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Absence of La Crosse Virus in the Presence of Aedes Triseriatus on The

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Absence of La Crosse Virus in the Presence of Aedes Triseriatus on The Mancu, 1986 J. Ar"r. Mosq. CoNrrol Assoc. JJ ABSENCEOF LA CROSSEVIRUS IN THE PRESENCEOF AEDESTRISERIATUS ON THE DELMARVA PENINSULA1 GARY G. CLARK, C. L. CRABBS euo BROOKE T. ELIAS Department of Arboviral Entomology, US Army Medical Research Institute of Infectious Diseases,Fort Detrick, Frederick, MD 2 I 701- 50 I I ABSTRACT. Between 1980 and 1984, field studies were conducted in 2 areas on the Delmarva Peninsula ro identify the presence of La Crosse (LAC) virus. Ovitraps were used to collect eggs of Aedes triseriatus complex mosquitoes. No virus was obtained from 969 pools containing,22,37O adult mosquitoes reared from eggs. Only I of 143 raccoon serum samples had neutralizing antibody to LAC virus; 36 sentinel domestic goats, and 99 other wild mammal serum samples were negative. The apparent absence of LAC virus may be due to the uncommon occurrence of the eastern chipmunk, a species which has been shown to be an amplifying host for this virus, on the Peninsul;r. INTRODUCTION North America for the period from 1943 to 1970, Sudia et al. (1971) reported 3 LAC virus La Crosse (LAC) virus, a member of the isolations from Georgia where recent human California virus serogroup, is a human patho- infections have been documented by Sikes et al. gen (Kappus et al. 1983) transmitted primarily (1984). In south Florida, Prather (1969) re- by the mosquito Aedes triserintus (Say) (Watts et ported thatSSVoof343 native Indians surveyed al. 1972). Grimstad and coworkers (1977) de- "the had neutralizing (N) anribody to LAC virus. lineated endemic region" for LAC virus in although LAC virus has never been isolated in the United States. This crescent-shaped area this area. Kappus et al. (1983) also report a extended eastward from southeastern Min- single California encephalitis casefrom Florida. nesota around the southern edge of the Great In contrast to LAC virus activity documented Lakes up to western New York state. However, to the north and the south, isolation of LAC with increasing interest in this arbovirus, virus virus has not been reported from the mid- isolations and human caseshave been identified Atlantic states of Delaware, Maryland and Vir- from areas well beyond this region. ginia (Sudia et al. 1971, Parkin et al. 1972),and In northeastern U.S., following confirmation no human caseshave been recorded from this of human encephalitis cases and recovery of area (Calisher 1983). Serological evidence of virus, a LAC virus endemic area was identified California group virus infection was reported in extreme east-central New York (Gravson et from Virginia (Parkin et al. 1972), but the spe- al. 1983). Evidehce of three human LAC virus cific viral etiology was not determined. infections has been reported from nearby In the area in which we were working, northern NewJersey (Kappus et al. 1983), and the Delmarva Peninsula, no LAC virus was isolated in southeastern Pennsylvania a serosurvey of from relatively small numbers,637 (Saugstad approximately 1,000 people suggested the ex- et al. 1972) and 347 (LeDuc et al. 1975), of Ae. istence of a LAC-endemic area there (Anony- triseriatus tested from 1969 to 1972, Serologic mous 1970). In southeastern U.S., evidence of tests for N antibody to LAC virus LAC virus activity has been reported from from sera of 25 research personnel involved in field studies North Carolina (Kappus et al. 1983), based on on the Peninsula were negative (Watts both occurrence of human cases and isolation et al. 1982). As a result of the expansion in the rec- of virus, and from South Carolina, where a ognized geographic distribution [,AC human case occurred in 1982 (Centers for Dis- of virus in the U.S., intensive efforts were easeControl, 1983).In summarizing California made to de- termine if this virus, which can be transovarially group virus isolations from mosquitoes in maintained, occurred on the Delmarva Penin, sula. I The views of the authors do not purport to rellect the positions of the Department of the Army or the METHODS AND MATERIALS Department of Defense. Mosqun'ors. In 1980, ovirraps with black In conducting the research described in this report, "Guide flannel liners (Loor and DeFoliart 1969) were the investigator adhered to the for the Care placed ground and Use of Laboratory Animals," as promulgated by at level on trees in the Pocomoke the Committee on Care and Use of Laboratory Ani- Cypress Swamp (PCS), Worcester Counry, rnals of the Institute of [,aboratory Animal Resources. Maryland, and approximately 36 km east- National Research Council. The faciliries are fully southeast of the PCS in the Chincoteague Na- accredited by the American Associarionfor Accredi- tional Wildlife Refuge (CNWR) ar the sourhern tation of Laboratory Animal Care. end of Assateague Island, Accomack County, 34 J. Ar"r. Mosg. ConrRoL Assoc. Vol.2, No. I Virginia. The PCS and its vegetation have been imately 2 km and 15 km, respectively. Ar- described by Saugstad et al. (1972), while thropods were collected 2-3 times per week Buescher et al. (1970) have characterized and goats were bled via venipuncture of the CNWR. In 1983, ovitraps, with the balsa sub- jugular vein once per week. Blood was pro- strate modification of Novak and Peloquin cessed as described above. At the PCS, 5 goats (1981), were again used in these two study were in a corral and had a Magoon trap with the areas. As part of a study of the oviposition pat- door removed for shelter. Arthropods and terns of Ae, triseria,tru and Aed,eshendersani Cock- goats were sampled as at CNWR. In 1984, erell (Clark and Craig 1985), individual ovi- goats were again utilized at PCS and the goat- traps were placed at 0, 3 and 6 m on 20 trees at baited traps were moved from CNWR and both the PCS and CNWR. A total of 120 traps situated near brackish water marshes of the were utilized. Ovitrap inserts were removed Chesapeake Bay in Somerset County, Mary- and replaced after 2 weeks in the field. land, and Accomack County, Virginia, on the In the laboratory, eggs were hatched and western border of the Delmarva Peninsula. The reared to the adult stage. Adult mosquitoes final serum sample, obtained from each goat in were stored at -70"C and identified on a chill late October, was assayed for N antibody to table at 4oC as being members of the Ae. LAC virus. triseriatus complex. Mosquito pools containing a Mammalian sera were tested for N antibody maximum of 25 individuals were sorted by sex to LAC, Jamestown Canyon (fC), and Keystone and site and date of collection. Mosquito pools (KEY) viruses in Vero cells. The LAC stock were triturated in prechilled tissue grinders virus used in the plaque reduction neutraliza- containing medium 199, supplemented with tion (PRNT) was originally received from Dr. glutamine, 20% fetal bovine serum (FBS), and Wayne H. Thompson at the University of Wis- antibioitics (200 units/ml penicillin, 200 p.glrr:'l consin and has undergone 8 passagesin suck- streptomycin, and l0 pglml fungizone). One ml ling mouse brain and I Vero cell passage.The was used for pools with I to 12 mosquitoes and JC stock virus was isolated from a pool of Aedes 2 ml for pools with 13 to 25 mosquitoes. Sus- canadensis (Theobald) collected at the PCS in pensions were clarified by centrifugation at 800 1979 and had undergone 2 passagesin tissue x g for 20 min at 4"C and assayedimmediately culture. The KEY stock virus was isolated from for virus or stored at -70"C until assayed.Sam- Aedes atlanticus Dyar and Knab collected at the ples were tested for virus by plaque assay in PCS in 1972 and had been passed 3 times in Vero cells. Aliquots of 0.1 ml of each supernat- tissue culture. Sera were heat treated at 56oC ant were inoculated in triplicate onto Vero 12- for 30 min and a l:10 dilution was prepared in well trays, adsorbed for I hr at 35'C, and then the medium described above. Mixtures of an overlaid with 2 ml of an agar overlay medium equal volume of each diluted serum and a virus consisting of equal volumes of 1.5% agarose suspension containing 50-100 PFU were incu- and 2X Eagle's basal medium in Earle's salts bated for I hr at room temperature and inocu- with Hepes buffer (4 g/liter), l0% hear- lated onto Vero cells (0. I ml per each of 3 inactivated FBS and antibiotics. Cell cultures cultures). Subsequent assay procedures were were incubated in a humidified atmosphere according to those described above. A reduc- with 5% COz for plaque development. After 4 tion of 80Voor more of the virus dose by a l: l0 days, the overlay medium (l ml/well), contain- serum dilution was our criterion for the pres- ing a 77o solution (stock l:300) of neutral red, ence of N antibody. was added to the cells. Cultures were incubated for 24 hr and examined for plaques. RESULTS Melrvet.s. During 1983 and 1984, wild mammals were trapped alive at CNWR, anes- A total of 22,370 Ae. triserh,tus complex mos- thetized, ear-tagged and bled via cardiac quitoes in 969 pools from PCS and CNWR was puncture. With recaptured mammals, the last assayed as adults for virus (Table l). No virus sample collected was assayed. Fluid was ob- was obtained from these mosquitoes. Most of tained from the thoracic cavity of white-tailed the mosquitoes (73Voof 16,349) were collected deer brought to state deer hunter checking sta- and assayed in 1983, with the remaining 6,021 tions. Blood specimens were allowed to clot, collected from the same localities in 1980.
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