RESUMEN
Mosquitoes are hematophagous insects that carry-on and transmit many human viruses. However, little information is available regarding the common mechanisms underlying the infection of mosquitoes by these viruses. In this study, we reveal that the hematophagous nature of mosquitoes contributes to arboviral infection after a blood meal, which suppresses antiviral innate immunity by activating the GABAergic pathway. dsRNA-mediated interruption of the GABA signaling and blockage of the GABAA receptor by the specific inhibitors both significantly impaired arbovirus replication. Consistently, inoculation of GABA enhanced arboviral infection, indicating that GABA signaling facilitates the arboviral infection of mosquitoes. The ingestion of blood by mosquitoes resulted in robust GABA production from glutamic acid derived from blood protein digestion. The oral introduction of glutamic acid increased virus acquisition by mosquitoes via activation of the GABAergic system. Our study reveals that blood meals enhance arbovirus replication in mosquitoes through activation of the GABAergic system.
Asunto(s)
Aedes/inmunología , Arbovirus/metabolismo , Sangre/inmunología , Culex/inmunología , Inmunidad Innata/inmunología , Replicación Viral/inmunología , Ácido gamma-Aminobutírico/inmunología , Animales , Virus Bunyamwera/metabolismo , Virus del Dengue/metabolismo , Virus de la Encefalitis de California/metabolismo , Virus de la Encefalitis Japonesa (Especie)/metabolismo , Antagonistas de Receptores de GABA-A/farmacología , Humanos , Mosquitos Vectores/inmunología , ARN Bicatenario/metabolismo , Receptores de GABA-A/metabolismo , Virus de los Bosques Semliki/metabolismo , Transducción de Señal , Virus Sindbis/metabolismo , Replicación Viral/efectos de los fármacos , Replicación Viral/fisiología , Ácido gamma-Aminobutírico/metabolismo , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
This paper presents selected results of the studies on emerging and reemerging infections caried out in D I Ivanovsky Research Institute of Virology with special reference to comprehensive ecological, virological, and molecular-genetic analysis of the following viruses: California encephalaitis serocomplex, West Nile fever, highly virulent avian influenza A virus (H5N 1), and new pandemic influenza A vires (HIN1). Special attention is given to the role of emerging and reemerging infections at the territory of Northern Eurasia in national and world-wide epidemiological cataclysms and their prognostication for minimizing their consequences based on monitoring pathogen evolution.
Asunto(s)
Virus de la Encefalitis de California , Encefalitis de California , Genoma Viral , Subtipo H5N1 del Virus de la Influenza A , Gripe Humana , Fiebre del Nilo Occidental , Virus del Nilo Occidental , Animales , Aves , Control de Enfermedades Transmisibles/organización & administración , Enfermedades Transmisibles Emergentes/epidemiología , Enfermedades Transmisibles Emergentes/prevención & control , Enfermedades Transmisibles Emergentes/virología , Desastres , Reservorios de Enfermedades/virología , Vectores de Enfermedades , Virus de la Encefalitis de California/genética , Virus de la Encefalitis de California/metabolismo , Virus de la Encefalitis de California/patogenicidad , Encefalitis de California/epidemiología , Encefalitis de California/transmisión , Monitoreo del Ambiente , Monitoreo Epidemiológico , Europa (Continente)/epidemiología , Humanos , Subtipo H5N1 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/metabolismo , Subtipo H5N1 del Virus de la Influenza A/patogenicidad , Gripe Aviar/epidemiología , Gripe Aviar/transmisión , Gripe Humana/epidemiología , Gripe Humana/transmisión , Internacionalidad , Pandemias/prevención & control , Pandemias/estadística & datos numéricos , Siberia/epidemiología , Fiebre del Nilo Occidental/epidemiología , Fiebre del Nilo Occidental/transmisión , Virus del Nilo Occidental/genética , Virus del Nilo Occidental/metabolismo , Virus del Nilo Occidental/patogenicidadRESUMEN
California (CAL) serogroup Bunyaviruses are significant agents of arboviral encephalitis in humans. They are maintained and transmitted in nature by mosquitoes to preferred vertebrate amplifying hosts. The G2 envelope glycoprotein of La Crosse virus (LAC) was proposed by Ludwig et al. to be a determinant for virus attachment to mosquito midgut cells. Monoclonal antibodies to G2 neutralize the infectivity of pronase-treated virus for mosquito cells. We determined the location of antigenic sites on the LAC G2. We showed that antigenic areas present on the LAC G2 protein are conserved among viruses in the California encephalitis and Melao subgroups of the CAL serogroup, but not in trivatattus virus, nor within the BUN serogroup. A comparison of the G2 exodomain amino acid sequences of eight CAL and three BUN viruses with monoclonal antibodies (MAb) binding data predicted the possible location of the antigenic sites. We used in vitro mutagenesis of the LAC G2 gene to construct a set of G2 genes with replacement sequences in the coding regions for the suspected MAb binding sites. The native and mutated proteins were expressed in Hela cells and the ability of MAbs to bind to the expressed proteins was tested. Four discontinuous amino acid sequences, conserved among eight CAL serogroup viruses, were identified as contributing to two conformational binding domains for neutralizing LAC G2 MAbs.
Asunto(s)
Antígenos Virales/inmunología , Secuencia Conservada , Virus de la Encefalitis de California/inmunología , Mapeo Epitopo , Proteínas del Envoltorio Viral/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Antígenos Virales/química , Chlorocebus aethiops , Virus de la Encefalitis de California/genética , Virus de la Encefalitis de California/metabolismo , Encefalitis de California/virología , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutagénesis , Células Vero , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genéticaRESUMEN
The following reviews the role of mRNA stability in the regulation of both viral and cellular gene expression in virus-infected cells. Indeed, several eukaryotic viruses, including the human immunodeficiency virus, HIV-1, regulate cellular protein synthesis via such control mechanisms. The following systems will be discussed: (i) the degradation of viral and cellular mRNAs in cells infected by herpes simplex virus (HSV) and advances made using the HSV virion host shutoff mutant; (ii) the degradation of viral and cellular mRNA and ribosomal RNA in cells infected by vaccinia virus and the possible role of the oligoadenylate synthetase-RNase L pathways; (iii) the turnover of RNAs in cells infected by encephalomyocarditis virus, reovirus, and La Crosse virus; and finally (iv) recent studies from our laboratory on the degradation of cellular mRNAs in cells infected by HIV-1.
Asunto(s)
Células Eucariotas/metabolismo , Regulación de la Expresión Génica , ARN/metabolismo , Virosis/metabolismo , Virus/metabolismo , Animales , Virus de la Encefalitis de California/metabolismo , Virus de la Encefalomiocarditis/metabolismo , Células Eucariotas/microbiología , VIH-1/metabolismo , Humanos , ARN Viral/metabolismo , Reoviridae/metabolismo , Simplexvirus/metabolismo , Virus Vaccinia/metabolismoRESUMEN
Within 24 to 48 h of La Crosse virus infection of mosquito cells, greater than 75% of the S mRNA was found to band in CsCl density gradients at the position of genome or antigenome nucleocapsids. The encapsidation of the S mRNA correlates with the repression of N protein synthesis in vivo, and the encapsidated S mRNA cannot be translated in vitro. Unlike genome and antigenome assembly, S mRNA assembly is a relatively slow process, which is not coupled to its synthesis. Within the encapsidated S mRNA population, three forms could be distinguished, those with intact primers which were or were not also assembled with N protein and those in which the primer and up to 3 template bases had been lost. We suggest that genome replication, but not transcription, is down regulated with time in mosquito cells for reasons that are unclear. The pool of unassembled N protein then increased to the point at which it began to interact with its own mRNA, as this mRNA also contains what is considered to be the assembly site, i.e., the conserved sequences at the 5' ends of all genome and antigenome chains. This lead to the assembly of the entire mRNA, except for the nontemplate primer. Some of the primers were then also assembled with N protein, whereas others were digested to produce truncated mRNAs.
Asunto(s)
Bunyaviridae/metabolismo , Cápside/metabolismo , Virus de la Encefalitis de California/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Proteínas del Núcleo Viral/metabolismo , Animales , Secuencia de Bases , Cápside/biosíntesis , Línea Celular , Culicidae , Sondas de ADN , Virus de la Encefalitis de California/genética , Cinética , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Viral/metabolismo , Proteínas del Núcleo Viral/biosíntesisRESUMEN
The translational requirement to prevent premature termination during La Crosse virus S mRNA synthesis was found to be cell-type dependent. This requirement was present in the BHK, HEL, and Vero cell lines we examined, but not in C6/36 mosquito cells. The cell-dependent translational requirement could be reproduced in vitro by using either cell extracts or purified virions of BHK and C6/36 cells. In the BHK reactions, the polymerase terminated predominantly at nucleotide 175 in the absence of concurrent translation and required translation to read through this position. In the C6/36 reactions, however, the polymerase reads through nucleotide 175 efficiently independent of translation. Reconstitution studies suggested that the translational requirement was due to a factor(s) present in BHK, but not in C6/36, cells.
Asunto(s)
Bunyaviridae/genética , Virus de la Encefalitis de California/genética , Biosíntesis de Proteínas , ARN Mensajero/biosíntesis , Animales , Línea Celular , Culicidae , ARN Polimerasas Dirigidas por ADN/metabolismo , Virus de la Encefalitis de California/metabolismo , Humanos , Cinética , Conejos , Transcripción Genética , Células Vero , Virión/genética , Virión/metabolismoRESUMEN
Efficient transmission, amplification, and dissemination of arboviruses require viral replication in vertebrate and invertebrate hosts. As a result, virions are exposed to two significantly different environments. Exposure of LaCrosse virus (LACV) to proteolytic enzymes, such as those that may be found in the mosquito midgut, increases virus affinity for mosquito cells. These enzymes remove the major envelope glycoprotein (G1) while leaving the second glycoprotein (G2) intact. Processing of LACV glycoproteins in the mosquito midgut may be necessary to expose attachment proteins on the virion surface before attachment to, and infection of, midgut cells can occur. This model may suggest answers to questions regarding the molecular basis for midgut infection barriers and species susceptibility to arbovirus infection in nature.
Asunto(s)
Bunyaviridae/metabolismo , Virus de la Encefalitis de California/metabolismo , Péptido Hidrolasas/farmacología , Receptores Virales/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Animales , Línea Celular , Encefalitis de California/fisiopatología , Técnicas In Vitro , Glicoproteínas de Membrana/metabolismo , Modelos Biológicos , Células VeroRESUMEN
Ongoing protein synthesis is required for La Crosse S-mRNA synthesis in vivo, and complete S-mRNA can be made in vitro only in the presence of an active rabbit reticulocyte lysate. Using in vitro systems based on the polymerase activity of purified virions, we further support the notion that it is translation of the nascent mRNA that is required for complete transcription. Since replacement of guanosine with inosine in the nascent mRNA substitutes for the translational requirement, it appears that translation is required to prevent interactions of the nascent chain from taking place, which, if not prevented, lead to premature termination. These interactions appear to be between the nascent mRNA chain and its nucleocapsid template. A model for the translational requirement for complete S-mRNA synthesis is presented.
Asunto(s)
Bunyaviridae/genética , Virus de la Encefalitis de California/genética , Biosíntesis de Proteínas , ARN Mensajero/biosíntesis , ARN Viral/biosíntesis , Transcripción Genética , Virus de la Encefalitis de California/metabolismo , Genes Virales , Guanosina Trifosfato/metabolismo , Inosina Trifosfato/metabolismo , Modelos Biológicos , Hibridación de Ácido Nucleico , Proteínas Virales/biosíntesisRESUMEN
The effect of drugs such as puromycin and cycloheximide, which inhibit protein synthesis, on the accumulation of La Crosse virus S genome RNAs in vivo has been examined. We have found that if these drugs are added to the cultures before infection, minuscule amounts of S-mRNA can be detected late in infection. Genome replication, on the other hand, cannot be detected at any time. When these drugs are added later in infection when RNA synthesis is well established, S-mRNA accumulation decreases in a dose-dependent manner proportional to the effect of these drugs on protein synthesis. This decrease cannot be accounted for by increased turnover of the mRNA in the presence of the drug. S genome replication, curiously, was found to be hypersensitive to the effects of these drugs. Our results confirm those of Abraham and Pattnaik (1983) that ongoing protein synthesis is required for the accumulation of complete bunyavirus S-mRNA.
Asunto(s)
Bunyaviridae/genética , Cicloheximida/farmacología , Virus de la Encefalitis de California/genética , Puromicina/farmacología , ARN Mensajero/biosíntesis , ARN Viral/biosíntesis , Anisomicina/farmacología , Virus de la Encefalitis de California/efectos de los fármacos , Virus de la Encefalitis de California/metabolismo , Genes Virales/efectos de los fármacos , Pactamicina/farmacología , Biosíntesis de Proteínas , Tricodermina/farmacologíaRESUMEN
The cellular site of La Crosse virus S genome mRNA synthesis was examined by pulse-labeling infected cells for various times and determining the amount of labeled S mRNA in both the cytoplasmic and nuclear fractions. With pulse times as short as 3 min, La Crosse virus S genome transcription was found to be localized in the cytoplasm.
Asunto(s)
Bunyaviridae/genética , Citoplasma/metabolismo , Virus de la Encefalitis de California/genética , ARN Mensajero/genética , ARN Viral/genética , Transcripción Genética , Animales , Línea Celular , Núcleo Celular/metabolismo , Cricetinae , Virus de la Encefalitis de California/metabolismo , ARN Mensajero/biosíntesis , ARN Viral/biosíntesisRESUMEN
The envelope of the bunyavirus La Crosse contains two glycoproteins, G1 (120 000 mol. wt.) and G2 (38 000 mol. wt.). When incubated with trypsin or plasmin, the G1 glycoprotein of virus grown in cell culture was cleaved, leaving two different sized polypeptides in the envelope (67 000 and 95 000 mol. wt.). Chymotrypsin cleaved G1 leaving polypeptides of 70 000 and 100 000 mol. wt. G2, however, was not altered by these enzymes. When used in antibody neutralization studies, these proteolytically modified viruses were neutralized approximately 1 to 2 log10 units in 60 min while control virus was neutralized by over 4 log10 units in 20 min. Because antibody to G1, but not G2, was involved in La Crosse virus neutralization, cleavage of G1 appeared to be directly responsible for these altered kinetics of neutralization. Antibody did bind to the polypeptides remaining associated with the envelope resulting in infectious virus-antibody complexes. This indicated that a critical site in terms of antibody neutralization was removed from G1 by proteolytic enzymes.
Asunto(s)
Anticuerpos Antivirales/análisis , Bunyaviridae/metabolismo , Virus de la Encefalitis de California/metabolismo , Glicoproteínas/metabolismo , Péptido Hidrolasas/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Animales , Sitios de Unión de Anticuerpos , Cricetinae , Electroforesis en Gel de Poliacrilamida , Virus de la Encefalitis de California/inmunología , Glicoproteínas/inmunología , Pruebas de Inhibición de Hemaglutinación , Ratones , Peso Molecular , Pruebas de Neutralización , Proteínas del Envoltorio Viral/inmunología , Cultivo de VirusRESUMEN
The effect of monensin, a monovalent ionophore, on La Crosse virus particle formation and polypeptide synthesis was examined. Monensin inhibited the release of virus particles (both infectious and non-infectious) from infected BHK-21 cells. Monensin had no detectable effect on the synthesis of polypeptides G1, G2, and N.
Asunto(s)
Bunyaviridae/efectos de los fármacos , Virus de la Encefalitis de California/efectos de los fármacos , Furanos/farmacología , Monensina/farmacología , Proteínas Virales/biosíntesis , Animales , Línea Celular , Cricetinae , Virus de la Encefalitis de California/crecimiento & desarrollo , Virus de la Encefalitis de California/metabolismo , Tunicamicina/farmacología , Replicación Viral/efectos de los fármacosRESUMEN
In the present investigation, the effect of colchicine on La Crosse virus production and export was tested. Colchicine-treated, La Crosse virus-infected cells: (1) had decreased mean virus titers compared with those of control cells; (2) had a ratio of released to cell-associated virus of 1-1.9 whereas control cells had a ratio of 13. A colchicine-sensitive step, possibly involving microtubules, may be involved in virus production and/or release from the cell.
Asunto(s)
Bunyaviridae/crecimiento & desarrollo , Colchicina/farmacología , Virus de la Encefalitis de California/crecimiento & desarrollo , Animales , Línea Celular , Cricetinae , Virus de la Encefalitis de California/efectos de los fármacos , Virus de la Encefalitis de California/metabolismo , Microtúbulos/fisiologíaAsunto(s)
Desoxiazúcares/farmacología , Desoxiglucosa/farmacología , Virus de la Encefalitis de California/efectos de los fármacos , Virus de la Encefalitis/efectos de los fármacos , Glucosamina/análogos & derivados , Glucosamina/farmacología , Tunicamicina/farmacología , Proteínas Virales/biosíntesis , Relación Dosis-Respuesta a Droga , Virus de la Encefalitis de California/metabolismo , Biosíntesis de Péptidos , TemperaturaRESUMEN
Tryptic peptide digests of the two viral glycoproteins (G1 and G2) of snowshow hare (SSH) virus, La Crosse, La Crosse (LAC) virus, and an SSH/LAC recombinant virus which has a large (L)/medium (M)/small (S) RNA segment genome composition of SSH/LAC/SSH were analyzed by ion-exchange column chromatography. The analyses prove that the M RNA species of bunyaviruses codes for the two viral glycoproteins.