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The Kunjin strain of West Nile virus (WNV KUN ) 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 WNV KUN 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. WNV KUN 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 WNV KUN and MVEV transmission by FTA TM 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 WNV KUN or MVEV were obtained from mosquitoes collected on Darwin crocodile farms immediately following the FTA TM card detections, we did isolate another flavivirus, Kokobera virus (KOKV), from Culex annulirostris mosquitoes. Our studies support the use of the FTA TM card system as a sensitive and accurate method to monitor the transmission of WNV KUN 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 FTA TM 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.

Arbovirus surveillance is crucial for the implementation of vector-borne disease control measures. Recently, it has been demonstrated that mosquitoes with a disseminated arbovirus infection excrete viral RNA, which can be detected by molecular methods. Thereby, mosquito excreta has been proposed as a sample type that could be utilized for arbovirus surveillance. In this study, we evaluated if West Nile virus (Kunjin strain, WNVKUN) RNA in Culex annulirostris Skuse (Diptera: Culicidae) excreta deposited on different substrates could be detected after storage for up to 2 wk at tropical conditions of high heat and humidity. No significant drop in relative quantity of WNVKUN RNA (determined by comparison of Ct values) in excreta deposited on Flinders Associate Technologies (FTA) cards was observed over 14 d, suggesting that RNA was stable for that time. There was no significant difference in relative quantity of WNVKUN RNA in excreta deposited on FTA cards or polycarbonate substrates after 24 h. However, after 7 and 14 d, there was a significant decline in the relative quantity of viral RNA in the excreta stored on polycarbonate substrates. For incorporation in arbovirus surveillance programs, we recommend the use of polycarbonate substrates for excreta collection in mosquito traps deployed overnight, and the integration of FTA cards in traps serviced weekly or fortnightly. Polycarbonate substrates facilitate the collection of the majority of excreta from a trap, and while FTA cards offer limited area coverage, they enable preservation of viral RNA in tropical conditions for extended periods of time., (© The Author(s) 2019. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Emerging and re-emerging arthropod-borne viruses (arboviruses) cause human and animal disease globally. Field and laboratory investigation of mosquito-borne arboviruses requires analysis of mosquito samples, either individually, in pools, or a body component, or secretion such as saliva. We assessed the applicability of mosquito excreta as a sample type that could be utilized during studies of Ross River and West Nile viruses, which could be applied to the study of other arboviruses., Methodology/principal Findings: Mosquitoes were fed separate blood meals spiked with Ross River virus and West Nile virus. Excreta was collected daily by swabbing the bottom of containers containing batches and individual mosquitoes at different time points. The samples were analyzed by real-time RT-PCR or cell culture enzyme immunoassay. Viral RNA in excreta from batches of mosquitoes was detected continuously from day 2 to day 15 post feeding. Viral RNA was detected in excreta from at least one individual mosquito at all timepoints, with 64% and 27% of samples positive for RRV and WNV, respectively. Excretion of viral RNA was correlated with viral dissemination in the mosquito. The proportion of positive excreta samples was higher than the proportion of positive saliva samples, suggesting that excreta offers an attractive sample for analysis and could be used as an indicator of potential transmission. Importantly, only low levels of infectious virus were detected by cell culture, suggesting a relatively low risk to personnel handling mosquito excreta., Conclusions/significance: Mosquito excreta is easily collected and provides a simple and efficient method for assessing viral dissemination, with applications ranging from vector competence experiments to complementing sugar-based arbovirus surveillance in the field, or potentially as a sample system for virus discovery., Competing Interests: The authors have declared that no competing interests exist.

Background: Although 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., Methods: The 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., Results: The 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 (t 6 = 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., Conclusions: These 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.

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.