Molecular Epizootiology and Evolution of Vesicular Stomatitis

Transcription

1 JOURNAL OF VROLOGY, Apr. 1987, p X/87/4129-8$2./ Copyright 1987, Americn Society for Microbiology Vol. 61, No. 4 Moleculr Epizootiology nd Evolution of Vesiculr Stomtitis Virus New Jersey STUART T. NCHOL Cell nd Moleculr Biology Progrm, School of Veterinry Medicine, nd Deprtment of Microbiology, School of Medicine, University of Nevd, Reno, Nevd Received 9 October 1986/Accepted 3 December 1986 Vesiculr stomtitis virus (VSV) hs been shown previously to be cpble of undergoing rpid muttionl chnge during sequentil experimentl infections in vrious tissue culture cell systems (J. Hollnd, K. Spindler, F. Horodyski, E. Grbu, S. Nichol, nd S. Vndepol, Science 215: , 1982). The present study ws undertken to determine the degree of genetic diversity nd evolution of the virus under nturl infection conditions nd to gin insight into the epizootiology of the disese. Between 1982 nd 1985, numerous outbreks of VSV of the New Jersey serotype were reported throughout regions of the United Sttes nd Mexico. A Ti RNse fingerprint nlysis ws performed on the RNA genomes of 43 virus isoltes from res of epizootic nd enzootic virus ctivity. This indictes tht virus popultions were geneticlly reltively homogeneous within successive U.S. virus epizootics. The dt included virus isoltes from different epizootic stges, geogrphicl loctions, host nimls, nd host lesion sites. n contrst, only distnt genome RNA Ti fingerprint similrities were observed mong viruses of the different U.S. epizootics. However, Mexicn viruses isolted before or concurrent with U.S. epizootics hd very similr RNA genome fingerprints, suggesting tht Mexico my hve been the possible origin of virus inititing recent U.S. VSV New Jersey outbreks. Comprison of Ti fingerprints of viruses within enzootic disese res reveled greter extent of virus genetic diversity in these res reltive to tht observed in epizootic res. The evolutionry significnce of these findings nd their reltionship to experimentl dt on VSV evolution re discussed. The vesiculr stomtitis viruses (VSV) re members of the fmily Rhbdoviride nd re divided into two mjor serotypes, ndin (N) nd New Jersey (NJ) (8). VSV N hs been extensively studied t the moleculr level; however, VSV NJ is the more importnt cuse of disese, being responsible for periodic nd unpredictble mjor disese epizootics in cttle, horses, nd swine throughout the Americs (9, 1, 15). Historiclly, VSV NJ epizootics in the United Sttes hve ppered t pproximte 1-yer intervls, virus ctivity first ppering in erly summer nd then disppering with the first frost in erly winter. Extensive enzootic virus ctivity lso exists in regions of Mexico, Centrl Americ, nd South Americ (15). n these res, virus ctivity persists, with regulr ppernce of the disese in cttle popultions. n ddition, nnul identifiction in the southestern United Sttes of VSV NJ-seropositive wildlife, prticulrly ferl swine, hs indicted tht limited enzootic virus ctivity lso exists in this region (6, 11, 24, 25). The nturl virus reservoir, the mechnism of mintennce of virus in enzootic regions, nd the mode of virus trnsmission re still unidentified. Possible insect involvement in the trnsmission nd mintennce of the disese hs been suggested bsed on the typicl sesonl nture of VSV NJ epizootics; numerous VSV isoltions from insects (7, 14, 22, 27); demonstrtion of experimentl trnsmission of VSV N by horseflies, mosquitos, nd sndflies (5, 28); nd trnsovril trnsmission of VSV N in sndflies (28). However, viremis sufficiently high for virus trnsmission hve not been demonstrted in VSV-infected nimls. n ddition, the spred of recent mjor epizootic which occurred in the western United Sttes from 1982 to 1983 continued throughout the winter months in the pprent bsence of insect vectors (16). Recent VSV outbreks hve lso been unusul 129 in their frequency. Unlike the historic pproximte 1-yer cycles of VSV ctivity, fter the lrge 1982 to 1983 western U.S. epizootic in cttle nd horses, virus ctivity ws confirmed in cttle in Texs in 1984 nd nother epizootic of cttle nd horses occurred in, New Mexico, nd Arizon in Epizootic nd enzootic virus ctivity lso occurred in vrious prts of Mexico during the sme time period. A number of possibilities exist s to the origin of the virus inititing these recent VSV NJ outbreks in the United Sttes. These include synchronous eruption of preexisting persistent or inpprent infections, initition by virus preexisting in the U.S. wildlife popultions of epizootic or enzootic res, nd introduction of the virus from outside the United Sttes. Considerble informtion is vilble concerning the moleculr evolution of VSV N under vrious conditions of tissue culture cell infections. t hs been demonstrted tht under conditions of seril undiluted virus pssge nd virus persistent infection, rpid ccumultion of virus RNA genome muttions cn occur (12, 13, 18, 2, 23, 29-31). However, seril pssge of the virus t low multiplicity of infection leds to the ccumultion of very few, if ny, genome muttions (23). The bility of the virus to evolve rpidly under certin tissue culture conditions my be owing to the inherent very high error rte of the virus RNA polymerse (26). An extensive genetic study of VSV field isoltes from vrious VSV NJ outbreks nd stges of epizootics from 1982 to 1985 ws undertken to clrify the epizootiology of VSV NJ nd to compre the virus RNA genome evolution under conditions of nturl infection with tht observed experimentlly.

2 13 NCHOL J. VROL. TABLE 1. VSV NJ isoltes nlyzed by Ti fingerprinting soltion dte Origin Host or species Host lesion site Nomenclture USA /6/82 8/13/82 8/14/82 8/3/82 9/8/82 9/9/82 9/1/82 9/16/82 9/2/82 3/22/83 5/27/83 11/2/83 7/27/83 2/13/84 2/13/84 6/28/85 6/29/85 7/3/85 7/19/85 7/26/85 7/27/85 7/28/85 7/3/85 8/4/85 8/7/85 9/4/85 Mexico 11/3/82 11/29/82 2/17/83 11/22/83 1/18/84 1/26/84 1/31/84 6/22/84 7/29/84 8/19/84 8/27/84 9/28/84 11/15/84 1/15/85 1/16/85 5/18/85 6/6/85 Ogden, Uth Hzelhurst, G. Wyoming dho dho Montn dho New Mexico Nebrsk Cliforni Ossbw slnd, G. Texs Texs New Mexico New Mexico Arizon New Mexico Vercruz Vercruz Vercruz Michocn Michocn Sonor Sonor Oxc Hidlgo Nuevo Lredo Morelos Sonor Lesion site is indicted in cses for which it is known. Porcine Culicoides Blck fly House fly Porcine (ferl) Porcine Porcine Tongue Tet Snout Tet Tet Tongue Snout Udder Tongue Tongue Udder?/49-UT-B?/52-GA-P 8/82-CO-B 8/82-WY-B 8/82-CO-C 8/82-D-B 9/82-D-Bl 9/82-CO-BF 9/82-MT-E 9/82-CO-HF 9/82-D-B2 3/83-NM-B 5/83-NE-B 11/83-CA-B 7/83-GA-P 2/84-TX-Bl 2/84-TX-B2 6/85-NM-E 6/85-NM-B 7/85-AZ-E 7/85-NM-B 7/85-CO-Bl 7/85-CO-El 7/85-CO-E2 7/85-CO-B2 8/85-CO-E 8/85-CO-B 9/85-CO-E 11/82-VC-B1 11/82-VC-B2 2/83-VC-B 11/83-MH-B 1/84-MH-B 1/84-SN-P1 1/84-SN-P2 6/84-CH-B 7/84-OA-B 8/84-CH-B1 8/84-CH-B2 9/84-CH-B 11/84-HD-B 1/85-NL-B 1/85-MR-B 5/85-SN-B 6/85-CH-B MATERALS AND METHODS Virus isoltes. VSV NJ field isoltes from the United Sttes were provided by the U.S. Deprtment of Agriculture Ntionl Veterinry Services Lbortory, Ames, ow. nsect VSV NJ isoltes were provided by the Centers for Disese Control, Fort Collins, Colo., nd the U.S. Deprtment of Agriculture Arthropod-Borne Animl Disese Reserch Lbortory, Lrmie, Wyo. Mexicn VSV NJ field isoltes were provided by the Mexico-U.S. Commission for the Prevention of Foot nd Mouth Disese, Mexico City, Mexico. The viruses were identified s VSV NJ by tissue complement fixtion nd the serum neutrliztion test before their disptch from these lbortories. Viruses were nlyzed fter minimum number of low-multiplicity virus pssges in tissue culture. n most cses, this involved two or three virus pssges from the field mteril, with no virus plque purifiction methods used. Virus growth nd Ti RNse fingerprinting technique. Viruses were grown t 37 C in BHK-21 cells in miniml essentil Egle medium contining 32P-lbeled inorgnic phosphte (8,uCi/ml). Virus ws hrvested nd purified, nd RNA ws extrcted s described previously (17). The Ti fingerprinting technique used ws modifiction of erlier techniques (4). Purified 32P-lbeled virus RNA (contining 1,ug of crrier trna) ws thoroughly suspended in 8,ul of digest buffer (8.4 M ure, 12.5 mm sodium citrte [ph 5], 1.25 mm EDTA,.5% bromophenol blue,.5% xylene cynol), boiled 1 min, nd rpidly cooled on wet ice; 2,ul of Ti RNse ws dded, nd the mixture ws incubted for 15 min t 5 C. The Ti-digested RNA ws loded directly onto.8-mm stndrd first-dimension gel nd electrophoresed t

3 VOL. VOL. 61, 61, ~~~MOLECULAR EPZOOTOLOGY AND EVOLUTON OF VSV NJ 131 TABLE 2. Ctlog of Ti spot differences mong VSV field isoltes T Spotb Viros 1813sol91 2t52 7e o % )C 8/82-VT /82-D /82-D-U /82-CO-NP /82-CO * /83-NE /82-CO-C /82-CO-F /82-1D-B /82-M-Kf /83-NM-B /83-CA /86-TX /86-TX /85-NM /85-NM-E ~ + 7/85-AZ-E /85-NM /85-CO-B /85-CO-K /85-CO-E /85-CO /85-CO-K /85-CO /85-CO-E /85-sN /85-Cl /82-VC /86-MNf /86-Cl /86-NO /86-Cl /86-Cl-B /86-CN /83-M /86-N-l /84-SN-P /85-L /85-MN See Tble 1 for nomenclture. bsee Fig. 1A. Only spots in which differences were seen re listed. +,Presence of spot. pproximtely 25 V for 18 h. The gel trck ws then excised nd trnsferred to.4-mm stndrd second-dimension gel nd electrophoresed t 475 V for 18 h. The reproducibility of ech virus RNA fingerprint ws checked t lest once (usully twice) for ech virus isolte. The comprison nd ctloging of Ti fingerprint spot differences ws ided by the use of n BM PC-XT microcomputer. RESULTS Ti RNse fingerprinting nlysis ws crried out on 45 VSV NJ isoltes from the United Sttes nd Mexico (Tble 1). The isoltes included two commonly studied stndrd virus lb strins, Ogden nd Hzelhurst, representing the two previously defined subtypes of VSV NJ (19). A totl of 43 were ctul field isoltes from the period 1982 to U.S. isoltes consisted of 12 virus isoltes from the 1982 to 1983 VSV epizootic in the western United Sttes, including 3 insect isoltes, one from Culicoides veriipennis midges, nother from Simulium bivttum blck flies, nd the other from Musc domestic house flies; 1983 isolte from ferl swine on Ossbw slnd, G., region of pprent enzootic virus ctivity (6); 2 isoltes from 1984 VSV outbrek in Texs; nd 11 isoltes from the 1985 VSV epizootic in Arizon, New Mexico, nd. Mexicn isoltes included 17 viruses from vrious epizootic nd enzootic disese regions from 1982 through These virus RNA fingerprints were compred, nd spot differences nd similrities were indexed nd correlted (Tble 2 nd Fig. 1A). Tble 3 shows the number of Ti spot differences between virus isoltes. The exct number of Ti spot differences is not given for viruses with genome RNA fingerprints demonstrting little similrity (more thn 3 spot differences) to the other viruses nlyzed. Comprison of RNA fingerprints of virus isoltes from the mjor VSV NJ epizootic of the western United Sttes in 1982 to 1983 (Tble 3) indictes high degree of genetic similrity mong the isoltes (e.g., Fig. ib). The results re represented digrmmticlly (Fig. 2). Four isoltes hd identicl fingerprints, nd mximum of four spot differences ws observed between closest pirings of the other viruses of this epizootic. The viruses from this epizootic included isoltes from cttle, horses, nd insects, demonstrting tht within this epizootic the viruses were reltively geneticlly homogeneous, despite the fct tht viruses were isolted from res of considerble geogrphicl seprtion over 15- month period. This group of isoltes hd completely different fingerprints from those of historic VSV NJ isoltes, Ogden nd Hzelhurst, which represent the two existing subtypes of the VSV NJ serotype (Fig. le nd J). Previous work hs demonstrted tht seril dilute pssge of VSV nd the pssge of VSV strins for mny yers in different lbortories led to very few Ti spot chnges in the virus fingerprint (3, 23). Thus, it is unlikely tht the differences in Ti fingerprints observed between the historic nd recent VSV isoltes re due to the long period of mintennce of the historic isoltes in tissue culture. n ddition, the VSV NJ isolte from the region of enzootic virus ctivity in ferl swine on Ossbw slnd, G., lso hd completely different fingerprint (Fig. if). However, 1982 isolte from Vercruz, Mexico, ws only 12 spots different from the min group of U.S to 1983 VSV NJ isoltes (Fig. ig nd Fig. 2). The reltionships mong the two 1984 VSV NJ Texs isoltes, representtive isolte of the preceding U.S. outbrek, nd the 1984 Mexicn isoltes re illustrted in Fig. 3. Clerly, the Texs isoltes re closely relted to ech other nd to the 1984 Mexicn isoltes. The bovine isolte from Michocn (1/84-MH-B) hd the closest reltionship to the Texs isoltes, being only two Ti spots different, nd ws isolted 1 month before the ppernce of the disese in the United Sttes. A considerbly more distnt reltionship (t lest 2 spots different) is evident between the 1984 Texs virus isoltes nd isoltes from the preceding VSV NJ U.S. outbrek between 1982 nd 1983 (Fig. lb nd C).

4 132 NCHOL J. VROL. w i i. ; 1 * v 1 t is # if c C) C) bi.y b#. "' pi C A 4-' C/D L. r g (W N ' ) O F- c 6 9,. # e A 4, % UJ r~~~~~' w mt# i.t 4 4 * A!,. P't,~ ': il ) r-_ m x co C m * # co' * S., in@ * * %., :, Al *9 * up{,it. *4 9 4 to 4- C. l) * * ttv _ >5 CM s 4- *> rc Cd w+sx~o > m ȱ 4-9 *p ) ~~~.s r Q 6 4*~~~ 'S t _- L,se S,i ; 1 4 & O _ * 6D %.4 *g C_, -4= co 1%4 LL

5 VOL. 61, 1987 MOLECULAR EPZOOTOLOGY AND EVOLUTON OF VSV NJ 133?/49-UT-B? /52-GA-P 8/82-WY-B 8/82-D-B 9/82-D-Bl 9/82-CO-HF 8/82-CO-B 5/83-NE-B 8/82-CO-C 9/82-CO-BF 9/82-D-B2 9/82-MT-E 3/83-NM-B 11/83-CA-B 7/83-GA-P 2/84-TX-B 2/84-TX-B2 6/85-NM-B 6/85-NM-E 7/85-AZ-E 7/85-NM-B 7/85-CO-Bl 7/85-CO-El 7/85-CO-E2 7/85-CO-B2 8/85-CO-E 8/85-CO-B 9/85-CO-E 5/85-SN-B 6/85-CH-B 1 1/82-VC-B1 1/84-NH-B 9/84-CH-B 11/84-ND-B 6/84-CH-B 8/84-CH-B1 8/84-CE-B2 11/83-MH-B 1/84-SNiPl 1 /84-SN-P2 1/85-NL-B 1 /85-Ml-B 1 1/82-VC-B2 2/83-VC-B 7/84-OA-B TABLE 3. > > > > > > > > > > > > >3 333 > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > Number of Ti spot differences mong RNA genome fingerprints of VSV isoltes > > 1 > 11 1 > 11 1 > > > > > > > > > > > > > > > > > > > > > > > s e > e < < u n e u G u N e w > > > > > > > > > O > > > > > > > > > 6 > > > > > > > > > > > O b-b e. co O-4 so oo U U h 1- D 1 UvE C D\ CO-fz no j _ - >, More thn 3 spot differences. The reltionships between VSV NJ isoltes from the 1985 epizootic in, New Mexico, nd Arizon re represented in Fig. 4. All these isoltes were found to hve identicl or extremely similr Ti fingerprints (e.g., Fig. 1D), despite the fct tht the viruses were isolted from both cttle nd horses. Comprison of the Ti fingerprints of these B..s P: > i- <-,,, - _. viruses with those isolted from this region during the 1982 to 1983 U.S. epizootic indictes distnt reltionship, the closest member being 22 spots different. n contrst, 1985 Mexicn isoltes hd very similr Ti fingerprints (Fig. 4). Two isoltes hd fingerprints identicl to the 1985 U.S. isoltes, including one, 5/85-SN-B, which ws isolted 1 4 9/82-CO-BF 3/ 83-NM-B 3 4 8/82-CO-C /82-WY-B 9/82-CO-HF 3 8/82-CO-B 8/82-D-B 9/82-D-S 5/83-NE-B 9182-D-B2 //2-MT-E 11/83-CA-B?/52-GA-S?/49-UT-B 7/83-GA-P (HAZELHURST) (OGDEN) (OSSABAW) FG. 2. Digrmmtic representtion of the reltionships of the Ti fingerprints of viruses of the 1982 to 1983 western U.S. VSV epizootic. Solid lines indicte close similrity. Numbers indicte the number of Ti spot differences mong isoltes.

6 134 NCHOL 1 1/84-HD-B 8/82-D-B 1 2 1/84-MH-B 2n 2/84-TX-B2 = 2/84-TX-B 1 8/84-CH-B 1 f 8/84-CH-B2 FG. 3. Digrmmtic representtion of the reltionships of the Ti fingerprints of viruses of the 1984 Texs VSV outbrek. month before the ppernce of the disese in the United Sttes (Fig. 4). Anlysis of Ti fingerprints of VSV NJ isoltes from epizootic nd enzootic regions of Mexico between 1982 nd 1985 revels different pttern (Fig. 5). Two viruses (11/82- VC-B1 nd 11/82-VC-B2) isolted the sme month from the stte of Vercruz (n enzootic region) hd very different Ti fingerprints (Fig. 1G nd H). An isolte (7/84-OA-B) from Oxc, nother enzootic region, lso hd Ti fingerprint with mny spot differences reltive to other VSV NJ isoltes (Fig. 1). n contrst, other isoltes hd clerly closely relted Ti fingerprints. For instnce, 11/82-VC-B2 nd 2/83- VC-B hd very similr fingerprints, nd the mjority of the other Mexicn isoltes were closely similr to one nother (Fig. 5). Agin, this ws evident even for isoltes from different host species, in this cse, cttle nd swine. DSCUSSON n nlyzing the 1982 to 1983, 1984, nd 1985 U.S. VSV NJ outbreks, common pttern emerges in which within given outbrek the viruses were ll closely relted, hving identicl or very similr Ti fingerprints. This pttern ws observed even for viruses isolted from different epizootic stges; seprte geogrphicl loctions; different host species, including cttle, horses, nd insects; nd from different J. VROL. host lesion sites. This suggests tht within given epizootic the virus popultion is geneticlly reltively homogeneous. No evidence ws found to suggest tht very geneticlly distinct viruses were involved in the infection of nimls nd insects or in different host nimls or lesion sites. n contrst to the very close Ti fingerprint reltionships observed mong isoltes within U.S. epizootics, only distnt reltionships were observed mong viruses from the three different outbreks nlyzed. These dt provide n indiction of the possible origin of virus inititing these VSV NJ outbreks. t is highly unlikely tht these U.S. epizootics rose owing to the synchronous eruption of preexisting persistent infections in these res. t hs been demonstrted erlier tht VSV undergoes rpid nd rndom evolution during experimentl persistent infections (12, 13). Synchronous eruption of persistent infections preexisting in ech ffected re would not led to such homogeneous virus popultions s observed here for ech virus outbrek. n ddition, the completely dissirnilr Ti fingerprint of the virus isolte from ferl swine on Ossbw slnd, G. (the only well-documented region of enzootic virus ctivity in the United Sttes), provided no evidence to support its being the source of virus inititing the 1982 to 1983 VSV NJ epizootic in the western United Sttes. n contrst, for ech U.S. VSV NJ outbrek, close reltionship to erlier or concurrent Mexicn virus isoltes cn be demonstrted. These dt suggest tht Mexico my be the source of virus inititing recent VSV NJ outbreks in the United Sttes. This is supported by epidemiologicl investigtions indicting northwrds spred of virus ctivity during the erly stges of recent VSV NJ epizootics (2, 16). However, it is still uncler how the virus is trnsmitted during n epizootic. The isoltion of virus from insects (during the 1982 to 1983 U.S. epizootic) which hd few, if ny, Ti fingerprint differences from cttle nd horse virus isoltes demonstrtes tht insects were involved in the epizootic but does not indicte their exct role in virus trnsmission. Clerly, other fctors must lso ply role. Direct-contct trnsmission hs been suggested s the mens of trnsmission, t lest in the bsence of insects during winter months (16). Movement of infected cttle my lso ply role. The proposed involvement of cttle dispersl sle in dho in the continued spred of VSV during the 1982 to 1983 U.S. epizootic (16) correltes well with Ti fingerprint reltionships of viruses from this epizootic (Fig. 2). Comprison of Ti fingerprints of the RNA genomes of VSV NJ isoltes from enzootic nd epizootic regions indi- 8/82-CO-C FG. 4. 7/85-AZ-E 2 2 7/8 5-NM-B 7/85-CO-B 1 6-7/5-UO-E2 1 7 _OD^_ 1 6/8 5-~N M-B 2 7/85-CO-E2 8 6/85-NM-E-7/85-CO-B2 i2/84-tx-b2 8/85-CO-E 8/85-CO-B 9/85-CO-E 5/85-SN-B 6/85-CH-B Digrmmtic representtion of the reltionships of the Ti fingerprints of viruses of the 1985 U.S. VSV epizootic.

7 VOL. 61, 1987 MOLECULAR EPZOOTlOLOGY AND EVOLtJTON OF VSV NJ /82-VC-B2 1/182-VC-B1 12 2/83-VC-B 7/84-OA-B 7 8/84-CH-B 1 1/83-MH-B 8/84-CH-81 6/ 5 1/84-MH-B 1/84-SN-P1 7 1/84-SN-P2 9/84-CH-B 4 1/84-HD-B _5/85-SN-B 6/85-CH-B ~ ~ ~ ~ ~ /8-C-B 8/4CHB 1/85-NL-B ii 1 /85-MR-B FG. 5. Digrmmtic representtion of the reltionships of the Ti fingerprints of VSV isoltes fronm Mexico from 1982 to ctes tht much greter genetic diversity exists mong virus isoltes from enzootic res. t ppers tht, unlike epizootic regions, enzootic regions cn exhibit more thn one distinct type of VSV NJ. Preliminry fingerprint nlysis of VSV NJ isoltes from enzootic regions of Centrl Americ confirms this observtion; these isoltes form severl different groups on the bsis of their fingerprints nd show little similrity to the U.S. nd Mexicn isoltes (unpublished observtion). t is currently uncler how the virus is mintined in enzootic regions. The possible involvement of persistent infections in the mintennce of the virus in enzootic regions would be consistent with the dt. Since VSV RNA genomes quickly ccumulte muttions during experimentl persistent infections, this type of infection process could explin the extensive virus genetic diversity observed in res of enzootic virus ctivity. The dt presented here identify t lest six distinct VSV NJ subgroups bsed on virus RNA genome Ti fingerprints. Two re the currently defined groups represented here by the Ogden nd Hzelhurst VSV NJ isoltes. The other four include the min group of U.S. nd Mexicn isoltes which hve fingerprints vrying from identicl to more distntly relted: the Ossbw slnd, G., isolte, 7183-GA-P; the Vercruz isoltes 11/82-VC-B2 nd 2/83-VC-B; nd the Oxc isolte, 7/84-OA-B. Virus RNA genomes which shre less thn 25% of their Ti oligonucleotides by comprison of their Ti fingerprints re pproximtely less thn 9% homologous (1). Consequently, it is not possible to determine the exct reltionship of these more distntly relted VSV NJ subgroups bsed on their Ti fingerprints. Previous hybridiztion studies hve demonstrted tht the western U.S. isolte 9/82-CO-BF is more closely relted to the Hzelhurst subgroup thn to the Ogden subgroup (21). This result would plce the mjority of the U.S. nd Mexicn VSV NJ isoltes studied here in the Hzelhurst subgroup nd would extend the host rnge of tht group to include cttle, house flies, nd culicoides midges in ddition to swine, horses, nd blck flies. Whether the other three fingerprint groups defined here represent new subgroups or more distntly relted members of the existing two subgroups remins to be determined. Although the degree of virus genetic diversity in enzootic regions ppers to be greter thn tht in epizootic regions, isoltes within n epizootic re not ll identicl. The bility of VSV to undergo rpid evolution, quickly ccumulting genome muttions under vrious experimentl conditions (12, 13, 18, 2, 23, 29-31), nd the inherent high error rte of VSV RNA polymerse (26) suggest tht virus genome vrition within VSV NJ epizootic isoltes represents the reltively rpid nturl evolution of the virus during the spred of the disese. RNA sequencing nd monoclonl ntibody rectivity studies re under wy to determine more precisely the genomic nd ntigenic vrition of these nturlly occurring virus vrints. ACKNOWLEDGMENTS thnk Ed Crbrey, Gene Erickson, nd Mike Snyder of the U.S. Deprtment of Agriculture Ntionl Veterinry Services Lbortory, Ames, ow, nd John Mson, Jun Gy Gutierrez, nd Willim Schroeder of the Mexico-U.S. Commission for the Prevention of Foot nd Mouth Disese, Mexico City, Mexico, for ssistnce in providing VSV field isoltes for this study. nsect isoltes were kindly provided by Bruce Frncy of the Centers for Disese Control Division of Vector-Borne Virl Diseses, Fort Collins, Colo., nd Tom Wlton of the U.S. Deprtment of Agriculture Arthropod-Borne Animl Diseses Reserch Lbortory, Lrmie, Wyo. thnk Bob Tesh of the Yle Arbovirus Reserch Unit for providing the Hzelhurst VSV NJ isolte nd for helpful comments. Excellent technicl ssistnce ws provided by Jon Rowe, Terri Willims, nd Sonny Rosenberg. Finlly, thnk John Hollnd for suggesting this project nd for invluble encourgement, support, nd insightful comments. This work ws supported by U.S. Deprtment of Agriculture Biotechnology competitive reserch grnt no. 85-CRCR LTERATURE CTFD 1. Aronson, R. P., J. F. Young, nd P. Plese Oligonucleotide mpping: evlution of its sensitivity by computer-

8 136 NCHOL simultion. Nucleic Acids Res. 1: Crbrey, E. A Vesiculr stomtitis in the United Sttes , epizootic or enzootic? p n J. Mson (ed.), Proceedings of n nterntionl Conference on Vesiculr Stomtitis, Mexico City. Mexico-U.S. Commission for the Prevention of Foot nd Mouth Disese, Mexico City. 3. Clewley, J. P., D. H. L. Bishop, C.-Y. Kng, J. Coffin, W. M. Schnitzlein, M. E. Reichmnn, nd R. E. Shope Oligonucleotide fingerprints of RNA species obtined from rhbdoviruses belonging to the vesiculr stomtitis virus subgroup. J. Virol. 23: De Wtcher, R., nd W. Fiers Preprtive twodimensionl polycrylmide gel electrophoresis of 32P-lbeled RNA. Anl. Biochem. 49: Ferris, D., R. P. Hnson, R. J. Dicke, nd R. H. Robers Experimentl trnsmission of vesiculr stomtitis virus by dipter. J. nfect. Dis. 96: Fletcher, W. O., D. E. Stllknecht, nd E. W. Jenney Serologic surveillnce for vesiculr stomtitis virus on Ossbw slnd, Georgi. J. Wildl. 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