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RNA interference targeting virion core protein ORF095 inhibits Goatpox virus replication in Vero cells.

Zhao Z, Wu G, Zhu X, Yan X, Dou Y, Li J, Zhu H, Zhang Q, Cai X - Virol. J. (2012)

Bottom Line: The results showed that the ORF095-specific siRNA-70 effectively down-regulated the expression of ORF095.When Vero cells were transfected with shRNA expression vectors (p61/GFP, p70/GFP, p165/GFP and p296/GFP) and then infected with GTPV, GTPV-ORF095-70 was found to be the most effective inhibition site in decreasing cytopathic effect (CPE) induced by GTPV.The results presented here indicated that DNA-based siRNA could effectively inhibit the replication of GTPV (approximately 463. 5-fold reduction of viral titers) on Vero cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Key Laboratory of Animal virology of the Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences, Gansu, PR China.

ABSTRACT

Background: Goatpox is an economically important disease in goat and sheep-producing areas of the world. Many vaccine strategies developed to control the disease are not yet completely successful. Hairpin expression vectors have been used to induce gene silencing in a large number of studies on viruses. However, none of these studies has been attempted to study GTPV. In the interest of exploiting improved methods to control goat pox, it is participated that RNAi may provide effective protection against GTPV. In this study we show the suppression of Goatpox virus (GTPV) replication via knockdown of virion core protein using RNA interference.

Results: Four short interfering RNA (siRNA) sequences (siRNA-61, siRNA-70, siRNA-165 and siRNA-296) against a region of GTPV ORF095 were selected. Sense and antisense siRNA-encoding sequences separated by a hairpin loop sequence were designed as short hairpin RNA (shRNA) expression cassettes under the control of a human U6 promoter. ORF095 amplicon was generated using PCR, and then cloned into pEGFP-N1 vector, named as p095/EGFP. p095/EGFP and each of the siRNA expression cassettes (p61, p70, p165 and p296) were co-transfected into BHK-21 cells. Fluorescence detection, flow cytometric analysis, retro transcription PCR (RT-PCR) and real time PCR were used to check the efficiency of RNAi. The results showed that the ORF095-specific siRNA-70 effectively down-regulated the expression of ORF095. When Vero cells were transfected with shRNA expression vectors (p61/GFP, p70/GFP, p165/GFP and p296/GFP) and then infected with GTPV, GTPV-ORF095-70 was found to be the most effective inhibition site in decreasing cytopathic effect (CPE) induced by GTPV. The results presented here indicated that DNA-based siRNA could effectively inhibit the replication of GTPV (approximately 463. 5-fold reduction of viral titers) on Vero cells.

Conclusions: This study demonstrates that vector-based shRNA methodology can effectively inhibit GTPV replication on Vero cells. Simultaneously, this work represents a strategy for controlling goatpox, potentially facilitating new experimental approaches in the analysis of both viral and cellular gene functions during of GTPV infection.

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Cytopathic effect (CPE) analysis of GTPV on Vero cells transfected with shRNA-expressing vectors (2.5 μg each). (A):Cells just were infected GTPV; (B)-(G):p61/GFP, p70/GFP, p165/GFP, p296/GFP, pC/GFP and untreated cells, respectively. Except(G), all cells were infected with GTPV(A/Goat/Qinghai/SV40/2006) isolate at a multiplicity of infection (MOI) of 0.01. Pictures were taken at 6 days post-infection with an Olympus digital camera (Olympus, Japan) at a magnification of × 40 with an exposure time of 1/8 s.
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Figure 5: Cytopathic effect (CPE) analysis of GTPV on Vero cells transfected with shRNA-expressing vectors (2.5 μg each). (A):Cells just were infected GTPV; (B)-(G):p61/GFP, p70/GFP, p165/GFP, p296/GFP, pC/GFP and untreated cells, respectively. Except(G), all cells were infected with GTPV(A/Goat/Qinghai/SV40/2006) isolate at a multiplicity of infection (MOI) of 0.01. Pictures were taken at 6 days post-infection with an Olympus digital camera (Olympus, Japan) at a magnification of × 40 with an exposure time of 1/8 s.

Mentions: To investigate whether or not knockout of ORF095 relieves cytopathic effect (CPE) induced by GTPV, Vero cells were transfected by plasmids expressing ORF095 protein-targeted shRNAs (p61/GFP, p70/GFP, p165/GFP and p296/GFP), respectively. Nonspecific shRNA expression vector pC/GFP was transfected in parallel. 4 h post-transfection, the cultures were infected with GTPV and checked daily. Six days later, we found that cells pre-transfected with p70/GFP exhibited less CPE, whereas other shRNA-treated cells and the empty vector control demonstrated the same typical GTPV-induced CPE as cells infected only with virus, as shown in Figure 5.


RNA interference targeting virion core protein ORF095 inhibits Goatpox virus replication in Vero cells.

Zhao Z, Wu G, Zhu X, Yan X, Dou Y, Li J, Zhu H, Zhang Q, Cai X - Virol. J. (2012)

Cytopathic effect (CPE) analysis of GTPV on Vero cells transfected with shRNA-expressing vectors (2.5 μg each). (A):Cells just were infected GTPV; (B)-(G):p61/GFP, p70/GFP, p165/GFP, p296/GFP, pC/GFP and untreated cells, respectively. Except(G), all cells were infected with GTPV(A/Goat/Qinghai/SV40/2006) isolate at a multiplicity of infection (MOI) of 0.01. Pictures were taken at 6 days post-infection with an Olympus digital camera (Olympus, Japan) at a magnification of × 40 with an exposure time of 1/8 s.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3298800&req=5

Figure 5: Cytopathic effect (CPE) analysis of GTPV on Vero cells transfected with shRNA-expressing vectors (2.5 μg each). (A):Cells just were infected GTPV; (B)-(G):p61/GFP, p70/GFP, p165/GFP, p296/GFP, pC/GFP and untreated cells, respectively. Except(G), all cells were infected with GTPV(A/Goat/Qinghai/SV40/2006) isolate at a multiplicity of infection (MOI) of 0.01. Pictures were taken at 6 days post-infection with an Olympus digital camera (Olympus, Japan) at a magnification of × 40 with an exposure time of 1/8 s.
Mentions: To investigate whether or not knockout of ORF095 relieves cytopathic effect (CPE) induced by GTPV, Vero cells were transfected by plasmids expressing ORF095 protein-targeted shRNAs (p61/GFP, p70/GFP, p165/GFP and p296/GFP), respectively. Nonspecific shRNA expression vector pC/GFP was transfected in parallel. 4 h post-transfection, the cultures were infected with GTPV and checked daily. Six days later, we found that cells pre-transfected with p70/GFP exhibited less CPE, whereas other shRNA-treated cells and the empty vector control demonstrated the same typical GTPV-induced CPE as cells infected only with virus, as shown in Figure 5.

Bottom Line: The results showed that the ORF095-specific siRNA-70 effectively down-regulated the expression of ORF095.When Vero cells were transfected with shRNA expression vectors (p61/GFP, p70/GFP, p165/GFP and p296/GFP) and then infected with GTPV, GTPV-ORF095-70 was found to be the most effective inhibition site in decreasing cytopathic effect (CPE) induced by GTPV.The results presented here indicated that DNA-based siRNA could effectively inhibit the replication of GTPV (approximately 463. 5-fold reduction of viral titers) on Vero cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Key Laboratory of Animal virology of the Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences, Gansu, PR China.

ABSTRACT

Background: Goatpox is an economically important disease in goat and sheep-producing areas of the world. Many vaccine strategies developed to control the disease are not yet completely successful. Hairpin expression vectors have been used to induce gene silencing in a large number of studies on viruses. However, none of these studies has been attempted to study GTPV. In the interest of exploiting improved methods to control goat pox, it is participated that RNAi may provide effective protection against GTPV. In this study we show the suppression of Goatpox virus (GTPV) replication via knockdown of virion core protein using RNA interference.

Results: Four short interfering RNA (siRNA) sequences (siRNA-61, siRNA-70, siRNA-165 and siRNA-296) against a region of GTPV ORF095 were selected. Sense and antisense siRNA-encoding sequences separated by a hairpin loop sequence were designed as short hairpin RNA (shRNA) expression cassettes under the control of a human U6 promoter. ORF095 amplicon was generated using PCR, and then cloned into pEGFP-N1 vector, named as p095/EGFP. p095/EGFP and each of the siRNA expression cassettes (p61, p70, p165 and p296) were co-transfected into BHK-21 cells. Fluorescence detection, flow cytometric analysis, retro transcription PCR (RT-PCR) and real time PCR were used to check the efficiency of RNAi. The results showed that the ORF095-specific siRNA-70 effectively down-regulated the expression of ORF095. When Vero cells were transfected with shRNA expression vectors (p61/GFP, p70/GFP, p165/GFP and p296/GFP) and then infected with GTPV, GTPV-ORF095-70 was found to be the most effective inhibition site in decreasing cytopathic effect (CPE) induced by GTPV. The results presented here indicated that DNA-based siRNA could effectively inhibit the replication of GTPV (approximately 463. 5-fold reduction of viral titers) on Vero cells.

Conclusions: This study demonstrates that vector-based shRNA methodology can effectively inhibit GTPV replication on Vero cells. Simultaneously, this work represents a strategy for controlling goatpox, potentially facilitating new experimental approaches in the analysis of both viral and cellular gene functions during of GTPV infection.

Show MeSH
Related in: MedlinePlus