The role of mosquitoes in the emergence of Japanese encephalitis virus in Australia

van den Hurk, Andrew Francis. (2002). The role of mosquitoes in the emergence of Japanese encephalitis virus in Australia PhD Thesis, School of Molecular and Microbial Sciences, The University of Queensland.

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Author van den Hurk, Andrew Francis.
Thesis Title The role of mosquitoes in the emergence of Japanese encephalitis virus in Australia
School, Centre or Institute School of Molecular and Microbial Sciences
Institution The University of Queensland
Publication date 2002
Thesis type PhD Thesis
Supervisor John Mackenzie
Scott Ritchie
Total pages 300
Collection year 2002
Language eng
Subjects L
321010 Infectious Diseases
730101 Infectious diseases
Formatted abstract Japanese encephalitis (JE) virus is a leading cause of viral encephalitis in Southeast Asia, responsible for over 15,000 deaths annually. In 1995, Japanese encephalitis emerged as a disease entity in northern Australia with an outbreak in the Torres Strait, resulting in 3 cases and 2 deaths. With the exception of 1999, virus activity has occurred every year, with the 1998 outbreak extending onto Cape York Peninsula, resulting in the first mainland case of Japanese encephalitis. Virus isolation studies suggest that Culex annulirostris Skuse is the primary potential vector of JE virus in the Australasian region, while a single isolate has been obtained from Ochlerotatus vigilax (Skuse). The research reported in this thesis used a combination of vector competence experiments, virus isolation studies and analysis of host feeding patterns, to determine the relative role of Australian mosquito species in potential transmission cycles.

Vector competence experiments were undertaken to assess the ability of Australian mosquitoes to become infected with and transmit a Torres Strait strain of JE virus. Colony or field collected mosquitoes were infected with 104.5 PS-EK TCID50/mosquito of the TS3306 strain of JE virus via a membrane feeder. Infection and transmission rates of Cx. annulirostris were 100% and 81% respectively for a southeast Queensland colony strain and 91% and 61% respectively for a north Queensland field strain, confirming its status as the primary vector of JE virus in Australia. High infection and transmission rates were also obtained with Culex sitiens Wiedemann, Culex quinquefasciatus Say and Culex gelidus Theobald suggesting that they are efficient vectors. The results suggest that Oc. vigilax, Ochlerotatus notoscriptus (Skuse), Ochlerotatus kochi (Dönitz), Verrallina funerea (Theobald) and Aedes aegypti Linnaeus are relatively inefficient vectors of JE virus. Virus titres in Cx. annulirostris and Cx. sitiens were 105.5 PS-EK TCID50/mosquito and 104.4PS-EK TCID50/mosquito respectively at day 10 post infection. Vertical transmission of JE virus was not demonstrated in F1 progeny of Oc. vigilax.

Virus isolation studies were undertaken in three regions of north Queensland. Mosquitoes were collected using CDC light traps baited with CO2 and octenol and processed for virus isolation using a tissue culture enzyme immunoassay (TCEIA). As part of the response to the 1998 outbreak, a total of 35,235 mosquitoes were processed from collections from Cape York Peninsula. No isolates of JE virus were obtained from these mosquitoes. However, 18 isolates of Kokobera virus were obtained, with 12 coming from western Cape York Peninsula and six from the Northern Peninsula Area.

A total of 153,529 mosquitoes were processed for virus isolation from the Gulf Plains region of northwest Queensland. No isolates of JE virus were obtained. However, four isolates of Murray Valley encephalitis, two Kunjin, seven Ross River, two Sindbis and one Edge Hill virus isolates were obtained. Identification of the species present in virus-positive pools of Cx. sitiens subgroup mosquitoes using PCR-RFLP analysis revealed that the majority of isolates were from pools containing mainly Cx. annulirostris, and low numbers of Cx. palpalis.

During the 2000 Badu Island outbreak, mosquitoes were collected for virus isolation and to examine the host feeding patterns of potential vectors. A total of 94,285 mosquitoes were processed for virus isolation, from which a single isolate of JE virus was obtained from Cx. gelidus. This is the first isolate of JE virus from this species in the Australasian region. No isolates of JE virus were obtained from Cx. annulirostris. To determine the host feeding patterns of Badu Island mosquitoes, a total of 1,197 blood meals, including 573 from Cx. annulirostris, were tested using a panel of polyclonal antisera in a gel diffusion assay. The results suggested that the proportion of Cx. annulirostris feeding on pigs had been significantly reduced following the removal of the pigs from the Badu Island Community. Consequently, contact between amplifying hosts and vectors has been limited, potentially reducing the risk of JE virus transmission to humans.

The final component of this thesis examined the role of mosquito host feeding patterns in the emergence of JE virus in northern Australia and Papua New Guinea. Blood engorged mosquitoes were collected using CDC light traps baited with CO2 and octenol from 15 locations ranging from the Balimo region of PNG to Mt Isa in northwest Queensland. A total of 3,658 blood meals, comprising primarily Cx. annulirostris, were tested using the gel diffusion assay. Highest rates of pig feeding (>30%) were obtained from locations where domestic pigs were kept, such as Badu Island and Baa's Yard on Cape York Peninsula. At most locations on mainland Australia, significantly more Cx annulirostris had obtained their blood meals from marsupials than from other pigs. This suggests that despite the presence of large populations of feral pigs in northern Australia, marsupials are the preferred blood meal host of Cx. annulirostris.

The results presented in this thesis confirm the status of Cx. annulirostris as the primary vector of JE virus in northern Australia. The results also suggest that other Culex spp. could potentially play a role in transmission cycles. The virus isolation studies suggest that there are numerous other flaviviruses circulating on mainland Australia that may compete with JE virus for susceptible amplifying hosts. In addition, analysis of host feeding patterns suggest that the propensity of Cx. annulirostris to feed on marsupials and not pigs may suppress JE virus transmission. Despite the fact that Australia possesses very efficient vectors of JE virus, it remains to be seen what influence these mosquitoes will have on the ultimate establishment and spread of JE virus in Australia.
Keyword Mosquitoes
Japanese B encephalitis

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