Cloning and further sequence analysis of the ORF3 gene of wild- and attenuated-type porcine epidemic diarrhea viruses.
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.
Park
Seong-Jun
SJ
Department of Veterinary Medicine Virology Lab, College of Veterinary Medicine, Seoul National University, 599 Gwanangno, Gwanak-gu, Seoul, 151-742, Korea.
Moon
Hyoung-Joon
HJ
Luo
Yuzi
Y
Kim
Hye-Kwon
HK
Kim
Eun-Mi
EM
Yang
Jeong-Sun
JS
Song
Dae-Sub
DS
Kang
Bo-Kyu
BK
Lee
Chul-Seung
CS
Park
Bong-Kyun
BK
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D018352:Coronavirus Infections / Q000662:veterinary / Q000821:virology; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics*; D053485:Porcine epidemic diarrhea virus / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity*; D016415:Sequence Alignment; D017384:Sequence Deletion; D013552:Swine / Q000821:virology; D013553:Swine Diseases / Q000821:virology; D014709:Vero Cells; D014765:Viral Vaccines
D014765:Viral Vaccines