Development of a regional climate change model for Aedes vigilax and Aedes camptorhynchus (Diptera: Culicidae) in Perth, Western Australia.

Mosquito-borne disease is a significant public health issue and within Australia Ross River virus (RRV) is the most reported. This study combines a mechanistic model of mosquito development for two mosquito vectors; Aedes vigilax and Aedes camptorhynchus, with climate projections from three climate models for two Representative Concentration Pathways (RCPs), to examine the possible effects of climate change and sea-level rise on a temperate tidal saltmarsh habitat in Perth, Western Australia. The projections were run under no accretion and accretion scenarios using a known mosquito habitat as a case study. This improves our understanding of the possible implications of sea-level rise, accretion and climate change for mosquito control programmes for similar habitats across temperate tidal areas found in Southwest Western Australia. The output of the model indicate that the proportion of the year mosquitoes are active increases. Population abundances of the two Aedes species increase markedly. The main drivers of changes in mosquito population abundances are increases in the frequency of inundation of the tidal wetland and size of the area inundated, increased minimum water temperature, and decreased daily temperature fluctuations as water depth increases due to sea level changes, particularly under the model with no accretion. The effects on mosquito populations are more marked for RCP 8.5 when compared to RCP 4.5 but were consistent among the three climate change models. The results indicate that Ae. vigilax is likely to be the most abundant species in 2030 and 2050, but that by 2070 Aedes camptorhynchus may become the more abundant species. This increase would put considerable pressure on existing mosquito control programmes and increase the risk of mosquito-borne disease and nuisance biting to the local community, and planning to mitigate these potential impacts should commence now.

Genome-Scale Phylogeny and Evolutionary Analysis of Ross River Virus Reveals Periodic Sweeps of Lineage Dominance in Western Australia, 1977-2014.

Ross River virus (RRV), an alphavirus of the Togaviridae family, is the most medically significant mosquito-borne virus of Australia. Past RRV phylogenetic and evolutionary analyses have been based on partial genome analyses only. Three geographically distinct RRV lineages, the Eastern, the Western, and the supposedly extinct North-Eastern lineage, were classified previously. We sought to expand on past phylogenies through robust genome-scale phylogeny to better understand RRV genetic diversity and evolutionary dynamics. We analyzed 106 RRV complete coding sequences, which included 13 genomes available on NCBI and 94 novel sequences derived for this study, sampled throughout Western Australia (1977-2014) and during the substantial Pacific Islands RRV epidemic (1979-1980). Our final data set comprised isolates sampled over 59 years (1959-2018) from a range of locations. Four distinct genotypes were defined, with the newly described genotype 4 (G4) found to be the contemporary lineage circulating in Western Australia. The prior geographical classification of RRV lineages was not supported by our findings, with evidence of geographical and temporal cocirculation of distinct genetic groups. Bayesian Markov chain Monte Carlo (MCMC) analysis revealed that RRV lineages diverged from a common ancestor approximately 94 years ago, with distinct lineages emerging roughly every 10 years over the past 50 years in periodic bursts of genetic diversity. Our study has enabled a more robust analysis of RRV evolutionary history and resolved greater genetic diversity that had been previously defined by partial E2 gene analysis.IMPORTANCE Ross River virus (RRV) causes the most common mosquito-borne infection in Australia and causes a significant burden of suffering to infected individuals as well as being a large burden to the Australian economy. The genetic diversity of RRV and its evolutionary history have so far only been studied using partial E2 gene analysis with a limited number of isolates. Robust whole-genome analysis has not yet been conducted. This study generated 94 novel near-whole-genome sequences to investigate the evolutionary history of RRV to better understand its genetic diversity through comprehensive whole-genome phylogeny. A better understanding of RRV genetic diversity will enable better diagnostics, surveillance, and potential future vaccine design.

Metatranscriptomic Sequencing of Medically Important Mosquitoes Reveals Extensive Diversity of RNA Viruses and Other Microbial Communities in Western Australia.

Mosquitoes harbor a wide diversity of microorganisms, including viruses that are human pathogens, or that are insect specific. We used metatranscriptomics, an unbiased high-throughput molecular approach, to describe the composition of viral and other microbial communities in six medically important mosquito species from across Western Australia: Aedes vigilax, Culex annulirostris, Cx. australicus, Cx. globocoxitus, Cx. pipiens biotype molestus, and Cx. quinquefasciatus. We identified 42 viral species, including 13 novel viruses, from 19 families. Culex mosquitoes exhibited a significantly higher diversity of viruses than Aedes mosquitoes, and no virus was shared between the two genera. Comparison of mosquito populations revealed a heterogenous distribution of viruses between geographical regions and between closely related species, suggesting that geography and host species may play a role in shaping virome composition. We also detected bacterial and parasitic microorganisms, among which Wolbachia bacteria were detected in three members of the Cx. pipiens complex, Cx. australicus, Cx. pipiens biotype molestus, and Cx. quinquefasciatus. In summary, our unbiased metatranscriptomics approach provides important insights into viral and other microbial diversity in Western Australian mosquitoes that vector medically important viruses.

A Multi-Species Simulation of Mosquito Disease Vector Development in Temperate Australian Tidal Wetlands Using Publicly Available Data.

Worldwide, mosquito monitoring and control programs consume large amounts of resources in the effort to minimise mosquito-borne disease incidence. On-site larval monitoring is highly effective but time consuming. A number of mechanistic models of mosquito development have been developed to reduce the reliance on larval monitoring, but none for Ross River virus, the most commonly occurring mosquito-borne disease in Australia. This research modifies existing mechanistic models for malaria vectors and applies it to a wetland field site in Southwest, Western Australia. Environmental monitoring data were applied to an enzyme kinetic model of larval mosquito development to simulate timing of adult emergence and relative population abundance of three mosquito vectors of the Ross River virus for the period of 2018-2020. The model results were compared with field measured adult mosquitoes trapped using carbon dioxide light traps. The model showed different patterns of emergence for the three mosquito species, capturing inter-seasonal and inter-year variation, and correlated well with field adult trapping data. The model provides a useful tool to investigate the effects of different weather and environmental variables on larval and adult mosquito development and can be used to investigate the possible effects of changes to short-term and long-term sea level and climate changes.

Optimising predictive modelling of Ross River virus using meteorological variables.

BACKGROUND: Statistical models are regularly used in the forecasting and surveillance of infectious diseases to guide public health. Variable selection assists in determining factors associated with disease transmission, however, often overlooked in this process is the evaluation and suitability of the statistical model used in forecasting disease transmission and outbreaks. Here we aim to evaluate several modelling methods to optimise predictive modelling of Ross River virus (RRV) disease notifications and outbreaks in epidemiological important regions of Victoria and Western Australia. METHODOLOGY/PRINCIPAL FINDINGS: We developed several statistical methods using meteorological and RRV surveillance data from July 2000 until June 2018 in Victoria and from July 1991 until June 2018 in Western Australia. Models were developed for 11 Local Government Areas (LGAs) in Victoria and seven LGAs in Western Australia. We found generalised additive models and generalised boosted regression models, and generalised additive models and negative binomial models to be the best fit models when predicting RRV outbreaks and notifications, respectively. No association was found with a model's ability to predict RRV notifications in LGAs with greater RRV activity, or for outbreak predictions to have a higher accuracy in LGAs with greater RRV notifications. Moreover, we assessed the use of factor analysis to generate independent variables used in predictive modelling. In the majority of LGAs, this method did not result in better model predictive performance. CONCLUSIONS/SIGNIFICANCE: We demonstrate that models which are developed and used for predicting disease notifications may not be suitable for predicting disease outbreaks, or vice versa. Furthermore, poor predictive performance in modelling disease transmissions may be the result of inappropriate model selection methods. Our findings provide approaches and methods to facilitate the selection of the best fit statistical model for predicting mosquito-borne disease notifications and outbreaks used for disease surveillance.

The Diversity and Distribution of Viruses Associated with Culex annulirostris Mosquitoes from the Kimberley Region of Western Australia.

Metagenomics revealed an impressive breadth of previously unrecognized viruses. Here, we report the virome of the Culex annulirostris Skuse mosquito, an important vector of pathogenic arboviruses in Australia. Mosquitoes were collected from three sites in the Kimberley region of Western Australia. Unbiased high-throughput sequencing (HTS) revealed the presence of 16 novel viral sequences that share less than 90% identity with known viruses. None were closely related to pathogenic arboviruses. Viruses were distributed unevenly across sites, indicating a heterogeneous Cx. annulirostris virome. Polymerase chain reaction assays confirmed HTS data and identified marked variation between the virus prevalence identified at each site.

Phylogenetic and Timescale Analysis of Barmah Forest Virus as Inferred from Genome Sequence Analysis.

Barmah Forest virus (BFV) is a medically important mosquito-borne alphavirus endemic to Australia. Symptomatic disease can be a major cause of morbidity, associated with fever, rash, and debilitating arthralgia. BFV disease is similar to that caused by Ross River virus (RRV), the other major Australian alphavirus. Currently, just four BFV whole-genome sequences are available with no genome-scale phylogeny in existence to robustly characterise genetic diversity. Thirty novel genome sequences were derived for this study, for a final 34-taxon dataset sampled over a 44 year period. Three distinct BFV genotypes were characterised (G1-3) that have circulated in Australia and Papua New Guinea (PNG). Evidence of spatio-temporal co-circulation of G2 and G3 within regions of Australia was noted, including in the South West region of Western Australia (WA) during the first reported disease outbreaks in the state's history. Compared with RRV, the BFV population appeared more stable with less frequent emergence of novel lineages. Preliminary in vitro assessment of RRV and BFV replication kinetics found that RRV replicates at a significantly faster rate and to a higher, more persistent titre compared with BFV, perhaps indicating mosquitoes may be infectious with RRV for longer than with BFV. This investigation resolved a greater diversity of BFV, and a greater understanding of the evolutionary dynamics and history was attained.

Discovery of Jogalong virus, a novel hepacivirus identified in a Culex annulirostris (Skuse) mosquito from the Kimberley region of Western Australia.

The discovery of hepaciviruses in non-human hosts has accelerated following the advancement of high-throughput sequencing technology. Hepaciviruses have now been described in reptiles, fish, birds, and an extensive array of mammals. Using metagenomic sequencing on pooled samples of field-collected Culex annulirostris mosquitoes, we discovered a divergent hepacivirus-like sequence, named Jogalong virus, from the Kimberley region in northern Western Australia. Using PCR, we screened the same 300 individual mosquitoes and found just a single positive sample (1/300, 0.33%). Phylogenetic analysis of the hepacivirus NS5B protein places Jogalong virus within the genus Hepacivirus but on a distinct and deeply rooted monophyletic branch shared with duck hepacivirus, suggesting a notably different evolutionary history. Vertebrate barcoding PCR targeting two mitochondrial genes, cytochrome c oxidase subunit I and cytochrome b, indicated that the Jogalong virus-positive mosquito had recently fed on the tawny frogmouth (Podargus strigoides), although it is currently unknown whether this bird species contributes to the natural ecology of this virus.

The forecasting of dynamical Ross River virus outbreaks: Victoria, Australia.

Ross River virus (RRV) is Australia's most epidemiologically important mosquito-borne disease. During RRV epidemics in the State of Victoria (such as 2010/11 and 2016/17) notifications can account for up to 30% of national RRV notifications. However, little is known about factors which can forecast RRV transmission in Victoria. We aimed to understand factors associated with RRV transmission in epidemiologically important regions of Victoria and establish an early warning forecast system. We developed negative binomial regression models to forecast human RRV notifications across 11 Local Government Areas (LGAs) using climatic, environmental, and oceanographic variables. Data were collected from July 2008 to June 2018. Data from July 2008 to June 2012 were used as a training data set, while July 2012 to June 2018 were used as a testing data set. Evapotranspiration and precipitation were found to be common factors for forecasting RRV notifications across sites. Several site-specific factors were also important in forecasting RRV notifications which varied between LGA. From the 11 LGAs examined, nine experienced an outbreak in 2011/12 of which the models for these sites were a good fit. All 11 LGAs experienced an outbreak in 2016/17, however only six LGAs could predict the outbreak using the same model. We document similarities and differences in factors useful for forecasting RRV notifications across Victoria and demonstrate that readily available and inexpensive climate and environmental data can be used to predict epidemic periods in some areas. Furthermore, we highlight in certain regions the complexity of RRV transmission where additional epidemiological information is needed to accurately predict RRV activity. Our findings have been applied to produce a Ross River virus Outbreak Surveillance System (ROSS) to aid in public health decision making in Victoria.

The Highs and Lows of Making a Bucket List-Quantifying Potential Mosquito Breeding Habitats in Metropolitan Backyards.

While the development of land for residential housing along the Swan and Canning Rivers in Perth, WA, Australia has reduced natural mosquito breeding sites, the role of backyard container breeding remains a relatively unknown factor. Local Governments responsible for these areas focus management and control efforts on low lying, tidally driven mosquito habitats to control Aedes vigilax (Skuse) and Aedes camptorhynchus (Thomson) mosquitoes in an effort to reduce both the nuisance and disease risk to residents. In spite of their efforts, Local Governments continue to receive complaints regarding mosquito nuisance, even when environmental conditions do not favor hatching and development of the two species in the Swan River tidal flats. In this study, 150 backyard inspections were conducted in the residential suburb of Bassendean, Perth, WA, Australia, situated in close proximity to the Swan River tidal plain. The occurrence and species composition of the mosquito fauna found in residential backyards was documented. Of the backyards inspected, 94% were found to possess containers capable of breeding mosquitoes, although only 3% contained mosquito larvae. Nine species of mosquito were collected from containers ranging in capacity from 0.05 to 50 L across the study area. Additionally, encephalitis virus surveillance trapping was conducted within residential properties and compared to the tidally driven natural habitat at Ashfield Flats and a tidally influenced brackish creekline at Bindaring Park. The species composition of the fauna at the three habitat types differed significantly, with Aedes notoscriptus (Skuse) dominating residential lots and A. vigilax more prevalent at the saltmarsh site. Bindaring Park had an adult composition at the mid-point of these two habitats, reflecting its proximity to both the Swan River and residential lots.

A Survey of Knowledge, Attitudes, and Practices in Relation to Mosquitoes and Mosquito-Borne Disease in Western Australia.

On average, more than 1,000 individuals will acquire a mosquito-borne disease in Western Australia (WA) each year. Knowledge, attitudes, and practices (KAP) in relation to mosquitoes and mosquito-borne disease have not yet been investigated within Australia. A randomized telephone survey of 2,500 households across 12 regions in WA was undertaken between February and May 2014. The aim of the survey was to obtain baseline KAP data surrounding mosquitoes and mosquito-borne diseases in different regions of WA, across a range of age groups and between males and females. The results of this survey indicate that the majority of respondents are aware of the potential for mosquitoes in WA to transmit Ross River virus, while awareness of other endemic mosquito-borne diseases remains limited. Common misconceptions exist in relation to exotic mosquito-borne diseases, with respondents incorrectly identifying malaria and dengue as endemic diseases in WA. The survey also highlighted a range of important issues, such as limited awareness of the potential for backyard breeding in domestic containers, occupational exposure to mosquitoes in regions with a large employment base in the mining and resources sector, increased exposure to mosquitoes as a result of participation in outdoor recreational activities in the north of the State, and reduced awareness of mosquito-borne disease in individuals aged 18-34 years. The results of this study will be used to inform the development of a new communication strategy by the Department of Health, to further raise awareness of mosquito-borne disease in WA. The data will then provide a baseline against which to compare future survey results, facilitating the rigorous evaluation of new communication efforts.

Deployable Molecular Detection of Arboviruses in the Australian Outback.

The most common causes of human infection from the arboviruses that are endemic in Australia are the arthritogenic alphaviruses: Ross River virus (RRV) and Barmah Forest virus (BFV). The most serious infections are caused by the neurotropic flaviviruses, Murray Valley encephalitis virus (MVEV) and the Kunjin subtype of West Nile virus. The greatest individual risk of arbovirus infection occurs in tropical/subtropical northern Australia because of the warm, wet summer conditions from December to June, where conventional arbovirus surveillance is difficult due to a combination of low population density, large distances between population centers, poor roads, and seasonal flooding. Furthermore, virus detection requires samples to be sent to Perth up to 2,000 km away for definitive analysis, causing delays of days to weeks before test results are available and public health interventions can be started. We deployed a portable molecular biology laboratory for remote field detection of endemic arboviruses in northern Queensland, then in tropical Western Australia and detected BFV, MVEV, and RRV RNA by polymerase chain reaction (PCR) assays of extracts from mosquitoes trapped in Queensland. We then used a field-portable compact real-time thermocycler for the samples collected in the Kimberley region of Western Australia. Real-time field PCR assays enabled concurrent endemic arbovirus distribution mapping in outback Queensland and Western Australia. Our deployable laboratory method provides a concept of operations for future remote area arbovirus surveillance.

Proximity to mosquito breeding habitat and Ross River virus risk in the Peel region of Western Australia.

It is intuitive that vector-borne disease exposure risk is related to proximity to sources of vector breeding, but this aspect rarely receives empirical testing. The population of Western Australia (WA) is increasing rapidly, with many new residential developments proposed in close proximity to mosquito breeding habitat. However, potential mosquito-borne disease risks for future residents are given little consideration by planning authorities. The Peel region is one of the fastest growing regions in WA and regularly experiences a large number of cases of the mosquito-borne Ross River virus (RRV) disease with epidemics occuring in the region every few years. A spatial analysis of RRV disease data in the Peel region was undertaken to determine the risk associated with proximity to a mosquito breeding habitat. Geographic Information Systems (GIS) software was used to create buffers between 1 and 6 km from the breeding habitat. The number of cases per 1000 dwellings in each buffer was calculated between 2002/03 to 2011/12 for years with >100 cases across all buffers (n=5) in addition to the cumulative rate over the entire period in each buffer. Residents living within 1 km of a mosquito breeding habitat had a significantly higher rate of RRV disease compared to the background rate across the Peel region in all individual years investigated. The cumulative data over the 10-year study period showed that residents in the 1- and 2-km buffers had a significantly higher rate, whereas those living between 3 and 6 km away did not. This study demonstrates an increased mosquito-borne disease risk associated with living in close proximity to a mosquito breeding habitat in a rapidly expanding region of WA and highlights the importance of considering mosquito-borne disease risks when planning authorities assess new residential development applications. Known mosquito breeding wetlands should be incorporated into land use planning scheme maps to ensure that they are accurately delineated and the implications are considered when planning decisions are made.

Ross River virus risk associated with dispersal of Aedes (Ochlerotatus) camptorhynchus (Thomson) from breeding habitat into surrounding residential areas: muddy lakes, Western Australia.

Rapid population growth in Western Australia has resulted in increased development of land for residential housing, and new developments are often proposed close to water because of intrinsic aesthetic values. However, this placement may place future residents at risk of mosquito-borne disease, of which Ross River virus (RRV) disease is the most common in Australia. Mosquito dispersal data were combined with a spatial analysis of human RRV cases to show that mosquitoes dispersed readily from larval habitat into surrounding low- and high-density residential areas and that residents living within 2 km of mosquito breeding habitat had a significantly higher rate of RRV disease. This finding highlights the importance of planning authorities in state and local governments to consider the implications of mosquito-borne disease risks when assessing residential development applications.