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Virus Genes

, Volume 55, Issue 6, pp 795–801 | Cite as

Short hairpin RNAs targeting M and N genes reduce replication of porcine deltacoronavirus in ST cells

  • Wen-yuan Gu
  • Yan Li
  • Bao-jing Liu
  • Jing Wang
  • Guang-fu Yuan
  • Shao-jie Chen
  • Yu-Zhu Zuo
  • Jing-Hui FanEmail author
Original Paper

Abstract

Porcine deltacoronavirus (PDCoV) is a recently identified coronavirus that causes intestinal diseases in neonatal piglets with diarrhea, vomiting, dehydration, and post-infection mortality of 50–100%. Currently, there are no effective treatments or vaccines available to control PDCoV. To study the potential of RNA interference (RNAi) as a strategy against PDCoV infection, two short hairpin RNA (shRNA)-expressing plasmids (pGenesil-M and pGenesil-N) that targeted the M and N genes of PDCoV were constructed and transfected separately into swine testicular (ST) cells, which were then infected with PDCoV strain HB-BD. The potential of the plasmids to inhibit PDCoV replication was evaluated by cytopathic effect, virus titers, and real-time quantitative RT-PCR assay. The cytopathogenicity assays demonstrated that pGenesil-M and pGenesil-N protected ST cells against pathological changes with high specificity and efficacy. The 50% tissue culture infective dose showed that the PDCoV titers in ST cells treated with pGenesil-M and pGenesil-N were reduced 13.2- and 32.4-fold, respectively. Real-time quantitative RT-PCR also confirmed that the amount of viral RNA in cell cultures pre-transfected with pGenesil-M and pGenesil-N was reduced by 45.8 and 56.1%, respectively. This is believed to be the first report to show that shRNAs targeting the M and N genes of PDCoV exert antiviral effects in vitro, which suggests that RNAi is a promising new strategy against PDCoV infection.

Keywords

Porcine deltacoronavirus RNA interference Short hairpin RNA Nucleocapsidgene Swine testicular cells 

Notes

Acknowledgements

This research was supported by the Program of the Modern Agriculture Industry Technology System Foundation of Hebei Province (HBCT2018110207), Science and Technology Innovation Program of Hebei Province for graduate students (CXZZBS2019095), and Key R & D projects in Hebei Province, China (19226622D).

Author contributions

WYG performed the main experiments and wrote the paper; YL participated in editing the paper; BJL was involved in executing the study and editing the paper; YZZ and JHF participated in experimental design; JW, GFY, and SJC revised the paper.

Compliance with ethical standards

Conflict of interests

The authors declare that they have no conflicting interests.

Ethical approval

This article does not contain any studies with animals performed by any of the authors.

References

  1. 1.
    Liu BJ, Zuo YZ, Gu WY, Luo SX, Shi QK, Hou LS, Zhong F, Fan JH (2018) Isolation and phylogenetic analysis of porcine deltacoronavirus from pigs with diarrhoea in Hebei province China. Transbound Emerg Dis 65(3):874–882.  https://doi.org/10.1111/tbed.12821 CrossRefPubMedGoogle Scholar
  2. 2.
    Li G, Chen Q, Harmon KM, Yoon KJ, Schwartz KJ, Hoogland MJ, Gauger PC, Main RG, Zhang J (2014) Full-length genome sequence of porcine deltacoronavirus strain USA/IA/2014/8734. Genome Announc.  https://doi.org/10.1128/genomeA.00278-14 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Song D, Zhou X, Peng Q, Chen Y, Zhang F, Huang T, Zhang T, Li A, Huang D, Wu Q, He H, Tang Y (2015) Newly emerged porcine deltacoronavirus associated with diarrhoea in swine in China: identification, prevalence and full-length genome sequence analysis. Transbound Emerg Dis 62(6):575–580.  https://doi.org/10.1111/tbed.12399 CrossRefPubMedGoogle Scholar
  4. 4.
    Woo PC, Lau SK, Lam CS, Lau CC, Tsang AK, Lau JH, Bai R, Teng JL, Tsang CC, Wang M, Zheng BJ, Chan KH, Yuen KY (2012) Discovery of seven novel Mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus. J Virol 86(7):3995–4008.  https://doi.org/10.1128/JVI.06540-11 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Wang L, Byrum B, Zhang Y (2014) Detection and genetic characterization of deltacoronavirus in pigs, Ohio, USA. Emerg Infect Dis 20(7):1227–1230.  https://doi.org/10.3201/eid2007.140296 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Hu H, Jung K, Vlasova AN, Chepngeno J, Lu Z, Wang Q, Saif LJ (2015) Isolation and characterization of porcine deltacoronavirus from pigs with diarrhea in the United States. J Clin Microbiol 53(5):1537–1548.  https://doi.org/10.1128/JCM.00031-15 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Lee S, Lee C (2015) Functional characterization and proteomic analysis of the nucleocapsid protein of porcine deltacoronavirus. Virus Res 208:136–145.  https://doi.org/10.1016/j.virusres.2015.06.013 CrossRefPubMedGoogle Scholar
  8. 8.
    Zhai SL, Wei WK, Li XP, Wen XH, Zhou X, Zhang H, Lv DH, Li F, Wang D (2016) Occurrence and sequence analysis of porcine deltacoronaviruses in southern China. Virol J 13:136.  https://doi.org/10.1186/s12985-016-0591-6 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Lee S, Lee C (2014) Complete genome characterization of korean porcine deltacoronavirus strain KOR/KNU14-04/2014. Genome Announc.  https://doi.org/10.1128/genomeA.01191-14 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Zhang Z, Chen J, Shi H, Chen X, Shi D, Feng L, Yang B (2012) Identification of a conserved linear B-cell epitope in the M protein of porcine epidemic diarrhea virus. Virol J 9:225.  https://doi.org/10.1186/1743-422X-9-225 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Arndt AL, Larson BJ, Hogue BG (2010) A conserved domain in the coronavirus membrane protein tail is important for virus assembly. J Virol 84:11418–11428.  https://doi.org/10.1128/JVI.01131-10 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Fan JH, Zuo YZ, Li JH, Pei LH (2012) Heterogeneity in membrane protein genes of porcine epidemic diarrhea viruses isolated in China. Virus Genes 45(1):113–117.  https://doi.org/10.1007/s11262-012-0755-4 CrossRefPubMedGoogle Scholar
  13. 13.
    Chang CK, Sue SC, Yu TH, Hsieh CM, Tsai CK, Chiang YC, Lee SJ, Hsiao HH, Wu WJ, Chang WL, Lin CH, Huang TH (2006) Modular organization of SARS coronavirus nucleocapsid protein. J Biomed Sci 13(1):59–72.  https://doi.org/10.1007/s11373-005-9035-9 CrossRefPubMedGoogle Scholar
  14. 14.
    Molenkamp R, Spaan WJ (1997) Identification of a specific interaction between the coronavirus mouse hepatitis virus A59 nucleocapsid protein and packaging signal. Virology 239(1):78–86.  https://doi.org/10.1006/viro.1997.8867 CrossRefPubMedGoogle Scholar
  15. 15.
    Shen H, Zhang C, Guo P, Liu Z, Zhang J (2015) Effective inhibition of porcine epidemic diarrhea virus by RNA interference in vitro. Virus Genes 51(2):252–259.  https://doi.org/10.1007/s11262-015-1242-5 CrossRefPubMedGoogle Scholar
  16. 16.
    Sui HY, Zhao GY, Huang JD, Jin DY, Yuen KY, Zheng BJ (2009) Small interfering RNA targeting M2 gene induces effective and long term inhibition of influenza A virus replication. PLoS ONE 4(5):e5671.  https://doi.org/10.1371/journal.pone.0005671 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Wang L, Dai X, Song H, Yuan P, Yang Z, Dong W, Song Z (2017) Inhibition of porcine transmissible gastroenteritis virus infection in porcine kidney cells using short hairpin RNAs targeting the membrane gene. Virus Genes 53(2):226–232.  https://doi.org/10.1007/s11262-016-1409-8 CrossRefPubMedGoogle Scholar
  18. 18.
    Zhou F, Liang S, Chen AH, Singh CO, Bhaskar R, Niu YS, Miao YG (2012) A transgenic Marc-145 cell line of piggyBac transposon-derived targeting shRNA interference against porcine reproductive and respiratory syndrome virus. Vet Res Commun 36(2):99–105.  https://doi.org/10.1007/s11259-012-9519-9 CrossRefPubMedGoogle Scholar
  19. 19.
    Homwong N, Jarvis MC, Lam HC, Diaz A, Rovira A, Nelson M, Marthaler D (2016) Characterization and evolution of porcine deltacoronavirus in the United States. Prev Vet Med 123:168–174.  https://doi.org/10.1016/j.prevetmed.2015.11.001 CrossRefPubMedGoogle Scholar
  20. 20.
    Madapong A, Saeng-Chuto K, Lorsirigool A, Temeeyasen G, Srijangwad A, Tripipat T, Wegner M, Nilubol D (2016) Complete genome sequence of porcine deltacoronavirus isolated in Thailand in 2015. Genome Announc.  https://doi.org/10.1128/genomeA.00408-16 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Zhang J (2016) Porcine deltacoronavirus: overview of infection dynamics, diagnostic methods, prevalence and genetic evolution. Virus Res 226:71–84.  https://doi.org/10.1016/j.virusres.2016.05.028 CrossRefPubMedGoogle Scholar
  22. 22.
    Fouad AM, Soliman H, Abdallah ESH, Ibrahim S, El-Matbouli M, Elkamel AA (2018) In-vitro inhibition of spring viremia of carp virus replication by RNA interference targeting the RNA-dependent RNA polymerase gene. J Virol Methods 263:14–19.  https://doi.org/10.1016/j.jviromet.2018.10.008 CrossRefPubMedGoogle Scholar
  23. 23.
    Man DK, Chow MY, Casettari L, Gonzalez-Juarrero M, Lam JK (2016) Potential and development of inhaled RNAi therapeutics for the treatment of pulmonary tuberculosis. Adv Drug Deliv Rev 102:21–32.  https://doi.org/10.1016/j.addr.2016.04.013 CrossRefPubMedGoogle Scholar
  24. 24.
    Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, Conklin DS (2002) Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev 16:948–958.  https://doi.org/10.1101/gad.981002 CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Dong N, Fang L, Yang H, Liu H, Du T, Fang P, Wang D, Chen H, Xiao S (2016) Isolation, genomic characterization, and pathogenicity of a Chinese porcine deltacoronavirus strain CHN-HN-2014. Vet Microbiol 196:98–106.  https://doi.org/10.1016/j.vetmic.2016.10.022 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Wen-yuan Gu
    • 1
    • 2
  • Yan Li
    • 1
  • Bao-jing Liu
    • 1
  • Jing Wang
    • 1
  • Guang-fu Yuan
    • 1
  • Shao-jie Chen
    • 1
  • Yu-Zhu Zuo
    • 1
  • Jing-Hui Fan
    • 1
    Email author
  1. 1.College of Veterinary MedicineAgricultural University of HebeiBaodingPeople’s Republic of China
  2. 2.Animal Diseases Control Center of HebeiShijiazhuangChina

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