Development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex PCR and a fluid microbead-based assay.

Virology laboratories historically have used direct fluorescent-antibody assay (DFA) and culture to detect six or seven respiratory viruses. Following the discovery of five new human respiratory viruses since 2000, there is an increasing need for diagnostic tests to detect these emerging viruses. We have developed a new test that can detect 20 different respiratory virus types/subtypes in a single 5-h test. The assay employs multiplex PCR using 14 virus-specific primer pairs, followed by a multiplexed target-specific primer extension (TSPE) reaction using 21 primers for specific respiratory virus types and subtypes. TSPE products were sorted and identified by using a fluid microsphere-based array (Universal Array; TmBioscience Corporation, Toronto, Canada) and the Luminex x-MAP system. The assay detected influenza A and B viruses; influenza A virus subtypes H1, H3, and H5 (including subtype H5N1 of the Asian lineage); parainfluenza virus types 1, 2, 3, and 4; respiratory syncytial virus types A and B; adenovirus; metapneumovirus; rhinovirus; enterovirus; and coronaviruses OC43, 229E, severe acute respiratory syndrome coronavirus, NL63, and HKU1. In a prospective evaluation using 294 nasopharyngeal swab specimens, DFA/culture detected 119 positives and the respiratory virus panel (RVP) test detected 112 positives, for a sensitivity of 97%. The RVP test detected an additional 61 positive specimens that either were not detected by DFA/culture or were positive for viruses not tested for by DFA/culture. After resolution of discordant results by using a second unique PCR assay and by using a combined reference standard of positivity, the RVP test detected 180 of 183 true positives, for a sensitivity of 98.5%, whereas DFA and culture detected only 126 of 183 true positives, for a sensitivity of 68.8%. The RVP test should improve the capabilities of hospital and public health laboratories for diagnosing viral respiratory tract infections and should assist public health agencies in identifying etiologic agents in respiratory tract infection outbreaks.

Novel signal amplification technology with applications in DNA and protein detection systems.

A non-enzymatic approach to signal amplification has practical advantages over conventional target amplification methods. We have designed a simple, cost-efficient signal amplification system that can be used to enhance the detection of nucleic acids or protein. The signal amplification process requires initial capture of analyte by a specific probe, which, depending on the analyte, can be an oligomer or an antibody. Once the analyte is captured, amplification moieties are applied to significantly enhance the sensitivity of analyte detection. Nucleic acid amplification is typically greater than 1000-fold, increasing the sensitivity of target detection to less than 1 amol/100 microL. This amplification strategy presents a very flexible system with components that are easily altered to accommodate diverse assay requirements.

Hybridization of single-stranded DNA targets to immobilized complementary DNA probes: comparison of hairpin versus linear capture probes.

A microtiter-based assay system is described in which DNA hairpin probes with dangling ends and single-stranded, linear DNA probes were immobilized and compared based on their ability to capture single-strand target DNA. Hairpin probes consisted of a 16 bp duplex stem, linked by a T(2)-biotin.dT-T(2) loop. The third base was a biotinylated uracil (U(B)) necessary for coupling to avidin coated microtiter wells. The capture region of the hairpin was a 3' dangling end composed of either 16 or 32 bases. Fundamental parameters of the system, such as probe density and avidin adsorption capacity of the plates were characterized. The target DNA consisted of 65 bases whose 3' end was complementary to the dangling end of the hairpin or to the linear probe sequence. The assay system was employed to measure the time dependence and thermodynamic stability of target hybridization with hairpin and linear probes. Target molecules were labeled with either a 5'-FITC, or radiolabeled with [gamma-(33)P]ATP and captured by either linear or hairpin probes affixed to the solid support. Over the range of target concentrations from 10 to 640 pmol hybridization rates increased with increasing target concentration, but varied for the different probes examined. Hairpin probes displayed higher rates of hybridization and larger equilibrium amounts of captured targets than linear probes. At 25 and 45 degrees C, rates of hybridization were better than twice as great for the hairpin compared with the linear capture probes. Hairpin-target complexes were also more thermodynamically stable. Binding free energies were evaluated from the observed equilibrium constants for complex formation. Results showed the order of stability of the probes to be: hairpins with 32 base dangling ends > hairpin probes with l6 base dangling ends > 16 base linear probes > 32 base linear probes. The physical characteristics of hairpins could offer substantial advantages as nucleic acid capture moieties in solid support based hybridization systems.

The cDNA sequence and the deduced amino acid sequence of human transcobalamin II show homology with rat intrinsic factor and human transcobalamin I.

The cellular uptake of cobalamin (Cbl, vitamin B12) is mediated by transcobalamin II (TCII), a plasma protein that binds Cbl and is secreted by human umbilical vein endothelial (HUVE) cells. These cells synthesize and secrete TCII and, therefore, served as the source of the complementary DNA (cDNA) library from which the TCII cDNA was isolated. This full-length cDNA consists of 1866 nucleotides that code for a leader peptide of 18 amino acids, a secreted protein of 409 amino acids, a 5'-untranslated segment of 37 nucleotides, and a 3'-untranslated region of 548 nucleotides. A single 1.9-kilobase species of mRNA corresponding to the size of the cDNA was identified by Northern blot analysis of the RNA isolated from HUVE cells. TCII has 20% amino acid homology and greater than 50% nucleotide homology with human transcobalamin I (TCI) and with rat intrinsic factor (R-IF). TCII has no homology with the amino-terminal region of R-IF that has been reported to have significant primary as well as secondary structural homology with the nucleotide-binding domain of NAD-dependent oxidoreductases. The regions of homology that are common to all three proteins are located in seven domains of the amino acid sequence. One or more of these conserved domains is likely to be involved in Cbl binding, a function that is common to all three proteins. However, the difference in the affinity of TCII, TCI, and R-IF for Cbl and Cbl analogues indicates, a priori, that structural differences in the ligand-binding site of these proteins exist and these probably resulted from divergence of a common ancestral gene.

A base substitution at a splice site in the COL3A1 gene causes exon skipping and generates abnormal type III procollagen in a patient with Ehlers-Danlos syndrome type IV.

The dermis of a child with Ehlers-Danlos syndrome type IV (EDS-IV) contained about 11% of the normal amount of type III collagen and cultured dermal fibroblasts produced a reduced amount of type III procollagen which was secreted poorly. Type III collagen produced by these cells contained normal and abnormal alpha-chains and cyanogen bromide peptides. The site of the structural defect in the abnormal alpha 1 (III) chains was localized to the region of Met797, which is at the junction of the two carboxyl-terminal CB5 and CB9 cyanogen bromide peptides. Chemical cleavage of heteroduplexes formed between EDS-IV mRNA and a normal cDNA clone covering the CB5 and CB9 region showed that about 100 nucleotides were mismatched. Sequencing of amplified and cloned cDNA spanning the mutant region revealed a 108 nucleotide deletion corresponding to amino acid residues Gly775 to Lys810. The deleted nucleotide sequence corresponded to sequences that, by analogy to the organization of the type I collagen genes, should be precisely encoded by exon 41 of the COL3A1 gene. Sequencing of amplified genomic DNA, prepared using disimilar amounts of primers specific for exons 41 and 42, displayed a base substitution (G-to-A) in the highly conserved GT dinucleotide of the 5' splice site of intron 41. Normal sequences were also obtained from the normal allele. It is likely that the GT-to-AT transition at the splice donor site of intron 41 generated an abnormally spliced mRNA in which sequences of exon 40 and 42 were joined together with maintenance of the reading frame. The corresponding peptide deletion included the cyanogen bromide cleavage site Met797-Pro798 and the mammalian collagenase cleavage site at Gly781-Ile782. These losses account for the resistance of EDS-IV collagen to cyanogen bromide and mammalian collagenase digestion. Cultured fibroblasts produced normal homotrimer, mutant homotrimer, and mixed heterotrimer type III collagen molecules. The mutant homotrimer molecules were the major pepsin-resistant species and about 69% of the alpha 1(III) mRNA was in the mutant form.

Hormonal regulation of levels of the messenger RNA encoding hepatic P450 2c (IIC11), a constitutive male-specific form of cytochrome P450.

A cDNA clone for rat hepatic cytochrome P450 2c (gene product IIC11) was isolated and used to study the sex specificity, expression during development, and hormonal regulation of the mRNA encoding this protein in rat liver. P450 2c mRNA levels were about 16-fold higher in males than in females and were only slightly increased in male rats after administration of phenobarbital, a drug that dramatically raises the levels of mRNAs encoding several other members of the P450 II family. In contrast to the mRNA encoding P450 f (gene product IIC7), which increases gradually over the first 6 weeks of life, P450 2c mRNA showed a dramatic increase at puberty, between 4.5-5.5 weeks of life. The roles of sex steroids and GH in controlling this male-specific, developmentally regulated mRNA were then examined. A dependence on adult androgen was demonstrated by the 2- to 4-fold decrease in P-450 2c mRNA levels after castration of adult male rats and their restoration to normal by administration of the synthetic androgen methyltrienolone. Prolonged treatment (15 days) of ovariectomized female rats with this androgen also increased the levels of P450 2c mRNA and its encoded testosterone 16 alpha-hydroxylase to those of intact males. In male rats treated with estradiol valerate, mRNAs for P450 2c and alpha 2u-globulin, a major male-specific hepatic secretory protein that is under complex hormonal control, fell to negligible levels. None of these hormonal perturbations had a detectable effect on the levels of PB-1 (gene product IIC6) mRNA, which is not expressed in a sex-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the mechanism of entry of vaccinia virus in animal cells.

In order to study the mechanism of entry of vaccinia virus into cells the fate of virion associated polypeptides was investigated during infection of african green monkey kidney (BSC-40) cells with 35 S-methionine labelled virus. Approximately 12-15 percent of the virion polypeptides were degraded to acid-soluble products by 3 hours post-infection. Proteolysis was inhibited (50 percent) by methylamine, suggesting a lysosomal site of degradation. Neither methylamine or chloroquine inhibited virus infectivity or uncoating indicating a non-acid endocytic mechanism of entry. Subcellular fractionation studies on density gradients indicated that the bulk of the input virion polypeptides were associated with the plasma membrane fraction. In addition, input virion DNA was partially resolved from the membrane fraction. The results are most consistent with a mechanism of entry involving fusion of the virus with the plasma membrane.

Isolation and characterization of neutralizing monoclonal antibodies to vaccinia virus.

Cells producing neutralizing monoclonal antibodies (mAbs) to UV-inactivated vaccinia virus strain WR were derived by fusion of hyperimmunized mouse spleen cells with mouse myeloma cells. Three mAbs that reacted strongly with purified virus envelopes as determined by enzyme-linked immunosorbent assay were studied. The three mAbs recognized a 14,000-molecular-weight (14K) envelope protein of vaccinia virus and were shown to be immunoglobulin G2b (mAbC3 and mAbB11) and immunoglobulin M (mAbF11). By using ascites, one of the antibodies, mAbC3, neutralized (50%) virus infectivity with a titer of about 10(-4), whereas the others exhibited lower neutralization titers of 10(-2) to 10(-3). The binding of the mAbs to vaccinia virus did not alter virus attachment to cells. However, virus uncoating was extensively blocked by mAbC3, whereas mAbB11 and mAbF11 had little or no effect. The three mAbs recognized a similar 14K protein in cowpox, rabbitpox, and vaccinia Elstree strains, indicating a high degree of protein conservation among orthopoxviruses. Based on the binding of mAbs to V-8 protease cleavage products of the 14K protein, the extent of protein recognition for other poxviruses, and differences in the degree of virus neutralization and of virus uncoating into cells, we suggest that the three mAbs recognize different domains of vaccinia 14K viral envelope protein. Furthermore, our findings indicate that the 14K protein may play a role in virus penetration.

Endocytosis, proteolysis, and exocytosis of exogenous proteins by cultured myotubes.

The endocytic activity of chick myotubes in cultures was investigated. Differentiated myotubes internalized the fluid-phase marker horseradish peroxidase in membrane-bound particles which typically displayed reaction product at the inner surface of the vesicle. Pulse-chase experiments demonstrated a rapid decrease in the number of horseradish peroxidase-containing vesicles and a redistribution from a uniform to a perinuclear pattern. Horseradish peroxidase uptake was extensively inhibited by incubation at 0-4 degrees C consistent with an endocytic mechanism. To further characterize the process, the fate of labeled protein was investigated. Following uptake [3H] hemoglobin A was extensively degraded (40-50%) to acid-soluble products within 10 h. Degradation displayed a biphasic pattern with a rapid early phase followed by a much slower second phase. The decreasing rate of proteolysis can be accounted for, in part, by a simultaneous exocytosis of a substantial fraction (25-30%) of acid-insoluble label from myotubes. The lysosomotropic agents methylamine, monensin, and chloroquine significantly inhibited (23-75%) proteolysis, indicating a lysosomal site of degradation. Part of the inhibitory effect results from an increase in exocytosis in the presence of these agents. Degradation of endocytosed [3H]hemoglobin A was not inhibited by insulin. In contrast degradation of endogenous myotube proteins was inhibited (40%) by insulin and blocked by methylamine. These results suggest that cultured myotubes possess a coupled endocytic/exocytic pathway for macromolecules and that a fraction of the internalized substrate is degraded by an insulin-insensitive lysosomal pathway.

Inhibition of intracellular proteolysis in muscle cultures by multiplication-stimulating activity. Comparison of effects of multiplication-stimulating activity and insulin on proteolysis, protein synthesis, amino acid uptake, and sugar transport.

The effects of the insulin-like growth factor, multiplication-stimulating activity (MSA), on chick myotube cultures were investigated. In serum-free media, MSA at levels reported to be present in fetal serum (5 ng/ml) significantly inhibited overall rates of protein degradation and stimulated protein synthesis and amino acid uptake. Half-maximal effects on protein degradation (-30%), synthesis (+25%), and amino acid uptake (+50%) occurred at approximately 0.05 micrograms/ml. In contrast, 10(2)-10(3)-fold higher concentrations (5 micrograms/ml) were required to stimulate transport of the glucose analog 2-deoxyglucose. The results indicate that MSA is an effective anabolic agent regulating protein metabolism and amino acid uptake, but not sugar transport in these cells. Parallel studies conducted with insulin demonstrated similar size effects on protein metabolism and amino acid uptake in serum-free media. However, unlike MSA, insulin levels (10(-2) units/ml) well in excess of its normal physiological range were required to produce significant effects. In addition, the relative sensitivity of sugar transport with respect to protein metabolic effects differed for insulin and MSA. Thus, 2-deoxyglucose transport was approximately 10 times more sensitive to insulin than protein synthesis, proteolysis, or amino acid uptake in contrast to MSA where the reverse was true. However, despite the relatively higher sensitivity of sugar transport to insulin, supraphysiological levels (10(-3) units/ml) of this hormone were still required for significant stimulation. These results suggest a generally low insulin sensitivity in cultured chick myotubes relative to adult tissues. In contrast, the effects of MSA are consistent with a possible role of this or similar factors in regulating growth and development of embryonic muscle.