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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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Related in: MedlinePlus

Detection of PRRSVORF7copy numbers in rAd-transduced and/or exosome-incubated PAMs. Primary PAMs were transduced with single or double rAds and/or incubated for 48 h with single or double amiRNA-containing exosomes. The cells were infected with PRRSV strain VR-2332 and the total RNA was extracted 24 h post infection for ORF7 copy number detection by real time quantitative RT-PCR.
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Fig5: Detection of PRRSVORF7copy numbers in rAd-transduced and/or exosome-incubated PAMs. Primary PAMs were transduced with single or double rAds and/or incubated for 48 h with single or double amiRNA-containing exosomes. The cells were infected with PRRSV strain VR-2332 and the total RNA was extracted 24 h post infection for ORF7 copy number detection by real time quantitative RT-PCR.

Mentions: We used two different quantitative assays to evaluate the additive anti-PRRSV effect between Sn and CD163 receptor-targeted amiRNAs: quantitative RT-PCR and viral titration. For the quantitative RT-PCR, primary PAMs were transduced with rAd-amiRcon, rAd-amiRSn and/or rAd-amiRCD163 as described, and infected with PRRSV strain VR2332 (MOI 0.2) 48 h after transduction. At 24 h post infection, the total RNA was extracted for quantitative RT-PCR using PRRSV ORF7-specific primers (Table 2). Compared to that (5.4 log10) in rAd-amiRcon-transduced cells, the ORF7 copy number in rAd-amiRSn and/or rAd-amiRCD163 transduced cells was decreased by 3.5, 2.3 or 2.6 log10 (Figure 5). Next, primary PAMs were incubated with the exosomes derived from rAd-amiRSn- and/or rAd-amiRCD163-transduced PK-15 cells, infected with PRRSV and the total RNA was extracted for quantitative RT-PCR as described. Compared to that (5.4 log10) in the control group, the ORF7 copy number in amiRSn- and/or amiRCD163-containing exosome-incubated cells was decreased by 3.3, 2.0 or 2.2 log10 (Figure 5). Finally, primary PAMs were transduced with different rAds, incubated with different amiRNA-containing exosomes, infected with PRRSV and total RNA was extracted for RT-PCR as described. Compared to that (5.4 log10) in the control group, the ORF7 copy number in double rAd-transduced and double amiRNA-containing exosome-incubated cells was decreased by 4.2 log10, while the ORF7 copy number in single rAd-transduced and single amiRNA-containing exosome-incubated cells was decreased by 3.3 or 3.6 log10 (Figure 5). Figure 5


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)

Detection of PRRSVORF7copy numbers in rAd-transduced and/or exosome-incubated PAMs. Primary PAMs were transduced with single or double rAds and/or incubated for 48 h with single or double amiRNA-containing exosomes. The cells were infected with PRRSV strain VR-2332 and the total RNA was extracted 24 h post infection for ORF7 copy number detection by real time quantitative RT-PCR.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: Detection of PRRSVORF7copy numbers in rAd-transduced and/or exosome-incubated PAMs. Primary PAMs were transduced with single or double rAds and/or incubated for 48 h with single or double amiRNA-containing exosomes. The cells were infected with PRRSV strain VR-2332 and the total RNA was extracted 24 h post infection for ORF7 copy number detection by real time quantitative RT-PCR.
Mentions: We used two different quantitative assays to evaluate the additive anti-PRRSV effect between Sn and CD163 receptor-targeted amiRNAs: quantitative RT-PCR and viral titration. For the quantitative RT-PCR, primary PAMs were transduced with rAd-amiRcon, rAd-amiRSn and/or rAd-amiRCD163 as described, and infected with PRRSV strain VR2332 (MOI 0.2) 48 h after transduction. At 24 h post infection, the total RNA was extracted for quantitative RT-PCR using PRRSV ORF7-specific primers (Table 2). Compared to that (5.4 log10) in rAd-amiRcon-transduced cells, the ORF7 copy number in rAd-amiRSn and/or rAd-amiRCD163 transduced cells was decreased by 3.5, 2.3 or 2.6 log10 (Figure 5). Next, primary PAMs were incubated with the exosomes derived from rAd-amiRSn- and/or rAd-amiRCD163-transduced PK-15 cells, infected with PRRSV and the total RNA was extracted for quantitative RT-PCR as described. Compared to that (5.4 log10) in the control group, the ORF7 copy number in amiRSn- and/or amiRCD163-containing exosome-incubated cells was decreased by 3.3, 2.0 or 2.2 log10 (Figure 5). Finally, primary PAMs were transduced with different rAds, incubated with different amiRNA-containing exosomes, infected with PRRSV and total RNA was extracted for RT-PCR as described. Compared to that (5.4 log10) in the control group, the ORF7 copy number in double rAd-transduced and double amiRNA-containing exosome-incubated cells was decreased by 4.2 log10, while the ORF7 copy number in single rAd-transduced and single amiRNA-containing exosome-incubated cells was decreased by 3.3 or 3.6 log10 (Figure 5). Figure 5

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