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1.
Gene ; 775: 145448, 2021 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-33482281

RESUMO

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 , Suínos
2.
Virology ; 552: 43-51, 2021 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-33059319

RESUMO

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 , Desmame
3.
Arch Virol ; 165(11): 2471-2478, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32772174

RESUMO

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.


Assuntos
Infecções por Coronavirus/veterinária , Surtos de Doenças , Genoma Viral , Vírus da Diarreia Epidêmica Suína/genética , Glicoproteína da Espícula de Coronavírus/genética , Doenças dos Suínos/epidemiologia , Animais , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/virologia , Evolução Molecular , Variação Genética , Genótipo , Filogenia , Vírus da Diarreia Epidêmica Suína/classificação , Vírus da Diarreia Epidêmica Suína/patogenicidade , Recombinação Genética , República da Coreia/epidemiologia , Suínos , Doenças dos Suínos/transmissão , Doenças dos Suínos/virologia , Sequenciamento Completo do Genoma
4.
Arch Virol ; 165(10): 2279-2289, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32719955

RESUMO

In the early stage of virus infection, the pattern recognition receptor (PRR) signaling pathway of the host cell is activated to induce interferon production, activating interferon-stimulated genes (ISGs) that encode antiviral proteins that exert antiviral effects. Viperin is one of the innate antiviral proteins that exert broad-spectrum antiviral effects by various mechanisms. Porcine epidemic diarrhea virus (PEDV) is a coronavirus that causes huge losses to the pig industry. Research on early antiviral responses in the gastrointestinal tract is essential for developing strategies to prevent the spread of PEDV. In this study, we investigated the mechanisms of viperin in PEDV-infected IPEJ-C2 cells. Increased expression of interferon and viperin and decreased replication of PEDV with a clear reduction in the viral load were observed in PEDV-infected IPEC-J2 cells. Amino acids 1-50 of porcine viperin contain an endoplasmic reticulum signal sequence that allows viperin to be anchored to the endoplasmic reticulum and are necessary for its function in inhibiting PEDV proliferation. The interaction of the viperin S-adenosylmethionine domain with the N protein of PEDV was confirmed via confocal laser scanning microscopy and co-immunoprecipitation. This interaction might interfere with viral replication or assembly to reduce virus proliferation. Our results highlight a potential mechanism whereby viperin is able to inhibit PEDV replication and play an antiviral role in innate immunity.


Assuntos
Antivirais/metabolismo , Interações entre Hospedeiro e Microrganismos/fisiologia , Proteínas do Nucleocapsídeo/fisiologia , Vírus da Diarreia Epidêmica Suína/fisiologia , Animais , Linhagem Celular , Infecções por Coronavirus/genética , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/imunologia , Imunidade Inata , Interferons/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Proteínas do Nucleocapsídeo/antagonistas & inibidores , Proteínas do Nucleocapsídeo/química , Vírus da Diarreia Epidêmica Suína/imunologia , Vírus da Diarreia Epidêmica Suína/patogenicidade , Domínios e Motivos de Interação entre Proteínas , Proteínas/química , Proteínas/genética , Proteínas/fisiologia , Interferência de RNA , Suínos , Replicação Viral
5.
Virus Res ; 286: 198045, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32502552

RESUMO

Porcine epidemic diarrhea virus (PEDV), a member of the genus Alphacoronavirus in the family Coronaviridae, causes acute diarrhea and/or vomiting, dehydration and high mortality in neonatal piglets. Two different genogroups of PEDV, S INDEL [PEDV variant containing multiple deletions and insertions in the S1 subunit of the spike (S) protein, G1b] and non-S INDEL (G2b) strains were detected during the diarrheal disease outbreak in US swine in 2013-2014. Similar viruses are also circulating globally. Continuous improvement and update of biosecurity and vaccine strains and protocols are still needed to control and prevent PEDV infections worldwide. Although the non-S INDEL PEDV was highly virulent and the S INDEL PEDV caused milder disease, the latter has the capacity to cause illness in a high number of piglets on farms with low biosecurity and herd immunity. The main PEDV transmission route is fecal-oral, but airborne transmission via the fecal-nasal route may play a role in pig-to-pig and farm-to-farm spread. PEDV infection of neonatal pigs causes fecal virus shedding (alongside frequent detection of PEDV RNA in the nasal cavity), acute viremia, severe atrophic enteritis (mainly jejunum and ileum), and increased pro-inflammatory and innate immune responses. PEDV-specific IgA effector and memory B cells in orally primed sows play a critical role in sow lactogenic immunity and passive protection of piglets. This review focuses on the etiology, transmission, pathogenesis, and prevention and control of PEDV infection.


Assuntos
Infecções por Coronavirus/patologia , Infecções por Coronavirus/transmissão , Mucosa Intestinal/virologia , Vírus da Diarreia Epidêmica Suína/patogenicidade , Doenças dos Suínos/transmissão , Aerossóis , Animais , Infecções por Coronavirus/prevenção & controle , Progressão da Doença , Imunidade Humoral/imunologia , Mucosa Intestinal/patologia , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/imunologia , Receptores Virais/metabolismo , Suínos , Doenças dos Suínos/patologia , Doenças dos Suínos/virologia , Tropismo Viral/fisiologia , Viremia/sangue
6.
J Virol ; 94(16)2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32461321

RESUMO

The 5' cap methylation of viral RNA plays important roles in RNA stability, efficient translation, and immune evasion. Thus, RNA cap methylation is an attractive target for antiviral discovery and development of new live attenuated vaccines. For coronaviruses, RNA cap structure is first methylated at the guanine-N-7 (G-N-7) position by nonstructural protein 14 (nsp14), which facilitates and precedes the subsequent ribose 2'-O methylation by the nsp16-nsp10 complex. Using porcine epidemic diarrhea virus (PEDV), an Alphacoronavirus, as a model, we showed that G-N-7 methyltransferase (G-N-7 MTase) of PEDV nsp14 methylated RNA substrates in a sequence-unspecific manner. PEDV nsp14 can efficiently methylate RNA substrates with various lengths in both neutral and alkaline pH environments and can methylate cap analogs (GpppA and GpppG) and single-nucleotide GTP but not ATP, CTP, or UTP. Mutations to the S-adenosyl-l-methionine (SAM) binding motif in the nsp14 abolished the G-N-7 MTase activity and were lethal to PEDV. However, recombinant rPEDV-D350A with a single mutation (D350A) in nsp14, which retained 29.0% of G-N-7 MTase activity, was viable. Recombinant rPEDV-D350A formed a significantly smaller plaque and had significant defects in viral protein synthesis and viral replication in Vero CCL-81 cells and intestinal porcine epithelial cells (IPEC-DQ). Notably, rPEDV-D350A induced significantly higher expression of both type I and III interferons in IPEC-DQ cells than the parental rPEDV. Collectively, our results demonstrate that G-N-7 MTase activity of PEDV modulates viral replication, gene expression, and innate immune responses.IMPORTANCE Coronaviruses (CoVs) include a wide range of important human and animal pathogens. Examples of human CoVs include severe acute respiratory syndrome coronavirus (SARS-CoV-1), Middle East respiratory syndrome coronavirus (MERS-CoV), and the most recently emerged SARS-CoV-2. Examples of pig CoVs include porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine enteric alphacoronavirus (SeACoV). There are no vaccines or antiviral drugs for most of these viruses. All known CoVs encode a bifunctional nsp14 protein which possesses ExoN and guanine-N-7 methyltransferase (G-N-7 MTase) activities, responsible for replication fidelity and RNA cap G-N-7 methylation, respectively. Here, we biochemically characterized G-N-7 MTase of PEDV nsp14 and found that G-N-7 MTase-deficient PEDV was defective in replication and induced greater responses of type I and III interferons. These findings highlight that CoV G-N-7 MTase may be a novel target for rational design of live attenuated vaccines and antiviral drugs.


Assuntos
Exorribonucleases/metabolismo , Interferon Tipo I/biossíntese , Interferons/biossíntese , Vírus da Diarreia Epidêmica Suína/fisiologia , Capuzes de RNA/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Chlorocebus aethiops , Exorribonucleases/genética , Expressão Gênica , Guanina/metabolismo , Imunidade Inata , Metilação , Mutação , Vírus da Diarreia Epidêmica Suína/enzimologia , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/patogenicidade , RNA Viral/metabolismo , S-Adenosilmetionina/metabolismo , Suínos , Células Vero , Proteínas não Estruturais Virais/genética , Replicação Viral
7.
PLoS One ; 15(3): e0228983, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32182247

RESUMO

Failing to mitigate propagation of disease spread can result in dire economic consequences for agricultural networks. Pathogens like Porcine Epidemic Diarrhea virus, can quickly spread among producers. Biosecurity is designed to prevent infection transmission. When considering biosecurity investments, management must balance the cost of protection versus the consequences of contracting an infection. Thus, an examination of the decision making processes associated with investment in biosecurity is important for enhancing system wide biosecurity. Data gathered from experimental gaming simulations can provide insights into behavioral strategies and inform the development of decision support systems. We created an online digital experiment to simulate outbreak scenarios among swine production supply chains, where participants were tasked with making biosecurity investment decisions. In Experiment One, we quantified the risk associated with each participant's decisions and delineated three dominant categories of risk attitudes: risk averse, risk tolerant, and opportunistic. Each risk class exhibited unique approaches in reaction to risk and disease information. We also tested how information uncertainty affects risk aversion, by varying the amount of visibility of the infection as well as the amount of biosecurity implemented across the system. We found evidence that more visibility in the number of infected sites increases risk averse behaviors, while more visibility in the amount of neighboring biosecurity increased risk taking behaviors. In Experiment Two, we were surprised to find no evidence for differences in behavior of livestock specialists compared to Amazon Mechanical Turk participants. Our findings provide support for using experimental gaming simulations to study how risk communication affects behavior, which can provide insights towards more effective messaging strategies.


Assuntos
Infecções por Coronavirus/veterinária , Surtos de Doenças/prevenção & controle , Doenças dos Suínos/virologia , Animais , Infecções por Coronavirus/prevenção & controle , Tomada de Decisões , Surtos de Doenças/veterinária , Abastecimento de Alimentos , Jogos Experimentais , Humanos , Modelos Teóricos , Vírus da Diarreia Epidêmica Suína/patogenicidade , Suínos , Doenças dos Suínos/prevenção & controle , Jogos de Vídeo
8.
Vet Microbiol ; 240: 108511, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31902508

RESUMO

Since late 2010, highly virulent PEDV G2-genotype strains have emerged globally extracting heavy losses on the pork industries of numerous countries. We investigated the characteristics of a field strain of PEDV (PEDV strain SH) isolated from a piglet with severe diarrhea on a farm in Shanghai China. Whole genome sequencing and analysis revealed that the SH strain belonged to subtype G2b and has a unique 12-aa deletion (aa 399-410) including the antigenic epitope NEP-1C9 (aa 398-406) of the N protein. PEDV SH strain is highly pathogenic to challenged newborn piglets, resulting in 100 % morbidity and mortality. Pathological examination revealed significant villus atrophy in the jejuna of infected piglets. Mice inoculated with inactivated PEDV SH produced antibodies against the N protein, but no antibodies against the deletions. These results illustrated that deletion of the NEP-1C9 epitope had no effect on the immunogenicity or pathogenicity of PEDV, providing evidence of the necessity to monitor the genetic diversity of the virus. Our study also contributes to development of candidate for vaccines and diagnostics that could differentiate pigs seropositive due to vaccination by conventional strains from wild virus infection.


Assuntos
Infecções por Coronavirus/veterinária , Diarreia/veterinária , Proteínas do Nucleocapsídeo/genética , Vírus da Diarreia Epidêmica Suína/imunologia , Vírus da Diarreia Epidêmica Suína/patogenicidade , Animais , Animais Domésticos/virologia , Animais Recém-Nascidos/virologia , Anticorpos Antivirais/sangue , Infecções por Coronavirus/virologia , Diarreia/virologia , Epitopos , Genoma Viral , Genótipo , Camundongos , Proteínas do Nucleocapsídeo/imunologia , Filogenia , Vírus da Diarreia Epidêmica Suína/genética , Deleção de Sequência , Suínos , Doenças dos Suínos/virologia , Virulência , Sequenciamento Completo do Genoma
9.
Microb Pathog ; 140: 103922, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31838173

RESUMO

BACKGROUND: Highly virulent variants of porcine epidemic diarrhea virus (PEDV) have been closely associated with recent outbreaks of porcine epidemic diarrhea (PED) in China, which have resulted in severe economic losses to the pork industry. METHODS: In the current study, the variant PEDV strain HM2017 was isolated and purified and a viral growth curve was constructed according to the median tissue culture infective dose (TCID50). HM2017 were amplify with RT-PCR and analyzed by phylogeny analysis. Animal pathogenicity experiment was carried to evaluate the HM2017 clinical assessment. RESULTS: Genome-based phylogenetic analysis revealed that PEDV strain HM2017 was clustered into the variant subgroup GII-a that is currently circulating in pig populations in China. The highest median tissue culture infectious dose of strain HM2017 after 15 passages in Vero cells was 1.33 × 107 viral particles/mL. Strain HM2017 was highly virulent to suckling piglets, which exhibited clinical symptoms at 12 h post-infection (hpi) (i.e., weight loss at 12-84 hpi, increased body temperatures at 24-48 hpi, high viral loads in the jejunum and ileum, and 100% mortality by 84 hpi). CONCLUSION: The present study reports a variant subgroup GII-a PEDV HM2017 strain in China and characterize its pathogenicity. PEDV strain HM2017 of subgroup GII-a presents a promising vaccine candidate for the control of PED outbreaks in China.


Assuntos
Infecções por Coronavirus/veterinária , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Animais , China/epidemiologia , Chlorocebus aethiops , Surtos de Doenças/prevenção & controle , Genoma Viral , Filogenia , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/imunologia , Vírus da Diarreia Epidêmica Suína/patogenicidade , Suínos , Doenças dos Suínos/virologia , Células Vero , Vacinas Virais/imunologia
10.
Transbound Emerg Dis ; 67(1): 65-79, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31381232

RESUMO

A strain of porcine epidemic diarrhoea virus (PEDV), namely HLJBY, was isolated in Heilongjiang province, China. To provide insight into the understanding of the phylogenetic and the current epidemiological status of PEDV, PEDV HLJBY was compared with CV777 and other PEDV strains deposited in the GenBank. The homology between the entire genomic nucleotide sequences of PEDV HLJBY and CV777 was 97.7%. The homology of M gene was the highest (99.0%). However, the homology of ORF3 gene was 97.7%, and protein of ORF3 was 90.1%. In addition, HLJBY showed the highest nucleotide identity (99.9%) with PEDV-SX/China/2017 strain and lowest similarity (91.2%) to PEDV/Belgorod/dom/2008 strain. We analysed the changes in S gene and its protein of PEDV HLJBY with 65 historic PEDV strains. The highest nucleotide identity was 99.9% compared with PEDV-SX/China/2017 strain, and the lowest nucleotide identity was 60.0% compared with PEDV/Belgorod/dom/2008 strain. The length of deduced amino acid sequences of S proteins varied from 1,372 to 1,390 amino acids (aa). Compared with most aa sequences of S proteins, HLJBY exhibited 5 aa deletions (position 55, 59-61, 144). Analysis and comparison of open reading frame 3 (ORF3) proteins between HLJBY strain and other PEDV strains were also focused in this study. We revealed that the length of deduced amino acid sequences of ORF3 proteins was 80-224 aa among tested strains and the identity of HLJBY ORF3 amino acids with other PEDV strains was 71.4%-98.9%. ORF3 protein of both HLJBY strain and PEDV-SX/China/2017 strain consists of 91 aa, with 133 aa deletions at their C' end in relation to the other tested PEDV strains. The phylogenetic tree based on different proteins or genes resulted in different phylogenetic groups. For pathogenicity evaluation of PEDV HLJBY strain, colostrum deprivation piglets were challenged with PEDV HLJBY, and PEDV reference strain CV777 as a control, the results showed that animals challenged with either of these PEDV strains developed diarrhoea, and histopathological examination of small intestines of challenged animals showed acute viral enteritis with villous atrophy in either PEDV HLJBY-P10 or PEDV CV777-P8 inoculated piglets.


Assuntos
Infecções por Coronavirus/veterinária , Diarreia/veterinária , Genoma Viral/genética , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/genética , Doenças dos Suínos/virologia , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Evolução Biológica , China/epidemiologia , Chlorocebus aethiops , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Diarreia/epidemiologia , Diarreia/patologia , Diarreia/virologia , Intestino Delgado/patologia , Intestino Delgado/virologia , Fases de Leitura Aberta/genética , Filogenia , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/patogenicidade , Suínos , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/patologia , Células Vero
11.
Virol J ; 16(1): 121, 2019 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-31660993

RESUMO

BACKGROUND: Porcine epidemic diarrhea virus (PEDV) has caused enormous economic losses to the global pig industry. Currently available PEDV vaccine strains have limited protective effects against PEDV variant strains. METHODS: In this study, the highly virulent epidemic virus strain CT was serially passaged in Vero cells for up to 120 generations (P120). Characterization of the different passages revealed that compared with P10 and P64, P120 had a higher viral titer and more obvious cytopathic effects, thereby demonstrating better cell adaptability. RESULTS: Pathogenicity experiments using P120 in piglets revealed significant reductions in clinical symptoms, histopathological lesions, and intestinal PEDV antigen distribution; the piglet survival rate in the P120 group was 100%. Furthermore, whole-genome sequencing identified 13 amino acid changes in P120, which might be responsible for the attenuated virulence of P120. CONCLUSIONS: Thus, an attenuated strain was obtained via cell passaging and that this strain could be used in preparing attenuated vaccines.


Assuntos
Infecções por Coronavirus/veterinária , Vírus da Diarreia Epidêmica Suína/patogenicidade , Doenças dos Suínos/virologia , Animais , Chlorocebus aethiops , Infecções por Coronavirus/patologia , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/virologia , Genoma Viral/genética , Mutação , Filogenia , Vírus da Diarreia Epidêmica Suína/classificação , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/crescimento & desenvolvimento , Inoculações Seriadas/veterinária , Taxa de Sobrevida , Suínos , Doenças dos Suínos/patologia , Doenças dos Suínos/prevenção & controle , Vacinas Atenuadas , Células Vero , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Virulência/genética , Eliminação de Partículas Virais
12.
Vet Microbiol ; 235: 220-228, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31383305

RESUMO

The highly virulent porcine epidemic diarrhea virus (PEDV) variants cause the death of mainly neonatal piglets, but how the viruses spread within the gastro-intestinal tract in a temporal and spatial manner has remained poorly characterized but is critical to understand the viral pathogenesis. In this study, we used the Chinese PEDV epidemic strain BJ2011C as a model organism and took advantage of the newly developed RNAscope in situ hybridization technology to investigate the tempo-spatial infection dynamics in neonatal piglets. We found that the PEDV strain BJ2011C could quickly colonize the small intestine, which occurred in just 6 h post infection, with virus shedding starting at 6 hpi and peaking at 24 hpi. Jejunum was the first target tissue for infection and then ileum, followed by infrequent infection of duodenum. In these tissues, the virus nucleic acids were mainly present in the villous epithelial cells but not in crypt cells. Interestingly, the viral RNAs were not detectable by RNAscope in large intestines although tissue damages could be discerned by H & E staining. Overall, our results provide useful information about spread dynamics and tissue preference of PEDV epidemic strain BJ2011C.


Assuntos
Infecções por Coronavirus/patologia , Intestino Delgado/virologia , Vírus da Diarreia Epidêmica Suína/patogenicidade , RNA Viral/isolamento & purificação , Doenças dos Suínos/patologia , Animais , Animais Recém-Nascidos , Diarreia/veterinária , Diarreia/virologia , Células Epiteliais/virologia , Hibridização in Situ Fluorescente , Jejuno/citologia , Jejuno/virologia , Suínos , Doenças dos Suínos/virologia , Eliminação de Partículas Virais
13.
Vet Microbiol ; 235: 209-219, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31383304

RESUMO

Porcine epidemic diarrhea virus (PEDV), the causative agent of PED, is an enveloped, positive-stranded RNA virus in the genus Alphacoronavirus, family Coronaviridae, order Nidovirales. PEDV non-structural accessory protein ORF3 is an ion channel related to viral infectivity and pathogenicity. Our previous study showed that PEDV ORF3 has expression characteristic of aggregation in cytoplasm, but its biological function remains elusive. Thus in this study, we initiated the construction of various vectors to express ORF3, and found ORF3 localized in the cytoplasm in the aggregation manner. Subsequently, confocal microscopy analysis showed that the aggregated ORF3 localized in endoplasmic reticulum (ER) to trigger ER stress response via up-regulation of GRP78 protein expression and activation of PERK-eIF2α signaling pathway. In addition, our results showed that PEDV ORF3 could induce the autophagy through inducing conversion of LC3-I to LC3-II, but couldn't influence the apoptosis. In contrast, conversion of LC3-I/LC3-II could be significantly inhibited by 4-PBA, an ER stress inhibitor, indicating that ORF3-induced autophagy is dependent on ER stress response. This work not only provides some new findings for the biological function of the PEDV ORF3 protein, but also help us for the further understanding the molecular interaction between PEDV ORF3 protein and cells.


Assuntos
Autofagia , Estresse do Retículo Endoplasmático , Retículo Endoplasmático/virologia , Fases de Leitura Aberta , Vírus da Diarreia Epidêmica Suína/patogenicidade , Proteínas Virais/genética , Animais , Chlorocebus aethiops , Retículo Endoplasmático/patologia , Vetores Genéticos , Células HEK293 , Células HeLa , Proteínas de Choque Térmico/genética , Humanos , Transdução de Sinais , Suínos , Células Vero , Replicação Viral
14.
Viruses ; 11(8)2019 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-31349683

RESUMO

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus causing high morbidity and mortality in porcine herds worldwide. Although both inactivated and live attenuated vaccines have been extensively used, the emergence of highly virulent strains and the recurrent outbreaks even in vaccinated farms highlight the need of effective vaccines. Engineering of genetically defined live attenuated vaccines is a rational approach for novel vaccine development. In this line, we engineered an attenuated virus based on the transmissible gastroenteritis virus (TGEV) genome, expressing a chimeric spike protein from a virulent United States (US) PEDV strain. This virus (rTGEV-RS-SPEDV) was attenuated in highly-sensitive five-day-old piglets, as infected animals did not lose weight and none of them died. In addition, the virus caused very minor tissue damage compared with a virulent virus. The rTGEV-RS-SPEDV vaccine candidate was also attenuated in three-week-old animals that were used to evaluate the protection conferred by this virus, compared with the protection induced by infection with a virulent PEDV US strain (PEDV-NVSL). The rTGEV-RS-SPEDV virus protected against challenge with a virulent PEDV strain, reducing challenge virus titers in jejunum and leading to undetectable challenge virus RNA levels in feces. The rTGEV-RS-SPEDV virus induced a humoral immune response specific for PEDV, including neutralizing antibodies. Altogether, the data indicated that rTGEV-RS-SPEDV is a promising vaccine candidate against virulent PEDV infection.


Assuntos
Gastroenterite Suína Transmissível/prevenção & controle , Vírus da Diarreia Epidêmica Suína/genética , Doenças dos Suínos/prevenção & controle , Vírus da Gastroenterite Transmissível/genética , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Genoma Viral , Vírus da Diarreia Epidêmica Suína/patogenicidade , Recombinação Genética , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/virologia , Estados Unidos , Vacinas Atenuadas/imunologia , Vacinas Sintéticas/imunologia , Vacinas Virais/genética
15.
Vet Microbiol ; 233: 21-27, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31176408

RESUMO

Swine enteric coronavirus (CoV) is an important group of pathogens causing diarrhea in piglets. At least four kinds of swine enteric CoVs have been identified, including transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and the emerging HKU2-like porcine enteric alphacoronavirus (PEAV). The small intestines, particularly the jejunum and ileum, are the most common targets of these four CoVs in vivo, and co-infections by these CoVs are frequently observed in clinically infected pigs. This study was conducted to investigate the susceptibility of the porcine ileum epithelial cell line, IPI-2I, to different swine enteric CoVs. We found that IPI-2I cells are highly susceptible to TGEV, PDCoV, and PEAV, as demonstrated by cytopathic effect and virus multiplication. However, only a small number of cells could be infected by PEDV, possibly due to the heterogeneity of IPI-2I cells. A homogeneous cell line, designated IPI-FX, obtained from IPI-2I cells by sub-cloning with limited serial dilutions, was found to be highly susceptible to PEDV. Furthermore, IPI-FX cells were also highly susceptible to TGEV, PDCoV, as well as PEAV. Thus, this sub-cloned IPI-FX cell line is an ideal cell model to study the mechanisms of infection, particularly co-infections of swine enteric CoVs.


Assuntos
Técnicas de Cultura de Células/veterinária , Coronavirus/patogenicidade , Células Epiteliais/virologia , Intestino Delgado/citologia , Vírus da Diarreia Epidêmica Suína/patogenicidade , Animais , Linhagem Celular , Diarreia/virologia , Fezes/virologia , Filogenia , Vírus da Diarreia Epidêmica Suína/genética , Suínos , Doenças dos Suínos/virologia , Replicação Viral
16.
Transbound Emerg Dis ; 66(5): 1894-1909, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31055885

RESUMO

Since the 2013-2014 incursion of the virulent G2b porcine epidemic diarrhoea virus (PEDV) pandemic strains in South Korea, frequent moderate-scale regional outbreaks have recurred. In particular, areas of Jeju Island with extensive swine production have faced repeated epidemics since the re-emergence in 2014. The current study reports the complete genome sequences and molecular characterization of the representative PEDV strains responsible for the 2018 endemic outbreaks on Jeju Island. All isolates were determined to belong genetically to the highly pathogenic pandemic G2b group. Full-length genome sizes of four isolates differed from that of the G2b epidemic field strain due to insertion or deletion (DEL) mutations in the non-structural protein (nsp)- or spike (S) protein-coding regions. The 2018 Jeju isolates shared 96.7%-98.7% and 98.5%-99.4% identity at the S gene and whole-genome levels, respectively, compared to global G2b PEDV strains. Genetic and phylogenetic analyses indicated that the 2018 isolates were closest to the 2014 G2b re-emergent Jeju strains, but appeared to have undergone substantial rapid independent evolution. Among the isolates, a notable nsp3 DEL variant strain, KOR/KNU-1807/2018, was isolated and propagated by continuous passages in Vero cells, and displayed typical PEDV-induced syncytia formation. Genomic sequencing identified a unique 8-nt DEL in the extreme C-terminal region of the S gene at the 4th passage (KNU-1807-P4) compared to its original sample. This DEL resulted in the premature termination of S by nine amino acid residues (EVFEKVHVQ), which contained a KxHxx motif that is a potential endoplasmic reticulum retrieval signal. In vivo animal studies showed that variant strain KNU-1807 had decreased virulence in suckling piglets. These results advance our knowledge regarding the genetic variation and pathogenicity of the G2b PEDV endemic strains prevalent in Jeju swine herds in South Korea.


Assuntos
Infecções por Coronavirus/veterinária , Genoma Viral/genética , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/patogenicidade , Glicoproteína da Espícula de Coronavírus/genética , Doenças dos Suínos/virologia , Sequência de Aminoácidos , Animais , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Filogenia , República da Coreia , Alinhamento de Sequência , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Sus scrofa , Suínos , Células Vero , Virulência
17.
PLoS One ; 14(5): e0217236, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31141512

RESUMO

The porcine epidemic diarrhoea virus (PEDV) devastates the health of piglets but may not infect piglets whose CMP-N-glycolylneuraminic acid hydroxylase (CMAH) gene is mutated (knockouts, KO) by using CRISPR/Cas9 gene editing techniques. This hypothesis was tested by using KO piglets that were challenged with PEDV. Two single-guide RNAs targeting the CMAH gene and Cas9 mRNA were microinjected into the cytoplasm of newly fertilized eggs. Four live founders generated and proven to be biallelic KO, lacking detectable N-glycolylneuraminic acid (NGNA). The founders were bred, and homozygous offspring were obtained. Two-day-old (in exps. I, n = 6, and III, n = 15) and 3-day-old (in exp. II, n = 9) KO and wild-type (WT, same ages in respective exps.) piglets were inoculated with TCID50 1x103 PEDV and then fed 20 mL of infant formula (in exps. I and II) or sow's colostrum (in exp. III) every 4 hours. In exp. III, the colostrum was offered 6 times and was then replaced with Ringer/5% glucose solution. At 72 hours post-PEDV inoculation (hpi), the animals either deceased or euthanized were necropsied and intestines were sampled. In all 3 experiments, the piglets showed apparent outward clinical manifestations suggesting that infection occurred despite the CMAH KO. In exp. I, all 6 WT piglets and only 1 of 6 KO piglets died at 72 hpi. Histopathology and immunofluorescence staining showed that the villus epithelial cells of WT piglets were severely exfoliated, but only moderate exfoliation and enterocyte vacuolization was observed in KO piglets. In exp. II, delayed clinical symptoms appeared, yet the immunofluorescence staining/histopathologic inspection (I/H) scores of the two groups differed little. In exp. III, the animals exhibited clinical and pathological signs after inoculation similar to those in exp. II. These results suggest that porcine CMAH KO with nullified NGNA expression are not immune to PEDV but that this KO may lessen the severity of the infection and delay its occurrence.


Assuntos
Citidina Monofosfato/análogos & derivados , Predisposição Genética para Doença/genética , Vírus da Diarreia Epidêmica Suína/genética , Animais , Sistemas CRISPR-Cas , Infecções por Coronavirus/virologia , Citidina Monofosfato/genética , Diarreia/virologia , Suscetibilidade a Doenças/metabolismo , Enterócitos/patologia , Feminino , Regulação da Expressão Gênica/genética , Ácidos Neuramínicos , Vírus da Diarreia Epidêmica Suína/patogenicidade , Gravidez , Suínos , Doenças dos Suínos/virologia
19.
J Virol ; 93(15)2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31118255

RESUMO

Porcine epidemic diarrhea virus (PEDV) causes high mortality in neonatal piglets; however, effective and safe vaccines are still not available. We hypothesized that inactivation of the 2'-O-methyltransferase (2'-O-MTase) activity of nsp16 and the endocytosis signal of the spike protein attenuates PEDV yet retains its immunogenicity in pigs. We generated a recombinant PEDV, KDKE4A, with quadruple alanine substitutions in the catalytic tetrad of the 2'-O-MTase using a virulent infectious cDNA clone, icPC22A, as the backbone. Next, we constructed another mutant, KDKE4A-SYA, by abolishing the endocytosis signal of the spike protein of KDKE4A Compared with icPC22A, the KDKE4A and KDKE4A-SYA mutants replicated less efficiently in vitro but induced stronger type I and type III interferon responses. The pathogenesis and immunogenicities of the mutants were evaluated in gnotobiotic piglets. The virulence of KDKE4A-SYA and KDKE4A was significantly reduced compared with that of icPC22A. Mortality rates were 100%, 17%, and 0% in the icPC22A-, KDKE4A-, and KDKE4A-SYA-inoculated groups, respectively. At 21 days postinoculation (dpi), all surviving pigs were challenged orally with a high dose of icPC22A. The KDKE4A-SYA- and KDKE4A-inoculated pigs were protected from the challenge, because no KDKE4A-SYA- and one KDKE4A-inoculated pig developed diarrhea whereas all the pigs in the mock-inoculated group had severe diarrhea, and 33% of them died. Furthermore, we serially passaged the KDKE4A-SYA mutant in pigs three times and did not find any reversion of the introduced mutations. The data suggest that KDKE4A-SYA may be a PEDV vaccine candidate.IMPORTANCE PEDV is the most economically important porcine enteric viral pathogen and has caused immense economic losses in the pork industries in many countries. Effective and safe vaccines are desperately required but still not available. 2'-O-MTase (nsp16) is highly conserved among coronaviruses (CoVs), and the inactivation of nsp16 in live attenuated vaccines has been attempted for several betacoronaviruses. We show that inactivation of both 2'-O-MTase and the endocytosis signal of the spike protein is an approach to designing a promising live attenuated vaccine for PEDV. The in vivo passaging data also validated the stability of the KDKE4A-SYA mutant. KDKE4A-SYA warrants further evaluation in sows and their piglets and may be used as a platform for further optimization. Our findings further confirmed that nsp16 can be a universal target for CoV vaccine development and will aid in the development of vaccines against other emerging CoVs.


Assuntos
Infecções por Coronavirus/veterinária , Vírus da Diarreia Epidêmica Suína/imunologia , Doenças dos Suínos/prevenção & controle , Vacinas Virais/imunologia , Animais , Animais Recém-Nascidos , Infecções por Coronavirus/patologia , Infecções por Coronavirus/prevenção & controle , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/patogenicidade , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Análise de Sobrevida , Suínos , Doenças dos Suínos/patologia , Resultado do Tratamento , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/efeitos adversos , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/isolamento & purificação , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Vacinas Virais/administração & dosagem , Vacinas Virais/efeitos adversos , Vacinas Virais/isolamento & purificação , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
20.
PLoS One ; 14(4): e0215227, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30973929

RESUMO

Surfactin has antiviral activity against various enveloped viruses by inhibiting viral membrane fusion. However, the potential utility of surfactin as an antiviral drug is limited by its cytotoxicity. In this study, 10 surfactin analogues were obtained by chemical synthesis and evaluated to determine their anti-PEDV activities, hemolytic activities, and critical micelle concentrations. The main goal of our study was to develop a safer drug; a surfactin analogue with high anti-PEDV activity and low hemolytic activity. Compared with surfactin, one of the analogues we developed, SLP5, has lower hemolytic activity, with the same antiviral activity. The selectivity index of SLP5 is 52, while the SI for surfactin is 4, in other words, the safe and effective concentration range of SLP5 is 12 times greater than that of surfactin. Like surfactin, SLP5 has a direct antiviral effect on PEDV. Structurally, SLP5 is a linear lipopeptide with three carboxyl groups. Surfactin derivatives similar to SLP5 could be obtained by lactone bond hydrolyzation of surfactin, as well as total synthesis.


Assuntos
Antivirais/farmacologia , Lipopeptídeos/farmacologia , Peptídeos Cíclicos/farmacologia , Vírus da Diarreia Epidêmica Suína/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Antivirais/síntese química , Antivirais/química , Chlorocebus aethiops , Desenho de Fármacos , Hemólise/efeitos dos fármacos , Lipopeptídeos/síntese química , Lipopeptídeos/química , Micelas , Estrutura Molecular , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/química , Vírus da Diarreia Epidêmica Suína/patogenicidade , Vírus da Diarreia Epidêmica Suína/fisiologia , Sus scrofa , Células Vero , Internalização do Vírus/efeitos dos fármacos
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