A.K. Broom

Arboviruses causing human disease in the Australasian zoogeographic region

SummaryOver 65 arboviruses have been reported from countries in the Australasian zoogeographic region, but only a few have been implicated in human disease. These include the flaviviruses Murray Valley encephalitis (MVE), Kunjin (KUN), Kokobera (KOK), and dengue, particularly types 1 and 2; the alphaviruses Ross River (RR), Barmah Forest (BF), and Sindbis (SIN); and the bunyaviruses, Gan Gan and Trubanaman. In this paper recent epidemiological and clinical results pertaining to these viruses are reviewed, with major emphasis on MVE and RR viruses. The extensive early studies of Australian arboviruses have been reviewed by Doherty [49, 50], and their ecology and vectors more recently by Kay and Standfast [87]. In addition, the biology of MVE and KUN [113] and RR [87, 114] viruses have been the subjects of more detailed reviews.The Australasian zoogeographic region is defined as countries east of the Wallace and Weber lines, two hypothetical lines in the Indo-Australian archipelago where the fauna of the Australasian and Oriental regions meet. Seroepidemiological studies of human arboviral infections have suggested that the Japanese encephalitis flavivirus and the chikungunya alphavirus occur only in the Oriental region, whereas the related MVE and RR viruses, respectively, are restricted to the Australasian region [85, 148]. Serological results from Wallacea, the zone between the Wallace and Weber lines, are not so clear-cut [85]. This review is therefore restricted to countries east of Wallacea, specifically New Guinea and Australia.

The ecology and epidemiology of Kunjin virus.

Kunjin (KUN) virus has long been considered an arbovirus of minor medical and veterinary significance in Australia, with human infections associated with a mild febrile illness and rare reports of encephalitis in both horses and man. However its close relationship to a more virulent Australian arbovirus, Murray Valley encephalitis (MVE) virus, in terms of epidemiology, ecology and cross-reactivity in traditional serological diagnostic assays has necessitated that the activity of both viruses be carefully monitored in surveillance of mosquito-borne viruses in Australia. For a thorough discussion on the history, ecology and epidemiology of MVE and KUN viruses the reader is referred to an extensive review by Marshall (1988).Of more recent relevance, are the outbreaks of a fatal viral encephalitis in Europe, Russia, North America and the Middle East caused by strains of West Nile (WN) virus shown to be genetically closely related to KUN virus (Tsai et al. 1998; Briese et al. 1999; Jia et al. 1999; Lanciotti et al. 1999; Platonov et al. 2001; Hindiyeh et al. 2001). These events have stimulated additional interest in WN virus and its relationship to KUN virus with respect to taxonomy, and clinical and ecological comparisons. The purpose of this report is to review briefly the ecology and epidemiology of KUN virus, and to discuss methods of surveillance, diagnosis and control, with pertinent comparisons to WN and MVE viruses. Brief reference will also be made to the recent changes in the phylogeny and taxonomy of KUN in relation to WN virus; however, this aspect is covered in depth by Scherret et al., in this volume.

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.

Immunisation with gamma globulin to Murray Valley encephalitis virus and with an inactivated Japanese encephalitis virus vaccine as prophylaxis against Australian encephalitis: Evaluation in a mouse model

In northwestern Australia, the flavivirus Murray Valley encephalitis (MVE) poses a significant health risk to infants in some aboriginal communities, particularly during each wet season. While there are too few cases to warrant the development of a vaccine against MVE, a safe, effective prophylaxis for these children is still urgently required. The use of passive transfer of human gamma globulin to MVE or immunisation with a vaccine to the closely related Japanese encephalitis (JE) virus were investigated as potential strategies. When 40 μg of IgG was purified from MVE‐immune human sera and transferred to 3‐week‐old mice, the animals were protected from lethal IP inoculation with MVE virus while still producing a detectable immune response to the virus. Similarly, sera from adult mice infected sublethally with MVE or JE virus provided significant protection against MVE infection. However, sera from mice sublethally infected with the related Kunjin or immunised with the inactivated JE vaccine (Biken) provided no protection against MVE challenge. In fact, mice immunised passively with the latter appeared to succumb to MVE challenge more rapidly than mice that received serum from unimmunised animals, suggesting that antibody to the vaccine had accelerated the progression of disease. These preliminary trials in mice indicate that passive immunisation with human gamma globulin has the greatest potential as a strategy for MVE prophylaxis, whilst the apparent enhancement of MVE by antibodies to the JE vaccine requires further investigation, with particular reference to current vaccination programs in areas of Australia and Papua New Guinea, where both JE and MVE occur. J. Med. Virol. 61:259–265, 2000.

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

Investigation of the southern limits of Murray Valley encephalitis activity in Western Australia during the 2000 wet season.

Western Australia experienced its worst-ever outbreak of the mosquito-borne Murray Valley encephalitis (MVE) virus during the 2000 wet season. Highest-on-record rainfall throughout much of the state during the 2000 wet season gave rise to extensive mosquito breeding and increased MVE virus transmission, resulting in nine cases of encephalitis. Activity of MVE virus in Western Australia is monitored by detecting MVE virus-specific antibodies in serum from sentinel chickens, located at towns and communities throughout the north of the state. However, during 2000, all 28 flocks of chickens seroconverted to MVE virus, including a flock located >600 km further south than MVE virus activity had ever previously been recorded. Furthermore, the majority of the nine cases of encephalitis occurred outside the enzootic Kimberley region. We therefore undertook a major serosurvey of domestic chicken flocks both south and east of the previously defined regions of virus activity. The results suggest that MVE virus activity extended as far south as the Midwest and northern Goldfields during 2000. A new southern limit of activity of MVE virus is therefore proposed. The results have implications for managing outbreaks of MVE virus in Western Australia and have enabled us to locate additional sentinel flocks as part of the MVE surveillance program for future years.

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.

Genetic and phenotypic differences between isolates of Murray Valley encephalitis virus in Western Australia, 1972-2003

Murray Valley encephalitis virus (MVEV) is a medically important mosquito-borne flavivirus found in Australia and Papua New Guinea (PNG). Partial envelope gene nucleotide sequences of 28 isolates of MVEV from Western Australia (WA) between 1972 and 2003 were aligned and compared phylogenetically with the prototype MVE-1-51 from Victoria in 1951 and isolates from northern Queensland and PNG. Monoclonal antibody-binding patterns were also investigated. Results showed that the majority of isolates of MVEV from widely disparate locations in WA were genetically and phenotypically homogeneous. Furthermore, isolates of MVEV from WA and northern Queensland were almost identical, confirming results from earlier studies. Recent isolates of MVEV from Western Province in PNG were more similar to Australian isolates of MVEV than to isolates from PNG in 1956 and 1966, providing further evidence for the movement of flaviviruses between PNG and Australia. Additional representatives of a unique variant of MVEV (OR156) from Kununurra in the northeast Kimberley region of WA were also detected. This suggests that the OR156 lineage is still intermittently active but may be restricted to a small geographic area in northern WA, possibly due to altered biological characteristics.

Molecular Phylogeny of Edge Hill Virus Supports its Position in the Yellow Fever Virus Group and Identifies a New Genetic Variant

Edge Hill virus (EHV) is a mosquito-borne flavivirus isolated throughout Australia during mosquito surveillance programs. While not posing an immediate threat to the human population, EHV is a taxonomically interesting flavivirus since it remains the only member of the yellow fever virus (YFV) sub-group to be detected within Australia. Here we present both an antigenic and genetic investigation of collected isolates, and confirm taxonomic classification of the virus within the YFV-group. Isolates were not clustered based on geographical origin or time of isolation, suggesting that minimal genetic evolution of EHV has occurred over geographic distance or time within the EHV cluster. However, two isolates showed significant differences in antigenic reactivity patterns, and had a much larger divergence from the EHV prototype (19% nucleotide and 6% amino acid divergence), indicating a distinct subtype or variant within the EHV subgroup.

Epitope-Blocking Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Ross River Virus in Vertebrate Sera

ABSTRACT We describe the development of an epitope-blocking enzyme-linked immunosorbent assay (ELISA) for the sensitive and rapid detection of antibodies to Ross River virus (RRV) in human sera and known vertebrate host species. This ELISA provides an alternative method for the serodiagnosis of RRV infections.