Correlation between serum HCV RNA and aminotransferase levels in patients with chronic HCV infection.

Cross-sectional studies on the correlation between serum hepatitis C virus (HCV) RNA and alanine aminotransferase (ALT) levels in patients with chronic hepatitis C have yielded conflicting results. We conducted a longitudinal study to examine the correlation between HCV viremia and serum ALT levels in individual patients over time. Serial samples (mean 9) from 25 patients with chronic HCV infection, including interferon-treated and untreated immunocompetent and immunosuppressed patients, collected over a period of 1-4.8 years (mean 2.6 years) were tested for HCV RNA and ALT levels using a highly reproducible quantitative (bDNA) assay. A significant correlation was found between serum HCV RNA and ALT levels in the patients who received IFN therapy, but no correlation was observed in the untreated patients. Among the untreated patients, the immunosuppressed patients had significantly higher HCV RNA levels (39 +/- 4 vs 3.6 +/- 8 Meq/ml, P < 0.0001) but significantly lower ALT (56 +/- 11 vs 97 +/- 12 units/liter, P = 0.03) levels when compared to the immunocompetent ones. In summary, we found no correlation between serum HCV RNA and ALT levels in chronic hepatitis C patients who are not receiving interferon therapy. Immunosuppression results in higher HCV RNA but lower ALT levels.

Effect of human immunodeficiency virus infection on hepatitis C virus infection in hemophiliacs.

Chronic liver disease due to hepatitis C virus (HCV) infection is a major problem in hemophiliacs. Recent reports suggested that hemophiliacs coinfected with hepatitis C virus and human immunodeficiency virus (HIV) have an increased incidence of liver failure but the mechanism of accelerated liver injury is not clear. We tested plasma from 100 hemophiliacs for anti-HCV by second generation ELISA, anti-HIV by EIA, and HCV RNA and HIV RNA by branched DNA and polymerase chain reaction assays to determine if hemophiliacs coinfected with HCV and HIV have higher HCV RNA levels and more active liver disease. Seventy-nine (79%) patients were anti-HCV positive, of whom 85% were HCV RNA positive. None of the anti-HCV-negative patients had detectable HCV RNA in plasma. Forty-two (42%) patients were anti-HIV positive, of whom 47% had detectable HIV RNA. All the anti-HIV-positive patients were also anti-HCV positive. The prevalence of both anti-HCV and anti-HIV increased significantly with age. There was no difference in HCV RNA levels between anti-HIV-positive and anti-HIV-negative patients (mean: 21 +/- 4 vs 18 +/- 5 Meq/ml), although HCV RNA levels were significantly higher in anti-HIV-positive patients with CD4 counts < 200/mm3 (P = 0.008). There was an inverse correlation between HCV RNA levels and CD4 counts but no correlation was found between HCV RNA and serum aminotransferase levels. We found a high prevalence of HCV and HIV coinfection in our hemophiliacs. Hepatitis C virus replication appears to be increased in patients with severe immunodeficiency secondary to progressive HIV infection. However, there was no correlation between HCV RNA and serum ALT level, suggesting that HCV is not directly cytopathic.

Accurate quantification of hepatitis C virus (HCV) RNA from all HCV genotypes by using branched-DNA technology.

In studies monitoring disease progression and therapeutic response, it is essential that the method used for hepatitis C virus (HCV) quantification not be influenced by genotypic variability. The branched DNA assay provides a reliable method for the quantification of HCV RNA. A modified set of oligonucleotide probes for the branched DNA assay was developed to enhance the efficiency of binding to genotypic variants of HCV. The improved branched DNA assay (HCV RNA 2.0) yielded highly reproducible quantification of hepatitis C virus RNA and displayed a nearly 600-fold dynamic range in quantification up to 120 Meq of HCV RNA per ml. The quantification limit was set at 0.2 Meg of HCV RNA per ml to ensure a specificity of > or = 95%. With this lowered quantification limit and the enhanced hybridization of the probes, the HCV RNA 2.0 assay exhibited a high level of sensitivity (96%) and was virtually unaffected by the genotypic variability of HCV. The HCV RNA 2.0 assay may be a useful tool for following HCV RNA levels throughout the course of disease, selecting patients for therapy, and evaluating therapeutic response.

Evaluation of branched DNA signal amplification for the detection of hepatitis C virus RNA.

There is an increasing need for a practical assay to measure HCV RNA to assess the viral burden in chronic hepatitis C virus (HCV) infection as viral load relates to transmission and therapeutic response. This study evaluates branched DNA (bDNA) signal amplification, a technique that avoids many of the pitfalls of polymerase chain reaction (PCR). The bDNA assay uses a microtitre well format and a series of capture, target and amplification probes that bind RNA to the well and then successively bind oligonucleotides to the RNA and branched DNA molecules to the oligonucleotides. Enzyme-labelled probes are bound to the arms of the bDNA and light output from a chemiluminescent substrate is directly proportional to the amount of starting HCV RNA. Appropriate standards provide direct quantitation. Whereas PCR amplifies the HCV genome, bDNA amplifies the hybridization signal. In testing a standardized, coded panel, bDNA showed 100% specificity and detected five of six sera proven to transmit hepatitis C to the chimpanzee; PCR detected all six infectious sera. Serial samples were measured in two acute and five chronic cases of transfusion-associated hepatitis and in three commercial seroconversion panels. In acute cases, 10(7)-10(8) molecular equivalents per ml (eq per ml) of HCV RNA were detected prior to peak alanine aminotransferase (ALT) activity and then rapidly declined to non-detectable levels. Similar levels of HCV RNA were observed early in the course of two patients who progressed to chronic hepatitis; the chronic course was characterized by diminished, fluctuating and sometimes non-detectable levels of HCV RNA. In two chronic cases, HCV RNA was not detected, or only transiently detected by bDNA, but was present when assayed by PCR. In one chronic case, the periodicity of HCV RNA levels closely paralleled the fluctuations of ALT suggesting a relationship between viral replication and subsequent hepatocellular injury. In testing 50 blood donors whose anti-HCV reactivity was confirmed by a recombinant immunoblot assay (RIBA), HCV RNA was detected by bDNA in 41 (81%), while PCR was positive in 45 (90%); the overall concordance between bDNA and PCR in 100 anti-HCV enzyme immunoassays (EIA) reactive donor samples was 96%.(ABSTRACT TRUNCATED AT 400 WORDS)

Rapid nucleic acid assay for detection of bacteria with tetM-mediated tetracycline resistance.

A novel nucleic acid assay has been developed to screen bacterial populations for the presence of the tetM structural gene. The method involves the specific hybridization of several synthetic oligonucleotides to the gene in a crude bacterial lysate solution. As few as 1.5 x 10(4) CFU can be detected with the assay.

Application of a rapid non-radioisotopic nucleic acid analysis system to the detection of sexually transmitted disease-causing organisms and their associated antimicrobial resistances.

We devised a versatile method for detecting nucleic acids in crude lysates of biological samples. A controlled network of nucleic acid hybrids composed of the target fragment, several oligonucleotide probes, branched DNA amplifiers, and labeled oligonucleotides is produced on a solid phase to ultimately incorporate 60 to 300 molecules of alkaline phosphatase, which are detected with a chemiluminescent substrate. The visible light output can be recorded on a luminometer or on instant black-and-white film. Assays have been developed for the detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and for genes conferring penicillin and tetracycline resistance. Conducted much like ELISAS, the assays are performed in about 4 h (for 96 samples) in microliter dishes. The molecular detection limit of approximately 50,000 molecules of double-stranded DNA has permitted us to detect 1 to 10 x 10(3) of C. trachomatis and N. gonorrhoeae with specific probe sequences. Both plasmid and genomic target sequences can be detected by the same procedure. All of the assay components, except for a set of unmodified oligonucleotide probes, are universally applicable for all targets.

Rapid chemiluminescent nucleic acid assays for detection of TEM-1 beta-lactamase-mediated penicillin resistance in Neisseria gonorrhoeae and other bacteria.

Two new assays for the detection of TEM-1 beta-lactamase-mediated bacterial penicillin resistance were developed that involve the use of specific nucleic acid hybridization. Both techniques are based on a solution-phase hybridization of oligonucleotide probes to the target DNA sequence, solid-phase capture of the probe-target complex, and an amplified chemiluminescent labeling method. One configuration of hybridization probes detected the presence of TEM-1 in Neisseria gonorrhoeae (45 strains), Haemophilus spp., Escherichia coli, Shigella sonnei and Salmonella typhi. A second configuration (TEM-1NH) detected TEM-1 beta-lactamase-mediated penicillin resistance only in N. gonorrhoeae (97 strains) and Haemophilus (6 strains) isolates in which TEM-1 is inserted in a pFA7-type plasmid. Both methods were 100 times more sensitive than a commercially available colorimetric beta-lactamase activity test and approximately 5 times more sensitive than radioisotopic dot blot screening for the gene. The assays are particularly well suited to the analysis of large numbers of samples, can be performed in a total of 4 h, and are sensitive to 10(4) to 10(5) CFU.

Diversity of Chlamydia trachomatis major outer membrane protein genes.

Genomic DNA libraries were constructed for Chlamydia trachomatis serovars B and C by using BamHI fragments, and recombinants that contained the major outer membrane protein (omp1) gene for each serovar were identified and sequenced. Comparisons between these gene sequences and the gene from serovar L2 demonstrated fewer base pair differences between serovars L2 and B than between L2 and C; this finding is consistent with the serologic and antigenic relationships among these serovars. The translated amino acid sequence for the major outer membrane proteins (MOMPs) contained the same number of amino acids for serovars L2 and B, whereas the serovar C MOMP contained three additional amino acids. The antigenic diversity of the chlamydial MOMP was reflected in four sequence-variable domains, and two of these domains were candidates for the putative type-specific antigenic determinant. The molecular basis of omp1 gene diversity among C. trachomatis serovars was observed to be clustered nucleotide substitutions for closely related serovars and insertions or deletions for distantly related serovars.

The complete nucleotide sequence of the glnALG operon of Escherichia coli K12.

The nucleotide sequence of the E. coli glnALG operon has been determined. The glnL (ntrB) and glnG (ntrC) genes present a high homology, at the nucleotide and aminoacid levels, with the corresponding genes of Klebsiella pneumoniae. The predicted aminoacid sequence for glutamine synthetase allowed us to locate some of the enzyme domains. The structure of this operon is discussed.

Determination of the nucleotide sequence for the glutamate synthase structural genes of Escherichia coli K-12.

We have determined the complete nucleotide sequence of a 6.3-kb chromosomal HpaI-EcoRI fragment, that contains the structural genes for both the large and small subunits of the Escherichia coli K-12 glutamate synthase (GOGAT) enzyme, as well as the 5'- and 3'-flanking and intercistronic DNA regions. The Mrs of the two subunits, as deduced from the nucleotide (nt) sequence, were estimated as 166,208 and 52,246. Partial amino acid sequence of the GOGAT enzyme revealed that the large subunit starts with a cysteine residue that is probably generated by a proteolytic cleavage. Northern blotting experiments revealed a transcript of approximately 7300 nt, that at least contains the cistrons for both subunits. A transcriptional start point and a functional promoter were identified in the 5' DNA flanking region of the large subunit gene. The messenger RNA nontranslated leader region has 120 nt and shares identity with the leader regions of E. coli ribosomal operons, in particular around the so-called boxA sequence implicated in antitermination. Other possible regulatory sequences are described.

Molecular characterization of human immunodeficiency virus from Zaire: nucleotide sequence analysis identifies conserved and variable domains in the envelope gene.

To examine the genetic relatedness of human immunodeficiency viruses (HIV) from different geographic locations, we molecularly cloned the genome of HIV isolated from a Zairian AIDS patient. Restriction mapping of the recombinant clone, designated HIV-Zr6, revealed both common (as observed in other HIV isolates) and unique restriction sites. The DNA clone of HIV-Zr6, shown to give rise to infectious cytopathic virus after transfection of cultured lymphoid cells, was sequenced in several regions. The long terminal repeat (LTR), open reading frame 1 (ORF1), C-terminal envelope (env) gene domain, and ORF2 showed less than 6% difference in nucleotide sequence when compared to other HIV isolates including human T-lymphotropic virus-type III (HTLV-III) clone B10, lymphadenopathy-associated virus-1 (LAV-1), and AIDS-associated retrovirus-2 (ARV-2). About 15% difference in nucleotide sequences was noted in the N-terminal env gene domain. Alignments of env gene sequences revealed conserved, moderately variable, and hypervariable stretches in the predicted amino acid sequences. This model provides a basis for assessing the significance of sequence variation on properties controlled by the viral Env glycoproteins such as cell tropism and immunogenicity.

High level expression of proinsulin in the yeast, Saccharomyces cerevisiae.

Human proinsulin (PI) has been expressed to a high level (100 mg/liter) as a human superoxide dismutase-PI fusion protein in the yeast, Saccharomyces cerevisiae. At the junction of the two proteins is a methionine residue, allowing PI to be released from the fusion by reaction with cyanogen bromide. The fusion is expressed using a regulated, hybrid promoter containing the regulatory region of the alcohol dehydrogenase II promoter and the 3' end of a glyceraldehyde-3-phosphate dehydrogenase promoter, allowing the recombinant yeast cells to be stably maintained. Production of the fusion protein is induced by growth in medium lacking a fermentable carbon source. The heterologous fusion protein is probably insoluble within the cell, since electron microscopy reveals the presence of 'inclusion bodies'. In a cell-free extract the fusion protein is also insoluble, but can be solubilized with sodium dodecyl sulfate, and cleaved with cyanogen bromide. The PI that is produced contains incorrect disulfide bonds. After sulfitolysis, the product can be easily purified, renatured, and processed to yield insulin.

Sequence analysis of the major outer membrane protein gene from Chlamydia trachomatis serovar L2.

The structural gene for the major outer membrane protein (MOMP) from Chlamydia trachomatis was cloned and sequenced. A lambda gt11 recombinant (lambda gt11/L2/33) that contains a portion of the MOMP coding sequence was used to probe a lambda 1059 library constructed from DNA obtained from C. trachomatis serovar L2. Selected lambda 1059 recombinants were mapped with endonuclease restriction enzymes. The MOMP gene was mapped to the 5' end of a BamHI fragment of approximately 9 kilobases. Contiguous endonuclease restriction fragments identified within this region permitted the selection of specific fragments for subcloning and DNA sequencing. The MOMP gene consisted of a 1,182-base-pair open reading frame that encoded 394 amino acids and ended with three stop codons. The known amino-terminal amino acid was preceded by 22 amino acids whose sequence was compatible with a leader or signal sequence. The primary structure of MOMP determined from the translated DNA sequence demonstrated nine cysteine residues and a remarkably homogeneous distribution of charged and hydrophobic residues.

Human epidermal growth factor precursor: cDNA sequence, expression in vitro and gene organization.

Complementary DNA clones encoding the human kidney epidermal growth factor (EGF) precursor have been isolated and sequenced. They predict the sequence of a 1,207 amino acid protein which contains EGF flanked by polypeptide segments of 970 and 184 residues at its NH2- and COOH-termini, respectively. The structural organization of the human EGF precursor is similar to that previously described for the mouse protein and there is 66% identity between the two sequences. Transfection of COS-7 cells with the human EGF precursor cDNA linked to the SV40 early promoter indicate that it can be synthesized as a membrane protein with its NH2-terminus external to the cell surface. The human EGF precursor gene is approximately 110 kilobase pairs and has 24 exons. Its exon-intron organization revealed that various domains of the EGF precursor are encoded by individual exons. Moreover, 15 of the 24 exons encode protein segments that are homologous to sequences in other proteins. Exon duplication and shuffling appear to have played an important role in determining the present structure of this protein.

Differential antibody responses of individuals infected with AIDS-associated retroviruses surveyed using the viral core antigen p25gag expressed in bacteria.

Infection with the retrovirus that is the etiological agent of acquired immune deficiency syndrome (AIDS) is characterized by the development of antiviral antibodies. To generate reagents for studying immune responses to individual viral proteins, we have produced viral antigens in microorganisms by recombinant DNA techniques. Large amounts of the major core protein (p25gag) of an isolate of the AIDS retrovirus (AIDS-associated retrovirus; ARV-2) have been directly expressed in Escherichia coli. Recombinant p25gag (R-p25gag) has been purified and used in an enzyme-linked immunosorbent assay (ELISA) for antibodies to p25gag. Serum samples obtained from 100 individuals with AIDS, AIDS-related complex (ARC), or potential exposure to the virus through sexual contact with AIDS or ARC patients (contacts) were tested first in an ELISA with disrupted whole virus to determine which of the subjects had mounted an antibody response to the virus (virus seropositive) and then in the p25gag ELISA to determine if they had antibodies to this particular viral antigen. We observed a decrease in the proportion of virus seropositive individuals with antibodies to p25gag among patients groups in which the disease was more advanced; contacts were often positive (71%), ARC patients less frequently positive (48%), and AIDS patients only rarely positive (16%). Our results suggest that monitoring p25gag seropositivity of infected individuals may be useful for predicting either the prognosis or the stage of the disease.

Recombinant polypeptide from the endonuclease region of the acquired immune deficiency syndrome retrovirus polymerase (pol) gene detects serum antibodies in most infected individuals.

Sera from the majority of individuals that were positive in an enzyme-linked immunosorbent assay (ELISA) retrovirus (ARV), an isolate of the for antibodies to acquired immune deficiency syndrome (AIDS)-associated retrovirus (ARV), an isolate of the retrovirus identified as the etiologic agent of AIDS, were found to react with a 31,000-dalton protein (p31) in virus Western blot assays. To determine if this 31,000-dalton immunoreactive species originated from the putative endonuclease region of the polymerase (pol) gene of ARV, we cloned this portion of pol into bacterial expression vectors for direct expression and for expression as a fusion protein with human superoxide dismutase. Transformants from both constructions expressed immunoreactive protein detected in immunoblots with an AIDS patient's serum. Extracts from transformants expressing these sequences competed with the binding of antibodies from AIDS patients' sera to the 31,000-dalton protein in virus immunoblots, confirming that viral p31 originated from the endonuclease domain of the ARV polymerase gene. The superoxide dismutase-p31 fusion protein was purified, and an ELISA for detecting antibodies to p31 was developed. The majority (95%) of serum samples obtained from individuals seropositive in the virus ELISA were also positive in the p31 antibody ELISA.

Isolation of the human insulin-like growth factor genes: insulin-like growth factor II and insulin genes are contiguous.

Overlapping recombinant clones that encompass the insulin-like growth factor (IGF) I and II genes have been isolated from a human genomic DNA library. Each gene is present once per haploid genome; the IGF-I gene spans greater than 35 kilobase pairs (kbp) and the IGF-II gene is at least 15 kbp. The exon-intron organization of these genes is similar, each having four exons, which is one more than the related insulin gene. Comparison of the restriction endonuclease cleavage maps of the IGF-II and insulin genes, including their flanking regions and hybridization with an IGF-II cDNA probe, revealed that they are adjacent to one another. The IGF-II and insulin genes have the same polarity and are separated by 12.6 kbp of intergenic DNA that includes a dispersed middle repetitive Alu sequence. The order of the genes is 5'-insulin-IGF-II-3'.

Cassette of eight exons shared by genes for LDL receptor and EGF precursor.

The amino acid sequences of the human low-density lipoprotein (LDL) receptor and the human precursor for epidermal growth factor (EGF) show 33 percent identity over a stretch of 400 residues. This region of homologous is encoded by eight contiguous exons in each respective gene. Of the nine introns that separate these exons, five are located in identical positions in the two protein sequences. This finding suggests that the homologous region may have resulted from a duplication of an ancestral gene and that the two genes evolved further by recruitment of exons from other genes, which provided the specific functional domains of the LDL receptor and the EGF precursor.