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1.
Viruses ; 15(5)2023 04 25.
Article in English | MEDLINE | ID: covidwho-20233862

ABSTRACT

Porcine deltacoronavirus (PDCoV) is an emergent swine coronavirus which infects cells from the small intestine and induces watery diarrhea, vomiting and dehydration, causing mortality in piglets (>40%). The aim of this study was to evaluate the antigenicity and immunogenicity of the recombinant membrane protein (M) of PDCoV (rM-PDCoV), which was developed from a synthetic gene obtained after an in silico analysis with a group of 138 GenBank sequences. A 3D model and phylogenetic analysis confirmed the highly conserved M protein structure. Therefore, the synthetic gene was successfully cloned in a pETSUMO vector and transformed in E. coli BL21 (DE3). The rM-PDCoV was confirmed by SDS-PAGE and Western blot with ~37.7 kDa. The rM-PDCoV immunogenicity was evaluated in immunized (BLAB/c) mice and iELISA. The data showed increased antibodies from 7 days until 28 days (p < 0.001). The rM-PDCoV antigenicity was analyzed using pig sera samples from three states located in "El Bajío" Mexico and positive sera were determined. Our results show that PDCoV has continued circulating on pig farms in Mexico since the first report in 2019; therefore, the impact of PDCoV on the swine industry could be higher than reported in other studies.


Subject(s)
Coronavirus Infections , Swine Diseases , Swine , Animals , Mice , Membrane Proteins , Phylogeny , Genes, Synthetic , Escherichia coli
2.
J Virol Methods ; 318: 114755, 2023 Aug.
Article in English | MEDLINE | ID: covidwho-20240515

ABSTRACT

Porcine epidemic diarrhea virus (PEDV) is a highly contagious intestinal virus. However, the current PEDV vaccine, which is produced from classical strain G1, offers low protection against recently emerged strain G2. This study aims to develop a better vaccine strain by propagating the PS6 strain, a G2b subgroup originating from Vietnam, on Vero cells until the 100th passage. As the virus was propagated, its titer increased, and its harvest time decreased. Analysis of the nucleotide and amino acid variation of the PS6 strain showed that the P100PS6 had 11, 4, and 2 amino acid variations in the 0 domain, B domain, and ORF3 protein, respectively, compared to the P7PS6 strain. Notably, the ORF3 gene was truncated due to a 16-nucleotide deletion mutation, resulting in a stop codon. The PS6 strain's virulence was evaluated in 5-day-old piglets, with P7PS6 and P100PS6 chosen for comparison. The results showed that P100PS6-inoculated piglets exhibited mild clinical symptoms and histopathological lesions, with a 100% survival rate. In contrast, P7PS6-inoculated piglets showed rapid and typical clinical symptoms of PEDV infection, and the survival rate was 0%. Additionally, the antibodies (IgG and IgA) produced from inoculated piglets with P100PS6 bound to both the P7PS6 and P100PS6 antigens. This finding suggested that the P100PS6 strain was attenuated and could be used to develop a live-attenuated vaccine against highly pathogenic and prevalent G2b-PEDV strains.


Subject(s)
Coronavirus Infections , Porcine epidemic diarrhea virus , Swine Diseases , Chlorocebus aethiops , Swine , Animals , Vero Cells , Porcine epidemic diarrhea virus/genetics , Virulence , Serial Passage , Vaccines, Attenuated/genetics , Coronavirus Infections/epidemiology , Diarrhea/veterinary
3.
Viruses ; 15(5)2023 04 29.
Article in English | MEDLINE | ID: covidwho-20238821

ABSTRACT

Porcine deltacoronavirus (PDCoV) causes diarrhea and vomiting in neonatal piglets worldwide and has the potential for cross-species transmission. Therefore, virus-like particles (VLPs) are promising vaccine candidates because of their safety and strong immunogenicity. To the best of our knowledge, the present study reported for the first time the generation of PDCoV VLPs using a baculovirus expression vector system, and electron micrograph analyses revealed that PDCoV VLPs appeared as spherical particles with a diameter similar to that of the native virions. Furthermore, PDCoV VLPs effectively induced mice to produce PDCoV-specific IgG and neutralizing antibodies. In addition, VLPs could stimulate mouse splenocytes to produce high levels of cytokines IL-4 and IFN-γ. Moreover, the combination of PDCoV VLPs and Freund's adjuvant could improve the level of the immune response. Together, these data showed that PDCoV VLPs could effectively elicit humoral and cellular immunity in mice, laying a solid foundation for developing VLP-based vaccines to prevent PDCoV infections.


Subject(s)
Coronavirus Infections , Coronavirus , Swine Diseases , Animals , Mice , Swine , Baculoviridae/genetics , Antibodies, Neutralizing , Coronavirus/genetics , Immunity , Coronavirus Infections/prevention & control , Coronavirus Infections/veterinary
4.
PLoS One ; 18(5): e0282767, 2023.
Article in English | MEDLINE | ID: covidwho-20238544

ABSTRACT

The global distribution and constant evolution are challenges for the control of porcine reproductive and respiratory syndrome virus (PRRSV), one of the most important viruses affecting swine worldwide. Effective control of PRRSV benefits from genotyping, which currently relies on Sanger sequencing. Here we developed and optimized procedures for real-time genotyping and whole genome sequencing of PRRSV directly from clinical samples based on targeted amplicon- and long amplicon tiling sequencing using the MinION Oxford Nanopore platform. Procedures were developed and tested on 154 clinical samples (including lung, serum, oral fluid and processing fluid) with RT-PCR Ct values ranging from 15 to 35. The targeted amplicon sequencing (TAS) approach was developed to obtain sequences of the complete ORF5 (main target gene for PRRSV genotyping) and partial ORF4 and ORF6 sequences of both PRRSV-1 and PRRSV-2 species. After only 5 min of sequencing, PRRSV consensus sequences with identities to reference sequences above 99% were obtained, enabling rapid identification and genotyping of clinical PRRSV samples into lineages 1, 5 and 8. The long amplicon tiling sequencing (LATS) approach targets type 2 PRRSV, the most prevalent viral species in the U.S. and China. Complete PRRSV genomes were obtained within the first hour of sequencing for samples with Ct values below 24.9. Ninety-two whole genome sequences were obtained using the LATS procedure. Fifty out of 60 sera (83.3%) and 18 out of 20 lung samples (90%) had at least 80% of genome covered at a minimum of 20X sequence depth per position. The procedures developed and optimized in this study here are valuable tools with potential for field application during PRRSV elimination programs.


Subject(s)
Nanopore Sequencing , Porcine respiratory and reproductive syndrome virus , Animals , Swine , Genotype , Chemoradiotherapy , China
5.
Arch Virol ; 168(6): 166, 2023 May 22.
Article in English | MEDLINE | ID: covidwho-20238472

ABSTRACT

Clostridium perfringens is a constituent of the normal gut microbiome in pigs; however, it can potentially cause pre- and post-weaning diarrhea. Nevertheless, the importance of this bacterium as a primary pathogen of diarrhea in piglets needs to be better understood, and the epidemiology of C. perfringens in Korean pig populations is unknown. To study the prevalence and typing of C. perfringens, 203 fecal samples were collected from diarrheal piglets on 61 swine farms during 2021-2022 and examined for the presence of C. perfringens and enteric viruses, including porcine epidemic diarrhea virus (PEDV). We determined that the most frequently identified type of C. perfringens was C. perfringens type A (CPA; 64/203, 31.5%). Among the CPA infections, single infections with CPA (30/64, 46.9%) and coinfections with CPA and PEDV (29/64, 45.3%) were the most common in diarrheal samples. Furthermore, we conducted animal experiments to investigate the clinical outcome of single infections and coinfections with highly pathogenic (HP)-PEDV and CPA in weaned piglets. The pigs infected with HP-PEDV or CPA alone showed mild or no diarrhea, and none of them died. However, animals that were co-inoculated with HP-PEDV and CPA showed more-severe diarrheal signs than those of the singly infected pigs. Additionally, CPA promoted PEDV replication in coinfected piglets, with high viral titers in the feces. A histopathological examination revealed more-severe villous atrophy in the small intestine of coinfected pigs than in singly infected pigs. This indicates a synergistic effect of PEDV and CPA coinfection on clinical disease in weaned piglets.


Subject(s)
Coinfection , Coronavirus Infections , Porcine epidemic diarrhea virus , Swine Diseases , Swine , Animals , Clostridium perfringens , Coinfection/epidemiology , Coinfection/veterinary , Weaning , Coronavirus Infections/epidemiology , Coronavirus Infections/veterinary , Coronavirus Infections/pathology , Diarrhea/epidemiology , Diarrhea/veterinary , Diarrhea/pathology , Swine Diseases/epidemiology , Patient Acuity
6.
Xenotransplantation ; 30(3): e12805, 2023.
Article in English | MEDLINE | ID: covidwho-20237587

ABSTRACT

After a significant hiatus imposed by the COVID-19 pandemic, we hereby restart Xenotransplantation literature updates. With the recently performed clinical xenotransplantation cases and the much-heightened interest in the field, we have determined that this is an optimal time to reinstate this section. There has been an invigorated focus on unique challenges posed by pig-to-human xenotransplantation, and specific attention will be given to this aspect. In this issue, we aimed to cover the gap and compiled the most relevant publications from March 2021 to March 2023.


Subject(s)
COVID-19 , Pandemics , Humans , Animals , Swine , Transplantation, Heterologous/methods
7.
Front Immunol ; 14: 1196031, 2023.
Article in English | MEDLINE | ID: covidwho-20236991

ABSTRACT

Swine acute diarrhoea syndrome coronavirus (SADS-CoV), which is a recently discovered enteric coronavirus, is the major aetiological agent that causes severe clinical diarrhoea and intestinal pathological damage in pigs, and it has caused significant economic losses to the swine industry. Nonstructural protein 5, also called 3C-like protease, cleaves viral polypeptides and host immune-related molecules to facilitate viral replication and immune evasion. Here, we demonstrated that SADS-CoV nsp5 significantly inhibits the Sendai virus (SEV)-induced production of IFN-ß and inflammatory cytokines. SADS-CoV nsp5 targets and cleaves mRNA-decapping enzyme 1a (DCP1A) via its protease activity to inhibit the IRF3 and NF-κB signaling pathways in order to decrease IFN-ß and inflammatory cytokine production. We found that the histidine 41 and cystine 144 residues of SADS-CoV nsp5 are critical for its cleavage activity. Additionally, a form of DCP1A with a mutation in the glutamine 343 residue is resistant to nsp5-mediated cleavage and has a stronger ability to inhibit SADS-CoV infection than wild-type DCP1A. In conclusion, our findings reveal that SADS-CoV nsp5 is an important interferon antagonist and enhance the understanding of immune evasion by alpha coronaviruses.


Subject(s)
Alphacoronavirus , Coronavirus , Interferon Type I , Animals , Swine , Alphacoronavirus/genetics , Alphacoronavirus/metabolism , Coronavirus/metabolism , Endopeptidases , Interferon Type I/metabolism
8.
J Virol ; 97(6): e0058923, 2023 Jun 29.
Article in English | MEDLINE | ID: covidwho-20236657

ABSTRACT

The inflammasome pathway is a critical early response mechanism of the host that detects pathogens, initiates the production of inflammatory cytokines, and recruits effector cells to the infection site. Nonetheless, the mechanism of inflammasome activation in coronavirus infection and its biological functions in host defense remain unclear. Transmissible gastroenteritis virus (TGEV), a member of the genus Alphacoronavirus, is a significant pathogen that mainly infects piglets and causes intestinal inflammation and inflammatory cell infiltration. Here, we investigated the mechanism of inflammasome activation in intestinal epithelial cells (IECs) infected with TGEV. We observed a substantial increase in interleukin 1ß (IL-1ß) and IL-18 levels in both IECs and TGEV-infected porcine intestinal tissues. Furthermore, TGEV infection resulted in increased activation of caspase-1 and the NLRP1 (NOD-like receptor [NLR]-containing pyrin domain [PYD]) inflammasome. Our findings revealed that TGEV infection impeded the interaction between porcine NLRP1 (pNLRP1) and porcine dipeptidyl peptidases 9 (pDPP9), yet it did not reduce the expression of pDPP9. Importantly, the ZU5 domain, not the function-to-find domain (FIIND) reported in human NLRP1, was identified as the minimal domain of pNLRP1 for pDPP9 binding. In addition, the robust type I IFN expression induced by TGEV infection also upregulated pNLRP1 expression and pNLRP1 itself acts as an interferon-stimulated gene to counteract TGEV infection. Our data demonstrate that pNLRP1 has antiviral capabilities against coronavirus infection, which highlights its potential as a novel therapeutic target for coronavirus antiviral therapy. IMPORTANCE Coronavirus primarily targets the epithelial cells of the respiratory and gastrointestinal tracts, leading to damage in both humans and animals. NLRP1 is a direct sensor for RNA virus infection which is highly expressed in epithelial barrier tissues. However, until recently, the precise molecular mechanisms underlying its activation in coronavirus infection and subsequent downstream events remained unclear. In this study, we demonstrate that the alphacoronavirus TGEV induces the production of IL-1ß and IL-18 and upregulates the expression of pNLRP1. Furthermore, we found that pNLRP1 can serve as an interferon-stimulated gene (ISG) to inhibit the infection of enterovirus TGEV. Our research highlights the crucial role of NLRP1 as a regulator of innate immunity in TGEV infection and shows that it may serve as a potential therapeutic target for the treatment of coronavirus infection.


Subject(s)
Gastroenteritis, Transmissible, of Swine , Inflammasomes , NLR Proteins , Transmissible gastroenteritis virus , Animals , Inflammasomes/immunology , Interferon Type I , Interleukin-18 , NLR Proteins/immunology , Swine , Gastroenteritis, Transmissible, of Swine/immunology , Gastroenteritis, Transmissible, of Swine/transmission
9.
Viruses ; 15(5)2023 05 17.
Article in English | MEDLINE | ID: covidwho-20236544

ABSTRACT

Since SARS-CoV-2 caused the COVID-19 pandemic, records have suggested the occurrence of reverse zoonosis of pets and farm animals in contact with SARS-CoV-2-positive humans in the Occident. However, there is little information on the spread of the virus among animals in contact with humans in Africa. Therefore, this study aimed to investigate the occurrence of SARS-CoV-2 in various animals in Nigeria. Overall, 791 animals from Ebonyi, Ogun, Ondo, and Oyo States, Nigeria were screened for SARS-CoV-2 using RT-qPCR (n = 364) and IgG ELISA (n = 654). SARS-CoV-2 positivity rates were 45.9% (RT-qPCR) and 1.4% (ELISA). SARS-CoV-2 RNA was detected in almost all animal taxa and sampling locations except Oyo State. SARS-CoV-2 IgGs were detected only in goats from Ebonyi and pigs from Ogun States. Overall, SARS-CoV-2 infectivity rates were higher in 2021 than in 2022. Our study highlights the ability of the virus to infect various animals. It presents the first report of natural SARS-CoV-2 infection in poultry, pigs, domestic ruminants, and lizards. The close human-animal interactions in these settings suggest ongoing reverse zoonosis, highlighting the role of behavioral factors of transmission and the potential for SARS-CoV-2 to spread among animals. These underscore the importance of continuous monitoring to detect and intervene in any eventual upsurge.


Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Humans , Swine , SARS-CoV-2/genetics , Nigeria/epidemiology , COVID-19/epidemiology , COVID-19/veterinary , Pandemics , RNA, Viral/genetics , Zoonoses/epidemiology , Animals, Domestic , Goats
10.
J Virol ; 97(6): e0054923, 2023 Jun 29.
Article in English | MEDLINE | ID: covidwho-20245375

ABSTRACT

Porcine epidemic diarrhea virus (PEDV) has caused huge economic losses to the global pig industry. The swine enteric coronavirus spike (S) protein recognizes various cell surface molecules to regulate viral infection. In this study, we identified 211 host membrane proteins related to the S1 protein by pulldown combined with liquid-chromatography tandem mass spectrometry (LC-MS/MS) analysis. Among these, heat shock protein family A member 5 (HSPA5) was identified through screening as having a specific interaction with the PEDV S protein, and positive regulation of PEDV infection was validated by knockdown and overexpression tests. Further studies verified the role of HSPA5 in viral attachment and internalization. In addition, we found that HSPA5 interacts with S proteins through its nucleotide-binding structural domain (NBD) and that polyclonal antibodies can block viral infection. In detail, HSPA5 was found to be involved in viral trafficking via the endo-/lysosomal pathway. Inhibition of HSPA5 activity during internalization would reduce the subcellular colocalization of PEDV with lysosomes in the endo-/lysosomal pathway. Together, these findings show that HSPA5 is a novel PEDV potential target for the creation of therapeutic drugs. IMPORTANCE PEDV infection causes severe piglet mortality and threatens the global pig industry. However, the complex invasion mechanism of PEDV makes its prevention and control difficult. Here, we determined that HSPA5 is a novel target for PEDV which interacts with its S protein and is involved in viral attachment and internalization, influencing its transport via the endo-/lysosomal pathway. Our work extends knowledge about the relationship between the PEDV S and host proteins and provides a new therapeutic target against PEDV infection.


Subject(s)
Coronavirus Infections , Porcine epidemic diarrhea virus , Swine Diseases , Animals , Swine , Chlorocebus aethiops , Porcine epidemic diarrhea virus/physiology , Spike Glycoprotein, Coronavirus/chemistry , Virus Internalization , Chromatography, Liquid , Tandem Mass Spectrometry , Lysosomes/metabolism , Vero Cells
11.
J Virol ; 97(6): e0068923, 2023 Jun 29.
Article in English | MEDLINE | ID: covidwho-20245290

ABSTRACT

Goblet cells and their secreted mucus are important elements of the intestinal mucosal barrier, which allows host cells to resist invasion by intestinal pathogens. Porcine deltacoronavirus (PDCoV) is an emerging swine enteric virus that causes severe diarrhea in pigs and causes large economic losses to pork producers worldwide. To date, the molecular mechanisms by which PDCoV regulates the function and differentiation of goblet cells and disrupts the intestinal mucosal barrier remain to be determined. Here, we report that in newborn piglets, PDCoV infection disrupts the intestinal barrier: specifically, there is intestinal villus atrophy, crypt depth increases, and tight junctions are disrupted. There is also a significant reduction in the number of goblet cells and the expression of MUC-2. In vitro, using intestinal monolayer organoids, we found that PDCoV infection activates the Notch signaling pathway, resulting in upregulated expression of HES-1 and downregulated expression of ATOH-1 and thereby inhibiting the differentiation of intestinal stem cells into goblet cells. Our study shows that PDCoV infection activates the Notch signaling pathway to inhibit the differentiation of goblet cells and their mucus secretion, resulting in disruption of the intestinal mucosal barrier. IMPORTANCE The intestinal mucosal barrier, mainly secreted by the intestinal goblet cells, is a crucial first line of defense against pathogenic microorganisms. PDCoV regulates the function and differentiation of goblet cells, thereby disrupting the mucosal barrier; however, the mechanism by which PDCoV disrupts the barrier is not known. Here, we report that in vivo, PDCoV infection decreases villus length, increases crypt depth, and disrupts tight junctions. Moreover, PDCoV activates the Notch signaling pathway, inhibiting goblet cell differentiation and mucus secretion in vivo and in vitro. Thus, our results provide a novel insight into the mechanism underlying intestinal mucosal barrier dysfunction caused by coronavirus infection.


Subject(s)
Coronavirus Infections , Goblet Cells , Receptors, Notch , Swine Diseases , Animals , Coronavirus , Coronavirus Infections/pathology , Coronavirus Infections/veterinary , Goblet Cells/cytology , Signal Transduction , Swine , Swine Diseases/pathology , Swine Diseases/virology , Stem Cells/cytology , Cell Differentiation , Receptors, Notch/metabolism
12.
Infect Genet Evol ; 112: 105463, 2023 08.
Article in English | MEDLINE | ID: covidwho-20244841

ABSTRACT

Recent reports on identification of canine coronavirus (CCoV) in humans have emphasized the urgency to strengthen surveillance of animal CoVs. The fact that recombinations between CCoV with feline, porcine CoVs brought about new types of CoVs indicated that more attention should be paid to domestic animals like dogs, cats and pigs, and the CoVs they carried. However, there are about ten kinds of CoVs that infect above animals, and thus representative CoVs with zoonotic potentials were considered in this study. Multiplex RT-PCR against CCoV, Feline coronavirus (FCoV), porcine deltacoronavirus and porcine acute diarrhea syndrome coronavirus was developed to investigate the prevalence of CoVs from domestic dogs in Chengdu, Southwest China. Samples from a total of 117 dogs were collected from a veterinary hospital, and only CCoV (34.2%, 40/117) was detected. Therefore, this study focused on CCoV and its characteristics of S, E, M, N and ORF3abc genes. Compared with CoVs that are capable of infecting humans, CCoV strains showed highest nucleotide identity with the novel canine-feline recombinant detected from humans (CCoV-Hupn-2018). Phylogenetic analysis based on S gene, CCoV strains were not only clustered with CCoV-II strains, but also closely related to FCoV-II strains ZJU1617 and SMU-CD59/2018. As for assembled ORF3abc, E, M, N sequences, CCoV strains had the closest relationship with CCoV-II (B203_GZ_2019, B135_JS_2018 and JS2103). What's more, specific amino acid variations were found, especially in S and N proteins, and some mutations were consistent with FCoV, TGEV strains. Altogether, this study provided a novel insight into the identification, diversification and evolution of CoVs from domestic dogs. It is of top priority to recognize zoonotic potential of CoVs, and continued comprehensive surveillance will help better understand the emergence, spreading, and ecology of animal CoVs.


Subject(s)
Coronavirus Infections , Coronavirus, Canine , Dog Diseases , Animals , Dogs , Cats , Humans , Swine , Coronavirus, Canine/genetics , Coronavirus Infections/epidemiology , Coronavirus Infections/veterinary , Reverse Transcriptase Polymerase Chain Reaction , Phylogeny , Molecular Epidemiology , Mutation , Animals, Domestic , China/epidemiology , Dog Diseases/epidemiology
13.
Virulence ; 13(1): 1697-1712, 2022 12.
Article in English | MEDLINE | ID: covidwho-20244441

ABSTRACT

Autophagy plays an important role in defending against invading microbes. However, numerous viruses can subvert autophagy to benefit their replication. Porcine epidemic diarrhoea virus (PEDV) is an aetiological agent that causes severe porcine epidemic diarrhoea. How PEDV infection regulates autophagy and its role in PEDV replication are inadequately understood. Herein, we report that PEDV induced complete autophagy in Vero and IPEC-DQ cells, as evidenced by increased LC3 lipidation, p62 degradation, and the formation of autolysosomes. The lysosomal protease inhibitors chloroquine (CQ) or bafilomycin A and Beclin-1 or ATG5 knockdown blocked autophagic flux and inhibited PEDV replication. PEDV infection activated AMP-activated protein kinase (AMPK) and c-Jun terminal kinase (JNK) by activating TGF-beta-activated kinase 1 (TAK1). Compound C (CC), an AMPK inhibitor, and SP600125, a JNK inhibitor, inhibited PEDV-induced autophagy and virus replication. AMPK activation led to increased ULK1S777 phosphorylation and activation. Inhibition of ULK1 activity by SBI-0206965 (SBI) and TAK1 activity by 5Z-7-Oxozeaenol (5Z) or by TAK1 siRNA led to the suppression of autophagy and virus replication. Our study provides mechanistic insights into PEDV-induced autophagy and how PEDV infection leads to JNK and AMPK activation.


Subject(s)
Porcine epidemic diarrhea virus , AMP-Activated Protein Kinases/genetics , AMP-Activated Protein Kinases/metabolism , Animals , Autophagy , Beclin-1 , Chloroquine , MAP Kinase Kinase Kinases , Porcine epidemic diarrhea virus/physiology , Protease Inhibitors , RNA, Small Interfering , Swine , Virus Replication
14.
Virulence ; 13(1): 1315-1330, 2022 12.
Article in English | MEDLINE | ID: covidwho-20238606

ABSTRACT

Porcine rotavirus (PoRV) is an important pathogen, leading to the occurrence of viral diarrhoea . As the infection displays obvious enterotropism, intestinal mucosal immunity is the significant line of defence against pathogen invasion. Moreover, as lactic acid bacteria (LAB) show acid resistance, bile salt resistance and immune regulation, it is of great significance to develop an oral vaccine. Most traditional plasmid delivery vectors use antibiotic genes as selective markers, easily leading to antibiotic accumulation. Therefore, to select a food-grade marker in genetically engineering food-grade microorganisms is vital. Based on the CRISPR-Cas9D10A system, we constructed a stable auxotrophic Lactobacillus paracasei HLJ-27 (Lactobacillus △Alr HLJ-27) strain. In addition, as many plasmids replicate in the host bacteria, resulting in internal gene deletions. In this study,we used a temperature-sensitive gene editing plasmidto insert the VP4 gene into the genome, yielding the insertion mutant strains VP4/△Alr HLJ-27, VP4/△Alr W56, and VP4/W56. This recombinant bacterium efficiently induced secretory immunoglobulin A (SIgA)-based mucosal and immunoglobulin G (IgG)-based humoral immune responses. These oral mucosal vaccines have the potential to act as an alternative to the application of antibiotics in the future and induce efficient immune responses against PEDV infection.


Subject(s)
Capsid Proteins , Lactobacillus , Animals , Anti-Bacterial Agents , Capsid Proteins/genetics , Clustered Regularly Interspaced Short Palindromic Repeats , Lactobacillus/genetics , Rotavirus , Swine
15.
Microb Pathog ; 181: 106185, 2023 Aug.
Article in English | MEDLINE | ID: covidwho-20231019

ABSTRACT

Porcine Epidemic Diarrhoea (PED) is an acute, extremely infectious intestinal disease of pigs caused by the Porcine Epidemic Diarrhoea Virus (PEDV). The virus can affect pigs of all breeds and age groups and shows varying degrees of symptoms, with piglets, in particular, being infected with mortality rates of up to 100%. PEDV was first identified in China in the 1980s and in October 2010 a large-scale PED outbreak caused by a variant of PEDV occurred in China, resulting in huge economic losses. Initially, vaccination can effectively prevent the classical strain, but since December 2010, the PEDV variant has caused "persistent diarrhoea" with severe vomiting, watery diarrhoea, and high morbidity and mortality in newborn piglets as the dominant clinical features, with a significant increase in morbidity and mortality. This indicates that PEDV strains have mutated during evolution and that traditional vaccines no longer provide effective cross-immune protection, so it is necessary to optimize immunization programs and find effective treatments through epidemiological surveys of PEDV to reduce the economic losses caused by infections with mutated strains. This article reviews the progress of research on the aetiology, epidemiological characteristics, genotyping, pathogenesis, transmission routes, and comprehensive control of PEDV infection in China.


Subject(s)
Coronavirus Infections , Dysentery , Porcine epidemic diarrhea virus , Swine Diseases , Animals , Swine , Porcine epidemic diarrhea virus/genetics , Genotype , Coronavirus Infections/epidemiology , Coronavirus Infections/prevention & control , Coronavirus Infections/veterinary , Diarrhea , China/epidemiology , Swine Diseases/epidemiology , Swine Diseases/prevention & control
16.
Int J Biol Macromol ; 242(Pt 4): 125190, 2023 Jul 01.
Article in English | MEDLINE | ID: covidwho-20230951

ABSTRACT

Porcine deltacoronavirus (PDCoV) is a novel swine enteropathogenic coronavirus that, because of its broad host range, poses a potential threat to public health. Here, to identify the neutralizing B-cell epitopes within the S1-CTD protein, we generated three anti-PDCoV monoclonal antibodies (mAbs). Of these, the antibody designated 4E-3 effectively neutralized PDCoV with an IC50 of 3.155 µg/mL. mAb 4E-3 and one other, mAb 2A-12, recognized different linear B-cell epitopes. The minimal fragment recognized by mAb 4E-3 was mapped to 280FYSDPKSAV288 and designated S280-288, the minimal fragment recognized by mAb 2A-12 was mapped to 506TENNRFTT513, and designated S506-513. Subsequently, alanine (A)-scanning mutagenesis indicated that Asp283, Lys285, and Val288 were the critical residues recognized by mAb 4E-3. The S280-288 epitope induces PDCoV specific neutralizing antibodies in mice, demonstrating that it is a neutralizing epitope. Of note, the S280-288 coupled to Keyhole Limpet Hemocyanin (KLH) produces PDCoV neutralizing antibodies in vitro and in vivo, in challenged piglets it potentiates interferon-γ responses and provides partial protection against disease. This is the first report about the PDCoV S protein neutralizing epitope, which will contribute to research of PDCoV-related pathogenic mechanism, vaccine design and antiviral drug development.


Subject(s)
Epitopes, B-Lymphocyte , Immunodominant Epitopes , Animals , Swine , Mice , Spike Glycoprotein, Coronavirus/chemistry , Antibodies, Neutralizing
17.
Vector Borne Zoonotic Dis ; 23(7): 397-400, 2023 07.
Article in English | MEDLINE | ID: covidwho-2317952

ABSTRACT

Background: Serological evidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been reported in white-tailed deer (WTD) in the United States and Canada. Even though WTD are susceptible to SARS-CoV-2 infection, there is no evidence of infection by this virus in other mammalian species that might interact with WTD in nature. Similar to WTD, feral swine are widely distributed and generally occupy the same range as WTD in Texas. The objective of this study was to determine the prevalence of SARS-CoV-2 neutralizing antibody in WTD during 2020 and 2021 and determine the prevalence of SARS-CoV-2 neutralizing antibody in feral swine during 2018 (prepandemic period) and from March 2020 to February 2021 (pandemic period) in Travis County, Texas. Materials and Methods: Sera samples were collected from hunter-killed WTD and feral swine during the prepandemic and pandemic period and tested for SARS-CoV-2 antibody by a plaque reduction neutralization assay in Vero cells. Results: SARS-CoV-2 antibody was not detected in any of the 166 feral swine sera samples, including 24 samples collected during the prepandemic and 142 samples collected during the pandemic period. Furthermore, SARS-CoV-2 antibody was not detected in the 115 WTD samples collected during late 2020, but antibody was detected in WTD in early 2021. Conclusions: The results indicated that SARS-CoV-2 infection of WTD occurred during early 2021 in Travis County, Texas, but serological evidence of SARS-CoV-2 infection was not detected in the feral swine samples collected from the same locality and during the same time period of the collection of WTD samples.


Subject(s)
COVID-19 , Deer , Swine Diseases , Chlorocebus aethiops , Animals , Swine , Texas/epidemiology , SARS-CoV-2 , Vero Cells , COVID-19/epidemiology , COVID-19/veterinary , Antibodies, Viral , Antibodies, Neutralizing , Swine Diseases/epidemiology
18.
Arch Virol ; 168(5): 152, 2023 May 04.
Article in English | MEDLINE | ID: covidwho-2317672

ABSTRACT

Porcine epidemic diarrhea (PED) virus (PEDV) is a highly contagious virus. PED was first identified in 2008 and has greatly affected the Vietnamese pig production economy. The aim of this study was to investigate the epidemiological and genetic characteristics of PEDV in piglet herds in the Mekong Delta, Vietnam. Diarrheal stool and intestinal samples from 2262 piglets from 191 herds in five provinces were collected to test for the presence of PEDV. Ten PEDV strains were randomly selected for sequencing, and four genes encoding PEDV structural proteins were analyzed. The rates of herds and samples positive for PEDV were 27.23% and 27.72%, respectively. In positive herds, the morbidity and mortality of PEDV-positive piglets were 97.97% and 79.06%, respectively, with most of the infected piglets under 7 days of age. Phylogenetic analysis showed that the 10 PEDV strains from this study clustered with genotype G2 strains from Vietnam and neighboring countries. Many amino acid substitutions were identified in important antigenic regions in the spike protein of the 10 strains when compared to four PEDV vaccine strains. This study provides novel insights into the epidemiology and genetic diversity of circulating PEDV strains, which could facilitate the development of an appropriate and proactive strategy for controlling PED.


Subject(s)
Coronavirus Infections , Porcine epidemic diarrhea virus , Swine Diseases , Animals , Swine , Porcine epidemic diarrhea virus/genetics , Phylogeny , Vietnam/epidemiology , Coronavirus Infections/epidemiology , Coronavirus Infections/veterinary , Molecular Epidemiology , Diarrhea/epidemiology , Diarrhea/veterinary , Swine Diseases/epidemiology
19.
Virology ; 584: 9-23, 2023 07.
Article in English | MEDLINE | ID: covidwho-2317224

ABSTRACT

Porcine epidemic diarrhea virus (PEDV) is a porcine enteropathogenic coronavirus causing severe watery diarrhea, vomiting, dehydration, and death in piglets. However, most commercial vaccines are developed based on the GI genotype strains, and have poor immune protection against the currently dominant GII genotype strains. Therefore, four novel replication-deficient human adenovirus 5-vectored vaccines expressing codon-optimized forms of the GIIa and GIIb strain spike and S1 glycoproteins were constructed, and their immunogenicity was evaluated in mice by intramuscular (IM) injection. All the recombinant adenoviruses generated robust immune responses, and the immunogenicity of recombinant adenoviruses against the GIIa strain was stronger than that of recombinant adenoviruses against the GIIb strain. Moreover, Ad-XT-tPA-Sopt-vaccinated mice elicited optimal immune effects. In contrast, mice immunized with Ad-XT-tPA-Sopt by oral gavage did not induce strong immune responses. Overall, IM administration of Ad-XT-tPA-Sopt is a promising strategy against PEDV, and this study provides useful information for developing viral vector-based vaccines.


Subject(s)
Adenoviruses, Human , Coronavirus Infections , Porcine epidemic diarrhea virus , Swine Diseases , Viral Vaccines , Animals , Swine , Mice , Humans , Antibodies, Viral , Porcine epidemic diarrhea virus/genetics , Vaccines, Synthetic/genetics , Viral Vaccines/genetics , Coronavirus Infections/prevention & control , Coronavirus Infections/veterinary , Genotype , Spike Glycoprotein, Coronavirus/genetics
20.
J Virol ; 97(5): e0037523, 2023 05 31.
Article in English | MEDLINE | ID: covidwho-2316566

ABSTRACT

Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus that has the potential to infect humans. Histone deacetylase 6 (HDAC6) is a unique type IIb cytoplasmic deacetylase with both deacetylase activity and ubiquitin E3 ligase activity, which mediates a variety of cellular processes by deacetylating histone and nonhistone substrates. In this study, we found that ectopic expression of HDAC6 significantly inhibited PDCoV replication, while the reverse effects could be observed after treatment with an HDAC6-specific inhibitor (tubacin) or knockdown of HDAC6 expression by specific small interfering RNA. Furthermore, we demonstrated that HDAC6 interacted with viral nonstructural protein 8 (nsp8) in the context of PDCoV infection, resulting in its proteasomal degradation, which was dependent on the deacetylation activity of HDAC6. We further identified the key amino acid residues lysine 46 (K46) and K58 of nsp8 as acetylation and ubiquitination sites, respectively, which were required for HDAC6-mediated degradation. Through a PDCoV reverse genetics system, we confirmed that recombinant PDCoV with a mutation at either K46 or K58 exhibited resistance to the antiviral activity of HDAC6, thereby exhibiting higher replication compared with wild-type PDCoV. Collectively, these findings contribute to a better understanding of the function of HDAC6 in regulating PDCoV infection and provide new strategies for the development of anti-PDCoV drugs. IMPORTANCE As an emerging enteropathogenic coronavirus with zoonotic potential, porcine deltacoronavirus (PDCoV) has sparked tremendous attention. Histone deacetylase 6 (HDAC6) is a critical deacetylase with both deacetylase activity and ubiquitin E3 ligase activity and is extensively involved in many important physiological processes. However, little is known about the role of HDAC6 in the infection and pathogenesis of coronaviruses. Our present study demonstrates that HDAC6 targets PDCoV-encoded nonstructural protein 8 (nsp8) for proteasomal degradation through the deacetylation at the lysine 46 (K46) and the ubiquitination at K58, suppressing viral replication. Recombinant PDCoV with a mutation at K46 and/or K58 of nsp8 displayed resistance to the antiviral activity of HDAC6. Our work provides significant insights into the role of HDAC6 in regulating PDCoV infection, opening avenues for the development of novel anti-PDCoV drugs.


Subject(s)
Coronavirus Infections , Coronavirus , Swine Diseases , Animals , Antiviral Agents/pharmacology , Antiviral Agents/metabolism , Coronavirus/metabolism , Histone Deacetylase 6/genetics , Histone Deacetylase 6/metabolism , Lysine/metabolism , Swine , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitination , Virus Replication
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