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.

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.

On September 26, 2012, a woman aged 35 years from Kosrae in the Federated States of Micronesia (FSM) was hospitalized with fever, headache, muscle pain, vomiting, leukopenia, and thrombocytopenia. A rapid diagnostic test (RDT) (Dengue Duo, Standard Diagnostics Inc.) was positive for dengue virus (DENV) nonstructural protein-1 (NS1). During the next week, seven more persons with suspected dengue were tested with the RDT, of whom three were RDT-positive for NS1 or anti-DENV immunoglobulin M (IgM). During October, the Kosrae State Department of Health Services, with support from the FSM Department of Health and Social Affairs and the World Health Organization (WHO), responded to the outbreak with enhanced surveillance, training in clinical management, analysis of hospital surge capacity, a rapid mosquito survey to identify species and distributions, and control measures. By March 14, 2013, approximately 3.7% of Kosrae State residents had been hospitalized with suspected dengue. The outbreak consumed scarce medical and public health services, including outpatient, inpatient, and laboratory services, resulting in redirection of human and material resources from other important medical and public health activities. Because the health consequences of dengue can be substantial in resource-limited settings, Pacific Island nations might wish to consider preparedness measures for dengue outbreaks such as developing and testing outbreak response plans and ensuring adequate capacity for epidemiologic surveillance and laboratory testing.

The WHO estimates there may be 50 million dengue virus (DENV) infections worldwide every year, with the disease being endemic in more than 100 countries [1]. There has been a dramatic rise in the incidence of dengue in recent decades, making this an arbovirus of major international public health concern. Dengue viruses belong to the family Flaviviridae and are transmitted between humans via infected female Aedes mosquitoes, particularly Aedes aegypti. In the state of Queensland, Australia, infected travellers from overseas have facilitated numerous DENV outbreaks [2], [3]. However, these outbreaks are limited to the far north of the state, the only area of Australia where Ae. aegypti occurs [4]. There have been case reports of non-vector, healthcare-associated transmission of DENVs—four cases of percutaneous transmission via needlestick injuries, mucocutaneous transmission through a blood splash to the face, vertical transmission, and transmission via bone marrow transplant (summarised in [5]). We report the first case to our knowledge of DENV infection acquired by a laboratory scientist conducting mosquito infection and transmission experiments.

Hendra virus (HeV) was first isolated in 1994, from a disease outbreak involving at least 21 horses and two humans in the Brisbane suburb of Hendra, Australia. The affected horses and humans all developed a severe but unidentified respiratory disease that resulted in the deaths of one of the human cases and the deaths or putting down of 14 of the horses. The virus, isolated by culture from a horse and the kidney of the fatal human case, was initially characterised as a new member of the genus Morbillivirus in the family Paramyxoviridae. Comparative sequence analysis of part of the matrix protein gene of the virus and the discovery that the virus had an exceptionally large genome subsequently led to HeV being assigned to a new genus, Henipavirus, along with Nipah virus (a newly emergent virus in pigs).The regular outbreaks of HeV-related disease that have occurred in Australia since 1994 have all been characterised by acute respiratory and neurological manifestations, with high levels of morbidity and mortality in the affected horses and humans. The modes of transmission of HeV remain largely unknown. Although fruit bats have been identified as natural hosts of the virus, direct bat-horse, bat-human or human-human transmission has not been reported. Human infection can occur via exposure to infectious urine, saliva or nasopharyngeal fluid from horses. The treatment options and efficacy are very limited and no vaccine exists.Reports on the outbreaks of HeV in Australia are collated in this review and the available data on the biology, transmission and detection of the pathogen are summarized and discussed.

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.

We report the development of a flavivirus immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (MAC-ELISA) which improves the determination of an infecting flavivirus serotype over that by current serological methods. A panel of 165 IgM-positive sera from flavivirus patients with specific diagnostic results was tested by the flavivirus MAC-ELISA using a panel of 10 antigens. For 134 of these sera (81.2%), the highest reactivity was demonstrated against the infecting virus, which was consistent with the original diagnostic result. Specific antibody reactions inconsistent with the original diagnosis were found for six sera (3.6%). In our experience, the flavivirus-serotyping ELISA provides a rapid and accurate alternative to other serological tests, such as hemagglutination inhibition, for the specific diagnosis of flavivirus infections.

In mid-January 2000, the reappearance of Japanese encephalitis (JE) virus activity in the Australasian region was first demonstrated by the isolation of JE virus from 3 sentinel pigs on Badu Island in the Torres Strait. Further evidence of JE virus activity was revealed through the isolation of JE virus from Culex gelidus mosquitoes collected on Badu Island and the detection of specific JE virus neutralizing antibodies in 3 pigs from Saint Pauls community on Moa Island. Nucleotide sequencing and phylogenetic analyses of the premembrane and envelope genes were performed which showed that both the pig and mosquito JE virus isolates (TS00 and TS4152, respectively) clustered in genotype I, along with northern Thai, Cambodian, and Korean isolates. All previous Australasian JE virus isolates belong to genotype II, along with Malaysian and Indonesian isolates. Therefore, for the first time, the appearance and transmission of a second genotype of JE virus in the Australasian region has been demonstrated.