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PubMed1999

Sequence variation within the capsid protein of Australian isolates of feline calicivirus.

The capsid protein of Australian feline calicivirus (FCV) isolates is demonstrably different from the prototype strain F9. Five Australian isolates of FCV, dating from 1970 to 1989, were analysed by western blotting and immunoprecipitation. Varying reactivity to a panel of F9 specific monoclonal antibodies (MAbs) was observed. DNA sequencing of RT-PCR generated clones supported the observation of variation between capsid proteins. Predicted amino acid sequences varied by 11 to 17.5% across the whole capsid when compared to the published F9 sequence. Differences in amino acid sequence were most apparent in previously described hypervariable regions (C and E). Within hypervariable region E differences of 22 to 34% were observed compared to F9. The observed lack of reactivity to F9 MAbs correlated with amino acid changes within previously characterized binding sites within region E.

Amino Acid Sequence↗
PubMed1992

The susceptibility sequence to rheumatoid arthritis is a cross-reactive B cell epitope shared by the Escherichia coli heat shock protein dnaJ and the histocompatibility leukocyte antigen DRB10401 molecule.

Immunological responses to bacterial heat shock proteins have been implicated in the pathogenesis of arthritis in animals and humans. The predicted amino acid sequence of dnaJ, a heat shock protein from Escherichia coli, contains an 11-amino acid segment that is homologous to the third hypervariable region of the human histocompatibility antigen (HLA) DRB10401 (formerly known as HLA Dw4), the part of the molecule that carries susceptibility to rheumatoid arthritis. To test the biological significance of this finding, we expressed and purified recombinant dnaJ (rdnaJ), and determined its immunologic cross-reactivity with HLA DRB10401. A rabbit antipeptide antiserum raised against the sequence of the third hypervariable region of HLA DRB10401 specifically bound to 'dnaJ, thus confirming that a similar sequence is expressed on the bacterial protein. Of greater consequence, an antiserum to the 'dnaJ protein recognized not only a peptide from the third hypervariable region of HLA DRB10401, but also the intact HLA DRB10401 polypeptide. Furthermore, the antibody to 'dnaJ reacted with HLA DRB10401 homozygous B lymphoblasts, but not with HLA DRB11501, DRB10101, DRB10301, and DRB10701 (formerly known as HLA Dw2, DR 1, DR 3, and DR 7, in the same order) homozygous cells. These results demonstrate that exposure to a bacterial heat shock protein can elicit antibodies against the rheumatoid arthritis susceptibility sequence in the third hypervariable region of HLA DRB10401.

Amino Acid Sequence↗
PubMed1998

Small subunit ribosomal RNA genes of tabanids and hippoboscids (Diptera: Brachycera): evolutionary relationships and comparison with other Diptera.

The small subunit ribosomal RNA (SSU rRNA) genes of hippoboscid (Ornithoica vicina Walker) and tabanid (Chrysops niger Macquart) Diptera were sequenced to determine their phylogenetic position within the order and to determine whether or not extensive hypervariable regions in this gene are widespread in the Diptera. A parsimony analysis of an alignment containing 8 dipteran sequences produced a single most parsimonious tree that placed O. vicina as sister group to Drosophila melanogaster Meigen. The tabanid Chrysops niger was sister group to the asilomorphan taxa, and the sister group to the Brachycera was a Tipula sp. although this relationship was not supported by bootstrap analysis. The hippoboscid and tabanid sequences contain extensive hypervariable regions in the V2, V4, V6, and V7 regions as do other Diptera. When these regions of the alignment were excluded from the phylogenetic analysis, a single most parsimonious tree was found. This tree had an identical overall topology to the tree obtained from the total data set. The hypervariable regions in parts of the dipteran SSU rRNA genes were more extensive in the nematocerous dipteran sequences used in this study than in the other dipteran representatives; these hypervariable regions may be of more utility in inferring relationship among species and subspecies than at the suprageneric level.

Animals↗
PubMed2002

Genetic variability and stability of Anaplasma phagocytophila msp2 (p44).

Anaplasma (Ehrlichia) phagocytophila's major immunodominant surface protein antigen, Msp2 (P44, 44-kDa antigen), is encoded by a family of paralogous genes characterized by conserved sequences flanking a hypervariable region. The antigenic profiles of most strains of A. phagocytophila are different, and the differences are principally related to Msp2 expression. To date, multiple unique msp2 gene paralogs have been found in A. phagocytophila isolates, but the overall number in the genome of a single strain is not yet known. Changes in msp2 expression may be related to antigenic variability; thus, we examined the minimal complement of msp2 genes or pseudogenes in two strains of A. phagocytophila and the number of transcriptionally active msp2 gene paralogs during low-passage, steady-state, in vitro propagation. Of 15 BDS strain clones, 1 had a hypervariable region identical to the region in a clone obtained from a BDS strain genomic library previously prepared from organisms after only two horse passages. When 124 Webster strain clones were examined, 18 unique hypervariable regions were identified. Of 64 Webster strain cDNA clones, 56 (87.5%) were derived from a single gene, and transcripts from six additional msp2 genes were also identified. The sequences of several hypervariable regions that were > or = 97% similar to regions present in other strains were identified by performing a BLAST analysis of sequences deposited in the GenBank database. These findings suggest that antigenic variability results from transcription of one or a few of the multiple paralogs and not from genetic instability that results in random accumulated mutations, although the possibility that gene recombination plays a role cannot be eliminated. The predominant Msp2 pattern in vitro is determined by transcription from a single gene.

Anaplasma↗
PubMed1993

Hypervariable-control-region sequences reveal global population structuring in a long-distance migrant shorebird, the Dunlin (Calidris alpina).

Hypervariable segments of the control region of mtDNA as well as part of the cytochrome b gene of Dunlins were amplified with PCR and sequenced directly. The 910 base pairs (bp) obtained for each of 73 individuals complete another of the few sequencing studies that examine the global range of a vertebrate species. A total of 35 types of mtDNA were detected, 33 of which were defined by the hypervariable-control-region segments. Thirty of the latter were specific to populations of different geographic origin in the circumpolar breeding range of the species. The remaining three types indicate dispersal between populations in southern Norway and Siberia, but female-mediated flow of mtDNA apparently is too low to overcome the effects of high mutation rates of the control-region sequences, as well as population subdivision associated with historical range disjunctions. A genealogical tree relating the types grouped them into five populations: Alaska, West Coast of North America, Gulf of Mexico, western Europe, and the Taymyr Peninsula. The Dunlin is thus highly structured geographically, with measures of mutational divergence approaching 1.0 for fixation of alternative types in different populations. High diversity of types within populations as well as moderate long-term effective population sizes argue against severe population bottlenecks in promoting this differentiation. Instead, population fragmentation in Pleistocene refuges is the most plausible mechanism of mtDNA differentiation but at a much earlier time scale than suggested previously with morphometric data.

Animals↗
PubMedDate not supplied

Molecular characterization of seven field isolates of infectious bursal disease virus obtained from commercial broiler chickens.

Specific-pathogen-free sentinel birds were used as an initial biological system to isolate infectious bursal disease virus (IBDV) field isolates from commercial broiler farms exhibiting recurrent respiratory problems and poor performance. Reverse transcription (RT)-polymerase chain reaction (PCR) was used to amplify a 248-bp product encompassing the hypervariable region of the IBDV VP2 gene. Restriction fragment length polymorphism (RFLP) analysis of the RT-PCR products was performed with the restriction endonucleases DraI, SadI, TaqI, StyI, BstNI, and SspI. Two isolates (619 and 850) exhibited a RFLP pattern characteristic of Delaware variant E IBDV. Restriction enzyme digestion for four isolates (625, 849, 853, and 11,153) revealed unmatched RFLP patterns when compared with reference IBDV strains. Nucleotide and deduced amino acid sequence analyses of the VP2 hypervariable region for these six isolates revealed identity (96.3% up to 98%) with Delaware E variant IBDV strain. However, serine at position 254, which is characteristic of Delaware variant strains, was substituted by asparagine in these six isolates. The seventh IBDV isolate (9109) also exhibited a unique RFLP pattern, which included the SspI restriction site, which is characteristic of very virulent (vv) IBDV strains. Nucleotide and amino acid sequence analyses of the hypervariable region for this isolate revealed identity (90%) with the standard challenge strain. However, the leucine residue at position 294 was substituted by isoleucine. This substitution corresponds to one of the amino acids that are conserved in the vvIBDV strains. Antigenic index studies of the predicted amino acid sequence of the hypervariable region of VP2 from isolates 619, 625, 849, 850, 853, and 11,153 exhibited a profile almost identical to variant E, whereas the isolate 9109 exhibited a profile characteristic of standard IBDV strains.

Amino Acid Sequence↗
PubMed2000

High-affinity interaction between gram-negative flagellin and a cell surface polypeptide results in human monocyte activation.

Flagella from diverse gram-negative bacteria induce tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta) synthesis by human monocytes (F. Ciacci-Woolwine, P. F. McDermott, and S. B. Mizel, Infect. Immun. 67:5176-5185, 1999). In this study, we establish that purified flagellin (FliC or FljB), the major filament protein from Salmonella enterica serovar Enteritidis, S. enterica serovar Typhimurium, and Pseudomonas aeruginosa, is an extremely potent inducer of TNF-alpha production by human monocytes and THP-1 myelomonocytic cells. Fifty percent of maximal TNF-alpha production (EC(50)) was obtained with 1.5 x 10(-11) M flagellin (0.75 ng/ml). Mutagenesis studies revealed that the central hypervariable region of flagellin is essential for the TNF-alpha-inducing activity of the protein. Although less active than the wild-type protein, a Salmonella flagellin mutant composed of only the central hypervariable region retained substantial TNF-alpha-inducing activity at nanomolar concentrations. In contrast, the conserved amino- and carboxy-terminal regions are inactive. Mutational analysis of the hypervariable region revealed that it contains two equally active TNF-alpha-inducing domains. The ability of THP-1 cells to respond to purified flagellins is dramatically reduced by mild trypsin treatment of the cells. Taken together, our results demonstrate that the cytokine-inducing activity of flagellins from gram-negative bacteria results from the interaction of these proteins with high-affinity cell surface polypeptide receptors on monocytes.

Cells, Cultured↗
PubMed1991

DNA typing for class II HLA antigens with allele-specific or group-specific amplification. V. Typing for subsets of HLA-DR1 and DR'Br'.

Three DRB1 alleles of the DR1 group including DRB1*0101, DRB1*0102, and DRB1*0103 are currently recognized. The first two of these are defined as HLA-DR1 by serologic typing and as either Dw1 or Dw20 by typing with T cells. DRB1*0103, previously called DR'Br' or DR'BON', is not detectable by serology. Little information exists about the population frequencies of DRB1*0102 and DRB1*0103. In the present study we have used the polymerase chain reaction (PCR) and allele-specific oligonucleotide probes to determine these alleles. To avoid cross-hybridization with other DRB genes having the same DNA sequences as those of the region to be analyzed, we performed group-specific PCR to amplify only DR1 DRB1 genes. This was accomplished using a 21-nucleotide-long primer, homologous to the first hypervariable region common to the DR1 DRB1 genes, and which under appropriate conditions amplified only DRB1 genes of the DR1 group. Five oligonucleotide probes, one matching the second hypervariable region, two spanning the third hypervariable region, and two covering codons 82 through 89, were used to determine the three alleles. DRB1*0101 (Dw1) was found to be the major type of DR1 in North American Caucasians. In North American black and Brazilian mestizo populations DRB1*0102 (Dw20) was more prevalent. DRB1*0103 (DR'Br') was detected in only six individuals in the present study.

Alleles↗
PubMed1988

Genetic susceptibility to rheumatoid arthritis and human leukocyte antigen class II polymorphism. The role of shared conformational determinants.

Genetic susceptibility for rheumatoid arthritis has been associated with both human leukocyte antigen (HLA)-DR4 and HLA-DR1, depending on the ethnic origin of the population under study. Furthermore, various subtypes of DR4 exist, only some of which appear to be associated with rheumatoid arthritis. DNA sequence analysis of the DR-beta chain genes encoding the DR4 subtypes as well as DR1 has led to the observation that similar third hypervariable region sequences are found on rheumatoid arthritis-associated DR-beta chain alleles. The data indicate that third hypervariable region sequence polymorphisms strongly influence T cell recognition as well as risk for rheumatoid arthritis. This has led to the hypothesis that genetic susceptibility for rheumatoid arthritis is due to a group of similar third hypervariable region sequences that may share conformational determinants important in antigen presentation and/or immune regulation.

Alleles↗
PubMed1990

Structure of the Bordetella pertussis gene coding for the serotype 3 fimbrial subunit.

Bordetella pertussis strains contain at least three distinct genes coding for fimbrial subunits, designated fim2, fim3, and fimX. The sequences of the fim2 and fimX genes have been published. Here we present the sequence of the fim3 gene. Proximal and distal to the fim3 gene, regions were observed that could function as rho-independent terminators, suggesting that the gene is not part of a larger operon. Comparison of the putative promoter regions of the fim2 and fim3 genes revealed a conserved region containing a stretch of approximately 13 C's. This region may be involved in fimbrial phase variation. A comparison of the deduced amino acid sequences of the three fimbrial subunits revealed conserved, variable, and hypervariable regions. The hypervariable regions coincided with predicted antigenic determinants. Peptides derived from the conserved regions may be incorporated into a future pertussis vaccine to induce antibodies which confer protection against strains producing different fimbrial serotypes.

Amino Acid Sequence↗
PubMed2002

Phylogeography of the African buffalo based on mitochondrial and Y-chromosomal loci: Pleistocene origin and population expansion of the Cape buffalo subspecies.

Population genetics and phylogeography of the African buffalo (Syncerus caffer) are inferred from genetic diversity at mitochondrial D-loop hypervariable region I sequences and a Y-chromosomal microsatellite. Three buffalo subspecies from different parts of Africa are included. Nucleotide diversity of the subspecies Cape buffalo at hypervariable region I is high, with little differentiation between populations. A mutation rate of 13-18% substitutions/million years is estimated for hypervariable region I. The nucleotide diversity indicates an estimated female effective population size of 17 000-32 000 individuals. Both mitochondrial and Y-chromosomal diversity are considerably higher in buffalo from central and southwestern Africa than in Cape buffalo, for which several explanations are hypothesized. There are several indications that there was a late middle to late Pleistocene population expansion in Cape buffalo. This also seems to be the period in which Cape buffalo evolved as a separate subspecies, according to the net sequence divergence with the other subspecies. These two observations are in agreement with the hypothesis of a rapid evolution of Cape buffalo based on fossil data. Additionally, there appears to have been a population expansion from eastern to southern Africa, which may be related to vegetation changes. However, as alternative explanations are also possible, further analyses with autosomal loci are needed.

Africa↗
PubMed1999

Emergence of Anaplasma marginale antigenic variants during persistent rickettsemia.

Anaplasma marginale is an ehrlichial pathogen of cattle, in the order Rickettsiales, that establishes persistent cyclic rickettsemia in the infected host. Within each rickettsemic cycle, A. marginale expressing antigenically variant major surface protein 2 (MSP2) emerge. By cloning 17 full-length msp2 transcripts expressed during cyclic rickettsemia, we determined that emergent variants have a single, central hypervariable region encoding variant B-cell epitopes. The N- and C-terminal regions are highly conserved among the expressed A. marginale variants, and similar sequences define the MSP2 homologues in the agent of human granulocytic ehrlichiosis (HGE). This is in contrast to the MSP2 homologues in ehrlichial genogroup I pathogens, Ehrlichia chaffeensis, Ehrlichia canis, and Cowdria ruminantium, that have multiple hypervariable regions. By defining the variable and conserved regions, we were able to show that the single hypervariable region of A. marginale MSP2 encodes epitopes that are immunogenic and induce variant-specific antibody responses during persistent infection. These findings demonstrate that the MSP2 structural variants that emerge during each cycle of persistent rickettsemia are true antigenic variants, consistent with MSP2 antigenic variation as a mechanism of A. marginale persistence.

Amino Acid Sequence↗
PubMed1997

Striking sequence similarity in inter- and intra-specific comparisons of class I SLG alleles from Brassica oleracea and Brassica campestris: implications for the evolution and recognition mechanism.

Self-incompatibility in Brassica is controlled by a single multi-allelic locus (S locus), which contains at least two highly polymorphic genes expressed in the stigma: an S glycoprotein gene (SLG) and an S receptor kinase gene (SRK). The putative ligand-binding domain of SRK exhibits high homology to the secretory protein SLG, and it is believed that SLG and SRK form an active receptor kinase complex with a self-pollen ligand, which leads to the rejection of self-pollen. Here, we report 31 novel SLG sequences of Brassica oleracea and Brassica campestris. Sequence comparisons of a large number of SLG alleles and SLG-related genes revealed the following points. (i) The striking sequence similarity observed in an inter-specific comparison (95.6% identity between SLG14 of B. oleracea and SLG25 of B. campestris in deduced amino acid sequence) suggests that SLG diversification predates speciation. (ii) A perfect match of the sequences in hypervariable regions, which are thought to determine S specificity in an intra-specific comparison (SLG8 and SLG46 of B. campestris) and the observation that the hypervariable regions of SLG and SRK of the same S haplotype were not necessarily highly similar suggests that SLG and SRK bind different sites of the pollen ligand and that they together determine S specificity. (iii) Comparison of the hypervariable regions of SLG alleles suggests that intragenic recombination, together with point mutations, has contributed to the generation of the high level of sequence variation in SLG alleles. Models for the evolution of SLG/SRK are presented.

Alleles↗
PubMed1980

Nucleotide sequence of immunoglobulin heavy chain joining segments between translocated VH and mu constant regions genes.

To investigate the mechanism of recombination of immunoglobulin heavy chain variable and constant region genes, we have determined the nucleotide sequence of a large portion of the recombination region between an active C mu gene and its associated VH gene, isolated from an IgM-secreting mouse plasmacytoma, HPC76. By comparison with the sequence of the mu mRNA, we determined the exact boundaries of the intervening sequence between the VH76 and C mu genes. The rearranged VH76 gene encodes up to amino acid 116 without interruption, the 3' 39 nucleotides (the JH76 region) being derived from an embryonic JH segment (JH315) whose sequence was recently determined [Early, P., Huang, H., Davis, M., Calame, K. & Hood, L. (1980) Cell 195, 981-992]. The active JH76 does not use the first two codons of the embryonic JH315 from which it is derived. This indicates that V-J recombination is important in generating diversity within the third hypervariable region of heavy chains. We have identified another JH segment (JHA4), located 336 nucleotides 3' to the rearranged JH76 segment. This JH segment is expressed in the heavy chains of anti-levan myeloma proteins, which have truncated third hypervariable regions. We propose that the nucleotide sequence 5' to JHA4 is important for generating V region genes with shortened third hypervariable regions.

Animals↗
PubMed1995

Hepatitis C viral quasispecies in hepatitis C virus carriers with normal liver enzymes and patients with type C chronic liver disease.

Hepatitis C virus (HCV) has been reported to conform to a quasispecies nature, which is most evident in hypervariable regions of the putative envelope 2 domain. The aim of this study was to determine the relationship between the nucleotide complexity and diversity of hypervariable region 1 and various stages of the carrier states. The subjects studied were 20 HCV carriers with normal alanine aminotransferase (ALT) levels, 50 patients with chronic hepatitis who showed elevated ALT levels, 22 with cirrhosis, and 24 with hepatocellular carcinoma. The quasispecies complexity was analyzed by means of polymerase chain reaction-mediated single strand conformation polymorphism (PCR-SSCP). The value of nucleotide diversity was calculated by PCR cloning and sequencing. The number of SSCP bands ranged from 1 to 7, with no significant differences in the mean numbers among the stages of HCV infection. There was no correlation between the amounts of serum HCV RNA and the numbers of SSCP bands. No significant difference was found in the values of nucleotide diversity between carriers with normal ALT levels (mean, 6.6 x 10(-2) per site) and patients with chronic hepatitis (7.7 x 10(-2). These findings suggest that the quasispecies complexity of hypervariable region 1 is independent of the stage of chronic HCV infection.

Adolescent↗
PubMed1993

Sequence variation in the large envelope glycoprotein (E2/NS1) of hepatitis C virus during chronic infection.

Sequence variation in the putative large surface glycoprotein (E2/NS1) of hepatitis C virus (HCV) was analyzed over 18 months in 4 patients with chronic relapsing non-A, non-B liver disease, of whom 2 were treated with lymphoblastoid interferon-alpha. Sequence analysis showed marked heterogeneity between isolates from different patients at the hypervariable region mapping to the amino-terminus of E2/NS1 (amino acid position, 386-411) and the extended sequence analyzed again confirmed the existence of two types, HCV1 and HCV2. Sequential nucleotide analysis of the hypervariable region over 1 year for each patient showed 0-9 amino acid changes, more common in the untreated than in the interferon-alpha-treated patients (3 and 9 vs. 1 and 0). Peaks of raised serum transaminases in individual patients showed no consistent association with mutations within this region. However, results do not exclude the possibility that viral persistence may be due to the emergence of "escape mutants" in the hypervariable region.

Adult↗
PubMed1997

Prediction of effect of interferon on chronic hepatitis C.

Clinical, pathological, and virological analysis including hypervariable region-1 of hepatitis C virus (HCV) was performed to predict the effect of interferon (IFN) on 41 patients with chronic hepatitis type C. The low virus load, low frequency of the mutation in the hypervariable region-1 as the change of amino acid and high level of serum aminotransferase make one estimate the good effect of IFN on patients with HCV. Mutation in the hypervariable region-1 of HCV measured by fast assay fluorescence single-stranded conformational polymorphism was more frequent in nonresponders to IFN than responders. The most frequently mutated position was amino acid number 406. This indicates that the specific mutation site might affect the response of IFN.

Amino Acid Sequence↗
PubMed2000

Cell specificity of the transcription-factor repertoire used by a lentivirus: motifs important for expression of equine infectious anemia virus in nonmonocytic cells.

The equine infectious anemia virus (EIAV) long-terminal repeat (LTR) has been identified as highly variable, both in infected horses and in cell culture. This nucleotide hypervariation is localized to the LTR enhancer region. The EIAV LTR has been implicated in controlling both the cell tropism and virulence of the virus and it is postulated that the enhancer-region hypervariation may be responsible for the LTR effects. Our previous studies have demonstrated that the presence of DNA motifs bound by the ets transcription-factor family member PU.1 are critically important for EIAV expression in equine macrophages. Here we identify and characterize the EIAV LTR enhancer motifs PEA-2, Lvb, Oct, and CRE, that bind to fibroblast nuclear extracts. Three of these four motifs, PEA-2, Oct, and CRE, were determined to be important for expression of the LTR in a fibroblast cell line that supports productive infection of EIAV. These motifs that are important for expression of the LTR in fibroblasts were found to be interdigitated between the PU.1 sites. We hypothesize that the combination of motif interdigitation and cell-specific usage of these motifs may be responsible for the observed EIAV LTR enhancer-region hypervariation.

Animals↗