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PubMed2002

Circular viral DNA and anomalous junction sequence in PBMC of HIV-infected individuals with no detectable plasma HIV RNA.

Closed circular (cc) forms of extrachromosomal HIV DNA are detected in patients with high viral loads; however, it is unclear whether these forms remain if virus replication is suppressed to undetectable levels by combination antiretroviral therapy. A nested primer polymerase chain reaction amplification assay was used to detect the presence of ccDNA containing two long terminal repeat sequences (2-LTR) in PBMC of patients with low or undetectable plasma HIV RNA. Fifty percent of patients with plasma RNA levels <50 copies/ml of blood had detectable 2-LTR DNA. Sequencing of the products identified normal LTR--LTR junctions in the minority of cases with the majority containing anomalies including deletions and insertions. The persistence of HIV ccDNA in patients with no detectable plasma RNA could be consistent with ongoing de novo infection of dividing cells or with stability of this form of DNA in nondividing cells.

Base Sequence↗

PubMed2000

Endogenously produced nitric oxide inhibits endothelial cell growth as demonstrated using novel antisense cell lines.

Proliferation of endothelial cells is a vital component of vascular repair and angiogenesis. The endothelial cell mediator, nitric oxide (NO) has been reported both to inhibit and to promote endothelial cell proliferation. In this study we have generated cell lines which constitutively express antisense RNA to a region of inducible nitric oxide synthase (iNOS) from a murine endothelial cell line, sEnd-1. In response to stimulation with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) these antisense cells had no detectable RNA for endogenous iNOS, barely detectable iNOS protein and produced 82% less NO than did the control transfected line. Stimulation of the control transfected line caused significant NO production and inhibition of cell growth whereas for the antisense line, producing little NO in response to stimulation, proliferation remained the same as for unstimulated cells. No differences in cell death were observed between unstimulated and LPS/IFN-gamma stimulated cells. The data presented in this study directly demonstrate that NO derived endogenously from iNOS inhibits proliferation of endothelial cells. This approach overcomes problems in other studies where NO donors or non-isoform specific inhibitors of NO synthase have been used.

Animals↗

PubMed2002

Diagnostic methods for detection of respiratory RNA viruses.

This article is a comprehensive description of diagnostic methods for detection of RNA respiratory viruses - respiratory syncytial virus RSV, influenza A and B viruses, parainfluenza 1, 2 and 3 viruses, coronaviruses and rhinoviruses--from cell culture to molecular biology methods. Both patients and medical personnel appear to be at risk of viral infection, specially during the winter season. Moreover, many health care units lack viral diagnostic facilities; therefore, it is essential for medical personnel to have an understanding of the etiology, mechanisms of transmission and of all disposable today diagnostic methods of RNA respiratory viruses. Patients at greatest risk of acquiring nosocomial viral respiratory disease are children, patients with immunodeficiency and patients treated in intensive care.

Antibodies, Viral↗

PubMed1992

Detection of enterovirus RNA in patients with idiopathic dilated cardiomyopathy by polymerase chain reaction.

The pathogenic role of enterovirus in patients with idiopathic dilated cardiomyopathy has been determined through a molecular biologic approach. Sensitivity in the detection of viral genomes in tissues varied between conventional nucleic acid hybridization and polymerase chain gene amplification. To improve diagnosis, we developed a strategy for reverse transcription polymerase chain reaction (RT-PCR) to detect viral RNA. We synthesized two sequence-specific oligonucleotides, primer 1 (5'dACCGACGAATACCACTGTTA3') and primer 2 (5'dCCTCCGGCCCCTGAATGCGGCTAAT3'), complementary to the 5' conserved viral genomic fragments. Viral RNA was amplified by double PCR with these two primers and hybridized with a 32-P labeled inter-primer probe (5'dATGAAACCCACAGGCACAAAG3'). Using this strategy, we detected as little as 10(-8) micrograms of coxsackievirus B3 RNA after amplification with RT-PCR, but detected none in the plasma of eight healthy adults. Among 15 patients with idiopathic dilated cardiomyopathy, viral RNA could be detected in one out of 12 plasmas (8%) and three out of four explanted heart tissues (75%). In contrast, no viral RNA could be detected in six samples of myocardial tissue from patients with other heart diseases. The only patient who had viral RNA in his plasma also had viral RNA in his myocardium. Thus, the high incidence of viral RNA in these patients suggests a possible etiologic link between them. Correct selection of specific PCR primers and the application of double PCR can improve chances of diagnosing enteroviral infection.

Adolescent↗

PubMed1986

Detection of bovine herpesvirus type 1 RNA in trigeminal ganglia of latently infected rabbits by in situ hybridization.

At times after conjunctival inoculation with bovine herpesvirus type 1 (BHV-1), representing the acute and latent phases of infection, rabbit trigeminal ganglia were examined for the presence of BHV-1 nucleic acids by in situ hybridization using a 3H-labelled BHV-1 DNA probe. During the acute phase of virus infection, both BHV-1 DNA and RNA were detected in ganglionic neurons and occasionally in adjacent satellite cells. However, during the latent phase of infection only viral RNA was detectable in involved neurons. Viral RNA appeared restricted to the nucleus of latently infected cells and was present in varying amounts in individual cells. These results indicate that the BHV-1 genome is transcriptionally active in ganglionic neurons during latent infection.

Animals↗

PubMed1997

Improved method to prepare RNA-free DNA from mammalian cells.

To isolate DNA for nucleoside analog incorporation studies, many investigators use RNase A to remove RNA from total cellular nucleic acid. We observed persistence of ribonucleotides from RNA in nucleic acid samples treated with RNase A alone. Although incubation of [5-3H]uridine-labeled nucleic acid with 50 microg/ml RNase A decreased tritium by 97%, HPLC analysis of the resulting DNA preparation digested to nucleosides revealed high levels of ribonucleosides. Increasing RNase A 10-fold (500 microg/ml) effected only a 1.7-fold reduction in ribonucleosides. Overall, the level of ribonucleosides was one-fourth that of the deoxynucleosides, primarily due to the high levels of guanosine. It was hypothesized that the ribonucleosides originated from guanosine-rich tracts of RNA since RNase A cuts preferentially 3' to pyrimidine monophosphates and to some extent after AMP. The addition of 0.05 microg/ml RNase T1, which preferentially cleaves RNA 3' to GMP, decreased total ribonucleosides by nearly 20-fold. In conclusion, we have developed a rapid method which removes greater then 99% of cellular RNA from nucleic acid extracts and a reversed-phase HPLC procedure that detects RNA contamination more sensitively than [5-3H]uridine labeling. These methods are useful for the determination of analog incorporation into DNA, especially for agents which incorporate into both DNA and RNA.

Animals↗

PubMed1973

Virus-specific messenger RNA and nascent polypeptides in polyribosomes of cells replicating murine sarcoma-leukemia viruses.

We present evidence that virus-specific RNA is present in polyribosomes of transformed cells replicating the murine sarcoma-leukemia virus complex and that it serves as messenger RNA for the synthesis of viral-coded proteins. Both virus-specific RNA (detected by hybridization with the [(3)H]DNA product of the viral RNA-directed DNA polymerase) and nascent viral polypeptides (measured by precipitation with antiserum to purified virus) were found in membrane-bound and free polyribosomes. Membrane-bound polyribosomes contained a higher content of both virus-specific RNA and nascent viral polypeptides. From 60 to 70% of viral RNA sequences were released from polyribosomes with EDTA, consistent with a function as messenger RNA. Maximum amounts of both virus-specific RNA and nascent viral polypeptides were found in the polyribosome region sedimenting at about 350 S.

Animals↗

PubMed2003

No role for Epstein-Barr virus in Dutch hepatocellular carcinoma: a study at the DNA, RNA and protein levels.

Epstein-Barr virus (EBV) has been suggested to play a role in hepatocellular carcinoma (HCC). However, reports on detailed EBV transcript analyses in HCCs are limited. It was shown recently that expression of the transforming BARF1 (BamHI A rightward open reading frame 1) gene of EBV is restricted to latently EBV-infected epithelial malignancies, i.e. nasopharyngeal carcinoma and gastric carcinoma. The aim of this study was to test the presence of EBV in Dutch HCCs. A semiquantitative DNA PCR-enzyme immunoassay (PCR-EIA) for the BamHI W fragment of EBV was used to assess the presence of EBV in frozen and paraffin-embedded tissues of 16 HCCs. In addition, several RNA detection techniques, i.e. nucleic acid sequence-based amplification (NASBA), RT-PCR, RNA in situ hybridization (RISH) and immunohistochemistry (IHC), were applied. Five of 16 HCCs and two of four hepatitis C virus hepatitis samples were weakly positive for EBV DNA by PCR-EIA. Using sensitive RNA transcription techniques, no transcripts were found for BARF1, EBNA-1 and BARTs (BamHI A rightward transcripts) in any of the liver tissues tested. In addition, RISH for EBER1/2 and BARTs and IHC for EBNA-1, LMP-1 and ZEBRA, performed on the paraffin-embedded tissue of the PCR-EIA-positive cases and on adjacent non-neoplastic liver tissues, were negative. The absence of epithelial-specific BARF1 transcripts and other EBV transcripts and proteins in the EBV DNA PCR-positive cases argues strongly against a role for EBV in HCC.

Carcinoma, Hepatocellular↗

PubMed1995

Demonstration and distribution of HCV RNA sequences by in situ hybridization and HCV-related proteins by immunohistochemistry in the liver tissue of patients with chronic HCV infection.

Nonisotopic in situ cytohybridization of HCV RNA was attempted in liver specimens from 12 chronically hepatitis C virus (HCV) infected patients. Oligonucleotides deduced from 5'-noncoding and core regions of the HCV genome were labeled with digoxigenin and used on paraformaldehyde-fixed frozen liver sections. The hybrids were visualized immunohistochemically with alkaline phosphatase-conjugated anti-digoxigenin and alkaline phosphatase substrate. These findings were correlated with the results of tissue immunohistochemistry for HCV antigens identified with specific mouse monoclonal antibodies developed against c22-3 antigen (Ag), a core-encoded protein, and c100-3 Ag, a NS4-encoded protein, and histologic assessment of each liver. HCV RNA detected in the above assay was predominantly cytoplasmic; it was detected in all 12 patients and in none of the controls. Tissue HCV RNA was associated with the presence of cytoplasmic (c100-3 Ag) and membrane (c22-3 Ag) expression of viral proteins in all 9 patients with histological evidence of chronic progressive liver disease as judged by the presence of piecemeal necrosis, and lobular and portal tract inflammation. Despite the presence of abundant HCV RNA, none of 3 patients without histological evidence of chronic liver disease showed intrahepatocyte expression of viral proteins. These findings support the view that tissue HCV antigens are markers of progressive damage and demonstrate that active liver disease does not occur without such markers. It is proposed that synthesis of viral proteins and membrane accumulation of c22-3 Ag may be involved in the pathogenesis of hepatocyte injury in chronic hepatitis C infection.

Adult↗

PubMed1994

Treatment of chronic hepatitis D virus infection with low and high doses of interferon-alpha 2a: utility of polymerase chain reaction in monitoring antiviral response.

We examined the efficacy of decreasing high doses (beginning at 18 MU/day) of interferon-alpha 2a vs. that of daily low doses (3 MU) in the treatment of chronic hepatitis delta virus infection. Patients treated with 18 MU had a somewhat higher frequency of normalization of serum ALT levels than patients treated with low doses (31% and 12%, respectively, on an intention-to-treat basis). A decrease in the percentage of hepatitis D virus RNA positivity was observed in both groups at the end of treatment. Thus, whereas in baseline samples 10 (62%) of the patients in each group were positive for hepatitis D virus RNA in serum on slot-blot hybridization, these numbers decreased to 5 (31%) and 4 (25%) patients in groups 1 and 2, respectively, at the end of therapy. However, hepatitis D virus RNA, detected by means of nested polymerase chain reaction, remained in all but two (one in each group) patients who completed the treatment. Finally, during posttreatment follow-up, hepatitis D virus RNA levels returned to baseline values, and only one patient remained negative for this marker. The beneficial effect of interferon-alpha was only transient. Only two patients (one from each treatment group) had persistently normal serum ALT levels after 18 mo of follow-up. Finally, the presence of serum hepatitis D virus RNA at the end of therapy, detected with nested polymerase chain reaction, might be a good marker for the prediction of viral replication relapse.

Adult↗

PubMed2004

Titration of hepatitis C virus in chimpanzees for determining the copy number required for transmission.

OBJECTIVE: To determine the copy number of hepatitis C virus (HCV) RNA, determined by nucleic acid amplification test (NAT) for screening blood units in Japan, that can transmit infection to chimpanzees. METHODS: Fresh-frozen plasma with markers of HCV infection, as well as inocula pedigreed from 1 of them, were evaluated for the infectious activity in chimpanzees. RESULTS: One unit each (273-282 ml) of fresh-frozen plasma from 2 blood donors or a pool from 13 donors to make a unit, which contained high-titered antibody to HCV but without HCV RNA detectable by NAT, did not infect any of 3 chimpanzees. Two chimpanzees were infected, however, when they were inoculated with 1 ml of serum from a blood donor in the 'window period' of HCV infection and containing 7.0 x 10(6) copies/ml of HCV RNA. The preacute phase serum from 1 of them harvested 7 weeks after the inoculation was titrated in 2 chimpanzees, and an inoculum containing approximately 2 x 10(1) copies of HCV RNA could transmit infection to both of them. CONCLUSION: Approximately 20 copies of HCV can transmit infection to recipients, which needs to be taken into consideration in planning the screening of blood units for HCV RNA by NAT. Although the sensitivity of present NAT could be improved further, there would be a limit of it in detecting a low-level HCV RNA in the window period of donors with the infectious capacity in recipients.

Animals↗

PubMed1973

Synthesis of globin ribonucleic acid from duck-reticulocyte chromatin in vitro.

The proteins of chromatin serve to restrict the transcription of DNA. The relevance of these findings to the control of gene expression is contingent upon the demonstration that this restriction is specific and mirrors the patterns of RNA synthesis observed in vivo. In this study we demonstrate by RNA-DNA hybridization that the vast majority of the chromatin-directed RNA is synthesized from the unique regions of the reticulocyte genome. Furthermore, by use of the DNA complement of globin mRNA as a probe in annealing reactions, de novo synthesis of globin RNA was detected in RNA transcripts from duck reticulocyte chromatin. No globin sequences were detected in similar preparations of RNA in vitro either from liver chromatin or from DNA freed of protein. These results show that the proteins of chromatin serve to restrict transcription in a very specific manner and provide convincing evidence for the existence of transcriptional control factors in eukaryotes.

Animals↗

PubMed1997

Comparison of HCV RNA assays for the detection and quantification of hepatitis C virus RNA levels in serum of patients with chronic hepatitis C treated with interferon.

Detection and quantification of hepatitis C virus (HCV) RNA levels by using the standardized qualitative Amplicor HCV and quantitative Amplicor HCV Monitor assays (Roche Molecular Systems) were evaluated in 48 patients with chronic hepatitis C treated with interferon. Results were compared with an in-house reverse transcription and polymerase chain reaction (RT-PCR) assay and the branched DNA (bDNA) assay (Quantiplex, version 1.0, Chiron Diagnostics). Concordance of the qualitative results with the Amplicor HCV and in-house RT-PCR assays occurred in 82% of the samples. All but one of the discrepant specimens were found positive by the Amplicor HCV assay and negative by the in-house RT-PCR. Among the samples with HCV RNA levels measurable with the Amplicor HCV Monitor assay, 22% had HCV RNA titers below the detection limit of the Quantiplex assay. A statistically significant correlation was found between the 2 quantitative assays, although lower titers were obtained with the Amplicor HCV Monitor assay. More important, a good correlation was observed in the evolution of viremia as measured by the 2 assays during interferon therapy. During follow-up of interferon treatments, with the Amplicor HCV Monitor assay, persisting viremia was still detected in 27% of the patients who normalised alanine aminotransferase (ALT), emphasizing the bioclinical relevance of the assay. Pre-treatment serum HCV RNA levels above 10(5) copies/ml were found more frequently in nonresponders than in responders (76% vs. 44%; P < 0.05). Given their great sensitivity and the significant correlations, the Amplicor HCV qualitative and quantitative assays appear useful for the diagnosis and management of hepatitis C infection, and especially for monitoring of therapy.

Adult↗

PubMed1993

Structure of the maize mitochondrial replicon RNA b and its relationship with other autonomously replicating RNA species.

The mitochondria of maize plants with S-type cytoplasm possess a family of single and double-stranded RNA molecules, termed RNA plasmids, that replicate in a DNA-independent manner typical of RNA viruses. We have determined the sequence of the smallest and most abundant member of this family, a single-stranded RNA termed RNA b. The 719 nucleotide sequence of RNA b lacks open reading frames of significant length. Probes complementary to the determined (+) strand sequence identified two larger RNA species of 2900 and > 4000 nucleotides in S-type maize mitochondria, the smaller of which corresponds to a previously identified member of the RNA plasmid family designated RNA a. These probes also unexpectedly identified a low abundance form of RNA b in N-type maize mitochondria. (+) strand probes identified corresponding (-) strand forms in S cytoplasm that likely represent components of replication intermediates. Primer extension experiments demonstrated that RNA b is an internally deleted form of the larger RNA plasmid, RNA a, and that it has a discrete and homogeneous 5' terminus. The solubility of RNA b in 2 M-LiCl as well as structural modelling studies indicated that it has a very high degree of secondary structure. Analysis of specific cleavage products generated by treatment of RNA b-oligonucleotide duplexes with RNase H indicated that the only identifiable forms of RNA b were linear molecules with relatively homogeneous 3' termini; no circular forms were detected. RNA b was found to share a 13 nucleotide sequence with the circular cadang-cadang viroid; 11 of these nucleotides fall within the central conserved region of viroid RNAs. In addition, RNA b shares certain structural similarities with phage Q beta and "variant" RNAs that serve as in vitro templates for the Q beta replicase. Collectively, our results indicate that RNA b is a "defective-interfering" member of an RNA family representing a new and apparently distinct class of RNA replicons.

Base Sequence↗

PubMed1975

The ribonucleic acids of the mammary gland of the guinea pig.

1. 32-P-labelled polyribosome preparations were made from the mammary glands of lactating and late-pregnant guinea pigs after injection of (32-P)i into the animals. 2. The RNA of polyribosomes, ribosomal subunits and that released from polyribosomes by EDTA were analysed by zone velocity centrifugation and by polyacrylamide-gel electrophoresis. 3. RNA species which have the physical properties expected for the milk protein mRNA were detected. RNA species of a size which could code for the caseins were present in lactating but not in pre-lactating mammary-gland polyribosomes.

Animals↗

PubMed1965

A CYTOCHEMICAL STUDY OF CYTOPLASMIC BASIC PROTEINS IN THE ASCIDIAN OOCYTE.

The cytoplasm of young oocytes of the ascidians contains high concentrations of proteins which are stainable with alkaline fast green at pH 8.1 and above. These proteins cannot be stained even with acid dyes at low pH unless RNA is removed. Deamination and formalin blockage of amino groups is incapable of destroying the net positive charge on these protein molecules in the presence of RNA, but these treatments destroy the charge if RNA is removed. It is therefore concluded that basic proteins and RNA exist as a nucleoprotein complex in the ribosomes of these young oocytes. The detectable RNA of the mature oocytes and unfertilized eggs shows no evidence of being associated with basic proteins.

Animals↗

PubMed1991

Identification of the viral haemorrhagic disease virus of rabbits as a calicivirus.

Liver tissue from rabbits which had died of viral haemorrhagic disease (VHD) was used to identify the causative agent of the disease. After extraction of liver homogenates and density gradient ultracentrifugation, viral particles with buoyant densities between 1.32 to 1.38 g/ml and estimated sedimentation coefficients between 100 S and 175 S were obtained. The isolated virions were determined to have a diameter of 40 nm. The particles morphologically resembled those of the Caliciviridae family, had positive reactions both in ELISA and haemagglutination assays, and were infective for rabbits. By immunoblotting, a major structural protein with a molecular weight of 60 kDa was identified. RNA of high purity and of approximately 8 kb was isolated from virions. Labelled cDNA of virion RNA detected two RNAs of 8 kb and 2 kb in Northern blots of liver RNA from animals infected with the VHD virus (VHDV). Finally, isolated virion RNA injected directly into the liver or rabbits produced a disease with clinical symptoms and pathological findings typical of VHD. A calicivirus originally designated "rabbit haemorrhagic disease virus (RHDV)" was thus identified as the causative agent of VHD. The agent causing the European brown hare syndrome (EBHS) is also a calicivirus (EBHSV). VHDV and EBHSV are different members of the Caliciviridae family; their relationship can be clearly demonstrated by using different approaches of investigation.

Animals↗

PubMed1999

Hepatitis C virus transmission in family members of Egyptian patients with HCV related chronic liver disease.

Hepatitis C virus (HCV) infection in Egypt has become a major public health problem. In the present study, sexual and intrafamilial transmission of HCV infection were assessed in the family members of 200 Egyptian patients (index patients) with HCV-RNA positive and biopsy ascertained chronic hepatitis C. Index patients were 139 men (mean age 55+/-11 years) and 61 women (mean age 48+/-8 years). Family members consisted of 200 spouses; 139 women (mean age 45+/-12 years) and 61 men (mean age 58+/-9 years); and 355 children (183 males and 172 females, mean age 11.8+/-10 years). All the family members were tested for the presence of antibodies to HCV in their sera. Thereafter, HCV-RNA detection by PCR and HCV serotype determination were performed in antibody positive contacts. Hepatitis C virus antibodies were detected in 28 (14%) spouses, all of them were also positive for HCV-RNA. Hepatitis C virus serotypes were identical in HCV seropositive patient-spouse pairs (Serotype 4). None of the 355 children involved in this study showed HCV antibodies in their sera. No significant difference was found between the prevalence of male-to-female and female-to-male transmission of HCV. A highly significant association was found between both the age of the spouse and the duration of marriage to index patient and HCV seropositivity in spouses. Moreover, HCV seropositivity in spouses was significantly related to increased serum ALT and HCV-RNA levels, histological severity of chronic hepatitis C and to a history of dental care, as a risk factor for HCV acquisition, in index patients. It was concluded that spouses of patients with HCV viremia and chronic liver disease have an increased risk for acquiring HCV, while intrafamilial acquisition of HCV in non sexual contacts seems to be rare. The authors suggest that spouses of HCV viraemic patients should be followed routinely for markers of HCV infection and liver disease.

Age Distribution↗