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Objective(s): Oncolytic Herpes simplex virus type 1 (HSV-1) has emerged as a promising strategy for cancer therapy. However, development of novel oncolytic mutants has remained a major challenge owing to low efficiency of conventional genome editing methods. Recently, CRISPR-Cas9 has revolutionized genome editing.Materials and Methods: In this study, we aimed to evaluate the capability of CRISPR-Cas9 to manipulate the UL39 gene to create oncolytic HSV-1. Herein, three sgRNAs were designed against the UL39 gene and transfected into HEK-293 cell line followed by infection with HSV-1 KOS.Results: After three rounds of plaque purification, several HSV-1 mutants were identified by PCR analysis and sequencing. One of these mutations in which 55 nucleotides were deleted resulted in a frameshift mutation that in turn produced a truncated protein with only 167 amino acids from 1137 amino acids. Functional analysis in Vero and primary fibroblast cells revealed that viral replication was significantly lower and plaque size was smaller in the HSV-1 mutant compared with HSV-1 KOS. Moreover, the relative amount of viral genome present in the supernatants of infected cells (Vero and primary fibroblast cells) with HSV-1 mutant was significantly decreased compared with those of HSV-1 KOS.Conclusion: Our data revealed that targeting UL39 with CRISPR-Cas9 could develop oncolytic HSV-1.

Objective(s): Here, a reporter cell line containing two reporter vectors were developed, in order to monitor the Human T-Lymphotropic Virus type1(HTLV-1) infectivity and the cell viability simultaneously. Materials and Methods: The reporter cell line was constructed by stably transfected baby hamster’s kidney cell line (BHK-21), with the genomes expressing two different reporters in separate plasmids. The first reporter gene is transactivated by the HTLV-1 tax protein, while the second reporter is continuously expressed when introduced into a mammalian cell. In order to show its functionality, the effect of the drug mix on HTLV-1 was assayed by this system and was compared to the results obtained by other methods. Results: HTLV-1 reporter cell line was found to produce high level of luciferase when co-cultured with MT-2 and Hut-102 cells but not with Jurkat cell. Moreover, the combination therapy against HTLV-1 can reduce luciferase expression of the cell when co-cultured with MT-2 and Hut-102 comparable to the ELISA (R=0.932, P-value =0.002). In addition, the results revealed the superiority of the present system over the molecular methods. Conclusion: The results demonstrated that the biological assay system is a beneficial tool for the medium-throughput anti-HTLV-1 drug screening and inhibitory effect.