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

Nathan D Grubaugh,John S Lee,Scott S Mcmenamy,Michael J Turell

doi:10.1371/journal.pntd.0002349

Abstract

BackgroundArthropod-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 FindingsThe 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/SignificanceWe 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.

Highlights

Arthropod-borne viruses are important human and veterinary pathogens that are biologically transmitted to vertebrates by hematophagous arthropod vectors, such as female mosquitoes
Mosquito-borne flaviviruses are phylogenetically divided into two groups: those transmitted by Aedes species mosquitoes, such as dengue virus (DENV) and yellow fever virus (YFV), and those transmitted by Culex species mosquitoes, such as Japanese encephalitis virus (JEV) and West Nile virus (WNV)
Half of the world’s population is at risk of viral, mosquito-borne illness such as dengue, yellow fever, Japanese encephalitis, Rift Valley fever, and chikungunya. These viruses have been regarded as pathogens of the tropics; they are emerging as global causes of illness

Summary

Introduction

Arthropod-borne viruses (arboviruses) are important human and veterinary pathogens that are biologically transmitted to vertebrates by hematophagous (blood feeding) arthropod vectors, such as female mosquitoes. The diverse group of mosquito-borne RNA viruses primarily includes flaviviruses (Flaviviridae: Flavivirus), alphaviruses (Togaviridae: Alphavirus), orthobunyaviruses (Bunyaviridae: Orthobunyavirus), and phleboviruses (Bunyaviridae: Phlebovirus) [1]. Alphavirus genomes are positive-sense, single-stranded RNA molecules, approximately 11.7 kb in length that translate into four nonstructural and three structural proteins [10]. They are classified in two geographically isolated groups, Old World alphaviruses, such as chikungunya virus (CHIKV) that is found in parts of Africa, Asia, and Europe, and New World alphaviruses, such as eastern equine encephalitis and Venezuelan equine encephalitis (VEEV) viruses that circulate in the Americas. 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)

Methods

Results

Discussion

Conclusion