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PubMed2000

Assessment of genotype and molecular evolution of hepatitis C virus in formalin-fixed paraffin-embedded liver tissue from patients with chronic hepatitis C virus infection.

Drawbacks of hepatitis C virus (HCV) RNA detection in paraffin-embedded liver tissue have satisfactorily been solved by RT-PCR amplification of the 5'non-coding region (5'NCR). However, detection of this highly conserved region does not provide information on epidemiological or pathogenetic aspects of HCV infection. This study explores whether other functionally important genetic regions of HCV, such as the hypervariable region 1 (HVR-1) and the interferon sensitivity-determining region (ISDR), can be retrieved from paraffin-embedded liver specimens by RT-PCR, and whether the amplified material is suitable for further molecular analyses. RT-PCR amplification of 5'NCR, HVR-1, and ISDR was assessed in RNA extracted from 50 formalin-fixed, paraffin-embedded liver specimens, including 23 needle liver biopsies (11 from patients with non-A, non-B chronic hepatitis diagnosed between 1971 and 1985, 8 from subjects with normal liver histology and 4 from sequential biopsies from a patient with HCV recurrence after liver transplantation), and 27 liver explants from patients undergoing transplantation between 1988 and 1996 (16 with HCV-related cirrhosis and 11 with other disorders). The 5'NCR was successfully amplified in 8 of 11 (73%) non-A, non-B chronic hepatitis biopsies and in all of the specimens from patients with serological documentation of HCV infection. There were no false-positive results. HCV genotype was identified by RFLP analysis of the 5'NCR in the 13 cases analyzed. HVR-1 and ISDR were amplified in 24 of 28 (86%) samples, which were positive for the 5'NCR. Efficient amplification was inversely related to the time of storage. The evolutionary changes of HVR-1 and ISDR were successfully analyzed by direct sequencing of amplificates from the explanted liver and from the sequential liver biopsies in a patient with HCV infection recurrence after transplantation. These observations indicate that paraffin-embedded liver tissue, even when stored for more than 20 years, is appropriate for advanced studies on the molecular biology of HCV.

Amino Acid Sequence↗
PubMed2001

Sequence analysis of feline caliciviruses isolated from the oral cavity of clinically normal domestic cats (Felis catus) in Florida.

Four isolates (7.3 per cent) of feline calicivirus (FCV), from oropharyngeal swabs taken from 55 unvaccinated apparently healthy cats, were identified by electron microscopy and reverse transcription-polymerase chain reaction (RT-PCR). A 671-bp fragment, comprising part of region B, all of regions C, D, and E and part of region F of the FCV capsid protein gene, was amplified from each isolate by RT-PCR and cloned for sequence analysis. Amino acid sequence comparison of these regions revealed significant sequence divergence from the F9 vaccine strain within regions C, E, and F. The hypervariable region E of the four Florida isolates and the NADC isolate contained three fewer amino acids than the commonly used F9 vaccine strain. This work provides support to the idea that currently circulating FCV strains may differ substantially from presently used vaccine strains.

Amino Acid Sequence↗
PubMed1981

Sequences of the joining region genes for immunoglobulin heavy chains and their role in generation of antibody diversity.

To assess the contribution to immunoglobulin heavy chain diversity made by recombination between variable region (VH) genes and joining region (JH) genes, we have determined the sequence of about 2000 nucleotides spanning the rearranged JH gene cluster associated with the VH gene expressed in plasmacytoma HPC76. The active VH76 gene has recombined with the second germ-line JH gene. The region we have studied contains two other JH genes, designated JH3 and JH4. No other JH gene was found within the region 1000 nucleotides downstream from JH4. Between JH3 and JH4 there is a pseudo-JH sequence with substantial homology to the authentic JH genes. The four JH genes whose sequences now are known can account for all known JH amino acid sequences. The JH genes are more divergent than the Jk genes and vary in length, encoding either 15 or 17 amino acid residues. Because JH regions comprise part of the third hypervariable region (HV3), combinatorial VH--JH joining substantially augments VH diversity. Moreover, a VH gene can recombine with each JH gene at several positions, and either one or two germ-line JH codons can be excised. This JH truncation markedly reduces the length of HV3 and hence must alter antigen-binding specificity. We have also determined the sequence of the JH4 region in two different gamma 2a mRNAs and have found that each has suffered a point mutation (aspartate to asparagine) which would alter the charge of the antigen-binding site.

Amino Acid Sequence↗
PubMed1986

Diversity and structure of human T-cell receptor beta-chain variable region genes.

The nucleotide sequences of 27 T-cell receptor beta cDNA clones isolated from a human peripheral lymphocyte library were determined and compared to five additional published sequences. These cDNA clones represent 22 distinct T-cell receptor beta-chain variable region (V beta) gene segment sequences, which fall into 15 different V beta gene subfamilies, each containing six or fewer members. From this analysis, we estimate that the repertoire of expressed human V beta genes is less than 59, apparently much smaller than the immunoglobulin heavy chain and light chain variable region (VH and VL) repertoires. Variability plots comparing these human V beta regions with each other and with published mouse V beta regions provide evidence for only four hypervariable regions homologous to those seen in comparisons of immunoglobulin V regions. Somatic hypermutation appears to be used infrequently, if at all, in these V beta genes.

Amino Acid Sequence↗
PubMed1992

Molecular mimicry of hepatitis B surface antigen by an anti-idiotype-derived synthetic peptide.

Monoclonal antibody 2F10 is an "internal-image" anti-idiotype (anti-id) antibody capable of mimicking the group-specific "a" determinant of human hepatitis B surface antigen (HBsAg). By mRNA sequencing and computer-assisted molecular modeling of monoclonal antibody 2F10, we identified a 15-amino acid region of the heavy-chain hypervariable region that has partial residue homology with sequences of the "a" determinant epitopes of HBsAg. We have established that a linear 15-mer peptide from a contiguous region on the anti-id antibody can (i) generate anti-HBsAg-specific antibodies when injected into mice, (ii) prime murine lymph node cells for in vitro HBsAg-specific T-cell proliferative responses, and (iii) stimulate in vitro human CD4+ T cells that were primed in vivo to HBsAg by natural infection with hepatitis B virus or vaccination with a commercially available HBsAg vaccine. Significantly, this peptide could also stimulate CD4+ T cells of human hepatitis B virus carriers. We conclude that a 15-mer peptide derived from the anti-id sequence can duplicate the B- and T-cell stimulatory activity of the intact anti-id antibody and the antigen that is mimicked, HBsAg.

Amino Acid Sequence↗
PubMed2004

A combination of mutations in the S1 part of the spike glycoprotein gene of coronavirus MHV-A59 abolishes demyelination.

The A59 strain of coronavirus, mouse hepatitis virus (MHV), produces acute hepatitis, meningoencephalitis, and chronic demyelination. The authors have previously shown that the spike (S) glycoprotein gene of MHV contains determinants of virulence, hepatitis, and demyelination. They then identified viruses containing mutations in the S gene that exhibit alterations in viral pathogenesis. In the present study, the authors produced new recombinant viruses with each one of these S gene mutations by site-directed mutagenesis and targeted recombination and studied the effect of each individual mutation on the pathogenesis of the virus. They identified a combination of mutations in the S1 gene (I375M and L652I) that abolishes demyelination. Individual mutation and other combinations of mutations in the S gene only interfere with virulence and hepatitis and only reduce demyelination (I375M), but do not abolish demyelination completely. Thus, demyelination determinants exist within genomic regions on both sides of the hypervariable region, downstream from the receptor-binding domain in the S1 part of the MHV spike glycoprotein gene. The structure and precise function of these regions awaits further investigation.

Animals↗
PubMed2004

Comprehensive detection and serotyping of human adenoviruses by PCR and sequencing.

Human adenoviruses are common pathogens associated with many diseases, including respiratory, gastrointestinal, and ocular infections. Because they are now being increasingly recognized as agents of life-threatening disseminated infection in immunocompromised patients, robust and sensitive laboratory detection methods are needed for their rapid diagnosis. We describe here a PCR assay using a single primer pair, targeting a region of the hexon gene containing hypervariable region 7, that can detect all known human adenovirus serotypes and allows for serotype determination through the analysis of the nucleotide sequence. This comprehensive assay has proven effective for diagnosing adenoviruses at the serotype level in a broad range of patient specimens, including conjunctival, nasopharyngeal, stool, blood, and urine specimens.

Adenoviruses, Human↗
PubMed1999

Evidence of avian leukosis virus subgroup E and endogenous avian virus in measles and mumps vaccines derived from chicken cells: investigation of transmission to vaccine recipients.

Reverse transcriptase (RT) activity has been detected recently in all chicken cell-derived measles and mumps vaccines. A study of a vaccine manufactured in Europe indicated that the RT is associated with particles containing endogenous avian retrovirus (EAV-0) RNA and originates from the chicken embryonic fibroblasts (CEF) used as a substrate for propagation of the vaccine. We investigated the origin of RT in measles and mumps vaccines from a U.S. manufacturer and confirm the presence of RT and EAV RNA. Additionally, we provide new evidence for the presence of avian leukosis virus (ALV) in both CEF supernatants and vaccines. ALV pol sequences were first identified in particle-associated RNA by amplification with degenerate retroviral pol primers. ALV RNA sequences from both the gag and env regions were also detected. Analysis of hypervariable region 2 of env revealed a subgroup E sequence, an endogenous-type ALV. Both CEF- and vaccine-derived RT activity could be blocked by antibodies to ALV RT. Release of ALV-like virus particles from uninoculated CEF was also documented by electron microscopy. Nonetheless, infectivity studies on susceptible 15B1 chicken cells gave no evidence of infectious ALV, which is consistent with the phenotypes of the ev loci identified in the CEF. PCR analysis of ALV and EAV proviral sequences in peripheral blood mononuclear cells from 33 children after measles and mumps vaccination yielded negative results. Our data indicate that the sources of RT activity in all RT-positive measles and mumps vaccines may not be similar and depend on the particular endogenous retroviral loci present in the chicken cell substrate used. The present data do not support transmission of either ALV or EAV to recipients of the U.S.-made vaccine and provide reassurance for current immunization policies.

Animals↗
PubMed2000

Application of the partial 16S rDNA sequence as an index for rapid identification of species in the genus Bacillus.

A comparison of 16S rRNA gene (rDNA) sequences was made among type strains of 69 Bacillus species approved in the International Journal of Systematic Bacteriology (IJSB) until 1998. The results suggested that 5' end region (approx. 275 bp) was the hypervariant region (HV region) in the gene and was highly specific for each type strain. Furthermore, a sequence analysis of the HV region of Bacillus strains revealed that this region was highly conserved within the species. These results indicate that the HV region is a useful index for the identification or grouping of Bacillus species.

Journal Article↗
PubMedDate not supplied

Molecular epidemiology of infectious bronchitis virus isolates from China and Southeast Asia.

In order to trace the origin and evolution of avian infectious bronchitis virus (IBV) isolates in China and Southeast Asia, genomic sequencing was used for molecular characterization of 24 IBV isolates and two reference strains in comparison with the published sequences. The 5' region of the S1 genes, containing hypervariable regions I and II, and 3' region of the nucleocapsid genes, containing cytotoxic T lymphocyte epitopes, were used to construct phylogenetic trees for analysis. The results showed that the 24 isolates could be divided into three distinct groups, that is, American, Asian, and European. Some isolates formed a distinct Asian phylogenetic group, suggesting that IBV has existed for some time in Asia. Our results also showed that in vivo recombination of IBV may have occurred at a rather high frequency, contributing to the diversity of these IBV isolates. Importantly, recombination events have probably occurred between vaccine strains and field strains in the natural condition.

Animals↗
PubMed1989

Structure and expression of the rice glutelin multigene family.

A near full-length cDNA and three genomic clones for rice (Oryza sativa L.) glutelin were isolated and studied. Based on nucleic acid sequence and Southern blot analyses, the three isolated glutelin genomic clones were representative members of three gene subfamilies each containing five to eight copies. A comparison of DNA sequences displayed by relevant regions of these genomic clones showed that two subfamilies, represented by clones Gt1 and Gt2, were closely related and evolved by more recent gene duplication events. The 5'-flanking and coding sequences of Gt1 and Gt2 displayed at least 87% homology. In contrast, Gt3 showed little or no homology in the 5'-flanking sequences upstream of the putative CAAT boxes and exhibited significant divergence in all other portions of the gene. Conserved sequences in the 5'-flanking regions of these genes were identified and discussed in light of their potential regulatory role. The derived primary sequences of all three glutelin genomic clones showed significant homology to the legume 11 S storage proteins indicating a common gene origin. A comparison of the derived glutelin primary sequences showed that mutations were clustered in three peptide regions. One peptide region corresponded to the highly mutable hypervariable region of legume 11 S storage proteins, a potential target area for protein modification. Expression studies indicated that glutelin mRNA transcripts are differentially accumulated during endosperm development. Promoters of Gt2 and Gt3 were functional as they direct transient expression of chloramphenicol acetyltransferase in cultured plant cells.

Amino Acid Sequence↗
PubMed1988

Linkage disequilibrium in the human insulin/insulin-like growth factor II region of human chromosome II.

Caucasian (N = 128) and Chinese (N = 84) subjects were typed for RFLPs in the insulin (INS)/insulin-like growth factor II (IGF2) region of chromosome 11. Both the analysis of extended haplotypes and the pairwise measures of linkage disequilibrium among the RFLPs indicate that there is extensive linkage disequilibrium in the INS/IGF2 region. The disequilibrium extends across the hypervariable region (HVR) located just 5' to the INS gene and encompasses a region of at least 40 kbp. Previous studies had suggested that linkage disequilibrium in the INS region was negligible and that this region may therefore contain a "recombinational hotspot" (Chakravarti et al. 1986). However, results of this and another recent study (Thompson et al. 1988) highlight the importance of the frequencies of associated alleles in the ability to detect linkage disequilibrium. Thus, the previous failure to detect disequilibrium in the INS region may have reflected a lack of power, rather than a true absence of disequilibrium in this region.

Alleles↗
PubMed1996

Genomic quasispecies associated with the initiation of infection and disease in ponies experimentally infected with equine infectious anemia virus.

Equine infectious anemia virus (EIAV) provides a uniquely dynamic system in which to study the mechanism and role of genomic variation in lentiviral persistence and pathogenesis. We have used a Shetland pony model of infection to investigate the association of specific long terminal repeat (LTR) and env gene genomic sequences with the initiation of infection and the onset of disease. We analyzed viral RNA isolated from a pathogenic stock of virus (EIAV PV) and from plasma taken during the first disease episode from two ponies infected with EIAV PV. Overall sequence variation within gp90 was low in EIAV PV and only slightly higher in plasma virus samples isolated from ponies during the first disease episode. However, a high proportion of mutations were localized to the principal neutralizing domain in EIAV PV and to the principal neutralizing domain and the gp90 hypervariable region in the two pony-derived samples. The rate of fixation of mutations was analyzed and determined to be approximately 4 x 10(-2) mutations per site per year. Sequence diversity within the U3 region of the LTR was extremely low, which suggested that the previously reported hypervariability of this region may be a consequence of selection for replication of EIAV in different host cells. The predominant EIAV PV env and LTR sequences were used to construct chimeric viruses so that the contribution of these sequences to viral pathogenicity could be examined. The chimeras replicated in cultured equine monocytes to the same extent as the parental nonpathogenic virus and did not cause disease in Shetland ponies by 120 days postinfection, suggesting that the EIAV genomic determinants of pathogenesis are complex.

Amino Acid Sequence↗
PubMed1992

A consensus repeat sequence from the human insulin gene linked polymorphic region adopts multiple quadriplex DNA structures in vitro.

A hypervariable region consisting of repeats of a 14 base pair (bp) consensus sequence ACAGGGGT(G/C)(T/C)GGGG is located 363 bp upstream of the human insulin gene. Different repeat numbers of this oligonucleotide give rise to a polymorphism, and so this region is commonly known as the insulin gene linked polymorphic region (ILPR). Here we present evidence, based on the mobility in non-denaturing polyacrylamide gels of two dissimilarly sized oligonucleotides containing the ILPR consensus sequence, that this sequence can adopt a number of quadriplex DNA structures in vitro.

Base Sequence↗
PubMed1996

Novel unconventional binding site in the variable region of immunoglobulins.

The variable immunoglobulin (Ig) domains contain hypervariable regions that are involved in the formation of the antigen binding site. Besides the canonical antigen binding site, so-called unconventional sites also reside in the variable region that bind bacterial and viral proteins. Docking to these unconventional sites does not typically interfere with antigen binding, which suggests that these sites may be a part of the biological functions of Igs. Herein, a novel unconventional binding site is described. The site is detected with 8-azidopurine nucleotide photoaffinity probes that label antibodies efficiently and under mild conditions. Tryptic peptides were isolated from photolabeled monoclonal antibodies and aligned with the variable antibody domains of heavy and light chains. The structure of a variable Ig fragment was used to model the binding of the purine nucleotide to invariant residues in a hydrophobic pocket of the Ig molecule at a location distant from the antigen binding site. Monoclonal and polyclonal antibodies were biotinylated with the photoaffinity linker and used in fluorescence-activated cell sorter and ELISA analyses. The data support the utility of this site for tethering diagnostic and therapeutic agents to the variable Ig fragment region without impairing the structural and functional integrity of antibodies.

Affinity Labels↗
PubMed2005

Molecular investigation of interspousal transmission of hepatitis C virus in two Japanese patients who acquired acute hepatitis C after 40 or 42 years of marriage.

A 65-year-old woman (C1I) and a 65-year-old man (C2I) contracted acute hepatitis C 40 or 42 years after marriage, respectively, in Japan. They had no discernible risk factors for acquiring hepatitis C virus (HCV) infection, except that they had monogamous sexual relationships with their spouses (C1S [66-year-old] with hepatocellular carcinoma and C2S [64-year-old] with liver cirrhosis, respectively) who were infected with HCV of the same genotype (1b) and had a high-titer HCV RNA in the serum (bDNA probe assay, 17 Meq/ml [C1S] and 15 Meq/ml [C2S]). The HCV isolates from Patients C1I and C1S and those from Patients C2I and C2S shared identity of 99.9% and 99.1%, respectively, in the 1,087-nucleotide (nt) sequence of the NS5B region, although these four isolates were only 91.7%-96.2% identical to the 94 reported genotype 1b isolates including those from Japanese patients. To confirm the high degree of genetic relatedness among ten HCV clones from each spouse within each pair of spouses, the E1 and E2 junctional region sequence (268 or 271 nt) including hypervariable region 1 (HVR-1) was analyzed. There was a close relationship between clones obtained from each spouse within each couple. Regarding the HVR-1 amino acid sequence, nine of the ten C1I clones were 100% identical with six of the ten C1S clones, and one each of the C2I and C2S clones differed by only one amino acid residue. This study indicates that two Japanese patients with acute hepatitis C had acquired HCV infection most probably by interspousal sexual transmission during a long-lasting marriage.

Acute Disease↗
PubMed2001

Both morphological and molecular characters support speciation of South American siskins by sexual selection.

South American siskin radiation was studied by both mitochondrial cytochrome b (mt cyt b) DNA sequencing and homologous phenotypic characters; the latter were coded separately according to sex. Mixed phenetic and molecular (total evidence) dendrograms were constructed and the corresponding analyses suggest that speciation started in the South American siskin group with a north to south separation (Carduelis notata/C. barbata) along the Andean spine. A second split may have taken place around the Peruvian Andean mountains, corresponding to the present distribution pattern of C. olivacea. The most recent speciation events seem to have occurred in three sister species pairs: (i) C. xanthogastra/C. atrata, (ii) C. magellanica/C. yarrellii, (iii) C. cucullata/C. crassirostris. Accumulation of consistent characters in both morphological and molecular data at the basal nodes of the dendrograms indicate that speciation events occurred within a short period of time. Our data also suggest that speciation probably occurred by sexual selection through female mating choice in this radiation. Additionally, studies of variable amino acid residues in the mt cyt b molecule show that the three variable amino acids found are placed in the mitochondrial transmembrane region, which is also part of the hypervariable region in mammals. Each of the three amino acid changes occur in each of the three postulated evolutionary groups.

Amino Acid Sequence↗
PubMed1999

Mutations in mitochondrial control region DNA in gastric tumours of Japanese patients.

The non-coding control region of mitochondrial DNA (mtDNA), containing the hypervariable regions HV1 and HV2 and the D-loop region, was screened for mutations in 45 gastric tumours (15 tumours each of adenoma, differentiated adenocarcinoma and undifferentiated carcinoma). We found mutations in two of the 45 tumours (4%); a 1 bp A deletion at nucleotide position 248 in a differentiated adenocarcinoma and a G to A transition at nucleotide position 16,129 in an adenoma. We also observed 10 polymorphisms, four of which were not previously recorded. Both mtDNA mutations were present in replication error negative (RER-) tumours. Short mono- or dinucleotide repeats in the control region, such as (C)7, (A)5 or (CA)5, were not altered regardless of nuclear genetic instability. In summary, mtDNA is mutated in a subset of benign and malignant gastric tumours, but, disruption of the mtDNA repair system appears not to be significantly involved in gastric tumours of Japanese patients.

DNA Repair↗