Cloning and further sequence analysis of the spike gene of attenuated porcine epidemic diarrhea virus DR13

Abstract

The spike (S) gene of the attenuated porcine epidemic diarrhea virus (PEDV) DR13 was cloned and sequenced to further explore the functions of wild type PEDV and attenuated PEDV. Sequencing revealed a single large ORF of 4,149 nucleotides encoding a protein of 1,382 amino acids with predicted M(r) of 151 kDa. The coding region of the S gene of attenuated PEDV DR13 had 20 nucleotide changes that appeared to be significant determinants of function in that they produced changes in its predicted amino acid sequence. Notably, attenuated PEDV DR13 has previously been found to exhibit reduced pathogenicity in pigs. The regions containing these 20 nucleotide changes may therefore be crucial for PEDV pathogenicity. The attenuated PEDV DR13 S protein contains 28 Asn-Xaa-Ser/Thr sequons, 21 asparagines that are predicted to be N-glycosylated and a stretch of highly hydrophobic residues at positions 1,327-1,347, which is predicted to form an alpha-helix and to function as a membrane anchor. One (from N to K at 378) of the changes in the deduced amino acid sequence destroyed N-linked glycosylation sites, while another change (from N to S at 114) created a new one at a different location. These alterations in N-linked glycosylation sites reflected 3 nucleotide changes, which were related to the above-mentioned nucleotide changes and are suggested to influence the pathogenicity of attenuated PEDV DR13. Attenuated PEDV DR13 has 96.5, 96.4, 96.1, 93.9, 93.5 and 96.6% DNA sequence identities with CV777, Br1/87, JS-2004-2, Spk1, Chinju99 and parent DR13, respectively. Likewise, it shares 95.7, 95.4, 95.6, 92.0, 91.6 and 95.7% identity with those genes at the deduced amino acid sequence level. Phylogenetic analysis suggested that attenuated PEDV DR13 is closely related to CV777, Br1/87, JS-2004-2 and parent DR13, rather than to Spk1 and Chinju99 and is especially close to the Chinese PEDV strain JS-2004-2.

Mutations in the spike gene of porcine epidemic diarrhea virus associated with growth adaptation in vitro and attenuation of virulence in vivo

“…Previously, Park et al [27] reported the S gene sequences of a live attenuated DR13 vaccine strain of PEDV that was established by a serial passage of the parental virus in Vero cells. To examine whether the 83P-5 and DR13 viruses were subjected to a similar selection pressure during serial passage in Vero cells, the S protein sequences of the parent and the serially passaged 83P-5 were aligned with those of the parent and the attenuated DR13 (Fig.…”

Complete Genome Sequence of an Avian-Origin H3N2 Canine Influenza Virus Isolated from Dogs in South Korea

“…The RT-PCR product, corresponding to each of eight genome segments, was purified, cloned (10), and sequenced on an automated DNA sequencer (ABI system 3700; Applied Biosystems, Inc.) by using universal primer sets (9) with slight modification and newly designed segment-specific primers spanning the ligation junction of the circularized RNAs to generate both ends of the respective genome segments. The lengths of the 3= and 5= NCRs of the viral RNA of A/canine/ Korea/01/2007 (H3N2) were variable (19 to 45 and 20 to 58 nt at the 3= and 5= NCRs, respectively) in the different genome segments, but the terminal 12 and 13 nt of the 3= and 5= ends, respectively, were highly conserved [3=UCG(C/U)UUUCGUCC-and -GGAACAAAGAUGA5=] among all eight genome segments, which is consistent with previous studies (2,12).…”

Complete Genome Sequence of a Mammalian Species-Infectious and -Pathogenic H6N5 Avian Influenza Virus without Evidence of Adaptation

“…The PCR products produced by RNA ligation-mediated reverse transcriptase PCR (RT-PCR) were purified, cloned (9), and sequenced to determine its exact complete genome sequence containing 3= and 5= NCRs on an automated DNA sequencer (ABI system 3700; Applied Biosystems Inc.) by utilizing universal primers (7) with simple modifications and newly designed segmentspecific primers.…”