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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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rAd transduction efficiencies for pig cells. (A) The schematic structure of Ad vector for amiRNA expression. ITR, inverted terminal repeat of adenovirus; PCMV, immediate early promoter of cytomegalovirus; miR, miR-flanking sequences of mouse BIC non-coding mRNA; Pre-amiRNA, double–stranded oligonucleotide for amiRNA; IRES, internal ribosome entry sequence; GFP, green fluorescent protein coding sequence; SV40 pA, poly(A) signal of SV40 virus. (B) PK-15 cells and (C) primary PAMs were transduced with different doses of rAd-amiRSn and the GFP-positive cell numbers were measured by flow cytometry 48 h after transduction. (D) Primary PAMs and PK-15 cells were mock-transduced or transduced with each rAd and analyzed by fluorescent microscopy (40×) 48 h after transduction.
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Fig2: rAd transduction efficiencies for pig cells. (A) The schematic structure of Ad vector for amiRNA expression. ITR, inverted terminal repeat of adenovirus; PCMV, immediate early promoter of cytomegalovirus; miR, miR-flanking sequences of mouse BIC non-coding mRNA; Pre-amiRNA, double–stranded oligonucleotide for amiRNA; IRES, internal ribosome entry sequence; GFP, green fluorescent protein coding sequence; SV40 pA, poly(A) signal of SV40 virus. (B) PK-15 cells and (C) primary PAMs were transduced with different doses of rAd-amiRSn and the GFP-positive cell numbers were measured by flow cytometry 48 h after transduction. (D) Primary PAMs and PK-15 cells were mock-transduced or transduced with each rAd and analyzed by fluorescent microscopy (40×) 48 h after transduction.

Mentions: We subcloned amiRSn-2, amiR163-2 or amiRcon expression cassette into Ad vector pShuttle-IRES-hrGFP, and three rAds, namely rAd-amiRSn, rAd-amiR163 and rAd-amiRcon, were generated by transfecting AAV-293 cells with the rAd vectors (Figure 2A). Primary PAMs and PK-15 cells were transduced with different doses of rAds, and the cell cultures were analyzed for GFP+ cells since the GFP reporter gene was included in the rAd vectors. Compared to 100% transduction efficiency for PK-15 cells at MOI 100 (Figure 2B), only 62% transduction efficiency was achieved for PAMs at MOI 800 (Figure 2C), which was not increased further by using higher MOI. The different rAd transduction efficiencies for the two pig cell types were confirmed by fluorescent microscopy (Figure 2D). Therefore, the further experiments were carried out at MOI 800 for PAM transduction and MOI 100 for PK-15 cell transduction.Figure 2


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)

rAd transduction efficiencies for pig cells. (A) The schematic structure of Ad vector for amiRNA expression. ITR, inverted terminal repeat of adenovirus; PCMV, immediate early promoter of cytomegalovirus; miR, miR-flanking sequences of mouse BIC non-coding mRNA; Pre-amiRNA, double–stranded oligonucleotide for amiRNA; IRES, internal ribosome entry sequence; GFP, green fluorescent protein coding sequence; SV40 pA, poly(A) signal of SV40 virus. (B) PK-15 cells and (C) primary PAMs were transduced with different doses of rAd-amiRSn and the GFP-positive cell numbers were measured by flow cytometry 48 h after transduction. (D) Primary PAMs and PK-15 cells were mock-transduced or transduced with each rAd and analyzed by fluorescent microscopy (40×) 48 h after transduction.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: rAd transduction efficiencies for pig cells. (A) The schematic structure of Ad vector for amiRNA expression. ITR, inverted terminal repeat of adenovirus; PCMV, immediate early promoter of cytomegalovirus; miR, miR-flanking sequences of mouse BIC non-coding mRNA; Pre-amiRNA, double–stranded oligonucleotide for amiRNA; IRES, internal ribosome entry sequence; GFP, green fluorescent protein coding sequence; SV40 pA, poly(A) signal of SV40 virus. (B) PK-15 cells and (C) primary PAMs were transduced with different doses of rAd-amiRSn and the GFP-positive cell numbers were measured by flow cytometry 48 h after transduction. (D) Primary PAMs and PK-15 cells were mock-transduced or transduced with each rAd and analyzed by fluorescent microscopy (40×) 48 h after transduction.
Mentions: We subcloned amiRSn-2, amiR163-2 or amiRcon expression cassette into Ad vector pShuttle-IRES-hrGFP, and three rAds, namely rAd-amiRSn, rAd-amiR163 and rAd-amiRcon, were generated by transfecting AAV-293 cells with the rAd vectors (Figure 2A). Primary PAMs and PK-15 cells were transduced with different doses of rAds, and the cell cultures were analyzed for GFP+ cells since the GFP reporter gene was included in the rAd vectors. Compared to 100% transduction efficiency for PK-15 cells at MOI 100 (Figure 2B), only 62% transduction efficiency was achieved for PAMs at MOI 800 (Figure 2C), which was not increased further by using higher MOI. The different rAd transduction efficiencies for the two pig cell types were confirmed by fluorescent microscopy (Figure 2D). Therefore, the further experiments were carried out at MOI 800 for PAM transduction and MOI 100 for PK-15 cell transduction.Figure 2

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