Discovery of a novel Tymoviridae-like virus in mosquitoes from Mexico.

A novel Tymoviridae-like virus, designated Ek Balam virus, was isolated from male Culex quinquefasciatus mosquitoes collected in Yucatan, Mexico. The genome was fully sequenced and shown to have no more than 69% nt sequence identity to its closest known relative. Mosquito cells were permissive to Ek Balam virus replication, but mammalian and avian cells were refractory, suggesting that vertebrates are not involved in the maintenance of the virus in nature.

Detection of novel and recognized RNA viruses in mosquitoes from the Yucatan Peninsula of Mexico using metagenomics and characterization of their in vitro host ranges.

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.

Merida virus, a putative novel rhabdovirus discovered in Culex and Ochlerotatus spp. mosquitoes in the Yucatan Peninsula of Mexico.

Sequences corresponding to a putative, novel rhabdovirus [designated Merida virus (MERDV)] were initially detected in a pool of Culex quinquefasciatus collected in the Yucatan Peninsula of Mexico. The entire genome was sequenced, revealing 11 798 nt and five major ORFs, which encode the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (L). The deduced amino acid sequences of the N, G and L proteins have no more than 24, 38 and 43 % identity, respectively, to the corresponding sequences of all other known rhabdoviruses, whereas those of the P and M proteins have no significant identity with any sequences in GenBank and their identity is only suggested based on their genome position. Using specific reverse transcription-PCR assays established from the genome sequence, 27 571 C. quinquefasciatus which had been sorted in 728 pools were screened to assess the prevalence of MERDV in nature and 25 pools were found positive. The minimal infection rate (calculated as the number of positive mosquito pools per 1000 mosquitoes tested) was 0.9, and similar for both females and males. Screening another 140 pools of 5484 mosquitoes belonging to four other genera identified positive pools of Ochlerotatus spp. mosquitoes, indicating that the host range is not restricted to C. quinquefasciatus. Attempts to isolate MERDV in C6/36 and Vero cells were unsuccessful. In summary, we provide evidence that a previously undescribed rhabdovirus occurs in mosquitoes in Mexico.

Ecuador Paraiso Escondido Virus, a New Flavivirus Isolated from New World Sand Flies in Ecuador, Is the First Representative of a Novel Clade in the Genus Flavivirus.

UNLABELLED: A new flavivirus, Ecuador Paraiso Escondido virus (EPEV), named after the village where it was discovered, was isolated from sand flies (Psathyromyia abonnenci, formerly Lutzomyia abonnenci) that are unique to the New World. This represents the first sand fly-borne flavivirus identified in the New World. EPEV exhibited a typical flavivirus genome organization. Nevertheless, the maximum pairwise amino acid sequence identity with currently recognized flaviviruses was 52.8%. Phylogenetic analysis of the complete coding sequence showed that EPEV represents a distinct clade which diverged from a lineage that was ancestral to the nonvectored flaviviruses Entebbe bat virus, Yokose virus, and Sokoluk virus and also the Aedes-associated mosquito-borne flaviviruses, which include yellow fever virus, Sepik virus, Saboya virus, and others. EPEV replicated in C6/36 mosquito cells, yielding high infectious titers, but failed to reproduce either in vertebrate cell lines (Vero, BHK, SW13, and XTC cells) or in suckling mouse brains. This surprising result, which appears to eliminate an association with vertebrate hosts in the life cycle of EPEV, is discussed in the context of the evolutionary origins of EPEV in the New World. IMPORTANCE: The flaviviruses are rarely (if ever) vectored by sand fly species, at least in the Old World. We have identified the first representative of a sand fly-associated flavivirus, Ecuador Paraiso Escondido virus (EPEV), in the New World. EPEV constitutes a novel clade according to current knowledge of the flaviviruses. Phylogenetic analysis of the virus genome showed that EPEV roots the Aedes-associated mosquito-borne flaviviruses, including yellow fever virus. In light of this new discovery, the New World origin of EPEV is discussed together with that of the other flaviviruses.