RESUMO
BACKGROUND: Zika fever has been a global health security threat, especially in the tropical and subtropical regions where most of the cases occur. The disease is caused by Zika virus (ZIKV), which belongs to the family Flaviviridae, genus Flavivirus. The virus is transmitted by Aedes mosquitoes, mostly by Aedes aegypti, during its blood meal. In this study we present a descriptive analysis, by transmission electron microscopy (TEM), of ZIKV infection in A. aegypti elected tissues at the 3rd day of infection. ZIKV vertical transmission experiments by oral infection were conducted to explore an offspring of natural infection. RESULTS: Gut and ovary tissues harbored a higher number of viral particles. The ZIKV genome was also detected, by RT-qPCR technique, in the organism of orally infected female mosquitoes and in their eggs laid. CONCLUSIONS: The data obtained suggest that the ovary is an organ susceptible to be infected with ZIKV and that virus can be transmitted from mother to a fraction of the progeny.
Assuntos
Aedes/virologia , Mosquitos Vetores/virologia , Zika virus/fisiologia , Animais , Feminino , Intestinos/virologia , Microscopia Eletrônica de Transmissão , Ovário/virologia , Óvulo/virologia , RNA Viral/genética , Vírion/ultraestrutura , Zika virus/ultraestrutura , Infecção por Zika virus/transmissão , Infecção por Zika virus/virologiaRESUMO
During infection by a flavivirus (FV), cells accumulate noncoding subgenomic flavivirus RNAs (sfRNAs) that interfere with several antiviral pathways. These sfRNAs are formed by structured RNA elements in the 3' untranslated region (UTR) of the viral genomic RNA, which block the progression of host cell exoribonucleases that have targeted the viral RNA. Previous work on these exoribonuclease-resistant RNAs (xrRNAs) from mosquito-borne FVs revealed a specific three-dimensional fold with a unique topology in which a ring-like structure protectively encircles the 5' end of the xrRNA. Conserved nucleotides make specific tertiary interactions that support this fold. Examination of more divergent FVs reveals differences in their 3' UTR sequences, raising the question of whether they contain xrRNAs and if so, how they fold. To answer this, we demonstrated the presence of an authentic xrRNA in the 3' UTR of the Tamana bat virus (TABV) and solved its structure by X-ray crystallography. The structure reveals conserved features from previously characterized xrRNAs, but in the TABV version these features are created through a novel set of tertiary interactions not previously seen in xrRNAs. This includes two important A-C interactions, four distinct backbone kinks, several ordered Mg2+ ions, and a C+-G-C base triple. The discovery that the same overall architecture can be achieved by very different sequences and interactions in distantly related flaviviruses provides insight into the diversity of this type of RNA and will inform searches for undiscovered xrRNAs in viruses and beyond.
Assuntos
Flaviviridae/ultraestrutura , Interações Hospedeiro-Patógeno/genética , Dobramento de RNA , RNA não Traduzido/química , RNA Viral/química , Regiões 3' não Traduzidas , Animais , Pareamento de Bases , Sequência de Bases , Cátions Bivalentes , Cristalografia por Raios X , Vírus da Encefalite do Vale de Murray/genética , Vírus da Encefalite do Vale de Murray/metabolismo , Vírus da Encefalite do Vale de Murray/ultraestrutura , Exorribonucleases/química , Exorribonucleases/metabolismo , Flaviviridae/genética , Flaviviridae/metabolismo , Magnésio/química , Magnésio/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Vírus não Classificados/genética , Vírus não Classificados/metabolismo , Vírus não Classificados/ultraestrutura , Zika virus/genética , Zika virus/metabolismo , Zika virus/ultraestruturaRESUMO
The endoplasmic reticulum (ER) is a complex subcellular organelle composed of diverse structures such as tubules, sheets and tubular matrices. Flaviviruses such as Zika virus (ZIKV) induce reorganization of ER membranes to facilitate viral replication. Here, using 3D super resolution microscopy, ZIKV infection is shown to induce the formation of dense tubular matrices associated with viral replication in the central ER. Viral non-structural proteins NS4B and NS2B associate with replication complexes within the ZIKV-induced tubular matrix and exhibit distinct ER distributions outside this central ER region. Deep neural networks trained to distinguish ZIKV-infected versus mock-infected cells successfully identified ZIKV-induced central ER tubular matrices as a determinant of viral infection. Super resolution microscopy and deep learning are therefore able to identify and localize morphological features of the ER and allow for better understanding of how ER morphology changes due to viral infection.
Assuntos
Aprendizado Profundo , Retículo Endoplasmático/metabolismo , Microscopia/métodos , Zika virus/fisiologia , Encéfalo/patologia , Encéfalo/virologia , Linhagem Celular Tumoral , Retículo Endoplasmático/ultraestrutura , Matriz Extracelular/metabolismo , Humanos , Organoides/metabolismo , Organoides/ultraestrutura , Organoides/virologia , RNA de Cadeia Dupla/metabolismo , Proteínas não Estruturais Virais/metabolismo , Zika virus/ultraestrutura , Infecção por Zika virus/virologiaRESUMO
Zika virus (ZIKV), a mosquito-borne human flavivirus that causes microcephaly and other neurological disorders, has been a recent focus for the development of flavivirus vaccines and therapeutics. We report here a 4.0 Å resolution structure of the mature ZIKV in complex with ADI-30056, a ZIKV-specific human monoclonal antibody (hMAb) isolated from a ZIKV infected donor with a prior dengue virus infection. The structure shows that the hMAb interactions span across the E protein dimers on the virus surface, inhibiting conformational changes required for the formation of infectious fusogenic trimers similar to the hMAb, ZIKV-117. Structure-based functional analysis, and structure and sequence comparisons, identified ZIKV residues essential for neutralization and crucial for the evolution of highly potent E protein crosslinking Abs in ZIKV. Thus, this epitope, ZIKV's "Achilles heel", defined by the contacts between ZIKV and ADI-30056, could be a suitable target for the design of therapeutic antibodies.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Coinfecção , Reações Cruzadas/imunologia , Infecções por Flavivirus/imunologia , Flavivirus/imunologia , Infecção por Zika virus/imunologia , Zika virus/imunologia , Sequência de Aminoácidos , Animais , Chlorocebus aethiops , Dengue/imunologia , Vírus da Dengue/imunologia , Mapeamento de Epitopos , Epitopos/química , Epitopos/imunologia , Infecções por Flavivirus/virologia , Humanos , Imageamento Tridimensional , Modelos Moleculares , Testes de Neutralização , Conformação Proteica , Células Vero , Zika virus/ultraestrutura , Infecção por Zika virus/virologiaRESUMO
Zika virus (ZIKV) is an emerging mosquito borne flavivirus and a major public health concern causing severe disease. Due to the presence of a lipid membrane and structural heterogeneity, attaining an atomic resolution structure is challenging, but important to understand virus assembly and life cycle mechanisms that offer distinct targets for therapeutic intervention. We here use subvolume refinement to achieve a 3.4 Å resolution structure and identify two distinct lipid moieties. The first arises from the inner leaflet and is coordinated by hydrophobic residues of the M and E transmembrane helices that form a binding pocket not previously characterized. The second lipid arises from the outer leaflet coordinate between two E protein helices. Structure-based mutagenesis identifies critical hydrophobic interactions and their effect on the virus life cycle. Results show that lipids play an essential role in the ZIKV assembly pathway revealing a potential target of lipid based antiviral drug development.
Assuntos
Montagem de Vírus/fisiologia , Zika virus/fisiologia , Animais , Chlorocebus aethiops , Células HEK293 , Humanos , Células Vero , Vírion/ultraestrutura , Zika virus/isolamento & purificação , Zika virus/ultraestruturaRESUMO
Previous flavivirus (dengue and Zika viruses) studies showed largely spherical particles either with smooth or bumpy surfaces. Here, we demonstrate flavivirus particles have high structural plasticity by the induction of a non-spherical morphology at elevated temperatures: the club-shaped particle (clubSP), which contains a cylindrical tail and a disc-like head. Complex formation of DENV and ZIKV with Fab C10 stabilize the viruses allowing cryoEM structural determination to ~10 Å resolution. The caterpillar-shaped (catSP) Fab C10:ZIKV complex shows Fabs locking the E protein raft structure containing three E dimers. However, compared to the original spherical structure, the rafts have rotated relative to each other. The helical tail structure of Fab C10:DENV3 clubSP showed although the Fab locked an E protein dimer, the dimers have shifted laterally. Morphological diversity, including clubSP and the previously identified bumpy and smooth-surfaced spherical particles, may help flavivirus survival and immune evasion.
Assuntos
Anticorpos Antivirais/metabolismo , Vírus da Dengue/ultraestrutura , Proteínas do Envelope Viral/metabolismo , Zika virus/ultraestrutura , Aedes , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Antivirais/imunologia , Linhagem Celular , Microscopia Crioeletrônica , Dengue/imunologia , Dengue/terapia , Dengue/virologia , Vacinas contra Dengue/imunologia , Vírus da Dengue/imunologia , Vírus da Dengue/metabolismo , Evasão da Resposta Imune , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Mesocricetus , Multimerização Proteica , Propriedades de Superfície , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/ultraestrutura , Ligação Viral , Zika virus/imunologia , Zika virus/metabolismo , Infecção por Zika virusRESUMO
During flavivirus infection, some viral proteins move to the nucleus and cellular components are relocated from the nucleus to the cytoplasm. Thus, the integrity of the main regulator of the nuclear-cytoplasmic transport, the nuclear pore complex (NPC), was evaluated during infection with dengue virus (DENV) and Zika virus (ZIKV). We found that while during DENV infection the integrity and distribution of at least three nucleoporins (Nup), Nup153, Nup98, and Nup62 were altered, during ZIKV infection, the integrity of TPR, Nup153, and Nup98 were modified. In this work, several lines of evidence indicate that the viral serine protease NS2B3 is involved in Nups cleavage. First, the serine protease inhibitors, TLCK and Leupeptin, prevented Nup98 and Nup62 cleavage. Second, the transfection of DENV and ZIKV NS2B3 protease was sufficient to inhibit the nuclear ring recognition detected in mock-infected cells with the Mab414 antibody. Third, the mutant but not the active (WT) protease was unable to cleave Nups in transfected cells. Thus, here we describe for the first time that the NS3 protein from flavivirus plays novel functions hijacking the nuclear pore complex, the main controller of the nuclear-cytoplasmic transport.
Assuntos
Vírus da Dengue/metabolismo , Poro Nuclear/metabolismo , Serina Endopeptidases/metabolismo , Proteínas Virais/metabolismo , Zika virus/metabolismo , Transporte Ativo do Núcleo Celular , Dengue/metabolismo , Dengue/virologia , Vírus da Dengue/ultraestrutura , Immunoblotting , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Membrana Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Zika virus/ultraestrutura , Infecção por Zika virus/metabolismo , Infecção por Zika virus/virologiaRESUMO
The functioning of proteins requires highly specific dynamics, which depend critically on the details of how amino acids are packed. Hinge motions are the most common type of large motion, typified by the opening and closing of enzymes around their substrates. The packing and geometries of residues are characterized here by graph theory. This characterization is sufficient to enable reliable hinge predictions from a single static structure, and notably, this can be from either the open or the closed form of a structure. This new method to identify hinges within protein structures is called PACKMAN. The predicted hinges are validated by using permutation tests on B-factors. Hinge prediction results are compared against lists of manually curated hinge residues, and the results suggest that PACKMAN is robust enough to reproduce the known conformational changes and is able to predict hinge regions equally well from either the open or the closed forms of a protein. A group of 167 protein pairs with open and closed structures has been investigated Examples are shown for several additional proteins, including Zika virus nonstructured (NS) proteins where there are 6 hinge regions in the NS5 protein, 5 hinge regions in the NS2B bound in the NS3 protease complex and 5 hinges in the NS3- helicase protein. Results obtained from this method can be important for generating conformational ensembles of protein targets for drug design. PACKMAN is freely accessible at (https://PACKMAN.bb.iastate.edu/).
Assuntos
Enzimas/ultraestrutura , Conformação Proteica , Proteínas/ultraestrutura , Proteínas não Estruturais Virais/ultraestrutura , Algoritmos , Simulação por Computador , Enzimas/química , Simulação de Dinâmica Molecular , Proteínas/química , Proteínas não Estruturais Virais/química , Zika virus/química , Zika virus/ultraestruturaRESUMO
Zika virus (ZIKV) infection has caused global concern because of its association with fetal microcephaly and serious neurological complications in adults since 2016. Currently, no specific anti-ZIKV therapy is available to control ZIKV infection. During the last couple of years, the intensive investigation of ZIKV structure has provided significant information for structure-based vaccine and drug design. In this review, we summarized the research progress on the structures of ZIKV and its component proteins. We analyzed the structure identity and the differences between ZIKV and other flaviviruses. This information is crucial to guiding structure-based anti-ZIKV inhibitors and vaccine discovery.
Assuntos
Flavivirus/ultraestrutura , Proteínas Virais/química , Zika virus/ultraestrutura , Flavivirus/química , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Modelos Moleculares , Nucleocapsídeo/química , Nucleocapsídeo/efeitos dos fármacos , Conformação Proteica , Relação Estrutura-Atividade , Vacinas Virais/química , Vacinas Virais/farmacologia , Zika virus/química , Zika virus/efeitos dos fármacosRESUMO
BACKGROUND: During pregnancy, the Zika virus (ZIKV) replicates in the placenta and central nervous system (CNS) of infected fetuses; nevertheless, the ability of ZIKV to replicate in other fetal tissues has not been extensively characterized. METHODS: We researched whether dissemination of congenitally-acquired ZIKV outside the CNS exists by searching for the accumulation of the viral envelope protein, ZIKV ribonucleic acid (RNA), and infectious viral particles in different organs of a deceased newborn with Congenital Zika Syndrome. A real-time qualitative polymerase chain reaction (qPCR) was used to detect ZIKV RNA in the brain, thymus, lungs, kidneys, adrenal glands, spleen, liver, and small intestine. The same tissues were analyzed by indirect immunofluorescence and immunoperoxidase assays using the monoclonal antibody 4G2 to detect ZIKV envelope antigens. Isolation of infectious ZIKV in a cell culture was carried out using brain and kidney samples. RESULTS: A postmortem, virological analysis of multiple organs, such as the kidneys (epithelial cells in the renal tubules), lungs (bronchial epithelia), thymus (epithelial cells inside the Hassall's corpuscles), and brain (neurons, ependymal cells, and macrophages) revealed the presence of ZIKV RNA and envelope antigens. Other tissues of the deceased newborn tested positive by qPCR for Epstein-Barr virus and human herpesvirus 6, including the brain cortex (Epstein-Barr) and the thymus, kidneys, and adrenal glands (human herpesvirus 6). The kidneys were identified as a significant niche for viral replication, given that infectious particles were successfully isolated from renal tissues. CONCLUSIONS: Our findings demonstrate the ability of congenitally-acquired ZIKV to produce disseminated infections and the viral tropism towards epithelial cells.
Assuntos
Complicações Infecciosas na Gravidez/diagnóstico , Complicações Infecciosas na Gravidez/virologia , Infecção por Zika virus/diagnóstico , Infecção por Zika virus/virologia , Zika virus/genética , Antígenos Virais , Autopsia , Biópsia , Coinfecção , Feminino , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Transmissão Vertical de Doenças Infecciosas , Nefropatias/patologia , Nefropatias/virologia , México/epidemiologia , Gravidez , Complicações Infecciosas na Gravidez/epidemiologia , Vigilância em Saúde Pública , RNA Viral , Adulto Jovem , Zika virus/imunologia , Zika virus/ultraestrutura , Infecção por Zika virus/epidemiologia , Infecção por Zika virus/transmissãoRESUMO
We followed the presence of Zika virus (ZIKV) in four healthy adults (two men and two women), for periods ranging from 78 to 298 days post symptom onset. The patients were evaluated regarding the presence of the virus in different body fluids (blood, saliva, urine and semen), development of immune responses (including antibodies, cytokines and chemokines), and virus genetic variation within samples collected from semen and urine during the infection course. The analysis was focused primarily on the two male patients who shed the virus for up to 158 days after the initial symptoms. ZIKV particles were detected in the spermatozoa cytoplasm and flagella, in immature sperm cells and could also be isolated from semen in cell culture, confirming that the virus is able to preserve integrity and infectivity during replication in the male reproductive system (MRS). Despite the damage caused by ZIKV infection within the MRS, our data showed that ZIKV infection did not result in infertility at least in one of the male patients. This patient was able to conceive a child after the infection. We also detected alterations in the male genital cytokine milieu, which could play an important role in the replication and transmission of the virus which could considerably increase the risk of ZIKV sexual spread. In addition, full genome ZIKV sequences were obtained from several samples (mainly semen), which allowed us to monitor the evolution of the virus within a patient during the infection course. We observed genetic changes over time in consensus sequences and lower frequency intra-host single nucleotide variants (iSNV), that suggested independent compartmentalization of ZIKV populations in the reproductive and urinary systems. Altogether, the present observations confirm the risks associated with the long-term replication and shedding of ZIKV in the MRS and help to elucidate patterns of intra-host genetic evolution during long term replication of the virus.
Assuntos
Evolução Molecular , Interações Hospedeiro-Patógeno , Infecção por Zika virus/virologia , Zika virus/fisiologia , Brasil/epidemiologia , Citocinas/metabolismo , Feminino , Genitália Masculina/virologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Masculino , Sêmen/metabolismo , Sêmen/virologia , Zika virus/classificação , Zika virus/ultraestrutura , Infecção por Zika virus/epidemiologia , Infecção por Zika virus/imunologia , Infecção por Zika virus/transmissãoRESUMO
In response to the 2016 global public health emergency of international concern announced by the World Health Organization surrounding Zika virus (ZIKV) outbreaks, we developed a purified inactivated Zika virus vaccine (PIZV) candidate from ZIKV strain PRVABC59, isolated during the outbreak in 2015. The virus isolate was plaque purified, creating six sub-isolated virus stocks, two of which were selected to generate PIZV candidates for preclinical immunogenicity and efficacy evaluation in mice. The alum-adjuvanted PIZV candidates were highly immunogenic in both CD-1 and AG129 mice after a 2-dose immunization. Further, AG129 mice receiving 2 doses of PIZV formulated with alum were fully protected against lethal ZIKV challenge and mouse immune sera elicited by the PIZV candidates were capable of neutralizing ZIKVs of both African and Asian genetic lineages in vitro. Additionally, passive immunization of naïve mice with ZIKV-immune serum showed strong positive correlation between neutralizing ZIKV antibody (NAb) titers and protection against lethal challenge. This study supported advancement of the PIZV candidate toward clinical development.
Assuntos
Vacinas de Produtos Inativados/imunologia , Vacinas Virais/imunologia , Infecção por Zika virus/imunologia , Infecção por Zika virus/prevenção & controle , Zika virus/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linhagem Celular , Chlorocebus aethiops , Modelos Animais de Doenças , Imunização , Imunização Secundária , Imunogenicidade da Vacina/imunologia , Camundongos , Vacinas de Produtos Inativados/administração & dosagem , Células Vero , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Vacinas Virais/administração & dosagem , Zika virus/genética , Zika virus/ultraestrutura , Infecção por Zika virus/virologiaRESUMO
Flaviviruses assemble initially in an immature, noninfectious state and undergo extensive conformational rearrangements to generate mature virus. Previous cryo-electron microscopy (cryo-EM) structural studies of flaviviruses assumed icosahedral symmetry and showed the concentric organization of the external glycoprotein shell, the lipid membrane, and the internal nucleocapsid core. We show here that when icosahedral symmetry constraints were excluded in calculating the cryo-EM reconstruction of an immature flavivirus, the nucleocapsid core was positioned asymmetrically with respect to the glycoprotein shell. The core was positioned closer to the lipid membrane at the proximal pole, and at the distal pole, the outer glycoprotein spikes and inner membrane leaflet were either perturbed or missing. In contrast, in the asymmetric reconstruction of a mature flavivirus, the core was positioned concentric with the glycoprotein shell. The deviations from icosahedral symmetry demonstrated that the core and glycoproteins have varied interactions, which likely promotes viral assembly and budding.
Assuntos
Glicoproteínas/química , Nucleocapsídeo/ultraestrutura , Proteínas do Envelope Viral/química , Vírus do Nilo Ocidental/ultraestrutura , Zika virus/ultraestrutura , Animais , Chlorocebus aethiops , Microscopia Crioeletrônica , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Células Vero , Montagem de Vírus/fisiologia , Liberação de Vírus/fisiologiaRESUMO
Among the several arthropod-borne human flaviviral diseases, the recent outbreak of Zika virus (ZIKV) has caused devastating birth defects and neurological disorders, challenging the world with another major public health concern. We report here the refined structure of the mature ZIKV at a resolution of 3.1 Å as determined by cryo-electron microscopic single-particle reconstruction. The improvement in the resolution, compared with previous enveloped virus structures, was the result of optimized virus preparation methods and data processing techniques. The glycoprotein interactions and surface properties of ZIKV were compared with other mosquito-borne flavivirus structures. The largest structural differences and sequence variations occur at the glycosylation loop associated with receptor binding. Probable drug binding pockets were identified on the viral surface. These results also provide a structural basis for the design of vaccines against ZIKV.
Assuntos
Flavivirus/química , Zika virus/química , Zika virus/ultraestrutura , Microscopia Crioeletrônica , Desenho de Fármacos , Flavivirus/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Ligação Proteica , Imagem Individual de Molécula/métodos , Relação Estrutura-Atividade , Estruturas Virais/química , Vacinas Virais/química , Vacinas Virais/farmacologia , Zika virus/metabolismoRESUMO
Zika virus (ZIKV) is an emerging flavivirus that caused thousands of human infections in recent years. Compared to other human flaviviruses, ZIKV replication is not well understood. Using fluorescent, transmission electron, and focused ion beam-scanning electron microscopy, we examined ZIKV replication dynamics in Vero 76 cells and in the brains of infected laboratory mice. We observed the progressive development of a perinuclear flaviviral replication factory both in vitro and in vivo. In vitro, we illustrated the ZIKV lifecycle from particle cell entry to egress. ZIKV particles assembled and aggregated in an induced convoluted membrane structure and ZIKV strain-specific membranous vesicles. While most mature virus particles egressed via membrane budding, some particles also likely trafficked through late endosomes and egressed through membrane abscission. Interestingly, we consistently observed a novel sheet-like virus particle array consisting of a single layer of ZIKV particles. Our study further defines ZIKV replication and identifies a novel hallmark of ZIKV infection.
Assuntos
Membrana Celular/ultraestrutura , Vírion/ultraestrutura , Infecção por Zika virus/virologia , Zika virus/química , Zika virus/ultraestrutura , Animais , Encéfalo/citologia , Encéfalo/virologia , Membrana Celular/virologia , Chlorocebus aethiops , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Microscopia/instrumentação , Microscopia/métodos , RNA Viral/genética , RNA Viral/isolamento & purificação , Células Vero , Montagem de Vírus , Internalização do Vírus , Liberação de Vírus , Replicação Viral , Zika virus/fisiologia , Infecção por Zika virus/fisiopatologiaAssuntos
Proteínas do Capsídeo/ultraestrutura , Capsídeo/ultraestrutura , Genoma Viral , Infecção por Zika virus/virologia , Zika virus/genética , Zika virus/ultraestrutura , Capsídeo/química , Proteínas do Capsídeo/química , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação Proteica , RNA Viral/genética , Zika virus/químicaRESUMO
Zika virus (ZIKV) is a mosquito-borne flavivirus that has re-emerged and is associated with many debilitating clinical manifestations. Research is currently being conducted to develop a prophylactic vaccine against the virus; however, there has not been any licensed ZIKV vaccine. Recent studies have identified potential B-cell epitopes (amino acids 241-259, 294-315, 317-327, 346-361, 377-388 and 421-437) on the envelope protein of ZIKV, which could be explored to develop peptide vaccines against ZIKV infection. Nevertheless, the immunogenicity of these epitopes has never been assessed. Here, we displayed these epitopes on highly immunogenic bacteriophage virus-like particles (VLPs; MS2, PP7 and Qß) platforms and assessed their immunogenicity in mice. Mice immunized with a mixture of VLPs displaying ZIKV envelope B-cell epitopes elicited anti-ZIKV antibodies. Although, immunized mice were not protected against a high challenge dose of ZIKV, sera - albeit at low titers - from immunized mice neutralized (in vitro) a low dose of ZIKV. Taken together, these results show that these epitopes are B-cell epitopes and they are immunogenic when displayed on a Qß VLP platform. Furthermore, the results also show that immunization with VLPs displaying a single B-cell epitope minimally reduces ZIKV infection whereas immunization with a mixture of VLPs displaying a combination of the B-cell epitopes neutralizes ZIKV infection. Thus, immunization with a mixture of VLPs displaying multiple ZIKV B-cell epitopes is a good strategy to enhance ZIKV neutralization.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Bacteriófagos/imunologia , Técnicas de Visualização da Superfície Celular , Epitopos de Linfócito B/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Infecção por Zika virus/imunologia , Zika virus/imunologia , Animais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Epitopos de Linfócito B/genética , Haplorrinos , Imunização , Imunogenicidade da Vacina , Camundongos , Testes de Neutralização , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/ultraestrutura , Zika virus/ultraestrutura , Infecção por Zika virus/prevenção & controleRESUMO
The growing number of zika virus (ZIKV) infections plus a 20-fold increase in neonatal microcephaly in newborns in Brazil have raised alarms in many countries regarding the threat to pregnant women. Instances of microcephaly and central nervous system malformations continue to increase in ZIKV outbreak regions. ZIKV is a small enveloped positive-strand RNA virus belonging to the Flavivirus genus of the Flaviviridae family. High-resolution ZIKV structures recently identified by cryo-electron microscopy indicate that the overall ZIKV structure is similar to those of other flaviviruses. With its compact surface, ZIKV is more thermally stable than the dengue virus (DENV). ZIKV E proteins have a characteristic "herringbone" structure with a single glycosylation site. The ZIKV E protein, the major protein involved in receptor binding and fusion, is formed as a head-to-tail dimer on the surfaces of viral particles. The E monomer consists of three distinct domains: DI, DII, and DIII. The finger-like DII contains a fusion loop (FL) that is inserted into the host cell endosomal membrane during pH-dependent conformational changes that drive fusion. Quaternary E:E dimer epitopes located at the interaction site of prM and E dimers can be further divided into two dimer epitopes. To date, more than 50 ZIKV vaccine candidates are now in various stages of research and development. Candidate ZIKV vaccines that are currently in phase I/II clinical trials include inactivated whole viruses, recombinant measles viral vector-based vaccines, DNA and mRNA vaccines, and a mosquito salivary peptide vaccine. Stabilized forms of ZIKV E:E dimer proteins have been successfully obtained either by introducing additional inter-subunit disulfide bond(s) in DII or via the direct assembly of E:E dimer proteins by immobilization with monomeric E proteins. The VLP-based approach is another alternative method for presenting native E:E dimer antigens among the vaccine components. Several forms of ZIKV VLPs have been reported featuring the co-expression of the prM-E, prM-E-NS1, C-prM-E, and NS2B/NS3 viral genes in human cells. To minimize the effect of the cross-reactive ADE-facilitating antibodies between ZIKV and DENV, several novel mutations have been reported either in or near the FL of DII or DIII to dampen the production of cross-reactive antibodies. Future ZIKV vaccine design efforts should be focused on eliciting improved neutralizing antibodies with a reduced level of cross-reactivity to confer sterilizing immunity.
Assuntos
Vacinas Virais , Infecção por Zika virus , Zika virus , Anticorpos Antivirais/imunologia , Humanos , Modelos Moleculares , Vacinas de DNA , Proteínas Virais/genética , Proteínas Virais/imunologia , Zika virus/química , Zika virus/imunologia , Zika virus/ultraestrutura , Infecção por Zika virus/prevenção & controle , Infecção por Zika virus/virologiaRESUMO
Zika virus (ZIKV) infection has been documented within Central and South America, Asia, and Africa. Here we report the isolation of virus from a patient infected with ZIKV returning to Japan from the Dominican Republic. The ZIKV strain was imaged by electron microscopy and its complete genome sequence was analyzed. Phylogenetic analysis and molecular characterization revealed that the strain was of Asian lineage, and carried 2 unique mutations in its NS5 region. These mutations are characteristic of strains that originated in the Dominican Republic and the USA in 2016.
Assuntos
Genoma Viral/genética , Infecção por Zika virus/epidemiologia , Zika virus/genética , Adulto , República Dominicana/epidemiologia , Feminino , Humanos , Japão/epidemiologia , Microscopia Eletrônica , Mutação/genética , Filogenia , Viagem , Zika virus/isolamento & purificação , Zika virus/ultraestrutura , Infecção por Zika virus/virologiaRESUMO
The emergence of Zika virus (ZIKV) as a major public health threat has focused research on understanding virus biology and developing a suite of strategies for disease intervention. Recent advances in cryoelectron microscopy have accelerated structure-function studies of flaviviruses and of ZIKV in particular. Structures of the mature and immature ZIKV have demonstrated its similarity with other known flaviviruses such as dengue and West Nile viruses. However, ZIKV's unique pathobiology demands an explanation of how its structure, although similar to its flavivirus relatives, is sufficiently unique to address questions of receptor specificity, transmission, and antigenicity. Progress in defining the immunodominant epitopes and how neutralizing antibodies bind to them will provide great insight as vaccines progress through clinical trials. Identification of host receptors will substantially illuminate the interesting ZIKV tropism and provide insights into pathogenesis. Although the answers to all of these questions are not yet available, rapid progress in combining structural biology with other techniques is revealing the similarities and the differences in virion structure and function between ZIKV and related flaviviruses.