Genetic Heterogenity of Bvdv in Sa

8/3/2019 Genetic Heterogenity of Bvdv in Sa

http://slidepdf.com/reader/full/genetic-heterogenity-of-bvdv-in-sa 1/16

Virus Research 52 (1997) 205–220

Genetic heterogeneity of bovine viral diarrhoea viruses isolatedin 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 V eterinary M icrobiology, Section of Virology,

Biomedical Center , Box 585 , S -751 23 Uppsala, Swedenb Uni6ersity of Pretoria, Faculty of Veterinary Science, Department of Tropical Diseases, Pri6ate Bag X 04 ,

Onderstepoort 0110 , South Africac Central Veterinary Laboratory ( W eybridge ) , New Haw, Addlestone, Surrey KT 15 3 N B, 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, derivedfrom 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 [email protected] 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



C . Baule et al. / Virus Research 52 (1997) 205–220 206

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; Phy-

logeny

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 impor-

tant syndromes (Perdrizet et al., 1987). BVDV

belongs to the Pesti6irus genus, which also in-

cludes classical swine fever virus (CSFV) and

border disease virus (BDV), within the Fla6i6iri-

dae family (Horzinek, 1991; Collett, 1992; Wen-

gler et al., 1995).The genome of pestiviruses consists of a single

stranded, positive sense RNA molecule, approxi-

mately 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 spe-

cific 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; Hof-mann 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 corre-

sponding to nucleotides 1 –73 (I), 209– 223 (II)

and 284–323 (III) in the genome of BVDV refer-

ence strain NADL . Nucleotide substitutions ac-

counting 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 andBrock, 1993).

Two genotypes of BVDV have been discrimi-

nated 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 BVD V strain s iso lated so far. G en otyp e I I

(BVDV II) has strain 890 as reference and com-prises mainly isolates associated with haemor-

rhagic syndrome of cattle, a form of BVDVinfection recently described in North America

(Ridpath et al., 1994; Pellerin et al., 1994). BVDVII 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 trans-

mission of pestiviruses between various host ani-mal species, as well as the recent results of

monoclonal antibody typing and of comparativegenome analysis, a new classification of the mem-

bers of the Pesti6irus genus has been suggested(Becher et al., 1995; Pat on, 1995; Vilcek et al.,

1997). The new grouping is based on grounds of

antigenic and genomic relationship rather thanthe species of origin. The proposed classification,

which is still under discussion, divides the genusinto 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 pigisolates with characteristics of ‘true BDV’ viruses;

and genotype 4 would encompass isolates of cattleand sheep currently grouped as BVDV II (Becher

et al., 1995; Vilcek et al., 1997).The occurrence of h eterogeneous strains a mong

BVDV I has been revealed by nucleotide sequenc-ing and nucleic acid hybridisation (Kwang et al.,

1991; Lewis et al., 1991; Ridpath and Bolin,1991a,b) suppor ting evidence previously shown by

polyclonal and monoclonal antibody analysis

(Howard et al., 1987; Bolin et al., 1988, 1991).

The practical significance of the heterogeneityamong BVDV strains is still under assessment.However, it is considered to have implications in

the design of broad reactive diagnostic assays




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 knowl-

edge of the type of strains present and the epi-demiological profiles of the infections they cause.

In Southern Africa, BVDV has been detected

since the early seventies (Thomson and Black-

burn, 1972; Theodoridis and Boshoff, 1974) and is

found in association with diarrhoea, mucosal dis-

ease, 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). Consider-

ing the implications of the genomic diversity inthe diagnosis, epidemiology and control of BVDV

infections it deemed important to characterise the

BVD viruses occurring in the region.

2. Materials and methods

2.1. Virus strains

At the Veterinary Research Institute in Ma-

puto, 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 Eagle’s 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 Veteri-

nary Diseases, Faculty of Veterinary Science, Uni-

versity of Pretoria. The identification and originof the isolates and the predominant clinical syn-

drome present in cattle from which the specimen

were collected are listed in Table 1.

2.2. RN A extraction and cDNA synthesis

BVDV RN A was extracted from cell culture

lysates by the guanidinium-thiocyanate phenol/

chloroform method described by Chomiczynski

and Sacchi (1987), with minor modifications.

Briefly, 150 vl of cell culture lysates were vigor-

ously mixed with 450 vl of 6 M GuScn. Five-hun-dred microlitres of these specimens were extracted

twice with equal volume of a 1:1 v/v mixture of

acidic p henol:chloroform a nd once with chloro-

form. The aqueous phase was precipitated in two

volumes of 95% ethanol with 0.1 volume of 3 M

sodium acetate at −20°C. RNA was pelleted by

centrifugation for 30 min a t 10000×g and the

pellets were resuspended in 20 vl diethyl pyrocar-

bonate (DEPC) treated water.

Synthesis of cDNA was performed in 25 vl

fin al vo lu me u sin g r an do m h examer s and

Moloney Murine Leukaemia Virus Reverse Tran-scriptase (M-MLV RT) (Gibco, BRL, Bethesda,

MD). The cDNA was either used immediately for

the PCR or kept at −70°C until use.

2.3. Polymerase chain reaction ( PC R )

The primers were selected from highly con-

served stretches within the 5%NCR of the BVDV

genome, based on published sequences of refer-

ence strains NADL (Collett et al., 1988), SD-1

(Deng and Brock, 1992) and Osloss (De Moer-

looze et al., 1993). The sequences were as follows:

Primer 5A (forward) 5%-GCAGAATTCCTAGC-

CATGCCCTTAGTAGGACTAG-3 % (position

102–126 of NADL, incorporating an Eco R I

cloning site) and primer 5B (reverse) 5%-GCAAA-

GCTTATCAACTCCATGTGCCATGTACAGC-

3% (position 396–372 of NADL, incorporating a

HindIII cloning site). The PCR was performed in

50 vl final 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,

C A) and 5 vl o f cD N A , o ver laid with twodroplets of mineral oil. The cycling profile was

run as follows: five cycles with denaturation at

94°C for 45 s, annealing at 55°C for 45 s and




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 Respiratory syndromea Othersa

27 isolatesb 15 isolatesb31 isolatesb

+M140B/91 −M590A/93 +M1118-27CK/95 −M265A/91−M388A/90

−M1118-32CK/95 −M597A/93−M557A/90 −M181B/91 −M867A/93

−M085B/93 −M36CK/96+M1194A/90 −M065B/93 −M667A/93

−M427C/92 −M199CH /94+MSN50CK/95−M099B/93+M1515A/90

−M169B/93 −M432C/92 +M65CK/96 −M657GX/95+M105A/91

+MV39CB/95 +M12-73GX/96−M245A/91 +1114J/93 +M12-43GX/96

+M40-14GX/96+MV69CB/95 −M346T/96−S-ALT1/K+M278A/91

−M217GX/95 −S-ALT2/K+M279A/91 +S-ALT5/K +MV98CB/95

−S-ALT3/K−M17IN/95−M-116-28I /95−S-ALT10/K+M390A/91

−M116-53I /95 −M233IN /93−M398A/92 +S-BFL/W93 −S-ALT4/K

+M1117-38CK7/95 +M1096-5IN/95+M549A/92 +S-ALT6/W

−S-ALT7/K+M1096-16IN/95+M1117-49CK/95−M583A/92

+S-ALT8/K+S-063W/95−M589A/92 +M1118-8CK/95

+S-IFK2/W95+M839A/92 −S-ALT9/K

+S-ALT11/K+M840A/92

+M841A/92

−M567A/93

+M723A/93+M725A/93

−M079B/91

+M139B/91

+ Cytopathogenic; − Non-cytopatogenica 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, abno rmal percurssion sounds, sneezing, coughing; ot her symptoms includes poor development, abor tions/repro-

ductive failure.b Isolate identification: M —Mozambique, S—South 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;

J—dairy farm in Beira, Sofala Province; K —Kwazulu Natal; W—Western Cape.

extension at 72°C for 1 min, followed by 35 cycles

with denaturation at 94°C for 45 s, annealing at

50°C for 45 s and extension at 72°C for 1 min. A

final extension step at 72°C for 7 min was in-

cluded. Precautions to avoid contaminations were

followed throughout the RT-PCR, as described

by Belak an d Ba llagi-Pord an y (1993). PC R pro d-

ucts were visualised by ethidium bromide staining,

after electrophoresis on 2% agarose gel.

2.4. Cloning

The amplicons were purified from low meltingagarose using the Qiagen DNA purification Kit,

according to the manufacturer’s instructions.

Purified products were digested with Eco RI an d

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 DN A were isolated from multi-

plied bacteria using the Wizard mini-prep system

(Promega, Madison, WI), according to the manu-

facturer’s instructions.

2.5. Sequencing strategy and methods

Cloned DNA was sequenced by dideoxynucle-

otide chain-termination, using a T7 polymerase-based DN A sequencing kit (Sequenase, version

2.0, USB), following the manufacturer’s instruc-

tions. The Universal and Reverse M 13 primers



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

Fig. 1.




were used as sequencing primers. Sequencing re-

actions were separated in 6% acrylamide gels in-

corporating 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.

F or direct sequencing, P CR products were gen-

erated with primers 13C (forward) 5%-AGCCAT-GCCCTTAGTAGGACT-3 % (position 104–124 of

NADL) and BV3 (reverse) 5%-TCAACTCCAT-

GTGCCATGTACA-3% (position 395–374 of

NADL). The amplicons were purified by using

microconcentrato rs (Amicon, Beverly, MA) and

the DNA concentration adjusted to 30 ng/v l. The

same primers as for the PCR were used in the

automated sequencing of both strands with the

ABI200 system (Model 377).

2.6. Phylogenetic analysis

Nucleotide sequence comparisons and phyloge-

netic 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 reliabil-

ity 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 pes-

tiviruses of bovine, porcine and ovine origin, pub-

lished by other groups. These included BVDV I

strains NADL, Osloss and SD-1; CSFV strains

Alfort (Meyers et al., 1989) and Brescia (Moor-

mann et al., 1990); BDV strains Moredun cp and

ncp and BVDV II strain 890 in Fig. 1, and BVDV

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 Ha-

rasawa (1995) in Fig. 2. For comparisons in the

NS3 (Fig. 4A) and NS2–NS3 (Fig. 4B) regions,

sequences from the GenBank with the following

accession numbers were used: L35850, L35851,L35852, Z54333, A22708 and PTU96334, in addi-

tion to sequences of previously mentioned BVDV

strains. The position of the African isolates in

relation to other pestiviruses was a nalysed 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 epidemi-

ological features.

3. Results

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 se-

quences from the 5%NCR obtained in this study

and those published by others were compared.

The 73 isolates analysed could be a ssigned t o 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 an d ex-

panding 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 field 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 NAD L, O sloss, SD1), type 2 (CSFV Brescia and Alfort/Tubingen), type 3 (BDV strain Moredunncp 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.



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

Fig. 2.




(only the last two are shown in the phylogenetic

tree of F ig. 1). The sequence homology within the

cluster ranged from 85 to 100%. Cluster Ib inte-

grated isolates sharing a sequence identity of 92–

100%. In relation to BVDV I reference strains, the

cluster showed highest sequence homology with

BVDV Osloss, 91–98%. Cluster Ic comprised iso-

lates sharing a sequence homology of 94–100%. Asearch 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 homol-

ogy 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 aggre-

gated highly related isolates, as illustrated on the

tree. A search and comparison with sequences

available in the database showed the publishedsequence 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 represen-

tatives 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).

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 pub-

lished sequences from other groups. To allow the

data to be used, 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. Fig. 2 shows that: our cluster Ia and Ib

sequences correspond, respectively, to subgroups

Ia and Ib of Pellerin’s subgrouping, while no

corresponding subgroup was found for our Ic

cluster. Our Group B sequences appear to showcloser relation to the Ib subgroup in the 90 bp

nucleotide stretch included in this comparison.

The previously mentioned similarity between se-

quences 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 t he

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 out-

side these regions. No indication can be given

about the sequences in variable region I which

was not sequenced in this study. In variable re-gion II, distinctive patt erns of nucleotide substitu-

tions were formed, 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 F ig. 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).

Fig. 2. Phylogenetic tree from part (90 bp) of the 5%NCR of the BVDV genome, including ten sequences representing th e groups an d

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 thecorrespondence 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 Qi’s and Hofmann’s strains, which form additional

separate clusters. Our Id cluster appear to be a variant of the Ib subgroup in t his 90 bp tree. The similarity between isolate

SN50CK /95 and strain 88753 is shown on the tree.




Fig. 3. Alignment of 245 bp nucleotide sequences representatives of cluster Ia– Id isolates, compared to BVDV strain NADL

(positions 127–371) in the first 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

defined 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 t owards the5% part of the fragment which excluded the 90 bp

fragment (shown as full bar in Fig. 3). F or in-

stance, in the first 142 bases, for the viruses

forming the second Id division (233IN /95-1096-

5IN /95) there are six unique nucleotide positions

as opposed to one in the remainder of the frag-

ment, compared to the Group A sequences. Vari-

a ble r egio n I I seem s p ar ticu la rly r ich in

informative bases whilst variable region III (whichincludes much of the 90 bp fragment) seemed to

contain little information that supported the 245

bp tree.




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. A nalysis 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, grouped according to the predomi-

nant type of clinical syndrome observed in the

affected animals from which the viruses were iso-

lated. The biotype of the isolates and the location

of origin are also shown. The relationship be-

tween clinical syndrome and phylogenetic group-

ing 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 fail-

ure 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 relationshipcould be established. The isolates from t he groups

and clusters defined included both non-cytopat ho-

genic and cytopathogenic isolates.




Fig. 4. Phylogenetic trees derived from the N S3 (Fig 4A) and the N S2–N S3 (Fig 4B) gene regions of t he 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 G roup A. In Fig. 4B, Group B viruses (cluster Id) segregated into a distinct subset within the BVDV I

genotype, consistent with Fig. 1.

When ana lysing the groupings in relation t o the

geographical origin of the isolates, it could be

seen that some of the defined groupings werewidespread while others involved isolates from a

common place of origin. T he 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 first subdivision of cluster Id




composed isolates from the same location in Xai-

Xai (Central Gaza Province), while the isolates in

the remaining subdivisions originated from In-

hambane 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%N C R o f t he

genome in 73 isolates originating from Mozam-

bique and South Africa. The isolates were dis-

criminated into t wo groups within t he 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 confidence level of 95.8% bythe bootstrap analysis.

In Group A, three clusters could be defined, 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

p laced them in su bgr ou p I a o f BVD V I , as

defined by Pellerin et al. (1994). The Ia intra-clus-

ter variation, of 85–100%, is reflected in the wide

distribution of these isolates in the phylogenetic

trees of Figs. 1 and 2, where highly homologous

isolates were discriminated into further aggre-

gates. 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 com-

prised isolates that were related to the knownreference strains but branched separately, forming

a distinct subset within the BVDV I genotype.

There was a large difference between the Ia –Ic

and the I d isolates in t he region sequenced, which

suggested the presence of group rather than sub-

group relationships between the respective iso-

lates. In the comparative alignments made with

sequences available in the database, the only se-

quence which showed similarity to this group was

that of strain 88753C, reported by De Moerlooze

et al. (1993). In the comparison of Fig. 4B, involv-ing the NS2–NS3 region, the segregation of

group B viruses (clusters Id) in relation to sub-

group Ia and Ib strains was consistent with F ig. 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

NS2–NS3. 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 mayindicate 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 dis-

tributed more widely.

The nucleotide substitutions, indicative o f t he

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 appar-

ently 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 vari-

able region III seems to have few sequence pat-

terns 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 muta-

tion.

The fact that the present studies revealed the

existence of t wo distinct groups within the BVDV

I genotype, branching into different clusters indi-cates 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




greater diversity was observed, suggests th at 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 contin-

uous 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 significance of

this heterogeneity seen in BVDV I strains still

remains to be clearly assessed.

The high conserved nature of the 5%NCR facili-

tates 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 phylogeneticanalysis. 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) a dditionally considered the

polymerase-coding gene (NS5B) o f 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 ap-

pears, however, that by extending the analysis to

a larger p ar t o f th e 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 phy-

logenetic 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 reflect the introduction and establishment

of these virus variants in the local cattle popula-

tion. 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 poten-

tially infected products such as vaccines and se-

men . I n farms A and B (M o zamb iq ue), fo rinstance, 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. Addition-

ally, regional movements of cattle and products

may also explain the spread of similar virus vari-

ants 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 epidemiologi-

cal profiles revealed an apparent link between the

occurrence of certain virus variants and a pre-

dominant type of clinical syndrome. Group A

variants were found associated with different clin-

ical symptoms, consistent with descriptions of in-

fections caused by BVDV viruses studied so far,

i.e. diarrhoea, mucosal disease, respiratory infec-

tions, abortions/reproductive failure, persistent in-

fections (Perdrizet et al., 1987). Group B virus

variants, however, were isolated predominantly incases where the respiratory form of infection was

the most consistent clinical feature. Further stud-

ies comparing biological features of the viruses

from both groups would be required to establish a

definite connection between genetic variants of the

virus a nd the induction of a particular clinical

syndrome. Before any conclusions are made we

would have to investigate the effect of host ge-

netic profiles or farm management practices upon

the virulence an d pathogenesis of these viruses.

For example, the semi-intensive type of cattle

rearing, used in dairy farms in Mozambique andin South Africa utilises a comparatively closed-in

system more likely to favour the establishment of

a cycle of enteric infections than is the extensive




type of cattle rearing practised in the other loca-

tions where samples for this study originated

from, i.e. Northern part of Maputo, G aza and

Inhambane Provinces.

5. Conclusion

In conclusion, the present studies revealed that

different genetic variants o f 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 t he 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 h ave

evolved separately. Further studies are required toestablish 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 importa-

tion of cattle and/or the use of infected products

in addition t o regular cattle movements. The p res-

ence 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 th e present

study of viruses from two countries in Southern

Africa, suggests th at 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.

Acknowledgements

We thank Prof Bror Morein for valuable dis-

cussions and for the critical reading of the

manuscript. We also thank Adriana James (Aller-ton Laboratory, Department of Agriculture,

Kwazulu Natal, South Africa) for providing sev-

eral BVDV isolates; Jacinto Banze and Carlos

Quembo for sample collection and screening inMozambique. A special appreciation to all themembers of the Research and Development Sec-tion of the Department of Virology for assistancein different parts of this work and interestingdiscussions. This study was supported by a grantfrom the Swedish Agency for Research Coopera-

tion with Developing Countries (SAREC) and bya reaseach grant of the National Veterinary Insti-tute, Uppsala, Sweden.

References

Baule, C., Banze, J., 1994. Bovine virus diarrhoea virus infec-

tions in calves from selected farms in Mozambique. Bull.

Anim. Health Prod. Afr. 42, 279–286.

Becher, P., K on ig, M., Paton, D.J., Thiel, H .-J., 1995. F urther

characterisation of border disease virus isolates: evidence

for the presence of more than three species within the

genus Pestivirus. Virology 209, 200–206.

Becher, P., Orlich, M., Shannon, A.D., Horner, G., Konig,M., Thiel, H.-J., 1997. Phylogenetic analysis of pestiviruses

from domestic and wild ruminants. J. Gen. Virol. 78,

1357–1366.

Belak , S., Ballagi-Pordany, A., 1993. Experiences on the appli-

cation of t he polymerase chain reaction in a diagnostic

laboratory. Mol. Cell. Probes 7, 241–248.

Bolin, S.R., Moennig, V., Kelso Gourley, N.E., Ridpath, J.F.,

1988. M onoclonal an tibodies with n eutralising activity seg-

regate isolates of bovine viral diarrhea virus into groups.

Arch. Virol. 99, 117–124.

Bolin, S.R., Littledike, E.T., Ridpath, J.F., 1991. Serological

detection and practical consequences of antigenic d iversity

among bovine viral diarrhea viruses in a vaccinated herd.

Am. J. Vet. Res. 53, 1033–1037.

Boye, M ., Kamstrup, S., Dalsgaard, K., 1991. Specific se-quence amplification of bovine virus diarrhea virus

(BVDV) and hog cholera virus and sequencing of BVDV

nucleic acid. Vet. Microbiol. 29, 1–3.

Chomiczynski, P., Sacchi, N., 1987. Single-step method of

RNA isolation by acid guanidinium thiocyanate-phenol-

chloroform extraction. Anal. Biochem. 162, 156–159.

Collett, M.S., Larson, R., Gold, C., Strick, C.D., Anderson,

D.K., Purchio, A.F., 1988. Molecular cloning and nucle-

otide sequence of the pestivirus bovine viral diarrh ea virus.

Virology 165, 191–199.

Collett, M., 1992. M olecular genetics of pestiviruses. Comp.

Immunol. Microbiol. Infect. Dis. 15, 145–154.

De Moerlooze, L., Lecomte, C., Brown-Shimmer, S., Schmetz,

D., Guiot, C., Vandenbergh, D., Allaer, D., Rossius, M.,

Chappuis, G., Dina, D., Renard, A., Martial, J.A., 1993.Nucleotide sequence of the bovine viral diarrhoea virus

Osloss strain: comparison with related viruses and identifi-

cation of specific DNA probes in the 5% untranslated

region. J. Gen. Virol. 74, 1433–1438.




Deng, R ., Brock, K.V., 1992. Molecular cloning and nucle-

otide sequence of a pestivirus genome, noncytopathic

bovine viral diarrhea virus strain SD-1. Virology 191,

867–879.

Deng, R., Brock, K.V., 1993. 5% and 3% untranslated regions of

pestivirus genome: primary and secondary structure analy-

ses. Nucleic Acids Res. 21, 1949–1957.

Depner, K., Hubschle, O.J.B., Liess, B., 1991. Pr evalence of

ruminant pestivirus infections in N amibia. OnderstepoortJ. Vet. Res. 58, 107–109.

Harasawa, R., Tomiyama, T., 1994. Evidence of pestivirus

RNA in human virus vaccines. J. Clin. Microbiol. 32,

1604–1605.

Harasawa, R ., 1995. Adventitious pestivirus R NA in live virus

vaccines against bovine an d swine diseases. Vaccine 13,

100–103.

Hofmann, M.A., Brechtbuhl, K., Stauber, N., 1994. Rapid

characterisation of new pestivirus strains by direct sequenc-

ing of PCR-amplified cDNA from the 5% noncoding region.

Arch. Virol. 139, 217–219.

Horzinek, M ., 1991. Pestiviruses—ta xonomic perspectives. In :

Liess, B., Moennig, C., Pohlenz, J., Trautwein, G. (Eds.),

Ruminant Pestivirus Infections. Virology, Pathogenesisand Perspectives of Prophylaxis. Arch. Virol. (Suppl. 3).

Springer, Vienna, pp. 1–5.

Howard, C.J., Brownlie, J., Clark, M .C., 1987. Comparison by

the neutralization assay of pairs of noncytopathogenic and

cytopathogenic strains of bovine virus diarrhea virus from

cases of mucosal disease. Vet. Microbiol. 13, 361–369.

Kwang, J., Littledike, E.T., Bolin, S.R., Collett, M.S., 1991.

Efficiency of various cloned cDNA probes for detection of

bovine viral diarrhea viruses. Vet. M icrobiol. 28, 279–288.

Lewis, T.L., R idpath, J.F., Bolin, S.R., Berry, E.S., 1991.

Detection of BVD viruses using synthetic oligonucleotides.

Arch. Virol. 117, 269–278.

Lowings, P., Ibata, G., Needham, J., Paton, D., 1996. Classi-

cal swine fever virus diversity and evolution. J . G en. Virol.77, 1311–1321.

Meyers, G., Rumenapf, T., Thiel, H.-J., 1989. Molecular

cloning and nucleotide sequence of the genome of hog

cholera virus. Virology 171, 555–567.

Moormann, R.J.M., Warmedam, P.A.M., Van der Meer, B.,

Schaaper, W.M.M., Wensvoort, G., Hulst, M., 1990.

Molecular cloning and nucleotide sequence of hog cholera

virus strain Brescia and mapping of the genomic region

encoding envelope protein E1. Virology 177, 184–198.

Muvavarirwa, P., Mudenge, D., Moyo, D., Javangwe, S.,

1995. Detection of bovine virus diarrhoea virus antibodies

in cattle with an enzyme-linked immunosorbent assay.

Onderstepoort J. Vet. Res. 62, 241–244.

Paton, D.J., Sands, J.J., Edwards, S., 1994. Border diseasevirus: delineation by monoclonal antibodies. Arch. Virol.

135, 241–252.

Paton, D.J., 1995. Pestivirus diversity. J. Comp. Path. 112,

215–236.

Paton, D .J., Sands, J.J., Lowings, J.P., Smith, J.E., Ibata, G.,

Edwards, S., 1995. A proposed d ivision of the p estivirus

genus using monoclonal antibodies, supported by cross-

neutralisation assays an d genetic sequencing. Vet. Res. 26,

92–109.

Pellerin, C., Van Den Hurk, J., Lecomte, J., Tijssen, P., 1994.

Identification of a new group of bovine viral diarrhea virus

strains associated with severe outbreaks and high mortali-

ties. Virology 203, 260–268.Perdrizet, J.A., Rebhun, W.C., Dubovi, E.J., D onis, R.O.,

1987. Clinical syndromes induced by bovine viral diarrhea

virus. Cornell Vet. 77, 46–74.

Qi, F., Gustad, T., Lewis, T.L., Berry, E.S., 1993. The nucle-

otide sequence of the 5 %-untraslated region of bovine viral

diarrhoea virus: its use as a probe in rapid detection of

bovine viral diarrhoea viruses and border disease viruses.

Mol. Cell. Probes 7, 349–356.

Ridpath, J.F., Bolin, S.R., 1991a. Antigenic and genomic

comparison between non-cytopathic and cytopathic bovine

viral diarrhoea viruses isolated from cattle that had spon-

taneous mucosal disease. J. Gen. Virol. 72, 725–729.

Ridpath, J.F., Bolin, S.R., 1991b. Hybridization analysis of

genomic variability among isolates of bovine viral di-

arrhoea virus using cDN A probes. Mol. Cell. Probes 5,

291–298.

Ridpath, J.F., Bolin, S.R., Katz, J., 1993. Comparison of

nucleic acid hybridisation and nucleic acid amplification

using conserved sequences from the 5% noncoding region

for detection of bovine viral diarrhea virus. J. Clin. Micro-

biol. 31, 986–989.

Ridpath, J.F., Bolin, S.R., Dubovi, E.J., 1994. Segregation of

bovine viral diarrhea virus into genotypes. Virology 205,

66–74.

Sambrock, J., Fritsch, E.F., Maniatis, T., 1989. Molecular

Cloning: a Laboratory Manual. Cold Spring Harbor Labo-

ratory, Cold Spring Harbor, NY.

Theodoridis, A., Boshoff, S.E.T., Botha, M.J., 1973. Mucosal

disease in southern Africa. J. South Afr. Vet. Assoc. 44,61–63.

Theodoridis, A., Boshoff, S.E.T., 1974. Isolation of cytopatho -

genic strains of bovine viral diarrhoea virus in southern

Africa. J. South Afr. Vet. Assoc. 445, 203–205.

Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. Clustal

W: improving the sensitivity of progressive multiple se-

quence aligmnent through sequence weighting, position

specific gap penalties and weight matrix choice. N ucleic

Acids Res. 22, 4673–4680.

Thomson, G.R., Blackburn, N.K., 1972. Bovine virus di-

arrhoea-mucosal disease in Rhodesian cattle. Rhodesian

Vet. J. 3, 15–19.

Van Vuuren, M., 1991. The microbiological diagnosis of res-

piratory tract infections in feedlot cattle in South Africa.MMedVet thesis. University of Pretoria, Pretoria.

Vilcek, S., Stadejek, T., Ballagi-Pordany, A., Lowings, J.P.,

Pato n, D .J., Belak, S., 1996. Genetic variab ility of classical

swine fever virus. Virus Res. 43, 137–147.




Vilcek, S., N ettleton, P.F., Pa ton, D.J., Belak, S., 1997. M olec-

ular characterisation o f ovine pestiviruses. J. Gen. Virol.

78, 725–735.

Ward, P., Misra, V., 1991. Detection of bovine viral diarrhea

virus, using degenerate oligonucleotide primers and the

polymerase chain reaction. Am. J. Vet. Res. 52, 1231–

1236.

Wengler, G., Bradley, D.W., Collett, M.S., Heinz, F.X.,

Schlesinger, R.W., Strauss, J.H., 1995. Family Flaviviridae.

In: Murphy, F.A., Fauquet, C.M., Bishop, D.H.L.,

Ghabrial, S.A., Jarvis, A.W., M artelli, G.P., M ayo, M .A.,

Summers, M.D. (Eds), Virus Taxonomy. Sixth Interna-

tional Report of the Committee on the Taxonomy of

Viruses. Springer-Verlag, Vienna, pp. 415–427.

.

Documents

Genetic Heterogenity of Bvdv in Sa