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1.
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 , Retículo Endoplasmático/patologia , Vetores Genéticos , Células HEK293 , Células HeLa , Proteínas de Choque Térmico/genética , Humanos , Transdução de Sinais , Suínos , Células Vero , Replicação Viral
2.
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
3.
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
4.
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
6.
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 , 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
7.
Microb Pathog ; 132: 73-79, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31026494

RESUMO

Caused by porcine epidemic diarrhea virus (PEDV), porcine epidemic diarrhea (PED) is an acute infectious disease which causes damage to the intestine including intestinal villus atrophy and shedding, leading to serious economic losses to the pig industry worldwide. In order to obtain detailed information about the pathogenesis and host immune response in a PEDV-infected host for first In vivo study we used high-throughput sequencing to analyze the gene expression differences of the small intestinal mucosa after infection with PEDV. Transcripts obtained were over 65,525,000 clean reads after reassembly were 22,605 genes detected, of which 22,248 were known genes and 371 new genes were predicted. Moreover, 3168 genes expression was up-regulated and 3876 genes down-regulated. (Gene Ontology) GO annotation and functional enrichment analysis indicated that all of the DEGs (differentially expressed genes) were annotated into biological process, cellular component and molecular function. Most of these unigenes are annotated in cellular processes, the cell and binding. KEGG analysis of the DEGs showed that a total of 7044 DEGs unigenes were annotated into 323 pathways classified into 6 main categories. Most of these unigenes are annotated were related to immune system response to the infectious diseases pathways. In addition, 20 DEGs were verified by quantitative real-time PCR. As the first, in vivo, RNAseq analysis of piglets and PEDV infection, our study provides knowledge about the transcriptomics of intestinal mucosa in PEDV-infected piglets, from which a complex molecular pathways and pathogenesis-related biological processes are involved in PEDV interaction with piglet intestinal mucosa.


Assuntos
Disenteria/imunologia , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Mucosa Intestinal/imunologia , Vírus da Diarreia Epidêmica Suína/patogenicidade , Doenças dos Suínos/imunologia , Animais , Infecções por Coronavirus/genética , Infecções por Coronavirus/imunologia , Modelos Animais de Doenças , Disenteria/patologia , Disenteria/virologia , Ontologia Genética , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Patógeno/imunologia , Sistema Imunitário/imunologia , Mucosa Intestinal/patologia , Mucosa Intestinal/virologia , Intestinos/patologia , Intestinos/virologia , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/genética , Suínos , Doenças dos Suínos/patologia , Doenças dos Suínos/virologia
8.
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 , Desenho de Drogas , 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
9.
Arch Virol ; 164(5): 1287-1295, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30859476

RESUMO

Since 2010, continual outbreaks of highly virulent variants of porcine epidemic diarrhea virus (PEDV) belonging to genotype GII have led to serious economic losses for the Chinese swine industry. To better understand the biological characteristics and pathogenicity of the current prevalent Chinese PEDV field strains, in this study, a highly virulent Chinese genotype GIIa PEDV strain, CH/HBXT/2018, was isolated and serially propagated using Vero cells. Sequencing and phylogenetic analysis showed that strain CH/HBXT/2018 contained novel insertion and deletion mutations in the S gene region relative to the classical strain and belonged to the genotype GIIa, similar to other recently isolated PEDV strains from China and the United States. Pig infection studies indicated that the CH/HBXT/2018 strain was highly virulent in suckling piglets, and the median pig diarrhea dose (PDD50) was 8.63 log10PDD50/3 mL at 7 days postinfection (DPI). The results of the present study are important for future PEDV challenge studies and the development of new PEDV vaccines based on prevalent field strains for the prevention and control of PED in China.


Assuntos
Infecções por Coronavirus/veterinária , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/patogenicidade , Glicoproteína da Espícula de Coronavírus/genética , Animais , Linhagem Celular , China , Infecções por Coronavirus/virologia , Surtos de Doenças , Genótipo , Mutagênese Insercional/genética , Filogenia , Vírus da Diarreia Epidêmica Suína/classificação , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Deleção de Sequência/genética , Suínos , Doenças dos Suínos/virologia , Células Vero , Vacinas Virais/imunologia , Virulência/genética
10.
PLoS One ; 14(3): e0213153, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30840679

RESUMO

The porcine epidemic diarrhea virus (PEDV) that emerged and spread throughout Taiwan in 2014 triggered significant concern in the country's swine industry. Acknowledging the absence of a thorough investigation at the geographic level, we used 2014 outbreak sequence information from the Taiwan government's open access databases plus GenBank records to analyze PEDV dissemination among Taiwanese pig farms. Genetic sequences, locations, and dates of identified PEDV-positive cases were used to assess spatial, temporal, clustering, GIS, and phylogeographic factors affecting PEDV dissemination. Our conclusion is that S gene sequences from 2014 PEDV-positive clinical samples collected in Taiwan were part of the same Genogroup 2 identified in the US in 2013. According to phylogenetic and phylogeographic data, viral strains collected in different areas were generally independent of each other, with certain clusters identified across different communities. Data from GIS and multiple potential infection factors were used to pinpoint cluster dissemination in areas with large numbers of swine farms in southern Taiwan. The data indicate that the 2014 Taiwan PEDV epidemic resulted from the spread of multiple strains, with strong correlations identified with pig farm numbers and sizes (measured as animal concentrations), feed mill numbers, and the number of slaughterhouses in a specifically defined geographic area.


Assuntos
Infecções por Coronavirus/transmissão , Surtos de Doenças/veterinária , Vírus da Diarreia Epidêmica Suína/classificação , Doenças dos Suínos/virologia , Matadouros , Animais , Infecções por Coronavirus/veterinária , Filogenia , Filogeografia , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/patogenicidade , Análise de Sequência de RNA/veterinária , Glicoproteína da Espícula de Coronavírus/genética , Suínos , Doenças dos Suínos/transmissão , Taiwan/epidemiologia
11.
Vet Microbiol ; 230: 278-282, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30827401

RESUMO

Although highly virulent GII-genotype PEDV strains have become pandemic in the swine population worldwide, little is known about the differences in immunogenicity and cross-protective efficacy between the GIIa and GIIb subgenotypes. Hence, in the present study, we vaccinated suckling piglets with GIIa (CH/HBXT/2018) and GIIb (CH/HNPJ/2017) PEDV strain-based inactivated vaccine candidates and compared their immunogenicity and cross-protective efficacy. The results showed that both vaccine candidates induced high levels of PEDV-specific IgG antibodies and IFN-γ and reduced the levels of neutralizing antibodies at 21 dpv in suckling piglets. The GIIa-based inactivated vaccine protected all piglets (8/8) against virulent homologous and heterologous virus challenge, while the GIIb strain-based inactivated vaccine protected only 2/4 and 1/4 piglets against virulent homologous and heterologous virus challenge, respectively. Furthermore, antibodies against the GIIa and GIIb strains cross-reacted and cross-neutralized both strains in vitro. Taken together, the data presented in this study indicate that GIIa strain-based inactivated vaccine candidates are more promising than GIIb-based candidates for the development of an effective vaccine against the current highly virulent pandemic PEDV strains.


Assuntos
Anticorpos Antivirais/sangue , Infecções por Coronavirus/veterinária , Proteção Cruzada/imunologia , Imunogenicidade da Vacina , Doenças dos Suínos/prevenção & controle , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Técnicas de Cultura de Células , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Genótipo , Imunoglobulina G/sangue , Interferon gama/imunologia , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/patogenicidade , Suínos , Doenças dos Suínos/imunologia , Vacinas de Produtos Inativados/imunologia , Vacinas Virais/administração & dosagem , Eliminação de Partículas Virais
12.
BMC Vet Res ; 15(1): 26, 2019 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-30634958

RESUMO

BACKGROUND: Porcine epidemic diarrhea virus (PEDV) causes diarrhea in all ages of pigs with 50-100% mortality rates in neonatal piglets. In the United States, inactivated and subunit PEDV vaccines for pregnant sows are available, but fail to induce sufficient protection in neonatal piglets farrowed from PEDV naïve sows. A safe and efficacious live attenuated vaccine that can prime mucosal immune responses is urgently needed. In this study, we evaluated the safety and efficacy of two attenuated PEDV vaccine candidates, the emerging non-S INDEL PEDV strain PC22A at the 100th cell culture passage level - Clone no. 4 (P100C4) and at the 120th passage level (P120), in weaned pigs. RESULTS: Four groups of 40-day-old weaned pigs were inoculated orally with PEDV PC22A-P3 (virulent), -P100C4, -P120, and mock, respectively, and challenged with the P3 virus at 24 days post-inoculation (dpi). After inoculation, P3 caused diarrhea in all pigs with a high level of fecal viral RNA shedding. P100C4 and P120 did not cause diarrhea in pigs, although viral RNA was detected in feces of all pigs, except for one P100C4-inoculated pig. Compared with the P120 group, P3- and P100C4-inoculated pigs had higher serum PEDV-specific IgG and viral neutralizing (VN) antibody (Ab) titers at 14 dpi. After the challenge, no pigs in the P3 group but all pigs in the P100C4, P120, and mock groups had diarrhea. Compared with the P120 group, pigs in the P100C4 group had a more rapid decline in fecal PEDV RNA shedding titers, higher titers of serum PEDV-specific IgG, IgA, and VN Abs, and higher numbers of intestinal IgA Ab-secreting cells. CONCLUSIONS: PEDV PC22A P100C4 and P120 were fully attenuated in weaned pigs but failed to elicit protection against virulent P3 challenge. P100C4 induced higher PEDV-specific antibody responses than P120 post inoculation resulting in a greater anamnestic response post challenge. Therefore, P100C4 potentially could be tested as a priming vaccine or be further modified using reverse genetics. It also can be administered in multiple doses or be combined with inactivated or subunit vaccines and adjuvants as a PEDV vaccination regimen, whose efficacy can be tested in the future.


Assuntos
Infecções por Coronavirus/veterinária , Imunogenicidade da Vacina , Vírus da Diarreia Epidêmica Suína/imunologia , Vacinas Virais/imunologia , Animais , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Diarreia/imunologia , Diarreia/prevenção & controle , Diarreia/veterinária , Vírus da Diarreia Epidêmica Suína/patogenicidade , Distribuição Aleatória , Suínos , Vacinas Atenuadas/imunologia , Desmame
13.
Curr Opin Virol ; 34: 39-49, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30654269

RESUMO

Porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome-coronavirus (SADS-CoV) are emerging/reemerging coronaviruses (CoVs). They cause acute gastroenteritis in neonatal piglets. Sequence analyses suggest that PEDV and SADS-CoV may have originated from bat CoVs and PDCoV from a sparrow CoV, reaffirming the interspecies transmission of CoVs. The clinical signs and pathogenesis of the three viruses are similar. Necrosis of infected intestinal epithelial cells occurs, causing villous atrophy that results in malabsorptive diarrhea. The severe diarrhea and vomiting may lead to dehydration and death of piglets. Natural infection induces protective immunity, but there is no cross-protection among the three viruses. Besides strict biosecurity measures, individual vaccines are needed for each virus for disease prevention and control.


Assuntos
Infecções por Coronavirus/veterinária , Coronavirus/patogenicidade , Diarreia/veterinária , Vírus da Diarreia Epidêmica Suína/patogenicidade , Doenças dos Suínos/virologia , Animais , Coronavirus/classificação , Infecções por Coronavirus/virologia , Diarreia/virologia , Genoma Viral , Humanos , Filogenia , Suínos , Doenças dos Suínos/transmissão
14.
Infect Genet Evol ; 69: 153-165, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30677534

RESUMO

In recent years, the outbreaks of porcine epidemic diarrhea (PED) caused by the highly virulent porcine epidemic diarrhea virus (PEDV) variants occurred frequently in China, resulting in severe economic impacts to the pork industry. In this study, we selected and analyzed the genetic evolution of 15 PEDV representative strains that were identified in fecal samples of diarrheic piglets in 10 provinces and cities during 2011-2017. The phylogenetic analysis indicated that all the 15 PEDV isolates clustered into G2 genotype associated with the current circulating strains. Compared with the genome of the prototype strain CV777, these strains had 103-120 amino acid mutations in their S proteins, most of which were in the N terminal domain of S1 (S1-NTD). We also found 37 common mutations in all these 15 strains, although these strains shared 96.9-99.7% nucleotide homology and 96.3-99.8% amino acid homology in the S protein compared with the other original pandemic strains. Computational analysis showed that these mutations may lead to remarkable changes in the conformational structure and asparagine (N)-linked glycosylation sites of S1-NTD, which may be associated with the altered pathogenicity of these variant PEDV strains. We evaluated the pathogenicity of the PEDV strain FJzz1 in piglets through oral and intramuscular infection routes. Compared with oral infection, intramuscular infection could also cause typical clinical signs but with a slightly delayed onset, confirming that the variant PEDV isolate FJzz1 was highly pathogenic to suckling piglets. In conclusion, we analyzed the genetic variation and pathogenicity of the emerging PEDV isolates of China, indicating that G2 variant PEDV strains as the main prevalent strains that may mutate continually. This study shows the necessity of monitoring the molecular epidemiology and the etiological characteristics of the epidemic PEDV isolates, which may help better control the PED outbreaks.


Assuntos
Infecções por Coronavirus/veterinária , Evolução Molecular , Vírus da Diarreia Epidêmica Suína/genética , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/virologia , Substituição de Aminoácidos , Animais , Linhagem Celular , China/epidemiologia , Fezes/virologia , Genes Virais , Variação Genética , Genótipo , História do Século XXI , Epidemiologia Molecular , Mutação , Filogenia , Vírus da Diarreia Epidêmica Suína/classificação , Vírus da Diarreia Epidêmica Suína/patogenicidade , RNA Viral , Análise de Sequência de DNA , Suínos , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/história
15.
Virus Genes ; 55(2): 174-181, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30637608

RESUMO

Griffithsin is a lectin with potent antiviral activity against enveloped viruses. The objective of this study was to assess Griffithsin's inhibitory effect on porcine epidemic diarrhea virus (PEDV). The results showed that Griffithsin reduced PEDV infection of Vero cells by approximately 82.8%. Moreover, using time-of-addition assays and RT-qPCR, we found that delayed addition of Griffithsin had a weaker inhibitory effect on PEDV than earlier treatment. The mechanism of Griffithsin's action against PEDV involved both preventing viral attachment to host cells and disrupting cell-to-cell transmission; its dual mode of action distinguished Griffithsin from most other antiviral drugs. In conclusion, Griffithsin was identified as a potent PEDV inhibitor and may represent a candidate drug for preventing PEDV infection.


Assuntos
Antivirais/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Lectinas de Plantas/farmacologia , Vírus da Diarreia Epidêmica Suína/efeitos dos fármacos , Animais , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Vírus da Diarreia Epidêmica Suína/patogenicidade , Suínos/virologia , Células Vero/efeitos dos fármacos , Células Vero/virologia , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
16.
Arch Virol ; 164(1): 83-90, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30284628

RESUMO

Porcine epidemic diarrhea (PED) is a highly contagious, acute enteric tract infectious disease of pigs (Sus domesticus) caused by porcine epidemic diarrhea virus (PEDV). PED is characterized by watery diarrhea, dehydration, weight loss, vomiting and death. PEDV damages pig intestinal epithelial tissue, causing intestinal hyperemia and atrophy of intestinal villi, with formation of intestinal epithelial cell cytoplasmic vacuoles. Since pig small intestinal epithelial cells (IECs) are target cells of PEDV infection, IEC cells were utilized as a model for studying changes in cellular activities post-PEDV infection. Monitoring of Na+-K+-ATPase and Ca2+-Mg2+-ATPase activities demonstrated that PEDV infection decreased these activities. In addition, IECs proliferation was shown to decrease after PEDV infection using an MTT assay. Moreover, IECs apoptosis detected by flow cytometry with propidium iodide (PI) staining was clearly shown to increase relative to the control group. Meanwhile, animal experiments indicated that PEDV virulence for IEC cells was greater than viral virulence for Vero cells, although this may be due to viral attenuation after numerous passages in the latter cell line. Collectively, these studies revealed viral pathogenic mechanisms in PEDV-infected IECs and offer a theoretical basis for PEDV prevention and control.


Assuntos
Infecções por Coronavirus/veterinária , Células Epiteliais/virologia , Mucosa Intestinal/citologia , Intestino Delgado/patologia , Vírus da Diarreia Epidêmica Suína/patogenicidade , Animais , ATPase de Ca(2+) e Mg(2+)/metabolismo , Sobrevivência Celular , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Células Epiteliais/patologia , Intestino Delgado/virologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Suínos , Células Vero , Virulência
17.
J Virol ; 93(2)2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30404797

RESUMO

Porcine epidemic diarrhea virus (PEDV) causes high mortality in neonatal piglets. The PEDV spike (S) protein contains two intracellular sorting motifs, YxxΦ (tyrosine-based motif YEVF or YEAF) and KVHVQ at the cytoplasmic tail, yet their functions have not been fully elucidated. Some Vero cell-adapted and/or attenuated PEDV variants contain ablations in these two motifs. We hypothesized that these motifs contribute to viral pathogenicity. By transiently expressing PEDV S proteins with mutations in the motifs, we confirmed that the motif KVHVQ is involved in retention of the S proteins in the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC). In addition, we showed that the YxxΦ motif triggers endocytosis of S proteins. These two motifs synergistically regulate the level of S expressed on the cell surface. To investigate their role in viral pathogenicity, we generated three recombinant PEDVs by introducing deletions or a mutation in the two motifs of the infectious clone of PEDV PC22A strain (icPC22A): (i) icΔ10aa (ΔYxxΦEKVHVQ), (ii) icΔ5aa (ΔKVHVQ), and (iii) icYA (Y1378A, to an inactivated motif, AEVF). Infection of Vero cells with icΔ10aa resulted in larger syncytia and more virions, with reduced numbers of S protein projections on the surface compared with icPC22A. Furthermore, we orally inoculated five groups of 5-day-old gnotobiotic piglets with the three mutants, icPC22A, or a mock treatment. Mutant icΔ10aa caused less severe diarrhea rate and significantly milder intestinal lesions than icPC22A, icΔ5aa, and icYA. These data suggest that the deletion of both motifs can reduce the virulence of PEDV in piglets.IMPORTANCE Many coronaviruses (CoVs) possess conserved motifs YxxΦ and/or KxHxx/KKxx in the cytoplasmic tail of the S protein. The KxHxx/KKxx motif has been identified as the ER retrieval signal, but the function of the YxxΦ motif in the intracellular sorting of CoV S proteins remains controversial. In this study, we showed that the YxxΦ of PEDV S protein is an endocytosis signal. Furthermore, using reverse genetics technology, we evaluated its role in PEDV pathogenicity in neonatal piglets. Our results explain one attenuation mechanism of Vero cell-adapted PEDV variants lacking functional YxxΦ and KVHVQ motifs. Knowledge from this study may aid in the design of efficacious live attenuated vaccines against PEDV, as well as other CoVs bearing the same motif in their S protein.


Assuntos
Vírus da Diarreia Epidêmica Suína/patogenicidade , Deleção de Sequência , Glicoproteína da Espícula de Coronavírus/química , Doenças dos Suínos/virologia , Motivos de Aminoácidos , Animais , Endocitose , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Vírus da Diarreia Epidêmica Suína/genética , Glicoproteína da Espícula de Coronavírus/genética , Suínos , Células Vero , Virulência
18.
Transgenic Res ; 28(1): 21-32, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30315482

RESUMO

The alphacoronaviruses, transmissible gastroenteritis virus (TGEV) and Porcine epidemic diarrhea virus (PEDV) are sources of high morbidity and mortality in neonatal pigs, a consequence of dehydration caused by the infection and necrosis of enterocytes. The biological relevance of amino peptidase N (ANPEP) as a putative receptor for TGEV and PEDV in pigs was evaluated by using CRISPR/Cas9 to edit exon 2 of ANPEP resulting in a premature stop codon. Knockout pigs possessing the null ANPEP phenotype and age matched wild type pigs were challenged with either PEDV or TGEV. Fecal swabs were collected daily from each animal beginning 1 day prior to challenge with PEDV until the termination of the study. The presence of virus nucleic acid was determined by PCR. ANPEP null pigs did not support infection with TGEV, but retained susceptibility to infection with PEDV. Immunohistochemistry confirmed the presence of PEDV reactivity and absence of TGEV reactivity in the enterocytes lining the ileum in ANPEP null pigs. The different receptor requirements for TGEV and PEDV have important implications in the development of new genetic tools for the control of enteric disease in pigs.


Assuntos
Aminopeptidases/genética , Animais Geneticamente Modificados/genética , Infecções por Coronavirus/genética , Coronavirus/patogenicidade , Aminopeptidases/deficiência , Animais , Animais Geneticamente Modificados/virologia , Sistemas CRISPR-Cas , Coronavirus/genética , Infecções por Coronavirus/virologia , Enterócitos/enzimologia , Enterócitos/virologia , Vírus da Diarreia Epidêmica Suína/patogenicidade , Suínos , Vírus da Gastroenterite Transmissível/patogenicidade
19.
Vet Microbiol ; 228: 20-25, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30593368

RESUMO

PEDV is mainly transmitted by the oro-fecal route although PEDV shedding in semen has already been shown for an S-non-InDel PEDV strain infection. The aim of this study was to determine if PEDV can be shed in semen from SPF (specific pathogens free) boars infected by a French S-InDel PEDV strain (PEDV/FR/001/2014) and in case of positive semen to determine the infectivity of that semen. Both infected boars had diarrhea after inoculation and shed virus in feces. PEDV genome was also detected by RT-qPCR in the sperm-rich fraction of semen (6.94 × 103 and 4.73 × 103 genomic copies/mL) from the two boars infected with the S-InDel PEDV strain but only once at 7DPI. In addition, PEDV RNA in Peyer's patches and in mesenteric lymph nodes was also present for the two inoculated boars. The PEDV positive semen (S-non-InDel and S-InDel) sampled during a previous trial and in this boar trial were inoculated to six SPF weaned pigs. The inoculated piglets did not seroconvert and did not shed virus throughout the duration of the study except for one pig at 18 DPI. But, PEDV could be detected in intestinal tissues such as duodenum, jejunum and jejunum Peyer's patches by RT-qPCR except for one pig. Even if PEDV genome has been detected in semen, experimental infection of piglets with positive semen failed to conclude to the infectivity of the detected PEDV.


Assuntos
Infecções por Coronavirus/veterinária , Diarreia/veterinária , Vírus da Diarreia Epidêmica Suína/fisiologia , Doenças dos Suínos/virologia , Animais , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Diarreia/epidemiologia , Diarreia/virologia , Modelos Animais de Doenças , Fezes/virologia , Intestinos/virologia , Masculino , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/imunologia , Vírus da Diarreia Epidêmica Suína/patogenicidade , Sêmen/virologia , Organismos Livres de Patógenos Específicos , Suínos , Doenças dos Suínos/epidemiologia , Eliminação de Partículas Virais
20.
Vet Microbiol ; 228: 202-212, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30593369

RESUMO

Porcine epidemic diarrhea virus (PEDV) variants having a large deletion in the N-terminal domain of the S1 subunit of spike (S) protein were designated as S1 NTD-del PEDVs. They replicate well in experimentally infected pigs. However, on farms they often co-infect pigs with the PEDV containing an intact S protein (S-intact PEDV). We aimed to characterize viral replication and pathogenesis in neonatal gnotobiotic pigs infected simultaneously with the two types of PEDV using two recombinant PEDVs: icPC22A and its S1 NTD-del form icPC22A-S1Δ197. Additionally, viral replication was compared in Vero and IPEC-DQ cells at the presence of bovine mucin (BM), porcine gastric mucin (PGM), swine bile and bile acids during inoculation. In the pigs coinfected with icPC22A and icPC22A-S1Δ197, icPC22A replicated to a higher peak titer than its infection of pigs without the presence of icPC22A-S1Δ197. The severity of diarrhea and intestinal atrophy were similar between icPC22A and the coinfection groups, but were significantly higher than icPC22A-S1Δ197 group. In Vero and IPEC-DQ cells, certain concentrations of BM, PGM, bile and bile acids increased significantly the infectivity of icPC22A but had no or negative effects on icPC22A-S1Δ197. These results indicated that the replication of the S-intact PEDV was enhanced during coinfection in piglets. This observation may be explained partially by the fact that mucin, bile and bile acids in gastrointestinal tract had facilitating effects on the infection of S-intact PEDV, but no/inhibition effects on S1 NTD-del PEDV.


Assuntos
Infecções por Coronavirus/veterinária , Diarreia/veterinária , Vírus da Diarreia Epidêmica Suína/fisiologia , Glicoproteína da Espícula de Coronavírus/genética , Doenças dos Suínos/virologia , Replicação Viral , Animais , Coinfecção/veterinária , Infecções por Coronavirus/virologia , Diarreia/virologia , Fazendas , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/imunologia , Vírus da Diarreia Epidêmica Suína/patogenicidade , Deleção de Sequência , Suínos , Células Vero
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