Determination of mosquito (Diptera: Culicidae) bloodmeal sources in Western Australia: implications for arbovirus transmission.

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.

Antibody-dependent enhancement of Murray Valley encephalitis virus virulence in mice.

Enhancement of flavivirus infection in vitro in the presence of subneutralizing concentrations of homologous or heterologous antiserum has been well described. However, the importance of this phenomenon in the enhancement of flavivirus infection in vivo has not been established. In order to study antibody-mediated enhancement of flavivirus infection in vivo, we investigated the effect of passive immunization of mice with Japanese encephalitis virus (JE) antiserum on the outcome of infection with Murray Valley encephalitis virus (MVE). We show that prior treatment of mice with subneutralizing concentrations of heterologous JE antiserum resulted in an increase in viraemia titres and in mortality following challenge with wild-type MVE. Our findings support the hypothesis that subneutralizing concentrations of antibody may enhance flavivirus infection and virulence in vivo. These findings are of potential importance for the design of JE vaccination programs in geographic areas in which MVE co-circulates. Should subneutralizing concentrations of antibody remain in the population following JE vaccination, it is possible that enhanced disease may be observed during MVE epidemics.

The relationships between West Nile and Kunjin viruses.

Until recently, West Nile (WN) and Kunjin (KUN) viruses were classified as distinct types in the Flavivirus genus. However, genetic and antigenic studies on isolates of these two viruses indicate that the relationship between them is more complex. To better define this relationship, we performed sequence analyses on 32 isolates of KUN virus and 28 isolates of WN virus from different geographic areas, including a WN isolate from the recent outbreak in New York. Sequence comparisons showed that the KUN virus isolates from Australia were tightly grouped but that the WN virus isolates exhibited substantial divergence and could be differentiated into four distinct groups. KUN virus isolates from Australia were antigenically homologous and distinct from the WN isolates and a Malaysian KUN virus. Our results suggest that KUN and WN viruses comprise a group of closely related viruses that can be differentiated into subgroups on the basis of genetic and antigenic analyses.

Murray Valley encephalitis virus surveillance and control initiatives in Australia. National Arbovirus Advisory Committee of the Communicable Diseases Network Australia.

Mechanisms for monitoring Murray Valley encephalitis (MVE) virus activity include surveillance of human cases, surveillance for activity in sentinel animals, monitoring of mosquito vectors and monitoring of weather conditions. The monitoring of human cases is only one possible trigger for public health action and the additional surveillance systems are used in concert to signal the risk of human disease, often before the appearance of human cases. Mosquito vector surveillance includes mosquito trapping for speciation and enumeration of mosquitoes to monitor population sizes and relative composition. Virus isolation from mosquitoes can also be undertaken. Monitoring of weather conditions and vector surveillance determines whether there is a potential for MVE activity to occur. Virus isolation from trapped mosquitoes is necessary to define whether MVE is actually present, but is difficult to deliver in a timely fashion in some jurisdictions. Monitoring of sentinel animals indicates whether MVE transmission to vertebrates is actually occurring. Meteorological surveillance can assist in the prediction of potential MVE virus activity by signalling conditions that have been associated with outbreaks of Murray Valley encephalitis in humans in the past. Predictive models of MVE virus activity for south-eastern Australia have been developed, but due to the infrequency of outbreaks, are yet to be demonstrated as useful for the forecasting of major outbreaks. Surveillance mechanisms vary across the jurisdictions. Surveillance of human disease occurs in all States and Territories by reporting of cases to health authorities. Sentinel flocks of chickens are maintained in 4 jurisdictions (Western Australia, the Northern Territory, Victoria and New South Wales) with collaborations between Western Australia and the Northern Territory. Mosquito monitoring complements the surveillance of sentinel animals in these jurisdictions. In addition, other mosquito monitoring programs exist in other States (including South Australia and Queensland). Public health control measures may include advice to the general public and mosquito management programs to reduce the numbers of both mosquito larvae and adult vectors. Strategic plans for public health action in the event of MVE virus activity are currently developed or being developed in New South Wales, the Northern Territory, South Australia, Western Australia and Victoria. A southern tri-State agreement exists between health departments of New South Wales, Victoria and South Australia and the Commonwealth Department of Health and Aged Care. All partners have agreed to co-operate and provide assistance in predicting and combatting outbreaks of mosquito-borne disease in south-eastern Australia. The newly formed National Arbovirus Advisory Committee is a working party providing advice to the Communicable Diseases Network Australia on arbovirus surveillance and control. Recommendations for further enhancement of national surveillance for Murray Valley encephalitis are described.

The molecular epidemiology of Kokobera virus.

We describe herein the molecular epidemiology and phylogeny of Kokobera (KOK) virus, a flavivirus found in Australia and Papua New Guinea. We sequenced a region encompassing the 200 nucleotides of the 3' terminus of the NS5 gene, and the first 300 nucleotides of the 3' untranslated region (UTR). The study included 25 isolates of the virus, including an isolate from PNG, and several recent isolates from the south-west of Western Australia (WA), where the virus had not previously been detected. We found that the KOK isolates clustered according to geographic location and time of isolation into three distinct topotypes: one covering Queensland and New South Wales; another represented by the single isolate from PNG; and a third covering the Northern Territory and WA. This latter group was further subdivided into northern and south-west isolates. This molecular epidemiology is significantly different from other Australian flaviviruses, such as Murray Valley encephalitis (MVE) and Kunjin (KUN) viruses, which exist as single genetic types across the entire Australian continent. However, it is similar to the molecular epidemiology of the alphavirus Ross River (RR) virus. This may be explained by the fact that MVE and KUN viruses are known to have birds as their main vertebrate hosts, whereas RR virus utilises macropods, which have also been implicated as the vertebrate host for KOK virus. In addition, the south-west isolates exhibited a degree of sequence heterogeneity, including one isolate that has a nine nucleotide deletion in the 3'UTR. This suggests that KOK virus has been in the south-west of WA for some time, and was not recently introduced.

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 microg 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.

Murray Valley encephalitis in Western Australia in 2000, with evidence of southerly spread.

We describe the epidemiological and clinical features of human Murray Valley encephalitis (MVE) and Kunjin (KUN) virus infections in Western Australia (WA) during March to July 2000. A case series was performed. For laboratory-confirmed cases, travel histories and clinical details were collected from patients, family members, friends or treating physicians. Surveillance data from the sentinel chicken program and climatic conditions were reviewed. Nine encephalitic cases of MVE were recorded. Eight were non-Aboriginal adults (age range, 25 to 79 years; 5 male, 3 female) and 1 was an Aboriginal boy. Four cases acquired infection in the Murchison and Midwest regions of WA from which no human cases of MVE have been reported previously. One of the 9 cases was fatal and 3 had severe neurological sequelae. Five non-encephalitic infections were also recorded, 3 MVE and 2 KUN. Encephalitis caused by MVE virus remains a serious problem with no improvement in clinical outcomes in the last 25 years. Excessive rainfall with widespread flooding in the northern two-thirds of WA provided ideal conditions for mosquito breeding and favoured southerly spread of the virus into new and more heavily populated areas. Surveillance in WA with sentinel chickens and mosquito trapping needs expansion to define the boundaries of MVE virus activity. To enable timely warnings to the public, and to institute mosquito control where feasible, continued surveillance in all Australian areas at risk is indicated.

Identification of Australian arboviruses in inoculated cell cultures using monoclonal antibodies in ELISA.

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.

Arboviruses in the Australian region, 1990 to 1998.

Arboviruses continue to be major human pathogens in the Australian region. This report provides a summary of the activities of these viruses over the past eight years, and comments on new findings relevant to their respective ecologies. Of particular interest and concern is the propensity of these viruses to spread. The examples discussed include the initiation of dengue epidemics in north Queensland by virus imported in viraemic travellers; the spread of Japanese encephalitis virus to the Australasian region and its probable enzootic establishment in the south-west of Papua New Guinea; the potential spread of Ross River virus to other countries, as demonstrated by the 1979-80 outbreak in the South Pacific, and the recent occurrence in military personnel from the United States of America after an exercise; and the recent spread of Barmah Forest virus into Western Australia.

Communicable diseases surveillance

Communicable diseases surveillance highlights (vaccine preventable diseases, arboviruses, respiratory viruses), and surveillance outcomes from several reporting series as follows: National Notifiable Diseases Surveillance System tables, 14 October to 10 November 1998; Laboratory Serology and Virology Reporting Scheme tables, 8 October to 4 November 1998; Australian Sentinel Practice Research Network report tables, weeks 40 to 43, 1999; Gonococcal surveillance, reporting period 1 April to 30 June 1998; Sentinel chicken surveillance program, reporting period September 1998; HIV and AIDS surveillance, reporting period 1 to 30 June 1998, assessed as at 30 September 1998; Australian childhood immunisation coverage, 1 April to 30 June 1998 cohort, assessed as at 30 June 1998.

Genetic stability among temporally and geographically diverse isolates of Barmah Forest virus.

An increase in the incidence of polyarthritis caused by Barmah Forest (BF) virus, and its recent emergence into Western Australia, prompted a study of the molecular epidemiology of this Australian mosquito-borne alphavirus. The nucleotide sequence of a 500-basepair region of the 3' end of the envelope (E2) gene of the prototype BF virus strain (BH2193) was compared with other members of the alphavirus genus, and to a panel of isolates of BF virus collected more for than 20 years from different geographic regions of Australia. The BF virus was shown to be genetically distinct from other members of the alphavirus genus. A high degree of sequence homology (98-100%) was found between the BF isolates, with no evidence of geographic or temporal divergence. This nucleotide homogeneity was similar to that observed with other Australian mosquito-borne viruses with avian vertebrate hosts, such as Sindbis, Murray Valley, and Kunjin viruses, but it contrasts to the heterogeneity reported for Ross River virus, an alphavirus with mammalian vertebrate hosts.

Australian X disease, Murray Valley encephalitis and the French connection.

Epidemics of a severe encephalitis occurred in eastern Australia between 1917 and 1925, in which over 280 cases were reported with a fatality rate of 68%. The disease had not been described previously and was called Australian X disease. The next epidemic occurred in south-east Australia in the summer of 1950-51. The disease was given its name of Murray Valley encephalitis as this was the area from which most cases were reported. A virus was isolated by Eric French in Victoria, and about the same time by John Miles and colleagues in South Australia. The virus Murray Valley encephalitis (MVE) virus, was shown to be a Group B arbovirus (flavivirus) which was related to, but distinct from, Japanese encephalitis virus. Early seroepidemiological studies showed that the most likely vertebrate hosts were water birds. MVE virus was first isolated from Culex annulirostris mosquitoes in 1960. The most recent epidemic of Murray Valley encephalitis occurred in 1974, at which time it was renamed Australian encephalitis. Since 1974, however, all cases have been confined to northern Australia, particularly the north of Western Australia. Indeed, the Kimberley region of Western Australia contains the only confirmed enzootic foci of virus activity. A closely related flavivirus, Kunjin virus, has also been shown to be an aetiological agent of Australian encephalitis. Since the first isolation of MVE and Kunjin viruses, considerable information has been accumulated on their ecology and epidemiology, some aspects of which are briefly described.

Specific enzyme immunoassays for the rapid detection of Ross River virus in cell cultures inoculated with infected mosquito homogenates.

BACKGROUND: Ross River (RR) virus is a mosquito-borne alphavirus and one of the aetiological agents of epidemic polyarthritis in humans. Early detection of increased virus activity in mosquito populations enables public health authorities to implement measures to reduce the number of human infections during epidemics. However, current surveillance techniques require a minimum of four weeks for viruses to be isolated and identified. OBJECTIVES: This study was carried out to assess the use of enzyme immunoassays (EIA) as rapid alternatives to traditional cell culture techniques for detection of RR virus in mosquitoes. STUDY DESIGN: Enzyme immunoassays and immunoperoxidase assays were developed using RR-specific monoclonal antibodies and compared to traditional methods for detection of RR virus in field-caught mosquito samples. RESULTS: By inoculation of C6/36 cell cultures with mosquito homogenates and testing monolayers and culture supernatant by EIA, RR virus was detected and identified in all infected samples within 6 days. CONCLUSIONS: The use of EIA provides a rapid, sensitive and specific alternative to traditional methods for the detection of RR virus in mosquito vectors.

Two possible mechanisms for survival and initiation of Murray Valley encephalitis virus activity in the Kimberley region of Western Australia.

Two possible mechanisms are described for the initiation of Murray Valley encephalitis (MVE) virus activity in arid, epizootic regions of tropical Australia. Virus isolations were made from mosquitoes trapped shortly after the first heavy wet season rains and flooding in the east Kimberley, which followed approximately nine months of drought. A number of isolates of MVE virus were obtained, including isolates from pools of blood-engorged Culex annulirostris mosquitoes and from a single pool of male Aedes tremulus mosquitoes. The results strongly suggested that MVE virus activity was due both to its introduction in viremic vertebrate hosts, from which first-generation mosquitoes became infected following blood meals, and also to reactivation of vertically transmitted virus from desiccation-resistant eggs of Ae. tremulus. Both mechanisms are discussed with respect to environmental conditions.

Immunodominant epitopes on the NS1 protein of MVE and KUN viruses serve as targets for a blocking ELISA to detect virus-specific antibodies in sentinel animal serum.

Two mosquito-borne flaviviruses, Murray Valley encephalitis (MVE) and Kunjin (KUN), are the aetiological agents of Australian encephalitis. MVE causes a severe and potentially fatal form of the disease while KUN is responsible for only a few relatively mild cases. Therefore it is important that serological tests used in flavivirus surveillance differentiate between infections with these two viruses. However, this has been hampered in the past by the close antigenic relationships between flaviviruses in traditional serological assays. An epitope blocking ELISA using MVE-specific and KUN-specific monoclonal antibodies (mAb) reacting to the non-structural protein NS1 of these viruses and a flavivirus group-specific mAb reacting to the envelope (E) protein was assessed for testing sentinel animals for seroconversion to specific flavivirus infections. Using these assays we were able to detect serum antibodies to a variety of flavivirus in laboratory infected rabbits, and naturally infected chickens and in the case of primary infections, differentiate those caused by KUN or MVE. These assays are now used routinely in our laboratory for testing chicken sera from sentinel flocks in the Kimberley and Pilbara regions of north Western Australia.

Glycosylation and antigenic variation among Kunjin virus isolates.

Previous studies have found Kunjin (KUN) virus isolates from within Australia to be genetically homogenous and that the envelope protein of the type strain (MRM61C) was unglycosylated and lacked a potential glycosylation site. We investigated the extent of antigenic variation between KUN virus isolates from Australia and Sarawak using an immunoperoxidase assay and a panel of six monoclonal antibodies. The glycosylation status of the E protein of each virus was also determined by N glycosidase F (PNGase F) digestion and limited sequence analysis. The results showed that KUN viruses isolated within Australia oscillated between three antigenic types defined by two epitopes whose expression was influenced by passage history and host cell type. In contrast an isolate from Sarawak formed a stable antigenic type that was not influenced by passage history and was distinct from all Australian isolates. PNGase F digestions of KUN isolates indicated that 19 of the 33 viruses possessed a glycosylated E protein. Nucleotide sequence of the 5' third of the E gene of selected KUN isolates revealed that a single base change in PNGase F sensitive strains changed the tripeptide N-Y-F (amino acids 154-156 of the published sequence) to the potential glycosylation site N-Y-S. Further analysis revealed that passage history also had a significant influence on glycosylation.