Full-Length Genome Sequence of a Plaque-Cloned Virulent Porcine Epidemic Diarrhea Virus Isolate (USA/Iowa/18984/2013) from a Midwestern U.S. Swine Herd
Published: December 22, 2017
By: Hai Hoang 1; Mary L. Killian 1,2; Darin M. Madson 3; Paulo H. E. Arruda 3; Dong Sun 1; Kent J. Schwartz 3; Kyoungjin J. Yoon 3. / 1 Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, Iowa, USA; 2 Diagnostic Virology Laboratory, National Veterinary Services Laboratories, U.S. Department of Agriculture, APHIS/VS, Ames, Iowa, USA; 3 Department of Veterinary Diagnostic and Production Animal Medicine, Iowa State University, Ames, Iowa, USA.
Summary
Porcine epidemic diarrhea (PED) was recognized in U.S. swine for the first time in early 2013. A plaque-purified PED virus (PEDV) isolate (USA/Iowa/18984/2013) was obtained from a diarrheic piglet. The isolate is genetically close to other previously reported U.S. PEDVs and recent Chinese PEDVs and was virulent when inoculated into neonatal pigs.
Porcine epidemic diarrhea (PED), caused by a member of the genus Alphacoronavirus (1–3), was first identified in England in 1971 and later in other countries, such as Belgium, China, Hungary, Italy, Japan, Korea, and Thailand (4–11). In April of 2013, PED emerged in U.S. swine (12) and was detected in swine herds in 18 U.S. states by the end of October (http://www.aasv.org/pedv /PEDV_weekly_report_103013.pdf), causing considerable economic losses. An isolate of PED virus (PEDV) was sought for use in studies to address concerns regarding diagnosis, transmission, pathogenesis, and vaccination.
Virus isolation was attempted from field samples submitted to the Iowa State University Veterinary Diagnostic Laboratory that were positive for PEDV by a real-time reverse transcriptase PCR (RT-PCR) assay (13) using Vero cells (ATCC CCL-81), as previously described (14). A cytopathic virus with coronavirus morphology was isolated from intestinal tissues collected from a 1.5week-old piglet from a swine farm in Iowa. In the cells that were inoculated with the virus, the virus-induced syncytia and eventually cell death. The presence of PEDV in the inoculated cells was confirmed by immunofluorescence microscopy using a PEDV-specific monoclonal antibody, 6C8 (12). After 3 cell culture passages, the primary isolate was subjected to plaque purification. A plaque-cloned virus isolate was further propagated in the cells for 2 more passages, for a total of 6 cell passages (P6).
The plaque-cloned PEDV P6 isolate (USA/Iowa/18984/2013) was able to cause cytopathic effects at 12 to 18 h post inoculation (hpi) and reach a titer of 105.5 PFU/ml within 48 hpi. Caesareanborncolostrum-deprived piglets orally inoculated with the isolate at a rate of 103 PFU/ml developed severe watery diarrhea and dehydration within 24 hpi and eventually died. Microscopically, intensive immunohistochemical staining of almost all enterocytes for PEDV was observed at 18 hpi, leading to severe villous atrophy at a later time.
The entire genome of the isolate USA/Iowa/18984/2013 was sequenced using next-generation sequencing technology on the Ion Torrent platform (Life Technologies, Austin, TX) as per the manufacturer’s instructions, and the data were assembled using DNAStar NGen based on known PEDV sequences. The genomic RNA of the isolate is 28,039 nucleotides long, excluding the 3= poly(A) tail. The genomic organization of the isolate is similar to what was previously described (2, 15, 16) and includes a 5= untranslated region (5= UTR), open reading frame 1a (ORF1a)/ ORF1b, S, ORF3, E, M, N, and a 3=UTR with a slippery sequence (12610TTTAAAC12616) in ORF1. There is an insertion between nucleotides 20204 and 20205 in ORF1 that causes a reading frameshift, shortening the replicase polyprotein 1ab (6,649 amino acids long). Phylogenetically, the isolate is 99. 8 to 99.9% similar to other U.S. PEDVs reported earlier (12, 15, 17), 97.2 to 99.6% similar to recent Chinese PEDVs (18–27), with AH2012 (GenBank accession no. KC210145) being the closest, and 96.9% similar to the prototype PEDV strain CV777 (1).
In conclusion, the PEDV isolate USA/Iowa/18984/2013 is a virulent strain with a genetic profile similar to those of other U.S. PEDVs reported to date. Such a representative purified virulent PEDV isolate can be a valuable reagent for studying the pathogenesis and immunobiology of PEDV and developing diagnostic reagents and kits, as well as effective vaccines.
Nucleotide sequence accession number. The complete genome sequence of PEDV strain USA/Iowa/18984/2013 was submitted to GenBank under the accession no. KF804028.
ACKNOWLEDGMENTS
We are grateful to practicing swine veterinarians for submitting samples from suspect cases to the Iowa State University Veterinary Diagnostic Laboratory and procuring additional samples. We also thank Drew Magstadt (Veterinary Diagnostic Laboratory, Iowa State University, Ames, IA) and John Neil (Ruminant Diseases and Immunology Research Unit, National Animal Disease Center, USDA/ARS, Ames, IA) for assistance in animal work and laboratory support, respectively. The study was supported in part by funding from the Iowa Pork Producers Association (NPB no. 13-209).
This article was originally published in Genome Announcements. 1(6):e01049-13. doi:10.1128/genomeA.01049-13. This is an Open Access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license.
REFERENCES
1. Bridgen A, Duarte M, Tobler K, Laude H, Ackermann M. 1993. Sequence determination of the nucleocapsid protein gene of the porcine epidemic diarrhoea virus confirms that this virus is a coronavirus related to human coronavirus 229E and porcine transmissible gastroenteritis virus. J. Gen. Virol. 74(Pt 9):1795–1804.
2. Duarte M, Gelfi J, Lambert P, Rasschaert D, Laude H. 1993. Genome organization of porcine epidemic diarrhoea virus. Adv. Exp. Med. Biol. 342:55–60.
3. Tobler K, Bridgen A, Ackermann M. 1993. Sequence analysis of the nucleocapsid protein gene of porcine epidemic diarrhoea virus.Adv.Exp. Med. Biol. 342:49–54.
4. Oldham J. 1972. Letter to the editor. Pig Farming 1972 (October suppl): 72–73.
5. Pensaert MB, de Bouck P. 1978. A new coronavirus-like particle associated with diarrhea in swine. Arch. Virol. 58:243–247.
6. Chen JF, Sun DB, Wang CB, Shi HY, Cui XC, Liu SW, Qiu HJ, Feng L. 2008. Molecular characterization and phylogenetic analysis of membrane protein genes of porcine epidemic diarrhea virus isolates in China. Virus Genes 36:355–364.
7. Nagy B, Nagy G, Meder M, Mocsári E. 1996. Enterotoxigenic Escherichia coli, rotavirus, porcine epidemic diarrhoea virus, adenovirus and calicilike virus in porcine postweaning diarrhoea in Hungary. Acta Vet. Hung. 44:9–19.
8. Martelli P, Lavazza A, Nigrelli AD, Merialdi G, Alborali LG, Pensaert MB. 2008. Epidemic of diarrhoea caused by porcine epidemic diarrhoea virus in Italy. Vet. Rec. 162:307–310.
9. Takahashi K, Okada K, Ohshima K.1983.Anoutbreakofswinediarrhea of a new-type associated with corona virus-like particles in Japan. Nippon Juigaku Zasshi 45:829–832.
10. Chae C, Kim O, Choi C, Min K, Cho WS, Kim J, Tai JH. 2000. Prevalence of porcine epidemic diarrhoea virus and transmissible gastroenteritis virus infection in Korean pigs. Vet. Rec. 147:606–608.
11. Puranaveja S, Poolperm P, Lertwatcharasarakul P, Kesdaengsakonwut S, Boonsoongnern A, Urairong K, Kitikoon P, Choojai P, Kedkovid R, Teankum K, Thanawongnuwech R. 2009. Chinese-like strain of porcine epidemic diarrhea virus, Thailand. Emerg. Infect. Dis. 15:1112–1115.
12. Stevenson GW, Hoang H, Schwartz KJ, Burrough ER, Sun D, Madson D, Cooper VL, Pillatzki A, Gauger P, Schmitt BJ, Koster LG, Killian ML, Yoon KJ. 2013. Emergence of porcine epidemic diarrhea virus in the United States: clinical signs, lesions, and viral genomic sequences. J. Vet. Diagn. Invest. 25:649–654.
13. Kim SH, Kim IJ, Pyo HM, Tark DS, Song JY, Hyun BH. 2007. Multiplex real-time RT-PCR for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus. J. Virol. Methods 146:172–177.
14. Hofmann M, Wyler R. 1988. Propagation of the virus of porcine epidemic diarrhea in cell culture. J. Clin. Microbiol. 26:2235–2239.
15. Marthaler D, Jiang Y, Otterson T, Goyal S, Rossow K, Collins J. 2013. Complete genome sequence of porcine epidemic diarrhea virus strain USA/Colorado/2013 from the United States. Genome Announc. 1(4): e00555-13. doi:10.1128/genomeA.00555-13.
16. Song D, Park B. 2012. Porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines. Virus Genes 44:167–175.
17. Huang YW, Dickerman AW, Piñeyro P, Li L, Fang L, Kiehne R, Opriessnig T, Meng XJ. 2013. Origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the United States. mBio 4(5):e00737-13.
18. Bi J, Zeng S, Xiao S, Chen H, Fang L. 2012. Complete genome sequence of porcine epidemic diarrhea virus strain AJ1102 isolated from a suckling piglet with acute diarrhea in China. J. Virol. 86:10910–10911.
19. Chen J, Wang C, Shi H, Qiu HJ, Liu S, Shi D, Zhang X, Feng L. 2011. Complete genome sequence of a Chinese virulent porcine epidemic diarrhea virus strain. J. Virol. 85:11538–11539.
20. Chen J, Liu X, Shi D, Shi H, Zhang X, Feng L. 2012. Complete genome sequence of a porcine epidemic diarrhea virus variant. J. Virol. 86:3408. doi:10.1128/JVI.07150-11.
21. Fan H, Zhang J, Ye Y, Tong T, Xie K, Liao M. 2012. Complete genome sequence of a novel porcine epidemic diarrhea virus in south China. J. Virol. 86:10248–10249.
22. Gao Y, Kou Q, Ge X, Zhou L, Guo X, Yang H. 2013. Phylogenetic analysis of porcine epidemic diarrhea virus field strains prevailing recently in China. Arch. Virol. 158:711–715.
23. Li B, Liu H, He K, Guo R, Ni Y, Du L, Wen L, Zhang X, Yu Z, Zhou J, Mao A, Lv L, Hu Y, Yu Y, Zhu H, Wang X. 2013. Complete genome sequence of a recombinant porcine epidemic diarrhea virus strain from eastern China. Genome Announc. 1(2):e00105-13.doi:10.1128/genomeA .00105-13.
24. Luo Y, Zhang J, Deng X, Ye Y, Liao M, Fan H. 2012. Complete genome sequence of a highly prevalent isolate of porcine epidemic diarrhea virus in south China. J. Virol. 86:9551.
25. Wang XM, Niu BB, Yan H, Gao DS, Huo JY, Chen L, Chang HT, Wang CQ, Zhao J. 2013. Complete genome sequence of a variant porcine epidemic diarrhea virus strain isolated in central China. Genome Announc. 1(1):e00243-12. doi:10.1128/genomeA.00243-12.
26. Wei ZY, Lu WH, Li ZL, Mo JY, Zeng XD, Zeng ZL, Sun BL, Chen F, Xie QM, Bee YZ, Ma J-Y. 2012. Complete genome sequence of novel porcine epidemic diarrhea virus strain GD-1 in China. J. Virol. 86:13824–13825.
27. Zhao M, Sun Z, Zhang Y, Wang G, Wang H, Yang F, Tian F, Jiang S. 2012.Complete genome sequence of a Verocell-adapted isolate of porcine epidemic diarrhea virus in eastern China. J. Virol. 86:13858–13859.
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