The document discusses Rift Valley Fever virus, including its diagnosis and vaccines. It provides information on the virus structure and proteins. It then summarizes methods for laboratory diagnosis of Rift Valley Fever during outbreaks and for surveillance. Finally, it reviews currently available and experimental vaccines for Rift Valley Fever, discussing their advantages and disadvantages.

1. Rift Valley Fever Virus: Diagnosis
and Vaccines
M. Kariuki Njenga, BVM, PhD
Centers for Disease Control and Prevention
Nairobi, Kenya

2. RVF Virology
RVF virions (Neg stain) Virions in hepatocytes
By Geisbert TW, USAMRIID, MD. G1,G2
vRNA
S 1690 nt
M 3885 nt
271 nt L 6404 nt
NP NSs untranslated
(245aa) (264aa)
[26 kDa] [17 kDa]
NSm G1 G2
(135 aa) (534aa) (507aa) RdRp
[14 kDa) [58 kDa] [56 kDa] (2092 aa)
[237 kDa]
NSm
NP  Not essential for virus replication
 Most abundant component of virion  Shown to suppress virus-induced apoptosis in vitro
 complexes vRNA in virion, cRNA in infection  Other functions unknown
Needed for virus replication + packaging
 recN ELISA good diagnostic tool G1, G2
NSs  Envelope surface glycoproteins
 Not essential for virus replication  Integral membrane proteins
 Blocks production of interferon in vitro  Involved in virus attachment and tissue tropism
 Essential for virulence  Targets of neutralizing antibodies

3. Laboratory Diagnosis – RVF Case Confirmation
Specimen: Serum, whole blood, liver tissue, aborted fetus
Tests performed: VI, antigen ELISA, PCR, IgM sandwich ELISA, IgG ELISA, VN

4. Biosafety in diagnosis
There is no vaccination for humans
– Reduce the risks!
• Special caution when doing PM’s
• Inactivate the sample within the 1st step
• Handling of lab specimen (let serum clot....)
• Reduce pipetting and dilution steps
• Wash plates and equipment with caution
• Wear PPE

5. Laboratory Quality Assurance
Successful diagnosis depends largely on
the quality of the specimen and the transport
and storage conditions of the specimen before
it is processed in the laboratory

6. Strategies for diagnosis of RVF
Based on detection of:
 Live virus
 Viral antigens 1 – 10 days
 Viral nucleic acids
 Acute phase antibodies (IgM) 4 – 42 days
 Chronic phase antibodies (IgG) 7 - ?days

7. Short viremia following RVF infection
• Data shows that 1010 RNA copies/mL of serum in
sheep and 108 copies/mL in cattle and humans
• At day 9 post-infection, calves no longer viremic,
and RVF virus can be isolated only from the brain.

8. RVF Diagnosis
 during outbreak
 post-outbreak
 for routine surveillance*
 for return to trade
 sentinel animals
*For routine surveillance
- is the region endemic or RVF-free?
- has there been vaccination?

9. RVF Diagnosis (Endemic Region)
During outbreak and post-outbreak
- PCR, antigen and IgM
- Send samples for virus isolation
- IgG NOT useful
For returning to trade after outbreak
- PCR, antigen and IgM
- Send samples for virus isolation
- IgG NOT useful

10. RVF Diagnosis (Endemic Region)
For routine surveillance
- IgM, IgG for animals born in IEP
- Send samples for virus neutralization
For sentinel animals
- IgM, IgG
- Send samples for virus neutralization

11. RVF Diagnosis (RVF-free Region)
- IgG sufficient
- May do both IgM and IgG for confirmation
- Send samples for virus neutralization

12. Antibody profile in infected vs vaccinated
IgG IgM
Infected
Vaccinated
Paweska , J Virol Methods. 2003

13. RVF Seroprevalence
Domestic animals
– Cattle, sheep goat sandwich/indirect EIA, VN
– Camel inhibition EIA, VN
Sentinel animals
– Sheep sandwich/indirect EIA, VN
Wildlife
– Buffalos + others inhibition EIA, VN
Vaccination NONE

14. Available test
Commercially:
– BDSL.....
inhibition ELISA for detecting IgG (in all species).
capture ELISA for IgM (in specified species, bov. capr. ovi)
indirect ELISA for IgG (anti-species conjugate)
Sandwich ELISA for IgG (in specified species, bov. capr. ovi)
Through research links
– CDC/USA (S. Nichol) - not available commercially
– USDA/USA (W. Wilson) – still undergoing validation
– NICD/RSA (J. Paweska) – most available through BDSL

15. RVF Vaccines: Situations and control
approaches
RVF Situation Examples of countries Current Control Strategy
Endemic with regular Kenya, Tanzania, Vaccination at sign of outbreak
outbreaks Egypt, Senegal, Mali Egypt: continuous vaccination
No vaccination
Endemic with sporadic/re- South Africa, Saudi Arabia Continuous/yearly vaccination
occuring outbreaks
Free high risk Middle East, North Africa (Active) surveillance
Free low risk Europe, Americas Surveillance, talks of vaccine
banks
Limited continuous vaccination of livestock in Africa:
• Cost of yearly vaccination
• Safety concerns: difficulties to determine physiological stages of pregnant animals
• Irregularity of outbreaks (years without signs of outbreak)
• Policy aspects: vaccination not always covered by government
Courtesy: Baptiste Dungu, GALVmed

16. Ideal RVF vaccine (Product profile)…
• Generic characteristics • Endemic regions
– Safety – Continuous vaccination: yearly
• Safe to produce vaccination of susceptible livestock
• Safe to all physiological stages of animals • Need to know how many vaccinations
• No residual virulence may be required to build a life long
• No risk of introduction into the environment (shedding, immunity
persistence in animals etc.)
• No risk of spread to human or other species
– Efficacy
• Solid protective immunity after 1
– Efficacy vaccination
• Protection of all susceptible species
• Quick onset of protective immunity, including in young
animals
• Long lasting immunity • Free regions
• STOP TRANSMISSION: prevent amplification of RVFV • Quick onset of protective immunity
in ruminants
• Protective in young animals and possibly
– Vaccination newborn naïve animals
• Cost effective for producers and users • Sterilizing immunity
• Single vaccination
• DIVA
• Ease of application
• Suitable for stockpiling (vaccine or antigen bank) and
quick availability
Courtesy: Baptiste Dungu, GALVmed

17. Vaccination strategies to be considered
 Endemic regions  Free regions/ Prevent epidemics
– Yearly vaccination – Elimination of possible source of re-infection
- Intermittent multiyear vaccination* – Use of non-replicating antigen vaccine
– Multivalent or combination vaccine, – Early and rapid onset of immunity, even in
consisting of RVF antigen & antigen of a young animals
vaccine likely to be used regularly
RVF+LSD; RVF+ s/g pox; RVF + CBPP  DIVA
– Thermostability – Positive marker: export of animals from
– Use of sentinel animals: need for good endemic countries
diagnostics capability & effective – Negative marker: for detecting infection
– Role of veterinary services  Possible suitable candidates:
 Possible suitable candidates: – Replication deficient, deleted, marker vaccine
– Multivalents including a safe deleted
RVFV vaccine
 Set up regional vaccine bank
- FAO/ NGO/Private company
- Need storage facility
Suitable vaccination strategies more critical than improved vaccines
Modified from: Baptiste Dungu, GALVmed

18. RVF traditional vaccines
VACCINE STRAIN ADVANTAGES DISADVANTAGES
Inactivated Pathogenic ● Safe in pregnant ● Short term immunity
(OBP, VSVRI) field strain animals ● Multiple vaccinations required
● Can be used in ● Risk of handling virulent strain during
outbreak production
● Colostral immunity present but poor
● Sheep better protected than cattle
● 100 x more antigen required than for
live attenuated
● Longer production lead time
Live Smithburn ● Highly immunogenic ● Potential residual virulence
Attenuated (OBP, ● Single dose ● Teratogenic for foetus
KEVEVAPI) ● Good immunity ● Potential risk of reversion to virulence
(within 21days) ● Not advisable for use in outbreaks
● Effective and easy ● Theoretical possibility of transmission
L production by mosquitoes (?)
● Safer production
● Large batches: >4m
S doses
Courtesy: Baptiste Dungu, GALVmed

19. New candidates evaluated in target animals
VACCINE STRAIN ADVANTAGES DISADVANTAGES
Live MP12 ● Effective and good protective ● Teratogenic for foetus
attenuated immunity ● Abortion in early pregnancy
● Easy and safe to produce ● Not available commercially
● Better safety than Smithburn in
most species and age groups
Avirulent Clone 13 ● Good protective immunity in ●Only registered to date in South
natural sheep & cattle Africa & Namibia
mutant ● Safe in pregnant animals ●large scale field data in other regions
● Safe in outbreak needed
● Produced as standard freeze- ●No evidence of DIVA to date
dried live vaccine
● Safe, effective and easy to
produce
● Possible DIVA (NSs ELISA?)
●Registered 7 used extensively in
South Africa
Recombinant LSD ● Dual vaccine ● Only proof of concept to date
Lumpy skin Neethling ● Safe in all animals ● Currently grown in primary cells
virus strain ● DIVA ● Possible GMO regulation challenge
expressing expressing ● Long shelf life (LSD) (?)
RVF RVF ● More thermo-tolerant than others
glycoproteins ● Efficacy shown in animal trials

20. RVFV clone 13 deletion
RNA segments Proteins
Nucleocapsid protein (N)
Large (L) Viral RNA polymerase (L)
Medium (M)
Small (S) Glycoprotein G1
Glycoprotein G2
NSm 14 & 78 KDa
NSs
100 nm
Courtesy: Baptiste Dungu, GALVmed

21. Clone 13 sheep efficacy data
Experiment 3: average temperature per group
post-challenge
42
41
40
39
38
37
36
35
D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12 D13 D14
3A Early Chall 3A Late Chall
3B Early Chall 3B Late Chall
3C Early Chall 3C Late Chall
Early Chall control Late Chall Control
Dungu et al., 2010

22. Clone 13 cattle efficacy data
Mean Body Temperature
41.5
Mean Body Temperature
41
Body Temp (Deg. C.)
for each group through the
viral challenge phase. DPC: 40.5
Days post-challenge. 40
39.5 Vaccinated Group
39 Control Group
38.5
38
37.5
37
0
3
6
9
-3
12
15
18
21
24
27
Days Post- challenge
Clinical course for the unvaccinated control group
Animal ID Peak Fever Day PC Duration of Euthanasia
fever
1367 41.2 3 8 days (2-9) 9
1402 41.4 2 1 day (2) 3
1404 41.0 2 7 days (2–8) 11
1405 41.3 2 1 day (2) 3
1406 40.4 2 3 days (2-4) 11
Dungu et al., In Submission

23. Candidates not evaluated in target animals
VACCINE STRAIN ADVANTAGES DISADVANTAGES
Avirulent (lab R566: deletion in ● Safer due to deletions in all 3 ● Never tested in target animals
generated) the M and S segments, may never reassort ● More stringent regulatory
reassortant segments ● Protection in mice requirements for registration (?)
Virus- Canarypox- ● DIVA: Positive & Negative ● No registered vaccine yet available
vectored RVF expressing RVF marker ● No large scale field data yet
vaccines proteins ● Live vaccine available, although extensive
● Replication deficient analytical data generated
Heterologous ● Multivalent: suitable where ●Data to date showing low
virus expressing annual vaccination is a challenge immunogenicity
GP: Newcastle ● Potential for improved
disease virus as thermostability
vector
Virus like VLP made of ● Potentially very safe ● No proof of concept in target
particle (VLP) envelop proteins ● Immunity similar to live vaccine, animals
(GP) but no replication ● Large scale production might be a
Naslund et al., ● DIVA challenge
2009
DNA DNA priming + ● DIVA ● Only incomplete protection
inact. Vaccine ● Potentially long lasting demonstrated in mice
Lorenzo et al., immunity ● Production challenges
2009 ● Ability to enhance and modulate ● Regulatory challenges (use in food
induced immunity animals)
cDNA encoding
GP

24. New Candidate…
VACCINE STRAIN ADVANTAGES DISADVANTAGES
Recombinant- ● Reverse genetic ● Less prone to reassortment ● Not yet registered
multiple generating RVF ● Live vaccine
deletion virus virus with double ● DIVA: negative marker
deletions in NSs &
● Easy and safe to produce
NSm
Bird et al., 2008 ●Target animal efficacy & safety
data generated
RNA segments Proteins
Nucleocapsid protein (N)
Large (L) Viral RNA polymerase (L)
Medium (M)
Small (S)
Glycoprotein G1
Glycoprotein G2
NSm 14 & 78 KDa
100 nm NSs
Courtesy: Baptiste Dungu, GALVmed