First Isolation of Japanese Encephalitis Virus Genotype IV from Mosquitoes in Australia.

Introduction: Widespread transmission of Japanese encephalitis virus (JEV) genotype four (GIV) occurred across mainland Australia in 2022. This resulted in forty-five human cases, including seven deaths, and the identification of JEV infection in over 80 commercial piggeries. Materials and Methods: We collected mosquitoes which were trapped using CO2-baited light traps deployed near piggeries reporting disease or in regions linked to human cases in the Wide Bay region in the state of Queensland. Mosquitoes from four traps yielded JEV RNA by real-time RT-PCR. Pools containing RNA positive mosquitoes were inoculated onto mosquito cell monolayers. Discussion: A single isolate of JEV was obtained from a pool of mixed mosquito species. Near whole genome sequencing and phylogenetic analysis of the JEV isolate demonstrated its high genomic relatedness with JEV GIV pig sequences sampled from Queensland and the state of New South Wales in 2022. Conclusion: We report the first isolation of JEV GIV from mosquitoes collected in Australia. With only a few JEV GIV isolates available globally, the isolate we report will be essential for future research of JEV host interactions, evolution and disease markers, and development of effective therapies, vaccines, diagnostic assays, and mosquito control strategies.

Uncovering the genetic diversity within the Aedes notoscriptus virome and isolation of new viruses from this highly urbanised and invasive mosquito.

The Australian backyard mosquito, Aedes notoscriptus, is a highly urbanised pest species that has invaded New Zealand and the USA. Importantly, Ae. notoscriptus has been implicated as a vector of Ross River virus, a common and arthritogenic arbovirus in Australia, and is a laboratory vector of numerous other pathogenic viruses, including West Nile, yellow fever, and Zika viruses. To further explore live viruses harboured by field populations of Ae. notoscriptus and, more specifically, assess the genetic diversity of its virome, we processed 495 pools, comprising a total of 6,674 female Ae. notoscriptus collected across fifteen suburbs in Brisbane, Australia, between January 2018 and May 2019. Nine virus isolates were recovered and characterised by metagenomic sequencing and phylogenetics. The principal viral family represented was Flaviviridae. Known viruses belonging to the genera Flavivirus, Orbivirus, Mesonivirus, and Nelorpivirus were identified together with two novel virus species, including a divergent Thogoto-like orthomyxovirus and an insect-specific flavivirus. Among these, we recovered three Stratford virus (STRV) isolates and an isolate of Wongorr virus (WGRV), which for these viral species is unprecedented for the geographical area of Brisbane. Thus, the documented geographical distribution of STRV and WGRV, both known for their respective medical and veterinary importance, has now been expanded to include this major urban centre. Phylogenies of the remaining five viruses, namely, Casuarina, Ngewotan, the novel Thogoto-like virus, and two new flavivirus species, suggested they are insect-specific viruses. None of these viruses have been previously associated with Ae. notoscriptus or been reported in Brisbane. These findings exemplify the rich genetic diversity and viral abundance within the Ae. notoscriptus virome and further highlight this species as a vector of concern with the potential to transmit viruses impacting human or animal health. Considering it is a common pest and vector in residential areas and is expanding its global distribution, ongoing surveillance, and ecological study of Ae. notoscriptus, together with mapping of its virome and phenotypic characterisation of isolated viruses, is clearly warranted. Immanently, these initiatives are essential for future understanding of both the mosquito virome and the evolution of individual viral species.

Simulating an invasion: unsealed water storage (rainwater tanks) and urban block design facilitate the spread of the dengue fever mosquito, Aedes aegypti, in Brisbane, Australia.

Aedes aegypti (Linnaeus) was once highly prevalent across eastern Australia, resulting in epidemics of dengue fever. Drought conditions have led to a rapid rise in semi-permanent, urban water storage containers called rainwater tanks known to be critical larval habitat for the species. The presence of these larval habitats has increased the risk of establishment of highly urbanised, invasive mosquito vectors such as Ae. aegypti. Here we use a spatially explicit network model to examine the role that unsealed rainwater tanks may play in population connectivity of an Ae. aegypti invasion in suburbs of Brisbane, a major Australian city. We characterise movement between rainwater tanks as a diffusion-like process, limited by a maximum distance of movement, average life expectancy, and a probability that Ae. aegypti will cross wide open spaces such as roads. The simulation model was run against a number of scenarios that examined population spread through the rainwater tank network based on non-compliance rates of tanks (unsealed or sealed) and road grids. We show that Ae. aegypti tank infestation and population spread was greatest in areas of high tank density and road lengths were shortest e.g. cul-de-sacs. Rainwater tank non-compliance rates of over 30% show increased connectivity when compared to less than 10%, suggesting rainwater tanks non-compliance should be maintained under this level to minimize the spread of an invading Ae. aegypti population. These results presented as risk maps of Ae. aegypti spread across Brisbane, can assist health and government authorities on where to optimally target rainwater tank surveillance and educational activities. Supplementary Information: The online version contains supplementary material available at 10.1007/s10530-021-02619-z.

Impact of COVID-19 Mitigation Measures on Mosquito-Borne Diseases in 2020 in Queensland, Australia.

We describe the impact of COVID-19 mitigation measures on mosquito-borne diseases in Queensland, Australia, during the first half of 2020. Implementation of restrictions coincided with an atypical late season outbreak of Ross River virus (RRV) characterized by a peak in notifications in April (1173) and May (955) which were greater than 3-fold the mean observed for the previous four years. We propose that limitations on human movement likely resulted in the majority of RRV infections being acquired at or near the place of residence, and that an increase in outdoor activities, such as gardening and bushwalking in the local household vicinity, increased risk of exposure to RRV-infected mosquitoes. In contrast, the precipitous decline in international passenger flights led to a reduction in the number of imported dengue and malaria cases of over 70% and 60%, respectively, compared with the previous five years. This substantial reduction in flights also reduced a risk pathway for importation of exotic mosquitoes, but the risk posed by importation via sea cargo was not affected. Overall, the emergence of COVID-19 has had a varied impact on mosquito-borne disease epidemiology in Queensland, but the need for mosquito surveillance and control, together with encouragement of personal protective measures, remains unchanged.

Urban Landscape Features Influence the Movement and Distribution of the Australian Container-Inhabiting Mosquito Vectors Aedes aegypti (Diptera: Culicidae) and Aedes notoscriptus (Diptera: Culicidae).

Urban landscape features play an important role in the distribution and population spread of mosquito vectors. Furthermore, current insecticide and novel rear-and-release strategies for urban mosquito management rarely consider the spatial structure of the landscape when applying control practices. Here, we undertake a mark-recapture experiment to examine how urban features influence the movement and distribution of Australian container-inhabiting Aedes vectors. We pay attention to the role of semipermanent water storage containers, called rainwater tanks, and the influence of movement barriers, such as roads, on the spread and distribution of vector populations. Results suggest that Aedes aegypti (Linnaeus) (Diptera: Culicidae) were more likely to be captured around rainwater tanks, and that released males travel throughout residential blocks but do not cross roads. Conversely, female Aedes notoscriptus (Skuse) (Diptera: Culicidae) movement was uninhibited by roads and rainwater tanks did not influence female distribution or oviposition behavior. Using an isotropic Gaussian kernel framework, we show that vector movement is likely to be greater when applying a temporal effect, than when estimated by traditional methods. We conclude that a greater understanding on the role of urban features on vector movement will be important in the new age of rear-and-release mosquito control strategies, particularly those where estimations of movement are important for ensuring efficacy of application.

Epidemiologic, Entomologic, and Virologic Factors of the 2014-15 Ross River Virus Outbreak, Queensland, Australia.

Australia experienced its largest recorded outbreak of Ross River virus (RRV) during the 2014-15 reporting year, comprising >10,000 reported cases. We investigated epidemiologic, entomologic, and virologic factors that potentially contributed to the scale of the outbreak in Queensland, the state with the highest number of notifications (6,371). Spatial analysis of human cases showed that notifications were geographically widespread. In Brisbane, human case notifications and virus detections in mosquitoes occurred across inland and coastal locations. Viral sequence data demonstrated 2 RRV lineages (northeastern genotypes I and II) were circulating, and a new strain containing 3 unique amino acid changes in the envelope 2 protein was identified. Longitudinal mosquito collections demonstrated unusually high relative abundance of Culex annulirostris and Aedes procax mosquitoes, attributable to extensive freshwater larval habitats caused by early and persistent rainfall during the reporting year. Increased prevalence of these mosquitoes probably contributed to the scale of this outbreak.

Life on the margin: Rainwater tanks facilitate overwintering of the dengue vector, Aedes aegypti, in a sub-tropical climate.

A key determinant of insect persistence in marginal habitats is the ability to tolerate environmental extremes such as temperature. Aedes aegypti is highly invasive and little is known about the physiological sensitivity of the species to fluctuating temperature regimes at the lower critical threshold for development. A temperature that may limit the establishment and persistence of the species in sub-optimal regions. Daily winter temperatures were measured in common Australian larval habitats, replicated in environmental chambers and used to investigate the effect of fluctuating temperatures on the development and survival of tropical and subtropical strains of Australian Ae. aegypti. Development was slow for all treatments but both strains were able to complete development to the adult stage, suggesting previous models underestimate the potential for the species to persist in eastern Australia. Results suggested that thermal buffering in large volume habitats, and water that persists for greater than 32 days, will facilitate completion of the life cycle during sub-tropical winters. Furthermore, we provide a non-linear estimate of the lower critical temperature for Ae. aegypti development that suggests the current threshold may be incorrect. Our study demonstrates that the current re-introduction of water storage containers such as rainwater tanks, into major Australian population centres will increase the risk of Ae. aegypti establishment by permitting year-round development in locations south of its current distribution.

The new European invader Aedes (Finlaya) koreicus: a potential vector of chikungunya virus.

Arthropod-borne disease outbreaks, facilitated by the introduction of exotic mosquitoes, pose a significant public health threat. Recent chikungunya virus (CHIKV) epidemics in Europe highlight the importance of understanding the vector potential of invading mosquitoes. In this paper we explore the potential of Aedes koreicus, a mosquito new to Europe, to transmit CHIKV. Mosquitoes were challenged with CHIKV and maintained at two temperatures: 23 °C and a fluctuating temperature. Total CHIKV infection rates at 3, 10 and 14 days post-feeding were low for both temperature treatments (13.8% at 23 °C; 6.2% at fluctuating T). A low percentage (6.1%, n = 65) of mosquitoes maintained at a constant 23 °C showed dissemination of the virus to the wings and legs. Infection of mosquito saliva, with live virus, occurred in 2 mosquitoes. No dissemination was noted under the fluctuating temperature regime. Based on these results we conclude that CHIKV transmission by this species is possible.

The elimination of the dengue vector, Aedes aegypti, from Brisbane, Australia: The role of surveillance, larval habitat removal and policy.

Aedes aegypti (L.) (Diptera: Culicidae) is a highly invasive mosquito whose global distribution has fluctuated dramatically over the last 100 years. In Australia the distribution of Ae. aegypti once spanned the eastern seaboard, for 3,000 km north to south. However, during the 1900s this distribution markedly reduced and the mosquito disappeared from its southern range. Numerous hypotheses have been proffered for this retraction, however quantitative evidence of the mechanisms driving the disappearance are lacking. We examine historical records during the period when Ae. aegypti disappeared from Brisbane, the largest population centre in Queensland, Australia. In particular, we focus on the targeted management of Ae. aegypti by government authorities, that led to local elimination, something rarely observed in large cities. Numerous factors are likely to be responsible including the removal of larval habitat, especially domestic rainwater tanks, in combination with increased mosquito surveillance and regulatory enforcement. This account of historical events as they pertain to the elimination of Ae. aegypti from Brisbane, will inform assessments of the risks posed by recent human responses to climate change and the reintroduction of 300,000 rainwater tanks into the State over the past decade.

Laboratory colonization of the European invasive mosquito Aedes (Finlaya) koreicus.

BACKGROUND: Aedes (Finlaya) koreicus (Edwards) is a mosquito that has recently entered Europe from Asia. This species is considered a potential threat to newly colonized territories, but little is known about its capacity to transmit pathogens or ability to compete with native mosquito species. The establishment of a laboratory colony is a necessary first step for further laboratory studies on the biology, ecology and vector competence of Ae. koreicus. RESULTS: A self-mating colony was established at QIMR Berghofer Medical Research Institute (Brisbane, Australia) from eggs of the F1 progeny of individuals collected as free-living larvae in northeastern Italy (Belluno province). Mosquitoes are currently maintained on both defibrinated sheep blood provided via an artificial membrane system and human blood from volunteers. Larvae are maintained in rain water and fed with Tetramin® fish food (©2015 Spectrum Brands - Pet, Home and Garden Division, Tetra-Fish). Morphometric measurements related to body size were taken and a fecundity index, based on wing length, was calculated. An in vivo technique for differentiating male and female pupae has been optimized. Our findings provide the basis for further studies on the ecology and physiology of Ae. koreicus. CONCLUSION: We describe the establishment of an Ae. koreicus colony in the laboratory and identify critical requirements for the maintenance of this mosquito species under artificial conditions. The laboratory colony will facilitate studies investigating the vector potential of this species for human pathogens.

Reducing biting rates of Aedes aegypti with metofluthrin: investigations in time and space.

BACKGROUND: Indoor residual spraying is key to dengue control in Cairns and other parts of northern Queensland, Australia, where Aedes aegypti is prevalent, but the strategy faces challenges with regards to slow application time and, therefore, community coverage. A faster potential improvement might be the use of polyethylene netting impregnated with the volatile pyrethroid metofluthrin (SumiOne™). This formulation was assessed in rooms in three houses in Cairns, Australia. One emanator was placed in each room and cages of 10 female Aedes aegypti were exposed at distances of 1 and 3 m. Knockdown and landings on a human hand were counted before metofluthrin exposure and at 10, 30, 60, 90 and 120 min during exposure. In addition, two trials continued over 48 h of exposure to assess the long-term sublethal effects of metofluthrin on caged mosquitoes. RESULTS: Percentage landing rates fell to 0-2.5% in the first 10 min of exposure. Knockdown was most evident between 10 and 30 min (54% at 1 m and 33% at 3 m). Distance from the emanator strongly affected the results: mosquitoes at 3 m exhibited less knockdown and more landings than those at 1 m. As room volume increased, knockdown decreased and the number of landing increased. There is a cumulative mortality and landing inhibition and, for mosquitoes exposed to metofluthrin for > 48 h, mortality was 100% at 1 m and 90% at 3 m. Of those still alive, a small number continued to land and bite. After being removed from metofluthrin-treated rooms, exposed insect cages were found to reducing landing rates for up to 2 h. CONCLUSIONS: Despite only moderate levels of knockdown during the initial hours of exposure, metofluthrin emanators were effective in reducing mosquito landing rates, especially within 1 m, even when exposed on an open veranda. The evaluation methods and results described in this paper will help inform the optimal conditions of deployment of metofluthrin emanators. These devices have the potential to reduce contact between humans and urban disease vectors faster than indoor residual spraying so supplement our current arsenal of dengue control tools.

Field evaluation of the establishment potential of wMelPop Wolbachia in Australia and Vietnam for dengue control.

BACKGROUND: Introduced Wolbachia bacteria can influence the susceptibility of Aedes aegypti mosquitoes to arboviral infections as well as having detrimental effects on host fitness. Previous field trials demonstrated that the wMel strain of Wolbachia effectively and durably invades Ae. aegypti populations. Here we report on trials of a second strain, wMelPop-PGYP Wolbachia, in field sites in northern Australia (Machans Beach and Babinda) and central Vietnam (Tri Nguyen, Hon Mieu Island), each with contrasting natural Ae. aegypti densities. METHODS: Mosquitoes were released at the adult or pupal stages for different lengths of time at the sites depending on changes in Wolbachia frequency as assessed through PCR assays of material collected through Biogents-Sentinel (BG-S) traps and ovitraps. Adult numbers were also monitored through BG-S traps. Changes in Wolbachia frequency were compared across hamlets or house blocks. RESULTS: Releases of adult wMelPop-Ae. aegypti resulted in the transient invasion of wMelPop in all three field sites. Invasion at the Australian sites was heterogeneous, reflecting a slower rate of invasion in locations where background mosquito numbers were high. In contrast, invasion across Tri Nguyen was relatively uniform. After cessation of releases, the frequency of wMelPop declined in all sites, most rapidly in Babinda and Tri Nguyen. Within Machans Beach the rate of decrease varied among areas, and wMelPop was detected for several months in an area with a relatively low mosquito density. CONCLUSIONS: These findings highlight challenges associated with releasing Wolbachia-Ae. aegypti combinations with low fitness, albeit strong virus interference properties, as a means of sustainable control of dengue virus transmission.

Increased container-breeding mosquito risk owing to drought-induced changes in water harvesting and storage in Brisbane, Australia.

BACKGROUND: Extended drought conditions in south-east Queensland during the early 2000s have resulted in a culture of water harvesting and legislated water restrictions. Aedes notoscriptus is a container-breeding mosquito vector of Ross River and Barmah Forest viruses. METHODS: From 2008-2009, the larval habitats and seasonal abundance of domestic container-breeding mosquitoes were recorded from three suburbs of Brisbane. A knowledge, attitudes and practice questionnaire was administered to householders. A low-cost, desktop methodology was used to predict the proportion of shaded premises compared with front-of-property estimates. RESULTS: We highlight changes in the frequency of container categories for A. notoscriptus as a response to human behavioural changes to drought. Garden accoutrements, discarded household items and water storage containers accounted for 66.2% (525/793) of positive containers and 77.5% (73 441/94 731) of all immature mosquitoes. Of all household premises surveyed, 52.6% (550/1046) contained rainwater tanks and 29.4% (308/1046) harvested water in other containers, contrasting with a previous 1995 survey where neither category was observed. Both Premise Condition Index and shade directly correlated with positive premises. CONCLUSIONS: Human response to drought has resulted in new habitats for domestic container-breeding mosquitoes. This recent trend of prolific water storage is similar to earlier years (1904-1943) in Brisbane when Aedes aegypti was present and dengue epidemics occurred.

Effects of Beauveria bassiana on survival, blood-feeding success, and fecundity of Aedes aegypti in laboratory and semi-field conditions.

The fungus Beauveria bassiana reduces Aedes aegypti longevity in laboratory conditions, but effects on survival, blood-feeding behavior, and fecundity in realistic environmental conditions have not been tested. Adult, female Ae. aegypti infected with B. bassiana (FI-277) were monitored for blood-feeding success and fecundity in the laboratory. Fungal infection reduced mosquito-human contact by 30%. Fecundity was reduced by (mean ± SD) 29.3 ± 8.6 eggs per female per lifetime in the laboratory; egg batch size and viability were unaffected. Mosquito survival, blood-feeding behavior, and fecundity were also tested in 5 meter × 7 meter × 4 meter semi-field cages in northern Queensland, Australia. Fungal infection reduced mosquito survival in semi-field conditions by 59-95% in large cages compared with 61-69% in small cages. One semi-field cage trial demonstrated 80% reduction in blood-feeding; a second trial showed no significant effect. Infection did not affect fecundity in large cages. Beauveria bassiana can kill and may reduce biting of Ae. aegypti in semi-field conditions and in the laboratory. These results further support the use of B. bassiana as a potential biocontrol agent against Ae. aegypti.

Spore persistence and likelihood of aeroallergenicity of entomopathogenic fungi used for mosquito control.

Entomopathogenic fungi, such as Metarhizium anisopliae and Beauveria bassiana, are being researched as alternatives to chemical adulticides to control mosquito vectors of malaria and dengue. Two cited concerns of fungal control include conidial viability and risks fungal entomopathogens pose to human health. We measured spore viability of 10 fungal isolates over 26 weeks and found a range of persistence. Three B. bassiana isolates maintained > or = 50% viability 14 weeks after application. No M. anisopliae isolate persisted longer than 1 week. To help assess risk of conidia as potential human allergens, we measured airborne conidia in enclosed environments after simulated biopesticide treatment of M. anisopliae. Conidia were detectable immediately after treatment, with concentrations of ~7000/m(3), decreasing over 48 hours to 500 conidia/m(3). At most, Metarhizium conidia comprised 2% of total visible particulate matter, falling to 0.1% by 2 days. The implications for viability of biological control of adult mosquitoes are discussed.

Mycoplasma gallisepticum infection in house finches (Carpodacus mexicanus) affects mosquito blood feeding patterns.

Disease-induced lethargy can diminish host capacity to repel or kill biting mosquitoes. We exposed house finches (Carpodacus mexicanus) to mosquitoes (Culex pipiens pipiens), repeated the experiment after inoculating finches with Mycoplasma gallisepticum, and then repeated the experiment with the same birds after curing their infections. We videotaped avian behaviors before and during mosquito exposure, identifying hosts through blood meal DNA fingerprinting. Results revealed heterogeneity in mosquito preference regardless of infection. Mosquitoes choosing between two healthy finches were more likely to feed upon the same individual bird consistently. When one bird was sick, mosquitoes exhibited no preference. Sick birds made fewer total defensive behaviors than healthy birds, but only foot stomps were associated with reduced mosquito feeding success. Our results suggest that Mycoplasma and other avian infections that alter bird defensive behavior may influence mosquito feeding patterns and transmission of arthropod-borne pathogens such as West Nile virus.

Bird-baited traps for surveillance of West Nile mosquito vectors: effect of bird species, trap height, and mosquito escape rates.

Host-seeking mosquitoes were sampled in bird-baited traps at four sites in New York state in 2003-2004. Trap placement and efficacy of chickens, Gallus gallus domesticus L., as bait compared with house sparrows, Passer domesticus L., an important reservoir of West Nile virus (family Flaviviridae, genus Flavivirus, WNV), was evaluated. Each site had a chicken-baited trap near ground level (approximately 1.5 m) and in the tree canopy (approximately 9 m), and a house sparrow-baited trap at ground level and canopy level. Each trap allowed mosquito access to birds on one end, and an inner mesh screen blocked bird access on the other end. The two most abundant mosquitoes, Culex restuans Theobald and Culex pipiens pipiens L., were differentiated using molecular characters. In 2003, Cx. restuans and Cx. p. pipiens made up 88% of total mosquito catch. In 2004, Cx. restuans comprised 43% of total catch and Cx. p. pipiens comprised 33%. The remaining species representing at least 1% of total catch were Ochlerotatus trivittatus (Coquillett), Coquilletidia perturbans (Walker), and Culiseta morsitans (Theobald). Capture rates were similar for chicken and house sparrow-baited traps; however, significantly more mosquitoes were captured in the canopy for both bird species. Cx. restuans preferred canopy traps, whereas equal numbers of Cx. p. pipiens were captured at ground and canopy levels. Mosquitoes were more likely to escape (74%) when excluded from birds than when allowed free access to birds (54%). Sentinel bird surveillance for WNV can be improved by trapping in the tree canopy in addition to ground level to capture the most important avian vectors.

Assessing insecticide resistance and aversion to methomyl-treated toxic baits in Musca domestica L (Diptera: Muscidae) populations in southern California.

Progeny of house flies (Musca domestica L) from ten California poultry operations, three dairies and one horse-riding facility were tested for methomyl- and muscalure-treated bait resistance using up to three different assays: a topical assay, a no-choice feeding assay and a choice feeding assay. LD50 resistance factors from the topical assay, compared with a locally-derived susceptible colony, ranged between 1 and 4. LC50 resistance factors from the no-choice feeding assay ranged mostly between 2 and 5, with one value of 18. Measurable LT50 resistance ratios for female flies in the choice feeding assay ranged from 43 to 159; two populations had <10% mortality at 48h and could not be measured. LT50 resistance ratios for male flies in the choice feeding assay ranged from 26 to 96, and one population was too resistant to measure. A behavioral assay tested the feeding preference of male and female flies provided a dish of sugar and a dish of methomyl- and muscalure-treated bait. Of eight strains tested, females from seven strains and males from six strains showed significant preference for sugar over bait. Behavioral factors appear to be important in the severe resistance of house flies to baits in California.