Park Seong-Jun, Moon Hyoung-Joon, Luo Yuzi, Kim Hye-Kwon, Kim Eun-Mi, Yang Jeong-Sun, Song Dae-Sub, Kang Bo-Kyu, Lee Chul-Seung, Park Bong-Kyun
Department of Veterinary Medicine Virology Lab, College of Veterinary Medicine, Seoul National University, 599 Gwanangno, Gwanak-gu, Seoul, 151-742, Korea.
Virus Genes. 2008 Feb;36(1):95-104. doi: 10.1007/s11262-007-0164-2. Epub 2007 Oct 12.
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.
克隆并测序了亲本DR13、致弱DR13、KPED-9、P-5V以及12份田间样本的开放阅读框（ORF3）基因，以进一步探究野生型和致弱型猪流行性腹泻病毒（PEDV）的功能。测序结果显示，野生型PEDV的ORF3基因有一个675个核苷酸的单一开放阅读框，编码一个224个氨基酸的蛋白质，预测分子量（M（r））为25.1 - 25.3 kDa。致弱型PEDV的ORF3基因有一个624个核苷酸的单一开放阅读框，编码一个207个氨基酸的蛋白质，预测分子量（M（r））为23.4 kDa。包括致弱DR13、KPED-9和P-5V在内的致弱型PEDV的ORF3基因编码区有51个核苷酸缺失，而在包括CV777、Br1/87、LZ C、亲本DR13和12份田间样本在内的野生型PEDV的ORF3基因中未发现这种缺失。此外，此前已发现致弱型PEDV在猪体内的致病性降低。因此，51个核苷酸缺失似乎具有重要意义，可能对PEDV致病性至关重要，因为它们导致了致弱型PEDV预测氨基酸序列的变化。对包含51个核苷酸缺失的部分ORF3基因进行逆转录聚合酶链反应（RT-PCR），结果显示所有PEDV可分为野生型和致弱型两种类型。包含亲本DR13和12份田间样本的野生型PEDV的RT-PCR产物大小为245 bp，而包含PEDV疫苗株（致弱DR13、KPED-9、P-5V）的致弱型PEDV的产物大小为194 bp。此外，所有PEDV疫苗株均用作活病毒疫苗，因为它们此前在猪体内表现出较低的致病性。因此，由51个核苷酸缺失导致的包含17个氨基酸缺失的大片段缺失区域在所有PED活疫苗株中均可见，可能是PEDV致病性的重要位点，我们可以利用它来区分野生型和致弱型PEDV。
