3.01.03 Bluetongue

C H A P T E R 3.1 . 3.
B LU E TO N G U E
( I N F E C T I O N W I T H B LU E TO N G U E V I R U S )
SUMMARY
Bluetongue (BT) is an infectious, primarily vector-borne viral disease that affects wild and domestic
ruminants such as sheep, goats, cattle, buffaloes, deer and most species of African antelope, and
camelids as vertebrate hosts. The virus is primarily transmitted between susceptible vertebrate hosts
by competent species of Culicoides midges, and the global distribution of the disease is largely
determined by the distribution of competent vectors. Infection with bluetongue virus (BTV) is
inapparent in the vast majority of animals but can cause fatal disease in a proportion of infected
sheep, deer and wild ruminants. Infection of cattle is usually subclinical, with the exception of BTV-8
infection. Cattle are particularly significant in the epidemiology of the disease due to the prolonged
viraemia that occurs following infection. When apparent, clinical signs of BT are mainly attributable
to an increase in vascular permeability and include fever, hyperaemia and congestion, facial oedema
and haemorrhages, erosion of the mucous membranes, coronitis and laminitis, and pleural and
pericardial haemorrhages.
Detection of the agent: BTV is the type member of the Orbivirus genus of the family Reoviridae.
There are 27 recognised BTV serotypes and several recently isolated but as yet unclassified unique
‘atypical’ strains. Virus serotype identification traditionally requires isolation and amplification of the
virus in embryonated chicken eggs, Culicoides cells or other tissue culture and the subsequent
application of serogroup- and serotype-specific tests including virus neutralisation assays. The
reverse-transcription polymerase chain reaction (RT-PCR) assay has permitted rapid amplification of
BTV cDNA in clinical samples and well validated real-time RT-PCR-based systems for virus detection
are now routine, allowing for more rapid and sensitive diagnostic testing. The combination of RT-PCR
amplification and capillary sequencing or whole genome sequencing now offer relatively rapid
unequivocal agent identification at the genome level. Collectively, these procedures augment the
classical virological techniques to provide information on virus serogroup, serotype and topotype.
Serological tests: Serological responses appear 7–14 days after BTV infection. Infected animals
produce both neutralising and non-neutralising anti-BTV antibodies that are generally long-lasting.
Enzyme-linked immunosorbent assay (ELISA) and virus neutralisation (VN) are the most frequently
used serological tests. A monoclonal antibody-based competitive ELISA to specifically detect anti-
BTV (serogroup-specific) antibodies is the test recommended to certify animals as free from infection
prior to movement; the test is highly sensitive and specific (compared with the agar gel
immunodiffusion), quick, inexpensive and reliable. However, the ELISA may have reduced sensitivity
for some serotypes. Procedures to identify and quantify BTV serotype-specific antibodies are more
complex, being typically based on cell culture- and live virus-dependent neutralisation tests.
Requirements for vaccines: Vaccination is the preferred method of BT control in endemic regions.
Vaccination has been used successfully to limit direct losses, minimise the circulation of BTV,
eradicate BTV from country/regions and allow safe movement of animals. However, use of live
vaccines can be associated with adverse outcomes and live attenuated vaccine strains can be spread
by vectors, with eventual reversion to virulence or reassortment of vaccine virus genes with those of
wild-type virus strains. Inactivated vaccines are safer, but require multiple doses to become
efficacious and incur higher costs. Such vaccines have been very effective in combatting the spread
of BTV-8, BTV-1 and BTV-4 in Europe.
WOAH Terrestrial Manual 2021 1

Chapter 3.1.3. – Bluetongue (infection with bluetongue virus)
A. INTRODUCTION
1. Description of the disease
Bluetongue (BT) is an infectious, primarily vector-borne viral disease that affects wild and domestic ruminants.
Midges of an increasing number and geographical range of species (Carpenter et al., 2015) in the genus Culicoides
(the insect host) typically transmit bluetongue virus (BTV) among susceptible ruminants, having become infected
by feeding on viraemic animals (the vertebrate host). Other routes of transmission have been documented including
direct vertical, oral and possibly venereal transmission and indirect transmission through reused needles; however
the epidemiological significance of these routes remains uncertain (Belbis et al., 2017; Darpel et al., 2016; Kirkland
et al., 2004). The more recently recognised serotypes, such as BTV-25, BTV-26 and BTV-27 appear to be transmitted
exclusively by these vector-independent routes, and may result in persistent infection in goats (Belbis et al., 2017;
MacLachlan et al., 2015; Vogtlin et al., 2013).
The global distribution of BTV is determined by epidemiological systems (episystems) that are in turn primarily
delimited by specific vector species, the presence of susceptible host species and their collective natural history.
Observations in Europe, India and the USA indicate that strains of BTV can move between episystems through
movement of animals, wind dispersion of infected vectors and by adaptations of both vector species and virus
(Carpenter et al., 2015; Jacquot et al., 2019; Maan et al., 2015). The expanding global range of BTV infection has been
notable, particularly at the traditional northern- and southern-most limits of BTV distribution with incursions of
multiple serotypes into Europe, Australia, both North and South America, and Asia, in countries that had not
previously reported BTV infections (MacLachlan et al., 2015; MacLachlan & Mayo, 2013). In addition to detecting and
confirming an increasing number of serotypes, developing sequencing technologies, phenotyping software and
molecular assays have revealed that two major ancestral lineages, a Western (Africa, Europe, the Americas) and an
Eastern (Australia, and Asia) exist globally (Maan et al., 2015; Mertens et al., 2007).
In temperate parts of the world, infection has a seasonal occurrence (Verwoerd & Erasmus, 2004), whereas in
tropical regions BTV infection can occur year-round (MacLachlan et al., 2015). Survival of BTV in the environment is
associated with insect factors such as over-wintering (Mayo et al., 2016). Additionally, long-term persistence of BTV
in a region only occurs when multiple virus serotypes circulate (MacLachlan et al., 2015). The ability of the more
recently detected BTV serotypes 25, 26 and 27 to be transmitted in the absence of vector insects reduces the
likelihood of seasonality in their occurrence or dependence on the geographical distribution of vector species for
transmission.
The vertebrate hosts for BTV include both domestic and wild ruminants, such as sheep, goats, cattle, buffaloes,
deer, most species of African antelope and other Artiodactyla such as camels. Although detection of antibodies to
BTV antigen or viral nucleic acid or live virus has been demonstrated in some carnivores, black and white
rhinoceroses and elephants, the role of non-ruminant species in BTV epidemiology is considered minimal. The
outcome of infection ranges from subclinical in the vast majority of infected animals, especially wild African
ruminants, cattle and goats, to serious or fatal in a proportion of infected sheep, goats, deer and some wild
ruminants (Verwoerd & Erasmus, 2004). Infection in cattle and goats is typically subclinical and these species are
considered to be amplifying reservoir hosts in endemic regions, making control measures for BTV in those animals
important. However, a higher incidence and severity of clinical disease has been observed in naïve cattle infected
with BTV-8. As breeds of sheep have varying levels of susceptibility to disease, BTV infections of livestock can occur
unobserved and be detected only by active surveillance (Daniels et al., 2004).
Clinical signs of disease in sheep vary markedly in severity, influenced by the type or strain of the infecting virus,
husbandry factors and animal breed (Verwoerd & Erasmus, 2004). In severe cases there is an acute febrile response
characterised by hyperaemia and congestion, leading to oedema of the face, eyelids and ears, and haemorrhages
and erosions of the mucous membranes. The tongue may show intense hyperaemia and become oedematous,
protrude from the mouth and in severe cases become cyanotic. Hyperaemia may extend to other parts of the body
particularly the coronary band of the hoof, the groin, axilla and perineum (MacLachlan & Mayo, 2013). Sheep that
develop chronic disease often have severe muscle degeneration, and breaks in the wool may occur associated with
pathology in the follicles. A reluctance to move is common and torticollis may occur in severe cases (Maclachlan et
al., 2009). In fatal cases, the lungs may show interalveolar hyperaemia, severe alveolar oedema and the bronchial
tree may be filled with froth. The thoracic cavity and pericardial sac may contain varying quantities of plasma-like
fluid. Most cases show a distinctive haemorrhage near the base of the pulmonary artery (Verwoerd & Erasmus,
2004). In cases of vertical transmission of BTV-8, the central nervous system may be severely affected resulting in
a failure of the cerebral hemispheres to develop (dummy calf syndrome). With virulent strains, infection in naïve
sheep can result in mortality rates as high as 70%.
2 WOAH Terrestrial Manual 2021

Control of BTV in animals is covered in Chapter 8.3 of the WOAH Terrestrial Animal Health Code. Because of the
high percentage of subclinical infections, persistent viraemia, and challenges with vector control, traditional
methods of control and eradication of BTV, such as movement controls, stamping out and vector control have not
always been successful in controlling the disease. Thus, a safe and efficacious vaccine that meets the objectives of
control, eradication and prevention requirements is an important component of disease control in many settings
(MacLachlan & Mayo, 2013).
2. Nature and classification of the pathogen
Taxonomically, BTV is classified as the type species in the Orbivirus genus in the family Reoviridae, one of
22 recognised species in the genus that also includes epizootic haemorrhagic disease virus (EHDV), equine
encephalosis virus and African horse sickness virus. There is significant immunological cross-reactivity among
members of the BTV serogroup. Within species, individual BTV are differentiated on the basis of genotype and
neutralisation tests; currently 27 serotypes of BTV are recognised including Toggenburg Orbivirus (BTV-25), BTV-
26 from Kuwait and BTV-27 from Corsica. These most recently detected serotypes and several other as yet
serotypically unclassified unique BTV strains (Belbis et al., 2017) may exhibit quite distinct modes of transmission
associated with their respective abilities to infect Culicoides spp. and specific ruminant hosts (Breard et al., 2018,
Chaignat et al., 2009, Maan et al., 2015; Savini, 2015). Both genetic shift and genetic drift account for the diversity
and heterogenicity of the BTV strains in the field (Pritchard et al., 2004).
BTV particles are composed of three protein layers. The outer capsid layer contains two proteins, VP2 and VP5. VP2
is the major neutralising antigen and determinant of serotype specificity. Removal of the outer VP2/VP5 layer leaves
a bi-layered icosahedral core particle that comprises an outer layer composed entirely of capsomeres of VP7 and a
complete inner capsid shell (the subcore layer) comprising VP3, which surrounds 10 dsRNA genome segments and
minor structural proteins (VP1, VP4 and VP6). VP7 is a major determinant of serogroup specificity and the site of
epitopes used in competitive enzyme-linked immunosorbent assays (C-ELISAs) to detect anti-BTV antibodies
(Mertens et al., 2005). VP7 can also mediate attachment of BTV to insect cells.
Genetic sequencing of specific BTV genome segments and movement towards full genome analyses allows for
additional differentiation and analysis of strains apart from serotyping, with potential to identify multiple different
clades for each genome segment 1 (Gould, 1987; Jacquot et al., 2019). Even for strains within one serotype it is
therefore possible to identify the likely geographical origin (topotype) for each genome segment (Gould, 1987).
Identification of apparent associations between some genotypes of virus and some vector species has led to further
development of the concept of viral-vector episystems (Daniels et al., 2004). Movements of several BTV serotypes
between vector species and into new geographical regions, leading to multiple reassortment events and the
emergence of novel strains containing new combinations of genome segments from different origins (Jacquot et
al., 2019; Nomikou et al., 2015), indicate that a more complete identification of the genetic makeup of each strain
would contribute to a better understanding of BTV epidemiology.
3. Zoonotic potential and biosafety and biosecurity requirements
There is no known risk of human infection with BTV. Biocontainment measures should be determined by risk
analysis as described in Chapter 1.1.4 Biosafety and biosecurity: Standard for managing biological risk in the
veterinary laboratory and animal facilities.
4. Differential diagnosis
Depending on the clinical presentation, affected species and epidemiological data, differential diagnoses for
suspected cases of BTV infection may include other infectious diseases including but not limited to epizootic
haemorrhagic disease, foot and mouth disease, peste des petits ruminants, sheep and goat pox, vesicular stomatitis
and contagious ecthyma. In cases of dummy calf syndrome, bovine viral diarrhoea virus should be taken into
consideration. Non-infectious causes, including photosensitisation, should also be considered.
1 See BTV-Glue database http://btv-glue.cvr.gla.ac.uk

B. DIAGNOSTIC TECHNIQUES
Table 1. Test methods available for the diagnosis of bluetongue and their purpose
Purpose
Method Individual animal Immune status in

Population Contribute to Confirmation Prevalence of
freedom from individual animals
freedom from eradication of clinical infection –
infection prior to or populations
infection policies cases surveillance
movement post-vaccination
Detection of the agent(a)
Real-time
– +++ – +++ ++ –
RT-PCR
RT-PCR – +++ – +++ ++ –
Classical virus
– +++ – +++ – –
isolation
Detection of immune response
C-ELISA
(serogroup +++ +++ ++ – +++ ++
specific)
VN
(serotype + + ++ + ++ ++
specific)
AGID + + + – + +
Key: +++ = recommended for this purpose; ++ recommended but has limitations;
+ = suitable in very limited circumstances; – = not appropriate for this purpose.
RT-PCR = reverse-transcription polymerase chain reaction; C-ELISA = competitive enzyme-linked immunosorbent assay;
VN = virus neutralisation; AGID = agar gel immunodiffusion
(a)
A combination of agent identification methods applied on the same clinical sample is recommended.
1. Detection of the BTV agent
1.1. In-vitro and in-vivo cultures
Specimens for virus isolation can include unclotted blood (heparin or EDTA [ethylene diamine tetra-
acetic acid]-treated whole blood) from suspected viraemic animals, blood clots after separation of
serum, spleen or lymph nodes collected at necropsy of clinical cases, or midges. Several virus isolation
systems for BTV are in common use, including inoculation of embryonated chicken eggs (ECE) and
primary inoculation of cell cultures such as the KC cell line (a cell line derived from C. sonorensis midges).
The KC cell line has been proven to be very sensitive (McHolland & Mecham, 2003) and from an animal
ethics perspective reduces the use of embryonated eggs. Attempts to isolate virus in vitro in mammalian
cell culture systems may be considered more convenient, however the success rate is frequently much
lower than that achieved with embryonated chicken egg and KC cell systems. The same diagnostic
procedures are used for domestic and wild ruminants. Inoculation of sheep continues to be used for in-
vivo testing and amplification of virus but should be avoided wherever possible in accordance with
chapter 7.8 of the Terrestrial Animal Health Code.
1.1.1. Isolation in embryonated chicken eggs

i) Blood is collected from suspected viraemic animals into an anticoagulant such as EDTA,
heparin or sodium citrate, and the blood cells are washed three times with sterile phosphate-
buffered saline (PBS). Washed cells are re-suspended in PBS or isotonic sodium chloride

and either stored at 4°C or used immediately for attempted virus isolation. Tissue and midge
suspension can be also prepared and stored as described above or immediately used.
ii) For long-term storage at 4°C or where refrigeration is not possible for short periods of time,
blood samples are collected in oxalate–phenol–glycerine. Washed red blood cells and tissue
homogenates can be stored directly at –70°C. The virus is not stable at –20°C. BTV has
remained viable for several months or even years in whole blood in anticoagulant stored at 4°C.
iii) In fatal cases, spleen and lymph nodes are the preferred organs for virus isolation attempts.
Organs and tissues should be kept and transported at 4°C to a laboratory where they are
homogenised in PBS or isotonic saline (1/10), centrifuged at 1500 rpm for 10 minutes, and
filtered (0.2–0.4 µm). The tissue suspensions can be used as described below for blood cells.
iv) Washed blood cells are re-suspended 1/1 in distilled water (to disrupt whole red blood cells)
then diluted to 1/10 in PBS to ensure isotonic balance. 0.1 ml amounts are inoculated
intravascularly into 5–12 ECE that are 9–12 days old. This procedure requires skill and
practice. Details are provided by Clavijo et al. (2000).
v) The eggs are incubated in a humid chamber at 32–33.5°C and candled daily. Any embryo
deaths within the first 24 hours post-inoculation are regarded as nonspecific.
vi) Embryos that die between days 2 and 7 and embryos remaining alive at 7 days are retained
at 4°C overnight before harvesting. Infected embryos may have a haemorrhagic appearance.
Dead embryos and those that live to 7 days are homogenised as two separate pools. Whole
embryos, after removal of their heads, or pooled organs such as the liver, heart, spleen, lungs
and kidney, are homogenised and the debris removed by centrifugation at 1500 g for
10 minutes.
vii) Virus in the supernatant may be identified either directly as described in Section B.1.2 below
or after further amplification in cell culture, as described in Section B.1.1.2.
1.1.2. Isolation in cell culture

Virus isolation is best achieved by primary isolation (or amplification) of virus in ECE, followed by
a passage in the Aedes albopictus (AA) clone C6/36 insect cell culture or primary isolation in cells
derived from Culicoides sonorensis (free of BT viruses) and designated as KC or CuVa cells
(McHolland & Mecham, 2003). These two amplification steps are then followed by a passage in
mammalian cell lines such as baby hamster kidney (BHK 21) or African green monkey kidney
(Vero). Passages in BHK 21 and Vero will enable further replication of virus and visual confirmation
of virus isolation by cytopathic effect (CPE).
There have been occasions where CPE is not seen in a mammalian cell line, but BTV antigen has
been visualised when staining by direct immunofluorescence. Cell monolayers are incubated at
37°C in 5% CO2 with humidity and monitored for the appearance of CPE after 5–7 days. If no CPE
appears, a second passage is made in the mammalian cell culture.
Positive CPE or immunofluorescence detection or negative cell culture results for BTV must be
confirmed after each ECE, KC or cell culture passage by antigen detection ELISA or polymerase
chain reaction (PCR) techniques.
1.2. Virus detection and characterisation
The success of virus isolation techniques is assessed by testing for the presence of BTV in the cell culture
supernatants or embryo tissues using a variety of detection systems. Virus detection systems include
antigen-capture ELISA, direct immunofluorescence, reverse-transcription PCR (RT-PCR) or real-time RT-
PCR, as described in Section B.1.3 below. Currently, testing of the isolation media by real-time RT-PCR is
the preferred screening method.
Detection and characterisation are typically step-wise processes, with serogroup-specific tests used
initially to detect the presence of a BTV. Subsequent genotype and serotype identification of BTV isolates
provides valuable epidemiological information and is critical for the implementation of vaccines or for
vaccine development. RT-PCR assays employing serotype-specific primers will provide the most rapid

and specific information regarding isolate serotype (Mertens et al., 2007). Genotyping for molecular
epidemiology can be based on RT-PCR tests and sequencing of the amplicon. Different laboratories have
standardised several different gene sequences for this purpose. Where available, full genome
sequencing may also be performed to provide serotype, as well as other unique sequence information of
isolates.
Neutralisation procedures using individual serotype antisera may also be employed for serotyping,
although some serotypes are cross-reactive and interpretation can be difficult. For laboratories without
serotyping capabilities, BTV isolates may be submitted to any WOAH BT Reference Laboratory for
serotyping of isolates.
1.2.2. Immunological serogrouping of viruses

Orbivirus isolates are typically serogrouped on the basis of their reactivity with specific standard
antisera that detect proteins, such as VP7, that are conserved within each serogroup. The cross-
reactivity between members of the BT and EHD serogroups raises the possibility that an isolate
of EHDV could be mistaken for BTV on the basis of a weak immunofluorescence reaction with a
polyclonal anti-BTV antiserum. Polyclonal and monoclonal antibodies (MAbs) used for
serogrouping BTV isolates must be characterised as appropriate for the purpose. There exists
significant VP7 variation within BTV, as well as antigenic relatedness between other closely
related orbiviruses, such as EHDV, that will influence antibodies binding in different assay formats
(IFA, ELISA, AGID). For this reason, a BT serogroup-specific MAb can be used. A number of
laboratories have generated such serogroup-specific reagents. Commonly used methods for the
identification of viruses to serogroup level are as follows.
i) Immunofluorescence/immunoperoxidase staining
Monolayers of BHK or Vero cells in various tissue culture substrates (including chamber
slides, glass cover-slips, 96 or 24 well plates or other suitable formats) are infected with
either tissue culture-adapted virus or virus in ECE lysates. After 24–48 hours at 37°C, or after
the appearance of a mild CPE, infected cells are fixed with agents such as paraformaldehyde,
acetone or methanol, dried and viral antigen detected using anti-BTV antiserum or BTV-
specific MAbs and standard immunofluorescent procedures. Standard immunoperoxidase
staining procedures can also be used when fluorescent microscopes are not available for
reading immunofluorescence, as stained antigen can be read by eye or using light
microscope at 100× magnification.
ii) Antigen capture enzyme-linked immunosorbent assay

Viral antigen in ECE, culture medium harvests and infected insects may be detected directly
using an antigen-capture ELISA (Ag ELISA). In this technique, virus derived proteins are
captured by antibody adsorbed to an ELISA plate and bound materials detected using a
second antibody. The capture antibody may be polyclonal or a serogroup-specific MAb.
Serogroup-specific MAbs and polyclonal antibody raised to VP7 proteins or whole virus
have been used successfully to detect captured virus. Antigen detection from whole blood
is not always successful using the Ag ELISA.
1.2.2. Serotyping of isolates by virus neutralisation

Neutralisation tests are type specific for the currently recognised BTV serotypes that have been
isolated in culture. Reference antibodies can be used to serotype a virus. In the case of an untyped
isolate, the characteristic regional localisation of BTV serotypes can generally obviate the need to
attempt neutralisation by antisera to all isolated serotypes, particularly when endemic serotypes
are well known.
There is a variety of tissue culture-based methods available to aid in typing isolates. Cell lines
commonly used are BHK, Vero and L929. Three methods to serotype BTV isolates are outlined
briefly below. For antibody typing virus neutralisation methods, see Section B.2. below. There is
also a fluorescence inhibition test, not described. BTV serotype-specific antisera generated in
guinea-pigs or rabbits have been reported to have less serotype cross-reactivity than those made
in cattle or sheep. It is important that antiserum controls be included.

i) Plaque reduction
The virus to be serotyped is diluted to contain approximately 100 plaque-forming units
(PFU) and incubated with either no antiserum (virus control) or with serial dilutions of
individual standard antisera to a panel of BTV serotypes. Virus/antiserum mixtures are
added to monolayers of cells. After adsorption and removal of inoculum, monolayers are
overlaid with agarose or carboxy-methyl cellulose (medium viscosity) for easy removal of
semi-solid agar before staining. The neutralising antibody titres are determined as the
reciprocal of the serum dilution that causes a fixed reduction (e.g. 80%) in the number of
PFU. The unidentified virus is considered serologically identical to a standard serotype if the
latter is run in parallel with the untyped virus in the test and is similarly neutralised.
ii) Plaque inhibition

Tests are performed in 90 mm diameter Petri dishes containing confluent cell monolayers
that are infected with approximately 5 × 104 PFU standard or untyped virus. After adsorption
and removal of inoculum, monolayers are overlaid with agarose. Standard anti-BTV antisera
are added to individual filter paper discs and placed on the agarose surface. Dishes are
incubated for at least 4 days. A zone of virus neutralisation, with concomitant survival of the
cell monolayer, will surround the disc containing the homologous antiserum.
iii) Microtitre neutralisation

Approximately 100 TCID50 (50% tissue culture infective dose) of the standard or untyped
virus is added in 50 µl volumes to test wells of a flat-bottomed microtitre plate and mixed
with an equal volume of standard antiserum serially diluted in tissue culture medium.
Approximately 104 cells are added per well in a volume of 100 µl, and plates incubated for up
to 7 days, depending on the level of CPE observed in negative serum wells of the test. When
CPE has developed to 4+ (100%) in the negative control serum wells of each serotype tested,
the test is read using an inverted microscope. Wells are scored for the degree of CPE
observed. Those wells that contain cells only or cells and antiserum, should show no CPE. In
contrast, wells containing cells and virus should show 75–100% CPE. The unidentified virus
is considered serologically identical to a standard BTV serotype if both are neutralised in the
test to a similar extent.
1.3. Molecular methods
1.3.1. Detection of nucleic acid

RT-PCR techniques provide rapid identification of BT viral nucleic acid in blood and other tissues
of infected animals. Importantly, RT-PCR-based diagnostics should be interpreted with caution
because the RT-PCR procedure will detect virus-specific nucleic acid after the virus is no longer
viable and capable of establishing a new infection in either insects or mammalian hosts. Hence a
positive RT-PCR result, does not necessarily indicate the presence of infectious virus. In addition,
RNA originating from vaccine strains can be detected.
Multiple RT-PCR formats are available that can be used to detect BTV specifically to ‘serogroup’
Orbiviruses and to ‘serotype’ BTV. These molecular approaches are much more rapid than
traditional virological and immunological approaches, which may require up to 4 weeks to
generate information on serogroup and serotype. Developments in molecular assays, new
sequencing technologies and phenotyping software have identified increasing numbers of new
serotypes, methods of transmission, and host-specific susceptibility patterns.
The nucleic acid sequence of cognate BTV genes may differ depending on the geographical area
of virus isolation (Gould, 1987; Maan et al., 2015). This has provided a unique opportunity to
complement studies of BTV epidemiology by providing information on the potential geographical
origin of virus isolates, a process termed genotyping or topotyping. While sequencing of BTV
isolates from different parts of the world may allow detection of various clades for each genome
segment, which may permit finer discrimination of geographical origin, the relationship between
sequence and geographical origin is not absolute in all cases. Thus, BTV sequencing information
is vitally important and all data regarding BTV segment sequences should be made widely
available by submitting appropriately validated data to reputable sequence databases such as

GenBank (https://www.ncbi.nlm.nih.gov/genbank/). The BTV-Glue website (http://btv-

glue.cvr.gla.ac.uk) can provide phylogenetic tree analyses of BTV isolates based on the sequence
of RNA segments. These compiled data will provide a resource for epidemiological studies, the
identification of new isolates, as well as supporting in-silico analyses for maintenance and
validation of existing RT-PCR assays and the design of new primers probes for development of
additional assays, e.g. for novel/variant BTV types/strains.
The capacity of RT-PCR assays to detect very small numbers of nucleic acid molecules means
that such tests are exquisitely sensitive to contamination by extraneous nucleic acids leading to
false positive results. False negatives, due for example to poor nucleic acid preparation or
inappropriate primers, may also be encountered. This is covered in detail in Chapter 1.1.6
Validation of diagnostic assays for infectious diseases of terrestrial animals.
There are many RT-PCR assays currently in use that use different extraction methods, reverse
transcriptases, amplification enzymes, primers and conditions. Technology is changing rapidly,
and the genetic diversity of the BTV genes makes the choice and validation of RT-PCR assays
conditional on its application in a regional setting. Therefore, the procedures listed below are
examples only.
Two BTV RT-PCR assays are presented here: a real-time assay (Hofmann et al., 2008), targeting
the NS3 gene segment and a conventional nested assay targeting the NS1 gene segment, using
primers designed by Katz et al. (1993). The nested assay has been successfully used for over
20 years and can detect serotypes 1–24 and 26 (there are no reports of testing of other serotypes
from multiple species. The nested assay may be beneficial for laboratories without the capacity
to perform real-time RT-PCR. The real-time RT-PCR assay presented below has been tested at
several laboratories world-wide and has been found capable of detecting all 27 serotypes of BTV
(as well as other recently detected novel BTV strains), and equals or surpasses the sensitivity of
the nested assay while providing rapid, quantitative detection of BTV without the contamination
risks associated with nested RT-PCR assays.
1.3.1.1. Real-time reverse-transcription polymerase chain reaction

Real-time RT-PCR methods provide sensitive and rapid detection of BTV in a one-step
procedure. Advantages of real-time methods over traditional PCR methods include rapidity
of testing, quantitation of the virus present, and the reduced opportunity for contamination
to occur as no post-amplification handling such as gel electrophoresis is needed. The real-
time RT-PCR assays are the tests of choice for diagnosis.
The method presented here is an adaption from Hofmann et al. (2008) and is capable of
detecting all known BTV serotypes and strains currently circulating. The assay targets BTV
segment 10 (NS3). The procedure given may require modification to accommodate
individual laboratory or different RT-PCR kit requirements.
i) RNA extraction from blood, tissue samples, and midges

Commercial kits are widely available; the RNA extraction step can be performed according
to the procedures specified in each kit.
ii) Real-time reverse-transcription polymerase chain reaction

Kits for the one-step real-time PCR are available commercially. Below are some basic steps
as described by Hofmann et al. (2008), which can be modified depending upon local/case-
specific requirements, kits used and equipment available.
Primer and probe sequences for the detection of BTV species viruses:
BTV_IVI_F 5’-TGG-AYA-AAG-CRA-TGT-CAA-A-3’
BTV_IVI_R 5’-ACR-TCA-TCA-CGA-AAC-GCT-TC-3’
BTV_IVI_P 5’FAM-ARG-CTG-CAT-TCG-CAT-CGT-ACG- C-3’ BHQ1

a) Primer stock solutions are diluted to a working concentration of 20 pmol/µl, whereas

probe is diluted to a working concentration of 5 pmol/µl.
b) A test plate layout should be designed and loaded into the real-time PCR machine
software. Using the layout as a guide, 0.5 µl of each primer working stock (20 pmol/µl)
is added to each well that will go on to contain RNA samples, positive or negative
controls. The plate is held on ice.
Note: PCR plates can be replaced with tubes or strips as appropriate.
c) 2 µl of RNA samples, including test and positive and negative controls, are added to
appropriate wells of the plate following the layout (note: these wells already contain
primers from step b).
d) Heat denaturation: 95°C for 5 minutes, hold on ice for further 3 minutes.
e) An appropriate volume of real-time one-step RT-PCR master mix for the number of
samples to be tested is prepared, following the manufacturer’s instructions. Probe
should be included in the master mix to give a final concentration of 0.2 pmol/µl per
sample.
f) 22 µl of master mix is distributed in each well on the PCR plate containing the
denatured primers and RNA.
g) The plate is placed in a real-time thermal cycler programmed for reverse transcription
and cDNA amplification/fluorescence detection as suggested by the manufacturers.
The following thermal profile is an example:
48°C 30 minutes
95°C 2 minutes
50 cycles: 95°C 15 seconds

56°C 30 seconds
72°C ×30 seconds
1.3.1.2. Reverse-transcription polymerase chain reaction – nested PCR
Select RT-PCR (reverse-transcription and first stage amplification) and PCR (nested
amplification) kits are available to perform the nested assay. The assay presented below for
illustration uses the parameters associated with a specific kit. The PCR parameters should
be adjusted according to the manufacturer’s recommendations for the specific kits to be
used.
The nested assay employs the use of the following primers:
First stage amplification (outer) (dilute to 25 pmol/µl; final concentration in PCR is 0.6 µM
each):
FW: GTT-CTC-TAG-TTG-GCA-ACC-ACC
RV: AGG-CCA-GAC-TGT-TTC-CCG-AT
Nested amplification (dilute to 25 pmol/µl; final concentration in PCR is 0.5 µM each):
nFW: GCA-GCA-TTT-TGA-GAG-AGC-GA
nRV: CCC-GAT-CAT-ACA-TTG-CTT-CCT
i) Prepare the first stage amplification mixture (one-step RT-PCR kit) of the following reagents
(per sample):
Nuclease-free water 11.8 µl
5× one-step RT-PCR buffer 5.0 µl
dNTP mix 1.0 µl
Enzyme 1.0 µl
FW primer (25 pmol/µl 0.6 µl
RV primer (25 pmol/µl) 0.6 µl

ii) Dispense 20 µl of the mixture into each PCR tube included in the assay. Add 5 µl of sample
or control denatured RNA (described above) to the appropriate tube. Place tubes in a
thermal cycler and run the following programme:
Reverse transcription 50°C 30 minutes
Taq activation 95°C 15 minutes
Followed by 35 cycles of:
Denature: 94°C 45 seconds
Anneal: 58°C 45 seconds
Extension 72°C 60 seconds (final extension 10 minutes)
iii) Prepare a nested PCR mixture (HotStarTaq DNA Polymerase kit) of the following reagents
(per sample):
Nuclease-free water 40.75 µl
10×HotStar buffer 5.0 µl
dNTP mix 1.0 µl
HotStarTaq 0.25 µl
nFW primer (25 pmol/µl) 1.0 µl
nRV primer (25 pmol/µl) 1.0 µl
iv) Dispense 49 µl of the mixture into each PCR tube. Transfer 1.0 µl of the amplified DNA from
the first stage reaction (step 2) to the appropriate nested tube. Change gloves between
samples and use caution when transferring the DNA to avoid cross contamination of
samples. Place tubes in a thermal cycler and run the following programme:
Taq activation 95°C 15 minutes
Followed by 28 cycles of:
Denature: 94°C 45 seconds
Anneal: 62°C 45 seconds
Extension: 72°C 1 minute (final extension 10 minutes)
Perform gel electrophoresis followed by modern gel visualisation methods on the nested
PCR product. The positive control(s) and any positive samples will have a 101 base pair band.
Negative controls and negative samples should not have a visible band. Positive samples
may be sequenced for verification.
1.3.2. Nucleic acid sequencing

Although PCR-based methods can provide a rapid prospective determination of the serotype and
genotype of a BTV isolate, the nucleic sequence of specific BTV genome segments is required for
ultimate unequivocal identification. Through the exploitation of information provided by real-time
RT-PCR or serology, and the now extensive availability of BTV sequence databases that better
inform primer design, the combination of RT-PCR amplification and high throughput capillary
sequencing can offer a relatively rapid, confirmative diagnostic approach. The now more
economically viable option of whole genome sequencing (WGS) is also being increasingly
routinely applied to BTV diagnosis (Belbis et al., 2017). However, the concentration of BTV in field
isolations can provide limitations to the effectiveness of these sequencing approaches and prior
establishment of growth in cell culture may be required for some isolates. Care should be taken
concerning interpretation of results obtained with tissue culture grown virus samples, as the
process of adaptation to cell culture inevitably involves selection of those viruses that can infect
and replicate in the culture system used. The sequences generated may not therefore fully
represent the virus population present in the original diagnostic sample.
2. Serological tests
Serological responses appear 7–14 days after BTV infection. Infected animals produce both neutralising and non-
neutralising anti-BTV antibodies that are generally long-lasting. Anti-BTV antibodies generated in infected animals
can be detected in a variety of ways that vary in sensitivity and specificity. Both serogroup-specific and serotype-
specific antibodies are elicited and if the animal was not previously exposed to BTV, the neutralising antibodies
generated are specific for the serotype of the infecting virus. Multiple infections with different BTV serotypes lead
to the production of antibodies capable of neutralising serotypes to which the animal has not been exposed.

2.1. Competitive enzyme-linked immunosorbent assay
The BT competitive or blocking ELISA (C-ELISA) was developed to measure BTV-specific antibody
without detecting cross-reacting antibody to other Orbiviruses (Afshar et al., 1989; Lunt et al., 1988). The
specificity is the result of using BT serogroup-reactive MAbs. These antibodies were derived in separate
laboratories, and although possessing different properties or epitope specificities, all appear to bind to
the amino-terminal region of the major core protein VP7. In the C-ELISA, antibodies in test sera compete
with the MAbs for binding to antigen. The following procedure for the C-ELISA has been standardised
after comparative studies in a number of international laboratories. Importantly, monoclonal antibody-
based competitive ELISA formats have been known to not detect all serotypes of BTV (particularly some
BTV-15). Use of such ELISAs therefore requires an understanding of the test’s fitness for purpose, and the
assay must be validated accordingly to avoid missing detection of some BTV serotypes, including the
novel serotypes recently identified.
2.1.1. Test procedure

There are several test procedures described; this is an example of one BT ELISA procedure.
i) First, 96-well microtitre plates are coated at 4°C overnight or at 37°C for 1 hour with 50–100 µl
of either tissue culture-derived sonicated cell culture antigen or the major core antigen VP7
expressed in either Baculovirus (Oldfield et al., 1990) or yeast (Martyn et al., 1990) and diluted
in 0.05 M carbonate buffer, pH 9.6.
ii) The plates are washed five times with PBST (0.01 M PBS containing 0.05% or 0.1% Tween 20,
pH 7.2).
iii) Next, 50 µl of test sera is added in duplicate at a single dilution, either 1/5 (Afshar et al., 1989)
or 1/10 (Lunt et al., 1988) in PBST containing 3% bovine serum albumin (BSA).
iv) Immediately, 50 µl of a predetermined dilution of MAb diluted in PBST containing 3% BSA is
added to each well. MAb control wells contain diluent buffer in place of test serum.
v) Plates are incubated for 1 hour at 37°C or 3 hours at 25°C, with continuous shaking.
vi) After washing as described above, wells are filled with 100 µl of an appropriate dilution of
horseradish peroxidase-labelled rabbit anti-mouse IgG (H+L) in PBST containing 2% normal
bovine serum.
vii) Following incubation for 1 hour at 37°C, the conjugate solution is discarded and plates are
washed five times using PBS or PBST. Wells are filled with 100 µl substrate solution
containing 1.0 mM ABTS (2,2’-azino-bis-[3-ethylbenzothiazoline-6-sulphonic acid]), 4 mM
H2O2 in 50 mM sodium citrate, pH 4.0, and the plates are shaken at 25°C for 30 minutes.
(Other substrates may be used and the reaction continued with shaking for an appropriate
length of time to permit colour development.)
viii) The reaction is stopped by addition of a stopping reagent, such as sodium azide.
ix) After blanking the ELISA reader on wells containing substrate and stopping reagent, the
absorbance values are measured at 414 nm. Results are expressed as per cent inhibition and
are derived from the mean absorbance values for each sample by the following formula:
% inhibition = 100 – [(mean absorbance test sample)/(mean absorbance MAb control) ×
100].
NB: Some laboratories prefer to use a negative control serum that has previously been
shown to have a percentage inhibition of zero as an alternative to the MAb control.
x) Percentage inhibition values >50% are deemed positive. Inhibition between 40% and 50% is
considered to be suspicious. The results of the test sera duplicates can vary as long as the
results do not lie either side of the positive cut -off.
xi) Strong and weak positive sera and a negative serum should be included on each plate. The
weak positive should give 60–80% inhibition and the negative should give less than 40%
inhibition.

A number of commercially produced C-ELISAs based on recombinant VP7 and anti-VP7 MAb are
now available. These commercial assays are routinely used in many laboratories across the world
and have been proved to be fit for purpose in ring-trials (Batten et al., 2008). Formal acceptance
for trade purposes should depend on adoption of individual kits to the WOAH Register.
Genetic divergence of certain BTV strains (e.g. different regional groups or topotypes) may affect
the nature of serogroup-reactive antibodies. It is therefore possible that diagnostic
characteristics for antibody detection are not uniform for all viruses encompassed by the
serogroup. Diagnostic reagents and kits produced in one region may not have the same
performance characteristics when used in another region. This should be addressed in
considerations of fitness for purpose.
2.2. Indirect ELISA
An indirect ELISA for bulk milk samples has been shown to be reliable and useful for surveillance
purposes (Kramps et al., 2008). It should be validated for relevant serotypes before use. While an indirect
ELISA may have the disadvantage of cross reactivity with related viruses such as EHDV, it does have high
analytical sensitivity and could be a useful screening assay in some situations.
2.3. Virus neutralisation serology
VN serology can identify serotype-specific neutralising antibodies as well as determine their titre. It is an
important additional test in endemic areas where multiple serotypes are likely to be present. Its capability
to identify the serotype involved in an outbreak is essential for putting in place appropriate control
measures such as vaccination or animal movement restrictions. It is also useful for epidemiological
surveillance, transmission studies and for determining useful antibody response to vaccination. Cross-
neutralising antibodies can develop in animals that have experienced BTV infection. Importantly,
infection with a second or third serotype can broaden the neutralising antibody response to include
antibodies to serotypes to which the animal has not been exposed. The application of VN serology is
frequently most useful in conjunction with other virological investigations that, in combination, can
provide a more definitive basis for resolving serotype distribution. The use of the plaque reduction test
(modified from Section B.1.2.2) can improve resolution of serotype involvement, where this is unclear on
a standard VN test due to its quantitative nature.
2.3.1. Test procedure

Several methods to determine titre and serotype of BTV have been described; here the procedure
that has been standardised after comparative studies in various international laboratories is
briefly outlined. Indicator cell lines commonly used are BHK and Vero. It is important that well
characterised, reference positive and negative control antiserum be included in each test.
i) 50 µl of serial sera dilutions, from 1/10 to 1/1280, are added to each test well of flat-bottomed
microtitre plates and each well is mixed with an equal volume of medium containing
approximately 100 TCID50 of suitable, well characterised, BTV reference viruses. Note the
selection of reference strains used will be dependent on the circulating (or possibly
circulating) serotypes in the testing population.
ii) The plates are incubated in a humid chamber at 37°C in 5% CO2.
iii) After 1 hour of incubation, approximately 104 Vero or BHK-21 cells are added per well in a
volume of 100 µl of suitable media (containing) able to sustain growth of the chosen cells.
iv) Incubate at 37°C for up to 7 days, depending on the level of CPE obtained in normal serum
wells of the test. When CPE has developed to 100% in the normal serum wells of each
serotype tested, the test is read using a light microscope at 100× magnification to determine
presence of CPE.
v) Wells are scored for the degree of CPE observed. A sample is considered positive when it
nears full neutralisation, allowing for the acceptance of trace CPE at the lowest dilution (1/10).
The presence of trailing trace CPE may indicate evidence of partial neutralisation. In these
situations, the reference virus used may not be representative of contemporary circulating
viruses and further investigation may be required (for example, use of the plaque reduction

neutralisation test, which is superior in resolving cross reactivity between related serotypes).
The serum titre represents the highest serum dilution capable of near complete
neutralisation of 50% of replicate test wells. A fourfold difference in endpoint titre obtained
from different serotypes tested can indicate the serotype involved in an infection. Cross
reactions are known to occur between serotypes in the VN test even when only one serotype
is involved.
2.4. Agar gel immunodiffusion
It must be recognised that a major disadvantage of the AGID used for BT is its lack of specificity in that it
does not exclusively differentiate between antibodies to the BT and EHD serogroups. Hence, it cannot be
used definitively to detect antibodies to BTV as a positive reaction may have been the result of an
infection to another Orbivirus species. Notably, the AGID test is simple to perform and the antigen used
in the assay relatively easy to produce. Since 1982, the test has been one of the standard testing
procedures for international movement of ruminants, however, it is no longer considered sufficiently
accurate for use in the support of international trade. The assay does have a role in the investigation of
samples that give inconsistent results in other assays. AGID positive sera should be retested using a BT
serogroup-specific assay if BTV specificity is required. The preferred test, a C-ELISA, is described in
Section B.2.1.
C. REQUIREMENTS FOR VACCINES
1. Background
Vaccination with effective vaccines is the preferred method of BTV control in endemic countries. Vaccination has
been used successfully to limit direct losses, minimise the circulation of BTV, eradicate BTV from a region, and allow
safe movement of animals. Both live attenuated and inactivated BTV vaccines are currently available for use in
sheep and sheep and cattle, respectively. Recombinant BT vaccines based on various approaches are under
development (van Rijn, 2019), but none has been licensed and these vaccines will not be addressed here.
Live attenuated vaccines are inexpensive to produce in large quantities; they generate protective immunity after a
single inoculation and have proven effective in preventing clinical BT disease in the areas where they are used. In
South Africa, live attenuated vaccines have been used for over 50 years and are known to induce an effective and
lasting immunity. Live attenuated vaccines are produced by adapting BTV field isolates to growth in vitro through
serial passages in tissue culture or in ECEs. Stimulation of a strong antibody response by these vaccines is directly
correlated to their ability to replicate in the vaccinated host. However, live attenuated BTV vaccines suffer from a
variety of documented or potential adverse outcomes, including depressed milk production, abortion/embryonic
death, teratogenesis and congenital defects, and have been documented to be spread by vectors with considerable
potential for reversion to virulence and reassortment of vaccine virus genes with those of wild-type virus strains
(Ferrari et al., 2005; Ranjin et al., 2019; Savini et al., 2014). Under-attenuation, the impact of which may vary with
different breeds of sheep (Veronesi et al., 2010), and reassortment with other vaccine and field strains (Nomikou et
al., 2015) may also occur. The frequency and significance of these events remain poorly defined but transmission of
vaccine strains by vector midges has already been documented in the USA, South Africa and Europe (Ferrari et al.,
2005).
Inactivated vaccines containing tissue-culture grown and chemically inactivated vaccine strains are far safer but
require multiple doses to become efficacious and incur higher costs. Such vaccines have been very effective in
combatting the spread of BTV-8 in Europe. Whilst inactivated vaccines (as with live vaccines) are not compatible
with serological assays for detection of infection in vaccinated animals (DIVA strategies), surveillance can be
maintained after their use by RNA based (RT-PCR) assays.
Additionally, non-BTV vaccines for both target and non-target species have been found to be contaminated with
BTV leading to abortion, heart failure, respiratory distress and death in non-ruminant species. These drawbacks
have resulted in a great deal of research and development of BTV vaccines that are safe, efficacious against multiple
serotypes, inexpensive, are DIVA capable, only require a single dose with rapid onset of immunity, and block
viraemia. Recent efforts with reverse genetics and molecular technology has led to next generation vaccines that
balance these often competing requirements while meeting specific field situation needs.

Guidelines for the production of veterinary vaccines are given in Chapter 1.1.8 Principles of veterinary vaccine
production. The guidelines given below and in chapter 1.1.8 are intended to be general in nature and may be
supplemented by national and regional requirements.
2. Outline of production and minimum requirements for vaccines
2.1. Characteristics of the seed
See chapter 1.1.8 for general requirements for master seeds and allowable passages for vaccine
production.
2.1.1. Biological characteristics of the master seed

For live attenuated vaccines, the master or primary virus seed is prepared from a single plaque of
serially passaged, attenuated BTV. Vaccine viruses have been attenuated by either passage in
ECE, tissue culture cells or a combination of both. Each primary seed virus lot should also be
tested for transmissibility and reversion to virulence prior to vaccine manufacture. Samples of
vaccine prepared from secondary seed virus at the maximum permitted passage level should be
tested in sheep for avirulence, safety and immunogenicity.
For inactivated vaccines, the issues of attenuation do not apply, and the approach adopted has
been to use field strains of low passage level with the intent of achieving high antigenicity.
2.1.2. Quality criteria (sterility, purity, freedom from extraneous agents)

Primary seed virus must be free of contaminating bacteria, viruses, prions, fungi and
mycoplasmas, particularly pestivirus contamination. For the latter, attention should be paid to the
fetal bovine serum used in cell cultures, as it may be contaminated. Seed viruses must be shown
to have the desired serotype specificity. BTV seed lot viruses should be sequenced and the data
made available to relevant databases. Secondary seed lots, which are used as inocula for vaccine
production, are usually not more than three passages beyond the primary seed lot.
2.1.3. Validation as a vaccine strain

Live attenuated BT vaccines must be safe and efficacious, and a brief description of appropriate
tests for these parameters is given below. In addition, attenuated viruses should not revert to
virulence during replication in vaccinated animals or be able to be transmitted from such animals
by insect vectors. The latter criterion is very important because insect-mediated transmission of
attenuated virus from vaccinated to non-immune animals, with the subsequent replicative steps
in each host species, increases the possibility of reversion to virulence. Although tests for
reversion to virulence and transmissibility are rarely, if ever performed, a brief description of what
may be necessary is outlined.
There is a variation in BT susceptibility between breeds of sheep; it is important that sheep that
have been proven to be susceptible to infection with BTV be used for vaccine validation.
2.1.4. Emergency procedure for provisional acceptance of new master seed virus (MSV) in the
case of an epizootic
In a number of different circumstances (see Chapter 1.1.10 Vaccine banks) and in the event that a
new or different serotype or variant of BTV results in an emergency epizootic situation that cannot
be controlled by currently available vaccines, and where there is not enough time to fully test a
new MSV for all extraneous agents, provisional acceptance of the new strain could be based on a
risk analysis of the possibility of contamination of the antigen produced from the new MSV with
extraneous agents. This risk assessment should take into account the characteristics of the
process, including the nature and concentration of the inactivant for inactivated vaccines, before
allowing or not the early release of the new product.

2.2. Method of manufacture
2.2.1. Procedure
Attenuation of field isolates of BTV was first achieved by serial passage in ECE. Because of the
concern about transmission of the egg propagated attenuated virus, it has been recommended
that animals receiving vaccines produced in ECE should not be moved internationally. More
recently, it is accepted that passage in cultured cells will also result in attenuation of virulence. No
studies have been done to precisely relate passage number and extent of attenuation for
individual virus isolates or serotypes. To prepare attenuated virus, field isolates are adapted to cell
culture and passaged in vitro up to 40 times or more. Ideally, a number of plaque-purified viruses
are picked at this stage and each is examined to determine the level of viraemia they generate
and their ability to elicit a protective immune response in vaccinated sheep. The most suitable
virus is one that replicates to low titre but generates a protective immune response, and this may
represent the source of vaccine primary seed stock virus.
BTV for inactivated vaccines is produced in large-scale suspension cell systems under aseptic
and controlled conditions. Cell lines adapted for large scale industrial cultures are used and these
are proven to be free from contaminating microorganisms. When the viral suspension virus
reaches its maximum titre, followed by cell disruption, the culture is clarified and filtered.
Subsequently inactivation is performed according to processes adopted by the manufacturer,
such as by addition of binary ethyleneiminee (BEI) or other inactivating agents. The process must
comply with legislation relevant for the intended market, be validated to ensure complete
inactivation and supported by appropriate documentation. The inactivation process should not
significantly alter the immunogenic properties of the viral antigens. Purification is carried out by
chromatography. The inactivated virus is then concentrated by ultrafiltration and stored. The
inactivated, chromatography-purified and concentrated BTV antigens are made into vaccine by
dilution in a buffer solution and addition of adjuvants.
2.2.2. Requirements for ingredients

All ingredients of animal origin, including serum and cells must be checked for the presence of
viable bacteria, viruses, fungi or mycoplasma. For further details, see chapter 1.1.8 for general
guidance on ingredients of animal origin. Ingredients of animal origin should be sourced from a
country with negligible risk for transmissible spongiform encephalopathies.
2.2.3. In-process controls

Virus concentration of live attenuated vaccines is assessed by infectivity and ELISAs.
For inactivated vaccines, during inactivation of the virus, timed samples are taken at regular
intervals for the purpose of monitoring the rate and linearity of the inactivation process. Virus
titres in the samples are determined by inoculation of BHK-21 or other appropriate cell cultures.
At the end of the inactivation process, the vaccine is checked to ensure that there is no live virus.
2.2.4. Final product batch tests

i) Sterility
Every batch of vaccine should be tested for the presence of contaminant viruses, viable
bacterial, fungi or mycoplasma. For example, in South Africa a pool of ten randomly selected
ampoules are inoculated into soya broth and thioglycolate broth, and incubated at room
temperature and 37°C for 14 days, respectively. If contaminated, the batch is disqualified.
ii) Safety
Every batch of attenuated vaccine is safety tested in newborn and adult mice or guinea-pigs.
If any adverse reactions or significant signs are noted, the test is repeated. Any increase in
the body temperature of the test animal that is above the level expected for the strain of
attenuated virus under test should be regarded as symptomatic. If the results are
unsatisfactory after a second attempt, the batch is disqualified.
Safety testing of inactivated vaccines is conducted to ensure side effects are not observed.

iii) Potency
Each batch is tested by inoculation of susceptible sheep. Pre-vaccination, 21- and 28-day
post-vaccination sera are tested by VN assay to determine neutralising antibody levels. To
be passed, the antibody titre must be equal to or higher than a set standard based on
international vaccine standards.
iv) Duration of immunity

Studies with live attenuated BTV vaccine have shown that antibodies in sheep may appear
before day 10 post-vaccination, reach a maximum approximately 4 weeks later and persist
for well over a year. There is a temporal relationship between the increase in neutralising
antibody titre and clearance of virus from the peripheral circulation. Live attenuated BTV
vaccines have been in use for over 50 years and are known to induce an effective and lasting
immunity in sheep (Verwoerd & Erasmus, 2004). Many serotypes of BTV may be present in
endemic areas of South Africa, and polyvalent vaccines are used. The inclusion of
15 serotypes in three polyvalent vaccines that are administered sequentially sometimes
means that an effective immune response is not generated to all serotypes, presumably
because of the antigenic mass of individual serotype-specific antigens is small. In an
attempt to broaden the response, vaccination is repeated annually.
Initial studies with inactivated vaccines show that antibody against BTV can be detected by
day 7 post-vaccination and increase in titre to days 14–21. A second dose of vaccine boosts
the titre. Data to demonstrate the expected duration of immunity are being acquired.
v) Stability
Procedures have been developed for attenuated vaccines. Stability should be tested over a
period of 2 years. Vaccines in liquid and lyophilised forms are deemed to have shelf lives of
1 and 2 years, respectively. Each batch of vaccine is subjected to an accelerated shelf-life test
by storing it at 37°C for 7 days. It is then titrated and evaluated according to a set standard,
as determined in the initial testing of the vaccine. Shelf life of stock aliquots stored at +4°C
should be tested periodically.
2.3. Requirements for regulatory approval
2.3.1. Manufacturing process

For registration of vaccine, all relevant details concerning manufacture of the vaccine and quality
control testing (see Section C.2.1 and C.2.2) should be submitted to the authorities. This
information shall be provided from three consecutive vaccine batches with a volume not less than
1/3 of the typical industrial batch volume.
2.3.2. Safety requirements

All vaccines must be safety tested. Safety tests for attenuated vaccines do not address the issue
of their teratogenicity. Attenuated virus vaccines are teratogenic and should not be administered
to pregnant sheep during the first half of pregnancy as this may cause fetal abnormalities and
embryo death.
i) Target and non-target animal safety

Demonstration of avirulence is necessary for live, attenuated vaccines. A number of sheep
seronegative by an appropriate, sensitive serological test (that will reliably detect antibodies
even in vaccinated animals), are inoculated with the primary seed stock. Temperatures are
noted twice daily. The animals are monitored at regular intervals over a period of 28 days for
clinical signs and any local or systemic reactions to ensure avirulence and innocuity. Blood
samples removed at regular intervals can be used to measure level of viraemia and antibody
responses. The test shall be valid if all of the vaccinated sheep show evidence of virus
replication and do not display signs of disease other than mild transient illness. In South
Africa, a clinical reaction index is calculated for each animal between days 4 and 14 and must
be below a specific standard value.

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

Transmissibility is an issue with live attenuated vaccines but not with killed vaccines.
Procedures to determine if attenuated virus can be transmitted by insects that feed on
vaccinated, viraemic sheep are difficult to perform and analyse statistically, and
consequently, this criterion of vaccine validation is rarely sought. Laboratory data indicate
that laboratory-adapted viruses can be transmitted by insect vectors (Ferrari et al., 2005). A
suitable procedure to determine attenuated virus transmissibility requires that sheep be
vaccinated and, during viraemia, that they be exposed to competent, uninfected Culicoides,
which are then permitted to feed on uninfected animals that are monitored for the presence
of BTV and anti-BTV antibody. As the titre of attenuated virus in the blood of vaccinated
sheep is usually low, very large numbers of Culicoides may be needed and only a small
proportion of these would become infected and live long enough to feed on and potentially
transmit the virus to other uninfected sheep. It is difficult to design a laboratory experiment
that takes account of the large numbers of vaccinated sheep and insects that would be
present in field situations. Although virus titres in blood less than 103 TCID50/ml have
traditionally been considered a “safe” threshold, authentic instances of insects acquiring
BTV from animals with viraemic titres much less than 103 TCID50/ml have been reported.
Given the complex interaction of BTV, Culicoides vectors and animal hosts in the life cycle of
infection, virus titres induced by live attenuated vaccine should be kept to an absolute
minimum especially if field transmission of vaccine strains is a concern.
Current data indicate that during viraemia and in contrast to wild-type virus, laboratory-
adapted strains of BTV may be found in the semen of bulls and rams (Kirkland et al., 2004).
The implications of these observations for virus transmissibility are unclear. A recent study
of semen from rams vaccinated with BTV-2 live attenuated vaccine showed that even if BTV
was not detected in the semen, the vaccine caused a decrease in the quality of the semen
(Breard et al., 2007).
Validation studies confirm that attenuated viruses do not revert to virulence in vaccinated
sheep. However, if attenuated viruses can be transmitted from vaccinated animals, reversion
to virulence following several sheep-insect replication cycles becomes a distinct prospect.
The only appropriate way to monitor for reversion to virulence under these circumstances is
to compare the virulence of the vaccine virus with that which had been subject to several
sheep–insect replication cycles as described above. As indicated, this is difficult to achieve.
Consequently, the effect of sheep/insect passages on the virulence of attenuated viruses
has not been determined. In South Africa, it is accepted that if blood from vaccinated animals
during the viraemic stages is serially passaged three times in sheep without reversion to
virulence, the chances of reversion in the field will be infinitely small. In Europe, five passages
are required.
iii) Precautions (hazards)

Attenuated vaccines should be used in the cooler months when adult Culicoides vector
populations are at a minimum. They should not be used in ewes during the first half of
pregnancy and in rams 2 months before the breeding season
2.3.3. Efficacy requirements

Vaccinated and unvaccinated sheep known to be susceptible to BT disease should be challenged
with virulent homologous serotype. It is recommended that the challenge model preferably use
virus passaged only in ruminant animals and with no or limited ECE or cell culture passages.
Passage in such an isolation system results in viral cultures that might induce clinical BT disease
that is milder than the natural disease. Animals are monitored for clinical signs of BT disease,
rectal temperatures are taken twice daily and blood samples removed at regular intervals to
measure viraemia and antibody responses. Unvaccinated control sheep should show clinical
signs of BT disease and viraemia. However, it is difficult to be certain of the appearance of clinical
disease following inoculation of sheep with certain BTV serotypes and isolates, and consequently,
evidence of infection of unvaccinated control sheep may rest on the appearance of a temperature
rise above 40°C and a viraemia. As a further evidence of infection pre- and post-vaccination sera
are checked for the presence of neutralising antibody.

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

The live attenuated and inactivated products now commercially available do not allow any DIVA
strategy via serological testing.
RT-PCR may be used as DIVA assays, allowing surveillance to be maintained during inactivated
vaccination campaigns. DIVA strategies may be possible using PCR-based test systems in
populations in which inactivated vaccines have been applied. The inactivated vaccines may
generate a very weak RT-PCR signal although this disappears a few days post-vaccination. In
contrast, infected animals usually maintain a high RT-PCR signal that can last for several weeks to
months, even in the presence of neutralising antibodies, due to the haemagglutinin activity
associated with outer capsid protein VP2. A similar approach is not possible with currently
available live attenuated vaccines. The generation of weak signal due to very early infection
should also be considered if using this strategy.
2.3.5. Duration of immunity

Studies to determine a minimum duration of immunity should be conducted before the vaccine
receives final approval. Duration of immunity should be demonstrated in a manner similar to the
original efficacy study, challenging animals at the end of the claimed period of protection. At a
minimum, the duration should be for the length of the mosquito season in areas with seasonal
infections. It may be desirable to demonstrate longer immunity for animals at higher risk and in
infected areas with year-round mosquito activity.
2.3.6. Stability
Live and inactivated vaccines are typically assigned an initial dating of 18 or 24 months,
respectively, before expiry. Real-time stability studies should be conducted to confirm the
appropriateness of all expiration dating. Product labelling should specify proper storage
conditions.
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BELBIS G., ZIENTARA S., BREARD E., SAILLEAU C., CAIGNARD G., VITOUR D. & ATTOUI H. (2017). Bluetongue virus: From BTV-1 to
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BREARD E., POZZI N., SAILLEAU C., CATINOT V., DURAND B., DUMONT P., GUÉRIN B. & ZIENTARA S. (2007). Transient effect of
the attenuated bluetongue virus vaccine on the quality of the ram semen. Vet. Rec., 160, 431–435.
BREARD E., SHULZ C., SAILLEAU C., BERNELIN-COTTET C., VIAROUGE C., GUILLAUME B., CAIGNARD G., GORLIER A., ATTOUI H.,
GALLOIS M., HOFFMANN B., ZIENTARA S. & BEER M. (2018). Bluetongue virus serotype 27: Experimental infection of goats,
sheep and cattle with three BTV-27 variants reveal atypical characteristics and likely direct contact transmission of
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CARPENTER S., VERONESI E., MULLENS B. & VENTER G. (2015). Vector competence of Culicoides for arboviruses: three
major periods of research, their influence on current studies and future directions. Rev. Sci. Tech., 34, 97–112.
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CLAVIJO A., HECKERT R.A., DULAC G.C. & AFSHAR A. (2000). Isolation and identification of bluetongue virus. J. Virol.
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DARPEL K.E., BARBER J., HOPE A., WILSON A.J., GUBBINS S., HENSTOCK M. & MERTENS P.P.C. (2016). Using shared needles for
subcutaneous inoculation can transmit bluetongue virus mechanically between ruminant hosts. Sci. Rep., 6, 20627.
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SCICLUNA M.T., AMADDEO D., SCARAMOZZINO P. & AUTORINO G.L. (2005). Active circulation of bluetongue vaccine virus
serotype-2 among unvaccinated cattle in central Italy. Prev. Vet. Med., 68, 10–13.
GOULD A.R. (1987). The complete nucleotide sequence of bluetongue virus serotype 1 RNA3 and a comparison with
other geographic serotypes from Australia, South Africa and the United States of America, and with other orbivirus
isolates. Virus Res., 7, 169–183.
HOFMANN M., GRIOT C., CHAIGNAT V., PERLER L. & THÜR B. (2008). Bluetongue disease reaches Switzerland. Schweiz.
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JACQUOT M., RAO P.P., YADAV S., NOMIKOU K., MAAN S., JYOTHI Y.K., REDDY N., PUTTY K., HEMADRI D., SINGH K.P., MAAN N.S.,
HEGDE N.R., MERTENS P. & BIEK R. (2019). Contrasting selective patterns across the segmented genome of bluetongue
virus in a global reassortment hotspot. Virus Evol., 5 (2):vez027. doi: 10.1093/ve/vez027.
KATZ J., GUSTAFSON G., ALSTAD D., ADLER K. & MOSER K. (1993). Colorimetric diagnosis of prolonged bluetongue viremia
in sheep using an enzyme-linked oligonucleotide sorbent assay of amplified viral nucleic acids. Am. J. Vet. Res., 54,
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KIRKLAND P.D., MELVILLA L.F., HUNT N.T., WILLIAMS C.F. & DAVIS R.J. (2004). Excretion of bluetongue virus in cattle semen:
a feature of laboratory adapted virus. Vet. Ital., 40, 497–501.
KRAMPS J.A., VAN MAANEN K., MARS M.H., POPMA J.K. & VAN RIJN P.A. (2008). Validation of a commercial ELISA for the
detection of bluetongue virus (BTV)-specific antibodies in individual milk samples of Dutch dairy cows Vet.
Mircrobiol.,130, 80–87.
LUNT R.A., WHITE J.R. & BLACKSELL S.D. (1988). Evaluation of a monoclonal antibody blocking ELISA for the detection
of group-specific antibodies to bluetongue virus in experimental and field sera. J. Gen. Virol., 69, 2729–2740.
MAAN S., MAAN N.S., BELAGANAHALLI M.N., RAO P.P., SINGH K.P., HEMADRI D., PUTTY K., KUMAR A., BATRA K., KRISHNAJYOTHI Y.,
CHANDEL B.S., REDDY G.H., NOMIKOU K., REDDY Y.N., ATTOUI H., HEGDE N.R. & MERTENS P.P. (2015). Full-Genome
Sequencing as a Basis for Molecular Epidemiology Studies of Bluetongue Virus in India. PLoS One, 10(6):e0131257.
doi: 10.1371/journal.pone.0131257.
MACLACHLAN N.J., DREW C.P., DARPEL K.E. & WORWA G. (2009). The pathology and pathogenesis of Bluetongue. J.
Comp. Pathol., 141, 1–16.
MACLACHLAN N.J. & MAYO C.E. (2013). Potential strategies for control of bluetongue, a globally emerging, Culicoides-
transmitted viral disease of ruminant livestock and wildlife. Antiviral Research, 99, 79–90.
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329–340.
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MAYO C., MULLENS B., GIBBS E.P. & MACLACHLAN N.J. (2016). Overwintering of Bluetongue virus in temperate zones. Vet.
Ital., 52, 243–246.

MCHOLLAND L.E. & MECHAM J.O. (2003). Characterization of cell lines developed from field populations of Culicoides
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SAVINI G. (2015). Bluetongue: a disease that does not speak ‘one tongue’ only. Vet. Ital., 51, 247–248.
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VERWOERD D.W & ERASMUS B.J. (2004). Bluetongue. In: Infectious Diseases of Livestock, Second Edition, Coetzer
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*
* *
NB: There are WOAH Reference Laboratories for bluetongue

(please consult the WOAH Web site:
https://www.woah.org/en/what-we-offer/expertise-network/reference-laboratories/#ui-id-3).
Please contact the WOAH Reference Laboratories for any further information on
diagnostic tests, reagents and vaccines for bluetongue
NB: FIRST ADOPTED IN 1991. MOST RECENT UPDATES ADOPTED IN 2021.

3.01.03 Bluetongue

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C H A P T E R 3.1 . 3.

WOAH Terrestrial Manual 2021 1

1. Description of the disease

2 WOAH Terrestrial Manual 2021

2. Nature and classification of the pathogen

3. Zoonotic potential and biosafety and biosecurity requirements

1 See BTV-Glue database http://btv-glue.cvr.gla.ac.uk

WOAH Terrestrial Manual 2021 3

Method Individual animal Immune status in

Detection of the agent(a)

RT-PCR – +++ – +++ ++ –

Detection of immune response

1. Detection of the BTV agent

1.1. In-vitro and in-vivo cultures

1.1.1. Isolation in embryonated chicken eggs

4 WOAH Terrestrial Manual 2021

1.1.2. Isolation in cell culture

1.2. Virus detection and characterisation

WOAH Terrestrial Manual 2021 5

1.2.2. Immunological serogrouping of viruses

ii) Antigen capture enzyme-linked immunosorbent assay

1.2.2. Serotyping of isolates by virus neutralisation

6 WOAH Terrestrial Manual 2021

ii) Plaque inhibition

iii) Microtitre neutralisation

1.3. Molecular methods

1.3.1. Detection of nucleic acid

WOAH Terrestrial Manual 2021 7

GenBank (https://www.ncbi.nlm.nih.gov/genbank/). The BTV-Glue website (http://btv-

1.3.1.1. Real-time reverse-transcription polymerase chain reaction

i) RNA extraction from blood, tissue samples, and midges

ii) Real-time reverse-transcription polymerase chain reaction

BTV_IVI_P 5’FAM-ARG-CTG-CAT-TCG-CAT-CGT-ACG- C-3’ BHQ1

8 WOAH Terrestrial Manual 2021

a) Primer stock solutions are diluted to a working concentration of 20 pmol/µl, whereas

50 cycles: 95°C 15 seconds

The nested assay employs the use of the following primers:

Nested amplification (dilute to 25 pmol/µl; final concentration in PCR is 0.5 µM each):

WOAH Terrestrial Manual 2021 9

1.3.2. Nucleic acid sequencing

10 WOAH Terrestrial Manual 2021

2.1. Competitive enzyme-linked immunosorbent assay

2.1.1. Test procedure

WOAH Terrestrial Manual 2021 11

2.2. Indirect ELISA

2.3. Virus neutralisation serology

2.3.1. Test procedure

12 WOAH Terrestrial Manual 2021

2.4. Agar gel immunodiffusion

C. REQUIREMENTS FOR VACCINES

WOAH Terrestrial Manual 2021 13

2. Outline of production and minimum requirements for vaccines

2.1. Characteristics of the seed

2.1.1. Biological characteristics of the master seed

2.1.2. Quality criteria (sterility, purity, freedom from extraneous agents)

2.1.3. Validation as a vaccine strain

14 WOAH Terrestrial Manual 2021

2.2. Method of manufacture

2.2.2. Requirements for ingredients