Cheryl A. Johansen

Identification of australian arboviruses in inoculated cell cultures using monoclonal antibodies in ELISA

Summary An ELISA using a panel of specific monoclonal antibodies was developed to identify all alpha and flaviviruses isolated from mosquitoes caught throughout Australia. This technique is sensitive and rapid and is more specific than the traditional methods used to identify flaviviruses. The ability to identify unknown virus isolates from field‐caught mosquitoes quickly and accurately improves the efficiency of arbovirus surveillance programs and allows health authorities to give an early warning of an increased health risk from a mosquito‐borne virus in a particular region.Abbreviations: BF, Barmah Forest; CPE, cytopathic effect; Mab, monoclonal antibody; MVE, Murray Valley encephalitis; RR, Ross River.

Exploiting mosquito sugar feeding to detect mosquito-borne pathogens

Arthropod-borne viruses (arboviruses) represent a global public health problem, with dengue viruses causing millions of infections annually, while emerging arboviruses, such as West Nile, Japanese encephalitis, and chikungunya viruses have dramatically expanded their geographical ranges. Surveillance of arboviruses provides vital data regarding their prevalence and distribution that may be utilized for biosecurity measures and the implementation of disease control strategies. However, current surveillance methods that involve detection of virus in mosquito populations or sero-conversion in vertebrate hosts are laborious, expensive, and logistically problematic. We report a unique arbovirus surveillance system to detect arboviruses that exploits the process whereby mosquitoes expectorate virus in their saliva during sugar feeding. In this system, infected mosquitoes captured by CO2-baited updraft box traps are allowed to feed on honey-soaked nucleic acid preservation cards within the trap. The cards are then analyzed for expectorated virus using real-time reverse transcription-PCR. In field trials, this system detected the presence of Ross River and Barmah Forest viruses in multiple traps deployed at two locations in Australia. Viral RNA was preserved for at least seven days on the cards, allowing for long-term placement of traps and continuous collection of data documenting virus presence in mosquito populations. Furthermore no mosquito handling or processing was required and cards were conveniently shipped to the laboratory overnight. The simplicity and efficacy of this approach has the potential to transform current approaches to vector-borne disease surveillance by streamlining the monitoring of pathogens in vector populations.

A curious coincidence: mosquito biodiversity and the limits of the Japanese encephalitis virus in Australasia

BackgroundThe mosquito Culex annulirostris Skuse (Diptera: Culicidae) is the major vector of endemic arboviruses in Australia and is also responsible for the establishment of the Japanese encephalitis virus (JEV) in southern Papua New Guinea (PNG) as well as its incursions into northern Australia. Papua New Guinea and mainland Australia are separated by a small stretch of water, the Torres Strait, and its islands. While there has been regular JEV activity on these islands, JEV has not established on mainland Australia despite an abundance of Cx. annulirostris and porcine amplifying hosts. Despite the public health significance of this mosquito and the fact that its adults show overlapping morphology with close relative Cx. palpalis Taylor, its evolution and genetic structure remain undetermined. We address a hypothesis that there is significant genetic diversity in Cx. annulirostris and that the identification of this diversity will shed light on the paradox that JEV can cycle on an island 70 km from mainland Australia while not establishing in Australia itself.ResultsWe sequenced 538 bp of the mitochondrial DNA cytochrome oxidase I gene from 273 individuals collected from 43 localities in Australia and the southwest Pacific region to describe the phylogeography of Cx. annulirostris and its sister species Cx. palpalis. Maximum Likelihood and Bayesian analyses reveal supporting evidence for multiple divergent lineages that display geographic restriction. Culex palpalis contained three divergent lineages geographically restricted to southern Australia, northern Australia and Papua New Guinea (PNG). Culex annulirostris contained five geographically restricted divergent lineages, with one lineage restricted to the Solomon Islands and two identified mainly within Australia while two other lineages showed distributions in PNG and the Torres Strait Islands with a southern limit at the top of Australias Cape York Peninsula.ConclusionThe existence of divergent mitochondrial lineages within Cx. annulirostris and Cx. palpalis helps explain the difficulty of using adult morphology to identify Cx. annulirostris and its ecological diversity. Notably, the southern limit of the PNG lineages of Cx. annulirostris coincides exactly with the current southern limit of JEV activity in Australasia suggesting that variation in these COI lineages may be the key to why JEV has not yet established yet on mainland Australia.The mosquito Culex annulirostris Skuse (Diptera: Culicidae) is the major vector of endemic arboviruses in Australia and is also responsible for the establishment of the Japanese encephalitis virus (JEV) in southern Papua New Guinea (PNG) as well as its incursions into northern Australia. Papua New Guinea and mainland Australia are separated by a small stretch of water, the Torres Strait, and its islands. While there has been regular JEV activity on these islands, JEV has not established on mainland Australia despite an abundance of Cx. annulirostris and porcine amplifying hosts. Despite the public health significance of this mosquito and the fact that its adults show overlapping morphology with close relative Cx. palpalis Taylor, its evolution and genetic structure remain undetermined. We address a hypothesis that there is significant genetic diversity in Cx. annulirostris and that the identification of this diversity will shed light on the paradox that JEV can cycle on an island 70 km from mainland Australia while not establishing in Australia itself. We sequenced 538 bp of the mitochondrial DNA cytochrome oxidase I gene from 273 individuals collected from 43 localities in Australia and the southwest Pacific region to describe the phylogeography of Cx. annulirostris and its sister species Cx. palpalis. Maximum Likelihood and Bayesian analyses reveal supporting evidence for multiple divergent lineages that display geographic restriction. Culex palpalis contained three divergent lineages geographically restricted to southern Australia, northern Australia and Papua New Guinea (PNG). Culex annulirostris contained five geographically restricted divergent lineages, with one lineage restricted to the Solomon Islands and two identified mainly within Australia while two other lineages showed distributions in PNG and the Torres Strait Islands with a southern limit at the top of Australias Cape York Peninsula. The existence of divergent mitochondrial lineages within Cx. annulirostris and Cx. palpalis helps explain the difficulty of using adult morphology to identify Cx. annulirostris and its ecological diversity. Notably, the southern limit of the PNG lineages of Cx. annulirostris coincides exactly with the current southern limit of JEV activity in Australasia suggesting that variation in these COI lineages may be the key to why JEV has not yet established yet on mainland Australia.

Domestic pigs and Japanese encephalitis virus infection, Australia.

To determine whether relocating domestic pigs, the amplifying host of Japanese encephalitis virus (JEV), decreased the risk for JEV transmission to humans in northern Australia, we collected mosquitoes for virus detection. Detection of JEV in mosquitoes after pig relocation indicates that pig relocation did not eliminate JEV risk.

Evolution of Mosquito-Based Arbovirus Surveillance Systems in Australia

Control of arboviral disease is dependent on the sensitive and timely detection of elevated virus activity or the identification of emergent or exotic viruses. The emergence of Japanese encephalitis virus (JEV) in northern Australia revealed numerous problems with performing arbovirus surveillance in remote locations. A sentinel pig programme detected JEV activity, although there were a number of financial, logistical, diagnostic and ethical limitations. A system was developed which detected viral RNA in mosquitoes collected by solar or propane powered CO2-baited traps. However, this method was hampered by trap-component malfunction, microbial contamination and large mosquito numbers which overwhelmed diagnostic capabilities. A novel approach involves allowing mosquitoes within a box trap to probe a sugar-baited nucleic-acid preservation card that is processed for expectorated arboviruses. In a longitudinal field trial, both Ross River and Barmah Forest viruses were detected numerous times from multiple traps over different weeks. Further refinements, including the development of unpowered traps and use of yeast-generated CO2, could enhance the applicability of this system to remote locations. New diagnostic technology, such as next generation sequencing and biosensors, will increase the capacity for recognizing emergent or exotic viruses, while cloud computing platforms will facilitate rapid dissemination of data.

Emergence of Barmah Forest virus in Western Australia.

To the editor: Barmah Forest (BF) virus is a mosquito-borne alphavirus, found only in Australia, which causes outbreaks of polyarthritis in humans. The disease is very similar to epidemic polyarthritis caused by infection with Ross River virus, another Australian alphavirus. BF virus was first isolated from mosquitoes in the State of Western Australia in 1989. After this, small clusters of human cases were diagnosed in the arid northern and central regions of Western Australia in 1992, and the first substantial outbreak of human disease due to infection with BF virus (BF virus disease) occurred in the southwestern region of the state during the spring and summer (September-March) of 1993-94 (2). No evidence of BF virus activity had been found in these regions before these events, which suggests that the virus had only recently been introduced to Western Australia. This report describes the timing and distribution of BF virus disease in humans and the isolation of the virus from mosquitoes in Western Australia, which corroborate the view that BF virus is an emerging virus in this state. The ecology of Australian arboviruses that cause human disease, including BF virus, has recently been reviewed (3). BF virus was first isolated from Culex annulirostris mosquitoes collected at the Barmah Forest in northern Victoria (southeastern Australia) in 1974 (4). It was first shown to infect humans in New South Wales (central-eastern and southeastern Australia) in 1986 (5) and was reported as a cause of clinical disease in humans in 1988 (6). The most common clinical features include polyarthritis, arthralgia, myalgia, fever, rash, and lethargy (7); in some cases, symptoms may persist for more than 6 months (2). Although the symptoms are similar to those caused by infection with Ross River virus, there is little cross-reaction between the two viruses in serologic tests (8), and differentiating between infections caused by either is generally not difficult. The first true outbreak of BF virus disease occurred concurrently with an outbreak of Ross River virus infection at Nhulunbuy in the Northern Territory in early 1992 (9). The principal vectors of BF are believed to be mosquitoes, and although the vertebrate hosts of BF virus are not known, serologic surveys in eastern Australia have suggested that marsupials are involved in the natural cycle. BF virus was first detected in Western Australia in 1989. Since then, 73 isolates of the virus have been obtained from mosquitoes trapped in several different regions of Western Australia (Table 1). The first human cases of BF virus disease in Western Australia were reported in 1992, and 67 serologically confirmed cases have now been diagnosed. The locations of towns where human cases have occurred or where mosquitoes that yielded BF virus were collected are shown in Figure 1. Eight isolates of the virus were obtained from five different mosquito species (Table 1) collected at Billiluna, a small, remote aboriginal community in an arid area in the southeastern Kimberley region in April 1989 (10). The infected mosquitoes were collected 3 weeks after heavy local rains. Only moderate wet season rains were recorded in the remainder of the Kimberley region, and no cases of BF virus disease were reported from any region in Western Australia that year. There have been no subsequent isolations of BF virus from mosquitoes collected at Billiluna, despite annual collections in the region. No human cases have been reported from Billiluna. The first cases of BF virus disease in Western Australian were reported almost 3 years later, either individually or in small clusters from towns in the arid East Kimberley, Pilbara, Gascoyne, Murchison and Southeast (Goldfields) regions between April and September (Autumn-Spring) 1992. Most activity was reported from the towns of Exmouth (six cases) and Carnarvon (four cases). All of these cases occurred during or just after much larger outbreaks of disease caused by Ross River virus. This suggested that BF and Ross River viruses may have similar mosquito vectors and require similar environmental conditions for successful transmission. The main environmental factor contributing to the 1992 outbreaks of Ross River virus disease was extremely heavy rain in these normally arid regions during autumn and winter (11). BF virus was isolated from five species of mosquito in the Fortescue region of the Pilbara and from three species in the West Gascoyne, just prior to, and during, these arid-region outbreaks. In coastal regions of the Pilbara, the main vector of BF virus appears to be Aedes vigilax, a salt marsh-breeding species. Large numbers of this species develop after very high tides or heavy rains on salt marshes. It is also the main vector of Ross River virus in these regions (12). Several other temporary freshwater ground pool-breeding species in the subgenus Ochlerotatus, particularly Ae. eidsvoldensis and Ae. EN Marks’ species #85, were found to be infected with the virus in inland areas or coastal areas where such pools develop. These preliminary investiga1 This report is adapted from and expands on a previous bulletin. (1) Dispatches

Field Evaluation of a Sentinel Mosquito (Diptera: Culicidae) Trap System to Detect Japanese Encephalitis in Remote Australia

Abstract Incursions of Japanese encephalitis (JE) virus into northern Queensland are currently monitored using sentinel pigs. However, the maintenance of these pigs is expensive, and because pigs are the major amplifying hosts of the virus, they may contribute to JE transmission. Therefore, we evaluated a mosquito-based detection system to potentially replace the sentinel pigs. Single, inactivated JE-infected Culex annulirostris Skuse and C. sitiens Wiedemann were placed into pools of uninfected mosquitoes that were housed in a MosquitoMagnet Pro (MM) trap set under wet season field conditions in Cairns, Queensland for 0, 7, or 14 d. JE viral RNA was detected (cycling threshold [CT] = 40) in 11/12, 10/14, and 2/5 pools containing 200, 1,000, and 5,000 mosquitoes, respectively, using a TaqMan real-time reverse transcription-polymerase chain reaction (RT-PCR). The ability to detect virus was not affected by the length of time pools were maintained under field conditions, although the CT score tended to increase with field exposure time. Furthermore, JE viral RNA was detected in three pools of 1,000 mosquitoes collected from Badu Island using a MM trap. These results indicated that a mosquito trap system employing self-powered traps, such as the MosquitoMagnet, and a real-time PCR system, could be used to monitor for JE in remote areas.

A novel insect-specific flavivirus replicates only in Aedes-derived cells and persists at high prevalence in wild Aedes vigilax populations in Sydney, Australia

To date, insect-specific flaviviruses (ISFs) have only been isolated from mosquitoes and increasing evidence suggests that ISFs may affect the transmission of pathogenic flaviviruses. To investigate the diversity and prevalence of ISFs in Australian mosquitoes, samples from various regions were screened for flaviviruses by ELISA and RT-PCR. Thirty-eight pools of Aedes vigilax from Sydney in 2007 yielded isolates of a novel flavivirus, named Parramatta River virus (PaRV). Sequencing of the viral RNA genome revealed it was closely related to Hanko virus with 62.3% nucleotide identity over the open reading frame. PaRV failed to grow in vertebrate cells, with only Aedes-derived mosquito cell lines permissive to replication, suggesting a narrow host range. 2014 collections revealed that PaRV had persisted in A. vigilax populations in Sydney, with 88% of pools positive. Further investigations into its mode of transmission and potential to influence vector competence of A. vigilax for pathogenic viruses are warranted.

Determination of Mosquito (Diptera: Culicidae) Bloodmeal Sources in Western Australia: Implications for Arbovirus Transmission

ABSTRACT A double-antibody enzyme-linked immunosorbent assay was used to determine the bloodmeal sources of adult mosquitoes (Diptera: Culicidae) collected in encephalitis vector surveillance mosquito traps in Western Australia between May 1993 and August 2004. In total, 2,606 blood-fed mosquitoes, representing 29 mosquito species, were tested, and 81.7% reacted with one or more of the primary antibodies. Aedes camptorhynchus (Thomson) and Culex annulirostris Skuse were the most common species tested, making up 47.2% (1,234) and 35.6% (930), respectively. These species obtained bloodmeals from a variety of vertebrate hosts but particularly marsupials and cows. In contrast, Culex pullus Theobald (72.7%; 24/33), Culiseta atra (Lee) (70.0%; 7/10), Culex globocoxitus Dobrotworsky (54.5%; 12/22), and Culex quinquefasciatus Say (39.3%; 22/56) often obtained bloodmeals from birds. Although Ae. camptorhynchus and Cx. annulirostris are well established vectors of arboviruses, other mosquitoes also may have a role in enzootic and/or epizootic transmission.

Application of satellite precipitation data to analyse and model arbovirus activity in the tropics

BackgroundMurray Valley encephalitis virus (MVEV) is a mosquito-borne Flavivirus (Flaviviridae: Flavivirus) which is closely related to Japanese encephalitis virus, West Nile virus and St. Louis encephalitis virus. MVEV is enzootic in northern Australia and Papua New Guinea and epizootic in other parts of Australia. Activity of MVEV in Western Australia (WA) is monitored by detection of seroconversions in flocks of sentinel chickens at selected sample sites throughout WA.Rainfall is a major environmental factor influencing MVEV activity. Utilising data on rainfall and seroconversions, statistical relationships between MVEV occurrence and rainfall can be determined. These relationships can be used to predict MVEV activity which, in turn, provides the general public with important information about disease transmission risk. Since ground measurements of rainfall are sparse and irregularly distributed, especially in north WA where rainfall is spatially and temporally highly variable, alternative data sources such as remote sensing (RS) data represent an attractive alternative to ground measurements. However, a number of competing alternatives are available and careful evaluation is essential to determine the most appropriate product for a given problem.ResultsThe Tropical Rainfall Measurement Mission (TRMM) Multi-satellite Precipitation Analysis (TMPA) 3B42 product was chosen from a range of RS rainfall products to develop rainfall-based predictor variables and build logistic regression models for the prediction of MVEV activity in the Kimberley and Pilbara regions of WA. Two models employing monthly time-lagged rainfall variables showed the strongest discriminatory ability of 0.74 and 0.80 as measured by the Receiver Operating Characteristics area under the curve (ROC AUC).ConclusionsTMPA data provide a state-of-the-art data source for the development of rainfall-based predictive models for Flavivirus activity in tropical WA. Compared to ground measurements these data have the advantage of being collected spatially regularly, irrespective of remoteness. We found that increases in monthly rainfall and monthly number of days above average rainfall increased the risk of MVEV activity in the Pilbara at a time-lag of two months. Increases in monthly rainfall and monthly number of days above average rainfall increased the risk of MVEV activity in the Kimberley at a lag of three months.