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. 2008 Feb;36(1):95-104.
doi: 10.1007/s11262-007-0164-2. Epub 2007 Oct 12.

Cloning and further sequence analysis of the ORF3 gene of wild- and attenuated-type porcine epidemic diarrhea viruses

Seong-Jun Park  1 , Hyoung-Joon Moon, Yuzi Luo, Hye-Kwon Kim, Eun-Mi Kim, Jeong-Sun Yang, Dae-Sub Song, Bo-Kyu Kang, Chul-Seung Lee, Bong-Kyun Park
Affiliations

Cloning and further sequence analysis of the ORF3 gene of wild- and attenuated-type porcine epidemic diarrhea viruses

Seong-Jun Park et al. Virus Genes. 2008 Feb.

Abstract

The open reading frame (ORF3) genes of the parent DR13, attenuated DR13, KPED-9, P-5V, and 12 field samples were cloned and sequenced to further explore the functions of wild- and attenuated-type porcine epidemic diarrhea viruses (PEDVs). Sequencing revealed that wild-type PEDVs ORF3 genes had a single ORF of 675 nucleotides encoding a protein of 224 amino acids with a predicted M (r) of 25.1-25.3 kDa. Attenuated-type PEDVs ORF3 genes had a single ORF of 624 nucleotides encoding a protein of 207 amino acids with a predicted M (r) of 23.4 kDa. The coding region of the ORF3 gene of attenuated-type PEDVs including attenuated DR13, KPED-9, and P-5V had 51 nucleotide deletions that were not found in the ORF3 genes of wild-type PEDVs including CV777, Br1/87, LZC, parent DR13, and 12 field samples. In addition, attenuated-type PEDVs have previously been found to exhibit reduced pathogenicity in pigs. Therefore, 51 nucleotide deletions appear to be meaningful and may be significant for PEDV pathogenicity, because they lead to changes in the predicted amino acid sequences of attenuated-type PEDVs. Reverse transcriptase-polymerase chain reaction (RT-PCR) on the partial ORF3 gene including 51 nucleotide deletions revealed that all PEDVs fell into two types, wild- and attenuated-type PEDVs. Wild-type PEDVs containing parent DR13 and 12 field samples had RT-PCR products of 245 bp in size, while attenuated-type PEDVs containing PEDV vaccine strains (attenuated DR13, KPED-9, P-5V) had products of 194 bp. In addition, all PEDV vaccine strains were used as live virus vaccine, because they previously exhibited a reduced pathogenicity in pigs. Therefore, large deletion region, which is comprise 17 amino acid deletions caused by 51 nucleotide deletions and is seen in all PED live vaccine strains, may be important site for PEDV pathogenicity, and we can use it for differentiation of wild- and attenuated-type PEDVs.

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Figures

Fig. 1
Fig. 1
Construction of cDNA clones for the full-length ORF3 gene of wild- and attenuated-type PEDVs by RT-PCR using a pair of sense (ORF3-1) and antisense (ORF3-2) primers: diagrammatic representation of the ORF3 gene of viral RNA (long solid rectangle) show primer-binding sites (small open rectangles) and large nucleotide deletion legion (stripe rectangles); 16 DNA fragments of wild- and attenuated-type PEDVs amplified by RT-PCR and cloned into the pDrive Cloning Vector are denoted as recombinant DNA clones Ofrag I–XVI. Wild-type PEDVs: Ofrag I (Parent DR13), Ofrag V (DBI825) Ofrag VI (BI976), Ofrag VII (BI1166), Ofrag VIII (M1763), Ofrag IX (M1764), Ofrag X (e1834), Ofrag XI (e2540), Ofrag XII (BI2804), Ofrag XIII (e3991), Ofrag XIV (PF4275), Ofrag XV (M4758), Ofrag XVI (e8066) Attenuated-type PEDVs: Ofrag II (Attenuated DR13), Ofrag III (KPED-9), Ofrag IV (P-5V)
Fig. 2
Fig. 2
Comparison of the (a) nucleotide and (b) deduced amino acid sequences of the ORF3 gene of wild- and attenuated-type PEDVs. Asterisks represent (a) nucleotides and (b) amino acids that are identical to those in the attenuated DR13 (GenBank accession No. EU054930). Dashed lines represent missing (a) nucleotides and (b) amino acids compared to the PEDV CV777 (Br1/87) (EMBL accession No. Z24733), LZC (GenBank accession No. EF185992), parent DR13 (GenBank accession No. EU054929) and 12 field samples. Start codon ATG and stop codon TGA are underlined. Regions corresponding to the primers used for cloning and differentiation of wild- and attenuated-type PEDVs are underlined and labeled above the sequence as ORF3-1-2 and PEDO1-2. Three variable regions previously reported are underlined and labeled above the sequence as Roman numbers, I–III. PEDVs in parentheses below had identical nucleotide and amino acid sequences with that in front of parentheses in the ORF3 genes and especially DBI825 and M4758 underlined below had different nucleotide but identical amino acid sequences with parent DR13. Wild-type PEDVs: CV777, Br1/87, LZC, Parent DR13, DBI825, BI976, BI1166, M1763 (M1764), e1834 (e2540), BI2804, e3991, PF4275, M4758, e8066 Attenuated-type PEDVs: Attenuated DR13 (KPED-9, P-5V)
Fig. 2
Fig. 2
Comparison of the (a) nucleotide and (b) deduced amino acid sequences of the ORF3 gene of wild- and attenuated-type PEDVs. Asterisks represent (a) nucleotides and (b) amino acids that are identical to those in the attenuated DR13 (GenBank accession No. EU054930). Dashed lines represent missing (a) nucleotides and (b) amino acids compared to the PEDV CV777 (Br1/87) (EMBL accession No. Z24733), LZC (GenBank accession No. EF185992), parent DR13 (GenBank accession No. EU054929) and 12 field samples. Start codon ATG and stop codon TGA are underlined. Regions corresponding to the primers used for cloning and differentiation of wild- and attenuated-type PEDVs are underlined and labeled above the sequence as ORF3-1-2 and PEDO1-2. Three variable regions previously reported are underlined and labeled above the sequence as Roman numbers, I–III. PEDVs in parentheses below had identical nucleotide and amino acid sequences with that in front of parentheses in the ORF3 genes and especially DBI825 and M4758 underlined below had different nucleotide but identical amino acid sequences with parent DR13. Wild-type PEDVs: CV777, Br1/87, LZC, Parent DR13, DBI825, BI976, BI1166, M1763 (M1764), e1834 (e2540), BI2804, e3991, PF4275, M4758, e8066 Attenuated-type PEDVs: Attenuated DR13 (KPED-9, P-5V)
Fig. 2
Fig. 2
Comparison of the (a) nucleotide and (b) deduced amino acid sequences of the ORF3 gene of wild- and attenuated-type PEDVs. Asterisks represent (a) nucleotides and (b) amino acids that are identical to those in the attenuated DR13 (GenBank accession No. EU054930). Dashed lines represent missing (a) nucleotides and (b) amino acids compared to the PEDV CV777 (Br1/87) (EMBL accession No. Z24733), LZC (GenBank accession No. EF185992), parent DR13 (GenBank accession No. EU054929) and 12 field samples. Start codon ATG and stop codon TGA are underlined. Regions corresponding to the primers used for cloning and differentiation of wild- and attenuated-type PEDVs are underlined and labeled above the sequence as ORF3-1-2 and PEDO1-2. Three variable regions previously reported are underlined and labeled above the sequence as Roman numbers, I–III. PEDVs in parentheses below had identical nucleotide and amino acid sequences with that in front of parentheses in the ORF3 genes and especially DBI825 and M4758 underlined below had different nucleotide but identical amino acid sequences with parent DR13. Wild-type PEDVs: CV777, Br1/87, LZC, Parent DR13, DBI825, BI976, BI1166, M1763 (M1764), e1834 (e2540), BI2804, e3991, PF4275, M4758, e8066 Attenuated-type PEDVs: Attenuated DR13 (KPED-9, P-5V)
Fig. 3
Fig. 3
RT-PCR on the partial ORF3 gene including large deletion region. From left to right: Lane M, 25/100 bp DNA ladder; Lane 1, parent DR13; Lane 2, attenuated DR13; Lane 3, negative control
Fig. 4
Fig. 4
RT-PCR on the commercial vaccine strains used in Korea. From left to right: Lane M, 25/100 bp DNA ladder; Lane 1, parent DR13; Lane 2, attenuated DR13 strain of the Korean PED oral vaccine; Lane 3, KPED-9 strain of the Korean PED live virus vaccine; Lane 4, P-5V strain of the Japanese PED live virus vaccine; Lane 5, negative control
Fig. 5
Fig. 5
RT-PCR on the 12 field samples. From left to right; Lane M, 25/100 bp DNA ladder; Lane 1, parent DR13; Lane 2, attenuated DR13; Lane 3, negative control, Lane 4, DBI825; Lane 5, BI976; Lane 6, BI1166; Lane 7, M1763; Lane 8, M1764; Lane 9, e1834; Lane 10, e2540; Lane 11, BI2804, Lane 12, e3991; Lane 13, PF4275; Lane 14, M4758; Lane 15 e8066
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References

    1. Pensaert M.B., de Bouck P. Arch. Virol. 1978;58:243–247. doi: 10.1007/BF01317606. - DOI - PMC - PubMed
    1. Debouck P., Pensaert M. Am. J. Vet. Res. 1980;41:219–223. - PubMed
    1. Kweon C.H., Kwon B.J., Jung T.S., Kee Y.J., Hur D.H., Hwang E.K., Rhee J.C., An S.H. Korean J. Vet. Rec. 1993;33:249–254.
    1. Debouck P., Pensaert M., Coussement W. Vet. Microbiol. 1981;6:157–165. doi: 10.1016/0378-1135(81)90007-9. - DOI
    1. M.B. Pensaert, S. G. Yeo, in Disease of swine, ed. by B.E. Straw, J.J. Zimmerman, S. D’Allaire, D.J. Taylor (The Blackwell Publishing Professional, Ames, IA, 2006), pp. 367–372

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