Exploiting Mosquito Sugar Feeding to Detect Mosquito-Borne Pathogens

Exploiting mosquito sugar feeding to detect mosquito-borne pathogens

Sonja Hall-Mendelina, Scott A. Ritchieb,c, Cheryl A. Johansend, Paul Zborowskia, Giles Cortise, Scott Dandridgef, Roy A. Halla, and Andrew F. van den Hurkg,a,1

aSchool of Chemistry and Molecular Biosciences, University of Queensland, St. Lucia, Queensland 4072, Australia; bSchool of Public Health and Tropical Medicine, James Cook University, Cairns, Queensland 4870, Australia; cTropical Population Health Unit Network, Queensland Health, Cairns, Queensland 4870, Australia; dDiscipline of Microbiology and Immunology, School of Biomedical, Biomolecular and Chemical Sciences, University of Western Australia, Nedlands, Western Australia 6009, Australia; ePrivate Contracting Engineer; fThe Shire of Harvey, Australind, Western Australia 6233, Australia; and gVirology, Queensland Health Forensic and Scientific Services, Coopers Plains, Queensland 4108, Australia

Edited* by Barry J. Beaty, Colorado State University, Fort Collins, CO, and approved April 29, 2010 (received for review February 22, 2010)

Arthropod-borne viruses (arboviruses) represent a global public viral RNA in, mosquitoes collected with a variety of trapping health problem, with dengue viruses causing millions of infections techniques. annually, while emerging arboviruses, such as West Nile, Japanese Although these surveillance strategies provide important infor- encephalitis, and chikungunya viruses have dramatically expanded mation regarding the distribution of arboviruses, each has limita- their geographical ranges. Surveillance of arboviruses provides tions. For instance, disease surveillance systems are hampered by vital data regarding their prevalence and distribution that may a similarity of disease signs and symptoms induced by different be utilized for biosecurity measures and the implementation of pathogens, the potentially low clinical to subclinical disease ratio, disease control strategies. However, current surveillance methods and the recognition of cases only after an outbreak has com- that involve detection of virus in mosquito populations or sero- menced. The disadvantages of sentinel animals include the need conversion in vertebrate hosts are laborious, expensive, and logis- for intensive animal husbandry and risk of injury to staff bleeding tically problematic. We report a unique arbovirus surveillance animals (7). Furthermore, sentinel animals may themselves be system to detect arboviruses that exploits the process whereby amplifying hosts of the virus, thus contributing to the transmission SCIENCES mosquitoes expectorate virus in their saliva during sugar feeding. cycle, and some closely related arboviruses are difficult to

In this system, infected mosquitoes captured by CO2-baited updraft distinguish using current serological tests. Finally, processing APPLIED BIOLOGICAL box traps are allowed to feed on honey-soaked nucleic acid thousands of mosquitoes for virus detection is labor-intensive, preservation cards within the trap. The cards are then analyzed especially when presorting of mosquitoes is required, and often for expectorated virus using real-time reverse transcription-PCR. requires a cold-chain to preserve viral integrity in arthropods In field trials, this system detected the presence of Ross River collected from the field (7). and Barmah Forest viruses in multiple traps deployed at two loca- Tocircumvent these issues, we developed a unique surveillance tions in Australia. Viral RNA was preserved for at least seven days strategy for the detection of arboviruses that exploits the process on the cards, allowing for long-term placement of traps and con- whereby mosquitoes expel virus in their saliva during sugar feed- tinuous collection of data documenting virus presence in mosquito ing (8, 9). In this system, mosquitoes attracted to and captured by populations. Furthermore no mosquito handling or processing was specialized traps were provided with a sugar source in the form of required and cards were conveniently shipped to the laboratory a honey-soaked card, which preserves nucleic acids. Viral RNA, overnight. The simplicity and efficacy of this approach has the expectorated from any infected mosquito, was subsequently potential to transform current approaches to vector-borne disease detected by real-time reverse-transcriptase (RT)—PCR. The surveillance by streamlining the monitoring of pathogens in vector novelty of this concept lies in the detection of viral RNA directly populations. from the cards, rather than the mosquitoes, thus eliminating the costly and time-consuming analysis of mosquitoes. Furthermore, arboviruses ∣ disease control ∣ honey ∣ saliva ∣ surveillance traps were designed to run continuously, and the cards preserved viral RNA for at least 7 d and inactivated live virus, demonstrat- rthropod-borne viruses (arboviruses) are responsible for ing the suitability of this system for surveillance in remote areas. Asignificant global morbidity and mortality, with many ree- In this paper, we describe the laboratory development and field merging or appearing in new geographic regions. The continued evaluation of the system and prove its utility in the detection of disease burden of dengue throughout the tropics, the widespread the alphaviruses, Ross River virus (RRV), Barmah Forest virus establishment of West Nile virus (WNV) in North America fol- (BFV), and CHIKV, as well as the flavivirus, WNV virus (Kunjin lowing its introduction in 1999, and the chikungunya virus subtype). (CHIKV) pandemic that afflicted nations in the western Indian Ocean, India, and Italy between 2004 and 2007 graphically illus- Results trate the impact of arboviruses on human and animal populations Detection of Viral RNA in Honey-Soaked Substrates Fed on by Infected (1, 2). The implementation of timely and effective control Mosquitoes. It was essential to use a substrate that preserved viral strategies, such as mosquito control and/or vaccination, can be RNA for at least 7 d under field conditions and rapidly inacti- highly dependent on data generated by surveillance systems. vated infectious virus to ensure integrity of the samples and then Accordingly, a number of strategies have been employed to detect arbovirus activity, but most are based on clinical diagnosis of Author contributions: S.H.-M., S.A.R., C.A.J., R.A.H., and A.F.v.d.H. designed research; symptoms and/or detection of the virus or virus-specific antibo- S.H.-M., P.Z., S.D., and A.F.v.d.H. performed research; S.H.-M., S.A.R., C.A.J., R.A.H., and dies in vertebrates or detection of the virus in arthropod vectors A.F.v.d.H. analyzed data; G.C. contributed new reagents/analytic tools; and S.H.-M., (3). Sentinel animals, such as chickens for WNV, St. Louis ence- S.A.R., C.A.J., R.A.H., and A.F.v.d.H. wrote the paper. phalitis virus, and Murray Valley encephalitis virus surveillance, The authors declare no conflict of interest. or pigs for Japanese encephalitis virus surveillance, are often *This Direct Submission article had a prearranged editor. deployed to provide an early warning system for an impending 1To whom correspondence should be addressed: E-mail: [email protected]. – outbreak or to detect an incursion (4 6). Virus surveillance using This article contains supporting information online at www.pnas.org/lookup/suppl/ arthropods is based on virus isolation from, or detection of doi:10.1073/pnas.1002040107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1002040107 PNAS Early Edition ∣ 1of5 Downloaded by guest on October 1, 2021 Table 1. Detection of West Nile, Ross River and chikungunya viruses on honey-baited substrates (FTA® cards or FP cards) and saliva collected from infected Cx. annulirostris and Ae. aegypti % surviving mosquitoes that % substrates had fed on % positive that had % detection Virus Substrate type positive* substrate† fed on substrate‡ in saliva§ West Nile FTA® card 83 (25∕30)60(6∕10) 100 (6∕6)90(9∕10) FP card 83 (25∕30)83(19∕23)89(17∕19) 100 (23∕23) Ross River FTA® card 90 (27∕30)56(9∕16) 100 (9∕9)69(11∕16) FP card 70 (21∕30)100(13∕13) 100 (13∕13)31(4∕13) Chikungunya FTA® card 75 (21∕28)0(0∕28)0(0∕0)52(14∕27) Mosquitoes were processed 12 days post exposure for WNV and RRV in Cx. annulirostris, and 14 days for CHIKV in Ae. aegypti. *Percentage of the total number of substrates positive for virus (number positive/number infected mosquitoes). †Percentage of surviving mosquitoes in which blue food dye was observed, indicating that the mosquito had imbibed honey from the substrate (number with blue dye/number surviving). ‡Percentage of mosquitoes that had imbibed honey from the substrate and expectorated virus (number positive/number that had imbibed the honey). §Percentage of saliva expectorates from surviving mosquitoes in which viral RNA was detected by TaqMan RT-PCR (number saliva expectorates positive/ number tested).

safe handling by field staff. Flinders Technology Associates filter or WNV (P ¼ 1.000, Fisher’s exact test). The viral detection rates paper (FTA®) cards have previously been used to inactivate and in saliva expectorates collected using a standard capillary tube preserve viral RNA, including rabies viruses (10) and infectious method (12) ranged from 31% to 100% and were not significantly bursal disease virus (11). Preliminary experiments demonstrated different from the detection rates for either of the honey-soaked that both honey-soaked FTA® cards and untreated filter paper substrates (P>0.05, Fisher’s exact test), except in the case of (FP) cards were able to bind RRV RNA and preserve it at 23 °C RRV from the FP cards (P ¼ 0.022, Fisher’s exact test). for at least 28 d (Table S1). Furthermore, the FTA® cards inac- Interestingly, in some cases, virus was detected on the tivated both RRVand WNVon contact, while infectious virus was ≤6 honey-baited substrates, although there was no evidence that still present on the FP cards hr post inoculation (SI Text). the mosquito had imbibed any of the honey (i.e., blue color The ability to detect viral RNA expectorated on these honey- was not observed in the mosquito). This was particularly evident soaked substrates by individual infected mosquitoes was assessed. Culex annulirostris were infected with WNVor RRV via intrathor- in Ae. aegypti, where despite blue dye not being observed in any acic inoculation, and Aedes aegypti were offered an infectious mosquitoes, CHIKV RNA was detected in 75% of FTA® cards CHIKV blood meal. Ten to 12 d after exposure, >30 mosquitoes processed. from each cohort were contained individually and allowed to feed on honey-soaked FTA® cards or FP cards for 48 hr. The honey Field Trials. ACO2-baited updraft box trap was developed to col- was colored with blue food dye and internalized blue color within lect and house mosquitoes, and it incorporated a mechanism by the mosquito confirmed whether a mosquito had fed on the card. which the FTA® cards or FP cards could be offered to trapped All three viruses were subsequently detected on the honey- mosquitoes (Fig. 1 A–C). In a field efficacy trial conducted in soaked FTA® cards and FP cards at rates ≥70% (Table 1). There Cairns, far north Queensland, Australia, updraft box traps col- was no significant difference between the FTA® cards and FP lected 1.72 times as many mosquitoes than CO2-baited Centers cards for the detection of RRV (P ¼ 0.104, Fisher’s exact test) for Disease Control miniature light traps (Table S2), and 77–95%

Fig. 1. Updraft box trap use to collect and house mosquitoes. (A) Trap components and direction of airflow depicted by solid arrows. Mosquitoes are sucked into the trap at the bottom, then become trapped within the plastic box (B) where they can feed on honey-soaked FTA® cards (C). The front of the trap has been removed for photographic purposes.

2of5 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1002040107 Hall-Mendelin et al. Downloaded by guest on October 1, 2021 of collected mosquitoes fed on the honey-soaked substrates while four separate traps were positive for BFV, while eight mosquito in the trap (Tables S3 and S4). pools from a total of five trap collections were positive (Fig. 2B). Longitudinal field trials to detect arboviruses were conducted To assess the level of virus activity at the WA study sites, at two locations with history of RRV and BFV activity: near CO2-baited light traps were used to trap mosquitoes that were Bunbury in Western Australia (WA) and near Cairns (13–15). then processed for RRV and BFV using a cell culture enzyme Mosquitoes and FTA® cards were removed weekly and for- immunoassay. A total of 3,994 mosquitoes in 230 pools were processed from six fortnightly collections obtained from two sites warded to the laboratory for RRV and BFV detection using near the trial locations. No RRV was isolated, but three isolates real-time RT-PCR. Over the 11-week trial period in WA, RRV of BFV were obtained, with one obtained on week five and two was detected on FTA® cards removed from at least one trap on week seven, which coincided with virus detection on in five separate weeks (Fig. 2A). Overall, 22 FTA® cards from FTA® cards. seven different trap collections and two pools of mosquitoes, re- In Cairns field trials, RRV was detected in three of the 14 moved from traps that simultaneously yielded positive FTA® weeks from nine FTA® cards from a total of five traps (Fig. 2C). cards, were positive. When analyzed for BFV, FTA® cards from Two mosquito pools yielded RRV (one each in weeks three and SCIENCES APPLIED BIOLOGICAL

Fig. 2. Temporal depiction of the detection of arboviruses in honey-soaked FTA® cards and mosquito pools collected from duplicate CO2-baited updraft box traps in the field: (A) Ross River virus and (B) Barmah Forest virus from the Leschenault Peninsula near Bunbury in Western Australia, and (C) Ross River virus from Cairns, far north Queensland. Each square represents a single FTA® card that was processed in each week, and black squares indicate a card from which viral RNA was detected. Detections of viral RNA in mosquitoes removed from the updraft box traps at the same time as the FTA® cards are represented by a mosquito.

Hall-Mendelin et al. PNAS Early Edition ∣ 3of5 Downloaded by guest on October 1, 2021 four). BFV was not detected in any mosquito pool or from FTA® (20), and thus our technique could be expanded to include other cards in Cairns. pathogens such as Plasmodium. For instance, should Anopheles spp. expel Plasmodium sporozoites during sugar feeding and if Discussion Plasmodium DNA was likewise preserved on the FTA® cards, Monitoring viral prevalence in mosquito vectors or vertebrate this system could be used as a surveillance tool for malaria. hosts provides vital data regarding the activity and spread of Additionally, this system could be adapted for surveillance of emerging and reemerging arboviruses for biosecurity purposes. pathogens transmitted by other hematophagous arthropods that However, current surveillance methods are laborious, expensive, are attracted to similar traps, such as Culicoides spp, which are and vary in their sensitivity and specificity, which greatly reduces vectors of bluetongue virus (26). Finally, honey-soaked FTA® their efficacy as a surveillance system. The unique system de- cards or FP cards could also be used to demonstrate pathogen scribed here circumvents the issues with sentinel animals and transmission in the laboratory, bypassing the need to use animal eliminates the need to handle or sort mosquitoes prior to virus models (27) or methods that sacrifice the mosquito, such as in detection, which is a major limitation of current arthropod-based vitro saliva collection (12). systems (7). This will result in a reduction in the turnaround time between sample collection and output of results, thus enhancing Materials and Methods its application as an early warning system. Finally, the utility of Laboratory Experiments. Honey-soaked substrates. As substrates we used the system was proven under both temperate (WA) and tropical FTA® cards (Whatman International Ltd, Maidstone, UK), which are filter (Cairns) climatic conditions. papers impregnated with proprietary chemicals to retain and preserve The relative simplicity of this system will facilitate the deploy- RNA and DNA. Untreated filter paper (FP) cards (Bio-Rad, Hercules, CA) were ment of a large number of traps, resulting in a wider geographical used for comparison. Manuka honey (Manuka, bee vital active 5þ; Capillano, Richlands, Austra- area that can be included in the surveillance program. Once a lia) was selected as food source due to its antibacterial and antifungal proper- virus of interest has been detected, more comprehensive trapping ties (24, 25). Because quarantine restrictions prevented the use of manuka in a given location can be implemented to obtain more informa- honey in Western Australia, Medihoney™ Antibacterial Medical Honey™ tion on virus ecology. For instance, further trapping for virus iso- (Medihoney®, Richlands, Australia) was used as the honey bait in Western lation can incriminate mosquito vectors and allow for calculation Australia. Blue food coloring (Queen, Alderley, Queensland, Australia) was of infection rates (16, 17), while blood meal analysis may provide added to honey to differentiate between mosquitoes that had imbibed an important indication of vertebrate hosts that may be involved the honey and those that were unfed. The blue color was visible in the mos- in transmission cycles (18, 19). To increase the capacity for quito crop, diverticula, and midgut post-feeding. Blue honey-soaked FTA® this system to detect unexpected or novel arboviruses/pathogens, cards and/or FP cards were used in all experiments. Dry substrates without universal primer sets and/or probes could be employed in the mo- honey were used as negative controls. lecular assays (20). Alternatively, a multiplexed assay may further enhance the capabilities of this system to rapidly detect a greater Mosquitoes. Colonized Cx. annulirostris were obtained from the Australian range of pathogens. Army Malaria Institute, Brisbane, Australia. The colony originated from adults collected from the Boondall Wetlands, Australia, in 1998 and were Sugar is an important energy source to facilitate mosquito >F50 generation. Aedes aegypti eggs were collected from ovitraps set in flight, mating, and egg production (21). We developed the surveil- Cairns, Australia, in 2009, and F2 adults were used in the experiments. lance system to exploit the phenomenon whereby mosquitoes expectorate virus while they sugar feed (9). Honey was used as the Viruses. West Nile virus (Kunjin subtype; 2002–1412) was isolated from Cx. sugar source on FTA® cards and FP cards because it possesses a annulirostris collected from Burketown in 2002 and had been passaged twice number of unique biochemical properties. Honey is composed in C6/36 (Aedes albopictus) cells and once in porcine stable-equine kidney primarily of sugars, predominately glucose and fructose (22), (PS-EK) cells. Ross River virus (389A) was isolated from mosquitoes collected which are attractive to mosquitoes (21). Indeed, observed feeding from Cairns in 1998 and passaged three times in C6/36 cells. Chikungunya rates of trapped mosquitoes on the honey-baited FTA® cards virus was isolated from a patient from Mauritius who had visited Melbourne, during the field trial (as indicated by the presence of blue dye Australia, in 2006 and was passaged three times in African green monkey in the mosquito abdomen) were 42% and 73% in WA and Cairns, kidney (Vero) cells. The WNV, RRV, and CHIKV stocks had final titers 108 109 108 ð Þ ∕ respectively, although there was variation in rates between spe- of , , and Vero tissue culture infectious dose TCID 50 mL, respec- cies. However, the results of the laboratory experiments suggest tively. that virus is expectorated in the saliva irrespective of whether the mosquitoes imbibe the honey or not. This is not surprising, Mosquito infection. Five to 7 d old Cx. annulirostris were intrathoracically μ 105.0 ∕ because probing by an infected mosquito is sufficient to transmit inoculated with 0.2 L of a suspension containing TCID50 mL of WNV or the virus to a susceptible host (23). Therefore, the presence of RRV diluted in growth media (GM; Opti-MEM (Gibco, Invitrogen Corporation, Grand Island, NY) containing 3% foetal bovine serum (FBS), and antibiotics blue dye in the mosquito abdomen may underestimate the true and antimycotics). Aedes aegypti were exposed to CHIKV diluted in washed expectoration rate for some mosquito species. defibrinated sheep blood maintained at 37 °C within a membrane feeding Once dispensed onto the FTA® cards, the honey remained apparatus. Following virus exposure, all mosquitoes were maintained at moist for the weekly trapping period, ensuring that trapped mos- 28 °C, 75% relative humidity and 12 h∶12 h light:day cycle. quitoes maintained access to liquid sugars. This was particularly important because feeding and survival rates are significantly Exposure of mosquitoes to honey-baited substrates. After 10 d for WNV lower when mosquitoes are maintained on substrates coated in and RRV or 12 d for CHIKV, individual mosquitoes were placed in separate dried sugar, compared with substrates that remain moistened 50 mL vials. A 1 cm2 square section of honey-soaked FTA® card or FP card with sugar solution. Another advantage of honey, and, in parti- was placed over a hole that had been cut in the lid of each vial. After cular, Manuka honey, is its antibacterial properties (24, 25), 48 h, all mosquitoes, whether dead or alive, were microscopically examined which could reduce the inhibitory effects of RNAses produced for visible blue food dye, indicative of feeding on the substrates. To compare by bacteria. Combined with the proprietary chemicals on the the honey-bait system with a standard in vitro capillary tube technique (12), cards, this potentially helped to preserve viral RNA expectorated saliva from surviving mosquitoes was collected in capillary tubes containing GM þ 20% FBS. All FTA® cards, FP cards, mosquitoes and saliva were stored by mosquitoes in the field trial. at −80 °C. Although the utility of the sugar-baited system for detecting arboviruses has been demonstrated here, there are many other Virus Assay. Individual mosquitoes were homogenized in 1 mL of potential applications for this strategy. Sugar feeding, primarily GM þ 3% FBS, filtered through a 0.2 μm filter (Pall Corporation, Ann in the form of nectar, is widespread within mosquito vectors Arbor, MI), before being inoculated in duplicate onto confluent Vero cell

4of5 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1002040107 Hall-Mendelin et al. Downloaded by guest on October 1, 2021 monolayers within a 96 well microtiter plate. Plates were incubated at 37 °C into the trap (32). CO2 was released at 450–500 mL∕ min using an adjustable and 5% CO2 for 7 d. Plates were then examined for cytopathic effect and regulator with a 0.25 mm orifice (Pacific Biologics, Scarborough, Australia). fixed in PBS/acetone. Infection was confirmed using a cell culture enzyme Four 8 cm diameter holes were cut into the portion of the PVC pipe inside the immunoassay and the monoclonal antibodies, 10A1 for WNV, and B10 for holding compartment. A plastic baffle inside the PVC pipe shunted mosqui- RRV and CHIKV (28). toes through the unscreened bottom two holes into the holding compart- The FTA® cards and filter papers were placed into a 5 mL tube, and 1 mL ment. The upper two holes allowed the air to exhaust through the top of of GM þ 3% FBS was added. Samples were kept on ice and vortexed every the trap. These upper holes were covered with 1 mm fly screen to prevent 5 min for 20 min (11). The eluate was either extracted immediately or stored mosquitoes from being drawn back into the pipe from the holding compart- at −80 °C to await analysis. ment. To prevent access by ants when deployed in the field, petroleum jelly Viral RNA was extracted from the FTA® card and FP card eluates, and was applied to the suspending chain, battery cable, and CO2 outlet line. saliva expectorates with a Bio Robot Universal System (Qiagen, Hilden, Ger- To produce the honey-baited FTA® card, blue honey was dispensed onto many) using the QIAamp® Virus BioRobot® MDx Kit (Qiagen, Clifton Hill, the wound pad of a Primapore Dressing (Smith & Nephew, 8.3 × 6 cm) and Australia) according to manufacturer’s instructions. Viral RNA was detected the FTA® card was laid over the honey and left to absorb overnight. Traps using real-time TaqMan RT-PCR assays specific for WNV, CHIKV, and RRV were run continuously during the study period, and FTA® cards and mosqui- (29–31). toes were removed weekly. Upon collection, FTA® cards were placed between layers of Parafilm M (Alcan Packaging, Neenah, WI) within a Field Trials. Study locations. Field trials were undertaken on the Leschenault snap-lock bag, and mosquitoes were placed in 50 mL jars, before being Peninsula near Bunbury in Western Australia from October 14 to December dispatched by overnight courier to the laboratory in Brisbane, Australia, 24, 2008, and at a site near Cairns in far north Queensland from January 21 to for analysis. April 28, 2009. The Leschenault Peninsula is a narrow stretch of land between Mosquitoes were sorted into pools of ≤500, to which five beads and 5 mL the Indian Ocean and the Leschenault Inlet, approximately 18 km north of of GM þ 3% FBS was added. Pools were homogenized, clarified by centrifu- the city of Bunbury in the southwest of Western Australia. It is characterized gation and filtered through a 0.8∕0.2 μm dual filter (Pall Corporation, Ann by coastal heath, tuart tree (Eucalyptus gomphocephala), and peppermint Arbor, MI), before being stored at −80 °C. The FTA® cards were cut into small tree (Agonis flexuosa) woodland and tidal salt marshes (Sarcocornia sp.). pieces, before being eluted as described previously. The FTA® card eluates The site near Cairns is bordered by sugar cane fields, tidal salt marshes, and mosquito pools were extracted and analyzed using an in-house BFV and Melaleuca swamps, and it is in close proximity to waste transfer and and the RRV real-time TaqMan RT-PCR assays. Pools collected during sewerage treatment facilities. concurrent trapping were processed for virus isolation using the methods of Johansen and others (33). Trap design. An updraft box trap was designed that combines an updraft

fan to maximize mosquito collections (32) and a plastic box to contain ACKNOWLEDGMENTS. The authors are grateful for the advice and assistance SCIENCES captured mosquitoes (Fig. 1A). A 32 x 21 x 19 cm clear plastic container with of Alyssa Pyke, Cassie Jansen, and Frederick Moore. We also thank Haydn

removable lid was used to hold mosquitoes and provide access to the honey- Jones for assistance in the field, and laboratory staff in the Arbovirus Surveil- APPLIED BIOLOGICAL soaked FTA® cards (Fig. 1 B and C). A computer cooling fan (12 V, 0.27 A lance and Research Laboratory who conducted the concurrent trapping and virus isolation work in WA. In addition, we thank Donna Mackenzie, Stephen brushless motor) was fitted to a 44 cm section of 10 cm diameter polyvi- Frances, and Petrina Johnson for providing mosquitoes for the laboratory nylchloride (PVC) pipe that was inserted into the middle of the box. A experiments, and Julian Druce for providing the CHIKV isolate. This work 19 cm diameter, 8 cm deep clear plastic bowl was fitted to the other end was funded by the Australian Biosecurity Cooperative Research Centre for of the PVC pipe to enhance mosquito capture. A section of wire fitted to Emerging Infectious Disease. Additional funding for the Western Australian the bowl was used to hold the outlet tube from a CO2 cylinder 10 cm below study was provided by the Western Australian Department of Health and a the bowl, the minimal distance that released gas would not be sucked back University of Western Australia Small Research Grant.

1. Gould EA, Solomon T (2008) Pathogenic flaviviruses. Lancet 371:500–509. 18. Apperson CS, et al. (2002) Host-feeding habits of Culex and other mosquitoes (Diptera: 2. Staples JE, Breiman RF, Powers AM (2009) Chikungunya fever: An epidemiological Culicidae) in the Borough of Queens in New York City, with characters and techniques review of a re-emerging infectious disease. Clin Infect Dis 49:942–948. for identification of Culex mosquitoes. J Med Entomol 39:777–785. 3. Eisen L, Beaty BJ, Morrison AC, Scott TW (2009) Proactive vector control strategies and 19. Jansen CC, et al. (2009) Blood sources of mosquitoes collected from urban and improved monitoring and evaluation practices for dengue prevention. J Med Entomol peri-urban environments in eastern Australia with species-specific molecular analysis 46:1245–1255. of avian blood meals. Am J Trop Med Hyg 81:849–857. 4. Reisen WK, et al. (2008) Persistent West Nile virus transmission and the apparent 20. Scaramozzino N, et al. (2001) Comparison of Flavivirus universal primer pairs and displacement of St. Louis encephalitis virus in southeastern California, 2003–2006. development of a rapid, highly sensitive heminested reverse transcription-PCR assay J Med Entomol 45:494–508. for detection of flaviviruses targeted to a conserved region of the NS5 gene sequences. 5. Hanna JN, et al. (1999) Japanese encephalitis in north Queensland, Australia, 1998. J Clin Microbiol 39:1922–1927. Med J Aust 170:533–536. 21. Foster WA (1995) Mosquito sugar feeding and reproductive energetics. Annu Rev 6. Broom AK, Lindsay MD, Harrington SA, Smith DW (2002) Investigation of the southern Entomol 40:443–474. limits of Murray Valley encephalitis activity in Western Australia during the 2000 wet 22. Anklam E (1998) A review of the analytical methods to determine the geographical season. Vector-Borne Zoonot Dis 2:87–95. and botanical origin of honey. Food Chem 63:549–562. 7. Ritchie SA, et al. (2007) Operational trial of a remote mosquito trap system for 23. Gould DJ, Barnett HC, Suyemoto W (1962) Transmission of Japanese encephalitis virus Japanese encephalitis virus surveillance in the Torres Strait, Australia. Vector-Borne by Culex gelidus Theobald. T Roy Soc Trop Med H 56:429–435. Zoonot Dis 7:497–506. 24. Weston RJ, Brocklebank LK, Lu Y (2000) Identification and quantitative levels of 8. Doggett SL, Klowden MJ, Russell RC (2001) Are vector competence experiments antibacterial components of some New Zealand honeys. Food Chem 70:427–435. competent vector experiments?. Arbovirus Res Aust 8:126–130. 25. Lusby PE, Coombes AL, Wilkinson JM (2005) Bactericidal activity of different honeys 9. van den Hurk AF, et al. (2007) Expectoration of flaviviruses during sugar feeding by against pathogenic bacteria. Arch Med Res 36:464–467. mosquitoes (Diptera: Culicidae). J Med Entomol 44:845–850. 26. Mellor PS, Boorman J, Baylis M (2000) Culicoides biting midges: Their role as arbovirus 10. Picard-Meyer E, Barrat J, Cliquet F (2007) Use of filter paper (FTA®) technology for vectors. Annu Rev Entomol 45:307–340. sampling, recovery and molecular characterisation of rabies viruses. J Virol Methods 27. van den Hurk AF, et al. (2003) Vector competence of Australian mosquitoes (Diptera: 140:174–182. Culicidae) for Japanese encephalitis virus. J Med Entomol 40:82–90. 11. Purvis LB, Villegas P, Perozo F (2006) Evaluation of FTA paper and phenol for storage, 28. Broom AK, et al. (1998) Identification of Australian arboviruses in inoculated cell extraction and molecular characterization of infectious bursal disease virus. J Virol cultures using monoclonal antibodies in ELISA. Pathology 30:286–288. Methods 138:66–69. 29. Pyke AT, et al. (2004) Detection of Australasian flavivirus encephalitic viruses using 12. Aitken THG (1977) An in vitro feeding technique for artificially demonstrating virus rapid fluorogenic TaqMan RT-PCR assays. J Virol Methods 117:161–167. transmission by mosquitoes. Mosq News 37:130–133. 30. van den Hurk AF, Hall-Mendelin S, Pyke AT, Smith GA, Mackenzie JS Vector compe- 13. Lindsay M, et al. (1996) An outbreak of Ross River virus disease in southwestern tence of Australian mosquitoes for chikungunya virus. Vector-Borne Zoonot Dis Ahead Australia. Emerg Infect Dis 2:117–120. of print. doi:10.1089/vbz.2009.0106. 14. Lindsay MDA, Johansen CA, Broom AK, Smith DW, Mackenzie JS (1995) Emergence of 31. Hall RA, Prow N, Pyke A (2010) Molecular diagnostics for Ross River virus. Molecular Barmah Forest virus in Western Australia. Emerg Infect Dis 1:22–26. Detection of Human Viral Pathogens, ed D Liu (CRC Press, Boca Raton, FL), Chapter 75. 15. Harley DO, Ritchie SA, Phillips DA, van den Hurk AF (2000) Mosquito isolates of Ross In press. River virus from Cairns, Queensland, Australia. Am J Trop Med Hyg 62:561–565. 32. Ritchie SA, Zborowski P, Banks D, Walsh I, Davis J (2008) Efficacy of novel updraft traps 16. Nasci RS, et al. (2001) West Nile virus isolates from mosquitoes in New York and New for the collection of mosquitoes in Cairns, Australia. J Am Mosq Control Assoc Jersey, 1999. Emerg Infect Dis 7:626–630. 24:520–527. 17. Ritchie SA, et al. (1997) Isolation of Japanese encephalitis virus from Culex annulirostris 33. Johansen CA, et al. (2005) Surveillance of arboviruses in mosquitoes from the south- in Australia. Am J Trop Med Hyg 56:80–84. west of Western Australia between 2000 and 2004. Arbovirus Res Aust 9:159–163.

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