Genetic and antigenic characterization of an atypical pestivirus isolate, a putative member of a novel pestivirus species

Horst Schirrmeier, Günther Strebelow, Klaus Depner, Bernd Hoffmann and Martin Beer

Friedrich-Loeffler-Institut, Institute of Diagnostic Virology, Boddenblick 5a, 17493 Greifswald-Insel Riems, Germany

Correspondence
Martin Beer
beer{at}rie.bfav.de


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The genus Pestivirus within the family Flaviviridae currently consists of four different main species: Classical swine fever virus, Bovine viral diarrhea virus types 1 and 2 and Border disease virus. A fifth tentative species is represented by an isolate from a giraffe. In this study, a completely new pestivirus, isolated from a batch of fetal calf serum that was collected in Brazil, is described. It is proposed that the isolate D32/00_‘HoBi’ may constitute a novel sixth pestivirus species, because it is genetically, as well as antigenically, markedly different from all other pestiviruses. Based on the entire Npro- and E2-encoding sequences, identities of <70 % to all other pestivirus species were determined. Similarly, cross-neutralization and binding studies using antisera and mAbs revealed marked antigenic differences between D32/00_‘HoBi’ and all other pestiviruses.

The GenBank/EMBL/DDBJ accession numbers for the 5'-UTR, Npro, E2, NS3 and 3'-UTR sequences of isolate D32/00_‘HoBi’ reported in this paper are AY489116, AY489117, AY604725, AY713481 and AY604726, respectively.


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The genus Pestivirus belongs to the family Flaviviridae and consists of four separate virus species: Bovine viral diarrhea virus types 1 and 2 (BVDV-1 and -2), Classical swine fever virus (CSFV) and Border disease virus (BDV). A tentative fifth species is defined by an atypical pestivirus that was isolated from a giraffe (van Regenmortel et al., 2000). All pestiviruses are genetically and structurally related and serological cross-reactivity between all members of the genus has been reported. The natural hosts of pestiviruses are domestic and wild ruminants, as well as porcines (Thiel et al., 1996). The host ranges of BVDV and BDV are not restricted to cattle and sheep, but include a wide range of hosts in the Artiodactyla (Plowright, 1969; Hamblin & Hedger, 1979; Doyle & Heuschele, 1983; Nettleton, 1990; Becher et al., 1997), whereas CSFV has only been isolated from pigs (Dahle et al., 1987; Thiel et al., 1996). The pestivirus virion is enveloped and contains a genome that consists of a single-stranded RNA of approximately 12·5 kb and positive polarity (Collett et al., 1988; Meyers et al., 1989; Ridpath & Bolin, 1995; Becher et al., 1998). It contains a single ORF that is flanked by 5' and 3' untranslated regions (UTRs) and encodes the structural proteins capsid, ERNS, E1 and E2, as well as the non-structural proteins Npro, p7, NS2–3 (NS2, NS3), NS4A, NS4B, NS5A and NS5B (Donis, 1995). For genetic analyses, the 5'-UTR (Vilcek et al., 1994; Wolfmeyer et al., 1997; Beer et al., 2002) and the regions encoding Npro (Becher et al., 1997, 2003), E2 (van Rijn et al., 1997) and NS3 (Ridpath et al., 1994; Pellerin et al., 1995) have primarily been utilized and the Npro-encoding region is thought to yield a precise distinction of different pestivirus types and subtypes (Becher et al., 1997, 2003; Avalos-Ramirez et al., 2001). However, the highly conserved 5'-UTR gives nearly identical results, particularly concerning the allocation of pestivirus isolates to species or genotypes (Vilcek et al., 2001). Important criteria for the classification of pestiviruses are their similarity at the nucleotide level, as well as their reactivity in binding assays with mAbs and cross-neutralization tests with homologous and heterologous polyclonal antisera. By using these techniques, the giraffe isolate was shown to differ clearly from all other known pestiviruses (Harasawa et al., 2000; Avalos-Ramirez et al., 2001), leading to a new, tentative pestivirus species, Pestivirus of giraffe (van Regenmortel et al., 2000). In addition, new and previously unclassified pestiviruses from reindeer (Reindeer-1), pig (Gifhorn) and sheep (Stolpe, Chemnitz) have recently been demonstrated to form distinct genotypes within BDV species (BDV-2 and -3) (Becher et al., 2003; H. Schirrmeier, K. Depner, G. Strebelow & M. Beer, unpublished data). In this study, we report the genetic and antigenic characterization of a new atypical pestivirus that was isolated from a batch of fetal calf serum (FCS) originating from Brazil. The marked differences of the investigated virus isolate from other pestiviruses led to the conclusion that the virus strain designated D32/00_‘HoBi’ may be a member of a novel pestivirus species.

D32/00_‘HoBi’ was isolated from fetal sheep thymus cells ‘SFT-R’ [RIE43, Collection of Cell Lines in Veterinary Medicine (CCLV), Insel Riems] that were grown in Dulbecco's modified Eagle's medium supplemented with 10 % FCS batch 547 from Brazil (Biochrom). PCR analysis directly from the FCS sample, as well as inoculation of KOP-R cells, a diploid bovine oesophageal cell line (RIE244, CCLV), demonstrated clearly that this particular batch (547) was the source of contamination with D32/00_‘HoBi’. Most efficient replication of the virus was observed with bovine cell lines. In contrast, no efficient virus propagation was detectable after inoculation of porcine kidney PK-15 cells (data not shown). D32/00_‘HoBi’-infected KOP-R cells were tested in binding assays with a panel of pestivirus-specific mAbs directed against NS2–3, ERNS and E2 (Table 1; Peters et al., 1986; Moennig et al., 1987; Edwards et al., 1988, 1991; Paton et al., 1994, 1995). Immunofluorescence (IF) and immunoperoxidase analyses were performed as described previously (Depner et al., 2001). It could be demonstrated that three of the five NS2–3-specific mAbs (C16, 103/105 and 435) were able to detect D32/00_‘HoBi’-infected cells (Table 1). Interestingly, the BVDV-1- and BDV-specific mAb 160 (Beer & Wolf, 1999) was unable to bind, and only mAbs 103/105 and C16 reacted with all strains tested (Table 1). In addition, only two (433 and 434) of the eight ERNS-specific mAbs tested, which normally detect BVDV-2 isolates, and none of the ten E2-specific mAbs reacted with D32/00_‘HoBi’ in the binding assay (Table 1). This markedly restricted reaction pattern of the newly isolated virus was significantly different from those of other pestiviruses. However, failure of mAb 160 to bind to D32/00_‘HoBi’-infected cells and interaction with the ERNS-specific mAbs 433 and 434 are related most closely to the reaction pattern of BVDV-2 and the Giraffe-1 strains (Table 1).


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Table 1. Reactivity of NS2–3-, ERNS- and E2-specific mAbs with members of different pestivirus species

Reactivity was measured by indirect immunoperoxidase assay. +, Positive reaction.

 
For sequencing, RNA was isolated from inoculated KOP-R cell cultures and directly from contaminated FCS by using TRIzol reagent (Invitrogen). Different parts of the D32/00_‘HoBi’ genome were amplified by RT-PCR as described previously (Pfeffer et al., 2000; Schaarschmidt et al., 2000). For amplification of a 5'-UTR fragment and the entire Npro-encoding region, the primer pairs BVD_II (sense; 5'-GGTAGCAACAGTGGTGAGTTC-3')/BVD_II_NTR51 (antisense; 5'-CAACTCCATGTGCCATGTAC-3') and PP235F (sense; 5'-AYGTGGACGAGGGCRYGCCCA-3')/PP1040R (antisense; 5'-CCYTTCTTYYTNACCTGGTA-3') were used. Amplification products generated with both primer pairs cover a genomic region that corresponds to nt 139–1059 of BVDV-1 strain NADL (Collett et al., 1988; GenBank accession no. NC_001461). In addition, the entire E2-encoding sequence and a 135 bp fragment of the 3'-UTR were amplified by using the primer pairs Hobi1300-F (sense; 5'-CATGGAATGATGGACGCTAG-3')/PP3450-R (antisense; 5'-TTBARCATGTATTGYTGGAAGTA-3'), Hobi 3400-F (sense; 5'-ATTCAACTGGACTGAGACGC-3')/PP4600-R (antisense; 5'-AYCTCTTCTATDATTTTCCTGTG-3') and PP3'-1F (sense; 5'-TGATGTAYTCHTGGAAYCC-3')/PP3'-3R (antisense; 5'-CTCWBMCAGCTAAAGTGCT-3'). The complete NS3-encoding sequence was amplified by using the primer pairs Hobi 4920-F (sense; 5'-GTAGGCCCATATCATGCGGA-3')/PP6200-R (antisense; 5'-GGGGCTATGAACTCYTCTAT-3') and Hobi 6120-F (sense; 5'-AGCTGGGTCGGTCACAAC-3')/PP7200-R (antisense; 5'-AACTGKAGRTGDGTKGTGTC-3').

The RT-PCR products were cloned directly and three independent plasmid clones were sequenced by using M13 universal and reverse primers. Subsequently, a consensus sequence of the amplified and cloned fragments was generated. Part of the 5'-UTR (184 bp fragment; GenBank accession no. AY489116), the entire Npro sequence (504 bp; accession no. AY489117), the entire E2 sequence (1119 bp, accession no. AY604725), the entire NS3 sequence (2049 bp; accession no. AY713481) and part of the 3'-UTR (135 bp, accession no. AY604726) were aligned with the sequences of standard strains from GenBank (GCG programs; CLUSTALW; HUSAR, DKFZ Heidelberg), followed by phylogenetic analysis based on the Npro, E2 and NS3 alignments (Fig. 1a–c). The trees were generated by using the PUZZLE software (Strimmer & von Haeseler, 1996). For maximum-likelihood tree reconstructions, 1000 puzzling steps and the Hasegawa model for nucleotide substitution were used. Dendrograms generated by PUZZLE were visualized by using the TREEVIEW software (Page, 1996). The Npro- and E2-based phylogenetic trees clearly showed six major branches corresponding to the four pestivirus species BVDV-1, BVDV-2, BDV and CSFV, the tentative species Pestivirus of giraffe and the novel strain D32/00_‘HoBi’ (Fig. 1a, b). Phylogenetic analysis of NS3 revealed a clear distinction of all different species. However, due to the markedly higher identities of the NS3-encoding sequences between all pestivirus species, hypothetical taxonomical units were generated for BVDV types 1 and 2, as well as for the tentative species Pestivirus of giraffe and D32/00_‘HoBi’ (Fig. 1c). The robustness of the obtained clades was supported by high probability values of 75–100 % at the internal branch-points dividing the different species (Fig. 1a–c). A comparison of sequence identities was obtained with the program GAP (HUSAR, DKFZ Heidelberg). Based on the entire Npro-encoding sequence, none of the selected pestivirus species had >67 % identity to D32/00_‘HoBi’ (64–67 %; Table 2). Identity values for the entire E2 nucleotide sequence ranged between 58 and 62 %. In addition, analyses using the 5'-UTR and 3'-UTR sequences resulted in an identical genotype/species classification, with identities of 66–75 % (5'-UTR) and 41–55 % (3'-UTR) (data not shown). The highest identity values were calculated for the extremely conserved NS3 nucleotide sequences (76–78 %). The calculated sequence identities within the Npro-encoding region compared with the other pestivirus species are similar to that reported for the giraffe strain (Avalos-Ramirez et al., 2001), but 4–11 % lower than those between the different BDV genotypes (Table 2).



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Fig. 1. (a–c) Phylogenetic trees generated from the entire Npro-, E2- and NS3-encoding nucleotide sequences of selected members of known species of the genus Pestivirus and D32/00_‘HoBi’. The sequences of the virus strains Osloss, NADL, PT810, Giessen-6, 890, Alfort187, Brescia, CSFV39, X818, Gifhorn, Reindeer-1 and Giraffe-1 were taken from GenBank (accession nos M96687, NC_001461, AY078406, AF144470, AF144612, U18059, X87939, AF091661, AF407339, NC_003679, AY163653, AY163660, NC_003677 and NC_003678). The Npro, E2 and NS3 sequences of D32/00_‘HoBi’ were generated in this study. The trees were constructed by using the software PUZZLE (Strimmer & von Haeseler, 1996) and TREEVIEW (Page, 1996). The numbers refer to the percentage support for the internal branches of the quartet-puzzling tree (1000 puzzling steps). Branch lengths are proportional to genetic distances. (d) Cross-neutralization assays of D32/00_‘HoBi’ with sera specific for members of the different species and major genotypes within the genus Pestivirus. D32/00_‘HoBi’ was not neutralized efficiently by heterologous sera (black bars), with titres ranging between <1 : 5 and 1 : 10. In contrast, homologous neutralization titres (grey bars) reached levels between 1 : 120 and 1 : 1280. Strains used for the generation of antisera are indicated.

 

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Table 2. Percentage nucleotide identity of members of different pestivirus species or major genotypes, based on the entire Npro sequence

Identity was determined by using GAP (HUSAR, DKFZ Heidelberg).

 
The results of the cross-neutralization assays corroborated the classification of D32/00_‘HoBi’ as the putative prototype of a novel species within the genus Pestivirus. By using well-defined reference sera from pigs (serum against CSFV strain 976/43; kindly provided by the EU reference laboratory for CSFV, TiHo Hannover, Germany) or rabbit hyperimmune sera (sera against BVDV-1 strain Paplitz, BVDV-2 strain Munich-2, BDV-1 strain Moredun, BDV-2 strain Reindeer-1 and BDV-3 strain Gifhorn; H. Schirrmeier, G. Strebelow & K. Depner, unpublished data), no or only low neutralizing titres against D32/00_‘HoBi’ could be determined (Fig. 1d). No neutralizing activity versus D32/00_‘HoBi’ was detectable with four of the six sera tested (neutralizing antibody titre <1 : 5), although homologous titres varied from 1 : 120 to 1 : 1280 (Fig. 1d). A D32/00_‘HoBi’-specific immune serum obtained from an inoculated calf with a homologous titre of >1 : 640 was used as a positive control (Fig. 1d). With this antiserum, a heterologous titre of 1 : 40 could be determined against the giraffe strain (data not shown).

As D32/00_‘HoBi’ was isolated from an FCS sample, its real host remains unidentified. Nevertheless, domestic or free-ranging ruminants, particularly of the genus Bos within the family Bovidae, are the most likely target species of D32/00_‘HoBi’. In a first animal experiment, pigs (n=2) and cattle (n=2) were infected intranasally with 2x108 TCID50 D32/00_‘HoBi’ per animal. No D32/00_‘HoBi’ virus could be reisolated from the inoculated pigs, none of the contact pigs seroconverted and no clinical symptoms or leukopenia could be detected. However, both inoculated pigs seroconverted versus D32/00_‘HoBi’. In contrast, viraemia was detected at day 5 post-infection in the infected calves, D32/00_‘HoBi’ virus was shed for several days (days 3–6 post-infection) and slight leukopenia, as well as a mild increase in body temperature, could be detected (data not shown). As observed for the inoculated pigs, the infected calves did not show clinical signs during the whole experiment.

Taken together, comparative sequence analyses, binding studies with mAbs and cross-neutralization assays revealed marked differences between D32/00_‘HoBi’ and all current pestivirus species. We propose that the low nucleotide sequence identities between D32/00_‘HoBi’ and selected members of all other species based on the 5'-UTR, Npro, E2 and 3'-UTR sequences support classification as a novel pestivirus species; the low sequence similarities are consistent with previous values that have been used for division into species (Vilcek et al., 1994; Becher et al., 1997, 2003; Avalos-Ramirez et al., 2001). Thus, D32/00_‘HoBi’ may be classified as a representative of a novel tentative species within the genus Pestivirus by analogy to the species Pestivirus of giraffe (van Regenmortel et al., 2000). In addition, the NS3 nucleotide sequence comparison generated the same identity values between D32/00_‘HoBi’ and all other pestivirus species. Interestingly, the tree based on the NS3 sequences revealed a slightly enhanced phylogenetic relationship between the tentative species Pestivirus of giraffe and D32/00_‘HoBi’, but also between both BVDV species. Therefore, a discussion about generally accepted criteria that are used for the classification of pestiviruses into species, genotypes and subtypes could be helpful, and D32/00_‘HoBi’ represents an important jigsaw piece.

Although isolated from a batch of FCS from Brazil, it has to be taken into consideration that D32/00_‘HoBi’ might be introduced into susceptible animals in Europe, e.g. via biological products containing FCS. It has previously been demonstrated that numerous animal cell lines contain BVDV (Bolin et al., 1994). Moreover, a Bovine herpesvirus 1 modified live vaccine was contaminated with virulent BVDV-2 (Falcone et al., 1999). Assuring the absence of pestiviruses is therefore an important part of the quality control of biological products such as vaccines. For the detection of D32/00_‘HoBi’ in cell cultures, IF staining with NS3-specific pan-pestivirus mAbs (e.g. 103/105 and C16) can be used. In addition, RT-PCR could be used for testing sera, media or master stocks. However, due to the large sequence differences, established RT-PCR protocols might fail to detect D32/00_‘HoBi’ RNA. In this context, it must be mentioned that the well-known pan-pestivirus primer pair 324/326 (Vilcek et al., 1994), which can be used for the amplification of a fragment of approximately 290 bp in the 5'-UTR region, did not detect D32/00_‘HoBi’ (data not shown). As a consequence, diagnostic assays used for the determination of pestiviruses in biological products should be tested for their ability to detect D32/00_‘HoBi’.


   ACKNOWLEDGEMENTS
 
We thank Birgit Meinke and Doreen Reichelt for excellent technical assistance. We are grateful to Dr J. Dedek, LVL Rostock, for providing the contaminated SFT-R cells and to Biochrom, Berlin, Germany, for making FCS batch 547 available.


   REFERENCES
Top
ABSTRACT
MAIN TEXT
REFERENCES
 
Avalos-Ramirez, R., Orlich, M., Thiel, H.-J. & Becher, P. (2001). Evidence for the presence of two novel pestivirus species. Virology 286, 456–465.[CrossRef][Medline]

Becher, P., Orlich, M., Shannon, A. D., Horner, G., König, M. & Thiel, H.-J. (1997). Phylogenetic analysis of pestiviruses from domestic and wild ruminants. J Gen Virol 78, 1357–1366.[Abstract]

Becher, P., Orlich, M. & Thiel, H.-J. (1998). Complete genomic sequence of border disease virus, a pestivirus from sheep. J Virol 72, 5165–5173.[Abstract/Free Full Text]

Becher, P., Avalos Ramirez, R., Orlich, M., Cedillo Rosales, S., König, M., Schweizer, M., Stalder, H., Schirrmeier, H. & Thiel, H.-J. (2003). Genetic and antigenic characterization of novel pestivirus genotypes: implications for classification. Virology 311, 96–104.[CrossRef][Medline]

Beer, M. & Wolf, G. (1999). Selection of BVDV genotype II isolates using a monoclonal antibody and FACS analysis. Berl Munch Tierarztl Wochenschr 112, 345–350 (in German).[Medline]

Beer, M., Wolf, G. & Kaaden, O.-R. (2002). Phylogenetic analysis of the 5'-untranslated region of German BVDV type II isolates. J Vet Med B Infect Dis Vet Public Health 49, 43–47.[Medline]

Bolin, S. R., Ridpath, J. F., Black, J., Macy, M. & Roblin, R. (1994). Survey of cell lines in the American Type Culture Collection for bovine viral diarrhea virus. J Virol Methods 48, 211–221.[CrossRef][Medline]

Collett, M. S., Larson, R., Gold, C., Strick, D., Anderson, D. K. & Purchio, A. F. (1988). Molecular cloning and nucleotide sequence of the pestivirus bovine viral diarrhea virus. Virology 165, 191–199.[Medline]

Dahle, J., Liess, B. & Frey, H. R. (1987). Transmission of pestiviruses between animal species: experimental infection of swine with the virus of bovine virus diarrhea (BVD) and of cattle with the virus of European swine fever (ESP). Dtsch Tierarztl Wochenschr 94, 590–594 (in German).[Medline]

Depner, K. R., Bouma, A., Koenen, F., Klinkenberg, D., Lange, E., de Smit, H. & Vanderhallen, H. (2001). Classical swine fever (CSF) marker vaccine. Trial II. Challenge study in pregnant sows. Vet Microbiol 83, 107–120.[CrossRef][Medline]

Donis, R. O. (1995). Molecular biology of bovine viral diarrhea virus and its interactions with the host. Vet Clin North Am Food Anim Pract 11, 393–423.[Medline]

Doyle, L. G. & Heuschele, W. P. (1983). Bovine viral diarrhea virus infection in captive exotic ruminants. J Am Vet Med Assoc 183, 1257–1259.[Medline]

Edwards, S., Sands, J. J. & Harkness, J. W. (1988). The application of monoclonal antibody panels to characterize pestivirus isolates from ruminants in Great Britain. Arch Virol 102, 197–206.[Medline]

Edwards, S., Moennig, V. & Wensvoort, G. (1991). The development of an international reference panel of monoclonal antibodies for the differentiation of hog cholera virus from other pestiviruses. Vet Microbiol 29, 101–108.[CrossRef][Medline]

Falcone, E., Tollis, M. & Conti, G. (1999). Bovine viral diarrhea disease associated with a contaminated vaccine. Vaccine 18, 387–388.[CrossRef][Medline]

Hamblin, C. & Hedger, R. S. (1979). The prevalence of antibodies to bovine viral diarrhoea/mucosal disease virus in African wildlife. Comp Immunol Microbiol Infect Dis 2, 295–303.[CrossRef][Medline]

Harasawa, R., Giangaspero, M., Ibata, G. & Paton, D. J. (2000). Giraffe strain of pestivirus: its taxonomic status based on the 5'-untranslated region. Microbiol Immunol 44, 915–921.[Medline]

Meyers, G., Rümenapf, T. & Thiel, H.-J. (1989). Molecular cloning and nucleotide sequence of the genome of hog cholera virus. Virology 171, 555–567.[Medline]

Moennig, V., Bolin, S. R., Coulibaly, C. O. Z., Gourley, N. E., Liess, B., Mateo, A., Peters, W. & Greiser-Wilke, I. (1987). Investigation of the antigenic structure of pestiviruses by means of monoclonal antibodies. Dtsch Tierarztl Wochenschr 94, 572–576 (in German).[Medline]

Nettleton, P. F. (1990). Pestivirus infections in ruminants other than cattle. Rev Sci Tech 9, 131–150.[Medline]

Page, R. D. M. (1996). TREEVIEW: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12, 357–358.[Medline]

Paton, D. J., Sands, J. J. & Edwards, S. (1994). Border disease virus: delineation by monoclonal antibodies. Arch Virol 135, 241–252.[Medline]

Paton, D. J., Sands, J. J., Lowings, J. P., Smith, J. E., Ibata, G. & Edwards, S. (1995). A proposed division of the pestivirus genus using monoclonal antibodies, supported by cross-neutralisation assays and genetic sequencing. Vet Res 26, 92–109.[Medline]

Pellerin, C., Moir, S., Lecomte, J. & Tijssen, P. (1995). Comparison of the p125 coding region of bovine viral diarrhea viruses. Vet Microbiol 45, 45–57.[CrossRef][Medline]

Peters, W., Greiser-Wilke, I., Moennig, V. & Liess, B. (1986). Preliminary serological characterization of bovine viral diarrhoea virus strains using monoclonal antibodies. Vet Microbiol 12, 195–200.[CrossRef][Medline]

Pfeffer, M., Freyburg, M. V., Kaaden, O.-R. & Beer, M. (2000). A universal ‘one-tube’ RT-PCR protocol for amplifying isolates of bovine viral diarrhoea virus. Vet Res Commun 24, 491–503.[CrossRef][Medline]

Plowright, W. (1969). Other virus diseases in relation to the JP 15 programme. In Joint Campaign Against Rinderpest. First Technical Review Meeting, Phase IV, Mogadiscio, pp. 19–23. Mogadiscio, Kenya: Organization of African Unity.

Ridpath, J. F. & Bolin, S. R. (1995). The genomic sequence of a virulent bovine viral diarrhea virus (BVDV) from the type 2 genotype: detection of a large genomic insertion in a noncytopathic BVDV. Virology 212, 39–46.[CrossRef][Medline]

Ridpath, J. F., Bolin, S. R. & Dubovi, E. J. (1994). Segregation of bovine viral diarrhea virus into genotypes. Virology 205, 66–74.[CrossRef][Medline]

Schaarschmidt, U., Schirrmeier, H., Strebelow, G. & Wolf, G. (2000). Detection of border disease virus in a sheep flock in Saxony. Berl Munch Tierarztl Wochenschr 113, 284–288 (in German).[Medline]

Strimmer, K. & von Haeseler, A. (1996). Quartet puzzling: a quartet maximum-likelihood method for reconstructing tree topologies. Mol Biol Evol 13, 964–969.[Free Full Text]

Thiel, H.-J., Plagemann, P. G. W. & Moennig, V. (1996). Pestiviruses. In Fields Virology, 3rd edn, vol. 1, pp. 1059–1073. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia, PA: Lippincott-Raven.

van Rijn, P. A., van Gennip, H. G. P., Leendertse, C. H., Bruschke, C. J. M., Paton, D. J., Moormann, R. J. M. & van Oirschot, J. T. (1997). Subdivision of the Pestivirus genus based on envelope glycoprotein E2. Virology 237, 337–348.[CrossRef][Medline]

van Regenmortel, M. H. V., Fauquet, C. M., Bishop, D. H. L. & 8 other editors (2000). Virus Taxonomy: Seventh Report of the International Committee on Taxonomy of Viruses. San Diego, CA: Academic Press.

Vilcek, S., Herring, A. J., Herring, J. A., Nettleton, P. F., Lowings, J. P. & Paton, D. J. (1994). Pestiviruses isolated from pigs, cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis. Arch Virol 136, 309–323.[Medline]

Vilcek, S., Paton, D. J., Durkovic, B. & 8 other authors (2001). Bovine viral diarrhoea virus genotype 1 can be separated into at least eleven genetic groups. Arch Virol 146, 99–115.[CrossRef][Medline]

Weiland, E., Stark, R., Haas, B., Rümenapf, T., Meyers, G. & Thiel, H.-J. (1990). Pestivirus glycoprotein which induces neutralizing antibodies forms part of a disulfide-linked heterodimer. J Virol 64, 3563–3569.[Medline]

Wolfmeyer, A., Wolf, G., Beer, M., Strube, W., Hehnen, H.-R., Schmeer, N. & Kaaden, O.-R. (1997). Genomic (5'UTR) and serological differences among German BVDV field isolates. Arch Virol 142, 2049–2057.[CrossRef][Medline]

Received 28 April 2004; accepted 25 August 2004.



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