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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 -
Virus Research Mar 2020Porcine deltacoronavirus (PDCoV) is the etiological agent of acute diarrhoea and vomiting in pigs, threatening the swine industry worldwide. Although several PDCoV...
Porcine deltacoronavirus (PDCoV) is the etiological agent of acute diarrhoea and vomiting in pigs, threatening the swine industry worldwide. Although several PDCoV studies have been conducted in China, more sequence information is needed to understand the molecular characterization of PDCoV. In this study, the partial ORF1a, spike protein (S) and nucleocapsid protein (N) were sequenced from Shandong Province between 2017 and 2018. The sequencing results for the S protein from 10 PDCoV strains showed 96.7 %-99.7 % nucleotide sequence identity with the China lineage strains, while sharing a lower level of nucleotide sequence identity, ranging from 95.7 to 96.8%, with the Vietnam/Laos/Thailand lineage strains. N protein sequencing analysis showed that these strains showed nucleotide homologies of 97.3%-99.3% with the reference strains. Phylogenetic analyses based on S protein sequences showed that these PDCoV strains were classified into the China lineage. The discontinuous 2 + 3 aa deletions at 400-401 and 758-760 were found in the Nsp2 and Nsp3 coding region in five strains, respectively, with similar deletions having been identified in Vietnam, Thailand, and Laos. Three novel patterns of deletion were observed for the first time in the Nsp2 and Nsp3 regions. Importantly, those findings suggest that PDCoV may have undergone a high degree of variation since PDCoV was first detected in China.
Topics: Animals; China; Coronavirus Infections; Deltacoronavirus; Diarrhea; Feces; Gene Deletion; Genome, Viral; Phylogeny; Prevalence; Swine; Swine Diseases; Viral Proteins
PubMed: 31962065
DOI: 10.1016/j.virusres.2020.197869 -
Vaccine Jul 2022Porcine deltacoronavirus (PDCoV) is a novel coronavirus that causes diarrhea in pigs of various ages, especially in suckling piglets, and there are no effective measures...
Porcine deltacoronavirus (PDCoV) is a novel coronavirus that causes diarrhea in pigs of various ages, especially in suckling piglets, and there are no effective measures to prevent and control PDCoV currently. In this study, two adjuvants Al(OH) and ODN2395 working through different mechanisms were used to prepare inactivated PDCoV vaccines, and the immune effects of PDCoV inactivated vaccines were assessed in mice. From the results, we found that both PDCoV/Al(OH) vaccine and PDCoV/2395 vaccine could induce IgG and neutralizing antibodies with high levels in mice. At the same time, cytokines of IFN-γ, IL-4 and chemokine ligand of CXCL13 in serum were significantly increased after immunization, and reached the highest levels in PDCoV/2395 vaccine group, which suggested that PDCoV/2395 could promote the production of both Th1 and Th2 polarized cytokines. In addition, histopathological observations showed that vaccination helped mice resist PDCoV infection. These results indicated that both the two inactivated vaccines have good immune effects. Moreover, the PDCoV/2395 vaccine worked better than the PDCoV/Al(OH) vaccine for PDCoV/2395 having the good ability to induce both humoral and cellular immunogenicity. The PDCoV/2395 inactivated vaccine developed in this study might be an effective tool for the prevention of PDCoV infection.
Topics: Animals; COVID-19; Cytokines; Deltacoronavirus; Mice; Swine; Swine Diseases; Vaccines, Inactivated
PubMed: 35691873
DOI: 10.1016/j.vaccine.2022.05.085 -
Veterinary Sciences Feb 2023Porcine deltacoronavirus (PDCoV) was first identified approximately a decade ago, but much is still obscure in terms of its pathogenesis. We aimed to further...
Detection of Porcine Deltacoronavirus RNA in the Upper and Lower Respiratory Tract and Biliary Fluid and the Effect of Infection on Serum Cholesterol Levels and Blood T Cell Population Frequencies in Gnotobiotic Piglets.
Porcine deltacoronavirus (PDCoV) was first identified approximately a decade ago, but much is still obscure in terms of its pathogenesis. We aimed to further characterize PDCoV infection by investigating the presence of virus in respiratory and biliary tissues or fluids; T cell population frequencies in blood; and altered serum cholesterol levels. Twelve, 6-day-old, gnotobiotic piglets were inoculated oronasally with PDCoV OH-FD22 (2.6 × 10 FFU/pig). Six control piglets were not inoculated. Rectal swab (RS), nasal swab (NS), nasal wash (NW), bronchoalveolar lavage (BAL), and biliary fluid (BF) samples were collected at 2, 4, and 7 days post-inoculation (DPI) and tested for PDCoV RNA by RT-qPCR. Blood T cell populations and serum cholesterol levels were determined by flow cytometry and a colorimetric assay, respectively. Moderate to high, and low to moderate titers of PDCoV RNA were detected in RS and in NS, NW, BAL, and BF samples, respectively, of inoculated piglets. There were trends toward decreased CD4+CD8-, CD4-CD8+, and CD4+CD8+ blood T cell frequencies in inoculated piglets. Furthermore, serum cholesterol levels were increased in inoculated piglets. Overall, we found that PDCoV infection does not exclusively involve the intestine, since the respiratory and biliary systems and cholesterol metabolism also can be affected.
PubMed: 36851421
DOI: 10.3390/vetsci10020117 -
Virology Jul 2021Porcine deltacoronavirus (PDCoV) is a novel swine enteropathogenic coronavirus that causes serious vomiting and diarrhea in piglets. Previous work demonstrated that...
Porcine deltacoronavirus (PDCoV) is a novel swine enteropathogenic coronavirus that causes serious vomiting and diarrhea in piglets. Previous work demonstrated that PDCoV infection inhibits type I interferon (IFN) production. Here, we found that ectopic expression of PDCoV nsp10 significantly inhibited Sendai virus (SeV)-induced IFN-β production by impairing the phosphorylation and nuclear translocation of two transcription factors, IRF3 and NF-κB p65 subunit. Interestingly, experiments with truncated mutants and site-directed mutagenesis revealed that PDCoV nsp10 mutants with missing or destroyed zinc fingers (ZFs) domains also impeded SeV-induced IFN-β production, suggesting that nsp10 does not require its ZF domains to antagonize IFN-β production. Further work found that co-expression of nsp10 with nsp14 or nsp16, two replicative enzymes, significantly enhanced the inhibitory effects of nsp10 on IFN-β. Taken together, our results demonstrate that PDCoV nsp10 antagonizes IFN via a ZF-independent mechanism and has a synergistic effect with nsp14 and nsp16 on inhibiting IFN-β production.
Topics: Animals; Cell Line; Deltacoronavirus; Host-Pathogen Interactions; Humans; Interferon Regulatory Factor-3; Interferon-beta; Mutation; Sendai virus; Signal Transduction; Swine; Transcription Factor RelA; Viral Nonstructural Proteins; Zinc Fingers
PubMed: 33813212
DOI: 10.1016/j.virol.2021.03.015 -
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 -
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 -
Journal of Virology Nov 2021Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes serious diarrhea in suckling piglets and has the potential for cross-species...
Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes serious diarrhea in suckling piglets and has the potential for cross-species transmission. Although extensive studies have been reported on the biology and pathogenesis of PDCoV, the mechanisms by which PDCoV enters cells are not well characterized. In this study, we investigated how PDCoV enters IPI-2I cells, a line of porcine intestinal epithelial cells derived from pig ileum. Immunofluorescence assays, small interfering RNA (siRNA) interference, specific pharmacological inhibitors, and dominant negative mutation results revealed that PDCoV entry into IPI-2I cells depended on clathrin, dynamin, and a low-pH environment but was independent of caveolae. Specific inhibition of phosphatidylinositol 3-kinase (PI3K) and the Na/H exchanger (NHE) revealed that PDCoV entry involves macropinocytosis and depends on NHE rather than on PI3K. Additionally, Rab5 and Rab7, but not Rab11, regulated PDCoV endocytosis. This is the first study to demonstrate that PDCoV uses clathrin-mediated endocytosis and macropinocytosis as alternative endocytic pathways to enter porcine intestinal epithelial cells. We also discussed the entry pathways of PDCoV into other porcine cell lines. Our findings reveal the entry mechanisms of PDCoV and provide new insight into the PDCoV life cycle. An emerging enteropathogenic coronavirus, PDCoV, has the potential for cross-species transmission, attracting extensive attenuation. Characterizing the detailed process of PDCoV entry into cells will deepen our understanding of the viral infection and pathogenesis and provide clues for therapeutic intervention against PDCoV. With the objective, we used complementary approaches to dissect the process in PDCoV-infected IPI-2I cells, a line of more physiologically relevant intestinal epithelial cells to PDCoV infection . Here, we demonstrate that PDCoV enters IPI-2I cells via macropinocytosis, which does not require a specific receptor, and clathrin-mediated endocytosis, which requires a low-pH environment and dynamin, while a caveola-mediated endocytic pathway is used by PDCoV to enter swine testicular (ST) cells and porcine kidney (LLC-PK1) cells. These findings provide a molecular detail of the cellular entry pathways of PDCoV and may direct us toward novel antiviral drug development.
Topics: Animals; Cell Line; Cell Survival; Clathrin; Coronavirus; Coronavirus Infections; Deltacoronavirus; Dynamins; Endocytosis; Epithelial Cells; Hydrogen-Ion Concentration; Ileum; Kidney; Phosphatidylinositol 3-Kinases; Pinocytosis; RNA, Small Interfering; Swine; Swine Diseases; Virus Internalization; rab5 GTP-Binding Proteins
PubMed: 34586858
DOI: 10.1128/JVI.01345-21 -
FEMS Microbiology Reviews Sep 2020Wild birds interconnect all parts of the globe through annual cycles of migration with little respect for country or continental borders. Although wild birds are... (Review)
Review
Wild birds interconnect all parts of the globe through annual cycles of migration with little respect for country or continental borders. Although wild birds are reservoir hosts for a high diversity of gamma- and deltacoronaviruses, we have little understanding of the ecology or evolution of any of these viruses. In this review, we use genome sequence and ecological data to disentangle the evolution of coronaviruses in wild birds. Specifically, we explore host range at the levels of viral genus and species, and reveal the multi-host nature of many viral species, albeit with biases to certain types of avian host. We conclude that it is currently challenging to infer viral ecology due to major sampling and technical limitations, and suggest that improved assay performance across the breadth of gamma- and deltacoronaviruses, assay standardization, as well as better sequencing approaches, will improve both the repeatability and interpretation of results. Finally, we discuss cross-species virus transmission across both the wild bird - poultry interface as well as from birds to mammals. Clarifying the ecology and diversity in the wild bird reservoir has important ramifications for our ability to respond to the likely future emergence of coronaviruses in socioeconomically important animal species or human populations.
Topics: Animals; Animals, Wild; Birds; Coronavirus; Coronavirus Infections; Disease Reservoirs; Gammacoronavirus; Host Specificity
PubMed: 32672814
DOI: 10.1093/femsre/fuaa026 -
Veterinary Microbiology Aug 2020Porcine deltacoronavirus (PDCoV) is a novel swine enteropathogenic coronavirus that causes watery diarrhea, vomiting and mortality in nursing piglets. Type III...
Porcine deltacoronavirus (PDCoV) is a novel swine enteropathogenic coronavirus that causes watery diarrhea, vomiting and mortality in nursing piglets. Type III interferons (IFN-λs) are the major antiviral cytokines in intestinal epithelial cells, the target cells in vivo for PDCoV. In this study, we found that PDCoV infection remarkably inhibited Sendai virus-induced IFN-λ1 production by suppressing transcription factors IRF and NF-κB in IPI-2I cells, a line of porcine intestinal mucosal epithelial cells. We also confirmed that PDCoV infection impeded the activation of IFN-λ1 promoter stimulated by RIG-I, MDA5 and MAVS, but not by TBK1 and IRF1. Although the expression levels of IRF1 and MAVS were not changed, PDCoV infection resulted in reduction of the number of peroxisomes, the platform for MAVS to activate IRF1, and subsequent type III IFN production. Taken together, our study demonstrates that PDCoV suppresses type III IFN responses to circumvent the host's antiviral immunity.
Topics: Animals; Cell Line; Coronavirus; Coronavirus Infections; Epithelial Cells; Host-Pathogen Interactions; Interferon Regulatory Factor-1; Interferons; Intestines; Kidney; NF-kappa B; Sendai virus; Signal Transduction; Swine; Swine Diseases; Interferon Lambda
PubMed: 32768229
DOI: 10.1016/j.vetmic.2020.108785