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.

A Case of Japanese Encephalitis with a Fatal Outcome in an Australian Who Traveled from Bali in 2019.

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.

A recombinant platform for flavivirus vaccines and diagnostics using chimeras of a new insect-specific virus.

Flaviviruses such as dengue, yellow fever, Zika, West Nile, and Japanese encephalitis virus present substantial global health burdens. New vaccines are being sought to address safety and manufacturing issues associated with current live attenuated vaccines. Here, we describe a new insect-specific flavivirus, Binjari virus, which was found to be remarkably tolerant for exchange of its structural protein genes (prME) with those of the aforementioned pathogenic vertebrate-infecting flaviviruses (VIFs). Chimeric BinJ/VIF-prME viruses remained replication defective in vertebrate cells but replicated with high efficiency in mosquito cells. Cryo-electron microscopy and monoclonal antibody binding studies illustrated that the chimeric BinJ/VIF-prME virus particles were structurally and immunologically similar to their parental VIFs. Pilot manufacturing in C6/36 cells suggests that high yields can be reached up to 109.5 cell culture infectious dose/ml or ≈7 mg/liter. BinJ/VIF-prME viruses showed utility in diagnostic (microsphere immunoassays and ELISAs using panels of human and equine sera) and vaccine applications (illustrating protection against Zika virus challenge in murine IFNAR-/- mouse models). BinJ/VIF-prME viruses thus represent a versatile, noninfectious (for vertebrate cells), high-yield technology for generating chimeric flavivirus particles with low biocontainment requirements.

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.