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Viruses are infectious agents that can infect various living organisms, including humans, animals, plants, and microorganisms. Understanding their molecular biology is crucial for developing effective prevention and treatment strategies. Alphaviruses are a group of mosquito-borne viruses that cause diseases such as rash, arthritis, and encephalitis. Barmah Forest virus (BFV) is an alphavirus that poses a public health threat in Australia. However, research on BFV is relatively limited compared to other alphaviruses. In this study, we focus on the expression of BFV replicons in cultured mammalian cells. Replicons are valuable tools for studying viral replication and developing antiviral agents. We review the successful generation of replicons for alphaviruses and Hepatitis C virus and propose a workflow for constructing BFV replicons in cultured mammalian cells.

During 2017–2018, Barmah Forest virus was recovered from mosquitoes trapped in military training areas in Australia and from a soldier infected at 1 of these areas. Phylogenies of the nucleotide sequences of the envelope glycoprotein gene E2 and the 3′ untranslated region suggest that 2 lineages are circulating in eastern Australia.

We report a case of Barmah Forest virus infection in a child from Central Province, Papua New Guinea, who had no previous travel history. Genomic characterization of the virus showed divergent origin compared with viruses previously detected, supporting the hypothesis that the range of Barmah Forest virus extends beyond Australia.

An arbovirus surveillance military exercise was conducted to assess the risk of Ross River virus (RRV) and Barmah Forest virus (BFV) in the Australian Defence Force (ADF) Wide Bay training area (WBTA), northeastern Australia, in April 2018. Of the 5,540 female mosquitoes collected, 3,702 were screened for RRV and BFV by quantitative reverse transcription–polymerase chain reaction in a field laboratory. One pool of Verrallina funerea was positive for RRV and 8 pools (7 pools of Aedes vigilax and 1 pool of Culex annulirostris) were positive for BFV. Phylogenetic analysis of the complete nucleotide sequence of the E2 protein subgrouped both RRV and BFV with viruses previously isolated from human infections, indicating the potential risk of RRV and BFV infection to ADF personnel while training in WBTA. This is the 1st time that both RRV and BFV have been detected in a military training area.

Barmah Forest virus (BFV) is an alphavirus that is mostly spread in Australia and it causes disease in humans upon infection that may manifest as arthritis, myalgia, fever and rash. For this reason, BFV is a cause for concern in puclic health and often a subject in alphavirus related research. In the current work, we observed that BFV replicase activates the type I IFN response in the absence of a viral template, and that mutations in viral replicase proteins may boost IFN production. Furthermore, we looked into the effect that these mutations may possess on the replication and transcription of the BFV replicase using the trans-complementation system. These experiments aid in understanding how alphaviruses influence host cells and vice versa, which could provide useful information in the research for antiviral agents.

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.

Arboviruses (ARthropods BOrne VIRUSES) are disease-causing viruses transmitted through the bite of hematophagous arthropods, such as mosquitoes and ticks. Among these, the alphavirus, genus of the Togaviridae family, is considered the most arthritogenic species, responsible for diseases such as chikungunya fever (CHIK), O'nyong-nyong virus fever, Ross River virus disease, Barmah Forest virus disease, Sindbis virus disease, and Mayaro fever. These arboviral diseases, especially CHIK, have impacted public health in recent decades, leading to devastating epidemics, particularly in developing countries, due to their high potential for chronicity, functional impairment, and great impact on the quality of life. In a similar way, chronic musculoskeletal symptoms have been described in all alphavirus infections. However, CHIK is the best studied. The purpose of this article is to review physiopathology, clinical manifestations, diagnosis, and treatment of alphaviruses, focusing on CHIK and chronic evolution of musculoskeletal symptoms.

Objective: To investigate the seroprevalence and associated risk factors of alphaviruses (Ross River virus (RRV), Barmah Forest virus (BFV) and Whataroa virus (WHAV)) in northern Queensland horses. Methods: A cross‐sectional study of alphavirus antibodies in horses (n = 287) from 147 properties in northern Queensland from September 2013 to June 2014 was conducted. Owners of sampled horses were interviewed on potential risk factors. Data were analysed for associations using multivariable logistic regression. Results: Antibody titres for RRV were demonstrated in samples from 134 properties (91%; 95% confidence interval (CI) 87–96%); 22 properties (15%) had BFV reactors (95% CI 12–18%) and 2 properties (1.4%) had WHAV reactors (95% CI −0.5–3.2%). The highest seroprevalence of RRV was in the Townsville–Burdekin region (93%; 95% CI 90–96%) followed by the Mackay–Whitsunday (90%; 95% CI 88–98%) and Far North Coast–Tableland (82%; 95% CI 74–90%) regions. No association (P ≤ 0.05) could be shown between any of the viruses and age groups, sexes, annual average temperature, degree of rainfall or proximity to wet environments. An association with reported large numbers of mosquitoes was seen for RRV but not BFV. A significant association between properties in close proximity to poultry and pigs was shown for BFV. Conclusion: RRV is endemic within the horse population of northern Queensland, but horses exhibit few clinical signs and could play a role as amplifying hosts in the tropics. Exposure of horses to BFV is significant in northern Queensland and it should be considered a differential diagnosis for RRV. WHAV warrants further study.

Infections with commonly occurring Australian arthropod-borne arboviruses such as Ross River virus (RRV) and Barmah Forest virus (BFV) are diagnosed routinely by pathology laboratories in Australia. Others, such as Murray Valley encephalitis (MVEV) and Kunjin (KUNV) virus infections may be diagnosed by specialist reference laboratories. Although Alfuy (ALFV), Edge Hill (EHV), Kokobera (KOKV), Sindbis (SINV), and Stratford (STRV) viruses are known to infect humans in Australia, all are considered ‘neglected.’ The aetiologies of approximately half of all cases of undifferentiated febrile illnesses (UFI) in Australia are unknown and it is possible that some of these are caused by the neglected arboviruses. The aims of this study were to determine the seroprevalence of antibodies against several neglected Australian arboviruses among residents of Queensland, north-east Australia, and to ascertain whether any are associated with UFI. One hundred age- and sex-stratified human plasma samples from blood donors in Queensland were tested to determine the prevalence of neutralising antibodies against ALFV, BFV, EHV, KOKV, KUNV, MVEV, RRV, SINV, and STRV. The seroconversion rates for RRV and BFV infections were 1.3 and 0.3% per annum, respectively. The prevalence of antibodies against ALFV was too low to enable estimates of annual infection rates to be determined, but the values obtained for other neglected viruses, EHV (0.1%), KOKV (0.05%), and STRV (0.05%), indicated that the numbers of clinical infections occurring with these agents are likely to be extremely small. This was borne out by the observation that only 5.7% of a panel of 492 acute phase sera from UFI patients contained IgM against any of these arboviruses, as detected by an indirect immunofluorescence assay. While none of these neglected arboviruses appear to be a cause of a significant number of UFIs in Australia at this time, each has the potential to emerge as a significant human pathogen if there are changes to their ecological niches.

Background: Ross River virus (RRV) and Barmah Forest virus (BFV) cause approximately 4000 and 1000 cases, respectively, of rheumatic disease in Australia every year. Confirmation of a diagnosis usually involves testing for virus-specific immunoglobulin (Ig) M and IgG by a National Association of Testing Authorities-accredited pathology facility. Objective: The aim of the article is to provide a logical framework by which clinicians can interpret paired RRV and BFV serology results in environments in which numerical antibody titres are no longer routinely provided. The traditional recommendation to look for an increase in titres is now largely obsolete. Discussion: Paired serology is clinical best practice but needs to be appropriately interpreted given the false positive and negative rates, the large number of asymptomatic infections and the long-term persistence of IgM in some individuals. An inappropriate interpretation risks a misdiagnosis.

Mosquito-borne viruses are well recognized as a global public health burden amongst humans, but the effects on non-human vertebrates is rarely reported. Australia, houses a number of endemic mosquito-borne viruses, such as Ross River virus, Barmah Forest virus, and Murray Valley encephalitis virus. In this review, we synthesize the current state of mosquito-borne viruses impacting non-human vertebrates in Australia, including diseases that could be introduced due to local mosquito distribution. Given the unique island biogeography of Australia and the endemism of vertebrate species (including macropods and monotremes), Australia is highly susceptible to foreign mosquito species becoming established, and mosquito-borne viruses becoming endemic alongside novel reservoirs. For each virus, we summarize the known geographic distribution, mosquito vectors, vertebrate hosts, clinical signs and treatments, and highlight the importance of including non-human vertebrates in the assessment of future disease outbreaks. The mosquito-borne viruses discussed can impact wildlife, livestock, and companion animals, causing significant changes to Australian ecology and economy. The complex nature of mosquito-borne disease, and challenges in assessing the impacts to non-human vertebrate species, makes this an important topic to periodically review.

Effective arbovirus surveillance is essential to ensure the implementation of control strategies, such as mosquito suppression, vaccination, or dissemination of public warnings. Traditional strategies employed for arbovirus surveillance, such as detection of virus or virus-specific antibodies in sentinel animals, or detection of virus in hematophagous arthropods, have limitations as an early-warning system. A system was recently developed that involves collecting mosquitoes in CO2-baited traps, where the insects expectorate virus on sugar-baited nucleic acid preservation cards. The cards are then submitted for virus detection using molecular assays. We report the application of this system for detecting flaviviruses and alphaviruses in wild mosquito populations in northern Australia. This study was the first to employ nonpowered passive box traps (PBTs) that were designed to house cards baited with honey as the sugar source. Overall, 20/144 (13.9%) of PBTs from different weeks contained at least one virus-positive card. West Nile virus Kunjin subtype (WNVKUN), Ross River virus (RRV), and Barmah Forest virus (BFV) were detected, being identified in 13/20, 5/20, and 2/20 of positive PBTs, respectively. Importantly, sentinel chickens deployed to detect flavivirus activity did not seroconvert at two Northern Territory sites where four PBTs yielded WNVKUN. Sufficient WNVKUN and RRV RNA was expectorated onto some of the honey-soaked cards to provide a template for gene sequencing, enhancing the utility of the sugar-bait surveillance system for investigating the ecology, emergence, and movement of arboviruses.

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.

The two most reported mosquito-borne diseases in Queensland, a northern state of Australia, are Ross River virus (RRV) disease and Barmah Forest virus (BFV) disease. Both diseases are endemic in Queensland and have similar clinical symptoms and comparable transmission cycles involving a complex inter-relationship between human hosts, various mosquito vectors, and a range of nonhuman vertebrate hosts, including marsupial mammals that are unique to the Australasian region. Although these viruses are thought to share similar vectors and vertebrate hosts, RRV is four times more prevalent than BFV in Queensland.We performed a retrospective analysis of BFV and RRV human disease notification data collected from 1995 to 2007 in Queensland to ascertain whether there were differences in the incidence patterns of RRV and BFV disease. In particular, we compared the temporal incidence and spatial distribution of both diseases and considered the relationship between their disease dynamics. We also investigated whether a peak in BFV incidence during spring was indicative of the following RRV and BFV transmission season incidence levels.Although there were large differences in the notification rates of the two diseases, they had similar annual temporal patterns, but there were regional variations between the length and magnitude of the transmission seasons. During periods of increased disease activity, however, there was no association between the dynamics of the two diseases.The results from this study suggest that while RRV and BFV share similar mosquito vectors, there are significant differences in the ecology of these viruses that result in different epidemic patterns of disease incidence. Further investigation is required into the ecology of each virus to determine which factors are important in promoting RRV and BFV disease outbreaks.

Between October 2012 and October 2013, unprecedented high numbers of Barmah Forest virus (BFV) disease cases were reported in the Northern Territory (NT). An investigation was launched by the NT Department of Health in cooperation with the Department of Primary Industry and Fisheries and the Department of Land Resource Management to investigate possible causes for this phenomenon. The investigation included virus isolations from mosquitoes collected in Darwin urban areas, BFV antibody testing in peri-urban small mammals and a human BFV disease case series investigation of recent cases. No BFV was isolated from the 4641 mosquitoes tested, none of the mammals tested positive for BFV antibodies, and the high BFV disease case numbers did not correlate with the relatively low mosquito vector numbers trapped in 2012-2013. It was estimated that up to 89% of the 79 human cases investigated did not have an acute arboviral illness and therefore had tested falsely positive. An Alere PanBio BFV immunoglobulin M enzyme-linked immunosorbent assay test kit is generally used to test for BFV, with the BFV disease case definition based on immunoglobulin M positives only. Other jurisdictions in Australia also reported high numbers of BFV disease cases, with the majority of the cases suspected to be false positives. Therefore, current testing methods need to be revised to reflect the true numbers of BFV disease cases occurring in Australia and to provide correct diagnoses for patients.

Host feeding patterns of mosquitoes were assessed through the identification of 865 blood meals collected from Brisbane during 2000-2001. Under natural conditions, mosquito feeding (including that of Culex annulirostris, Aedes vigilax, and Aedes notoscriptus) was primarily on dogs (37.4%), but also on birds (18.4%), horses (16.8%), brushtail possums (13.3%), humans (11.6%), and cats, flying foxes, and macropods, depending on site. From 1997 to 1999, sera (N=1706) were collected from dogs, cats, horses, flying foxes, and brushtail possums in the Brisbane area and were analyzed by microneutralization assay for antibodies to Ross River virus (RRV) and Barmah Forest virus (BFV). For RRV, all vertebrate species tested had been naturally infected, and seroprevalence varied from 10.5% to 25.5%, whereas for BFV, rates varied between 0% and 11.3%. Brushtail possums were often infected in the field, with 17.6% and 10.7% of wild individuals having antibodies to RRV and BFV, respectively. Horses and flying foxes also had a relatively high prevalence of antibodies to RRV. This study, therefore, provides data to indicate that brushtail possums play a role in the urban transmission of RRV in Brisbane and that horses, when they occur, also fill the same role.

Spatially and temporally accurate information about infectious mosquito distribution allows for pre-emptivepublic health interventions that can reduce the burden of mosquito-borne infections on human populations.However, the labile nature of arboviruses, the low prevalence of infection in mosquitoes, the expensive laborcosts for mosquito identification and sorting, and the specialized equipment required for arbovirus testing canobstruct arbovirus surveillance efforts. The recently developed techniques of testing mosquito expectorate usinghoney-baited nucleic acid preservation cards or sugar bait stations allows a sensitive method of testing forinfectious, rather than infected, mosquito vectors. Here we report the results from the first large-scale incorporationof honey-baited cards into an existing mosquito surveillance program. During 4 months of the peakvirus season ( January–April, 2014) for a total of 577 trap nights, we set CO2-baited encephalitis vector survey(EVS) light traps at 88 locations in South Australia. The collection container for the EVS trap was modified toallow for the placement of a honey-baited nucleic acid preservation card (FTATM card) inside. After collection,mosquitoes were maintained in a humid environment and allowed access to the cards for 1 week. Cards werethen analyzed for common endemic Australian arboviruses using a nested RT-PCR. Eighteen virus detections,including 11 Ross River virus, four Barmah Forest virus, and three Stratford virus (not previously reported fromSouth Australia) were obtained. Our findings suggest that adding FTA cards to an existing mosquito surveillanceprogram is a rapid and efficient way of detecting infectious mosquitoes with high spatial resolution. Refereed/Peer-reviewed

Verrallina funerea (Theobald) (Diptera: Culicidae) is a brackish water mosquito species found most commonly in Indonesia, Papua New Guinea, and the northeastern coastal regions of Australia. Aspects of the vector competence of this species for Barmah Forest virus (family Togaviridae, genus Alphavirus, BFV) and Ross River virus (family Togaviridae, genus Alphavirus, RRV), two medically important arboviruses in Australia, were investigated. Laboratory-reared Ve. funerea were moderately susceptible to experimental infection with BFV (median cell culture infectious dose of 10(3.6) per mosquito) and were capable of transmission to suckling mice (52% after a 9-12-d extrinsic incubation period). Maximum salivary gland infections for BFV (65%) and RRV (50%) were observed 8 and 10 d postinfection, respectively. To examine any regional differences in vector competence, field populations (separated by up to 200 km) of Ve. funerea and Aedes vigilax (Skuse) from southeastern Queensland and northern New South Wales were fed BFV and RRV, and subsequent infection rates were compared. For both viruses, no statistically significant variations in body, disseminated, or salivary gland infection rates were found in either Ve. funerea or Ae. vigilax. The results from this study indicate that Ve. funerea may have an important role as an amplification vector during outbreaks of both viruses and that local government authorities should rapidly treat brackish water habitats to control this species during periods of increased disease activity.

SUMMARY A series of five patients presented with eruptions beginning on the face. In each case the facial changes were characterized by urticated erythema with minimal epidermal change. The patients also demonstrated more widespread vesiculopapular, macular or purpuric eruptions. At presentation most patients were asymptomatic; however, several subsequently developed constitutional symptoms. Each of these patients was reactive for Barmah Forest virus immunoglobulin (Ig)M, and on repeat testing four were reactive for Barmah Forest virus IgG.

Shoalwater Bay military training area (SWBTA), 2,713 km2 of land located 50-80 km north of Rockhampton, Queensland, Australia, is used by Australian and allied forces for training purposes. Between March 1998 and February 2000, monthly collections of mosquitoes at 15 sites were conducted using carbon dioxide-baited traps to study the seasonal occurrence of mosquitoes and Ross River virus (RRV) and Barmah Forest virus (BFV) in mosquitoes. A total of 72,616 mosquitoes, comprising 3,897 pools were collected, and 2,428 pools were tested using a reverse transcriptase-polymerase chain reaction. A total of 15 pools of mosquitoes were positive for virus, 10 RRV and five BFV. Blood meals from an additional 763 mosquitoes were tested by a gel diffusion assay, and the majority (96%) of those identified were from kangaroo, which was the most common mammal in the study area. The results indicate that Culex annulirostris Skuse and Ochlerotatus vigilax (Skuse) are the main vectors of RRV at SWBTA.

Ross River virus and Barmah Forest virus are Australian arboviruses of the Alphavirus genus. Features of alphavirus infection include an increased permeability of cells to monovalent cations followed by virion budding. Virally encoded ion channels are thought to have a role in these processes. In this paper, the 6K proteins of Ross River virus and Barmah Forest virus are shown to form cation-selective ion channels in planar lipid bilayers. Using a novel purification method, bacterially expressed 6K proteins were inserted into bilayers with a defined orientation (i.e. N-terminal cis, C-terminal trans). Channel activity was reversibly inhibited by antibodies to the N and C termini of 6K protein added to the cis and trans baths, respectively. Channel conductances varied from 40-800 picosiemens, suggesting that the protein is able to form channels with a range of possible oligomerization states.

Objectives T-705, also known as favipiravir, is a small-molecule inhibitor that is currently in clinical development for the treatment of influenza virus infections. This molecule also inhibits the replication of a broad spectrum of other RNA viruses. The objective of this study was to investigate the antiviral effect of favipiravir on chikungunya virus (CHIKV) replication and to contribute to unravelling the molecular mechanism of action against this virus. Methods The anti-CHIKV effect of favipiravir was examined in cell culture and in a mouse model of lethal infection. A five-step protocol was used to select for CHIKV variants with reduced susceptibility to favipiravir. The resistant phenotype was confirmed in cell culture and the whole genome was sequenced. The identified mutations were reverse-engineered into an infectious clone to confirm their impact on the antiviral efficacy of favipiravir. Results Favipiravir inhibits the replication of laboratory strains and clinical isolates of CHIKV, as well as of a panel of other alphaviruses. Several favipiravir-resistant CHIKV variants were independently selected and all of them in particular acquired the unique K291R mutation in the RNA-dependent RNA polymerase (RdRp). Reverse-engineering of this K291R mutation into an infectious clone of CHIKV confirmed the link between the mutant genotype and the resistant phenotype. Interestingly, this particular lysine is also highly conserved in the RdRp of positive-stranded RNA viruses in general. Conclusions This study provides an important insight into the precise molecular mechanism by which favipiravir exerts its antiviral activity against (alpha)viruses, which may be of help in designing other potent broad-spectrum antivirals.

Alphaviruses including Barmah Forest virus (BFV) and Ross River virus (RRV) cause arthritis, arthralgia and myalgia in humans. The rheumatic symptoms in human BFV infection are very similar to those of RRV. Although RRV disease has been studied extensively, little is known about the pathogenesis of BFV infection. We sought to establish a mouse model for BFV to facilitate our understanding of BFV infectivity, tropism and pathogenesis, and to identify key pathological and immunological mechanisms of BFV infection that may distinguish between infections with BFV and RRV. Here, to the best of our knowledge, we report the first study assessing the virulence and replication of several BFV isolates in a mouse model. We infected newborn Swiss outbred mice with BFV and established that the BFV2193 prototype was the most virulent strain. BFV2193 infection resulted in the highest mortality among all BFV variant isolates, comparable to that of RRV. In comparison with RRV, C57BL/6 mice infected with BFV showed delayed onset, moderate disease scores and early recovery of the disease. BFV replicated poorly in muscle and did not cause the severe myositis seen in RRV-infected mice. The mRNAs for the inflammatory mediators TNF-α, IL-6, CCL2 and arginase-1 were highly upregulated in RRV- but not BFV-infected muscle. To our knowledge, this is the first report of a mouse model of BFV infection, which we have used to demonstrate differences between BFV and RRV infections and to further understand disease pathogenesis. With an increasing number of BFV cases occurring annually, a better understanding of the disease mechanisms is essential for future therapeutic development.

Evidence of Ross River (RR) virus infection in field-collected mosquitoes and data from laboratory vector competence experiments incriminated a range of mosquito species as important vectors of RR virus in Maroochy Shire, Queensland, Australia. Nine RR and 2 Barmah Forest virus isolates were recovered from 27,529 mosquitoes collected in light traps from Maroochy Shire during 1996. Nine of the 10 most abundant mosquito species collected in light traps were fed on blood containing the B94/20 strain RR isolated from Queensland in 1994. All species except for Culex sitiens Wiedemann were susceptible to experimental infection. Evidence of RR virus transmission to mice was found with Aedes vigilax (Skuse), Aedes funereus (Theobald), Aedes procax (Skuse), Culex annulirostris Skuse, Mansonia uniformis (Theobald) and Culex australicus Dobrotworsky & Drummond. Aedes notoscriptus (Skuse) and Aedes multiplex (Theobald) were susceptible to RR virus infection, although there was no evidence of virus transmission. Based on adult abundance and vector competence results, freshwater species such as Cx. annulirostris, Ae. procax, and Ae. funereus, and saltmarsh Ae. vigilax, appear to be important vectors of RR virus in Maroochy Shire and control programs should be revised to include these species.

In 1995, the largest recorded outbreak of human disease resulting from infection with the mosquito transmitted alphavirus Barmah Forest (BF) virus occurred along the south coast of New South Wales, Australia. The virus was first isolated in early January from mosquitoes collected at Batemans Bay and predisposed the recognition of 135 human clinical cases. The cases of BF virus were identified initially from Batemans Bay during late January, and the majority (30%) of all cases came from this town. After 5 wk, all major centers on the south coast had clinical patients. Aedes vigilax (Skuse) were especially abundant at Batemans Bay, with levels up to 8 times greater than normal. This species yielded 111 isolates and appeared to be the major vector of BF virus. Attempts to examine if BF virus was maintained in the field by vertical transmission within Ae. vigilax populations were unsuccessful; no evidence of vertical transmission with BF virus, nor any other arbovirus, was found in >17,000 adults emerging from field-collected larvae from the region following peak virus activity. In addition to BF virus, other viruses were recovered from field-collected adult mosquitoes, including Ross River (10 isolates), Edge Hill (21), and Stratford (10). Ae. vigilax again yielded the majority of these viral isolates. The BF virus outbreak appeared to be associated with several factors. A lack of recent BF virus activity in the region provided a highly susceptible human population, and unusual weather conditions of above average rainfall coupled with high tides resulted in extraordinarily large populations of Ae. vigilax.

Aedes notoscriptus (Skuse) mosquitoes colonized from Brisbane, Queensland, Australia, were fed on blood suspensions containing either Barmah Forest (BF) virus, dengue virus type 1 (DEN-1), dengue virus type 2 (DEN-2), dengue virus type 3 (DEN-3), or dengue virus type 4 (DEN-4). Ae. notoscriptus was shown to be moderately susceptible to oral infection with BF virus, ID50 = 10(3.3) CCID50 per mosquito, with titers in infected mosquitoes peaking 10 d after infection. BF virus transmission occurred between days 5 and 12, with an average rate of 45% (range, 40-50%). For DEN 1-4, Ae. notoscriptus showed low level susceptibility to oral infection, with maximum head and thorax squash infection rates of 8, 5, 13, and 3%, respectively. Rates of infection were notably higher in a north Queensland strain of Aedes aegypti (L.) used as a positive control, with maximum thorax squash infection rates of 58, 68, 78, and 60% for DEN 1-4, respectively. On the basis of these data, it is concluded that Ae. notoscriptus could act as an efficient vector of BF in urban environments, but it is unlikely to be an important vector of DEN.

Barmah Forest virus (BFV) and Ross River virus (RRV) are mosquito-borne viruses with similar vectors and environmental requirements. They cause diseases characterised by arthralgia, arthritis and myalgia, often accompanied by fever and rash. Arthritis is more common and more prominent in RRV disease and rash is more common and florid with BFV infection, although the diseases cannot be reliably distinguished by their clinical symptoms. Diagnosis is based on serological tests and a definite diagnosis of recent infection requires the demonstration of rising titres of IgG. Arthralgia, myalgia and lethargy may continue for at least six months in up to half of patients with RRV, but in only about 10% of patients with BFV. Both diseases are managed symptomatically.

Barmah Forest virus (BFV) is a mosquito borne (+) ssRNA alphavirus found only in Australia. It causes rash, myalgia and arthralgia in humans and is usually diagnosed serologically. We developed a real-time PCR assay to detect BFV in an effort to improve diagnosis early in the course of infection. The limit of detection was 16 genome equivalents with a specificity of 100%. Fifty five serum samples from BFV-infected patients were tested by the PCR. 52 of 53 antibody-positive samples were PCR negative. Two culture-positive (neutralizing antibody negative) samples were positive on first round PCR, while one sample (IgM and neutralizing antibody strongly positive, IgG negative) was positive on second round PCR, suggesting that viral RNA is detectable and transiently present in early infection. PCR can provide results faster than culture, is capable of high throughput and by sequencing the PCR product strain variants can be characterized.

BackgroundMosquito-borne diseases are climate sensitive and there has been increasing concern over the impact of climate change on future disease risk. This paper projected the potential future risk of Barmah Forest virus (BFV) disease under climate change scenarios in Queensland, Australia.Methods/principal findingsWe obtained data on notified BFV cases, climate (maximum and minimum temperature and rainfall), socio-economic and tidal conditions for current period 2000-2008 for coastal regions in Queensland. Grid-data on future climate projections for 2025, 2050 and 2100 were also obtained. Logistic regression models were built to forecast the otential risk of BFV disease distribution under existing climatic, socio-economic and tidal conditions. The model was applied to estimate the potential geographic distribution of BFV outbreaks under climate change scenarios. The predictive model had good model accuracy, sensitivity and specificity. Maps on potential risk of future BFV disease indicated that disease would vary significantly across coastal regions in Queensland by 2100 due to marked differences in future rainfall and temperature projections.Conclusions/significanceWe conclude that the results of this study demonstrate that the future risk of BFV disease would vary across coastal regions in Queensland. These results may be helpful for public health decision making towards developing effective risk management strategies for BFV disease control and prevention programs in Queensland.

Background Arthropod-borne viruses are important emerging pathogens world-wide. Viruses transmitted by mosquitoes, such as dengue, yellow fever, and Japanese encephalitis viruses, infect hundreds of millions of people and animals each year. Global surveillance of these viruses in mosquito vectors using molecular based assays is critical for prevention and control of the associated diseases. Here, we report an oligonucleotide DNA microarray design, termed ArboChip5.1, for multi-gene detection and identification of mosquito-borne RNA viruses from the genera Flavivirus (family Flaviviridae), Alphavirus (Togaviridae), Orthobunyavirus (Bunyaviridae), and Phlebovirus (Bunyaviridae). Methodology/Principal Findings The assay utilizes targeted PCR amplification of three genes from each virus genus for electrochemical detection on a portable, field-tested microarray platform. Fifty-two viruses propagated in cell-culture were used to evaluate the specificity of the PCR primer sets and the ArboChip5.1 microarray capture probes. The microarray detected all of the tested viruses and differentiated between many closely related viruses such as members of the dengue, Japanese encephalitis, and Semliki Forest virus clades. Laboratory infected mosquitoes were used to simulate field samples and to determine the limits of detection. Additionally, we identified dengue virus type 3, Japanese encephalitis virus, Tembusu virus, Culex flavivirus, and a Quang Binh-like virus from mosquitoes collected in Thailand in 2011 and 2012. Conclusions/Significance We demonstrated that the described assay can be utilized in a comprehensive field surveillance program by the broad-range amplification and specific identification of arboviruses from infected mosquitoes. Furthermore, the microarray platform can be deployed in the field and viral RNA extraction to data analysis can occur in as little as 12 h. The information derived from the ArboChip5.1 microarray can help to establish public health priorities, detect disease outbreaks, and evaluate control programs., Author Summary Approximately half of the world's population is at risk of viral, mosquito-borne illness such as dengue, yellow fever, Japanese encephalitis, Rift Valley fever, and chikungunya. In the past, these viruses have been regarded as pathogens of the tropics; however, they are emerging as global causes of illness. Very few effective drugs and vaccines have been developed for mosquito-borne viral infections and even less are available to people in resource-limited countries. An important aspect of disease prevention is mosquito surveillance to determine geographical range and seasonal prevalence of the associated viruses. However, there are hundreds of viruses transmitted by mosquitoes that are pathogenic to humans and animals. Using a portable microarray, we developed an assay with the ability to detect most of the known medically important viruses transmitted by mosquitoes. This assay was designed for use in conjunction with broad-range screening tools as a cost effective, rapid method to determine the identity of viruses from infected mosquitoes. To our knowledge, this is the most comprehensive assay to date for field surveillance of mosquito-borne viruses.

Objectives To describe the first reported outbreak of Barmah Forest (BF) virus disease in the south-west of Western Australia. Design Case series correlated with results of arbovirus surveillance. All patients with clinically suspected Ross River (RR) virus infection were serologically tested for antibodies to BF and RR viruses. Home address and date of presentation of patients with serologically confirmed recent infection were recorded. Mosquitoes collected from the districts before and during the BF virus outbreak were identified to species level and tested for virus. Results Twenty-two cases of BF disease were reported from the region between August 1992 and March 1994. Most occurred in the Peel region in the spring and early summer of 1993. Eighteen isolates of BF virus were obtained from three different species of mosquito trapped between January and October 1993. Fifteen were from mosquitoes in the Peel region and a single isolate was from the Perth metropolitan area. No isolates were obtained from the region before 1993. RR virus was not isolated from mosquitoes trapped in the region during the BF virus outbreak. Conclusions Most BF infections were acquired in the Peel region during spring and early summer of 1993. Aedes camptorhynchus mosquitoes were probably the main vectors. The lack of isolations from mosquitoes before 1993 suggests that the virus may have only recently been introduced (or reintroduced) to the region. It was transmitted under conditions that were apparently not conducive to transmission of RR virus.

Mosquito-transmitted alphaviruses causing human rheumatic disease are globally distributed and include chikungunya virus, Ross River virus, Barmah Forest virus, Sindbis virus, o'nyong-nyong virus and Mayaro virus. These viruses cause endemic disease and, occasionally, large epidemics; for instance, the 2004-2011 chikungunya epidemic resulted in 1.4-6.5 million cases, with imported cases reported in nearly 40 countries. The disease is usually self-limiting and characterized by acute and chronic symmetrical peripheral polyarthralgia-polyarthritis, with acute disease usually including fever, myalgia and/or rash. Arthropathy can be debilitating, usually lasts weeks to months and can be protracted; although adequate attention to differential diagnoses is recommended. The latest chikungunya virus epidemic was also associated with some severe disease manifestations and mortality, primarily in elderly patients with comorbidities and the young. Chronic alphaviral rheumatic disease probably arises from inflammatory responses stimulated by the virus persisting in joint tissues, despite robust antiviral immune responses. Serodiagnosis by ELISA is the standard; although international standardization is often lacking. Treatment usually involves simple analgesics and/or NSAIDs, which can provide relief, but better drug treatments are clearly needed. However, the small market size and/or the unpredictable and rapid nature of epidemics present major hurdles for development and deployment of new alphavirus-specific interventions.

Barmah Forest virus (BFV) disease is the second most common mosquito-borne disease in Australia, but the linkages of the wetlands and climate zones with BFV transmission remain unclear. We aimed to examine the relationship between the wetlands, climate zones and BFV risk in Queensland, Australia. Data on the wetlands, climate zones, population and BFV cases for the period 1992 to 2008 were obtained from relevant government agencies. BFV risk was grouped as low-, medium- and high-level based on BFV incidence percentiles. The buffer zones around each BFV case were made using 1, 5, 10, 15, 20, 25 and 50 km distances. We performed a discriminant analysis to determine the differences between wetland classes and BFV risk within each climate zone. The discriminant analyses show that saline 1, riverine and saline tidal influence were the most significant contributors to BFV risk in all climate and buffer zones, while lacustrine, palustrine, estuarine and saline 2 and saline 3 wetlands were less important. These models had classification accuracies of 76%, 98% and 100% for BFV risk in subtropical, tropical and temperate climate zones, respectively. This study demonstrates that BFV risk varies with wetland class and climate zone. The discriminant analysis is a useful tool to quantify the links between wetlands, climate zones and BFV risk.