Journal of the Ameican Control Association, 17(1):73--78,2OOl Copyright O 2001 by the American Association, Inc.

ARBOVIRUS SURVEILLANCE IN SOUTH CAROLINA, 1996-98

ARTHUR WOZNIAK,' HAROLD E. DOWDA,' MARSHA W. TOLSON,' NICK KARABATSOS,' DAVID R. VAUGHAN,I PEARL E. TURNER,' DIANA I. ORTIZ3 AND WILLIAM WILLS4

ABSTRACT. Arboviruses isolated and identified from mosquitoes in South Carolina (USA) are described, including new state records for eastern equine encephalitis (EEE), St. Louis encephalitis virus (SLE), Flanders virus, Tensaw virus (TEN), and a variant of Jamestown Canyon virus (JC). Mosquitoes were collected at 52 locations in 30 of 46 South Carolina counties beginning in June 1996, and ending in October 1998, and tested for arboviruses. Of 1,329 mosquito pools tested by virus isolation (85,806 mosquitoes representing 34 mosquito species or complexes), 15 pools were positive. Virus isolations included EEE from I pool each of Anopheles crucians complex and Culex erraticus; a variant of JC from I pool of An. crucians complex; a California serogroup virus from 1 pool of Aedes atlanticusltormentoriTEN from 5 pools of An. crucians complex and I pool each of Culex salinarius and Psorophora ciliata; Flanders virus from 1 pool of melanura; and Potosi virus from I pool each of Aedes vexans, Coquillettidia perturbans, and Psorophora columbiae. Of 300 mosquito pools tested by antigen-capture assay for EEE and SLE (14,303 mosquitoes representing 16 mosquito species or complexes), 2l were positive for EEE and I was positive for SLE. Positive EEE mosquito pools by antigen-capture assay included An. crucians complex (14 pools), Anopheles punctipennis (l pool), Anopheles quadrimaculatus (1 pool), Cq. perturbans (4 pools), and Cs. melanura (l pool). One pool of Cx. salinarius was positive for SLE by antigen-capture assay. Arbovirus-positive mosquito pools were identified from 12 South Carolina counties, all located in the Atlantic Coastal Plain, and from 4 of 8 Carolina bays surveyed.

KEY WORDS Mosquitoes, arboviruses, Anopheles crucians, eastern equine encephalitis virus, South Carolina

INTRODUCTION found EEE-neutralizing in 4 sera and St. Louis encephalitis virus (SlE)-neutralizing anti- Infections caused by eastern equine encephalitis bodies in 2 sera. In another study, Tidwell et al. virus (EEE) occur annually in South Carolina (1984) collected mosquitoes at 2 South Carolina (USA), most notably in animals of commercial im- coastal sites and reported no arbovirus isolations portance, such as horses, pheasants, donkeys, emus, from 41 mosquito pools comprising 1,913 mosqui- and quail. Historically, most of these animal cases toes and representing ll species. Before 1996, have occurred in the Atlantic Coastal Plain area of when we began our study, no statewide mosquito South Carolina (Tidwell et al. 1984, Wright 1993). collections had been performed to determine arbo- Cases of human disease caused by EEE also occur virus activity in South Carolina. The purposes of in South Carolina, but are infrequent. Two human our study were to identify the mosquito vectors of cases were confirmed, including 1 fatal case in arboviral diseases of public health importance and 1997. I nonfatal case was confirmed in 1996. and to identify naturally occurring endemic foci of ar- no cases were confirmed for the years 1991-95 and bovirus activity in South Carolina. 1998 (South Carolina Department of Health and Environmental Control, reportable disease case re- ports). All 3 of the 1996 and 1997 human cases MATERIALS AND METHODS were residents of counties located in the Atlantic Coastal Plain, specifically I case in Horry County Field sites: Fifty-two field sites were located in in 1996, and 2 cases in Charleston County in 1997. 3O of 46 South Carolina counties, including all 6 Although EEE is enzootic in South Carolina, nat- Atlantic Coast counties (Beaufort, Charleston, Col- ural history studies of arboviral diseases and mos- leton, Georgetown, Horry, and Jasper), 18 inland quito vectors in South Carolina have been limited Atlantic Coastal Plain counties (Aiken, Allendale, to a few field sites and relatively small sample siz- Bamberg, Barnwell, Berkeley, Calhoun, Chester- es. For example, Durden et al. (199'7) collected 121 field, Clarendon, Darlington, Dillon, Dorchester, avian sera at I South Carolina coastal site and Hampton, Kershaw, Lee, Marion, Orangeburg, Richland, and Sumter), 3 Piedmont counties (Fair- field, Lancaster, and McCormick), and 3 counties I Bureau of Laboratories, South Carolina Department of that extend from the Piedmont into the Blue Ridge Health and Environmental Control, 8231 Parklane Road, geologic province (Greenville, Oconee, and Pick- Columbia, SC 29223. ens) (Shimer 1972). 2 Arbovirus Diseases Branch, Division of Vector-Borne Ten of the field sites were located on private Infectious Diseases, National Center for Infectious Dis- land, including 7 farms, 2 barrier island commu- eases, Centers for Disease Control and Prevention, Fort Collins, CO 80522. nities, and I coastal residential suMivision. Mos- 3 Department of Biology, Jackson State University, quito collections were made at these 10 sites at the Jackson, MS 39217. invitation of the property owners or their commu- a 15 Chandler Court, Columbia, SC 2921O. nity representatives, and with the cooperation of lo- 74 JounN,ql- oF tgg AvrntcAN MoseulTo CoN'TRoL AssocrnrloN cal mosquito control personnel, all of whom wanted et al. 1987, Day and Stark 1996). The California to learn more about the mosquitoes in their area. group viral isolate was initially identified as being Forty-two field sites were state-owned or state- a member of the California (CAL) serogroup by managed lands, for example, parks, wildlife man- positive neutralization using mouse ascitic fluid agement areas, public utility company lands, Heri- containing to CE. Flanders virus was iden- tage Preserves, and university field research tified by an indirect immunofluorescent antibody centers. These 42 sites were selected for their fa- (IFA) test using MHIAF to protot)4)e Flanders vi- vorable mosquito breeding habitats and histories of rus. The Jamestown Canyon virus (JC) variant, Po- low pesticide pressure. Eight Carolina bays in 7 tosi virus, and Tensaw virus (TEN) isolates were counties were included as field sites: Cathedral Bay placed into antigenic groups initially by IFA using (Bamberg), Santee Coastal Reserve Carolina bay National Institutes of Health immune grouping flu- complex (Charleston), Dingle Pond (Clarendon), ids. Potosi virus and TEN isolates were then iden- Little Pee Dee Bay (Dillon), Cartwheel Bay (Hor- tified by 1-way neutralization tests with pretitered ry), Lewis Ocean Bay complex (Hony), Savage MHIAF for the 6 prototype domestic virus mem- Bay (Kershaw), and Woods Bay (Sumter). Six bers of the Bunyamwera serogroup. These included coastal marsh areas were also included as field Cache Valley, Tensaw, Potosi, Main Drain, Lokern, sites. and Northway . After placement of the JC Mosquito c o llec tions : Mosquitoes were collected isolate in the CAL serogroup, the isolate was iden- using COr-baited, Centers for Disease Control min- tifled in l-way neutralization assays using pretiter- iature light traps. Collections began in June 1996, ed single-injection antibodies for prototype Califor- and continued through October 1998. All of the nia encephalitis, Keystone, Jamestown Canyon, collections were made during the months of March LaCrosse, San Angelo, and snowshoe hare viruses. through October. Five to 13 mosquito traps were A MHIAF for trivittatus virus was also employed used at each field site for l-4 nights for a total of in l-way neutralization tests. Single-injection im- 1,431 trap nights. Although mosquitoes were col- mune serum to the Jamestown Canyon-like viral lected at some of the field sites once or twice an- isolate was prepared in adult mice and the virus and nually, no established time intervals were used to its antibody were compared in cross-neutralization determine when a particular field site was revisited. tests with prototype JC and its homologous single- The trap nets were collected at dawn, and placed injection antibody. Both the variant and prototype in chests containing dry ice to kill the mosquitoes. JCs also were tested against a monoclonal antibody The dead mosquitoes were placed in labeled, 150 to JC that had neutralizing activity (Artsob et al. X l5-mm polystyrene petri dishes, taped shut, and L99D. stored and transported to our laboratory at -70'C using dry ice. RESULTS Inboratory analyses: Female mosquitoes from each field site were identified and pooled by species A total of 1,329 mosquito pools were tested by in 100 X 15-mm polystyrene petri dishes. During virus isolation. These pools represented 34 mos- the identification and pooling process, petri dishes quito species or complexes and 85,806 mosquitoes. were placed in small styrofoam boxes containing Viral antigen-capture assays for EEE and SLE were dry ice to permit sorting at -7OoC while using a performed on 3OO mosquito pools representing 16 dissecting microscope to identify the mosquitoes to mosquito species or complexes and 14,303 mos- species. Virus isolations, and, for EEE and SLE, quitoes (Thble 1). Thirty-seven (37) mosquito pools antigen-capture assays were performed with tlle from 14 field sites in 12 counties, representing 11 species-specific mosquito pools following previous- mosquito species or complexes, tested positive for ly published methods (Tsai et al. 1987, Day and arboviruses by isolation or antigen-capture assay Stark 1996). Mosquito pools for virus isolation con- (Table 2). All 14 field sites were located in the At- sisted of 1-135 mosquitoes. Mosquito pools for the lantic Coastal Plain. Four of these 14 field sites viral antigen-capture assays consisted of 20-68 were Carolina bays, including Cathedral Bay in mosquitoes; one half of the homogenized volume Bamberg County, Dingle Pond in Clarendon Coun- of each pool was used for the EEE antigen-capture ty, Little Pee Dee Bay in Dillon County, and Woods assay and other half was used for the SLE antigen- Bay in Sumter County. All 37 positive mosquito capture assay. Vero cell cultures demonstrating cy- pools were collected during the months of March topathic effect were confirmed by virus neutrali- through September, Eastern equine encephalitis vi- zation testing, initially using a panel of mouse nts (Togaviridae: ) was most frequently hyperimmune ascitic fluids (MHIAFs) containing identified, with 2 positive virus isolations and 2l antibodies against EEE, SLE, California encepha- positive antigen-capture assays from 12 field sites. litis virus (CE), western equine encephalitis virus, Anopheles crucians complex was most frequent- and Highlands J virus. Mosquito pools testing pos- ly found positive for EEE, with I positive isolation itive by viral antigen-capture assay were confirmed and 14 positive antigen-capture assays from 8 field by antigen-capture inhibition testing using MHIAFs sites, including 3 Carolina bays. Two Carolina containing antibodies against EEE and SLE (Tsai bays, Dingle Pond and Woods Bay, yielded EEE- Mencu 2001 AnnovrnusssrN Sourn C.cnor-rNa

Table 1. Mosquito species from South Carolina tested for arboviruses, 1996-98

No. arbovirus-positive pools/no. pools tested (total number of mosquitoes tested) Viral antigen-capture Mosquito speciesr Virus culture assay Ae. albopictus ol2 (40) NT2 Ae. atlanticus/tormentor 1/27 (r,460) ol4 (2O2) Ae. atropalpus otl (5) NT Ae, canadensis 0/88 (s;144) ot20 (e82) Ae. fulvus pallens ot6 (71) NT Ae. hendersoni ot3 (30o) NT Ae. infirmatus ot30 (2,42r) ot3 (r47) Ae. mitchellae o/2 (44) NT Ae. sollicitans ot32 (r,803) olto (43e) Ae. sticticus o/2 (67) NT Ae. taeniorhynchus o/99 (7,887) ol25 (r,299) Ae. thibaulti o/2 (3) NT Ae. triseriseriatus o/2 (40) NT Ae. vexans 1t50 (3,377) ot14 (67r) An. barberi otl (2) NT An. crucians complex 7t364 (2s,388) t4t98 (4,636) An. punctipennis ol16 (648) t/3 (ls0) An. quadrimaculatus 0/38 (r,799) u6 (284) An. walkeri 0/l (l) NT Cq. perturbans t/171 (6.618) 4t26 (1,222) Cx. erraticus r/76 (3,7s7) ol12 (s84) Cx. nigripalpus 0/15 (72r) NT Cx. p ip i ens/q u i nq uefa s c iat us o/4 (222) 0/r (s0) Cx. restuans on2 (332) NT Cx. salinarius l/230 (18,071) U59 (2,789) Cx. territans ot2 (47) NT Cs. melanura U53 (3,354) r/15 (678) Or. signifera ol1 (l) NT Ps. ciliata t/16 (l l7) NT Ps. colurnbiae 1/18 (814) oll (50) Ps- cyanescens ol1 (2) NT Ps. ferox 0/10 (2r7) NT Ps. howardii o/3 (23) NT Ur. sapphirirut oltl (412) ol3 (r20) Totals 15/1,329 (8s,806) 22/300 (14,303) I Ae., Aedes; An., Arcpheles; Cq., Coquillettidiat Cx., Culex; Cs., Culiseta; Or-, Orthopodomyia; Ps., Psorophora; IJr., IJranotaenia- 'NT: not tested.

infected An. crucians complex mosquitoes in both positive mosquito species. Four additional mosqui- the springtime (March-April) and late summer to species were identified as positive for EEE, (August-September). One other pool of An. cru- including Anophe le s punctipennis (Say), Anophele s cians complex was isolation positive for a neutral- quadrimaculatzs Say, Culex enaticzs (Dyar and ization variant of IC (Bunyaviridae: Bunyavirus). Knab), and Culiseta melanura (Coqui[ett). In addition to the JC isolate, discussed above. a 2nd Potosi virus (Bunyaviridae: Bunyavirus) was iso- CAL serogroup virus was isolated from a pool of lated from a pool of Cq. perturbans and from a Aedes atlanticusltormentor. However, this isolate pool of Psorophora colurnbiae (Dyar and Knab). A became nonviable during passage and could not be Potosi-like virus was also isolated from a pool of further identified. Aedes vexans (Meigen). All 3 pools were collected Coquillettidia perturbans (Walker) was collected alongside the Waccamaw River in Horry County. at29 field sites and 111 pools (6,618 mosquitoes) Three other viruses were also isolated. Flanders were tested by virus isolation and 26 pools (1,022 virus, a mosquito-borne member of the Rhabdovir- mosquitoes) were tested by antigen-capture assay. idae, was isolated from a pool of Cs. melanura col- At I field site in Lee County, 4 of 4 pools (200 lected at Little Pee Dee Bay in Dillon County. Ten- mosquitoes) of Cq. perturbans were positive for saw virus (Bunyaviridae: Bunyavirus) was isolated EEE by antigen-capture assay. Coquillettidia per- from 5 pools of An. crucians complex and from I turbans was the 2nd most frequently found EEE- pool each of Culex salinarius Coquillett and, Pso- JounNel or rse AuenrceN Moseuno CoNrnor-Assocrerrou Vor-. 17, No. I

Table 2. Arbovirus-positive mosquito pools from South Carolina, 1996-98

Collection date County; habitat; no. Lab (month- positive pools/total pools Mosquitospeciesl Arbovirus2 method year) (no. mosquitoes)

Ae. at lantic us/to rmentor CAL Culture 8-96 Sumter: Carolina bay; l/5 (317) Ae. vexans Potosi-like Culture 9-98 Horry; riverside; l/9 (50O) An. crucians EEE Ag' 6-97 Allendale; mixed pine-hardwood wet- land; l/4 (180) An. crucians EEE Ag 4-97 Bamberg; Carolina bay; l/4 (195) An. crucians JC variant Culture 6-96 Barnwell; man-made ponds, mixed pine-hardwood; | /4 (2OO) An. crucians EEE Ag s-98 Beaufort; coastal subdivision; 1/4 (194) An. crucians EEE Ag 9-96 Clarendon; Carolina bay; l/l (4O) An. crucians EEE Ag 4-97 Clarendon; Carolina bay; ll5 (25O) An. crucians EEE Ag 4-98 Clarendon; Carolina bay:'213 (132) An. crucians EEE Ag 3-98 Colleton; swamp; 213 (135) An. crucians EEE Culture 7-6 Dillon; Carolinabay; lll (66) An. crucians EEE Ag 4-98 Horry; riverside: lll (45) An, crucians TEN Culture 9-98 Horry; riverside; 5/42 (21OO) An. crucians EEE Ag 8-96 Sumter; Carolina bay: I/14 (696) An. crucians EEE Ag 3-97 Sumter; Carolina bay;315 (25O) An. punctipennis EEE Ag 4-97 Hampton; mixed pine-hardwood wet- land; l/3 (150) An. quadrimaculatus EEE Ag 6-96 Charleston; coastal marsh; 1/3 (149) Cq. perturbans Potosi Culture 9-98 Horry; riverside; l/1 (50) Cq. perturbans EEE Ag 5-97 Lee; riverside/swamp; 4/4 (2OO) Cx. erraticus EEE Culture 7-96 Dillon; Carolina bay; l/3 (195) Cx. salinarius SLE Ag 4-98 Clarendon; Carolina bay; 1/l (45) Cx. salinarius TEN Culture 9-98 Horry; riverside; l/10 (500) Cs. melanura EEE Ag 4-98 Charleston; coastal rice field, marsh; l/2 (L49\ Cx. melanura Flanders Culture 7-96 Dillon; Carolina bay; l/l (58) Ps. columbiae Potosi Culture 9-98 Horry; riverside; l/2 (78) Ps. ciliata TEN Culture 9-98 Horry; riverside; l/2 (2O)

I Ae., Aedes: An., Anophelesl Cq-, Coquillettidia: Cx-, Culex; Cs., Culiseta; Ps., Psorophora. , CAL, Califomia serogroup virus; EEE, eastem equine encephalitis virus; JC variant, Jamestown Canyon virus variant; TEN, Tensaw virus; SLE, St. Louis encephalitis virus. 3 Ag, antigen-capture assay,

rophora ciliata (Fabicius). All 7 TEN-positive be an epidemic vector species in Florida. Our re- pools were collected alongside the Waccamaw Riv- sults include isolating EEE from mosquitoes for the er in Horry County. One pool of Cx. salinarias was lst time in South Carolina. Our identiflcation of positive for SLE (: Flavivirus) by an- EEE-infected An. crucians complex mosquitoes tigen-capture assay. The SlE-positive pool was from 8 South Carolina counties also suggests that collected at a Carolina bay, Dingle Pond in Clar- An. crucians complex may be a significant vector endon County. for EEE in South Carolina. Three anopheline spe- cies comprise the An. crucians complex: An. cru- cians, Anopheles georgianzs King, and Anopheles DISCUSSION bradleyi King. The adult stages of each are indis- In 1954, Chamberlain et al. reported laboratory tinguishable, and their natural ranges overlap in studies indicating that Anopheles crucians Wiede- South Carolina (Darsie and Ward l98l). Anopheles mann was not an efficient vector for EEE. Shortly crucians complex has been reported from every afterwards, Kissling et al. (1955) isolated EEE from South Carolina county except York (Weathersbee An. crucians collected in Georgia. Because vector and Arnold 1947). The preferred breeding sites of competence for virus transmission by mosquitoes mosquitoes of this complex are in acidic waters, and viral infection in mosquitoes are not synony- such as those found in the Carolina bays. Elliptical mous, An. crucians received little further consid- in shape and aligned along a northwest-southeast eration as a vector for EEE until recently when Day axis, Carolina bays are shallow and poorly drained and Stark (1996) studied arboviral infections in inland basins found in the Atlantic Coastal Plain, emus at a Florida ranch and isolated EEE from An. particularly in Virginia and the Carolinas, as single crucians, Cx. erraticus, and Culex nigripalpus bays or in clusters (Shimer 1972). We found EEE- Theobald. They suggested that each species might infected An. crucians complex mosquitoes at 4 of Mnncs 20Ol ARBovrRUsEsrN SourH Cenolrwl the 8 Carolina bays included in our study, suggest- pool was collected at Little Pee Dee Bay in Dillon ing that Carolina bays may be enzootic foci for County. EEE in South Carolina. Additionally, the flight Tensaw virus was lst described by Sudia et al. range for An. crucians may exceed 1 mi (1.6 km), in 1968. Chamberlain et al. (1969) collected mos- these mosquitoes will feed on , and these mos- quitoes in Georgia, Alabama, and Florida, and re- qutioes will enter shelters and feed on humans ported isolating TEN primarily from pools of An. (Horsfall 1955). Sabrosky (1946) has also reported crucians and Ps. columbiae, and. to a much lesser that 47.3Vo of the An. crucians complex mosquitoes extent, from pools of An. quadrimaculatus, Aedes in I South Carolina study had fed on equine blood. atlanticus Dyar and Knab, Aedes mitchellne (Dyar), These characteristics and descriptions provide ad- Cx. nigripalprs, and Cq. perturbans. Since then, ditional support for considering the members of the TEN has also been isolated from mosquitoes col- An. crucians complex as possible vectors for EEE, lected in Mississippi, Louisiana, and east Texas, and perhaps other arboviruses, such as JC and TEN, and Aedes taeniorhynchus (Wiedemann), Cx. sali- in South Carolina and in the southeastern United narius, and Cq. perturbans have also yielded TEN States. Our findings strongly suggest that An. cru- isolates (Calisher et al. 1986). We demonstrated the cians complex mosquitoes in the southeastern Unit- presence of TEN in mosquitoes for the lst time in ed States should be reexamined for their competen- South Carolina by isolating the virus from 5 pools cy to serve as arbovirus vectors. of An. crucians complex and from 1 pool each of Indeed, our findings include isolating a variant Cx. salirwrius and Ps. ciliata. All 7 pools were col- JC from an An. crucians complex pool for the lst lected at 1 site, alongside the Waccamaw River in time in South Carolina. The JC isolate was identi- Horry County. Antibodies to TEN have been de- fled as a neutralization variant. It was neutralized tected in , gray foxes, raccoons, dogs, cattle, significantly less effectively by homologous and and humans, but not in wild birds or sentinel chick- heterologous prototype JC antibodies, although it ens (Calisher et al. 1986). Calisher and Sever was neutralized by a JC monoclonal antibody at (1995) discussed the possible role of TEN as an least as well as the prototype virus was. etiologic agent of human congenital defects of the TWo other EEE-infected anopheline species were central nervous system, and additional virology and identified in our study, An. punctipennis and An. epidemiology studies seem to be indicated. quadrimaculatzs. Considering that both species Mitchell et al. (1996) reported isolating Potosi will feed on humans, and the latter's past role as a virus from a pool of Ps. columbiae collected in malaria vector in the southeastern United States Charleston, SC, in 1991. We also isolated Potosi (Horsfall 1955), neither species can be ignored as virus from a pool of Ps. columbiae, as well as from arbovirus vectors for human disease. 1 pool of Cq. perturbans. A Potosi-like virus was The possible role of Cx. erraticus as a vector for also isolated from a pool of Ae. vexans. All 3 Potosi EEE in Florida has been mentioned above (Day and virus-positive pools were collected from the same Stark 1996). We also isolated EEE from Cx. erra- site where the TEN-positive pools were collected, ticus collected at Little Pee Dee Bay, a Carolina alongside the Waccamaw River in Horry County. bay, in Dillon County. Additionally, we identified Potosi virus and TEN are distinct species but are 4 EEE antigen-positive pools of Cq. perturbans that serologically related (Mclean et al. 1996). The role were collected alongside Lynches River in Lee of Potosi virus as a human pathogen is not yet clear. County. The roles of Cq. perturbans and Cx. er- Using an antigen-capture assay, we identified raticus as enzootic or epidemic vectors of EEE in SLE virus for the lst time in South Carolina in a South Carolina are unclear and require further pool of Cx. salinarius collected at Dingle Pond, a study. Carolina bay, in Clarendon County. The SLE-pos- Culiseta melanura is frequently cited as a prin- itive antigen-capture assay was confirmed by an ciple enzootic vector of EEE in the eastern United SLE antigen-capture inhibition assay. However, States, especially north of the Carolinas (Scott and since then, West Nile (WN) virus has been found Weaver 1989). We collected Cs. melanura at 16 in the New York City metropolitan area (CDC field sites and tested 53 pools (3,354 mosquitoes) 1999). West Nile viral antigens and antibodies may by virus isolation and 15 pools (678 mosquitoes.l cross-react with some SLE enzyme immunoassays, by antigen-capture assay, but identified only I EEE- yielding false-positive SLE test results. We were positive Cs. melanura pool by antigen-capture as- unable to perform retrospective testing for WN on say. The role of Cs. melanura as a vector of EEE the SlE-positive Cx. salinaius pool, because this in South Carolina, and perhaps southwards as well, pool had been consumed in the SLE testing. seems to be diminished when compared to that of tlae An. crucians complex. The EEE-positive Cs. ACKNOWLEDGMENTS melanura pool was collected in an abandoned rice field on the coast in Charleston County. Flanders We thank our summer interns, Ronald Moak, virus, a nonhuman pathogen and member of the Aaron Wozniak, Suneeta Sahoni, and Timothy Fill- , was also isolated from a pool of Cs. more, for their untiring field work. We also thank melanura for the lst time in South Carolina. This Stan Hutto and the staff of the South Carolina De- JounNnI- oF THE AMERTCIN Mosqurro Corvrrol AssocrlrroN Vor-. 17, No. I partment of Parks, Recreation, and Tourism; Buddy sia County, Florida: 1992 through 1994. 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