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 field 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 amplified 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 confirmed 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

1995. termed I.b) supporting evidence previously shown by polyclonal and monoclonal antibody analysis (Howard et al. Paton. Genotype II (BVDV II) has strain 890 as reference and comprises mainly isolates associated with haemorrhagic syndrome of cattle. Collett. been the target region when studying differences between and within pestivirus species (Boye et al. Nucleotide substitutions accounting for differences between strains are located within these variable regions. and are to a great extent..V. Qi et al. Considering the natural transmission of pestiviruses between various host animal species. a new classification of the members of the Pesti6irus genus has been suggested (Becher et al. 1991. positive sense RNA molecule. 1997). Two genotypes of BVDV have been discriminated on basis of the 5%NCR analysis.5 Kb long. it is considered to have implications in the design of broad reactive diagnostic assays . BVDV belongs to the Pesti6irus genus. Ridpath et al. Introduction Bovine virus diarrhoea virus (BVDV) is a pathogen of cattle distributed world-wide and the causative agent of pre. 1994... Sequencing. 1991a. The proposed classification. © 1997 Published by Elsevier Science B. 1991. The 5% non-coding region (5%NCR) of the genome is considered to be highly conserved among pestiviruses. Ridpath et al. Vilcek et al. 1995)... therefore. Lewis et al. the detection of isolates apparently rare or absent from Europe and America may indicate the presence of African variants of BVDV I (Pestivirus 1). 1988.. within the Fla6i6iridae family (Horzinek. as well as the recent results of monoclonal antibody typing and of comparative genome analysis. These are located in positions corresponding to nucleotides 1 – 73 (I).. Wengler et al. 1994). 1994). Recent investigations have shown that the 5%NCR of pestiviruses is composed of highly conserved regions intercalated by three variable regions. Vilcek et al. a form of BVDV infection recently described in North America (Ridpath et al. 1997)... approximately 12. divides the genus into four types or genotypes: genotype 1 would include the present BVDV I strains.. Keywords: Pestivirus.. Becher et al... De Moerlooze et al.. Hofmann et al. BVDV. Pellerin et al. Bolin et al. II and III (Deng and Brock... 1987). and genotype 4 would encompass isolates of cattle and sheep currently grouped as BVDV II (Becher et al.and post-natal infections accounting for a variety of economically important syndromes (Perdrizet et al. Reverse-transcription polymerase chain reaction. 1993. The practical significance of the heterogeneity among BVDV strains is still under assessment. BVDV 5%NCR. Phylogeny 1.206 C. mainly of cattle origin. 1991). Genotype I (BVDV I) is represented by the reference strains NADL and Osloss and involves the majority of BVDV strains isolated so far. 1991. 1993). 1993). genotype 3 would include sheep and pig isolates with characteristics of ‘true BDV’ viruses. 1995. 1994. 1994. In addition. However. The new grouping is based on grounds of antigenic and genomic relationship rather than the species of origin... 1987. genotype 2 would involve isolates of CSFV. Vilcek et al.. / Virus Research 52 (1997) 205–220 through imports of cattle or through potentially infectious bovine products. of the covariant type compensating to preserve RNA secondary structure (Deng and Brock. Ridpath and Bolin. comprising one large open reading frame (ORF) which encodes about 4000 amino acids. 1995. which also includes classical swine fever virus (CSFV) and border disease virus (BDV). 209 – 223 (II) and 284–323 (III) in the genome of BVDV reference strain NADL. 1995.. Baule et al. 1993. 1997). 1992. The genome of pestiviruses consists of a single stranded.. 1991. which is still under discussion. 1993. It has. The occurrence of heterogeneous strains among BVDV I has been revealed by nucleotide sequencing and nucleic acid hybridisation (Kwang et al. allowing the selection of specific primers that amplify all known pestiviruses. BVDV II comprises also isolates of ovine origin (Paton et al.

Baule et al. Baule and Banze. The isolates were obtained from organ suspensions. ..1. A number of serological surveys have indicated that infections with BVDV are widespread in cattle. 1992) and Osloss (De Moerlooze et al. 50 mM KCl.. Synthesis of cDNA was performed in 25 vl final volume using random hexamers and Moloney Murine Leukaemia Virus Reverse Transcriptase (M-MLV RT) (Gibco. University of Pretoria. Polymerase chain reaction (PCR) 2.5 mM MgCl2. 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. Mozambique. 1995). mucosal disease. epidemiology and control of BVDV infections it deemed important to characterise the BVD viruses occurring in the region. Depner et al.3. Muvavarirwa et al. The identification and origin of the isolates and the predominant clinical syndrome present in cattle from which the specimen were collected are listed in Table 1. Lewis et al. The viruses were propagated on secondary bovine turbinate cells grown in Eagle’s Minimum Essential Medium supplemented with 10% foetal calf serum. 150 vl of cell culture lysates were vigorously mixed with 450 vl of 6 M GuScn. 0.0). annealing at 55°C for 45 s and 2.. 1991.1 volume of 3 M sodium acetate at −20°C. SD-1 (Deng and Brock.. 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).. 15 pmole of each primer. 1994). Virus strains At the Veterinary Research Institute in Maputo. / Virus Research 52 (1997) 205–220 207 based on serological and molecular methods (Kwang et al.C. 59 BVD viruses were isolated during the period 1990 – 1996. 2. MD). Fourteen BVDV isolates from South Africa were obtained from the Department of Tropical Veterinary Diseases.2. nasal swabs. lymphocytes or sera of calves and adult cattle with either clinical symptoms of BVDV infection or persistently infected with BVDV. The PCR was performed in 50 vl final volume. Bethesda.. Ridpath et al. Van Vuuren. 1991. overlaid with two droplets of mineral oil. 1991. The sequences were as follows: Primer 5A (forward) 5%-GCAGAATTCCTAGCCATGCCCTTAGTAGGACTAG-3% (position 102–126 of NADL. foetal and respiratory disease.. 1988).1% BSA. incorporating a HindIII cloning site). incorporating an EcoRI cloning site) and primer 5B (reverse) 5%-GCAAAGCTTATCAACTCCATGTGCCATGTACAGC3% (position 396–372 of NADL. Briefly. 1972. 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. 0. 2. 1994. BVDV has been detected since the early seventies (Thomson and Blackburn.. 1974) and is found in association with diarrhoea. 1991. sheep. 2. BRL. Faculty of Veterinary Science. Considering the implications of the genomic diversity in the diagnosis.2 mM of each dNTP (Pharmacia). goats and wild ruminants (Theodoridis et al. The development of effective strategies to control BVDV infections also rely on the knowledge of the type of strains present and the epidemiological profiles of the infections they cause. 1 U of Taq DNA polymerase (Perkin-Elmer Cetus. CA) and 5 vl of cDNA. 1991. Ward and Misra.. Norwalk. The cDNA was either used immediately for the PCR or kept at −70°C until use. with minor modifications. The aqueous phase was precipitated in two volumes of 95% ethanol with 0. in a reaction mix containing 10 mM Tris-HCl (pH 9. 1993). Both the cells and the serum were free from adventitious contamination with BVDV. Theodoridis and Boshoff. Materials and methods The primers were selected from highly conserved stretches within the 5%NCR of the BVDV genome. In Southern Africa. 1991) as well as in the development of vaccines conferring protection against a wide range of strains (Bolin et al. The cycling profile was run as follows: five cycles with denaturation at 94°C for 45 s. 1973. based on published sequences of reference strains NADL (Collett et al.

0. initials after case number or name indicate location of origin: A. Sofala Province. respiratory syndrome includes nasal discharge. 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. coli cells were transformed. according to the manufacturer’s instructions.4. respiratory distress. USB). WI). abortions/reproductive failure. C and T— farms from the Umbeluzi Dairy Basin in the southern part of Maputo Province. Precautions to avoid contaminations were followed throughout the RT-PCR. Enteric syndrome includes different forms of acute and chronic diarrhoea and mucosal disease. Plasmid DNA were isolated from multiplied bacteria using the Wizard mini-prep system (Promega. other symptoms includes poor development. screened and multiplied according to standard protocols (Sambrock et al. PCR prod´ ´ ucts were visualised by ethidium bromide staining. J—dairy farm in Beira. Madison.208 C. using a T7 polymerasebased DNA sequencing kit (Sequenase. sneezing. as described by Belak and Ballagi-Pordany (1993). abnormal percurssion sounds. Cloning The amplicons were purified from low melting agarose using the Qiagen DNA purification Kit. − Non-cytopatogenic a The separation is based on the predominance type of clinical symptoms showed by naturally infected animals. extension at 72°C for 1 min. IN — large scale farming project in Inhassune. followed by 35 cycles with denaturation at 94°C for 45 s. The Universal and Reverse M13 primers . annealing at 50°C for 45 s and extension at 72°C for 1 min. CB and CH — farms in northern part of Maputo Province. Inhambane Province. Baule et al. K—Kwazulu Natal. W — Western Cape. Sequencing strategy and methods 2. version 2. 1989). HindIII and ligated into similarly cut pUC19 plasmid using T4 DNA ligase. I — dairy farm in south Gaza Province. / Virus Research 52 (1997) 205–220 Table 1 List of BVDV isolates included in the present study in relation to the predominant clinical syndrome. following the manufacturer’s instructions. Purified products were digested with EcoRI and Cloned DNA was sequenced by dideoxynucleotide chain-termination. coughing. B. S—South Africa.. 2. Competent E. A final extension step at 72°C for 7 min was included. according to the manufacturer’s instructions. after electrophoresis on 2% agarose gel.5. b Isolate identification: M—Mozambique.

/ Virus Research 52 (1997) 205–220 209 Fig. 1. Baule et al. .C.

Phylogenetic analysis 3. WI) and with multiple programmes from the Clustal W package (Thompson et al. Phylogenetic tree showing the positioning of 73 field BVDV isolates from Mozambique and South Africa in relation to published sequences of pestiviruses. Baule et al. such as Oregon C24V. Osloss and SD-1. BDV strains Moredun cp and ncp and BVDV II strain 890 in Fig. 2. The groupings derived from the comparisons were evaluated in terms of relationships they bear within and between groups/clusters. MA) and the DNA concentration adjusted to 30 ng/vl.8%.6. (1994). (1994). Z54333. using strain 890 of BVDV II as an outgroup. 4B) regions. For direct sequencing. The tree was generated from comparative alignment of sequences from part (245 bp) of the 5%NCR of the BVDV genome. The bootstrap value for the branch separating the two groups was of 95. Sequencing reactions were separated in 6% acrylamide gels incorporating 8 M urea. L35852. The 73 isolates analysed could be assigned to two main groups. NADL and SD-1 Fig. 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. (1993). No known strains were found to group with cluster Ic. made with multiple programs of the Clustal W package. Results Nucleotide sequence comparisons and phylogenetic analysis were done with the DNASTAR software package (DNASTAR. A22708 and PTU96334. The amplicons were purified by using microconcentrators (Amicon. and BVDV 3. Ridpath et al. 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). CSFV strains Alfort (Meyers et al. Hofmann et al. 2. SD1). in cluster Id. and Harasawa (1995) in Fig. For comparisons in the NS3 (Fig. termed here as Group A and Group B... These included BVDV I strains NADL. At least two clones were sequenced both strands for each amplicon analysed. Only one published sequence (88753C) was found to be similar to that of isolate SN50CK/95 (*). published by other groups. The Southern African isolates branched into two distinct groups. Clusters Ia and Ib integrated the NADL-related and the Osloss-related viruses. (1993). 1994) as Ia.210 C. Singer. L35851. Genetic comparison of the 73 isolates and relationship between the groups and clusters The phylogenetic tree of Fig. The same primers as for the PCR were used in the automated sequencing of both strands with the ABI200 system (Model 377). respectively.. Osloss. 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. I sequences from De Moerlooze et al. Ib and Ic. origin of the isolates and epidemiological features. type 3 (BDV strain Moredun ncp and cp) and type 4 (BVDV strain 890). . Cluster Ia grouped with different strains of American origin. Paton et al. Harasawa and Tomiyama (1994). porcine and ovine origin. sequences from the GenBank with the following accession numbers were used: L35850. The position of the African isolates in relation to other pestiviruses was analysed by phylogenetic comparisons. Group A was further subdivided into three clusters. Qi et al. 1989) and Brescia (Moormann et al.. 1. 1. type 2 (CSFV Brescia and Alfort/Tubingen). Ia. differing in a maximum of 23% of sequence divergence. / Virus Research 52 (1997) 205–220 were used as sequencing primers. Independent PCR reactions and clones were run to clarify ambiguous readings. Ib and Ic and Group B composed cluster Id. Bolded names are representatives of Pestiviruses type 1 (BVDV strains NADL. Beverly. (1995). Group A was subdivided into three clusters. within the BVDV I genotype. (1994). 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. 1990). The numbers on each branch represent the number of times the group or subgroup was picked in 1000 reruns in the bootstrap analysis. Pellerin et al. Groups A and B.1. 4A) and NS2–NS3 (Fig. in addition to sequences of previously mentioned BVDV strains. which will be designated here following and expanding the proposed nomenclature for BVDV I (Pellerin et al. Madison.

2. . Baule et al. / Virus Research 52 (1997) 205–220 211 Fig.C.

Harasawa. Pellerin. 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. 1). De Moerlooze. The nucleotide substitutions were located in the stretches corresponding to the variable regions II and III (Deng and Brock. 1993). but also at discrete positions outside these regions. / Virus Research 52 (1997) 205–220 (only the last two are shown in the phylogenetic tree of Fig. Meyers. The sequence homology within the cluster ranged from 85 to 100%. 3. distinctive patterns of nucleotide substitutions were formed. A further observation was that the sequences from Qi et al. respectively. d.212 C. distinguishing the Group A (Ia–Ic) from the Group B (Id) sequences as well as the subdivisions of each group illustrated on the tree of Fig. to subgroups Ia and Ib of Pellerin’s subgrouping. ho. q. For consistency with nomenclature. including ten sequences representing the groups and clusters derived in the present study (indicated by arrows) and those published by others. (1994) fall outside our groups or those of Pellerin. it will be considered as cluster Id. Baule et al. The tree shows the correspondence of our clusters Ia and Ib to subgroups Ia and Ib of BVDV I. To allow the Fig. No other reported sequences were found to form clusters with the isolates from Group B. 88753C (De Moerlooze et al. shown as shaded bars in Fig. 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. which form additional separate clusters. 3.. When cluster Id sequences were compared to NADL. Analysis of di6ersity within the BVDV I genotype The phylogenetic tree on Fig. data to be used. In relation to BVDV I reference strains. 2 shows that: our cluster Ia and Ib sequences correspond. all sequences had to be shortened to the 90 bp fragment (positions 255–344 in BVDV I strain NADL) which was common to all isolates. r. No corresponding subgroup in the present BVDV I nomenclature was found for our Ic cluster. forming separate clusters. m. Fig. No indication can be given about the sequences in variable region I which was not sequenced in this study. further subdividing into groupings which aggregated highly related isolates. 2. . 2. respectively (Fig. as illustrated on the tree. b. h. while no corresponding subgroup was found for our Ic cluster. When compared to the positioning of representatives of pestiviruses types 1 – 4. mo. (1993) and those of Hofmann et al. Our Id cluster appear to be a variant of the Ib subgroup in this 90 bp tree. Paton. Cluster Ib integrated isolates sharing a sequence identity of 92 – 100%. Osloss and SD-1 was of 77 – 82%. Group B integrated the isolates where the sequence homology with BVDVs NADL. A search and comparison with sequences available in the database showed the published sequence of one strain. abbreviated as follows: p. Brock.3. Phylogenetic tree from part (90 bp) of the 5%NCR of the BVDV genome. Moormann. respectively. Cluster Ic comprised isolates sharing a sequence homology of 94 – 100%. Our Group B sequences appear to show closer relation to the Ib subgroup in the 90 bp nucleotide stretch included in this comparison. the cluster showed highest sequence homology with BVDV Osloss. 3.2. some sequence insertions or deletions were noted within variable regions II and III. 1. In variable region II. The similarity between isolate SN50CK/95 and strain 88753 is shown on the tree. Qi. 3). The previously mentioned similarity between sequences of strains 88753C and SN50CK/95 is shown in Fig. Ridpath. 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). Hofmann. 3. 2 was constructed from ten sequences representing the groups and the clusters derived in the present study and published sequences from other groups. 91–98%. A search and comparison with sequences available in the database did not show sequences which could group with this cluster of isolates. based on the comparison of the 245 bp nucleotide fragment. It composed isolates sharing 85– 100% of sequence homology. a. 1993) to be close to the sequence of isolate SN50CK/95. same as for some of the Qi’s and Hofmann’s strains.

. Nucleotide deletions are marked with an asterisk (*). compared to the Group A sequences. 2. / Virus Research 52 (1997) 205–220 213 Fig. in the first 142 bases. 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.C. 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. For instance. 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. Dots represent nucleotides that are identical to NADL. The patterns of nucleotide substitutions additionally contributing to the groupings defined in Fig. compared to BVDV strain NADL (positions 127 – 371) in the first and Osloss in the second lane. 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. The nucleotide substitutions and deletions were mainly located in the stretches corresponding to variable regions II and III. 3). which is almost fully conserved in the Ia – Ic sequences. 1 are seen upstream the 90bp fragment. The gaps are introduced by the program to optimise the alignment. 3. Baule et al. 3). Uppercase letters show nucleotide substitutions. 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. Alignment of 245 bp nucleotide sequences representatives of cluster Ia – Id isolates. In cluster Id some position substitutions occurred towards the 5% part of the sequenced fragment.

1. i. in this region of the NS3. 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. Analysis of the genetic grouping in relation to clinical profile and origin of the isolates Table 1 shows the list of isolates included in the present study.e. was 84% and 82% with the Ia and Ib subgroups. distinct from other BVDV I strains was consistent with Fig. The nucleotide sequence homology of cluster Ic isolates. The isolates from the groups and clusters defined included both non-cytopathogenic and cytopathogenic isolates. .4. The biotype of the isolates and the location of origin are also shown. 3. respectively. In Fig. grouped according to the predominant type of clinical syndrome observed in the affected animals from which the viruses were isolated. It was found that viruses in Group A were associated with a range of clinical syndromes. the segregation of group B viruses into a different cluster. No biotype/phylogenetic grouping relationship could be established. (Continued) In the phylogenetic tree on Fig. cluster Ic branched out separately from subgroup Ia and Ib strains. Id. enteric/mucosal disease (31 in 52). 3. respiratory (9 in 52). 4A. Baule et al. 4B. The relationship between clinical syndrome and phylogenetic grouping was investigated.214 C. / Virus Research 52 (1997) 205–220 Fig.

Phylogenetic trees derived from the NS3 (Fig 4A) and the NS2 – NS3 (Fig 4B) gene regions of the BVDV genome. it could be seen that some of the defined groupings were widespread while others involved isolates from a common place of origin. Sequences representing the groups and clusters derived in the present study are shown in relation to published sequences of BVDV (in bold). The first subdivision of cluster Id .C. consistent with Fig. In Fig. 4B. Group B viruses (cluster Id) segregated into a distinct subset within the BVDV I genotype. The isolates clustering with the standard American and European strains in clusters Ia and Ib. Xai-Xai and Beira (South and Central Mozambique) and in the Kwazulu Natal and Western Cape provinces in South Africa. / Virus Research 52 (1997) 205–220 215 Fig. 1. 4. Group and cluster denominations were used as in Fig. When analysing the groupings in relation to the geographical origin of the isolates. 4A. In Fig. Baule et al. and also viruses from Ic were found in locations corresponding to the Dairy Basins in Maputo. cluster Ic viruses grouped separately from strains of the Ia and the Ib subgroups. 1. in Group A.

was not found to group with any characterised BVDV strains. SD-1. This applied particularly to cluster Id sequences. The fact that the present studies revealed the existence of two distinct groups within the BVDV I genotype. Cluster Ia grouped with different strains of American origin. Ib and Ic. and other studies (Qi et al. Oregon C24V. In the comparative alignments made with sequences available in the database. 1993. The apparent relationship to BVDV reference strain Osloss. although clearly still BVDV I. 1. 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. but due to the relatively small sample numbers in this study could also be distributed more widely. The distinction between the two groups was supported at a confidence level of 95. differing from each other by a maximum of 23% in the nucleotide sequence. Clusters Ia and Ic included isolates found both in Mozambique and in South Africa and cluster Ib involved isolates from Mozambique. Hofmann et al. phylogenetic information could be lost rapidly both by mutation and back mutation. 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. Group B of the Southern African viruses comprised isolates that were related to the known reference strains but branched separately. 1 and 2 and Fig.. suggested by the 90 bp tree from the 5%NCR was not supported in this region of the NS2–NS3.8% by the bootstrap analysis. such as NADL. In the comparison of Fig. (1994). three clusters could be defined. when the comparison was limited to the 90 bp stretch that covers variable region III. as Ia. Cluster Ib showed high similarity to BVDV reference strain Osloss and falled under subgroup Ib of BVDV I. which placed them in subgroup Ia of BVDV I. The nucleotide substitutions. as shown in Figs. Ia may be too diverse to be considered a single cluster. 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 may provide an explanation for the apparently contradictory positioning of the Group B viruses in the phylogenetic tree of Fig. the segregation of group B viruses (clusters Id) in relation to subgroup Ia and Ib strains was consistent with Fig. 4A.216 C. Ic. 1994) where even . involving the NS2–NS3 region. All the Group B isolates were from Mozambique. 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. 4B. the only sequence which showed similarity to this group was that of strain 88753C. 4. 1 and 2. There was a large difference between the Ia – Ic and the Id isolates in the region sequenced. on the basis of nucleotide sequencing of the 5%NCR of the genome in 73 isolates originating from Mozambique and South Africa. It appears that cluster Ic represents strains that are rare or absent in America (North) and Europe. as defined by Pellerin et al. The third cluster. 2. Evidence from this. Discussion We have analysed the genetic diversity of BVDV strains from Southern Africa. The isolates were discriminated into two groups within the BVDV I genotype (Pestivirus type 1). reported by De Moerlooze et al. is reflected in the wide distribution of these isolates in the phylogenetic trees of Figs. Singer and C3. which suggested the presence of group rather than subgroup relationships between the respective isolates. 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. Baule et al. Therefore. forming a distinct subset within the BVDV I genotype.. / Virus Research 52 (1997) 205–220 composed isolates from the same location in XaiXai (Central Gaza Province). branching into different clusters indicates the presence of diverse BVDV strains in the region. In Group A. New York-1. of 85– 100%. where highly homologous isolates were discriminated into further aggregates. (1993). The Ia intra-cluster variation.

1. Despite the scarcity of data on BVDV infection in wildlife.e. Baule et al. The partial comparisons presented in Fig. 1994). Lowings et al.C.. 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. Additionally. Group A variants were found associated with different clinical symptoms. Vilcek et al. however. 1987).. (1997) suggested that the Npro region is more suitable for analysing genetic relationships within genotype. From our results. respiratory infections. there is a possibility of a connection between this practise and the occurrence of the same virus variants in both farms. For this reason it has been chosen by several groups as a target region to discriminate pestivirus genomes. It is possible that a vaccine batch was contaminated.. Studies of the relationship between the present groupings compared to clinical and epidemiological profiles revealed an apparent link between the occurrence of certain virus variants and a predominant type of clinical syndrome. Becher et al. 1996). 1996. analysis of this region alone might not give the highest resolution for phylogenetic analysis. / Virus Research 52 (1997) 205–220 217 greater diversity was observed.e. (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.. were found in cattle raising areas with history of cattle importation from Europe or use of potentially infected products such as vaccines and semen. 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 . however. the semi-intensive type of cattle rearing. Vilcek et al. If the tree derived from the 90 bp data is an accurate representation of variation. Lowings et al. The biological and evolutionary significance of this heterogeneity seen in BVDV I strains still remains to be clearly assessed. mucosal disease. The high conserved nature of the 5%NCR facilitates the design of primers capable of amplifying all known Pestivirus strains. Group B virus variants. Further studies comparing biological features of the viruses from both groups would be required to establish a definite connection between genetic variants of the virus and the induction of a particular clinical syndrome. it appears. suggests that the BVDV I group variation may be more extensive than the type Ia and Ib subgroups previously proposed (Pellerin et al. For example. consistent with descriptions of infections caused by BVDV viruses studied so far.. abortions/reproductive failure. 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. 1994. diarrhoea. BVDV I may possess a more continuous spectrum of variation in contrast to what appears to be found in CSFV (Hofmann et al. we consider the possibility of the involvement of a wildlife reservoir in the spread of BVD viruses. regional movements of cattle and products may also explain the spread of similar virus variants in certain regions. In farms A and B (Mozambique). i. This seems to be supported by the fact that variants bearing a high relation to those strains. Before any conclusions are made we would have to investigate the effect of host genetic profiles or farm management practices upon the virulence and pathogenesis of these viruses. a whole blood based vaccine was used for the immunisation of calves against Cowdria ruminantium. show that although providing useful data. Osloss. Findings by others. as opposed to the 5%NCR (the latter based on 130 bp fragment spanning the last two thirds of the 5%NCR). were isolated predominantly in cases where the respiratory form of infection was the most consistent clinical feature. persistent infections (Perdrizet et al. however. the NS2 – NS3). NADL and SD- 1 may reflect the introduction and establishment of these virus variants in the local cattle population. The high sequence similarity between isolates from Southern Africa and strains of European and American origin. (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. for instance. This supports our analysis based on the 245 bp fragment of the 5%NCR and the suitability of this region for phylogenetic segregation. i.e.

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