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.

Background Knockdown resistance (kdr) to dichlorodiphenyltrichloroethane (DDT) and pyrethroids is known to link amino acid substitutions in the voltage-gated sodium channel (VGSC) in Aedes aegypti . Dengue fever primarily transmitted by Ae . aegypti is an annual public health issue in Taiwan. Accordingly, pyrethroid insecticides have been heavily used for decades to control mosquito populations in the summer and autumn. In Taiwan, an Ae . aegypti population with two VGSC mutations, V1016G and D1763Y, was described previously. Methodology/Principal finding Aedes aegypti (G0) were collected in Tainan and Kaohsiung in southern Taiwan. The VGSC gene polymorphisms of the kdr mutations and the intron flanked by exons 20 and 21 were verified. The first generation offspring (G1) were used to measure the resistance level to cypermethrin, a pyrethroid insecticide currently used in Taiwan. In addition to V1016G and D1763Y, we describe two new mutations, S989P and F1534C, which have not been reported in Taiwan. Moreover, we also identify two types (groups A and B) of introns between exons 20 and 21. Intriguingly, the kdr mutations S989P, V1016G and D1763Y are strictly located on the haplotype harboring the group A intron, whereas F1534C links to the group B intron. When those data were taken together, we proposed the following six haplotypes for VGSC genes in Taiwan today: (i)S989-intron A-V1016-F1534-D1763, (ii)S989-intron A-V1016G-F1534-D1763, (iii)S989P-intron A-V1016G-F1534-D1763, (iv)S989-intron A-V1016G-F1534-D1763Y, (v)S989-intron B-V1016-F1534-D1763 and (vi)S989-intron B-V1016-F1534C-D1763. Triple heterozygous mutations of either S989P/V1016G/F1534C or V1016G/F1534C/D1763Y can be found in one single Ae . aegypti mosquito. The proportions of the VGSC mutations were relevant to cypermethrin resistance. Notably, the presence of S989P and V1016G in the population could be a helpful reference to predict the resistance level to cypermethrin. This is the first study to demonstrate the coexistence of four kdr mutations in a population of Ae . aegypti . Conclusions/Significance Four kdr mutations (S989P, V1016G, F1534C and D1763Y) and two intron forms (Group A and B) were commonly found in local Ae . aegypti populations in Taiwan.

We have previously demonstrated that vaccination with a subunit dengue vaccine containing a consensus envelope domain III with aluminum phosphate elicits neutralizing antibodies against all four serotypes of dengue virus in mice. In this study, we evaluated the immunogenicity of the subunit dengue vaccine in non-human primates. After vaccination, monkeys that received the subunit vaccine with aluminum phosphate developed a significantly strong and long-lasting antibody response. A specific T cell response with cytokine production was also induced, and this correlated with the antibody response. Additionally, neutralizing antibodies against serotype 2 were detected in two of three monkeys. The increase in serotype-2-specific antibody titers and avidity observed in these two monkeys suggested that a serotype-2-biased antibody response occurs. These data provide evidence that a protective neutralizing antibody response was successfully elicited in non-human primates by the dengue subunit vaccine with aluminum phosphate adjuvant.

Dengue has a major impact on global public health, and the use of dengue vaccine is very limited. In this study, we evaluated the immunogenicity and protective efficacy of a dengue vaccine made from a recombinant measles virus (MV) that expresses envelope protein domain III (ED3) of dengue-1 to 4. Following immunization with the MV-vectored dengue vaccine, mice developed specific interferon-gamma and antibody responses against dengue virus and MV. Neutralizing antibodies against MV and dengue viruses were also induced, and protective levels of FRNT50 ≥ 10 to 4 serotypes of dengue viruses were detected in the MV-vectored dengue vaccine-immunized mice. In addition, specific interferon-gamma and antibody responses to dengue viruses were still induced by the MV-vectored dengue vaccine in mice that were pre-infected with MV. This finding suggests that the pre-existing immunity to MV did not block the initiation of immune responses. By contrast, mice that were pre-infected with dengue-3 exhibited no effect in terms of their antibody responses to MV and dengue viruses, but a dominant dengue-3-specific T-cell response was observed. After injection with dengue-2, a detectable but significantly lower viremia and a higher titer of anti-dengue-2 neutralizing antibodies were observed in MV-vectored dengue vaccine-immunized mice versus the vector control, suggesting that an anamnestic antibody response that provided partial protection against dengue-2 was elicited. Our results with regard to T-cell responses and the effect of pre-immunity to MV or dengue viruses provide clues for the future applications of an MV-vectored dengue vaccine.

Dengue is the leading cause of mosquito-borne viral infections and no vaccine is available now. Envelope protein domain III (ED3) is the major target for the binding of dengue virus neutralizing antibodies; however, the ED3-specifc T-cell response is less well understood. To investigate the T-cell responses to four serotypes of dengue virus (DENV-1 to 4), we immunized mice using either a tetravalent ED3-based DNA or protein vaccine, or combined both as a DNA prime-protein boost strategy (prime-boost). A significant serotype-dependent IFN-γ or IL-4 response was observed in mice immunized with either the DNA or protein vaccine. The IFN-γ response was dominant to DENV-1 to 3, whereas the IL-4 response was dominant to DENV-4. Although the similar IgG titers for the four serotypes were observed in mice immunized with the tetravalent vaccines, the neutralizing antibody titers varied and followed the order of 2 = 3>1>4. Interestingly, the lower IFN-γ response to DENV-4 is attributable to the immunodominance change between two CD4+ T-cell epitopes; one T-cell epitope located at E349-363 of DENV-1 to 3 was more immunogenic than the DENV-4 epitope E313-327. Despite DENV-4 specific IFN-γ responses were suppressed by immunodominance change, either DENV-4-specific IFN-γ or neutralizing antibody responses were still recalled after DENV-4 challenge and contributed to virus clearance. Immunization with the prime-boost elicited both IFN-γ and neutralizing antibody responses and provided better protection than either DNA or protein immunization. Our findings shed light on how ED3-based tetravalent dengue vaccines sharpen host CD4 T-cell responses and contribute to protection against dengue virus.