The role of ORF3 accessory protein in replication of cell-adapted porcine epidemic diarrhea virus (PEDV).

The ORF3 accessory protein has been shown to impede reverse genetics of cell-culture-adapted porcine epidemic diarrhea virus (PEDV). Its absence or truncated variants are also associated with viral attenuation in vivo. Here, three ORF3 variants (ORF3NP12, ORF3NP14 and ORF3RB14) and their truncated counterparts were investigated for their regulatory role in recovery of cell-adapted PEDV in vitro. We demonstrate that ORF3NP12, but not the truncated form, can inhibit recovery of reverse-genetics-derived PEDV when expressed in trans. When testing with other RNA viruses, ORF3 was found to inhibit rescue of porcine respiratory and reproductive syndrome virus (PRRSV), but not of influenza virus. Interestingly, results from mutagenesis of ORF3NP12 suggest that F81 and M167 are responsible for impairing PEDV rescue in vitro. By changing specific residues of ORF3, the recombinant PEDV bearing the modified ORF3NP12 can be productively propagated in VeroE6-APN cells. These results may provide mechanistic insights into ORF3-mediated inhibition of PEDV replication in new host cells.

Detection and phylogenetic analysis of porcine epidemic diarrhea virus in central China based on the ORF3 gene and the S1 gene.

BACKGROUND: Porcine epidemic diarrhea (PED) has increased in severity in China since 2010. To investigate further the infectivity, genetic diversity and molecular epidemiology of its causative agent, the porcine epidemic diarrhea virus (PEDV), we assessed 129 clinical samples, which were the intestinal tissue of piglets with severe diarrhea, from 17 cities in central China. Both the spike (S) glycoprotein (S1, 1-789 amino acids (aa)) and the full-length ORF3 gene of 21 representative field strains from 21 farms in 11 cities were sequenced and analysed. METHODS: PEDV was detected by reverse transcription-polymerase chain reaction (RT-PCR), and S1 and ORF3 sequences were processed by the Clustal W method via DNAMAN 8 software, and phylogenetic trees were constructed by the neighbor-joining method using MEGA 6 software. RESULTS: The prevalence of PEDV was 92.25% and was detected in 119 of 129 samples, with 94.03% (63 of 67) of pig farms harbouring the disease. According to the phylogenetic analysis of the S1 genes, our isolates all fell into group G2 (variants) and showed a close relationship to isolates from Chinese (HN1303, CH/ZMDZY/11 and AJ1102), Korean (AD01), American (MN, IA1, IA2 and 13-019349) sources, and these isolates differed genetically from other Chinese (LZC, CH/HNZZ/2011 and SD-M) and Korean (SM98) strains as well Japanese (83-P5 and MK) strains. In addition, our isolates differed from attenuated vaccine strains, CV777 (used in China) and DR13 (used in Korea). According to our derived amino acid sequence analysis, we detected one novel variant PEDV, viz: CH/HNLY, with 4-aa insertion/deletion (RSSS/T) at position 375 and 1-aa (D) deletion at position 430 compared to the CV777 attenuated strain. These mutations were located on the receptor binding domain. Our ORF3 gene analyses showed that the prevalent PEDV isolates were variants, and the isolated strains differed genetically from the vaccine strains. CONCLUSIONS: These findings illustrated the existence of genetic diversity among geographically distinct PEDV strains, and our study has provided an impetus to conduct further research on the PEDV receptor binding protein and on the new and efficacious vaccines design.

Comprehensive analysis of synonymous codon usage patterns in orf3 gene of porcine epidemic diarrhea virus in China.

The ORF3 protein of porcine epidemic diarrhea virus (PEDV) is found to function as an ion channel which influences virus virulence and production. Taking consideration of the importance of PEDV orf3 gene, we have performed comprehensive analysis to investigate its synonymous codon usage patterns. In this study, the results of base composition analysis showed A/T rich and G/C poor in PEDV orf3 genes, and the most abundant base was nucleotide T. The relative synonymous codon usage value in each codon revealed that codon usage bias existed. The mean ENC value of each gene was 48.75, indicating a low codon usage bias, as well as a relatively instable change in PEDV orf3 genes. The general correlation analysis between base composition and codon usage bias indicated that mutational bias has an impact on the PEDV codon usage bias. Neutral analysis suggested that natural selection pressure takes a more important influence than mutational bias in shaping codon usage bias. Moreover, other factors including hydrophobicity and aromaticity have been also found to influence the codon usage variation among the PEDV orf3 genes. This study not only represents the most systematic analysis of codon usage patterns in PEDV orf3 genes, but also provides a basic shaping mechanism of the codon usage bias.

Genetic diversity of ORF3 and spike genes of porcine epidemic diarrhea virus in Thailand.

Porcine epidemic diarrhea virus (PEDV) has become endemic in the Thai swine industry, causing economic losses and repeated outbreaks since its first emergence in 2007. In the present study, 69 Thai PEDV isolates were obtained from 50 swine herds across Thailand during the period 2008-2012. Both partial and complete nucleotide sequences of the spike (S) glycoprotein and the nucleotide sequences of ORF3 genes were determined to investigate the genetic diversity and molecular epidemiology of Thai PEDV. Based on the analysis of the partial S glycoprotein genes, the Thai PEDV isolates were clustered into 2 groups related to Korean and Chinese field isolates. The results for the complete spike genes, however, demonstrated that both groups were grouped in the same cluster. Interestingly, both groups of Thai PEDV isolates had a 4-aa (GENQ) insertion between positions 55 and 56, a 1-aa insertion between positions 135 and 136, and a 2-aa deletion between positions 155 and 156, making them identical to the Korean KNU series and isolates responsible for outbreaks in China in recent years. In addition to the complete S sequences, the ORF3 gene analyses suggested that the isolates responsible for outbreaks in Thailand are not vaccine related. The results of this study suggest that the PEDV isolates responsible for outbreaks in Thailand since its emergence represent a variant of PEDV that was previously reported in China and Korea.

Infection of cats with atypical feline coronaviruses harbouring a truncated form of the canine type I non-structural ORF3 gene.

Feline and canine coronaviruses (FCoV and CCoV, respectively) are common pathogens of cats and dogs sometimes leading to lethal infections named feline infectious peritonitis (FIP) and canine pantropic coronavirus infection. FCoV and CCoV are each subdivided into two serotypes, FCoV-I/II and CCoV-I/II. A phylogenetic relationship is evident between, on one hand, CCoV-I/FCoV-I, and on the other hand, CCoV-II/FCoV-II, suggesting that interspecies transmission can occur. The aim of the present study was to evaluate the prevalence of coronavirus (CoV)-infected cats according to their contact with dogs and to genetically analyse the CoV strains infecting cats. From 2003 to 2009, we collected 88 faecal samples from healthy cats and 11 ascitic fluids from FIP cats. We investigated the possible contact with dog in the household and collected dogs samples if appropriate. Out of 99 cat samples, 26 were coronavirus positive, with six cats living with at least one dog, thus showing that contact with dogs does not appear as a predisposing factor for cats CoV infections. Molecular and phylogenetic analyses of FCoV strains were conducted using partial N and S sequences. Six divergent strains were identified with the N gene clustering with CCoV-I whereas the 3' end of S was related to FCoV-I. Further analysis on those six samples was attempted by researching the presence of the ORF3 gene, the latter being peculiar to CCoV-I to date. We succeeded to amplify the ORF3 gene in five samples out of six. Thus, our data strongly suggest the circulation of atypical FCoV strains harbouring the CCoV-I ORF3 gene among cats. Moreover, the ORF3 genes recovered from the feline strains exhibited shared deletions, never described before, suggesting that these deletions could be critical in the adaptation of these strains to the feline host.