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PubMed1981

Detection of RNA complementary to herpes simplex virus DNA in human cervical squamous cell neoplasms.

Nonneoplastic and neoplastic cervical biopsy specimens were examined by in situ hybridization to 125I-labeled DNA of herpes simplex virus (HSV), adenovirus, and bacteriophage lambda DNA's, and quantitative hybridization data were obtained using a Video Image Analyser. HSV-specific RNA was detected in 72% of cervical intraepithelial neoplasia, 60% of squamous cervical carcinomas, 2% of nonneoplastic cervices, and 9% of primary adenocarcinomas of the cervix. None of the tissues gave positive hybridization with adenovirus or lambda DNA probes. In paired biopsies of cervical intraepithelial neoplasia and nonneoplastic epithelium from 29 individuals, HSV-specific RNA was detected only in the epithelium of the neoplastic sample and not in the nonneoplastic control. Infectious HSV-2 was isolated from a low proportion (2%) of both ectocervical swabs and cell-free tissue extracts of patients examined, suggesting that the HSV-specific RNA detected in squamous cell neoplasms was not due to overt infections.

Antibodies, Viral↗
PubMed2001

Storage of bovine viral diarrhoea virus samples on filter paper and detection of viral RNA by a RT-PCR method.

Of four solid carriers tested, Whatman paper No 1 was the best for storing blood and serum samples for the diagnosis of bovine viral diarrhoea (BVD) by means of viral RNA detection. The filter papers were impregnated with 10 microl of blood or serum, followed by air drying. Samples collected in this way from persistently infected animals had lost infectivity within a few days, but viral RNA could still be detected by RT-PCR for up to 6 months. When investigated by RT-PCR, 12 blood and 10 serum samples selected at random from animals persistently infected with BVD virus showed the same results whether samples had been spotted onto filters or examined directly from the liquid state. The filters spotted with blood or serum are convenient for storage and transport of samples to a diagnostic laboratory without the need for cooling. Sequencing of amplified RNA can be used subsequently for genetic typing.

Animals↗
PubMed1991

Prediction of hepatitis C virus infectivity in seropositive Australian blood donors by supplemental immunoassays and detection of viral RNA.

The prevalence of anti-hepatitis C virus (HCV) enzyme immunoassay (EIA)-positive in 167,511 Australian volunteer blood donors from Adelaide, Melbourne, Perth, and Sydney was 0.78%. One thousand two-hundred and eighteen EIA-positive serum samples were assessed by supplemental tests including a blocking EIA and two peptide EIAs corresponding to major epitopes of the HCV C-100-3 antigen. Seven hundred and eighteen samples (59%) were negative by supplemental testing; no evidence of reactivity with other HCV gene products or HCV RNA detected by cDNA polymerase chain reaction was found in selected samples from this group. In contrast, of 43 samples randomly selected from 400 samples (32.8%) positive by supplemental testing, 88% were reactive with HCV 33-C or core antigens and 52% contained HCV RNA, suggesting contact with HCV and infectivity of most donors in this group. Most samples equivocal by supplemental testing reacted only with C-100 and not with other HCV antigens when tested by dot immunoblot assay. Only 21% had detectable HCV RNA. The battery of assays used in this study indicated that approximately 32% of HCV EIA repeatably reactive serum samples were serologically related to HCV, corresponding to a 0.25% prevalence of potentially infectious donors.

Adult↗
PubMed1992

Improved detection of hepatitis C virus RNA by reverse transcription and polymerase chain reaction.

The polymerase chain reaction with prior reverse transcription of RNA into cDNA was applied to hepatitis C virus RNA detection in human serum samples of different origin. In order to eliminate false negative results, the following steps were optimized: RNA extraction, reverse transcription, and oligonucleotide primer selection. We compared different RNA extraction methods using guanidinium salt/detergent and proteinase K digestion/phenol extraction, and tested virus particle enrichment with polyethylene glycol precipitation and ultracentrifugation. RNA extraction with guanidinium salt/detergent was the most efficient method. Ultracentrifugation of single samples did not improve hepatitis C virus RNA detection. Polyethylene glycol precipitation performed poorly. Recombinant thermostable reverse transcriptase produced cDNA from fewer samples than did Moloney murine leukaemia virus reverse transcriptase. Nested oligonucleotide primers from the 5'-terminal non-coding region of the hepatitis C virus genome amplified cDNA from more samples than did primers from the coding regions. Thirty six anti-hepatitis C virus antibody positive samples were tested; nested primers (nucleotides 6 to 327 and 15 to 288) yielded 21 amplificates, whereas primers from the coding region produced 16 amplificates (nucleotides 4684-5276) and 5 amplificates (nucleotides 5166-5270), respectively. The most efficient combination of steps was RNA extraction with guanidinium salt solution, reverse transcription with Moloney murine leukaemia virus reverse transcriptase and nested polymerase chain reaction primed with primers from the 5'-terminal non-coding region of the hepatitis C virus genome. Other combinations produced more false negative results. Three different groups of anti-hepatitis C virus antibody positive individuals had markedly different viraemia patterns: Hepatitis C virus RNA was detected in the sera of only 10% of anti-hepatitis C virus antibody positive blood donors, but in 90% of anti-hepatitis C virus antibody positive patients with clinically manifest hepatitis C, and 90% of anti-hepatitis C virus antibody positive haemophiliacs who had received plasma products in the past which had not been virus-inactivated. No hepatitis C virus RNA could be detected in the sera of 450 anti-hepatitis C virus antibody negative blood donors with elevated serum alanine aminotransferase catalytic concentrations.

Alanine Transaminase↗
PubMed1993

Amplification and detection of a single molecule of human immunodeficiency virus RNA.

Detection of plasma viremia in human immunodeficiency virus type 1 (HIV-1)-infected people is indispensable for the diagnosis of seronegative infection as well as for the evaluation of virus activities in vivo. The direct detection of HIV-1 RNA in circulation has been performed by means of reverse transcription followed by polymerase chain reaction (RT-PCR). As an attempt to establish a highly sensitive assay, we evaluated the effects of two-step amplification with nested primers and double priming of reverse transcription on the sensitivity of RT-PCR. The sensitivity of two-step amplification was 100 times higher than that of one-step amplification. The double priming of reverse transcription further increased the sensitivity of the following two-step amplification 100 times, which appeared to be enough to detect HIV-1 RNA from as little as a 2.2 x 10(-4) TCID50 unit equivalent of culture supernatant of HIV-1-infected cells and a single molecule of HIV-1 gag complementary RNA synthesized by in vitro transcription. By use of this most sensitive assay, we successfully detected HIV-1 RNA in serum or plasma from all 22 patients with acquired immune deficiency syndrome (AIDS) or AIDS-related complex (ARC) and 13 out of 14 untreated asymptomatic carriers. Of 43 asymptomatic carriers under the treatment with interferon-alpha or azidothymidine, 17 cases showed negative results, indicating that the virus activity was suppressed by the therapeutics. We also noted the inhibitory effect of heparin on RT-PCR.

Base Sequence↗
PubMed2004

A sensitive and quantitative single-tube real-time reverse transcriptase-PCR for detection of enteroviral RNA.

BACKGROUND: Enteroviruses (EVs) are significant human pathogens. Rapid and sensitive diagnostic techniques are desirable. OBJECTIVES: To develop a quantitative single-tube real-time reverse transcription-polymerase chain reaction (RT-PCR) for human enterovirus ribonucleic acid (RNA) (QPCR), with protection against amplimer contamination. STUDY DESIGN: The method was evaluated with serial dilutions of EV, 62 cerebrospinal fluid (CSF) specimens from meningitis patients, and the third and fourth European Union Concerted Action Enterovirus Proficiency Panels. A commercial EV PCR test was run in parallel. RESULTS: Optimisation included RNA extraction procedure, design and concentrations of primers and probes from the 5' non-coding region as well as recombinant Thermus thermophilus polymerase (rTth), Mn(OAc)(2) and thermolabile UNG concentrations. Of 62 CSF samples from cases of meningitis submitted for QPCR testing, 34 (76%) and 21 (47%) were positive by QPCR and a commercial EV RNA detection kit, respectively. The detection limit of QPCR was 0.001 TCID(50)/ml (50% tissue culture-infective dose per millilitre) for a coxsackievirus B2 preparation and <10 copies of a plasmid containing coxsackievirus B2 complementary deoxyribonucleic acid (cDNA). The relation between threshold cycle (C(t)) and amount of virus was linear (r = 0.99) over a range of 10(-3) to 10(4) TCID(50)/ml of coxsackievirus B2. CONCLUSIONS: The QPCR method allows a large number of samples to be screened rapidly. Its sensitivity, simplicity, and reproducibility make it a suitable tool for the routine laboratory.

5' Untranslated Regions↗
PubMed1990

Detection of HDV-RNA by PCR in serum of patients with chronic HDV infection.

In this paper, we studied the usefulness of polymerase chain reaction (PCR) in HDV-RNA detection. Using serial dilutions of serum samples of known concentrations of HDV-RNA, PCR was 10,000-times more sensitive than slot-blot hybridization. PCR was used for the detection of HDV-RNA in 33 serum samples negative to HDV-RNA by conventional slot-blot hybridization. HDV-RNA was detected in 18/33 (54%) of the samples included in this study using PCR. When positivity to a viral genome was related to other viral replication markers, it was found that among the 18 patients positive to the viral genome, 13 (72%) had hepatitis delta antigen in the liver, and five (28%) were negative. In conclusion, HDV-RNA detection by gene amplification is 10,000-times more sensitive than slot-blot hybridization, and allows the detection of viral replication in patients without other viral replication markers.

Alanine Transaminase↗
PubMed1994

Rapid detection of hepatitis C virus RNA by direct capture from blood.

A new diagnostic assay for hepatitis C virus RNA detection is described. HCV genomic RNA is captured onto streptavidin-coated magnetic beads by solution hybridization with biotinylated complementary oligonucleotides. The specificity of the capture assay is confirmed using different capture oligonucleotides as well as sera representing different types of HCV. Sensitivity was determined by testing serial dilutions of a HCV infected plasma. A panel of 50 sera was tested for anti-HCV by a Line Immunoassay and for HCV-RNA by both a conventional guanidinium extraction method and the new capture assay. The specificity of the capture assay was 95.8% and the sensitivity was 92.3% compared to the standard protocol. This method provides a rapid and simple alternative for HCV-RNA detection in blood samples.

Animals↗
PubMed1985

Family of developmentally regulated, maternally expressed Drosophila RNA species detected by a v-myc probe.

Drosophila melanogaster genomic sequences that hybridize with v-myc have been reported (B.-Z. Shilo and R. A. Weinberg, Proc. Natl. Acad. Sci. U.S.A. 78:6789-6792, 1981). We have detected Drosophila RNA sequences that also hybridize with v-myc. In an attempt to characterize these RNA sequences, we used v-myc hybridization probes to isolate Drosophila genomic segments. None of the Drosophila genomic or cDNA clones that we have isolated hybridize with the 3' exon of v-myc. Preliminary nucleotide sequence analyses have revealed sufficient homology to account for the observed hybridization between v-myc and the Drosophila clones but have failed to detect significant amino acid sequence homology. Thus is seems unlikely that the mRNA sequences or the genomic sequences that we have isolated by hybridization with v-myc represent homologs of the vertebrate myc gene. Despite the lack of structural homology between the cloned Drosophila sequences and v-myc, we have investigated the pattern of expression of those RNA species that hybridize with v-myc. Polyadenylic acid-containing transcripts of 2.7, 2.2, and 1.7 kilobases (kb) in embryos, pupae, adults, and Kc cells and an additional 1.4-kb transcript in adults were complementary to the Drosophila genomic clones and to v-myc. The 1.7- and 2.2-kb transcripts were localized on polyribosomes in Kc cells. The 1.7- and 2.2-kb transcripts were present after 45 min, 2 h, and 4 h of embryonic development, but by 16 h of development their levels had decreased by more than sixfold. During metamorphosis, two peaks of expression of the 1.7- and 2.2-kb transcripts were observed, at 6 and 72 h postpupariation. The 1.4-kb RNA species was first detected at 72 h postpupariation. In adults, the 1.7- and 2.2-kb transcripts were detected only in ovaries in females, whereas the 1.4-kb transcript was present in female nonovarian RNA and in males. These results suggest that the transcripts in early embryos are of maternal origin.

Age Factors↗
PubMed1993

Selective enzymatic depletion during RNA-PCR: improved detection of rare RNA forms.

A procedure that facilitates the detection of rare RNAs by RNA-polymerase chain reaction (RNA-PCR) when a large excess of competing template is present in the PCR is described. We have used this assay to detect spliced mRNAs of low abundance, which result from in vitro splicing of an intron derived from the rat fibronectin gene. RNA was isolated from splicing reactions, reverse transcribed, then PCR was performed, using primers based on exon sequences. After a limited number of cycles, amplified DNAs were incubated with a restriction enzyme which cleaved only within the intron, leaving the spliced two-exon product intact. This step selectively depleted the unspliced pre-mRNA, and subsequent rounds of PCR served to enrich for spliced product. The spliced product was not detected by performing PCR without restriction digestion to deplete pre-mRNA-derived species. The broader application of the enzymatic depletion strategy to detection of rare alternative mRNA isoforms is also demonstrated.

Animals↗
PubMed2005

[Detection of RNA interference in nasopharyngeal carcinoma cell lines using reporter genes].

BACKGROUND & OBJECTIVE: RNA interference (RNAi) technique is now widely used in studies of gene function, signal transduction pathway, and gene therapy because it can effectively and specifically inhibit gene expression. This study was designed to synthesize small interfering RNA (siRNA) by in vitro transcription, and construct retrovirus vectors to express small hairpin RNA (shRNA), detect RNAi in nasopharyngeal carcinoma cell lines, and to develop a RNAi technique platform. METHODS: siRNAs targeting green fluorescent protein (GFP) and luciferase (Luc) were synthesized by in vitro transcription, while shRNAs targeting GFP and Luc were constructed from pSUPER.retro. Cervical cancer cell line HeLa, nasopharyngeal carcinoma cell lines CNE1, CNE2, and 5-8F were co-transfected with siRNAs or shRNAs and reporter gene pEGFP-N1 or pGL3. The expression of GFP was detected by fluorescent microscopy and Western blot. The activity of luciferase was measured by Luciferase Enzyme Assay System. RESULTS: siRNA duplexes with 3' UU overhangs and shRNA specifically silenced GFP expression, while antisense RNA and siRNA without 3' UU overhangs did not trigger RNA interference of GFP. Quantitative luciferase activity analysis showed that siRNA inhibited Luc expression in HeLa, CNE1, CNE2, and 5-8F cell lines with inhibition rates of 91.43%, 78.01%, 90.30%, and 62.85%, respectively. Similarly, the inhibition rate was 78.22% when shRNA targeting Luc was co-transfected into HeLa cell line. CONCLUSIONS: Both siRNAs and shRNAs can induce RNAi. 3' UU overhangs of siRNA may play a role in RNAi. RNAi can be triggered in both nasopharyngeal carcinoma cell lines and HeLa cell line.

Cell Line, Tumor↗
PubMedDate not supplied

Detection of hepatitis C virus RNA by a reliable, optimized single-step reverse transcription polymerase chain reaction.

A single-step reverse transcription polymerase chain reaction (SRT-PCR) method was optimized for hepatitis C virus (HCV) RNA detection. Extraction procedures by proteinase K and guanidinium isothiocyanate gave similar results. The optimal MgCl2 concentration for the SRT-PCR method was 2 mM with 10 units of superscript M-MLV RNase H-reverse transcriptase and 1 unit of Taq polymerase. Shorter PCR cycling steps gave a 10-fold-increased PCR product compared with longer cycling steps. Twenty-five anti-HCV-positive sera from chronic hepatitis C patients were positive with SRT-PCR, whereas only 17 out of 25 were positive by dissociated RT and PCR (dRT/PCR). Specificity was assessed by twenty negative controls. SRT-PCR was 5-fold more sensitive (5 HCV RNA copies per assay) than dRT/PCR with an HCV RNA transcript. Our SRT-PCR method for HCV RNA detection appears fully adapted for routine use in a medical virology laboratory.

Base Sequence↗
PubMed1992

Rapid treatment of whole cells and RNA viruses for analysis of RNA by slot blot hybridization.

To avoid extensive manipulation for the purification of RNA from cells, several methods were evaluated for the direct release of RNA from influenza virus infected cells and supernatants using slot blot hybridization and non-radioactive probes. Treatment with an equal volume of 10 M aqueous guanidine hydrochloride produced the best hybridization signal. Less, but significant amounts of RNA were also released using the following treatments: dilute alkali (final concentration of 0.16 M NaOH) or 100 degrees C/5 min or RNA sample buffer containing formamide/formaldehyde, then heating at 65 degrees C/10 min. Despite the presence of large amounts of cell debris, RNA from guanidine hydrochloride treated whole cell extracts bound quantitatively to the positively charged nylon membranes. The sensitivity of RNA detection when whole cell extracts treated with guanidine hydrochloride were probed with a digoxigenin labelled cDNA probe was similar to the detection of RNA in highly purified, protein free samples. Three positively charged membranes were tested (from Amersham, ICN and Boehringer Mannheim) using two alkaline phosphatase substrates, NBT-X phos, and a chemiluminescent substrate, 3-(2'-spiroadamantane)-4-methoxy-4-(3'-phosphoyloxy)-phenyl- 1-1,2-dioextane (AMPPD) and a peroxidase substrate, tetramethylbenzidine (TMB). The Boehringer Mannheim membrane had the highest sensitivity for the alkaline phosphatase substrates, but the peroxidase reaction with the TMB substrate was the most consistently sensitive, irrespective of which membrane was used. The ability to quantitatively detect RNA from whole cells without any purification will allow the rapid screening of large numbers of samples for specific RNA species in research or diagnostic laboratories.

Animals↗
PubMed1997

Use of reverse-transcription polymerase chain reaction for detection of rubella virus RNA in cell cultures inoculated with clinical samples.

A recently developed reverse transcription-nested polymerase chain reaction (RT-nPCR) method for rubella virus (RV) RNA detection was assessed in a series of African green monkey kidney (AGMK) cell cultures inoculated with clinical samples from patients with suspected RV infection. Results were compared with those of conventional virus isolation/identification. The assay included an internal control of amplification consisting of a synthetic RNA molecule mimicking the RV E1 target sequence. A semiquantitation of RV RNA was achieved by comparing the relative band intensity of internal control and RV E1 fragment. RT-nPCR was positive in 15/16 (94%) RV isolation-positive cultures and in 12/60 (20%) RV isolation-negative cultures. All 27 cell cultures positive by RT-nPCR had been inoculated with clinical samples taken from patients with ascertained RV infection or given RV vaccination and consisted of cells harvested 1-10 days after primary inoculation of clinical samples. No RV RNA was found in any of the cell cultures inoculated with 14 clinical samples from 6 patients in whom RV infection was excluded. When considering the time-course of RV infection, it was found that RV RNA could be detected as early as 4 days p.i. in 10/21 (48%), and by 7-10 days p.i. in 27/28 (96%) cell cultures, whereas by the same time RV was isolated in only 7/16 (44%) cell cultures. Semiquantitation showed that: i) viral RNA amount progressively increased with time; ii) cell cultures containing very low levels of viral RNA one week p.i. either required a few blind passages for virus recovery or remained negative for RV isolation. Finally, PCR inhibitors were found in 10/164 (6%) cell cultures examined. In conclusion, RT-nPCR proved to be very sensitive and very specific and greatly reduced RV detection time in inoculated cell cultures.

Adult↗
PubMed2005

A method for aligning RNA secondary structures and its application to RNA motif detection.

BACKGROUND: Alignment of RNA secondary structures is important in studying functional RNA motifs. In recent years, much progress has been made in RNA motif finding and structure alignment. However, existing tools either require a large number of prealigned structures or suffer from high time complexities. This makes it difficult for the tools to process RNAs whose prealigned structures are unavailable or process very large RNA structure databases. RESULTS: We present here an efficient tool called RSmatch for aligning RNA secondary structures and for motif detection. Motivated by widely used algorithms for RNA folding, we decompose an RNA secondary structure into a set of atomic structure components that are further organized by a tree model to capture the structural particularities. RSmatch can find the optimal global or local alignment between two RNA secondary structures using two scoring matrices, one for single-stranded regions and the other for double-stranded regions. The time complexity of RSmatch is O(mn) where m is the size of the query structure and n that of the subject structure. When applied to searching a structure database, RSmatch can find similar RNA substructures, and is capable of conducting multiple structure alignment and iterative database search. Therefore it can be used to identify functional RNA motifs. The accuracy of RSmatch is tested by experiments using a number of known RNA structures, including simple stem-loops and complex structures containing junctions. CONCLUSION: With respect to computing efficiency and accuracy, RSmatch compares favorably with other tools for RNA structure alignment and motif detection. This tool shall be useful to researchers interested in comparing RNA structures obtained from wet lab experiments or RNA folding programs, particularly when the size of the structure dataset is large.

Algorithms↗
PubMed1989

Nonisotopic detection of RNA in an enzyme immunoassay using a monoclonal antibody against DNA-RNA hybrids.

A sensitive nonisotopic solution hybridization assay for detection of RNA is described and characterized using a pSP65 plasmid model system. The assay procedure is based on a hybridization reaction in solution between a biotinylated DNA probe and a target RNA. The biotin-labeled hybrids are captured on a microtiter plate coated with an antibody to biotin. Bound DNA-RNA hybrids are detected by an immunoreaction with an enzyme-labeled monoclonal antibody specifically directed against DNA-RNA heteropolymers and the hybrids are quantitatively measured with the addition of a fluorogenic substrate. Optimal conditions under which to perform the assay were hybridization time, 1000 min; temperature, 75 degrees C; probe concentration, 0.2 microgram/ml; extent of probe biotinylation, 6.7%; buffer stringency, 2x SSC. A bisulfite-modified DNA probe was compared to nick-translated probes synthesized with reporter groups of different lengths (bio-11-dUTP or bio-19-dUTP). All probes could detect 10 pg/ml of target RNA. The presence of nonhomologous DNA or RNA sequences reduced the sensitivity of RNA detection by one half-log to 32 pg/ml (1.6 pg/assay).

Animals↗
PubMedDate not supplied

Detection of HIV and HCV RNA in semen from Brazilian coinfected men using multiplex PCR before and after semen washing.

INTRODUCTION: Prolonged survival of patients under HAART has resulted in new demands for assisted reproductive technologies. HIV serodiscordant couples wish to make use of assisted reproduction techniques in order to avoid viral transmission to the partner or to the newborn. It is therefore essential to test the effectiveness of techniques aimed at reducing HIV and HCV loads in infected semen using molecular biology tests. METHODS: After seminal analysis, semen samples from 20 coinfected patients were submitted to cell fractioning and isolation of motile spermatozoa by density gradient centrifugation and swim-up. HIV and HCV RNA detection tests were performed with RNA obtained from sperm, seminal plasma and total semen. RESULTS: In pre-washing semen, HIV RNA was detected in 100% of total semen samples, whereas HCV RNA was concomitantly amplified in only one specimen. Neither HIV nor HCV were detected either in the swim-up or in the post-washing semen fractions. CONCLUSIONS: Reduction of HIV and/or HCV shedding in semen by density gradient centrifugation followed by swim-up is an efficient method. These findings lead us to believe that, although semen is rarely found to contain HCV, semen processing is highly beneficial for HIV/HCV coinfected individuals.

Adult↗
PubMed1992

Detection of hepatitis C viral RNA sequences in fresh and paraffin-embedded liver biopsy specimens of non-A, non-B hepatitis patients.

In this study methods of HCV-RNA detection in fresh frozen and formalin-fixed, paraffin-embedded liver biopsies are described. Of 22 untreated chronic non-A, non-B hepatitis patients and 6 control patients, a plasma sample and part of a liver biopsy were freshly frozen for hepatitis C virus (HCV) cDNA-PCR. From 16 of the same non-A, non-B hepatitis patients and from 5 of the same control patients formalin-fixed, paraffin-embedded liver tissue from the same biopsy was available also for HCV cDNA-PCR. In 13 of 22 non-A, non-B hepatitis patients HCV-RNA could be detected in plasma as well as in liver tissue. In the other 9 non-A, non-B hepatitis patients and in 6 control patients, no HCV-RNA was detectable in either plasma or liver tissue. The comparison between HCV cDNA-PCR results in fresh frozen versus formalin-fixed, paraffin-embedded liver biopsies showed that although detection of HCV-RNA in both correlated 100% the quantity of HCV-RNA was lower in the formalin-fixed, paraffin-embedded liver biopsies of 5 of 8 patients for whom end-point dilution titration of liver RNA was performed. We conclude that using the procedures described HCV-RNA can be reliably detected in both fresh-frozen and formalin-fixed, paraffin-embedded liver biopsies and that HCV cDNA-PCR in liver tissue may become an important assay, especially for monitoring anti-viral therapy.

Biopsy↗