A Wolbachia symbiont in Aedes aegypti limits infection with dengue, Chikungunya, and Plasmodium.

Wolbachia are maternally inherited intracellular bacterial symbionts that are estimated to infect more than 60% of all insect species. While Wolbachia is commonly found in many mosquitoes it is absent from the species that are considered to be of major importance for the transmission of human pathogens. The successful introduction of a life-shortening strain of Wolbachia into the dengue vector Aedes aegypti that halves adult lifespan has recently been reported. Here we show that this same Wolbachia infection also directly inhibits the ability of a range of pathogens to infect this mosquito species. The effect is Wolbachia strain specific and relates to Wolbachia priming of the mosquito innate immune system and potentially competition for limiting cellular resources required for pathogen replication. We suggest that this Wolbachia-mediated pathogen interference may work synergistically with the life-shortening strategy proposed previously to provide a powerful approach for the control of insect transmitted diseases.

Locally acquired lymphocytic choriomeningitis virus infections in South-East Queensland: an outbreak of a pathogen rarely described in Australia.

Lymphocytic choriomeningitis virus (LCMV) is a zoonotic virus that can cause clinically significant illnesses in humans. Although cases of LCMV infection are well described globally, and there is evidence that the virus is present in Australian rodent populations, there has been only one case of domestically acquired LCMV infection published previously. Here, we describe a cluster of LCMV infections in South-East Queensland identified in early 2021, and the diagnostic testing processes implemented. This identifies LCMV as an under-recognised human pathogen in Australia.

Epidemiologic, Entomologic, and Virologic Factors of the 2014-15 Ross River Virus Outbreak, Queensland, Australia.

Australia experienced its largest recorded outbreak of Ross River virus (RRV) during the 2014-15 reporting year, comprising >10,000 reported cases. We investigated epidemiologic, entomologic, and virologic factors that potentially contributed to the scale of the outbreak in Queensland, the state with the highest number of notifications (6,371). Spatial analysis of human cases showed that notifications were geographically widespread. In Brisbane, human case notifications and virus detections in mosquitoes occurred across inland and coastal locations. Viral sequence data demonstrated 2 RRV lineages (northeastern genotypes I and II) were circulating, and a new strain containing 3 unique amino acid changes in the envelope 2 protein was identified. Longitudinal mosquito collections demonstrated unusually high relative abundance of Culex annulirostris and Aedes procax mosquitoes, attributable to extensive freshwater larval habitats caused by early and persistent rainfall during the reporting year. Increased prevalence of these mosquitoes probably contributed to the scale of this outbreak.

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.

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.

Neutralizing monoclonal antibodies to the E2 protein of chikungunya virus protects against disease in a mouse model.

Chikungunya virus (CHIKV) recently caused the largest epidemic ever recorded for this virus involving an estimated 1.4-6.5million cases, with imported cased reported in over 40 countries. The number of monoclonal antibodies specific for this re-emerging alphavirus is currently limited. Herein we describe the generation and characterisation of five monoclonal antibodies specific for the E2 glycoprotein of CHIKV. The antibodies detected a range of CHIKV isolates in several assays including ELISA, Western blot, immunofluorescence assay (IFA) and immunohistochemistry (IHC) without evidence of cross-reactivity with other alphaviruses. Four antibodies also neutralised CHIKV in vitro, two of which provided complete protection against arthritis in a CHIKV mouse model when administered prior to infection. Given the current shortage of widely available reagents for CHIKV, these specific antibodies will be useful not only in research, but may also provide the basis for new diagnostics and treatments.

Genomic Analysis of Sindbis Virus Reveals Uncharacterized Diversity within the Australasian Region, and Support for Revised SINV Taxonomy.

Sindbis virus (SINV) is a widely dispersed mosquito-borne alphavirus. Reports of Sindbis disease are largely restricted to northern Europe and South Africa. SINV is frequently sampled in Australian mosquito-based arbovirus surveillance programs, but human disease has rarely been reported. Molecular epidemiological studies have characterized six SINV genotypes (G1-G6) based on E2 gene phylogenies, mostly comprising viruses derived from the African-European zoogeographical region and with limited representation of Australasian SINV. In this study, we conducted whole genome sequencing of 66 SINV isolates sampled between 1960 and 2014 from countries of the Australasian region: Australia, Malaysia, and Papua New Guinea. G2 viruses were the most frequently and widely sampled, with three distinct sub-lineages defined. No new G6 SINV were identified, confirming geographic restriction of these viruses to south-western Australia. Comparison with global SINV characterized large-scale nucleotide and amino acid sequence divergence between African-European G1 viruses and viruses that circulate in Australasia (G2 and G3) of up to 26.83% and 14.55%, respectively, divergence that is sufficient for G2/G3 species demarcation. We propose G2 and G3 are collectively a single distinct alphavirus species that we name Argyle virus, supported by the inapparent or mild disease phenotype and the higher evolutionary rate compared with G1. Similarly, we propose G6, with 24.7% and 12.61% nucleotide and amino acid sequence divergence, is a distinct alphavirus species that we name Thomson's Lake virus.

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.

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.

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.

Temperature modulates immune gene expression in mosquitoes during arbovirus infection.

The principal vector of dengue, Zika and chikungunya viruses is the mosquito Aedes aegypti, with its ability to transmit pathogens influenced by ambient temperature. We use chikungunya virus (CHIKV) to understand how the mosquito transcriptome responds to arbovirus infection at different ambient temperatures. We exposed CHIKV-infected mosquitoes to 18, 28 and 32°C, and found that higher temperature correlated with higher virus levels, particularly at 3 days post infection, but lower temperature resulted in reduced virus levels. RNAseq analysis indicated significantly altered gene expression levels in CHIKV infection. The highest number of significantly differentially expressed genes was observed at 28°C, with a more muted effect at the other temperatures. At the higher temperature, the expression of many classical immune genes, including Dicer-2, was not substantially altered in response to CHIKV. The upregulation of Toll, IMD and JAK-STAT pathways was only observed at 28°C. Functional annotations suggested that genes in immune response and metabolic pathways related to energy supply and DNA replication were involved in temperature-dependent changes. Time post infection also led to substantially different gene expression profiles, and this varied with temperature. In conclusion, temperature significantly modulates mosquito gene expression in response to infection, potentially leading to impairment of immune defences at higher temperatures.

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.

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.

Metagenomic Analysis of the Virome of Mosquito Excreta.

Traditional screening for arboviruses in mosquitoes requires a priori knowledge and the utilization of appropriate assays for their detection. Mosquitoes can also provide other valuable information, including unexpected or novel arboviruses, nonarboviral pathogens ingested from hosts they feed on, and their own genetic material. Metagenomic analysis using next-generation sequencing (NGS) is a rapidly advancing technology that allows us to potentially obtain all this information from a mosquito sample without any prior knowledge of virus, host, or vector. Moreover, it has been recently demonstrated that pathogens, including arboviruses and parasites, can be detected in mosquito excreta by molecular methods. In this study, we investigated whether RNA viruses could be detected in mosquito excreta by NGS. Excreta samples were collected from Aedes vigilax and Culex annulirostris experimentally exposed to either Ross River or West Nile viruses and from field mosquitoes collected across Queensland, Australia. Total RNA was extracted from the excreta samples, reverse transcribed to cDNA, and sequenced using the Illumina NextSeq 500 platform. Bioinformatic analyses from the generated reads demonstrate that mosquito excreta provide sufficient RNA for NGS, allowing the assembly of near-full-length viral genomes. We detected Australian Anopheles totivirus, Wuhan insect virus 33, and Hubei odonate virus 5 and identified seven potentially novel viruses closely related to members of the order Picornavirales (2/7) and to previously described, but unclassified, RNA viruses (5/7). Our results suggest that metagenomic analysis of mosquito excreta has great potential for virus discovery and for unbiased arbovirus surveillance in the near future.IMPORTANCE When a mosquito feeds on a host, it ingests not only its blood meal but also an assortment of microorganisms that are present in the blood, thus acting as an environmental sampler. By using specific tests, it is possible to detect arthropod-borne viruses (arboviruses) like dengue and West Nile viruses in mosquito excreta. Here, we explored the use of next-generation sequencing (NGS) for unbiased detection of RNA viruses present in excreta from experimentally infected and field-collected mosquitoes. We have demonstrated that mosquito excreta provide a suitable template for NGS and that it is possible to recover and assemble near-full-length genomes of both arboviruses and insect-borne viruses, including potentially novel ones. These results importantly show the direct practicality of the use of mosquito excreta for NGS, which in the future could be used for virus discovery, environmental virome sampling, and arbovirus surveillance.

Arboviruses isolated from mosquitoes collected from urban and peri-urban areas of eastern Australia.

To determine the presence of arboviruses in mosquito populations from major urban areas of eastern Australia, a total of 67,825 mosquitoes, representing -60 species, was collected and tested from Cairns, Brisbane, and Sydney between January 2005 and April 2008. Mosquito pools were screened by inoculation onto mosquito cell cultures and the detection of viral antigen using a panel of flavivirus and alphavirus monoclonal antibodies in an enzyme-linked immunosorbent assay. Suspect positive samples were confirmed using virus-specific real-time reverse transcriptase-polymerase chain reaction assays. No flaviviruses were detected, but 2 alphaviruses were isolated from mosquito pools collected from Cairns, with 1 Barmah Forest virus isolate from a pool of 100 Aedes vigilax and 1 Ross River virus isolate from a pool of 83 Verrallina carmenti. In addition, a single Aedes alternans collected from Sydney yielded an isolate most similar to Stretch Lagoon virus, a newly described virus from the genus Orbivirus. These results suggest that during the study, arboviruses were circulating at a low level in the areas sampled. The findings from this study will promote public health awareness of the risk of arboviruses in urban areas, leading to more informative public health campaigns to safeguard the Australian public.

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.

Erratum: Pyke, A.T. et al. On the Home Front: Specialized Reference Testing for Dengue in the Australasian Region. Trop. Med. Infect. Dis. 2018, 3, 75.

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

Japanese Encephalitis Virus in Australia: From Known Known to Known Unknown.

Japanese encephalitis virus (JEV) is a major cause of neurological disease in Asia. It is a zoonotic flavivirus transmitted between water birds and/or pigs by Culex mosquitoes; humans are dead-end hosts. In 1995, JEV emerged for the first time in northern Australia causing an unprecedented outbreak in the Torres Strait. In this article, we revisit the history of JEV in Australia and describe investigations of JEV transmission cycles in the Australian context. Public health responses to the incipient outbreak included vaccination and sentinel pig surveillance programs. Virus isolation and vector competence experiments incriminated Culex annulirostris as the likely regional vector. The role this species plays in transmission cycles depends on the availability of domestic pigs as a blood source. Experimental evidence suggests that native animals are relatively poor amplifying hosts of JEV. The persistence and predominantly annual virus activity between 1995 and 2005 suggested that JEV had become endemic in the Torres Strait. However, active surveillance was discontinued at the end of 2005, so the status of JEV in northern Australia is unknown. Novel mosquito-based surveillance systems provide a means to investigate whether JEV still occurs in the Torres Strait or is no longer a risk to Australia.

Chikungunya Virus Transmission at Low Temperature by Aedes albopictus Mosquitoes.

Aedes albopictus is an important vector of chikungunya virus (CHIKV). In Australia, Ae. albopictus is currently only known to be present on the islands of the Torres Strait but, should it invade the mainland, it is projected to spread to temperate regions. The ability of Australian Ae. albopictus to transmit CHIKV at the lower temperatures typical of temperate areas has not been assessed. Ae. albopictus mosquitoes were orally challenged with a CHIKV strain from either Asian or East/Central/South African (ECSA) genotypes (107 pfu/mL), and maintained at a constant temperature of either 18 °C or 28 °C. At 3- and 7-days post-infection (dpi), CHIKV RNA copies were quantified in mosquito bodies, and wings and legs using real time polymerase chain reaction (qRT-PCR), while the detection of virus in saliva (a proxy for transmission) was performed by amplification in cell culture followed by observation of cytopathic effect in Vero cells. Of the ≥95% of Ae. albopictus that survived to 7 dpi, all mosquitoes became infected and showed body dissemination of CHIKV at both temperatures and time points. Both the Asian and ECSA CHIKV genotypes were potentially transmissible by Australian Ae. albopictus at 28 °C within 3 days of oral challenge. In contrast, at 18 °C none of the mosquitoes showed evidence of ability to transmit either genotype of CHIKV at 3 dpi. Further, at 18 °C only Ae. albopictus infected with the ECSA genotype showed evidence of virus in saliva at 7 dpi. Overall, infection with the ECSA CHIKV genotype produced higher virus loads in mosquitoes compared to infection with the Asian CHIKV genotype. Our results suggest that lower ambient temperatures may impede transmission of some CHIKV strains by Ae. albopictus at early time points post infection.