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1.
PLoS Biol ; 22(2): e3002544, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38422166

RESUMEN

Ebolavirus (EBOV) belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans. EBOV replication requires the activity of the viral polymerase complex, which includes the cofactor and Interferon antagonist VP35. We previously showed that the covalent ubiquitination of VP35 promotes virus replication by regulating interactions with the polymerase complex. In addition, VP35 can also interact non-covalently with ubiquitin (Ub); however, the function of this interaction is unknown. Here, we report that VP35 interacts with free (unanchored) K63-linked polyUb chains. Ectopic expression of Isopeptidase T (USP5), which is known to degrade unanchored polyUb chains, reduced VP35 association with Ub and correlated with diminished polymerase activity in a minigenome assay. Using computational methods, we modeled the VP35-Ub non-covalent interacting complex, identified the VP35-Ub interacting surface, and tested mutations to validate the interface. Docking simulations identified chemical compounds that can block VP35-Ub interactions leading to reduced viral polymerase activity. Treatment with the compounds reduced replication of infectious EBOV in cells and in vivo in a mouse model. In conclusion, we identified a novel role of unanchored polyUb in regulating Ebola virus polymerase function and discovered compounds that have promising anti-Ebola virus activity.


Asunto(s)
Ebolavirus , Fiebre Hemorrágica Ebola , Proteínas de la Nucleocápside , Humanos , Animales , Ratones , Proteínas Reguladoras y Accesorias Virales , Ubiquitina , Replicación Viral , Ebolavirus/genética
2.
Nature ; 591(7849): 293-299, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33494095

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-a new coronavirus that has led to a worldwide pandemic1-has a furin cleavage site (PRRAR) in its spike protein that is absent in other group-2B coronaviruses2. To explore whether the furin cleavage site contributes to infection and pathogenesis in this virus, we generated a mutant SARS-CoV-2 that lacks the furin cleavage site (ΔPRRA). Here we report that replicates of ΔPRRA SARS-CoV-2 had faster kinetics, improved fitness in Vero E6 cells and reduced spike protein processing, as compared to parental SARS-CoV-2. However, the ΔPRRA mutant had reduced replication in a human respiratory cell line and was attenuated in both hamster and K18-hACE2 transgenic mouse models of SARS-CoV-2 pathogenesis. Despite reduced disease, the ΔPRRA mutant conferred protection against rechallenge with the parental SARS-CoV-2. Importantly, the neutralization values of sera from patients with coronavirus disease 2019 (COVID-19) and monoclonal antibodies against the receptor-binding domain of SARS-CoV-2 were lower against the ΔPRRA mutant than against parental SARS-CoV-2, probably owing to an increased ratio of particles to plaque-forming units in infections with the former. Together, our results demonstrate a critical role for the furin cleavage site in infection with SARS-CoV-2 and highlight the importance of this site for evaluating the neutralization activities of antibodies.


Asunto(s)
COVID-19/virología , Furina/metabolismo , Mutación , SARS-CoV-2/genética , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Neutralizantes/inmunología , COVID-19/patología , COVID-19/fisiopatología , Línea Celular , Chlorocebus aethiops , Cricetinae , Femenino , Humanos , Enfermedades Pulmonares/patología , Enfermedades Pulmonares/fisiopatología , Enfermedades Pulmonares/virología , Masculino , Ratones , Ratones Transgénicos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteolisis , SARS-CoV-2/química , SARS-CoV-2/metabolismo , Serina Endopeptidasas/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Células Vero , Replicación Viral/genética
3.
J Infect Dis ; 228(5): 604-614, 2023 08 31.
Artículo en Inglés | MEDLINE | ID: mdl-36869692

RESUMEN

The common marmoset (Callithrix jacchus) is increasingly recognized as an ideal nonhuman primate (NHP) at high biocontainment due to its smaller size and relative ease of handling. Here, we evaluated the susceptibility and pathogenesis of Nipah virus Bangladesh strain (NiVB) infection in marmosets at biosafety level 4. Infection via the intranasal and intratracheal route resulted in fatal disease in all 4 infected marmosets. Three developed pulmonary edema and hemorrhage as well as multifocal hemorrhagic lymphadenopathy, while 1 recapitulated neurologic clinical manifestations and cardiomyopathy on gross pathology. Organ-specific innate and inflammatory responses were characterized by RNA sequencing in 6 different tissues from infected and control marmosets. Notably, a unique transcriptome was revealed in the brainstem of the marmoset exhibiting neurological signs. Our results provide a more comprehensive understanding of NiV pathogenesis in an accessible and novel NHP model, closely reflecting clinical disease as observed in NiV patients.


Asunto(s)
Infecciones por Henipavirus , Virus Nipah , Edema Pulmonar , Animales , Callithrix , Bangladesh
4.
J Infect Dis ; 218(suppl_5): S438-S447, 2018 11 22.
Artículo en Inglés | MEDLINE | ID: mdl-30192975

RESUMEN

Marburg virus (MARV; family Filoviridae) causes sporadic outbreaks of Marburg hemorrhagic fever in sub-Saharan Africa with case fatality rates reaching 90%. Wild-type filoviruses, including MARV and the closely related Ebola virus, are unable to suppress the type I interferon response in rodents, and therefore require adaptation of the viruses to cause disease in immunocompetent animals. In the current study, we demonstrate that STAT2 knockout Syrian hamsters are susceptible to infection with different wild-type MARV variants. MARV Musoke causes a robust and systemic infection resulting in lethal disease. Histopathological findings share features similar to those observed in human patients and other animal models of filovirus infection. Reverse-transcription polymerase chain reaction analysis of host transcripts shows a dysregulation of the innate immune response. Our results demonstrate that the STAT2 knockout hamster represents a novel small animal model of severe MARV infection and disease without the requirement for virus adaptation.


Asunto(s)
Enfermedad del Virus de Marburg/etiología , Factor de Transcripción STAT2/fisiología , Animales , Cricetinae , Citocinas/biosíntesis , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Femenino , Masculino , Enfermedad del Virus de Marburg/inmunología , Enfermedad del Virus de Marburg/patología
5.
J Infect Dis ; 218(10): 1602-1610, 2018 10 05.
Artículo en Inglés | MEDLINE | ID: mdl-29912426

RESUMEN

Background: Nipah virus (NiV) is a paramyxovirus (genus Henipavirus) that can cause severe respiratory illness and encephalitis in humans. Transmission occurs through consumption of NiV-contaminated foods, and contact with NiV-infected animals or human body fluids. However, it is unclear whether aerosols derived from aforesaid sources or others also contribute to transmission, and current knowledge on NiV-induced pathogenicity after small-particle aerosol exposure is still limited. Methods: Infectivity, pathogenicity, and real-time dissemination of aerosolized NiV in Syrian hamsters was evaluated using NiV-Malaysia (NiV-M) and/or its recombinant expressing firefly luciferase (rNiV-FlucNP). Results: Both viruses had an equivalent pathogenicity in hamsters, which developed respiratory and neurological symptoms of disease, similar to using intranasal route, with no direct correlations to the dose. We showed that virus replication was predominantly initiated in the lower respiratory tract and, although delayed, also intensely in the oronasal cavity and possibly the brain, with gradual increase of signal in these regions until at least day 5-6 postinfection. Conclusion: Hamsters infected with small-particle aerosolized NiV undergo similar clinical manifestations of the disease as previously described using liquid inoculum, and exhibit histopathological lesions consistent with NiV patient reports. NiV droplets could therefore play a role in transmission by close contact.


Asunto(s)
Aerosoles/administración & dosificación , Infecciones por Henipavirus , Virus Nipah/patogenicidad , Administración por Inhalación , Animales , Cricetinae , Modelos Animales de Enfermedad , Infecciones por Henipavirus/diagnóstico por imagen , Infecciones por Henipavirus/patología , Infecciones por Henipavirus/transmisión , Infecciones por Henipavirus/virología , Luciferasas de Luciérnaga/genética , Luciferasas de Luciérnaga/metabolismo , Pulmón/diagnóstico por imagen , Pulmón/patología , Pulmón/virología , Mesocricetus , Imagen Óptica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
6.
J Virol ; 91(15)2017 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-28539439

RESUMEN

Nipah virus (NiV) is a zoonotic emerging paramyxovirus that can cause fatal respiratory illness or encephalitis in humans. Despite many efforts, the molecular mechanisms of NiV-induced acute lung injury (ALI) remain unclear. We previously showed that NiV replicates to high titers in human lung grafts in NOD-SCID/γ mice, resulting in a robust inflammatory response. Interestingly, these mice can undergo human immune system reconstitution by the bone marrow, liver, and thymus (BLT) reconstitution method, in addition to lung tissue engraftment, giving altogether a realistic model to study human respiratory viral infections. Here, we characterized NiV Bangladesh strain (NiV-B) infection of human lung grafts from human immune system-reconstituted mice in order to identify the overall effect of immune cells on NiV pathogenesis of the lung. We show that NiV-B replicated to high titers in human lung grafts and caused similar cytopathic effects irrespective of the presence of human leukocytes in mice. However, the human immune system interfered with virus spread across lung grafts, responded to infection by leukocyte migration to small airways and alveoli of the lung grafts, and accelerated oxidative stress in lung grafts. In addition, the presence of human leukocytes increased the expression of cytokines and chemokines that regulate inflammatory influx to sites of infection and tissue damage. These results advance our understanding of how the immune system limits NiV dissemination and contributes to ALI and inform efforts to identify therapeutic targets.IMPORTANCE Nipah virus (NiV) is an emerging paramyxovirus that can cause a lethal respiratory and neurological disease in humans. Only limited data are available on NiV pathogenesis in the human lung, and the relative contribution of the innate immune response and NiV to acute lung injury (ALI) is still unknown. Using human lung grafts in a human immune system-reconstituted mouse model, we showed that the NiV Bangladesh strain induced cytopathic lesions in lung grafts similar to those described in patients irrespective of the donor origin or the presence of leukocytes. However, the human immune system interfered with virus spread, responded to infection by leukocyte infiltration in the small airways and alveolar area, induced oxidative stress, and triggered the production of cytokines and chemokines that regulate inflammatory influx by leukocytes in response to infection. Understanding how leukocytes interact with NiV and cause ALI in human lung xenografts is crucial for identifying therapeutic targets.


Asunto(s)
Lesión Pulmonar Aguda/patología , Infecciones por Henipavirus/patología , Leucocitos/inmunología , Pulmón/patología , Virus Nipah/crecimiento & desarrollo , Estrés Oxidativo , Animales , Citocinas/análisis , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones SCID
7.
J Gen Virol ; 97(4): 839-843, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26781134

RESUMEN

Incorporation of reporter genes within virus genomes is an indispensable tool for interrogation of virus biology and pathogenesis. In previous work, we incorporated a fluorophore into a viral ORF by attaching it to the viral gene via a P2A ribosomal skipping sequence. This recombinant Nipah virus, however, was attenuated in vitro relative to WT virus. In this work, we determined that inefficient ribosomal skipping was a major contributing factor to this attenuation. Inserting a GSG linker before the P2A sequence resulted in essentially complete skipping, significantly improved growth in vitro, and WT lethality in vivo. To the best of our knowledge, this represents the first time a recombinant virus of Mononegavirales with integration of a reporter into a viral ORF has been compared with the WT virus in vivo. Incorporating the GSG linker for improved skipping efficiency whenever functionally important is a critical consideration for recombinant virus design.


Asunto(s)
Genes Reporteros , Ingeniería Genética/métodos , Infecciones por Henipavirus/genética , Virus Nipah/genética , Proteínas Ribosómicas/genética , Secuencia de Aminoácidos , Animales , Femenino , Regulación de la Expresión Génica , Infecciones por Henipavirus/mortalidad , Infecciones por Henipavirus/patología , Infecciones por Henipavirus/virología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Mesocricetus , Datos de Secuencia Molecular , Mutagénesis Insercional , Virus Nipah/patogenicidad , Faloidina/genética , Faloidina/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Análisis de Supervivencia , Transcripción Genética , Replicación Viral/genética , Proteína Fluorescente Roja
8.
J Virol ; 89(14): 7262-76, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-25948740

RESUMEN

UNLABELLED: Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to Africa and characterized by a high rate of abortion in ruminants and hemorrhagic fever, encephalitis, or blindness in humans. RVF is caused by Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus), which has a tripartite negative-stranded RNA genome (consisting of the S, M, and L segments). Further spread of RVF into countries where the disease is not endemic may affect the economy and public health, and vaccination is an effective approach to prevent the spread of RVFV. A live-attenuated MP-12 vaccine is one of the best-characterized RVF vaccines for safety and efficacy and is currently conditionally licensed for use for veterinary purposes in the United States. Meanwhile, as of 2015, no other RVF vaccine has been conditionally or fully licensed for use in the United States. The MP-12 strain is derived from wild-type pathogenic strain ZH548, and its genome encodes 23 mutations in the three genome segments. However, the mechanism of MP-12 attenuation remains unknown. We characterized the attenuation of wild-type pathogenic strain ZH501 carrying a mutation(s) of the MP-12 S, M, or L segment in a mouse model. Our results indicated that MP-12 is attenuated by the mutations in the S, M, and L segments, while the mutations in the M and L segments confer stronger attenuation than those in the S segment. We identified a combination of 3 amino acid changes, Y259H (Gn), R1182G (Gc), and R1029K (L), that was sufficient to attenuate ZH501. However, strain MP-12 with reversion mutations at those 3 sites was still highly attenuated. Our results indicate that MP-12 attenuation is supported by a combination of multiple partial attenuation mutations and a single reversion mutation is less likely to cause a reversion to virulence of the MP-12 vaccine. IMPORTANCE: Rift Valley fever (RVF) is a mosquito-transmitted viral disease that is endemic to Africa and that has the potential to spread into other countries. Vaccination is considered an effective way to prevent the disease, and the only available veterinary RVF vaccine in the United States is a live-attenuated MP-12 vaccine, which is conditionally licensed. Strain MP-12 is different from its parental pathogenic RVFV strain, strain ZH548, because of the presence of 23 mutations. This study determined the role of individual mutations in the attenuation of the MP-12 strain. We found that full attenuation of MP-12 occurs by a combination of multiple mutations. Our findings indicate that a single reversion mutation will less likely cause a major reversion to virulence of the MP-12 vaccine.


Asunto(s)
Fiebre del Valle del Rift/patología , Fiebre del Valle del Rift/virología , Virus de la Fiebre del Valle del Rift/genética , Virus de la Fiebre del Valle del Rift/fisiología , Vacunas Virales/genética , Vacunas Virales/inmunología , Animales , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Femenino , Ratones , Mutación Missense , Virus de la Fiebre del Valle del Rift/inmunología , Virus de la Fiebre del Valle del Rift/patogenicidad , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Virulencia
9.
J Virol ; 89(2): 1242-53, 2015 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-25392218

RESUMEN

UNLABELLED: Nipah virus (NiV) and Hendra virus (HeV) are closely related henipaviruses of the Paramyxovirinae. Spillover from their fruit bat reservoirs can cause severe disease in humans and livestock. Despite their high sequence similarity, NiV and HeV exhibit apparent differences in receptor and tissue tropism, envelope-mediated fusogenicity, replicative fitness, and other pathophysiologic manifestations. To investigate the molecular basis for these differences, we first established a highly efficient reverse genetics system that increased rescue titers by ≥3 log units, which offset the difficulty of generating multiple recombinants under constraining biosafety level 4 (BSL-4) conditions. We then replaced, singly and in combination, the matrix (M), fusion (F), and attachment glycoprotein (G) genes in mCherry-expressing recombinant NiV (rNiV) with their HeV counterparts. These chimeric but isogenic rNiVs replicated well in primary human endothelial and neuronal cells, indicating efficient heterotypic complementation. The determinants of budding efficiency, fusogenicity, and replicative fitness were dissociable: HeV-M budded more efficiently than NiV-M, accounting for the higher replicative titers of HeV-M-bearing chimeras at early times, while the enhanced fusogenicity of NiV-G-bearing chimeras did not correlate with increased replicative fitness. Furthermore, to facilitate spatiotemporal studies on henipavirus pathogenesis, we generated a firefly luciferase-expressing NiV and monitored virus replication and spread in infected interferon alpha/beta receptor knockout mice via bioluminescence imaging. While intraperitoneal inoculation resulted in neuroinvasion following systemic spread and replication in the respiratory tract, intranasal inoculation resulted in confined spread to regions corresponding to olfactory bulbs and salivary glands before subsequent neuroinvasion. This optimized henipavirus reverse genetics system will facilitate future investigations into the growing numbers of novel henipavirus-like viruses. IMPORTANCE: Nipah virus (NiV) and Hendra virus (HeV) are recently emergent zoonotic and highly lethal pathogens with pandemic potential. Although differences have been observed between NiV and HeV replication and pathogenesis, the molecular basis for these differences has not been examined. In this study, we established a highly efficient system to reverse engineer changes into replication-competent NiV and HeV, which facilitated the generation of reporter-expressing viruses and recombinant NiV-HeV chimeras with substitutions in the genes responsible for viral exit (the M gene, critical for assembly and budding) and viral entry (the G [attachment] and F [fusion] genes). These chimeras revealed differences in the budding and fusogenic properties of the M and G proteins, respectively, which help explain previously observed differences between NiV and HeV. Finally, to facilitate future in vivo studies, we monitored the replication and spread of a bioluminescent reporter-expressing NiV in susceptible mice; this is the first time such in vivo imaging has been performed under BSL-4 conditions.


Asunto(s)
Modelos Animales de Enfermedad , Virus Hendra/fisiología , Infecciones por Henipavirus/virología , Virus Nipah/fisiología , Internalización del Virus , Liberación del Virus , Animales , Prueba de Complementación Genética , Humanos , Ratones Noqueados , Recombinación Genética , Genética Inversa , Tropismo Viral
10.
PLoS Pathog ; 9(4): e1003297, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23637597

RESUMEN

LJ001 is a lipophilic thiazolidine derivative that inhibits the entry of numerous enveloped viruses at non-cytotoxic concentrations (IC50 ≤ 0.5 µM), and was posited to exploit the physiological difference between static viral membranes and biogenic cellular membranes. We now report on the molecular mechanism that results in LJ001's specific inhibition of virus-cell fusion. The antiviral activity of LJ001 was light-dependent, required the presence of molecular oxygen, and was reversed by singlet oxygen ((1)O2) quenchers, qualifying LJ001 as a type II photosensitizer. Unsaturated phospholipids were the main target modified by LJ001-generated (1)O2. Hydroxylated fatty acid species were detected in model and viral membranes treated with LJ001, but not its inactive molecular analog, LJ025. (1)O2-mediated allylic hydroxylation of unsaturated phospholipids leads to a trans-isomerization of the double bond and concurrent formation of a hydroxyl group in the middle of the hydrophobic lipid bilayer. LJ001-induced (1)O2-mediated lipid oxidation negatively impacts on the biophysical properties of viral membranes (membrane curvature and fluidity) critical for productive virus-cell membrane fusion. LJ001 did not mediate any apparent damage on biogenic cellular membranes, likely due to multiple endogenous cytoprotection mechanisms against phospholipid hydroperoxides. Based on our understanding of LJ001's mechanism of action, we designed a new class of membrane-intercalating photosensitizers to overcome LJ001's limitations for use as an in vivo antiviral agent. Structure activity relationship (SAR) studies led to a novel class of compounds (oxazolidine-2,4-dithiones) with (1) 100-fold improved in vitro potency (IC50<10 nM), (2) red-shifted absorption spectra (for better tissue penetration), (3) increased quantum yield (efficiency of (1)O2 generation), and (4) 10-100-fold improved bioavailability. Candidate compounds in our new series moderately but significantly (p≤0.01) delayed the time to death in a murine lethal challenge model of Rift Valley Fever Virus (RVFV). The viral membrane may be a viable target for broad-spectrum antivirals that target virus-cell fusion.


Asunto(s)
Antivirales/farmacología , Oxazoles/farmacología , Rodanina/análogos & derivados , Virus de la Fiebre del Valle del Rift/efectos de los fármacos , Tionas/farmacología , Acoplamiento Viral/efectos de los fármacos , Internalización del Virus/efectos de los fármacos , Animales , Antivirales/química , Disponibilidad Biológica , Membrana Celular/efectos de los fármacos , Membrana Celular/virología , Ratones , Ratones Endogámicos BALB C , Fosfolípidos/química , Fosfolípidos/metabolismo , Fármacos Fotosensibilizantes/farmacología , Rodanina/farmacología , Fiebre del Valle del Rift/tratamiento farmacológico , Oxígeno Singlete/química , Relación Estructura-Actividad
11.
Viruses ; 16(7)2024 Jul 09.
Artículo en Inglés | MEDLINE | ID: mdl-39066263

RESUMEN

Favipiravir is a ribonucleoside analogue that has been explored as a therapeutic for the treatment of Ebola Virus Disease (EVD). Promising data from rodent models has informed nonhuman primate trials, as well as evaluation in patients during the 2013-2016 West African EVD outbreak of favipiravir treatment. However, mixed results from these studies hindered regulatory approval of favipiravir for the indication of EVD. This study examined the influence of route of administration, duration of treatment, and treatment schedule of favipiravir in immune competent mouse and guinea pig models using rodent-adapted Zaire ebolavirus (EBOV). A dose of 300 mg/kg/day of favipiravir with an 8-day treatment was found to be fully effective at preventing lethal EVD-like disease in BALB/c mice regardless of route of administration (oral, intraperitoneal, or subcutaneous) or whether it was provided as a once-daily dose or a twice-daily split dose. Preclinical data generated in guinea pigs demonstrates that an 8-day treatment of 300 mg/kg/day of favipiravir reduces mortality following EBOV challenge regardless of route of treatment or duration of treatments for 8, 11, or 15 days. This work supports the future translational development of favipiravir as an EVD therapeutic.


Asunto(s)
Amidas , Antivirales , Modelos Animales de Enfermedad , Ebolavirus , Fiebre Hemorrágica Ebola , Ratones Endogámicos BALB C , Pirazinas , Animales , Amidas/uso terapéutico , Amidas/administración & dosificación , Amidas/farmacología , Cobayas , Pirazinas/administración & dosificación , Pirazinas/uso terapéutico , Fiebre Hemorrágica Ebola/tratamiento farmacológico , Ratones , Ebolavirus/efectos de los fármacos , Antivirales/administración & dosificación , Antivirales/uso terapéutico , Femenino , Vías de Administración de Medicamentos , Esquema de Medicación
12.
J Gen Virol ; 94(Pt 7): 1441-1450, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23515022

RESUMEN

Rift Valley fever is a mosquito-borne zoonotic disease endemic to sub-Saharan Africa. Rift Valley fever virus (RVFV; genus Phlebovirus, family Bunyaviridae) causes high rates of abortion and fetal malformation in pregnant ruminants, and haemorrhagic fever, neurological disorders or blindness in humans. The MP-12 strain is a highly efficacious and safe live-attenuated vaccine candidate for both humans and ruminants. However, MP-12 lacks a marker to differentiate infected from vaccinated animals. In this study, we originally aimed to characterize the efficacy of a recombinant RVFV MP-12 strain encoding Toscana virus (TOSV) NSs gene in place of MP-12 NSs (rMP12-TOSNSs). TOSV NSs promotes the degradation of dsRNA-dependent protein kinase (PKR) and inhibits interferon-ß gene up-regulation without suppressing host general transcription. Unexpectedly, rMP12-TOSNSs increased death in vaccinated outbred mice and inbred BALB/c or C57BL/6 mice. Immunohistochemistry showed diffusely positive viral antigens in the thalamus, hypothalamus and brainstem, including the medulla. No viral antigens were detected in spleen or liver, which is similar to the antigen distribution of moribund mice infected with MP-12. These results suggest that rMP12-TOSNSs retains neuroinvasiveness in mice. Our findings demonstrate that rMP12-TOSNSs causes neuroinvasion without any hepatic disease and will be useful for studying the neuroinvasion mechanism of RVFV and TOSV.


Asunto(s)
Encéfalo/virología , Enfermedades del Sistema Nervioso/virología , Fiebre del Valle del Rift/prevención & control , Virus de Nápoles de la Fiebre de la Mosca de los Arenales/genética , Virus de Nápoles de la Fiebre de la Mosca de los Arenales/patogenicidad , Vacunas Atenuadas/efectos adversos , Proteínas no Estructurales Virales/metabolismo , Vacunas Virales/efectos adversos , Animales , Línea Celular , Chlorocebus aethiops , Femenino , Humanos , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fiebre del Valle del Rift/inmunología , Virus de la Fiebre del Valle del Rift/inmunología , Virus de Nápoles de la Fiebre de la Mosca de los Arenales/inmunología , Vacunación , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/inmunología , Células Vero , Proteínas no Estructurales Virales/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/inmunología
13.
J Virol ; 86(14): 7650-61, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22573861

RESUMEN

Rift Valley fever virus (RVFV), belonging to the genus Phlebovirus, family Bunyaviridae, is endemic to sub-Saharan Africa and causes a high rate of abortion in ruminants and hemorrhagic fever, encephalitis, or blindness in humans. MP-12 is the only RVFV strain excluded from the select-agent rule and handled at a biosafety level 2 (BSL2) laboratory. MP-12 encodes a functional major virulence factor, the NSs protein, which contributes to its residual virulence in pregnant ewes. We found that 100% of mice subcutaneously vaccinated with recombinant MP-12 (rMP12)-murine PKRN167 (mPKRN167), which encodes a dominant-negative form of mouse double-stranded RNA (dsRNA)-dependent protein kinase (PKR) in place of NSs, were protected from wild-type (wt) RVFV challenge, while 72% of mice vaccinated with MP-12 were protected after challenge. rMP12-mPKRN167 induced alpha interferon (IFN-α) in sera, accumulated RVFV antigens in dendritic cells at the local draining lymph nodes, and developed high levels of neutralizing antibodies, while parental MP-12 induced neither IFN-α nor viral-antigen accumulation at the draining lymph node yet induced a high level of neutralizing antibodies. The present study suggests that the expression of a dominant-negative PKR increases the immunogenicity and efficacy of live-attenuated RVFV vaccine, which will lead to rational design of safe and highly immunogenic RVFV vaccines for livestock and humans.


Asunto(s)
Virus de la Fiebre del Valle del Rift/inmunología , Proteínas no Estructurales Virales/inmunología , Vacunas Virales/inmunología , eIF-2 Quinasa/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Línea Celular , Quimiocinas/sangre , Chlorocebus aethiops , Cricetinae , Células Dendríticas/inmunología , Interferón-alfa/sangre , Interferón gamma/sangre , Interleucina-17/sangre , Interleucina-5/sangre , Ratones , Mutación , Virus de la Fiebre del Valle del Rift/genética , Virus de la Fiebre del Valle del Rift/patogenicidad , Células Vero , eIF-2 Quinasa/antagonistas & inhibidores
14.
Mol Pharm ; 10(9): 3342-55, 2013 Sep 03.
Artículo en Inglés | MEDLINE | ID: mdl-23915419

RESUMEN

Pre-existing immunity (PEI) to human adenovirus serotype 5 (Ad5) worldwide is the primary limitation to routine clinical use of Ad5-based vectors in immunization platforms. Using systemic and mucosal PEI induction models in rodents (mice and guinea pigs), we assessed the influence of PEI on the type of adaptive immune response elicited by an Ad5-based vaccine for Ebola with respect to immunization route. Splenocytes isolated from vaccinated animals revealed that immunization by the same route in which PEI was induced significantly compromised Ebola Zaire glycoprotein (ZGP)-specific IFN-γ+ CD8+ T cells and ZGP-specific multifunctional CD8+ T cell populations. ZGP-specific IgG1 antibody levels were also significantly reduced and a sharp increase in serum anti-Ad5 neutralizing antibody (NAB) titers were noted following immunization. These immune parameters correlated with poor survival after lethal challenge with rodent-adapted Ebola Zaire virus (ZEBOV). Although the number of IFN-γ+ CD8+ T cells was reduced in animals given the vaccine by a different route from that used for PEI induction, the multifunctional CD8+ T cell response was not compromised. Survival rates in these groups were higher than when PEI was induced by the same route as immunization. These results suggest that antigen-specific multifunctional CD8(+) T cell and Th2 type antibody responses compromised by PEI to Ad5 are required for protection from Ebola. They also illustrate that methods for induction of PEI used in preclinical studies must be carefully evaluated for successful development of novel Ad5-based vaccines.


Asunto(s)
Adenoviridae/inmunología , Vacunas contra el Virus del Ébola/inmunología , Animales , Formación de Anticuerpos/inmunología , Linfocitos T CD8-positivos/inmunología , Ebolavirus/inmunología , Cobayas , Masculino , Ratones , Linfocitos T/inmunología
15.
Methods Mol Biol ; 2682: 219-229, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37610585

RESUMEN

Small rodent animal models that recapitulate the symptomology and pathology of the human disease caused by Nipah virus (NiV) are crucial not only to study virus-induced disease but also a critical component for the development of vaccine and therapeutic candidates. The Syrian golden hamster is the most commonly used small animal model for NiV and develops clinical symptoms and pathologies that closely resemble NiV disease in humans. In this chapter, we describe standard techniques used to infect hamsters and conduct evaluation of therapeutics and vaccine candidates.


Asunto(s)
Infecciones por Henipavirus , Animales , Cricetinae , Humanos , Mesocricetus , Modelos Animales
16.
Methods Mol Biol ; 2682: 149-157, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37610580

RESUMEN

In vivo imaging system (IVIS) is a powerful tool for the study of infectious diseases, providing the ability to non-invasively follow viral infection in an individual animal over time. Recombinant henipaviruses expressing bioluminescent or fluorescent reporter proteins can be used both to monitor the spatial and temporal progression of Nipah virus (NiV) infection in vivo as well as in ex vivo tissues. Virally produced luciferases react with systemically administered substrate to produce bioluminescence that can then be detected via IVIS imaging, while fluorescent reporters inherently generate detectable fluorescence without a substrate. Here we describe protocols applying bioluminescent or fluorescent reporter expressing recombinant viruses to in vivo or ex vivo imaging of NiV infection.


Asunto(s)
Infecciones por Henipavirus , Orthopoxvirus , Animales , Diagnóstico por Imagen , Colorantes , Modelos Animales
17.
Viruses ; 15(7)2023 07 21.
Artículo en Inglés | MEDLINE | ID: mdl-37515275

RESUMEN

Ebola virus (EBOV) causes Ebola virus disease (EVD), which is characterized by hemorrhagic fever with high mortality rates in humans. EBOV sexual transmission has been a concern since the 2014-2016 outbreak in Africa, as persistent infection in the testis and transmission to women was demonstrated. The only study related to establishing an intravaginal small animal infection model was recently documented in IFNAR-/- mice using wild-type and mouse-adapted EBOV (maEBOV), and resulted in 80% mortality, supporting epidemiological data. However, this route of transmission is still poorly understood in women, and the resulting EVD from it is understudied. Here, we contribute to this field of research by providing data from immunocompetent BALB/c mice. We demonstrate that progesterone priming increased the likelihood of maEBOV vaginal infection and of exhibiting the symptoms of disease and seroconversion. However, our data suggest subclinical infection, regardless of the infective dose. We conclude that maEBOV can infect BALB/c mice through vaginal inoculation, but that this route of infection causes significantly less disease compared to intraperitoneal injection at a similar dose, which is consistent with previous studies using other peripheral routes of inoculation in that animal model. Our data are inconsistent with the disease severity described in female patients, therefore suggesting that BALB/c mice are unsuitable for modeling typical EVD following vaginal challenge with maEBOV. Further studies are required to determine the mechanisms by which EVD is attenuated in BALB/c mice, using maEBOV via the vaginal route, as in our experimental set-up.


Asunto(s)
Ebolavirus , Fiebre Hemorrágica Ebola , Humanos , Masculino , Animales , Femenino , Ratones , Ratones Endogámicos BALB C , Vagina , Modelos Animales
18.
Viruses ; 15(2)2023 01 19.
Artículo en Inglés | MEDLINE | ID: mdl-36851495

RESUMEN

The genetic diversities of mammalian tick-borne flaviviruses are poorly understood. We used next-generation sequencing (NGS) to deep sequence different viruses and strains belonging to this group of flaviviruses, including Central European tick-borne encephalitis virus (TBEV-Eur), Far Eastern TBEV (TBEV-FE), Langat (LGTV), Powassan (POWV), Deer Tick (DTV), Kyasanur Forest Disease (KFDV), Alkhurma hemorrhagic fever (AHFV), and Omsk hemorrhagic fever (OHFV) viruses. DTV, AHFV, and KFDV had the lowest genetic diversity, while POWV strains LEIV-5530 and LB, OHFV, TBEV-Eur, and TBEV-FE had higher genetic diversities. These findings are compatible with the phylogenetic relationships between the viruses. For DTV and POWV, the amount of genetic diversity could be explained by the number of tick vector species and amplification hosts each virus can occupy, with low diversity DTV having a more limited vector and host pool, while POWV with higher genetic diversities has been isolated from different tick species and mammals. It is speculated that high genetic diversity may contribute to the survival of the virus as it encounters these different environments.


Asunto(s)
Virus de la Encefalitis Transmitidos por Garrapatas , Encefalitis Transmitida por Garrapatas , Animales , Filogenia , Virus de la Encefalitis Transmitidos por Garrapatas/genética , Mamíferos , Variación Genética
19.
Biomedicines ; 11(7)2023 Jun 22.
Artículo en Inglés | MEDLINE | ID: mdl-37509430

RESUMEN

Recent studies have confirmed that lung microvascular endothelial injury plays a critical role in the pathophysiology of COVID-19. Our group and others have demonstrated the beneficial effects of H2S in several pathological processes and provided a rationale for considering the therapeutic implications of H2S in COVID-19 therapy. Here, we evaluated the effect of the slow-releasing H2S donor, GYY4137, on the barrier function of a lung endothelial cell monolayer in vitro, after challenging the cells with plasma samples from COVID-19 patients or inactivated SARS-CoV-2 virus. We also assessed how the cytokine/chemokine profile of patients' plasma, endothelial barrier permeability, and disease severity correlated with each other. Alterations in barrier permeability after treatments with patient plasma, inactivated virus, and GYY4137 were monitored and assessed by electrical impedance measurements in real time. We present evidence that GYY4137 treatment reduced endothelial barrier permeability after plasma challenge and completely reversed the endothelial barrier disruption caused by inactivated SARS-CoV-2 virus. We also showed that disease severity correlated with the cytokine/chemokine profile of the plasma but not with barrier permeability changes in our assay. Overall, these data demonstrate that treatment with H2S-releasing compounds has the potential to ameliorate SARS-CoV-2-associated lung endothelial barrier disruption.

20.
J Virol ; 85(20): 10605-16, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-21849459

RESUMEN

The search for a safe and efficacious vaccine for Ebola virus continues, as no current vaccine candidate is nearing licensure. We have developed (i) replication-competent, (ii) replication-deficient, and (iii) chemically inactivated rabies virus (RABV) vaccines expressing Zaire Ebola virus (ZEBOV) glycoprotein (GP) by a reverse genetics system based on the SAD B19 RABV wildlife vaccine. ZEBOV GP is efficiently expressed by these vaccine candidates and is incorporated into virions. The vaccine candidates were avirulent after inoculation of adult mice, and viruses with a deletion in the RABV glycoprotein had greatly reduced neurovirulence after intracerebral inoculation in suckling mice. Immunization with live or inactivated RABV vaccines expressing ZEBOV GP induced humoral immunity against each virus and conferred protection from both lethal RABV and EBOV challenge in mice. The bivalent RABV/ZEBOV vaccines described here have several distinct advantages that may speed the development of inactivated vaccines for use in humans and potentially live or inactivated vaccines for use in nonhuman primates at risk of EBOV infection in endemic areas.


Asunto(s)
Vacunas contra el Virus del Ébola/inmunología , Vacunas Antirrábicas/inmunología , Animales , Anticuerpos Antivirales/sangre , Encéfalo/virología , Modelos Animales de Enfermedad , Vacunas contra el Virus del Ébola/administración & dosificación , Vacunas contra el Virus del Ébola/efectos adversos , Vacunas contra el Virus del Ébola/genética , Ebolavirus/genética , Ebolavirus/inmunología , Fiebre Hemorrágica Ebola/prevención & control , Ratones , Ratones Endogámicos BALB C , Rabia/prevención & control , Vacunas Antirrábicas/administración & dosificación , Vacunas Antirrábicas/efectos adversos , Vacunas Antirrábicas/genética , Virus de la Rabia/genética , Virus de la Rabia/inmunología , Enfermedades de los Roedores/prevención & control , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/efectos adversos , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Vacunas de Productos Inactivados/administración & dosificación , Vacunas de Productos Inactivados/efectos adversos , Vacunas de Productos Inactivados/genética , Vacunas de Productos Inactivados/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/efectos adversos , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Virulencia
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