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Bluetongue, caused by bluetongue virus (BTV), is a widespread arthropod-borne disease of ruminants that entails a recurrent threat to the primary sector of developed and developing countries. In this work, we report modified vaccinia virus Ankara (MVA) and ChAdOx1-vectored vaccines designed to simultaneously express the immunogenic NS1 protein and/or NS2-Nt, the N-terminal half of protein NS2 (NS21-180). A single dose of MVA or ChAdOx1 expressing NS1-NS2-Nt improved the protection conferred by NS1 alone in IFNAR(-/-) mice. Moreover, mice immunized with ChAdOx1/MVA-NS1, ChAdOx1/MVA-NS2-Nt, or ChAdOx1/MVA-NS1-NS2-Nt developed strong cytotoxic CD8+ T-cell responses against NS1, NS2-Nt, or both proteins and were fully protected against a lethal infection with BTV serotypes 1, 4, and 8. Furthermore, although a single immunization with ChAdOx1-NS1-NS2-Nt partially protected sheep against BTV-4, the administration of a booster dose of MVA-NS1-NS2-Nt promoted a faster viral clearance, reduction of the period and level of viremia and also protected from the pathology produced by BTV infection. IMPORTANCE Current BTV vaccines are effective but they do not allow to distinguish between vaccinated and infected animals (DIVA strategy) and are serotype specific. In this work we have develop a DIVA multiserotype vaccination strategy based on adenoviral (ChAdOx1) and MVA vaccine vectors, the most widely used in current phase I and II clinical trials, and the conserved nonstructural BTV proteins NS1 and NS2. This immunization strategy solves the major drawbacks of the current marketed vaccines.
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Vírus Bluetongue/imunologia , Bluetongue/prevenção & controle , Vetores Genéticos/genética , Vaccinia virus/genética , Proteínas não Estruturais Virais/genética , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vírus Bluetongue/classificação , Vetores Genéticos/imunologia , Imunidade Celular , Imunização , Imunogenicidade da Vacina , Sorogrupo , Ovinos , Vacinas de DNA/genética , Vacinas de DNA/imunologia , Vaccinia virus/imunologia , Proteínas não Estruturais Virais/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genéticaRESUMO
Borrelia burgdorferi causes Lyme disease, the most common tick-transmitted illness in North America. When Ixodes scapularis feed on an infected vertebrate host, spirochetes enter the tick gut along with the bloodmeal and colonize the vector. Here, we show that a secreted tick protein, I. scapularisprotein disulfide isomerase A3 (IsPDIA3), enhances B. burgdorferi colonization of the tick gut. I. scapularis ticks in which ispdiA3 has been knocked down using RNA interference have decreased spirochete colonization of the tick gut after engorging on B. burgdorferi-infected mice. Moreover, administration of IsPDIA3 antiserum to B. burgdorferi-infected mice reduced the ability of spirochetes to colonize the tick when feeding on these animals. We show that IsPDIA3 modulates inflammatory responses at the tick bite site, potentially facilitating spirochete survival at the vector-host interface as it exits the vertebrate host to enter the tick gut. These data provide functional insights into the complex interactions between B. burgdorferi and its arthropod vector and suggest additional targets to interfere with the spirochete life cycle.
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Borrelia burgdorferi/fisiologia , Ixodes/metabolismo , Doença de Lyme/transmissão , Isomerases de Dissulfetos de Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Vetores Aracnídeos/microbiologia , Linhagem Celular , Técnicas de Silenciamento de Genes , Humanos , Imunidade Humoral , Inflamação/enzimologia , Inflamação/genética , Inflamação/metabolismo , Ixodes/enzimologia , Ixodes/genética , Proteínas de Membrana/metabolismo , Camundongos , Filogenia , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/imunologia , Interferência de RNA , Proteínas Recombinantes , Alinhamento de Sequência , Spirochaetales/fisiologiaRESUMO
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.
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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
The development of vaccines against bluetongue, a prevalent livestock disease, has been focused on surface antigens that induce strong neutralizing antibody responses. Because of their antigenic variability, these vaccines are usually serotype restricted. We now show that a single highly conserved nonstructural protein, NS1, expressed in a modified vaccinia Ankara virus (MVA) vector can provide multiserotype protection in IFNAR-/- 129 mice against bluetongue virus (BTV) that is largely dependent on CD8 T cell responses. We found that the protective antigenic capacity of NS1 resides within the N terminus of the protein and is provided in the absence of neutralizing antibodies. The protective CD8 T cell response requires the presence of a specific peptide within the N terminus of NS1, since its deletion ablates the efficacy of the vaccine formulation. These data reveal the importance of the nonstructural protein NS1 in CD8 T cell-mediated protection against multiple BTV serotypes when vectorized as a recombinant MVA vaccine.IMPORTANCE Conventional vaccines have controlled or limited BTV expansion in the past, but they cannot address the need for cross-protection among serotypes and do not allow distinguishing between infected and vaccinated animals (DIVA strategy). There is a need to develop universal vaccines that induce effective protection against multiple BTV serotypes. In this work we have shown the importance of the nonstructural protein NS1, conserved among all the BTV serotypes, in CD8 T cell-mediated protection against multiple BTV serotypes when vectorized as a recombinant MVA vaccine.
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Vírus Bluetongue/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitopos Imunodominantes/imunologia , Receptor de Interferon alfa e beta/deficiência , Proteínas não Estruturais Virais/imunologia , Vacinas Virais/imunologia , Animais , Bluetongue/prevenção & controle , Células Cultivadas , Modelos Animais de Doenças , Portadores de Fármacos , ELISPOT , Vetores Genéticos , Interferon gama/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Knockout , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vaccinia virus/genética , Vacinas Virais/administração & dosagem , Vacinas Virais/genéticaRESUMO
The Aedes aegypti midgut is crucial for blood digestion, nutrition, reproduction, and pathogen interaction. Using single-cell RNA sequencing, we explored virus infection and transcriptomic changes at the cellular level. We identified 12 distinct cell clusters in the Ae. aegypti midgut post-Zika virus infection, including intestinal stem cells, enteroblasts, enteroendocrine cells (EE), and enterocytes (ECs). The virus was found mainly in specific subsets of ECs and EE. Infection altered transcriptional profiles related to metabolism, signaling, and immune responses. Functional studies highlighted three significantly differentially expressed genes in infected cells. Notably, silencing apolipophorin III reduced virus RNA copy number in the midgut, emphasizing the role of specific genes in viral infection. These findings enhance our understanding of mosquito midgut cell processes during Zika virus infection and suggest potential targets for vector control.
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Introduction: Bluetongue (BT), caused by bluetongue virus (BTV), is an important arthropod-borne livestock disease listed by the World Organization for Animal Health. Live-attenuated and inactivated vaccines have permitted to control BT but they do not simultaneously protect against the myriad of BTV serotypes. Recently, we identified the highly conserved BTV nonstructural protein NS1 and the N-terminal region of NS2 as antigens capable of conferring multiserotype protection against BTV. Methods: Here, we designed Modified Vaccinia Ankara (MVA) viral vectors that expressed BTV-4 proteins VP2 or VP7 along with NS1 and NS2-Nt as well as MVAs that expressed proteins VP2, VP7 or NS1 and NS2-Nt. Results: Immunization of IFNAR(-/-) mice with two doses of MVA-NS1-2A-NS2-Nt protected mice from BTV-4M infection by the induction of an antigen-specific T cell immune response. Despite rMVA expressing VP7 alone were not protective in the IFNAR(-/-) mouse model, inclusion of VP7 in the vaccine formulation amplified the cell-mediated response induced by NS1 and NS2-Nt. Expression of VP2 elicited protective non-cross-reactive neutralizing antibodies (nAbs) in immunized animals and improved the protection observed in the MVA-NS1-2A-NS2-Nt immunized mice when these three BTV antigens were co-expressed. Moreover, vaccines candidates co-expressing VP2 or VP7 along with NS1 and NS2-Nt provided multiserotype protection. We assessed protective efficacy of both vaccine candidates in sheep against virulent challenge with BTV-4M. Discussion: Immunization with MVA-VP7-NS1-2A-NS2-Nt partially dumped viral replication and clinical disease whereas administration of MVA-VP2-NS1-2A-NS2-Nt promoted a complete protection, preventing viraemia and the pathology produced by BTV infection.
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Vírus Bluetongue , Bluetongue , Proteínas do Capsídeo , Vetores Genéticos , Receptor de Interferon alfa e beta , Vaccinia virus , Proteínas não Estruturais Virais , Vacinas Virais , Animais , Vírus Bluetongue/imunologia , Vírus Bluetongue/genética , Proteínas não Estruturais Virais/imunologia , Proteínas não Estruturais Virais/genética , Bluetongue/prevenção & controle , Bluetongue/imunologia , Bluetongue/virologia , Camundongos , Vacinas Virais/imunologia , Vacinas Virais/genética , Vaccinia virus/genética , Vaccinia virus/imunologia , Receptor de Interferon alfa e beta/genética , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/genética , Camundongos Knockout , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/sangue , FemininoRESUMO
Bluetongue virus (BTV) is the causative agent of the important livestock disease bluetongue (BT), which is transmitted via Culicoides bites. BT causes severe economic losses associated with its considerable impact on health and trade of animals. By reverse genetics, we have designed and rescued reporter-expressing recombinant (r)BTV expressing NanoLuc luciferase (NLuc) or Venus fluorescent protein. To generate these viruses, we custom synthesized a modified viral segment 5 encoding NS1 protein with the reporter genes located downstream and linked by the Porcine teschovirus-1 (PTV-1) 2A autoproteolytic cleavage site. Therefore, fluorescent signal or luciferase activity is only detected after virus replication and expression of non-structural proteins. Fluorescence or luminescence signals were detected in cells infected with rBTV/Venus or rBTV/NLuc, respectively. Moreover, the marking of NS2 protein confirmed that reporter genes were only expressed in BTV-infected cells. Growth kinetics of rBTV/NLuc and rBTV/Venus in Vero cells showed replication rates similar to those of wild-type and rBTV. Infectivity studies of these recombinant viruses in IFNAR(-/-) mice showed a higher lethal dose for rBTV/NLuc and rBTV/Venus than for rBTV indicating that viruses expressing the reporter genes are attenuated in vivo. Interestingly, luciferase activity was detected in the plasma of viraemic mice infected with rBTV/NLuc. Furthermore, luciferase activity quantitatively correlated with RNAemia levels of infected mice throughout the infection. In addition, we have investigated the in vivo replication and dissemination of BTV in IFNAR (-/-) mice using BTV/NLuc and non-invasive in vivo imaging systems.IMPORTANCEThe use of replication-competent viruses that encode a traceable fluorescent or luciferase reporter protein has significantly contributed to the in vitro and in vivo study of viral infections and the development of novel therapeutic approaches. In this work, we have generated rBTV that express fluorescent or luminescence proteins to track BTV infection both in vitro and in vivo. Despite the availability of vaccines, BTV and other related orbivirus are still associated with a significant impact on animal health and have important economic consequences worldwide. Our studies may contribute to the advance in orbivirus research and pave the way for the rapid development of new treatments, including vaccines.
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Vírus Bluetongue , Vacinas , Chlorocebus aethiops , Animais , Camundongos , Vírus Bluetongue/genética , Genes Reporter , Células Vero , Proteínas Virais/genética , Luciferases/genéticaRESUMO
The Aedes aegypti mosquito is a vector of many infectious agents, including flaviviruses such as Zika virus. Components of mosquito saliva have pleomorphic effects on the vertebrate host to enhance blood feeding, and these changes also create a favorable niche for pathogen replication and dissemination. Here, we demonstrate that human CD47, which is known to be involved in various immune processes, interacts with a 34-kilodalton mosquito salivary protein named Nest1. Nest1 is up-regulated in blood-fed female A. aegypti and facilitates Zika virus dissemination in human skin explants. Nest1 has a stronger affinity for CD47 than its natural ligand, signal regulatory protein α, competing for binding at the same interface. The interaction between Nest1 with CD47 suppresses phagocytosis by human macrophages and inhibits proinflammatory responses by white blood cells, thereby suppressing antiviral responses in the skin. This interaction elucidates how an arthropod protein alters the human response to promote arbovirus infectivity.
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Aedes , Pele , Zika virus , Aedes/imunologia , Aedes/virologia , Animais , Humanos , Pele/imunologia , Pele/virologia , Zika virus/imunologia , Zika virus/fisiologia , Feminino , Proteínas de Insetos/imunologia , Infecção por Zika virus/imunologia , Proteínas e Peptídeos Salivares/imunologia , Mosquitos Vetores/imunologia , Mosquitos Vetores/virologia , Antígeno CD47RESUMO
Arthropod-borne viruses present important public health challenges worldwide. Viruses such as DENV, ZIKV, and WNV are of current concern due to an increasing incidence and an expanding geographic range, generating explosive outbreaks even in non-endemic areas. The clinical signs associated with infection from these arboviruses are often inapparent, mild, or nonspecific, but occasionally develop into serious complications marked by rapid onset, tremors, paralysis, hemorrhagic fever, neurological alterations, or death. They are predominately transmitted to humans through mosquito bite, during which saliva is inoculated into the skin to facilitate blood feeding. A new approach to prevent arboviral diseases has been proposed by the observation that arthropod saliva facilitates transmission of pathogens. Viruses released within mosquito saliva may more easily initiate host invasion by taking advantage of the host's innate and adaptive immune responses to saliva. This provides a rationale for creating vaccines against mosquito salivary proteins, especially because of the lack of licensed vaccines against most of these viruses. This review aims to provide an overview of the effects on the host immune response by the mosquito salivary proteins and how these phenomena alter the infection outcome for different arboviruses, recent attempts to generate mosquito salivary-based vaccines against flavivirus including DENV, ZIKV, and WNV, and the potential benefits and pitfalls that this strategy involves.
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The COVID-19 pandemic has underscored the importance of swift responses and the necessity of dependable technologies for vaccine development. Our team previously developed a fast cloning system for the modified vaccinia virus Ankara (MVA) vaccine platform. In this study, we reported on the construction and preclinical testing of a recombinant MVA vaccine obtained using this system. We obtained recombinant MVA expressing the unmodified full-length SARS-CoV-2 spike (S) protein containing the D614G amino-acid substitution (MVA-Sdg) and a version expressing a modified S protein containing amino-acid substitutions designed to stabilize the protein a in a pre-fusion conformation (MVA-Spf). S protein expressed by MVA-Sdg was found to be expressed and was correctly processed and transported to the cell surface, where it efficiently produced cell-cell fusion. Version Spf, however, was not proteolytically processed, and despite being transported to the plasma membrane, it failed to induce cell-cell fusion. We assessed both vaccine candidates in prime-boost regimens in the susceptible transgenic K18-human angiotensin-converting enzyme 2 (K18-hACE2) in mice and in golden Syrian hamsters. Robust immunity and protection from disease was induced with either vaccine in both animal models. Remarkably, the MVA-Spf vaccine candidate produced higher levels of antibodies, a stronger T cell response, and a higher degree of protection from challenge. In addition, the level of SARS-CoV-2 in the brain of MVA-Spf inoculated mice was decreased to undetectable levels. Those results add to our current experience and range of vaccine vectors and technologies for developing a safe and effective COVID-19 vaccine.
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Zika virus (ZIKV), an emerging arthropod-borne flavivirus, was first isolated in Uganda in 1947 from monkeys and first detected in humans in Nigeria in 1952; it has been associated with a dramatic burden worldwide. Since then, interventions to reduce the burden of ZIKV infection have been mainly restricted to mosquito control, which in the end proved to be insufficient by itself. Hence, the situation prompted scientists to increase research on antivirals and vaccines against the virus. These efforts are still ongoing as the pathogenesis and immune evasion mechanisms of ZIKV have not yet been fully elucidated. Understanding the viral disease mechanism will provide a better landscape to develop prophylactic and therapeutic strategies against ZIKV. Currently, no specific vaccines or drugs have been approved for ZIKV. However, some are undergoing clinical trials. Notably, different platforms have been evaluated for the design of vaccines, including DNA, mRNA, viral vectors, virus-like particles (VLPs), inactivated virus, live attenuated virus, peptide and protein-based vaccines, passive immunizations by using monoclonal antibodies (MAbs), and vaccines that target vector-derived antigens. These vaccines have been shown to induce specific humoral and cellular immune responses and reduce viremia and viral RNA titers, both in vitro and in vivo. This review provides a comprehensive summary of current advancements in the development of vaccines against Zika virus.
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Modified vaccinia virus Ankara (MVA) is employed widely as an experimental vaccine vector for its abortive replication in mammalian cells and high expression level of foreign/heterologous genes. Recombinant MVAs (rMVAs) are used as platforms for protein production as well as vectors to generate vaccines against a wide range of infectious diseases and other pathologies. The portrait of the virus combines desirable elements such as high-level biological safety, the ability to activate appropriate innate immune mediators upon vaccination , and the capacity to deliver substantial amounts of heterologous antigens. rMVAs encoding proteins of Bluetongue virus (BTV), an orbivirus that infects domestic and wild ruminants through transmission by biting midges of the Culicoides species, are excellent vaccine candidates against this virus. In this chapter, we describe the methods for the generation of rMVAs encoding VP2, NS1, and VP7 proteins of BTV . The included protocols cover the cloning of VP2, NS1, and VP7 BTV-4 genes in a transfer plasmid, the construction of rMVAs, the titration of virus working stocks, and the protein expression analysis by immunofluorescence and radiolabeling of rMVA infected cells as well as virus purification procedure.
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Vírus Bluetongue , Bluetongue , Vacinas Virais , Animais , Bluetongue/prevenção & controle , Vírus Bluetongue/genética , Proteínas do Capsídeo/genética , Mamíferos , Ovinos , Vaccinia virus/genéticaRESUMO
Bluetongue virus (BTV) and African horse sickness virus (AHSV) are widespread arboviruses that cause important economic losses in the livestock and equine industries, respectively. In addition to these, another arthropod-transmitted orbivirus known as epizootic hemorrhagic disease virus (EHDV) entails a major threat as there is a conducive landscape that nurtures its emergence in non-endemic countries. To date, only vaccinations with live attenuated or inactivated vaccines permit the control of these three viral diseases, although important drawbacks, e.g., low safety profile and effectiveness, and lack of DIVA (differentiation of infected from vaccinated animals) properties, constrain their usage as prophylactic measures. Moreover, a substantial number of serotypes of BTV, AHSV and EHDV have been described, with poor induction of cross-protective immune responses among serotypes. In the context of next-generation vaccine development, antigen delivery systems based on nano- or microparticles have gathered significant attention during the last few decades. A diversity of technologies, such as virus-like particles or self-assembled protein complexes, have been implemented for vaccine design against these viruses. In this work, we offer a comprehensive review of the nano- and microparticulated vaccine candidates against these three relevant orbiviruses. Additionally, we also review an innovative technology for antigen delivery based on the avian reovirus nonstructural protein muNS and we explore the prospective functionality of the nonstructural protein NS1 nanotubules as a BTV-based delivery platform.
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Dengue fever, caused by the dengue virus (DENV), is currently a threat to about half of the world's population. DENV is mainly transmitted to the vertebrate host through the bite of a female Aedes mosquito while taking a blood meal. During this process, salivary proteins are introduced into the host skin and blood to facilitate blood acquisition. These salivary proteins modulate both local (skin) and systemic immune responses. Several salivary proteins have been identified as immunogenic inducing the production of antibodies with some of those proteins also displaying immunomodulatory properties enhancing arboviral infections. IgG antibody responses against salivary gland extracts of a diverse number of mosquitoes, as well as antibody responses against the Ae. aegypti peptide, Nterm-34 kDa, have been suggested as biomarkers of human exposure to mosquito bites while antibodies against AgBR1 and NeSt1 proteins have been investigated for their potential protective effect against Zika virus (ZIKV) and West Nile virus infections. Thus, we were interested in evaluating whether IgG antibodies against AgBR1, NeSt1, Nterm-34 kDa peptide, and SGE were associated with DENV infections and clinical characteristics. For this, we tested samples from volunteers living in a dengue fever endemic area in Colombia in 2019 for the presence of IgG antibodies against those salivary proteins and peptides using an ELISA test. Results from this pilot study suggest an involvement of antibody responses against salivary proteins in dengue disease progression.
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Zika virus(ZIKV) is primarily spread by Aedes. aegyptimosquitoes. Infection with ZIKV can result in diverse clinical symptoms in humans, ranging from mild to severe. Previously, we demonstrated that passive immunization against A. aegypti AgBR1 or NeSt1 antiserum, two mosquito saliva proteins that are transmitted with the virus, conferred partial protection against ZIKV in mice. Each individual antiserum altered the early host response in the skin and reduced viremia. Here, we show that passive immunization with a combination of AgBR1- and NeSt1-specific antibodies enhanced survival and reduced the viral burden in blood, thereby protecting mice from mosquito-borne ZIKV infection. This finding suggests that targeting a combination of mosquito saliva proteins, with AgBR1 and NeSt1 as model antigens, may be used as a vaccine strategy to help prevent mosquito-borne ZIKV infection.
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Aedes , Infecção por Zika virus , Zika virus , Animais , Soros Imunes , Camundongos , Mosquitos Vetores , Saliva , Infecção por Zika virus/prevenção & controleRESUMO
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.
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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
Adiponectin-mediated pathways contribute to mammalian homeostasis; however, little is known about adiponectin and adiponectin receptor signaling in arthropods. In this study, we demonstrate that Ixodes scapularis ticks have an adiponectin receptor-like protein (ISARL) but lack adiponectin, suggesting activation by alternative pathways. ISARL expression is significantly upregulated in the tick gut after Borrelia burgdorferi infection, suggesting that ISARL signaling may be co-opted by the Lyme disease agent. Consistent with this, RNA interference (RNAi)-mediated silencing of ISARL significantly reduced the B. burgdorferi burden in the tick. RNA-seq-based transcriptomics and RNAi assays demonstrate that ISARL-mediated phospholipid metabolism by phosphatidylserine synthase I is associated with B. burgdorferi survival. Furthermore, the tick complement C1q-like protein 3 interacts with ISARL, and B. burgdorferi facilitates this process. This study identifies a new tick metabolic pathway that is connected to the life cycle of the Lyme disease spirochete.
Many countries around the world are seeing an increase in the number of patients diagnosed with Lyme disease, with often serious joint, heart, and neurologic complications. This illness is caused by species of 'spirochete' bacteria that live and multiply inside black-legged ticks, and get injected into mammals upon a bite. Ticks are not simply 'syringes' however, and a complex relationship is established between spirochetes and their host. This is particularly true since Lyme disease-causing bacteria such as Borrelia burgdorferi rely on ticks to obtain energy and nutrients. Tang, Cao et al. delved into these complex interactions by focusing on the molecular cascades (or pathways) involving adiponectin, a hormone essential for regulating sugar levels and processing fats. Analyses of gene and protein databases highlighted that ticks carry a receptor-like protein for adiponectin but not the hormone itself, suggesting that an alternative pathway is at play. This may involve B. burgdorferi, which gets its fats and sugars from its host. And indeed, experiments showed that ticks produced more of the adiponectin receptor-like protein when they carried B. burgdorferi; conversely, silencing the receptor reduced the number of surviving spirochetes inside the tick. Further exploration showed that the receptor mediates molecular cascades that help to process fat molecules; these are associated with spirochete survival. In addition, the receptor-like protein was activated by C1QL3, a 'complement 1q domain-contained' molecule which might be part of the tick energy-making or immune systems. Larger quantities of C1QL3 were found in ticks upon B. burgdorferi infection, suggesting that the spirochete facilitates an interaction that boosts activity of the adiponectin receptor-like protein. Overall, the work by Tang and Cao et al. revealed a new pathway which B. burgdorferi takes advantage of to infect their host and multiply. Targeting this molecular cascade could help to interfere with the life cycle of the spirochete, as well as fight Lyme disease and other insect-borne conditions.
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Borrelia burgdorferi/metabolismo , Ixodes/metabolismo , Ixodes/microbiologia , Receptores de Adiponectina/metabolismo , Animais , Proteínas de Artrópodes/metabolismo , Vetores Artrópodes/metabolismo , Vetores Artrópodes/microbiologia , Doença de Lyme/metabolismo , Doença de Lyme/microbiologia , Fosfolipídeos/metabolismo , Interferência de RNA , Receptores de Adiponectina/genética , TranscriptomaRESUMO
Zika Virus (ZIKV) is transmitted primarily by Aedes aegypti mosquitoes, resulting in asymptomatic infection, or acute illness with a fever and headache, or neurological complications, such as Guillain-Barre syndrome or fetal microcephaly. Previously, we determined that AgBR1, a mosquito salivary protein, induces inflammatory responses at the bite site, and that passive immunization with AgBR1 antiserum influences mosquito-transmitted ZIKV infection. Here, we show that the active immunization of mice with AgBR1 adjuvanted with aluminum hydroxide delays lethal mosquito-borne ZIKV infection, suggesting that AgBR1 may be used as part of a vaccine to combat ZIKV.
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African horse sickness (AHS) is a devastating disease caused by African horse sickness virus (AHSV) and transmitted by arthropods between its equine hosts. AHSV is endemic in sub-Saharan Africa, where polyvalent live attenuated vaccine is in use even though it is associated with safety risks. This review article summarizes and compares new strategies to generate safe and effective AHSV vaccines based on protein, virus like particles, viral vectors and reverse genetics technology. Manipulating the AHSV genome to generate synthetic viruses by means of reverse genetic systems has led to the generation of potential safe vaccine candidates that are under investigation.
Assuntos
Vírus da Doença Equina Africana/genética , Vírus da Doença Equina Africana/imunologia , Doença Equina Africana/prevenção & controle , Genética Reversa/métodos , Vacinas Virais/genética , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/imunologia , Cavalos , Camundongos , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologiaRESUMO
Bluetongue virus (BTV), the prototype member of the genus Orbivirus (family Reoviridae), is the causative agent of an important livestock disease, bluetongue (BT), which is transmitted via biting midges of the genus Culicoides. To date, up to 29 serotypes of BTV have been described, which are classified as classical (BTV 1-24) or atypical (serotypes 25-27), and its distribution has been expanding since 1998, with important outbreaks in the Mediterranean Basin and devastating incursions in Northern and Western Europe. Classical vaccine approaches, such as live-attenuated and inactivated vaccines, have been used as prophylactic measures to control BT through the years. However, these vaccine approaches fail to address important matters like vaccine safety profile, effectiveness, induction of a cross-protective immune response among serotypes, and implementation of a DIVA (differentiation of infected from vaccinated animals) strategy. In this context, a wide range of recombinant vaccine prototypes against BTV, ranging from subunit vaccines to recombinant viral vector vaccines, have been investigated. This article offers a comprehensive outline of the live viral vectors used against BTV.