Multi-gene detection and identification of mosquito-borne RNA viruses using an oligonucleotide microarray

PLoS Negl Trop Dis. 2013 Aug 15;7(8):e2349. doi: 10.1371/journal.pntd.0002349. eCollection 2013.

Abstract

Background: Arthropod-borne viruses are important emerging pathogens world-wide. Viruses transmitted by mosquitoes, such as dengue, yellow fever, and Japanese encephalitis viruses, infect hundreds of millions of people and animals each year. Global surveillance of these viruses in mosquito vectors using molecular based assays is critical for prevention and control of the associated diseases. Here, we report an oligonucleotide DNA microarray design, termed ArboChip5.1, for multi-gene detection and identification of mosquito-borne RNA viruses from the genera Flavivirus (family Flaviviridae), Alphavirus (Togaviridae), Orthobunyavirus (Bunyaviridae), and Phlebovirus (Bunyaviridae).

Methodology/principal findings: The assay utilizes targeted PCR amplification of three genes from each virus genus for electrochemical detection on a portable, field-tested microarray platform. Fifty-two viruses propagated in cell-culture were used to evaluate the specificity of the PCR primer sets and the ArboChip5.1 microarray capture probes. The microarray detected all of the tested viruses and differentiated between many closely related viruses such as members of the dengue, Japanese encephalitis, and Semliki Forest virus clades. Laboratory infected mosquitoes were used to simulate field samples and to determine the limits of detection. Additionally, we identified dengue virus type 3, Japanese encephalitis virus, Tembusu virus, Culex flavivirus, and a Quang Binh-like virus from mosquitoes collected in Thailand in 2011 and 2012.

Conclusions/significance: We demonstrated that the described assay can be utilized in a comprehensive field surveillance program by the broad-range amplification and specific identification of arboviruses from infected mosquitoes. Furthermore, the microarray platform can be deployed in the field and viral RNA extraction to data analysis can occur in as little as 12 h. The information derived from the ArboChip5.1 microarray can help to establish public health priorities, detect disease outbreaks, and evaluate control programs.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Culicidae / virology*
  • Oligonucleotide Array Sequence Analysis / methods*
  • RNA Viruses / classification
  • RNA Viruses / genetics
  • RNA Viruses / isolation & purification*
  • Thailand
  • Virology / methods*

Grants and funding

This research was funded by the Military Infectious Disease Research Program project # U0176_09_RD, U0321_12_RD (JSL), and supported in part by an appointment to the Postgraduate Research Participation Program at the U.S. Army Medical Research Institute of Infectious Disease administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and USAMRMC (NDG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.