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.

Laboratory methods supporting measles surveillance in Queensland, Australia, 2010-2017.

PURPOSE: Australia was officially recognised as having eliminated endemic measles transmission in 2014. Maintaining laboratory support for surveillance of vaccine-preventable diseases, such as measles, is an essential component of reaching and maintaining transmission-free status. METHODOLOGY: Real-time and conventional PCR-based tools were used to detect, differentiate from measles vaccine virus (MeVV), and sequence fragments of measles viruses (MeV) identified from specimens collected in Queensland. Specimens were mostly from travellers who had visited or returned to Queensland from international or interstate sites or been in contact with a case from either group. RESULTS: Between 2010 and 2017, 13 678 specimens were tested in our laboratory using real-time RT-PCR (RT-rPCR), identifying 533 positives. Most specimens were swabs (70.98 %) and urines (25.56 %). A MeVV RT-rPCR was used on request and identified 154 instances of MeVV. MeV-positive extracts were genotyped as required. Genotypes identified among sequenced specimens included B3, D4, D8, D9, G3, and H1 as well as members of clade A as expected from the detection of MeV among virus introductions due to global travel and vaccination. CONCLUSION: We describe the workflow employed and results from our laboratory between 2010 and 2017 for the sensitive detection of MeV infection, supporting high-quality surveillance to ensure the maintenance of Australia's measles-free status.

Diagnosis and typing of influenza using fluorescent barcoded probes.

In this work, we explore a new hybridization technology using barcoded probes which has large-scale multiplexing capability. We used influenza virus to test whether the technology has application in virus diagnostics. Typing of influenza virus strains is an important aspect of global health surveillance. Standard typing procedures use serological or amplification-based assays performed sequentially. By comparison, the hybridization technology was correctly able to detect, type and subtype influenza A and B virus strains directly from clinical samples in a single reaction without prior virus isolation or amplification. Whilst currently not as sensitive as amplification-based assays, these results are a first-step towards application of this technology to the detection and typing of influenza and other viruses.

Virulence and Evolution of West Nile Virus, Australia, 1960-2012.

Worldwide, West Nile virus (WNV) causes encephalitis in humans, horses, and birds. The Kunjin strain of WNV (WNVKUN) is endemic to northern Australia, but infections are usually asymptomatic. In 2011, an unprecedented outbreak of equine encephalitis occurred in southeastern Australia; most of the ≈900 reported cases were attributed to a newly emerged WNVKUN strain. To investigate the origins of this virus, we performed genetic analysis and in vitro and in vivo studies of 13 WNVKUN isolates collected from different regions of Australia during 1960-2012. Although no disease was recorded for 1984, 2000, or 2012, isolates collected during those years (from Victoria, Queensland, and New South Wales, respectively) exhibited levels of virulence in mice similar to that of the 2011 outbreak strain. Thus, virulent strains of WNVKUN have circulated in Australia for >30 years, and the first extensive outbreak of equine disease in Australia probably resulted from a combination of specific ecologic and epidemiologic conditions.

Australian Bat Lyssavirus in a child: the first reported case.

Human infection with Australian Bat Lyssavirus is extremely rare and has not previously been reported in a child. We describe a fatal case of Australian Bat Lyssavirus in an 8-year-old child, and review the literature pertaining to the diagnosis and management of lyssavirus infection with consideration of its applicability to this emerging strain.

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.

Sources of dengue viruses imported into Queensland, australia, 2002-2010.

To assess risk for importation of dengue virus (DENV) into Queensland, Australia, and sources of imported viruses, we sequenced the envelope region of DENV isolates from symptomatic patients with a history of travel during 2002-2010. The number of imported dengue cases greatly increased over the surveillance period, some of which were associated with domestic outbreaks. Patients reported traveling to (in order) Asia, Papua New Guinea, Pacific Island countries, and non-Asia-Pacific countries. By using phylogenetic methods, we assigned DENV isolates from returning residents and overseas visitors with viremia to a specific genotypic group. Genotypes circulating in Asia were extremely diverse. Genotyping and molecular clock analysis supported Asian origination of a strain that caused an outbreak of DENV-4 in Pacific Island countries during 2007-2009, and subsequently, in Innisfail, Australia, in 2009. Our findings indicate that Asia is a major source of DENVs that are imported into Australia, causing a risk for epidemics.

Transmission of Japanese Encephalitis virus from the black flying fox, Pteropus alecto, to Culex annulirostris mosquitoes, despite the absence of detectable viremia.

To determine the potential role of flying foxes in transmission cycles of Japanese encephalitis virus (JEV) in Australia, we exposed Pteropus alecto (Megachiroptera: Pteropididae) to JEV via infected Culex annulirostris mosquitoes or inoculation. No flying foxes developed symptoms consistent with JEV infection. Anti-JEV IgG antibodies developed in 6/10 flying foxes exposed to infected Cx. annulirostris and in 5/5 inoculated flying foxes. Low-level viremia was detected by real-time reverse transcriptase polymerase chain reaction in 1/5 inoculated flying foxes and this animal was able to infect recipient mosquitoes. Although viremia was not detected in any of the 10 flying foxes that were exposed to JEV by mosquito bite, two animals infected recipient mosquitoes. Likewise, an inoculated flying fox without detectable viremia infected recipient mosquitoes. Although infection rates in recipient mosquitoes were low, the high population densities in roosting camps, coupled with migratory behavior indicate that flying foxes could play a role in the dispersal of JEV.

Vector competence of Australian mosquito species for a North American strain of West Nile virus.

Since the establishment of West Nile virus (WNV) into the United States, concern has arisen that this virus may also pose a serious threat to Australian biosecurity. The vector competence of 19 Australian mosquito species for a North American strain of WNV was evaluated. Mosquitoes collected from Cairns, Brisbane, and Sydney were exposed to blood containing 10(4.0+/-0.3) cell culture infectious dose(50)/mosquito WNV that was isolated from a crow during the 1999 New York outbreak. Mosquitoes were tested 12-15 days later to determine their infection, dissemination, and transmission rates. A number of Culex spp. demonstrated a high vector competence for this virus, with some populations of Culex annulirostris, the primary Australian Kunjin virus vector, displaying transmission rates up to 84%. Similarly, Cx. quinquefasciatus and Cx. gelidus were highly competent, with infection and transmission rates of >80% and >50%, respectively. Common Aedes spp., including Aedes notoscriptus, Ae. vigilax, and Ae. procax, were moderately susceptible, and some Verrallina spp. and Coquillettidia spp. were relatively refractory to infection. Thus, Australia possesses a number of competent mosquito species that could facilitate local transmission of WNV, should it be introduced.

Short report: the first isolation of Japanese encephalitis virus from mosquitoes collected from mainland Australia.

In response to an incursion of Japanese encephalitis virus (JEV) on Cape York Peninsula, Australia, in 2005, 23,144 Culex mosquitoes were processed for virus detection. A single isolate of JEV was obtained from a pool of Culex sitiens subgroup mosquitoes. This is the first reported mosquito isolate of JEV from the Australian mainland.

Detection of Australasian Flavivirus encephalitic viruses using rapid fluorogenic TaqMan RT-PCR assays.

The development of single, sensitive, fluorogenic reverse transcriptase-polymerase chain reaction (TaqMan) assays were required for the rapid and specific detection of three encephalitic viruses found in the Australasian region, namely; Japanese encephalitis virus (JEV), Murray Valley encephalitis virus (MVEV), and Kunjin virus (KUNV). Primers and a fluorogenic probe were individually designed to be complementary to a nucleotide region encompassing the 3' terminus of the nonstructural (NS) 5 gene and a portion of the 3' untranslated region (NS5-3'UTR) of each of the viral genomes respectively. Synthetically produced primer and probe controls were developed to minimize the likelihood of contamination and generation of false positives. Viral RNA from singly infected mosquitoes could be detected in pools of 1000 mosquitoes and positive mosquito pools collected from the field have been identified using each assay, indicating a high level of sensitivity and suitability for use in mosquito surveillance programs. In addition, the JEV TaqMan assay has been used to detect successfully viral RNA in sentinel pig serum samples. These assays potentially offer superior and timely detection of encephalitic viruses from surveillance samples, which is essential for the rapid implementation of vector control measures and continued monitoring of virus activity in the Australasian region.

Field evaluation of a sentinel mosquito (Diptera: Culicidae) trap system to detect Japanese encephalitis in remote Australia.

Incursions of Japanese encephalitis (JE) virus into northern Queensland are currently monitored using sentinel pigs. However, the maintenance of these pigs is expensive, and because pigs are the major amplifying hosts of the virus, they may contribute to JE transmission. Therefore, we evaluated a mosquito-based detection system to potentially replace the sentinel pigs. Single, inactivated JE-infected Culex annulirostris Skuse and C. sitiens Wiedemann were placed into pools of uninfected mosquitoes that were housed in a MosquitoMagnet Pro (MM) trap set under wet season field conditions in Cairns, Queensland for 0, 7, or 14 d. JE viral RNA was detected (cycling threshold [CT] = 40) in 11/12, 10/14, and 2/5 pools containing 200, 1,000, and 5,000 mosquitoes, respectively, using a TaqMan real-time reverse transcription-polymerase chain reaction (RT-PCR). The ability to detect virus was not affected by the length of time pools were maintained under field conditions, although the CT score tended to increase with field exposure time. Furthermore, JE viral RNA was detected in three pools of 1,000 mosquitoes collected from Badu Island using a MM trap. These results indicated that a mosquito trap system employing self-powered traps, such as the MosquitoMagnet, and a real-time PCR system, could be used to monitor for JE in remote areas.

Single rapid TaqMan fluorogenic probe based PCR assay that detects all four dengue serotypes.

Public health laboratories require rapid diagnosis of dengue outbreaks for application of measures such as vector control. We have developed a rapid single fluorogenic probe-based polymerase chain reaction assay for the detection of all four dengue serotypes (FUDRT-PCR). The method employs primers and probe that are complementary to the evolutionarily conserved 3' untranslated region of the dengue genome. The assay detected viral RNA of strains of all four dengue serotypes but not of the flaviviruses Japanese encephalitis virus, Murray Valley encephalitis virus, Kunjin, Stratford, West Nile, Alfuy or Yellow fever. When compared to an existing nested-PCR assay for the detection of dengue on clinical samples, FUDRT-PCR detected dengue 1 (100%, n=14), dengue 2 (85%, n=13), dengue 3 (64%, n=14) and dengue 4 (100%, n=3) with the indicated sensitivities. FUDRT-PCR enables diagnosis of acute dengue infection in four hours from sample receipt. In addition, a single-test procedure should result in a reduction in the number of tests performed with considerable cost savings for diagnostic laboratories.