Arbovirus Detection in Insect Vectors by Rapid, High-Throughput Pyrosequencing

Abstract

BackgroundDespite the global threat caused by arthropod-borne viruses, there is not an efficient method for screening vector populations to detect novel viral sequences. Current viral detection and surveillance methods based on culture can be costly and time consuming and are predicated on prior knowledge of the etiologic agent, as they rely on specific oligonucleotide primers or antibodies. Therefore, these techniques may be unsuitable for situations when the causative agent of an outbreak is unknown.Methodology/Principal FindingsIn this study we explored the use of high-throughput pyrosequencing for surveillance of arthropod-borne RNA viruses. Dengue virus, a member of the positive strand RNA Flavivirus family that is transmitted by several members of the Aedes genus of mosquitoes, was used as a model. Aedes aegypti mosquitoes experimentally infected with dengue virus type 1 (DENV-1) were pooled with noninfected mosquitoes to simulate samples derived from ongoing arbovirus surveillance programs. Using random-primed methods, total RNA was reverse-transcribed and resulting cDNA subjected to 454 pyrosequencing.Conclusions/SignificanceIn two types of samples, one with 5 adult mosquitoes infected with DENV-1- and the other with 1 DENV-1 infected mosquito and 4 noninfected mosquitoes, we identified DENV-1 DNA sequences. DENV-1 sequences were not detected in an uninfected control pool of 5 adult mosquitoes. We calculated the proportion of the Ae. aegypti metagenome contributed by each infecting Dengue virus genome (pIP), which ranged from 2.75×10−8 to 1.08×10−7. DENV-1 RNA was sufficiently concentrated in the mosquito that its detection was feasible using current high-throughput sequencing instrumentation. We also identified some of the components of the mosquito microflora on the basis of the sequence of expressed RNA. This included members of the bacterial genera Pirellula and Asaia, various fungi, and a potentially uncharacterized mycovirus.

Insect-Specific Viruses Detected in Laboratory Mosquito Colonies and Their Potential Implications for Experiments Evaluating Arbovirus Vector Competence

“…1 Recent studies have shown that the microbiota of mosquitoes can affect their ability to become infected with and to vector certain pathogens. [2][3][4][5] There is a growing realization that mosquito populations are naturally infected with a wide range of bacteria, 6,7 fungi, 8 and viruses [9][10][11][12][13][14][15] ; and this recognition has raised interest in the potential effects of these microbes on mosquito ecology and on pathogen transmission.…”

Optimization of dengue virus genome assembling using GSFLX 454 pyrosequencing data: evaluation of assembling strategies

“…In the past, efforts have been made to use NGS-based approaches, in particular pyrosequencing, for obtaining genetic data from different viral pathogens, such as HIV; avian leukosis virus; lyssaviruses; enteric viruses, and few arboviruses, including DENV-1 and phleboviruses (Victoria et al, 2009;Bimber et al, 2010;Bishop-Lilly et al, 2010;Day et al, 2010;Hedskog et al, 2010;De Wolf et al, 2011;Schinazi et al, 2011;Abbate et al, 2011;De Benedictis et al, 2011;Palacios et al, 2011).…”

Next-Generation Sequencing for Pathogen Detection and Identification

“…Methods for removal of host nucleic acid have mostly focused on rRNA (O'Neil, Glowatz, & Schlumpberger, 2013). In the case of RNA-seq studies or detection of RNA viruses, these methods improve coverage of microbial genomes (Bishop-Lilly et al, 2010). However, especially in the case of clinical samples, often host nucleic acid is genomic DNA.…”