Epstein-Barr virus load in rheumatoid arthritis patients and normal controls: accurate quantification using real time PCR

Objective

For twenty years the Epstein-Barr Virus (EBV) has been suspected to contribute to the pathogenesis of rheumatoid arthritis (RA). RA is strongly associated with shared epitope positive HLA-DR alleles. EBV load has been extensively studied in RA patients, using semi-quantitative PCR. Inconsistent results reflect the lack of sensitivity and accuracy of this technique. We quantified EBV in peripheral blood mononuclear cells by real time PCR, to (1)determine whether EBV load is higher in RA patients compared to controls and (2)test whether HLA-DR alleles influence EBV load in RA patients and controls.

Methods

Fifty patients fulfulling the 1987 ACR criteria for RA were studied. Most patients were treated with DMARDs including methotrexate, leflunomide, etanercept or infliximab. Fifty healthy controls were chosen from bone marrow donors at the Marseille blood transfusion center. HLA-DR genotyping of patients and controls was performed by PCR-SSP. Real time PCR was performed using a Roche LightCycler. A 214 bp fragment from the highly conserved long Internal Repeat IR1 was amplified. IR1 is repeated eleven times in the EBV genome, increasing the sensitivity of detection. Two specific hybridization probes were used to recognize adjacent internal sequences within the target. EBV-positive Burkitt's lymphoma cell line was used as an external standard.

Results

EBV load is expressed in EBV genome copy number per microgramm of human genomic DNA. Preliminary results show a higher EBV load in RA patients (0–60 copies/μg) than in normal controls (0–10 copies/μg). We are currently testing the influence of HLA-DR genotypes on EBV load in controls.