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.

Differences in gene expression in field populations of Wolbachia-infected Aedes aegypti mosquitoes with varying release histories in northern Australia.

Aedes aegypti is the principal mosquito vector of dengue, yellow fever, Zika and chikungunya viruses. The wMel strain of the endosymbiotic bacteria Wolbachia pipientis was introduced into the vector as a novel biocontrol strategy to stop transmission of these viruses. Mosquitoes with Wolbachia have been released in the field in Northern Queensland, Australia since 2011, at various locations and over several years, with populations remaining stably infected. Wolbachia infection is known to alter gene expression in its mosquito host, but whether (and how) this changes over the long-term in the context of field releases remains unknown. We sampled mosquitoes from Wolbachia-infected populations with three different release histories along a time gradient and performed RNA-seq to investigate gene expression changes in the insect host. We observed a significant impact on gene expression in Wolbachia-infected mosquitoes versus uninfected controls. Fewer genes had significantly upregulated expression in mosquitoes from the older releases (512 and 486 from the 2011 and 2013/14 release years, respectively) versus the more recent releases (1154 from the 2017 release year). Nonetheless, a fundamental signature of Wolbachia infection on host gene expression was observed across all releases, comprising upregulation of immunity (e.g. leucine-rich repeats, CLIPs) and metabolism (e.g. lipid metabolism, iron transport) genes. There was limited downregulation of gene expression in mosquitoes from the older releases (84 and 71 genes from the 2011 and 2013/14 release years, respectively), but significantly more in the most recent release (509 from the 2017 release year). Our findings indicate that at > 8 years post-introgression into field populations, Wolbachia continues to profoundly impact expression of host genes, such as those involved in insect immune response and metabolism. If Wolbachia-mediated virus blocking is underpinned by these differential gene expression changes, our results suggest it may remain stable long-term.

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.

Japanese Encephalitis Virus: The Emergence of Genotype IV in Australia and Its Potential Endemicity.

A fatal case of Japanese encephalitis (JE) occurred in northern Australia in early 2021. Sequence studies showed that the virus belonged to genotype IV (GIV), a genotype previously believed to be restricted to the Indonesian archipelago. This was the first locally acquired case of Japanese encephalitis virus (JEV) GIV to occur outside Indonesia, and the second confirmed fatal human case caused by a GIV virus. A closely related GIV JEV strain subsequently caused a widespread outbreak in eastern Australia in 2022 that was first detected by fetal death and abnormalities in commercial piggeries. Forty-two human cases also occurred with seven fatalities. This has been the first major outbreak of JEV in mainland Australia, and geographically the largest virgin soil outbreak recorded for JEV. This outbreak provides an opportunity to discuss and document the factors involved in the virus' spread and its ecology in a novel ecological milieu in which other flaviviruses, including members of the JE serological complex, also occur. The probable vertebrate hosts and mosquito vectors are discussed with respect to virus spread and its possible endemicity in Australia, and the need to develop a One Health approach to develop improved surveillance methods to rapidly detect future outbreak activity across a large geographical area containing a sparse human population. Understanding the spread of JEV in a novel ecological environment is relevant to the possible threat that JEV may pose in the future to other receptive geographic areas, such as the west coast of the United States, southern Europe or Africa.

Not all mosquitoes are created equal: A synthesis of vector competence experiments reinforces virus associations of Australian mosquitoes.

The globalization of mosquito-borne arboviral diseases has placed more than half of the human population at risk. Understanding arbovirus ecology, including the role individual mosquito species play in virus transmission cycles, is critical for limiting disease. Canonical virus-vector groupings, such as Aedes- or Culex-associated flaviviruses, have historically been defined using virus detection in field-collected mosquitoes, mosquito feeding patterns, and vector competence, which quantifies the intrinsic ability of a mosquito to become infected with and transmit a virus during a subsequent blood feed. Herein, we quantitatively synthesize data from 68 laboratory-based vector competence studies of 111 mosquito-virus pairings of Australian mosquito species and viruses of public health concern to further substantiate existing canonical vector-virus groupings and quantify variation within these groupings. Our synthesis reinforces current canonical vector-virus groupings but reveals substantial variation within them. While Aedes species were generally the most competent vectors of canonical "Aedes-associated flaviviruses" (such as dengue, Zika, and yellow fever viruses), there are some notable exceptions; for example, Aedes notoscriptus is an incompetent vector of dengue viruses. Culex spp. were the most competent vectors of many traditionally Culex-associated flaviviruses including West Nile, Japanese encephalitis and Murray Valley encephalitis viruses, although some Aedes spp. are also moderately competent vectors of these viruses. Conversely, many different mosquito genera were associated with the transmission of the arthritogenic alphaviruses, Ross River, Barmah Forest, and chikungunya viruses. We also confirm that vector competence is impacted by multiple barriers to infection and transmission within the mesenteron and salivary glands of the mosquito. Although these barriers represent important bottlenecks, species that were susceptible to infection with a virus were often likely to transmit it. Importantly, this synthesis provides essential information on what species need to be targeted in mosquito control programs.

The Emergence of Japanese Encephalitis Virus in Australia in 2022: Existing Knowledge of Mosquito Vectors.

In early 2022, the Japanese encephalitis virus (JEV) was identified as the cause of stillborn and mummified piglets in pig farms in southeastern Australia. Human cases and additional pig farms with infected piglets were subsequently identified across a widespread area encompassing four states. To inform surveillance and control programs, we synthesized existing information on Australian vectors of JEV, much of which was generated in response to incursions of JEV into the northern state of Queensland between 1995 and 2005. Members of the Culex sitiens subgroup, particularly Culex annulirostris, should be considered the primary vectors of JEV in Australia, as they yielded >87% of field detections of JEV, were highly efficient laboratory vectors of the virus, readily fed on pigs and birds (the key amplifying hosts of the virus) when they were available, and are widespread and often occur in large populations. Three introduced species, Culex quinquefasciatus, Culex gelidus and Culex tritaeniorhynchus may also serve as vectors, but more information on their geographical distribution, abundance and bionomics in the Australian context is required. Mosquitoes from other genera, such as Aedes and Verrallina, whilst considered relatively poor vectors, could play a regional or supplemental role in transmission, especially facilitating vertical transmission as a virus overwintering mechanism. Additional factors that could impact JEV transmission, including mosquito survival, dispersal and genetics, are also discussed. Possible directions for investigation are provided, especially in the context of the virus emerging in a region with different mosquito fauna and environmental drivers than northern Australia.

Rapid molecular assays for the detection of the four dengue viruses in infected mosquitoes.

The pantropic emergence of severe dengue disease can partly be attributed to the co-circulation of different dengue viruses (DENVs) in the same geographical location. Effective monitoring for circulation of each of the four DENVs is critical to inform disease mitigation strategies. In low resource settings, this can be effectively achieved by utilizing inexpensive, rapid, sensitive and specific assays to detect viruses in mosquito populations. In this study, we developed four rapid DENV tests with direct applicability for low-resource virus surveillance in mosquitoes. The test protocols utilize a novel sample preparation step, a single-temperature isothermal amplification, and a simple lateral flow detection. Analytical sensitivity testing demonstrated tests could detect down to 1,000 copies/µL of virus-specific DENV RNA, and analytical specificity testing indicated tests were highly specific for their respective virus, and did not detect closely related flaviviruses. All four DENV tests showed excellent diagnostic specificity and sensitivity when used for detection of both individually infected mosquitoes and infected mosquitoes in pools of uninfected mosquitoes. With individually infected mosquitoes, the rapid DENV-1, -2 and -3 tests showed 100% diagnostic sensitivity (95% CI = 69% to 100%, n=8 for DENV-1; n=10 for DENV 2,3) and the DENV-4 test showed 92% diagnostic sensitivity (CI: 62% to 100%, n=12) along with 100% diagnostic specificity (CI: 48-100%) for all four tests. Testing infected mosquito pools, the rapid DENV-2, -3 and -4 tests showed 100% diagnostic sensitivity (95% CI = 69% to 100%, n=10) and the DENV-1 test showed 90% diagnostic sensitivity (55.50% to 99.75%, n=10) together with 100% diagnostic specificity (CI: 48-100%). Our tests reduce the operational time required to perform mosquito infection status surveillance testing from > two hours to only 35 minutes, and have potential to improve accessibility of mosquito screening, improving monitoring and control strategies in low-income countries most affected by dengue outbreaks.

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.

Physiology and ecology combine to determine host and vector importance for Ross River virus.

Identifying the key vector and host species that drive the transmission of zoonotic pathogens is notoriously difficult but critical for disease control. We present a nested approach for quantifying the importance of host and vectors that integrates species' physiological competence with their ecological traits. We apply this framework to a medically important arbovirus, Ross River virus (RRV), in Brisbane, Australia. We find that vertebrate hosts with high physiological competence are not the most important for community transmission; interactions between hosts and vectors largely underpin the importance of host species. For vectors, physiological competence is highly important. Our results identify primary and secondary vectors of RRV and suggest two potential transmission cycles in Brisbane: an enzootic cycle involving birds and an urban cycle involving humans. The framework accounts for uncertainty from each fitted statistical model in estimates of species' contributions to transmission and has has direct application to other zoonotic pathogens.

Impact of COVID-19 Mitigation Measures on Mosquito-Borne Diseases in 2020 in Queensland, Australia.

We describe the impact of COVID-19 mitigation measures on mosquito-borne diseases in Queensland, Australia, during the first half of 2020. Implementation of restrictions coincided with an atypical late season outbreak of Ross River virus (RRV) characterized by a peak in notifications in April (1173) and May (955) which were greater than 3-fold the mean observed for the previous four years. We propose that limitations on human movement likely resulted in the majority of RRV infections being acquired at or near the place of residence, and that an increase in outdoor activities, such as gardening and bushwalking in the local household vicinity, increased risk of exposure to RRV-infected mosquitoes. In contrast, the precipitous decline in international passenger flights led to a reduction in the number of imported dengue and malaria cases of over 70% and 60%, respectively, compared with the previous five years. This substantial reduction in flights also reduced a risk pathway for importation of exotic mosquitoes, but the risk posed by importation via sea cargo was not affected. Overall, the emergence of COVID-19 has had a varied impact on mosquito-borne disease epidemiology in Queensland, but the need for mosquito surveillance and control, together with encouragement of personal protective measures, remains unchanged.

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.

The Insect-Specific Parramatta River Virus Is Vertically Transmitted by Aedes vigilax Mosquitoes and Suppresses Replication of Pathogenic Flaviviruses In Vitro.

Insect-specific flaviviruses (ISFs) have been isolated from a range of mosquito species from different parts of the world. These viruses replicate efficiently in mosquitoes but do not appear to replicate in vertebrates. There is increasing evidence that ISFs persist in nature through vertical transmission, and that they interfere with the replication and transmission of pathogenic flaviviruses in the mosquito host. A novel ISF species, Parramatta River virus (PaRV), was previously shown to occur at high rates in Aedes (Ae.) vigilax mosquitoes collected from Sydney, Australia. We investigated whether vertical transmission was the mechanism of viral persistence in Ae. vigilax populations and whether PaRV affected replication of the pathogenic flaviviruses, West Nile virus (WNV), and dengue virus type 3 (DENV-3) in cultured mosquito cells. Progeny reared from eggs obtained from field-collected infected females had infection rates as high as 142 and 85 per 1000 for females and males, respectively. In vitro experiments showed that replication of both WNV and DENV-3 was significantly suppressed in Aedes albopictus (C6/36) cells persistently infected with PaRV. Our studies with PaRV support the findings of previous investigations that ISFs persist in nature through vertical transmission and that ISFs can suppress the replication of pathogenic flaviviruses in coinfected mosquito cells.

Wolbachia Genome Stability and mtDNA Variants in Aedes aegypti Field Populations Eight Years after Release.

A dengue suppression strategy based on release of Aedes aegypti mosquitoes infected with the bacterium Wolbachia pipientis is being trialed in many countries. Wolbachia inhibits replication and transmission of dengue viruses. Questions remain regarding the long-term stability of virus-suppressive effects. We sequenced the Wolbachia genome and analyzed Ae. aegypti mitochondrial DNA markers isolated from mosquitoes sampled 2-8 years after releases in the greater Cairns region, Australia. Few changes were detected when Wolbachia genomes of field mosquitoes were compared with Wolbachia genomes of mosquitoes obtained soon after initial releases. Mitochondrial variants associated with the initial Wolbachia release stock are now the only variants found in release sites, highlighting maternal leakage as a possible explanation for rare Wolbachia-negative mosquitoes and not migration from non-release areas. There is no evidence of changes in the Wolbachia genome that indicate selection against its viral-suppressive effects or other phenotypes attributable to infection with the bacterium.

Genetic, Morphological and Antigenic Relationships between Mesonivirus Isolates from Australian Mosquitoes and Evidence for Their Horizontal Transmission.

The Mesoniviridae are a newly assigned family of viruses in the order Nidovirales. Unlike other nidoviruses, which include the Coronaviridae, mesoniviruses are restricted to mosquito hosts and do not infect vertebrate cells. To date there is little information on the morphological and antigenic characteristics of this new group of viruses and a dearth of mesonivirus-specific research tools. In this study we determined the genetic relationships of recent Australian isolates of Alphamesonivirus 4 (Casuarina virus-CASV) and Alphamesonivirus 1 (Nam Dinh virus-NDiV), obtained from multiple mosquito species. Australian isolates of NDiV showed high-level similarity to the prototype NDiV isolate from Vietnam (99% nucleotide (nt) and amino acid (aa) identity). Isolates of CASV from Central Queensland were genetically very similar to the prototype virus from Darwin (95-96% nt and 91-92% aa identity). Electron microscopy studies demonstrated that virion diameter (≈80 nm) and spike length (≈10 nm) were similar for both viruses. Monoclonal antibodies specific to CASV and NDiV revealed a close antigenic relationship between the two viruses with 13/34 mAbs recognising both viruses. We also detected NDiV RNA on honey-soaked nucleic acid preservation cards fed on by wild mosquitoes supporting a possible mechanism of horizontal transmission between insects in nature.

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.

Zika virus noncoding RNA suppresses apoptosis and is required for virus transmission by mosquitoes.

Flaviviruses, including Zika virus (ZIKV), utilise host mRNA degradation machinery to produce subgenomic flaviviral RNA (sfRNA). In mammalian hosts, this noncoding RNA facilitates replication and pathogenesis of flaviviruses by inhibiting IFN-signalling, whereas the function of sfRNA in mosquitoes remains largely elusive. Herein, we conduct a series of in vitro and in vivo experiments to define the role of ZIKV sfRNA in infected Aedes aegypti employing viruses deficient in production of sfRNA. We show that sfRNA-deficient viruses have reduced ability to disseminate and reach saliva, thus implicating the role for sfRNA in productive infection and transmission. We also demonstrate that production of sfRNA alters the expression of mosquito genes related to cell death pathways, and prevents apoptosis in mosquito tissues. Inhibition of apoptosis restored replication and transmission of sfRNA-deficient mutants. Hence, we propose anti-apoptotic activity of sfRNA as the mechanism defining its role in ZIKV transmission.

A LAMP-based colorimetric assay to expedite field surveillance of the invasive mosquito species Aedes aegypti and Aedes albopictus.

BACKGROUND: Yellow fever, dengue, chikungunya and Zika viruses are responsible for considerable morbidity and mortality in humans. Aedes aegypti and Aedes albopictus are the most important mosquito vectors involved in their transmission. Accurate identification of these species is essential for the implementation of control programs to limit arbovirus transmission, during suspected detections at ports of first entry, to delimit incursions or during presence/absence surveillance programs in regions vulnerable to invasion. We developed and evaluated simple and rapid colorimetric isothermal tests to detect these two mosquito species based on loop-mediated isothermal amplification (LAMP) targeting the ribosomal RNA internal transcribed spacer 1 (ITS1). METHODOLOGY/PRINCIPAL FINDINGS: Samples were prepared by homogenizing and heating at 99 oC for 10 min before an aliquot was added to the LAMP reaction. After 40 min incubation at 65 oC, a colour change indicated a positive result. The tests were 100% sensitive and species-specific, and demonstrated a limit of detection comparable with PCR-based detection (TaqMan chemistry). The LAMP assays were able to detect target species for various life stages tested (adult, 1st instar larva, 4th instar larva and pupa), and body components, such as legs, wings and pupal exuviae. Importantly, the LAMP assays could detect Ae. aegypti DNA in mosquitoes stored in Biogents Sentinel traps deployed in the field for 14 d. A single 1st instar Ae. aegypti larva could also be detected in a pool of 1,000 non-target 1st instar Aedes notoscriptus, thus expediting processing of ovitrap collections obtained during presence/absence surveys. A simple syringe-sponge protocol facilitated the concentration and collection of larvae from the ovitrap water post-hatch. CONCLUSIONS/SIGNIFICANCE: We describe the development of LAMP assays for species identification and demonstrate their direct application for surveillance in different field contexts. The LAMP assays described herein are useful adjuncts to laboratory diagnostic testing or could be employed as standalone tests. Their speed, ease-of-use, low cost and need for minimal equipment and training make the LAMP assays ideal for adoption in low-resource settings without the need to access diagnostic laboratory services.

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.

Malaria surveillance from both ends: concurrent detection of Plasmodium falciparum in saliva and excreta harvested from Anopheles mosquitoes.

BACKGROUND: Malaria is the most important vector-borne disease in the world. Epidemiological and ecological studies of malaria traditionally utilize detection of Plasmodium sporozoites in whole mosquitoes or salivary glands by microscopy or serological or molecular assays. However, these methods are labor-intensive, and can over- or underestimate mosquito transmission potential. To overcome these limitations, alternative sample types have been evaluated for the study of malaria. It was recently shown that Plasmodium could be detected in saliva expectorated on honey-soaked cards by Anopheles stephensi, providing a better estimate of transmission risk. We evaluated whether excretion of Plasmodium falciparum nucleic acid by An. stephensi correlates with expectoration of parasites in saliva, thus providing an additional sample type for estimating transmission potential. Mosquitoes were exposed to infectious blood meals containing cultured gametocytes, and excreta collected at different time points post-exposure. Saliva was collected on honey-soaked filter paper cards, and salivary glands were dissected and examined microscopically for sporozoites. Excreta and saliva samples were tested by real time polymerase chain reaction (RT-rtPCR). RESULTS: Plasmodium falciparum RNA was detected in mosquito excreta as early as four days after ingesting a bloodmeal containing gametocytes. Once sporogony (the development of sporozoites) occurred, P. falciparum RNA was detected concurrently in both excreta and saliva samples. In the majority of cases, no difference was observed between the Ct values obtained from matched excreta and saliva samples, suggesting that both samples provide equally sensitive results. A positive association was observed between the molecular detection of the parasites in both samples and the proportion of mosquitoes with sporozoites in their salivary glands from each container. No distinguishable parasites were observed when excreta samples were stained and microscopically analyzed. CONCLUSIONS: Mosquito saliva and excreta are easily collected and are promising for surveillance of malaria-causing parasites, especially in low transmission settings or in places where arboviruses co-circulate.