540 J. AM. MO.SQ. COX'IKOI. A.S.SOC. Vol.. 1, No, 4 Huberi, A-. W. A. Rush. and.!. M. lli-enii.ni. 1954- ban', (Walker), ran^in^ from 77% of (he loial in Simplii'ied techniques for ihe, continuous rearing of July 10 44^ in September. From the 2.158 inos- Cuiexidr.viH.'- with addiiional noiesand observation;.. quitoe.s assayed from this area by (.he New New; Jer- Mosq. 14^75-78- sey State Dep;mmen! of Health, 2 isolates of McDon.ilcl, G.,J. R, Sinkh and (>. P. Shelden, 1977. EEE viru.s were obtained from 1.225 perlur- Laboratory re;n-iny oi' Cuif\ ffiiititliwli't.\ Skufc C/f. beins. These viral isolates, from collections made (Dip [era: Cu lie-id ;te). }- Ausi. Eniomol. Soc. 16'.:^5?-a58. on July 25 and September 7. reflected mini- Singh. K. R. P. and R. K. R;izd;m. 1977. Mas.s rein-ins mum field infection rate (MFIR) in this are;) of of Culc\ pifiifn'- fnligttiis tinder .nnbieni condition.1'. 1:612 for this species. Three additional EEE Indian J. Med. Res, 65 fSuppi.):86-90. virus isolates were obtained from 263 Culisefo meUmura (Coquiiletl) fMFIR 1:88). The first reported isolation of EEE virus from an arthropod was from Cq. perturbaiv collected in Georgia in 1948 (Howitt ei al. 1949) and EEE 127619 virus has since been isolated from this species in New York (Morris and Srihongse 1978) and New- Jersey fVeazey el al. 1980). Coquit'iettidw perlurbans ABSENCE OF EASTERN EQUINE was likelv responsible for transmission of EEE ENCEPHALITIS (EEE) VIRUS IN- virus to equines and humans in southwestern IMMATURE COQUILLETT1DIA Michigan in 1980 (Francy 1982) and has a high WITH PERTURBANS ASSOCIATED vector potential for this virus in Massachusetts EQUINE CASES OF FEE' (Nasci and Edman 1981). Field studies of the GARY G. CLARK.2 WAYNEJ. CRAW blood-feeding pattern for this species have linked C. L. CRAHBS^ it closely to large mammals, especially equines (Nasci and Edman 1981). During the summer of 1983, 5 fatal equine In laboratory Studies with Cq. perturbaiv and cases of eastern equine encephalitis (EEE) were EEE virus, Chamberlain et a!. (1954) demon- confirmed in New Jersey (Crans, unpublished strated virus transmission by this species and data). These cases were conspicuous by their gave it a "good vector potential rating." Schaef- occurrence at sites, ca. 30 kin from the nearest fer and Arnold (1954) found that EEE virus, salt marsh, in Ocean and Sussex counties- His- ". could be demonstrated in eggs iaid by in- torically, equine cases of EEE have been re- fected [Mansonw [Coyuilletlidui] perlurbans." peatediy diagnosed from several inland lo- Following oral infection of CQ. perturbans with calities in New Jersey (Goldfield and Sussman Venezuelan equine encephalitis (VEE) virus, 1968). Haves et a). (1962) indicated thai the Chamberlain et a!. (1956) recovered VEF. virus distance from coastal marshes made it unlikely from 7 of 16 egg rafts. In a similar study, they that the '.alt marsh mosquito, Aede^ sollicilaw found [hat 2 of 19 egg rafts laid by Cq. penur- (Walker), served as the vector for EEE virus in bdm, afier ingesting an infectious biood meal, these area'.. contained EEE virus. In an attempt to preclude Four of the 1983 cases occurred at a distinct transovum viral contamination. Chamberlain focus in Jackson Township, Ocean County. and coworkers rinsed the surface of the eggs After the initial case with an onset of July 8., they assayed with a serum-saline solution- The mosquito surveillance was initialed and con- difficulty associated with laboratory coloniza- centrated on 3 Jackson Township farms within tion of Cq. perturhuns has prevented any defi- a 5-km radius of each other. Subsequent cases nitive studies of its role as an overwintering host from this area had onsets ofJuly 23, August 12 for arboviruses. and August 26. The dominant mosquito species The association of EEE virus with equine collected at these farms was CoquiHeitidia pertur- fatalities in New Jersey during-1983 suggested a more important role for CQ. perim-bcif^ in the ecology and transmission of this virus. The The views of the .tinhors do purport reflect maintenance of the virus by larval mosquiioe-s (.he positions of the Department of [he Army me has been proposed as an overwintering mech- Department of Defense. New Jersey Agric. Exp!. Sla- anism during interepidemic periods. There- Pur.!. No. D40101-OI-85, si;pponecl In iunds from fore, we viewed this as an opportunity to ex- chc New Jersey Si;it<.' Mosquito Comro] Commission plore the hypothesis thai EEE virus was main- and the Haich Aci- tained through the winter in Cq. periurhan-, iar- Department of Arboviral Eni-omoiogy, US Army vae- Medical Research Insmme of Iniecuous Diseases. Pori Decrick, Frederick, MD 2I70]-50IL From October 1983 through March 1984, Mosquito Research and Control. Cook CoHe^e. immature Cq. perturlww were collected in areas P.O. Bo\ 231, New Brunswick, N.1 08903. ;Kl).icem co the sites where the 4 Ocean County DECEMBER, 1985 J. AM. MOSQ. CONTROL Assoc. 541 equine fatalities occurred. Additional collec- Table 1. Immature Coquillettidia perlurbans collected tions were made in Cape May and Gloucester in New Jersey and assayed for virus. counties where EEE virus transmission has been Colleaion Collection No. No. detected intermittently during prior years. Lar- period Stage assayed pools vae were removed from submerged roots of a variety of aquatic shrubs, herbaceous plants, Dennisville, Nov. 1983- 1.2 922 47 and grasses by utilizing a modified bilge pump Cape May Co.Jan.1984 1.3 701 70 70 according to the methods of Walker and Crans L4 7 Pupa 2 (personal communication). Larvae were sepa- Subtotal 1,695 125 rated with soil sieves and survey pooled by stage lona Lake, Nov. 1983 L2 120 6 in ml of diluent containing medium H-199 Gloucester Co. L3 70 7 supplemented with glutamine, 20% fetal bovine L4 10 serum (FBS), and antibiotics (200 units/ml Subtotal 200 14 penicillin, 200 ug/ml streptomycin, and 10 Jackson and Oct. 1983- L2 1,415 111 jiig/ml fungizone). In order to avoid a possible Mills Lake, Mar. 1984 L3 3,000 300 inhibitory effect on the virus by the larvae, as Ocean Co- L4 250 25 Subtotal 4,665 436 reported by Ksiazek et al. (1985) for several TOTAL 6,560 575 virus-vector systems and that was subsequently verified for Cq. perturbans and EEE virus (dark et al. unpublished data), 2nd stage larvae were imately 30 km northeast and northwest of this placed in pools of 20 while 3rd and 4th stage study site. In contrast to the previous year, were limited to 10 per pool. All pools were adult populations of Cq. perturbans were greatly sealed and stored on dry ice or at -70C until reduced in 1984, despite large larval collections assayed for virus. at these two localities. In 1984, 5 fatal equine Each pool of Cq. perturbans was ground in a cases of EEE were reported from Gloucester prechilted tissue grinder. Suspensions were County where only 200 immature Cq. perturbans clarified by centrifugation at 2,500 rpm for 20 were collected during November 1983. In gen- min at 4C, and the resultant supernatants were eral, there was substantial EEE virus activity assayed for virus by plaque assay in Vero cells. detected in these areas during the summer fol- Aliquots of 0.1 ml of each supernatant were lowing this study of immature Cq. perturbans. inoculated in triplicate into Vero 12-well trays This study of the overwintering stages ofCq. and adsorbed for hr at 35C and then overlaid perturbans, temporally and spatially associated with 2 ml of agar overlay media consisting of with equine fatalities of EEE and EEE virus- equal volumes of 1.5% agarose and 2x Eagle's infected female Cq. perturbans, failed to impli- basal medium in Earle's salts with Hepes buffer cate this species as an overwintering host for (4 g/liter), 10% heat-inactivated FBS, and PSF. EEE virus. With Keystone virus (LeDuc et at. Cell cultures were incubated in a humidified 1975), which is transovarially transmitted, the atmosphere with 5% COa for plaque develop- MFIR in reared adults approximates that found ment. After 4 days, mi of the overlay medium in larvae. If transovarial transmission was oc- containing a 7% solution (stock 1:300) of curring in the virus-vector system we investi- neutral red was added to each cell monolayer. gated and the 2 EEE virus isolates (MFIR These cultures were incubated for 24 hr and 1:612) were the result of transovarial transmis- examined for plaques. sion infections, we should have obtained 7 iso- Virus was not isolated from 6,560 immature lates from the Jackson Township collections. Cq. perturbans (Table I). The majority (71%) of Although Cq. perturbans appears to play a major these larvae originated from Ocean County role as an epizootic vector for EEE virus in near sites where the 4 equine fatalities had oc- freshwater habitats, we were not able to detect curred. transovarial or transstadiai maintenance of EEE Although EEE virus surveillance was initiated virus in this species. on June I, 1984, evidence of EEE virus We thank Drs. Michael D. Turell and Doug- transmission at the Jackson Township site las M. Watts for reviewing the manuscript. not detected until August 24. Virus transmis- sion continued through September 28, based References Cited on 7 EEE virus isolations from Cs. meianura during this period. At the Dennisville site in Chamberlain, R.W., R.K. Sikes and D.B. Nelson. Infection of Mansoma and Cape May County, EEE virus activity was indi- 1956.