The public health challenges from RNA viruses are at a tipping point, with dengue moving up Florida, human transmission of chickungunya now established in Florida, and, transmitted by different mechanism, Ebola. While these have long been a challenge, their incidence has been predominantly overseas. In addition, the health hazards of other RNA viruses better known in the United States remains unabated. Eastern equine encephalitis is again emerging in New England, with its 30% mortality rate. West Nile virus continues as an important health hazard. Further, given the speed of international travel and the ease with which the sequences of RNA viruses mutate, a public health official can easily have a mosquito that carries an RNA virus from one of these classes that is nevertheless not detected by PCR with standard primers. This creates a need for exactly the product that Firebird's technologies enable. These technologies are: 1. Self-avoiding molecular recognition systems (SAMRS). SAMRS primers do not interact with each other. This allows unlimited multiplexing of PCR. Further, SAMRS allows additional targets to be added to an assay without the multiplex collapsing. SAMRS also suppresses primer-dimers in isothermal amplification (e.g. RPA, HDA), making possible (in Phase 2) point of sampling kits. SAMRS therefore supports a highly adaptable molecular diagnostics able to detect dozens of RNA species for essentially the cost of detecting one. 2. Artificially expanded genetic information systems (AEGIS). AEGIS adds nucleotides to the four in standard DNA and RNA (collectively xNA). Thus, AEGIS generates primers that cannot complement any natural DNA, no matter how complex the sample, supporting assays with very low noise and very few false positives. 3. Universal base technology (Biversals). RNA viruses easily mutate; exemplifying the consequences to public health, recent surveillance of HIV-infected patients with the goal of measuring incidence found that the viruses in ~ 20% of the isolates were not PCR amplified by standard primers. Firebird's evolutionary analyses show that mutations often occur in silent sites in coding regions. Thus, Firebird scientist invented biversal nucleobases that prime on either A or G (the Y-biversal), or on either T or C (the R-biversal) Phase 1 will generate a kit to detect 22 common mosquito-borne RNA viruses, allowing public health staff to screen on a Luminex platform for all of these for less than $50, less than the cost of detecting any two RNA viruses separately. In Phase 2, these will be moved to simpler platforms. Firebird's pipeline investments allow us to prepare AEGIS and SAMRS building blocks in multi-gram amounts, and AEGIS and SAMRS oligonucleotides on demand. Because surveillance assays are not heavily regulated, and since its customers for those kits (public health staff) are well trained, this product fits well within Firebird's current product line. However, strong performance of a low cost, highly multiplexed, easily adaptable, and low false-positive surveillance product will undoubtedly help Firebird raise capital for patient-targeted applications of these technologies.

Public Health Relevance

The public health challenges presented by RNA viruses are this year reaching a tipping point, with dengue moving its way up the Florida peninsula, human to human transmission of chickungunya now established in the United States (which, until this year, was always imported from an exotic tropical locale), Eastern equine encephalitis (now being detected in New England, with a 30% mortality rate) and, of course operating by an entirely different mechanism of transmission, Ebola, which is uncontrolled at this moment in Western Africa. This creates a need for exactly the product that Firebird's technologies enable: a highly adaptable molecular diagnostics assay that is able to detect dozens of RNA species for essentially the same cost as detecting a single RNA virus, design to manage the rapid divergence of these species, and capable of extraordinarily few false positives. This project will develop kits that allow a single public health official, in a single step, to test real biological samples (for example, 200 mosquitoes caught in a single trap) for all RNA viruses at the same time and with the same total cost as a test for any one of them separately. Since such surveillance kits have few regulatory barriers, Firebird will be able to bring these kits to market on its own before the end of the Phase 2 project period.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Small Business Innovation Research Grants (SBIR) - Phase I (R43)
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Special Emphasis Panel (ZRG1-IMST-K (14))
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Maas, Stefan
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Firebird Biomolecular Sciences, LLC
United States
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Glushakova, Lyudmyla G; Alto, Barry W; Kim, Myong Sang et al. (2017) Detection of chikungunya viral RNA in mosquito bodies on cationic (Q) paper based on innovations in synthetic biology. J Virol Methods 246:104-111