Detection of arboviruses and other micro-organisms in experimentally infected mosquitoes using massively parallel sequencing

Abstract

Human disease incidence attributed to arbovirus infection is increasing throughout the world, with effective control interventions limited by issues of sustainability, insecticide resistance and the lack of effective vaccines. Several promising control strategies are currently under development, such as the release of mosquitoes trans-infected with virus-blocking Wolbachia bacteria. Implementation of any control program is dependent on effective virus surveillance and a thorough understanding of virus-vector interactions. Massively parallel sequencing has enormous potential for providing comprehensive genomic information that can be used to assess many aspects of arbovirus ecology, as well as to evaluate novel control strategies. To demonstrate proof-of-principle, we analyzed Aedes aegypti or Aedes albopictus experimentally infected with dengue, yellow fever or chikungunya viruses. Random amplification was used to prepare sufficient template for sequencing on the Personal Genome Machine. Viral sequences were present in all infected mosquitoes. In addition, in most cases, we were also able to identify the mosquito species and mosquito micro-organisms, including the bacterial endosymbiont Wolbachia. Importantly, naturally occurring Wolbachia strains could be differentiated from strains that had been trans-infected into the mosquito. The method allowed us to assemble near full-length viral genomes and detect other micro-organisms without prior sequence knowledge, in a single reaction. This is a step toward the application of massively parallel sequencing as an arbovirus surveillance tool. It has the potential to provide insight into virus transmission dynamics, and has applicability to the post-release monitoring of Wolbachia in mosquito populations.

Figure 1. Comparison of sequence coverage and depth of the virus genomes.

Products were generated using sequence-independent amplification from mosquitoes 1 and 2, which were infected with DENV-3 (A and B, respectively), mosquitoes 2 and 3, which were infected with CHIKV (C and D, respectively), and mosquito 5, which was infected with YFV and amplified either using sequence-independent amplification (E) or GenomiPhi amplification (F). These products were sequenced and assembled to a reference sequence as shown below the coverage diagram for each sample (DENV-3, NC_001475.2; CHIKV, DQ443544.2; YFV, NC_002031.1). The genome coverage (x-axis) and depth of coverage (y-axis) is shown.

Figure 2. Relationship of Wolbachia derived from Ae. albopictus and Ae. aegypti mosquitoes based on 16S ribosomal RNA sequence.

Ribosomal RNA sequences were assembled using a reference sequence, and a multiple alignment file was constructed. This was used to calculate a distance matrix and phylogenetic tree using the neighbour joining method. For comparison, Wolbachia 16S ribosomal RNA from field-caught Ae. albopictus (Accession number X61767.1) and D. melanogaster (Accession number AE017196.1) were included, and 16S ribosomal RNA from Rickettsia prowazekii (Accession number NC_017560.1) was used as an out-group. The numbers shown are the bootstrapping values for 1000 replicates.