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Adenovirus delivered short hairpin RNA targeting a conserved site in the 5' non-translated region inhibits all four serotypes of dengue viruses.

Korrapati AB, Swaminathan G, Singh A, Khanna N, Swaminathan S - PLoS Negl Trop Dis (2012)

Bottom Line: There is neither a preventive vaccine nor an effective antiviral therapy against dengue disease.We show that this shRNA matures to the cognate siRNA and is able to inhibit effectively antigen secretion, viral RNA replication and infectious virus production by all four DENV serotypes.The data demonstrate the feasibility of using AdV5-mediated delivery of shRNAs targeting conserved sites in the viral genome to achieve inhibition of all four DENV serotypes.

View Article: PubMed Central - PubMed

Affiliation: Recombinant Gene Products Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India.

ABSTRACT

Background: Dengue is a mosquito-borne viral disease caused by four closely related serotypes of Dengue viruses (DENVs). This disease whose symptoms range from mild fever to potentially fatal haemorrhagic fever and hypovolemic shock, threatens nearly half the global population. There is neither a preventive vaccine nor an effective antiviral therapy against dengue disease. The difference between severe and mild disease appears to be dependent on the viral load. Early diagnosis may enable timely therapeutic intervention to blunt disease severity by reducing the viral load. Harnessing the therapeutic potential of RNA interference (RNAi) to attenuate DENV replication may offer one approach to dengue therapy.

Methodology/principal findings: We screened the non-translated regions (NTRs) of the RNA genomes of representative members of the four DENV serotypes for putative siRNA targets mapping to known transcription/translation regulatory elements. We identified a target site in the 5' NTR that maps to the 5' upstream AUG region, a highly conserved cis-acting element essential for viral replication. We used a replication-defective human adenovirus type 5 (AdV5) vector to deliver a short-hairpin RNA (shRNA) targeting this site into cells. We show that this shRNA matures to the cognate siRNA and is able to inhibit effectively antigen secretion, viral RNA replication and infectious virus production by all four DENV serotypes.

Conclusion/significance: The data demonstrate the feasibility of using AdV5-mediated delivery of shRNAs targeting conserved sites in the viral genome to achieve inhibition of all four DENV serotypes. This paves the way towards exploration of RNAi as a possible therapeutic strategy to curtail DENV infection.

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Related in: MedlinePlus

The effect of rAd-mediated shRNA expression on DENV RNA accumulation.Vero cells were pre-infected either with rAdsh-5b (red bars) or rAdsh-scr (green bars) followed 24 hours later by infection with DENV-1, DENV-2, DENV-3 and DENV-4. Total RNA was isolated on days 2 (panels A and B) and 7 (panels C and D) post-DENV infection and analyzed for DENV ’minus’ (panels A and C) and ‘plus’ (panels B and d D) sense viral genomic RNAs by strand-specific real time PCR analyses. DENV RNA was normalized to GAP RNA in each sample analyzed. The data depict DENV RNA levels in rAdsh-5b treated cells relative to those in the corresponding rAdsh-scr treated cells. Each experiment was carried out in triplicate wells and the entire experiment repeated twice.
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pntd-0001735-g004: The effect of rAd-mediated shRNA expression on DENV RNA accumulation.Vero cells were pre-infected either with rAdsh-5b (red bars) or rAdsh-scr (green bars) followed 24 hours later by infection with DENV-1, DENV-2, DENV-3 and DENV-4. Total RNA was isolated on days 2 (panels A and B) and 7 (panels C and D) post-DENV infection and analyzed for DENV ’minus’ (panels A and C) and ‘plus’ (panels B and d D) sense viral genomic RNAs by strand-specific real time PCR analyses. DENV RNA was normalized to GAP RNA in each sample analyzed. The data depict DENV RNA levels in rAdsh-5b treated cells relative to those in the corresponding rAdsh-scr treated cells. Each experiment was carried out in triplicate wells and the entire experiment repeated twice.

Mentions: The results of a comparison based on real time RNA PCR assay of the relative DENV genomic RNA levels (normalized to GAP RNA levels) in DENV-infected cells between the rAdsh-scr pre-treated (control) and rAdsh-5b-pre-treated (test) samples are summarized in Figure 4. In this experiment the DENV RNA level in the test sample has been expressed relative to that in the corresponding control sample, which was arbitrarily set at 1.0. It is immediately apparent that test RNA levels are a fraction of the control RNA levels across the board, showing that rAdsh-5b exerted a uniformly inhibitory effect on all DENV serotypes. An analysis of sense viral RNA levels at day 2 post DENV infection is presented in panel B. DENV-1 and -2 sense RNA levels manifested very high inhibition (>80%) compared to their corresponding controls, while DENV-3 and DENV-4 sense RNA levels suffered relatively lesser degree of reduction (∼50–60%). By day 7, sense RNA was virtually indiscernible for DENV-1 and -4, and >70% reduced in DENV-3, with respect to their cognate controls. DENV-2 sense RNA levels did not manifest further reduction beyond ∼80% seen at day 2 post-DENV infection (panel D). The observed inhibition in the levels of sense viral RNA seen for all four DENV serotypes was mirrored in the antisense RNA levels as well (panels A and C), with the inhibition being relatively greater than that observed for sense RNA. Further, the reduction in antisense RNA levels was more pronounced on day 7 (panel C) compared to that on day 2 (panel A), as seen for sense RNA levels. The greater susceptibility of antisense DENV RNA to inhibition, compared to its sense counterpart, may be a reflection of the lower copy number of the former [3]. In all instances, the reduction in genomic RNA of the test samples, compared to the cognate reference samples, was significant (P<0.05).


Adenovirus delivered short hairpin RNA targeting a conserved site in the 5' non-translated region inhibits all four serotypes of dengue viruses.

Korrapati AB, Swaminathan G, Singh A, Khanna N, Swaminathan S - PLoS Negl Trop Dis (2012)

The effect of rAd-mediated shRNA expression on DENV RNA accumulation.Vero cells were pre-infected either with rAdsh-5b (red bars) or rAdsh-scr (green bars) followed 24 hours later by infection with DENV-1, DENV-2, DENV-3 and DENV-4. Total RNA was isolated on days 2 (panels A and B) and 7 (panels C and D) post-DENV infection and analyzed for DENV ’minus’ (panels A and C) and ‘plus’ (panels B and d D) sense viral genomic RNAs by strand-specific real time PCR analyses. DENV RNA was normalized to GAP RNA in each sample analyzed. The data depict DENV RNA levels in rAdsh-5b treated cells relative to those in the corresponding rAdsh-scr treated cells. Each experiment was carried out in triplicate wells and the entire experiment repeated twice.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3404111&req=5

pntd-0001735-g004: The effect of rAd-mediated shRNA expression on DENV RNA accumulation.Vero cells were pre-infected either with rAdsh-5b (red bars) or rAdsh-scr (green bars) followed 24 hours later by infection with DENV-1, DENV-2, DENV-3 and DENV-4. Total RNA was isolated on days 2 (panels A and B) and 7 (panels C and D) post-DENV infection and analyzed for DENV ’minus’ (panels A and C) and ‘plus’ (panels B and d D) sense viral genomic RNAs by strand-specific real time PCR analyses. DENV RNA was normalized to GAP RNA in each sample analyzed. The data depict DENV RNA levels in rAdsh-5b treated cells relative to those in the corresponding rAdsh-scr treated cells. Each experiment was carried out in triplicate wells and the entire experiment repeated twice.
Mentions: The results of a comparison based on real time RNA PCR assay of the relative DENV genomic RNA levels (normalized to GAP RNA levels) in DENV-infected cells between the rAdsh-scr pre-treated (control) and rAdsh-5b-pre-treated (test) samples are summarized in Figure 4. In this experiment the DENV RNA level in the test sample has been expressed relative to that in the corresponding control sample, which was arbitrarily set at 1.0. It is immediately apparent that test RNA levels are a fraction of the control RNA levels across the board, showing that rAdsh-5b exerted a uniformly inhibitory effect on all DENV serotypes. An analysis of sense viral RNA levels at day 2 post DENV infection is presented in panel B. DENV-1 and -2 sense RNA levels manifested very high inhibition (>80%) compared to their corresponding controls, while DENV-3 and DENV-4 sense RNA levels suffered relatively lesser degree of reduction (∼50–60%). By day 7, sense RNA was virtually indiscernible for DENV-1 and -4, and >70% reduced in DENV-3, with respect to their cognate controls. DENV-2 sense RNA levels did not manifest further reduction beyond ∼80% seen at day 2 post-DENV infection (panel D). The observed inhibition in the levels of sense viral RNA seen for all four DENV serotypes was mirrored in the antisense RNA levels as well (panels A and C), with the inhibition being relatively greater than that observed for sense RNA. Further, the reduction in antisense RNA levels was more pronounced on day 7 (panel C) compared to that on day 2 (panel A), as seen for sense RNA levels. The greater susceptibility of antisense DENV RNA to inhibition, compared to its sense counterpart, may be a reflection of the lower copy number of the former [3]. In all instances, the reduction in genomic RNA of the test samples, compared to the cognate reference samples, was significant (P<0.05).

Bottom Line: There is neither a preventive vaccine nor an effective antiviral therapy against dengue disease.We show that this shRNA matures to the cognate siRNA and is able to inhibit effectively antigen secretion, viral RNA replication and infectious virus production by all four DENV serotypes.The data demonstrate the feasibility of using AdV5-mediated delivery of shRNAs targeting conserved sites in the viral genome to achieve inhibition of all four DENV serotypes.

View Article: PubMed Central - PubMed

Affiliation: Recombinant Gene Products Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India.

ABSTRACT

Background: Dengue is a mosquito-borne viral disease caused by four closely related serotypes of Dengue viruses (DENVs). This disease whose symptoms range from mild fever to potentially fatal haemorrhagic fever and hypovolemic shock, threatens nearly half the global population. There is neither a preventive vaccine nor an effective antiviral therapy against dengue disease. The difference between severe and mild disease appears to be dependent on the viral load. Early diagnosis may enable timely therapeutic intervention to blunt disease severity by reducing the viral load. Harnessing the therapeutic potential of RNA interference (RNAi) to attenuate DENV replication may offer one approach to dengue therapy.

Methodology/principal findings: We screened the non-translated regions (NTRs) of the RNA genomes of representative members of the four DENV serotypes for putative siRNA targets mapping to known transcription/translation regulatory elements. We identified a target site in the 5' NTR that maps to the 5' upstream AUG region, a highly conserved cis-acting element essential for viral replication. We used a replication-defective human adenovirus type 5 (AdV5) vector to deliver a short-hairpin RNA (shRNA) targeting this site into cells. We show that this shRNA matures to the cognate siRNA and is able to inhibit effectively antigen secretion, viral RNA replication and infectious virus production by all four DENV serotypes.

Conclusion/significance: The data demonstrate the feasibility of using AdV5-mediated delivery of shRNAs targeting conserved sites in the viral genome to achieve inhibition of all four DENV serotypes. This paves the way towards exploration of RNAi as a possible therapeutic strategy to curtail DENV infection.

Show MeSH
Related in: MedlinePlus