Identification and Characterization of Two Novel RNA Viruses from Anopheles gambiae Species Complex Mosquitoes

Abstract

Mosquitoes of the Anopheles gambiae complex display strong preference for human bloodmeals and are major malaria vectors in Africa. However, their interaction with viruses or role in arbovirus transmission during epidemics has been little examined, with the exception of O'nyong-nyong virus, closely related to Chikungunya virus. Deep-sequencing has revealed different RNA viruses in natural insect viromes, but none have been previously described in the Anopheles gambiae species complex. Here, we describe two novel insect RNA viruses, a Dicistrovirus and a Cypovirus, found in laboratory colonies of An. gambiae taxa using small-RNA deep sequencing. Sequence analysis was done with Metavisitor, an open-source bioinformatic pipeline for virus discovery and de novo genome assembly. Wild-collected Anopheles from Senegal and Cambodia were positive for the Dicistrovirus and Cypovirus, displaying high sequence identity to the laboratory-derived virus. Thus, the Dicistrovirus (Anopheles C virus, AnCV) and Cypovirus (Anopheles Cypovirus, AnCPV) are components of the natural virome of at least some anopheline species. Their possible influence on mosquito immunity or transmission of other pathogens is unknown. These natural viruses could be developed as models for the study of Anopheles-RNA virus interactions in low security laboratory settings, in an analogous manner to the use of rodent malaria parasites for studies of mosquito anti-parasite immunity.

Fig 1. Readmaps and size distributions of small RNAs that align on the Anopheles Cypovirus reconstructed RdRp genomic sequence.

The four small RNA datasets were independently aligned to the sequence of Anopheles Cypovirus segment 1 (accession number KU169879) with bowtie allowing no mismatches. From the bowtie output, lattice maps and read size histograms were generated representing the positions and size of the small RNAs that align to the viral genomic segment.

Fig 2. AnCPV RNA dependent RNA polymerase and polyhedrin phylogeny.

(A) Neighbor-joining tree of RNA-dependent RNA polymerase (RdRp) sequences of Cypoviruses, (B) Neighbor-joining tree of polyhedrin sequences of Cypoviruses. Multiple protein alignment was performed with the longest ORF (corresponding to the polymerase) for each nucleic acid. NJ bootstrap values (above 0.75) are indicated above branches. Pairwise distances are indicated in S6 File.

Fig 3. Microscopic observation of Anopheles coluzzii larvae midguts.

Signs of Cypoviral infection are white-blue iridescent areas as indicated by the white arrows in each panel. Each panel corresponds to a different mosquito larva.

Fig 4. Readmaps and size distributions of small RNAs that align on the Anopheles C virus reconstructed genome.

The four small RNA datasets were independently aligned to the sequence of the Anopheles C virus (AnCV) genome (accession number KU169878) with bowtie allowing no mismatches. From the bowtie output, lattice maps and read size histograms were generated representing the positions and size of the small RNAs that align to the viral genome.

Fig 5. AnCV non-structural protein phylogeny.

Neighbor-joining tree of non-structural polyprotein sequences of Dicistroviruses. NJ bootstrap values (above 0.75) are indicated above branches. The two genera of the Dicistroviridae family are indicated on the tree. Pairwise genetic distances are indicated in S6 File.

Fig 6. AnCV IRES show conservation of structure with the Cripavirus IRES family (RF0458).

Multiple sequence alignment of AnCV potential IRES sequence (nucleotides 5800 to 6001) with the seven seed IRES of the RF0458 family. Panel (A) represents the most probable conserved helix and secondary structures of the IRES using the known RF0458 structure as a scaffold, and panel (B) depicts the covariance between the nucleotides in the known RF0458 structure, showing covariation of the nucleotides to conserve IRES structural integrity.

Fig 7. Northern blot of total RNA purified from pooled midguts of An. coluzzii adult mosquitoes shows the presence of the positive strand of AnCV genome and AnCPV segment 1.

Fig 8. Example of RT-PCR amplicons of AnCV and AnCPV non-structural regions separated by agarose gel for wild mosquitoes and positive and negative controls.

Mosquito ID suffix -S or -C stands for the country of collection, Senegal or Cambodia respectively.