ABSTRACT
Parvovirus B19 (B19V) is a minute ssDNA virus associated with a wide range of diseases from childhood erythema to fetal death. After primary infection, the viral genomes persist lifelong in solid tissues of most types. Quantification of the viral DNA is important in the timing of primary infection, assessment of tissue persistence and screening of blood donor plasma. In this study, we present a new PCR assay for detection and quantification as well as for differentiation of all three B19V genotypes. A new B19V qPCR was designed to target a 154-bp region of the NS1 area. Serum, plasma and solid tissue samples were suitable for testing in the assay. The WHO International Reference Panel for Parvovirus B19 Genotypes was utilized to validate the assay for detection of different genotypes of B19V in clinical material. Each panel member yielded, by the new qPCR, a quantity similar to the one reported by National Institute for Biological Standards and Control (NIBSC). The qPCR was specific for B19V and amplified and quantified all three genotypes with detection sensitivities of ≤10 copies/reaction. The differentiation of B19V genotypes was performed by Sanger sequencing of the amplified products.
Subject(s)
DNA, Viral/genetics , Erythema Infectiosum/diagnosis , Parvovirus B19, Human/classification , Real-Time Polymerase Chain Reaction/methods , Viral Nonstructural Proteins/genetics , DNA, Viral/analysis , Erythema Infectiosum/virology , Humans , Palatine Tonsil/virology , Parvovirus B19, Human/genetics , Reproducibility of Results , Sensitivity and Specificity , Tonsillitis/virologyABSTRACT
In man, the genes encoding the complement component C4 (C4A, C4B) of the immune system and the steroid 21-hydroxylase enzyme (CYP21A, CYP21B) of adrenal steroid biosynthesis are located in the major histocompatibility complex (MHC). Frequent gene deletions and duplications have been described in the C4 and CYP21 genes, particularly in patients with autoimmune diseases and congenital adrenal hyperplasia. Here we report the determination of deletion sizes in 11 chromosomes with six different deletions. The deletions spanned the C4A+CYP21A, C4B+CYP21A, and C4B+CYP21B gene pairs as determined by standard Southern blot analysis. The deletion size fell within the range of 30-38 kb in all the chromosomes, as determined by pulsed-field gel electrophoresis. Because the deletion sizes in most other gene clusters are more heterogeneous, the results suggest the involvement of a specific mechanism in the generation of C4+CYP21 deletions.
Subject(s)
Chromosome Deletion , Complement C4/genetics , Major Histocompatibility Complex , Steroid 21-Hydroxylase/genetics , Steroid Hydroxylases/genetics , DNA Probes , DNA Restriction Enzymes , Electrophoresis , Haplotypes , Humans , Hybridization, Genetic , PhenotypeABSTRACT
Fab fragments of a murine C3d-specific monoclonal antibody, clone 4C2, strongly inhibited the binding of H, and up to about 80% that of B, to C3b on sheep erythrocytes, as shown by using radiolabelled purified proteins. The inhibition was also detected in the fluid phase. Conversion of C3 by preformed solid phase alternative pathway C3 convertase was not affected by 4C2. Also, it did not affect the decay rate of the convertase, but reduced the decay-accelerating effect of H on it. The results suggest that the binding sites of B and H on C3b are close to each other, and their competition for binding to C3b is focused around the C3d region. In addition, 4C2 partially blocked noncovalent binding of C3b to human erythrocytes, which suggests that CR1 also binds close to the H- and B-binding region in C3b.
Subject(s)
Complement C3b Inactivator Proteins/metabolism , Complement C3b/metabolism , Complement Factor B/metabolism , Enzyme Precursors/metabolism , Receptors, Complement/metabolism , Antibodies, Monoclonal , Binding Sites , Complement C3b/immunology , Complement Factor H , Enzyme-Linked Immunosorbent Assay , Erythrocytes/immunology , Humans , Kinetics , Protein Binding , Receptors, Complement 3bABSTRACT
Incubation of Acanthamoeba castellanii (Neff strain) with FITC-ConA (15 micrograms/ml) resulted in the appearance of patches of fluorescence on the amoebae within 2 min of incubation. These patches disappeared following treatment of the amoebae with alpha-MeMan. Pretreatment of the amoebae with colchicine or cytochalasin B or with colchicine and cytochalasin B in combination did not significantly alter the distribution pattern of fluorescence in the amoebae. 2,4-Dinitrophenol and incubation at 4 degrees C on the other hand decreased the degree of patching of the amoebae. Pretreatment with 2,4-dinitrophenol and incubation at 4 degrees C also decreased the ConA-mediated agglutination of the amoebae. No effect on the ConA-mediated agglutination was, however, observed following pretreatment of the amoebae with colchicine and cytochalasin B neither alone nor in combination. Our results indicate that ConA-mediated agglutination and long-range ConA-receptor mobility in the Acanthamoeba are not under the control of structures sensitive to cytochalasin B or colchicine.
