The genus Orthopoxvirus contains several species of related viruses, including the causative agent of smallpox (Variola virus). In addition to smallpox, several other members of the genus are capable of causing human infection, including monkeypox, cowpox, and other zoonotic rodent-borne poxviruses. Therefore, a single assay that can accurately identify all orthopoxviruses could provide a valuable tool for rapid broad orthopovirus identification. We have developed a pan-Orthopoxvirus assay for identification of all members of the genus based on four PCR reactions targeting Orthopoxvirus DNA and RNA helicase and polymerase genes. The amplicons are detected using electrospray ionization-mass spectrometry (PCR/ESI-MS) on the Ibis T5000 system. We demonstrate that the assay can detect and identify a diverse collection of orthopoxviruses, provide sub-species information and characterize viruses from the blood of rabbitpox infected rabbits. The assay is sensitive at the stochastic limit of PCR and detected virus in blood containing approximately six plaque-forming units per milliliter from a rabbitpox virus-infected rabbit.

ABSTRACT We have developed a PCR/electrospray ionization mass spectrometry (PCR/ESI-MS) assay for the rapid detection, identification, and serotyping of human adenoviruses. The assay employs a high-performance mass spectrometer to “weigh” the amplicons obtained from PCR using primers designed to amplify known human adenoviruses. Masses are converted to base compositions and, by comparison against a database of the genetic sequences, the serotype present in a sample is determined. The performance of the assay was demonstrated with quantified viral standards and environmental and human clinical samples collected from a military training facility. Over 500 samples per day can be analyzed with sensitivities greater than 100 genomes per reaction. This approach can be applied to many other families of infectious agents for rapid and sensitive analysis.

Background. Effective influenza surveillance requires new methods capable of rapid and inexpensive genomic analysis of evolving viral species for pandemic preparedness, to understand the evolution of circulating viral species, and for vaccine strain selection. We have developed one such approach based on previously described broad-range reverse transcription PCR/electrospray ionization mass spectrometry (RT-PCR/ESI-MS) technology. Methods and Principal Findings. Analysis of base compositions of RT-PCR amplicons from influenza core gene segments (PB1, PB2, PA, M, NS, NP) are used to provide subspecies identification and infer influenza virus H and N subtypes. Using this approach, we detected and correctly identified 92 mammalian and avian influenza isolates, representing 30 different H and N types, including 29 avian H5N1 isolates. Further, direct analysis of 656 human clinical respiratory specimens collected over a seven-year period (1999-2006) showed correct identification of the viral species and subtypes with >97% sensitivity and specificity. Base composition derived clusters inferred from this analysis showed 100% concordance to previously established clades. Ongoing surveillance of samples from the recent influenza virus seasons (2005-2006) showed evidence for emergence and establishment of new genotypes of circulating H3N2 strains worldwide. Mixed viral quasispecies were found in approximately 1% of these recent samples providing a view into viral evolution. Conclusion/Significance. Thus, rapid RT-PCR/ESI-MS analysis can be used to simultaneously identify all species of influenza viruses with clade-level resolution, identify mixed viral populations and monitor global spread and emergence of novel viral genotypes. This high-throughput method promises to become an integral component of influenza surveillance.

We describe a new approach to the sensitive and specific identification of bacteria, viruses, fungi, and protozoa based on broad-range PCR and high-performance mass spectrometry. The Ibis T5000 is based on technology developed for the Department of Defense known as T.I.G.E.R. (Triangulation Identification for the Genetic Evaluation of Risks) for pathogen surveillance. The technology uses mass spectrometry—derived base composition signatures obtained from PCR amplification of broadly conserved regions of the pathogen genomes to identify most organisms present in a sample. The process of sample analysis has been automated using a combination of commercially available and custom instrumentation. A software system known as T-Track manages the sample flow, signal analysis, and data interpretation and provides simplified result reports to the user. No specialized expertise is required to use the instrumentation. In addition to pathogen surveillance, the Ibis T5000 is being applied to reducing health care—associated infections (HAIs), emerging and pandemic disease surveillance, human forensics analysis, and pharmaceutical product and food safety, and will be used eventually in human infectious disease diagnosis. In this review, we describe the automated Ibis T5000 instrument and provide examples of how it is used in HAI control.

Epidemic respiratory infections are responsible for extensive morbidity and mortality within both military and civilian populations. We describe a high-throughput method to simultaneously identify and genotype species of bacteria from complex mixtures in respiratory samples. The process uses electrospray ionization mass spectrometry and base composition analysis of PCR amplification products from highly conserved genomic regions to identify and determine the relative quantity of pathogenic bacteria present in the sample. High-resolution genotyping of specific species is achieved by using additional primers targeted to highly variable regions of specific bacterial genomes. This method was used to examine samples taken from military recruits during respiratory disease outbreaks and for follow up surveillance at several military training facilities. Analysis of respiratory samples revealed high concentrations of pathogenic respiratory species, including Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pyogenes. When S. pyogenes was identified in samples from the epidemic site, the identical genotype was found in almost all recruits. This analysis method will provide information fundamental to understanding the polymicrobial nature of explosive epidemics of respiratory disease.

In this work, we describe a strategy for the detection and characterization of microorganisms associated with a potential biological warfare attack or a natural outbreak of an emerging infectious disease. This approach, termed TIGER (Triangulation Identification for the Genetic Evaluation of Risks), relies on mass spectrometry-derived base composition signatures obtained from PCR amplification of broadly conserved regions of the microbial genome(s) in a sample. The sample can be derived from air filtration devices, clinical samples, or other sources. Core to this approach are “intelligent PCR primers” that target broadly conserved regions of microbial genomes that flank variable regions. This approach requires that high-performance mass measurements be made on PCR products in the 80–140bp size range in a high-throughput, robust modality. As will be demonstrated, the concept is equally applicable to bacteria and viruses and could be further applied to fungi and protozoa. In addition to describing the fundamental strategy of this approach, several specific examples of TIGER are presented that illustrate the impact this approach could have on the way biological weapons attacks are detected and the way that the etiologies of infectious diseases are determined. The first example illustrates how any bacterial species might be identified, using Bacillus anthracis as the test agent. The second example demonstrates how DNA-genome viruses are identified using five members of Poxviridae family, whose members includes Variola virus, the agent responsible for smallpox. The third example demonstrates how RNA-genome viruses are identified using the Alphaviruses (VEE, WEE, and EEE) as representative examples. These examples illustrate how the TIGER technology can be applied to create a universal identification strategy for all pathogens, including those that infect humans, livestock, and plants.

Use of a compound of formula (I): (see formula) or pharmaceutically acceptable salts thereof, wherein NonAr is a nonaromatic 5-7 membered ring containing 1 or 2 nitrogen ring atoms or an aza bicyclo octane ring; HetAr is a heteroaromatic ring of 5 or 6 members containing 1-3 nitrogen ring atoms, or isoxazolyl, thiazolyl, thiadiazolyl, quinolinyl, quinazolinyl, purinyl, pteridinyl, benzimidazolyl, pyrrolopyrimidinyl, or imidazopyridinyl; HetAr is optionally substituted with 1 or 2 substituents, each substituent independently is C1 - 4, C1 - 4, C2 - 4, trifluoromethyl, hydroxy, hydroxy C1 - 4, fluorine, chlorine, bromine, iodine, cyano, methylsulfanyl , cyclopropylethynyl-, phenylethynyl-, heteroarylethynyl, -N (C0 - 4) (C0 - 4), nitro, (C1 - 2) (C1 - 2) nCH2-, (C1 - 2) HNCH2- , Si (CH3) 3-C-, or NH2C (O) -; A is -C0 - 4-; B is aryl (CH2) 0-3-OC (O) -, heteroaryl (CH2) 1-3-OC (O) -, indanyl (CH2) 0-3-OC (O) -, aryl (CH 2) 1 - 3-C (O) -, aryl-cyclopropyl-C (O) -, heteroaryl-cyclopropyl-C (O) -, heteroaryl (CH2) 1-3-C (O) -, aryl (CH 2) 1 - 3-, heteroaryl (CH2) 1 - 3, aryl (CH2) 1-3-NH-C (O) -, aryl (CH2) 1-3-NH-C (NCN) -, aryl (CH 2) 1 - 3-SO2-, heteroaryl (CH2) 1-3-SO2-, wherein any aryl or heteroaryl is optionally substituted by 1-5 substituents, each substituent independently C1 - 4 alkyl, C3 - 6, C1 - 4, trifluoromethyl, bromo, fluoro, or chloro; and X is H, OH, F, C1 - 4, C1 - 4, NH2, or X taken with an adjacent bond is = O; for the manufacture of a medicament for the treatment of pain, migraines, depression, anxiety, schizophrenia, Parkinson's disease or stroke.

p38 is a member of the mitogen-activated protein (MAP) kinase family and is a critical enzyme in the proinflammatory cytokine pathway. Other MAP kinase group members that share both structural and functional homology to p38 include the c-Jun NH2-terminal kinases (JNKs or SAPKs) and the extracellular-regulated protein kinases (ERKs). In this study, we determined the molecular basis for p38alpha inhibitor specificity exhibited by five compounds in the diarylimidazole, triarylimidazole, and triarylpyrrole classes of protein kinase inhibitors. These compounds are significantly more potent inhibitors of p38 compared to the JNKs and ERKs. Three active site ATP-binding domain residues in p38, T106, M109, and A157, selected based on primary sequence alignment, molecular modeling, and X-ray crystal structure data, were mutated to assess their role in inhibitor binding and enzymatic catalysis. All mutants, with the exception of T106M, had kinase activity within 3-fold of wild-type p38. Mutation of T106 to glutamine, the residue present at the corresponding position in ERK-2, or methionine, the corresponding residue in p38gamma, p38delta, and the JNKs, rendered all five inhibitors ineffective. The diarylimidazoles had approximately a 6-fold decrease in potency toward M109A p38. For the mutant A157V, all diarylimidazoles and triarylimidazoles tested were 5-10-fold more potent compared with wild-type p38. In contrast, two triarylpyrroles were 15-40-fold less potent versus A157V p38. These results showed that the molecular basis for the specificity of the p38 inhibitors was attributed largely to threonine 106 in p38 and that methionine 109 contributes to increased binding affinity for imidazole based inhibitors.