Propagation of Semliki Forest virus in various human lymphoblastoid cell lines.
Semliki Forest virus (SFV) propagation was studied in one marmoset and eight human lymphoblastoid cell lines. In eight of these cell lines SFV propagated well. Only in the Daudi (human) cell line virus replication was suppressed. This suppression takes place after virus adsorption but before virus inhibitory effects on cell functions. (+info)
The clinical utility of CMV surveillance cultures and antigenemia following bone marrow transplantation.
At our institution, the cytomegalovirus (CMV) prophylaxis protocol for allogeneic bone marrow transplant (BMT) recipients who are CMV-seropositive or receive marrow from a CMV-seropositive donor consists of a surveillance bronchoscopy approximately 35 days posttransplant. Patients with a positive surveillance bronchoscopy for CMV receive pre-emptive ganciclovir. In order to determine the utility of other screening methods for CMV, we prospectively performed weekly CMV antigenemia, and blood, urine and throat cultures from time of engraftment to day 120 post-BMT in 126 consecutive patients. Pre-emptive ganciclovir was given to 11/81 patients (13.6%) because of a positive surveillance bronchoscopy for CMV. Results of CMV blood, urine and throat cultures and the antigenemia assay done prior to or at the time of the surveillance bronchoscopy were analyzed for their ability to predict the bronchoscopy result. The antigenemia test had the highest positive and negative predictive values (72% and 96%, respectively). The ability of these tests to predict CMV disease was evaluated in the 70 patients with a negative surveillance bronchoscopy who did not receive pre-emptive ganciclovir. Of 19 cases of active CMV disease, CMV antigenemia was positive in 15 patients (79%) a mean of 34 days preceding symptoms. Blood cultures were positive in 14/19 patients (74%) a mean of 31 days before onset of disease. CMV antigenemia is useful for predicting the surveillance bronchoscopy result, and also predicts the development of CMV disease in the majority of patients missed by the surveillance bronchoscopy. (+info)
Infection of apheresis cells by parvovirus B19.
Parvovirus B19 is the only member of the Parvoviridae family known to cause disease in humans. Owing to the high level of cell tropism the virus can only replicate in proliferating and differentiating erythroid precursor cells, which are present in human bone marrow and foetal liver. As human bone marrow is very difficult to obtain, an alternative in vitro system for the propagation of B19 virus has been developed, based on the application of mobilized haemapoietic progenitor (apheresis) cells. These cells are routinely harvested from cancer patients after treatment with recombinant human granulocyte/macrophage colony-stimulating factor. Replication of parvovirus B19 in vitro is possible in these cells after stimulation with erythropoietin. Therefore, this system is an easily, accessible alternative to the use of human bone marrow in parvovirus B19 infection assays. (+info)
Tracheal aspirate as a substrate for polymerase chain reaction detection of viral genome in childhood pneumonia and myocarditis.
BACKGROUND: Infectious respiratory disorders are important causes of childhood morbidity and mortality. Viral causes are common and may lead to rapid deterioration, requiring mechanical ventilation; myocardial dysfunction may accompany respiratory decompensation. The etiologic viral diagnosis may be difficult with classic methods. The purpose of this study was to evaluate polymerase chain reaction (PCR) as a diagnostic method for identification of causative agents. METHODS AND RESULTS: PCR was used to amplify sequences of viruses known to cause childhood viral pneumonia and myocarditis. Oligonucleotide primers were designed to amplify specific sequences of DNA virus (adenovirus, cytomegalovirus, herpes simplex virus, and Epstein-Barr virus) and RNA virus (enterovirus, respiratory syncytial virus, influenza A, and influenza B) genomes. Tracheal aspirate samples were obtained from 32 intubated patients and nucleic acid extracted before PCR. PCR results were compared with results of culture, serology, and antigen detection methods when available. In cases of myocarditis (n=7), endomyocardial biopsy samples were analyzed by PCR and compared with tracheal aspirate studies. PCR amplification of viral genome occurred in 18 of 32 samples (56%), with 3 samples PCR positive for 2 viral genomes. Amplified viral sequences included RSV (n=3), enterovirus (n=5), cytomegalovirus (n=4), adenovirus (n=3), herpes simplex virus (n=2), Epstein-Barr virus (n=1), influenza A (n=2), and influenza B (n=1). All 7 cases of myocarditis amplified the same viral genome from heart as found by tracheal aspirate. CONCLUSIONS: PCR is a rapid and sensitive diagnostic tool in cases of viral pneumonia with or without myocarditis, and tracheal aspirate appears to be excellent for analysis. (+info)
Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line.
An estimated 170 million persons worldwide are infected with hepatitis C virus (HCV), a major cause of chronic liver disease. Despite increasing knowledge of genome structure and individual viral proteins, studies on virus replication and pathogenesis have been hampered by the lack of reliable and efficient cell culture systems. A full-length consensus genome was cloned from viral RNA isolated from an infected human liver and used to construct subgenomic selectable replicons. Upon transfection into a human hepatoma cell line, these RNAs were found to replicate to high levels, permitting metabolic radiolabeling of viral RNA and proteins. This work defines the structure of HCV replicons functional in cell culture and provides the basis for a long-sought cellular system that should allow detailed molecular studies of HCV and the development of antiviral drugs. (+info)
Isolation and partial characterization of a lentivirus from talapoin monkeys (Myopithecus talapoin).
We have identified a novel lentivirus prevalent in talapoin monkeys (Myopithecus talapoin), extending previous observations of human immunodeficiency virus-1 cross-reactive antibodies in the serum of these monkeys. We obtained a virus isolate from one of three seropositive monkeys initially available to us. The virus was tentatively named simian immunodeficiency virus from talapoin monkeys (SIVtal). Despite the difficulty of isolating this virus, it was readily passed between monkeys in captivity through unknown routes of transmission. The virus could be propagated for short terms in peripheral blood mononuclear cells of talapoin monkeys but not in human peripheral blood mononuclear cells or human T cell lines. The propagated virus was used to infect a naive talapoin monkey, four rhesus macaques (M. mulatta), and two cynomolgus macaques (M. fascicularis). All animals seroconverted and virus could be reisolated during a short period after experimental infection. A survey of SIVtal-infected captive talapoin monkeys revealed a relative decrease in CD4(+) cell numbers in chronically (>2 years) infected animals. No other signs of immunodeficiency were observed in any of the infected animals. PCR amplification followed by DNA sequencing of two fragments of the polymerase gene revealed that SIVtal is different from the presently known lentiviruses and perhaps most related to the SIV from Sykes monkeys. (+info)
Quantification of endogenous viral polymerase, 3D(pol), in preparations of Mengo and encephalomyocarditis viruses.
Measurement of an antigenic response to the aphthovirus infection-associated antigen (VIA), the viral RNA polymerase 3D(pol), is frequently used as a discriminating assay for the extent of viral replication in animals. In practice, animals seropositive for VIA are assumed to have been exposed to live virus, although in fact it is suspected that endogenous 3D(pol) in commercial inactivated vaccines may occasionally stimulate analogous responses and result in false-positive tests for virus exposure. Cardiovirus infections in mice produce similar anti-VIA antibodies, and in view of recently developed attenuated Mengo vaccines and live Mengo vectors, these VIA responses are also under investigation as potential correlates of vaccine efficacy. We have purified recombinant Mengo 3D(pol), developed monoclonal antibodies to the protein, and used these reagents in highly sensitive Western blot assays to quantify the levels of endogenous 3D(pol) in Mengo and encephalomyocarditis virus (EMCV) preparations. The presence of 3D(pol) was detected at all stages of standard vaccine purification procedures, including materials purified by CsCl. Clarified suspensions of Mengo- or encephalomyocarditis virus-infected HeLa cells were found to contain very high quantities of 3D(pol), averaging approximately 1.2-1.5 micrograms of protein/micrograms of virus. Pelleting through 30% sucrose or purification by CsCl removed much of this material, but even these samples retained approximately 0.2-0.4 ng of 3D(pol)/micrograms virus. These ratios represent approximately 1 3D(pol) molecule/20 virus particles in the most highly purified materials and probably indicate that 3D(pol) is a contaminant on the particle surface rather than an intrinsically packaged molecule. In clarified cell lysates, which are commonly used as vaccine inocula, the protein to virus ratio was approximately 210:1, a level that could represent serious contamination problems for future VIA detection if such inocula are used without further purification. (+info)
Isolation of eastern equine encephalitis virus in A549 and MRC-5 cell cultures.
Eastern equine encephalitis (EEE) has been diagnosed either serologically or by virus isolation. Until now, the recovery of EEE virus has been delegated to reference laboratories with the expertise and resources needed to amplify the virus in a susceptible vertebrate host and/or to isolate and identify the virus in cell culture. We report a case in which EEE virus was recovered directly from a patient's cerebrospinal fluid in A549 and MRC-5 cell cultures. Many clinical virology laboratories routinely use these cells to recover adenovirus, herpes simplex virus, and enterovirus. To the best of our knowledge, this is the first report of isolation of EEE virus in A549 cell culture. This report demonstrates the possibility of recovery of EEE virus in cell culture without the necessity of bioamplification or maintaining unusual cell lines. (+info)