Rapid and accurate diagnosis of individuals exposed to and / or infected with NIAID Category A-C viral pathogens is critical because of the threat to national security and potential to spread and quickly disseminate in a vulnerable population. Currently available diagnostics are typically neither able to diagnose all known viral causes of outbreaks or bioterrorist threats in a single test, nor capable of identifying novel variants of known viruses. We have previously developed a DNA microarray (Virochip) as a broad-spectrum surveillance platform for detection of all known viruses. Since the Virochip does not rely on use of virus-specific priming, it is also well-suited for detection of novel viruses, and has been previously used to identify SARS coronavirus and 2009 novel H1N1 influenza A virus in the absence of a priori knowledge of the pathogen. In conjunction with Akonni Biosystems, we propose to develop and validate a Virochip-based diagnostic assay for Category A-C viral pathogens (Biothreat Detection Virochip) that is rapid (<2 hour turnaround time), sensitive / specific, portable, and cost-effective, yet capable of simultaneously detecting all Category A-C viruses as well as related novel variants. We will also develop and test a secondary Pan-Influenza Detection Virochip for rapid subtyping and characterization of novel influenza strains. We will rigorously assess the performance characteristics of these assays for detection of Category A-C viral pathogens using positive clinical specimens, cultured virus stocks, and cDNA plasmids / in vitro transcribed mRNA containing viral sequence. We will test viruses that are BSL-3/4 select biological agents using clinical specimens from infected humans as well as from non-human primates and rodents inoculated with live virus. Through an extensive network of worldwide collaborators in Canada, Mexico, the United States, India, and Africa, we will analyze surveillance and outbreak clinical specimens (n ~ 2,500) for Category A-C viral pathogens in cases of influenza-like illness, diarrhea, sepsis, and encephalitis. Our goal is a clinical sensitivity of >90% and specificity of >99% for detection of Category A-C viral pathogens relative to PCR methods and conventional laboratory testing, using direct viral sequence recovery as the gold standard for detection. We will analyze the remaining negative outbreak specimens by deep sequencing methods to discover novel viruses associated with outbreaks, and perform preliminary conventional and molecular epidemiology to assess whether these novel viruses are likely to be causal. This project is innovative because it translates a well-established pan-viral microarray platform that has the additional capacity to detect novel variants (Virochip) into a portable, sample-to-answer system (Akonni) for rapid detection of all Category A-C viruses. The significance of this research is that a rapid and comprehensive yet portable diagnostic assay for Category A-C viruses could be deployed in public health agencies, hospitals, and point-of-care sites to assist in outbreak and bioterrorist investigation. The use of panviral microarray and deep sequencing strategies will also facilitate efforts to discover novel viruses that may be previously unrecognized emerging pathogens or potential agents of bioterrorism.