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.

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.

Effective arbovirus surveillance is essential to ensure the implementation of control strategies, such as mosquito suppression, vaccination, or dissemination of public warnings. Traditional strategies employed for arbovirus surveillance, such as detection of virus or virus-specific antibodies in sentinel animals, or detection of virus in hematophagous arthropods, have limitations as an early-warning system. A system was recently developed that involves collecting mosquitoes in CO2-baited traps, where the insects expectorate virus on sugar-baited nucleic acid preservation cards. The cards are then submitted for virus detection using molecular assays. We report the application of this system for detecting flaviviruses and alphaviruses in wild mosquito populations in northern Australia. This study was the first to employ nonpowered passive box traps (PBTs) that were designed to house cards baited with honey as the sugar source. Overall, 20/144 (13.9%) of PBTs from different weeks contained at least one virus-positive card. West Nile virus Kunjin subtype (WNVKUN), Ross River virus (RRV), and Barmah Forest virus (BFV) were detected, being identified in 13/20, 5/20, and 2/20 of positive PBTs, respectively. Importantly, sentinel chickens deployed to detect flavivirus activity did not seroconvert at two Northern Territory sites where four PBTs yielded WNVKUN. Sufficient WNVKUN and RRV RNA was expectorated onto some of the honey-soaked cards to provide a template for gene sequencing, enhancing the utility of the sugar-bait surveillance system for investigating the ecology, emergence, and movement of arboviruses.

BackgroundAlthough sentinel animals are used successfully throughout the world to monitor arbovirus activity, ethical considerations and cross-reactions in serological assays highlight the importance of developing viable alternatives. Here we outline the development of a passive sentinel mosquito arbovirus capture kit (SMACK) that allows for the detection of arboviruses on honey-baited nucleic acid preservation cards (Flinders Technology Associates; FTA®) and has a similar trap efficacy as standard light traps in our trials.MethodsThe trap efficacy of the SMACK was assessed against Centers for Disease Control and Prevention (CDC) miniature light traps (standard and ultraviolet) and the Encephalitis Vector Survey (EVS) trap in a series of Latin square field trials conducted in North Queensland, Australia. The ability of the SMACK to serve as a sentinel arbovirus surveillance tool was assessed in comparison to Passive Box Traps (PBT) during the 2014 wet season in the Cairns, Australia region and individually in the remote Northern Peninsula Area (NPA) of Australia during the 2015 wet season.ResultsThe SMACK caught comparable numbers of mosquitoes to both CDC light traps (mean capture ratio 0.86: 1) and consistently outperformed the EVS trap (mean capture ratio 2.28: 1) when CO2 was supplied by either a gas cylinder (500 ml/min) or dry ice (1 kg). During the 2014 arbovirus survey, the SMACK captured significantly (t6 = 2.1, P = 0.04) more mosquitoes than the PBT, and 2 and 1 FTA® cards were positive for Ross River virus and Barmah Forest virus, respectively, while no arboviruses were detected from PBTs. Arbovirus activity was detected at all three surveillance sites during the NPA survey in 2015 and ca. 27 % of FTA® cards tested positive for either Murray Valley encephalitis virus (2 detections), West Nile virus (Kunjin subtype; 13 detections), or both viruses on two occasions.ConclusionsThese results demonstrate that the SMACK is a versatile, simple, and effective passive arbovirus surveillance tool that may also be used as a traditional overnight mosquito trap and has the potential to become a practical substitute for sentinel animal programs.