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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., Author summary Mosquito-borne diseases cause significant illness worldwide. Mosquito breeding, which leads to disease transmission, is driven by favorable climatic and meteorological events (e.g., rainfall and warm temperatures). Understanding the association meteorological conditions have with mosquito breeding aids in directing mosquito control activities when there is a likelihood of disease transmission. Predictive models are used in public health decision making and resource allocation to guide mosquito control programs. However, there are multiple modelling methods, all of which provide differing degrees of accuracy in their predictions and suitability to the disease transmission dynamics. This study aims to assess commonly used statistical models for predicting mosquito-borne disease notifications and outbreaks. We demonstrate that statistical model selection plays an important role in accurately forecasting mosquito-borne disease and poor predictive performance may be due to inappropriate model selection. Furthermore, a model suited to predicting disease notifications may not always be the best model to accurately predict the occurrence of disease outbreaks. The methods used here can aid in public health to establish suitable predictive mosquito-borne disease surveillance systems to help guide disease prevention and resource allocation, and mosquito control activities.

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&ndash, 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&rsquo, 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.

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.

Mosquito population data in the Perth Metropolitan area are limited. This research aimed to determine mosquito species abundance and composition within three local government areas of inner metropolitan Perth. A secondary aim was to determine whether mosquito species abundance and composition in the study area were related to proximity of known saltmarsh mosquito breeding sites along two major tidal estuary systems. Eleven mosquito species were collected, of which four were found in high abundance: Aedes vigilax, Culex quinquefasciatus, Culex annulirostris and Aedes notoscriptus. The majority of mosquitoes were collected within 1 km of the tidal estuary systems, suggesting that productive mosquito breeding habitat is likely to be located along the fringe of the estuaries. The implication for mosquito management is that saltmarsh mosquitoes are not the only type of mosquito species in the study area. Broadening surveillance and management methods to include freshwater and container breeding species will reduce nuisance concerns for the public and is also likely to reduce incidence of disease in the area.

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., 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.

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. Keywords: Arboviruses, Transmission, Mosquito-borne disease, Forecasting, Predicting epidemics, Epidemiology

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.

Mosquitoes harbor a high diversity of RNA viruses, including many that impact human health. Despite a growing effort to describe the extent and nature of the mosquito virome, little is known about how these viruses persist, spread, and interact with both their hosts and other microbes. To address this issue we performed a metatranscriptomics analysis of 12 Western Australian mosquito populations structured by species and geographic location. Our results identified the complete genomes of 24 species of RNA viruses from a diverse range of viral families and orders, among which 19 are newly described. Comparisons of viromes revealed a striking difference between the two mosquito genera, with viromes of mosquitoes of the Aedes genus exhibiting substantially less diversity and lower abundances than those of mosquitoes of the Culex genus, within which the viral abundance reached 16.87% of the total non-rRNA. In addition, there was little overlap in viral diversity between the two genera, although the viromes were very similar among the three Culex species studied, suggesting that the host taxon plays a major role in structuring virus diversity. In contrast, we found no evidence that geographic location played a major role in shaping RNA virus diversity, and several viruses discovered here exhibited high similarity (95 to 98% nucleotide identity) to those from Indonesia and China. Finally, using abundance-level and phylogenetic relationships, we were able to distinguish potential mosquito viruses from those present in coinfecting bacteria, fungi, and protists. In sum, our metatranscriptomics approach provides important insights into the ecology of mosquito RNA viruses. IMPORTANCE Studies of virus ecology have generally focused on individual viral species. However, recent advances in bulk RNA sequencing make it possible to utilize metatranscriptomic approaches to reveal both complete virus diversity and the relative abundance of these viruses. We used such a metatranscriptomic approach to determine key aspects of the ecology of mosquito viruses in Western Australia. Our results show that RNA viruses are some of the most important components of the mosquito transcriptome, and we identified 19 new virus species from a diverse set of virus families. A key result was that host genetic background plays a more important role in shaping virus diversity than sampling location, with Culex species harboring more viruses at higher abundance than those from Aedes mosquitoes.

The psyllid Leptynoptera sulfurea Crawford (Hemiptera: Psylloidea: Triozidae) is reported for the first time from the Cocos (Keeling) Islands located in the Indian Ocean. It is associated with the tree Calophyllum inophyllum L. (Calophyllaceae). It is the only species of psyllid currently known from these islands.

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.

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.

(1) Background: Vector-borne diseases are a significant public health problem in Western Australia. Mosquitoes are responsible for the transmission of a number of pathogens and may pose a serious nuisance problem. Prevention efforts in the State are multi-faceted and include physical, chemical, and cultural control methods for restricting mosquito breeding. This is less complex where breeding areas are located within public open spaces. In Australia’s developed urban areas, breeding sites are, however, frequently located within private residential landholdings, where the scope of public health officials to act is constrained by law and practicality. Consequently, mosquito prevention in these locations is predominantly the responsibility of the residents. This research addressed a gap, both in understanding the degree to which “backyard” mosquito breeding has the potential to contribute to local mosquito problems, and in assessing what residents “think and do” about mosquito control within their home environment. (2) Methods: The study was conducted in the Town of Bassendean, a metropolitan Local Government Area of Perth, Western Australia, in close proximity to two natural, productive mosquito breeding sites, namely Ashfield Flats and Bindaring Park. A total of 150 householders were randomly surveyed during the summer of 2015–2016, to gauge residents’ knowledge, attitudes, and practices (KAP (knowledge, attitudes, and practices) Survey) in regards to mosquitoes, their breeding and ecology, and avoidance or minimization strategies. The survey comprised nine questions covering residents’ knowledge (3 questions), attitudes (3 questions), and practices (3 questions), as well as additional questions regarding the basic demographics of the resident. Larvae were collected from backyard containers and reared to adults for species identification. A series of Encephalitis Vector Surveillance carbon dioxide (EVS CO2) traps were also deployed, to assess adult mosquito density and species composition. (3) Results: Aedes notoscriptus (Skuse), a known container-inhabiting species, accounted for just over 50% of all mosquitoes identified. Most residents were aware of mosquito-borne disease and its risk in their local area. While the majority (79%) of the sample correctly identified Ross River virus as the most common infection in WA, a significant gap in the general knowledge of residents in regards to mosquito biology and breeding habits, was noted. Furthermore, only 50% of residents reported using personal protective measures to reduce mosquito bites and only one in six residents undertook physical or chemical mosquito control around their home. Additionally, 60% of respondents believed that mosquito control was “a job for the council and the state government”, rather than for individual householders. (4) Conclusions: A significant gap in the knowledge of residents in the study area existed in regards to the general knowledge of mosquitoes and their breeding habits; types of treatments that could be employed within the home; and the residents’ responsibility for the management of mosquito breeding on their private property. A public education campaign has been deployed to educate the residents.

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.