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Virology ; 533: 77-85, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31128495


Emerging porcine epidemic diarrhea viruses (PEDVs) have caused large economic losses since 2010, and G2b is the prevalent globally epidemic genotype. Given the fastidious isolation of emerging PEDV in cell culture and difficulties in retaining the isolate infectivity upon further in vitro passage, highly attenuated recombinant vesicular stomatitis virus (rVSVMT) was used as a vector to express the PEDV spike (S) protein, aiming to develop a subunit vaccine against G2b viruses. An S protein with 19 of its cytoplasmic domain amino acids deleted could be incorporated into VSV particles, generating rVSVMT (VSVMT-SΔ19) with high efficiency. Our results suggest that VSVMT-SΔ19 could effectively induce PEDV-specific immunity in pigs via intramuscular, but not intranasal, immunization. Notably, immunizations of sows with VSV MT-SΔ19 provided protective lactogenic immunity against a virulent G2b PEDV challenge in piglets. Consequently, recombinant VSVMT may be a promising platform for preparing a subunit vaccine against PEDV.

Infecções por Coronavirus/veterinária , Vírus da Diarreia Epidêmica Suína/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Doenças dos Suínos/prevenção & controle , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/virologia , Feminino , Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Vírus da Diarreia Epidêmica Suína/classificação , Vírus da Diarreia Epidêmica Suína/genética , Glicoproteína da Espícula de Coronavírus/administração & dosagem , Glicoproteína da Espícula de Coronavírus/genética , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/virologia , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/metabolismo , Vacinas Virais/administração & dosagem , Vacinas Virais/genética
Vet Microbiol ; 219: 30-39, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29778202


Vesicular stomatitis virus (VSV) can cause serious vesicular lesions in pigs, and the matrix (M) protein is its predominant virulence factor. Dendritic cells (DCs) act as the bridge between innate and adaptive immune responses. However, the susceptibility of porcine DCs to VSV infection and the role of M protein in modulating the function of infected DCs are still poorly defined. Thus, this study aimed to determine the ability of virulent wild-type VSV(wtVSV) and two attenuated M protein variants (VSVΔM51 and VSVMT) to induce maturation of porcine monocyte-derived DCs (MoDCs) in vitro. It was found that both wtVSV and the M protein mutant VSVs could productively replicate in porcine MoDCs. Infection with wtVSV resulted in weak proinflammatory cytokine responses and interfered with DC maturation via downregulation of the costimulatory molecule complex CD80/86. Whilst VSVΔM51 could activate porcine MoDCs, VSVMT, a highly attenuated recombinant VSV with triple mutations in the M protein, induced a potent maturation of MoDCs, as evidenced by efficient cytokine induction, and upregulation of CD80/86 and MHC class II. Overall, our findings reveal that porcine MoDCs are differentially activated by VSV, dependent on the presence of a functional M protein. M protein plays a crucial role in modulating porcine DC-VSV interactions. The data further support the potential use of VSVMT as a vaccine vector for pigs.

Células Dendríticas/virologia , Monócitos/virologia , Vírus da Estomatite Vesicular Indiana/genética , Proteínas da Matriz Viral/farmacologia , Animais , Moléculas de Adesão Celular/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Interleucina-1beta/biossíntese , Interleucina-1beta/imunologia , Monócitos/imunologia , Monócitos/fisiologia , Proteínas Mutantes/genética , Proteínas Mutantes/imunologia , Proteínas Mutantes/farmacologia , Suínos , Estomatite Vesicular/virologia , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Vírus da Estomatite Vesicular Indiana/imunologia , Vírus da Estomatite Vesicular Indiana/patogenicidade , Proteínas da Matriz Viral/genética
Vaccine ; 33(46): 6268-76, 2015 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-26431989


Vesicular stomatitis virus (VSV) causes a serious vesicular disease responsible for economic losses in the livestock industry. Currently, there are no suitable vaccines to prevent VSV infection. Although the structural matrix (M) protein of VSV has been shown to be a virulence factor in rodent models, its role in the pathogenicity of VSV infection in livestock species is unknown. We hypothesized that VSV with mutations in the M protein represents a novel live attenuated vaccine candidate. To test this, we introduced mutations into VSV M protein using reverse genetics and assessed their attenuation both in vitro and in pigs, an important natural host of VSV. A recombinant VSV with a triple amino acid mutation in M protein (VSVMT) demonstrated a significantly reduced ability to inhibit the type I interferon (IFN) signaling pathway and to shutoff host gene expression compared to WT-VSV and a mutant virus with a single amino acid deletion (VSVΔM51). Inoculation of pigs with VSVMT induced no apparent vesicular lesions but stimulated virus-neutralizing antibodies and animals were protected against virulent VSV challenge infection. These data demonstrate that the M protein is an important virulence factor for VSV in swine and VSVMT represents a novel vaccine candidate for VSV infections in pigs.

Mutação de Sentido Incorreto , Infecções por Rhabdoviridae/veterinária , Doenças dos Suínos/prevenção & controle , Doenças dos Suínos/virologia , Vesiculovirus/imunologia , Proteínas da Matriz Viral/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Infecções por Rhabdoviridae/prevenção & controle , Suínos , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vesiculovirus/genética , Proteínas da Matriz Viral/genética , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Fatores de Virulência/genética , Fatores de Virulência/imunologia
Vaccine ; 30(7): 1313-21, 2012 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-22222871


Vesicular stomatitis virus (VSV) is a promising vector for vaccine and oncolysis, but it can also produce acute diseases in cattle, horses, and swine characterized by vesiculation and ulceration of the tongue, oral tissues, feet, and teats. In experimental animals (primates, rats, and mice), VSV has been shown to lead to neurotoxicities, such as hind limb paralysis. The virus matrix protein (M) and glycoprotein (G) are both major pathogenic determinants of wild-type VSV and have been the major targets for the production of attenuated strains. Existing strategies for attenuation included: (1) deletion or M51R substitution in the M protein (VSVΔM51 or VSVM51R, respectively); (2) truncation of the C-terminus of the G protein (GΔ28). Despite these mutations, recombinant VSV with mutated M protein is only moderately attenuated in animals, whereas there are no detailed reports to determine the pathogenicity of recombinant VSV with truncated G protein at high dose. Thus, a novel recombinant VSV (VSVΔM51-GΔ28) as well as other attenuated VSVs (VSVΔM51, VSV-GΔ28) were produced to determine their efficacy as vaccine vectors with low pathogenicity. In vitro studies indicated that truncated G protein (GΔ28) could play a more important role than deletion of M51 (ΔM51) for attenuation of recombinant VSV. VSVΔM51-GΔ28 was determined to be the most attenuated virus with low pathogenicity in mice, with VSV-GΔ28 also showing relatively reduced pathogenicity. Further, neutralizing antibodies stimulated by VSV-GΔ28 proved to be significantly higher than in mice treated with VSVΔM51-GΔ28. In conclusion, among different attenuated VSVs with mutated M and/or G proteins, recombinant VSV with only truncated G protein (VSV-GΔ28) demonstrated ideal balance between pathogenesis and stimulating a protective immune response. These properties make VSV-GΔ28 a promising vaccine vector and vaccine candidate for preventing vesicular stomatitis disease.

Glicoproteínas de Membrana/genética , Estomatite Vesicular/prevenção & controle , Vírus da Estomatite Vesicular Indiana/imunologia , Proteínas do Envelope Viral/genética , Vacinas Virais/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Linhagem Celular , Cricetinae , Feminino , Imunidade Ativa , Glicoproteínas de Membrana/química , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Deleção de Sequência , Vacinação , Vacinas Atenuadas , Estomatite Vesicular/imunologia , Estomatite Vesicular/virologia , Proteínas do Envelope Viral/química , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Vacinas Virais/genética
Cell Biol Toxicol ; 23(6): 445-57, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17484068


In order to reveal the metabolic reaction to the presence of fenvalerate mediated by P450 in insects, we used the trypan blue exclusion technique and 3-(4,5-dimethylthiazol)-2,5-diphenyltrazolium bromide (MTT) reduction assay to assess the vitality of Trichoplusia ni (Tn) cells treated with fenvalerate, and observed dose- and time-dependent changes in total cellular P450s. In addition, two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were used to identify the proteins involved in the fenvalerate reaction process. Finally, the cDNA of P450 fragments was cloned and real-time RT-PCR was performed. Our data showed that at the 0-15 mumol/L challenge concentration of fenvalerate, at which the vitality of Tn cells was not affected (p > 0.05), there was a tendency toward a dose- and time-response of total cellular P450s, which peaked at the 9 h (p < 0.05) and 12 h (p < 0.01) time points following 12.5 mumol/L stimulation with fenvalerate. The 2-DE assay detected more than 1300 protein spots in each two-dimensional gel, of which 33 spots displayed significant differences. Among the changed spots, three isoforms of P450 were identified. One of the three P450 cDNA fragments (CYP4L4) was cloned and sequenced, and its expression in treated Tn cells increased significantly (p < 0.01). It was found that fenvalerate induced the expression of P450s in insect cells. This suggests that fenvalerate could be metabolized by CYP4L4 through a hydroxylation reaction in insect cells.

Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Insetos/análise , Mariposas/citologia , Mariposas/enzimologia , Nitrilos/farmacologia , Piretrinas/farmacologia , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/genética , Eletroforese em Gel Bidimensional , Indução Enzimática/efeitos dos fármacos , Proteínas de Insetos/química , Mariposas/efeitos dos fármacos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz