Assessment of the Human Cytomegalovirus UL97 Gene to identify the Resistance to Ganciclovir in Kidney Transplant Recipients in Labbafi-Nejad Hospital

Cytomegalovirus (CMV) is one of the major opportunistic especially after transplantation, and variant resistance to antiviral therapies. Human cytomegalovirus (HCMV) as one of the common virus, is epidemiologically varies in different regions of the world and between socioeconomic and age groups (1, 2). Therefore, for the accurate treatment of HCMV, diagnosis in an early stage is especially necessary to avoid poor clinical outcomes (3-5). Ganciclovir is a nucleoside analogue antiviral drug which is widely used to treat systemic CMV disease. To exert antiviral activity as an inhibitor of viral DNA polymerase (UL54), Ganciclovir should be phosphorylated. The UL97 kinase, a virus-encoded product, can activate the drug by monophosphorylation (6, 7). The CMV resistance to ganciclovir is favored by prolonged therapy and it is mainly associated with the presence of mutations within the UL97 gene (8, 9). As an impairment of GCV monophosphorylation, mutations in the human cytomegalovirus (HCMV) UL97 phosphotransferase have been associated with ganciclovir (GCV) resistance (8). Resistance to the GCV has been evaluated by many researchers in previous studies, while GCV have been used as the first-line treatment in most of HCMV patients (10).

During recent years, to detect HCMV-resistant mutations, many studied have been done and different methods such as the plaque reduction assay, ELISA, and DNA hybridization assays (11-13) have been used based on phenotypic testing, but these tests are burdensome, time-consuming, and labor-intensive (14). As a result, there is a significant need for early-stage detection of the emerging mutants (15).

The aim of this study was to determine the GCV-resistant HCMV in renal transplant recipient patients.

Our study focused on kidney transplant recipients with high levels of CMV viral load after antiviral therapy. 5 mL of the blood was collected from patients. After blood collection from patients, their sera were separated and stored at -20°C. DNA extraction was performed using the Roche DNA Mini kit, and it was extracted according to the manufacturer’s instructions. For checking the quality of sample, they were checked by a spectrophotometer (Nanodrop 2000), and they were kept at -80°C. Samples were detected and sent to the Labbafi Nejad specialty laboratory for identification of UL97 gene.

Specific regions of the UL97 gene of HCMV in the samples were amplified using nested PCR. The sequence of the forward primer in the first round of PCR was UL97-F1 (5′-GTTTCATCACGACCAGTGGA-3′) and the reverse primer was UL97-R1 (5′-GGTCCTCCTCGCAGATTATG-3′). The second round primers were UL97-F2 (5′-TCATCACGACCAGTGGAAGCT-3′) and UL97-R2 (5′-GCGACACGAGGACATCTTGG-3′). The nested PCR was carried out in a 15 μL reaction mixture containing 7.5 μL master mix (Bie & Berntsen A/S), 1 μL of the equally-mixed primers, 1.5 μL DNA, and 5 μL ddH₂O. The following thermal cycling condition was used for PCR reaction: 94°C for 30 seconds, 54°C for 40 seconds, 72°C for 2 minutes for 40 cycles, and 72°C for 10 minutes.

All of the selected patients answered and completed questionnaires and informed consent forms for participation in this study.

During the observation period, 60 recipients developed CMV infection with or without tissue-invasive diseases. The specific nested PCR primers amplified the mutation region in the UL97 gene. The binding site of primers on the UL97 gene of HCMV (270 bp) were nucleotide numbers 400 - 670. Unfortunately, there was no positive sample.

Resistance of cytomegalovirus (CMV) to antiviral agents is the well-recognized phenomenon which has been observed in the laboratory and clinical environments (16, 17).

The emergence of drug-resistant HCMV strains especially in immunocompromised individuals with the active HCMV infection is a life-threatening condition (9). Drug-resistant HCMV was considered as a major problem in patients with acquired immunodeficiency syndrome until the recent introduction of highly active antiretroviral combination therapy, and unfortunately, we are faced a dramatically decrease in it’s the incidence in this clinical setting. Moreover, recently, HCMV antiviral drugs resistance in the transplantation is also known as an emerging problem. Ganciclovir-resistant CMV is mostly observed and caused clinical problems in solid-organ transplant recipients (18). One of the posttransplantation complications is resistance and it is predominantly observed among CMV-seronegative recipients of the organs from seropositive donors. Ganciclovir therapy length before CMV resistance ranges from 51 to 438 days in the kidney transplant recipients (19). However, resistance was identified after 3 months of treatment for most patients (19-21).

UL97 coding for a viral phosphotransferase and UL54 coding for the viral DNA polymerase are two key viral genes which have been elucidated in the molecular mechanisms of drug-resistant HCMV and rely on the selection during treatment of HCMV strains harboring mutations. Generally, drug resistance mutations appear after the extended ganciclovir therapy. This most likely is due to the proofreading function of the viral DNA polymerase which greatly reduces nucleotide misincorporation resulting in a low mutation rates. The most prevalent sites of ganciclovir resistance mutations are in the UL97 gene (22).

Approximately, 95% of the GCV-resistant HCMV strains contain one or more mutations in the UL97 gene (23). Mutations in three specific codons (460, 594, and 595), have observed in approximately 70% of the GCV-resistant HCMV strains (24). The rate of HCMV drug resistance is widely vary depending on several factors such as type of patients, and several risk factors which have already been identified including the patient and disease-related factors, treatment-related factors, and viral factors (9, 25).

In the previous study some deletions in an immunocompromised patient associated with clinical resistance to the GCV, especially in the codons of the UL97 gene have been reported. In another study, Hantz et al. explored a new GCV-resistance mutation in the kidney transplant patients which is a deletion of codon 601 of the UL97 gene (6). Our study could not present any mutation in a GCV-resistant HCMV isolate among renal transplant recipients patients.