Carmela T Taylor

A severe case of Japanese encephalitis virus (JEV) infection, resulting in fatality, occurred in an unvaccinated Australian male traveler from Bali, Indonesia, in 2019. During hospitalisation in Australia, patient cerebrospinal fluid (CSF) yielded JEV-specific IgM antibodies and RNA, and an isolate of the virus. Ongoing transmission of JEV in Bali underscores this pathogen as a public health risk and the importance of appropriate health, vaccination and mosquito avoidance advice to prospective travelers to the region.

The authors wish to make the following correction to Table 1 of this paper

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.

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.

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. 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 was10^6.4 tissue culture infectious dose₅₀ (TCID₅₀)/mL. Additionally, a threshold viral load within the mosquito of at least 10^5.1 TCID₅₀ equivalents/mL had to be reached before virus transmission occurred. 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. Zika virus was first isolated in Uganda in 1947 and exists in a transmission cycle between mosquitoes and non-human primates or humans. Whilst most clinical infections result in a self-limiting febrile illness, Zika virus has recently been linked to neurological syndromes, such as Guillain-Barré syndrome and congenital birth defects. Since 2007, Zika virus has undergone a dramatic range expansion, causing epidemics in nations and territories of the western Pacific and South America. To assess the emergence and transmission risk of Zika virus emerging in Australia, we evaluated the ability of local mosquitoes to become infected with and transmit the prototype African Zika virus strain. In agreement with its substantiated role in Zika virus transmission overseas, Australian Aedes aegypti were shown to be competent vectors. Coupled with its anthropophilic feeding behavior, this species should be considered the primary potential Zika virus vector in Australia. Although other common Australian species, such as Ae. notoscriptus and Ae. vigilax, were readily infected, they did not transmit the virus. The species of Culex tested were either refractory to infection or had a low infection rate. We also demonstrated that the Zika virus dose necessary to infect Ae. aegypti was higher than virus levels reported in infected humans. Finally, a high threshold level of virus circulating through the mosquito body was required before Ae. aegypti transmitted the virus. These results suggest that an outbreak of Zika virus in Australia would require high mosquito population densities and a susceptible human population.

Australian clinicians need to recognise Zika virus infection, given the potential for transmission in northern Australia

Dengue viruses (DENVs) are the leading cause of mosquito-borne viral disease of humans. They exist in both endemic and sylvatic ecotypes. In 2014, a viremic patient who had recently visited the rainforests of Brunei returned to Australia displaying symptoms consistent with DENV infection. A unique DENV strain was subsequently isolated from the patient, which we propose belongs to a new genotype within DENV serotype 1 (DENV-1). Bayesian evolutionary phylogenetic analysis suggests that the putative sylvatic DENV-1 Brunei 2014 (Brun2014) is the most divergent DENV-1 yet recorded and increases the time to the most recent common ancestor (MRCA) for DENV-1 from ≈120 years to ≈315 years. DENV-1 classification of the Brun2014 strain was further supported by monoclonal antibody serotyping data. Phenotypic characterization demonstrated that Brun2014 replication rates in mosquito cells and infection rates in Aedes aegypti mosquitoes were not significantly different from an epidemic DENV-1 strain. Given its ability to cause human illness and infect Ae. aegypti, potential urban spillover and clinical disease from further Brun2014 transmission cannot be discounted.

Chikungunya virus (CHIKV) is an arthropod-borne agent that causes severe arthritic disease in humans and is considered a serious health threat in areas where competent mosquito vectors are prevalent. CHIKV has recently been responsible for several millions of cases of disease, involving over 40 countries. The recent re-emergence of CHIKV and its potential threat to human health has stimulated interest in better understanding of the biology and pathogenesis of the virus, and requirement for improved treatment, prevention and control measures. In this study, we mapped the binding sites of a panel of eleven monoclonal antibodies (mAbs) previously generated towards the capsid protein (CP) of CHIKV. Using N- and C-terminally truncated recombinant forms of the CHIKV CP, two putative binding regions, between residues 1–35 and 140–210, were identified. Competitive binding also revealed that five of the CP-specific mAbs recognized a series of overlapping epitopes in the latter domain. We also identified a smaller, N-terminally truncated product of native CP that may represent an alternative translation product of the CHIKV 26S RNA and have potential functional significance during CHIKV replication. Our data also provides evidence that the C-terminus of CP is required for authentic antigenic structure of CP. This study shows that these anti-CP mAbs will be valuable research tools for further investigating the structure and function of the CHIKV CP.

A female resident of Townsville, Queensland, Australia has been diagnosed with Zika virus infection following a recent trip to the Cook Islands. An initial serum sample collected in March, 2014 was positive by two separate Zika virus TaqMan real-time RT-PCRs and a pan-Flavivirus RT-PCR. Nucleotide sequencing and phylogenetics of the complete Cook Islands Zika virus envelope gene revealed 99.1% homology with a previous Cambodia 2010 sequence within the Asian lineage. In addition, IgG and IgM antibody seroconversions were detected between paired acute and convalescent phase sera using recombinant Zika virus serology assays. This is the first known imported case of Zika virus infection into northern Queensland where the potential mosquito vector Aedes aegypti is present and only the second such reported case diagnosed within Australia.

Dengue outbreaks have increased in size and frequency in Australia, and transfusion-transmitted dengue poses a risk to transfusion safety. Using whole blood samples collected during the large 2008–2009 dengue epidemic, we estimated the risk for a dengue-infectious blood donation as ≈1 in 7,146 (range 2,218–50,021).