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Aedes aegypti is an important vector of dengue virus and other arboviruses that affect human health. After being ingested in an infectious bloodmeal, but before being transmitted from mosquito to human, dengue virus must disseminate from the vector midgut into the hemocoel and then the salivary glands. This process, the extrinsic incubation period, typically takes 6-14 days. Since older mosquitoes are responsible for transmission, understanding the age structure of vector populations is important. Transcriptional profiling can facilitate predictions of the age structures of mosquito populations, critical for estimating their potential for pathogen transmission. In this study, we utilized a two-gene transcript model to assess the age structure and daily survival rates of three populations (Key West, Marathon, and Key Largo) of Ae. aegypti from the Florida Keys, United States, where repeated outbreaks of autochthonous dengue transmission have recently occurred. We found that Key Largo had the youngest Ae. aegypti population with the lowest daily survival rate, while Key West had the oldest population and highest survival rate. Across sites, 22.67% of Ae. aegypti females were likely old enough to transmit dengue virus (at least 15 days post emergence). Computed estimates of the daily survival rate (0.8364 using loglinear and 0.8660 using non-linear regression), indicate that dengue vectors in the region experienced relatively low daily mortality. Collectively, our data suggest that Ae. aegypti populations across the Florida Keys harbor large numbers of older individuals, which likely contributes to the high risk of dengue transmission in the area.

Abstract Background Though persons of African descent have one of the widest genetic variability, genetic polymorphisms of drug-metabolising enzymes such as N-Acetyltransferase-2 (NAT2) are understudied. This study aimed to identify prevalent NAT2 single nucleotide polymorphisms (SNPs) and infer their potential effects on enzyme function among Kenyan volunteers with tuberculosis (TB) infection. Genotypic distribution at each SNP and non-random association of alleles were evaluated by testing for Hardy-Weinberg Equilibrium (HWE) and Linkage Disequilibrium (LD). Methods We isolated genomic DNA from cryopreserved Peripheral Blood Mononuclear Cells of 79 volunteers. We amplified the protein-coding region of the NAT2 gene by polymerase chain reaction (PCR) and sequenced PCR products using the Sanger sequencing method. Sequencing reads were mapped and aligned to the NAT2 reference using the Geneious software (Auckland, New Zealand). Statistical analyses were performed using RStudio version 4.3.2 (2023.09.1 + 494). Results The most frequent haplotype was the wild type NAT2 * 4 (37%). Five genetic variants: 282C > T (NAT2 * 13), 341 T > C (NAT2 * 5), 803A > G (NAT2*12), 590G > A (NAT2*6) and 481C > T (NAT2*11) were observed with allele frequencies of 29%, 18%, 6%, 6%, and 4% respectively. According to the bimodal distribution of acetylation activity, the predicted phenotype was 76% rapid (mainly consisting of the wildtype NAT2 * 4 and the NAT2*13A variant). A higher proportion of rapid acetylators were female, 72% vs 28% male (p = 0.022, odds ratio [OR] 3.48, 95% confidence interval [CI] 1.21 to 10.48). All variants were in HWE. NAT2 341 T > C was in strong complete LD with the 590G > A variant (D′ = 1.0, r2 = − 0.39) but not complete LD with the 282C > T variant (D′ = 0.94, r2 = − 0.54). Conclusion The rapid acetylation haplotypes predominated. Despite the LD observed, none of the SNPs could be termed tag SNP. This study adds to the genetic characterisation data of African populations at NAT2, which may be useful for developing relevant pharmacogenomic tools for TB therapy. To support optimised, pharmacogenomics-guided TB therapy, we recommend genotype-phenotype studies, including studies designed to explore gender-associated differences.

Insect-specific viruses (ISVs) include viruses that are restricted to the infection of mosquitoes and are spread mostly through transovarial transmission. Despite using a distinct mode of transmission, ISVs are often phylogenetically related to arthropod-borne viruses (arboviruses) that are responsible for human diseases and able to infect both mosquitoes and vertebrates. ISVs can also induce a phenomenon called “superinfection exclusion”, whereby a primary ISV infection in an insect inhibits subsequent viral infections of the insect. This has sparked interest in the use of ISVs for the control of pathogenic arboviruses transmitted by mosquitoes. In particular, insect-specific flaviviruses (ISFs) have been shown to inhibit infection of vertebrate-infecting flaviviruses (VIFs) both in vitro and in vivo. This has shown potential as a new and ecologically friendly biological approach to the control of arboviral disease. For this intervention to have lasting impacts for biological control, it is imperative that ISFs are maintained in mosquito populations with high rates of vertical transmission. Therefore, these strategies will need to optimise vertical transmission of ISFs in order to establish persistently infected mosquito lines for sustainable arbovirus control. This review compares recent observations of vertical transmission of arboviral and insect-specific flaviviruses and potential determinants of transovarial transmission rates to understand how the vertical transmission of ISFs may be optimised for effective arboviral control.

Vector surveillance of Plasmodium falciparum is critical for monitoring and reducing one of the most severe forms of malaria, which causes high morbidity and mortality in children under five and pregnant women. Here we developed a rapid and highly sensitive test for the detection of P. falciparum (Pf)-infected mosquitoes (Rapid Pf test), with high suitability for low-resource vector surveillance implementation. The Rapid Pf test had similar analytical sensitivity to laboratory-based tests, detecting down to 4 copies/μL of a 18S rRNA DNA standard. In addition, the Rapid Pf test could be completed in less than 30 minutes, and only required a liquid sample preparation reagent, pestle, tube, and 39°C heating block for operation, indicating amenability for low-resource implementation. Diagnostic testing was performed using Anopheles stephensi mosquitoes, either uninfected, or fed with P. falciparum gametocyte cultures. These P. falciparum fed mosquitoes were determined to have 79% infection prevalence based on parallel microscopy and qPCR testing on a subset of 19 mosquitoes. However, our Rapid Pf test determined a 90% positive test rate when testing individual infected mosquitoes (n=30), and did not detect 40 uninfected mosquitoes regardless of blood-fed status (n=40), suggesting the true prevalence of infection in the mosquitoes may have been higher than calculated by qPCR and microscopy. The Rapid Pf test was demonstrated to detect infection in individual mosquitoes (both fresh and frozen/thawed), as well as pools of 1 infected mosquito mixed with 19 known uninfected mosquitoes, and individual mosquitoes left in traps for up to 8 days. After testing on infected and uninfected mosquitoes (n=148) the Rapid Pf test was conservatively estimated to achieve 100% diagnostic sensitivity (95% confidence interval, CI: 91%-100%) and 97% diagnostic specificity (CI: 92%-99%) compared to the estimated prevalence from combined microscopy and qPCR results. These results indicate the Rapid Pf test could provide a highly effective tool for weekly surveillance of infected mosquitoes, to assist with P. falciparum monitoring and intervention studies.

Abstract The mosquito Aedes aegypti is the primary vector of a range of medically important viruses including dengue, Zika, West Nile, yellow fever, and chikungunya viruses. The endosymbiotic bacterium Wolbachia pipientis wAlbB strain is a promising biocontrol agent for blocking viral transmission by Ae. aegypti. To predict the long-term efficacy of field applications, a thorough understanding of the interactions between symbiont, host, and pathogen is required. Wolbachia influences host physiology in a variety of ways including reproduction, immunity, metabolism, and longevity. MicroRNAs (miRNAs) are highly conserved small non-coding RNAs that regulate gene expression in eukaryotes and viruses. Several miRNAs are known to regulate biological processes in Drosophila and mosquitoes, including facilitating Wolbachia maintenance. We generated the first chromosomal map of Ae. aegypti miRNAs, and compared miRNA expression profiles between a wAlbB-transinfected Ae. aegypti mosquito line and a tetracycline cleared derivative, using deep small RNA-sequencing. We found limited modulation of miRNAs in response to wAlbB infection. Several miRNAs were modulated in response to age, some of which showed greater upregulation in wAlbB-infected mosquitoes than in tetracycline cleared ones. By selectively inhibiting some differentially expressed miRNAs, we identified miR-2946-3p and miR-317-3p as effecting mosquito longevity in Wolbachia-infected mosquitoes.

The pantropic emergence of severe dengue disease can partly be attributed to the co-circulation of different dengue viruses (DENVs) in the same geographical location. Effective monitoring for circulation of each of the four DENVs is critical to inform disease mitigation strategies. In low resource settings, this can be effectively achieved by utilizing inexpensive, rapid, sensitive and specific assays to detect viruses in mosquito populations. In this study, we developed four rapid DENV tests with direct applicability for low-resource virus surveillance in mosquitoes. The test protocols utilize a novel sample preparation step, a single-temperature isothermal amplification, and a simple lateral flow detection. Analytical sensitivity testing demonstrated tests could detect down to 1,000 copies/µL of virus-specific DENV RNA, and analytical specificity testing indicated tests were highly specific for their respective virus, and did not detect closely related flaviviruses. All four DENV tests showed excellent diagnostic specificity and sensitivity when used for detection of both individually infected mosquitoes and infected mosquitoes in pools of uninfected mosquitoes. With individually infected mosquitoes, the rapid DENV-1, -2 and -3 tests showed 100% diagnostic sensitivity (95% CI = 69% to 100%, n=8 for DENV-1; n=10 for DENV 2,3) and the DENV-4 test showed 92% diagnostic sensitivity (CI: 62% to 100%, n=12) along with 100% diagnostic specificity (CI: 48–100%) for all four tests. Testing infected mosquito pools, the rapid DENV-2, -3 and -4 tests showed 100% diagnostic sensitivity (95% CI = 69% to 100%, n=10) and the DENV-1 test showed 90% diagnostic sensitivity (55.50% to 99.75%, n=10) together with 100% diagnostic specificity (CI: 48–100%). Our tests reduce the operational time required to perform mosquito infection status surveillance testing from > two hours to only 35 minutes, and have potential to improve accessibility of mosquito screening, improving monitoring and control strategies in low-income countries most affected by dengue outbreaks.

Biological control of mosquito vectors using the endosymbiotic bacteria Wolbachia is an emerging strategy for the management of human arboviral diseases. We recently described the development of a strain of Aedes aegypti infected with the Wolbachia strain wAlbB (referred to as the wAlbB2-F4 strain) through simple backcrossing of wild type Australian mosquitoes with a wAlbB infected Ae. aegypti strain from the USA. Field releases of male wAlbB2-F4 mosquitoes resulted in the successful suppression of wild populations of mosquitoes in the trial sites by exploiting the strain's Wolbachia-induced cytoplasmic incompatibility. We now demonstrate that the strain is resistant to infection by dengue and Zika viruses and is genetically similar to endemic Queensland populations. There was a fourfold reduction in the proportion of wAlbB2-F4 mosquitoes that became infected following a blood meal containing dengue 2 virus (16.7%) compared to wild type mosquitoes (69.2%) and a 6-7 fold reduction in the proportion of wAlbB2-F4 mosquitoes producing virus in saliva following a blood meal containing an epidemic strain of Zika virus (8.7% in comparison to 58.3% in wild type mosquitoes). Restriction-site Associated DNA (RAD) sequencing revealed that wAlbB2-F4 mosquitoes have > 98% Australian ancestry, confirming the successful introduction of the wAlbB2 infection into the Australian genomic background through backcrossing. Genotypic and phenotypic analyses showed the wAlbB2-F4 strain retains the insecticide susceptible phenotype and genotype of native Australian mosquitoes. We demonstrate that the Wolbachia wAlbB2-F4, in addition to being suitable for population suppression programs, can also be effective in population replacement programs given its inhibition of virus infection in mosquitoes. The ease at which a target mosquito population can be transfected with wAlbB2, while retaining the genotypes and phenotypes of the target population, shows the utility of this strain for controlling the Ae. aegypti mosquitoes and the pathogens they transmit.

The pantropic emergence of severe dengue disease can partly be attributed to the co-circulation of different dengue viruses (DENVs) in the same geographical location. Effective monitoring for circulation of each of the four DENVs is critical to inform disease mitigation strategies. In low resource settings, this can be effectively achieved by utilizing inexpensive, rapid, sensitive and specific assays to detect viruses in mosquito populations. In this study, we developed four rapid DENV tests with direct applicability for low-resource virus surveillance in mosquitoes. The test protocols utilize a novel sample preparation step, a single-temperature isothermal amplification, and a simple lateral flow detection. Analytical sensitivity testing demonstrated tests could detect down to 1,000 copies/µL of virus-specific DENV RNA, and analytical specificity testing indicated tests were highly specific for their respective virus, and did not detect closely related flaviviruses. All four DENV tests showed excellent diagnostic specificity and sensitivity when used for detection of both individually infected mosquitoes and infected mosquitoes in pools of uninfected mosquitoes. With individually infected mosquitoes, the rapid DENV-1, -2 and -3 tests showed 100% diagnostic sensitivity (95% CI = 69% to 100%, n=8 for DENV-1; n=10 for DENV 2,3) and the DENV-4 test showed 92% diagnostic sensitivity (CI: 62% to 100%, n=12) along with 100% diagnostic specificity (CI: 48–100%) for all four tests. Testing infected mosquito pools, the rapid DENV-2, -3 and -4 tests showed 100% diagnostic sensitivity (95% CI = 69% to 100%, n=10) and the DENV-1 test showed 90% diagnostic sensitivity (55.50% to 99.75%, n=10) together with 100% diagnostic specificity (CI: 48–100%). Our tests reduce the operational time required to perform mosquito infection status surveillance testing from > two hours to only 35 minutes, and have potential to improve accessibility of mosquito screening, improving monitoring and control strategies in low-income countries most affected by dengue outbreaks.

During 2015-2016, outbreaks of Zika virus (ZIKV) occurred in Southeast Asia and the Americas. Most ZIKV infections in humans are asymptomatic, while clinical manifestation is usually a self-limiting febrile disease with maculopapular rash. However, ZIKV is capable of inducing a range of severe neurological complications collectively described as congenital Zika syndrome (CZS). Notably, the scale and magnitude of outbreaks in Southeast Asia were significantly smaller compared to those in the Americas. Sequence comparison between epidemic-associated ZIKV strains from Southeast Asia with those from the Americas revealed a methionine to valine substitution at residue position 114 of the NS5 protein (NS5-M114V) in all the American isolates. Using an American isolate of ZIKV (Natal), we investigated the impact of NS5-M114V mutation on virus replication in cells, virulence in interferon (IFN) α/β receptor knockout (Ifnar-/-) mice, as well as replication and transmission potential in Aedes aegypti mosquitoes. We demonstrated that NS5-M114V mutation had insignificant effect on ZIKV replication efficiency in cells, its ability to degrade STAT2, and virulence in vivo, albeit viremia was slightly prolonged in mice. Furthermore, NS5-M114V mutation decreased mosquito infection and dissemination rates but had no effect on virus secretion into the saliva. Taken together, our findings support the notion that NS5-M114V mutation is unlikely to be a major determinant for virus replication and transmission potential.

Abstract Background Effective vector control measures are essential in a world where many mosquito-borne diseases have no vaccines or drug therapies available. Insecticidal tools remain the mainstay of most vector-borne disease management programmes, although their use for both agricultural and public health purposes has resulted in selection for resistance. Despite this, little is known about the fitness costs associated with specific insecticide-resistant genotypes and their implications for the management of resistance. In Aedes aegypti, the primary vector of dengue, chikungunya, and Zika, the best-characterised resistance mechanisms are single-point mutations that protect the voltage-gated sodium channel from the action of pyrethroids. Methods We evaluated the fitness cost of two co-occurring, homozygous mutations (V1016G and S989P) by back-crossing a resistant strain of A. aegypti from Timor-Leste into a fully susceptible strain from Queensland. The creation of the backcross strain allowed us to isolate these kdr mutations in an otherwise susceptible genetic background. Results In comparison to the susceptible strain, the backcrossed colony exhibited longer larval development times (5 days, P

The function on subgenomic flaviviral RNA (sfRNA) in the mosquito vector is not well understood. Here, Slonchak et al. show that sfRNA affects virus-induced apoptosis and dissemination of ZIKV in Aedes aegypti mosquitoes, suggesting a role of sfRNA in Zika virus replication and transmission.

Abstract Background Humans are the primary hosts of dengue viruses (DENV). However, sylvatic cycles of transmission can occur among non-human primates and human encroachment into forested regions can be a source of emergence of new strains such as the highly divergent and sylvatic strain of DENV2, QML22, recovered from a dengue fever patient returning to Australia from Borneo. The objective of the present study was to evaluate the vector competence of Australian Aedes aegypti mosquitoes for this virus. Methods Four- to five-day-old mosquitoes from two strains of Ae. aegypti from Queensland, Australia, were fed a meal of sheep blood containing 108 50% cell culture infectious dose per ml (CCID50/ml) of either QML22 or an epidemic strain of DENV serotype 2 (QML16) isolated from a dengue fever patient in Australia in 2015. Mosquitoes were maintained at 28 °C, 75% relative humidity and sampled 7, 10 and 14 days post-infection (dpi). Live virions in mosquito bodies (abdomen/thorax), legs and wings and saliva expectorates from individual mosquitoes were quantified using a cell culture enzyme-linked immunosorbent assay (CCELISA) to determine infection, dissemination and transmission rates. Results The infection and dissemination rates of the sylvatic DENV2 strain, QML22, were significantly lower than that for QML16. While the titres of virus in the bodies of mosquitoes infected with either of these viruses were similar, titres in legs and wings were significantly lower in mosquitoes infected with QML22 at most time points although they reached similar levels by 14 dpi. QML16 was detected in 16% (n = 25) and 28% (n = 25) of saliva expectorates at 10 and 14 dpi, respectively. In contrast, no virus was detected in the saliva expectorates of QML22 infected mosquitoes. Conclusions Australia urban/peri-urban Ae. aegypti species are susceptible to infection by the sylvatic and highly divergent DENV 2 QML22 but replication of QML22 is attenuated relative to the contemporary strain, QML16. A salivary gland infection or escape barrier may be acting to prevent infection of saliva and would prevent onward transmission of this highly divergent virus in Australia.

Abstract Background More than 70 arboviruses have been identified in Australia and the transmission cycles of most are poorly understood. While there is an extensive list of arthropods from which these viruses have been recovered, far less is known about the non-human hosts that may be involved in the transmission cycles of these viruses and the relative roles of different mosquito species in cycles of transmission involving different hosts. Some of the highest rates of human infection with zoonotic arboviruses, such as Ross River (RRV) and Barmah Forest (BFV) viruses, occur in coastal regions of north-eastern Australia. Methods Engorged mosquitoes collected as a part of routine surveillance using CO2-baited light traps in the Rockhampton Region and the adjoining Shire of Livingstone in central Queensland, north-eastern Australia, were analysed for the source of their blood meal. A 457 or 623 nucleotide region of the cytochrome b gene in the blood was amplified by PCR and the amplicons sequenced. The origin of the blood was identified by comparing the sequences obtained with those in GenBank®. Results The most common hosts for the mosquitoes sampled were domestic cattle (26/54) and wild birds (14/54). Humans (2/54) were an infrequent host for this range of mosquitoes that are known to transmit arboviruses causing human disease, and in an area where infections with human pathogens like RRV and BFV are commonly recorded. The blood meals identified in the most abundant vector analysed, Culex annulirostris, were from 10 different vertebrate hosts. The notable detection of chimpanzee blood in two mosquitoes, presumably obtained from a nearby zoo, extends the known range of hosts for this species. Culex quinquefasciatus and Cx. sitiens fed almost exclusively on a variety of bird species. Conclusions While human-mosquito-human transmission of arboviruses like RRV can occur, this study highlights the potential importance of zoonotic cycles of transmission, including avian species, of arboviruses that are indigenous to Australia. Further studies on larger samples of blood-engorged mosquitoes are required to validate the trends observed herein. Moreover, serological and virological evidence that the hosts on which the mosquitoes are feeding are being infected with arboviruses of interest are required.

Binjari virus (BinJV) is a lineage II or dual-host affiliated insect-specific flavivirus previously demonstrated as replication-deficient in vertebrate cells. Previous studies have shown that BinJV is tolerant to exchanging its structural proteins (prM-E) with pathogenic flaviviruses, making it a safe backbone for flavivirus vaccines. Here, we report generation by circular polymerase extension reaction of BinJV expressing zsGreen or mCherry fluorescent protein. Recovered BinJV reporter viruses grew to high titres (107−8 FFU/mL) in Aedes albopictus C6/36 cells assayed using immunoplaque assays (iPA). We also demonstrate that BinJV reporters could be semi-quantified live in vitro using a fluorescence microplate reader with an observed linear correlation between quantified fluorescence of BinJV reporter virus-infected C6/36 cells and iPA-quantitated virus titres. The utility of the BinJV reporter viruses was then examined in homologous and heterologous superinfection exclusion assays. We demonstrate that primary infection of C6/36 cells with BinJVzsGreen completely inhibits a secondary infection with homologous BinJVmCherry or heterologous ZIKVmCherry using fluorescence microscopy and virus quantitation by iPA. Finally, BinJVzsGreen infections were examined in vivo by microinjection of Aedes aegypti with BinJVzsGreen. At seven days post-infection, a strong fluorescence in the vicinity of salivary glands was detected in frozen sections. This is the first report on the construction of reporter viruses for lineage II insect-specific flaviviruses and establishes a tractable system for exploring flavivirus superinfection exclusion in vitro and in vivo.

The adaptive immune system in vertebrates has evolved to recognize non-self antigens, such as proteins expressed by infectious agents and mutated cancer cells. T cells play an important role in antigen recognition by expressing a diverse repertoire of antigen-specific receptors, which bind epitopes to mount targeted immune responses. Recent advances in high-throughput sequencing have enabled the routine generation of T-cell receptor (TCR) repertoire data. Identifying the specific epitopes targeted by different TCRs in these data would be valuable. To accomplish that, we took advantage of the ever-increasing number of TCRs with known epitope specificity curated in the Immune Epitope Database (IEDB) since 2004. We compared seven metrics of sequence similarity to determine their power to predict if two TCRs have the same epitope specificity. We found that a comprehensive k-mer matching approach produced the best results, which we have implemented into TCRMatch, an openly accessible tool (http://tools.iedb.org/tcrmatch/) that takes TCR β-chain CDR3 sequences as an input, identifies TCRs with a match in the IEDB, and reports the specificity of each match. We anticipate that this tool will provide new insights into T cell responses captured in receptor repertoire and single cell sequencing experiments and will facilitate the development of new strategies for monitoring and treatment of infectious, allergic, and autoimmune diseases, as well as cancer.

BackgroundThe development of insecticide resistance in mosquitoes can have pleiotropic effects on key behaviours such as mating competition and host-location. Documenting these effects is crucial for understanding the dynamics and costs of insecticide resistance and may give researchers an evidence base for promoting vector control programs that aim to restore or conserve insecticide susceptibility.Methods and findingsWe evaluated changes in behaviour in a backcrossed strain of Aedes aegypti, homozygous for two knockdown resistance (kdr) mutations (V1016G and S989P) isolated in an otherwise fully susceptible genetic background. We compared biting activity, host location behaviours, wing beat frequency (WBF) and mating competition between the backcrossed strain, and the fully susceptible and resistant parental strains from which it was derived. The presence of the homozygous kdr mutations did not have significant effects on blood avidity, the time to locate a host, or WBF in females. There was, however, a significant reduction in mean WBF in males and a significant reduction in estimated male mating success (17.3%), associated with the isolated kdr genotype.ConclusionsOur results demonstrate a cost of insecticide resistance associated with an isolated kdr genotype and manifest as a reduction in male mating success. While there was no recorded difference in WBF between the females of our strains, the significant reduction in male WBF recorded in our backcrossed strain might contribute to mate-recognition and mating disruption. These consequences of resistance evolution, especially when combined with other pleiotropic fitness costs that have been previously described, may encourage reversion to susceptibility in the absence of insecticide selection pressures. This offers justification for the implementation of insecticide resistance management strategies based on the rotation or alternation of different insecticide classes in space and time.

Japanese encephalitis (JE) is the leading cause of viral encephalitis in Asia. Until 2022, only six locally transmitted human JE cases had been reported in Australia; five in northern Queensland and one in the Northern Territory. Thus, JE was mainly considered to be a disease of travellers. On 4 March 2022, JE was declared a ‘Communicable Disease Incident of National Significance’ when a locally acquired human case was confirmed in southern Queensland. By 11 May 2022, 41 human JE cases had been notified in four states in Australia, in areas where JE has never been detected before. From this perspective, we discuss the potential reasons for the recent emergence of the JE virus in Australia in areas where JE has never been previously reported as well as the implications of and options for mass immunisation programs if the outbreak escalates in a JE virus-immunologically naïve population.

IntroductionMany arboviruses of public health significance are maintained in zoonotic cycles with complex transmission pathways. The presence of serum antibody against arboviruses in vertebrates provides evidence of their historical exposure but reveals nothing about the vector-reservoir relationship. Moreover, collecting blood or tissue samples from vertebrate hosts is ethically and logistically challenging. We developed a novel approach for screening the immune status of vertebrates against Ross River virus that allows us to implicate the vectors that form the transmission pathways for this commonly notified Australian arboviral disease.MethodsA micro-plaque reduction neutralisation test (micro-PRNT) was developed and validated on koala (Phascolarctos cinereus) sera against a standard PRNT. The ability of the micro-PRNT to detect RRV antibodies in mosquito blood meals was then tested using two mosquito models. Laboratory-reared Aedes aegypti were fed, via a membrane, on sheep blood supplemented with RRV seropositive and seronegative human sera. Aedes notoscriptus were fed on RRV seropositive and seronegative human volunteers. Blood-fed mosquitoes were harvested at various time points after feeding and their blood meals analysed for the presence of RRV neutralising antibodies using the micro-PRNT.ResultsThere was significant agreement of the plaque neutralisation resulting from the micro-PRNT and standard PRNT techniques (R2 = 0.65; PConclusionsThe small volumes of blood present in mosquito abdomens can be used to identify RRV antibodies and therefore host exposure to arbovirus infection. In tandem with the accurate identification of the mosquito, and diagnostics for the host origin of the blood meal, this technique has tremendous potential for exploring RRV transmission pathways. It can be adapted for similar studies on other mosquito borne zoonoses.

Due to their good mechanical properties, low density, and ease of processing polymer nanocomposites are of interest for a multitude of applications in the automotive, electronics, and leisure industry. Besides having an impact on short-term mechanical performance of polymers, the addition of nanoreinforcements can have also a significant effect on long-term properties such as the resistance to static (creep) and cyclic (fatigue) loadings. However, despite its significance there is a shortage of long-term mechanical performance data for thermoplastic-based polymer nanocomposites. Reason being that existing characterization methods for long-term performance and durability are time consuming and limited in their applicability. Here, an engineering approach to predict long-term time-to-failure of polycarbonate/carbon nanotube (PC/CNT) nanocomposites is presented based on short-term experimentation with an application to both creep and fatigue. Results showed that the addition of CNTs had an opposite effect on two important long-term failure mechanisms. Addition of CNTs lead to improvements in durability in the plasticity-controlled failure regime, whereas it had an adverse effect in the slow crack growth-controlled regime, meaning that in the latter regime nanocomposite performance was significantly less than that of the neat polymer matrix.

The anisotropy and strain rate dependence of the mechanical response of short-fiber-reinforced thermoplastics was studied using a straightforward micromechanical finite element analysis of representative volume elements (RVEs). RVEs are created based on the fiber orientation tensor, which quantifies the processing-induced fiber orientation distribution. The matrix is described by a strain rate-dependent constitutive model (the Eindhoven glassy polymer (EGP) model), which accurately captures the intrinsic response of amorphous polymers. The micromechanical results indicate that the influence of strain rate and that of the loading direction on the yield stress are multiplicatively decouplable, which confirms previous experimental observations. Moreover, it is demonstrated that the yield stress, to a good approximation, can be directly linked to the fiber orientation in the direction of loading. This leads to a new relation that uniquely links the rate dependence of the yield stress to the fiber orientation in loading direction.

Abstract Observational studies on smoking and risk of hay fever and asthma have shown inconsistent results. However, observational studies may be biased by confounding and reverse causation. Mendelian randomization uses genetic variants as markers of exposures to examine causal effects. We examined the causal effect of smoking on hay fever and asthma by using the smoking-associated single nucleotide polymorphism (SNP) rs16969968/rs1051730. We included 231,020 participants from 22 population-based studies. Observational analyses showed that current vs never smokers had lower risk of hay fever (odds ratio (OR) = 0·68, 95% confidence interval (CI): 0·61, 0·76; P

BACKGROUND:Recent epidemics of Zika virus (ZIKV) in the Pacific and the Americas have highlighted its potential as an emerging pathogen of global importance. Both Aedes (Ae.) aegypti and Ae. albopictus are known to transmit ZIKV but variable vector competence has been observed between mosquito populations from different geographical regions and different virus strains. Since Australia remains at risk of ZIKV introduction, we evaluated the vector competence of local Ae. aegypti and Ae. albopictus for a Brazilian epidemic ZIKV strain. In addition, we evaluated the impact of daily temperature fluctuations around a mean of 28°C on ZIKV transmission and extrinsic incubation period. METHODOLOGY/PRINCIPAL FINDINGS:Mosquitoes were orally challenged with a Brazilian ZIKV strain (8.8 log CCID50/ml) and maintained at either 28°C constant or fluctuating temperature conditions. At 3, 7 and 14 days post-infection (dpi), ZIKV RNA copies were quantified in mosquito bodies, as well as wings and legs, using qRT-PCR, while virus antigen in saliva (a proxy for transmission) was detected using a cell culture ELISA. Despite high body and disseminated infection rates in both vectors, the transmission rates of ZIKV in saliva of Ae. aegypti (50-60%) were significantly higher than in Ae. albopictus (10%) at 14 dpi. Both species supported a high viral load in bodies, with no significant differences between constant and fluctuating temperature conditions. However, a significant difference in viral load in wings and legs between species was observed, with higher titres in Ae. aegypti maintained at constant temperature conditions. For ZIKV transmission to occur in Ae. aegypti, a disseminated virus load threshold of 7.59 log10 copies had to be reached. CONCLUSIONS/SIGNIFICANCE:Australian Ae. aegypti are better able to transmit a Brazilian ZIKV strain than Ae. albopictus. The results are in agreement with the global consensus that Ae. aegypti is the major vector of ZIKV.

In the 2015 Paris Agreement, nations worldwide pledged emissions reductions (Nationally Determined Contributions-NDCs) to avert the threat of climate change, and agreed to periodically review these pledges to strengthen their level of ambition. Previous studies have analyzed NDCs largely in terms of their implied contribution to limit global warming, their implications on the energy sector or on mitigation costs. Nevertheless, a gap in the literature exists regarding the understanding of implications of the NDCs on countries' Energy-Water-Land nexus resource systems. The present paper explores this angle within the regional context of Latin America by employing the Global Change Assessment Model, a state-of-the-art integrated assessment model capable of representing key system-wide interactions among nexus sectors and mitigation policies. By focusing on Brazil, Mexico, Argentina and Colombia, we stress potential implications on national-level water demands depending on countries' strategies to enforce energy-related emissions reductions and their interplays with the land sector. Despite the differential implications of the Paris pledges on each country, increased water demands for crop and biomass irrigation and for electricity generation stand out as potential trade-offs that may emerge under the NDC policy. Hence, this study underscores the need of considering a nexus resource planning framework (known as "Nexus Approach") in the forthcoming NDCs updating cycles as a mean to contribute toward sustainable development.

Dengue virus (DENV) is the most important mosquito-borne viral pathogen of humans, comprising four serotypes (DENV-1 to -4) with a myriad of genotypes and strains. The kinetics of DENV replication within the mosquito following ingestion of a blood meal influence the pathogen’s ability to reach the salivary glands and thus the transmission potential. The influence of DENV serotype and strain diversity on virus kinetics in the two main vector species, Aedes aegypti and Ae. albopictus, has been poorly characterized. We tested whether DENV replication kinetics vary systematically among serotypes and strains, using Australian strains of the two vectors. Mosquitoes were blood fed with two strains per serotype, and sampled at 3, 6, 10 and 14-days post-exposure. Virus infection in mosquito bodies, and dissemination of virus to legs and wings, was detected using qRT-PCR. For both vectors, we found significant differences among serotypes in proportions of mosquitoes infected, with higher numbers for DENV-1 and -2 versus other serotypes. Consistent with this, we observed that DENV-1 and -2 generally replicated to higher RNA levels than other serotypes, particularly at earlier time points. There were no significant differences in either speed of infection or dissemination between the mosquito species. Our results suggest that DENV diversity may have important epidemiological consequences by influencing virus kinetics in mosquito vectors.

Similar to other flaviviruses, Zika virus (ZIKV) produces abundant subgenomic flavivirus RNA (sfRNA) derived from the 3’ untranslated region. The molecular mechanisms that determine the functions of sfRNA are currently not completely understood. Here, we created ZIKV mutants deficient in sfRNA production and employed them to investigate the role of this RNA in virus interactions with mammalian and insect hosts. We found that in mosquitoes, sfRNA facilitates virus replication and is required for ZIKV dissemination into saliva and virus transmission. The production of sfRNA was found to have no effect on the RNAi pathway, but instead downregulated the expression of genes involved in the regulation of apoptosis. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) of histological sections from infected mosquitoes confirmed that sfRNA prevents the apoptotic death of infected cells, thus identifying inhibition of apoptosis as a novel mechanism of sfRNA action in mosquitoes. We also found that sfRNA facilitates ZIKV replication in mammalian cells, mice, and human brain organoids. Moreover, ZIKV mutants deficient in sfRNA production were unable to form plaques, cause the death of human brain organoids, or establish infection in the mouse foetal brain. We then found that the proviral activity of sfRNA in mammalian cells relies on its ability to suppress type I interferon signalling. We showed that this is achieved via the inhibition of phosphorylation and the nuclear translocation of STAT1. In addition, we found that the production of sfRNA in the ZIKV infection of human brain organoids is associated with the suppression of multiple genes involved in brain development, indicating that sfRNA can be involved in the disruption of brain development associated with ZIKV infection.

This study investigated proteomic changes occurring in Anopheles gambiae and Anopheles stephensi during adult mosquito aging. These changes were evaluated using two-dimensional difference gel electrophoresis (2D-DIGE) and the identities of aging related proteins were determined using capillary high-pressure liquid chromatography (capHPLC) coupled with a linear ion-trap (LTQ)-Orbitrap XL hybrid mass spectrometry (MS). Here, we have described the techniques used to determine age associated proteomic changes occurring in heads and thoraces across three age groups; 1, 9 and 17 d old A. gambiae and 4 age groups; 1, 9, 17 and 34 d old A. stephensi. We have provided normalised spot volume raw data for all protein spots that were visible on 2D-DIGE images for both species and processed Orbitrap mass spectrometry data. For public access, mass spectrometry raw data are available via ProteomeXchange with identifier PXD002153. A detailed description of this study has been described elsewhere [1].

This article develops the notion that a government has a public responsibility to prevent electoral fraud in a way that extends beyond the protections conferred by an electorate’s directly correlative right to voting freedom. Focusing on electoral freedom and voter fraud in electoral systems, it presents theoretical arguments for holding governments responsible arising from the incomplete or unclear nature of juristic rights, powers, and duties. It holds that such public responsibilities are functionally necessary, in the interests of a truly inclusive participatory democracy. The article uses illustrations of fair elections globally, and in the United States in particular, including the divided 2014 US Supreme Court decision, US v. Texas, in which the majority denied the right to vote to prisoners and parolees who are disproportionately represented by ethnic minorities.

ABSTRACT Zika virus (ZIKV) has recently emerged and is the etiological agent of congenital Zika syndrome (CZS), a spectrum of congenital abnormalities arising from neural tissue infections in utero. Herein, we describe the de novo generation of a new ZIKV isolate, ZIKVNatal, using a modified circular polymerase extension reaction protocol and sequence data obtained from a ZIKV-infected fetus with microcephaly. ZIKVNatal thus has no laboratory passage history and is unequivocally associated with CZS. ZIKVNatal could be used to establish a fetal brain infection model in IFNAR−/− mice (including intrauterine growth restriction) without causing symptomatic infections in dams. ZIKVNatal was also able to be transmitted by Aedes aegypti mosquitoes. ZIKVNatal thus retains key aspects of circulating pathogenic ZIKVs and illustrates a novel methodology for obtaining an authentic functional viral isolate by using data from deep sequencing of infected tissues. IMPORTANCE The major complications of an ongoing Zika virus outbreak in the Americas and Asia are congenital defects caused by the virus’s ability to cross the placenta and infect the fetal brain. The ability to generate molecular tools to analyze viral isolates from the current outbreak is essential for furthering our understanding of how these viruses cause congenital defects. The majority of existing viral isolates and infectious cDNA clones generated from them have undergone various numbers of passages in cell culture and/or suckling mice, which is likely to result in the accumulation of adaptive mutations that may affect viral properties. The approach described herein allows rapid generation of new, fully functional Zika virus isolates directly from deep sequencing data from virus-infected tissues without the need for prior virus passaging and for the generation and propagation of full-length cDNA clones. The approach should be applicable to other medically important flaviviruses and perhaps other positive-strand RNA viruses.

Chorioamnionitis can induce pulmonary inflammation and promote bronchopulmonary dysplasia development, distinguished by alveolar simplification and impaired vascular growth. Chorioamnionitis is more common during the extremely preterm canalicular lung stage (crucial for vascular development); and increases the risk for subsequent sepsis. We hypothesized that single/combined exposure to chronic and/or acute inflammation induces pulmonary inflammatory responses and vascular changes.Ovine fetuses were intra-amniotically exposed to chronic Ureaplasma parvum (UP) at 24 days (d) before extreme preterm delivery at 94d (term 147d) and/or to lipopolysaccharide (LPS) 7 or 2d before delivery. Pulmonary inflammation, vascular remodeling and angiogenic factors were assessed.LPS exposure increased CD3-positive and myeloperoxidase-positive cells. Combined UP-LPS exposure increased pulmonary inflammation compared with 2d LPS or UP groups. The UP+2d LPS group had an increased adventitial fibrosis score when compared with UP-treated animals. A reduced wall-to-lumen ratio was found in the 7d LPS animals when compared to the 2d LPS-treated animals. Exposure to UP+2d LPS reduced VEGF and VEGFR-2 levels compared with 2d LPS-treated animals. Angiopoietin-1 (Ang1) and tunica interna endothelial cell kinase 2 (Tie-2) levels were decreased after UP+7d LPS as well as after 7d LPS, but not with UP alone.Chronic UP and subsequent LPS exposure increased pulmonary inflammation and decreased expression of angiogenic growth factors and receptors when compared to single hit-exposed animals.

Estimating the age distribution of mosquito populations is crucial for assessing their capacity to transmit disease and for evaluating the efficacy of available vector control programs. This study reports on the capacity of the near-infrared spectroscopy (NIRS) technique to rapidly predict the ages of the principal dengue and Zika vector, Aedes aegypti. The age of wild-type males and females, and males and females infected with wMel and wMelPop strains of Wolbachia pipientis were characterized using this method. Calibrations were developed using spectra collected from their heads and thoraces using partial least squares (PLS) regression. A highly significant correlation was found between the true and predicted ages of mosquitoes. The coefficients of determination for wild-type females and males across all age groups were R2 = 0.84 and 0.78, respectively. The coefficients of determination for the age of wMel and wMelPop infected females were 0.71 and 0.80, respectively (P< 0.001 in both instances). The age of wild-type female Ae. aegypti could be identified as < or ≥ 8 days old with an accuracy of 91% (N = 501), whereas female Ae. aegypti infected with wMel and wMelPop were differentiated into the two age groups with an accuracy of 83% (N = 284) and 78% (N = 229), respectively. Our results also indicate NIRS can distinguish between young and old male wild-type, wMel and wMelPop infected Ae. aegypti with accuracies of 87% (N = 253), 83% (N = 277) and 78% (N = 234), respectively. We have demonstrated the potential of NIRS as a predictor of the age of female and male wild-type and Wolbachia infected Ae. aegypti mosquitoes under laboratory conditions. After field validation, the tool has the potential to offer a cheap and rapid alternative for surveillance of dengue and Zika vector control programs.

The wMel strain of Wolbachia bacteria is known to prevent dengue and Zika virus transmission in the mosquito vector Aedes aegypti. Accordingly, the release of wMel-infected A. aegypti in endemic regions has been recommended by the World Health Organization as a potential strategy for controlling dengue and Zika outbreaks. However, the utility of this approach could be limited if high temperatures in the aquatic habitats where A. aegypti develop are detrimental to Wolbachia. We exposed wMel-infected A. aegypti eggs and larvae to fluctuating daily temperatures of 30-40°C for three, five, or seven days during their development. We found that Wolbachia levels in females emerging from heat treatments were significantly lower than in the controls that had developed at 20-30°C. Notably, seven days of high temperatures starting at the egg stage reduced Wolbachia levels in emerging females to less than 0.1% of the wMel control levels. However, after adult females returned to 20-30°C for 4-7 days, they experienced differing degrees of Wolbachia recovery. Our findings suggest that the spread of Wolbachia in wild A. aegypti populations and any consequent protection from dengue and Zika viruses might be limited in ecosystems that experience periods of extreme heat, but Wolbachia levels recover partially after temperatures return to normal.

The release of Wolbachia infected mosquitoes is likely to form a key component of disease control strategies in the near future. We investigated the potential of using near-infrared spectroscopy (NIRS) to simultaneously detect and identify two strains of Wolbachia pipientis (wMelPop and wMel) in male and female laboratory-reared Aedes aegypti mosquitoes. Our aim is to find faster, cheaper alternatives for monitoring those releases than the molecular diagnostic techniques that are currently in use. Our findings indicate that NIRS can differentiate females and males infected with wMelPop from uninfected wild type samples with an accuracy of 96% (N = 299) and 87.5% (N = 377), respectively. Similarly, females and males infected with wMel were differentiated from uninfected wild type samples with accuracies of 92% (N = 352) and 89% (N = 444). NIRS could differentiate wMelPop and wMel transinfected females with an accuracy of 96.6% (N = 442) and males with an accuracy of 84.5% (N = 443). This non-destructive technique is faster than the standard polymerase chain reaction diagnostic techniques. After the purchase of a NIRS spectrometer, the technique requires little sample processing and does not consume any reagents.

The survival characteristics of the mosquito Aedes aegypti affect transmission rates of dengue because transmission requires infected mosquitoes to survive long enough for the virus to infect the salivary glands. Mosquito survival is assumed to be high in tropical, dengue endemic, countries like Vietnam. However, the survival rates of wild populations of mosquitoes are seldom measured due the difficulty of predicting mosquito age. Hon Mieu Island in central Vietnam is the site of a pilot release of Ae. aegypti infected with a strain of Wolbachia pipientis bacteria (wMelPop) that induces virus interference and mosquito life-shortening. We used the most accurate mosquito age grading approach, transcriptional profiling, to establish the survival patterns of the mosquito population from the population age structure. Furthermore, estimations were validated on mosquitoes released into a large semi-field environment consisting of an enclosed house, garden and yard to incorporate natural environmental variability. Mosquito survival was highest during the dry/cool (January-April) and dry/hot (May-August) seasons, when 92 and 64% of Hon Mieu mosquitoes had survived to an age that they were able to transmit dengue (12 d), respectively. This was reduced to 29% during the wet/cool season from September to December. The presence of Ae. aegypti older than 12 d during each season is likely to facilitate the observed continuity of dengue transmission in the region. We provide season specific Ae. aegypti survival models for improved dengue epidemiology and evaluation of mosquito control strategies that aim to reduce mosquito survival to break the dengue transmission cycle.

We report on the accuracy of using near-infrared spectroscopy (NIRS) to predict the age of Anopheles mosquitoes reared from wild larvae and a mixed age-wild adult population collected from pit traps after exposure to pyrethroids. The mosquitoes reared from wild larvae were estimated as

The Confession of Belhar: A spirituality of reception in the local church. The GeneralSynod of the Dutch Reformed Church took the following decision during October 2011: �TheGeneral Synod decides to make the Confession of Belhar part of its confessional foundation,that is, in terms of its church ordinances, and commission the Moderamen to prepare thenecessary processes regarding ecclesiastical law.� This article deals with the perception of,the reception of and resistance against Belhar as confession in a local congregation, ElardusPark. The research also describes how this obstructs the development of missional focus. Themain contribution of this article is to argue that the ecumenical concept�s full reception shouldbe assessed within the broader framework of building up a missional local church where aspirituality of reception is fully developed in terms of a missional positioning in Africa.

Biomarkers of the age of mosquitoes are required to determine the risk of transmission of various pathogens as each pathogen undergoes a period of extrinsic incubation in the mosquito host. Using the 2-D Difference Gel Electrophoresis (2-D DIGE) procedure, we investigated the abundance of up to 898 proteins from the Yellow Fever and dengue virus vector, Aedes aegypti, during ageing. By applying a mixed-effects model of protein expression, we identified five common patterns of abundance change during ageing and demonstrated an age-related decrease in variance for four of these. This supported a search for specific proteins with abundance changes that remain tightly associated with ageing for use as ageing biomarkers. Using MALDI-TOF/TOF mass spectrometry we identified ten candidate proteins that satisfied strict biomarker discovery criteria (identified in two out of three multivariate analysis procedures and in two cohorts of mosquitoes). We validated the abundances of the four most suitable candidates (Actin depolymerising factor; ADF, Eukaryotic initiation factor 5A; eIF5A, insect cuticle protein Q17LN8, and Anterior fat body protein; AFP) using semi-quantitative Western analysis of individual mosquitoes of six ages. The redox-response protein Manganese superoxide dismutase (SOD2) and electron shuttling protein Electron transfer oxidoreductase (ETO) were subject to post-translational modifications affecting their charge states with potential effects on function. For the four candidates we show remarkably consistent decreases in abundance during ageing, validating initial selections. In particular, the abundance of AFP is an ideal biomarker candidate for whether a female mosquito has lived long enough to be capable of dengue virus transmission. We have demonstrated proteins to be a suitable class of ageing biomarkers in mosquitoes and have identified candidates for epidemiological studies of dengue and the evaluation of new disease reduction projects targeting mosquito longevity.

Autophagy plays a critical role in cell metabolism by degrading and recycling internal components when challenged with limited nutrients. This fundamental and conserved mechanism is based on a membrane trafficking pathway in which nascent autophagosomes engulf cytoplasmic cargo to form vesicles that transport their content to the lysosome for degradation. Based on this simple scheme, autophagy modulates cellular metabolism and cytoplasmic quality control to influence an unexpectedly wide range of normal mammalian physiology and pathophysiology. In this review, we summarise recent advancements in three broad areas of autophagy regulation. We discuss current models on how autophagosomes are initiated from endogenous membranes. We detail how the uncoordinated 51-like kinase (ULK) complex becomes activated downstream of mechanistic target of rapamycin complex 1 (MTORC1). Finally, we summarise the upstream signalling mechanisms that can sense amino acid availability leading to activation of MTORC1.