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Virus Research Jul 2024Porcine epidemic diarrhea (PED) is a highly contagious swine intestinal disease caused by PED virus (PEDV). Vaccination is a promising strategy to prevent and control...
Porcine epidemic diarrhea (PED) is a highly contagious swine intestinal disease caused by PED virus (PEDV). Vaccination is a promising strategy to prevent and control PED. Previous studies have confirmed that glycosylation could regulate the immunogenicity of viral antigens. In this study, we constructed three recombinant PEDVs which removed the glycosylation sites in RBD. Viral infection assays revealed that similar replication characteristics between the recombinant viruses and parental PEDV. Although animal challenging study demonstrated that the glycosylation sites in RBD do not affect the pathogenicity of PEDV, we found that removing the glycosylation sites on the RBD regions could promote the IgG and neutralization titer in vivo, suggesting deglycosylation in RBD could enhance the immunogenicity of PEDV. These findings demonstrated that removal of the glycosylation sites in RBD is a promising method to develop PEDV vaccines.
Topics: Animals; Porcine epidemic diarrhea virus; Glycosylation; Spike Glycoprotein, Coronavirus; Swine; Swine Diseases; Antibodies, Viral; Antibodies, Neutralizing; Viral Vaccines; Coronavirus Infections; Vero Cells; Chlorocebus aethiops; Immunoglobulin G; Immunogenicity, Vaccine; Mice
PubMed: 38679392
DOI: 10.1016/j.virusres.2024.199381 -
Veterinary Microbiology Jun 2024The infection of canine coronavirus (CCoV) causes a highly contagious disease in dogs with acute gastroenteritis. The efficient serological diagnostics is critical for...
The infection of canine coronavirus (CCoV) causes a highly contagious disease in dogs with acute gastroenteritis. The efficient serological diagnostics is critical for controlling the disease caused by CCoV. Nucleocapsid (N) protein of CCoV is an important target for developing serological approaches. However, little is known about the antigenic sites in the N protein of CCoV. In this study, we generated a monoclonal antibody (mAb) against the N protein of CCoV, designated as 13E8, through the fusion of the sp2/0 cells with the spleen cells from a mouse immunized with the purified recombinant GST-N protein. Epitope mapping revealed that mAb 13E8 recognized a novel linear B cell epitope in N protein at 294-314aa (named as EP-13E8) by using a serial of truncated N protein through Western blot and ELISA. Sequence analysis showed that the sequence of EP-13E8 was highly conserved (100 %) among different CCoV strains analyzed, but exhibited a low similarity (31.8-63.6 %) with the responding sequence in other coronaviruses of the same genus such as FCoV, PEDV and HCoV except for TGEV (95.5 % identity). Structural assay suggested that the epitope of EP-13E8 were located in the close proximity on the surface of the N protein. Overall, the mAb 13E8 against N protein generated and its epitope EP-13E8 identified here paid the way for further developing epitope-based serological diagnostics for CCoV.
Topics: Animals; Antibodies, Monoclonal; Epitopes, B-Lymphocyte; Dogs; Mice; Nucleocapsid Proteins; Coronavirus, Canine; Epitope Mapping; Antibodies, Viral; Mice, Inbred BALB C; Coronavirus Nucleocapsid Proteins; Dog Diseases; Coronavirus Infections; Amino Acid Sequence
PubMed: 38677126
DOI: 10.1016/j.vetmic.2024.110098 -
Veterinary Microbiology Jun 2024Porcine epidemic diarrhea virus (PEDV) is a devastating pathogen of acute- gastrointestinal infectious diseases, which can cause vomiting, diarrhea, dehydration and high...
Porcine epidemic diarrhea virus (PEDV) is a devastating pathogen of acute- gastrointestinal infectious diseases, which can cause vomiting, diarrhea, dehydration and high morbidity and mortality among neonatal piglets. Humoral immunity plays a vital role in the host anti-PEDV infection process, but the mechanism of PEDV-induced B-cell immune response remains unknown. In this study, the effects of PEDV infection on CD21 B cell activation were systematically analyzed through animal experiments. Enzyme-linked immunosorbent assays (ELISA) revealed that low levels of serum-specific IgA, IgM, or IgG were detected in piglets after PEDV infection, respectively. Serum interleukin (IL)-6 levels increased significantly at 4 d after infection, and the levels of IL-4, B-cell activating factor (BAFF), interferon (IFN)-γ, transforming growth factor (TGF)-β and IL-10 decreased at 7 d after infection. Fluorescence-activated cell sorting (FACS) showed that expression levels of CD21, MHC Ⅱ, CD40, and CD38 on B cell surfaces were significantly higher. In contrast, the proportions of CD21IgM B cells were decreased in peripheral blood mononuclear cells (PBMCs) from the infected piglets. No differences were found in the percentage of CD21CD80 and CD21CD27 B cells in PBMCs from the infected piglets. In addition, the number of CD21B cells in PBMCs stimulated with PEDV in vitro was significantly lower. No significant change in the mRNA expression of BCR molecules was found while the expression levels of paired immunoglobulin-like receptor B (PIR-B), B cell adaptor molecule of 32 kDa (Bam32) and BAFF were decreased. In conclusion, our research demonstrates that virulent strains of PEDV profoundly impact B cell activation, leading to alterations in phenotypic expression and BCR signaling molecules. Furthermore, this dysregulation results in compromised specific antibody secretion and perturbed cytokine production, highlighting the intricate immunological dysfunctions induced by PEDV infection.
Topics: Animals; Porcine epidemic diarrhea virus; Swine; B-Lymphocytes; Coronavirus Infections; Receptors, Complement 3d; Swine Diseases; Lymphocyte Activation; Cytokines; Antibodies, Viral; Immunoglobulin A; Immunoglobulin M
PubMed: 38663176
DOI: 10.1016/j.vetmic.2024.110087 -
The Journal of General Virology Apr 2024Porcine epidemic diarrhea (PED) is a serious disease in piglets that leads to high mortality. An effective measure that provides higher IgA levels in the intestine and...
Porcine epidemic diarrhea (PED) is a serious disease in piglets that leads to high mortality. An effective measure that provides higher IgA levels in the intestine and milk is required to decrease losses. Porcine epidemic diarrhea virus (PEDV) was dissolved in calcium alginate (Alg) and combined with chitosan (CS) via electrostatic interactions between cationic chitosan and anionic alginate to create a porous gel (Alg-CS+PEDV). The gel was used to immunize mice orally or in combination with subcutaneous injections of inactivated PEDV vaccine. At 12 and 24 days after immunization, levels of IgA and IgG in Alg-CS+PEDV were higher than with normal PEDV oral administration. At 24 days after immunization, the concentration of IFN-γ in Alg-CS+PEDV was higher than with normal PEDV oral administration. Furthermore, oral administration combining subcutaneous immunization induced higher levels of IgG and IgA than oral administration alone. Our study provides a new method for the preparation and administration of oral vaccines to achieve enhanced mucosal immunity against PEDV.
Topics: Animals; Administration, Oral; Porcine epidemic diarrhea virus; Alginates; Immunity, Mucosal; Chitosan; Mice; Viral Vaccines; Antibodies, Viral; Immunoglobulin A; Immunoglobulin G; Swine; Coronavirus Infections; Vaccines, Inactivated; Swine Diseases; Female; Gels; Mice, Inbred BALB C; Interferon-gamma; Glucuronic Acid; Hexuronic Acids
PubMed: 38656455
DOI: 10.1099/jgv.0.001979 -
Daru : Journal of Faculty of Pharmacy,... Jun 2024Identifying the molecular mechanisms behind SARS-CoV-2 disparities and similarities will help find new treatments. The present study determines networks' shared and...
Deciphering the similarities and disparities of molecular mechanisms behind respiratory epithelium response to HCoV-229E and SARS-CoV-2 and drug repurposing, a systems biology approach.
PURPOSE
Identifying the molecular mechanisms behind SARS-CoV-2 disparities and similarities will help find new treatments. The present study determines networks' shared and non-shared (specific) crucial elements in response to HCoV-229E and SARS-CoV-2 viruses to recommend candidate medications.
METHODS
We retrieved the omics data on respiratory cells infected with HCoV-229E and SARS-CoV-2, constructed PPIN and GRN, and detected clusters and motifs. Using a drug-gene interaction network, we determined the similarities and disparities of mechanisms behind their host response and drug-repurposed.
RESULTS
CXCL1, KLHL21, SMAD3, HIF1A, and STAT1 were the shared DEGs between both viruses' protein-protein interaction network (PPIN) and gene regulatory network (GRN). The NPM1 was a specific critical node for HCoV-229E and was a Hub-Bottleneck shared between PPI and GRN in HCoV-229E. The HLA-F, ADCY5, TRIM14, RPF1, and FGA were the seed proteins in subnetworks of the SARS-CoV-2 PPI network, and HSPA1A and RPL26 proteins were the seed in subnetworks of the PPI network of HCOV-229E. TRIM14, STAT2, and HLA-F played the same role for SARS-CoV-2. Top enriched KEGG pathways included cell cycle and proteasome in HCoV-229E and RIG-I-like receptor, Chemokine, Cytokine-cytokine, NOD-like receptor, and TNF signaling pathways in SARS-CoV-2. We suggest some candidate medications for COVID-19 patient lungs, including Noscapine, Isoetharine mesylate, Cycloserine, Ethamsylate, Cetylpyridinium, Tretinoin, Ixazomib, Vorinostat, Venetoclax, Vorinostat, Ixazomib, Venetoclax, and epoetin alfa for further in-vitro and in-vivo investigations.
CONCLUSION
We suggested CXCL1, KLHL21, SMAD3, HIF1A, and STAT1, ADCY5, TRIM14, RPF1, and FGA, STAT2, and HLA-F as critical genes and Cetylpyridinium, Cycloserine, Noscapine, Ethamsylate, Epoetin alfa, Isoetharine mesylate, Ribavirin, and Tretinoin drugs to study further their importance in treating COVID-19 lung complications.
Topics: Humans; Drug Repositioning; SARS-CoV-2; Protein Interaction Maps; Systems Biology; Coronavirus 229E, Human; Antiviral Agents; COVID-19 Drug Treatment; Nucleophosmin; Respiratory Mucosa; Gene Regulatory Networks; COVID-19
PubMed: 38652363
DOI: 10.1007/s40199-024-00507-0 -
New Zealand Veterinary Journal Jul 2024To isolate canine respiratory coronavirus (CRCoV) and canine pneumovirus (CnPnV) in cell culture and to compare partial genomic sequences of CRCoV and CnPnV from New...
AIMS
To isolate canine respiratory coronavirus (CRCoV) and canine pneumovirus (CnPnV) in cell culture and to compare partial genomic sequences of CRCoV and CnPnV from New Zealand with those from other countries.
METHODS
Oropharyngeal swab samples from dogs affected by canine infectious respiratory disease syndrome that were positive for CnPnV (n = 15) or CRCoV (n = 1) by virus-specific reverse transcriptase quantitative PCR (RT-qPCR) in a previous study comprised the starting material. Virus isolation was performed in HRT-18 cells for CRCoV and RAW 264.7 and Vero cells for CnPnV. The entire sequence of CnPnV G protein (1,266 nucleotides) and most (8,063/9,707 nucleotides) of the 3' region of CRCoV that codes for 10 structural and accessory proteins were amplified and sequenced. The sequences were analysed and compared with other sequences available in GenBank using standard molecular tools including phylogenetic analysis.
RESULTS
Virus isolation was unsuccessful for both CRCoV and CnPnV. Pneumovirus G protein was amplified from 3/15 (20%) samples that were positive for CnPnV RNA by RT-qPCR. Two of these (NZ-048 and NZ-049) were 100% identical to each other, and 90.9% identical to the third one (NZ-007). Based on phylogenetic analysis of the G protein gene, CnPnV NZ-048 and NZ-049 clustered with sequences from the USA, Thailand and Italy in group A, and CnPnV NZ-007 clustered with sequences from the USA in group B. The characteristics of the predicted genes (length, position) and their putative protein products (size, predicted structure, presence of N- and O-glycosylation sites) of the New Zealand CRCoV sequence were consistent with those reported previously, except for the region located between open reading frame (ORF)3 (coding for S protein) and ORF6 (coding for E protein). The New Zealand virus was predicted to encode 5.9 kDa, 27 kDa and 12.7 kDa proteins, which differed from the putative coding capacity of this region reported for CRCoV from other countries.
CONCLUSIONS
This report represents the first characterisation of partial genomic sequences of CRCoV and CnPnV from New Zealand. Our results suggest that the population of CnPnV circulating in New Zealand is not homogeneous, and that the viruses from two clades described overseas are also present here. Limited conclusions can be made based on only one CRCoV sequence, but the putative differences in the coding capacity of New Zealand CRCoV support the previously reported variability of this region. The reasons for such variability and its biological implications need to be further elucidated.
Topics: Animals; Dogs; New Zealand; Coronavirus, Canine; Dog Diseases; Pneumovirus; Genome, Viral; Phylogeny; Coronavirus Infections; Vero Cells; Chlorocebus aethiops
PubMed: 38650102
DOI: 10.1080/00480169.2024.2339845 -
Veterinary Microbiology Jun 2024Porcine epidemic diarrhea virus (PEDV) envelope protein (E) has been characterized as an important structural protein that plays critical roles in the interplay with its...
Porcine epidemic diarrhea virus (PEDV) envelope protein (E) has been characterized as an important structural protein that plays critical roles in the interplay with its host to affect the virus life cycle. Stress granules (SGs) are host translationally silent ribonucleoproteins, which are mainly induced by the phosphorylation of eIF2α in the PERK/eIF2α signaling pathway. Our previous study found that PEDV E protein caused endoplasmic reticulum stress response (ERS)-mediated suppression of antiviral proteins' translation. However, the link and the underlying mechanism by which PEDV induces SGs formation and suppresses host translation remain elusive. In this study, our results showed that PEDV E protein significantly elevated the expression of GRP78, CANX, and phosphorylation of PERK and eIF2α, indicating that the PERK/eIF2α branch of ERS was activated. PEDV E protein localized to the ER and aggregated into puncta to reconstruct ER structure, and further induced SGs formation, which has been caused through upregulating the G3BP1 expression level. In addition, a significant global translational stall and endogenous protein translation attenuation were detected in the presence of E protein overexpression, but the global mRNA transcriptional level remained unchanged, suggesting that the shutoff of protein translation was associated with the translation, not with the transcription process. Collectively, this study demonstrates that PERK/eIF2α activation is required for SGs formation and protein translation stall. This study is beneficial for us to better understand the mechanism by which PEDV E suppresses host protein synthesis, and provides us a new insight into the host translation regulation during virus infection.
Topics: Porcine epidemic diarrhea virus; Animals; eIF-2 Kinase; Eukaryotic Initiation Factor-2; Signal Transduction; Swine; Vero Cells; Protein Biosynthesis; Stress Granules; Chlorocebus aethiops; Endoplasmic Reticulum Chaperone BiP; Phosphorylation; Endoplasmic Reticulum Stress
PubMed: 38643723
DOI: 10.1016/j.vetmic.2024.110095 -
Journal of Hazardous Materials Jun 2024The effective removal of viruses from swine wastewater using anaerobic membrane bioreactor (AnMBR) is vital to ecological safety. However, most studies have focused only...
The effective removal of viruses from swine wastewater using anaerobic membrane bioreactor (AnMBR) is vital to ecological safety. However, most studies have focused only on disinfectants, whereas the capabilities of the treatment process have not been investigated. In this study, the performance and mechanism of an AnMBR in the removal of porcine hepatitis E virus (HEV), porcine kobuvirus (PKoV), porcine epidemic diarrhea virus (PEDV), and transmissible gastroenteritis coronavirus (TGEV) are systematically investigated. The results show that the AnMBR effectively removes the four viruses, with average removal efficiencies of 1.62, 3.05, 2.41, and 1.34 log for HEV, PKoV, PEDV and TGEV, respectively. Biomass adsorption contributes primarily to the total virus removal in the initial stage of reactor operation, with contributions to HEV and PKoV removal exceeding 71.7 % and 68.2 %, respectively. When the membrane is fouled, membrane rejection dominated virus removal. The membrane rejection contribution test shows the significant contribution of membrane pore foulants (23-76 %). Correlation analysis shows that the surface characteristics and size differences of the four viruses contribute primarily to their different effects on biomass adsorption and membrane rejection. This study provides technical guidance for viral removal during the treatment of high-concentration swine wastewater using an AnMBR.
Topics: Animals; Bioreactors; Wastewater; Swine; Membranes, Artificial; Anaerobiosis; RNA Viruses; Water Purification; Adsorption; Biomass; Porcine epidemic diarrhea virus; Waste Disposal, Fluid
PubMed: 38643574
DOI: 10.1016/j.jhazmat.2024.134296 -
Veterinary Microbiology Jun 2024IgA plays a vital role in defending against the infectious pathogens. However, the specific regulatory pathways involved in IgA secretion in the context of PEDV...
IgA plays a vital role in defending against the infectious pathogens. However, the specific regulatory pathways involved in IgA secretion in the context of PEDV infection have remained elusive. Therefore, in this study, we explore the molecular mechanisms underlying IgA secretion in response to infection, with a particular focus on PEDV, a devastating enteric virus affecting global swine production. Our investigation begins by examining changes in IgA concentrations in both serum and small intestinal contents following PEDV infection in 2- and 4-week-old pigs. Remarkably, a significant increase in IgA levels in these older pigs post-infection were observed. To delve deeper into the regulatory mechanisms governing IgA secretion in response to PEDV infection, isolated porcine intestinal B cells were co-cultured with monocytes derived DCs (Mo-DCs) in vitro. In the intestinal DC-B cell co-cultures, IgA secretion was found to increase significantly after PEDV infection, as well as upregulating the expression of AID, GLTα and PSTα reflecting isotype switching to IgA. In addition, the expression of TLR9 was upregulated in these cultures, as determined by RT-qPCR and western blotting. Moreover, our findings extend to in vivo observations, where we detected higher levels of TLR9 expression in the ileum of pig post PEDV infection. Collectively, our results highlight the ability of PEDV to stimulate the generation of IgA, particularly in elder pigs, and identify TLR9 as a critical mediator of IgA production within the porcine intestinal microenvironment during PEDV infection.
Topics: Animals; Swine; Porcine epidemic diarrhea virus; Swine Diseases; Intestine, Small; Immunoglobulin A; Toll-Like Receptor 9; Coronavirus Infections; B-Lymphocytes; Coculture Techniques; Dendritic Cells
PubMed: 38636174
DOI: 10.1016/j.vetmic.2024.110096 -
Microbial Pathogenesis Jun 2024Porcine viral diarrhea is a common ailment in clinical settings, causing significant economic losses to the swine industry. Notable culprits behind porcine viral...
Porcine viral diarrhea is a common ailment in clinical settings, causing significant economic losses to the swine industry. Notable culprits behind porcine viral diarrhea encompass transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and porcine rotavirus-A (PoRVA). Co-infections involving the viruses are a common occurrence in clinical settings, thereby amplifying the complexities associated with differential diagnosis. As a consequence, it is therefore necessary to develop a method that can detect and differentiate all four porcine diarrhea viruses (TGEV, PEDV, PDCoV, and PoRVA) with a high sensitivity and specificity. Presently, polymerase chain reaction (PCR) is the go-to method for pathogen detection. In comparison to conventional PCR, TaqMan real-time PCR offers heightened sensitivity, superior specificity, and enhanced accuracy. This study aimed to develop a quadruplex real-time RT-qPCR assay, utilizing TaqMan probes, for the distinctive detection of TGEV, PEDV, PDCoV, and PoRVA. The quadruplex real-time RT-qPCR assay, as devised in this study, exhibited the capacity to avoid the detection of unrelated pathogens and demonstrated commendable specificity, sensitivity, repeatability, and reproducibility, boasting a limit of detection (LOD) of 27 copies/μL. In a comparative analysis involving 5483 clinical samples, the results from the commercial RT-qPCR kit and the quadruplex RT-qPCR for TGEV, PEDV, PDCoV, and PoRVA detection were entirely consistent. Following sample collection from October to March in Guangxi Zhuang Autonomous Region, we assessed the prevalence of TGEV, PEDV, PDCoV, and PoRVA in piglet diarrhea samples, revealing positive detection rates of 0.2 % (11/5483), 8.82 % (485/5483), 1.22 % (67/5483), and 4.94 % (271/5483), respectively. The co-infection rates of PEDV/PoRVA, PEDV/PDCoV, TGEV/PED/PoRVA, and PDCoV/PoRVA were 0.39 %, 0.11 %, 0.01 %, and 0.03 %, respectively, with no detection of other co-infections, as determined by the quadruplex real-time RT-qPCR. This research not only established a valuable tool for the simultaneous differentiation of TGEV, PEDV, PDCoV, and PoRVA in practical applications but also provided crucial insights into the prevalence of these viral pathogens causing diarrhea in Guangxi.
Topics: Animals; Swine; Real-Time Polymerase Chain Reaction; Transmissible gastroenteritis virus; Sensitivity and Specificity; Porcine epidemic diarrhea virus; Swine Diseases; Rotavirus; Gastroenteritis, Transmissible, of Swine; Deltacoronavirus; Diarrhea; Coronavirus; Feces; Coronavirus Infections
PubMed: 38631414
DOI: 10.1016/j.micpath.2024.106646