Structure and dsRNA-binding activity of the Birnavirus Drosophila X Virus VP3 protein.

The Birnavirus multifunctional protein VP3 plays an essential role coordinating the virus life cycle, interacting with the capsid protein VP2, with the RNA-dependent RNA polymerase VP1 and with the dsRNA genome. Furthermore, the role of this protein in controlling host cell responses triggered by dsRNA and preventing gene silencing has been recently demonstrated. Here we report the X-ray structure and dsRNA-binding activity of the N-terminal domain of Drosophila X virus (DXV) VP3. The domain folds in a bundle of three α-helices and arranges as a dimer, exposing to the surface a well-defined cluster of basic residues. Site directed mutagenesis combined with Electrophoretic Mobility Shift Assays (EMSA) and Surface Plasmon Resonance (SPR) revealed that this cluster, as well as a flexible and positively charged region linking the first and second globular domains of DXV VP3, are essential for dsRNA-binding. Also, RNA silencing studies performed in insect cell cultures confirmed the crucial role of this VP3 domain for the silencing suppression activity of the protein. IMPORTANCE The Birnavirus moonlighting protein VP3 plays crucial roles interacting with the dsRNA genome segments to form stable ribonucleoprotein complexes and controlling host cell immune responses, presumably by binding to and shielding the dsRNA from recognition by the host silencing machinery. The structural, biophysical and functional data presented in this work has identified the N-terminal domain of VP3 as responsible for the dsRNA-binding and silencing suppression activities of the protein in Drosophila X virus.

Characterization of Port Bolivar Virus, a Novel Entomobirnavirus (Birnaviridae) Isolated from Mosquitoes Collected in East Texas, USA

This report describes and characterizes a novel entomobirnavirus, designated Port Bolivar virus (PTBV), that was isolated from a pool of Aedes sollicitans mosquitoes collected in a saltwater marsh in East Texas, USA. Full genome sequencing and phylogenetic analyses indicate that PTBV is distinct but genetically related to Drosophila X virus and mosquito X virus, which are assigned to species in the genus Entomobirnavirus, family Birnaviridae. PTBV produced cytopathic effect (CPE) in cultures of mosquito (C6/36) cells, but not in Vero cell cultures. Ultrastructural studies of PTBV in infected C6/36 cells demonstrated unenveloped virus particles about 55 nm in diameter.

Twenty five new viruses associated with the Drosophilidae (Diptera)

Drosophila melanogasteris an important laboratory model for studies of antiviral immunity in invertebrates, andDrosophilaspecies provide a valuable system to study virus host range and host switching. Here we use metagenomic RNA sequencing ofca. 1600 adult flies to discover 25 new RNA viruses associated with six different drosophilid hosts in the wild. We also provide a comprehensive listing of viruses previously reported from the Drosophilidae. The new viruses include Iflaviruses, Rhabdoviruses, Nodaviruses, and Reoviruses, and members of unclassified lineages distantly related to Negeviruses, Sobemoviruses and Poleroviruses, Flaviviridae, and Tombusviridae. Among these are close relatives ofDrosophila X virusandFlock House virus, which we find in association with wildDrosophila immigrans. These two viruses are widely used in experimental studies but have not previously been reported to naturally infectDrosophila. Although we detect no new DNA viruses, inD. immigransandD. obscurawe identify sequences very closely related toArmadillidium vulgareIridescent virus (Invertebrate Iridescent virus 31), bringing the total number of DNA viruses found in the Drosophilidae to three.

Presence of entomobirnaviruses in Chinese mosquitoes in the absence of Dengue virus co‐infection

Birnaviruses, including the genus Entomobirnavirus, are socio-economically important viruses. Currently, only Drosophila X virus has been formally assigned to the genus Entomobirnavirus, but two more viruses were recently isolated, Espirito Santo virus (ESV) and Culex Y virus. The host mosquito has been reported to carry many viruses, but seldom entomobirnaviruses. To discover potential pathogens in mosquitoes, we exploited small-RNAs high-throughput sequencing of three mosquito species caught in South China. A virus that genetically likes entomobirnavirus, Mosquito X virus (MXV), was identified from Anopheles sinensis and was 97 % identical to ESV, which co-infects with Dengue virus (DENV). However, the absence of DENV in the A. sinensis suggested the independence of MXV infection from dengue co-infection. Our discovery complements prior research on entomobirnaviruses and proved that MXV may be widespread in mosquitoes on different continents. This work also highlights the applying of high-throughput sequencing of small RNAs to survey viruses carried by insect vectors.

First isolation of an Entomobirnavirus from free-living insects

Drosophila X virus (DXV), the prototype Entomobirnavirus, is a well-studied RNA virus model. Its origin is unknown, and so is that of the only other entomobirnavirus, Espirito Santo virus (ESV). We isolated an entomobirnavirus tentatively named Culex Y virus (CYV) from hibernating Culex pipiens complex mosquitoes in Germany. CYV was detected in three pools consisting of 11 mosquitoes each. Full-genome sequencing and phylogenetic analyses suggested that CYV and ESV define one sister species to DXV within the genus Entomobirnavirus. In contrast to the laboratory-derived ESV, the ORF5 initiation codon AUG was mutated to 1927GUG in all three wild-type CYV isolates. Also in contrast to ESV, replication of CYV was not dependent on other viruses in insect cell culture. CYV could provide a wild-type counterpart in research fields relying on DXV and other cell culture-adapted strains.