RESUMO
Human herpesvirus 6 (HHV-6) and HHV-7 are closely related betaherpesviruses that encode a number of genes with no known counterparts in other herpesviruses. The product of one such gene is the HHV-6 glycoprotein gp82-105, which is a major virion component and a target for neutralizing antibodies. A 1.7-kb cDNA clone from HHV-7 was identified which contains a large open reading frame capable of encoding a predicted primary translational product of 468 amino acids (54 kDa) with 13 cysteine residues and 9 potential N-linked glycosylation sites. This putative protein, which we have termed gp65, was homologous to HHV-6 gp105 (30% identity) and contained a single potential membrane-spanning domain located near its amino terminus. Comparison of the cDNA sequence with that of the viral genome revealed that the gene encoding gp65 contains eight exons, spanning almost 6 kb of the viral genome at the right (3') end of the HHV-7 genome. Northern (RNA) blot analysis with poly(A)(+) RNA from HHV-7-infected cells revealed that the cDNA insert hybridized to a single major RNA species of 1.7 kb. Antiserum raised against a purified, recombinant form of gp65 recognized a protein of roughly 65 kDa in sucrose density gradient-purified HHV-7 preparations; treatment with PNGase F reduced this glycoprotein to a putative precursor of approximately 50 kDa. Gp65-specific antiserum also neutralized the infectivity of HHV-7, while matched preimmune serum did not do so. Finally, analysis of the biochemical properties of recombinant gp65 revealed a specific interaction with heparin and heparan sulfate proteoglycans and not with closely related molecules such as N-acetylheparin and de-N-sulfated heparin. At least two domains of the protein were found to contribute to heparin binding. Taken together, these findings suggest that HHV-7 gp65 may contribute to viral attachment to cell surface proteoglycans.
Assuntos
Glicoproteínas/genética , Heparina/metabolismo , Herpesvirus Humano 7/metabolismo , Proteínas do Envelope Viral/genética , Motivos de Aminoácidos , Baculoviridae/genética , Sequência de Bases , Células Cultivadas , DNA Complementar/genética , Éxons/genética , Glicoproteínas/química , Glicoproteínas/imunologia , Glicoproteínas/metabolismo , Herpesvirus Humano 7/genética , Humanos , Dados de Sequência Molecular , Testes de Neutralização , Testes de Precipitina , Splicing de RNA , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Transcrição Gênica , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo , Vírion/metabolismoRESUMO
Human immunodeficiency virus type 1 (HIV-1) is the etiologic agent of acquired immunodeficiency syndrome (AIDS). The pathogenesis of HIV-1-induced disease is complex and characterized by the interplay of both viral and host factors, which together determine the outcome of infection. An improved understanding of the pathogenic mechanisms of AIDS, combined with recent insights into the dynamics of viral infection may provide powerful new opportunities for therapeutic intervention against this virus.
Assuntos
Infecções por HIV , HIV-1/fisiologia , Vacinas contra a AIDS/uso terapêutico , Fármacos Anti-HIV/uso terapêutico , Previsões , Infecções por HIV/imunologia , Infecções por HIV/fisiopatologia , Infecções por HIV/terapia , Infecções por HIV/virologia , HIV-1/patogenicidade , HumanosAssuntos
Síndrome da Imunodeficiência Adquirida , HIV-1 , Síndrome de Imunodeficiência Adquirida dos Símios , Vírus da Imunodeficiência Símia , Síndrome da Imunodeficiência Adquirida/fisiopatologia , Síndrome da Imunodeficiência Adquirida/virologia , Animais , Humanos , Primatas , Síndrome de Imunodeficiência Adquirida dos Símios/fisiopatologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologiaRESUMO
The PBj14 isolate of the simian immunodeficiency virus SIVsmmPBj14 is unique among primate lentiviruses in its ability to induce lymphocyte proliferation and acutely lethal disease. The studies reported here show that viral induction of T-cell proliferation requires accessory cells, such as primary monocytes or Raji B-lymphoma cells, as well as the presence of a putative immunoreceptor tyrosine-based activation motif within the viral Nef protein. Addition of CTLA4-immunoglobulin fusion protein or anti-B7 antibodies to virally infected T cells led to substantial, but not complete, inhibition of monocyte-costimulated T-cell proliferation-suggesting that both CD28/B7-dependent and non-CD28-dependent pathways may contribute to the costimulation of virally induced lymphoproliferation. Finally, cyclosporin A, a specific inhibitor of the calcium-calmodulin-regulated phosphatase activity of calcineurin, which influences activation of the transcription factor nuclear factor of activated T cells, was shown to block virally mediated T-cell proliferation. Taken together, these findings suggest that the effect of SIVsmmPBj14 on T-cell activation may be functionally analogous, at least in part, to the effect of engagement of the T-cell receptor.
Assuntos
Imunoconjugados , Ativação Linfocitária , Vírus da Imunodeficiência Símia/imunologia , Abatacepte , Animais , Antígenos CD , Antígenos de Diferenciação/fisiologia , Antígeno B7-1/fisiologia , Antígenos CD28/fisiologia , Antígenos CD40/fisiologia , Antígeno CTLA-4 , Ciclosporina/farmacologia , Humanos , Macaca nemestrinaRESUMO
Simian immunodeficiency virus strain PBj14, SIVsmmPBj14, is unique among primate lentiviruses in its ability to trigger the proliferation of resting simian lymphocytes and to cause the rapid death of experimentally inoculated pigtailed macaques. Severe enteropathy, immune activation, and extensive apoptosis, particularly within gut-associated lymphoid tissue, characterize the acute disease syndrome associated with SIVsmmPBj14 infection. In the present study, we examined whether the ability of this virus to cause widespread apoptosis might be linked to the up-regulation of Fas ligand (CD95L) expression in virally infected cells. In vitro studies revealed that expression of the viral Nef protein, in the absence of any other viral gene product, was sufficient to up-regulate the transcriptional activity of the CD95L promoter and to cause cell surface expression of Fas ligand. This up-regulation was NFAT dependent (inhibited by cyclosporin A) and did not occur in cells that expressed a mutated derivative of the viral Nef protein, lacking a previously defined immunoreceptor tyrosine-based activation motif. These findings were corroborated by analysis of tissue sections from virally infected macaques. Immunohistochemical staining revealed that Fas ligand expression was efficiently up-regulated in the GALT of animals that had been experimentally infected with wild-type SIVsmmPBj14 but not in animals that were infected with a nonacutely pathogenic viral mutant lacking the Nef ITAM. Taken together, these results suggest that the ability of SIVsmmPBj14 to cause acutely lethal disease and to up-regulate FasL expression may be linked. Additional studies will be required to determine whether the induction of FasL expression is in itself important for acute disease pathogenesis.
Assuntos
Regulação da Expressão Gênica , Glicoproteínas de Membrana/genética , Proteínas Nucleares , Vírus da Imunodeficiência Símia/fisiologia , Vírus da Imunodeficiência Símia/patogenicidade , Animais , Membrana Celular/metabolismo , Clonagem Molecular , Proteínas de Ligação a DNA/metabolismo , Proteína Ligante Fas , Produtos do Gene nef/biossíntese , Produtos do Gene nef/genética , Genes Reporter , Genes nef , Humanos , Células Jurkat , Macaca , Macaca mulatta , Glicoproteínas de Membrana/biossíntese , Fatores de Transcrição NFATC , Fases de Leitura Aberta , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/biossíntese , Síndrome de Imunodeficiência Adquirida dos Símios/fisiopatologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , TransfecçãoRESUMO
Human immunodeficiency virus type 1 (HIV-1) is the etiologic agent of acquired immunodeficiency syndrome (AIDS) and is estimated to presently infect 24 million adults and 1.5 million children, worldwide. The pathogenesis of HIV-1-induced disease is complex and characterized by the interplay of both viral and host factors, which together determine the outcome of infection. An improved understanding of the pathogenic mechanisms of AIDS, combined with recent insights into the dynamics of viral infection, and the cellular co-receptors for HIV-1, may provide powerful new opportunities for therapeutic intervention against this virus.
Assuntos
Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/patogenicidade , Adulto , Fármacos Anti-HIV/química , Fármacos Anti-HIV/uso terapêutico , Criança , Quimioterapia Combinada , Infecções por HIV/imunologia , HIV-1/genética , HIV-1/fisiologia , Humanos , Receptores Virais/metabolismoRESUMO
Mutations in the 5' nontranslated RNA (5'NTR) of an attenuated, cell culture-adapted hepatitis A virus (HAV), HM175/P16, enhance growth in cultured African green monkey kidney (BS-C-1) cells but not in fetal rhesus monkey kidney (FRhK-4) cells (S. P. Day, P. Murphy, E. A. Brown, and S. M. Lemon, J. Virol. 66: 6533-6540, 1992). To determine whether these mutations enhance cap-independent translation directed by the HAV internal ribosomal entry site (IRES), we compared the translational activities of the 5'NTRs of wild-type and HM175/P16 viruses in two stably transformed cell lines (BT7-H and FRhK-T7) which constitutively express cytoplasmic bacteriophage T7 RNA polymerase and which are derived from BS-C-1 and FRhK-4 cells, respectively. Translational activity was assessed by monitoring expression of a reporter protein, chloramphenicol acetyltransferase (CAT), following transfection with plasmid DNAs containing bicistronic T7 transcriptional units of the form luciferase-5'NTR-CAT. In both cell types, transcripts containing the 5'NTR of HM175/P16 expressed CAT at levels that were 50- to 100-fold lower than transcripts containing the IRES elements of Sabin type 1 poliovirus or encephalomyocarditis virus, confirming the low activity of the HAV IRES. However, in BT7-H cells, transcripts containing the 5'NTR of wild-type virus. This translational enhancement was due to additive effects of a UU deletion at nucleotides 203 and 204 and a U-to-G substitution at nucleotide 687 of HM175/P16. These mutations did not enhance translation in FRhK-T7 or Huh-T7 cells (a T7 polymerase-expressing cell line derived from human hepatoblastoma cells) or in vitro in rabbit reticulocyte lysates. These results demonstrate that mutations in the 5'NTR of a cell culture-adapted HAV enhance viral replication by facilitating cap-independent translation in a cell-type-specific fashion and support the concept that picornaviral host range is determined in part by differences in cellular translation initiation factors.
Assuntos
Regulação Viral da Expressão Gênica , Hepatovirus/genética , Biossíntese de Proteínas , Capuzes de RNA/genética , RNA Viral/genética , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Chlorocebus aethiops , DNA Viral , RNA Polimerases Dirigidas por DNA/metabolismo , Humanos , Fígado/citologia , Macaca mulatta , Dados de Sequência Molecular , Mutação , Proteínas ViraisRESUMO
To investigate which hairpin structures within the 5' untranslated region of hepatitis C virus (HCV) are necessary for cap-independent translation, mutants were constructed that lack one or more hairpin structures. Here we demonstrate, by constructing precisely defined hairpin deletion mutants, that with the exception of the most 5' located hairpin structure, which on deletion shows an increase on translation, each of the predicted hairpins is found to be essential for cap-independent translation. In addition, we demonstrate that HCV 5'UTR driven translation is stimulated by poliovirus 2Apro co-expression.
Assuntos
Hepacivirus/genética , Hepacivirus/metabolismo , Biossíntese de Proteínas , Sequências Repetitivas de Ácido Nucleico , Animais , Linhagem Celular , Cloranfenicol O-Acetiltransferase/biossíntese , Chlorocebus aethiops , Primers do DNA , Expressão Gênica , Humanos , Neoplasias Laríngeas , Mutagênese , Conformação de Ácido Nucleico , Iniciação Traducional da Cadeia Peptídica , Poliovirus/genética , Poliovirus/metabolismo , Reação em Cadeia da Polimerase , Mapeamento por Restrição , Deleção de Sequência , Transfecção , Células Tumorais CultivadasRESUMO
To characterize in vivo the translational control elements present in the 5' nontranslated region (5'NTR) of hepatitis A virus (HAV) RNA, we created an HAV-permissive monkey kidney cell line (BT7-H) that stably expresses T7 RNA polymerase and carries out cytoplasmic transcription of uncapped RNA from transfected DNA containing the T7 promoter. The presence of an internal ribosomal entry site (IRES) within the 5'NTR of HAV was confirmed by using BT7-H cells transcribing bicistronic RNAs in which the 5'NTR was placed within the intercistronic space, controlling translation of a downstream reporter protein (bacterial chloramphenicol acetyltransferase). However, translation directed by the 5'NTR in these bicistronic transcripts and in monocistronic T7 transcripts in which the HAV 5'NTR was placed upstream of the chloramphenicol acetyltransferase coding sequence was very inefficient compared with the translation of monocistronic transcripts containing either the IRES of encephalomyocarditis (EMC) virus or a short nonpicornavirus 5' nontranslated leader sequence. A large deletion within the HAV IRES (delta 355-532) eliminated IRES activity in bicistronic transcripts. In contrast, larger deletions within the IRES in monocistronic transcripts (delta 1-354, delta 1-532, delta 1-633, and delta 158-633) resulted in 4- to 14-fold increases in translation. In the latter case, this was most probably due to a shift from IRES-directed translation to translation initiation by 5'-end-dependent scanning. Translation of RNAs containing either the EMC virus IRES or the nonpicornavirus leader was significantly enhanced by cotransfection of the reporter constructs with pEP2A, which directs transcription of RNA containing the EMC virus IRES fused to the poliovirus 2Apro coding region. This 2Apro enhancement of cap-independent translation suggests a greater availability of limiting cellular translation factors following 2Apro-mediated cleavage of the p220 subunit of the eukaryotic initiation factor eIF-4F and subsequent shutdown of 5' cap-dependent translation. In contrast, pEP2A cotransfection resulted in severe inhibition of translation directed by the HAV IRES in either monocistronic or bicistronic transcripts. This inhibition was due to competition from the EMC virus IRES present in pEP-2A transcripts, as well as the expression of proteolytically active 2Apro. 2Apro-mediated suppression of HAV translation was not seen with transcripts containing large deletions in the HAV IRES (delta 158-633, delta 1-532, or delta 1-633). These data suggest that the HAV IRES may have a unique requirement for intact p220 or that it may be dependent on active expression of another cellular translation factor which is normally present in severely limiting quantities.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Hepatovirus/genética , Íntrons , Biossíntese de Proteínas , RNA Viral/genética , Animais , Sequência de Bases , Linhagem Celular , DNA Viral , RNA Polimerases Dirigidas por DNA/metabolismo , Genes , Haplorrinos , Rim/citologia , Dados de Sequência Molecular , Ribossomos/química , Ribossomos/metabolismo , Deleção de Sequência , Transcrição Gênica , TransfecçãoRESUMO
Hepatitis A virus (HAV) exhibits several characteristics which distinguish it from other picornaviruses, including slow growth in cell culture even after adaptation, and lack of host-cell protein synthesis shut-down. Like other picornaviruses, HAV contains a long 5' nontranslated region (NTR) incorporating an internal ribosomal entry site (IRES), which directs cap-independent translation. We compared HAV IRES-initiated translation with translation initiated by the structurally similar encephalomyocarditis virus (EMCV) IRES, using plasmids in which each of the 5'NTRs is linked in-frame with the chloramphenicol acetyltransferase (CAT) gene. Translation was assessed in an HAV-permissive cell line which constitutively expresses T7 RNA polymerase and transcribes high levels of uncapped RNA from these plasmids following transfection. RNAs containing the EMCV IRES were efficiently translated in these cells, while those containing the HAV IRES were translated very poorly. Analysis of translation of these RNAs in the presence of poliovirus protein 2A, which shuts down cap-dependent translation, demonstrated that their translation was cap independent. Our results suggest that the HAV IRES may function poorly in these cells, and that inefficient translation may contribute to the exceptionally slow replication cycle characteristic of cell culture-adapted HAV.
Assuntos
Bacteriófago T7/enzimologia , RNA Polimerases Dirigidas por DNA/biossíntese , Hepatovirus/metabolismo , Biossíntese de Proteínas , Proteínas Virais , Animais , Bacteriófago T7/genética , Sequência de Bases , Linhagem Celular , Cloranfenicol O-Acetiltransferase , Cisteína Endopeptidases/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Dados de Sequência Molecular , Capuzes de RNA , Proteínas Recombinantes/biossíntese , Sequências Reguladoras de Ácido Nucleico/genética , TransfecçãoRESUMO
An IgG1 mouse monoclonal antibody, designated 1.646, is described which recognizes a cytoplasmic antigen of equine mononuclear phagocytes. Indirect fluorescent antibody staining of peripheral blood leukocytes reveals a granular cytoplasmic staining, predominantly in adherent blood mononuclear cells. Indirect fluorescent antibody staining is positive for alveolar and peritoneal macrophages. In some horses, a few neutrophils are also stained. In equine tissue samples stained by immunohistochemistry, the distribution of positive cells is consistent with the distribution of tissue macrophages. The most intense and reliable staining occurs with splenic and lymph node macrophages. Hepatic Kupffer cells also stain with antibody 1.646, although the intensity of that staining is somewhat variable between horses. A granular pattern of staining typical of lipofuscin deposition is also seen in liver sections. There is also pale staining of some biliary and renal tubular epithelium. Equine erythrocytes, platelets and lymphocytes are not recognized by this antibody, and neither are monocyte/macrophages of human, canine or feline origin. Antibody 1.646 recognizes two proteins (150 and 30 kDa) of equine monocyte-derived macrophages when assayed by Western immunoblot. Because of the distribution of staining (tissue mononuclear phagocytes, lipofuscin-containing storage granules, biliary and renal tubular epithelium, and some neutrophils) we hypothesize that antibody 1.646 recognizes a cytoplasmic antigen that is closely associated with lysosomal membranes.
Assuntos
Anticorpos Monoclonais/biossíntese , Cavalos/imunologia , Leucócitos Mononucleares/imunologia , Fagócitos/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/biossíntese , Especificidade de Anticorpos , Antígenos Virais/imunologia , Western Blotting/veterinária , Anemia Infecciosa Equina/etiologia , Anemia Infecciosa Equina/imunologia , Imunofluorescência/veterinária , Hibridomas , Imunoglobulina G/biossíntese , Imunoglobulina G/imunologia , Fígado/imunologia , Pulmão/imunologia , Linfonodos/imunologia , Macrófagos/imunologia , Camundongos , Baço/imunologiaRESUMO
The five viruses which classically cause hepatitis in man represent diverse families of viruses and share in common only a striking hepatotropism and substantial restrictions to replication in conventional cell cultures. Hepatitis A virus is unique among these viruses in that it is amenable to propagation in cell culture, but replication of this virus is much slower and less efficient than replication of other picornaviruses. This probably reflects less efficient cap-independent viral translation, as well as restrictions at other points in the replication cycle. We speculate that the significantly restricted replication of hepatitis viruses in cell culture reflects evolutionary forces controlling their transmission and propagation through human populations.
Assuntos
Vírus de Hepatite/fisiologia , Replicação Viral , Sequência de Bases , Evolução Biológica , Células Cultivadas , Vírus de Hepatite/genética , Hepatovirus/genética , Hepatovirus/fisiologia , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Biossíntese de Proteínas , RNA Viral/química , RNA Viral/genética , Cultura de Vírus , Replicação Viral/genéticaRESUMO
Virulent, wild-type equine infectious anemia virus (EIAV) is restricted in one or more early steps in replication in equine skin fibroblast cells compared with cell culture-adapted virus, which is fully competent for replication in this cell type. We compared the sequences of wild-type EIAV and a full-length infectious proviral clone of the cell culture-adapted EIAV and found that the genomes were relatively well conserved with the exception of the envelope gene region, which showed extensive sequence differences. We therefore constructed several wild-type and cell culture-adapted virus chimeras to examine the role of the envelope gene in replication in different cell types in vitro. Unlike wild-type virus, which is restricted by an early event(s) for replication in equine dermis cells, the wild-type outer envelope gene chimeras are replication competent in this cell type. We conclude that even though there are extensive sequence differences between wild-type and cell culture-adapted viruses in the surface envelope gene region, this domain is not a determinant of the differing in vitro cell tropisms.
Assuntos
Vírus da Anemia Infecciosa Equina/genética , Proteínas do Envelope Viral/genética , Animais , Sequência de Bases , Linhagem Celular , DNA Viral , Anemia Infecciosa Equina/microbiologia , Cavalos , Vírus da Anemia Infecciosa Equina/patogenicidade , Vírus da Anemia Infecciosa Equina/fisiologia , Cinética , Dados de Sequência Molecular , Fenótipo , Provírus/genética , Homologia de Sequência do Ácido Nucleico , Virulência/genética , Replicação ViralRESUMO
An adult horse infected with a virulent, cell culture-adapted strain of equine infectious anemia virus (EIAV) developed cyclical thrombocytopenia in which the nadir of platelet counts coincided with peak febrile responses. In order to investigate the mechanism of thrombocytopenia during acute febrile episodes, four adult horses were experimentally infected with the wild-type Wyoming strain of EIAV. Platelet counts decreased from baseline as rectal temperature increased. Serum reverse transcriptase activity increased above background levels in all horses, coincident with increase in rectal temperature. All horses developed an EIAV-specific immune response detectable by Western immunoblot by postinfection day 10. Increases in platelet-associated immunoglobulins G and M were detectable by direct fluorescent-antibody test and flow cytometric assay. Viral replication in bone marrow megakaryocytes was not detectable by in situ hybridization. Results suggest an immune-mediated mechanism of thrombocytopenia in horses infected with EIAV. Despite an inability to identify virion particles in association with platelet-bound antibody, the cyclical nature of the thrombocytopenia and the occurrence of a marked cell-free viremia concomitant with fever and thrombocytopenia suggest immune complex deposition on platelets. We propose that clearance of virus and antibody-coated platelets from the peripheral circulation by hepatic Kupffer cells and splenic macrophages may target infectious virus particles, in the form of immune complexes, to host cells most permissive for in vivo viral replication.
Assuntos
Anemia Infecciosa Equina/imunologia , Vírus da Anemia Infecciosa Equina/isolamento & purificação , Trombocitopenia/imunologia , Animais , Biomarcadores/sangue , Plaquetas/fisiologia , Western Blotting , Medula Óssea/microbiologia , Medula Óssea/patologia , Clonagem Molecular , Anemia Infecciosa Equina/sangue , Anemia Infecciosa Equina/microbiologia , Imunofluorescência , Genes gag , Cavalos , Vírus da Anemia Infecciosa Equina/genética , Hibridização de Ácido Nucleico , Contagem de Plaquetas , Sondas RNA , DNA Polimerase Dirigida por RNA/sangue , Mapeamento por RestriçãoRESUMO
A full-length molecular clone of equine infectious anemia virus (EIAV) was isolated from a persistently infected canine fetal thymus cell line (Cf2Th). Upon transfection of equine dermis cells, the clone, designated CL22, yielded infectious EIAV particles (CL22-V) that replicated in vitro in both Cf2Th cells and an equine dermis cell strain. Horses infected with CL22-V developed an antibody response to viral proteins and possessed viral DNA in peripheral blood mononuclear cells, as determined by polymerase chain reaction assays. In addition, horses infected with CL22-V became persistently infected and were capable of transmitting the infection by transfer of whole blood to uninfected horses. However, CL22-V, like the parental canine cell-adapted virus, did not cause clinical signs in infected horses. Reverse transcriptase assays of CL22-V- and virulent EIAV-infected equine mononuclear cell cultures indicated that the lack of virulence of CL22-V was not due to an inability to infect and replicate in equine mononuclear cells in vitro.
Assuntos
Anemia Infecciosa Equina/microbiologia , Genes Virais , Cavalos/microbiologia , Vírus da Anemia Infecciosa Equina/genética , Animais , Sequência de Bases , Células Cultivadas , Clonagem Molecular , Vírus da Anemia Infecciosa Equina/isolamento & purificação , Vírus da Anemia Infecciosa Equina/fisiologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Provírus/genética , Provírus/isolamento & purificação , Pele , Transfecção , Replicação ViralRESUMO
The temporal appearance of 4 viral proteins was detected in bluetongue virus (BTV)-infected Vero cells by indirect immunofluorescence staining with monoclonal antibodies (MAb) to BTV structural proteins VP2 and VP7 and nonstructural proteins NS1 and NS2. Bluetongue viral proteins were detected at distinct intervals after inoculation of Vero cells; VP7 was first detected 3 hours after inoculation, NS1 and NS2 at 5 hours after inoculation, whereas VP2 was not detected until 8 hours after inoculation. Patterns of fluorescence varied with the fixative used, but each MAb induced a distinct pattern of fluorescence in infected cells. Flow cytometry, which was used with each of the 4 MAb, proved to be an accurate and sensitive method of detecting BTV-infected P3 mouse myeloma cells. The temporal appearance of each viral protein in BTV-infected P3 cells was similar to that detected in BTV-infected Vero cells. Advantages of flow cytometry over conventional immunofluorescence staining to detect BTV-infected cells included: (1) enumeration of the proportion of infected cells in a population; (2) further characterization of infected cells, including estimates of their viability; and (3) computer-assisted storage and analysis of data obtained.
Assuntos
Anticorpos Monoclonais , Vírus Bluetongue , Reoviridae , Proteínas Virais/análise , Animais , Separação Celular , Citometria de Fluxo/métodos , Imunofluorescência , Fatores de Tempo , Células Vero , Proteínas Virais/biossíntese , Proteínas Estruturais Virais/análise , Proteínas Estruturais Virais/biossínteseRESUMO
Cultures of adherent and non-adherent bovine blood mononuclear cells were infected with bluetongue virus (BTV) serotype 10. Production of BTV proteins in mononuclear cell cultures was detected by immune precipitation of viral proteins from [35S]methionine-labelled extracts of these cells, by immunofluorescence staining of cells using monoclonal antibodies (MAbs) to BTV proteins VP7 and NS2, and by flow cytometry with MAbs to VP2, VP7, NS1 and NS2. BTV-infected cells were most numerous in cultures of adherent mononuclear cells; infected cells were initially identified as monocytes on the basis of their morphology, and size and scatter characteristics as determined by analysis with a fluorescence-activated cell sorter (FACS). The majority of adherent mononuclear cells with these scatter characteristics were confirmed to be monocytes by FACS analysis with a MAb specific for bovine monocytes. Identification of BTV-infected adherent mononuclear cells as monocytes was further established by double immunofluorescent labelling, as infected adherent cells reacted with the MAb specific for bovine monocytes, and with another MAb specific for class II antigen. Infection of adherent mononuclear cells was also confirmed by transmission electron microscopy, as BTV virions and tubules were present in lysates of cultures of BTV-infected adherent mononuclear cells and within the cytoplasm of adherent cells. In contrast, BTV proteins were detected in few cells identified as lymphocytes on the basis of their scatter characteristics, and mean fluorescence of such cells was considerably less than that of BTV-infected monocytes. Viraemia persisted until 35 days after inoculation of a colostrum-deprived calf inoculated with BTV. Virus was isolated from blood mononuclear cells at 1 week after infection of the calf, but not thereafter. BTV infection of blood mononuclear cells was demonstrated until 9 days after inoculation by indirect immunofluorescence staining of mononuclear cells. In contrast, virus was consistently isolated from erythrocyte-enriched preparations throughout viraemia in titres comparable to those in whole blood. These results indicate that although bovine monocytes are readily infected in vitro with this strain of BTV serotype 10, infection of blood monocytes is unlikely to be responsible for the prolonged viraemia that consistently occurs in BTV-infected cattle.
Assuntos
Vírus Bluetongue/crescimento & desenvolvimento , Monócitos/microbiologia , Reoviridae/crescimento & desenvolvimento , Animais , Anticorpos Monoclonais , Especificidade de Anticorpos , Autorradiografia , Bluetongue/sangue , Bluetongue/microbiologia , Vírus Bluetongue/imunologia , Vírus Bluetongue/ultraestrutura , Bovinos , Células Cultivadas , Citometria de Fluxo , Imunofluorescência , Monócitos/análise , Monócitos/ultraestrutura , Testes de PrecipitinaRESUMO
Infection of three calves with a highly plaque-purified strain of bluetongue virus (BTV) resulted in prolonged infections, during which virus and neutralizing antibodies co-circulated in peripheral blood. Oligonucleotide fingerprint analyses of the original challenge virus and of the final virus isolate obtained from each calf demonstrated the BTV genome to remain stable throughout prolonged infection as no differences in fingerprint patterns were detected. Six neutralizing monoclonal antibodies (MAbs), and a polyclonal rabbit antiserum, were produced against the challenge virus. This panel of MAbs recognized at least two distinct neutralizing epitopes as demonstrated by immune precipitation. Neutralizing epitopes remained stable through the prolonged infections, as all MAbs and the polyclonal rabbit antiserum neutralized the challenge virus and the final calf isolates to equivalent titres. These results suggest that antigenic drift is not the mechanism by which BTV is able to persist in cattle in spite of a strong humoral immune response.