EBV miRNA expression profiles in different infection stages: A prospective cohort study

Abstract

The Epstein-Barr virus (EBV) produces different microRNAs (miRNA) with distinct regulatory functions within the infectious cycle. These viral miRNAs regulate the expression of viral and host genes and have been discussed as potential diagnostic markers or even therapeutic targets, provided that the expression profile can be unambiguously correlated to a specific stage of infection or a specific EBV-induced disorder. In this context, miRNA profiling becomes more important since the roles of these miRNAs in the pathogenesis of infections and malignancies are not fully understood. Studies of EBV miRNA expression profiles are sparse and have mainly focused on associated malignancies. This study is the first to examine the miRNA profiles of EBV reactivation and to use a correction step with seronegative patients as a reference. Between 2012 and 2017, we examined the expression profiles of 11 selected EBV miRNAs in 129 whole blood samples from primary infection, reactivation, healthy carriers and EBV seronegative patients. Three of the miRNAs could not be detected in any sample. Other miRNAs showed significantly higher expression levels and prevalence during primary infection than in other stages; miR-BHRF1-1 was the most abundant. The expression profiles from reactivation differed slightly but not significantly from those of healthy carriers, but a specific marker miRNA for each stage could not be identified within the selected EBV miRNA targets.

Fig 1. Comparison of EBV miRNA presence and respective expression levels during different stages of infection.

(A) The number of samples with a positive miRNA PCR signal in the respective group is shown as a percentage. (B) Non-normalized averaged miRNA copy numbers for all 4 study groups. Calibration was performed for each miRNA species using synthetic miRNAs of identical sequence. (C) Relative expression levels were normalized to the SN group. Only detectable miRNAs are shown. Relative expression was calculated by the efficiency-corrected ΔΔCt method [35]. (D) Expression ratios in relation to the HC group, which represents the basal miRNA levels in EBV-positive patients. Expression levels are shown as the mean and standard deviation. Comparisons were performed using the Kruskal-Wallis test with Dunn’s multiple comparison test. Significance was expressed as asterisk (* indicates p < 0.05).

Fig 2. Pairwise correlations of the miRNAs and viral load in the different patient groups.

The grey scale as well as the line sizes correspond to the respective Spearman correlation coefficient (rs).

Fig 3. Comparison of the expression profiles of an HLH sample and two PTLD samples.

Samples were collected from one patient with haemophagocytic lymphohistiocytosis (HLH) and two patients with post-transplant lymphoproliferative disorder (PTLD) and separately analysed. Expression levels were calculated by the efficiency-corrected ΔΔCt method.