Contrasting patterns of gene expression indicate differing pyrethroid resistance mechanisms across the range of the New World malaria vector Anopheles albimanus

Decades of unmanaged insecticide use and routine exposure to agrochemicals have left many populations of malaria vectors in the Americas resistant to multiple classes of insecticides, including pyrethroids. The molecular basis of pyrethroid resistance is relatively uncharacterised in American malaria vectors, preventing the design of suitable resistance management strategies. Using whole transcriptome sequencing, we characterized the mechanisms of pyrethroid resistance in Anopheles albimanus from Peru and Guatemala. An. albimanus were phenotyped as either deltamethrin or alpha-cypermethrin resistant. RNA from 1) resistant, 2) unexposed, and 3) a susceptible laboratory strain of An. Albimanus was sequenced and analyzed using RNA-Seq. Expression profiles of the three groups were compared based on the current annotation of the An. albimanus reference genome. Several candidate genes associated with pyrethroid resistance in other malaria vectors were found to be overexpressed in resistant An. albimanus. In addition, gene ontology terms related to serine-type endopeptidase activity, extracellular activity and chitin metabolic process were also commonly overexpressed in the field caught resistant and unexposed samples from both Peru and Guatemala when compared to the susceptible strain. The cytochrome P450 CYP9K1 was overexpressed 14x in deltamethrin and 8x in alpha-cypermethrin-resistant samples from Peru and 2x in deltamethrin-resistant samples from Guatemala, relative to the susceptible laboratory strain. CYP6P5 was overexpressed 68x in deltamethrin-resistant samples from Peru but not in deltamethrin-resistant samples from Guatemala. When comparing overexpressed genes between deltamethrin-resistant and alpha-cypermethrin-resistant samples from Peru, a single P450 gene, CYP4C26, was overexpressed 9.8x (p<0.05) in alpha-cypermethrin-resistant samples. In Peruvian deltamethrin-resistant samples, the knockdown resistance mutation (kdr) variant alleles at position 1014 were rare, with approximately 5% frequency, but in the alpha-cypermethrin-resistant samples, the frequency of these alleles was approximately 15–30%. Functional validation of the candidate genes and the kdr mutation as a resistance marker for alpha-cypermethrin will confirm the role of these mechanisms in conferring pyrethroid resistance

Spatial and temporal patterns of locally-acquired dengue transmission in Northern Queensland, Australia, 1993-2012

Background: Dengue has been a major public health concern in Australia since it re-emerged in Queensland in 1992–1993. We explored spatio-temporal characteristics of locally-acquired dengue cases in northern tropical Queensland, Australia during the period 1993–2012.Methods: Locally-acquired notified cases of dengue were collected for northern tropical Queensland from 1993 to 2012. Descriptive spatial and temporal analyses were conducted using geographic information system tools and geostatistical techniques. Results: 2,398 locally-acquired dengue cases were recorded in northern tropical Queensland during the study period. The areas affected by the dengue cases exhibited spatial and temporal variation over the study period. Notified cases of dengue occurred more frequently in autumn. Mapping of dengue by statistical local areas (census units) reveals the presence of substantial spatio-temporal variation over time and place. Statistically significant differences in dengue incidence rates among males and females (with more cases in females) (χ2 = 15.17, d.f. = 1, p<0.01). Differences were observed among age groups, but these were not statistically significant. There was a significant positive spatial autocorrelation of dengue incidence for the four sub-periods, with the Moran's I statistic ranging from 0.011 to 0.463 (p<0.01). Semi-variogram analysis and smoothed maps created from interpolation techniques indicate that the pattern of spatial autocorrelation was not homogeneous across the northern Queensland.Conclusions: Tropical areas are potential high-risk areas for mosquito-borne diseases such as dengue. This study demonstrated that the locally-acquired dengue cases have exhibited a spatial and temporal variation over the past twenty years in northern tropical Queensland, Australia. Therefore, this study provides an impetus for further investigation of clusters and risk factors in these high-risk areas

Evaluation of a temperate climate mosquito, Ochlerotatus detritus (=Aedes detritus), as a potential vector of Japanese encephalitis virus

The U.K. has not yet experienced a confirmed outbreak of mosquito-borne virus transmission to people or livestock despite numerous autochthonous epizootic and human outbreaks of mosquito-borne diseases on the European mainland. Indeed, whether or not British mosquitoes are competent to transmit arboviruses has not been established. Therefore, the competence of a local (temperate) British mosquito species, Ochlerotatus detritus (=Aedes detritus) (Diptera: Culicidae) for transmission of a member of the genus Flavivirus, Japanese encephalitis virus (JEV) as a model for mosquito-borne virus transmission was assessed. The JEV competence in a laboratory strain of Culex quinquefasciatus (Diptera: Culicidae), a previously incriminated JEV vector, was also evaluated as a positive control. Ochlerotatus detritus adults were reared from field-collected juvenile stages. In oral infection bioassays, adult females developed disseminated infections and were able to transmit virus as determined by the isolation of virus in saliva secretions. When pooled at 7-21days post-infection, 13% and 25% of O.detritus were able to transmit JEV when held at 23 degrees C and 28 degrees C, respectively. Similar results were obtained for C.quinquefasciatus. To our knowledge, this study is the first to demonstrate that a British mosquito species, O.detritus, is a potential vector of an exotic flavivirus

Evaluation of a temperate climate mosquito,Ochlerotatus detritus(Aedes detritus), as a potential vector of Japanese encephalitis virus

The U.K. has not yet experienced a confirmed outbreak of mosquito-borne virus transmission to people or livestock despite numerous autochthonous epizootic and human outbreaks of mosquito-borne diseases on the European mainland. Indeed, whether or not British mosquitoes are competent to transmit arboviruses has not been established. Therefore, the competence of a local (temperate) British mosquito species, Ochlerotatus detritus (Aedes detritus) (Diptera: Culicidae) for transmission of a member of the genus Flavivirus, Japanese encephalitis virus (JEV) as a model for mosquito-borne virus transmission was assessed. The JEV competence in a laboratory strain of Culex quinquefasciatus (Diptera: Culicidae), a previously incriminated JEV vector, was also evaluated as a positive control. Ochlerotatus detritus adults were reared from field-collected juvenile stages. In oral infection bioassays, adult females developed disseminated infections and were able to transmit virus as determined by the isolation of virus in saliva secretions. When pooled at 7–21 days post-infection, 13% and 25% of O. detritus were able to transmit JEV when held at 23 °C and 28 °C, respectively. Similar results were obtained for C. quinquefasciatus. To our knowledge, this study is the first to demonstrate that a British mosquito species, O. detritus, is a potential vector of an exotic flavivirus

Japanese encephalitis virus tropism in experimentally infected pigs.

Pigs are considered to be the main amplifying host for Japanese encephalitis virus (JEV), and their infection can correlate with human cases of disease. Despite their importance in the ecology of the virus as it relates to human cases of encephalitis, the pathogenesis of JEV in pigs remains obscure. In the present study, the localization and kinetics of virus replication were investigated in various tissues after experimental intravenous infection of pigs. The data demonstrate a rapid and broad spreading of the virus to the central nervous system (CNS) and various other organs. A particular tropism of JEV in pigs not only to the CNS but also for secondary lymphoid tissue, in particular the tonsils with the overall highest viral loads, was observed. In this organ, even 11 days post infection, the latest time point of the experiment, no apparent decrease in viral RNA loads and live virus was found despite the presence of a neutralizing antibody response. This was also well beyond the clinical and viremic phase. These results are of significance for the pathogenesis of JEV, and call for further experimental studies focusing on the cellular source and duration of virus replication in pigs