DNA Microarray Platform for Detection and Surveillance of Viruses Transmitted by Small Mammals and Arthropods

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Abstract
Viruses transmitted by small mammals and arthropods serve as global threats to humans. Most emergent and re-emergent viral agents are transmitted by these groups; therefore, the development of high-throughput screening methods for the detection and surveillance of such viruses is of great interest. In this study, we describe a DNA microarray platform that can be used for screening all viruses transmitted by small mammals and arthropods (SMAvirusChip) with nucleotide sequences that have been deposited in the GenBank. SMAvirusChip was designed with more than 15,000 oligonucleotide probes (60-mers), including viral and control probes. Two SMAvirusChip versions were designed: SMAvirusChip v1 contains 4209 viral probes for the detection of 409 viruses, while SMAvirusChip v2 contains 4943 probes for the detection of 416 viruses. SMAvirusChip was evaluated with 20 laboratory reference-strain viruses. These viruses could be specifically detected when alone in a sample or when artificially mixed within a single sample. The sensitivity of SMAvirusChip was evaluated using 10-fold serial dilutions of dengue virus (DENV). The results showed a detection limit as low as 2.6E3 RNA copies/mL. Additionally, the sensitivity was one log10 lower (2.6E2 RNA copies/mL) than quantitative real-time RT-PCR and sufficient to detect viral genomes in clinical samples. The detection of DENV in serum samples of DENV-infected patients (n = 6) and in a whole blood sample spiked with DENV confirmed the applicability of SMAvirusChip for the detection of viruses in clinical samples. In addition, in a pool of mosquito samples spiked with DENV, the virus was also detectable. SMAvirusChip was able to specifically detect viruses in cell cultures, serum samples, total blood samples and a pool of mosquitoes, confirming that cellular RNA/DNA did not interfere with the assay. Therefore, SMAvirusChip may represent an innovative surveillance method for the rapid identification of viruses transmitted by small mammals and arthropods. Viruses transmitted by small mammals and blood-feeding insects represent global threats to humans. Most emergent viral agents are transmitted by these groups; therefore, the development of methods for the detection and surveillance of such viruses is of great interest. Here, we describe a DNA microchip platform (SMAvirusChip) that can be used for screening all known viruses that are transmitted by small mammals and blood-feeding insects. SMAvirusChip was evaluated using 20 laboratory reference-strain viruses, which were specifically detected when present alone in a sample or when artificially mixed within a single sample. SMAvirusChip had a detection limit as low as 2.6E3 RNA copies/mL, which was sufficient to detect viral genomes in clinical samples. The detection of DENV in serum samples of DENV-infected patients (n = 6) and in a whole blood sample artificially contaminated with DENV confirmed the applicability of SMAvirusChip for the detection of viruses in clinical samples. In addition, in a sample composed of a pool of mosquitoes that was spiked with DENV, virus was also detectable. Therefore, SMAvirusChip may serve as an innovative surveillance method for the rapid identification of viruses transmitted by small mammals and blood-feeding insects.
Funding Information
  • Fundação de Amparo à Pesquisa do Estado de São Paulo (2008/50617-6)
  • Fundação de Amparo à Pesquisa do Estado de São Paulo (2011/02045-6)
  • Conselho Nacional de Desenvolvimento Científico e Tecnológico (310735/2013-0)

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