Quantification of gp350/220 Epstein–Barr Virus (EBV) mRNA by Real-Time Reverse Transcription-PCR in EBV-Associated Diseases

Abstract

Quantification of the Epstein-Barr (EBV) DNA genome by quantitative real-time PCR has been useful for the management of EBV-associated diseases (1-4 ). Nevertheless, it gives no information on the various patterns of expression of the latent genes or on the presence of a lytic infection. EBV lytic mRNAs have been detected in infectious mononucleosis and some EBV-associated malignancies, but their relevance is still debated (5-7 ). The quantification of late EBV transcripts may bring additional information to EBV DNA quantification for the understanding and follow-up of EBV-associated disease, as already demonstrated for other herpes viruses (8 -11 ).In this study, we developed a method for quantifying a late transcript of the highly conserved EBV envelope glycoprotein gene, gp350/220 (12, 13 ), that uses real-time reverse transcription-PCR (RT-PCR), the Taqman ® technology, and serial dilutions of in vitro transcripts to quantify mRNA. gp350/220 mRNA was quantified in EBV-infected cell lines and was then applied to a series of clinical samples.Total RNA from cell lines (500 000 cells), clinical samples, or in vitro transcripts (5 L) was extracted with the High-Pure-RNA-Isolation-Kit Absolute quantification was performed with an external RNA calibration curve. The RNA calibrator was obtained by in vitro transcription of 1 g of a constructed plasmid for 2 h at 37°C (SP6/T7-Transcription-Kit ® ; Roche). The plasmid was obtained by cloning in pSPT18 the 200-bp gpU2/gpL2 RT-PCR product, obtained from B95-8 cell line RNA. Serial dilutions of the RNA calibrator were analyzed by real-time RT-PCR, and a wide linear range (50 to 5 ϫ 10 6 copies) was obtained, indicating the efficiency of the amplification and the sensitivity of the method.The amplification efficiency in gp350/220-negative samples was controlled by real-time RT-PCR of a reference gene using LightCycler-h-PBGD-HousekeepingGene-Set ® (Roche). The specificity of the method was confirmed by use of EBV-negative BJAB cells and cells infected by five other herpesvirus strains. A PCR without the reverse transcription step confirmed the absence of DNA contamination.We evaluated the intraassay repeatability of the method by quantifying in the same assay three aliquots of the calibrators and three aliquots of three biopsy specimens. The CV of the calculated log concentrations ranged from 0.18% to 4.2%. We assessed the interassay reproducibility by quantifying three other aliquots of the same biopsy extracts in three separate runs and five aliquots of the calibrators in five independent assays. The CV ranged from 0.68% to 3.8% (see the Data Supplement that accompanies the online version of this Technical Brief at http:// www.clinchem.org/content/vol50/issue10/). gp350/220 mRNAs was quantified in the EBV-infected cell lines P3HR1, B95-8, and Akata. The P3HR1 and B95-8 B-cell lines spontaneously release viral particles. Moreover, the EBV lytic cycle can be superinduced by treatment with 30 g/L 12-tetradecanoylphorbol 13-acetate and 3 mmol/L sodium butyrate. The nonprodu...

Epstein-Barr Virus Stimulates Torque Teno Virus Replication: A Possible Relationship to Multiple Sclerosis

“…The actual activity of the EBV DNA is less clear in the Ramos/EBV cell line and unknown for the ND1 cell line. We therefore quantified the EBV envelope glycoprotein gp350/220 mRNA as marker for EBV lytic activity in all EBV positive cell lines used in this study [79]. The results obtained showed a similar pattern as the EBV genome quantification.…”

“…EBV activity/replication was measured by quantifying mRNA production of a 200 bp fragment of the EBV envelope glycoprotein gene gp350/220 [79]. Primers and a hydrolysis probe used are listed in Table 1.…”

“…EBV gp350/220 gene expression was quantified using an external RNA calibration standard which was generated as follows: The EBV late envelope glycoprotein gp350/220 gene was transcribed from B95-8 cellular RNA with SuperScript II reverse transcriptase (Invitrogen) using specific primers containing restriction sites for BamHI and EcoRI ( Table 1). The resulting 200 bp cDNA amplicon was cloned into the pSPT18 vector (SP6/T7 Trancription Kit, Roche) as described previously [79]. Sequence analysis verified the sequence.…”

Methylation of Epstein–Barr virus Rta promoter in EBV primary infection, reactivation and lymphoproliferation

“…Regarding EBV latency and reactivation, several studies have tried to identify and understand the role of the EBV lytic cycle in EBV‐related cancers and in the oropharyngeal tissue of patients presenting with recurrent or chronic tonsillitis by measuring the EBV DNA load [Gupta et al, ; Germi et al, ; Hsieh et al, ; Dogan et al, ; Sahiner et al, ]. The EBV DNA load in a tumor biopsy may be higher than in a non‐malignant biopsy (Fig.…”

Inhibition of Epstein–Barr virus replication by small interfering RNA targeting the Epstein–Barr virus protease gene

“…Inhibition of Epstein-Barr virus replication by small interfering RNA targeting the Epstein-Barr virus protease gene Introduction nasopharyngeal carcinoma (NPC); immediate early, early or late transcripts or proteins are regularly reported in EBV-associated cancers [12][13][14][15][16]; the lytic cycle has been demonstrated to be important for B-cell immortalization and for the development of EBV-associated lymphoproliferative disease in animal models [17][18][19]; the EBV-associated hairy leukoplasia, a rare tongue lesion in immunodeficient patients, is characterized by an EBV replication in squamous epithelial cells that is sensitive to antiviral drugs [20]; and treatment with cytotoxic T-cells targeting the lytic antigen might be useful against EBV-associated post-transplantation lymphoproliferative diseases [21,22]. Thus, inhibition of the EBV lytic cycle might be valuable in the treatment of EBV-associated diseases.…”