Current Status and Future Perspectives on MRNA Drug Manufacturing.

The coronavirus disease of 2019 (COVID-19) pandemic launched an unprecedented global effort to rapidly develop vaccines to stem the spread of the novel severe acute respiratory syndrome coronavirus (SARS-CoV-2). Messenger ribonucleic acid (mRNA) vaccines were developed quickly by companies that were actively developing mRNA therapeutics and vaccines for other indications, leading to two mRNA vaccines being not only the first SARS-CoV-2 vaccines to be approved for emergency use but also the first mRNA drugs to gain emergency use authorization and to eventually gain full approval. This was possible partly because mRNA sequences can be altered to encode nearly any protein without significantly altering its chemical properties, allowing the drug substance to be a modular component of the drug product. Lipid nanoparticle (LNP) technology required to protect the ribonucleic acid (RNA) and mediate delivery into the cytoplasm of cells is likewise modular, as are technologies and infrastructure required to encapsulate the RNA into the LNP. This enabled the rapid adaptation of the technology to a new target. Upon the coattails of the clinical success of mRNA vaccines, this modularity will pave the way for future RNA medicines for cancer, gene therapy, and RNA engineered cell therapies. In this review, trends in the publication records and clinical trial registrations are tallied to show the sharp intensification in preclinical and clinical research for RNA medicines. Demand for the manufacturing of both the RNA drug substance (DS) and the LNP drug product (DP) has already been strained, causing shortages of the vaccine, and the rise in development and translation of other mRNA drugs in the coming years will exacerbate this strain. To estimate demand for DP manufacturing, the dosing requirements for the preclinical and clinical studies of the two approved mRNA vaccines were examined. To understand the current state of mRNA-LNP production, current methods and technologies are reviewed, as are current and announced global capacities for commercial manufacturing. Finally, a vision is rationalized for how emerging technologies such as self-amplifying mRNA, microfluidic production, and trends toward integrated and distributed manufacturing will shape the future of RNA manufacturing and unlock the potential for an RNA medicine revolution.

Accurate identification of Australian mosquitoes using protein profiling.

Australian mosquito species significantly impact human health through nuisance biting and the transmission of endemic and exotic pathogens. Surveillance programmes designed to provide an early warning of mosquito-borne disease risk require reliable identification of mosquitoes. This study aimed to investigate the viability of Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) as a rapid and inexpensive approach to the identification of Australian mosquitoes and was validated using a three-step taxonomic approach. A total of 300 mosquitoes representing 21 species were collected from south-eastern New South Wales and morphologically identified. The legs from the mosquitoes were removed and subjected to MALDI-TOF MS analysis. Fifty-eight mosquitoes were sequenced at the cytochrome c oxidase subunit I (cox1) gene region and genetic relationships were analysed. We create the first MALDI-TOF MS spectra database of Australian mosquito species including 19 species. We clearly demonstrate the accuracy of MALDI-TOF MS for identification of Australian mosquitoes. It is especially useful for assessing gaps in the effectiveness of DNA barcoding by differentiating closely related taxa. Indeed, cox1 DNA barcoding was not able to differentiate members of the Culex pipiens group, Cx. quinquefasciatus and Cx. pipiens molestus, but these specimens were correctly identified using MALDI-TOF MS.

Anncaliia algerae Microsporidial Myositis, New South Wales, Australia.

We describe the successful management of Anncaliia algerae microsporidial myositis in a man with graft versus host disease after hemopoietic stem cell transplantation. We also summarize clinical presentation and management approaches and discuss the importance of research into the acquisition of this infection and strategies for prevention.

New genotypes of Liao ning virus (LNV) in Australia exhibit an insect-specific phenotype.

Liao ning virus (LNV) was first isolated in 1996 from mosquitoes in China, and has been shown to replicate in selected mammalian cell lines and to cause lethal haemorrhagic disease in experimentally infected mice. The first detection of LNV in Australia was by deep sequencing of mosquito homogenates. We subsequently isolated LNV from mosquitoes of four genera (Culex, Anopheles, Mansonia and Aedes) in New South Wales, Northern Territory, Queensland and Western Australia; the earliest of these Australian isolates were obtained from mosquitoes collected in 1988, predating the first Chinese isolates. Genetic analysis revealed that the Australian LNV isolates formed two new genotypes: one including isolates from eastern and northern Australia, and the second comprising isolates from the south-western corner of the continent. In contrast to findings reported for the Chinese LNV isolates, the Australian LNV isolates did not replicate in vertebrate cells in vitro or in vivo, or produce signs of disease in wild-type or immunodeficient mice. A panel of human and animal sera collected from regions where the virus was found in high prevalence also showed no evidence of LNV-specific antibodies. Furthermore, high rates of virus detection in progeny reared from infected adult female mosquitoes, coupled with visualization of the virus within the ovarian follicles by immunohistochemistry, suggest that LNV is transmitted transovarially. Thus, despite relatively minor genomic differences between Chinese and Australian LNV strains, the latter display a characteristic insect-specific phenotype.

Discovery of new orbiviruses and totivirus from Anopheles mosquitoes in Eastern Australia.

Three new viruses classifiable within the Totivirus and Orbivirus genera were detected from Anopheles mosquito species collected in Eastern Australia. The viruses could not be isolated in C6/36 mosquito cell cultures but were shown to replicate in their mosquito hosts by small RNA analysis. The viruses grouped phylogenetically with other viruses recently detected in insects. These discoveries contribute to a better understanding of commensal viruses in Australian mosquitoes and the evolution of these viruses.

EFFECTS OF SUGAR CONCENTRATION ON FECUNDITY, BITING BEHAVIOR AND SURVIVABILITY OF FEMALE AEDES (STEGOMYIA) ALBOPICTUS (SKUSE).

This study was conducted to better understand the effect of different sucrose concentrations on Aedes albopictus fecundity, biting behavior and survival. Laboratory strain Ae. albopictus females were raised at four different sucrose concentrations (10%, 30%, 50%, and 70%) and their fecundity, host biting on and survival rates were determined. Mosquitoes fed on high (50% or 70%) showed higher mean fecundity rate compared to those on low (10% or 30%) sucrose concentration, and had higher daily biting rate. On the other hand, mosquitoes fed on the low (10% or 30%) sucrose concentrations recorded higher survival rate. These results suggest female mosquitoes deficient in nutrient intake during sugar feeding may regain nutrients needed during blood feeding, whereas those fed on high sucrose concentration have high fecundity due to high biting rate but have low survivability due to low sucrose intake during sugar feeding. Thus, Ae. albopictus females have a capability to regulate their metabolic needs based on sugar nutrient availability.

Anncaliia algerae microsporidial myositis.

The insect microsporidian Anncaliia algerae was first described in 2004 as a cause of fatal myositis in an immunosuppressed person from Pennsylvania, USA. Two cases were subsequently reported, and we detail 2 additional cases, including the only nonfatal case. We reviewed all 5 case histories with respect to clinical characteristics, diagnosis, and management and summarized organism life cycle and epidemiology. Before infection, all case-patients were using immunosuppressive medications for rheumatoid arthritis or solid-organ transplantation. Four of the 5 case-patients were from Australia. All diagnoses were confirmed by skeletal muscle biopsy; however, peripheral nerves and other tissues may be infected. The surviving patient received albendazole and had a reduction of immunosuppressive medications and measures to prevent complications. Although insects are the natural hosts for A. algerae, human contact with water contaminated by spores may be a mode of transmission. A. algerae has emerged as a cause of myositis, particularly in coastal Australia.

An evaluation of the landscape structure and La Niña climatic anomalies associated with Japanese encephalitis virus outbreaks reported in Australian piggeries in 2022.

The widespread activity of Japanese encephalitis virus (JEV) reported in previously unaffected regions of eastern and southern Australia in 2022 represents the most significant local arbovirus emergency in almost 50 years. Japanese encephalitis virus is transmitted by mosquitoes and maintained in wild ardeid birds and amplified in pigs, the latter of which suffer significant reproductive losses as a result of infection. The landscape epidemiology of JEV in mainland Australia is almost entirely unknown, particularly in the eastern and southern parts of the country where the virus has not been previously documented. Although other areas with endemic JEV circulation in the Indo-Pacific region have demonstrated the importance of wild waterbird-livestock interface in agricultural-wetland mosaics, no such investigation has yet determined the composition and configuration of pathogenic landscapes for Australia. Moreover, the recent emergence in Australia has followed substantial precipitation and temperature anomalies associated with the La Niña phase of the El Niño Southern Oscillation. This study investigated the landscape epidemiology of JEV outbreaks in Australian piggeries reported between January and April of 2022 to determine the influence of ardeid habitat suitability, hydrogeography, hydrology, land cover and La Niña-associated climate anomalies. Outbreaks of JEV in domestic pigs were associated with intermediate ardeid species richness, cultivated land and grassland fragmentation, waterway proximity, temporary wetlands, and hydrological flow accumulation. This study has identified the composition and configuration of landscape features that were associated with piggery outbreaks reported in 2022 in Australia. Although preliminary, these findings can inform actionable strategies for the development of new One Health JEV surveillance specific to the needs of Australia.

An efficient and biodegradable alginate-gelatin hydrogel beads as bait against Aedes aegypti and Aedes albopictus.

The effective control of Aedes mosquitoes using traditional control agents is increasingly challenging due to the presence of insecticide resistance in many populations of key mosquito vectors. An alternative strategy to insecticides is the use of toxic sugar baits, however it is limited due to short-term efficacy. Alginate-Gelatin hydrogel beads (AGHBs) may be an effective alternative by providing longer periods of mosquito attraction and control, especially of key vectors of dengue viruses such as Aedes aegypti and Aedes albopictus. Sodium alginate (ALG) and gelatin (GLN) are natural polymers, which can be a potential candidate to develop the AGHBs baits due to their biodegradability and environmental safety. Here we provide an assessment of the preparation of AGHBs optimized by varying the concentrations of ALG, GLN, and its cross-linking time (TIME). Fourier transform infrared spectroscopy (FTIR) analysis results in the determination of liquid bait loaded in the AGHBs. The evaluation of AGHBs' effectiveness as the potential baiting tool based on the mortality rate of mosquitoes after the bait consumption. The 100 % percent mortality of Aedes mosquitoes was obtained within 72 h of bait consumption. The field evaluation also justifies the applicability of AGHBs for outdoor applications. We conclude that the AGHBs are applicable as a baiting tool in carrying liquid bait in achieving mosquito mortality.

Non-canonical inflammasome activation mediates the adjuvanticity of nanoparticles.

The non-canonical inflammasome sensor caspase-11 and gasdermin D (GSDMD) drive inflammation and pyroptosis, a type of immunogenic cell death that favors cell-mediated immunity (CMI) in cancer, infection, and autoimmunity. Here we show that caspase-11 and GSDMD are required for CD8+ and Th1 responses induced by nanoparticulate vaccine adjuvants. We demonstrate that nanoparticle-induced reactive oxygen species (ROS) are size dependent and essential for CMI, and we identify 50- to 60-nm nanoparticles as optimal inducers of ROS, GSDMD activation, and Th1 and CD8+ responses. We reveal a division of labor for IL-1 and IL-18, where IL-1 supports Th1 and IL-18 promotes CD8+ responses. Exploiting size as a key attribute, we demonstrate that biodegradable poly-lactic co-glycolic acid nanoparticles are potent CMI-inducing adjuvants. Our work implicates ROS and the non-canonical inflammasome in the mode of action of polymeric nanoparticulate adjuvants and establishes adjuvant size as a key design principle for vaccines against cancer and intracellular pathogens.

Does the monomolecular film aquatain mosquito formula provide effective control of container-breeding mosquitoes in Australia?

The mosquito control potential of the silicone-based monomolecular film Aquatain Mosquito Formula (AMF) was investigated in field tests against the backyard mosquitoes Aedes notoscriptus and Culex quinquefasciatus. Plastic tubs, with and without emergent aquatic vegetation (Cyperus alternifolius), were sampled weekly for 2 wk prior to an application of Aquatain and up to 6 wk postapplication. The mean abundance of mosquito larvae and pupae was compared between pre- and postapplication periods as well as between treatment and control tubs. There was a significant reduction in the abundance of immature stages of both Ae. notoscriptus and Cx. quinquefasciatus within 48 h of application, and the mean weekly abundance of larvae of both species was significantly lower in treatment tubs compared with control tubs for up to 6 wk postapplication. Egg rafts, larvae, and pupae were not detected in treatment tubs until 5 wk postapplication. The results indicate that AMF holds great potential for mosquito control in backyard habitats.

The importance of males: larval diet and adult sugar feeding influences reproduction in Culex molestus.

Culex molestus is an obligatory autogenous mosquito that is closely associated with subterranean habitats in urban areas. The objective of our study was to investigate the influence of larval and adult nutrition on the role of males in determining the expression of autogeny in Cx. molestus. Mosquitoes raised at low and high larval diets had sex ratio, wing length, mating rates, autogenous egg raft size, and hatching rates recorded. There was a higher ratio of males to females when raised at a low larval diet. Mean wing lengths of both males and females were significantly greater when raised at the high larval diet regime. Regardless of larval or adult diet, males mated with only a single female. Mosquitoes raised at the higher larval diet regimes developed significantly more autogenous eggs. However, the egg raft size was reduced when adult females were denied access to sugar. The results of this study indicate that the performance of males in the reproductive process is influenced by both larval diet and adult sugar feeding.

Citizen Science Mosquito Surveillance by Ad Hoc Observation Using the iNaturalist Platform.

Citizen science mosquito surveillance has been growing in recent years due to both increasing concern about mosquito-borne disease and the increasing popularity of citizen science projects globally. Health authorities are recognising the potential importance of citizen science to expanding or enhancing traditional surveillance programs. Different programs have shown success in engaging communities to monitor species of medical importance through low-cost methods. The Mozzie Monitors project was established on iNaturalist-an open citizen science platform that allows participants to upload photos (i.e., observers) and assist identification (i.e., identifiers). This article describes the likelihood of citizen scientists submitting photos of mosquitoes, assesses user submission behaviour, and evaluates public health utility from these citizen science-derived data. From October 2018 to July 2021, the Mozzie Monitors project on iNaturalist received 2118 observations of 57 different species of mosquitoes across Australia. The number of observers in the system increased over time with more than 500 observers and 180 identifiers being active in the project since its establishment. Data showed species bias with large-bodied and colourful mosquitoes being over-represented. Analyses also indicate regional differentiation of mosquito fauna per state, seasonality of activity, and ecological information about mosquitoes. The iNaturalist citizen science platform also allows connectedness, facilitated communication and collaboration between overall users and expert entomologists, of value to medical entomology and mosquito management.

A biogeographical description of the wild waterbird species associated with high-risk landscapes of Japanese encephalitis virus in India.

Wild reservoirs of Japanese encephalitis virus are under-studied globally, which presents critical knowledge gaps for JEV epidemiology and infection ecology despite decades of received wisdom regarding this high-impact mosquito-borne virus. As a result, ardeid birds, generally understood to be the primary reservoirs for JEV, as well as other waterbirds occupying landscapes at high risk for spillover to humans, are frequently ignored by current surveillance mechanisms and infrastructure. This is particularly true in India, which experiences a high annual burden of human outbreaks. Incorporating wild reservoirs into surveillance of human and livestock populations is therefore essential but will first require a data-driven approach to target individual host species. The current study sought to identify preliminary waterbird target species for JEV surveillance development based on species' distributions in high-risk landscapes. Twenty-one target species were identified after adjusting species presence and abundance for the biotic constraints of sympatry. Furthermore, ardeid bird species richness demonstrated a strong non-linear association with the distribution of human JEV outbreaks, which suggested areas with the highest ardeid species richness corresponded to low JEV outbreak risk. No association was identified between JEV outbreaks and anatid or rallid richness. The lack of association between Anatidae and Rallidae family-level diversity and JEV outbreak risk notwithstanding, this study did identify several individual species among these two bird families in high-risk landscapes. The findings from this work provide the first data-driven evidence base to inform wildlife sampling for the monitoring of JEV circulation in outbreak hotspots in India and thus identify good preliminary targets for the development of One Health JEV surveillance.

High-risk landscapes of Japanese encephalitis virus outbreaks in India converge on wetlands, rain-fed agriculture, wild Ardeidae, and domestic pigs and chickens.

BACKGROUND: Japanese encephalitis virus (JEV) is a zoonotic mosquito-borne virus that causes a significant burden of disease across Asia, particularly in India, with high mortality in children. JEV circulates in wild ardeid birds and domestic pig reservoirs, both of which generate sufficiently high viraemias to infect vector mosquitoes, which can then subsequently infect humans. The landscapes of these hosts, particularly in the context of anthropogenic ecotones and resulting wildlife-livestock interfaces, are poorly understood and thus significant knowledge gaps in the epidemiology of JEV persist. This study sought to investigate the landscape epidemiology of JEV outbreaks in India over the period 2010-2020 to determine the influence of shared wetland and rain-fed agricultural landscapes and animal hosts on outbreak risk. METHODS: Using surveillance data from India's National Centre for Disease Control Integrated Disease Surveillance Programme, JEV outbreaks were modelled as an inhomogeneous Poisson point process and externally validated against independently sourced data. RESULTS: Outbreak risk was strongly associated with the habitat suitability of ardeid birds, both pig and chicken density, and the shared landscapes between fragmented rain-fed agriculture and both river and freshwater marsh wetlands. CONCLUSION: The results from this work provide the most complete understanding of the landscape epidemiology of JEV in India to date and suggest important One Health priorities for control and prevention across fragmented terrain comprising a wildlife-livestock interface that favours spillover to humans.

Japanese Encephalitis Virus Emergence in Australia: Public Health Importance and Implications for Future Surveillance.

Japanese encephalitis virus (JEV) continues to cause significant numbers of human infections and fatalities despite the availability of efficacious vaccines. It is regarded as an emerging mosquito-borne pathogen with the potential of introduction into many countries. In 2022, JEV was detected in Australia on a hitherto unprecedented scale, with local transmission by indigenous mosquitoes to amplifying swine hosts and to humans. In this study, we review this recent disease activity, propose possible routes of virus movement, ecological drivers of activity, and consider possible future transmission scenarios. Measures to enhance current surveillance systems and potential strategies for health authorities to minimize future risks are discussed.

Weather extremes associated with increased Ross River virus and Barmah Forest virus notifications in NSW: learnings for public health response.

OBJECTIVE: To examine the sequence of environmental and entomological events prior to a substantial increase in Ross River virus (RRV) and Barmah Forest virus (BFV) notifications with a view to informing future public health response. METHODS: Rainfall, tidal, mosquito and human arboviral notification data were analysed to determine the temporality of events. RESULTS: Following two extremely dry years, there was a substantial increase in the abundance of mosquitoes along coastal New South Wales (NSW) two weeks after a significant rainfall event and high tides in February 2020. Subsequently, RRV and BFV notifications in north east NSW began to increase eight and nine weeks respectively after the high rainfall, with RRV notifications peaking 12 weeks after the high rainfall. CONCLUSIONS: Mosquito bite avoidance messaging should be instigated within two weeks of high summer rainfall, especially after an extended dry period. IMPLICATIONS FOR PUBLIC HEALTH: Intense summertime rain events, which are expected to increase in frequency in south-east Australia with climate change, can lead to significant increases in arboviral disease. These events need to be recognised by public health practitioners to facilitate timely public health response. This has taken on added importance since the emergence of Japanese encephalitis virus in southeastern Australia in 2022.

Supporting urban ecosystem services across terrestrial, marine and freshwater realms.

The terrestrial, freshwater and marine realms all provide essential ecosystem services in urban environments. However, the services provided by each realm are often considered independently, which ignores the synergies between them and risks underestimating the benefits derived collectively. Greater research collaboration across these realms, and an integrated approach to management decisions can help to support urban developments and restoration projects in maintaining or enhancing ecosystem services. The aim of this paper is to highlight the synergies and trade-offs among ecosystem services provided by each realm and to offer suggestions on how to improve current practice. We use case studies to illustrate the flow of services across realms. In our call to better integrate research and management across realms, we present a framework that provides a 6-step process for conducting collaborative research and management with an Australian perspective. Our framework considers unifying language, sharing, and understanding of desired outcomes, conducting cost-benefit analyses to minimise trade-offs, using multiple modes of communication for stakeholders, and applying research outcomes to inform regulation. It can be applied to improve collaboration among researchers, managers and planners from all realms, leading to strategic allocation of resources, increased protection of urban natural resources and improved environmental regulation with broad public support.

Response of Mosquitoes Associated with Estuarine Wetlands to Bushfire in Australia.

The response of mosquitoes to bushfire is poorly understood. During the 2019-20 summer, many regions of Australia were impacted by devastating bushfires. An area of estuarine and brackish-water wetlands alongside the Georges River, Sydney, New South Wales, was burned in January 2020. Mosquito populations within the area were monitored as part of the local authority's mosquito management program, providing a unique opportunity to record the response of key mosquitoes of pest and public health concern to bushfire. Ground pools within a tidally influenced swamp oak forest dominated by Casuarina glauca and associated wetlands dominated by Phragmites australis and Bolboschoenus spp. had been identified as suitable habitat for a range of mosquitoes, including Aedes alternans, Ae. vigilax, and Verrallina funerea. Surveys of immature stages of mosquitoes within recently burned habitats inundated by tides demonstrated that mosquito eggs survived the direct and indirect impacts of fire and immature stages successfully completed development as reflected in concomitant changes in adult mosquito populations following the bushfire. This unique observation has implications for mosquito management following bushfire in Australia and internationally.