Molecular characterization of the first reported Aichivirus A in Australia.

A novel real-time reverse transcription polymerase chain reaction (RT-rPCR) assay was developed to detect Aichivirus A (AiV-A) based on four complete genomes. The assay successfully detected AiV-A in a sample from a patient with acute gastroenteritis in January 2008. Screening of 756 samples submitted for norovirus testing during May 2008 detected a further 23 AiV-A-positive samples from 18 individual patients. Genotyping using novel primers targeting the 3C-3D junction region identified AiV-A genotype B. Further sequencing of the VP1 region supported the 3C-3D result. All three assays proved useful to support foodborne outbreak investigations. This is the first report of AiV-A detection in Australia.

Applications of a sugar-based surveillance system to track arboviruses in wild mosquito populations.

Effective arbovirus surveillance is essential to ensure the implementation of control strategies, such as mosquito suppression, vaccination, or dissemination of public warnings. Traditional strategies employed for arbovirus surveillance, such as detection of virus or virus-specific antibodies in sentinel animals, or detection of virus in hematophagous arthropods, have limitations as an early-warning system. A system was recently developed that involves collecting mosquitoes in CO2-baited traps, where the insects expectorate virus on sugar-baited nucleic acid preservation cards. The cards are then submitted for virus detection using molecular assays. We report the application of this system for detecting flaviviruses and alphaviruses in wild mosquito populations in northern Australia. This study was the first to employ nonpowered passive box traps (PBTs) that were designed to house cards baited with honey as the sugar source. Overall, 20/144 (13.9%) of PBTs from different weeks contained at least one virus-positive card. West Nile virus Kunjin subtype (WNVKUN), Ross River virus (RRV), and Barmah Forest virus (BFV) were detected, being identified in 13/20, 5/20, and 2/20 of positive PBTs, respectively. Importantly, sentinel chickens deployed to detect flavivirus activity did not seroconvert at two Northern Territory sites where four PBTs yielded WNVKUN. Sufficient WNVKUN and RRV RNA was expectorated onto some of the honey-soaked cards to provide a template for gene sequencing, enhancing the utility of the sugar-bait surveillance system for investigating the ecology, emergence, and movement of arboviruses.

Expectoration of Flaviviruses during sugar feeding by mosquitoes (Diptera: Culicidae).

Biological transmission of arboviruses to a vertebrate host occurs when virions are expelled along with saliva during blood feeding by a hematophagous arthropod. We undertook experiments to determine whether mosquitoes expectorate flaviviruses in their saliva while sugar feeding. Batches of Culex annulirostris Skuse and Culex gelidus Theobald (Diptera: Culicidae) were orally infected with Japanese encephalitis (family Flaviviridae, genus Flavivirus, JEV), Kunjin (family Flaviviridae, genus Flavivirus, KUNV; a subtype of West Nile virus), and Murray Valley encephalitis (family Flaviviridae, genus Flavivirus, MVEV) viruses. After a 7-d extrinsic incubation, these mosquitoes were offered sucrose meals via cotton pledgets, which were removed daily and processed for viral RNA by using real-time TaqMan reverse transcriptase-polymerase chain reaction (RT-PCR) assays. JEV, MVEV, and KUNV RNA was detected in all pledgets removed from batches of Cx. gelidus on days 7-14 postexposure. In contrast, detection rates were variable for Cx. annulirostris, with KUNV detected in 0.3 M sucrose pledgets on all days postexposure, and JEV and MVEV detected on 57 and 50% of days postexposure, respectively. Higher concentrations of sucrose in the pledget did not increase virus detection rates. When individual JEV-infected Cx. gelidus were exposed to the sucrose pledget, 73% of mosquitoes expectorated virus with titers that were detectable by TaqMan RT-PCR. These results clearly show that flaviviruses are expectorated by infected mosquitoes during the process of sugar feeding on artificial pledgets. Potential applications of the method for arboviral bioassays and field surveillance are discussed.