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18 pages, 6347 KiB

Open AccessArticle

Transcriptome Profiling of Vero E6 Cells during Original Parental or Cell-Attenuated Porcine Epidemic Diarrhea Virus Infection

by Ouyang Peng, Yu Wu, Fangyu Hu, Yu Xia, Rui Geng, Yihui Huang, Siying Zeng, Guangli Hu, Chunyi Xue, Hao Zhang and Yongchang Cao

Viruses 2023, 15(7), 1426; https://doi.org/10.3390/v15071426 - 23 Jun 2023

Cited by 1 | Viewed by 2647

Abstract

Porcine epidemic diarrhea virus (PEDV) has led to significant economic losses in the global porcine industry since the emergence of variant strains in 2010. The high mutability of coronaviruses endows PEDV with the ability to evade the host immune response, which impairs the [...] Read more.

Porcine epidemic diarrhea virus (PEDV) has led to significant economic losses in the global porcine industry since the emergence of variant strains in 2010. The high mutability of coronaviruses endows PEDV with the ability to evade the host immune response, which impairs the effectiveness of vaccines. In our previous study, we generated a highly cell-passaged PEDV strain, CT-P120, which showed promise as a live attenuated vaccine candidate by providing satisfactory protection against variant PEDV infection in piglets. However, the mechanism by which the attenuated CT-P120 adapts to cells during passage, resulting in increased replication efficiency, remains unclear. To address this question, we conducted a comparative transcriptomic analysis of Vero E6 cells infected with either the original parental strain (CT-P10) or the cell-attenuated strain (CT-P120) of PEDV at 6, 12, and 24 h post-infection. Compared to CT-P10, CT-P120 infection resulted in a significant decrease in the number of differentially expressed genes (DEGs) at each time point. Functional enrichment analysis of genes revealed the activation of various innate immune-related pathways by CT-P10, notably attenuated during CT-P120 infection. To validate these results, we selected eight genes (TRAF3, IRF3, IFNL1, ISG15, NFKB1, MAP2K3, IL1A, and CCL2) involved in antiviral processes and confirmed their mRNA expression patterns using RT-qPCR, in line with the transcriptomic data. Subsequent protein-level analysis of selected genes via Western blotting and enzyme-linked immunosorbent assay corroborated these results, reinforcing the robustness of our findings. Collectively, our research elucidates the strategies underpinning PEDV attenuation and immune evasion, providing invaluable insights for the development of effective PEDV vaccines. Full article

(This article belongs to the Special Issue State-of-the-Art Livestock and Poultry Coronaviruses Research in China)

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10 pages, 1462 KiB

Open AccessArticle

Development and Primary Application of an Indirect ELISA Based on Rep Protein to Analyze Antibodies against Porcine Cocirvirus-like Virus (PCLV)

by Zheng Chen, Xifeng Hu, Xiangdong Wu, Yu Li, Zhen Ding, Qinghua Zeng, Tong Wan, Jingyi Yin and Huansheng Wu

Viruses 2022, 14(7), 1398; https://doi.org/10.3390/v14071398 - 27 Jun 2022

Cited by 2 | Viewed by 2195

Abstract

Porcine circovirus-like virus (PCLV) is a member of circovirus that contains a single-strand DNA genome, which may be one of the pathogens that causes diarrheal symptoms in pigs. The Rep protein encoded by the genome of PCLV may be responsible for viral genome [...] Read more.

Porcine circovirus-like virus (PCLV) is a member of circovirus that contains a single-strand DNA genome, which may be one of the pathogens that causes diarrheal symptoms in pigs. The Rep protein encoded by the genome of PCLV may be responsible for viral genome replication. The development of serological detection methods for PCLV is of great necessity for clinical diagnosis, as well as epidemiological investigations. Therefore, this study attempted to build an indirect enzyme-linked immunosorbent assay (ELISA) to examine antibodies against PCLV based on the His-tagged recombinant Rep protein. Full-length PCLV Rep protein was induced and expressed in E. coli and was purified as an antigen to establish an ELISA detection kit. The purified Rep protein was used to inject into mice to produce specific antibodies. There was no cross-reaction of Rep-based ELISA with antisera against other porcine viruses. The intra-assay and inter-assay coefficient variations (CVs) were 0.644–8.211% and 0.859–7.246%, respectively, indicating good repeatability. The non-cross-reaction with TGEV, PRRSV and PCV2 testing showed high sensitivity and high specificity for this ELISA assay. A total of 1593 serum samples collected from different pig farms in Jiangxi Province were tested for anti-PCLV Rep antibodies, and 284 (17.83%) of the 1593 samples were Rep antibody positive. Altogether, the indirect ELISA detection tool developed in this study could be applied to examine serum of PCLV antibodies with good repeatability, high sensitivity and high specificity. In addition, field sample detection results suggested that the PCLV antibody has a low prevalence in pig populations in Jiangxi Province of China. Full article

(This article belongs to the Special Issue State-of-the-Art Livestock and Poultry Coronaviruses Research in China)

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10 pages, 1840 KiB

Open AccessArticle

Poly(A)-Binding Protein Cytoplasmic 1 Inhibits Porcine Epidemic Diarrhea Virus Replication by Interacting with Nucleocapsid Protein

by Tingting Wu, Xiaona Wei, Shumei Zheng, Gaoli She, Zhenling Han, Zhichao Xu, Yongchang Cao and Chunyi Xue

Viruses 2022, 14(6), 1196; https://doi.org/10.3390/v14061196 - 31 May 2022

Cited by 3 | Viewed by 2417

Abstract

Porcine epidemic diarrhea virus (PEDV) is the etiological agent of porcine epidemic diarrhea (PED) characterized by vomit, watery diarrhea, dehydration and high mortality. Outbreaks of highly pathogenic variant strains of PEDV have resulted in extreme economic losses to the swine industry all over [...] Read more.

Porcine epidemic diarrhea virus (PEDV) is the etiological agent of porcine epidemic diarrhea (PED) characterized by vomit, watery diarrhea, dehydration and high mortality. Outbreaks of highly pathogenic variant strains of PEDV have resulted in extreme economic losses to the swine industry all over the world. The study of host–virus interaction can help to better understand the viral pathogenicity. Many studies have shown that poly(A)-binding proteins are involved in the replication process of various viruses. Here, we found that the infection of PEDV downregulated the expression of poly(A)-binding protein cytoplasmic 1 (PABPC1) at the later infection stage in Vero cells. The overexpression of PABPC1 inhibited the proliferation of PEDV at transcription and translation level, and siRNA-mediated depletion of PABPC1 promoted the replication of PEDV. Furthermore, mass spectrometry analysis and immunoprecipitation assay confirmed that PABPC1 interacted with the nucleocapsid (N) protein of PEDV. Confocal microscopy revealed the co-localizations of PABPC1 with N protein in the cytoplasm. Taken together, these results demonstrate the antiviral effect of PABPC1 against PEDV replication by interacting with N protein, which increases understanding of the interaction between PEDV and host. Full article

(This article belongs to the Special Issue State-of-the-Art Livestock and Poultry Coronaviruses Research in China)

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14 pages, 3770 KiB

Open AccessArticle

The Structure of the Porcine Deltacoronavirus Main Protease Reveals a Conserved Target for the Design of Antivirals

by Fenghua Wang, Cheng Chen, Zefang Wang, Xu Han, Peidian Shi, Kaixuan Zhou, Xiaomei Liu, Yunjie Xiao, Yan Cai, Jinhai Huang, Lei Zhang and Haitao Yang

Viruses 2022, 14(3), 486; https://doi.org/10.3390/v14030486 - 27 Feb 2022

Cited by 6 | Viewed by 2959

Abstract

The existing zoonotic coronaviruses (CoVs) and viral genetic variants are important microbiological pathogens that cause severe disease in humans and animals. Currently, no effective broad-spectrum antiviral drugs against existing and emerging CoVs are available. The CoV main protease (M^pro) plays an [...] Read more.

The existing zoonotic coronaviruses (CoVs) and viral genetic variants are important microbiological pathogens that cause severe disease in humans and animals. Currently, no effective broad-spectrum antiviral drugs against existing and emerging CoVs are available. The CoV main protease (M^pro) plays an essential role in viral replication, making it an ideal target for drug development. However, the structure of the Deltacoronavirus M^pro is still unavailable. Porcine deltacoronavirus (PDCoV) is a novel CoV that belongs to the genus Deltacoronavirus and causes atrophic enteritis, severe diarrhea, vomiting and dehydration in pigs. Here, we determined the structure of PDCoV M^pro complexed with a Michael acceptor inhibitor. Structural comparison showed that the backbone of PDCoV M^pro is similar to those of alpha-, beta- and gamma-CoV M^pros. The substrate-binding pocket of M^pro is well conserved in the subfamily Coronavirinae. In addition, we also observed that M^pros from the same genus adopted a similar conformation. Furthermore, the structure of PDCoV M^pro in complex with a Michael acceptor inhibitor revealed the mechanism of its inhibition of PDCoV M^pro. Our results provide a basis for the development of broad-spectrum antivirals against PDCoV and other CoVs. Full article

(This article belongs to the Special Issue State-of-the-Art Livestock and Poultry Coronaviruses Research in China)

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13 pages, 2677 KiB

Open AccessArticle

Antiviral Activities of Carbazole Derivatives against Porcine Epidemic Diarrhea Virus In Vitro

by Zheng Chen, Jinfeng Chen, Xiaodong Wei, Huiying Hua, Ruiming Hu, Nengshui Ding, Jinhua Zhang, Deping Song, Yu Ye, Yuxin Tang, Zhen Ding and Shaoyong Ke

Viruses 2021, 13(12), 2527; https://doi.org/10.3390/v13122527 - 16 Dec 2021

Cited by 8 | Viewed by 3269

Abstract

Porcine epidemic diarrhea virus (PEDV), an enteric coronavirus, causes neonatal pig acute gastrointestinal infection with a characterization of severe diarrhea, vomiting, high morbidity, and high mortality, resulting in tremendous damages to the swine industry. Neither specific antiviral drugs nor effective vaccines are available, [...] Read more.

Porcine epidemic diarrhea virus (PEDV), an enteric coronavirus, causes neonatal pig acute gastrointestinal infection with a characterization of severe diarrhea, vomiting, high morbidity, and high mortality, resulting in tremendous damages to the swine industry. Neither specific antiviral drugs nor effective vaccines are available, posing a high priority to screen antiviral drugs. The aim of this study is to investigate anti-PEDV effects of carbazole alkaloid derivatives. Eighteen carbazole derivatives (No.1 to No.18) were synthesized, and No.5, No.7, and No.18 were identified to markedly reduce the replication of enhanced green fluorescent protein (EGFP) inserted-PEDV, and the mRNA level of PEDV N. Flow cytometry assay, coupled with CCK8 assay, confirmed No.7 and No.18 carbazole derivatives displayed high inhibition effects with low cell toxicity. Furthermore, time course analysis indicated No.7 and No.18 carbazole derivatives exerted inhibition at the early stage of the viral life cycle. Collectively, the analysis underlines the benefit of carbazole derivatives as potential inhibitors of PEDV, and provides candidates for the development of novel therapeutic agents. Full article

(This article belongs to the Special Issue State-of-the-Art Livestock and Poultry Coronaviruses Research in China)

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16 pages, 2966 KiB

Open AccessArticle

Construction, Characterization and Application of Recombinant Porcine Deltacoronavirus Expressing Nanoluciferase

by Puxian Fang, Huichang Zhang, He Sun, Gang Wang, Sijin Xia, Jie Ren, Jiansong Zhang, Liyuan Tian, Liurong Fang and Shaobo Xiao

Viruses 2021, 13(10), 1991; https://doi.org/10.3390/v13101991 - 4 Oct 2021

Cited by 13 | Viewed by 3062

Abstract

Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes diarrhoea in suckling piglets and has the potential for cross-species transmission. No effective PDCoV vaccines or antiviral drugs are currently available. Here, we successfully generated an infectious clone of PDCoV strain CHN-HN-2014 using a combination [...] Read more.

Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes diarrhoea in suckling piglets and has the potential for cross-species transmission. No effective PDCoV vaccines or antiviral drugs are currently available. Here, we successfully generated an infectious clone of PDCoV strain CHN-HN-2014 using a combination of bacterial artificial chromosome (BAC)-based reverse genetics system with a one-step homologous recombination. The recued virus (rCHN-HN-2014) possesses similar growth characteristics to the parental virus in vitro. Based on the established infectious clone and CRISPR/Cas9 technology, a PDCoV reporter virus expressing nanoluciferase (Nluc) was constructed by replacing the NS6 gene. Using two drugs, lycorine and resveratrol, we found that the Nluc reporter virus exhibited high sensibility and easy quantification to rapid antiviral screening. We further used the Nluc reporter virus to test the susceptibility of different cell lines to PDCoV and found that cell lines derived from various host species, including human, swine, cattle and monkey enables PDCoV replication, broadening our understanding of the PDCoV cell tropism range. Taken together, our reporter viruses are available to high throughput screening for antiviral drugs and uncover the infectivity of PDCoV in various cells, which will accelerate our understanding of PDCoV. Full article

(This article belongs to the Special Issue State-of-the-Art Livestock and Poultry Coronaviruses Research in China)

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17 pages, 4336 KiB

Open AccessArticle

Transcriptional Landscape of Vero E6 Cells during Early Swine Acute Diarrhea Syndrome Coronavirus Infection

by Siying Zeng, Ouyang Peng, Ruipu Sun, Qiuping Xu, Fangyu Hu, Yan Zhao, Chunyi Xue, Yongchang Cao and Hao Zhang

Viruses 2021, 13(4), 674; https://doi.org/10.3390/v13040674 - 14 Apr 2021

Cited by 10 | Viewed by 2907

Abstract

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a newly emerged and highly pathogenic virus that is associated with fatal diarrhea disease in piglets, causing significant economic losses to the pig industry. At present, the research on the pathogenicity and molecular mechanisms of host-virus [...] Read more.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a newly emerged and highly pathogenic virus that is associated with fatal diarrhea disease in piglets, causing significant economic losses to the pig industry. At present, the research on the pathogenicity and molecular mechanisms of host-virus interactions of SADS-CoV are limited and remain poorly understood. Here, we investigated the global gene expression profiles of SADS-CoV-infected Vero E6 cells at 12, 18, and 24 h post-infection (hpi) using the RNA-sequencing. As a result, a total of 3324 differentially expressed genes (DEG) were identified, most of which showed a down-regulated expression pattern. Functional enrichment analyses indicated that the DEGs are mainly involved in signal transduction, cellular transcription, immune and inflammatory response, and autophagy. Collectively, our results provide insights into the changes in the cellular transcriptome during early infection of SADS-CoV and may provide information for further study of molecular mechanisms. Full article

(This article belongs to the Special Issue State-of-the-Art Livestock and Poultry Coronaviruses Research in China)

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Review

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11 pages, 467 KiB

Open AccessReview

Host Antiviral Responses against Avian Infectious Bronchitis Virus (IBV): Focus on Innate Immunity

by Yun Zhang, Zhichao Xu and Yongchang Cao

Viruses 2021, 13(9), 1698; https://doi.org/10.3390/v13091698 - 26 Aug 2021

Cited by 18 | Viewed by 4313

Abstract

Avian infectious bronchitis virus (IBV) is an important gammacoronavirus. The virus is highly contagious, can infect chickens of all ages, and causes considerable economic losses in the poultry industry worldwide. In the last few decades, numerous studies have been published regarding pathogenicity, vaccination, [...] Read more.

Avian infectious bronchitis virus (IBV) is an important gammacoronavirus. The virus is highly contagious, can infect chickens of all ages, and causes considerable economic losses in the poultry industry worldwide. In the last few decades, numerous studies have been published regarding pathogenicity, vaccination, and host immunity-virus interaction. In particular, innate immunity serves as the first line of defense against invasive pathogens and plays an important role in the pathogenetic process of IBV infection. This review focuses on fundamental aspects of host innate immune responses after IBV infection, including identification of conserved viral structures and different components of host with antiviral activity, which could provide useful information for novel vaccine development, vaccination strategies, and intervention programs. Full article

(This article belongs to the Special Issue State-of-the-Art Livestock and Poultry Coronaviruses Research in China)

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21 pages, 2445 KiB

Open AccessReview

Functions of Coronavirus Accessory Proteins: Overview of the State of the Art

by Puxian Fang, Liurong Fang, Huichang Zhang, Sijin Xia and Shaobo Xiao

Viruses 2021, 13(6), 1139; https://doi.org/10.3390/v13061139 - 13 Jun 2021

Cited by 33 | Viewed by 5083

Abstract

Coronavirus accessory proteins are a unique set of proteins whose genes are interspersed among or within the genes encoding structural proteins. Different coronavirus genera, or even different species within the same coronavirus genus, encode varying amounts of accessory proteins, leading to genus- or [...] Read more.

Coronavirus accessory proteins are a unique set of proteins whose genes are interspersed among or within the genes encoding structural proteins. Different coronavirus genera, or even different species within the same coronavirus genus, encode varying amounts of accessory proteins, leading to genus- or species-specificity. Though accessory proteins are dispensable for the replication of coronavirus in vitro, they play important roles in regulating innate immunity, viral proliferation, and pathogenicity. The function of accessory proteins on virus infection and pathogenesis is an area of particular interest. In this review, we summarize the current knowledge on accessory proteins of several representative coronaviruses that infect humans or animals, including the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with an emphasis on their roles in interaction between virus and host, mainly involving stress response, innate immunity, autophagy, and apoptosis. The cross-talking among these pathways is also discussed. Full article

(This article belongs to the Special Issue State-of-the-Art Livestock and Poultry Coronaviruses Research in China)

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