Heterogeneous expression of the non-structural protein p80/p125 in cells infected with different pestiviruses

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1 Journal of General Virology (1992), 73, Printed in Great Britain 47 Heterogeneous expression of the non-structural protein p80/p125 in cells infected with different pestiviruses I. Greiser-Wilke, 1. K. E. Dittmar, 2 B. Liess I and V. Moennig 1 i Institute of Virology, Hannover Veterinary School, Bischofsholer Damm 15, D-3000 Hannover 1 and 2 Gesellschaft f~r Biotechnologische Forschung mbh, Maseheroder Weg 1, 3300 Braunsehweig, Germany In order to analyse the expression of the non-structural (ns) protein p80/p125 in cells infected with different pestiviruses at the protein level, radioimmunoprecipitations with the pestivirus-specific monoclonal antibody (MAb) BVD/C16 were performed. Cell lysates infected with cytopathic (cp) and non-cytopathic (ncp) bovine viral diarrhoea (BVD) virus strains and isolates, and with hog cholera (HC) virus strains were analysed. From cpbvd virus-infected cells, the MAb precipitated one or more proteins corresponding to ns p125, displaying a marked size heterogeneity. In contrast, the lower Mr ns p80 proteins from all cpbvd virus strains and isolates analysed had identical electrophoretic motility. The ncpbvd virus strains displayed either one single band or a doublet of the p 125 protein and no p80 cleavage products. The p125 proteins precipitated from HC virus-infected cells showed no size heterogeneity. The possibility is discussed that multiple recombination events, including both insertions or deletions in the genomes of ncpbvd viruses, may lead to the heterogeneous expression of the ns p125 in cpbvd virus populations. Introduction Bovine viral diarrhoea (BVD) virus, an enveloped positive-strand RNA virus, together with border disease (BD) virus of sheep and hog cholera (HC) virus, holds generic status in the family Flaviviridae (Horzinek, 1991; Collett et al., 1988a, b; Stark et al., 1990). In general, pestiviruses are not cytolytic. However, since several BD and BVD viruses induce cytopathic (cp) effects in cultured ovine and bovine cells, a noncytopathic (ncp) biotype is distinguished from the cp biotype. BVD viruses of the latter induce cell shrinkage, cytoplasmic profusions, vacuolation and cell death after inoculation of susceptible monolayer cell cultures (Lee & Gillespie, 1957; Gillespie et al., 1960). The cp biotype plays a key role in the pathogenesis of BVD virusinduced late onset mucosal disease (Brownlie et al., 1984; Bolin et al., 1985; Moennig et el., 1990). At the genomic and polypeptide levels, markers of cpbvd viruses have been identified. Some cpbvd virus strains have been reported to contain insertions highly homologous to cellular sequences in their genome (Meyers et al., 1989, 1991). However, these findings could not be confirmed for other strains (Desport & Brownlie, 1991). A regular biotype-specific feature is the appearance of the additional non-structural (ns) protein p80, apparently a cleavage product of p125, in cells infected with cpbvd viruses (Purchio et al., 1984; Pocock et al., 1987; Donis & Dubovi, 1987). In order to analyse whether possible insertions in the genome are reflected at the protein level, the electrophoretic mobilities of the ns proteins p80/p 125 (nomenclature according to Collett et al., 1989) from different pestiviruses were compared. For this, cell lysates from cells infected with ncp- and cpbvd viruses and with HC viruses were radioimmunoprecipitated with the pestivirus-specific monoclonal antibody (MAb) BVD/C16 (Cay et al., 1989). Methods Cell cultures. BVD virus strains and isolates were propagated using foetal bovine kidney cells (FBKC) and stored at -80 C. For radioactive labelling, BVD virus strains and isolates were grown in the permanent Madin Darby bovine kidney (MDBK) cell line. HC virus strains were propagated in PK(15) cells as described earlier (Greiser- Wilke et al., 1990). All cell cultures and media were proven to be free of BVD virus and detectable mycoplasma contaminations. Virus strains andfiem isolates. The BVD virus strains were obtained from the National Animal Disease Center, Ames, Iowa, U.S.A. (strains Indiana, 7443, Auburn, Nebraska and TGAN), or from the Institute of Virology, Hannover, Germany (strains NADL, Oregon C24V and Singer). The field isolates were cultured from blood or faeces taken from animals hospitalized in the cattle clinic of the Hannover Veterinary School (Greiser-Wilke et al., 1991). Before being used for SGM

2 48 I. Greiser-Wilke and others labelling, they were passaged three times on FBKC. Isolates P913 and P 1142 (ncp) and 3486 (cp) were isolated from the same calf (H.-R. Frey, personal communication). The HC virus strains Alfort/187, 331 and Glentorf were from stocks maintained in the Institute of Virology, Hannover. Virus titres were determined in microassays (Holm-Jensen, 1981; Hyera et al., 1987). 200K MAb. Isolation and initial characterization of MAb BVD/C16 has been described earlier (Peters et al., 1986). The homologous antigen for the hybridoma was the cpbvd virus strain NADL. Hybridoma cells were propagated in Dulbecco's modification of Eagle's medium supplemented with 10% donor horse serum (Biochrom, Berlin). The MAbs were purified by immunoaffinity column chromatography over a column containing goat anti-mouse IgG covalently coupled to CNBr-activated Sepharose 4B as indicated by the manufacturers (Pharmacia). Isotopic labelling and preparation of cell extracts. MDBK cells were seeded into 10 cm diameter tissue culture dishes (Becton-Dickinson) at a concentration of cells per dish and incubated for 24 h. The cells were infected with BVD virus (m.o.i. = 2) or mock-infected with medium only. After 48 h, the monolayers were rinsed three times with PBS ph 7.2 and overlaid with methionine-free medium (Gibco-BRL). After 2 h incubation at 37 C, labelling was started by the addition of 150 gci L-[3sS]methionine (Amersham) in 2 ml of methionine-free medium. Cultures were incubated for 5 h at 37 C. The cells were then rinsed three times with PBS. Cells were lysed in 1 ml of TNE buffer (50 mm-tris-hc1 ph 8.0, 500 mm-nac1, 5 mm-edta) containing 1% Triton X-100, 0.1% SDS and 50 gm-pmsf. Cell debris was pelleted by centrifugation (10000 g, 30 min) and supernatants were frozen at -80 C. Labelled lysates of HC virus-infected PK(15) cells were prepared as described earlier (Greiser-Wilke et al, 1990) Radioimmunoprecipitation (RIP). This was performed as described earlier (Greiser-Wilke et al., 1990). SDS-PAGE. SDS PAGE (Laemmli, 1970) was performed in 7.5% acrylamide resolving gels with 5% acrylamide stacking gels. Mr standards were 14C-methylated proteins (Amersham). After electrophoresis, gels were fixed in 25% isopropanol-10% acetic acid and dried under vacuum. For autoradiography, dried gels were exposed for 7 days on Kodak DEF-2 direct exposure films (Eastman Kodak). Results Comparison of the electrophoretic mobility of the ns p80/p125 proteins from cells infected with different pestivirus laboratory strains The MAb BVD/C16 was used for RIP of lysates from cells infected with four cpbvd virus, four ncpbvd virus and three HC virus strains (Fig. 1 and 2). As negative controls, lysates from BVD virus- (Fig. 1, lane 7) and HC virus- (Fig. 2, lanes 1 and 2) infected cells were precipitated with the unrelated MAb BM/40 (specific for Brucella melitensis lipopolysaccharide). From lysates from cells infected with cpbvd virus strains, two bands were specifically precipitated (Fig. 1). The lower band (p80) precipitated from the four cpbvd virus-infected cell lysates had almost identical Mr values between 82K and 83K, but the upper band (p125) displayed a considerable heterogeneity in size (Table 1). 97.4K-- 69K-- 46K-- Fig. 1. RIP of the ns protein p80/p125 from 35S-labelled lysates from cells infected with different BVD and HC virus laboratory strains with the MAb BVD/CI6. Lane 1, PK(15) cells infected with HCV-strain Alfort. Lanes 2 to 6, MDBK cells infected with BVD virus strains 7443 (ncp), Oregon (cp), Indiana (cp), Singer (cp) and NADL (cp), respectively. Lane 7, negative control; NADL-infected cells precipitated with an unrelated MAb (BM/40). Lane 8, positive control; NADL-infected cells precipitated with polyclonal swine BVD/HC virus-specific serum. Mr standards (Amersham) are indicated. Arrows indicate (from bottom to top) viral proteins gp48, gp53, p80 and p125. The estimated Mr ranged from l14k (Fig. 1, lane 3) to 128K (Fig. 1, lane 6). In contrast, from cell lysates infected with ncpbvd viruses only one band (Fig. 2, lanes 3 and 5), or occasionally a doublet of high Mr (p 125), was precipitated. The doublet precipitated from the lysate of cells infected with strain Auburn (Fig. 2, lane 4) had Mr values of about 120K for the slower and 116K for the faster migrating band (Table 1). From cell lysates infected with HC viruses, a single prominent band (p125) with an apparent Mr of 120K in all cases, and a faint band of about 89K were precipitated (Fig. 1, lane 1 ; Fig. 2, lanes 6 and 7). Comparison of the electrophoretic mobility of the ns p80/p125 proteins from cells infected with different B VD field virus isolates To exclude the possibility that the observed heterogeneity of the p125 protein both in size (cpbvd virus) as well as in the number of precipitated bands (ncpbvd virus) was due to the fact that only cloned BVD virus

3 Expression of pestiviral protein p80/p Table 1. Apparent Mr values of the ns proteins p80/p125 precipitated with the MAb BVD/C16 from cells infected with different HC and BVD virus strains and isolates Apparent Mr of protein (x 10-3) * Virus p125 (I) p125 (II) p80 Fig. 2. RIP of the ns protein p125 from 3sS-labelled lysates from cells infected with different BVD and HC virus laboratory strains with the MAb BVD/C 16. Lanes 1 and 2, negative control; Alfort-infected cells precipitated with an unrelated MAb. Lanes 3 to 5, MDBK ceils infected with BVD virus strains TGAN (ncp), Auburn (ncp) and Nebraska (ncp). Lanes 6 and 7, PK(15) cells infected with HC virus strains 331 and Glentorf. Mr standards are indicated. Arrows indicate p125 (top) and a band precipitated by the MAb BVD/C16 from lysates from cells infected with HC virus (Mr about 89K). laboratory strains were used, eight uncloned cp- and nine ncpbvd virus isolates from naturally occurring cases of mucosal disease were also analysed. One cp and two ncp isolates were derived from the same animal. RIP analyses of the lysates from cells infected with cpbvd viruses confirmed the size heterogeneity of p125 observed with the cp strains (Table 1). The MAb precipitated a protein with an apparent Mr of about 116K from all cp isolates analysed, but two of the isolates displayed two (Fig. 3, lanes 2 and 6), and one of the isolates a smear of three or four (Fig. 3, lane 5) additional proteins with apparent Mr values ranging from 120K to 127K (Table 1). Confirming the results obtained with the cloned cpbvd virus strains, the p80 proteins from these isolates all had a consistent apparent Mr of about 83K. No marked differences in apparent Mr values were observed in the RIP with lysates from cells infected with ncpbvd virus isolates. From five of these isolates cpbvd strains Oregon 114 ND? 83 Indiana 122 ND 82 Singer 120 ND 83 NADL 128 ND 83 cpbvd isolates ND ND ND ND 83 ncpbvd strains 7443 l 16 ND ND TGAN 117 ND ND Auburn ND Nebraska 117 ND ND ncpbvd isolates ND ND ND ND ND ND ND ND ND HC strains Alfort 120 ND (89) ND (89) Glentorf 120 ND (89) Matching pair (BVD) ncpp ND ND ncpp ND ND cp ND 83 * The apparent Mr values were calculated from the R F values (S.E.M. = + 2K). The standards were as indicated in Fig. 1. From several strains and isolates, one [p125 (I)] or more [p125 (II)] proteins were precipitated. The protein from HC virus-infected cells which coprecipitated migrating at about 89K is in parentheses as its identity could not be established. t ND, Not detected. doublets with apparent Mr values of about 115K to 117K and 119K to 122K, respectively, were precipitated (Fig. 4; Table 1). Depending on the isolate used, either the faster (Fig. 4, lanes 4, 7 and 8) or the slower (Fig. 3, lane 9; Fig. 4, lane 6) migrating bands were predominant. The two isolates derived from the same animal (Fig. 4, lanes 2 and 3) showed the faster migrating band (l15k) only. The corresponding cp isolate (Fig. 4, lane 1) displayed the same single p125 band in addition to p80. From the remaining two ncp isolates (Fig. 3, lane 8; Fig. 4, lane 5) a single band was precipitated.

4 50 I. Greiser-Wilke and others 46K Fig. 3. RIP of the ns protein p80/p125 from 35S-labelled lysates from cells infected with different cpbvd virus field isolates with the MAb BVD/C16. Lanes 1 to 9, MDBK cells infected with BVD virus isolates cp4468, cp4250, cp4410, cp5367, cp742, cp5316, cp5069, ncp5368 and ncp5412, respectively. Lanes 5 to 7, negative controls. The corresponding lysates were precipitated with an unrelated MAb. Mr standards are indicated. Arrows indicate p125 (top) and p80 (bottom) proteins precipitated by the MAb BVD/C16 from lysates from cells infected with BVD virus isolates. 200K- 97.4K- 69K- 46K Fig. 4. RIP of the ns protein p80/p125 from 35S-labelled lysates from cells infected with different ncpbvd virus field isolates and with three isolates derived from one calf (lanes 1 to 3) with the MAb BVD/C16. Lanes 1 to 8, MDBK cells infected with BVD virus isolates cp3486, ncppl142, ncpp913, ncp2510, ncp7367, ncp6707, ncp6547 and ncp6544, respectively. Lanes 5 to 7, negative controls. The corresponding lysates were precipitated with an unrelated MAb. Mr standards are indicated. Arrows indicate p125 (top) and p80 (bottom) proteins precipitated by the MAb BVD/C16 from lysates from ceils infected with BVD virus isolates. Discussion The experimental induction of mucosal disease (Brownlie et al., 1984; Bolin et al., 1985; Moennig et al., 1990) supports the hypothesis that both cpbvd and ncpbvd viral biotypes are involved in the pathogenesis of this late onset disease. Whether superinfection of cattle persistently infected with ncpbvd virus with an immunologically closely related cp strain (McClurkin et al., 1985; Bolin et al., 1985), or a transition to the cp biotype induced by a mutation (Howard et al., 1987), triggers the disease is not clear. At the molecular level, comparison of protein profiles obtained by RIP showed differences between lysates of cp- and ncpbvd virus-infected cells. Although two ns proteins (p125 and p80) are precipitated from cpbvd virus-infected cells, the p80 is missing in lysates from ncpbvd virus-infected cells (Pocock et al., 1987; Donis & Dubovi, 1987; Magar et al., 1988). By tryptic peptide mapping it has been shown that p125 and p80 are structurally related (Purchio et al., 1984). The pestivirusspecific MAb BVD/C16 (Cay et al., 1989) immunoprecipitated the p125 protein from lysates of cells infected with BVD and HC viruses. In addition, it precipitated the cpbvd virus-specific p80 protein. From these results we have concluded that the MAb is directed against a conserved epitope on the C-terminal part of p125. The reaction of the MAb with p80 and p125 presents further evidence that p80 is a cleavage product of p125. The RIP with lysates from cells infected with four ncpand four cpbvd laboratory virus strains, nine ncp- and eight cpbvd field virus isolates and three HC virus strains revealed that the p125 proteins were heterogeneous in size. This confirms the results obtained in RIP performed with lysates from cells infected with the Moredun cp and ncp BD virus strains (Dutia et al., 1990). The Mr values of the p125 proteins from cpbvd virus ranged between about 114K (Oregon) and about 128K (NADL). In contrast, the proteins from the 13 ncpbvd virus-infected cell lysates had identical Mr (about 117K); a second protein of about 120K was precipitated from six of these strains. The size difference of 11K between the faster migrating p 125 proteins of lysates of cells infected with ncp- or cpbvd virus NADL corresponds to about 200 to 300 nucleotides in genomic size. This is in agreement with the length of the insert found for this strain (Meyers et al., 1989). Based on the migration profiles, an insert of about 125 nucleotides could be predicted for Indiana, and of about 75 nucleotides for Singer. On the other hand, the faster migration of the p125 from cpbvd virus strain Oregon (l14k) as compared to ncp- and the other cpbvd viral proteins might indicate that apart from insertion of cellular sequences, deletion of viral sequences may also be a

5 Expression of pestiviral protein p80/p possible mechanism for the altered processing of p125 in cells infected with cpbvd virus strains. Discussion concerning the global existence of cellular inserts in cpbvd virus strains has arisen recently as more data from different laboratories have become available (Meyers et al., 1991 ; Desport and Brownlie, 1991). Only the sequencing of genomes from a larger number of BVD virus strains and isolates will finally answer this question. The migration profiles of the p 125 from the three cell lysates infected by the HC virus strains tested indicated a similar size (about 120K), thus resembling ncpbvd viruses. An additional weak band migrating somewhat faster corresponded to a glycosylated protein of unknown origin (not shown) of about 89K. Because of the conserved size of the p125 protein, and in agreement with the genomic analysis of HC virus strains Alfort (Meyers et al., 1989) and Brescia (Moormann et al., 1990), we postulate that the HCV strains analysed do not contain genomic inserts. In addition, RIP with the MAb BVD/C16 indicates that the HC viral p125 protein is not processed to yield p80. Although the p125 proteins of the different cpbvd virus strains analysed were heterogeneous in size, the processed p80 proteins displayed identical migration properties, with Mr values of about 83K. From this observation we concluded that the insert must be localized in the p54 cleavage product, which should consequently be heterogeneous in size. We were unable to perform these experiments, since this protein is not precipitated by polyclonal BVD virus-specific sera. So far, it has only been detected by a specific anti-peptide serum (Collett et al., 1991). In addition, it is tempting to speculate that the cleavage site at the N-terminal part of the p80 protein is specific and conserved among pestiviruses, but is not accessible for processing in cells infected with ncpbvd or with HC virus strains. Another possible mechanism would be the acquisition of a new cleavage site. This would support the hypothesis that an ncpbvd virus biotype mutates to cpbvd virus by gaining cellular sequences (Meyers et al., 1989) or, alternatively, by losing viral sequences during a recombination event, thus acquiring or exposing the cleavage site for further processing of p125. RIP analysis of uncloned cpbvd virus isolates showed that the p125 proteins not only differed in size but also showed variation in the number of precipitated proteins. This strongly indicates that these isolates consisted of two or even more different virus populations; in addition, it can not be excluded that a certain subpopulation is of the ncp biotype. In this case, it has been shown that both the ncp as well as the cp virus proteins are expressed (Ridpath & Bolin, 1990). It is unlikely that these highly heterogeneous virus isolates arise by superinfection of persistently infected calves with a single antigenically related cp biotype virus. However, our knowledge is far from complete; when cp isolates are cloned and passaged in the laboratory, there seems to be selection for subpopulations expressing a single p125 protein (Fig. 1). In contrast, the ncpbvd virus strains and isolates seemed to be more homogeneous when analysed by RIP. Since no high Mr precursor for the ns p125 protein seems to exist (Collett et al., 1991), the fact that some strains expressed two p125 molecules with different apparent Mr strengthens the notion that the recombination events may occur frequently in infected cells, but with only few of them finally leading to the appearance of cp virus (sub) populations. The authors wish to thank S. Aydogdu, M. Kaps, R. Neth and G. Miiller for excellent technical assistance. References BOLIN, S. R., MCCLURKIN, A. W., CUTLIP, R. C. & CORIA, M. F. (1985). Severe clinical disease induced in cattle persistently infected with noncytopathic bovine viral diarrhea virus by superinfection with cytopathic bovine viral diarrhea virus. American Journal of Veterinary Research 46, BROWNLm, J., CLARKE, M. C. & HOWARD, C. J. (1984). Experimental production of fatal mucosal disease in cattle. Veterinary Record 114, CAY, B., CHAPPUtS, G., COULIBALY, C., DINTER, Z., EDWARDS, S., GREISER-WILKE, I., GUNN, M., HAVE, P., HESS, G., JUNTTI, N., L ~ss, B., MATEO, A., MCHUGH, P., MOENNIG, V., NETrLETON, P. & WENSVOORT, G. (1989). Comparative analysis of monoclonal antibodies against pestiviruses: report of an international workshop. Veterinary Microbiology 20, COLLETT, M. S., LARSON, R., BELZER, S. K. & RETZEL, E. (1988a). Proteins encoded by bovine viral diarrhea virus: the genomic organization of a pestivirus. Virology 165, COLLETT, M. S., LARSON, R., GOLD, C., STRICK, D., ANDERSON, D. K. & PURCHIO, A. F. (1988b). Molecular cloning and nucleotide sequence of the pestivirus bovine viral diarrhea virus. Virology 165, COLLETT, M. S., MOENNIG, V. & HORZlNEK, M. C. (1989). Recent advances in pestivirus research. Journal of General Virology 70, COLLETI', M. S., WISKERCHEN, M. A., WELNIAK, E. & BELZER, S. K. (1990. Bovine viral diarrhea virus genomic organization. Archives of Virology supplement 3, 19~7. DESPORT, i. & BROWNLIE, J. (1991). Molecular characterisation of the coding region for the p125 from homologous BVDV biotypes. Archives of Virology supplement 3, DONIS, R. O. & DUBOVI, E. J. (1987). Differences in-virus-induced polypeptides in cells infected by cytopathic and noncytopathic biotypes of bovine virus diarrhea-mucosal disease virus. Virology 158, DUTIA, B. i., ENTRICAN, G. & NETI'LETON, P. (1990). Cytopathic and non-cytopathic biotypes of border disease virus induce polypeptides of different molecular weight with common antigenic determinants. Journal of General Virology 71, GILLESPIE, J. H., BAKER, J. A. & McENTEE, K. (1960). A cytopathogenic strain of virus diarrhea virus. Cornell Veterinarian 50, GREISER-WILKE, I., MOENNIG, V., COULIBALY, C. O. Z., DAHLE, J., LEDER, L. & LIESS, B. (1990). Identification of conserved epitopes on a hog cholera virus protein. Archives of Virology 111,

6 52 I. Greiser-Wilke and others GREISER-WlLKE, I., LIESS, B., SCHEI'ERS, J, STAHL-HENNIG, C. & MOENNIG, V. (1991). Correlation of bovine viral diarrhoea virus induced cytopathic effects with expression of a biotype-specific marker. Archives of Virology supplement 3, HOLM-JENSEN, i. (1981). Detection of antibodies against hog cholera virus and bovine viral diarrhea virus in porcine serum. A comparative examination using CF, PLA and NPLA assays. Acta veterinaria scandinaviea 22, HORZlNEK, M. C. (1991). Pestiviruses - taxonomic perspectives. Archives of Virology supplement 3, I-5. HOWARD, C. J., BROWNLIE, J. & CLARKE, M. C. (i987). Comparison by the neutralization assay of pairs of non-cytopathogenic and cytopathogenic strains of bovine virus diarrhoea virus isolated from cases of mucosal disease. Veterinary Microbiology 13, HYERA, J. i. K., DAHLE, J., LIESS, B., MOENNIG, V. X, FREY, H.-R. (1987). Production of potent antisera raised in pigs by anamnestic response and use for direct immunofluorescent and immunoperoxidase techniques. In Agriculture. Pestivirus Infections of Ruminants, pp Edited by J. W. Harkness. Brussels: Commission of the European Communities, publication CD-NA EN-C. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227, LEE, K. M. & GILLESPlE, J. H. (1957). Propagation of virus diarrhea virus of cattle in tissue culture. American Journal of Veterinary Research 18, McCLURKIN, A. W., BOLIN, S. R. & CORIA, M. F. (1985). Isolation of cytopathic and noncytopathic bovine viral diarrhea virus from the spleen of cattle acutely and chronically affected with bovine viral diarrhea. Journal of the American Veterinary Medical Association 186, MAGAR, R., MINOCHA, H. C., MONTPETIT, C., CARMAN, P. S. & LECOMTE, J. (1988). Typing of cytopathic and noncytopathic bovine viral diarrhea virus reference and Canadian field strains using a neutralizing monoclonal antibody. Canadian Journal of Veterinary Research 52, MEYEr, G., RUEMENAPF, T. & THIEL, H. J. (1989). Ubiquitin in a togavirus. Nature, London 341, 491. MEYERS, G., TAUTZ, N., DUBOVI, E. J. & THIEL, H.-J. (1991). Viral cytopathogenicity correlated with integration of ubiquitin-coding sequences. Virology 180, MOENNIG, V., FREY, H.-R., L1EBLER, E., POHLENZ, J. & LIESS, B. (1990). Reproduction of mucosal disease with cytopathogenic bovine viral diarrhoea virus selected in vitro. Veterinary Record 127, MOORMANN, R. J. M., WARMERDAM, P. A. M., VAN DER MEER, B., SCI-IA~PER, W. M. M., WENSVOORT, G. & HULST, M. M. (1990). Molecular cloning and nucleotide sequence of hog cholera virus strain Brescia and mapping of the genomic region encoding envelope protein El. Virology 177, PETERS, W., GREISER-WILKE, I., MOENNIG, V. & LIESS, B. (1986). Preliminary serological characterization of bovine viral diarrhoea virus strains using monoclonal antibodies. Veterinary Microbiology 12, POCOCK, D. H., HOWARD, C. J., CLARKE, M. C. & BROWNLIE, J. (1987). Variation in the intracellular polypeptide profiles from different isolates of bovine virus diarrhoea virus. Archives of Virology 94, PURCHIO, A. F., LARSON, R. & COLLETr, M. S. (1984). Characterization of bovine viral diarrhea virus proteins. Journal of Virology 50, RIDPATH, J. F. & BOLIN, S. R. (1990). Viral protein production in homogeneous and mixed infections of cytopathic and noncytopathic BVD virus. Archives of Virology 111, STARK, R., RUEMENAPF, T., MEYERS, G. & THIEL, H.-J. (1990). Genomic localization of hog cholera virus glycoproteins. Virology 174, (Received 17 December 1990; Accepted 19 September 1991)