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

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.

Genetic manipulation of porcine epidemic diarrhoea virus recovered from a full-length infectious cDNA clone

“…2a) and some cell-adapted PEDV strains display internal truncation of the ORF3 gene (Park et al, 2008), we speculated that functional ORF3 might play an important role in regulating PEDV replication in vitro. To test this hypothesis, we engineered the PEDV AVCT12 infectious clone to express full-length ORF3 derived from the Chinese strain JS-2004-2 (RgPEDV wtORF3 ) using the strategy depicted in Fig.…”

A novel strain of porcine epidemic diarrhea virus in Vietnamese pigs

“…PEDV was detected in fecal specimens by RT-PCR as described previously [16,17], with primers specific for the spike and ORF3 regions of PEDV. Products of the expected size were cloned using pGEM-T Vector System II (Promega, Cat.…”

Isolation and genetic analysis of a variant porcine epidemic diarrhea virus in China

“…At present, RT-PCR based on the amplification of ORF3, M, S, and N gene has been used for the clinical diagnosis of PEDV (Lee et al 2003, Park et al 2008, Ogawa et al 2009). However, morphological methods are still useful for the identification of an unknown virus.…”