CMV INFECTION

L.F. Lisboa¹, D. Kumar¹, X. Pang², L. Tyrrell³, J. Huang¹, A. Åsberg⁴, A. Hartmann⁵, A. Humar^1
1Transplant Infectious Diseases, University of Alberta, Edmonton/AB/CANADA, ²Laboratory Medicine & Pathology, University of Alberta, Edmonton/AB/CANADA, ³Medical Microbiology And Immunology, University of Alberta, Edmonton/AB/CANADA, ⁴School Of Pharmacy, University of Oslo, Oslo/NORWAY, ⁵, Rikshospitalet-Radiumhospitalet HF, Oslo/NORWAY

Body: Introduction
MicroRNAs (miRNAs) are small nucleotides involved in post-transcriptional regulation of gene expression. Cytomegalovirus (CMV) expresses 14 mature miRNAs, some of which are implicated in immune evasion mechanisms and would be expected to promote viral replication. Other CMV miRNAs are thought to inhibit viral replication by targeting viral RNAs. CMV miRNA expression in vivo is largely uncharacterized and their potential use as biomarkers for prediction of virologic and clinical endpoints of CMV disease in transplant recipients has not been assessed. Methods In vivo expression profiling of 9-CMV miRNAs was performed in a large cohort of SOT recipients with CMV disease (VICTOR Study) using real-time quantitative PCR of whole blood samples. Absolute quantification was obtained by generation of synthetic miRNA oligonucleotide standard curves. Patient samples were assayed at the onset of CMV disease (day-0). All patients received standard oral or intravenous antiviral therapy for 3-weeks followed by 1-month of prophylaxis. All patients were followed with regular virologic sampling until 6-months after disease onset. Expression of CMV miRNAs on day-0 and plasma viral load data were tested for their potential as predictors of response to treatment, virologic and clinical recurrence endpoints. Results A total of 205 transplant patients with CMV disease were assessed. Quantitative expression of the following CMV miRNAs was determined: miR-US5-1, miR-UL22, miR-UL36, miR-US5-2, miR-US25-1, miR-US25-2-3p, miR-US25-2-5p, miR-US33-3p and miR-UL112. Detectable viral miRNAs were found ranging from 18.5% of patients (miR-US5-2) up to 79.3% (miR-UL22) of patients. Quantitative levels ranged from 1.66E3 to 9.57E6 copies/ug of input RNA. Overall, viral miRNA expression levels were significantly and positively correlated with baseline CMV viral load (ranging from Spearman’s rho 0.177 p=0.01 for miR-UL112 to 0.689 p<0.001 for miR-UL22). Day-0 detection of all miRNAs, except miR-US5-2 and miR-UL112, was significantly associated with residual detectable CMV viral load at the end of antiviral treatment (day 21) in the univariate analysis (p<0.03 to <0.001). Virologic recurrence within 6 months post-CMV disease was associated with detection of miR-UL22 and/or miR-US36 at baseline (p=0.005 and p=0.007, respectively) in the univariate analysis. In a multivariate analysis, including baseline viral load, miRNA expression was not predictive of treatment success or failure by day 21. However, expression of miR-UL22 was independently associated with virologic recurrence within 6-months from the initial CMV disease episode (p=0.008, OR 7.593, CI 1.709 – 33.251). Symptomatic disease recurrence within 6-months was not associated with the expression of any of the miRNAs analyzed. Conclusion The in vivo differential expression of 9 CMV miRNAs in transplant patients with CMV disease was characterized. We demonstrate that viral miRNAs are readily detectable in these patients. No negative effect on viral clearance kinetics was observed suggesting they do not play an important in vivo role in inhibiting viral replication. In keeping with a potential immune evasion role, detectable levels of miR-UL22 at diagnosis were associated with higher rates of virologic recurrence within 6-months post initial CMV disease episode, independent of the baseline viral load. This viral microRNA emerges as a potentially useful baseline biomarker in transplant patients with CMV disease.

Disclosure: All authors have declared no conflicts of interest.