Sandra A. W. M. Verkuijlen

Rapid and Sensitive Routine Detection of All Members of the Genus Enterovirus in Different Clinical Specimens by Real-Time PCR

ABSTRACT We developed a rapid and sensitive method for the routine detection of all members of the enterovirus genus in different clinical specimens by using real-time TaqMan quantitative PCR. Multiple primer and probe sets were selected in the highly conserved 5′-untranslated region of the enterovirus genome. Our assay detected all 60 different enterovirus species tested, whereas no reactivity was observed with the viruses from the other genera of the picornaviridae family, e.g., hepatovirus and parechovirus. Weak cross-reactivity was observed with 7 of the 90 different high-titer rhinovirus stocks but not with rhinovirus-positive clinical isolates. Analysis of a well-characterized reference panel containing different enteroviruses at various concentrations demon-strated that the enterovirus real-time TaqMan PCR is as sensitive as most of the currently used molecular detection assays. Evaluation of clinical isolates demonstrated that the assay is more sensitive than the “gold standard” method, i.e., viral culture. Moreover, the PCR assay can be used on different clinical specimens, such as plasma, serum, nose and throat swabs, cerebrospinal fluid, and bronchoalveolar lavage, without apparent inhibition. Our data demonstrate that the real-time TaqMan PCR is a rapid and sensitive assay for the detection of enterovirus infection. The assay has a robust character and is easily standardized, which makes it an excellent alternative for the conventional time-consuming viral culture.

Diagnostic Value of Measuring Epstein-Barr Virus (EBV) DNA Load and Carcinoma-Specific Viral mRNA in Relation to Anti-EBV Immunoglobulin A (IgA) and IgG Antibody Levels in Blood of Nasopharyngeal Carcinoma Patients from Indonesia

ABSTRACT Nasopharyngeal carcinoma (NPC) is a prevalent malignancy in Southeast Asia and is strongly associated with Epstein-Barr virus (EBV). We investigated the primary diagnostic value of circulating EBV DNA and anti-EBV immunoglobulin G (IgG) and IgA levels in Indonesian NPC patients (n = 149). By a 213-bp Epstein-Barr virus nuclear antigen 1 (EBNA1)-based real-time LightCycler PCR, 72.5% of patients were positive for EBV DNA in whole blood, with 29.5% having levels above a previously determined clinical cutoff value (COV) of 2,000 EBV DNA copies/ml, the upper level in healthy carriers. In a 99-bp LightCycler PCR, 85.9% of patients were positive and 60.4% had levels above the COV. This assay quantified a significantly higher EBV load than the 213-bp PCR assay (P < 0.0001), suggesting that circulating EBV DNA is fragmented. Using data from 11 different studies, we showed a significant inverse correlation between PCR amplicon size and the percentage of patients positive for circulating EBV DNA (Spearmans rho = −0.91; P < 0.0001). EBV DNA loads were unrelated to anti-EBV IgG or IgA levels, as measured by VCA-p18 and EBNA1-specific synthetic peptide-based enzyme-linked immunosorbent assays. The presence of circulating tumor cells was assessed by amplification of BamHI-A rightward frame 1 (BARF1) mRNA, a viral oncogene abundantly expressed in EBV-carrying carcinomas but virtually absent from EBV-associated lymphomas. Despite high EBV DNA loads and the presence of EBNA1 and human U1A small nuclear ribonucleoprotein mRNA, BARF1 mRNA was never detected in blood. We conclude that amplicon size significantly influences EBV DNA load measurement in NPC patients. The circulating EBV DNA load is independent of serological parameters and does not reflect intact tumor cells. The primary diagnostic value of the EBV DNA load for the detection of NPC is limited.

Cytolytic Virus Activation Therapy for Epstein-Barr Virus–Driven Tumors

Purpose: Nasopharyngeal carcinoma (NPC) is causally linked to Epstein–Barr virus (EBV) infection. Because all tumor cells carry EBV, the virus itself is a potential target for therapy. In these tumor cells, EBV hides in a latent state and expresses only a few non-immunogenic proteins for EBV maintenance and contributes to tumor growth. We developed a cytolytic virus activation (CLVA) therapy for NPC treatment, reactivating latent EBV, triggering immune recognition, and inducing susceptibility to antiviral therapy. Experimental Design: CLVA therapy combines gemcitabine (GCb) and valproic acid (VPA) for virus activation and tumor clearance with (val)ganciclovir (GCV) as the antiviral drug to block virus replication and kill proliferating virus-infected cells. CLVA treatment was optimized and validated in NPC cell lines and subsequently tested in 3 Dutch patients with NPC that was refractory to conventional treatment. Results: In NPC cell lines, both GCb and VPA can induce the lytic cycle of EBV. Their combination resulted in a strong synergistic effect. The addition of GCV resulted in higher cytotoxicity compared with chemotherapy alone, which was not observed in EBV-negative cells. CLVA therapy was analyzed in 3 patients with end-stage NPC. Patients developed increased levels of viral DNA in the circulation originating from apoptotic tumor cells, had disease stabilization, and experienced improved quality of life. Conclusions: Our results in the initial CLVA-treated patients indicate that the therapy had a biological effect and was well tolerated with only moderate transient toxicity. This new virus-specific therapy could open a generic approach for treatment of multiple EBV-associated malignancies. Clin Cancer Res; 18(18); 5061–70. ©2012 AACR.

Comparison of Quantitative Competitive PCR with LightCycler-Based PCR for Measuring Epstein-Barr Virus DNA Load in Clinical Specimens

ABSTRACT The aim of this study was to develop a LightCycler-based real-time PCR assay for monitoring the Epstein-Barr virus (EBV) DNA load in unfractionated whole blood. This assay was compared with quantitative competitive PCR (Q-PCR) for EBV. The LightCycler-based assay was highly sensitive and reproducible when quantifying plasmid DNA in either the presence or absence of healthy donor blood DNA. Amplifying plasmid DNA in DNA backgrounds from different donors slightly increased the variation of quantification, indicating that clinical specimen DNA has an influence on quantification. In most transplant recipients, a good correlation was observed between EBV DNA load dynamics determined by LightCycler and Q-PCR in follow-up samples, although the correlation between absolute values of EBV DNA loads was weak and occasional samples were false negative in the LightCycler assay. In 253 cross-sectional blood samples from patients with Burkitts lymphoma, infectious mononucleosis, or human immunodeficiency virus infection, a weak but significant correlation between the two methods was found (r2 = 0.37, P < 0.001). Our results indicate that the clinical specimen DNA background may influence the absolute values of EBV DNA load in LightCycler analyses but that this effect is rare. LightCycler PCR is very well suited for monitoring of EBV DNA load dynamics, and its diagnostic value is comparable to that of Q-PCR. To avoid false negativity or underestimation of viral load, future internal calibration of the LightCycler is recommended. This would also enhance EBV load assay standardization and interinstitute comparisons.

Sensing of latent EBV infection through exosomal transfer of 5′pppRNA

Significance Increasing evidence suggests that the exosomal messenger pathway warns neighboring cells against cellular stress and infection. Recent studies have shown that viruses and cancer cells exploit exosomes to transmit functional RNAs. Our studies reveal that a viral small RNA signal for innate immunity Epstein–Barr virus (EBV)-EBER1 is produced by latent EBV-infected B cells and recognized by noninfected dendritic cells activating an inflammatory response. We detected high amounts of EBV-EBER1 transcripts and EBV-microRNAs in inflamed skin lesions of autoimmune patients that are infiltrated with dendritic cells. Importantly, we found virtually no EBV-DNA present in these tissues, suggesting that continuous cell–cell EBER1 transmission via exosomes occurs in humans. We propose that innate sensing of latent EBV in predisposed individuals may be more harmful than previously thought. Complex interactions between DNA herpesviruses and host factors determine the establishment of a life-long asymptomatic latent infection. The lymphotropic Epstein–Barr virus (EBV) seems to avoid recognition by innate sensors despite massive transcription of immunostimulatory small RNAs (EBV-EBERs). Here we demonstrate that in latently infected B cells, EBER1 transcripts interact with the lupus antigen (La) ribonucleoprotein, avoiding cytoplasmic RNA sensors. However, in coculture experiments we observed that latent-infected cells trigger antiviral immunity in dendritic cells (DCs) through selective release and transfer of RNA via exosomes. In ex vivo tonsillar cultures, we observed that EBER1-loaded exosomes are preferentially captured and internalized by human plasmacytoid DCs (pDCs) that express the TIM1 phosphatidylserine receptor, a known viral- and exosomal target. Using an EBER-deficient EBV strain, enzymatic removal of 5′ppp, in vitro transcripts, and coculture experiments, we established that 5′pppEBER1 transfer via exosomes drives antiviral immunity in nonpermissive DCs. Lupus erythematosus patients suffer from elevated EBV load and activated antiviral immunity, in particular in skin lesions that are infiltrated with pDCs. We detected high levels of EBER1 RNA in such skin lesions, as well as EBV-microRNAs, but no intact EBV-DNA, linking non–cell-autonomous EBER1 presence with skin inflammation in predisposed individuals. Collectively, our studies indicate that virus-modified exosomes have a physiological role in the host–pathogen stand-off and may promote inflammatory disease.

Epstein-Barr Virus DNA Load in Nasopharyngeal Brushings and Whole Blood in Nasopharyngeal Carcinoma Patients before and after Treatment

Purpose: Nasopharyngeal carcinoma (NPC) is consistently associated with Epstein-Barr virus (EBV) and highly prevalent in Indonesia. EBV-DNA load can be used for early diagnosis and may have prognostic value. In this study, EBV-DNA load was evaluated in minimal invasive nasopharyngeal (NP) brushings and whole blood for initial diagnosis and therapy assessment against the standard-of-care diagnosis by biopsy with EBV-RISH and standard EBV-IgA serology. Experimental Design: NP brushings and blood samples were collected from 289 consecutive ENT patients suspected of NPCs and 53 local healthy controls. EBV-DNA load was quantified by real-time PCR and serology by peptide-based EBV-IgA ELISA. Tissue biopsies were examined by routine histochemistry and by EBER RNA in situ hybridization. Results: Repeated NP brushing was well tolerated by patients and revealed high viral load in the 228 NPC cases at diagnosis than 61 non-NPC cancer cases and healthy controls (P < 0.001). The diagnostic value of EBV-DNA load in blood and EBV-IgA serology was inferior to the NP brush results. The level of EBV-DNA load in brushes of patients with NPC was not related to T, N, or M stage, whereas elevated EBV-DNA load in blood correlated with N and M stage. EBV-DNA levels in brushings and whole blood showed a significant reduction at 2 months after treatment (P = 0.001 and P = 0.005, respectively), which was not reflected in EBV-IgA serology. Conclusions: NP brush sampling combined with EBV-DNA load analysis is a minimal invasive and well-tolerated diagnostic procedure, suited for initial diagnosis and follow-up monitoring of NPCs. Clin Cancer Res; 19(8); 2175–86. ©2013 AACR.

Quantitative detection of Epstein-Barr virus DNA in clinical specimens by rapid real-time PCR targeting a highly conserved region of EBNA-1.

Here we describe a LightCycler-based real-time PCR for quantitative detection of EBV DNA in clinical samples such as unfractionated whole blood, serum, or plasma. This assay is based on amplification of a highly conserved 213-bp region of the EBNA-1 gene, a single-copy gene of EBV required for maintenance of the EBV genome within the infected host cell. For real-time detection of amplicons, two internal hybridization probes are added, labeled with the fluoregenic dyes fluorescein and LCRed640, respectively. Simultaneous hybridization of these probes to the amplification products brings them in close proximity. Subsequent excitation of the fluorescein label by filtered excitation light from a light source in the LightCycler device will lead to fluorescence energy transfer (FRET) from the fluorescein label to the LCRed640 label. The light emitted from the LCRed640 label is then measured and correlates to the amount of product generated. The cycle at which the fluorescence exceeds the background is designated the threshold cycle. By comparing the threshold cycle of a clinical specimen with those of standard curve samples, the amount of EBV DNA in clinical samples can be determined. This real-time PCR approach is extremely rapid owing to efficient heat conduction by using glass capillaries, small reaction volumes, and air as heating medium. The closed-tube system eliminates the risk of PCR contamination by product carryover and also the need for post-PCR detection.

Aberrant Epstein-Barr virus persistence in HIV carriers is characterized by anti-Epstein-Barr virus IgA and high cellular viral loads with restricted transcription.

Objective:Epstein–Barr virus (EBV)-positive lymphomas in HIV carriers are paralleled by elevated EBV-DNA loads in the circulation. Approximately 20% of asymptomatic HIV carriers also show elevated circulating EBV-DNA loads. We aimed to characterize the nature of this EBV DNA and to determine the transcriptional phenotype of EBV in blood, in relation to serological parameters. Design:A total of 197 random asymptomatic HIV carriers, representing 2% of the Dutch HIV-positive population, were sampled for blood, peripheral blood mononuclear cells and plasma. In addition, 39 EBV-DNA carriers were sampled twice, with a 5-year interval. Methods:EBV-DNA loads were determined by LightCycler-based real-time polymerase chain reaction (PCR). EBV transcription was studied by nucleic acid sequence-based amplification and reverse transcriptase PCR. IgA and IgG antibodies to EBV antigens EBNA1 and VCA-p18 were quantified by synthetic peptide-based enzyme-linked immunosorbent assay. Results:Elevated EBV-DNA loads were found in whole blood of 19.3% of the tested HIV population, which were persistent in 82%. Plasma samples were EBV-DNA negative and circulating EBV DNA could be attributed to the B-cell compartment. Transcription of only LMP2 and (non-translated) transcripts from the BamHI-A region of the EBV genome was found, whereas EBNA1, LMP1 and lytic EBV transcripts were absent despite high cellular EBV-DNA loads. IgA-reactivity to VCA-p18 was seen in 69%. IgG to VCA-p18 was significantly higher in high EBV-DNA load carriers. Conclusion:Asymptomatic HIV carriers show aberrant EBV persistence in the circulation, characterized by elevated, B-cell-associated EBV-DNA loads. EBV transcription is restricted, arguing for EBV gene shutdown in circulating EBV-carrying B cells. Increased IgA and IgG reactive to VCA-p18 is indicative of increased lytic EBV replication, possibly occurring at mucosal lymphoid sites but not in the circulation.

Conserved mutation of Epstein-Barr virus-encoded BamHI-A Rightward Frame-1 (BARF1) gene in Indonesian nasopharyngeal carcinoma

BackgroundBamHI-A rightward frame-1 (BARF1) is a carcinoma-specific Epstein-Barr virus (EBV) encoded oncogene. Here we describe the BARF1 sequence diversity in nasopharyngeal carcinoma (NPC), other EBV-related diseases and Indonesian healthy EBV carriers in relation to EBV genotype, viral load and serology markers. Nasopharyngeal brushings from 56 NPC cases, blood or tissue from 15 other EBV-related disorders, spontaneous B cell lines (LCL) from 5 Indonesian healthy individuals and several prototype EBV isolates were analysed by PCR-direct sequencing.ResultsMost NPC isolates revealed specific BARF1 nucleotide changes compared to prototype B95-8 virus. At the protein level these mutations resulted in 3 main substitutions (V29A, W72G, H130R), which are not considered to cause gross tertiary structure alterations in the hexameric BARF1 protein. At least one amino acid conversion was detected in 80.3% of NPC samples compared to 33.3% of non-NPC samples (p < 0.001) and 40.0% of healthy LCLs (p = 0.074). NPC isolates also showed more frequent codon mutation than non-NPC samples. EBV strain typing revealed most isolates as EBV type 1. The viral load of either NPC or non-NPC samples was high, but only in non- NPC group it related to a particular BARF1 variant. Serology on NPC sera using IgA/EBNA-1 ELISA, IgA/VCA-p18 ELISA and immunoblot score showed no relation with BARF1 sequence diversity (p = 0.802, 0.382 and 0.058, respectively). NPC patients had variable antibody reactivity against purified hexameric NPC-derived BARF1 irrespective of the endogenous BARF1 sequence.ConclusionThe sequence variation of BARF1 observed in Indonesian NPC patients and controls may reflect a natural selection of EBV strains unlikely to be predisposing to carcinogenesis. The conserved nature of BARF1 may reflect an important role in EBV (epithelial) persistence.

Can Epstein-Barr virus DNA load in nasopharyngeal brushings or whole blood predict recurrent nasopharyngeal carcinoma in a non-endemic region? A prospective nationwide study of the Dutch Head and Neck Oncology Cooperative Group

This study estimated the value of quantitative measurements of EBV markers in the clinical management of nasopharyngeal carcinoma in a non-endemic area. The aim was to predict prognosis and detect recurrent and residual disease. In 72 patients, EBV DNA load in blood and nasopharyngeal brushes, and IgA VCA-p18 and EBNA1 in plasma were measured at different time points. At diagnosis and post-treatment, a cut-off value was used for detecting disease [positive (PPV) and negative (NPV) predictive value]. The markers were correlated as a continuous variable with tumor stage, disease-free survival (DFS) and overall survival (OS). The Cox hazard ratio model assessed hazard ratios. At diagnosis, the markers were above the COV in 45, 92, 85 and 83xa0% of the patients, respectively. Post-treatment, DNA load test in blood and brush had the best discriminating power (blood DNA load test: PPV 39xa0% and NPV 97xa0%, brush for local disease: PPV 75xa0% and NPV 99xa0%). Post-treatment, DNA load in blood was the best predictor for OS and DFS [hazard ratio 3.2 (95xa0% CI 1.51–3.5) and 2.3 (95xa0% CI 1.72–5.8)]. Assessing the EBV DNA load in blood has significant prognostic value, although the clinical value is for discussion. The EBV DNA load in the brush might improve early detection of local failures post-treatment.