Journal of the American Mosquito Control Association, 19(4):392-396,2003 Copyright O 2003 by the American Mosquito Control Association,Inc,

FLAVIVIRUS ISOLATIONS FROM MOSQUITOES COLLECTED FROM WESTERNCAPE YORK PENINSULA, , 1999-2OOO

CHERYL A. JOHANSEN,' DEBRA J. NISBEX' PAUL ZBOROWSKI,' ANDREW E VAN DEN HURK.I SCOTT A. RITCHIE, AND JOHN S. MACKENZIEI

ABSTRACT' After the lst appearance of Japanese encephalitis virus (JE) on mainland Australia in 1998, a study was undertaken to investigate whether JE had become established in enzootic transmission cycles on western Cape York Peninsula. Adult mosquitoes were collected during the late wet season from Kowanyama and Pormpuraaw in April 1999, and Pormpuraaw and Barr's Yard in April 2000. Despite processing 269,270 mosquitoes for virus isolation, no isolates of JE were obtained. However, other flaviviruses comprising Murray Valley encephalitis virus, Kunjin virus, Alfuy virus, and Kokobera virus (KOK) were isolated. lsolaies of the alphaviruses , Barmah Forest virus (BF), and Sindbis virus (SIN) also were obtained. The majority (88Vo) of isolates were from members of the Culex slllens subgroup. Single isolates of KOK, B[ and SIN were obtained from Ochlerotatus vigilar, Oc. normanensls, and Anopheles bancroftii, respectively. The isolations of flaviviruses during the late wet season indicate that conditions were suitable for flavivirus activity in the area. No evidence was found to suggest that JE has become established in enzootic transmission cycles on western Cape York, although study sites and field trips were limited.

KEY WORDS Flavivirus, alphavirus, Murray Valley encephalitis virus, Kunjin virus, , Australia

INTRODUCTION 11'S and l4l"3O'E to 146'E (Fig. 1). The peninsula The lst appearance of Japanese encephalitis vi- is bordered by the to the west rus (JE) on mainland Australia occurred during a and the to the east, and encompasses an widespread outbreak in northern Queensland and area of approximately L37,2OO km,. The main riv- the Torres Strait in 1998 (Hanna et al. 1999). In ers are the Mitchell and the Gilbert, which empty addition to a confirmed human case on Badu Island into the Gulf of Carpentaria. The climate is mon- in the Torres Strait, a 2nd human case was serolog- soonal. The average annual rainfall on westem ically confirmed in a fisherman working near the Cape York Peninsula is 1,200 mm (Biggs and Phil- mouth of the Mitchell River on western baoe York lip 1995). More than SOVoof the rain occurs during Peninsula. Seroconversions to JE were detected in the wet season. between December and March. sentinel pig herds on Badu Island and northern Temperatures are usually warm to hot, with maxi- Cape York Peninsula (Hanna et al. 1999), and 43 mum temperatures of >40'C recorded during sum- isolates of JE were obtained from mosquitoes col- mer. The study sites included the communities in Iected on Badu Island (Johansen et al. 2001). Fur- the Mitchell River area of western Cape York Pen- thermore, JE activity on western Cape York was insula: Kowanyama (previously known as the confirmed when antibodies to JE were detected in Mitchell River Mission), Pormpuraaw (Edward young domestic pigs at Barr's Yard, a prison farm River), and Barr's Yard. The population near the Mitchell River (Hanna et al. 1999). How- of western ever, no other evidence of JE activity was detected Cape York Peninsula in 1995 was approximately (King in a widespread serosurvey of humans, and JE was 1,450 1995). not isolated from mosquitoes collected from north- Mosquito collections were carried out between ern and western Cape York Peninsula soon after the Apnl 2O-27, 1999, and April 3-7, 2000, when human case was recognized (van den Hurk et al. adult vector mosquitoes were likely to be most 2001a). This paper describes entomological and vi- abundant. Adult mosquitoes were collected by us- rological investigations undertaken on western ing Centers for Disease Control (CDC)-type light Cape York Peninsula during the late wet season in traps baited with I kg of dry ice and octenol (re- 1999 and 2000, to examine indigenous flavivirus lease rate ca. 5 mg/h). Mosquitoes were identified activity and the possibility that JE may have be- to species on a refrigerated table and placed into come established in enzootic transmission cycles in pools of s100. The closely related Culex annuli- the region. roslris Skuse, Cx. palpalis (Taylor), and Cx. sitiens Wiedemann were pooled as Cx. sitiens subgroup MATERIALS AND METHODS mosquitoes for analysis because of overlapping Cape York Peninsula is located in far northern morphology, which makes identification of field- Queensland, Australia, and extends from 16'5 to collected specimens difficult (Chapman et al. 2000). Pools were homogenized and inoculated onto 96-well monolayers of C6136 cells. After 6 r Department of Microbiology and Parasitology, School days of incubation the monolayers were fixed with of Molecular and Microbial Sciences, The University of Queensland, St. Lucia, Queensland 4072, Australia. acetone and assayed by tissue culture enzyme im- 'Tiopical Public Health Unit, 19 Aplin Street, Cairns, munoassay (TC/EIA) (Broom et al. 1998) by using Queensland 4870, Australia. fl avivirus group-reactive monoclonal antibody 4G2 Dscpvsen 2003 FLlvrvnus IsoLATIoNspnorvr C.lpe Yonr, Austnl-r,c 393

Anopheles bancroftii Glles (26.4Vo), Ochlerotatus PAPUA (3.3Vo), Laveran 6 normanensis (Taylor) An. farauti ' -saibai (Giles) (l-8vo), k sensu lato (3.27o), Cx. whitmorei Tones : .. Mansonia uniformis (Theobald) (l.1%o). . ' and - Str4i, were obtained from mosquitoes ^o? I"t|"h No isolates of JE collected from western Cape York Peninsula in 67, 1999 and 2000. However, other Japanese encepha- litis serogroup viruses were isolated' including 17 Northern Kunjin virus (KUN), 3 Murray Valley encephalitis PeninsuJa (MVE), Alfuy virus (ALF) isolates (Ta- At{pa virus and 5 Ared SB ble 2). One isolate of Kokobera virus (KOK) (a Nu&| member of the Kokobera serogroup) was obtained, as were 4 isolates of Sindbis virus (SIN) and single (RR) and Barmah For- otu loohil isolates of Ross River virus est virus (BF). Twenty-eight (87.57o) of the isolates were obtained from members of the Cx. sitiens sub- Soatb group. Kunjin virus was the only arbovirus ob- P6frr collected in both years of Western iained from mosquitoes Ocean virus cee the study period. Murray Valley encephalitis and ALF were only isolated from mosquitoes col- 'ffi lected in 1999, from both Kowanyama and Porm- Cape puraaw. The KOK isolate was obtained from a pool of Ochlerotatus vigilax (Skuse) collected at Porm- L**"G puraaw, and was the only flavivirus isolated from mosquitoes not belonging to the Cx. sitiens sttb- ct4/ group. Pools of Oc. normanensis yielded single iso- of York lates of BF and SIN and 1 isolate of SIN was ob- tained from An. bancrofiii. Alphaviruses were only collected in April 2000. Cairns isolated from mosquitoes Peninsula The overall infection rate of mosquitoes was gen- erally low, with the highest MIR of 0.35 per 1'000 mosquitoes obtained with KUN isolated from Cx. siriens subgroup mosquitoes collected from Ko- Fig. 1. Locations of adult mosquito collection siteson wanyama in April 1999, western Cape York Peninsula, 1999-2OOO. DISCUSSION

(Henchal et al. 1982) for detection of flaviviruses. No isolates of JE were obtained from mosquitoes All isolates were conflrmed by reisolation from the collected on western Cape York Peninsula during original homogenate and stocks were grown on the late wet season in 1999 and 2000, despite pro- porcine-stable equine kidney (PSEK) cells. Isolates cessing 269,270 mosquitoes for virus isolation- were identified by inoculation of virus stocks onto Laboratory studies have revealed that Cx. sitiens 96-well monolayers of C6136 cells, incubation for subgroup mosquitoes are vector competent for JE 6 days, and subsequent assay by TC/EIA with a (van den Hurk et al. 2OO3), and it is likely that JE panel of flavivirus-specific monoclonal antibodies would have been detected had JE activity been pre- (Johansen et al. 2000). Mosquito homogenates that sent. The absence of JE isolations indicates either caused cytopathic effect but were not identified as that the virus has not become established since ini- a flavivirus were subsequently assayed by TC/EIA tially detected in 1998 (Hanna et al. 1999), or that with alphavirus-specific monoclonal antibodies (Jo- JE was not active in the study site at the time mos- hansen et al. 2000). Minimum infection rates quitoes were collected. More sustained sampling (MIRs) were calculated by using a formula derived over an extensive areafiray be required to elucidate from Chiang and Reeves (1962). this. Nonetheless, 4 other flaviviruses comprising MVE, KUN, ALR and KOK were isolated, indi- cating that conditions were favorable for transmis- RESULTS sion of flaviviruses during the study period. Infec- A total of 269,27O adult mosquitoes collected tion rates in mosquitoes were similar to those from western Cape York Peninsula in 1999 and observed in mosquitoes collected from the Gulf 2000, comprising 27 recognizable taxonomic units, Plains region of northern Queensland in 2000 (van were processed for virus isolation (Table 1). The den Hurk et al.2OO2). most abundant species processed for virus isolation This is not the 1st time isolates of these viruses were members of the Cx. sitiens subgroup (59.8Vo), have been recorded from mosquitoes collected in 194 JounNar-op rtrn AusnrceN Mosqurro CoNrnol Assocrenon Vor-.19, No. 4

Table l. Number of female mosquitoesprocessed for virus isolation from western Cape York, Australia, 1999- 2000.

No. processed (7o) Species 1999 Aedeomyia catasticta Knab 0 (o.0) l9 (<0.1) l9 (<0.1) A ede s line ato p enn i s (Ludlow) 66(<0.1) 1,719(1.5) 1,785(0.7) Anopheles amictus Edwards 318(0.2) 54 (<0.1) 372 (O.t) An. annulipes Walker sensu lato 104(0.1) 0 (0.0) r04 (<0.1) An. bancroftii Giles 4s,s9o(29.r) 25,494(22.7) 7r,084 (26.4) An. farauti Laveran sensu lato 4,237(2;7) 4,363 (3.9) 8,600 (3.2) An. meraukensis Venhuis 0 (0.0) 5 (<0.1) 5 (<0.r) Co qu ille ttidia xant ho gas ter (Edwards) 86(0.1) 43 (<0.r) 129(<0.1) Culex bitaeniorhynchus Glles 137(0.1) 249 (O.2) 386(0.1) Cx. gelidus Theobald l (<0.1) r (<0.1) 2 (<0.1) Cx. pullus Theobald 20(<0.r) 2 (<0.r) 22 (

o toes during this study may also indicate enzootic z transmission. (t The majority of flavivirus isolates were from Cx. V siliezs subgroup mosquitoes, providing further ev- d v idence for the role of these species in transmission of flaviviruses. Culex annulirostris is a confirmed F vector of MVE and KUN (Kay et al. 1984)' and Cx. annuliros- A the vector competence of Australian o tris and Cx. sitiens for a Torres Strait JE strain has c.t (van den I recently been confirmed in the laboratory J Hurk et al. 2003). Other species of mosquitoes col- lected from western Cape York Peninsula that are s d g c- confirmed or suspected vectors of IE include Culex H bitaeniorhynchus Giles (Banerjee and Deshmukh 1987), Cx. whitmorei and Ma. uniformis (Peiris et (Vy- ho @ o al. 1992), and Aedes lineatopennis (Ludlow) # z thilingam et al. 1997). Mansonia uniformis can and could po- dc{ al reach pest proportions in Australia, o = of JE on Cape (! tentially have a role in transmission (.) in \o York Peninsula. In contrast Cx. bitaeniorhynchus F collected in large num- q and Cx. whitmorei were not N a minor role in JE trans- o bers, and may have only B Ei mission. However, the collection of Culex gelidus +:nn\O 60\ O:(o ol - Theobald, a newly recognized mosquito in Austra- -iA-i-i-i ,; lia (Ritchie et al. 2001), on western Cape York may iN6or+ e{ al have implications for flavivirus transmission. Al- #E E though only a single conflrmed Cx' gelidus was col- ifia*3SDRil8ERE3S lected in the westem Cape York, large numbers of .9e= cl- -c{Nic.l*cl lE (Taylor) were collected that may have - z li Culex vicinus 2\ lp been misidentified Cx. gelidus (Ritchie et al. 2001). tv k I possible that Cx. gelidus is not well es- lx It is also ff q-n09nqoq\\q'1 ld locations where adult mosquitoes 9e eP\oroc.lc'.lNO:oo\Oo l\o tablished at the F-\Or|rlu.) \O\OOlr)- lo stttdy- Culex gelidus has gig€g le were sampled during this lo been collected in large numbers in CDC-type traps &o. t> to (Johansen et al. 2003)' in- Eqr lo in northern Queensland RK:8$fl$fiF38 readily attracted to the mosquito o ;9E'Z66@6+C.l<-f-6O\N dicating they are zH:* +ddoiviiriddic.iri t6 collection traps used in this study- Furthermore, JE E.o 6 Ni -C.l -ei*ci I o*'- lb0 was isolated from Cx. gelidus collected on Badu P o Island in 2000 (van den Hurk et al. 2001b), and is q q td considered an important vector of JE in southeast 6.4 Asia (Vaughn and Hoke 1992). lf JE reappears on o t'; d'e'eeETEEFEFo Cape York Peninsula, further collections of mos- o P-8 I z quitoes for virus isolation should be undertaken to -s8-8.-SS*3'8 i a AAAZ7'r e; ! e! to examine the potential for virus establishment, and o ? 3 = ! 3 3 3 3s 3 3to .o a iinit€$c-Gs the subsequent implementation of control programs te, to be con- s.c::qqqqh:%huqxE:! x:sE s.s x to protect residents from infection needs P k k k k \o P k> k ! t- sidered. sssssRss \\sRto l8 The isolations of the alphaviruses RR and SIN are de(jdddde€d6t-t not surprising. Sindbis virus is the arbovirus most to commonly isolated from mosquitoes in Australia 1.. AAAAS$=s==v,vi-==- has the c.i FF999V (Mackenzie et al. 1994), and Queensland . .NolNen lH o:c-lco\nv.) . -. . highest incidence of RR disease in Australia (Harley Nala.lololel(or)r)s r.(g et at. 2001). Barmah Forest virus is also a common L!!LLL!LHE t5 cause of arboviral disease in Queensland (Hills and t- Sheridan 1997). However, disease caused by SIN in- fection is rare in Australia (Mackenzie et al. 1994). E3i >: FFFt- t^) ACKNOWLEDGMENTS FF^6 *trF rl g;rna!Et?e We would like to acknowledge the assistance of Alistair Hart (Tiopical Public Health Unit, Cairns) 396 JoUnNer-or rns AvBmcAN Moseurro Corlrnol AssocnnoN VoL. 19.No.4

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