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Frontiers in Microbiology 2022Porcine deltacoronavirus (PDCoV) cause diarrhea and dehydration in newborn piglets and has the potential for cross-species transmission. Rapid and early diagnosis is...
Porcine deltacoronavirus (PDCoV) cause diarrhea and dehydration in newborn piglets and has the potential for cross-species transmission. Rapid and early diagnosis is important for preventing and controlling infectious disease. In this study, two monoclonal antibodies (mAbs) were generated, which could specifically recognize recombinant PDCoV nucleocapsid (rPDCoV-N) protein. A colloidal gold immunochromatographic assay (GICA) strip using these mAbs was developed to detect PDCoV antigens within 15 min. Results showed that the detection limit of the GICA strip developed in this study was 10 TCID/ml for the suspension of virus-infected cell culture and 0.125 μg/ml for rPDCoV-N protein, respectively. Besides, the GICA strip showed high specificity with no cross-reactivity with other porcine pathogenic viruses. Three hundred and twenty-five fecal samples were detected for PDCoV using the GICA strip and reverse transcription-quantitative real-time PCR (RT-qPCR). The coincidence rate of the GICA strip and RT-qPCR was 96.9%. The GICA strip had a diagnostic sensitivity of 88.9% and diagnostic specificity of 98.5%. The specific and efficient detection by the strip provides a convenient, rapid, easy to use and valuable diagnostic tool for PDCoV under laboratory and field conditions.
PubMed: 36687576
DOI: 10.3389/fmicb.2022.1074513 -
Journal of Virology Feb 2023Members of deltacoronavirus (DCoV) have mostly been identified in diverse avian species as natural reservoirs, though the porcine DCoV (PDCoV) is a major swine...
Members of deltacoronavirus (DCoV) have mostly been identified in diverse avian species as natural reservoirs, though the porcine DCoV (PDCoV) is a major swine enteropathogenic virus with global spread. The important role of aminopeptidase N (APN) orthologues from various mammalian and avian species in PDCoV cellular entry and interspecies transmission has been revealed recently. In this study, comparative analysis indicated that three avian DCoVs, bulbul DCoV HKU11, munia DCoV HKU13, and sparrow DCoV HKU17 (Chinese strain), and PDCoV in the subgenera are grouped together at whole-genome levels; however, the spike (S) glycoprotein and its S1 subunit of HKU17 are more closely related to night heron DCoV HKU19 in . Nevertheless, the S1 protein of HKU11, HKU13, or HKU17 bound to or interacted with chicken APN (chAPN) or porcine APN (pAPN) by flow cytometry analysis of cell surface expression of APN and by coimmunoprecipitation in APN-overexpressing cells. Expression of chAPN or pAPN allowed entry of pseudotyped lentiviruses with the S proteins from HKU11, HKU13 and HKU17 into nonsusceptible cells and natural avian and porcine cells, which could be inhibited by the antibody against APN or anti-PDCoV-S1. APN knockdown by siRNA or knockout by CRISPR/Cas9 in chicken or swine cell lines significantly or almost completely blocked infection of these pseudoviruses. Hence, we demonstrate that HKU11, HKU13, and HKU17 with divergent S genes likely engage chAPN or pAPN to enter the cells, suggesting a potential interspecies transmission from wild birds to poultry and from birds to mammals by certain avian DCoVs. The receptor usage of avian deltacoronaviruses (DCoVs) has not been investigated thus far, though porcine deltacoronavirus (PDCoV) has been shown to utilize aminopeptidase N (APN) as a cell receptor. We report here that chicken or porcine APN also mediates cellular entry by three avian DCoV (HKU11, HKU13, and HKU17) spike pseudoviruses, and the S1 subunit of three avian DCoVs binds to APN and in the surface of avian and porcine cells. The results fill the gaps in knowledge about the avian DCoV receptor and elucidate important insights for the monitoring and prevention of potential interspecies transmission of certain avian DCoVs. In view of the diversity of DCoVs, whether this coronavirus genus will cause novel virus to emerge in other mammals from birds, are worthy of further surveillance and investigation.
Topics: Animals; CD13 Antigens; Chickens; Coronavirus Infections; Deltacoronavirus; Swine; Virus Internalization; Spike Glycoprotein, Coronavirus; Lentivirus
PubMed: 36656013
DOI: 10.1128/jvi.01947-22 -
Applied Microbiology and Biotechnology Feb 2023Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus that caused diarrhea and/or vomiting in neonatal piglets worldwide. Coronaviruses...
Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus that caused diarrhea and/or vomiting in neonatal piglets worldwide. Coronaviruses nucleocapsid (N) protein is the most conserved structural protein for viral replication and possesses good antigenicity. In this study, three monoclonal antibodies (mAbs), 3B4, 4D3, and 4E3 identified as subclass IgG2aκ were prepared using the lymphocytic hybridoma technology against PDCoV N protein. Furthermore, the B-cell epitope recognized by mAb 4D3 was mapped by dozens of overlapping truncated recombinant proteins based on the western blotting. The polypeptide QFRGNGVPLNSAIKPVE (EP-4D3) in the N-terminal of PDCoV N protein was identified as the minimal linear epitope for binding mAb 4D3. And the EP-4D3 epitope's amino acid sequence homology study revealed that PDCoV strains are substantially conserved, with the exception of the Alanine substitution Valine in the China lineage, the Early China lineage, and the Thailand, Vietnam, and Laos lineage. The epitope sequences shared high similarity (94.1%) with porcine coronavirus HKU15-155 (PorCoV HKU15), Asian leopard cats coronavirus (ALCCoV), sparrow coronavirus HKU17 (SpCoV HKU17), and sparrow deltacoronavirus. In contrast, the epitope sequences shared a very low homology (11.8 to 29.4%) with other porcine CoVs (PEDV, TGEV, PRCV, SADS-CoV, PHEV). Overall, the study will enrich the biological function of PDCoV N protein and provide foundational data for further development of diagnostic applications. KEY POINTS: • Three monoclonal antibodies against PDCoV N protein were prepared. • Discovery of a novel B-cell liner epitope (QFRGNGVPLNSAIKPVE) of PDCoV N protein. • The epitope EP-4D3 was conserved among PDCoV strains.
Topics: Swine; Animals; Deltacoronavirus; Epitopes, B-Lymphocyte; Nucleocapsid Proteins; Swine Diseases; Coronavirus; Coronavirus Infections; Antibodies, Monoclonal
PubMed: 36602561
DOI: 10.1007/s00253-022-12348-5 -
Virology Jan 2023Porcine deltacoronavirus is an evolving coronavirus that primarily infects the intestine and may lead to intestinal disease in piglets. Up to now, no commercial...
Porcine deltacoronavirus is an evolving coronavirus that primarily infects the intestine and may lead to intestinal disease in piglets. Up to now, no commercial vaccination is readily accessible to protect against the spread of PDCoV. Lactococcus lactis has been shown to have good immune efficacy and safety and can be used as a genetically engineered vaccine to deliver antigens. In this research, we utilized L. lactis NZ9000 to provide the S1 protein orally and improved the delivery efficiency by connecting the M cell targeting ligand Co1 with the S1 protein of PDCoV in tandem to obtain the recombinant protein S1-Co1. We successfully constructed two recombinant strains capable of expressing PDCoV-S1 and PDCoV-S1-Co1 proteins (i.e., L. lactis NZ9000-S1 and L. lactis NZ9000-S1-Co1), and their immunogenic capacity was evaluated in mice. Our study shows that Lactococcus is an advantageous bacterial live vector vaccine and is anticipated as a potential PDCoV vaccination option.
Topics: Animals; Mice; Swine; Lactococcus lactis; Immunity, Mucosal; Vaccination; Recombinant Proteins; Administration, Oral
PubMed: 36586181
DOI: 10.1016/j.virol.2022.12.010 -
BMC Veterinary Research Dec 2022The emergence and rapid spread of the acute respiratory syndrome coronavirus-2 have confirmed that animal coronaviruses represent a potential zoonotic source. Porcine...
The emergence and rapid spread of the acute respiratory syndrome coronavirus-2 have confirmed that animal coronaviruses represent a potential zoonotic source. Porcine deltacoronavirus is a worldwide evolving enteropathogen of swine, detected first in Hong Kong, China, before its global identification. Following the recent detection of PDCoV in humans, we attempted in this report to re-examine the status of PDCoV phylogenetic classification and evolutionary characteristics. A dataset of 166 complete PDCoV genomes was analyzed using the Maximum Likelihood method in IQ-TREE with the best-fitting model GTR + F + I + G4, revealing two major genogroups (GI and GII), with further seven and two sub-genogroups, (GI a-g) and (GII a-b), respectively. PDCoV strains collected in China exhibited the broadest genetic diversity, distributed in all subgenotypes. Thirty-one potential natural recombination events were identified, 19 of which occurred between China strains, and seven involved at least one China strain as a parental sequence. Importantly, we identified a human Haiti PDCoV strain as recombinant, alarming a possible future spillover that could become a critical threat to human health. The similarity and recombination analysis showed that PDCoV spike ORF is highly variable compared to ORFs encoding other structural proteins. Prediction of linear B cell epitopes of the spike glycoprotein and the 3D structural mapping of amino acid variations of two representative strains of GI and GII showed that the receptor-binding domain (RBD) of spike glycoprotein underwent a significant antigenic drift, suggesting its contribution in the genetic diversity and the wider spread of PDCoV.
Topics: Humans; Swine; Animals; Phylogeny; COVID-19; Biological Evolution; Glycoproteins; Swine Diseases
PubMed: 36550483
DOI: 10.1186/s12917-022-03554-4 -
Animals : An Open Access Journal From... Nov 2022Coronaviruses (CoVs) are part of the family, and the genera Gamma (γ) and Delta (δ) are found mostly in birds. Migratory birds have an enormous potential for...
Coronaviruses (CoVs) are part of the family, and the genera Gamma (γ) and Delta (δ) are found mostly in birds. Migratory birds have an enormous potential for dispersing pathogenic microorganisms. Ducks (order Anseriformes) can host CoVs from birds, with pathogenic expression and high economic impact. This study aimed to identify and characterize the diversity of CoVs in migratory ducks from Portugal. Duck stool samples were collected using cloacal swabs from 72 individuals (, and ). Among the 72 samples tested, 24 showed amplicons of the expected size. Twenty-three were characterized as and one as (accession numbers ON368935-ON368954; ON721380-ON721383). The sequences showed greater similarities to those obtained in ducks () from Finland and Poland, duck from the USA, and mute swans from Poland. Birds can occupy many habitats and therefore play diverse ecological roles in various ecosystems, especially given their ability to migrate exceptional distances, facilitating the dispersal of microorganisms with animal and/or human impact. There are a considerable number of studies that have detected CoVs in ducks, but none in Portugal. The present study assessed the circulation of CoVs in wild ducks from Portugal, being the first description of CoVs for these animals in Portugal.
PubMed: 36496804
DOI: 10.3390/ani12233283 -
Journal of Virology Dec 2022Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, not only causes diarrhea in piglets but also possesses the potential to infect humans. To...
Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, not only causes diarrhea in piglets but also possesses the potential to infect humans. To better understand host-virus genetic dependencies and find potential therapeutic targets for PDCoV, we used a porcine single-guide RNA (sgRNA) lentivirus library to screen host factors related to PDCoV infection in LLC-PK1 cells. The solute carrier family 35 member A1 (SLC35A1), a key molecule in the sialic acid (SA) synthesis pathway, was identified as a host factor required for PDCoV infection. A knockout of SLC35A1 caused decreases in the amounts of cell surface sialic acid (SA) and viral adsorption; meanwhile, trypsin promoted the use of SA in PDCoV infection. By constructing and assessing a series of recombinant PDCoV strains with the deletion or mutation of possible critical domain or amino acid residues for SA binding in the S1 N-terminal domain, we found that S T182 might be a PDCoV SA-binding site. However, the double knockout of SLC35A1 and amino peptidase N (APN) could not block PDCoV infection completely. Additionally, we found that different swine enteric coronaviruses, including transmissible gastroenteritis coronavirus, porcine epidemic diarrhea virus, and swine acute diarrhea syndrome coronavirus, are differentially dependent on SA. Overall, our study uncovered a collection of host factors that can be exploited as drug targets against PDCoV infection and deepened our understanding of the relationship between PDCoV and SA. Identifying the host factors required for replication will be helpful to uncover the pathogenesis mechanisms and develop antivirals against the emerging coronavirus porcine deltacoronavirus (PDCoV). Herein, we performed a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 knockout screen, the results of which revealed that the solute carrier family 35 member A1 (SLC35A1) is a host factor required for PDCoV infection that acts by regulating cell surface sialic acid (SA). We also identified the T182 site in the N-terminal domain of PDCoV S1 subunit as being associated with the SA-binding site and found that trypsin promotes the use of cell surface SA by PDCoV. Furthermore, different swine enteric coronaviruses use SLC35A1 differently for infection. This is the first study to screen host factors required for PDCoV replication using a genome-wide CRISPR-Cas9 functional knockout, thereby providing clues for developing antiviral drugs against PDCoV infection.
Topics: Animals; Humans; Adsorption; Coronavirus; Coronavirus Infections; CRISPR-Cas Systems; N-Acetylneuraminic Acid; Nucleotide Transport Proteins; Swine; Swine Diseases; Trypsin; Host Microbial Interactions; Protein Domains; Binding Sites
PubMed: 36453883
DOI: 10.1128/jvi.01626-22 -
Frontiers in Veterinary Science 2022Porcine enteric coronaviruses are pathogens that cause viral diarrhea in pigs and are widely prevalent worldwide. Moreover, studies have shown that some porcine enteric...
Porcine enteric coronaviruses are pathogens that cause viral diarrhea in pigs and are widely prevalent worldwide. Moreover, studies have shown that some porcine enteric coronaviruses can infect humans and poultry. In order to effectively monitor these viruses, it is necessary to establish a multiple detection method to understand their prevalence and conduct in-depth research. Common porcine enteric coronaviruses include Porcine epidemic diarrhea virus (PEDV), Porcine transmissible gastroenteritis virus (TGEV), Porcine delta coronavirus (PDCoV), and Swine acute diarrhea syndrome coronavirus (SADS-CoV). Pigs infected with these viruses have the common clinical symptoms that are difficult to distinguish. A quadruplex RT-PCR (reverse transcription-polymerase chain reaction) method for the simultaneous detection of PEDV, PDCoV, TGEV and SADS-CoV was developed. Four pairs of specific primers were designed for the PEDV gene, PDCoV gene, TGEV gene and SADS-CoV gene. Multiplex RT-PCR results showed that the target fragments of PDCoV, SADS-CoV, PEDV and TGEV could be amplified by this method. and the specific fragments with sizes of 250 bp, 368 bp, 616 bp and 801 bp were amplified, respectively. This method cannot amplify any fragment of nucleic acids of Seneca Valley virus (SVV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Atypical Porcine Pestivirus (APPV), and has good specificity. The lowest detection limits of PDCoV, PEDV, TGEV and SADS-CoV were 5.66 × 10 copies/μL, 6.48 × 10 copies/μL, 8.54 × 10 copies/μL and 7.79 × 10 copies/μL, respectively. A total of 94 samples were collected from pig farms were analyzed using this method. There were 15 positive samples for PEDV, 3 positive samples for mixed infection of PEDV and PDCoV, 2 positive samples for mixed infection of PEDV and TGEV, and 1 positive sample for mixed infection of PEDV, TGEV, and PDCoV. Multiplex RT-PCR method could detect four intestinal coronaviruses (PEDV, PDCoV, TGEV, and SADS-CoV) in pigs efficiently, cheaply and accurately, which can be used for clinical large-scale epidemiological investigation and diagnosis.
PubMed: 36425116
DOI: 10.3389/fvets.2022.1033864 -
Veterinary Sciences Nov 2022Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus...
Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV) are four identified porcine enteric coronaviruses. Pigs infected with these viruses show similar manifestations of diarrhea, vomiting, and dehydration. Here, a quadruplex real-time quantitative PCR (qRT-PCR) assay was established for the differential detection of PEDV, TGEV, PDCoV, and SADS-CoV from swine fecal samples. The assay showed extreme specificity, high sensitivity, and excellent reproducibility, with the limit of detection (LOD) of 121 copies/μL (final reaction concentration of 12.1 copies/μL) for each virus. The 3236 clinical fecal samples from Guangxi province in China collected between October 2020 and October 2022 were evaluated by the quadruplex qRT-PCR, and the positive rates of PEDV, TGEV, PDCoV, and SADS-CoV were 18.26% (591/3236), 0.46% (15/3236), 13.16% (426/3236), and 0.15% (5/3236), respectively. The samples were also evaluated by the multiplex qRT-PCR reported previously by other scientists, and the compliance rate between the two methods was more than 99%. This illustrated that the developed quadruplex qRT-PCR assay can provide an accurate method for the differential detection of four porcine enteric coronaviruses.
PubMed: 36423083
DOI: 10.3390/vetsci9110634 -
Scientific Reports Nov 2022Porcine deltacoronavirus (PDCoV) and porcine epidemic diarrhea virus (PEDV) infect the small intestine and cause swine enteric coronavirus disease. The mucosal innate...
Porcine deltacoronavirus (PDCoV) and porcine epidemic diarrhea virus (PEDV) infect the small intestine and cause swine enteric coronavirus disease. The mucosal innate immune system is the first line of defense against viral infection. The modulatory effect of PDCoV and PEDV coinfection on antiviral signaling cascades of the intestinal mucosa has not been reported. Here, we investigate the gene expression levels of pattern recognition receptors, downstream inflammatory signaling pathway molecules, and associated cytokines on the intestinal mucosa of neonatal piglets either infected with a single- or co-infected with PDCoV and PEDV using real-time PCR. The results demonstrate that single-PEDV regulates the noncanonical NF-κB signaling pathway through RIG-I regulation. In contrast, single-PDCoV and PDCoV/PEDV coinfection regulate proinflammatory and regulatory cytokines through TRAF6-mediated canonical NF-κB and IRF7 signaling pathways through TLRs. Although PDCoV/PEDV coinfection demonstrated an earlier modulatory effect in these signaling pathways, the regulation of proinflammatory and regulatory cytokines was observed simultaneously during single viral infection. These results suggested that PDCoV/PEDV coinfection may have synergistic effects that lead to enhanced viral evasion of the mucosal innate immune response.
Topics: Animals; Swine; Porcine epidemic diarrhea virus; NF-kappa B; TNF Receptor-Associated Factor 6; Coinfection; Coronavirus Infections; Swine Diseases; Signal Transduction; Cytokines; Diarrhea
PubMed: 36376395
DOI: 10.1038/s41598-022-24190-w