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At least 361 records · Page 20Linked to original sources
PubMed1991

Enteroviral RNA sequences detected by polymerase chain reaction in muscle of patients with postviral fatigue syndrome.

OBJECTIVE: To determine the presence of enteroviral sequences in muscle of patients with the postviral fatigue syndrome. DESIGN: Detection of sequences with the polymerase chain reaction in a well defined group of patients with the syndrome and controls over the same period. SETTING: Institute of Neurological Sciences, Glasgow. SUBJECTS: 60 consecutive patients admitted to the institute with the postviral fatigue syndrome who had undergone extensive investigation to exclude other conditions. 41 controls from the same catchment area without evidence of fatigue, all undergoing routine surgery. MAIN OUTCOME MEASURES: Routine investigations, serological screen for antibodies to a range of viruses, and presence of enteroviral RNA sequences in muscle biopsy specimens. RESULTS: 15 (25%) patients and 10 (24.4%) controls had important serological findings. 12 patients had neutralising antibody titres of greater than or equal to 256 to coxsackieviruses B1-5 (six positive for enteroviral RNA sequences, six negative); three were positive for Epstein-Barr virus specific IgM (two positive, one negative). Six controls had similar neutralising antibody titres to coxsackieviruses (all negative); one was positive for Epstein-Barr virus specific IgM (negative); and three had titres of complement fixing antibody greater than or equal to 256 to cytomegalovirus (all negative). Overall, significantly more patients than controls had enteroviral RNA sequences in muscle (32/60, 53% v 6/41, 15%; odds ratio 6.7, 95% confidence interval 2.4 to 18.2). This was not correlated with duration of disease, patient and age, or to raised titres of antibodies to coxsackieviruses B1-5. CONCLUSIONS: Persistent enteroviral infection of muscle may occur in some patients with postviral fatigue syndrome and may have an aetiological role.

Adolescent↗
PubMed2001

Influenza RNA not detected in archival brain tissues from acute encephalitis lethargica cases or in postencephalitic Parkinson cases.

Encephalitis lethargica (EL) was a mysterious epidemic. temporally associated with the 1918 Spanish influenza pandemic. Numerous symptoms characterized this disease, including headache, diplopia, fever, fatal coma, delirium, oculogyric crisis, lethargy, catatonia, and psychiatric symptoms. Many patients who initially recovered subsequently developed profound, chronic parkinsonism. The etiologic association of influenza with EL is controversial. Five acute EL autopsies and more than 70 postencephalitic parkinsonian autopsies were available in the Armed Forces Institute of Pathology (AFIP) tissue repository. Two of these 5 acute EL cases had histopathologic changes consistent with that diagnosis. The remaining 3 cases were classified as possible acute EL cases as the autopsy material was insufficient for detailed histopathologic examination. RNA lysates were prepared from 29 CNS autopsy tissue blocks from the 5 acute cases and 9 lysates from blocks containing substantia nigra from 2 postencephalitic cases. RNA recovery was assessed by amplification of beta-2-microglobulin mRNA and 65% of the tissue blocks contained amplifiable RNA. Reverse transcription-polymerase chain reaction (RT-PCR) for influenza matrix and nucleoprotein genes was negative in all cases. Thus, it is unlikely that the 1918 influenza virus was neurotropic and directly responsible for the outbreak of EL.

Adult↗
PubMed1993

Cryoglobulinemia with vasculitis associated with hepatitis C virus infection.

Essential mixed cryoglobulinemia is frequently associated with chronic hepatitis. This report presents four cases of cryoglobulinemia with vasculitis associated with chronic hepatitis related to hepatitis C virus infection. Hepatitis C virus infection was ascertained in the four patients by both the presence in the serum of anti-HCV antibodies detected by the four-antigen recombinant immunoblot assay and of HCV RNA detected by polymerase chain reaction. In two patients tested, anti-HCV antibodies were not detected after centrifugation in the purified cryoglobulin but were detected in the supernatant. HCV RNA was detected in the purified cryoglobulin in all four patients and was detected in the supernatant in three patients. In one patient receiving recombinant interferon alfa, serum aminotransferases normalized and cryoglobulin disappeared; in another patient receiving recombinant interferon alfa, serum aminotransferases remained high and the cryoglobulin persisted. The presence of HCV RNA in the cryoglobulin and the parallelism of the changes of the cryoglobulinemia and of the serum aminotransferases during recombinant interferon alfa administration suggest that HCV infection is responsible for the production of cryoglobulinemia and vasculitis. It is proposed that HCV infection is a cause of cryoglobulinemia associated with chronic hepatitis.

Adult↗
PubMed2003

A decontamination and sterilization protocol employed during reuse of cardiac electrophysiology catheters inactivates human immunodeficiency virus.

OBJECTIVE: To assess the effect of a standard decontamination and sterilization protocol employed during reuse of cardiac electrophysiology (EP) catheters on human immunodeficiency virus (HIV). SETTING: Public health viral research laboratory. METHODS: Studies were performed on distal, electrode-containing segments of 40 EP catheters previously used in up to 10 clinical EP procedures. EP catheter segments were immersed for 1 hour in blood contaminated with a high titer of HIV. After air drying for 2 hours, subgroups of 8 EP catheters were subjected to either (1) no treatment, (2) washing in general purpose detergent, (3) washing in enzyme cleaner, (4) sterilization in ethylene oxide, or (5) the full protocol of sequential detergent-enzyme cleaner-ethylene oxide exposure. HIV infectivity after treatment was determined by measuring HIV RNA and, in cell culture studies, assessing HIV-induced cytopathic effects (CPEs) and supernatant HIV-specific p24 antigen content RESULTS: With no treatment, all catheters had high HIV RNA levels associated with CPEs and high p24 antigen levels. After washing in detergent, 5 of 8 catheters had HIV RNA detected, but without CPEs or p24 antigen. HIV RNA was detected in all catheters after washing in enzyme cleaner, with CPEs and a high p24 antigen level in 1 of 8 catheters. HIV RNA, CPEs, and p24 antigen were absent after ethylene oxide. After the full protocol, HIV RNA levels were undetectable (n = 7) or low (n = 1), without evidence of CPEs or p24 antigen. CONCLUSION: Appropriate decontamination and sterilization of EP catheters during reuse is highly effective in inactivating HIV.

Antigens, Viral↗
PubMed2003

Inhibition of hepatitis C virus serine protease in living cells by RNA aptamers detected using fluorescent protein substrates.

Hepatitis C virus is one of the causative agents of non-A non-B hepatitis. Since one of viral proteins, NS3, has serine protease activity indispensable for virus maturation. NS3 serine protease is considered to be a suitable target for anti-HCV reagents. We report an assay of HCV NS3 protease in living cells. We designed peptide substrates bearing one of the sequences of HCV NS3 protease cleavage sites sandwiched with fluorescent proteins CFP and YFP. Substrates were expressed and cleaved efficiently in HeLa cells by cotransfection with HCV NS3 protease. The relationship between the progress of cleavage reaction and the change in fluorescence of the substrate emitted from living cells was confirmed. As a group of candidates for inhibitor of HCV NS3 protease, we chose RNA aptamers, nucleic acid ligands selected from a completely random RNA pool by in vitro selection. We found that 3 classes of aptamers, G9-I, II and III, bound NS3 protease specifically and inhibited cleavage in vitro. We studied the effect of RNA aptamers introduced into HeLa cells. The addition of G9-II RNA in the medium at a concentration of 2.5 micro g/ml reduced cleavage by one-third that of control.

Blotting, Western↗
PubMed1995

Detection of Coxsackievirus B3 RNA in mouse myocarditis by nested polymerase chain reaction.

BACKGROUND: A majority of cases of viral myocarditis are associated with group B Coxsackieviruses (CVB) and the persistence of these viruses in the myocardium is associated with the progression of acute myocarditis to chronic dilated cardiomyopathy. A highly sensitive nested polymerase chain reaction (NPCR) is required to study the mechanisms of viral persistence in the myocardium. OBJECTIVES: To develop an enterovirus group-specific NPCR system, to compare it to the reverse-transcription PCR (RT-PCR) plus Southern hybridisation and to investigate the dynamics of viral RNA in a murine model of myocarditis induced by CVB3. STUDY DESIGN: Primers corresponding to the conserved sequences in the 5'-nontranslated region of enteroviruses were designed to ensure a broad specificity. The specificity of PCR products was confirmed by Southern hybridisation. The sensitivity of RT-PCR or NPCR was assessed using reconstructed infected muscle samples. The myocardial samples of the SWR murine model of CVB3-myocarditis were collected from day 1 to 30 after infection. The presence of viral RNA was detected by the RT-PCR or NPCR and infectious virus was isolated by cell culture. RESULTS: Both RT-PCR and NPCR could detect all 11 representative enteroviruses. The NPCR could detect as few as 0.01 plaque forming unit of virus, 100 times more sensitive than the RT-PCR. Virus was isolated from the myocardium in acute phase, but was no longer recoverable after 9 days. Viral RNA was detected by the NPCR technique throughout the studied period. CONCLUSIONS: An enterovirus group-specific NPCR system was developed and was much more sensitive than the RT-PCR technique. It can replace the Southern hybridisation of RT-PCR products. The presence of viral RNA in the myocardium after acute phase indicates a possibility of CVB3 shifting to persistent infection in the SWR mice.

Journal Article↗
PubMed1996

Collaborative study to assess the suitability of an HCV RNA reference sample for detection of an HCV RNA in plasma pools by PCR.

A collaborative study was undertaken to examine the sensitivity and reproducibility of hepatitis C virus (HCV) RNA PCR assays of plasma pools in order to establish a reference sample for HCV PCR testing of plasma pools. Samples consisting of an HCV-RNA-positive donation diluted tenfold in an HCV-RNA-negative cryosupernatant were sent to 16 participating laboratories (including blood product manufacturers and control authorities). The results of the in-house assays indicate that a 10(-4) dilution of the positive donation in the cryosupernatant could be used as the reference sample as this was the highest dilution of the positive donation that was detected in all assays. In contrast, the results obtained with a commercially available assay, designed for use with single-donation plasma or serum (Roche Amplicor TM HCV test), were not so clear-cut and the assay appeared to be ten-fold less sensitive than the in-house assays.

Hepacivirus↗
PubMed1992

The natural history of community-acquired hepatitis C in the United States. The Sentinel Counties Chronic non-A, non-B Hepatitis Study Team.

BACKGROUND: Chronic liver disease develops in more than half of patients with post-transfusion hepatitis C, but little is known about the natural history of community-acquired hepatitis C. METHODS: In 1985 and 1986 we identified adults with acute non-A, non-B hepatitis in four counties in the United States and followed them prospectively. We used three markers to detect hepatitis C virus (HCV) infection in stored samples of serum: antibody to HCV (anti-HCV) detected by second-generation serologic assays; HCV RNA detected by polymerase-chain-reaction assay; and antibody to HCV antigen (anti-HCVAg) detected by fluorescent-antibody-blocking assay. RESULTS: Of 130 patients with non-A, non-B hepatitis, 106 (82 percent) had HCV infection, 93 were positive for anti-HCV, and 13 were positive only for HCV RNA or anti-HCVAg. Chronic hepatitis developed in 60 (62 percent) of 97 HCV-infected patients followed for 9 to 48 months, with no relation to the risk factors for infection. Ten of the 30 patients who had liver biopsies had chronic active hepatitis. In samples collected 42 to 48 months after the onset of hepatitis, HCV RNA was detected in 12 of 13 tested patients with chronic hepatitis and in all 15 tested patients with hepatitis that had resolved. Anti-HCV persisted in all but two of the initially positive patients, for a rate of antibody loss of 0.6 per 100 person-years. CONCLUSIONS: Patients with community-acquired hepatitis C have a high rate of chronic hepatitis. HCV may be a major cause of chronic liver disease in the United States, and in most patients HCV infection seems to persist for at least several years, even in the absence of active liver disease.

Adolescent↗
PubMed1998

[Clinical evaluation of RT-PCR method for detection of HCV-RNA: second report availability of RT-PCR system using PCR internal control for detection of HCV-RNA and the influence of interfering substances on the detection].

The COBAS Amplicor system is an automated diagnostic PCR system which contains a PCR internal control (P. I. C.) template to monitor the amplification. The applicability of COBAS Amplicor HCV was examined using sera of patients with hepatitis. Furthermore, the effects of possible interfering substance (total protein, triglyceride, hemoglobin, glucose, total bilirubin, heparin, lysis reagent including guanidium) on HCV-RNA detection were investigated. The sensitivity of COBAS Amplicor HCV was equivalent to the manual method of Amplicor HCV, moreover all of the results in 54 clinical samples analyzed on both COBAS Amplicor HCV and Amplicor HCV were in agreement. Detection sensitivity of HCV-RNA decreased in the presence of total bilirubin and heparin. Ten and 25mg/dl of total bilirubin affected HCV-RNA detection but did not affect P. I. C. This result suggested that total bilirubin interfered with the protein denature caused by the lysis reagent. Fifteen U/ml of heparin in the sample completely inhibited amplification both of the HCV-RNA and P. I. C. One U/ml of heparin did not affect amplification, but heparinized blood samples should not be used for the detection of HCV-RNA. To examine the effect of possible carry over contamination on the lysis reagent which contains guanidium, various concentrations of lysis reagent in P. I. C. were tested. RT-PCR was inhibited by 1/500 volume contamination of lysis reagent in specimen diluent. Other substances did not affect the sensitivity. Our results indicate that the carryover contamination of lysis reagent cause more "false negative" results than interfering substances in sera. In conclusion, HCV-RNA detection system containing P. I. C., such as COBAS Amplicor HCV, will become a very useful to differentiate "false negative" and "true negative" result.

Bilirubin↗
PubMed2003

Molecular epidemiological and clinical aspects of hepatitis D virus in a unique triple hepatitis viruses (B, C, D) endemic community in Taiwan.

The molecular epidemiological and clinical aspects of hepatitis D virus (HDV) in a unique HBV, HCV, and HDV triple virus endemic community in southern Taiwan were investigated. A total of 2,909 residents aged 45 or older were screened for hepatitis B surface antigen (HBsAg), anti-HCV antibody, and anti-HDV antibody (specifically for HBsAg-positive carriers). Factors that might be associated with HDV infection, viral nucleic acid detection, and genotyping of HBV, HCV, and HDV were investigated. The prevalence of HBsAg and anti-HCV were 12.6% (366/2,909) and 41.6% (1,227/2,909), respectively. For HBsAg carriers, 15.3% (56/366) were positive for anti-HDV assay. Living in a higher endemic district of HCV infection (odds ratio [OR] = 3.2; 95% confidence interval [CI] = 1.7-6.3), male gender (OR = 1.9; 95% CI = 1.1-3.6) and co-infection with HCV (OR = 1.8; 95% CI = 1.0-3.3) were significantly independent factors associated with HDV infection. The detection rate of HDV RNA among anti-HDV-positive patients was only 12.7% (7/55). The mean HBV titer of triple infection group was significantly lower than in the HBV/HDV co-infection group (2.23 vs 3.05 in log(10), copies/ml, P = 0.046). HCV RNA detection among the triple infection group showed 47.4% (9/19) viremia rate and viral loads of 579,121 IU/ml in median (16,803-1,551,190 IU/ml). The prevalent genotype of HBV was type B (23/25); HCV was 1b (7/9) and HDV was IIa/IIb (4/4). Only the presence of HCV RNA predicted the presence of elevated ALT significantly (OR = 25.0; 95% CI = 3.39-184.6). In conclusion, the geographical aggregation of HDV infection paralleled that of HCV infection in this community. HCV suppressed the replication of HBV among triple vital infection patients. HBV and HDV lapsed into a remission or nonreplicative phase in most cases, and HCV acted as a dominant factor in triple viral-infected individuals. Only the presence of HCV RNA was associated with elevated ALT values, but not HBV or HDV.

Female↗
PubMed1987

Primary muscle cells cultivated in medium conditioned by spinal cord cells show changes in messenger RNA as detected by translation in ovo accompanied by synthesis of extracellular matrix components.

Primary muscle cell cultures were prepared from rat embryos and maintained in normal (unconditioned) and spinal cord (conditioned) media. In order to relate translational regulation to the morphological changes associated with each culture condition, mRNA was isolated from both culture variants and subjected to in ovo translation. Xenopus oocytes were injected with mRNA and their incubation media checked for the presence of newly formed proteins coded by the mRNAs and secreted into the medium. Electrophoretograms indicated mRNA-dependent synthesis of several proteins in the molecular mass range of 30-260 kD. To further characterize these proteins, antisera directed against several extracellular matrix proteins were used for immunoprecipitation: antigens recognized by anti-heparan-sulfate-proteoglycan and anti-fibronectin were enhanced in conditioned cells, whereas laminin was found to be reduced.

Animals↗
PubMed1995

Detection of enterovirus-specific RNA sequences in explanted myocardium biopsy specimens from patients with dilated or ischemic cardiomyopathy.

Enteroviral RNA (EV-RNA) was detected in endomyocardial tissue by means of retrotranscription and polymerase chain reaction (RT-PCR) followed by slot-blot hybridization. The myocardial biopsy specimens studied were taken at the time of heart transplantation from 15 patients with dilated cardiomyopathy (DCM) and from 10 patients with ischemic cardiomyopathy (ICM). Specimens from 18 (72%) of the 25 patients were positive for EV-RNA, whereas no control specimens (myocardial specimens from 29 healthy organ donors and atrial specimens from 15 patients with acute myocardial infarction treated by anatomic bypass) yielded evidence of EV-RNA. The rates of EV-RNA detection for the two groups requiring heart transplantation did not differ significantly (66.7% vs. 80.0%; chi 2 test). Our findings support a link between enteroviral infection in both DCM and ICM and suggest a pathogenic role for the enteroviruses.

Adult↗
PubMed1995

Variant estrogen receptor messenger RNA species detected in human primary hepatocellular carcinoma.

The development of hepatocellular carcinoma (HCC) in addition to cirrhosis affects males in a significantly higher proportion than females. Liver estrogen receptors increase when HCC develops in males; however, these tumors usually respond poorly to antiestrogens. We have, therefore, hypothesized that, similar to breast cancer, estrogen receptors in males with HCC may be mutated. Variant estrogen receptor transcripts (lacking exon 5 of the hormone binding domain) were investigated by reverse transcription-PCR in 14 patients (7 males and 7 females) with HCC. While females mostly displayed the wild-type transcript (both in peritumoral and in tumor liver tissue), males showed both transcripts in the cirrhotic tissue and almost only the variant in the tumor. As the variant ER transcripts when translated could give rise to truncated receptors still able to constitutively activate transcription, they may be key factors in favoring deregulated proliferation in the male liver.

Blotting, Northern↗
PubMed1999

[Detection and proliferation of hemorrhagic fever virus in chigger mites].

OBJECTIVE: To study the impact of Leptotromhidium scutellaris as a vector in transmission of hemorrhagic fever. METHODS: Reverse transcription-polymerase chain reaction (PCR) and in situ hybridization technique were used to detect RNA of hemorrhagic fever with renal syndrome virus (HFRSV). TCID(50)/ml of HFRSV were titrated for larva and nymph of chigger mites periodically. RESULTS: RNA of HFRSV was detected in the tissues of chigger mites, such as ovary cells, etc. by PCR and in situ hybridization. Titration of TCID(50)/ml showed that HFRSV could be transmitted transstadially and proliferated in chigger mites. CONCLUSION: It provides direct evidence at molecular level for the role of chigger mite as a vector in transmission of HFRSV with theoretical and practical importance in prevention of HFRS.

Animals↗
PubMed2001

[HCV-PCR-Hybrid.ELISA for the detection of HCV RNA in serum samples from hepatitis patients].

BACKGROUND: To detect HCV RNA in serum samples from hepatitis patients by using the methods of HCV-Reverse transcription polymerase chain reaction and hybrid ELISA. METHODS: HCV RNA in serum samples was amplified by RT-PCR with one of the primer labeled with biotin. PCR amplified products were detected with microtiter plate hybridization with HCV specific probe, then determined with steptavidin-HRP assay. Anti-HCV in these samples was also detected with ELISA simultaneously. RESULTS: The positive rates of HCV RNA were 81.8% (72/88) in anti-HCV positive group and 57.9% (88/152) in the whole patients group. Serum HCV RNA and anti-HCV positive rates were compared and the consistent rate was 78.9% (120/152, P>0.05). The positive rate of HCV RNA was higher in H.ELISA (57.9%, 88/152) than that in electrophoresis (35.5%, 54/152)(P<0.01). CONCLUSIONS: The HCV-PCR-H.ELISA was a sensitive, specific, stable, semi-quantitative and easy to operate method. This HCV RNA detection was especially beneficial to early diagnosis during viramia period and was helpful for the evaluation of anti-HCV therapy.

Adolescent↗
PubMed1997

Hepatitis C virus infection in mixed cryoglobulinemia and B-cell non-Hodgkin's lymphoma: evidence for a pathogenetic role.

We investigated the pathogenetic relevance of hepatitis C virus (HCV) infection in mixed cryoglobulinemia (MC) with or without complicating B-cell Non-Hodgkin's lymphoma (NHL) in comparison with other immunological and lymphoproliferative disorders. The following groups of patients were studied: A) 25 patients with MC in 7 cases evolved into B-cell NHL; B) 25 healthy subjects; C) 22 patients with different systemic immune diseases; D) 24 patients with chronic HCV infection without MC; E) 25 patients with B-cell idiopathic NHL. Methods used included: i) Polymerase chain reaction (PCR) for HCV RNA detection in serum and peripheral blood mononuclear cells (PBMC) (uncultured or mitogen-stimulated); ii) Branched DNA (b-DNA) for HCV RNA quantification; iii) HCV genotyping by genotype-specific primers localized in the core region and by hybridization of amplification products of the 5' untranslated region (5'UTR), obtained with universal primers, using genotype-specific probes. Serum anti-HCV and HCV RNA were detected in 88% and 73% of MC patients, respectively, and in a significantly lower percentage of healthy controls and patients with autoimmune diseases. HCV RNA concentration was significantly lower in supernatants than in corresponding whole sera (p < 0.001). Plus-strand HCV RNA was detected in 81% of peripheral blood mononuclear cell (PBMC) samples and minus-strand in the majority of fresh or mitogen stimulated cells. All MC patients with NHL had HCV RNA sequences in PBMC. HCV genotype 2a/III was detected in MC patients with a prevalence that was significantly higher than in HCV infected patients without MC. Surprisingly, HCV markers (anti-HCV and/or HCV RNA) were found in 32% of patients with idiopathic NHL. These data suggest that HCV infection is involved in the pathogenesis of MC through both direct participation in the immune complex related vasculitis and by triggering the lymphoproliferative disorder underlying the disease. This latter disorder seems to be related to HCV lymphotropism which could also be responsible for the evolution of MC to malignant lymphoma. This study also suggests that HCV infection may be involved in the pathogenesis of idiopathic B-cell NHL through a similar pathogenetic mechanism.

Aged↗
PubMed2007

Simple proteomic checks for detecting noncoding RNA.

Proper validation can accelerate sequence-based discovery of proteins and protein-coding genes. Databases currently contain a backlog of experimentally unverified gene models and tentative assignments of observed transcripts to coding or noncoding RNA. We present and apply a general principle, founded on base composition and the genetic code and validated here by bulk 2-D gels, that can improve the reliability of such classifications and of the algorithms or pipelines that lead to them.

Base Composition↗