Aedes Anphevirus: an Insect-Specific Virus Distributed Worldwide in Aedes aegypti Mosquitoes That Has Complex Interplays with Wolbachia and Dengue Virus Infection in Cells

Abstract

Insect-specific viruses (ISVs) of the yellow fever mosquito Aedes aegypti have been demonstrated to modulate transmission of arboviruses such as dengue virus (DENV) and West Nile virus by the mosquito. The diversity and composition of the virome of A. aegypti, however, remains poorly understood. In this study, we characterized Aedes anphevirus (AeAV), a negative-sense RNA virus from the order Mononegavirales AeAV identified from Aedes cell lines was infectious to both A. aegypti and Aedes albopictus cells but not to three mammalian cell lines. To understand the incidence and genetic diversity of AeAV, we assembled 17 coding-complete and two partial genomes of AeAV from available transcriptome sequencing (RNA-Seq) data. AeAV appears to transmit vertically and be present in laboratory colonies, wild-caught mosquitoes, and cell lines worldwide. Phylogenetic analysis of AeAV strains indicates that as the A. aegypti mosquito has expanded into the Americas and Asia-Pacific, AeAV has evolved into monophyletic African, American, and Asia-Pacific lineages. The endosymbiotic bacterium Wolbachia pipientis restricts positive-sense RNA viruses in A. aegypti Reanalysis of a small RNA library of A. aegypti cells coinfected with AeAV and Wolbachia produces an abundant RNA interference (RNAi) response consistent with persistent virus replication. We found Wolbachia enhances replication of AeAV compared to a tetracycline-cleared cell line, and AeAV modestly reduces DENV replication in vitro The results from our study improve understanding of the diversity and evolution of the virome of A. aegypti and adds to previous evidence that shows Wolbachia does not restrict a range of negative-strand RNA viruses.IMPORTANCE The mosquito Aedes aegypti transmits a number of arthropod-borne viruses (arboviruses), such as dengue virus and Zika virus. Mosquitoes also harbor insect-specific viruses that may affect replication of pathogenic arboviruses in their body. Currently, however, there are only a few insect-specific viruses described from A. aegypti in the literature. Here, we characterize a novel negative-strand virus, AeAV. Meta-analysis of A. aegypti samples showed that it is present in A. aegypti mosquitoes worldwide and is vertically transmitted. Wolbachia-transinfected mosquitoes are currently being used in biocontrol, as they effectively block transmission of several positive-sense RNA viruses in mosquitoes. Our results demonstrate that Wolbachia enhances the replication of AeAV and modestly reduces dengue virus replication in a cell line model. This study expands our understanding of the virome in A. aegypti as well as providing insight into the complexity of the Wolbachia virus restriction phenotype.

Keywords: Aedes aegypti; Mononegavirales; Wolbachia; anphevirus; insect viruses; mosquito; virome.

FIG 1

Presence of AeAV in insect cell lines and genome organization and phylogeny of the virus. (A) RT-PCR analysis of Aedes cell lines Aag2, Aag2.wMelPop-CLA (Pop), wMelPop-CLA.Tet (Pop-T), Aa20, RML-12, and C6/36 for the presence of AeAV. RPS17 was used as a control. (B) Genome organization of the Cali, Colombia, AeAV strain and subgenomic gene transcription profile. Transmembrane domains (TMD) are depicted as boxes with dashed lines, and the signal peptide is depicted as a blue box. NP, nucleoprotein; G, glycoprotein; ZnF, zinc-like finger; RdRP, RNA-dependent RNA polymerase. (C) AeAV is a member of the Anphevirus genus (red), related to members of the Nyamiviridae (pink) and Bornaviridae (purple) in an unassigned family within the order Mononegavirales. A multiple-sequence alignment of the RNA-dependent RNA polymerase and the mRNA capping domain was used to create a maximum likelihood phylogeny. The phylogeny is arbitrarily rooted. One thousand bootstraps were performed, and branches with bootstrap values greater than 85% are highlighted. Branch lengths represent expected numbers of substitutions per amino acid site. Viruses used (GenBank protein accession numbers) are the following: Bolahun virus variant 1 (AOR51366.1), Culex mononega-like virus 1 (CMLV1) (ASA47369.1), Culex mononega-like virus 2 (CMLV2) (ASA47322.1), Gambie virus (AOR51378.1), Xincheng mosquito virus (XcMV) (YP_009302387.1), Borna disease virus (YP_009269418.1), canary bornavirus 1 (YP_009268910.1), Loveridges garter snake virus 1 (YP_009055063.1), parrot bornavirus 1 (AEG78314.1), variegated squirrel bornavirus 1 (SBT82903.1), Midway nyavirus (YP_002905331.1), Nyamanini nyavirus (YP_002905337.1), Sierra Nevada virus (YP_009044201.1), soybean cyst nematode socyvirus (YP_009052467.1), Farmington virus (YP_009091823.1), Beihai rhabdo-like virus 3 (APG78650.1), Beihai rhabdo-like virus 5 (YP_009333422.1), Beihai rhabdo-like virus 6 (YP_009333413.1), Drosophila unispina virus 1 (AMK09260.1), Hubei diptera virus 11 (YP_009337182.1), Hubei orthoptera virus 5 (YP_009336728.1), Hubei rhabdo-like virus 7 (YP_009337121.1), Orinoco virus (ANQ45640.1), Sanxia water strider virus 4 (YP_009288955.1), Shuangao fly virus 2 (AJG39135.1), Wenling crustacean virus 12 (YP_009336618.1), Wenzhou crab virus 1 (YP_009304558.1), Wenzhou tapeworm virus 1 (YP_009342311.1), and Wuchan romanomermis nematode virus 2 (YP_009342285.1).

FIG 2

Conservation of GATA-like zinc finger (ZnF) domain and small transmembrane domain-containing protein between tentative members of the Anphevirus taxon. (A) Genome orientation of previously discovered viruses within the Anphevirus taxon. (B) Two viruses within a closely related clade. Predicted ORF encoding the ZnF domain is indicated by a black square. Predicted ORFs containing transmembrane domains are indicated by dashed lines. GenBank accession numbers are shown below virus names. NP, nucleoprotein; G, glycoprotein; ZnF, zinc-like finger; RdRp, RNA-dependent RNA polymerase. (C) Alignment of predicted GATA-like ZnF protein sequence [C-X(2)-C-X (17–20)-C-X(2)-C] between three representative strains of AeAV (Miami, Florida, USA; Pune, India; Rabai, Kenya) and predicted ZnF domain proteins from panels A and B.

FIG 3

AeAV cis-regulatory elements. (A) Location and orientation of predicted cis-regulatory element in AeAV indicated by numbered red arrows, with down indicating genome and up indicating antigenome. (B) Predicted minimum free energy (MFE) RNA structure of the region surrounding the motif for each element using the RNAfold web server. Color indicates probability of base pairing, and motif is indicated by the black line. (C) Sequence of the conserved motif as predicted by MEME as well as location and the statistical confidence of the motif. Sequences are written 3′ to 5′, and antigenome motif sequences 1 and 7 are depicted as reverse complement for visual clarity.

FIG 4

AeAV has worldwide distribution in A. aegypti laboratory colonies, cell lines, and wild-caught mosquitoes. Locations of mosquito collection from RNA-Seq data that were positive for AeAV are shown (see Table S1). Points refer to collection sites from American (orange), Asia-Pacific (blue), and African (green) locations.

FIG 5

AeAV strains have evolved into African, Asia-Pacific, and American lineages. (A) Maximum likelihood phylogeny (PhyML) between AeAV strains using a GTR + G + T model with 1,000 bootstraps. Branch lengths represent expected numbers of substitutions per nucleotide site. For visual clarity, the RML-12 clade and Miami clades were collapsed and single examples are shown. (B) Evolutionary history of worldwide sampling of A. aegypti, adapted from references and , from 1,504 SNP species. Bootstrapped neighbor-joining network based on population pairwise chord distances with node support over 90% is shown on relevant branches. New World (American) populations are in yellow, and Asia-Pacific populations are shown in light blue. We have truncated the tree and rooted it to A. aegypti formosus (Aef), shown as a red branch.

FIG 6

Genomic context for anphevirus-like insertions into the A. aegypti genome. A 21,242-nt portion of chromosome 2 depicting anphevirus insertions (red) with predicted ORFs that encode LTR retrotransposase elements (yellow).

FIG 7

AeAV is infectious to Aedes cell lines but does not replicate in Huh-7, Vero, and BSR vertebrate cell lines. (A) RT-qPCR of AeAV genome and anti-genome in a 5-day time course in A. aegypti Aa20 cells and A. albopictus C6/36 cells. Error bars represent the standard errors of the means (SEM) from three biological replicates. (B) RT-PCR of AeAV genome in a 7-day time course in human hepatocellular carcinoma cells (Huh-7), African green monkey cells (Vero), and baby hamster kidney (BSR). M, mock-infected cells.

FIG 8

AeAV genome replication is enhanced by Wolbachia infection in A. aegypti cells and produces abundant vsiRNAs and vpiRNAs. (A) RT-qPCR of the AeAV genomic (gRNA) and antigenomic RNA in tetracycline-cured Aag2.wMelPop-CLA cells (Pop-tet) and Aag2.wMelPop-CLA cells (Pop) relative to RPS17. Error bars represent the SEM from six (genome) and three (antigenome) biological replicates. n.s, not significant; **, P < 0.01. (B) Mapping profile of pooled small RNA fraction in Aag2.wMelPop-CLA cells. (C) Alignment of the 21-nt sRNA reads (representing siRNAs) and (D) the 26- to 31-nt reads (representing piRNAs) mapped to the AeAV antigenome (blue) and genome (red) in Aag2.wMelPop-CLA cells. Relative nucleotide frequency and conservation of the 28-nt small RNA reads that mapped to the genome (E) and the antigenome (F) of AeAV in Aag2.wMelPop-CLA cells.

FIG 9

AeAV reduces dengue virus replication in Aa20 cells. Aa20 cells persistently infected with AeAV were infected with DENV-2 at MOIs of 0.1 (A) and 1 (B). Total RNA was extracted at 0, 1, 3, and 5 days following DENV-2 inoculation and analyzed by RT-qPCR. (C) RT-qPCR analysis of AeAV persistently infected Aa20 cells infected with DENV-2 at MOIs of 0.1 and 1 using primers specific to the AeAV genome. Error bars represent the SEM from three biological replicates. n.s, not significant; *, P < 0.05; **, P < 0.01.

FIG 10

AeAV is potentially vertically transmitted. (A) Diagram showing the parental (K14 and K27) and hybrid strains (GP1, GP2, HP1, and HP2) from reference . (B) Table showing assembly statistics and BLASTN similarity of AeAV genomes assembled from K14 and K27 hybrid strains.