RESUMO
Mosquito-borne dengue disease is caused by the dengue virus serotype-1 to serotype-4. The contemporary dengue outbreaks in the southwestern Indian ocean coincided with the widespread of dengue virus serotype 2 genotype II (Cosmopolitan), including epidemic viral strains DES-14 and RUN-18 isolated in Dar es Salaam (Tanzania) in 2014 and La Reunion Island (France) in 2018, respectively. Heterodimeric interaction between prM (intracellular precursor of surface structural M protein) and envelope E proteins is required during the initial stage of dengue virus assembly. Amino acid 127 of DES-14 prM protein (equivalent to M36) has been identified as an infrequent valine whereas RUN-18 has a common isoleucine. In the present study, we examined the effect of M-I36V mutation on the expression of a recombinant RUN-18 E protein co-expressed with prM in human epithelial A549 cells. The M ectodomain of dengue virus serotype 2 embeds a pro-apoptotic peptide referred as D2AMP. The impact of M-I36V mutation on the death-promoting capability of D2AMP was assessed in A549 cells. We showed that valine at position M36 affects expression of recombinant RUN-18 E protein and potentiates apoptosis-inducing activity of D2AMP. We propose that the nature of M residue 36 influences the virological characteristics of dengue 2 M and E proteins belonging to genotype II that contributes to global dengue burden.
Assuntos
Vírus da Dengue , Dengue , Animais , Humanos , Vírus da Dengue/genética , Sorogrupo , Tanzânia/epidemiologia , GenótipoRESUMO
Tumor cells exhibit two different modes of individual cell movement. Mesenchymal-type movement is characterized by an elongated cellular morphology and requires extracellular proteolysis. In amoeboid movement, cells have a rounded morphology, are less dependent on proteases, and require high Rho-kinase signaling to drive elevated levels of actomyosin contractility. These two modes of cell movement are interconvertible. We show that mesenchymal-type movement in melanoma cells is driven by activation of the GTPase Rac through a complex containing NEDD9, a recently identified melanoma metastasis gene, and DOCK3, a Rac guanine nucleotide exchange factor. Rac signals through WAVE2 to direct mesenchymal movement and suppress amoeboid movement through decreasing actomyosin contractility. Conversely, in amoeboid movement, Rho-kinase signaling activates a Rac GAP, ARHGAP22, that suppresses mesenchymal movement by inactivating Rac. We demonstrate tight interplay between Rho and Rac in determining different modes of tumor cell movement, revealing how tumor cells switch between different modes of movement.
Assuntos
Movimento Celular , Melanoma/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Actomiosina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular Tumoral , Quimerina 1/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Proteínas do Tecido Nervoso/metabolismo , Fosfoproteínas/metabolismo , Transdução de Sinais , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
Mosquito-borne Zika virus (ZIKV) became a real threat to human health due to the lack of vaccine and effective antiviral treatment. The virus has recently been responsible for a global outbreak leading to millions of infected cases. ZIKV complications were highlighted in adults with Guillain-Barré syndrome and in newborns with increasing numbers of congenital disorders ranging from mild developmental delays to fatal conditions. The ability of ZIKV to establish a long-term infection in diverse organs including the kidneys has been recently documented but the consequences of such a viral infection are still debated. Our study aimed to determine whether the efficiency of ZIKV growth in kidney cells relates to glucose concentration. Human kidney HK-2 cells were infected with different ZIKV strains in presence of normal and high glucose concentrations. Virological assays showed a decrease in viral replication without modifying entry steps (viral binding, internalization, fusion) under high glucose conditions. This decrease replication was associated with a lower virus progeny and increased cell viability when compared to ZIKV-infected HK-2 cells in normal glucose concentration. In conclusion, we showed for the first time that an elevated glucose level influences ZIKV replication level with an effect on kidney cell survival.
Assuntos
Glucose/farmacologia , Rim/efeitos dos fármacos , Replicação Viral , Infecção por Zika virus/prevenção & controle , Zika virus/crescimento & desenvolvimento , Células Cultivadas , Humanos , Rim/virologia , Edulcorantes/farmacologia , Ligação Viral , Zika virus/efeitos dos fármacos , Zika virus/isolamento & purificação , Infecção por Zika virus/virologiaRESUMO
Mosquito-borne Zika virus (ZIKV) is an emerging flavivirus of medical concern associated with neurological disorders. ZIKV utilizes apoptosis as a mechanism of cell killing. The structural M protein may play a role in flavivirus-induced apoptosis. The death-promoting capability of M has been restricted to an oligopeptide representing the residues M-32/40. Here, we evaluated the apoptosis inducing ability of the residues M-31/41 of ZIKV. The ZIKV M oligopeptide was associated to a soluble form of GFP (sGFP) and the resulting sGFP-M31/41 construct was assessed in Huh7 cells. Expression of sGFP-M31/41 can trigger apoptosis in Huh7 cells through caspase-3/7 activation. The translocation of sGFP-M31/41 in the endoplasmic reticulum was a prerequisite for apoptosis induction. The residues M-33/35/38 may play a critical role in the death-promoting activity of sGFP-M31/41. The effect of ZIKV M oligopeptide defined as ZAMP (for Zika Apoptosis M Peptide) on expression of a tumor-associated antigen was assayed on megakaryocyte-potentiating factor (MPF). Expression of MPF-ZAMP construct resulted in caspase-associated apoptosis activation in A549 and Huh7 cells. ZIKV has been proposed as an oncolytic virus for cancer therapy. The ability of the Zika M oligopeptide to confer death-promoting capability to MPF opens up attractive perspectives for ZAMP as an innovative anticancer agent.
Assuntos
Antígenos de Neoplasias/metabolismo , Apoptose , Proteínas Ligadas por GPI/metabolismo , Oligopeptídeos/metabolismo , Proteínas da Matriz Viral/química , Zika virus/química , Células A549 , Antígenos de Neoplasias/genética , Caspase 3/metabolismo , Caspase 7/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas Ligadas por GPI/genética , Células HEK293 , Humanos , Mesotelina , Oligopeptídeos/química , Oligopeptídeos/genéticaRESUMO
Interferon-induced viperin (VP) was identified as playing an important role in the innate immune response against Zika virus (ZIKV). The 361 amino acid long human VP protein comprises of a highly conserved C-terminal region, which has been associated with VP antiviral properties against ZIKV. In the present study, we sought to determine whether the very last C-terminal amino-acid residues of VP might play a role in VP-mediated ZIKV inhibition. To address this issue, a recombinant human viperin (rVPwt) was overexpressed by transfection in human epithelial A549 cells. We confirmed that transient overexpression of rVPwt prior to ZIKV infection dramatically reduced viral replication in A549 cells. Deletion of the last 17 C-terminal amino acids of VP resulted in a higher expression level of mutant protein compared to wild-type VP. Mutational analysis revealed that residue substitution at positions 356 to 360 with five alanine led to the same phenotype. The charged residues Asp356, Lys358, and Asp360 were then identified to play a role in the weak level of VPwt protein in A549 cells. Mutant VP bearing the D360A substitution partially rescued ZIKV growth in A549 cells. Remarkably, a single Lys-to-Arg substitution at position 358 was sufficient to abrogate VP antiviral activity against ZIKV. In conclusion, our study showed that Asp356, Lys358, and Asp360 may have an influence on biochemical properties of VP. Our major finding was that Lys358 was a key amino-acid in VP antiviral properties against ZIKV.
Assuntos
Substituição de Aminoácidos , Antivirais/farmacologia , Proteínas Mutantes/metabolismo , Proteínas/genética , Zika virus/efeitos dos fármacos , Células A549 , Sequência de Aminoácidos , Animais , Chlorocebus aethiops , Humanos , Proteínas Mutantes/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Proteínas/química , Proteínas/metabolismo , Proteínas Recombinantes/farmacologia , Células Vero , Replicação Viral/efeitos dos fármacosRESUMO
The medical importance of Zika virus (ZIKV) was fully highlighted during the recent epidemics in South Pacific islands and Americas due to ZIKV association with severe damage to fetal brain development and neurological complications in adult patients. A worldwide research effort has been undertaken to identify effective compounds to prevent or treat ZIKV infection. Fruits and vegetables may be sources of compounds with medicinal properties. Flavonoids are one class of plant compounds that emerge as promising antiviral molecules against ZIKV. In the present study, we demonstrated that flavonoid isoquercitrin exerts antiviral activity against African historical and Asian epidemic strains of ZIKV in human hepatoma, epithelial, and neuroblastoma cell lines. Time-of-drug addition assays showed that isoquercitrin acts on ZIKV entry by preventing the internalisation of virus particles into the host cell. Our data also suggest that the glycosylated moiety of isoquercitrin might play a role in the antiviral effect of the flavonoid against ZIKV. Our results highlight the importance of isoquercitrin as a promising natural antiviral compound to prevent ZIKV infection.
Assuntos
Antivirais/uso terapêutico , Flavonoides/uso terapêutico , Quercetina/análogos & derivados , Infecção por Zika virus/prevenção & controle , Butiratos , Humanos , Quercetina/uso terapêutico , SulfonasRESUMO
Zika virus (ZIKV) is a mosquito-borne flavivirus that recently emerged in the South Pacific, Americas, and Caribbean islands, where the larger epidemics were documented. ZIKV infection in humans is responsible for neurological disorders and microcephaly. Flavivirus NS1 is a non-structural glycoprotein that is expressed on the cell surface and secreted as a hexameric lipoprotein particle. Intracellular NS1 exists as a dimer that is required for viral replication, whereas the secreted NS1 hexamer interacts with host factors, leading to pathophysiological conditions. In an effort to dispose of specific anti-ZIKV NS1 immune serum, Vero cells were transduced with a lentiviral vector containing the NS1 gene from an epidemic strain of ZIKV. We showed that stably transduced Vero/ZIKV NS1 cell clone was efficient in the secretion of recombinant NS1 oligomer. Immunization of adult rat with purified extracellular NS1 developed anti-ZIKV antibodies that specifically react with the NS1 dimer produced in human cells infected with African and Asian strains of ZIKV. The rat antibody against ZIKV NS1 dimer is a reliable biological tool that enables the immunological detection of secreted NS1 from host-cells infected with ZIKV.
Assuntos
Soros Imunes/imunologia , Multimerização Proteica/imunologia , Proteínas Recombinantes/metabolismo , Proteínas não Estruturais Virais/imunologia , Proteínas não Estruturais Virais/metabolismo , Células A549 , Animais , Chlorocebus aethiops , Clonagem Molecular , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Imunização , Lentivirus/genética , Ratos , Células VeroRESUMO
Cell cycle is an ordered sequence of events that occur in a cell preparing for cell division . The cell cycle is a four-stage process in which the cell increases in size, copies its DNA , prepares to divide, and divides. All these stages require a coordination of signaling pathways as well as adequate levels of energy and building blocks. These specific signaling and metabolic switches are tightly orchestrated in order for the cell cycle to occur properly. In this book chapter, we will provide information on the basis of metabolism and cell cycle interplay, and we will finish by an unexhaustive list of metabolomics approaches available to study the reciprocal control of metabolism and cell cycle.
Assuntos
Metabolômica , Transdução de Sinais , Ciclo Celular , Divisão Celular , DNARESUMO
In addition to its role in controlling cell cycle progression, the tumor suppressor protein p53 can also affect other cellular functions such as cell migration. In this study, we show that p53 deficiency in mouse embryonic fibroblasts cultured in three-dimensional matrices induces a switch from an elongated spindle morphology to a markedly spherical and flexible one associated with highly dynamic membrane blebs. These rounded, motile cells exhibit amoeboid-like movement and have considerably increased invasive properties. The morphological transition requires the RhoA-ROCK (Rho-associated coil-containing protein kinase) pathway and is prevented by RhoE. A similar p53-mediated transition is observed in melanoma A375P cancer cells. Our data suggest that genetic alterations of p53 in tumors are sufficient to promote motility and invasion, thereby contributing to metastasis.
Assuntos
Movimento Celular/fisiologia , Matriz Extracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Invasividade Neoplásica , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Supressora de Tumor p53/deficiência , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Forma Celular/fisiologia , Células Cultivadas , Colágeno/metabolismo , Combinação de Medicamentos , Eletroporação , Embrião de Mamíferos , Fibroblastos/citologia , Fibroblastos/metabolismo , Deleção de Genes , Laminina/metabolismo , Camundongos , Proteoglicanas/metabolismo , Transfecção , Proteína Supressora de Tumor p53/genética , Quinases Associadas a rho , Proteína rhoA de Ligação ao GTP/análiseRESUMO
Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that rapidly became a major medical concern worldwide. We have recently reported that a high glucose level decreases the rate of Zika virus (ZIKV) replication with an impact on human kidney HK-2 cell survival. However, the mechanisms by which cells cultured in a high glucose medium inhibit ZIKV growth remain unclear. Viperin belongs to interferon-stimulated genes (ISG) and its expression is highly up-regulated upon viral infection, leading to antiviral activity against a variety of viruses, including flaviviruses. As such, viperin has been shown to be a major actor involved in the innate immune response against Zika virus (ZIKV). Our present study aims to further characterize the involvement of viperin in ZIKV growth inhibition under high glucose concentration (HK-2HGC). We show for the first time that endogenous viperin is over-expressed in HK-2 cells cultured under high glucose concentration (HK-2HGC), which is associated with ZIKV growth inhibition. Viperin knockdown in HK-2HGC rescues ZIKV growth. In addition, our results emphasize that up-regulated viperin in HK-2HGC leads to ZIKV growth inhibition through the stimulation of IFN-ß production. In summary, our work provides new insights into the ZIKV growth inhibition mechanism observed in HK-2 cells cultured in a high glucose environment.
RESUMO
The newly identified coronavirus SARS-CoV-2 is responsible for the worldwide pandemic COVID-19. Considerable efforts have been devoted for the development of effective vaccine strategies against COVID-19. The SARS-CoV-2 spike protein has been identified as the major antigen candidate for the development of COVID-19 vaccines. The Pfizer-BioNTech COVID-19 vaccine COMIRNATY is a lipid nanoparticle-encapsulated mRNA encoding a full-length and prefusion-stabilized SARS-CoV-2 spike protein. In the present study, synthetic peptide-based ELISA assays were performed to identify linear B-cell epitopes into the spike protein that contribute to elicitation of antibody response in COMIRNATY-vaccinated individuals. The synthetic S2P6 peptide containing the spike residues 1138/1169 and to a lesser extent, the synthetic S1P4 peptide containing the spike residues 616/644 were recognized by the immune sera from COMIRNATY vaccine recipients but not COVID-19 recovered patients. We assume that the synthetic S2P6 peptide and to a lesser extent the synthetic S1P4 peptide, could be of interest to measure the dynamic of antibody response to COVID-19 mRNA vaccines. The S2P6 peptide has been identified as immunogenic in adult BALB/c mice that received protein-peptide conjugates in a prime-boost schedule. This raises the question on the role of the B-cell epitope peptide containing the SARS-CoV-2 spike residues 1138/1169 in protective efficacy of the Pfizer-BioNTech COVID-19 vaccine COMIRNATY.
Assuntos
Anticorpos Antivirais , Vacinas contra COVID-19 , COVID-19 , Epitopos de Linfócito B , Glicoproteína da Espícula de Coronavírus , Animais , Anticorpos Antivirais/imunologia , Vacina BNT162 , COVID-19/prevenção & controle , Vacinas contra COVID-19/imunologia , Humanos , Lipossomos , Camundongos , Nanopartículas , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
BACKGROUND: Tumor cells can move in a three-dimensional (3D) environment in either mesenchymal-type or amoeboid modes. In mesenchymal-type movement, cells have an elongated morphology with Rac-induced protrusions at the leading edge. Amoeboid cells have high levels of actomyosin contractility, and movement is associated with deformation of the cell body through the matrix without proteolysis. Because signaling pathways that control the activation of GTPases for amoeboid movement are poorly understood, we sought to identify regulators of amoeboid movement by screening an siRNA library targeting guanine nucleotide exchange factors (GEFs) for Rho-family GTPases. RESULTS: We identified DOCK10, a Cdc42 GEF, as a key player in amoeboid migration; accordingly, we find that expression of activated Cdc42 induces a mesenchymal-amoeboid transition and increases cell invasion. Silencing DOCK10 expression promotes conversion to mesenchymal migration and is associated with decreased MLC2 phosphorylation and increased Rac1 activation. Consequently, abrogating DOCK10 and Rac1 expression suppresses both amoeboid and mesenchymal migration and results in decreased invasion. We show that the Cdc42 effectors N-WASP and Pak2 are required for the maintenance of the rounded-amoeboid phenotype. Blocking Cdc42 results in loss of mesenchymal morphology, arguing that Cdc42 is also involved in mesenchymal morphology through different activation and effector pathways. CONCLUSIONS: Previous work has identified roles of Rho and Rac signaling in tumor cell movement, and we now elucidate novel roles of Cdc42 signaling in amoeboid and mesenchymal movement and tumor cell invasion.
Assuntos
Movimento Celular , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Melanoma/metabolismo , Invasividade Neoplásica , Proteína cdc42 de Ligação ao GTP/metabolismo , Linhagem Celular Tumoral , Forma Celular , Humanos , Melanoma/patologia , Melanoma/fisiopatologia , Transdução de Sinais , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
The neurological complications of infection by the mosquito-borne Zika virus (ZIKV) include Guillain-Barré syndrome (GBS), an acute inflammatory demyelinating polyneuritis. GBS was first associated with recent ZIKV epidemics caused by the emergence of the ZIKV Asian lineage in South Pacific. Here, we hypothesize that ZIKV-associated GBS relates to a molecular mimicry between viral envelope E (E) protein and neural proteins involved in GBS. The analysis of the ZIKV epidemic strains showed that the glycan loop (GL) region of the E protein includes an IVNDT motif which is conserved in voltage-dependent L-type calcium channel subunit alpha-1C (Cav1.2) and Heat Shock 70 kDa protein 12A (HSP70 12A). Both VSCC-alpha 1C and HSP70 12A belong to protein families which have been associated with neurological autoimmune diseases in central nervous system. The purpose of our in silico analysis is to point out that IVNDT motif of ZIKV E-GL region should be taken in consideration for the development of safe and effective anti-Zika vaccines by precluding the possibility of adverse neurologic events including autoimmune diseases such as GBS through a potent mimicry with Heat Shock 70 kDa protein 12A (HSP70 12A).
RESUMO
The p53 isoform, Δ133p53ß, is critical in promoting cancer. Here we report that Δ133p53ß activity is regulated through an aggregation-dependent mechanism. Δ133p53ß aggregates were observed in cancer cells and tumour biopsies. The Δ133p53ß aggregation depends on association with interacting partners including p63 family members or the CCT chaperone complex. Depletion of the CCT complex promotes accumulation of Δ133p53ß aggregates and loss of Δ133p53ß dependent cancer cell invasion. In contrast, association with p63 family members recruits Δ133p53ß from aggregates increasing its intracellular mobility. Our study reveals novel mechanisms of cancer progression for p53 isoforms which are regulated through sequestration in aggregates and recruitment upon association with specific partners like p63 isoforms or CCT chaperone complex, that critically influence cancer cell features like EMT, migration and invasion.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Agregação Patológica de Proteínas , Proteína Supressora de Tumor p53/genética , Animais , Linhagem Celular Tumoral , Humanos , Células MCF-7 , Camundongos , Modelos Moleculares , Mutação , Invasividade Neoplásica , Neoplasias/metabolismo , Neoplasias/patologia , Agregados Proteicos , Conformação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Desdobramento de Proteína , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismoRESUMO
Mosquito-borne Zika virus (ZIKV) causes a severe congenital syndrome and neurological disorders in humans. With the aim to develop a live-attenuated ZIKV strain, we generated a chimeric viral clone ZIKALIVax with African MR766-NIID strain as backbone and the envelope E protein of epidemic Brazilian BeH810915 strain. The MR766-NIID residues E-T152/I156/Y158 were introduced into BeH810915 E protein leading to a nonglycosylated ZIKALIVax. Recently, we reported that the residues E-152/156/158 that are part of ZIKV glycan loop (GL) region might have an impact on the availability of neutralizing antibody epitopes on ZIKV surface. In the present study, we evaluated the antigenic reactivity of a synthetic 20-mer peptide representing the ZIKALIVax GL region. The GL-related peptide was effective for the detection of GL-reactive antibody in mouse anti-ZIKALIVax immune serum. We showed that the residue E-158 influences the antigenic reactivity of GL-related peptide. The ZIKALIVax peptide was effective in generating mouse antibodies with reactivity against a recombinant E domain I that encompasses the GL region. The GL peptide-reactive antibodies revealed that antigenic reactivity of E-domain I may be impacted by both residues E-152 and E-156. In conclusion, we proposed a role for the residues E-152/156/158 as key antigenic determinants of ZIKV glycan loop region.
Assuntos
Anticorpos Antivirais/sangue , Epitopos/imunologia , Peptídeos/imunologia , Polissacarídeos/imunologia , Zika virus/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Zika virus/genética , Infecção por Zika virus/imunologiaRESUMO
Zika virus (ZIKV) dramatically emerged in French Polynesia and subsequently in the Americas where it has been associated with severe neurological complications in adults and newborns, respectively. Although plasmacytoid dendritic cells (pDCs) are a key sensor of viral infection and are critical for initiating an antiviral response, little is known about the impact of ZIKV infection on pDCs. Here, we investigated the susceptibility of human pDCs to infection with multiple strains of ZIKV and further investigated the impact of infection on pDCs functions. We observed that pDCs were refractory to cell-free ZIKV virions but were effectively infected when co-cultured with ZIKV-infected cells. However, exposure of pDCs to ZIKV-infected cells resulted in limited maturation/activation with significant down regulation of CD303 expression, a severe impairment of inflammatory cytokine production, and an inability to mount an IFN-α response. We show that ZIKV developed a strategy to inhibit the IFN-α response in primary human pDCs likely mediated through NS1-dependent CD303 signaling, thus suggesting a new mechanism of immune evasion.
Assuntos
Células Dendríticas/imunologia , Interferon-alfa/imunologia , Lectinas Tipo C/imunologia , Glicoproteínas de Membrana/imunologia , Receptores Imunológicos/imunologia , Transdução de Sinais/imunologia , Infecção por Zika virus/imunologia , Zika virus/imunologia , Animais , Linhagem Celular , Chlorocebus aethiops , Citocinas/imunologia , Regulação para Baixo/imunologia , Humanos , Inflamação/imunologia , Células VeroRESUMO
Zika virus (ZIKV) is an emerging arthropod-borne virus of major public health concern. ZIKV infection is responsible for congenital Zika disease and other neurological defects. Antibody-mediated virus neutralization is an essential component of protective antiviral immunity against ZIKV. In the present study, we assessed whether our GFP reporter ZIKV derived from African viral strain MR766 could be useful for the development of a flow cytometry neutralization test (FNT), as an alternative to the conventional plaque-reduction neutralization test (PRNT). To improve the efficacy of GFP-expressing MR766, we selected virus variant MR766GFP showing a high level of GFP signal in infected cells. A MR766GFP-based FNT was assayed with immune sera from adult mice that received ZIKBeHMR-2. The chimeric ZIKV clone ZIKBeHMR-2 comprises the structural protein region of epidemic strain BeH819015 into MR766 backbone. We reported that adult mice inoculated with ZIKBeHMR-2 developed high levels of neutralizing anti-ZIKV antibodies. Comparative analysis between MR766GFP-based FNT and conventional PRNT was performed using mouse anti-ZIKBeHMR-2 immune sera. Indistinguishable neutralization patterns were observed when compared with PRNT50 and FNT50. We consider that the newly developed MR766GFP-based FNT is a valid format for measuring ZIKV-neutralizing antibodies in serum specimens.
RESUMO
Reunion Island is currently experiencing an epidemic caused by Dengue virus type-2 (DENV-2) resulting in over 6,763 cases from austral summer 2017 to winter 2018. Phylogenetic analyses on two non-imported cases of dengue infection from Reunion Island highlight a regional circulation of DENV-2 Cosmopolitan lineage 1 virus on both Reunion Island and the Seychelles.
RESUMO
The Zika virus (ZIKV) was first isolated in Africa in 1947. It was shown to be a mild virus that had limited threat to humans. However, the resurgence of the ZIKV in the most recent Brazil outbreak surprised us because it causes severe human congenital and neurologic disorders including microcephaly in newborns and Guillain-Barré syndrome in adults. Studies showed that the epidemic ZIKV strains are phenotypically different from the historic strains, suggesting that the epidemic ZIKV has acquired mutations associated with the altered viral pathogenicity. However, what genetic changes are responsible for the changed viral pathogenicity remains largely unknown. One of our early studies suggested that the ZIKV structural proteins contribute in part to the observed virologic differences. The objectives of this study were to compare the historic African MR766 ZIKV strain with two epidemic Brazilian strains (BR15 and ICD) for their abilities to initiate viral infection and to confer neurocytopathic effects in the human brain's SNB-19 glial cells, and further to determine which part of the ZIKV structural proteins are responsible for the observed differences. Our results show that the historic African (MR766) and epidemic Brazilian (BR15 and ICD) ZIKV strains are different in viral attachment to host neuronal cells, viral permissiveness and replication, as well as in the induction of cytopathic effects. The analysis of chimeric viruses, generated between the MR766 and BR15 molecular clones, suggests that the ZIKV E protein correlates with the viral attachment, and the C-prM region contributes to the permissiveness and ZIKV-induced cytopathic effects. The expression of adenoviruses, expressing prM and its processed protein products, shows that the prM protein and its cleaved Pr product, but not the mature M protein, induces apoptotic cell death in the SNB-19 cells. We found that the Pr region, which resides on the N-terminal side of prM protein, is responsible for prM-induced apoptotic cell death. Mutational analysis further identified four amino-acid residues that have an impact on the ability of prM to induce apoptosis. Together, the results of this study show that the difference of ZIKV-mediated viral pathogenicity, between the historic and epidemic strains, contributed in part the functions of the structural prM-E proteins.
Assuntos
Neuroglia/virologia , Proteínas do Envelope Viral/genética , Ligação Viral , Infecção por Zika virus/patologia , Zika virus/patogenicidade , África , Apoptose , Encéfalo/citologia , Encéfalo/virologia , Brasil , Surtos de Doenças , Epidemias , Humanos , Mutação , Neuroglia/imunologia , Replicação Viral , Zika virus/classificaçãoRESUMO
Zika virus (ZIKV) is an emerging mosquito-borne flavivirus which is of major public health concern. ZIKV infection is recognized as the cause of congenital Zika disease and other neurological defects, with no specific prophylactic or therapeutic treatments. As the humoral immune response is an essential component of protective immunity, there is an urgent need for effective vaccines that confer protection against ZIKV infection. In the present study, we evaluate the immunogenicity of chimeric viral clone ZIKBeHMR-2, in which the region encoding the structural proteins of the African strain MR766 backbone was replaced with its counterpart from the epidemic strain BeH819015. Three amino-acid substitutions I152T, T156I, and H158Y were introduced in the glycan loop of the E protein (E-GL) making ZIKBeHMR-2 a non-glycosylated virus. Adult BALB/c mice inoculated intraperitoneally with ZIKBeHMR-2 developed anti-ZIKV antibodies directed against viral proteins E and NS1 and a booster dose increased antibody titers. Immunization with ZIKBeHMR-2 resulted in a rapid production of neutralizing anti-ZIKV antibodies. Antibody-mediated ZIKV neutralization was effective against viral strain MR766, whereas epidemic ZIKV strains were poorly sensitive to neutralization by anti-ZIKBeHMR-2 immune sera. From our data, we propose that the three E-GL residues at positions E-152, E-156, and E-158 greatly influence the accessibility of neutralizing antibody epitopes on ZIKV.