RESUMO
Symptoms of acute febrile respiratory tract infection are often unspecific, but the rapid identification of pathogens allows optimised patient management. The objective of this study was to evaluate a novel multiplex polymerase chain reaction (PCR) suspension microarray which detects 19 viral and four atypical bacterial targets. A comprehensive set of sensitive monoplex real-time PCR assays was used for each pathogen as the gold standard. A panel of archived as well as 300 prospectively collected clinical samples was analysed by both methods. At least one target was detected in 165/300 (55 %) samples by monoplex PCR and in 140/300 (46 %) samples by multiplex PCR, respectively. The positivity rate was significantly higher in paediatric patients compared to adults [126/154 (82 %) vs. 39/146 (27 %) by monoplex and 114/154 (74 %) vs. 26/146 (18 %) by multiplex PCR, respectively]. Among all samples, 17/300 (5.6 %) were positive for atypical bacteria by monoplex and 8/300 (2.6 %) by multiplex PCR, respectively. Multiple detections were recorded in 35/300 (11.6 %) samples by monoplex and 26/300 (8.7 %) by multiplex PCR. For the most common pathogens, the sensitivity ranged from 57 to 93 % and the specificity ranged from 95 to 100 %. The overall concordance between both methods was 77 % [95 % confidence interval (CI) 72-81 %]. False-negative results by multiplex PCR were mainly due to the low target concentration. Compared to monoplex PCR, the novel microarray assay proved its principle but displayed overall lower sensitivities, potentially restricting its use to paediatric patients. For some targets, only small numbers of positive samples were available, requiring larger studies to firmly assess the sensitivity and specificity.
Assuntos
Bactérias/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex/métodos , Doenças Nasofaríngeas/diagnóstico , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Vírus/isolamento & purificação , Adulto , Bactérias/classificação , Infecções Bacterianas/diagnóstico , Infecções Bacterianas/microbiologia , Criança , Pré-Escolar , Intervalos de Confiança , Humanos , Lactente , Doenças Nasofaríngeas/microbiologia , Doenças Nasofaríngeas/virologia , Nasofaringe/microbiologia , Nasofaringe/virologia , Estudos Prospectivos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Estatísticas não Paramétricas , Viroses/diagnóstico , Viroses/virologia , Vírus/classificação , Adulto JovemRESUMO
Quantitative measurement of anti-HBs is used to evaluate the response to hepatitis B vaccination in health care workers and to optimize postexposure management. The different guidelines for hepatitis B vaccination and booster policy imply that the measurement of anti-HBs levels by different assays is accurate and consistent, yielding comparable quantitative results. We measured anti-HBs levels in 200 serum samples from patients and health care professionals by nine different anti-HBs assays and compared the quantitative results and the performance characteristics of the different test systems. The assay specificity ranged between 96.8 and 100% when sera from individuals without a vaccination history and with negative anti-HBc status were defined as true negatives. Sensitivity ranged between 93.5 and 100%. A high number of sera showed discrepancies between measurements by the different systems. The mean coefficient of variation between the different measurements was 47.1% (range, 15.0 to 201.0%), and the factors of multiplication ranged from 2.8 to 105. Hemolysis or lipemia did not seem to influence the measurement, and there was no difference between anti-HBc-positive and -negative individuals. The classical enzyme immunoassays tend to find lower anti-HBs levels than the automated systems, with higher values by the Abbott AXSYM assay. The serial dilution of the international standard preparation was measured accurately by most of the assays. In conclusion, the quantitative measurement of anti-HBs levels is not reliable, even though an international standard is used for the calibration of the systems. Some systems showed specific problems that should be addressed by the manufacturers.
Assuntos
Anticorpos Anti-Hepatite B/sangue , Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Imunoensaio/métodos , Medições Luminescentes/métodos , Kit de Reagentes para Diagnóstico , Automação , Reações Falso-Negativas , Humanos , Técnicas Imunoenzimáticas , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeAssuntos
Bocavirus/isolamento & purificação , DNA Viral/análise , Infecções por Parvoviridae/virologia , Reação em Cadeia da Polimerase/métodos , Infecções Respiratórias/virologia , Sequência de Bases , Bocavirus/genética , Pré-Escolar , Hospitalização , Humanos , Lactente , Masculino , Dados de Sequência MolecularRESUMO
Human metapneumovirus (HMPV) was isolated from a 63-year-old multiple myeloma patient who had undergone hematopoietic stem cell transplantation and who presented with lower respiratory tract infection several weeks prior to the diagnosis of lung cancer. The isolate was phylogenetically and biologically characterized and compared to HMPV prototypes and recent pediatric isolates. Remarkably, it belonged to the novel genomic subgroup A2b.
Assuntos
Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Neoplasias Pulmonares/complicações , Metapneumovirus/isolamento & purificação , Mieloma Múltiplo/complicações , Infecções por Paramyxoviridae/virologia , Infecções Respiratórias/virologia , Animais , Linhagem Celular , Chlorocebus aethiops , Evolução Fatal , Humanos , Masculino , Metapneumovirus/classificação , Metapneumovirus/genética , Pessoa de Meia-Idade , Infecções por Paramyxoviridae/diagnóstico , Filogenia , Infecções Respiratórias/diagnóstico , Células Vero , Replicação ViralAssuntos
Infecções por Citomegalovirus/transmissão , Granulócitos/transplante , Transfusão de Leucócitos/efeitos adversos , Leucemia-Linfoma Linfoblástico de Células Precursoras/complicações , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Adolescente , Transplante de Medula Óssea/métodos , Infecções por Citomegalovirus/prevenção & controle , Feminino , Fator Estimulador de Colônias de Granulócitos , Humanos , Doadores de Tecidos , Transplante HomólogoRESUMO
Foamy viruses (FVs) are common apathogenic retroviruses readily spread by horizontal transmission in nonhuman primate and some other mammalian host populations. Primate FV infections have been known for half a century, i.e., 15 years before the definition of retroviruses and another 15 years before the detection of primate immune deficiency viruses. The emerging interest in human retroviruses included primate FV, and although the role of human hosts for FV was greatly overestimated temporarily, enthusiastic researchers compiled invaluable data on molecular biology and classic as well as molecular epidemiology of these viruses. It has been shown that lytic FV infection in a wide range of cell cultures is in great contrast to the silent state of the infection in animals. Once transmitted by saliva via biting, FVs reside in all tissues as DNA copies, but their replication is untraceable except in oral submucosal cells, which are thought to supply the virus for transmission. FVs have not definitely been associated with any disease, regardless of viral phylogenetic differences. Various primate and nonprimate species have been used for studies on the natural carrier state and primary infection. Experimental infections have mostly proven to be inefficient in primates as well as lower laboratory animals. However, investigation of the immune response in FV-infected animals has only partly explained the control of FV replication in the animal host. Thus, the biological role of FV remains an enigma to be resolved in the future.
Assuntos
Infecções por Retroviridae/veterinária , Spumavirus/fisiologia , Animais , Linhagem Celular , Haplorrinos , Camundongos , Modelos Animais , Filogenia , Coelhos , Infecções por Retroviridae/epidemiologia , Infecções por Retroviridae/imunologia , Infecções por Retroviridae/transmissão , Spumavirus/classificação , Spumavirus/isolamento & purificação , Replicação ViralRESUMO
BACKGROUND: The aim of this study was to evaluate pp65 antigen-guided antiviral therapy in preventing human cytomegalovirus (HCMV) infection in solid organ transplant recipients. METHODS: Ten kidney and two liver transplant recipients with asymptomatic HCMV infection were randomized either for i.v. ganciclovir or placebo treatment in a prospective, double-blind study. All patients were positive by HCMV pp65 antigen test at levels >5 positive cells/2 x 10(5) investigated cells. RESULTS: No cases of HCMV end-organ disease occurred. In contrast to patients on placebo (5/7), none of the patients on ganciclovir (0/5) developed HCMV-associated symptoms (P=0.01). However, because of the small number of patients, all three high-risk patients (donor seropositive, recipient seronegative) were randomized to placebo and all three developed symptoms. CONCLUSIONS: Preemptive antiviral therapy guided by the pp65 antigen test seems to have a beneficial effect on preventing HCMV-associated symptoms in kidney and liver transplant recipients.
Assuntos
Antivirais/uso terapêutico , Infecções por Citomegalovirus/prevenção & controle , Sistemas de Liberação de Medicamentos , Ganciclovir/administração & dosagem , Transplante de Rim , Transplante de Fígado , Fosfoproteínas/uso terapêutico , Proteínas da Matriz Viral/uso terapêutico , Método Duplo-Cego , Ganciclovir/uso terapêutico , Humanos , Estudos ProspectivosRESUMO
Due to various advantageous features there is current interest in retroviral vectors derived from primate foamy viruses (PFVs). Two PFV cis-acting sequences have been mapped in the 5' region of the RNA (pre-)genome and in the 3' pol genomic region. In order to genetically separate PFV packaging constructs from vector constructs, we investigated the effect of deletions in the 5' untranslated region (UTR) of PFV packaging constructs and vectors on gene expression and RNA incorporation into viral particles. Our results indicate that the 5' UTR serves different previously unknown functions. First, the R region of the long terminal repeat was found to be required for PFV gag gene expression. This regulation of gene expression appeared to be mainly posttranscriptional. Second, constructs with sequence deletions between the R region and the gag gene start codon packaged as much viral mRNA into particles as the undeleted construct, and RNA from such a 5'-UTR-deleted packaging construct was copackaged into vector-virus particles, together with vector RNA which was preferentially packaged. Finally, in the U5 region a sequence was identified that was required to allow cleavage of the Gag precursor protein by the pol gene-encoded protease, suggesting a role of RNA in PFV particle formation. Taken together, the results indicate that complex interactions of the viral RNA, capsid, and polymerase proteins take place during PFV particle formation and that a clear separation of PFV vector and packaging construct sequences may be difficult to achieve.
Assuntos
Regiões 5' não Traduzidas/genética , Regulação Viral da Expressão Gênica , RNA Viral/genética , Deleção de Sequência , Spumavirus/genética , Animais , Produtos do Gene gag/genética , Primatas , Sequências Repetitivas de Ácido NucleicoRESUMO
The genetic variability of the envelope surface domain (SU) of simian foamy virus (FV) of African green monkeys was studied. To assess the interindividual diversity of FV, isolates were obtained from 19 animals living together in a monkey house. The monkeys had been imported from Kenya prior to being placed in long-term housing in the research institute. In addition, a simian FV isolate and proviral DNA were obtained from an animal caretaker infected in this setting. DNA of the complete SU (1779 to 1793 bp) was analyzed by PCR and sequencing. The sequences revealed four clusters with high homologies (>95%). Between the clusters, divergencies ranged from 3 to 25%. Obviously, the clusters reflect four different strains or subtypes of simian FV type 3 that were prevalent in the colony. In contrast to lentiviruses, hypervariable regions could not be detected in the FV SU. Furthermore, to analyze the intraindividual diversity of FV, we investigated the virus population within an individual monkey at a given time point and its evolution over 13 years. For this purpose, 22 proviral SU clones generated by PCR from one oral swab and seven isolates obtained from the same animal between 1982 and 1995 were examined. These sequences revealed exceptionally high homology rates (99.5 to 100%), and only a minimal genetic drift was recognized within the series of isolates. In conclusion, the low in vivo divergency of FV SU suggests that genetic variability is not important for the maintenance of FV persistence.
Assuntos
Chlorocebus aethiops/virologia , Variação Genética , Doenças dos Macacos/virologia , Infecções por Retroviridae/veterinária , Spumavirus/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Clonagem Molecular , Genes env/genética , Humanos , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Estrutura Terciária de Proteína , Infecções por Retroviridae/virologia , Análise de Sequência de DNA , Spumavirus/isolamento & purificação , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genéticaRESUMO
Foamy viruses (FVs) make use of a replication strategy which is unique among retroviruses and shows analogies to hepadnaviruses. The presence of an integrase (IN) and obligate provirus integration distinguish retroviruses from hepadnaviruses. To clarify whether a functional IN is required for FV replication, a mutant in the highly conserved DD35E motif of the active centre was analysed. This mutant was found to be able to express Gag and Pol protein precursors and cleavage products and to generate and deliver cDNA. However, this mutant was replication-deficient. The junctions of individual foamy proviruses with cellular DNA were sequenced. The findings suggest that FV integration is asymmetrical, because the proviruses started with what is believed to be the U3 end of the free linear DNA to generate the conventional TG dinucleotide, while apparently two nucleotides from the U5 end were cleaved to create the complementary CA dinucleotide. Alignment of known FV genome sequences indicated that this mechanism of integration is not restricted to the two FV isolates from which integrates were studied, but appears to be a common feature of this retrovirus subfamily. In conclusion, with respect to the necessity of a functionally active IN for virus replication FVs behave like other retroviruses; their mechanism of integration, however, is probably unique.
Assuntos
Integrases/genética , Integrases/metabolismo , Spumavirus/fisiologia , Replicação Viral , Animais , Sequência de Bases , Southern Blotting , Linhagem Celular , DNA Viral/análise , Humanos , Dados de Sequência Molecular , Mutação , Provírus/genética , Análise de Sequência de DNA , Spumavirus/enzimologia , Spumavirus/genética , Transfecção , Integração ViralRESUMO
Foamy viruses (FV), retroviruses of the genus Spumavirus, are able to infect a wide variety of animal species and replicate in nearly all types of cultured cells. To identify the cells targeted by FV in the natural host and define the sites of viral replication, multiple organs of four African green monkeys naturally infected with simian FV type 3 were investigated for the presence of FV proviral DNA and viral transcripts. All organs contained significant amounts of FV proviral DNA. In addition to proviruses containing the complete transactivator gene taf, proviral genomes carrying a specific 295-bp deletion in the taf gene were detected in all monkeys. As in the case of human foamy virus the deletion leads to the formation of the bet gene that is regarded to be instrumental in the regulation of viral persistence. FV RNA was detected by RT-PCR and in situ hybridization only in the oral mucosa of one monkey. No other samples contained detectable levels of viral transcripts. Histopathological changes were not observed in any of the tissue samples analyzed. Our results show that the natural history of FV is characterized by latent infection in all organs of the host and by minimal levels of harmless viral replication in the oral mucosa. The broad host cell range in vivo further encourages the development of FV-derived vectors for therapeutic gene delivery.
Assuntos
Chlorocebus aethiops/virologia , Mucosa Bucal/virologia , Provírus , Spumavirus/fisiologia , Latência Viral , Replicação Viral , Animais , DNA Viral/análise , Proteínas de Ligação a DNA/genética , Humanos , Hibridização In Situ , Reação em Cadeia da Polimerase/veterinária , RNA Viral/análise , Proteínas dos Retroviridae/genética , Spumavirus/patogenicidade , Transativadores/genéticaAssuntos
Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Doenças do Sistema Digestório/virologia , Herpes Simples/complicações , Infecções Oportunistas/complicações , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Adulto , Evolução Fatal , Feminino , HumanosRESUMO
The activation of human peripheral blood lymphocytes by mitogens or by triggering the T-cell receptor with anti-CD3 antibodies leads to the production of a potent soluble inhibitory activity against foamy virus-induced cytopathic effects in vitro. The inhibitory activity acts in a species-specific manner. As a consequence, the isolation of foamy viruses from blood lymphocytes of infected humans is accelerated in a heterologous coculture system. Antibodies against gamma interferon (IFN-gamma) are able to suppress most of the inhibitory activity, suggesting that IFN-gamma is the dominant component.
Assuntos
Interferon gama/imunologia , Linfócitos/imunologia , Spumavirus/imunologia , Células 3T3 , Animais , Linhagem Celular , Cricetinae , Efeito Citopatogênico Viral , Haplorrinos , Humanos , Camundongos , Spumavirus/fisiologiaRESUMO
Studying the transmission of simian retroviruses to humans can help define the importance of these infections to public health. We identified a substantial prevalence (4/231, 1.8%) of infection with simian foamy viruses (SFV) among humans occupationally exposed to nonhuman primates. Evidence of SFV infection included seropositivity, proviral DNA detection and isolation of foamy virus. The infecting SFV originated from an African green monkey (one person) and baboons (three people). These infections have not as yet resulted in either disease or sexual transmission, and may represent benign endpoint infections.
Assuntos
Doenças Profissionais/epidemiologia , Doenças dos Primatas , Infecções por Retroviridae/transmissão , Spumavirus , Zoonoses/epidemiologia , Animais , Anticorpos Antivirais/sangue , Chlorocebus aethiops , Humanos , Linfócitos/virologia , Macaca mulatta , Masculino , Exposição Ocupacional , Pan troglodytes , Papio , Filogenia , Reação em Cadeia da Polimerase , Prevalência , Retroviridae/isolamento & purificação , Infecções por Retroviridae/epidemiologia , Infecções por Retroviridae/veterinária , Spumavirus/genética , Spumavirus/isolamento & purificaçãoRESUMO
Evidence for natural foamy virus (FV) infections in humans is still lacking. However, accidental infections of humans with simian FV have been demonstrated by serology and PCR, but all previous attempts to recover infectious virus in such cases have failed. Here we describe the isolation of a simian FV from peripheral blood mononuclear cells (PBMC) of a healthy animal caretaker, who acquired the virus 20 years ago from an African green monkey (AGM) bite. Properties of the human isolate such as host range in cell cultures including human PBMC and ability to induce neutralizing antibodies in the primate host proved to be similar to those of FV obtained from AGM. The genomic sequence of the isolate was found to be virtually identical to the proviral sequence present in the host lymphocytes and related to AGM isolates but distinct from those of all FV isolates handled in the laboratory. For successful virus isolation, it was essential to stimulate the host lymphocytes by phytohemagglutinin and interleukin-2 for 2 weeks prior to cocultivation with permissive cells. In contrast to the situation found in FV-infected monkeys, virus isolation from the saliva of the animal caretaker was not possible, and no evidence for FV transmission to family contacts was obtained. We conclude that, in contrast to active infection in monkeys, FV persists in a state of latency following accidental infection of humans.
Assuntos
Chlorocebus aethiops/microbiologia , Infecções por Retroviridae/microbiologia , Spumavirus/isolamento & purificação , Acidentes , Animais , Anticorpos Antivirais/análise , Mordeduras e Picadas/microbiologia , Células Cultivadas , Humanos , Exposição Ocupacional , Filogenia , Infecções por Retroviridae/etiologia , Homologia de Sequência do Ácido Nucleico , Fatores de TempoRESUMO
With the intention to evaluate the frequency of asymptomatic infections with TBE virus and B. burgdorferi clinical data and serum specimens were collected from 393 individuals living in an area endemic for both agents (Freiburg, southern Germany). Sera were examined by ELISA. Borderline and positive results were checked by immunoblotting. Only specific antibodies detected by immunoblotting (B. burgdorferi: 22 kDa, 31 kDa, 34 kDa, 39 kDa, 83 kDa; TBE: glycoprotein E) were assessed as positive findings. Specific antibodies to B. burgdorferi were detected in 17/105 individuals with possible symptoms of borreliosis (16%) and in 36/288 individuals without current or previous symptoms of borreliosis (12.5%). Antibody to TBE virus was demonstrated in 34/361 individuals (9.4%) without clinical symptoms of TBE or vaccination against TBE. Thirty individuals had been immunised against TBE (10.6%) and two had clinical TBE one year ago. Antibodies against both agents were detected only in 1.5% of all subjects. Considering the low seroprevalence, antibody screening is not recommended prior to TBE vaccination.
Assuntos
Anticorpos Antibacterianos/sangue , Anticorpos Antivirais/sangue , Encefalite Transmitida por Carrapatos/sangue , Doenças Endêmicas , Doença de Lyme/sangue , Encefalite Transmitida por Carrapatos/imunologia , Feminino , Alemanha/epidemiologia , Humanos , Doença de Lyme/imunologia , Masculino , Prevalência , Estudos ProspectivosRESUMO
Simian and human foamy virus (FV) DNA can be readily detected in peripheral blood leukocytes. However, it is unknown which leukocyte populations harbor the virus in vivo. We, therefore, analyzed blood samples from nine African green monkeys, four chimpanzees, and two humans for the presence of foamy virus proviral DNA in different FACS-purified leukocyte populations, using a highly sensitive nested polymerase chain reaction (PCR). The CD8+ lymphocytes were PCR positive in all 15 samples and the average viral burden was highest in this population. FV DNA was detected in 10 of 15 cell samples enriched for B lymphocytes, and 4 of 9 CD4+ lymphocyte, 3 of 13 CD14+ monocyte, and 4 of 13 polymorphonuclear leukocyte samples. A highly sensitive reverse transcriptase PCR was performed to detect viral transcripts in peripheral blood leukocytes. All samples were negative. In conclusion, lymphocytes, and especially CD8+ T lymphocytes, were found to be a major target for foamy virus in the peripheral blood, but viral gene expression was not detected.
Assuntos
Leucócitos/virologia , Spumavirus/isolamento & purificação , Animais , Linfócitos B/virologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/virologia , Chlorocebus aethiops , DNA Viral/análise , Humanos , Leucócitos Mononucleares , Receptores de Lipopolissacarídeos , Monócitos/virologia , Neutrófilos/virologia , Pan troglodytes , Células VeroRESUMO
Simian foamy virus type 3 (SFV-3), a member of the spumavirus genus of retroviruses, has a complex genome organization and encodes two open reading frames (ORFs), in addition to the structural genes gag, pol, and env. ORF-1 encodes a viral transcriptional transactivator designated Taf (transactivator of foamy viruses) which augments transcription from the viral long terminal repeat (LTR). It was recently shown that human foamy virus, as well as the simian viruses SFV-1 and SFV-3, contains a second internal transcriptional promoter in the transmembrane domain of the env gene; this promoter also is transactivated by Taf. Here we report the characterization of the internal promoter of SFV-3. The transcriptional start site of this promoter has been mapped in two different SFV-3-infected cell lines to nt position 9761 in the proviral genome of SFV-3. All cis-regulatory elements required for transactivation by Taf are located between -202 and -32 (+1 representing the transcription initiation site in the internal promoter). Analysis of hybrid promoter constructs and deletion mutants in transient expression assays revealed that this region contains two elements which are independently responsive to Taf. In addition, we employed an in vivo DNA competition assay to determine whether the transactivation mechanisms of both SFV-3 promoters are similar or different. The differences observed utilizing this competition assay suggest that Taf transactivates the internal promoter and the LTR through different cellular transcription factors.
Assuntos
Regiões Promotoras Genéticas , Sequências Repetitivas de Ácido Nucleico , Spumavirus/genética , Ativação Transcricional , Animais , Sequência de Bases , Sítios de Ligação , Chlorocebus aethiops , Mapeamento Cromossômico , DNA Viral , Regulação Viral da Expressão Gênica , Haplorrinos/virologia , Humanos , Dados de Sequência Molecular , Iniciação Traducional da Cadeia Peptídica , Transativadores/metabolismo , Células VeroRESUMO
Infection of hematopoietic progenitor cells with the human cytomegalovirus (HCMV) has been proposed as an explanation for the cytopenias associated with HCMV-related disease. To test this hypothesis, CD34+ cells, which include the hematopoietic progenitors, as well as mature leukocyte populations were purified on a fluorescence-activated cell sorter and analyzed for HCMV DNA by polymerase chain reaction (PCR). A total of 33 samples from 31 immunosuppressed as well as immunocompetent HCMV-seropositive individuals were studied. CD34+ cells were PCR-positive in four of seven bone marrow aspirates from allogeneic bone marrow transplant recipients, in three of eight aspirates from patients with acquired immunodeficiency syndrome, and in the first of two bone marrow samples from an immunocompetent patient with primary HCMV disease. CD34+ cells purified from peripheral blood for autologous and allogeneic transplantation were also analyzed, and 4 of 13 samples were HCMV DNA-positive. Interestingly, two of the four HCMV-positive samples were from healthy allogeneic donors. Among the mature leukocyte populations, the monocytes were most frequently found to be HCMV DNA-positive. No HCMV DNA was detected in the total bone marrow leukocytes of 13 healthy seropositive bone marrow donors or in the CD34+ cell fraction of three further seropositive donors. In conclusion, the data provide strong evidence that CD34+ hematopoietic progenitor cells can be infected with HCMV in immunosuppressed patients, while this cell population was not identified as a major viral reservoir in healthy HCMV-seropositive individuals.
Assuntos
Antígenos CD34/metabolismo , Citomegalovirus/isolamento & purificação , DNA Viral/isolamento & purificação , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/virologia , Leucócitos/imunologia , Leucócitos/virologia , Síndrome da Imunodeficiência Adquirida/imunologia , Síndrome da Imunodeficiência Adquirida/virologia , Sequência de Bases , Transplante de Medula Óssea/imunologia , Separação Celular , Citomegalovirus/genética , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/virologia , Primers do DNA/genética , DNA Viral/sangue , DNA Viral/genética , Citometria de Fluxo , Humanos , Tolerância Imunológica , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/estatística & dados numéricos , Sensibilidade e Especificidade , Viremia/imunologia , Viremia/virologiaRESUMO
Infectious proviral clones of simian foamy virus isolated from chimpanzee (SFVcpz) were generated by long PCR. Two overlapping fragments representing the complete provirus were amplified from genomic DNA of infected cells. Four 8.8-kbp amplimers extending from base 1 of the provirus into the env gene and five 4.45-kbp amplimers reaching from env to the end of the 3'-LTR were cloned into pCR II. Subsequently, the proviral fragments were combined in a chessboard manner to generate 20 plasmids containing full-length proviral DNA. Four plasmids produced infectious virus after transfection of susceptible cells. A distinct proviral form bearing a deletion in the transactivator gene joining both exons of a second regulatory gene present in wild-type foamy virus-infected cells started to emerge 48 hr after transfection of BHK cells with infectious SFVcpz DNA. This observation supports a novel hypothesis to explain establishment of foamy virus latency. The transactivator protein Taf of SFVcpz transcomplemented for the homologous protein Bel-1 of the unique human foamy virus isolate (HFV) and Bel-1 exhibited the reciprocal activity, suggesting that HFV could represent a variant of chimpanzee foamy virus.