Pteropine Orthoreovirus, PRV7S (Sikamat Virus) Demonstrates Oncolysis in Nasopharyngeal Carcinoma Cell Lines

Abstract

Background: Oncolytic properties had been demonstrated in Mammalian Orthoreovirus (MRV) and Avian Orthorevirus (ARV). Besides MRV and ARV, Pteropine Orthoreovirus (PRV) is also categorized under the genus Orthoreovirus. PRV7S (Sikamat virus) is an orthoreovirus isolated in Malaysia. Present study aims to investigate the oncolytic effects of PRV7S on ranges of nasopharyngeal carcinoma (NPC) cells through apoptosis in comparison to MRV3. Methods: Non-cancerous nasopharyngeal (NCNP) and NPC cells were infected by PRV7S and MRV3. The effects of PRV7S on the proliferation inhibition and apoptotic activity of NPC cells was examined using MTT assay and flow cytometry. Additionally, western blot assay was performed to analyze the expression of RAS and apoptotic protein. Lastly, qPCR assay was performed to demonstrate that PRV7S and MRV3 replicated in infected-NPC and infected-NCNP cells. Results: The proliferation of NPC cells were significantly inhibited after PRV7S infection in a time dependent manner in comparison to infected-NCPC cells. Flow cytometry analysis showed that PRV7S infection was able to induce apoptosis on NPC cells at 48 hpi. Western blot results showed that upon PRV7S infection, N/H/K RAS protein expression was reduced, whereas caspase-3 protein expression increased in NPC cells. qPCR assay showed higher viral load of PRV7S found in infected-NPC compared to infected-NCNP cells. Conclusions: PRV7S inhibits the proliferation and induces apoptosis of NPC cells similar to MRV3. Therefore, PRV7S is a potential oncolytic virus.

Surveillance, isolation and genomic characterization of Pteropine orthoreovirus of probable bat origin among patients with acute respiratory infection in Malaysia

“…Real‐time PCR was performed using SensiFAST SYBR kit (Bioline). Modified primers sequences of PRVSig1F2 5′‐TGCTGATTGGAAYGCTGACT‐3′ and PRVSig1R2 5′‐CKGAAAAGGYTTGAKACGCC‐3′ were used for quantification of PRV by targeting the Sigma 1/A gene 8,19 . The qPCR protocol started with 95°C for 3 min followed by 40 cycles of 95°C for 10 s then 57°C for 30 s and the last step was 72°C for 50 s, with data collection and real‐time analysis by iQ5 real‐time PCR detection system (Bio‐Rad Laboratories).…”

“…Cells were observed for cytopathic effect (CPE) for 5 days postinfection and the titers were expressed as TCID 50 /ml. Multiplicity of infection (MOI) was calculated based on the TCID 50 as previously described 19 . For virus kinetic study, OKF‐6, NP69 and AC16 cells were infected with 12MYKLU1034, PRV3M or PRV4K, respectively, at a MOI of 0.1.…”

“…Multiplicity of infection (MOI) was calculated based on the TCID 50 as previously described. 19 For virus kinetic study, OKF-6, NP69 and AC16 cells were infected with 12MYK-LU1034, PRV3M or PRV4K, respectively, at a MOI of 0.1. After adsorption for 1 h, the inocula were removed, and the cells were incubated with the appropriate complete media at 37°C and 5% CO 2 , except for AC16 cells which were cultured in DMEM/F12 with 2% FBS with 1% penicillin/streptomycin.…”

The role of Epstein–Barr virus in nasopharyngeal carcinoma

“…Oncolytic viruses have been genetically engineered to express cytokines, costimulatory molecules, immune checkpoint inhibitors, or bispecific T-cell engager (BiTE) molecules to boost anti-tumour immune response and suppress tumour tolerance. For instance, an oncolytic adenovirus MG1 encoding HPV oncogenes E6 and E7 demonstrated tumour-specific responses in preclinical studies and is currently undergoing a phase I clinical trial involving patients with HPV-associated malignancies ( Wang et al, 2011 ; Loh et al, 2022 ; Tan et al, 2022 ). Thus, oncolytic viruses represent attractive targets for the development of therapeutic tools against EBV-associated NPC.…”