In 1997, the State of Connecticut established its first comprehensive arbovirus surveillance program as part of a state-wide Mosquito Management Program (Andreadis, 1997; Capotosto, 1997). The program relies on the trapping and testing of mosquitoes for eastern equine encephalitis (EEE) and other arboviruses at 37 locations throughout the state. Locations include fresh water swamp sites (mostly red maple/white cedar) known or suspected to support mosquito populations that have historically tested positive for EEE, are capable of supporting such populations, or are proximate to locations where EEE-related equine or emu deaths have occurred. The results of the second full year of this program are presented herein.

Mosquito Collections. Thirty six of the 37 permanent locations identified in 1997 (Andreadis, 1997) were again selected to trap mosquitoes for virus testing. Due to low trap catches in 1997, the Stamford site was eliminated. One additional site in Waterford (Waterford Country School), where 3 EEE-related emu deaths occurred in 1997, was added. Trapping was conducted from June 1 through October 23 with CO₂-baited CDC miniature light traps. Traps were routinely set once every 10 days at each location on a regular rotation. One trap per site per night was used. Mosquitoes were transported live to the laboratory where they were immediately frozen on dry ice and then identified microscopically on a chill table using the keys of Carpenter and LaCasse (1955), Darsie and Ward (1981) and Means (1979, 1987). Specimens were pooled by species, site, and collection date. The number of mosquitoes per pool was 50. Specimens were stored at -80^oC.

Virus Assays. All of the virus isolation work was conducted in a newly renovated laboratory at The Connecticut Agricultural Experiment Station using the same protocols established at the Arbovirus Research Laboratory at Yale University in 1997. In most cases, mosquitoes were processed for virus the day after collection.

Each frozen mosquito pool was homogenized in phosphate buffered saline containing 0.5% gelatin, 30% rabbit serum, antibiotic, and antimycotic. The homogenate was centrifuged for 10 min at 520 g to clear the mixture of mosquito debris. A 0.1-ml aliquot of each supernatant then was inoculated into a 25-cm² flask containing a monolayer of Vero cells and incubated at 37^oC in 5% CO₂ for up to 7 d (Tesh et al., 1992). One uninoculated flask was kept as a negative control. The remainder of the supernatant was stored at -70^oC.

Flasks were examined daily for cytopathic effect. If cytopathic effect was noted, the cells were scraped from the flask and a cell lysate antigen was prepared (Ansari et al., 1993). Isolates were identified by enzyme immunoassay using reference antibodies that were prepared in mice and provided by the World Health Organization Center for Arbovirus Research and Reference, Yale Arbovirus Research Unit, Department of Epidemiology and Public Health, Yale University School of Medicine. These included: Cache Valley (CV), EEE, Highlands J (HJ), Jamestown Canyon (JC), La Crosse, and St. Louis encephalitis virus antibodies. Positive and negative control cell lysates were included in each test. Highlands J and eastern equine encephalitis antibodies crossreact in the enzyme immunoassay, but were distinguishable on the basis of titer.

Mosquito Collections. A total of 66,383 female mosquitoes, representing 28 species and 8 genera were collected, identified and tested for arboviruses (Table 1). This represented over 20,000 more mosquitoes than in 1997. The increased numbers were attributed to the excessive amount of rainfall the region received in June that resulted in increased numbers of flood-water Aedes, Anopheles and Culex spp. The most abundant species were Coquillettidia perturbans, Aedes canadensis and Culiseta melanura.. Thirteen additional Aedes species were collected, among which, Aedes trivittatus, Aedes cinereus, and Aedes vexans were the most numerous. Aedes abserratus was the most frequently caught univoltine "snow pool" Aedes. Culex pipiens and Culex restuans were equally abundant, and relatively large numbers of Anopheles punctipennis and Uranotaenia sapphirina were trapped.

Eastern Equine Encephalitis. Increased EEE virus activity was seen in 1998. Eight isolations from four different mosquito species in five locations were obtained (Tables 1 and 2). The first EEE isolations were made on September 29 from two pools of Ae. vexans and Cs. melanura mosquitoes trapped at Barn Island in Stonington (New London County) (Table 3). In response to this, a Phase II: Public Health Alert of the State Contingency Plan for Eastern Equine Encephalitis was implemented. Pesticide applications (truck-mounted ULV) were ordered by the Governor and subsequently implemented by the Department of Environmental Protection (DEP) in the affected region. Additional traps were set and no further EEE isolations were made at that specific location.

Six more EEE isolations were obtained from mosquitoes collected on October 6 and 7 from four widely scattered locations in Chester (Middlesex County), Newtown, Ridgefield (Fairfield County) and Voluntown (New London County). A Phase II Public Health Alert with pesticide spraying by the DEP was similarly implemented at Cockaponset State Forest in Chester due to the isolation of the virus from two species of human-biting mosquitoes Ae. canadensis and An. punctipennis. However, because the virus was limited Cs. melanura in Newtown, Ridgefield and Voluntown, a Phase I Public Health Notification (no pesticide sprays) was implemented in those regions. No further EEE isolations were made from mosquitoes that were collected in additional traps that were set in any of the four locations.

Five equines with clinical symptoms consistent with EEE infection were tested (Table 4). Specimens were obtained from the Connecticut Diagnostic Laboratory, Department of Pathobiology, University of Connecticut. EEE virus was isolated from the brain of a donkey that had died on October 16 in Canterbury. The animal was housed in an open barn surrounded by a wetland swamp. Previous history suggests the animal contracted the infection at this location which was approximately 3.7 miles from the trap site in Plainfield. No EEE isolations were made from any other the other four animals.

Results obtained in 1998 once again reinforce the highly focal nature of EEE which can be limited to mosquitoes and birds in a single swamp. The isolation of EEE from mosquitoes collected in Newtown and Ridgefield are particularly noteworthy as this area of the state has historically been considered to be at low risk for EEE. However, our findings now suggest that the EEE virus may be more widespread than had been previously thought. The isolation of EEE from mosquitoes in Stonington for three consecutive years (Andreadis, 1997; Andreadis et al., 1998) suggests that this region of the state is a focal center for the virus. The deaths of the donkey in 1998 and the 3 emus in 1997 clearly indicate that the present strain of EEE in Connecticut is a potentially serious public health threat and further emphasize the need for continued trapping and testing of mosquitoes in all areas of the state.

Highlands J. Twenty-three isolations of HJ virus were obtained from nine species of mosquitoes: Cs. melanura (10), Cs. morsitans (3), Ae. canadensis (3), Ae. vexans (2), Ae. stimulans, Ae. triseriatus, An. punctipennis, Cx. pipiens and Cx. restuans (Table 1). These mosquitoes were collected from nine different locations in five towns (Ledyard, Lyme, North Stonington, Stonington and Voluntown) all of which were located in the southeastern corner (New London County) of the state (Table 2). Both the number and geographic location of these isolates were similar to results obtained in 1997. The first isolation was made on August 5 and the last on October 15. Highlands J virus isolations were made in only two (Stonington and Voluntown) of the five locations where EEE was isolated, once again calling to question its usefulness as a reliable predictor of pending EEE activity in Connecticut.

Cache Valley. Twenty-two isolations of CV virus were made from seven species of mosquitoes (An. punctipennis, An. quadrimaculatus, An. walkeri, Ae. canadensis Ae. cinereus, Cq. perturbans and Cs. melanura) collected in 13 towns throughout all regions of the state, from August 19 through September 17 (Tables 1 and 2). More than half (14) of these isolations were obtained from An. punctipennis. This represents only the second isolation of this virus from Connecticut mosquitoes. The first isolation was made in 1979 from Ae. triseriatus (Calisher et al., 1986). This virus has been isolated from at least six genera of mosquitoes and is now recognized as the most widely spread Bunyamwera serogroup virus in North America, occurring in much of North America except the extreme southeastern states and southern Mexico (Calisher et al., 1986). Cache Valley virus has been isolated from large wild and domestic animals and has been associated with congenital malformations in sheep. In 1995, CV virus was isolated from a 28-year old male residing in North Carolina and presenting with severe encephalitis and multiorgan failure that ultimately resulted in death (Sexton et al., 1997). The broad distribution and relatively high prevalence of CV virus in mosquitoes known to feed on humans in Connecticut suggest wide spread exposure of humans to this virus which may be an etiology of unknown disease.

Jamestown Canyon. Six isolations of JC virus were obtained from four species of mosquitoes (Ae. canadensis, Ae. stimulans, Ae. trivittatus and An. punctipennis) collected in four widely distributed towns in four counties (Fairfield, Litchfield, Middlesex and New London) from June 25 through August 3 (Table 1 and 2). Three of the six isolations were made from Ae. trivittatus. These results were similar to those obtained in 1997 where seven isolations from five mosquito species were made from June 30 - July 14 (Andreadis, 1997). Results obtained from this and other studies on mosquitoes (Andreadis et al., 1994) and white-tailed deer (Zamparo and Andreadis, 1997), indicate that JC virus is widely distributed throughout the state and appears to be mostly vectored by early summer Aedes mosquitoes.

We wish to acknowledge the assistance of John Shepard, Jodi Corriea, Bonnie Hamid, Colleen Scott, Susana Cantu, J.R. Dubicki, Ronald Ferrucci, Arwen Mohr, John Russo, Kristina Steiff (The Connecticut Agricultural Experiment Station); Shirley Tirrell-Peck (Yale University); and Lt. David Florin (U.S. Navy).

Andreadis, T. G. 1997.

Mosquito arbovirus surveillance in Connecticut, 1997. Proc. 43^rd Ann. Meet. Northeastern Mosq. Control Assoc. pp. 10-12.

Andreadis, T. G., J. F. Anderson, and S. J. Tirrell-Peck. 1998.

Multiple isolations of Eastern equine encephalitis and Highlands J viruses from mosquitoes (Diptera: Culicidae) during a 1996 epizootic in southeastern Connecticut. J. Med. Entomol. 35:296-302.

Andreadis, T. G., P. M. Capotosto, R. E. Shope, and S. J. Tirrell. 1994.

Mosquito and arbovirus surveillance in Connecticut, 1991-1992. J. Am. Mosq. Control Assoc. 10: 556-564.

Ansari, M. Z., R. E. Shope, and S. Malik. 1993.

Evaluation of Vero cell lysate antigen for ELISA of flaviviruses. J. Clin. Lab. Anal. 7: 230-237.

Capotosto, P. 1997.

Connecticut's new mosquito management unit- first year. Proc. 43^rd Ann. Meet. Northeastern Mosq. Control Assoc. pp. 8-9.

Carpenter, S. J., and W. J. LaCasse. 1955.

Mosquitoes of North America (North of Mexico). University of California Press, Berkeley.

Calisher, C. H., D. B. Francy, G. C. Smith, D. J. Muth, J. S. Lazuick, N. Karabatsos, W. L. Jakob and R. G. McLean. 1986.

Distribution of bunyamwera serogroup viruses in North America, 1956-1984. Am. J. Trop. Med. Hyg. 35: 429-43.

Darsie, R. F., Jr., and R. A. Ward. 1981. Identification and geographic distribution of mosquitoes of North America, north of Mexico. Mosq. Syst. Suppl. 1: 1-313.

Means, R. G. 1979.

Mosquitoes of New York. Part I. The genus Aedes Meigen with identification keys to genera of Culicidae. N. Y. State Mus. Bull. 430a.

Means, R. G. 1987.

Mosquitoes of New York. Part II. Genera of Culicidae other than Aedes occurring in New York. New York State Mus. Bull. 430b.

Sexton, D. J., P. E. Rollin, E. B. Breitschwerdt, G. R. Corey, S. A. Myers, M.R. Dumais, M. D. Bowen, C. S. Goldsmith, S. R. Zaki, S. T. Nichol, C. J. Peters and T. G. Ksiazek. 1997.

Brief Report.: Life-threatening Cache Valley virus infection. New Eng. J. Med. 336: 547-549.

Tesh, R. B., J. Lubroth, and H. Guzman. 1992.

Simulation of arbovirus overwintering: survival of Toscana virus (Bunyaviridae: Phlebovirus) in its natural sand fly vector Phlebotomus perniciosus. Am. J. Trop. Med. Hyg. 47: 574-581.

Zamparo, J. M., T. G. Andreadis, R. E., Shope, and S. J. Tirrell. 1997.

Serological evidence of Jamestown Canyon virus infection in white-tailed deer populations in Connecticut. J. Wildlife Dis. 33:623-627.

Table 1. Total Number Mosquitoes Trapped and Tested for Arboviruses in Connecticut, 1998

Mosquito species	No.	No.	No. Virus Isolations
	Mosquitoes	Pools	EEE	HJ	CV	JC
Aedes abserratus	1,379	79
Aedes atropalpus	1	1
Aedes aurifer	494	54
Aedes canadensis	14,005	503	1	2	1	1
Aedes cantator	134	31
Aedes cinereus	5,645	341			3
Aedes communis	119	12
Aedes excrucians	301	47
Aedes sollicitans	13	11
Aedes sticticus	1,033	81
Aedes stimulans	539	87		1		1
Aedes taeniorhynchus	8	3
Aedes triseriatus	406	144		1
Aedes trivittatus	5,848	225				3
Aedes vexans	1,981	213	1	2
Anopheles punctipennis	1,644	275	1	1	14	1
Anopheles quadrimaculatus	194	51			1
Anopheles walkeri	386	75			1
Coquillettidia perturbans	15,946	485			1
Culex pipiens	4,334	256		1
Culex restuans	3,133	236		1
Culex salinarius	10	6
Culex territans	16	14
Culiseta melanura	6,441	398	5	10	1
Culiseta morsitans	708	130		3
Orthopodomyia signifera	2	2
Psorophora ferox	355	53
Uranotaenia sapphirina	1,308	168
TOTALS	66,383	3,981	8	23	22	6

Table 2. Locations of Arbovirus Isolations Made From Mosquitoes in Connecticut, 1998

Town	Location	No. Mosquitoes	No. Virus Isolations
			EEE	HJ	CV	JC
Barkhamsted	Hoyt Hayes Swamp	633
Bethany	Bethany Bog	288
Canaan	Robin's Swamp	9,237			1	2
Chester	Cockaponset St. For.	3,813	3
Cornwall	Mohawk Pond	2,166
Cromwell	Cromwell Meadows	3,173			3
Fairfield	Catamount Rd.	872
Farmington	Shade Swamp	4,243			1
Franklin	Wildlife Refuge Area	2,483			1
Groton	U. S. Sub Base	711
Haddam	Little City Rd.	172			1
Hampton	Fiske Rd.	1,338			1
Killingworth	Chittenden Rd.	699				1
Ledyard	Cedar Swamp	1,736		1	1	1
Litchfield	White Memorial	1,942			1
Lyme	Cedar Lake	1,069		1
Madison	Rt. 80 Cedar Swamp	586
Newtown	Key Rock Rd.	2,536	1			1
New Canaan	Hoyts Swamp	2,875
North Stonington	Assekonk Swamp	596
	Bell Cedar Swamp	2,313		3
	Exit 93	957		6
	Pawcatuck River	2,141		1
	Wyassup Lake	1,546		1	1
Old Lyme	Great Island	3,583
Plainfield	Cedar Swamp	1,584			5
Redding	Lyons Swamp	369			1
Ridgefield	Great Swamp	468	1		2
Stonington	Barn Island	1,156	2	8
	High School	2,690		1
Stafford	Nipmuck St. Forest	477
Tolland	Bolton Lake	1,581
Voluntown	Mt. Misery	2,300	1	1
Waterford	Country School	446			1	1
	Great Neck	2,418		1
Windam	Bass Rd.	738		1	2
Willington	Pinney Hill Rd.	448
TOTALS		66,383	8	23	22	6

Table 3. Eastern Equine Encephalitis Virus Isolations from Mosquitoes in Connecticut, 1998

Date

Town

Location

Mosquito Species

Pool size

September 29	Stonington	Barn Island	Ae. vexans	23
			Cs. melanura	15
October 6	Voluntown	Mt . Misery	Cs. melanura	5
October 7	Chester	Cockaponset	Ae. canadensis	3
			An. punctipennis	1
			Cs. melanura	2
October 7	Newtown	Key Rock Rd.	Cs. melanura	3
October 7	Ridgefield	Great Swamp	Cs. melanura	1

Table 4. Equine Testing for Eastern Equine Encephalitis Virus in Connecticut, 1998

Date

Town

Breed

Tissue

Results

September 18	Marlborough	Horse	Brain	Negative
October 16	Canterbury	Donkey	Brain	Positive
October 28	Canterbury	Thoroughbred	Brain	Negative
October 28	Woodbury	American Quarter Horse	Brain	Negative
November 17	Lebanon	Appaloosa	Brain	Negative