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Veterinarni Medicina Mar 2023Porcine deltacoronavirus (PDCoV) and porcine sapelovirus (PSV) are two viruses that can cause diarrhoea in pigs and bring great economic loss to the pig industry....
Development and application of a low-priced duplex quantitative PCR assay based on SYBR Green I for the simultaneous detection of porcine deltacoronavirus and porcine sapelovirus.
Porcine deltacoronavirus (PDCoV) and porcine sapelovirus (PSV) are two viruses that can cause diarrhoea in pigs and bring great economic loss to the pig industry. In this research, a duplex real-time quantitative polymerase chain reaction (qPCR) assay based on SYBR Green I was developed to simultaneously detect PDCoV and PSV. No specific melting peaks were found in other porcine diarrhoea-associated viruses, indicating that the method developed in this study had good specificity. The detection limits of PDCoV and PSV were 1.0 × 10 copies μl and 1.0 × 10 copies μl, respectively. The duplex real-time qPCR assay tested two hundred and three (203) intestinal and faecal samples collected from diarrhoeal and asymptomatic pigs. The positive rates of PDCoV and PSV were 20.2% and 23.2%, respectively. The co-infection rate of PDCoV and PSV was 13.8%. To evaluate the accuracy of the developed method, conventional PCR and singular TaqMan real-time qPCR assays for PDCoV/PSV were also used to detect the samples. The results showed that the duplex real-time qPCR assay was consistent with the singular assays, but its sensitivity was higher than conventional PCR methods. This duplex real-time qPCR assay provides a rapid, sensitive and reliable method in a clinic to simultaneously detect PDCoV and PSV.
PubMed: 37981902
DOI: 10.17221/79/2022-VETMED -
Microbiology Spectrum Dec 2023Porcine deltacoronavirus (PDCoV) is a newly emerged enteric virus threatening pig industries worldwide. Our previous work showed that PDCoV enters porcine kidney (PK-15)...
Porcine deltacoronavirus (PDCoV) is a newly emerged enteric virus threatening pig industries worldwide. Our previous work showed that PDCoV enters porcine kidney (PK-15) cells through a caveolae-dependent pathway, but the entry mechanism for PDCoV into swine testicle (ST) cells remains unclear. Mechanisms of virus entry can be different with different virus isolates and cell types. Here, we determined that PDCoV enters ST cells via clathrin-mediated endocytosis. Additionally, we found that PDCoV entry does not require Rab5, Rab7, or Rab11. These findings provide additional understanding of the entry mechanisms of PDCoV and possible antiviral targets.
Topics: Animals; Swine; Endocytosis; Deltacoronavirus; Virus Internalization; Clathrin; Swine Diseases; Coronavirus Infections
PubMed: 37962380
DOI: 10.1128/spectrum.02553-23 -
Viruses Sep 2023Emerging and re-emerging swine coronaviruses (CoVs), including porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea... (Review)
Review
Emerging and re-emerging swine coronaviruses (CoVs), including porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome-CoV (SADS-CoV), cause severe diarrhea in neonatal piglets, and CoV infection is associated with significant economic losses for the swine industry worldwide. Reverse genetics systems realize the manipulation of RNA virus genome and facilitate the development of new vaccines. Thus far, five reverse genetics approaches have been successfully applied to engineer the swine CoV genome: targeted RNA recombination, in vitro ligation, bacterial artificial chromosome-based ligation, vaccinia virus -based recombination, and yeast-based method. This review summarizes the advantages and limitations of these approaches; it also discusses the latest research progress in terms of their use for virus-related pathogenesis elucidation, vaccine candidate development, antiviral drug screening, and virus replication mechanism determination.
Topics: Animals; Swine; Coronavirus; Reverse Genetics; Coronavirus Infections; Porcine epidemic diarrhea virus; RNA; Diarrhea; Swine Diseases
PubMed: 37896780
DOI: 10.3390/v15102003 -
Journal of Virology Nov 2023As a member of the δ-coronavirus family, porcine deltacoronavirus (PDCoV) is a vital reason for diarrhea in piglets, which can contribute to high morbidity and...
As a member of the δ-coronavirus family, porcine deltacoronavirus (PDCoV) is a vital reason for diarrhea in piglets, which can contribute to high morbidity and mortality rates. Initially identified in Hong Kong in 2012, the virus has rapidly spread worldwide. During PDCoV infection, the virus employs evasion mechanisms to evade host surveillance, while the host mounts corresponding responses to impede viral replication. Our research has revealed that PDCoV infection down-regulates the expression of PGAM5 to promote virus replication. In contrast, PGAM5 degrades PDCoV N through autophagy by interacting with the cargo receptor P62 and the E3 ubiquitination ligase STUB1. Additionally, PGAM5 interacts with MyD88 and TRAF3 to activate the IFN signal pathway, resulting in the inhibition of viral replication.
Topics: Animals; Coronavirus Infections; Interferon Type I; Signal Transduction; Swine; Swine Diseases; Ubiquitin-Protein Ligases; Virus Replication; Coronavirus Nucleocapsid Proteins; Deltacoronavirus; Phosphoprotein Phosphatases; Mitochondrial Proteins; Down-Regulation; Immune Evasion; RNA-Binding Proteins; Proteolysis
PubMed: 37882521
DOI: 10.1128/jvi.01470-23 -
Journal of Virology Nov 2023As an emerging porcine enteropathogenic coronavirus that has the potential to infect humans, porcine deltacoronavirus (PDCoV) is receiving increasing attention. However,...
As an emerging porcine enteropathogenic coronavirus that has the potential to infect humans, porcine deltacoronavirus (PDCoV) is receiving increasing attention. However, no effective commercially available vaccines against this virus are available. In this work, we designed a spike (S) protein and receptor-binding domain (RBD) trimer as a candidate PDCoV subunit vaccine. We demonstrated that S protein induced more robust humoral and cellular immune responses than the RBD trimer in mice. Furthermore, the protective efficacy of the S protein was compared with that of inactivated PDCoV vaccines in piglets and sows. Of note, the immunized piglets and suckling pig showed a high level of NAbs and were associated with reduced virus shedding and mild diarrhea, and the high level of NAbs was maintained for at least 4 months. Importantly, we demonstrated that S protein-based subunit vaccines conferred significant protection against PDCoV infection.
Topics: Animals; Female; Humans; Mice; Coronavirus; Coronavirus Infections; Deltacoronavirus; Swine; Swine Diseases; Vaccines, Subunit
PubMed: 37846983
DOI: 10.1128/jvi.00958-23 -
Journal of Virology Oct 2023Retrograde transport has been reported to be closely associated with normal cellular biological processes and viral replication. As an emerging enteropathogenic...
Retrograde transport has been reported to be closely associated with normal cellular biological processes and viral replication. As an emerging enteropathogenic coronavirus with zoonotic potential, porcine deltacoronavirus (PDCoV) has attracted considerable attention. However, whether retrograde transport is associated with PDCoV infection remains unclear. Our present study demonstrates that retromer protein VPS35 acts as a critical host factor that is required for PDCoV infection. Mechanically, VPS35 interacts with PDCoV NS6, mediating the retrograde transport of NS6 from endosomes to the Golgi and preventing it from lysosomal degradation. Recombinant PDCoVs with an NS6 deletion display resistance to VPS35 deficiency. Our work reveals a novel evasion mechanism of PDCoV that involves the manipulation of the retrograde transport pathway by VPS35, providing new insight into the mechanism of PDCoV infection.
Topics: Animals; Coronavirus; Coronavirus Infections; Deltacoronavirus; Swine; Swine Diseases; Virus Replication; Viral Regulatory and Accessory Proteins; Vesicular Transport Proteins
PubMed: 37815351
DOI: 10.1128/jvi.00957-23 -
PLoS Pathogens Oct 2023Coronaviruses (CoVs) are a family of the largest RNA viruses that typically cause respiratory, enteric, and hepatic diseases in animals and humans, imposing great...
Coronaviruses (CoVs) are a family of the largest RNA viruses that typically cause respiratory, enteric, and hepatic diseases in animals and humans, imposing great threats to the public safety and animal health. Porcine deltacoronavirus (PDCoV), a newly emerging enteropathogenic coronavirus, causes severe diarrhea in suckling piglets all over the world and poses potential risks of cross-species transmission. Here, we use PDCoV as a model of CoVs to illustrate the reciprocal regulation between CoVs infection and host antiviral responses. In this study, downregulation of DNA polymerase delta interacting protein 3 (POLDIP3) was confirmed in PDCoV infected IPEC-J2 cells by isobaric tags for relative and absolute quantification (iTRAQ) and Western blotting analysis. Overexpression of POLDIP3 inhibits PDCoV infection, whereas POLDIP3 knockout (POLDIP3-/-) by CRISPR-Cas9 editing significantly promotes PDCoV infection, indicating POLDIP3 as a novel antiviral regulator against PDCoV infection. Surprisingly, an antagonistic strategy was revealed that PDCoV encoded nonstructural protein 5 (nsp5) was responsible for POLDIP3 reduction via its 3C-like protease cleavage of POLDIP3 at the glutamine acid 176 (Q176), facilitating PDCoV infection due to the loss of antiviral effects of the cleaved fragments. Consistent with the obtained data in IPEC-J2 cell model in vitro, POLDIP3 reduction by cleavage was also corroborated in PDCoV infected-SPF piglets in vivo. Collectively, we unveiled a new antagonistic strategy evolved by PDCoV to counteract antiviral innate immunity by nsp5-mediated POLDIP3 cleavage, eventually ensuring productive virus replication. Importantly, we further demonstrated that nsp5s from PEDV and TGEV harbor the conserved function to cleave porcine POLDIP3 at the Q176 to despair POLDIP3-mediated antiviral effects. In addition, nsp5 from SARS-CoV-2 also cleaves human POLDIP3. Therefore, we speculate that coronaviruses employ similar POLDIP3 cleavage mechanisms mediated by nsp5 to antagonize the host antiviral responses to sustain efficient virus infection.
Topics: Animals; Humans; Swine; Immunity, Innate; Virus Replication; Antiviral Agents; Coronavirus Infections; Swine Diseases; RNA-Binding Proteins
PubMed: 37801439
DOI: 10.1371/journal.ppat.1011702 -
International Journal of Molecular... Sep 2023Porcine deltacoronavirus (PDCoV) is an emerging virus that poses a significant threat to the global swine industry. Its membrane (M) protein is crucial for virion...
Porcine deltacoronavirus (PDCoV) is an emerging virus that poses a significant threat to the global swine industry. Its membrane (M) protein is crucial for virion assembly and virus-host interactions. We selected the hydrophilic region of M protein for prokaryotic expression, purification, and recombinant protein production. Utilizing hybridoma technology, we prepared the monoclonal antibody (mAb) 24-A6 against M protein. The mAb 24-A6 was shown to be suitable for use in immunofluorescence assays, western blotting, and immunoprecipitation, with specificity for PDCoV and no cross-reactivity with other five porcine viruses. The M protein was observed to be expressed as early as 3 h after PDCoV infection, increasing its expression over the duration of infection. Notably, the antigenic epitope of the M protein identified as SPESRL recognized by mAb 24-A6 was found within a conserved structural domain (SWWSFNPETNNL) of the coronavirus M protein, indicating a crucial overlap between a functionally important viral assembly region and a region recognized by the immune system. Our findings provide valuable insights into mAb 24-A6 targeting the antigenic epitope of M protein and may contribute to the development of diagnostic tools for PDCoV infection and fundamental research into the function of PDCoV M protein.
Topics: Animals; Swine; Membrane Proteins; Antibodies, Monoclonal; Deltacoronavirus; Epitopes
PubMed: 37762237
DOI: 10.3390/ijms241813934 -
BMC Veterinary Research Sep 2023Porcine deltacoronavirus (PDCoV) is a novel coronavirus that causes enteric diseases in pigs leading to substantial financial losses within the industry. The absence of...
Porcine deltacoronavirus (PDCoV) is a novel coronavirus that causes enteric diseases in pigs leading to substantial financial losses within the industry. The absence of commercial vaccines and limited research on PDCoV vaccines presents significant challenges. Therefore, we evaluated the safety and immunogenicity of recombinant pseudorabies virus (PRV) rPRVXJ-delgE/gI/TK-S through intranasal mucosal immunization in weaned piglets and SPF mice. Results indicated that rPRVXJ-delgE/gI/TK-S safely induced PDCoV S-specific and PRV gB-specific antibodies in piglets, with levels increasing 7 days after immunization. Virus challenge tests demonstrated that rPRVXJ-delgE/gI/TK-S effectively improved piglet survival rates, reduced virus shedding, and alleviated clinical symptoms and pathological damage. Notably, the recombinant virus reduced anti-inflammatory and pro-inflammatory responses by regulating IFN-γ, TNF-α, and IL-1β secretion after infection. Additionally, rPRVXJ-delgE/gI/TK-S colonized target intestinal segments infected with PDCoV, stimulated the secretion of cytokines by MLVS in mice, stimulated sIgA secretion in different intestinal segments of mice, and improved mucosal immune function. HE and AB/PAS staining confirmed a more complete intestinal mucosal barrier and a significant increase in goblet cell numbers after immunization. In conclusion, rPRVXJ-delgE/gI/TK-S exhibits good immunogenicity and safety in mice and piglets, making it a promising candidate vaccine for PDCoV.
Topics: Animals; Mice; Swine; Immunity, Mucosal; Administration, Intranasal; COVID-19; Vaccines, Synthetic; Intestines; Antibodies, Viral; Swine Diseases
PubMed: 37741960
DOI: 10.1186/s12917-023-03739-5 -
Scientific Reports Sep 2023The coronaviruses (CoV) are ubiquitous pathogens found in wide variety of hosts that constantly pose a threat to human and animal health as a result of their enormous...
The coronaviruses (CoV) are ubiquitous pathogens found in wide variety of hosts that constantly pose a threat to human and animal health as a result of their enormous capacity to generate genetic changes. Constant monitoring of virus reservoirs can constitute an early-warning tool and control the spread and evolution of the virus. Coronaviruses are common in wild birds, globally, and birds of the Charadriiformes in particular have been demonstrated to be carriers of delta- (dCoV) and gammacoronaviruses (gCoV). In this paper, we present the genetic characterisation of five CoV strains from black-headed (Chroicocephalus ridibundus) and common (Larus canus) gulls. Whole genome sequence analysis showed high similarity of detected dCoV in gulls to previously identified strains from falcon, houbara, pigeon and gulls from Asia (UAE, China). However, phylogenetic analysis revealed bifurcation within a common branch. Furthermore, the accumulation of numerous amino acid changes within the S-protein was demonstrated, indicating further evolution of dCoV within a single gull host. In turn, phylogenetic analysis for the most of the structural and non-structural genes of identified gCoV confirmed that the strain belongs to the duck coronavirus 2714 (DuCoV2714) species within Igacovirus subgenera, while for the spike protein it forms a separate branch not closely related to any gCoV species known to date. The current study provides new and significant insights into the evolution and diversification of circulating coronaviruses in members of Laridae family.
Topics: Animals; Humans; Charadriiformes; Deltacoronavirus; Phylogeny; Columbidae; Coronavirus; Coronavirus Infections; Gammacoronavirus
PubMed: 37704675
DOI: 10.1038/s41598-023-42241-8