Use of Denaturing High-Performance Liquid Chromatography To Identify Bacillus anthracis by Analysis of the 16S-23S rRNA Interspacer Region and gyrA Gene

Denaturing high-performance liquid chromatography (DHPLC) was evaluated as a method for identifying Bacillus anthracis by analyzing two chromosomal targets, the 16S-23S intergenic spacer region (ISR) and the gyrA gene. The 16S-23S ISR was analyzed by this method with 42 strains of B. anthracis, 36 strains of Bacillus cereus, and 12 strains of Bacillus thuringiensis; the gyrA gene was analyzed by this method with 33 strains of B. anthracis, 27 strains of B. cereus, and 9 strains of B. thuringiensis. Two blind panels of 45 samples each were analyzed to evaluate the potential diagnostic capability of this method. Our results show that DHPLC is an efficient method for the identification of B. anthracis

Transiently Transfected Mammalian Cell Cultures: An Adaptable and Effective Platform for Virus-like Particle-Based Vaccines against Foot-and-Mouth Disease Virus

RNA viruses, such as foot-and-mouth disease virus (FMDV), have error-prone replication resulting in the continuous emergence of new viral strains capable of evading current vaccine coverage. Vaccine formulations must be regularly updated, which is both costly and technically challenging for many vaccine platforms. In this report, we describe a plasmid-based virus-like particle (VLP) production platform utilizing transiently transfected mammalian cell cultures that combines both the rapid response adaptability of nucleic-acid-based vaccines with the ability to produce intact capsid epitopes required for immunity. Formulated vaccines which employed this platform conferred complete protection from clinical foot-and-mouth disease in both swine and cattle. This novel platform can be quickly adapted to new viral strains and serotypes through targeted exchanges of only the FMDV capsid polypeptide nucleic acid sequences, from which processed structural capsid proteins are derived. This platform obviates the need for high biocontainment manufacturing facilities to produce inactivated whole-virus vaccines from infected mammalian cell cultures, which requires upstream expansion and downstream concentration of large quantities of live virulent viruses

Comparative Evaluation of the Foot-and-Mouth Disease Virus Permissive LF-BK α_Vβ₆ Cell Line for Senecavirus A Research

Senecavirus A (SVA) is a member of the family Picornaviridae and enzootic in domestic swine. SVA can induce vesicular lesions that are clinically indistinguishable from Foot-and-mouth disease, a major cause of global trade barriers and agricultural productivity losses worldwide. The LF-BK αVβ6 cell line is a porcine-derived cell line transformed to stably express an αVβ6 bovine integrin and primarily used for enhanced propagation of Foot-and-mouth disease virus (FMDV). Due to the high biosecurity requirements for working with FMDV, SVA has been considered as a surrogate virus to test and evaluate new technologies and countermeasures. Herein we conducted a series of comparative evaluation in vitro studies between SVA and FMDV using the LF-BK αVβ6 cell line. These include utilization of LF-BK αVβ6 cells for field virus isolation, production of high virus titers, and evaluating serological reactivity and virus susceptibility to porcine type I interferons. These four methodologies utilizing LF-BK αVβ6 cells were applicable to research with SVA and results support the current use of SVA as a surrogate for FMDV

Detection of the Bacillus anthracis gyrA Gene by Using a Minor Groove Binder Probe

Identification of chromosomal markers for rapid detection of Bacillus anthracis is difficult because significant chromosomal homology exists among B. anthracis, Bacillus cereus, and Bacillus thuringiensis. We evaluated the bacterial gyrA gene as a potential chromosomal marker for B. anthracis. A real-time PCR assay was developed for the detection of B. anthracis. After analysis of the unique nucleotide sequence of the B. anthracis gyrA gene, a fluorescent 3′ minor groove binding probe was tested with 171 organisms from 29 genera of bacteria, including 102 Bacillus strains. The assay was found to be specific for all 43 strains of B. anthracis tested. In addition, a test panel of 105 samples was analyzed to evaluate the potential diagnostic capability of the assay. The assay showed 100% specificity, demonstrating the usefulness of the gyrA gene as a specific chromosomal marker for B. anthracis