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Frontiers in Microbiology 2024Porcine viral diarrhea is a common clinical disease, which results in high mortality and economic losses in the pig industry. Porcine epidemic diarrhea virus (PEDV),...
INTRODUCTION
Porcine viral diarrhea is a common clinical disease, which results in high mortality and economic losses in the pig industry. Porcine epidemic diarrhea virus (PEDV), porcine rotavirus (PoRV), and porcine deltacoronavirus (PDCoV) are important diarrhea viruses in pig herds. The similarities of their clinical symptoms and pathological changes make it difficult to distinguish these three viruses clinically. Therefore, there is a need for a highly sensitive and specific method to simultaneously detect and differentiate these viruses.
METHODS
A multiplex real-time PCR assay using TaqMan probes was developed to simultaneously detect PEDV, PoRV, and PDCoV. To assess the efficacy of the established assay, 30 clinical samples with diarrhea symptoms were used to compare the results obtained from the multiplex real-time PCR assay with those obtained from commercial singleplex real-time PCR kit. Importantly, a total of 4,800 diarrhea samples were tested and analyzed to validate the utility of the assay.
RESULTS
This multiplex real-time PCR assay showed high sensitivity, specificity, and excellent repeatability with a detection limit of 1 × 10 copies/μL. Comparing the results of the commercial singleplex real-time PCR kit and the multiplex real-time PCR method for detecting PEDV, PoRV, and PDCoV, there was complete agreement between the two approaches. Clinical data revealed single infection rates of 6.56% for PEDV, 21.69% for PoRV, and 6.65% for PDCoV. The co-infection rates were 11.83% for PEDV + PoRV, 0.29% for PEDV + PDCoV, 5.71% for PoRV + PDCoV, and 1.29% for PEDV + PDCoV + PoRV, respectively.
DISCUSSION
The multiplex real-time PCR method established in this study is a valuable diagnostic tool for simultaneously differentiating PEDV, PoRV, and PDCoV. This method is expected to significantly contribute to prevent and control the spread of infectious diseases, as well as aid in conducting epidemiological investigations.
PubMed: 38690365
DOI: 10.3389/fmicb.2024.1380849 -
Veterinary Microbiology Jun 2024Stress granules (SGs), the main component is GTPase-activating protein-binding protein 1 (G3BP1), which are assembled during viral infection and function to sequester...
Stress granules (SGs), the main component is GTPase-activating protein-binding protein 1 (G3BP1), which are assembled during viral infection and function to sequester host and viral mRNAs and proteins, are part of the antiviral responses. In this study, we found that porcine deltacoronavirus (PDCoV) infection induced stable formation of robust SGs in cells through a PERK (protein kinase R-like endoplasmic reticulum kinase)-dependent mechanism. Overexpression of SGs marker proteins G3BP1 significantly reduced PDCoV replication in vitro, while inhibition of endogenous G3BP1 enhanced PDCoV replication. Moreover, PDCoV infected LLC-PK1 cells raise the phosphorylation level of G3BP1. By overexpression of the G3BP1 phosphorylated protein or the G3BP1 dephosphorylated protein, we found that phosphorylation of G3BP1 is involved in the regulation of PDCoV-induced inflammatory response. Taken together, our study presents a vital aspect of the host innate response to invading pathogens and reveals attractive host targets for antiviral target.
Topics: Animals; Swine; RNA Recognition Motif Proteins; Phosphorylation; Poly-ADP-Ribose Binding Proteins; RNA Helicases; DNA Helicases; Inflammation; Virus Replication; Coronavirus; Cell Line; Swine Diseases; Immunity, Innate
PubMed: 38593624
DOI: 10.1016/j.vetmic.2024.110070 -
Virology Jun 2024Coronaviruses (CoVs) comprise a group of important human and animal pathogens that threaten public health because of their interspecies transmission potential to humans....
Coronaviruses (CoVs) comprise a group of important human and animal pathogens that threaten public health because of their interspecies transmission potential to humans. However, virus-like particles (VLPs) constitute versatile tools in CoVs vaccine development due to their favorable immunological characteristics. Here, we engineered the VLPs composed of the spike (S), membrane (M), and envelope (E) structural proteins of the Porcine deltacoronavirus (PDCoV) and examined their immune responses in mice. Neutralization assays and flow Cytometry demonstrated that PDCoV VLPs induced highly robust neutralizing antibodies (NAbs) and elicited cellular immunity. To assess the protective efficacy of VLPs in newborn piglets, pregnant sows received vaccinations with either a PDCoV-inactivated vaccine or VLPs at 40 and 20 days before delivery. Five days post-farrowing, piglets were orally challenged with the PDCoV strain. Severe diarrhea, high viral RNA copies, and substantial intestinal villus atrophy were detected in piglets born to unimmunized sows. However, piglets from sows immunized with VLPs exhibited high NAbs titers and markedly reduced microscopic damage to the intestinal tissues, with no piglet showing diarrhea. Hence, the results indicate that the VLPs are a potential clinical candidate for PDCoV vaccination, while the strategy may serve as a platform for developing other coronavirus vaccines.
PubMed: 38917690
DOI: 10.1016/j.virol.2024.110150 -
Veterinary Microbiology Jan 2024The discovery of antiviral molecules is crucial for controlling porcine deltacoronavirus (PDCoV). Previous studies have provided evidence that the IFN-inducible...
The discovery of antiviral molecules is crucial for controlling porcine deltacoronavirus (PDCoV). Previous studies have provided evidence that the IFN-inducible transmembrane protein 3 (IFITM3), which is coded by an interferon-stimulated gene, prevents the infections of a number of enveloped viruses. Nevertheless, the involvement of IFITM3 in PDCoV infection remains unexplored. In this study, it was observed that the overexpression of IFITM3 successfully restrictes the infection of PDCoV in cell cultures. Conversely, the suppression of IFITM3 facilitates the infection of PDCoV in IPI-2I and IPEC-J2 cells. Further studies revealed that IFITM3 limits the attachment phase of viral infection by interacting with the S1 subunit of the PDCoV Spike (S) protein. In addition, IFITM3 is verified as a member of the CD225 family, the GxxxG conserved motif of this family is important for it to limit PDCoV infection. In summary, this study reveals the mechanism of IFITM3 as an antiviral molecule to inhibit PDCoV infection, and also provides theoretical supports for screening effective anti-PDCoV drugs.
Topics: Swine; Animals; Coronavirus; Coronavirus Infections; Spike Glycoprotein, Coronavirus; Antiviral Agents; Swine Diseases
PubMed: 38118371
DOI: 10.1016/j.vetmic.2023.109953 -
Journal of Virology Mar 2024Continuously emerging highly pathogenic coronaviruses remain a major threat to human and animal health. Porcine deltacoronavirus (PDCoV) is a newly emerging enterotropic...
Continuously emerging highly pathogenic coronaviruses remain a major threat to human and animal health. Porcine deltacoronavirus (PDCoV) is a newly emerging enterotropic swine coronavirus that causes large-scale outbreaks of severe diarrhea disease in piglets. Unlike other porcine coronaviruses, PDCoV has a wide range of species tissue tropism, including primary human cells, which poses a significant risk of cross-species transmission. Nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain-containing 1 (NLRP1) has a key role in linking host innate immunity to microbes and the regulation of inflammatory pathways. We now report a role for NLRP1 in the control of PDCoV infection. Overexpression of NLRP1 remarkably suppressed PDCoV infection, whereas knockout of NLRP1 led to a significant increase in PDCoV replication. A mechanistic study revealed that NLRP1 suppressed PDCoV replication in cells by upregulating IL-11 expression, which in turn inhibited the phosphorylation of the ERK signaling pathway. Furthermore, the ERK phosphorylation inhibitor U0126 effectively hindered PDCoV replication in pigs. Together, our results demonstrated that NLRP1 exerted an anti-PDCoV effect by IL-11-mediated inhibition of the phosphorylation of the ERK signaling pathway, providing a novel antiviral signal axis of NLRP1-IL-11-ERK. This study expands our understanding of the regulatory network of NLRP1 in the host defense against virus infection and provides a new insight into the treatment of coronaviruses and the development of corresponding drugs.IMPORTANCECoronavirus, which mainly infects gastrointestinal and respiratory epithelial cells , poses a huge threat to both humans and animals. Although porcine deltacoronavirus (PDCoV) is known to primarily cause fatal diarrhea in piglets, reports detected in plasma samples from Haitian children emphasize the potential risk of animal-to-human spillover. Finding effective therapeutics against coronaviruses is crucial for controlling viral infection. Nucleotide-binding oligomerization-like receptor (NLR) family pyrin domain-containing 1 (NLRP1), a key regulatory factor in the innate immune system, is highly expressed in epithelial cells and associated with the pathogenesis of viruses. We demonstrate here that NLRP1 inhibits the infection of the intestinal coronavirus PDCoV through IL-11-mediated phosphorylation inhibition of the ERK signaling pathway. Furthermore, the ERK phosphorylation inhibitor can control the infection of PDCoV in pigs. Our study emphasizes the importance of NLRP1 as an immune regulatory factor and may open up new avenues for the treatment of coronavirus infection.
Topics: Animals; Child; Humans; Coronavirus Infections; Deltacoronavirus; Diarrhea; Haiti; Interleukin-11; NLR Proteins; Nucleotides; Phosphorylation; Signal Transduction; Swine; Swine Diseases; Zoonoses
PubMed: 38411106
DOI: 10.1128/jvi.01982-23 -
Nature Communications Jun 2024Porcine deltacoronavirus (PDCoV) is an emerging enteric pathogen that has recently been detected in humans. Despite this zoonotic concern, the antigenic structure of...
Porcine deltacoronavirus (PDCoV) is an emerging enteric pathogen that has recently been detected in humans. Despite this zoonotic concern, the antigenic structure of PDCoV remains unknown. The virus relies on its spike (S) protein for cell entry, making it a prime target for neutralizing antibodies. Here, we generate and characterize a set of neutralizing antibodies targeting the S protein, shedding light on PDCoV S interdomain crosstalk and its vulnerable sites. Among the four identified antibodies, one targets the S1A domain, causing local and long-range conformational changes, resulting in partial exposure of the S1B domain. The other antibodies bind the S1B domain, disrupting binding to aminopeptidase N (APN), the entry receptor for PDCoV. Notably, the epitopes of these S1B-targeting antibodies are concealed in the prefusion S trimer conformation, highlighting the necessity for conformational changes for effective antibody binding. The binding footprint of one S1B binder entirely overlaps with APN-interacting residues and thus targets a highly conserved epitope. These findings provide structural insights into the humoral immune response against the PDCoV S protein, potentially guiding vaccine and therapeutic development for this zoonotic pathogen.
Topics: Spike Glycoprotein, Coronavirus; Animals; Antibodies, Neutralizing; Swine; Antibodies, Viral; Epitopes; Humans; Deltacoronavirus; CD13 Antigens; Coronavirus Infections; Protein Domains; Protein Binding; Swine Diseases; HEK293 Cells
PubMed: 38909062
DOI: 10.1038/s41467-024-49693-0 -
Microorganisms Apr 2024This study was conducted to elucidate the intestinal damage induced by the IPEC-J2 cell culture-passaged PDCoV. The results showed that PDCoV disrupted the intestinal...
This study was conducted to elucidate the intestinal damage induced by the IPEC-J2 cell culture-passaged PDCoV. The results showed that PDCoV disrupted the intestinal structure and increased intestinal permeability, causing abnormalities in mucosal pathology. Additionally, PDCoV induced an imbalance in the intestinal flora and disturbed its stability. Microbial community profiling revealed bacterial enrichment (e.g., Proteobacteria) and reduction (e.g., Firmicutes and Bacteroidetes) in the PDCoV-inoculated piglet model. In addition, metabolomics analysis indicated that 82 named differential metabolites were successfully quantified, including 37 up-regulated and 45 down-regulated metabolites. Chenodeoxycholic acid, sphingosine, and oleanolic aldehyde levels were reduced in PDCoV-inoculated piglets, while phenylacetylglycine and geranylgeranyl-PP levels were elevated. Correlation analysis indicated a negative correlation between and choline, succinic acid, creatine, phenyllactate, and hippuric acid. Meanwhile, was positively correlated with acetylcholine, L-Glutamicacid, and N-Acetylmuramate. , , , and were negatively and positively correlated with sphingosine, respectively. These data suggested PDCoV-inoculated piglets exhibited significant taxonomic perturbations in the gut microbiome, which may result in a significantly altered metabolomic profile.
PubMed: 38792704
DOI: 10.3390/microorganisms12050874 -
Veterinary Microbiology Feb 2024Porcine deltacoronavirus (PDCoV) infection in piglets can cause small intestinal epithelial necrosis and atrophic enteritis, which leads to severe damages to host cells,...
Porcine deltacoronavirus (PDCoV) infection in piglets can cause small intestinal epithelial necrosis and atrophic enteritis, which leads to severe damages to host cells, and result in diarrhea. In this study, we investigated the relationship between miR-361, SLC9A3(Solute carrier family 9, subfamily A, member 3), and NHE3(sodium-hydrogen exchanger member 3) in in porcine intestinal epithelial cells (IPI-2I) cells after PDCoV infection. Our results showed that the ssc-miR-361-3p expression inhibits the mRNA level of SLC9A3 gene which lead to the descending of NHE3 protein expression, and the NHE3 activity was suppressed. NHE3 activity was suppressed via down-regulation expression of SLC9A3 mRNA by transfection with siRNA. Ssc-miR-361-3p mimics and inhibitors were used to change the expression of ssc-miR-361-3p in IPI-2I cells. Ssc-miR-361-3p overexpression reduced the mRNA level of SLC9A3 gene, the level of NHE3 protein expression and NHE3 activity in IPI-2I cells, while ssc-miR-361-3p inhibits NHE3. Furthermore, luciferase reporter assay showed that SLC9A3 gene was a direct target of ssc-miR-361-3p. Ssc-miR-361-3p inhibition restored NHE3 activity in PDCoV infected IPI-2I cells by up-regulating SLC9A3 mRNA expression and NHE3 protein expression. These results demonstrate that the PDCoV infection can inhibit NHE3 activity through miR-361-3p/SLC9A3 regulatory axis. The relevant research is reported for the first time in PDCoV, which has significance in exploring the pathogenic mechanism of PDCoV and can provide a theoretical basis for its prevention and control. suggesting that NHE3 and ssc-miR-361-3p may be potential therapeutic targets for diarrhea in infected piglets.
Topics: Swine; Animals; Coronavirus; Sodium-Hydrogen Exchanger 3; Coronavirus Infections; Epithelial Cells; Diarrhea; RNA, Messenger; MicroRNAs; Swine Diseases
PubMed: 38159369
DOI: 10.1016/j.vetmic.2023.109916 -
Archives of Virology Mar 2024Porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV) cause intestinal diseases with similar...
A multiplex real-time RT-qPCR assay for simultaneous detection of porcine epidemic diarrhea virus, porcine deltacoronavirus, and swine acute diarrhea syndrome coronavirus.
Porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV) cause intestinal diseases with similar manifestations in suckling piglets. In this study, we developed a multiplex real-time PCR for differential diagnosis of PEDV, PDCoV, and SADS-CoV. The assay demonstrated high specificity with a detection limit of 5 copies/µl for each virus. The assay specifically detected PEDV, PDCoV, and SADS-CoV and excluded all other swine pathogens circulating in pigs. Furthermore, the assay exhibited satisfactory performance in analyzing clinical samples. The data indicate that the newly developed multiplex real-time PCR method can be applied for differential diagnosis of porcine enteric coronaviruses.
Topics: Animals; Swine; Porcine epidemic diarrhea virus; Diarrhea; Swine Diseases; Sensitivity and Specificity; Coronavirus Infections; Alphacoronavirus; Deltacoronavirus
PubMed: 38520595
DOI: 10.1007/s00705-024-06003-9 -
Veterinary Research Jun 2024Porcine deltacoronavirus (PDCoV) is an enteropathogenic coronavirus that has been reported to use various strategies to counter the host antiviral innate immune...
Porcine deltacoronavirus (PDCoV) is an enteropathogenic coronavirus that has been reported to use various strategies to counter the host antiviral innate immune response. The cGAS-STING signalling pathway plays an important role in antiviral innate immunity. However, it remains unclear whether PDCoV achieves immune evasion by regulating the cGAS-STING pathway. Here, we demonstrated that the nonstructural protein 2 (nsp2) encoded by PDCoV inhibits cGAS-STING-mediated type I and III interferon (IFN) responses via the regulation of porcine STING (pSTING) stability. Mechanistically, ectopically expressed PDCoV nsp2 was found to interact with the N-terminal region of pSTING. Consequently, pSTING was degraded through K48-linked ubiquitination and the proteasomal pathway, leading to the disruption of cGAS-STING signalling. Furthermore, K150 and K236 of pSTING were identified as crucial residues for nsp2-mediated ubiquitination and degradation. In summary, our findings provide a basis for elucidating the immune evasion mechanism of PDCoV and will contribute to the development of targets for anti-coronavirus drugs.
Topics: Animals; Swine; Viral Nonstructural Proteins; Deltacoronavirus; Swine Diseases; Membrane Proteins; Coronavirus Infections; Interferon Type I; Immunity, Innate; HEK293 Cells; Immune Evasion; Ubiquitination
PubMed: 38886840
DOI: 10.1186/s13567-024-01330-w