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

2019 ◽  
Vol 16 (1) ◽  
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.

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

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.

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

Viruses ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 391 ◽  
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.

scholarly journals Porcine Epidemic Diarrhea Virus 3C-Like Protease-Mediated Nucleocapsid Processing: Possible Link to Viral Cell Culture Adaptability

2016 ◽  
Vol 91 (2) ◽  
Author(s):  
Peera Jaru-Ampornpan ◽  
Juggragarn Jengarn ◽  
Asawin Wanitchang ◽  
Anan Jongkaewwattana
Keyword(s):  
Cell Culture ◽  
Growth Retardation ◽  
Porcine Epidemic Diarrhea Virus ◽  
Vaccine Development ◽  
Wild Type Virus ◽  
Economic Losses ◽  
Swine Industry ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea

ABSTRACT Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea and high mortality rates in newborn piglets, leading to massive losses to the swine industry worldwide during recent epidemics. Intense research efforts are now focusing on defining viral characteristics that confer a growth advantage, pathogenicity, or cell adaptability in order to better understand the PEDV life cycle and identify suitable targets for antiviral or vaccine development. Here, we report a unique phenomenon of PEDV nucleocapsid (N) cleavage by the PEDV-encoded 3C-like protease (3Cpro) during infection. The identification of the 3Cpro cleavage site at the C terminus of N supported previous observations that PEDV 3Cpro showed a substrate requirement slightly different from that of severe acute respiratory syndrome coronavirus (SARS-CoV) 3Cpro and revealed a greater flexibility in its substrate recognition site. This cleavage motif is present in the majority of cell culture-adapted PEDV strains but is missing in emerging field isolates. Remarkably, reverse-genetics-derived cell culture-adapted PEDVAVCT12 harboring uncleavable N displayed growth retardation in Vero E6-APN cells compared to the wild-type virus. These observations altogether shed new light on the investigation and characterization of the PEDV nucleocapsid protein and its possible link to cell culture adaptation. IMPORTANCE Recurrent PEDV outbreaks have resulted in enormous economic losses to swine industries worldwide. To gain the upper hand in combating this disease, it is necessary to understand how this virus replicates and evades host immunity. Characterization of viral proteins provides important clues to mechanisms by which viruses survive and spread. Here, we characterized an intriguing phenomenon in which the nucleocapsids of some PEDV strains are proteolytically processed by the virally encoded main protease. Growth retardation in recombinant PEDV carrying uncleavable N suggests a replication advantage provided by the cleavage event, at least in the cell culture system. These findings may direct us to a more complete understanding of PEDV replication and pathogenicity.

scholarly journals Molecular Characterization of a Variant Subgroup GII-a Porcine Epidemic Diarrhea Virus Strain in China

2020 ◽  
Author(s):  
Zhige Tian ◽  
Yuexiao Lian ◽  
Xiaoliang Hu ◽  
Yue Shi ◽  
Feng Cong
Keyword(s):  
Phylogenetic Analysis ◽  
Porcine Epidemic Diarrhea Virus ◽  
Genetic Characterization ◽  
Economic Losses ◽  
Recombination Analysis ◽  
Essential Information ◽  
Diarrhea Virus ◽  
Newborn Piglets ◽  
Porcine Epidemic Diarrhea

Abstract Background: Since 2010, variant porcine epidemic diarrhea virus (PEDV) has emerged in several provinces of China. The variant strain causes high mortality of newborn piglets, which has resulted in severe economic losses to the pork breeding industry.Results: A variant PEDV strain, SC-YB73, was isolated and identified in China. Pathological observation showed atrophy of villi and edema in the lamina propria. Sequence analysis indicated that six nucleotides were inserted in the E gene, which was first detected in PEDV strains. Furthermore, fifty nucleotide sites were unique in SC-YB73, when compared with another 28 PEDV strains. Phylogenetic analysis based on the complete genome showed that SC-YB73 was clustered in the variant subgroup GII-a, which is widely prevalent in the pig population in China. Recombination analysis suggested that SC-YB73 originated from the recombination of GDS47, TW/Yunlin550/2018, and COL/Cundinamarca/2014.Conclusion: The present study involved the isolation and genetic characterization of a variant PEDV strain. It will provide essential information for the control of PED outbreaks in China.

scholarly journals Molecular characteristics and pathogenicity of porcine epidemic diarrhea virus in some areas of China from 2015 to 2018

2020 ◽  
Author(s):  
Linyang Yu ◽  
Jianguo Dong ◽  
Shuangyun Wang ◽  
Yanling Liu ◽  
Leyi Zhang ◽  
...  
Keyword(s):  
Small Intestine ◽  
Phylogenetic Tree ◽  
Porcine Epidemic Diarrhea Virus ◽  
Evolutionary Relationship ◽  
Vero Cells ◽  
Economic Losses ◽  
Molecular Characteristics ◽  
Genetic Characteristics ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea

Abstract Background Since 2010, porcine epidemic diarrhea virus (PEDV) has caused severe diarrhea disease in piglets, which has led to large economic losses in China. To understand the genetic characteristics of PEDV strains that have been circulating in some provinces of China from 2015 to 2018, 362 feces and small intestine samples in pigs were collected and tested. Methods The S gene of PEDV in collected samples were amplified by RT-PCR. A phylogenetic tree was constructed using MEGA6.0 software with the neighbor-joining method to analyze the evolutionary relationship. Nucleotide and deduced amino acid (AA) sequences of S were aligned using the MegAlign program of DNASTAR7.1 software to determine sequence homology. PEDV GDgh16 strain isolation, IFA identification and titer detection were performed in Vero cells. Six 4-day-old healthy colostrum deprived suckling piglets were used for challenging experiment of PEDV GDgh16 strain. Virus copies from the small intestine were detected by RT-qPCR. The other section was stained with the anti-N protein McAb at 1:1000 dilution for immunohistochemical (IHC) examinations. Results The results showed that 160 samples tested positive and the PEDV-positive prevalence was 44.20%. Phylogenetic tree analysis of entire S genes showed that these strains were clustered into four subgroups, G1-b, G1-c, G2-a and G2-b, and that the G2-b strains had become dominant in recent years. Compared with previous strains, these strains had multiple variations in the SP and S1-NTD domains and in the neutralizing epitope of the S protein. Furthermore, we successfully isolated and identified a new virulent G2-b strain, GDgh16, which was well adapted to Vero cells and had a high mortality rate in piglets through challenge experiments. Conclusions Our study provides full insights into the genetic characteristics of prevalent PEDV strains in parts of China, suggesting that the development of novel effective vaccines is necessary and urgent.

Rapid host response to an infection with Coronavirus. Study of transcriptional responses with Porcine Epidemic Diarrhea Virus

2020 ◽  
Author(s):  
Wei Hou ◽  
Fei Liu ◽  
Wim H.M. van der Poel ◽  
Marcel M. Hulst
Keyword(s):  
Porcine Epidemic Diarrhea Virus ◽  
Clinical Symptoms ◽  
Salt Water ◽  
Vero Cells ◽  
The Body ◽  
Protease Gene ◽  
Transcriptional Responses ◽  
Mucus Production ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea

AbstractThe transcriptional response in Vero cells (ATCC® CCL-81) infected with the coronavirus Porcine Epidemic Diarrhea Virus (PEDV) was measured by RNAseq analysis 4 and 6 hours after infection. Differential expressed genes (DEGs) in PEDV infected cells were compared to DEGs responding in Vero cells infected with Mammalian Orthoreovirus (MRV). Functional analysis of MRV and PEDV DEGs showed that MRV increased the expression level of several cytokines and chemokines (e.g. IL6, CXCL10, IL1A, CXCL8 [alias IL8]) and antiviral genes (e.g. IFI44, IFIT1, MX1, OASL), whereas for PEDV no enhanced expression was observed for these “hallmark” antiviral and immune effector genes. Pathway and Gene Ontology “enrichment analysis” revealed that PEDV infection did not stimulate expression of genes able to activate an acquired immune response, whereas MRV did so within 6h. Instead, PEDV down-regulated the expression of a set of zinc finger proteins with putative antiviral activity and enhanced the expression of the transmembrane serine protease gene TMPRSS13 (alias MSPL) to support its own infection by virus-cell membrane fusion (Shi et al, 2017, Viruses, 9(5):114). PEDV also down-regulated expression of Ectodysplasin A, a cytokine of the TNF-family able to activate the canonical NFKB-pathway responsible for transcription of inflammatory genes like IL1B, TNF, CXCL8 and PTGS2. The only 2 cytokine genes found up-regulated by PEDV were Cardiotrophin-1, an IL6-type cytokine with pleiotropic functions on different tissues and types of cells, and Endothelin 2, a neuroactive peptide with vasoconstrictive properties. Furthermore, by comprehensive datamining in biological and chemical databases and consulting related literature we identified sets of PEDV-response genes with potential to influence i) the metabolism of biogenic amines (e.g. histamine), ii) the formation of cilia and “synaptic clefts” between cells, iii) epithelial mucus production, iv) platelets activation, and v) physiological processes in the body regulated by androgenic hormones (like blood pressure, salt/water balance and energy homeostasis). The information in this study describing a “very early” response of epithelial cells to an infection with a coronavirus may provide pharmacologists, immunological and medical specialists additional insights in the underlying mechanisms of coronavirus associated severe clinical symptoms including those induced by SARS-CoV-2. This may help them to fine-tune therapeutic treatments and apply specific approved drugs to treat COVID-19 patients.

scholarly journals Caerin1.1 Suppresses the Growth of Porcine Epidemic Diarrhea Virus In Vitro via Direct Binding to the Virus

Viruses ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 507 ◽  
Author(s):  
Nan Guo ◽  
Bingzhou Zhang ◽  
Han Hu ◽  
Shiyi Ye ◽  
Fangzhou Chen ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Porcine Epidemic Diarrhea Virus ◽  
Antiviral Drug ◽  
Vero Cells ◽  
Economic Losses ◽  
Diarrhea Virus ◽  
Direct Binding ◽  
Porcine Epidemic Diarrhea ◽  
And Control

Porcine epidemic diarrhea (PED) has re-emerged in recent years and has already caused huge economic losses to the porcine industry all over the world. Therefore, it is urgent for us to find out efficient ways to prevent and control this disease. In this study, the antiviral activity of a cationic amphibian antimicrobial peptide Caerin1.1 against porcine epidemic diarrhea virus (PEDV) was evaluated by an in vitro system using Vero cells. We found that even at a very low concentration, Caerin1.1 has the ability to destroy the integrity of the virus particles to block the release of the viruses, resulting in a considerable decrease in PEDV infections. In addition, Caerin1.1 showed powerful antiviral activity without interfering with the binding progress between PEDV and the receptor of the cells, therefore, it could be used as a potential antiviral drug or as a microbicide compound for prevention and control of PEDV.

scholarly journals Gene Variations in Cis-Acting Elements between the Taiwan and Prototype Strains of Porcine Epidemic Diarrhea Virus Alter Viral Gene Expression

Genes ◽  
2018 ◽  
Vol 9 (12) ◽  
pp. 591
Author(s):  
Tsung-Lin Tsai ◽  
Chen-Chang Su ◽  
Ching-Chi Hsieh ◽  
Chao-Nan Lin ◽  
Hui-Wen Chang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Porcine Epidemic Diarrhea Virus ◽  
The Body ◽  
Economic Losses ◽  
Viral Gene ◽  
Sequence Motif ◽  
Cis Acting ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea ◽  
In Cis

In 2013, the outbreak of porcine epidemic diarrhea (PED) in Taiwan caused serious economic losses. In this study, we examined whether the variations of the cis-acting elements between the porcine epidemic diarrhea virus (PEDV) Taiwan (TW) strain and the prototype strain CV777 alter gene expression. For this aim, we analyzed the variations of the cis-acting elements in the 5’ and 3’ untranslated regions (UTRs) between the PEDV TW, CV777, and other reference strains. We also determined the previously unidentified transcription regulatory sequence (TRS), a sequence motif required for coronavirus transcription, and found that a nucleotide deletion in the TW strain, in comparison with CV777 strain, immediately downstream of the leader core sequence alters the identity between the leader TRS and the body TRS. Functional analyses using coronavirus defective interfering (DI) RNA revealed that such variations in cis-acting elements for the TW strain compared with the CV777 strain have an influence on the efficiency of gene expression. The current data show for the first time the evolution of PEDV in terms of cis-acting elements and their effects on gene expression, and thus may contribute to our understanding of recent PED outbreaks worldwide.

scholarly journals Characterization of a pathogenic full-length cDNA clone of a virulent porcine epidemic diarrhea virus strain AH2012/12 in China

Virology ◽  
2017 ◽  
Vol 500 ◽  
pp. 50-61 ◽  
Author(s):  
Baochao Fan ◽  
Zhengyu Yu ◽  
Fengjiao Pang ◽  
Xiangwei Xu ◽  
Baimeng Zhang ◽  
...  
Keyword(s):  
Virus Strain ◽  
Cdna Clone ◽  
Porcine Epidemic Diarrhea Virus ◽  
Full Length ◽  
Diarrhea Virus ◽  
Full Length Cdna ◽  
Porcine Epidemic Diarrhea

scholarly journals Engineering a Live Attenuated Porcine Epidemic Diarrhea Virus Vaccine Candidate via Inactivation of the Viral 2'-O-Methyltransferase and the Endocytosis Signal of the Spike Protein

2019 ◽  
Vol 93 (15) ◽  
Author(s):  
Yixuan Hou ◽  
Hanzhong Ke ◽  
Jineui Kim ◽  
Dongwan Yoo ◽  
Yunfang Su ◽  
...  
Keyword(s):  
Porcine Epidemic Diarrhea Virus ◽  
Vaccine Development ◽  
Vaccine Candidate ◽  
Spike Protein ◽  
Economic Losses ◽  
High Dose ◽  
Type I ◽  
Viral Pathogen ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea

ABSTRACT Porcine epidemic diarrhea virus (PEDV) causes high mortality in neonatal piglets; however, effective and safe vaccines are still not available. We hypothesized that inactivation of the 2′-O-methyltransferase (2′-O-MTase) activity of nsp16 and the endocytosis signal of the spike protein attenuates PEDV yet retains its immunogenicity in pigs. We generated a recombinant PEDV, KDKE4A, with quadruple alanine substitutions in the catalytic tetrad of the 2′-O-MTase using a virulent infectious cDNA clone, icPC22A, as the backbone. Next, we constructed another mutant, KDKE4A-SYA, by abolishing the endocytosis signal of the spike protein of KDKE4A. Compared with icPC22A, the KDKE4A and KDKE4A-SYA mutants replicated less efficiently in vitro but induced stronger type I and type III interferon responses. The pathogenesis and immunogenicities of the mutants were evaluated in gnotobiotic piglets. The virulence of KDKE4A-SYA and KDKE4A was significantly reduced compared with that of icPC22A. Mortality rates were 100%, 17%, and 0% in the icPC22A-, KDKE4A-, and KDKE4A-SYA-inoculated groups, respectively. At 21 days postinoculation (dpi), all surviving pigs were challenged orally with a high dose of icPC22A. The KDKE4A-SYA- and KDKE4A-inoculated pigs were protected from the challenge, because no KDKE4A-SYA- and one KDKE4A-inoculated pig developed diarrhea whereas all the pigs in the mock-inoculated group had severe diarrhea, and 33% of them died. Furthermore, we serially passaged the KDKE4A-SYA mutant in pigs three times and did not find any reversion of the introduced mutations. The data suggest that KDKE4A-SYA may be a PEDV vaccine candidate. IMPORTANCE PEDV is the most economically important porcine enteric viral pathogen and has caused immense economic losses in the pork industries in many countries. Effective and safe vaccines are desperately required but still not available. 2′-O-MTase (nsp16) is highly conserved among coronaviruses (CoVs), and the inactivation of nsp16 in live attenuated vaccines has been attempted for several betacoronaviruses. We show that inactivation of both 2′-O-MTase and the endocytosis signal of the spike protein is an approach to designing a promising live attenuated vaccine for PEDV. The in vivo passaging data also validated the stability of the KDKE4A-SYA mutant. KDKE4A-SYA warrants further evaluation in sows and their piglets and may be used as a platform for further optimization. Our findings further confirmed that nsp16 can be a universal target for CoV vaccine development and will aid in the development of vaccines against other emerging CoVs.

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