Detection of Porcine Epidemic Diarrhea Virus Using Polymerase Chain Reaction and Comparison of the Nucleocapsid Protein Genes among Strains of the Virus.

Kubota, Shuichi; Sasaki, Osamu; Amimoto, Katsuhiko; Okada, Nobutaka; Kitazima, Takashi; Yasuhara, Hisao

doi:10.1292/jvms.61.827

Cited by 30 publications

(23 citation statements)

References 15 publications

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“…Although PEDV is known to have only one serotype at present, the genome of PEDV is widely to show genetic diversity, according to the analysis results using molecular biological methods [21][22][23]. The M protein genes of four Chinese strains (LJB/03, JS-2004-2, LZC, and QH) have been sequenced and reported.…”

Section: Discussionmentioning

confidence: 99%

Molecular characterization and phylogenetic analysis of membrane protein genes of porcine epidemic diarrhea virus isolates in China

et al. 2008

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Six porcine epidemic diarrhea viruses (PEDVs) were isolated from the fecal samples of piglets infected with PEDV in 2006 in China. The membrane (M) protein genes of six PEDV isolates were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR), then cloned, sequenced, and compared with each other as well as those ten PEDV reference strains. The M protein genes of six Chinese PEDV isolates consisted of 692 nucleotides containing a single open reading frame (ORF) of 681 nucleotides, which encoded a 226aa-long peptide. The conserved intergenic motif (ATAAAC), as previously recognized in Br1/87, was found in the 5 nucleotides upstream of the initiator ATG of M protein genes of six Chinese PEDV isolates. The hexamer motif was also found in CV777, JMe2, LZC, and QH. The M protein of six isolates had three main transmembrane domains (aa20-38, aa43-65, aa75-97). The M protein of one isolate, CH/IMT/06, had one potential glycosylation site, but those of the other five isolates had two. The glycosylation sequence Asn-Phe-Thr was highly conserved in the M proteins of six PEDV isolates. The six PEDV isolates showed nucleotide sequence homology between 98.8 and 100% and deduced amino acid sequence homology between 98.2 and 100% with each other. The nucleotide and amino acid identity of M protein genes between the six PEDV isolates and ten reference PEDV strains varied from 97.2 to 99.4% and 96.9 to 100%, respectively. On the basis of the phylogenetic relationship of M protein genes, six Chinese PEDV isolates composed of a separate cluster including one Chinese strain JS-2004-02, however, not including the Chinese strain LJB/03. These results demonstrated that there was a new genotype of PEDV prevailing in China.

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Section: Discussionmentioning

confidence: 99%

Molecular characterization and phylogenetic analysis of membrane protein genes of porcine epidemic diarrhea virus isolates in China

et al. 2008

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“…Similarly, although PEDV is known to have only one serotype at present, genome of PEDV is widely known to show genetic diversity, according to analysis results using molecular biological methods [3,[28][29][30]. Based on sequence analyses of M glycoprotein gene and N phosphoprotein gene, Korean PEDV isolates are different from foreign PEDV strains.…”

Section: Discussionmentioning

confidence: 99%

Sequence analysis of the partial spike glycoprotein gene of porcine epidemic diarrhea viruses isolated in Korea

et al. 2007

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Porcine epidemic diarrhea virus (PEDV) causes a devastating enteric disease with acute diarrhea, dehydration and significant mortality in swine, thereby incurring heavy economic losses in Korea. Spike (S) glycoprotein has been suggested as an important determinant for PEDV biological properties. In this study, the nucleotide and deduced amino acid sequences of the partial S glycoprotein genes of Korean PEDV isolates, including epitope region that is capable of inducing PEDV-neutralizing antibodies, were determined. The partial S glycoprotein genes were amplified by RT-PCR, cloned, sequenced, and compared with each other as well as with reference PEDV strains. By phylogenetic analysis, the Korean PEDV isolates were divided into three groups (G1, G2, G3), which had three subgroups (G1-1, G1-2, G1-3). Group1 (G1) Korean PEDV isolates were highly homologous to CV777, Br1/87, JS-2004-2, KPED-9, P-5V, SM98-1, parent DR13, and attenuated DR13, group2 (G2) Korean PEDV isolates were highly homologous to Spk1, and group3 (G3) was Chinju99 at the nucleotide and deduced amino acid sequence levels. In addition, the G1 Korean PEDV isolates didn't had several specific nucleotides and amino acids which were found in the G2 and G3 Korean PEDV isolates, and especially the G1-1 Korean PEDV isolates had specific nucleotides and amino acids which were not found in the G1-2, G1-3, G2, and G3 Korean PEDV isolates. It was suggested that many Korean PEDV isolates are closely related to the G1 including CV777, Br1/87, JS-2004-2, KPED-9, P-5 V, SM98-1, parent DR13, and attenuated DR13 rather than to the G2 and G3 including Spk1 and Chinju99, and notably more prevalent PEDVs isolated in Korea are especially close to the Chinese PEDV strain JS-2004-2 rather than Korean PEDV strains Spk1, Chinju99, KPED-9, SM98-1, parent DR13, and attenuated DR13.

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“…Porcine epidemic diarrhea virus (PEDV), a member of the family Coronaviridae, causes acute diarrhea, dehydration, and high mortality in piglets [1,2], which incurs significant economic losses in European and Asian swine industries [3][4][5][6]. PEDV has a single-stranded positive-sense RNA genome of approximately 28 kb in size that encodes four structural proteins (spike [S], envelope, membrane [M], and nucleocapsid [N] protein) and four nonstructural proteins (1a, 1b, 3a, and 3b).…”

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confidence: 99%

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

et al. 2011

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Previously, we have reported that a serial passage of 83P-5 strain of porcine epidemic diarrhea virus (PEDV) in Vero cells resulted in a growth adaptation of the virus in cultured cells at the 22nd passage. In this study, we further maintained the 83P-5 in Vero cells up to the 100th passage and analyzed changes in the spike (S), membrane (M), and nucleocapsid (N) gene sequences and pathogenicity of the virus at the 34th, 61st, and 100th passage levels. Sequence analyses revealed a strong selection for the S gene of 83P-5 in Vero cells, and virtually all mutations occurring at the 34th and 61st passages had been carried over to the 100th-passaged virus. In contrast, the viral M and N genes showed a strong conservation during the serial passage. Pigs experimentally infected with the 34th- or 61st-passaged virus, but not the 100th-passaged virus, exhibited diarrhea, indicating an attenuation of the 83P-5 at the 100th passage. Interestingly, S protein of the attenuated 100th-passaged 83P-5 showed a remarkable sequence similarity to that of previously reported DR-13 strain of attenuated PEDV that also had been established by serial passage in Vero cells. Further studies will be required to define whether the mutations in the S gene of 83P-5 that had been selected and accumulated during the serial passages are indeed the causalities of the growth adaptation in vitro and the attenuation of virulence in vivo.

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Detection of Porcine Epidemic Diarrhea Virus Using Polymerase Chain Reaction and Comparison of the Nucleocapsid Protein Genes among Strains of the Virus.

Cited by 30 publications

References 15 publications

Molecular characterization and phylogenetic analysis of membrane protein genes of porcine epidemic diarrhea virus isolates in China

Molecular characterization and phylogenetic analysis of membrane protein genes of porcine epidemic diarrhea virus isolates in China

Sequence analysis of the partial spike glycoprotein gene of porcine epidemic diarrhea viruses isolated in Korea

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

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