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Inhibition of porcine reproductive and respiratory syndrome virus infection by recombinant adenovirus- and/or exosome-delivered the artificial microRNAs targeting sialoadhesin and CD163 receptors.

Zhu L, Song H, Zhang X, Xia X, Sun H - Virol. J. (2014)

Bottom Line: Both PRRSV ORF7 copy number and viral titer were reduced significantly by transduction of PAMs with the two rAds and/or by treatment with the two amiRNA-containing exosomes.The additive anti-PRRSV effect between the two amiRNAs was relatively long-lasting (96 h) and effective against three different viral strains.These results suggested that Sn- and CD163-targeted amiRNAs had an additive anti-PRRSV effect against different viral strains.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, China. 1029765408@qq.com.

ABSTRACT

Background: The current vaccines failed to provide substantial protection against porcine reproductive and respiratory syndrome (PRRS) and the new vaccine development faces great challenges. Sialoadhesin (Sn) and CD163 are the two key receptors for PRRS virus (PRRSV) infection of porcine alveolar macrophages (PAMs), but the artificial microRNA (amiRNA) strategy targeting two viral receptors has not been described.

Methods: The candidate miRNAs targeting Sn or CD163 receptor were predicted using a web-based miRNA design tool and validated by transfection of cells with each amiRNA expression vector plus the reporter vector. The amiRNA-expressing recombinant adenoviruses (rAds) were generated using AdEasy Adenoviral Vector System. The rAd transduction efficiencies for pig cells were measured by flow cytometry and fluorescent microscopy. The expression and exosome-mediated secretion of amiRNAs were detected by RT-PCR. The knock-down of Sn or CD163 receptor by rAd- and/or exosome-delivered amiRNA was detected by quantitative RT-PCR and flow cytometry. The additive anti-PRRSV effect between the two amiRNAs was detected by quantitative RT-PCR and viral titration.

Results: All 18 amiRNAs validated were effective against Sn or CD163 receptor mRNA expression. Two rAds expressing Sn- or CD163-targeted amiRNA were generated for further study. The maximal rAd transduction efficiency was 62% for PAMs at MOI 800 or 100% for PK-15 cells at MOI 100. The sequence-specific amiRNAs were expressed efficiently in and secreted from the rAd-transduced cells via exosomes. The expression of Sn and CD163 receptors was inhibited significantly by rAd transduction and/or amiRNA-containing exosome treatment at mRNA and protein levels. Both PRRSV ORF7 copy number and viral titer were reduced significantly by transduction of PAMs with the two rAds and/or by treatment with the two amiRNA-containing exosomes. The additive anti-PRRSV effect between the two amiRNAs was relatively long-lasting (96 h) and effective against three different viral strains.

Conclusion: These results suggested that Sn- and CD163-targeted amiRNAs had an additive anti-PRRSV effect against different viral strains. Our findings provide new evidence supporting the hypothesis that exosomes can also serve as an efficient small RNA transfer vehicle for pig cells.

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Knock-down of Sn or CD163 receptor by the rAd- and/or exosome-delivered amiRNA. (A) Primary PAMs were transduced with each rAd and/or incubated with each amiRNA-containing exosomes derived from the rAd-transduced PK-15 cells. At 48 h after incubation, the total RNA was extracted for Sn or CD163 mRNA detection by real-time quantitative RT-PCR. (B) The rAd-transduced and/or exosome-incubated cells were stained with Sn- or CD163-specific antibody and analyzed by flow cytometry.
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Fig4: Knock-down of Sn or CD163 receptor by the rAd- and/or exosome-delivered amiRNA. (A) Primary PAMs were transduced with each rAd and/or incubated with each amiRNA-containing exosomes derived from the rAd-transduced PK-15 cells. At 48 h after incubation, the total RNA was extracted for Sn or CD163 mRNA detection by real-time quantitative RT-PCR. (B) The rAd-transduced and/or exosome-incubated cells were stained with Sn- or CD163-specific antibody and analyzed by flow cytometry.

Mentions: The sufficient knock-down of Sn and CD163 receptors is the precondition for the objective evaluation of anti-PRRSV effects of the receptor-targeted amiRNAs. We compared the knock-down efficiencies of the two viral receptors using three different strategies: rAd transduction, exosome treatment and rAd transduction plus exosome treatment. First, primary PAMs were transduced with rAd-amiRSn, rAd-amiR163 or rAd-amiRcon. After incubation for 48 h, the total RNA was extracted for mRNA detection by quantitative RT-PCR and the cell culture was analyzed for Sn+ or CD163+ cell number by flow cytometry. Compared to that in rAd-amiRcon-transduced cell cultures, Sn or CD163 receptor mRNA expression in the rAd-amiRSn- or rAd-amiRCD163-transduced cells was decreased by 0.51 or 0.54 fold (Figure 4A), while Sn+ and CD163+ cell numbers were decreased from 48.7% to 22.7% and from 49.9% to 27.6%, respectively (Figure 4B). Next, primary PAMs were incubated with the exosomes (0.5 mg protein/ml) derived from rAd-amiRSn- or rAd-amiRCD163-transduced PK-15 cells. At 48 h after incubation, the total RNA was extracted for quantitative RT-PCR and cell cultures were harvested for flow cytometry analysis as described. Compared to that in the two control groups, Sn or CD163 mRNA expression in the amiRNA-containing exosome-incubated cells was decreased by 0.41 or 0.46 fold (Figure 4A), while Sn+ and CD163+ cell numbers were decreased to 33.4% and 36.5%, respectively (Figure 4B). Finally, primary PAMs were transduced first with each rAd, incubated for 48 h with each amiRNA-containing exosomes, the total RNA was extracted for quantitative RT-PCR and the cell cultures were harvested for flow cytometry analysis as described. Compared to that in the two control groups, Sn or CD163 mRNA expression was decreased by 0.73 or 0.73 fold (Figure 4A), while Sn+ and CD163+ cell numbers were decreased to 18.9% and 19.1%, respectively (Figure 4B).Figure 4


Inhibition of porcine reproductive and respiratory syndrome virus infection by recombinant adenovirus- and/or exosome-delivered the artificial microRNAs targeting sialoadhesin and CD163 receptors.

Zhu L, Song H, Zhang X, Xia X, Sun H - Virol. J. (2014)

Knock-down of Sn or CD163 receptor by the rAd- and/or exosome-delivered amiRNA. (A) Primary PAMs were transduced with each rAd and/or incubated with each amiRNA-containing exosomes derived from the rAd-transduced PK-15 cells. At 48 h after incubation, the total RNA was extracted for Sn or CD163 mRNA detection by real-time quantitative RT-PCR. (B) The rAd-transduced and/or exosome-incubated cells were stained with Sn- or CD163-specific antibody and analyzed by flow cytometry.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4279792&req=5

Fig4: Knock-down of Sn or CD163 receptor by the rAd- and/or exosome-delivered amiRNA. (A) Primary PAMs were transduced with each rAd and/or incubated with each amiRNA-containing exosomes derived from the rAd-transduced PK-15 cells. At 48 h after incubation, the total RNA was extracted for Sn or CD163 mRNA detection by real-time quantitative RT-PCR. (B) The rAd-transduced and/or exosome-incubated cells were stained with Sn- or CD163-specific antibody and analyzed by flow cytometry.
Mentions: The sufficient knock-down of Sn and CD163 receptors is the precondition for the objective evaluation of anti-PRRSV effects of the receptor-targeted amiRNAs. We compared the knock-down efficiencies of the two viral receptors using three different strategies: rAd transduction, exosome treatment and rAd transduction plus exosome treatment. First, primary PAMs were transduced with rAd-amiRSn, rAd-amiR163 or rAd-amiRcon. After incubation for 48 h, the total RNA was extracted for mRNA detection by quantitative RT-PCR and the cell culture was analyzed for Sn+ or CD163+ cell number by flow cytometry. Compared to that in rAd-amiRcon-transduced cell cultures, Sn or CD163 receptor mRNA expression in the rAd-amiRSn- or rAd-amiRCD163-transduced cells was decreased by 0.51 or 0.54 fold (Figure 4A), while Sn+ and CD163+ cell numbers were decreased from 48.7% to 22.7% and from 49.9% to 27.6%, respectively (Figure 4B). Next, primary PAMs were incubated with the exosomes (0.5 mg protein/ml) derived from rAd-amiRSn- or rAd-amiRCD163-transduced PK-15 cells. At 48 h after incubation, the total RNA was extracted for quantitative RT-PCR and cell cultures were harvested for flow cytometry analysis as described. Compared to that in the two control groups, Sn or CD163 mRNA expression in the amiRNA-containing exosome-incubated cells was decreased by 0.41 or 0.46 fold (Figure 4A), while Sn+ and CD163+ cell numbers were decreased to 33.4% and 36.5%, respectively (Figure 4B). Finally, primary PAMs were transduced first with each rAd, incubated for 48 h with each amiRNA-containing exosomes, the total RNA was extracted for quantitative RT-PCR and the cell cultures were harvested for flow cytometry analysis as described. Compared to that in the two control groups, Sn or CD163 mRNA expression was decreased by 0.73 or 0.73 fold (Figure 4A), while Sn+ and CD163+ cell numbers were decreased to 18.9% and 19.1%, respectively (Figure 4B).Figure 4

Bottom Line: Both PRRSV ORF7 copy number and viral titer were reduced significantly by transduction of PAMs with the two rAds and/or by treatment with the two amiRNA-containing exosomes.The additive anti-PRRSV effect between the two amiRNAs was relatively long-lasting (96 h) and effective against three different viral strains.These results suggested that Sn- and CD163-targeted amiRNAs had an additive anti-PRRSV effect against different viral strains.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, China. 1029765408@qq.com.

ABSTRACT

Background: The current vaccines failed to provide substantial protection against porcine reproductive and respiratory syndrome (PRRS) and the new vaccine development faces great challenges. Sialoadhesin (Sn) and CD163 are the two key receptors for PRRS virus (PRRSV) infection of porcine alveolar macrophages (PAMs), but the artificial microRNA (amiRNA) strategy targeting two viral receptors has not been described.

Methods: The candidate miRNAs targeting Sn or CD163 receptor were predicted using a web-based miRNA design tool and validated by transfection of cells with each amiRNA expression vector plus the reporter vector. The amiRNA-expressing recombinant adenoviruses (rAds) were generated using AdEasy Adenoviral Vector System. The rAd transduction efficiencies for pig cells were measured by flow cytometry and fluorescent microscopy. The expression and exosome-mediated secretion of amiRNAs were detected by RT-PCR. The knock-down of Sn or CD163 receptor by rAd- and/or exosome-delivered amiRNA was detected by quantitative RT-PCR and flow cytometry. The additive anti-PRRSV effect between the two amiRNAs was detected by quantitative RT-PCR and viral titration.

Results: All 18 amiRNAs validated were effective against Sn or CD163 receptor mRNA expression. Two rAds expressing Sn- or CD163-targeted amiRNA were generated for further study. The maximal rAd transduction efficiency was 62% for PAMs at MOI 800 or 100% for PK-15 cells at MOI 100. The sequence-specific amiRNAs were expressed efficiently in and secreted from the rAd-transduced cells via exosomes. The expression of Sn and CD163 receptors was inhibited significantly by rAd transduction and/or amiRNA-containing exosome treatment at mRNA and protein levels. Both PRRSV ORF7 copy number and viral titer were reduced significantly by transduction of PAMs with the two rAds and/or by treatment with the two amiRNA-containing exosomes. The additive anti-PRRSV effect between the two amiRNAs was relatively long-lasting (96 h) and effective against three different viral strains.

Conclusion: These results suggested that Sn- and CD163-targeted amiRNAs had an additive anti-PRRSV effect against different viral strains. Our findings provide new evidence supporting the hypothesis that exosomes can also serve as an efficient small RNA transfer vehicle for pig cells.

Show MeSH
Related in: MedlinePlus