Novel flaviviruses detected in different species of mosquitoes in Spain

Abstract

We report the characterization of three novel flaviviruses isolated in Spain. Marisma Mosquito virus, a novel mosquito borne virus, was isolated from Ochlerotatus caspius mosquitoes; Spanish Ochlerotatus flavivirus and Spanish Culex flavivirus, two novel insect flaviviruses, were isolated from Oc. caspius and Culex pipiens, respectively. During this investigation, we designed a sensitive RT-nested polymerase chain reaction method that amplifies a 1019bp fragment of the flavivirus NS5 gene and could be directly used in clinical or environmental samples for flavivirus characterization and surveillance. Analysis of the sequence generated from that amplicon contains enough phylogenetic information for proper taxonomic studies. Moreover, the use of this tool allowed the detection of additional flavivirus DNA forms in Culex, Culiseta, and Ochlerotatus mosquitoes.

FIG. 1.

Phylogenetic tree based on partial NS5 gene of new group Marisma Mosquito virus (MMV) detected in Ochlerotatus caspius mosquitoes from Spain. Phylogenetic analysis is based on the 860 nucleotides fragment of 54 sequences. The tree is unrooted and was displayed by using the program Mega4, Neighbor-Joining method and Tamura-Nei model with gamma parameter: 0.6990 and 1000 bootstrap replicates. GenBank accession numbers for the different groups of mosquito-borne flavivirus (MBV) sequences are indicated in the tree. Accession numbers for MMV sequences: JF737835, JF737836, JF737837, JF737838, JF737839, and JN603190.

FIG. 2.

Phylogenetic tree based on partial NS5 gene of new groups Spanish Ochlerotatus Flavivirus (SOcFV) and Spanish Culex Flavivirus (SCxFV) detected in Oc. caspius and Culex mosquitoes from Spain. Phylogenetic analysis is based on the 860 nucleotides fragment of 85 sequences. The tree is un-rooted and was displayed by using the program Mega4, Neighbor-Joining method, and Tamura-Nei model with gamma parameter: 0.6617 and 1000 bootstrap replicates. GenBank accession numbers for the insect flavivirus sequences are indicated in the tree. Accession numbers for SOcFV sequences: JF707790, JF707791, JF707792, JF707793, JF707794, JF707795, JF707796, JF707797, JF707798, JF707799, JF707800, JF707800, JF707801, JF707802, JF707803, JF707804, JF707805, JF707806, JF707807, JF707808, JF707809, and JF707810; accession numbers for SCxFV sequences: JF707811, JF707812, JF707813, JF707814, JF707815, JF707816, JF707817, JF707818, JF707819, JF707820, JF707821, JF707822, JF707823, JF707824, JF707825, JF707826, JF707827, JF707828, JF707829, JF707830, JF707831, JF707832, JF707833, JF707834, JF707835, JF707836, JF707837, JF707838, JF707839, JF707840, JF707841, JF707842, JF707843, JF707844, JF707845, JF707846, JF707847, JF707848, JF707849, JF707850, JF707851, JF707852, JF707853, JF707854, and JF707855; accession numbers for DNA forms sequences: JF707856, JF707857, JF707858, JF707859, and JF707860.