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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. (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 isolates seroconvertedto 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 , 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 , 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 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 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. not tested.

(0.6%) eastern chipmunks, 5/31 (16%) $ay Field Station, Middleboro; personal communi- squirrels and,8/144 (6%) white-tailed deer had cation). neutralizing antibody to LAC virus. Positive Results of the serosurvey of eastern chip- animals came from widely separatedsites. munks, gray squirrels and white-tailed deer showedlow rates of exposureof these mammals to CAL viruses. Use of LAC virus as antigen in DISCUSSION our neutralization tests on sera from these ani- Jamestown Canyon virus has been isolated mals would detect antibody specifically to LAC from a variety of biting , including Tabani- virus or SSH virus, or possibly to heterotypic dae and Culicidae (T\rrrell and LeDuc 1983, CAL viruses. Thus, we cannot conclude defini- Grimstad 1988). In the eastern U.S., JC virus tively that the seropositive mammals had been has been associatedwith the exposed to LAC virus. Considering that LAC (De Geer) group (i.e., "dark-legged" spring virus was not isolated from mosquitoescollected Aedes),Ae, abserratusandAe. stimulans, as well in areawhere seropositivechipmunks and squir- as Anophclcs spp. and Ae. triseriatus (Grimstad rels were trapped, we think it more likely that 1988). Recent studies in northern Michigan they were exposedto another CAL virus, prob- (Heard et al. 1990)and New York State (Borom- ably SSH. isa and Grayson 1990) implicate Aedes prouo- Western Massachusettsseems to be a likely cons (Walker) as a vector of JC virus; minimum area for CAL viruses, including LAC virus, to field infection rates for that specieswere high occur. Forest regrowth and maturation (Mac- in both of those studies, ranging from 1:27 to Connell 1975) created extensive woodland hab- 1:714,depending upon the site and year. We did itat for spring Aedes and Aedes triseriatus, as not isolate JC virus ftom 2,497 Ae. prouocans, well as for sciurid rodents and white-tailed deer. eventhough collectionswere madeat siteswhere AIso, western Massachusettsabuts the CAL vi- JC virus was isolated from Ae. intntdens and. rus endemic areasof eastern New York State. Ae. absenatusfpunctor. Our data indicate that Ae. intrud.ens and.Ae. abserratusfpunrtor may ACKNOWLEDGMENTS be enzootic vectors of JC virus in western Mas- sachusetts.Heard et al. (1990) isolated JC virus We thank Wren Withers, Chris Gates, Lynn from Ae. intntdens in northern Michigan, while Thoma and Jane Bain for field assistance.Rob- Main et al. (1979) isolated JC virus from Ae. ert Boromisa for help in identifying the virus absematusin Connecticut. However, the mini- isolates, and Paul Grimstad and Tracy Smith mum field infection rates we obtained were con- for review of the manuscript. This study was siderably lower than in those studies. supportedby NIH grant NIAID 13981,the Uni- Isolation of SSH virus from Ae. stimulans versity of Massachusetts Agricultural Experi- group mosquitoesin our study is consistent with ment Station, and a scholarshipfrom the North- surveys in New York, where this virus was iso- east Association to E. D. lated numerous times ftom Ae. stimulans group Walker. and Ae. cana.densis(Grayson et al. 1983). Pre- viously, SSH virus was isolated from Ae. cana- densls in Bristol County in southeastern Mas- REFERENCES CITED sachusetts,in 1968 (H. K. Maxfield, Massachu- Boromisa,R. D. and M. A. Grayson.1990. Incrimi- setts Department of Public Health, Encephalitis nation of Aedes prouocans as a vector of Jamestown 134 JounNnl oF THE AranRrceNMosqumo CoNTRoLAssocrenor.r Vor,.9,No. 2

Canyon virus in an enzootic focus of northeastern serogroup) from Aedes mosquitoes in an enzootic New York. J. Am. Mosq. Control Assoc.6:504-509. focus in Michigan. J. An. Mosq. Control Assoc. Deibel, R., S. Srihongse,M. A. Grayson, P. R. Grim- 6:461-468. stad, J. S. Mahdy, H. Artsob and C. H. Calisher. Lenette, E. H. and N. J. Schmidt. 1969. Diagnostic 1983.Jamestown Canyon virus: the etiologic agent proceduresfor viral and rickettsial infections. Am. of an emerging human disease,pp. 313-325. .In: C. Public Health Assoc.,New York. H. Calisher and W. H. Thompson (eds.),California Lindsey, H. S., C. H. Calisher and J. H. Mathews. serogroupviruses. A. R. Liss, New York. 1976.Serum dilution neutralization test for Califor- Grayson, M. A., S. Srihongse, R. Deibel and C. H. nia group virus identification and serology.J. Clin. Calisher. 1983.California serogroupviruses in New Microbiol. 4:503-510. York State: a retrospectiveanalysis of subtype pat- MacOonnell, W. P. 19?5.Remote sensing 20 years of terns and their epidemiologic significance, 1965- changein Hampshire County, Massachusetts,1952- 1981, pp. 257-267. In: C. H. Calisher and W. H. 1972.Mass. Agric. Exp. Stn. Res. Bull. 628. Thompson (eds.), California serogroupviruses. A. Main, A. J., S. E. Brown, R. D. Wallis and J. Elston. R. Liss, New York. 1979. surveillancein Connecticut. II. Cal- Grimstad, P. R. 1988. California group virus disease, ifornia serogroup.Mosq. News 39:552-559. pp. 99-136.1n.'T. P. Monath (ed.),The : Srihongse, S., M. A. Grayson and R. Deibel. 1984. epidemiologyand ecology,vol. 2. CRC Press, Boca California serogtoupviruses in New York State: the Raton, FL. role of subtypesin human infections. Am. J. Trop. Grimstad, P. R., C. L. Shabino, C. H. Calisher and R. Med. Hyg. 33:1218-1227. J. Waldman. 1982.A caseof encephalitisin a human Turrell, M. J. and J. W. LeDuc. 1983. The role of associatedwith a serologicalrise to JamestownCan- mosquitoesin the natural history of California ser- yon virus. Am. J. Trop. Med. Hyg. 3l:1238-1244. ogroupviruses, pp.43-55. In: C. H. Calisher and W. Heard, P. B., M. Zhang and P. R. Grimstad. 1990. H. Thompson (eds.), California serogroupviruses. Isolation of Jamestown Canyon virus (California A. R. Liss, New York.