Molecular cloning and nucleotide sequence of the pestivirus bovine viral diarrhea virus

VIROLOGY

165, 191-199 (1988)

Molecular Cloning and Nucleotide Sequence of the Pestivirus Bovine Viral Diarrhea Virus MARC S . COLLETT,' RUBY LARSON, CONRAD GOLD, DAVID STRICK, DENNIS K . ANDERSON, ANn A . F . PURCHIO 2 Molecular Genetics, Inc., 10320 Bren Road East, Minnetonka, Minnesota 55343 Received December 8, 1987; accepted February 1, 1988 The RNA genome of the cytopathic NADL isolate of bovine viral diarrhea virus (BVDV) has been molecularly cloned and the nucleotide sequence determined . The cloned sequence was 12,573 nucleotides in length, corresponding to a molecular weight of 4.3 X 106 , having a base composition of 32 .2% A, 25 .7% G, 22 .1 % U, and 20 .0% C . However, the sequences at the 5' and 3' termini of the RNA have not been unequivocally established . A single major open reading frame extending the length of the molecule was found in the viral-sense (positive polarity) sequence . This open reading frame was capable of encoding 3988 amino acids, representing 449 kDa of protein . 1J 1988 Academic Prose, inc.

INTRODUCTION

1987) . Together, the results from all these studies have created a rather unclear picture of the precise molecular features of BVDV . Similarly unclear is the taxonomic classification of BVDV . BVDV, a member of the Pestivirus genus, is currently grouped in the Togaviridae family (Westaway et al., 1985a) . However, based on certain characteristics of the BVDV-specific RNA found in virus-infected cells, we previously pointed out significant dissimilarities with togaviruses and, more important, similarities with flaviviruses (Purchio et al., 1983) . Flavivirus now constitutes a genus within the recently established family Flaviviridae (Westaway et al ., 1985b) . Establishing the correct taxonomic classification of BVDV, although significant in itself, may have far greater import on our overall view of the etiologic agent and its pathogenic mechanisms . We have been interested in developing rational strategies for the control and prevention of BVDV infection and the resultant diseases associated with BVDV . Our approach has been to first gain a clear understanding of the molecular, biochemical, and immunologic features of BVDV . Toward this end, we have undertaken work to precisely define the genetic material of this virus and the gene products encoded therein . Here, we describe the molecular cloning, and present the nucleotide sequence, of the cytopathic NADL isolate of BVDV . In the following communication (Collett etat, 1988), we report on our progress toward defining the viral polypeptide products encoded by the BVDV genome .

Bovine viral diarrhea virus (BVDV) is an economically important pathogen of cattle throughout the world, causing a wide range of clinical syndromes (Brownlie, 1986 ; Baker, 1987) . Infection during pregnancy can result in abortion, teratogenic defects, or birth of persistently infected animals . Persistently infected animals are viremic with a ''noncytopathic" biotype of BVDV, to which they are immunologically tolerant, Such animals serve as reservoirs for the maintenance and spread of BVDV within a herd . Upon superinfection with an antigenically closely related ''cytopathic'' biotype BVDV, these animals succumb to the generally fatal mucosal disease (Brownlie et al., 1984 ; Bolin et al., 1 985a,b) . BVDV also contributes to a variety of illnesses in adult cattle, from subclinical or mild disease to involvement in bovine respiratory disease (BRD) complex (Potgieter et at, 1 984a,b) . Greater understanding of the complicated pathogenesis of this agent is necessary . To date, the molecular characterization of BVDV has been limited . This may reflect, in part, the numerous difficulties associated with the appropriate cell culture propagation of this virus . The genome of BVDV has been shown to be an infectious, positive-strand RNA (Diderholm and Dinter, 1966), variously estimated at 2 .9 to 4 .4 x 106 Da in size (Pritchett et at, 1975 ; Purchio et al., 1983 ; Renard et al., 1985) . Numerous reports on the number and size of BVDV-specific polypeptides have appeared (Pritchett and Zee, 1975 ; Matthaeus, 1979 ; Coria at al., 1983 ; Purchio et at, 1984 ; Donis and Dubovi, 1987a,b,c ; Pocock et at,

MATERIALS AND METHODS Virus and cells The National Animal Disease Laboratory (NADL) isolate of BVDV was three-times plaque purified and

1 To whom requests for reprints should be addressed . 2 Present address : Oncogen, Seattle, WA 98121 . 191

0042-6822/88 $3 .00 Copyright ® 1988 by Academic Press, Inc . All rights o reproduction in any form reserved .



1 92

COLLETT ET AL .

passaged twice at low multiplicity (m .o .i . of 0 .1) in Madin-Darby bovine kidney (MDBK) cells to establish virus stock . This virus was cytopathic to cells in culture and caused a mild fever and leukopenia when administered to colostrum-deprived calves . The MDBK cell line was obtained from the National Veterinary Services Laboratory (NVSL, Ames, )A) . These cells were demonstrated to be free of BVDV by immunofluorescent, radioimmunoprecipitation, and nucleic acid analyses . Cells were cultured in Dulbecco's modified Eagle's medium containing 5% horse serum . Cultures were periodically checked by the above assays to ensure and reaffirm their BVDV-free nature . Preparation of viral RNA Cells infected at an m .o .i, of 0 .2 were harvested at 40-48 hr postinfection, washed in STE (0 .15 M NaCl, 0 .02 M Tris-HCI, pH 7 .2, 1 mM EDTA), and resuspended in STE containing 0 .1% sodium dodecyl sulfate (SDS) . This suspension was extracted with STEsaturated phenol, and the aqueous phase was reextracted two times with phenol and once with chloroform . After ethanol precipitations, the RNA was treated with DNase as previously described (Purchio et al., 1983) . This "total" cytoplasmic RNA was then adjusted to 1 mg/ml in 10 mM EDTA, 1% SDS, 2 M LiCl 2 . After 16 hr at -20°, the RNA was centrifuged at 11 ,000 g for 30 min at 4° . The LiCl 2 supernatant fraction RNA was then ethanol precipitated . BVDV RNA was further purified by gel electrophoresis . LiCl 2 supernatant RNA in 10 mM Tris-HCI, pH 7 .2, 1 mM EDTA was heated at 65° for 5 min prior to loading onto a preparative 0 .7% low-melting-point agarose gel (Maniatis etal., 1982) . After electrophoresis, the BVDV RNA was localized by ethidium bromide fluorescence, excised, and extracted from the molten agarose gel pieces by phenol extraction (Maniatis at al., 1982) . Electrophoresis of RNA in methylmercuric hydroxide gels was performed as described (Maniatis et al., 1982) . Molecular size standards for RNA included an RNA ladder (Bethesda Research Laboratories, Gaithersburg, MD) and RNA extracted from purified Sindbis virus (provided by J . M . Dalrymple, Frederick, MD) . Molecular cloning and nucleotide sequencing of the BVDV genome LiCl 2 supernatant RNA (60 gg total RNA, approximately 10 µg BVDV RNA) in 50,ul of water was boiled for 90 sec, quenched on ice, and adjusted to firststrand reaction buffer (Collett et al., 1985) in a final volume of 100 Al . Reverse transcriptase (35 units ; Life Sciences, Inc ., St . Petersburg, FL) was added, and after 90 min at 37°, the reaction mixture was phenol

extracted . The aqueous phase was chromatographed on a Sepharose CL-6B column . The material in the void volume was pooled, ethanol precipitated, and then used for second-strand synthesis according to the procedures of Gubler and Hoffman (1983) . The duplex cDNA was inserted by dC-dG homopolymeric tailing (Maniatis at at, 1982) into the Pstl site of plasmid pUC9, and used to transform competent Escherichia coli K12 . E. colt strains used were HB101, RR1, and NF1829 (Maniatis et al., 1982 ; Weis et al., 1983) . Ampicillin-resistant transformants were screened for BVDV-specific inserts by colony hybridization (Grunstein and Hogness, 1975) with 32 P-labeled gel-purified BVDV genomic RNA . This probe was prepared as follows . Gel-purified BVDV RNA was fragmented with alkali (50 mM Na 2 CO 3 ; 65° for 10 min), neutralized, and then labeled with [-y 32 P]ATP and polynucleotide kinase, Plasmids from hybridization-positive bacteria were isolated and further characterized by restriction enzyme mapping and by cross-hybridization . Their BVDV-specific nature was established from the results of Northern format hybridization to uninfected and BVDV-infected cellular RNA (data not shown), The sequence of the cDNA inserts was determined using the methods of Maxam and Gilbert (1980) . All sequence was obtained from both strands . RESULTS Genomic RNA of BVDV Several reports have appeared that describe viralspecific RNA in BVDV-infected cells (Pritchett at al., 1975 ; Purchio etal., 1983 ; Renard etal., 1985) . By gel electrophoresis, only a single, high-molecular-weight RNA band was observed ; no subgenomic virus-specific RNA species have ever been detected (Purchio et al., 1983 ; Renard at al., 1985) . This RNA lacked a poly(A) stretch and had certain characteristics that suggested it possessed an unusually high degree of secondary structure ; yet it could be distinguished from classical replicative form (RF) RNA (Purchio et al ., 1983) . This single BVDV-specific RNA band, however, may yet be composed of unresolvable genomic RNA and some form of replicative-intermediate RNA . We previously estimated the size of the BVDV-specific RNA band to be approximately 8 .2 kilobases (kb) (Purchio at al., 1983) . We have reevaluated the size of this RNA using appropriate molecular length standards and have found this measurement to be in error . As shown in Fig . 1, the BVDV RNA migrated in a methylmercuric hydroxide agarose gel as a single band just slightly slower than the Sindbis virus RNA standard . Given the low resolution of RNAs in this size range, we could only estimate the BVDV RNA to be on

1 93

CLONING AND SEQUENCE OF BOVINE VIRAL DIARRHEA VIRUS

the order of 12-13 kb in length . This measurement is in agreement with that reported by Renard et al. (1985) for the Osloss isolate of BVDV . Similar analysis of the RNA of other isolates of BVDV, including both cytopathic and noncytopathic viruses, showed their genomes to be indistinguishable in electrophoretic mobility (data not shown) . As is obvious from Fig . 1, track 5, the BVDV RNA represents a very minor RNA species within virus-infected cells . Attempts to enrich for the viral RNA by purification of virions were unsuccessful, probably due to the close association of this virus with cellular material in infected cultures . Therefore, to obtain RNA for molecular cloning, we took advantage of the physical properties (secondary structural features) of the BVDV RNA . By adjusting preparations of total cytoplasmic RNA from BVDV-infected cells to 2 M LiCl 2 , in a simple precipitation step we were able to separate the BVDV RNA from all cellular RNA, except for several low-molecular-weight RNA species (Fig . 1, track 4) . A small portion (ca . 10%) of the BVDV RNA did appear in the LiCl 2 precipitate (not shown) . The nature of this RNA is currently under investigation . The BVDV RNA in the LiCl 2 supernatant fraction could be purified further by either sucrose gradient sedimentation (not shown) or gel electrophoresis (Fig . 1, track 3) .

1

2

3

4

5

6

FIG . 1 . Agarose gel analysis of BVDV RNA . Total cellular cytoplasmic RNA was extracted from BVDV-infected MDBK cells as described under Materials and Methods . After DNase treatment, the RNA was fractionated by LiCl 2 precipitation, and the LiC1 2 supernatant fraction was subjected to gel purification, all as described in the text. An aliquot of RNA from each step was electrophoresed in a CH 2 HgOH agerose gel in parallel with RNA markers of known size . The gel was stained with ethidium bromide and photographed under uv illumination . Track 1, RNA markers ; tracks 2 and 6, Sindbis virus RNA ; track 3, gel-purified BVDV RNA ; track 4, LiC1 2 supernatant RNA ; track 5, total cellular cytoplasmic RNA (DNase treated) . The numbers represent nucleotide length (in kilobases) . The arrow indicates the position of the BVDV RNA .

TABLE 1 BASE COMPOSITION OF

A G U C

BVDV

GENOMIC

RNA

RNA'

cDNA°

30 .8 23 .7 23 .1 22 .4

32 .2 25 .7 22 .1 20 .0

'As previously determined by direct compositional analysis of purified viral RNA (Purchio et at, 1983) . ° Determined from the nucleotide sequence of the molecularly cloned cDNAs .

Molecular cloning of BVDV RNA As BVDV RNA lacked a poly(A) tract, several approaches for priming reverse transcription were explored . We attempted to add a poly(A) tail to the 3' terminus of the BVDV RNA using E. coli poly(A) polymerase . We found, as did Renard et al . (1985), that poly(A) polymerase was unable to use the 3' end of BVDV RNA as a substrate, regardless of the reaction conditions tested . As expected, gel-purified BVDV RNA (Fig . 1, track 3) served as a poor substrate for reverse transcriptase, likely due to a lack of priming function . Addition of calf thymus primers (Taylor et al., 1976) improved cDNA synthesis of this RNA, but still reverse transcription was inefficient . The direct use of the LiCl 2 supernatant RNA fraction (Fig . 1, track 4), however, proved to be quite efficient for BVDV-specific cDNA synthesis . Presumably, the low-molecularweight cellular RNAs in this preparation served as competent random primers on the BVDV RNA . The observation that these RNAs were much more efficient at priming DNA synthesis than calf thymus DNA oligonucleotides may indicate a general utility of RNA random primers for preparation of cDNA libraries . The cDNA inserts of plasmids containing BVDV-specific DNA were aligned by cross-hybridization analyses and overlapping restriction site patterns . Multiple independent cDNA clones encompassing the entire genome were characterized . From initial DNA sequence data of several regions along the length of the aligned cDNAs, sets of complementary oligonucleotides (18 to 24 mers) were synthesized . These strand-specific oligonucleotides were radiolabeled, and then individually hybridized in a Northern format to BVDV RNA . The results of these hybridizations allowed establishment of the orientation of the aligned cDNA clones with respect to the viral RNA (data not shown) . The DNA sequence of the BVDV inserts was determined for both strands . Furthermore, approximately 78% of the genome was sequenced from at least two

1 94

COLLETT ET AL,

independent cDNA clones . This was done to address the potential for heterogeneity in the viral RNA population and to minimize the possibility of artifacts or mistakes introduced by the cloning procedures . We found several nucleotide differences in this process, most representing silent third base codon changes . However, in a few instances, the differences resulted in amino acid changes . In these cases, additional cDNA clones encompassing the region were analyzed to establish the "predominant" sequence . The nucleotide sequence of the BVDV genome, NADL isolate, is presented in Fig . 2 . The sequence is 12,573 nucleotides in length . This is in line with the estimated size of the BVDV RNA (Renard etal., 1985 ; this report) . However, we are at this time unable to state that this sequence represents the complete nucleotide sequence of the genome . Two clones encompassing the 5' end of the genome differed by several nucleotides at their termini . And, even though the terminal sequence of two cDNA clones from the Tend of the genome was identical, until we determine the sequence of the termini of the genomic RNA by direct RNA sequencing, the complete sequence will not be established . Features of the BVDV sequence Using purified viral RNA, we had previously determined the base composition of the BVDV genome (Purchio et al., 1983) . These values differ slightly from those calculated from the cloned cDNA sequence (Table 1) . The discrepancy may be due to contaminants in the RNA preparation used for the direct compositional analysis . In fact, the presence of a small amount of replicative intermediate form RNA in with the genomic RNA could be responsible for this difference . Figure 3 shows the distribution of translation termination codons within the sequence for all three phases in both RNA orientations . A single large open reading frame (ORF) extending nearly the entire length of the RNA in the positive polarity was found . There are scattered throughout the sequence several small ORFs with methionine codons appropriately positioned for potential translation initiation of small polypeptides . At this time, however, there is no reason to think these ORFs have any functional significance . Given the single large ORF, translation initiation would be expected to occur at the first methionine codon at the 5' end of the ORF . This initiation codon

occurs at nucleotide positions 386-388 . There are, however, upstream of this position, two methionine codons near the 5' ends of two small ORFs . One (ATG at nucleotides 131-133) is in the same phase as the large ORF and could potentially initiate translation of a 45 amino acid polypeptide . The second (ATG at nucleotides 249-251) is in a different phase from the large ORF and could yield a 48 amino acid product . Whether or not these small ORFs 5' of the large OFF are functional remains to be determined . From the methionine codon at nucleotides 386-388, the large OFF extends uninterrupted to nucleotide 12,349, when a TGA termination codon is encountered (nucleotides 12,350-12,352) . Thus, the BVDV large ORF consists of 3988 codons capable of encoding 449 kDa of protein . The amino acid translation of this ORF is provided in Fig . 2 . Further analyses of the protein coding sequences of this ORF and the features of its gene products are presented in the following report (Collett et al., 1988) . The 3' untranslated region continues for 223 nucleotides to the end of the cloned sequence at position 12,573 . As we have yet to determine the sequence of the 3' terminus of the BVDV genome by direct RNA sequencing, we do not know the actual length of the 3' untranslated segment . Inspection of the sequence within this region revealed interesting repeats . Shortly after the TGA termination codon, within a 60-base A-T-rich sequence, there was an identical 8-nucleotide repeat (TGTATATA ; underlined in Fig . 2) . This sequence does not appear elsewhere in the genome, but interestingly, the subsequence GTATA appeared in a two-times repeated 7-nucleotide sequence at the 5' end of the genome (underlined in Fig . 2) . A second, more extensive repeated sequence was noted in the 3' untranslated region . This involved two closely spaced 50-base stretches in which 30 nucleotides were identical, with the remainder representing transition changes . Whether or not these repeats have any functional significance in virus replication is unknown . DISCUSSION BVDV is an important disease agent of cattle, directly causing or significantly contributing to a wide range of clinical syndromes . The pathogenesis of disease by BVDV is complicated, and interactions between BVDV biotypes are poorly understood . A greater understanding of the virus itself is necessary

Fio . 2 . Nucleotide sequence of the genome of the NADL isolate of BVDV . The deduced amino acid sequence of the major open reading frame (nucleotides 386 to 12,349) is provided . Repeated nucleotide sequences in the untranslated regions are underlined .



CLONING AND SEQUENCE OF BOVINE VIRAL DIARRHEA VIRUS A

CTAG

TACGACMTTACMMGGCAtn !C A1ACGIATTGGGAATTAM

195 45

49 AATATMIlACCh'TAf.Er.MCMA7000TCTCAGCGMr4CGMAAGAGGC7AGCCATCCCCTTACTACGACTAGCATMTGAGWGCGTAGCMCACTGGTWCTTCCT7GGTCCCIIAAGCCCTCAG7ACACCCTAC

19,

192 TCGICACTGCITCCA000CTTGGAATMACCTCTCCACATCCCACCTCCACCACCCCATCCCCAMECACALCTTMCCTCAG CMrM,TCOCGCAOGTAMACGOTTTiMCCGCTOTTACGMTACAGCCTGIAGGGT

334

335 4CIGCACA000CCACTOYATTOUIACTAMMY CTOTGCTGTACATGGGAC Ale GAG TIC ATC ACA MT GM CTT TEA Too AM ACA TAG MA CM AM CCC GIC CGC OTC CC GM OCT MET GIU Len lie Thr Asn We Len Lau lye Lys Thr Tyr Lys Gin Lys Pro Val Gly Val Clu Clu Pro

454 23

455 OTT TAT GAT GAG GCA GOT WI CCC TEA ITT MI GM AM GGA GCA GTC CAC MI CM TCC ACC CTA AM CTC CCA CAC MC AGA MC GA CCC CAT GET CCA ACC MC 24 Val Tyr Asp GIn Ala Cly ASP Pro Lou The Gly GIu Arg Gly Ale Val His Pro CIA Bar Ihr Lou Lys Lou Pro His Lys Are Cry Clu Are Asp Val Pro The Man

562 59

563 T7G GCA 7CC YEA CCA AAA AGA BUT GAG IGC AM TOO GUT MT AM AGA CCA CUT GEE ACC COG AIC TAG OTC MC CCA CCG GOA CIA EYE TAG GAG On TAT SAME MY 670 60 Lev Al . See Lou Pro Lys Arg Gly Asp Cys Arg Bar Gly Asn Bar Arg Gly Pro US I Bee Gly IIe Tyr Lou Lys Pro Cly Pro Lou Phe Tyr Gin UP Tyr Lys Oly 95 671 CCC OTC TAT CAC ACC GM CCC GIG GAG CIC 7TT GAG CAC MA TOO Alt 701 WAS ACE ACT AAA CGG AIA GM AGA GTA ACI GCA AGE GAG MA MG GIG ISO CAC MET 778 96 Pro Val Tyr His Art Ale Pro Lou Glu Lou Phe Glu Glu CIy Bar MET Cya Clu Thr Thr Lys Arg Ile CIy Arg Val The Cly Sar Asp Ely Lye Lou Tyr 1111 It . 131 779 TAT GTG TOT ATA CAT CCA YET ATA ATA ATA MA ACT 4CC ACC AP ACT TAC GM AGO GEO EYE ACE TEE OTC CAT MT AM CTT PC 7GC MI CIA YOU OTC ACA ACT B06 132 lye VII Cys 110 ASP Gly Oys Ile lie Ile Lys Bar Ale Thr Are Bar Tyr Gin Are Val Phe Are Irp Val His Asn Arg Lou Asp Gas Pro Lou Cep Val Thr Bar 167 SET IGC TOM GAG AM MA GM GAG MA MA ACA AAA AAG MA ACA CAC AM CCC CAC AGA CIA EM AGO OCO MA ATG AM ATA GTG CCC AM CAA ICI CAA AM GAG ACC 160 Cys Bar Asp Thr Lys Glu Gin Gly Ala Ihr Lys Lys Lys Thr Gin Lys Pro Asp ACT, Leo Clu Arg Ciy Lys MET Lys 114 Vol Pro Lys Glu Bar CIV Lys Asp Bar

994 203

995 OR ACT AA OCT WO GAT MI ACA ATA COG GIG CM MA GTC AM TAC CAC GIG AGO ME MC MA AM ACC An ACT AAA ABC ACT CPU GAC GAM TIC TAG CAT An 204 Lys Thr Lys Pro Pro Asp Ala Thr Ile Val Val Clu Gly US l Lys Tyr Gin Val Arg Lys Lys Ciy Lys Thr Lys So, Lys Asp Thr Gin Asp BY, Lee Tyr His Mn

1102 239

1103 AAA AAC MA CCT CAC CAA TCA CCC AAG MA CIE GAA AAA CCA ITO TIC GM 7CC OCA AEA ATA MI AlA OTT TTC TTT CM GET ACA ATC MA CM MC AEA ACA CAC 240 Lys Man Lys Pro Gin Cle Sor Arg Lys Lys Lou Clu Lys Ala Lou LOU Ala lrp Ale lie Ile Ale lie Val Lou PINT Gin Vol Thr MET Gly Clu Asn IIe Thr Gin

1210 275

1211 TM AAC CTA CAA GAT MT MG ACG GAA GGC ATA CAA CM MA ATC TTC CM AGO MI GTG AAE ACA ACT TEA CAT MA ATC TOG CCA GAG AM ATC TOT ACT GGC GTC 276 irp Ann Len Gin Asp Man Gly Thr Glu Cly lie Gin Arg Ala NEI Ph . Gin Arg Cly Val Man Arg Bar Lau His Gly ill TPp Pro Gill Lys lie Cys Ihr Gly Vol

131B 311

1319 OCT 7CC CAT CIA CCC ACC GAT MA GM CTA MA ACA ATT CAT MI ATC ATG GAT GCA ACT GAG MG ACC MC TAG ACC TOT TOO ACA CIT CAA CCC CAT CC TO An 312 Pro Bar His Lou Ala Thr Asp lie Glu Lou Lys Thr lie HIS Gly MET MET Asp Ale Bar GlU Lys Thr Asn Tyr Thr Cys Cys ACT, Leu Gin Arg HIS CIU Trp Mn

1426 347

1427 AAC CAT CCT ICC TOO AAC YOU TAC MI Ail -CAA CCC ECC MET CIA CEC AIC MI ACA ACC CAA CCC M7 CTC ACT GAG MA CM CCA MA AM GAG IGC GCA GEC ACT 34B Lys His Cly Trp Cys Asn Trp lye Man Ile Gin Pro Trp Ile Lev Val MET Asn Arg Thr Gin Ale Mn Lou Thr Glu CIy Gin Pro Pro Arg Gin Cys Ala Val Ihr

1534 3B3

1535 IGT ACC EAT CAT AGO CUT ACE CAC ETA AAC CTC CIA ACA CAA MI ACA GAT ACC CCC ACA CCC ETA ACA GOT TOO AAG MA GGA MG AMC ITO TCC 7TT MA CCC AEA 1642 384 Cys Arg Eyr Asp Arg Ala Bar Asp Len Asn Val Val EM Gin Ala Arg Asp Bar Pro Thr Pro LOU Thr Gly Cye Lys Lys Gly Lys Man PRO Bar Phe Ala Gly Ile 419 1643 TIC AIC CGG GCC CCC TOO An TIT CAA AlA MI MA ACT CAT CTA ETA ITC AM CM CAT GAA COO All ACT A10 ITC CPU CAT ACC ACT CET TAG CTT CET CAC CCC 420 Len MET Arg Gly Pro Cys Asn Phe Clu lie Ale Ala Bar Asp Val Lou Phe Lys CIU Rig Clu Arg Ile Bar MET Phe Gin Asp Ihr lhr Len Tyr Lou Val Asp Gly

1750 455

1751 tTO ACC AAC TOO CIA GM GOT CEO AGA CM GGA ACC OCT AAA GIG ACA ACC ECC TEA CCC An CAC CTC GM ATA CIA GGA MA AAG TIC GAA AAC An AMY An AM 456 Len Thr Man Bar Lou Glu Gly Ala Arg Glen Gly Thr Ala Lys Leu Thr Thr Erp LOU Ely Lys Gin Lou Cly IIe Lou Gly Lys Lys Lou GlU Man Lys Bar Lys Thr

1058 491

1859 TOG ITT EGA GCA TAG MI OCT ECC MI In TOT GAT GTC CAT CCC AA ATE CCC TAC AlA TOO TAT ACA AM ME YOU ACC MI GCC IGC 11A CCC An AAC ACA AM 492 lrp Put Gly Ala Tyr Ala Ale Bar Pro Tyr Cys Asp Vel Asp Arg Lys lie CIy lye lie Erp Tyr The Lys Men Cys Thr Pro Ale Cys Lev Pro Lys AAA The Lys

1966 527

1967 MET GTC GM CCT GM AAA ITT CGC PLC AAE GCA GAG GAG WC An ATA IIA CAT GAG AEO GGG GUT CAC TIC 7CC GAG CIA CIA CIA CTT TOT 11A GTC CTC C1G ECC 520 lie Val Gly Pro Gly Lys Phe Gly THE Man Ala Gill Asp Gly Lys lie Len His Glu MET Gly Gly His Len Bar Clu Val Lou tell Leu Bar Lou Val Val Lou Bar

2074 563

2075 CAC IEC MA CCG GAA ACA GCI ACT GTA Ale IAC CIA ATC CIA CAT ITT TOO ATG CCA CM ACT CAC GET CAT CTA ATG CAT TOT CAT MG AGO CAG TIC An GIG ACA 564 Asp Phe Ala Pro Oli Thr Ale Bar Val MET Tyr Lou 110 Lau His Phe Bar Ile Pro 010 Bar His Val Asp Up l MET Asp Cys Asp Lys Ehr Gin Leu Asn LOU Ihr

2182 599

2183 OTC GAC GIG ACA ACA OCT CAA CIA ATA CCA CCC ECC OTC TCC MI CTA CCC AM TAT GTA TOT AEA AGA CCA MAY EGG TOG CCT TAT GAG AGA ACT CIA CEC ITC CCA 600 Val GIU Len EM Thr Ale Clu Val Ile Pro CIy Bar Vat lep Man Lau CIy Lys lye Val Cys lle Arg Pro Man Erp Trp Pro lye Glu ME lhr Val Val Lou Al .

2290 635

2291 ITT GM CC GIG AGE GAG GIG GIG AAG TIA GIG EEG AGO GCA CIC AGA CAT TEA ACA CGC MIT TOO AAC CUT MA ACA ACE ACE MI III ETA CIA ICC CTT GET An 636 The Clu Clu Val Bar Gin Val Val Lys Lou Val Leu Arg Ala Lou Arg Asp Lev Thr Arg IIe Trp Mn Ala Ale Ihr Thr Thr Ala Phe Lou Vol Cys Leu Vol Lys

2399 671

2399 ATA OTC AGE MCC CAC AEG CIA CAG GM ATT CIO YOU CIA CTA TIC MA ACA CM GTA CAA CM CAC TIC G1 ECC AM OCT CM TTC ECC TAT CCC ATA GCA MC CAC 672 11 . Val Arg GI, Gin MET Val GIn ely 11e Lt . Trp Lou Lou Lau lie Ehr Ciy Val Gin Gly His Lou Asp Cys Lys Pro Glu Phe Bar Tyr AIR lie Ale Lys Asp

2506 707

2507 GAA AGA MIT MI CM GIG CM OCT GA COG CTT PLC ACC ACT TM An CAA In TCA OCT GG Ale MC CTC GM On ACA AEC OIC All CAI TOG TOO GM CAT MG 700 Glu Arg lie Gly Gin Lou Gly Ale Glu GIy Lou THE Thr Ehr Trp Lys Gin Tyr Bar Pro GIy MET Lys Lou Glu Asp Thr MET Vel lie Ale Trip Cya Gin Asp ely

2614 743

2615 MC TEA ATC TAC CEC CM AGA ECC ACC ACA GM ACC ACA TAE GIG CCA ATC TIC CAT ACA AGA CCC TTE CCC ACC ACT GIG CIA TIC MA AM CT, ITT GAT (AC CGA 744 Lys Len MET Tyr Leo Gin Are CVs Thr Arg Glu Ehr Arg Tyr Lou Ala lie Len His Thr Arg Ale Leu Pro Inc Bar Val Val Phe Lys Lys Lou Phe Asp Gly Are

2722 779

2723 An GAA GAG GAT CIA GTC GM ATG ME GAG MC TTT GM ITT GGA GIG TM CCA TOT GAT GM MA CM AlA CIA ACA ME Audi EIC ME ACA ACC CTC GIG AAC GGA 780 Lys Gln Gin Asp Val Val Glu ME I AAA Asp Man Phe CIU Phe ely Lou Cys Pro Cys Asp Al . Lys Pro lie V .1 Arg Ciy Lys Phe Man Thr Thr Len Lou Asn Ciy

2030 615

2831 CCC CCC TTC GAG AIC CTA TM CCC ATA CCA TOO ACA WC ACT ETA AGO TOE ACG ECA TIC MI AEG GAG ACC ETA GM AGA ACT GIG CIA CGG ACA TAE ACA ACC TOT 016 Pro Al . The Gin MET Vel Cys Pro lie CIy Trip Ehr Cly Thr Vii Bar Cys 7M Bar Phe Mn MET Asp The LOU Ala Thr Thr Val Val Arg Thr Tyr Mg Are Sar

1930 051

2939 AAA UP TIC MI CAT AM CAA GGC 7G7 ATC ACC CAA An APL GIG GM GAG GAT OTC CAT AAC TOO AID CTT MA CCA M7 ECC ACT YET GIG OCT GOA GC CAA CIA 052 Lys Pro The Pro His Arg Gin Gly Cys Ile Thr Gin Lye Ann Lou Gly Gin Asp Lou His Mn Gys Ile Lau Gly Coy Man Imp Thr Cys Vat Pro Ely Asp GIn Lou

3046 887

3047 CIA TAG AAA CGC GGC TOT MET GA YET ECC AAC ECC TOT CCC EAT GA Ill MA PC ACE GAG CGA CTA CCA GAG TAC CM MET GGC AAC TOT Me ETO GAG AAC GAG B60 Lou Tyr Lys Gly Gly Bar Ile Gin Bar Gys Lys Trp GYM Gly lye Gin Phe Lys Clu Bar Gin Gly Leu Pro His Tyr Pro 110 GIy Lys Oya Lys Len GIV Aan Gin

3154 923

3155 ACT COT lAG AGO CTA ETA GAG ACT ACC TOT TOO AA7 AP CAA CCT CTC CCC ATA CIA CCA CM OGG ACA ITS MG IGC An ATA GG AAA CA ACT CIA CAC GTC AlA 924 Thr GIy lye Are Lou Val Asp Bar The Bar Cys Man Arg CIU Gly Val Ale lie Val Pro Gin Ciy Thr Lou Lys Cys Lys Ile GIy Lys Thr THE Vei Gin Val lie

3262 959

3263 OCT Ale CAT ACC MA CTC MA OCT ATE CCT TOO AGA CCA TAT CAA ATG AEA ICA ACE GAG CCC OCT CIA CM MC ACA CCG TOT ACT TIC AAC TAU ACT MG ACA ETA 960 Ale MET Asp Thr Lys Lou CIy Pro MET Pro Cyn Arg Pro Tyr Glu IIa lie Bar Bar Clu GIy Pro Vol Clu Lys Thr Ale Cys Thr PM Ash Tyr Thr Lys Thr Lou

3370 995

3311 AM AAE MG TAT Ill GAG CCC ACA CAC AM EAC III CC CAA EAC ATG CIA MA MA GAG TAT CM In EGG Ill GAG GIG GAG GIG ACE CAC CAT CAC CCC GI TAG 996 Lys Ann Lys lye The Gin Pro Arg Asp Bar lye Phe 01A CIA lye MET Len Lys Gly Glu lye Gln Tyr Trp Pat Asp Lou Gin Val Thr Asp His His Are Asp lye

3470 1031

3479 TIC OCT GAG TCC AlA TEA GIG GIG CIA CIA GCC CEC TEO MI WC ACA 1A1 ETA GET ECC TEA CEC CIT MOP EAC ATC GIG EEA TEA CAA GAG An GCC TTA GOO All 3556 1032 The Ala CIS Bar lie Len VII Val Val Val Ala Lou Len Gly Gly Arg Tyr Vol te n irp Lev Len Val The lye MET Val Lau Bar Gin Gin Lys Aid Leo Gly Ile 1067 3507 GAG TAT GCA TCA WE GM OTC OTC AIC Ale ME AAC 710 CIA AGO CAT AAC AAE MIT CM GIG OTC ACA TAC EEC ITC CTC CEC TAC CIA CIG OTC AGO GAG GAG ACC 3694 1068 Gin Tyr Gly Bar Gly Gin Val Vat MET MET CIy Man Lou Len Thr HIs Man Ash lie Glu Val Val The lye Put Lou Lou Len Tyr Len Lou Lou Arg Gin Clu Bar 1103 3695 CIA AAC An ECC GIG TTA C70 TTA IAC CAC AEG TEA GIG CIA CAC CCA PLC MA TOT CTA All GEC ATC CTA GIG ATG MET MG CAT CEC CIA MAC CCC CAT TOM EGG 3002 1104 Val Lys Lys Trip Val Len Len Len lye His lie Lou Val Val His Pro lie Lye Bar Val Ile Val [to Lou Leu ME E Ile Gly Asp Vol Val Lys Ale Asp Bar Cly 1139 3803 ME CAA GAC TAC TTC CGC AM Ale GAG OTC TOY 777 ACA AGA CIA CIA CIA AIC OTC AlA GOT TTA ALL Are OCT AGE MI GAG CG ACE ALA GIG CCA CEO CIA ACA 3910 1100 Ely Gin Clu Tyr Len GIy Lys Ile Asp Len Cys Phe Thr Thr Val Val Len Ile Val lie Gly Len Ile lie Ale Arg Arg Asp Pro Thr Ile Val Pro Len Val Thr 1175 3911 CIA API CCA WA CTC AGO OTC ACT GAA CIO ACC CAC CAG MI GGA Gil PC ATC CUT GIG CCC CIC AEG ACT AEA ACC CTA CTC AIC CEE AM TAT GIG ACA CAT TAT 1176 lie MET Ala Ala Len Arg Val lhr Gin Lou Thr His Gin Pro GIy Val Asp Ile Ale Val Ala Val MET TM Ile Thr Len Len MET Val Bar lye Val lhr Asp lye

4016 1211

4019 III ACA TAT AM AAA TOO 11A GAG TOO A77 CTC AGO CIG CIA TCT GGG GIG ITO TIC AEA AGA ACC CIA ATA TAG CIA GOT ACA ATC GAG At0 GCA GAG CIA ACT ATC 4126 1212 Phe Are lye Lys Lys Trp Leu Cln Cys Ile Leu Bar Leu Val Bar Gly Val The Len lie Arg Bar Lou Ile Tyr Len GIy Are lie Gin MET Pro GIu VAT ihr IIe 1247 4127 CCA AAC IGG AGA GCA CIA ACT tTA ATA CIA IM TAT TIC AEC ICA ACA ACA MIT CIA ACC ACC 7CC AAC GTT CAC GEC MI CGC CIA TTE TIC CAA 161 GIG OCT AIC 1248 Pro Asn Trp Arg Pro Len ihr Leu lie Lou LOU Tyr Lau Ile Bar Thr Thr lie Val Thr Arg Trip Lys Val Asp Val Ala Cr y L en Len L en Gin Cys Val Pro ii .

4234 1203

4235 TEA TIC CEC CEC ACA ACC ETC ECC CCC GAG ITO ITA ACG CIA AlA CTG AIC CIG OCT ACC TAI CAA TIC GET AM LTA TAG EAT CIG MA ACE CTT ACE ACT OAT ACA 4242 1284 Lau Leo Len Val ihr TIRE Leu Trp Ala Asp Phe Lau EM Len IIe Lou lie Lou Pro Thr Tyr CTU Len Val Lye Lou Tyr lye Lau Lys Thr Val Arg Ehr Asp Ihr 1319



196

COLLETT ET AL



CLONING AND SEQUENCE OF BOVINE VIRAL DIARRHEA VIRUS C

197

0555 CIA ALA TAO CAC GIG TAT GM GTT TAG TAG MA GOT TOG CC GCG ASO GM CTA 101 GAG AM ACA WA CCC AGA MC TEP TLC ACA TUG ALA AEG ITT GM GM ITC 2724 Lee lie Iyr Mi . Lou Tyr GI, Val Tyr Tyr Lye Cly Trip CIu Ale Lys GIu Lou See CIu Arg Thr Ala Gly Arp Mn Lou Phi The LOU Ile MET Phe CIu Ale Pole

0662 2759

$663 GAG ITS TTA COG AIG CAL ICA CM GM . MA ALA AGO ASC GIG TOG CGA M7 TAG All OTG CAT ITC ATA TAG GM CIA CAC ASC CM ATC AAL AGA CCC GIG AAG MA 2760 CIU Leu Lou Gly AT Asp Per Cl . Gly Lys II . Arg Asn Lou See Gly ASn Tyr Ile Lou Up LOU Ila Tyr Cly Lou His Lye Gin Ile Mn Arg Gly LOU Lye Lye

6770 2795

$171 ATC CIA CTG COG TOG CCC OCT COA CCC III AGE TOT GC TOG ACC OCT ACT GAG GAG AM ALC ACA TIC CCA ACA GAG AAC TAT TIC AGO CIA GAP AM AGO ICC CCA 996 MET Val Lou Gly Trp Ala Pro Ale Pro Phi See eye Asp Tell Thr Pro See Up CIu Are II • Are Lou Pro Thr Asp Asn Tyr LOU Art, Vol Glu The Arg eye Pro

1870 2631

0679 LGT GGC TAT GAG AUTO AM MI TIG AAA MI OTA MI GGC AAA LTT ACC MA OTC CAG GAG ACC CCC CCT TIC CTA TOY AGA Me AGA CCL GG7 SCOTT MA CG GLC AAC 2032 Cys Gly lyr GIU MET Lys Ale Phi Lye Asn Vol Gly Gly Lye Lou The Lys Val Glu Glu Sir Gly Pro Polo Lou eye Are Asn Arg Pro Gly Arg Gly Pro Val Mn

8906 1067

0907 TAG AGA GIG AM AAG TAT TAG CAT GAG AAC GIG AGA GAG A1A MA COO CIA MA AAC TIC GM GOB CAG CIA GAG CAC TAG TAG AM CCC GIG ALA MA MA All GAC 9094 2060 Tyr Are Vs I The Lys Tyr lyr Asp Mp Amu Lou Arg Glv Ile Lys Pro Val Ale Lye Leo C10 Cly GIn Val GIU HIS Tyr Tyr Lys Gly Val The Ala Lys II . Asp 2903 9095 TAG AGE AAA GGA MA ATO CIO 1EG COG ACT GAG AAG TOG GAG GIG GAA CAT MI GIG ALA AGO AM ITS GLT MG CA TAT ACT COG CTC CCC TTC M7 MI MA In 2904 Tyr See Lye Gly LyS MET Lou LOU Ala The Asp Lys Trip CIu Val Clu His Gly Val Ile The Arg Lou Ale Lys Mg Tyr The Gly Vsl Gly Polo son Cly Ma Tyr

9202 2939

9201 LTA CUT CC GAG CCC MI CAG CUT CUT CIA OTC GAG AGO CAC TOT CCA ACT ATA ACC MA AC ACA CIA CAC TTT CIA AM ATC AM MG CG7 TOY GCG ITC ACC TAT 2940 Lou Gly Asp COO Pro ASP His Arg Al . Lee Val Gin Are Asp Cys Al . The 118 The Lys Asp the Val Gin Phe Lou Lys MET Lys Lys MY Cys Ala The The Tyr

9310 2975

9311 GAG GIG ACC AEC TOG MI CTO ACC AGO GIG AIC GAP CIA CIA GAO ACC AAC AAT CIT CAA GAG Me GAS AlA CCC ACC OCT ACC OTC ACC ACA TOO CIA OCT TAG ACC 9418 2976 Asp Lau The Ile See Aso Lou The Arg Lou Ile CIu Lou Vol His Arg Asn Ash Lou Clu Cite Lys Glu Ile Pro The Al . The Val The The Tell Lou Ala Tyr the 3011 9419 TTC GIG MI CAP GAG CIA GGG ACT AIA AAA GCA CIA CIA MA GAG AGA CIA AIC CCC GAG COO CIA GTE GAT AIG AAL ITS CM MA GAG GIG CAA GIG GAG AGO ICA 9526 Arg Val IIe Pro Abp Pro Val Val Asp Its Asn Lou Gin Pro Glu Val Gin Val Asp the Ser 3047 3012 Pho Val Mn GIU ASP Vol Gly The Ile Lys Pro USE Lou Gly

alt

9527 GAG GTL GM ATC AGA ALA 1T1 GGA ACC CM ACC GIG ALC ACA ACC MA CIO ACA OCT CTC TIC CAA MA CIA GAG CCl GAG CCC AGO GAC AAC CM AAC TOG GIG AAC 3048 Glu Val Gly Ile Thr Ile lie Gly Arg CIu The LOU MET The The Gly Val The Pro Val Lau Glu Lys Val Glu Pro Asp Ala See Asp Asn Gin Asn See Val Lys

9634 3083

9615 AIC 0CC TIC GAT CAG UGT ME In CCA Gam'. OCT CCA ATA CAC AGA CAT ACA CTA AGA CM GM ATA CAC MC AGG CAT GCG AGO CCC ITO ATC ATG ATC CTC CM ICA 9742 3004 110 Oly Lou Asp Clu Gly Asp Tyr Pro Cly Pro Cly Ile Gin The His The Lou Thr CIu GIu Ile His Asn Arg Up Al . Arg Pro Phi 110 HOT Its Lou coy Mr 3119 9743 AGO OAT TOG ATA ECA OAT ACC CCA AAG ACT CUT ALA MY SEA MT GIG TAG ACA COO MT GAL CCC AGO GM ALA GUS GAG PIG ALG OCT GOP GGG CGC A1C ITS CIA 3120 Arg Asn See Ile See A50 Arg Ala Lys The Al . Mg Asp lie AGO Lou Tyr The GIy Ago Asp Pro Arg Glu Ila Arg Asp Lau MET Ale Ale Gly Art, MET Lou Val

9050 3155

9051 CIA CCA CIC AGO GAT OTC GAG OCT GAG CTG TCI GM ATG G1C GAT ITC MG MG ACT III ITS CAT AM GAG GM CIO GAG GOT CIA ACT CTC CCC CM OCT MA 6CC 9958 3156 Val Ala Lou Are Asp Val Asp Pro GOP Lou 5, CIu MET Val Asp Phe Lys Gly ihr Phe Lou Asp Arg Got Ale Lou CIu Ale LOU Sir LOU Cly Gin Pro Lye Pro 3191 9959 MG CAG GTT ACC MO GAA OCT CTT ACC ME ITC ALA GM CAC MA MA CAT CTG CAC Ale CCT AAL TOO III GCA TCA CAT CAC CLA CIA III GIG CM GIG CM ITS 10066 7192 Lys Gin Val the Lys ON Ale Val Arg Asn Leo 11e Clu Gin Lys Lys Asp Val Clu Ile Pro Asn Lrp PM Ale See Up Asp Pro Val Phe Lou CIu Vol Ale Lou 3227 10067 MA APT CAT AAG TAG 'AC TTA CIA MA CAT CTT GGA GAG CIA MP GI CM GOT MP MA OII GGG GCC ACC GAT CAG ACA CA ATE ATA MG GAG CIA WC ICA AM 10174 3228 Lys Ash Asp Lye Tyr Tyr Lou Val GI, Asp Vol Gly GIu Val Lys Asp Gin Ale Lys Ale Lau Gly Ale The Asp Gin The Arg Ile Ile Lys GIu Val Cly Our Arg 3263 10175 ACC TAT GCC AEG MG CIA TCT AGO TOG TTC CTC MG GCA ECA PAL MA CAC AIC ACT ITS ACT CCA CIO TTT GAG CM TIC TTC CIA CM TOG Me CUT CCA ACT MG 10262 3264 The Tyr Ala MET Lys Lou See See Irp The Lou Lys Ale See Asn Lys Gin HE I See Lou The Pro Lou The GIu CIu Lau Lau Lou Are Cys Pro Pro Ale Lhr Lye 3299 10303 AM PR MG MG GAG AIG GCA ICA GCT TAG CAA TIC GCA CC MI AC TOG GAG CCC CLC CUT TOO GM GIG CAC CIA OCT ACA ATA CCA CCC ACA AGO GIG AC ATA 10390 3335 3300 Ser Ash Lye Gly HIS MET Ale Sir Ala Lyr Gin Lau Alm Gin Cly Ass Trp Clu Pro Lau Cly Cys Gly lop His Lou Gly The Ile Pro Ala Are Art, Val Lys Ile 10391 GAC CCA TAT GAA OCT TAG CTC AAC TIC MA CAT TTC AlA GM GM GAP GAG MG MA CC' AGO GTI AAG CAT ACA CIA AlA AGA GAG GAG ML MA LOG A1A CTT MA 10490 3371 3336 His Pro Tyr Glu Ale Tyr Lou Lys Lou Lys Asp Phe 110 Olu Olu Glu GIu Lye Lye Pro Arg Val Lys Asp The Val Ile Arg GIu HIS Aen Lye Lrp 110 Leo Lye 10499 AAA ATA AGO III CM MA AAC CLC AAC ACC MG AAA AIC CTC AAC MA CLC MA CTA YET GM CAG ITC GAG AGO GAG GM ME AC AGO AAL ATC TAG ASO CAC CAC 10606 3372 Lys Ile Arg Phe Gin Gly Asn Loll Asn The Lys Lys MET Leu Asn Pro Gly Lye Lou Ser Gin Gin Lou Asp Are GIu GIy Are Lye Are Asn Ila Tyr Aan Mi . OIn 3407 1060? All MI ACT ATA AEO ICA ACT CUP GM AlA AGO C1G GAG AAA TIC CCA ATA 01G AUG GCC CAA ACC CAC ACC AM ACC ITT CAT GAG GUS ATA ACA CAT ASC ALA GAG 10714 3400 Ile Gly Thr Ile MET Ser See Ale GIy 118 Art, Lau GIG Lys Lau Pro lie Val Arg Ale Gin Thr Asp The Lys The Phe His Coo Ale Ile Are Asp Lys Ile Up 3443 10715 AAC ACT CM AAC MY CAA AA7 CCA CAA TIC CAC AAC MA TIC TIC GAG ALT TIC CAL ACC CIA CCC CM CM ACC CTC MA CAL ACC TAC COT GAG GIG AM TCC GC 10023 3444 Lys See GIu ASn Arg Gin Asn Pro GIu Lou His Asn Lys Lau Lou Glu Ile Phe His Thr Ile AIa Gin Pro Thr Lou Lys His The Tyr Gly CIu Val Ihr Trip Glu 3479 10023 LM CII GAG GCG 000 CIA AAT AGA AAG GGG MA CCA GGC 01C CTG GAG AAG MG SAC ATC GCA GAP CIA TIC GAT IG GM MG CAC GIG CIA GAS CAA LTG GIG AGO 10930 3480 Gin Lou Olu Ale Cly Val ASn AT, Lye GI, Ale AIa Gly Phe Lou 010 Lys Lys Mn Ile Gly Glu Val Lou Asp See GIG Lys His Lau Val GIU Gin Lou Vet Arg 3515 10931 GAT CTG AAC CCC 0GG AGA MG ALA MA TAT TAT GAA ACT GCA AIA CCA AM M7 GAG AAG AGA CAT GIC AM CAI GAL 100 CAG WA COG CAC CIG GIG GTE GAG ASC 11030 3516 Asp Lou Lys Ale Gly Arg Lys Ile Lys Tyr Tyr Glu The Ala Ile Pro Lys Asn CIu Lys Are Asp Val Ser ASP Asp Irp Gin Ale GIy Asp Lou Val Val Glu L's 3551 11039 ACG CCA AGA CTT ATC CM TAG COI OM WC MG ACA AGO CIA CCC AMC ACT MG 01C A10 TAT AAL LOG GIG MA CAG CC CCC GEL G00 All CCA GUS TAT GM GGA 11146 3552 Arg Pro Arg VeI Ile Gin lyr Pro ON Ala Lys The Arg LOU Ala lie The Lys Val MET Tyr Asp Trip Vol Lys Gin Gin Pro Val Val Ile Pro Giy Lyr ON GIy 3587 11147 MG ACC CCC TIC TIC AAL ATC TIT CAT MA GIG ACA MG CM ICC GAG TOG ETC MT GAG CCA GIG GCC CIA ACT III GAG ACC MA WC TOG GAG ACT CM GIG ACT 11254 3500 Lys The Pro Lou Phe Asn 11 . Phi Pup Lys VeI Arg Ly6 Glu Trp Up Ser Phi Asn Ulu Pro Val Ale Val See Phe Asp The Lys Ale Trp Pop The Gin Vat Thr 3623 11255 ACT MG CAT CTG CAA CTT ATE CCA GAA ATC GAG AM TAT In TAT MG AAG GAG TOG CPC AC TTC AlT CAC ACC ATC AGO GC CAC PTO AGA GM CIA CCA GTT ALA 11362 3624 See Lys Asp Lau Gin Lau Ile GIy GIu Ile Gin Lys Tyr Tyr Lyr Lys Lys GIu Trip His Lys Phu Ile Asp The Ile The Up HIS ME I The CIu Val Pro Val 110 3659 11363 AGA COO CAT GOT CAA GIA TAT AlA AGA ML We CAC AGA Me ACC LCC CAG CCA GAG AGA ACT OCT CM MC ACC ATC TTA MI OTC CIO ACA ATC ATC TAG CCC TIC 11470 3660 The Ala Asp Gly Glu Val Tyr Ile Arg Asn GIy Gin Arg GIy See Cly Gin Pro Asp The Our Ale Gly Asn Ser MET Lau Asn Val Lou The MET MET Tyr Ala Phe 3695 11471 TOO CSA ACC ACA GGG CIA COG TAG AAG ACT TLC AC AGO GIG CCA AGO Alt CAC GIG TOT GGG GAT CAL GM TIC ITS ATA ACT GAS AM GCC ITA GM C10 SM III 11578 3696 Cys GIu See The Cly Val Pro Tyr Lys See The Asn Arg Val Ale Ar, IIe His Val Cys Cly Asp Asp Cl, Polo Lou II . The CIU Lys G19 Lou Cly Lou Lye Phe 3731 11579 CUT MG MA CGG ALC CC PIT OTT CAI GAA GCA WC AM LCI CAG AAG AIA PCG GM COG GAA MG ATO AM GTE OCC TAT AGA III GAG GAT AIA GAG TIC TOT TOY 11606 3732 Ale Asn Lye Guy MET Gin lie Lou Mi . Glu Al . Cl9 Lys Pro Gin Lys Ile The Clu 019 Clu Lye MET Lye Val All Tyr Arg Phe C1u Asp 118 Glu Phe Cys Sir 3767 1160? CAT ACG MA CTC OCT CTT AGC TOO TCL CAC AC ACC ACT MI CAC ATG GCC TIM AGA GAG ACC OCT GIG ATA CIA TGA AM ATG GCA ACA AGA 010 CAT IG ACT MA 11798 3768 His The Pro Val Pro Vol Pry Trp Sir Asp Mn The See Per Mi . HEY Ala Ely Arg ASP 1hr Ale Val Ile Lou Sir LyS MET Ale Ihr Are Lou Asp SO, So, 61, 3803 11795 GAG ACC MI ACC AGA WA TAT GS MA CCC CIA CCC 7TC ACT TIC TIC CTC ATC TAT TOG ICC PAC Me CTT OTT ACC AM ATE TOG CTC ITC GTC CTT TOG CAA CAC 11902 3004 Clot Arg Gly The The Ala Tyr Clu Lys Ale Val Ale Ph0 Our Phi Leo Lou MET Tyr See Trip Asn Pro Lou Val Arg Are Ile eye Lau Lou Val Lou See Gin Gin 3639 11903 CGA CC AGA CAL LCA TOO AAA CAT CCC ACT TAT TAT TAG AAA OCT GAT CCA AIA CCC CCC TAT MA CAT GIA ATA CUT CCG MI LIA ACT GM CEO AAG AGA AGA GCC 12010 3075 3040 Pro GIU The Asp Pro See Lys His AT* The Tyr Tyr Tyr Lys Gig Asp Pro 110 GIy Ala Tyr Lye Asp Val lie GIy Arg Asn Lau See Glu Lou Lye Arg The Gly 12011 YET GAG MA TIC GOA OAT CTA AAC CTA AGO CTG TOG AGO ITG GCG GIG EGG ACT AAG CPC ACA ACC AM AGA AlA ATT CC GAC TCI GTT GCC AOl GM MA GM GAG 12110 3876 Phe Glu Lys Lou Ale Asn Lau Asn Lou See Lev See 1St Lou Gly Val Thr Lys Him The See Lys Are Ile Ile Gin Asp Lys Val Al . ]IS Gly Lye Olu GI. 3911

lrp

12119 COG AAC LCC CIA CT7 AAG CCC GAG ALL CTG AlA TOG AGO AAA ACT CCC CAC TEA ICC AlA OCT GAT MA OTC ITT AGA TTA LM MA AC CAT TAT GAG GM GIG CC 12226 3912 Gly Asn Trip Lou Val Lys Pro Asp Are Lou Ile See See Lys The Gly His Lou Tyr Ile Pro Asp Lys 01y Pro The Lou Gin Gly Lys His Tyr Glu Gin Lou GIn 3947 12227 CTA ACA ACA CC ACA A4C CCC GIG AIG GM GIT MG ACT GAG AGA TAG APO TTA GOT LCC ALA GIG MI GIG CIO CLS AG AGO IGO AAA All GIG CIC ATG AGO CCC 12334 3940 LOU Arg Thr Glu Thr Mn Pro Val MET Gly Val Gly the GIG Are Tyr Lys Lou GIy Pro Ile Val Asn LOU Lou LOU Arg Arg Lou Lys Ile Lou Leo MET ihr A's 3903 12335 GIG COG OTC AM ACC IG CACMM TGTATA_A LTCTAMTMATTMTCCATGTA 3904 Val Gly Val See See "'

TACTOTATATMATATMTTWC-r

.TCCACLTCMCMGCGCACCCCGACACPLAGSCCTMSGGTMTCAM. 17471

12472 AIIAIOTACCLC,MGTAMIGTAC ATTTMTGCACGAGCACITTAGCIGTATCAGGIA000CCGACCTCLALAGTTMACTAGWACACCTOTMCAC

FIG . 2-Continued.

12573



COLLETT ET AL.

198

(+)

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11 11 Ii1I111IIII11I11Iii11111 1111IIIIIIIIIIIIIMI 11111 /III 11

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12000

FIG . 3 . Distribution of translation termination codons in the BVDV RNA sequence . Vertical lines represent the positions of stop codons (UAA, UAG, UGA) in all three phases of both the viral (+) and viral complementary (-) RNA polarities .

before further inroads can be made into disease prevention . However, studies in this area have been hampered by the inherent low level growth of BVDV in vitro and by the compromising effects of contamination of cell cultures and reagents with unsuspected BVDV or antibodies to BVDV . In this report, we describe the molecular cloning, and present the nucleotide sequence, of the cytopathic NADL isolate of BVDV . The information derived therefrom should allow for significant advancement in our understanding of the molecular and pathogenic features of BVDV . Several difficulties had to be overcome to successfully complete the molecular cloning of the BVDV genome . First, it was necessary to obtain sufficient quantities of purified viral RNA . We observed that substantially more viral RNA was associated with virus-infected cells than was found in cell-free virus particles . Enrichment of the BVDV RNA from total cellular cytoplasmic RNA was accomplished by a simple salt precipitation step . This means of fractionation exploited the structural features of BVDV RNA previously described (Purchio et al., 1983) . Although the viral RNA at this stage was significantly contaminated with lowmolecular-weight cellular RNAs, it could be readily purified to homogeneity by either density gradient sedimentation or gel electrophoresis . However, the low-molecular-weight RNA contamination proved fortuitous by providing a solution to the next problem in cloning : priming reverse transcription of the viral RNA . BVDV RNA lacks a 3' poly(A) tract and was refractory to enzymatic poly(A) addition . Apparently, however, the low-molecular-weight RNA species present with the BVDV RNA in the LiC1 2 supernatant fraction created relatively efficient template-primer complexes for cDNA synthesis . The resultant cDNA library contained clones with inserts having overlapping sequences from the entire length of the BVDV RNA . From the range of insert endpoints, it appeared reverse transcription initiated

randomly along the RNA template . Although numerous independent clones were obtained for every region of the genome, not all areas of the RNA were equally represented in the cDNA library . Of note was the region between nucleotides 4200 and 5200, which was particularly underrepresented . This may indicate that this area was infrequently primed or poorly reverse transcribed, possibly due to structural features in this region of the RNA . Alternatively, these sequences may not be tolerated well in E. colt. The final nucleotide sequence obtained was 12,573 bases . This number is in line with estimates of the size of the BVDV RNA (Renard at al., 1985 ; this report) . However, establishment of the complete sequence of the BVDV genome must await direct analyses of the termini of the viral RNA . Within the BVDV nucleotide sequence presented in Fig . 2 was found a single OFF in the viral RNA sense spanning the entire length of the genome . Further description of this ORF, its coding capability, and identification of encoded viral polypeptides, is presented in the following report (Collett et al., 1988) . ACKNOWLEDGMENTS We thank Susan Belzer, Nicole Meade Chartrain, Kimberly Glassman, Bill Hays, Laura Torborg Kakach, David Knight, Gary Opperman, Ernest Retzel, Linda Simmons, and Gayle Strum for their help during the course of this work, and Joel Dalrymple for providing purified Sindbis virus . Manuscript preparation by Sherri Pierson is greatly appreciated . This work was supported in part by USDA Grant 86-CRCR-1-2249 . We are cooperating with GENBANK to expedite entry of the sequence into the database . However, parties interested in obtaining the sequence in computer readable form in advance of the GENBANK release may contact the authors in writing . We anticipate having the data available as ARC II text files for both IBM-PC and Apple Macintosh .

REFERENCES

BAKER, J . C . (1987) . Bovine viral diarrhea virus : A review . J. Amer. Vet. Med. Assoc . 190, 1449-1458 . BouN, S . R ., McCLuRKIN, A . W., CUTUP, R . C ., and CORIA, M . F .

CLONING AND SEQUENCE OF BOVINE VIRAL DIARRHEA VIRUS (1985a) . Severe clinical disease induced in cattle persistently infected with noncytopathic bovine viral diarrhea virus by superinfection with cytopathic bovine viral diarrhea virus . Amer. J. Vet. Res. 46, 573-576 . BOLIN, S . R ., MCCLURKIN, A . W ., CUTUP, R . C ., and CORIA, M . F . (I 985b) . Response of cattle persistently infected with noncytopathic bovine viral diarrhea virus to vaccination for bovine viral diarrhea and to subsequent challenge exposure with cytopathic bovine viral diarrhea virus . Amer. J. Vet. Res . 46, 2467-2470 . BROwNLIE, J . (1985) . Clinical aspects of the bovine virus diarrhea/ mucosal disease complex in cattle . In Pract. 7, 195-202 . BROWNLE, I ., CLARKE, M . C ., and HOWARD, C . J . (1984) . Experimental production of fatal mucosal disease in cattle . Vet. Rec . 114, 535-536 . COLLETT, M . S ., LARSON, R ., BELZER, S . K ., and RETZEL, E . (1988). Proteins encoded by bovine viral diarrhea virus : The genomic organization of a Pestivirus . Virology 165, 200-208 .

COLLETT, M . S ., PURCHio, A . F ., KEEGAN, K ., FRAZIER, S ., HAYS, W ., ANDERSON, D . K ., PARKER, M . D ., SCHMAUOHN, C ., SCHMIDT, J ., and DALRYMPLE, J . M . (1985) . Complete nucleotide sequence of the M RNA segment of Rift Valley fever virus . Virology 144, 228-245 . CORIA, M . F ., SCHMERR, M . J . F ., and MCCLURKIN, A . W . (1983). Characterization of the major structural proteins of purified bovine viral diarrhea virus . Arch . Viral. 76, 335-339 . DIDERHOLM, H ., and DINTER, Z . (1966) . Infectious RNA derived from bovine viral diarrhea virus . Zbl. 8akt. 1. Abt. Orig. 201, 270-272 . DONIS, R . 0 ., and Dueovl, E . J . (1987a) . Characterization of bovine viral diarrhoea-mucosal disease virus specific proteins in bovine cells . J. Gen . Viral. 68, 1597-1605 .

DONIS, R . O ., and Dueovl, E . J . (1 987b) . Glycoproteins of bovine viral diarrhoea-mucosal disease virus in infected bovine cells . J. Gen . Viral. 68, 1607-1616 . DONIS, R . 0 ., and Dueovl, E . J . (1 987c) . Differences in virus-induced polypeptides in cells infected by cytopathic and noncytopathic bictypes of bovine virus diarrhea-mucosal disease virus . Virology 158,168-173 . GRUNSTEIN, M ., and HoGNESS, D . (1975) . Colony hybridization : A

method for the isolation of cloned DNAs that contain a specific gene . Proc . Natl. Aced. Sci. USA 72, 3961-3965 . GuDLER, U ., and HOFFMAN, B . J . (1983) . A simple and very efficient method for generating eDNA libraries . Gene 25, 263-269 .

MANIATIS, T ., FRITSCH, E . F ., and SAMBROOK, J . (1982) . ''Molecular Cloning . A Laboratory Manual ." Cold Spring Harbor Laboratory, Cold Spring Harbor, NY .

1 99

MATTHAEUS, W . (1979) . Detection of three polypeptides in preparations of bovine viral diarrhoea virus . Arch. Viral, 59, 299-305 . MAxAM, A ., and GILBERT, W . (1980) . Sequencing end-labeled DNA with base specific chemical cleavages . M "Methods in Enzymology" (L . Grossman and K . Moldave, Eds .), Vol . 65, pp . 499-560 . Academic Press, New York .

POCOCK, D . H ., HOWARD, C . J ., CLARKE, M . C ., and BROWNLIE, J . (1987) . Variation in the intracellular polypeptide profiles from different isolates of bovine virus diarrhea virus . Arch . Viral. 94, 43-53 .

POTGIETER, L . N . D ., MCCRACKEN, M . D ., HOPKINS, F . M ., and Guy, 1 . S . (1984a) . Comparison of the pneumopathogenicity of two strains of bovine virus diarrhea virus . Amer . J. Vet . Res . 46, 151-153 . POTGIETER, L . N . D ., MCCRACKEN, M . D ., HOPKINS, F . M ., WALKER, R . D ., and Guy, 1 . S . (1984b) . Experimental production of bovine respiratory tract disease with bovine viral diarrhea virus . Amer. J. Vet. Res . 45, 1582-1585 . PRITCHETT, R ., MANNING, J . S ., and ZEE, Y . C . (1975) . Characterization of bovine viral diarrhea virus RNA . J. Viral. 15, 1342-1347 . PRITCHETT, R . F ., and ZEE, Y . C . (1975) . Structural proteins of bovine

viral diarrhea virus . Amer. J. Vet. Res . 36, 1731--1734 . PURCHIO, A . F ., LARSON, R ., and COLLETT, M . 5 . (1983) . Characterization of virus-specific RNA synthesized in bovine cells infected with bovine viral diarrhea virus . J. Viral, 48, 320-324 .

PURCHIO, A . F ., LARSON, R ., and COLLETT, M . S . (1984). Characterization of bovine viral diarrhea viral proteins . J. Viral . 50, 666-669 .

RENARD, A ., GUIOT, C ., ScHMETZ, D ., DAGENAIS, L., PASTORET, P . P ., DINA, D ., and MARTIAL, J . A. (1985) . Molecular cloning of bovine viral diarrhea virus sequences . DNA 4, 429-438 . TAYLOR, J . M ., ILLMENSEE, R ., and SUMMERS, 1 . (1976) . Efficient transcription of RNA into DNA by avian sarcoma virus polymerase .

Biochim. Biophys. Acta 442, 324-330 .

WEIS, 1 . H ., ENOUIST, L . W ., SALSTROM, J . S ., and WATSON, R .1 . (1983) . An immunologically active chimeric protein containing herpes simplex type 1 glycoprotein D . Nature (London) 302, 72-74 . WESTAwAY, E . G ., BRINTON, M . A ., GAIDAMOVICH, S . Y., HORZINEK, M . C ., IGARASHI, A ., K&&RIAINEN, L ., and Lvov, D . K . (1985a) . Togaviridae . Intervirology 24, 125-139 . WESTAwAY, E . G ., BRINTON, M . A., GAIOAMOvICH, S . Y ., HORZINEK, M . C ., IGARASHI, A ., KAARIAINEN, L ., Lvov, D . K ., and PORTERFIELD, J . S . (1 985b). Flaviviridae. Intervirology 24, 183-192 .