RESUMO
Retrospective analysis of archived tissue samples from bird deaths in the Tuscany region of Italy in 1996 identified Usutu virus. Partial sequencing confirmed identity with the 2001 Vienna strain and provided evidence for a much earlier introduction of this virus into Europe than previously assumed.
Assuntos
Doenças das Aves/virologia , Vírus da Encefalite Japonesa (Subgrupo)/genética , Encefalite por Arbovirus/veterinária , Infecções por Flavivirus/veterinária , Aves Canoras/virologia , Animais , Doenças das Aves/epidemiologia , Encéfalo/patologia , Encéfalo/virologia , Vírus da Encefalite Japonesa (Subgrupo)/metabolismo , Encefalite por Arbovirus/epidemiologia , Encefalite por Arbovirus/virologia , Infecções por Flavivirus/epidemiologia , Infecções por Flavivirus/virologia , Itália/epidemiologia , Tipagem Molecular , Filogenia , RNA Viral/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Estudos RetrospectivosRESUMO
The Japanese encephalitis virus (JEV) is the most common cause of neurodegenerative disease in Southeast Asia and the Western Pacific region; approximately 1.15 billion people are at risk, and thousands suffer from permanent neurological disorders across Asian countries, with 10-15 thousand people dying each year. JEV crosses the blood-brain barrier (BBB) and forms a complex with receptors on the surface of neurons. GRP78, Src, TLR7, caveolin-1, and dopamine receptor D2 are involved in JEV binding and entry into the neurons, and these receptors also play a role in carcinogenic activity in cells. JEV binds to GRP78, a member of the HSP70 overexpressed on malignant cells to enter neurons, indicating a higher chance of JEV infection in cancer patients. However, JEV enters human brain microvascular endothelial cells via an endocytic pathway mediated by caveolae and the ezrin protein and also targets dopamine-rich areas for infection of the midbrain via altering dopamine levels. In addition, JEV complexed with CLEC5A receptor of macrophage cells is involved in the breakdown of the BBB and central nervous system (CNS) inflammation. CLEC5A-mediated infection is also responsible for the influx of cytokines into the CNS. In this review, we discuss the neuronal and macrophage surface receptors involved in neuronal death.
Assuntos
Vírus da Encefalite Japonesa (Espécie) , Vírus da Encefalite Japonesa (Subgrupo) , Encefalite Japonesa , Doenças Neurodegenerativas , Humanos , Vírus da Encefalite Japonesa (Espécie)/fisiologia , Células Endoteliais/metabolismo , Chaperona BiP do Retículo Endoplasmático , Dopamina , Doenças Neurodegenerativas/patologia , Sistema Nervoso Central , Vírus da Encefalite Japonesa (Subgrupo)/metabolismo , Receptores de Superfície Celular , Lectinas Tipo C/metabolismoRESUMO
Alfuy (ALFV) is an attenuated flavivirus related to the Murray Valley encephalitis virus (MVEV). We previously identified markers of attenuation in the envelope (E) protein of the prototype strain (ALFV3929), including the hinge region (E273-277) and lack of glycosylation at E154-156. To further determine the mechanisms of attenuation we assessed ALFV3929 binding to glycosaminoglycans (GAG), a known mechanism of flaviviruses attenuation. Indeed, ALFV3929 exhibited reduced binding to GAG-rich cells in the presence of heparin; however, low-passage ALFV isolates were relatively unaffected. Sequence comparisons between ALFV strains and structural modelling incriminated a positively-charged residue (K327) in ALFV3929 as a GAG-binding motif. Substitution of this residue to the corresponding uncharged residue in MVEV (L), using a previously described chimeric virus containing the prM & E genes of ALFV3929 in the backbone of MVEV (MVEV/ALFV-prME), confirmed a role for K327 in enhanced GAG binding. When the wild type residues at E327, E273-277 and E154-156 of ALFV3929 were replaced with the corresponding residues from virulent MVEV, it revealed each motif contributed to attenuation of ALFV3929, with the E327/E273-277 combination most dominant. These data demonstrate that attenuation of ALFV3929 is multifactorial and provide new insights for the rational design of attenuated flavivirus vaccines.
Assuntos
Vírus da Encefalite do Vale de Murray/patogenicidade , Vírus da Encefalite Japonesa (Subgrupo)/patogenicidade , Encefalite por Arbovirus/virologia , Infecções por Flavivirus/virologia , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Motivos de Aminoácidos , Animais , Encéfalo/patologia , Encéfalo/virologia , Linhagem Celular , Vírus da Encefalite do Vale de Murray/química , Vírus da Encefalite do Vale de Murray/metabolismo , Vírus da Encefalite Japonesa (Subgrupo)/química , Vírus da Encefalite Japonesa (Subgrupo)/crescimento & desenvolvimento , Vírus da Encefalite Japonesa (Subgrupo)/metabolismo , Encefalite por Arbovirus/patologia , Infecções por Flavivirus/patologia , Glicosaminoglicanos/metabolismo , Glicosilação , Heparina/farmacologia , Camundongos , Mutação , Domínios Proteicos , Inoculações Seriadas , Proteínas do Envelope Viral/genética , Ensaio de Placa Viral , VirulênciaRESUMO
Initial experiments using whole cells have shown that there were specific and saturable interactions between kunjin (KUN) virus and receptor molecules on the Vero cell surfaces. Solubilisation of Vero cell plasma membranes with octyl-D-glucoside (OG) yielded an extract which also interacted specifically with KUN virus. This was proven using electron microscopy. When the virus-OG-extract complex was exposed onto Vero cell monolayers, no KUN virus was observed to enter into the whole cells. This would imply that there was virus-receptor interaction with the OG-extract leaving no free virus to attach to the whole cells. The attachment kinetics of KUN virus was studied further using the Scatchard analysis which indicated the involvement of more than one interactive macromolecule in the attachment event.
Assuntos
Membrana Celular/virologia , Detergentes/química , Vírus da Encefalite Japonesa (Subgrupo)/metabolismo , Glucosídeos/química , Receptores Virais/metabolismo , Animais , Extratos Celulares , Chlorocebus aethiops , Vírus da Encefalite Japonesa (Subgrupo)/ultraestrutura , Cinética , Células VeroRESUMO
The binding and entry of Japanese encephalitis virus (JEV) to established cell lines were analyzed using radiolabeled virion. A JEV high-susceptibility cell line, Vero cells, adsorbed and internalized much more JEV than did NRK cells, which is a low-susceptibility cell line. NRK cells injected with viral genomic RNA produced infectious virions in culture medium. Protease treatment of cells reduced the binding of JEV to Vero cells stronger than that to NRK cells. JEV bound to a 74 KD molecule present in membrane fraction of Vero cells and this binding was inhibited by a hemagglutinin-inhibiting monoclonal antibody against JEV (MAb 301). MAb 301 also inhibited the binding of JEV to Vero cells much more strongly than to the NRK cells. The 74 KD molecule was not detected in the membrane fraction of NRK cells. These results suggest that the process of binding and entry of JEV determine the JEV susceptibility of cells at least to a certain extent, and in particular suggest that the 74 KD molecule may be a possible candidate or component of the cellular receptor for JEV.
Assuntos
Vírus da Encefalite Japonesa (Subgrupo)/metabolismo , Receptores Virais/metabolismo , Adsorção , Animais , Sítios de Ligação , Encéfalo/microbiologia , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/microbiologia , Suscetibilidade a Doenças , Vírus da Encefalite Japonesa (Subgrupo)/fisiologia , Feminino , Rim/microbiologia , Masculino , Ligação ProteicaRESUMO
Using the RNA replication machinery of Japanese encephalitis virus (JEV), the authors have established and characterized three strategies for the expression of foreign genes. Initially, approximately 11 kb genomic RNA was engineered to express heterologous genes of various sizes by preferentially inserting a new cistron at the beginning of the 3' nontranslated variable region. RNA transfection yielded recombinant viruses that initiated foreign gene expression after infecting permissive cells. JEV was capable of packaging recombinant genomes as large as approximately 15 kb. However, larger genome size was inversely correlated with RNA replication efficiency and cytopathogenicity, with no significant change in infectivity. Second, a variety of self-replicating propagation-deficient viral replicons were constructed by introducing one to three in-frame deletions into the ectodomains of all the structural proteins of JEV. These replicons displayed a spectrum of RNA replication efficiency upon transfection, suggesting that remnant transmembrane domains play a suppressive role in this process. Third, the authors generated a panel of stable packaging cell lines (PCLs) providing all three JEV structural proteins in trans. These PCLs efficiently packaged viral replicon RNAs into single-round infectious viral replicon particles. These JEV-based virus/vector systems may provide useful tools for a variety of biological applications, including foreign gene expression, antiviral compound screening, and genetic immunization.
Assuntos
Vírus da Encefalite Japonesa (Subgrupo)/genética , Genoma Viral , RNA Viral/metabolismo , Replicon , Montagem de Vírus/genética , Replicação Viral/fisiologia , Linhagem Celular , Vírus da Encefalite Japonesa (Subgrupo)/metabolismo , Vírus da Encefalite Japonesa (Subgrupo)/fisiologia , Expressão Gênica , Técnicas de Transferência de Genes , Engenharia Genética , Vetores Genéticos , RNA Viral/genética , Recombinação GenéticaRESUMO
The subcellular locations in infected Vero cells of Kunjin (KUN) virus core protein C and NS4B were analyzed by immunofluorescence (IF) and by immunoelectron microscopy using monospecific antibodies. Selection of appropriate fixation methods for IF showed that both proteins were associated at all times with perinuclear membranes spreading outward in a reticular pattern and they entered the nucleus late during the latent period. Subsequently NS4B was also dispersed through the nucleoplasm, while C appeared in the nucleolus and the nucleoplasm. These nuclear locations were confirmed by immunogold labeling of cryosections of infected cells at 24 hr postinfection. Labeling of NS4B in cryosections was especially enriched in the perinuclear membranes of the endoplasmic reticulum. When C and NS4B were each expressed separately in stably transformed cell lines, both cytoplasmic and nuclear localization was observed by IF and confirmed by immunoelectron microscopy. Thus the two proteins translocated to the nucleus independently of each other and of other viral proteins. Dual IF with antibodies to double-stranded RNA showed that cytoplasmic locations of C and NS4B were apparently associated in part with the sites of viral RNA synthesis which were resistant to solubilization by Triton X-100.
Assuntos
Núcleo Celular/virologia , Vírus da Encefalite Japonesa (Subgrupo)/metabolismo , Proteínas do Core Viral/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Especificidade de Anticorpos , Transporte Biológico , Linhagem Celular Transformada , Chlorocebus aethiops , Técnica Indireta de Fluorescência para Anticorpo , Cobaias , Microscopia Imunoeletrônica , RNA de Cadeia Dupla , Coelhos , Frações Subcelulares , Células Vero , Proteínas do Core Viral/ultraestrutura , Proteínas não Estruturais Virais/ultraestruturaRESUMO
This report focuses mainly on the characterization of a Vero cell line stably expressing the flavivirus Kunjin (KUN) replicon C20SDrep (C20SDrepVero). We showed by immunofluorescence and cryoimmunoelectron microscopy that unique flavivirus-induced membrane structures, termed convoluted membranes/paracrystalline structures, were induced in the C20SDrepVero cells. These induced cytoplasmic foci were immunolabeled with KUN virus anti-NS3 antibodies and with antibodies to the cellular markers ERGIC53 (for the intermediate compartment) and protein disulfide isomerase (for the rough endoplasmic reticulum). However, in contrast to the large perinuclear inclusions observed by immunofluorescence with anti-double-stranded (ds)RNA antibodies in KUN virus-infected cells, the dsRNA in C20SDrepVero cells was localized to small isolated foci scattered throughout the cytoplasm, which were coincident with small foci dual-labeled with the trans-Golgi specific marker GalT. Importantly, persistent expression of the KUN replicons in cells did not produce cytopathic effects, and the morphology of major host organelles (including Golgi, mitochondria, endoplasmic reticulum, and nucleus) was apparently unaffected. The amounts of plus- and minus-sense RNA synthesis in replicon cells were similar to those in KUN virus-infected cells until near the end of the latent period, but subsequently increases of about 10- and fourfold, respectively, occurred in infected cells. Virus-specified protein synthesis in C20SDrepVero cells was also about 10-fold greater than that in infected cells. When several KUN replicon cell lines were compared with respect to membrane induction, the relative efficiencies increased in parallel with increases in viral RNA and protein synthesis, consistent with the increases observed during the virus infectious cycle. Based on these observations, cell lines expressing less-efficient replicons may provide a useful tool to study early events in flavivirus RNA replication, which are difficult to assess in virus infections.
Assuntos
Vírus da Encefalite Japonesa (Subgrupo)/metabolismo , Vírus da Encefalite Japonesa (Subgrupo)/ultraestrutura , Corpos de Inclusão Viral/ultraestrutura , Membranas Intracelulares/ultraestrutura , Replicon , Replicação Viral , Animais , Linhagem Celular , Chlorocebus aethiops , Microscopia Crioeletrônica/métodos , Imunofluorescência , Imuno-Histoquímica , RNA Helicases , RNA de Cadeia Dupla/metabolismo , RNA Viral/metabolismo , Replicon/genética , Replicon/fisiologia , Serina Endopeptidases , Transfecção , Células Vero , Proteínas não Estruturais Virais/biossíntese , Replicação Viral/fisiologiaRESUMO
The susceptibility of fourteen established cell lines to infection with Japanese encephalitis virus (JEV) was assayed using an indirect fluorescent antibody technique. In kinetic studies, the degree of binding and internalization of JEV allowed the identification of high susceptibility and low-susceptibility cells. Scatchard analysis showed that JEV specifically bound to high-susceptibility Vero cells with greater affinity than to low-susceptibility NRK cells. Microinjection of viral genomic RNA into NRK cells induced highly efficient production of viral antigen and infectious virions. A hemagglutinin-inhibiting monoclonal antibody against JEV (MAb 301) inhibited the binding of JEV to the Vero and NRK cells. JEV was found to bind to a 74K molecule present in the membrane fraction of Vero cells and this binding was inhibited by MAb 301. Importantly, the 74K molecule was not detected in the membrane faction of NRK cells. These results suggest that early events in the JEV-cell interaction influence the susceptibility of cells to infection, and in particular suggests that the 74K molecule may be a possible candidate or component of the cellular receptor for JEV.
Assuntos
Membrana Celular/virologia , Vírus da Encefalite Japonesa (Subgrupo)/metabolismo , Proteínas de Membrana/metabolismo , Receptores Virais/metabolismo , Replicação Viral , Animais , Anticorpos Monoclonais , Linhagem Celular , Chlorocebus aethiops , Vírus da Encefalite Japonesa (Subgrupo)/fisiologia , Imunofluorescência , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Microinjeções , RNA Viral/genética , Ratos , Células Vero , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismoRESUMO
Kunjin virus (KUN) C is a typical flavivirus core protein which is truncated in vivo to a mature form of 105 residues enriched in lysine and arginine. In order to study the possible association of KUN C with RNA in vitro, we prepared several recombinant C proteins with specific deletions, each fused at the amino-terminus to glutathione-S-transferase (GST) and expressed in E. coli. They were reacted with KUN RNA probes transcribed in vitro from cDNA representing the 5' untranslated region (5' UTR, 93 to 96 nucleotides), the 3' UTR (624 nucleotides), and the 5' UTR plus most of the C coding region (5' core, 440 nucleotides). Fusion protein C107 (incorporating mature C) bound strongly to all KUN RNA probes with apparent specificity, being completely resistant to inhibition by 800 mM NaCl, and to competition by a large excess of tRNA. In reactions with labelled KUN RNA probes putative binding sites were identified in the isolated amino-terminal (32 residues) and carboxy-terminal (26 residues) basic amino acid domains; this binding was strongly competed by unlabelled KUN UTR probes but weakly or not at all by tRNA. These small domains probably acted co-operatively in binding of mature C to KUN RNA probes. The KUN RNA-core protein binding reactions are similar to those reported with other viral coat or capsid proteins and viral RNAs.