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Construction of a multiple targeting RNAi plasmid that inhibits target gene expression and FMDV replication in BHK-21 cells and suckling mice

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Foot-and-mouth disease (FMD) is a highly contagious disease that afflicts cloven-hoofed animals. The etiological agent of FMD is foot-and-mouth disease virus (FMDV). The VP1 gene of FMDV is essential during the life cycle of the virus and plays a key role in the attachment of the virus to susceptible cells. We constructed a plasmid, pCWN11, that expresses siRNAs multiple-targeting the VP1 genes of FMDV. We evaluated the gene silencing efficiency of the plasmid using an enhanced green fluorescent protein (EGFP) reporter system in BHK-21 cells. The antiviral potential of the plasmid in BHK-21 cells and suckling mice were investigated. The results indicate that cotransfection of pCWN11 with any one of three serotypes VP1-EGFP plasmids resulted in a reduction in the EGFP signal relative to the control. Moreover, the antiviral potential induced by pCWN11 was evident during challenge with one FMDV isolate of either serotype O (HKN/2002) or serotype Asia I (YNBS/58), and the inhibition extended to almost 40 h. Furthermore, subcutaneous injection of pCWN11 in the neck made suckling mice significantly less susceptible to FMDV serotype O and Asia I.

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  1. Bachrach HL (1968) Foot-and-mouth disease virus. Annu. Rev. Microbiol. 22: 201−224.

  2. Bernstein E, Caudy AA, Hammond SM, Hannon GJ (2001a) Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409: 363–366.

  3. Bernstein E, Denli AM, Hannon GJ (2001b) The rest is silence. RNA 7: 1509–1521.

  4. Brummelkamp TR, Bernards R, Agami R (2002) Asystem for stable expression of short interfering RNAs in mammalian cells. Science 296: 550–553.

  5. Chen W, Yan W, Du Q, Fei L, Liu M, Ni Z, Sheng Z, Zheng Z (2004) RNA interference targeting VP1 inhibits foot-and-mouth disease virus replication in BHK-21 cells and suckling mice. J. Virol. 78: 6900–6907.

  6. Chang LJ, Liu X, He J (2005) Lentiviral siRNAs targeting multiple highly conserved RNA sequences of human immunodeficiency virus type 1. Gene Ther. 12: 1133–1144.

  7. Dumbell KR, Jarrett JO, Mautner V, Minson AC, Pereira HG, Pereira MS, Porterfield JS, Wilkinson PJ (1989) Picornaviridae. In: Porterfield JS (ed) Andrewes’ viruses of vertebrates, 5th edn. Cambridge University Press, Cambridge, United Kingdom, pp 120–145.

  8. Ding H, Schwarz DS, Keene A, Affar EB, Fenton L, Xia X, Shi Y, Zamore PD, Xu Z (2003) Selective silencing by RNAi of a dominant allele that causes amyotrophic lateral sclerosis. Aging Cell 2: 209–217.

  9. Das AT, Brummelkamp TR, Westerhout EM, Vink M, Madiredjo M, Bernards R, Berkhout B (2004) Human Immunodeficiency Virus type 1 escapes from RNA interference-mediated inhibition. J. Virol. 78: 2601–2605.

  10. Dave RS, Pomerantz RJ (2004) Antiviral effects of Human Immunodeficiency Virus type 1-specific small interfering RNAs against targets conserved in select neurotropic viral strains. J. Virol. 78: 13687–13696.

  11. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001a) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411: 379 494–498.

  12. Elbashir SM, Lendeckel W, Tuschl T (2001b) RNA interference is mediated by 21-and 22-nucleotide RNAs. Genes Dev. 15: 188–200.

  13. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391: 806–811.

  14. Gitlin L, Andino R (2003) Nucleic acid-based immune system: the antiviral potential of mammalian RNA silencing. J. Virol. 77: 7159–7165.

  15. Gitlin L, Stone JK, Andino R (2005) Poliovirus escape from RNA interference: short interfering RNA-target recognition and implications for therapeutic approaches. J. Virol. 79: 1027–1035.

  16. Grubman M, Santos T (2005) Rapid control of foot-and-mouth disease outbreaks:is RNAi a possible solution? TRENDS in Immunology 26: 65–68.

  17. Geisbert TW, Hensley LE, Kagan E, Yu EZ, Geisbert JB, Daddario-DiCaprio K, Fritz EA, Jahrling PB, McClintock K, Phelps JR, Lee ACH, Judge A, Jeffs LB, MacLachlan I (2006) Postexposure protection of guinea pigs against a lethal Ebola virus challenge is conferred by RNA interference. J. Infect. Dis. 193: 1650–1657.

  18. Jackson AL, Bartz SR, Schelter J, Kobayashi SV, Burchard J, Mao M, Li B, Cavet G, Linsley PS (2003) Expression profiling reveals off-target gene regulation by RNAi. Nat. Biotechnol. 21: 635–637.

  19. Kennerdell JR, Carthew RW (2000) Heritable gene silencing in Drosophila using double-stranded RNA. Nat. Biotechnol. 18: 896–898.

  20. Knight SW, Bass BL (2001) A Role for the RNase III Enzyme DCR-1 in RNA Interference and Germ Line Development in Caenorhabditis elegans. Science 293: 2269–2271.

  21. Kahana R, Kuznetzova L, Rogel A, Shemesh M, Hai D, Yadin H, Stram Y (2004) Inhibition of foot-and-mouth disease virus replication by small interfering RNA. J. Gen. Virol. 85: 3213–3217.

  22. Liu M, Chen W, Ni Z, Yan W, Fei L, Jiao Y, Zhang J, Du Q, Wei X, Chen J, Liu Y, Zheng Z (2005) Cross-inhibition to heterologous foot and- mouth disease virus infection induced by RNA interference targeting the conserved regions of viral genome. Virology 336: 51–59.

  23. Macpherson I, Stoker M (1962) Polyoma transformation of hamster cell clones—an investigation of genetic factors affecting cell competence. Virology 16: 147–151.

  24. Mohapatra JK, Sanyal A, Hemadri D, Tosh C, Kumar RM, Bandyopadhyay SK (2005) Evaluation of in vitro inhibitory potential of small interfering RNAs directed against various regions of foot-and-mouth disease virus genome. Biochem. Biophys. Res. Commun. 329: 1133–1138.

  25. Nishitsuji H, Kohara M, Kannagi M, Masuda T (2006) Effective suppression of human immunodeficiency virus type 1 through a combination of short- or long-hairpin RNAs targeting essential sequences for retroviral integration. J. Virol. 80: 7658–7666.

  26. Paul CP, Good PD, Winer I, Engelke DR (2002) Effective expression of small interfering RNA in human cells. Nat. Biotechnol. 20: 505–508.

  27. Phipps KM, Martinez A, Lu J, Heinz BA, Zhao G (2004) Small interfering RNA molecules as potential anti-human rhinovirus agents: in vitro potency, specificity, and mechanism. Antiviral Res. 61: 49–55.

  28. Reed LJ, Muench HA (1938) A simple method of estimating fifty percent end points. Am. J. Hyg. 27: 493–497.

  29. Sharp PA (2001) RNA interference—2001. Genes Dev. 15: 485–490.

  30. Sui G, Soohoo C, Affar B, Gay F, Shi Y, Forrester WC, Shi Y (2002) A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc. Natl. Acad. Sci. USA 99: 5515–5520.

  31. Song E, Lee SK, Dykxhoorn DM, Novina C, Zhang D, Crawford K, Cerny J, Sharp PA, Lieberman J, Manjunath N, Shankar P (2003) Sustained small interfering RNA-mediated human immunodeficiency virus type 1 inhibition in primary macrophages. J. Virol. 77: 7174–7181.

  32. Tavernarakis N, Wang SL, Dorovkov M, Ryazanov A, Driscoll M (2000) Heritable and inducible genetic interference by double-stranded RNA encoded by transgenes. Nat. Genet. 24: 180–183.

  33. Wu KL, Zhang X, Zhang J, Yang Y, Mu YX, Liu M, Lu L, Li Y, Zhu Y, Wu J (2005) Inhibition of hepatitis B virus gene expression by single and dual small interfering RNA treatment. Virus Res. 112: 100–107.

  34. Wu KL, Mu Y, Hu J, Lu L, Zhang X, Yang Y, Li Y, Liu F, Song D, Zhu Y, Wu J (2007) Simultaneously inhibition of HIV and HBV replication through a dual small interfering RNA expression system. Antiviral Res. 74: 142–149.

  35. Zhang H, Kolb FA, Brondani V, Billy E, Filipowicz W (2002) Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP. EMBO J. 21: 5875–5885.

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We thank Peng Hao, Xuecen Xie, Lijun Wang, Yanhong Zhang, Yanqi Sun and Tailiang Sun for technical assistance.

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Correspondence to Zhaoxin Zheng.

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Cong, W., Cui, S., Chen, J. et al. Construction of a multiple targeting RNAi plasmid that inhibits target gene expression and FMDV replication in BHK-21 cells and suckling mice. Vet Res Commun 34, 335–346 (2010). https://doi.org/10.1007/s11259-010-9360-y

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  • FMDV
  • VP1 gene
  • RNA interference (RNAi)
  • siRNA
  • Multiple targeting plasmid