Archives of Virology

, Volume 163, Issue 11, pp 2997–3004 | Cite as

Preparation and characterization of an attenuated porcine epidemic diarrhea virus strain by serial passaging

  • Tao Jie
  • Li Benqiang
  • Cheng Jinghua
  • Shi Ying
  • Liu HuiliEmail author
Original Article


Porcine epidemic diarrhea virus (PEDV) is prevalent in most parts of the world. Owing to its antigenic variation, prevention of the diseases caused by this virus is difficult. In this study, two PEDV isolates with similar growth kinetics were successfully propagated in Vero cells. Complete genome sequence analysis showed that they have a 49nt deletion in the ORF3 gene and were classified into Group 1, the same group that includes the classical CV777 strain. Recombination analysis revealed that the event had occurred in the ORF1a gene, at 3596–6819 nt, among the two PEDV isolates and the CV777 and DR13 strains. During their continuous propagation, 14 nonsynonymous mutations occurred in the spike (S) gene of strain JS-2/2014 between generations G5 and G90, but there were no changes between G90 and G100. We assumed that strain JS-2/2014 might be attenuated by the 90th generation. Piglets orally fed with JS-2/2014 G90 showed no clinical symptoms, and no virus was detected in the feces and nasal fluid. In conclusion, JS-2/2014 was successfully identified by screening, was attenuated after propagation in Vero cells, and may serve as a candidate virus for vaccine preparations.



Porcine epidemic diarrhea virus


Transmissible gastroenteritis virus


Open reading frame








Days of postinfection



This study was funded by the National Key Research and Development Program of China (No. 2018YFD0500100), Shanghai Agriculture Applied Technology Development Program of China (No.T20170111), and the Youth Talent Development Plan of Shanghai Municipal Agricultural System of China (No. 20160135).

Compliance with ethical standards

Conflict of interest

There are no conflicts of interest associated with this article.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. Furthermore, this article does not contain any experiments with human subjects or animals performed by any of the authors.


  1. 1.
    Jung K, Saif LJ (2015) Porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis. Vet J 204(2):134–143CrossRefPubMedGoogle Scholar
  2. 2.
    Wang L, Byrum B, Zhang Y (2014) New variant of porcine epidemic diarrhea virus, United States. Emerg Infect Dis 20(5):917–919CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Pasick J, Berhane Y, Ojkic D et al (2014) Investigation into the role of potentially contaminated feed as a source of the first-detected outbreaks of porcine epidemic diarrhea in Canada. Transbound Emerg Dis 61(5):397–410CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Stadler J, Zoels S, Fux R et al (2015) Emergence of porcine epidemic diarrhea virus in southern Germany. BMC Vet Res 11:142CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Masuda T, Murakami S, Takahashi O et al (2015) New porcine epidemic diarrhoea virus variant with a large deletion in the spike gene identified in domestic pigs. Arch Virol 160(10):2565–2568CrossRefPubMedGoogle Scholar
  6. 6.
    Li W, Li H, Liu Y et al (2012) New variants of porcine epidemic diarrhea virus, China, 2011. Emerg Infect Dis 18(8):1350–1353CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Wang E, Guo D, Li C et al (2016) Molecular characterization of the ORF3 and S1 genes of porcine epidemic diarrhea virus non S-INDEL strains in seven regions of China, 2015. PLoS One 11(8):e0160561CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Gao X, Li D, Zhao J et al (2016) Complete genome sequence of PEDV from an outbreak in a vaccinated farm in Shandong, China. Genome Announc 4(4):e00619–16CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Li R, Qiao S, Yang Y et al (2016) Genome sequencing and analysis of a novel recombinant porcine epidemic diarrhea virus strain from Henan, China. Virus Genes 52(1):91–98CrossRefPubMedGoogle Scholar
  10. 10.
    Wang K, Xie C, Zhang J et al (2016) The identification and characterization of two novel epitopes on the nucleocapsid protein of the porcine epidemic diarrhea virus. Sci Rep 6:39010CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Sun D, Wang X, Wei S et al (2016) Epidemiology and vaccine of porcine epidemic diarrhea virus in China: a mini-review. J Vet Med Sci 78(3):355–363CrossRefPubMedGoogle Scholar
  12. 12.
    Chen F, Zhu Y, Wu M et al (2015) Comparative genomic analysis of classical and variant virulent parental/attenuated strains of porcine epidemic diarrhea virus. Viruses 7(10):5525–5538CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Jaru-Ampornpan P, Jengarn J, Wanitchang A et al (2017) Porcine epidemic diarrhea virus (PEDV) 3C-like protease-mediated nucleocapsid processing: a possible link to viral cell-culture adaptability. J Virol 91(2):e01660–16CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Pan Y, Tian X, Li W et al (2012) Isolation and characterization of a variant porcine epidemic diarrhea virus in China. Virol J 9:195CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Sun RQ, Leng ZM, Zhai SL et al (2014) Genetic variability and phylogeny of current Chinese porcine epidemic diarrhea virus strains based on spike, ORF3, and membrane genes. Sci World J 2014:208439Google Scholar
  16. 16.
    LaBarre DD, Lowy RJ (2011) Improvements in methods for calculating virus titer estimates from TCID50 and plaque assays. J Virol Methods 96(2):107–126CrossRefGoogle Scholar
  17. 17.
    Song DS, Yang JS, Oh JS, Han JH, Park BK (2003) Differentiation of a vero cell adapted porcine epidemic diarrhea virus from Korean field strains by restriction fragment length polymorphism analysis of ORF 3. Vaccine 21:1833–1842CrossRefPubMedGoogle Scholar
  18. 18.
    Ni JQ, Yuan L, Wang J et al (2017) Epidemiological survey on porcine diarrhea in swine breeding farms in China during 2013–2015. China Anim Health Insp 34(4):5–9Google Scholar
  19. 19.
    Chen J, Wang C, Shi H et al (2010) Molecular epidemiology of porcine epidemic diarrhea virus in China. Arch Virol 155(9):1471–1476CrossRefPubMedGoogle Scholar
  20. 20.
    Trudeau MP, Verma H, Sampedro F et al (2017) Environmental persistence of porcine coronaviruses in feed and feed ingredients. PLoS One 12(5):e0178094CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Chang TC, Chen JF, Feng L et al (2016) Epidemiological survey of porcine epidemic diarrhea virus in 2014. Chin J Prev Vet Med 38(4):335–338Google Scholar
  22. 22.
    Hu XY, Zhang SX, Jin ZQ et al (2016) The epidemiological investigation on viral diarrhea of piglets in Guizhou region. Chin J Prev Vet Med 38(7):542–545Google Scholar
  23. 23.
    Zhu WX, Guo HY, Chen LG et al (2012) Isolation and identification of porcine epidemic diarrhea virus strain HBMC2012 and its pathogenicity in piglet. Chin J Prev Vet Med 38(12):934–938Google Scholar
  24. 24.
    Li W, van Kuppeveld FJ, He Q et al (2016) Cellular entry of the porcine epidemic diarrhea virus. Virus Res 226:117–127CrossRefPubMedGoogle Scholar
  25. 25.
    Park SJ, Moon HJ, Luo Y et al (2008) Cloning and further sequence analysis of the ORF3 gene of wild- and attenuated-type porcine epidemic diarrhea viruses. Virus Genes 36(1):95–104CrossRefPubMedGoogle Scholar
  26. 26.
    McGoldrick A, Lowings JP, Paton DJ (1999) Characterisation of a recent virulent transmissible gastroenteritis virus from Britain with a deleted ORF 3a. Arch Virol 144(4):763–770CrossRefPubMedGoogle Scholar
  27. 27.
    Park SJ, Song DS, Ha GW, Park B (2007) Cloning and further sequence analysis of the spike gene of attenuated porcine epidemic diarrhea virus DR13. Virus Genes 35(1):55–64CrossRefPubMedGoogle Scholar
  28. 28.
    Lin CM, Saif LJ, Marthaler D, Wang Q (2016) Evolution, antigenicity and pathogenicity of global porcine epidemic diarrhea virus strains. Virus Res 226:20–39CrossRefPubMedGoogle Scholar
  29. 29.
    Li R, Qian S, Yang Y et al (2016) Genome sequencing and analysis of a novel recombinant porcine epidemic diarrhea virus strain from Henan, China. Virus Genes 52(1):91–98CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Authors and Affiliations

  • Tao Jie
    • 1
    • 2
    • 3
  • Li Benqiang
    • 1
    • 2
    • 3
  • Cheng Jinghua
    • 1
    • 2
    • 3
  • Shi Ying
    • 1
    • 2
    • 3
  • Liu Huili
    • 1
    • 2
    • 3
    Email author
  1. 1.Institute of Animal Science and Veterinary MedicineShanghai Academy of Agricultural SciencesShanghaiChina
  2. 2.Shanghai Key Laboratory of Agricultural Genetic BreedingShanghaiChina
  3. 3.Shanghai Engineering Research Center of Pig BreedingShanghaiChina

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