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J Virol. May 2012; 86(10): 5964.
PMCID: PMC3347302

Complete Genome Sequences of a Korean Virulent Porcine Epidemic Diarrhea Virus and Its Attenuated Counterpart

Abstract

A virulent porcine epidemic diarrhea virus (PEDV) strain, DR13, was obtained from suckling pigs suspected of having porcine epidemic diarrhea in 1999 in Korea, and its attenuated counterpart was derived from virulent strain DR13 by serial propagation in Vero cells. This report describes the first complete genome sequences of virulent PEDV and its attenuated counterpart, which will provide important insights into the molecular basis of the attenuation of PEDV.

GENOME ANNOUNCEMENT

Porcine epidemic diarrhea virus (PEDV), first reported in 1978 (6), is an enveloped, single-stranded RNA virus belonging to the family Coronaviridae. PEDV causes an acute and highly contagious enteric disease characterized by severe diarrhea, dehydration, and a high mortality rate in swine. A virulent strain, DR13, was obtained from suckling pigs suspected of having porcine epidemic diarrhea in 1999, and an attenuated counterpart was derived from virulent strain DR13 by serial propagation in Vero cells (7). To date, the complete genome sequences of a virulent and attenuated PEDV strain pair have not been reported. Therefore, to provide important insights into the molecular basis of PEDV attenuation, it is necessary to analyze the complete genome sequences of virulent DR13 and its attenuated counterpart.

Both ends of the genomes of the virulent and attenuated PEDV strain pair were confirmed by a system for the rapid amplification of cDNA ends (Invitrogen Corp.). The other parts were generated by analyzing ~28 overlapping cDNA fragments to complete the entire genomes and were determined by primer-walking sequencing. The genome of virulent DR13 is 28,029 nucleotides (nt) long after exclusion of the poly(A) tail, whereas that of attenuated DR13 contains 27,931 nt [excluding the poly(A) tail], which is 95 to 102 nt shorter than those of other sequenced virulent PEDVs (1, 4). The organization of the genomes of the virulent and attenuated PEDV strain pair is similar to that of other reported PEDV genomes (1, 4), with the characteristic gene order 5′-replicase (1a/1b)-S-ORF3-E-M-N-3′, and both their 5′ (292 nt) and 3′ (334 and 333 nt for virulent and attenuated DR13, respectively) ends contain untranslated regions (UTRs). Downstream of the 5′ UTR, the PEDV genome contains two large open reading frames (ORF1a and ORF1ab) encoding two large nonstructural precursor polyproteins (replicases pp1a and pp1ab), the latter of which is expressed by programmed translational frameshifting (3). The precursor polyproteins are proteolytically ~15 replicase proteins (5). While the sizes of ORF1a and ORF1ab of virulent DR13 are 12,354 nt (nt 293 to 12,646) and 20,346 nt (nt 293 to 12,601 and 12601 to 20,637), those of attenuated DR13 are 12,330 nt (nt 293 to 12622) and 20,322 nt (nt 293 to 12,577 and 12,577 to 20,613). Moreover, and surprisingly, attenuated DR13 has 4,149-, 276-, and 210-nt ORFs in its S, ORF3 and E genes, which are 3, 399, and 21 nt shorter than those of its virulent counterpart.

The virulent and attenuated DR13 strains have hexameric XUA(A/G)AC motifs at sites immediately upstream of their respective genes (CUAGAC, GUAAAC, AUAAAC, and CUAAAC sequences are present 85 [46, 10], 8, 11, and 15 nt upstream from the initiator ATG of the ORF1 [ORF3, E], S, M, and N genes) that are identical to those found at the same sites of CV777 (2). These motifs are common to coronaviruses and have been proposed to be start sites for the transcription of subgenomic mRNAs (5).

This is the first report of the complete genome sequences of a virulent PEDV strain and its attenuated counterpart. We hope that these data will help create a better understanding of the molecular basis of the attenuation of PEDV, as well as other coronaviruses.

Nucleotide sequence accession numbers.

The complete genome sequences of the virulent and attenuated DR13 strains described here have been deposited in GenBank under accession no. JQ023161 and JQ023162.

ACKNOWLEDGMENT

This work was supported by a grant (code no. 20070401034009) from the BioGreen 21 Program, Rural Development Administration, Republic of Korea.

REFERENCES

1. Chen J, et al. 2011. Complete genome sequence of a Chinese virulent porcine epidemic diarrhea virus strain. J. Virol. 85:11538–11539 [PMC free article] [PubMed]
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 [PubMed]
3. Farabaugh PJ. 1996. Programmed translational frameshifting. Annu. Rev. Genet. 30:507–528 [PubMed]
4. Kocherhans R, Bridgen A, Ackermann M, Tobler K. 2001. Completion of the porcine epidemic diarrhoea coronavirus (PEDV) genome sequence. Virus Genes 23:137–144 [PubMed]
5. Masters PS. 2006. The molecular biology of coronaviruses. Adv. Virus Res. 66:193–292 [PubMed]
6. Pensaert MB, de Bouck P. 1978. A new coronavirus-like particle associated with diarrhea in swine. Arch. Virol. 58:243–247 [PubMed]
7. 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 21:1833–1842 [PubMed]

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