OIE Reference Laboratory Reports
Activities in 2011
Name of disease (or topic) for
which you are a designated
OIE Reference Laboratory:
Rift Valley fever
Address of laboratory:
100 Old Soutpan Road
Onderstepoort 0110
Tshwane
SOUTH AFRICA
Tel.:
Tel.: (+27 12) 529 9117
Fax:
Fax: (+2712) 529 9418
e-mail address:
LubisiA@arc.agric.za
website:
http://www.arc.agric.za
Name (including Title and
Position) of Head of
Laboratory (Responsible
Official):
Dr Baratang Alison Lubisi
Name(including Title and
Position) of OIE Reference
Expert:
Dr Baratang Alison Lubisi
Name (including Title and
Position) of writer of this
report
(if different from above):
Dr Baratang Alison Lubisi
Annual reports of OIE Reference Centres, 2011
1
Rift Valley fever
Part I: Summary of general activities related to the disease
The OIE Rift Valley Fever (RVF) reference laboratory at Agricultural Research Council’s - Onderstepoort
Veterinary Institute (ARC-OVI) aligns its activities with the ARC’s strategic objective of enhancing the ability of
the agricultural sector to manage and mitigate agricultural risks. South Africa experienced sporadic outbreaks of
the disease in 2011. Laboratory activities were focused on disease diagnoses, sero surveillance and disease
certification of live animals and embryo donors for trade and breeding purposes (Table 1). The animal species
tested included bushbuck, blesbuck, gemsbok, impala, duiker, buffalo, sable, nyala, oryx, kudu, wildebeest, roan,
springbok, giraffe, alpaca, rhinoceros, elephant, ovines, bovines, caprines, canines and felines.
Validation of an in-house IgM ELISA assay and research projects aimed at studying RVF virus population
dynamics (temporal/spatial), host/tissue specificity and the role of mutations in virulence applying reverse genetics
techniques were embarked on.
Table 1 Diagnostic results for tests conducted on South African samples
Province
1.
No. of specimens
Positive results per test conducted
rRT-PCR
IgM ELISA
IgG ELISA
Gauteng
1384
11/57
7/516
22/811
Limpopo
203
0/21
0/17
1/165
North West
342
0/19
24/67
3/256
Northern Cape
495
3/73
4/8
3/72
Kwa-Zulu Natal
41
1/8
0/6
1/27
Free State
532
10/215
17/56
2/261
Eastern Cape
1362
98/266
17/165
21/931
Mpumalanga
670
2/18
2/385
1/267
Western Cape
327
61/188
1/11
7/128
Test(s) in use/or available for the specified disease/topic at your laboratory
Test
For
Specificity
Total
IgG ELISA
Antibody
Group
4835
IgM ELISA
Antibody
Group
4425
Serum Neutralisation (SN)
Antibody
Group
10
Real time hydrolysis probe
RT-PCR
Genomic material
Group
867
BHK or Vero cell
inoculations
Virus isolation
Group
13 submissions with varying
numbers of samples
Diagnostics project leaders: Drs BA Lubisi – BVMCh.MSc (classical virological methods) and Marco
Romito – BVSc.MSc (molecular based diagnostic tests)
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Annual reports of OIE Reference Centres, 2011
Rift Valley fever
2.
Production and distribution of diagnostic reagents
Type of reagent
Amount supplied nationally
(including for own use)
Amount supplied to other
countries
RVFV recombinant
nucleoprotein
One batch from 1 litre culture - 50 ml
purified antigen: for routine diagnostics
None
Rift Valley Fever virus negative
and positive antisera
None
Eight vials (1ml each) were sent to
NAHDIC, Sebeta, Ethiopia for
inter-laboratory testing
RVFV IgG and IgM ELISA kits
None
Five plates per kit were dispatched
to: Dr George Michuki
International Livestock Research
Institute (ILRI); P.O. Box 30709,
Kabete Campus, Old Naivasha
Road. Nairobi 00100, Kenya
IgM and IgG RVFV ELISA kits
for RVFV diagnostics
None
Three plates per kit were supplied
to each of the European countries
which participated in the ring trial
for RVFV ELISAs mentioned
under point 3.II below
Reagents for RVF rapid test
development
None
i. 43 mg purified antigen
ii. 40 mg purified anti-RVFv IgG
iii. Rabbit anti-sheep mu chain
antibodies
iv. Five positive sheep sera
v. Five negative sheep sera
vi. Four positive bovine sera
vii. One negative bovine serum:
Sent to Dr Oscar Landeta
CERTEST BIOTEC
CEEI Aragón, Nave 16
María de Luna 11
E-50018 Zaragoza (SPAIN)
Second batch of reagents to Dr Dr
María de Luna 11 ; E-50018 Zaragoza (SPAIN)
Type of reagent
Oscar
Landeta
CERTEST
BIOTEC ;
CEEI
Aragón,
Nave
Amount supplied nationally
(including for own use)
Amount supplied to other
countries
N/A
vials – 20ml each
(animal number 4027)
RVFV IgG positive antisera Bovine
16
vials – 10ml each
(animal number 107)
vials – 20ml each
(animal number 114)
RVFV IgG and IgM positive
antisera - Ovine
Annual reports of OIE Reference Centres, 2011
N/A
vials – 100ml (total volume
(animal number 452))
3
Rift Valley fever
Type of reagent
Amount supplied nationally
(including for own use)
Amount supplied to other
countries
IgM ELISA plates:
N/A
5
Positive and negative serum:
N/A
1 vial each
Conjugate:
N/A
1 vial
Substrate:
N/A
1 bottle 60 ml
IgG ELISA plates
N/A
5
Conjugate:
N/A
1 bottle: 60 ml
Substrate:
N/A
1 bottle 60 ml
Stop solution:
N/A
1 bottle 60 ml
Project leader, recombinant RVFV nucleoproprotein production: Dr Charlotte Ellis (PhD); coworker: Ms
Vuyo Mareledwane (MSc).
Part II: Activities specifically related to the mandate
of OIE Reference Laboratories
3.
International harmonisation and standardisation of methods for diagnostic testing or the
production and testing of vaccines
a)
Establishment and maintenance of a network with other OIE Reference Laboratories
designated for the same pathogen or disease and organisation of regular interlaboratory proficiency testing to ensure comparability of results
Not done.
b)
Organisation of inter-laboratory proficiency testing with laboratories other than OIE
Reference Laboratories for the same pathogens and diseases to ensure equivalence of
results
Evaluation of ARC-OVI’s in-house developed indirect ELISAs in an international ring trial
Our IgG and IgM ELISA assay performances were evaluated in a ring-trial arranged by Dr Jeroen
Kortekaas (CVI, Netherlands) as part of the ENCRAD project on RVF. The following ELISAs were
tested in the same trial: BDSL (Ayrshire, Scotland, UK), ID-VET (Montpellier, France). And the IgG
and IgM ELISA from ARC-OVI. The following labs participated: Department of Virology, Central
Veterinary Institute of Wageningen University and Research Centre, P. O. Box 65, 8200 AB Lelystad,
The Netherlands; CIRAD, UMR Contrôle des maladies, 34398 Montpellier cedex 5, France; Anses,
Laboratoire de Lyon 31 Avenue Tony Garnier, 69394 Lyon Cedex 07, France; Wildlife Zoonoses and
Vector Borne Disease Research Group, Animal Health and Veterinary Laboratories Agency (AHVLA),
Weybridge New Haw, Surrey, UK; Institute for Novel and Emerging Infectious Diseases at the
Friedrich-Loeffler-Institut (FLI), Federal Research Institute for Animal Health, D-17493 GreifswaldInsel Riems, Germany; Centro de Investigación en Sanidad Animal (CISA-INIA), Carretera de
Valdeolmos-El Casar s/n, Valdeolmos, Madrid, Spain 28130. In conclusion, the combined results of this
European ring trial suggest that the evaluated ELISAs are suitable for monitoring RVFV infections in
Europe, provided that surveillance protocols include validated confirmation assays.
Researchers involved from ARC-OVI: Dr Charlotte Ellis (PhD); Ms. Vuyo Mareledwane (MSc); Phelix
Majiwa (PhD).
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Annual reports of OIE Reference Centres, 2011
Rift Valley fever
ARC-OVI and the National Animal Health Diagnostic and Investigation Centre (NAHDIC), Sebeta,
Ethiopia, embarked on a serological inter-laboratory testing exercise (Table 2), aimed at determining the
harmony among the various diagnostic tests used in the two.
Table 2 Results of a RVF serology-inter-laboratory exercise between ARC-OVI, South Africa and
NAHDIC, Ethiopia carried out in April 2011
ARC-OVI, South Africa
NAHDIC Sebeta, Ethiopia
In-house: recombinant NP based
ID VET (Innovative Diagnostics)
Positive: S/P>/=10; Doubtful: S/P 5-9;
Negative: S/P <5
Cut-off values not provided
Result
Result
Interpretation
Interpretation
IgM
Sample 1
2.58
Negative
5.55
Negative
Sample 2
51.4
Positive
132.1
Positive
Sample 3
0.73
Negative
3.8
Negative
Sample 4
108.8
Positive
154.3
Positive
Sample 5
-0.53
Negative
4.4
Negative
Sample 6
4.26
Negative
7.8
Negative
Sample 7
101.5
Positive
143.2
Positive
Sample 8
157.93
Positive
161.9
Positive
IgG ELISA
Sample 1
2.34
Negative
117.7
Negative
Sample 2
38.2
Positive
24.5
Positive
Sample 3
2.04
Negative
105.9
Negative
Sample 4
72.8
Positive
5.5
Positive
Sample 5
0.79
Negative
108
Negative
Sample 6
0.49
Negative
110
Negative
Sample 7
23.3
Positive
7.5
Positive
Sample 8
26.4
Positive
10.6
Positive
A satisfactory series of results with 100% correlation of sample designation was observed between the two
laboratories. The noticeable large S/P ratio difference for sample 2 in the IgM ELISA between the two labs
could most likely be due to pippetting and washing inconsistencies, as concentrations of the capture antigen
and conjugate; environmental temperature; incubation time, and substrate quality would have affected other
samples as well.
4.
Preparation and supply of international reference standards for diagnostic tests or vaccines
No.
Item
Description
Quntity
Amount dispatched
1
Virus
RVF
Approximately 50 X 1 ml
None
2
Antiserum
Anti-RVFV
Approximately 5 0X 1 ml
None
Annual reports of OIE Reference Centres, 2011
5
Rift Valley fever
5.
Research and development of new procedures for diagnosis and control
I. Improvement of RVFV cell culturing
Various studies aimed at improving the growth of RVFV in cell culture were conducted in 2011.
Preliminary results showed that using a MOI of 1 in BHK cells, DMEM/F12 / 2% FCS medium and
harvesting after 5 days post-infection, produced the highest infectious titres.
Participants in the project were: Dr David Wallace (PhD); Mr Maanda Phosiwa (MSc) and Ms. Rachel
Maluleka (MSc).
II. Sequencing of South African RVF isolates
The purpose of this project is to sequence the whole genomes of RVF viruses (RVFV isolated during
outbreaks in South Africa (SA), from 1949 to 2010. The expected outputs are full genome sequences and
phylogenetic trees of these isolates, to assist in determining the relationships between the different outbreak
strains. Naturally occurring mutations in the genomes could be identified and selected for further studies
using the reverse genetics system.
Fifty one viruses were selected for the study. Full genome cDNAs of all the selected viruses were prepared
and sent to the International Livestock Research Institute (ILRI) for sequencing. Full genome sequences of
14 viruses have been produced and their analysis is under way.
Researchers involved in the project are: Mr Maanda N. Phosiwa (Msc); Ms Rachel Maluleka (MSc) and
Drs Phelix Majiwa (PhD) and David Wallace (PhD) from ARC-OVI; and Dr George Michuki (PhD) from
the ILRI.
III. Virulence of RVF virus
The purpose of this study is to apply reverse genetics techniques to investigate the impact of naturally
occurring mutations on the virulence of RVFV.
The long term expected output of this study is the development of a South African strain based vaccine, or an
improvement of currently available vaccines (e.g. clone13-like vaccines based on a SA strain).
Mr Maanda Phosiwa has since the inception of the project received training on RVFV reverse genetics
systems from the Animal Science Group - Central Veterinary Institute (ASG-CVI), The Netherlands,
between 08 May and 08 June 2011.
The naturally occurring mutation in the glycoprotein (GnGc) of the SA-2008 RVFV isolate was introduced
into a transcription plasmid.
Sequencing of the transcription plasmid to confirm introduction of the mutations, creation of more mutations
(if identified through sequence analysis of the virus), rescue of the recombinant viruses and performance of
virulence studies on the rescued viruses in cell culture and small animal models, is planned for 2012 and
beyond.
Researchers involved in this project are: Mr Maanda N. Phosiwa (MSc); Dr Phelix Majiwa (PhD) – ARCOVI; and Drs Jeroen Kortekaas (PhD) and Rob Moorman (PhD) – Animal Science Group - Central
Veterinary Institute (ASG-CVI), The Netherlands.
IV. Evaluation of a marker-free recombinant LSDV-vectored RVF vaccine
The project on this construct is on-going. One animal (sheep) trial was conducted late in 2011 using a primeboost regime. Results of sample testing and analysis are pending.
Project leader: Dr David Wallace (PhD)
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Annual reports of OIE Reference Centres, 2011
Rift Valley fever
6.
Collection, analysis and dissemination of epizootiological data relevant to international
disease control
Not done.
7.
Maintenance of a system of quality assurance, biosafety and biosecurity relevant to the
pathogen and the disease concerned
All diagnostic tests are performed by competent personnel; the laboratories engage in inter-laboratory tests,
and verified controls are employed. Tests are performed in biohazard cabinets, and personnel wear protective
clothing in addition to exercising good laboratory practice. Some of the staff members were vaccinated
against RVFV. Biological waste is autoclaved and incinerated.
8.
Provision of consultant expertise to OIE or to OIE Member Countries
Not done.
9.
Provision of scientific and technical training to personnel from other OIE Member Countries
The following candidates from the NAHDIC, Sebeta, Ethiopia, received training on RVF antibody detection
using the indirect ELISA assays between 17 and 21 October 2011:
i. Mr Tadiwos Kassa
ii. Mr Laikemariam Teshome
iii. Mr Tsehaye Siyum
Final year veterinary students, Mr Sam Beza Phiri and Jackson Katampi from Zambia, also received training
on RVF antibody detection using the indirect ELISA techniques between 4 and 5 July 2011, as part of their
state veterinarian electives.
10. Provision of diagnostic testing facilities to other OIE Member Countries
Country
No. of
specimens
Test
Tentative
Confirmatory
Results
Positive results
reported to OIE
Uganda
100
Yes
No
14
N/A
Botswana
444
Yes
No
90
N/A
No
Yes
0
N/A
Yes
No
5
N/A
No
Yes
0
N/A
Namibia
8
N/A - not applicable because the testes conducted were not confirmatory,
or if confirmatory, the results were negative
11. Organisation of international scientific meetings on behalf of OIE or other international
bodies
Not done.
Annual reports of OIE Reference Centres, 2011
7
Rift Valley fever
12. Participation in international scientific collaborative studies
ARC-OVI, South Africa is collaborating with various international institutions on the following projects:
i).
RVF Rapid Test development: Collaborator: CERTEST BIOTEC CEEI Aragón, Spain
ii). ENCRAD project on RVF
Co-workers; Central Veterinary Institute of Wageningen University and Research Centre, The
Netherlands; CIRAD, UMR Contrôle des maladies, France ; Anses, Laboratoire de Lyon France;
Wildlife Zoonoses and Vector Borne Disease Research Group, Animal Health and Veterinary
Laboratories Agency (AHVLA), UK; Institute for Novel and Emerging Infectious Diseases at the
Friedrich-Loeffler-Institut (FLI), Federal Research Institute for Animal Health, Germany; Centro de
Investigación en Sanidad Animal (CISA-INIA), Spain
iii). Sequencing of RVF isolates: Collaborator: ILRI
iv). Determination of RVFV virulence. International institution involved: ASG-CVI, Netherland
13. Publication and dissemination of information relevant to the work of OIE (including list of
scientific publications, internet publishing activities, presentations at international
conferences)

Presentations at international conferences and meetings
Dr David Wallace was an invited expert at the FAO RVF Diagnosis and Control workshop, where he gave an
oral presentation:
D. Wallace, 2011. Capripoxvirus-vectored recombinant Rift Valley fever vaccines. FAO RVF Diagnosis and
Control workshop, 19-21 January 2011, Rome, Italy.
Dr Lubisi attended a meeting organised by Global Alliance for Livestock Veterinary Medicines (GALVmed)
where she presented a talk: BA Lubisi*; M Romito; C Ellis; R. Maluleke; M Phosiwa; P Kegakilwe & P
Majiwa, 2011. Rift Valley Fever situation in South Africa: the OVI reference laboratory perspective.
GALVmed organised meeting on the technical feasibility of the establishment of a RVF vaccine bank, 15 -16
December 2011, Pretoria, South Africa.

Scientific publications in peer-reviewed journals
i) One article was published in a peer reviewed journal: Williams R, Ellis CE, Smith SJ, Potgieter CA,
Wallace D, Mareledwane VE, Majiwa PA, 2011. Validation of an IgM antibody capture ELISA based on a
recombinant nucleoprotein for identification of domestic ruminants infected with Rift Valley fever virus.
J. Virol. Methods. 177(2):140 -146
Articles or chapters reviewed:
ii) Dr Lubisi reviewed a RVF article for the OIE’s Scientific and Technical Review
iii) Dr Charlotte Ellis and Dr Roy Williams reviewed the first draft of the article: European ring trial to
evaluate Rift Valley fever virus ELISAs J. Kortekaasa, J. Kanta, C. Cêtre-Sossahb, P. Marianneauc, A. C.
Banyardd, M. Eidene, A. Brunf
a
Department of Virology, Central Veterinary Institute of Wageningen University and Research Centre, P. O.
Box 65, 8200 AB Lelystad, The Netherlands
b
CIRAD, UMR Contrôle des maladies, 34398 Montpellier cedex 5, France
c
Anses, Laboratoire de Lyon 31 Avenue Tony Garnier, 69394 Lyon Cedex 07, France
d
Wildlife Zoonoses and Vector Borne Disease Research Group, Animal Health and Veterinary Laboratories
Agency (AHVLA), Weybridge New Haw, Surrey, UK
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Annual reports of OIE Reference Centres, 2011
Rift Valley fever
e
Institute for Novel and Emerging Infectious Diseases at the Friedrich-Loeffler-Institut (FLI), Federal
Research Institute for Animal Health, D-17493 Greifswald-Insel Riems, Germany
f
Centro de Investigación en Sanidad Animal (CISA-INIA), Carretera de Valdeolmos-El Casar s/n,
Valdeolmos, Madrid, Spain 28130.

Other communications
i). RVFV (M35/74) was prepared and supplied to Onderstepoort Biological Products (OBP) for use as
challenge virus in their vaccine trial. The reference strain was prepared by Dr David Wallace (PhD)
ii). Dr Lubisi formed part of an expert team that convened at the OIE headquarters between 6 and 8
December 2011, to revise the Rift Valley Fever chapter, Chapter 2.1.14 of the OIE Manual of Diagnostics
and Vaccines for Terrestrial Animals, 6th Edition of 2008. The group comprised the following
individuals: Drs Jeroen Kortekaas -,Central Veterinary Institute of Wageningen, University Research;
Michèle Bouloy - Département de Virologie Institut Pasteur; Amadou Alpha Sall - of Institut Pasteur de
Dakar Unité des Arbovirus et Virus de fièvres hémorragiques ; Pierre Rollin - Special Pathogens Branch
National Center for Zoonotic, Vector-borne, and Enteric Diseases Centers for Disease Control and
Prevention ; Medhi El Harrak - Chef Département Virologie, Biopharma Laboratory; Baptiste Dungu GALVmed ; Danny Goovaerts - Global R&D Government Regulated Diseases MSD Animal Health;
Stéphane de La Rocque - OIE- Sub-Regional Representation for Europe.
_______________
Annual reports of OIE Reference Centres, 2011
9