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Brazilian Journal of Microbiology :... Sep 2023Porcine epidemic diarrhea virus (PEDV) is a virus that can cause diarrhea in pigs, resulting in significant economic losses to the pig industry. The mutation of the...
Phylogenetic analysis and molecular characterization of the co-infection of the new variant of the porcine epidemic diarrhea virus and the novel porcine kobuvirus isolated from piglets with diarrhea.
Porcine epidemic diarrhea virus (PEDV) is a virus that can cause diarrhea in pigs, resulting in significant economic losses to the pig industry. The mutation of the virus and its co-infection with other enteroviruses leads to poor control of PEDV infection. In this study, we found that the diarrhea outbreak in a pig farm in Shandong Province was mainly caused by PEDV infection. Through high-throughput sequencing, we also detected one other diarrhea-related virus (porcine kobuvirus). In the phylogenetic analysis and molecular characterization of the detected PEDV S gene and PKV, it was found that the S gene of the PEDV strain detected in this study (named SD22-2) had more mutations than the CV777 strain. The highest homology between PKV (named SD/2022/China) detected in this study and other strains was only 89.66%. Based on polyprotein, we divided SD/2022/China strains into a new grouping (designated group 4) and detected recombination signals. In summary, SD22-2 detected in this study is a new PEDV variant strain, and SD/2022/China strain might be a novel PKV strain. We also found the co-infection of the new PEDV variant and the novel PKV isolated from piglets with diarrhea. Our data suggested the importance of continuous surveillance of PEDV and PKV.
Topics: Animals; Swine; Phylogeny; Porcine epidemic diarrhea virus; Kobuvirus; Swine Diseases; Coinfection; Coronavirus Infections; Diarrhea; China
PubMed: 37344656
DOI: 10.1007/s42770-023-01025-y -
Viruses Dec 2023Porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) are the two most prevalent swine enteric coronaviruses worldwide. They commonly cause natural...
Porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) are the two most prevalent swine enteric coronaviruses worldwide. They commonly cause natural coinfections, which worsen as the disease progresses and cause increased mortality in piglets. To better understand the transcriptomic changes after PEDV and PDCoV coinfection, we compared LLC porcine kidney (LLC-PK) cells infected with PEDV and/or PDCoV and evaluated the differential expression of genes by transcriptomic analysis and real-time qPCR. The antiviral efficacy of interferon-stimulated gene 20 (ISG20) against PDCoV and PEDV infections was also assessed. Differentially expressed genes (DEGs) were detected in PEDV-, PDCoV-, and PEDV + PDCoV-infected cells at 6, 12, and 24 h post-infection (hpi), and at 24 hpi, the number of DEGs was the highest. Furthermore, changes in the expression of interferons, which are mainly related to apoptosis and activation of the host innate immune pathway, were found in the PEDV and PDCoV infection and coinfection groups. Additionally, 43 ISGs, including GBP2, IRF1, ISG20, and IFIT2, were upregulated during PEDV or PDCoV infection. Furthermore, we found that ISG20 significantly inhibited PEDV and PDCoV infection in LLC-PK cells. The transcriptomic profiles of cells coinfected with PEDV and PDCoV were reported, providing reference data for understanding the host response to PEDV and PDCoV coinfection.
Topics: Animals; Swine; Porcine epidemic diarrhea virus; Coinfection; Deltacoronavirus; Gene Expression Profiling; Interferons
PubMed: 38257774
DOI: 10.3390/v16010074 -
BMC Veterinary Research Sep 2023Porcine epidemic diarrhea virus (PEDV) and porcine delta-coronavirus (PDCoV) are economically important pathogens that cause diarrhea in sows and acute death of newborn...
BACKGROUND
Porcine epidemic diarrhea virus (PEDV) and porcine delta-coronavirus (PDCoV) are economically important pathogens that cause diarrhea in sows and acute death of newborn piglets. Moreover, the emerging PDCoV was reported to infect children. The current situation is that vaccine prevention has not met expectations, and emergency containment strategies following outbreaks cannot prevent the damages and losses already incurred. Therefore, a more sensitive detection method, that is both convenient and enables accurate and effective sequencing, that will provide early warning of PEDV and PDCoV is necessary. This will enable active, effective, and comprehensive prevention and control, which will possibly reduce disease occurrences.
RESULTS
Duplex nested RT-PCR (dnRT-PCR) is an ideal method to achieve early warning and monitoring of PEDV and PDCoV diseases, and to additionally investigate any molecular epidemiological characteristics. In this study, two pairs of primers were designed for each virus based upon the highly conserved N protein sequences of both PEDV and PDCoV strains retrieved from the NCBI Genbank. After optimization of the reaction conditions, the dnRT-PCR assay amplified a 749-bp fragment specific to PEDV and a 344-bp fragment specific to PDCoV. Meanwhile, the specificity and sensitivity of the primers and clinical samples were tested to verify and establish this dnRT-PCR method. The limit of detection (LoD)for both PEDV and PDCoV was 10 copies/µL. The results showed that among 251 samples, 1 sample contained PEDV infection, 19 samples contained a PDCoV infection, and 8 samples were infected with both viruses, following the use of dnRT-PCR. Subsequently, the positive samples were sent for sequencing, and the sequencing results confirmed that they were all positive for the viruses detected using dnRT-PCR, and conventional RT-PCR detection was conducted again after the onset of disease. As these results were consistent with previous results, a detection method for PEDV and PDCoV using dnRT-PCR was successfully established. In conclusion, the dnRT-PCR method established in this study was able to detect both PEDV and PDCoV, concomitantly.
CONCLUSIONS
The duplex nested RT-PCR method represents a convenient, reliable, specific, sensitive and anti-interference technique for detecting PEDV and PDCoV, and can additionally be used to simultaneously determine the molecular epidemiological background.
Topics: Animals; Swine; Female; Coronavirus; Porcine epidemic diarrhea virus; Reverse Transcriptase Polymerase Chain Reaction; Coronavirus Infections; Polymerase Chain Reaction; DNA Primers
PubMed: 37684673
DOI: 10.1186/s12917-023-03708-y -
Pharmaceutics Dec 2023This study aimed to develop a holobiont tablet with rapid dispersibility to provide regulation of the microbiota, virucidal activity, and skin barrier protection.
OBJECTIVE
This study aimed to develop a holobiont tablet with rapid dispersibility to provide regulation of the microbiota, virucidal activity, and skin barrier protection.
METHODS
A 2 factorial experiment was planned to define the best formulation for the development of the base tablet, using average weight, hardness, dimensions, swelling rate, and disintegration time as parameters to be analyzed. To produce holobiont tablets, the chosen base formulation was fabricated by direct compression of prebiotics, postbiotics, and excipients. The tablets also incorporated solid lipid nanoparticles containing postbiotics that were obtained by high-pressure homogenization and freeze-drying. The in vitro virucidal activity against alpha-coronavirus particles (CCoV-VR809) was determined in VERO cell culture. In vitro analysis, using monolayer cells and human equivalent skin, was performed by rRTq-PCR to determine the expression of interleukins 1, 6, 8, and 17, aquaporin-3, involucrin, filaggrin, FoxO3, and SIRT-1. Antioxidant activity and collagen-1 synthesis were also performed in fibroblast cells. Metagenomic analysis of the skin microbiome was determined in vivo before and after application of the holobiont tablet, during one week of continuous use, and compared to the use of alcohol gel. Samples were analyzed by sequencing the V3-V4 region of the 16S rRNA gene.
RESULTS
A handrub tablet with rapid dispersibility was developed for topical use and rinse off. After being defined as safe, the virucidal activity was found to be equal to or greater than that of 70% alcohol, with a reduction in interleukins and maintenance or improvement of skin barrier gene markers, in addition to the reestablishment of the skin microbiota after use.
CONCLUSIONS
The holobiont tablets were able to improve the genetic markers related to the skin barrier and also its microbiota, thereby being more favorable for use as a hand sanitizer than 70% alcohol.
PubMed: 38140133
DOI: 10.3390/pharmaceutics15122793 -
Frontiers in Immunology 2024CpG oligodeoxynucleotides (CpG ODNs) boost the humoral and cellular immune responses to antigens through interaction with Toll-like receptor 9 (TLR9). These CpG ODNs...
CpG oligodeoxynucleotides (CpG ODNs) boost the humoral and cellular immune responses to antigens through interaction with Toll-like receptor 9 (TLR9). These CpG ODNs have been extensively utilized in human vaccines. In our study, we evaluated five B-type CpG ODNs that have stimulatory effects on pigs by measuring the proliferation of porcine peripheral blood mononuclear cells (PBMCs) and assessing interferon gamma (IFN-γ) secretion. Furthermore, this study examined the immunoenhancing effects of the MF59 and CpG ODNs compound adjuvant in mouse and piglet models of porcine epidemic diarrhea virus (PEDV) subunit vaccine administration. The screening revealed that the CpG ODN named CpG5 significantly stimulated the proliferation of porcine PBMCs and elevated IFN-γ secretion levels. In the mouse vaccination model, CpG5 compound adjuvant significantly bolstered the humoral and cellular immune responses to the PEDV subunit vaccines, leading to Th1 immune responses characterized by increased IFN-γ and IgG2a levels. In piglets, the neutralizing antibody titer was significantly enhanced with CpG5 compound adjuvant, alongside a considerable increase in CD8+ T lymphocytes proportion. The combination of MF59 adjuvant and CpG5 exhibits a synergistic effect, resulting in an earlier, more intense, and long-lasting immune response in subunit vaccines for PEDV. This combination holds significant promise as a robust candidate for the development of vaccine adjuvant.
Topics: Animals; Swine; Mice; Humans; Porcine epidemic diarrhea virus; Leukocytes, Mononuclear; Adjuvants, Immunologic; Immunity; Vaccines, Subunit; Adjuvants, Pharmaceutic; Oligodeoxyribonucleotides; Polysorbates; Squalene
PubMed: 38322258
DOI: 10.3389/fimmu.2024.1336239 -
Virology Journal Jul 2023Porcine epidemic diarrhea virus (PEDV) is an α-coronavirus that causes highly contagious intestinal infectious disease, involving clinically characterized by diarrhea,...
BACKGROUND
Porcine epidemic diarrhea virus (PEDV) is an α-coronavirus that causes highly contagious intestinal infectious disease, involving clinically characterized by diarrhea, dehydration, vomiting, and high mortality to suckling piglets. As a strategy for antiviral therapy, artificial microRNA (amiRNA) mediated suppression of viral replication has recently become increasingly important. In this study, we evaluated the advantages of using an amiRNA vector against PEDV.
METHODS
In this study, we evaluated the advantages of using an amiRNA vector against PEDV. We designed two single amiRNA sequences for different conserved sequences of the PEDV S and N genes, and tested their inhibitory effects on PEDV in Vero cells.
RESULTS
It was obvious from the CCK-8 results that the transient transfection of amiRNA was non-toxic to the cells. In addition, our results showed that the transient expression of two amiRNAs (amiRNA-349 and amiRNA-1447) significantly reduced the expression of viral RNA and protein in the cells. The TCID results showed that the release of virus particles into the culture supernatant was significantly reduced, with an effect as high as 90%. To avoid virus mutation escape, the above two single amiRNA sequences were tandem in this study (amiRNA-349 + 1447), enabling a single microRNA to be expressed simultaneously. The real-time PCR and Western blot results showed that the inhibitory effect was significantly enhanced in each of the different time periods. The TCID results showed that the release of virus particles in the culture supernatant was significantly reduced at the different time periods.
CONCLUSIONS
In summary, these results suggest that an RNAi based on amiRNA targeting the conserved region of the virus is an effective method to improve PEDV nucleic acid inhibitors and provide a novel treatment strategy for PEDV infection.
Topics: Animals; Swine; Chlorocebus aethiops; MicroRNAs; Porcine epidemic diarrhea virus; Vero Cells; RNA Interference; Coronavirus
PubMed: 37488599
DOI: 10.1186/s12985-023-02129-5 -
BioRxiv : the Preprint Server For... Sep 2023Phosphodiesterases (PDEs) encoded by viruses are putatively acquired by horizontal transfer of cellular PDE ancestor genes. Viral PDEs inhibit the OAS-RNase L antiviral...
Phosphodiesterases (PDEs) encoded by viruses are putatively acquired by horizontal transfer of cellular PDE ancestor genes. Viral PDEs inhibit the OAS-RNase L antiviral pathway, a key effector component of the innate immune response. Although the function of these proteins is well-characterized, the origins of these gene acquisitions is less clear. Phylogenetic analysis revealed at least five independent PDE acquisition events by ancestral viruses. We found evidence that PDE-encoding genes were horizontally transferred between coronavirus genera. Three clades of viruses within : merbecoviruses (MERS-CoV), embecoviruses (OC43), and toroviruses encode independently acquired PDEs, and a clade of rodent alphacoronaviruses acquired an embecovirus PDE via recent horizontal transfer. Among rotaviruses, the PDE of Rotavirus A was acquired independently from Rotavirus B and G PDEs, which share a common ancestor. Conserved motif analysis suggests a link between all viral PDEs and a similar ancestor among the mammalian AKAP7 proteins despite low levels of sequence conservation. Additionally, we used ancestral sequence reconstruction and structural modeling to reveal that sequence and structural divergence are not well-correlated among these proteins. Specifically, merbecovirus PDEs are as structurally divergent from the ancestral protein and the solved structure of human AKAP7 PDE as they are from each other. In contrast, comparisons of Rotavirus B and G PDEs reveal virtually unchanged structures despite evidence for loss of function in one, suggesting impactful changes that lie outside conserved catalytic sites. These findings highlight the complex and volatile evolutionary history of viral PDEs and provide a framework to facilitate future studies.
PubMed: 37745432
DOI: 10.1101/2023.05.12.540623 -
Viruses Mar 2024Feline infectious peritonitis (FIP) is a multisystemic, generally lethal immuno-inflammatory disease of domestic cats caused by an infection with a genetic variant of...
Feline infectious peritonitis (FIP) is a multisystemic, generally lethal immuno-inflammatory disease of domestic cats caused by an infection with a genetic variant of feline coronavirus, referred to as the FIP virus (FIPV). We leveraged data from four different antiviral clinical trials performed at the University of California, Davis. Collectively, a total of 60 client-owned domestic cats, each with a confirmed diagnosis of naturally occurring FIP, were treated with a variety of antiviral compounds. The tested therapies included the antiviral compounds GS-441524, remdesivir, molnupiravir and allogeneic feline mesenchymal stem/stroma cell transfusions. Four client-owned cats with FIP did not meet the inclusion criteria for the trials and were not treated with antiviral therapies; these cats were included in the data set as untreated FIP control cats. ELISA and Western blot assays were performed using feline serum/plasma or ascites effusions obtained from a subset of the FIP cats. Normalized tissue/effusion viral loads were determined in 34 cats by a quantitative RT-PCR of nucleic acids isolated from either effusions or abdominal lymph node tissue. Twenty-one cats were PCR "serotyped" (genotyped) and had the S1/S2 region of the coronaviral gene amplified, cloned and sequenced from effusions or abdominal lymph node tissue. In total, 3 untreated control cats and 14 (23.3%) of the 60 antiviral-treated cats died or were euthanized during (13) or after the completion of (1) antiviral treatment. Of these 17 cats, 13 had complete necropsies performed (10 cats treated with antivirals and 3 untreated control cats). We found that anticoronaviral serologic responses were persistent and robust throughout the treatment period, primarily the IgG isotype, and focused on the viral structural Nucleocapsid and Membrane proteins. Coronavirus serologic patterns were similar for the effusions and serum/plasma of cats with FIP and in cats entering remission or that died. Viral RNA was readily detectable in the majority of the cats in either abdominal lymph node tissue or ascites effusions, and all of the viral isolates were determined to be serotype I FIPV. Viral nucleic acids in cats treated with antiviral compounds became undetectable in ascites or abdominal lymph node tissue by 11 days post-treatment using a sensitive quantitative RT-PCR assay. The most common pathologic lesions identified in the necropsied cats were hepatitis, abdominal effusion (ascites), serositis, pancreatitis, lymphadenitis, icterus and perivasculitis. In cats treated with antiviral compounds, gross and histological lesions characteristic of FIP persisted for several weeks, while the viral antigen became progressively less detectable.
Topics: Humans; Cats; Animals; Feline Infectious Peritonitis; Ascites; Coronavirus Infections; Coronavirus, Feline; RNA, Viral; Antiviral Agents
PubMed: 38543827
DOI: 10.3390/v16030462 -
Virus Research Jan 2024Porcine epidemic diarrhea (PED) is a contagious intestinal disease caused by α-coronavirus porcine epidemic diarrhea virus (PEDV). At present, no effective vaccine is...
Porcine epidemic diarrhea (PED) is a contagious intestinal disease caused by α-coronavirus porcine epidemic diarrhea virus (PEDV). At present, no effective vaccine is available to prevent the disease. Therefore, research for novel antivirals is important. This study aimed to identify the antiviral mechanism of Veratramine (VAM), which actively inhibits PEDV replication with a 50 % inhibitory concentration (IC) of ∼5 µM. Upon VAM treatment, both PEDV-nucleocapsid (N) protein level and virus titer decreased significantly. The time-of-addition assay results showed that VAM could inhibit PEDV replication by blocking viral entry. Importantly, VAM could inhibit PEDV-induced phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) activity and further suppress micropinocytosis, which is required for PEDV entry. In addition, PI3K inhibitor LY294002 showed anti-PEDV activity by blocking viral entry as well. Taken together, VAM possessed anti-PEDV properties against the entry stage of PEDV by inhibiting the macropinocytosis pathway by suppressing the PI3K/Akt pathway. VAM could be considered as a lead compound for the development of anti-PEDV drugs and may be used during the viral entry stage of PEDV infection.
Topics: Animals; Chlorocebus aethiops; Coronavirus Infections; Phosphatidylinositol 3-Kinases; Porcine epidemic diarrhea virus; Proto-Oncogene Proteins c-akt; Swine; Swine Diseases; Veratrum Alkaloids; Vero Cells; Virus Internalization
PubMed: 37923169
DOI: 10.1016/j.virusres.2023.199260 -
Pathogens (Basel, Switzerland) Aug 2023It is important to be able to detect and differentiate between distinct porcine enteric coronaviruses that can cause similar diseases. However, the existence of...
It is important to be able to detect and differentiate between distinct porcine enteric coronaviruses that can cause similar diseases. However, the existence of naturally occurring recombinant coronaviruses such as swine enteric coronavirus (SeCoV) can give misleading results with currently used diagnostic methods. Therefore, we have developed and validated three duplex real-time quantitative RT-PCR assays for the simultaneous detection of, and differentiation between, porcine epidemic diarrhea virus (PEDV) and SeCoV. Transmissible gastroenteritis virus (TGEV) is also detected by two out of these three assays. In addition, a novel triplex assay was set up that was able to detect and differentiate between these alphacoronaviruses and the porcine deltacoronavirus (PDCoV). The validated assays have low limits of detection, close to 100% efficiency, and were able to correctly identify the presence of PEDV and SeCoV in 55 field samples, whereas 20 samples of other pathogens did not give a positive result. Implementing one or more of these multiplex assays into the routine diagnostic surveillance for PEDV will ensure that the presence of SeCoV, TGEV, and PDCoV will not go unnoticed.
PubMed: 37624000
DOI: 10.3390/pathogens12081040