We describe emergence of Buruli ulcer in urban Geelong, Victoria, Australia, and examine timing and proximity of human cases to detection of Mycobacterium ulcerans DNA in possum feces. M. ulcerans-positive feces preceded human cases by up to 39 months, constituting an early warning of impending risk for Buruli ulcer.

Current arbovirus surveillance strategies in Australia involve mosquito collection, species identification, and virus detection. These processes are labour-intensive, expensive, and time-consuming and can lead to delays in reporting. Mosquito excreta has been proposed as an alternative sample type to whole mosquito collection, with potential to streamline the virus surveillance pipeline. In this study, we investigated the feasibility of Aedes aegypti excreta as a sample type in the detection of Dengue virus serotype 2 (DENV2). DENV2 could be detected from as little as one DENV2-infected mosquito excreta spot, with virus levels in individual excreta spots varying within and between mosquitoes and depending highly on mosquito viral load. Detectability was improved by pooling up to 20 DENV2-infected mosquitoes and collecting excreta into liquid substrate, followed by virus concentration using magnetic nanoparticles. Virus concentration improves quantification accuracy in comparison to unconcentrated samples and increases the amount of material available for detection, expanding detection capabilities to techniques with higher limits of detection. Mosquito excreta as a sample type, coupled with magnetic virus concentration, expands the current detection toolbox for DENV2 and has the potential to improve arbovirus surveillance strategies in Australia.

In April 2024, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was expanded by 1 new order, 1 new family, 6 new subfamilies, 34 new genera and 270 new species. One class, two orders and six species were renamed. Seven families and 12 genera were moved; ten species were renamed and moved; and nine species were abolished. This article presents the updated taxonomy of Negarnaviricota as currently accepted by the ICTV, providing an essential annual update on the classification of members of this phylum that deepen understandings of their evolution, and supports critical public health measures for virus identification and tracking.

Graphical abstract A metatranscriptomics study of a dominant bluetongue virus vector, Culicoides brevitarsis from Australia, identified 10 complete/near-complete viral genomes including 7 novel species.

Abstract Buruli ulcer (BU) is a necrotizing disease of skin and soft tissue caused by the bacterium Mycobacterium ulcerans (MU). In Australia, where the disease is emerging in new geographic areas and human case numbers are increasing, native possum species act as reservoir hosts. To better understand the life history of MU in one of its natural hosts, we conducted intra-dermal challenge of six wild caught, MU-naïve common ringtail possums (Pseudocheirus peregrinus). All six animals developed BU disease consistent with that observed in naturally infected ringtail possums. Time to ulceration varied between 49 and 77 days (mean = 61.8 days). Molecular evidence of systemic infection was detected in five animals and was supported by consistent histopathological findings in four animals. Pathological findings included random, multifocal, granulomatous hepatitis in four possums, one of which also had a mild, multifocal, interstitial granulomatous pneumonia. Acid-fast bacilli were only evident in inflammatory foci beyond the primary inoculation site in one possum. The ringtail possum model of MU infection is an important tool for the investigation of bacterial transmission dynamics, pathogenesis and immune response in a natural host. Data from this model may improve disease risk modelling and help identify intervention points to stop zoonotic transmission and disease spread.

Buruli ulcer (BU) endemicity is changing in Victoria, Australia. This study describes the emergence of BU cases in urban Geelong and examines timing and proximity of human cases to environmental Mycobacterium ulcerans DNA detection in possum faeces. From 2011-2022, 80 BU cases were reported, with clusters emerging in new endemic areas from 2019 onwards. Temporal analysis revealed cases occurred near M. ulcerans positive possum faeces, that preceded human cases by up to 39 months. Median diagnosis delay in these emerging areas was 7 weeks, longer than in established BU endemic areas. The findings emphasise the geographic and temporal association between BU emergence and possum faecal M. ulcerans presence, suggesting monitoring possum faeces as a viable early warning sign of increased BU risk in such areas. This proactive approach can enable early action to improve clinician and community BU awareness, or development of innovative control strategies, to reduce disease burden.### Competing Interest StatementThe authors have declared no competing interest.### Funding StatementThe data collection and analysis of the possum faecal surveys were funded as part of the NHMRC Partnership Project Grant 2018-2020 (GNT1152807).### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:Barwon Health Human Research Ethics Committee gave ethical approval for this work.I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesSharing of de-identified human case data used in this publication may be requested from the Department of Health Victoria (infectious.diseases@health.vic.gov.au), contingent on approval of an analysis plan by this Department and relevant ethical approval. Possum faecal data may be shared, after publication, on a collaborative basis upon reasonable request made to Dr Kim Blasdell (kim.blasdell@csiro.au).

The novel coronavirus disease (COVID-19) and any future coronavirus outbreaks will require more affordable, effective and safe treatment options to complement current ones such as Paxlovid . Drug repurposing can be a promising approach if we are able to find a rapid, robust and reliable way to down-select and screen candidates using in silico and in vitro approaches. With repurposed drugs, ex vivo models could offer a rigorous route to human clinical trials with less time invested into nonclinical animal ( in vivo ) studies. We have previously shown the value of commercially available ex vivo/3D airway and alveolar tissue models, and this paper takes this further by developing and validating human nasal epithelial model and embryonic stem cells derived cardiomyocyte model. Five shortlisted candidates (fluvoxamine, everolimus, pyrimethamine, aprepitant and sirolimus) were successfully compared with three control drugs (remdesivir, molnupiravir, nirmatrelvir) when tested against key variants of the SARS-CoV-2 virus including Delta and Omicron, and we were able to reconfirm our earlier finding that fluvoxamine can induce antiviral efficacy in combination with other drugs. Scalability of this high-throughput screening approach has been demonstrated using a liquid handling robotic platform for future ‘Disease-X’ outbreaks. ### Competing Interest Statement The authors have declared no competing interest.

Ticks are important medical and veterinary parasites that represent a substantial health threat to humans, companion animals, and livestock. Ixodiphagus wasps (Hymenoptera; Encyrtidae) are known endoparasitoids of ixodid (hard) and argasid (soft) ticks, with potential utility as natural biocontrol agents. Two species, Ixodiphagus brunneus and Ixodiphagus mysorensis, are previously recorded from Australia, however, the genus lacks formal revisionary work in Australia, and the validity and host ranges of these species remain uncertain. This work aimed to investigate the diversity of Ixodiphagus in Australasia and provide a molecular data resource for future work on these understudied endoparasitoids. We extracted DNA from archival Ixodiphagus specimens from Australian and New Zealand insect collections and performed high-throughput sequencing which resulted in complete or mostly complete mitochondrial genome sequences from 11 specimens, including I. brunneus, Ixodiphagus taiaroaensis, and a novel Ixodiphagus sp. reared from Rhipicephalus linnaei from Townsville, Australia. In addition, approximately 70% of the genome of the Wolbachia endosymbiont of I. brunneus was recovered. Finally, we screened 178 recently collected pooled tick samples from southern New South Wales, Australia, for Ixodiphagus spp. using 28S rRNA and cytochrome c oxidase subunit 1(COI) gene PCR, and recovered 14 positive samples. Phylogenetic analysis of Australasian Ixodiphagus spp. based on 28S rRNA and complete mitochondrial genome sequences determined that members of the Australasian fauna are distinct from Ixodiphagus hookeri (the only other Ixodiphagus species for which genetic data exists), and that at least two distinct species are present in Australia; I. brunneus identified from Ixodes holocyclus and Haemaphysalis bancrofti ticks, and an uncharacterised Ixodiphagus sp. found in Rhipicephalus linnaei ticks from northern Queensland. Furthermore, there was substantial genetic diversity at the 28S rRNA loci among I. brunneus samples, which may represent normal genetic variability or a secondary cryptic species. The molecular data generated here represents the first known for the genus Ixodiphagus in Australasia, doubling that of the world fauna, and provides the first known complete mitochondrial genomes for these important tick parasitoids.

Japanese encephalitis virus (JEV) is transmitted by Culex species of mosquitoes. In 2022, JEV belonging to a previously unrecognized lineage of genotype IV (GIV) caused a major outbreak of JE in South-eastern Australia, resulting in human cases and affecting piggeries. Cx. annulirostris has previously been implicated as the major vector of JEV in northern Australia where the virus has circulated since its first detection in 1995. Here, we showed that experimental infection of a laboratory colony of Australian Cx. annulirostris with the isolate JEV NSW/22 resulted in a 100% mosquito infection rate, with 87% of mosquito saliva samples testing positive by RT-qPCR at 14 days post-infection. Immunohistochemistry confirmed the presence of a replicating virus in the mosquito midgut and dissemination throughout the body, including the salivary glands. Our results also showed evidence of transovarial transmission of this virus; however, transstadial transmission from the eggs to the adult stage was not found. Comparison with an Indonesian isolate of GIV JEV and previous Australian isolates belonging to genotypes I and II showed that infection with JEV NSW/22 resulted in higher viral titres in the early stage of infection and higher proportions of mosquitoes with JEV-positive saliva, indicating a greater transmission potential compared to other isolates. This study provides compelling experimental evidence that Australian Cx. annulirostris is a highly efficient vector for the 2022 Australian JEV GIV outbreak strain.

Buruli ulcer, a chronic subcutaneous infection caused by Mycobacterium ulcerans , is increasing in prevalence in southeastern Australia. Possums are a local wildlife reservoir for M. ulcerans and, although mosquitoes have been implicated in transmission, it remains unclear how humans acquire infection. We conducted extensive field survey analyses of M. ulcerans prevalence among mosquitoes in the Mornington Peninsula region of southeastern Australia. PCR screening of trapped mosquitoes revealed a significant association between M. ulcerans and Aedes notoscriptus . Spatial scanning statistics revealed overlap between clusters of M. ulcerans -positive Ae. notoscriptus , M. ulcerans -positive possum excreta and Buruli ulcer cases, and metabarcoding analyses showed individual mosquitoes had fed on humans and possums. Bacterial genomic analysis confirmed shared single-nucleotide-polymorphism profiles for M. ulcerans detected in mosquitoes, possum excreta and humans. These findings indicate Ae. notoscriptus probably transmit M. ulcerans in southeastern Australia and highlight mosquito control as a Buruli ulcer prevention measure.

Tick-borne haemoparasites, including piroplasms and trypanosomes, are almost ubiquitous in Australian wildlife, with some associated with health impacts to individual animals and declining wildlife populations. An array of ecologically distinct piroplasm and trypanosome species occur throughout Australia although many of these species and their sylvatic ecologies are poorly characterised. Between May 2022 and October 2023, an anecdotally reported localised eastern grey kangaroo (Macropus giganteus) morbidity/mortality event occurred in coastal southern New South Wales, Australia, characterised by animals presenting with blindness, emaciation, lethargy, ataxia, and astasia. Here we used molecular techniques to identify tick-borne piroplasms (Babesia and Theileria) and trypanosomes in affected animals. Blood (n=89) and liver (n=19) samples were collected after the humane euthanasia of wild animals due to welfare concerns, and brief notes on the animal’s health were recorded. In total, 20 (22.5%) animals were infected with tick-borne haemoparasites, including a novel Theileria sp. nov. (14, 15.7%), Babesia macropus (2, 2.2%), Trypanosoma gilletti (5, 5.6%), and Trypanosoma vegrandis (1, 1.1%). Liver samples were also screened for Wallal and Warego viruses due to animals’ blindness, but were negative. This is the first report of T. gilletti and T. vegrandis in eastern grey kangaroos, although they have been previously reported in high numbers in ticks which commonly parasites this host. The novel Theileria sp. was previously reported in questing Ixodes holocyclus and in ticks from an opportunistically collected eastern grey kangaroo and red-necked wallaby (Notamacropus rufogriseus). However, we show for the first time this Theileria sp. can occur widely in eastern grey kangaroos. Ultimately, this small study did not intend, and is not able to draw inference regarding the pathogenicity of these haemoparasites to eastern grey kangaroos and it is likely that other factors, such as chronic Phalaris grass toxicity, had a role in this localised mortality/morbidity event.

Mosquitoes of the Culex genus are responsible for a large burden of zoonotic virus transmission globally. Collectively, they play a significant role in the transmission of medically significant diseases such as Japanese encephalitis virus and West Nile virus. Climate change, global trade, habitat transformation and increased urbanisation are leading to the establishment of Culex mosquitoes in new geographical regions. These novel mosquito incursions are intensifying concerns about the emergence of Culex-transmitted diseases and outbreaks in previously unaffected areas. New mosquito control methods are currently being developed and deployed globally. Understanding the complex interaction between pathogens and mosquitoes is essential for developing new control strategies for Culex species mosquitoes. This article reviews the role of Culex mosquitos as vectors of zoonotic disease, discussing the transmission of viruses across different species, and the potential use of Wolbachia technologies to control disease spread. By leveraging the insights gained from recent successful field trials of Wolbachia against Aedes-borne diseases, we comprehensively discuss the feasibility of using this technique to control Culex mosquitoes and the potential for the development of next generational Wolbachia-based control methods.

In April 2023, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by one new family, 14 new genera, and 140 new species. Two genera and 538 species were renamed. One species was moved, and four were abolished. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.

Buruli ulcer (BU) is a flesh-eating infection caused by Mycobacterium ulcerans that leads to significant community morbidity, with an estimated cost to Victoria in 2016 of more than $2.5 million.1 It was first recognised in Victoria, Australia, in 1948.2 Until the turn of the century, it was only reported in low numbers and a few localised regions, but the BU epidemic in Victoria has shown a consistent upward trend in recent years. In 2022, reported case numbers increased to 340, the equal highest annual case load recorded and more than five times the number reported 10 years previously. Alarmingly, the disease is also moving into new areas — in the past 5 years it has moved into a coastal region south of Geelong (Surf Coast), with urban outbreaks in the regional city of Geelong and in the inner north-western suburbs of Melbourne. The proportion of severe cases at diagnosis is also on the rise — since 2010, the proportion of people with infection presenting with World Health Organization category 2 or 3 lesions has increased from 20% to about 30%.3 Efforts to implement public health interventions to address this have been hindered by the lack of a clear understanding of the environmental reservoir and transmission mechanisms of M. ulcerans.1 An important discovery in the mid-2000s identified that possum faecal specimens (both common ringtail [Pseudocheirus peregrinus] and common brushtail [Trichosurus vulpecula] possums) were M. ulcerans DNA positive in the newly endemic area of Point Lonsdale, Victoria.4 On further assessment, 33% of captured possums either had evidence of clinical disease or were asymptomatic but had positive excreta. The rate of BU lesions or positive excreta was higher in ringtail (38%) than in brushtails (24%) possums. Interestingly, M. ulcerans isolates from possums were genetically indistinguishable from those from Victorian BU patients.5 More comprehensive One Health surveillance has found that the presence of M. ulcerans positive possum faecal samples correlates geographically with the occurrence of human BU cases, and the detection of positive samples in a new area can herald the onset of human cases in the next one to two years.6 Further evidence for the role of possums in BU transmission comes from a large comprehensive case–control study across Victoria's endemic regions from 2018 to 2020.7 This showed a greatly increased risk of BU among people reporting the presence of possums on their property, with a dose–response effect suggested by more possums reported correlating with a higher risk of BU. Additionally, when environmental samples were collected from case and control properties, a high proportion of possum faeces (mainly ringtail) was found to be M. ulcerans DNA positive, and a large percentage was shown to contain viable M. ulcerans that suggests transmissibility.8 Other environmental features associated with possums, such as known food sources, habitat and mobility, were also associated with detection of M. ulcerans at the property. Finally, to support possums carrying viable M. ulcerans organisms that can be transmitted to humans, a case was recently reported where a man bitten while helping a sick ringtail possum developed a BU lesion at the bite site within 6 months.9 The case for possums being a zoonotic reservoir and being involved in the transmission of M. ulcerans is now strong in Victorian endemic areas. Considering the urgent need to deal with the worsening Victorian BU epidemic, the challenge is how we can utilise this knowledge to reduce M. ulcerans transmission. Preventing contact between possums and humans via public education campaigns to increase awareness of potential risks, especially if possums are sick or unwell, may help. However, possums have adapted well to suburban settings, including gardens where their faeces can cover much of the ground, making limiting contact (direct or indirect) very difficult. Culling is not an appropriate solution as they are a protected species in Victoria and form an important part of the natural ecosystem, with unintended consequences likely to result if their social structure is disrupted. Culling would also likely result in significant public anger. We propose that research be undertaken to explore the effectiveness and feasibility of vaccinating ringtail possums with an oral bait bacille Calmette–Guérin (BCG) vaccine in the wild. If effective in reducing BU disease in possums, it should hopefully simultaneously reduce the occurrence in humans. Furthermore, it would benefit possums, as BU can lead to death, and this would be a safe intervention that could reduce both morbidity and mortality in possums. The aim would be to manage this zoonotic pathogen in its reservoir possum host with benefits for human and animal health, without damaging the local ecosystem. Oral bait vaccines have been used to control other diseases in wildlife such as rabies in Europe and North America and classical swine flu virus in Europe.10 Specifically, the use of oral bait BCG vaccine has been successfully employed to control bovine tuberculosis in wildlife such as badgers in Ireland and the United Kingdom, deer in North America, and wild boars in Spain.11 Most importantly, researchers in New Zealand, where bovine tuberculosis is a major problem in non-native Australian brushtail possums, have shown that they can successfully vaccinate possums with oral bait BCG and induce immunity to bovine tuberculosis lasting more than two years.10 This was effective both in laboratory studies and in wide-scale field trials, with up to a 95% reduction in the prevalence of bovine tuberculosis in vaccinated wild possum populations.12 It is reasonable to expect that the intervention would be equally effective in both ringtail and brushtail possums. Furthermore, BCG vaccine has been shown to be safe for numerous livestock and wildlife species. Whether BCG vaccination of ringtail possums could protect them against M. ulcerans infection has not been evaluated, but mice vaccinated with BCG are protected against BU.13 Two large randomised controlled trials of BCG vaccination in humans for the prevention of BU conducted in Uganda during the late 1960s and early 1970s found significant but short-lasting protection against BU.14, 15 In a recent case–control study in Victoria, a history of BCG vaccine was associated with a 40% reduction in the risk of BU (odds ratio, 0.6; 95% CI, 0.4–0.9).7 Although BCG vaccination of humans in endemic areas could also be considered, vaccinating possums would have the added benefits of protecting everyone in endemic areas regardless of vaccine status, protecting visitors living outside the area (Victorian endemic regions are high tourism areas), and providing health benefits for possums. Additionally, as possums likely play a role in introducing the disease into new areas, it may stop disease spread. Furthermore, the uptake of oral bait BCG by non-target species could be beneficial as M. ulcerans infection has been detected in a wide range of mammalian species in Australia.1 The accidental vaccination of these animals could therefore contribute to overall host immunity within the endemic ecosystem and subsequently reduce the risk to humans. The proposed research would involve several stages. The first would be to establish housing and husbandry conditions for ringtail possums in a research facility. Once established, this would be followed by attempts to develop a M. ulcerans infection model using both high dose (~ 103 colony-forming units) and low dose (~ 20–30 colony-forming units) M. ulcerans inocula, with monitoring of excreta and clinical signs of disease to determine infective dose and susceptibility to infection, incubation periods, immunological responses, and the routes and magnitude of M. ulcerans shedding. If successful, possums would then be vaccinated with BCG before M. ulcerans challenge, to assess the level and durability of BU protection by comparing vaccine with control groups. Blood samples would be collected to measure the immune responses to BCG vaccination, which would be correlated with immune protection. If shown to be effective, attempts would be made to optimise the palatability and feasibility of oral bait BCG delivery. Finally, testing the effectiveness of oral bait BCG vaccine against M. ulcerans in ringtail possums would be performed in laboratory and real-life field settings. Data obtained from these studies could be used to assess whether possum vaccination would be cost-effective in controlling BU, provide information on the epidemiology of infection in possums, and help develop disease transmission models. Studies could also be extended to brushtail possums. Although mosquitoes are considered to play a role in transmission,1 and mosquito prevention measures might have an impact on human case numbers, evidence for their effectiveness is lacking. It is therefore important to explore other interventions that may reduce transmission, such as possum vaccination.7 In south-eastern Australia, BU is a One Health challenge involving a complex interaction between the environment, insects, wildlife and humans, with many knowledge gaps remaining. However, there are currently no proven public health interventions to address the worsening epidemic of BU in this region. A proposal to vaccinate possums in the wild with oral bait BCG provides hope that an acceptable, safe, feasible and cost-effective intervention can be found that would benefit both human and possum populations by reducing the transmission of M. ulcerans and the incidence of BU in the region. Open access publishing facilitated by The University of Melbourne, as part of the Wiley – The University of Melbourne agreement via the Council of Australian University Librarians. No relevant disclosures. Not commissioned; externally peer reviewed.

Background:Buruli ulcer (BU) is a neglected tropical disease caused by infection of subcutaneous tissue with Mycobacterium ulcerans. BU is commonly reported across rural regions of Central and West Africa but has been increasing dramatically in temperate southeast Australia around the major metropolitan city of Melbourne, with most disease transmission occurring in the summer months. Previous research has shown that Australian native possums are reservoirs of M. ulcerans and that they shed the bacteria in their fecal material (excreta). Field surveys show that locales where possums harbor M. ulcerans overlap with human cases of BU, raising the possibility of using possum excreta surveys to predict the risk of disease occurrence in humans.Methods:We thus established a highly structured 12 month possum excreta surveillance program across an area of 350 km2 in the Mornington Peninsula area 70 km south of Melbourne, Australia. The primary objective of our study was to assess using statistical modeling if M. ulcerans surveillance of possum excreta provided useful information for predicting future human BU case locations.Results:Over two sampling campaigns in summer and winter, we collected 2,282 possum excreta specimens of which 11% were PCR positive for M. ulcerans-specific DNA. Using the spatial scanning statistical tool SaTScan, we observed non-random, co-correlated clustering of both M. ulcerans positive possum excreta and human BU cases. We next trained a statistical model with the Mornington Peninsula excreta survey data to predict the future likelihood of human BU cases occurring in the region. By observing where human BU cases subsequently occurred, we show that the excreta model performance was superior to a null model trained using the previous year’s human BU case incidence data (AUC 0.66 vs 0.55). We then used data unseen by the excreta-informed model from a new survey of 661 possum excreta specimens in Geelong, a geographically separate BU endemic area to the southwest of Melbourne, to prospectively predict the location of human BU cases in that region. As for the Mornington Peninsula, the excreta-based BU prediction model outperformed the null model (AUC 0.75 vs 0.50) and pinpointed specific locations in Geelong where interventions could be deployed to interrupt disease spread.Conclusions:This study highlights the One Health nature of BU by confirming a quantitative relationship between possum excreta shedding of M. ulcerans and humans developing BU. The excreta survey-informed modeling we have described will be a powerful tool for the efficient targeting of public health responses to stop BU.Funding:This research was supported by the National Health and Medical Research Council of Australia and the Victorian Government Department of Health (GNT1152807 and GNT1196396).

Escalating vector disease burdens pose significant global health risks, as such innovative tools for targeting mosquitoes are critical. CRISPR-Cas technologies have played a crucial role in developing powerful tools for genome manipulation in various eukaryotic organisms. Although considerable efforts have focused on utilizing class II type II CRISPR-Cas9 systems for DNA targeting, these modalities are unable to target RNA molecules, limiting their utility against RNA viruses. Recently, the Cas13 family has emerged as an efficient tool for RNA targeting; however, the application of this technique in mosquitoes, particularly Aedes aegypti, has yet to be fully realized. In this study, we engineered an antiviral strategy termed REAPER (vRNA Expression Activates Poisonous Effector Ribonuclease) that leverages the programmable RNA-targeting capabilities of CRISPR-Cas13 and its potent collateral activity. REAPER remains concealed within the mosquito until an infectious blood meal is uptaken. Upon target viral RNA infection, REAPER activates, triggering programmed destruction of its target arbovirus such as chikungunya. Consequently, Cas13-mediated RNA targeting significantly reduces viral replication and viral prevalence of infection, and its promiscuous collateral activity can even kill infected mosquitoes within a few days. This innovative REAPER technology adds to an arsenal of effective molecular genetic tools to combat mosquito virus transmission.

Hendra virus (HeV) and Nipah virus (NiV) are henipaviruses that can cause fatal encephalitis in humans. Many animal models have been used to study henipavirus pathogenesis. In the mouse, HeV infection has previously shown that intranasal challenge can lead to neurological infection, however mice similarly challenged with NiV show no evidence of virus infecting the brain. We generated recombinant HeV (rHeV) and NiV (rNiV) where selected proteins were switched to examine their role in neuroinvasion in the mouse. These viruses displayed similar growth kinetics when compared to wildtype in vitro. In the mouse, infection outcomes with recombinant virus did not differ to infection outcomes of wildtype viruses. Virus was detected in the brain of 5/30 rHeVchallenged mice, but not rNiV-challenged mice. To confirm the permissiveness of mouse neurons to these viruses, primary mouse neurons were successfully infected in vitro, suggesting that other pathobiological factors contribute to the differences in disease outcomes in mice.

To examine protective and risk factors for Buruli ulcer (BU), we conducted a case-control study of 245 adult BU cases and 481 postcode-matched controls across BU-endemic areas of Victoria, Australia. We calculated age- and sex-adjusted odds ratios for socio-environmental, host, and behavioral factors associated with BU by using conditional logistic regression. Odds of BU were >2-fold for persons with diabetes mellitus and persons working outdoors who had soil contact in BU-endemic areas (compared with indoor work) but were lower among persons who had bacillus Calmette-Guerin vaccinations. BU was associated with increasing numbers of possums and with ponds and bore water use at residences. Using insect repellent, covering arms and legs outdoors, and immediately washing wounds were protective; undertaking multiple protective behaviors was associated with the lowest odds of BU. Skin hygiene/ protection behaviors and previous bacillus Calmette-Guerin vaccination might provide protection against BU in BU-endemic areas.

AbstractIn temperate southeastern Australia over the past two decades there has been a marked progressive increase in human cases of Buruli ulcer, an infection of subcutaneous tissue caused byMycobacterium ulcerans. Native possums are the major local environmental reservoir ofM. ulceransas they not only develop Buruli lesions but they also shedM. ulceransin their excreta. However the way humans acquireM. ulceransfrom possums has not been determined. Previous case-control studies, insect field surveys and vector competence studies have suggested a role for mosquitoes inM. ulceranstransmission between possums and humans. To explore these links we conducted an extensive, 4-month structured mosquito field survey and fourad hocfield surveys across an area of 350km2on the Mornington Peninsula, an area endemic for Buruli ulcer to the south of the major metropolitan city of Melbourne. We then compared spatial and temporal patterns ofM. ulcerans-positive mosquito occurrence withM. ulcerans-positive possums (established by previous possum excreta surveys) and human Buruli ulcer cases across the region. We used metabarcoding to assess mosquito blood-feeding host preference and to reconstructM. ulceransgenomes from positive mosquitoes to test epidemiological inferences. We collected 66,325 mosquitoes spanning 26 different species from 180 repeatedly sampled traps over a 4-month period.Culex molestusandAedes notoscriptuswere the dominant species (42% and 35% of trapped mosquitoes, respectively). PCR screening 25% of trapped mosquitoes revealed a significant association betweenM. ulceransandAe. notoscriptus(p<0.0001) with a maximum likelihood estimate (MLE) of 5.88M. ulceranspositive mosquitoes per 1,000 tested. Using spatial scanning statistics, we also observed significant overlap between clusters ofM. ulcerans-positiveAe. notoscriptus,M. ulcerans-positive possum excreta and human Buruli ulcer cases. Metabarcoding analyses of blood-fedAe. notoscriptusshowed individual mosquitoes had fed both on humans and native possums. Enrichment genome sequencing from PCR-positive mosquitoes confirmed sharedM. ulceransgenome single-nucleotide polymorphism (SNP) profiles between mosquitoes, possum excreta and clinical human isolates within the same regions. These findings indicate thatAe. notoscriptuslikely transmitM. ulceransin southeastern Australia and highlight mosquito control as a plausible means to control the Buruli ulcer epidemic in our region.

The repurposing of licenced drugs for use against COVID-19 is one of the most rapid ways to develop new and alternative therapeutic options to manage the ongoing pandemic. Given circa 7817 licenced compounds available from Compounds Australia that can be screened, this paper demonstrates the utility of commercially available ex vivo/3D airway and alveolar tissue models. These models are a closer representation of in vivo studies than in vitro models, but retain the benefits of rapid in vitro screening for drug efficacy. We demonstrate that several existing drugs appear to show anti-SARS-CoV-2 activity against both SARS-CoV-2 Delta and Omicron Variants of Concern in the airway model. In particular, fluvoxamine, as well as aprepitant, everolimus, and sirolimus, has virus reduction efficacy comparable to the current standard of care (remdesivir, molnupiravir, nirmatrelvir). Whilst these results are encouraging, further testing and efficacy studies are required before clinical use can be considered.