**Annex 14. Item 5.1. – Chapter 3.8.1. Border disease**

###### 2	MEETING OF THE BIOLOGICAL STANDARDS COMMISSION

3	**Paris, 4–8 September 2023**

4

5	**SEC T I O N  3. 8.**

# 6	~~OV I D A E A N D~~C A P R I N AE

7	**CHAP T E R  3 . 8 . 1 .**

# 8	B OR D E R D I SE A S E

9	**SUMMARY**

10 *Border disease*(*BD*) *is a viral disease of sheep and goats first reported in sheep in 1959 from the border*

11 *region of England and Wales, and since recorded world-wide. Prevalence rates in sheep vary from 5% to*

12 *50% between countries and from region to region within countries. Clinical signs include barren ewes,*

13 *abortions, stillbirths and the birth of small weak lambs. Affected lambs can show ~~and~~a fine tremor, abnormal*

14 *body conformation and hairy fleeces*(*so-called ‘hairy-shaker’ or ‘fuzzy’ lambs*). *Consequently,**the disease*

15 *has sometimes been referred to as ‘hairy shaker disease’. Vertical transmission plays an important role in*

16 *the epidemiology of the disease. Infection of fetuses can result in the birth of persistently infected*(*PI*) *lambs.*

17 *These PI lambs are viraemic, antibody negative and constantly excrete virus. The virus spreads from sheep*

18 *to sheep, with PI animals being the most potent source of infection. Infection in goats is less common with*

19 *abortion being the main presenting sign.*

20 *BD is caused by the*Pestivirus *border disease virus*(*BDV*), *but in some parts of the world, especially where*

21 *there is close contact between sheep or goats and cattle, the same clinical signs may be caused by infection*

22 *with bovine viral diarrhoea virus*(*BVDV*). *Therefore the genetic and antigenic differences between BDV and*

23 *BVDV need to be taken into consideration when investigating disease outbreaks or certifying animals or*

24 *germplasm for international movement. It is important to identify the viraemic PI animals so that they will not*

25 *be used for breeding or trading purposes. Serological testing is insufficient. However, it is generally*

26 *considered that serologically positive, nonviraemic sheep ~~are ‘safe’,~~do not present a risk as latent infections*

27 *are not known to occur in recovered animals. Pregnant seropositive, nonviraemic animals may, however,*

28 *present a risk by carrying a PI fetus that cannot be detected until after parturition.*

29 ***Identification of the agent:****BDV is a species of*Pestivirus (Pestivirus ovis)*in the family*Flaviviridae *and is*

30 *closely related to classical swine fever virus*(Pestivirus suis)*and BVDV viruses, which are classified in the*

31 *distinct species*Pestivirus bovis (*commonly known as BVDV type 1*), Pestivirus tauri (*formerly BVDV type 2*)

32 *and*Pestivirus brazilense (*BVDV type 3 or Hobi-like pestivirus*). *Nearly all isolates of BDV are*

33 *noncytopathogenic in cell culture. There are no defined serotypes but virus isolates exhibit considerable*

34 *antigenic diversity. A number of separate genotypes, have been identified.*

35 *Apparently healthy PI sheep resulting from congenital infection can be identified by direct detection of virus*

36 *or nucleic acid in blood or tissues or by virus isolation in cell culture followed by immunostaining to detect*

37 *the noncytopathogenic virus.*

38 ***Diagnostic methods:****The demonstration of virus by culture and antigen detection may be less reliable in*

39 *lambs younger than 2 months that have received colostral antibody. Acute infection is usually subclinical*

40 *and viraemia is transient and difficult to detect. The isolation of virus from tissues of aborted or stillborn*

41 *lambs is often difficult but virus can be detected by sensitive reverse transcriptase polymerase chain reaction*

42 *methods that are able to detect residual nucleic acid. However, tissues and blood from PI sheep more than*

43 *a few months old contain high levels of virus, which can be easily identified by isolation and direct methods*

44 *to detect antigens or nucleic acids. As sheep may be infected with BVDV, it is preferable to use diagnostic*

45 *assays that are ‘pan-pestivirus’ reactive and will readily detect all strains of BDV and BVDV.*

46 ***Serological tests:****Acute infection with BDV is best confirmed by demonstrating seroconversion using paired*

47 *or sequential samples from several animals in the group. The enzyme-linked immunosorbent assay and*

48 *virus neutralisation test*(*VNT*) *are the most commonly used antibody detection methods. Due to the antigenic*

49 *differences between BDV and BVDV, assays for the detection of antibodies to BDV, especially by VNT,*

50 *should preferably be based on a strain of BDV.*

51 ***Requirements for vaccines:****There is no standard vaccine for BDV, but a commercial killed whole-virus*

52 *vaccine has been produced. Ideally, such a vaccine should be suitable for administration to females before*

53 *breeding for prevention of transplacental infection. The use of BVDV vaccines has been advocated, but the*

54 *antigenic diversity of BD viruses must be considered. In many instances, the antigenic diversity of BDV*

55 *strains is sufficiently different to BVDV that a BVDV vaccine is unlikely to provide protection.*

56 *BD viruses have contaminated several modified live veterinary vaccines produced in sheep cells or*

57 *containing sheep serum. This potential hazard should be recognised by manufacturers of biological*

58 *products.*

## 59	A. INTRODUCTION

60 Border disease virus (BDV) is a *Pestivirus*of the family *Flaviviridae*and is closely related to classical swine fever virus

61 (CSFV) and bovine viral diarrhoea virus (BVDV). There are ~~four~~a number of officially recognised species, namely – BDV

62 (*Pestivirus ovis*) CSFV (*Pestivirus suis*), BVDV types 1 and 2 (taxonomically known as *Pestivirus bovis*and *Pestivirus tauri*,

63 respectively) and ~~BDV (ICTV, 2016)~~BVDV 3 or Hobi-like pestivirus (*Pestivirus brazilense*), but a number of other

64 pestiviruses that are considered to be distinct species have been reported. While CSF viruses are predominantly restricted

65 to pigs, ~~examples of~~there are situations where the other ~~three~~species have ~~all~~been recovered from sheep. While the

66 majority of isolates have been identified as BD viruses in areas where sheep or goats are raised in isolation from other

67 species (Vilcek *et al*., 1997), in regions where there is close contact between small ruminants and cattle, BVDV may be

68 frequently identified (Carlsson, 1991). Nearly all virus isolates of BDV are noncytopathogenic, although occasional

69 cytopathic viruses have been isolated (Vantsis *et al*., 1976). BDV spreads naturally among sheep by the oro-nasal route

70 and by vertical transmission. It is principally a cause of congenital disease in sheep and goats, but can also cause acute

71 and persistent infections. Infection is less common in goats, in which persistent infection is rare as abortion is the main

72 presenting sign. Pigs may also be infected by pestiviruses other than CSFV and antibodies to BDV in pigs may interfere

73 with tests for the diagnosis of CSF (Oguzoglu *et al*., 2001). Several genotypes of BD viruses from sheep, goats and

74 Pyrenean chamois (*Rupicapra pyrenaica pyrenaica*) have been described. Phylogenetic analysis using computer-assisted

75 nucleotide sequence analysis suggests that genetic variability among BD viruses is greater than within each of the other

76 *Pestivirus*species. Four distinguishable genogroups of BDV have been described as well as putative novel *Pestivirus*

77 genotypes from Tunisian sheep and a goat (Becher *et al*., 2003; Vilcek & Nettleton, 2006). The chamois BD virus is similar

78 to isolates from sheep in the Iberian Peninsula (Valdazo-Gonzalez *et al*., 2007). This chapter describes BDV infection in

79 sheep. Chapter 3.4.7 *Bovine viral diarrhoea*should also be consulted for related diagnostic methods.

#### 80 1.	Acute infections

81 Healthy newborn and adult sheep exposed to BDV usually experience only mild or inapparent disease. Slight fever and a

82 mild leukopenia are associated with a short-lived viraemia detectable between days 4 and 11 post-infection, after which

83 virus neutralising antibody appears in the serum (Thabti *et al*., 2002).

84 Acute infections are best diagnosed serologically using paired sera from a representative number of sheep. Occasional

85 BDV isolates have been shown to produce high fever, profound and prolonged leukopenia, anorexia, conjunctivitis, nasal

86 discharge, dyspnoea and diarrhoea, and 50% mortality in young lambs. One such isolate was recovered from a severe

87 epidemic of BD among dairy sheep in 1984 (Chappuis *et al*., 1986). A second such isolate was a BDV contaminant of a

88 live CSFV vaccine (Wensvoort & Terpstra, 1988).

#### 89 2.	Fetal infection

90 The main clinical signs of BD are seen following the infection of pregnant ewes. While the initial maternal infection is

91 subclinical or mild, the consequences for the fetus are serious. Fetal death may occur at any stage of pregnancy, but is

92 more common in fetuses infected early in gestation. Small dead fetuses may be resorbed or their abortion may pass

93 unnoticed as the ewes continue to feed well and show no sign of discomfort. As lambing time approaches, the abortion of

94 larger fetuses, stillbirths and the premature births of small, weak lambs will be seen. Confirmation that an abortion or

95 stillbirth is due to BDV is often difficult to establish, but virus may be isolated from fetal tissues in some cases. The use of

96 an appropriate real-time reverse-transcription polymerase chain reaction (RT-PCR) assay may give a higher level of

97 success because of the advantages of high sensitivity and the ability to detect genome from non-infectious virus. In aborted

98 fetuses, it is also possible to detect virus by immunohistochemistry of brain, thyroid and other tissues (Thur *et al*., 1997).

99 Samples of fetal fluids or serum should be tested for BDV antibody.

100 During lambing, an excessive number of barren ewes will become apparent, but it is the diseased live lambs that present

101 the main clinical features characteristic of BD. The clinical signs exhibited by BD lambs are very variable and depend on

102 the breed of sheep, the virulence of the virus and the time at which infection was introduced into the flock. Affected lambs

103 are usually small and weak, many being unable to stand. Nervous signs and fleece changes are often apparent. The

104 nervous signs of BD are its most characteristic feature. The tremor can vary from violent rhythmic contractions of the

105 muscles of the hindlegs and back, to barely detectable fine trembling of the head, ears, and tail. Fleece abnormalities are

106 most obvious in smooth-coated breeds, which develop hairy fleeces, especially on the neck and back. Abnormal brown or

107 black pigmentation of the fleece may also be seen in BD-affected lambs. Blood samples to be tested for the presence of

108 BDV or antibody should be collected into anticoagulant from suspect lambs before they have received colostrum. Once

109 lambs have ingested colostrum, it is difficult to isolate virus until they are 2 months old and maternal antibody levels have

110 waned. However, during this period, it may be possible to detect viral antigen in skin biopsies, by immunohistochemistry,

111 in washed leukocytes by enzyme-linked immunosorbent assay (ELISA) or by real-time RT-PCR. ELISAs directed at

112 detection of the Erns antigen appear to be less prone to interference by maternal antibodies and can often be used to

113 detect antigen in serum.

114 With careful nursing, a proportion of BD lambs can be reared, although deaths may occur at any age. The nervous signs

115 gradually decline and can disappear by 3–6 months of age. Weakness, and swaying of the hind-quarters, together with

116 fine trembling of the head, may reappear at times of stress. Affected lambs often grow slowly and under normal field

117 conditions many will die before or around weaning time. In cases where losses at lambing time have been low and no

118 lambs with obvious signs of BD have been born, this can be the first presenting sign of disease.

119 Some fetal infections occurring around mid-gestation can result in lambs with severe nervous signs, locomotor

120 disturbances and abnormal skeletons. Such lambs have lesions of cerebellar hypoplasia and dysplasia, hydranencephaly

121 and porencephaly resulting from necrotising inflammation. The severe destructive lesions appear to be immune mediated,

122 and lambs with such lesions frequently have high titres of serum antibody to BDV. Most lambs infected in late gestation

123 are normal and healthy and are born free from virus but with BDV antibody. Some such lambs can be weak and may die

124 in early life (Barlow & Patterson, 1982).

#### 125 3.	Persistent viraemia

126 When fetuses survive an infection that occurs before the onset of immune competence, they are born with a persistent

127 viraemia. The ovine fetus can first respond to an antigenic stimulus between approximately 60 and 85 days of its 150-day

128 gestation period. In fetuses infected before the onset of immune competence, viral replication is uncontrolled and 50%

129 fetal death is common. In lambs surviving infection in early gestation, virus is widespread in all organs. Such lambs appear

130 to be tolerant of the virus and have a persistent infection, usually for life. A precolostral blood sample will be virus positive

131 and antibody negative. Typically, there is no inflammatory reaction and the most characteristic pathological changes are

132 in the central nervous system (CNS) and skin. Throughout the CNS, there is a deficiency of myelin, and this causes the

133 nervous signs. In the skin, primary wool follicles increase in size and the number of secondary wool follicles decreases,

134 causing the hairy or coarse fleece.

135 Persistently viraemic sheep can be identified by the detection of viral antigens, nucleic acids or infectious virus in a blood

136 sample. Viraemia is readily detectable by testing of serum at any time except within the first 2 months of life, when virus

137 may be masked by colostral antibody and, possibly, in animals older than 4 years, some of which develop low levels of

138 anti-BDV antibody (Nettleton *et al*., 1992). Methods other than virus isolation may be preferred to avoid interference from

139 antibodies. When the presence of colostral antibodies is suspected, the virus may be detected in washed leukocytes and

140 in skin by using sensitive ELISAs. Although virus detection in blood during an acute infection is difficult, persistent viraemia

141 should be confirmed by retesting animals after an interval of at least 3 weeks. The use of real-time RT-PCR should be

142 considered at all times and for any sample type due to its high analytical sensitivity and the lack of interference from

143 antibodies in a sample.

144 Some viraemic sheep survive to sexual maturity and are used for breeding. Lambs born to these infected dams are always

145 persistently viraemic. Persistently viraemic sheep are a continual source of infectious virus to other animals and their

146 identification is a major factor in any control programme. Sheep being traded should be screened for the absence of BDV

147 viraemia.

148 Usually persistently infected (PI) rams have poor quality, highly infective semen and reduced fertility. All rams used for

149 breeding should be screened for persistent BDV infection on a blood sample. Semen samples can also be screened for

150 virus, but virus isolation is much less satisfactory than from blood because of the toxicity of semen for cell cultures. Real-

151 time RT-PCR for detection of pestivirus nucleic acid would usually overcome toxicity problems, and thus this assay should

152 be useful for testing semen from rams.

#### 153 4.	Late-onset disease in persistently viraemic sheep

154 Some PI sheep housed apart from other animals spontaneously develop intractable diarrhoea, wasting, excessive ocular

155 and nasal discharges, sometimes with respiratory distress. At necropsy such sheep have gross thickening of the distal

156 ileum, caecum and colon resulting from focal hyperplastic enteropathy. Cytopathic BDV can be recovered from the gut of

157 these lambs. With no obvious outside source of cytopathic virus, it is most likely that such virus originates from the lamb's

158 own virus pool, similar to what occurs with BVDV. Other PI sheep in the group ~~do~~may not develop the disease. This

159 syndrome, which has been produced experimentally and recognised in occasional field outbreaks of BD, has several

160 similarities with bovine mucosal disease (Nettleton *et al*., 1992).

## 161	B. DIAGNOSTIC TECHNIQUES

162	***Table 1.****Test methods available for diagnosis of border disease and their purpose*

163	Key: +++ = recommended for this purpose; ++ recommended but has limitations;

164	+ = suitable in very limited circumstances; – = not appropriate for this purpose.

165 ELISA = enzyme-linked immunosorbent assay; ~~IHC = immunohistochemistry;~~NA = nucleic acid; RT-PCR = reverse-transcription

166 polymerase chain reaction; ISH = *in-situ*hybridisation; VN = virus neutralisation.

167 (a)A combination of agent identification methods applied on the same clinical sample is recommended.

#### 168 1.	Identification of the agent

169 There is no designated WOAH Reference Laboratory for BDV, but the reference laboratories for BVDV or CSFV will be

170 able to provide advice 42. One of the most sensitive proven methods for identifying BDV remains virus isolation. However,

171 a broadly reactive real-time RT-PCR assay (preferably pan-pestivirus reactive) will usually provide higher analytical

172 sensitivity than virus isolation, can be used to test samples that are difficult to manage by virus isolation and can be

173 performed in a few hours. Antigen-detection ELISA and immunohistochemical techniques on tissue sections are also

174 valuable methods for identifying BDV-infected animals.

##### 175 1.1.	Virus isolation

176 It is essential that laboratories undertaking virus isolation have a guaranteed supply of pestivirus-free susceptible

177 cells and bovine serum, or equivalent, that contain no anti-pestivirus activity and no contaminating virus. It is important

178 that a laboratory quality assurance programme be in place. Chapter 3.4.7 provides detailed methods for virus isolation

179 in either culture tubes or microplates for the isolation of pestiviruses from sheep or goat samples, including serum,

180 whole blood, semen and tissues. The principles and precautions outlined in that chapter for the selection of cell

181 cultures, medium components and reagents are equally relevant to this chapter. Provided proven pan-pestivirus

182 reactive reagents (e.g. monoclonal antibodies [MAbs], primers and probes for real-time RT-PCR) are used for antigen

183 or nucleic acid detection, the principal difference is the selection of appropriate cell cultures.

184 BD virus can be isolated in a number of primary or secondary ovine cell cultures (e.g. kidney, testes, lung). Ovine cell

185 lines for BDV growth are rare. Semicontinuous cell lines derived from fetal lamb muscle (FLM), whole embryo (Thabti

186 *et al*., 2002) or sheep choroid plexus can be useful, but different lines vary considerably in their susceptibility to the

187 virus. Ovine cells have been used successfully for the isolation and growth of BD viruses and BVDV types 1 and 2

188 from sheep. In regions where sheep may become infected with BVD viruses from cattle, a virus isolation system using

189 both ovine and bovine cells could be optimal. However, bovine cells have lower sensitivity for the primary isolation

190 and growth of some BD viruses, so reliance on bovine cells alone is inadvisable. Details of suitable bovine cell cultures

191 are provided chapter 3.4.7. The precautions outlined in that chapter for the establishment of cells and medium

192 components that are free from contamination with either pestiviruses or antibodies, and measures to ensure that the

193 cells are susceptible to a wide range of local field strains are equally relevant to systems for detection of BDV.

194 From live animals, serum is the most frequently used sample to be tested for the presence of infectious virus.

195 However, for difficult cases, the most sensitive way to confirm pestivirus viraemia is to wash leukocytes repeatedly

196 (at least three times) in culture medium before co-cultivating them with susceptible cells in either cell culture tubes or

197 microplates. After culture for 5–7 days, the cultures should be frozen and thawed once and an aliquot of diluted culture

198 fluid passaged onto further susceptible cells grown in microplates or on chamber slides to allow antigen detection by

199 immunocytochemistry. Staining for noncytopathic pestiviruses will usually detect virus at the end of the primary

200 passage, but to detect slow-growing viruses in poorly permissive cells two passages are desirable. It is recommended

201 that the culture supernatant used as inoculum for the second passage is diluted approximately 1/100 in new culture

202 medium because some high titred field isolates will replicate poorly if passaged undiluted (i.e. at high multiplicity of

203 infection – moi).

204 Tissues should be collected from dead animals in virus transport medium. In the laboratory, the tissues are ground

205 to give a 10–20% (w/v) suspension, centrifuged to remove debris, and the supernatant passed through 0.45 μm

206 filters. Spleen, lung, thyroid, thymus, kidney, brain, lymph nodes and gut lesions are the best organs for virus isolation.

207 Semen can be examined for the presence of BDV, but raw semen is strongly cytotoxic and must be diluted, usually

208 at least 1/10 in culture medium. As the major threat of BDV-infected semen is from PI rams, blood is a more reliable

209 clinical sample than semen for identifying such animals. There are many variations in virus isolation procedures. All

210 should be optimised for maximum sensitivity using a standard reference virus preparation and, whenever possible,

211 recent BDV field isolates. Most of the limitations of virus isolation for the detection of BDV in serum or blood, tissues

212 or semen can be overcome by the use of a proven, sensitive pan-pestivirus reactive real-time RT-PCR. Some

213 laboratories screen samples by real-time RT-PCR and undertake virus isolation on positive samples to collect BDV

214 strains for future reference or research purposes.

215 For specific technical details of virus isolation procedures, including immunoperoxidase staining, refer to chapter

216 3.4.7.

42 Please consult the WOAH Web site: https://www.woah.org/en/what-we-offer/expertise-network/reference-laboratories/#ui-id-3

##### 217 1.2.	Nucleic acid detection methods

218 The complete genomic sequences of three BD viruses have been determined and compared with those of other

219 pestiviruses (Becher *et al*., 1998; Ridpath & Bolin, 1997). Phylogenetic analysis shows BD viruses to be more closely

220 related to CSFV than to BVDV (Becher *et al*., 2003; Van Rijn *et al*., 1997; Vilcek & Nettleton, 2006; Vilcek *et al*.,

221 1997). Real-time RT-PCR for diagnosing pestivirus infection is now used widely and a number of formats have been

222 described. Real-time RT-PCR assays have the advantages of being able to detect both infectious virus and residual

223 nucleic acid, the latter being of value for investigating abortions and lamb deaths. Furthermore, the presence of virus-

224 specific antibodies in a sample will have no adverse effect on the sensitivity of the real-time RT-PCR assay. These

225 assays are also useful for screening semen and, when recommended nucleic acid extraction protocols are followed,

226 are less affected by components of the semen compared with virus isolation. Because of the potential for small

227 ruminants to be infected with genetically different strains of BDV or with strains of BVDV, a ~~proven~~pan-pestivirus

228 reactive real-time RT-PCR with proven high sensitivity should be used. To ensure that the genetic spectrum of BDV

229 strains is sufficiently covered, it may be necessary to apply a broadly reactive BDV specific real time RT-PCR in

230 parallel to maximise diagnostic sensitivity. Suitable protocols for both nucleic acid extraction as well as the real-time

231 RT-PCR are described in chapter 3.4.7. All precautions to minimise laboratory contamination should be followed

232 closely.

233 After testing samples in a pan-pestivirus reactive assay, samples giving ~~positive results can~~any level of reactivity

234 should be investigated further by the application of a BDV-specific real-time RT-PCR (Willoughby *et al*., 2006). It is

235 important to note however that different genotypes of BDV may be circulating in some populations, especially wild

236 ruminants such as chamois and deer, and may be transferred to sheep. An assay that is specific for the detection of

237 BDV should be used with some caution as variants or previously unrecognised genotypes may not be detected,

238 hence the value of initially screening samples with a pan-pestivirus reactive real-time RT-PCR. Nevertheless, there

239 are also situations where a pan-pestivirus reactive real-time RT-PCR may have lower analytical sensitivity.

240 Consequently, in any situation where BDV infection is suspected, the application of several diagnostic methods is

241 recommended. Maternal serology can also play an important role as negative results should exclude the potential

242 involvement of a pestivirus.

##### 243 1.3.	Enzyme-linked immunosorbent assay for antigen detection

244 ELISAs for the direct detection of pestivirus antigen in blood and tissues of infected animals have proven to be

245 extremely useful for the detection of PI animals and the diagnosis of disease. The first ELISA for pestivirus antigen

246 detection was described for detecting viraemic sheep and was later modified into a double MAb capture ELISA for

247 use in sheep and cattle (Entrican *et al.,*1994). The test is most commonly employed to identify PI viraemic sheep

248 using washed, detergent-lysed blood leukocytes. The sensitivity is close to that of virus isolation and it is a practical

249 method for screening large numbers of blood samples. As with virus isolation, high levels of colostral antibody can

250 mask persistent viraemia. The ELISA is more effective than virus isolation in the presence of antibody, but may give

251 false-negative results in viraemic lambs younger than 2 months old. The ELISA is usually not sensitive enough to

252 detect acute BDV infections on blood samples. As well as for testing leukocytes, the antigen ELISA can also be used

253 on tissue suspensions, especially spleen, from suspected PI sheep and, as an alternative to immunofluorescence

254 and immunoperoxidase methods, on cell cultures. Several pestivirus ELISA methods have been published but there

255 are at present no commercially available kits that have been fully validated for detecting BDV. Prior to use for

256 regulatory purposes, these kits should be validated in the region where they are to be used to ensure that a wide

257 range of field strains of BDV can be detected and that they are suitable for the sample types to be tested.

##### 258 1.4.	Immunohistochemistry

259 Viral antigen demonstration is possible in most of the tissues of PI animals (Braun *et al*., 2002; Thur *et al*., 1997)

260 although this is not a method that is routinely used for diagnostic purposes. This should be done on acetone-fixed

261 frozen tissue sections (cryostat sections) or paraffin wax embedded samples using appropriate antibodies. Pan-

262 pestivirus reactive antibodies with NS2-3 specificity are suitable. Tissues with a high amount of viral antigen are brain,

263 thyroid gland, lung and oral mucosa. Skin biopsies have been shown to be useful for *in-vivo*diagnosis of persistent

264 BDV infection.

#### 265 2.	Serological tests

266 Antibody to BDV is usually detected in sheep sera using VN or an ELISA. The less sensitive agar gel immunodiffusion test

267 is not recommended. Control positive and negative reference sera must be included in every test. These should give results

268 within predetermined limits for the test to be considered valid. Single sera can be tested to determine the prevalence of

269 BDV in a flock, region or country. For diagnosis, however, acute and convalescent sera are the best samples for confirming

270 acute BDV infection. Repeat sera from one animal should always be tested alongside each other on the same plate to

271 provide a reliable comparison of titres.

##### 272 2.1.	Virus neutralisation test

273 Due to antigenic diversity among pestiviruses the choice of test virus is difficult (Dekker *et al*., 1995; Nettleton *et al*.,

274 1998). No single strain of BDV is ideal. A local strain that gives the highest antibody titre with a range of positive

275 sheep sera should be used.

276 Because there are few cytopathogenic strains of BDV available, to achieve optimal analytical sensitivity, it is more

277 usual to employ a representative local non-cytopathogenic strain and read the assay after immunoperoxidase staining

278 of the cells. Proven highly sensitive, pestivirus-free sheep cells such as lamb testis or kidney cells are suitable and

279 can be maintained as cryogenically frozen stocks for use over long periods of time. The precautions outlined for

280 selection of pestivirus-free medium components are equally applicable to reagents to be used in VN tests. A

281 recommended procedure follows.

###### 282 2.1.1.	Test procedure

283 i)	The test sera are heat-inactivated for 30 minutes at 56°C.

284 ii)	From a starting dilution of 1/4, serial twofold dilutions of the test sera are made in a cell-culture

285 grade flat-bottomed 96-well microtitre plate, using cell culture medium as diluent. For each

286 sample, three or four wells are used at each dilution depending on the degree of precision

287 required. Also, for each sample and at each serum dilution, one well is left without virus to

288 monitor for evidence of sample toxicity that could mimic viral cytopathology or interfere with

289 virus replication. Control positive and negative sera should also be included in each batch of

290 tests.

291 iii)	An equal volume (e.g. 50 μl) of a stock of BDV containing 100 TCID50 (50% tissue culture

292 infective dose) is added to each well. A back titration of virus stock is also done in some spare

293 wells to check the potency of the virus (acceptance limits ~~30~~80–300 TCID50).

294 iv)	The plate is incubated for 1 hour at 37°C.

295 v)	A flask of suitable cells (e.g. ovine testis or kidney cells) is trypsinised and the cell concentration

296 is adjusted to 2 × 105/ml. 100 μl of the cell suspension is added to each well of the microtitre

297 plate.

298 vi)	The plate is incubated at 37°C for 4–5 days, either in a 5% CO2 atmosphere or with the plate

299 sealed.

300 vii)  The wells are examined microscopically to ensure that there is no evidence of toxicity or

301 cytopathic effect (CPE), then fixed and stained by immunoperoxidase staining using an

302 appropriate MAb. The VN titre for each serum is the dilution at which the virus is neutralised in

303 50% of the wells. This can be calculated by the Spearman–Kärber or Reed Muench methods.

304 A seronegative animal will show no neutralisation at the lowest dilution of serum (i.e. 1/4),

305 equivalent to a final dilution of 1/8. For accurate comparison of antibody titres, and particularly

306 to demonstrate significant (more than fourfold) changes in titre, samples should be tested in

307 parallel in the same test

308 viii) Occasionally there may be a need to determine whether antibody in a flock is against a virus

309 belonging to a particular *Pestivirus*serogroup. A differential VN test can be used in which sera

310 are titrated out against representative viruses from each of the four *Pestivirus*groups, i.e. BDV,

311 BVDV types 1 and 2, and CSFV. Maximum titre will identify the infecting serotype and the

312 spectrum of cross-reactivity with the other serotypes will also be revealed.

##### 313 2.2.	Enzyme-linked immunosorbent assay

314 An MAb-capture ELISA for measuring BDV antibodies has been described. Two pan-pestivirus MAbs that detect

315 different epitopes on the immunodominant nonstructural protein NS 2/3 are used to capture detergent-lysed cell-

316 culture grown antigen. The results correlate qualitatively with the VN test (Fenton *et al*., 1991).

###### 317 2.2.1.	Antigen preparation

318 Use eight 225 cm2 flasks of newly confluent FLM cells; four flasks will be controls and four will be

319 infected. Wash the flasks and infect four with a 0.01–0.1 m.o.i. of Moredun cytopathic BDV. Allow the

320 virus to adsorb for 2 hours at 37°C. Add maintenance media containing 2% FBS (free from BDV

321 antibody), and incubate cultures for 4–5 days until CPE is obvious. Pool four control flask

322 supernatants and separately pool four infected flask supernatants. Centrifuge at 3000 ***g***for

323 15 minutes to pellet cells. Discard the supernatants. Retain the cell pellets. Wash the flasks with

324 50 ml of PBS and repeat the centrifugation step as above. Pool all the control cell pellets in 8 ml PBS

325 containing 1% Nonidet P40 and return 2 ml to each control flask to lyse the remaining attached cells.

326 Repeat for infected cells. Keep the flasks at 4°C for at least 2 hours agitating the small volume of fluid

327 on the cells vigorously every 30 minutes to ensure total cell detachment. Centrifuge the control and

328 infected antigen at 12,000 ***g***for 5 minutes to remove the cell debris. Supernatant antigens are stored

329 at –70°C in small aliquots.

###### 330 2.2.2.	Test procedure

The two MAbs are diluted to a predetermined dilution in 0.05 M bicarbonate buffer, pH 9.6. All wells of a suitable ELISA-grade microtitre plate (e.g. Nunc maxisorb, Greiner 129b) are coated overnight at 4°C.
331

i)

332

333

334

ii)

335

336

iii)

337

338

339

iv)

340

341

v)

342

343

vi)

344

345

346

347

348

349

350

vii)

351

352

353

354

355

After washing three times in PBST, a blocking solution of PBST containing 10% horse serum (PBSTH) is added to all wells, which are incubated at 37°C for 1 hour.

The antigen is diluted to a predetermined dilution in PBSTH and alternate rows of wells are coated with virus and control antigens for 1 hour at 37°C. The plates are then washed three times in PBST before addition of test sera.

Test sera are diluted 1/50 in PBSTH and added to duplicate virus and duplicate control wells for 1 hour at 37°C. The plates are then washed three times in PBST.

Anti-ovine IgG peroxidase conjugate is diluted to a predetermined dilution in PBSTH and added to all wells for 1 hour at 37°C. The plates are washed three times in PBST.

A suitable activated enzyme substrate/chromogen, such as ortho-phenylene diamine (OPD) or tetramethyl benzidine (TMB), is added). After colour development, the reaction is stopped with sulphuric acid and the absorbance read on an ELISA plate reader. The mean value of the two control wells is subtracted from the mean value of the two virus wells to give the corrected absorbance for each serum. Results are expressed as corrected absorbance with reference to the corrected absorbance of known positive and negative sera. Alternatively, ELISA titres can be extrapolated from a standard curve of a dilution series of a known positive reference serum.

If antigens of sufficient potency can be produced the MAb capture stage can be omitted. In this case alternate rows of wells are coated with virus and control antigen diluted to a predetermined dilution in 0.05 M bicarbonate buffer, pH 9.6, overnight at +4°C. The plates are washed and blocked as in step ii above. After washing, diluted test sera are added and the test proceeds from step iv as above.

## C. REQUIREMENTS FOR VACCINES

#### 356 1.	Background

357 To be useful, a BDV vaccine should be effective when administered to female sheep before breeding to prevent

358 transplacental infection. Experimental and commercial inactivated whole virus BDV vaccines have been produced in

359 Europe (Brun *et al*., 1993; Vantsis *et al*., 1980). Unlike vaccines for BVDV, there is limited demand for vaccines against

360 BDV and those produced have only been inactivated products. No live attenuated or recombinant subunit vaccines for

361 BDV have been produced commercially.

362 Pestivirus contaminants of modified live virus vaccines have been found to be a cause of serious disease following their

363 use in pigs, cattle, sheep and goats. Contaminated vaccines have included those used for the control of Aujesky’s disease,

364 CSF, rotavirus, coronavirus, rinderpest, sheep pox and contagious pustular dermatitis. The insidious ability of pestiviruses

365 to cross the placenta, and thus establish PI animals, gives them the potential to contaminate vaccines through cells, serum

366 used as medium supplement, or seed stock virus. As nearly all isolates of pestiviruses are noncytopathic, they will remain

367 undetected unless specific tests are carried out. Although such contamination should be less likely to be a problem with

368 an inactivated vaccine, nevertheless steps should be taken to ensure that materials used in production are not

369 contaminated.

##### 370 1.1.	Characteristics of a target product profile

371 Traditionally, pestivirus vaccines fall into two classes: modified live or inactivated virus vaccines. The essential

372 requirement for both types is to afford a high level of fetal infection. Only inactivated vaccines have been produced

373 for BDV. Properly formulated inactivated vaccines are very safe to use but, to obtain satisfactory levels of immunity,

374 they usually require booster vaccinations, which may be inconvenient. Because of the propensity for antigenic

375 variability, the vaccine should contain strains of BDV that are closely matched to viruses found in the area in which

376 they are used. This may present particular challenges with BDV in regions where several antigenic types have been

377 found. Due to the need to customise vaccines for the most commonly encountered strains within a country or region,

378 it is not feasible to produce a vaccine antigen bank that can be drawn upon globally

379 Guidance for the production of veterinary vaccines is given in Chapter 1.1.8 *Principles of veterinary vaccine*

380 *production*. The guidelines given here and in chapter 1.1.8 are intended to be general in nature and may be

381 supplemented by national and regional requirements.

#### 382 2.	Outline of production and minimum requirements for vaccines

##### 383 2.1.	Characteristics of the seed

384 An ideal vaccine should contain a strain or strains of virus that give protection against all sheep pestiviruses. This

385 may be challenging however, because of the range of pestiviruses with which sheep can be infected. There is

386 considerable antigenic variation across these viruses – both between viruses that have been classified in the BDV

387 genogroup as well as between viruses in the BVDV1 and BVDV2 genotypes (Becher *et al*., 2003; Vilcek & Nettleton,

388 2006; Wensvoort *et al*., 1989). Infection of sheep with the putative BVDV-3 genotype has also been described (Decaro

389 *et al.,*2012). It is likely that the antigenic composition of a vaccine will vary from region to region to provide an

390 adequate antigenic match with dominant virus strains. Cross-neutralisation studies are required to establish optimal

391 combinations. Nevertheless it would appear that any BDV vaccine should contain at least a representative of the BDV

392 and BVDV (type 1) groups. Characterisation of the biologically cloned vaccine viruses should include typing with

393 MAbs and genotyping (Paton *et al*., 1995).

###### 394 2.1.1.	Quality criteria (sterility, purity, freedom from extraneous agents)

395 It is crucial to ensure that all materials used in the preparation of the bulk antigens have been

396 extensively screened to ensure freedom from extraneous agents. This should include master and

397 working seeds, the cell cultures and all medium supplements such as bovine serum. Some bovine

398 viruses and particularly BVDV can readily infect small ruminants such as sheep. Therefore, it is

399 particularly important to ensure that any serum used that is of bovine origin is free of both adventitious

400 BVDV and antibodies against BVDV strains because low levels of either virus or antibody can mask

401 the presence of the other. Materials and vaccine seeds should be tested for sterility and freedom

402 from contamination with other agents, especially viruses as described in the chapter 1.1.8 and

403 Chapter 1.1.9 *Tests for sterility and freedom from contamination of biological materials intended for*

404 *veterinary use*.

405 If a vaccine passes basic tests, the efficacy of vaccination should ultimately be measured by the

406 capacity to prevent transplacental transmission. Effective challenge of vaccinated pregnant ewes at

407 50–60 days gestation has been achieved by intranasal installation of virus or by mixing with PI sheep

408 (Brun *et al.,*1993). Usually this reliably produces persistently viraemic offspring in non-immune ewes.

409 In regions where multiple genotypes of BDV viruses are commonly encountered, efficacy in protecting

410 against multiple strains should be measured.

##### 411 2.2.	Method of manufacture

###### 412 2.2.1.	Procedure

413 Inactivated vaccines have been prepared using conventional laboratory techniques with stationary or

414 rolled cell cultures. Inactivants have included formalin and beta-propriolactone. Adjuvants have

415 included aluminium hydroxide and oil (Brun *et al*., 1993; Vantsis *et al*., 1980). Optimal yields depend

416 on the cell type and isolate used. A commercial BDV vaccine containing two strains of virus has been

417 prepared on ovine cell lines (Brun *et al*., 1993). Cells must be produced according to a seed-lot

418 system from a master cell seed (MCS) that has been shown to be free from all contaminating

419 microorganisms. Vaccine should only be produced in cells fewer than 20 passages from the MCS.

420 Control cells from every passage should be checked for pestivirus contamination. Standard

421 procedures may be used, with the expectation for harvesting noncytopathic virus on days 4–7 after

422 inoculation of cultures. The optimal yield of infectious virus will depend on several factors, including

423 the cell culture, isolate used and the initial seeding rate of virus. These factors should be taken into

424 consideration and virus replication kinetics investigated to establish the optimal conditions for large-

425 scale virus production. Whether a live or inactivated vaccine, the essential aim will be to produce a

426 high-titred virus stock. This bulk antigen preparation can subsequently be prepared according to the

427 type of vaccine being considered.

###### 428 2.2.2.	Requirements for ingredients

429 BDV vaccines have usually been grown in cell cultures of ovine origin that are frequently

430 supplemented with medium components of animal origin. The material of greatest concern is bovine

431 serum due to the potential for contamination with BVD viruses and antibodies to these viruses. These

432 adventitious contaminants not only affect the efficiency of production but also may mask the presence

433 of low levels of infectious BVDV that may have undesirable characteristics. In addition to the virus

434 seeds, all materials should be tested for sterility and freedom from contamination with other agents,

435 especially viruses as described in chapters 1.1.8 and 1.1.9. Furthermore, materials of bovine or ovine

436 origin should originate from a country with negligible risk for transmissible spongiform

437 encephalopathies (see chapter 1.1.9).

###### 438 2.2.3.	In-process controls

439 In-process controls are part of the manufacturing process. Cultures should be inspected regularly to

440 ensure that they remain free from gross bacterial contamination, and to monitor the health of the cells

441 and the development or absence of CPE, as appropriate. While the basic requirement for efficacy is

442 the capacity to induce an acceptable neutralising antibody response, during production, target

443 concentrations of antigen required to achieve an acceptable response may be monitored indirectly

444 by assessment of the quantity of infectious virus or antigen mass that is produced. Rapid diagnostic

445 assays such as the ELISA are useful for monitoring BVDV antigen production. Alternatively, the

446 quality of a batch of antigen may be determined by titration of the quantity of infectious virus present,

447 although this may underestimate the quantity of antigen. For inactivated vaccines, infectivity is

448 evaluated before inactivation. For inactivated vaccines the inactivation kinetics should be established

449 so that a suitable safety margin can be determined and incorporated into the routine production

450 processes. At the end of production, *in-vitro*cell culture assays should be undertaken to confirm that

451 inactivation has been complete. These innocuity tests should include a sufficient number of passages

452 and volume of inoculum to ensure that very low levels of infectious virus would be detected if present.

###### 453 2.2.4.	Final product batch tests

454 i)  Sterility

455 Tests for sterility and freedom from contamination of biological materials intended for veterinary use

456 may be found in chapter 1.1.9.

457 ii) Identity

458 Identity tests should demonstrate that no other strain of BDV is present when several strains are

459 propagated in a facility producing multivalent vaccines.

460 iii) Safety

461 Samples from inactivated vaccines should be tested rigorously for viable virus. Samples of the

462 product should be passaged for a minimum of three passages in sensitive cell cultures to ensure

463 absence of live BDV. This *in-vitro*monitoring can be augmented by injecting two BDV-seronegative

464 sheep with 20 doses of unformulated antigen as part of a standard safety test. Presence of live virus

465 will result in the development of a more convincing serological response than will occur with

466 inactivated virus alone. The sheep sera can also be examined for antibody to other prescribed agents.

467 Safety tests shall also consist of detecting any abnormal local or systemic adverse reactions to the

468 vaccine by all vaccination route(s). Batch-to-batch safety tests are required unless safety of the

469 product is demonstrated and approved in the registration dossier and production is consistent with

470 that described in chapter 1.1.8. Vaccines must either be demonstrated to be safe in pregnant sheep

471 (i.e. no transmission to the fetus), or should be licensed with a warning not to use them in pregnant

472 animals.

473 iv) Batch potency

474 Vaccine potency is best tested in seronegative sheep in which the development and level of antibody

475 is measured. BVD vaccines must be demonstrated to produce adequate immune responses when

476 used in their final formulation according to the manufacturer’s published instructions. The minimum

477 quantity of infectious virus or antigen required to produce an acceptable immune response should be

478 determined. An indirect measure of potency is given by the level of virus infectivity prior to inactivation.

479 *In-vitro*assays should be used to monitor individual batches during production. The antigen content

480 following inactivation can be assayed by MAb-capture ELISA and related to the results of established

481 *in-vivo*potency results. It should be demonstrated that the lowest recommended dose of vaccine can

482 prevent transplacental transmission of BDV in pregnant sheep.

##### 483 2.3.	Requirements for authorisation/registration/licensing

###### 484 2.3.1.	Manufacturing process

485 For registration of a vaccine, all relevant details concerning manufacture of the vaccine and quality

486 control testing should be submitted to the relevant authorities. Unless otherwise specified by the

487 authorities, information should be provided from three consecutive vaccine batches with a volume

488 not less than 1/3 of the typical industrial batch volume.

489 There is no standard method for the manufacture of a BDV vaccine, but conventional laboratory

490 techniques with stationary, rolled or suspension (micro-carriers) cell cultures may be used.

491 Inactivated vaccines can be prepared by conventional methods, such as binary ethylenimine, formalin

492 or beta-propiolactone inactivation (Park & Bolin, 1987). A variety of adjuvants may be used.

###### 493 2.3.2.	Safety requirements

494 *In-vivo*tests should be undertaken using repeat doses (taking into account the maximum number of

495 doses for primary vaccination and, if appropriate, the first revaccination/booster vaccination) and

496 contain the maximum permitted antigen load and, depending on the formulation of the vaccine, the

497 maximum number of vaccine strains.

498 i)  Target and non-target animal safety

499 The safety of the final product formulation of inactivated vaccines should be assessed in susceptible

500 young sheep that are free of maternally derived antibodies and in pregnant ewes. They should be

501 checked for any local reactions following administration, and, in pregnant ewes, for any effects on

502 the unborn lamb.

503 ii) Reversion-to-virulence for attenuated/live vaccines and environmental considerations

504 In the event that a live virus vaccine was developed for BDV, virus seeds that have been passaged

505 at least up to and preferably beyond the passage limit specified for the seed should be inoculated

506 into young lambs to confirm that there is no evidence of disease. If a live attenuated vaccine has

507 been registered for use in pregnant animals, reversion to virulence tests should also include pregnant

508 animals. Live attenuated vaccines should not be transmissible to unvaccinated ‘in-contact’ animals.

509 iii) Precautions (hazards)

510 BDV is not considered to be a human health hazard. Standard good microbiological practice should

511 be adequate for handling the virus in the laboratory. While the inactivated virus in a vaccine should

512 be identified as harmless for people administering the product, adjuvants included in the vaccine may

513 cause injury to people. Manufacturers should provide adequate warnings that medical advice should

514 be sought in the case of self-injection (including for adjuvants, oil-emulsion vaccine, preservatives,

515 etc.) with warnings included on the product label/leaflet so that the vaccinator is aware of any danger.

###### 516 2.3.3.	Efficacy requirements

517 The potency of the vaccine should be determined by inoculation into seronegative and virus negative

518 lambs, followed by monitoring of the antibody response. Antigen content can be assayed by infectivity

519 titration prior to inactivation and subsequently by ELISA and adjusted as required to a standard level

520 for the particular vaccine. Standardised assay protocols applicable to all vaccines do not exist. Live

521 vaccine batches may be assayed by infectivity titration. Each production batch of vaccine should

522 undergo potency and safety testing as batch release criteria. BVD vaccines must be demonstrated

523 to produce adequate immune responses, as outlined above, when used in their final formulation

524 according to the manufacturer’s published instructions.

###### 525 2.3.4.	Vaccines permitting a DIVA strategy (detection of infection in vaccinated animals)

526 To date, there are no commercially available vaccines for BDV that support use of a true DIVA

527 strategy.

###### Duration of immunity
528

**2.3.5.**

529

530

531

532

533

534

535

536

**2.3.6.**

537

538

539

540

541

542

543

Inactivated vaccines are unlikely to provide sustained levels of immunity and it is likely that after an initial course of two or three injections annual booster doses may be required. Insufficient information is available to determine any correlation between vaccinal antibody titres in the dam and fetal protection. As there are likely to be different commercial formulations and these involve a range of adjuvants, there are likely to be different periods of efficacy. Consequently, duration of immunity data must be generated separately for each commercially available product by undertaking challenge tests at the end of the period for which immunity has been claimed.

###### Stability

There are no accepted guidelines for the stability of BDV vaccines, but it can be assumed that an inactivated virus vaccine should remain potent for at least 1 year if kept at 4°C and probably longer. Lower temperatures could prolong shelf life but adjuvants in a killed vaccine may preclude this. Bulk antigens that have not been formulated into finished vaccine can be reliably stored frozen at low temperatures, but the antigen quality should be monitored with *in-vitro*assays prior to incorporation into a batch of vaccine.

### REFERENCES

544 BARLOW R.M. & PATTERSON D.S.P. (1982). Border disease of sheep: a virus-induced teratogenic disorder. *Adv. Vet. Med.*

545 (*Suppl. J. Vet. Med.*), **36**, 1–87.

546 BECHER P., AVALOS-RAMIREZ R., ORLICH M., CEDILLO ROSALES S., KONIG, M., SCHWEIZER M., STALDER H., SCHIRRMEIR H & THIEL

547 H.-J. (2003). Genetic and antigenic characterisation of novel pestivirus genotypes; Implications for classification. *Virology,*

548 **311**, 96–104.

549 BECHER P., ORLICH M. & THIEL H.-J. (1998). Complete genomic sequence of border disease virus a pestivirus from sheep.

550 *J. Virol*., **72**, 5165–5173.

551 BRAUN U., HILBE M., EHRENSPERGER F., SALIS F., ALTHER P., STRASSER M., STALDER H.P. & PETERHANS E. (2002). Border

552 Disease in einem Schafbetrieb. *Schweiz. Arch. Tierheilk*., **144**, 419–426.

553 BRUN A., LACOSTE F., REYNAUD G., KATO F. & SAINT-MARC B. (1993). Evaluation of the potency of an inactivated vaccine

554 against border disease pestivirus infection in sheep. *In:*Proceedings of the Second Symposium on Pestiviruses, Edwards

555 S., ed. Fondation Marcel Merieux, Annecy, France, 1–3 October 1992, 257–259

556 CARLSSON U. (1991). Border disease in sheep caused by transmission of virus from cattle persistently infected with bovine

557 virus diarrhoea virus. *Vet. Rec*., **128**, 145–147.

558 CHAPPUIS G., BRUN A., KATO F., DAUVERGNE M., REYNAUD G. & DURET C. (1986). Etudes serologiques et immunologiques

559 realisees a la suite de I’isolement d’un pestivirus dans un foyer ovina chez des moutons de L’Aveyron. *In:*Pestiviroses des

560 Ovins et des Bovins, Espinasse J. & Savey M. eds. Ste Françoise de Buiatrie, Paris, France, **55**, 66.

561 DECARO N., MARI V., LUCENTE M., SCIARRETTA R., MORENO A., ARMENISE C., LOSURDO M., CAMERO M., LORUSSO E., CORDIOLI

562 P., & BUONAVOGLIA C. (2012). Experimental infection of cattle, sheep and pigs with ‘Hobi’-like pestivirus. *Vet. Microbiol*.,

563 **155**, 165–171.

564 DEKKER A., WENSVOORT G. & TERPSTRA C. (1995). Six antigenic groups within the genus pestivirus as identified by cross-

565 neutralisation assays. *Vet. Microbiol*., **47**, 317–329.

566 ENTRICAN G., DAND A. & NETTLETON P.F. (1994). A double monoclonal-antibody ELISA for detecting pestivirus antigen in

567 the blood of viraemic cattle and sheep. *Vet. Microbiol*., **43**, 65–74.

568 FENTON A., SINCLAIR J.A., ENTRICAN G., HERRING J.A. & NETTLETON P.F. (1991). A monoclonal antibody capture ELISA to

569 detect antibody to border disease virus in sheep sera. *Vet. Microbiol*., **28**, 327–333.

570 ~~INTERNATIONAL	COMMITTEE	ON	TAXONOMY	OF	VIRUSES	(2016).	Virus	Taxonomy	2015	release.~~

571 ~~http://www.ictvonline.org/virusTaxonomy.asp~~

572 NETTLETON P.F., GILMOUR J.S., HERRING J.A. & SINCLAIR J.A. (1992). The production and survival of lambs persistently

573 infected with border disease virus. *Comp. Immunol. Microbiol. infect. Dis*., **15**, 179–188.

574 NETTLETON P.F., GILRAY J.A., RUSSO P. & DLISSI E. (1998). Border disease of sheep and goats *Vet. Res.,***29**, 327–340.

575 OGUZOGLU T.C., FLOEGEL-NIESMANN G., FREY H.R. & MOENNIG V. (2001). Differential diagnosis of classical swine fever and

576 border disease: seroepidemiological investigation of a pestivirus infection on a mixed sheep and swine farm. *Dtsch*

577 *Tierarztl. Wochenschr.*, **108**, 210–213.

578 PARK B.K. & BOLIN S.R. (1987). Molecular changes of bovine viral diarrhea virus polypeptides treated with binary

579 ethylenimine, beta-propiolactone and formalin. *Res. Rep. Rural Dev. Admin.*(*L&V*) *Korea*, **29**, 99–103.

580 PATON D.J., SANDS J.J., LOWINGS J.P., SMITH J.E., IBATA G. & EDWARDS S. (1995). A proposed division of the pestivirus genus

581 into subgroups using monoclonal antibodies, supported by cross-neutralization assays and genetic sequencing. *Vet. Res*.,

582 **26**, 92–109.

583 RIDPATH J.F. & BOLIN S.R. (1997). Comparison of the complete genomic sequence of the border disease virus, BD31, to

584 other pestiviruses. *Virus Res.,***50**, 237–243.

585 THABTI F., FRONZAROLI L., DLISSI E., GUIBERT J.M., HAMMAMI S., PEPIN M. & RUSSO P. (2002). Experimental model of border

586 disease virus infection in lambs: comparative pathogenicity of pestiviruses isolated in France and Tunisia. *Vet. Res.,***33**,

587 35–45.

588 THUR B., HILBE M., STRASSER M. & EHRENSPERGER F. (1997). Immunohistochemical diagnosis of pestivirus infection

589 associated with bovine and ovine abortion and perinatal death. *Am. J. Vet. Res.,***58**, 1371–1375.

590 VALDAZO-GONZALEZ B., ALVAREZ-MARTINEZ M. & SANDVIK T. (2007). Genetic and antigenic typing of border Disease virus

591 isolates in sheep from the Iberian peninsula. *Vet. J*., **174**, 316–324.

592 VAN RIJN P.A., VAN GENNIP H.G.P., LEENCLERSE C.H., BRUSCHKE C.J.M., PATON D.J., MOORMANN R.J.M. & VAN OIRSCHOT J.T.

593 (1997). Subdivision of the pestivirus genus based on envelope glycoprotein E2 *Virology*, **237**, 337–348.

594 VANTSIS J.T., BARLOW R.M., FRASER J. & MOULD D.L. (1976). Experiments in border disease VIII. Propagation and properties

595 of a cytopathic virus. *J. Comp. Pathol*., **86**, 111–120.

596 VANTSIS J.T., RENNIE J.C., GARDINER A.C., WELLS P.W., BARLOW R.M. & MARTIN W.B. (1980). Immunisation against Border

597 disease. *J. Comp. Path*., **90**, 349–354.

598 VILCEK S. & NETTLETON P.F. (2006). Pestiviruses in wild animals *Vet. Microbiol.,***116**, 1–12.

599 VILCEK S., NETTLETON P.F., PATON D.J. & BELAK S. (1997). Molecular characterization of ovine pestiviruses. *J. Gen. Virol.,*

600 **78**, 725–735.

601 WENSVOORT G. & TERPSTRA C. (1988). Bovine viral diarrhoea virus infection in piglets born to sows vaccinated against

602 swine fever with contaminated virus. *Res. Vet. Sci.,***45**, 143–148.

603 WENSVOORT G., TERPSTRA C. & DE KLUYVER E.P. (1989). Characterisation of porcine and some ruminant pestiviruses by

604 cross-neutralisation. *Vet. Microbiol.*, **20**, 291–306.

605 WILLOUGHBY K., VALDAZO-GONZALEZ, B., MALEY M., GILRAY J. & NETTLETON P.F. (2006). Development of a real time RT-PCR

606 to detect and type ovine pestiviruses. *J. Virol. Methods,***132,**187–194*.*

607	*

608	* *

609	**NB:**At the time of publication (2017) there were no WOAH Reference Laboratories

610	for border disease (please consult the WOAH Web site:

611	https://www.woah.org/en/what-we-offer/expertise-network/reference-laboratories/#ui-id-3).

612	**NB**: FIRST ADOPTED IN 1996. MOST RECENT UPDATES ADOPTED IN 2017.