Dataset Information

Analysis of the spike, ORF3, and nucleocapsid genes of porcine epidemic diarrhea virus circulating on Thai swine farms, 2011-2016.

ABSTRACT: Porcine epidemic diarrhea virus (PEDV) outbreaks on pig farms have caused significant economic loss in the swine industry since it was first reported in Thailand a decade ago. Anecdotal evidence suggests that PEDV is now endemic in this region, therefore genome information of circulating PEDV is important for molecular surveillance and evaluation of potential benefits of field vaccination. Here, we characterized PEDV infection on commercial Thai swine farms by screening 769 samples of feces and small intestinal contents from pigs with diarrhea between 2011 and 2016. Using reverse-transcription polymerase chain reaction targeting the spike (S) gene, 153 PEDV-positive samples were further subjected to analysis of the open reading frame 3 and nucleocapsid (N) genes. Comparison of 95 samples in which nucleotide sequencing was successfully obtained for all three genes revealed evolutionary diversity among the Thai PEDV strains. Phylogenetic analyses suggest that although some Thai strains changed little from years past, others resembled more closely to the recent strains reported in China. Interestingly, eight Thai PEDV strains possessed amino acid deletions in the N protein. The PEDV sequence divergence may be responsible for driving periodic outbreaks and continued persistence of PEDV on commercial swine farms. Our findings provide important insight into regional PEDV strains in circulation, which may assist future inclusions of suitable strains for future PEDV vaccines.

SUBMITTER: Tuanthap S

PROVIDER: S-EPMC6499054 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Analysis of the spike, ORF3, and nucleocapsid genes of porcine epidemic diarrhea virus circulating on Thai swine farms, 2011-2016.

Tuanthap Supansa S Vongpunsawad Sompong S Phupolphan Cherdpong C Duang-In Ausanee A Wattanaphansak Suphot S Assavacheep Pornchalit P Theamboonlers Apiradee A Luengyosluechakul Supol S Amonsin Alongkorn A Poovorawan Yong Y

PeerJ 20190430

Porcine epidemic diarrhea virus (PEDV) outbreaks on pig farms have caused significant economic loss in the swine industry since it was first reported in Thailand a decade ago. Anecdotal evidence suggests that PEDV is now endemic in this region, therefore genome information of circulating PEDV is important for molecular surveillance and evaluation of potential benefits of field vaccination. Here, we characterized PEDV infection on commercial Thai swine farms by screening 769 samples of feces and ...[more]

PMID: 31106060

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Project description:The porcine epidemic diarrhea virus (PEDV) causes an acute and highly contagious enteric disease characterized by severe enteritis, vomiting, watery diarrhea, and a high mortality rate in seronegative neonatal piglets. In the last few years, PED had a large economic impact on the swine industries in Asia and the US, and in 2014, the PEDV also re-emerged in Europe. Two main PEDV variants circulate worldwide but only the S INDEL variant, considered a mild strain, is spreading in Europe. To gain insights into the pathogenicity of this variant, its viral load and temporal shedding pattern were evaluated in piglets from infected farms. Quantitative real-time PCR (qPCR) targeting the spike gene, was validated according to the minimum information for quantitative real-time PCR experiments guidelines. The qPCR was applied to longitudinal studies conducted in four swine farms naturally infected with the PEDV S INDEL variant. Clinical data, fecal swabs, and blood samples were collected from 103 piglets at 15-30-day intervals for 2-5 months. On all four farms, diarrhea was observed in sows during gestation and in farrowing units, and the mortality rates of piglets were 18, 25, 30, and 35%. Different clinical pictures (0-50% of diarrhea positivity), viral titer levels (mean 5.3-7.2 log10 genome copies/mL), and antibody conditions (30-80% of positivity) were registered among sows on the four farms. The percentage of qPCR positive piglets varied greatly from the beginning (63-100%) to the end (0%) of the infection course. Clinical signs were present in 96% of the qPCR positive animals. Viral loads ranged from 8.5 log10 to 4 log10 genome copies/mL in suckling pigs at 3-6 days of age and were not statistically different among farms, despite the different patterns observed in sows. After 2-3 weeks, only a few piglets still showed detectable viral levels and clinical signs, and they developed antibody responses. Moreover, co-infections with other pathogens and biosecurity procedures limiting the circulation of the virus could have influenced the severity of PED infection. QPCR and clinical data were useful in understanding the dynamics of PEDV infections and, therefore, in implementing appropriate control measures.

Project description:Porcine epidemic diarrhea virus is a swine pathogen that has been responsible for significant animal and economic losses worldwide in recent years. In this manuscript, we report the generation of a reverse genetics system C(RGS) for the highly virulent US PEDV strain Minnesota (PEDV-MN; GenBank accession number KF468752), which was based on the assembly and cloning of synthetic DNA, using vaccinia virus as a cloning vector. Viral rescue was only possible following the substitution of 2 nucleotides within the 5'UTR and 2 additional nucleotides within the spike gene, based on the sequence of the cell culture-adapted strains. Besides displaying a highly pathogenic phenotype in newborn piglets, in comparison with the parental virus, the rescued recombinant PEDV-MN was used to confirm that the PEDV spike gene has an important role in PEDV virulence and that the impact of an intact PEDV ORF3 on viral pathogenicity is modest. Moreover, a chimeric virus with a TGEV spike gene in the PEDV backbone generated with RGS was able to replicate efficiently in vivo and could be readily transmitted between piglets. Although this chimeric virus did not cause severe disease upon the initial infection of piglets, there was evidence of increasing pathogenicity upon transmission to contact piglets. The RGS described in this study constitutes a powerful tool with which to study PEDV pathogenesis and can be used to generate vaccines against porcine enteric coronaviruses. IMPORTANCE PEDV is a swine pathogen that is responsible for significant animal and economic losses worldwide. Highly pathogenic variants can lead to a mortality rate of up to 100% in newborn piglets. The generation of a reverse genetics system for a highly virulent PEDV strain originating from the United States is an important step in phenotypically characterizing PEDV. The synthetic PEDV mirrored the authentic isolate and displayed a highly pathogenic phenotype in newborn piglets. With this system, it was possible to characterize potential viral virulence factors. Our data revealed that an accessory gene (ORF3) has a limited impact on pathogenicity. However, as it is also now known for many coronaviruses, the PEDV spike gene is one of the main determinants of pathogenicity. Finally, we show that the spike gene of another porcine coronavirus, namely, TGEV, can be accommodated in the PEDV genome background, suggesting that similar viruses can emerge in the field via recombination.

Project description:Accessory genes occurring between the S and E genes of coronaviruses have been studied quite intensively during the last decades. In porcine epidemic diarrhea virus (PEDV), the only gene at this location, ORF3, encodes a 224-residue membrane protein shown to exhibit ion channel activity and to enhance virus production. However, little is known about its intracellular trafficking or about its function during PEDV infection. In this study, two recombinant PEDVs were rescued by targeted RNA recombination, one carrying the full-length ORF3 gene and one from which the gene had been deleted entirely. These viruses as well as a PEDV encoding a naturally truncated ORF3 protein were employed to study the ORF3 protein's subcellular trafficking. In addition, ORF3 expression vectors were constructed to study the protein's independent transport. Our results show that the ORF3 protein uses the exocytic pathway to move to and accumulate in the Golgi area of the cell similarly in infected and transfected cells. Like the S protein, but unlike the other structural proteins M and N, the ORF3 protein was additionally observed at the surface of PEDV-infected cells. In addition, the C-terminally truncated ORF3 protein entered the exocytic pathway but it was unable to leave the endoplasmic reticulum (ER) and ER-to-Golgi intermediate compartment (ERGIC). Consistently, a YxxØ motif essential for ER exit was identified in the C-terminal domain. Finally, despite the use of sensitive antibodies and assays no ORF3 protein could be detected in highly purified PEDV particles, indicating that the protein is not a structural virion component.IMPORTANCE Coronaviruses typically express several accessory proteins. They vary in number and nature, and only one is conserved among most of the coronaviruses, pointing at an important biological function for this protein. PEDV is peculiar in that it expresses just this one accessory protein, termed the ORF3 protein. While its analogs in other coronaviruses have been studied to different extents, and these studies have indicated that they share an ion channel property, little is still known about the features and functions of the PEDV ORF3 protein except for its association with virulence. In this investigation, we studied the intracellular trafficking of the ORF3 protein both in infected cells and when expressed independently. In addition, we analyzed the effects of mutations in five sorting motifs in its C-terminal domain and investigated whether the protein, found to follow the same exocytic route by which the viral structural membrane proteins travel, is also incorporated into virions.

Project description:Three strains of porcine epidemic diarrhea virus (PEDV) were isolated from dead or diseased pigs at different swine farms in Guangdong during 2011-2013, and their S genes were sequenced. In the same period, seven PEDV strains were also isolated in Guangdong by other laboratories. The spike sequences of 10 Guangdong isolates were compared with vaccine strains and reference pathogenic isolates using six bioinformatics tools. The results revealed that 10 Guangdong strains, excluding strain GDS03, had distinct characteristics in terms of primary structure, secondary structure, high-specificity N-glycosylation sites, potential phosphorylation sites, and palmitoylation sites. Phylogenetic analysis also confirmed these findings and revealed that all PEDV strains were clustered into three distinct groups. Ten Guangdong strains, not including GDS03, belong to Group 1, whereas four vaccine strains and GDS03 belong to Group 3, which is evolutionarily distant from Group 1. Alignment analysis of the neutralizing region amino acid sequences indicated that the amino acid substitutions of Y/D766S, T549S, and G594S that are present in the Guangdong strains, not including GDS03, were a sign of predominant genetic changes among the isolated strains. GDS03 is closely related to the 83P-5 vaccine strain, which suggests that it might represent re-isolation of the vaccine strain or vaccine variants. Taken together, these results indicate that there have been predominant new strains circulating in Guangdong from 2011 to 2013, and the circulating PEDV strains have a genetic composition that is distant from reference strains, especially the vaccine strains; however, the vaccinations might also provide some level of cross-protection, as there have been no changes in the neutralizing epitopes of SS2 and 2C10. This explains why there have been constant but infrequent outbreaks recently in comparison to late 2010 in which PEDV outbreaks were more frequent and severe. In addition, the USA-Colorado-2013 strain had the same amino acid substitutions in the neutralizing regions as the Guangdong strains except GDS03, which suggests that the information and strategies in this study may play role in PEDV variant research in other countries.

Dataset Information

Analysis of the spike, ORF3, and nucleocapsid genes of porcine epidemic diarrhea virus circulating on Thai swine farms, 2011-2016.

Publications

Analysis of the spike, ORF3, and nucleocapsid genes of porcine epidemic diarrhea virus circulating on Thai swine farms, 2011-2016.

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