title: Inhibition of porcine reproductive and respiratory syndrome virus infection by recombinant adenovirus- and/or exosome-delivered the artificial microRNAs targeting sialoadhesin and CD163 receptors.
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.
  
  
  CONCLUSIONS
  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.
authors.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.
authors.forename: Li
authors.initials: L
authors.lastname: Zhu
authors.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. hqsong@yzu.edu.cn.
authors.forename: Hongqin
authors.initials: H
authors.lastname: Song
authors.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. zxy@yzu.edu.cn.
authors.forename: Xinyu
authors.initials: X
authors.lastname: Zhang
authors.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. xlxia@yzu.edu.cn.
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Xia
authors.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. sunh@yzu.edu.cn.
authors.forename: Huaichang
authors.initials: H
authors.lastname: Sun
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D015703:Antigens, CD / Q000235:genetics; D015214:Antigens, Differentiation, Myelomonocytic / Q000235:genetics; D000998:Antiviral Agents / Q000378:metabolism*; D002478:Cells, Cultured; D004337:Drug Carriers; D055354:Exosomes / Q000378:metabolism*; D005434:Flow Cytometry; D015870:Gene Expression; D020869:Gene Expression Profiling; D055785:Gene Knockdown Techniques; D008264:Macrophages / Q000821:virology; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D019316:Porcine respiratory and reproductive syndrome virus / Q000254:growth & development*; D060888:Real-Time Polymerase Chain Reaction; D011956:Receptors, Cell Surface / Q000037:antagonists & inhibitors* / Q000235:genetics; D011991:Receptors, Virus / Q000037:antagonists & inhibitors / Q000235:genetics; D063267:Sialic Acid Binding Ig-like Lectin 1 / Q000037:antagonists & inhibitors* / Q000235:genetics; D013552:Swine; D014161:Transduction, Genetic; D019562:Viral Load; D000098902:CD163 Antigen
chemical_list: D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D000998:Antiviral Agents; D000098902:CD163 Antigen; D004337:Drug Carriers; D035683:MicroRNAs; D011956:Receptors, Cell Surface; D011991:Receptors, Virus; D063267:Sialic Acid Binding Ig-like Lectin 1