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Pawtucket, RI, United States

Opal S.M.,Center for Biodefense and Emerging Pathogens | Opal S.M.,Brown University
Human Vaccines and Immunotherapeutics

Since December 2011, influenza virologists and biosecurity experts have been engaged in a controversial debate over research on the transmissibility of H5N1 influenza viruses. Influenza virologists disagreed with the NSABB's recommendation not to publish experimental details of their findings, whereas biosecurity experts wanted the details to be withheld and future research restricted. The virologists initially declared a voluntary moratorium on their work, but later the NSABB allowed their articles to be published, and soon transmissibility research will resume. Throughout the debate, both sides have had understandable views, but both have overlooked the more important question of whether anything could be done if one of these experimentally derived viruses or a naturally occurring and highly virulent influenza virus should emerge and cause a global pandemic. This is a crucial question, because during the 2009 H1N1 influenza pandemic, more than 90% of the world's people had no access to timely supplies of affordable vaccines and antiviral agents. Observational studies suggest that inpatient statin treatment reduces mortality in patients with laboratory-confirmed seasonal influenza. Other immunomodulatory agents (glitazones, fibrates and AMPK agonists) improve survival in mice infected with influenza viruses. These agents are produced as inexpensive generics in developing countries. If they were shown to be effective, they could be used immediately to treat patients in any country with a basic health care system. For this reason alone, influenza virologists and biosecurity experts need to join with public health officials to develop an agenda for laboratory and clinical research on these agents. This is the only approach that could yield practical measures for a global response to the next influenza pandemic. © 2013 Landes Bioscience. Source

Artenstein A.W.,Center for Biodefense and Emerging Pathogens | Artenstein A.W.,Brown University | Opal S.M.,Center for Biodefense and Emerging Pathogens | Opal S.M.,Brown University
Clinical Infectious Diseases

Anthrax continues to generate concern as an agent of bioterrorism and as a natural cause of sporadic disease outbreaks. Despite the use of appropriate antimicrobial agents and advanced supportive care, the mortality associated with the systemic disease remains high. This is primarily due to the pathogenic exotoxins produced by Bacillus anthracis as well as other virulence factors of the organism. For this reason, new therapeutic strategies that target events in the pathogenesis of anthrax and may potentially augment antimicrobials are being investigated. These include anti-toxin approaches, such as passive immune-based therapies; non-antimicrobial drugs with activity against anthrax toxin components; and agents that inhibit binding, processing, or assembly of toxins. Adjunct therapies that target spore germination or downstream events in anthrax intoxication are also under investigation. In combination, these modalities may enhance the management of systemic anthrax. © 2012 The Author. Source

Kerby M.B.,Brown University | Kerby M.B.,Stanford University | Sarma A.A.,Brown University | Sarma A.A.,Harvard University | And 8 more authors.
Applied Biochemistry and Biotechnology

Rapid diagnostic identification of the human H5 influenza virus is a strategic cornerstone for outbreak prevention. We recently reported a method for direct detection of viral RNA from a highly pathogenic human H5 influenza strain (A/Hanoi/30408/2005(H5N1)), which necessarily was transcribed in vitro from non-viral sources. This article provides an in-depth analysis of the reaction conditions for in vitro transcription (IVT) of full-length influenza H5 RNA, which is needed for diagnostic RNA production, for the T7 and SP6 phage promoter systems. Gel analysis of RNA transcribed from plasmids containing the H5 sequence between a 5′ SP6 promoter and 3′ restriction site (BsmBI) showed that three sequence-verified bands at 1,776, 784, and 591 bases were consistently produced, whereas only one 1,776-base band was expected. These fragments were not observed in H1 or H3 influenza RNA transcribed under similar conditions. A reverse complement of the sequence produced only a single band at 1,776 bases, which suggested either self-cleavage or early termination. Aliquots of the IVT reaction were quenched with EDTA to track the generation of the bands over time, which maintained a constant concentration ratio. The H5 sequence was cloned with T7 and SP6 RNA polymerase promoters to allow transcription in either direction with either polymerase. The T7 transcription product from purified, restricted plasmids in the vRNA direction only produced the 1,776-base full-length sequence and the 784-base fragment, instead of the three bands generated by the SP6 system, suggesting an early termination mechanism. Additionally, the T7 system produced a higher fraction of full-length vRNA transcripts than the SP6 system did under similar reaction conditions. By sequencing we identified a type II RNA hairpin loop terminator, which forms in a transcription direction-dependent fashion. Variation of the magnesium concentration produced the greatest impact on termination profiles, where some reaction mixtures were unable to produce full-length transcripts. Optimized conditions are presented for the T7 and SP6 phage polymerase systems to minimize these early termination events during in vitro transcription of H5 influenza vRNA. © 2010 Springer Science+Business Media, LLC. Source

Ong C.,Brown University | Tai W.,Brown University | Sarma A.,Brown University | Opal S.M.,Brown University | And 4 more authors.
Journal of Molecular Diagnostics

This work presents a novel method for detecting nucleic acid targets using a ligation step along with an isothermal, exponential amplification step. We use an engineered ssDNA with two variable regions on the ends, allowing us to design the probe for optimal reaction kinetics and primer binding. This two-part probe is ligated by T4 DNA Ligase only when both parts bind adjacently to the target. The assay demonstrates that the expected 72-nt RNA product appears only when the synthetic target, T4 ligase, and both probe fragments are present during the ligation step. An extraneous 38-nt RNA product also appears due to linear amplification of unligated probe (P3), but its presence does not cause a false-positive result. In addition, 40 mmol/L KCl in the final amplification mix was found to be optimal. It was also found that increasing P5 in excess of P3 helped with ligation and reduced the extraneous 38-nt RNA product. The assay was also tested with a single nucleotide polymorphism target, changing one base at the ligation site. The assay was able to yield a negative signal despite only a single-base change. Finally, using P3 and P5 with longer binding sites results in increased overall sensitivity of the reaction, showing that increasing ligation efficiency can improve the assay overall. We believe that this method can be used effectively for a number of diagnostic assays. © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology. Source

McCalla S.E.,Brown University | Ong C.,Brown University | Sarma A.,Brown University | Opal S.M.,Brown University | And 4 more authors.
Journal of Molecular Diagnostics

We present a novel and simple method for amplifying RNA targets (named by its acronym, SMART), and for detection, using engineered amplification probes that overcome existing limitations of current RNA-based technologies. This system amplifies and detects optimal engineered ssDNA probes that hybridize to target RNA. The amplifiable probe-target RNA complex is captured on magnetic beads using a sequence-specific capture probe and is separated from unbound probe using a novel microfluidic technique. Hybridization sequences are not constrained as they are in conventional target-amplification reactions such as nucleic acid sequence amplification (NASBA). Our engineered ssDNA probe was amplified both off-chip and in a microchip reservoir at the end of the separation microchannel using isothermal NASBA. Optimal solution conditions for ssDNA amplification were investigated. Although KCl and MgCl 2 are typically found in NASBA reactions, replacing 70 mmol/L of the 82 mmol/L total chloride ions with acetate resulted in optimal reaction conditions, particularly for low but clinically relevant probe concentrations (≤100 fmol/L). With the optimal probe design and solution conditions, we also successfully removed the initial heating step of NASBA, thus achieving a true isothermal reaction. The SMART assay using a synthetic model influenza DNA target sequence served as a fundamental demonstration of the efficacy of the capture and microfluidic separation system, thus bridging our system to a clinically relevant detection problem. Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved. Source

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