Biological Threat Agents Research Articles

DEVELOPMENT OF A BIOLOGICAL AEROSOL DETECTOR USING LASER-INDUCED FLUORESCENCE AND A PARTICLE COLLECTION SYSTEM

A real-time biological aerosol fluorescence detector was designed for monitoring aerosols in the atmosphere. The operating principles of the current biological aerosol monitoring system (BAMS) include the measurement of optically determined single particle properties, two fluorescence bands, and a scattering signal. Furthermore, the development of an algorithm to significantly increase the biological aerosol discrimination is described, which uses the results of the experimental evaluation of a number of aerosols in an evaluation chamber in the laboratory. The detector was applied to seasonal pollen, Asian dust, seawater environment, industrial zones, and residential areas in Korea. Applications of the device include all aspects of biological aerosol monitoring, including use as a biological agent trigger and as a hazardous environment monitor. The detectors were used in various laboratories as an ambient biological aerosol monitor and in various fields as a fast biological threat agent trigger in the 2010 G20 Seoul Summit and 2012 Nuclear Security Summit.

Bis-indolic compounds as potential new therapeutic alternatives for tularaemia

Francisella tularensis is the etiological agent of tularaemia and a CDC class A biological threat agent. Few antibiotic classes are currently useful in treating tularaemia, including the aminoglycosides gentamicin and streptomycin, fluoroquinolones, and tetracyclines. However, treatment failures and relapses remain frequent and F. tularensis strains resistant to antibiotics have been easily selected in vitro. In this study, we evaluated the activity of new synthetic bis-indole derivatives against this pathogen. Minimum inhibitory concentrations (MICs) of four compounds (dcm01 to dcm04) were determined for the reference strains F. tularensis subsp. holarctica LVS NCTC10857, F. tularensis subsp. novicida CIP56.12 and F. philomiragia ATCC25015, and for 41 clinical strains of F. tularensis subsp. holarctica isolated in France. Minimal bactericidal concentrations (MBCs) were determined for the dcm02 and dcm04 compounds for the LVS and two clinical strains. Killing curves were also determined for the same three strains exposed to dcm04. All tested bis-indole compounds were bacteriostatic against F. tularensis subsp. holarctica strains, with a MIC90 of 8 μg/mL for dcm01, dcm02, and dcm03, and 2 μg/mL for dcm04. Only one strain was resistant to both dcm01 and dcm03, with MICs > 32 μg/mL. In contrast, F. tularensis subsp. novicida was resistant to all derivatives and F. philomiragia was only susceptible to dcm02 and dcm04, with MICs of 16 and 4 μg/mL, respectively. MBC and killing curve experiments revealed significant bactericidal activity (i.e., 3-log reduction of the bacterial inoculum) of the dcm02 and dcm04 compounds only for the LVS strain. In conclusion, we have identified novel synthetic bis-indole compounds that are active against F. tularensis subsp. holarctica. They may be drug candidates for the development of new therapeutic alternatives for tularaemia treatment. Their further characterization is needed, especially identification of their bacterial targets.

Label free detection of pseudorabies virus infection in Vero cells using laser force analysis

The rapid and robust identification of viral infections has broad implications for a number of fields, including medicine, biotechnology and biodefense. Most detection systems rely on specific molecules, such as nucleic acids or proteins, to identify the target(s) of interest. These molecules afford great specificity, but are often expensive, labor-intensive, labile and limited in scope. Label free detection methods seek to overcome these limitations by instead using detection methods that rely on intrinsic properties as a basis for identifying and separating species of interest and thus do not rely on specific prior knowledge of the target. Optical chromatography, one such technique, uses the balance between optical and fluidic drag forces within a microfluidic channel to determine the optical force on cells or particles. Here we present the application of individual optical force measurements as a means of investigating pseudorabies virus infection in Vero cells. Optical force differences are seen between cells from uninfected and infected populations at a multiplicity of infection as low as 0.001 and as soon as 2 hours post infection, demonstrating the potential of this technique as a means of detecting viral infection. Through the application of a pattern recognition neural network, individual cell size data are combined with optical force as a means of classifying cell populations. Potential applications include the early detection of bloodborne pathogens for the prevention of sepsis and other diseases as well as the detection of biological threat agents.

Temporal Variability of the Bioaerosol Background at a Subway Station: Concentration Level, Size Distribution, and Diversity of Airborne Bacteria

Naturally occurring bioaerosol environments may present a challenge to biological detection-identification-monitoring (BIODIM) systems aiming at rapid and reliable warning of bioterrorism incidents. One way to improve the operational performance of BIODIM systems is to increase our understanding of relevant bioaerosol backgrounds. Subway stations are enclosed public environments which may be regarded as potential bioterrorism targets. This study provides novel information concerning the temporal variability of the concentration level, size distribution, and diversity of airborne bacteria in a Norwegian subway station. Three different air samplers were used during a 72-h sampling campaign in February 2011. The results suggested that the airborne bacterial environment was stable between days and seasons, while the intraday variability was found to be substantial, although often following a consistent diurnal pattern. The bacterial levels ranged from not detected to 10(3) CFU m(-3) and generally showed increased levels during the daytime compared to the nighttime levels, as well as during rush hours compared to non-rush hours. The airborne bacterial levels showed rapid temporal variation (up to 270-fold) on some occasions, both consistent and inconsistent with the diurnal profile. Airborne bacterium-containing particles were distributed between different sizes for particles of >1.1 μm, although ∼50% were between 1.1 and 3.3 μm. Anthropogenic activities (mainly passengers) were demonstrated as major sources of airborne bacteria and predominantly contributed 1.1- to 3.3-μm bacterium-containing particles. Our findings contribute to the development of realistic testing and evaluation schemes for BIODIM equipment by providing information that may be used to simulate operational bioaerosol backgrounds during controlled aerosol chamber-based challenge tests with biological threat agents.

Vaccines and Therapeutics for the Encephalitic Alphaviruses

This article is a review of vaccines and therapeutics in development for the encephalitic alphaviruses, which includes eastern equine encephalitis virus, western equine encephalitis virus and Venezuelan equine encephalitis virus. The encephalitic alphaviruses are endemic within regions in North and South America. Hosts are normally exposed after being bitten by infectious mosquitoes, and infection can develop into encephalitis in equines and humans with severe rates of morbidity and mortality. These viruses are also potential biological threat agents, being highly infectious via an aerosol route of exposure. In humans, equine encephalitis virus and western equine encephalitis virus are neurotropic viruses targeting the CNS and causing encephalitis. Mortality rates are 50 and 10%, respectively, for these viruses. On the other hand, Venezuelan equine encephalitis virus produces a systemic influenza-like illness with pathogenesis in the lungs and lymphoid tissue in adults and older children. The incidence of encephalitis is less than 5% in younger children with a case–mortality rate of 1%. The host response to virus infectivity is briefly discussed, along with a number of promising therapeutic and prophylactic approaches. These approaches can be broadly classified as: virus-specific, including vaccines, antibody therapy and gene-silencing oligonucleotides; or broad-spectrum, including interferon and activation of the host‘s innate immunity.

A Systematic Screen of FDA-Approved Drugs for Inhibitors of Biological Threat Agents

BackgroundThe rapid development of effective medical countermeasures against potential biological threat agents is vital. Repurposing existing drugs that may have unanticipated activities as potential countermeasures is one way to meet this important goal, since currently approved drugs already have well-established safety and pharmacokinetic profiles in patients, as well as manufacturing and distribution networks. Therefore, approved drugs could rapidly be made available for a new indication in an emergency.Methodology/Principal FindingsA large systematic effort to determine whether existing drugs can be used against high containment bacterial and viral pathogens is described. We assembled and screened 1012 FDA-approved drugs for off-label broad-spectrum efficacy against Bacillus anthracis; Francisella tularensis; Coxiella burnetii; and Ebola, Marburg, and Lassa fever viruses using in vitro cell culture assays. We found a variety of hits against two or more of these biological threat pathogens, which were validated in secondary assays. As expected, antibiotic compounds were highly active against bacterial agents, but we did not identify any non-antibiotic compounds with broad-spectrum antibacterial activity. Lomefloxacin and erythromycin were found to be the most potent compounds in vivo protecting mice against Bacillus anthracis challenge. While multiple virus-specific inhibitors were identified, the most noteworthy antiviral compound identified was chloroquine, which disrupted entry and replication of two or more viruses in vitro and protected mice against Ebola virus challenge in vivo.Conclusions/SignificanceThe feasibility of repurposing existing drugs to face novel threats is demonstrated and this represents the first effort to apply this approach to high containment bacteria and viruses.

A chimeric Sindbis-based vaccine protects cynomolgus macaques against a lethal aerosol challenge of eastern equine encephalitis virus

Eastern equine encephalitis virus (EEEV) is a mosquito-borne alphavirus that causes sporadic, often fatal disease outbreaks in humans and equids, and is also a biological threat agent. Two chimeric vaccine candidates were constructed using a cDNA clone with a Sindbis virus (SINV) backbone and structural protein genes from either a North (SIN/NAEEEV) or South American (SIN/SAEEEV) strain of EEEV. The vaccine candidates were tested in a nonhuman primate (NHP) model of eastern equine encephalitis (EEE). Cynomolgus macaques were either sham-vaccinated, or vaccinated with a single dose of either SIN/NAEEEV or SIN/SAEEEV. After vaccination, animals were challenged by aerosol with a virulent North American strain of EEEV (NA EEEV). The SIN/NAEEEV vaccine provided significant protection, and most vaccinated animals survived EEEV challenge (82%) with little evidence of disease, whereas most SIN/SAEEEV-vaccinated (83%) and control (100%) animals died. Protected animals exhibited minimal changes in temperature and cardiovascular rhythm, whereas unprotected animals showed profound hyperthermia and changes in heart rate postexposure. Acute inflammation and neuronal necrosis were consistent with EEEV-induced encephalitis in unprotected animals, whereas no encephalitis-related histopathologic changes were observed in the SIN/NAEEEV-vaccinated animals. These results demonstrate that the chimeric SIN/NAEEEV vaccine candidate protects against an aerosol EEEV exposure.

Biosensor technology: recent advances in threat agent detection and medicine

Biosensors are of great significance because of their capability to resolve a potentially large number of analytical problems and challenges in very diverse areas such as defense, homeland security, agriculture and food safety, environmental monitoring, medicine, pharmacology, industry, etc. The expanding role of biosensing in society and a real-world environment has led to an exponential growth of the R&D efforts around the world. The world market for biosensor devices, according to Global Industry Analysts, Inc., is expected to reach $12 billion by 2015. Such expedient growth is driven by several factors including medical and health problems, such as a growing population with a high risk of diabetes and obesity, and the rising incidence of chronic diseases such as heart disease, stroke, cancer, chronic respiratory diseases, tuberculosis, etc.; significant problems with environmental monitoring; and of course serious challenges in security and military applications and agriculture/food safety. A review paper in the biosensor technology area may be structured based on (i) the principles of detection, such as the type of transducer platform, bioanalytical principles (affinity or kinetic), and biorecognition elements origin/properties (i.e. antibodies, enzymes, cells, aptamers, etc.), and (ii) the application area. This review follows the latter strategy and focuses on the applications. This allows discussion on how different sensing strategies are brought to bear on the same problem and highlights advantages/disadvantages of these sensing strategies. Given the broad range of biosensor related applications, several particularly relevant areas of application were selected for review: biological threat agents, chemical threat agents, and medicine.

Assessment of the bio-preparedness and of the training of the French hospital laboratories in the event of biological threat

A national laboratory network 'Biotox-Piratox' was created in 2003 in France with the purpose of detecting, confirming and reporting potential biological and chemical threat agents. This network is divided into three levels: Level 1 is dedicated to the evaluation of risks (biological, chemical, radiological), to sampling and packing. Level 2 consists of university and military hospitals, who deal with biological specimens, and of environmental and veterinary laboratories, who deal with environmental and animal samples. Level 3 comprises national reference laboratories and the Jean Mérieux biosafety level (BSL)-4 laboratory in Lyon. This report presents the results of four bio-preparedness exercises to check critical points in the processing of samples. These exercises took place in 2007, 2009, 2010 and 2011. Each of them consisted of two parts. The first part was the identification of an unknown bacterial strain and its susceptibility to antibiotics used as a default in case of a bioterrorist event. The second part was the detection of Class III microorganisms, mainly by molecular techniques. The main lesson learnt in these exercises was that the key to successful detection of biological agents in case of a biological threat was standardisation and validation of the methods implemented by all the laboratories belonging to the network.

Two protein lysine methyltransferases methylate outer membrane protein B from Rickettsia.

Rickettsia prowazekii, the etiologic agent of epidemic typhus, is a potential biological threat agent. Its outer membrane protein B (OmpB) is an immunodominant antigen and plays roles as protective envelope and as adhesins. The observation of the correlation between methylation of lysine residues in rickettsial OmpB and bacterial virulence has suggested the importance of an enzymatic system for the methylation of OmpB. However, no rickettsial lysine methyltransferase has been characterized. Bioinformatic analysis of genomic DNA sequences of Rickettsia identified putative lysine methyltransferases. The genes of the potential methyltransferases were synthesized, cloned, and expressed in Escherichia coli, and expressed proteins were purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. The methyltransferase activities of the purified proteins were analyzed by methyl incorporation of radioactively labeled S-adenosylmethionine into recombinant fragments of OmpB. Two putative recombinant methyltransferases (rRP789 and rRP027-028) methylated recombinant OmpB fragments. The specific activity of rRP789 is 10- to 30-fold higher than that of rRP027-028. Western blot analysis using specific antibodies against trimethyl lysine showed that both rRP789 and rRP027-028 catalyzed trimethylation of recombinant OmpB fragments. Liquid chromatography-tandem mass spectrometry (LC/MS-MS) analysis showed that rRP789 catalyzed mono-, di-, and trimethylation of lysine, while rRP027-028 catalyzed exclusively trimethylation. To our knowledge, rRP789 and rRP027-028 are the first biochemically characterized lysine methyltransferases of outer membrane proteins from Gram-negative bacteria. The production and characterization of rickettsial lysine methyltransferases provide new tools to investigate the mechanism of methylation of OmpB, effects of methylation on the structure and function of OmpB, and development of methylated OmpB-based diagnostic assays and vaccine candidates.

High Sensitivity ELISA for Detection of Botulinum Neurotoxin Serotype F

Botulinum neurotoxins (BoNTs) are classified as category A biological threat agents by the Centers for Disease Control and Prevention (CDC) in the United States for its hazardous and potential bioterrorist threat to the public. About 1% naturally occurring botulisms are caused by Botulinum neurotoxin serotype F (BoNT/F). Most of the immunoassays for detecting BoNTs focus on the serotypes A and B, but few methods have been established for the detection of BoNT/F. Recently, the recombinant Hc subunit of botulinum neurotoxin type F (rFHc) was expressed as an effective vaccine against BoNT/F, indicating that this rFHc could be an effective immunogen to raise monoclonal antibodies (MAbs) for the detection and neutralization of BoNT/F. Here we present a novel sandwich enzyme-linked immunosorbent assay (ELISA) based on two MAbs against rFHc, which were FMMU-BTF-8 and FMMU-BTF-29 as capture antibody and detection antibody, respectively. The limit of detection (LOD) of this ELISA reached 12.09 pg/mL, much less than that of the other reported immunoassays. A simple, sensitive ELISA for detecting and quantifying BoNT/F was established, which can be used as a valuable method to detect and quantify BoNT/F.

Characterization of potential virulence factors in Rickettsia prowazekii as lysine methyltransferases

Rickettsia prowazekii, the etiologic agent of epidemic typhus, is a potential biological threat agent. Lysine methylation of OmpB (outer membrane protein) of Rickettsia correlates with the bacterial virulence. However, no lysine methyltransferase from Rickettsia has been characterized. Bioinformatic analysis of the Rickettsia prowazekii genome revealed six potential lysine methyltransferases. Codon‐optimized cDNAs of the six potential methyltransferases were synthesized and subcloned into pET28a. The six proteins were then expressed in E. coli BL21(DE3) and purified by affinity chromatography on Ni‐NTA agarose. The methyltransferase activity was assayed by the methyl incorporation of radioactively labeled S‐adenosyl‐methionine. Two of the six putative methyltransferases showed time‐ and enzyme‐concentration‐dependent methylation of the recombinant AN and K fragments of OmpB from R. typhi. The stoichiometry of the methyl incorporation suggests methylation at multiple sites of both fragments. Western blot analysis using trimethyl‐lysine specific antibodies confirmed the trimethylation of lysines in recombinant fragments of OmpB by the methyltransferases. Enzymatic methylation of OmpB delineates the functions of these proteins, and may provide tools for the development of diagnostic reagents

Characterization of Airborne Bacteria at an Underground Subway Station

The reliable detection of airborne biological threat agents depends on several factors, including the performance criteria of the detector and its operational environment. One step in improving the detector's performance is to increase our knowledge of the biological aerosol background in potential operational environments. Subway stations are enclosed public environments, which may be regarded as potential targets for incidents involving biological threat agents. In this study, the airborne bacterial community at a subway station in Norway was characterized (concentration level, diversity, and virulence- and survival-associated properties). In addition, a SASS 3100 high-volume air sampler and a matrix-assisted laser desorption ionization-time of flight mass spectrometry-based isolate screening procedure was used for these studies. The daytime level of airborne bacteria at the station was higher than the nighttime and outdoor levels, and the relative bacterial spore number was higher in outdoor air than at the station. The bacterial content, particle concentration, and size distribution were stable within each environment throughout the study (May to September 2010). The majority of the airborne bacteria belonged to the genera Bacillus, Micrococcus, and Staphylococcus, but a total of 37 different genera were identified in the air. These results suggest that anthropogenic sources are major contributors to airborne bacteria at subway stations and that such airborne communities could harbor virulence- and survival-associated properties of potential relevance for biological detection and surveillance, as well as for public health. Our findings also contribute to the development of realistic testing and evaluation schemes for biological detection/surveillance systems by providing information that can be used to mimic real-life operational airborne environments in controlled aerosol test chambers.

Genome wide search for identification of potential drug targets in Bacillus anthracis

With the heightened interest in Bacillus anthracis as a potential biological threat agent, novel drug targets identification is of great importance in drug discovery. This study considered a genome-wide approach to identify 270 non-redundant, non-human homologous genes and 103 essential genes of the bacteria as putative drug targets. Sub-cellular localisation of each drug target was annotated using PSORTb 3.0 and confirmation by a hybrid support vector machine analysis identified 16 membrane-bound genes with reliability index ≥4. SPAAN analysis predicted 3 adhesion-like proteins and BLAST against the MEROPS database identified 7 peptidases with inhibitors. As a case study, a homology model was built for the ptsG gene using Modeller 9v8. The work reported here identified a small subset of potential drug targets involved in vital aspects of the metabolism of pathogen, persistence, virulence and cell wall biosynthesis. Thus, this manifold workflow can speed up the process of drug target discovery.

Botulinum toxin: Therapeutic agent to cosmetic enhancement to lethal biothreat

The clinical effects of Clostridium botulinum and its extremely potent neurotoxin have been known for two centuries. The disease threat and the clinical uses are now well established. What’s changed is the potential for botulinum neurotoxin to be used as a biological threat agent. The recent upsurge of illegal trafficking of reagent-grade toxin could, if bought in large enough quantities, be as serious a threat as other biothreat agents such as anthrax and smallpox, which have received much more attention. Fortunately, effective countermeasures are available.

Disease profile development methodology for syndromic surveillance of biological threat agents

Disease profile development methodology for syndromic surveillance of biological threat agents

Reliable identification at the species level of Brucella isolates with MALDI-TOF-MS

BackgroundThe genus Brucella contains highly infectious species that are classified as biological threat agents. The timely detection and identification of the microorganism involved is essential for an effective response not only to biological warfare attacks but also to natural outbreaks. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a rapid method for the analysis of biological samples. The advantages of this method, compared to conventional techniques, are rapidity, cost-effectiveness, accuracy and suitability for the high-throughput identification of bacteria. Discrepancies between taxonomy and genetic relatedness on the species and biovar level complicate the development of detection and identification assays.ResultsIn this study, the accurate identification of Brucella species using MALDI-TOF-MS was achieved by constructing a Brucella reference library based on multilocus variable-number tandem repeat analysis (MLVA) data. By comparing MS-spectra from Brucella species against a custom-made MALDI-TOF-MS reference library, MALDI-TOF-MS could be used as a rapid identification method for Brucella species. In this way, 99.3% of the 152 isolates tested were identified at the species level, and B. suis biovar 1 and 2 were identified at the level of their biovar. This result demonstrates that for Brucella, even minimal genomic differences between these serovars translate to specific proteomic differences.ConclusionsMALDI-TOF-MS can be developed into a fast and reliable identification method for genetically highly related species when potential taxonomic and genetic inconsistencies are taken into consideration during the generation of the reference library.

Rapid detection of ribosome inactivating protein toxins by mass-spectrometry-based functional assays

A family of structurally related protein toxins extracted from bacteria or plants have a similar mechanism of action, namely, blocking protein synthesis by ribosome inactivation. Members of this family of ribosome-inactivating proteins (RIP), classified simultaneously as chemical and biological threat agents, include ricin, abrin, and shiga toxins. We have adapted and streamlined mass spectrometry based functional assays, described by others, to develop a rapid, robust, broad-band and fieldable protocol with the primary goal of RIP toxin detection in unknown powder materials. Complementary to intact protein detection and proteomics methods, this protocol involves monitoring the depurination of selected DNA substrates by mass spectrometry. It is being implemented on the CB-TOF system for rapid detection of bio-threats by MALDI MS. Here we illustrate its performance on the example of a plant (ricin) and a bacterial (shiga) RIP toxin.

Microbial Inactivation for Safe and Rapid Diagnostics of Infectious Samples

The high risk associated with biological threat agents dictates that any suspicious sample be handled under strict surety and safety controls and processed under high-level containment in specialized laboratories. This study attempted to find a rapid, reliable, and simple method for the complete inactivation of a wide range of pathogens, including spores, vegetative bacteria, and viruses, while preserving microbial nucleic acid fragments suitable for PCRs and proteinaceous epitopes for detection by immunoassays. Formaldehyde, hydrogen peroxide, and guanidium thiocyanate did not completely inactivate high titers of bacterial spores or viruses after 30 min at 21°C. Glutaraldehyde and sodium hypochlorite showed high microbicidal activity but obliterated the PCR or enzyme-linked immunosorbent assay (ELISA) detection of bacterial spores or viruses. High-level inactivation (more than 6 log(10)) of bacterial spores (Bacillus atrophaeus), vegetative bacteria (Pseudomonas aeruginosa), an RNA virus (the alphavirus Pixuna virus), or a DNA virus (the orthopoxvirus vaccinia virus) was attained within 30 min at 21°C by treatment with either peracetic acid or cupric ascorbate with minimal hindrance of subsequent PCR tests and immunoassays. The data described here should provide the basis for quickly rendering field samples noninfectious for further analysis under lower-level containment and considerably lower cost.

The NYC Native Air Sampling Pilot Project: Using HVAC Filter Data for Urban Biological Incident Characterization

Native air sampling (NAS) is distinguished from dedicated air sampling (DAS) devices (eg, BioWatch) that are deployed to detect aerosol disseminations of biological threat agents. NAS uses filter samples from heating, ventilation, and air conditioning (HVAC) systems in commercial properties for environmental sampling after DAS detection of biological threat agent incidents. It represents an untapped, scientifically sound, efficient, widely distributed, and comparably inexpensive resource for postevent environmental sampling. Calculations predict that postevent NAS would be more efficient than environmental surface sampling by orders of magnitude. HVAC filter samples could be collected from pre-identified surrounding NAS facilities to corroborate the DAS alarm and delineate the path taken by the bioaerosol plume. The New York City (NYC) Native Air Sampling Pilot Project explored whether native air sampling would be acceptable to private sector stakeholders and could be implemented successfully in NYC. Building trade associations facilitated outreach to and discussions with property owners and managers, who expedited contact with building managers of candidate NAS properties that they managed or owned. Nominal NAS building requirements were determined; procedures to identify and evaluate candidate NAS facilities were developed; data collection tools and other resources were designed and used to expedite candidate NAS building selection and evaluation in Manhattan; and exemplar environmental sampling playbooks for emergency responders were completed. In this sample, modern buildings with single or few corporate tenants were the best NAS candidate facilities. The Pilot Project successfully demonstrated that in one urban setting a native air sampling strategy could be implemented with effective public-private collaboration.