-negative tumors. One infected cancer selectively failed to amplify the LMP2 gene because of a point mutation, whereas another cancer had an atypical pattern of Q-PCR positivity suggesting deletion of large segments of the EBV genome. Three different viral latency profiles were observed in the cancers based on constant expression of EBER and focal or variable expression of LMP1 or LMP2, without lytic protein expression. We conclude that EBV DNA levels generally reflect EBER status, and a panel of at least two Q-PCR assays is recommended for sensitive identification of infected cancers.

Cytomegalovirus DNA measurement in blood and plasma using Roche LightCycler CMV quantification reagents.

Cytomegalovirus (CMV) is a common cause of morbidity and mortality in immunosuppressed patients. Newly available commercial systems facilitate the measurement of CMV DNA in whole blood or plasma, as a means of detecting and monitoring active disease. We evaluated the performance characteristics of a quantitative polymerase chain reaction that relies on analyte-specific reagents and instruments from Roche Diagnostics. DNA was extracted using a MagNaPure instrument from blood and matched plasma specimens of patients with active CMV disease (defined by another molecular assay) and from controls. Viral load was measured against Roche's standards on a LightCycler instrument, followed by melt curve analysis to confirm product specificity. Dual hybridization probes targeted the CMV UL54 gene and a control sequence that was spiked before extraction. Accuracy and linearity were established using spiked DNA from the Towne-strain CMV. The assay was linear across 6 logs, and it detected CMV DNA in 67/70 blood samples (96%) from patients who were considered to have an active CMV infection, including all 67 viral loads above 208 CMV copies/mL, suggesting that it was sensitive enough to detect clinically significant infections in immunosuppressed patients. Virus levels in plasma correlated reasonably well with the levels in whole blood (r=0.5756), suggesting that levels in either plasma or blood level were indicative of active infection. It was important to verify the calculated values by visualizing the amplification plot and using the melt curve analysis to resolve discrepancies. The LightCycler CMV assay is rapid, sensitive, and linear for quantifying CMV viral load, and it seems to be useful in the diagnosis and monitoring of affected patients.

Validation of Roche LightCycler Epstein-Barr virus quantification reagents in a clinical laboratory setting.

Epstein-Barr virus (EBV) is associated with a wide range of benign and malignant diseases, including infectious mononucleosis, lymphoma, posttransplant lymphoproliferative disorder, and nasopharyngeal carcinoma. Measurement of EBV viral load in plasma is increasingly used for rapid assessment of disease status. We evaluated the performance characteristics of an EBV polymerase chain reaction assay that uses commercial reagents and instruments from Roche Diagnostics (Indianapolis, IN). DNA was extracted from plasma using a MagNaPure instrument, and viral load was measured by real-time polymerase chain reaction on a LightCycler. Analyte-specific reagents included primers and hybridization probes targeting the EBV LMP2 gene and a spiked control sequence. Accuracy and reproducibility were established using DNA from three cell lines. The assay was sensitive to approximately 750 copies of EBV DNA per milliliter of plasma and was linear across at least four orders of magnitude. The assay detected EBV DNA in three of five samples from nasopharyngeal carcinoma patients, seven of nine infectious mononucleosis samples, and 34/34 samples from immunosuppressed patients with clinically significant EBV-related disease, whereas EBV DNA was undetectable in plasma from 21 individuals without EBV-related disease. In conclusion, this LightCycler EBV assay is rapid, sensitive, and linear for quantifying EBV viral load. The assay appears to be useful for measuring clinically significant EBV levels in immunodeficient patients.

Distribution of Epstein-Barr viral load in serum of individuals from nasopharyngeal carcinoma high-risk families in Taiwan.

The utility of EBV load as a tumor marker in nasopharyngeal carcinoma (NPC) patients suggests that it might also serve as a screening test for individuals who are at high risk for developing NPC. We previously demonstrated that unaffected individuals from high-risk families had elevated anti-EBV antibody levels compared to community controls. In this study, we measured EBV load using 2 different real-time PCR assays (targeting BamH1W and polymerase gene sequences, respectively) carried out in 2 independent research labs in serum samples from 19 untreated NPC cases, 11 healthy community controls and 100 unaffected individuals from families in which 2 or more individuals were affected with NPC. EBV genomes were detectable in 68% of NPC cases by the EBV BamH1W assay and in 74% by the EBV polymerase assay (kappa = 0.64). Patients with stage III or IV disease had significantly higher EBV load compared to those with stage I or II disease (p = 0.008). EBV DNA was detected in a single community control sample by the EBV BamH1W assay and in none of the samples by the EBV polymerase assay. Only one of 100 unaffected family members tested positive by both assays. An additional 14 were positive by only one of the 2 EBV load assays used and usually in only one of the duplicate wells tested, all with very low viral loads (3-50 copies/ml). In addition, EBV load did not correlate with EBV serology results (anti-VCA, anti-DNase, anti-EBNA-1) among these unaffected family members. In conclusion, our study suggests limited clinical utility of the EBV load test, in its current configuration, to screen individuals from high-risk families. Should a more sensitive or specific molecular assay be developed that is capable of detecting and distinguishing tumor-derived EBV genomes or gene products from true negatives, it could be evaluated as a possible screening tool for asymptomatic and early-stage NPC.

Real-time PCR measures Epstein-Barr Virus DNA in archival breast adenocarcinomas.

The role of Epstein-Barr Virus (EBV) in breast cancer pathogenesis remains controversial. Fifty-five cases of paraffin-embedded, formalin-fixed invasive breast cancer were screened for the presence of EBV using quantitative polymerase chain reaction (PCR) directed at five different targets within the EBV genome (BamH1W, LMP1, EBNA1, LMP2, and BZLF1 regions). In four tumors (7%), low level EBV DNA was detected by at least one of the assays, with levels of up to 11 copies of EBV DNA per 100,000 cells. Immunohistochemisty for viral BMRF1 and BZLF1 and in situ hybridization for lytic gene transcripts showed no evidence of replicative EBV gene expression. Lymphocytes and malignant cells were also negative for latent infection by EBER in situ hybridization. Laser capture microdissection followed by quantitative real-time PCR was not useful in localizing EBV DNA to malignant cells or bystander lymphocytes. In conclusion, EBV DNA is detectable in a fraction of breast cancer specimens using real-time PCR as a screening tool, albeit at quite low levels, which suggests that only rare cells are infected. The low levels probably confounded our ability to localize the virus to particular cell types or to characterize viral gene expression.