Stefanie L. Hurt

A metagenomics approach was used to detect novel and recognized RNA viruses in mosquitoes from the Yucatan Peninsula of Mexico. A total of 1359 mosquitoes of 7 species and 5 genera (Aedes, Anopheles, Culex, Mansonia and Psorophora) were sorted into 37 pools, homogenized and inoculated onto monolayers of Aedes albopictus (C6/36) cells. A second blind passage was performed and then total RNA was extracted and analysed by RNA-seq. Two novel viruses, designated Uxmal virus and Mayapan virus, were identified. Uxmal virus was isolated from three pools of Aedes (Ochlerotatus) taeniorhynchus and phylogenetic data indicate that it should be classified within the recently proposed taxon Negevirus. Mayapan virus was recovered from two pools of Psorophora ferox and is most closely related to unclassified Nodaviridae-like viruses. Two recognized viruses were also detected: Culex flavivirus (family Flaviviridae) and Houston virus (family Mesoniviridae), with one and two isolates being recovered, respectively. The in vitro host ranges of all four viruses were determined by assessing their replicative abilities in cell lines of avian, human, monkey, hamster, murine, lepidopteran and mosquito (Aedes, Anopheles and Culex) origin, revealing that all viruses possess vertebrate replication-incompetent phenotypes. In conclusion, we report the isolation of both novel and recognized RNA viruses from mosquitoes collected in Mexico, and add to the growing plethora of viruses discovered recently through the use of metagenomics.

The genomic organization and in vitro host range of a novel mosquito-associated orbivirus, designated Skunk River virus, is described. The virus was isolated from Aedes trivittatus collected in Iowa in the United States. Three recognized viruses were also recovered: Culex flavivirus (family Flaviviridae), Houston virus (family Mesoniviridae) and Umatilla virus (family Reoviridae). The genome of Skunk River virus contains 10 segments and its organization is characteristic of viruses in the genus Orbivirus (family Reoviridae). The coding region of each segment was fully sequenced, revealing that the greatest nucleotide identity was to the corresponding regions of Big Cypress orbivirus and Sathuvachari virus, two recently described mosquito-associated orbiviruses. The phylogenetic inference is in agreement with these findings. In vitro host range experiments revealed that Aedes, Anopheles and Culex cell lines, and select lepidopteran and rodent cell lines, are permissive to Skunk River virus replication. In conclusion, we provide evidence of a novel mosquito-associated orbivirus in Iowa.