Veterinary Microbiology, 29 ( 1991 ) 1-13 Elsevier Science Publishers B.V., Amsterdam
Specific sequence amplification of bovine virus diarrhea virus (BVDV) and hog cholera virus and sequencing of BVDV nucleic acid M. Boye, S. Kamstrup I and K. Dalsgaard Animal Biotechnology Research Centre, State Veterinary Institute for Virus Research, Lindholm, DK 4 771 Kalvehave, Denmark (Accepted 5 April 1991 )
ABSTRACT Boye, M., Kamstrup, S. and Dalsgaard, K., 1991. Specific sequence amplification of bovine virus diarrhea virus (BVDV) and hog cholera virus and sequencing of BVDV nucleic acid. Vet. Microbiol., 29: 1-13. The pestiviruses are small enveloped RNA viruses and are causative agents of economically important animal diseases in cattle, swine, sheep and goats worldwide. We used the polymerase chain reaction to amplify one common fragment of several different strains of both hog cholera virus and bovine virus diarrhea virus (BVDV). The fragment is located at the 5'-end of the genome immediately upstream of the open reading frame. This is a highly conserved region among the different published pestivirus sequences. An internal restriction digest of the amplified fragment with XhoI and Pst! was performed in order to confirm specificity of the amplified fragment. The fragment was sequenced for a number of different BVDV strains, and the sequences obtained were compared to those published and used to deduce genetic relationships between strains. Apart from this common fragment we have amplified several other fragments of the Danish BVDV strain Ug59 and obtained specific amplification fragments of the expected size.
INTRODUCTION
The genus Pestivirus holds generic status in the nonarthropod-borne family Togaviridae (Matthews, 1982 ). The pestiviruses are causative agents of economically important animal diseases such as bovine viral diarrhea (BVD) and mucosal disease (MD) of cattle (Brownlie et al., 1984; Baker, 1987), hog cholera (HC) of swine (van Oirschot, 1986) and border disease (BD) of sheep and goats (Fenner et al., 1987). The genomes of BVDV and HCV consist of positive-strand RNA of approximately 12 500 nucleotides in length. This RNA has a single open reading ~Author for correspondence.
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M. BOYE ET AL.
frame (ORF) (Renard et al., 1985; Collett et al., 1988; Meyers at al. 1989a; Moormann et al., 1990). The recent molecular cloning and sequencing of the genome of the BVDV strains NADL (Collett et al,, 1988 ) and Osloss (Renard et al., 1985 ) and the HCV strains Alfort (Meyers et al., 1989a) and Brescia (Moormann et al., 1990) has provided new possibilities for detection of the viruses. The viruses are structurally and antigenically closely related (Osburn et al., 1973 ). The polymerase chain reaction (PCR) is an in vitro method for primerdirected enzymic amplification of specific DNA sequences (Saiki et al., 1985 ). PCR provides a technique by which part of the nucleic acid of the pathogen in a sample can be specifically amplified up'to 106-fold. When the starting material is RNA an initial reverse transcriptase step is necessary to generate cDNA prior to amplification by PCR. In this paper we present the amplification of one fragment common for several different pestivirus strains, including both HCV and BVDV, and the amplification of several other fragments of the BVDV strain Ug59. MATERIALS AND METHODS
B ~"DV strains The BVDV strains used for PCR were Ug59, 258, 2890, NADL, Singer (all cytopathogenic) and 619, 1795 and Ry (all noncytopathogenic). BVDV strain Ug59 was the first strain from which infeo.tious, positive stranded RNA was isolated (Diderholm and Dinter, 1966). Except for NADL and Singer, all BVDV strains used in this study are Danish field isolates. HCV strains The hog cholera virus strains used for PCR were Alfort, Aid and Brescia. Cell cultures For propagation of BVDV strains we used primary calf kidney cells, grown in Eagles MEM supplemented with 5% calf serum (free of BVDV and BVDV antibodies) and antibiotics (neomycin 50 rag/1 and streptomycin 100 mg/ i). For propagation of HCV we used PK-15A cells grown in Eagles MEM (Glasgow moalficat~o~) wlth 5% serum and antibiotics as above. .~"
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Virus propagation Confluent cell monolayers were used for virus propagation. Prior to infection the medium wa~ discarded and replaced with serum-free medium. For infecting one 80 cm-' bottle we used typically l 0 6 TCIDso of virus in 0.5 ml of medium.
SEQUENCE AMPLIFICATIONOF BVDV
3
RNA purification We have tried using both infected cells and pelleted virions as source of RNA. To obtain virus pellets, the growth medium was centrifuged (200 g, 30 rain, 5 °C) to remove cells released from the flask and large particulate cellular debris, and thereafter the virus was pelleted by high-speed centrifugation (30 000 g, 2 h, 5°C). RNA was purified from this pellet using standard protocols for isolation of total RNA from mammalian cells (Sambrook et al., 1989), with the following modifications: Triton X-100 was used instead of Nonidet P-40, RNAse inhibitors were omitted, and proteinase K digestion was for two hours. RNA was purified from infected cells using the same procedure.
Oligonucleotide primers Oligonucleotide primers were purchased form Genetic Engineering Group, Technical University of Denmark, DK-2800 Lyngby, Denmark. The sequences of the primers are shown in Table I. Amplified fragments are designated with the primer used for preparing cDNA (anti-template sense), followed by a slash and the other primer (template sense). For example P3/PI is a fragment obtained by reverting viral sense RNA with P3 and amplification of the resulting cDNA using P3 and P 1, whereas P 1/P3 is obtained by reverting anti-sense RNA (formed during virus replication) and amplifying with P3 and P 1. The fragment is the same, but the template is different.
Reverse transcription The reverse transcription mixture contained 1 X PCR-buffer ( l0 mM Tris, 50 mM KC1, 1.5 mM MgCI2, 0.01% w/v gelatin), 125/zM each of dATP, dGTP, dCTP and d'[TP, 20 nM of the oligonucleotide primer and 10/zl RNA ternTABLE 1 Nucleotide sequence and location on the NADL genome of the primers used for PCR. The underlined sequence of primer P3 shows the (anti-sense) initiation codon of the open reading frame Primer
Sequence
NADL-Iocation Nucleotide Nos.
PI P2 P3 P5 P6 P 15 P 19 P25 P26
5'-GAGGCTAGCCATGCCCTTAGT-3' 5'-GTAGCAACAGTGGTGAGTTCGTTGG-3' 5'-TCAACTCCATGTGCCATGTACAGCA-3' 5'-TGCCTTGCAGACCATATGAAATCAT-3" 5'-ATGATTTCATATGGTCTGCAAGGCA-3' 5'-GGACTCAGCGAAGTAATCCC-3' 5'-ATTGGTGGCCTTATGAGACA-3' 5'-GGGCATACCATCTGGAAGGC-3' 5'-GCCTTCCAGATGGTATGCCC-3'
98- ! 19 140- 164 3 7 i - 395 3288-3312 3288-3312 3471-3490 2256-2275 2834-2853 2834-2853
4
M. BOYE ET AL.
plate in a final volume of 20 pl. The mixture was heated at 55 °C for 5 s i n , cooled on ice and 40 units M-MLV-reverse transcriptase (BRL) was added. The mixture was incubated at 37°C for 2.5 h. After incubation the reaction was stopped by heating at 95 °C for 5 s i n and cooled on ice. Polymerase chain reaction Amplification was performed on the reverse transcription cDNA product (20/~l) in a final volume of 100pl containing: 200/~M each ofdATP, dGTP, dCTP and dTTP, 100 nM of each of the two oligonucleotide palmers, 1 X PCRbuffer and 2.5 units Taq polymerase (AmpliTaq, Perkin-Elmer Cetus). The samples were overlaid with 100 pl of mineral oil (Perkin-E!mer Cetus) to prevent evaporation. Amplification was performed over 35 cycles in a PerkinElmer Cetus Thermocycler by denaturation at 95°C for l rain, primer-annealing at 60°C for l rain and primer-extension at 72°C for l min. The fiaal extension step was at 72 °C for 5 min. Gel electrophoresis o f the amplified product Amplified DNA ( 10 ~1) was mixed with stop-mix (a saturated sucrose solution containing 2% SDS, 100 mM EDTA and 0.25% bromphenolblue) and analyzed by I. 5% agarose gel electrophoresis (SeaKem GTG, FMC Bio Products) in 2×TBE (TBE consists of 89 mM Tds, 89 mM boric acid, 2 mM EDTA pH 0.8). The band was visualized by ethidium bromide staini~g. A 123 bp. DNA-ladder (BRL) was used as a size marker. Restriction enzyme digestion ,'x',t_ _ . . . . . . ~". . . . ..,l ~ ,+.'k~ l~t"~l~ ..,~,~.+.....~, t~r~¢l-+_ L ne restriction e n z y m e U...I i" g~ e_ 3 ,tt l".O. .l l. . Wi~b p C l IUI III~U 111 [11• • ~I~.'IIIIAI.UI~,~ W'I~,IIout any prior purification. Digestion was at 37°C for 2 h. The restriction enzymes used were PstI and Xhol (New England Biolabs ). PCR-mixture (9 pl ) was added to a pl of the appropriate 10 × enzyme-buffer and 2 units of enzyme, and digested at 37°C for 2 h. For control of the digestion by XhoI of the HCV strains Aid and Alfort the PCR reaction mixture was extracted with phenol-chloroform extraction and precipitated in ethanol according to standard protocols. The DNA was resuspended in 1/4 TE (TE consists of 10 mM Tris, 1 mM EDTA pH 8.0). Samples were electrophoresed as described above. Asymmetric PCR Asymmetric PCR was ,~,~..¢ w , , o. . .,. - , ~ ,-1 ., ~. . ., h. a. . . .~.v.m. .~. l. . . .PER . . . . . . .~ulth . . . . . t h e following modifications: 50 pM each of dATP, dCTP, dGTP, dTTP, 100 nM of the excess primer and 2 nM of the limiting primer. Amplification was performed . . ~ d 1¢1. .~ l.l l.~.l l.t * .... by adding~ 90 pl of 4 M in 40 cycles. The o- m v-':=~ It ~o n ~ lr ~ ~otignl ti~l tn~tue d ammonium acetate and two volumes of isopropanol. The precipitate was redissolved in 20 pl 1/4 TE buffer. ~
SEQUENCE AMPLIFICATION OF BVDV
5
Sequencing A 10pl aliquot of the redissolved asymmetric PCR fragment was sequenced using the TAQence DNA sequencing kit (United States Biochemical Corporation) according to manufacturers instructions using 35S dATP as labelling reagent. Electrophoresis was performed according to Lang and Burger (1990). RESULTS
Oligonucleotide primers to the 5'-region of the BVDV genome were used to reverse transcribe and amplify a 298 base pair (bp) fragment from the BVDV strains NADL, Ug59, 619, 1795, 2890, 258, and Ry and the HCV
Fig. I. Electrophoresis on 1.5% agarose gel of PCR amplified P 3 / P 1 fragments of different strains of BVDV and HCV. From left to right: 1, Size marker; 2, uninfected PK-I 5A cells (no visible ~ I -'--'LI 1~ marker);3, ~-o~ ,I,~ "~'4, "~" 5, ole~:l~t, " - -^: . 6, uniniected . . . wsmte t, ,~u, calf kidney cells (no marker); 7, ~xy; 8, 258; 9, 2890; 10, 1795; 1 !, 619; 12, Ug59; 13, Size marker.
6
M. BOYE ET AL.
Fig. 2. Electrophoresis on 1.5% agarose gel of XhoI digest og P3/PI amplified fragments of different strains of BVDV and HCV. XhoI is expected to have a recognition site at 127 bp in the 298 bp fragment. The HCV strains AIfort and Aid remained undigested while the HCV strain Brescia and all the BVDV strains were digested. From left to right: I, Size marker; 2, Aid; 3, Alfort; 4, Brescia: 5, Ug59; 6, 258; 7, Ry; 8, 2890; 9, 619; 10, 1795; l 1, Size marker.
strains Aifort, Aid, and Brescia using the PCR technique. We used both P3 ,"-'~ m u r~"t to prime ",..~ ~ reverse transcription, uz~,. . . . w. .e ~ a P ! ¢,,, . ~ . preparing cDNA we obtained less amplification product than when P3 was used to prime the reverse transcription• Figure 1 shows a gel electrophoresis of the fragment amplified by P 3 / P I for different BVDV strains and HCV strains. A specific fragment of the correct size is observed for all strains. However, the phenol chloroform extraction of RNA failed to give suitable RNA for the NADL strain, but using guanidinium isothiocyanate extraction we obtained RNA suitable for template in PCR. We made a restriction enzyme digest of the P 3 / P I amplified fragment. It was not necessa~ to purify the amplified DNA before digestion with Xhol and Pstl. Digestion of P3/P1 with Pstl gave the expected digestion products for all BVDV and "n~'~,r stralns" ~~ ~*~ not ~ n ) As shown in Fig. 2, digestion o f P 3 / P l with Xhol gave the expected pattern for all the BVDV strains and the HCV strain Brescia, but the P 3 / P I fragments of the HCV strains Alfoff and Aid remain undigested. Wc ~ , , . v . . . . ~ ~ : ~ ~,,~t~,~.~.~ther~.. . fragments of the BVDV strain Ug59. As shown in Fig. 3 we obtained specific amplification fragments of the expected size. •
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SEQUENCEAMPLIFICATIONOF BVDV
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Fig. 3. Electrophoresis on 1.5% agarose gel of PCR amplified fiagments of the BVDV strain Ug59. From left to right: 1, Size marker, 2, P3/PI 298 bp; 3, P3/P2 256 bp: 4, P 2 5 / P I 9 598 bp; 5, P6/P26 479 bp: 6, P 6 / P I 9 1057 bp; 7, PI5/P26 657 bp: 8, PI5/P5 203 bp: 9, Size marker.
For amplification of fragments greater than 1.1 kb we found that 45 cycles of amplification were necessary in our protocol. However this increased the risk of non-specific amplification products (data not shown). We have sequenced a portion of the P3/PI fragment from the BVDV isolates NADL, Singer, Ug59 and 10039. The NADL isolate sequence shows complete homology to the sequence already published (Collett et al., 1988). A comparison between the published sequences and the sequences obtained by us is shown in Fig. 4. For clarity, only the differences between the strains and NADL are indicated. The sequenced nucleotides are numbers 156-287 on the NADL genome. The development of infection with the BVDV strain Ug59 in cell cultures . . .u,,, . time for harwas examined h y t h ~ P ~ R t ~ o h n i c t , , ~ *,~ d,~t,~,-m;na ,r~ . . . . . ...,~ V" .... ~
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Fig. 4. Differences in nucleotide sequence between BVDV strains, using the NADL strain as reference strain. The region shown is nucleotides 156-287 on the NADL genome. A horizontal line represents any number of nucleotides identical to the NADL sequence. See text for full details.
vesting nucleic acids. Determinations were based on a comparison of band intensities of PCR products after35 cycles (fragment P3/P l ) separated on a 1.5% agarose gel. When extracting RNA from monolayer cells the optimum time of harvest ~vas found to be one day, whereas from pelleted virions it was found to be three days (data not shown). These results reflect the initial amplification of viral nucleic acid in the cells, and the subsequent release in viral particles. Also, the number of cells remaining attached to the flask decreases after infection due to the cytopathic effects of Ug59. DISCUSSION
Pestiviruses are causative agents of important diseases in cattle, pigs, sheep and goats worldwide. Therefore there is a great demand for information on the nature of these viruses. Traditional serological investigations can now be supplemented with information obtained by analysis of the nucleic acid. PCR has the potential of being a very sensitive and rapid method for detection of viral nucleic acid in clinical material. Subsequent sequencing of amplified fragments of the viral genome can provide detailed information on the degree ofamino acid homology between different strains. Such information could be relevant in epidemiology or for surveillance of vaccination areas for introduction of new strains, potentially refractory to the vaccine used. For this reason we have concentrated our PCR efforts on the conserved regions upstream to the open reading frame and the region coding for the protein gp53, the only protein known to carry neutralizing epitopes (Donis et al., 1988; Magar et al., 1988). This paper describes the amplification of one fragment common for several different pestivirus strains, including both HCV and BVDV, sequencing of this fragment from some strains of BVDV, and the amplification of several other fragments of the BVDV strain Ug59. This is the first report on PCR mediated amplification of HCV nucleic acid. By comparison of published sequences of the BVDV strains NADL and Osloss and the HCV strain Alfort we have chosen the primers P l and P3 from the conserved regions, immediately upstream of the open reading frame. A
SEQUENCE AMPLIFICATION OF BVDV
9
125 nucleotide region spanning the start of the open reading frame (nucleotides 386-388 ) shows a very high homology with only one mismatch between the BVDV strains NADL and Osloss. Although the sequence of the HCV strain Alfort is less homologous to the two BVDV strains, this region shows considerable homology when compared to the rest of the HCV sequence. We therefore chose primers specific for this region in order to have a broad-reactive fragment. A comparison among the primers P 1 and P3 and the corresponding published sequences is shown in Table 2. The expected length of the fragments amplified by the primer pairs used and the location on the NADL genome are shown in Table 3 and Fig. 5. In order to minimize the risk of false negative reactions with the PCR technique, it is crucial to choose priming regions showing the highest possible degree of conservation between strains. In a previous report on PCR amplification of BVDV no such considerations were made (Schroeder and BalassuChan, 1990). In fact, one of the primers chosen lies within the insert of cellderived RNA. The homology between primers P 1 and P3 and the corresponding published sequences shown in Table 2 illustrates the degree of homology which we use in our PCR assay. Proper selection of primers should also make it possible to discriminate between HCV and BVDV, based on the sequences published until now (Renard et al., 1985; Collett et al., 1988; Meyers et al., 1989a; Moormann et al., 1990). We are currently investigating this aspect. Virus-specific amplification products of the expected size were found with several virus isolates tested. The method used for RNA preparation in this report is laborious and difficult to use for a large number of samples, and it failed to give suitable RNA from the BVDV strain NADL. Therefore, if PCR is to be used as a routine diagnostic tool, it is necessary to use a more simple method of sample preparation prior to the amplification. Studies regarding this aspect are currently under way. Demonstration of the digestion ability of PstI and Xhol in the PCR reaction mixture facilitates the identification of BVDV specific sequences, by eliminating false positives due to non-specific amplification of sequences of similar size. The inclusion of more than these two restriction enzymes may be necessary for this purpose. From the published sequences of the BVDV strains NADL and Osloss and the HCV strains Alfort and Brescia, it is expected that XhoI and Pstl each have one recognition site in the P3/Pi amplified fragment. However, as shown in Fig. 2 the P3/PI fragment of the HCV strains Alfort and Aid remains undigested. This cannot be ascribed to failure to digest in the PCR-mixture for the following reasons: ( l ) Digestiori of other fragments with XhoI and PstI using this protocol consistently showed complete digestion, and (2) X hol was not able to digest puri fled P3/P l fragments of the HCV strains Alfort and Aid.
Primer 3 P3 NADL Osloss Alfort Brescia
Primer I PI NADL Osloss Alfort Brescia
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5'-G 5'-5'-5'-T 5'-T
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Comparison of primers P1 and P3 with the corresponding published sequences. Dots indicate identical nuclcotides
TABLE 2
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SEQUENCE AMPLIFICATION OF BVDV
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TABLE 3
The expected length of the amplified fragment by the primer pairs used and the location on the BVDV strain NADL Primers pairs
Expected length of amplified product (base pairs)
NADL location (nucleotide Nos. )
P3/PI P3/P2 PI5/P5 PI5/P26 P25/PI9 P6/P26 P6/PI9
298 256 203 657 598 479 1057
98- 395 140- 395 3288-3490 2834-3490 2256-2853 2834-3312 2256-3312
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Fig. 5. Position of primers and amplified fragments on the ~VDV strain NADL genome. The position ofglycoprotein gp53 indicated.
Asymmetric PCR has successfully been used to generate template DNA for sequencing of BVDV strains. The comparison in Fig. 5 shows the presence of markers shared between strains, as well as strain-specific markers. The distribution of markers has been used to propose the genetic relationship shown in Fig. 6. The high similarity between the American isolates NADL and Singer is remarkable, but corresponds well to similar neutralizing properties of the two virus strains (L. Ronsholt, personal communication 1990). The shaded boxes show two markers shared by the European isolates, but not by the American isolates. The most remarkable finding is perhaps the extensive homology between the Danish Ug59 and the German Osloss strain (unshaded boxes), suggesting a close genetic relationship between these two isolates. At present we do not know whether the two strains have other similarities, as for instance neutralization properties. The marker common for Osloss and 10039,
12
M. BOYEETAL.
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Fig. 6. Preliminary pedigree showing the genetic relationship between the BVDV strains NADL, Singer, 10039, Osloss and Ug59, as deduced from the genetic map shown in Figure 5. The question marks represents hypothetical intermediate forms.
marked by an asterisk does not follow the general pattern and may have arisen independently in the two strains or been lost in Ug59. In conclusion we may say that, even with a relatively small number of nucleotides sequenced, we have identified markers which discriminate the European from the American isolates, and which link two of the European isolates genetically. At a symposium held in Hannover on June 8-9 1990 by the European society for Veterinary Virology data were presented by different groups working on BVDV, using PCR as a method for detecting and analyzing pestivirus nucleic acid. One of the reports focused on the gene coding for the p54 protein with the aim of detecting possible insertions in this gene as su~ested by Meyers et al. (1989b). No such insertions were found. Two other reports (presented by Hertig et al. and K.V. Brock) mainly focused on the use of PCR for detection of BVDV and comparing the method with traditional methods such as dot blot and Southern blot. In contrast to these reports we have optimized the choice of primers in order to obtain broad-reactive fragments. In this report we have demonstrated the usefulness of PCR of BVDV and HCV nucleic acids, thereby creating a platform for further inroads to the study of the molecular biology of these viruses. Traditional strategies, involving molecular cloning can now for many applications be replaced by this simple method for preparing DNA suitable for sequencing. Thus, PCR is a helpful tool for the study of the genomic variation (restriction enzyme pattern or sequence) of these important viruses.
REFERENCES Baker, J.C., 1987. Bovine viral diarrhea virus: a review. J. Am. Vet. Med. Assoc., 190: 14491458.
SEQUENCE AMPLIFICATION OF BVDV
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Brownlie, J., Clarke, M.C. and Howard, C.J., 1984. Experimental production of fatal mucosal disease in cattle. Vet. Rec., 114: 535-536. Collett, M.S., Larson, R., Gold, C., Strick, D., Anderson, D.K. and Purchio, A.F., 1988. Molecular cloning and nucleotide sequence of the pestivirus bovine viral diarrhea virus. Virology, 165: 191-199. Diderholm, H. and Dinter, A., 1966. Infectious RNA from bovine virus diarrhea virus. Zentralbl. Bakteriolo Pasrasitenhvd. lnfektionshr. Hgg. Abt. 1:Orig., 201: 270-272. Donis, R.O., Corapi, W., Dubovi, E.J., 1988. Neutralizing monoclonal antibodies to bovine viral diarrhoea virus bind to the 56K to 58K glycoprotein. J. Gen. Virol., 69: 77-86. Fenner, F., Bachmann, P.A., Gibbs, E.P.J., Murphy, F.A., Studdert, M.J. and White, D.O., 1987. Diseases caused by togaviruses. In: Veterinary Virology, Academic Press Inc., 289-300, 458469. Lang, B.F. and Burger, G., 1990. A rapid, high resolution DNA sequencing gel system. Anal. Biochem., 188: 176-180. Magar, R., Minocha, H.C. and Lecomte, J., 1988. Bovine viral diarrhea virus proteins: Heterogeneity ofcytopathogenic and noncytopathogenic strains and evidence of a 53K glycoprotein neutralization epitope. Vet. Microbiol., 16: 303-314. Matthews, R.E.F., 1982. Classification and nomenclature of viruses: fourth report of the international committee on taxonomy of viruses. Intervirology, 17: 97-10. Meyers, G., Riimenapf, T. and Thiel, H.J., 1989a. Molecular cloning and nucleotide sequence of the genome of hog cholera virus. Virology, 171: 555-567. Meyers, G., Riimenapf, T. and Thiel, H.J., 1989b. Ubiquitin in a togavirus. Nature, 341: 49 I. Moormann, R.J.M., Warmerdam, P.A.M., Meet, B. van de Schaaper, W.M.M., Wenswoort, G. and Hulst, M.M., 1990. Molecular cloning and nucleotide sequence of hog cholera virus strain Brescia and mapping of the genomic region encoding envelope protein E I. Virology, 177: 184-198. van Oirschot, J., 1986. Hog cholera. In: A.D. Leman, B. Shaw, R.D., Glock, W.I. Mengeling, P.A.G. Penny and E. Scholl (Editors), Diseases of Swine 6th edn., Iowa State Univ. Press, Ames, 289-300. Osburn, B.I., Clarke, G.L., Stewart, W.C. and Sawer, M., 1973. Border disease-like syndrome in lambs: antibodies to hog cholera and bovine viral diarrhoea virus. J. Virol., 48: 320-324. Renard, A., Guiot, C., Schmetz, D., Dagenais, L., Pastoret, P.P. and Martial, J.A., 1985. Molecular cloning of bovine viral diarrhea viral sequences. DNA, 4: 429-438. Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn, G.T., Erlich, H.A and Arnheim, N., 1985. Enzymatic amplification of B-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science, 230:1350-1354. Sambrook, J., Fritsch, E.E, Maniatis, T., 1989. Molecular cloning, A laboratory manual. Second edn., Cold Spring Harbor, Laboratory, Cold Spring Harbor, NY. Schroeder, B.A. and Balassu-Chan, T.C., 1990. Specific sequence amplification of bovine viral diarrhoea virus nucleic acid. Arch. Virol., 111: 239-246.
Specific sequence amplification of bovine virus diarrhea virus (BVDV) and hog cholera virus and sequencing of BVDV nucleic acid M. Boye, S. Kamstrup I and K. Dalsgaard Animal Biotechnology Research Centre, State Veterinary Institute for Virus Research, Lindholm, DK 4 771 Kalvehave, Denmark (Accepted 5 April 1991 )
ABSTRACT Boye, M., Kamstrup, S. and Dalsgaard, K., 1991. Specific sequence amplification of bovine virus diarrhea virus (BVDV) and hog cholera virus and sequencing of BVDV nucleic acid. Vet. Microbiol., 29: 1-13. The pestiviruses are small enveloped RNA viruses and are causative agents of economically important animal diseases in cattle, swine, sheep and goats worldwide. We used the polymerase chain reaction to amplify one common fragment of several different strains of both hog cholera virus and bovine virus diarrhea virus (BVDV). The fragment is located at the 5'-end of the genome immediately upstream of the open reading frame. This is a highly conserved region among the different published pestivirus sequences. An internal restriction digest of the amplified fragment with XhoI and Pst! was performed in order to confirm specificity of the amplified fragment. The fragment was sequenced for a number of different BVDV strains, and the sequences obtained were compared to those published and used to deduce genetic relationships between strains. Apart from this common fragment we have amplified several other fragments of the Danish BVDV strain Ug59 and obtained specific amplification fragments of the expected size.
INTRODUCTION
The genus Pestivirus holds generic status in the nonarthropod-borne family Togaviridae (Matthews, 1982 ). The pestiviruses are causative agents of economically important animal diseases such as bovine viral diarrhea (BVD) and mucosal disease (MD) of cattle (Brownlie et al., 1984; Baker, 1987), hog cholera (HC) of swine (van Oirschot, 1986) and border disease (BD) of sheep and goats (Fenner et al., 1987). The genomes of BVDV and HCV consist of positive-strand RNA of approximately 12 500 nucleotides in length. This RNA has a single open reading ~Author for correspondence.
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M. BOYE ET AL.
frame (ORF) (Renard et al., 1985; Collett et al., 1988; Meyers at al. 1989a; Moormann et al., 1990). The recent molecular cloning and sequencing of the genome of the BVDV strains NADL (Collett et al,, 1988 ) and Osloss (Renard et al., 1985 ) and the HCV strains Alfort (Meyers et al., 1989a) and Brescia (Moormann et al., 1990) has provided new possibilities for detection of the viruses. The viruses are structurally and antigenically closely related (Osburn et al., 1973 ). The polymerase chain reaction (PCR) is an in vitro method for primerdirected enzymic amplification of specific DNA sequences (Saiki et al., 1985 ). PCR provides a technique by which part of the nucleic acid of the pathogen in a sample can be specifically amplified up'to 106-fold. When the starting material is RNA an initial reverse transcriptase step is necessary to generate cDNA prior to amplification by PCR. In this paper we present the amplification of one fragment common for several different pestivirus strains, including both HCV and BVDV, and the amplification of several other fragments of the BVDV strain Ug59. MATERIALS AND METHODS
B ~"DV strains The BVDV strains used for PCR were Ug59, 258, 2890, NADL, Singer (all cytopathogenic) and 619, 1795 and Ry (all noncytopathogenic). BVDV strain Ug59 was the first strain from which infeo.tious, positive stranded RNA was isolated (Diderholm and Dinter, 1966). Except for NADL and Singer, all BVDV strains used in this study are Danish field isolates. HCV strains The hog cholera virus strains used for PCR were Alfort, Aid and Brescia. Cell cultures For propagation of BVDV strains we used primary calf kidney cells, grown in Eagles MEM supplemented with 5% calf serum (free of BVDV and BVDV antibodies) and antibiotics (neomycin 50 rag/1 and streptomycin 100 mg/ i). For propagation of HCV we used PK-15A cells grown in Eagles MEM (Glasgow moalficat~o~) wlth 5% serum and antibiotics as above. .~"
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Virus propagation Confluent cell monolayers were used for virus propagation. Prior to infection the medium wa~ discarded and replaced with serum-free medium. For infecting one 80 cm-' bottle we used typically l 0 6 TCIDso of virus in 0.5 ml of medium.
SEQUENCE AMPLIFICATIONOF BVDV
3
RNA purification We have tried using both infected cells and pelleted virions as source of RNA. To obtain virus pellets, the growth medium was centrifuged (200 g, 30 rain, 5 °C) to remove cells released from the flask and large particulate cellular debris, and thereafter the virus was pelleted by high-speed centrifugation (30 000 g, 2 h, 5°C). RNA was purified from this pellet using standard protocols for isolation of total RNA from mammalian cells (Sambrook et al., 1989), with the following modifications: Triton X-100 was used instead of Nonidet P-40, RNAse inhibitors were omitted, and proteinase K digestion was for two hours. RNA was purified from infected cells using the same procedure.
Oligonucleotide primers Oligonucleotide primers were purchased form Genetic Engineering Group, Technical University of Denmark, DK-2800 Lyngby, Denmark. The sequences of the primers are shown in Table I. Amplified fragments are designated with the primer used for preparing cDNA (anti-template sense), followed by a slash and the other primer (template sense). For example P3/PI is a fragment obtained by reverting viral sense RNA with P3 and amplification of the resulting cDNA using P3 and P 1, whereas P 1/P3 is obtained by reverting anti-sense RNA (formed during virus replication) and amplifying with P3 and P 1. The fragment is the same, but the template is different.
Reverse transcription The reverse transcription mixture contained 1 X PCR-buffer ( l0 mM Tris, 50 mM KC1, 1.5 mM MgCI2, 0.01% w/v gelatin), 125/zM each of dATP, dGTP, dCTP and d'[TP, 20 nM of the oligonucleotide primer and 10/zl RNA ternTABLE 1 Nucleotide sequence and location on the NADL genome of the primers used for PCR. The underlined sequence of primer P3 shows the (anti-sense) initiation codon of the open reading frame Primer
Sequence
NADL-Iocation Nucleotide Nos.
PI P2 P3 P5 P6 P 15 P 19 P25 P26
5'-GAGGCTAGCCATGCCCTTAGT-3' 5'-GTAGCAACAGTGGTGAGTTCGTTGG-3' 5'-TCAACTCCATGTGCCATGTACAGCA-3' 5'-TGCCTTGCAGACCATATGAAATCAT-3" 5'-ATGATTTCATATGGTCTGCAAGGCA-3' 5'-GGACTCAGCGAAGTAATCCC-3' 5'-ATTGGTGGCCTTATGAGACA-3' 5'-GGGCATACCATCTGGAAGGC-3' 5'-GCCTTCCAGATGGTATGCCC-3'
98- ! 19 140- 164 3 7 i - 395 3288-3312 3288-3312 3471-3490 2256-2275 2834-2853 2834-2853
4
M. BOYE ET AL.
plate in a final volume of 20 pl. The mixture was heated at 55 °C for 5 s i n , cooled on ice and 40 units M-MLV-reverse transcriptase (BRL) was added. The mixture was incubated at 37°C for 2.5 h. After incubation the reaction was stopped by heating at 95 °C for 5 s i n and cooled on ice. Polymerase chain reaction Amplification was performed on the reverse transcription cDNA product (20/~l) in a final volume of 100pl containing: 200/~M each ofdATP, dGTP, dCTP and dTTP, 100 nM of each of the two oligonucleotide palmers, 1 X PCRbuffer and 2.5 units Taq polymerase (AmpliTaq, Perkin-Elmer Cetus). The samples were overlaid with 100 pl of mineral oil (Perkin-E!mer Cetus) to prevent evaporation. Amplification was performed over 35 cycles in a PerkinElmer Cetus Thermocycler by denaturation at 95°C for l rain, primer-annealing at 60°C for l rain and primer-extension at 72°C for l min. The fiaal extension step was at 72 °C for 5 min. Gel electrophoresis o f the amplified product Amplified DNA ( 10 ~1) was mixed with stop-mix (a saturated sucrose solution containing 2% SDS, 100 mM EDTA and 0.25% bromphenolblue) and analyzed by I. 5% agarose gel electrophoresis (SeaKem GTG, FMC Bio Products) in 2×TBE (TBE consists of 89 mM Tds, 89 mM boric acid, 2 mM EDTA pH 0.8). The band was visualized by ethidium bromide staini~g. A 123 bp. DNA-ladder (BRL) was used as a size marker. Restriction enzyme digestion ,'x',t_ _ . . . . . . ~". . . . ..,l ~ ,+.'k~ l~t"~l~ ..,~,~.+.....~, t~r~¢l-+_ L ne restriction e n z y m e U...I i" g~ e_ 3 ,tt l".O. .l l. . Wi~b p C l IUI III~U 111 [11• • ~I~.'IIIIAI.UI~,~ W'I~,IIout any prior purification. Digestion was at 37°C for 2 h. The restriction enzymes used were PstI and Xhol (New England Biolabs ). PCR-mixture (9 pl ) was added to a pl of the appropriate 10 × enzyme-buffer and 2 units of enzyme, and digested at 37°C for 2 h. For control of the digestion by XhoI of the HCV strains Aid and Alfort the PCR reaction mixture was extracted with phenol-chloroform extraction and precipitated in ethanol according to standard protocols. The DNA was resuspended in 1/4 TE (TE consists of 10 mM Tris, 1 mM EDTA pH 8.0). Samples were electrophoresed as described above. Asymmetric PCR Asymmetric PCR was ,~,~..¢ w , , o. . .,. - , ~ ,-1 ., ~. . ., h. a. . . .~.v.m. .~. l. . . .PER . . . . . . .~ulth . . . . . t h e following modifications: 50 pM each of dATP, dCTP, dGTP, dTTP, 100 nM of the excess primer and 2 nM of the limiting primer. Amplification was performed . . ~ d 1¢1. .~ l.l l.~.l l.t * .... by adding~ 90 pl of 4 M in 40 cycles. The o- m v-':=~ It ~o n ~ lr ~ ~otignl ti~l tn~tue d ammonium acetate and two volumes of isopropanol. The precipitate was redissolved in 20 pl 1/4 TE buffer. ~
SEQUENCE AMPLIFICATION OF BVDV
5
Sequencing A 10pl aliquot of the redissolved asymmetric PCR fragment was sequenced using the TAQence DNA sequencing kit (United States Biochemical Corporation) according to manufacturers instructions using 35S dATP as labelling reagent. Electrophoresis was performed according to Lang and Burger (1990). RESULTS
Oligonucleotide primers to the 5'-region of the BVDV genome were used to reverse transcribe and amplify a 298 base pair (bp) fragment from the BVDV strains NADL, Ug59, 619, 1795, 2890, 258, and Ry and the HCV
Fig. I. Electrophoresis on 1.5% agarose gel of PCR amplified P 3 / P 1 fragments of different strains of BVDV and HCV. From left to right: 1, Size marker; 2, uninfected PK-I 5A cells (no visible ~ I -'--'LI 1~ marker);3, ~-o~ ,I,~ "~'4, "~" 5, ole~:l~t, " - -^: . 6, uniniected . . . wsmte t, ,~u, calf kidney cells (no marker); 7, ~xy; 8, 258; 9, 2890; 10, 1795; 1 !, 619; 12, Ug59; 13, Size marker.
6
M. BOYE ET AL.
Fig. 2. Electrophoresis on 1.5% agarose gel of XhoI digest og P3/PI amplified fragments of different strains of BVDV and HCV. XhoI is expected to have a recognition site at 127 bp in the 298 bp fragment. The HCV strains AIfort and Aid remained undigested while the HCV strain Brescia and all the BVDV strains were digested. From left to right: I, Size marker; 2, Aid; 3, Alfort; 4, Brescia: 5, Ug59; 6, 258; 7, Ry; 8, 2890; 9, 619; 10, 1795; l 1, Size marker.
strains Aifort, Aid, and Brescia using the PCR technique. We used both P3 ,"-'~ m u r~"t to prime ",..~ ~ reverse transcription, uz~,. . . . w. .e ~ a P ! ¢,,, . ~ . preparing cDNA we obtained less amplification product than when P3 was used to prime the reverse transcription• Figure 1 shows a gel electrophoresis of the fragment amplified by P 3 / P I for different BVDV strains and HCV strains. A specific fragment of the correct size is observed for all strains. However, the phenol chloroform extraction of RNA failed to give suitable RNA for the NADL strain, but using guanidinium isothiocyanate extraction we obtained RNA suitable for template in PCR. We made a restriction enzyme digest of the P 3 / P I amplified fragment. It was not necessa~ to purify the amplified DNA before digestion with Xhol and Pstl. Digestion of P3/P1 with Pstl gave the expected digestion products for all BVDV and "n~'~,r stralns" ~~ ~*~ not ~ n ) As shown in Fig. 2, digestion o f P 3 / P l with Xhol gave the expected pattern for all the BVDV strains and the HCV strain Brescia, but the P 3 / P I fragments of the HCV strains Alfoff and Aid remain undigested. Wc ~ , , . v . . . . ~ ~ : ~ ~,,~t~,~.~.~ther~.. . fragments of the BVDV strain Ug59. As shown in Fig. 3 we obtained specific amplification fragments of the expected size. •
~
g
~
•
~ r i ~
~.
~
*
SEQUENCEAMPLIFICATIONOF BVDV
7
Fig. 3. Electrophoresis on 1.5% agarose gel of PCR amplified fiagments of the BVDV strain Ug59. From left to right: 1, Size marker, 2, P3/PI 298 bp; 3, P3/P2 256 bp: 4, P 2 5 / P I 9 598 bp; 5, P6/P26 479 bp: 6, P 6 / P I 9 1057 bp; 7, PI5/P26 657 bp: 8, PI5/P5 203 bp: 9, Size marker.
For amplification of fragments greater than 1.1 kb we found that 45 cycles of amplification were necessary in our protocol. However this increased the risk of non-specific amplification products (data not shown). We have sequenced a portion of the P3/PI fragment from the BVDV isolates NADL, Singer, Ug59 and 10039. The NADL isolate sequence shows complete homology to the sequence already published (Collett et al., 1988). A comparison between the published sequences and the sequences obtained by us is shown in Fig. 4. For clarity, only the differences between the strains and NADL are indicated. The sequenced nucleotides are numbers 156-287 on the NADL genome. The development of infection with the BVDV strain Ug59 in cell cultures . . .u,,, . time for harwas examined h y t h ~ P ~ R t ~ o h n i c t , , ~ *,~ d,~t,~,-m;na ,r~ . . . . . ...,~ V" .... ~
,
~
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~ a . . a ~ t . ~ . ,
I,~.~
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8
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M. BOYE ET AL.
-
-
A .
.
.
.
.
.
.
.
.
.
.
-
-. ~
::::i:
"
~
~
Fig. 4. Differences in nucleotide sequence between BVDV strains, using the NADL strain as reference strain. The region shown is nucleotides 156-287 on the NADL genome. A horizontal line represents any number of nucleotides identical to the NADL sequence. See text for full details.
vesting nucleic acids. Determinations were based on a comparison of band intensities of PCR products after35 cycles (fragment P3/P l ) separated on a 1.5% agarose gel. When extracting RNA from monolayer cells the optimum time of harvest ~vas found to be one day, whereas from pelleted virions it was found to be three days (data not shown). These results reflect the initial amplification of viral nucleic acid in the cells, and the subsequent release in viral particles. Also, the number of cells remaining attached to the flask decreases after infection due to the cytopathic effects of Ug59. DISCUSSION
Pestiviruses are causative agents of important diseases in cattle, pigs, sheep and goats worldwide. Therefore there is a great demand for information on the nature of these viruses. Traditional serological investigations can now be supplemented with information obtained by analysis of the nucleic acid. PCR has the potential of being a very sensitive and rapid method for detection of viral nucleic acid in clinical material. Subsequent sequencing of amplified fragments of the viral genome can provide detailed information on the degree ofamino acid homology between different strains. Such information could be relevant in epidemiology or for surveillance of vaccination areas for introduction of new strains, potentially refractory to the vaccine used. For this reason we have concentrated our PCR efforts on the conserved regions upstream to the open reading frame and the region coding for the protein gp53, the only protein known to carry neutralizing epitopes (Donis et al., 1988; Magar et al., 1988). This paper describes the amplification of one fragment common for several different pestivirus strains, including both HCV and BVDV, sequencing of this fragment from some strains of BVDV, and the amplification of several other fragments of the BVDV strain Ug59. This is the first report on PCR mediated amplification of HCV nucleic acid. By comparison of published sequences of the BVDV strains NADL and Osloss and the HCV strain Alfort we have chosen the primers P l and P3 from the conserved regions, immediately upstream of the open reading frame. A
SEQUENCE AMPLIFICATION OF BVDV
9
125 nucleotide region spanning the start of the open reading frame (nucleotides 386-388 ) shows a very high homology with only one mismatch between the BVDV strains NADL and Osloss. Although the sequence of the HCV strain Alfort is less homologous to the two BVDV strains, this region shows considerable homology when compared to the rest of the HCV sequence. We therefore chose primers specific for this region in order to have a broad-reactive fragment. A comparison among the primers P 1 and P3 and the corresponding published sequences is shown in Table 2. The expected length of the fragments amplified by the primer pairs used and the location on the NADL genome are shown in Table 3 and Fig. 5. In order to minimize the risk of false negative reactions with the PCR technique, it is crucial to choose priming regions showing the highest possible degree of conservation between strains. In a previous report on PCR amplification of BVDV no such considerations were made (Schroeder and BalassuChan, 1990). In fact, one of the primers chosen lies within the insert of cellderived RNA. The homology between primers P 1 and P3 and the corresponding published sequences shown in Table 2 illustrates the degree of homology which we use in our PCR assay. Proper selection of primers should also make it possible to discriminate between HCV and BVDV, based on the sequences published until now (Renard et al., 1985; Collett et al., 1988; Meyers et al., 1989a; Moormann et al., 1990). We are currently investigating this aspect. Virus-specific amplification products of the expected size were found with several virus isolates tested. The method used for RNA preparation in this report is laborious and difficult to use for a large number of samples, and it failed to give suitable RNA from the BVDV strain NADL. Therefore, if PCR is to be used as a routine diagnostic tool, it is necessary to use a more simple method of sample preparation prior to the amplification. Studies regarding this aspect are currently under way. Demonstration of the digestion ability of PstI and Xhol in the PCR reaction mixture facilitates the identification of BVDV specific sequences, by eliminating false positives due to non-specific amplification of sequences of similar size. The inclusion of more than these two restriction enzymes may be necessary for this purpose. From the published sequences of the BVDV strains NADL and Osloss and the HCV strains Alfort and Brescia, it is expected that XhoI and Pstl each have one recognition site in the P3/Pi amplified fragment. However, as shown in Fig. 2 the P3/PI fragment of the HCV strains Alfort and Aid remains undigested. This cannot be ascribed to failure to digest in the PCR-mixture for the following reasons: ( l ) Digestiori of other fragments with XhoI and PstI using this protocol consistently showed complete digestion, and (2) X hol was not able to digest puri fled P3/P l fragments of the HCV strains Alfort and Aid.
Primer 3 P3 NADL Osloss Alfort Brescia
Primer I PI NADL Osloss Alfort Brescia
5'--
5'-T 5'-5'o5,oo
5'-G 5'-5'-5'-T 5'-T
G
G G
A
C
G
T
G
O
C
T
T
A
A
C
G
A
C
T
C
G
A
G
T
(7
G
A
C
C
C
A
C
T
A A
T
G
C
T
G
A
A
G
G
T
T
A
T
-3' -3' -3' -3' -3'
Comparison of primers P1 and P3 with the corresponding published sequences. Dots indicate identical nuclcotides
TABLE 2
G
A
°3~
-3' -3' -3' o3'
SEQUENCE AMPLIFICATION OF BVDV
| 1
TABLE 3
The expected length of the amplified fragment by the primer pairs used and the location on the BVDV strain NADL Primers pairs
Expected length of amplified product (base pairs)
NADL location (nucleotide Nos. )
P3/PI P3/P2 PI5/P5 PI5/P26 P25/PI9 P6/P26 P6/PI9
298 256 203 657 598 479 1057
98- 395 140- 395 3288-3490 2834-3490 2256-2853 2834-3312 2256-3312
~.~'
5'Enrlof .11
e
~-
=.
~
~-
I
I
I
I
I
g~Bloc~e
~
P3/P1
~
~"
Nt'~L~o~e
~
~
42~
P25/P19 P15/P26 P6p~9 ~
P6/P26 ~
P15/P5
Fig. 5. Position of primers and amplified fragments on the ~VDV strain NADL genome. The position ofglycoprotein gp53 indicated.
Asymmetric PCR has successfully been used to generate template DNA for sequencing of BVDV strains. The comparison in Fig. 5 shows the presence of markers shared between strains, as well as strain-specific markers. The distribution of markers has been used to propose the genetic relationship shown in Fig. 6. The high similarity between the American isolates NADL and Singer is remarkable, but corresponds well to similar neutralizing properties of the two virus strains (L. Ronsholt, personal communication 1990). The shaded boxes show two markers shared by the European isolates, but not by the American isolates. The most remarkable finding is perhaps the extensive homology between the Danish Ug59 and the German Osloss strain (unshaded boxes), suggesting a close genetic relationship between these two isolates. At present we do not know whether the two strains have other similarities, as for instance neutralization properties. The marker common for Osloss and 10039,
12
M. BOYEETAL.
f
Fig. 6. Preliminary pedigree showing the genetic relationship between the BVDV strains NADL, Singer, 10039, Osloss and Ug59, as deduced from the genetic map shown in Figure 5. The question marks represents hypothetical intermediate forms.
marked by an asterisk does not follow the general pattern and may have arisen independently in the two strains or been lost in Ug59. In conclusion we may say that, even with a relatively small number of nucleotides sequenced, we have identified markers which discriminate the European from the American isolates, and which link two of the European isolates genetically. At a symposium held in Hannover on June 8-9 1990 by the European society for Veterinary Virology data were presented by different groups working on BVDV, using PCR as a method for detecting and analyzing pestivirus nucleic acid. One of the reports focused on the gene coding for the p54 protein with the aim of detecting possible insertions in this gene as su~ested by Meyers et al. (1989b). No such insertions were found. Two other reports (presented by Hertig et al. and K.V. Brock) mainly focused on the use of PCR for detection of BVDV and comparing the method with traditional methods such as dot blot and Southern blot. In contrast to these reports we have optimized the choice of primers in order to obtain broad-reactive fragments. In this report we have demonstrated the usefulness of PCR of BVDV and HCV nucleic acids, thereby creating a platform for further inroads to the study of the molecular biology of these viruses. Traditional strategies, involving molecular cloning can now for many applications be replaced by this simple method for preparing DNA suitable for sequencing. Thus, PCR is a helpful tool for the study of the genomic variation (restriction enzyme pattern or sequence) of these important viruses.
REFERENCES Baker, J.C., 1987. Bovine viral diarrhea virus: a review. J. Am. Vet. Med. Assoc., 190: 14491458.
SEQUENCE AMPLIFICATION OF BVDV
.
13
Brownlie, J., Clarke, M.C. and Howard, C.J., 1984. Experimental production of fatal mucosal disease in cattle. Vet. Rec., 114: 535-536. Collett, M.S., Larson, R., Gold, C., Strick, D., Anderson, D.K. and Purchio, A.F., 1988. Molecular cloning and nucleotide sequence of the pestivirus bovine viral diarrhea virus. Virology, 165: 191-199. Diderholm, H. and Dinter, A., 1966. Infectious RNA from bovine virus diarrhea virus. Zentralbl. Bakteriolo Pasrasitenhvd. lnfektionshr. Hgg. Abt. 1:Orig., 201: 270-272. Donis, R.O., Corapi, W., Dubovi, E.J., 1988. Neutralizing monoclonal antibodies to bovine viral diarrhoea virus bind to the 56K to 58K glycoprotein. J. Gen. Virol., 69: 77-86. Fenner, F., Bachmann, P.A., Gibbs, E.P.J., Murphy, F.A., Studdert, M.J. and White, D.O., 1987. Diseases caused by togaviruses. In: Veterinary Virology, Academic Press Inc., 289-300, 458469. Lang, B.F. and Burger, G., 1990. A rapid, high resolution DNA sequencing gel system. Anal. Biochem., 188: 176-180. Magar, R., Minocha, H.C. and Lecomte, J., 1988. Bovine viral diarrhea virus proteins: Heterogeneity ofcytopathogenic and noncytopathogenic strains and evidence of a 53K glycoprotein neutralization epitope. Vet. Microbiol., 16: 303-314. Matthews, R.E.F., 1982. Classification and nomenclature of viruses: fourth report of the international committee on taxonomy of viruses. Intervirology, 17: 97-10. Meyers, G., Riimenapf, T. and Thiel, H.J., 1989a. Molecular cloning and nucleotide sequence of the genome of hog cholera virus. Virology, 171: 555-567. Meyers, G., Riimenapf, T. and Thiel, H.J., 1989b. Ubiquitin in a togavirus. Nature, 341: 49 I. Moormann, R.J.M., Warmerdam, P.A.M., Meet, B. van de Schaaper, W.M.M., Wenswoort, G. and Hulst, M.M., 1990. Molecular cloning and nucleotide sequence of hog cholera virus strain Brescia and mapping of the genomic region encoding envelope protein E I. Virology, 177: 184-198. van Oirschot, J., 1986. Hog cholera. In: A.D. Leman, B. Shaw, R.D., Glock, W.I. Mengeling, P.A.G. Penny and E. Scholl (Editors), Diseases of Swine 6th edn., Iowa State Univ. Press, Ames, 289-300. Osburn, B.I., Clarke, G.L., Stewart, W.C. and Sawer, M., 1973. Border disease-like syndrome in lambs: antibodies to hog cholera and bovine viral diarrhoea virus. J. Virol., 48: 320-324. Renard, A., Guiot, C., Schmetz, D., Dagenais, L., Pastoret, P.P. and Martial, J.A., 1985. Molecular cloning of bovine viral diarrhea viral sequences. DNA, 4: 429-438. Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn, G.T., Erlich, H.A and Arnheim, N., 1985. Enzymatic amplification of B-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science, 230:1350-1354. Sambrook, J., Fritsch, E.E, Maniatis, T., 1989. Molecular cloning, A laboratory manual. Second edn., Cold Spring Harbor, Laboratory, Cold Spring Harbor, NY. Schroeder, B.A. and Balassu-Chan, T.C., 1990. Specific sequence amplification of bovine viral diarrhoea virus nucleic acid. Arch. Virol., 111: 239-246.
