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Porcine Epidemic Diarrhea Virus (PEDV) ORF3 Enhances Viral Proliferation by Inhibiting Apoptosis of Infected Cells.

ABSTRACT: The genomes of coronaviruses carry accessory genes known to be associated with viral virulence. The single accessory gene of porcine epidemic diarrhea virus (PEDV), ORF3, is dispensable for virus replication in vitro, while viral mutants carrying ORF3 truncations exhibit an attenuated phenotype of which the underlying mechanism is unknown. Here, we studied the effect of ORF3 deletion on the proliferation of PEDV in Vero cells. To this end, four recombinant porcine epidemic diarrhea viruses (PEDVs) were rescued using targeted RNA recombination, three carrying the full-length ORF3 gene from different PEDV strains, and one from which the ORF3 gene had been deleted entirely. Our results showed that PEDVs with intact or naturally truncated ORF3 replicated to significantly higher titers than PEDV without an ORF3. Further characterization revealed that the extent of apoptosis induced by PEDV infection was significantly lower with the viruses carrying an intact or C-terminally truncated ORF3 than with the virus lacking ORF3, indicating that the ORF3 protein as well as its truncated form interfered with the apoptosis process. Collectively, we conclude that PEDV ORF3 protein promotes virus proliferation by inhibiting cell apoptosis caused by virus infection. Our findings provide important insight into the role of ORF3 protein in the pathogenicity of PEDV.

SUBMITTER: Si F

PROVIDER: S-EPMC7077256 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Porcine Epidemic Diarrhea Virus (PEDV) ORF3 Enhances Viral Proliferation by Inhibiting Apoptosis of Infected Cells.

Si Fusheng F Hu Xiaoxia X Wang Chenyang C Chen Bingqing B Wang Ruiyang R Dong Shijuan S Yu Ruisong R Li Zhen Z

Viruses 20200214 2

The genomes of coronaviruses carry accessory genes known to be associated with viral virulence. The single accessory gene of porcine epidemic diarrhea virus (PEDV), ORF3, is dispensable for virus replication in vitro, while viral mutants carrying ORF3 truncations exhibit an attenuated phenotype of which the underlying mechanism is unknown. Here, we studied the effect of ORF3 deletion on the proliferation of PEDV in Vero cells. To this end, four recombinant porcine epidemic diarrhea viruses (PEDV ...[more]

PMID: 32075094

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Project description:Background:Porcine epidemic diarrhea virus (PEDV) is an enteric disease of swine that has emerged as a worldwide threat to swine herd health and production. Substantial research has been conducted to assess viability of the virus on surfaces of vehicles and equipment, in feed and water, and on production building surfaces, but little is known about the persistence in PEDV-infected carcasses and effective disposal methods thereof. This study was conducted to quantify the persistence of PEDV RNA via quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) at various time-temperature combinations and in infected piglet carcasses subjected to composting. Although this method does not distinguish between infectious and noninfectious virus, it is a rapid and sensitive test to evaluate materials for evidence of virus genome. Results:In the first study, PEDV was suspended in cell culture media at 1 × 105 TCID50 per sample (1 mL sample size) and subjected to various time and temperature combinations in triplicate including temperatures of 37, 45, 50, 55, 60, 65, 70 °C and exposure times of 0, 1, 2, 3, 4, 5, 7, and 14 days. At all temperatures, viral RNA copies declined over time, with the decline most marked and rapid at 65 and 70 °C. Detectable RNA did persist throughout the trial in all but the most extreme condition, where two of three samples incubated at 70 °C yielded undetectable viral RNA after 14 days. In the second study, PEDV-infected piglet carcasses were subjected to two cycles of composting lasting 36 and 37 days, respectively, for a total compost time of 73 days. Composting was performed in triplicate windrow sections housed inside biosecure, climate-controlled rooms using insulated bins designed to represent a continuous windrow compost pile. Temperatures reached 35-57 °C for 26 days of cycle 1 and 35-45 °C for 3 days of cycle 2. Samples consisting of carbon material with or without decomposed tissue as available per sample site collected at ten locations throughout the cross-section of each windrow section following the primary and secondary compost cycles yielded no detectable viral RNA. Conclusions:Composting appears to be an effective disposal method for PEDV-infected piglet carcasses under the conditions examined. The combination of time and high temperature of the compost cycle effectively degraded viral RNA in cell culture media that should provide optimum stability. Complex compost material matrices collected from windrow sections yielded undetectable PEDV RNA by qRT-PCR after one 36-day compost cycle despite incomplete decomposition of soft tissue.

Project description:Puerarin has been reported to be an excellent antioxidant, anti-inflammatory and antimicrobial agent, but the potential effect of puerarin on porcine epidemic diarrhea virus (PEDV) is unclear. This study aimed to determine whether puerarin could alleviate intestinal injury in piglets infected with PEDV. A PEDV (Yunnan province strain) infection model was applied to 7-day-old piglets at 104.5 TCID50 (50% tissue culture infectious dose). Piglets were orally administered with puerarin at the dosage of 0.5 mg/kg body weight from day 5 to day 9. On day 9 of the trial, piglets were inoculated orally with PEDV. Three days later, jugular vein blood and intestinal samples were collected. Results showed puerarin reduced morbidity of piglets infected with PEDV. In addition, puerarin reduced the activities of aspartate aminotransferase and alkaline phosphatase, the ratio of serum aspartate aminotransferase to serum alanine aminotransferase, the number of white blood cells and neutrophils, and the plasma concentrations of interleukin-6, interleukin-8 and tumor necrosis factor-α, as well as protein abundances of heat shock protein-70 in PEDV-infected piglets. Moreover, puerarin increased D-xylose concentration but decreased intestinal fatty acid-binding protein concentration and diamine oxidase activity in the plasma of piglets infected with PEDV. Puerarin increased the activities of total superoxide dismutase, glutathione peroxidase and catalase, while decreasing the activities of myeloperoxidase and concentration of hydrogen peroxide in both the intestine and plasma of PEDV-infected piglets. Puerarin decreased mRNA levels of glutathione S-transferase omega 2 but increased the levels of nuclear factor erythroid 2-related factor 2. Furthermore, puerarin increased the abundance of total eubacteria (16S rRNA), Enterococcus genus, Lactobacillus genus and Enterobacteriaceae family in the intestine, but reduced the abundance of Clostridium coccoides in the caecum. These data indicate puerarin improved intestinal function in piglets infected by PEDV and may be a promising supplement for the prevention of PEDV infection.

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.

Dataset Information

Porcine Epidemic Diarrhea Virus (PEDV) ORF3 Enhances Viral Proliferation by Inhibiting Apoptosis of Infected Cells.

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Porcine Epidemic Diarrhea Virus (PEDV) ORF3 Enhances Viral Proliferation by Inhibiting Apoptosis of Infected Cells.

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