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SID 5 Research Project Final Report

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NoteIn line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The SID 5 (Research Project Final Report) is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra website. A SID 5 must be completed for all projects.

This form is in Word format and the boxes may be expanded or reduced, as appropriate.

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Project identification

1. Defra Project code R16 (comissioned research) (SE2613)

2. Project title

Molecular characterisation of the bluetongue virus genome to develop and support improved diagnostic methods and molecular epidemiology studies in relation to control of infection.

3. Contractororganisation(s)

Pirbright Laboratory, Institute forAnimal Health,Ash Road,Pirbright, Woking ,Surrey,UK GU240NF

54. Total Defra project costs £ 587165.52(agreed fixed price)

5. Project: start date................ 01 July 2006

end date................. 30 June 2009

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6. It is Defra’s intention to publish this form. Please confirm your agreement to do so...................................................................................YES NO (a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They

should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow.Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer.In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.

(b) If you have answered NO, please explain why the Final report should not be released into public domain

Executive Summary7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the

intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.

The work of this research project builds on previous sequencing studies of the bluetongue virus genome that were carried out at IAH Pirbright (including the previous Defra funded project SE2612 (2003-2006) and will be continued in contract SE2617). Novel cDNA synthesis methods were initially developed (Maan et al 2007a) making it much easier to synthesise and sequence full length cDNA copies

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of both dsRNA and ssRNA virus genomes (Maan et al 2007b; Attoui et al 2007). Using these new techniques, full-length sequence data were generated for multiple genome segments, particularly genome segment 2 (Seg-2), from different isolates of the 24 established BTV (sero)types. During the project BTV genome segment 2 (Seg-2) has been completely sequenced (full length) from 134 BTV isolates from around the world (including particularly multiple strains from the BT outbreaks that have occurred in Europe - since 1998) (Objective 1).

The availability of full length, high quality nucleotide sequence data for multiple genome segments, in some cases for the entire genomes, of different BTV isolates has made it possible to clearly identify ‘geographic’ separation between eastern (India, Malaysia, Australia) and western (Africa, and the Americas) groups of viruses. Some evidence for further sub-grouping was also detected (e.g. between western strains from African and American, or eastern and far-eastern sub-groups). These studies have shown that Europe now represents a ‘cross-roads’ for bluetongue viruses, containing a unique mixture of both eastern and western lineages, providing unparalleled opportunities for genome segment exchange (reassortment) and the potential for the emergence of novel virus strains. As one example two distinct lineages of BTV-1 were detected within Europe, belonging to eastern and western groups respectively.

Analysis of the sequence data that were generated, have confirmed that BTV Seg-2 varies in a type-specific manner, and that the outer capsid protein ’VP2’ (which it encodes) is the major determinant of (sero)type (Maan et al 2007b; 2008; Mertens et al 2007). These sequences (together with the sequences generated for other BTV genome segments – Objective 3) provide a basis for identification of the different BTV types, as well as molecular epidemiology studies of the virus, making it possible to track the lineage and origins of any new isolates more accurately/reliably than was previously possible by serological methods. As the most variable region of the BTV genome, Seg-2 is particularly suited as a target for these studies and can be used to differentiate between closely related virus strains, making it possible to track the movements of the virus even within a single disease outbreak.

Sequence analyses of the entire genomes (including the more ‘conserved’ segments 1, 3, 4, 5, 6 and 9) of selected European field strains and the vaccine strains of BTV-1, 2, 4, 9 and 16 (Maan et al 2008 - Objective 5), have supported the design of BTV species/serogroup-specific, conventional and real time RT-PCR assays (Anthony et al 2007, Shaw et al 2007 – Objective 4). These assays can be used for the rapid detection and reliable identification of BTV RNA in tissue cultures material, blood or other tissue samples, and are now widely accepted as standard diagnostic assays for virus detection ad identification. Similarly sequence data for Seg-2 were used to develop RT-PCR assays, to identify individual BTV types more reliably and much more rapidly than is possible by conventional serological methods (hours rather than weeks) see: www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/ReoID/btv-S2-primers.htm. (Mertens et al 2007 – Objective 2)

As a consequence of widespread outbreaks of BTV across Europe, and associated vaccination campaigns, the detection of BTV serogroup specific antibodies (e.g. by ELISA) has lost much of its previous importance as an epidemiological tool for surveillance, to detect recent BTV infections/introductions in the region. However, primers (targeting Seg-2) were also designed from the sequence data generated during the project, for the identification and differentiation of vaccine and field strains of all BTV serotypes, as well as eastern and western geographic variants (topotypes) of each of the European BTV types (Mertens et al 2007 – Objective 2). The rapid identification of BTV serotype is essential to detect incursion of new BTV strains into Europe or the UK and is required for selection and deployment of the homologous vaccine type. These assays therefore make a significant contribution to current control programmes / strategies and epidemiological studies for BTV.

The real-time serogroup and type specific RT-PCR assays were evaluated / validated and are in the process of commercialisation and are being established within the Community Reference laboratory for BTV at IAH Pirbright as standard diagnostic procedures (Objective 6). In many cases these methods have removed the need for virus isolation prior to BTV identification and typing (see typing assays above) assays. Together with the development of alternative isolation methods for BTV in a Culicoides cell line (KC cells), this has significantly reduced the need to use animals (particularly embryonated chicken eggs) for BTV diagnosis.

The methods and databases developed by the project have provided a primary basis for the identification of multiple incursions of BTV into Europe since 1998, helping to identify four separate routes of entry into the region. These events have included 12 distinct virus strains/lineages belonging to nine different serotypes of BTV (types 1, 2, 4, 6, 8, 9, 11, 16 and 25). During 2006 BTV-8 was identified for the first time in Europe, at the start of the northern European outbreak (Maan et al 2008). These methods were also used to identify BTV-1 in North Africa (which subsequently spread to Spain, Italy and France), as well as BTV-4 and 15 in Israel. During 2007, 2008 and 2009 these techniques were used to identify and track the incursion and spread of BTV-8 in the UK and many other European countries, as

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well as novel incursions BTV-9 into Europe and BTV-24 in Israel. The appearance of these strains, which had not previously been detected in the Mediterranean region represent further potential threats to European animals. In late 2008 / early 2009, strains of BTV-6 and BTV-11 were also identified in the Netherlands/Germany and Belgium respectively, which were shown to be derived from the South African live- BTV-vaccine strains (Objective 5). Several of the modified live vaccines had been shown to replicate to high levels, causing significant/severe disease in naive northern European animals, and consequently represent a disease risk in their own right (Veronesi et al 2005; 2009). Indeed previous outbreaks caused by vaccine strains of BTV-2, 9 or 16 had been detected after their use in Southern Europe. However the source /route of introduction of these BTV-6 and 11 vaccine strains remains uncertain, as neither vaccine strain has ‘officially’ been approved or used in the region.

Sequencing studies of ‘Toggenburg virus’ from goats in Switzerland, and comparisons to the sequence database generated by this project, confirmed that although it is a BTV, it does not belong to any of the 24 BTV types previously recognised, and it therefore represents the first identification of a distinct and novel 25th serotype (Hoffman et al 2008). This conclusion was confirmed by analysis of serum samples from infected animals showing that they did not neutralise any of the existing 24 types.

The entire genome the first isolate of BTV-8 from the northern European outbreak in 2006 (represented by strain NET2006/04) was sequenced (Maan et al 2008) (Objective 3). Each genome segment was compared to other BTV isolates from Europe and around the world, including vaccine strains of BTV-1, 2, 4, 9 and 16. In each case it was shown that NET2006/04 belongs to a ‘western group’ (topotype) of viruses, but (using data for Seg-2) is distinct from the live attenuated vaccine strain of BTV-8 that was originally developed in South Africa. NET2006/04 was also shown to be most closely related to an isolate of BTV-8 from Nigeria that was made in 1982 (NIG1982/07). However, it has not been possible to determine exactly how BTV-8 first arrived in northern Europe in 2006.

The molecular diagnostic methods developed as part of this project to identify the 24 BTV serotypes, were also used to assist USDA at Ames, Iowa in identification of previously untyped isolates of BTV made in the USA since 1999. Eight BTV serotypes (types 1, 3, 5, 6, 14 19, 22 and 24) were identified which had not previously been detected in the North-American continent. With the recent appearance of BTV-2 & BTV-7 in Australia, this suggests that the emergence of exotic BTV strains into new areas associated with climate-change is a global phenomenon (Purse et al 2008).

The reference collection at IAH Pirbright was expanded during the project with the addition of 324 further isolates of BTV, many of them from the European outbreak of BTV-8, including many samples / isolates from the UK. Full-length sequences of genome segment 2 from 134 BTV strains, belonging to a range of different BTV serotypes and topotypes, were added to the molecular epidemiology data base held at IAH Pirbright during this year alone (milestone 7). This has provided additional information concerning the geographic variability, and global distribution / grouping of these viruses.

Genome segment 2 of BTV-8 was analysed from over 37 different blood samples (milestone 7), identifying a series of individual point mutations. This ‘single nucleotide polymorphism’ (SNPs) analysis, can be used to ‘track’ virus movements (Cottam et al 2008), and has provided additional information concerning the spread of BTV-8 from site to site during the 2006-8 BT-outbreaks in northern Europe and the UK (Maan et al in preparation). In addition to the sequence data for 134 examples of genome segment 2 (Objective 1), sequence data were also generated for other BTV genome segments, from multiple isolates belonging to different BTV serotypes and topotypes (Objective 3), including the vaccine strains of BTV-2, 4, 9 and 16 previously used in Europe (Objective 5). These included: 27 different examples of genome segment 1; 43 examples of segment 3(giving a total of 94); 45 examples of segment 4; 117 examples of segment 5; 30 examples of segment 6 (giving a total of 97); 122 examples of segment 7; 67 examples of segment 8; 54 examples of segment 9; & 85 examples of segment 10 (with 20 further examples of segment 10 specifically from strains of serotype 2). These data were generated using multiple sequencing primers, that can also be used to specifically detect and identify individual genome segments from viruses that belong to either eastern or western BTV topotypes (or both) (milestone 8 and 10). These sequence data have provided an initial assessment of the frequency of genome segment exchange (reassortment) between different BTV strains within Europe, suggesting that it is both a widespread and frequent event. They have also shown that the BTV-16 strain from Italy 2002 is a reassortant between the BTV-2 and BTV-16 vaccine strains (Batten et al 2008). Both of these vaccine viruses were used for several years as part of a multivalent live vaccination campaign in Israel, which may have allowed the initial reassortment event to occur. The recent BTV-6 strain from the Netherlands 2008 is also a reassortant, between the BTV-6 vaccine strain and another unknown virus (in segment 10).

Project Report to Defra

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http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/btv-8.htm#NIG1982/07

http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/btv-8.htm#NET2006/04

8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the scientific objectives as set out in the contract; the extent to which the objectives set out in the contract have been met; details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability; the main implications of the findings; possible future work; and any action resulting from the research (e.g. IP, Knowledge Transfer).

The Scientific Objectives as set out in the original contract

Objective 1: Generate sequence data for genome segment 2 of additional BTV isolates, for molecular epidemiology studies and to extend the existing database.

Objective 2: Design and test oligonucleotide primers, for RT-PCR assays to detect and identify each of the 24 BTV types.

Objective 3: Generate sequence data from other genome segments (specifically segments 3, 6, 7 and 10) for additional BTV isolates, to provide information on genetic variation and gene reassortment

Objective 4: Design oligonucleotide primers that can be used in RT-PCR assays to detect and identify any strain of BTV and distinguish them from isolates of other Orbivirus species (e.g. AHSV, EHDV, and EEV).

Objective 5 : Generate additional sequence data for the European vaccine strains.

Objective 6: Validate primers designed in objectives 2 and 4 and provide these to the BTV reference laboratory at IAH Pirbright

The extent to which the Objectives have been metThe work of this research project builds on previous sequencing studies of the bluetongue virus genome

segments (including the previous Defra funded project SE2612 (2003-2006)). The project has been very successful, achieving or exceeding all of its stated objectives and milestones within the timescales specified. Using novel cDNA synthesis methods developed at IAH Pirbright (Maan et al 2007a), full length nucleotide sequence data have been generated and analysed for multiple individual isolates of the different BTV serotypes (Maan et al 2007b). These data have been used to design oligonucleotide primers for use in conventional and real time RT-PCR assays to detect and identify BTV as well as each of the 24 BTV serotypes more reliably and more rapidly than ever before (Shaw et al 2007; Anthony et al 2007; Mertens et al 2007) see: www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/ReoID/btv-S2-primers.htm. These methods (which are in a process of commercialisation as assay kits through Qiagen, and LSI) and the associated sequence databases now represent the principle tools used to detect and identify existing and novel incursions of BTV in the UK reference laboratory at Pirbright, and in many other laboratories in Europe and around the world.

cDNA synthesis and sequencing methodsNovel methods for cDNA synthesis (using ligation of an ‘anchor primer’ to the 3’ ends of each

dsRNA segment) and cDNA sequence analyses (using terminal ‘phased’ primers), were initially developed at IAH Pirbright as part of this and the previous Defra project (SE2612 2003-2006) ( Maan et al 2007a). These methods have proved to be suitable not only for the full length amplification and sequencing of the genome segments of BTV and related orbiviruses (Maan et al 2007b, Nomikou et al 2009; Anthony et al 2009a, b, c, d; Ozkul et al 2009; Yadin et al 2007) but also for other dsRNA viruses (Green et al 2006, 2007, 2008; Green et al 2006, 2007 Mohd Jaafar et al 2007) and even ssRNA viruses (Attoui et al 2007).

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The sequencing studies of different genome segments of different BTV strains, belonging to different serotypes and from different geographic origins (topotypes), have primarily used virus isolates from the BTV reference collection that was set up at IAH Pirbright, specifically to support these studies (see http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/Virus-nos.htm ). During the period of the project (2006-2009), a total of 324 new isolates/samples of BTV, derived from well documented sources (locations / dates / species) were added to the reference collection (see:http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/virus-nos-by-country.htm). These included viruses from around the world, particularly multiple isolates/samples from European outbreak of BTV-8 (191 samples) and strains of both established and previously exotic serotypes from the USA (54 isolates) supplied by Dr. D.Johnson as part of collaborative studies with USDA (Aimes, Iowa).

Objective 1: Generate sequence data for genome segment 2 of additional BTV isolates, for molecular epidemiology studies and to extend the existing database:

During the project, full length sequence data were generated for genome segment 2 from a total of 134 bluetongue virus isolates / samples. With previous data this gives a total of 219 full length seg-2 sequences for different BTV strains, and included the reference strains of the 24 established BTV serotypes (Maan et al 2007b).

Analyses of this initial data set showed that Seg-2 separated into 24 distinct clades, representing the individual BTV ‘types’ Figure 1. Further comparisons, of Seg-2, from multiple BTV isolates of each serotype from around the world (different topotypes), demonstrated that although a significant levels of variation exists between different topotypes within a single serotype, they consistently group together. This indicates that the different BTV serotypes existed, before individual strains of each serotype became geographically separated, then accumulated further mutations to give rise to different topotypes.

Figure1: Phylogenetic comparison of full-length Seg-2 nucleotide sequences for the reference strains of the different BTV serotypes. The bluetongue viruses that have invaded Europe since 1998 (types 1, 2, 4, 6, 8, 9, 11 and 16) are indicated. 24 distinct clades were detected that correlate perfectly with virus serotype. Molecular comparisons of Seg-2 can therefore be used to identity BTV serotype more rapidly and more accurately than by conventional serological methods (Maan et al 2007a)

. The sequence data generated for Seg-2 showed that some of the individual BTV types are more closely related than others, forming into a number of distinct ‘nucleotypes’ (figure 2: A-E). These groupings also reflect serological relationships between types, as previously detected in serum neutralisation assays (figure 3 A-E) , providing further evidence for a close correlation between variations in Seg- 2 and the serological properties (serotype) of individual BTV isolates.

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http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/virus-nos-by-country.htm

http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/Virus-nos.htm

Figure 2: Unrooted neighbour-joining tree showing relationships between nucleotide sequences of Seg-2 from the 24 BTV types: This NJ tree was constructed using MEGA2 (Kumar, et. al., 2001) with the default parameters and the full-length genome seg-2 (VP2 gene) sequences of the 24 BTV types. The nine evolutionary branching points are indicated by black dots (along with their bootstrap values) on the tree which correlate with the 10 'Nucleotypes’ designated as A-J. Nucleotype is <35% difference in Seg-2 nucleotide sequences. (From Maan et al 2007b)

Figure 3: Schematic illustrating the serological relationships between BTV serotypes (Adapted from Erasmus B.J., 1990): The thicker lines shown between serotypes (represented by appropriate numbers) indicate stronger serological relationships, as detected in plaque-reduction assays. The thinner lines represent relationships that are only evident as cross or heterotypic antibody responses in cross-protection assays. The black dotted lines represent interrelationships that are very weak. The different BTV nucleotypes (A – J) that were identified by phylogenetic analyses are represented by ovals. Serotype 24 is phylogenetically related to serotypes 4, 10, 11, 17 and 20 but the serological data concerning relationships between this and other types is limited. We have therefore included it within the oval shown for nucleotype ‘A’ but without a line connecting it to other serotypes. (From Maan et al 2007b)

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The close correlation that was demonstrated between variations in BTV Seg-2 sequences and the virus serotype, indicated that it would be possible to identify individual BTV ‘types’ by sequence analyses and phylogenetic comparisons of Seg-2.

The sequences of Seg-2 from the 24 reference strains of different serotypes, provides an initial ‘reference’ dataset that can be used for BTV ‘type’ identification. Comparisons of the entire Seg-2 data set (including over 220 different isolates from around the world), were used to set initial values for the levels of variation detected in the coding and non-coding regions of Seg-2 and amino acid sequences of VP2, within different topotypes, serotypes, nucleotypes and different Orbivirus species, as a guide to the identification and classification of new isolates of BTV and other orbiviruses (figure 4).

Figure 4, panels A-D: Sequence identity in Seg-2 and outer capsid protein VP2: The range of amino acid and nucleotide sequence identities that were detected within and between BTV serotypes, topotypes and nucleotypes, or between BTV and other Orbivirus species, are shown schematically. Estimates of the level of similarity within a single serotype are based on multiple datasets for widely distributed isolates of types 1, 2, 4, 9 and 16 (the European serotypes) (Maan et. al., 2007). The comparison of Seg-2 and VP2 from different Orbivirus species included available data for EHDV-1 (Ac. no. D10767); EHDV-2 (Ac. no. AB030735); Chuzan virus (Ac. no. AB014725); AHSV-1 (Ac. no. AY163329); AHSV-5 (Ac. no. AY163331); BRDV-2 (Ac. no. M87875) and SCRV (Ac. no. AF133432). (From “Bluetongue” a Monograph Chapter 7 Maan et al 2009)

The geographic/topotypic variations that were detected between different isolates of the same serotype indicated that analyses of Seg-2 sequences could also be used for ‘molecular epidemiology studies’ of BTV strains, to determine their spread, movements and geographic origins. The largest of the topotypic variations detected in Seg-2 (as well as in other more conserved segments of the viral genome: see below) separates the viruses into ‘eastern’ and ‘western’ groups (representing Australia, the Middle East, India and Indonesia -- or Africa and the Americas, respectively), with evidence of still further sub-groupings. By creating a database of Seg-2 sequences from a large number of different well documented BTV isolates from around the world (from the IAH reference collection), this project

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27

78

% Amino acid identity

22

7459

70

Between Orbivirus species

BTV: Same serotype, different topotype

BTV: Different serotype & nucleotypeBTV: Different serotype; same nucleotype

0%

100%

5.5

BTV: Same serotype, same topotype9393

41

% nucleotide identity

40 61BTV: Same serotype, different topotype


0%

100%

71696729


BTV: Same serotype, same topotype

87

% nucleotide identity in 3’NCR



BTV: Different serotype & nucleotype

BTV: Different serotype; same nucleotype

0%

100%

9736 59 77 7821 84







0% 100%

94

38 62 696723.5

82


81.4

75

88.2

27

78

% Amino acid identity

22

7459

70




0%

100%

5.5

BTV: Same serotype, same topotype9393

41

% nucleotide identity

40 61BTV: Same serotype, different topotype


0%

100%

71696729



87






0%

100%

9736 59 77 7821 84







0% 100%

94

38 62 696723.5

82


81.4

75

88.2

has made it possible to identify the origins of individual virus lineages/isolates with greater precision, by simple phylogenetic comparisons.

Sequence analyses of Seg- 2 from the original index case of BTV-8, from the outbreak in northern Europe (isolate NET2006/04 from the Netherlands 2006 – Maan et al 2008) and comparisons to isolates of BTV-8 from the Netherlands and the UK in 2007, demonstrated that their sequences were very similar. However, a small number of point mutations changes were identified that could be used to differentiate between different isolates from the outbreak. The two viruses analysed from the Netherlands contained a total of 5 nucleotide changes in Seg-2, four of which were ‘conservative’ in terms of their amino acid coding. This ‘single nucleotide polymorphisms’ (SNPs) analysis, indicated that both of the Netherlands strains had arrived at the same farm in East-Anglia during August September 2007. The virus then spread within the East of England, via cycles of infection in midges and ruminant hosts. During transmission a number of further point mutations were identified that had accumulated within Seg-2. This initial analysis provided indications of the early movements of BTV-8 during the UK outbreak (figure 5), and suggest that further analyses of BTV-8 strains could be used to track the viruses movement across Europe and help to identify any further ‘new’ incursions into the UK.

Similar studies conducted with samples from the UK outbreak of foot and mouth disease during 2007, clearly showed the routes of transmission from farm to farm and even help predict the existence of individual infected premises (Cottam et al 2007).

Figure 5, SNPs analyses of Seg-2 from BTV-8 isolated in the Netherlands and the UK during 2006-2007: Five nucleotide differences (four of which are conservative) were detected in genome segment 2, between BTV-8 isolates from the Netherlands in 2006 and 2007. Comparisons of these sequences with those obtained for BTV-8 isolates from the UK in 2007, indicate that both of the Dutch strains arrived simultaneously in the UK in August 2007, infecting two different highland cows (Lorraine and Debbie) in the same field at Balyham. These two Seg-2 lineages accumulated a number of further point mutations during the early stages of virus spread in the UK, suggesting routes of movement (as indicated).

During 2008 several goats in Switzerland, that were due to be exported, were shown to be positive for BTV by RT-PCR, although subsequent attempts to type the virus were unsuccessful. Sequence analyses of genome segment 2 and comparisons to our the database generated by this project indicated that the virus does not belong to one of the 24 conventional BTV types, but in fact represents an entirely novel ‘25th’ serotype (Hofman et al 2008) (figure 6). These conclusions were confirmed by serological studies in the Reference Laboratory at IAH Pirbright, showing that antisera from animals

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http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/btv-8.htm#NET2006/04

infected with the virus did not neutralise any of the 24 reference strains of BTV. Sequencing studies of eight of the BTV-25 genome segments, confirmed its identity as a bluetongue virus but also indicated that it does not belongs to the same topotype as any of the other viruses we have analysed.,

Figure 6: Neighbour joining tree showing the relationship of Toggenburg orbivirus (BTV-25) to the other BTV serotypes in Seg-2. The BTV-25 sequence has a distinct branching point from the other viruses in nucleotype A indicating that it also represents a further distinct nucleotype (nucleotype ‘F’).

Objective 2: Design and test oligonucleotide primers, for RT-PCR assays to detect and identify each of the 24 BTV types.

The confirmation that the nucleotide sequence of BTV genome segment 2 shows a close correlation with virus serotype (see above), indicated that it would be possible to design nucleic-acid based ‘type-specific’ assays, using either conventional / real-time RT-PCRs, or ‘chip’ based strategies.

Full-length Seg-2 sequences for the 24 BTV reference strains were used to design type-specific oligonucleotide primers targeting Seg-2, which were initially used for further sequencing studies (table 1). The arrival of an unknown strain of BTV in northern Europe in 2006 (subsequently identified as BTV-8) prompted the use of these primer sets for virus ‘type’ identification by RT-PCR) (figure 7).

The availability of sequence data for multiple isolates for individual serotypes, from different geographic locations, facilitated the design and validation of improved typing primers for use in conventional RT-PCR assays. These assays can now be used to identify and distinguish all 24 BTV types, as well as different topotypes and even field and vaccine strains of the European BTV types) (see: http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/BTV-S2-Primers-Eurotypes.htm) (Mertens et al 2008). An important feature of this method is that it can be used to detect and identify BTV RNA in nucleic acid extracted directly from a blood sample, in many cases making it unnecessary to isolate the virus in cell culture prior to typing. This considerably speeds up the diagnostic process. The assays themselves are also much faster than conventional serum neutralisation tests, which can take several weeks, giving results in a matter of hours. In addition RT-PCR based typing assays do not use standardised serotype specific antisera against each of the BTV serotypes, which are in limited supply and are therefore expensive.

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http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/BTV-S2-Primers-Eurotypes.htm

During 2006 these RT-PCR based assays were used at IAH Pirbright for the first identification of BTV-8 in northern Europe (the first BTV strain ever detected or isolated in the region - Figure 7). Sequence analyses and comparisons of the cDNA amplicons generated for Seg-2 of the first Netherlands and UK BTV isolates confirmed that they all belong to type 8 (figure 8-9).

Table 1: The initial set of oligo nucleotide Primers used for identification of BTV serotypes by conventional Seg-2 Rt-PCR

The northern European strain of BTV-8 was shown to belong to a sub-Saharan / African (western) lineage but is distinct from recent South African virus isolates and is clearly distinct from both the South African reference and vaccine strains of type 8 (partial sequences provided by Dr. AC. Potgieter : OVI Onderstepoort) Genome segmentv 2 of the Netherlands isolate of BTV-8 (NET2006/04) was most closely related to that of BTV-8 from Nigeria (isolated in 1982) (figure 9 www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/BTV-8-Seg-2-tree.htm).

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http://www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/BTV-8-Seg-2-tree.htm

Figure 7: The first identification of BTV-8 in northern Europe, using conventional RT-PCR assays, targeting genome segment 2 of the 24 BTV serotypes (Maan et al 2008, Mertens et al 2008). The ‘type’ of each primer sets used is indicated at the bottom of the gel.

Other strains of BTV were also identified using these methods including, BTV-1 in North Africa (2006 – which subsequently spread into Europe); BTV-4, 15 and 24 in Israel (2006); as well as novel incursions (of distinct strains) of BTV-9 in North Africa (2008); and BTV 1 and BTV-8 into Oman (2009).

Further studies were also conducted in collaboration with Dr D. Johnson and Dr E. Ostlund (from USADA Aimes Iowa), using these methods to characterise multiple ‘untyped’ BTV strains that had been isolated from the south–eastern United States since 1999. These studies identified a total of eight serotypes that were previously regarded as exotic to the USA (BTV-1, 3, 5, 6, 14, 19, 22, 24 - see: http://www.reoviridae.org/dsRNA_virus_proteins/btv-serotype-distribution.htm) and are being prepared for publication.

The development of BTV type-specific assays has continued throughout the project and into project SE2617 (2009-2012), with the design of real-time RT-PCR typing assays. Real-time assays are faster; less labour intensive and better suited for high throughput diagnostic systems. Since they use a ‘closed-tube’ format they also pose less of a risk of cross-contamination and false positive results. Real-time RT-PCR assays have been designed for the European serotypes 1, 2, 4, 6, 8, 9, 11 and 16 and have been ‘evaluated’ with very good levels of specificity and sensitivity. These assays will be available as a diagnostic kit on August 1st 2009 (produced by LSI (Laboratoire Service International) – 69380 Lissieu, France).

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http://www.reoviridae.org/dsRNA_virus_proteins/btv-serotype-distribution.htm

Figure 8: Comparison of the Seg-2 nucleotide sequences from NET2006/01 and UKG2007/01 with those from the to the 24 BTV reference strains, confirming their identity as BTV-8

Figure 9: Seg-2 comparison of the Netherlands isolate NET2006/01 to other BTV-8 strains : A neighbor joining tree showing comparisons of nucleotide sequences of Seg-2 from different BTV-8 strains,

The appearance of different BTV types, which had not previously been detected in the Mediterranean region, suggests that they represent further potential threats to European animals.

In late 2008 / early 2009, strains of BTV-6 and BTV-11 were also identified in the Netherlands/Germany and Belgium, respectively (initially by conventional Rt-PCR and then confirmed by sequence analyses of genome segment 2). Sequence comparisons of all ten genome segments of the type 6 strain and eight segments of the type 11 strain (see below) showed that they were very recently derived from the South African live-vaccine strains of these types. The BTV-6 strain has also been isolated from both Netherlands and German samples and is included in the IAH reference collection (isolates NET2008/04 to NET2008/06 and GER2008/01 to GER2008/04). However attempts to isolate the BTV-11 strain were unsuccessful, suggesting that it reflects an earlier infection during 2008.

Objective 3: Generate sequence data from other genome segments (specifically segments 3, 6, 7 and 10) for additional BTV isolates, to provide information on genetic variation and gene reassortment

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http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/BTV-6.htm#GER2008/04

http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/BTV-6.htm#GER2008/01

http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/BTV-6.htm#NET2008/06

http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/BTV-6.htm#NET2008/04

This objective was initially intended to generate sequence data for representatives of genome segments 3, 6, 7 and 10, from multiple BTV isolates, in order to provide further information concerning serotype, topotype and strain variation. It was also anticipated that this would provide evidence concerning the frequency of genome segment reassortment in the field. However, since reassortment can involve any individual, or even multiple segments, the study was extended to include all of the BTV genome segments.

During the project, in addition to the sequence data for 134 examples of genome segment 2 (with earlier data, giving a total of 219 full-length sequences), sequence data were also generated for 27 different examples of genome segment 1; 43 examples of segment 3 (giving a total of 94); 45 examples of segment 4; 117 examples of segment 5; 30 examples of segment 6 (giving a total of 97); 122 examples of segment 7; 67 examples of segment 8; 54 examples of segment 9; & 85 examples of segment 10 (with 20 further examples of segment 10 specifically from strains of serotype 2). These data have been used to create an initial database (which will be expanded) that can be used to analyse / study genome reassortment in specific virus isolates.

The data obtained for this part of the project demonstrated (as expected from earlier work) that genome segments 2 and 6 (encoding BTV outer capsid proteins VP2 and VP5) are the most variable regions / segments of the virus genome (Figure 10). Six of the remaining genome segments (Seg-1, 3, 4, 9, 5 and 8 – encoding virus-core proteins VP1, VP3, VP4 and VP6, or non-structural proteins NS1 and NS2 respectively) are much more highly conserved (see figure 10).

However, like Seg-2 and 5 these conserved segments also showed clear evidence of topotypic separation, primarily into eastern and western groups, with further sub-groups (figure 10). The two remaining genome segments (Seg-7 and Seg-10 – coding for the core surface protein VP7 and the smallest non-structural proteins NS2 and NS2a) showed intermediate levels of variation, with some evidence of separation into a number of different eastern and western groups.

VP7 can mediate cell attachment and entry, particularly in insect vector cells, while NS3 can mediate cell exit by budding, which may be particularly important for virus release in insect vector cell systems that become persistently infected. It is considered possible that the levels of variation detected in Seg-7 and 10 may be linked to variations in the insect vector species / populations that are responsible for transmitting these viruses in different locations. The variations observed in Seg-7 also show a higher relative percentage of conservative base changes (in codon position three) than in the other genome segments, suggesting that this segment, although conserved has been accumulating

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changes over a longer time period. This may reflect the maintenance of different variants of VP7 in the virus population, possibly to make use of different vectors for transmission.

Sequence analyses of multiple genome segments from BTV-25 (TOV) indicated that it represents a separate group, possible indicating another distinct geographic origin / topotype, although the levels of variation detected in these analyses confirmed that TOV should be classified within the Bluetongue virus species (figure 11).

Figure 11: A neighbor joining tree comparing the nucleotide sequence of Seg-5 (the NS1 gene) from Toggenburg orbivirus (BTV-25) with Seg-5 from different eastern and western strains of BTV-8 and EHDV.

The bluetongue viruses that have arrived or have been detected in the field in Europe, or have been released as vaccines since 1998, represent a unique combination of eastern strains (BTV-1, BTV-9 and BTV-16), western strains (BTV-1, BTV-2, BTV-4 and BTV-8), live attenuated vaccine strains (BTV-2, BTV-4, BTV-6 BTV-9, BTV-11 and BTV-16) and even the novel BTV-25 originally detected in Switzerland. This mixture provides many opportunities for genome segment reassortment / exchange between different virus lineages, and the potential for emergence of novel progeny strains that may have distinct biological characteristics. Our initial studies of reassortment based on the sequence analyses of multiple genome segments from individual virus isolates, have shown that a strain BTV-2 from Italy 2002 is a reassortant, containing genome segment 5 derived from the BTV-16 vaccine strain. (Batten et al 2008).

Indeed our studies indicate that all of the BTV-16 strains isolated throughout Europe share a recent common ancestry with the BTV-16 vaccine from South Africa, suggesting that their emergence in the region may be linked to the use of this strain for many years, as part of an annual live-vaccination campaign in Israel.

Our Initial analyses of the genome segments from multiple European BTV isolates, indicate that reassortment is a widespread phenomenon in the field within Europe. Indeed the exchange of individual genome segments and the gradual selection of those variants of each segment that are most suited to the local ecosystem, could play a part in the emergence / evolution of a typically ‘European’

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combination of specific variants of each segment, potentially leading towards a distinct European ‘topotype’.

Objective 4: Design oligonucleotide primers that can be used in RT-PCR assays to detect and identify any strain of BTV and distinguish them from isolates of other Orbivirus species (e.g. AHSV, EHDV, and EEV).

By identifying the six genome segments that show the highest levels of conservation within the BTV species, the sequencing studies described here have also provided an opportunity for the design of novel nucleic acid based ‘serogroup / virus-species-specific’ assays, using either conventional RT-PCR, real-time RT-PCR, or chip based technologies.

VP7 (encoded by genome segment 7) is the immunodominant serogroup specific antigen of BTV. Seg-7 was therefore chosen as an initial target for the development of conventional group-specific primers and assays (Anthony et al 2007), although entirely successful this has subsequently been superseded by a real-time Rt-PCR assay targeting genome segment 1 (which encodes the highly conserved viral polymerase – VP1) (Shaw et al 2007). This second assay has become one of the primary diagnostic assays for BTV, both in the UK and across many other countries in Europe. It is particularly useful during the current period when inactivated virus vaccines have been widely used, as it provides an effective mechanism to detect recently infected animals, distinguishing them from seropositive individuals that have been vaccinated. This assay has allowed the Reference Laboratory at IAH Pirbright and other reference laboratories using such assays, to maintain surveillance for import/export purposes and for outbreak detection.

The BTV group-specific PCR assays were validated against a panel of RNA samples from other orbiviruses and different host species, showing very high levels of sensitivity and specificity, exceeding those of the ELISA assays previously used for diagnosis. The real-time RT-PCR assay targeting Seg-1 will be available as a diagnostic assay kit produced by Qiagen (Germany) in August 2009 .

Objective 5: Generate additional sequence data for the European vaccine strains.Our studies are increasingly moving towards full genome analyses, for at least representative strains from each new virus lineages/outbreak. Vaccine strains of BTV-2, 4, 9 and 16 have previously been used in southern Europe (Papadopoulos et al (2008) Chapter 21 In: “Bluetongue” a Monograph). As part of this project and to help identify vaccine derived outbreaks and reassortment events involving vaccine strains, the entire genomes of these strains has been sequenced. The detection of BTV-6 and BTV-11 sequences in blood samples from cattle in the Netherlands and Belgium respectively during late 2008 / early 2009 was unexpected, as neither of these vaccine types had previously been detected in Europe.

Sequence analyses of the entire genome of BTV-6, both directly from a blood sample and from a subsequent virus isolate, not only confirmed its type, but also showed a very close relationship (99-100% sequence identity) to the South African live-vaccine strain of BTV-6 (from Onderstepoort Biological products - OBP) in eight of the genome segments, demonstrating that it was primarily derived from this source. However, genome segment 10 showed a lower level of similarity to the BTV-6 vaccine and higher similarity (although not identity) to the vaccine strain of BTV-2. This suggested that the Netherlands virus is in fact a reassortant, possibly between BTV-6 and another (as yet unidentified) vaccine strain. This in turn indicates that it may have been derived from the use of a multivalent vaccine preparation (as produced by OBP). The BTV-6 sequences from the blood sample also showed differences in Seg-7, with >99% similarity to the local field strain of BTV-8, indicating that further reassortment events were occurring.

Sequence data were only obtained for eight of the BTV-11 genome segments from the Belgium samples. However this was clearly sufficient to show that the virus was also derived from the BTV-11 OBP vaccine strain. In this case no evidence of reassortment was detected.

These studies, which were greatly facilitated by the availability of sequence data for BTV vaccine strains supplied by Dr. A.C. Potgeiter from the Onderstepoort Veterinary Research Institute. The studies carried out during this project have shown that three distinct BTV strains have arrived in Belgium or the Netherlands since 2006. Although our studies can identify the lineages and origins of individual virus strains, they cannot show exactly how the viruses were transported or gained access to the region. However, the data generated suggest that other BTV strains, and even other orbiviruses, such as African horse sickness virus (AHSV), Equine encephalosis virus (EEV), or Epizootic hemorrhagic disease virus (EHDV), could arrive by the same route into northern Europe, where we clearly have both susceptible host populations and vector-competent insect species.

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Objective 6: Validate primers designed in objectives 2 and 4 and provide these to the BTV reference laboratory at IAH Pirbright

The primers (and probes) that were originally designed for both conventional and real-time RT-PCR assays, to detect members of the BTV serogroup / virus-species (Seg-1 and Seg-7 group specific assays), have been widely evaluated for their specificity and sensitivity, with consistently good results. These assays are more sensitive, faster and more reliable as diagnostic tools than any of the previous BTV specific serological assays. They now form the primary tools used for BTV diagnosis and surveillance in the Bluetongue Reference Laboratory at IAH Pirbright. The real-time assay is also in the process of being commercialised as a diagnostic kit, by Qiagen. Any results obtained can be confirmed by sequencing and further phylogenetic comparisons of Seg-2.

A set of serotype-specific conventional RT-PCR primers has been evaluated and has been supplied to the Bluetongue Reference Laboratory for each of the 24 established BTV serotypes. These primers have been and continue to be used by the staff employed on this project, to type novel virus isolates, not only as part of ongoing sequencing studies but also to provide further technical support for the Bluetongue Reference Laboratory.

Although more rapid and reliable than previous methods for the initial typing of BTV, these conventional Rt-PCR assays are already starting to be replaced by real-time RT-PCR typing assays, which are even faster, more sensitive and less prone to cross-contamination, or therefore false positive results.

Real-time assays for BTV types 1, 2, 4, 6, 8, 9, 11 and 16 have already been evaluated and the designs details for both primers and probes have been provided to the Bluetongue Reference Laboratory. These assays, which are also in the process of being commercialised as a diagnostic kit by LSI (available 1st August 2009), now form the primary diagnostic methods used for identification of the European BTV serotypes at IAH.

The main implications of the findings The studies outlined in this report have clearly been very successful, meeting or exceeding all of

the objectives and milestones in the original project proposal / contract. They have also specifically addressed and met the pressing-need for more rapid and reliable diagnostic and typing assays, in the face of the first recorded outbreaks of BTV in northern Europe and the UK.

Indeed the provision of rapid and reliable epidemiological data by the reference and research groups of the Arthropod Transmitted Pathogens Programme at IAH Pirbright, using the methods and databases developed by this project, has made a significant contribution to information given to Defra and other government agencies across Europe and the Mediterranean region. This advice, concerning the movement of existing viruses in the region and the emergence of novel strains / serotypes, informed Defra’s decision to purchase and deploy the BTV-8 inactivated vaccine at the start of the Culicoides vector season in May 2008. It is likely that the timing and implementation of these vaccines are the primary factors why the UK was alone amongst the European countries affected by BTV-8 in controlling the disease during 2008. Indeed the prevention of a massive ‘second year’ outbreak of BT-8 in the UK can be regarded as one of the major successes of recent veterinary medicine, saving many millions of farm animals, with major economic implications.

The assay systems and molecular epidemiology studies that were implemented as part of this study, continue to be of primary importance in the detection and typing of BTV in any infected animals imported to the UK, helping to keep the country free of further incursions / outbreaks. Possible future work

It is clear that continuing work will be needed to maintain and expand the BTV reference collection and associated sequence databases at IAH Pirbright, to ensure that they remain relevant and as comprehensive as possible for the identification of novel virus isolates. It would be very useful to fill in current gaps in the collection (addressing particularly the lack of viruses from the far-east, and more recent isolates from India and central Africa). By generating additional sequence data, particularly for genome segment 2 of these viruses, it may be possible to identify the origins of outbreak strains in Europe and elsewhere with even greater precision (by phylogenetic and molecular epidemiological studies).

The studies outlined above, and from a previous Defra contact (SE2612), have helped demonstrate BTV entry routes into Europe from the east (via Turkey) and from North Africa into Italy and the Mediterranean Islands, or into Iberia (from Morocco). In particular it would be valuable to have access to additional virus samples from eastern Turkey and neighbouring countries, as well as from

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Egypt and central Africa to help assess the risks of further European incursion involving different BTV strains / types.

Further sequence data needs to be generated for additional genome segments from viruses already in the IAH reference collection, to help explore the frequency and significance of genome segment reassortment in the field within Europe, and its role in virus evolution. The detection of reassortant viruses can also provide some information concerning the history and origins of virus isolates (as seen with the 2002 strain of BTV-2 from Italy (Batten et al 2008) and the Netherlands strain of BTV-6 from 2008 (- see above)).

It would be particular valuable to generate full genome sequence data for an early representative isolate from new outbreaks, to help determine their origins and provide further reference data to determine the origins of subsequent outbreaks. Our initial intention is to generate full genome sequence data for 100 selected virus isolates from around the world, as a reference dataset.

SNPS analyses of additional isolates from the BTV-8 outbreak (particularly genome segment 2), will help to track the movements of the virus during 2006-2009 and beyond. This may give more specific details concerning movement routes and mechanisms within Europe that may help to inform decisions concerning useful preventative measures.

Further work will be needed to complete the design and evaluation of real-time RT-PCR primers and probes to detect the different serotypes of BTV not currently in Europe. There may also be some further design / evaluation work required to maintain the current set of assays already developed for the European types.

The phylogenetic studies that have been completed, or are in progress for BTV, could also be carried out with the other orbiviruses that represent a threat to Europe, leading to developments of new generation diagnostic assays, databases and molecular epidemiology capabilities for these viruses. In particular the development of novel serogroup specific and typing assays for EHDV, will be valuable. Some of the initial sequencing work has been started for EHDV (Anthony et al 2009 a, b, c, d) including a reference collection of different strains (see:http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/EHDV-isolates.htm). Similar studies will also be needed for AHSV and EEV. and a full genome analysis of at least one representative of each of 22 different Orbivirus species, would help in the design of discriminatory and diagnostic assays.

The real-time RT-PCR assays for BTV species and type that have been and are still being developed are very effective. They have been particularly useful at a time when widespread vaccination has invalidated the group-specific, competition ELISA previously used to detect BTV antibodies as a diagnostic tool. However, there are still other technologies, such as chip based systems, that could give a positive identification for any of the 25 BTV types, in a single tube reaction. Development of these systems will give further improvements in speed and throughput of testing.

Actions resulting from the work (e.g. IP, knowledge transfers)An agreement has been reached with Qiagen for commercialisation of an improved BTV group-

specific real-time RT-PCR assay targeting genome segment 1, as originally described by Shaw et al (2007). It is anticipated that this will be available during August 2009.

Agreements have been reached with LSI for commercialisation of BTV type-specific real-time RT-PCR assays targeting genome segment 2. It is anticipated that the first of these, targeting BTV-1, 2, 4, 6, 8, 9, 11, 16, will be generally available on the 1st of August 2009.

An agreement has also been reached with LSI for commercialisation of an EHDV group-specific real-time RT-PCR assay. It is anticipated that this will be generally available on the 1st of August 2009.

The BTV reference collection (see http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/BTV-isolates.htm ) has become an internationally recognised resource, supplying well characterised BTV strains for both vaccine development and challenge studies. Viruses have been supplied to Merial, Intervet, Fort-Dodge, and Pfizer UK for these purposes. A significant number of refereed publications, book chapters and ProMed reports have also been submitted concerning the results and conclusions of these studies.

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http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/BTV-isolates.htm

http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/BTV-isolates.htm

http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/EHDV-isolates.htm

http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/EHDV-isolates.htm

References to published material9. This section should be used to record links (hypertext links where possible) or references to other

published material generated by, or relating to this project.

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32. Anthony S.J; Darpel, K.E; Maan, S; Sutton, G; Attoui, H; Mertens, P.P.C (2009d) The core protein VP6 of epizootic haemorrhagic disease virus (EHDV) exists as two exclusive re: Virus Genes (in press)

31. Anthony, S.J; Darpel, K.E; Maan, S; Sutton, G; Attoui, H; Mertens, P.P.C (2009a) ''The core protein VP6 of epizootic haemorrhagic disease virus (EHDV) exists as two exclusive homologues'' Virus Genes (In Press).

30. Anthony, S.J., Maan, N., Maan, S., Sutton, G., Attoui, H., Mertens, P.P.C., (2009b) Genetic and phylogenetic analysis of the core proteins VP1, VP3, VP4, VP6 and VP7 of epizootic haemorrhagic disease virus (EHDV). (Virus Research in press).

29. Anthony, S.J., Maan, N., Maan, S., Sutton, G., Attoui, H., Mertens, P.P.C., (2009c) Genetic and phylogenetic analysis of the Outer Capsid Proteins VP2 and VP5 of epizootic haemorrhagic disease virus (EHDV). (Virus Research in press).

28. Behzad Hemati, Vanessa Contreras, Céline Urien, Michel Bonneau, Haru-Hisa Takamatsu, Peter Mertens, Emmanuel Bréard, Corinne Sailleau, Stéphan Zientara and Isabelle Schwartz-Cornil (2009) Conventional dendritic cells disseminate the Bluetongue hemorrhagic fever virus via skin lymph. Journal of Virology (in press)

27. Bernd Hoffmann, Martin Beer, Scott M. Reid, Peter Mertens , Chris A. L. Oura Piet A. van Rijn, Marek J. Slomka, Jill Banks, Ian H. Brown, Dennis J. Alexander, Donald P. King (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 (in Press) 2009 May 6. [Epub ahead of print].

26. Kyriaki Nomikou , Chrysostomos Ι. Dovas, Sushila Maan, Simon J. Anthony, Alan R. Samuel, Maria Papanastassopoulou, Narender S. Maan, Olga Mangana, Peter P . C . Mertens (2009) Evolution and phylogenetic analysis of full-length VP3 genes of eastern Mediterranean bluetongue virus isolates. Plos One (in Press).

25. Ozkul A, Erturk A, Caliskan E, Sarac F, Ceylan C, Mertens P , Kabakli O, Dincer E, Cizmeci SG. (2009) Segment 10 based molecular epidemiology of bluetongue virus (BTV) isolates from Turkey: 1999-2001. Virus Res. 142, 134-139. Epub 2009 Feb 21. PMID: 19428746.

24. I. Schwartz-Cornil, N. J. MacLachlan, V. Contreras, Behzad Hemati, F. Pascale, P. P. C. Mertens, P. S. Mellor, E. Bréard and S.Zientara (2008) Bluetongue virus: Virology, pathogenesis and immunity Veterinary Research (in Press).

23. Cottam EM, Wadsworth J, Shaw AE, Rowlands RJ, Goatley L, Maan S, Maan NS, Mertens PP , Ebert K, Li Y, Ryan ED, Juleff N, Ferris NP, Wilesmith JW, Haydon DT, King DP, Paton DJ, Knowles NJ. (2008) Transmission pathways of foot-and-mouth disease virus in the United Kingdom in 2007. PLoS Pathog. 4 :e1000050.

22. Batten, C.A., Shaw, A.E, Maan, S. , Maan, N., and Mertens P. P. C . (2008) Genome segment reassortment in the field between a European field strain of bluetongue virus serotype 16 and a serotype 2 vaccine strain. Virus Research 137, 56-63. Epub 2008 Jul 29.

21. S. Maan, N.S. Maan, N. Ross-Smith, C.A. Batten, A.E. Shaw, S.J. Anthony, A.R. Samuel, K.E. Darpel, E.Veronesi, C.A.L. Oura, K.P. Singh, K. Nomikou, A.C. Potgieter, H. Attoui, E.van Rooij, P. van Rijn, K. De Clercq, F.Vandenbussche, S. Zientara, E.Bréard, C. Sailleau, M.Beer, B. Hoffman, P.S. Mellor & P.P.C. Merten s (2008) Sequence analysis of bluetongue virus serotype 8 from the Netherlands 2006 and comparison to other European strains. Virology 377, 308-318.

20. Purse BV, Brown HE, Harrup L, Mertens PP, Rogers DJ. 2008 Invasion of bluetongue and other orbivirus infections into Europe: the role of biological and climatic processes. Rev Sci Tech.427-442.

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http://www.ncbi.nlm.nih.gov/pubmed/18819670?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum











19.Jelle Matthijnssens, Max Ciarlet, Mustafizur Rahman, Houssam Attoui, Krisztián Bányai, Mary K. Estes, Jon R. Gentsch, Miren Iturriza-Gómara, Carl Kirkwood, Vito Martella, Peter P.C. Mertens , Osamu Nakagomi, John T. Patton, Franco M. Ruggeri, Linda J. Saif, Norma Santos, Andrej Steyer, Koki Taniguchi, Ulrich Desselberger, and Marc Van Ranst (2008). Recommendations for the classification of group A rotaviruses using all 11 genomic RNA segments. Virus Research; 153, 1621-9.

18. Mellor PS, Carpenter S, Harrup L, Baylis M, Mertens PP C (2008) Bluetongue in Europe and the Mediterranean Basin before 2006. Special issue of Preventive Veterinary Medicine 87, 4–20.

17. Y adin, H., Brenner, J., Gelman, B., Bumbrov, V., Oved, Z., Stram, Y., Galon, N., Klement, E., Perl, S., Batten, C., Anthony, S. & Mertens, P.P.C. (2008) Epizootic hemorrhagic disease virus type 7 infection in cattle in Israel, August- October 2006 Vet Record 162, 53-56.

16. Graham RI, Rao S, Sait SM, Attoui H, Mertens PP, Hails RS, Possee RD. (2008) Sequence analysis of a reovirus isolated from the winter moth Operophtera brumata (Lepidoptera: Geometridae) and its parasitoid wasp Phobocampe tempestiva (Hymenoptera: Ichneumonidae). Virus Res. Jul;135(1):42-7. Epub 2008 Apr 10.

15. . Graham RI, Rao S, Sait SM, Mertens PP, Hails RS, Possee RD. (2007) Characterisation and partial

sequence analysis of two novel cypoviruses isolated from the winter moth Operophtera brumata (Lepidoptera: Geometridae).Virus Genes. Oct;35(2):463-71. Epub 2007 Jun 2.

14. Veronesi E, Mertens PPC, Shaw AE, Brownlie J, Mellor PS, Carpenter S. (2007). Quantifying bluetongue virus in adult Culicoides biting midges (Diptera: Ceratopogonidae). Journal of Medical Entomology 45, 129-132..

13. Szmaragd C, Wilson A, Carpenter S, Mertens PPC, Mellor PS & Gubbins S (2007). Mortality and case fatality during the recurrence of BTV-8 in northern Europe in 2007. Veterinary Record, 161,

571-572. 12. K.E Darpel, C.A Batten, E. Veronesi, A.E Shaw, S. Anthony, K. Bachanek-Bankowska, L.Kgosana, A.

bin-Tarif, S. Carpenter, U.U. Müller-Doblies, H.-H. Takamatsu, P.S. Mellor, P.P.C. Mertens, C.A.L. Oura (2007) Clinical signs and pathology shown by British sheep and cattle infected with bluetongue virus serotype 8 derived from the 2006 outbreak in northern Europe.. Veterinary Record 161, 253-261.

11. Mertens, P.P.C., Maan N. S., Prasad, G., Samuel A.R., Shaw A., Potgieter, A.C., Anthony, S. J., and Maan S . (2007). The design of primers and use of RT-PCR assays for typing European BTV isolates: Differentiation of field and vaccine strains Journal of general Virology 88, 2811–2823.

10. Shaw A.E. , Monaghan, P. , Alpar, H.O. , Anthony, S. , Darpel, K.E. a, Batten, C.A. , Carpenter, S., Jones, H. , Oura, C.A.L. , King, D.P. , Elliot, H., Mellor P.S. Mertens, P.P.C. (2007) Development and validation of a real-time RT-PCR assay to detect genome bluetongue virus segment 1 Journal of Virological Methods. 145, 115-26.

9. Maan S, Rao S, Maan NS, Anthony SJ, Attoui H, Samuel AR, Mertens PPC. (2007a), Rapid cDNA synthesis and sequencing techniques for the genetic study of bluetongue and other dsRNA viruses. Journal of Virological Methods 143:132-139.

8. Breard, E., Sailleau, C., Nomikou, K., Hamblin, C., Mertens, P.P.C, Mellor, P.S., El Harrak, M., Zientara, S. (2007) Molecular epidemiology of bluetongue virus serotype 4 isolated in the Mediterranean Basin between 1979 and 2004. Virus Research 125:191-197.

7. Terry B. Green, Susan White, Shujing Rao, Peter P. C. Mertens , Peter H. Adler, James J. Becnel, Jimmy Becnel (2007) Biological and Molecular Studies of a Cypovirus from the Blackfly Simulium ubiquitum (Diptera: Simuliidae) J Invertebr Pathol. May;95(1):26-32. Epub 2007 Jan 16)

6. Maan S., Maan N.S, Samuel A.R., Rao S, Attoui, H., & Mertens P.P.C (2007b) Analysis and Phylogenetic Comparisons of Full-Length VP2 Genes of the Twenty-Four Bluetongue Virus Serotypes. Journal of General Virology 88:621-630.

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http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17251581&query_hl=1&itool=pubmed_docsum



http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17280733&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum





http://www.ncbi.nlm.nih.gov/pubmed/17872535?ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum









http://veterinaryrecord.bvapublications.com/cgi/content/full/162/2/53?view=long&pmid=18192658

http://veterinaryrecord.bvapublications.com/cgi/content/full/162/2/53?view=long&pmid=18192658





5. Houssam Attoui, Corinne Sailleau, Fauziah Mohd Jaafar, Mourad Belhouchet, Philippe Biagini, Jean François Cantaloube, Philippe de Micco, Peter Mertens and Stephan Zientara (2007) The complete nucleotide sequence of Middelburg virus isolated from the spleen of a horse with severe clinical disease in Zimbabwe. Journal of General Virology 88, 3078-3088

4. Mohd Jaafar, F., Goodwin 2 A. E., Belhouchet, M., Merry, G. Fang, Q. Cantaloube, J.-F. Biagini, P. de Micco, P., Mertens, P. P.C. and Attoui, H., (2007) Complete sequence analysis of the American grass carp reovirus genome, a new species of Aquareovirus (family Reoviridae): evidence for an evolutionary link between the aquareoviruses and coltiviruses Submitted to the Journal of General Virology

3. S. Anthony, H. Jones, K.E Darpel, H. Elliott, S. Maan, A. Samuel, P. S. Mellor, P. P. C. Mertens. S. Anthony, H. Jones, K.E Darpel, H. Elliott, S. Maan, A. Samuel, P. S. Mellor, P. P. C. Mertens (2007). A duplex RT-PCR assay for detection of genome segment 7 (VP7 gene) from 24 BTV serotypes. Journal of Virological Methods 141: 188-197.

2. Green TB, Shapiro A, White S, Rao S, Mertens PP, Carner G, Becnel JJ. (2006) Molecular and biological characterization of a Cypovirus from the mosquito Culex restuans. J. Invertebr. Pathol. 2006 91 :27-34.

1. Graham RI, Rao S, Possee RD, Sait SM, Mertens PP, Hails RS. (2006) Detection and characterisation of three novel species of reovirus (Reoviridae), isolated from geographically separate populations of the winter moth Operophtera brumata (Lepidoptera: Geometridae) on Orkney. J Invertebr Pathol. 91:79-87.

Chapters in books

Philip S Mellor, Matthew Baylis and Peter P C Mertens “Introduction” Chapter 1. In: “Bluetongue”, (eds. Mellor PS, Baylis M & Mertens PPC), Elsevier, London (pp1-6).

Attoui, H., Maan, S., Anthony, S. and Mertens, P.P.C. (2008). The relationships and taxonomy of bluetongue and related orbviviruses. Chapter 5. In: “Bluetongue”, (eds. Mellor PS, Baylis M & Mertens PPC), Elsevier, London (pp23-52).

Natalie Ross-Smith, Karin E. Darpel, Paul Monaghan and Peter P.C. Mertens “Bluetongue virus: Cell biology” Chapter 7. In: “Bluetongue”, (eds. Mellor PS, Baylis M & Mertens PPC), Elsevier, London (pp77-100).

Peter P.C. Mertens, Natalie Ross-Smith, Jon Diprose and Houssam Attoui “The structure of bluetongue virus core and proteins” Chapter 8 In: “Bluetongue”, (eds. Mellor PS, Baylis M & Mertens PPC), Elsevier, London (pp101-134).

Maan, S. Maan N. S., Nomikou, K., Anthony, S., Ross-Smith, N., Singh, K.P., Samuel, A.R., Shaw, E.A. and Mertens, P.P.C., (2008). Molecular epidemiology of Bluetongue virus in Europe. Chapter 9 In: “Bluetongue”, (eds. Mellor PS, Baylis M & Mertens PPC), Elsevier, London (pp135-166).

Prasad G., Sreenivasulu, D. Singh, K.P. Mertens, P.P.C., Maan, S., (2008). “Bluetongue on the Indian subcontinent.” Chapter 10 In: “Bluetongue”, (eds. Mellor PS, Baylis M & Mertens PPC), Elsevier, London (pp167-189).

Philip S Mellor, Simon Carpenter, Lara Harrup, Matthew Baylis Anthony Wilson and Mertens, P.P.C., “Bluetongue in Europe and the Mediterranean Basin” Chapter 13 In: “Bluetongue”, (eds. Mellor PS, Baylis M & Mertens PPC), Elsevier, London (pp235-264).

Karin E. Darpel, Paul Monaghan, Simon Anthony, Haru-Hisa Takamatsu, Peter P.C. Mertens (2008) “BTV in the mammalian host & the induced immune response” Chapter 14 In: “Bluetongue”, (eds. Mellor PS, Baylis M & Mertens PPC), Elsevier, London (pp265-284).

Mertens, P.P.C., Maan, S. and other authors (2008). Diagnosis of Bluetongue and related orbiviruses. Chapterr 19 In: “Bluetongue”, (eds. Mellor PS, Baylis M & Mertens PPC), Elsevier, London (pp343-364).

H. Oya Alpar, Vincent W. Bramwell, Eva Veronesi, Karin E. Darpel, Paul-Pierre Pastoret, and Peter P. C. Mertens. (2008) Bluetongue virus vaccines past and present” Chapter 20 In: “Bluetongue”, (eds. Mellor PS, Baylis M & Mertens PPC), Elsevier, London (pp397-428).

Orestis Papadopoulos, Philip S. Mellor and Peter P.C. Mertens, (2008) “Bluetongue control strategies”

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Chapter 21 In: “Bluetongue”, (eds. Mellor PS, Baylis M & Mertens PPC), Elsevier, London (pp429-452).

S Mellor, Peter P C Mertens and Matthew Baylis (2008) “Conclusions” Chapter 22 In: “Bluetongue”, (eds. Mellor PS, Baylis M & Mertens PPC), Elsevier, London (pp453-458).

Mellor PS and Mertens PPC (2008). African horse sickness viruses. Encyclopaedia of Virology, (eds Mahy BWJ & Van Regenmortel MHV), Elsevier, Oxford, pp. 37-43.

Mertens PPC, Attoui H, Mellor PS, Orbiviruses. Encyclopedia of Virology (eds Mahy BWJ & Van Regenmortel MHV), Elsevier, Oxford, pp. 454-465.

Peter P. C. Mertens and Houssam Attoui (In Press) “Orbiviruses” In the Encyclopedia of Life Sciences

Talks, Posters and Abstracts

1. Peter Mertens: “Bluetongue – Vaccines past present and future” Invited Plenary Lecture 26th November 2008, Prague.

2. Peter Mertens “The European Outbreak of BTV-8” Invited Plenary Lecture, BTV-8 Symposium Denver USA July 10th 2008

3. Peter Mertens “Bluetongue” Invited Plenary Lecture Veterinary Products Committee Horizon Scanning Meeting on Blue Tongue. 23th July 2008 Guilford, Surrey.

4. Peter Mertens “ Bluetongue” Invited Plenary Lecture 28th August 2008, Institute of Virology andImmuno-prophylaxis (IVI) Switzerland

5. Peter Mertens “Bluetongue: The virus and its emergence in Europe 1998 – 2008” Invited Seminar to Sardinian veterinarians, in Sardinia organised by Emilio Gil of Esteve. June 2008-06-26.

6. S. Maan, N. S. Maan, C. A. Batten, A. E. Shaw, K. E. Darpel, K. Nomikou, H. Attoui, N. Ross-smith, Eugene van Rooij, Piet van Rijn, A.C Potgieter, S. J. Anthony and P. P.C. Mertens. (2008). Sequence analysis of bluetongue virus serotype 8 from the Netherlands 2006 and comparison to other European strains. Poster and Abstract: presented at the 2nd annual meeting of EPIZONE “The need for speed” June 4th-6th 2008 Brescia, Italy and Bluetongue Satellite Symposium "Bluetongue in Europe, back to the future !!” June 7th 2008, Brescia, Italy.

7. Peter Mertens Summary and closing remarks (discussion) Bluetongue Satellite Symposium "Bluetongue in Europe, back to the future !!” June 7th 2008, Brescia, Italy.

8. Mertens, P.P.C. N. S. Maan, K. Nomikou, A. Shaw, C. A. Batten, E. Veronesi, K. Darpel, S. Anthony, N. Ross-Smith, H. Attoui, and S. Maan. (2008). Movement of bluetongue virus strains during the disease outbreak caused by BTV-8 in northern Europe during 2006-2008; as revealed by sequence analysis of genome segment 2. Poster and Abstract: presented at the 2nd annual meeting of EPIZONE “The need for speed” June 4th-6th 2008 Brescia, Italy and Bluetongue Satellite Symposium "Bluetongue in Europe, back to the future !!” June 7th 2008, Brescia, Italy.

9. Narender S. Maan, S. Maan, D.J. Johnson, E.N. Ostlund, K. Nomikou, and P. P.C. Mertens (2008). Sequence analysis of exotic bluetongue virus serotype 5 isolated from ruminants in the Southeastern US in 2003. Poster and Abstract: presented at the 2nd annual meeting of EPIZONE “The need for speed” June 4th-6th 2008 Brescia, Italy and Bluetongue Satellite

10. Symposium "Bluetongue in Europe, back to the future !!” June 7th 2008, Brescia, Italy.

11. Nomikou, K., S. Maan, N. S. Maan, C.A. Batten, H. Attoui, N. Ross-smith and P.P.C. Mertens. (2008). Molecular epidemiology of BTV strains in Europe since 1998. Talk and Abstract: presented at the 2nd annual meeting of EPIZONE “The need for speed” June 4th-6th 2008 Brescia, Italy and Bluetongue Satellite Symposium "Bluetongue in Europe, back to the future !!” June 7th 2008, Brescia, Italy.

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12. Batten, C.A., Kasia Bankowska, Anna Swain, Abid Bin-Tarif, Sushila Maan, Narender Maan, Peter P. C. Mertens and Chris Oura (2008). An update on bluetongue virus in the United Kingdom. Talk and Abstract: presented at the 2nd annual meeting of EPIZONE “The need for speed” June 4th-6th 2008 Brescia, Italy and Bluetongue Satellite Symposium "Bluetongue in Europe, back to the future !!” June 7th 2008, Brescia, Italy.

13. Peter Mertens, Narender Maan, Kiki Nomikou and Sushila Maan, “The Molecular Epidemiology of BTV” Training course held at Pirbright May 2008.

14. Peter Mertens “Bluetongue virus: Structure, replication and molecular epidemiology” Invited Seminar Glasgow University Veterinary School 29th April 2008.

15. Peter Mertens, Karin Darpel, Eva Veronesi: “Bluetongue virus: Structure, replication and molecular epidemiology“ Invited Seminar At Intervet Boxmeer. 29th April 2008.

16. Chiam, R., Sharp, E., Maan, S. Mertens, P., Paillot, R., Elton, D., Wood, J., Blacklaws, B., and Castillo-Olivares, J. (2008). Use of a prime/ boost vaccination strategy to evaluate the immunogenicity of African horse sickness virus (AHSV) non-structural proteins in the natural host. In 162nd SGM Meeting, Edinburgh, 31 March - 3 April 2008.

17. Peter Mertens, Narender Maan, Kiki Nomikou Dr. Sushila Maan, “Bluetongue molecular epidemiology & development of molecular diagnostic assays Work package 6: Sequencing and molecular epidemiology of BT virus isolates from Europe”. CVDL grant review by Defra 16th

March 2008.

18. Peter Mertens “Bluetongue virus type 8 vaccines” Invited Seminar at Veterinary Medicines Directorate (Surrey) 4th March 2008

19. Peter Mertens “Real time RT-PCR for detection of BTV RNA” Talk presented to a review of vaccines and diagnosis at the EU Commission in Brussels 2nd March 2008-06-26.

20. Peter Mertens “Bluetongue virus type 8 vaccines (our last hope!!)” Invited Lecture Vet Research Club February 2008.

21. Peter Mertens and Chris Oura. Bluetongue vaccines Presentation to NFU meeting at IAH Compton. 17.01.2008

22. Peter Mertens and Chris Oura “ Evidence Vertical transmission of Bluetongue virus type 8” Talk presented to a review of vaccines and diagnosis at the EU Commission in Brussels, February 2008-06-26.

23. Peter Mertens "Identification of Individual BTV-8 strains in Northern Europe 2006-2007" Talk at MedReoNet December 2007.

24. Peter Mertens, Simon Anthony, Sushila Maan, Narender Maan "Molecular Epidemiology studies of EHDV and AHSV" Talk at MedReoNet December 2007.

25. Sushila Maan, N.S. Maan, S.J. Anthony, K.E. Darpel, A. E. Shaw, C. A. Batten, H. Attoui and Peter P.C. Mertens* "MOLECULAR EPIDEMIOLOGY OF BLUETONGUE VIRUS TYPE 8 FROM THE NETHERLANDS 2006" Talk at MedReoNet December 2007.

26. Peter Mertens "Sequencing and RT-PCR assays for genome segment 2 of the bluetongue virus" Talk at MedReoNet December 2007

27. Peter Mertens "MedReoNet Work Package 3: Molecular Epidemiology Recommendations / opportunities" Talk at MedReoNet December 2007.

28. Peter Mertens: "Sequencing and RT-PCR assays for genome segment 2 of the bluetongue virus": Defra Review 4th December 2007.

29. Maan, S., Maan, N.S. Anthony, S.J. Darpel, K.E. Shaw, A.E. Batten, C.A. Attoui, H., &

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Mertens, P.P.C. (2007). Molecular epidemiology of bluetongue virus type 8 from the Netherlands 2006. Talk and Abstract: presented at World Association of Veterinary Laboratory Diagnosticians (WAVLD) – 13th International Symposium Proceeding. Melbourne, Australia, 12-14 November 2007, p 54.

30. Mertens, P.P.C. Maan, N.S. Johnson, D.J. Ostlund, E.N. and Maan, S. (2007). Sequencing and RT-PCR assays for genome segment 2 of the 24 bluetongue virus serotypes: identification of exotic serotypes in the southeastern USA (1999-2006). Talk and Abstract: presented at World Association of Veterinary Laboratory Diagnosticians (WAVLD) – 13th International Symposium Proceeding. Melbourne, Australia, 12-14 November 2007, p 55.

31. Anthony, S.J., Maan, N., Maan, S., Attoui, H., Darpel, K., and Mertens, P.P.C. (2007). Complete sequence analyses of the genome of Epizootic Haemorrhagic Disease Virus (EHDV): the design of diagnostic assays. Poster and Abstract: presented at World Association of Veterinary Laboratory Diagnosticians (WAVLD) – 13th International Symposium Proceeding. Melbourne, Australia, 12-14 November 2007, p 103.

32. Anthony, S.J., Maan, N., Batten, C., Maan, S., Kgosana, L., Bachanek-Bankowska, K., Darpel, K., and Mertens, P.P.C. (2007). Nucleotide sequence analyses of Epizootic Haemorrhagic Disease Virus (EHDV) genome segments encoding the outer capsid proteins: a serological and genetic re-evaluation of serotypes. Talk and Abstract: presented at World Association of Veterinary Laboratory Diagnosticians (WAVLD) – 13th International Symposium Proceeding. Melbourne, Australia, 12-14 November 2007, p 149.

33. E. Veronesi , P. P.C. Mertens, P. S. Mellor, S. Carpenter "Validation of a diagnostic technique for the detection and quantification of bluetongue virus in adult biting midges (Culicoides Spp: Diptera: Ceratopogonidae)." WAVLD meeting Australia November 2007.

34. Karin E. Darpel, Paul Monaghan, Jennifer Simpson, Harriet W. Brooks, Simon J. Anthony, Eva Veronesi, Joe Brownlie, Haru H. Takamatsu, Philip Mellor and Peter P.C. Mertens "Replication of bluetongue virus in the skin of infected sheep" WAVLD meeting Australia November 2007.

35. Batten, C.A. Sushila Maan, Kasia Bankowska, Andrew Shaw, Abid Bin-Tarif, Simon Anthony, Chris Oura and Peter P. C. Mertens (2007). An update on bluetongue virus in Europe. Talk and Abstract: presented at World Association of Veterinary Laboratory Diagnosticians (WAVLD) – 13th International Symposium Proceeding. Melbourne, Australia, 12-14 November 2007.

36. Donna J. Johnson, Peter P. C. Mertens, Sushila Maan, Eileen N. Ostlund "Exotic Bluetongue Viruses Identified from Ruminants in the South-eastern U.S. from 1999-2006" AAVLD meeting USA September 2007 .

37. Peter Mertens, Javier Castillo Olivares Sushila Maan "The Molecular Epidemiology of BTV & AHSV vaccine studies" Defra review July 2007

38. Peter Mertens "Bluetongue in Europe (1998- 06) (an update)" Invited lecture the Moredun June 2007

39. Peter Mertens (2008), Bluetongue in Europe 1998 – 2008: ‘Ten Years’ : An update at The University of Florida Department of Veterinary Medicine, Gainsville 14th November 2008.

40. Peter Mertens (2008) Bluetongue vaccines, Past, Present and Future: Informa Life Sciences, Vaccines meeting: Prague, Czech Republic 25th November 2008.

41. Peter Mertens (2008) Bluetongue in Europe 1998 – 2008: ‘Ten Years’ : An update and Vaccines. Epizone meeting, El Escorial, Madrid 23rd October 2008.

42. Peter Mertens (2008) “Bluetongue: The virus and its emergence in Europe 1998 – 2008” Invited Seminar to Sardinian veterinarians, in Sardinia organised by Emilio Gil of Esteve. June 26th June 2008.

43. Peter Mertens (2008) Meeting summary and closing remarks an discussion: Bluetongue Satellite Symposium "Bluetongue in Europe, back to the future !!” June 5th - 7th 2008, Brescia, Italy.

44. Peter Mertens , Karin Darpel, Eva Veronesi, Camille Szmaragd (2008) BTV vaccines Invited Seminar At Intervet Boxmeer. 23rd April 2008.

45. Peter Mertens , Karin Darpel, Eva Veronesi (2008) “Bluetongue virus: Structure, replication and molecular epidemiology“ Invited Seminar At Intervet Boxmeer. 24th April 2008.

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46. Peter Mertens (2008) “Real time RT-PCR for detection of BTV RNA” Talk presented to a review of vaccines and diagnosis at the EU Commission in Brussels 2nd March 2008.

47. Mertens PPC (2008) Bluetongue vaccination (our last hope). Invited talk The Veterinary Research Club London 15th February 2008.

ProMed reports11-FEB-09 PRO/AH> Bluetongue - Europe (04): Belgium, BTV-11, lab report 20090211.0622

31-JAN-09 PRO/AH/EDR> Epizootic hemorrhagic disease, bovine - Turkey 2007 20090131.0434

30-JAN-09 PRO/AH/EDR> Bluetongue, ovine, bovine, caprine - Israel (02): typed 20090130.0412

31-OCT-08 PRO/AH/EDR> Bluetongue - Europe (69): Netherlands, BTV-6 vaccine strain update 20081031.3431

25-OCT-08 PRO/AH/EDR> Bluetongue - Europe (65): Netherlands, BTV-6, diagnostic update 20081025.3371

24-JUN-08 PRO/AH/EDR> Bluetongue - Europe (31): BTV-8, transmission routes 20080624.1949

26-SEP-07 PRO/AH/EDR> Bluetongue, bovine - UK (England) (XX): BTV-8 20070926.3196

27-JUN-07 PRO/AH> African horse sickness - Senegal: serotype 2, sequence (2) 20070627.2074

14-DEC-06 PRO/AH> Epizootic hemorrhagic disease, cattle - Morocco, Algeria, Israel: serotyped 20061214.3513

25-NOV-06 PRO/AH> Bluetongue, ovine - Morocco (03): BTV-1 20061125.3348

24-NOV-06 PRO/AH> Bluetongue - Europe (21): Bulgaria, BTV-8 susp 20061124.3347

Consultancies Advice provided to Veterinary Medicines Directorate during 2007 and 2008 concerning dossiers for BTV inactivated vaccines.

Advice has also been provided to Defra on a regular basis via the ‘Bluetongue expert group’ meetings over

Popular Press

During 2007 and 2008 there were a large number of interviews with journalists from the national press, with Sky News, the BBC, ITN and Australian television. These are not recoded individually

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