Novel genome sequences of cell-fusing agent virus allow comparison of virus phylogeny with the genetic structure of Aedes aegypti populations

Abstract

Flaviviruses encompass not only medically relevant arthropod-borne viruses (arboviruses) but also insect-specific flaviviruses (ISFs) that are presumably maintained primarily through vertical transmission in the insect host. Interestingly, ISFs are commonly found infecting important arbovirus vectors such as the mosquito Aedes aegypti. Cell-fusing agent virus (CFAV) was the first described ISF of mosquitoes more than four decades ago. Despite evidence for widespread CFAV infections in A.aegypti populations and for CFAV potential to interfere with arbovirus transmission, little is known about CFAV evolutionary history. Here, we generated six novel CFAV genome sequences by sequencing three new virus isolates and subjecting three mosquito samples to untargeted viral metagenomics. We used these new genome sequences together with published ones to perform a global phylogenetic analysis of CFAV genetic diversity. Although there was some degree of geographical clustering among CFAV sequences, there were also notable discrepancies between geography and phylogeny. In particular, CFAV sequences from Cambodia and Thailand diverged significantly, despite confirmation that A.aegypti populations from both locations are genetically close. The apparent phylogenetic discrepancy between CFAV and its A.aegypti host in Southeast Asia indicates that other factors than host population structure shape CFAV genetic diversity.

Keywords: Aedes aegypti; insect-specific virus; phylogenetic analysis.

Figure 1.

Global distribution of CFAV. CFAV detection and isolation history are represented on the world map. Symbols represent detection or isolation events annotated with the country of mosquito origin and the year of the event. Novel isolations and detections from this study are shown in red symbols.

Figure 2.

Limited geographical structuring of the CFAV phylogeny. Consensus tree of 1,000 ultrafast bootstrap replicate ML trees represents phylogenetic relationships between all CFAV sequences based on the nearly full ORF alignment. Tips are color-coded according to the geographic origin of the sequences (red: Africa; green: Americas; blue: Asia-Pacific). Tips in gray represent CFAV sequences derived from the Aag2 cell line. Tips indicate the GenBank or ENA accession number (if any), virus isolate/strain name, country of origin, and the year of detection/isolation. The tree is midpoint rooted. Node support values include both ultrafast bootstrap proportion (%, first value) and posterior probability derived from the Bayesian maximum clade credibility tree (%, second value). The scale bar represents branch length in substitutions per site.

Figure 3.

Lack of phylogenetic congruence between CFAV and A.aegypti in Southeast Asia. Left: consensus tree of 1,000 ultrafast bootstrap replicate ML trees represents phylogenetic relationships between CFAV sequences based on the nearly full ORF alignment. The tree is midpoint rooted. Node support values include both ultrafast bootstrap proportion (%, first value) and posterior probability derived from the Bayesian maximum clade credibility tree (%, second value). The scale bar represents branch length in substitutions per site. Right: midpoint rooted consensus tree of 1,000 bootstrap replicates trees based on the Edwards’ genetic distances calculated from microsatellite allele frequencies among A.aegypti populations. Node support values represent bootstrap proportions (%). Lines between the trees connect the tips sharing the same geographic origin.

Figure 4.

Differing topology of cISF phylogenetic trees based on structural vs. nonstructural ORF regions. Consensus trees of 1,000 ultrafast bootstrap replicate ML trees (A, B) represent phylogenetic relationships among all cISFs based on the concatenated amino-acid alignment of C, prM, and E (A) and NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 (B). Both consensus trees are rooted with an outgroup composed of three non-cISF sequences shown at the bottom of the tree. Node support values represent ultrafast bootstrap proportions (%) and are only shown if <100% next to the open red circles. All nodes represented by filled red circles have 100 per cent support values. The scale bar represents branch length in substitutions per site. Tree tips indicate cISF species and number of sequences per species (n) if n > 1. The star symbol points to CFAV. Color highlighting of the tree leaves indicates the predominant mosquito host (red = Anopheles-associated cISFs; yellow = Aedes- and Ochlerotatus-associated cISFs; cyan =Culex- and Culiseta-associated cISFs; gray = Mansonia-, Coquillettidia-, and Sabethes-associated cISFs). The same color-coding is used in the schematic tree representation of evolutionary relationships between mosquito genera (C) based on limited phylogenetic studies (Shepard, Andreadis, and Vossbrinck 2006; Harbach 2007; Reidenbach et al. 2009; Chu et al. 2018; Aragao et al. 2019). In (C), the multifurcating dashed branches indicate uncertain clade assignment and the branch length is arbitrary.