Inactivation of West Nile virus in serum with heat, ionic detergent, and reducing agent for proteomic applications.

Research involving biosafety level 3 pathogens such as West Nile virus (WNV) is often limited by the limited space and technical constraints of these environments. To conduct complex analytical studies outside of high containment, robust and reliable inactivation methods are needed that maintain compatibility with downstream assays. Here we report the inactivation of WNV in spiked serum samples using a commercially available SDS-PAGE sample buffer for proteomic studies. Using this method, we demonstrate its utility by identification proteins differentially expressed in the serum of mice experimentally infected with WNV.

Real-time reverse transcriptase polymerase chain reaction assays for Middle East Respiratory Syndrome.

Here we designed and tested two highly specific quantitative TaqMan(®)-MGB-based reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assays for Middle East Respiratory Syndrome (MERS). The primers and probes for these assays were evaluated and found to have a limit of detection (LOD) of 0.005 plaque-forming units/PCR (pfu/PCR).

Demonstration of the Pre-Emergency Use Authorization Path Using 3 Minor Groove Binder-Hydrolysis Probe Assays to Detect Escherichia coli O104:H4.

BACKGROUND: The Department of Defense (DoD) and the Food and Drug Administration (FDA) have collaboratively worked on a pre-Emergency Use Authorization (pre-EUA) process for in vitro diagnostic (IVD) devices, using FDA's regulatory flexibilities under the EUA authorities. The pre-EUA process enables FDA review of data in anticipation of a request for an EUA, advancing US government public health emergency preparedness efforts. METHODS: The IVD device developed to detect Escherichia coli O104:H4, for which an EUA has not been issued, serves as an example to illustrate that process. Specifically, DoD designed real-time PCR assays to target the virulent E. coli strain O104:H4 (etiological agent of the 2011 German outbreak) including: fliC (flagellin), Agg3C (AAF), and rfb (wbwC) on the basis of the published sequences. RESULTS: After development and optimization of these 3 specific assays, a defined protocol was followed to determine and document the sensitivity and specificity of each assay analytically. CONCLUSIONS: FDA reviewed these data and returned commentary on additional required experiments to complete the pre-EUA process and expedite the use of the device should there be an emergency need for an IVD device to detect this virulent E. coli strain before such a test is cleared by FDA.

Finished Genome Assembly of Yersinia pestis EV76D and KIM 10v.

Here, we sequenced the completed genome of Yersinia pestis EV76D and KIM 10v, two genomes used as references in assay development, to improved high-quality draft status.

Finished Genome Assembly of Warm Spring Isolate Francisella novicida DPG 3A-IS.

We sequenced the complete genome of Francisella novicida DPG 3A-IS to closed and finished status. This is a warm spring isolate recovered from Hobo Warm Spring (Utah, USA). The final assembly is available in NCBI under accession number CP012037.

Genome sequencing of 18 francisella strains to aid in assay development and testing.

Francisella tularensis is a highly infectious bacterium with the potential to cause high fatality rates if infections are untreated. To aid in the development of rapid and accurate detection assays, we have sequenced and annotated the genomes of 18 F. tularensis and Francisella philomiragia strains.

Thirty-Two Complete Genome Assemblies of Nine Yersinia Species, Including Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica.

The genus Yersinia includes three human pathogens, of which Yersinia pestis is responsible for >2,000 illnesses each year. To aid in the development of detection assays and aid further phylogenetic elucidation, we sequenced and assembled the complete genomes of 32 strains (across 9 Yersinia species).

Complete genome sequences for 35 biothreat assay-relevant bacillus species.

In 2011, the Association of Analytical Communities (AOAC) International released a list of Bacillus strains relevant to biothreat molecular detection assays. We present the complete and annotated genome assemblies for the 15 strains listed on the inclusivity panel, as well as the 20 strains listed on the exclusivity panel.

Complete genome sequences for 59 burkholderia isolates, both pathogenic and near neighbor.

The genus Burkholderia encompasses both pathogenic (including Burkholderia mallei and Burkholderia pseudomallei, U.S. Centers for Disease Control and Prevention Category B listed), and nonpathogenic Gram-negative bacilli. Here we present full genome sequences for a panel of 59 Burkholderia strains, selected to aid in detection assay development.

Draft Genome Assembly of Acinetobacter baumannii ATCC 19606.

Acinetobacter baumannii is an emerging nosocomial pathogen, and therefore high-quality genome assemblies for this organism are needed to aid in detection, diagnostic, and treatment technologies. Here we present the improved draft assembly of A. baumannii ATCC 19606 in two scaffolds. This 3,953,621-bp genome contains 3,750 coding regions and has a 39.1% G+C content.

Integrating high-content imaging and chemical genetics to probe host cellular pathways critical for Yersinia pestis infection.

The molecular machinery that regulates the entry and survival of Yersinia pestis in host macrophages is poorly understood. Here, we report the development of automated high-content imaging assays to quantitate the internalization of virulent Y. pestis CO92 by macrophages and the subsequent activation of host NF-κB. Implementation of these assays in a focused chemical screen identified kinase inhibitors that inhibited both of these processes. Rac-2-ethoxy-3 octadecanamido-1-propylphosphocholine (a protein Kinase C inhibitor), wortmannin (a PI3K inhibitor), and parthenolide (an IκB kinase inhibitor), inhibited pathogen-induced NF-κB activation and reduced bacterial entry and survival within macrophages. Parthenolide inhibited NF-κB activation in response to stimulation with Pam3CSK4 (a TLR2 agonist), E. coli LPS (a TLR4 agonist) or Y. pestis infection, while the PI3K and PKC inhibitors were selective only for Y. pestis infection. Together, our results suggest that phagocytosis is the major stimulus for NF-κB activation in response to Y. pestis infection, and that Y. pestis entry into macrophages may involve the participation of protein kinases such as PI3K and PKC. More importantly, the automated image-based screening platform described here can be applied to the study of other bacteria in general and, in combination with chemical genetic screening, can be used to identify host cell functions facilitating the identification of novel antibacterial therapeutics.

Rapid real-time PCR assays for detection of Klebsiella pneumoniae with the rmpA or magA genes associated with the hypermucoviscosity phenotype: screening of nonhuman primates.

The relationship of mucoviscosity-associated (magA) and/or regulator of mucoid phenotype (rmpA) genes to the Klebsiella pneumoniae hypermucoviscosity (HMV) phenotype has been reported. We previously demonstrated that rmpA+ K. pneumoniae can cause serious disease in African green monkeys and isolated rmpA+ and magA+ HMV K. pneumoniae from other species of non-human primates. To rapidly screen African green monkeys/non-human primates for these infections, we developed three real-time PCR assays. The first was K. pneumoniae-specific, targeting the khe gene, while the others targeted rmpA and magA. Primer Express 2 was used with the three K. pneumoniae genes to generate sequence-specific TaqMan/TaqMan-Minor Groove Binder assays. Oral/rectal swabs and necropsy samples were collected; swabs were used for routine culture and DNA extraction. K. pneumoniae colonies were identified on the Vitek 2 with DNA tested using the K. pneumoniae-specific assays. Testing of 45 African green monkeys resulted in 19 khe+ samples from 14 animals with none positive for either rmpA or magA. Of these 19 khe+ samples, five were culture-positive, but none were HMV "string test"-positive. Subsequent testing of 307 non-human primates resulted in 64 HMV K. pneumoniae isolates of which 42 were rmpA+ and 15 were magA+. Non-human primate testing at the U.S. Army Medical Research Institute of Infectious Diseases demonstrated the ability to screen both live and necropsied animals for K. pneumoniae by culture and real-time PCR to determine HMV genotype.