Characterization and Mosquito infection of the Tengah Isolate of Japanese encephalitis virus

Japanese encephalitis (JE) is a brain infection caused by JE virus (JEV). It has an estimated worldwide incidence of 68,000 cases and 10,000 to 15,000 deaths annually. Despite large effective immunization campaigns, Japanese encephalitis remains a disease of global health concern, because the virus is spreading. There are five genotypes of JEV (genotype I – V), each associated with different geographical areas and associated epidemiology. The Muar strain of JEV, the fifth genotype, is believed to represent the oldest lineage from which genotype I - IV evolved. Muar was isolated in Singapore in 1952. At the same time as Muar was isolated; the Tengah strain of JEV was also isolated from a nearby location. However, Tengah and the characteristics of Tengah have largely remained unknown. Muar was considered the only known representative of genotype-V prior to 2009. Vector competence studies have examined genotype-I, II and III of JEV. However, genotype-IV and genotype-V have never been investigated in vector competence studies. Therefore, the infectivity of these viruses to mosquitoes is unknown. The competence of non-Asian mosquitoes to JEV has been demonstrated suggesting potential for emergence in some other regions. In Great Britain, JEV is considered a potential threat to animals and public health. However, the level of competence of British mosquitoes to any arbovirus is not known. The overall objective of this thesis was to characterise Tengah, investigate molecular and mosquito factors that might relate to the lack of circulation of genotype-V isolates and assess the potential of arbovirus (JEV) emergence in Great Britain. Molecular characterization of Tengah strain showed that it is another isolate of genotype-V, with 99% sequence similarity to Muar. Evolutionary analysis performed using the Bayesian Evolutionary analysis of Sampling Trees (BEAST) program estimated that JEV is evolving at a rate of 3.53 × 104 nucleotide substitution per site per year. Vector competence studies demonstrated Culex quinquefasciatus mosquitoes are able to transmit Muar with transmission rates of 23% at 21-days post infection. Comparison of transmission between Muar (genotype V) and Nakayama (genotype III) found no significant difference between the two genotypes. Ochlerotatus detritus, a British mosquito, was susceptible to JEV at both 23°C and 28°C as determined by the detection of virus in the saliva 7 days post infection. The overall transmission rate was 13% at 23°C and 25% at 28°C. There was no significant difference between the two temperatures. Infection rates for Ochlerotatus detritus and Culex quinquefaciatus were similar. This thesis has shown that on account of its similarity to the Muar isolate, Tengah represents a variant of Muar and is another genotype-V isolate. Evolutionary analysis showed that JEV originated from its ancestral virus in the year 1120 while the Time to Most Recent Common Ancestor for genotype V was in the year 1840. Muar has the ability to infect, replicate and be transmitted in Culex quinquefasciatus, suggesting that the limited distribution, isolation and circulation of genotype-V is probably not explained solely by mosquito factors. Ochlerotatus detritus is competent to transmit JEV and would therefore pose a threat should this virus occur in Great Britain

A whole transcriptomic approach provides novel insights into the molecular basis of organophosphate and pyrethroid resistance in Anopheles arabiensis from Ethiopia.

The development of insecticide resistance in malaria vectors is of increasing concern in Ethiopia because of its potential implications for vector control failure. To better elucidate the specificity of resistance mechanisms and to facilitate the design of control strategies that minimize the likelihood of selecting for cross-resistance, a whole transcriptomic approach was used to explore gene expression patterns in a multi-insecticide resistant population of Anopheles arabiensis from Oromia Region, Ethiopia. This field population was resistant to the diagnostic doses of malathion (average mortality of 71.9%) and permethrin (77.4%), with pools of survivors and unexposed individuals analyzed using Illumina RNA-sequencing, alongside insecticide susceptible reference strains. This population also demonstrated deltamethrin resistance but complete susceptibility to alpha-cypermethrin, bendiocarb and propoxur, providing a phenotypic basis for detecting insecticide-specific resistance mechanisms. Transcriptomic data revealed overexpression of genes including cytochrome P450s, glutathione-s-transferases and carboxylesterases (including CYP4C36, CYP6AA1, CYP6M2, CYP6M3, CYP6P4, CYP9K1, CYP9L1, GSTD3, GSTE2, GSTE3, GSTE4, GSTE5, GSTE7 and two carboxylesterases) that were shared between malathion and permethrin survivors. We also identified nineteen highly overexpressed cuticular-associated proteins (including CYP4G16, CYP4G17 and chitinase) and eighteen salivary gland proteins (including D7r4 short form salivary protein), which may be contributing to a non-specific resistance phenotype by either enhancing the cuticular barrier or promoting binding and sequestration of insecticides, respectively. These findings provide novel insights into the molecular basis of insecticide resistance in this lesser well-characterized major malaria vector species

Pyrethroid resistance in the New World malaria vector Anopheles albimanus is mediated by cytochrome P450 CYP6P5

Pyrethroid resistance in the malaria vector Anopheles albimanus presents an obstacle to malaria elimination in the Americas. Here, An. albimanus CYP6P5 (the most overexpressed P450 in a Peruvian population) was functionally characterized. Recombinant CYP6P5 metabolized the type II pyrethroids, deltamethrin and α cypermethrin with comparable affinities (KM of 3.3 μM ± 0.4 and 3.6 μM ± 0.5, respectively), but exhibited a 2.7-fold higher catalytic rate for α-cypermethrin (kcat of 6.02 min− 1 ± 0.2) versus deltamethrin (2.68 min− 1 ± 0.09). Timecourse assays revealed progressive depletion of the above pyrethroids with production of four HPLCdetectable metabolites. Low depletion was obtained with type I pyrethroid, permethrin. Transgenic expression in Drosophila melanogaster demonstrated that overexpression of CYP6P5 alone conferred type II pyrethroid resistance, with only 16% and 55.3% mortalities in flies exposed to 0.25% α-cypermethrin and 0.15% deltamethrin, respectively. Synergist bioassays using P450 inhibitor piperonylbutoxide significantly recovered susceptibility (mortality = 73.6%, p < 0.001) in synergized flies exposed to 4% piperonylbutoxide, plus 0.25% α-cypermethrin, compared to non-synergized flies (mortality = 4.9%). Moderate resistance was also observed towards 4% DDT. These findings established the preeminent role of CYP6P5 in metabolic resistance in An. albimanus, highlighting challenges associated with deployment of insecticide-based control tools in the Americas

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

Transcriptomic analysis of Anopheles gambiae from Benin reveals overexpression of salivary and cuticular proteins associated with cross-resistance to pyrethroids and organophosphates

Background: Insecticide resistance (IR) is one of the major threats to malaria vector control programs in endemic countries. However, the mechanisms underlying IR are poorly understood. Thus, investigating gene expression patterns related to IR can offer important insights into the molecular basis of IR in mosquitoes. In this study, RNA-Seq was used to characterize gene expression in Anopheles gambiae surviving exposure to pyrethroids (deltamethrin, alphacypermethrin) and an organophosphate (pirimiphos-methyl). Results: Larvae of An. gambiae s.s. collected from Bassila and Djougou in Benin were reared to adulthood and phenotyped for IR using a modified CDC intensity bottle bioassay. The results showed that mosquitoes from Djougou were more resistant to pyrethroids (5X deltamethrin: 51.7% mortality; 2X alphacypermethrin: 47.4%) than Bassila (1X deltamethrin: 70.7%; 1X alphacypermethrin: 77.7%), while the latter were more resistant to pirimiphos-methyl (1.5X: 48.3% in Bassila and 1X: 21.5% in Djougou). RNA-seq was then conducted on resistant mosquitoes, non-exposed mosquitoes from the same locations and the laboratory-susceptible An. gambiae s.s. Kisumu strain. The results showed overexpression of detoxification genes, including cytochrome P450s (CYP12F2, CYP12F3, CYP4H15, CYP4H17, CYP6Z3, CYP9K1, CYP4G16, and CYP4D17), carboxylesterase genes (COEJHE5E, COE22933) and glutathione S-transferases (GSTE2 and GSTMS3) in all three resistant mosquito groups analyzed. Genes encoding cuticular proteins (CPR130, CPR10, CPR15, CPR16, CPR127, CPAP3-C, CPAP3-B, and CPR76) were also overexpressed in all the resistant groups, indicating their potential role in cross resistance in An. gambiae. Salivary gland protein genes related to ‘salivary cysteine-rich peptide’ and ‘salivary secreted mucin 3’ were also over-expressed and shared across all resistant groups. Conclusion: Our results suggest that in addition to metabolic enzymes, cuticular and salivary gland proteins could play an important role in cross-resistance to multiple classes of insecticides in Benin. These genes warrant further investigation to validate their functional role in An. gambiae resistance to insecticides

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

Potential vectors of equine arboviruses in the UK

There is growing concern about the increasing risk of disease outbreaks caused by arthropod-borne viruses (arboviruses) in both humans and animals. There are several mosquito-borne viral diseases that cause varying levels of morbidity and mortality in horses and that can have substantial welfare and economic ramifications. While none has been recorded in the UK, vector species for some of these viruses are present, suggesting that UK equines may be at risk. We undertook, therefore, the first study of mosquito species on equine premises in the UK. Mosquito Magnet traps and red-box traps were used to sample adults, and larvae were collected from water sources such as tyres, buckets, ditches and pools. Several species which are known to be capable of transmitting important equine infectious arboviruses were trapped. The most abundant, with a maximum catch of 173 in 72 hours was Ochlerotatus detritus, a competent vector of some flaviviruses; the highest densities were found near saltmarsh habitats. The most widespread species, recorded at >75% of sites, was Culiseta annulata. This study demonstrates that potential mosquito vectors of arboviruses, including those known to be capable of infecting horses, are present and may be abundant on equine premises in the UK

The challenges posed by equine arboviruses

Equine populations worldwide are at increasing risk of infection by viruses transmitted by biting arthropods including mosquitoes, biting midges (Culicoides), sandflies and ticks. These include the flaviviruses (Japanese encephalitis, West Nile and Murray Valley encephalitis), alphaviruses (eastern, western and Venezuelan encephalitis) and the orbiviruses (African horse sickness and equine encephalosis). This review provides an overview of the challenges faced in the surveillance, prevention and control of the major equine arboviruses, particularly in the context of these viruses emerging in new regions of the world

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