This study investigated potential Ross River virus (RRV) exposure sites in Greater Brisbane during the Queensland coronavirus disease 2019 lockdown (January-July 2020). Using RRV notifications, cluster identification techniques, and mobile phone data for movement network analysis, the study examined 993 RRV cases and 9 million movement trajectories from residential RRV cluster areas (hot spots). The findings revealed that population movement was a key risk factor to RRV incidence within hot spots, whereby highly interconnected areas had more RRV cases during lockdown. While environmental conditions within RRV hot spots were less significant compared with their connectivity, areas with higher vegetation density had fewer RRV cases. The study also noted that individuals from RRV hot spots spent less time in green areas before lockdown than during and after lockdown. The results suggest that population movement significantly influenced the 2020 RRV outbreak. These insights can help adapt current vector control and surveillance protocols to target areas identified in this study.

Japanese encephalitis virus (JEV) is a multi-vector, multi-host pathogen maintained in circulation between Culex mosquitoes and waterbirds, with occasional spillover to humans. In Australia, sporadic local transmission of JEV was historically confined to northern Australia until 2021 after which outbreaks occurred for the first time in temperate southern regions in 2022 and 2023, respectively. Following this outbreak, there have been concerns that JEV has potentially become endemic in Australia, posing an ongoing public health risk exacerbated by shifting climatic and environmental factors. We developed and applied a spatially explicit spillover modelling framework which focuses on estimating i) an ecological suitability index for JEV in Australia and, ii) the spillover potential to human populations if endemic transmission is established. To calculate ecological suitability and the potential number of humans that could be exposed, we integrated the ecological and epidemiological conditions that would allow JEV to circulate in Australia and spillover to human populations. An ecological suitability index was calculated by combining the presence of hosts (Ardeidae birds, domestic piggeries, feral pigs) and vectors (Culex annulirostris and Culex quinquefasciatus), host-vector contact rates, and vector infection and transmission potential for JEV at a 1km resolution. JEV spillover potential was estimated by multiplying the ecological suitability index with human population density. We used this estimate to calculate the total population that could be exposed to JEV at the Local Government Area (LGA) level and State/Territory level. We validated our estimates by calculating a population-weighted mean value for each LGA and compared the values between LGAs with confirmed JEV clinical cases to those without. We found an elevated ecological suitability along the east coast and south-western coast of Australia, inland from the northern centre of the country, and surrounding the Murray River Basin (along the border of New South Wales and Victoria). If JEV becomes established in ecologically suitable areas, high spillover potential to humans would exist along Australia's eastern coast. This exposure potential extends inland to areas like the Murray River Basin, which provide ample habitat for enzootic hosts and vector species. High spillover potential also exists in the Northern Territory, along the southwestern coast of Western Australia, and in South Australia. LGAs with historically confirmed clinical cases in humans had a statistically higher population-weighted mean value compared to those without confirmed cases, supporting the model's capability to differentiate highly suitable areas. By integrating vector-host dynamics and human population density into a spatially explicit framework, we identified areas with high JEV ecological suitability and the potential for spillover into human populations if transmission of JEV were to become established. The results were driven by interactions between vectors, hosts and vector competence. These findings provide insights for targeted surveillance and vector control strategies in Australia. Proactive and sustained interventions are essential to mitigate JEV's growing threat and to protect vulnerable populations in the face of ongoing environmental changes.

An identification guide is provided for female adults of the mosquito groups, complexes or species that can be morphologically differentiated and that are likely to transmit arboviruses (e.g., dengue, Zika, chikungunya, Ross River and Japanese encephalitis) or parasites (e.g., Plasmodium spp. and Wuchereria bancrofti ) in the Pacific Islands, countries and territories. This dichotomous key is adapted, with permissions, from a variety of text and image sources to facilitate the identification of disease vectors by individuals with limited taxonomic training including Pacific island country Vector Surveillance and Environmental Health officers, other public health officials and students.

BackgroundSpatial emanators (SE) are innovative tools for controlling indoor Aedes aegypti due to their relatively easy use and high efficacy. Large-scale implementation challenges include community adoption, particularly ensuring proper installation and timely replacement as SE efficacy wanes.Methodology and principal findingsWe conducted a three-arm, open-label entomological cluster randomized controlled trial with a crossover design, involving 588 households, to assess the entomological effect of the community use of metofluthrin emanators. Arms were: "no treatment"; "community-led deployment" (CD), where the households were responsible for installing and replacing SE with minimal guidance; and "managed deployment" (MD), where the research team handled SE installation and replacement. Emanators were replaced every 3 weeks across four deployment cycles, followed by a crossover between the CD and MD arms. Indoor resting mosquitoes were collected using Prokopack aspirators, and human landing counts (HLCs) were conducted in a subset of 12 houses (4 by arm) at the first, fourth, fifth, and eighth SE replacement rounds. Values of each endpoint during all sampling periods were compared using generalized linear mixed effects models (GLMM), the coefficients of the best-fitting model estimated that SE intervention reduced the number of Ae. aegypti per house by 32.7% (95%CI = 16.2-46.0%) in the CD arm and 36.8% (21.1-49.3%) in the MD arm. HLCs accounted 74-94% efficacy (MD) and 35-79% (CD). The crossover analysis found no significant difference between periods and arms, demonstrating the community's ability to manage SE as effectively as research team, even without prior training.Conclusions/significanceThis trial suggests that safe, portable SE are suited to deployment by householders as a rapid response to local Aedes-borne disease outbreaks even in the presence of high pyrethroid resistance in the local Aedes population. In urban areas where effective coverage and resourcing is a challenge to control campaigns, community "ownership" of SE products may enhance the impact of insecticidal interventions.

Background: Japanese encephalitis virus is a leading cause of viral encephalitis in Asia, with high case-fatality rate and morbidity. Although the live recombinant Japanese encephalitis chimeric vaccine (Imojev (R)) offers strong initial immunity, data on long-term efficacy beyond 5 years remain limited. Methods: We conducted a cross-sectional study on adults vaccinated with Imojev (R) at a specialist travel clinic in Brisbane, Australia. Participants were stratified based on the time since vaccination: 2-5 years and >5 years. Neutralizing antibody titres were measured using the plaque reduction neutralization test (PRNT50), with titres >= 10 indicating seropositivity. Results: Of the 103 participants, 47 were vaccinated 2-5 years prior and 56 were vaccinated >= 5 years prior to enrolment. All participants vaccinated within 5 years remain seropositive, whilst 52 of 56 (92.9%) vaccinated >= 5 years ago were seropositive. Four participants (7.1%) were seronegative post-vaccination, with time since vaccination ranging from 5 to 9 years. These seronegative individuals were vaccinated a median of 9.2 years ago, compared to 5.1 years for seropositive participants (P-value = 0.037). Aside from time since vaccination, no other factors (e.g. age, sex) were associated with seronegativity. Conclusions: Imojev (R) provides durable immunity, with seropositivity exceeding 90% up to 10 years post-vaccination. However, waning immunity in a small proportion of individuals suggests that booster doses may be beneficial for high-risk travellers vaccinated over 5 years ago.

Next-generation sequencing (NGS) has the potential to transform mosquito-borne disease surveillance but remains under-utilised. This study introduces a comprehensive multi-loci metabarcoding-based MX (molecular xenomonitoring) approach to mosquito and arbovirus surveillance, enabling parallel identification of mosquito vectors, circulating arboviruses, and vertebrate hosts from bulk mosquito collections. The feasibility of this approach was demonstrated through its application to a large set (n = 110) of bulk field collections. This set was complemented by a number (n = 28) of single-species mosquito pools that had previously been screened for viruses using quantitative reverse transcription PCR (RT-qPCR) and metatranscriptomics. Universal alphavirus and flavivirus primer sets were used to screen for arboviruses in the resulting metabarcoding library. Viral amplicons were then indexed and combined with mosquito-specific (ITS2), universal invertebrate (COI), and vertebrate (Cyt b) barcode amplicons prior to sequencing. This approach confirmed the presence of all previously identified mosquito species, as well as those commonly misidentified morphologically, and enabled a degree of quantification regarding their relative physical abundance in each collection. Additionally, the developed approach identified a diverse vertebrate host community (18 species), demonstrating its potential for defining host preferences and, in tandem with the viral screens and associated vector data, understanding disease transmission pathways. Importantly, metabarcoding detected a diversity of regionally prevalent arboviruses and insect-specific viruses, with all three viral diagnostics demonstrating a similar sensitivity and specificity in detecting Ross River virus and Barmah Forest virus, Australia's most common arboviruses. In summary, multi-loci metabarcoding is an affordable and efficient MX tool that enables complete mosquito-borne disease surveillance.

Zoonotic diseases whose transmission processes are sensitive to environmental characteristics represent an important public health burden in Australia, particularly in Queensland. This study aimed to analyse the epidemiology of hospitalisations from the four main environmental zoonotic diseases-leptospirosis, melioidosis, Q fever, and Ross River virus-from 2012 to 2019 in Queensland. Our analyses reveal an increasing trend of hospitalisation incidence for melioidosis, stable incidence for Q fever and Ross River virus infection, and a declining trend for leptospirosis. We identified sex and age disparities in hospitalisations, with males being more likely to be hospitalised for leptospirosis, melioidosis, and Q fever compared to females. We also uncovered discrepancies between hospitalisation and notification data, which could be attributed to diagnostic and reporting criteria. The findings of this study show that the epidemiological patterns of hospitalisation are different to the notification for the same diseases and underscore the importance of accurate recording and reporting of zoonoses-related hospitalisations to inform environmental public health interventions.

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 wAlbB2-F4). The new Ae. aegypti strain wAlbB2-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, wAlbB2-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 wAlbB2-Dhaka strain could be used for Ae. aegypti suppression or replacement strategies for dengue management in Bangladesh.

Arbovirus surveillance of wild-caught mosquitoes is an affordable and sensitive means of monitoring virus transmission dynamics at various spatial-temporal scales, and emergence and re-emergence during epidemic and interepidemic periods. A variety of molecular diagnostics for arbovirus screening of mosquitoes (known as xeno-monitoring) are available, but most provide limited information about virus diversity. PCR-based screening coupled with metatranscriptomics is an increasingly affordable and sensitive pipeline for integrating complete viral genome sequencing into surveillance programs. This enables large-scale, high-throughput arbovirus screening from diverse samples. We collected mosquitoes in CO2-baited light traps from five urban parks in Brisbane from March 2021 to May 2022. Mosquito pools of ≤200 specimens were screened for alphaviruses and flaviviruses using virus genus-specific primers and reverse transcription quantitative PCR (qRT-PCR). A subset of virus-positive samples was then processed using a mosquito-specific ribosomal RNA depletion method and then sequenced on the Illumina NextSeq. Overall, 54,670 mosquitoes, representing 26 species were screened in 382 pools. Thirty detections of arboviruses were made in 28 pools. Twenty of these positive pools were further characterised using meta-transcriptomics generating 18 full-length genomes. These full-length sequences belonged to four medically relevant arboviruses: Barmah Forest, Ross River, Sindbis-like and Stratford viruses. Phylogenetic and evolutionary analyses revealed the evolutionary progression of arbovirus lineages over the last 100 years, highlighting long-distance dispersal across the Australian continent and continuous circulation characterised by constant turnover of virus lineages. ### Competing Interest Statement The authors have declared no competing interest.

Arbovirus surveillance of wild-caught mosquitoes is an affordable and sensitive means of monitoring virus transmission dynamics at various spatial-temporal scales, and emergence and re-emergence during epidemic and interepidemic periods. A variety of molecular diagnostics for arbovirus screening of mosquitoes (known as xeno-monitoring) are available, but most provide limited information about virus diversity. Polymerase chain reaction (PCR)-based screening coupled with RNA sequencing is an increasingly affordable and sensitive pipeline for integrating complete viral genome sequencing into surveillance programs. This enables large-scale, high-throughput arbovirus screening from diverse samples. We collected mosquitoes in CO2-baited light traps from five urban parks in Brisbane from March 2021 to May 2022. Mosquito pools of <= 200 specimens were screened for alphaviruses and flaviviruses using virus genus-specific primers and reverse transcription quantitative PCR (qRT-PCR). A subset of virus-positive samples was then processed using a mosquito-specific ribosomal RNA depletion method and then sequenced on the Illumina NextSeq. Overall, 54,670 mosquitoes representing 26 species were screened in 382 pools. Thirty detections of arboviruses were made in 28 pools. Twenty of these positive pools were further characterized using RNA sequencing generating 18 full-length genomes. These full-length sequences belonged to four medically relevant arboviruses: Barmah Forest, Ross River, Sindbis-like, and Stratford viruses. Phylogenetic and evolutionary analyses revealed the evolutionary progression of arbovirus lineages over the last 100 years, demonstrating that different epidemiological, immunological, and evolutionary processes may actively shape the evolution of Australian arboviruses. These results underscore the need for more genomic surveillance data to explore the complex evolutionary pressures acting on arboviruses. Overall, our findings highlight the effectiveness of our methodology, which can be applied broadly to enhance arbovirus surveillance in various ecological contexts and improve understanding of transmission dynamics.

Human infections with the Japanese encephalitis virus (JEV) are a leading cause of viral encephalitis. An unprecedented outbreak of JEV genotype 4 was recently reported in Australia, with an isolate (JEVNSW/22) obtained from a stillborn piglet brain. Herein we conduct a thorough characterization of JEVNSW/22 in three different mouse strains and in human cortical brain organoids (hBOs), and determined the ability of JEVNSW/22 to be neutralized by sera from humans vaccinated with IMOJEV. JEVNSW/22 was less virulent than JEVFU (genotype 2) and JEVNakayama (genotype 3) in C57BL/6J mice and in interferon regulatory factor 7 deficient (Irf7-/-) mice, with infection of wild-type and knockout murine embryonic fibroblasts indicating JEVNSW/22 is more sensitive to type I interferon responses. Irf7-/- mice provide a new model for JEVNSW/22, showing higher viremia levels compared to C57BL/6J mice, and allowing for lethal neuroinvasive infection. All JEV strains were universally lethal in Ifnar-/- mice by day 3, with histological signs of brain hemorrhage, but no other lesions. There were no indications of brain infection in Ifnar-/- mice, with viral protein detected in blood vessels, but not neurons. All JEV isolates showed robust cytopathic infection of human cortical brain organoids, albeit lower for JEVNSW/22. IMOJEV vaccination in humans induced antibodies capable of neutralizing JEVNSW/22, although, for all JEV strains, cross-neutralization titers declined with increasing divergence from IMOJEV in the envelope amino acid sequences. Overall, our study establishes JEVNSW/22 mouse and hBO models of infection, allowing for possible lethal neuroinvasive infection in mice that was rarer than for other JEV genotypes. JEV vaccination regimens may afford protection against this newly emerged JEV genotype 4 strain, although neutralizing antibody responses are sub-optimal.

The World Health Organization recently declared a global initiative to control arboviral diseases. These are mainly caused by pathogenic flaviviruses (such as dengue, yellow fever and Zika viruses) and alphaviruses (such as chikungunya and Venezuelan equine encephalitis viruses). Vaccines represent key interventions for these viruses, with licensed human and/or veterinary vaccines being available for several members of both genera. However, a hurdle for the licensing of new vaccines is the epidemic nature of many arboviruses, which presents logistical challenges for phase III efficacy trials. Furthermore, our ability to predict or measure the post-vaccination immune responses that are sufficient for subclinical outcomes post-infection is limited. Given that arboviruses are also subject to control by the immune system of their insect vectors, several approaches are now emerging that aim to augment antiviral immunity in mosquitoes, including Wolbachia infection, transgenic mosquitoes, insect-specific viruses and paratransgenesis. In this Review, we discuss recent advances, current challenges and future prospects in exploiting both vertebrate and invertebrate immune systems for the control of flaviviral and alphaviral diseases. In this Review, the authors discuss recent advances, current challenges and future prospects in exploiting both vertebrate and invertebrate immune systems for the control of flaviviral and alphaviral diseases.

Great advances in automated identification systems, or ‘smart traps’, that differentiate insect species have been made in recent years, yet demonstrations of field-ready devices under free-flight conditions remain rare. Here, we describe the results of mixed-species identification of female mosquitoes using an advanced optoacoustic smart trap design under free-flying conditions. Point-of-capture classification was assessed using mixed populations of congeneric (Aedes albopictus and Aedes aegypti) and non-congeneric (Ae. aegypti and Anopheles stephensi) container-inhabiting species of medical importance. Culex quinquefasciatus, also common in container habitats, was included as a third species in all assessments. At the aggregate level, mixed collections of non-congeneric species (Ae. aegypti, Cx. quinquefasciatus, and An. stephensi) could be classified at accuracies exceeding 90% (% error = 3.7–7.1%). Conversely, error rates increased when analysing individual replicates (mean % error = 48.6; 95% CI 8.1–68.6) representative of daily trap captures and at the aggregate level when Ae. albopictus was released in the presence of Ae. aegypti and Cx. quinquefasciatus (% error = 7.8–31.2%). These findings highlight the many challenges yet to be overcome but also the potential operational utility of optoacoustic surveillance in low diversity settings typical of urban environments.

The impact of the programmatic use of larvicides for mosquito control on native stingless bees (e.g., Apidae, Meliponini) is a growing concern in Australia due to heightened conservation awareness and the growth of hobbyist stingless bee keeping. In Australia, the two most widely used mosquito larvicides are the bacterium Bacillus thuringiensis var. israelensis (Bti) and the insect hormone mimic methoprene (as S-methoprene). Each has a unique mode of action that could present a risk to stingless bees and other pollinators. Herein, we review the potential impacts of these larvicides on native Australian bees and conclude that their influence is mitigated by their low recommended field rates, poor environmental persistence, and the seasonal and intermittent nature of mosquito control applications. Moreover, evidence suggests that stingless bees may display a high physiological tolerance to Bti similar to that observed in honey bees (Apis mellifera), whose interactions with B. thuringiensis-based biopesticides are widely reported. In summary, neither Bti or methoprene is likely to pose a significant risk to the health of stingless bees or their nests. However, current knowledge is limited by regulatory testing requirements that only require the use of honey bees as toxicological models. To bridge this gap, we suggest that regulatory testing is expanded to include stingless bees and other nontarget insects. This is imperative for improving our understanding of the potential risks that these and other pesticides may pose to native pollinator conservation.

The surveillance and detection of zoonotic pathogens in animals is essential for predicting disease transmission pathways and the risks of spillover, but challenges include the costs, ethics and technical expertise required for vertebrate trapping, serum sampling and antibody or virus screening. Surveillance using haematophagous arthropods as a sampling tool offers a unique opportunity to obtain blood samples from a wide range of vertebrate species, allowing the study of host-mosquito associations, and host exposure to pathogens. We explored vertebrate diversity and potential Ross River virus (RRV) transmission pathways by analysing blood-fed mosquitoes collected in Brisbane, Australia. Host origins were identified using barcode sequencing, and host exposure to RRV was assessed using a modified plaque reduction neutralisation test. In total, 480 blood-fed mosquitoes were collected between February 2021 and May 2022. The host origins of 346 (72%) bloodmeals were identified, with humans (73%) and cattle (9%) comprising the dominant hosts. RRV seroprevalence was high in both vertebrate species with evidence of RRV exposure in 70% (21/30) of cattle and 52% (132/253) of humans. This is a novel, non-invasive method of estimating seroprevalence in vertebrate host populations. Our results highlight the potential of blood-fed mosquitoes to provide species-specific insights into pathogen transmission dynamics.

BACKGROUND With no effective drugs or widely available vaccines, dengue control in Bangladesh is dependent on targeting the primary vector Aedes aegypti with insecticides and larval source management. Despite these interventions, the dengue burden is increasing in Bangladesh, and the country experienced its worst outbreak in 2019 with 101,354 hospitalized cases. This may be partially facilitated by the presence of intense insecticide resistance in vector populations. Here, we describe the intensity and mechanisms of resistance to insecticides commonly deployed against Ae. aegypti in Dhaka, Bangladesh. RESULTS Dhaka Ae. aegypti colonies exhibited high-intensity resistance to pyrethroids. Using CDC bottle assays, we recorded 2 - 24% mortality (recorded at 24 hours) to permethrin and 48 - 94% mortality to deltamethrin, at 10x the diagnostic dose. Bioassays conducted using insecticide-synergist combinations suggested that metabolic mechanisms were contributing to pyrethroid resistance, specifically multi-function oxidases, esterases, and glutathione S-transferases. In addition, kdr alleles were detected, with a high frequency (78-98%) of homozygotes for the V1016G mutation. A large proportion (≤ 74%) of free-flying and resting mosquitoes from Dhaka colonies survived exposure to standard applications of pyrethroid aerosols in an experimental free-flight room. Although that exposure affected Ae. aegypti's immediate host-seeking behavior, the effect was transient in surviving mosquitoes. CONCLUSION The intense resistance characterized in this study is likely compromising the operational effectiveness of pyrethroids against Ae. aegypti in Dhaka. Switching to alternative chemical classes may offer a medium-term solution, but ultimately a more sustainable and effective approach to controlling dengue vectors is required. This article is protected by copyright. All rights reserved.