Development and evaluation of a species diagnostic polymerase chain reaction-restriction fragment-length polymorphism procedure for cryptic members of the Culex sitiens (Diptera : Culicidae) subgroup in Australia and the southwest Pacific

Members of the Culex sitiens subgroup are important vectors of arboviruses, including Japanese encephalitis virus, Murray Valley encephalitis virus and Ross River virus. Of the eight described species, Cx. annulirostris Skuse, Cx. sitiens Wiedemann, and Cx. palpalis Taylor appear to be the most abundant and widespread throughout northern Australia and Papua New Guinea (PNG). Recent investigations using allozymes have shown this subgroup to contain cryptic species that possess overlapping adult morphology. We report the development of a polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) procedure that reliably separates these three species. This procedure utilizes the sequence variation in the ribosomal DNA ITS1 and demonstrates species-specific PCR-RFLP profiles from both colony and field collected material. Assessment of the consistency of this procedure was undertaken on mosquitoes sampled from a wide geographic area including Australia, PNG, and the Solomon Islands. Overlapping adult morphology was observed for Cx. annulirostris and Cx. palpalis in both northern Queensland and PNG and for all three species at one site in northwest Queensland.

Enhancement or modulation of the vector competence of ochlerotatus vigilax (Diptera : Culicidae) for Ross River virus by temperature

Two different doses of Ross River virus (1111) were fed to Ochlerotatus vigilax (Skuse), the primary coastal vector in Australia; and blood engorged females were held at different temperatures up to 35 d. After ingesting 10(4.3) CCID50/Mosquito, mosquitoes reared at 18 and 25degreesC (and held at the same temperature) had higher body remnant and head and salivary gland titers than those held at 32degreesC, although infection rates were comparable. At 18, 25, and 32degreesC, respectively, virus was first detected in the salivary glands on days 3, 2, and 3. Based on a previously demonstrated 98.7% concordance between salivary gland infection and transmission, the extrinsic incubation periods were estimated as 5, 4, and 3 d, respectively, for these three temperatures. When Oc. vigilax reared at 18, 25, or 32degreesC were fed a lower dosage of 10(3.3) CCID50 RR/mosquito, and assayed after 7 d extrinsic incubation at these (or combinations of these) temperatures, infection rates and titers were similar. However, by 14 d, infection rates and titers of those reared and held at 18 and 32degreesC were significantly higher and lower, respectively. However, this process was reversible when the moderate 25degreesC was involved, and intermediate infection rates and titers resulted. These data indicate that for the strains of RR and Oc. vigilax used, rearing temperature is unimportant to vector competence in the field, and that ambient temperature variations will modulate or enhance detectable infection rates only after 7 d: extrinsic incubation. Because of the short duration of extrinsic incubation, however, this will do little to influence RR epidemiology, because by this time some Oc. vigilax could be seeking their third blood meal, the latter two being infectious.

DNA vaccine coding for the full-length infectious Kunjin virus RNA protects mice against the New York strain of West Nile virus

A plasmid DNA directing transcription of the infectious full-length RNA genome of Kunjin (KUN) virus in vivo from a mammalian expression promoter was used to vaccinate mice intramuscularly. The KUN viral cDNA encoded in the plasmid contained the mutation in the NS1 protein (Pro-250 to Leu) previously shown to attenuate KUN virus in weanling mice. KUN virus was isolated from the blood of immunized mice 3-4 days after DNA inoculation, demonstrating that infectious RNA was being transcribed in vivo; however, no symptoms of virus-induced disease were observed. By 19 days postimmunization, neutralizing antibody was detected in the serum of immunized animals. On challenge with lethal doses of the virulent New York strain of West Nile (WN) or wild-type KUN virus intracerebrally or intraperitoneally, mice immunized with as little as 0.1-1 mug of KUN plasmid DNA were solidly protected against disease. This finding correlated with neutralization data in vitro showing that serum from KUN DNA-immunized mice neutralized KUN and WN,viruses with similar efficiencies. The results demonstrate that delivery of an attenuated but replicating KUN virus via a plasmid DNA vector may provide an effective vaccination strategy against virulent strains of WN virus.

Cross-protective and Infection-Enhancing Immunity in Mice Vaccinated Against Flaviviruses Belonging to the Japanese Encephalitis Virus Serocomplex

The Japanese encephalitis virus serocomplex is a group of mosquito-borne flaviviruses that cause severe encephalitic disease in humans. The recent emergence of several members of this serocomplex in geographic regions where other closely related flaviviruses are endemic has raised urgent human health issues. Thus, the impact of vaccination against one of these neurotropic virus on the outcome of infection with a second, serologically related virus is unknown. We show here that immunity against Murray Valley encephalitis virus in vaccinated mice can cross-protect but also augment disease severity following challenge with Japanese encephalitis virus. Immunepotentiation of heterologous flavivirus disease was apparent in animals immunized with a 'killed' virus preparation when humoral antiviral immunty of low magnitude was elicited. (C) 2002 Elsevier Science Ltd. All rights reserved.

Vector competence of Australian mosquitoes (Diptera : Culicidae) for Japanese encephalitis virus

Australian mosquitoes were evaluated for their ability to become infected with and transmit a Torres Strait strain of Japanese encephalitis virus. Mosquitoes, which were obtained from either laboratory colonies and collected using Centers for Disease Control and Prevention light traps baited with CO2 and octenol or reared from larvae, were infected by feeding on a blood/sucrose solution containing 10(4.5+/-0.1) porcine stable-equine kidney (PS-EK) tissue culture infectious dose(50)/ mosquito of the TS3306 virus strain. After 14 d, infection and transmission rates of 100% and 81%, respectively, were obtained for a southeast Queensland strain of Culex annulirostris Skuse, and 93% and 61%, respectively, for a far north Queensland strain. After 13 or more days, infection and transmission rates of > 90% and greater than or equal to 50%, respectively, were obtained for southeast Queensland strains of Culex sitiens Wiedemann and Culex quinquefasciatus Say, and a far north Queensland strain of Culex gelidus Theobald. Although infection rates were > 55%, only 17% of Ochlerotatus vigilax (Skuse) and no Cx. quinquefasciatus, collected from far north Queensland, transmitted virus. North Queensland strains of Aedes aegypti L., Ochlerotatus kochi (Donitz), and Verrallina funerea (Theobald) were relatively refractory to infection. Vertical transmission was not detected among 673 F, progeny of Oc. vigilax. Results of the current vector competence study, coupled with high field isolation rates, host feeding patterns and widespread distribution, confirm the status of Cx. annulirostris as the major vector of Japanese encephalitis virus in northern Australia. The relative roles of other species in potential Japanese encephalitis virus transmission cycles in northern Australia are discussed.

Mosquito host-feeding patterns and implications for Japanese encephalitis virus transmission in northern Australia and Papua New Guinea

Japanese encephalitis (JE) virus spread to northern Australia during the 1990s, transmitted by Culex annulirostris Skuse and other mosquitoes (Diptera: Culicidae). To determine the relative importance of various hosts for potential vectors of JE virus, we investigated the host-feeding patterns of mosquitoes in northern Australia and Western Province of Papua New Guinea, with particular attention to pigs, Sus scrofa L. - the main amplifying host of JE virus in South-east Asia. Mosquitoes were collected by CDC light traps baited with dry ice and 1-octen-3-ol, run 16.00-08.00 hours, mostly set away from human habitations, if possible in places frequented by feral pigs. Bloodmeals of 2569 mosquitoes, representing 15 species, were identified by gel diffusion assay. All species had fed mostly on mammals: only 30%) were trapped where domestic pigs were kept close to human habitation. From seven of eight locations on the Australian mainland, the majority of Cx. annulirostris had obtained their bloodmeals from marsupials, probably the Agile wallaby Macropus agilis (Gould). Overall proportions of mosquito bloodmeals identified as marsupial were 60% from the Gulf Plains region of Australia, 78% from the Cape York Peninsula and 64% from the Daru area of Papua New Guinea. Thus, despite the abundance of feral pigs in northern Australia, our findings suggest that marsupials divert host-seeking Cx. annulirostris away from pigs. As marsupials are poor JE virus hosts, the prevalence of marsupials may impede the establishment of JE virus in Australia.

Numerical simulations of wind and temperature structure within an Alpine lake basin, Lake Tekapo, New Zealand

High-resolution numerical model simulations have been used to study the local and mesoscale thermal circulations in an Alpine lake basin. The lake (87 km(2)) is situated in the Southern Alps, New Zealand and is located in a glacially excavated rock basin surrounded by mountain ranges that reach 3000 m in height. The mesoscale model used (RAMS) is a three-dimensional non-hydrostatic model with a level 2.5 turbulence closure scheme. The model demonstrates that thermal forcing at local (within the basin) and regional (coast-to-basin inflow) scales drive the observed boundary-layer airflow in the lake basin during clear anticyclonic summertime conditions. The results show that the lake can modify (perturb) both the local and regional wind systems. Following sunrise, local thermal circulations dominate, including a lake breeze component that becomes embedded within the background valley wind system. This results in a more divergent flow in the basin extending across the lake shoreline. However, a closed lake breeze circulation is neither observed nor modelled. Modelling results indicate that in the latter part of the day when the mesoscale (coast-to-basin) inflow occurs, the relatively cold pool of lake air in the basin can cause the intrusion to decouple from the surface. Measured data provide qualitative and quantitative support for the model results.

Flavivirus isolations from mosquitoes collected from western Cape York Peninsula, Australia, 1999-2000

After the 1st appearance of Japanese encephalitis virus (JE) on mainland Australia in 1998, a study was undertaken to investigate whether JE had become established in enzootic transmission cycles on western Cape York Peninsula. Adult mosquitoes were collected during the late wet season from Kowanyama and Pormpuraaw in April 1999, and Pormpuraaw and Barr's Yard in April 2000. Despite processing 269,270 mosquitoes for virus isolation, no isolates of JE were obtained. However, other flaviviruses comprising Murray Valley encephalitis virus, Kunjin virus, Alfuy virus, and Kokobera virus (KOK) were isolated. Isolates of the alphaviruses Ross River virus, Barmah Forest virus (BF), and Sindbis virus (SIN) also were obtained. The majority (88%) of isolates were from members of the Culex sitiens subgroup. Single isolates of KOK, BF and SIN were obtained from Ochlerotatus vigilax, Oc. normanensis, and Anopheles bancroftii, respectively. The isolations of flaviviruses during the late wet season indicate that conditions were suitable for flavivirus activity in the area. No evidence was found to suggest that JE has become established in enzootic transmission cycles on western Cape York, although study sites and field trips were limited.