METHODS: Six unique siRNAs with three each targeting different regions of IE2 (ie2a, ie2b and ie2c) and DNA polymerase (dpa, dpb and dpc) were prepared and tested for antiviral activities. The efficacy as an antiviral was determined in in-vitro by measuring TCID50 virus titer, severity of virus-induced cytopathic effect (CPE), intracellular viral genome loads by droplet digital PCR, the degree of apoptosis in siRNA-treated cells and relative expression of viral mRNA in infected Rat Embryo Fibroblast (REF) cells.
FINDINGS: Remarkably, the siRNAs: dpa, dpb and IE2b, significantly reduced virus yield (approximately >90%) compared to control group at day 18 post infection (p.i). Changes in CPE indicated that DNA polymerase siRNAs were capable of protecting cells against CMV infection at day 14 p.i with higher efficiency than GCV (at the concentration of 300 pmol). Gene expression analysis revealed a marked down regulation of the targeted DNA polymerase gene (73.9%, 96.0% and 90.7% for dpa, dpb and dpc siRNA, respectively) and IE2 gene (50.8%, 49.9% and 15.8% for ie2a, ie2b and ie2c siRNA, respectively) when measured by RT-qPCR. Intracellular viral DNA loads showed a significant reduction for all the DNA polymerase siRNAs (dpa: 96%, dpb: 98% and dpc:92) compared to control group (P
METHODOLOGY: A quasi-experimental study was conducted at three time-points (pre-intervention, post-intervention and one-month after working as a HO) on the level of confidence and readiness of medical graduates. The intervention was the Medicorp module, which included information and training needed for the HO such as common clinical cases in the wards, case referrals, experience sharing and hands on clinical training. We recruited eligible participants undergoing the course between April-November 2018. The adapted IMU Student Competency Survey was used to measure the confidence and readiness levels, which were scored from a Likert scale of 1-5. The higher score indicated higher levels of confidence or readiness.
RESULTS: A total of 239 participants were recruited at baseline (90% response rate). They were mostly female (77.8%), Malays (79.1%), single (90.0%), graduated overseas (73.6%), in 2018 (65.3%). The mean (SE) confidence scores significantly increased from 2.18 (1.00) pre-course to 3.50 (0.75) immediately after course and 3.79 (0.92) after one-month of work (p <0.001, η2 = 0.710). The mean (SE) readiness scores at pre-course, immediately and one-month post work were 2.36 (1.03), 3.46(0.78) and 3.70(0.90), respectively (p< 0.001, η2 = 0.612).
CONCLUSION: The HO Preparatory Course module was effective in increasing levels of confidence and readiness for medical graduates, most of whom are overseas graduates; namely Egypt, Russia and Indonesia.
METHODS: The rat embryo fibroblast (REF) cells were transfected with multi siRNA before infecting with CMV strain ALL-03. Viral growth inhibition was measured by tissue culture infectious dose (TCID50), cytopathic effect (CPE) and droplet digital PCR (ddPCR) while IE2 and DNA polymerase gene knockdown was determined by real-time PCR. Ganciclovir was deployed as a control to benchmark the efficacy of antiviral activities of respective individual siRNAs.
RESULTS: There was no significant cytotoxicity encountered for all the combinations of siRNAs on REF cells analyzed by MTT colorimetric assay (P > 0.05). Cytopathic effects (CPE) in cells infected by RCMV ALL-03 had developed significantly less and at much slower rate compared to control group. The expression of targeted genes was downregulated successfully resulted in significant reduction (P DNA copies (dpb + dpc: 79%, 68%; dpb + ie2b: 68%, 60%; dpb + dpc + ie2b: 48%, 42%). Flow cytometry analysis showed a greater percentage of viable and early apoptosis of combined siRNAs-treated cells compared to control group. Notably, the siRNAs targeting gene regions were sequenced and mutations were not encountered, thereby avoiding the formation of mutant with potential resistant viruses.
CONCLUSIONS: In conclusion. The study demonstrated a tremendous promise of innovative approach with the deployment of combined siRNAs targeting at several genes simultaneously with the aim to control CMV replication in host cells.