Evaluation of Thymidine Kinase and DNA polymerase Genes of Herpes Simplex Viruses type 1 Isolated from Patients: a phenotypic and genotypic acyclovir Susceptibility Study

Background and objectives: Mutations in Herpes Simplex Virus Thymidine kinase (TK, UL23) and DNA polymerase (pol, UL30) genes may confer resistance to acyclovir (ACV). Phenotypic resistance has to be determined along with genotypic resistance to achieve a complete acyclovir susceptibility. The aim of this study was to determine Acyclovir susceptibility patterns among HSV clinical isolates collected from 33 patients between 2016 and 2019, referred to the Clinical Microbiology Research Center, Namazi Hospital Shiraz, Iran. Materials and Methods: The samples were collected from Oro-pharyngeal, facial, eyes and other parts of skin body’s immunocompetent and immunocompromised individuals. Age of patients ranged between 2 and 67 years (mean 43.0 ± 17.3 years) all HSV stains were isolated and propagated in Vero cells. Viral stocks were tittered by plaque assay and the average titers were 107 PFU/ml. phenotypic susceptibility was determined by using three concentrations of ACV (0.01, 0.5 and 5 μg/ml) and based on it, the results were expressed as ability to reduce fifty percent virus plaques. Genotyping analysis was carried out by the amplification of viral TK and DNA Pol genes by PCR and upon purification of DNA fragments, sequencing was done and aligned based on the HSV-1 reference strain 17 (GenBank Accession No. X14112) and phylogenic tree was drawn with CLC Sequence Viewer software for all sequences. Results: All tested isolates were sensitive to 0.01 μg/ml concentration of ACV. On the other hand, there were several nucleotides variations through TK and Pol genes, including single nucleotides substitution, deletion or insertion when in comparison with HSV 1 reference strain. Conclusion: This study presents the results of phenotyping and genotyping 33 HSV-1 isolates obtained from immunocompetent and immunocompromised individuals within a time interval of 3 years. All samples of HSV-1 isolates were sensitive to the nucleoside analog ACV. Synonymous mutations were detected in the HSV-1 TK and Pol genes from whole samples have been described as natural polymorphisms. While data showed a high genetic variability of TK and DNA Pol of HSV-1 but none of them determined phenotypic resistance.