CHAPTER 3.1.24.

VESICULAR STOMATITIS

SUMMARY
Vesicular stomatitis (VS) is a vesicular disease of horses, cattle and pigs caused by vesiculoviruses of
the family Rhabdoviridae. This disease is clinically indistinguishable in relevant susceptible species
from foot and mouth disease (FMD), vesicular exanthema of swine (VES), or swine vesicular disease
(SVD). Sheep, goats and many other wild species can be infected. Humans are also susceptible. The
disease is endemic in the Americas, but has occasionally spread to other continents.
Virus is transmitted directly by the transcutaneous or transmucosal route and has been isolated from
sandflies and mosquitoes. Experimental transmission has been shown from black flies to both pigs
and cattle. There is seasonal variation in the occurrence of VS: it disappears at the end of the rainy
season in tropical areas, and at the first frosts in temperate zones. The pathogenesis of the disease is
unclear, and it has been observed that the specific circulating antibodies do not always prevent
infection with different VS serogroups.
Although VS may be suspected when horses are involved as well as pigs and cattle, prompt
differential diagnosis is essential because the clinical signs of VS are indistinguishable from FMD
when cattle and pigs are affected, and from SVD, VES and senecavirus A when only pigs are affected.
Diagnosis of VS is by virus isolation or by the demonstration of VS viral antigen or nucleic acid in
samples of tissue or fluid. Detection of virus-specific antibody against structural proteins, in paired
serum, can also be used as an indicator of infection.
Detection of the agent: Virus can be readily isolated by the inoculation of several tissue culture
systems, or embryonated chicken eggs. Viral RNA can be detected from epithelial tissue and vesicular
fluid by conventional and real-time reverse transcription polymerase chain reaction (RT-PCR). Viral
antigen can be identified by an indirect sandwich enzyme-linked immunosorbent assay (IS-ELISA) – this
is the least expensive and most rapid test. The complement fixation test (CFT) is also a good alternative.
The virus neutralisation test (VNT) may be used, but it is elaborate and time-consuming.
Serological tests: Convalescent animals develop specific antibodies within 4–8 days of infection that
are demonstrated by a liquid-phase blocking ELISA (LP-ELISA), a competitive ELISA (C-ELISA) and
VNT. Other described tests are CFT, agar gel immunodiffusion and counter immuno-electrophoresis.
The demonstration of specific antibodies to structural proteins in nonvaccinated animals is indicative
of prior infection with VS virus.
Requirements for vaccines: Inactivated virus vaccines with aluminium hydroxide or oil as adjuvants
have been tested in the United States of America and in Colombia, respectively. Both vaccines
generated high levels of specific antibodies in the sera of vaccinated cattle. However, it is not yet clear
if serum antibodies would prevent the disease. An attenuated virus vaccine has been used in the field
with unknown efficacy.

A. INTRODUCTION
Vesicular stomatitis (VS) was described in the United States of America (USA) by Oltsky et al. (1926) and Cotton
(1927) as a vesicular disease of horses, and subsequently of cattle and pigs. Vesicles are caused by virus on the
tongue, lips, buccal mucosa, teats, and in the coronary band epithelium of cattle, horses, pigs, and many other
species of domestic and wild animals. Natural disease in sheep and goats is rare, although both species can be
experimentally infected. Mixed infections of foot and mouth disease (FMD) and VS viruses have occurred in the
same herds of cattle and can be induced experimentally. Many species of laboratory animals are also susceptible.
The disease is endemic in the Americas, but has occasionally spread to other continents.

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 Chapter 3.1.24. – Vesicular stomatitis

Influenza-like signs, normally without vesicles, have been observed in humans who are in contact with animals with
VS or who handle infective virus. Laboratory manipulations involving live virus, including handling infective
materials from animals, should be carried out at an appropriate biosafety and containment level determined by risk
analysis (see Chapter 1.1.4 Biosafety and biosecurity: Standard for managing biological risk in the veterinary
laboratory and animal facilities).

The pathogenic agents causing vesicular stomatitis belong to the Rhabdoviridae family, genus Vesiculovirus with
four species as follows: Indiana vesiculovirus – VSIV (formerly IND-1), Cocal vesiculovirus – COCV (formerly IND-2),
Alagoas vesiculovirus – VSAV (formerly IND-3) and New Jersey vesiculovirus – VSNJV. They have been extensively
studied at the molecular level (Fowler et al., 2016; ICTV, 2020; Pauszek et al., 2011). Several other closely related
rhabdoviruses have been isolated from sick animals over the past decades. The VSIV, Cocal virusand Alagoas virus
are serologically related (Federer et al., 1967). Strains of the VSNJV and VSIV are endemic in livestock in areas of
southern Mexico, Central America, Venezuela, Colombia, Ecuador and Peru, with VSVNJ causing the majority of the
clinical cases. Sporadic activity of VSNJV and VSIV has been reported in northern Mexico and the western United
States. Cocal vesiculovirus was isolated from domestic animals only in Argentina and Brazil and only from horses
(Salto-Argentina/63, Maipú-Argentina/86, Rancharia-Brazil/66, Ribeirão-Brazil/79) (Alonso et al., 1991; Alonso
Fernandez & Sondahl, 1985). This finding confirms the first descriptions, in 1926 and 1927 (Cotton, 1927; Oltsky et
al., 1926) of the VSNJV and VSIV, COCV, VSAV in horses, and subsequently in cattle and pigs; this same predilection
has been observed in other VS outbreaks. The Alagoas virus, (Alagoas-Brazil/64), has been identified, endemically
in North-eastern Brazil, in cattle, horses, swine, sheep and goats (Alonso et al., 1991; Alonso Fernandez & Sondahl,
1985; Cargnelutti et al., 2014; PANAFTOSA-OPS/OMS, 2019; Rocha et al., 2020).

The mechanism of transmission of the virus is unclear. The viruses have been isolated from sandflies, mosquitoes,
and other insects (Comer et al., 1992; Francy et al., 1988; Mason, 1978). Experimental transmission of VSNJV has
been demonstrated to occur from black flies (Simulium vittatum) to domestic swine and cattle (Mead et al., 2004;
2009). During the 1982 epizootic in the western USA, there were a number of cases where there was direct
transmission from animal to animal (Sellers & Maarouf, 1990). VSV has historically been considered to be endemic
in feral pigs on Ossabaw Island, Georgia, USA (Boring & Smith, 1962), but subsequently may have disappeared from
the island (Killmaster et al., 2011).

The incidence of disease can vary widely among affected herds. Usually 10–15% of the animals show clinical signs.
Clinical cases are mainly seen in adult animals. Cattle and horses under 1 year of age are rarely affected. Mortality is
close to zero in both species. However, high mortality rates in pigs affected by VSNJV have been observed. Sick
animals recover in about 2 weeks. The most common complications of economic importance are mastitis and loss
of production in dairy herds (Lauerman et al., 1962). Recent VS outbreaks in the USA have been associated primarily
with horses and VSNJV.

B. DIAGNOSTIC TECHNIQUES
VS cannot be reliably differentiated clinically from the other vesicular diseases in the relevant susceptible species,
such as FMD, vesicular exanthema of swine (VES), and swine vesicular disease (SVD). An early laboratory diagnosis
of any suspected VS case is therefore a matter of urgency.

The sample collection and technology used for the diagnosis of VS must be in concordance with the methodology
used for the diagnosis of FMD (chapter 3.1.8), VES and SVD (chapter 3.9.8), in order to facilitate the differential
diagnosis of these vesicular diseases.

Vesicle fluid, epithelium covering unruptured vesicles, epithelial flaps of freshly ruptured vesicles, or swabs of the
ruptured vesicles are the best diagnostic samples. These samples can be collected from mouth lesions, as well as
from the feet and any other sites of vesicle development. It is recommended to sedate the animal before collection
of samples to avoid injury to animals and people Tissue and fluid samples, including swabs from all species should
be placed in containers of Tris-buffered tryptose broth (TBTB) minimal essential medium (MEM) or 0.08 M
phosphate buffer (with phenol red and antibiotics [1000 units/ml penicillin, 100 units/ml nystatin, 100 units/ml
neomycin, and 50 units/ml polymyxin B], and adjusted to pH 7.2–7.6. Tissue samples can also be placed in
glycerol/phosphate buffer with phenol red pH 7.2–7.6. (Note: glycerol is toxic to cell cultures and decreases the
sensitivity of virus isolation.) Samples should be sent to the laboratory on ice packs if they can arrive at the
laboratory within 48 hours after collection. If samples require more than 48 hours transit time, they should be sent
frozen on dry ice with precautions to protect the sample from direct contact with CO2. There are special packaging
requirements for shipping samples with dry ice (see Chapter 1.1.2 Collection, submission and storage of diagnostic
specimens for further information on shipping of diagnostic samples).

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Considering the need for differential diagnosis with FMD, if it is not possible to collect epithelium from unruptured
or freshly ruptured vesicles or vesicular fluid, oesophageal–pharyngeal (OP) fluid samples, can be collected by
probang cups from ruminants only, as an alternative source of virus. Mix probang fluid with an equal volume of
transport fluid (see Chapter 3.1.8 Foot and mouth disease). The container should be capable of withstanding
freezing above dry ice (solid carbon dioxide) or liquid nitrogen (Kitching & Donaldson, 1987).

When it is not possible to collect samples for identification of the agent, serum samples can be used for detecting
and quantifying specific antibodies. Paired sera from the same animals, collected 7–14 days apart, may be needed
depending on the serological assay being used and prior history of vesicular stomatitis in the animals.

Specific reagents for VS diagnosis are not commercially available and each laboratory must produce its own or
obtain them from a Reference Laboratory. The WOAH Reference Laboratories for vesicular stomatitis 1 produce and
distribute diagnostic reagents on request.

Table 1. Test methods available for the diagnosis of vesicular stomatitis and their purpose

Purpose

Population Individual animal Contribute Immune status in

Method Confirmation Prevalence of
freedom freedom from to individual animals or
of clinical infection –
from infection prior to eradication populations post-
cases surveillance
infection movement policies vaccination(a)

Detection of the agent(b)

Virus isolation(C) – + – +++ – –

IS-ELISA(C) – + – +++ – –

CFT(C) – + – ++ – –

RT-PCR(C) – + – ++ – –

Detection of immune response(d)

LP-ELISA(e) ++ ++ ++ ++ ++ ++

C-ELISA(e) +++ ++ ++ – +++ ++

VNT(e) +++ +++ +++ ++ ++ +++

CFT(e) – + + ++ + –

Key: +++ = recommended for this purpose; ++ recommended but has limitations;
+ = suitable in very limited circumstances; – = not appropriate for this purpose.
IS-ELISA = indirect sandwich enzyme-linked immunosorbent assay; CFT = complement fixation test; RT-PCR = reverse transcription
polymerase chain reaction; LP-ELISA = liquid-phase blocking ELISA; C-ELISA = competitive ELISA; VNT = virus neutralisation test.
(a)
Indicates the presence of antibodies only; does not indicate protection from infection.
(b)
A combination of agent identification methods applied on the same clinical sample is recommended.
(C)
Should only be used on animals demonstrating clinical signs compatible with VSV. A positive result is meaningful. A negative result could
mean the animal is no longer shedding virus, the virus level is too low to detect, or, for virus isolation samples that the samples were not
maintained at appropriate temperatures and received in an appropriate time period following collection for virus isolation (virus inactivated).
(d)
One of the listed serological tests is sufficient
(e)
The presence of VSV antibodies only indicates prior exposure to VSV. It does not determine whether the antibodies are due to current
or past infection. Paired sera from the same animals, collected at least 7–14 days apart, may be needed to evaluate seroconversion
depending on the serological assay being used and prior history of VS. Interpretation of results needs to be based on serological results,
clinical presentation, and epidemiology. CF antibody duration in an animal is generally less than 1 year. Antibodies detected by the VNT
and competitive ELISAs can be detected for years following infection. The difference in sensitivity of the serological assays has an effect
on detection during the acute phase of infection; combination testing, such as C-ELISA and CF or paired sampling showing four-fold
titre change (CFT, VNT, LP-ELISA), is therefore necessary when an animal presents with acute clinical signs of VS.

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

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 Chapter 3.1.24. – Vesicular stomatitis

1. Detection of the agent

1.1. Direct visualisation

Due to the different morphological characteristics of the rhabdovirus (VS serogroup viruses),
picornavirus (FMD virus, SVD virus and senecavirus A), calicivirus (VES) and the large number of virus
particles present in vesicular fluids and epithelial tissues, electron microscopy can be a useful diagnostic
tool for differentiating the virus family involved.

1.2. Virus isolation in cell culture

For identification of VS viruses and the differential diagnosis of vesicular diseases, clarified suspensions
of field samples suspected to contain virus should be submitted for testing. For virus isolation, the
samples are inoculated into appropriate cell cultures. The inoculation of African green monkey kidney
(Vero), baby hamster kidney (BHK-21) and IB-RS-2 cell cultures with the same sample permits
differentiation of the vesicular diseases: VS viruses cause a cytopathic effect (CPE) in all three cell lines;
FMD virus causes a CPE in BHK-21 and in IB-RS-2, while SVD virus causes a CPE in IB-RS-2 only. Many
other cell lines, as well as most primary cell cultures of animal origin, are susceptible to VS serogroup
viruses.

1.2.1. Test procedure

i) Where tissue has been collected in phosphate-buffered saline (PBS)/glycerol solution, it
should be blotted dry on absorbent paper to reduce the glycerol content, which is toxic for
cell cultures, and weighed. A suspension is then prepared using a tissue grinder or by
grinding the sample in sterile sand in a sterile pestle and mortar with a small volume of tissue
culture medium and antibiotics. Further medium should be added until a final volume of nine
times that of the original sample has been added, giving a 10% suspension. For swabs
samples, a 10% solution is made using the cell culture medium. The sample is clarified on a
bench centrifuge at 2000 g for at least 10 minutes.
ii) The clarified suspension of tissues, swab or vesicular fluid from field samples suspected to
contain VSV are inoculated onto cell culture vessels (BHK-21, IB-RS-2 or Vero cells).

iii) Incubate inoculated cell cultures at 37°C for 1 hour (adsorption).

iv) Discard inoculum and wash cell cultures three times with cell culture medium and replace
with cell culture medium containing 2.5% fetal bovine serum (FBS).
v) Incubate plates, plastic tubes or flasks cell cultures at 35–37°C and observe for cytopathic
effect (CPE). The cell cultures should be examined for CPE for 48–72 hours. If, after 72 hours,
no CPE has been detected, a blind passage must be made. The cell culture is freeze–thawed
and clarified by centrifugation, and the supernatant is used for inoculation of fresh
monolayers or cell suspension. The sample is considered to be negative if there is no
evidence of a CPE after on average of three blind passages in cell cultures; longer passages
of up to 7 days may be conducted using fewer passages.
vi Reverse-transcription polymerase chain reaction (RT-PCR) may be used to identify virus
recovery, using appropriate sets of primers (Rainwater-Lovett et al., 2007; Sepulveda et al.,
2007). Alternatively, immunological methods for the identification of the viral antigens can
be used such as enzyme-linked immunosorbent assay (ELISA) (Alonso et al., 1991; Ferris &
Donaldson, 1988), the complement fixation test (CFT) (Alonso et al., 1991; Jenny et al., 1958)
or fluorescent antibody staining. The virus neutralisation test (VNT), with known positive
antisera against VSNJV and VSIV, may be used in tissue cultures or embryonated eggs, but
it is more time-consuming.

1.3. In-ovo testing

The virus replicates and can be isolated in 8- to 10-day-old chicken embryos by inoculation into the
allantoic sac.

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1.4. Enzyme-linked immunosorbent assay

The indirect sandwich ELISA (IS-ELISA) (Alonso et al., 1991; Ferris & Donaldson, 1988) is a common
diagnostic method of choice for identification of VS and other vesicular diseases. Specifically, the ELISA
procedure with a set of polyvalent rabbit/guinea-pig antisera, prepared against virions for representative
strains of VSIV, COCV, and VSAV (Alonso et al., 1991). For detection of VSNJV, a monovalent set of
rabbit/guinea-pig antisera is suitable (Alonso et al., 1991; Ferris & Donaldson, 1988).

1.4.1. Test procedure

i) Solid phase: ELISA plates are coated either for 1 hour at 37°C or overnight at 4°C with rabbit
antisera and normal rabbit serum (as described in Alonso et al., 1991), and optimally diluted
in carbonate/bicarbonate buffer, pH 9.6. Subsequently, the plates are washed once with
PBS and blocked for 1 hour at room temperature with 1% ovalbumin Grade V (grade of
purification) in PBS. After washing, the plates can be used immediately or stored at
–20°C for future use.
ii) Test samples: Antigen suspensions of test samples (10–20% epithelial tissue suspension, in
PBS or MEM or undiluted clarified cell culture supernatant fluid) are deposited in the
corresponding wells and the plates are incubated for 1 hour at 37°C on an orbital shaker.
iii) Detector: Monovalent guinea-pig antisera to VSNJV and polyvalent guinea-pig antisera to
VSIV, COCV and VSAV, that are homologous to coated rabbit serum and that have been
diluted appropriately in PBS containing 0.05% Tween 20, 1% ovalbumin Grade II, 2% normal
rabbit serum, and 2% normal bovine serum (PBSTB) are added to the corresponding wells
and left to react for 30–60 minutes at 37°C on an orbital shaker.
iv) Conjugate: Peroxidase/rabbit or goat IgG anti-guinea-pig Ig conjugate, diluted in PBSTB, is
added and left to react for 30–60 minutes at 37°C on an orbital shaker.
v) Substrate: H2O2-activated substrate is added and left to react at room temperature for
15 minutes, followed by the addition of sulphuric acid to stop the reaction. Absorbance
values are measured using an ELISA reader.
Throughout the test, 50 µl reagent volumes are used. The plates are washed three–five
times between each stage with physiological saline solution or PBS containing 0.05% Tween
20. Controls for the reagents used are included.
vi) Interpretation of the results: Absorbance values of positive and negative antigen control
wells should be within specified values for acceptance. Sample wells giving an absorbance
≥0.3 are considered to be positive for the corresponding virus subtype. Absorbance values
<0.3–0.2 are considered suspicious and values <0.2 are considered negative for the
corresponding virus subtype. Suspicious and negative samples should be inoculated in cell
culture and passages re-tested in ELISA.

1.5. Complement fixation test

The IS-ELISA is preferable to the CFT because it is more sensitive and it is not affected by pro- or anti-
complementary factors. When ELISA reagents are not available, however, the CFT may be performed.
The CFT in U-bottomed microtitre plates, using the reagents titrated by CF50% test, is described.

1.5.1. Test procedure

i) Antisera: Monovalent guinea-pig antisera to VSNJV and polyvalent guinea-pig antisera to
VSIV, COCV and VSAV, diluted in barbital buffer or an alternative CF buffer at a dilution
containing 2.5 CFU50 (50% complement fixation units) against homologous virus, are
deposited in plate wells. Those antisera are the detectors used in ELISA.
ii) Test samples: The antigen suspension of test samples, prepared as described for IS-ELISA,
is added to the wells with serum.
iii) Complement: 4 CHU50 (50% complement haemolytic units) are added to the serum and
antigen. (An alternative is to use 7.5, 10 and 20 CHU50 with the goal of reaching 4 CHU50 in
the test.) The mixture of antisera, test samples and complement is incubated at 37°C for
60 minutes.

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iv) Haemolytic system: A suspension of sheep red blood cells (SRBC) in CF buffer, sensitised
with 10 HU50 (50% haemolytic units) of rabbit anti-SRBC serum, is added to the wells. The
haemolytic system has an absorbance of 0.66 read at 545 nm, in the proportion of two
volumes of haemolytic system + three volumes of distilled water. The mixture is incubated
for 30 minutes at 37°C. Subsequently, the plates are centrifuged and the reaction is
observed visually.
Volumes of 25 µl for antisera, test samples and complement, and 50 µl of haemolytic system,
are required. Appropriate controls for the antisera, antigens, complement and haemolytic
system are included.
It is possible to perform the CF50% test in tubes (Alonso et al., 1991) using reagent volumes
of 200 µl (eight times greater than those indicated for the CFT in microtitre plates). With the
CF50% test, the reaction can be expressed as absorbance read spectrophotometrically at
545 nm.
v) Interpretation of the results: When controls react as expected, samples with haemolysis
<20% for one antiserum in comparison with the other antiserum and controls are considered
to be positive for the corresponding type.
Field samples that are negative by the ELISA or CFT should be inoculated into cell culture. If
there is no evidence of viral infection after three passages, the specimen is considered to be
negative for virus.

1.6. Molecular methods

The RT-PCR can be used to amplify small genomic areas of the VS virus (Hofner et al., 1994; Hole et al.,
2010; Rodriguez et al., 1993; Wilson et al., 2009). This technique will detect the presence of virus RNA in
tissue and vesicular fluid samples and cell culture, but cannot determine if the virus is infectious.

The diagnosis of VS in samples of epithelium, vesicular fluid and cell culture by molecular methods is the
most suitable for confirmation of the disease. No screening protocol that allows the detection of all
species of vesiculovirus of economic interest has been described. However, there are some multiplex
tests described below that allow the detection of more than one viral species. Therefore, for a differential
diagnosis, different protocols are needed to detect all VS viruses.

Extraction of nucleic acid from the sample must be done according to the manufacturer’s instructions.

Methods for detecting and typing the four VS viruses have been described using both real-time and
conventional RT-PCR.

1.6.1. Real-time RT-PCR detection and typing

VSNJV, VSIV, COCV and VSAV can be detected and typed using the real-time RT-PCR methods
described by Hole et al. (2010) with adaptations, de Oliveira et al. (2018) and Sales et al. (2020),
and the primers and probes described in Table 2.

Hole et al. (2010) developed a real-time RT-PCR assay that allows differentiation between VSIV
and VSNJV with good performance. Primer concentrations must be 0.2 mM except the reverse
VSNJV, which is 0.8 mM, and probe concentrations are 0.2 mM for VSNJV and 0.1 mM for VSIV.
The real-time RT-PCR protocol consists of reverse transcription cycles at 50°C for 30 minutes
and denaturation at 95°C for 1 minute, followed by 45 cycles of 95°C for 15 seconds, 54°C for
30 seconds and 72°C for 60 seconds. The reaction is performed according to the kit
manufacturer’s instructions, adding MgSO4 (4 mM).

Due to the extent of genetic variation of this virus in some regions, de Oliveira et al. (2018)
developed a method for detecting VSAV by multiplex real-time RT-PCR with excellent sensitivity
and specificity. The test is performed according to the kit manufacturer’s instructions and the
concentration of the primers in a final volume of 25 µl was 0.4 µM. The real-time RT-PCR protocol
consists of reverse transcription cycles at 50°C for 10 minutes and denaturation at 95°C for
5 minutes followed by 45 cycles of 95°C for 10 seconds and 60°C for 1 minute.

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Sales et al. (2020) described a real-time RT-PCR protocol for the diagnosis of COCV. The test is
carried out according to the kit manufacturer’s instructions, with the concentration of each primer
0.1 µM and probe 0.4 µM in a final volume of 25 µl. The real-time RT-PCR protocol consists of
reverse transcription cycles of 45°C for 10 minutes and denaturation of 95°C for 5 minutes,
followed by 45 cycles of 95°C for 10 seconds and 60°C for 1 minute.

Table 2. Oligonucleotides target for real-time RT-PCR of VSV

Real-time RT-PCR for vesicular stomatitis

Multiplex New Jersey vesiculovirus and Indiana vesiculovirus

Name Sequence (5 ’→ 3’) Reference

VSNJV 7230-7254 Forward: TGA-TTC-AAT-ATA-ATT-ATT-TTG-GGA-C

VSNJV 7476-7495 R Reverse: AGG-CTC-AGA-GGC-ATG-TTC-AT

VSNJV 7274-7296 P 1 Probe: FAM-TTT-ATG-CAT-GAC-CCW-GCA-ATA-AG-NFQ-MGB

VSNJV 7334-7353 P 2 Probe: FAM-TTG-CAC-ACC-AGA-ACA-TTC-AA-BHQ1 Hole et al., 2010

VSIV 7230-7254 IN F Forward: TGA-TAC-AGT-ACA-ATT-ATT-TTG-GGA-C

VSIV 7433-7456 IN R Reverse: GAG-ACT-TTC-TGT-TAC-GGG-ATC-TGG

VSIV 7274-7290 P Probe: VIC-ATG-ATG-CAT-GAT-CCA-GC-NFQ-MGB

Alagoas vesiculovirus
Name Sequence (5’ → 3’) Reference

VSAV-3.GP.95.F Forward: GGG-TWA-ACA-TCC-GTG-CTA

VSAV-3.GP.95.R Reverse: GTC-ACA-AGT-GGT-GAT-CCA

VSAV-3.GP.95.S Probe: FAM-cac+Atc+Cat+Cca+Tcagc-IowaBlack

VSAV.LP.78.F Forward: GTC-CAT-CAA-CCC-ATT-GTT-CC

VSAV.LP.78.R Reverse: ATC-AAT-CCA-TCT-GCG-ACT-CC de Oliveira et al.,

2018
Probe: FAM-CGC-GAT-TCT-TAA-GTG-AGT-TCA-AAT-CAG-GA-
VSAV.LP.78.S
IowaBlack

VSAV.GP.87.F Forward: GAG-TGT-GGA-TCA-ACC-CAG

VSAV.GP.87.R Reverse: CTG-TGG-CTT-GAA-CRA-TCA

VSAV.GP.87.S Probe: FAM-CTGC+GGTTATG+CC+TCCA-IowaBlack

Cocal vesiculovirus
Name Sequence (5’ → 3’) Reference

COCV.GP.81.F Forward: CGT-TGC-TGT-GAT-TGT-YCA

COCV.GP.81.R Reverse: GGG-AAC-TGG-GAG-TCA-ATC Sales et al., 2020
COCV.GP.81.S Probe: FAM-ctc+Atc+Cac+Caa+Cacat -BHQ1

1.6.2. Conventional RT-PCR detection and typing

VSNJV, VSIV, COCV and VSAV can also be detected and typed using the RT-PCR methods
described by Rodriguez et al. (1993) and Pauszek et al. (2008; 2011), and the primers described in
Table 3. After obtaining the reverse transcription cDNA, the conventional PCR is undertaken. The
protocol for all RT-PCR described consists of cycles of denaturation at 94°C for 3 minutes
followed by 40 cycles of 94°C for 1 minute, 50°C for 1 minute and 72°C for 1 minute, and the
extension step should be 72°C for 5 minutes.

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 Chapter 3.1.24. – Vesicular stomatitis

de Oliveira et al. (2018) developed an RT-PCR to detect Cocal vesiculovirus. The test is performed
according to the kit manufacturer’s instructions, with the concentration of each primer being 1 µM
in a final volume of 20 µl. The RT-PCR protocol consists of reverse transcription cycles at 50°C for
30 minutes and denaturation at 95°C for 15 minutes followed by 40 cycles of 94°C for 1 minute,
54°C for 1 minute and 72°C for 1 minute.

Table 3. Primers and references for conventional RT-PCR of VSV

Conventional RT-PCR for vesicular stomatitis virus

New Jersey vesiculovirus and Indiana vesiculovirus

Name Sequence (5’ → 3’) Reference

VSNJV P102 Forward: GAG-AGG-ATA-AAT-ATC-TCC

VSNJV P744 Reverse: GGG-CAT-ACT-GAA-GAA-TA
Rodriguez et al., 1993
VSNJV P179 Forward: GCA-GAT-GAT-TCT-GAC-AC

VSNJV P793 Reverse: GAC-TCT-(C/T)GC-CTG-(A/G)TT-GTA

Cocal vesiculovirus and Alagoas vesiculovirus

Name Sequence (5’ → 3’) Reference
COCV P66 Forward: AAT-TGG-ATG-ACG-CMG-TCC-A

COCV P711 Reverse: CCT-CCD-ACH-GAR-ATG-AAY-TCT-CC

Pauszek et al., 2008
VSAV PJX Forward: TAT-GAA-AAA-AAI-TAA-CAG-IIA-TC

VSAV P711 Reverse: CCT-CCD-ACH-GAR-ATG-AAY-TCT-CC

Nested PCR for Cocal vesiculovirus and Alagoas vesiculovirus

Name Sequence (5’ → 3’) Reference

COCV P169 Forward: TTA-CCA-AAA-TCA-GGA-GGA-TGA

COCV P686 Reverse: GCC-TCC-CAC-CGA-GAT-G

Pauszek et al., 2011
VSAV P163 Forward: AGA-GCA-GCT-CCY-TCT-TAT-TAT
VSAV P691 Reverse: TCA-TCA-TTC-CAT-TTC-CTC

One step RT-PCR for Alagoas vesiculovirus

Name Sequence (5’ → 3’) Reference

VASV - P.722 F Forward: GGG-GCC-ATT-CAA-GAG-ATA-GA

de Oliveira et al., 2018
VASV -P.722 R Reverse: TGA-TAT-CTC-ACT-CTG-GCC-TGA-TTA-T

2. Serological tests

For the identification and quantification of specific antibodies in serum, the ELISA and the VNT are preferable. The
CFT may be used for quantification of early antibodies. Antibody can usually be detected between 5 and 8 days
post-infection; the length of time antibody persists has not been accurately determined for the three tests but is
thought to be relatively short for the CFT and for extended periods for the VNT and ELISA (Katz et al., 1997).

The competitive ELISA (C-ELISA) may be preferable to the CFT because it is more sensitive and it is not affected by
pro- or anti-complementary factors; however during an outbreak where there may be previously exposed animals,
appropriate assay selection, and paired serum samples collected at least 7–14 days apart are important to ensure
ability to distinguish recent from past exposure. When C-ELISA reagents are not available however, the CFT may
be performed. The CFT in U-bottomed microtitre plates, using the reagents titrated by CF50% test, is described.

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2.1. Liquid phase blocking enzyme-linked immunosorbent assay

The liquid-phase blocking ELISA (LP-ELISA) is a method for the detection and quantification of
antibodies to VS serogroup viruses. The use of viral glycoproteins as antigen is recommended because
they are not infectious, allow the detection of neutralising antibodies, and give fewer false-positive
results than the VNT (Allende et al., 1992).

2.1.1. Test procedure

i) Solid phase: As described above in Section B.1.5 for the IS-ELISA.
ii) Liquid phase: Duplicate, two- to five-fold dilution series of each test serum, starting at 1/4,
are prepared in U-bottomed microtitre plates. An equal volume of VSNJV or VSIV
glycoprotein, in a predetermined dilution, is added to each well and the plates are incubated
for 1 hour at 37°C. 50 µl of these mixtures is then transferred to the ELISA plates with the
solid phase and left to react for 30 minutes at 37°C on an orbital shaker.
iii) Detector, conjugate and substrate: The same steps described for the IS-ELISA are
performed using monovalent antisera homologous to the test antigen, as detectors
iv) Interpretation of the results: 50% end-point titres are expressed in log10 in reference to the
50% OD of the antigen control, according to the Spearmann–Kärber method. Titres of >1.0
(1/10) are considered to be positive.

2.2. Competitive enzyme-linked immunosorbent assay

A C-ELISA for detection of antibodies has also been developed. The procedure described here is based
on a procedure described by Afshar et al. (1993). It uses vesicular stomatitis VSNJV and VSIV
recombinant antigens as described by Katz et al. (1995).

2.2.1. Test procedure

i) Solid phase: Antigens are diluted in carbonate/bicarbonate buffer, pH 9.6, and 75 µl is added
to each well of a 96-well ELISA plate. The plates are incubated overnight at 4°C; coated
plates can be frozen, with antigen in situ, at –70°C for up to 30 days. The plates are thawed,
antigen is decanted, and 100 µl of blocking solution (5% nonfat dry milk powder solution in
PBS [for example, 5 g dry milk powder dissolved in 95 ml PBS) is added. The plates are then
incubated at 25°C for 15–30 minutes and blocking solution is decanted. The plates are
washed three times with PBS/0.05% Tween 20 solution.

ii) Liquid phase: 50 µl of serum diluted 1/8 in 1% nonfat dry milk in PBS is added to each of the
duplicate wells for each sample. A positive and negative control serum for each virus
species should be included on each ELISA plate. The plates are incubated at 37°C for
30 minutes. Without washing, 50 µl of bioreactor fluid is added to each well and plates are
incubated at 37°C for 30 minutes.

iii) Detector: The plates are washed three times, and 50 µl of goat anti-mouse horseradish-
peroxidase conjugate diluted in 1% nonfat dry milk with 10% normal goat serum is added to
each well. The plates are incubated at 37°C for 30 minutes, washed three times, and 50 µl of
tetramethyl-benzidine (TMB) substrate solution is added to each well. The plates are
incubated at 25°C for 5–10 minutes and then 50 µl of 0.05 M sulphuric acid is added to each
well. The plates are read at 450 nm and the optical density of the diluent control wells must
be > 1.0.

iv) Interpretation of the results: A sample is positive if the absorbance is ≤50% of the
absorbance of the diluent control. Note that horses naturally infected with VSNJV virus have
been known to test positive by this assay for at least 8 years following infection.

2.3. Virus neutralisation test

Virus and cells: VSIV, COCV, VSAV and VSNJV are propagated in BHK, IB-RS-2 or Vero cell monolayers
and stored in liquid nitrogen or frozen at –70°C (Allende et al., 1992).

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2.3.1. Test procedure

i) The test sera are heat-inactivated, including control standard sera, for 30 minutes at 56°C.
ii) Starting from 1/8 dilution, sera are diluted twofold or fourfold in cell culture medium, and the
dilution series is continued in a cell-culture grade flat-bottomed 96-well microtitre plate
using at least two rows of wells, preferably four rows (depending on the degree of precision
required) and a volume of 50 μl per well. An extra well with 1/8 dilution test serum is used for
toxicity control of sera. Dilutions of control sera with known titres (positive, weak positive
and negative) are also included in the test.
iii) Add 50 μl per well of the VSV strain (VSIV, COCV, VSAV and VSNJV) stock at a dilution in
culture medium calculated to provide 100 TCID50 (50% tissue culture infective dose) per
well. In the toxicity control wells, add 50 μl of culture medium in place of virus. Add 100 μl of
culture medium to one row of empty wells as cell controls. A back titration of virus stock is
also undertaken, at least four wells per dilution, to check the potency of the virus (dose
acceptance limits 32–320 TCID50 per well). An alternative protocol can be a viral dose of
1000 TCID50 per well (tolerance range between 750 and 1270 TCID50) to increase the
specificity of the test.
iv) Incubate the plates for 60 minutes at 37°C in a 3–5% CO2 atmosphere to allow viral
neutralisation.
v) Add 100 μl per well of the BHK, IB-RS-2 or Vero cell suspension at 3 × 105 ml, containing 10%
FBS for cell growth.
vi) Incubate the plates for 48–72 hours at 37°C, either in a 3–5% CO2 atmosphere or seed the
plate with pressure-sensitive tape and incubate.
vii) Check the cell cultures for the onset of CPE and read the results: positive monolayer wells –
where the virus has been neutralised, have no CPE and the cells remain intact (blue-stained
cells sheets); negative wells – where virus has not been neutralised, have a CPE (empty
cavity – no staining). If staining is used fix the cells with 10% formol/saline for 30 minutes.
For staining, the plates are immersed in 0.1% crystal violet in 1 % ethanol and 5 % buffered
formalin for 30 minutes.
vii) Validate the test by checking the back titration of the virus (which should give a value of
100 TCID50 per well with a permissible range of 32–320 TCID50), the control sera and the cell
control wells. The positive control serum should give a titre of twofold dilution (±0.3 log10
units) from its target value. The weak positive serum should be positive. The negative serum
should give no neutralisation. In the cell control wells, the monolayers should be intact.
iv) Interpretation of the results: Virus neutralising titres of serum antibody responses (titres are
expressed as the final dilution of serum present in the serum/virus mixture where 50% of
wells are protected). This can be calculated by the Spearman–Kärber or Reed Muench
methods. The 50% neutralisation titre of each serum is expressed as log 10. When only two
repetitions per dilution are performed, the highest inverse of the dilution that neutralised
100% of the cavities can be considered. In general, a titre of ≥32 (1.5) or more of the final
serum dilution in the serum/virus mixture is regarded as positive for VSV antibodies.
Laboratories are encouraged to verify this cut-off internally, with reagents provided by a
WOAH Reference Laboratory, where available. If cytotoxicity is observed in the control wells,
the sample is reported to be toxic (no result) unless neutralisation of the virus without
cytotoxicity is observed at higher dilutions and a titre can be read without ambiguity
(Allende et al., 1992).
Note: Seroconversion is considered when there is a four-fold increase in antibody titre between
paired serum samples collected with a minimum interval of 7–14 days between the first (acute
phase of the disease) and the second blood collection (convalescent phase).

2.4. Complement fixation test

A detailed description of this test is given in Section B.1.5. This is modified as follows. The CFT may be
used for quantification of early antibodies, mostly IgM. For this purpose, twofold serum dilutions are
mixed with 2 CFU50 of known antigen and with 5% normal bovine or calf sera included in 4 CHU50 of
complement. The mixture is incubated for 3 hours at 37°C or overnight at 4°C. Subsequently, the
haemolytic system is added followed by incubation for 30 minutes at 37°C. The serum titre is the highest

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dilution in which no haemolysis is observed. Titres of 1/5 or greater are positive. This CFT has low
sensitivity and is frequently affected by anticomplementary or nonspecific factors.

C. REQUIREMENTS FOR VACCINES

1. Background

1.1. Rationale and intended use of the product

VSV infections can have significant impacts on the health and production aspects of animals, resulting
in considerable economic losses for producers. Reduced feed intake caused by oral lesions can result in
weight loss and delays to market. Lesions on the feet can cause temporary locomotor problems affecting
the ability of an animal to obtain food and water, and permanent foot problems that result in the animal
being culled. Lesions of the mammary gland can impact the ability of the dam to nurse her offspring and
for harvesting milk for sale. Animals may be culled if mammary or teat lesions are severe. Although
vaccination is not widely practised, vaccine is used to reduce the severity of clinical signs and the
economic impacts of the disease.

Attenuated virus vaccines have been tested in the field in the USA, Panama, Guatemala, Peru and
Venezuela (Lauerman et al., 1962; Mason, 1978) with unknown efficacy. Killed vaccines for VSIV and
VSNJV are manufactured in Colombia and Venezuela (2002 WOAH vaccine survey). Although a
commercial vaccine combining VS and FMD antigens in a single emulsion for Andean countries has been
tested in vaccination–challenge experimentation and published (House et al., 2003), the vaccine is not
produced/applied routinely.

Guidelines for the production of veterinary vaccines are given in Chapter 1.1.8 Principles of veterinary
vaccine production. The guidelines given here 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 conventional vaccines

2.1. Characteristics of the seed

2.1.1. Biological characteristics

Identity of the seed and the source of the serum used in growth and passage of the virus should
be well documented, including the source and passage history of the organism.

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

The purity of the seed and cells to be used for vaccine production must be demonstrated. The
master seed virus (MSV) should be free from adventitious agents, bacteria, or Mycoplasma, using
tests known to be sensitive for detection of these microorganisms. The test aliquot should be
representative of a titre adequate for vaccine production, but not such a high titre that
hyperimmune antisera are unable to neutralise seed virus during purity testing. Seed virus is
neutralised with monospecific antiserum or monoclonal antibody against the seed virus and the
virus/antibody mixture is cultured on several types of cell line monolayers. A cell line highly
permissive for bovine viral diarrhoea virus, types 1 and 2, is recommended as one of the cell lines
chosen for evaluation of the MSV. Bovine viral diarrhoea virus is a potential contaminant
introduced using FBS in cell culture systems. Cultures are subpassaged at 7-day intervals for a
total of at least 14 days, then tested for adventitious viruses that may have infected the cells or
seed during previous passages.

2.2. Method of manufacture

2.2.1. Procedure
Once the vaccine is shown to be efficacious, and the proposed conditions for production are
acceptable to regulatory authorities, approval may be granted to manufacture vaccine. Virus

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 Chapter 3.1.24. – Vesicular stomatitis

seed can be grown in cell culture. Selection of a cell type for culture is dependent on the degree
of virus adaptation, growth in medium, and viral yield in the specific culture system. Vaccine
products should be limited to the number of passages from the MSV that can be demonstrated
to be effective. Generally, large-scale monolayer or suspension cell systems are operated under
strict temperature-controlled, aseptic conditions and defined production methods, to assure lot-
to-lot consistency. Dose of virus used to inoculate cell culture should be kept to a minimum to
reduce the potential for viral defective interfering particles. When the virus has reached its
appropriate titre, as determined by CPE, fluorescent antibody assay, or other approved
technique, the virus is clarified, filtered, and inactivated (for killed vaccines).

2.2.2. Requirements for substrates and media

Cell cultures should be demonstrated free of adventitious viruses. All animal origin products used
in the production and maintenance of cells (i.e. trypsin, FBS) and growth of virus should be free of
adventitious agents, with special attention paid to the presence of bovine viral diarrhoea virus.

2.2.3. In-process controls

Cell cultures should be checked macroscopically for abnormalities or signs of contamination and
discarded if unsatisfactory. Virus concentration can be assessed using antigenic mass or
infectivity assays.

An inactivation kinetics study should be conducted using the approved inactivating agent (β-
propiolactone or 1 ml for 100 ml of viral suspension of 0.1 M binary ethyleneimine [BEI during
24 hours]) on each viral lot with a titre greater than the maximum production titre and grown using
the approved production method. This study should demonstrate that the inactivation method is
adequate to assure complete inactivation of virus. Samples taken at regular timed intervals
during inactivation, then inoculated on to a susceptible cell line, should indicate a linear and
complete loss of titre by the end of the inactivation process.

During production, antigen content is measured to establish that minimum bulk titres have been
achieved. Antigen content is generally measured before inactivation (if killed vaccine) and prior
to further processing.

2.2.4. Final product batch tests

Vaccine candidates should be shown to be pure, safe, potent, and efficacious.

i) Sterility and purity

During production, tests for bacteria, Mycoplasma, and fungal contamination should be
conducted on both inactivated and live vaccine harvest lots and confirmed on the
completed product (see Chapter 1.1.9 Tests for sterility and freedom from contamination of
biological materials intended for veterinary use).

ii) Safety
The use of target animal batch release safety tests or laboratory animal batch release safety
tests should be avoided wherever possible.

iii) Batch potency

Potency is examined on the final formulated product. Mirroring what is done for the potency
test in FMD vaccines, a vaccination–challenge test has being proposed for testing VSV
vaccines (House et al., 2003). The gaps in knowledge regarding the pathogenesis of VSV
infection and the immune mechanism that affords protection against viral infection are
limitations for development and implementation of a validated protocol for a challenge test.
However, for batch release, indirect tests can also be used for practicability and animal
welfare considerations, as long as correlation has been validated to protection in the target
animal during efficacy tests. Frequently indirect potency tests include antibody titration
after vaccination of target species. Ideally, indirect tests are carried out for each strain for
one species and each formulation of vaccine to establish correlation between the indirect
test results and the vaccine efficacy test results.

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Relative potency could be used to determine antigen content in final product. It is necessary
to confirm the sensitivity, specificity, reproducibility, and ruggedness of such assays.

2.3. Requirements for regulatory approval

2.3.1. Safety requirements

i) Target and non-target animal safety
Final product may be evaluated in the host animal using two animals of the minimum age
recommended for use, according to the instructions given on the label; the animals are
observed for 21 days. Field safety studies conducted on vaccinates, in at least three
divergent geographical areas, with at least 300 animals per area, are also recommended.

For killed and modified live virus (MLV) vaccines product safety will be based on an absence
of adverse reactions such as shock, abscesses at site of inoculation, etc. In the specific case
of MLV vaccines, it would not be expected to see clinical signs. If clinical signs of vesicular
stomatitis virus are observed, use of the vaccine should be reconsidered. Residual virus
should be evaluated for prior to mixing the antigen with adjuvant. Initial safety is evaluated
in a few animals for 21 days under close observation to assess for gross safety issues. If the
vaccine passes this first safety test, the vaccine is used in the field in a larger number of
animals to evaluate if subtle safety issues are present: adverse reactions/swelling,
abscesses, shock, etc.

ii) Reversion-to-virulence for attenuated/live vaccines

Reversion to virulence for live viral vaccines is often demonstrated by back passage through
susceptible species. Virus is isolated from the vaccinated animal and the isolated virus is
then used to inoculate additional animals. Sequential passage through animals should show
that animals remain clinically healthy with no demonstration of typical vesicular stomatitis
lesions.

iii) Environmental consideration

Inactivated vesicular stomatitis vaccines probably present no special danger to the user,
although accidental inoculation may result in an adverse reaction caused by the adjuvant
and secondary components of the vaccine. MLV vaccines may pose a hazard to the user
depending on the level of inactivation of the virus.

Preservatives should be avoided if possible, and where not possible, should be limited to the
lowest concentration possible. Vaccine bottles, syringes, and needles may pose an
environmental hazard for vaccines using adjuvants or preservatives and for MLV vaccines.
Instructions for disposal should be included within the vaccine packaging information and
based on current environmental regulations in the country of use.

2.3.2. Efficacy requirements

The gaps in knowledge regarding the pathogenesis of VSV infection and the immune mechanism
that affords protection against viral infection are limitations for the development and
implementation of a validated protocol for an efficacy test. Ideally vaccine efficacy should be
estimated in vaccinated animals directly by evaluating their resistance to live virus challenge.
Vaccine efficacy should be established for every strain to be authorised for use in the vaccine.

Live reference VSV viruses corresponding to the virus strains circulating in the region are stored
at ultralow temperatures. Each challenge virus is prepared as follows. Tongue tissue infected by
VSV should be obtained from original field case of VS and received at the Reference Laboratory
in glycerol buffer as described in Section B. Diagnostic Techniques.

The preparation of cattle challenge virus follows the process described in Chapter 3.1.8 Foot and
mouth disease, Section B.1.1 Virus isolation, with the view of obtaining a sterile 10% suspension in
Eagles minimal essential medium with 10% sterile FBS.

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 Chapter 3.1.24. – Vesicular stomatitis

The preparation of the stock of challenge virus to be aliquoted is prepared starting from lesions
collected in two cattle over 6 months of age, previously recognised to be free of VSV antibodies.
These animals are tranquillised, for example using xylazine 100 mg/ml (follow instructions for
use), then inoculated intradermally (i.d.) in the tongue with the suspension in about 20 sites, 0.1 ml
each. The vesiculated tongue tissue is harvested at the peak of the lesions, approximately 2 days
later.

A 2% suspension is prepared as above and filtered through a 0.2 µm filter, aliquoted and frozen in
the gas phase of liquid nitrogen, and constitutes the stock of challenge virus. The infective titres
of this stock are determined both in cell culture (TCID50) and in two cattle (BID50: 50% bovine
infective dose). These two cattle that have been tranquilised using xylazine, are injected
intradermally in the tongue with tenfold dilutions (1/10 through 1/10,000), using four sites per
dilution (Henderson, 1949). The cattle titrations are read 2 days later. Most frequently, titres are
above 106 TCID50 for 0.1 ml and above 105 BID50 for 0.1 ml calculated using the Spearmann–Kärber
method. The dilution for use in cattle challenge test is 10 000 DIB50 in a total volume of 4× 0.1 ml
by intralingual injection for both the 50% protective dose (PD50) test and the PGP (protection
against generalised foot infection) test (House et al., 2003).

i) Vaccination–challenge method
For this experimental method, a group of 12 VSV sero-negative cattle of at least 6 months of
age are vaccinated with a bovine dose by the route and in the volume recommended by the
manufacturer at day 0 and day 40. These animals and a control group of two non-vaccinated
animals are challenged 2 weeks or more after the second vaccination. The challenge strain
is a suspension of bovine virus that is fully virulent and appropriate to the virus types in the
vaccine under test by inoculating a total of 10,000 BID50 intradermally into four sites (0.1 ml
per site) on the upper surface of the tongue. Animals are observed at 7–8 days after
challenge.

It was proposed that vaccinated animals showing no lesion on the tongue should be
considered fully protected. Vaccinated animals showing lesions at one, two, or three
inoculation sites should be considered partially protected, and animals showing lesions at
four sites are considered not protected (House et al., 2003). Control animals must develop
lesions at four sites. Vaccine should fully protect at least nine animals out of 12 vaccinated
(75% protection), the remaining animals being partially or not protected. This test gives a
certain measure of the protection following the injection of two commercial bovine doses of
vaccine in a limited cattle population.

Although the vaccination-challenge method has been described and published (House et
al., 2003) data on the validation under field conditions for the efficacy of released vaccine
are not available.

ii) Efficacy in other species

Efficacy tests in other target species, such as horses, are not yet described or standardised.
In general, a successful test in cattle should be considered to be sufficient evidence of the
quality of a VS vaccine to endorse its use in other species.

2.3.3. Duration of immunity

The duration of immunity (D.O.I) of a VS vaccine will depend on the efficacy (formulation and
antigen payload). As part of the approval procedure the manufacturer should be required to
demonstrate the D.O.I. of a given vaccine by either challenge or the use of a validated alternative
test, such as serology at the end of the claimed period of protection.

2.3.4. Stability
The stability of all vaccines including oil emulsion vaccines should be demonstrated as part of the
shelf-life determination studies for approval. Vaccines should never be frozen or stored above the
target temperature.

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Vaccines should be stored at 4–8°C, with minimal exposure to light. The shelf life should be
determined by use of the approved potency test (Section C.2.2.4.iii) over the proposed period of
viability.

i) For animal production

Virus(es) used in vaccine production should be antigenically relevant to virus(es) circulating
in the field. A vaccination/challenge study in the species for which the vaccine will be used
will indicate the degree of protection afforded by the vaccine. Species used in
vaccination/challenge studies should be free of antibodies against vesicular stomatitis.
Vaccination/challenge studies should be conducted using virus produced by the intended
production method, at the maximum viral passage permitted, and using an experimental
animal model. It is necessary to confirm the sensitivity, specificity, reproducibility, statistical
significance and confidence level of such experimental model.

Antibody levels after vaccination measured in vitro could be used to assess vaccine efficacy
provided a statistically significant correlation study has been made. For vaccines containing
more than one virus (for example, VSNJV and VSIV), the efficacy of the different components
of these vaccines must each be established independently and then as a combination in
case interference between different viruses exists.

The duration of immunity and recommended frequency of vaccination of a vaccine should

be determined before a product is approved. Initially, such information is acquired directly
using host animal vaccination/challenge studies. The period of demonstrated protection, as
measured by the ability of vaccinates to withstand challenge in a valid test, can be
incorporated into claims found on the vaccine label.

If the vaccine is to be used in horses, swine, cattle, or other ruminants destined for market
and intended for human consumption, a withdrawal time consistent with the adjuvant used
(generally 21 days) should be established by such means as histopathological examination
submitted to the appropriate food safety regulatory authorities.

ii) For control

The same principles apply as for animal production usage. In addition, it should be noted
that antibody responses in vaccinated animals may not be differentiated from animals
exposed to field virus. Therefore, vaccinated animals will need to be clearly identified if
serological methods will be used in conjunction with compatible clinical signs to assess field
virus exposure.

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 Chapter 3.1.24. – Vesicular stomatitis

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WILSON W.C., LETCHWORKTH G.J., JIMENEZ C., HERRERO M.V., NAVARRO R., PAZ P., CORNISH T.E., SMOLIGA G., PAUSZEK S.J.,
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*
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NB: There are WOAH Reference Laboratories for vesicular stomatitis

(please consult the WOAH Web site:
https://www.woah.org/en/what-we-offer/expertise-network/reference-laboratories/#ui-id-3).
Please contact WOAH Reference Laboratories for any further information on
diagnostic tests, reagents and vaccines for vesicular stomatitis

NB: FIRST ADOPTED IN 1990. MOST RECENT UPDATES ADOPTED IN 2021.

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