Reaction of nitrous acid with viral nucleic acids in stitu.
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Absorption↗
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Nucleic acid homology studies of viral nucleic acids in idiopathic Parkinson's disease.
Reassociation kinetics analyses with radioiodinated herpes simplex type 1 DNA and influenza A/NWS RNA were performed in the presence of tissue nucleic acids from defined loci of the brains of nine patients with idiopathic Parkinson's disease, one normal control brain, and the brains of uninfected mice or mice infected with either herpes simplex type 1 virus or influenza A/NWS virus. Herpes simplex type 1 DNA was detected by an increased reassociation rate in the herpes simplex type 1 virus-infected mouse brains. Influenza A/NWS RNA was detected by reassociation in the influenza A/NWS virus-infected mouse brains. Experimental limits for the detection of homologous nucleic acids are given for each separate experiment with human or mouse tissue. Within these detection limits, nucleic acids complementary to herpes simplex type 1 DNA or influenza A/NWS RNA were not detected in any of the brains of patients with idiopathic Parkinson's disease.
Brain↗
The Vpr protein of human immunodeficiency virus type 1 influences nuclear localization of viral nucleic acids in nondividing host cells.
The replication of human immunodeficiency virus type 1 (HIV-1) in nondividing host cells such as those of macrophage lineage is an important feature of AIDS pathogenesis. The pattern of HIV-1 replication is dictated, in part, by the nucleophilic property of the viral gag matrix (MA) protein, a component of the viral preintegration complex that facilitates nuclear localization of viral nucleic acids in the absence of mitosis. We now identify the accessory viral protein Vpr, as a second nucleophilic component that influences nuclear localization of viral nucleic acids in nondividing cells. Reverse transcription and nuclear localization of viral nucleic acids following infection of cells by viruses lacking Vpr or viruses containing mutations in a gag MA nuclear localization sequence were indistinguishable from the pattern observed in cells infected by wild-type HIV-1. These viruses retained the ability to replicate in both dividing and nondividing host cells including monocyte-derived macrophages. In contrast, introduction of both gag MA and Vpr mutations in HIV-1 attenuated nuclear localization of viral nucleic acids in nondividing cells and virus replication in monocyte-derived macrophages. These studies demonstrate redundant nucleophilic determinants of HIV-1 that independently permit nuclear localization of viral nucleic acids and virus replication in nondividing cells such as monocyte-derived macrophages. In addition, these studies provide a defined function for an accessory gene product of HIV-1.
Base Sequence↗
In situ hybridization: detecting viral nucleic acid in formalin-fixed, paraffin-embedded tissue samples.
In situ hybridization is a method for detecting specific nucleic acid sequences within individual cells. This technique permits visualization of viral nucleic acid or gene expression in individual cells within their histologic context. In situ hybridization is based on the complementary binding of a labeled nucleic acid probe to complementary sequences in cells or tissue sections, followed by visualization of target sequences within the cells. It has been used widely for the detection of viral nucleic acid sequences within individual cells. This review will define the technical approaches of in situ hybridization and its current application to detect viral nucleic acids within formalin-fixed, paraffin-embedded tissue samples, with special reference to the Epstein-Barr virus.
DNA, Viral↗
Polymerase chain reaction (PCR) search for viral nucleic acid sequences in schizophrenia.
BACKGROUND: Previous studies looking for evidence of viral infection in schizophrenics have yielded conflicting results. We searched for viral nucleic acids to test the hypothesis of the viral aetiology of schizophrenia. METHOD: We used the polymerase chain reaction (PCR) to search for cytomegalovirus (CMV), human immunodeficiency virus (HIV), influenza A, Borna disease virus (BDV), and bovine viral diarrhoea virus (BVDV) in: hippocampus from three schizophrenic and three non-schizophrenic subjects; cerebrospinal fluid (CSF) from 48 schizophrenic patients; CSF and peripheral blood mononuclear cells (PBMC) from nine sets of identical twins discordant for schizophrenia; and SK-N-SHEP cells co-cultured with schizophrenic and non-schizophrenic brain homogenates. All patients met DSM-III-R criteria. RESULTS: Virus-specific nucleic acids were not found in any of the samples tested. CONCLUSIONS: The absence of viral nucleic acids in the samples tested suggest that, in these patients, schizophrenia is not associated with a persistent or latent infection due to these viruses.
Adult↗
Quest for a reliable, valid, and sensitive in situ hybridization procedure to detect viral nucleic acids in the central nervous system.
In situ hybridization (ISH) to detect and to quantitate viral nucleic acid sequences in cryopreserved central nervous system (CNS) tissue is a reliable, valid and sensitive molecular technique. On the other hand, utilization of formaldehyde fixed paraffin embedded (FFPE) tissue to improve cytomorphology requires fundamental changes in the procedure since it is necessary to cleave the elaborate protein network cross-linked by formaldehyde using elevated concentrations of proteinases in order to permit diffusion of complementary DNA probes to the targets (genomic viral nucleic acid sequences and/or viral mRNA). Adversely, this procedure hydrolyzed the proteinaceous glues generally used to fix tissue to glass slides resulting in loss of tissue sections during the ISH protocol. This report describes the application of a novel procedure utilizing a silano-organic compound to covalently bond to glass slides FFPE sections as well as cryopreserved tissue sections and cultured cells with and without virus infections. This covalent bonding procedure has permitted optimization of the ISH procedure for virus detection and quantification, especially for exploratory studies of specificity and wash stringency in relation to the Tm of the hybridized product. Progressive multifocal leucoencephalopathy (PML) caused by an opportunistic papovavirus (JC) was chosen because of the ready availability of tissue, stability of papovavirus nucleic acids, and specificity of 3H- and 35S-radiolabeled JC cloned DNA probes. Further, this laboratory is utilizing the optimized sensitive procedure to search for several virus etiologies in human diseases such as multiple sclerosis, temporal lobe epilepsy, Alzheimer's disease, schizophrenia, and Parkinson's disease, as well as normal aging.(ABSTRACT TRUNCATED AT 250 WORDS)
Adhesives↗
Comparative usefulness of tissue fixatives for in situ viral nucleic acid hybridization.
Traditionally tissues for in situ hybridization of viral nucleic acid have been small pieces obtained from laboratory rodents, and fixatives that are designed for electron microscopy, such as periodate-lysine-paraformaldehyde (PLP) can handle them adequately. However, these fixatives have limited penetrating ability and may produce no appreciable hardening, so alternative fixation methods were evaluated. The intention was to determine whether fixatives adequate for bulky tissues such as whole or halved pig and cow brains would also be compatible with in situ hybridization. Various fixatives were evaluated using a system of intracranial inoculation of BALB/c mice with pseudorabies virus (PRV) followed by in situ hybridization of brain tissue sections with a 35S-labeled PRV DNA probe. Loss of tissue sections was a major problem, particularly with PLP and formalin, but positive results were obtained with five fixatives tested. Cellular morphology was especially good with PLP and with a modification of Carnoy's fluid, MOCA fixative. An incidental but important observation was that formalin is compatible with in situ hybridization. Retroactive studies of viral diseases using routinely processed blocks of tissue (formalin-fixed, paraffin-embedded) are therefore conceivable.
Acetates↗
Stabilized viral nucleic acids in plasma as an alternative shipping method for NAT.
BACKGROUND: Preservation of the integrity of viral nucleic acids in blood specimens during shipping and handling is crucial for NAT and viral load monitoring. An economical and convenient method is described for nucleic acid stabilization by using an RNA stabilizing solution (RNAlater, Ambion) in plasma that is designed for the shipment of samples to tropical countries. STUDY DESIGN AND METHODS: HCV, HIV, and HBV FFP were compared with RNAlater-treated plasma and dried plasma spots (DPSs) after incubation at 37 degrees C, which was chosen as an upper limit of ambient shipping temperature, for up to 28 days. HCV-infected chimpanzee plasma was shipped at either room temperature after RNAlater treatment or as frozen plasma in liquid nitrogen from Liberia to New York City. They were then compared for HCV RNA levels. The nucleic acid stabilities were determined by quantitative PCR by using a molecular beacon assay on a sequence detection system (ABI 7700, PE-Biosystems) and by visualizing the PCR components on an acrylamide gel. RESULTS: Quantitative PCR data showed that a 60:40 or greater ratio of RNAlater:plasma volume successfully stabilized HCV RNA and HIV RNA in plasma for up to 28 days at 37 degrees C. HBV DNA in plasma was stable for up to 14 days at 37 degrees C without any stabilizing solution. DPSs on filter paper stabilized viral nucleic acids, but the recoveries were 3 to 10 times less than those with frozen plasma. The integrity of the 5' UTR region of HCV RNA in RNA later-treated chimpanzee plasma was intact when its PCR component was viewed on an acrylamide gel. CONCLUSION: The DPS method stabilized nucleic acids, at least with the extraction method used, was less sensitive than use of RNAlater, and required tedious manual handling. RNAlater provides a convenient way of stabilizing viral nucleic acid in plasma at ambient temperature during sample transportation.
5' Untranslated Regions↗
[Recognition of viral nucleic acids and regulation of type I IFN expression].
Recognition of viral nucleic acids with pattern recognition receptors (PRRs) is the first step to induce innate immune system. Type I interferons (IFNs), central mediators in the antiviral innate immunity, are responsible for induction of cytokines and chemokines that disrupt virus replication. Recent studies indicated that there are at least two distinct pathways for the induction of type I IFN by viral infection. Toll-like receptors (TLRs) are extracellular and endosomal PRRs for microbial pathogens whereas retinoic acid inducible gene-I(RIG-I) and melanoma differentiation-associated gene 5(MDA5) are novel intracellular PRRs for viral dsRNA. In this report we describe the distinct mechanisms inducing type I IFNs through TLRs and RIG-I/MDA5 pathways.
Animals↗
[Effect of actinomycin D and daunomycin on the biosynthesis of nucleic acids in cells transformed by oncornavirus and on the biosynthesis of viral nucleic acids (author's transl)].
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Animals↗
Detection of persistent vegetative bacteria and amplified viral nucleic acid from in-use testing of gastrointestinal endoscopes.
Hospital-acquired infection attributed to inadequate decontamination of gastrointestinal endoscopes prompted an in use evaluation of recommended procedures. Specimens were obtained from the internal channels of 123 endoscopes before, during and after decontamination by flushing with saline and brushing with a sterile brush, and examined for vegetative bacteria by broth and plate culture. Four endoscopy units were tested; the chemical disinfectants used were: 2% glutaraldehyde in Centres 1 and 2 (automated) and Centre 3 (manual); peracetic acid in Centre 4 (automated). Samples from patients in Centre 1 with known chronic hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV-1) infection were also examined for viral nucleic acid by ultracentrifugation, nucleic acid extraction, reverse transcription (for RNA) and polymerase chain reaction (PCR). No persistent vegetative bacteria were found following standard manual cleaning and disinfection for 20 min in 2% glutaraldehyde in Centres 2 and 3 (N = 37). At Centre 1, while plate culture yielded no growth, 34% of samples (10/29) grew vegetative bacteria in broth culture after cleaning and disinfection for 20 min in 2% glutaraldehyde. Investigation revealed an error in manual cleaning; no bacteria were detected in 37 samples taken after this was corrected. At Centre 4, despite the use of peracetic acid as a sterilant, three out of 20 (15%) of post decontamination samples grew bacteria; one contained persistent bacteria. HBV and HCV PCR analysis detected viral nucleic acid in three out of four and four out of six samples from viraemic patients undergoing endoscopy in Centre 1 during the period of improper manual washing. After proper cleaning was instituted, samples from nine out of nine HCV viraemic patients were negative. HIV RNA was detected in five of 14 samples taken from endoscopes after use on HIV positive patients but all post decontamination samples were negative. Detection of bacteria in washes from endoscope channels is a useful warning of a breakdown in decontamination practice. Inadequate brushing of internal channels may result in persistent HCV and HBV viral nucleic acid, the significance of which is not clear. These results reinforce the importance of adequate manual cleaning of endoscopes before chemical disinfection.
Bacteria↗
Studies on the infectivity of viral nucleic acids performed in the "Stefan S. Nicolau" Institute of Virology.
The investigations on viral nucleic acids (NA) performed in the Institute of Virology since 1957 refer to the infectivity of NA (RNA or DNA) from large, complex viruses, from RNA viruses (ECHO, MM Coxsackie, etc.), from modified viruses, as well as to the biological activity (oncogenicity and infectivity) of NA from tumors and leukemic tissues of human, murine, bovine and avian origin. Studies are reviewed as regards the action of physical and chemical agents on the behaviour of viral and cellular NA and the implications of the use of such agents in viral vaccine preparation are discussed.
Animals↗
PCR detection of viral nucleic acid in fatal asthma: is the lower respiratory tract a reservoir for common viruses?
BACKGROUND: There is indirect evidence implicating viral respiratory tract infections in the pathogenesis of fatal asthma. However, it is unknown whether viruses are present within the lower respiratory tract in fatal asthma. OBJECTIVES: To apply a nine-virus polymerase chain reaction (PCR) panel to postmortem specimens of lower airway secretions and compare the prevalence of viral nucleic acid among patients who died of asthma, asthmatic patients who died of other causes and persons who died without lung disease. PATIENTS AND METHODS: Postmortem specimens of lower airway secretions from patients who died of asthma (fatal asthma [n=10]), asthmatic patients who died of other causes (n=4) and nonasthma controls (n=6) underwent PCR for nine common respiratory viruses. The prevalence of each virus was compared among the three groups. RESULTS: PCR was positive for at least one virus in 19 of 20 cases, and multiple viruses were detected in 14 of 20 cases. The prevalence of each virus was similar in the three groups studied. CONCLUSIONS: In fatal asthma, lower airway secretions do not show a specific pattern of viral nucleic acid. Intriguingly, these results suggest that the lower respiratory tract may act as a potential reservoir for common respiratory viruses.
Adenoviridae↗
Ultrastructural localization of viral nucleic acid by in situ hybridization.
In situ hybridization has become a standard technique in the localization of viral nucleic acids in tissue sections and cytologic preparations at the light microscopic level. We have extended this technique to the electron microscopic level using human cytomegalovirus (CMV) infection in cultured human foreskin fibroblasts, and have shown for the first time that colloidal gold can be used to study intranuclear localization of viral replication. CMV-infected fibroblasts exhibiting early (4-day) and late (18-day) cytopathic effect were fixed in formalin, gently permeabilized with detergent and protease, and hybridized with a biotinylated CMV DNA probe. Hybridized sequences were localized by a pre-embedding technique using streptavidin-conjugated 15 to 20 nm colloidal gold particles. Ultrastructural nuclear and cytoplasmic architecture were well preserved through permeabilization and hybridization steps. Viral DNA was clearly detected in fibroblast nuclei containing nascent and well-formed electron-dense viral inclusions. Gold particles were localized to the periphery of electron-dense nuclear inclusions, occasionally in association with 70 nm nuclear dense bodies, but not with complete viral nucleocapsids. DNA hybridization was abolished by pretreatment of infected cells with DNase. Cross-hybridization of CMV DNA sequences with human DNA or with herpes simplex virus genome was not observed. The ultrastructural findings suggest that CMV DNA replication may occur at the margins of electron-dense regions in maturing viral inclusions, and that viral DNA associated with core dense bodies is available for hybridization with complementary nucleic acid sequences. This technique can be useful in studies of viral pathogenesis.
Cells, Cultured↗
Frequent detection of viral nucleic acids in heart valve tissue.
Due to a paucity of published data concerning the prevalence of viral nucleic acid in homografts, we analyzed tissue from 30 donor hearts for the presence of viral genome sequences of enteroviruses, adenoviruses, human cytomegalovirus, and influenza virus using different PCR techniques. Viral DNA was amplified in 64 and 52% of the subvalvular myocardial tissue and non-coronary valve samples, respectively. These findings, compared with clinical history and histologic and serologic analysis, demonstrate the importance of viral safety measures in heart valve banking.
Adolescent↗
Viral nucleic acid synthesis and antigen accumulation in pancreas and kidney of Pekin ducks infected with duck hepatitis B virus.
Liver, pancreas, and kidney from Pekin ducks infected with duck hepatitis B virus (DHBV) were assayed for the presence of both viral antigen and replication-specific forms of viral nucleic acid. In young congenitally infected ducks, antigen was detectable in hepatocytes and bile duct epithelia, in kidney glomeruli and tubular epithelia, and in cells localized to pancreatic acini. In older experimentally infected ducks, antigen was detectable in hepatocytes, in glomeruli and tubular epithelia, and in cells localized to presumptive pancreatic alpha-islets. All but the glomeruli-associated viral antigen appeared to be localized to the cytoplasm of antigen-positive cells. Much of the glomeruli-associated antigen appeared to be extracellular and was detected in glomeruli that were positive for the accumulation of immunoglobulin, observations suggestive of the deposition of viral antigen-antibody complexes. As analyzed with bulk tissue, replication-specific forms of viral nucleic acid were detectable in liver and pancreas from the young congenitally infected ducks and in liver and kidney from the older experimentally infected ducks.
Animals↗
A novel method for liberating viral nucleic acid for assay of water samples with cDNA probes.
Rapid and sensitive methods are needed for the detection of enteric viruses to ensure proper drinking water quality. Gene probes have been shown to be useful for this purpose. Previously, samples to be assayed were treated with a series of phenol-chloroform extractions to release the viral nucleic acid. We have developed a more rapid procedure for liberating or exposing the genome of poliovirus for probing. In this study, a poliovirus model was used to test the ability of heat (65 degrees C for 30 min) for release or exposure of viral nucleic acid. Several different RNase inhibitors were tested for their ability to prevent viral RNA degradation. A comparison of the two methods indicates phenol-chloroform extraction is not necessary before probing. In addition to saving 2-4 h of time, maximum sensitivity levels were consistently obtained using this novel procedure.
DNA↗
Evaluation of an automated high-volume extraction method for viral nucleic acids in comparison to a manual procedure with preceding enrichment.
BACKGROUND AND OBJECTIVES: Nucleic acid extraction still harbours the potential for improvements in automation and sensitivity of nucleic acid amplification technology (NAT) testing. This study evaluates the feasibility of a novel automated high-volume extraction protocol for NAT minipool testing in a blood bank setting. MATERIALS AND METHODS: The chemagic Viral DNA/RNA Kit special for automated purification of viral nucleic acids from 9.6 ml of plasma by using the chemagic Magnetic Separation Module I was investigated. Analytical sensitivity for hepatitis C virus (HCV), human immunodeficiency virus-1 (HIV-1), hepatitis B virus (HBV), hepatitis A virus (HAV) and parvovirus B19 (B19) was compared to our present manual procedure that involves virus enrichment by centrifugation. RESULTS: Chemagic technology allows automation of the viral DNA/RNA extraction process. Viral nucleic acids were bound directly to magnetic beads from 9.6-ml minipools. By combining the automated magnetic beads-based extraction technology with our in-house TaqMan polymerase chain reaction (PCR) assays, 95% detection limits were 280 IU/ml for HCV, 4955 IU/ml for HIV-1, 249 IU/ml for HBV, 462 IU/ml for HAV and 460 IU/ml for B19, calculated for an individual donation in a pool of 96 donors. The detection limits of our present method were 460 IU/ml for HCV, 879 IU/ml for HIV-1, 90 IU/ml for HBV, 203 IU/ml for HAV and 314 IU/ml for B19. CONCLUSIONS: The 95% detection limits obtained by using the chemagic method were within the regulatory requirements for blood donor screening. The sensitivities detected for HCV, HBV, HAV and B19 were found to be in a range similar to that of the manual purification method. Sensitivity for HIV-1, however, was found to be inferior for the chemagic method in this study.
Automation↗