We found a new flavivirus that is widespread in Culex pipiens and other Culex mosquitoes in Japan. The virus isolate, named Culex flavivirus (CxFV), multiplied only in mosquito cell lines producing a moderate cytopathic effect, but did not grow in mammalian cells. The CxFV genome is single-stranded RNA, 10,834 nt in length and containing a single open reading frame encoding a polyprotein of 3362 aa with 5' and 3' untranslated regions (UTRs) of 91 and 657 nt, respectively. Phylogenetic analyses revealed that CxFV is closely related to the insect flaviviruses associated with Aedes mosquitoes, Cell fusing agent (CFA) and Kamiti River virus (KRV). The 3' UTR of CxFV contains four tandem repeats, which have sequence similarities to the two direct repeats in the CFA and KRV 3' UTRs. These results suggest that CxFV may be a new group of insect flaviviruses.

Introduction Aedes albopictus (Skuse) is an important vector of arboviral diseases, including dengue, chikungunya and Zika virus disease. Monitoring insecticide resistance and mechanisms by which the mosquito develops resistance is crucial to minimise disease transmission. Aim To determine insecticide resistance status and mechanisms in Ae. albopictus from different geographical regions. Methods We sampled 33 populations of Ae. albopictus from Asia, Europe and South America, and tested these for susceptibility to permethrin, a pyrethroid insecticide. In resistant populations, the target site for pyrethroids, a voltage-sensitive sodium channel ( Vssc ) was genotyped. Three resistant sub-strains, each harbouring a resistance allele homozygously, were established and susceptibilities to three different pyrethroids (with and without a cytochrome P450 inhibitor) were assayed. Results Most populations of Ae. albopictus tested were highly susceptible to permethrin but a few from Italy and Vietnam (4/33), exhibited high-level resistance. Genotyping studies detected a knockdown resistance ( kdr ) allele V1016G in Vssc for the first time in Ae. albopictus . Two previously reported kdr alleles, F1534C and F1534S, were also detected. The bioassays indicated that the strain homozygous for the V1016G allele showed much greater levels of pyrethroid resistance than other strains harbouring F1534C or F1534S. Conclusion The V1016G allele was detected in both Asian and Italian Ae. albopictus populations, thus a spread of this allele beyond Italy in Europe cannot be ruled out. This study emphasises the necessity to frequently and regularly monitor the V1016G allele in Ae. albopictus , particularly where this mosquito species is the main vector of arboviruses.

We isolated a new flavivirus from Aedes albopictus mosquito and a related species in Japan. The virus, designated Aedes flavivirus (AEFV), only replicated in a mosquito cell line and produced a mild cytopathic effect. The AEFV genome was positive-sense, single-stranded RNA, 11,064 nucleotides in length and contained a single open reading frame encoding a polyprotein of 3341 amino acids with 5' and 3' untranslated regions (UTRs) of 96 and 945 nucleotides, respectively. Genetic and phylogenetic analyses classified AEFV with the insect flavivirus, but distinct from Cell fusing agent (CFA), Kamiti river virus and Culex flavivirus. Interestingly, a partial sequence of AEFV showed significant similarity to that of Cell silent agent (CSA), the insect flavivirus-related nucleotide sequence integrated in the genome of A. albopictus. These results suggest that AEFV is a new member of the insect flaviviruses, which are intimately associated with Aedes mosquitoes and may share a common origin with CSA.

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To evaluate the vectorial capacity of mosquitoes for viruses in Japan, the host-feeding habits of the mosquitoes were analyzed by sequencing polymerase chain reaction-amplified fragments of the cytochrome b and 16S ribosomal RNA regions of the mitochondrial DNA of 516 mosquitoes of 15 species from seven genera that were collected from residential areas during 2003-2006. Culex pipiens L. and Aedes albopictus Skuse were the most commonly collected species in urban and suburban residential areas. Anautogenous Culex pipiens pallens Coquillett was distinguished from the autogenous Cx. pipiens form molestus Forskal using a polymerase chain reaction-based identification method. Both Cx. p. pallens and Cx. p. form molestus exhibited similar host-feeding habits, broadly preferring avian (50.0 and 42.5% of avian, respectively) and mammalian (38.6 and 45.0% of avian, respectively) hosts, such as tree sparrows, ducks, and humans. Conversely, Ae. albopictus exhibited a highly mammalophilic and anthropophilic feeding pattern, with 84.2% feeding on mammalian hosts and 68.5% of these on humans. We concluded that in Japan, Cx. pipiens might play a significant role in the avian-to-mammal transmission of viruses, such as West Nile virus, whereas Ae. albopictus might play a role in the human-human transmission of dengue and Chikungunya viruses.