Multiple gene targeting siRNAs for down regulation of Immediate Early-2 (Ie2) and DNA polymerase genes mediated inhibition of novel rat Cytomegalovirus (strain All-03)

Abstract Background: Cytomegalovirus (CMV) is an opportunistic pathogen that causes severe complications in congenitally infected newborns and non-immunocompetent individuals. Developing an effective vaccine is a major public health priority and current drugs are fronting resistance and side effects on recipients. In the present study, with the aim of exploring new strategies to counteract CMV replication, several anti-CMV siRNAs targeting IE2 and DNA polymerase gene regions were characterized and used as in combinations for antiviral therapy. 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<0.05) of viral mRNA and 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.

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Multiple gene targeting siRNAs for down regulation of Immediate Early-2 (Ie2) and DNA polymerase genes mediated inhibition of novel rat Cytomegalovirus (strain All-03)

Virology Journal ◽

10.1186/s12985-020-01436-5 ◽

2020 ◽

Vol 17 (1) ◽

Author(s):

Krishnan Nair Balakrishnan ◽

Ashwaq Ahmed Abdullah ◽

Jamilu Abubakar Bala ◽

Faez Firdaus Abdullah Jesse ◽

Che Azurahanim Che Abdullah ◽

...

Keyword(s):

Dna Polymerase ◽

Colorimetric Assay ◽

Opportunistic Pathogen ◽

Digital Pcr ◽

Host Cells ◽

Effective Vaccine ◽

Control Group ◽

Polymerase Gene ◽

Infectious Dose ◽

Dna Polymerase Gene

Abstract Background Cytomegalovirus (CMV) is an opportunistic pathogen that causes severe complications in congenitally infected newborns and non-immunocompetent individuals. Developing an effective vaccine is a major public health priority and current drugs are fronting resistance and side effects on recipients. In the present study, with the aim of exploring new strategies to counteract CMV replication, several anti-CMV siRNAs targeting IE2 and DNA polymerase gene regions were characterized and used as in combinations for antiviral therapy. 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 < 0.05) of viral mRNA and 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.

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Multiple gene targeting siRNAs for down regulation of Immediate Early-2 (Ie2) and DNA polymerase genes mediated inhibition of novel rat Cytomegalovirus (strain All-03)

10.21203/rs.3.rs-44829/v1 ◽

2020 ◽

Author(s):

Krishnan Nair Balakrishnan ◽

Ashwaq Ahmed Abdullah ◽

Jamilu Abubakar Bala ◽

Faez Firdaus Abdullah Jesse ◽

Che Azurahanim Che Abdullah ◽

...

Keyword(s):

Dna Polymerase ◽

Colorimetric Assay ◽

Opportunistic Pathogen ◽

Digital Pcr ◽

Host Cells ◽

Effective Vaccine ◽

Control Group ◽

Polymerase Gene ◽

Antiviral Activities ◽

Dna Polymerase Gene

Abstract Background Cytomegalovirus (CMV) is an opportunistic pathogen that causes severe complications in congenitally infected newborns and non-immunocompetent individuals. Developing an effective vaccine is a major public health priority and current drugs are fronting resistance and side effects on recipients. In the present study, with the aim of exploring new strategies to counteract CMV replication, several anti-CMV siRNAs targeting IE2 and DNA polymerase gene regions were characterized and used as in combinations for antiviral therapy. 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 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 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 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 < 0.05) of viral mRNA and 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 no any mutation was identified, 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.

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Site of the base change in the vaccinia virus DNA polymerase gene which confers aphidicolin resistance.

Journal of Virology ◽

10.1128/jvi.63.9.4060-4063.1989 ◽

1989 ◽

Vol 63 (9) ◽

pp. 4060-4063 ◽

Cited By ~ 12

Author(s):

F M DeFilippes

Keyword(s):

Vaccinia Virus ◽

Dna Polymerase ◽

Base Change ◽

Polymerase Gene ◽

Dna Polymerase Gene

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The silent presence of Mycoplasma hominis in patients with prostate cancer

Pathogens and Disease ◽

10.1093/femspd/ftaa037 ◽

2020 ◽

Vol 78 (7) ◽

Author(s):

Saman Saadat ◽

Pezhman Karami ◽

Mohammad Jafari ◽

Mahdi Kholoujini ◽

Zahra Rikhtegaran Tehrani ◽

...

Keyword(s):

Prostate Cancer ◽

Mycoplasma Hominis ◽

Opportunistic Pathogen ◽

Host Cells ◽

Prostate Tissue ◽

Control Group ◽

P Value ◽

Retrospective Case ◽

Formalin Fixed Paraffin Embedded ◽

Pcr Targeting

ABSTRACT Background Mycoplasma hominis, an opportunistic pathogen in human genitourinary tract, can cause chronic infection in the prostate. Intracellular survival of M. hominis leads to a prolonged presence in the host cells that can affect the cell's biological cycle. In the present study, we aimed to evaluate the frequency of M. hominis DNA in prostate tissue of Iranian patients with prostate cancer (PCa) in comparison to a control group with benign prostatic hyperplasia (BPH). Methods This research was a retrospective case-control study using 61 archived formalin-fixed paraffin-embedded (FFPE) blocks of prostate tissue from patients with PCa and 70 FFPE blocks of patients with BPH. Real-time PCR, targeting two different genes, 16S rRNA and yidC, in the M. hominis genome was performed for all specimens. Results Out of 61 blocks of prostate biopsy from patients with PCa, eight samples (13%) were positive for M. hominis, while the bacterium was not detected in any of the 70 blocks of patients with BPH (P value, 0.002). Conclusions The high frequency of M. hominis in patients with PCa likely shows a hidden role of the organism in prostate cancer during its chronic, apparently silent and asymptomatic colonization in prostate.

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