A novel approach to interrogating the effects of chemical warfare agent exposure using organ-on-a-chip technology and multiomic analysis.

Organ-on-a-chip platforms are utilized in global bioanalytical and toxicological studies as a way to reduce materials and increase throughput as compared to in vivo based experiments. These platforms bridge the infrastructure and regulatory gaps between in vivo animal work and human systems, with models that exemplify active biological pathways. In conjunction with the advent of increased capabilities associated with next generation sequencing and mass spectrometry based '-omic' technologies, organ-on-a-chip platforms provide an excellent opportunity to investigate the global changes at multiple biological levels, including the transcriptome, proteome and metabolome. When investigated concurrently, a complete profile of cellular and regulatory perturbations can be characterized following treatment with specific agonists. In this study, global effects were observed and analyzed following liver chip exposure to the chemical warfare agent, VX. Even though the primary mechanism of action of VX (i.e. acetylcholinesterase inhibition) is well characterized, recent in vivo studies suggest additional protein binding partners that are implicated in metabolism and cellular energetic pathways. In addition, secondary toxicity associated with peripheral organ systems, especially in human tissues, is not well defined. Our results demonstrate the potential of utilizing an organ-on-a-chip platform as a surrogate system to traditional in vivo studies. This is realized by specifically indicating significant dysregulation of several cellular processes in response to VX exposure including but not limited to amino acid synthesis, drug metabolism, and energetics pathways.

A Rapid, Whole Genome Sequencing Assay for Detection and Characterization of Novel Coronavirus (SARS-CoV-2) Clinical Specimens Using Nanopore Sequencing.

A new human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged at the end of 2019 in Wuhan, China that caused a range of disease severities; including fever, shortness of breath, and coughing. This disease, now known as coronavirus disease 2019 (COVID-19), quickly spread throughout the world, and was declared a pandemic by the World Health Organization in March of 2020. As the disease continues to spread, providing rapid characterization has proven crucial to better inform the design and execution of control measures, such as decontamination methods, diagnostic tests, antiviral drugs, and prophylactic vaccines for long-term control. Our work at the United States Army's Combat Capabilities Development Command Chemical Biological Center (DEVCOM CBC) is focused on engineering workflows to efficiently identify, characterize, and evaluate the threat level of any potential biological threat in the field and more remote, lower resource settings, such as forward operating bases. While we have successfully established untargeted sequencing approaches for detection of pathogens for rapid identification, our current work entails a more in-depth sequencing analysis for use in evolutionary monitoring. We are developing and validating a SARS-CoV-2 nanopore sequencing assay, based on the ARTIC protocol. The standard ARTIC, Illumina, and nanopore sequencing protocols for SARS-CoV-2 are elaborate and time consuming. The new protocol integrates Oxford Nanopore Technology's Rapid Sequencing Kit following targeted RT-PCR of RNA extracted from human clinical specimens. This approach decreases sample manipulations and preparation times. Our current bioinformatics pipeline utilizes Centrifuge as the classifier for quick identification of SARS-CoV-2 and RAMPART software for verification and mapping of reads to the full SARS-CoV-2 genome. ARTIC rapid sequencing results, of previous RT-PCR confirmed patient samples, showed that the modified protocol produces high quality data, with up to 98.9% genome coverage at >1,000x depth for samples with presumably higher viral loads. Furthermore, whole genome assembly and subsequent mutational analysis of six of these sequences identified existing and unique mutations to this cluster, including three in the Spike protein: V308L, P521R, and D614G. This work suggests that an accessible, portable, and relatively fast sample-to-sequence process to characterize viral outbreaks is feasible and effective.

Draft Whole-Genome Sequences of Ciprofloxacin-Resistant Derivatives of a Bacillus anthracis ΔANR Strain Lacking pXO1 and pXO2 Plasmids.

Mutants of an attenuated Bacillus anthracis (ΔANR) strain conferring increasing levels of ciprofloxacin resistance have been described. Here, we report the draft genome sequences of the parent strain (ΔANR pXO1-, pXO2-) and its derivatives conferring low (step 1; 0.5 µg/ml), medium (step 2; 8 to 16 µg/ml), and high (step 3; 32 to 64 µg/ml) levels of ciprofloxacin resistance.

Use of Unamplified RNA/cDNA-Hybrid Nanopore Sequencing for Rapid Detection and Characterization of RNA Viruses.

Nanopore sequencing, a novel genomics technology, has potential applications for routine biosurveillance, clinical diagnosis, and outbreak investigation of virus infections. Using rapid sequencing of unamplified RNA/cDNA hybrids, we identified Venezuelan equine encephalitis virus and Ebola virus in 3 hours from sample receipt to data acquisition, demonstrating a fieldable technique for RNA virus characterization.

Whole-Genome Sequencing in Microbial Forensic Analysis of Gamma-Irradiated Microbial Materials.

Effective microbial forensic analysis of materials used in a potential biological attack requires robust methods of morphological and genetic characterization of the attack materials in order to enable the attribution of the materials to potential sources and to exclude other potential sources. The genetic homogeneity and potential intersample variability of many of the category A to C bioterrorism agents offer a particular challenge to the generation of attributive signatures, potentially requiring whole-genome or proteomic approaches to be utilized. Currently, irradiation of mail is standard practice at several government facilities judged to be at particularly high risk. Thus, initial forensic signatures would need to be recovered from inactivated (nonviable) material. In the study described in this report, we determined the effects of high-dose gamma irradiation on forensic markers of bacterial biothreat agent surrogate organisms with a particular emphasis on the suitability of genomic DNA (gDNA) recovered from such sources as a template for whole-genome analysis. While irradiation of spores and vegetative cells affected the retention of Gram and spore stains and sheared gDNA into small fragments, we found that irradiated material could be utilized to generate accurate whole-genome sequence data on the Illumina and Roche 454 sequencing platforms.

Pangenome Analysis of Burkholderia pseudomallei: Genome Evolution Preserves Gene Order despite High Recombination Rates.

The pangenomic diversity in Burkholderia pseudomallei is high, with approximately 5.8% of the genome consisting of genomic islands. Genomic islands are known hotspots for recombination driven primarily by site-specific recombination associated with tRNAs. However, recombination rates in other portions of the genome are also high, a feature we expected to disrupt gene order. We analyzed the pangenome of 37 isolates of B. pseudomallei and demonstrate that the pangenome is 'open', with approximately 136 new genes identified with each new genome sequenced, and that the global core genome consists of 4568±16 homologs. Genes associated with metabolism were statistically overrepresented in the core genome, and genes associated with mobile elements, disease, and motility were primarily associated with accessory portions of the pangenome. The frequency distribution of genes present in between 1 and 37 of the genomes analyzed matches well with a model of genome evolution in which 96% of the genome has very low recombination rates but 4% of the genome recombines readily. Using homologous genes among pairs of genomes, we found that gene order was highly conserved among strains, despite the high recombination rates previously observed. High rates of gene transfer and recombination are incompatible with retaining gene order unless these processes are either highly localized to specific sites within the genome, or are characterized by symmetrical gene gain and loss. Our results demonstrate that both processes occur: localized recombination introduces many new genes at relatively few sites, and recombination throughout the genome generates the novel multi-locus sequence types previously observed while preserving gene order.

Genomic sequences of six botulinum neurotoxin-producing strains representing three clostridial species illustrate the mobility and diversity of botulinum neurotoxin genes.

The whole genomes for six botulinum neurotoxin-producing clostridial strains were sequenced to provide references for under-represented toxin types, bivalent strains or unusual toxin complexes associated with a bont gene. The strains include three Clostridium botulinum Group I strains (CDC 297, CDC 1436, and Prevot 594), a Group II C. botulinum strain (Eklund 202F), a Group IV Clostridium argentinense strain (CDC 2741), and a Group V Clostridium baratii strain (Sullivan). Comparisons of the Group I genomic sequences revealed close relationships and conservation of toxin gene locations with previously published Group I C. botulinum genomes. The bont/F6 gene of strain Eklund 202F was determined to be a chimeric toxin gene composed of bont/F1 and bont/F2. The serotype G strain CDC 2741 remained unfinished in 20 contigs with the bont/G located within a 1.15Mb contig, indicating a possible chromosomal location for this toxin gene. Within the genome of C. baratii Sullivan strain, direct repeats of IS1182 insertion sequence (IS) elements were identified flanking the bont/F7 toxin complex that may be the mechanism of bont insertion into C. baratii. Highlights of the six strains are described and release of their genomic sequences will allow further study of unusual neurotoxin-producing clostridial strains.

Detection of Burkholderia pseudomallei O-antigen serotypes in near-neighbor species.

BACKGROUND: Burkholderia pseudomallei is the etiological agent of melioidosis and a CDC category B select agent with no available effective vaccine. Previous immunizations in mice have utilized the lipopolysaccharide (LPS) as a potential vaccine target because it is known as one of the most important antigenic epitopes in B. pseudomallei. Complicating this strategy are the four different B. pseudomallei LPS O-antigen types: A, B, B2, and rough. Sero-crossreactivity is common among O-antigens of Burkholderia species. Here, we identified the presence of multiple B. pseudomallei O-antigen types and sero-crossreactivity in its near-neighbor species. RESULTS: PCR screening of O-antigen biosynthesis genes, phenotypic characterization using SDS-PAGE, and immunoblot analysis showed that majority of B. mallei and B. thailandensis strains contained the typical O-antigen type A. In contrast, most of B. ubonensis and B. thailandensis-like strains expressed the atypical O-antigen types B and B2, respectively. Most B. oklahomensis strains expressed a distinct and non-seroreactive O-antigen type, except strain E0147 which expressed O-antigen type A. O-antigen type B2 was also detected in B. thailandensis 82172, B. ubonensis MSMB108, and Burkholderia sp. MSMB175. Interestingly, B. thailandensis-like MSMB43 contained a novel serotype B positive O-antigen. CONCLUSIONS: This study expands the number of species which express B. pseudomallei O-antigen types. Further work is required to elucidate the full structures and how closely these are to the B. pseudomallei O-antigens, which will ultimately determine the efficacy of the near-neighbor B serotypes for vaccine development.