Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 9 de 9
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
J Virol ; 94(24)2020 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-32999019

RESUMO

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus associated with debilitating arthralgia in humans. RNA secondary structure in the viral genome plays an important role in the lifecycle of alphaviruses; however, the specific role of RNA structure in regulating CHIKV replication is poorly understood. Our previous studies found little conservation in RNA secondary structure between alphaviruses, and this structural divergence creates unique functional structures in specific alphavirus genomes. Therefore, to understand the impact of RNA structure on CHIKV biology, we used SHAPE-MaP to inform the modeling of RNA secondary structure throughout the genome of a CHIKV isolate from the 2013 Caribbean outbreak. We then analyzed regions of the genome with high levels of structural specificity to identify potentially functional RNA secondary structures and identified 23 regions within the CHIKV genome with higher than average structural stability, including four previously identified, functionally important CHIKV RNA structures. We also analyzed the RNA flexibility and secondary structures of multiple 3'UTR variants of CHIKV that are known to affect virus replication in mosquito cells. This analysis found several novel RNA structures within these 3'UTR variants. A duplication in the 3'UTR that enhances viral replication in mosquito cells led to an overall increase in the amount of unstructured RNA in the 3'UTR. This analysis demonstrates that the CHIKV genome contains a number of unique, specific RNA secondary structures and provides a strategy for testing these secondary structures for functional importance in CHIKV replication and pathogenesis.IMPORTANCE Chikungunya virus (CHIKV) is a mosquito-borne RNA virus that causes febrile illness and debilitating arthralgia in humans. CHIKV causes explosive outbreaks but there are no approved therapies to treat or prevent CHIKV infection. The CHIKV genome contains functional RNA secondary structures that are essential for proper virus replication. Since RNA secondary structures have only been defined for a small portion of the CHIKV genome, we used a chemical probing method to define the RNA secondary structures of CHIKV genomic RNA. We identified 23 highly specific structured regions of the genome, and confirmed the functional importance of one structure using mutagenesis. Furthermore, we defined the RNA secondary structure of three CHIKV 3'UTR variants that differ in their ability to replicate in mosquito cells. Our study highlights the complexity of the CHIKV genome and describes new systems for designing compensatory mutations to test the functional relevance of viral RNA secondary structures.


Assuntos
Regiões 3' não Traduzidas/genética , Vírus Chikungunya/genética , RNA Viral/química , RNA Viral/genética , Animais , Linhagem Celular , Febre de Chikungunya/virologia , Chlorocebus aethiops , Culicidae , Efeito Citopatogênico Viral , Genoma Viral , Mutação , Conformação de Ácido Nucleico , Análise de Sequência , Células Vero , Replicação Viral/genética
2.
Nucleic Acids Res ; 46(7): 3657-3670, 2018 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-29361131

RESUMO

Alphaviruses are mosquito-borne pathogens that cause human diseases ranging from debilitating arthritis to lethal encephalitis. Studies with Sindbis virus (SINV), which causes fever, rash, and arthralgia in humans, and Venezuelan equine encephalitis virus (VEEV), which causes encephalitis, have identified RNA structural elements that play key roles in replication and pathogenesis. However, a complete genomic structural profile has not been established for these viruses. We used the structural probing technique SHAPE-MaP to identify structured elements within the SINV and VEEV genomes. Our SHAPE-directed structural models recapitulate known RNA structures, while also identifying novel structural elements, including a new functional element in the nsP1 region of SINV whose disruption causes a defect in infectivity. Although RNA structural elements are important for multiple aspects of alphavirus biology, we found the majority of RNA structures were not conserved between SINV and VEEV. Our data suggest that alphavirus RNA genomes are highly divergent structurally despite similar genomic architecture and sequence conservation; still, RNA structural elements are critical to the viral life cycle. These findings reframe traditional assumptions about RNA structure and evolution: rather than structures being conserved, alphaviruses frequently evolve new structures that may shape interactions with host immune systems or co-evolve with viral proteins.


Assuntos
Vírus da Encefalite Equina Venezuelana/genética , RNA/genética , Sindbis virus/genética , Replicação Viral/genética , Alphavirus/química , Alphavirus/genética , Alphavirus/patogenicidade , Animais , Encefalite/genética , Encefalite/virologia , Vírus da Encefalite Equina Venezuelana/química , Vírus da Encefalite Equina Venezuelana/patogenicidade , Genoma Viral/genética , Cavalos/virologia , Humanos , Conformação de Ácido Nucleico , RNA/química , Sindbis virus/química , Sindbis virus/patogenicidade
3.
mBio ; 8(6)2017 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-29138302

RESUMO

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus responsible for several significant outbreaks of debilitating acute and chronic arthritis and arthralgia over the past decade. These include a recent outbreak in the Caribbean islands and the Americas that caused more than 1 million cases of viral arthralgia. Despite the major impact of CHIKV on global health, viral determinants that promote CHIKV-induced disease are incompletely understood. Most CHIKV strains contain a conserved opal stop codon at the end of the viral nsP3 gene. However, CHIKV strains that encode an arginine codon in place of the opal stop codon have been described, and deep-sequencing analysis of a CHIKV isolate from the Caribbean identified both arginine and opal variants within this strain. Therefore, we hypothesized that the introduction of the arginine mutation in place of the opal termination codon may influence CHIKV virulence. We tested this by introducing the arginine mutation into a well-characterized infectious clone of a CHIKV strain from Sri Lanka and designated this virus Opal524R. This mutation did not impair viral replication kinetics in vitro or in vivo Despite this, the Opal524R virus induced significantly less swelling, inflammation, and damage within the feet and ankles of infected mice. Further, we observed delayed induction of proinflammatory cytokines and chemokines, as well as reduced CD4+ T cell and NK cell recruitment compared to those in the parental strain. Therefore, the opal termination codon plays an important role in CHIKV pathogenesis, independently of effects on viral replication.IMPORTANCE Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes significant outbreaks of viral arthralgia. Studies with CHIKV and other alphaviruses demonstrated that the opal termination codon within nsP3 is highly conserved. However, some strains of CHIKV and other alphaviruses contain mutations in the opal termination codon. These mutations alter the virulence of related alphaviruses in mammalian and mosquito hosts. Here, we report that a clinical isolate of a CHIKV strain from the recent outbreak in the Caribbean islands contains a mixture of viruses encoding either the opal termination codon or an arginine mutation. Mutating the opal stop codon to an arginine residue attenuates CHIKV-induced disease in a mouse model. Compared to infection with the opal-containing parental virus, infection with the arginine mutant causes limited swelling and inflammation, as well as dampened recruitment of immune mediators of pathology, including CD4+ T cells and NK cells. We propose that the opal termination codon plays an essential role in the induction of severe CHIKV disease.


Assuntos
Artrite/patologia , Febre de Chikungunya/patologia , Vírus Chikungunya/patogenicidade , Códon de Terminação , Mutação , Proteínas não Estruturais Virais/genética , Fatores de Virulência/genética , Animais , Arginina/genética , Artrite/virologia , Febre de Chikungunya/virologia , Vírus Chikungunya/fisiologia , Modelos Animais de Doenças , Camundongos , Replicação Viral
4.
J Virol ; 90(1): 433-43, 2016 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-26491151

RESUMO

UNLABELLED: Chikungunya virus (CHIKV) is an alphavirus responsible for causing epidemic outbreaks of polyarthralgia in humans. Because CHIKV is initially introduced via the skin, where γδ T cells are prevalent, we evaluated the response of these cells to CHIKV infection. CHIKV infection led to a significant increase in γδ T cells in the infected foot and draining lymph node that was associated with the production of proinflammatory cytokines and chemokines in C57BL/6J mice. γδ T cell(-/-) mice demonstrated exacerbated CHIKV disease characterized by less weight gain and greater foot swelling than occurred in wild-type mice, as well as a transient increase in monocytes and altered cytokine/chemokine expression in the foot. Histologically, γδ T cell(-/-) mice had increased inflammation-mediated oxidative damage in the ipsilateral foot and ankle joint compared to wild-type mice which was independent of differences in CHIKV replication. These results suggest that γδ T cells play a protective role in limiting the CHIKV-induced inflammatory response and subsequent tissue and joint damage. IMPORTANCE: Recent epidemics, including the 2004 to 2007 outbreak and the spread of CHIKV to naive populations in the Caribbean and Central and South America with resultant cases imported into the United States, have highlighted the capacity of CHIKV to cause explosive epidemics where the virus can spread to millions of people and rapidly move into new areas. These studies identified γδ T cells as important to both recruitment of key inflammatory cell populations and dampening the tissue injury due to oxidative stress. Given the importance of these cells in the early response to CHIKV, this information may inform the development of CHIKV vaccines and therapeutics.


Assuntos
Febre de Chikungunya/imunologia , Vírus Chikungunya/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/análise , Linfócitos T/imunologia , Animais , Peso Corporal , Modelos Animais de Doenças , Membro Posterior/patologia , Histocitoquímica , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Antígenos de Linfócitos T gama-delta/genética , Linfócitos T/química
5.
Curr Trop Med Rep ; 2(1): 13-21, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26366337

RESUMO

Chikungunya virus (CHIKV) is an arbovirus responsible for causing epidemic outbreaks of human disease characterized by painful and often debilitating arthralgia. Recently CHIKV has moved into the Caribbean and the Americas resulting in massive outbreaks in naïve human populations. Given the importance of CHIKV as an emerging disease, a significant amount of effort has gone into interpreting the virus-host interactions that contribute to protection or virus-induced pathology following CHIKV infection, with the long term goal of using this information to develop new therapies or safe and effective anti-CHIKV vaccines. This work has made it clear that numerous distinct host responses are involved in the response to CHIKV infection, where some aspects of the host innate and adaptive immune response protect from or limit virus-induced disease, while other pathways actually exacerbate the virus-induced disease process. This review will discuss mechanisms that have been identified as playing a role in the host response to CHIKV infection and illustrate the importance of carefully evaluating these responses to determine whether they play a protective or pathologic role during CHIKV infection.

6.
J Virol ; 88(7): 3719-32, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24429363

RESUMO

UNLABELLED: Ross River virus (RRV) is one of a group of mosquito-transmitted alphaviruses that cause debilitating, and often chronic, musculoskeletal disease in humans. Previously, we reported that replacement of the nonstructural protein 1 (nsP1) gene of the mouse-virulent RRV strain T48 with that from the mouse-avirulent strain DC5692 generated a virus that was attenuated in a mouse model of disease. Here we find that the six nsP1 nonsynonymous nucleotide differences between strains T48 and DC5692 are determinants of RRV virulence, and we identify two nonsynonymous nucleotide changes as sufficient for the attenuated phenotype. RRV T48 carrying the six nonsynonymous DC5692 nucleotide differences (RRV-T48-nsP1(6M)) was attenuated in both wild-type and Rag1(-/-) mice. Despite the attenuated phenotype, RRV T48 and RRV-T48-nsP1(6M) loads in tissues of wild-type and Rag1(-/-) mice were indistinguishable from 1 to 3 days postinoculation. RRV-T48-nsP1(6M) loads in skeletal muscle tissue, but not in other tissues, decreased dramatically by 5 days postinoculation in both wild-type and Rag1(-/-) mice, suggesting that the RRV-T48-nsP1(6M) mutant is more sensitive to innate antiviral effectors than RRV T48 in a tissue-specific manner. In vitro, we found that the attenuating mutations in nsP1 conferred enhanced sensitivity to type I interferon. In agreement with these findings, RRV T48 and RRV-T48-nsP1(6M) loads were similar in mice deficient in the type I interferon receptor. Our findings suggest that the type I IFN response controls RRV infection in a tissue-specific manner and that specific amino acid changes in nsP1 are determinants of RRV virulence by regulating the sensitivity of RRV to interferon. IMPORTANCE: Arthritogenic alphaviruses, including Ross River virus (RRV), infect humans and cause debilitating pain and inflammation of the musculoskeletal system. In this study, we identified coding changes in the RRV nsP1 gene that control the virulence of RRV and its sensitivity to the antiviral type I interferon response, a major component of antiviral defense in mammals. Furthermore, our studies revealed that the effects of these attenuating mutations are tissue specific. These findings suggest that these mutations in nsP1 influence the sensitivity of RRV to type I interferon only in specific host tissues. The new knowledge gained from these studies contributes to our understanding of host responses that control alphavirus infection and viral determinants that counteract these responses.


Assuntos
Infecções por Alphavirus/virologia , Interações Hospedeiro-Patógeno , Interferon Tipo I/imunologia , Mutação de Sentido Incorreto , Ross River virus/patogenicidade , Proteínas não Estruturais Virais/metabolismo , Fatores de Virulência/metabolismo , Infecções por Alphavirus/patologia , Estruturas Animais/virologia , Animais , Análise Mutacional de DNA , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ross River virus/imunologia , Carga Viral , Proteínas não Estruturais Virais/genética , Virulência , Fatores de Virulência/genética
7.
Methods Mol Biol ; 1031: 97-100, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23824892

RESUMO

Footpad injection is an important route of inoculation in mouse models of disease and immunology. Although commonly used to deliver antigens as a means of eliciting an efficacious immunological response, herein, we describe a protocol for inoculating mice via footpad injection using a hands-free method to deliver infectious material. These procedures allow for efficient delivery of infectious agents in a manner that is safe for both the researcher and animal.


Assuntos
Antígenos/administração & dosagem , Injeções/métodos , Vacinação/métodos , Animais , Modelos Animais de Doenças , Camundongos
8.
J Virol ; 87(10): 5697-706, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23487448

RESUMO

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus responsible for recent epidemic outbreaks of debilitating disease in humans. Alphaviruses are known to interact with members of the C-type lectin receptor family of pattern recognition proteins, and given that the dendritic cell immunoreceptor (DCIR) is known to act as a negative regulator of the host inflammatory response and has previously been associated with rheumatoid arthritis, we evaluated DCIR's role in response to CHIKV infection. Although we observed an increase in the proportion of dendritic cells at the site of CHIKV infection at 24 to 36 h postinfection, these cells showed decreased cell surface DCIR, suggestive of DCIR triggering and internalization. In vitro, bone marrow-derived dendritic cells from DCIR-deficient (DCIR(-/-)) mice exhibited altered cytokine expression following exposure to CHIKV. DCIR(-/-) mice exhibited more severe disease signs than wild-type C57BL6/J mice following CHIKV infection, including a more rapid and more severe onset of virus-induced edema and enhanced weight loss. Histological examination revealed that DCIR-deficient animals exhibited increased inflammation and damage in both the fascia of the inoculated foot and the ankle joint, and DCIR deficiency skewed the CHIKV-induced cytokine response at the site of infection at multiple times postinfection. Early differences in virus-induced disease between C57BL6/J and DCIR(-/-) mice were independent of viral replication, while extended viral replication correlated with enhanced foot swelling and tissue inflammation and damage in DCIR(-/-) compared to C57BL6/J mice at 6 to 7 days postinfection. These results suggest that DCIR plays a protective role in limiting the CHIKV-induced inflammatory response and subsequent tissue and joint damage.


Assuntos
Vírus Chikungunya/imunologia , Vírus Chikungunya/patogenicidade , Células Dendríticas/imunologia , Células Dendríticas/virologia , Lectinas Tipo C/metabolismo , Infecções por Alphavirus/imunologia , Infecções por Alphavirus/patologia , Animais , Articulação do Tornozelo/patologia , Febre de Chikungunya , Modelos Animais de Doenças , Pé/patologia , Histocitoquímica , Lectinas Tipo C/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...