Porcine Epidemic Diarrhea Virus Induces Vero Cell Apoptosis via the p53-PUMA Signaling Pathway

Porcine Epidemic Diarrhea Virus (PEDV) is the causative agent of swine epidemic diarrhea. In order to study the pathogenic mechanism of PEDV, PEDV was inoculated into Vero cells cultured in vitro, and the total RNA of Vero cells was extracted to construct a library for Illumina high-throughput sequencing and screening of differentially expressed genes (p < 0.05). Five differentially expressed genes for qRT-PCR verification analysis were randomly selected, and the verification results were consistent with the transcriptome sequencing results. The Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathway enrichment analysis was performed on the differentially expressed genes screened above. The results showed that the target gene annotations of differentially expressed genes in the African green monkey genome were mainly enriched in the TNF signaling pathway, the P53 signaling pathway, the Jak-STAT signaling pathway, the MAPK signaling pathway, and immune inflammation. In addition, it has been reported that Puma can promote apoptosis and is a key mediator of P53-dependent and non-dependent apoptosis pathways. However, there is no report that PEDV infection can activate Puma and induce apoptosis in a P53-dependent pathway. It was found by flow cytometry that PEDV infection induced apoptosis, and by Western Blotting detection, PEDV infection significantly increased the expression of p53, BAX, and Puma apoptosis-related proteins. Treatment Vero cells with the p53 inhibitor, PFT-α, could significantly inhibit PEDV-induced apoptosis. Studies have shown that PEDV infection can activate Puma and induce apoptosis in a P53-dependent pathway. These findings provide data support for further elucidating the pathogenic mechanism of PEDV and developing an effective vaccine against PEDV.

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

Viruses ◽

10.3390/v12020214 ◽

2020 ◽

Vol 12 (2) ◽

pp. 214 ◽

Cited By ~ 3

Author(s):

Fusheng Si ◽

Xiaoxia Hu ◽

Chenyang Wang ◽

Bingqing Chen ◽

Ruiyang Wang ◽

...

Keyword(s):

Porcine Epidemic Diarrhea Virus ◽

Underlying Mechanism ◽

Vero Cells ◽

Orf3 Gene ◽

Accessory Gene ◽

Orf3 Protein ◽

Diarrhea Virus ◽

Viral Virulence ◽

Porcine Epidemic Diarrhea

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.

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Characterization and pathogenicity of Vero cell-attenuated porcine epidemic diarrhea virus CT strain

Virology Journal ◽

10.1186/s12985-019-1232-7 ◽

2019 ◽

Vol 16 (1) ◽

Cited By ~ 2

Author(s):

Yu Wu ◽

Wei Li ◽

Qingfeng Zhou ◽

Qunhui Li ◽

Zhichao Xu ◽

...

Keyword(s):

Porcine Epidemic Diarrhea Virus ◽

Clinical Symptoms ◽

Vero Cells ◽

Economic Losses ◽

Protective Effects ◽

Diarrhea Virus ◽

Cytopathic Effects ◽

Porcine Epidemic Diarrhea ◽

Piglet Survival

Abstract Background Porcine epidemic diarrhea virus (PEDV) has caused enormous economic losses to the global pig industry. Currently available PEDV vaccine strains have limited protective effects against PEDV variant strains. Methods In this study, the highly virulent epidemic virus strain CT was serially passaged in Vero cells for up to 120 generations (P120). Characterization of the different passages revealed that compared with P10 and P64, P120 had a higher viral titer and more obvious cytopathic effects, thereby demonstrating better cell adaptability. Results Pathogenicity experiments using P120 in piglets revealed significant reductions in clinical symptoms, histopathological lesions, and intestinal PEDV antigen distribution; the piglet survival rate in the P120 group was 100%. Furthermore, whole-genome sequencing identified 13 amino acid changes in P120, which might be responsible for the attenuated virulence of P120. Conclusions Thus, an attenuated strain was obtained via cell passaging and that this strain could be used in preparing attenuated vaccines.

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The dynamics of Chinese variant porcine epidemic diarrhea virus production in Vero cells and intestines of 2-day old piglets

Virus Research ◽

10.1016/j.virusres.2015.06.009 ◽

2015 ◽

Vol 208 ◽

pp. 82-88 ◽

Cited By ~ 7

Author(s):

Yanhui Wang ◽

Xiaojing Gao ◽

Yali Yao ◽

Yunjing Zhang ◽

Chaochao Lv ◽

...

Keyword(s):

Porcine Epidemic Diarrhea Virus ◽

Virus Production ◽

Vero Cells ◽

Diarrhea Virus ◽

Porcine Epidemic Diarrhea

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Clathrin- and serine proteases-dependent uptake of porcine epidemic diarrhea virus into Vero cells

Virus Research ◽

10.1016/j.virusres.2014.07.022 ◽

2014 ◽

Vol 191 ◽

pp. 21-29 ◽

Cited By ~ 21

Author(s):

Jung-Eun Park ◽

Deu John M. Cruz ◽

Hyun-Jin Shin

Keyword(s):

Porcine Epidemic Diarrhea Virus ◽

Serine Proteases ◽

Vero Cells ◽

Diarrhea Virus ◽

Porcine Epidemic Diarrhea

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Molecular Characteristics and Pathogenicity of Porcine Epidemic Diarrhea Virus Isolated in Some Areas of China in 2015–2018

Frontiers in Veterinary Science ◽

10.3389/fvets.2020.607662 ◽

2020 ◽

Vol 7 ◽

Author(s):

Linyang Yu ◽

Yanling Liu ◽

Shuangyun Wang ◽

Leyi Zhang ◽

Pengshuai Liang ◽

...

Keyword(s):

Porcine Epidemic Diarrhea Virus ◽

Vero Cells ◽

Economic Losses ◽

Molecular Characteristics ◽

Phylogenetic Tree Analysis ◽

Genetic Characteristics ◽

Diarrhea Virus ◽

Severe Diarrhea ◽

Positive Rate ◽

Porcine Epidemic Diarrhea

Since 2010, Porcine epidemic diarrhea virus (PEDV) has caused severe diarrhea disease in piglets in China, resulting in large economic losses. To understand the genetic characteristics of the PEDV strains that circulated in some provinces of China between 2015 and 2018, 375 samples of feces and small intestine were collected from pigs and tested. One hundred seventy-seven samples tested positive and the PEDV-positive rate was 47.20%. A phylogenetic tree analysis based on the entire S gene showed that these strains clustered into four subgroups, GI-a, GI-b, GII-a, and GII-b, and that the GII-b strains have become dominant in recent years. Compared with previous strains, these strains have multiple variations in the SP and S1-NTD domains and in the neutralizing epitopes of the S protein. We also successfully isolated and identified a new virulent GII-b strain, GDgh16, which is well-adapted to Vero cells and caused a high mortality rate in piglets in challenge experiments. Our study clarifies the genetic characteristics of the prevalent PEDV strains in parts of China, and suggests that the development of effective novel vaccines is both necessary and urgent.

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Successive Passage In Vitro Led to Lower Virulence and Higher Titer of A Variant Porcine Epidemic Diarrhea Virus

Viruses ◽

10.3390/v12040391 ◽

2020 ◽

Vol 12 (4) ◽

pp. 391 ◽

Cited By ~ 1

Author(s):

Pengwei Zhao ◽

Song Wang ◽

Zhi Chen ◽

Jiang Yu ◽

Rongzhi Tang ◽

...

Keyword(s):

Amino Acids ◽

Porcine Epidemic Diarrhea Virus ◽

Vero Cells ◽

Economic Losses ◽

Diarrhea Virus ◽

S Gene ◽

Successive Passage ◽

Porcine Epidemic Diarrhea

A highly virulent porcine epidemic diarrhea virus (PEDV) appeared in China and spread rapidly to neighbor countries, which have led to great economic losses to the pig industry. In the present study, we isolated a PEDV using Vero cells and serially propagated 100 passages. PEDV SDSX16 was characterized in vitro and in vivo. The viral titers increased to 107.6 TCID50/mL (100th) by serial passages. The spike (S) gene and the whole gene of the SDSX16 virus was fully sequenced to assess the genetic stability and relatedness to previously identified PEDV. Along with successive passage in vitro, there were 18 nucleotides (nt) deletion occurred in the spike (S) gene resulting in a deletion of six amino acids when the SDSX16 strain was passaged to the 64th generation, and this deletion was stable until the P100. However, the ORF1a/b, M, N, E, and ORF3 genes had only a few point mutations in amino acids and no deletions. According to growth kinetics experiments, the SDSX16 deletion strain significantly enhanced its replication in Vero cells since it was passaged to the 64th generation. The animal studies showed that PEDV SDSX16-P10 caused more severe diarrhea and vomiting, fecal shedding, and acute atrophic enteritis than SDSX16-P75, indicating that SDSX16-P10 is enteropathogenic in the natural host, and the pathogenicity of SDSX16 decreased with successive passage in vitro. However, SDSX16-P10 was found to cause lower levels of cytokine expression than SDSX16-P75 using real-time PCR and flow cytometry, such as IL1β, IL6, IFN-β, TNF-α, indicating that SDSX16-P10 might inhibit the expression of cytokines. Our data indicated that successive passage in vitro resulted in virulent attenuation in vivo of the PEDV variant strain SDSX16.

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