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PubMedDate not supplied

[PCR system for the detection of the RNA of hepatitis A virus].

The detection of the causative agent of hepatitis A in the patient's body is the necessary element of the diagnostics of this disease. In this work the PCR system for the analysis of the RNA of hepatitis A virus in the patient's blood is presented and characterized. The method of the detection of the RNA of hepatitis A virus is based on the "nest" principle and consists of two consecutive reactions. In the first reaction the reverse transcription and amplification with the external pair of primers are carried out. The product thus obtained is used as material for the second reaction with the internal pair of primers. This method was used for the study of 44 blood samples from hepatitis A patients and 23 blood samples from healthy donors. The detection rate of the RNA of hepatitis A virus in blood samples from the patients was 82%. Viral RNA could be detected in the serum in 72% of cases, both in the serum and in mononuclear blood cells in 20% of cases, in mononuclear blood cells only in 8% of cases.

DNA Primers↗
PubMed1993

Rapid diagnosis of enterovirus infection by magnetic bead extraction and polymerase chain reaction detection of enterovirus RNA in clinical specimens.

We describe a rapid method for extraction and detection of enterovirus RNA in clinical samples. By using magnetic bead technology, enterovirus RNA was efficiently and rapidly extracted from cerebrospinal fluid, stool, saliva, blood, pericardial fluid, urine, and cryopreserved or formalin-fixed solid tissue. Enterovirus RNA was then detected by reverse transcription followed by polymerase chain reaction amplification with primers designed to allow detection of most enterovirus serotypes. For detection of enteroviruses in specimens from patients with acute enteroviral disease, the overall sensitivity of enzymatic RNA amplification was greater than that of cell culture isolation, especially in blood specimens and in stool specimens from patients with acute cardiac disease. Enterovirus RNA was also detected in cryopreserved and archival formalin-fixed myocardial tissue from patients with acute myocarditis and chronic dilated cardiomyopathy. The ability to study archival specimens is of particular value in conducting retrospective investigation. The RNA extraction procedure used was considerably faster than extraction methods using organic reagents, used less hazardous reagents, and was of similar sensitivity. This detection protocol may therefore be useful both for the diagnosis of enterovirus infection and in studying the pathogenesis of acute and chronic enterovirus-induced disease.

Adult↗
PubMed1997

Frequent detection of hepatitis A viral RNA in serum during the early convalescent phase of acute hepatitis A.

The diagnosis of type A hepatitis is performed mainly by immunoglobulin M (IgM) anti-hepatitis A antibody assay, but it has not been established whether there is a correlation between changes in viremia and the clinical course of type A hepatitis. We examined hepatitis A virus (HAV) RNA in the sera from type A and non-A, non-B, non-C acute hepatitis and analyzed the relation of HAV viremia with alanine aminotransferase (ALT) and IgM-HA levels. Two hundred sera from 38 patients with type A acute hepatitis and 20 patients with non-A, non-B, non-C acute hepatitis were examined for the presence of HAV RNA. HAV RNA was detected by nested reverse transcription-polymerase chain reaction (RT-PCR) with primers located at the 5' non-translated region of HAV. HAV RNA was detected in 35 of 38 (92%) type A hepatitis patients and in 60 of 156 (38%) serum samples. In contrast, it was detected in none of 44 serum specimens from 20 non-A, non-B, non-C acute hepatitis patients. In type A hepatitis, the mean ALT level in HAV RNA positive specimens was 1,481 +/- 2,042 (range, 20-10,370) IU/L and that in HAV RNA negative specimens 186 +/- 330 (range, 8-1,698). The positivity of HAV RNA was correlated with the level of transaminase at the time of sample collection. The mean duration from the onset of symptoms to disappearance of HAV RNA was 18 +/- 14 days. The mean titer of IgM-HA in HAV RNA positive cases was 5.0 +/- 1.4, in negative cases 5.7 +/- 1.1, with no statistical difference. Our results indicate that HAV RNA in serum is detectable in the majority of type A hepatitis cases in their early convalescent phase by nested RT-PCR.

Acute Disease↗
PubMed1993

Contributions of the brome mosaic virus RNA-3 3'-nontranslated region to replication and translation.

Sequences upstream of the 3'-terminal tRNA-like structure of brome mosaic virus RNAs have been predicted to fold into several stem-loop and pseudoknot structures. To elucidate the functional role of this upstream region, a series of deletions was made in cDNA clones of RNA-3, a genomic component not required for replication. These deletion mutants were transcribed in vitro and cotransfected with RNA-1 and RNA-2 into barley protoplasts. Deletion of single stem-loop structures gave progeny retaining near-wild-type accumulation levels. Constructions representing deletion of two or three stem-loops substantially lowered the accumulation of progeny RNA-3 relative to wild-type levels. RNA-3 mutants bearing deletions of longer sequences or of the entire region (delta PsKs RNA-3) replicated poorly, yielding no detectable RNA-3 or RNA-4 progeny. Levels of RNA-1 and RNA-2, in the presence of a mutant RNA-3, were found to increase relative to the accumulation observed in a complete wild-type transfection. The stability of delta PsKs RNA-3 in protoplasts was somewhat lower than that of wild-type RNA during the first 3 h postinoculation. Little difference in translatability in vitro of wild-type and RNA-3 constructs bearing deletions within the stem-loop region was observed, and Western immunoblot analysis of viral coat protein produced in transfected protoplasts showed that protein accumulation paralleled the amount of RNA-4 message produced from the various sequences evaluated. These results indicate that the RNA-3 pseudoknot region plays a minor role in translational control but contributes substantially to the overall replication of the brome mosaic virus genome.

Base Sequence↗
PubMed1995

Transfection of a differentiated human hepatoma cell line (Huh7) with in vitro-transcribed hepatitis C virus (HCV) RNA and establishment of a long-term culture persistently infected with HCV.

T7 RNA polymerase transcripts of a putative full-length cDNA clone of hepatitis C virus type 1 (HCV-1) were used to transfect a differentiated human hepatoma cell line, Huh7. The transfected genome replicated in cells, as evidenced by the appearance of progeny HCV RNA, detection of negative-strand viral RNA, and incorporation of [3H]uridine into the viral genome. Incubation of naive Huh7 cells with conditioned medium from transfected cells resulted in a new HCV infection, suggesting the production of biologically active virus in the inoculum. Maintenance of the transfected cells under serum-free culture conditions resulted in the selection of persistently infected cells which displayed a distinctive cellular morphology. This is the first demonstration that HCV RNA produced from cloned HCV cDNA is infectious and replication competent. This approach should provide a valuable system for studying HCV replication, persistence, and pathogenicity.

Base Sequence↗
PubMed1998

A single-tube RT-PCR method for the detection of Borna disease viral genomic RNA.

For detecting Borna disease virus (BDV) genomic stranded RNA, single-tube reverse transcription-polymerase chain reaction (St RT-PCR) was developed to equal the sensitivity of RT-nested PCR but with reduced risk of contamination. BDV-genomic stranded RNA was synthesized in vitro using plasmid cDNA of BDV p24 region as a template and RNA was also extracted from BDV-persistently infected MDCK (MDCK/BDV) cells. Both RNAs were amplified by St RT-PCR in which a single round of RT and a single round of PCR were performed in the same tube. Ten copies of synthesized RNA could be amplified by St RT-PCR, indicating that St RT-PCR method is as sensitive as the ordinary RT-nested PCR method. Furthermore, this method was applied to quantify the exact copy number of genomic RNA in MDCK/BDV cells. Signals were obtained from the samples containing more than 1 pg total cellular RNA. From the results, approximately 100 copies of BDV genomic RNA exist in one MDCK/BDV cell. BDV genomic RNA from the in vivo RNA samples using St RT-PCR, indicating this method is applicable for the epidemiological study of BDV without contamination.

Animals↗
PubMed1993

Identification of bovine glutamate dehydrogenase as an RNA-binding protein.

Two RNA binding activities were demonstrated in bovine liver homogenate. One binding protein was isolated by a simple ion exchange and gel filtration protocol and was shown by N-terminal protein sequence analysis to be glutamate dehydrogenase. Using identical RNA substrate and assay conditions, no detectable RNA binding was observed with equimolar amounts of other representative dehydrogenases and proteins. Furthermore, excesses of tRNA, salmon testis DNA, or each of the four homoribopolymers were unable to compete for the RNA-binding site. Total cytosolic RNA, however, successfully prevented binding of radiolabeled RNA substrate. These data are consistent with glutamate dehydrogenase containing a binding site for heteropolymeric RNA with highest affinity for an as yet undefined nucleotide consensus sequence or structure. The potential physiological relevance of these observations is discussed.

Animals↗
PubMed1994

Detection of hepatitis C virus RNA in hemodialysis patients.

The clinical significance of the high prevalence of antibodies to hepatitis C virus (HCV) in dialysis patients remains undefined. In order to assess the relationship between seropositivity and potential infectivity, 63 patients undergoing maintenance hemodialysis were evaluated between April and May 1990. The mean duration of maintenance hemodialysis was 45 mo (range, 13 to 144). Eighty-two percent (52 of 63) had received blood transfusions, and 16% (10 of 63) had a history of iv drug abuse. Serum samples were analyzed by HCV-cDNA polymerase chain reaction; antibodies to HCV structural (core) and nonstructural regions NS3 and NS4 were determined by enzyme immunoassay. Specimens repeatedly reactive for anti-HCV and HCV-RNA-positive samples were tested by HCV MATRIX dot immunoblot assay and HBV-DNA PCR. Twenty-five percent (16 of 63) were anti-HCV-positive. Of the 16 anti-HCV-positive patients, HCV-RNA was detected in 5 (31%) with the NS3 primers and in 12 (75%) with 5'-noncoding primers. Among the anti-HCV-negative patients, HCV-RNA was detected in 2 (4.3%) of 47 patients. Eleven of the 18 patients with HCV infection (anti-HCV and/or HCV-RNA-positive) had evidence of additional present or past viral infections (human immunodeficiency virus and/or hepatitis B virus). In summary, HCV-RNA is present in at least 75% of anti-HCV-positive patients, suggesting that they may be infectious. The detection of HCV-RNA in anti-HCV-negative patients may indicate early or chronic HCV infection not detected by current antibody assays or the inability of these patients to mount or sustain a significant antibody response.

Adult↗
PubMed1988

Flow cytometric detection of ribosomal RNA in suspended cells by fluorescent in situ hybridization.

A method using flow cytometry and fluorescent in situ hybridization (ISH) to detect RNA in cells is described. L1210 murine leukemia cells were fixed with 1% formaldehyde in HEPES buffered Hank's balanced salt solution (HH) followed by 70% ethanol. Endogenous RNAses were blocked by diethylpyrocarbonate treatment. Single-stranded sense and antisense RNA probes, labeled with biotin-11-UTP, were transcribed from a 2.1 kb 28S ribosomal RNA (rRNA) gene fragment subcloned into the pGEM2 plasmid. For good results, it was essential that the probes were degraded to 100-150 nucleotides before use. Hybridization was performed at 45 degrees C in 50% formamide, 5 x SSC, 0.5% SDS. Hybrids were detected with streptavidin-FITC by flow cytometry. Antisense rRNA probe signal was 100 times higher than the background. The hybrids were largely resistant to RNAse and melted at high temperature. The sense probe also gave a signal (5 times background), which was not RNAse resistant and was attributed to the presence of internal inverted repeats in the ribosomal RNA. When sufficient background reduction can be achieved, it is expected that as few as ten mRNA molecules per cell can be detected with the fluorescent in situ hybridization method.

Animals↗
PubMed1997

Indeterminate results of the second-generation hepatitis C virus (HCV) recombinant immunoblot assay: significance of high-level c22-3 reactivity and influence of HCV genotypes.

A second-generation recombinant immunoblot assay (RIBA 2.0) is used in the United States to confirm infection with hepatitis C virus (HCV) in samples that are anti-HCV (enzyme immunoassay) positive. In some cases, indeterminate results of RIBA 2.0, which are defined as reactivity to a single antigen species or reactivity limited to two proteins derived from the same coding region of the HCV genome, are encountered. This study was performed to establish the significance of indeterminate RIBA 2.0 results in relation to HCV RNA detection, high positivity for the c22-3 band, and the HCV genotype as determined by direct DNA sequencing. Ninety-six samples with indeterminate RIBA 2.0 results were studied. HCV RNA was detected in 21 of 34 (62%) samples with high reactivity to c22-3 and in 8 of 62 (13%) samples with low reactivity to c22-3. The HCV genotype distribution in samples that were RIBA 2.0 indeterminate and HCV RNA positive was significantly different from that in samples of a control group with positive results for both the RIBA 2.0 and HCV PCR. These results suggest that highly positive c22-3 samples are likely to be associated with HCV viremia and that infection with less common HCV genotypes is more commonly associated with indeterminate RIBA 2.0 results.

Antigens, Viral↗
PubMedDate not supplied

Tests for RNA tumor viruses in cell substrates used in virus vaccine production.

Test to detect RNA tumor viruses in cell substrates were reviewed with respect to advantages and disadvantages. Viral induction, electron microscopy, reverse transcriptase assays, density gradient centrifugation, and radioimmunoassays were performed on several cell substrates now in use or being considered for use in vaccine production. RNA tumor viruses were not detected by any of the methods except in the positive control cultures.

Animals↗
PubMed1976

Search for scrapie-specific RNA and attempts to detect an infectious DNA or RNA.

Preparations of in vivo labelled RNA from normal and scrapie infected mouse brains were fractionated by polyacrylamide gel electrophoresis. No appreciable differences were detected between the two preparations. The biological activity of preparations of RNA and DNA from scrapie brain was also examined. In each case the nucleic acid was obtained by one method envolving the use of phenol and one without phenol. Attempts were made to bind the nucleic acid to normal membrane by three different methods. In no instance was a single case of scrapie obtained by inoculating nucleic acid extracts from scrapie brain either alone or associated with normal membranes. It is concluded that a putative scrapie-specific nucleic acid must either be present in scrapie brain in such an unusual from that it is not extracted readily by methods suitable for the preparation of most virus nucleic acids; or, if it is extracted, that it has no detectable biological activity. In either case, the scrapie agent differs substantially in its properties from the plant viroids.

Animals↗
PubMed1993

Replication of hepatitis delta virus RNA: effect of mutations of the autocatalytic cleavage sites.

Hepatitis delta virus (HDV) contains a circular RNA genome of 1.7 kb. HDV RNA replication is thought to proceed via a rolling-circle mechanism that is dependent on autocatalytic cleavage and ligation reactions. However, it has never been established that these ribozyme activities are indeed involved in HDV RNA replication. To investigate the possible biological significance of HDV RNA self-cleavage, we constructed several HDV dimer cDNAs containing single-base substitutions of the 3' nucleotide of the genomic and the antigenomic self-cleavage sites. These mutations were known to affect self-cleavage in vitro to various extents. The effects of these mutations on HDV RNA replication were examined in hepatic and nonhepatic cell lines. The results showed that all of the mutants which had lost the in vitro self-cleavage activity could not replicate. The only mutant which retained full cleavage activity replicated as efficiently as the wild-type RNA. Thus, this study established that self-cleavage activity is required for HDV RNA replication in cells. Interestingly, the level of HDV RNA detected in cells transfected with this replication-competent mutant and that detected in cells transfected with the wild-type construct were similar in COS-7 cells but vastly different in HepG2 and Huh-7 cells, suggesting that HDV RNA self-cleavage activity may be modulated by cell-specific factors. We also compared the effects of mutations when the primary transcripts of these constructs were of either genomic or antigenomic sense. In constructs which synthesize primary transcripts of genomic sense, all of the antigenomic self-cleavage mutants produced as much hepatitis delta antigen (HDAg) as did the wild-type construct, even in the absence of detectable HDV RNA replication, whereas the genomic self-cleavage mutants produced very little HDAg. These and other data suggest that (i) the primary HDV RNA transcripts of both genomic and antigenomic polarities must first be processed to serve as a template for HDV RNA transcription, (ii) efficient cleavage at the antigenomic self-cleavage site is not required for HDAg expression, and (iii) HDV RNA replication most likely occurs by a double-rolling-circle mechanism.

Animals↗
PubMed2000

Detection of chemical-induced differential expression of rat hepatic cytochrome P450 mRNA transcripts using branched DNA signal amplification technology.

The importance of the cytochrome P450 (CYP) enzyme family in xenobiotic metabolism, as well as their differential expression and activity in response to a wide range of environmental chemicals and pharmaceuticals, is well documented. The objective of this study was to evaluate the specificity of the branched DNA (bDNA) signal amplification technique for the detection of multiple rat CYPs from hepatocellular RNA. Oligonucleotide probe sets were designed to various chemically inducible rat CYP mRNA transcripts, including CYP1A1, CYP1A2, CYP2B1/2, CYP2E1, CYP3A1/23, and CYP4A2/3. The robustness of the bDNA assay was assessed with the CYP2B1/2-specific probe set, and total RNA was isolated from control and phenobarbital (PB)-treated rats. Analysis of these RNA samples by bDNA signal amplification resulted in a linear quantifiable range of RNA detection that spanned three orders of magnitude (0.1-100 microg of total RNA). The fidelity of the bDNA assay was evaluated within a single assay and between assays where repeated measurements of a single sample were reproduced reliably. The specificity of individual CYP probe sets was evaluated with five typical CYP-inducing chemicals on the expression of specific hepatic CYP mRNA transcripts. Male Sprague-Dawley rats were administered 3-methylcholanthrene, PB, isoniazid, pregnenolone-16alpha-carbonitrile, or clofibric acid to induce transcription of CYP1A1, CYP1A2, CYP2B1/2, CYP2E1, CYP3A1/23, and CYP4A2/3 mRNA, respectively. Analysis of chemical-induced differences in gene expression by bDNA signal amplification indicated that 3-methylcholanthrene induced CYP1A1 and CYP1A2 mRNA levels 670- and 11-fold, respectively; PB induced CYP2B1/2 expression 71-fold; pregnenolone-16alpha-carbonitrile induced CYP3A1/23 expression 34-fold; and clofibric acid induced CYP4A2/3 expression 4.7-fold. Overall, these data support the use of bDNA signal amplification technology as a robust, reproducible, and efficient means of monitoring the differential expression of multiple isoforms of the CYP enzyme family.

Animals↗
PubMed2001

[Detection of enterovirus RNA in cerebrospinal fluid from patients with aseptic meningitis and encephalitis and its clinical significance].

BACKGROUND: To study the pathogenicity of enterovirus (EV) infection in central nervous system and the method for its detection. METHODS: Using reverse transcription polymerase chain reaction (RT-PCR) and viral culture techniques to detect EV from 46 cerebrospinal fluid (CSF) samples of aseptic meningitis and encephalitis patients. The sensitivity and specificity of RT-PCR for EV RNA detection was affirmed by specific RNA identification of 40 strains of prototype enterovirus. RESULTS: By the virus culture method, 14 out of the 46 CSF sample were tested to beEV positive (26.1%) including 6 Echovirus type 2 (ECV2), 2 Coxackie virus type B2 (CVB2), 2CVB6, 1 ECV3 and 1 ECV11. By RT-PCR method, 31 of 46 CSF sample were tested EV RNA positive (67.4%) through amplified product agarose electrophoresis and northern blot hybridizition. The positive CSF included 14 samples that had been tested positive by virus culture and 17 samples that accounted for 53.9% of the 32 CSF samples which were negetive by virus culture. The sensitivity of RT-PCR was statistically higher than that of the virus culture method, Chi(2) =12.57, P <0.01. CONCLUSIONS: EV is known as important etiological agents of aseptic meningitis and encephalitis. RT-PCR is sensitivity, specificity and rapidity, it may be popularized as an effective method to the detection of enterovirus.

Adolescent↗
PubMedDate not supplied

Radionuclide angiography and Doppler sonography to detect patients with cerebrovascular disease. A correlation with radiographic angiography.

Cerebral radionuclide angiography with 99mTc pertechnetate (RNA) and directional Doppler sonography (DS) were employed to study patients with cerebrovascular disease (CBVD). The 86 patients investigated were divided following radiographic angiography (RGA) into normals (n = 26) and into patients with intracranial (n = 22) and extracranial (n = 38) vascular lesions. Of the patients with angiographically demonstrated CBVD, RNA detected 90%, DS 53.3%. The combined evaluation had a sensitivity of 93.3%. If intracranial arterial disease was excluded, the sensitivity of the studies was 92.1% for RNA and 84.2% for DS and combined evaluation had a sensitivity of 97.4%. The diagnostic accuracy by combined evaluation was 93.8% for the extracranial arterial lesions if the clinical findings were also used in patients with normal RGA pattern. RNA and DS complement each other as RNA contributes to the detection of intracranial blood flow alterations indicating vascular changes in either the extra- or intra-cranial vessels and helps confirm and complete DS findings.

Arterial Occlusive Diseases↗
PubMed1997

Frequency and natural history of rhinovirus infections in adults during autumn.

Human rhinovirus (HRV) accounts for a significant portion of common-cold illness, with the peak incidence being in the early fall. Three hundred forty-six adults who had self-diagnosed colds of 48 h or less were enrolled in a study during September and October 1994 to determine the frequency and clinical course of HRV infections. Nasal wash specimens for viral culture and reverse transcription-PCR (RT-PCR) for HRV RNA and human coronavirus OC43 and 229E RNA detection were collected on enrollment, and participants recorded their symptoms twice daily for 14 days. Middle ear pressure (MEP) was measured with a digital tympanometer on days 1 and 7. Picornaviruses (224 HRV and 7 enterovirus isolates) were detected by culture in 67% (231 of 346) of the subjects. Among 114 samples negative by culture, HRV was detected by RT-PCR in 52 (46%) for an overall picornavirus infection rate of 82% (283 of 346 subjects). Among the remaining 62 negative samples, human coronavirus RNA was detected by RT-PCR in 5 patients, so that 288 (83%) of patients had documented viral infection. The first symptom noticed most often was sore throat (40%) in HRV culture- or PCR-positive patients and stuffy nose in HRV-negative patients (27%). No differences in symptom scores over time or in the presence of individual symptoms were noted between groups. The median duration of the cold episodes was 11 days in HRV culture-positive patients, 9.5 days in HRV RT-PCR-positive patients, and 11.5 days in HRV-negative patients. On enrollment, abnormal MEPs (< or = -100 or > or = +100 mm of H2O) were found for 21% of HRV culture-positive patients, 14% of HRV RT-PCR-positive patients, and 10% of HRV-negative patients. No important differences in the clinical course of HRV culture-positive, HRV culture-negative and RT-PCR-positive, or HRV-negative colds were found. These results represent the highest frequency of virologically confirmed natural colds to date and document the importance of rhinoviruses as the cause of colds during fall months.

Adolescent↗
PubMed2002

Factors related to the chronicity and evolution of hepatitis C infection in patients co-infected by the human immunodeficiency virus.

OBJECTIVES: This work analyses the influence of immune status, serum human immunodeficiency virus (HIV) load and hepatitis C virus (HCV) genotypes on the probability of resolution of HCV infection in HIV-co-infected patients, as well as the evolution of HCV viremia after antiretroviral therapy. PATIENTS AND METHODS: Forty-five patients with anti-HIV and anti-HCV antibodies were classified into two groups as a function of the positivity or persistent negativity of HCV RNA detection (active or recovered HCV infection, respectively). They were treated with highly active antiretroviral therapy (HAART). Serum HCV RNA was quantified by the reverse transcription-polymerase chain reaction. HCV genotypes were detected by line probe assay or by detection of type-specific antibodies. RESULTS: HCV RNA was detectable in 30 (66.6%) out of 45 HIV-infected patients. CD4+ T-cell counts, HIV viremia, or HCV genotypes were similar in patients with active or recovered HCV infection. Patients with active HCV infection had a non-significant decrease of HCV viremia during a follow-up of 12 months (from 6.15 +/- 6.32 to 5.96 +/- 6.05 log copies/mL). This was not influenced by baseline HCV or HIV viral load, HCV genotype, or CD4+ T-cell count. The non-significant decrease was present in patients with or without an immunological response to HAART. CONCLUSION: HCV genotypes, immune status, or serum HIV load did not influence the resolution or chronicity of HCV infection in HIV-co-infected individuals. A non-significant decrease of HCV viremia in these patients treated with combinations including antiproteases could be expected.

Adult↗