RESUMO
Saliva from the mosquito vector of flaviviruses is capable of changing the local immune environment, leading to an increase in flavivirus-susceptible cells at the infected bite site. In addition, an antibody response to specific salivary gland (SG) components changes the pathogenesis of flaviviruses in human populations. To investigate whether antigenic SG proteins are capable of enhancing infection with Zika virus (ZIKV), a reemerging flavivirus primarily transmitted by the Aedes aegypti mosquito, we screened for antigenic SG proteins using a yeast display library and demonstrate that a previously undescribed SG protein we term neutrophil stimulating factor 1 (NeSt1) activates primary mouse neutrophils ex vivo Passive immunization against NeSt1 decreases pro-interleukin-1ß and CXCL2 expression, prevents macrophages from infiltrating the bite site, protects susceptible IFNAR-/- IFNGR-/- (AG129) mice from early ZIKV replication, and ameliorates virus-induced pathogenesis. These findings indicate that NeSt1 stimulates neutrophils at the mosquito bite site to change the immune microenvironment, allowing a higher level of early viral replication and enhancing ZIKV pathogenesis.IMPORTANCE When a Zika virus-infected mosquito bites a person, mosquito saliva is injected into the skin along with the virus. Molecules in this saliva can make virus infection more severe by changing the immune system to make the skin a better place for the virus to replicate. We identified a molecule that activates immune cells, called neutrophils, to recruit other immune cells, called macrophages, that the virus can infect. We named this molecule neutrophil-stimulating factor 1 (NeSt1). When we used antibodies to block NeSt1 in mice and then allowed Zika virus-infected mosquitoes to feed on these mice, they survived much better than mice that do not have antibodies against NeSt1. These findings give us more information about how mosquito saliva enhances virus infection, and it is possible that a vaccine against NeSt1 might protect people against severe Zika virus infection.
Assuntos
Aedes/virologia , Neutrófilos/metabolismo , Neutrófilos/virologia , Infecção por Zika virus/imunologia , Zika virus/imunologia , Aedes/imunologia , Animais , Arbovírus , Quimiocina CCL2 , Quimiocina CXCL2/metabolismo , Modelos Animais de Doenças , Feminino , Imunidade , Interleucina-1/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mosquitos Vetores/virologia , Precursores de Proteínas/metabolismo , Células RAW 264.7 , Saliva/virologia , Glândulas Salivares/virologia , Replicação Viral , Zika virus/patogenicidade , Infecção por Zika virus/virologiaRESUMO
Human metapneumovirus (HMPV), a member of the Paramyxoviridae family, is a leading cause of lower respiratory illness. Although receptor binding is thought to initiate fusion at the plasma membrane for paramyxoviruses, the entry mechanism for HMPV is largely uncharacterized. Here we sought to determine whether HMPV initiates fusion at the plasma membrane or following internalization. To study the HMPV entry process in human bronchial epithelial (BEAS-2B) cells, we used fluorescence microscopy, an R18-dequenching fusion assay, and developed a quantitative, fluorescence microscopy assay to follow virus binding, internalization, membrane fusion, and visualize the cellular site of HMPV fusion. We found that HMPV particles are internalized into human bronchial epithelial cells before fusing with endosomes. Using chemical inhibitors and RNA interference, we determined that HMPV particles are internalized via clathrin-mediated endocytosis in a dynamin-dependent manner. HMPV fusion and productive infection are promoted by RGD-binding integrin engagement, internalization, actin polymerization, and dynamin. Further, HMPV fusion is pH-independent, although infection with rare strains is modestly inhibited by RNA interference or chemical inhibition of endosomal acidification. Thus, HMPV can enter via endocytosis, but the viral fusion machinery is not triggered by low pH. Together, our results indicate that HMPV is capable of entering host cells by multiple pathways, including membrane fusion from endosomal compartments.
Assuntos
Metapneumovirus/fisiologia , Infecções por Paramyxoviridae/metabolismo , Mucosa Respiratória/virologia , Internalização do Vírus , Brônquios/virologia , Linhagem Celular , Endossomos/metabolismo , Citometria de Fluxo , Humanos , Microscopia Confocal , RNA Interferente Pequeno , Transfecção , Proteínas Virais de Fusão/metabolismoRESUMO
Acute viral infections typically generate functional effector CD8(+) T cells (TCD8) that aid in pathogen clearance. However, during acute viral lower respiratory infection, lung TCD8 are functionally impaired and do not optimally control viral replication. T cells also become unresponsive to Ag during chronic infections and cancer via signaling by inhibitory receptors such as programmed cell death-1 (PD-1). PD-1 also contributes to TCD8 impairment during viral lower respiratory infection, but how it regulates TCD8 impairment and the connection between this state and T cell exhaustion during chronic infections are unknown. In this study, we show that PD-1 operates in a cell-intrinsic manner to impair lung TCD8. In light of this, we compared global gene expression profiles of impaired epitope-specific lung TCD8 to functional spleen TCD8 in the same human metapneumovirus-infected mice. These two populations differentially regulate hundreds of genes, including the upregulation of numerous inhibitory receptors by lung TCD8. We then compared the gene expression of TCD8 during human metapneumovirus infection to those in acute or chronic lymphocytic choriomeningitis virus infection. We find that the immunophenotype of lung TCD8 more closely resembles T cell exhaustion late into chronic infection than do functional effector T cells arising early in acute infection. Finally, we demonstrate that trafficking to the infected lung alone is insufficient for TCD8 impairment or inhibitory receptor upregulation, but that viral Ag-induced TCR signaling is also required. Our results indicate that viral Ag in infected lungs rapidly induces an exhaustion-like state in lung TCD8 characterized by progressive functional impairment and upregulation of numerous inhibitory receptors.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Metapneumovirus/imunologia , Infecções por Paramyxoviridae/imunologia , Infecções Respiratórias/imunologia , Doença Aguda , Animais , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Análise por Conglomerados , Perfilação da Expressão Gênica/métodos , Interações Hospedeiro-Patógeno/imunologia , Humanos , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/virologia , Metapneumovirus/fisiologia , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Infecções por Paramyxoviridae/genética , Infecções por Paramyxoviridae/virologia , Fenótipo , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Infecções Respiratórias/genética , Infecções Respiratórias/virologia , Baço/imunologia , Baço/metabolismo , Baço/virologia , Transcriptoma/genética , Transcriptoma/imunologiaRESUMO
Beginning in 2015, concern over a new global epidemic has spread in the media, governmental agencies, legislative bodies and the public at large. This newly emerging threat has been reported to cause symptoms ranging from mild fever, rash, and body aches, to severe birth defects and acute onset paralysis. The causative agent of this disease, Zika virus, is closely related to two other important human pathogens, dengue and West Nile Virus (WNV), but has some distinguishing features that has raised alarms from the scientific community. Like its two close relatives, this virus is a member of the Flaviviridae family, a class of single stranded RNA viruses with a positive sense genome and is spread primarily via the bite of an infected mosquito. However, this virus has demonstrated another route of transmission that is particularly concerning for people outside of the regions where the main mosquito vector for this virus is present. Sexual transmission of Zika virus has been increasingly reported, from both infected males and females to their partner, which has resulted in the World Health Organization (WHO) and the Center for Disease Control (CDC) issuing warnings to those living in or travelling to areas of Zika transmission to practice abstinence and/or avoid unprotected sexual contact for up to six months after infection with this virus. This perspective will outline the evidence for sexual transmission and persistence of viral infection in semen and vaginal secretions as well as review the animal models for sexual transmission of Zika virus.
Assuntos
Doenças Virais Sexualmente Transmissíveis/transmissão , Infecção por Zika virus/transmissão , Zika virus/fisiologia , Aedes/virologia , Animais , Modelos Animais de Doenças , Feminino , Genitália/virologia , Humanos , Masculino , Camundongos , Mosquitos Vetores/virologia , Doenças Virais Sexualmente Transmissíveis/virologiaRESUMO
UNLABELLED: Type I IFN signaling, which is initiated through activation of the alpha interferon receptor (IFNAR), regulates the expression of proteins that are crucial contributors to immune responses. Paramyxoviruses, including human metapneumovirus (HMPV), have evolved mechanisms to inhibit IFNAR signaling, but the specific contribution of IFNAR signaling to the control of HMPV replication, pathogenesis, and adaptive immunity is unknown. We used IFNAR-deficient (IFNAR(-/-)) mice to assess the effect of IFNAR signaling on HMPV replication and the CD8(+) T cell response. HMPV-infected IFNAR(-/-) mice had a higher peak of early viral replication but cleared the virus with kinetics similar to those of wild-type (WT) mice. However, IFNAR(-/-) mice infected with HMPV displayed less airway dysfunction and lung inflammation. CD8(+) T cells of IFNAR(-/-) mice after HMPV infection expressed levels of the inhibitory receptor programmed death 1 (PD-1) similar to those of WT mice. However, despite lower expression of inhibitory programmed death ligand 1 (PD-L1), HMPV-specific CD8(+) T cells of IFNAR(-/-) mice were more functionally impaired than those of WT mice and upregulated the inhibitory receptor Tim-3. Analysis of the antigen-presenting cell subsets in the lungs revealed that the expansion of PD-L1(low) dendritic cells (DCs), but not PD-L1(high) alveolar macrophages, was dependent on IFNAR signaling. Collectively, our results indicate a role for IFNAR signaling in the early control of HMPV replication, disease progression, and the development of an optimal adaptive immune response. Moreover, our findings suggest an IFNAR-independent mechanism of lung CD8(+) T cell impairment. IMPORTANCE: Human metapneumovirus (HMPV) is a leading cause of acute respiratory illness. CD8(+) T cells are critical for clearing viral infection, yet recent evidence shows that HMPV and other respiratory viruses induce CD8(+) T cell impairment via PD-1-PD-L1 signaling. We sought to understand the role of type I interferon (IFN) in the innate and adaptive immune responses to HMPV by using a mouse model lacking IFN signaling. Although HMPV titers were higher in the absence of type I IFN, virus was nonetheless cleared and mice were less ill, indicating that type I IFN is not required to resolve HMPV infection but contributes to pathogenesis. Further, despite lower levels of the inhibitory ligand PD-L1 in mice lacking type I IFN, CD8(+) T cells were more impaired in these mice than in WT mice. Our data suggest that specific antigen-presenting cell subsets and the inhibitory receptor Tim-3 may contribute to CD8(+) T cell impairment.
Assuntos
Regulação da Expressão Gênica/imunologia , Interferon Tipo I/metabolismo , Metapneumovirus/imunologia , Infecções por Paramyxoviridae/imunologia , Transdução de Sinais/imunologia , Replicação Viral/fisiologia , Análise de Variância , Animais , Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Citometria de Fluxo , Receptor Celular 2 do Vírus da Hepatite A , Humanos , Interferon Tipo I/genética , Metapneumovirus/patogenicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oximetria , Infecções por Paramyxoviridae/patologia , Reação em Cadeia da Polimerase em Tempo Real , Receptores Virais/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Reinfections with respiratory viruses are common and cause significant clinical illness, yet precise mechanisms governing this susceptibility are ill defined. Lung Ag-specific CD8(+) T cells (T(CD8)) are impaired during acute viral lower respiratory infection by the inhibitory receptor programmed death-1 (PD-1). To determine whether PD-1 contributes to recurrent infection, we first established a model of reinfection by challenging B cell-deficient mice with human metapneumovirus (HMPV) several weeks after primary infection, and found that HMPV replicated to high titers in the lungs. A robust secondary effector lung TCD8 response was generated during reinfection, but these cells were more impaired and more highly expressed the inhibitory receptors PD-1, LAG-3, and 2B4 than primary T(CD8). In vitro blockade demonstrated that PD-1 was the dominant inhibitory receptor early after reinfection. In vivo therapeutic PD-1 blockade during HMPV reinfection restored lung T(CD8) effector functions (i.e., degranulation and cytokine production) and enhanced viral clearance. PD-1 also limited the protective efficacy of HMPV epitope-specific peptide vaccination and impaired lung T(CD8) during heterotypic influenza virus challenge infection. Our results indicate that PD-1 signaling may contribute to respiratory virus reinfection and evasion of vaccine-elicited immune responses. These results have important implications for the design of effective vaccines against respiratory viruses.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Paramyxoviridae/imunologia , Receptor de Morte Celular Programada 1/imunologia , Infecções Respiratórias/imunologia , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Linfócitos B/imunologia , Linfócitos B/patologia , Linfócitos T CD8-Positivos/patologia , Linfócitos T CD8-Positivos/virologia , Degranulação Celular/imunologia , Regulação da Expressão Gênica , Humanos , Evasão da Resposta Imune , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Contagem de Linfócitos , Metapneumovirus/imunologia , Camundongos , Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Infecções por Paramyxoviridae/genética , Infecções por Paramyxoviridae/prevenção & controle , Infecções por Paramyxoviridae/virologia , Receptor de Morte Celular Programada 1/genética , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Infecções Respiratórias/genética , Infecções Respiratórias/patologia , Infecções Respiratórias/virologia , Transdução de Sinais , Família de Moléculas de Sinalização da Ativação Linfocitária , Carga Viral , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologia , Replicação Viral , Proteína do Gene 3 de Ativação de LinfócitosRESUMO
BACKGROUND: Human metapneumovirus (HMPV) is a leading cause of acute respiratory tract infection, with significant morbidity and mortality. No licensed vaccines or therapeutic agents exist. Monoclonal antibodies (mAbs) are effective at preventing other infectious diseases and could be used against HMPV in high-risk hosts. METHODS: In vitro assays were performed to assess the neutralizing activity and affinity kinetics of human mAb 54G10. A new mouse model was developed to assess prophylactic and therapeutic efficacy in vivo. The epitope of 54G10 was identified by generating mAb-resistant mutants (MARMs). RESULTS: At low concentrations, 54G10 neutralized all 4 subgroups of HMPV in vitro and had subnanomolar affinity for the fusion protein. DBA/2 mice were permissive for all 4 HMPV subgroups, and 54G10 was effective both prophylactically and therapeutically against HMPV in vivo. Sequencing of HMPV MARMs identified the 54G10 epitope, which was similar to an antigenic site on respiratory syncytial virus (RSV). 54G10 also exhibited in vitro neutralizing activity and in vivo protective and therapeutic efficacy against RSV. CONCLUSIONS: Human mAb 54G10 has broad neutralizing activity against HMPV and could have prophylactic and therapeutic utility clinically. The conserved epitope could represent a structural vaccine target for HMPV and RSV.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/uso terapêutico , Metapneumovirus/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Animais , Anticorpos Monoclonais Humanizados/isolamento & purificação , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Quimioprevenção/métodos , Modelos Animais de Doenças , Feminino , Humanos , Imunoterapia/métodos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Testes de Neutralização , Infecções por Paramyxoviridae/prevenção & controle , Infecções por Paramyxoviridae/terapia , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Infecções por Vírus Respiratório Sincicial/terapia , Resultado do TratamentoRESUMO
UNLABELLED: Human metapneumovirus (HMPV) is a leading cause of respiratory disease in infants, children, and the elderly worldwide, yet no licensed vaccines exist. Live-attenuated vaccines present safety challenges, and protein subunit vaccines induce primarily antibody responses. Virus-like particles (VLPs) are an attractive alternative vaccine approach because of reduced safety concerns compared with live vaccines. We generated HMPV VLPs by expressing viral proteins in suspension-adapted human embryonic kidney epithelial (293-F) cells and found that the viral matrix (M) and fusion (F) proteins were sufficient to form VLPs. We previously reported that the VLPs resemble virus morphology and incorporate fusion-competent F protein (R. G. Cox, S. B. Livesay, M. Johnson, M. D. Ohi, and J. V. Williams, J. Virol. 86:12148-12160, 2012), which we hypothesized would elicit F-specific antibody and T cell responses. In this study, we tested whether VLP immunization could induce protective immunity to HMPV by using a mouse model. C57BL/6 mice were injected twice intraperitoneally with VLPs alone or with adjuvant and subsequently challenged with HMPV. Mice were euthanized 5 days postinfection, and virus titers, levels of neutralizing antibodies, and numbers of CD3(+) T cells were quantified. Mice immunized with VLPs mounted an F-specific antibody response and generated CD8(+) T cells recognizing an F protein-derived epitope. VLP immunization induced a neutralizing-antibody response that was enhanced by the addition of either TiterMax Gold or α-galactosylceramide adjuvant, though adjuvant reduced cellular immune responses. Two doses of VLPs conferred complete protection from HMPV replication in the lungs of mice and were not associated with a Th2-skewed cytokine response. These results suggest that nonreplicating VLPs are a promising vaccine candidate for HMPV. IMPORTANCE: Human metapneumovirus (HMPV) is a leading cause of acute respiratory infection in infants, children, and the elderly worldwide, yet no licensed vaccines exist. Live-attenuated vaccines present safety challenges, and protein subunit vaccines induce primarily antibody responses. Virus-like particles (VLPs) are an attractive alternative vaccine approach. We generated HMPV VLPs by expressing the viral matrix (M) and fusion (F) proteins in mammalian cells. We found that mice immunized with VLPs mounted an F-specific antibody response and generated CD8(+) T cells recognizing an F protein-derived epitope. VLP immunization induced a neutralizing-antibody response that was enhanced by the addition of either TiterMax Gold or α-galactosylceramide adjuvant. Two doses of VLPs conferred complete protection against HMPV replication in the lungs of mice and were not associated with a Th2-skewed cytokine response. These results suggest that nonreplicating VLPs are a promising vaccine candidate for HMPV.
Assuntos
Anticorpos Neutralizantes/imunologia , Linfócitos B/imunologia , Linfócitos T CD8-Positivos/imunologia , Imunidade Celular/imunologia , Metapneumovirus/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Análise de Variância , Animais , Western Blotting , Ensaio de Imunoadsorção Enzimática , ELISPOT , Citometria de Fluxo , Galactosilceramidas , Células HEK293 , Humanos , Imuno-Histoquímica , Pulmão/imunologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Poloxaleno , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vacinas de Partículas Semelhantes a Vírus/ultraestruturaRESUMO
Dengue virus (DENV) is a flavivirus that causes marked human morbidity and mortality worldwide, and is transmitted to humans by Aedes aegypti mosquitoes. Habitat expansion of Aedes, mainly due to climate change and increasing overlap between urban and wild habitats, places nearly half of the world's population at risk for DENV infection. After a bloodmeal from a DENV-infected host, the virus enters the mosquito midgut. Next, the virus migrates to, and replicates in, other tissues, like salivary glands. Successful viral transmission occurs when the infected mosquito takes another blood meal on a susceptible host and DENV is released from the salivary gland via saliva into the skin. During viral dissemination in the mosquito and transmission to a new mammalian host, DENV interacts with a variety of vector proteins, which are uniquely important during each phase of the viral cycle. Our study focuses on the interaction between DENV particles and protein components in the A. aegypti vector. We performed a mass spectrometry assay where we identified a set of A. aegypti salivary gland proteins which potentially interact with the DENV virion. Using dsRNA to silence gene expression, we analyzed the role of these proteins in viral infectivity. Two of these candidates, a synaptosomal-associated protein (AeSNAP) and a calcium transporter ATPase (ATPase) appear to play a role in viral replication both in vitro and in vivo, observing a ubiquitous expression of these proteins in the mosquito. These findings suggest that AeSNAP plays a protective role during DENV infection of mosquitoes and that ATPase protein is required for DENV during amplification within the vector.
Assuntos
Aedes/genética , Aedes/virologia , ATPases Transportadoras de Cálcio/metabolismo , Vírus da Dengue/fisiologia , Mosquitos Vetores/genética , Mosquitos Vetores/virologia , Animais , ATPases Transportadoras de Cálcio/genética , Linhagem Celular , Clonagem Molecular , Dengue/transmissão , Dengue/virologia , Regulação Enzimológica da Expressão Gênica , Técnicas de Silenciamento de Genes , Polimorfismo de Nucleotídeo Único , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Glândulas Salivares/virologiaRESUMO
West Nile virus (WNV) is transmitted by mosquitoes and can cause severe disease, including meningoencephalitis. AgBR1 is a mosquito salivary protein that enhances Aedes aegypti mosquito-borne Zika virus pathogenesis in mice. Here, we show that AgBR1 antibodies reduce the initial West Nile viral load and delay lethal infection after feeding by an infected Aedes aegypti mosquito. Targeting AgBR1 may therefore be incorporated into strategies to prevent mosquito-transmitted West Nile virus infection.
RESUMO
The TAM receptor, Axl, has been implicated as a candidate entry receptor for Zika virus (ZIKV) infection but has been shown as inessential for virus infection in mice. To probe the role of Axl in murine ZIKV infection, we developed a mouse model lacking the Axl receptor and the interferon alpha/beta receptor (Ifnar-/-Axl-/-), conferring susceptibility to ZIKV. This model validated that Axl is not required for murine ZIKV infection and that mice lacking Axl are resistant to ZIKV pathogenesis. This resistance correlates to lower pro-interleukin-1ß production and less apoptosis in microglia of ZIKV-infected mice. This apoptosis occurs through both intrinsic (caspase 9) and extrinsic (caspase 8) manners, and is age dependent, as younger Axl-deficient mice are susceptible to ZIKV pathogenesis. These findings suggest that Axl plays an important role in pathogenesis in the brain during ZIKV infection and indicates a potential role for Axl inhibitors as therapeutics during viral infection.
RESUMO
A recent epidemic of Zika virus in the Americas, affecting well over a million people, caused substantial mortality and morbidity, including Guillain-Barre syndrome, microcephaly and other fetal developmental defects1,2. Preventive and therapeutic measures that specifically target the virus are not readily available. The transmission of Zika virus is predominantly mosquito-borne, and Aedes aegypti mosquitoes serve as a key vector for Zika virus3. Here, to identify salivary factors that modulate mosquito-borne Zika virus infection, we focused on antigenic proteins in mice that were repeatedly bitten by mosquitoes and developed antibodies against salivary proteins. Using a yeast surface display screen, we identified five antigenic A. aegypti salivary proteins in mice. Antiserum against one of these five proteins-A. aegypti bacteria-responsive protein 1 (AgBR1)-suppressed early inflammatory responses in the skin of mice bitten by Zika-virus-infected mosquitoes. AgBR1 antiserum also partially protected mice from lethal mosquito-borne-but not needle-injected-Zika virus infection. These data suggest that AgBR1 is a target for the prevention of mosquito-transmitted Zika virus infection.
Assuntos
Aedes/imunologia , Mosquitos Vetores/imunologia , Proteínas e Peptídeos Salivares/imunologia , Infecção por Zika virus/imunologia , Zika virus/patogenicidade , Aedes/virologia , Animais , Bactérias , Modelos Animais de Doenças , Feminino , Inativação Gênica , Mordeduras e Picadas de Insetos/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mosquitos Vetores/virologia , Proteínas Recombinantes/imunologia , Saliva/imunologia , Glândulas Salivares , Proteínas e Peptídeos Salivares/genética , Pele/imunologia , Pele/patologia , Infecção por Zika virus/prevenção & controle , Infecção por Zika virus/transmissãoRESUMO
Few animal models of Zika virus (ZIKV) infection have incorporated arthropod-borne transmission. Here, we establish an Aedes aegypti mosquito model of ZIKV infection of mice, and demonstrate altered vector competency among three strains, (Orlando, ORL, Ho Chi Minh, HCM, and Patilas, PAT). All strains acquired ZIKV in their midguts after a blood meal from infected mice, but ZIKV transmission only occurred in mice fed upon by HCM, and to a lesser extent PAT, but not ORL, mosquitoes. This defect in transmission from ORL or PAT mosquitoes was overcome by intrathoracic injection of ZIKV into mosquito. Genetic analysis revealed significant diversity among these strains, suggesting a genetic basis for differences in ability for mosquito strains to transmit ZIKV. The intrathoracic injection mosquito-mouse transmission model is critical to understanding the influence of mosquitoes on ZIKV transmission, infectivity and pathogenesis in the vertebrate host, and represents a natural transmission route for testing vaccines and therapeutics.
Assuntos
Modelos Animais de Doenças , Vetores de Doenças , Mosquitos Vetores/virologia , Infecção por Zika virus/transmissão , Zika virus/fisiologia , Aedes/fisiologia , Aedes/virologia , Animais , Comportamento Alimentar , Humanos , Camundongos , Mosquitos Vetores/fisiologia , Receptores de Interferon/deficiência , Receptores de Interferon/genética , Saliva/virologia , Zika virus/classificação , Zika virus/genética , Zika virus/patogenicidade , Infecção por Zika virus/virologiaRESUMO
Circadian rhythms refer to biologic processes that oscillate with an approximate 24-h period. These rhythms direct nearly all aspects of animal behavior and physiology. The aim of our study was to determine if Toll-like receptor (TLR) expression and responsiveness exhibit time-of-day dependent differences. Therefore, we isolated an adherent splenocyte population, which consisted primarily of B cells, dendritic cells, and macrophages, over the course of a 24-h light-dark period and measured daily changes in Tlr1-8 mRNA levels and cytokine expression after cells were challenged at Zeitgeber time (ZT) 1 or ZT13 with a TLR ligand. In addition, we assessed TLR3 protein levels in adherent splenocytes over the 24-h light-dark period and challenged mice at ZT1 or ZT13 with poly(I:C), the TLR3 ligand. Our study revealed that in this adherent cell population, all Tlrs exhibited rhythmic expression except Tlr2 and Tlr5, and all TLRs, except TLR8, demonstrated daily variations in responsiveness after challenge with their respective ligand. We also revealed that TLR3 protein levels fluctuate over the daily light-dark cycle in adherent splenocytes and mice exhibit a time-of-day dependent immune response when challenged with poly(I:C). Finally, we demonstrated that mRNA levels of Tlr2 and Tlr6 display rhythmic expression in splenic macrophages. Taken together, these findings could have important implications for TLR-directed therapeutics.
RESUMO
Zika virus (ZIKV) infection during pregnancy is associated with adverse fetal outcomes, including microcephaly, growth restriction, and fetal demise. Type I interferons (IFNs) are essential for host resistance against ZIKV, and IFN-α/ß receptor (IFNAR)-deficient mice are highly susceptible to ZIKV infection. Severe fetal growth restriction with placental damage and fetal resorption is observed after ZIKV infection of type I IFN receptor knockout (Ifnar1-/-) dams mated with wild-type sires, resulting in fetuses with functional type I IFN signaling. The role of type I IFNs in limiting or mediating ZIKV disease within this congenital infection model remains unknown. In this study, we challenged Ifnar1-/- dams mated with Ifnar1+/- sires with ZIKV. This breeding scheme enabled us to examine pregnant dams that carry a mixture of fetuses that express (Ifnar1+/-) or do not express IFNAR (Ifnar1-/-) within the same uterus. Virus replicated to a higher titer in the placenta of Ifnar1-/- than within the Ifnar1+/- concepti. Yet, rather unexpectedly, we found that only Ifnar1+/- fetuses were resorbed after ZIKV infection during early pregnancy, whereas their Ifnar1-/- littermates continue to develop. Analyses of the fetus and placenta revealed that, after ZIKV infection, IFNAR signaling in the conceptus inhibits development of the placental labyrinth, resulting in abnormal architecture of the maternal-fetal barrier. Exposure of midgestation human chorionic villous explants to type I IFN, but not type III IFNs, altered placental morphology and induced cytoskeletal rearrangements within the villous core. Our results implicate type I IFNs as a possible mediator of pregnancy complications, including spontaneous abortions and growth restriction, in the context of congenital viral infections.
Assuntos
Morte Fetal/etiologia , Interferon Tipo I/imunologia , Complicações Infecciosas na Gravidez/imunologia , Complicações Infecciosas na Gravidez/virologia , Infecção por Zika virus/imunologia , Zika virus/imunologia , Animais , Modelos Animais de Doenças , Feminino , Retardo do Crescimento Fetal/imunologia , Retardo do Crescimento Fetal/virologia , Feto/imunologia , Feto/virologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Placenta/imunologia , Placenta/virologia , Gravidez , Receptor de Interferon alfa e beta/imunologia , Útero/imunologia , Útero/virologia , Infecção por Zika virus/virologiaRESUMO
Plasmodium infection begins with the bite of an anopheline mosquito, when sporozoites along with saliva are injected into a vertebrate host. The role of the host responses to mosquito saliva components in malaria remains unclear. We observed that antisera against Anopheles gambiae salivary glands partially protected mice from mosquito-borne Plasmodium infection. Specifically, antibodies to A. gambiae TRIO (AgTRIO), a mosquito salivary gland antigen, contributed to the protection. Mice administered AgTRIO antiserum showed lower Plasmodium liver burden and decreased parasitemia when exposed to infected mosquitoes. Active immunization with AgTRIO was also partially protective against Plasmodium berghei infection. A combination of AgTRIO antiserum and antibodies against Plasmodium circumsporozoite protein, a vaccine candidate, further decreased P. berghei infection. In humanized mice, AgTRIO antiserum afforded some protection against mosquito-transmitted Plasmodium falciparum. AgTRIO antiserum reduced the movement of sporozoites in the murine dermis. AgTRIO may serve as an arthropod-based target against Plasmodium to combat malaria.
Assuntos
Anopheles/imunologia , Imunização Passiva , Proteínas de Insetos/imunologia , Malária/prevenção & controle , Proteínas e Peptídeos Salivares/imunologia , Animais , Modelos Animais de Doenças , Proteínas de Insetos/administração & dosagem , Fígado/parasitologia , Fígado/patologia , Malária/parasitologia , Malária/patologia , Camundongos , Carga Parasitária , Parasitemia/parasitologia , Parasitemia/prevenção & controle , Plasmodium berghei/imunologia , Plasmodium falciparum , Proteínas e Peptídeos Salivares/administração & dosagem , Resultado do TratamentoRESUMO
Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that has recently been found to cause fetal infection and neonatal abnormalities, including microcephaly and neurological dysfunction. ZIKV persists in the semen months after the acute viremic phase in humans. To further understand the consequences of ZIKV persistence in males, we infected Ifnar1-/- mice via subcutaneous injection of a pathogenic but nonlethal ZIKV strain. ZIKV replication persists within the testes even after clearance from the blood, with interstitial, testosterone-producing Leydig cells supporting virus replication. We found high levels of viral RNA and antigen within the epididymal lumen, where sperm is stored, and within surrounding epithelial cells. Unexpectedly, at 21 days post-infection, the testes of the ZIKV-infected mice were significantly smaller compared to those of mock-infected mice, indicating progressive testicular atrophy. ZIKV infection caused a reduction in serum testosterone, suggesting that male fertility can be affected. Our findings have important implications for nonvector-borne vertical transmission, as well as long-term potential reproductive deficiencies, in ZIKV-infected males.
Assuntos
RNA Viral/biossíntese , Testículo , Testosterona/sangue , Replicação Viral/fisiologia , Infecção por Zika virus , Zika virus/fisiologia , Animais , Atrofia , Masculino , Camundongos , Camundongos Knockout , RNA Viral/genética , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/metabolismo , Testículo/metabolismo , Testículo/virologia , Infecção por Zika virus/sangue , Infecção por Zika virus/genética , Infecção por Zika virus/patologiaRESUMO
Tyro3, Axl, and Mertk (TAM) receptors are candidate entry receptors for infection with the Zika virus (ZIKV), an emerging flavivirus of global public health concern. To investigate the requirement of TAM receptors for ZIKV infection, we used several routes of viral inoculation and compared viral replication in wild-type versus Axl-/-, Mertk-/-, Axl-/-Mertk-/-, and Axl-/-Tyro3-/- mice in various organs. Pregnant and non-pregnant mice treated with interferon-α-receptor (IFNAR)-blocking (MAR1-5A3) antibody and infected subcutaneously with ZIKV showed no reliance on TAMs for infection. In the absence of IFNAR-blocking antibody, adult female mice challenged intravaginally with ZIKV showed no difference in mucosal viral titers. Similarly, in young mice that were infected with ZIKV intracranially or intraperitoneally, ZIKV replication occurred in the absence of TAM receptors, and no differences in cell tropism were observed. These findings indicate that, in mice, TAM receptors are not required for ZIKV entry and infection.
Assuntos
Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Infecção por Zika virus/metabolismo , Zika virus/fisiologia , Animais , Animais Recém-Nascidos , Feminino , Feto/virologia , Injeções Intraperitoneais , Camundongos , Placenta/virologia , Gravidez , Tropismo , Vagina/virologia , Replicação Viral , c-Mer Tirosina Quinase , Receptor Tirosina Quinase AxlRESUMO
Human metapneumovirus (HMPV) is a major cause of morbidity and mortality from acute lower respiratory tract illness, with most individuals seropositive by age five. Despite the presence of neutralizing antibodies, secondary infections are common and can be severe in young, elderly, and immunocompromised persons. Preclinical vaccine studies for HMPV have suggested a need for a balanced antibody and T cell immune response to enhance protection and avoid lung immunopathology. We infected transgenic mice expressing human HLA-A*0201 with HMPV and used ELISPOT to screen overlapping and predicted epitope peptides. We identified six novel HLA-A2 restricted CD8(+) T cell (TCD8) epitopes, with M39-47 (M39) immunodominant. Tetramer staining detected M39-specific TCD8 in lungs and spleen of HMPV-immune mice. Immunization with adjuvant-formulated M39 peptide reduced lung virus titers upon challenge. Finally, we show that TCD8 from HLA-A*0201 positive humans recognize M39 by IFNγ ELISPOT and tetramer staining. These results will facilitate HMPV vaccine development and human studies.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Antígeno HLA-A2/imunologia , Metapneumovirus/imunologia , Infecções por Paramyxoviridae/imunologia , Adulto , Animais , Humanos , Pulmão/imunologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Baço/imunologiaRESUMO
Type I interferon (IFN) is a key mediator of antiviral immunity. Human metapneumovirus (HMPV) inhibits IFN signaling, but does not encode homologues of known IFN antagonists. We tested the hypothesis that a specific viral protein prevents type I IFN signaling by targeting signal transducer and activator of transcription-1 (STAT1). We found that human airway epithelial cells (capable of expressing IFNs) became impaired for STAT1 phosphorylation even without direct infection due to intrinsic negative feedback. HMPV-infected Vero cells (incapable of expressing IFN) displayed lower STAT1 expression and impaired STAT1 phosphorylation in response to type I IFN treatment compared to mock-infected cells. Transient overexpression of HMPV small hydrophobic (SH) protein significantly inhibited STAT1 phosphorylation and signaling, and recombinant virus lacking SH protein was unable to inhibit STAT1 phosphorylation. Our results indicate a role for the SH protein of HMPV in the downregulation of type I IFN signaling through the targeting of STAT1.