SEPTEMBER1990 As. puncnnn tNo OtHnn Vpctons or CHIK VIRUS 4t5 AEDES FURCIFERAND OTHER MOSQUITOESAS VECTORSOF CHIKUNGUNYAVIRUS AT MICA. NORTHEASTERNTRANSVAAL, SOUTH AFRICA P. G. JUPP ANDB. M. McINTOSH Arbouirus Ilnit, National lrctitute for Virolagy and, Department of Virolngy, Uniuersity of Witwatersrand, Priuate Bag X4, Sandringham 2131,South Africa ABSTRACT. From 1977to 1981,studies were conductedon a farm at Mica wherc Aedesfurcifer had. been a vector during an epidemic of chikungunya virus in 1976to determine whether the virus persisted in this mosquito,the likelihood of vertical transmission, and whether any other Aedesspecies could have been vectors. Aedes furcifer/cord,ellieri was the only prevalent tree hole Aedes which fed readily on monkeysand humans and occurredthrough the summer until the onset of winter. Virus was not isolated from 7,241females and 4,052 males of this group, which were largely Ae. furcifer and which included a sample of the first post-epidemicpopulation. Five additional Aedes specieswere prevalent in bamboo pots, 3 of which (Ae. aegypti,Ae. fulgens and.Ae. uittatus) were shown to be competentlaboratory vectors. Virus was not isolated from a sample of 13,029such newly emergedmosquitoes representing the first post-epidemicpopulation. It is concludedthat Ae. furcifer is an epidemic-epizooticvector which doesnot maintain the virus at Mica and that no other mosquito speciescould have been important vectors. INTRODUCTION Ae. furcifer was highly susceptibleto infection with the virus and a moderately efficient trans- (CHIK) Chikungunya virus occurs in the mitter (Jupp et al. 1981). tropical region of southern Africa, which in After the epidemic, field observations were South Africa comprises the eastern Transvaal continued at Mica on the farm "Hope," chosen lowveld and coastalnorthern Natal. Human out- becausethe highest post-epidemicimmune rates breaks of the virus have been infrequent and were recorded there for both the human and always related to ample rains in wooded sa- baboonpopulations. A seriesofmosquito collec- vanna. Infections in man have been recognized tions with monkey and baboonbaits was carried in the easternTransvaal in 1956,1975, 1976 and out in March 1977during the summer following 1977(Gear and Reid 1957, Mclntosh et al. 1977. the outbreak (Mclntosh et al. 1977). The Ae. Morrison 1979). A large epidemic occurred in group, composed mainly of the Zimbabwean lowveld in 1962 (Mclntosh furcifer/cordellieri et Ae. was the predominant speciestaken al. 1963a)and an epizootic among furcifer, vervet mon- on wild primate bait and 8- to lO-fold greater keys (Cercopithccus aethinps) in northern Natal numbers were collected in the understory than in 1964(Mclntosh 1970).Studies done in rela- on the ground. tion to these outbreaks, especiallythe outbreak Further observations were made during the at Mica in the northeastern Transvaal in 1976 summer in 1977, 1978, 1980 and 1981 which (Mclntosh et al. 1977),have shown that vervet were designedto answer 2 questions. The first monkeys and baboons (Papio ursinus) are the was whether the virus remained in the local Ae. primary vertebrate hosts,while the primary vec- population, with this speciesacting as a tor is the Aedes group furcifer furcifer/cordellierir of reservoir vector, or whether it disappeared.Sec- mosquitoes. ond, could other mosquito species have been During the rural epidemic at Mica in March- involved in the transmission of virus during the April 1976,Aedes was furcifer/cordellieri by far outbreak which escapeddetection at the time? the most prevalent speciescollected off human Answers were sought as follows: 1) the mosqui- bait and yielded16 isolations of (Mclntosh virus toes at "Hope" were monitored for virus infec- et aI. 1977).The identification of male mosqui- tion, including post-epidemic survival of virus toes also taken in the catchesindicated that Ae. through the dry winter by vertical transmission, was comprised largely of Ae. furcifer/cordellieri 2) the relative abundance of mosquito species (Edwards) and was probably the princi- furcifer and their feeding preference for man and wild pal speciesin the epidemic. In the laboratory primates were determined, and 3) the vector competenceof the 5 most common tree hole breeding aedine mosquitoesapart from Ae. fur- I Taxonomic examination of the Ae. furcifer gtoup cifer was evaluated. Transmission experiments in South Africa in the light of Huang's (1986)revision had been conductedwith 3 of these speciespre- indicates that Ae. taylori is not present but Ae. cordel- viously but not with the "Hope" mosquito pop- Iieri Huane occursthere. ulations. Furthermore, more tests were needed JounNer, oF THE Avnnrcen Mosqurro CoNrRor, AssocrATroN Vol.6, No.3 on these speciesbefore a 50% infection thresh- the eggs were hatched and adults reared. The old could be determined. pots were alternately flooded and dried twice so that the majority of eggswere hatched. Mosquitoes collected as adults were killed MATERIALS AND METHODS with hydrogencyanide, pooled accordingto spe- cies and stored in liquid nitrogen. Adults reared Human-baited catches:In these collections 2 from pots and bottles were discardedafter iden- volunteers used test tubes to collect mosquitoes tification, except those from the 1976-77 sum- which alighted on their bare legs fot 2-3 h after mer which were stored according to speciesin sunset. At night, flashlights shaded with red liquid nitrogen and selectedspecies which were cloth were used. Forty-nine man-hours of col- kept alive for vector competencestudies. Iecting were done for 12 daysduring March 1977 Attempted uirus isolation from mosquitoes: and February 1978. Mosquito suspensionsprepared from pools of Monkcy-baited surtion traps: In the March eachspecies were inoculated intracerebrally into 1977collection series (Mclntosh et al. 1977),a 1- to 2-day-old mice for attempted virus isola- single anesthetized baboon or monkey was tion. placed on a wire mesh platform with two 12- Vector competencetests: Specimensof the 5 volt, 8-watt fans suspendedbelow. These fans most prevalent Aedes speciesaged 1-14 days, sucked mosquitoes attracted to the bait down- and reared from the bamboo pots, were used for ward into organdie cages (Jupp 1978). Subse- vector competencetests. Experiments were con- quently, the traps differed slightly in that 2 ducted in an insectary where the mosquitoes unanesthetizedvervet monkeys were housed in were maintained at 75-80% RH 24-26'C. The a wire meshcage (60 x 45 x 30 cm) under which virus used was the H817 strain of CHIK virus the 2 suction fans were hung. Traps were sus- at the third mouse-passagelevel. Attempts to pended10 m abovethe ground in the understory infect mosquitoes were made by feeding them of 3 trees for 2 h after sunsetduring March 1977, on vervet monkeys while these monkeys were February 1978and January to June 1980. viremic after inoculation of virus 48 h previ- Carbon dioxide-baited light traps: The light ously. Monkeys were anesthetizedand exposed traps used had a 5-mm wire mesh filter fitted to mosquitoesheld in 10-cmdiam canisterswith over the trap entrance and COz was released mesh-coveredends strapped to the monkey's through 3-mm bore polythene tubing close to chest and abdomen.Immediately before feeding the opening (Jupp et at. 1980).The sourceofthe began, a blood sample was collected from each COz was a tin insulated with corrugated card- animal to determine the viremia. Virus titra- board containing about 2 kg of dry ice. The traps tions were done in infant mice inoculated intra- were set overnight during February, March and cerebrally and titers refer to logrolDso/ml. The April 1977and February, March and June 1980 infection rate, i.e., the proportion of mosquitoes with a total of 63 trap-nights. feeding which becameinfected, was determined Artificial ouiposition sites in trees: To collect by testing them individually for virus by inocu- tree hole breeding speciesunsampled as adults lation of infant mice. Thesedeterminations were by the 3 collecting methods described above, done 15-16 days after the infective meal and 1- bamboo oviposition pots were exposedso as to 2 days after the transmission feeds. Infected include all or part of 4 summers:November 1976 mosquitoeswere held for 13 or 14 days before to April 1977, December 1977 to April 1978, transmissionswere attempted. These were done February to June 1980, and February to June by feeding groups of potentially infected mos- 1981. During 1980, 560-ml plastic bottles, quitoeson 1 or 2 Syrian hamsters.Transmission painted black on the outside, containing 2 of virus was determined by testing for hemag- wooden paddles ("tongue depressors") for ovi- glutination-inhibition antibodies in the serum position were exposed in addition to the pots. of hamsters 21 days after the transmission was Pots and bottles were suspendedfrom trees at attempted. The titer of virus needed to infect varying heights up to 6 m above the ground. In 50% of the mosquitoeswas estimated from the 1980 and 1981, the openings of about half the infection rates. pots were reduced by closing them partly with "Hope" board. During each visit to a sample of any larvae present in pots or bottles was pre- RESULTS served for subsequent identifrcation and the paddles were changedin the caseof the plastic Adult mosquitocollections: At least 15 species bottles, the exposedones being returned to the were collected off human bait (Table 1) but Ae. laboratory under humidification. At the end of furcifer/ cordellieri represented,84% of the total summer,pots and bottles with their severalsets catch with a high biting late of 40.2mosquitoes ofpaddles were returned to the laboratory where per man-hour. Other specieswhich occurred in SnprnMspn 1990 Ap. punarsn lno Otnnn VEcroRS or CHIK VIRUs 4t7 Table 1. Mosouito collectionsusine 3 different baits. Monkey-baited suction Human bait at ground Light traps with CO2at traps 10 m above ground IeveI' ground level" in trees' No. as % No./ No. as % No./trap No. as % No./trap Species No. of total man-hour No. of total hour No.