Evaluation of variable number tandem repeat (VNTR) loci in molecular typing of Mycobacterium bovis isolates from Ireland.

Various sets of short tandem repeats such as the exact tandem repeats (ETRs), mycobacterial interspersed repetitive units (MIRUs) and variable number tandem repeat (VNTR) loci, have recently been described as effective tools in strain typing M. tuberculosis complex isolates, representative of global diversity. This study extends our previous study, evaluating the discrimination of a further 17 MIRU_VNTR loci individually and comparing the resolution of published VNTR sets and spoligotyping using a panel of 47 local M. bovis field isolates, including known epidemiologically linked isolates and 9 M. tuberculosis complex reference isolates. Individual loci differed greatly in their discrimination. The discriminatory capacity of novel combinations of the most discriminating VNTR loci was also assessed. In the panel of 47 M. bovis isolates, 17 unique profiles were resolved using VNTR set 1, whilst the MIRUs and ETRs resolved the panel into 11 and 6 profiles, respectively. A novel combination of 10 highly discriminatory VNTRs was determined, which resolved 30 unique profiles. The configuration of a multi-locus VNTR-based assay and its ability to provide a flexible, convenient and high-resolution genotyping method is discussed. We suggest a panel of VNTR markers which may be widely suitable for molecular epidemiological studies of M. bovis. However, the number and combination of informative VNTR markers selected needs to be determined empirically with reference to locally prevalent strains and will depend on the epidemiological study requirements.

Molecular basis for estrogen receptor alpha deficiency in BRCA1-linked breast cancer.

BACKGROUND: BRCA1-mutant breast tumors are typically estrogen receptor alpha (ER alpha) negative, whereas most sporadic tumors express wild-type BRCA1 and are ER alpha positive. We examined a possible mechanism for the observed ER alpha-negative phenotype of BRCA1-mutant tumors. METHODS: We used a breast cancer disease-specific microarray to identify transcripts that were differentially expressed between paraffin-embedded samples of 17 BRCA1-mutant and 14 sporadic breast tumors. We measured the mRNA levels of estrogen receptor 1 (ESR1) (the gene encoding ER alpha), which was differentially expressed in the tumor samples, by quantitative polymerase chain reaction. Regulation of ESR1 mRNA and ER alpha protein expression was assessed in human breast cancer HCC1937 cells that were stably reconstituted with wild-type BRCA1 expression construct and in human breast cancer T47D and MCF-7 cells transiently transfected with BRCA1-specific short-interfering RNA (siRNA). Chromatin immunoprecipitation assays were performed to determine if BRCA1 binds the ESR1 promoter and to identify other interacting proteins. Sensitivity to the antiestrogen drug fulvestrant was examined in T47D and MCF-7 cells transfected with BRCA1-specific siRNA. All statistical tests were two-sided. RESULTS: Mean ESR1 gene expression was 5.4-fold lower in BRCA1-mutant tumors than in sporadic tumors (95% confidence interval [CI] = 2.6-fold to 40.1-fold, P = .0019). The transcription factor Oct-1 recruited BRCA1 to the ESR1 promoter, and both BRCA1 and Oct-1 were required for ER alpha expression. BRCA1-depleted breast cancer cells expressing exogenous ER alpha were more sensitive to fulvestrant than BRCA1-depleted cells transfected with empty vector (T47D cells, the mean concentration of fulvestrant that inhibited the growth of 40% of the cells [IC40] for empty vector versus ER alpha: >10(-5) versus 8.0 x 10(-9) M [95% CI = 3.1 x 10(-10) to 3.2 x 10(-6) M]; MCF-7 cells, mean IC40 for empty vector versus ER alpha: >10(-5) versus 4.9 x 10(-8) M [95% CI = 2.0 x 10(-9) to 3.9 x 10(-6) M]). CONCLUSIONS: BRCA1 alters the response of breast cancer cells to antiestrogen therapy by directly modulating ER alpha expression.

Serological diagnosis of goose circovirus infections.

Infections with goose circovirus (GoCV) are associated with growth retardation and developmental problems in farmed geese. An indirect immunofluorescence assay for detecting virus-specific serum antibody was developed for diagnostic and epidemiological purposes. In the absence of a method for growing GoCV in cell culture, the assay was based on the reaction of antibodies with the GoCV capsid protein produced within baby hamster kidney cells using the eukaryotic Semliki forest virus expression vector. Using an optimized test that involved screening sera at 1:50 dilution and the use of a fluorescein isothiocyanate anti-duck immunoglobulin conjugate, GoCV-specific antibody was detected in 141 (88.6%) of 159 samples obtained from 27 of 28 breeder flocks aged from 1 to 6 years. Testing also showed the presence of GoCV-specific antibody in 85 (40.9%) of 208 serum samples from birds aged 30 weeks or less. Although maternally derived antibody was detected in birds when 1 and 4 days old, actively acquired antibody was first detected in birds aged 53 days. Following experimental inoculation of 21-day-old geese with tissue homogenate containing GoCV, virus-specific antibody was detected in serum samples collected at 27 and 34 days post inoculation. It is concluded that the SFV expression vector approach may prove useful for developing serological tests for other viruses, including other avian circoviruses, that do not grow in cell culture.

Molecular basis of the attenuation exhibited by molecularly cloned highly passaged chicken anemia virus isolates.

Chimeric virus experiments indicated that the pathogenicity and monoclonal antibody reactivity differences between two molecularly cloned, highly passaged chicken anemia virus isolates could be attributed to the VP1 amino acid change at residue 89. The introduction of this change into a pathogenic cloned low-passage isolate was not sufficient to cause attenuation.

Investigation of the unstable attenuation exhibited by a chicken anaemia virus isolate.

An attenuated chicken anaemia virus (CAV) isolate, cloned isolate 10, which was molecularly cloned from the Cuxhaven-1 CAV after 173 cell-culture passages, was shown previously to recover pathogenicity following 10 passages in young chicks. The consensus nucleotide sequence of the 'revertant' (Rev) virus, present as a tissue homogenate, differed from cloned isolate 10 at a single nucleotide residue (nucleotide 1739) that changed amino acid 287 of the capsid protein from alanine to aspartic acid. Subjecting Rev virus to 10 cell-culture passages reselected viruses with an alanine at this amino acid position. Experimental infections using a molecularly cloned Rev virus isolate demonstrated that the mutation at nucleotide 1739 was not in itself responsible for the recovery of pathogenicity exhibited by the Rev virus. Additional sequence analyses of cloned amplicons provided evidence that the Rev virus population comprised minor, genetically different subpopulations, and provided an indication of CAV's potential for genetic change.

Discrimination of Mycobacterium tuberculosis complex bacteria using novel VNTR-PCR targets.

The lack of a convenient high-resolution strain-typing method has hampered the application of molecular epidemiology to the surveillance of bacteria of the Mycobacterium tuberculosis complex, particularly the monitoring of strains of Mycobacterium bovis. With the recent availability of genome sequences for strains of the M. tuberculosis complex, novel PCR-based M. tuberculosis-typing methods have been developed, which target the variable-number tandem repeats (VNTRs) of minisatellite-like mycobacterial interspersed repetitive units (MIRUs), or exact tandem repeats (ETRs). This paper describes the identification of seven VNTR loci in M. tuberculosis H37Rv, the copy number of which varies in other strains of the M. tuberculosis complex. Six of these VNTRs were applied to a panel of 100 different M. bovis isolates, and their discrimination and correlation with spoligotyping and an established set of ETRs were assessed. The number of alleles varied from three to seven at the novel VNTR loci, which differed markedly in their discrimination index. There was positive correlation between spoligotyping, ETR- and VNTR-typing. VNTR-PCR discriminates well between M. bovis strains. Thirty-three allele profiles were identified by the novel VNTRs, 22 for the ETRs and 29 for spoligotyping. When VNTR- and ETR-typing results were combined, a total of 51 different profiles were identified. Digital nomenclature and databasing were intuitive. VNTRs were located both in intergenic regions and annotated ORFs, including PPE (novel glycine-asparigine-rich) proteins, a proposed source of antigenic variation, where VNTRs potentially code repeating amino acid motifs. VNTR-PCR is a valuable tool for strain typing and for the study of the global molecular epidemiology of the M. tuberculosis complex. The novel VNTR targets identified in this study should additionally increase the power of this approach.