Ross River Virus Research Articles

Ross River Virus Seroprevalence, French Polynesia, 2014-2015.

Ross River virus (RRV), spread by Aedes and Culex mosquitoes, is the most commonly transmitted arbovirus in Australia. A serosurvey of blood donors in French Polynesia during 2011–2013 suggested that RRV circulated without being detected. We report RRV circulation in French Polynesia based on further screening of blood samples collected during 2014–2015.

Fine-temporal forecasting of outbreak probability and severity: Ross River virus in Western Australia.

Health warnings of mosquito-borne disease risk require forecasts that are accurate at fine-temporal resolutions (weekly scales); however, most forecasting is coarse (monthly). We use environmental and Ross River virus (RRV) surveillance to predict weekly outbreak probabilities and incidence spanning tropical, semi-arid, and Mediterranean regions of Western Australia (1991-2014). Hurdle and linear models were used to predict outbreak probabilities and incidence respectively, using time-lagged environmental variables. Forecast accuracy was assessed by model fit and cross-validation. Residual RRV notification data were also examined against mitigation expenditure for one site, Mandurah 2007-2014. Models were predictive of RRV activity, except at one site (Capel). Minimum temperature was an important predictor of RRV outbreaks and incidence at all predicted sites. Precipitation was more likely to cause outbreaks and greater incidence among tropical and semi-arid sites. While variable, mitigation expenditure coincided positively with increased RRV incidence (r 2 = 0·21). Our research demonstrates capacity to accurately predict mosquito-borne disease outbreaks and incidence at fine-temporal resolutions. We apply our findings, developing a user-friendly tool enabling managers to easily adopt this research to forecast region-specific RRV outbreaks and incidence. Approaches here may be of value to fine-scale forecasting of RRV in other areas of Australia, and other mosquito-borne diseases.

Metagenomic arbovirus detection using MinION nanopore sequencing

With its small size and low cost, the hand-held MinION sequencer is a powerful tool for in-field surveillance. Using a metagenomic approach, it allows non-targeted detection of viruses in a sample within a few hours. This study aimed to determine the ability of the MinION to metagenomically detect and characterise a virus from an infected mosquito. RNA was extracted from an Aedes notoscriptus mosquito infected with Ross River virus (RRV), converted into cDNA and sequenced on the MinION. Bioinformatic analysis of the MinION reads led to detection of full-length RRV, with reads of up to 2.5kb contributing to the assembly. The cDNA was also sequenced on the MiSeq sequencer, and both platforms recovered the RRV genome with >98% accuracy. This proof of concept study demonstrates the metagenomic detection of an arbovirus, using the MinION, directly from a mosquito with minimal sample purification.

Specific inhibition of NLRP3 in chikungunya disease reveals a role for inflammasomes in alphavirus-induced inflammation.

Mosquito-borne viruses can cause severe inflammatory diseases and there are limited therapeutic solutions targeted specifically at virus-induced inflammation. Chikungunya virus (CHIKV), a re-emerging alphavirus responsible for several outbreaks worldwide in the past decade, causes debilitating joint inflammation and severe pain. Here, we show that CHIKV infection activates the NLRP3 inflammasome in humans and mice. Peripheral blood mononuclear cells isolated from CHIKV-infected patients showed elevated NLRP3, caspase-1 and interleukin-18 messenger RNA expression and, using a mouse model of CHIKV infection, we found that high NLRP3 expression was associated with peak inflammatory symptoms. Inhibition of NLRP3 activation using the small-molecule inhibitor MCC950 resulted in reduced CHIKV-induced inflammation and abrogated osteoclastogenic bone loss and myositis, but did not affect in vivo viral replication. Mice treated with MCC950 displayed lower expression levels of the cytokines interleukin-6, chemokine ligand 2 and tumour necrosis factor in joint tissue. Interestingly, MCC950 treatment abrogated disease signs in mice infected with a related arthritogenic alphavirus, Ross River virus, but not in mice infected with West Nile virus-a flavivirus. Here, using mouse models of alphavirus-induced musculoskeletal disease, we demonstrate that NLRP3 inhibition in vivo can reduce inflammatory pathology and that further development of therapeutic solutions targeting inflammasome function could help treat arboviral diseases.

Ross River virus disease clinical presentation, pathogenesis and current therapeutic strategies

Ross River virus (RRV) is an arthitogenic alphavirus capable of causing outbreaks of debilitating musculoskeletal inflammatory disease in humans. RRV is the most common mosquito-borne disease in Australia, with outbreaks of RRV generally occurring during seasonal wet and warm conditions. Patients with Ross River virus disease (RRVD) typically present with fever, polyarthralgia, myalgia and a maculopapular erythematous rash. Treatment of the disease is usually palliative with no licensed vaccines or antiviral therapies currently available. In an effort to better inform therapeutic design, much progress has been made to understand the pathogenesis of RRVD. Progress has been largely driven by clinical evaluations supported by research using established murine models of RRVD, able to accurately replicate human disease. In this review we describe RRVD pathogenesis and the role of the host immune response, with particular focus on insights from studying animal models. We also discuss prospects for effective vaccines, preclinical development of therapeutic strategies and raise important questions for future RRV research.

The ecology and epidemiology of Ross River and Murray Valley encephalitis viruses in Western Australia: examples of One Health in Action.

Arboviruses are maintained and transmitted through an alternating biological cycle in arthropods and vertebrates, with largely incidental disease in humans and animals. As such, they provide excellent examples of One Health, as their health impact is inextricably linked to their vertebrate hosts, their arthropod vectors and the environment. Prevention and control requires a comprehensive understanding of these interactions, and how they may be effectively and safely modified. This review concentrates on human disease due to Ross River and Murray Valley encephalitis viruses, the two major arboviral pathogens in Australia. It describes how their pattern of infection and disease is influenced by natural climatic and weather patterns, and by anthropogenic activities. The latter includes human-mediated environmental manipulations, such as water impoundment infrastructures, human movements and migration, and community and social changes, such as urban spread into mosquito larval habitats. Effective interventions need to be directed at the environmental precursors of risk. This can best be achieved using One Health approaches to improve collaboration and coordination between different disciplines and cross-sectoral jurisdictions in order to develop more holistic mitigation and control procedures, and to address poorly understood ecological issues through multidisciplinary research.

Effective mosquito and arbovirus surveillance using metabarcoding.

Effective vector and arbovirus surveillance requires timely and accurate screening techniques that can be easily upscaled. Next‐generation sequencing (NGS) is a high‐throughput technology that has the potential to modernize vector surveillance. When combined with DNA barcoding, it is termed ‘metabarcoding.’ The aim of our study was to establish a metabarcoding protocol to characterize pools of mosquitoes and screen them for virus. Pools contained 100 morphologically identified individuals, including one Ross River virus (RRV) infected mosquito, with three species present at different proportions: 1, 5, 94%. Nucleic acid extracted from both crude homogenate and supernatant was used to amplify a 269‐bp section of the mitochondrial cytochrome c oxidase subunit I (COI) locus. Additionally, a 67‐bp region of the RRV E2 gene was amplified from synthesized cDNA to screen for RRV. Amplicon sequencing was performed using an Illumina MiSeq, and bioinformatic analysis was performed using a DNA barcode database of Victorian mosquitoes. Metabarcoding successfully detected all mosquito species and RRV in every positive sample tested. The limits of species detection were also examined by screening a pool of 1000 individuals, successfully identifying the species and RRV from a single mosquito. The primers used for amplification, number of PCR cycles and total number of individuals present all have effects on the quantification of species in mixed bulk samples. Based on the results, a number of recommendations for future metabarcoding studies are presented. Overall, metabarcoding shows great promise for providing a new alternative approach to screening large insect surveillance trap catches.

Mosquito distribution in a saltmarsh: determinants of eggs in a variable environment

Two saltmarsh mosquitoes dominate the transmission of Ross River virus (RRV, Togoviridae: Alphavirus), one of Australia's most prominent mosquito-borne diseases. Ecologically, saltmarshes vary in their structure, including habitat types, hydrological regimes, and diversity of aquatic fauna, all of which drive mosquito oviposition behavior. Understanding the distribution of vector mosquitoes within saltmarshes can inform early warning systems, surveillance, and management of vector populations. The aim of this study was to identify the distribution of Ae. camptorhynchus, a known vector for RRV, across a saltmarsh and investigate the influence that other invertebrate assemblage might have on Ae. camptorhynchus egg dispersal. We demonstrate that vegetation is a strong indicator for Ae. camptorhynchus egg distribution, and this was not correlated with elevation or other invertebrates located at this saltmarsh. Also, habitats within this marsh are less frequently inundated, resulting in dryer conditions. We conclude that this information can be applied in vector surveillance and monitoring of temperate saltmarsh environments and also provides a baseline for future investigations into understanding mosquito vector habitat requirements.

Ly6Chi monocytes mediate control of acute alphavirus infection by MAVS-dependent production of type I IFN

Abstract Chikungunya virus (CHIKV) and Ross River virus (RRV) are mosquito-borne alphaviruses that cause epidemics of debilitating musculoskeletal disease. Monocytes have been implicated in the pathogenesis of these infections, but their specific roles are less clear. To assess the role of monocytes in alphavirus pathogenesis, we used CCR2-DTR transgenic mice to deplete CCR2+ cells by administering diphtheria toxin (DT). DT-treated CCR2-DTR+ mice showed reduced weight gain and more severe disease following infection and had fewer Ly6Chi monocytes and NK cells in circulation compared to DT-treated WT mice. Also, depletion of CCR2+ cells, but not NK cells alone, restored virulence and increased viral loads in the muscle tissue of mice infected with RRV encoding attenuating mutations in nsP1 to levels seen in monocyte-depleted mice infected with fully virulent RRV. A similar increase in disease severity and viral loads was observed in DT-treated CCR2-DTR+;Rag1−/− mice infected with the nsP1 mutant virus, confirming that these effects are independent of adaptive immunity. In vitro, culture of Ly6Chi monocytes with RRV-infected cells resulted in induction of IFNα gene expression in monocytes that was MAVS-dependent. IFNα expression in monocytes also required new virus particle production from infected cells. Furthermore, monocytes sorted from the circulation and muscle of infected mice had elevated expression of Irf7. Finally, viral loads of the attenuated nsP1 mutant virus were equivalent to those of WT RRV in the muscle tissue of MAVS−/− mice. Collectively, these data suggest that MAVS-dependent production of type I IFN by monocytes is critical for control of acute alphavirus infection and that determinants in nsP1 counteract this response.

Increased Proinflammatory Cytokine Levels in Prolonged Arthralgia in Ross River Virus Infection.

Ross River virus, a mosquitoborne alphavirus, causes epidemic polyarthritis in Australia and the Pacific region. We analyzed serum cytokine, chemokine, and growth factor levels in travelers returning to Germany from Australia. Serum samples showed elevated concentrations in the acute phase of the illness and, more pronounced, in the long-lasting convalescent phase.

"Looking over the Backyard Fence": Householders and Mosquito Control.

(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.

Mutation of the N-Terminal Region of Chikungunya Virus Capsid Protein: Implications for Vaccine Design.

ABSTRACTMosquito-transmitted chikungunya virus (CHIKV) is an arthritogenic alphavirus of the Togaviridae family responsible for frequent outbreaks of arthritic disease in humans. Capsid protein, a structural protein encoded by the CHIKV RNA genome, is able to translocate to the host cell nucleolus. In encephalitic alphaviruses, nuclear translocation induces host cell transcriptional shutoff; however, the role of capsid protein nucleolar localization in arthritogenic alphaviruses remains unclear. Using recombinant enhanced green fluorescent protein (EGFP)-tagged expression constructs and CHIKV infectious clones, we describe a nucleolar localization sequence (NoLS) in the N-terminal region of capsid protein, previously uncharacterized in CHIKV. Mutation of the NoLS by site-directed mutagenesis reduced efficiency of nuclear import of CHIKV capsid protein. In the virus, mutation of the capsid protein NoLS (CHIKV-NoLS) attenuated replication in mammalian and mosquito cells, producing a small-plaque phenotype. Attenuation of CHIKV-NoLS is likely due to disruption of the viral replication cycle downstream of viral RNA synthesis. In mice, CHIKV-NoLS infection caused no disease signs compared to wild-type CHIKV (CHIKV-WT)-infected mice; lack of disease signs correlated with significantly reduced viremia and decreased expression of proinflammatory factors. Mice immunized with CHIKV-NoLS, challenged with CHIKV-WT at 30 days postimmunization, develop no disease signs and no detectable viremia. Serum from CHIKV-NoLS-immunized mice is able to efficiently neutralize CHIKV infection in vitro. Additionally, CHIKV-NoLS-immunized mice challenged with the related alphavirus Ross River virus showed reduced early and peak viremia postchallenge, indicating a cross-protective effect. The high degree of CHIKV-NoLS attenuation may improve CHIKV antiviral and rational vaccine design.

New evidence for endemic circulation of Ross River virus in the Pacific Islands and the potential for emergence

An epidemic of Ross River virus (RRV) occurred in the South Pacific in 1979-1980, but RRV has not been thought to occur endemically outside Australia and Papua New Guinea. A seroprevalence study was conducted to determine whether RRV has circulated in American Samoa since 1980. RRV ELISA IgG was performed on 200 serum samples collected in American Samoa in 2010; seroneutralization tests were performed on 60 representative samples. Of 196 available ELISA IgG results, 145 (74%, 95% confidence interval 67-80%) were seropositive. Of the 60 samples subjected to seroneutralization testing, none of the 15 ELISA IgG-negative and 16 of the 45 ELISA IgG-positive samples neutralized RRV. ELISA IgG seroprevalence was higher in persons born before/during the 1979-1980 RRV outbreak (78.3%), but was also high (63.0%) in people born after the outbreak who had lived their entire lives in American Samoa. This study provides serological evidence that RRV circulation is likely to have occurred in American Samoa after 1980. Considering there are no marsupials in American Samoa, this finding implies that other species are capable of acting as reservoir hosts and indicates the potential for RRV to circulate in a much wider area than those currently recognized.

A comparative analysis of three vector-borne diseases across Australia using seasonal and meteorological models

Ross River virus (RRV), Barmah Forest virus (BFV), and dengue are three common mosquito-borne diseases in Australia that display notable seasonal patterns. Although all three diseases have been modeled on localized scales, no previous study has used harmonic models to compare seasonality of mosquito-borne diseases on a continent-wide scale. We fit Poisson harmonic regression models to surveillance data on RRV, BFV, and dengue (from 1993, 1995 and 1991, respectively, through 2015) incorporating seasonal, trend, and climate (temperature and rainfall) parameters. The models captured an average of 50–65% variability of the data. Disease incidence for all three diseases generally peaked in January or February, but peak timing was most variable for dengue. The most significant predictor parameters were trend and inter-annual periodicity for BFV, intra-annual periodicity for RRV, and trend for dengue. We found that a Temperature Suitability Index (TSI), designed to reclassify climate data relative to optimal conditions for vector establishment, could be applied to this context. Finally, we extrapolated our models to estimate the impact of a false-positive BFV epidemic in 2013. Creating these models and comparing variations in periodicities may provide insight into historical outbreaks as well as future patterns of mosquito-borne diseases.

A model for the dynamics of Ross River Virus in the Australian environment

Ross River Disease is a mosquito-borne viral condition that affects pockets of the Australian human population, and can be debilitating in some instances. The evidence is that the virus reservoirs in marsupials, such as kangaroos, and this may account for the unpredictable outbreaks of the disease in humans. Accordingly, we present here a new model for the dynamics of Ross River Virus (RRV) in populations of mosquitoes and kangaroos. We calculate steady-state populations for the sub-groups in each species and demonstrate that naturally occurring oscillations in the populations (limit cycles) do not occur. When seasonal forcing of vector populations and kangaroo birth rates is taken into account, however, the model may predict multi-annual outbreaks and chaos, perhaps explaining the unpredictability of some RRV disease epidemics, particularly across southern Australia. Detailed results in this case are presented.

A Model for the Dynamics of Ross River Virus in the Australian Environment

A Model for the Dynamics of Ross River Virus in the Australian Environment

Exotic mosquito threats require strategic surveillance and response planning.

Mosquito-borne diseases caused by endemic pathogens such as Ross River, Barmah Forest and Murray Valley encephalitis viruses are an annual concern in New South Wales (NSW), Australia. More than a dozen mosquito species have been implicated in the transmission of these pathogens, with each mosquito occupying a specialised ecological niche that influences their habitat associations, host feeding preferences and the environmental drivers of their abundance. The NSW Arbovirus Surveillance and Mosquito Monitoring Program provides an early warning system for potential outbreaks of mosquito-borne disease by tracking annual activity of these mosquitoes and their associated pathogens. Although the program will effectively track changes in local mosquito populations that may increase with a changing climate, urbanisation and wetland rehabilitation, it will be less effective with current surveillance methodologies at detecting or monitoring changes in exotic mosquito threats, where different surveillance strategies need to be used. Exotic container-inhabiting mosquitoes such as Aedes aegypti and Ae. albopictus pose a threat to NSW because they are nuisance-biting pests and vectors of pathogens such as dengue, chikungunya and Zika viruses. International movement of humans and their belongings have spread these mosquitoes to many regions of the world. In recent years, these two mosquitoes have been detected by the Australian Government Department of Agriculture and Water Resources at local airports and seaports. To target the detection of these exotic mosquitoes, new trapping technologies and networks of surveillance locations are required. Additionally, incursions of these mosquitoes into urban areas of the state will require strategic responses to minimise substantial public health and economic burdens to local communities.

A review of recommendations on the safe and effective use of topical mosquito repellents.

Mosquito-borne disease is an annual problem in Australia, with endemic pathogens such as Ross River virus infecting thousands of people each year. The recent emergence of Zika virus in South America and the Pacific, together with ongoing outbreaks of dengue viruses in Southeast Asia, generated great community interest in the most effective strategies to avoid mosquito bites. Large-scale mosquito control programs are not common in Australia and are limited in New South Wales (NSW). The use of topical insect repellents is a key recommendation by health authorities to prevent mosquito-borne disease. All products sold in Australia purporting to repel mosquitoes must be registered with the Australian Pesticides and Veterinary Medicines Authority. Despite around 100 commercial products registered as repelling mosquitoes, there are relatively few active ingredients used across these formulations. The most common are diethyltoluamide (DEET), picaridin, p-menthane-3,8-diol (PMD) and a range of plant-derived products (e.g. melaleuca, eucalyptus, citronella oils). Research has shown that each of these active ingredients varies in the duration of protection provided against biting mosquitoes. Recommendations by health authorities are informed by this research, but inconsistencies between recommendations and available repellent formulations and their concentration of active ingredients can cause confusion in the community. There are conflicts between the data resulting from scholarly research, marketing promotion by manufacturers and recommendations provided by overseas health authorities. A review was undertaken of NSW Health's current recommendations on choosing and using insect repellents, taking into consideration recent research and currently registered topical repellents.

Disease surveillance based on Internet-based linear models: an Australian case study of previously unmodeled infection diseases.

Effective disease surveillance is critical to the functioning of health systems. Traditional approaches are, however, limited in their ability to deliver timely information. Internet-based surveillance systems are a promising approach that may circumvent many of the limitations of traditional health surveillance systems and provide more intelligence on cases of infection, including cases from those that do not use the healthcare system. Infectious disease surveillance systems built on Internet search metrics have been shown to produce accurate estimates of disease weeks before traditional systems and are an economically attractive approach to surveillance; they are, however, also prone to error under certain circumstances. This study sought to explore previously unmodeled diseases by investigating the link between Google Trends search metrics and Australian weekly notification data. We propose using four alternative disease modelling strategies based on linear models that studied the length of the training period used for model construction, determined the most appropriate lag for search metrics, used wavelet transformation for denoising data and enabled the identification of key search queries for each disease. Out of the twenty-four diseases assessed with Australian data, our nowcasting results highlighted promise for two diseases of international concern, Ross River virus and pneumococcal disease.

Epidemic host community contribution to mosquito-borne disease transmission: Ross River virus.

Most vector-borne diseases infect multiple host species, but disentangling the relative importance of different host species to transmission can be complex. Here we study how host species' abundance and competence (duration and titre of parasitaemia) influence host importance during epidemic scenarios. We evaluate this theory using Ross River virus (RRV, family Togaviridae, genus Alphavirus), a multi-host mosquito-borne disease with significant human health impacts across Australia and Papua New Guinea. We used host contribution models to find the importance of key hosts (possums, wallabies, kangaroos, horses, humans) in typical mammal communities around five Australian epidemic centres. We found humans and possums contributed most to epidemic RRV transmission, owing to their high abundances, generally followed by macropods. This supports humans as spillover hosts, and that human-mosquito and possum-mosquito transmission is predominant during epidemics. Sensitivity analyses indicate these findings to be robust across epidemic centres. We emphasize the importance of considering abundance and competence in identifying key hosts (during epidemics in this case), and that competence alone is inadequate. Knowledge of host importance in disease transmission may help to equip health agencies to bring about greater effectiveness of disease mitigation strategies.