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1.
Microb Pathog ; 162: 105358, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34902539

RESUMO

Chandipura vesiculovirus (CHPV) is a fast-emerging virus that causes acute encephalitis with a high death rate. Because of its extensive prevalence in African and Asian countries, this infection has become a global hazard, and there is an urgent need to create an effective and non-allergenic vaccine or appropriate treatment to combat it. A vaccine candidate is offered utilizing a computational technique in this study. To build a potential vaccine candidate, viral protein sequences were acquired from the National Center for Biotechnology Information database and evaluated with several bioinformatics techniques to identify B-cell and T-cell epitopes. V1 was shown to be superior in terms of various physicochemical qualities, as well as highly immunogenic and non-allergic. Molecular docking revealed that the CHPV vaccine construct had a greater binding affinity with human Toll-like receptors (TLR-3 and TLR-8) and that it was stable in molecular dynamics simulations. MEC-CHPV was in silico cloned in the pET28a (+) expression vector using codon optimization. The current research identifies potential antigenic epitopes that could be used as vaccine candidates to eradicate the CHPV. This in-silico development of a CHPV vaccine with multiple epitopes could open the path for future rapid laboratory tests.


Assuntos
Epitopos de Linfócito B , Vacinas , Biologia Computacional , Epitopos de Linfócito B/genética , Epitopos de Linfócito T/genética , Humanos , Simulação de Acoplamento Molecular , Vacinas de Subunidades , Vesiculovirus/genética
2.
Mol Med ; 27(1): 105, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34503440

RESUMO

BACKGROUND: Vaccination programs have been launched worldwide to halt the spread of COVID-19. However, the identification of existing, safe compounds with combined treatment and prophylactic properties would be beneficial to individuals who are waiting to be vaccinated, particularly in less economically developed countries, where vaccine availability may be initially limited. METHODS: We used a data-driven approach, combining results from the screening of a large transcriptomic database (L1000) and molecular docking analyses, with in vitro tests using a lung organoid model of SARS-CoV-2 entry, to identify drugs with putative multimodal properties against COVID-19. RESULTS: Out of thousands of FDA-approved drugs considered, we observed that atorvastatin was the most promising candidate, as its effects negatively correlated with the transcriptional changes associated with infection. Atorvastatin was further predicted to bind to SARS-CoV-2's main protease and RNA-dependent RNA polymerase, and was shown to inhibit viral entry in our lung organoid model. CONCLUSIONS: Small clinical studies reported that general statin use, and specifically, atorvastatin use, are associated with protective effects against COVID-19. Our study corroborrates these findings and supports the investigation of atorvastatin in larger clinical studies. Ultimately, our framework demonstrates one promising way to fast-track the identification of compounds for COVID-19, which could similarly be applied when tackling future pandemics.


Assuntos
Antivirais/farmacologia , Atorvastatina/farmacologia , COVID-19/tratamento farmacológico , Pulmão/efeitos dos fármacos , Organoides/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , Antivirais/química , Atorvastatina/química , COVID-19/prevenção & controle , Linhagem Celular , Proteases 3C de Coronavírus/química , RNA-Polimerase RNA-Dependente de Coronavírus/química , Doxiciclina/farmacologia , Aprovação de Drogas , Reposicionamento de Medicamentos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Pulmão/virologia , Modelos Biológicos , Simulação de Acoplamento Molecular , Organoides/virologia , Cloridrato de Raloxifeno/química , Cloridrato de Raloxifeno/farmacologia , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/genética , Trifluoperazina/química , Trifluoperazina/farmacologia , Estados Unidos , United States Food and Drug Administration , Vesiculovirus/genética , Internalização do Vírus/efeitos dos fármacos
3.
Arch Virol ; 166(11): 3143-3150, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34533641

RESUMO

Chandipura virus (CHPV), belonging to the genus Vesiculovirus of the family Rhabdoviridae, has been identified as one of the causes of pediatric encephalitis in India. Currently, neither vaccines nor therapeutic drugs are available against this agent. Considering that the disease progresses very fast with a high mortality rate, working towards the development of potential therapeutics against it will have a public health impact. Although the use of viral inhibitors as antiviral agents is the most common way to curb virus replication, the mutation-prone nature of viruses results in the development of resistance to antiviral agents. The recent development of proteomic platforms for analysis of purified viral agents has allowed certain upregulated host proteins that are involved in the morphogenesis and replication of viruses to be identified. Thus, the alternative approach of inhibition of host proteins involved in the regulation of virus replication could be explored for their therapeutic effectiveness. In the current study, we have evaluated the effect of inhibition of cyclophilin A (CypA), an immunophilin with peptidyl-prolyl cis/trans-isomerase activity, on the replication of CHPV. Treatment with cyclosporin A, used in vitro for the inhibition of CypA, resulted in a 3-log reduction in CHPV titer and an undetectable level of CypA in comparison to an untreated control. An in silico analysis of the interaction of the CHPV nucleoprotein with the human CypA protein showed stable interaction in molecular docking and molecular dynamics simulations. Overall, the results of this study suggest a possible role of CypA in facilitating CHPV replication, thus making it one of the potential host factors to be explored in future antiviral studies.


Assuntos
Ciclofilina A/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Infecções por Rhabdoviridae/virologia , Vesiculovirus/patogenicidade , Ciclofilina A/antagonistas & inibidores , Ciclofilina A/química , Ciclosporina/farmacologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/metabolismo , Vesiculovirus/efeitos dos fármacos , Vesiculovirus/fisiologia , Replicação Viral/efeitos dos fármacos
4.
J Virol ; 95(20): e0079021, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34346765

RESUMO

Rabies is a fatal zoonosis that causes encephalitis in mammals, and vaccination is the most effective method to control and eliminate rabies. Virus-like vesicles (VLVs), which are characterized as infectious, self-propagating membrane-enveloped particles composed of only Semliki Forest virus (SFV) replicase and vesicular stomatitis virus glycoprotein (VSV-G), have been proven safe and efficient as vaccine candidates. However, previous studies showed that VLVs containing rabies virus glycoprotein (RABV-G) grew at relatively low titers in cells, impeding their potential use as a rabies vaccine. In this study, we constructed novel VLVs by transfection of a mutant SFV RNA replicon encoding RABV-G. We found that these VLVs could self-propagate efficiently in cell culture and could evolve to high titers (approximately 108 focus-forming units [FFU]/ml) by extensive passaging 25 times in BHK-21 cells. Furthermore, we found that the evolved amino acid changes in SFV nonstructural protein 1 (nsP1) at positions 470 and 482 was critical for this high-titer phenotype. Remarkably, VLVs could induce robust type I interferon (IFN) expression in BV2 cells and were highly sensitive to IFN-α. We found that direct inoculation of VLVs into the mouse brain caused reduced body weight loss, mortality, and neuroinflammation compared with the RABV vaccine strain. Finally, it could induce increased generation of germinal center (GC) B cells, plasma cells (PCs), and virus-neutralizing antibodies (VNAs), as well as provide protection against virulent RABV challenge in immunized mice. This study demonstrated that VLVs containing RABV-G could proliferate in cells and were highly evolvable, revealing the feasibility of developing an economic, safe, and efficacious rabies vaccine. IMPORTANCE VLVs have been shown to represent a more versatile and superior vaccine platform. In previous studies, VLVs containing the Semliki Forest virus replicase (SFV nsP1 to nsP4) and rabies virus glycoprotein (RABV-G) grew to relatively low titers in cells. In our study, we not only succeeded in generating VLVs that proliferate in cells and stably express RABV-G, but the VLVs that evolved grew to higher titers, reaching 108 FFU/ml. We also found that nucleic acid changes at positions 470 and 482 in nsP1 were vital for this high-titer phenotype. Moreover, the VLVs that evolved in our studies were highly attenuated in mice, induced potent immunity, and protected mice from lethal RABV infection. Collectively, our study showed that high titers of VLVs containing RABV-G were achieved, demonstrating that these VLVs could be an economical, safe, and efficacious rabies vaccine candidate.


Assuntos
Vacinas Antirrábicas/imunologia , Raiva/imunologia , Vacinação/métodos , Animais , Anticorpos Antivirais/sangue , Linfócitos B/imunologia , Modelos Animais de Doenças , Feminino , Engenharia Genética/métodos , Centro Germinativo/imunologia , Glicoproteínas/genética , Imunização/métodos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Raiva/metabolismo , Vacinas Antirrábicas/metabolismo , Vacinas Antirrábicas/farmacologia , Vírus da Raiva/imunologia , Vírus da Floresta de Semliki/imunologia , Vesiculovirus/genética , Proteínas Virais/genética
5.
J Biomed Sci ; 28(1): 52, 2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34233673

RESUMO

BACKGROUND: Chandipura virus (CHPV) is a negative single-stranded RNA virus of the Rhabdoviridae family. CHPV infection has been reported in Central and Western India. CHPV causes acute encephalitis with a case fatality rate of 70 % and mostly affects children below 15 years of age. CHPV infection in brain leads to neuronal apoptosis and activation of the microglial cells. The microRNAs (miRNAs) are small endogenous non-coding RNA that regulate the gene expression. Viral infections perturb the expression pattern of cellular miRNAs, which may in turn affect the expression pattern of downstream genes. This study aims to investigate hsa-miR-21-5p mediated regulation of PTEN, AKT, NF-ĸBp65, IL-6, TNF-α, and IL-1ß, in human microglial cells during CHPV infection. METHODS: To understand the role of hsa-miR-21-5p in CHPV infection, the human microglial cells were infected with CHPV (MOI-0.1). Real-time PCR, western blotting, Luciferase assay, over-expression and knockdown techniques were used to understand the role of hsa-miR-21-5p in the regulation of PTEN, AKT and, NF-ĸBp65, IL-6, TNF-α, and IL-1ß in this study. RESULTS: The hsa-miR-21-5p was found to be upregulated during CHPV infection in human microglial cells. This led to the downregulation of PTEN which promoted the phosphorylation of AKT and NF-ĸBp65. Over-expression of hsa-miR-21-5p led to the decreased expression of PTEN and promoted further phosphorylation of AKT and NF-ĸBp65 in human microglial cells. However, the inhibition of hsa-miR-21-5p using hsa-miR-21-5p inhibitor restored the expression. CONCLUSIONS: This study supports the role of hsa-miR-21-5p in the regulation of pro-inflammatory genes in CHPV infected human microglial cells.


Assuntos
MicroRNAs/genética , Microglia/metabolismo , NF-kappa B/genética , Vesiculovirus/fisiologia , Humanos , MicroRNAs/metabolismo , NF-kappa B/metabolismo
6.
Sci Rep ; 11(1): 15290, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315959

RESUMO

Vaccination can prevent viral infections via virus-specific T cells, among other mechanisms. A goal of oncolytic virotherapy is replication of oncolytic viruses (OVs) in tumors, so pre-existing T cell immunity against an OV-encoded transgene would seem counterproductive. We developed a treatment for melanomas by pre-vaccinating against an oncolytic vesicular stomatitis virus (VSV)-encoded tumor antigen. Surprisingly, when the VSV-vectored booster vaccine was administered at the peak of the primary effector T cell response, oncolysis was not abrogated. We sought to determine how oncolysis was retained during a robust T cell response against the VSV-encoded transgene product. A murine melanoma model was used to identify two mechanisms that enable this phenomenon. First, tumor-infiltrating T cells had reduced cytopathic potential due to immunosuppression. Second, virus-induced lymphopenia acutely removed virus-specific T cells from tumors. These mechanisms provide a window of opportunity for replication of oncolytic VSV and rationale for a paradigm change in oncolytic virotherapy, whereby immune responses could be intentionally induced against a VSV-encoded melanoma-associated antigen to improve safety without abrogating oncolysis.


Assuntos
Vírus Oncolíticos/genética , Transgenes , Vesiculovirus/genética , Vacinas Virais/imunologia , Animais , Camundongos , Vacinas Virais/genética
7.
Elife ; 102021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34110282

RESUMO

Nuclear factor 90 (NF90) is a novel virus sensor that serves to initiate antiviral innate immunity by triggering stress granule (SG) formation. However, the regulation of the NF90-SG pathway remains largely unclear. We found that Tim-3, an immune checkpoint inhibitor, promotes the ubiquitination and degradation of NF90 and inhibits NF90-SG-mediated antiviral immunity. Vesicular stomatitis virus (VSV) infection induces the up-regulation and activation of Tim-3 in macrophages, which in turn recruit the E3 ubiquitin ligase TRIM47 to the zinc finger domain of NF90 and initiate a proteasome-dependent degradation via K48-linked ubiquitination at Lys297. Targeted inactivation of Tim-3 enhances the NF90 downstream SG formation by selectively increasing the phosphorylation of protein kinase R and eukaryotic translation initiation factor 2α, the expression of SG markers G3BP1 and TIA-1, and protecting mice from VSV challenge. These findings provide insights into the crosstalk between Tim-3 and other receptors in antiviral innate immunity and its related clinical significance.


Assuntos
Receptor Celular 2 do Vírus da Hepatite A , Imunidade Inata/imunologia , Proteínas do Fator Nuclear 90 , Ubiquitinação/imunologia , Viroses/imunologia , Animais , Grânulos Citoplasmáticos/imunologia , Grânulos Citoplasmáticos/metabolismo , Receptor Celular 2 do Vírus da Hepatite A/imunologia , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Proteínas do Fator Nuclear 90/imunologia , Proteínas do Fator Nuclear 90/metabolismo , Infecções por Rhabdoviridae/imunologia , Vesiculovirus
8.
Sci Rep ; 11(1): 13253, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34168211

RESUMO

Chandipura virus (CHPV, a member of the Rhabdoviridae family) is an emerging pathogen that causes rapidly progressing influenza-like illness and acute encephalitis often leading to coma and death of the human host. Given several CHPV outbreaks in Indian sub-continent, recurring sporadic cases, neurological manifestation, and high mortality rate of this infection, CHPV is gaining global attention. The 'dark proteome' includes the whole proteome with special emphasis on intrinsically disordered proteins (IDP) and IDP regions (IDPR), which are proteins or protein regions that lack unique (or ordered) three-dimensional structures within the cellular milieu. These proteins/regions, however, play a number of vital roles in various biological processes, such as cell cycle regulation, control of signaling pathways, etc. and, therefore, are implicated in many human diseases. IDPs and IPPRs are also abundantly found in many viral proteins enabling their multifunctional roles in the viral life cycles and their capability to highjack various host systems. The unknown abundance of IDP and IDPR in CHPV, therefore, prompted us to analyze the dark proteome of this virus. Our analysis revealed a varying degree of disorder in all five CHPV proteins, with the maximum level of intrinsic disorder propensity being found in Phosphoprotein (P). We have also shown the flexibility of P protein using extensive molecular dynamics simulations up to 500 ns (ns). Furthermore, our analysis also showed the abundant presence of the disorder-based binding regions (also known as molecular recognition features, MoRFs) in CHPV proteins. The identification of IDPs/IDPRs in CHPV proteins suggests that their disordered regions may function as potential interacting domains and may also serve as novel targets for disorder-based drug designs.


Assuntos
Proteínas Intrinsicamente Desordenadas/metabolismo , Infecções por Rhabdoviridae/metabolismo , Vesiculovirus/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Genoma Viral/genética , Humanos , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Fosfoproteínas/metabolismo , Proteoma , Infecções por Rhabdoviridae/virologia , Alinhamento de Sequência , Vesiculovirus/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo
9.
Vet Q ; 41(1): 202-209, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33985418

RESUMO

BACKGROUND: Rabies is a viral disease that causes severe neurological manifestations both in humans and various mammals. Although inactivated and/or attenuated vaccines have been developed and widely used around the world, there are still concerns with regard to their safety, efficacy, and costs. OBJECTIVE: As demand has grown for a new rabies vaccine, we have developed a new vesicular stomatitis viruses (VSVs) based rabies vaccine that replaces glycoproteins with rabies virus (RABV) glycoprotein (GP), or so-called VSV/RABV-GP. METHODS: VSV/RABV-GP production was measured by sandwich ELISA. The generation of VSV/RABV-GP was evaluated with GP-specific antibodies and reduced transduction with GP-specific neutralizing antibodies. Virus entry was quantified by measuring the luciferase levels at 18-h post-transduction. BALB/c mice (three groups of six mice each) were intraperitoneally immunized with PBS, RABA, or VSV/RABV-GP at 0 and 14 days. At 28 days post-immunization serology was performed. Statistical significance was calculated using the Holm-Sidak multiple Student's t test. RESULTS: Mice immunized with VSV/RABV-GP produced IgM and IgG antibodies, whereas IgM titers were significantly higher in mice immunized with VSV/RABV-GP compared to inactivated RABV. The secretion profiles of IgG1 and IgG2a production suggested that VSV/RAVB-GP induces the T helper cell type-2 immune bias. In addition, the average (±SD; n = 3) serum neutralization titers of the inactivated RABV and VSV/RABV-GP groups were 241 ± 40 and 103 ± 54 IU/mL, respectively. CONCLUSION: Our results confirm that VSV/RABV-GP could be a new potential vaccination platform for RABV.


Assuntos
Glicoproteínas/imunologia , Imunogenicidade da Vacina , Vacinas Antirrábicas/imunologia , Vírus da Raiva/imunologia , Vesiculovirus/genética , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , Glicoproteínas/genética , Imunoglobulina G , Imunoglobulina M , Camundongos Endogâmicos BALB C , Raiva/imunologia , Raiva/virologia , Vírus da Raiva/genética , Vacinas Sintéticas , Proteínas Virais , Replicação Viral/genética
10.
Front Immunol ; 12: 667478, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34025669

RESUMO

Viral encephalitis is the most common cause of encephalitis. It is responsible for high morbidity rates, permanent neurological sequelae, and even high mortality rates. The host immune response plays a critical role in preventing or clearing invading pathogens, especially when effective antiviral treatment is lacking. However, due to blockade of the blood-brain barrier, it remains unclear how peripheral immune cells contribute to the fight against intracerebral viruses. Here, we report that peripheral injection of an antibody against human Tim-3, an immune checkpoint inhibitor widely expressed on immune cells, markedly attenuated vesicular stomatitis virus (VSV) encephalitis, marked by decreased mortality and improved neuroethology in mice. Peripheral injection of Tim-3 antibody enhanced the recruitment of immune cells to the brain, increased the expression of major histocompatibility complex-I (MHC-I) on macrophages, and as a result, promoted the activation of VSV-specific CD8+ T cells. Depletion of macrophages abolished the peripheral injection-mediated protection against VSV encephalitis. Notably, for the first time, we found a novel post-translational modification of MHC-I by Tim-3, wherein, by enhancing the expression of MARCH9, Tim-3 promoted the proteasome-dependent degradation of MHC-I via K48-linked ubiquitination in macrophages. These results provide insights into the immune response against intracranial infections; thus, manipulating the peripheral immune cells with Tim-3 antibody to fight viruses in the brain may have potential applications for combating viral encephalitis.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Anticorpos Neutralizantes/administração & dosagem , Células Apresentadoras de Antígenos/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encefalite Viral/prevenção & controle , Receptor Celular 2 do Vírus da Hepatite A/antagonistas & inibidores , Macrófagos/efeitos dos fármacos , Infecções por Rhabdoviridae/prevenção & controle , Vesiculovirus/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Células Apresentadoras de Antígenos/virologia , Encéfalo/imunologia , Encéfalo/metabolismo , Encéfalo/virologia , Chlorocebus aethiops , Modelos Animais de Doenças , Encefalite Viral/imunologia , Encefalite Viral/metabolismo , Encefalite Viral/virologia , Células HEK293 , Receptor Celular 2 do Vírus da Hepatite A/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Injeções Intraperitoneais , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Células RAW 264.7 , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/metabolismo , Infecções por Rhabdoviridae/virologia , Ubiquitinação , Células Vero , Vesiculovirus/patogenicidade , Carga Viral
11.
J Virol ; 95(16): e0059421, 2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34037421

RESUMO

Snakehead vesiculovirus (SHVV), a kind of fish rhabdovirus isolated from diseased hybrid snakehead fish, has caused great economic losses in snakehead fish culture in China. The large (L) protein, together with its cofactor phosphoprotein (P), forms a P/L polymerase complex and catalyzes the transcription and replication of viral genomic RNA. In this study, the cellular heat shock protein 90 (Hsp90) was identified as an interacting partner of SHVV L protein. Hsp90 activity was required for the stability of SHVV L because Hsp90 dysfunction caused by using its inhibitor destabilized SHVV L and thereby suppressed SHVV replication via reducing viral RNA synthesis. SHVV L expressed alone was detected mainly in the insoluble fraction, and the insoluble L was degraded by Hsp90 dysfunction through the proteasomal pathway, while the presence of SHVV P promoted the solubility of SHVV L and the soluble L was degraded by Hsp90 dysfunction through the autophagy pathway. Collectively, our data suggest that Hsp90 contributes to the maturation of SHVV L and ensures the effective replication of SHVV, which exhibits an important anti-SHVV target. This study will help us to understand the role of Hsp90 in stabilizing the L protein and regulating the replication of negative-stranded RNA viruses. IMPORTANCE It has long been proposed that cellular proteins are involved in viral RNA synthesis via interacting with the viral polymerase protein. This study focused on identifying cellular proteins interacting with the SHVV L protein, studying the effects of their interactions on SHVV replication, and revealing the underlying mechanisms. We identified Hsp90 as an interacting partner of SHVV L and found that Hsp90 activity was required for SHVV replication. Hsp90 functioned in maintaining the stability of SHVV L. Inhibition of Hsp90 activity with its inhibitor degraded SHVV L through different pathways based on the solubility of SHVV L due to the presence or absence of SHVV P. Our data provide important insights into the role of Hsp90 in SHVV polymerase maturation, which will help us to understand the polymerase function of negative-stranded RNA viruses.


Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Vesiculovirus/fisiologia , Proteínas Virais/metabolismo , Replicação Viral , Animais , Células Cultivadas , Peixes , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Fosfoproteínas/metabolismo , Estabilidade Proteica , RNA Viral/biossíntese , Infecções por Rhabdoviridae/veterinária , Infecções por Rhabdoviridae/virologia , Vesiculovirus/metabolismo
12.
mBio ; 12(2)2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33849978

RESUMO

Mammalian cells detect microbial molecules known as pathogen-associated molecular patterns (PAMPs) as indicators of potential infection. Upon PAMP detection, diverse defensive responses are induced by the host, including those that promote inflammation and cell-intrinsic antimicrobial activities. Host-encoded molecules released from dying or damaged cells, known as damage-associated molecular patterns (DAMPs), also induce defensive responses. Both DAMPs and PAMPs are recognized for their inflammatory potential, but only the latter are well established to stimulate cell-intrinsic host defense. Here, we report a class of DAMPs that engender an antiviral state in human epithelial cells. These DAMPs include oxPAPC (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine), PGPC (1-palmitoyl-2-glutaryl phosphatidylcholine), and POVPC [1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphatidylcholine], oxidized lipids that are naturally released from dead or dying cells. Exposing cells to these DAMPs prior to vesicular stomatitis virus (VSV) infection limits viral replication. Mechanistically, these DAMPs prevent viral entry, thereby limiting the percentage of cells that are productively infected and consequently restricting viral load. We found that the antiviral actions of oxidized lipids are distinct from those mediated by the PAMP Poly I:C, in that the former induces a more rapid antiviral response without the induction of the interferon response. These data support a model whereby interferon-independent defensive activities can be induced by DAMPs, which may limit viral replication before PAMP-mediated interferon responses are induced. This antiviral activity may impact viruses that disrupt interferon responses in the oxygenated environment of the lung, such as influenza virus and SARS-CoV-2.IMPORTANCE In this work, we explored how a class of oxidized lipids, spontaneously created during tissue damage and unprogrammed cell lysis, block the earliest events in RNA virus infection in the human epithelium. This gives us novel insight into the ways that we view infection models, unveiling a built-in mechanism to slow viral growth that neither engages the interferon response nor is subject to known viral antagonism. These oxidized phospholipids act prior to infection, allowing time for other, better-known innate immune mechanisms to take effect. This discovery broadens our understanding of host defenses, introducing a soluble factor that alters the cellular environment to protect from RNA virus infection.


Assuntos
Alarminas/farmacologia , Antivirais/farmacologia , Vírus de RNA/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Células A549 , Morte Celular/efeitos dos fármacos , Humanos , Imunidade Inata , Interferons/genética , Interferons/metabolismo , Cinética , Padrões Moleculares Associados a Patógenos/farmacologia , Fosfatidilcolinas/farmacologia , Vírus de RNA/fisiologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Vesiculovirus/efeitos dos fármacos , Vesiculovirus/fisiologia , Carga Viral
13.
Biotechnol Bioeng ; 118(7): 2649-2659, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33837958

RESUMO

The Vero cell line is the most used continuous cell line in viral vaccine manufacturing. This adherent cell culture platform requires the use of surfaces to support cell growth, typically roller bottles, or microcarriers. We have recently compared the production of rVSV-ZEBOV on Vero cells between microcarrier and fixed-bed bioreactors. However, suspension cultures are considered superior with regard to process scalability. Therefore, we further explore the Vero suspension system for recombinant vesicular stomatitis virus (rVSV)-vectored vaccine production. Previously, this suspension cell line was only able to be cultivated in a proprietary medium. Here, we expand the adaptation and bioreactor cultivation to a serum-free commercial medium. Following small-scale optimization and screening studies, we demonstrate bioreactor productions of highly relevant vaccines and vaccine candidates against Ebola virus disease, HIV, and coronavirus disease 2019 in the Vero suspension system. rVSV-ZEBOV, rVSV-HIV, and rVSVInd -msp-SF -Gtc can replicate to high titers in the bioreactor, reaching 3.87 × 107 TCID50 /ml, 2.12 × 107 TCID50 /ml, and 3.59 × 109 TCID50 /ml, respectively. Furthermore, we compare cell-specific productivities, and the quality of the produced viruses by determining the ratio of total viral particles to infectious viral particles.


Assuntos
Reatores Biológicos/virologia , Técnicas de Cultura de Células/métodos , Vacinas contra Ebola , Vesiculovirus/genética , Animais , Vacinas contra COVID-19 , Chlorocebus aethiops , Meios de Cultura Livres de Soro , Células Vero , Vacinas Virais
14.
Viruses ; 13(3)2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33804519

RESUMO

Australian bat lyssavirus (ABLV) is a rhabdovirus that circulates in four species of pteropid bats (ABLVp) and the yellow-bellied sheath-tailed bat (ABLVs) in mainland Australia. In the three confirmed human cases of ABLV, rabies illness preceded fatality. As with rabies virus (RABV), post-exposure prophylaxis (PEP) for potential ABLV infections consists of wound cleansing, administration of the rabies vaccine and injection of rabies immunoglobulin (RIG) proximal to the wound. Despite the efficacy of PEP, the inaccessibility of human RIG (HRIG) in the developing world and the high immunogenicity of equine RIG (ERIG) has led to consideration of human monoclonal antibodies (hmAbs) as a passive immunization option that offers enhanced safety and specificity. Using a recombinant vesicular stomatitis virus (rVSV) expressing the glycoprotein (G) protein of ABLVs and phage display, we identified two hmAbs, A6 and F11, which completely neutralize ABLVs/ABLVp, and RABV at concentrations ranging from 0.39 and 6.25 µg/mL and 0.19 and 0.39 µg/mL respectively. A6 and F11 recognize overlapping epitopes in the lyssavirus G protein, effectively neutralizing phylogroup 1 lyssaviruses, while having little effect on phylogroup 2 and non-grouped diverse lyssaviruses. These results suggest that A6 and F11 could be effective therapeutic and diagnostic tools for phylogroup 1 lyssavirus infections.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Reações Cruzadas/imunologia , Lyssavirus/classificação , Lyssavirus/imunologia , Filogenia , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Austrália , Mordeduras e Picadas , Técnicas de Visualização da Superfície Celular , Quirópteros/virologia , Epitopos/imunologia , Células HEK293 , Cavalos , Humanos , Lyssavirus/genética , Testes de Neutralização , Profilaxia Pós-Exposição , Raiva/prevenção & controle , Vacinas Antirrábicas/imunologia , Vírus da Raiva/imunologia , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/prevenção & controle , Infecções por Rhabdoviridae/terapia , Vesiculovirus/genética
15.
J Biol Chem ; 296: 100701, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33895135

RESUMO

The acid sphingomyelinase/ceramide system has been shown to be important for cellular infection with at least some viruses, for instance, rhinovirus or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Functional inhibition of the acid sphingomyelinase using tricyclic antidepressants prevented infection of epithelial cells, for instance with SARS-CoV-2. The structure of ambroxol, that is, trans-4-[(2,4-dibromanilin-6-yl)-methyamino]-cyclohexanol, a mucolytic drug applied by inhalation, suggests that the drug might inhibit the acid sphingomyelinase and thereby infection with SARS-CoV-2. To test this, we used vesicular stomatitis virus pseudoviral particles presenting SARS-CoV-2 spike protein on their surface (pp-VSV-SARS-CoV-2 spike), a bona fide system for mimicking SARS-CoV-2 entry into cells. Viral uptake and formation of ceramide localization were determined by fluorescence microscopy, activity of the acid sphingomyelinase by consumption of [14C]sphingomyelin and ceramide was quantified by a kinase method. We found that entry of pp-VSV-SARS-CoV-2 spike required activation of acid sphingomyelinase and release of ceramide, events that were all prevented by pretreatment with ambroxol. We also obtained nasal epithelial cells from human volunteers prior to and after inhalation of ambroxol. Inhalation of ambroxol reduced acid sphingomyelinase activity in nasal epithelial cells and prevented pp-VSV-SARS-CoV-2 spike-induced acid sphingomyelinase activation, ceramide release, and entry of pp-VSV-SARS-CoV-2 spike ex vivo. The addition of purified acid sphingomyelinase or C16 ceramide restored entry of pp-VSV-SARS-CoV-2 spike into ambroxol-treated epithelial cells. We propose that ambroxol might be suitable for clinical studies to prevent coronavirus disease 2019.


Assuntos
Ambroxol/farmacologia , Antivirais/farmacologia , SARS-CoV-2/efeitos dos fármacos , Esfingomielina Fosfodiesterase/genética , Vesiculovirus/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Administração por Inalação , Animais , Transporte Biológico , Ceramidas/metabolismo , Chlorocebus aethiops , Reposicionamento de Medicamentos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Células Epiteliais/virologia , Expectorantes , Expressão Gênica , Humanos , Cultura Primária de Células , Vírus Reordenados/efeitos dos fármacos , Vírus Reordenados/fisiologia , SARS-CoV-2/fisiologia , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Esfingomielina Fosfodiesterase/metabolismo , Esfingomielinas/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero , Vesiculovirus/fisiologia
16.
Nat Commun ; 12(1): 1362, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33649317

RESUMO

Therapeutic application of RNA viruses as oncolytic agents or gene vectors requires a tight control of virus activity if toxicity is a concern. Here we present a regulator switch for RNA viruses using a conditional protease approach, in which the function of at least one viral protein essential for transcription and replication is linked to autocatalytical, exogenous human immunodeficiency virus (HIV) protease activity. Virus activity can be en- or disabled by various HIV protease inhibitors. Incorporating the HIV protease dimer in the genome of vesicular stomatitis virus (VSV) into the open reading frame of either the P- or L-protein resulted in an ON switch. Here, virus activity depends on co-application of protease inhibitor in a dose-dependent manner. Conversely, an N-terminal VSV polymerase tag with the HIV protease dimer constitutes an OFF switch, as application of protease inhibitor stops virus activity. This technology may also be applicable to other potentially therapeutic RNA viruses.


Assuntos
Vírus de RNA/genética , Vírus de RNA/fisiologia , Replicação Viral/genética , Animais , Linhagem Celular Tumoral , Genoma Viral , Protease de HIV/química , Protease de HIV/metabolismo , Inibidores da Protease de HIV/farmacologia , Humanos , Camundongos Endogâmicos NOD , Fosfoproteínas/metabolismo , Multimerização Proteica , Vírus de RNA/efeitos dos fármacos , Vesiculovirus/efeitos dos fármacos , Vesiculovirus/genética , Vesiculovirus/fisiologia , Replicação Viral/efeitos dos fármacos
17.
Viruses ; 13(3)2021 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-33669141

RESUMO

Viruses are highly dependent on the host they infect. Their dependence triggers processes of virus-host co-adaptation, enabling viruses to explore host resources whilst escaping immunity. Scientists have tackled viral-host interplay at differing levels of complexity-in individual hosts, organs, tissues and cells-and seminal studies advanced our understanding about viral lifecycles, intra- or inter-species transmission, and means to control infections. Recently, it emerged as important to address the physical properties of the materials in biological systems; membrane-bound organelles are only one of many ways to separate molecules from the cellular milieu. By achieving a type of compartmentalization lacking membranes known as biomolecular condensates, biological systems developed alternative mechanisms of controlling reactions. The identification that many biological condensates display liquid properties led to the proposal that liquid-liquid phase separation (LLPS) drives their formation. The concept of LLPS is a paradigm shift in cellular structure and organization. There is an unprecedented momentum to revisit long-standing questions in virology and to explore novel antiviral strategies. In the first part of this review, we focus on the state-of-the-art about biomolecular condensates. In the second part, we capture what is known about RNA virus-phase biology and discuss future perspectives of this emerging field in virology.


Assuntos
Interações Hospedeiro-Patógeno/fisiologia , Fenômenos Fisiológicos Virais , Animais , Fenômenos Biofísicos , HIV/fisiologia , Humanos , Vírus da Influenza A/fisiologia , Morbillivirus/fisiologia , Organelas/virologia , SARS-CoV-2/fisiologia , Vesiculovirus/fisiologia , Viroses/virologia , Internalização do Vírus
18.
Fish Shellfish Immunol ; 113: 24-34, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33757800

RESUMO

An increasing important area in immunology is the process cell death mechanism, enabling the immune system triggered thru extrinsic or intrinsic signals to effectively remove unwanted or virus infected cells called apoptosis. A recently isolated infectious Snakehead fish vesiculovirus (SHVV), comprising negative strand RNA and encoded viral matrix (M) proteins, is responsible for causing cytopathic effects in infected fish cells. However, the mechanism by which viral M protein mediates apoptosis has not been elucidated. Therefore, in the present experiments, it was investigated the regulatory potential of apoptosis signals during SHVV infection. By employing the model of SHVV infection in SSN-1 cells, the accelerated apoptosis pathway involves an intrinsic pathway requiring the activation of caspase-9 but not caspase-3 or -8. In the groups of infection (SHVV) or treatment (hydrogen peroxide) were induced apoptotic morphological changes and indicated the activation of the main caspases, i.e.; executioner caspase-3, initiators caspase-8 and caspase-9 using colorimetric assays. Turning to the role of viral M protein when it was overexpressed in SSN-1 cells, it was indicated that the viral M gene alone has the ability to induce apoptosis. To elucidate the mechanism of apoptosis in SSN-1 cells, the activation inhibitors of main caspases were used showing that inhibiting of caspase-3 or caspase-8 activation did not seize induction of apoptosis in virus-infected SSN-1 cells. However, the inhibiting of caspase-9 activation reduced significantly the apoptosis initiation process and sharply the expression of viral M gene, suggesting that SHVV plays a major role in the early induction of apoptosis by caspase-9. Interestingly, there were also differences in the mitochondrial membrane potential after the apoptotic induction of caspases, which confirm that caspase-9 is primarily responsible for the cleavage of caspases during apoptosis. Taken together, these findings can therefore be assumed that viral M protein induces apoptosis via the intrinsic apoptotic pathway in SHVV infecting SSN-1 cells.


Assuntos
Apoptose , Doenças dos Peixes/imunologia , Peixes , Infecções por Rhabdoviridae/veterinária , Transdução de Sinais/imunologia , Vesiculovirus/fisiologia , Proteínas da Matriz Viral/fisiologia , Animais , Linhagem Celular , Doenças dos Peixes/virologia , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/virologia
19.
Emerg Microbes Infect ; 10(1): 651-663, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33719915

RESUMO

ABSTRACTThe recent impact of Ebola virus disease (EVD) on public health in Africa clearly demonstrates the need for a safe and efficacious vaccine to control outbreaks and mitigate its threat to global health. ERVEBO® is an effective recombinant Vesicular Stomatitis Virus (VSV)-vectored Ebola virus vaccine (VSV-EBOV) that was approved by the FDA and EMA in late 2019 for use in prevention of EVD. Since the parental virus VSV, which was used to construct VSV-EBOV, is pathogenic for livestock and the vaccine virus may be shed at low levels by vaccinated humans, widespread deployment of the vaccine requires investigation into its infectivity and transmissibility in VSV-susceptible livestock species. We therefore performed a comprehensive clinical analysis of the VSV-EBOV vaccine virus in swine to determine its infectivity and potential for transmission. A high dose of VSV-EBOV resulted in VSV-like clinical signs in swine, with a proportion of pigs developing ulcerative vesicular lesions at the nasal injection site and feet. Uninoculated contact control pigs co-mingled with VSV-EBOV-inoculated pigs did not become infected or display any clinical signs of disease, indicating the vaccine is not readily transmissible to naïve pigs during prolonged close contact. In contrast, virulent wild-type VSV Indiana had a shorter incubation period and was transmitted to contact control pigs. These results indicate that the VSV-EBOV vaccine causes vesicular illness in swine when administered at a high dose. Moreover, the study demonstrates the VSV-EBOV vaccine is not readily transmitted to uninfected pigs, encouraging its safe use as an effective human vaccine.


Assuntos
Vacinas contra Ebola/efeitos adversos , Vacinas contra Ebola/imunologia , Ebolavirus/imunologia , Estomatite Vesicular/transmissão , Estomatite Vesicular/virologia , Vírus da Estomatite Vesicular Indiana/imunologia , Vesiculovirus/imunologia , África , Animais , Chlorocebus aethiops , Ebolavirus/genética , Feminino , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/virologia , Humanos , Masculino , Modelos Animais , RNA Viral , Suínos , Vacinação/métodos , Vacinas Sintéticas/efeitos adversos , Vacinas Sintéticas/imunologia , Células Vero , Vesiculovirus/genética
20.
Dis Aquat Organ ; 143: 169-188, 2021 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-33629660

RESUMO

Spring viremia of carp virus (SVCV) ia a carp sprivivirus and a member of the genus Sprivivirus within the family Rhabdoviridae. The virus is the etiological agent of spring viremia of carp, a disease of cyprinid species including koi Cyprinus carpio L. and notifiable to the World Organisation for Animal Health. The goal of this study was to explore hypotheses regarding inter-genogroup (Ia to Id) SVCV infection dynamics in juvenile koi and contemporaneously create new reverse-transcription quantitative PCR (RT-qPCR) assays and validate their analytical sensitivity, specificity (ASp) and repeatability for diagnostic detection of SVCV. RT-qPCR diagnostic tests targeting the SVCV nucleoprotein (Q2N) or glycoprotein (Q1G) nucleotides were pan-specific for isolates typed to SVCV genogroups Ia to Id. The Q2N test had broader ASp than Q1G because Q1G did not detect SVCV isolate 20120450 and Q2N displayed occasional detection of pike fry sprivivirus isolate V76. Neither test cross-reacted with other rhabdoviruses, infectious pancreatic necrosis virus or co-localizing cyprinid herpesvirus 3. Both tests were sensitive with observed 50% limits of detection of 3 plasmid copies and high repeatability. Test analysis of koi immersed in SVCV showed that the virus could be detected for at least 167 d following exposure and that titer, prevalence, replicative rate and persistence in koi were correlated significantly with virus virulence. In this context, high virulence SVCV isolates were more prevalent, reached higher titers quicker and persisted in koi for longer periods of time relative to moderate and low virulence isolates.


Assuntos
Carpas , Doenças dos Peixes , Infecções por Rhabdoviridae , Animais , Doenças dos Peixes/diagnóstico , Infecções por Rhabdoviridae/diagnóstico , Infecções por Rhabdoviridae/veterinária , Vesiculovirus , Viremia/veterinária
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