Detection of rhinoviruses by tissue culture and two independent amplification techniques, nucleic acid sequence-based amplification and reverse transcription-PCR, in children with acute respiratory infections during a winter season.

Five hundred seventeen consecutive nasopharyngeal aspirates were collected between October 1998 and May 1999 for episodes of acute respiratory tract infections in children presenting at the University Hospital of Antwerp. Culture and nucleic acid amplification techniques--nucleic acid sequence-based amplification (NASBA) and reverse transcription-PCR (RT-PCR)--were applied to detect rhinoviruses (RVs). Other respiratory viruses were detected by immunofluorescence (IF) analysis of the specimens and IF analysis of shell vial cultures. Among the 517 specimens, 219 viral agents were identified. They were, in decreasing order, rhinoviruses (93 [18.0%]), respiratory syncytial virus (76 [14.7%]), adenoviruses (16 [3.1%]), influenza viruses (15 [2.9%]), enteroviruses (15 [2.9%]), and herpes simplex virus (4 [0.8%]). For the evaluation of rhinovirus detection, culture positivity and/or a positive reaction in the two independent amplification methods was used as an expanded "gold standard." Based on this standard, the sensitivity, specificity, positive predictive value, and negative predictive value of culture were 44.7, 100, 100, and 99.8%, and those of NASBA and RT-PCR were 85.1, 98.3, 83.3, and 98.5% and 82.9, 93.4, 55.7, and 98.2%, respectively. NASBA and RT-PCR produced comparable results and were significantly more sensitive than virus culture. RVs showed the highest incidence in acute respiratory tract infections in children.

Improved detection of rhinoviruses by nucleic acid sequence-based amplification after nucleotide sequence determination of the 5' noncoding regions of additional rhinovirus strains.

The isothermal nucleic acid sequence-based amplification (NASBA) system was applied for the detection of rhinoviruses using primers targeted at the 5' noncoding region (5' NCR) of the viral genome. The nucleotide sequence of the 5' NCRs of 34 rhinovirus isolates was determined to map the most conserved regions and design more appropriate primers and probes. The assay amplified RNA extracted from 30 rhinovirus reference strains and 88 rhinovirus isolates, it did not amplify RNA from 49 enterovirus isolates and other respiratory viruses. The assay allows one to discriminate between group A and B rhinoviruses. Sensitivities for the detection of group B and group A rhinoviruses was 20 and 200 50% tissue culture infective doses, respectively.

Detection of Mycoplasma pneumoniae in spiked clinical samples by nucleic acid sequence-based amplification.

Isothermal nucleic acid sequence-based amplification (NASBA) was applied to the detection of Mycoplasma pneumoniae. M. pneumoniae RNA prepared from a plasmid construct was used to assess the sensitivity of the assay, and an internal control for the detection of inhibitors was constructed. The sensitivity of the NASBA assay was 10 molecules of wild-type M. pneumoniae RNA generated in vitro and 5 color-changing units (CCU) of M. pneumoniae. An appropriate specimen preparation procedure was developed: after protease treatment of the respiratory specimens, guanidine thiocyanate lysis solution (4.7 M guanidine thiocyanate [Sigma-Aldrich NV], 46 mM Tris-HCl [Merck, Darmstadt, Germany], 20 mM EDTA [Sigma-Aldrich NV], 1.2% [wt/vol] Triton X-100 [Sigma-Aldrich NV], pH 6.2.) was added. With spiked throats, nasopharyngeal aspirates, bronchoalveolar lavage specimens, and sputum specimens, the sensitivity of the NASBA assay in the presence of the internal control was 2 x 10(4) molecules of in vitro-generated RNA or 5 CCU of M. pneumoniae. The sensitivity of the NASBA assay was comparable to that of a PCR targeted to the P1 adhesin gene. Fifteen clinical specimens positive for M. pneumoniae by PCR were also positive by NASBA. These results indicate that the sensitivity of detection of M. pneumoniae in spiked respiratory samples by NASBA is high. Together with the use of the internal control, the assay merits evaluation as a diagnostic tool.

Evaluation of a second-generation nucleic acid sequence-based amplification assay for quantification of HIV type 1 RNA and the use of ultrasensitive protocol adaptations.

Accurate assessment of plasma HIV RNA levels at low concentrations is clinically important. We evaluated a second-generation quantitative HIV RNA assay (NucliSens HIV-1 QT), and three simple adaptations of the NucliSens standard protocol to lower the lower cutoff level. The assays were evaluated in constructed panels with known HIV RNA concentrations and in clinical samples. Results were compared with those obtained with the first generation (NASBA HIV-1 QT) and with two other commercially available assays: the Amplicor HIV Monitor test and the Quantiplex assay. In a constructed panel, results obtained by NASBA QT were on average 0.13 log(10) copies/ml (SD 0.15) higher than those of NucliSens. The NucliSens assay could quantify HIV RNA in at least 50% of the samples down to 518 (2.71 log(10)) copies/ml and NASBA QT to 5.80 x 10(3) (3.76 log(10)) copies/ml). Both assays correlated well with the known input (R NucliSens = 0.99; R NASBA QT = 0.996), but results were more variable at lower input levels. With the three different ultrasensitive NucliSens adaptations, HIV RNA could be quantified in at least 50% of the samples down to 100 (2.00 log(10)), 46 (1.66 log(10)), and 10 (1.00 log(10)) copies/ml, respectively. In patient samples, Amplicor results were on average 0.11 (SD 0.20) log(10) copies/ml above, NucliSens 0.02 (SD 0.29) copies/ml above, and Quantiplex 0.13 (SD 0.19) copies/ml below the mean of the three assay results per sample. The variation remained the same over the range of RNA levels with all three assays. The NucliSens assay can quantify HIV RNA at lower levels than the NASBA QT and is comparable to other commercially available assays. The lower cutoff of the NucliSens can be lowered down to 10 copies/ml.

Design and evaluation of a human immunodeficiency virus type 1 RNA assay using nucleic acid sequence-based amplification technology able to quantify both group M and O viruses by using the long terminal repeat as target.

Currently available human immunodeficiency virus type 1 (HIV-1) RNA quantification assays can detect most viruses of the group M subtypes, but a substantial number are missed or not quantified reliably. Viruses of HIV-1 group O cannot be detected by any commercially available assay. We developed and evaluated a quantitative assay based on nucleic acid sequence-based amplification (NASBA) technology, with primers and probes located in the conserved long terminal repeat (LTR) region of the HIV-1 genome. In 68 of 72 serum samples from individuals infected with HIV-1 subtypes A to H of group M, viruses could be detected and quantified. In serum samples from two patients infected with HIV-1 group O viruses, these viruses as well could be detected and quantified. In contrast, the currently used gag-based assay underestimated the presence of subtype A viruses and could not detect subtype G and group O viruses. The discrepancy between the results of the two assays may be explained by the number of mismatches found within and among the probe and primer regions of the subtype isolates. These data indicate that LTR-based assays, including the NASBA format chosen here, are better suited to monitoring HIV-1 therapy than are gag-based assays in an era in which multiple HIV-1 subtypes and groups are spreading worldwide.

Vernier zone residue 4 of mouse subgroup II kappa light chains is a critical determinant for antigen recognition.

BACKGROUND: During the conversion of murine monoclonal antibodies directed against the human chorionic gonadotropin (hCG) into bacterially expressed single chain fragments (scFv), we found a major reduction of binding activity upon introduction of a primer encoded mutation. OBJECTIVES: In this study we tried to determine which mutation was responsible and on what manner this mutation affected antigen binding (structural effect versus direct involvement of the residue in binding). RESULTS: No binding could be detected, when the wild type residue methionine at position 4 within the Framework region 1 of the Vkappa light chain was substituted by serine in two antibodies with a subgroup II kappa light chain. However, a similar replacement within an anti-hCG antibody with a subgroup IV kappa light chain and thereby having leucine as wild type residue, did not affect the binding characteristics. The mutant scFv's derived from both AB-s sensitive for substitution by serine never reacted with antigen in ELISA. Analysis with surface plasmon resonance revealed a residual binding only on a sensorchip with a high density coating of antigen; however, an increased dissociation, relative to that of the wild type scFv and the absence of reactivity in ELISA suggest a drastically altered affinity. CONCLUSION: A structural explanation for the changed binding characteristics can be the influence of the position 4 residue, as being a constituent of the Vernier zone, on the position of the CDR1 loop of Vkappa, which might harbour residues that directly bind to antigen, or indirectly positions other variable loops of the binding pocket. An increased sensitivity for trypsin digestion supported the hypothesis of a local conformational change in the serine mutant of the subgroup II kappa containing antibody.

Absolute conservation of residue 6 of immunoglobulin heavy chain variable regions of class IIA is required for correct folding.

While studying the expression of single-chain antibodies (scFv) derived from several murine monoclonal antibodies, we found that residue 6 in Framework region 1 of the heavy chain variable domain plays a crucial role in antibody folding. Binding activity of three murine antibodies with a heavy chain variable region (VH) subgroup IIA was completely lost when at this position the wild-type residue glutamine (Q) was substituted by glutamate (E). Increased sensitivity towards trypsin digestion of soluble scFv suggested that the lack of binding activity was caused by incorrect folding of Q6E mutants. Grafting of the three additional class IA derived FR1 residues, based upon the comparison between both classes of VH sequences, on to the 'defect' subgroup IIA sequence, partially restored the antigen binding activity of the Q6E-containing scFv. Our results suggest that residue 6 of the heavy chain may be part of a folding nucleus, involving the first two beta-strands of Framework region 1. The evolutionary conservation of either glutamine or glutamate at position 6 in different antibody families may well indicate that within immunoglobulin VH domains, different family specific folding nuclei have evolved.

Selection of human anti-human immunodeficiency virus type 1 envelope single-chain antibodies from a peripheral blood cell-based phage repertoire.

Monoclonal antibodies play an important role in the development of diagnostic assays. Instead of using hybridoma technology to isolate human immunodeficiency virus type 1-specific antibodies, a phage-displayed antibody library was generated from a small number (10(7)) of peripheral blood lymphocytes from a seropositive donor. Two families of single-chain antibodies (scFvs) were selected by biopanning with the envelope precursor gp160. ELISA and competition in the BIAcore system revealed that one antibody family recognized a conformation-sensitive epitope within gp120, while the other antibody family was gp41-specific. The latter group had sequence similarity to antibodies recognizing the cluster III epitope of gp41. Binding of scFvs to gp160 could be inhibited with the donor's serum antibodies, indicating that antibodies with a similar specificity were circulating in the donor's blood. Competition experiments suggested that the epitope of the anti-gp41 antibodies was recognized by a broad range of patients' sera: 21 out of 22 sera from North American and all 20 sera from African seropositive patients inhibited binding of scFvs. In contrast, three sera from this panel did not react with the epitope of the anti-gp120 antibodies. These data indicate that, because of the conserved nature of its epitope, the anti-gp41 antibody will be suitable for diagnostic applications.

Stability of HIV-1 RNA in blood during specimen handling and storage prior to amplification by NASBA-QT.

The influence of different storage temperatures and anticoagulation conditions on the HIV-1 RNA load as measured by NASBA-QT was examined. Blood specimens from 14 HIV-1 infected individuals were processed within 2 h after collection. The HIV-1 RNA load remained stable for at least 6 months when samples were frozen directly at -70 degrees C in lysis buffer. This lysis buffer fully inactivated the virus. When whole EDTA blood was stored, the HIV-1 RNA load was stable for 72 h at 25 degrees C, but it declined within 24 h at 4 degrees C. The HIV-1 RNA load in whole heparinized blood declined significantly after 24 h at both 4 and 25 degrees C. It was slightly lower (average of 0.18 log ml-1) than in whole EDTA blood. At 4 degrees C, the HIV-RNA load in serum and EDTA-plasma stored with lysis buffer did not decline up to 14 days. At + 30 degrees C, however, the load declined significantly after 2 days. Of clinical significance, the mean load in EDTA plasma was 0.5 log ml-1 higher than in serum. This difference was patient dependent (range 0.1-0.7 log ml-1). We thus recommend, for quantifying HIV-1 RNA by NASBA, to use preferably EDTA blood which is kept at room temperature until plasma separation. When using heparinized blood, the plasma should be stored frozen within 8 h.

Antibody responses to the hepatitis C virus E2 protein: relationship to viraemia and prevalence in anti-HCV seronegative subjects.

A small proportion of patients with chronic hepatitis C virus (HCV) infection show no serological responses to the HCV polypeptides incorporated in commercial III generation immunoassays. To determine whether sera from these subjects contain antibodies to the highly immunoreactive second envelope polypeptide E2, which is not included in current anti-HCV assays, we studied 59 anti-HCV negative subjects who were found consistently to be HCV RNA positive by polymerase chain reaction (PCR). Controls included 167 anti-HCV seropositive patients with or without serum HCV RNA and normal subjects. Antibodies to the E2 region were sought for by ELISA using the following antigens: a full length E2 protein expressed in insect cells using a baculovirus vector and extracted under denaturing conditions (dE2), and a C-terminal truncated soluble E2 (sE2) protein (a.a. 390-683), also expressed with a baculovirus vector, containing a signal peptide of rabies virus G protein which allows its secretion into the culture supernatant. Sera from only two (3.4%) of the 59 anti-HCV negative, HCV RNA positive patients recognised sE2 and none dE2. In sharp contrast, 82% of seropositive, viraemic patients recognised sE2 and 60% dE2, the difference in immunoreactivity being statistically significant (P < 0.0003). A significantly lower proportion of sera from anti-HCV positive, HCV RNA negative subjects recognised either sE2 or dE2 (16% and 13%, respectively, P < 0.000001). Healthy controls were consistently negative. These results indicate that antibody responses to predominantly conformational epitopes on the HCV E2 protein are common in patients with chronic HCV infection and are strictly related to the presence of circulating viral genomes. In contrast, only a minor proportion of HCV RNA positive patients, but anti-HCV seronegative by commercial immunoassays, have humoral immune responses to the HCV E2 region.

A hybrid baculovirus-bacteriophage T7 transient expression system.

A hybrid recombinant baculovirus-bacteriophage T7 expression system was developed for transient expression in insect cells of plasmids with foreign genes provided with a T7 promoter. The coding sequence for T7 RNA polymerase, with or without a nuclear localization signal, was inserted into the genome of Autographa californica nuclear polyhedrosis virus. Recombinant viruses stably expressed T7 RNA polymerase in insect cells. Upon transfection of infected insect cells with plasmids containing the genes for chloramphenicol acetyltransferase (CAT), the hepatitis B virus precore-, core- or e- antigens under control of the T7 promoter, transient expression of these genes was detected by ELISA. The results obtained indicate that this baculovirus/T7 system provides a simple and widely applicable tool for transient gene expression studies.

Significance of the immune response to a major, conformational B-cell epitope on the hepatitis C virus NS3 region defined by a human monoclonal antibody.

The nonstructural protein NS3 of hepatitis C virus (HCV) possesses two enzymatic domains which are thought to be essential for the virus life cycle: an N-terminal serine-type proteinase, responsible for the processing of nonstructural polypeptides, and a C-terminal nucleoside triphosphatase/helicase, presumably involved in the unwinding of the viral genome. The human antibody response to NS3 usually appears early in the course of HCV infection and is predominantly directed against the carboxyl-terminal portion; however, its fine specificity and clinical significance are largely unknown. We have generated a human monoclonal antibody (hMAb), designated CM3.B6, from a cloned B-cell line obtained from the peripheral blood of a patient with chronic HCV infection, which selectively recognized the purified NS3 protein expressed in bacteria or in eukaryotic cells transfected with full-length or NS3 cDNA. Fine-specificity studies revealed that CM3.B6 recognized a 92-amino-acid sequence (clone 8, amino acids 1363 to 1454) selected from an NS3 DNase fragment library but failed to bind to 12-mer peptides synthesized from the same region, suggesting recognition of a conformational B-cell epitope. Experiments using deletion mutants of clone 8 and competitive inhibition studies using a panel of NS3 peptide-specific murine MAbs indicated that limited N-terminal and C-terminal deletions resulted in a significant reduction of hMAb binding to clone 8, thus identifying a minimal antibody binding domain within clone 8. Competition experiments showed that binding of CM3.B6 to the NS3 protein was efficiently inhibited by 39 of 44 (89%) sera from HCV-infected patients, suggesting that the hMAb recognized an immunodominant epitope within the NS3 region. More importantly, recognition of the sequence defined by CM3.B6 appeared to accurately discriminate between viremic and nonviremic anti-HCV positive sera, suggesting potentially relevant clinical applications in the diagnosis and treatment of HCV infection.

An initiation signal in the 5' untranslated leader sequence of encephalomyocarditis virus RNA.

A 593 nucleotide fragment of the 5' leader of encephalomyocarditis virus RNA (EMCV-RNA) was linked to the SP6 promoter and inserted upstream of the reporter gene chloramphenicol acetyltransferase (CAT). The presence of the 5'-UTR of EMCV-RNA in the RNA transcripts, made in vitro with the SP6 polymerase, resulted in a strong translational enhancement when tested in the micrococcal nuclease-treated reticulocyte lysate. The transcripts were equally active with or without a 5' methylated capstructure as expected, since EMCV-RNA is one of the mRNAs capable of internal initiation. We searched for a signal in the 5' leader that allows the 43S preinitiation complex to bind internally and localized a hairpin containing a unique nucleotide sequence, CUUUA, present in a domain conserved among cardio- and aphtoviral RNAs. Replacing this sequence into AGCU resulted in a 50% loss of translational activity. A second mutation involving a U-G change in the stem of that hairpin resulted in an almost complete loss of initiation.

Mitochondrial biogenesis: recent developments and insights.

Biosynthesis of a functional mitochondrion requires the coordinate expression of genes in both mitochondrial and nuclear DNAs. In yeast, three mitochondrial genes are split and RNA splicing plays a pivotal role in their expression. The recent finding that some introns are capable of self-splicing activity in vitro has permitted analysis of the mechanisms involved in RNA catalysis and may eventually shed light on the evolution of splicing mechanisms in general. Most mitochondrial proteins are encoded by nuclear genes, synthesized in the cytoplasm and imported by the organelle. The availability of cloned genes coding for several constituent subunits of the ubiquinol-cytochrome c reductase, which are imported by mitochondria, has allowed study of selected steps in the addressing of proteins to mitochondria and their intercompartmental sorting within the organelle. Recent developments are discussed.

Subunit II of yeast QH2:cytochrome-c oxidoreductase. Nucleotide sequence of the gene and features of the protein.

The nucleotide sequence of a 2.5 X 10(3)-base segment of yeast nuclear DNA, containing the structural gene for the 40-kDa subunit II of the ubiquinol:cytochrome-c oxidoreductase, has been determined. The region contains only one single reading frame of length sufficient to encode a protein of the size of subunit II. The mature protein is predicted to have a length of 352 amino acids, with a molecular mass of 38714 Da. It is predominantly hydrophilic, with an overall polarity of 45%. Comparison of the sequence of the reading frame with that derived from direct sequence analysis of the N terminus of the mature 40-kDa protein shows that subunit II is synthesized as a longer precursor and shows that the extension is N-terminal. The presequence is 16 amino acids long and it contains a number of positively charged residues and lacks acidic ones. It is also rich in neutral, polar amino acids. S1 nuclease protection analysis of DNA X RNA hybrids identifies two major and one minor transcript of the gene, whose 5' termini map approximately 55, 65 and 75 nucleotides upstream of the initiation codon. Sequences 5' of these termini lack obvious homology to the regulatory sequences of other imported mitochondrial proteins, whose synthesis is controlled by oxygen and by catabolite repression. A mutant lacking a functional subunit II gene has been constructed by a one-step gene-disruption procedure. This mutant grows only slowly on glycerol and still displays a low level of QH2: cytochrome-c oxidoreductase activity (approx. 5% of that of wild type). The implications of this finding for the possible role of subunit II in the complex are discussed.

Targeting efficiency of a mitochondrial pre-sequence is dependent on the passenger protein.

The mitochondrial matrix enzyme manganese superoxide dismutase (SOD) of Saccharomyces cerevisiae is encoded in the nucleus. It is synthesized as a precursor with an NH2-terminal extension of 26 amino acids which is cleaved off during import into the mitochondrion. Fusions between the NH2-terminal 34 amino acids of SOD and the cytosolic proteins invertase of yeast and mouse dihydrofolate reductase (DHFR) were tested for in vitro binding and import into mitochondria. Efficient translocation over the mitochondrial membranes takes place in the case of the SOD-DHFR fusion. The SOD-invertase fusion protein does not get translocated and binds to the organelle with only low efficiency. Yeast transformants harbouring the SOD-invertase fusion gene accumulate approximately 95% of the hybrid protein in the cytosol. The remaining material is found in the interior of the mitochondrion, loosely attached to the inner membrane. We conclude that the pre-sequence of SOD is able to deliver a passenger protein to the mitochondrion. The efficiency of protein delivery and translocation across the membrane is, however, influenced by the passenger protein.

Nucleotide sequence analysis of the nuclear gene coding for manganese superoxide dismutase of yeast mitochondria, a gene previously assumed to code for the Rieske iron-sulphur protein.

We have previously reported the isolation of the gene coding for a 25-kDa polypeptide present in a purified yeast QH2:cytochrome c oxidoreductase preparation, which was thus identified as the gene for the Rieske iron-sulphur protein [Van Loon et al. (1983) Gene 26, 261-272]. Subsequent DNA sequence analysis reported here reveals, however, that the encoded protein is in fact manganese superoxide dismutase, a mitochondrial matrix protein. Comparison with the known amino acid sequence of the mature protein indicates that it is synthesized with an N-terminal extension of 27 amino acids. In common with the N-terminal extensions of other imported mitochondrial proteins, the presequence has several basic residues but lacks negatively charged residues. The function of these positive charges and other possible topogenic sequences are discussed. Sequences 5' of the gene contain two elements that may be homologous to the suggested regulatory sites, UAS 1 and UAS 2 in the yeast CYC1 gene [Guarente et al. (1984) Cell 36, 503-511]. The predicted secondary structures in manganese superoxide dismutase appear to be very similar to those reported for iron superoxide dismutase, suggesting similar three-dimensional structures. Making use of the known three-dimensional structure of the Fe enzyme, the Mn ligands are predicted.