RESUMO
Zika virus (ZIKV) has recently emerged as a pandemic associated with severe neuropathology in newborns and adults. There are no ZIKV-specific treatments or preventatives. Therefore, the development of a safe and effective vaccine is a high priority. Messenger RNA (mRNA) has emerged as a versatile and highly effective platform to deliver vaccine antigens and therapeutic proteins. Here we demonstrate that a single low-dose intradermal immunization with lipid-nanoparticle-encapsulated nucleoside-modified mRNA (mRNA-LNP) encoding the pre-membrane and envelope glycoproteins of a strain from the ZIKV outbreak in 2013 elicited potent and durable neutralizing antibody responses in mice and non-human primates. Immunization with 30 µg of nucleoside-modified ZIKV mRNA-LNP protected mice against ZIKV challenges at 2 weeks or 5 months after vaccination, and a single dose of 50 µg was sufficient to protect non-human primates against a challenge at 5 weeks after vaccination. These data demonstrate that nucleoside-modified mRNA-LNP elicits rapid and durable protective immunity and therefore represents a new and promising vaccine candidate for the global fight against ZIKV.
Assuntos
RNA Mensageiro/administração & dosagem , RNA Mensageiro/química , Vacinas Virais/imunologia , Infecção por Zika virus/prevenção & controle , Zika virus/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/genética , Antígenos Virais/imunologia , Feminino , Glicoproteínas/genética , Glicoproteínas/imunologia , Injeções Intradérmicas , Macaca mulatta/imunologia , Macaca mulatta/virologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Nanopartículas/administração & dosagem , Nanopartículas/química , Estabilidade de RNA , RNA Mensageiro/genética , RNA Viral/administração & dosagem , RNA Viral/química , RNA Viral/genética , Fatores de Tempo , Vacinação , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Vacinas Virais/administração & dosagem , Zika virus/química , Zika virus/genética , Infecção por Zika virus/imunologiaRESUMO
The discovery of an odd virus from hematophagous arthropods 40 years ago by Stollar and Thomas described cell fusing agent virus in cells derived from Aedes aegypti mosquitoes. Then came the report of Kamiti River virus from Ae. macintoshi in 1999, followed by worldwide reports of the discovery of other viruses of mosquitoes, ticks, and midges that replicate only in arthropods and not in vertebrates or in vertebrate cells. These viruses (now totaling at least 64 published) have genomes analogous to viruses in various families that include arboviruses and nonarboviruses. It is likely that some of these viruses have been insufficiently studied and may yet be shown to infect vertebrates. However, there is no doubt that the vast majority are restricted to arthropods alone and that they represent a recently recognized clade. Their biology, modes of transmission, worldwide distribution (some have been detected in wild-caught mosquitoes in both Asia and the United States, for example), molecular characteristics of their genomes, and potential for becoming vertebrate pathogens, or at least serving as virus reservoirs, are fascinating and may provide evidence useful in understanding virus evolution. Because metagenomics studies of arthropods have shown that arthropod genomes are the sources of arthropod virus genomes, further studies may also provide insights into the evolution of arthropods. More recently, others have published excellent papers that briefly review discoveries of arthropod viruses and that characterize certain genomic peculiarities, but, to now, there have been no reviews that encompass all these facets. We therefore anticipate that this review is published at a time and in a manner that is helpful for both virologists and entomologists to make more sense and understanding of this recently recognized and obviously important virus group. This review focuses specifically on arthropod viruses in hematophagous arthropods.
Assuntos
Vetores Artrópodes/virologia , Artrópodes/virologia , Animais , Arbovírus/genética , Evolução BiológicaRESUMO
BACKGROUND: Dengue virus (DENV) is a mosquito-borne flavivirus that causes significant human disease and mortality in the tropics and subtropics. By examining the effects of virus infection on gene expression, and interactions between virus and vector, new targets for prevention of infection and novel treatments may be identified in mosquitoes. We previously performed a microarray analysis of the Aedes aegypti transcriptome during infection with DENV and found that mosquito ubiquitin protein Ub3881 (AAEL003881) was specifically and highly down-regulated. Ubiquitin proteins have multiple functions in insects, including marking proteins for proteasomal degradation, regulating apoptosis and mediating innate immune signaling. METHODS: We used qRT-PCR to quantify gene expression and infection, and RNAi to reduce Ub3881 expression. Mosquitoes were infected with DENV through blood feeding. We transfected DENV protein expression constructs to examine the effect of Ub3881 on protein degradation. We used site-directed mutagenesis and transfection to determine what amino acids are involved in Ub3881-mediated protein degradation. Immunofluorescence, Co-immunoprecipitation and Western blotting were used to examine protein interactions and co-localization. RESULTS: The overexpression of Ub3881, but not related ubiquitin proteins, decreased DENV infection in mosquito cells and live Ae. aegypti. The Ub3881 protein was demonstrated to be involved in DENV envelope protein degradation and reduce the number of infectious virions released. CONCLUSIONS: We conclude that Ub3881 has several antiviral functions in the mosquito, including specific viral protein degradation. GENERAL SIGNIFICANCE: Our data highlights Ub3881 as a target for future DENV prevention strategies in the mosquito transmission vector.
Assuntos
Aedes/metabolismo , Vírus da Dengue/metabolismo , Dengue/metabolismo , Dengue/virologia , Ubiquitina/metabolismo , Proteínas do Envelope Viral/metabolismo , Vírion/metabolismo , Aedes/genética , Animais , Apoptose/genética , Linhagem Celular , Dengue/genética , Dengue/prevenção & controle , Vírus da Dengue/genética , Regulação para Baixo/genética , Expressão Gênica/genética , Imunidade Inata/genética , Imunoprecipitação/métodos , Insetos Vetores/genética , Insetos Vetores/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Transcriptoma/genética , Proteínas do Envelope Viral/genética , Vírion/genéticaRESUMO
Dengue virus (DENV) is a mosquito-borne flavivirus that causes serious human disease and mortality worldwide. There is no specific antiviral therapy or vaccine for DENV infection. Alterations in gene expression during DENV infection of the mosquito and the impact of these changes on virus infection are important events to investigate in hopes of creating new treatments and vaccines. We previously identified 203 genes that were ≥5-fold differentially upregulated during flavivirus infection of the mosquito. Here, we examined the impact of silencing 100 of the most highly upregulated gene targets on DENV infection in its mosquito vector. We identified 20 genes that reduced DENV infection by at least 60% when silenced. We focused on one gene, a putative cysteine rich venom protein (SeqID AAEL000379; CRVP379), whose silencing significantly reduced DENV infection in Aedes aegypti cells. Here, we examine the requirement for CRVP379 during DENV infection of the mosquito and investigate the mechanisms surrounding this phenomenon. We also show that blocking CRVP379 protein with either RNAi or specific antisera inhibits DENV infection in Aedes aegypti. This work identifies a novel mosquito gene target for controlling DENV infection in mosquitoes that may also be used to develop broad preventative and therapeutic measures for multiple flaviviruses.
Assuntos
Aedes/virologia , Vírus da Dengue/patogenicidade , Dengue/metabolismo , Proteínas de Insetos/metabolismo , Insetos Vetores/virologia , Animais , Venenos de Artrópodes/metabolismo , Western Blotting , Cisteína , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Inativação Gênica , Insetos Vetores/metabolismo , Reação em Cadeia da Polimerase , RNA Interferente Pequeno , TransfecçãoRESUMO
UNLABELLED: Chikungunya virus (CHIKV) is an alphavirus transmitted by mosquitoes that is known to cause severe arthritis and myositis in affected patients. The ongoing epidemic began in eastern Africa in 2004 and then spread to islands of the Indian Ocean, India, and Southeast Asia, ultimately afflicting millions. During this outbreak, more severe disease manifestations, including fatalities, have been documented. The reasons for this change in pathogenesis are multifactorial but likely include mutations that have arisen in the viral genome which could alter disease pathogenesis. To test this hypothesis, we used a murine model of CHIKV to compare the disease pathogeneses of two recombinant strains of CHIKV, the first derived from the La Reunion outbreak in 2006 (LR2006 OPY1) and the second isolated from Senegal in 1983 (37997). While the two strains exhibited similar growth in mammalian cells in vitro, we observed more severe clinical disease and pathology in mice infected with the LR2006 OPY1 strain of CHIKV, which included prolonged viremia and elevated viral titers and persistence in the muscle, resulting in devastating myonecrosis. Both CHIKV strains infected connective tissue fibroblasts of the muscle, but only the LR2006 OPY1 strain replicated within myofibers in vivo, despite similar growth of the two strains in these cell types in vitro. However, when the 37997 strain was administered directly into muscle, myofiber infection was comparable to that in LR2006 OPY1-infected mice. These results indicate that differences in the ability of the strain of CHIKV to establish infection in myofibers may contribute to the increased disease severity. IMPORTANCE: CHIKV is an emerging pathogen that causes significant morbidity. Little is known about the pathogenesis of the disease, and this study suggests that the ability of a recent epidemic strain to infect myofibers results in increased disease severity. Better understanding of how CHIKV causes disease contributes to the ultimate goal of creating therapeutics to alleviate the impact of this debilitating virus.
Assuntos
Infecções por Alphavirus/virologia , Vírus Chikungunya/fisiologia , Fibras Musculares Esqueléticas/virologia , Infecções por Alphavirus/metabolismo , Animais , Febre de Chikungunya , Vírus Chikungunya/efeitos dos fármacos , Vírus Chikungunya/patogenicidade , Citocinas/metabolismo , Modelos Animais de Doenças , Fibroblastos/virologia , Genoma Viral , Mediadores da Inflamação/metabolismo , Interferon beta/biossíntese , Interferon beta/farmacologia , Camundongos , Camundongos Knockout , Fibras Musculares Esqueléticas/patologia , Recombinação GenéticaRESUMO
UNLABELLED: Chikungunya virus (CHIKV) is a reemerging mosquito-transmitted alphavirus that causes epidemics of debilitating polyarthritis in humans. A prior study identified two anti-CHIKV monoclonal antibodies ([MAbs] CHK-152 and CHK-166) against the E2 and E1 structural proteins, which had therapeutic efficacy in immunocompetent and immunocompromised mice. Combination MAb therapy was required as administration of a single MAb resulted in the rapid selection of neutralization escape variants and treatment failure in mice. Here, we initially evaluated the efficacy of combination MAb therapy in a nonhuman primate model of CHIKV infection. Treatment of rhesus macaques with CHK-152 and CHK-166 reduced viral spread and infection in distant tissue sites and also neutralized reservoirs of infectious virus. Escape viruses were not detected in the residual viral RNA present in tissues and organs of rhesus macaques. To evaluate the possible significance of MAb resistance, we engineered neutralization escape variant viruses (E1-K61T, E2-D59N, and the double mutant E1-K61T E2-D59N) that conferred resistance to CHK-152 and CHK-166 and tested them for fitness in mosquito cells, mammalian cells, mice, and Aedes albopictus mosquitoes. In both cell culture and mosquitoes, the mutant viruses grew equivalently and did not revert to wild-type (WT) sequence. All escape variants showed evidence of mild clinical attenuation, with decreased musculoskeletal disease at early times after infection in WT mice and a prolonged survival time in immunocompromised Ifnar1(-/-) mice. Unexpectedly, this was not associated with decreased infectivity, and consensus sequencing from tissues revealed no evidence of reversion or compensatory mutations. Competition studies with CHIKV WT also revealed no fitness compromise of the double mutant (E1-K61T E2-D59N) neutralization escape variant in WT mice. Collectively, our study suggests that neutralization escape viruses selected during combination MAb therapy with CHK-152 plus CHK-166 retain fitness, cause less severe clinical disease, and likely would not be purified during the enzootic cycle. IMPORTANCE: Chikungunya virus (CHIKV) causes explosive epidemics of acute and chronic arthritis in humans in Africa, the Indian subcontinent, and Southeast Asia and recently has spread to the New World. As there are no approved vaccines or therapies for human use, the possibility of CHIKV-induced debilitating disease is high in many parts of the world. To this end, our laboratory recently generated a combination monoclonal antibody therapy that aborted lethal and arthritogenic disease in wild-type and immunocompromised mice when administered as a single dose several days after infection. In this study, we show the efficacy of the antibody combination in nonhuman primates and also evaluate the significance of possible neutralization escape mutations in mosquito and mammalian cells, mice, and Aedes albopictus vector mosquitoes. Our experiments show that escape viruses from combination antibody therapy cause less severe CHIKV clinical disease, retain fitness, and likely would not be purified by mosquito vectors.
Assuntos
Aedes/virologia , Infecções por Alphavirus/virologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Antivirais/uso terapêutico , Vírus Chikungunya/fisiologia , Imunoterapia/métodos , Replicação Viral , Infecções por Alphavirus/terapia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Febre de Chikungunya , Vírus Chikungunya/imunologia , Vírus Chikungunya/isolamento & purificação , Vírus Chikungunya/patogenicidade , Modelos Animais de Doenças , Feminino , Proteínas de Homeodomínio , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sobrevida , VirulênciaRESUMO
BACKGROUND: The RGD motif in the mosquito-borne flaviviruses envelope protein domain III (EDIII) FG loop was shown to bind negatively charged cellular molecules and mediate virus entry in mammals. However, its importance in virus entry in the mosquito has not yet been defined. The sequences of RGD motifs are conserved in JEV-serocomplex members primarily transmitted by Culex mosquitoes but absent from members of the DENV serocomplex, which utilize Aedes mosquitoes as vectors. Interestingly, the RGD sequence is present in the attenuated 17D strain of yellow fever virus as a result of the T380R mutation in the EDIII of Asibi strain following extensive in vitro passage in mice and chicken embryos and was found to contribute to the more rapid clearance in mice challenged with 17D. However, viral infectivity and dissemination in mosquitoes had not been evaluated for this mutant. FINDINGS: The study utilized the reverse genetics system of YFV and Ae. aegypti RexD WE mosquitoes to assess the impact of a T380R mutation in YFV Asibi and 17D/Asibi M-E chimera. The T380R mutation led to higher infection rates but similar dissemination rates when introduced into the YFV Asibi strain and 17D/Asibi M-E chimera. CONCLUSIONS: While the increase of the positive charge in EDIII may reduce the virulence of YFV in mice, this mutation favored the establishment of the viral infection in Ae. aegypti. However, such gain in viral infectivity did not increase dissemination in infected mosquitoes.
Assuntos
Mutação de Sentido Incorreto , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus , Vírus da Febre Amarela/fisiologia , Aedes , Animais , Análise Mutacional de DNA , Camundongos , Mutagênese , Genética Reversa , Vírus da Febre Amarela/genéticaRESUMO
BACKGROUND: Millions of human infections caused by arthropod-borne pathogens are initiated by the feeding of an infected mosquito on a vertebrate. However, interactions between the viruses and the mosquito vector, which facilitates successful infection and transmission of virus to a subsequent vertebrate host, are still not fully understood. FINDING: Here we describe early chikungunya virus (CHIKV) infectious events in cells derived from one of the most important CHIKV vectors, Aedes albopictus. We demonstrated that CHIKV infection of mosquito cells depended on acidification of the endosome as indicated by significant inhibition following prophylactic treatment with the lysosomotropic drugs chloroquine, ammonium chloride, and monensin, which is consistent with observations in mammalian cells. While all three agents inhibited CHIKV infection in C6/36 cells, ammonium chloride was less toxic to cells than the other agents. CONCLUSION: The observation of similar mechanisms for inhibition of CHIKV infection in mosquito and mammalian cell lines suggests that conserved entry pathways are utilized by CHIKV for vertebrate and invertebrate cell types.
Assuntos
Aedes/virologia , Vírus Chikungunya/fisiologia , Endossomos/virologia , Internalização do Vírus , Animais , Antivirais/metabolismo , Linhagem Celular , Endossomos/química , Endossomos/efeitos dos fármacos , Concentração de Íons de HidrogênioRESUMO
INTRODUCTION: Approximately 100 million confirmed infections and 20,000 deaths are caused by Dengue virus (DENV) outbreaks annually. Global warming and rapid dispersal have resulted in DENV epidemics in formally non-endemic regions. Currently no consistently effective preventive measures for DENV exist, prompting development of transgenic and paratransgenic vector control approaches. Production of transgenic mosquitoes refractory for virus infection and/or transmission is contingent upon defining antiviral genes that have low probability for allowing escape mutations, and are equally effective against multiple serotypes. Previously we demonstrated the effectiveness of an anti-viral group I intron targeting U143 of the DENV genome in mediating trans-splicing and expression of a marker gene with the capsid coding domain. In this report we examine the effectiveness of coupling expression of ΔN Bax to trans-splicing U143 intron activity as a means of suppressing DENV infection of mosquito cells. RESULTS: Targeting the conserved DENV circularization sequence (CS) by U143 intron trans-splicing activity appends a 3' exon RNA encoding ΔN Bax to the capsid coding region of the genomic RNA, resulting in a chimeric protein that induces premature cell death upon infection. TCID50-IFA analyses demonstrate an enhancement of DENV suppression for all DENV serotypes tested over the identical group I intron coupled with the non-apoptotic inducing firefly luciferase as the 3' exon. These cumulative results confirm the increased effectiveness of this αDENV-U143-ΔN Bax group I intron as a sequence specific antiviral that should be useful for suppression of DENV in transgenic mosquitoes. Annexin V staining, caspase 3 assays, and DNA ladder observations confirm DCA-ΔN Bax fusion protein expression induces apoptotic cell death. CONCLUSION: This report confirms the relative effectiveness of an anti-DENV group I intron coupled to an apoptosis-inducing ΔN Bax 3' exon that trans-splices conserved sequences of the 5' CS region of all DENV serotypes and induces apoptotic cell death upon infection. Our results confirm coupling the targeted ribozyme capabilities of the group I intron with the generation of an apoptosis-inducing transcript increases the effectiveness of infection suppression, improving the prospects of this unique approach as a means of inducing transgenic refractoriness in mosquitoes for all serotypes of this important disease.
Assuntos
Apoptose/genética , Vírus da Dengue/genética , Expressão Gênica , Íntrons , Domínios e Motivos de Interação entre Proteínas/genética , Proteína X Associada a bcl-2/genética , Animais , Linhagem Celular , Culicidae , Dengue/virologia , Vírus da Dengue/classificação , Éxons , Ordem dos Genes , Vetores Genéticos , Regiões Promotoras Genéticas , Sorogrupo , Trans-Splicing , Replicação Viral/genética , Proteína X Associada a bcl-2/químicaRESUMO
Adaptation of RNA viruses to a new host or vector species often results in emergence of new viral lineages. However, lineage-specific restrictions on the adaptive processes remain largely unexplored. Recently, a Chikungunya virus (CHIKV) lineage of African origin emerged to cause major epidemics of severe, persistent, debilitating arthralgia in Africa and Asia. Surprisingly, this new lineage is actively replacing endemic strains in Southeast Asia that have been circulating there for 60 y. This replacement process is associated with adaptation of the invasive CHIKV strains to an atypical vector, the Aedes albopictus mosquito that is ubiquitously distributed in the region. Here we demonstrate that lineage-specific epistatic interactions between substitutions at amino acid positions 226 and 98 of the E1 envelope glycoprotein, the latter of which likely resulted from a founder effect, have for 60 y restricted the ability of endemic Asian CHIKV strains to adapt to this new vector. This adaptive constraint appears to be allowing invasion of the unoccupied vector niche by Ae. albopictus-adapted African strains. These results underscore how different adaptive landscapes occupied by closely related viral genotypes can profoundly affect the outcome of viral evolution and disease emergence.
Assuntos
Vírus Chikungunya/fisiologia , Vírus Chikungunya/patogenicidade , Adaptação Fisiológica , Aedes/virologia , Infecções por Alphavirus/epidemiologia , Infecções por Alphavirus/transmissão , Infecções por Alphavirus/virologia , Substituição de Aminoácidos , Animais , Sudeste Asiático , Sequência de Bases , Febre de Chikungunya , Vírus Chikungunya/genética , Doenças Transmissíveis Emergentes/epidemiologia , Doenças Transmissíveis Emergentes/transmissão , Doenças Transmissíveis Emergentes/virologia , DNA Viral/genética , Epistasia Genética , Evolução Molecular , Proteínas de Fluorescência Verde/genética , Humanos , Insetos Vetores/virologia , Camundongos , Modelos Moleculares , Filogenia , Proteínas Recombinantes de Fusão/genética , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genéticaRESUMO
Zika virus (ZIKV) is an emerging flavivirus associated with several neurological diseases such as Guillain-Barré syndrome in adults and microcephaly in newborn children. Its distribution and mode of transmission (via Aedes aegypti and Aedes albopictus mosquitoes) collectively cause ZIKV to be a serious concern for global health. High genetic homology of flaviviruses and shared ecology is a hurdle for accurate detection. Distinguishing infections caused by different viruses based on serological recognition can be misleading as many anti-flavivirus monoclonal antibodies (mAbs) discovered to date are highly cross-reactive, especially those against the envelope (E) protein. To provide more specific research tools, we produced ZIKV E directed hybridoma cell lines and characterized two highly ZIKV-specific mAb clones (mAbs A11 and A42) against several members of the Flavivirus genus. Epitope mapping of mAb A11 revealed glycan loop specificity in Domain I of the ZIKV E protein. The development of two highly specific mAbs targeting the surface fusion protein of ZIKV presents a significant advancement in research capabilities as these can be employed as essential tools to enhance our understanding of ZIKV identification on infected cells ex vivo or in culture.
Assuntos
Aedes , Flavivirus , Infecção por Zika virus , Zika virus , Animais , Recém-Nascido , Humanos , Proteínas do Envelope Viral , Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos AntiviraisRESUMO
In the past few decades, several emerging/re-emerging mosquito-borne flaviviruses have resulted in disease outbreaks of public health concern in the tropics and subtropics. Due to cross-reactivities of antibodies recognizing the envelope protein of different flaviviruses, serosurveillance remains a challenge. Previously we reported that anti-premembrane (prM) antibody can discriminate between three flavivirus infections by Western blot analysis. In this study, we aimed to develop a serological assay that can discriminate infection or exposure with flaviviruses from four serocomplexes, including dengue (DENV), Zika (ZIKV), West Nile (WNV) and yellow fever (YFV) viruses, and explore its application for serosurveillance in flavivirus-endemic countries. We employed Western blot analysis including antigens of six flaviviruses (DENV1, 2 and 4, WNV, ZIKV and YFV) from four serocomplexes. We tested serum samples from YF-17D vaccinees, and from DENV, ZIKV and WNV panels that had been confirmed by RT-PCR or by neutralization assays. The overall sensitivity/specificity of anti-prM antibodies for DENV, ZIKV, WNV, and YFV infections/exposure were 91.7%/96.4%, 91.7%/99.2%, 88.9%/98.3%, and 91.3%/92.5%, respectively. When testing 48 samples from Brazil, we identified multiple flavivirus infections/exposure including DENV and ZIKV, DENV and YFV, and DENV, ZIKV and YFV. When testing 50 samples from the Philippines, we detected DENV, ZIKV, and DENV and ZIKV infections with a ZIKV seroprevalence rate of 10%, which was consistent with reports of low-level circulation of ZIKV in Asia. Together, these findings suggest that anti-prM antibody is a flavivirus serocomplex-specific marker and can be employed to delineate four flavivirus infections/exposure in regions where multiple flaviviruses co-circulate.
Assuntos
Vírus da Dengue , Dengue , Infecções por Flavivirus , Flavivirus , Infecção por Zika virus , Zika virus , Animais , Flavivirus/genética , Infecção por Zika virus/diagnóstico , Infecção por Zika virus/epidemiologia , Zika virus/genética , Vírus da Dengue/genética , Estudos Soroepidemiológicos , Anticorpos Antivirais , Infecções por Flavivirus/diagnóstico , Infecções por Flavivirus/epidemiologia , Vírus da Febre Amarela , Reações CruzadasRESUMO
West Nile (WNV), dengue (DENV) and yellow fever (YFV) viruses are (re)emerging, mosquito-borne flaviviruses that cause human disease and mortality worldwide. Alterations in mosquito gene expression common and unique to individual flaviviral infections are poorly understood. Here, we present a microarray analysis of the Aedes aegypti transcriptome over time during infection with DENV, WNV or YFV. We identified 203 mosquito genes that were ≥ 5-fold differentially up-regulated (DUR) and 202 genes that were ≥ 10-fold differentially down-regulated (DDR) during infection with one of the three flaviviruses. Comparative analysis revealed that the expression profile of 20 DUR genes and 15 DDR genes was quite similar between the three flaviviruses on D1 of infection, indicating a potentially conserved transcriptomic signature of flaviviral infection. Bioinformatics analysis revealed changes in expression of genes from diverse cellular processes, including ion binding, transport, metabolic processes and peptidase activity. We also demonstrate that virally-regulated gene expression is tissue-specific. The overexpression of several virally down-regulated genes decreased WNV infection in mosquito cells and Aedes aegypti mosquitoes. Among these, a pupal cuticle protein was shown to bind WNV envelope protein, leading to inhibition of infection in vitro and the prevention of lethal WNV encephalitis in mice. This work provides an extensive list of targets for controlling flaviviral infection in mosquitoes that may also be used to develop broad preventative and therapeutic measures for multiple flaviviruses.
Assuntos
Aedes/virologia , Vírus da Dengue/fisiologia , Transcriptoma/genética , Vírus do Nilo Ocidental/fisiologia , Vírus da Febre Amarela/fisiologia , Aedes/genética , Animais , Linhagem Celular , Vírus da Dengue/genética , Regulação para Baixo , Feminino , Infecções por Flavivirus/genética , Infecções por Flavivirus/prevenção & controle , Perfilação da Expressão Gênica , Regulação Viral da Expressão Gênica , Proteínas de Insetos/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Vírus do Nilo Ocidental/genética , Vírus da Febre Amarela/genéticaRESUMO
Chikungunya virus (CHIKV) is a re-emerging alphavirus that has caused significant disease in the Indian Ocean region since 2005. During this outbreak, in addition to fever, rash and arthritis, severe cases of CHIKV infection have been observed in infants. Challenging the notion that the innate immune response in infants is immature or defective, we demonstrate that both human infants and neonatal mice generate a robust type I interferon (IFN) response during CHIKV infection that contributes to, but is insufficient for, the complete control of infection. To characterize the mechanism by which type I IFNs control CHIKV infection, we evaluated the role of ISG15 and defined it as a central player in the host response, as neonatal mice lacking ISG15 were profoundly susceptible to CHIKV infection. Surprisingly, UbE1Lâ»/â» mice, which lack the ISG15 E1 enzyme and therefore are unable to form ISG15 conjugates, displayed no increase in lethality following CHIKV infection, thus pointing to a non-classical role for ISG15. No differences in viral loads were observed between wild-type (WT) and ISG15â»/â» mice, however, a dramatic increase in proinflammatory cytokines and chemokines was observed in ISG15â»/â» mice, suggesting that the innate immune response to CHIKV contributes to their lethality. This study provides new insight into the control of CHIKV infection, and establishes a new model for how ISG15 functions as an immunomodulatory molecule in the blunting of potentially pathologic levels of innate effector molecules during the host response to viral infection.
Assuntos
Infecções por Alphavirus/imunologia , Vírus Chikungunya/fisiologia , Citocinas/imunologia , Interferon Tipo I/imunologia , Enzimas Ativadoras de Ubiquitina/imunologia , Ubiquitinas/imunologia , Infecções por Alphavirus/diagnóstico , Infecções por Alphavirus/metabolismo , Animais , Animais Recém-Nascidos , Febre de Chikungunya , Vírus Chikungunya/patogenicidade , Citocinas/metabolismo , Modelos Animais de Doenças , Humanos , Interferon Tipo I/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Recombinação Genética , Estudos Retrospectivos , Enzimas Ativadoras de Ubiquitina/metabolismo , Ubiquitinas/metabolismoRESUMO
BACKGROUND: Chikungunya virus (CHIKV) is a reemerging mosquito-borne virus that has been responsible for a number of large-scale epidemics as well as imported cases covering a wide geographical range. As a blood-borne virus capable of mounting a high-titer viremia in infected humans, CHIKV was included on a list of risk agents for transfusion and organ transplant by the AABB Transfusion-Transmitted Diseases Committee. Therefore, we evaluated the ability of the Mirasol pathogen reduction technology (PRT) system (CaridianBCT Biotechnologies) to inactivate live virus in contaminated plasma and platelet (PLT) samples. STUDY DESIGN AND METHODS: Plasma, PLTs, and phosphate-buffered saline controls were spiked with CHIKV and treated with riboflavin and varying doses of ultraviolet (UV) light using the Mirasol PRT system. Samples were tested before and after treatment for cytotoxicity, interference, and virus titer by titration and quantitative real-time reverse transcription-polymerase chain reaction. RESULTS: A significant reduction in CHIKV titer of greater than 99% was recorded after treatment of plasma or PLTs with the Mirasol PRT system, and the titer reduction was directly proportional to the UV dose delivered to the samples. No cytotoxicity of interference was observed in any sample at any treatment dose. CONCLUSION: These data indicate that the Mirasol PRT system efficiently inactivated live CHIKV in plasma and PLTs and could therefore potentially be used to prevent CHIKV transmission through the blood supply.
Assuntos
Plaquetas/virologia , Vírus Chikungunya/efeitos da radiação , Processos Fotoquímicos , Plasma/virologia , Inativação de Vírus , Animais , Animais Lactentes , Plaquetas/efeitos da radiação , Células Cultivadas , Vírus Chikungunya/fisiologia , Chlorocebus aethiops , Cricetinae , Humanos , Luz , Camundongos , Segurança do Paciente , Plasma/efeitos da radiação , Transfusão de Plaquetas/métodos , Células VeroRESUMO
Cache Valley virus (CVV) is a mosquito-borne bunyavirus that is enzootic throughout the new world. Although CVV is known as an important agricultural pathogen, primarily associated with embryonic lethality and abortions in ruminants, it has recently been recognized for its expansion as a zoonotic pathogen. With the increased emergence of bunyaviruses with human and veterinary importance, there have been significant efforts dedicated to the development of bunyavirus vaccines. In this study, the immunogenicity of a candidate live-attenuated vaccine (LAV) for CVV, which contains the deletion of the nonstructural small (NSs) and nonstructural medium (NSm) genes (2delCVV), was evaluated and compared with an autogenous candidate vaccine created through the inactivation of CVV using binary ethylenimine (BEI) with an aluminum hydroxide adjuvant (BEI-CVV) in sheep. Both 2delCVV and BEI-CVV produced a neutralizing antibody response that exceeds the correlate of protection, that is, plaque reduction neutralization test titer >10. However, on day 63 postinitial immunization, 2delCVV was more immunogenic than BEI-CVV. These results warrant further development of 2delCVV as a candidate LAV and demonstrate that the double deletion of the NSs and NSm genes can be applied to the development of vaccines and as a common attenuation strategy for orthobunyaviruses.
Assuntos
Vírus Bunyamwera , Vacinas Virais , Gravidez , Feminino , Animais , Humanos , Ovinos , Vírus Bunyamwera/fisiologia , Vacinas Atenuadas , Vacinas de Produtos Inativados , Anticorpos NeutralizantesRESUMO
Rift Valley fever virus (RVFV) is an emerging arbovirus that affects both ruminants and humans. RVFV causes severe and recurrent outbreaks in Africa and the Arabian Peninsula with a significant risk for emergence into new locations. Although there are a variety of RVFV veterinary vaccines for use in endemic areas, there is currently no licensed vaccine for human use; therefore, there is a need to develop and assess new vaccines. Herein, we report a live-attenuated recombinant vaccine candidate for RVFV, based on the previously described genomic reconfiguration of the conditionally licensed MP12 vaccine. There are two general strategies used to develop live-attenuated RVFV vaccines, one being serial passage of wild-type RVFV strains to select attenuated mutants such as Smithburn, Clone 13, and MP12 vaccine strains. The second strategy has utilized reverse genetics to attenuate RVFV strains by introducing deletions or insertions within the viral genome. The novel candidate vaccine characterized in this report contains a two-segmented genome that lacks the medium viral segment (M) and two virulence genes (nonstructural small and nonstructural medium). The vaccine candidate, named r2segMP12, was evaluated for the production of neutralizing antibodies to RVFV in outbred CD-1 mice. The immune response induced by the r2segMP12 vaccine candidate was directly compared to the immune response induced by the rMP12 parental strain vaccine. Our study demonstrated that a single immunization with the r2segMP12 vaccine candidate at 105 plaque-forming units elicited a higher neutralizing antibody response than the rMP12 vaccine at the same vaccination titer without the need for a booster.
Assuntos
Febre do Vale de Rift , Vírus da Febre do Vale do Rift , Vacinas Virais , Humanos , Animais , Camundongos , Vírus da Febre do Vale do Rift/genética , Febre do Vale de Rift/prevenção & controle , Febre do Vale de Rift/epidemiologia , Vacinas Atenuadas/genética , Vacinas Virais/genética , Anticorpos NeutralizantesRESUMO
Background: Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic flavivirus and the leading cause of pediatric encephalitis in the Asian Pacific region. The transmission cycle primarily involves Culex spp. mosquitoes and Ardeid birds, with domestic pigs (Sus scrofa domestica) being the source of infectious viruses for the spillover of JEV from the natural endemic transmission cycle into the human population. Although many studies have concluded that domestic pigs play an important role in the transmission cycle of JEV, and infection of humans, the role of feral pigs in the transmission of JEV remains unclear. Since domestic and feral pigs are the same species, and because feral pig populations in the United States are increasing and expanding geographically, the current study aimed to test the hypothesis that if JEV were introduced into the United States, feral pigs might play a role in the transmission cycle. Materials and Methods: Sinclair miniature pigs, that exhibit the feral phenotype, were intradermally inoculated with JEV genotype Ib. These pigs were derived from crossing miniature domestic pig with four strains of feral pigs and were used since obtaining feral swine was not possible. Results: The Sinclair miniature pigs became viremic and displayed pathological outcomes similar to those observed in domestic swine. Conclusion: Based on these findings, we conclude that in the event of JEV being introduced into the United States, feral pig populations could contribute to establishment and maintenance of a transmission cycle of JEV and could lead to the virus becoming endemic in the United States.
Assuntos
Culex , Culicidae , Vírus da Encefalite Japonesa (Espécie) , Encefalite Japonesa , Animais , Suínos , Humanos , Criança , Vírus da Encefalite Japonesa (Espécie)/genética , Encefalite Japonesa/epidemiologia , Encefalite Japonesa/veterinária , Porco Miniatura , Aves , FenótipoRESUMO
In the past few decades, several emerging/re-emerging mosquito-borne flaviviruses have resulted in disease outbreaks of public health concern in the tropics and subtropics. Due to cross-reactivities of antibodies recognizing the envelope protein of different flaviviruses, serosurveillance remains a challenge. Previously we reported that anti-premembrane (prM) antibody can discriminate between three flavivirus infections by Western blot analysis. In this study, we aimed to develop a serological assay that can discriminate infection or exposure with flaviviruses from four serocomplexes, including dengue (DENV), Zika (ZIKV), West Nile (WNV) and yellow fever (YFV) viruses, and explore its application for serosurveillance in flavivirus-endemic countries. We employed Western blot analysis including antigens of six flaviviruses (DENV1, 2 and 4, WNV, ZIKV and YFV) from four serocomplexes. We tested serum samples from YF-17D vaccinees, and from DENV, ZIKV and WNV panels that had been confirmed by RT-PCR or by neutralization assays. The overall sensitivity/specificity of anti-prM antibodies for DENV, ZIKV, WNV, and YFV infections/exposure were 91.7%/96.4%, 91.7%/99.2%, 88.9%/98.3%, and 91.3%/92.5%, respectively. When testing 48 samples from Brazil, we identified multiple flavivirus infections/exposure including DENV and ZIKV, DENV and YFV, and DENV, ZIKV and YFV. When testing 50 samples from the Philippines, we detected DENV, ZIKV, and DENV and ZIKV infections with a ZIKV seroprevalence rate of 10%, which was consistent with reports of low-level circulation of ZIKV in Asia. Together, these findings suggest that anti-prM antibody is a flavivirus serocomplex-specific marker and can be employed to delineate four flavivirus infections/exposure in regions where multiple flaviviruses co-circulate.
RESUMO
The flavivirus envelope protein is a class II fusion protein that drives flavivirus-cell membrane fusion. The membrane fusion process is triggered by the conformational change of the E protein from dimer in the virion to trimer, which involves the rearrangement of three domains, EDI, EDII, and EDIII. The movement between EDI and EDII initiates the formation of the E protein trimer. The EDI-EDII hinge region utilizes four motifs to exert the hinge effect at the interdomain region and is crucial for the membrane fusion activity of the E protein. Using West Nile virus (WNV) NY99 strain derived from an infectious clone, we investigated the role of eight flavivirus-conserved hydrophobic residues in the EDI-EDII hinge region in the conformational change of E protein from dimer to trimer and viral entry. Single mutations of the E-A54, E-I130, E-I135, E-I196, and E-Y201 residues affected infectivity. Importantly, the E-A54I and E-Y201P mutations fully attenuated the mouse neuroinvasive phenotype of WNV. The results suggest that multiple flavivirus-conserved hydrophobic residues in the EDI-EDII hinge region play a critical role in the structure-function of the E protein and some contribute to the virulence phenotype of flaviviruses as demonstrated by the attenuation of the mouse neuroinvasive phenotype of WNV. Thus, as a proof of concept, residues in the EDI-EDII hinge region are proposed targets to engineer attenuating mutations for inclusion in the rational design of candidate live-attenuated flavivirus vaccines.