Antigenic and genetic characterisation of border disease viruses isolated from UK cattle R. Strong, S.A. La Rocca, G. Ibata, T. Sandvik
To cite this version:
R. Strong, S.A. La Rocca, G. Ibata, T. Sandvik. Antigenic and genetic characterisation of border disease viruses isolated from UK cattle. Veterinary Microbiology, Elsevier, 2010, 141 (3-4), pp.208. 10.1016/j.vetmic.2009.09.010. hal-00570024
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Title: Antigenic and genetic characterisation of border disease viruses isolated from UK cattle
Authors: R. Strong, S.A. La Rocca, G. Ibata, T. Sandvik
PII: S0378-1135(09)00411-8 DOI: doi:10.1016/j.vetmic.2009.09.010 Reference: VETMIC 4567
To appear in: VETMIC
Received date: 20-5-2009 Revised date: 18-8-2009 Accepted date: 4-9-2009
Please cite this article as: Strong, R., La Rocca, S.A., Ibata, G., Sandvik, T., Antigenic and genetic characterisation of border disease viruses isolated from UK cattle, Veterinary Microbiology (2008), doi:10.1016/j.vetmic.2009.09.010
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1 Antigenic and genetic characterisation of border disease viruses isolated
2 from UK cattle
3
4
5
* 6 R. Strong, S. A. La Rocca, G. Ibata, T. Sandvik
7
8
9 Veterinary Laboratories Agency - Weybridge, New Haw, Addlestone, Surrey, KT15 3NB,
10 United Kingdom
11
12
13
14 *Corresponding author:
15
16 Telephone: +44 1932 34111
17 Telefax: +44 1932 347046
18 e-mail: [email protected]
20 21 Accepted Manuscript
1 Page 1 of 24 22 Abstract
23 Available empirical data on the natural occurrence of ruminant pestiviruses has shown that
24 bovine viral diarrhoea virus (BVDV) is nearly exclusively found in cattle, whereas both
25 border disease virus (BDV) and BVDV can be isolated from sheep. During routine genetic
26 typing of pestivirus RNA from UK cattle diagnosed as BVDV positive between 2006 and
27 2008, five samples that were classified as BDV positive yielded positive virus isolates in cell
28 cultures. The samples originated from animals that had shown signs typical for BVD.
29 Phylogenetic analysis of the bovine BDVs showed that two belonged to the BDV-1a group
30 and three to the BDV-1b group, thereby matching the genetic diversity seen for previously
31 described UK ovine BDVs. Antigenic typing with a set of monoclonal antibodies (MABs)
32 showed that all bovine BDVs lacked one or more epitopes conserved among ovine BDV-1
33 isolates, and that they had gained reactivity with at least one BVDV-1 specific MAB. Serial
34 passaging of two of the virus isolates in ovine cell cultures did not change the epitope
35 expression pattern. These findings suggest that the presumed natural resistance of cattle
36 against infection with BDV no longer holds. A consequence of this is that BVD diagnostic
37 assays should be checked for their ability to also detect BDV, and also highlights the need for
38 monitoring of the BDV status in sheep that may be in contact with cattle in areas with
39 organised BVD control programmes.
40
41
42 43 Accepted Manuscript 44 Keywords: Pestivirus; Border disease virus; Cattle; Genetic typing; Monoclonal antibody
2 Page 2 of 24 45 1. Introduction
46 Pestiviruses cause economically important diseases in domestic ruminants and pigs
47 worldwide. There are currently four recognized members of the genus Pestivirus, family
48 Flaviviridae, three of which infect ruminants naturally; bovine viral diarrhoea virus (BVDV)
49 1, BVDV-2 and border disease virus (BDV) (Thiel et al., 2005). Acute BVDV infection in
50 cattle can vary from sub-clinical infection to mild clinical disease with inappetence and
51 transient fever, or with more specific signs such as diarrhoea, respiratory or reproductive
52 dysfunction. Vertical transmission of virus in pregnant animals to their unborn young that can
53 result in abortion, mummification of the foetus, congenital defects, stillbirth or the birth of
54 persistently infected calves immunotolerant to BVDV (PI). PI animals have been shown to
55 excrete high levels of infectious virus in the absence of clinical disease. Identification of
56 persistently infected animals within the herd is obviously of great economic importance. The
57 presence of BVDV-1 infection in cattle has been detected worldwide. In contrast BVDV-2
58 infection has only been seen since the 1990s when it was detected in cattle in North America
59 (Pellerin et al., 2000). More recently, BVDV-2 isolates have been detected in Europe, Japan,
60 Korea and South America (Vilcek et al., 2001; Nagai et al., 2001; Flores et al., 2000).
61
62 Border disease is principally a disease of sheep that shares some of the characteristics of BVD
63 in cattle, but with more pronounced emphasis on reproductive disease such as abortion, and
64 birth of stillborn or mummified fetuses. It can also cause the birth of small weak lambs that
65 suffer with „hairy-shaker‟ syndrome named due to the hairy fleeces and tremors suffered by
66 the lambs (NettletonAccepted et al., 1992). Birth of PI animals Manuscript is common in sheep but more rare in
67 goats.
68
69 The Pestivirus genome consists of a single stranded positive-sense RNA of approx 12.3 kb in
70 length, encoding for a single open reading frame that is flanked by a 5‟ and 3‟ untranslated
71 regions (UTR). The genome codes for 4 structural proteins, the capsid (C) and three envelope
3 Page 3 of 24 rns 72 proteins (E , E1 and E2), plus several non-structural proteins (Thiel et al., 2005).
73 Phylogenetic studies generally classify virus isolates based on sequences generated from the
pro 74 5'-UTR or N . BDV isolates have been divided into six phylogenetic groups; BDV-1 has
75 been detected in sheep from the USA (Sullivan et al., 1997), the UK (Vilcek et al, 1997),
76 Australia (Becher et al., 1994) and New Zealand (Vilcek et al, 1998); BDV-2 in ruminants in
77 Germany (Becher et al., 2003); BDV-3 in Switzerland (Stalder et al., 2005) and Austria
78 (Krametter-Froetscher et al., 2007), BDV-4 in Spain (Valdazo-Gonzalez et al., 2007) and
79 BDV-5 and -6 in France (Dubois et al., 2008). Isolates from Turkey form a possible seventh
80 group, BDV-7, that remains to be fully characterised (Oguzoglu et al., 2008).
81
82 Historically pestiviruses were named after the animal host species they were isolated from.
83 However, the ability of interspecies transmissibility has been shown both naturally and
84 experimentally (Paton et al. 1995; 1997). For instance, BVDV also infects sheep, goats, wild
85 ruminants and pigs (Becher et al., 1997). In sheep, BVDV-1 has been detected in Germany,
86 Sweden, UK and the United States (Becher et al, 1994; Vilcek et al, 1997; Willoughby et al.,
87 2006) and BVDV-2 in sheep in Germany, UK and the US (Becher et al., 1995, Sullivan et al.,
88 1997, Vilcek et al., 1997). Conversely, classical swine fever virus (CSFV) has never been
89 isolated from naturally infected cattle, although an experimental study has shown cattle to be
90 susceptible to CSFV (Dahle et al., 1987).
91
92 Although BVDV is commonly seen in sheep there is very little evidence of natural infection
93 with BDV in cattle.Accepted One exception is the Australian Manuscript V/TOB isolate, which W.A. Snowdon
94 (1973) described as antigenically different from other bovine isolates, and that later was
95 genotyped as BDV by Becher et al. (1997). More recently, one case of BDV in cattle was
96 identified during routine testing in Austria as part of their BVDV control program (Hornberg
97 et al., 2009).
4 Page 4 of 24 98 During recent screening for exotic pestiviruses in UK cattle samples positive for BVDV
99 antigen, six cases were found to be BDV positive by TaqMan RT-PCR. Here we describe the
100 genetic and antigenic typing of five bovine BDV isolates from the UK.
101
102 2. Material and methods
103 2.1 Origin of BDV isolates
104 Three suspect BVD cases were identified as being caused by BDV by a routine TaqMan RT-
105 PCR genotyping as reported by Cranwell et al. (2007). Three further cases have tested
106 positive for BDV; one case in 2006 by RT-PCR of spleen tissue from an aborted foetus, and
107 in 2008 two more cases initially diagnosed as BVDV positive by antigen ELISA (Serelisa
108 BVD/MD Ag, Synbiotics, France) were typed as BDV positive by the TaqMan RT-PCR
109 (Table 1).
110
111 Information on the case histories and potential epidemiological links between cattle and sheep
112 on the farms where the isolates came from was obtained either by farm visits or sought from
113 the private veterinary surgeons attending the cases.
114
115 2.2 Virus isolation
116 Either plasma or clarified supernatant from homogenized tissue samples were set up for virus
117 isolation by inoculation onto pestivirus-free foetal bovine turbinate (fBT) cells, maintained in
118 minimum essential medium containing 10% foetal bovine serum proven free from
119 pestiviruses andAccepted pestivirus neutralizing antibodies. ManuscriptAfter a further virus passage on fBT cells,
120 samples were fixed and immunostained using an unconjugated pool of pan-pestivirus reacting
121 monoclonal antibodies (MABs) (WB103/WB105/WB112) and a peroxidase-linked secondary
122 mouse antibody as described by Paton et al. (1994).
123
5 Page 5 of 24 124 2.3 Genetic typing
125 RNA was extracted from infected fBT cells using Trizol (Invitrogen, UK) and RNA was
126 eluted in 100 l nuclease-free water. Reverse transcription (RT) was carried out using the
127 Superscript II Reverse Transcriptase kit (Invitrogen, UK) as per the manufacturer‟s
128 protocol. A 242-246 nucleotide (nt) fragment of the 5‟-UTR was amplified using primers
pro 129 324/326 (Vilcek et al., 1994) and Taq DNA polymerase (Promega, UK). The N gene was
130 amplified with primers 320F (5'-GCCTGATAGGGTGYWGCAGAG-3‟, forward) and
131 1040R (5'-TTYCCTTTCTTCTTYACCTGGTA-3‟, reverse). Sequencing reactions based on
132 purified amplicons were set up in both directions using both the PCR primers and the 356F /
133 1027R pair (Valdazo-Gonzalez et al., 2007) using a chain termination protocol based on
134 fluorochrome labeled dideoxynucleotides (Cogenics, UK). Assembled sequences were
135 aligned with matching sequences of BDV and BVDV reference strains using Clustal X
136 (Thompson et al., 1997). Transition/transversion ratios were calculated with Puzzle4
137 (Strimmer and von Haeseler, 1997) and phylograms were calculated using components of the
138 PHYLIP package (Felsenstein, 1989). Phylogenetic analyses were carried out using the
139 maximum likelihood distance matrix method using 1000 replicates for calculation of
140 bootstrap values. Trees were drawn with TREEVIEW (Page, 1996) and edited with
141 Freelance Graphics. Nucleotide sequences of the new BDV strains have been submitted to
142 GenBank; accession numbers are given in Table 1.
143 144 2.4 Antigenic typingAccepted Manuscript 145 fBT cells infected with each virus isolate were acetone fixed and immunostained using a
146 selection of monoclonal antibodies (MABs) raised against different pestiviruses (BDV,
147 BVDV-1, BVDV-2 and CSFV) (Edwards et al., 1988; Edwards and Sands 1990; Paton et al.,
148 1994). The staining intensity was graded as negative, weak positive and strong positive with
149 staining of less than 50% of the monolayer also being recorded. Ten serial passages were
6 Page 6 of 24 150 carried out on two of the isolates using pestivirus-free sheep choroid plexus (SCP) cells
151 maintained in minimum essential medium containing 10% foetal bovine serum proven free
152 from pestiviruses and neutralizing antibodies to pestiviruses. Each passage was fixed,
153 immunostained and results recorded as described for the isolates grown in fBT cells.
154
155 3. Results
156 3.1 Case histories
157 The available case history information was variable. For the adult animals the presentation
158 was undistinguishable from typical signs seen in animals PI with low-virulent BVDVs (Table
159 1). Four of the cases came from farms that also kept sheep, but the degree of contact
160 appeared to have been variable. There was no record of recent border disease among the
161 sheep on any of the farms involved, nor any obvious signs of BVD in other cattle.
162
163 3.2 BDV isolates
164 A pestivirus was isolated from five of the six cases that had been recorded positive by RT-
165 PCR. The sixth sample remained negative after two virus isolation attempts with spleen
166 samples from the aborted foetus, probably due to the cytotoxicity of the available sample.
167 When cultured on fBT cells, all five BDV isolates viruses were of the non-cytopathogenic
168 biotype.
169
170 3.3 Genetic typing
171 The genetic analysisAccepted of partial 5‟-UTR sequences Manuscriptshowed that all five bovine BDV isolates
172 grouped with other UK ovine BDV-1 sequences. Three isolates clustered within the BDV-1b
173 viruses, together with isolate K1729/3 from 1987, whilst the other two isolates were allocated
174 to different clusters within the BDV-1a viruses (Fig. 1). Pairwise sequence comparison with
175 the closest matching ovine BDV 5'-UTR sequence showed between two to five nucleotide
176 substitutions, corresponding to sequence identities between 97.9 and 99.2 %. Within the
7 Page 7 of 24 177 group of bovine BDVs sequence identities ranged from 97.1 to 88.5 %. A partial 5'-UTR
178 nucleotide sequence (140 nucleotides) derived from a PCR amplicon of the virus isolation
179 negative case showed greatest similarity to that of isolate 1505744, but with a significantly
180 lower sequence identity (81%).
181
pro 182 Phylogenetic analysis based on full length N sequences resulted in the same classification
183 of the five bovine BDVs as obtained with the partial 5'-UTR sequences, but with much higher
184 bootstrap values (Fig. 2). Pairwise sequence identities for the group of bovine BDVs ranged
185 from 98.4 to 75.4 %, with the same order of sequence identity as seen for the partial 5'-UTR
186 sequences.
187
188 3.4 Antigenic typing
189 The five bovine BDV isolates were most similar to ovine BDVs by immunostaining, although
190 an unusual reactivity pattern was observed compared to the BDV reference strains (Fig. 3).
191 The otherwise broadly reactive BDV MAB WS381 failed to detect four of the five bovine
192 BDV isolates. Similarly, MAB WB160 did not recognise three of the bovine BDVs. Most of
193 the bovine BDVs were immunostained by MABs WB214, CA1, and CA39, which have been
194 known as BVDV-1 specific. In general, the staining intensity with the anti-BDV MABs was
195 more variable for the bovine BDVs than for the ovine BDVs. The immunostaining pattern of
196 the bovine isolate 1505744 was more similar to BDV reference strains than the other four
197 bovine BDVs.
198 Two of the bovineAccepted BDV isolates, 1062689 and Manuscript 1118212, were maintained for up to 10
199 passages on SCP cells to assess any changes in the MAB reactivity pattern. No change in the
200 MAB reactivity pattern was seen for either of these isolates (data not shown).
201
8 Page 8 of 24 202 4. Discussion
203 It has been well documented that BVDV not only infects cattle but also other members of the
204 Order Artiodactyla, whilst BDV has mainly only been found in small ruminants. This study
205 describes antigenic and genetic properties of the first BDVs isolated from UK cattle.
206 Previous in vivo transmission studies of BVDV between cattle and sheep showed host animal
207 dependent reversible changes in the MAB reactivity pattern (Paton et al., 1997). To avoid
208 artificial induction of an ovine antigenic profile, the cattle BDV isolates were maintained in
209 bovine cell cultures for the antigenic characterization. Although variable, the MAB reactivity
210 pattern of the cattle isolates revealed an antigenic profile most similar to ovine BDV
211 reference strains. The most striking exception was that nearly all the bovine BDVs expressed
212 the BVDV-1 specific WB214, CA1 and CA39 epitopes, which reside within the major
213 envelope protein E2. Interestingly, these epitopes were the only ones that were expressed
214 exclusively in a bovine environment in the study by Paton et al. (1997). This may indicate
215 that the WB214, CA1 and CA39 epitopes are directed against cell surface receptors important
216 for replication in bovine, but not ovine cells. These changes, along with the general lower
217 reactivity against BDV specific MABs indicate that BDVs benefit from adapting
218 antigenically when shifting to a new host environment. However, this would not explain why
219 antigenic differences in non-structural proteins were seen – whilst the NS3 specific MAB
220 WB160 has been known to recognise most BVDV-1 and BDV isolates, three of the five
221 bovine BDVs were not immunostained with this MAB. The observation that the “bovine
222 MAB reactivity pattern” did not alter after 10 passages in ovine cell cultures is consistent
223 with the previouslyAccepted observed delayed reversion toManuscript an ovine antigenic pattern of a bovine
224 BVDV after transfer from cattle to sheep (Paton et al., 1997). This indicates that the antigenic
225 adaptation after host animal changes may be a clonal selection process that takes some time,
226 in the absence of immune selective pressure.
227
9 Page 9 of 24 228 Phylogenetic analysis showed that the bovine BDV isolates grouped together with previously
229 described UK ovine BDV-1 isolates (Vilcek et al 1997). This genetic classification is not
230 unlikely considering the expected continued field circulation of UK BDVs since they were
231 isolated 15-30 years ago. Based on the partial 5'-UTR sequences, the best sequence match for
232 the bovine BDVs was for the group of three BDV-1b‟s with a Scottish isolate obtained 20
233 years ago, whereas the remaining two bovine BDVs were allocated to two different groups of
pro 234 BDV-1a‟s. The same genotyping pattern was seen in the N based phylogram, with
235 sufficient statistical support to verify the 5'-UTR based classification. Direct comparison of
pro 236 N sequences of the bovine BDVs with their closest matches in the 5'-UTR phylogram was
pro 237 not possible since the latter N sequences were not available from GenBank. Interestingly,
238 the Australian V-TOB BDV isolate, reported to have originated from a case of acute mucosal
239 disease, and being antigenically distinct from contemporary BVDV isolates (Snowdon, 1973),
240 is also a BDV-1a, but genetically different from the bovine BDVs characterised in this paper.
241
242 Based on the relatively high genetic similarity of the isolates 1062689, 1502304 and 1505744,
243 it is possible that they might have emerged from the same parental virus strain, although the
244 differences in the MAB reactivity patterns did not support this. On the other hand, from the
245 more similar MAB profile of isolates 1118212 and 1376527 one might assume they are more
246 closely related than the bovine BDV-1b isolates, but the genotyping does not support this
247 either. However, it should be noted that the genetic typing is based on non-coding nucleotide
248 sequences, as opposed to antigenic properties that to a large extent reside in structural
249 glycoproteins involvedAccepted with host cell receptor binding. Manuscript
250
251 Recently a wide genetic diversity of BDV isolates has been described. Although all current
252 BDV reference strains belong to the BDV-1 cluster (Thiel et al., 2005), several new genetic
253 groups have now been described, most recently in southern Europe and around the
254 Mediterranean (De Mia et al., 2005; Thabti et al., 2005; Valdazo-Gonzalez et al., 2007;
10 Page 10 of 24 255 Dubois et al., 2008; Oguzoglu et al., 2009). To some extent there appears to be a
256 geographical bias in the distribution of BDVs in sheep. In the UK, BDV-1 is the only genetic
257 group that has been found (Vilcek et al., 1997), but this group has also been identified in
258 Australasia and the Americas. The genetic groups BDV-2 to -6 have only been described
259 regionally in Germany, Switzerland and Austria, Spain and France, respectively (Becher et
260 al., 2003; Stalder et al., 2005; Krametter-Froetscher et al., 2007; Valdazo-Gonzalez et al.,
261 2007; Dubois et al., 2008). Of these, only BDV-1 and BDV-3 have been isolated from cattle
262 in Australia, the UK, Italy and Austria (Snowdon, 1973; Cranwell et al., 2007; Schirrmeier et
263 al., 2008; Hornberg et al., 2009), and on each occasion the bovine BDV isolates were of the
264 common local genotype. Thus it seems like the BDV strain is less important than how sheep
265 and cattle are managed. A sufficiently close epidemiological contact between the two animal
266 species appears to be the most important risk factor for interspecies transfer. In the UK, sheep
267 and cattle are kept in relatively close contact on many farms, including the majority of the
268 cases investigated here. It is not unlikely that this also is a contributing factor to the high
269 prevalence of BVDV in sheep in the UK (Vilcek et al., 1997); if so, detection of BVDV in
270 sheep in other countries could be an indicator of likely presence of local BDVs in cattle as
271 well.
272
273 When a pestivirus is isolated from particularly a novel host animal, it is important to ensure
274 that the virus actually originated from the animal and is not a laboratory contamination. Cell
275 cultures and foetal calf serum used to supplement cell culture medium have frequently been
276 shown to be contaminatedAccepted with BVDV (Bolin et al.,Manuscript 1994; Sandvik et al., 1997; Schirrmeier
277 et al., 2004). This may be one explanation of the general high prevelence of pestivirus
278 infections worldwide, and also of some instances where the origin of sets of isolates has been
279 questioned (Vilcek et al., 1997; Vilcek and Paton, 2000). This potential problem can be
280 overcome by detection of pestiviruses by RT-PCR directly in original sample material, and
281 with identity confirmation by partial sequencing of PCR products. Following this approach,
11 Page 11 of 24 282 and demonstrating that the five bovine BDVs characterised in this study although genetically
283 distinguishable were allocated to three different phylogenetic clusters, it is highly likely that
284 BDV has jumped host species from sheep to cattle on at least three different occasions in the
285 UK. This route of infection is supported by the findings of Schirrmeier et al. (2008) and
286 Hornberg et al. (2009), who both also detected BDV-3 directly in blood samples by RT-PCR
287 and sequencing.
288
289 Detection of natural infection of cattle with BDV highlights the needs for BVDV diagnostic
290 tests that are able to detect all three ruminant pestiviruses; BVDV-1 and -2 as well as BDV.
291 The majority of the cases described here were originally diagnosed as BVDV antigen ELISA
292 positive, using an assay specific for the NS2-3 antigen. Being the most conserved of all
293 pestivirus proteins, NS2-3 is the most likely viral antigen to be detected by broadly reactive
rns 294 MABs. Recently BVDV antigen ELISAs targeting the E glycoprotein have become more
rns 295 popular. It may be difficult to know to what extent E ELISAs will detect bovine BDVs,
296 since the antigenic profile of the latter differed from BDV reference strains. Diagnostic RT-
297 PCR assays generally have a high sensitivity, and most assays targeting the 5‟-UTR by use of
298 Pestivirus generic primers should be able to amplify BDV cDNA, but it is important to ensure
299 that the fluorogenic detection probes in use also recognise BDV.
300
301 Lastly, from a disease control point of view, it would be of interest to assess to what extent
302 inactivated BVDV vaccines are able to provide immunoprotection against BDVs, of both
303 ovine and bovineAccepted origin. Since sheep previously have Manuscript been known to be infected with BVDV,
304 sheep should not be considered a greater risk for BVD control programmes after natural
305 infection of cattle with BDV has been described, since all domestic inter-ruminant contact
306 should be considered a risk for successful eradication of BVD.
307
308 5. Conclusion
12 Page 12 of 24 309 Molecular characterisation of five BDV-1s from naturally infected cattle has indicated that
310 they are likely to have jumped host species from sheep to cattle within the UK. These bovine
311 BDVs showed an antigenic profile differing from that of ovine BDVs, with lower reactivity to
312 BDV specific MABs, whilst they were expressing epitopes normally only seen in BVDV-1.
313 The previously assumed natural resistance of cattle against infection with BDV therefore no
314 longer holds.
315
316 Acknowledgements
317
318 Thanks to the following VLA veterinary investigation officers for following up and obtaining
319 case history data; M.C. Cranwell, C. Featherstone, K.L. Foyle, R. Hogg, P. Holmes, J.P.
320 Hutchinson, A. Otter and K. Whitaker. Also to J. Errington (VLA-Penrith) for the initial
321 identification of cases, and for providing partial sequence data for the virus isolation negative
322 case, to J. Peake (VLA Weybridge) for assistance with MAB typing, and DEFRA for funding
323 this work under research contract OD0345.
324
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Accepted Manuscript
19 Page 19 of 24 479 Table 1. Case history details of bovine border disease virus isolates characterised in this study.
480
481
Isolate Year County Clinical signs, age1 Contact with sheep Sample GenBank Accession no.
5'-UTR Npro
1062689 2006 West Yorkshire Diarrhoea / MD, 30 Not on farm of origin Blood EU887952 EU887957
1118212 2006 Clywd Diarrhoea / wasting, 12 Pet lambs Blood EU887953 EU887958
1376527 2007 Powys Neonatal death, 0 250 ewes on farm Thymus EU887954 EU887959
Buckinghamshir 1502304 2008 Diarrhoea / wasting, 15 2000 ewes on farm Blood EU887955 EU887960 e 1505744 2008 Durham Diarrhoea / wasting, 24 Sheep on farm Blood EU887956 EU887961
482
1 483 in months 484 Accepted Manuscript
20 Page 20 of 24 485 Figure legends
486
487 Fig. 1.
488 Phylogenetic tree constructed from partial 5‟UTR sequences (241-247 nucleotides) of five
489 UK bovine Border disease viruses (BDV) (bold & underlined typeface), BDV and Bovine
490 viral diarrhoea virus (BVDV) reference strains (bold typeface), previously described UK
491 ovine BDVs (Vilcek et al., 1997), and representatives for other BDV genetic groups
492 (Valdazo-Gonzalez et al., 2007; Dubois et al., 2008). BVDV strain NADL was used as
493 outgroup species.
494
495 Fig. 2.
pro 496 Unrooted phylogenetic tree based on full N sequences (504 nucleotides) of five UK bovine
497 Border disease viruses (BDV) (bold & underlined typeface) plus representatives of other
498 BDV genetic groups as well as Classical swine fever virus, Bovine viral diarrhoea virus and
499 various other pestivirus.. Literature references to sequences not included in Fig. 1 have been
500 described in Valdazo-Gonzalez et al., (2007), plus in Dubois et al., (2008). The BDV-1
501 branch is reproduced at 200% scale for clarity.
502
503 Fig. 3.
504 Reactivity of MABs to bovine BDV isolates, ovine BVD isolates, BVDV-1 and BVDV-2.
505 MABs used were raised against various epitopes; anti-E2 (WS381, WS384, WB166, WB214,
rns 506 WH303); anti-EAccepted (WS363, WS368); anti-NS2/3 (WB160,Manuscript WA437, WA443). Black boxes
507 indicate positive reaction between the MAB and virus and a grey box indicates a weak or
508 partial positive reaction.
509
21 Page 21 of 24 Figure 1
1502304 UK-08
1062689 UK-06 K1729/3 UK-87
1505744 UK-08
G1305 UK-86 137/4 UK-87
V2377/12 UK-93 V2536/2 UK-93 BDV-1b 135 661 UK-86 53 V1414 UK-93
170 337 UK-87 88 100 Q1488/1 UK-90 63 A841/1 UK-80
D1586/2 UK-82 37 G2048 UK-86
V3196/1 UK-93 T1789/1 UK-80
T1802/1 UK-92
Moredun UK-80 74 A1870 UK-80
62 1118212 UK-06
1376527 UK-07
39 L991 UK-88 Q1673/2 UK-88 38 X818 AU-87 BDV-1a 37 36 JH2816 UK-85
80 BD31 US-78
80 5809 NZ-91 53 8320-31 NZ-90 42 AcceptedFrijters NL-95Manuscript
72 M3 ES-01 85-F-0488 FR-85 BDV-4, 5, 6 90-F-6335 FR-90
100 06-F-0083 FR-06 90-F-6227 FR-90 BDV-3 Reindeer DE-96 BDV-2 NADL US-62
0.1
Page 22 of 24 Figure 2
Hobi BR-00
CSFV
Paderborn Giraffe-1 KE-69 DE-96 SN1T TN-95 Alfort 187 FR-68 BM01 TN-95 PG-2 afr.-90?
712-02 IT-02 100 100 100
BDV-2 890 US-90 Reindeer-1 DE-96 100 17385 DE-00 81 BVDV-2 61 466/T DE-85
CH-BD1 CH- BDV-3 100 Osloss DE-65 88 100 06-F-0083 FR-06
55 BVDV-1 Gifhorn DE-99 89 100 84 F-488 FR-85 NADL US-62 BDV-5 F-5415 FR-89
BU-CRA22 ES-02 M3 ES-01 100 0.1 BDV-4 Chamois AD-02 F-7446-1 FR-94 F-6335 FR-90 BDV-6 100
100 Pronghorn US- 0.05 100
99 87/6 UK-87 137/4 UK-87 V2536 UK-94 T1802/1 UK-92 1376527 UK-07 1502304 UK-08 Moredun UK-76 L83-84 X818 1062689 UK-06 b AU-87 1118212 UK-06 V-TOB 1505744 UK-08 AU-73?
BD 31 US-78 a BDV-1
Accepted Manuscript
Page 23 of 24 Figure 3
Protein specificity: E2 E2 Erns Erns Erns Erns Erns NS2-3 E2 E2 E2 E2 NS2-3 NS2-3 NS2-3 E2 E2 NS2-3 NS2-3 E2 E2
MOREDUN Ovine BDV 137/4 isolates AVERYON
1062689
1118212 Bovine BDV 1376527 isolates 1502304
1505744
BVDV-1 C24V
BVDV-2 166312
MAB parental virus: BDV (87/6) BVDV-1: (NADL, 7443) BVDV-2: (VOSGES, 59386) CSFV (86/2) Accepted Manuscript positive staining faint staining no staining
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