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PubMed2004

Detection of foot and mouth disease virus by RT-PCR and microplate hydridization assay using inactivated viral antigens.

A single step RT-PCR was tested for detection of foot and mouth disease virus (FMDV) and immunoenzymatic determination of amplified products in a microplate hybridization assay. Inactivated reference strains (ELISA antigen) of all seven serotypes were used to optimize the test. Oligonucleotide primers were selected from two different genomic regions coding for RNA polymerase and VP1 protein, respectively. The RT-PCR used to amplify the polymerase gene specific RNA detected FMDV strains A, C, O, Asial and SAT1, and the identity of the fragments obtained was confirmed with a specific internal biotin-labelled capture probe. For the amplification of the VP1 genome region, two sets of oligonucleotide primers were used. One primer pair was successfully applied for the detection of serotypes A, C, O and Asial and a second one for serotypes SAT1, SAT2, SAT3. The specific probe enabled the detection of all the amplified products in a PCR ELISA test. By comparison with antigen ELISA, the PCR ELISA method allowed the detection of smaller amounts of FMDV in the inactivated material examined. The application of molecular diagnostic methods to inactivated antigens offers a good alternative procedure for developing and optimizing a sensitive method for detection of FMDV in laboratories that are not allowed to work with viable FMDV.

Animals↗
PubMed2002

Hepatitis C virus viremia in HIV-infected individuals with negative HCV antibody tests.

Hepatitis C virus (HCV) viremia may occur in persons without detectable HCV antibodies and has been reported in as many as 5.5% of HIV-positive persons. To better characterize serosilent HCV infection, the authors prospectively tested 131 HIV-positive persons and 102 HIV-negative control subjects with diabetes for the presence of HCV antibody (Ab) and HCV RNA. Thirty of 31 HCV Ab-positive (AbP) HIV-positive people tested positive for HCV RNA as did both HCV AbP, HIV-negative control subjects. Similarly, none of the 100 HIV-negative, HCV Ab-negative (AbN) control subjects was HCV RNA positive (p<.001). In contrast, 19 of 100 HIV-positive, HCV AbN persons met stringent criteria for HCV viremia, and 9 of these 19 people were HCV RNA positive when tested by a commercially available HCV RNA detection method. The mean duration of HCV viremia in HCV AbN people was 26.8 months (range, 1-99 months). None of the subjects developed HCV antibody during the study. The HIV-positive, HCV AbP, and RNA-positive group was significantly more likely to have acquired HIV parenterally (p<.001), have higher initial CD4 counts (p=.029), and have higher ALT values than the HCV AbN group (p<.002). In summary, HCV infection appears to occur more frequently among HIV-infected, HCV-seronegative persons than appreciated, especially if HIV acquisition was through sexual as opposed to parenteral risk factors and was associated with a lower initial CD4 count and lower ALT values.

Adult↗
PubMed2005

Development of a microcapillary column for detecting targeted messenger RNA molecules.

A capillary column in a rapid-flow system has been developed for detecting targeted messenger RNA (mRNA) molecules. The column has a structure made of two beds-one bed of porous microbeads and one bed of microbeads with a polythymidine base sequence. The targeted eukaryotic mRNA molecules are detected by two-step hybridization (sandwich hybridization) composed of polyadenosine selection of mRNA molecules and formation of a probe-target (targeted mRNA) hybrid. The sandwich hybridization, which is accomplished within 1 h, was tested using synthetic polydeoxynucleotides. Ten picomoles of the targeted polydeoxynucleotide were detected.

Base Sequence↗
PubMed1995

Chemiluminescent detection of caprine arthritis encephalitis virus with a PCR-generated single stranded nonradiolabelled probe.

A 814-bp digoxigenin-labelled single stranded DNA probe was produced and utilized in slot-blot hybridization for detection of caprine arthritis encephalitis virus (CAEV) in goat synovial membrane (GSM) cell culture infected with CAEV. The sensitivity of a PCR-generated probe was compared with a random primer labelled probe. The probe with digoxigenin-dUTP incorporated in the PCR reaction mixture was more sensitive for RNA detection than the random primer probe and it was much simpler to use. The probe was applied for detection of CAEV by blot blot hybridization in peripheral blood mononuclear cells (PBMC) and macrophage cultures obtained from naturally infected goats. This technique was not sufficiently sensitive to detect the viral nucleic acid directly from PBMC or cultured macrophages. When macrophages were cultured in vitro and then cocultured with susceptible GSM cells, samples gave a positive signal in the slot-blot hybridization technique. The use of slot-blot RNA hybridization permits more convenient and rapid confirmation of CAEV isolation in susceptible cells than the conventional identification by syncytia formation.

Animals↗
PubMed1998

Search for hepatitis C virus extrahepatic replication sites in patients with acquired immunodeficiency syndrome: specific detection of negative-strand viral RNA in various tissues.

The existence of extrahepatic reservoirs of hepatitis C virus (HCV) replication remains highly controversial. We searched for the presence of HCV-RNA negative strand in various tissues from eight HCV-infected patients who died of acquired immunodeficiency syndrome (AIDS)-related complications. Negative-strand RNA was detected by a Tth-based reverse-transcriptase polymerase chain reaction (RT-PCR), which was optimized for sensitivity and strand specificity on synthetic RNA templates. This assay was capable of detecting about 10(2) genomic Eq molecules of the correct strand while unspecifically detecting >/=10(8) genomic Eq molecules of the incorrect strand. Negative-strand viral RNA was detected in all but one liver, in lymph nodes (5 cases), in pancreas (5 cases), in adrenal gland (2 cases), in thyroid (2 cases), in bone marrow (1 case), and in spleen (1 case). These data suggest a possible presence of HCV replication sites outside the liver, at least in AIDS patients. Whether these findings relate to various extrahepatic manifestations of HCV infection remains to be determined.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Early and late gene expression in UT-7 cells infected with B19 parvovirus.

UT-7, a human megakaryocytoblastoid cell line, can be persistently infected with B19 parvovirus. We performed detailed serial analysis of parvovirus DNA replication and RNA transcription of synchronized cells. RNA transcription appeared as an early event following infection, with viral RNA detected about 6 hr after infection. In contrast, dimer-replicative intermediate forms of parvovirus DNA did not appear until more than 16 hr after infection. Northern analysis of specific transcripts showed an earlier appearance of nonstructural protein RNA (6 hr) compared to capsid protein RNA (24 hr). The addition of an inhibitor of protein synthesis to block synthesis of nonstructural protein abolished capsid protein RNA transcription as well as DNA replication. Primer extension analysis confirmed the initiation of all transcription from the single P6 promoter. RNA transcription precedes DNA replication of B19 parvovirus in these cells, and RNA processing may have a major role in regulating gene expression.

Base Sequence↗
PubMed1997

cDNA sequencing of the 5' noncoding region (5' NCR) to determine hepatitis C genotypes in patients with chronic hepatitis C.

Reports suggest that response to interferon-alpha therapy is influenced by both hepatitis C viral genotype and titer. Our aim was to determine if direct, automated, cycle sequencing of the PCR product from an HCV RNA detection assay could be used to reliably determine HCV genotype. In addition, the approach was used to determine the HCV genotype distribution in our patient population and to learn if there was a correlation between HCV genotype and RNA titer that could be used to predict response to treatment. In all 143 consecutive patients were tested for both HCV RNA titer and genotype. Automated, cycle sequencing of PCR product was highly effective and failed to yield a genotype in only 3 (2%) patients. The distribution of HCV genotypes was: 1a (40%), 1b (39%), 2a (2%), 2b (6%), 3a (4%). There were significant differences in the median HCV RNA titers between genotypes 1, 2, and 3. High HCV RNA titers >4.4 x 10(6) copies/ml were only seen in genotype 1. However, the HCV RNA level should not be used as a surrogate marker of genotype because of a significant overlap of titers within the genotypes.

Base Sequence↗
PubMed2001

Marfan syndrome caused by a mutation in FBN1 that gives rise to cryptic splicing and a 33 nucleotide insertion in the coding sequence.

We have studied a patient with Marfan syndrome whose mutation was not detected by heteroduplex analysis. Primary cultured patient fibroblasts were metabolically labelled and found to secrete fibrillin-1 defectively when compared with an age-matched control. Sequencing of patient cDNA, isolated by reverse transcription-polymerase chain reaction of patient fibroblast RNA, detected a 33-bp insertion. The reading frame of the mutant allele was maintained and predicted the insertion of 11 amino acids at the beginning of calcium-binding epidermal growth factor-like domain 29. Direct sequencing of genomic DNA detected a heterozygous G+1-->A transversion in intron 46 of FBN1. The 11 amino acid insertion was the consequence of the usage of a cryptic splice site 33-bp downstream of the mutation. This is the first reported case of a splicing defect in FBN1 leading to the production of a full-length fibrillin-1 transcript containing a large amino acid insertion.

Adult↗
PubMed2004

STR2: a structure to string approach for locating G-box riboswitch shapes in pre-selected genes.

Traditional sequence-based search methods such as BLAST and FASTA can be used to identify sequence similarities. Recently, there is a growing interest in performing RNA shape similarity searches inside selected genes to locate RNA structure motifs that are known to possess functionally important roles. For example, in the newly discovered RNA genetic control elements called "riboswitches", the box domain is known to be highly conserved among various bacterial species in both its nucleotide composition and shape. However, in non-bacterial species, shape conservation is likely to become more important than sequence conservation when searching for riboswitch patterns. For this purpose, we present an approach tailored for detecting RNA shape similarities. We extend the Structure to String (ST R2) method that was initially proposed to locate shape similarities in proteins to identify predicted secondary structures of RNAs. The ST R2 for RNAs is a translation of a secondary structure to a string of characters, after which known sequence-based search algorithms with an efficient implementation are being used. We validate that the ST R2 succeeds to locate G-box riboswitches in prokaryotes, as expected. Subsequently we show running examples when attempting to detect G-box riboswitch candidates in eukaryotes.

Algorithms↗
PubMed1982

In vitro transcription of vesicular stomatitis virus: initiation with GTP at a specific site within the N cistron.

In vitro transcripts of vesicular stomatitis virus (VSV) were either 5'-terminally labeled by incorporation of [beta-(32)P]GTP or were selected on Hg-agarose after incorporation of gamma-thio-GTP. Capped RNAs ranged in size from 23 nucleotides, the shortest capped RNA detected, to full-length message size. The 5'-terminal sequences corresponded to those of N message and to a small amount of NS message. Approximately 14% of the capped N gene transcripts were terminated at positions 86 to 90 of the VSV genome, giving rise to specific, 36 to 40-nucleotide-long, capped RNA species. The GTP-initiated RNAs were short with a predominant 28-nucleotide-long RNA species. A minor portion was as large as mRNAs. Nucleotide sequence analyses of the short RNA revealed that it was specifically initiated at positon 91 of the VSV genome, 41 nucleotides within the N cistron. This corresponds exactly to the site where transcription of the 40-nucleotide-long, capped RNA terminated. Initiation with GTP at position 91 occurred at approximately the same frequency as termination of the capped RNA at position 90, suggesting that intracistronic initiation at position 91 may depend upon termination of transcription of the 5'-proximal region and therefore may be sequential. This unique RNA represents the first transcript of VSV which was initiated at an intracistronic site with GTP, and may also represent the first example of a transcript derived from a stop/start mechanism of VSV transcription in vitro. Although initiation occurred frequently at the beginning of the N cistron yielding 11 to 14-nucleotide-long, [beta-(32)P]ATP-labeled transcripts (D. F. Pinney and S. U. Emerson, J. Virol. 42:889-896, 1982), capping of these short RNAs was not detected. This suggests that transcripts may have to be 15 to 23 nucleotides long to be accepted as substrates by the guanyltransferase.

Base Sequence↗
PubMed1989

Cloning and expression of cDNA encoding ovine trophoblastin: its identity with a class-II alpha interferon.

The cDNAs encoding ovine trophoblastin (oTP) were isolated from an ovine embryo cDNA lambda gt 11 library by screening with a synthetic 29-mer oligodeoxynucleotide corresponding to amino acid (aa) residues 34 to 43 of oTP. The cDNA contained an open reading frame of 595 bp and the deduced amino acid sequence indicates a protein precursor of 195 aa. Nucleotide and amino acid sequence comparisons establish that oTP shares extensive homology with alpha-interferon (IFN-alpha) but is more closely related to the IFN-alpha sII subfamily. When the oTP cDNA was cloned into an eukaryotic expression vector and transfected in monkey COS cells, a high level of antiviral activity was detected. RNA blot analyses of total RNA reveal that the oTP-coding gene is expressed during a relatively short period (eleven to 21 days). The abundant expression of oTP mRNA corresponds closely to the time at which the embryo acts to extend luteal lifespan. RNAs homologous to oTP were also detected in goat and cow embryos at equivalent periods of their development, but not in the pig.

Amino Acid Sequence↗
PubMed2002

Detection and prevention of hepatitis C in dialysis patients and renal transplant recipients. A long-term follow up (1989-January 1997).

BACKGROUND: Hepatitis C is frequent problem in dialysis wards. DESIGN: A long time (1989-97) follow up of hepatitis C virus (HCV) infection in a Swedish nephrology unit was performed with anti-HCV screening, confirmatory antibody tests, viral RNA detection and molecular characterization. Case histories were reviewed with focus, onset of infection, liver morbidity and mortality. RESULTS: In October 1991, 10% (19 of 184) of the patients in the unit (haemodialysis-, peritoneal dialysis and transplanted patients) were verified or suspected HCV carriers, whilst the number at the end of 1996 was 8%, (13 of 157). Most patients were infected before 1991 but only in one case from a known HCV-infected blood donor. No new HCV infections associated with haemodialysis occurred during the study period. A total of 13 of 24 viremic patients had HCV genotype 2b, a pattern suggesting nosocomial transmission. This was further supported by phylogenetic analysis of HCV viral isolates in seven. HCV viremia was also common in patients with an incomplete anti-HCV antibody pattern as 8 of the 12 indeterminant sera were HCV-RNA positive. CONCLUSIONS: Awareness, prevention, identification of infected patients and donor testing limited transmission. Indeterminant recombinant immunoblot assays (RIBA)-results should be regarded with caution as a result of the relative immunodeficiency in uremic patients. Our data indicate nosocomial transmission in several patients.

Adolescent↗
PubMed1998

Nonradioactive in situ hybridization for detection of hydrophobin mRNA in the phytopathogenic fungus Claviceps purpurea during infection of rye.

Hydrophobins are unique fungal extracellular proteins that produce amphipathic films at interfaces, mediate contact to hydrophobic surfaces and are known to be important in phytopathogenicity. In the pathogenic ascomycete Claviceps purpurea, causing ergot disease in grasses and cereals and ergotism in livestock, a gene encoding an extraordinary type of hydrophobin has been detected, which appeared to be induced during alkaloid synthesis in axenic culture of an ergot-alkaloid producing strain of Claviceps (V. Garre and P. Tudzynski, pers. communication; Arntz and Tudzynski, 1997, Curr. Genet. 31, 357-360). To elucidate presence and function of this hydrophobin during infection of rye, the nonradioactive in situ hybridization technique was successfully adapted to the fungal organism and optimized in the pathogenic interaction system. Semithin cryosections proved to be suitable for microscopical gene expression analysis using immune-mediated alkaline-phosphatase staining for detection of digoxigenin-labeled cRNA probes. Specific hybridization of the prepared antisense riboprobe to hydrophobin mRNA was confirmed in nonradioactive Northern blots. While permeabilization by proteinase K had only a minor effect, the inclusion of detergent into the hybridization solutions enhanced specific RNA-RNA hybridization under maximum stringency. Hydrophobin mRNA was found in fungal cells, growing in axenic culture. In the disease cycle, hydrophobin transcripts were localized in abundance during vegetative fructification in conidiophores that actively produced conidia. No signals were observed in sclerotial hyphae during formation of the alkaloid-containing ergots, although they fluoresced intensely during total RNA detection using acridine orange. Notably, in situ hybridization experiments resulted in specific signals during early infection and colonization phases in the external mycelia and in hyphae penetrating the host epidermal layer. The presumed role of the hydrophobin gene product in ergot pathogenicity is discussed with respect to the described spatio-temporal distribution of the hydrophobin transcripts.

Claviceps↗
PubMed1996

Nuclear transplantation of ectodermal cells in pig oocytes: ultrastructure and radiography.

This study investigated the changes in nucleolar fine structure and the synthesis of both heterogeneous nuclear RNA (hnRNA) and ribosomal RNA (rRNA) in pig reconstructed embryos obtained by electrofusion of a single pig ectodermal cell to an enucleated metaphase-II oocyte. The nucleolar morphological changes and the pattern of transcription were examined in the ectodermal cells before fusion and in the nuclear transfer reconstructed embryos 16-18 hr after fusion. Before fusion the ectodermal cells exhibited a reticulated nucleolus with active RNA synthesis. In the reconstructed embryos, modifications of the nucleolar structure were observed, as assessed by the presence of either round-shaped, compact, dense nucleolar precursor bodies, or reticulated nucleoli. However, in both cases there was no RNA synthesis. Blebbing activity of the nuclear envelope was not observed. These results indicate that the nucleus of pig ectodermal cells exhibited either a complete or an incomplete remodelling when transferred to an enucleated metaphase-II oocyte, with no detectable RNA synthesis. Cell-cycle synchronization of ectodermal cell donor nuclei may play an important role in nuclear reprogramming after fusion.

Animals↗
PubMed1971

Selective precipitation of ribonucleic acid from a mixture of total cellular nucleic acids extracted from cultured mammalian cells.

A simple and reproducible method is described for precipitating RNA selectively from total mammalian-cell nucleic acids extracted by the phenol-sodium dodecyl sulphate procedure at pH8.0. Under specified conditions bulk RNA is precipitated almost quantitatively whereas bulk DNA remains in solution. Minor components of RNA (detected by pulse-labelling and chromatography on methylated albumin-kieselguhr) and rapidly labelled components of DNA containing single-stranded regions are also precipitated. The usefulness of the method is discussed in the context of isolating separately both RNA and DNA from cultured cells that are difficult to obtain in quantity.

Animals↗
PubMed2003

Liver biopsy, viral kinetics, and the impact of viremia on severity of hepatitis C virus recurrence.

1. Nearly all recipients who are chronically infected with hepatitis C virus (HCV) at the time of liver transplantation will have HCV RNA detectable postoperatively. 2. HCV replication can begin as early as the first postoperative week. 3. HCV levels fall significantly during the anhepatic phase of liver transplantation and continue to fall during the first 12-24 hours posttransplantation. 4. Serum HCV RNA levels typically increase rapidly from the second week posttransplantation and peak by the fourth postoperative month. HCV RNA levels at one year posttransplantation are 10-20 fold greater than pretransplant levels. 5. Corticosteroid treatment for acute cellular rejection is associated with large increases in HCV RNA levels. 6. HCV RNA levels do not appear to vary with choice of calcineurin inhibitor. 7. Early HCV RNA levels are predictive of subsequent histological severity of recurrence. 8. A correlation between early levels of viremia and subsequent allograft injury suggests that initiation of antiviral therapy early in the posttransplant course might be desirable.

Cyclosporine↗
PubMed2004

Interferon alfa-2b [correction of alpha-2b]and ribavirin for patients with chronic hepatitis C and normal ALT.

OBJECTIVES: Most studies establishing the role of antiviral therapy in patients with chronic hepatitis C (CHC) excluded the patients with normal ALT levels. Small trials with interferon monotherapy suggested a limited efficacy and/or de novo ALT elevations. We sought to evaluate the efficacy of two doses of interferon alfa-2b (IFN) with ribavirin (RBV) in patients with normal ALT [correction]. METHODS: Patients with biopsy-proven CHC with detectable HCV RNA and at least two normal ALT levels three or more months apart were randomized to receive either 3 or 5 million units of IFN thrice a week plus RBV 1,000-1,200 mg. Therapy was stopped at 24 wk if HCV RNA remained detectable and continued for an additional 24 wk if HCV RNA was undetectable. A final HCV RNA level was obtained 24 wk after discontinuation of therapy. RESULTS: Fifty-six patients were randomized and received at least one dose of treatment. The overall rate of sustained virologic response (SVR) was 32%. SVR rates were higher in genotype 2 and 3 patients (80%) than in genotype 1 patients (24%, p = 0.002). There was a tendency toward higher SVR in genotype 1 patients treated with the higher IFN dose (36%vs 10%, p = 0.07). Five patients had mild, transient ALT elevations. No sustained ALT elevations were noted. CONCLUSIONS: Patients with normal ALT had a rate of SVR comparable to that reported in patients with elevated ALT. Higher dose of interferon tended to be more effective in genotype 1 infected patients. De novo ALT elevations were transient and not clinically significant. Patients with CHC should not be excluded from treatment on the basis of ALT alone. Combination therapy with pegylated interferon and ribavirin should be evaluated in these patients.

Administration, Oral↗
PubMed2005

An electrochemical RNA hybridization assay for detection of the fecal indicator bacterium Escherichia coli.

Monitoring waters for indicator bacteria is required to protect the public from exposure to fecal pollution. Our proof-of-concept study describes a method for detecting fecal coliforms. The coliform Escherichia coli was used as a model fecal indicator. DNA probe-coated magnetic beads in combination with the electrochemical monitoring of the oxidation state of guanine nucleotides should allow for direct detection of bacterial RNA. To demonstrate this concept, we used voltammetry in connection with pencil electrodes to detect isolated E. coli 16S rRNA. Using this approach, 10(7) cells of E. coli were detected in a quantitative, reproducible fashion in 4h. Detection was achieved without a nucleic acid amplification step. The specificity of the assay for coliforms was demonstrated by testing against a panel of bacterial RNA. We also show that E. coli RNA can be detected directly from cell extracts. The method could be used for on-site detection and shows promise for adaptation into automated biosensors for water-quality monitoring.

Biological Assay↗