RESUMO
Porcine epidemic diarrhea (PED), caused by the porcine epidemic diarrhea virus (PEDV), emerges annually in several Asian countries. Its major symptoms include watery diarrhea, vomiting, anorexia, and dehydration. PED outbreaks incur significant economic losses. The efficacy of vaccines is limited by viral mutations and insufficient intestinal mucosal immunity. Therefore, new vaccines against these recent variants are urgently needed. Herein, we isolated and genetically characterized a novel Korean PEDV strain using NGS. Comparative genomic analysis demonstrated that the CKK1-1 strain belonged to genogroup 2. The isolated strain was cultured in sodium-glycochenodeoxycholic acid for 180 passages. Typically, PEDV isolation and passage require proteases, such as trypsin. However, the CKK1-1 strain adapted to this atypical culture condition, achieving a high titer of 8.83 ± 0.14 log TCID50/mL. In vitro biological analysis revealed no cell syncytium formation without trypsin; however, a cell-lysis-type cytopathic effect was noted. Notably, pathogenicity evaluation showed that CKK1-1 p0 exhibited naturally weakened virulence in five-day-old piglets, while piglets administered with CKK1-1 p180 exhibited 100% survival and reduced clinical symptoms. Collectively, our data demonstrate that this Korean PEDV strain, attenuated through atypical culture conditions with Na-glycochenodeoxycholic acid, has potential as a vaccine candidate, providing valuable insights into the genetic variation in and pathogenicity of PEDV.
Assuntos
Infecções por Coronavirus , Vírus da Diarreia Epidêmica Suína , Doenças dos Suínos , Animais , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Vírus da Diarreia Epidêmica Suína/patogenicidade , Vírus da Diarreia Epidêmica Suína/classificação , Suínos , República da Coreia , Doenças dos Suínos/virologia , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Virulência , Filogenia , Genoma Viral , Chlorocebus aethiops , Células VeroRESUMO
Practice of inoculating porcine epidemic diarrhea virus (PEDV) in piglets generating feedback material might influence the genetic evolution and attenuation of PEDV. The study was conducted to evaluate evolutionary rate and attenuation following serial in vitro and in vivo propagation. In the study, PED-JPFP0-PJ, Passage 0 (P0), was isolated from infected pigs and serially passaged in Vero cells for 5 consecutive times, P1-P5. P0, P2 and P5 were then subjected to orally inoculate 3-day-old piglets. At 24 h post inoculation, intestines of each passage (F1), were collected, and subsequently sub-passaged in piglets for 2 additional passages (F2-F3). Virus titration, PEDV genomic copies number, VH:CD ratios, and immunohistochemistry were evaluated. S and ORF3 genes were characterized. The results of the study demonstrated that virus titer and virulence were negatively correlated with increased passages, both in vitro and in vivo. Increased substitution rate was observed in higher passages. The evolutionary rate of S gene was higher than that of ORF3. Seven aa changes at positions 223, 291, 317, 607, 694, 1114 and 1199, with reduced N-linked glycan were observed in P5F3. In conclusion, serial passage of PEDV, both in vitro and in vivo, influence the genetic development and the attenuation of PEDV.
Assuntos
Infecções por Coronavirus , Vírus da Diarreia Epidêmica Suína , Doenças dos Suínos , Animais , Vírus da Diarreia Epidêmica Suína/patogenicidade , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/fisiologia , Suínos , Virulência , Células Vero , Chlorocebus aethiops , Doenças dos Suínos/virologia , Infecções por Coronavirus/virologia , Infecções por Coronavirus/veterinária , Evolução Molecular , Inoculações Seriadas , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
Recent epidemiological studies have discovered that a lot of cases of porcine epidemic diarrhea virus (PEDV) infection are frequently accompanied by porcine kobuvirus (PKV) infection, suggesting a potential relationship between the two viruses in the development of diarrhea. To investigate the impact of PKV on PEDV pathogenicity and the number of intestinal lymphocytes, piglets were infected with PKV or PEDV or co-infected with both viruses. Our findings demonstrate that co-infected piglets exhibit more severe symptoms, acute gastroenteritis, and higher PEDV replication compared to those infected with PEDV alone. Notably, PKV alone does not cause significant intestinal damage but enhances PEDV's pathogenicity and alters the number of intestinal lymphocytes. These results underscore the complexity of viral interactions in swine diseases and highlight the need for comprehensive diagnostic and treatment strategies addressing co-infections.
Assuntos
Coinfecção , Infecções por Coronavirus , Intestinos , Kobuvirus , Linfócitos , Vírus da Diarreia Epidêmica Suína , Doenças dos Suínos , Animais , Vírus da Diarreia Epidêmica Suína/patogenicidade , Vírus da Diarreia Epidêmica Suína/fisiologia , Suínos , Doenças dos Suínos/virologia , Coinfecção/virologia , Coinfecção/veterinária , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Linfócitos/virologia , Kobuvirus/patogenicidade , Kobuvirus/genética , Intestinos/virologia , Diarreia/virologia , Diarreia/veterinária , Replicação Viral , Gastroenterite/virologia , Gastroenterite/veterinária , Infecções por Picornaviridae/veterinária , Infecções por Picornaviridae/virologiaRESUMO
Non-structural protein 2 (nsp2) exists in all coronaviruses (CoVs), while its primary function in viral pathogenicity, is largely unclear. One such enteric CoV, porcine epidemic diarrhea virus (PEDV), causes high mortality in neonatal piglets worldwide. To determine the biological role of nsp2, we generated a PEDV mutant containing a complete nsp2 deletion (rPEDV-Δnsp2) from a highly pathogenic strain by reverse genetics, showing that nsp2 was dispensable for PEDV infection, while its deficiency reduced viral replication in vitro. Intriguingly, rPEDV-Δnsp2 was entirely avirulent in vivo, with significantly increased productions of IFNB (interferon beta) and IFN-stimulated genes (ISGs) in various intestinal tissues of challenged newborn piglets. Notably, nsp2 targets and degrades TBK1 (TANK binding kinase 1), the critical kinase in the innate immune response. Mechanistically, nsp2 induced the macroautophagy/autophagy process and recruited a selective autophagic receptor, NBR1 (NBR1 autophagy cargo receptor). NBR1 subsequently facilitated the K48-linked ubiquitination of TBK1 and delivered it for autophagosome-mediated degradation. Accordingly, the replication of rPEDV-Δnsp2 CoV was restrained by reduced autophagy and excess productions of type I IFNs and ISGs. Our data collectively define enteric CoV nsp2 as a novel virulence determinant, propose a crucial role of nsp2 in diminishing innate antiviral immunity by targeting TBK1 for NBR1-mediated selective autophagy, and pave the way to develop a new type of nsp2-based attenuated PEDV vaccine. The study also provides new insights into the prevention and treatment of other pathogenic CoVs.Abbreviations: 3-MA: 3-methyladenine; Baf A1: bafilomycin A1; CoV: coronavirus; CQ: chloroquine; dpi: days post-inoculation; DMVs: double-membrane vesicles; GABARAP: GABA type A receptor-associated protein; GFP: green fluorescent protein; GIGYF2: GRB10 interacting GYF protein 2; hpi: hours post-infection; IFA: immunofluorescence assay; IFIH1: interferon induced with helicase C domain 1; IFIT2: interferon induced protein with tetratricopeptide repeats 2; IFITM1: interferon induced transmembrane protein 1; IFNB: interferon beta; IRF3: interferon regulatory factor 3; ISGs: interferon-stimulated genes; mAb: monoclonal antibody; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; NBR1: NBR1 autophagy cargo receptor; nsp2: non-structural protein 2; OAS1: 2'-5'-oligoadenylate synthetase 1; PEDV: porcine epidemic diarrhea virus; PRRs: pattern recognition receptors; RIGI: RNA sensor RIG-I; RT-qPCR: reverse transcription quantitative polymerase chain reaction; SQSTM1: sequestosome 1; TBK1: TANK binding kinase 1; TCID50: 50% tissue culture infectious doses; VSV: vesicular stomatitis virus.
Assuntos
Autofagia , Imunidade Inata , Vírus da Diarreia Epidêmica Suína , Proteínas Serina-Treonina Quinases , Proteínas não Estruturais Virais , Replicação Viral , Animais , Proteínas não Estruturais Virais/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Autofagia/genética , Suínos , Vírus da Diarreia Epidêmica Suína/patogenicidade , Vírus da Diarreia Epidêmica Suína/imunologia , Chlorocebus aethiops , Humanos , Virulência , Células Vero , Ubiquitinação , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Interferon beta/metabolismo , Fatores de Virulência/metabolismo , Fatores de Virulência/genética , Células HEK293RESUMO
IMPORTANCE: Porcine epidemic diarrhea (PED) caused by PED virus (PEDV) remains a big threat to the swine industry worldwide. Vaccination with live attenuated vaccine is a promising method to prevent and control PED, because it can elicit a more protective immunity than the killed vaccine, subunit vaccine, and so on. In this study, we found two obvious deletions in the genome of a high passage of AH2012/12. We further confirmed the second deletion which contains seven amino acids at the carboxy-terminus of the S2 gene and the start codon of ORF3 can reduce its pathogenicity in vivo. Animal experiments indicated that the recombinant PEDV with deleted carboxy-terminus of S gene showed higher IgG, IgA, neutralization antibodies, and protection effects against virus challenge than the killed vaccine. These data reveal that the engineering of the carboxy-terminus of the S2 gene may be a promising method to develop live attenuated vaccine candidates of PEDV.
Assuntos
Infecções por Coronavirus , Vírus da Diarreia Epidêmica Suína , Doenças dos Suínos , Animais , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Diarreia , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/patogenicidade , Suínos , Doenças dos Suínos/virologia , Vacinas Atenuadas/genética , Vacinas de Produtos Inativados , Vacinas Virais/genética , VirulênciaRESUMO
We evaluated differences in the pathology and humoral immune status in one- and two-month-old weaned pigs infected with virulent Chinese genotype GIIa and GIIb strains of porcine epidemic diarrhea virus (PEDV). All pigs infected with the GIIa strain developed severe diarrhea (100%), while the morbidity of the GIIb strain in one- and two-month-old weaned pigs was 80% (4/5) and 40% (2/5), respectively. There was no significant difference in IgA, IgG, or virus-neutralizing (VN) antibody levels associated with GIIa and GIIb in one-month-old weaned pigs (P > 0.05), but in two-month-old weaned pigs, the IgA, IgG, and VN antibody levels associated with GIIa were significantly higher than those associated with GIIb (P < 0.05).
Assuntos
Vírus da Diarreia Epidêmica Suína , Doenças dos Suínos , Animais , Diarreia/veterinária , Diarreia/virologia , Genótipo , Imunoglobulina A , Imunoglobulina G , Vírus da Diarreia Epidêmica Suína/patogenicidade , Suínos , Doenças dos Suínos/virologia , VirulênciaRESUMO
Coinfection caused by various genotypes of porcine epidemic diarrhea virus (PEDV) is a new disease situation. We previously reported the coexistence of PEDV strains containing different ORF3 genotypes in China. In this study, the PEDV strains 17GXCZ-1ORF3d and 17GXCZ-1ORF3c were isolated and plaque-purified from the same piglet, which had a natural large deletion at the 172-554 bp position of the ORF3 gene or possessed a complete ORF3 gene, respectively. Meanwhile, 17GXCZ-1ORF3d had >99% nt identity with 17GXCZ-1ORF3c in the 5'UTR, ORF1a/1b, S, E, M, N and 3'UTR regions but only demonstrated low nucleotide identities (80.5%) in the ORF3 gene. To elucidate the pathogenicity, 7-day-old piglets were infected. Piglets infected with these two PEDV strains exhibited severe clinical signs and shed the virus at the highest level within 96 hpi. Compared with the piglets inoculated with the 17GXCZ-1ORF3c strain, the piglets inoculated with the 17GXCZ-1ORF3d strain had higher mortality rates (75% vs. 50%), an earlier onset of clinical signs with a significantly higher diarrhea score, lower VH:CD ratios and a higher percentage of PEDV-positive enterocytes. This study is the first to report PEDV coinfections with different ORF3 genotypes, and a PEDV strain with a large deletion in the ORF3 gene might have the advantage of a potential genetic marker, which would be useful during vaccine development.
Assuntos
Genótipo , Fases de Leitura Aberta/genética , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/patogenicidade , Animais , Chlorocebus aethiops , Coinfecção/virologia , Infecções por Coronavirus/virologia , Diarreia/virologia , Filogenia , Vírus da Diarreia Epidêmica Suína/classificação , Suínos , Doenças dos Suínos/virologia , Células Vero , VirulênciaRESUMO
The recent prevalence of coronavirus (CoV) poses a serious threat to animal and human health. Currently, porcine enteric coronaviruses (PECs), including the transmissible gastroenteritis virus (TGEV), the novel emerging swine acute diarrhoea syndrome coronavirus (SADS-CoV), porcine delta coronavirus (PDCoV), and re-emerging porcine epidemic diarrhoea virus (PEDV), which infect pigs of different ages, have caused more frequent occurrences of diarrhoea, vomiting, and dehydration with high morbidity and mortality in piglets. PECs have the potential for cross-species transmission and are causing huge economic losses in the pig industry in China and the world, which therefore needs to be urgently addressed. Accordingly, this article summarises the pathogenicity, prevalence, and diagnostic methods of PECs and provides an important reference for their improved diagnosis, prevention, and control.
Assuntos
Infecções por Coronavirus/veterinária , Doenças dos Suínos/virologia , Alphacoronavirus/genética , Alphacoronavirus/patogenicidade , Animais , China/epidemiologia , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/prevenção & controle , Deltacoronavirus/genética , Deltacoronavirus/patogenicidade , Humanos , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/patogenicidade , Prevalência , Suínos , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/prevenção & controle , Vírus da Gastroenterite Transmissível/genética , Vírus da Gastroenterite Transmissível/patogenicidadeRESUMO
Porcine Epidemic Diarrhea Virus (PEDV) is the causative agent of swine epidemic diarrhea. In order to study the pathogenic mechanism of PEDV, PEDV was inoculated into Vero cells cultured in vitro, and the total RNA of Vero cells was extracted to construct a library for Illumina high-throughput sequencing and screening of differentially expressed genes (p < 0.05). Five differentially expressed genes for qRT-PCR verification analysis were randomly selected, and the verification results were consistent with the transcriptome sequencing results. The Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathway enrichment analysis was performed on the differentially expressed genes screened above. The results showed that the target gene annotations of differentially expressed genes in the African green monkey genome were mainly enriched in the TNF signaling pathway, the P53 signaling pathway, the Jak-STAT signaling pathway, the MAPK signaling pathway, and immune inflammation. In addition, it has been reported that Puma can promote apoptosis and is a key mediator of P53-dependent and non-dependent apoptosis pathways. However, there is no report that PEDV infection can activate Puma and induce apoptosis in a P53-dependent pathway. It was found by flow cytometry that PEDV infection induced apoptosis, and by Western Blotting detection, PEDV infection significantly increased the expression of p53, BAX, and Puma apoptosis-related proteins. Treatment Vero cells with the p53 inhibitor, PFT-α, could significantly inhibit PEDV-induced apoptosis. Studies have shown that PEDV infection can activate Puma and induce apoptosis in a P53-dependent pathway. These findings provide data support for further elucidating the pathogenic mechanism of PEDV and developing an effective vaccine against PEDV.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Vírus da Diarreia Epidêmica Suína/patogenicidade , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Chlorocebus aethiops , Vírus da Diarreia Epidêmica Suína/genética , Suínos , Proteína Supressora de Tumor p53/genética , Células VeroRESUMO
This article is aimed at analyzing the structure and function of the spike (S) proteins of porcine enteric coronaviruses, including transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV) by applying bioinformatics methods. The physical and chemical properties, hydrophilicity and hydrophobicity, transmembrane region, signal peptide, phosphorylation and glycosylation sites, epitope, functional domains, and motifs of S proteins of porcine enteric coronaviruses were predicted and analyzed through online software. The results showed that S proteins of TGEV, PEDV, SADS-CoV, and PDCoV all contained transmembrane regions and signal peptide. TGEV S protein contained 139 phosphorylation sites, 24 glycosylation sites, and 53 epitopes. PEDV S protein had 143 phosphorylation sites, 22 glycosylation sites, and 51 epitopes. SADS-CoV S protein had 109 phosphorylation sites, 20 glycosylation sites, and 43 epitopes. PDCoV S protein had 124 phosphorylation sites, 18 glycosylation sites, and 52 epitopes. Moreover, TGEV, PEDV, and PDCoV S proteins all contained two functional domains and two motifs, spike_rec_binding and corona_S2. The corona_S2 consisted of S2 subunit heptad repeat 1 (HR1) and S2 subunit heptad repeat 2 (HR2) region profiles. Additionally, SADS-CoV S protein was predicted to contain only one functional domain, the corona_S2. This analysis of the biological functions of porcine enteric coronavirus spike proteins can provide a theoretical basis for the design of antiviral drugs.
Assuntos
Infecções por Coronavirus/epidemiologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/ultraestrutura , Alphacoronavirus/metabolismo , Alphacoronavirus/patogenicidade , Animais , Biologia Computacional/métodos , Coronavirus/imunologia , Coronavirus/ultraestrutura , Bases de Dados Genéticas , Deltacoronavirus/metabolismo , Deltacoronavirus/patogenicidade , Epitopos/imunologia , Vírus da Diarreia Epidêmica Suína/metabolismo , Vírus da Diarreia Epidêmica Suína/patogenicidade , Glicoproteína da Espícula de Coronavírus/metabolismo , Suínos/virologia , Doenças dos Suínos/virologia , Vírus da Gastroenterite Transmissível/metabolismo , Vírus da Gastroenterite Transmissível/patogenicidadeRESUMO
Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus causing acute intestinal infection in pigs, with high mortality often seen in neonatal pigs. The newborns rely on innate immune responses against invading pathogens because of lacking adaptive immunity. However, how PEDV disables the innate immunity of newborns toward severe infection remains unknown. We found that PEDV infection led to reduced expression of histone deacetylases (HDACs), especially HDAC1, in porcine IPEC-J2 cells. HDACs are considered important regulators of innate immunity. We hypothesized that PEDV interacts with certain host factors to regulate HDAC1 expression in favor of its replication. We show that HDAC1 acted as a negative regulator of PEDV replication in IPEC-J2 cells, as shown by chemical inhibition, gene knockout, and overexpression. A GC-box (GCCCCACCCCC) within the HDAC1 promoter region was identified for Sp1 binding in IPEC-J2 cells. Treatment of the cells with Sp1 inhibitor mithramycin A inhibited HDAC1 expression, indicating direct regulation of HDAC1 expression by Sp1. Of the viral proteins that were overexpressed in IPEC-J2 cells, the N protein was found to be present in the nuclei and more inhibitory to HDAC1 transcription. The putative nuclear localization sequence 261PKKNKSR267 contributed to its nuclear localization. The N protein interacted with Sp1 and interfered with its binding to the promoter region, thereby inhibiting its transcriptional activity for HDAC1 expression. Our findings reveal a novel mechanism of PEDV evasion of the host responses, offering implications for studying the infection processes of other coronaviruses. IMPORTANCE The enteric coronavirus porcine epidemic diarrhea virus (PEDV) causes fatal acute intestinal infection in neonatal pigs that rely on innate immune responses. Histone deacetylases (HDACs) play important roles in innate immune regulation. Our study found PEDV suppresses HDAC1 expression via the interaction of its N protein and porcine Sp1, which identified a novel mechanism of PEDV evasion of the host responses to benefit its replication. This study suggests that other coronaviruses, including SARS-CoV and SARS-CoV-2, also make use of their N proteins to intercept the host immune responses in favor of their infection.
Assuntos
Infecções por Coronavirus/veterinária , Células Epiteliais/virologia , Histona Desacetilase 1/antagonistas & inibidores , Mucosa Intestinal/virologia , Fator de Transcrição Sp1/metabolismo , Doenças dos Suínos/virologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Animais , Células Cultivadas , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Vírus da Diarreia Epidêmica Suína/patogenicidade , Fator de Transcrição Sp1/genética , Suínos , Doenças dos Suínos/metabolismo , Doenças dos Suínos/patologia , Proteínas não Estruturais Virais/genéticaRESUMO
Infection induces the production of proinflammatory cytokines and chemokines such as interleukin-8 (IL-8) and IL-6. Although they facilitate local antiviral immunity, their excessive release leads to life-threatening cytokine release syndrome, exemplified by the severe cases of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In this study, we investigated the roles of the integrated stress response (ISR) and activator protein-1 (AP-1) family proteins in regulating coronavirus-induced IL-8 and IL-6 upregulation. The mRNA expression of IL-8 and IL-6 was significantly induced in cells infected with infectious bronchitis virus (IBV), a gammacoronavirus, and porcine epidemic diarrhea virus, an alphacoronavirus. Overexpression of a constitutively active phosphomimetic mutant of eukaryotic translation initiation factor 2α (eIF2α), chemical inhibition of its dephosphorylation, or overexpression of its upstream double-stranded RNA-dependent protein kinase (PKR) significantly enhanced IL-8 mRNA expression in IBV-infected cells. Overexpression of the AP-1 protein cJUN or its upstream kinase also increased the IBV-induced IL-8 mRNA expression, which was synergistically enhanced by overexpression of cFOS. Taken together, this study demonstrated the important regulatory roles of ISR and AP-1 proteins in IL-8 production during coronavirus infection, highlighting the complex interactions between cellular stress pathways and the innate immune response.
Assuntos
Infecções por Coronavirus/metabolismo , Estresse do Retículo Endoplasmático/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Interleucina-8/metabolismo , Resposta a Proteínas não Dobradas/genética , Alphacoronavirus/metabolismo , Alphacoronavirus/patogenicidade , Animais , Linhagem Celular , Chlorocebus aethiops , Infecções por Coronavirus/genética , Gammacoronavirus/metabolismo , Gammacoronavirus/patogenicidade , Regulação da Expressão Gênica , Humanos , Imunidade Inata , Vírus da Bronquite Infecciosa/metabolismo , Vírus da Bronquite Infecciosa/patogenicidade , Interleucina-8/genética , Fosforilação , Vírus da Diarreia Epidêmica Suína/metabolismo , Vírus da Diarreia Epidêmica Suína/patogenicidade , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Transdução de Sinais/genética , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Regulação para Cima , Células Vero , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismoRESUMO
As the most abundant cell type in the blood, red blood cells (RBCs) are serving for transporting oxygen. However, the mechanism by which RBCs binding virus remains largely unknown. Here, we demonstrated that porcine epidemic diarrhea virus (PEDV), a kind of coronavirus, could hijack RBCs and cause typical diarrhea in neonatal piglets. In an epidemiology investigation of PEDV, the RBCs samples from diarrheic pigs in several pig farms were found to be PEDV-positive. PEDV could bind to neonatal RBCs through CD71 and clathrin-mediated endocytosis, and its viability was maintained for 12 h. PEDV-loaded RBCs could transfer the virus to CD3+ T cells by conjugation and reach the intestine mucosa, where it caused infection. Finally, a further animal challenge revealed that transfusing with PEDV-loaded RBCs could cause intestinal epithelial cells (IECs) infection and typical diarrhea symptom. Therefore, our studies illustrated the mechanism by which PEDV could cause intestinal infection through hijacking RBCs, further providing a novel insight into the role of RBCs as potential cells for viral transmission in coronavirus pathogenesis.
Assuntos
Transfusão de Sangue/veterinária , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/veterinária , Diarreia/veterinária , Eritrócitos/virologia , Vírus da Diarreia Epidêmica Suína/patogenicidade , Doenças dos Suínos/transmissão , Animais , Animais Recém-Nascidos , Chlorocebus aethiops , Diarreia/virologia , Mucosa Intestinal/patologia , Mucosa Intestinal/virologia , Suínos , Doenças dos Suínos/virologia , Linfócitos T/virologia , Células Vero , Ligação ViralRESUMO
Coinfection of porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) is one of common findings in diarrheal piglets that cause massive economic losses to the pig industry globally. However, the mechanism of the co-infection is unclear. In this study, neonatal non-colostrum-fed piglets were exposed orally with a single infection of PDCoV or PEDV, or coinfection of PDCoV and PEDV. Clinically all viral infected piglets developed watery diarrhea and dehydration in 24 h post-exposure (hpe) and were succumbed to viral diarrhea disease and euthanized at 72 hpe. Histopathologically, acute gastroenteritis is evident in all viral infected piglet. Immunohistochemistry, RNAscope and RT-qPCR demonstrated that PEDV tropism changes from epithelial cells of small intestine to gastric epithelial cells and macrophages in Peyer's patches in the ileum. These findings suggest that coinfection of PDCoV and PEDV can alter PEDV tropism that may affect the outcome of viral disease in piglets. This animal model can be used for the pathogenesis and vaccination of viral coinfection in piglet in the future.
Assuntos
Coinfecção/virologia , Infecções por Coronavirus/veterinária , Deltacoronavirus/patogenicidade , Trato Gastrointestinal/virologia , Vírus da Diarreia Epidêmica Suína/patogenicidade , Tropismo Viral , Animais , Infecções por Coronavirus/virologia , Diarreia/virologia , Modelos Animais de Doenças , Células Epiteliais/virologia , Íleo/virologia , SuínosRESUMO
Porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) cause an enteric disease characterized by diarrhea clinically indistinguishable. Both viruses are simultaneously detected in clinical cases, but a study involving the co-infection has not been reported. The study was therefore conducted to investigate the disease severity following a co-infection with PEDV and PDCoV. In the study, 4-day-old pigs were orally inoculated with PEDV and PDCoV, either alone or in combination. Following challenge, fecal score was monitored on a daily basis. Fecal swabs were collected and assayed for the presence of viruses. Three pigs per group were necropsied at 3 and 5 days post inoculation (dpi). Microscopic lesions and villous height to crypt depth (VH:CD) ratio, together with the presence of PEDV and PDCoV antigens, were evaluated in small intestinal tissues. Expressions of interferon alpha (IFN-α) and interleukin 12 (IL12) were investigated in small intestinal mucosa. The findings indicated that coinoculation increased the disease severity, demonstrated by significantly prolonged fecal score and virus shedding and decreasing VH:CD ratio in the jejunum compared with pigs inoculated with either PEDV or PDCoV alone. Notably, in single-inoculated groups, PEDV and PDCoV antigens were detected only in villous enterocytes wile in the coinoculated group, PDCoV antigen was detected in both villous enterocytes and crypts. IFN-α and IL12 were significantly up-regulated in coinoculated groups in comparison with single-inoculated groups. In conclusion, co-infection with PEDV and PDCoV exacerbate clinical signs and have a synergetic on the regulatory effect inflammatory cytokines compared to a single infection with either virus.
Assuntos
Deltacoronavirus/patogenicidade , Diarreia/genética , Interferon-alfa/genética , Interleucina-12/genética , Vírus da Diarreia Epidêmica Suína/patogenicidade , Animais , Coinfecção/genética , Coinfecção/veterinária , Coinfecção/virologia , Infecções por Coronavirus/genética , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Deltacoronavirus/genética , Deltacoronavirus/isolamento & purificação , Diarreia/veterinária , Diarreia/virologia , Fezes/virologia , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Índice de Gravidade de Doença , Suínos , Doenças dos Suínos/genética , Doenças dos Suínos/virologiaRESUMO
PURPOSE: The expression level of aminopeptidase N (APN) is evidently correlated with porcine epidemic diarrhea virus (PEDV) infectivity. This study aims to examine the mechanisms regulating APN expression level in response to PEDV infection. METHODS: Quantitative real time PCR was performed herein to detect gene expression dynamics at various timepoints after PEDV infection. Subsequently, CRISPR/Cas9 gene editing technology was used to generate a APN-knockout IPEC-J2 cell line, exploring the effects of APN on cell proliferation by propidium iodide staining and anti-PEDV activity by indirect immunofluorescence assay. Ultimately, the effects of single nucleotide polymorphisms (SNPs) and methylation in the APN promoter region on gene expression were analyzed by using bisulfite sequencing PCR and dual luciferase reporter gene assay. RESULTS: APN expression was significantly upregulated within 4-24 h post-infection. The cytoactivity of the APN-knockout IPEC-J2 cell line was markedly suppressed at different timepoints. Further, cell cycle analyses indicated an increase in the number of G1-phase cells and a significant decrease in that of S-phase cells. Moreover, key cyclical factors regulating the G1 phase were highly expressed in APN-knockout cells. The RNA copies of viral particles and mRNA levels of antiviral genes and inflammatory cytokines in APN-knockout cells were markedly decreased within 24 h of PEDV infection. Similarly, indirect immunofluorescence assay confirmed that the number of PEDV particles was significantly decreased. Sequence analysis revealed two CpG islands in the APN promoter region. However, there was no evident correlation between the methylation status of APN promoter and mRNA levels. Dual luciferase reporter gene assay showed that the SNP rs326030589 (G/A) significantly increased the promoter activity of APN. CONCLUSIONS: These results suggested that APN knockout enhanced the resistance of IPEC-J2 cells to PEDV. Moreover, rs326030589 in the APN promoter region participated in gene transcription regulation. Our results provide a reference for studying the mechanisms regulating APN and may contribute to the application of APN gene in resistance breeding of swine epidemic diarrhea.
Assuntos
Antígenos CD13/genética , Resistência à Doença , Polimorfismo de Nucleotídeo Único , Vírus da Diarreia Epidêmica Suína/patogenicidade , Regulação para Cima , Animais , Antígenos CD13/metabolismo , Sistemas CRISPR-Cas , Ciclo Celular , Linhagem Celular , Proliferação de Células , Metilação de DNA , Técnicas de Inativação de Genes , Vírus da Diarreia Epidêmica Suína/genética , Regiões Promotoras Genéticas , RNA Viral/genética , SuínosRESUMO
This study focused on intestinal restitution including phenotype switching of absorptive enterocytes and the abundance of different enterocyte subtypes in weaned pigs after porcine epidemic diarrhea virus (PEDV) infection. At 10 days post-PEDV-inoculation, the ratio of villus height to crypt depth in both jejunum and ileum had restored, and the PEDV antigen was not detectable. However, enterocytes at the villus tips revealed epithelial-mesenchymal transition (EMT) in the jejunum in which E-cadherin expression decreased while expression of N-cadherin, vimentin, and Snail increased. Additionally, there was reduced expression of actin in microvilli and Zonula occludens-1 (ZO-1) in tight junctions. Moreover, the protein concentration of transforming growth factor ß1 (TGFß1), which mediates EMT and cytoskeleton alteration, was increased. We also found a decreased number of Peyer's patch M cells in the ileum. These results reveal incomplete restitution of enterocytes in the jejunum and potentially impaired immune surveillance in the ileum after PEDV infection.
Assuntos
Infecções por Coronavirus/veterinária , Enterócitos/patologia , Transição Epitelial-Mesenquimal , Gastroenterite Suína Transmissível/patologia , Nódulos Linfáticos Agregados/patologia , Vírus da Diarreia Epidêmica Suína/patogenicidade , Animais , Caderinas/metabolismo , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Gastroenterite Suína Transmissível/imunologia , Gastroenterite Suína Transmissível/virologia , Íleo/imunologia , Íleo/patologia , Mucosa Intestinal/patologia , Jejuno/imunologia , Jejuno/patologia , Microvilosidades/patologia , Suínos , Junções Íntimas/patologia , Fator de Crescimento Transformador beta1/metabolismo , DesmameRESUMO
Porcine epidemic diarrhea virus (PEDV), the causative agent of PED, belongs to the genus Alphacoronavirus in the family Coronaviridae. Reactive oxygen species (ROS), endoplasmic reticulum (ER) stress, and autophagy play crucial roles in regulating a variety of cellular processes during viral infection. However, the precise role of autophagy in PEDV-infected Vero cells remains largely elusive. To elucidate how PEDV infection induces autophagy, this study ascertained whether ER stress was present in PEDV-infected Vero cells. The results showed PEDV infection significantly increased the expression of GRP78 and LC3â ¡. Treatment with the ER stress inhibitor 4-phenylbutyrate (4-PBA) could significantly inhibit PEDV-induced autophagy. Antioxidants, such as N-acetylcysteine (NAC), could significantly inhibit PEDV-induced ER stress and autophagy, indicating that ROS act as an upstream regulator of ER stress-mediated autophagy. Further research found that activation of ER stress triggered the unfolded protein response (UPR) through PERK, IRE1, and ATF6 pathways during PEDV infection. However, treatment with the PERK inhibitor GSK2606414, IRE1 inhibitor STF-083010 but not ATF6 inhibitor AEBSF reversed PEDV-induced autophagy. Taken together, the results of this study showed that accumulated ROS played an essential role in regulating ER stress-mediated autophagy during PEDV infection. We also found that PERK and IER1 pathways of UPR signalling were involved in PEDV-induced autophagy. Furthermore, PEDV induced autophagy to promote viral replication via PERK and IER1 pathways in Vero cells. These results provide the mechanism of PEDV-induced ROS-dependent ER stress-mediated autophagy in Vero cells through activating PERK and IRE1 pathways.
Assuntos
Autofagia , Estresse do Retículo Endoplasmático/fisiologia , Vírus da Diarreia Epidêmica Suína/patogenicidade , Proteínas Serina-Treonina Quinases/fisiologia , Espécies Reativas de Oxigênio/metabolismo , eIF-2 Quinase/fisiologia , Animais , Chlorocebus aethiops , Redes e Vias Metabólicas , Transdução de Sinais , Resposta a Proteínas não Dobradas , Células Vero , Replicação Viral , eIF-2 Quinase/genéticaRESUMO
Porcine epidemic diarrhea virus (PEDV) causes an emerging and re-emerging coronavirus disease characterized by vomiting, acute diarrhea, dehydration, and up to 100% mortality in neonatal suckling piglets, leading to huge economic losses in the global swine industry. Vaccination remains the most promising and effective way to prevent and control PEDV. However, effective vaccines for PEDV are still under development. Understanding the genomic structure and function of PEDV and the influence of the viral components on innate immunity is essential for developing effective vaccines. In the current review, we systematically describe the recent developments in vaccine against PEDV and the roles of structural proteins, non-structural proteins and accessory proteins of PEDV in affecting viral virulence and regulating innate immunity, which will provide insight into the rational design of effective and safe vaccines for PEDV or other coronaviruses.
Assuntos
Infecções por Coronavirus/veterinária , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/imunologia , Doenças dos Suínos/imunologia , Doenças dos Suínos/virologia , Vacinas Virais/imunologia , Animais , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/virologia , Imunidade Inata , Vírus da Diarreia Epidêmica Suína/patogenicidade , Suínos , Doenças dos Suínos/prevenção & controle , Vacinação/veterinária , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia , Proteínas Virais/genética , Vacinas Virais/administração & dosagem , VirulênciaRESUMO
Porcine epidemic diarrhea virus (PEDV) is a fatal epizootic swine coronavirus that presents a financial threat to the global swine industry. Since the discovery of the low-pathogenic genotype 1b (G1b) in 2014, it has been responsible for sporadic outbreaks in South Korea. In this study, we identified novel G1b variants arising from the natural recombination of a major pandemic-like G2b virus and a minor G1b virus currently circulating in the domestic field. The whole-genome sequences of two 2018-19 G1b recombinants, KNU-1808 and KNU-1909, were determined. A genomic comparison showed that these two viruses share the highest nucleotide sequence similarity with the 2017 G1b strain but share less similarity with the 2014 G1b emergent strain KNU-1406. However, the putative recombination breakpoints spanning the first 1,170 nucleotides of the spike (S) gene were almost identical among the emergent and contemporary G1b strains. Recombination detection indicated that the inter-subgroup G1b recombinant first emerged in 2017 by introducing the N-terminal domain of S from KNU-1406 into the backbone of KNU-1703, possibly leading to antigenic shift. It then evolved into KNU-1808 and KNU-1909 through genetic drift, moving toward a more G2b-like genotype. Phylogenetic analysis revealed that the 2018-2019 G1b recombinants belong to a cluster containing other G1b strains but form a new branch. This study provides an important advance warning in regard to the emergence and prevalence of new genotypes or variants that can result from genetic recombination between two different PEDV genotypes circulating in endemic areas and continuous non-lethal mutations essential for viral fitness in the host environment.