RESUMO
Japanese encephalitis is frequent in Asia, with a severe prognosis, but rare in travelers. Culex mosquitoes transmit Japanese encephalitis virus. Risk factors are destination, duration of stay, summer and fall seasons, outdoor activities, and type of accommodation. We report the case of a French traveler to Nepal with neutralization-based serological confirmed Japanese encephalitis. He presented classical clinical (viral syndrome before an encephalitis status with behavioral disorder, global hypotonia, mutism, movement disorders, seizure, and coma), radiological (lesions of thalami, cortico-spinal tracts, and brainstem) and biological features (lymphocytic meningitis). Nowadays, the presence of Japanese encephalitis virus in Nepal, including mountain areas, is established but Japanese encephalitis remains rare in travelers returning from this area and neurologist physicians need to become familiar with this. We recommend vaccination for travelers spending a long period of time in Nepal and having at-risk outdoor activities.
Assuntos
Encefalite Japonesa/patologia , Encefalite Japonesa/fisiopatologia , Viagem , Encefalite Japonesa/epidemiologia , Infecções por HIV/complicações , Humanos , Masculino , Nepal , População Branca , Adulto JovemRESUMO
The chikungunya virus (CHIKV) has become a substantial global health threat due to its massive re-emergence, the considerable disease burden and the lack of vaccines or therapeutics. We discovered a novel class of small molecules ([1,2,3]triazolo[4,5-d]pyrimidin-7(6H)-ones) with potent in vitro activity against CHIKV isolates from different geographical regions. Drug-resistant variants were selected and these carried a P34S substitution in non-structural protein 1 (nsP1), the main enzyme involved in alphavirus RNA capping. Biochemical assays using nsP1 of the related Venezuelan equine encephalitis virus revealed that the compounds specifically inhibit the guanylylation of nsP1. This is, to the best of our knowledge, the first report demonstrating that the alphavirus capping machinery is an excellent antiviral drug target. Considering the lack of options to treat CHIKV infections, this series of compounds with their unique (alphavirus-specific) target offers promise for the development of therapy for CHIKV infections.
Assuntos
Antivirais/farmacologia , Vírus Chikungunya/genética , Pirimidinonas/farmacologia , Proteínas não Estruturais Virais/genética , Substituição de Aminoácidos , Animais , Antivirais/química , Vírus Chikungunya/efeitos dos fármacos , Vírus Chikungunya/metabolismo , Chlorocebus aethiops , Farmacorresistência Viral/efeitos dos fármacos , Encefalomielite Equina/virologia , Cavalos , Estrutura Molecular , Pirimidinonas/química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Células Vero , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismoRESUMO
Jaagsiekte (JS), a contagious cancer affecting the lungs of sheep has been called many names over the years. At a recent workshop in Missilac, France it was agreed that the disease would be called ovine pulmonary adenocarcinoma (OPA). The disease is caused by an infectious retrovirus called jaagsiekte sheep retrovirus (JSRV). This chapter focuses on the early research that led up to the isolation, cloning and sequencing of the exogenous infectious form of JSRV and the demonstration that it has an endogenous counter part that is present in all sheep. As there was no in vitro production source of the virus much of the early research focused on the in vivo production and purification of the virus to obtain sufficient material to use to identify the viral proteins and purify the viral genetic material. Typically, new born lambs were inoculated intra-tracheally with concentrated lung lavage from previously infected sheep lungs. The optimal purification involved the concentration of lung lavage of freshly slaughtered sheep, an extraction with organic solvent, and final purification by both rate zonal and isopycnic centrifugation. Monoclonal and polyclonal antibodies were made against the purified fractions. The polyclonal antibodies were not very specific and the monoclonal antibodies proved to be against antigens expressed in high concentrations in response to any lung pathology. The genomic RNA of the virus was isolated from ex vivo purified materials, and cloned as a collection of cDNAs. The full length sequence was assembled by walking through the cDNA clones. The genome of the exogenous virus is 7462 bases and has the classical gag, pol, env genome arrangement and is flanked by a long terminal repeat (LTR) on each end. An additional open reading frame (ORF) was observed in the viral genome and has been called orfX. A function has not been determined for this ORF. JSRV is classified as a betaretrovirus, with gag and pol closely related to D type retrovirus, whereas env is related to the B type viruses such as the human endogenous retrovirus HERV-K. An interesting finding was that the exogenous infectious virus had an endogenous counter part which is present in the genomes of all sheep and goats. It is estimated that there are between 15 and 20 endogenous loci per sheep genome. No circulating antibodies have been found in OPA-affected sheep. It is suggested that the endogenous JSRV transcripts are expressed at an early age and are cause for the clonal elimination of JSRV specific T cells during T-cell ontogeny. Histopathologically the sheep disease resembles human bronchiolar alveolar carcinoma and has been identified as a natural out bred animal model that could be used to study the human disease.
Assuntos
Retrovirus Jaagsiekte de Ovinos/genética , Adenomatose Pulmonar Ovina/história , Animais , Anticorpos Monoclonais/história , Sequência de Bases , Clonagem Molecular , DNA Viral/química , DNA Viral/história , Genoma Viral , História do Século XIX , História do Século XX , Retrovirus Jaagsiekte de Ovinos/imunologia , Retrovirus Jaagsiekte de Ovinos/isolamento & purificação , Retrovirus Jaagsiekte de Ovinos/patogenicidade , Filogenia , Adenomatose Pulmonar Ovina/epidemiologia , Adenomatose Pulmonar Ovina/patologia , Adenomatose Pulmonar Ovina/virologia , OvinosRESUMO
An indirect microELISA test was performed for detection of maedi-visna antibodies in ovine and caprine species. The antigen consisted in viral particles, highly purified by successive ultracentrifugations. By comparative testing of 934 sera in ELISA and gel immunodiffusion, we found a good correlation between these two tests, and moreover, ELISA revealed another 11.3% of positive samples. The precocity of this ELISA was shown by experimental infection of sheep with different strains of maedi-visna: positive sera were detected 7 weeks post-infection, instead 4-5 months with gel immunodiffusion. The complement fixation test was compared with gel immunodiffusion and was found the less sensitive. This ELISA test appeared to be satisfactory, and may be used for early diagnosis of maedi-visna infection.
Assuntos
Anticorpos Antivirais/análise , Testes de Fixação de Complemento , Ensaio de Imunoadsorção Enzimática , Imunodifusão , Técnicas Imunoenzimáticas , Vírus Visna-Maedi/imunologia , Animais , Cabras/imunologia , Ovinos/imunologiaRESUMO
In the course of attempts to grow the jaagsiekte retrovirus in cell culture, a typical lentivirus was isolated for the first time in South Africa from adenomatous lungs. Morphologically the virus could not be distinguished from other lentiviruses, but serologically it was shown to be more closely related to visna virus than to caprine arthritis-encephalitis virus. However, a preliminary restriction enzyme analysis of the linear proviral DNA of this new lentivirus (SA-DMVV) revealed that it is significantly district from visna virus and CAEV and therefore may represent a third type of lentivirus. Antibodies to the virus were demonstrated in a number of sheep in various parts of the country, but a direct link to a disease condition was not found. Attempts to produce lung lesions by intratracheal injection of the virus have been unsuccessful to date but a transient arthritis was produced by intraarticular inoculation. Viral replication seems to be enhanced in jaagsiekte lungs.
Assuntos
Pulmão/microbiologia , Adenomatose Pulmonar Ovina/microbiologia , Vírus Visna-Maedi/isolamento & purificação , Animais , Anticorpos Antivirais/análise , Células Cultivadas , Pulmão/patologia , Macrófagos/microbiologia , Adenomatose Pulmonar Ovina/patologia , Ovinos/imunologia , Replicação Viral , Vírus Visna-Maedi/classificação , Vírus Visna-Maedi/imunologia , Vírus Visna-Maedi/patogenicidade , Vírus Visna-Maedi/fisiologiaRESUMO
The nucleotide sequence analysis of the visna-related South African Ovine Maedi Visna virus (SA-OMVV) demonstrates extensive genetic polymorphism among ovine lentiviruses. Differences between visna virus and SA-OMVV proteins range from 8.5 to 35% mismatched amino acids. Moreover, there is a new open reading frame (orf W) in the central part of the genome. A phylogenetic history calibrated by the divergence and isolation dates of these two ovine lentiviruses shows that radiation of the lentiviridae family is a recent event. Visna virus and SA-OMVV evolved independent of each other for about 42 years. The inferred molecular clock was used to calculate the minimal time elapsed since the divergence of some lentiviruses: 93 years for ovine and caprine lentiviruses, 430 years for ungulate and primate lentiviruses, and roughly 200 years for HIV-1, HIV-2, and SIVAGM. BRU, ELI, and MAL HIV-1 isolates diverged in the early 1960s.
Assuntos
DNA Viral/genética , Filogenia , Proteínas dos Retroviridae/genética , Vírus Visna-Maedi/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Produtos do Gene env/genética , Produtos do Gene gag/genética , Produtos do Gene pol/genética , Dados de Sequência Molecular , Provírus/genética , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Retroviridae/genética , Homologia de Sequência do Ácido Nucleico , SoftwareRESUMO
In 1991, we demonstrated, using electrophoretic mobility shift assays, that 3 different factors (termed B1, B2 and B3) with affinity for the KB-enhancer target sequence were specifically detected in nuclear extracts from HIV1-infected monocytes and macrophages. The B2 factor was induced in the nuclei of these cells only upon HIV1 infection. The B3 factor was only slightly evident in nuclei of uninfected cells but was readily detectable in nuclei of infected monocytes. Its expression remained very low in nuclei of HIV1-infected macrophages. In this paper, we demonstrate that the B2 factor is expressed in the cytosol of monocytes and macrophages as a DNA-binding protein, indicating that it is not associated with an inhibitor (IKB). This factor remained clustered in the cytosol and was translocated to the nuclei only after HIV1 infection. The B3 factor is detected in the cytosol only when cells are HIV1-infected. The role of HIV1 infection in the expression and the translocation of these factors is discussed.
Assuntos
HIV-1/fisiologia , Macrófagos/microbiologia , Monócitos/microbiologia , NF-kappa B/metabolismo , Sequência de Bases , Transporte Biológico , Núcleo Celular/metabolismo , Células Cultivadas , Citosol/metabolismo , Desoxirribonucleotídeos , Eletroforese , Humanos , Macrófagos/metabolismo , Dados de Sequência Molecular , Monócitos/metabolismo , Replicação ViralRESUMO
The genome of the jaagsiekte (JS) retrovirus (JSRV), the etiological agent of sheep pulmonary adenomatosis (jaagsiekte), has been identified, isolated, and partly cloned. The JSRV genome is ca. 8.7 kb long. cDNA of the genomic RNA was synthesized and cloned. A clone, JS 46.1, was isolated and characterized. It has an insert of 2.1 kb which hybridizes to the same 8.7-kb RNA in all the JSRV-infected sheep lung washes tested but does not hybridize to maedi-visna virus, a sheep lentivirus often found coinfecting JSRV-infected lungs. Comparison of the amino acid sequence encoded by JS 46.1 with those encoded by other retroviruses revealed that JSRV has homology to the type D and B oncoviruses and to human endogenous retrovirus.
Assuntos
Produtos do Gene env/genética , Adenomatose Pulmonar Ovina/microbiologia , RNA Viral/genética , Retroviridae/genética , Sequência de Aminoácidos , Animais , Northern Blotting , Centrifugação Isopícnica , Clonagem Molecular , DNA/genética , Lentivirus/isolamento & purificação , Pulmão/microbiologia , Dados de Sequência Molecular , RNA Viral/isolamento & purificação , Retroviridae/isolamento & purificação , OvinosRESUMO
The lentivirus caprine arthritis-encephalitis virus (CAEV) is closely related by nucleotide sequence homology to visna virus and other sheep lentiviruses and shows less similarity to the other animal and human lentiviruses. The genomic organization of CAEV is very similar to that of visna virus and the South African ovine maedi visna virus (SA-OMVV) as well as to those of other primate lentiviruses. The CAEV genome includes the small open reading frames (ORF) between pol and env which are the hallmarks of the lentivirus genomes. The most striking difference in the organization of CAEV is in the env gene. The Env polyproteins of visna virus and the related SA-OMVV contain 20 amino acids between the translational start and the signal peptide not present in CAEV. In addition to nucleotide sequence analysis, the transcriptional products of CAEV were determined by Northern analysis. The viral mRNA present in cells transfected with the infectious clone reveal a pattern characteristic of the mRNAs observed in other lentivirus infections. The putative tat ORF of CAEV could be identified by genomic location and amino acid homology to the visna virus tat gene. However, the CAEV rev gene could not be identified in a similar fashion. Thus, to determine the location of the rev ORF cDNA clones were obtained by PCR amplification of the mRNA from infected cells. To determine if a Rev response element was contained in the CAEV genome, secondary structural analysis of the viral RNA was performed. A stable stem loop structure which is similar in location, stability, and configuration to that determined for the Rev response element of HIV was found.
Assuntos
Vírus da Artrite-Encefalite Caprina/genética , Genes Virais , Transcrição Gênica , Sequência de Aminoácidos , Animais , Vírus da Artrite-Encefalite Caprina/patogenicidade , Sequência de Bases , Northern Blotting , Células Cultivadas , Clonagem Molecular , Produtos do Gene gag/genética , Produtos do Gene pol/genética , Produtos do Gene tat , Genes Reguladores , Cabras , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , RNA Viral/genética , RNA Viral/isolamento & purificação , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Membrana Sinovial/citologia , Transfecção , Proteínas do Envelope Viral/genéticaRESUMO
During the early step of the lytic cycle, visna provirus is first transcribed into two small multispliced mRNAs of 1.6 and 1.2 kilobases which may encode factors regulating the replication of visna virus (R. Vigne, V. Barban, G. Quérat, V. Mazarin, I. Gourdou, and N. Sauze, Virology 161:218-227, 1987). By cDNA cloning and nucleotide sequencing, we determined that the 1.2-kilobase mRNA is 1,174 nucleotides long without the 3'-polyadenylated tail and is composed of four exons, two of which originated from the 5' and 3' ends, respectively, of the env gene region. Two overlapping open reading frames are present in each of these two exons. They were translated in vitro and gave rise to three proteins, two of 19 and 17 kilodaltons, termed VEP1, and one of 16.5 kilodaltons, termed STM. Only the VEP1 proteins were recognized by a hyperimmune anti-visna virus serum of infected sheep. Transient-expression assays performed in eucaryotic cells demonstrated that the cDNA clone described here has a trans-acting effect on transcription of the visna virus genes.
Assuntos
DNA Viral/genética , RNA Mensageiro/genética , RNA Viral/genética , Vírus Visna-Maedi/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Células Cultivadas , Clonagem Molecular , Éxons , Regulação da Expressão Gênica , Genes Virais , Dados de Sequência Molecular , Biossíntese de Proteínas , Transcrição GênicaRESUMO
The complete genome of the jaagsiekte sheep retrovirus (JSRV), the suspected etiological agent of ovine pulmonary carcinoma, has been cloned from viral particles secreted in lung exudates of affected animals and sequenced. The genome is 7,462 nucleotides long and exhibits a genetic organization characteristic of the type B and D oncoviruses. Comparison of the amino acid sequences of JSRV proteins with those of other retrovirus proteins and phylogenetic studies suggest that JSRV diverged from its type B and D lineage after the type B mouse mammary tumor virus but before the type D oncoviruses captured the env gene of a reticuloendotheliosislike virus. Southern blot studies show that closely related sequences are present in sheep and goat normal genomic DNA, indicating that JSRV could be endogenous in ovine and caprine species.
Assuntos
Genoma Viral , Cabras/microbiologia , Retroviridae/genética , Ovinos/microbiologia , Proteínas Virais/genética , Animais , Sequência de Bases , Códon/genética , DNA Viral/genética , Humanos , Pulmão/microbiologia , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Fases de Leitura Aberta , Filogenia , Reação em Cadeia da Polimerase/métodos , RNA Viral/genética , Fases de Leitura , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Retroviridae/isolamento & purificação , Homologia de Sequência do Ácido NucleicoRESUMO
Visna lentivirus persists in sheep under a restricted form. Following induction events not yet defined at the molecular level, visna virus is activated to replicate productively through a short lytic cycle, the usual expression of visna virus in tissue culture. In an attempt to understand the relationship between latency and lytic replication, we characterized the transcripts of visna virus during its lytic growth by Northern blotting and S1 mapping analyses. The viral transcription pattern is relatively complex with a sequential expression in two steps: (i) an early (24 hr postinfection) expression of two multispliced mRNAs of 1.6 and 1.2 kb, which contain sequences from the 5' end of the genome, sequences from the central part of the genome from the 3' end of pol to the 5' end of env, and 3'-terminal sequences, and (ii) a late (72 hr postinfection) expression of both small mRNAs plus that of four large mRNAs of 9.4, 4.8, 4.3, and 3.7 kb. Except for the 9.4-kb RNA which is the genomic transcript, the three other large transcripts arise by a single splicing event joining 5'-terminal sequences to sequences located at positions 3' to the pol gene. This two-step expression of early and late genes of visna virus represents a novel important feature of the replicative cycle of lentiviruses.
Assuntos
Regulação da Expressão Gênica , RNA Mensageiro/genética , RNA Viral/genética , Transcrição Gênica , Vírus Visna-Maedi/genética , Animais , Linhagem Celular , Endonucleases , Éxons , Genes Virais , Hibridização de Ácido Nucleico , Endonucleases Específicas para DNA e RNA de Cadeia SimplesRESUMO
Soon after infection of ovine cell cultures, visna virus expression is first indicated by the accumulation of two multi-spliced transcripts of 1.2 and 1.6 kb that at present we have renamed 1.4 and 1.7 kb according to their exact length. The early 1.4-kb mRNA encodes for a protein which increases the level of transcripts directed from visna virus long terminal repeat (trans-activation). This trans-activating protein was previously called VEP1 and at present is renamed as the product of the rev gene according to significant amino acid sequence homologies between this protein and the rev gene products of simian immunodeficiency virus and human immunodeficiency virus type 2. In this study, the 1.7-kb mRNA was cloned, sequenced, and in vitro translated. It is 1491 nucleotides long, contains two short open reading frames, (orfs), tat (previously orf S) and rev which is the bipartite trans-acting gene specific for the early 1.4-kb mRNA. The tat gene of visna virus encodes for a protein of 11 kDa which in transient expression assays has a positive transacting effect on transcription as the rev gene product does.
Assuntos
Genes Virais , Proteínas dos Retroviridae/genética , Fatores de Transcrição/genética , Vírus Visna-Maedi/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA/genética , Regulação da Expressão Gênica , Dados de Sequência Molecular , Solubilidade , Transcrição GênicaRESUMO
The importance of the virally encoded dUTPase for CAEV replication, invasiveness, pathogenesis, and genetic stability was investigated in goats infected by viruses with single point (DU-G) and deletion (DU-1) mutations of the dUTPase gene (DU gene). The DU gene was found to be dispensable for CAEV replication in vivo as judged by times taken to seroconvert, frequencies of viral isolation, and tissue distribution of viral RNAs. DU- reversion at week 34 in one of three goats infected with the single point mutant DU-G, however, suggested that the viral dUTPase confers some advantages for replication in vivo. Moreover, we show that dUTPase is necessary for the timely development of bilateral arthritic lesions of the carpus. Finally, dUTPase was shown to efficiently prevent accumulation of G-to-A transitions in the viral genome.
Assuntos
Vírus da Artrite-Encefalite Caprina/enzimologia , Infecções por Lentivirus/microbiologia , Pirofosfatases/deficiência , Animais , Vírus da Artrite-Encefalite Caprina/patogenicidade , Sequência de Bases , DNA Viral/genética , Genes Virais , Doenças das Cabras/microbiologia , Doenças das Cabras/patologia , Cabras/microbiologia , Infecções por Lentivirus/patologia , Dados de Sequência Molecular , Monócitos/microbiologia , Mutação Puntual , Provírus/genética , Membrana Sinovial/ultraestrutura , Distribuição Tecidual , Proteínas Estruturais Virais/genética , Replicação ViralRESUMO
The virion-associated dUTPase activities of caprine arthritis-encephalitis virus (CAEV) and visna virus were determined by using an assay which measure the actual ability of the dUTPase to prevent the dUTP misincorporations into cDNA during reverse transcription. We showed that the CAEV molecular clone from the Cork isolate was dUTPase defective as a result of a single amino acid substitution. Using this point mutant and deletion mutants of CAEV as well as a deletion mutant of visna virus, we demonstrated that dUTPase-deficient viruses replicate similarly to wild-type viruses in dividing cells but show delayed replication in nondividing primary macrophages.
Assuntos
Vírus da Artrite-Encefalite Caprina/fisiologia , Pirofosfatases/metabolismo , Replicação Viral/fisiologia , Vírus Visna-Maedi/fisiologia , Animais , Vírus da Artrite-Encefalite Caprina/enzimologia , Sequência de Bases , Células Cultivadas , DNA Viral , Humanos , Macrófagos/citologia , Dados de Sequência Molecular , Mutação , Pirofosfatases/genética , Homologia de Sequência de Aminoácidos , Nucleotídeos de Timina/metabolismo , Vírus Visna-Maedi/enzimologiaRESUMO
Visna virus is a retrovirus which replicates in fibroblast-like cells of the sheep choroid plexus through a lytic cycle. Visna virions contain three major low-molecular-weight proteins (p30, p16, and p14) which, together with the genomic RNA and several molecules of reverse transcriptase, constitute the core structure of the virions. The core is surrounded by an envelope containing a major glycoprotein (gp135). By analogy with the oncoviruses, these three groups of structural proteins (i.e., the internal proteins, the envelope glycoprotein, and the reverse transcriptase) are probably encoded by the gag, env, and pol genes, respectively. To elucidate the genetic organization of the visna virus genome and its expression, we studied the synthesis of viral proteins in infected sheep choroid plexus cells. Intracellular viral proteins were detected by immunoprecipitation of pulse-labeled cell extracts with monospecific sera raised against p30, p16, and gp135 and resolution of the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoprecipitation with anti-p30 and anti-p16 sera allowed the characterization of the 55,000-dalton polypeptide precursor to internal virion proteins p30, p16, and p14 (Pr55(gag)). Tryptic peptide mapping confirmed the precursor-product relationship between Pr55(gag) and the three internal proteins. In addition, a gag-related polypeptide of 150,000 daltons was also detected. This polypeptide, which was less abundant than Pr55(gag), is a likely precursor to the viral reverse transcriptase (Pr150(gag-pol)). Pr55(gag) and Pr150(gag-pol) are not glycosylated. The precursor related to major envelope protein gp135 is a glycosylated polypeptide with an average molecular weight of 150,000 (gPr150(env)). Pulse-chase experiments indicated that gPr150(env) matures into glycoprotein gp135 intracellularly; however, gp135 was never preponderant in cell extracts. The non-glycosylated from of gPr150(env), which accumulated in the presence of 2-deoxy-d-glucose, appeared as a polypeptide of about 100,000 daltons. These results indicated that visna virus codes for the largest non-glycosylated env-related precursor among all of the retroviruses and therefore probably contains the largest env gene.
Assuntos
Precursores de Proteínas/metabolismo , Proteínas Virais/biossíntese , Vírus Visna-Maedi/metabolismo , Animais , Linhagem Celular , Plexo Corióideo , Glicoproteínas/biossíntese , Precursores de Proteínas/análise , DNA Polimerase Dirigida por RNA/biossíntese , Ovinos , Proteínas do Envelope Viral , Proteínas Virais/análise , Vírus Visna-Maedi/genéticaRESUMO
Sheep choroid plexus cells infected with visna virus produce intracytoplasmic viral ribonucleoprotein complexes with sedimentation values of 120S to 200S and buoyant densities of 1.29 to 1.32 g/cm3. These ribonucleoprotein complexes display an endogenous RNA-directed DNA polymerase activity and contain all of the species of RNA associated with polysomes. An analysis of the polypeptides present in the ribonucleoproteins allowed us to identify the mature internal virion core proteins and their precursor, Pr55gag, as well as the glycosylated envelope precursor gPr150env and small amounts of mature glycoprotein gp135. Ultracentrifugation-purified ribonucleoproteins could infect sheep choroid plexus cells and led to a normal lytic cycle with virus production. Our results suggest that visna virus can propagate by means of intracellular infectious particles.
Assuntos
Nucleoproteínas/fisiologia , Ribonucleoproteínas/fisiologia , Proteínas Virais/fisiologia , Vírus Visna-Maedi/crescimento & desenvolvimento , Animais , Linhagem Celular , Centrifugação com Gradiente de Concentração , Microscopia Eletrônica , RNA Viral/análise , DNA Polimerase Dirigida por RNA/análise , Ribonucleoproteínas/análise , Ribonucleoproteínas/isolamento & purificação , Ovinos , Proteínas Virais/análise , Proteínas Virais/isolamento & purificação , Vírus Visna-Maedi/análiseRESUMO
Ovine and caprine lentiviruses share the capacity to induce slowly progressive and inflammatory diseases of the central nervous system (leukoencephalitis or visna), lungs (progressive pneumonia or maedi), and joints (arthritis) in their natural hosts. Studies on their replication indicated that ovine lentiviruses and caprine arthritis-encephalitis virus (CAEV) recently isolated in the United States establish persistent infection in ovine and caprine fibroblasts, whereas older prototype ovine lentiviruses such as Icelandic visna virus or American progressive pneumonia virus irreversibly lyse fibroblast cultures. Since all of the recent isolates were found to be persistent, Narayan et al. (J. Gen. Virol. 59:345-356, 1982) concluded that the highly lytic viruses were only tissue-culture-adapted strains. In the present report, we isolated new ovine lentiviruses from French sheep with naturally occurring progressive pneumonia which are either highly lytic (five isolates), as are the Icelandic strains of visna virus, or persistent (one isolate), as are CAEV or American persistent ovine lentiviruses. Protein and nucleic acid content analyses of these new highly lytic (type I) and persistent (type II) isolates indicated that type I and type II ovine lentiviruses were genetically distinct, type I and type II viruses being closely related to the Icelandic strains of visna virus and to CAEV, respectively. We conclude that (i) highly lytic ovine lentiviruses, such as the Icelandic prototype strains of visna virus and persistent lentiviruses more related to CAEV, are naturally present in the ovine species, and (ii) irreversible cell lysis induced by highly lytic viruses does not result from a tissue culture adaptation of field isolates that were originally persistent but is instead the consequence of a genetic content distinct from that of persistent viruses.
Assuntos
Pneumonia Intersticial Progressiva dos Ovinos/microbiologia , Vírus Visna-Maedi/patogenicidade , Animais , Linhagem Celular , Enzimas de Restrição do DNA , DNA Viral/genética , DNA Viral/isolamento & purificação , Hibridização de Ácido Nucleico , Biossíntese de Proteínas , Ovinos , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação , Vírus Visna-Maedi/genética , Vírus Visna-Maedi/isolamento & purificaçãoRESUMO
Long-term ovine fibroblast cultures contain replicative-competent lentiviruses in a latent form. This in vitro phenomenon, never described previously for lentiviruses, was clearly demonstrated by activating the expression of latent viruses with various inducing cell treatments, some of which were efficient in inducing endogenous retroviruses or latent herpesviruses. Activated lentiviruses were highly lytic in ovine fibroblasts (type I), or they established persistent infections (type II) as described previously for field isolates from sheep with progressive pneumonia (Quérat et al., J. Virol. 52:671-678, 1984).
Assuntos
Ovinos/microbiologia , Vírus Visna-Maedi/isolamento & purificação , Animais , Linhagem Celular , Transformação Celular Viral , Células Cultivadas , Plexo Corióideo/microbiologia , Córnea/microbiologia , Embrião de Mamíferos , Feminino , Fibroblastos/microbiologia , Gravidez , Biossíntese de Proteínas , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação , Vírus Visna-Maedi/genéticaRESUMO
The genome of the sheep visna lentivirus contains an open reading frame, Q, which has a coding potential of 230 amino acid residues. This paper reports the identification and the subcellular localization of the Q ORF-encoded protein detected in lysates of visna virus-infected sheep choroid plexus cells. Sera from sheep either experimentally or naturally infected with visna virus reacted with the bacterially synthesized Q protein indicating that the in vivo expressed Q product is immunogenic. Antibodies raised against a synthetic N-terminal peptide, reacted with either the bacterial Q or the in vitro translated Q protein as well as with the Q protein expressed during cellular infection. This 29 kDa protein is detectable late in the lytic viral cycle, i.e., 72 hr postinfection, and this expression correlates with the late transcription of its 4.8-kb mRNA. These results provide evidence for the first time that the Q ORF is a late gene of visna virus and that the Q protein is located in the cytosol compartment, without evidence of accumulation at the cell membrane, or in cell-free virion particles.