The impact of arthritogenic viruses in oral tissues.

Arthritis and periodontitis are inflammatory diseases that share several immunopathogenic features. The expansion in the study of virus-induced arthritis has shed light on how this condition could impact other parts of the human body, including the mouth. Viral arthritis is an inflammatory joint disease caused by several viruses, most notably the alphaviruses Chikungunya virus (CHIKV), Sindbis virus (SINV), Ross River virus (RRV), Mayaro virus (MAYV), and O'nyong'nyong virus (ONNV). These viruses can induce an upsurge of matrix metalloproteinases and immune-inflammatory mediators such as Interleukin-6 (IL6), IL-1ß, tumor necrosis factor, chemokine ligand 2, and receptor activator of nuclear factor kappa-B ligand in the joint and serum of infected individuals. This can lead to the influx of inflammatory cells to the joints and associated muscles as well as osteoclast activation and differentiation, culminating in clinical signs of swelling, pain, and bone resorption. Moreover, several data indicate that these viral infections can affect other sites of the body, including the mouth. The human oral cavity is a rich and diverse microbial ecosystem, and viral infection can disrupt the balance of microbial species, causing local dysbiosis. Such events can result in oral mucosal damage and gingival bleeding, which are indicative of periodontitis. Additionally, infection by RRV, CHIKV, SINV, MAYV, or ONNV can trigger the formation of osteoclasts and upregulate pro-osteoclastogenic inflammatory mediators, interfering with osteoclast activation. As a result, these viruses may be linked to systemic conditions, including oral manifestations. Therefore, this review focuses on the involvement of alphavirus infections in joint and oral health, acting as potential agents associated with oral mucosal inflammation and alveolar bone loss. The findings of this review demonstrate how alphavirus infections could be linked to the comorbidity between arthritis and periodontitis and may provide a better understanding of potential therapeutic management for both conditions.

Weather extremes associated with increased Ross River virus and Barmah Forest virus notifications in NSW: learnings for public health response.

OBJECTIVE: To examine the sequence of environmental and entomological events prior to a substantial increase in Ross River virus (RRV) and Barmah Forest virus (BFV) notifications with a view to informing future public health response. METHODS: Rainfall, tidal, mosquito and human arboviral notification data were analysed to determine the temporality of events. RESULTS: Following two extremely dry years, there was a substantial increase in the abundance of mosquitoes along coastal New South Wales (NSW) two weeks after a significant rainfall event and high tides in February 2020. Subsequently, RRV and BFV notifications in north east NSW began to increase eight and nine weeks respectively after the high rainfall, with RRV notifications peaking 12 weeks after the high rainfall. CONCLUSIONS: Mosquito bite avoidance messaging should be instigated within two weeks of high summer rainfall, especially after an extended dry period. IMPLICATIONS FOR PUBLIC HEALTH: Intense summertime rain events, which are expected to increase in frequency in south-east Australia with climate change, can lead to significant increases in arboviral disease. These events need to be recognised by public health practitioners to facilitate timely public health response. This has taken on added importance since the emergence of Japanese encephalitis virus in southeastern Australia in 2022.

Complete genetic dissection and cell type-specific replication of old world alphaviruses, getah virus (GETV) and sagiyama virus (SAGV).

Getah virus (GETV), which was first isolated in Malaysia in 1955, and Sagiyama virus (SAGV), isolated in Japan in 1956, are members of the genus Alphavirus in the family Togaviridae. It is a consensus view that SAGV is a variant of GETV. In the present study, we determined the complete sequences of the prototype GETV MM2021 and SAGV M6-Mag132 genomic RNA extracted from plaque-purified viruses. The MM2021 genome was 11,692 nucleotides (nt) in length in the absence of 3' poly(A) tail, and the length of M6-Mag132 genome was 11,698 nt. Through sequence alignment of MM2021 and M6-Mag132, we located all the amino acid differences between these two strains, which were scattered in all the encoded proteins. Subsequently, we validated the close evolutionary relationship between GETV and SAGV by constructing phylogenetic trees based on either complete genomes or structural genomes. We eventually analyzed the growth kinetics of GETV and SAGV as well as other representative alphaviruses in various mammalian and insect cell lines. It was shown that human-oriented cell lines such as HEK-293T and Hela cells were relatively resistant to GETV and SAGV infection due to absence of proviral factors or species-specific barrier. On the other hand, both GETV and SAGV replicated efficiently in non-human cell lines. Our results provide essential genetic information for future epidemiological surveillance on Alphaviruses and lay the foundation for developing effective interventions against GETV and SAGV.

Altered Spatial and Temporal Gait Parameters in Mice Infected with Ross River Virus.

Infection with mosquito-borne arthritogenic alphaviruses, such as Ross River virus (RRV) and Barmah Forest virus (BFV), can lead to long-lasting rheumatic disease. Existing mouse models that recapitulate the disease signs and immunopathogenesis of acute RRV and BFV infection have consistently shown relevance to human disease. However, these mouse models, which chiefly model hindlimb dysfunction, may be prone to subjective interpretation when scoring disease. Assessment is therefore time-consuming and requires experienced users. The DigiGait system provides video-based measurements of movement, behavior, and gait dynamics in mice and small animals. Previous studies have shown DigiGait to be a reliable system to objectively quantify changes in gait in other models of pain and inflammation. Here, for the first time, we determine measurable differences in the gait of mice with infectious arthritis using the DigiGait system. Statistically significant differences in paw area and paw angle were detected during peak disease in RRV-infected mice. Significant differences in temporal gait parameters were also identified during the period of peak disease in RRV-infected mice. These trends were less obvious or absent in BFV-infected mice, which typically present with milder disease signs than RRV-infected mice. The DigiGait system therefore provides an objective model of variations in gait dynamics in mice acutely infected with RRV. DigiGait is likely to have further utility for murine models that develop severe forms of infectious arthritis resulting in hindlimb dysfunction like RRV. IMPORTANCE Mouse models that accurately replicate the immunopathogenesis and clinical disease of alphavirus infection are vital to the preclinical development of therapeutic strategies that target alphavirus infection and disease. Current models rely on subjective scoring made through experienced observation of infected mice. Here, we demonstrate how the DigiGait system, and interventions on mice to use this system, can make an efficient objective assessment of acute disease progression and changes in gait in alphavirus-infected mice. Our study highlights the importance of measuring gait parameters in the assessment of models of infectious arthritis.

How do local differences in saltmarsh ecology influence disease vector mosquito populations?

Saltmarsh breeding mosquitoes are an important source of vectors for arboviral transmission. In southern Australia, the most prominent vector borne disease, Ross River virus (Togaviridae: Alphavirus) (RRV), is transmitted by the saltmarsh mosquito (Diptera: Culicidae) Aedes camptorhynchus (Thomson). However, the factors driving the abundance of this mosquito within and among saltmarshes are poorly understood. To predict the abundance of this mosquito within saltmarshes, the environmental conditions and aquatic invertebrate ecology of three temperate saltmarshes habitats were monitored over two seasons. Up to 44% of first-instar mosquito numbers and 21% of pupal numbers were accounted for by environmental variables. Samphire vegetation cover was a common predictor of first-instar numbers across sites although, between saltmarshes, aquatic factors such as high salinity, temperatures less than 22 °C and water body volume were important predictors. The identified predictors of pupal numbers were more variable and included high tides, waterbody volume and alkalinity. The composition of invertebrate functional feeding groups differed between saltmarshes and showed that an increased diversity led to fewer mosquitoes. It was evident that apparently similar saltmarshes can vary markedly in invertebrate assemblages, water availability and conditions through tidal inundations, rainfall or waterbody permanency. The present study advances insight into predictors of vector mosquito numbers that drive the risk of RRV outbreaks.

Flooding and Arboviral Disease: Predicting Ross River Virus Disease Outbreaks Across Inland Regions of South-Eastern Australia.

Flood frequency is expected to increase across the globe with climate change. Understanding the relationship between flooding and arboviral disease can reduce disease risk and associated costs. South-eastern Australia is dominated by the flood-prone Murray-Darling River system where the incidence of Australia's most common arboviral disease, Ross River virus (RRV), is high. This study aimed to determine the relationship between riverine flooding and RRV disease outbreaks in inland south-eastern Australia, specifically New South Wales (NSW). Each study month from 1991 to 2013, for each of 37 local government areas (LGAs) was assigned 'outbreak/non-outbreak' status based on long-term trimmed-average age-standardized RRV notification rates and 'flood/non-flood' status based on riverine overflow. LGAs were grouped into eight climate zones with the relationship between flood and RRV outbreak modeled using generalized estimating equations. Modeling adjusted for rainfall in the previous 1-3 mo. Spring-summer flooding increased the odds of summer RRV outbreaks in three climate zones before and after adjusting for rainfall 1, 2, and 3 mo prior to the outbreak. Flooding at any time of the year was not predictive of RRV outbreaks in the remaining five climate zones. Predicting RRV disease outbreaks with flood events can assist with more targeted mosquito spraying programs, thereby reducing disease transmission and mosquito resistance.

Bioluminescent Ross River Virus Allows Live Monitoring of Acute and Long-Term Alphaviral Infection by In Vivo Imaging.

Arboviruses like chikungunya and Ross River (RRV) are responsible for massive outbreaks of viral polyarthritis. There is no effective treatment or vaccine available against these viruses that induce prolonged and disabling arthritis. To explore the physiopathological mechanisms of alphaviral arthritis, we engineered a recombinant RRV expressing a NanoLuc reporter (RRV-NLuc), which exhibited high stability, near native replication kinetics and allowed real time monitoring of viral spread in an albino mouse strain. During the acute phase of the disease, we observed a high bioluminescent signal reflecting viral replication and dissemination in the infected mice. Using Bindarit, an anti-inflammatory drug that inhibits monocyte recruitment, we observed a reduction in viral dissemination demonstrating the important role of monocytes in the propagation of the virus and the adaptation of this model to the in vivo evaluation of treatment strategies. After resolution of the acute symptoms, we observed an increase in the bioluminescent signal in mice subjected to an immunosuppressive treatment 30 days post infection, thus showing active in vivo replication of remnant virus. We show here that this novel reporter virus is suitable to study the alphaviral disease up to the chronic phase, opening new perspectives for the evaluation of therapeutic interventions.

Role of heparan sulfate in entry and exit of Ross River virus glycoprotein-pseudotyped retroviral vectors.

Variants of Ross River virus (RRV) that bind to heparan sulfate (HS) were previously selected by serial passaging in cell culture. To explore the effects of mutations that convey HS utilization, we pseudotyped Moloney murine leukemia virus (MoMLV), with the RRV envelope. We substituted amino-acid residues 216 and 218 on RRV-E2-envelope glycoprotein with basic amino-acid residues, because these mutations confer affinity for HS upon RRV. However, T216R-RRV- and N218R-RRV-pseudotyped viruses possessed lower transduction titers, and we demonstrated that HS-affinity impeded release of pseudotyped virus from producer cells. Addition of heparinase to HS-expressing target cells reduces the transduction efficiency of the T216R-RRV- and N218R-RRV-pseudotyped viruses, whereas no such effect is seen in cells lacking HS. Under appropriate conditions, these T216R-RRV- and N218R-RRV-pseudotyped viruses have enhanced capacities for transducing HS-expressing cells. General principles concerning viral adaptation to the use of attachment factors and design of pseudotyped viral vectors are discussed.

Ecological and life history traits are associated with Ross River virus infection among sylvatic mammals in Australia.

BACKGROUND: Ross River virus (RRV) is Australia's most important arbovirus given its annual burden of disease and the relatively large number of Australians at risk for infection. This mosquito-borne arbovirus is also a zoonosis, making its epidemiology and infection ecology complex and cryptic. Our grasp of enzootic, epizootic, and zoonotic RRV transmission dynamics is imprecise largely due to a poor understanding of the role of wild mammalian hosts in the RRV system. METHODS: The current study applied a piecewise structural equation model (PSEM) toward an interspecific comparison of sylvatic Australian mammals to characterize the ecological and life history profile of species with a history of RRV infection relative to those species with no such history among all wild mammalian species surveyed for RRV infection. The effects of species traits were assessed through multiple causal pathways within the PSEM framework. RESULTS: Sylvatic mammalian species with a history of RRV infection tended to express dietary specialization and smaller population density. These species were also characterized by a longer gestation length. CONCLUSIONS: This study provides the first interspecific comparison of wild mammals for RRV infection and identifies some potential targets for future wildlife surveys into the infection ecology of this important arbovirus. An applied RRV macroecology may prove invaluable to the epidemiological modeling of RRV epidemics across diverse sylvatic landscapes, as well as to the development of human and animal health surveillance systems.

Mosquito and Virus Surveillance as a Predictor of Human Ross River Virus Infection in South-West Western Australia: How Useful Is It?

Mosquito and virus surveillance systems are widely used in Western Australia (WA) to support public health efforts to reduce mosquito-borne disease. However, these programs are costly to maintain on a long-term basis. Therefore, we aimed to assess the validity of mosquito numbers and Ross River virus (RRV) isolates from surveillance trap sites as predictors of human RRV cases in south-west WA between 2003 and 2014. Using negative binomial regression modeling, mosquito surveillance was found to be a useful tool for predicting human RRV cases. In eight of the nine traps, when adjusted for season, there was an increased risk of RRV cases associated with elevated mosquito numbers detected 1 month before the onset of human cases for at least one quartile compared with the reference group. The most predictive urban trap sites were located near saltmarsh mosquito habitat, bushland that could sustain macropods and densely populated residential suburbs. This convergence of environments could allow enzootic transmission of RRV to spillover and infect the human population. Close proximity of urban trap sites to each other suggested these sites could be reduced. Ross River virus isolates were infrequent at some trap sites, so ceasing RRV isolation from mosquitoes at these sites or where isolates were not predictive of human cases could be considered. In future, trap sites could be reduced for routine surveillance, allowing other environments to be monitored to broaden the understanding of RRV ecology in the region. A more cost-effective and efficient surveillance program may result from these modifications.

Temperature explains broad patterns of Ross River virus transmission.

Thermal biology predicts that vector-borne disease transmission peaks at intermediate temperatures and declines at high and low temperatures. However, thermal optima and limits remain unknown for most vector-borne pathogens. We built a mechanistic model for the thermal response of Ross River virus, an important mosquito-borne pathogen in Australia, Pacific Islands, and potentially at risk of emerging worldwide. Transmission peaks at moderate temperatures (26.4°C) and declines to zero at thermal limits (17.0 and 31.5°C). The model accurately predicts that transmission is year-round endemic in the tropics but seasonal in temperate areas, resulting in the nationwide seasonal peak in human cases. Climate warming will likely increase transmission in temperate areas (where most Australians live) but decrease transmission in tropical areas where mean temperatures are already near the thermal optimum. These results illustrate the importance of nonlinear models for inferring the role of temperature in disease dynamics and predicting responses to climate change.

The non-human reservoirs of Ross River virus: a systematic review of the evidence.

Understanding the non-human reservoirs of zoonotic pathogens is critical for effective disease control, but identifying the relative contributions of the various reservoirs of multi-host pathogens is challenging. For Ross River virus (RRV), knowledge of the transmission dynamics, in particular the role of non-human species, is important. In Australia, RRV accounts for the highest number of human mosquito-borne virus infections. The long held dogma that marsupials are better reservoirs than placental mammals, which are better reservoirs than birds, deserves critical review. We present a review of 50 years of evidence on non-human reservoirs of RRV, which includes experimental infection studies, virus isolation studies and serosurveys. We find that whilst marsupials are competent reservoirs of RRV, there is potential for placental mammals and birds to contribute to transmission dynamics. However, the role of these animals as reservoirs of RRV remains unclear due to fragmented evidence and sampling bias. Future investigations of RRV reservoirs should focus on quantifying complex transmission dynamics across environments.

Alphavirus-induced hyperactivation of PI3K/AKT directs pro-viral metabolic changes.

Virus reprogramming of cellular metabolism is recognised as a critical determinant for viral growth. While most viruses appear to activate central energy metabolism, different viruses have been shown to rely on alternative mechanisms of metabolic activation. Whether related viruses exploit conserved mechanisms and induce similar metabolic changes is currently unclear. In this work we investigate how two alphaviruses, Semliki Forest virus and Ross River virus, reprogram host metabolism and define the molecular mechanisms responsible. We demonstrate that in both cases the presence of a YXXM motif in the viral protein nsP3 is necessary for binding to the PI3K regulatory subunit p85 and for activating AKT. This leads to an increase in glucose metabolism towards the synthesis of fatty acids, although additional mechanisms of metabolic activation appear to be involved in Ross River virus infection. Importantly, a Ross River virus mutant that fails to activate AKT has an attenuated phenotype in vivo, suggesting that viral activation of PI3K/AKT contributes to virulence and disease.

Lower temperatures reduce type I interferon activity and promote alphaviral arthritis.

Chikungunya virus (CHIKV) belongs to a group of mosquito-borne alphaviruses associated with acute and chronic arthropathy, with peripheral and limb joints most commonly affected. Using a mouse model of CHIKV infection and arthritic disease, we show that CHIKV replication and the ensuing foot arthropathy were dramatically reduced when mice were housed at 30°C, rather than the conventional 22°C. The effect was not associated with a detectable fever, but was dependent on type I interferon responses. Bioinformatics analyses of RNA-Seq data after injection of poly(I:C)/jetPEI suggested the unfolded protein response and certain type I interferon responses are promoted when feet are slightly warmer. The ambient temperature thus appears able profoundly to effect anti-viral activity in the periphery, with clear consequences for alphaviral replication and the ensuing arthropathy. These observations may provide an explanation for why alphaviral arthropathies are largely restricted to joints of the limbs and the extremities.

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.

Effectiveness of S-Methoprene Briquets and Application Method for Mosquito Control in Urban Road Gullies/Catch Basins/Gully Pots in a Mediterranean Climate: Implications for Ross River Virus Transmission.

Floating emergence traps were used in 15 road gullies to determine the effectiveness and longevity of S-methoprene briquets over 124 days. Samples were taken monthly from October 2014 to March 2015. Two treatment methods were assessed: application of briquet using a float, and application without a float. These methods were compared with untreated control gullies. Mosquito emergence peaked in early November, and decreased by February. Effectiveness of the briquet was not impacted significantly by the presence or absence of a float (P = 0.329). Gullies yielded a mean of 108 mosquitoes per day per gully over the season. Culex quinquefasciatus and Aedes notoscriptus were the most abundant species. The maximum number of Cx. quinquefasciatus emerging could exceed 1,600 per day per gully. Aedes notoscriptus numbers could exceed 70 adults per day per gully. Treatment with S-methoprene was highly effective against both species for at least 70 days and partially effective for up to 120 days. Treatment provided no control by day 124. S-methoprene provided 90% control over 124 days. Road gullies have been confirmed as a significant larval habitat and are likely to be increasing the potential for Ross River virus transmission in the area.

The growth of arthralgic Ross River virus is restricted in human monocytic cells.

Alphaviruses such as Chikungunya and Ross River (RRV) viruses are associated with persistent arthritis and arthralgia in humans. Monocytes and macrophages are believed to play an important role in alphaviral arthritides. In this study, we evaluated RRV permissiveness of the human acute leukemia MM6 cell line. Viral growth analysis showed that RRV infection of MM6 cells resulted in a very low virus progeny production with daily output. Using recombinant RRV expressing the reporter gene Renilla luciferase, a weak viral replication level was detected in infected cells at the early stages of infection. The infection restriction was not associated with type-I interferon and pro-inflammatory cytokines release. Apoptosis hallmarks (i.e. mitochondrial BAX localisation and PARP cleavage) were observed in infected MM6 cells indicating that RRV can trigger apoptosis at late infection times. The long-term persistence of RRV genomic RNA in surviving MM6 cells identifies human monocytic cells as potential cellular reservoirs of viral material within the infected host.

Improving public health intervention for mosquito-borne disease: the value of geovisualization using source of infection and LandScan data.

Epidemiological studies use georeferenced health data to identify disease clusters but the accuracy of this georeferencing is obfuscated by incorrectly assigning the source of infection and by aggregating case data to larger geographical areas. Often, place of residence (residence) is used as a proxy for the source of infection (source) which may not be accurate. Using a 21-year dataset from South Australia of human infections with the mosquito-borne Ross River virus, we found that 37% of cases were believed to have been acquired away from home. We constructed two risk maps using age-standardized morbidity ratios (SMRs) calculated using residence and patient-reported source. Both maps confirm significant inter-suburb variation in SMRs. Areas frequently named as the source (but not residence) and the highest-risk suburbs both tend to be tourist locations with vector mosquito habitat, and camping or outdoor recreational opportunities. We suggest the highest-risk suburbs as places to focus on for disease control measures. We also use a novel application of ambient population data (LandScan) to improve the interpretation of these risk maps and propose how this approach can aid in implementing disease abatement measures on a smaller scale than for which disease data are available.

Virus-specific thermostability and heat inactivation profiles of alphaviruses.

Serological diagnosis is a critical component for disease surveillance and is important to address the increase in incidence and disease burden of alphaviruses, such as the chikungunya (CHIKV) and Ross River (RRV) viruses. The gold standard for serological diagnosis is the plaque reduction neutralization test (PRNT), which demonstrates the neutralizing capacity of serum samples after the removal of complement activity and adventitious viruses. This procedure is normally performed following inactivation of the virus at 56°C for 30min. Although this protocol has been widely accepted for the inactivation of envelope RNA viruses, recent studies have demonstrated that prolonged heat inactivation is required to completely inactivate two alphaviruses, Western equine encephalitis virus and CHIKV. Incomplete inactivation of viruses poses a laboratory biosafety risk and can also lead to spurious test results. Despite its importance in ensuring the safety of laboratory personnel as well as test integrity, systematic investigation on the thermostability of alphaviruses has not been performed. In this study, the temperature tolerance and heat inactivation profiles of RRV, Barmah Forest, and o'nyong-nyong viruses were determined. Variations in thermostability were observed within the Semliki forest serocomplex. Therefore, evidence-based heat inactivation procedures for alphaviruses are recommended.

Development of a SYBR green I-based quantitative RT-PCR for Ross River virus: Application in vector competence studies and antiviral drug evaluation.

Ross River virus (RRV) is an emerging Alphavirus and is presently endemic in many parts of Oceania. Keeping in mind its emergence, we developed a molecular detection system and utilized it to study vector competence and evaluate activity of antiviral compounds against RRV. A SYBR Green I-based quantitative RT-PCR for detection of RRV was developed targeting the E2 gene, with a detection limit of 100 RNA copies/reaction. The specificity was confirmed with closely related Alphaviruses and Flaviviruses. The assay was applied to study the vector competence of Indian Aedes aegypti for RRV, which revealed 100% infection and dissemination rate with 75% transmission rate. Viral RNA was found in saliva as early as 3day post infection (dpi). Further application of the assay in antiviral drug evaluation revealed the superior in vitro activity of ribavirin compared to chloroquine in Vero cells. Successful demonstration of this assay to detect RRV in low titre mosquito samples makes it a sensitive tool in vector surveillance. This study also showed that Indian Ae. aegypti are well competent to transmit RRV highlighting the risk of its introduction to naïve territories across continents. Further validation of this assay, revealed its utility in screening of potential antivirals against RRV.