RESUMO
The human parainfluenza virus type 3 (HPIV3) phosphoprotein (P) gene is unusual as it contains an editing site where nontemplated ribonucleotide residues can be inserted. This RNA editing can lead to the expression of the viral P, PD, putative W, and theoretical V protein from a single gene. Although the HPIV3 PD protein has been detected, its function and those of the W and V proteins are poorly understood. Therefore, we first used reverse genetics techniques to construct and rescue a recombinant (r)HPIV3 clone with a polyhistidine sequence at the 5' end of the P gene for tagged protein detection. Western blot analysis demonstrated the presence of the P, PD, and W proteins, but no V protein was detected. Then, we functionally studied the D domain of the PD protein by constructing two rHPIV3 knockout clones that are deficient in the expression of the D domain. Results from growth kinetic studies with infected MA-104 and A596 cells showed that viral replication of the two knockout viruses (rHPIV3-ΔES and rHPIV3-ΔD) was comparable to that of the parental virus in both cell lines. However, viral mRNA transcription and genomic replication was significantly reduced. Furthermore, cytokine/chemokine profiles of A549 cells infected with either knockout virus were unchanged or showed lower levels compared to those from cells infected with the parental virus. These data suggest that the D domain of the PD protein may play a luxury role in HPIV3 RNA synthesis and may also be involved in disrupting the expression of beta interferon.
Assuntos
Interferon beta/genética , Vírus da Parainfluenza 3 Humana/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , RNA Viral/genética , Infecções por Respirovirus/genética , Proteínas Virais/química , Proteínas Virais/metabolismo , Linhagem Celular , Regulação para Baixo , Humanos , Interferon beta/imunologia , Vírus da Parainfluenza 3 Humana/química , Vírus da Parainfluenza 3 Humana/genética , Fosfoproteínas/genética , Estrutura Terciária de Proteína , RNA Viral/metabolismo , Infecções por Respirovirus/imunologia , Infecções por Respirovirus/virologia , Deleção de Sequência , Proteínas Virais/genéticaRESUMO
Bluetongue virus (BTV) genome contains ten double-stranded RNA segments. The sequence of the plus strand of each of the BTV genomic double-stranded RNAs is the same as that of its mRNA, which encodes for a single viral protein, except the smallest S4 segment which can encode for two nonstructural proteins, primarily for the release assistance of the viral progeny. The separation and isolation of each BTV dsRNA segment and viral protein have provided extensive data related to its viral infection, pathology, suppression of host cellular functions, and eventual apoptosis of the infected host cells. This cytoplasmic virus is also an animal killer that costs the U.S. livestock industry at least $125 million yearly. However, this virus has no known effect on humans. Thus, it is very safe to carry out investigation with the virus, preferably in a BSL-2 laboratory.
Assuntos
Vírus Bluetongue/química , Vírus Bluetongue/genética , RNA Viral/isolamento & purificação , Proteínas Virais/isolamento & purificação , Virologia/métodos , Animais , Contenção de Riscos Biológicos , Humanos , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/isolamento & purificação , RNA Viral/genética , Proteínas Virais/genéticaRESUMO
Humans are sero-negative toward bluetongue viruses (BTVs) since BTVs do not infect normal human cells. Infection and selective degradation of several human cancer cell lines but not normal ones by five US BTV serotypes have been investigated. We determined the susceptibilities of many normal and human cancer cells to BTV infections and made comparative kinetic analyses of their cytopathic effects, survival rates, ultra-structural changes, cellular apoptosis and necrosis, cell cycle arrest, cytokine profiles, viral genome, mRNAs, and progeny titers. The wild-type US BTVs, without any genetic modifications, could preferentially infect and degrade several types of human cancer cells but not normal cells. Their selective and preferential BTV-degradation of human cancer cells is viral dose-dependent, leading to effective viral replication, and induced apoptosis. Xenograft tumors in mice were substantially reduced by a single intratumoral BTV injection in initial in vivo experiments. Thus, wild-type BTVs, without genetic modifications, have oncolytic potentials. They represent an attractive, next generation of oncolytic viral approach for potential human cancer therapy combined with current anti-cancer agents and irradiation.
RESUMO
Urtica dioica agglutinin (UDA) is a small plant monomeric lectin, 8.7 kDa in size, with an N-acetylglucosamine specificity that inhibits viruses from Nidovirales in vitro. In the current study, we first examined the efficacy of UDA on the replication of different SARS-CoV strains in Vero 76 cells. UDA inhibited virus replication in a dose-dependent manner and reduced virus yields of the Urbani strain by 90% at 1.1 ± 0.4 µg/ml in Vero 76 cells. Then, UDA was tested for efficacy in a lethal SARS-CoV-infected BALB/c mouse model. BALB/c mice were infected with two LD50 (575 PFU) of virus for 4 h before the mice were treated intraperitoneally with UDA at 20, 10, 5 or 0 mg/kg/day for 4 days. Treatment with UDA at 5 mg/kg significantly protected the mice against a lethal infection with mouse-adapted SARS-CoV (p < 0.001), but did not significantly reduce virus lung titers. All virus-infected mice receiving UDA treatments were also significantly protected against weight loss (p < 0.001). UDA also effectively reduced lung pathology scores. At day 6 after virus exposure, all groups of mice receiving UDA had much lower lung weights than did the placebo-treated mice. Thus, our data suggest that UDA treatment of SARS infection in mice leads to a substantial therapeutic effect that protects mice against death and weight loss. Furthermore, the mode of action of UDA in vitro was further investigated using live SARS-CoV Urbani strain virus and retroviral particles pseudotyped with SARS-CoV spike (S). UDA specifically inhibited the replication of live SARS-CoV or SARS-CoV pseudotyped virus when added just before, but not after, adsorption. These data suggested that UDA likely inhibits SARS-CoV infection by targeting early stages of the replication cycle, namely, adsorption or penetration. In addition, we demonstrated that UDA neutralizes the virus infectivity, presumably by binding to the SARS-CoV spike (S) glycoprotein. Finally, the target molecule for the inhibition of virus replication was partially characterized. When UDA was exposed to N-acetylglucosamine and then UDA was added to cells just prior to adsorption, UDA did not inhibit the virus infection. These data support the conclusion that UDA might bind to N-acetylglucosamine-like residues present on the glycosylated envelope glycoproteins, thereby preventing virus attachment to cells.
Assuntos
Antivirais/administração & dosagem , Lectinas de Plantas/administração & dosagem , Síndrome Respiratória Aguda Grave/tratamento farmacológico , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/efeitos dos fármacos , Animais , Peso Corporal , Chlorocebus aethiops , Modelos Animais de Doenças , Feminino , Injeções Intramusculares , Camundongos , Camundongos Endogâmicos BALB C , Doenças dos Roedores/tratamento farmacológico , Doenças dos Roedores/mortalidade , Doenças dos Roedores/patologia , Doenças dos Roedores/virologia , Síndrome Respiratória Aguda Grave/mortalidade , Síndrome Respiratória Aguda Grave/patologia , Síndrome Respiratória Aguda Grave/virologia , Análise de Sobrevida , Células Vero , Replicação Viral/efeitos dos fármacosRESUMO
Interferons (IFNs) are a first line of defense against viral infection. Herein we describe the use of an adenovirus vectored mouse IFN alpha gene (mDEF201) as a prophylactic and treatment countermeasure in a SARS-CoV-infected BALB/c mouse model. Complete survival protection was observed in mice given a single dose of mDEF201 administered intranasally 1, 3, 5, 7, or 14 days prior to lethal SARS-CoV challenge (p<0.001), and body weights of these treated mice were unaffected by the challenge. In addition, low doses of mDEF201 protected lungs in a dose dependent manner as measured by a reduction in gross pathology. Intranasal treatment with mDEF201 ranging from 10(6) to 10(8)PFU significantly protected mice against a lethal SARS-CoV infection in a dose dependent manner up to 12h post infection (p<0.001). The data suggest that mDEF201 is a new class of antiviral agent further development as treatment for SARS-CoV infections.
Assuntos
Antivirais/administração & dosagem , Produtos Biológicos/administração & dosagem , Interferon-alfa/administração & dosagem , Síndrome Respiratória Aguda Grave/prevenção & controle , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Adenoviridae/genética , Administração Intranasal , Animais , Antivirais/imunologia , Produtos Biológicos/genética , Produtos Biológicos/imunologia , Peso Corporal , Chlorocebus aethiops , Modelos Animais de Doenças , Feminino , Vetores Genéticos , Humanos , Interferon-alfa/genética , Interferon-alfa/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Síndrome Respiratória Aguda Grave/mortalidade , Síndrome Respiratória Aguda Grave/patologia , Análise de Sobrevida , Células VeroRESUMO
The ability to rescue an infectious, recombinant RNA virus from a cDNA clone has led to new opportunities for measuring viral replication from a viral expressed reporter gene. In this protocol, the process of inserting the enhanced green fluorescent protein (EGFP) gene into the human parainfluenza virus type 3 (HPIV-3) antigenome and rescuing a recombinant, infectious virus is described. The first step in this process includes the generation of a cDNA clone copied from viral RNA isolated from an HPIV-3 wild-type infection. Next, the EGFP gene is inserted into the viral genome so that it is expressed independently during virus replication. Third, the viral support genes that are responsible for viral replication are cloned into T7 expression plasmids. Finally, an infectious, rHPIV3-EGFP virus is rescued from the cDNA clone with assistance from the viral support genes. Ultimately, cells infected with the rHPIV3-EGFP virus will emit green fluorescence that can be photographed and quantitated.
Assuntos
Proteínas de Fluorescência Verde/metabolismo , Mutagênese Insercional , Vírus da Parainfluenza 3 Humana/fisiologia , Coloração e Rotulagem/métodos , Replicação Viral , DNA Complementar/genética , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Humanos , Vírus da Parainfluenza 3 Humana/genética , Vírus da Parainfluenza 3 Humana/isolamento & purificação , Plasmídeos , RNA Viral/genéticaRESUMO
The ability to rescue an infectious, recombinant, negative-stranded, RNA virus from a complementary DNA (cDNA) clone, has led to new opportunities for measuring viral replication from a viral expressed reporter gene. In this study, the enhanced green fluorescent protein (EGFP) gene was inserted into the human parainfluenza virus type 3 (HPIV-3) antigenome and a recombinant, infectious virus was rescued. Maximum EGFP expression levels, measured by fluorescence, were seen at day 3. Comparison of a 3-day, viral expressed EGFP fluorescence assay to a 7-day, neutral red assay, based on complete cell destruction in virus infected MA-104 cells, yielded Z'-factor values of 0.83 and 0.70, respectively. A 3-day, endpoint EGFP-based antiviral assay and a 7-day, endpoint neutral red based antiviral assay were run in parallel to establish antiviral sensitivity profiles of 23 compounds based on selective index (SI) values. Using an SI threshold of 10, the EGFP-based antiviral assay had a sensitivity of 100% and a specificity of 54%. Thus, the use of an EGFP-based antiviral assay for testing potential antiviral compounds against HPIV-3 in a high-throughput format may be justified.
Assuntos
Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Proteínas de Fluorescência Verde/metabolismo , Vírus da Parainfluenza 3 Humana/efeitos dos fármacos , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Medições Luminescentes , Vírus da Parainfluenza 3 Humana/genética , Vírus da Parainfluenza 3 Humana/fisiologia , Replicação ViralRESUMO
Phenothiazine and derivatives were tested for inhibition of SARS-CoV replication. Phenothiazine slightly inhibited SARS-CoV replication in a neutral red (NR) uptake assay. Adding a propylamino group to give promazine reduced virus yields (VYR assay) with an EC(90)=8.3+/-2.8 microM, but without selectivity. Various substitutions in the basic phenothiazine structure did not promote efficacy. Phenazine ethosulfate was the most potent compound by VYR assay (EC(90)=6.1+/-4.3 microM). All compounds were toxic (IC(50)=6.6-74.5 microM) except for phenoxathiin (IC(50)=858+/-208 microM) and 10-(alpha-diethylamino-propionyl) phenothiazine.HCl (IC(50)=195+/-71.2 microM). Consequently, none were selective inhibitors of SARS-CoV replication (SI values <1-3.3 microM). These data portended the poor efficacy of promazine in a SARS-CoV mouse lung replication model. Intraperitoneal treatment with promazine using a prophylactic (-4h)/therapeutic regimen of 1, 10, or 50mg/(kg day) did not reduce virus lung titers at day 3, yet prolonged virus replication to 14 days. Similar therapeutic promazine doses were not efficacious. Thus, promazine did not affect SARS-CoV replication in vitro or in vivo, nor were any other phenothiazines efficacious in reducing virus replication. Therefore, treating SARS infections with compounds like promazine is not warranted.
Assuntos
Antivirais/farmacologia , Fenotiazinas/farmacologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/efeitos dos fármacos , Replicação Viral , Animais , Antivirais/administração & dosagem , Antivirais/uso terapêutico , Antivirais/toxicidade , Sobrevivência Celular , Quimioprevenção , Chlorocebus aethiops , Citocinas/análise , Feminino , Pulmão/patologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Vermelho Neutro/metabolismo , Fenotiazinas/administração & dosagem , Fenotiazinas/uso terapêutico , Fenotiazinas/toxicidade , Síndrome Respiratória Aguda Grave/tratamento farmacológico , Síndrome Respiratória Aguda Grave/prevenção & controle , Células VeroRESUMO
Bluetongue viruses (BTVs) infect primarily domestic cattle and wild ruminants but have never been shown to infect normal human cells. Thus, humans are sero-negative towards BTVs. The selective and differential effects of BTV serotype 10 (BTV-10) infection were investigated with five cell lines including primary human embryo lung fibroblast (HEL) and primary murine embryos fibroblast(MEF), human hepatic carcinoma 3B cell line (Hep-3B), human lung carcinoma cell line (A549) and mouse fibroblast cell line (NIH 3T3). In this study, comparative analyses of differential cytopathic effects (CPEs), survival rates using different Multiplicities of Infection (MOI), ultra-structural changes by transmission electron microscopy, and the preferential cell cycle changes of infected cells by flow cytometry were made among these cells. Detection of the presence of BTV genome and kinetic analysis of virus titers in TCID50 were also made. We provided the first analytical demonstration and evidence that BTV-10 could selectively infect and degrade human cancer cells but not cultured primary normal cells. No CPE or viral mRNAs could be detected within these normal cells, while various degrees of CPE could be found in Hep-3B and A549, as well as in NIH 3T3 under similar conditions. Before death, BTV-infected human cancer cells were directly arrested in the sub-G1 phase and the diversity of BTV infection as shown by the MTT method had significant difference (F = 95.635, p < 0.01). Above results suggested that this viral dose-dependent cytotoxic effect is caused by both effective virion amplification and induced apoptosis. Cellular distinctive transformation status may contribute to the selectivity. Thus, selective degradation of human cancer cells but not normal diploid cells by the newly discovered oncolytic potential of BTV would provide a very attractive approach for cancer therapy in the future.
Assuntos
Apoptose , Vírus Bluetongue , Fase G1 , Terapia Viral Oncolítica , Vírus Oncolíticos , Animais , Carcinoma/virologia , Carcinoma Hepatocelular/virologia , Bovinos , Linhagem Celular , Linhagem Celular Tumoral , Fibroblastos/virologia , Citometria de Fluxo , Humanos , Técnicas In Vitro , Neoplasias Hepáticas/virologia , Neoplasias Pulmonares/virologia , Camundongos , Microscopia Eletrônica de Transmissão , Células NIH 3T3RESUMO
Because of the conflicting data concerning the SARS-CoV inhibitory efficacy of ribavirin, an inosine monophosphate (IMP) dehydrogenase inhibitor, studies were done to evaluate the efficacy of ribavirin and other IMP dehydrogenase inhibitors (5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (EICAR), mizoribine, and mycophenolic acid) in preventing viral replication in the lungs of BALB/c mice, a replication model for severe acute respiratory syndrome (SARS) infections (Subbarao, K., McAuliffe, J., Vogel, L., Fahle, G., Fischer, S., Tatti, K., Packard, M., Shieh, W.J., Zaki, S., Murphy, B., 2004. Prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus (SARS-CoV) in the respiratory tract of mice. J. Virol. 78, 3572-3577). Ribavirin given at 75 mg/kg 4 h prior to virus exposure and then given twice daily for 3 days beginning at day 0 was found to increase virus lung titers and extend the length of time that virus could be detected in the lungs of mice. Other IMP dehydrogenase inhibitors administered near maximum tolerated doses using the same dosing regimen as for ribavirin were found to slightly enhance virus replication in the lungs. In addition, ribavirin treatment seemed also to promote the production of pro-inflammatory cytokines 4 days after cessation of treatment, although after 3 days of treatment ribavirin inhibited pro-inflammatory cytokine production in infected mice, significantly reducing the levels of the cytokines IL-1alpha, interleukin-5 (IL-5), monocyte chemotactic protein-1 (MCP-1), and granulocyte-macrophage colony stimulating factor (GM-CSF). These findings suggest that ribavirin may actually contribute to the pathogenesis of SARS-CoV by prolonging and/or enhancing viral replication in the lungs. By not inhibiting viral replication in the lungs of infected mice, ribavirin treatment may have provided a continual source of stimulation for the inflammatory response thought to contribute to the pathogenesis of the infection. Our data do not support the use of ribavirin or other IMP dehydrogenase inhibitors for treating SARS infections in humans.