JUNE 1993 Cer-rronrrl SpnocnoupVrnusns rt Mosqurrons ISOLATION OF JAMESTOWN CANYON AND SNOWSHOEHARE VIRUSES (CALIFORNIA SEROGROUP)FROM AEDES MOSQUITOES IN WESTERN MASSACHUSETTS EDWARD D. WALKER.I'' MARGARET A. GRAYSON3 eno JOHN D. EDMANT ABSTRACT. Three isolates of Jamestown Canyon virus and one isolate of snowshoehare virus (California serogroup)were obtained from adult Aedes females collected in western Massachusettsin 1982. Jamestown Canyon virus was isolated from Aedes abserratusfpunctor once, and fuom Aedes intrudens twice. Snowshoehare virus was isolated from Aedes stimulanwgtoup mosquitoes.La Crosse encephalitis (LAC) virus was not isolated from 1,552 adult Aedes triseriatus, nor from 22,557Aedes triseriatus larvae. However, sera from 1/178 eastern chipmunks, 5/31 gray squirrels, and 8/144 white- tailed deer had neutralizing antibody to LAC virus. No sentinel rabbits placed at sites yielding virus isolatesseroconverted to CAL viruses in either vear. INTRODUCTION Hampden and Berkshire counties in western Massachusetts during 1982. Mosquitoes were California (CAL) viruses have serogroup been killed by fteezing, sorted by speciesinto pools intensively increased recog- studied becauseof of 100 or fewer individuals on a chill table and nition of their role as etiologic agents of human stored at -85"C. Aedes triseriatus (Say) larvae disease (Grimstad 1988). Although La Crosse were collected from tree holes and tires at the encephalitis (LAC) virus has been the major sites, rearedto the fourth instar, pooled into lots CAL virus of concernin the easternU.S.. James- of 100and also storedat -85'C. town Canyon (JC) virus has also beenassociated Pools were triturated in 7.5% bovine plasma with human illness in Michigan (Grimstad et al. albumin (BSA) in sterile phosphate-bufferedsa- 1982),New York and Ontario (Deibel et al. 1983, line (PBS, pH 7.6) supplemented with antibi- Srihongse et al. 1984). There has been no sys- otics, then centrifuged. Suckling mice (2-4 days tematic survey for CAL viruses in western Mas- old) were inoculated intracerebrally (i.c.) with sachusetts, although snowshoe hare (SSH), 0.025 ml of supernatant and observed daily Keystone and severaluntyped CAL viruses were thereafter for signs of illness. Brains of sick mice isolated from mosquitoesduring surveillancefor were harvested, in BSA eastern equine encephalitis virus in southeast triturated, 0.75% to (w/v) passed Massachusetts(H. K. Maxfield, Massachusetts make l07o seeds,centrifuged and (20% Department of Public Health, Encephalitis i.c. to suckling mice. Crude antigens w/v) prepared Field Station, Middleboro; personal communi- were by trituration ofharvested mouse cation). brains in borate-buffered saline and incubation The purpose ofthis study was to survey west- at 4'C overnight, then centrifuged at 10,000for ern Mqssachusettsfor CAL viruses. Given the t h at 4'C. After initial isolation, viruses were proximity of this region to the LAC and JC virus re-isolated from the original pools for purposes endemicareas in easternNew York State (Gray- of confirmation. son et al. 1983, Boromisa and Grayson 1990), Virus isolates were identified first by screen- we made a special effort to search for these ing with an indirect immunofluorescent anti- viruses in mosquito, sciurid rodent, and white- body (IFA) procedure described by Boromisa tailed deer populations. and Grayson (1990). Identifications were con- firmed by serum dilution plaque reduction neu- tralization (SDPRN) tests done on Vero cell MATERIALS AND METHODS monolayers, described by Lindsey et al. (1976) (1990). Mosquito collection, uirus isolation and uirus as modified by Boromisa and Grayson identificatiou Adult female mosquitoeswere col- Virus stocks were inoculated onto Vero cell cul- lected at human bait or with dry-ice baited CDC ture monolayers.Virus strains used were a New (74-32813) light traps at 17 sites in Franklin, Hampshire, York strain of LAC virus or proto- type strains of LAC, JC, SSH, trivittatus (TVT) and Keystone (KEY) viruses (supplied by the I Centersfor DiseaseControl, Fort Collins, CO). Department of Entomology, University of Massa- In the IFA tests, virus identification was deter- chusetts,Amherst, MA 01003. mined by a 2-fold or greater in 2 Current address: Department of Entomology, difference the Michigan State University, East Lansing, MI 48824. intensity of immunofluorescence (1-4+) from 3Wadsworth Center for Laboratories and Research, that of heterologousantibodies. Reference im- New York State Department of Health, Albany, NY mune sera used in IFA and SDPRN tests were 7220r. California serogroup virus/type-specific im- r32 Jounter, oF THE ArrapmclN MosQuIro CoNtnol Assoctnuon VoL.9,No. 2 mune mouseascitic fluids (IMAFs), and a mono- Table 1. Mosquito collections from Franklin, clonal antibody specific for SSH virus (no, Hampshire, Hampden and Berkshire counties, 2BgA-2), suppliedby the CDC. western Massachusetts.1981-82 Serosurveyof wild mamrnalsand sentinel rab- No, mos- bits.' Eastern chipmunks (Tamias striatus) and Species quitoes No. pools (Sciurr^ls gray squirrels carolincnsis) were live- Ae. abserratttsfpunctor 3,844 68 trapped, anesthetizedand bled from the orbita, Ae. aurifer 95 IJ capillary nexus or heart at 9 sites in Franklin, Ae. canadensis 1.222 54 Hampshire, and Berkshire counties in 1980*81. Ae. cinereus 151 19 Animals were releasedafter bleeding. Blood of Ae. communis 6,001 87 white-tailed deet (Odocoilcusuirginianr:rs) was Ae. diantew/dectirus 370 26 obtained by aspirating the pool of blood in the Ae. implicatus 3 1 57 abdominal cavity of shot deer brought to check- Ae. intru.dens 3,060 prouocans 2,497 45 the 1980 Ae. ing stations in Berkshire County during Ae. stimulnns grortp 2,264 53 shotgut season(December 1-6). Sentinel rabbits Ae. triseriatus adtlts 1,552 40 were set out in securedmetal cagesat 8 ofthe 9 Ae. triserintus lawae 22,557 243 trapping sites. In 1981, 14 rabbits were placed Ae. triuittatus 88 t2 in early July and removed in late September;in Ae. uexans 461 23 1982, 10 rabbits were placed in early May and An. quadrirnaculatus 2 2 n removed in early July. The rabbits were bled An. punrtipennis 12 (ear vein or heart) before placement and weekly An. walheri 1 1 perturbans l7 6 thereafter. Cq. Cx. pipicns/restuans 47 2 Mammal blood was centrifuged, and the sera Cs.marsitans 3 2 -40"C. Sera were separated and stored at Total 44,247 76r screenedfor antibody to CAL viruses in neu- tralization tests done in suckling mice using a New York strain of LAC virus as antigen. Rabbit and Young) group (includrng Aedes punctor sera were also screenedin neutralization tests (Kirby) collected on June 22, L982,in Lawrence with this LAC virus isolate and a New York Swamp); and from Ae. intrudens collected on (78-30641) State isolate of JC virus as antigen. June 29, 1982,in Warwick State Forest, Frank- Sera that were positive in screenings were ti- Iin County. AII isolateswere reisolatedand iden- trated in neutralization index tests in suckling tified from the original pools. mice, using a constant-serum, varying-virus di- A summary of virus isolation and identifica- lution method (Lennette and Schmidt 1969). tion from these mosquito pools is given in Table Sera neutralizing at least 1.7 logs of virus were 2. In IFA screentests, three isolates(nos.247, consideredpositive. 471 and 555) gave strongly positive (3-4+) re- actions with JC IMAF; negative or weak reac- RESULTS tions (<1+) were obtained with the other refer- ence IMAFs. Isolate no. 235 exhibited positive Viru.s isolations from rnosquitoes:The results reactions (2-3+) with LAC and SSH IMAF of mosquito collections are shown in Table 1. In (which were strongly cross-reactive)but did not 1981, 1,732 mosquitoes were collected in 92 react with IMAFs to JC, KEY or TVT viruses. pools. In t982,42,515 mosquitoeswere collected In the SDPRN tests (Table 2), the number of in 669 pools. Collections consisted mainly of plaques formed in control wells ranged from 82 spring Aedes and Aed,estriseriatus larvae and to 139. The titer of the JC IMAF for 3 isolates adults. Many specimens were rubbed, so that (nos. 247,471 and 555) was at least 4-fold higher some similar specieswere lumped into species than the other CAL serogroupIMAFs, confirm- groups (seeTable 1). ing their identity as JC viruses. For isolate no. Four CAL viruses were isolated from mosqui- 235. there was cross-reactionbetween LAC and toes (Table 2). Snowshoehare was isolated once SSH IMAF at a titer of 1:160.However, the from Aedes stimulans (Walker) group, which SSH monoclonalantibody had a titer of 1:2,560 probably also included Aed.es excrurinns for this isolate in the SDPRN test, while this (Walker) and Aedes fitchii (Felt and Young). antibody had <1:20 titers with the heterologous These mosquitoes were collected on June 9, CAL viruses and with New York LAC strain no. 1982, in the Lawrence Swamp Conservation 74-32813.We concludethat isolate no. 235 was Area, Amherst, Hampshire County. Jamestown SSH virus. Canyon virus was isolated 3 times: ftom Aed.es Serosurueyof wild tnammalsand sentinel rab- intrudens Dyar collected on June 10, 1982, in bits.'No sentinel rabbits seroconvertedto LAC Lawrence Swamp; from Aedes absenatus (Felt or JC virusesin either 1981or 1982.One of 178 JUNE 1993 Cnr.rronnreSpnocnoup Vrnusss IN MoseuIToES 133 Table 2. Isolations of Jamestown Canyon and snowshoehare viruses from Aedesmosquitoes collected in Western Massachusetts,1982. Results of serum dilution plaque reduction neutralization tests, given as rectp.ocattrt€rs tn r Reciprocal serum dilution plaque reduction neutrali- zation titers Isolate Pool Mosquito species no. size MFIR JC TVT LAC SSH SSHMab KEY N Ae. stimulans grotp 235 100 I:.2,264 <20 20 160 160 2,560 40 <20 Ae. intrudcns 247 55 1:1,530 80 20 <20 <20 NT <20 <20 Ae. absenatusfputrctor 47t 17 1:3,844 80 20 <20 <20 NT <20 <20 Ae. intruderu 555 100 1:1,530 80 20 20 <20 NT <20 <20 MFIR, minimum freld infection rate; JC, Jamestown Canyon; TVT, trivittatus; LAC, La Crosse; SSH, snowshoehare; KEY, Keystone; N, no viral antigen used (i.e., negative control); Mab, monoclonal antibody; NT.