RESUMO
Nipah virus (NiV) is a highly pathogenic zoonotic paramyxovirus that causes fatal encephalitis and respiratory disease in humans. There is currently no approved therapeutic for human use against NiV infection. Griffithsin (GRFT) is high-mannose oligosaccharide binding lectin that has shown in vivo broad-spectrum activity against viruses, including severe acute respiratory syndrome coronavirus, human immunodeficiency virus 1, hepatitis C virus, and Japanese encephalitis virus. In this study, we evaluated the in vitro antiviral activities of GRFT and its synthetic trimeric tandemer (3mG) against NiV and other viruses from 4 virus families. The 3mG had comparatively greater potency than GRFT against NiV due to its enhanced ability to block NiV glycoprotein-induced syncytia formation. Our initial in vivo prophylactic evaluation of an oxidation-resistant GRFT (Q-GRFT) showed significant protection against lethal NiV challenge in Syrian golden hamsters. Our results warrant further development of Q-GRFT and 3mG as potential NiV therapeutics.
Assuntos
Antivirais/farmacologia , Infecções por Henipavirus/tratamento farmacológico , Vírus Nipah/efeitos dos fármacos , Lectinas de Plantas/farmacologia , Internalização do Vírus/efeitos dos fármacos , Animais , Antivirais/uso terapêutico , Chlorocebus aethiops , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Células HEK293 , Células HeLa , Infecções por Henipavirus/virologia , Humanos , Mesocricetus , Vírus Nipah/isolamento & purificação , Lectinas de Plantas/uso terapêutico , Células VeroRESUMO
Rift Valley fever virus (RVFV) is an important mosquito-borne veterinary and human pathogen that has caused large outbreaks of severe disease throughout Africa and the Arabian Peninsula. Currently, no licensed vaccine or therapeutics exists to treat this potentially deadly disease. The explosive nature of RVFV outbreaks and the severe consequences of its accidental or intentional introduction into RVFV-free areas provide the impetus for the development of novel vaccine candidates for use in both livestock and humans. Rationally designed vaccine candidates using reverse genetics have been used to develop deletion mutants of two known RVFV virulence factors, the NSs and NSm genes. These recombinant viruses were demonstrated to be protective and immunogenic in rats, mice, and sheep, without producing clinical illness in these animals. Here, we expand upon those findings and evaluate the single deletion mutant (ΔNSs rRVFV) and double deletion mutant (ΔNSs-ΔNSm rRVFV) vaccine candidates in the common marmoset (Callithrix jacchus), a non-human primate (NHP) model resembling severe human RVF disease. We demonstrate that both the ΔNSs and ΔNSs-ΔNSm rRVFV vaccine candidates were found to be safe and immunogenic in the current study. The vaccinated animals received a single dose of vaccine that led to the development of a robust antibody response. No vaccine-induced adverse reactions, signs of clinical illness or infectious virus were detected in the vaccinated marmosets. All vaccinated animals that were subsequently challenged with RVFV were protected against viremia and liver disease. In summary, our results provide the basis for further development of the ΔNSs and ΔNSs-ΔNSm rRVFV as safe and effective human RVFV vaccines for this significant public health threat.
Assuntos
Febre do Vale de Rift/prevenção & controle , Vírus da Febre do Vale do Rift/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/imunologia , Callithrix/imunologia , Callithrix/virologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Humanos , Febre do Vale de Rift/imunologia , Febre do Vale de Rift/virologia , Vírus da Febre do Vale do Rift/genética , Deleção de Sequência , Vacinação , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Proteínas não Estruturais Virais/administração & dosagem , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genéticaRESUMO
Brincidofovir (BCV) is the 3-hexadecyloxy-1-propanol (HDP) lipid conjugate of the acyclic nucleoside phosphonate cidofovir (CDV). BCV has established broad-spectrum activity against double-stranded DNA (dsDNA) viruses; however, its activity against RNA viruses has been less thoroughly evaluated. Here, we report that BCV inhibited infection of Ebola virus in multiple human cell lines. Unlike the mechanism of action for BCV against cytomegalovirus and other dsDNA viruses, phosphorylation of CDV to the diphosphate form appeared unnecessary. Instead, antiviral activity required the lipid moiety and in vitro activity against EBOV was observed for several HDP-nucleotide conjugates.
Assuntos
Antivirais/química , Antivirais/farmacologia , Citosina/análogos & derivados , Ebolavirus/efeitos dos fármacos , Doença pelo Vírus Ebola/tratamento farmacológico , Organofosfonatos/química , Organofosfonatos/farmacologia , Animais , Linhagem Celular Tumoral , Chlorocebus aethiops , Cidofovir , Citosina/química , Citosina/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Células HeLa , Doença pelo Vírus Ebola/prevenção & controle , Doença pelo Vírus Ebola/virologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Lipídeos/química , Lipídeos/farmacologia , Masculino , Relação Estrutura-Atividade , Células Vero , Replicação Viral/efeitos dos fármacosRESUMO
Previous studies have demonstrated the potential application of reverse genetics technology in studying a broad range of aspects of viral biology, including gene regulation, protein function, cell entry, and pathogenesis. Here, we describe a highly efficient reverse genetics system used to generate recombinant Ebola virus (EBOV) based on a recent isolate from a human patient infected during the 2014-2015 outbreak in Western Africa. We also rescued a recombinant EBOV expressing a fluorescent reporter protein from a cleaved VP40 protein fusion. Using this virus and an inexpensive method to quantitate the expression of the foreign gene, we demonstrate its potential usefulness as a tool for screening antiviral compounds and measuring neutralizing antibodies.
Assuntos
Ebolavirus/genética , Proteínas de Fluorescência Verde/biossíntese , Genética Reversa/métodos , África Ocidental , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Antivirais/isolamento & purificação , Avaliação Pré-Clínica de Medicamentos/métodos , Ebolavirus/isolamento & purificação , Genes Reporter , Proteínas de Fluorescência Verde/genética , Doença pelo Vírus Ebola/virologia , Humanos , Dados de Sequência Molecular , RNA Viral/genética , Análise de Sequência de DNA , Coloração e RotulagemRESUMO
Nipah virus (NiV) outbreaks have occurred in Malaysia, India, and Bangladesh, and the virus continues to cause annual outbreaks of fatal human encephalitis in Bangladesh due to spillover from its bat host reservoir. Due to its high pathogenicity, its potential use for bio/agro-terrorism, and to the current lack of approved therapeutics, NiV is designated as an overlap select agent requiring biosafety level-4 containment. Although the development of therapeutic monoclonal antibodies and soluble protein subunit vaccines have shown great promise, the paucity of effective antiviral drugs against NiV merits further exploration of compound libraries using rapid quantitative antiviral assays. As a proof-of-concept study, we evaluated the use of fluorescent and luminescent reporter NiVs for antiviral screening. We constructed and rescued NiVs expressing either Renilla luciferase or green fluorescent protein, and characterized their reporter signal kinetics in different cell types as well as in the presence of several inhibitors. The 50% effective concentrations (EC50s) derived for inhibitors against both reporter viruses are within range of EC50s derived from virus yield-based dose-response assays against wild-type NiV (within 1Log10), thus demonstrating that both reporter NiVs can serve as robust antiviral screening tools. Utilizing these live NiV-based reporter assays requires modest instrumentation, and circumvents the time and labor-intensive steps associated with cytopathic effect or viral antigen-based assays. These reporter NiVs will not only facilitate antiviral screening, but also the study of host cell components that influence the virus life cycle.
Assuntos
Antivirais/isolamento & purificação , Avaliação Pré-Clínica de Medicamentos/métodos , Genes Reporter , Luciferases de Renilla/análise , Vírus Nipah/efeitos dos fármacos , Animais , Antivirais/farmacologia , Linhagem Celular , Humanos , Luciferases de Renilla/genética , Medições Luminescentes , Vírus Nipah/genéticaRESUMO
Marburg virus (MARV) and Ebola virus (EBOV), members of the family Filoviridae, represent a significant challenge to global public health. Currently, no licensed therapies exist to treat filovirus infections, which cause up to 90% mortality in human cases. To facilitate development of antivirals against these viruses, we established two distinct screening platforms based on MARV and EBOV reverse genetics systems that express secreted Gaussia luciferase (gLuc). The first platform is a mini-genome replicon to screen viral replication inhibitors using gLuc quantification in a BSL-2 setting. The second platform is complementary to the first and expresses gLuc as a reporter gene product encoded in recombinant infectious MARV and EBOV, thereby allowing for rapid quantification of viral growth during treatment with antiviral compounds. We characterized these viruses by comparing luciferase activity to virus production, and validated luciferase activity as an authentic real-time measure of viral growth. As proof of concept, we adapt both mini-genome and infectious virus platforms to high-throughput formats, and demonstrate efficacy of several antiviral compounds. We anticipate that both approaches will prove highly useful in the development of anti-filovirus therapies, as well as in basic research on the filovirus life cycle.
Assuntos
Antivirais/isolamento & purificação , Avaliação Pré-Clínica de Medicamentos/métodos , Ebolavirus/efeitos dos fármacos , Marburgvirus/efeitos dos fármacos , Genética Reversa/métodos , Animais , Antivirais/farmacologia , Linhagem Celular , Ebolavirus/genética , Ebolavirus/fisiologia , Genes Reporter , Luciferases/análise , Luciferases/genética , Marburgvirus/genética , Marburgvirus/fisiologia , Replicação Viral/efeitos dos fármacosRESUMO
Zaire ebolavirus causes large outbreaks of severe and usually fatal hemorrhagic disease in humans for which there is no effective treatment or cure. To facilitate examination of early critical events in viral pathogenesis and to identify antiviral compounds, a recombinant Zaire ebolavirus was engineered to express a foreign protein, eGFP, to provide a rapid and sensitive means to monitor virus replication in infected cells. This genetically engineered virus represents the first insertion of a foreign gene into ebolavirus. We show that Ebola-eGFP virus (EboZ-eGFP) infects known early targets of human infections and serves as an ideal model to screen antiviral compounds in less time than any previously published assay.