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Laboratory Transmission of SSH Virus by Ment Againstthe Coevolutionofvirus and Vector Ae JounNar, oF THE AveRrclr.r Mosqulto CoNrRor, AssocrerroN Vol. 7, No. 1 LABORATORYTRANSMISSION OF JAMESTOWNCANYON AND SNOWSHOEHARE VIRUSES (BUNYAVIRIDAE:CALIFORNIA SEROGROUP)ey SEVERAL SPECTESOF MOSQUITOES1 PHILIP B. HEARD, MINGBAO ZHANG eNo PAUL R. GRIMSTAD Laboratory for Arbouirus Researchand Surueillance,Department of BiologicalSciences, Uniuersity of Notre Dame, Notre Dame, IN 46556 ABSTRACT' The ability of 14speciesof mosquitoes to biologicallytransmit JamestownCanyon virus wastested. Four speciesnot previouslydfscribed as vectorsof that-virus transmittedto;*kii;t-i;;. Among membrane-fed mosquitoeswith disseminatedinfections, field-colleiled eeae" canaaei;F 1iJij, Aryoplglespunctipennis (l/!2), Coquillettidiaperturbans (2/14).and a laboratory strain of Ae.epactii (19/67) transmitted virus. Two specieswerelested for their ability to transmit snowshoehare virus: field-c!llected A e-prouocdns (4/20) and.Ae. abserratus--punctor(2/20i successfullytransmitted to suckiinj mice.Evidence regarding the role of thesespecies as field vectorsis'summarized. INTRODUCTION gions of the United States,with the exception of one laboratorystrain that originated Oral transmissionof JamestownCanyon (JC) from El Salvador. virus has been describedfor B mosquito species: Aedesstimulans (Walker) (Boromisa Large field collections of 2 communrs group and Grim- members, stad 1986),Aedes prouocans (Walker) (Boromisa Ae. prouocans and Ae. abserratus- punctor,in Michigan in 1989made possible and Grayson1991) andAedes albopictus (Skuse) it to test the vector competenceof these species (Grimstad et al. 1989). There is evidenceto for a secondstrain of JC virus from suggestthat Ae. stimulans and,Ae. prouocansate Quebecwhere both mosquito speciesare found (Darsie natural vectors (Boromisaand Giimstad 1986. and Ward 1981).We also took this opportunity Boromisaand Grayson19g0). Jamestown Can- to test for snowshoehare (SSH) virus (Bunyaviri- yon virus isolations have been made from at dae:California serogroup) least 19 additionalspecies of mosquitoes(Grim- transmission by these species.Snowshoe hare virus has been isolated stad 1988,Campbell 1990) for which transmis- from numerousmosquito species, including Ae. sion data are lacking.Included among these are punctor and other members of the communis Aedesabserratus (Felt and Young)and otherAe. group (Belloncik et al. 1983, Grimstad 1988). communisgroup members that are suspectedof Oral transmissionof this virus has been dem- transmitting on the basis of high field infection onstratedin Ae. comrnunrs(De Geer) (Mcl-ean rates (Sudiaet al. 1971,Main et al. 1929,Grav- 1983).In addition, this virus is relevant to our son et al. 1983,Campbell i990). There is also ongoing studies of multiple California serogroup interest in Anophelesspecies, which may be virus infections of white-tailed deer (Odocoileus involved in early spring and late fall transmis- uirginianus),the primary vertebratehost for sion in the Midwest (DeFoliartet al. 1986). JC virus (Grimstad 1988),in the upper Midwest. In an effort to address questions of vector competencefor JC virus, we conductedtrans- mission trials with 14 species(18 geographic MATERIALS AND METHODS strains) of mosquitoes.Species were selectedon Ten species(11 geographicstrains) were col- the basis of their apparent potential as vectors lected and tested directly from the field and 4 and their availability for testing. Nine species species(7 strains) were colonized. Field-col- were from an enzootic focus of JC virus located lected mosquitoes were taken as adults from in the Lower Peninsulaof Michigan (Grimstad Coz-baitedtent traps at sites in northcentral et al. 1987).The remainingspecies/strains came Michigan and northwesternIndiana (Table 1). from northern Indiana and other enzootic re- Porter Flanch,in the Lower Peninsulaof Mich- igan, lies within an enzootic focus of JC virus (Grimstad et al. 1987, Heard et al. 1990). Snowshoehare virus is undocumentedat the 1 part Supported in by National Institutes of Health site; however, its range likely includes Michi- Grants AI-19679 and AI-07030. In conductine the gan's Lower Peninsula (Calisher 1983). research described in this report. the investigators All "Guide adhered to the for the Care and Use of Labo- snowmelt Aedesspecies were collected at Porter ratory Animals" as promulgated by the National Re- Ranch in the late spring, within days of their search Council and used facilities accredited by the emergence;others specieswere collected as in- American Association for the Accreditation of Labo- dicated in Table 1. Potato Creek and Spicer ratory Animal Care. Lake are in northern Indiana in a region where Mencu 1991 Moseurro TRarslrIssIoN oF JC AND SSH Vlnusns Table 1. Mosquito strains used in transmission trials with Jamestown Canyon and Snowshoe Hare viruses. Laboratory Origin and generation Mosquito species Strain tested collection date tested Aedes canadensis PORTER RANCH MissaukeeCo., MI, May 1988 0t cinereus PORTER RANCH MissaukeeCo., MI, May 1988 0 excrucLans PORTER RANCH MissaukeeCo., MI, May 1988 0 fitchii PORTER RANCH MissaukeeCo., MI, May 1988 0 prouocans PORTER RANCH MissaukeeCo., MI, May 1988 0 proDocan^9 PORTER RANCH MissaukeeCo., MI, May 1989 0 intrudens PORTER RANCH MissaukeeCo., MI, May 1989 0 abserratus fpunctor PORTER RANCH MissaukeeCo., MI, May 1989 0 triseriatus VERO Indian River Co.,FL, 1977 30+ triseriatus SALADO Bexar Co.,TX, 1972 40+ triseriatus ALA Alabama,prior to 1958 75+ stirnuLans POTATO CREEK St. JosephCo., IN, June 1988 0 hendersoni U.VILLAGE St. JosephCo., IN, 1981 20+ epactius SANSAL Matapan,El Salvador,1968 40+ epactius GRAND Grand Co..Utah. 1968 40+ Anopheles punctipennis PORTER RANCH MissaukeeCo., MI, April 1988 0 punctipennis PORTER RANCH MissaukeeCo., MI, August 1988 0 quadrimaculatus SAVANNA Savanna,GA, acquired in 7971' 100+ Coquillettid.ia perturbans PORTER RANCH MissaukeeCo.. MI. June 1988 0 perturbans SPICER LAKE St. JosephCo., IN, July 1988 0 l These strains were tested as field-collected,newly emergedfemales. 2This strain was acquired as colony stock in 19?1.Atl other strains were originally obtained as field stock and maintained in the Vector Biology Laboratory from the year of collection listed until the time of use in this studv. a high prevalenceof antibodyto JC virus occurs 19'C. Brain extracts from sick and moribund in all surveyeddeer populations (Boromisa and mice were tested for the presence of virus. Grimstad 1987). Specimenswere carried from Plaque assaysfor the detection of virus in mos- the field in ice-cooledboxes and housedat 19'C quitoes and mouse brain tissue were modified in 3.8-literplastic buckets. Additional handling from Grimstad and Haramis (1983).The Vero measureswere describedby Grimstad et al. cell cultures used for this purpose were main- (1e8e). tained in medium 199with a 10%serum supple- Laboratory-reared mosquitoes were taken ment (Seru-Max 2, Sigma Chemical Co., St. from coloniesat the University of Notre Dame Louis, MO), 2.2 g NaHCO3 per liter, 75.0 mg (Table 1). Larvae were reared according to gentamicinper liter, and 125.0mg amphotericin Grimstad et al. (1989), and adults were main- B per liter. For mosquito assays,the concentra- tained in the samemanner as wild-caughtmos- tions of antibioticswere increased 2 and 4 times, quitoes,with the exceptionthat the Aedestris- respectively.Samples were applied to Vero cell eriatus(Say) VERO strain was kept at 27"C. monolayersin 6-well plates,and replaced,after Virusesused in the trials were as follows: 1) a 1-h incubation at 37'C and 5% CO2,with an JC virus strain 800245,passage 3 in suckling overlay consisting of culture medium supple- mousebrain (SMB), was isolatedfrom a pool of mented with 0.8% gum tragacanth (Sigma Ae. stimulans males in Indiana and is horizon- ChemicalCo.). After 3-4 additional days of in- tally transmitted by that species(Boromisa and cubation, the plates were fixed and stained with Grimstad 1986);2) JC virus strain 136A3,pas- a crystal violet-formalin solution and examined sage 3 in Vero cells, passage1 in SMB, was for plaques. isolatedfrom mosquitoesin Quebec,Canada (H. Infectious blood meal (IBM) titers were de- Artsob, personalcommunication); and 3) SSH termined from observationsof cytopathic effects virus, "original" strain, was suppliedin lyophi- in Vero cellsgrown in 96-wellplates as described lized form, passage20 in SMB, from the Centers by Grimstadand Haramis (1983).Median tissue for DiseaseControl. culture infectious doses (TCIDun) were calcu- Mosquitoes were orally infected at a mem- lated using the method of Reed and Muench brane feeder and individually tested for trans- (1938).The valuesin Table 2 representan av- missionto newbornmice as describedby Grim- erage of titers taken at the beginning and end stad et al. 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