Am. J. Trop. Med. Hyg., 32(4), 1983, pp. 877—885 Copyright ©1983 by The American Society of Tropical Medicine and Hygiene . IDENTIFICATION OF ARBOVIRUSES HITHERTO FROM UNRECOGNIZED ECUADOR: MEMBERS OF SEROGROUPS B, C, BUNYAMWERA, PATOIS, AND MINATITLAN CHARLES H. CALISHER,* ERNESTO GUTIERREZ V.,t D. BRUCE FRANCY,8 ARACELY ALAVA A.,t DAVID J. MUTH,* @DJOHN S. LAZUICK* *Division of Vector-Borne Viral Diseases, Centers for Disease Control, Fort Collins, Colorado 80522, and tlnstituto Nacional de Higiene, Guayaquil, Ecuador Abstract. Three hundred seventy-nine virus isolates were obtained from mosquitoes cob lected and sentinel hamsters exposed in coastal Ecuador from 1974 to 1978. These included four alphaviruses [Venezuelan equine encephalitis lB (1), Venezuelan equine encephalitis 1D (35), western equine encephalitis (1) and eastern equine encephalitis (4)]; two flaviviruses [St. Louis encephalitis (3) and Nananjal (6)1; 11 bunyaviruses [Maguari (243), Playas (3), Vinces (33), Tunbock (2), Abras (5), Babahoyo (3), Acana (2), Guajara (3), San Juan (6), Pueblo Viejo (3), 18 unspecified Gamboa serogroup viruses, Palestina (7)]; and one vesiculovirus (vesicular stomatitis New Jersey). All but Venezuelan equine encephalitis virus were new to Ecuador, and Naranjal (scrognoup B), Playas (Bunyamwera serogroup), Vinces (scrognoup C), Abras and Babahoyo (Patois scrogroup), San Juan and Pueblo Viejo (Gamboa scrogroup) and Pa lestina (Minatitban serogroup) are newly recognized viruses. These isolates have enabled us to 1) expand our knowledge of the geographic distribution of recognized viruses, 2) expand our knowledge of the members of certain scrogroups and 3) establish two new serogroups (Gamboa and Minatitlan). In January 1969, an epidemic of Venezuelan equine encephalitis (VEE) occurred in Guayas Province, Ecuador, affecting humans and equines. Retrospective studies suggested that this had been an extension and expansion of an earlier outbreak of VEE further inland.' Since the enzootic pncs ence of epizootic VEE virus subtypes could have accounted fon periodic appearances of VEE in coastal Ecuador and since, subsequent to 1969, VEE spread northward from Ecuador and Cen tnal America to south Texas,2 a cooperative in vestigation of arbovirus transmission and ecology in the coastal lowlands of Ecuador was initiated to determine whether cnzootic transmission of epi zootic strains was occurring there. From 1974 to 1978, mosquitoes and other arthropods were col bected and sentinel hamsters exposed at various sites, maximizing the number of virus isolates ob tamed. Ecologic descriptions of the study areas will be published elsewhere, as will extensive tabulations of arthropod collections and definitive idcntifica tions of mosquitoes.3 This article describes the identifications of virus isolates obtained from an Accepted 24 November 1982. thnopods, brates. sentinel Among hamsters, the 379 isolates alphaviruscs, two flaviviruses, and one vesiculovirus. These newly recognized viruses. MATERIALS and other obtained verte were four 11 bunyaviruscs, 18 included eight AND METHODS Collection and processing of specimens Adult hamsters were exposed in the field for up to 10 days, after which they were returned to the laboratories in Guayaquib and observed for signs of illness. Sick hamsters were tested for virus by inoculating clarified 10% suspensions of their tis sues (whole blood, brain, heart, kidney, skeletal muscle on liver-lung-spleen pools) intracnanially into separate bitters of 2- to 4-day-old suckling mice (SM). Mice were observed daily for 2 weeks for signs of illness; the brains of those which appeared ill were passed as 10% suspensions to other SM to establish strains for subsequent virus identifi cations. Other mammals and birds were shot on live-trapped in the field and their tissues tested for virus by the same methods used for virus isolation from sentinel hamsters. Mosquitoes and other arthropods were collected 877 878 CALISHERET AL. with CDC light traps4 supplemented with dry ice (CO2), using methods published previously.5 Ar thropods were sent frozen on dry ice (—60°C) to the laboratories in Fort Collins, Colorado, where they were sorted, pooled by species, identified and ground in Medium 199 with heated (56°C/30')20% fetal bovine serum, using sterile abundum and chilled mortars and pestles.5 Each of the resulting suspensions was inoculated onto monolayer cub tures of continuously propagated Veno and pri many duck embryo cell cultures. The cell cultures were overlaycd with a medium containing agar and examined daily for plaque formation by the method of Hayes et al.5 When plaques were ob served, subsequent passage to a litter of SM served to establish the isolate. Virus identification and characterization dropped onto a monolayer culture of appropriate cells and the cells were incubated for 45 mm at 37°Cand overhayed with nutrient medium con taming agar. For viruses producing plaques in 1— 3 days, a single overlay, containing !:40,000 neu trab red, was used; a double overlay—the second, containing 1:25,000 neutral red, applied on the 3rd day after inoculation—was used for viruses which produced plaques 4 or more days after in oculation of cell cultures. Virus diluent for passage of tissue suspensions, harvested plaques, and stock viruses was Medium 199 with 5% heated fetal bovine serum supple mented with antibiotics. Stock viruses were prepared as !0% SM brain suspensions in phosphate-buffered saline (pH 7.4) with 4% bovine albumin. For production of an tibody, mice were hypcrimmunized according to the schedule suggested by Brandt et al. , 12and as citic fluids were elicited by the method of Tika singh et al. i:t @ Complement fixation (CF) tests were performed by the microtiter adaptation method of Casey,6 using crude, alkaline extracts of SM When sucrose-acetone extracted antigens were required, RESULTS for example, with crude antigens of low titer, or when definitive (reference comparison) reactivitics Nine flavivirus strains were isolated from sen were needed, extracts were prepared by the meth tinel hamsters and mosquitoes. Three isolates of St. Louis encephalitis (SLE) virus were obtained ods of Clarke and Casals.8 The same sucrose-ace (76V-! 177, 24684 and 78V-5682), two from Culex tone extracted antigens also were used for hem agglutination and hemagglutination-inhibition (HI) nigripalpus mosquitoes and one from a sentinel hamster. In addition, six strains of another flavi teSts.―For preliminary serologic identifications, a virus were isolated from Culex (Melanoconion) battery of grouping fluids, prepared by the Ar bovirus Reference Branch, or obtained from the adamesi and Cx. (Mel. ) spp. mosquitoes and the heart and skeletal muscle of a sentinel hamster. Reference Resources Branch, U.S. National In stitutes of Health, was used. This battery of im Strain 25008, isolated from a sentinel hamster, was chosen as the prototype for this virus because munc mouse ascitic fluids (MAF) represented more it was isolated, identified, and characterized first. than 200 recognized viruses. When an antigen was This virus titered 6. 7 and 7. 1 hog,,,PFU/0. 1 ml and found to react with one or more of these reagents, formed 1- and 4-mm indistinct, irregular plaques additional tests, using MAF for individual virus in duck embryo and Vcro cells, respectively. Strain es, were performed. effects in C6/36 (Aedes A test of sensitivity to the dchipidizing action of 25008 causes cytopathic albopictus mosquito) cells 6 days after inoculation sodium deoxycholatc (SDC) was performed in mice by the method of Thciler9 and in cell ‘°and kills SM on the 5th day after intracranial in To determine virulence by the peripheral route of oculation but does not kill weaned or older mice inoculated intrapenitoneally. inoculation, 0. ! ml of a 1:100 suspension of virus Strain 25008 was shown by HI and CF tests to was inoculated intraperitoncally into five or six be a flavivirus and identified as a newly recog 8-week-old mice. nized flavivirus by N tests (Table 1). Hemagglu Virus titrations were by plaque assay in cell cul tination of goose crythrocytes by strain 25008 oc tures. For definitive virus identifications, a serum curned at pH 6. 1 to 7.0 with optimum titers at pH dilution-plaque reduction neutralization (N) test 6. 7—6.9. was used.― Briefly, 200 plaque-forming units (PFU) of virus were mixed with an equal 0. 1-mb Thirty-three Group C virus strains were iso volume of antibody. After overnight incubation at bated, three from Cx. (Mel. ) vomerifer mosquitoes 4°C, 0. 1 ml of the serum-virus mixture was and 30 from tissues of 12 sentinel hamsters. In CF IDENTIFICATION OF ARBOVIRUSES FROM ECUADOR 879 TABLE! Results of hemag.glutination-inhibition (HI), complement-fixation (CF) and serum dilution-plaque tralization (N) tests with Ecuadorian strain 25008 and other fiaviviruses to:25008 NHICFNBussuquara Antigenorvirus StrainTiter HI CF of antibody NBSQSLEHICF —!60——BeAn4O7880 256 25008160 @2,56016St. 10——160@1,0241,280Rocio Louis enc. TBH-28— ——864Yellow SPH-34675!0 fever 17D20 —108Aroa !680816Ilheus VENA 1809320 —40864Jutiapa Onig.80 reduction 64 64040256 !6080@!,024 16 32 8 !6 8108—Tamana JG-128!0 154—8—Antigen bat Tr 27 to:ROCYFAROAHI or virus NHICFNBussuquara StrainTiter CFNHI 2500880 —108BeAn 32—40 6420 25610 407880 TBH-2840 SPH-34675640 fever 17D@80 160108Aroa VENA !809320 —320@‘!,024Ilheus Onig.1,280 —4032Jutiapa —10256St. —1064Rocio Louis enc. ——64Yellow —108Tamana of antibody CF — @‘1,0241,280— 64160 256 6440 51280 JG-!2820 3210 @27!54——8Antigen bat Tr to:ILHJUTTB CFNBussuquara or virus StrainTiter CFNRI CFNHI 8St. 8Rocio Louis enc. 4078640 TBH-28160 !6—20 6410 !28— 8Yellow SPH-34675160 512— 8—tBeAn 25008640 8Aroa 8Ilheus 8Jutiapa fever !7D320 VENA !8092,560 Orig.@!0,480 @!,024Tamana 154——S bat tested.t Blank signifies JG-!2880 Tr 27 not —= <10 HI, or N, <8 CF. 6410 6440 @!,02464080 —2,560 of antibody HI neu 880 CALISHER ET AL. TABLE 2 TABLE 3 Results of serum dilution-plaque reduction neutraliza tion (N) tests with Ecuadorian strain 75V-807 and 12 Results of serum dilution-plaque reduction neutraliza tion (N) tests with two Bunyamwera serogroup viruses other Group C viruses from Ecuador viruses and 22 other Bunyamwera serogroup 75V-807VirusStrainHomologous 75V-3066vi@sStrainHomologous bodyBruconha75V-807 titerVirusAnti body75V-3066160160160MaguariE4-34841,28040@eMaguariBeAr titervi@Anti 80OssaBT-182032020320RestanTr-5!!44@5,120320—“ApeuBeAn 6401,280 77V-148141,280@5,!201,280 727232040—BataiMM-2222@2,560160—BehiefeUGMP-68302,56020—ileshaKO-2@5 84816040—CaraparuBeAn 399416040—MaritubaBeAn 1532040—MurutucuBeAn 974@64040—Gumbo Valley6V-63316040—Tlacotalpan6!D-24032020—TensawA9-!7 !H160—40OribocaBeAn LimboFe3-7 17a'64020—ItaquiBeAn lb64040—BunyamweraRI-!a1,28020—GermistonSAAr-!0502,560——ShokweSAAr-4 12797@640——NepuyoTr-!846280——Madrid40308320——a— signifies <20. RosaM2-14931,280——LokernFMS-43322,56040—Northway0234160——Taia 67!320——WyeomyiaOriginal64020—AnhembiSPAr-298464020—SororocaBeAr tests all 33 strains reacted to within twofold of the homologous titer (1: 128) of the prototype strain (75V-807) and were glc Group C virus. considered Strain as strains 75V-807 of a sin formed plaques in Veno (1 mm, distinct on day 4) but not duck embryo cells, and was pathogenic for 3- to 4-week old mice inoculated intraperitoneally; these mice died 4—6days after inoculation by this route. Strain 78V-807 was tested by N with 12 other Group C viruses (Table 2). Most of the viruses isolated during these studies arc members of the Bunyamwera senogroup. Aedes scapularis and Aedes spp. mosquitoes were the source of 243 isolates of Maguari virus (topotype E4-3484), but three Bunyamwera scrogroup iso hates were obtained from Aedes taeniorhynchus mosquitoes. These three (75V-3066, 75V-5758 and 75V-5938) had been isolated from mosquitoes cob hected 8 March 1975. By N tests (results not shown) all three were identical to each other but distinct from the prototype Maguari virus (BeAr 7272). For definitive identifications both E4-3484 (Maguari virus) and the second virus (prototype 75V-3066) were tested by N with all recognized Bunyamwena senogroup viruses (Table 3). Eight viruses related by CF to members of the Patois serogroup were isolated, six from Culex (Mel. ) paracrybda, one from Cx. (Mel.) adamesi mosquitoes and one from the blood of a sentinel hamster. Since preliminary 149@2,56020—Main 32 DrainBFS-50151,280——KairiTr-8900320——GuaroaJC-216040—TucundubaBeAr tests indicated shared CF antigens, N tests were performed. First, the 278160——a sjg@@fles<20. eight strains were tested among themselves. Re sults of these tests showed that five viruses, rep resented by prototype strain 75V-1 183, were iden tical to each other but distinct from three other viruses, represented by prototype strain 75V-2858. Subsequent N tests included the four recognized Patois senogroup viruses and both Ecuadonian prototypes. The results showed that both Ecua dorian viruses were distinct from the four necog nized Patois serogroup viruses (Table 4). All eight Ecuadorian Patois serogroup viruses were tested in SM and Vero cells for pathogenicity and in vitro characteristics. The results revealed similar characteristics in both hosts by senotype; strain 75V-1 183 and viruses identical to it did not cause CPE but strain 75V-2858 and viruses iden tical to it did (days 5—7).The eight field strains killed SM in 6—10days and titered i0@°to 1064 in Veno cells by plaque assay, with 1-mm plaques appearing 5—7days after inoculation. Five isolates, two from Cx. (Mel.) paracrybda, and one each from Cx. (Aedinus) amazonensis and IDENTIFICATION OF ARBOVIRUSES TABLE FROM ECUADOR 881 4 Results of serum dilution-plaque reduction neutralization (N) tests with two viruses from Ecuador and four other Patois serogroup viruses virusStrain75v-118375v-2858PATSRPAHZEG75V-1!83@.i2:?.@:c@—*————7SV-28S880@:P40———PatoisBT-497 1——160———Shark RiverFE4-!R——40@!,280——PahayokeeFE3-S2F————320—ZeglaBT-S0!2————40320a signifies <20. Uranotaenia spp. mosquitoes and the blood of a sentinel hamster, were shown by CF tests to be related to members of the Capim senogroup. CrossCF tests revealed such extensive antigen sharing that N tests were required for definitive identifications. Preliminary N tests showed that strains strain E4-2535 is a strain of Acara virus and strain 183 15 is a variant of Guajara virus (Table 5). As strain E4-2535 was serially passed in SM, its vir ulence for and titer in these animals decreased until, after six passages, it did not kill SM; its titer by plaque assay in Vero cells remained stable rc E4-2535 gardless and 76V-885 1 were identical of passage level. Strain 71U-344 is most closely related to Bcnfica virus, and strains 7 1U-253 and 7 1U-350 most closely related to Guajara virus. Twenty-seven virus isolates from Aedeomyia serognoup 7 1U- squamipennis ham- ed by CF and N tests to the previously viruses 253, 7 1U-344 @ and distinct from strains 183 15, 76V-2585 and 76V-3029, which arc also identical. Therefore, for definitive identifications, N tests were performed with strains E4-2535 and 18315. In addition, three Capim from Guatemala and 7 1U-350, (strains all from sentinel sters) were received from W. F. Schercr'4 and included in these studies. The results indicated that bunyavirus, lishcd TABLE Results ofserum dilution-plaque reduction neutralization Acara ACA 2535) (MOR) BSB BeAn27639 .@2 320 E4-2S3S 640!60 Moriche TrS7896 80 —320 BEN 344)GJA18315350)353) — — — — —640 7!U-344 — — — Guajara BeAn!06!S 640 320 18315 640 320 7!U-3S0 320 80 640 640 160 320 BeAn !S3S64 Capim BeAn8S82 S _ = <20. — B@S CAP — — 20 160 640 7!U-253 — JD —* BeAn8438! Benevides ungrouped (N) tests with 13 Capim serogroup bunyaviruses TR 2668 MARU8S63 to be rehat 5 BushBush JuanDiaz shown Gamboa virus. Further studies, pub revealed that at least two new Benfica @ were Titer ofN antibody to:(E4-(71U-(71U-(71U-virus Strain mosquitoes — 320 — — 320 — — — 320 320 — — — 80 320 20 40 20 — — — 640 640 — 640 640 — 640 CALISHER ET AL. 882 TABLE 6 Results of complement-fixation (CF) and serum dilution-plaque reduction neutralization (N) tests with strain 76V- l5o5from Ecuador and Minatitlan and Mirim viruses to:76v-ls6sMNTMIR antibody Antigen or virusStrainTiterof NCFNCFNMinatitlan <8 CF, <20 256 16 840 M67U5 BeAn 7722.@: Mirim76V-!565 320C = CF —!28 virus and three of Pueblo Viejo virus were identified; 18 isolates were not identified more specifically than as members of the newly necog nized Gamboa senogroup. Seven isolates were shown by both CF and N tests to be strains of a single virus (76V-1565); three were isolated from Culex (Mel. ) paracrybda mos quitocs 256— —8 N. ly recognized agents, San Juan and Pueblo Viejo viruses, were present in Ecuador. Six strains of San Juan — 8 2,560 8—@ and four from tissues of sentinel Cross-CF tests with a representative virus of the six, strain 76V-1565, as well as Minatitlan and Mirim viruses, showed that strain 76V-1565 cross reacts significantly with Minatitlan but not Minim viruses. Additional N tests confirmed the relation ship of strain 76V-1565 with Minatitlan but not Minim viruses (Table 6). A single strain of vesicular stomatitis New Jer sey (VSNJ) virus was isolated from a pool of Man sonia indubitans mosquitoes. The virus was iden tified in CF tests with a vesicular stomatitis grouping MAF and with viruses and immune MAF for other vesicular stomatitis senogroup viruses (Table 7). hamsters. In preliminary CF tests using the battery of im mune MAFs used throughout this study, these iso lates reacted only with the NIH Polyvalent #2 MAF, which contains antibodies to M.inatitlan, Jurona, Ahajucla,'5 Gamboa, and Belem viruses. Additional CF tests showed that the six Ecuador ian isolates reacted to titer with MAF for Mina titlan and Minim viruses; weakly with MAF for Patois, Capim, and Guama viruses; and not at all with MAF for Junona, Abajucla, Gamboa, and Belem viruses. DISCUSSION The six flavivinus strains shown to be distinct from SLE virus appear to be isolates of a single newly recognized virus, for which the name Nan anjal virus is proposed (prototype strain 25008). TABLE 7 Results of complement-fixation viruses (CF) tests with Ecuadorian strain 76V-4357 and other vesicular stomatitis serogroup of CF antibody to:76V-4357vSNJvslCHPCOC1SFPIRYvSA@76V-4357!,0241288—t———VS vims5trainTiter JerseyHazelhurst1,0241,024—VS New strain81,0248Chandipura1-6535 IndianaLab 1481,024CocalTr-402331625616IsfahanOriginal16512PiryBeAn 2423216!@ C @ @5 (Alagoas): antiserum kindly restricted and was not available t —signifies <8. Blank indicates not tested. provided to us. by Dr. J. J. Callis, U.S. Department of Agriculture, Plum Island, Greenport, NY; use of live virus is IDENTIFICATION It is most closely related to Bussuquara OF ARBOVIRUSES FROM ECUADOR virus but is cleanly distinct from it by both CF and N tests. Strain 75V-807, the prototype Group C bun yavinus from Ecuador, @ is distinct from all other recognized Group C viruses; the name rus is proposed for this agent. Vinces vi Bunyamwera serogroup strains 75V-3066, 75V5758, and 75V-5938 are three isolates of a single, newly recognized virus for which the name Playas virus is proposed. The prototype, strain 75V-3066, was easily distinguished from others in the seno group, including the Ecuadorian topotype of Ma guam virus which represented 243 of 379 (64.1%) strains isolated during these studies. Previous studies of Bunyamwera senognoup viruses in the Americas, as elsewhere, have shown that they are isolated from mosquitoes, such as Aedes scapula ris @ @ and other Aedes spp. which feed predomi nantly on mammals. Of some apparent signifi cance are the accumulated pieces of information regarding equine pathogenicity of certain mem bers of this serogroup; Maguari virus has been isolated from horses in Guyana'6 and Argentina (M. Sabattini, personal communication, 1978) and Cache Valley virus from a horse in and antibody to Bunyamwcna scrogroup viruses is regularly found in equines distributed through out the In a serosurvey of 157 Ecuadorian equines 106 (67.5%) had antibody to the Ecua dorian Maguani virus topotype, strain E4-3484 (E. Gutienrez V. and C. H. Calisher, and 25024) caused cytopathic effects identical to strain 183 15. Since recombination be tween and among closely related bunyaviruses is recognized, such may be an explanation for this phenomenon; previous studies with the Gamboa serogroup have shown similar antigen These speculations notwithstanding, Schercr (pen sonal communication, 1982) has suggested that the area in Guatemala from which these three isolates were obtained is not likely to provide sufficient Capim scrognoup virus amplification to support a milieu in which viral persistence and recombina tion could take place on a regular basis. By the tests summarized in Table 5, Juan Diaz and Benevides viruses arc indistinguishable. We compared Ecuadonian strain 76V-1565, Minatitlan and Mirim viruses by both CF and N tests. Mirim virus has been shown by R. E. Shope (personal communication, 1978) to be rebated to Minatitban virus. In our tests, strain 76V-1565 is distinct from both Minatitban and Minim viruses; we suggest the name Palcstina virus for this newly recognized agent. Furthermore, the identification of a virus closely related to, but distinct from, the heretofore ungrouped Minatitlan virus is sufficient grounds to warrant establishment of the Minati than scrogroup of bunyaviruses. Minim virus is cx cluded from those in the Minatitban serognoup be cause (1) it is not sufficiently close antigenically and (2) in other studies Minim virus was shown to be properly classified as a member of the Guama ‘9 unpublished data). A comparison of the two prototype Patois scro group bunyavirus strains (75V-1 183 and 75V-2858) with the four previously recognized members of the Patois serogroup showed that both Ecuador ian viruses arc distinct, newly recognized viruses. The name Abras virus is suggested for viruses rep resented by strain 75V-1 183 and Babahoyo virus for those identical to strain 75V-2858. Interesting by, the three strains of Babahoyo virus (75V-2858, 76V-440 883 in Vero cells, whereas the five strains of Abras virus (75V-1285) did not. The significance, if any, of such an observation is unknown. Two Capim serogroup bunyaviruses were iso lated. Strains E4-2535 and 786V-885 1 are two iso lates of Acara virus; and strains 18315, 76V-2585 and 76V-3029 arc variants of Guajara virus. However, while strains 183 15 and 7 1U-350 are strains of Guajara virus, they are distinct from each other; and strain 7 1U-253, a subtype of Gua jara virus, is also a subtype of strain 7 1U-350 but The isolation of vesicular stomatitis (New Jer scy) virus, a known pathogen of cattle and hu mans, has potential significance in Ecuador; this virus should be considered in future arbovirus Sc rosurveys of the area. As the pressures of human populations cause expansion into the Amazon re gion, a similarly expanding cattle industry will probably emerge to feed the people necessary for conducting exploration and mining of minerals and other resources. Since alterations of the ecosystem of this area will occur, the natural equilibrium will be concomitantly altered, and VSNJ and other viruses currently maintained cnzootically could result in infection and perhaps disease outbreaks in settlers in these areas. A summary of virus isolations from these stud ics in Ecuador is presented in Table 8. In all, at least 18 distinct agents were isolated, eight of which arc newly recognized. In the absence of serosurvey data on prevalence of antibodies to these viruses on their isolations from clinical specimens collected from humans or 884 CALISHER TABLE Summary ofvirus 8 isolationsfrom Ecuador, 1974 —1978 ET AL. Viral Diseases, Centers for Disease Control, for his constant comments and continuing advice. NumberofSerogroupVirusisolatesA REFERENCES equine encephalitis lB Venezuelan 1. Gutierrez V. , E. , Monath, T. P. , Alava A. , A., Uriguen, equine 35 encephalitis !D Western equine 1 encephalitis Eastern equine B. D., Arzuba, R. M., and Chamber lain, R. W. , 1975. Epidemiologic investigations of the 1969 epidemic of Venezuelan encephalitis in Ecuador. Am. J. Epidemiol., 102: 400—4!3. 2. Sudia, W. D. , Newhouse, V. Miller, D. L., Johnston, encephalitis BVenezuelan St. Louis encephalitis 6BunyamweraMaguari Naranjal1 4 3 L. D., J. G., Jr., Young, F. , Beadle, R., Calisher, C. H. , and Maness, K. , 1975. Epidem ic Venezuelan equine encephalitis in North Amer ica in !97!: Vector studies. Am. J. Epidemiol., 101: 17—35. 3. Francy, D. B. Manuscript in preparation. 4. Sudia, W. D. , and Chamberlain, R. W. , 1962. 3CVinces33TurhockTurlock2PatoisAbras Playas243 Battery-operated light trap, an improved model. 3CapirnAcara Mosq. News, 22: 126—129. BabahoyoS S. Hayes, Guajara2 Juan 3GamboaSan Pueblo Viejo 3 18MinatitlanPalestina7Vesicular Unspecified6 stomatitis (New Jersey) stornatitis TotalsVesicuhar 181 R. 0. , Francy, D. B. , Lazuick, Public Health Service Publication 379 J. S. , Smith, G. C. , and Jones, R. H. , 1976. Arbovirus sur veiblance in six states during 1972. Am. J. Trop. Med. Hyg., 25: 463—476. 6. Casey, H. L. , 1965. Standardized diagnostic corn phemcnt-fixation method and adaptation to micro test, part H. Adaptation of LBCF method to rni cro technique. Public Health Monograph No. 74, No. 1228, U.S. Govt. Printing Office, Washington, D.C. , pp. 3!— 34. 7. Calisher,C. H., and Maness, K. S. C., 1975. Lab oratory studies of Venezuelan equine encephalitis domestic animals, conclusions regarding their in dividual or collective significance would be pre mature. However, such a large and diverse viro logical burden may, indeed, impact upon some facet of the local vertebrates—be they humans, animals of domestic importance or wild mammals or birds. No evidence of such impact is presently available. ACKNOWLEDGMENTS The authors are grateful to many individuals whose efforts in the field and the laboratory made this work possible. We would bike to extend par ticular thanks to the following for their advice, expert technical assistance and persistence in com pleting these virus identifications: R. E. Shope, Yale University School of Medicine, New Haven, Conn. ; 0. Garcia giene, Guayaquih; P. , National and Institute R. Bohin, W. of Hy L. Jakob, J. France, A. Hunt, D. Inghish, N. Karabatsos, J. Trimble, and K. Wolff, Centers for Disease Control, Fort Collins, Cob. We also thank Dr. T. P. Monath, Director, Division of Vector-Borne virus in equines, Texas, biol., 2: 198—205. 8. Clarke, D. H., and 197!. Casals, J., J. Clin. Micro 1958. Techniques for hemaggbutination and hemagglutination-in hibition with arthropod-borne viruses. Am. J. Trop. Med. Hyg., 7: 561—573. 9. Theiler, M. , 1957. Action of sodium deoxychohate on arthropod-borne viruses. Proc. Soc. Exp. Biol. Med., 96: 380—382. 10. Calisher, C. H., and Maness, K. S. C., 1973. Use of the sodium deoxycholate sensitivity test in cell cultures. Acta Virol., 17: 26!—262. 11. Lindsey, H. S. , Calisher, C. H. , and Mathews, J. H. , 1976. Serum dilution neutralization test for California group virus identification and serobo gy. J. Clin. Microbiol., 4: 503—5 10. 12. Brandt, W. E. , Buescher, E. L. , and Hetrick, F. M. , 1967. Production and characterization of ar bovirus antibody in mouse ascitic fluid. Am. J. Trop. Med. Hyg., 16: 339—347. 13. Tikasingh, E. S. , Aitken, T. H. G. , Worth, C. B., Spence, L., and Mongul, F. E., 1965. An out break of eastern equine encephalomyehitis on the Courantyne Coast of British Guiana. West In dian Med. J., 14: 158—166. 14. Ordonez, J. V. , Scherer, W. F. , and Dickerman, R. W. , 1976. Studies of possible movement of Venezuelan encephalitis virus from an enzootic focus in Guatemala during 1971—1974. Am. J. Trop. Med. Hyg., 25: 163—172. IDENTIFICATION 15. Calisher, C. H. , La.zuick, J. S. , Justines, OF ARBOVIRUSES G. , Fran cy, D. B., Monath, T. P., Gutierrez V., E., Sa battini, M. 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