Identification of Transcriptionally Active Open Reading Frames within the RD1 Genomic Segment of <i>Mycobacterium tuberculosis</i>

ABSTRACT By different approaches, we characterized the birnavirus blotched snakehead virus (BSNV). The sequence of genomic segment A revealed the presence of two open reading frames (ORFs): a large ORF with a 3,207-bp-long nucleotide sequence and a 417-nucleotide-long small ORF located within the N-terminal half of the large ORF, but in a different reading frame. The large ORF was found to encode a polyprotein cotranslationally processed by the viral protease VP4 to generate pVP2 (the VP2 precursor), a 71-amino-acid-long peptide ([X]), VP4, and VP3. The two cleavage sites at the [X]-VP4 and VP4-VP3 junctions were identified by N-terminal sequencing. We showed that the processing of pVP2 generated VP2 and several small peptides (amino acids [aa] 418 to 460, 461 to 467, 468 to 474, and 475 to 486). Two of these peptides (aa 418 to 460 and 475 to 486) were positively identified in the viral particles with 10 additional peptides derived from further processing of the peptide aa 418 to 460. The results suggest that VP4 cleaves multiple Pro-X-Ala↓Ala motifs, with the notable exception of the VP4-VP3 junction. Replacement of the members of the predicted VP4 catalytic dyad (Ser-692 and Lys-729) confirmed their indispensability in the polyprotein processing. The genomic segment B sequence revealed a single large ORF encoding a putative polymerase, VP1. Our results demonstrate that BSNV should be considered a new aquatic birnavirus species, slightly more related to IBDV than to IPNV.

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Profiling Antibodies to Mycobacterium tuberculosis by Multiplex Microbead Suspension Arrays for Serodiagnosis of Tuberculosis

Clinical and Vaccine Immunology ◽

10.1128/cvi.00354-07 ◽

2007 ◽

Vol 15 (3) ◽

pp. 433-438 ◽

Cited By ~ 59

Author(s):

Imran H. Khan ◽

Resmi Ravindran ◽

JoAnn Yee ◽

Melanie Ziman ◽

David M. Lewinsohn ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Simultaneous Detection ◽

Open Reading Frames ◽

Antibody Detection ◽

Primate Model ◽

Large Numbers ◽

Luminex Technology ◽

Novel Target ◽

Disease Antibody ◽

Reading Frames

ABSTRACT Tuberculosis (TB) is a serious global disease. The fatality rate attributed to TB is among the highest of infectious diseases, with approximately 2 million deaths occurring per year worldwide. Identification of individuals infected with Mycobacterium tuberculosis and screening of their immediate contacts is crucial for controlling the spread of TB. Current methods for detection of M. tuberculosis infection are not efficient, in particular, for testing large numbers of samples. We report a novel and efficient multiplex microbead immunoassay (MMIA), based on Luminex technology, for profiling antibodies to M. tuberculosis. Microbead sets identifiable by unique fluorescence were individually coated with each of several M. tuberculosis antigens and tested in multiplex format for antibody detection in the experimental nonhuman primate model of TB. Certain M. tuberculosis antigens, e.g., ESAT-6, CFP-10, and HspX, were included to enhance the specificity of the MMIA, because these antigens are absent in nontuberculous mycobacteria and the vaccine strain Mycobacterium bovis bacillus Calmette-Guérin. The MMIA enabled simultaneous detection of multiple M. tuberculosis plasma antibodies in several cohorts of macaques representing different stages of infection and/or disease. Antibody profiles were defined in early and latent/chronic infection. These proof-of-concept findings demonstrate the potential clinical use of the MMIA. In addition, the MMIA serodetection system has a potential for mining M. tuberculosis open reading frames (about 4,000) to discover novel target proteins for the development of more-comprehensive TB serodiagnostic tests.

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Computational prediction and experimental verification of novel IdeR binding sites in the upstream sequences of Mycobacterium tuberculosis open reading frames

Bioinformatics ◽

10.1093/bioinformatics/bti375 ◽

2005 ◽

Vol 21 (10) ◽

pp. 2161-2166 ◽

Cited By ~ 28

Author(s):

P. Prakash ◽

S. Yellaboina ◽

A. Ranjan ◽

S. E. Hasnain

Keyword(s):

Mycobacterium Tuberculosis ◽

Experimental Verification ◽

Binding Sites ◽

Computational Prediction ◽

Open Reading Frames ◽

Reading Frames

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Identification of Novel Mycobacterium tuberculosis Antigens with Potential as Diagnostic Reagents or Subunit Vaccine Candidates by Comparative Genomics

Infection and Immunity ◽

10.1128/iai.70.12.6996-7003.2002 ◽

2002 ◽

Vol 70 (12) ◽

pp. 6996-7003 ◽

Cited By ~ 108

Author(s):

P. J. Cockle ◽

S. V. Gordon ◽

A. Lalvani ◽

B. M. Buddle ◽

R. G. Hewinson ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Comparative Genomics ◽

Subunit Vaccine ◽

Open Reading Frames ◽

Vaccine Candidates ◽

Purified Protein Derivative ◽

Research Priority ◽

Independent Review ◽

Reading Frames

ABSTRACT An independent review for the British government has concluded that the development of a cattle vaccine against Mycobacterium bovis holds the best long-term prospects for tuberculosis control in British herds. The development of complementary diagnostic tests to differentiate between vaccinated and infected animals is necessary to allow the continuation of test-and-slaughter-based control policies alongside vaccination. Vaccination with M. bovis bacillus Calmette-Guérin (BCG), the only available vaccine, results in tuberculin purified protein derivative sensitivity and has shown varying vaccine efficacies in cattle. Thus, identification of more-specific reagents to distinguish between vaccination and infection, as well as the identification of subunit vaccine candidates for improved tuberculosis vaccines, is a research priority. In the present study, we applied comparative genomics to identify M. bovis-Mycobacterium tuberculosis antigens whose genes had been deleted in BCG Pasteur. In total, 13 open reading frames (ORFs) from the RD1, RD2, and RD14 regions of the M. tuberculosis genome were selected. Pools of overlapping peptides spanning these ORFs were tested in M. bovis-infected (n = 22), BCG-vaccinated (n = 6), and unvaccinated (n = 10) control cattle. All were recognized in infected cattle, with responder frequencies varying between 16 and 86%. In particular, eight highly immunogenic antigens were identified whose potentials as diagnostic reagents or as subunit vaccines warrant further study (Rv1983, Rv1986, Rv3872, Rv3873, Rv3878, Rv3879c, Rv1979c, and Rv1769).

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