Bluetongue

Activities in 2009

Bluetongue

Dr Chris Oura, Dr Carrie Batten,Prof Peter Mertens and Prof Philip Mellor

IAH- Pirbright, Ash Rd, PirbrightGU24 0NF, United Kingdom

Tel.: (+44 1483) 23441, Fax: (+44 1483) 23 24 48

e-mail:,,,

websites: and

Summary of general activities related to the disease

  1. Test(s) in use/or available for the specified disease at your laboratory

Test / For / Specificity / Total
ELISA / Antibody / Group / 1801

SNT

/ Virus / Type / 77

PCR (gel based and realtime)

/ Virus / Type / 115 (against multiple serotypes)

Real time PCR

/ Virus / Group / 24015

Sequence analysis and Phylogenetic comparison of Seg 2

/ Seg 2 / 9

Egg inoculation

/ Virus isolation / 4 (generally use KC cells)

BHK-21 cell culture

/ Virus isolation / 17*

KC cell culture

/ Virus isolation / 40*
* number successful isolations

2.Production and distribution of diagnostic reagents:

1. 2009 ring-trial samples (10 EDTA blood and 8 serum): Sent to 35 countries including all EU National reference laboratories.

2.BTV- reference serum: BTV-1, 6, 8 and 11 antiserawere supplied to the Central Veterinary Institute, Netherlands. BTV-1, 2, 4, 6, 8, 9, 11 and 16 reference antisera were supplied to the Institute for Diagnostics and Animal Health, Romania. BTV-1, 2, 4, 6, 8, 9, 11 and 16 reference antisera were supplied to the Austrian agency for Health and food safety. BTV-1 to 24 reference antisera were supplied to Biopharma, Morocco. BTV-1 to 24 reference antisera were sent to the Scientific Institute of Veterinary Medicine, Serbia and the Russian National Institute of Veterinary Virology. BTV- 4, 9 and 16 reference antisera were supplied to Turkey.

3.BTV-reference viruses: BTV-6 and 8 sent to Central Veterinary Institute, The Netherlands. BTV-1 was supplied to Biobest, Scotland and the CzechRepublic. BTV-8 was supplied to Estonia. BTV-1, 2, 4, 6, 8, 9, 11 and 16 reference strains supplied to the Institute for Diagnostics and Animal Health, Romania. BTV-1 to 24 reference viruses were supplied to Biopharma, Morocco. BTV-1 to 24 reference viruses were sent to the Russian National Institute of Veterinary Virology.

4.RNA from BTV Reference strains: Supplied to the Department of Diagnostic Research, Netherlands.

5.BTV field isolates: NET2008/05 BTV-6 was supplied to the National Veterinary Institute, Norway. MOR2009/01 BTV-1 and MOR2009/07 BTV-4 were supplied to Spain. BTV-1 vaccine challenge strain were sent to Intervet (Netherlands). NET2008/05 BTV-6 strain for vaccine development supplied to Merial (France).

6BTV monoclonal antibody (Mab): Sent to Laboratoire Service International (LSI), France.

7.BTV positive control serum: Supplied to the Danish Institute for Food and Veterinary Research and Veterinary laboratory Agency, UK.

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.

In our capacity as the Community Reference Laboratory (CRL) for Bluetongue (BT) and OIE Reference Laboratory for BT, we are continuing to strengthen contacts and improve harmonisation in testing between the National Reference Laboratories of Member States of the EU and Member Countries of OIE. To help achieve this goal a panel of eight test sera were prepared in 2009 and were distributed to 36 National BT Laboratories in July – September 2009 as part of an intra-laboratory comparison test for ELISA. In addition a panel of 10 EDTA blood samples for use in PCR testing were sent out to 30 participating laboratories. Laboratories were asked to serotype the virus if serotyping capabilities existed. Intra-laboratory results were reported at a meeting of national bluetongue reference labs held in Brussels in November 2009.

4.Preparation and supply of international reference standards for diagnostic tests or vaccines.

Reference standards of viruses are prepared as part of an ongoing programme of maintenance of virus and reagent stocks and for QA purposes.Antiserum is regularly supplied to provide control material for ELISA and SNT.

5.Research and development of new procedures for diagnosis and control.

  1. Real time RT-PCR assays for the European serotypes of BTV, BTV-1, 2, 4, 6, 8, 9, 11 16 and 25 have been validated and commercialised with Laboratoire Service International (LSI), France. In addition prototype assays for the remaining BTV serotypes have been developed and evaluated. Primer and probe sequences for BTV-15 and BTV-24 have been passed to LSI for commercialisation.
  2. A BTV group-specific real-time RT-PCR assay has been commercialised with Qiagenthat can be used to detect both Eastern and Western topotypes of the virus. The identification of different virus lineages, using the BTV sequence database at IAH, can now provide more detailed information concerning the geographic origins and movements of individual virus strains. This provides valuable information concerning the routes of movement and risks posed by BTV outbreaks in locations around the world, particularly in countries on the borders of Europe.
  3. A reporting system has been established to provide detailed information concerning molecular characterisation of BTV isolates that are sent to the reference lab for analyses.These reports, that are sent to the submitting laboratory, provide both similarity data and phylogenetic trees to related field, vaccine and reference strains.
  4. Challenge experiments were carried out to assess the efficacy of the inactivated BTV vaccine that was used to vaccinate UK livestock in 2008. These experiments proved a high level of vaccine efficacy which lasted for up to 9 months in sheep vaccinated once. Further challenge experiments proved that lambs, born from sheep vaccinated twice with the second booster vaccine given one month prior to lambing, were clinically protected for up to 14 weeks of age by colostral antibodies.

6.Collection, analysis and dissemination of epizootiological data relevant to international disease control

The OIE reference laboratory at Pirbright responded to a request from the Belgiumreference laboratory to serotype samples from animals that they had found to be positive for BTV, but not serotype 1, 6or 8 which were circulating in Europe at the time. The virus was rapidly serotyped as BTV-11 by serum neutralisation tests (SNT) and confirmed by conventional RT-PCR. This is the first report of BTV-11 inEurope.Unfortunately due to the condition of the blood sample and its high Ct value no virus could be isolated,although the sequence data that was generated for genome segment 2 was identical to that of the South African BTV-11 attenuated vaccine strain. However, these data do not explain how the virus arrived in Northern Europe.

BTV was confirmed in Oman,Israel, Morocco and Greece. The isolates from Omanwere serotyped as BTV-1, 4, 8 and 16 using both serotype specific conventional RT-PCR assays and real-time RT-PCR assays. However, sequence analyses of these viruses indicated that they had not been derived from the outbreaks in Europe and the Mediterranean region but represented distinct introductions of viruses from African lineages (most likely from Sub Saharan Africa).Using the same set of assays, Israeli samples were found to contain BTV-4, 5, 8, 16 and 24. This was the first time BTV-5, 8 and 24 were found in Israel. Greek samples were serotyped as BTV-8 and BTV-16and Moroccan samples were confirmed to contain BTV-1 and BTV-4.

Representative isolates from each of these outbreaks were sequenced and molecular epidemiology reports detailing the VP2 sequences as well as phylogenetic comparisons with related viruses were supplied to the submitting country.

7.Provision of consultant expertise to OIE or to OIE Members

Staff within the OIE reference laboratory at Pirbright reviewed the bluetongue chapter in the‘Manual of Diagnostic Testsand Vaccines for Terrestrial Animals’ (2009).

Staff attended the second meeting of OIE Bluetongue Reference laboratories in Teramo, Italy in June 2009.

Expertise has been provided to Merial, Fort-Dodge and Intervet, concerning the use of different strains of BTV for vaccine development and testing.Expertise has also been provided to the Veterinary Medicines Directorate (at New Haw, in Surrey UK) concerning the evaluation of vaccine dossiers for inactivated BTV vaccines.

8.Provision of scientific and technical training to personnel from other OIE Members

In April a BTV training course was held at IAH Pirbright, six participants from four countries took part (Morocco, Malaysia, Jordan and China). The course consisted of lectures, practical demonstrations and hands on practical work. Topics covered included BTV diagnosis, insect vector identification and molecular epidemiology.

As part of an ongoing OIE twinning project with the Moroccan National Reference laboratory staff from the OIE reference laboratory, Pirbright have supplied reference viruses and antisera for all 24 serotypes of BTV to the laboratory. In addition staff visited the Moroccan laboratory and helped set up KC cell culture for virus isolation and serotype specific real time RT-PCR assays for BTV.In addition two scientists from the Moroccan National Reference laboratory visited the IAH with samples from a BTV experiment performed in Morocco. The scientists were trained in all the techniques required to analyse the samples.

9.Provision of diagnostic testing facilities to other OIE Members

Diagnosis of BTV and serotype identification by serology and real-time RT-PCR was performed for Belgium, Greece, Oman, Israel and Morocco.

Numerous serology and real-time RT-PCR assays have been performed on behalf of the UK’s farming community for pre-movement and pre-export testing.

10.Organisation of international scientific meetings on behalf of OIE or other international bodies

The OIE Reference Laboratory, under its remit as CRL organised a BT Ring Trial meeting in Brussels (30th November 2009) for the benefit of national and international reference laboratories and other concerned parties.

Members of staff participated in the organisation of the MedReoNet (an EU funded FP7 network concerning BTV) that was held in Lisbon during December 2009. Members of staff presented numerous papers and posters.

11.Participation in international scientific collaborative studies

An OIE-accredited twinning arrangement was successfully set up in October 2008 with the Moroccan National Reference laboratory for BT and AHS. Staff from the OIE reference laboratory, Pirbright have visited the Moroccan laboratory in 2009 and given advice on test selection and virus isolation techniques.

12.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

Peter Mertens - Bluetongue in Northern Europe: an update – Invited Symposium Lecture, Esteve Veterinaria(in New York) 28th February 2009

Batten, C.A., Darpel, K.E., Veronesi, E., Williamson, S., Anderson, P., Dennison, M., Clifford, S., Smith, C., Philips, L,. Bachanek-Bankowska, K., Sanders, A.,Bin-Tarif, A.,Wilson, A., Gubbins, S., Mertens, P. P. C., Oura C. A. L.,and P. S. Mellor. Transplacental transmission of Bluetongue Serotype 8 (BTV-8) in the United Kingdom (2007-2008).Epizone, 12th -14th May 2009, Antalya, Turkey.

Peter Mertens-The emergence and spread of Bluetongue virus across Europe:Climate change, insect vectors and vaccination -Invited Symposium Lecture Society of General Microbiology Annual Virology Symposium (the Legacy of Fleming), Harrogate 29th March to 2nd April 2009

Peter Mertens - What does the future hold? Emerging diseases/new vaccines -Bluetongue in Northern Europe - National Office of Animal Health. Food Chain Conference. ,Invited symposium talk at the Royal Scociety, 25th March2009

Peter Mertens - Bluetongue virus in Northern Europe: Are we over the worst? -Invited Plenary lecture: Glasgow Annual Virology Symposium 30th January 2009

Batten, C.A., Darpel, K.E., Veronesi, E., Williamson, S., Anderson, P., Dennison, M., Clifford, S., Smith, C., Philips, L,. Bachanek-Bankowska, K., Sanders, A.,Bin-Tarif, A.,Wilson, A., Gubbins, S., Mertens, P. P. C., Oura C. A. L.,and P. S. Mellor. Transplacental transmission of Bluetongue Serotype 8 (BTV-8) in the United Kingdom (2007-2008).WALVD, 17th – 20th June 2009, Madrid, Spain.

Mertens PPC,The emergence and spread of bluetongue virus in Europe 1998-2009: Med-Vet-Netmeeting El Escorial, Spain 2nd June 2009

Mertens PPC "The emergence and spread of Bluetongue virus across Europe:The impact of climate change, insect vectors and vaccination" Invited Keynote Lecture: 10th dsRNA Virus Symposium,HamiltonIsland21st June 2009.

Sushila Maan, Narender S. Maan, Kyriaki Nomikou, A. C. Potgieter, Piet van Rijn and Peter P.C. Mertens (oral presentation) Full genome characterization of bluetongue virus serotype 6 from the Netherlands 2008 and comparison to other strains. 10th dsRNA Virus Symposium,HamiltonIsland21st June 2009 .

Narender S. Maan, Kyriaki Nomikou, Sushila Maan, and Peter P.C. Mertens. Identification of reassortant bluetongue viruses from the western Mediterranean region,10th dsRNA Virus Symposium,HamiltonIsland21st June 2009.

Kyriaki Nomikou, Sushila Maan, Narender S. Maan, and Peter P.C. Mertens. Genome segment reassortment between two serotypes of bluetongue virus circulating in the eastern Mediterranean region. 10th dsRNA Virus Symposium, HamiltonIsland 21st June 2009.

S.Gold, P. Monaghan, S. Berryman, H.O.Alpar, P.P.C. Mertens and T.Jackson. Cell Entry of Bluetongue Virus.10th dsRNA Virus Symposium, HamiltonIsland 21st June 2009.

Andrew E. Shaw, Paul Monaghan, H. Oya Alpar and Peter P.C. Mertens. The Role of Bluetongue virus Untranslated Regions in viral mRNA Translation 10th dsRNA Virus Symposium, HamiltonIsland 21st June 2009.

Chris Oura, James Wood, Toby Floyd and Carrie Batten. 8th International Congress of Veterinary Virology, Budapest: Bluetongue virus in Northern Europe: Vaccination strategies and protection in sheep. 24th -26th August.

Peter Mertens, Sushila Maan, Narender Maan, Kyriaki Nomikou, Kasia Bankowska,Christiann Potgieter, Houssam Attoui , Alan Samuel :‘Equine encephalosis virusand other Equine Orbiviruses : Current status and diagnostic RT-PCr development.’ US Animal Health (USAH) meeting and American Association of Veterinary Laboraory Diagnosticians (AAVLD) meeting, San Diego October 2009.

Peter Mertens, Sushila Maan, Christiaan Potgeiter, Kyriaki Nomikou and Narender Maan (2009) ‘The molecular epidemiology of bluetongue virus infection in Europe & the impact of vaccination’US Animal Health(USAH) meeting and American Association of Veterinary Laboraory Diagnosticians (AAVLD) meeting, San Diego October 2009.

Peter Mertens, Sushila Maan, Narender Maan, Kyriaki Nomikou, Kasia Bankowska,Pottie Potgieter, Houssam Attoui , Alan Samuel(2009) Orbivirus Diseases of Horses: African and Peruvian horse sickness, encephalosis, and beyond –Department of Veterinary Medicine UC Davis, University of California,October 2009.

Mellor PS “Climatic effects on arbovirus occurrence with special reference to bluetongue and AHS”.Keynote address, Nordic-Baltic Veterinary Contingency Group, Riga, Latvia, 14th October 2009.

Mellor PS: “Climatic effects on arbovirus occurrence”. The annual Margary lecture to a combined meeting of the Royal Meteorological Society and the Royal Entomological Society, ImperialCollege.

K.E.Darpel, K.F. Langner, G.Venter, L.Hunt, D. Kurian, A.C. Potgieter, H. Attoui, P.Mellor, H.H.Takamatsu AND P.P.C. Mertens (2009)‘Investigation of salivaproteins from different Culicoides species and their interaction with the bluetongue virus particle’.MedReoNet meeting Lisbon December 2009:

S. Maan, Maan, N.S., Bankowska, K., Nomikou, K.and P. P.C. Mertens(2009) ‘Development of novel diaganostic assays for Orbiviral diseases of domestic and wild animals.’ MedReoNet meeting Lisbon December 2009:

Nomikou, K., Maan, S., Maan, N.S., and P. P.C. Mertens(2009) Molecular Epidemiology of BTV-9 in the Mediterranean region ’. MedReoNet meeting Lisbon December 2009:

Houssam Attoui, Fauziah Mohd Jaafar, Sushila Maan, and Peter Mertens (2009) Reverse genetics for dsRNA viruses. MedReoNet meeting Lisbon December 2009.

Chris Oura (2009) Epidemiology in Northern Europe: Bluetongue symposium entitled: Bluetongue control in Europe – New Challenges and Achievments, Amsterdam, NL. 2-3rd March 2009.

Chris Oura (2009) Bluetongue – the present epidemiological situation in Europe and opportunities tocontrol the disease.The International Scientific Conference in Poland organized by the Department of Ruminant Diseases, National Veterinary Institute in Pulawy together with Polish Buiatrics Society and Buiatrics Section. Pulawy, Poland. April 2009.

Chris Oura (2009) Information dissemination and the response of the UK to Bluetongue. The second meeting of OIE Bluetongue Reference laboratories in Teramo, Italy 1-2nd June 2009.

Chris Oura (2009) Bluetongue – a true transboundary disease.VLA International Conference - Animal Diseases 2009 at RoyalHollowayUniversity, Surrey, UK2nd -3rd Sept 2009.

Scientific publications in peer-reviewed journals

Batten,C.A., Sanders, A.J.,Bachanek-Bankowska, K., Bin-Tarif, A., and Oura, C.A.L (2009). Bluetongue virus: European Community proficiency test (2007) to evaluate ELISA and RT-PCR detection methods with special reference to pooling of samples. Veterinary Microbiology, 135, (3-4), 380-383

BattenCA, van Rijn PA and OuraCAL (2009). Detection of the European “field” strain of bluetongue virus serotype 6 by real-time RT-PCR. Veterinary Microbiology, e-publication: (

Burgin L, Gloster J and Mellor PS (2009). Why were there no outbreaks of bluetongue in the UK during 2008? Veterinary Record, 164, 384-387.

C.A.L Oura, J.L.N.Wood, A.J Sanders, A. Bin-Tarif, M. Henstock, L. Edwards, T. Floyd, H. Simmons, and C.A Batten (2009). Seroconversion, Neutralising Antibodies and Protection in Bluetongue Serotype 8 Vaccinated Sheep. Vaccine, 27, 7326-7330.

Carpenter S, Wilson A & Mellor PS (2009). Bluetongue virus and Culicoides in the UK: The impact of research on policy. Outlooks on Pest Management, 20, 161-164.

Carpenter, S., Wilson, A and Mellor PS. (2009). Culicoides and the emergence of bluetongue virus in northern Europe. Trends in Microbiology, 17, 172-178.

Darpel, KE, Batten CA, Veronesi E, Williamson S, Anderson P, Dennison M, Clifford S, Smith C, Philips L, Bidewell C, Bachanek-Bankowska K, Sanders A, Bin-Tarif A, Wilson A, Gubbins S, Mertens PPC, Oura CA & Mellor PS (2009). Evidence for a high transplacental transmission rate of the north European BTV-8 strain from dams to calves in British cattle. Emerging Infectious Diseases,15(12):2025-8.

De Clercq K., Mertens PPC., , De Leeuw, Oura CAL, Houdart P, Maan S, Hooyberghs J, Batten C, Vandemeulebroucke E, Maan N, Sanders A, Vanderstede Y, Nomikou K, Raymaekers M, Bin-Tarif A, Henstock M, Bréard E, Dubois E, Zientara S, Vandenbussche F (2009) Emergence of bluetongue serotypes in Europe, Part 2: the occurrence of a BTV-11 strain in Belgium. Transbound Emerg Dis. 2009 Dec;56(9-10):355-61.

Hemati B, Contreras V,Urien C, Bonneau M, Takamatsu H-H, Mertens PPC, Bréard E, Sailleau C, Zientara S and Schwartz-Cornil I (2009) Conventional dendritic cells disseminate the bluetongue hemorrhagic fever virus via skin lymph.Journal of Virology, 83, 8789-8799.

Hoffmann B, Beer M, Reid SM, Mertens PPC, Oura CAL, van Rijn PA, Slomka MJ,Banks J, Brown IH, Alexander DJ and King DP (2009) A review of RT-PCR technologies used in veterinary virology and disease control: Sensitive and specific diagnosis of five livestock diseases notifiable to the World Organisation for Animal Health. Veterinary Microbiology, 139(1-2):1-23.