RESUMO
Enteric coronaviruses (CoVs) are major pathogens that cause diarrhea in piglets. To date, four porcine enteric CoVs have been identified: transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and HKU2-like porcine enteric alphacoronavirus (PEAV). In this study, we investigated the replicative capacity of these four enteric CoVs in LLC-PK1 cells, a porcine kidney cell line. The results showed that LLC-PK1 cells are susceptible to all four enteric CoVs, particularly to TGEV and PDCoV infections, indicating that LLC-PK1 cells can be applied to porcine enteric CoV research in vitro, particularly for coinfection studies.
Assuntos
Deltacoronavirus/crescimento & desenvolvimento , Gastroenterite Suína Transmissível/virologia , Vírus da Diarreia Epidêmica Suína/crescimento & desenvolvimento , Vírus da Gastroenterite Transmissível/crescimento & desenvolvimento , Replicação Viral/fisiologia , Animais , Linhagem Celular , Chlorocebus aethiops , Suscetibilidade a Doenças , Técnica Indireta de Fluorescência para Anticorpo , Intestino Delgado/virologia , Células LLC-PK1 , Suínos , Doenças dos Suínos/virologia , Células VeroRESUMO
The intestinal organoid culture system is a pathbreaking working model for investigating pathogen-host interactions in the intestines. However, due to the limitations of the first generation of intestinal organoids, basal-out structure and growth in Matrigel, most pathogens can rarely attach to the apical membrane directly and hardly initiate infection. In this study, we first developed a next-generation porcine intestinal organoid culture system, characterized by an apical membrane on the surface, called apical-out. To investigate the infectivity and antiviral immune responses of this apical-out porcine intestinal organoid, a swine enteric virus, transmissible gastroenteritis virus (TGEV), was employed to inoculate the culture system. Both reverse transcription-quantitative PCR (RT-qPCR) and immunofluorescence assay (IFA) analysis demonstrated that TGEV replicated in the apical-out porcine intestinal organoid culture system. Additionally, our results illustrated that TGEV infection significantly upregulated the expression levels of alpha interferon (IFN-α), IFN-λ1, interferon-stimulated gene 15 (ISG15), ISG58, tumor necrosis factor alpha (TNF-α), and interleukin 6 (IL-6) in this culture system. Hence, we successfully developed a porcine intestinal apical-out organoid culture system, which will facilitate the investigation of pathogen-host interactions in pig intestines.IMPORTANCE Intestinal organoids are a newly developed culture system for investigating pathogen-host interactions. Intestinal organoid models have been widely used since their development, because the results obtained from this type of culture model better represent physiological conditions than those from well-established cell lines. The three-dimensional (3D) porcine intestinal organoid model was reported in 2018 and 2019 for the investigation of intestinal pathogens. However, those organoid culture models were basal-out intestinal organoids, which are not suitable for porcine enteric virus research because they invade the intestines via the apical side of epithelial cells on villi. In this study, we developed a porcine apical-out intestinal organoid culture system and verified its infectivity, type I and type III interferon (IFN) antiviral responses, and inflammatory responses following infection by a swine enteric virus. Our results imply that this apical-out porcine intestinal organoid culture system is an ideal model for the investigation of interactions between swine enteric viruses and the intestines.
Assuntos
Células Epiteliais/imunologia , Gastroenterite Suína Transmissível/imunologia , Interações Hospedeiro-Patógeno/imunologia , Mucosa Intestinal/imunologia , Organoides/imunologia , Vírus da Gastroenterite Transmissível/imunologia , Animais , Bioensaio , Quimiocina CXCL10/genética , Quimiocina CXCL10/imunologia , Células Epiteliais/patologia , Células Epiteliais/virologia , Gastroenterite Suína Transmissível/genética , Gastroenterite Suína Transmissível/virologia , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Interferon-alfa/genética , Interferon-alfa/imunologia , Interferons/genética , Interferons/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Mucosa Intestinal/patologia , Mucosa Intestinal/virologia , Organoides/patologia , Organoides/virologia , Suínos , Vírus da Gastroenterite Transmissível/crescimento & desenvolvimento , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Ubiquitinas/genética , Ubiquitinas/imunologia , Replicação ViralRESUMO
Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and porcine deltacoronavirus (PDCoV) share tropism for swine intestinal epithelial cells. Whether mixing of viral components during co-infection alters pathogenic outcomes or viral replication is not known. In this study, we investigated how different coronavirus nucleocapsid (CoV N) proteins interact and affect PEDV replication. We found that PDCoV N and TGEV N can competitively interact with PEDV N. However, the presence of PDCoV or TGEV N led to very different outcomes on PEDV replication. While PDCoV N significantly suppresses PEDV replication, overexpression of TGEV N, like that of PEDV N, increases production of PEDV RNA and virions. Despite partial interchangeability in nucleocapsid oligomerization and viral RNA synthesis, endogenous PEDV N cannot be replaced in the production of infectious PEDV particles. Results from this study give insights into functional compatibilities and evolutionary relationship between CoV viral proteins during viral co-infection and co-evolution.
Assuntos
Interações Microbianas , Proteínas do Nucleocapsídeo/metabolismo , Vírus da Diarreia Epidêmica Suína/crescimento & desenvolvimento , Animais , Chlorocebus aethiops , Coinfecção/virologia , Coronavirus/crescimento & desenvolvimento , Proteínas do Nucleocapsídeo de Coronavírus , Células Epiteliais/virologia , Células HEK293 , Humanos , Vírus da Gastroenterite Transmissível/crescimento & desenvolvimento , Células VeroRESUMO
Because membrane fusion is a crucial step in the process by which enveloped viruses invade host cells, membrane fusion inhibitors can be effective drugs against enveloped viruses. We found that surfactin from Bacillus subtilis can suppress the proliferation of porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) in epithelial cells at a relatively low concentration range (15 to 50 µg/ml), without cytotoxicity or viral membrane disruption. Membrane fusion inhibition experiments demonstrate that surfactin treatment significantly reduces the rate at which the virus fuses to the cell membrane. Thermodynamic experiments show that the incorporation of small amounts of surfactin hinders the formation of negative curvature by lamellar-phase lipids, suggesting that surfactin acts a membrane fusion inhibitor. A fluorescent lipopeptide similar to surfactin was synthesized, and its ability to insert into the viral membrane was confirmed by spectroscopy. In vivo experiments have shown that oral administration of surfactin to piglets protects against PEDV infection. In conclusion, our study indicates that surfactin is a membrane fusion inhibitor with activity against enveloped viruses. As the first reported naturally occurring wedge lipid membrane fusion inhibitor, surfactin is likely to be a prototype for the development of a broad range of novel antiviral drugs.IMPORTANCE Membrane fusion inhibitors are a rapidly emerging class of antiviral drugs that inhibit the infection process of enveloped viruses. They can be classified, on the basis of the viral components targeted, as fusion protein targeting or membrane lipid targeting. Lipid-targeting membrane fusion inhibitors have a broader antiviral spectrum and are less likely to select for drug-resistant mutations. Here we show that surfactin is a membrane fusion inhibitor and has a strong antiviral effect. The insertion of surfactin into the viral envelope lipids reduces the probability of viral fusion. We also demonstrate that oral administration of surfactin protects piglets from PEDV infection. Surfactin is the first naturally occurring wedge lipid membrane fusion inhibitor that has been identified and may be effective against many viruses beyond the scope of this study. Understanding its mechanism of action provides a foundation for the development of novel antiviral agents.
Assuntos
Antivirais/farmacologia , Lipopeptídeos/farmacologia , Peptídeos Cíclicos/farmacologia , Vírus da Diarreia Epidêmica Suína/crescimento & desenvolvimento , Vírus da Gastroenterite Transmissível/crescimento & desenvolvimento , Ligação Viral/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Animais , Bacillus subtilis/metabolismo , Linhagem Celular , Membrana Celular/virologia , Células Epiteliais/virologia , Camundongos , Camundongos Endogâmicos BALB C , SuínosRESUMO
To gain a better understanding of the replication, proliferation and infection characteristics of porcine transmissible gastroenteritis virus (TGEV) in porcine intestinal epithelial cells (IECs), this study established a cell model of IECs infected with the Chongqing (CQ) strain of TGEV. The morphogenesis and proliferative rule of TGEV in porcine IECs were investigated using transmission electron microscopy, indirect immunofluorescence assays and real-time fluorescence quantitative PCR. Observations under the TEM indicated that the enveloped viral particles were roughly spherical, with diameters of between 80 and 120nm. The virions entered porcine IECs by membrane fusion and the mature viruses in the vacuoles were transported to the cell membrane before release. The results also showed that from 0 to 12h after TGEV infection of porcine IECs, the intracellular viral RNA content did not change significantly. Logarithmic growth occurred from 12 to 36h, after which it gradually decreased. Moreover, the extracellular RNA content began to rise at 24h after inoculation and then reduced gradually at approximately 48h. This study provided a theoretical foundation for further study on the infection characteristics of TGEV in target cells.
Assuntos
Células Epiteliais/virologia , Mucosa Intestinal/citologia , Vírus da Gastroenterite Transmissível/fisiologia , Vírus da Gastroenterite Transmissível/ultraestrutura , Animais , Membrana Celular/virologia , Células Epiteliais/ultraestrutura , Mucosa Intestinal/virologia , Microscopia Eletrônica de Transmissão , RNA Viral/análise , Suínos , Vírus da Gastroenterite Transmissível/crescimento & desenvolvimento , Virologia/métodos , Replicação ViralRESUMO
Transmissible gastroenteritis virus (TGEV), a porcine enteropathogenic coronavirus, causes lethal watery diarrhea and severe dehydration in piglets. In this study, liquid chromatography-tandem mass spectrometry coupled to isobaric tags for relative and absolute quantification labeling was used to quantitatively identify differentially expressed cellular proteins after TGEV infection in PK-15 cells. In total, 162 differentially expressed cellular proteins were identified, including 60 upregulated proteins and 102 downregulated proteins. These differentially expressed proteins were involved in the cell cycle, cellular growth and proliferation, the innate immune response, etc. Interestingly, many upregulated proteins were associated with interferon signaling, especially signal transducer and activator of transcription 1 (STAT1) and interferon-stimulated genes (ISGs). Immunoblotting and real-time quantitative reverse transcription polymerase chain reaction demonstrated that TGEV infection induces STAT1 phosphorylation and nuclear translocation, as well as ISG expression. This study for the first time reveals that TGEV induces interferon signaling from the point of proteomic analysis.
Assuntos
Janus Quinases/metabolismo , Proteoma/análise , Proteômica/métodos , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Vírus da Gastroenterite Transmissível/crescimento & desenvolvimento , Animais , Western Blotting , Linhagem Celular , Cromatografia Líquida , Expressão Gênica , Interações Hospedeiro-Patógeno , Janus Quinases/genética , Rim/citologia , Rim/metabolismo , Rim/virologia , Microscopia Confocal , Fosforilação , Proteoma/genética , Proteoma/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT1/genética , Suínos , Espectrometria de Massas em Tandem , Vírus da Gastroenterite Transmissível/fisiologiaRESUMO
The enhanced effect of cytokine combinations has been assessed empirically, based on their immunobiological mechanisms. However, far less is known of the enhanced protection of practical cytokine combinations against viral infection in the livestock industry, due to cost and production issues associated with mass administration. This study demonstrates the enhanced protection of oral co-administration of swine interferon-α (swIFN-α) and interleukin-18 (swIL-18) against infection with transmissible gastroenteritis virus (TGEV) in piglets using attenuated Salmonella enterica serovar Typhimurium as carrier of cytokine proteins. A single oral co-administration of S. enterica serovar Typhimurium expressing swIFN-α and swIL-18 induced enhanced alleviation of the severity of diarrhea caused by TGEV infection, compared to piglets administered S. enterica serovar Typhimurium expressing swIFN-α or swIL-18 alone. This enhancement was further observed by the reduction of TGEV shedding and replication, and the expression of IFN-stimulated gene products in the intestinal tract. The results suggest that the combined administration of the swIFN-α and swIL-18 cytokines using attenuated S. enterica serovar Typhimurium as an oral carrier provides enhanced protection against intestinal tract infection with TGEV.
Assuntos
Gastroenterite Suína Transmissível/prevenção & controle , Imunidade Ativa , Interferon-alfa/imunologia , Interleucina-18/imunologia , Intestinos/imunologia , Salmonella typhimurium/genética , Vírus da Gastroenterite Transmissível/efeitos dos fármacos , Vacinação , Vacinas Atenuadas/administração & dosagem , Administração Oral , Animais , Feminino , Gastroenterite Suína Transmissível/imunologia , Gastroenterite Suína Transmissível/metabolismo , Gastroenterite Suína Transmissível/virologia , Interferon-alfa/genética , Interleucina-18/genética , Intestinos/virologia , Gado , Camundongos , Plasmídeos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Salmonella typhimurium/química , Salmonella typhimurium/imunologia , Suínos , Transfecção , Vírus da Gastroenterite Transmissível/crescimento & desenvolvimento , Vírus da Gastroenterite Transmissível/imunologia , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Carga Viral/efeitos dos fármacos , Carga Viral/imunologia , Replicação Viral/efeitos dos fármacos , Replicação Viral/imunologiaRESUMO
Porcine aminopeptidase N (pAPN) is a cellular receptor of transmissible gastroenteritis virus (TGEV), a porcine coronavirus. Interaction between the spike (S) protein of TGEV and pAPN initiates cell infection. Small molecules, especially peptides are an expanding area for therapy or diagnostic assays for viral diseases. Here, the peptides capable of binding the pAPN were, for the first time, identified by biopanning using a random 12-mer peptide library to the immobilized protein. Three chemically synthesized peptides recognizing the pAPN showed effective inhibition ability to TGEV infection in vitro. A putative TxxF motif was identified in the S protein of TGEV. Phages bearing the specific peptides interacted with the pAPN in ELISA. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays confirmed the protective effect of the peptides on cell infection by TGEV. Moreover, the excellent immune responses in mice induced by the identified phages provided the possibility to develop novel phage-based vaccines.
Assuntos
Antivirais/farmacologia , Antígenos CD13/antagonistas & inibidores , Infecções por Coronavirus/prevenção & controle , Inibidores Enzimáticos/farmacologia , Peptídeos/farmacologia , Vírus da Gastroenterite Transmissível/efeitos dos fármacos , Animais , Antivirais/isolamento & purificação , Linhagem Celular , Inibidores Enzimáticos/isolamento & purificação , Camundongos , Biblioteca de Peptídeos , Peptídeos/isolamento & purificação , Ligação Proteica , Receptores Virais/antagonistas & inibidores , Suínos , Vírus da Gastroenterite Transmissível/crescimento & desenvolvimento , Carga Viral , Vacinas Virais/imunologia , Ligação Viral/efeitos dos fármacosAssuntos
Infecções por Coronavirus/transmissão , Coronavirus , Transmissão de Doença Infecciosa , Pessoal de Saúde , Roupa de Proteção/virologia , Equipamentos de Proteção/virologia , Animais , Linhagem Celular , Coronavirus/crescimento & desenvolvimento , Coronavirus/isolamento & purificação , Infecções por Coronavirus/virologia , Contaminação de Equipamentos , Humanos , Medição de Risco , Suínos , Vírus da Gastroenterite Transmissível/crescimento & desenvolvimento , Vírus da Gastroenterite Transmissível/isolamento & purificação , Ativação ViralRESUMO
A recombinant transmissible gastroenteritis coronavirus (rTGEV) in which E gene was deleted (rTGEV-DeltaE) has been engineered. This deletion mutant only grows in cells expressing E protein (E(+) cells) indicating that E was an essential gene for TGEV replication. Electron microscopy studies of rTGEV-DeltaE infected BHK-pAPN-E(-) cells showed that only immature intracellular virions were assembled. These virions were non-infectious and not secreted to the extracellular medium in BHK-pAPN-E(-) cells. RNA and protein composition analysis by RNase-gold and immunoelectron microscopy showed that rTGEV-DeltaE virions contained RNA and also all the structural TGEV proteins, except the deleted E protein. Nevertheless, full virion maturation was blocked. Studies of the rTGEV-DeltaE subcellular localization by confocal and immunoelectron microscopy in infected E(-) cells showed that in the absence of E protein virus trafficking was arrested in the intermediate compartment. Therefore, the absence of E protein in TGEV resulted in two actions, a blockade of virus trafficking in the membranes of the secretory pathway, and prevention of full virus maturation.
Assuntos
Deleção de Genes , Genes Essenciais , Vírus da Gastroenterite Transmissível/crescimento & desenvolvimento , Vírus da Gastroenterite Transmissível/fisiologia , Proteínas do Envelope Viral/genética , Animais , Linhagem Celular , Cricetinae , Células LLC-PK1/virologia , Suínos , Vírus da Gastroenterite Transmissível/genética , Vírus da Gastroenterite Transmissível/metabolismo , Proteínas do Envelope Viral/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Vírion/crescimento & desenvolvimento , Vírion/metabolismo , Replicação ViralRESUMO
A novel multiplex reverse transcription polymerase chain reaction (multiplex RT-PCR) that can detect porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine group A rotavirus (GAR) was developed. The 3 viruses (PEDV, TGEV, and porcine GAR) are major agents in viral enteric diseases of piglets. As the clinical signs of these diseases are similar, including watery diarrhea, differential detection is required for etiologic diagnosis. A mixture of 3 pairs of published primers was used for amplification of viral nucleic acids, yielding 3 different amplicons with sizes of 859 bp, 651 bp, and 309 bp for TGEV, PEDV, and porcine GAR, respectively. A total of 157 specimens (78 fecal and 79 intestinal samples) from piglets with acute gastroenteritis were collected in Korea between January 2004 and May 2005. They were tested for the presence of 3 viruses by multiplex RT-PCR. Coinfections with PEDV and porcine GAR were identified in 16 farms (43.2%). PEDV, porcine GAR, and TGEV infection were 26.3%, 13.2%, and 2.7% respectively. The relative sensitivity and specificity of multiplex RT-PCR were evaluated, with results suggesting that this assay is equal in quality to conventional single-agent RT-PCR assays (sensitivity:100%, 92.9%, 100% for TGEV, PEDV, GARs; specificity: 100% for all 3 viruses). This multiplex RT-PCR is a simple assay and may be a potentially useful for rapid, sensitive, and cost-effective etiological diagnostic tool for acute viral gastroenteritis in piglets.
Assuntos
Infecções por Coronavirus/veterinária , Coronavirus/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Infecções por Rotavirus/veterinária , Rotavirus/genética , Doenças dos Suínos/virologia , Animais , Animais Recém-Nascidos , Coronavirus/crescimento & desenvolvimento , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Diagnóstico Diferencial , Diarreia/diagnóstico , Diarreia/veterinária , Diarreia/virologia , Fezes/virologia , Gastroenterite Suína Transmissível/diagnóstico , Gastroenterite Suína Transmissível/virologia , Coreia (Geográfico) , RNA Viral/química , RNA Viral/genética , Distribuição Aleatória , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Rotavirus/crescimento & desenvolvimento , Infecções por Rotavirus/diagnóstico , Infecções por Rotavirus/virologia , Sensibilidade e Especificidade , Suínos , Doenças dos Suínos/diagnóstico , Vírus da Gastroenterite Transmissível/genética , Vírus da Gastroenterite Transmissível/crescimento & desenvolvimentoRESUMO
Assuming a synergistic or additive effect of Chlamydiaceae in coexistence with other enteropathogenic agents, the viral/bacterial interaction between a cell culture adapted porcine epidemic diarrhea virus (ca-PEDV) and different Chlamydiaceae strains was studied in vitro. Vero cells were dually infected with ca-PEDV and one of the three chlamydial strains Chlamydia trachomatis S45, Chlamydophila abortus S26/3 or Chlamydophila pecorum 1710S. Three experimental protocols were designed varying the inoculation sequence. Cell layers were first inoculated with Chlamydiaceae and 20 h later with ca-PEDV in protocol one. In protocol two, both agents were administered concurrently, whereas in protocol three, ca-PEDV was applied 20 h in advance of the Chlamydiaceae. Immunofluorescence techniques, immunohistochemical (IH) staining and electron microscopy were subsequently employed to investigate the cell layers. Using indirect immunofluorescence (IF) labeling, all mixed infections revealed dually infected cells, however, only incidentally and in low numbers. Characteristically, ca-PEDV syncytia with one or more chlamydial inclusions were detected but dually infected single cells were absent. Some syncytial cells contained enlarged C. abortus or C. pecorum inclusions with abnormally large developmental forms. In comparison with simultaneously conducted monoinfections, larger chlamydial inclusions were observed in dually infected cell layers. Experiments with C. trachomatis showed significantly increased numbers of chlamydial inclusions in dually infected cell layers compared to monoinfected ones. These findings indicate an influence of ca-PEDV on the chlamydial developmental cycle and in the case of C. trachomatis, a positive effect on chlamydial colonization in mixed infections.
Assuntos
Técnicas de Cultura de Células/veterinária , Chlamydiaceae/crescimento & desenvolvimento , Vírus da Gastroenterite Transmissível/crescimento & desenvolvimento , Animais , Técnicas de Cultura de Células/métodos , Chlamydiaceae/fisiologia , Chlorocebus aethiops , Técnicas de Cocultura/métodos , Técnicas de Cocultura/veterinária , Técnica Indireta de Fluorescência para Anticorpo/veterinária , Imuno-Histoquímica/veterinária , Microscopia Eletrônica de Transmissão/veterinária , Fatores de Tempo , Vírus da Gastroenterite Transmissível/fisiologia , Células VeroRESUMO
The effect of storage temperature was determined for the detection of porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) in fecal samples from experimentally and naturally infected pigs by multiplex reverse transcription-polymerase chain reaction (RT-PCR). To examine the effect of storage temperature on the ability to detect PEDV and TGEV RNA by multiplex RT-PCR, fecal samples were stored for different temperatures (4, 21, 36, and 45 C) before extracting viral RNA. The PEDV and TGEV nucleic acids in fecal samples were stable up to 3 days at 4 C. The PEDV and TGEV nucleic acids were consistently detected in fecal samples up to 60 hours at 21 C and 24 hours only at 36 and 45 C. Thereafter, the number of positive signals declined. Positive signals were detected in fecal samples stored at 4 C by 240 hours. The PEDV and TGEV nucleic acids were consistently detected in fecal samples up to 60 hours at 21 C and 24 hours only at 36 and 45 C. The results of this study suggested that storage temperature has significant effect on the detection of PEDV and TGEV nucleic acids from fecal samples by multiplex RT-PCR.
Assuntos
Diarreia/veterinária , Fezes/virologia , Gastroenterite Suína Transmissível/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Vírus da Gastroenterite Transmissível/crescimento & desenvolvimento , Animais , Diarreia/diagnóstico , Diarreia/virologia , Gastroenterite Suína Transmissível/diagnóstico , RNA Viral/química , RNA Viral/genética , Distribuição Aleatória , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Manejo de Espécimes/métodos , Manejo de Espécimes/veterinária , Suínos , Temperatura , Vírus da Gastroenterite Transmissível/genéticaRESUMO
The genome of the coronavirus transmissible gastroenteritis virus (TGEV) has been engineered as an expression vector with an infectious cDNA. The vector led to the efficient (>40 micro g/10(6) cells) and stable (>20 passages) expression of a heterologous gene (green fluorescent protein [GFP]), driven by the transcription-regulating sequences (TRS) of open reading frame (ORF) 3a inserted in the site previously occupied by the nonessential ORFs 3a and 3b. Expression levels driven by this TRS were higher than those of an expression cassette under the control of regulating sequences engineered with the N gene TRS. The recombinant TGEV including the GFP gene was still enteropathogenic, albeit with a 10- to 10(2)-fold reduction in enteric tissue growth. Interestingly, a specific lactogenic immune response against the heterologous protein has been elicited in sows and their progeny. The engineering of an additional insertion site for the heterologous gene between viral genes N and 7 led to instability and to a new genetic organization of the 3' end of the recombinant viruses. As a consequence, a major species of subgenomic mRNA was generated from a TRS with the noncanonical core sequence 5'-CUAAAA-3'. Extension of the complementarity between the TRS and sequences at the 3' end of the viral leader was associated with transcriptional activation of noncanonical core sequences. The engineered vector led to expression levels as high as those of well-established vectors and seems very promising for the development of vaccines and, possibly, for gene therapy.
Assuntos
Vetores Genéticos , Genoma Viral , Vírus da Gastroenterite Transmissível/genética , Vírus da Gastroenterite Transmissível/imunologia , Animais , Sequência de Bases , Linhagem Celular , Colostro/imunologia , Cricetinae , DNA Viral/genética , Feminino , Gastroenterite Suína Transmissível/imunologia , Gastroenterite Suína Transmissível/prevenção & controle , Engenharia Genética , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Dados de Sequência Molecular , Fases de Leitura Aberta , Gravidez , RNA Mensageiro/genética , RNA Viral/genética , Proteínas Recombinantes/genética , Sus scrofa , Suínos , Vírus da Gastroenterite Transmissível/crescimento & desenvolvimentoRESUMO
A systematic method was developed to assemble functional full-length genomes of large RNA and DNA viruses. Coronaviruses contain the largest single-stranded positive-polarity RNA genome in nature. The approximately 30-kb genome, coupled with regions of genomic instability, has hindered the development of a full-length infectious cDNA construct. We have assembled a full-length infectious construct of transmissible gastroenteritis virus (TGEV), an important pathogen in swine. Using a novel approach, six adjoining cDNA subclones that span the entire TGEV genome were isolated. Each clone was engineered with unique flanking interconnecting junctions which determine a precise systematic assembly with only the adjacent cDNA subclones, resulting in an intact TGEV cDNA construct of approximately 28.5 kb in length. Transcripts derived from the full-length TGEV construct were infectious, and progeny virions were serially passaged in permissive host cells. Viral antigen production and subgenomic mRNA synthesis were evident during infection and throughout passage. Plaque-purified virus derived from the infectious construct replicated efficiently and displayed similar plaque morphology in permissive host cells. Host range phenotypes of the molecularly cloned and wild-type viruses were similar in cells of swine and feline origin. The recombinant viruses were sequenced across the unique interconnecting junctions, conclusively demonstrating the marker mutations and restriction sites that were engineered into the component clones. Full-length infectious constructs of TGEV will permit the precise genetic modification of the coronavirus genome. The method that we have designed to generate an infectious cDNA construct of TGEV could theoretically be used to precisely reconstruct microbial or eukaryotic genomes approaching several million base pairs in length.
Assuntos
Genoma Viral , Vírus da Gastroenterite Transmissível/metabolismo , Montagem de Vírus , Animais , Linhagem Celular , Clonagem Molecular , DNA Viral/genética , Imunofluorescência , Marcadores Genéticos , Mutagênese , Mutação , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Suínos , Transfecção , Vírus da Gastroenterite Transmissível/crescimento & desenvolvimento , Vírus da Gastroenterite Transmissível/isolamento & purificaçãoRESUMO
The prevailing hypothesis is that the intracellular site of budding of coronaviruses is determined by the localization of its membrane protein M (previously called E1). We tested this by analyzing the site of budding of four different coronaviruses in relation to the intracellular localization of their M proteins. Mouse hepatitis virus (MHV) and infectious bronchitis virus (IBV) grown in Sac(-) cells, and feline infectious peritonitis virus (FIPV) and transmissible gastroenteritis virus (TGEV) grown in CrFK cells, all budded exclusively into smooth-walled, tubulovesicular membranes located intermediately between the rough endoplasmic reticulum and Golgi complex, identical to the so-called budding compartment previously identified for MHV. Indirect immunofluorescence staining of the infected cells showed that all four M proteins accumulated in a perinuclear region. Immunogold microscopy localized MHV M and IBV M in the budding compartment; in addition, a dense labeling in the Golgi complex occurred, MHV M predominantly in trans-Golgi cisternae and trans-Golgi reticulum and IBV M mainly in the cis and medial Golgi cisternae. The corresponding M proteins of the four viruses, when independently expressed in a recombinant vaccinia virus system, also accumulated in the perinuclear area. Quantitative pulse-chase analysis of metabolically labeled cells showed that in each case the majority of the M glycoproteins carried oligosaccharide side chains with Golgi-specific modifications within 4 h after synthesis. Immunoelectron microscopy localized recombinant MHV M and IBV M to the same membranes as the respective proteins in coronavirus-infected cells, with the same cis-trans distribution over the Golgi complex. Our results demonstrate that some of the M proteins of the four viruses are transported beyond the budding compartment and are differentially retained by intrinsic retention signals; in addition to M, other viral and/or cellular factors are probably required to determine the site of budding.
Assuntos
Coronaviridae/crescimento & desenvolvimento , Complexo de Golgi/microbiologia , Proteínas da Matriz Viral/biossíntese , Animais , Gatos , Linhagem Celular , Coronaviridae/metabolismo , Coronaviridae/ultraestrutura , Coronavirus Felino/crescimento & desenvolvimento , Coronavirus Felino/metabolismo , Coronavirus Felino/ultraestrutura , Feto , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Humanos , Vírus da Bronquite Infecciosa/crescimento & desenvolvimento , Vírus da Bronquite Infecciosa/metabolismo , Vírus da Bronquite Infecciosa/ultraestrutura , Rim , Microscopia Eletrônica , Microscopia Imunoeletrônica , Vírus da Hepatite Murina/crescimento & desenvolvimento , Vírus da Hepatite Murina/metabolismo , Vírus da Hepatite Murina/ultraestrutura , Recombinação Genética , Suínos , Vírus da Gastroenterite Transmissível/crescimento & desenvolvimento , Vírus da Gastroenterite Transmissível/metabolismo , Vírus da Gastroenterite Transmissível/ultraestrutura , Células Tumorais Cultivadas , Proteínas da Matriz Viral/análiseAssuntos
Vírus da Gastroenterite Transmissível/isolamento & purificação , Animais , Animais Recém-Nascidos , Linhagem Celular , Células Cultivadas/microbiologia , Embrião de Mamíferos , Fezes/microbiologia , Técnicas Imunoenzimáticas , Rim , Inoculações Seriadas , Suínos , Vírus da Gastroenterite Transmissível/crescimento & desenvolvimento , Vírus da Gastroenterite Transmissível/patogenicidade , Virulência , Cultura de Vírus/métodosRESUMO
Twenty-four field isolates of transmissible gastroenteritis virus (TGEV) were isolated and examined for antigenic and biological characteristics. Most TGEV isolates produced a typical cytopathic effect (CPE) in swine testis (ST) cell culture, which included a ballooning or lifting away of the infected cells from the cell monolayer with heavy granulation evident. Minor variations in CPE were observed with one isolate, IA-145. Protein profiles of the TGEV isolates as determined by SDS-PAGE were essentially identical, with the exception of the isolate IA-101. The TGEV isolate IA-101 presented a higher molecular mass M protein and lacked an N protein doublet that was present in all other TGEV isolates. The TGEV isolates were shown to be closely related antigenically by using hyperimmune sera in a virus neutralization (VN) test. Some antigenic diversity was detected by utilizing monoclonal antibodies (mAbs) in a VN test. Titers of the mAbs were highest with the homologous Miller TGEV, and one virus isolate, IA-156, was very poorly neutralized with the mAbs used in this study. Indirect immunofluorescence assay (IFA) results were similar to those obtained by the VN test. These studies show that some biologic and antigenic diversity exists among TGEV isolates.
Assuntos
Variação Antigênica , Gastroenterite Suína Transmissível/microbiologia , Vírus da Gastroenterite Transmissível/imunologia , Animais , Antígenos Virais/análise , Linhagem Celular , Efeito Citopatogênico Viral , Imunofluorescência , Masculino , Testes de Neutralização , Ensaio de Radioimunoprecipitação , Suínos , Testículo/citologia , Testículo/microbiologia , Vírus da Gastroenterite Transmissível/classificação , Vírus da Gastroenterite Transmissível/crescimento & desenvolvimento , Proteínas Virais/análiseRESUMO
The CPK cells derived from swine kidney were infected with the attenuated TO-163 strain of transmissible gastroenteritis (TGE) virus, and fused with uninfected Vero cells in the presence of polyethylene glycol. Repeated cocultivation of the fused cells with uninfected Vero cells rendered the virus to grow in Vero cells. The Vero cell-adapted virus acquired the ability to infect and produce cytopathic effects in several other non-permissive cell lines of non-porcine origin. No major differences in viral polypeptides were shown between the Vero cell-adapted TO-163 strain and its parent strain by indirect immunofluorescence and Western blotting using monoclonal and polyclonal antibodies to TGE virus.
Assuntos
Vírus da Gastroenterite Transmissível/crescimento & desenvolvimento , Cultura de Vírus , Adaptação Fisiológica , Animais , Linhagem Celular , Humanos , Polietilenoglicóis , Inoculações Seriadas , Células VeroRESUMO
Plaque formation, replication and related cytopathic function of 9 strains of transmissible gastroenteritis (TGE) virus were examined in primary cells and cell lines such as CPK, IB-RS-2, ESK, and PK-15 originated from porcine kidney and the effects of trypsin on the replication of TGE virus were examined in CPK cells. All strains produced a cytopathic effect and grew well in CPK cells as well as in primary porcine kidney cells. The effect of trypsin on the plaque formation was different from strains. The number of plaques produced by strains TO-163, Ukiha and Niigata increased from 2.6 to 3.52 times when trypsin was present in the medium during incubation at 37 degrees C for 1 hr after adsorption of the virus at 4 degrees C for 2 hr. The plaque sizes of TO-163, h-5, Ukiha and Niigata became larger from 1.4 to 1.7 times, when trypsin was present in the agar MEM overlay.