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PubMed1994

Transmission of hepatitis C virus by organ transplantation in the United Kingdom.

This study employed a second-generation anti-HCV ELISA, and a second-generation recombinant immunoblot assay and hepatitis C virus RNA detection by polymerase chain reaction to investigate the anti-HCV prevalence in 554 British organ donors and the transmission of hepatitis C virus to heart, liver and kidney recipients between 1984 and 1991. Serum samples from six (1.08%) donors were reactive in the second-generation anti-HCV ELISA and four (67%) of these gave positive or indeterminate results in the recombinant immunoblot assay-2. Of the 15 recipients of these organs from hepatitis C virus-confirmed positive/indeterminate donors, 14 (93%) acquired hepatitis C virus infection and seven (47%) had evidence of hepatitis C virus-related liver disease after transplantation and no evidence of blood transfusion-related transmission. Only six of the 15 (40%) recipients had detectable anti-HCV after transplantation, while 12 of 14 (86%) patients tested had hepatitis C virus RNA in their serum detectable by "nested" polymerase chain reaction. These data indicate a very high rate of transmission with a major risk of the development of liver disease. We believe our study supports the testing of all British organ donors for anti-HCV and that organs from anti-HCV-positive patients should not be transplanted unless the recipient has life-threatening disease and there is a donor shortage, when their use may be justified. Since there are time constraints on organ donor testing, which may frequently be done on call during unsocial hours, we would recommend second-generation ELISA as the current screening test of choice.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzyme-Linked Immunosorbent Assay↗
PubMed2004

Prediction of sustained virological response in liver transplant recipients with recurrent hepatitis C virus following combination pegylated interferon alfa-2b and ribavirin therapy using tissue hepatitis C virus reverse transcriptase polymerase chain reaction testing.

The optimal duration of therapy for pegylated interferon combined with ribavirin in recurrent Hepatitis C virus (HCV) following liver transplantation is not known. We wanted to determine if testing for HCV in liver tissue by reverse transcriptase polymerase chain reaction (RT-PCR) was superior in predicting sustained virological response (SVR) in comparison to standard HCV ribonucleic acid (RNA) detection in the serum. All recipients received combination pegylated alpha-2b interferon (1.5 mcg/kg) and ribavirin (200-600 mg/d) therapy for at least 48 weeks of therapy and were found to have nondetectable HCV RNA by PCR serum testing at the end of therapy. Sustained virological response (SVR) was defined as nondetectable serum HCV RNA at 6 months post treatment withdrawal. Ten liver transplant recipients were included in the study; mean time from transplantation was 29.2 months. All had nondetectable serum HCV RNA by RT-PCR. In hepatic tissue 7/10 patients HCV RNA was found to be positive by RT-PCR while 3/10 had nondetectable HCV RNA in their liver by RT-PCR. SVR was attained in all 3/10 that were hepatic tissue HCV PCR negative after 12 months of combination therapy. In conclusion, direct detection of HCV RNA by RT-PCR of liver tissue appears to more effectively predict SVR following pegylated interferon and ribavirin therapy than the conventional use of serum.

Antiviral Agents↗
PubMed1969

Formation and detection of RNA-DNA hybrid molecules in cytological preparations.

A technique is described for forming molecular hybrids between RNA in solution and the DNA of intact cytological preparations. Cells in a conventional tissue squash are immobilized under a thin layer of agar. Next they are treated with alkali to denature the DNA and then incubated with tritium-labeled RNA. The hybrids are detected by autoradiography. The technique is illustrated by the hybridization of ribosomal RNA to the amplified ribosomal genes in oocytes of the toad Xenopus. A low level of gene amplification was also detected in premeiotic nuclei (oogonia).

Animals↗
PubMedDate not supplied

Infection of human hepatocyte cell lines with hepatitis C virus in vitro.

Hepatocyte related cell lines, namely human embryonic hepatocyte cell line (WRL68) and hepatoblastoma cell line (Hep G2), were tested for their ability to support hepatitis C virus (HCV) replication in vitro. The replicative intermediate of the minus strand HCV RNA was newly transcribed from the inoculated virus, and was stable in WRL68 cells over a period of 62 days. HCV RNA detected in the culture medium at 62 days after infection suggested a long period of virus secretion from this cell line. In Hep G2, transcription of the minus strand HCV RNA was also detected until 39 days after infection, but transcription and secretion of viral progeny could not be demonstrated. Although HCV infection of WRL68 was not high, this cell line might prove to be a useful tool for studying HCV replication in vitro.

Base Sequence↗
PubMed2004

Measles virus prevalence in otosclerotic stapes footplate samples.

HYPOTHESIS: The cause of otosclerosis is still unknown. Persistent measles virus infection of the otic capsule is supposed to be one of the etiologic factors in otosclerosis. Chronic viral antigen expression on the surface of infected cells can induce a secondary autoimmune reaction against the otic capsule. BACKGROUND: In the past 15 years, some reports proposed the possible etiologic role of measles virus in otosclerosis. The presence of measles virus was shown in otosclerotic patients by reverse-transcriptase polymerase chain reaction amplification of the viral RNA, detecting the viral proteins by immunohistochemistry and detecting antimeasles immunoglobulin G in the perilymph samples. Many concerns were elicited by these results. METHODS: Nucleic acid was extracted from pulverized, frozen stapes footplate samples of otosclerotic patients. Measles virus RNA was amplified by reverse-transcriptase polymerase chain reaction: reverse transcription and the first round polymerase chain reaction amplification was performed by heat stable recombinant Thermus thermophilus polymerase, whereas in the nested round, polymerase chain reaction Taq-polymerase was used. Measles virus nucleoprotein RNA-specific oligonucleotide primers were used in these reactions. An Edmonston-type measles virus served as a positive control and cortical bone fragments or stapes superstructures served as negative controls. RESULTS: Among 34 otosclerotic patients, 20 stapes footplate samples contained measles virus RNA. Measles virus RNA was not detected in other bone specimens of the patients. CONCLUSION: The etiologic role of measles virus in the pathogenesis of otosclerosis should be considered. The 14 negative samples may be genetically determined otosclerotic cases.

Adult↗
PubMed1998

Automatic detection of conserved RNA structure elements in complete RNA virus genomes.

We propose a new method for detecting conserved RNA secondary structures in a family of related RNA sequences. Our method is based on a combination of thermodynamic structure prediction and phylogenetic comparison. In contrast to purely phylogenetic methods, our algorithm can be used for small data sets of approximately 10 sequences, efficiently exploiting the information contained in the sequence variability. The procedure constructs a prediction only for those parts of sequences that are consistent with a single conserved structure. Our implementation produces reasonable consensus structures without user interference. As an example we have analysed the complete HIV-1 and hepatitis C virus (HCV) genomes as well as the small segment of hantavirus. Our method confirms the known structures in HIV-1 and predicts previously unknown conserved RNA secondary structures in HCV.

Algorithms↗
PubMed2001

The clinic study on synchronous detection of HCV RNA in the plasma and PBMC of hepatitis C patients.

In order to increase the positive detection rate of HCV RNA in the patients with chronic hepatitis C (CHC), RT-PCR was used to synchronously detect HCV RNA in the plasma and peripheral blood mononuclear cells (PBMC) of 583 CHC patients with a continuously elevated level of ALT for more than one year. The results showed that the positive detection rate of HCV RNA in the plasma of the CHC patients was 19.2%, while 24.5% in PBMC. It was demonstrated that the positive detection rate for HCV RNA in PBMC was obviously higher than that detected in plasma. To synchronously detect HCV RNA in PBMC by using RT-PCR can increase the positive detection rate of HCV RNA in the CHC patients.

Adult↗
PubMed1999

Borna disease virus infection in domestic cats: evaluation by RNA and antibody detection.

Borna disease virus (BDV) infection has been suggested to cause spontaneous neurological disease in cats referred to as staggering disease. However the evaluation of BDV infection in neurologically asymptomatic cats remained unclear. In the present study, BDV infected, asymptomatic cats in Tokyo were surveyed both by the presence of plasma antibodies against BDV-p24 and -p40 and by RNA detection in peripheral blood mononuclear cells. Seven of 32 domestic cats (21.9%) were serologically or genetically judged to be BDV-infected. Six cats were positive for anti-BDV antibody and two cats were positive for BDV RNA. Within the 2 RNA-positive cats, only one was positive for anti-BDV antibodies. Furthermore, the findings of anti-BDV-p40 and anti-BDV-p24 antibody-positive cats did not completely overlap. These results suggest that there are neurologically asymptomatic domestic cats infected with BDV present in the Tokyo area.

Animals↗
PubMed2001

High frequencies of HGV and TTV infections in blood donors in Hangzhou.

AIM: To determine the frequencies of HGV and TTV infections in blood donors in Hangzhou. METHODS: RT-nested PCR for HGV RNA detection and semi-nested PCR for TTV DNA detection in the sera from 203 blood donors, and nucleotide sequence analysis were performed. RESULTS: Thirty-two (15.8%) and 30 (14.8%) of the 203 serum samples were positive for HGV RNA and TTV DNA, respectively. And 5 (2.5%) of the 203 serum samples were detectable for both HGV RNA and TTV DNA. Homology of the nucleotide sequences of HGV RT-nested PCR products and TTV semi-nested PCR products from 3 serum samples compared with the reported HGV and TTV sequences was 89.36%, 87.94%, 88.65% and 63.51%, 65.77% and 67.12%, respectively. CONCLUSION: The infection rates of HGV and/or TTV in blood donors are relatively high, and to establish HGV and TTV examinations to screen blood donors is needed for transfusion security. The genomic heterogeneity of TTV or HGV is present in the isolates from different areas.

Base Sequence↗
PubMed1969

A nondefective (competent) adenovirus-SV40 hybrid isolated from the AD.2-SV40 hybrid population.

A new nondefective hybrid virus has been plaque-isolated from the Ad.2-SV40 hybrid population. This virus replicates efficiently with one-hit kinetics in both human embryonic kidney and African green monkey kidney cells, induces an SV40 specific antigen which is detectable by immunofluorescence and complement-fixation using sera from SV40 tumor-bearing hamsters, and produces SV40-specific RNA detectable by DNA-RNA hybridization. The SV40-specific antigen induced by this virus is heat-stable, sensitive to inhibitors of DNA synthesis, serologically different from SV40 T and viral antigens, and is an unrecognized SV40 antigen.

Adenoviridae↗
PubMed2002

Detection of measles virus genomic RNA in tear samples from a patient with measles keratitis.

PURPOSE: To report a case of measles keratitis, in which measles virus genomic RNA was detected in tear samples. METHODS: Samples of tears and pharyngeal mucus were obtained from a 19-year-old man with active systemic measles on day 7 after illness onset. These specimens were then subjected to reverse-transcriptase polymerase chain reaction (RT-PCR), using measles-specific primers. RESULTS: One week after systemic measles outbreak, the patient presented with a chief complaint of irritation in both eyes. Slit-lamp examination revealed bilateral superficial punctate keratopathy in the central cornea with small diffuse subepithelial infiltrations, and RT-PCR detected measles virus genomic RNA in tears and pharyngeal mucus. The patient was treated with topical 0.1% fluorometholone and 0.5% levofloxacin three times a day for 1 week, and the keratopathy resolved without sequelae. CONCLUSION: This report demonstrates for the first time the presence of measles virus genomic RNA in the tears of a patient with measles keratitis. It also highlights the utility of RT-PCR for rapid detection of measles RNA in tear or mucus samples. Furthermore, the presence of virus in bodily fluids and secretions signifies the potential contagiousness of this type of keratopathy.

Adult↗
PubMed1990

Evidence for RNA-Oligonucleotides in Plant Vacuoles Isolated from Cultured Tomato Cells.

We have shown that highly purified vacuoles of suspension-cultured tomato (Lycopersicon esculentum) cells contain RNA-oligonucleotides, using two different approaches to label and detect RNA: (a) in vivo labeling of cellular RNA with [5-(3)H]uridine, followed by preparation of vacuoles from protoplasts and by quantification of radioactively labeled material; and (b) in vitro labeling and analysis on sequencing gels of nucleic acids prepared from tomato vacuoles and their identification as RNA. The intravacuolar location of the RNA found in vacuolar preparations was concluded from analyzing for RNA intact organelles after repeated flotation steps as well as ribonuclease A treatment. About 3% of the RNA in protoplasts was localized within vacuoles, exceeding by severalfold the contribution made by contamination with unlysed protoplasts and subcellular organelles. Investigation of the size distribution of vacuolar RNA revealed an oligonucleotide pattern strikingly different from that which would arise from contaminating protoplasts; vacuolar RNA fragments are considerably shorter than 80 nucleotides. Characterization of these oligoribonucleotides (3'-phosphorylated termini; relatively rich in pyrimidines) as possible products of tomato vacuolar ribonuclease I action, and, in addition, enzymatic hydrolysis of vacuolar RNA by inherent enzyme activities in lysed vacuole preparations support the hypothesis that plant vacuoles are involved in cellular nucleolytic processes.

Journal Article↗
PubMed2003

3' nontranslated RNA signals required for replication of hepatitis C virus RNA.

We describe a mutational analysis of the 3' nontranslated RNA (3'NTR) signals required for replication of subgenomic hepatitis C virus (HCV) RNAs. A series of deletion mutants was constructed within the background of an HCV-N replicon that induces the expression of secreted alkaline phosphatase in order to examine the requirements for each of the three domains comprising the 3'NTR, namely, the highly conserved 3' terminal 98-nucleotide (nt) segment (3'X), an upstream poly(U)-poly(UC) [poly(U/UC)] tract, and the variable region (VR) located at the 5' end of the 3'NTR. Each of these domains was found to contribute to efficient replication of the viral RNA in transiently transfected hepatoma cells. Replication was not detected when any of the three putative stem-loop structures within the 3'X region were deleted. Similarly, complete deletion of the poly(U/UC) tract abolished replication. Replacement of a minimum of 50 to 62 nt of poly(U/UC) sequence was required for detectable RNA replication when the native sequence was restored in a stepwise fashion from its 3' end. Lengthier poly(U/UC) sequences, and possibly pure homopolymeric poly(U) tracts, were associated with more efficient RNA amplification. Finally, while multiple deletion mutations were tolerated within VR, each led to a partial loss of replication capacity. The impaired replication capacity of the deletion mutants could not be explained by reduced translational activity or by decreased stability of the RNA, suggesting that each of these mutations may impair recognition of the RNA by the viral replicase during an early step in negative-strand RNA synthesis. The results indicate that the 3'-most 150 nt of the HCV-N genome [the 3'X region and the 3' 52 nt of the poly(U/UC) tract] contain RNA signals that are essential for replication, while the remainder of the 3'NTR plays a facilitating role in replication but is not absolutely required.

3' Untranslated Regions↗
PubMed1987

Differential expression of cellular genes in Rauscher leukaemia.

The methods of hybridization in solution and blot hybridization showed that spleen cells from BALB/c mice contain "silent" genes which can amplify and change their structure after infection by Rauscher leukaemia virus. The "silent" gene product is nuclear 35S RNA detectable by comparative electrophoretic analysis of the heterogeneous nuclear RNA from leukaemic and normal cells. About 7% of this 35S RNA is represented by the virus-specific sequences, but a major part is represented by the cellular sequences. In order to study the expression of the sequences homologous to 35S RNA in leukaemic and normal cells, hybridization in solution was used. Expression of the complete copies of 35S RNA was observed in nuclei of virus-infected cells, whereas this RNA in the cytoplasm is represented by the incomplete copies. The expression of the sequences homologous to this 35S RNA in normal mouse spleen cells was not revealed.

Animals↗
PubMed2005

Characterization of cancer stroma markers: in silico analysis of an mRNA expression database for fibroblast activation protein and endosialin.

Standardized, high-throughput RNA detection with microarray chips allows for the construction of genome-wide databases for tissue specimens suitable for in silico electronic Northern blot (eNorthern) analysis of marker genes. We used the BioExpress database, which contains transcriptional profiles of normal and cancer samples, to examine two putative markers of cancer stroma: fibroblast activation protein-alpha (FAP-alpha) and endosialin. Analyses for FAP-alpha showed that normal tissues generally lack RNA signals, with the exception of endometrium. Typing of tumors revealed prominent FAP-alpha signals in cancer types marked by desmoplasia, and localization of FAP-alpha in reactive cancer stroma was confirmed by immunohistochemistry. A subset of sarcomas displayed prominent FAP-alpha signals localizing to the malignant cells. For endosialin, eNorthern analyses showed low to moderate RNA signals in many normal organs, whereas immunohistochemistry revealed endosialin in only some tissues, such as endometrium. Endosialin was detected at the RNA and protein level in sarcomas, notably malignant fibrous histiocytomas. Low to moderate endosialin RNA signals were found in epithelial cancer types for which immunostaining identifies expression in subsets of tumor capillaries or fibroblasts. These findings extend the FAP-alpha and endosialin profiling in silico to an unbiased tumor database and place both molecules in a novel context of endometrial biology and sarcoma subtyping. Our findings suggest that BioExpress can be searched directly for tumor stroma markers but may need prior enrichment for markers with narrow cellular representation, such as endosialin. Constructing databases from microdissected cancer tissues may be an essential step for tumor stroma-targeted therapies.

Adult↗
PubMedDate not supplied

[Detection of the spring viremia of carp virus by hybridization with nonradioactive DNA probes].

In order to detect spring viraemia of carp virus, DNA probes have been constructed using reverse transcription-PCR amplification technique and cDNA cloning in plasmid and phage vectors. The sensitivity and specificity of viral RNA detection was assessed using nonradioactive probes in infected FHM cell culture and in tissues from dead fish. Viral RNA was more frequently detected in the brain and gill than in the abdominal organs.

Animals↗
PubMed2000

Detection and quantitation of human immunodeficiency virus type 1 in the female genital tract. The Division of AIDS Treatment Research Initiative 009 Study Group.

Human immunodeficiency virus type 1 (HIV-1) was detected in the genital tracts of 59% of 225 women by RNA PCR and in 7% of the women by culture. In a comparison of two sampling methods, endocervical swabs were more sensitive than cervicovaginal lavage for HIV-1 RNA detection by PCR but not by culture and their sensitivity was independent of the concentration of HIV-1 RNA.

Cross-Sectional Studies↗
PubMed2001

In situ detection of messenger RNA using digoxigenin-labeled oligonucleotides and rolling circle amplification.

The detection of specific RNA molecules in situ is routinely performed using haptenated probes, which are detected by either enzymatic amplification or direct fluorescence. A drawback of fluorescence labeling has been the reduced sensitivity relative to that of methods that use enzymes as signal generators. Reliable fluorescence detection methods often require the use of multiple oligonucleotide probes for each gene target. Here, we demonstrate that single haptenated DNA probes specific for actin mRNA may be detected in situ using antibody-coupled rolling circle amplification (immuno-RCA). This fluorescence-based detection method offers remarkable sensitivity due to the use of signal amplification and yet retains the ability to count hybridization signals as discrete objects. We demonstrate the detection of actin-specific immuno-RCA signals in the cytoplasm and use 3D image deconvolution of multiple z axis sections to show that there are hundreds of signals per cell. With some modifications, this method may be adaptable to the simultaneous detection of several RNA species, including low-copy-number mRNA.

5' Untranslated Regions↗