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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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Titration of PRRSV titers in rAd-transduced and/or exosome-incubated PAMs. (A) Primary PAMs were transduced with different rAds and infected with PRRSV strain VR2332 48 h after transduction. At different time points after infection, the cells were harvested for PRRSV titration on MARC-145 cells. (B) Primary PAMs were incubated for 48 h with different amiRNA-containing exosomes and then infected with PRRSV strain VR2332 before PRRSV infection and titration. (C) Primary PAMs were transduced with different rAds and then incubated for 48 h with different amiRNA-containing exosomes before PRRSV infection and titration. (D) Primary PAMs were transduced with different rAds and then incubated for 48 h with different amiRNA-containing exosomes. The cells were infected with three different PRRSV strains and harvested for PRRSV titration 72 h after infection.
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Fig6: Titration of PRRSV titers in rAd-transduced and/or exosome-incubated PAMs. (A) Primary PAMs were transduced with different rAds and infected with PRRSV strain VR2332 48 h after transduction. At different time points after infection, the cells were harvested for PRRSV titration on MARC-145 cells. (B) Primary PAMs were incubated for 48 h with different amiRNA-containing exosomes and then infected with PRRSV strain VR2332 before PRRSV infection and titration. (C) Primary PAMs were transduced with different rAds and then incubated for 48 h with different amiRNA-containing exosomes before PRRSV infection and titration. (D) Primary PAMs were transduced with different rAds and then incubated for 48 h with different amiRNA-containing exosomes. The cells were infected with three different PRRSV strains and harvested for PRRSV titration 72 h after infection.

Mentions: For the viral titration assay, primary PAMs were transduced with different rAds and infected with PRRSV strain VR2332 as described. At different time points post infection, the cells were harvested for PRRSV titration on MARC-145 cells. Compared to that (4.3 log10 TCID50) in the mock- or rAd-amiRcon-transduced cells, the PRRSV titer in rAd-amiRSn- and/or rAd-amiRCD163-transduced cells was decreased by 1.5, 0.9 or 1.0 log10 at 24 h post infection (Figure 6A). Next, primary PAMs were incubated with amiRSn- and/or amiRCD163-containing exosomes, infected with PRRSV and harvested for PRRSV titration as described. Compared to that (4.3 or 4.2 log10 TCID50) in the mock- or amiRcon-containing exosome-treated cells, PRRSV titer in amiRSn- and/or amiRCD163-containing exosome-incubated cells was decreased by 1.2, 0.6 or 1.0 log10 (Figure 6B). Then, primary PAMs were transduced with different rAds, incubated with different amiRNA-containing exosomes, infected with PRRSV and harvested for PRRSV titration as described. Compared to that (4.3 or 4.4 log10 TCID50) in the two control groups, the PRRSV titer in double rAd-transduced and double amiRNA-containing exosome-incubated cells was decreased by 2.0 log10, while the viral titer in single rAd-transduced and single amiRNA-containing exosome-incubated cells was decreased by 1.1 log10 (Figure 6C). The similar additive anti-PRRSV effect between the two amiRNAs lasted for at least 96 h (Figure 6A,B or C). Finally, primary PAMs were transduced with different rAds, incubated with different amiRNA-containing exosomes and infected with PRRSV strain JX-A1, CH-1R or VR2332 as described. At 72 h post infection, the infected cells were harvested for PRRSV titration. Compared to that (4.9-6.2 log10 TCID50) in the two control groups, the PRRSV titers in three viral strain-infected cells were decreased by 2.7, 2.1 and 2.6 log10, respectively (Figure 6D).Figure 6


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)

Titration of PRRSV titers in rAd-transduced and/or exosome-incubated PAMs. (A) Primary PAMs were transduced with different rAds and infected with PRRSV strain VR2332 48 h after transduction. At different time points after infection, the cells were harvested for PRRSV titration on MARC-145 cells. (B) Primary PAMs were incubated for 48 h with different amiRNA-containing exosomes and then infected with PRRSV strain VR2332 before PRRSV infection and titration. (C) Primary PAMs were transduced with different rAds and then incubated for 48 h with different amiRNA-containing exosomes before PRRSV infection and titration. (D) Primary PAMs were transduced with different rAds and then incubated for 48 h with different amiRNA-containing exosomes. The cells were infected with three different PRRSV strains and harvested for PRRSV titration 72 h after infection.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4279792&req=5

Fig6: Titration of PRRSV titers in rAd-transduced and/or exosome-incubated PAMs. (A) Primary PAMs were transduced with different rAds and infected with PRRSV strain VR2332 48 h after transduction. At different time points after infection, the cells were harvested for PRRSV titration on MARC-145 cells. (B) Primary PAMs were incubated for 48 h with different amiRNA-containing exosomes and then infected with PRRSV strain VR2332 before PRRSV infection and titration. (C) Primary PAMs were transduced with different rAds and then incubated for 48 h with different amiRNA-containing exosomes before PRRSV infection and titration. (D) Primary PAMs were transduced with different rAds and then incubated for 48 h with different amiRNA-containing exosomes. The cells were infected with three different PRRSV strains and harvested for PRRSV titration 72 h after infection.
Mentions: For the viral titration assay, primary PAMs were transduced with different rAds and infected with PRRSV strain VR2332 as described. At different time points post infection, the cells were harvested for PRRSV titration on MARC-145 cells. Compared to that (4.3 log10 TCID50) in the mock- or rAd-amiRcon-transduced cells, the PRRSV titer in rAd-amiRSn- and/or rAd-amiRCD163-transduced cells was decreased by 1.5, 0.9 or 1.0 log10 at 24 h post infection (Figure 6A). Next, primary PAMs were incubated with amiRSn- and/or amiRCD163-containing exosomes, infected with PRRSV and harvested for PRRSV titration as described. Compared to that (4.3 or 4.2 log10 TCID50) in the mock- or amiRcon-containing exosome-treated cells, PRRSV titer in amiRSn- and/or amiRCD163-containing exosome-incubated cells was decreased by 1.2, 0.6 or 1.0 log10 (Figure 6B). Then, primary PAMs were transduced with different rAds, incubated with different amiRNA-containing exosomes, infected with PRRSV and harvested for PRRSV titration as described. Compared to that (4.3 or 4.4 log10 TCID50) in the two control groups, the PRRSV titer in double rAd-transduced and double amiRNA-containing exosome-incubated cells was decreased by 2.0 log10, while the viral titer in single rAd-transduced and single amiRNA-containing exosome-incubated cells was decreased by 1.1 log10 (Figure 6C). The similar additive anti-PRRSV effect between the two amiRNAs lasted for at least 96 h (Figure 6A,B or C). Finally, primary PAMs were transduced with different rAds, incubated with different amiRNA-containing exosomes and infected with PRRSV strain JX-A1, CH-1R or VR2332 as described. At 72 h post infection, the infected cells were harvested for PRRSV titration. Compared to that (4.9-6.2 log10 TCID50) in the two control groups, the PRRSV titers in three viral strain-infected cells were decreased by 2.7, 2.1 and 2.6 log10, respectively (Figure 6D).Figure 6

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