Jamie McMahon

Field Validation of the Gravid Aedes Trap (GAT) for Collection of Aedes aegypti (Diptera: Culicidae)

ABSTRACT Current surveillance methods for adult Aedes aegypti (L.) are expensive, require electrical power (e.g., the BG-Sentinel trap, BGS), are labor intensive (aspirators), or require difficult to use and costly adhesives (sticky ovitraps). Field trials were conducted in Cairns (Australia) to compare the efficacy of the newly designed Gravid Aedes Trap (GAT) against existing sticky ovitraps (MosquiTRAP and double sticky ovitrap) and the BGS. Latin square design trials confirmed that alarge GAT using a 9.2-liters bucket treated with Mortein Barrier Outdoor Surface Spray ([AI] 0.3 g/kg imiprothrin and 0.6 g/kg deltamethrin) outperformed a smaller 1.2-liters GAT and collected, on average, 3.7X and 2.4 X more female Ae. aegypti than the MosquiTRAP and double sticky ovitrap, respectively. Field trials showed that the GAT collected 10–50% less female Ae. aegypti than the BGS trap but 30% more gravid mosquitoes than the BGS. Trials using the BGS and the GAT indicated that there was no difference in capture rates between female Ae. aegypti uninfected and infected with the wMel strain of Wolbachia, and wMel infection rates were nearly identical at >90% to field captured Ae. aegypti. The potential for the GAT to be used for dengue virus surveillance was also demonstrated with dengue virus type 3 RNA detected in five-sixths and six-sixths pools of Ae. aegypti stored in a GAT held at 28°C and 60% relative humidity for 7 and 14 d, respectively. Mosquito knock down in GATs treated with Mortein surface spray set in 30, 70, and 99% shade was comparable for up to 2 mo, with only ≈10% of adults escaping. The GAT is therefore a useful tool for capturing adult Ae. aegypti and may be suitable for other container-inhabiting species such as Aedes albopictus (Skuse) and Culex quinquefasciatus Say. The low cost and practicality of operation make the GAT suitable for vector surveillance and projects requiring monitoring of mosquitoes for Wolbachia and arboviruses, especially in developing countries.

Assessment of local mosquito species incriminates Aedes aegypti as the potential vector of Zika virus in Australia

Background Within the last 10 years Zika virus (ZIKV) has caused unprecedented epidemics of human disease in the nations and territories of the western Pacific and South America, and continues to escalate in both endemic and non-endemic regions. We evaluated the vector competence of Australian mosquitoes for ZIKV to assess their potential role in virus transmission. Methodology/Principal Findings Mosquitoes were exposed to infectious blood meals containing the prototype African ZIKV strain. After 14 days incubation at 28°C and high relative humidity, infection, dissemination and transmission rates were assessed. Infection in Culex annulirostris and Cx. sitiens could not be detected. 8% of Cx. quinquefasciatus were infected, but the virus did not disseminate in this species. Despite having infection rates > 50%, Aedes notoscriptus and Ae. vigilax did not transmit ZIKV. In contrast, Ae. aegypti had infection and transmission rates of 57% and 27%, respectively. In susceptibility trials, the virus dose required to infect 50% (ID50) of Ae. aegypti was106.4 tissue culture infectious dose50 (TCID50)/mL. Additionally, a threshold viral load within the mosquito of at least 105.1 TCID50 equivalents/mL had to be reached before virus transmission occurred. Conclusions/Significance We confirmed Ae. aegypti to be the most likely mosquito vector of ZIKV in Australia, although the restricted distribution of this species will limit the receptive zone to northern Queensland where this species occurs. Importantly, the role in ZIKV transmission of Culex and other Aedes spp. tested will be negligible. Despite being the implicated vector, the relatively high ID50 and need for a high titer disseminated infection in Ae. aegypti suggest that high mosquito population densities will be required to facilitate epidemic ZIKV transmission among the currently immunologically naïve human population 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.

No Evidence of Prolonged Hendra Virus Shedding by 2 Patients, Australia

To better understand the natural history of Hendra virus infection and its tendency to relapse, 2 humans infected with this virus were monitored after acute infection. Virus was not detected in blood samples when patients were followed-up at 2 and 6 years. Thus, no evidence was found for prolonged virus shedding.

Isolation of Zika Virus Imported from Tonga into Australia.

Introduction: The globally emergent Zika virus (ZIKV) is a threat to Australia, given the number of imported cases from epidemic regions and the presence of competent mosquito vectors. We report the isolation of ZIKV from a female traveler who recently returned from Tonga to Brisbane, Queensland, Australia in 2016. Methods: A specific TaqMan real-time reverse transcriptase polymerase chain reaction assay (RT-PCR) assay was used to detect ZIKV in serum and urine samples. Conventional cell culture techniques and suckling mice were employed in an attempt to isolate ZIKV from serum and urine. Results: A ZIKV isolate (TS17-2016) was recovered from the serum sample after one passage in suckling mouse brains and harvested 11 days post inoculation. Phylogenetic analysis of complete envelope (E) gene sequences demonstrated TS17-2016 shared 99.9% nucleotide identity with other contemporary sequences from Tonga 2016, Brazil 2015 and French Polynesia 2013 within the Asian lineage. Discussion: This is the first known report of successful isolation of ZIKV from a human clinical sample in Australia and the first from a traveler from Tonga. This study highlights the potential difficulties in isolating ZIKV from acute clinical samples using conventional cell culture techniques, particularly in non-endemic countries like Australia where access to samples of sufficient viral load is limited. The successful isolation of TS17-2016 will be essential for continued investigations of ZIKV transmission and pathogenicity and will enable the advancement of new preventative control measures extremely relevant to the Australian and Pacific region.

Genetic characterization of archived bunyaviruses and their potential for emergence in Australia

Genetic relationships between bunyaviruses from Australia and pathogenic bunyaviruses from elsewhere indicate emergence potential.

Rapid surveillance for vector presence (RSVP): development of a novel system for detecting Aedes aegypti and Aedes albopictus

Background The globally important Zika, dengue and chikungunya viruses are primarily transmitted by the invasive mosquitoes, Aedes aegypti and Aedes albopictus. In Australia, there is an increasing risk that these species may invade highly urbanized regions and trigger outbreaks. We describe the development of a Rapid Surveillance for Vector Presence (RSVP) system to expedite presence- absence surveys for both species. Methodology/Principal findings We developed a methodology that uses molecular assays to efficiently screen pooled ovitrap (egg trap) samples for traces of target species ribosomal RNA. Firstly, specific real-time reverse transcription-polymerase chain reaction (RT-PCR) assays were developed which detect a single Ae. aegypti or Ae. albopictus first instar larva in samples containing 4,999 and 999 non-target mosquitoes, respectively. ImageJ software was evaluated as an automated egg counting tool using ovitrap collections obtained from Brisbane, Australia. Qualitative assessment of ovistrips was required prior to automation because ImageJ did not differentiate between Aedes eggs and other objects or contaminants on 44.5% of ovistrips assessed, thus compromising the accuracy of egg counts. As a proof of concept, the RSVP was evaluated in Brisbane, Rockhampton and Goomeri, locations where Ae. aegypti is considered absent, present, and at the margin of its range, respectively. In Brisbane, Ae. aegypti was not detected in 25 pools formed from 477 ovitraps, comprising ≈ 54,300 eggs. In Rockhampton, Ae. aegypti was detected in 4/6 pools derived from 45 ovitraps, comprising ≈ 1,700 eggs. In Goomeri, Ae. aegypti was detected in 5/8 pools derived from 62 ovitraps, comprising ≈ 4,200 eggs. Conclusions/Significance RSVP can rapidly detect nucleic acids from low numbers of target species within large samples of endemic species aggregated from multiple ovitraps. This screening capability facilitates deployment of ovitrap configurations of varying spatial scales, from a single residential block to entire suburbs or towns. RSVP is a powerful tool for surveillance of invasive Aedes spp., validation of species eradication and quality assurance for vector control operations implemented during disease outbreaks.

Detection of Specific ZIKV IgM in Travelers Using a Multiplexed Flavivirus Microsphere Immunoassay

Zika virus (ZIKV) has spread widely in the Pacific and recently throughout the Americas. Unless detected by RT-PCR, confirming an acute ZIKV infection can be challenging. We developed and validated a multiplexed flavivirus immunoglobulin M (IgM) microsphere immunoassay (flaviMIA) which can differentiate ZIKV-specific IgM from that due to other flavivirus infections in humans. The flaviMIA bound 12 inactivated flavivirus antigens, including those from ZIKV and yellow fever virus (YFV), to distinct anti-flavivirus antibody coupled beads. These beads were used to interrogate sera from patients with suspected ZIKV infection following travel to relevant countries. FlaviMIA results were validated by comparison to the ZIKV plaque reduction neutralization test (PRNT). The results highlight the complexity of serological ZIKV diagnosis, particularly in patients previously exposed to, or vaccinated against, other flaviviruses. We confirmed 99 patients with ZIKV infection by a combination of RT-PCR and serology. Importantly, ZIKV antibodies could be discriminated from those ascribed to other flavivirus infections. Serological results were sometimes confounded by the presence of pre-existing antibodies attributed to previous flavivirus infection or vaccination. Where RT-PCR results were negative, testing of appropriately timed paired sera was necessary to demonstrate seroconversion or differentiation of recent from past infection with or exposure to ZIKV.

New insights into chikungunya virus emergence and spread from Southeast Asia

Chikungunya virus (CHIKV) is a mosquito-borne virus of the family Togaviridae, genus Alphavirus, which causes a debilitating polyarthritic disease syndrome characterised by fever, arthralgia, myalgia, headache and rash. Circulation of the virus predominantly occurs in urban transmission cycles between humans and mosquitoes and can rapidly escalate into large-scale epidemics causing high rates of morbidity. The virus was originally isolated in 1953 in Tanzania, however clinical descriptions of previous disease outbreaks in the Caribbean (St. Thomas) and southeast coastal regions of the United States, suggest CHIKV epidemics may have occurred earlier in the nineteenth century. Classified according to their original geographical associations, three evolutionary distinct CHIKV genotypes, namely, the Asian, the East/Central/South African (ECSA) and the West African have been defined. However, increased human travel, commercial trade and expanding habitats of the primary mosquito vectors Aedes aegypti and Ae. albopictus, have directly influenced genotype distribution and contributed to global spread and transmission in new locations. Exploding in the western Indian Ocean during 2005–2006, the ECSA genotype has caused autochthonous outbreaks in India, Italy (2007), Papua New Guinea (PNG) (2012) and several Southeast Asian countries. Outbreaks on La Réunion Island (2005–2006), in Italy (2007), and in PNG in 2012, were driven by Ae. albopictus mosquitoes and involved a highly adapted CHIKV strain containing a unique amino-acid change, A226V, in the fusion envelope glycoprotein, E1. An outbreak of the ECSA genotype has also been recorded in Brazil (2014) in the east-central region of Feira de Santana, possibly imported from Angola. CHIKV also re-emerged from Asia and rapidly spread to Pacific regions including New Caledonia (2007), Yap, Federated States of Micronesia (2013), Samoa, American Samoa and French Polynesia (2014), and Kiribati (2015). In December, 2013, the Asian lineage was detected on the Caribbean Island, Saint Martin and has since caused widespread epidemics, involving millions of cases, in North, Central and South America. Previous phylogenetic analyses suggested that reported Pacific and American Asian CHIKV strains were most closely related to 2012 strains from the Philippines and China, and that the Saint Martin, Caribbean 2013 strain may have originated from the South Pacific. However, the exact route of CHIKV introduction into the Americas is unknown and is largely influenced by the limited availability of whole-genome sequences, particularly from the Pacific region. We performed whole-genome sequencing of 11 CHIKV strains (GenBank accession numbers MF773559-MF773569) imported into Australia by patients with travel histories from Southeast Asia, the Pacific and the Americas between 2010 and 2017, and phylogenetically compared the coding regions with 546 globally available CHIKV sequences retrieved from GenBank (including sequences from humans, and mosquito vector Ae. aegypti and Ae. albopictus host species). The CHIKV isolates were recovered following inoculation of C6/36 cell monolayers with patient serum previously positive for CHIKV RNA using a real-time TaqMan RTPCR assay. Viral RNA was extracted from passage 1 or passage 2 cultures using the QIAamp viral RNA extraction kit (Qiagen) without carrier RNA and whole-genome sequencing was performed as previously described using the Illumina NextSeq 500 sequencing system (Illumina, San Diego). Sample sequence outputs ranged between

On the Home Front: Specialised Reference Testing for Dengue in the Australasian Region

Reference laboratories are vital for disease control and interpreting the complexities and impact of emerging pathogens. The role of these centralized facilities extends beyond routine screening capabilities to provide rapid, specific, and accurate diagnoses, advanced data analysis, consultation services, and sophisticated disease surveillance and monitoring. Within the Australasian region, the Public Health Virology Laboratory (PHV), Forensic and Scientific Services, Department of Health, Queensland Government, Australia, and the Institute of Environmental Science and Research Limited (ESR), New Zealand (NZ) perform specialised reference testing and surveillance for dengue viruses (DENVs) and other emerging arthropod-borne viruses (arboviruses), including chikungunya virus (CHIKV) and Zika virus (ZIKV). With a focus on DENV, we review the reference testing performed by PHV (2005 to 2017) and ESR (2008 to 2017). We also describe how the evolution and expansion of reference-based methodologies and the adoption of new technologies have provided the critical elements of preparedness and early detection that complement frontline public health control efforts and limit the spread of arboviruses within Australasia.