European Review for Medical and Pharmacological Sciences 2020; 24: 1454-1459 In vitro effect of some nucleoside reverse transcriptase inhibitors against HSV-1 replication R. NAHID SAMIEI¹, S. EBRAHIMI¹, M. FANI¹, S. GHAFARI², M. MAKVANDI¹, N. KHODADAD¹, M. TABASI¹, A. TEIMOORI¹,3 1Infectious and Tropical Diseases Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran 2Infectious Diseases Research Center, Birjand University of Medical Sciences, Birjand, Iran 3Department of Virology, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran Abstract. – OBJECTIVE: The current study diseases, especially in developing countries. Despite was designed to investigate the effects of some the remarkable advances in anti-HSV therapies, re- nucleoside reverse transcriptase inhibitors (NR- current infections are frequently found among im- TIs) on HSV-1 infection. munocompromised patients, and there is not yet a MATERIALS AND METHODS: Initially, the 3,4 SwissTargetPrediction server was used to predict cure for latent infections . the interactions between HSV-1 thymidine kinase Nucleoside analogs have had impressive im- and acyclovir, stavudine, zidovudine, didanosine, pacts on health development and care as the cor- and entecavir. The effect of each component on nerstone of anticancer and antiviral medications. Vero cell viability was assessed by the MTT as- For example, antiviral therapy of neonatal HSV say. After treatment, the cell supernatants were infections using nucleoside analogs, including collected, and HSV-1 replication was analyzed by high-dose acyclovir (ACV), has led to a signifi- quantitative real-time PCR. RESULTS: The qPCR results revealed that viral cant reduction in infant mortality (from 50% to titers were reduced 41, 40, 19, 44, and 31-fold in 4%). Acyclovir has been used for more than three the presence of acyclovir, zidovudine, stavudine, decades to treat herpes simplex virus infections. didanosine, and entecavir, respectively. Despite the widespread application of acyclovir CONCLUSIONS: Our findings indicate that NR- worldwide, the prevalence of acyclovir resistance TIs significantly reduce HSV-1 replication in cell in immunocompetent patients remains stable at culture. approximately 0.3%. In immunocompromised Key Words: patients, the prevalence of resistant virus has sim- Herpes Simplex Virus (HSV), NRTIs, Acyclovir (ACV), ilarly remained stable but at a higher level, typi- Stavudine (STV), Zidovudine (ZDV), Didanosine (ddI), 5 Entecavir (ETV). cally 4% to 7% . Therefore, new antiviral drugs alone or in combination with acyclovir should be developed to control resistance. Nucleoside analogue reverse transcriptase inhibitors (NR- Introduction TIs), for example, stavudine (STV)/d4T, zidovu- dine (ZDV)/AZT, didanosine (ddI), and entecavir Herpes simplex virus type 1 (HSV-1) is one (ETV), are commonly used in the treatment of of the most prevalent, incurable virus infections human immunodeficiency virus (HIV) infections worldwide, belonging to the Herpesviridae fami- and other reverse transcriptase coding viruses ly1. The ability to establish and maintain latent and such as hepatitis B virus (HBV)6-8. Given the se- recurrent infections are characteristic of all herpes vere consequences of HSV recurrence in patients viruses. Following the primary infection in epithe- with a suppressed immune system, including can- lial cells, the latent infection leads to life-long virus cer, organ transplantation, and HIV-infected indi- persistence in sensory nerve ganglia2. HSV-1 is fre- viduals, it is essential and inevitable to search for quently associated with oral and perioral infections. novel and combination therapies that can target It leads to severe infections, such as keratitis, corne- both HSV and HIV with minimum side effects. al blindness, and encephalitis. The advent of potent Alongside this, the current study was aimed to anti-inflammatory drugs and their extensive use in evaluate the effect of some NRTIs against HSV-1 medical wards have led to increased HSV-1-related replication in Vero cell line. 1454 Corresponding Author: Ali Teimoori, MD; e-mail: [email protected] In vitro effect of some nucleoside reverse transcriptase inhibitors against HSV-1 replication Materials and Methods Cytotoxicity Assay Cell toxicity of each nucleoside analog and Bioinformatics Analysis optimum concentrations for cell viability were All NRTIs were analyzed regarding their measured using MTT assay9. The 3-(4,5-Dimeth- mechanism of action using http://www.expasy.org ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (as well as the SwissTargetPrediction database (MTT; Sigma-Aldrich; St. Louis, MO, USA) pow- (STP). The NRTIs with thymidine kinase (TK) der was dissolved in PBS at the concentration of 5 interactions such as stavudine (STV), zidovudine mg/ml. Vero cells were plated in 96-well plates at (ZDV), didanosine (ddI), entecavir (ETV), and an initial density of 1×104 cells/well to reach 70- acyclovir (ACV, as a control group) were selected. 80% confluency. On the next day, the cells were treated with different concentrations of each nu- Cell Lines and Drugs cleoside analogs. Approximately 48 h after drug The African green monkey kidney (Vero) treatments, the medium was replaced with a fresh cell line was obtained from the Pasteur Institute medium containing 0.5 mg/ml MTT solution and of Iran. The cell line was cultured in Dulbec- incubated for 4 h at 37°C. After incubation, cul- co’s Modified Eagle’s Medium (DMEM; Gibco, ture media were discarded. Afterward, 100 µL Grand Island, NY, USA) supplemented with 10% of dimethylsulfoxide (DMSO) was added to each fetal bovine serum (FBS; Thermo Fisher Scien- well. The blue formazan crystals were dissolved tific, Waltham, MA, USA), IU/ml penicillin, and in DMSO, and the optical density (OD) was mea- 1 µg/ml streptomycin (Gibco, Grand Island, NY, sured at 570 nm wavelength via ELx800 micro- USA). All cells were incubated at 37°C in 5% plate reader (BioTeck, Winooski, VT, USA). CO2 and 90% humidity. Didanosine (D0162) and stavudine (D1413) drugs were purchased from Cell Treatment with HSV-1 and NRTIs Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, The cells were cultured in 24-well plates to USA), and acyclovir was obtained from Com- reach 70-80% confluency. The cells were cultured bino Pharm (Combino Pharm S.L., Barcelona, in 24-well plates to reach 70-80% confluency and Spain) as sodium salts. Entecavir and zidovudine were infected with HSV-1 at MOI 0.01 and appro- were obtained from Bakhtar Bioshimi (Bakhtar priate concentration of each drug (ACV: 13 μM, BioShimi Co., Kermanshah, Iran). ddI: 5 μM, STV: 10 μM, ZDV: 1 μM, ETV: 1.7 µM). About 72 h after infection, the supernatant Plaque Assay of cell cultures was centrifuged (8000 g, 5 min, Pre-cultured vero cells were seeded 5 × 105 4°C) and stored at -20ºC until DNA extraction. cells into six well plates to reach a confluent HSV-1 DNA was extracted using a High Pure Vi- monolayer. Then, 10-fold dilutions of HSV-1 ral Nucleic Acid kit (Roche, Mannheim, Germa- stock (obtained from the Pasteur Institute of Iran) ny) according to the manufacturer’s protocol. were prepared in DMEM, and a 500 µl volume of each dilution was added to each well. The plates Primers and Probe were placed at 37°C with 5% CO2 for 1 h. The In the present study, the HSV-1 primers and viral inoculum was replaced with a 1:1 mixture of probe set (Metabion, GmbH, Plannegg, Germany) 2x DMEM and agarose (1.5% in Milli-Q® water) were designed in the NCBI primer BLAST to spe- with 2% FBS. The plates were incubated at 37°C cifically detect the UL27 region, which encodes with 5% CO2 for 48 h. The cells were fixed with the viral glycoprotein B (gB). The sequence and 10% formaldehyde and stained with 1% crystal characteristics of the oligonucleotides used in this violet in ethanol. study are shown in Table I. Table I. The sequences of primers and probe used in the study. Oligonucleotide Sequence Region in Amplicon gB gene size Probe 5′-6-FAM-ATCACCACCGTCAGCACCTTCATCG-BHQ-1-3′ 702-726 Sense primer 5′-TCCAGCATGGTGATGTTGAG-3′ 680-996 96 bp Antisense primer 5′-CGTGTACTTCGAGGAGTA CG-3′ 756-775 1455 R. Nahid Samiei, S. Ebrahimi, M. Fani, S. Ghafari, M. Makvandi, N. Khodadad, M. Tabasi, A. Teimoori Real-time PCR have been plotted against the serial dilutions of Real-time PCR was performed using the virus concentrations, was drawn to evaluate QuantiNova Probe PCR kit (Qiagen, GmbH, primers and qPCR reactions. Reaction efficacy Hilden, Germany). Each reaction contained 2 μl was about 100%, which is acceptable for compar- template DNA, 1 μl Rox, 0.2 µM probe, 0.4 µM ative qPCR between treated and non-treated sam- of each primer, 10 μl Qiagen Taqman Master Mix ple groups (Figure 2). (2x), and up to 20 µl water. The amplification was NRTIs treatment significantly reduced the vi- carried out as follows: initial incubation at 95°C ral titers in comparison with the non-treatment for 2 min, 35 cycles of 95°C for 10 s, and 60°C control. Viral titers were reduced 40, 39.7, 43.3, for 15 sec. The standard curve was drawn using 29.7, and 17.7-fold in the presence of acyclovir, 10-fold serial dilutions of HSV-1 sample to deter- zidovudine, didanosine, entecavir, and stavudine, mine the reaction’s efficiency. respectively. Our findings indicate that there is a significant difference in viral titers between Statistical Analysis NRTIs treatment groups and the non-treatment The statistical analysis was performed with control (p <0.05, ANOVA). The Tukey’s test Prism V 5.01 (GraphPad Software Inc., San Di- showed that didanosine (p <0.06) and zidovu- ego, CA, USA). To compare the variables, we car- dine (p <0.99) act similarly to acyclovir, while ried out the ANOVA test, followed by the Tukey entecavir and stavudine had no advantage over post hoc test.