RESUMO
Vaccines to generate durable humoral immunity against antigenically evolving pathogens such as the influenza virus must elicit antibodies that recognize conserved epitopes. Analysis of single memory B cells from immunized human donors has led us to characterize a previously unrecognized epitope of influenza hemagglutinin (HA) that is immunogenic in humans and conserved among influenza subtypes. Structures show that an unrelated antibody from a participant in an experimental infection protocol recognized the epitope as well. IgGs specific for this antigenic determinant do not block viral infection in vitro, but passive administration to mice affords robust IgG subtype-dependent protection against influenza infection. The epitope, occluded in the pre-fusion form of HA, is at the contact surface between HA head domains; reversible molecular "breathing" of the HA trimer can expose the interface to antibody and B cells. Antigens that present this broadly immunogenic HA epitope may be good candidates for inclusion in "universal" flu vaccines.
Assuntos
Anticorpos Antivirais/imunologia , Epitopos/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Imunoglobulina G/imunologia , Vírus da Influenza A/imunologia , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae , Adulto , Animais , Cães , Feminino , Humanos , Células Madin Darby de Rim Canino , Masculino , Camundongos , Pessoa de Meia-Idade , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/prevenção & controleRESUMO
Human B cell antigen-receptor (BCR) repertoires reflect repeated exposures to evolving influenza viruses; new exposures update the previously generated B cell memory (Bmem) population. Despite structural similarity of hemagglutinins (HAs) from the two groups of influenza A viruses, cross-reacting antibodies (Abs) are uncommon. We analyzed Bmem compartments in three unrelated, adult donors and found frequent cross-group BCRs, both HA-head directed and non-head directed. Members of a clonal lineage from one donor had a BCR structure similar to that of a previously described Ab, encoded by different gene segments. Comparison showed that both Abs contacted the HA receptor-binding site through long heavy-chain third complementarity determining regions. Affinities of the clonal-lineage BCRs for historical influenza-virus HAs from both group 1 and group 2 viruses suggested that serial responses to seasonal influenza exposures had elicited the lineage and driven affinity maturation. We propose that appropriate immunization regimens might elicit a comparably broad response.
Assuntos
Anticorpos Antivirais/imunologia , Linfócitos B/imunologia , Vírus da Influenza A/imunologia , Adulto , Técnicas de Cultura de Células , Reações Cruzadas/imunologia , Feminino , Citometria de Fluxo , Hemaglutininas Virais/imunologia , Humanos , Interferometria , MasculinoRESUMO
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
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/químicaRESUMO
BACKGROUND: Chikungunya virus (CHIKV) is a mosquito transmitted alphavirus that recently caused several large scale outbreaks/epidemics of arthritic disease in tropics of Africa, Indian Ocean basin and South-East Asia. This re-emergence event was facilitated by genetic adaptation (E1-A226V substitution) of CHIKV to a newly significant mosquito vector for this virus; Aedes albopictus. However, the molecular mechanism explaining the positive effect of the E1-A226V mutation on CHIKV fitness in this vector remains largely unknown. Previously we demonstrated that the E1-A226V substitution is also associated with attenuated CHIKV growth in cells depleted by cholesterol. METHODS: In this study, using a panel of CHIKV clones that varies in sensitivity to cholesterol, we investigated the possible relationship between cholesterol dependence and Ae. albopictus infectivity. RESULTS: We demonstrated that there is no clear mechanistic correlation between these two phenotypes. We also showed that the E1-A226V mutation increases the pH dependence of the CHIKV fusion reaction; however, subsequent genetic analysis failed to support an association between CHIKV dependency on lower pH, and mosquito infectivity phenotypes. CONCLUSION: the E1-A226V mutation probably acts at different steps of the CHIKV life cycle, affecting multiple functions of the virus.
Assuntos
Adaptação Biológica , Aedes/virologia , Vírus Chikungunya/fisiologia , Colesterol/metabolismo , Internalização do Vírus , Substituição de Aminoácidos/genética , Animais , Vírus Chikungunya/genética , Vírus Chikungunya/crescimento & desenvolvimento , Concentração de Íons de Hidrogênio , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
Persistent West Nile virus (WNV) infection in the mosquito Culex quinquefasciatus Say (Diptera: Culicidae) is associated with pathological changes in the salivary glands, including apoptotic cell death and a corresponding reduction in virus transmission over time. The vector host response to WNV infection and the molecular basis of WNV pathogenesis in Cx. quinquefasciatus was investigated using oligonucleotide microarrays designed to detect differences in the salivary gland transcriptome between WNV-infected mosquitoes and uninfected controls. Transcripts with increased abundance in infected salivary glands included those related to immunity, transcription, protein transport and degradation, amino acid and nucleotide metabolism, signal transduction, and cellular detoxification. Microarray-based analysis detected a decrease in transcript levels of a Culex inhibitor of apoptosis gene (IAP-1) and a decrease in abundance of 11 transcripts encoding salivary gland proteins. Transcript levels for an endonuclease, a proline-rich mucin, and several D7 protein family members also decreased. Transcripts with the greatest change in abundance during infection had either no similarity to sequences found in GenBank, VectorBase, and FlyBase, or were similar to sequences with uncharacterized protein products. These transcripts represent exciting targets for future analysis. Results from this study suggest that WNV infection influences transcriptional changes in an invertebrate host target tissue that may confer an advantage to the replicating virus, induce a host defense response, and alter the composition of vector saliva. The ramifications of these changes are discussed in terms of mosquito vector competence and WNV pathogenesis.
Assuntos
Culex/genética , Perfilação da Expressão Gênica , Glândulas Salivares/fisiologia , Transcrição Gênica , Febre do Nilo Ocidental/transmissão , Vírus do Nilo Ocidental/patogenicidade , Ração Animal , Animais , Culex/virologia , DNA Complementar/genética , Regulação para Baixo , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Regulação para CimaRESUMO
Chikungunya virus (CHIKV) is an emerging arbovirus associated with several recent large-scale epidemics. The 2005-2006 epidemic on Reunion island that resulted in approximately 266,000 human cases was associated with a strain of CHIKV with a mutation in the envelope protein gene (E1-A226V). To test the hypothesis that this mutation in the epidemic CHIKV (strain LR2006 OPY1) might influence fitness for different vector species, viral infectivity, dissemination, and transmission of CHIKV were compared in Aedes albopictus, the species implicated in the epidemic, and the recognized vector Ae. aegypti. Using viral infectious clones of the Reunion strain and a West African strain of CHIKV, into which either the E1-226 A or V mutation was engineered, we demonstrated that the E1-A226V mutation was directly responsible for a significant increase in CHIKV infectivity for Ae. albopictus, and led to more efficient viral dissemination into mosquito secondary organs and transmission to suckling mice. This mutation caused a marginal decrease in CHIKV Ae. aegypti midgut infectivity, had no effect on viral dissemination, and was associated with a slight increase in transmission by Ae. aegypti to suckling mice in competition experiments. The effect of the E1-A226V mutation on cholesterol dependence of CHIKV was also analyzed, revealing an association between cholesterol dependence and increased fitness of CHIKV in Ae. albopictus. Our observation that a single amino acid substitution can influence vector specificity provides a plausible explanation of how this mutant virus caused an epidemic in a region lacking the typical vector. This has important implications with respect to how viruses may establish a transmission cycle when introduced into a new area. Due to the widespread distribution of Ae. albopictus, this mutation increases the potential for CHIKV to permanently extend its range into Europe and the Americas.
Assuntos
Aedes/virologia , Infecções por Alphavirus/transmissão , Vírus Chikungunya/genética , Vírus Chikungunya/patogenicidade , Insetos Vetores/virologia , Mutação , Infecções por Alphavirus/epidemiologia , Animais , Animais Lactentes , Chlorocebus aethiops , Cricetinae , Modelos Animais de Doenças , Feminino , Genoma Viral , Humanos , Camundongos , Reunião/epidemiologia , Sensibilidade e Especificidade , Células VeroRESUMO
Laboratory rodent influenza infection models have been and continue to be a critical tool for understanding virus-host interactions during infection. The incidence of seasonal influenza infections coupled with the need for novel therapeutics and universal vaccines highlights the need to uncover novel mechanisms of pathogenesis and protection. Mouse models are extremely useful for the evaluation of influenza vaccines and provide an invaluable tool to probe the immune response. This chapter describes the technique of intranasal inoculation of male C57BL/6J mice with an H1N1 strain of influenza (A/Puerto Rico/8/1934) and methods for assessing the optimum dose for infection, viral titers in lung tissue, and severity of disease.
Assuntos
Pulmão/imunologia , Infecções por Orthomyxoviridae/imunologia , Administração Intranasal , Animais , Modelos Animais de Doenças , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/uso terapêutico , Pulmão/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/virologia , Vacinação/métodosRESUMO
Hundreds of cellular host factors are required to support dengue virus infection, but their identity and roles are incompletely characterized. Here, we identify human host dependency factors required for efficient dengue virus-2 (DENV2) infection of human cells. We focused on two, TTC35 and TMEM111, which we previously demonstrated to be required for yellow fever virus (YFV) infection and others subsequently showed were also required by other flaviviruses. These proteins are components of the human endoplasmic reticulum membrane protein complex (EMC), which has roles in ER-associated protein biogenesis and lipid metabolism. We report that DENV, YFV and Zika virus (ZIKV) infections were strikingly inhibited, while West Nile virus infection was unchanged, in cells that lack EMC subunit 4. Furthermore, targeted depletion of EMC subunits in live mosquitoes significantly reduced DENV2 propagation in vivo. Using a novel uncoating assay, which measures interactions between host RNA-binding proteins and incoming viral RNA, we show that EMC is required at or prior to virus uncoating. Importantly, we uncovered a second and important role for the EMC. The complex is required for viral protein accumulation in a cell line harboring a ZIKV replicon, indicating that EMC participates in the complex process of viral protein biogenesis.
Assuntos
Infecções por Flavivirus/virologia , Flavivirus/patogenicidade , Interações Hospedeiro-Patógeno , Proteínas de Membrana/metabolismo , Biossíntese de Proteínas , Internalização do Vírus , Replicação Viral , Animais , Chlorocebus aethiops , Culicidae/virologia , Retículo Endoplasmático , Humanos , Proteínas de Membrana/genética , Células Tumorais Cultivadas , Células VeroRESUMO
Viral glycoproteins are under constant immune surveillance by a host's adaptive immune responses. Antigenic variation including glycan introduction or removal is among the mechanisms viruses have evolved to escape host immunity. Understanding how glycosylation affects immunodominance on complex protein antigens may help decipher underlying B cell biology. To determine how B cell responses can be altered by such modifications, we engineered glycans onto the influenza virus hemagglutinin (HA) and characterized the molecular features of the elicited humoral immunity in mice. We found that glycan addition changed the initially diverse antibody repertoire into an epitope-focused, genetically restricted response. Structural analyses showed that one antibody gene family targeted a previously subdominant, occluded epitope at the head interface. Passive transfer of this antibody conferred Fc-dependent protection to influenza virus-challenged mice. These results have potential implications for next-generation viral vaccines aimed at directing B cell responses to preferred epitope(s).
Assuntos
Anticorpos Antivirais/imunologia , Epitopos/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Polissacarídeos/metabolismo , Animais , Anticorpos Antivirais/metabolismo , Cristalografia por Raios X , Epitopos/química , Epitopos/genética , Epitopos/metabolismo , Glicosilação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Imunização Passiva , Vírus da Influenza A Subtipo H3N2/genética , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/prevenção & controle , Ligação Proteica , Conformação Proteica , Análise de SobrevidaRESUMO
Most FDA-approved adjuvants for infectious agents boost humoral but not cellular immunity, and have poorly-understood mechanisms. Stimulator of interferon genes (STING, also known as MITA, MPYS, or ERIS) is an exciting adjuvant target due to its role in cyclic dinucleotide (CDN)-driven anti-viral immunity; however, a major hindrance is STING's cytosolic localization which requires intracellular delivery of its agonists. As a result, STING agonists administered in a soluble form have elicited suboptimal immune responses. Delivery of STING agonists via particle platforms has proven a more successful strategy, but the opportunity for improved formulations and bioactivity remains. In this study we evaluated the adjuvant activity of the potent STING agonist, CDN 3'3'-cGAMP (cGAMP), encapsulated in acid-sensitive acetalated dextran (Ace-DEX) polymeric microparticles (MPs) which passively target antigen-presenting cells for intracellular release. This formulation was superior to all particle delivery systems evaluated and maintained its bioactivity following a sterilizing dose of gamma irradiation. Compared to soluble cGAMP, the Ace-DEX cGAMP MPs enhanced type-I interferon responses nearly 1000-fold in vitro and 50-fold in vivo, caused up to a 104-fold boost in antibody titers, increased Th1-associated responses, and expanded germinal center B cells and memory T cells. Furthermore, the encapsulated cGAMP elicited no observable toxicity in animals and achieved protective immunity against a lethal influenza challenge seven months post-immunization when using CDN adjuvant doses up to 100-fold lower than previous reports. For these reasons, Ace-DEX MP-encapsulated cGAMP represents a potent vaccine adjuvant of humoral and cellular immunity.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Portadores de Fármacos/administração & dosagem , Proteínas de Membrana/imunologia , Nucleotídeos Cíclicos/administração & dosagem , Animais , Células Cultivadas , Dextranos/administração & dosagem , Feminino , Imunidade Celular , Imunidade Humoral , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/administração & dosagem , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/administração & dosagem , VacinaçãoRESUMO
To evaluate the potential for nonviremic transmission (NVT) of West Nile virus (WNV) to occur in nature, we examined the effect of increasing spatial and temporal separation between co-feeding mosquitoes on the efficiency of nonviremic transmission and the potential of a West Nile virus bridge vector species, Aedes albopictus, to be infected via nonviremic transmission. West Nile virus-infected (donor) Culex pipiens quinquefasciatus were allowed to feed on a mouse for 5 minutes followed by non-infected (recipient) mosquitoes with increasing spatial (0, 10, 20, 30, 40, or 50 mm) or temporal (0, 15, 30, 45, or 60 min) separation from the site or time of donor feeding, respectively. Recipients became infected when feeding up to 40 mm from the donor and up to 45 minutes after donor feeding. Additionally, nonviremic transmission of West Nile virus from Cx. p. quinquefasciatus to Ae. albopictus was observed.
Assuntos
Aedes/virologia , Culex/virologia , Insetos Vetores/virologia , Viremia/transmissão , Febre do Nilo Ocidental/transmissão , Animais , Glândulas Salivares/virologia , Fatores de TempoRESUMO
The effect of long-term West Nile virus (WNV) infection on Culex salivary gland morphology and viability was evaluated by transmission electron microscopy during a four week period post-blood feeding. These studies showed that apoptosis and other cytopathologic changes occurred more frequently in WNV-infected mosquitoes compared with uninfected controls. The effect of long-term infection on WNV transmission was evaluated by titering virus in saliva over the same time period. Although the mean titer of WNV in mosquito saliva did not change significantly over time, the percentage of saliva samples containing WNV decreased. Because of the importance of saliva in blood meal acquisition and virus delivery, salivary gland pathology has the potential to affect mosquito feeding behavior and virus transmission. Results from this study add to a growing body of evidence that arbovirus infections in mosquito vectors can be cytopathic, and offer a potential mechanism for virus-induced cell death in mosquitoes.
Assuntos
Culex/virologia , Efeito Citopatogênico Viral , Glândulas Salivares/citologia , Glândulas Salivares/virologia , Febre do Nilo Ocidental/transmissão , Vírus do Nilo Ocidental/fisiologia , Animais , Morte Celular , Feminino , Febre do Nilo Ocidental/patologia , Febre do Nilo Ocidental/veterináriaRESUMO
BACKGROUND: Antipyretics reduce fever following childhood vaccinations; after inactivated influenza vaccine (IIV) they might ameliorate fever and thereby decrease febrile seizure risk, but also possibly blunt the immune response. We assessed the effect of antipyretics on immune responses and fever following IIV in children ages 6 through 47 months. METHODS: Over the course of three seasons, one hundred forty-two children, receiving either a single or the first of 2 recommended doses of IIV, were randomized to receive either oral acetaminophen suspension (nâ¯=â¯59) or placebo (nâ¯=â¯59) (double-blinded) or ibuprofen (nâ¯=â¯24) (open-label) immediately following IIV and every 4-8 h thereafter for 24 h. Blood samples were obtained at enrollment and 4 weeks following the last recommended IIV dose. Responses to IIV were assessed by hemagglutination inhibition assay (HAI). Seroprotection was defined as an HAI titer ≥1:40 and seroconversion as a titer ≥1:40 if baseline titer <1:10 or four-fold rise if baseline titer ≥1:10. Participants were monitored for fever and other solicited symptoms on the day of and day following IIV. RESULTS: Significant differences in seroconversion and post-vaccination seroprotection were not observed between children included in the different antipyretic groups and the placebo group for the vaccine antigens included in IIV over the course of the studies. Frequencies of solicited symptoms, including fever, were similar between treatment groups and the placebo group. CONCLUSIONS: Significant blunting of the immune response was not observed when antipyretics were administered to young children receiving IIV. Studies with larger sample sizes are needed to definitively establish the effect of antipyretics on IIV immunogenicity.
Assuntos
Antipiréticos/administração & dosagem , Febre/tratamento farmacológico , Imunidade Ativa/efeitos dos fármacos , Vacinas contra Influenza/imunologia , Vacinas de Produtos Inativados/imunologia , Acetaminofen/administração & dosagem , Acetaminofen/efeitos adversos , Acetaminofen/sangue , Anticorpos Antivirais/sangue , Antipiréticos/efeitos adversos , Antipiréticos/sangue , Antipiréticos/imunologia , Pré-Escolar , Feminino , Testes de Inibição da Hemaglutinação , Humanos , Imunização Secundária , Lactente , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/efeitos adversos , Influenza Humana/prevenção & controle , Masculino , Convulsões Febris/tratamento farmacológico , Convulsões Febris/prevenção & controle , Vacinação , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/efeitos adversosRESUMO
Four chimeric yellow fever (YF) 17D-dengue (DEN) candidate vaccine viruses (ChimeriVax-DEN; Acambis, Cambridge, MA) were characterized in Aedes aegypti and Ae. albopictus mosquitoes collected from Thailand. The four vaccine viruses contained the relevant prM and E genes of wild-type dengue viruses (DENV; serotypes 1-4) substituted for the equivalent genes in the YF vaccine virus (17D) backbone. Each chimera conferred protection against the homologous DENV serotype; a tetravalent mix of all four chimeras stimulates an immune response against all serotypes. Field-collected mosquitoes from Thailand were fed on blood containing each of the viruses under study and held 21 days after infection. Infection and dissemination rates were based on antigen detection in the body or head tissues, respectively. All four wild-type DENV serotypes infected and disseminated, but the candidate vaccine viruses were highly attenuated in mosquitoes with respect to infection and especially with respect to dissemination. Considering the low level viremias anticipated in humans vaccinated with these viruses, it is predicted that the risks of infection and transmission by mosquitoes in nature is minimal.
Assuntos
Aedes/virologia , Quimera , Vírus da Dengue/fisiologia , Dengue/prevenção & controle , Vacinas contra o Vírus do Nilo Ocidental/administração & dosagem , Animais , Vírus da Dengue/genética , Vírus da Dengue/crescimento & desenvolvimento , Vírus da Dengue/imunologia , Insetos Vetores/virologia , Tailândia , Replicação ViralRESUMO
The recent outbreak of Chikungunya virus (CHIKV) on several islands in the Indian Ocean and in India has focused attention on this reemerging virus and highlighted the need for development of new tools to study vector-virus-host interactions. We have constructed and characterized, in cell culture, Aedes aegypti and Ae. albopictus mosquitoes, infectious cDNA clones of CHIKV using a recent isolate from La Réunion Island. Comparison of the growth kinetics and infection rates of the viral isolate CHIKV strain LR2006 OPY1 (CHIKV-LR) and a full-length infectious clone (CHIKV-LR ic) indicate that the infectious clone has retained the viral phenotypes of the original isolate. Infectious clones that express green fluorescent protein (GFP) were also produced and characterized in cell culture and in Aedes mosquitoes. The CHIKV-LR 5'GFP infected Ae. aegypti and Ae. albopictus mosquitoes at a similar rate to the original virus and to the full length infectious clone. The CHIKV-LR 3'GFP only infected Ae. albopictus mosquitoes at similar rates. The development of these authentic infectious clones will enable targeted studies of the molecular determinants of infection, pathogenesis and transmission competence by Ae. aegypti and Ae. albopictus mosquitoes.
Assuntos
Aedes/virologia , Infecções por Alphavirus/virologia , Vírus Chikungunya/patogenicidade , Genoma Viral , Insetos Vetores/virologia , Infecções por Alphavirus/epidemiologia , Infecções por Alphavirus/prevenção & controle , Infecções por Alphavirus/transmissão , Animais , Vírus Chikungunya/classificação , Vírus Chikungunya/genética , Surtos de Doenças , Genótipo , Proteínas de Fluorescência Verde/genética , Humanos , Cinética , Filogenia , Reunião/epidemiologiaRESUMO
Modified vaccinia Ankara virus (MVA) is a smallpox vaccine candidate. This study was performed to determine if MVA vaccination provides long-term protection against rabbitpox virus (RPXV) challenge, an animal model of smallpox. Two doses of MVA provided 100% protection against a lethal intranasal RPXV challenge administered 9 months after vaccination.
Assuntos
Vacina Antivariólica/administração & dosagem , Vacina Antivariólica/imunologia , Varíola/prevenção & controle , Vaccinia virus/imunologia , Animais , Modelos Animais de Doenças , Feminino , Esquemas de Imunização , Coelhos , Análise de SobrevidaRESUMO
Chikungunya virus (CHIKV) is a mosquito-transmitted RNA virus that causes acute febrile infection associated with polyarthralgia in humans. Mechanisms of protective immunity against CHIKV are poorly understood, and no effective therapeutics or vaccines are available. We isolated and characterized human monoclonal antibodies (mAbs) that neutralize CHIKV infectivity. Among the 30 mAbs isolated, 13 had broad and ultrapotent neutralizing activity (IC50 < 10 ng/ml), and all of these mapped to domain A of the E2 envelope protein. Potent inhibitory mAbs blocked post-attachment steps required for CHIKV membrane fusion, and several were protective in a lethal challenge model in immunocompromised mice, even when administered at late time points after infection. These highly protective mAbs could be considered for prevention or treatment of CHIKV infection, and their epitope location in domain A of E2 could be targeted for rational structure-based vaccine development.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/uso terapêutico , Febre de Chikungunya/terapia , Vírus Chikungunya/imunologia , Imunização Passiva/métodos , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/isolamento & purificação , Quimioprevenção/métodos , Vírus Chikungunya/fisiologia , Modelos Animais de Doenças , Humanos , Concentração Inibidora 50 , Camundongos , Ligação Proteica , Análise de Sobrevida , Resultado do Tratamento , Proteínas do Envelope Viral/imunologia , Internalização do Vírus/efeitos dos fármacosRESUMO
Oxidative tissue injury often accompanies viral infection, yet there is little understanding of how it influences virus replication. We show that multiple hepatitis C virus (HCV) genotypes are exquisitely sensitive to oxidative membrane damage, a property distinguishing them from other pathogenic RNA viruses. Lipid peroxidation, regulated in part through sphingosine kinase-2, severely restricts HCV replication in Huh-7 cells and primary human hepatoblasts. Endogenous oxidative membrane damage lowers the 50% effective concentration of direct-acting antivirals in vitro, suggesting critical regulation of the conformation of the NS3-4A protease and the NS5B polymerase, membrane-bound HCV replicase components. Resistance to lipid peroxidation maps genetically to transmembrane and membrane-proximal residues within these proteins and is essential for robust replication in cell culture, as exemplified by the atypical JFH1 strain of HCV. Thus, the typical, wild-type HCV replicase is uniquely regulated by lipid peroxidation, providing a mechanism for attenuating replication in stressed tissue and possibly facilitating long-term viral persistence.
Assuntos
Hepacivirus/enzimologia , Peroxidação de Lipídeos , Estresse Oxidativo , RNA Polimerase Dependente de RNA/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/genética , Antivirais/farmacologia , Linhagem Celular , Membrana Celular/patologia , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Hepatite C/tratamento farmacológico , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas não Estruturais Virais/genéticaRESUMO
West Nile virus NS4B is a small hydrophobic nonstructural protein approximately 27 kDa in size whose function is poorly understood. Amino acid substitutions were introduced into the NS4B protein primarily targeting two distinct regions; the N-terminal domain (residues 35 through 60) and the central hydrophobic domain (residues 95 through 120). Only the NS4B P38G substitution was associated with both temperature-sensitive and small-plaque phenotypes. Importantly, this mutation was found to attenuate neuroinvasiveness greater than 10,000,000-fold and lower viremia titers compared to the wild-type NY99 virus in a mouse model. Full genome sequencing of the NS4B P38G mutant virus revealed two unexpected mutations at NS4B T116I and NS3 N480H (P38G/T116I/N480H), however, neither mutation alone was temperature sensitive or attenuated in mice. Following incubation of P38G/T116I/N480H at 41°C, five mutants encoding compensatory substitutions in the NS4B protein exhibited a reduction in the temperature-sensitive phenotype and reversion to a virulent phenotype in the mouse model.