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Virus Research 52 (1997) 205 220

Genetic heterogeneity of bovine viral diarrhoea viruses isolated in Southern Africa1


C. Baule 2,a, M. van Vuuren b, J.P. Lowings c, S. Belak d,*
a

Swedish Uni6ersity of Agricultural Sciences, Veterinary Faculty, Department of Veterinary Microbiology, Section of Virology, Biomedical Center, Box 585, S-751 23 Uppsala, Sweden b Uni6ersity of Pretoria, Faculty of Veterinary Science, Department of Tropical Diseases, Pri6ate Bag X04, Onderstepoort 0110, South Africa c Central Veterinary Laboratory (Weybridge), New Haw, Addlestone, Surrey KT15 3NB, UK d The National Veterinary Institute, Department of Virology, Box 585 Biomedical Center, S-751 23 Uppsala, Sweden Received 21 June 1997; received in revised form 23 September 1997; accepted 23 September 1997

Abstract Seventy three eld isolates of bovine viral diarrhoea virus (BVDV), obtained from cattle in Mozambique and South Africa, were characterised by comparative nucleotide sequence analysis of part of the 5% non-coding region (5%NCR) of the viral genome. The target region was amplied by reverse transcription-polymerase chain reaction (RT-PCR). The amplicons were cloned in pUC 19 plasmid and both strands were sequenced by T7 polymerase dideoxynucleotide chain-termination sequencing or directly by cycle sequencing. The 245 base pair (bp) nucleotide sequences, derived from the 5%NCR, were aligned and compared to the corresponding positions of published sequences of BVDV type I and II strains and of pestiviruses of ovine and porcine origin. The phylogenetic trees, generated from those comparisons, allowed the Southern African isolates to be assigned to two main groups within the BVDV I genotype. Group A could be subdivided into three clusters, two of which grouped with BVDV strains of European and American origin. The third cluster did not group with any known BVDV I strains and it was conrmed in a comparison from the NS3 coding region. Group B contained more divergent isolates which differed by 18 23%, from BVDV I reference strains NADL, Osloss and SD-1 and comprised another distinct subset within the BVDV I genotype. This grouping was consistent in a comparison involving the NS2 NS3 region. It was concluded that BVD viruses occurring in Southern Africa are genetically diverse, comprising different variants within the BVDV I genotype. They include viruses similar to BVDVs found in Europe and America and others apparently rare or absent in those continents, termed here as BVDV Ic and Id. The co-existence of BVDV strains of European and American origin in certain areas both in Mozambique and South Africa, indicates a probable introduction of those viruses
* Corresponding author. Tel.: +46 18 674135; fax: + 46 18 4714520; e-mail sandor.belak@bmc.uu.se 1 The GenBank accession numbers of the sequences from the 5%NCR reported in this paper are U97409 U97481. 2 Present address: Veterinary Research Institute, P.O. Box 1922, Maputo, Mozambique. 0168-1702/97/$17.00 1997 Published by Elsevier Science B.V. All rights reserved. PII S 0 1 6 8 - 1 7 0 2 ( 9 7 ) 0 0 1 1 9 - 6

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through imports of cattle or through potentially infectious bovine products. In addition, the detection of isolates apparently rare or absent from Europe and America may indicate the presence of African variants of BVDV I (Pestivirus 1). 1997 Published by Elsevier Science B.V. Keywords: Pestivirus; BVDV; BVDV 5%NCR; Reverse-transcription polymerase chain reaction; Sequencing; Phylogeny

1. Introduction Bovine virus diarrhoea virus (BVDV) is a pathogen of cattle distributed world-wide and the causative agent of pre- and post-natal infections accounting for a variety of economically important syndromes (Perdrizet et al., 1987). BVDV belongs to the Pesti6irus genus, which also includes classical swine fever virus (CSFV) and border disease virus (BDV), within the Fla6i6iridae family (Horzinek, 1991; Collett, 1992; Wengler et al., 1995). The genome of pestiviruses consists of a single stranded, positive sense RNA molecule, approximately 12.5 Kb long, comprising one large open reading frame (ORF) which encodes about 4000 amino acids. The 5% non-coding region (5%NCR) of the genome is considered to be highly conserved among pestiviruses, allowing the selection of specic primers that amplify all known pestiviruses. It has, therefore, been the target region when studying differences between and within pestivirus species (Boye et al., 1991; De Moerlooze et al., 1993; Qi et al., 1993; Ridpath et al., 1993; Hofmann et al., 1994). Recent investigations have shown that the 5%NCR of pestiviruses is composed of highly conserved regions intercalated by three variable regions, termed I, II and III (Deng and Brock, 1993). These are located in positions corresponding to nucleotides 1 73 (I), 209 223 (II) and 284323 (III) in the genome of BVDV reference strain NADL. Nucleotide substitutions accounting for differences between strains are located within these variable regions, and are to a great extent, of the covariant type compensating to preserve RNA secondary structure (Deng and Brock, 1993). Two genotypes of BVDV have been discriminated on basis of the 5%NCR analysis. Genotype I

(BVDV I) is represented by the reference strains NADL and Osloss and involves the majority of BVDV strains isolated so far. Genotype II (BVDV II) has strain 890 as reference and comprises mainly isolates associated with haemorrhagic syndrome of cattle, a form of BVDV infection recently described in North America (Ridpath et al., 1994; Pellerin et al., 1994). BVDV II comprises also isolates of ovine origin (Paton et al., 1994; Ridpath et al., 1994; Becher et al., 1995; Vilcek et al., 1997). Considering the natural transmission of pestiviruses between various host animal species, as well as the recent results of monoclonal antibody typing and of comparative genome analysis, a new classication of the members of the Pesti6irus genus has been suggested (Becher et al., 1995; Paton, 1995; Vilcek et al., 1997). The new grouping is based on grounds of antigenic and genomic relationship rather than the species of origin. The proposed classication, which is still under discussion, divides the genus into four types or genotypes: genotype 1 would include the present BVDV I strains, mainly of cattle origin; genotype 2 would involve isolates of CSFV; genotype 3 would include sheep and pig isolates with characteristics of true BDV viruses; and genotype 4 would encompass isolates of cattle and sheep currently grouped as BVDV II (Becher et al., 1995; Vilcek et al., 1997). The occurrence of heterogeneous strains among BVDV I has been revealed by nucleotide sequencing and nucleic acid hybridisation (Kwang et al., 1991; Lewis et al., 1991; Ridpath and Bolin, 1991a,b) supporting evidence previously shown by polyclonal and monoclonal antibody analysis (Howard et al., 1987; Bolin et al., 1988, 1991). The practical signicance of the heterogeneity among BVDV strains is still under assessment. However, it is considered to have implications in the design of broad reactive diagnostic assays

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based on serological and molecular methods (Kwang et al., 1991; Lewis et al., 1991; Ward and Misra, 1991) as well as in the development of vaccines conferring protection against a wide range of strains (Bolin et al., 1991; Ridpath et al., 1994). The development of effective strategies to control BVDV infections also rely on the knowledge of the type of strains present and the epidemiological proles of the infections they cause. In Southern Africa, BVDV has been detected since the early seventies (Thomson and Blackburn, 1972; Theodoridis and Boshoff, 1974) and is found in association with diarrhoea, mucosal disease, foetal and respiratory disease. A number of serological surveys have indicated that infections with BVDV are widespread in cattle, sheep, goats and wild ruminants (Theodoridis et al., 1973; Depner et al., 1991; Van Vuuren, 1991; Baule and Banze, 1994; Muvavarirwa et al., 1995). Considering the implications of the genomic diversity in the diagnosis, epidemiology and control of BVDV infections it deemed important to characterise the BVD viruses occurring in the region.

2.2. RNA extraction and cDNA synthesis


BVDV RNA was extracted from cell culture lysates by the guanidinium-thiocyanate phenol/ chloroform method described by Chomiczynski and Sacchi (1987), with minor modications. Briey, 150 vl of cell culture lysates were vigorously mixed with 450 vl of 6 M GuScn. Five-hundred microlitres of these specimens were extracted twice with equal volume of a 1:1 v/v mixture of acidic phenol:chloroform and once with chloroform. The aqueous phase was precipitated in two volumes of 95% ethanol with 0.1 volume of 3 M sodium acetate at 20C. RNA was pelleted by centrifugation for 30 min at 10 000 g and the pellets were resuspended in 20 vl diethyl pyrocarbonate (DEPC) treated water. Synthesis of cDNA was performed in 25 vl nal volume using random hexamers and Moloney Murine Leukaemia Virus Reverse Transcriptase (M-MLV RT) (Gibco, BRL, Bethesda, MD). The cDNA was either used immediately for the PCR or kept at 70C until use.

2.3. Polymerase chain reaction (PCR)


2. Materials and methods The primers were selected from highly conserved stretches within the 5%NCR of the BVDV genome, based on published sequences of reference strains NADL (Collett et al., 1988), SD-1 (Deng and Brock, 1992) and Osloss (De Moerlooze et al., 1993). The sequences were as follows: Primer 5A (forward) 5%-GCAGAATTCCTAGCCATGCCCTTAGTAGGACTAG-3% (position 102126 of NADL, incorporating an EcoRI cloning site) and primer 5B (reverse) 5%-GCAAAGCTTATCAACTCCATGTGCCATGTACAGC3% (position 396372 of NADL, incorporating a HindIII cloning site). The PCR was performed in 50 vl nal volume, in a reaction mix containing 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 0.1% BSA, 0.2 mM of each dNTP (Pharmacia), 15 pmole of each primer, 2.5 mM MgCl2, 1 U of Taq DNA polymerase (Perkin-Elmer Cetus, Norwalk, CA) and 5 vl of cDNA, overlaid with two droplets of mineral oil. The cycling prole was run as follows: ve cycles with denaturation at 94C for 45 s, annealing at 55C for 45 s and

2.1. Virus strains


At the Veterinary Research Institute in Maputo, Mozambique, 59 BVD viruses were isolated during the period 1990 1996. The isolates were obtained from organ suspensions, nasal swabs, lymphocytes or sera of calves and adult cattle with either clinical symptoms of BVDV infection or persistently infected with BVDV. The viruses were propagated on secondary bovine turbinate cells grown in Eagles Minimum Essential Medium supplemented with 10% foetal calf serum. Both the cells and the serum were free from adventitious contamination with BVDV. Fourteen BVDV isolates from South Africa were obtained from the Department of Tropical Veterinary Diseases, Faculty of Veterinary Science, University of Pretoria. The identication and origin of the isolates and the predominant clinical syndrome present in cattle from which the specimen were collected are listed in Table 1.

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Table 1 List of BVDV isolates included in the present study in relation to the predominant clinical syndrome, biotype and location of origin Enteric syndromea 31 isolatesb M388A/90 M557A/90 +M1194A/90 +M1515A/90 +M105A/91 M245A/91 +M278A/91 +M279A/91 +M390A/91 M398A/92 +M549A/92 M583A/92 M589A/92 +M839A/92 +M840A/92 +M841A/92 M567A/93 +M723A/93 +M725A/93 M079B/91 +M139B/91 +M140B/91 M181B/91 M065B/93 M099B/93 M169B/93 +1114J/93 S-ALT1/K +S-ALT5/K S-ALT10/K +S-BFL/W93 Respiratory syndromea 27 isolatesb M590A/93 M867A/93 M085B/93 M427C/92 M432C/92 +MV39CB/95 +MV69CB/95 +MV98CB/95 M-116-28I/95 M116-53I/95 +M1117-38CK7/95 +M1117-49CK/95 +M1118-8CK/95 +M1118-27CK/95 M1118-32CK/95 M36CK/96 +MSN50CK/95 +M65CK/96 +M12-73GX/96 +M40-14GX/96 M217GX/95 M17IN/95 M233IN/93 +M1096-5IN/95 +M1096-16IN/95 +S-063W/95 +S-IFK2/W95 Othersa 15 isolatesb M265A/91 M597A/93 M667A/93 M199CH/94 M657GX/95 +M12-43GX/96 M346T/96 S-ALT2/K S-ALT3/K S-ALT4/K +S-ALT6/W S-ALT7/K +S-ALT8/K S-ALT9/K +S-ALT11/K

+ Cytopathogenic; Non-cytopatogenic a The separation is based on the predominance type of clinical symptoms showed by naturally infected animals. Enteric syndrome includes different forms of acute and chronic diarrhoea and mucosal disease; respiratory syndrome includes nasal discharge, respiratory distress, abnormal percurssion sounds, sneezing, coughing; other symptoms includes poor development, abortions/reproductive failure. b Isolate identication: MMozambique, SSouth Africa; initials after case number or name indicate location of origin: A, B, C and T farms from the Umbeluzi Dairy Basin in the southern part of Maputo Province; CB and CH farms in northern part of Maputo Province; I dairy farm in south Gaza Province; IN large scale farming project in Inhassune, Inhambane Province; Jdairy farm in Beira, Sofala Province; KKwazulu Natal; W Western Cape.

extension at 72C for 1 min, followed by 35 cycles with denaturation at 94C for 45 s, annealing at 50C for 45 s and extension at 72C for 1 min. A nal extension step at 72C for 7 min was included. Precautions to avoid contaminations were followed throughout the RT-PCR, as described by Belak and Ballagi-Pordany (1993). PCR prod ucts were visualised by ethidium bromide staining, after electrophoresis on 2% agarose gel.

HindIII and ligated into similarly cut pUC19 plasmid using T4 DNA ligase. Competent E. coli cells were transformed, screened and multiplied according to standard protocols (Sambrock et al., 1989). Plasmid DNA were isolated from multiplied bacteria using the Wizard mini-prep system (Promega, Madison, WI), according to the manufacturers instructions.

2.5. Sequencing strategy and methods 2.4. Cloning


The amplicons were puried from low melting agarose using the Qiagen DNA purication Kit, according to the manufacturers instructions. Puried products were digested with EcoRI and Cloned DNA was sequenced by dideoxynucleotide chain-termination, using a T7 polymerasebased DNA sequencing kit (Sequenase, version 2.0, USB), following the manufacturers instructions. The Universal and Reverse M13 primers

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Fig. 1.

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were used as sequencing primers. Sequencing reactions were separated in 6% acrylamide gels incorporating 8 M urea. At least two clones were sequenced both strands for each amplicon analysed. Independent PCR reactions and clones were run to clarify ambiguous readings. For direct sequencing, PCR products were generated with primers 13C (forward) 5%-AGCCATGCCCTTAGTAGGACT-3% (position 104 124 of NADL) and BV3 (reverse) 5%-TCAACTCCATGTGCCATGTACA-3% (position 395 374 of NADL). The amplicons were puried by using microconcentrators (Amicon, Beverly, MA) and the DNA concentration adjusted to 30 ng/vl. The same primers as for the PCR were used in the automated sequencing of both strands with the ABI200 system (Model 377).

I sequences from De Moerlooze et al. (1993), Qi et al. (1993), Hofmann et al. (1994), Pellerin et al. (1994), Ridpath et al. (1994), Harasawa and Tomiyama (1994), Paton et al. (1995), and Harasawa (1995) in Fig. 2. For comparisons in the NS3 (Fig. 4A) and NS2NS3 (Fig. 4B) regions, sequences from the GenBank with the following accession numbers were used: L35850, L35851, L35852, Z54333, A22708 and PTU96334, in addition to sequences of previously mentioned BVDV strains. The position of the African isolates in relation to other pestiviruses was analysed by phylogenetic comparisons. The groupings derived from the comparisons were evaluated in terms of relationships they bear within and between groups/clusters, origin of the isolates and epidemiological features.

2.6. Phylogenetic analysis


3. Results Nucleotide sequence comparisons and phylogenetic analysis were done with the DNASTAR software package (DNASTAR, Madison, WI) and with multiple programmes from the Clustal W package (Thompson et al., 1994). The reliability of the phylogenetic tree obtained for the 5%NCR region was evaluated by running a 1000 replicas in the bootstrap test and the consensus tree was plotted, using strain 890 of BVDV II as an outgroup. The nucleotide sequences derived for the 245 base amplicons from the 5%NCR of the 73 BVDV isolates were aligned and compared to the corresponding region of sequences of pestiviruses of bovine, porcine and ovine origin, published by other groups. These included BVDV I strains NADL, Osloss and SD-1; CSFV strains Alfort (Meyers et al., 1989) and Brescia (Moormann et al., 1990); BDV strains Moredun cp and ncp and BVDV II strain 890 in Fig. 1, and BVDV

3.1. Genetic comparison of the 73 isolates and relationship between the groups and clusters
The phylogenetic tree of Fig. 1 shows the groups and clusters that were derived when sequences from the 5%NCR obtained in this study and those published by others were compared. The 73 isolates analysed could be assigned to two main groups, termed here as Group A and Group B, differing in a maximum of 23% of sequence divergence. The bootstrap value for the branch separating the two groups was of 95.8%. Group A was subdivided into three clusters, which will be designated here following and expanding the proposed nomenclature for BVDV I (Pellerin et al., 1994) as Ia, Ib and Ic. Cluster Ia grouped with different strains of American origin, such as Oregon C24V, Singer, NADL and SD-1

Fig. 1. Phylogenetic tree showing the positioning of 73 eld BVDV isolates from Mozambique and South Africa in relation to published sequences of pestiviruses. The tree was generated from comparative alignment of sequences from part (245 bp) of the 5%NCR of the BVDV genome, made with multiple programs of the Clustal W package. The numbers on each branch represent the number of times the group or subgroup was picked in 1000 reruns in the bootstrap analysis. Bolded names are representatives of Pestiviruses type 1 (BVDV strains NADL, Osloss, SD1), type 2 (CSFV Brescia and Alfort/Tubingen), type 3 (BDV strain Moredun ncp and cp) and type 4 (BVDV strain 890). The Southern African isolates branched into two distinct groups, Groups A and B, within the BVDV I genotype. Group A was further subdivided into three clusters, Ia, Ib and Ic and Group B composed cluster Id. Clusters Ia and Ib integrated the NADL-related and the Osloss-related viruses, respectively. No known strains were found to group with cluster Ic. Only one published sequence (88753C) was found to be similar to that of isolate SN50CK/95 (*), in cluster Id.

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Fig. 2.

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(only the last two are shown in the phylogenetic tree of Fig. 1). The sequence homology within the cluster ranged from 85 to 100%. Cluster Ib integrated isolates sharing a sequence identity of 92 100%. In relation to BVDV I reference strains, the cluster showed highest sequence homology with BVDV Osloss, 9198%. Cluster Ic comprised isolates sharing a sequence homology of 94 100%. A search and comparison with sequences available in the database did not show sequences which could group with this cluster of isolates. Group B integrated the isolates where the sequence homology with BVDVs NADL, Osloss and SD-1 was of 77 82%. For consistency with nomenclature, it will be considered as cluster Id. It composed isolates sharing 85 100% of sequence homology, further subdividing into groupings which aggregated highly related isolates, as illustrated on the tree. A search and comparison with sequences available in the database showed the published sequence of one strain, 88753C (De Moerlooze et al., 1993) to be close to the sequence of isolate SN50CK/95. No other reported sequences were found to form clusters with the isolates from Group B, based on the comparison of the 245 bp nucleotide fragment. When compared to the positioning of representatives of pestiviruses types 1 4, the analysed African viruses were all BVDV I (pestivirus type 1) and were positioned distinctly from pestiviruses of porcine and ovine origin (true BDV) as well as from BVDV II (pestivirus type 4).

data to be used, all sequences had to be shortened to the 90 bp fragment (positions 255344 in BVDV I strain NADL) which was common to all isolates. Fig. 2 shows that: our cluster Ia and Ib sequences correspond, respectively, to subgroups Ia and Ib of Pellerins subgrouping, while no corresponding subgroup was found for our Ic cluster. Our Group B sequences appear to show closer relation to the Ib subgroup in the 90 bp nucleotide stretch included in this comparison. The previously mentioned similarity between sequences of strains 88753C and SN50CK/95 is shown in Fig. 2. A further observation was that the sequences from Qi et al. (1993) and those of Hofmann et al. (1994) fall outside our groups or those of Pellerin, forming separate clusters.

3.3. Analysis of cluster Ia to Id sequences and additional data on the 5 %NCR


In order to pinpoint the differences displayed at the nucleotide level, 26 sequences representative of isolates forming clusters Ia to Id were aligned with those of NADL/Osloss and the changes are shown in relation to the NADL sequence in Fig. 3. The nucleotide substitutions were located in the stretches corresponding to the variable regions II and III (Deng and Brock, 1993), shown as shaded bars in Fig. 3, but also at discrete positions outside these regions. No indication can be given about the sequences in variable region I which was not sequenced in this study. In variable region II, distinctive patterns of nucleotide substitutions were formed, distinguishing the Group A (IaIc) from the Group B (Id) sequences as well as the subdivisions of each group illustrated on the tree of Fig. 1. When cluster Id sequences were compared to NADL, some sequence insertions or deletions were noted within variable regions II and III, respectively (Fig. 3).

3.2. Analysis of di6ersity within the BVDV I genotype


The phylogenetic tree on Fig. 2 was constructed from ten sequences representing the groups and the clusters derived in the present study and published sequences from other groups. To allow the

Fig. 2. Phylogenetic tree from part (90 bp) of the 5%NCR of the BVDV genome, including ten sequences representing the groups and clusters derived in the present study (indicated by arrows) and those published by others, abbreviated as follows: p, Pellerin; ho, Hofmann; r, Ridpath; d, De Moerlooze; m, Meyers; mo, Moormann; q, Qi; h, Harasawa; a, Paton; b, Brock. The tree shows the correspondence of our clusters Ia and Ib to subgroups Ia and Ib of BVDV I, respectively. No corresponding subgroup in the present BVDV I nomenclature was found for our Ic cluster, same as for some of the Qis and Hofmanns strains, which form additional separate clusters. Our Id cluster appear to be a variant of the Ib subgroup in this 90 bp tree. The similarity between isolate SN50CK/95 and strain 88753 is shown on the tree.

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Fig. 3. Alignment of 245 bp nucleotide sequences representatives of cluster Ia Id isolates, compared to BVDV strain NADL (positions 127 371) in the rst and Osloss in the second lane. Dots represent nucleotides that are identical to NADL. Uppercase letters show nucleotide substitutions. Nucleotide deletions are marked with an asterisk (*). The gaps are introduced by the program to optimise the alignment. Shaded bars identify variable regions II and III and the full bar shows the 90 bp fragment used to construct the phylogenetic tree of Fig. 2. The nucleotide substitutions and deletions were mainly located in the stretches corresponding to variable regions II and III. The patterns of nucleotide substitutions additionally contributing to the groupings dened in Fig. 1 are seen upstream the 90bp fragment. In cluster Id some position substitutions occurred towards the 5% part of the sequenced fragment, which is almost fully conserved in the Ia Ic sequences.

To investigate the apparent contradictory grouping of some viruses when the 90 bp tree was compared to the 245 bp tree we examined the location of base substitutions along the 245 bp fragment (Fig. 3). We found what appeared to be a bias in informative base distribution towards the 5% part of the fragment which excluded the 90 bp fragment (shown as full bar in Fig. 3). For instance, in the rst 142 bases, for the viruses

forming the second Id division (233IN/95-10965IN/95) there are six unique nucleotide positions as opposed to one in the remainder of the fragment, compared to the Group A sequences. Variable region II seems particularly rich in informative bases whilst variable region III (which includes much of the 90 bp fragment) seemed to contain little information that supported the 245 bp tree.

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Fig. 3. (Continued)

In the phylogenetic tree on Fig. 4A, cluster Ic branched out separately from subgroup Ia and Ib strains. The nucleotide sequence homology of cluster Ic isolates, in this region of the NS3, was 84% and 82% with the Ia and Ib subgroups, respectively. In Fig. 4B, the segregation of group B viruses into a different cluster, Id, distinct from other BVDV I strains was consistent with Fig. 1.

3.4. Analysis of the genetic grouping in relation to clinical prole and origin of the isolates
Table 1 shows the list of isolates included in the present study, grouped according to the predominant type of clinical syndrome observed in the

affected animals from which the viruses were isolated. The biotype of the isolates and the location of origin are also shown. The relationship between clinical syndrome and phylogenetic grouping was investigated. It was found that viruses in Group A were associated with a range of clinical syndromes, i.e. enteric/mucosal disease (31 in 52), respiratory (9 in 52), abortions/reproductive failure and poor development (12 in 52) while the majority of isolates in Group B (18 in 21) were associated with respiratory infections. No biotype/phylogenetic grouping relationship could be established. The isolates from the groups and clusters dened included both non-cytopathogenic and cytopathogenic isolates.

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Fig. 4. Phylogenetic trees derived from the NS3 (Fig 4A) and the NS2 NS3 (Fig 4B) gene regions of the BVDV genome. Sequences representing the groups and clusters derived in the present study are shown in relation to published sequences of BVDV (in bold). Group and cluster denominations were used as in Fig. 1. In Fig. 4A, cluster Ic viruses grouped separately from strains of the Ia and the Ib subgroups, in Group A. In Fig. 4B, Group B viruses (cluster Id) segregated into a distinct subset within the BVDV I genotype, consistent with Fig. 1.

When analysing the groupings in relation to the geographical origin of the isolates, it could be seen that some of the dened groupings were widespread while others involved isolates from a common place of origin. The isolates clustering with the standard American and European strains

in clusters Ia and Ib, and also viruses from Ic were found in locations corresponding to the Dairy Basins in Maputo, Xai-Xai and Beira (South and Central Mozambique) and in the Kwazulu Natal and Western Cape provinces in South Africa. The rst subdivision of cluster Id

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composed isolates from the same location in XaiXai (Central Gaza Province), while the isolates in the remaining subdivisions originated from Inhambane Province and from farming areas in the Northern part of Maputo and Southern part of Gaza Provinces in Mozambique.

4. Discussion We have analysed the genetic diversity of BVDV strains from Southern Africa, on the basis of nucleotide sequencing of the 5%NCR of the genome in 73 isolates originating from Mozambique and South Africa. The isolates were discriminated into two groups within the BVDV I genotype (Pestivirus type 1), differing from each other by a maximum of 23% in the nucleotide sequence. The distinction between the two groups was supported at a condence level of 95.8% by the bootstrap analysis. In Group A, three clusters could be dened, as Ia, Ib and Ic. Cluster Ia grouped with different strains of American origin, such as NADL, SD-1, Oregon C24V, New York-1, Singer and C3, which placed them in subgroup Ia of BVDV I, as dened by Pellerin et al. (1994). The Ia intra-cluster variation, of 85 100%, is reected in the wide distribution of these isolates in the phylogenetic trees of Figs. 1 and 2, where highly homologous isolates were discriminated into further aggregates. Therefore, Ia may be too diverse to be considered a single cluster. Cluster Ib showed high similarity to BVDV reference strain Osloss and falled under subgroup Ib of BVDV I. The third cluster, Ic, although clearly still BVDV I, was not found to group with any characterised BVDV strains, as shown in Figs. 1 and 2 and Fig. 4A. It appears that cluster Ic represents strains that are rare or absent in America (North) and Europe. Clusters Ia and Ic included isolates found both in Mozambique and in South Africa and cluster Ib involved isolates from Mozambique. Group B of the Southern African viruses comprised isolates that were related to the known reference strains but branched separately, forming a distinct subset within the BVDV I genotype. There was a large difference between the Ia Ic

and the Id isolates in the region sequenced, which suggested the presence of group rather than subgroup relationships between the respective isolates. In the comparative alignments made with sequences available in the database, the only sequence which showed similarity to this group was that of strain 88753C, reported by De Moerlooze et al. (1993). In the comparison of Fig. 4B, involving the NS2NS3 region, the segregation of group B viruses (clusters Id) in relation to subgroup Ia and Ib strains was consistent with Fig. 1. The apparent relationship to BVDV reference strain Osloss, suggested by the 90 bp tree from the 5%NCR was not supported in this region of the NS2NS3. It appears that the Group B viruses make up a subset of divergent BVDV I strains different from the majority of strains analysed so far. That only one virus sequence (from Europe) was found with similar characteristics in part of the genome region compared in this study may indicate that viruses from this Group are rare or absent in Europe. All the Group B isolates were from Mozambique, but due to the relatively small sample numbers in this study could also be distributed more widely. The nucleotide substitutions, indicative of the described phylogenetic groupings occurred mainly in the variable region II of the 5%NCR but also at discrete positions upstream of this variable region. This applied particularly to cluster Id sequences. This may provide an explanation for the apparently contradictory positioning of the Group B viruses in the phylogenetic tree of Fig. 2, when the comparison was limited to the 90 bp stretch that covers variable region III. The fact that the variable region III seems to have few sequence patterns which correspond to the groupings of the viruses on the 245 bp tree could indicate that this region is extremely unstable (but possibly within constraints). If so, phylogenetic information could be lost rapidly both by mutation and back mutation. The fact that the present studies revealed the existence of two distinct groups within the BVDV I genotype, branching into different clusters indicates the presence of diverse BVDV strains in the region. Evidence from this, and other studies (Qi et al., 1993; Hofmann et al., 1994) where even

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greater diversity was observed, suggests that the BVDV I group variation may be more extensive than the type Ia and Ib subgroups previously proposed (Pellerin et al., 1994). If the tree derived from the 90 bp data is an accurate representation of variation, BVDV I may possess a more continuous spectrum of variation in contrast to what appears to be found in CSFV (Hofmann et al., 1994; Lowings et al., 1996; Vilcek et al., 1996). The biological and evolutionary signicance of this heterogeneity seen in BVDV I strains still remains to be clearly assessed. The high conserved nature of the 5%NCR facilitates the design of primers capable of amplifying all known Pestivirus strains. For this reason it has been chosen by several groups as a target region to discriminate pestivirus genomes. Findings by others, however, show that although providing useful data, analysis of this region alone might not give the highest resolution for phylogenetic analysis. Lowings et al. (1996) recognised that the analysis of the E2 gene region provides better resolution in discriminating CSFV strains that appeared identical on basis of the 5%NCR analysis. Vilcek et al. (1996) additionally considered the polymerase-coding gene (NS5B) of CSFV more appropriate for phylogenetic discrimination of strains which appeared closely related on basis of a different region of the E2 gene. Becher et al. (1997) suggested that the Npro region is more suitable for analysing genetic relationships within genotype, as opposed to the 5%NCR (the latter based on 130 bp fragment spanning the last two thirds of the 5%NCR). From our results, it appears, however, that by extending the analysis to a larger part of the 5%NCR the discrimination capacity of this region is comparable to other regions of the genome (i.e. the NS2 NS3). The partial comparisons presented in Fig. 4A and 4B show a consistent clustering pattern and a clear distinction of clusters Ic and Id from the Ia and the Ib subgroups as found in Fig. 1. This supports our analysis based on the 245 bp fragment of the 5%NCR and the suitability of this region for phylogenetic segregation. The high sequence similarity between isolates from Southern Africa and strains of European and American origin, i.e. Osloss, NADL and SD-

1 may reect the introduction and establishment of these virus variants in the local cattle population. This seems to be supported by the fact that variants bearing a high relation to those strains, were found in cattle raising areas with history of cattle importation from Europe or use of potentially infected products such as vaccines and semen. In farms A and B (Mozambique), for instance, a whole blood based vaccine was used for the immunisation of calves against Cowdria ruminantium; there is a possibility of a connection between this practise and the occurrence of the same virus variants in both farms. It is possible that a vaccine batch was contaminated. Additionally, regional movements of cattle and products may also explain the spread of similar virus variants in certain regions. Despite the scarcity of data on BVDV infection in wildlife, we consider the possibility of the involvement of a wildlife reservoir in the spread of BVD viruses. Studies of the relationship between the present groupings compared to clinical and epidemiological proles revealed an apparent link between the occurrence of certain virus variants and a predominant type of clinical syndrome. Group A variants were found associated with different clinical symptoms, consistent with descriptions of infections caused by BVDV viruses studied so far, i.e. diarrhoea, mucosal disease, respiratory infections, abortions/reproductive failure, persistent infections (Perdrizet et al., 1987). Group B virus variants, however, were isolated predominantly in cases where the respiratory form of infection was the most consistent clinical feature. Further studies comparing biological features of the viruses from both groups would be required to establish a denite connection between genetic variants of the virus and the induction of a particular clinical syndrome. Before any conclusions are made we would have to investigate the effect of host genetic proles or farm management practices upon the virulence and pathogenesis of these viruses. For example, the semi-intensive type of cattle rearing, used in dairy farms in Mozambique and in South Africa utilises a comparatively closed-in system more likely to favour the establishment of a cycle of enteric infections than is the extensive

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type of cattle rearing practised in the other locations where samples for this study originated from, i.e. Northern part of Maputo, Gaza and Inhambane Provinces.

5. Conclusion In conclusion, the present studies revealed that different genetic variants of BVDV I, including viruses similar to those found in Europe and America and others apparently rare or absent from those continents are present and involved in BVDV infections in Southern Africa. Although the analysis of the 5%NCR of the genome did not establish the existence of any African type of BVDV, the viruses from clusters Ic and Id were rather divergent from those of European and American strains, suggesting that they might have evolved separately. Further studies are required to establish the full extent of genetic variability of these viruses in Southern Africa. The presence of isolates belonging to different clusters coexisting in certain areas is consistent with the possibility of multiple virus introductions through the importation of cattle and/or the use of infected products in addition to regular cattle movements. The presence of similar viruses geographically separated could also be explained by the above factors in addition to the possible presence of BVDV I in a wildlife reservoir. The fact that different genetic variants within BVDV I were found in the present study of viruses from two countries in Southern Africa, suggests that an even greater variability might be expected in a survey involving more variable regions of the pestivirus genome and isolates from larger geographic areas of the African continent.

Quembo for sample collection and screening in Mozambique. A special appreciation to all the members of the Research and Development Section of the Department of Virology for assistance in different parts of this work and interesting discussions. This study was supported by a grant from the Swedish Agency for Research Cooperation with Developing Countries (SAREC) and by a reaseach grant of the National Veterinary Institute, Uppsala, Sweden.

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Acknowledgements We thank Prof Bror Morein for valuable discussions and for the critical reading of the manuscript. We also thank Adriana James (Allerton Laboratory, Department of Agriculture, Kwazulu Natal, South Africa) for providing several BVDV isolates; Jacinto Banze and Carlos

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