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

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

Improved detection of flaviviruses in Australian mosquito populations via replicative intermediates.

Mosquito-borne flaviviruses are significant contributors to the arboviral disease burdens both in Australia and globally. While routine arbovirus surveillance remains a vital exercise to identify known flaviviruses in mosquito populations, novel or divergent and emerging species can be missed by these traditional methods. The MAVRIC (monoclonal antibodies to viral RNA intermediates in cells) system is an ELISA-based method for broad-spectrum isolation of positive-sense and double-stranded RNA (dsRNA) viruses based on detection of dsRNA in infected cells. While the MAVRIC ELISA has successfully been used to detect known and novel flaviviruses in Australian mosquitoes, we previously reported that dsRNA could not be detected in dengue virus-infected cells using this method. In this study we identified additional flaviviruses which evade detection of dsRNA by the MAVRIC ELISA. Utilising chimeric flaviviruses we demonstrated that this outcome may be dictated by the non-structural proteins and/or untranslated regions of the flaviviral genome. In addition, we report a modified fixation method that enables improved detection of flavivirus dsRNA and inactivation of non-enveloped viruses from mosquito populations using the MAVRIC system. This study demonstrates the utility of anti-dsRNA monoclonal antibodies for identifying viral replication in insect and vertebrate cell systems and highlights a unique characteristic of flavivirus replication.

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.

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 a novel iflavirus sequence in the eastern paralysis tick Ixodes holocyclus.

Ixodes holocyclus, the eastern paralysis tick, is a significant parasite in Australia in terms of animal and human health. However, very little is known about its virome. In this study, next-generation sequencing of I. holocyclus salivary glands yielded a full-length genome sequence which phylogenetically groups with viruses classified in the Iflaviridae family and shares 45% amino acid similarity with its closest relative Bole hyalomma asiaticum virus 1. The sequence of this virus, provisionally named Ixodes holocyclus iflavirus (IhIV) has been identified in tick populations from northern New South Wales and Queensland, Australia and represents the first virus sequence reported from I. holocyclus.

Understanding the role of microRNAs in the interaction of Aedes aegypti mosquitoes with an insect-specific flavivirus.

The Flavivirus genus contains some of the most prevalent vector-borne viruses, such as the dengue, Zika and yellow fever viruses that cause devastating diseases in humans. However, the insect-specific clade of flaviviruses is restricted to mosquito hosts, albeit they have retained the general features of the genus, such as genome structure and replication. The interactions between insect-specific flaviviruses (ISFs) and their mosquito hosts are largely unknown. Pathogenic flaviviruses are known to modulate host-derived microRNAs (miRNAs), a class of non-coding RNAs that are important in controlling gene expression. Alterations in miRNAs may represent changes in host gene expression and promote understanding of virus-host interactions. The role of miRNAs in ISF-mosquito interactions is largely unknown. A recently discovered Australian ISF, Palm Creek virus (PCV), has the ability to suppress medically relevant flaviviruses. Here, we investigated the potential involvement of miRNAs in PCV infection using the model mosquito Aedes aegypti. By combining small-RNA sequencing and bioinformatics analysis, differentially expressed miRNAs were determined. Our results indicated that PCV infection hardly affects host miRNAs. Out of 101 reported miRNAs of Ae. aegypti, only aae-miR-2940-5p had a significantly altered expression over the course of infection. However, further analysis of aae-miR-2940-5p revealed that this miRNA does not have any direct impact on PCV replication in vitro. Thus, overall the results suggest that PCV infection has a limited effect on the mosquito miRNA profile and therefore miRNAs may not play a significant role in the PCV-Ae. aegypti interaction.

Genetic Characterization of Archived Bunyaviruses and their Potential for Emergence in Australia.

To better understand the diversity of bunyaviruses and their circulation in Australia, we sequenced 5 viruses (Gan Gan, Trubanaman, Kowanyama, Yacaaba, and Taggert) isolated and serologically identified 4 decades ago as members of the family Bunyaviridae. Gan Gan and Trubanaman viruses almost perfectly matched 2 recently isolated, purportedly novel viruses, Salt Ash and Murrumbidgee viruses, respectively. Kowanyama and Yacaaba viruses were identified as being related to members of a large clade containing pathogenic viruses. Taggert virus was confirmed as being a nairovirus; several viruses of this genus are pathogenic to humans. The genetic relationships and historical experimental infections in mice reveal the potential for these viruses to lead to disease emergence.

Virulence and Evolution of West Nile Virus, Australia, 1960-2012.

Worldwide, West Nile virus (WNV) causes encephalitis in humans, horses, and birds. The Kunjin strain of WNV (WNVKUN) is endemic to northern Australia, but infections are usually asymptomatic. In 2011, an unprecedented outbreak of equine encephalitis occurred in southeastern Australia; most of the ≈900 reported cases were attributed to a newly emerged WNVKUN strain. To investigate the origins of this virus, we performed genetic analysis and in vitro and in vivo studies of 13 WNVKUN isolates collected from different regions of Australia during 1960-2012. Although no disease was recorded for 1984, 2000, or 2012, isolates collected during those years (from Victoria, Queensland, and New South Wales, respectively) exhibited levels of virulence in mice similar to that of the 2011 outbreak strain. Thus, virulent strains of WNVKUN have circulated in Australia for >30 years, and the first extensive outbreak of equine disease in Australia probably resulted from a combination of specific ecologic and epidemiologic conditions.

Programmed ribosomal frameshift alters expression of west nile virus genes and facilitates virus replication in birds and mosquitoes.

West Nile virus (WNV) is a human pathogen of significant medical importance with close to 40,000 cases of encephalitis and more than 1,600 deaths reported in the US alone since its first emergence in New York in 1999. Previous studies identified a motif in the beginning of non-structural gene NS2A of encephalitic flaviviruses including WNV which induces programmed -1 ribosomal frameshift (PRF) resulting in production of an additional NS protein NS1'. We have previously demonstrated that mutant WNV with abolished PRF was attenuated in mice. Here we have extended our previous observations by showing that PRF does not appear to have a significant role in virus replication, virion formation, and viral spread in several cell lines in vitro. However, we have also shown that PRF induces an over production of structural proteins over non-structural proteins in virus-infected cells and that mutation abolishing PRF is present in ∼11% of the wild type virus population. In vivo experiments in house sparrows using wild type and PRF mutant of New York 99 strain of WNV viruses showed some attenuation for the PRF mutant virus. Moreover, PRF mutant of Kunjin strain of WNV showed significant decrease compared to wild type virus infection in dissemination of the virus from the midgut through the haemocoel, and ultimately the capacity of infected mosquitoes to transmit virus. Thus our results demonstrate an important role for PRF in regulating expression of viral genes and consequently virus replication in avian and mosquito hosts.

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.

A simple non-powered passive trap for the collection of mosquitoes for arbovirus surveillance.

Mosquitoes often are collected as part of an arbovirus surveillance program. However, trapping and processing of mosquitoes for arbovirus detection is often costly and difficult in remote areas. Most traps, such as the gold standard Center for Disease control light trap, require batteries that must be charged and changed overnight. To overcome this issue we have developed several passive traps for collection of mosquitoes that have no power requirements. The passive traps capture mosquitoes as they follow a CO2 plume up a polyvinyl chloride pipe leading to a clear chamber consisting of a plastic crate. We believe the translucent, clear windows created by the crate inhibits escape. Once inside the crate mosquitoes readily feed on honey-treated Flinders Technology Associates cards that then can be processed by polymerase chain reaction for viral ribonucleic acid. Of the two designs tested, the box or crate-based passive trap (passive box trap, PBT) generally caught more mosquitoes than the cylinder trap. In Latin square field trials in Cairns and Florida, PBTs collected mosquitoes at rates of 50 to 200% of Center for Disease Control model 512 light traps. Mosquito collections by PBTs can be increased by splitting the CO2 gas line so it services two traps, or by placing an octenol lure to the outside of the box. Very large collections can lead to crowding at honey-treated cards, reducing feeding rates. Addition of fipronil to the honey killed mosquitoes and did not impact feeding rates nor the ability to detect Kunjin viral ribonucleic acid by polymerase chain reaction; this could be used to minimize crowding affects on feeding caused by large collections. The passive traps we developed are made from inexpensive, commonly available materials. Passive traps may thus be suitable for collection of mosquitoes and potentially other hematophagous dipterans for pathogen surveillance.

Exploiting mosquito sugar feeding to detect mosquito-borne pathogens.

Arthropod-borne viruses (arboviruses) represent a global public health problem, with dengue viruses causing millions of infections annually, while emerging arboviruses, such as West Nile, Japanese encephalitis, and chikungunya viruses have dramatically expanded their geographical ranges. Surveillance of arboviruses provides vital data regarding their prevalence and distribution that may be utilized for biosecurity measures and the implementation of disease control strategies. However, current surveillance methods that involve detection of virus in mosquito populations or sero-conversion in vertebrate hosts are laborious, expensive, and logistically problematic. We report a unique arbovirus surveillance system to detect arboviruses that exploits the process whereby mosquitoes expectorate virus in their saliva during sugar feeding. In this system, infected mosquitoes captured by CO(2)-baited updraft box traps are allowed to feed on honey-soaked nucleic acid preservation cards within the trap. The cards are then analyzed for expectorated virus using real-time reverse transcription-PCR. In field trials, this system detected the presence of Ross River and Barmah Forest viruses in multiple traps deployed at two locations in Australia. Viral RNA was preserved for at least seven days on the cards, allowing for long-term placement of traps and continuous collection of data documenting virus presence in mosquito populations. Furthermore no mosquito handling or processing was required and cards were conveniently shipped to the laboratory overnight. The simplicity and efficacy of this approach has the potential to transform current approaches to vector-borne disease surveillance by streamlining the monitoring of pathogens in vector populations.

Evolution of mosquito-based arbovirus surveillance systems in Australia.

Control of arboviral disease is dependent on the sensitive and timely detection of elevated virus activity or the identification of emergent or exotic viruses. The emergence of Japanese encephalitis virus (JEV) in northern Australia revealed numerous problems with performing arbovirus surveillance in remote locations. A sentinel pig programme detected JEV activity, although there were a number of financial, logistical, diagnostic and ethical limitations. A system was developed which detected viral RNA in mosquitoes collected by solar or propane powered CO2-baited traps. However, this method was hampered by trap-component malfunction, microbial contamination and large mosquito numbers which overwhelmed diagnostic capabilities. A novel approach involves allowing mosquitoes within a box trap to probe a sugar-baited nucleic-acid preservation card that is processed for expectorated arboviruses. In a longitudinal field trial, both Ross River and Barmah Forest viruses were detected numerous times from multiple traps over different weeks. Further refinements, including the development of unpowered traps and use of yeast-generated CO2, could enhance the applicability of this system to remote locations. New diagnostic technology, such as next generation sequencing and biosensors, will increase the capacity for recognizing emergent or exotic viruses, while cloud computing platforms will facilitate rapid dissemination of data.

Culex annulirostris (Diptera: Culicidae) host feeding patterns and Japanese encephalitis virus ecology in northern Australia.

Japanese encephalitis virus (JEV) transmission in northern Australia has, in the past, been facilitated by Culex annulirostris Skuse feeding on domestic pigs, the primary amplifying hosts of the virus. To further characterize mosquito feeding behavior in northern Australia, 1,128 bloodmeals from Cx. annulirostris were analyzed using a double-antibody enzyme-linked immunosorbent assay. Overall, Cx. annulirostris obtained > 94% of blood meals from mammals, comprising marsupials (37%), pigs (20%), dogs (16%), and cows (11%), although the proportion feeding on each of these host types varied between study locations. Where JEV activity was detected, feeding rates on pigs were relatively high. At the location that yielded the first Australian mainland isolate of JEV from mosquitoes, feral pigs (in the absence of domestic pigs) accounted for 82% of bloodmeals identified, representing the first occasion that feeding on feral pigs has been associated with JEV transmission in Australia. Interestingly, < 3% of Cx. annulirostris had fed on pigs at locations on Badu Island where JEV was detected in multiple pools of mosquitoes in a concurrent study. This suggests that either alternative hosts, such as birds, which comprised 21% of blood meals identified, or infected mosquitoes immigrating from areas where domestic pigs are housed, may have contributed to transmission at this location. Because Cx. annulirostris is both an opportunistic feeder and the primary JEV vector in the region, environmental characteristics and host presence can determine JEV transmission dynamics in northern Australia.

Arthropod-Borne Virus Surveillance as a Tool to Study the Australian Mosquito Virome.

Mosquitoes (n = 4381 in 198 pools) were collected in March and April 2018 to survey the presence of West Nile virus Kunjin strain in mosquito populations around crocodile farms in the Darwin region of the Northern Territory (NT) of Australia. While no Kunjin virus was detected in these mosquitoes, we applied our viral replicative intermediates screening system termed monoclonal antibodies to viral RNA intermediates in cells or MAVRIC to this set of samples. This resulted in the detection of 28 pools with virus replicating in C6/36 mosquito cells and the identification of three insect viruses from three distinct virus classes. We demonstrate the persistence of the insect-specific flavivirus Palm Creek virus in Coquillettidia xanthogaster mosquitoes from Darwin over almost a decade, with limited genetic drift. We also detected a novel Hubei macula-like virus 3 strain in samples from two mosquito genera, suggesting the virus, for which the sequence was originally detected in spiders and soybean thrips, might be involved in a horizontal transmission cycle between arthropods and plants. Overall, these data demonstrate the strength of the optimized MAVRIC system and contribute to our general knowledge of the mosquito virome and insect viruses.

Nucleic Acid Preservation Card Surveillance Is Effective for Monitoring Arbovirus Transmission on Crocodile Farms and Provides a One Health Benefit to Northern Australia.

The Kunjin strain of West Nile virus (WNVKUN) is a mosquito-transmitted flavivirus that can infect farmed saltwater crocodiles in Australia and cause skin lesions that devalue the hides of harvested animals. We implemented a surveillance system using honey-baited nucleic acid preservation cards to monitor WNVKUN and another endemic flavivirus pathogen, Murray Valley encephalitis virus (MVEV), on crocodile farms in northern Australia. The traps were set between February 2018 and July 2020 on three crocodile farms in Darwin (Northern Territory) and one in Cairns (North Queensland) at fortnightly intervals with reduced trapping during the winter months. WNVKUN RNA was detected on all three crocodile farms near Darwin, predominantly between March and May of each year. Two of the NT crocodile farms also yielded the detection of MVE viral RNA sporadically spread between April and November in 2018 and 2020. In contrast, no viral RNA was detected on crocodile farms in Cairns during the entire trapping period. The detection of WNVKUN and MVEV transmission by FTATM cards on farms in the Northern Territory generally correlated with the detection of their transmission to sentinel chicken flocks in nearby localities around Darwin as part of a separate public health surveillance program. While no isolates of WNVKUN or MVEV were obtained from mosquitoes collected on Darwin crocodile farms immediately following the FTATM card detections, we did isolate another flavivirus, Kokobera virus (KOKV), from Culex annulirostris mosquitoes. Our studies support the use of the FTATM card system as a sensitive and accurate method to monitor the transmission of WNVKUN and other arboviruses on crocodile farms to enable the timely implementation of mosquito control measures. Our detection of MVEV transmission and isolation of KOKV from mosquitoes also warrants further investigation of their potential role in causing diseases in crocodiles and highlights a "One Health" issue concerning arbovirus transmission to crocodile farm workers. In this context, the introduction of FTATM cards onto crocodile farms appears to provide an additional surveillance tool to detect arbovirus transmission in the Darwin region, allowing for a more timely intervention of vector control by relevant authorities.

Extended characterisation of five archival tick-borne viruses provides insights for virus discovery in Australian ticks.

BACKGROUND: A subset of Australians who have been bitten by ticks experience a complex of chronic and debilitating symptoms which cannot be attributed to the known pathogenic species of bacteria present in Australia. As a result, there has been a renewed effort to identify and characterise viruses in Australian terrestrial ticks. Recent transcriptome sequencing of Ixodes and Amblyomma ticks has revealed the presence of multiple virus sequences. However, without virus isolates our ability to understand the host range and pathogenesis of newly identified viruses is limited. We have established a successful method for high-throughput virus discovery and isolation in mosquitoes using antibodies to double-stranded RNA. In this study we sought to characterise five archival tick-borne viruses to adapt our virus discovery protocol for Australian ticks. METHODS: We performed virus characterisation using a combination of bioinformatic sequence analysis and in vitro techniques including replication kinetics, antigenic profiling, virus purification and mass spectrometry. RESULTS: Our sequence analysis of Nugget virus, Catch-me-Cave virus and Finch Creek virus revealed marked genetic stability in isolates collected from the same location approximately 30 years apart. We demonstrate that the Ixodes scapularis-derived ISE6 cell line supports replication of Australian members of the Flaviviridae, Nairoviridae, Phenuiviridae and Reoviridae families, including Saumarez Reef virus (SREV), a flavivirus isolated from the soft tick Ornithodoros capensis. While antibodies against double-stranded RNA could be used to detect replication of a tick-borne reovirus and mosquito-borne flavivirus, the tick-borne flaviviruses Gadgets Gully virus and SREV could not be detected using this method. Finally, four novel virus-like sequences were identified in transcriptome sequencing of the Australian native tick Ixodes holocyclus. CONCLUSIONS: Genetic and antigenic characterisations of archival viruses in this study confirm that three viruses described in 2002 represent contemporary isolates of virus species first identified 30 years prior. Our findings with antibodies to double-stranded RNA highlight an unusual characteristic shared by two Australian tick-borne flaviviruses. Finally, comparative growth kinetics analyses of Australian tick-borne members of the Flaviviridae, Nairoviridae, Phenuiviridae and Reoviridae families in ISE6 and BSR cells will provide a useful resource for isolation of Australian tick-borne viruses using existing cell lines.

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.

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.

Utilising a novel surveillance system to enhance field screening activities for the leishmaniases.

Over the last decade, an arbovirus surveillance system based on the preservation of nucleic acids (RNA/DNA) has been developed using Flinders Technology Associates (FTAⓇ) cards. Soaked in honey, FTAⓇ cards are applied in the field to detect arboviruses expectorated during mosquito sugar feeding. This technique has been shown to be inexpensive and efficient, and the implementation of this system for detecting parasites could be of international importance. As Leishmania parasites are highly prevalent in developing countries, FTAⓇ cards may offer an alternative inexpensive tool to enhance field surveillance activities for leishmaniasis. The simple approach of applying the cards in programs can substitute the necessary extensive training of personnel. In our hands, Leishmania macropodum DNA was shown to be stable on FTAⓇ cards during a 10-week time course, supporting their suitability for projects where direct access to laboratories is unobtainable and samples require storage prior to processing. This method may benefit programs in remote areas where accessibility to laboratory facilities are limited and samples need to be stored long-term.•This study found that FTA cards could be a valuable tool in the surveillance of leishmaniasis.•The method is based on the long-term preservation and detection of Leishmania DNA expectorated during insect sugar feeding.•The application of FTA cards can preclude the need to screen large samples and analysis of insect populations to provide evidence of disease transmission.