We propose to develop a portable, self-contained, microfabricated device for extraction of genomic information from RNA or DNA viruses. Initially, we choose as a model system and as an important target the hemagglutinin HA1 of influenza A virus. Influenza is a prevalent human pathogen with an RNA genome. Mutations in the hemagglutinin (HA1) domains of influenza regularly produce new virulent forms that are responsible for 6 percent of annual mortalities in the U.S.A. Seasonal changes in influenza HA1 have a major impact on influenza epidemics and public health, and pose an on-going threat of world-wide pandemic. From analyses of influenza virus evolution, 18 of the most dangerous mutation sites have been identified. A present need is a reliable means to rapidly survey domestic and foreign populations for the emergence of new mutations. A self-contained, inexpensive, microfabricated device that can rapidly detect viral mutations using a small amount of sample would address this need. To expedite development of such a device we will perform research to achieve the following specific aims:
Aim 1 - Determine the Influenza-A RNA purity requirements for Aims 2-4 by preparing samples of three levels: (a) cultured viral-infected cells, (b) purified whole viral particles, and (c) purified viral RNA.
Aim 2 - On a microfabricated device, reverse transcribe and amplify the HA1 hemagglutinin domain of Influenza A using reverse-transcription PCR to produce double-stranded complementary DNA.
Aim 3 - On a microfabricated device, perform fluorescent primer extension reactions on double-stranded DNA produced in Aim 2 to detect variations in bases in codons from the HA1 domain of hemagglutinin that have been involved in past viral mutations.
Aim 4 - On a microfabricated device, separate primer-extended DNA products by gel electrophoresis and identify the locations of the base variations.
Aim 5 - Integrate RNA separation, RT-PCR, primer extension reactions, electrophoretic separation, and (if necessary) RNA purification on a single microfabricated device.
Aim 6 - Develop a silica gel RNA-adsorption column for purification of RNA on a microfabricated device.
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