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RNA Interference Induces Effective Inhibition of mRNA Accumulation and Protein Expression of SHEV ORF3 Gene In vitro

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Abstract

RNA interference (RNAi) provides a powerful promising approach to inhibit viral infection specifically. To explore the possibility of using RNAi as a strategy against HEV infection, which is a serious public health problem in developing countries of Asia, Middle East, Africa, and in Mexico, after the fusion protein expression plasmids pEGFP-ORF3 which contain the EGFP reporter gene and SHEV ORF3 as silencing target, were constructed; EGFP-ORF3 fusion protein expressing HEK 293 cell lines were established; and four siRNAs targeting ORF3 gene were designed, synthesized, and used to transfect the stable cell lines. At 24, 48, and 72 h after transfection, flow cytometry, real-time quantitative PCR, and Western blot were used to assess the expression level of ORF3. The results demonstrated that specific siRNAs which are sequence dependant effectively inhibited mRNA accumulation and protein expression of SHEV ORF3 in HEK 293 cells. These findings provide useful information for the development of RNAi-based prophylaxis and therapy for SHEV infection.

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References

  1. Aggarwal R, Jameel S (2008) Hepatitis E vaccine. Hepatol Int 2:308–315

    Article  PubMed  Google Scholar 

  2. Choi IS, Kwon HJ, Shin NR et al (2003) Identification of swine hepatitis E virus (HEV) and prevalence of anti-HEV antibodies in swine and human populations in Korea. J Clin Microbiol 41:3602–3608

    Article  PubMed  CAS  Google Scholar 

  3. Cullen BR (2002) RNA interference: antiviral defence and genetic tool. Nat Immunol 3:597–599

    Article  PubMed  CAS  Google Scholar 

  4. Eick A, Ticehurst J, Tobler S et al (2010) Hepatitis E seroprevalence and seroconversion among US military service members deployed to Afghanistan. J Infect Dis 202:1302–1308

    Article  PubMed  Google Scholar 

  5. Faramawi MF, Johnson E, Chen S et al (2010) The incidence of hepatitis E virus infection in the general population of the USA. Epidemiol Infect 21:1–6

    Article  Google Scholar 

  6. Graff J, Nguyen H, Yu C et al (2005) The open reading frame 3 gene of hepatitis E virus contains a cis-reactive element and encodes a protein required for infection of macaques. J Virol 79:6680–6689

    Article  PubMed  CAS  Google Scholar 

  7. Hannon GJ (2002) RNA interference. Nature 418:244–251

    Article  PubMed  CAS  Google Scholar 

  8. Huang F, Hua X, Yang S et al (2009) Effective inhibition of hepatitis E virus replication in A549 cells and piglets by RNA interference (RNAi) targeting RNA-dependent RNA polymerase. Antiviral 83:274–281

    Article  CAS  Google Scholar 

  9. Huang F, Zhou J, Zhang W et al (2010) RNA interference effectively inhibits mRNA accumulation and protein synthesis of hepatitis E virus in A549 cells. Vet Microbiol 142:261–267

    Article  PubMed  CAS  Google Scholar 

  10. Husain MM, Srivastava R, Akondy R et al (2010) Evidence of hepatitis E virus exposure among seronegative healthy residents of an endemic area. Intervirology 54:139–143

    Article  PubMed  Google Scholar 

  11. Kar-Roy A, Korkaya H, Oberoi R et al (2004) The hepatitis E virus open reading frame 3 protein activates ERK through binding and inhibition of the MAPK phosphatase. J Biol Chem 279:28345–28357

    Article  PubMed  CAS  Google Scholar 

  12. Khuroo MS, Teli MR, Skidmore S et al (1981) Incidence and severity of viral hepatitis in pregnancy. Am J Med 70:252–255

    Article  PubMed  CAS  Google Scholar 

  13. Korkaya H, Jameel S, Gupta D et al (2001) The ORF3 protein of hepatitis E virus binds to Src homology 3 domains and activates MAPK. J Biol Chem 276:42389–42400

    Article  PubMed  CAS  Google Scholar 

  14. Krawczynski K (1993) Hepatitis E. Hepatology 17:932–941

    Article  PubMed  CAS  Google Scholar 

  15. Kumar A, Panda SK, Durgapal H et al (2010) Inhibition of Hepatitis E virus replication using short hairpin RNA (shRNA). Antiviral Res 85:541–550

    Article  PubMed  CAS  Google Scholar 

  16. Li TC, Chijiwa K, Sera N et al (2005) Hepatitis E virus transmission from wild boar meat. Emerg Infect Dis 11:1958–1960

    PubMed  Google Scholar 

  17. Lindemann ML, Gabilondon G, Romero B et al (2010) Low prevalence of hepatitis E infection among pregnant women in Madrid, Spain. J Med Virol 82:1666–1668

    Article  PubMed  Google Scholar 

  18. Liu T, Du L, Wang F et al (2009) Development of an indirect ELISA method for detection of antibody against recombinant ORF3 protein of swine hepatitis E virus genotype IV. Vet Sci China 39:310–315

    CAS  Google Scholar 

  19. Moin SM, Panteva M, Jameel S (2007) The hepatitis E virus ORF3 protein protects cells from mitochondrial depolarization and death. J Biol Chem 282:21124–21133

    Article  PubMed  CAS  Google Scholar 

  20. Panda SK, Nanda SK, Zafrullah M et al (1995) An Indian strain of Hepatitis E Virus (HEV): cloning, sequence, and expression of structural region and antibody responses in sera from Individuals from an area of high-level HEV endemicity. J Clin Microbiol 10:2653–2659

    Google Scholar 

  21. Pei Y, Yoo D (2002) Genetic characterization and sequence heterogeneity of a Canadian isolate of swine hepatitis E virus. J Clin Microbiol 40:4021–4029

    Article  PubMed  CAS  Google Scholar 

  22. Qu Y, Zhu G, Jin N et al (2008) Prokaryotic expression and identification of recombinant swine hepatitis E virus capsid protein. Chin J Biologicals 21:181–184

    CAS  Google Scholar 

  23. Shao Z, Li J, Zheng Y et al (2009) Epidemiological screening for hepatitis E Virus in bile specimens from livestock in Northwest China. J Clin Microbiol 47:814–816

    Article  PubMed  Google Scholar 

  24. Sun Z, Jin N, Zhu G et al (2006) Partial nucleotide sequencing and molecular epidemiology of hepatitis E viruses from swine and patients with hepatitis E infection in Changchun of Jilin province. Chin J Zoonoses 22:941–945

    CAS  Google Scholar 

  25. Tam AW, Smith MM, Guerra ME et al (1991) Hepatitis E virus (HEV): molecular cloning and sequencing of the full-length viral genome. Virology 185:120–131

    Article  PubMed  CAS  Google Scholar 

  26. Tei S, Kitajima N, Takahashi K et al (2003) Zoonotic transmission of hepatitis E virus from deer to human beings. Lancet 362:371–373

    Article  PubMed  Google Scholar 

  27. Tyagi S, Jameel S, Lal SK (2001) Self-association and mapping of the interaction domain of hepatitis E virus ORF3 protein. J Virol 75:2493–2498

    Article  PubMed  CAS  Google Scholar 

  28. Watson J (1979) Continuous proliferation of murine antigen-specific helper T lymphocytes in cu lture. J Exp Med 150:1510–1519

    Article  PubMed  CAS  Google Scholar 

  29. Xia N, Zhang J, Zheng Y et al (2004) Transfusion of plasma from a blood donor induced hepatitis E in Rhesus monkey. Vox Sang 86:45–47

    Article  PubMed  Google Scholar 

  30. Zafrullah M, Ozdener MH, Panda SK et al (1997) The ORF3 protein of hepatitis E virus is a phosphoprotein that associates with the cytoskeleton. J Virol 7:9045–9053

    Google Scholar 

  31. Zhang W, He Y, Wang H et al (2010) Hepatitis E virus genotype diversity in eastern China. Emerg Infect Dis 16:1630–1632

    Article  PubMed  Google Scholar 

Download references

Acknowledgment

This study was financially supported by the National Natural Science Foundation of China (NO. 30660139).

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Authors and Affiliations

  1. Hainan Key Lab of Tropical Animal Reproduction & Breeding and Epidemic Disease Research (Construction Period), Animal Genetic Engineering Key Lab of Haikou, College of Agriculture, Hainan University, Haidian Island, Haikou, 570228, People’s Republic of China

    Tao Liu, Ming Lei, Hanwei Jiao, Li Du, Ying Cheng, Donglin Zhang, Yongchang Hao, Churiga Man & Fengyang Wang

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  1. Tao Liu
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  2. Ming Lei
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  3. Hanwei Jiao
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  4. Li Du
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  5. Ying Cheng
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  6. Donglin Zhang
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  7. Yongchang Hao
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  8. Churiga Man
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  9. Fengyang Wang
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Corresponding author

Correspondence to Fengyang Wang.

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Tao Liu, Ming Lei, and Hanwei Jiao contributed equally to this work.

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Liu, T., Lei, M., Jiao, H. et al. RNA Interference Induces Effective Inhibition of mRNA Accumulation and Protein Expression of SHEV ORF3 Gene In vitro. Curr Microbiol 62, 1355–1362 (2011). https://doi.org/10.1007/s00284-010-9863-3

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  • DOI: https://doi.org/10.1007/s00284-010-9863-3

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