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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 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 w AlbB 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 w AlbB-transinfected Ae. aegypti mosquito line and a tetracycline cleared derivative, using deep small RNA-sequencing. We found limited modulation of miRNAs in response to w AlbB infection. Several miRNAs were modulated in response to age, some of which showed greater upregulation in w AlbB-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.

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.

Flaviviruses, including Zika virus (ZIKV), utilise host mRNA degradation machinery to produce subgenomic flaviviral RNA (sfRNA). In mammalian hosts, this noncoding RNA facilitates replication and pathogenesis of flaviviruses by inhibiting IFN-signalling, whereas the function of sfRNA in mosquitoes remains largely elusive. Herein, we conduct a series of in vitro and in vivo experiments to define the role of ZIKV sfRNA in infected Aedes aegypti employing viruses deficient in production of sfRNA. We show that sfRNA-deficient viruses have reduced ability to disseminate and reach saliva, thus implicating the role for sfRNA in productive infection and transmission. We also demonstrate that production of sfRNA alters the expression of mosquito genes related to cell death pathways, and prevents apoptosis in mosquito tissues. Inhibition of apoptosis restored replication and transmission of sfRNA-deficient mutants. Hence, we propose anti-apoptotic activity of sfRNA as the mechanism defining its role in ZIKV transmission. 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.

Dengue cases in Bangladesh have surged in recent years. The existing insecticide-based control program is challenged by issues of insufficient household coverage and high levels of insecticide resistance in the primary dengue virus (DENV) vector, Aedes aegypti. A more sustainable, effective alternative could be the implementation of a Wolbachia -mediated disease management strategy. Hence, we created and characterised a Wolbachia -infected Ae. aegypti strain with a Dhaka wild-type genetic background, and compared its reproductive compatibility, maternal inheritance, fitness, and virus-blocking ability to the parental strains (Dhaka wild-type and w AlbB2-F4). The new Ae. aegypti strain w AlbB2-Dhaka demonstrated complete cytoplasmic incompatibility with the wild-type and complete maternal transmission, retaining levels of pyrethroid resistance of the Dhaka wild-type. No significant fitness costs were detected during laboratory comparison. Compared to the wild-type, w AlbB2-Dhaka mosquitoes demonstrated a significantly reduced genome copies of DENV in the bodies (44.4%, p  = 0.0034); a two-fold reduction in dissemination to legs and wings (47.6%, p  < 0.0001); and > 13-fold reduction of DENV in saliva expectorates (proxy of transmission potential) (92.7%, p  < 0.0001) 14 days after ingesting dengue-infected blood. Our work indicates that the w AlbB2-Dhaka strain could be used for Ae. aegypti suppression or replacement strategies for dengue management in Bangladesh.

Background Chikungunya virus (CHIKV) is a mosquito-transmitted, arthritogenic alphavirus that causes sporadic outbreaks of often debilitating rheumatic disease. The recently approved CHIKV vaccine, IXCHIQ, is based on a live-attenuated CHIKV strain (VLA1553), with viraemic vaccine recipients theoretically able to transmit VLA1553 to mosquitoes with ensuing onward transmission. We thus evaluated VLA1553 transmission from artificial blood meals to Aedes albopictus mosquitoes, and onward transmission to mice. Methods Female A. albopictus mosquitoes were fed on defibrinated sheep blood containing wild-type CHIKV (viral titre: 7.50 log 10 CCID 50 /mL) or VLA1553 (viral titres: 7.85, 5.72, 4.58, and 3.79 log 10 CCID 50 /mL). Viral titres in mosquito bodies and saliva were determined using CCID 50 assays 7–8 days after the blood meal. After providing CHIKV or VLA1553 (viral titres ~ 7–8 log 10 CCID 50 /mL) in blood meals to mosquitoes, infected mosquitoes were fed on highly susceptible Irf3/7 −/− mice ( n  = 3 per group). Data were re-analysed using the same reverse transcription quantitative polymerase chain reaction (RT-qPCR) as for an earlier VLA1553 phase 1 clinical trial, to allow correlations between blood meal titres and viraemia in vaccine recipients. Results Mosquito body viral titres were significantly higher ( P  < 0.0001) for CHIKV versus VLA1553-fed mosquitoes at blood meal viral titres of ~ 7–8 log 10 CCID 50 /mL. Mosquito body VLA1553 titres decreased with reducing blood meal titres, but there was no dose-dependent effect on saliva viral titres. No dissemination to salivary glands was seen at blood meal titres ≤ 3.875 log 10 CCID 50 /mL. CHIKV-fed mosquitoes were able to transmit virus, and induce viraemia in, 3/3 Irf3/7 −/− mice via mosquito bites. In contrast, 0/3 Irf3/7 −/− mice became infected after bites from VLA1553-fed mosquitoes. RT-qPCR comparisons with phase 1 clinical data for VLA1553-vaccinated individuals indicated that VLA1553 viraemia was at or below the aforementioned threshold for transmission. Conclusions The evidence presented herein argue that the low viraemia in VLA1553-vaccinated individuals would mitigate against transmission. In addition, replication of VLA1553 in mosquito bodies was also significantly attenuated. Overall, mosquito-borne transmission of VLA1553 from vaccinated individuals to others appears improbable. Graphical Abstract

Many 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. A 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. There was significant agreement of the plaque neutralisation resulting from the micro-PRNT and standard PRNT techniques (R.sup.2 = 0.65; P<0.0001) when applied to RRV antibody detection in koala sera. Sensitivity and specificity of the micro-PRNT assay were 88.2% and 96%, respectively, in comparison with the standard PRNT. Blood meals from mosquitoes fed on sheep blood supplemented with RRV antibodies, and on blood from RRV seropositive humans neutralised the virus by [greater than or equal to]50% until 48 hr post feeding. The vertebrate origin of the blood meal was also ascertained for the same samples, in parallel, using established molecular techniques. The 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.

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. 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. 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.

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.

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 w AlbB (referred to as the w AlbB2-F4 strain) through simple backcrossing of wild type Australian mosquitoes with a w AlbB infected Ae . aegypti strain from the USA. Field releases of male w AlbB2-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 w AlbB2-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 w AlbB2-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 w AlbB2-F4 mosquitoes have > 98% Australian ancestry, confirming the successful introduction of the w AlbB2 infection into the Australian genomic background through backcrossing. Genotypic and phenotypic analyses showed the w AlbB2-F4 strain retains the insecticide susceptible phenotype and genotype of native Australian mosquitoes. We demonstrate that the Wolbachia w AlbB2-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 w AlbB2, 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.