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1.
Sci Rep ; 12(1): 700, 2022 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-35027600

RESUMO

Venezuelan equine encephalitis virus (VEEV) is a mosquito borne alphavirus which leads to high viremia in equines followed by lethal encephalitis and lateral spread to humans. In addition to naturally occurring outbreaks, VEEV is a potential biothreat agent with no approved human vaccine or therapeutic currently available. Single domain antibodies (sdAb), also known as nanobodies, have the potential to be effective therapeutic agents. Using an immune phage display library derived from a llama immunized with an equine vaccine that included inactivated VEEV, five sdAb sequence families were identified that showed varying ability to neutralize VEEV. One of the sequence families had been identified previously in selections against chikungunya virus, a related alphavirus of public health concern. A key advantage of sdAb is the ability to optimize properties such as neutralization capacity through protein engineering. Neutralization of VEEV was improved by two orders of magnitude by genetically linking sdAb. One of the bivalent constructs showed effective neutralization of both VEEV and chikungunya virus. Several of the bivalent constructs neutralized VEEV in cell-based assays with reductions in the number of plaques by 50% at protein concentrations of 1 ng/mL or lower, making future evaluation of their therapeutic potential compelling.

2.
PLoS Pathog ; 17(11): e1009433, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34752502

RESUMO

Arthropod-borne viruses (arboviruses) require replication across a wide range of temperatures to perpetuate. While vertebrate hosts tend to maintain temperatures of approximately 37°C-40°C, arthropods are subject to ambient temperatures which can have a daily fluctuation of > 10°C. Temperatures impact vector competence, extrinsic incubation period, and mosquito survival unimodally, with optimal conditions occurring at some intermediate temperature. In addition, the mean and range of daily temperature fluctuations influence arbovirus perpetuation and vector competence. The impact of temperature on arbovirus genetic diversity during systemic mosquito infection, however, is poorly understood. Therefore, we determined how constant extrinsic incubation temperatures of 25°C, 28°C, 32°C, and 35°C control Zika virus (ZIKV) vector competence and population dynamics within Aedes aegypti and Aedes albopictus mosquitoes. We also examined fluctuating temperatures which better mimic field conditions in the tropics. We found that vector competence varied in a unimodal manner for constant temperatures peaking between 28°C and 32°C for both Aedes species. Transmission peaked at 10 days post-infection for Aedes aegypti and 14 days for Aedes albopictus. Conversely, fluctuating temperature decreased vector competence. Using RNA-seq to characterize ZIKV population structure, we identified that temperature alters the selective environment in unexpected ways. During mosquito infection, constant temperatures more often elicited positive selection whereas fluctuating temperatures led to strong purifying selection in both Aedes species. These findings demonstrate that temperature has multiple impacts on ZIKV biology, including major effects on the selective environment within mosquitoes.

3.
Vaccines (Basel) ; 9(10)2021 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-34696250

RESUMO

Vaccination remains critical for viral disease outbreak prevention and control, but conventional vaccine development typically involves trade-offs between safety and immunogenicity. We used a recently discovered insect-specific flavivirus as a vector in order to develop an exceptionally safe, flavivirus vaccine candidate with single-dose efficacy. To evaluate the safety and efficacy of this platform, we created a chimeric Zika virus (ZIKV) vaccine candidate, designated Aripo/Zika virus (ARPV/ZIKV). ZIKV has caused immense economic and public health impacts throughout the Americas and remains a significant public health threat. ARPV/ZIKV vaccination showed exceptional safety due to ARPV/ZIKV's inherent vertebrate host-restriction. ARPV/ZIKV showed no evidence of replication or translation in vitro and showed no hematological, histological or pathogenic effects in vivo. A single-dose immunization with ARPV/ZIKV induced rapid and robust neutralizing antibody and cellular responses, which offered complete protection against ZIKV-induced morbidity, mortality and in utero transmission in immune-competent and -compromised murine models. Splenocytes derived from vaccinated mice demonstrated significant CD4+ and CD8+ responses and significant cytokine production post-antigen exposure. Altogether, our results further support that chimeric insect-specific flaviviruses are a promising strategy to restrict flavivirus emergence via vaccine development.

4.
Cell ; 184(17): 4392-4400.e4, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34289344

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic underscores the need to better understand animal-to-human transmission of coronaviruses and adaptive evolution within new hosts. We scanned more than 182,000 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes for selective sweep signatures and found a distinct footprint of positive selection located around a non-synonymous change (A1114G; T372A) within the spike protein receptor-binding domain (RBD), predicted to remove glycosylation and increase binding to human ACE2 (hACE2), the cellular receptor. This change is present in all human SARS-CoV-2 sequences but not in closely related viruses from bats and pangolins. As predicted, T372A RBD bound hACE2 with higher affinity in experimental binding assays. We engineered the reversion mutant (A372T) and found that A372 (wild-type [WT]-SARS-CoV-2) enhanced replication in human lung cells relative to its putative ancestral variant (T372), an effect that was 20 times greater than the well-known D614G mutation. Our findings suggest that this mutation likely contributed to SARS-CoV-2 emergence from animal reservoirs or enabled sustained human-to-human transmission.


Assuntos
COVID-19/virologia , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Substituição de Aminoácidos , Enzima de Conversão de Angiotensina 2 , Animais , Linhagem Celular , Quirópteros/virologia , Chlorocebus aethiops , Reservatórios de Doenças , Evolução Molecular , Genoma Viral , Humanos , Modelos Moleculares , Mutação , Filogenia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero
5.
Viruses ; 13(6)2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34205098

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible RNA virus that is the causative agent of the Coronavirus disease 2019 (COVID-19) pandemic. Patients with severe COVID-19 may develop acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) and require mechanical ventilation. Key features of SARS-CoV-2 induced pulmonary complications include an overexpression of pro-inflammatory chemokines and cytokines that contribute to a 'cytokine storm.' In the current study an inflammatory state in Calu-3 human lung epithelial cells was characterized in which significantly elevated transcripts of the immunostimulatory chemokines CXCL9, CXCL10, and CXCL11 were present. Additionally, an increase in gene expression of the cytokines IL-6, TNFα, and IFN-γ was observed. The transcription of CXCL9, CXCL10, IL-6, and IFN-γ was also induced in the lungs of human transgenic angiotensin converting enzyme 2 (ACE2) mice infected with SARS-CoV-2. To elucidate cell signaling pathways responsible for chemokine upregulation in SARS-CoV-2 infected cells, small molecule inhibitors targeting key signaling kinases were used. The induction of CXCL9, CXCL10, and CXCL11 gene expression in response to SARS-CoV-2 infection was markedly reduced by treatment with the AKT inhibitor GSK690693. Samples from COVID-19 positive individuals also displayed marked increases in CXCL9, CXCL10, and CXCL11 transcripts as well as transcripts in the AKT pathway. The current study elucidates potential pathway specific targets for reducing the induction of chemokines that may be contributing to SARS-CoV-2 pathogenesis via hyperinflammation.


Assuntos
COVID-19/imunologia , Quimiocina CXCL10/genética , Quimiocina CXCL11/genética , Quimiocina CXCL9/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Regulação para Cima , Enzima de Conversão de Angiotensina 2/genética , Animais , Linhagem Celular , Quimiocina CXCL10/imunologia , Quimiocina CXCL11/imunologia , Quimiocina CXCL9/imunologia , Síndrome da Liberação de Citocina/genética , Síndrome da Liberação de Citocina/imunologia , Células Epiteliais/imunologia , Células Epiteliais/virologia , Feminino , Humanos , Inflamação , Pulmão/citologia , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia
6.
Viruses ; 13(6)2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-34070524

RESUMO

SARS-CoV-2 emerged in 2019 as a devastating viral pathogen with no available preventative or treatment to control what led to the current global pandemic. The continued spread of the virus and increasing death toll necessitate the development of effective antiviral treatments to combat this virus. To this end, we evaluated a new class of organometallic complexes as potential antivirals. Our findings demonstrate that two pentamethylcyclopentadienyl (Cp*) rhodium piano stool complexes, Cp*Rh(1,3-dicyclohexylimidazol-2-ylidene)Cl2 (complex 2) and Cp*Rh(dipivaloylmethanato)Cl (complex 4), have direct virucidal activity against SARS-CoV-2. Subsequent in vitro testing suggests that complex 4 is the more stable and effective complex and demonstrates that both 2 and 4 have low toxicity in Vero E6 and Calu-3 cells. The results presented here highlight the potential application of organometallic complexes as antivirals and support further investigation into their activity.


Assuntos
Antivirais/farmacologia , Compostos Organometálicos/farmacologia , SARS-CoV-2/efeitos dos fármacos , Animais , Antivirais/química , COVID-19/virologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Compostos Organometálicos/química , SARS-CoV-2/fisiologia , Células Vero , Replicação Viral/efeitos dos fármacos
7.
Nat Commun ; 12(1): 2290, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33863888

RESUMO

Arthropod-borne viruses pose a major threat to global public health. Thus, innovative strategies for their control and prevention are urgently needed. Here, we exploit the natural capacity of viruses to generate defective viral genomes (DVGs) to their detriment. While DVGs have been described for most viruses, identifying which, if any, can be used as therapeutic agents remains a challenge. We present a combined experimental evolution and computational approach to triage DVG sequence space and pinpoint the fittest deletions, using Zika virus as an arbovirus model. This approach identifies fit DVGs that optimally interfere with wild-type virus infection. We show that the most fit DVGs conserve the open reading frame to maintain the translation of the remaining non-structural proteins, a characteristic that is fundamental across the flavivirus genus. Finally, we demonstrate that the high fitness DVG is antiviral in vivo both in the mammalian host and the mosquito vector, reducing transmission in the latter by up to 90%. Our approach establishes the method to interrogate the DVG fitness landscape, and enables the systematic identification of DVGs that show promise as human therapeutics and vector control strategies to mitigate arbovirus transmission and disease.


Assuntos
Antivirais/administração & dosagem , Vírus Defeituosos/genética , Mosquitos Vetores/efeitos dos fármacos , Infecção por Zika virus/tratamento farmacológico , Zika virus/genética , Aedes/efeitos dos fármacos , Aedes/virologia , Animais , Chlorocebus aethiops , Biologia Computacional , Evolução Molecular Direcionada , Modelos Animais de Doenças , Feminino , Aptidão Genética , Genoma Viral/genética , Células HEK293 , Humanos , Camundongos , Controle de Mosquitos/métodos , Mosquitos Vetores/virologia , Fases de Leitura Aberta/genética , RNA Viral/genética , Células Vero , Infecção por Zika virus/transmissão , Infecção por Zika virus/virologia
8.
Front Med (Lausanne) ; 8: 626028, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33585527

RESUMO

A single domain antibody (clone CC3) previously found to neutralize a vaccine strain of the chikungunya virus (PRNT50 = 2. 5 ng/mL) was found to be broadly neutralizing. Clone CC3 is not only able to neutralize a wild-type (WT) strain of chikungunya virus (CHIKV), but also neutralizes WT strains of Mayaro virus (MAYV) and Ross River virus (RRV); both arthralgic, Old World alphaviruses. Interestingly, CC3 also demonstrated a degree of neutralizing activity against the New World alphavirus, Venezuelan equine encephalitis virus (VEEV); albeit both the vaccine strain, TC-83, and the parental, WT Trinidad donkey strain had PRNT50 values ~1,000-fold higher than that of CHIKV. However, no neutralization activity was observed with Western equine encephalitis virus (WEEV). Ten CC3 variants designed to possess a range of isoelectric points, both higher and lower, were constructed. This approach successfully identified several lower pI mutants which possessed improved thermal stabilities by as much as 10°C over the original CC3 (Tm = 62°C), and excellent refolding abilities while maintaining their capacity to bind and neutralize CHIKV.

9.
J Virol ; 2021 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-33536175

RESUMO

Zika virus (ZIKV) has the unusual capacity to circumvent natural alternating mosquito-human transmission and be directly transmitted human-to-human via sexual and vertical routes. The impact of direct transmission on ZIKV evolution and adaptation to vertebrate hosts is unknown. Here we show that molecularly barcoded ZIKV rapidly adapted to a mammalian host during direct transmission chains in mice, coincident with the emergence of an amino acid substitution previously shown to enhance virulence. In contrast, little to no adaptation of ZIKV to mice was observed following chains of direct transmission in mosquitoes or alternating host transmission. Detailed genetic analyses revealed that ZIKV evolution in mice was generally more convergent and subjected to more relaxed purifying selection than in mosquitoes or alternate passages. These findings suggest that prevention of direct human transmission chains may be paramount to resist gains in ZIKV virulence.Importance We used experimental evolution to model chains of direct and indirect Zika virus (ZIKV) transmission by serially passaging a synthetic swarm of molecularly barcoded ZIKV within and between mosquitoes and mice. We observed that direct mouse transmission chains facilitated a rapid increase in ZIKV replication and enhanced virulence in mice. These findings demonstrate that ZIKV is capable of rapid adaptation to a vertebrate host and indicate that direct human-to-human transmission could pose a greater threat to public health than currently realized.

10.
PLoS Pathog ; 17(2): e1009110, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33556143

RESUMO

Defective viral genomes (DVGs) are truncated and/or rearranged viral genomes produced during virus replication. Described in many RNA virus families, some of them have interfering activity on their parental virus and/or strong immunostimulatory potential, and are being considered in antiviral approaches. Chikungunya virus (CHIKV) is an alphavirus transmitted by Aedes spp. that infected millions of humans in the last 15 years. Here, we describe the DVGs arising during CHIKV infection in vitro in mammalian and mosquito cells, and in vivo in experimentally infected Aedes aegypti mosquitoes. We combined experimental and computational approaches to select DVG candidates most likely to have inhibitory activity and showed that, indeed, they strongly interfere with CHIKV replication both in mammalian and mosquito cells. We further demonstrated that some DVGs present broad-spectrum activity, inhibiting several CHIKV strains and other alphaviruses. Finally, we showed that pre-treating Aedes aegypti with DVGs prevented viral dissemination in vivo.


Assuntos
Aedes/virologia , Antivirais/farmacologia , Febre de Chikungunya/transmissão , Vírus Chikungunya/genética , Vírus Defeituosos/genética , Genoma Viral , Replicação Viral , Animais , Febre de Chikungunya/imunologia , Febre de Chikungunya/virologia , Vírus Chikungunya/crescimento & desenvolvimento , Vírus Chikungunya/isolamento & purificação , Humanos , Mosquitos Vetores/virologia
11.
Virology ; 555: 64-70, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33454558

RESUMO

Usutu virus (USUV; Flavivirus) has caused massive die-offs in birds across Europe since the 1950s. Although rare, severe neurologic disease in humans has been reported. USUV is genetically related to West Nile virus (WNV) and shares an ecological niche, suggesting it could spread from Europe to the Americas. USUV's risk of transmission within the United States is currently unknown. To this end, we exposed field-caught Aedes japonicus, Culex pipiens pipiens, and Culex restuans-competent vectors for WNV-to a recent European isolate of USUV. While infection rates for each species varied from 7%-21%, no dissemination or transmission was observed. These results differed from a 2018 report by Cook and colleagues, who found high dissemination rates and evidence of transmission potential using a different USUV strain, U.S. mosquito populations, temperature, and extrinsic incubation period. Future studies should evaluate the impact of these experimental conditions on USUV transmission by North American mosquitoes.

12.
Virology ; 554: 28-36, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33352463

RESUMO

Usutu virus (USUV; genus Flavivirus; family Flaviviridae) is a mosquito-borne, positive-sense RNA virus that is currently causing significant die-offs in numerous bird species throughout Europe and has caused infections in humans. Currently, there are no molecular clones for USUV, hence, hindering studies on the pathogenesis and transmission of USUV. Here, we demonstrate the development and characterization of infectious clones for two modern strains of USUV isolated from Europe and Africa. We show that the infectious clone-derived viruses replicated similarly to the parental strains in mammalian and insect cells. Additionally, we observed similar levels of replication and disease in two mouse models. These clones will aid the study of USUV infection, transmission, diagnostics, and vaccines.

13.
J Virol ; 95(6)2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33328311

RESUMO

Zika virus (ZIKV; Flaviviridae, Flavivirus) is an arthropod-borne infection that can result in severe outcomes, particularly in fetuses infected in utero It has been assumed that infection by ZIKV, as well as other viruses, is largely initiated by individual virus particles binding to and entering a cell. However, recent studies have demonstrated that multiple virus particles are frequently delivered to a cell simultaneously and that this collective particle delivery enhances infection. ZIKV is maintained in nature between Aedes aegypti mosquitos and vertebrate hosts, including humans. Human infection is initiated through the injection of a relatively small initial inoculum comprised of a genetically complex virus population. Since most mutations decrease virus fitness, collective particle transmission could benefit ZIKV and other arthropod-borne diseases by facilitating the maintenance of genetic complexity and adaptability during infection or through other mechanisms. Therefore, we utilized a barcoded ZIKV to quantify the number of virus genomes that initiate a plaque. We found that individual plaques contain a mean of 10 infecting viral genomes (range, 1 to 212). Few plaques contained more than two dominant genomes. To determine whether multigenome infectious units consist of collectively transmitting virions, infectious units of ZIKV were then separated mechanically by centrifugation, and heavier fractions were found to contain more genomes per plaque-forming unit, with larger diameters. Finally, larger/heavier infectious units reformed after removal. These data suggest that ZIKV populations consist of a variety of infectious unit sizes, likely mostly made up of aggregates, and only rarely begin with a single virus genome.IMPORTANCE The arthropod-borne Zika virus (ZIKV) infects humans and can cause severe neurological sequelae, particularly in fetuses infected in utero How this virus has been able to spread across vast geological ranges and evolve in new host populations is not yet understood. This research demonstrates a novel mechanism of ZIKV transmission through multigenome aggregates, providing insight into ZIKV evolution, immunologic evasion, and better future therapeutic design. This study shows that ZIKV plaques result from collections of genomes rather than individual genomes, increasing the potential for interactions between ZIKV genotypes.


Assuntos
Genoma Viral/genética , Polimorfismo Genético , Infecção por Zika virus/virologia , Zika virus/genética , Aedes/virologia , Animais , Linhagem Celular , Variações do Número de Cópias de DNA , Tamanho do Genoma , Genótipo , Humanos , Mosquitos Vetores/virologia , Temperatura , Vírion/metabolismo , Replicação Viral , Zika virus/crescimento & desenvolvimento , Infecção por Zika virus/transmissão
14.
PLoS One ; 15(11): e0241592, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33180795

RESUMO

Superinfection exclusion (SIE) is a process by which a virally infected cell is protected from subsequent infection by the same or a closely related virus. By preventing cell coinfection, SIE favors preservation of genome integrity of a viral strain and limits its recombination potential with other viral genomes, thereby impacting viral evolution. Although described in virtually all viral families, the precise step(s) impacted by SIE during the viral life cycle have not been systematically explored. Here, we describe for the first time SIE triggered by chikungunya virus (CHIKV), an alphavirus of public health importance. Using single-cell technologies, we demonstrate that CHIKV excludes subsequent infection with: CHIKV; Sindbis virus, a related alphavirus; and influenza A, an unrelated RNA virus. We further demonstrate that SIE does not depend on the action of type I interferon, nor does it rely on host cell transcription. Moreover, exclusion is not mediated by the action of a single CHIKV protein; in particular, we observed no role for non-structural protein 2 (nsP2), making CHIKV unique among characterized alphaviruses. By stepping through the viral life cycle, we show that CHIKV exclusion occurs at the level of replication, but does not directly influence virus binding, nor viral structural protein translation. In sum, we characterized co-infection during CHIKV replication, which likely influences the rate of viral diversification and evolution.


Assuntos
Febre de Chikungunya/virologia , Vírus Chikungunya/fisiologia , Superinfecção/virologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Animais , Células Cultivadas , Vírus Chikungunya/genética , Vírus Chikungunya/patogenicidade , Chlorocebus aethiops , Genoma Viral , Vírus da Influenza A/patogenicidade , Camundongos , Vírus Sindbis/patogenicidade , Células Vero , Proteínas não Estruturais Virais/genética
15.
Virology ; 551: 58-63, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33032077

RESUMO

Alphaviruses (genus Alphavirus; family Togaviridae) are a medically relevant family of viruses that include chikungunya virus and Mayaro virus. Infectious cDNA clones of these viruses are necessary molecular tools to understand viral biology. Traditionally, rescuing virus from an infectious cDNA clone requires propagating plasmids in bacteria, which can result in mutations in the viral genome due to bacterial toxicity or recombination and requires specialized equipment and knowledge to propagate the bacteria. Here, we present an alternative- rolling circle amplification (RCA), an in vitro technology. We demonstrate that the viral yield of transfected RCA product is comparable to midiprepped plasmid, albeit with a slight delay in kinetics. RCA, however, is cheaper and less time-consuming. Further, sequential RCA did not introduce mutations into the viral genome, subverting the need for glycerol stocks and retransformation. These results indicate that RCA is a viable alternative to traditional plasmid-based approaches to viral rescue.


Assuntos
Alphavirus , Técnicas de Amplificação de Ácido Nucleico/métodos , Alphavirus/genética , Alphavirus/isolamento & purificação , Animais , Chlorocebus aethiops , Cricetinae , DNA Complementar , Fibroblastos , Genoma Viral , Células HEK293 , Humanos , Células Vero
16.
BMC Biol ; 18(1): 106, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32854687

RESUMO

BACKGROUND: Dengue virus (DENV) is estimated to infect 390 million people annually. However, few host factors that alter disease severity are known. Malnutrition, defined as both over- and undernutrition, is a growing problem worldwide and has long been linked to dengue disease severity by epidemiological and anecdotal observations. Accordingly, we sought to establish a mouse model to assess the impact of nutritional status on DENV disease severity. RESULTS: Using transiently immunocompromised mice, we established a model of mild dengue disease with measurable viremia. We then applied it to study the effects of healthy weight, obese, and low-protein diets representing normal, over-, and undernutrition, respectively. Upon infection with DENV serotype 2, obese mice experienced more severe morbidity in the form of weight loss and thrombocytopenia compared to healthy weight groups. Additionally, obesity altered cytokine expression following DENV infection. Although low protein-fed mice did not lose significant weight after DENV2 infection, they also experienced a reduction in platelets as well as increased spleen pathology and viral titers. CONCLUSIONS: Our results indicate that obese or undernourished mice incur greater disease severity after DENV infection. These studies establish a role for nutritional status in DENV disease severity.


Assuntos
Peso Corporal/fisiologia , Dengue/virologia , Dieta Hiperlipídica/efeitos adversos , Dieta com Restrição de Proteínas/efeitos adversos , Estado Nutricional , Animais , Dengue/fisiopatologia , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Viremia/fisiopatologia
17.
Antiviral Res ; 172: 104642, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31678479

RESUMO

Chikungunya virus (CHIKV) is a rapidly emerging mosquito-borne RNA virus that causes epidemics of debilitating disease in tropical and sub-tropical regions with autochtonous transmission in regions with temperate climate. Currently, there is no licensed vaccine or specific antiviral drug available against CHIKV infection. In this study, we examine the role, in the CHIKV viral cycle, of fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD1), two key lipogenic enzymes required for fatty acid production and early desaturation. We show that both enzymes and their upstream regulator PI3K are required for optimal CHIKV infection. We demonstrate that pharmacologic manipulation of FASN or SCD1 enzymatic activity by non-toxic concentrations of cerulenin or CAY10566 decreases CHIKV genome replication. Interestingly, a similar inhibitory effect was also obtained with Orlistat, an FDA-approved anti-obesity drug that targets FASN activity. These drugs were also effective against Mayaro virus (MAYV), an under-studied arthritogenic Old world Alphavirus endemic in South American countries with potential risk of emergence, urbanization and dispersion to other regions. Altogether, our results identify FASN and SCD1 as conserved druggable cofactors of Alphavirus genome replication and support the broad-spectrum activity of drugs targeting the host fatty acids metabolism.


Assuntos
Alphavirus/efeitos dos fármacos , Ácido Graxo Sintases/metabolismo , Estearoil-CoA Dessaturase/metabolismo , Replicação Viral/efeitos dos fármacos , Alphavirus/genética , Infecções por Alphavirus/tratamento farmacológico , Animais , Antivirais/farmacologia , Linhagem Celular , Cerulenina/farmacologia , Febre de Chikungunya/tratamento farmacológico , Vírus Chikungunya/efeitos dos fármacos , Vírus Chikungunya/genética , Chlorocebus aethiops , Ácido Graxo Sintases/efeitos dos fármacos , Genoma Viral , Células HEK293 , Humanos , Orlistate/farmacologia , Estearoil-CoA Dessaturase/efeitos dos fármacos , Células Vero
18.
PLoS Pathog ; 15(11): e1008089, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31710653

RESUMO

Malnourishment, specifically overweight/obesity and undernourishment, affects more than 2.5 billion people worldwide, with the number affected ever-increasing. Concurrently, emerging viral diseases, particularly those that are mosquito-borne, have spread dramatically in the past several decades, culminating in outbreaks of several viruses worldwide. Both forms of malnourishment are known to lead to an aberrant immune response, which can worsen disease outcomes and reduce vaccination efficacy for viral pathogens such as influenza and measles. Given the increasing rates of malnutrition and spread of arthropod-borne viruses (arboviruses), there is an urgent need to understand the role of host nutrition on the infection, virulence, and transmission of these viruses. To address this gap in knowledge, we infected lean, obese, and undernourished mice with arthritogenic arboviruses from the genus Alphavirus and assessed morbidity, virus replication, transmission, and evolution. Obesity and undernourishment did not consistently influence virus replication in the blood of infected animals except for reductions in virus in obese mice late in infection. However, morbidity was increased in obese mice under all conditions. Using Mayaro virus (MAYV) as a model arthritogenic alphavirus, we determined that both obese and undernourished mice transmit virus less efficiently to mosquitoes than control (lean) mice. In addition, viral genetic diversity and replicative fitness were reduced in virus isolated from obese compared to lean controls. Taken together, nutrition appears to alter the course of alphavirus infection and should be considered as a critical environmental factor during outbreaks.


Assuntos
Aedes/virologia , Infecções por Alphavirus/etiologia , Infecções por Alphavirus/transmissão , Alphavirus/patogenicidade , Evolução Biológica , Estado Nutricional , Obesidade/virologia , Infecções por Alphavirus/patologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Mosquitos Vetores/virologia , Obesidade/patologia , Virulência , Replicação Viral
19.
Heliyon ; 5(10): e02660, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31692696

RESUMO

Mosquitoes are the most important arthropods from the point of view of public health, due to the fact that they can transmit a large number of pathogens which can cause diseases to humans and animals. Aedes aegypti (L.) is one of the most important vector species in the world, since it can transmit numerous pathogens such as dengue, Zika, and chikungunya. Therefore, studies involving the molecular aspects of this and other mosquitoes species are currently increasing. In this report, we describe the comparison between two DNA extraction techniques, Chelex and cetyltrimethylammonium bromide (CTAB), for carrying out DNA extraction in larvae, pupae and adult female of Ae. aegypti. The Chelex technique was superior in the amount and purity of DNA as compared to the CTAB technique in the three life stages we tested.

20.
J Virol ; 93(18)2019 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-31270226

RESUMO

Chikungunya virus (CHIKV) is a reemerged arbovirus, a member of the Togaviridae family. It circulates through mosquito vectors mainly of the Aedes family and a mammalian host. CHIKV causes chikungunya fever, a mild to severe disease characterized by arthralgia, with some fatal outcomes described. In the past years, several outbreaks mainly caused by enhanced adaptation of the virus to the vector and ineffective control of the contacts between infected mosquito populations and the human host have been reported. Vaccines represent the best solution for the control of insect-borne viruses, including CHIKV, but are often unavailable. We designed live attenuated CHIKVs by applying a rational genomic design based on multiple replacements of synonymous codons. In doing so, the virus mutational robustness (capacity to maintain phenotype despite introduction of mutations to genotype) is decreased, driving the viral population toward deleterious evolutionary trajectories. When the candidate viruses were tested in the insect and mammalian hosts, we observed overall strong attenuation in both and greatly diminished signs of disease. Moreover, we found that the vaccine candidates elicited protective immunity related to the production of neutralizing antibodies after a single dose. During an experimental transmission cycle between mosquitoes and naive mice, vaccine candidates could be transmitted by mosquito bite, leading to asymptomatic infection in mice with compromised dissemination. Using deep-sequencing technology, we observed an increase in detrimental (stop) codons, which confirmed the effectiveness of this genomic design. Because the approach involves hundreds of synonymous modifications to the genome, the reversion risk is significantly reduced, rendering the viruses promising vaccine candidates.IMPORTANCE Chikungunya fever is a debilitating disease that causes severe pain to the joints, which can compromise the patient's lifestyle for several months and even in some grave cases lead to death. The etiological agent is chikungunya virus, an alphavirus transmitted by mosquito bite. Currently, there are no approved vaccines or treatments against the disease. In our research, we developed novel live attenuated vaccine candidates against chikungunya virus by applying an innovative genomic design. When tested in the insect and mammalian host, the vaccine candidates did not cause disease, elicited strong protection against further infection, and had low risk of reversion to pathogenic phenotypes.


Assuntos
Vírus Chikungunya/genética , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Imunidade Adaptativa/imunologia , Aedes/virologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linhagem Celular , Febre de Chikungunya/genética , Febre de Chikungunya/virologia , Vírus Chikungunya/metabolismo , Chlorocebus aethiops , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mosquitos Vetores/virologia , Mutação , Células Vero , Vacinas Virais/genética , Vacinas Virais/imunologia
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