Multi-Gene Detection and Identification of Mosquito-Borne RNA Viruses Using an Oligonucleotide Microarray

<div><p>Background</p><p>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 <i>Flavivirus</i> (family <i>Flaviviridae</i>), <i>Alphavirus</i> (<i>Togaviridae</i>), <i>Orthobunyavirus</i> (<i>Bunyaviridae</i>), and <i>Phlebovirus</i> (<i>Bunyaviridae</i>).</p><p>Methodology/Principal Findings</p><p>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, <i>Culex</i> flavivirus, and a Quang Binh-like virus from mosquitoes collected in Thailand in 2011 and 2012.</p><p>Conclusions/Significance</p><p>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.</p></div

PCR amplification and microarray detection of flaviviruses.

<p>Viral RNA derived from cell culture or infected mosquitoes were PCR amplified using GSPs. Amplicons were analyzed using the ArboChip5.1 microarray. JE, Japanese encephalitis; MVE, Murray Valley encephalitis; SLE, St. Louis encephalitis; L1, lineage 1; L2, lineage 2; POS, positive PCR amplification; NEG, negative PCR amplification; yes, detected by microarray; no, not detected by microarray; n.t., not tested.</p>a<p>Viruses propagated in cell culture and identified by microarray to species unless otherwise noted.</p>b<p>Virus-infected mosquito used for microarray evaluations, microarray detected at least one target for each virus.</p>c<p>PCR amplification produced a weak visible band.</p>d<p>ArboChip5.1 does not include probes specific to the target.</p>e<p>Microarray detection with genus-level probes only.</p

PCR amplification and microarray detection of phleboviruses.

<p>Viral RNA derived from cell culture or infected mosquitoes were PCR amplified using GSPs. Amplicons were analyzed using the ArboChip5.1 microarray. POS, positive PCR amplification; NEG, negative PCR amplification; yes, detected by microarray; no, not detected by microarray; n.t., not tested.</p>a<p>Viruses propagated in cell culture and identified by microarray to species unless otherwise noted.</p>b<p>Virus-infected mosquito used for microarray evaluations, microarray detected at least one target for each virus.</p>c<p>ArboChip5.1 does not include probes specific to the target.</p>d<p>Microarray detection with genus-level probes only.</p>e<p>Not a target virus on ArboChip5.1.</p

PCR amplification and microarray detection of alphaviruses.

<p>Viral RNA derived from cell culture or infected mosquitoes were PCR amplified using GSPs. Amplicons were analyzed using the ArboChip5.1 microarray. VEE, Venezuelan equine encephalitis; WEE, western equine encephalomyelitis; POS, positive PCR amplification; NEG, negative PCR amplification; yes, detected by microarray; no, not detected by microarray; n.t., not tested.</p>a<p>Viruses propagated in cell culture and identified by microarray to species unless otherwise noted.</p>b<p>Virus-infected mosquito used for microarray evaluations, microarray detected at least one target for each virus.</p>c<p>PCR amplification produced a weak visible band.</p>d<p>ArboChip5.1 does not include probes specific to the target.</p>e<p>Microarray detection with genus-level probes only.</p>f<p>Target was detected but could not be differentiated between BBKV, OCKV, and SINV.</p>g<p>Target was detected but could not be differentiated between ONNV and IOV.</p

Lower limits of detection from virus-infected mosquito pools.

<p>The lower limits of detection determined for infected mosquitoes were evaluated by using 10-fold serial dilutions of RNA extracted from one infected mosquito pooled with 24 uninfected mosquitoes. PFUe, plaque forming unit equivalents; qPCR, real-time PCR, Conv. PCR, conventional PCR.</p

Visual analysis of cell-culture derived RVFV strain ZH548.

<p><i>Phlebovirus</i> gene-specific PCR amplicons were identified using oligonucleotide microarray probes grouped by virus genus and segment, (A) <i>Phlebovirus</i> S segment, (B) <i>Phlebovirus</i> M segment, and (C) <i>Phlebovirus</i> L segment, and sorted into subgroups based on phylogenetic clade and target virus(es). ECD signals were converted into z-scores. Subgroups with average z-scores greater than 10 were considered positive and used for virus identification. The plotted maximum z-scores represent the greatest individual probe z-score within a subgroup and were used to determine cross-hybridization. The PCR amplicons hybridized with probes in the RVFV subgroups for all three segment targets and <i>Phlebovirus</i> generic subgroup for L segment. The virus abbreviations are defined in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002349#pntd.0002349.s003" target="_blank">Table S1</a>.</p

Microarray reproducibility.

<p>Microarray analysis using cell-cultured derived viruses were performed in four replicates. The z-scores of the virus-specific probes were averaged and listed for each run. The mean and standard deviation (SD) was calculated to determine the 95% confidence interval (CI) and coefficient of variance (CV) for each virus.</p

Microarray assay workflow.

<p>Microarray assay workflow.</p

Visual analysis of <i>Ae. aegypti</i> mosquito pool Th9-0122 collected in Thailand.

<p>Screening methods indicated that the mosquito pool contained flavivirus RNA and cDNA from the pool was PCR amplified with <i>Flavivirus</i> (A) E, (B) NS3, and (C) NS5 GSPs. The PCR amplicons hybridized only with probes in the DENV3 subgroups for the (A) E and (C) NS5 gene targets. For the (B) NS3 gene, the amplicons hybridized with probes in the DENV clade and DENV2/3 (hybridizes with DENV2 and DENV3) subgroups, but did not hybridize with the DENV2-specific subgroup. The mosquito pool was identified as containing DENV-3 RNA.</p

PCR amplification and microarray detection of orthobunyaviruses.

<p>Viral RNA derived from cell culture or infected mosquitoes were PCR amplified using GSPs. Amplicons were analyzed using the ArboChip5.1 microarray. POS, positive PCR amplification; NEG, negative PCR amplification; yes, detected by microarray; no, not detected by microarray; n.t., not tested.</p>a<p>Viruses propagated in cell culture and identified by microarray to species unless otherwise noted.</p>b<p>Virus-infected mosquito used for microarray evaluations, microarray detected at least one target for each virus.</p>c<p>ArboChip5.1 does not include probes specific to the target.</p>d<p>GSP set not specific for this clade.</p>e<p>CEV clade detection only.</p