RESUMO
Ad CFTR, a replication-deficient adenovirus expressing the human cystic fibrosis transmembrane conductance regulator (CFTR), was administered by aerosolization in a single escalating dose to three pairs (cohorts) of cystic fibrosis (CF) patients. Buffer only was administered to the nose and lungs 9-14 days before nasal instillation of virus followed the day after by aerosolization of Ad CFTR to the lung. Nasal doses (defined in terms of viral plaque forming units, pfu) were 10(5), 10(7), and 4 x 10(8), whereas aerosolized doses were 10(7), 10(8), 5.4 x 10(8) for each cohort, respectively. No acute toxic effects were observed in the first 4 weeks after virus treatment. Shedding of infectious Ad CFTR was never detected, whereas detection of vector DNA sequences and CFTR expression demonstrated DNA transfer to the nose and airways of patients. No significant deviations in immunological and inflammatory parameters were observed in serum and in bronchoalveolar lavage (BAL). Importantly, for all patients, the serum anti-adenovirus antibody levels did not change significantly from baseline and no antibodies against adenovirus were found in BAL.
Assuntos
Adenoviridae/metabolismo , Aerossóis/uso terapêutico , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Terapia Genética , Adolescente , Adulto , Southern Blotting , Lavagem Broncoalveolar , Regulador de Condutância Transmembrana em Fibrose Cística/análise , Regulador de Condutância Transmembrana em Fibrose Cística/genética , DNA/análise , Feminino , Expressão Gênica/genética , Vetores Genéticos/genética , Humanos , Imuno-Histoquímica , Masculino , Mucosa Nasal/citologia , Mucosa Nasal/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro/análiseRESUMO
OBJECTIVE: To investigate the possible association of persistent enterovirus (EV) infection with the development of ALS. BACKGROUND: Although ALS is a clinically well-defined motor neuron disease, little is known about the etiology and pathogenesis of the sporadic cases. Among the different causes that have been hypothesized, conflicting results have been reported about the possible role of persistent enteroviral infection. METHODS: Reverse transcriptase-PCR (RT-PCR) and direct RT in situ PCR (RT-IS-PCR) were performed in formaldehyde-fixed spinal cord samples of 17 patients with confirmed ALS and 29 control subjects with no history of motor neuron disease. When obtained, PCR products were sequenced subsequently. RESULTS: Using direct RT-IS-PCR, EV nucleic acid sequences were detected in 15 (88.3%) of 17 patients with ALS compared to 1 (3.4%) of 29 control subjects. PCR products were located in neuronal cell bodies of the anterior horns of the spinal cord. The RT-PCR products obtained in 13 of the 17 patients with ALS showed between 94% and 86% homology with echovirus 7 sequences. CONCLUSION: The 88.3% rate of detection of enterovirus (EV) nucleic acids in the neuronal cell bodies within the gray matter of the spinal cord of patients with ALS strongly suggests association between persistent EV RNA and ALS. Further work is required to confirm that the persisting EV sequences we detected are somehow involved in the development of ALS.
Assuntos
Esclerose Lateral Amiotrófica/virologia , Enterovirus/genética , RNA Viral/metabolismo , Medula Espinal/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Sequência de Bases/genética , Cadáver , Enterovirus Humano B/genética , Humanos , Dados de Sequência Molecular , Doenças do Sistema Nervoso/metabolismo , Doenças do Sistema Nervoso/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Homologia de Sequência do Ácido Nucleico , Distribuição TecidualRESUMO
The study aimed at analyzing the role of CMV infection as a risk factor for rejection occurring after CMV infection because of the clinical consequences of the prevention of CMV infection that might lead to the decrease in rejection episodes. Two hundred forty-two consecutive renal transplant patients were prospectively checked for the occurrence of CMV infection. CMV infection was defined virologically by a positive viremia or/and a positive viruria or/and a seroconversion or/and a significant rise of the anti-CMV antibody titers. Viremia, viruria, and serology were performed weekly for the first month and then at day 90, day 180, and every 6 months, and moreover if clinical symptoms related to a viral infection occurred. Rejection episode was defined by a creatininemia rise of 25%, after cyclosporine nephrotoxicity and urological complications had been discarded, and by the response to the antirejection therapy, steroids, or OKT3 in case of steroid-resistant rejection. The outcome factor was rejection episode occurring from day 4 after the diagnosis of CMV infection. A patient undergoing "a rejection episode after CMV infection" could also be exposed to other potential confounding factors that can be considered as risk factors of rejection among our patients. Rejection occurring before CMV infection was the main factor because it was linked both to CMV infection itself and to "rejection after." Thus infected and noninfected patients were randomly paired off. To the noninfected patient of the pair was attributed the date of a fictitious CMV infection that was the date of the CMV infection of the infected member of the pair. Therefore, "rejection after" and "rejection before" were defined in infected and noninfected patients of the pair according to the time of onset of CMV infection of the infected member of the pair. The incidence of CMV infection was 65%, 157 of the 242 patients were infected, and 85 not infected. Thus 85 pairs of infected-noninfected patients were studied. The incidence of "rejection after" the diagnosis of CMV infection was significantly higher in the group of patients with CMV infection: 45% among infected (38/85) versus 10.60% among noninfected (9/85) (P < 0.0001). Among the 85 pairs, 48 pairs were concordant in which patient of the pair evinced the same outcome factor: 43 showed no rejection after, and 5 showed one.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Infecções por Citomegalovirus/complicações , Rejeição de Enxerto/etiologia , Transplante de Rim/imunologia , Adulto , Infecções por Citomegalovirus/epidemiologia , Rejeição de Enxerto/epidemiologia , Rejeição de Enxerto/imunologia , Antígenos HLA-A/análise , Antígenos HLA-B/análise , Antígenos HLA-DR/análise , Humanos , Pessoa de Meia-Idade , Transplante de Pâncreas/imunologia , Estudos Prospectivos , Análise de Regressão , Doadores de TecidosRESUMO
The pathophysiology of chronic hepatitis in rabbits infected with coxsackievirus B5 (CVB5), (strain Mitchell) was investigated. Three-week-old male New Zealand White rabbits were inoculated intraperitoneally with 1 x 10(5) plaque forming units of virus. Every 3 months for 15 months postinoculation (p.i.) groups of animals were sacrificed for the following tests: interleukin (IL)-6, tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-beta cytokine levels by enzyme-linked immunosorbent assay (ELISA); splenic natural killer (NK) cell function; sequence of a 154-bp section of the 5' noncoding region; antihepatocyte autoantibodies; histologic examination; in situ polymerase chain reaction (ISPCR) of the liver; neutralizing antibody response to CVB5; and viral cultures of liver, spleen, blood, brain, heart, skeletal muscle, and pancreas samples. Histologic evidence of hepatocyte necrosis was evident at each time point, although few inflammatory cells were seen. Liver samples were positive at each time by ISPCR, with viral nucleic acid localized to hepatocyte cytoplasm. Other cells in the liver did not stain. No hepatocyte autoantibodies were detected, and there was no elevation of intrahepatic cytokine levels compared to uninfected controls. There were no mutations in the virus over time. A vigorous neutralizing antibody response to CVB5, Mitchell was generated, but splenic natural killer (NK) function and numbers of splenic NK cells were significantly decreased. Virus culture was positive at 3 months, but negative at further time points. Cultures were negative at 3 months for the other tissues tested. Thus, CVB5, Mitchell causes a chronic hepatitis in rabbits, with virus limited to hepatocyte cytoplasm and no evidence of autoimmunity.
Assuntos
Infecções por Coxsackievirus/imunologia , Enterovirus Humano B , Hepatite Crônica/imunologia , Hepatite Viral Animal/imunologia , Fígado/imunologia , Animais , Autoanticorpos/análise , Infecções por Coxsackievirus/patologia , Infecções por Coxsackievirus/virologia , Enterovirus Humano B/imunologia , Enterovirus Humano B/isolamento & purificação , Enterovirus Humano B/fisiologia , Hepatite Crônica/patologia , Hepatite Crônica/virologia , Hepatite Viral Animal/patologia , Hepatite Viral Animal/virologia , Células Matadoras Naturais , Fígado/patologia , Fígado/virologia , Masculino , Reação em Cadeia da Polimerase/métodos , RNA Viral/análise , Coelhos , Baço/imunologiaRESUMO
Epidemiological studies were carried out in Yaoundé and in Ngaoundéré from 1984 to 1986, in an attempt to develop adequate methods of collecting blood from small children and of diagnosing measles appropriate to conditions in the field. Alternative methods were necessary since classical methods used in modern laboratories are unsuitable in rural regions. Each study was carried out on a representative sample of 6- to 36-months-old infected children seen at consultation. This group was chosen because it suffers the highest mortality rate. The blood was obtained by digital puncture on blotting paper because venepuncture required sterile equipment and also the establishment of a cold chain for transporting the samples to the laboratory. The first criteria examined were the relative titers of sera taken by finger prick. Four serological techniques were used: indirect immunofluorescence (IIF), indirect immunoperoxidase (IIP), hemagglutination inhibition (HAI) and ELISA. Antimeasles antibodies were detected in 12% of infected children using IIF and in 18% using IIP. When sera were examined by HAI the percentage of positives was 54% and by ELISA 75%. These results clearly indicate that ELISA is the most effective and practical technique for diagnosing measles under field conditions.
Assuntos
Sarampo/epidemiologia , Anticorpos Antivirais/sangue , Camarões/epidemiologia , Criança , Ensaio de Imunoadsorção Enzimática , Estudos de Avaliação como Assunto , Imunofluorescência , Testes de Inibição da Hemaglutinação , Humanos , Técnicas Imunoenzimáticas , Vírus do Sarampo/imunologia , População Rural , Estudos Soroepidemiológicos , Virologia/métodosRESUMO
Both HA and NA proteins elicit antibodies which have been shown to be capable of altering the course of infection. Nevertheless, while influenza virus vaccine standardization involves hemagglutinin (HA) and neuraminidase (NA) in terms of antigenic characterization, only HA protein quantitation is undertaken. An immunocapture ELISA (EIA) is described for N2 NA quantitation, based on the use of a highly specific monoclonal antibody (MAb) for capturing NA and an anti-NA antiserum for antigen detection. The amounts of NA in samples were deduced from the standard curve established by using purified NA. The NA-EIA is specific and detects as a little as 7 ng/ml. The capture and detector antibodies directed against A/Beijing/32/92 NA were shown to react with H3N2 prototype strains used in current influenza vaccines, provided that an antigenically matched reference NA is used as standard.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Vírus da Influenza A Subtipo H3N2 , Vírus da Influenza A/química , Neuraminidase/análise , Proteínas Virais/análise , Animais , Anticorpos Monoclonais/imunologia , Reações Cruzadas , Vírus da Influenza A/imunologia , Vírus da Influenza A/isolamento & purificação , Masculino , Coelhos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
A new and rapid method for co-purification of haemagglutinin (HA) and neuraminidase (NA) proteins from influenza A/H3N2 viruses is described. Surface glycoproteins were first solubilized using a non-ionic detergent under high ionic strength conditions, then they were separated by chromatography on sepharose previously bound to monoclonal antibodies (MAbs) directed either against HA (IaH-chromatography) or against NA (IaN-chromatography). Depending on the protein specificity of the MAb immobilized on the column, HA or NA was bound to sepharose and the counterpart protein was free in the flow-through volume. IaH-chromatography and IaN-chromatography proved equally efficient in term of recoveries (> 75%) and purity (> or = 99%) of both HA and NA but differences appeared when considering functional and antigenic properties of pure proteins. Those properties were highly retained in IaH- and IaN-derived HA as well as in IaH-derived NA while IaN-NA was partially degraded. IaH-chromatography allowed the co-purification of HA and NA proteins in heterologous antigen-antibody system with a 50% rate of cross reactivity. IaH-HA and IaH-NA may be suitable for immunity studies, standardization of influenza vaccine and for diagnostic purposes.
Assuntos
Antígenos Virais/isolamento & purificação , Cromatografia/métodos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/isolamento & purificação , Neuraminidase/isolamento & purificação , Animais , Anticorpos Monoclonais , Antígenos de Superfície/isolamento & purificação , Humanos , Vírus da Influenza A/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Coelhos , SefaroseRESUMO
The Directigen Flu-A is an enzyme immunoassay for detecting in 15 min the influenza A nucleoproteinic antigen directly from specimens after passive adsorption on a cellulose membrane. The test was assessed using 160 frozen (-20 degrees C) specimens collected during the 1988-1989 A/H1N1 influenza epidemic and the 1989-1990 A/H3N2 epidemic. Compared to the ELISA immunocapture test, the sensitivity of the commercial test was 87.8% and the specificity was 97.6%. When compared to isolation of viruses on LLCMK2 cells and/or chicken embryo, the sensitivity was 84%. No cross-reaction was found with other respiratory disease viruses. The feasibility, practicability and rapidity of the test make it a test of choice for rapid diagnosis of influenza A.
Assuntos
Ensaio de Imunoadsorção Enzimática , Técnicas Imunoenzimáticas , Vírus da Influenza A , Influenza Humana/diagnóstico , Antígenos Virais , Humanos , Vírus da Influenza A/isolamento & purificação , Sensibilidade e EspecificidadeRESUMO
A dot blot hybridization test for enteroviruses is described using non-radioactive digoxigenin-labelled probes and a chemiluminescent detection. The use of a 5' non-coding riboprobe which detects all enteroviruses and a VP1 probe that detects the three serotypes of polioviruses allows the rapid detection of polioviruses and non-polio enteroviruses in human specimens or environmental water samples. The assay is strictly enterovirus specific and sensitive (800 fg RNA) and offers several advantages over conventional diagnosis or radioactive probes.
Assuntos
Capsídeo/genética , Infecções por Enterovirus/diagnóstico , Enterovirus/isolamento & purificação , RNA Viral/isolamento & purificação , Microbiologia da Água , Proteínas do Capsídeo , Digoxigenina , Enterovirus/genética , Infecções por Enterovirus/epidemiologia , Infecções por Enterovirus/genética , França/epidemiologia , Humanos , Medições Luminescentes , Hibridização de Ácido Nucleico/métodos , Poliovirus/genética , Poliovirus/isolamento & purificação , Sondas RNARESUMO
During an outbreak of meningitis in France (in the Lyon area), from June to October 1982, serum and stool samples were collected from 227 patients. An enzyme-linked immunosorbent assay (ELISA) for titrating IgG and IgM antibodies anti-echovirus type 33 was developed and compared with the virus isolation technique, and with the titration of neutralizing antibodies. In 39 patients excreting echovirus 33 in faeces, the ELISA test allowed a positive serodiagnosis in 85% of the cases by detection of specific IgM (64% of the cases) and by seroconversion (21%). Compared with the neutralization (Nt) test, ELISA was found to be more sensitive. The antibody titres in ELISA were over 50 times higher and detected earlier than the neutralizing antibodies. This early immune response allowed a rapid diagnosis by specific IgM detection in the acute sera collected within 8 days after the appearance of the clinical symptoms in more than 50% of the 97 patients examined, whereas the Nt test allowed a positive serodiagnosis in only 32% of the patients. The use of a caesium chloride purified antigen insured the specificity of the reactions.
Assuntos
Anticorpos Antivirais/análise , Infecções por Echovirus/diagnóstico , Ensaio de Imunoadsorção Enzimática/métodos , Técnicas Imunoenzimáticas , Imunoglobulina M/análise , Meningite Viral/diagnóstico , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Testes de Neutralização , Testes SorológicosRESUMO
Three non-neutralizing monoclonal antibodies were produced and selected against echovirus type 25 JV-4 prototype strain. They were used in an ELISA to investigate the intratypic differentiation of 28 wild isolates. Clinical isolates fell into seven different groups according to their reactivity patterns in ELISA. Two of the non-neutralizing monoclonal antibodies, 9E4 and 6D3, were highly specific, while the third, 6C9, may recognize an epitope common to other types of echoviruses. In contrast, mouse polyclonal antiserum exhibited large cross-reactivities among echovirus serotypes. The reactivity patterns and the geographical origin of the isolates were generally not correlated and, in the same area, four major antigenic variants sometimes coexisted, especially in the south of France. Moreover, reactivity patterns found with ELISA were hardly ever correlated with those observed in a previous study when neutralization tests were used. These results again underline the non-correlation between structure and biological function in the Picornavirus family.
Assuntos
Anticorpos Monoclonais , Infecções por Echovirus/microbiologia , Enterovirus Humano B/isolamento & purificação , Ensaio de Imunoadsorção Enzimática/métodos , Animais , Criança , Pré-Escolar , Enterovirus Humano B/classificação , Enterovirus Humano B/imunologia , Epitopos , Humanos , Lactente , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Especificidade da EspécieRESUMO
An immunocapture ELISA test for the diagnosis of human and animal influenza A and/or B is described. A monoclonal anti-nucleoprotein (NP) antibody was used to capture the NP antigen and the captured antigen was detected by an anti-NP polyclonal rabbit antiserum. Compared with the usual diagnostic method by cultivation in embryonated eggs, this test had a high specificity (97%) and sensitivity when used for diagnosis using clinical nasopharyngeal samples obtained from patients and animals. Immunocapture ELISA permitted an easier reading than the indirect immunofluorescence technique. It also permitted diagnosis in frozen samples (-20 degrees C) or in infected LLCMK2 cells mixed with uninfected nasopharyngeal cells and kept at 20 degrees C for one week. This test can be carried out in 3 h.
Assuntos
Autoantígenos/análise , Ensaio de Imunoadsorção Enzimática , Influenza Humana/diagnóstico , Nucleoproteínas/análise , Animais , Anticorpos Monoclonais , Autoantígenos/imunologia , Furões , Imunofluorescência , Humanos , Vírus da Influenza A/imunologia , Vírus da Influenza A/isolamento & purificação , Influenza Humana/microbiologia , Influenza Humana/veterinária , Camundongos , Nucleoproteínas/imunologia , Coelhos , Suínos , TemperaturaRESUMO
Adolescent female mice were inoculated intraperitoneally with coxsackievirus B3 Nancy strain, sacrificed 3 and 5 days later and the livers harvested. A protocol for direct reverse transcriptase in situ PCR (RT-ISPCR) detection of enteroviral RNA in paraffin-embedded liver tissues was developed. The optimal conditions for the assay were determined. The best results were obtained when the tissue was fixed in formalin, prior to being embedded in paraffin, then cut in 5 micron thick sections, and mounted onto silanized slides. After deparaffination the slides were incubated in 1 microgram/m1 Proteinase K for 10 min and cDNA synthesis was carried out. For successful RT-ISPCR 40-50 cycles of amplification were necessary. The optimal concentrations of dNTP, primers and Taq Polymerase for RT-ISPCR were determined by serial dilution assays. Primers were selected from highly conserved sequences in the 5' non-coding region (5'NTR). To detect the viral RNA in the liver, digoxigenin-dUTP was incorporated during amplification, subsequently bound with an antidigoxigenin antibody conjugated to alkaline phosphatase (AP), followed by colorimetric detection with nitroblue tetrazolium salt (NBT) and 5-brom-4chloro-3indolyl-phosphate (BCIP). The result was a blue precipitate in the cytoplasm of hepatocytes from infected mice. Fibroblasts, endothelial cells, lymphocytes and the nuclei of hepatocytes were negative. Thus, RT-ISPCR is a specific method for the detection of enterovirus RNA in the hepatocytes of infected mice, and can be of use for the determination of EV liver disease in man.
Assuntos
Enterovirus/genética , Fígado/fisiologia , Reação em Cadeia da Polimerase/métodos , DNA Polimerase Dirigida por RNA/metabolismo , Animais , Feminino , Genoma Viral , Hibridização In Situ , Fígado/química , Fígado/virologia , Hepatopatias/diagnóstico , Camundongos , DNA Polimerase Dirigida por RNA/genética , Sensibilidade e EspecificidadeRESUMO
Rapid diagnosis of cytomegalovirus (CMV) infection may be obtained by molecular techniques, such as the polymerase chain reaction (PCR) and hybridization assays. The optimal technique to detect CMV in clinical samples was assessed. Two different PCR assays were used, targeting either the major immediate early 1 (MIE 1) or the HXLF 4 gene. The PCR products were detected by gel electrophoresis, dot blotting and an easy to use, rapid, solid phase hybridization assay, DNA enzyme immunoassay (DEIA). Standard tissue culture was also used. Cerebrospinal fluids (18), liver biopsies (9) from hepatic transplant recipients, amniotic fluids (7) from mothers with suspected peripartum infection, and samples (6) of miscellaneous origin (brain and fundus biopsy, pericardial and pleural fluid) were tested. Among the 40 samples, CMV was detected in 19 cases. Three were positive by both molecular techniques and tissue culture, 14 by molecular methods and 2 by culture. 16/19 or 9/19 CMV-positive samples were detected by PCR amplification of the HXLF 4 or MIE 1 gene, respectively and 14/16 HXLF 4-positive samples were detected using either dot-blot or DEIA, compared to 9/16 using gel electrophoresis. Thus, the most sensitive assays for the detection of CMV in clinical samples using the methods compared in the current study were PCR amplification of the HXLF 4 gene followed by dot-blot or DEIA hybridization.
Assuntos
Infecções por Citomegalovirus/diagnóstico , Citomegalovirus/genética , Citomegalovirus/isolamento & purificação , DNA Viral/genética , DNA Viral/isolamento & purificação , Hibridização de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase/métodos , Líquido Amniótico/virologia , Biópsia , Encéfalo/virologia , Células Cultivadas , Infecções por Citomegalovirus/líquido cefalorraquidiano , DNA Viral/imunologia , Eletroforese em Gel de Ágar , Feminino , Fibroblastos , Genes Precoces/genética , Humanos , Imunoensaio/métodos , Immunoblotting/métodos , Fígado/virologia , Transplante de Fígado , Derrame Pericárdico/virologia , Derrame Pleural/virologia , Gravidez , Complicações Infecciosas na Gravidez/virologia , Sensibilidade e EspecificidadeRESUMO
ELISA capture technique (ELISAc) was carried out using a rabbit hyperimmune serum attached to a solid phase for capturing mumps antigens in cerebrospinal fluid (CSF) in patients with meningitis and/or in supernatants of infected Vero cells. A biotin-labelled rabbit serum prepared from the previous serum was added and the reaction was read by an enzymatic (avidine-peroxidase) reaction by automated reading. The cut-off was calculated in 100 CSFs negative for viruses by conventional diagnosis. The specificity was evaluated in Vero cells infected with 22 CSFs collected from vaccinated children (URABE AM9 attenuated vaccine) who developed meningitis. A guinea pig hyperimmune serum confirmed the specificity. Results in culture correlated with the ELISA capture technique (ELISAc). No cross-reactivity was observed with parainfluenza 1, 2, 3 human reference strains. At least 2.5 ngs of purified mumps proteins were detected corresponding to 10(1.5) infectious particles per ml. ELISAc applied directly to 14 CSFs collected from unvaccinated children with meningitis diagnosed five positive cases, whereas in four cases conventional diagnosis had to be undertaken twice. ELISAc permitted the diagnosis of one additional patient. The test can be carried out in 3 h.
Assuntos
Ensaio de Imunoadsorção Enzimática , Meningite Viral/diagnóstico , Caxumba/diagnóstico , Animais , Biotina/metabolismo , Criança , Pré-Escolar , Chlorocebus aethiops , Estudos de Avaliação como Assunto , Cobaias , Humanos , Meningite Viral/sangue , Meningite Viral/imunologia , Caxumba/sangue , Caxumba/imunologia , Estudos Prospectivos , Coelhos , Reprodutibilidade dos Testes , Estudos Retrospectivos , Sensibilidade e Especificidade , Células VeroRESUMO
The pathogenesis of the postpolio syndrome (PPS) remains unclear. In this study we looked for poliovirus (PV) persistence in the CSF of 20 patients with PPS, in a control group including 20 patients with unrelated neurological diseases, and in 7 patients with stable poliomyelitis sequelae. CSF samples and sera were examined using reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of PV or other enterovirus genomes; this assay allows the detection from as little as 1 fg viral RNA. Sequencing of amplified products from 5 patients was performed. PV genomic sequences were detected in the CSF of 11 of 20 patients with PPS and in none of the control group. Sequencing in the 5' untranslated region confirmed the presence of mutated PV sequences. These findings suggest that PPS is related to the persistence of PV in the central nervous system.
Assuntos
Poliovirus/isolamento & purificação , Síndrome Pós-Poliomielite/virologia , Adulto , Idoso , Estudos de Casos e Controles , Infecções do Sistema Nervoso Central/virologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Síndrome Pós-Poliomielite/líquido cefalorraquidiano , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVE: To determine the incidence of cytomegalovirus in the ejaculates of infertile men who were seropositive for IgG antibodies to cytomegalovirus. DESIGN: Prospective study. PATIENT(S): We tested cytomegalovirus infection in the semen of men participating in an IVF-ET program. MAIN OUTCOME MEASURE(S): IgG and IgM antibodies to cytomegalovirus were measured in sera. We used polymerase chain reaction (PCR) and cell culture to look for both cytomegalovirus DNA and infectious virus in the semen of 70 men with cytomegalovirus-specific antibodies detected in sera. RESULT(S): Of the infertile couples, 13.5% exhibited "mismatching" serology (i.e., detection of IgG antibodies to cytomegalovirus in male serum only and not in female serum) and constituted a potential risk for cytomegalovirus transmission. Cytomegalovrius was identified in the semen of two patients who were positive for IgG antibodies to cytomegalovirus. Cytomegalovirus DNA also was detected in one positive sample after centrifugation through a three-layer Percoll gradient. CONCLUSION(S): Human cytomegalovirus was present in the semen from a population of infertile men. Rapid detection can be achieved by molecular techniques such as PCR combined with a hybridization assay. Even though cytomegalovirus was infrequently detected in semen, these data must be considered in determining the risk of transmission and developmental anomalies in infected fetuses.
Assuntos
Citomegalovirus/isolamento & purificação , Infertilidade Masculina/virologia , Sêmen/virologia , Adulto , Anticorpos Antivirais/sangue , Células Cultivadas , Citomegalovirus/genética , Citomegalovirus/imunologia , DNA Viral/análise , Eletroforese em Gel de Ágar , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , Reação em Cadeia da Polimerase , Estudos ProspectivosRESUMO
The sodium salts of 2-difluoromethyl-phenyl-α-ketoside of N-acetyl-neuraminic acid (compound 1) and of 4-difluoromethyl-2-methoxy-phenyl-α-ketoside of N-acetylneuraminic acid (compound 2) were designed as potential mechanism-based inhibitors of sialidase. In vitro both of these compounds competitively inhibited the sialidases of Clostridium perfringens and of influenza virus A/HK/1/68. Inhibition was irreversible with the sialidase of Clostridium perfringens whereas it was reversible with that of A/HK/1/68. Compound 2 did not inhibit the hemagglutinin of the virus but exhibited significant anti-influenza activity when added to the medium of Madin-Darby canine kidney (MDCK) cells infected by influenza virus. In non-infected MDCK cells no inhibition of cellular sialidase was observed. Compound 2 did not block primary infection, but inhibited the release of progeny virus from infected cells. Even after 8 passages in its presence, no resistant strains were detected. Because of its high Ki (8 × 10-5M) compared to the low Ki (1 × 1-10 M) of 4 guanidino-Neu 5 Ac 2en and its reversible inhibition of viral sialidase, its development as an anti-influenza agent is no longer envisaged. Nevertheless, as a mechanism-based irreversible inhibitor of the bacterial enzyme, it could at least be useful for investigating the intrinsic role of sialidase in infections caused by this strain.
RESUMO
Little is known of the epidemiology of influenza C virus infections in western Europe and of the exact role of this agent in acute viral respiratory infections. Several tests may be used for detecting antibodies against this agent but the significance of their respective results is not clear. A total of 301 samples of serum was collected from persons aged from 4 months to 88 years living in France in 1988. The samples were tested for the presence of antibodies to influenza C virus by haemagglutination-inhibition (HI) tests and ELISA. The specificity of the results was checked by immunoblotting and by antibody absorption with staphylococcal protein A. Significant HI activity was found in 61% of the 301 samples tested, titres ranging from 20-320; 70% were positive by ELISA with titres ranging from 500 to 32,000. The population tested was divided into four age groups: 0-15 years; 16-30 years; 31-50 years and 51-88 years. The highest rates for positive samples were found in the 16-30 year group (76 and 79% by HI tests and ELISA respectively) as well as significant HI and ELISA geometric mean titres. Positive samples were less common in young children (46 and 50% by HI tests and ELISA respectively) and in the oldest group (44 and 54% respectively). The 31-50 years age group formed an intermediate class. The high prevalence of antibody as well as the significant titres indicate intense circulation of influenza C virus, especially among young adults.
Assuntos
Gammainfluenzavirus , Infecções por Orthomyxoviridae/epidemiologia , Adolescente , Adulto , Fatores Etários , Idoso , Anticorpos Antivirais/sangue , Criança , Ensaio de Imunoadsorção Enzimática , França/epidemiologia , Testes de Inibição da Hemaglutinação , Testes de Hemaglutinação , Humanos , Imunoglobulina G/metabolismo , Lactente , Recém-Nascido , Gammainfluenzavirus/imunologia , Pessoa de Meia-Idade , Infecções por Orthomyxoviridae/imunologia , Sensibilidade e Especificidade , Proteína Estafilocócica A/isolamento & purificação , Proteína Estafilocócica A/metabolismoRESUMO
The emergence of cross-resistance to various antiviral drugs was investigated both in vivo and in vitro for herpes simplex virus type 1 (HSV1) resistant to idoxuridine (IUdR 0.24%) obtained by seven successive passages (from P0 to P7) in rabbit keratitis treated by IUdR. The viral population obtained at the seventh IUdR passage (P7) showed an activity of the thymidine kinase (TK) reduced to 5.6% of the parental strain (PO); moreover, most of the clones of P7 showed an altered TK phenotype determined by the [125I]iododeoxycytidine (IDC) procedure. In rabbit keratitis, IUdR-resistant viral population P7 showed cross-resistance to bromovinyl desoxyuridine (BVDU) (0.5%) and to acyclovir (ACV) (3%). Under trifluorothymidine (1%) treatment, P7 showed an intermediate sensitivity. HSV1 at P7 remained sensitive to adenine arabinoside (Ara A) (3%) and to dihydroxy-propoxymethylguanine used at high concentration (3%). The in vitro sensitivity determination to various antiviral drugs was investigated by dye-uptake assay for the initial viral population PO and for HSV1 collected under IUdR treatment at the third (P3) and the seventh (P7) passages. Cross-resistance to TK-dependent drugs, such as IDC, BVDU, and ACV were found at P7. P7 remained sensitive to Ara A and to phosphonoformic acids antiviral drugs known not to be dependent on viral TK.