Molecular Cloning and Nucleotide Sequence of a Pestivirus...

VIROLOGY 191, 867-879 (1992)

Molecular Cloning and Nucleotide Sequence of a Pestivirus Genome, Noncytopathic Bovine Viral Diarrhea Virus Strain SD-l

RUITANG DENG AND KENNY V. BROCK’

Ohio Agricultural Research And Development Center, Food Animal Health Research Program, 1680 Madison Avenue, Wooster, Ohio 4469 1

Received July 10, 1992; accepted August 12. 1992

Genomic RNA of noncytopathic (NCP) bovine viral diarrhea virus (BVDV) strain SD-l was extracted directly from serum obtained from a persistently infected animal. cDNA was synthesized and amplified by polymerase chain reaction (PCR) before cloning. The complete genomic nucleotide sequence was determined by sequencing at least two different clones from independent PCR reactions. The 5’ and 3’ end sequences of the SD-1 genome was determined from 5-3 ligation clones. The complete genome sequence was comprised of 12,308 nucleotides containing one large open reading frame which encodes an amino acid sequence of 3898 residues with a calculated molecular weight of 438 kDa. In contrast to cytopathic (CP) BVDV strain NADL, which contains a cellular RNA insert of 270 nucleotides and CP BVDV strain Osloss, which has an inserted ubiquitin RNA sequence of 228 nucleotides, the NCP strain SD-l had no insertion along the genome. Sequence comparison with other pestiviruses revealed that the overall nucleotide sequence homologies of SD-1 are 88.6% with NADL, 78.3% with Osloss, 67.1 O/o with HoCV Alfort, and 67.2% with HoCV Brescia. The overall deduced amino acid sequence homologies of SD-1 are 92.7% with NADL, 86.2% with Osloss, 72.5% with HoCV Alfort, and 71.2% with HoCV Brescia. The most conserved nucleotide and amino acid sequences are located in the 5’ untranslated region (S’UTR) and nonstructural protein p80 region, respectively. The viral glycoproteins, particularly gp53, and nonstructural proteins p54 and p58 have the lowest homology comparing both nucleotide and amino acid sequences between SD-l and other pestiviruses. Extensive analyses of amino acid sequences for the viral structural proteins and nonstructural protein p54 regions from five pestiviruses led to the identification of four conserved domains (designated as Cl, C2, C3, C4) and three highly variable domains (designated as Vl, V2, V3) within this region. The Cl, C2, and C3 domains are located in the capsid protein ~14, glycoprotein gp48, and gp25, respectively. The C4 domain is located in the junction between gp53 and ~54. Interestingly, out of three variable domains, two (Vl, V2) are located in the same glycoprotein gp53. The third variable domain is located in the nonstructural protein ~54. Q 1992 Academic Press. Inc

INTRODUCTION

Bovine viral diarrhea virus (BVDV), a small enveloped virus, is one of the most important viral pathogens of cattle (Duffel1 and Harkness, 1985). BVDV infection can result in a variety of clinical diseases in cattle, such as abortion, persistent infection, and mucosal disease (MD). Its genome is a single-stranded RNA with positive polarity and consists of about 12,500 nucleotides (Renard et al., 1987; Collett et al., 1988a). Together with the other two serologically and structurally related viruses: hog cholera virus (HoCV) of swine and border diseases virus (BDV) of sheep, BVDV belongs to the Pestivirus group. Based on their similarities of genome organization and strategy of gene expression with that of the Flaviviruses, Pestiviruses were recently reclassi- fied into the Flaviviridae family (Collett et a/., 1988c; Francki et a/., 1991; Horzinek, 1991). However, differences in virion composition with the Flaviviridae (Thiel

Sequence data from this article have been deposited with the EMBUGenBank Data Libraries under Accession No. M96751.

’ To whom reprint requests should be addressed.

et a/., 199 1) and the absence of a 5’ cap structure of its RNA genome (Brock et al., 1992) reflecting different mechanisms of viral RNA translation, are some objec- tions to this reclassification. Based on the cytopathogenicity in cell culture, BVDV has been divided into two biotypes: cytopathic (CP) BVDV and noncytopathic (NCP) BVDV (Bolin et a/., 1985). Only NCP BVDV is capable of establishing persistent infections in cattle following in utero infection (Brownlie et a/., 1984). Fur- thermore, mucosal disease, a severe clinical syn- drome, occurs only in persistently infected animals when they are superinfected with a second, antigeni- tally indistinguishable CP BVDV (Brownlie et al., 1984; Bolin et a/., 1985; Corapi et al., 1988). These observations led to the hypothesis that CP BVDV may originate from NCP BVDV by genomic mutation (Corapi et al., 1988).

To date, two CP strains of BVDV: NADL (Collett et al., 1988a) and Osloss (Renard et a/., 1987) and two strains of HoCV: Alfort (Meyers et a/., 1989a) and Bre- scia (Moormann et al., 1990) have been cloned and sequenced. Recently, a partial genomic sequence located in the ~125 region of a pair of BVDV, CP BVDV

867 0042.6822/92 $5.00 CopyrIght 0 1992 by Academic Press. Inc. All rtghts of reproduction in any form reserved.

868 DENG AND BROCK

strain CPl (about 5.7 kb), and NCP BVDV strain NCPl (about 3.2 kb) were published (Meyers et a/., 1991). Comparison of the genomic sequence between BVDV and HoCV led to the finding of cellular sequence inserts in a region coding for the N-part of ~125 in the CP BVDV (Meyers eta/., 198913; Collett eta/., 1989). There- fore, a hypothesis was proposed by Meyers et a/. (1991) that the insertion of a cellular RNA sequence by RNA recombination into the NCP BVDV genome was responsible for the development of CP BVDVfrom NCP BVDV. However, this hypothesis was challenged by observations that some CP BVDV strains lack the insertion in their genomes (Moerlooze et a/., 1990; De- sport eta/., 1991) which suggests that the understand- ing of BVDV cytopathogenicity is far from complete.

In this report, the complete nucleotide sequence of NCP BVDV, strain SD-l, is presented for the first time. In addition, analyses and comparisons of the nucleotide and amino acid sequences are made with those of other pestiviruses.

MATERIALS AND METHODS

Persistently infected animal and virus

A persistently infected heifer was maintained in an isolation facility to prevent exposure and infection with other BVDV strains. The virus isolated from this heifer was a NCP BVDV and was designated as strain SD-l. Blood was collected from the persistently infected heifer and serum recovered for virus purification and extraction of viral RNA.

Virus purification and RNA extraction

Partial purification of virus and viral RNA extraction by the guanidine thiocyanate method were performed as described previously (Brock et a/., 1992).

cDNA synthesis, PCR amplification, and cloning

cDNA synthesis using random primer and genomic viral RNA extracted from partially purified viral particles was done as previously reported (Brock et al., 1992). Following the first-strand cDNA synthesis, PCR was carried out to amplify the defined viral segments. The two primers used to amplify the first SD-l fragment were designed based on the sequence of the NADL strain in the nonstructural protein region. After determination of the nucleotide sequence of the first SD-l cDNA clone, some of the primers were designed based on SD-l sequence. The size of amplified segments was chosen to be 1.5 to 2.0 kb in orderto obtain optimal amplification. Primer length was chosen to be 18 to 24 bases in order to maintain the T, value above 55”. PCR amplification was performed with Taq poly-

merase (Perkin-Elmer Cetus, Norwalk, CT) for 30 to 35 cycles. The working profiles were as follows: 94” for 1 min to denature the DNA, 42 to 60°C for 1.5 min to allow primer annealing and 72” for 2 to 5 min for DNA extension. All the primers were purchased from either Biochemical Instrument Center of The Ohio State Uni- versity (Columbus, OH) or Genosys (Woodlands, TX). Purification and C-tailing of the PCR products was done as described previously (Brock et al., 1992). C- tailed PCR products were cloned into G-tailed pUC9 plasmid and used to transform competent Escherichia co/i strain JM 109 (Hanahan, 1983). Colony blots were done using nitrocellulose membranes and positive clones were screened by the corresponding [“PI- dCTP-labeled NADL cDNA fragments. The cloning of 5’ and 3’ end sequences of viral RNA was done by 5’-3’ ligation and PCR as previously described (Mandl et a/., 1991; Brock e2 a/., 1992).

Sequencing of cDNA clones

Prior to nucleotide sequencing, restriction enzyme mapping was performed to determine the appropriate restriction enzyme sites in the cDNA clones for subcloning of restriction fragments into pGEM-3Z vector (Promega, Madison, WI). The alkaline lysis and PEG precipitation method was used to extract and purify plasmid DNA (Birnboim, 1983; Lis and Schleif, 1975) for double-stranded DNA sequencing (Chen and See- burg, 1985). The nucleotide sequence of the inserts was determined by the dideoxy chain termination method (Sanger et al., 1977) using sequencing kits (United State Biochemicals, Cleveland, OH). The nucleotide sequences of the clones that lacked the appropriate restriction enzyme sites for subcloning were determined by a progressive oligonucleotide primer method (Sambrook et a/., 1989). Considering the potential of sequence errors created by Taq polymerase the entire genomic sequence of NCP BVDV SD-l was determined by completely sequencing a minimum of two clones from independent PCR reactions for each region. If a different nucleotide sequence was obtained from the two clones, the consensus nucleotide sequence was verified by sequencing a third or even fourth clone from other independent PCR reactions. The 5’ and 3’ end sequences were confirmed by sequencing nine independent 5’-3’ ligation clones.

Computer analysis

Nucleotide sequence comparison and analysis were made with HIBIO DNASIS (Hitachi Software Engineer- ing Co., Ltd., Brisbane, CA) (Lipman and Pearson, 1985; Needleman and Wunsch, 1970). The predicted amino acid sequence was analyzed and compared by

NCP BVDV STRAIN SD-l 869

using HIBIO PROSIS (Hitachi Software Engineering Co., Ltd., Brisbane, CA) (Kyte and Doolittle, 1982; Lip- man and Pearson, 1985).

RESULTS

Molecular cloning of BVDV SD-l RNA

To determine the nucleotide sequence, viral RNA was directly extracted from serum obtained from a persistently infected heifer. The virus titer in the serum was 1 03-1 O4 CCID,,Jml. Viral RNA extracted from 1 to 5 ml of serum was enough to carry out the PCR amplification and cDNA cloning. To optimize PCR amplification and minimize the nonspecific priming, PCR profiles varied for each set of primers. The annealing tempera- tures ranged from 42 to 60°C depending on the T, value of the primer and the homology between the primer and the sequence to be amplified. Several cloning methods were tried to clone PCR products. Com- pared with either blunt-end ligation or AT annealing and ligation cloning methods for PCR products, the GC-tailing method had a higher cloning efficiency. In order to eliminate the potential sequence errors created by Taq polymerase, repeat cDNA cloning of three independent PCR reactions for each viral RNA region was done. The corresponding restriction fragments of NADL cDNA clones were used as probes for identification of positive cDNA clones of SD-l genomic RNA. To determine the extreme 5’ and 3’ end sequences of the genome, genomic RNA ligation was performed before PCR amplification and cloning. Based on previous results suggesting that there is no cap structure at the 5’ end of BVDV genome (Brock et al., 1992), genomic RNA of SD-l was directly ligated without the treatment of pyrophosphatase to remove a 5’ cap structure. After restriction enzyme mapping, about 70% of inserts in the original pUC9 vector were subcloned into pGEM vectors for sequencing. A total of 29 cDNA clones that almost overlapped the whole genome three times were used to determine the nucleotide sequence of SD-l genome (Fig. 1).

Nucleotide sequence of NCP BVDV strain SD-l

The complete SD-l sequence was determined by sequencing at least two clones from independent PCR reactions. Seventy percent of the sequence was determined from both strands of cDNA clones. The remainder was obtained from multiple determinations on a single strand. A total of 36 nucleotides were different by comparison of the two independent nucleotide sequences in the cDNA sequence of about 30,000 nucleotides. In those cases, the consensus nucleotide sequence was obtained by sequencing a third or even

Okb 2kb 4kb 6kb 8kb 10kb 12kb

5’ ! , , ’ , , ,‘, , , ‘, I I I II I , ’ 3’

pPpBp MfvlMpMKA B N HNK K

H P - - - - - ---- ---

--+ -- - - __ *- - --- *----- --

FIG. 1. Distribution of cDNA clones of NCP BVDV strain SD-l along the genome of 12,308 nucleotides. Only the clones used to determine the nucleotide sequence are shown here. The 5’-3’ ligation clones are indicated by stars. The restriction enzyme sites of cDNA clones utilrzed to perform subcloning are shown: A, Accl; B, BarnHI; H, HindIll; K. Kpnl; M, I\/lbol; N, Nhel; P, Pstl.

fourth independent clone. Interestingly, one of the errors found in one of the cDNA clones has been previously described for BVDV NADL (Collett et al., unpub- lished data) and HoCV Brescia cDNA clones (Moor- mann eta/., 1990) involving a stretch of five sequential adenosines, where six adenosines were the final correct nucleotide sequence.

The 5’ and 3’ end sequences of the SD-l genome were determined from the 5’-3’ ligation clones. Of nine clones sequenced, four clones have the 3’ end sequence of 5’. CAGCCCCC 3’, four clones have 5’

CAGCCCC 3’, and one clone has 5’ . . . CAGE 3’. Therefore, the dominant and longest sequence, 5’. CAGCCCCC 3’, is the authentic 3’end sequence of SD-l genome, which is identical to the 3’ end sequence of NADL. The complete nucleotide sequence consists of 12,308 nucleotides (Fig. 2), which is 270 bases less than CP BVDV strain NADL (12,578 nucleotides) and 124 bases less than Osloss genomes (12,430 nucleotides), but closer to HoCV strain Alfort and Brescia genomes (12,284 and 12,283 nucleotides, respectively). Base composition of the entire genome of SD-l is 32.2% A, 22.00/o U, 25.6% G, and 20.2% C and is similar to NADL RNA (Collett et a/., 1988a).

Analysis of the SD-l sequence revealed one transla- tional open reading frame (ORF) in the second phase of one strand. The other two reading phases of this strand and the three reading phases of thecomplemen- tary strand contain multiple stop codons throughout the sequence. No other significant ORF can be predicted in these reading phases. The large ORF starts with the AUG at position 386 to 388 and ends with a stop codon UGA at position 12,080 to 12,082. This ORF is capable of encoding a polyprotein of 3898 amino acids with the calculated molecular weight of 438 kDa (Fig. 2). The predicted amino acid sequence of the large ORF is given in Fig. 2. The 5’ untranslated region (5’UTR) preceding the large ORF consists of 385 nucleotides, including six AUG start codons and sev-

DENG AND BROCK

1 C TAT ACG AGA ACT AGA TAA AAT ACT 25 26 CGT ATA CAT ATT GGA CAA CAG AAA ATA ACT ATT AGG CCT AGG GAA TGA ATC CCT CTC AGC GAA GGC CGA AAG GAG GCT AGC CAT CCC CTT 115

116 ACT AGG ACT AGC ATA ATG AGG GGG GTA GCA ACA GTG GTG AGT TCG TTG GAT GGC TTA AGC CCT GAG TAC AGG GTA GTC GTC AGT GGT TCG 205 206 ACG CCT CGG TAT AAA GGT CTC GAG ATG CCA CGT GGA CGA GGG CAC GCC CAA AGC ACA TCT TAA CCT GAG CGG GGG TCG CCC AGG CAA AAG 295 296 CAG ATC GAC CAA TCT GTT ACG AAT ACA GCC TGA TAG GGT GCT GCA GAG GCC CAC TGT ATT GCT ACT AAA AAT CTC TGC TGT ACA TGG CAC 385

386 ATG GAG TTG ATT ACA AAT GAA CTT TTA TAC AAA ACA TAC AAA CAA AAA CCC GTC GGG GTG GAG GAA CC7 GTT TAC CAT CAG GCA GGT AAT 1 met Glu Leu Ile Thr Asn Glu Leu Leu Tyr Lys Thr Tyr Lys Gln Lys Pro Val Gly Val Glu Glu Pro Val Tyr Asp Gln Ala Gly Asn

475 30

476 CCC TTA TTT GGT GAA AGG GGA GCG ATC CAC CCT CAA TCG ACG CTA AAG CTC CCA CAC AAG AGA GGG GAA CCC AAT GTC CCC ACC AGT TTG 31 Pro Leu Phe Gly Glu Arg Gly Ala Ile His Pro Gln Ser Thr Leu Lys Leu Pro His Lys Arg Gly Glu Arg Asn Val Pro Thr Ser Leu

565 60

566 GCG TCT TTG CCA AAA AGA GGT GAC TGC AGG TCG GGT AAC AGC AAA GGA CCT GTG AGT GGT ATC TAC CTG AAG CCA GGG CCA CTA TTT TAC 61 Ala Ser Leu Pro Lys Arg Gly Asp Cys Arg Ser Gly Asn Ser Lys Gly Pro Val Ser Gly tie Tyr Leu Lys Pro Gly Pro Leu Phe Tyr

655 90

656 CAG GAC TAT AAA GGT CCC GTC TAC CAC AGG GCC CCA CTG GAG CTC TTT GAG GAG GGA TCT ATG TGT GAA ACA ACT AAA CCC ATA GGA AGG 91 Gln Asp Tyr Lys Gly Pro Val Tyr His Arg Ala Pro Leu Glu Leu Phe Glu Glu Gly Ser Met Cys Glu Thr Thr Lys Arg Ile Gly Arg

745 120

746 GTA ACT CCC ACT GAC GGC AAG CTG TAC CAC ATT TAT ATA TGT ATA GAT GGA TGT ATA ACA GTG AAG AGT GCC ACG AGA AGT CAC CAA AGG 121 Val Thr Gly Ser Asp Gly Lys Leu Tyr His Ile Tyr Ile Cys Ile Asp Gly Cys I1e Thr Val Lys Ser Ala Thr Arg Ser His Gln Arg

a35 150

a36 GTA CTT AGG TGG GTC cAc AAT AGG cTc GAC TGC ccc TTA TGG GTC ACA AGC TGC TCA GAT ACA AAA GAA GAA GGA GCA ACA AAG AAG AAA 151 Val Leu Arg Trp Vat His Am Arg Leu Asp Cys Pro Leu Trp Val Thr Ser Cys Ser Asp Thr Lys Glu Glu Gly Ala Thr Lys Lys Lys

925 180

926 CAA CAA AAA CCC GAC AGA TTA GAG AAA GGG AGG ATG AAG ATA GTG CCT AAA GAA TCT GAG AAA GAT AGT AAG ACC AAA CCC CCT CAT GCT 1;;; 181 Gln Gln Lys Pro Asp Arg Leu Glu Lys Gly Arg Met Lys Ile Val Pro Lys Glu Ser Glu Lys Asp Ser Lys Thr Lys Pro Pro Asp Ala

1016 ACA ATA GTG GTA CAT CGA GTC AAA TAC CAA GTA AAG AAG AAG GGG AAA GTC AAG ACT AAA AAC ACA CAA GAC GGT TTA TAC CAT AAC AAA 1;:; 211 Thr Ile Vat Val Asp Gly Val Lys Tyr Gln Val Lys Lys Lys Gly Lys Val Lys Ser Lys Am Thr Gln Asp Gly Leu Tyr His Asn Lys

1106 AAT AAG CCC CCA GAA TCA CGC AAG AAA CTT GAG AAA GCA TTG TTG GCA TGG GCA ATA TTG GCC GTA GTC TTG ATT GAA GTT ACA ATG CGA 1195 241 Asn Lys Pro Pro Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala Trp Ala Ile Leu Ala Val Val Leu Ile Glu Val Thr Met Gly 270

1196 GAA AAT ATA ACA CAG TGG AAC TTA CAA GAC AAT CCC ACA GAA GGG ATA CAA CGG GCA ATG TTT CAA AGG GGG GTG AAC AGA ACT CTA CAC 1:;; 271 Glu Asn Ile Thr Gln Trp Asn Leu Gin Asp Asn Glv Thr GIu Gly Ile Gln Arg Ala Met Phe Gln Arg Gly Val Am Arg Ser Leu His

1286 CGA ATC TGG CCA GAG AAG ATC TGT ACA GGT GTC ccT Tee CAT CTA Gee Act CAT GTG GAG CTA AAA ACA ATC CAT GGT ATG ATG GAT GCA 1375 301 Gly Ile Trp Pro G1u Lys Ile Cys Thr Gly Val Pro Ser His Leu Ala Thr Asp Val Glu Leu Lys Thr Ile His Gly Met Met Asp Ala 330

1376 ACT GAA AAG ACC AAC TAC ACG TGC TGC AGA CTT CAA CGC CAT GAG TGG AAC AAG CAT GGT TGG TGC AAC TGG TAC AAT ATT GAG CCT TGG 1465 331 Ser Glu Lys Thr Asn Tyr Thr Cys Cys Arg Leu Gln Arg His Glu Trp Am Lys His Gly Trp Cys Asn Trp Tyr Am Ile Glu Pro Trp 360

1466 ATT TTA ATC ATG AAT AGA ACC CAA GCC AAT CTC ACT'GAG GGT CAA CCA CCA AGA GAA TGT GCA GTC ACG TGC AGG TAT GAT AGG GAT AGT 1555 361 Ile Leu Ile Met Asn Arg Thr Gin Ala Am Leu Thr Glu Gly Gln Pro Pro Arg Glu Cys Ala Val Thr Cys Arg Tyr Asp Arg Asp Ser 390

1556 GAC CTA AAT GTG GTA ACA CAA GCT AGA GAC AGT CCC ACG CCA TTA ACA GGC TGC AAG AAA CGA AAA AAC TTC TCT TTT GCT GGC GTA CTG '64; 391 Asp Leu Am Val Val Thr Gin Ala Arg Asp SW Pro Thr Pm Leu Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe Ala Gly Val Leu

1646 ACG CGG GGT CCT TGC AAC TTT GAA ATA GCT GCG AGT GAT GTA TTG TTC AAA GAA CAT GAA TGC ACT GGT GTG TTT CAG GAT ACT GCT CAT 1735 421 Thr Arg Gly Pro Cys Asn Phe Glu Ile Ala Ala Ser Asp Val Leu Phe Lys Glu His Glu Cys Thr Gly Val Phe Gln Asp Thr Ala His 450

1736 TAC CTT GTT GAC GGG GTG ACC AAT TCA TTG GAG AGT GCC AGA CAA GGG ACA GCT AAA TTG ACA ACC TGG TTA GGC AAA CAG CTC GGG ATC 1;;; 451 Tyr Leu Val Asp Gly Val Thr Asn Ser Leu Glu Ser Ala Arg Gln Gly Thr Ala Lys Leu Thr Thr Trp Leu Gly Lys Gln Leu Gly Ile

1826 CTA GGG AAA AAG TTG GAA AAT AAA AGC AAG ACA TGG TTT GGA GCT TAT GCA GCT TCC CCT TAC TGT GAT GTC CAT CGA AAA ATT GGC TAC 1915 481 Leu Gly Lys Lys Leu Glu Am LYS Ser Lys Thr Trp Phe Gly Ala Tyr Ala Ala Ser Pro Tyr Cys Asp Val Asp Arg Lys Ile Gly Tyr 510

1916 ATA TGG TTT ACA AAA AAC TGC ACC CCT GCT TGC TTG CCT AAG AAC ACA AAA ATC ATC GGC CCT GGG AAG TTT GAC ACC AAT GCA GAG GAT 2005 511 Ile Trp Phe Thr Lys Am CYS Thr Pro Ala Cys Leu Pro Lys Am Thr Lys Ile Ile Gly Pro Gly Lys Phe Asp Thr Am Ala Glu Asp 540

2006 GGC AAG ATA CTG CAT GAG ATG GGG GGT CAC TTG TCG GAG GTA CTA CTA CTT TCT TTA GTA GTG TTG TCT GAC TTT GCA CCA GAA ACG GCT 2095 541 Gly Lys Ile Leu His Glu Met Gly Gly His Leu Ser Glu Val Leu Leu Leu Ser Leu Val Val Leu Ser Asp Phe Ala Pro Glu Thr Ala 570

2096 AGC GCA ATG TAT CTG ATC CTA CAT TTT TCC ATC CCG CAA AGC CAC GTT CAT ATA ACG GAC TGT GAT AAG ACC CAG CTG AAC CTC ACC ATA 2185 571 Ser Ala Met Tyr Leu Ile Leu His Phe Ser Ile Pro Gln Ser His Val Asp Ile Thr Asp Cys Asp Lys Thr Gln Leu Am Leu Thr Ile 600

2186 GAG CTT ACA ACA GCA CAT GTA ATA CCA GGG TCG GTC TGG AAT TTA GGC AAA TAT GTC TGC ATA AGA CCA GAT TGG TGG CCT TAT GAG ACA 2:;; 601 Glu Leu Thr Thr Ala Asp Val Ile Pro Gly Ser Val Trp Asn Leu Gly Lys Tyr Val Cys Ile Arg Pro Asp Trp Trp Pro Tyr Glu Thr

2276 GCT GCA GTG CTG GCA TTC GAA GAG GTG GGT CAG GTG GTA AAA ATA GTG CTG AGG GCA CTT AGA CAT CTG ACA CGC ATT TGG AAC GCT GCC 2365 631 Ala Ala Val Leu Ala Phe Glu Glu Val Gly Gln Val Val Lys Ile Val Leu Arg Ala Leu Arg Asp Leu Thr Arg Ile Trp Asn Ala Ala 660

2366 ACG ACC ACA GCA TTC TTA GTG TGC CTA ATT AAG ATG GTC AGG GGC CAG GTG GTA CAG GGC ATT CTG TGG TTA CTA CTG ATA ACA GGG GTA 2455 661 Thr Thr Thr Ala Phe Leu Val Cys Leu Ile Lys Met Val Arg Gly Gln Val Val Gln Gly Ile Leu Trp Leu Leu Leu Ile Thr Gly Val 690

2455 CAA GGG CAC CTA GAC TGC AAA CCT GAA TAC TCA TAC GCC ATA GCC AAG AAT GAT AGA GTT GGC CCA CTA GGA GCT GAA GGA CTT ACC ACC 2545 691 Gln Gly His Leu Asp Cys Lys Pro Glu Tyr Ser Tyr Ala Ile Ala Lys Asn Asp Arg Vat Gly Pro Leu Gly Ala Glu Gly Leu Thr Thr 720

2546 GTT TGG AAG GAC TAT TCA CAT GAA ATG AAG CTG GAA GAC ACA ATG GTT ATA GCT TGG TGC AAA GGT GGC AAG TTT ACG TAC CTC TCA AGG 2635 721 Val Trp Lys Asp Tyr Ser His Glu Met Lys Leu Glu Asp Thr Met Val Ile Ala Trp Cys Lys Gly Gly Lys Phe Thr Tyr Leu Ser Arg 750

2636 TGC ACA AGA GAA ACC AGA TAT CTC GCT ATC TTA CAT TCA AGA GCC TTA CCC ACC AGT GTG GTG TTC AAA AAA CTT TTT GAA GGG CAA AAG 2725 751 Cys Thr Arg Glu Thr Arg Tyr Leu Ala Ile Leu His Ser Arg Ala Leu Pro Thr Ser Val Val Phe Lys Lys Leu Phe Glu Gly Gln Lys 780

2726 CAA GAG GAC ACA GTC GAA ATG GAT GAC GAC TTT GAA TTT GGT CTT TGC CCA TGC GAT CCC AAA CCT ATA GTA AGA GGG AAG TTC AAT ACA 2815 781 Gln Glu Asp Thr Val Glu Met Asp Asp Asp Phe Glu Phe Gly Leu Cys Pro Cys Asp Ala Lys Pro Ile Val Arg Gly Lys Phe Am Thr ai0

2816 ACA CTG CTA AAC GGA CCG GCC TTC CAA ATG GTA TGC CCC ATA GGA TGG ACA GGG ACT GTG AGC TGT ATG TTA GCT AAT AGG CAT ACC CTA 2905 811 E Leu Leu Asn Gly Pro Ala Phe Gln Met Val Cys Pro Ile Gly Trp Thr Gly Thr Val Ser Cys Met Leu Ala Asn Arg Asp Thr Leu 840

2906 GAC ACA GCA GTA GTA CCC ACG TAC AGG AGG TCC GTA CCA TTC CCC TAT AGG CAG GGC TGT ATC ACC CAA AAA ACT CTG GGG GAG CAT CTC 2995 841 Asp Thr Ala Vat Val Arg Thr Tyr Arg Arg Ser Val Pro Phe Pro Tyr Arg Gin Gly Cys Ile Thr Gln Lys Thr Leu Gly Glu Asp Leu 870

2996 TAT GAC TGT GCC CTC CGA GGA AAC TGG ACT TGC GTG ACT GGG GAC CAG TCA CGA TAC ACA GGA GGC CTT ATC GAA TCC TGT AAG TGG TGT 33; 871 Tyr Asp Cys Ala Leu Gly Gly Am TrD Thr Cys Val Thr Gly Asp Gln Ser Arg Tyr Thr Gly Gly Leu 11e Glu Ser Cys Lys Trp Cys

3086 GGT TAT AAA TTT CAA AAA AGT GAG GGG TTA CCL; CAC TAC CCT ATT GGT AAG TGT AGG TTG AAT AAT GAA ACT GGC TAC AGA TTA GTA GAT 3175 901 Gly Tyr Lys Phe Gin Lys Ser Glu Gly Leu Pro His Tyr Pro Ile Gly Lys Cys Arg Leu Asn Asn Glu Thr Gly Tyr Arg Leu Vat Asp 930

FIG. 2. The complete genomic nucleotide sequence of the genomic RNA of NCP BVDV strain SD-1 The deduced amino acid sequence of the large ORF is shown in three-letter code below the nucleotide sequence. Boxed sequence at the N-terminal of viral glycoprotein indicates the viral signal sequence. The putative glycosylation sites in the viral glycoprotein region are underlined. The double-underlined sequence is the cysteine-rich stretch. AAA indicates the catalytic triad of His, Asp, and Ser residues of the viral p80 proteinase. 000 denotes the tripeptide sequence highly conserved in viral RNA dependent RNA polymerases.

NCP BVDV STRAIN SD-1 871

3176 GAC ACC TCT TGC GAC AGA GAA GGT GTG GCC ATA GTA CCA CAT GGG CTG GTA AAG TGT AAG ATA GGG GAC ACA ACT GTA CAG GTC A,TA GCT 3265 931 Asp Thr Ser Cys Asp Arg GLU G1y Va1 Ala Ile Va1 Pro His G1y Leu Va1 Lys Cys Lys 11e C1y Asp Thr Thr Va1 G1n Va1 11-z A1a 960

3266 ACT GAC ACC AAA CTT GGG CC1 ATG CCT TGC AAA CCA CAT GAG ATC ATA TCA AGT GAG GGG CCT ATA GAA AAG ACA GCA TGC ACC TTC AAT 961 Thr Asp Thr Lys Leu G1y Pro Met Pro Cys Lys Pro His Glu 11e 11e Ser Ser Glu G1y Pro 11e G1u Lys Thr ALa Cys Thr Phe m

3355 990

3356 TAC ACG AGG ACA TTA AAA AAT AAA TAT TTT GAA CCC AGA GAC ACT TAC TTC CAG CAA TAC ATG CTA AAG GGA GAT TAT CAA TAC TGG TTT 991 Tyr Thr Arg Thr Leu Lys Asn Lys Tyr Phe Glu Pro Arg Asp Ser Tyr Phe Cln Gin Tyr Met Leu Lys Gly Asp Tyr Gln Tyr Trp Phe

344s 1020

3446 GAC CTG GAG GTC ACT CAC CAT CAT CGG GAT TAC TTT GCC GAG TCC ATA TTA GTG GTG GTG GTA GCT CT1 CTA GGT GGT AGA TAT GTA CTC 1021 Asp Leu Glu Va1 Thr Asp His His Arg Asp Tyr Phe Ala G1u Ser 11e LeU Va1 Va1 Va1 Val Ala Leu Leu Gly G1y Arg Tyr Va1 Leu %5

3536 TGG TTA CTG GTC ACA TAC ATG GTT CTA TCA GAA CAA AAG GCC TCA GGG GCC CAG TAT GGG GCA GGG GAG GTG GTG ATG ATG GGC AAC TTG 3625 1051 Trp Leu Leu Vat Thr Tyr Met Vat Leu Ser Glu Gin Lys Ala Ser G1y Ala Gln Tyr G1y Ala Gly G1u Va1 Va1 Met Met Gly Asn Leu 1080

3626 CTA ACA CAT CAT AAT GTT GAA GTA GTG ACA TAT TTC TTC TTA CTA TAC CTG CTG CTA AGA GAG GAG AGT GTA AAG AAG TGG GTC TTA CTC 1081 Leu Thr His Asp Am Va1 Glu Val Va1 Thr Tyr Phe Phe Leu Leu Tyr Leu Leu Leu Arg G1u Glu Ser Va1 Lys Lys Trp Val Leu Leu

3715 1110

3716 TTA TAC CAC ATC TTG GTG GCA CAC CCA TTA AAG TCA GTG ATA GTG ATC CTA TTA ATG ATT GGG CAT GTG GTG AAG GCT GAC CCA GGG GGC 1111 Leu Tyr His Ile Leu Va1 Ala His Pro Leu Lys Ser Va1 11e Val Ile Leu Leu Met 11e G1y Asp Vat Va1 Lys Ala Asp Pro G1y Gly

3805 1140

3806 CAA GGC TAC CTG GGT CAG ATA GAT CTC TGC TTC ACA ATG GTT GTA AlA ATC ATA ATA GGT TTG ATC ATA GCC AGG CC1 GAT CCC ACC ATA 1141 G1n G1y Tyr Leu G1y G1n Ile Asp Val Cys Phe Thr Met Vat Val Ile 11e 11e ILe Gly Leu Ile 11e A1a Arg Arg Asp Pro Thr Ile

3895 1170

3896 GTA CCA CTG ATC ACA ATA GTA GCA TCG CTG AGG GTC ACT GGA TTG ACC TAC AGC CC1 GGC GTG GA1 GCA CCA ATG GCA GTC ATA ACC ATA 1171 Va1 Pro Leu Ile Thr ILe Va1 Ala Ser Leu Arg Val Thr Gly Leu Thr Tyr Ser Pro Gly Val Asp A1a A1a Met A1a Val Ile Thr Ile

3905 1200

3906 ACC TTG CTG ATG GTT AGC TAT GTG ACA GAT TAC TTC AGA TAT AAA AGA TGG CTG CAG TGC ATC CTC AGC CTG GTA TCA GGG GTG TTC TTG 4075 1201 Thr Leu Leu Met Va1 Ser Tyr Val Thr Asp Tyr Phe Arg Tyr Lys Arg Trp Leu Gln Cys 11e Leu Ser Leu Va1 Ser Gly Va1 Phe Leu 1230

4076 ATA AGA TGC CTA AlA CAC CTA GGT AGA ATT GAG ACG CCA GAG GTG ACC ATC CCA AAC TGG AGA CCA CTA ACC TTA ATA CTG TTT TAT CTG 4165 1231 Ile Arg Cys Leu 11e His Leu Gly Arg Ile Glu Thr Pro Glu Val Thr Ile Pro Asn Trp Arg Pro Leu Thr Leu Ile Leu Phe Tyr Leu 1260

4166 ATT TCA ACA ACA GTT GTT ACA ATG TGG AAC ATT GAC TTG CCC GGC CTA TTA TTG CAA GGT GTG CCT ATC TTA TTG CTG ATC ACG ACC CTG 4255 1261 ILe Ser Thr Thr Va1 Va1 Thr Met Trp Lys Ile Asp Leu Ala Gly Leu Leu Leu Gln Gly Va1 Pro Ile Leu Leu Leu ILe Thr Thr Leu 1290

4256 TGG CCC GAC TTT TTA ACC CTC ATA CTG ATA CTG CCA ACC TAT GAA CTG GTT AAG TTA TAC TAC TTG AAA ACT ATT AAG ACT GAT ATA GAA 4345 1291 Trp Ala Asp Phe Leu Thr Leu Ile Leu 11e Leu Pro Thr Tyr Glu Leu Val Lys Leu Tyr Tyr Leu Lys Thr Ile Lys Thr Asp Ile Glu 1320

4346 AAA AGC TGG CTG GGG GGG TTA GAC TAT AAG AGA GTT GAC TCC ATC TAC GAT GTT GA1 GAG AGT GGA GAG GGC GTA TAT CTT TTC CCA TCT 1321 Lys Ser Trp Leu G1y Gly Leu Asp Tyr Lys Arg Va1 Asp Ser Ile Tyr Asp Va1 Asp Glu SW G1y Glu Gly Va1 Tyr Leu Phe Pro Ser

4436 AGG CAG AAG GCA CAG AAA AAC TTC TCC ATG CT1 TTG CCT CTT GTG AGA GCA ACA CTG ATA AGT TGT GTC AGT AGT AAA TGG CAG CTA ATA 1351 Arg Gin Lys Ala G1n Lys Asn Phe Ser Met Leu Leu Pro Leu Va1 Arg ALa Thr Leu I1e Ser Cys Va1 Ser SW Lys Trp Gln Leu Ile

4526 TAC ATG GCT TAC TTA TCT GTG GAC TTT ATG TAC TAC ATG CAC AGG AAA GTT ATA GAG GAG ATC TCA GGA GGC ACT AAC ATG ATA TCC AGG 1381 Tyr Met Ala Tyr Leu Ser Val Asp Phe Met Tyr Tyr Met His Arg Lys Val Ile Glu Glu Ile Ser Gly GLy Thr Am Met Ile Ser Arg

4616 ATA GTG GCG GCA CTT ATA GAG CTG AAT TGG TCC ATG GAA GAA GAG GAA AGT AAA GGC TTG AAG AAG TTT TAT TTA TTA TCT GGA AGG CTG 1411 Ile Va1 Ala A1a Leu Ile Glu Leu Am Trp Ser Met G1u G1u G1u G1u SW Lys Gly Leu Lys Lys Phe Tyr Leu Leu SW Gly Arg Leu

4706 AGA AAC CTA ATA ATA AAA CAC AAA GTA AGA AAT GAG ACC GTG CCC GGC TGG TAC GGA GAG GAG GAA GTC TAC GGC ATG CCA AAG ATC ATG 1441 Arg Am Leu Ile Ile Lys His Lys Va1 Arg Am G1u Thr Va1 A1a Gly Trp Tyr G1y G1u GLU G1u Va1 Tyr Gly Met Pro Lys Ile Met

4796 ACC ATA ATC AAG CC1 AGT ACG CTG AAT AAG AAT AAA CAC TGC ATA ATA TGC ACT GTA TGT GAA GGC CGG AAG TGG AAA GGT GGT ACC TGC 1471 Thr 11e Ile Lys A1a Ser Thr Leu Am Lys Asn Lys His ms Thr Va1 Cys Glu G1y Arg LYS 1~ Lys Gly G1y Thr Cys

4886 CCA AAA TGT GGA CGT CAT GGG AAA CCC ATA ACG TGT GGG ATG TCA CTG GCA GAT TTT GAA GAA AGG CAT TAT AAA AGG ATC TTC ATA AGG 1501 Pro Lys Cys C1y Arg Hi&LVs Pro I1e Thr Cys Gly Met Ser Leu Ala Asp Phe G1u G1u Arg His Tyr Lys Arg ILe Phe Ile Arg

4976 GAA GGC AAC TTT GAA GGC CCG TTC AGA CAA GAA TAT AAT GGC TTT ATC CAA TAT ACC GCT AGG GGG CAA TTG TTT TTG AGA AAC TTG CCC 5065 1531 G1u G1y Asn Phe G1u G1y Pro Phe Arg G1n G1u Tyr Asn G1y Phe I1e Gln Tyr Thr A1a Arg G1y Gln Leu Phe Leu Arg Am Leu Pro 1560

5066 ATA CTG GCA ACA AAA GTA AAA ATG CTC ATG GTG GGT AAC CT1 CGA GAA GAA GTT GGG GAC CTA GAA CAC CTT GGG TGG ATC CTA AGG GGG 1561 Ile Leu A1a Thr Lys Va1 Lys Met Leu Met Va1 Gly Am Leu Gly Glu G1u Val Gly Asp Leu G1u His Leu Gly Trp Ile Leu Arg G1y

5155 1590

5156 CC1 CCC GTG TGT AAG AAG ATC ACA GAA CAC GAG AGA TGC CAC ATC AAC ATA TTA GAT AAA CTA ACT GCA TTT TTC GGG ATC ATG CCA AGA 5245 1591 Pro Ala Va1 Cys Lys Lys 11e Thr G1u His GLu Arg Cys His Ile Asn Ile Leu Asp Lys Leu Thr A1a Phe Phe G1y 11e Met Pro Arg 1620

5246 GGG ACC ACA CCC AGA CCC CCA GTG AGG TTT CCT ACG AGT TTG TTA AAA GTG AGG AGG GGT CTA GAG ACT GGT TGG GCT TAT ACA CAC CAA 5335 1621 G1y Thr lhr Pro Arg A1a Pro Val Arg Phe Pro Thr Ser Leu Leu Lys Val Arg Arg GLy Leu G1u Thr Gly Trp Ata Tyr Thr His G1n 1650

5336 GGT GGG AlA ACT TCA GTC GAC CAT GTA ACC GCA GGC AAA GA1 CTA CTG GTC TGT GAC ACT ATG CGA AGA ACT AGA GTG GTC TGC CAG AGC 1651 G1y Gly Ile Ser Ser Val Asp His Va1 Thr Ala Gly Lys Asp Leu Leu Vat Cys Asp Ser Met G1y Arg Thr Arg Val Va1 Cys Gln Ser

. . .

5425 1680

5426 AAC AAT AAG TTA ACT CAT GAG ACA GAG TAT GGT GTC AAG ACT GAC TCA GGA TGC CCA GAT GGT GCC AGA TGT TAC GTG CTG AA1 CCA GAG 1681 Am Asn Lys Leu Thr Asp Glu Thr G1u Tyr G1y Va1 Lys Thr Asp Ser G1y Cys Pro Asp G1y Ala Arg Cys Tyr Va1 Leu Am Pro G1u

1.1

5515 1710

5516 CCC GTC AAC ATA TCG GGT TCC AAA GGG GCA GTC GTC CAC CTC CAA AAG ACA GGT GGG GAA TTC ACG TGT GTC ACC GCA TCA GGC ACA CCG 5605 1711 Ala Va1 Am 11e Ser G1y Ser Lys G1y A1a Val Va1 His Leu Gin Lys Thr G1y G1y G1u Phe Thr Cys Va1 Thr A1a Ser Gly Thr Pro 1740

5606 GCT TTC TTT GAT CTA AAA AAC TTG AAG GGA TGG TCG GGC CTA CCT ATA TTT GAA GCC TCC AGC GGA AGG GTG GTT GGT AGA GTT AAG GTG 1741 A1a Phe Phe Asp Leu Lys Asn Leu Lys G1y Trp Ser G1y Leu Pro Ile Phe G1u Ala St-r Ser Gly Arg Vat Vat Gly Arg Va1 Lys Va1

I..

5695 1770

5696 GGG AAG AAT GAA GAA TCC AAA CCC ACG AAG ATA ATG ACT GGC ATC CAG ACC GTC TCA AAG AAC ACA GCA GA1 CTG ACC GAG ATG GTC AAG 5785 1771 Gly Lys Am Glu G1u Ser Lys Pro Thr Lys Ile Met Ser G1y 11e G1n Thr Va1 SW Lys Asn Thr Ala Asp Leu Thr G1u Met Va1 Lys 1800

5786 AAG ATA ACT AGT ATG AAT AGG GGA GAC TTC AAG CAG ATA ACA TTA GCA ACA GGG GCA GGA AAG ACC ACA GAA CT1 CCA AAG GCA GTG ATA 1801 Lys Ile Thr Ser Met Asn Arg Gly Asp Phe Lys Gln Ile Thr Leu A1a Thr G1y A1a G1y Lys Thr Thr Glu Leu Pro Lys Ala Va1 Ile

5875 1830

5076 GAG GAG AlA CGA AGA CAT AAG CGA GTG CTA GTC CTT ATA CCA CTG AGG GCA GCA GCA GAG TCA GTC TAC CAG TAT ATG AGA TTG AAA CAC 5965 1831 G1u Glu Ile Gly Arg His Lys Arg Vat Leu Va1 Leu Ile Pro Leu Arg A1a Ala Ala G1u Ser Val Tyr Gin Tyr Met Arg Leu Lys His 1860

5966 CCA AGT ATC TCT TTT AAC CTA AGG ATA GGG GAT ATG AAG GAG GGG GAC ATG GCA ACT GGG ATA ACC TAT GCA TCA TAC GGG TAC TTC TGC 6055 1861 Pro Ser Ile Ser Phe Asn Leu Arg 11e Gly Asp Met Lys Glu Gly Asp Met A1a Thr G1y Ile Thr Tyr Ala Ser Tyr Gly Tyr Phe Cys 1890

6056 CAG ATG CCT CAA CCA AAG CT1 AGA GCA GCT ATG GTT GAA TAC TCG TAC ATA TTC CTA GA1 GAA TAC CAC TGT GCC ACT CCT GAA CAG TTG 6145 1891 Gin Met Pro Gln Pro Lys Leu Arg Ala A1a Met Va1 Glu Tyr Ser Tyr ILe Phe Leu Asp Glu Tyr His Cys Ala Thr Pro Glu Gln Leu 1920

6146 GCA ATT AK GGA AAA ATC CAC AGA TTT TCA GAG AGT ATA AGA GTG GTT GCC ATG ACT CC1 ACC CCA GCA GGG TCG GTA ACC ACA ACA CGA 6235 1921 Ala Ile Ile Gly Lys Ile His Arg Phe Ser Glu Ser Ile Arg Va1 Val A1a Met Thr Ala Thr Pro Ala Gly Ser Val Thr Thr Thr Gly 1950

FIG. 2-Continued

4435 1350

4705 1440

4795 1470

4975 1530

a72 DENG AND BROCK

6236 CAA AAG CAC CCA ATA GAG GAA TTT ATA GCC CCC GAG GTG ATG GAA GGG GAA CAT CTA GGC AGC CAG TTC CTT CAT ATA GCG GGG CTA AM 6325 1951 G1n Lys His Pro Ile Glu Glu Phe Ile Ala Pro Glu Vat Met Glu Gly Glu Asp Leu Gly Ser Gin Phe Leu Asp Ile Ala Gly Leu Lys 1980

6326 ATC CCC GTG CAT GAG ATG AAG GGT AAC ATG TTG GTT TTC GTG CCC ACG AGA AAC ATG GCA GTT GAG GTG GCT AAG AAG CTA AAA GCT AAG 6415 1981 Ile Pro Vat Asp GLu Net Lys Gly Am Met Leu Val Phe Val Pro Thr Arg Am Met Ala Vat Glu Val Ala Lys Lys Leu Lys Ala Lys 2010

6416 GGC TAC AAT TCT GGG TAC TAT TAC ACT CGA GAG GAT CCA GCT AAT CTG AGA GTT GTA ACA TCA CAG TCC CCC TAT GTA ATT GTG CCC ACG 6505 2011 Gly Tyr Asn Ser G1y Tyr Tyr Tyr Ser Gly Glu Asp Pro Ala Asn Leu Arg Val Val Thr Ser Gin Ser Pro Tyr Vat Ile Val Ala Thr 2040

6506 AAT CCC ATC GAG TCG CGA GTG ACA TTA CCA GAC TTG GAC ACA GTC GTG CAT ACA GGG TTG AAA TGT GAA AAG AGA GTC AGG GTG TCG TCA 6595 2041 Asn Ala Ile Glu Ser G1y Val Thr Leu Pro Asp Leu Asp Thr Val Val Asp Thr Gly Leu Lys Cys G1u Lys Arg Val Arg Vat Ser Set 2070

6596 AAG ATA CCA TTC ATC GTA ACA CCC CTT AAG AGG ATG GCT GTA ACT GTG GGT GAA CAG CCC CAA CGC AGG GGT AGA GTA GGT AGA GTG AAA 2071 Lys ILe Pro Phe Ile Vel Thr Gly Leu Lys Arg Met Ala Vat Thr Val Gly Glu Gin Ala Gln Arg Arg Gly Arg Val Gly Arg Vel Lys

6685 2100

6686 CCC GGG AGA TAT TAT AGG AGC CAA GAA ACA GCA ACC GGG TCA AAG CAC TAT CAC TAT GAC CTC TTG CAG GCA CAG AGA TAC GGT ATT GAG 2101 Pro Gly Arg Tyr Tyr Arg Ser Gln Glu Thr Ala Thr Gly Ser Lys Asp Tyr His Tyr Asp Leu Leu Gin ALa Gin Arg Tyr Gly Ile Glu

6776 CAT GGA ATC AAC GTA ACA AAA TCC TTC AGG GAA ATG AAT TAC GAC TGG ACT CTA TAC GAG GAG GAC AGC CTG CTA ATA ACC CAG TTA GAA 6865 2131 Asp Gly Ile Asn Vat Thr Lys Ser Phe Arg Glu Met Am Tyr Asp Trp Ser Leu Tyr Glu Glu Asp Ser Leu Leu Ile Thr Gin Leu G1u 2160

6866 ATA CTA AAT AAC TTA CTC ATT TCA GAA GAC TTG CCG GCC GCT GTC AAG AAC ATA ATG GCC AGG ACT CAT CAC CCA GAG CCC ATC CAA CTT 2161 Ile Leu Asn Asn Leu Leu Ile SW Glu Asp Leu Pro Ala Ala Vat Lys Am lie Met ALa Arg Thr Asp His Pro Glu Pro Ile Gln Leu

6956 GCA TAC AAC AGC TAC GAG GTC CAG GTC CCA GTC CTG TTC CCA AAA ATA AGG AAT GGA GAA GTC ACA GAC ACC TAT GAA AAC TAC TCA TTT 2191 Ala Tyr Asn Ser Tyr Glu Val Gin Val Pro Val Leu Phe Pro Lys Ile Arg Am Gly Glu Va1 Thr Asp Thr Tyr Glu Asn Tyr Ser Phe

7045 2220

7046 CTA AAC GCT AGA AAA TTA CGA GAG CAT GTG CCT GTT TAC ATC TAT CCC ACT GAA CAT GAG GAC CTG GCA GTT GAC CTA CTA GGG CTA GAC 7135 2221 Leu Am Ala Arg Lys Leu Gly Glu Asp Va1 Pro Val Tyr Ile Tyr A1a Thr Glu Asp G1u Asp Leu Ala Val Asp Leu Leu Gly Leu Asp 2250

7136 TGG CCC CAT CCT GGG AAC CAA CAG GTT GTG GAA ACT GGT AAA GCA CTA AAG CAA GTG CCC GGG TTG TCC TCA GCT GAG AAT CCC CTG CTC 7225 2251 Trp Pro Asp Pro Gly Am Gln Gln Val Val Glu Thr Gly Lys Ala Leu Lys Gin Val Ala Gly Leu Ser Ser Ala Glu Asn Ala Leu Leu 2280

7226 GTG GCT TTA TTC GGG TAT GlA GGT TAC CAA GCT TTA TCA AAG AGA CAL: GTC CCA ATG ATC ACA CAT ATA TAC ACC ATA GAG GAC CAG AGA 7315 2281 Val Ala Leu Phe Gly Tyr Vat Gly Tyr Gin Ala Leu Ser Lys Arg His Val Pro Met Ile Thr Asp Ile Tyr Thr Ile Glu Asp Gln Arg 2310

7316 CTA GAA GAC ACC ACC CAC CTC CAG TAT GCA CCC AAC GCC ATA AAA ACT GAG GGA ACA GAA ACT GAG CTA AAG GAA TTG GCG TCG GGC GAC 7405 2311 Leu Glu Asp Thr Thr His Leu Gin Tyr Ala Pro Am Ala Ile Lys Thr Glu Gly Thr Glu Thr Glu Leu Lys Glu Leu Ala Ser Gly Asp 2340

7406 GTG GAA AAA ATA ATG CGA GCC ATT TCA GAT TAT GCA GCT GGG GGA CTG CAT TTT GTA AAA TCT CAA GCA GAA AAG ATA AAA ACA GCC CCT 7495 2341 Val Glu Lys Ile Met Gly Ala Ile Ser Asp Tyr Ala Ala Gly G1y Leu Asp Phe Val Lys Ser G1n Ala Glu Lys Ile Lys Thr Ala Pro 2370

7496 TTG TTT AAG GAA AAT GTA GAA GCT GCA AGG GGG TAT GTC CAA AAA CTC ATT GAC TCA TTA ATT GAG CAT AAA GAT GTA ATA ATC AGA TAT 7585 2371 Leu Phe Lys Glu Asn Val Glu Ala ALa Arg Gly Tyr Val Gin Lys Leu Ile Asp Ser Leu Ile Glu Asp Lys Asp Val lie Ile Arg Tyr 2400

7586 GCC TTA TGG GGA ACA CAT ACA GCG CTC 1AT AAA AGC ATA GCT GCA AGA TTG GGG CAT GAA ACA GCG TTT GCC ACA CTA GTG TTG AAA TGG 7675 2401 Gly Leu Trp GLy Thr His Thr Ala Leu Tyr Lys Ser Ile Ala ALa Arg Leu Gly His Glu Thr Ala Phe Ale Thr Leu Val Leu Lys Trp 2430

7676 CTA GCC TTT GGA GGG GAG ACG GTG TCA GAC CAC ATC AGA CAG GCG GCA GTT GAT TTA GTG GTC TAT TAT GTG ATG AAC AAG CCT TCC TTC 7765 2431 Leu ALa Phe Gly Gly Glu Thr Val Ser Asp His Ile.Arg Gin Ale Ala Val Asp Leu Val Val Tyr Tyr Val Met Asn Lys Pro Ser Phe 2460

7766 CCA CCC CAT ACT GAA ACT CAG CAA GAA GGG AGG CCC TTT GTC GCA AGC CTG TTC ATC TCC GCA TTG GCA ACT TAC ACG TAC AAA ACC TGG 2461 Pro Gly Asp Thr GLu Thr Gin Gin Glu Gly Arg Arg Phe Vat Ala Ser Leu Phe Ile Ser Ala Leu Ala Thr Tyr Thr Tyr Lys Thr Trp

7856 AAT TAT AAT AAT CTC TCA AAA GTG GTG GAA CCG GCC CTG GCA TAC CTC CCC TAT CCC ACC AGC GCA CTA AAA ATG TTC ACC CCA ACG AGA 7945 2491 Asn Tyr Asn Asn Leu Ser Lys Val Val Glu Pro Ala Leu Ala Tyr Leu Pro Tyr Ala Thr Ser Ala Leu Lys Met Phe Thr Pro Thr Arg 2520

7946 CTG GAA AGC GTG GTG ATA CTA AGT ACC ACT ATA TAT AAA ACA TAC CTC TCC ATA AGA AAA GGG AAG AGT GAT GGA TTG CTG GGT ACG GGG 8035 2521 Leu Glu Ser Val Val Ile Leu Ser Thr Thr Ile Tyr Lys Thr Tyr Leu Ser Ile Arg Lys Gly Lys Ser Asp Gly Leu Leu GLy Thr Gly 2550

8036 ATC ACT GCA GCT ATG GAG ATC CTG TCA CAA AAC CCA GTA TCG GTA GGT ATA TCC GTG ATG CTG GGG GTA GGG GCT ATT GCT GCG CAC AAC 8125 2551 Ile Ser Ale Ala Met GLu Ile Leu Ser Gin Asn Pro Val Ser Val Gly Ile Ser Val Met Leu Gly Val Gly Ala Ile Ala Ala His Asn 2580

8126 CCC ATT GAA TCC AGC GAG CAG AAA AGG ACC CTA CTT ATG AAG GTG TTT GTA AAG AAC TTC TTA GAT CAG GCC GCA ACG GA1 GAG CTG GTT 2581 Ala Ile GLu Ser Ser G1u GLn Lys Arg Thr Leu Leu Met Lys Val Phe Val Lys Asn Phe Leu Asp Gin Ala Ala Thr Asp Glu Leu Val

8216 AAA GAA AAC CCA GAA AAA ATC ATA ATG GCT CTA TTT GAA GCA GTA CAG ACA ATA GGC AAC CCC TTG AGA TTA ATA TAC CAC CTG TAT GGG 8305 2611 Lys Glu Asn Pro Glu Lys I(e Ile Met Ala Leu Phe Glu Ala Val Gln Thr Ile Gty Am Pro Leu Arg Leu Ile Tyr His Leu Tyr Gly 2640

8306 GTT TAC TAC AAG GGC TGG GAG GCC AAG GAA CTA TCT GAG AGG ACA GCA GGT AGA AAC TTA TTC ACT CTG ATA ATG TTT GAA CCC TTC GAA 8395 2641 Val Tyr Tyr Lys Gly Trp Glu ALa Lys Glu Leu Ser Glu Arg Thr Ala Gly Arg Asn Leu Phe Thr Leu Ile Met Phe Glu Ala Phe Glu 2670

8396 TTA CTA GGG ATG GAT TCA GAA CGA AAA ATA AGA AAC CTG TCC CGA AAC TAC ATT TTG CAT CTG ATC CAT CGA TTA CAT AAA CAG ATC AAC 8485 2671 Leu Leu Gly Met Asp Ser Glu Gly Lys Ile Arg Asn Leu Ser Gly Asn Tyr lie Leu Asp Leu Ile His G1y Leu His Lys Gin Ile Asn 2700

8486 AGA GGG CTG AAA AAG ATA GTA CTG GGA TGG GCC CCT GCA CCC TTC AGC TGT GAC TGG ACT CCT AGT GAC GAG AGG ATC AGA TTG CCA ACG 8575 2701 Arg Gly Leu Lys Lys Ile Val Leu GLy Trp A1a Pro Ala Pro Phe Ser Cys Asp Trp Thr Pro Ser Asp Glu Arg Ile Arg Leu Pro Thr 2730

8576 GAC AGC TAT CTA AGG GTG GAA ACT AAA TGC CCA TGC GGC TAT GAG ATG AAA GCA TTA AAG AAT GTA AGT GGT AAG CTT ACC AAG GTG GAG 8665 2731 Asp Ser Tyr Leu Arg Val Glu Thr Lys Cys Pro Cys Gly Tyr Glu Met Lys Ala Leu Lys Asn Val Ser Gly Lys Leu Thr Lys Val Glu 2760

8666 GAA AGC GGG CCT TTC TTA TGT AGG AAC AGA CCT GGT AGA GGG CCA GTC AAC TAC AGA GTC ACC AAA TAT TAT CAT CAT AAT CTC AGA GAG 2761 Glu Ser Gly Pro Phe Leu Cys Arg Asn Arg Pro Gly Arg Gly Pro Vat Asn Tyr Arg Val Thr Lys Tyr Tyr Asp Asp Am Leu Arg G1u

8756 ATA AGA CCA GTG GCA AAG TTG GAA GGA CAG GTG GAG CAC TAC TAC AAA GGG GTC ACG GCA AGA ATT GAC TAC AGT AAA GGA AAA ACA CTC 8845 2791 Ile Arg Pro Val Ala Lys Leu Glu Gly Gin Val Glu His Tyr Tyr Lys Gly Val Thr Ala Arg Ile Asp Tyr Ser Lys Gly Lys Thr Leu 2820

8846 TTA GCT ACT GAT AAA TGG GAG GTG GAA CAT GGT ACC TTA ACC AGG CTG ACT AAG AGA TAT ACT GGG GTC GGG TTC CGT GGT GCA TAC CTA 8935 2821 Leu Ala Thr Asp Lys Trp Glu Vat Glu His Gly Thr Leu Thr Arg Leu Thr Lys Arg Tyr Thr Gly Val Gly Phe Arg Gly Ala Tyr Leu 2850

8936 GGC GAC GAG CCC AAT CAC CGT CAT CTA GTG GAG AGG GAC TGT GCA ACT ATA ACT AAA AAC ACT GTG CAG TTT CTA AAA ATG AAG AAG GGG 2851 Gly Asp Glu Pro Asn His Arg Asp Leu Vat Glu Arg Asp Cys A1a Thr Ile Thr Lys Asn Tht Vat Gin Phe Leu Lys Met Lys Lys GLy

9026 TGC GCT TTC ACC TAC GAC CTG ACC ATC TCC AAT TTG ACC AGG CTT ATT GAA CTA GTA CAC AGA AAC AAC CTT GAA GAG AAG GAA ATA CCC 9115 2881 Cys Ala Phe Thr Tyr Asp Leu Thr Ile Ser Asn Leu Thr At-g Leu Ile Glu Leu Val His Arg Asn Am Leu Glu Glu Lys Glu Ile Pro 2910

9116 ACC GCT ACA GTC ACT ACA TGG CTA GCT TAC ACC TTT GTT AAT GAA GAT GTG GGG ACT ATA AAA CCA GTG CTA GGA GAG AGA GTA ATC CCC 9205 2911 Thr Ala Thr Val Thr Thr Trp Leu Ala Tyr Thr Phe Vat Asn Glu Asp Vat Gly Thr Ile Lys Pro Vat Leu Gly Glu Atg Vat Ile Pro 2940

9206 GAC CCT GTA GTT CAT ATT AAT TTA CAA CCA GAA GTC CAA GTG GAC ACA TCA GAG GTT GGG ATC ACA ATA ATT GGA AAG GAA GCC GTG ATG 2941 Asp Pro Val Vat Asp Ile Asn Leu GLn Pro Glu Val Gln Vat Asp Thr Ser Glu Vat Gly Ile Thr Ile Ile Gly Lys Glu Ala Val Met

FIG. 2-Continued

NCP BVDV STRAIN SD-l

9296 ACA ACA CGA GTG ACA CCC GTA ATG GAA AAG GTG GAG CC7 GAC ACT GAC AAC AAC CAA AGC TCG GTG AAG ATC CGA CTG GAi GAA GGT AAT 9385 2971 Thr Thr Gly Val Thr Pro Val Met Glu Lys Val Glu Pro Asp Thr Asp Am Asn Gin Ser Ser Vat Lys Ile Gly Leu Asp Glu Gly Asn 3000

9385 TAC CCA GGG ccc GGC GTA CAG ACA c~c ACA CTA GTT GAA cm ATA cAc AA~ AAG GAC CCC AGG ccc TTT ATT ATG GTC c7~ GGC TCG AAG 9475 3001 Tyr Pro Gly Pro Gly Val Gin Thr His Thr Leu Val Glu Glu I1e His Asn Lys Asp Ala Arg Pro Phe Ile Met Vat Leu Gly Ser Lys 3030

9476 ACT TCC ATG TCA AAT AGA GCA AAG ACA GCT AGG AAT ATA AAC CTG TAT ACA CGA AAT GAT CCC AGG GAA ATA AGA GAC TTG ATG GCA GM 9565 3031 Ser Set Met Ser Asn Arg Ala Lys Thr Ala Arg Asn Il'e Asn Leu Tyr Thr Gly Asn Asp PrO Arg Glu Ile Arg Asp Leu Met Ala Glu 3060

9566 GGG CCC ATA TTG GTG GTA GCA TTA AGG GAC ATT GAC CCT CAT CTA TCT GAA CTT GTC GAC TTC AAA GGG ACC TTT TTA GAT AGG GAA GCC 9655 3061 Gly Arg Ile Leu Val Val Ala Leu Arg Asp Ile Asp Pro Asp Leu Ser Glu Leu Val Asp Phe Lys Gly Thr Phe Leu Asp Arg Glu Ala 3090

9656 TTA GAG GCT CTG AGT CTT GGG CAG CCT AAA CCA AAG CAG GTT ACA AAA GCA GCA ATC AGA GAT TTA TTG AAA GAG GAG AGA CAG GTG GAG 9745 3091 Leu Glu Ala Leu Ser Leu Gly Gln Pro Lys Pro Lys Gln Val Thr Lys Ala Ala Ite Arg Asp Leu Leu Lys Glu Glu Arg Gin Val Glu 3120

9746 ATC CCT GAC TGG TTC ACA TCA GAC GAC CCC GTA TTT TTG GAC ATA GCC ATG AAA AAA CAT AAG TAC CAC TTG ATA GGA CAT GTA GTA GAA 9835 3121 Ile Pro Asp Trp Phe Thr Ser Asp Asp Pro Val Phe Leu Asp Ile Ala Met Lys Lys Asp Lys Tyr His Leu Ile Gly Asp Vat Va1 Glu 3150

9836 GTG AAG CAT CAA GCT AAA CCC. CTG GGG GCT ACG GAC CAA ACA AGG ATT GTA MC GAG GTA GGC TCA AGG ACG TAT ACC ATG AAA TTG TCT 9925 3151 Val Lys Asp Gin Ala Lys Ala Leu Gly Ala Thr Asp Gin Thr Arg Ile Val Lys Glu Vat Gly Ser Arg Thr Tyr Thr Met Lys Leu Ser 3180

9926 AGC TGG TTC CTT CAA GCA TCA AGC AAA CAG ATG AGC CTG ACT CCA CTA TTT GAG GAA TTG CTG TTA CCC TGC CCG CCT GCA ACT AAG AGC 10015 3181 Ser Trp Phe Leu Gln Ala Ser Ser Lys Gln Met Ser Leu Thr Pro Leu Phe Glu Glu Leu Leu Leu Arg Cys Pro Pro Ala Thr Lys Ser 3210

10016 AAT AAA GGG CAC ATG GCA TCA GCC TAC CAA TTG GCA CAG GGC AAC TGG GAA CCC CTC GGT TGC GGG GTA CAC CTA GGT ACC GTG CCA CCC 10105 3211 Asn Lys Gly His Met Ala Ser Ala Tyr Gin Leu Ala Gln Gly Asn Trp Glu Pro Leu Gly Cys Gly Vat His Leu Gly Thr Val Pro Ala 3240

10106 AGA AGA GTG AAG ATG CAC CCA TAT GAG GCC TAC CTG AAG CTG AAA GAC CTC GTA GAA GAA GAG GAA AAG AAA CCA AGA ATT AGG CAT ACA 10195 3241 Arg Arg Val Lys Met His Pro Tyr Glu Ala Tyr Leu Lys Leu Lys Asp Leu Val Glu Glu Glu Glu Lys Lys Pro Arg Ile Arg Asp Thr 3270

10196 GTA ATA AGG GAG CAC AAC AAA TGG ATT CTT AAA AAA ATA AAG TTC CAA GGG AAT CTC AAC ACT AAG AAA ATG CTC AAC CCC GGG AAA CTA 10285 3271 Val Ile Arg Glu His Am Lys Trp Ile Leu Lys Lys Ile Lys Phe Gln G1y Asn Leu Asn Thr Lys Lys Met Leu Asn Pro Gly Lys Leu 3300

10286 TCT GAA CAG CTG GAC AGA GAG GGG CAC AAG AGA AAC ATC TAC AAT AAC CAG ATT AGC ACC GTG ATG TCA ACT GCA GGC ATA AGG CTG GAA 10375 3301 Ser Glu Gin Leu Asp Arg Glu Gly His Lys Arg Asn 11e Tyr Asn Asn Gln Ite Ser Thr Vat Met Ser Ser Ala Gly Ile Arg Leu Glu 3330

10376 AAA TTG CCA ATA GTG AGG CCC CAA ACC GAC ACT AAA AGC TTC CAT GAG GCA ATA AGA CAT AAG ATA GAT AAG AAT GAG AAC CGG CAA AAT 10465 3331 Lys Leu Pro Ile Val Arg Ala Gin Thr Asp Thr Lys Ser Phe His Glu Ala 11e Arg Asp Lys Ile Asp Lys Asn Glu Asn Arg Gln Asn 3360

10466 CCG GAA TTG CAC AAC AAA CTG TTG GAA ATC TTT CAC ACA ATA GCT GAC CCC TCC TTA AAA CAC ACC TAT GGT GAA GTG ACG TGG GAG CAA 10555 3361 Pro Glu Leu His Asn Lys Leu Leu Glu Ile Phe His Thr Ile Ala Asp Pro Ser Leu Lys His Thr Tyr Gly Glu Val Thr Trp Glu GLn 3390

10556 CTT GAG GCA GGG ATA AAC AGG AAA GGG GCC GCA GGC TTC CTA GAA AAG AAG AAT ATC CCC GAA GTG TTG CAT TCA GAA AAA CAC CTG GTA 10645 3391 Leu Glu Ala Gly Ile Asn Arg Lys Gly Ala Ala Gly Phe Leu Glu Lys Lys Asn Ile Gly Glu Val Leu Asp Ser Glu Lys His Leu Val 3420

10646 GAG CAA TTG GTC AGG GAT CTG AAG CCC GGG AGA AAG ATA AGG TAT TAT GAA ACA GCA ATA CCA AAA AAT GAA AAA AGA CAT GTC AGC GAC 10735 3421 Glu Gin Leu Val Arg Asp Leu Lys Ala Gly Arg Lys Ile Arg Tyr Tyr Glu Thr Ala Ile Pro Lys Asn Glu Lys Arg Asp Val Ser Asp 3450

10736 GAC TGG CAG GCT GGG GAC CTG GTG GAT GAG AAG AAG CCA AGA GTT ATC CAA TAC CCC GAA GCC AAG ACA AGG TTA CCC ATC ACC AAG GTC 10825 3451 Asp Trp Gin Ala Gly Asp Leu Val Asp Glu Lys Lys Pro Arg Val Ile Gin Tyr Pro Glu Ala Lys Thr Arg Leu Ala Ile Thr Lys Val 3480

10826 ATG TAC AAC TGG GTG AAG CAG CAA CCC GTT GTG ATT CCA GGA TAC GAG GGT AAG ACC CCC TTG TTC AAC AK TTT AAT AAA GTG AGA AAG 10915 3481 Met Tyr Asn Trp Val Lys Gln Gln Pro Val Val Ile Pro Gly Tyr Glu Gly Lys Thr Pro Leu Phe Asn Ile Phe Asn Lys Val Arg Lys 3510

10916 GAA TGG GAT TTG TTC AAT GAA CCA GTG GCC GTA ACT TTT GAT ACC AAG CCC TGG GAT ACC CAA GTG ACT AGC AGG GAT CTG CAC CTT ATT 11005 3511 Glu Trp Asp Leu Phe Asn Glu Pro Val Ala Val Ser Phe Asp Thr Lys Ala Trp Asp Thr Gin Val Thr Set Arg Asp Leu His Leu Ile 3540

11006 GGT GAA ATC CAG AAA TAT TAC TAT AGG AAG GAA TGG CAT AAG TTC ATT GAC ACT ATC ACT GAC CAC ATG GTA GAA GTG CCA GTA ATA ACA 11095 3541 Gly Glu Ile Gin Lys Tyr Tyr Tyr Arg Lys Glu Trp His Lys Phe Ile Asp Thr Ile Thr Asp His Met Val Glu Val Pro Val Ile Thr 3570

11096 GCA CAT GGT GAA GTG TAT ATA AGA AAT GGG CAG AGG GGG AGT GGC CAG CCA GAC ACA AGT GCA GGC AAC ACT ATG CTA AAT GTC CTA ACA 11185 3571 Ala Asp Gly Glu Val Tyr Ile Arg Asn Gly Gln Arg Gly Ser Gly Gln Pro Asp Thr Ser Ala Gly Asn Ser Met Leu Asn Val Leu Thr 3600

11186 ATG ATC TAT GCT TTC TGT GAA AGC ACA GGG GTC CCA TAC AAG AGC TTC AAC AGA GTG GCA AAG ATC CAC GTT TGT GGG GAT GAT GGC UC 11275 3601 Met Ile Tyr Ala Phe Cys Glu Ser Thr Gly Val Pro Tyr Lys Ser Phe Am Arg Val Ala Lys Ile His Val Cys Gl Asp Asp Gly Phe 3630

bbr; bbb bbb 11276 CTA ATA ACC GAA AAA GGG TTA GGG CTA AAA TTT TCC AAC AAA GGG ATG CAA ATT CTT CAC GAA GCC GGC AAG CCT CAG AAA CTA ACG GAA 11365

3631 Leu Ile Thr Glu Lys Gly Leu Gly Leu Lys Phe Ser Am Lys Gly Met Gin Ile Leu His Glu Ala Gly Lys Pro Gln Lys Leu Thr Glu 3660

11366 GGG GAA AAA ATG AAA GTT CCC TAC AAA TTT GAA GAT ATA GAG TTT TGC TCC CAT ACC CCA GTC CCT GTT AGG TGG TCT GAT AAC ACC ACT 11455 3661 Gly Glu Lys Met Lys Val Ala Tyr Lys Phe Glu Asp Ile Glu Phe Cys Ser His Thr Pro Val Pro Val Arg Trp Ser Asp Asn Thr Ser 3690

11456 AGT TAC ATG GCT GGT AGA GAC ACT GCC GTG ATA CTA TCA AAG ATG GCA ACA AGA TTG GAC TCA AGT GGG GAG AGG GGT ACC ACG GCG TAC 11545 3691 Ser Tyr Met Ala Gly Arg Asp Thr Ala Val Ile Leu Ser Lys Met Ala Thr Arg Leu Asp Ser Ser Gly Glu Arg Gly Thr Thr Ala Tyr 3720

11546 GAA AAA GCC GTG CCC TTT AGT TTC TTA CTG ATG TAT 'ICC TGG AAC CCG CTT GTT AGG AGG ATC TGC CTT TTG GTC CTT KC CAG CGA CCG 11635 3721 Glu Lys Ala Val Ala Phe Ser Phe Leu Leu Met Tyr Ser Trp Am Pro Leu Val Arg Arg Ile Cys Leu Leu Vat Leu Ser Gln Arg Pro 3750

11636 GAG ACA GCT CCA TCA ACA CAG ACC ACT TAT TAT TAC AAA GGT GAT CCA ATA GGG GCA TAT AAA GAT GTG ATA GGC CCC MC TTA AGT GAA 11725 3751 Glu Thr Ala Pro Ser Thr Gln Thr Thr Tyr Tyr Tyr Lys Gly Asp Pro Ile Gly Ala Tyr Lys Asp Val Ile Gly Arg Asn Leu Ser Glu 3780

11726 CTG AAG AGA ACA GGC TTT GAG AAA TTG GCA AAT CTA AAC TTG AGT CTG TCC ACT CTA GGG ATC TGG ACT AAA CAC ACA ACT AAA AGA ATA 11815 3781 Leu Lys Arg Thr Gly Phe Glu Lys Leu Ala Asn Leu Asn Leu Ser Leu Ser Thr Leu Gly Ile Trp Thr Lys His Thr Ser Lys Arg Ile 3810

11816 ATT CAA GAC TGC GTG GCC ATT GGA AAG GAA GAG GGA AAC TGG CTA GTA AAT CCC GAT AGG CTA ATA TCC AGC AAA ACT GGC CAC TTA TAC 11905 3811 Ile Gln Asp Cys Val Ala Ile Gly Lys Glu Glu Gly Am Trp Leu Val Am Ala Asp Arg Leu Ile Ser Ser Lys Thr G1y His Leu Tyr 3840

11906 ATA CCT GAC AAA GGC TTT ACA TTA CAA GGA AAG CAT TAT GAG CAG CTG CAG 'ITA GGA GCA GAG ACC AAC CCG GTT ATG GGT GTG GGG ACT 11995 3841 Ile Pro Asp Lys Gly Phe Thr Leu Gin Gly Lys His Tyr Glu Gln Leu Gin Leu Gly Ala Glu Thr Am Pro Vat Met Gly Val Gly Thr 3870

11996 GAA AGG TAC AAA TTA GGT CCC ATA GTC AAT CTG CTG TTG AGA AGG TTA AAG GTC CTG CTC ATG GCG GCT GTC GGC GCC AGC AGC TGA GAC 12085 3871 G1u Arg Tyr Lys Leu G1y Pro Ile Vat Asn Leu Leu Leu Arg Arg Leu Lys Val Leu Leu Met Ala Ala Val Gly Ala Ser Ser 3898

12086 AAA ATG TAT ATA TTA TAA ATA GAA TTA ACC CTT GTA CAT ATT GTA TAT AAG CAT AGT GGG GTT CAT CTA CCT CAA AAG GCT ATA TAC TCA 1217-j

12176 ACA TAC ACA GCT AAA CAG TAG TTG AGA TTA TCT ACC TCA AGA TAA CAC TAC ACT CIA CGC ACA CAG CAC TTT AGC TGT ATG AGG ATA CAC 12265

12266 CCG ACG TCT ATA GTT CGA CTA CCC AAG ACC TCT AAC AGC CCC C 12308

FIG. 2-Continued

a74 DENG AND BROCK

era1 small ORFs. Following the stop codon of the large ORF, the 3’ untranslated region (3’UTR) continues for another 229 nucleotides.

Amino acid sequence of NCP BVDV strain SD-l

Hydrophobicity analysis of the entire amino acid sequence revealed two characteristic regions. The first region, located at the N-terminal of the polyprotein (the first 250 amino acid residues), is highly hydrophilic, particularly in the region from residues 165 to 250 which was reported to encode the viral capsid protein pl4 (Thiel et a/., 1991). Of 85 amino acid residues, 25 are positively charged residues (22 lysines, 3 arginines) and 12 are negatively charged residues (6 aspartic acids, 6 glutamic acids). The second region located at position 1036 to 1301 consists of 266 amino acid residues and is highly hydrophobic. This region contains 51 leucines, 35 valines, 29 isoleucines, and 20 threo- nines.

Twenty-eight potential N-linked glycosylation sequences (Asn-X-Ser or Asn-X-Thr) were predicted along the polyprotein. Fourteen sites are located in the glycoprotein region between residues 271 and 992 (Fig. 2). Of the 14 sites, 8 were conserved among all the pestiviruses and 3 other sites were conserved among the BVDV strains. A cysteine-rich stretch, reported to conform to a “zinc finger” binding domain (Moerlooze et al., 1990), was also found in the SD-l polyprotein at amino acid residues 1484 to 1512 (Fig. 2). The tripeptide sequence, Gly-Asp-Asp, highly conserved in viral RNA dependent RNA polymerases (Kamer and Argos, 1984), was uniquely identified in the nonstructural protein p75 region at position 3626 to 3628 (Fig. 2). This finding supports the proposal that this protein is a candidate BVDV replicase (Collett et al., 1991). Sequence comparisons and structural pat- tern analysis predicted the BVDV p80 protein to be a trypsin-like serine proteinase (Bazan and Fletlerick, 1989; Gorbalenya et a/., 1989a; Moormann et al., 1990). Recently, Wiskerchen and Collett (1991) experi- mentally demonstrated that this protein is a viral proteinase and is responsible for all the nonstructural protein processing. The catalytic triad of His, Asp, and Ser residues was also observed in the SD-l sequence and located at positions 1658, 1659, and 1752, respectively (Fig. 2).

Comparison of nucleotide and amino acid sequences

The inserts identified in CP BVDV Osloss (Meyers et a/., 1988c) and NADL (Collett et al., 1989) and a deletion of 41 nucleotides in the 3’UTR of Osloss (Deng and Brock, in preparation) are responsible for the variation of genome sizes (Fig. 3). There is a 270-nucleotide cellular RNA insert in NADL between nucleotides 4992

and 4993 of SD-l and a 228-base ubiquitin RNA insert in Osloss between nucleotides 5152 and 5153 of SD- 1. Our results reveal that, in contrast to CP BVDV, NADL and Osloss, NCP BVDV SD-l has no RNA insertions along its genome.

The overall nucleotide sequence homologies of SD- 1 are 88.6% with NADL, 78.3% with Osloss, 67.1% with HoCV Alfort and 67.2% with HoCV Brescia. The most conserved region is located in the 5’UTR in which the degree of homologies of SD-l sequence are 93% with NADL, 86% with Osloss, 74% with HoCV Alfort and Brescia (Fig. 3). A moderately high homology is found along most of the viral nonstructural protein region. The viral structural proteins (~14, gp48, gp25, and gp53) and nonstructural proteins p54 and p58 have the lowest homology between SD-l and other pestivirus sequences (Fig. 3).

The predicted amino acid sequence is more conserved than nucleotide sequence among all the pestiviruses. The overall amino acid sequence homologies of SD-l are 92.7% with NADL, 86.2% with Osloss, 72.5% with HoCV Alfort, and 71.2% with HoCV Bre- scia. The p80 is the most conserved viral protein with sequence homologies of SD-l with NADL of 98%, with Osloss of 95%, and with HoCV Alfort and Brescia of 86% (Fig. 3). Similar to the nucleotide sequence, the amino acid sequence of the viral structural proteins and nonstructural proteins p54 and p58 is more variable than other regions. Extensive analysis of amino acid sequence led to the identification of four conserved domains (designated Cl, C2, C3, C4) and three highly variable domains (designated Vl, V2, V3) in the viral structural proteins and nonstructural protein p54 region. The regions that had average amino acid sequence homologies of above 90% between SD-l and other pestiviruses were defined as conserved domains. The average percentages of homology between SD-1 and other pestiviruses in Cl, C2, C3, and C4 domains are 95, 92, 94, and 90%, respectively. The regions that had average amino acid homologies of below 61% between SD-l and other pestiviruses were defined as variable domains. The average percentages of homology between SD-l and other pestiviruses in Vl, V2, and V3 domains are 54, 61, and 48%, respectively. A comparison of amino acid sequence in those domains is shown in Fig. 4. The Cl, C2, and C3 domains are located in the viral capsid protein P14, glycoproteins gp48 and gp25, respectively. The C4 domain is located in the junction between gp53 and ~54. Of three highly variable domains, two (Vl and V2) are located in the same viral glycoprotein gp53 (Fig. 5). The hydrophobicity analysis of the amino acid sequence in this area show that the Vl and V2 domains identified in gp53 are hydrophilic (Fig. 5). The third variable domain,

NCP BVDV STRAIN SD-1 a75

SIJTR ~20~14 QP46 QP26 QP63 P54 PBO PlQ P? P66 P75 3’UTR

SD-I 5 I I I I ! 3’BVDV

93 92 64 90 ,94)(92) (94) ,::I 85 (65) (ii, (ii, (9”:) (ii, 07 08

(89) (E,

NADL 5’ ! ’ ’ I

! 3’BVDV

86 81 85 82

(85),93) (91) :897) (Ts”, :729)

83 80 62 76 62 72

(95) (94) ,961 ,811 (63)

I I I I I I I ! O’BVDV

74 65 73 66 70 62 63 783 72 70 60

(70) (76) (74) (79) (‘31) (62) (64) (80) (85;) (R,

3’ tlocv

74 66 71 66 68 64 72 70 70 63

(72) (~0) (72) (W (62) I I I

(*‘) (79)

BRESCIA 5 I (74)

1 3’HOCV

FIG. 3. Comparison of nucleotide and amino acid sequences of NCP BVDV SD-l with those of other pestiviruses: CP BVDV NADL and Osloss, HoCV Alfort and Brescia. Sequences were compared on basis of the genome organizations of BVDV (Collett eta/., 1988b) and HoCV (Thiel eta/., 1991). The percentages of homology of nucleotide sequences are given in the upper line and the percentages of homology between amino acid sequences are given in the next line parentheticallv. The black bars below the sequences of NADL and Osloss indicate the cellular sequence insertion within the ~54 region.

the largest one, is located in the N-terminal of nonstructural protein ~54.

DISCUSSION

Strain SD-l is a NCP BVDV obtained from a persistently infected heifer maintained in isolation. In order to eliminate the possibility of the adaptive selection of virus during cell culture passage, which may change the dominance of virus in the population, and to eventu- ally evaluate the naturally occurring mutation rate of the viral genome in the persistently infected animal in viva, the persistently infected animal was the source of virus for cDNA cloning. However, the virus titer in serum obtained from the persistently infected heifer was approximately 1 O3 logs lower than that in infected cell culture supernatants. Initially, direct cloning of cDNA from SD-l RNA extracted from serum was at- tempted. Because of low concentrations of viral RNA in the preparations, no positive clones of SD-l were obtained after screening of the cDNA library. To over- come this problem, PCR was used to amplify the lower viral RNA levels in the serum before cloning. Based on dot-blot hybridization results (Brock et a/., 1992), there was a high homology between SD-1 and NADL nucleotide sequences. It was also reported that the high- est sequence homology was located in the nonstructural protein region, particularly in the p80 region (Meyers et a/., 1989a; Moormann et a/., 1990). There- fore, the first two primers used to amplify SD-l RNA were designed based on NADL sequence in the p80 region, which is located in the middle of the genome. Because of the lower homology between NADL and SD-l nucleotide sequence in the regions of the viral glycoproteins and the nonstructural protein ~58, NADL

sequence primers were not able to amplify SD-l sequence in these regions. These two gaps, therefore, were filled in following cloning and determination of the 5’ and 3’UTR nucleotide sequences of SD-1 and the use of specific SD-l sequence primers. The cDNA sequence of SD-l reported represents the genomic sequence of the virus in viva, which may be different from that of the virus obtained in vitro due to the adaptive selection of virus during cell culture passage.

It was reported that the error rate of Taq polymerase during the DNA polymerization is about 2 X 10m5 errors/nucleotide/cycle under standard conditions (Eckert and Kunkel, 1990; Lundberg et al., 1991). In this study, the actual error rate was 4 X 1 Om5 errors/nucleotide/cycle, which is two times higher-than the theo- retical error rate of Taq polymerase. Therefore, out of 36 “errors,” some may have been created by either reverse transcriptase or DNA polymerase. In addition, some nucleotide differences may have been due to the sequence heterogeneity in the viral RNA population.

The cDNA sequence of NCP BVDV strain SD-l RNA was determined to be 12,308 nucleotides in length using a 5’-3’ ligation strategy to determine the extreme 5’ and 3’ end sequence. Therefore, it is reasonable to state that the sequence of our cDNA clones represents the complete nucleotide sequence of NCP BVDV SD-1 genome. Of nine 5’-3’ligation clones sequenced, four had one nucleotide and one had two nucleotides shorter than the authentic 3’ end sequence. Whether this variation represents the heterogeneity in the viral RNA population or indicates the presence of a low level of RNA exonuclease activity in the viral RNA preparations, resulting in cleavage of 1 or 2 bases at the 3’end of viral RNA prior to the 5’-3’ ligation, is unknown.

876 DENG AND BROCK

A SD-1 NADL OSLOSS ALFORT BRKSCIA

SD-1 NADL OSLOSS ALFORT BRESCIA




SD-l NADL OSLOSS ALFORT BRESCIA



Vl Reeion 609aa 755aa VQGHLDCKPEYSYAIAKNDRVGPLGAEGLTTVWKDYSHEMKLEDTMVIAWCRGGKFTYLSRCTRET ----------F------DE-I-Q------T--T--E--pG------------ED--~-- ______ A--LPV---GFY------NEI-----T---- P A--R-A--ED-R---SSTNEI-L--------

-YE--DG-R-Q--G-VV-----EIK-- T-E--A -D-GT-K-V-TA-S-KVTALNVVSR -D-GT-K-I-MA-S-KVTALNVVSR

833aa V2 Region

90Baa MLANRDTLDTAWRTYRRSVPFPYRQGCITQKTLGEDLYDCALGGNWTCVTGDQSRYTGGLIESCKWCGYKF KSE TSF-I---A-T--------K---H--------N-----HN-I--------P---LL-K--S---------Q- E-m s HWS-K---AMT-----K-HR---F--------VI-G----------LDLC---IL--VD-PV----------H---

TAVSPT--R-E--K-F--DK---H-VD-V-TIVEK---FH-K--------K--PVT-K-- TAVSPT--R-E--K-F--EK-----RD-V-TTVEN---FY-KW-------K-EPVT----

1065aa V3 Region

1140aa SGAQYQAGEVVHMGNLLTHDNVEVVTYFFLLYLLLREESVPGG L-I---S------------N-I------L-----------------------V--I-----------------S-- --RP~---I----------SI------L---------NI----I-I---I-M------T-----V-G~-E--A A-L-L- ----LI---I--TDN---V--L----VI-D-PI---I---F-~TNN-"-TIT-A----SG-A-GGKID A-L-L- ----LI---I--TDI---V--L----~-D-PI---I---F-~TNN-V-TIT-A---VSG-A-GGKID 8 1141aa 1200aa QGY**LG IDVCFTMVVIIIIGLIIARRDPTIVPLITIVASLRVTGLTYSPGVDAAMAVITI -E-,c--- :! --L---T--L-V---------------V----E--H ----I-"--M--

8 -SF*w*-E-"-LS-S-ITL-VV--"-------V---V----A------GF ----V----L-L LAIA-VVVVVMLL-K----TF--VIT--T--TAKI-NGFST-LVI-TVSA LALT--VVAVMLL-KK---T---VIT--T--TAKI-NGLST-L-I-TVST

200aa Cl Region

25Baa EKDSKTKPPDATIVV~GVKYQVKKKGKVKSKNTQDGLYHNKNKPPESRKKLEKALLAWA ---------------E------K---------- ---------------------------I------ ---------------E-----I-------G---- ----R----------E-------------G----

--- ___ ___ ___

293aa C2 Region

357aa RGVNRSLHGIWPEKICTGVPSHLATDVELKTIHGMMDASEKTNYTCCRLQRHEWNKHGWCNWYNI --------------------------I-------------------------------------- --------------------------I---A---.------------------------------ ----------------K---T-----T---E---------R------------------------ ---S------------K---T----T--RE-Q--RE-Q---V---G------K-----------------

51baa C3 Region

578aa NCTPACLPKNTKIIGPGKFDTNAEDGKILHEMGGHLSEVLLLSLVVLSDFAPETASAPnLILH -------------V-----G------------------------------------V------ -------------V---R-----------------.-------V------------W-----

984aa C4 Region

105baa KTACTFNYTRTLKNKYFEPRDSYFOOYnLKGDYOYWFDLEVTDHHRDYFAESILVWVALLGGRYVLWLLVTY

FIG. 4. Comparison of the predicted amino acid sequence of SD-1 in the three variable domains and the four conserved domains within the region of viral structural proteins and nonstructural protein p54 with that of other pestiviruses: BVDV strains NADL and Osloss, HoCV strains Alfort and Brescia. (A) The amino acid sequences in the three variable domains which are indicated by Vl , V2, V3 domains. (B) The amino acid sequences in the four conserved domains which are indicated by Cl, C2, C3, C4 domains. One-letter symbols for amino acids was used. The sequence shown in the top line is for SD-l. Hyphens indicate the identity with SD-l sequence. Stars indicate the deletions compared with each other. Their positions in the large ORF are indicated by numbers above the sequences.

Comparison of the nucleotide sequence between CP BVDV and HoCV led to the identification of a cellular RNA and a ubiquitin sequence insert within the region coding for the p54 in CP BVDV NADL and Osloss, respectively (Meyers et a/., 1989c; Collett et al., 1989). Because the NADL and Osloss strains are cytopathic, it has been proposed that cellular RNA insertion into NCP BVDV genome is responsible for the development of cytopathogenicity (Meyers et al., 1989c). Recently, Meyers et a/. (1991) cloned and determined the partial genomic nucleotide sequence in the ~125 region of a pair of CP BVDV strain CPl and NCP BVDV strain NCPl. A ubiquitin insert and p80 nucleotide sequence

duplicate were found in the CPl, but not in the NCPl within this region, which supports the previous hypothesis. However, whether other differences are present in the remainder of the genome is unknown. Compari- son of the complete nucleotide sequence of NCP BVDV SD-l with that of CP BVDV NADL and Osloss revealed that the most remarkable difference is the absence of an inserted cellular RNA sequence in the ~125 region.

It has been reported that the p80 protein, generated by either cleavage of ~125 or expression from a p80 duplicated region, is the only marker of CP BVDV (Meyers et a/., 1991). Therefore, the release of the p80

NCP BVDV STRAIN SD-l a77

FIG. 5. Location and hydrophobicity of three variable domains and four conserved domains identified among the pestiviruses in the region of viral structural proteins and nonstructural protein ~54. The gene organization given was proposed by Collett er al. (1998b) and Thiel et al. (1991). The black box and symbol V indicate the variable domain. The shadow box and symbol C indicate the conserved domain. The hydrophobicity plot in this region is proportionally given above the viral proteins. Solid and dotted lines present the hydrophobicity of SD-1 and NADL sequences, respectively. The star indicates the insertion region in NADL. The amino acid number is given at the bottom of the figure. S indicates the signal sequence.

protein from the viral polyprotein is the key event associated with the development of cytopathogenicity. Al- though the ~125 proteins of the different CP BVDV strains were heterogeneous in size, the processed p80 had the identical size (Akkina, 1991; Greiser-Wilke et a/., 1992). These results indicate that the size variation of ~125 is determined in the ~54 region and the cleavage site at the N-terminal of p80 is present in the authentic BVDV sequence rather than in the cellular inserts (Greiser-Wilke eta/., 1992). Some of the CP BVDV strains had ~125 almost identically sized with that of NCP BVDV (Akkina, 1991; Greiser-Wilke et al., 1992) suggesting that minor base changes in CP BVDV, such as base substitutions, small insertions, deletions, and gene duplication (Meyers et a/., 1991) may be also responsible for the release of p80 from the polyprotein. Comparison of NCP BVDV SD-l nucleotide sequence with CP BVDV NADL and Osloss at the N-terminal part of ~125 revealed that, in addition to the obvious difference of cellular RNA insertion, there are many base substitutions in NADL and Osloss, which result in the change of amino acid sequence, particularly in the V3 domain. Secondary structure prediction of amino acid sequence in this region reveals that some of the amino acid changes alter the secondary structure of the N- terminal portion of ~125 (data not shown). Therefore, in addition to the insertion, the base substitutions resulting in conformational change of the N-terminal part of ~125 may also contribute to the release of p80 from the polyprotein. It is tempting to propose that the mutations that occurred in NCP BVDV, such as insertion, deletion, duplication, and substitution, result in conformational changes in the N-terminal portion of ~125, which also may affect its electrophoretic mobility. Fol- lowing the conformational change, the cleavage site at the N-terminus of p80 becomes functional and the p80

is released from the viral polyprotein, resulting in the development of CP BVDV from NCP BVDV. The con- struction of an infectious cDNA clone and the following experimental conversion of both biotypes will help to elucidate the mechanism for cytopathogenicity of BVDV.

The most conserved nucleotide and amino acid sequences are located in the 5’UTR and nonstructural protein p80 respectively, which reflect the functional importance of these two regions for either virus replication or viral RNA translation or viral polyprotein processing. The p80 was reported to possess protease activity which is responsible for the processing of all the viral nonstructural proteins (Wiskerchen and Collett, 1991). In addition, a helicase motif was also predicted within the p80 region (Gorbalenya et a/., 1989b; and Moor- mann et a/., 1990). The possible regulatory elements and the secondary structure of 5’UTR of BVDV RNA will be analyzed and reported elsewhere (Deng and Brock, in preparation).

The comparison of both nucleotide and amino acid sequence of SD-l with that of other pestiviruses revealed the high heterogeneity in the region of viral structural proteins and nonstructural proteins p54 and ~58. It is reasonable to assume that the conserved domains identified in this region are under highly functional selection and are critical for either viral RNA packaging or virion assembly or important for viral in- teraction with receptors on infected cells. However, the functions of these domains remain to be determined. It has been reported repeatedly that neutralizing monoclonal antibodies against BVDV bound to and immunoprecipitated glycoprotein gp53 (Magar et al., 1988; Donis et al., 1988; Bolin et a/., 1988; Xue et a/., 1990; Weiland et al., 1990). Antigenic variation of this protein among BVDV isolates was demonstrated by

878 DENG AND BROCK

the fact that none of the monoclonal antibodies neutral- ized all the BVDV isolates tested (Magar et al., 1988; Xue et a/., 1990). The amino acid sequence analysis revealed that two hypervariable domains (Vl , V2) are located in the viral glycoprotein gp53. The hydrophobicity analysis indicated that these two domains are hydrophilic, suggesting that Vl and V2 domains are present on the outer surface of virions. Therefore, it is proposed that Vl and V2 domains in gp53 may repre- sent the protective epitopes of this protein, which are the important targets for the immune response of the host, hence under high immunological pressure. The high diversity of amino acid sequence within the protective epitopes of gp53 among BVDV and HoCV im- plies the difficulty in developing effective vaccine for preventing pestivirus infections by expression of this immunodominant glycoprotein. Recently, Weiland et a/. (1992) reported that antibodies to a second envelope glycoprotein gp44/48 were able to mediate neutralization of HoCV. Whether the corresponding glycoprotein gp48 in BVDV has the same ability to induce neutralizing antibodies remains to be investigated. Amino acid sequence analyses indicated that glycoprotein gp48 was more conserved than gp53 among pestiviruses. Furthermore, a highly conserved domain (C2) was observed within this protein. Therefore, gp48 may be a candidate for a subunit vaccine as well. Simi- lar to pestiviruses, the variable and hypervariable domains were also identified in the regions of Hepatitis C

ment Center, The Ohio State University. Journal article no. 200-92. This work was supported in part by Cooperative Research Agree- ment 88-341 16-3653 from the U.S. Department of Agricultural Science and Education Administration. The authors thank Dr. M. S. Collett for kindly providing BVDV NADL cDNA clones, Sylva Riblet and Jerry Meitzler for excellent technical assistance, and Fengmin Tian for typing the manuscript.

REFERENCES

AKKINA, R. K. (1991). Pestivirus bovine viral diarrhea virus polypep- tides: Identification of new precursor proteins and alternative cleavage pathways. Virus Res. 19, 67-82.

BAZAN, J. F., and FLETTERICK, R. J. (1989). Detection of a trypsin-like serine protease domain in flaviviruses and pestiviruses. Virology 171, 637-639.

BIRNBOIM, H. C. (1983). A rapid alkaline extraction method for the isolation of plasmid DNA. Methods Enzymol. 100, 243-255.

BOLIN, S. R., MCCLURKIN, A. W., CUTLIP. R. C., and CORIA, M. F. (1985). Severe clinical disease induced in cattle persistently infected with noncytopathic bovine viral diarrhea virus by superin- fection with cytopathic bovine viral diarrhea virus. Am. J. Vet Res. 46, 573-576.

BOLIN, S. R., MOENNIG, V., KELSO GOURLEY, N. E., and RIDPATH, J. (1988). Monoclonal antibodies with neutralizing activity segregate isolates of bovine viral diarrhea virus into groups. Arch. Viral. 99, 117-123.

BROCK, K. V., DENG, R., and RIBLET, S. (1992). Nucleotide sequencing of 5’and 3’termini of bovine viral diarrhea virus by RNA ligation and PCR. J. Virol. Methods 38, 39-46.

BROCK, K. V., RIDPATH, J. F.. and DENG, R. (1992). Comparative hybridization and nucleotide sequence information from two noncytopathic isolates of bovine viral diarrhea virus. Vet. Microbial., sub- mitted for publication.

virus (HCV) corresponding to the gp53 of pestivirus

regions have been reported in the corresponding envelope E protein and NSl protein of flaviviruses (Chu et

(Weiner et a/., 199 1). However, no similar hypervariable

a/., 1989; Deubel eta/., 1986; Lobigs eta/., 1988; Hahn et al., 1988; lrie et al., 1989).

The largest variable domain was identified within the

BROWNLIE, J., CLARKE, M. C., and HOWARD, C. J. (1984). Experimental

536. CHEN, E. Y., and SEEBURG, P. H. (1985). Supercoil sequencing: A fast

production of fatal mucosal disease in cattle. Vet. Rec. 114, 535-

and simple method for sequencing plasmid DNA. DNA 4, 165- 170.

CHU, M. C., O’ROURKE, E. J., and TRENT, D. W. (1989). Genetic related- ness among structural protein genes of dengue 1 virus strains. J.

first nonstructural protein ~54, the N-part of precursor protein ~125. The characteristic feature of this protein is that a highly hydrophobic stretch including 266 amino acids is present in this protein and it was usually not detected by immunoprecipitation (Moennig and Plagemann, 1992). The V3 domain is located in the highly hydrophobic stretch of this protein. Although the primary amino acid sequence is hypervariable, the hydrophobic character of this domain is maintained in all the pestiviruses, indicating that the hydrophobicity rather than the primary sequence of this protein is under the functional selection. Although a membrane- associated function of this protein was proposed (Wis- kerchen and Collett, 1991), further work is needed to elucidate its function.

ACKNOWLEDGMENTS

Salaries and research support were provided by state and federal funds appropriated to the Ohio Agricultural Research and Develop-

Gen. Viral. 70, 1701-1712. COLLET, M. S., LARSON, R., GOLD, C., STRICK, D., ANDERSON, D. K.,

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Molecular Cloning and Nucleotide Sequence of a Pestivirus...

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