OIE Reference Laboratory Reports s3

OIE Reference Laboratory Reports Activities in 2011

Name of disease (or topic) for Rift Valley fever which you are a designated OIE Reference Laboratory:

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: [email protected]

website: http://www.arc.agric.za

Name (including Title and Dr Baratang Alison Lubisi Position) of Head of Laboratory (Responsible Official):

Name(including Title and Dr Baratang Alison Lubisi Position) of OIE Reference Expert:

Name (including Title and Dr Baratang Alison Lubisi Position) of writer of this report (if different from above):

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 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

1. 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 Genomic material Group 867 RT-PCR

BHK or Vero cell Virus isolation Group 13 submissions with varying inoculations numbers of samples

Diagnostics project leaders: Drs BA Lubisi – BVMCh.MSc (classical virological methods) and Marco Romito – BVSc.MSc (molecular based diagnostic tests)

2 Annual reports of OIE Reference Centres, 2011 Rift Valley fever

2. Production and distribution of diagnostic reagents

Amount supplied nationally Amount supplied to other Type of reagent (including for own use) countries

RVFV recombinant One batch from 1 litre culture - 50 ml None nucleoprotein purified antigen: for routine diagnostics

Rift Valley Fever virus negative None Eight vials (1ml each) were sent to and positive antisera 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 None Three plates per kit were supplied for RVFV diagnostics 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 None i. 43 mg purified antigen development 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 Oscar Landeta CERTEST BIOTEC ; CEEI Aragón, Nave 16 María de Luna 11 ; E-50018 Zaragoza (SPAIN)

RVFV IgG positive antisera - N/A vials – 20ml each Bovine (animal number 4027)

vials – 10ml each (animal number 107)

vials – 20ml each (animal number 114)

RVFV IgG and IgM positive N/A vials – 100ml (total volume antisera - Ovine (animal number 452))

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 inter- laboratory 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 Greifswald- Insel 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).

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; Cut-off values not provided Negative: S/P <5

Result Interpretation Result Interpretation

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

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) – ARC- OVI; 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 prime- boost regime. Results of sample testing and analysis are pending.

Project leader: Dr David Wallace (PhD)

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 Test Results Positive results specimen reported to OIE s Tentative Confirmatory

Uganda 100 Yes No 14 N/A

Botswana 444 Yes No 90 N/A

No Yes 0 N/A

Namibia 8 Yes No 5 N/A

No Yes 0 N/A

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.

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

aDepartment of Virology, Central Veterinary Institute of Wageningen University and Research Centre, P. O. Box 65, 8200 AB Lelystad, The Netherlands bCIRAD, UMR Contrôle des maladies, 34398 Montpellier cedex 5, France cAnses, Laboratoire de Lyon 31 Avenue Tony Garnier, 69394 Lyon Cedex 07, France dWildlife Zoonoses and Vector Borne Disease Research Group, Animal Health and Veterinary Laboratories Agency (AHVLA), Weybridge New Haw, Surrey, UK eInstitute for Novel and Emerging Infectious Diseases at the Friedrich-Loeffler-Institut (FLI), Federal Research Institute for Animal Health, D-17493 Greifswald-Insel Riems, Germany

fCentro 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.

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Annual reports of OIE Reference Centres, 2011 9