NB: Ve rsion a dopted by the Worl d A ssembly of De legates of the OIE in May 2014

CHAPTER 2.1.14.

RIFT VALLEY FEVER

SUMMARY
Rift Valley fever (RVF) is a peracute or acute zoonotic disease of domestic ruminants. The virus is
currently confined to the African continent and Arabian Peninsula. It is caused by a single serotype
of a mosquito-borne virus of the Bunyaviridae family (genus Phlebovirus). The disease occurs in
climatic conditions favouring the breeding of mosquito vectors and is characterised by abortion,
neonatal mortality and liver damage. The disease is most severe in sheep, goats and cattle. Older
non-pregnant animals, although susceptible to infection, are more resistant to clinical disease.
There is considerable variation in the susceptibility to RVF of animals of different species. Camels
usually have an inapparent infection with RVF virus (RVFV), but sudden mortality, neonatal
mortality and abortion occurs and abortion rates can be as high as in cattle.
Humans are susceptible to infection and are contaminated through contact with infected animal
material (body fluids or tissues) or mosquito bites. RVFV has also caused serious infections in
laboratory workers and must be handled with biosafety and biocontainment measures. It is
recommended that laboratory workers be vaccinated if possible.
Identification of the agent: RVFV consists of a single serotype of Phlebovirus that has
morphological and physicochemical properties typical of this genus.
Identification of RVFV can be achieved by virus isolation, antigen-detection enzyme-linked
immunosorbent assay (ELISA) or immunopathology. Viral RNA can be detected by reversetranscription polymerase chain reaction.
The virus can be isolated from blood, preferably collected with anticoagulant, during the febrile
stage of the disease, or from organs (e.g. liver, spleen and brain tissues) of animals that have died
and from the organs of aborted fetuses. Primary isolations are usually made on cell cultures of
various types, such as African green monkey kidney (Vero) and baby hamster kidney (BHK) cells.
Alternatively, sucking mice may be used for primary virus isolation.
Serological tests: Identification of specific antibodies is mostly achieved by ELISA or the virus
neutralisation test.
Requirements for vaccines: Live attenuated or inactivated vaccines can be used in countries
where RVF is endemic or that are at risk of its introduction. These vaccines should preferably be
prepared from attenuated strains of RVFV grown in cell cultures.
In RVF-free countries, vaccines and diagnostic tests should preferably be limited to those using
inactivated virus. Work with live virus should be performed by trained personnel in biocontainment
facilities following appropriate biosafety procedures.
There are two OIE Reference Laboratories for RVF (see Table given in Part 4 of this Terrestrial
Manual).

A. INTRODUCTION
Rift Valley fever (RVF) is a peracute or acute, febrile, mosquito-borne, zoonotic disease caused by a virus of the
family Bunyaviridae, genus Phlebovirus. It is usually present in epizootic form over large areas of a country
following heavy rains and flooding, and is characterised by high rates of abortion and neonatal mortality, primarily
in sheep, goats, cattle and camels. The susceptibility of different species and breeds to RVF may vary
considerably. Some animals may have inapparent infections, while others have severe clinical disease with
mortality and abortion. Susceptible, older non-pregnant animals often do not show signs of disease.

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Chapter 2.1.14. Rift Valley fever

Signs of the disease tend to be nonspecific, rendering it difficult to recognise individual cases during epidemics
(Coackley et al., 1967; Coetzer, 1982; Coetzer & Barnard, 1977; Easterday, 1965; Gerdes, 2004; Meegan &
Bailey, 1989; Swanepoel & Coetzer, 1994; Weiss, 1957); however, the occurrence of numerous abortions and
mortalities among young animals, together with disease in humans, is characteristic of RVF. RVF has a short
incubation period of about 1236 hours in lambs. A biphasic fever of up to 41C may develop, and the fever
remains high until shortly before death. Affected animals are listless, disinclined to move or feed, and may show
enlarged superficial lymph nodes and evidence of abdominal pain. Lambs rarely survive longer than 36 hours
after the onset of signs of illness. Animals older than 2 weeks may die peracutely, acutely or may recover or
develop an inapparent infection. Some animals may regurgitate ingesta and may show melaena or bloody, foulsmelling diarrhoea and bloodstained mucopurulent nasal discharge. Icterus may sometimes be observed,
particularly in cattle. In addition to these signs, adult cattle may show lachrymation, salivation and dysgalactia. In
pregnant sheep, the mortality and abortion rates vary from 5% to almost 100% in different outbreaks and between
different flocks. The death rate in cattle is usually less than 10%. Camels have been regularly involved in the RVF
epidemics in East Africa, Egypt and more recently Mauritania. Clinical disease is usually not seen in adult camels,
but sudden deaths, abortion and some early post-natal deaths have been observed. Differential diagnosis
includes: bluetongue, Wesselsbron disease, enterotoxemia of sheep, ephemeral fever, brucellosis, vibriosis,
trichomonosis, Nairobi sheep disease, heartwater, ovine enzootic abortion, toxic plants, bacterial septicaemias,
peste des petits ruminants, anthrax and Schmallenberg disease.
The hepatic lesions of RVF are very similar in all species, varying mainly with the age of the infected individual
(Coetzer, 1982). The most severe lesion, occurring in aborted fetuses and newborn lambs, is a moderately to
greatly enlarged, soft, friable liver with a yellowish-brown to dark reddish-brown colour with irregular congested
patches. Numerous greyish-white necrotic foci are invariably present in the parenchyma, but may not be clearly
discernible. In adult sheep, the lesions are less severe and pinpoint reddish to greyish-white necrotic foci are
distributed throughout the parenchyma. Haemorrhage and oedema of the wall of the gallbladder are common.
Hepatic lesions in lambs are almost invariably accompanied by numerous small haemorrhages in the mucosa of
the abomasum. The contents of the small intestine and abomasum can be dark chocolate-brown as a result of the
presence of partially digested blood. In all animals, the spleen and peripheral lymph nodes can be enlarged,
oedematous and may have petechiae.
Microscopically, hepatic necrosis is the most obvious lesion of RVF in both animals and humans. In fetuses and
neonates of cattle and sheep, foci of necrosis consist of dense aggregates of cellular and nuclear debris, some
fibrin and a few inflammatory cells. There is a severe lytic necrosis of most hepatocytes and the normal
architecture of the liver is lost. In about 50% of affected livers, intranuclear inclusion bodies that are eosinophilic
and oval or rod-shaped are found. Mineralisation of necrotic hepatocytes is also seen. In adult animals, hepatic
necrosis is less diffuse, and in sheep, icterus is more common than in lambs (Coetzer, 1982; Swanepoel &
Coetzer, 1994).
In humans, RVF infections are usually inapparent or associated with a moderate to severe, nonfatal, influenza-like
illness (Madani et al., 2003; McIntosh et al., 1980; Meegan, 1981). A minority of patients may develop retinal
lesions, encephalitis, or severe hepatic disease with haemorrhagic manifestations, which is generally fatal. RVF
virus (RVFV) has caused serious human infections in laboratory workers. Staff should be vaccinated when a
vaccine is available. An inactivated vaccine has been developed for human use. However, this vaccine is not
licensed and is not commercially available. It has been used experimentally to protect veterinary and laboratory
personnel at high risk of exposure to RVF. Further information about the disease and vaccination in humans is
available from WHO1. RVFV is classed in Risk Group 3 for human infection and should be handled with
appropriate measures as described in Chapter 1.1.3 Biosafety and biosecurity in the veterinary microbiology
laboratory and animal facilities. Biocontainment measures should be determined by risk analysis as described in
Chapter 1.1.3a Standard for managing biorisk in the veterinary laboratory and animal facilities. Particular care
needs to be exercised when working with infected animals or when performing post-mortem examinations.
RVFV consists of a single serotype of the Bunyaviridae family (genus Phlebovirus) and has morphological and
physicochemical properties typical of bunyaviruses. The virus is enveloped, spherical and 80120 nm in diameter.
Glycoprotein spikes project through a bilayered lipid envelope. The virus is readily inactivated by lipid solvents
and acid conditions below pH 6. RVFV has a three-segmented, single-stranded, negative-sense RNA genome
and consists of the following segments: L (large), M (medium) and S (small), each of which is contained in a
separate nucleocapsid within the virion. The S segment is an ambisense RNA, i.e. has bi-directional coding
(Giorgi, 1991).
No significant antigenic differences have been demonstrated between RVF isolates and laboratory-passaged
strains from many countries, but differences in pathogenicity between genotypes have been shown (Bird et al.,
2007; Swanepoel et al., 1986).

http://www.who.int/mediacentre/factsheets/fs207/en/

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Chapter 2.1.14. Rift Valley fever

RVFV is endemic in many African countries and may involve several countries in the region at the same time or
progressively expand geographically over the course of a few years. In addition to Africa, large outbreaks have
been observed in the Arabian Peninsula and some Indian Ocean Islands. These generally, but not
exclusivelyfollow the periodic cycles of unusually heavy rainfall, which may occur at intervals of several years, or
the flooding of wide areas favouring the proliferation of mosquitoes.
Rainfall facilitates mosquito eggs to hatch. Aedes mosquitoes acquire the virus from feeding on infected animals,
and may potentially vertically transmit the virus, so that new generations of infected mosquitoes may hatch from
their eggs (Linthicum et al., 1985). This provides a potential mechanism for maintaining the virus in nature, as the
eggs of these mosquitoes may survive for periods of up to several years in dry conditions. Once livestock is
infected, a wide variety of mosquito species may act as the vector for transmission of RVFV and can spread the
disease.
Low level RVF activity may take place during inter-epizootic periods. RVF should be suspected when exceptional
flooding and subsequent abundant mosquito populations are followed by the occurrence of abortions together
with fatal disease marked by necrosis and haemorrhages in the liver that particularly affect newborn lambs, kids
and calves, potentially concurrent with the occurrence of an influenza-like illness in farm workers and people
handling raw meat.
During an outbreak, preventive measures to protect workers from infection should be employed when there are
suspicions that RVFV-infected animals or animal products are to be handled.

B. DIAGNOSTIC TECHNIQUES
The collection of specimens and their transport should comply with the Chapter 1.1.1 Collection, submission and
storage of diagnostic specimens and Chapter 1.1.2 Transport of specimens of animal origin of this Terrestrial
Manual.
Proper diagnosis should always use a combination of techniques based on history, the purpose of the testing and
the stage of the suspected infection. For a definitive interpretation, combined epidemiological, clinical and
laboratory information should be evaluated carefully.
All the test methods described below should be validated in each laboratory using them (see Chapter 1.1.5
Principles and methods of validation of diagnostic assays for infectious diseases of this Terrestrial Manual). The
OIE Reference Laboratories for RVF should be contacted for technical support. Table 1 provides a general
guidance summary on the use of the diagnostic tests methods. More detailed aspects are addressed in the test
descriptions that follow.
Table 1. Test methods available for diagnosis of Rift Valley fever and their purposes
Purpose
Method

Population
freedom from
infection (nonvaccinated
animals)

Individual
animal
freedom from
infection prior
to movement

Contribute to
eradication
policies

Confirmation
of clinical
cases2

Prevalence
of infection
surveillance

Immune status
in individual
animals or
populations
post-vaccination

Agent identification3

Virus isolation in
cell culture

+++

Virus isolation in
sucking mice

RT-PCR

+++

Laboratory confirmation of clinical cases should require a combination of at least two positive results from two different
diagnostic test methods: either positive for virus/viral RNA and antibodies or positive for IgM and IgG with demonstration
of rising titres between paired sera samples collected 24 weeks apart. Depending of the stage of the disease, virus
and/or antibodies will be detected.
A combination of agent identification methods applied on the same clinical sample is recommended.

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Chapter 2.1.14. Rift Valley fever

Purpose
Population
freedom from
infection (nonvaccinated
animals)

Individual
animal
freedom from
infection prior
to movement

Contribute to
eradication
policies

Confirmation
of clinical
cases2

Prevalence
of infection
surveillance

Immune status
in individual
animals or
populations
post-vaccination

Antigen detection

++

Histopathology
with immunohistochemistry

++

Method

Detection of immune response4

ELISA

+++

++

+++

++

+++

+++

VN

+++

+++

+++

++

++

+++

Key: +++ = recommended method; ++ = suitable method; + = may be used in some situations, but cost, reliability, or other
factors severely limits its application; = not appropriate for this purpose.
Although not all of the tests listed as category +++ or ++ have undergone formal validation, their routine nature and the fact that
they have been used widely without dubious results, makes them acceptable.
RT-PCR = reverse-transcription polymerase chain reaction;
ELISA = enzyme-linked immunosorbent assay; VN = Virus neutralisation.

1.

Identification of the agent

RVFV may be isolated from serum but preferentially from plasma or blood collected with anticoagulant during the
febrile stage of the disease in live animals, or from liver, spleen and brain of animals that have died, or from
aborted fetuses. Primary isolation is usually performed in cell cultures of various types or by intracerebral
inoculation of sucking mice.

1.1. Specimen collection

Using appropriate protective equipment to ensure biosafety of the staff, approximately 5 ml of blood
with anticoagulant (preferably ethylene diamine tetra-acetic acid [EDTA]) collected during the febrile
stage of the disease or approximately 1 cm3 of liver, spleen, brain or abortion products collected postmortem should be submitted for virus isolation. The samples should be kept at 04C during transit. If
transport to the laboratory is likely to take more than 24 hours, the samples should be frozen and sent
on dry ice or frozen cold pack. In the case of a blood sample, plasma should be collected and frozen
for transport.

1.2. Isolation in cell culture

A variety of cell line monolayers including African green monkey kidney (Vero), baby hamster kidney
(BHK) and AP61 mosquito cells (Digoutte et al., 1989) may be used. They are inoculated with 1/10
dilution of the sample and incubated at 37C for 1 hour (with mosquito cell lines, the incubation should
be done at 27C for 1 hour). It is advisable to also inoculate some cultures with a further 1/100 dilution
of the inoculum. This is to avoid the production of defective particles, which follows the use of very high
virus inoculaum. The inoculum is removed and the monolayer is washed with phosphate-buffered
saline (PBS) or culture medium. The wash solution is removed, replaced by fresh culture medium and
incubated at an appropriate temperature. The cultures are observed for 56 days. Mammalian cell lines
are preferably used as RVFV induces a consistent cytopathic effect (CPE) characterised by slight
rounding of cells followed by destruction of the whole cell sheet within 1224 hours. Confirmation of
virus isolation should be performed preferably by immunostaining or reverse-transcription polymerase
chain reaction (RT-PCR).

1.3. Isolation in sucking mice

For reasons of animal welfare and biosafety, this method should be avoided if possible. Approximately
1 g of homogenised tissue is suspended 1/10 in cell culture medium or buffered saline, pH 7.5,

One of the listed serological tests is sufficient.

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Chapter 2.1.14. Rift Valley fever

containing sodium penicillin (1000 International Units [IU]/ml), streptomycin sulphate (1 mg/ml),
mycostatin (100 IU/ml), or fungizone (2.5 g/ml). The suspension is centrifuged at 1000 g for
10 minutes and the supernatant fluid is injected intracerebrally into 1- to 5-day-old mice. Sucking mice
will either die or be obviously ill by day 2 post-inoculation.
Confirmation of virus isolation should be performed preferably by immunostaining or PCR.

1.4. Reverse-transcription polymerase chain reaction

A rapid diagnosis can also be made by detection of viral RNA (Sall et al., 2001) using validated
conventional or real-time RT-PCR (Bird et al., 2007; Drosten et al., 2002; Garcia et al., 2001; Sall et al.,
2001). These techniques were very useful during RVF outbreaks in Africa. It may also be used to
detect RVFV in mosquito pools (Jupp et al., 2000).
This technique should be followed by sequencing selected samples. Below are proposed protocols for
conventional and real-time RT-PCR. For information on specific procedures consult the OIE Reference
Laboratories.

1.4.1. Agarose gel-based RT-PCR assay

This procedure is used by the OIE Reference Laboratories. The RT-PCR assay consists of the
three successive procedures of (a) extraction of template RNA from the test or control sample
followed by (b) RT of the extracted RNA, (c) PCR amplification of the RT product and
(d) detection of the PCR products by agarose gel electrophoresis.
i)

Test procedure
RNA is extracted by an appropriate chemical method according to the procedure
recommended by the manufacturer of the commercial kit. When the procedure is finished,
keep the extracted RNA samples on ice if the RT step is about to be performed. Otherwise
store at 20C or 70C. For RT-PCR, the protocol from Sall et al. (2001) is used. For the
first RT-PCR step, NSca (5-CCT-TAA-CCT-CTA-ATC-AAC-3) and NSng (5-TA-TCATGG-ATT-ACT-TTC-C-3) primers are used.
a)

Prepare the PCR mix described below for each sample. It is recommended to
prepare the mix in bulk for the number of samples to be tested plus one extra
sample.
Nuclease-free water (15.5 l); RT-PCR reaction buffer, 5 conc (10 l); MgCl2,
25 mM (1 l); dNTPs, 10 mM mixture each of dATP, dCTP, dGTP, dTTP (1 l);
primer NSca, 10 M (2.5 l); primer NSng 10 M (2.5 l): Enzyme Mix, 5 units/l
(0.25 l).

b)

Add 40 l of PCR reaction mix to a well of a PCR plate or to a microcentifuge tube for
each sample to be assayed followed by 10 l of the RNA (prepared in step i) to give
a final reaction volume of 50 l.

c)

Spin the plate or tubes for 1 minute in a suitable centrifuge to mix the contents of
each well.

d)

Place the plate in a thermal cycler for PCR amplification and run the following
programme:
45C for 30 minutes: 1 cycle;
95C for 2 minutes: 1 cycle;
94C for 30 seconds, 44C for 30 seconds, 72C for 1 minute: 40 cycles;
72C for 5 minutes: 1 cycle.

e)

Mix a 20 l aliquot of each PCR reaction product with 4 l of staining solution and
load onto a 1.2% agarose gel. After electrophoresis a positive result is indicated by
the presence of a 810 bp (242 bp for Clone 13) band corresponding to RVFV
sequence in the NSs coding region of the S segment of the genome.
For the nested RT-PCR step, NS3a (5-ATG-CTG-GGA-AGT-GAT-GAG-CG-3) and
NS2g (5-GAT-TTG-CAG-AGT-GGT-CGT-C-3) are used.

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Chapter 2.1.14. Rift Valley fever

f)

Prepare the PCR mix described below for each sample. It is recommended to
prepare the mix in bulk for the number of samples to be tested plus one extra
sample.
Nuclease-free water (35.5 l); RT-PCR reaction buffer, 10 conc (5 l); MgCl2,
25 mM (1.25 l); dNTPs, 10 mM mixture each of dATP, dCTP, dGTP, dTTP (1 l);
primer NS3a
(5-ATG-CTG-GGA-AGT-GAT-GAG-CG-3),
10
M
(2.5 l);
primer NS2g (5-GAT-TTG-CAG-AGT-GGT-CGT-C-3), 10 M (2.5 l): Enzyme Mix,
5 units/l (0.25 l).

g)

Add 49 l of PCR reaction mix to a well of a PCR plate or to a microcentifuge tube for
each sample to be assayed followed by 1 l of the amplicon obtained from RT-PCR
reaction with NSca and NSng to give a final reaction volume of 50 l.

h)

Spin the plate or tubes for 1 minute in a suitable centrifuge to mix the contents of
each well.

i)

Place the plate in a thermal cycler for PCR amplification and run the following
programme:
95C for 2 minutes: 1 cycle;
94C for 1 minute, 55C for 1 minute, 72C for 1 minute: 25 cycles;
72C for 5 minutes: 1 cycle.

j)

Mix a 20 l aliquot of each PCR reaction product with 4 l of staining solution and
load onto a 1.2% agarose gel. After electrophoresis a positive result is indicated by
the presence of a 668 bp (129 bp for Clone 13) band corresponding to RVFV
sequence in the NSs coding region of the S segment of the genome.

1.4.2. Real-time RT-PCR assay

The real-time RT-PCR assay can use the same procedures for extraction of total RNA from the
test or control sample followed by RT of the extracted RNA as for the conventional procedure.
The protocol is adapted from Drosten et al. (2002). If commercial kits are used the
manufacturers method should be followed.
i)

Test procedure
a)

Prepare the PCR mix described below for each sample. Again it is recommended to
prepare the mix in bulk for the number of samples to be tested plus one extra
sample.
Nuclease-free water (1.4 l); RT-PCR reaction master mix, 2 conc. (10 l); real-time
PCR forward primer RVS: 5-AAA-GGA-ACA-ATG-GAC-TCT-GGT-CA-3, 10 M
(2 l); real-time PCR reverse primer RVAs: 5-CAC-TTC-TTA-CTA-CCA-TGT-CCTCCA-AT-3, 10 M (2 l); RVP: FAM 5-AAA-GCT-TTG-ATA-TCT-CTC-AGT-GCCCCA-A-3 TAMRA 20 M (0.2 l).

b)

Add 17 l PCR reaction mix to a well of a real-time PCR plate for each sample to be
assayed followed by 3 l of the prepared RNA to give a final reaction volume of 20 l.

c)

Spin the plate for 1 minute in a suitable centrifuge to mix the contents of each well.

d)

Place the plate in a real-time PCR machine for PCR amplification and run the
following programme:
45C for 30 minutes: 1 cycle;
95C for 5 minutes: 1 cycle;
95C for 5 seconds, 57C for 35 seconds: 45 cycles.

e)

Reading the results: assign a threshold cycle (CT) value to each PCR reaction from
the amplification plots (fluorescence signal versus cycle number); different cut-off
values may be appropriate for different sample types. The CT values used to assign
samples as either RVFV positive or negative should be defined by individual
laboratories using appropriate reference material.

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Chapter 2.1.14. Rift Valley fever

1.5. Antigen detection

The antigen detection enzyme-linked immunosorbent assay (ELISA) is an immunocapture test.
Samples are tested at different dilutions with appropriate positive and negative controls. This test has
been used for human and animal samples during outbreaks in Saudi Arabia and Kenya (Madani et al.,
2003; Munyua et al., 2010).

1.5.1. Test procedure

The controls and antisera used in the performance of this assay should have been treated to kill
any RVFV that they may have contained at the time of production. These products are safe,
within the limits of our ability to detect viable virus. The material to be tested for the presence of
RVF viral antigen is potentially contaminated with viable RVFV or other agents for which a
differential determination is being sought. At the very least, good laboratory practices should be
used. Samples could be inactivated using appropriate detergent and heat inactivation.
i)

The basic approach is that of a double antibody sandwich capture assay in which the
antigen is captured by antibody on a solid phase and then detected by a second antibody.
A detection system using horseradish peroxidase (HRPO)ABTS (2,2-azino-di-(3-ethylbenzthiazoline)-6-sulphonic acid) is then applied to determine how much of the detection
antibody has been retained on the solid phase of the system.
a)

Capture (coating) antibody (diluted 1/2000 in PBS [no Tween], pH 7.4 and coated
overnight at 4C; control wells are coated with a similar dilution of normal fluid)
Plates are coated with a specific anti-viral antibody (available in OIE or WHO
Reference Laboratories) capable of capturing viral antigen from the test sample.
Normal serum is added to rows to serve as controls used to determine the
nonspecific background or noise of the system. In this instance it is a hyperimmune
mouse ascitic fluid (HMAF) (it could also be monoclonal antibodies) specific for RVF
viruses.

b)

Suspect samples and control antigen (diluted 1/4 and then diluted four-fold, down the
plate)
These are added in serum diluent to allow specific viral antigens to bind to the
capture antibody. The serum diluent (PBS, 0.01 M, pH 7.4, with or without
thiomersal) contains 5% skim milk and 0.1% Tween 20 to reduce nonspecific binding.

c)

Detection antibody
An antibody, high titred for specific viral antigen, is added to allow detection of the
bound viral antigen. In this experiment, it is an anti-RVF hyperimmune rabbit serum
(available in OIE or WHO Reference Laboratories) that has a high titre against RVF
viruses.

d)

Anti-rabbit conjugated to HRPO (commercial product)

This is used to detect the rabbit anti-RVF that binds to the antigen.

e)

Criteria for determining positives

A standard control antigen has been provided and will be run in a standard dilution
series. This, in effect, provides a standard curve that will determine the limits of
detection of the assay. A group of normal tissues or samples, uncontaminated with
antigen, are tested to determine the background of the assay and the limit at which
the standard was positive. The values of these normal controls are used to generate
the mean and standard deviation of the random background to be expected with
negative samples. A sample is considered positive if its optical density (OD) value
exceeds the mean plus 3 standard deviations of these normal controls.

1.6. Histopathology
Histopathological examination of the liver of affected animals will reveal characteristic cytopathology,
and immunostaining will allow the specific identification of RVF viral antigen in tissue (Coetzer, 1982;
Swanepoel et al., 1986). This is an important diagnostic tool because liver or other tissue placed in
neutral buffered formaldehyde in the field is inactivated and does not require a cold chain, which
facilitates handling and transport from remote areas .

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Chapter 2.1.14. Rift Valley fever

2.

Serological tests

Samples collected from animals for antibody testing may contain live virus and appropriate inactivation steps
should be put in place. A combination of heat and chemical inactivation has been described (Van Vuren &
Paweska, 2010). Immunofluorescence assays are still used, although cross-reactions may occur between RVFV
and other phleboviruses. Techniques such as the agar gel immunodiffusion (AGID), radioimmunoassays,
haemagglutination inhibition (HI), and complement fixation are no longer used.
Several assays are available for detection of anti-RVFV antibodies in a variety of animal species. Currently the
most widely used technique is the ELISA for the detection of IgM and IgG. Virus neutralisation tests (VNT) have
been used to detect antibodies against RVFV in the serum of a variety of species. Neutralisation tests are the
most specific diagnostic serological tests, but these tests can only be performed with live virus and are not
recommended for use outside endemic areas or in laboratories without appropriate biosecurity facilities and
vaccinated personnel. However, alternative neutralisation assays not requiring handling of highly virulent RVFV
and not requiring high containment, are being developed and validated.

2.1. Enzyme-linked immunosorbent assay

The ELISA is a reliable and sensitive test to detect antibodies against RVFV. Both IgG and IgM ELISAs
are available for most species. IgM-capture ELISA allows diagnosis of recent infections.
A number of ELISAs using different formats are commercially available and others are under
development (Afetine et al., 2007; Cettre-Sossah et al., 2009; Jansen Van Vuren et al., 2007; Madani
et al., 2003; Munyua et al., 2010; Paweska et al., 2003; 2005; Van Vuren & Paweska, 2010). They are
used routinely in many countries for single case diagnosis, outbreak management, and surveillance.
Below are two such tests used at the OIE Reference Laboratory in South Africa. Both assays use 10%
non-fat milk/Tris salt Tween (NFM/TST) as blocking and dilution buffer, and TST buffer (50 mM Tris;
150 mM NaCl; 0.1% Tween 20) as washing buffer (pH 8.0). The reactions are stopped with 2 N H2SO4.
The recombinant nucleoprotein (rN) of RVFV is produced and purified as described by Williams et al.
(2011). Conjugation of the protein to HRPO is performed following the Nakane & Akira Kawaoi (1974)
protocol. The rN antigen is stable for up to 1 year at 4C.
To prepare plates for immediate use, make a checkerboard titration of the capture antibody or antigen
against the conjugate in a 96-well ELISA plate to determine the minimum reagent concentration that
would give an OD value of 0.50.6 when read at 650 nm after an incubation period of 20 minutes. This
will determine how the antibody/antigen and conjugate must be diluted for coating of the plates and
detection of the antigen/antibody binding in the test.

2.1.1. IgM capture ELISA

i)

Test procedure
a)

Coat each well of the 96-well ELISA plates with 100 l of the capture antibody
(affinity purified rabbit anti-sheep IgM1) diluted to 1 g/ml in PBS (that is a 1/1000
dilution if so determined by the titration), and incubate overnight at room temperature
in a humid chamber.

b)

Wash the plates three times with wash buffer.

c)

Block the plates with 300 l blocking buffer and incubate for 1 hour at 37C.

d)

Wash the plates again three times with wash buffer.

e)

Dilute both control (positive and negative) and test sera 1/100 in blocking buffer and
add each serum in a designated well at volumes of 100 l /well.

f)

Incubate the plates for 1 hour at 37C. Avoid drying by putting plates in a humid
chamber.

g)

Following the incubation step, wash the ELISA plates with wash buffer three times.

h)

Dilute the rN-HRP conjugate 1/6000 and add 100 l of this in each well. Use blocking
buffer as the conjugate control.

i)

The plates are then incubated for 60 minutes at 37C.

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Chapter 2.1.14. Rift Valley fever

j)

The plates are washed, as in step b above. Ready-to-use tetramethyl benzidine

(TMB) substrate at 100 l quantities is then transferred to each well, and the plates
allowed to stand at room temperature for a few minutes, until development of a
colour change or OD values of 0.5 when the plates are read at 650 nm. Exposure to
direct light should be avoided.

k)

Stop the reaction with 100 l stop solution, and read the OD values using an ELISA
plate reader at 450 nm.

l)

Interpretation of results: results are expressed as percentage of the positive serum

control (PP) using the formula:
(mean OD of duplicate test serum)/(mean OD of positive control) 100
where positive and negative cut-off values are determined by receiver operating
characteristic (ROC) curve analysis.

It should be noted that the cut-off value for an ELISA can be adjusted for different target
populations as well as for different diagnostic purposes (Jacobson, 1998). The cut-off values
determined by the validation exercise at the OIE Reference Laboratory in South Africa are the
following: PP (%) values: negative <4; suspicious 45; positive>6.

2.1.2. Indirect IgG ELISA

i)

Test procedure
a)

Coat each well of the 96-well ELISA plate with 100 l of rN diluted in 50 mM of
carbonate buffer (pH 9.6) using the dilution ratio determined by prior titration as
explained above; incubate overnight at room temperature in a humid chamber.

b)

Wash the plates three times with approximately 300 l wash buffer per well.

c)

Block the plates with approximately 300 l blocking buffer and incubate for 1 hour at
37C.

d)

Wash the plates again three times with nearly 300 l of wash buffer per well.

e)

Dilute both control (positive and negative) and test sera 1/100 in blocking buffer.

f)

Add 100 l of the diluted sera in designated wells in duplicate.

g)

Incubate the plates for 1 hour at 37C. Avoid drying by putting plates in a humid
chamber.

h)

Following the incubation step, wash the ELISA plates with wash buffer three times.

i)

Dilute protein G-HRP conjugate 1/32,000 in blocking buffer and add 100 l of the
conjugate in each well.

j)

Incubate for 60 minutes at 37C.

k)

The plates are washed, as in step b above. Add 100 l of ready to use TMB
substrate to each well and allow the plates to stand at room temperature for a few
minutes, while avoiding exposure to direct light. The plates are read at 650 nm to
determine if OD of 0.40.6 has been reached.

l)

Stop the reaction with 100 l stop solution, and read the plates using ELISA plate
reader at 450 nm.

m)

Interpretation of results: results are expressed as percentage of the positive serum

(PP) using the formula:
(mean OD of duplicate test serum)/(mean OD of positive control) 100
where positive and negative cut-off values are determined by receiver operating
characteristic (ROC) curve analysis.

It should be noted that the cut-off value for an ELISA can be adjusted for different target
populations as well as for different diagnostic purposes (Jacobson, 1998). The cut-off values
determined by the validation exercise at the OIE Reference Laboratory in South Africa are the
following: PP values (%): negative <4; suspicious 46; positive>7.

2.2. Virus neutralisation (the prescribed test for international trade)

The VN test may be used to determine the presence of antibodies in naturally infected animals and in
vaccinated animals. The test is highly specific and can be used to test serum of any species. It is

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Chapter 2.1.14. Rift Valley fever

generally used to measure vaccine efficacy. The Smithburn neurotropic mouse brain strain of highly
attenuated RVFV (Smithburn, 1949) or any other, preferably attenuated, RVFV, is used as challenge
virus. The virus is stored at 80C, or 4C in freeze-dried form. The stock is titrated to determine the
dilution that will give 100 TCID50 (50% tissue culture infective dose) in 25 l under the conditions of the
test.

2.2.1. Test procedure

i)

Inactivate the test sera for 30 minutes in a water bath at 56C.

ii)

Add 25 l of cell culture medium with 5% RVF-negative serum and antibiotics to each well
of a 96-well cell culture plate.

iii)

Add 25 l of test serum to the first well of each row and make twofold dilutions. Titrate
each serum in duplicate from 1/10 to 1/80 for screening purposes or in quadruplet and to
higher dilutions for determination of end-point titres. Include known positive and negative
control sera.

iv)

Add 25 l per well of RVFV (diluted in cell culture medium and calculated to provide
100 TCID50 per well) to each well that contains diluted test serum and to wells in rows
containing negative and positive control serum. In addition, make twofold dilutions of
challenge virus in at least two rows each containing cell culture medium only.

v)

Incubate for 30 minutes at 37C.

vi)

Add 50 l per well of Vero, BHK or any other suitable cell suspension at 3 105 cells/ml or
at a dilution known to produce a confluent monolayer within 12 hours.

vii)

Incubate the plates in an atmosphere of 35% CO2 for 35 days.

viii) Using an inverted microscope, the monolayers are examined daily for evidence of CPE.
There should be no CPE in rows containing positive control serum and clear evidence of
CPE in rows containing negative control serum indicating the presence of virus. Determine
the results by the SpearmanKrber method.

C. REQUIREMENTS FOR VACCINES

1.

Background

Currently available RVF vaccines are either live attenuated or inactivated vaccines.
Table 2. Summary of the current RVF vaccine strains

10

Smithburn live
attenuated virus
vaccines

Clone-13 live
attenuated
virus vaccine

Inactivated
virus vaccines

TSIGSD200 inactivated
human vaccine
(presently not available)

Origin of the
isolate

Mosquito isolate,
Uganda, 1948

Human isolate,
1974

Field strains
(South Africa and
Egypt) used

Mosquito isolate, Uganda,

1944

Attenuation

More than 200

passages in murine
brain

Natural deletion in
NSs gene

Not applicable

Not applicable

Production
substrate

BHK cell line

Vero cell line

BHK cell line

Diploid fetal rhesus lung cell

line

Target

livestock

livestock

livestock

human

DIVA policy

No

No

No

Not applicable

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Chapter 2.1.14. Rift Valley fever

1.1. The live attenuated Smithburn RVF vaccine

The vaccine virus is derived from Smithburns original neurotropic strain. This strain is not lethal to
adult mice inoculated intraperitoneally and is safe for use in all breeds of cattle, sheep and goats
(Barnard, 1979; Smithburn, 1949). However, it may cause fetal abnormalities or abortion in pregnant
animals. The Smithburn RVF vaccine has been used for decades in the control of RVF in Eastern and
Southern Africa and in the Middle East, and is still used to date in different endemic region.

1.2. The Clone 13 RVF vaccine

Clone 13 is a naturally attenuated strain characterised by a large deletion of the gene encoding for the
main virulent factor, the NSs (Muller et al., 1995). The risk of reversion is considered unlikely. No
abortion or side effects have been seen in experimental vaccine trials (Dungu et al., 2010; Hunter &
Bouloy, 2001). It was recently introduced in South Africa for use in sheep and cattle using a single
injection regimen.

1.3. The inactivated RVF vaccine

The currently produced formalin-inactivated vaccines derived from a field strain of RVFV adapted to
growth in cell culture (Barnard, 1979; Barnard & Botha, 1977). These vaccines are currently adjuvanted
in aluminium hydroxide. However inactivated RVF vaccines need a booster 36 months following initial
vaccination, followed by yearly boosters. Inactivated RVF vaccine is also used in outbreak situations,
and in pregnant animals as the attenuated Smithburn vaccine is not suitable for this group.

1.4. Inactivated experimental human vaccine

Formerly produced by the Salk Institute (USA), the inactivated experimental human vaccine is no
longer available (Meadors, 1986).
Many other candidate vaccines are either being developed and evaluated in target animals or are in early stage of
development (Food and Agriculture Organization of the United Nations [FAO], 2011).
There are a number of product characteristics that it would be preferable to have in an effective and safe RVF
vaccine, and that should be used to define a target product profile. Elements of a target product profile for a RVF
vaccine should preferably meet recommendation 2 of the report of the FAO meeting, 2011 (FAO, 2011) and as
indicated below.
The main purpose of a RVF vaccine is to prevent epizootics and epidemics in species of economic interest
(susceptible livestock species [ruminants] and, potentially, camelids), and limit the impact on animal and public
health. In addition to the potential economic impact, it could also have some implications in international
movements of animals. It is relevant to distinguish specific requirements for endemic regions and regions free of
the disease.

1.5. Endemic region

The objective is the prevention and control of epizootics and epidemics in endemic areas and to
contribute to the improvement of livestock production in endemic areas. In order of priorities,
characteristics of the vaccines are:
i)

preferably one dose, resulting in a long-lasting immunity;

ii)

preferably a life-long immunity after a limited number of doses.

1.6. Free or non-endemic region

Vaccines would be used either for the prevention of, or the response to an introduction of the virus. The
expected characteristics of the vaccines are: safe with a quick onset of protective immunity and
protection in animals of all ages and physiological status. Although DIVA (detection of infection in
vaccinated animals) is an important property of any future vaccine, a requirement for DIVA should not
hinder or block the development or licensing of an effective RVF vaccine.

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In all the cases, the vaccines should be:

i)

Safe for the staff involved in the production of the vaccines and for the users, safe to all physiological stages
of animals, and with minimal risk of introduction into the environment (potential vectors);

ii)

Protective in multispecies and if possible in all susceptible species of economic importance, to prevent
infection and transmission;

iii)

Cost effective for producers and users preferentially with a single-dose vaccination;

iv)

Easy to use (e.g. preferably needle-free delivery), suitable for stockpiling (vaccine bank) and quick
availability.

Staff handling virulent RVFV should preferably work in high containment facilities and be vaccinated, if vaccines
are available, to minimise the risk of infection.
Guidelines for the production of veterinary vaccines are given in Chapter 1.1.6 Principles of veterinary vaccine
production. The guidelines given below and in chapter 1.1.6 are intended to be general in nature and may be
supplemented by national and regional requirements.
In the following description of vaccine production, information is given on live vaccine production adjacent to
information on inactivated vaccine production.

2.

Outline of production and minimum requirements for conventional vaccines

2.1. Characteristics of the seed virus
2.1.1. Biological characteristics of the master seed virus
The exact source of the isolate should be recorded and should include the type of material from
which the virus was derived. The in-vitro passage history of the virus and details of the
ingredients should be recorded in accordance with chapter 1.1.6. The master seed virus (MSV)
should be tested for identity, purity (freedom from adventitious agents) and safety.
Characterisation of the MSV should be done using biological or genetic parameters, as relevant.
Assuming adequate immunogenicity and for obvious safety reasons, it is highly recommended
that attenuated virus strains be used for the production of inactivated vaccines. The number of
virus passages from the MSV stock to the final product should not exceed five (European
Pharmacopoeia, 2012).

2.1.2. Quality criteria

The purity of the MSV and cells to be used for vaccine production must be maintained during
the process. The seed virus should be free from adventitious agents, bacteria and Mycoplasma,
using tests known to be sensitive for detection of these microorganisms (see Chapter 1.1.7
Tests for sterility and freedom from contamination of biological materials). The aliquot to be
tested 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 a RVFV
different from the seed virus, and the virus/antibody mixture is cultured on several types of cell
line monolayers. Neutralised cultures should be passaged and tested for adventitious viruses
that may have infected the cells or virus seed during previous passages. As an example, bovine
viral diarrhoea virus (BVDV) is a potential contaminant introduced through the use of fetal
bovine serum in cell culture systems. A cell line highly permissive for BVDV types 1 and 2 is
recommended as one of the cell lines chosen for evaluation of the MSV. Products of bovine
origin should be obtained from countries with negligible bovine spongiform encephalopathy risk.

2.1.3. Validation as a vaccine strain

The vaccine derived from the MSV must be shown to be satisfactory with respect to safety and
efficacy for the species for which it is intended.

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2.2. Method of manufacture

2.2.1. Procedure
i)

Live vaccines
Virus seed is produced 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 and
should be restricted to five. 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 defective interfering viral
particles. When the virus has reached its appropriate titre, as determined by CPE or other
approved technique, the harvest can be clarified. Generally, the vaccine is freeze-dried,
preferably in the presence of a suitable stabiliser.

ii)

Inactivated vaccines
Antigens used in inactivated vaccines are generally prepared in a similar way to live
vaccines. The virus present in the virus maintenance medium is inactivated using a
validated inactivation method then can be eventually concentrated/purified and formulated
with a suitable adjuvant.
In the case where a virulent RVFV is used for inactivated vaccine production, staff
handling the live virus should be vaccinated, if vaccines are available, and the facilities and
practices should conform with high containment level minimising the risk of infection of the
staff and release into the environment.

2.2.2. Requirements for ingredients

Cell lines used for cell culture should be demonstrated free of extraneous agents. All animal
origin products used in the production and maintenance of cells (i.e. trypsin, fetal bovine sera)
and growth of virus should be free of extraneous agents, with special attention paid to the
presence of BVDV.

2.2.3. In-process controls

Yield can be assessed using antigenic mass or infectivity assays. Sterility of antigens should be
checked throughout the process.
A validated inactivation control method is used to assure complete inactivation of the bulk
material of each batch. For inactivated vaccines, samples taken at regular timed intervals during
inactivation, then inoculated into a susceptible cell line (as used for production), should indicate
a complete loss of titre by 2/3 of the total duration of the inactivation process.
For tests in cell cultures, not less than 150 cm2 of cell culture monolayer is inoculated with
1.0 ml of inactivated harvest. The product complies with the test if no evidence of the presence
of any live virus or other micro-organism is observed.
At the end of the production, antigen content is measured to establish that minimum bulk titres
or antigenic mass have been achieved.

2.2.4. Final product batch tests

i)

Sterility
The final products should be tested for absence of bacteria, Mycoplasma and fungal
contamination (see chapter 1.1.7).

ii)

Identity
The bulk live attenuated virus or the inactivated antigen as well as the final formulated
product (freeze-dried or liquid) should undergo identity testing before release to
demonstrate that the relevant RVF strain is present.

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iii)

Safety
Unless consistent safety of the product is demonstrated and approved in a registration
dossier and the production process is approved for consistency in accordance with the
standard requirements referred to in chapter 1.1.6, batch safety testing is to be performed.
The final product batch safety test is designed to detect any abnormal local or systemic
adverse reactions.
If batch safety testing needs to be performed, each of at least two healthy sero-negative
target animals have to be inoculated by the recommended route of administration with the
recommended dose of vaccine. The animals are observed for local and systemic reactions
to vaccination for no fewer than 14 days. Any adverse reaction attributable to the vaccine
should be assessed and may prevent acceptance of the batch. If the potency test is
performed in the target species, observation of the safety during this test can also be
considered as an alternative to the batch safety test described here.

iv)

Batch potency
For live vaccines, potency is usually based on live virus titre. For batch release of
inactivated vaccines, indirect tests can be used for practicability and animal welfare
considerations, as long as correlation has been validated to the percentage of protection in
the target animal. Frequently indirect potency tests include antibody titration after
vaccination of suitable species. Alternative methods (antigen mass) could be used if
suitably validated.

v)

Moisture content
The moisture content of the lyophilised attenuated vaccine should not exceed 5%.

2.3. Requirements for authorisation/registration/licensing

2.3.1. Manufacturing process
For registration of vaccine, all relevant details concerning manufacture of the vaccine and
quality control testing (see Section C.2.2.1 to C.2.2.4 of this chapter) should be submitted to the
Regulatory 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.
The in-process controls are part of the manufacturing process.

2.3.2. Safety requirements

For the purpose of gaining regulatory approval, the following safety tests should be performed
satisfactorily. In addition of these tests, the vaccines should be tested for safety in the field (see
chapter 1.1.6 on field tests [safety and efficacy]).
i)

Live vaccines
Vaccines should be tested for any pathogenic effects in each of the target species claimed
on the label.
a)

Safety test (overdose) in young animals

Carry out the test for each recommended route of application using in each young
target animal not older than the minimum age recommended for vaccination. Use
vaccine virus at the least attenuated passage level that will be present in a batch of
the vaccine.
Use no fewer than eight healthy young target animals without antibodies against
RVFV. Administer to each animal a quantity of the vaccine virus equivalent to not
less than 10 times the maximum virus titre likely to be contained in 1 dose of the
vaccine. Observe the animals daily for at least 14 days. The body temperature of
each vaccinated animal is measured on at least the 3 days preceding administration
of the vaccine, at the time of administration, 4 hours after and then daily for at least
14 days. The vaccine complies with the test if the average body temperature
increase for all animals does not exceed 1.5C, no animal shows a temperature rise
greater than 1.5C for a period exceeding 3 consecutive days, and no animal shows
notable signs of disease or dies from causes attributable to the vaccine.

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b)

Safety test in pregnant animals

Safety at different stages of gestation should be demonstrated if the product is to be
used in pregnant animals.
Carry out the test with vaccination by a recommended route using no fewer than
16 healthy animals of the same age and origin and that do not have antibodies
against RVFV: eight in the first third of gestation and eight in the second third
(periods of time where the teratogenic risk of RVF is the highest [Botros, 2006;
Hunter, 2002]). Use vaccine virus at the least attenuated passage level that will be
present in a batch of the vaccine.
Administer to each group a quantity of the vaccine virus equivalent to not less than
the maximum virus titre likely to be contained in 1 dose of the vaccine. Clinical
observation of animals is carried out daily until parturition. Blood samples should be
taken from newborn animals before ingestion of colostrum.
The test is invalid if the vaccinated animals do not seroconvert before parturition. The
vaccine virus complies with the test if no abnormalities in the gestation or in the
animals are noted. No animal shows notable signs of disease or dies from causes
attributable to the vaccine.
Vaccine virus must not be present in blood samples from newborn animals.

c)

Non-transmissibility
This test should be performed in the most susceptible species for RVF, which is
sheep.
Keep together no fewer than 12 healthy lambs, at the minimum age recommended
for vaccination and of the same origin, and that do not have antibodies against
RVFV. Use vaccine virus at the lowest passage level that will be present between the
MSV and a batch of the vaccine. Administer by a recommended route to no fewer
than six lambs a quantity of the vaccine virus equivalent to not less than the
maximum virus titre likely to be contained in 1 dose of the vaccine.
Maintain no fewer than six lambs as contact controls. The mixing of vaccinated lambs
and contact lambs is done 24 hours after vaccination.
After 45 days, euthanise all lambs. Carry out appropriate tests on the lambs to detect
antibodies against RVF virus and on the control lambs to detect RVFV in the spleen
and liver. The vaccine complies with the test if antibodies are found in all vaccinated
lambs and if no antibodies and no virus are found in the control lambs.

d)

Reversion-to-virulence
This test is carried out using the master seed lot. If the quantity of the master seed lot
sufficient for performing the test is not available, the lowest passage material used for
the production that is available in sufficient quantity may be used. At the time of
inoculation, the animals in all groups are of an age suitable for recovery of the strain.
Serial passages are carried out in target animals using five groups of animals, unless
there is justification to carry out more passages or unless the strain disappears from
the test animal sooner. In-vitro propagation may not be used to expand the passage
inoculum.
The passages are carried out using animals most appropriate to the potential risk
being assessed.
Initially, the vaccine is administered by the recommended route most likely to lead to
reversion-to-virulence, using an initial inoculum containing the maximum release titre.
After this, no fewer than four further serial passages through animals of the target
species are undertaken. The passages are undertaken by the route of administration
most likely to lead to reversion-to-virulence. If the properties of the strain allow
sequential passage via natural spreading, this method may be used, otherwise
passage of the virus is carried out and the virus that is recovered at the final passage
is tested for increase in virulence. For the first four groups, a minimum of two animals
is used. The last group consists of a minimum of eight animals. At each passage, the

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presence of living vaccine-derived virus in the material used for passage is

demonstrated. Care must be taken to avoid contamination by virus from previous
passages. When the virus is not recovered from any intermediate in-vivo passage,
the passage is repeated in ten animals using in-vivo passaged material from the last
passage in which the virus was recovered. The virus recovered is used as the
inoculum for the next passage. If the vaccine virus is not recovered, the experiment is
considered to be completed with the conclusion that the vaccine virus does not show
an increase in virulence.
General clinical observations are made during the study. Animals in the last group
are observed for 21 days unless otherwise justified. These observations include all
relevant parameters typical for the disease that could indicate increase in virulence.
Compare the clinical signs and other relevant parameters with those observed in the
animals used in the test for safety of the administration of 1 dose. If the last group of
animals shows no evidence of an increase in virulence, further testing is not required.
Otherwise, material used for the first passage and the virus recovered at the final
passage level are used in a separate experiment using at least eight animals per
group, to compare directly the clinical signs and other relevant parameters. This
study is carried out using the route of administration that was used for previous
passages. An alternative route of administration may be used if justified.
Unless otherwise justified and authorised, the product complies with the test if no
animal dies or shows signs attributable to the vaccine strain and no indication of
increased virulence is observed in the animals of the last group.
e)

Environmental considerations
A risk assessment should be prepared where potential spread or risk of live vaccines
to non-target species or spread by vector is considered.

f)

Precautions (hazards)
Modified live virus vaccines may pose a hazard to the vaccinator depending on the
strain and level of attenuation of the virus. Manufacturers should provide adequate
warnings that medical advice should be sought in case of self-injection of vaccine.

ii)

Inactivated vaccines
a)

Safety test (of one dose and a repeated dose)

For the purposes of gaining regulatory approval, a trial batch of inactivated vaccine
should be tested for local and systemic safety by each recommended route of
administration in an in-vivo test in eight animals of each target species. Single dose
and repeat dose tests using vaccines formulated to contain the maximum permitted
payload should be conducted. The repeat dose test should correspond to the primary
vaccination schedule (e.g. two injections) plus the first revaccination (i.e. a total of
three injections). The animals are observed for local and systemic reactions to
vaccination for no fewer than 14 days after each injection. Any undue reaction
attributable to the vaccine should be assessed and may prevent acceptance of the
vaccine.

b)

Safety test in pregnant animals

Safety at different stages of gestation should be demonstrated if the product is to be
used in pregnant animals.
Carry out the test with vaccination by a recommended route using no fewer than
16 healthy animals of the same age and origin and without antibodies against RVFV:
eight in the first third of gestation and eight in the second third.
Administer to each group a quantity of the vaccine equivalent to not less than the
maximum antigen mass likely to be contained in 1 dose of the vaccine. Clinical
observation of animals is carried out daily until parturition.
The test is invalid if the vaccinated animals do not seroconvert before parturition. The
vaccine complies with the test if no abnormalities in the gestation or in the animals
are noted. No animal shows notable signs of disease or dies from causes attributable
to the vaccine.

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c)

Precautions (hazards)
Inactivated RVFV vaccines present no danger to vaccinators, although accidental
inoculation may result in an adverse reaction caused by the adjuvant and secondary
components of the vaccine. Manufacturers should provide adequate warnings that
medical advice should be sought in case of self-injection of vaccine.

2.3.3. Efficacy requirements

Vaccine efficacy is estimated in vaccinated animals directly by evaluating their resistance to live
virus challenge. In general, a successful test in lamb is considered to be sufficient evidence of
the quality of a vaccine to endorse its use in other species. Under circumstances where a
vaccine is produced for use primarily in a species other than lamb, it may be more appropriate
to test the efficacy of the vaccine in that same species. However, except for cattle, efficacy tests
in other target species, such as goats or camelids have not been developed yet.
i)

Immunogenicity test in young animals

The following test is applicable to sheep. For other species, appropriate modifications
could be made.
A test is carried out for each route and method of administration recommended for
vaccination using in each case lambs of the minimum age to be recommended. The
quantity of vaccine to be administered to each lamb for a live vaccine is not greater than
the minimum virus titre to be stated on the label and the virus is at the most attenuated
passage level that will be present in a batch of vaccine. For inactivated vaccines, a
minimum antigenic dose should be used according to the recommended vaccination
schedule.
For the test use no fewer than 16 lambs without antibodies against RVF.
For live vaccine, collect sera from the lambs before vaccination, 7 and 14 days after
vaccination and just before challenge. For inactivated vaccine, collect sera from the lambs
before the first and second injection of the primo vaccination and at the time of the
challenge.
Vaccinate no fewer than eight lambs, according to the recommended schedule. Maintain
no fewer than eight lambs as controls. For live vaccines, challenge each lamb after 20
22 days by an appropriate route with a virulent RVFV. In the case of inactivated vaccines,
challenge each lamb 14 days after completion of primo vaccination. Observe the lambs at
least daily for 14 days after challenge and monitor for clinical signs and viral load by virus
isolation and quantitative RT-PCR in blood.
The test is invalid if antibodies against RVFV in the sera of the control animals indicate
that there was intercurrent infection with the virus during the test.
The vaccine complies with the test if, during the observation period after challenge, there
is a significant reduction in duration and titre of viraemia, and a notable reduction in clinical
signs (if the challenge virus used produces such signs) in vaccinated lambs compared with
controls.

ii)

Immunogenicity test in pregnant animals

The following test is applicable to sheep. For other species, appropriate modifications
should be made, e.g. the most sensitive gestation period for challenge.
Use 16 ewes without antibodies against RVFV, randomly allocated to either the vaccine
group (n = 8) or the control group (n = 8).
A test should be carried out for each of the recommended routes and methods of
administration. The vaccine administered to each ewe is of minimum potency.
Vaccinate ewes free from RVFV and without antibodies against RVFV, before pregnancy,
according to the recommended schedule. For the test use no fewer than 16 pregnant ewes
(eight vaccinated and eight controls). Keep all the animals as one group. Take a blood
sample from non-vaccinated animals shortly before challenge. Challenge each animal
between day 40 and day 60 of gestation. Two test models for the challenge may be

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performed with a sufficient quantity of virulent RVFV (observe until lambing or harvest of
fetuses at 28 days). Observe the animals clinically at least daily from challenge, and either
until the end of gestation or until harvest of fetuses after 28 days. If abortion occurs,
examine the aborted fetus for the presence of the RVFV. If animals are observed until
lambing, immediately after birth and prior to ingestion of colostrum, examine all lambs for
viraemia and antibodies against RVFV. If fetuses are harvested 28 days after challenge,
examine the fetuses for RVFV by suitable methods. Transplacental infection is considered
to have occurred if virus is detected in fetal organs or in the blood of newborn lambs or if
antibodies are detected in precolostral sera of lambs.
The test is invalid if any of the control animals have neutralising antibody before challenge.
The test should show significant difference in protection and transplacental transmission
between the vaccinated and control groups of animals.

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

There is currently no DIVA strategy available for the existing RVF vaccines.

2.3.5. Duration of immunity

As part of the authorisation procedure the manufacturer should demonstrate the duration of
immunity 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.

2.3.6. Stability
The stability of all vaccines should be demonstrated as part of the shelf-life determination
studies for authorisation.
The period of validity of a batch of lyophilised RVF vaccine or a batch of liquid inactivated
vaccine should not be less than 1 year.

REFERENCES
AFETINE J.M., TIJHAAR E., PAWESKA J.T., NEVES L.C., HENDRIKS J., SWANEPOEL R., COETZER J.A., EGBERINK H.F. &
RUTTEN V.P. (2007). Cloning and expression of Rift Valley fever virus nucleocapsid (N) protein and evaluation of
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*
* *
NB: There are OIE Reference Laboratories for Rift Valley fever
(see Table in Part 4 of this Terrestrial Manual or consult the OIE Web site for the most up-to-date list:
http://www.oie.int/en/our-scientific-expertise/reference-laboratories/list-of-laboratories/ ).
Please contact the OIE Reference Laboratories for any further information on
diagnostic tests, reagents and vaccines for Rift Valley fever

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