First Isolation of Japanese Encephalitis Virus Genotype IV from Mosquitoes in Australia.

Introduction: Widespread transmission of Japanese encephalitis virus (JEV) genotype four (GIV) occurred across mainland Australia in 2022. This resulted in forty-five human cases, including seven deaths, and the identification of JEV infection in over 80 commercial piggeries. Materials and Methods: We collected mosquitoes which were trapped using CO2-baited light traps deployed near piggeries reporting disease or in regions linked to human cases in the Wide Bay region in the state of Queensland. Mosquitoes from four traps yielded JEV RNA by real-time RT-PCR. Pools containing RNA positive mosquitoes were inoculated onto mosquito cell monolayers. Discussion: A single isolate of JEV was obtained from a pool of mixed mosquito species. Near whole genome sequencing and phylogenetic analysis of the JEV isolate demonstrated its high genomic relatedness with JEV GIV pig sequences sampled from Queensland and the state of New South Wales in 2022. Conclusion: We report the first isolation of JEV GIV from mosquitoes collected in Australia. With only a few JEV GIV isolates available globally, the isolate we report will be essential for future research of JEV host interactions, evolution and disease markers, and development of effective therapies, vaccines, diagnostic assays, and mosquito control strategies.

Uncovering the genetic diversity within the Aedes notoscriptus virome and isolation of new viruses from this highly urbanised and invasive mosquito.

The Australian backyard mosquito, Aedes notoscriptus, is a highly urbanised pest species that has invaded New Zealand and the USA. Importantly, Ae. notoscriptus has been implicated as a vector of Ross River virus, a common and arthritogenic arbovirus in Australia, and is a laboratory vector of numerous other pathogenic viruses, including West Nile, yellow fever, and Zika viruses. To further explore live viruses harboured by field populations of Ae. notoscriptus and, more specifically, assess the genetic diversity of its virome, we processed 495 pools, comprising a total of 6,674 female Ae. notoscriptus collected across fifteen suburbs in Brisbane, Australia, between January 2018 and May 2019. Nine virus isolates were recovered and characterised by metagenomic sequencing and phylogenetics. The principal viral family represented was Flaviviridae. Known viruses belonging to the genera Flavivirus, Orbivirus, Mesonivirus, and Nelorpivirus were identified together with two novel virus species, including a divergent Thogoto-like orthomyxovirus and an insect-specific flavivirus. Among these, we recovered three Stratford virus (STRV) isolates and an isolate of Wongorr virus (WGRV), which for these viral species is unprecedented for the geographical area of Brisbane. Thus, the documented geographical distribution of STRV and WGRV, both known for their respective medical and veterinary importance, has now been expanded to include this major urban centre. Phylogenies of the remaining five viruses, namely, Casuarina, Ngewotan, the novel Thogoto-like virus, and two new flavivirus species, suggested they are insect-specific viruses. None of these viruses have been previously associated with Ae. notoscriptus or been reported in Brisbane. These findings exemplify the rich genetic diversity and viral abundance within the Ae. notoscriptus virome and further highlight this species as a vector of concern with the potential to transmit viruses impacting human or animal health. Considering it is a common pest and vector in residential areas and is expanding its global distribution, ongoing surveillance, and ecological study of Ae. notoscriptus, together with mapping of its virome and phenotypic characterisation of isolated viruses, is clearly warranted. Immanently, these initiatives are essential for future understanding of both the mosquito virome and the evolution of individual viral species.

Replication Kinetics of B.1.351 and B.1.1.7 SARS-CoV-2 Variants of Concern Including Assessment of a B.1.1.7 Mutant Carrying a Defective ORF7a Gene.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, is a readily transmissible and potentially deadly pathogen which is currently re-defining human susceptibility to pandemic viruses in the modern world. The recent emergence of several genetically distinct descendants known as variants of concern (VOCs) is further challenging public health disease management, due to increased rates of virus transmission and potential constraints on vaccine effectiveness. We report the isolation of SARS-CoV-2 VOCs imported into Australia belonging to the B.1.351 lineage, first described in the Republic of South Africa (RSA), and the B.1.1.7 lineage originally reported in the United Kingdom, and directly compare the replication kinetics of these two VOCs in Vero E6 cells. In this analysis, we also investigated a B.1.1.7 VOC (QLD1516/2021) carrying a 7-nucleotide deletion in the open reading frame 7a (ORF7a) gene, likely truncating and rendering the ORF7a protein of this virus defective. We demonstrate that the replication of the B.1.351 VOC (QLD1520/2020) in Vero E6 cells can be detected earlier than the B.1.1.7 VOCs (QLD1516/2021 and QLD1517/2021), before peaking at 48 h post infection (p.i.), with significantly higher levels of virus progeny. Whilst replication of the ORF7a defective isolate QLD1516/2021 was delayed longer than the other viruses, slightly more viral progeny was produced by the mutant compared to the unmutated isolate QLD1517/2021 at 72 h p.i. Collectively, these findings contribute to our understanding of SARS-CoV-2 replication and evolutionary dynamics, which have important implications in the development of future vaccination, antiviral therapies, and epidemiological control strategies for COVID-19.

Infection, Dissemination, and Replication of Urban and Sylvatic Strains of Dengue Virus Type 2 (Flaviviridae: Flavivirus) in Australian Aedes aegypti (Diptera: Culicidae).

The dengue viruses (DENVs) occur throughout tropical and subtropical regions of the world where they infect 100s of millions of people annually. In Australia, the dengue receptive zone is confined to the northern state of Queensland where the principal vector Aedes aegypti (L.) is present. In the current study, two populations of Ae. aegypti from north Queensland were exposed to two urban outbreak strains and one sylvatic strain of dengue virus type 2 (DENV-2). The titer of virus required to infect 50% of mosquitoes was between 105 and 106 50% tissue culture infectious dose (TCID)50/ml and was influenced by the combination of the origin of Ae. aegypti population and virus strain. When exposed to infectious bloodmeal titers > 106 TCID50/ml, infection and dissemination rates were all > 50% and were significantly affected by the origin of the mosquito population but not by the strain of DENV-2. Replication of DENV-2 was also significantly affected by the mosquito population and the titer of the infectious bloodmeal that mosquitoes were exposed to. The results of this study are discussed in the context of DENV transmission dynamics in northern Australia and the relative fitness of the sylvatic virus strain in urban Ae. aegypti populations.

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.

Genome Sequences of Chikungunya Virus Strains from Bangladesh and Thailand.

We sequenced the genomes of two chikungunya virus isolates obtained from viremic patients who had traveled to Australia. The first patient acquired the infection in Bangladesh in 2017, and the second was infected in Thailand in 2019. Phylogenetic sequence analysis demonstrated that both isolates belonged to the East/Central/South African genotype.

Inflammatory responses to a pathogenic West Nile virus strain.

BACKGROUND: West Nile virus (WNV) circulates across Australia and was referred to historically as Kunjin virus (WNVKUN). WNVKUN has been considered more benign than other WNV strains circulating globally. In 2011, a more virulent form of the virus emerged during an outbreak of equine arboviral disease in Australia. METHODS: To better understand the emergence of this virulent phenotype and the mechanism by which pathogenicity is manifested in its host, cells were infected with either the virulent strain (NSW2012), or less pathogenic historical isolates, and their innate immune responses compared by digital immune gene expression profiling. Two different cell systems were used: a neuroblastoma cell line (SK-N-SH cells) and neuronal cells derived from induced pluripotent stem cells (iPSCs). RESULTS: Significant innate immune gene induction was observed in both systems. The NSW2012 isolate induced higher gene expression of two genes (IL-8 and CCL2) when compared with cells infected with less pathogenic isolates. Pathway analysis of induced inflammation-associated genes also indicated generally higher activation in infected NSW2012 cells. However, this differential response was not paralleled in the neuronal cultures. CONCLUSION: NSW2012 may have unique genetic characteristics which contributed to the outbreak. The data herein is consistent with the possibility that the virulence of NSW2012 is underpinned by increased induction of inflammatory genes.

First Complete Genome Sequences of Dengue Virus Serotype 2 Strains from the Solomon Islands and Vanuatu.

Isolates of dengue virus serotype 2 (DENV-2) were recovered from a female resident of the Solomon Islands in 2016 and another female patient who had traveled from Vanuatu to Australia in 2017. Here, we describe the first complete genome sequences of DENV-2 strains from Vanuatu and the Solomon Islands.

First Reported Complete Genome Sequence of a Dengue Virus Serotype 4 Strain from Papua New Guinea.

A male patient in his 50s who traveled from Papua New Guinea (PNG) to Australia in 2016 was diagnosed with a dengue virus serotype 4 (DENV-4) infection, and the virus was isolated from his acute-phase serum. Here, we describe the first complete genome sequence of a DENV-4 strain from PNG.

Genome Sequences of Coxsackievirus B5 Isolates from Two Children with Meningitis in Australia.

Two coxsackievirus B5 (CVB5) strains were isolated from two children with aseptic meningitis in Australia. Their genomes were sequenced and found to be divergent from the previously reported CVB5 genome sequences, with both having 84% and 97% identities to the closest strains at the nucleotide and amino acid levels, respectively.

FTA Cards Facilitate Storage, Shipment, and Detection of Arboviruses in Infected Aedes aegypti Collected in Adult Mosquito Traps.

AbstractThe utility of applying infected Aedes aegypti to Flinders Technology Associates (FTA®) cards for storage, transport, and detection of dengue, Zika, and Barmah Forest viruses was assessed in laboratory-based experiments. The mosquitoes had been removed from Gravid Aedes Traps maintained under conditions of high temperature and humidity. RNA of all viruses could be detected in infected mosquitoes on FTA cards either individually or in pools with uninfected mosquitoes, and stored for up to 28 days. Importantly, there was only a minimal decrease in RNA levels in mosquitoes between days 0 and 28, indicating that viral RNA was relatively stable on the cards. FTA cards thus provide a mechanism for storing potentially infected mosquitoes collected in the field and transporting them to a central diagnostic facility for virus detection.