Stabilization of the RNA Complement of Enveloped Viruses: Beta Testing and Manufacturing Scale-up of a Novel Filter Paper Product for the Field Collection of Dried Blood Spots

Abstract. In 1960, filter paper based sample collection with Guthrie Cards revolutionized blood-based screening of small molecule biomarkers of birth defect: via the collection of heal stick blood directly onto filter paper, to form a dried blood spot (DBS). In the 2000andapos;s, Guthrie Card (Whatman 903) DBS sample collection was extended (by this Team and many others) to DNA biomarkers and to analysis of blood-borne bacteria and DNA viruses. That substantial success with DNA, has shifted research to the “loftier” goal of collecting and preserving the highly unstable RNA complement of DBS: with emphasis on tools to enable the refrigeration-free collection of blood. This supports screening of RNA virus infections. Numerous publications have subsequently emerged, to establish the use of Whatman 903 DBS for HIV, Zika and other emerging RNA viruses. The Good News is, those several published studies have confirmed viral RNA can be recovered by quantitative RT-PCR (qRT-PCR) from DBS obtained from a finger prick, if collected and stored “appropriately”. The Bad News is, published studies have shown that adequate RNA stability can be obtained only under unrealistic conditions (4C, -20C storage). Under conditions of tropical ambient temperatures (30C-40C) those studies have concluded that DBS collection and transport does not work: i.e. RNA degrades beyond the qRT-PCR detection limit within less than a week. This Phase IIB SBIR is focused on beta testing of a pair of fundamentally new filter paper based products (GT-A andamp; GT-B) created by the Applicant Team. For the first time, it will be possible to collect/ship viremic blood under realistic tropical conditions; 2 weeks of continuous 40C storage, yielding RNA for standard qRT-PCR analysis. The core technology exploited in this Phase IIB, originally developed by GenTegra with DARPA funding, uses two different approaches to viral RNA preservation: GT-A, to stabilize the structure of the virus in a DBS, thus exploiting the natural protection afforded by that intact structure; and GT-B, to instantly lyse the virus, simultaneously introducing into the DBS, a novel panel of nuclease and free radical inhibitors. Phase IIB beta test is to be validated by two top labs: ATCC (for Zika) and Stanford (for HIV-1), for both technologies. GenTegra will in parallel test with other viruses. Upon completion of this Phase IIB, one or both products will be ready for international sales andamp; marketing, supported by the Existing collaboration between GenTegra and Ahlstrom-Munksjö.Project Narrative A Phase IIB SBIR is described, which is focused on testing and validation of two filter paper based products, GT-A and GT-B, invented by GenTegra, to preserve the blood borne RNA viruses that may be collected in the field as a dried blood spot, delivered from a finger prick. HIV-1 and Zika will be the first test cases to be validated, at two top international laboratories: the ATCC (Zika) and Stanford (HIV-1). It is proposed that both products will be unique in molecular epidemiology as they will be the first to stabilize RNA viruses, in the face of extremely primitive field conditions (no cold storage capability), to support advanced methods of RNA based viral detection and sequencing.