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E L S E V I E R Virus Research 38 (1995) 231-239

Virus Research

A nested polymerase chain reaction assay to differentiate pestiviruses

D a n i e l G . S u l l i v a n , R a m e s h K. A k k i n a *

Department of Pathology, Colorado State University, Fort Collins, CO 80523, USA

Received 26 April 1995; revised 8 June 1995; accepted 12 June 1995

Abstract

Viruses that comprise the Pestivirus genus cause significant losses to the livestock industry. Based on sequence analysis, currently 4 distinct genotypes are identified of which 3 infect cattle and sheep. Distinguishing between bovine and ovine isolates by serological tests has often been difficult because of a high degree of cross reactivity. In this study, a nested polymerase chain reaction (PCR) assay was developed to identify and distinguish between bovine viral diarrhea virus (BVDV) type I, BVDV type II, as well as border disease virus (BDV) genotypes. Consensus oligonucleotide primers were designed to amplify a 826-bp product from any of the 3 pestivirus types in a reverse transcription-PCR (RT-PCR). This product was subjected to a second round of nested PCR with type-specific primers which yielded DNA products of unique size characteristic for each pestivirus genotype. Using this assay, we were able to rapidly characterize several viral isolates and determine that all 3 genotypes can be found among ovine isolates.

Keywords: Pestivirus; Border disease virus; Bovine viral diarrhea virus; Nested polymerase chain reaction (PCR); Pestiviral genotype

1. Introduction

T h e Pestivirus genus is c o m p o s e d of 3 i m p o r t a n t l ivestock viral pa thogens tha t a re r e spons ib le for subs tan t ia l economic losses in ca t t le , swine, and sheep fa rming ( M o e n n i g and P l agemann , 1992). T h e s e a re bovine viral d i a r r h e a virus (BVDV) , classical swine fever virus (CSFV) , and b o r d e r d i sease virus (BDV), which cause

* Corresponding author. Fax: + 1 (970) 491 0603.

0168-1702/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0168-1702(95)00065-8

232 D.G. Sullit'an and R.K. Akkina / ~qrus Research 38 (1995) 231-239

bovine viral diarrhea and mucosal disease in domestic and wild ruminants, hog cholera or classical swine fever in pigs and wild boar, and border disease in sheep, respectively (Terpstra, 1985; Brownlie, 1990; Collett, t992; Moennig, 1992; Sawyer, 1992). Recently, a new group of pestiviruses has been identified that causes severe thrombocytopenia and hemorrhaging in cattle (Corapi et al., 1989; Bolin and Ridpath, 1992; Pellerin et al., 1994). This new type of pestivirus is genetically distinct from the classical BVDV strains (Pellerin et al., 1994; Ridpath et al., 1994). Comparison of the published sequences of the 5'-untranslated region (UTR) from these viruses with that of an ovine pestivirus isolate (BD78) which we have sequenced revealed a high degree of homology, suggesting that this distinct virus type can also infect sheep (Sullivan et al., 1994). Preliminary analysis of these pestiviruses has led investigators to refer to them as BVDV type II viruses which is the term we will use in this report (Pellerin et al., 1994; Ridpath et al., 1994).

The complete genomic sequence of 5 pestiviruses has been determined. Three of these are bovine isolates, NADL, Osloss and SD1 (Collett et al., 1988; Deng and Brock, 1992; De Moerlooze et al., 1993). The other two are swine isolates, Alfort and Brescia (Meyers et al., 1989; Moormann et al., 1990). Additionally, partial genomic sequences have been determined for 6 ovine isolates, BD78, X818, L83/84, R27/27, 59386 and SCP (Becher et al., 1994, 1995; Sullivan et al., 1994). Phylogenetic analysis of the common regions sequenced for these 11 pestiviral isolates reveals the existence of at least 4 genetically distinct types or genotypes of pestiviruses. One type is represented by CSFV and includes the isolates Alfort and Brescia, the second type is represented by the classical BVDV or BVDV type I and includes NADL, Osloss, SD1 and R27/27, the third is represented by BVDV type II and includes BD78, 59386 and SCP and the fourth is represented by BDV and includes X818 and L83/84 (Sullivan et al., 1994; Becher et al., 1995).

Currently, detection of pestiviruses is based on virus isolation in cell culture and detection of viral antigen by immunohistochemistry or immunoassay (Pearson, 1992). Differentiation of CSFV from BVDV and BDV is done using a panel of monoclonal antibodies against the major envelope protein (Wensvoort et al., 1989). However, the distinction between BVDV type I, BVDV type II and BDV is less clear and there is no diagnostic method currently available to distinguish these 3 viruses. The differentiation of all 4 pestiviruses from each other is important in understanding the viral epidemiology and in developing disease control measures.

The introduction of the reverse transcription-polymerase chain reaction (RT- PCR) has led to the development of rapid and sensitive tests that can detect and differentiate between viruses of the same genus (Lanciotti et al., 1992; Borchers and Slater, 1993). Detection of pestiviruses by RT-PCR has been reported by a number of investigators (Boye et al., 1991; Liu et al., 1991; Lopez et al., 1991; Hooft van Iddekinge et al., 1992; Katz et al., 1993; Wirz et al., 1993; Vilcek et al., 1994). There has also been reports of using RT-PCR to detect and differentiate between BVDV and CSFV (Katz et al., 1993; Wirz et al., 1993). Vilcek et al. has used RT-PCR and restriction endonuclease analysis to subgroup pestiviruses into 3 genotypes (Vilcek et al., 1994). In this paper, we report the development of a nested RT-PCR that amplifies a common fragment from the genomes of BVDV

D.G. Sullivan and R.K. Akkina / Vitrus Research 38 (1995) 231-239 233

type I and type II viruses, as well as BDV. For subsequent differentiation of these 3 types of viruses, an internal fragment is amplified which is specific for each virus type.

2. Materials and methods

2.1. Viruses

A collection of 16 sheep and 33 cattle isolates was tested. These viruses comprise of pestiviral isolates obtained from different regions of the United States. Viruses were propagated in bovine turbinate (BT) cells (ATCC) infected at a multiplicity of infection of 1 and harvested 48 h post infection. Laboratory reference strains used in this study were NADL (Collett et al., 1988), BVD890 (Ridpath et al., 1994), BD78 (Akkina and Raisch, 1990; Sullivan et al., 1994), and have been previously characterized with regard to their nucleotide sequence and phylogenetic classification. Additionally, we have determined the nucleotide se- quence of the E0 and E1 coding regions for 3 additional reference strains referred to as BD31, BDSC and OV97 (unpublished data). Phylogenetic analysis of these sequences places BD31 and BDSC within the BDV group, and OV97 within the BVDV type I group.

2.2. R N A extraction

Viral RNA was isolated by a modified method of the procedure described by Chomczynski and Sacchi (Chomczynski and Sacchi, 1987). Briefly, supernatant culture fluid from virus-infected cells was mixed with an equal volume of guanidine isothiocyanate lysis buffer (8 M guanidine isothiocyanate, 50 mM sodium citrate, 100 mM 2-mercaptoethanol, 1% Sarkosyl, and 1 /~g of yeast tRNA per ml). The solution was sequentially mixed with the following (all added in relation to the final volume of sample plus lysis buffer): 1/10 volume of 2 M sodium acetate (pH 4), equal volume of phenol (pH 4.3), and 2/10 volume of chloroform. The mixture was centrifuged at 16,000 g for 15 min, and the aqueous phase was removed and combined with an equal volume of isopropanol to precipitate the RNA. After centrifugation, the resulting RNA pellet was washed with 75% ethanol and dissolved in water.

2.3. Primer design

The targeted region of the pestiviral genome is shown in Fig. 1. Pestivirus consensus primers P1 and P2 (Table 1) were designed based on published se- quences with the aid of the sequence analysis program MacVector (IBI-A Kodak Co., New Haven, CT) (Collett et al., 1988; Meyers et al., 1989; Moormann et al., 1990; Deng and Brock, 1992; De Moerlooze et al., 1993; Becher et al., 1994; Sullivan et al., 1994). Consensus primers were designed to share maximum homol-

234 D.G. Sullivan and R.& Akkina / Virus Research 38 (1995) 231-239

S'UTR p20 p14 EO E1 E2 ~ pSO plO p30

i ~J I I I I I I I 5' ---'l IIIIIIIIII / - - -

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

TS1 ~ > 566bp

TS2

TS3 ~ >

p58 pTS

I 3'UTR

~_~.

223 bp

Fig. 1. Schematic representation of the nested RT-PCR assay designed to detect and differentiate pestiviruses. The shaded region within the pestivirus genome (at top) shows the target for amplification. Primers P1/P2 are designed to amplify a 826-bp region from all types of pestiviruses. Nested PCR is then performed on this product and includes primers TS1, TS2, TS3 and P2. Primer TS1 is specific for only BDV, primer TS2 is specific for BVDV type II viruses and primer TS3 is specific for BVDV type I viruses.

ogy wi th all 3 types o f pes t iv i ruses ( B V D V type I and type II, and B D V ) and have

no h o m o l o g y to o t h e r r eg ions o f the pes t iv i rus g e n o m e s . T h e type - spec i f i c p r i m e r s

TS1, T S 2 and TS3 ( shown in T a b l e 1) w e r e d e s i g n e d to a n n e a l speci f ica l ly to e a c h

o f the i r r e spec t i ve g e n o m e s .

2.4. PCR amplification o f pestivirus R N A

T h e a m p l i f i c a t i o n r e a c t i o n was p e r f o r m e d by c o m b i n i n g the r eve r se t r ansc r ip -

t ion o f v i ra l R N A and the s u b s e q u e n t Taq p o l y m e r a s e amp l i f i c a t i on in a s ingle

Table l Oligonucleotide primers used to amplify and differentiate pestiviruses

Primer a Sequences Genome Size of amplified position b DNA product (bp)

P 1 5'-AACAAACATGGTTGGTGCAACTGGT-3' 1424-1449 826 P2 5'-CTTACACAGACATATTTGCCTAGGT- 2221-2250 826

TCCA-3'

TSI 5'-TATATTATTTGGAGACAGTGAATGTA- 1684-1716 566 (TS1 and P2) GTAGCT-3'

TS2 5'-TGGTTAGGGAAGCAATTAGG-3' 1802-1821 448 (TS2 and P2) TS3 5'-GGGGGTCACTTGTCGGAGG-3' 2027-2045 223 (TS3 and P2)

'~ Primer set P1/P2 were designed to amplify all pestiviruses. Pestivirus type-specific primers TS1/P2, TS2/P2 and TS3/P2 were designed to amplify BDV, BVDV type II, and BVDV type I, respectively. b The genome positions of each primer are based on the sequence of the BVDV strain NADL (Collett et. al., 1988).

D.G. Sullivan and R.K. Akkina / Virus Research 38 (1995) 231-239 235

reaction vessel. Primers and viral RNA (approximately 1 ng) were incubated at 75°C for 10 min and quickly cooled on ice for 10 min to allow annealing of the downstream primer to the genomic RNA. The RT-PCR reaction contained 1 × PCR buffer (10 × buffer contains 500 mM KC1; 100 mM Tris-HC1, pH 8.3; 15 mM MgC12), 0.25 mM dithiothreitol, 1 mM of each deoxynucleotide (dATP, dCTP, dGTP, dTTP), 50 pmol each of primer P1 and P2, 40 U of RNasin (Promega Corp., Madison, WI), 2.5 U of RAV-2 reverse transcriptase (Amersham Corp., Arlington Heights, IL), and 2.5 U of Amplitaq (Perkin-Elmer Cetus Corp., Norwalk, CT) in a reaction volume of 100/zl. The RT reaction was carried out at 50°C for 30 min followed by an initial denaturation at 94°C for 2 min. The PCR reaction was 30 cycles with the following reaction parameters: template denatura- tion 94°C for 1 min, primer annealing 55°C for 1 min, and extension at 72°C for 1 min. A single final extension step was done at 72°C for 10 min to complete the amplification reaction. Amplified products were analyzed on 1.5% agarose gels (Sambrook et al., 1989).

2.5. Pestivirus typing by second-round amplification with type-specific primers (nested PCR)

A second amplification reaction was performed with 1 /~1 of diluted material (1:500 in sterile distilled water) from the initial RT-PCR. The reaction mixture contained all the components described for the initial RT-PCR with the following exceptions: primer P1 was substituted with 50 pmol of each of the pestivirus type-specific primers TS1, TS2 and TS3. Dithiothreitol, RNasin and RAV-2 were omitted. The samples were subjected to 25 cycles of denaturation (94°C, 1 min), primer annealing (50°C, 45 s), and primer extension (72°C, 45 s). A 15-~1 sample of the reaction product was electrophoresed on a 2% composite agarose gel (NuSieve 3 : 1; FMC BioProducts, Rockland, ME). The position of priming with each of the pestivirus type-specific primers was designed such that the size of the nested DNA product was characteristic for each pestivirus type (Table 1).

3. Results

RNA isolated from each of the 6 pestivirus reference strains was subjected to the RT-PCR assay. DNA products of the expected size (826 bp) were obtained for all of the pestiviruses after amplification with the consensus primers P1 and P2 (Fig. 2A). The DNA products from the RT-PCR were then subjected to a second round of amplification with the nested, type-specific primers. The correctly sized products were obtained for each of the 3 pestivirus types (Fig. 2B). The assay was then tested on an additional 44 pestivirus isolates (Table 2): 13 from ovine and 31 from bovine origin. All 44 isolates were detected with the Pestivirus genus consensus primers P1 and P2. Of the 13 ovine isolates, 6 were amplified with the BVDV type I-specific primers (TS3), 4 were amplified with the BVDV type II-specific primers (TS2), and 3 were amplified by the BDV-specific primers (TS1).

236 D. G. Sullit,an and R.K. Akkina / Virus Research 38 (1995) 231-239

A M 1 2 3 4 5 6 7 8 9 10

B

M 1 2 3 4 5 6 7 8 9 10

i

Fig. 2. Agarose gel analysis of the DNA product from RT-PCR samples isolated from pestiviruses. A: amplification products with consensus primers P1 and P2. B: second-round amplification products with type-specific primers TSI, TS2 and TS3. One hundred-base pair marker is shown on the left; DNA sizes are given in base pairs. Lanes show amplification of RNA from the following viral isolates: 1, NADL; 2, OV97: 3, 190C; 4, BD31; 5, BDSC: 6, BDCB#2; 7, BD78; 8, BVD890: 9, BD21800-2; 10, negative control.

Table 2 Results of RT-PCR and nested PCR amplification of BDV, BVDV type I1 and BVDV type 1

Host species No. of isolates of viral isolate tested

Amplification result for each primer set a,b

P1 /P2 TS1/P2 TS2/P2 TS3/P2

Bocine Laboratory reference strains 2 2 0 1 1 Diagnostic isolates 31 31 0 2 29

Ocine Laboratory reference strains 4 4 2 1 1 Diagnostic isolates 13 13 3 4 6

~t Values indicate number of isolates yielding predicted specific amplification product. b Primer set P1/P2 was designed to amplify all pestiviruses. Pestivirus type-specific primers TSI /P2 , TS2/P2 and TS3/P2 were designed to amplify BDV, BVDV type If, and BVDV type I, respectively.

D.G. Sullivan and R.K. Akkina /l/irus Research 38 (1995) 231-239 237

Of the 31 bovine isolates, 29 were amplified with the BVDV type I specific primers (TS3), 2 were amplified with the BVDV type II-specific primers (TS2) and none were amplified with the BDV type-specific primers (TS1). A single, DNA product of expected size was amplified for each of the pestivirus isolates in the nested, type-specific PCR. The specificity of the amplified products was confirmed by sequencing (about 350 nucleotides) the amplified products of two isolates from each of the 3 viral types and comparison of these sequences with those of previously published pestiviruses (data not shown).

4. Discussion

Historically, differentiating between ovine and bovine pestiviruses has been difficult for several reasons. First, BVDV and BDV can infect both ovine and bovine species and may cause disease symptoms in each (Carlsson, 1991; Loken et al., 1991). Second, both viruses are serologically related and often cross-neutralize with each other (Akkina and Raisch, 1990; Moennig and Plagemann, 1992; Pearson, 1992). Furthermore, until recently the lack of adequate sequence data of various genotypes of BVDV and BDV has hampered the development of geno- type-specific PCR techniques. In this report, we describe the development of a nested PCR assay for differentiating between 3 types of pestiviruses, that is, BDV, BVDV type I, and BVDV type II. The steps of reverse transcription and PCR are combined in a single reaction vessel reducing the assay time, lowering the risk of contamination problems and facilitating the handling of large numbers of samples. The use of primers homologous to conserved regions of the pestivirus genomic sequences ensures that all strains of pestiviruses will be amplified in the first-round amplification reaction. The fact that all 50 isolates were amplified with the consensus primers indicates their broad reactivity.

The specificity of our assay relies on the ability of the type-specific primers to recognize genomic sequences unique to each of the 3 pestivirus genotypes. Al- though all 3 type-specific primers were included during the second amplification, only a single amplified product was obtained. Thus, no cross-reactivity was de- tected between the type-specific primers and heterologous pestivirus genotypes. This nested PCR method for typing pestiviruses is faster and simpler than hybridization since correct typing requires only electrophoresis of the amplified product on an agarose gel. In contrast, filter hybridization requires labeling, purification, and standardization of probes which are time consuming and labori- ous.

Several investigators have previously reported PCR-based assays to detect and /o r differentiate BVDV and CSFV (Boye et al., 1991; Liu et al., 1991; Lopez et al., 1991; Hooft van Iddekinge et al., 1992; Katz et al., 1993; Wirz et al., 1993; Vilcek et al., 1994). However, none of these were designed to differentiate between BVDV type I, type II and BDV. The use of the present assay has permitted us to categorize a large number of ovine and bovine pestivirus isolates in a short amount of time in our laboratory. Historically, all sheep pestivirus isolates

238 D.G. Sullivan and R.K. Akkina / Virus Research 38 (1995) 231-239

were re fe r red to as BDV, however the avai labi l i ty of sequence da ta on an increas ing n u m b e r of ovine pest ivi ruses has m a d e it poss ib le to ca tegor ize them genotypical ly . Of the 13 pest ivi ruses i sola ted f rom sheep, 6 were ident i f ied as B V D V type I, 4 were ident i f ied as B V D V type II, and 3 were ident i f ied as B D V in this study. There fo re , these resul ts indica te for the first t ime that ovine isolates can be seg rega ted into 3 types using this nes ted P C R protocol . As more pest ivirus isola tes f rom di f fe ren t par t s of the world are genet ica l ly charac te r ized , it is l ikely that add i t iona l geno types may be recognized. I so la t ion and cha rac te r i za t ion of unique pest ivi ruses will have signif icant impl ica t ions for ep idemio log ica l and vaccine d e v e l o p m e n t studies. The fact that var ious genotypes can be found in d i f ferent hosts unde r sco re s the need for d iagnost ic assays that can d i f fe ren t ia te the known geno types of pest iviruses.

Acknowledgements

The au thors would like to thank Gwong-Jen Chang for helpful discussion and cri t ical review of the manuscr ip t . W e would also like to thank P e t e r Just ice and Sherry Becker for p r e p a r a t i o n of viral stocks. W o r k r e p o r t e d here is s u p p o r t e d by grants f rom Un i t ed Sta tes D e p a r t m e n t of Agr i cu l tu re and the Col lege R e s e a r c h Counci l , Col lege of Ve te r ina ry Medic ine , Co lo rado S ta te Universi ty.

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