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Venue Menorca 21st-22nd May 2002.

Dr Peter Mertens. Welcomed the participants to the meeting and told them that the 1^st annual report had been accepted by the EU in Brussels. He said that the financial report is outstanding and is being prepared by IAH=s financial department. The next sum of money for the project would not be forthcoming until this report was submitted and Dr Mertens told the project partners that if this was going to cause them financial problems then they should contactthe IAH financial department.

Dr Mertens said that a mid-grant meeting will need to be held in six months time and it is likely that the EU will send one or two representatives to this meeting and so the presentations will need to be more formal. This meeting could be held in conjunction with Dr Mellor who is project holder for a sister grant. The commision may also be approached for support for an international meeting at the end of the project.

Dr Mellor introduced the sister project to the REO-ID project.

1. Risk analysis of culicoides borne diseases to Europe. Eleven partners and is EU funded.
2. Development of a safe and efficacious BTV vaccination strategy for Europe.EU funded.
3. Effect of temperature and region of origin on the vector capacity of British and European culicoides species for BTV. DEFRA funded.

Dr Mellors talk gave an overview of the current situation of BTV in Europe, South America and Japan. He described how it was possible that a vector species Ajump@ had occurred. He also described research on a novel overwintering mechanism involving γΔT-cells which would have facilitated the survival of BTV at temperatures too low to have allowed this to previously happen.

Methods for sequence analysis of BTV isolates and molecular epidemiology.

Nick gave a talk on new sequences for members of the Reoviridae and an update on what was available. Nick discussed ways that information on Reoviruses can be accessed from a database so that phylogenetic analysis can be made easier. This could be tied in to better information on the isolate whether it is partial or full.

Peter Mertens then spoke about the REO-ID website as a long term resource. Peter then went on to discuss what is currently available and asked the partners for input on what they would like to be available.

Paco suggested that perhaps as the website is updated that interested partners should be automatically e-mailed.

Evolution, taxonomy and protein expression of selected members of the family Reoviridae.

Less than 20% differentiates polymerase genes of the various Reovirus genera.

5^th Speaker

A discussion was held on the availability of segment 2 sequences and a table on the dsRNA website chronicling this was discussed.

Dr Zientara stated that within the last three weeks that vaccine virus had been sequenced in segment 2 in field viruses that had been received in France.

Sequencing of Aquareovirus genomes.

Expression of VP6 in HS cells and production of Mabs and polyclonal antibodies for diagnostic suitability were discussed. Paco said that obtaining samples had been difficult.

Paco and Houssam agreed to trade their contacts who have virus isolates. Paco stated that requests to researchers in America was not usually fruitful. Professor Mertens said that he knew Jim Winton and could contact him and that it might help.

Kiki gave a talk on the validation of an Rt-PCR for the serotype determination of Greek isolates of BTV. Kiki also had said that BTV-1 was present in the north west of Greece. Dr Samuel asked if it was possible to obtain further samples.

Prof.. Mertens said that in work package one that D19 was probaly the most diffiult objective ie obtaining viruses for the collections. He stressed that having good information was paramount. It was also agreed that the coltivirus and aquareovirus collections could also be put on the website. Prof.. Mertens also said that negotiations were underway with the Australians with regard to obtaining some viruses. Prof.. Mertens also said that Onderstepoort had also agreed that we could have the whole of their collections which comprised of many hundreds of isolates. Mr Knowles said that perhaps initially specific isolates could be requested.

There was a discussion on the use of software for tracking samples within laboratories and for managing stocks of viruses and sera in freezers. Dr Samuel was looking at some commercial software and will report on this for the next meeting. Action: Alan Samuel

Paco Rodriguez said that he will hold a meeting with Xavier and Houssam on obtaining Coltivirus and Aquareovirus samples. It was discussed who could be contacted for the acquisition of different samples. Prof. Mertens said that links with Peter Kirkland in Australia might help to obtain Chinese isolates.

Nick Knowles suggested that making a brochure outlining the reference laboratories aims and offering a limited molecular typing/epidemiology service for selected samples to be sent out to the veterinary authorities in various countries might be helpful.

Paco Rodriguez suggested that the web pages should be linked to the main virology web servers and Office International Epizooties. It was agreed that this would be a good idea.and should be investigated. Action: Peter Mertens.

Nick Knowles also suggested that perhaps simpler names might be beneficial ie www.reovirus or www.Bluetongue

Dr Attoui said that nearly all the objectives had been met. He said that getting further isolates from the USA had been particularly difficult. Dr Attoui stated that as regards seadornaviruses that they have good and active collaboration with the Chinese.

D16 has been achieved. The epidemiology in D18 would need further isolates. Paco Rodriguez said that developing PCR technology would be the focus for the next six months to facilitate the isolation of new viruses. This would also help the epidemiological studies. Paco said that obtaining samples from other sources is a problem and Prof. Mertens suggested that he could contact Jeff |Cowley in Australia on his behalf.

Professor Mertens said that this is an Orbivirus species for which there is no full sequence data available. He says that there are full lengh EEV clones which will be sequenced in the near future. It was suggested that partners 1 & 2 should hold talks so that a co-ordinated approach could be made to this work package. The information gaineed would be put onto the website. Which genes to be tackled first was discussed ie a group specific gene to facilitate the development of diagnostic PCR was considered important. It was said that looking at the polymerase gene was a good way of looking at the genera. Work already underway on BTV VP7 group specific PCR was discussed. By Using the groups collective expertise (ie Dr Zientara=s on AHS) could help develop these type of diagnostics for other Orbiviruses.

The difficulty in obtaining isolates was discussed again, particularly samples of Great Island Virus. Dr Attoui thought that he would be able to obtain some samples of GIV though.. It was thought that these would be particularly interesting since they were Tick-borne and were able to infect humans.

Prof. Mertens made the point that the methodology being developed (particularly at Pirbright) meant that clones would not be generated. The Greek partners said that they had been concentrating their efforts on segment 20 but over the coming year were going to focus more on segment 3. Dr Zientara suggested that partners should share sequence data on the website to i) co-ordinate efforts & ii) to facilitate sequence alignments etc. It was suggested that intentions to sequence a particular gene would help to avoid duplication of effort. Dr Zientara also suggested that if the information on the isolates was put on the web then partners could check against this information.

Prof. Mertens re-iterated the importance of the segment 10 studies particularly in the light of the the suspicion that a jump in vectors had occurred. It was also mentioned that it was extremely important to obtain the Turkish vaccine strain for comparative purposes. It was agreed between the partners that the main genes of interest were 2, 5, pol, VP3, VP7 and segment 10 and efforts should focus on these.

Work Package 5. Structural studies, identifying proteins, electron microscopy and crystallography.

Dr Mellor said that the community reference laboratory needed to acquire specific antibodies to BTV for diagnostic tests. To this end, Mr Nick Burroughs has been employed on a DEFRA grant to use his expertise for one year to grow and purify BTV. He has already prepared types 1, 4, 5 & 10 and these were awaiting inoculation into rabbits. Dr Mellor said that these are extremely pure preparations and are to be used in eight sequential injections every two weeks and that this meant that there was no anti cell response and resulted in very high titred antibody

EEV and Pallyam antibodies have already been raised and the diagnostic tests are undergoing validation. It is hoped that in the future his expertise could be used to produce antigens for EHD etc. subject to funding from DEFRA. Nick Knowles asked whether having sera to other viruses would be useful for differential diagnosis. Dr Mellor said that that as they were not list A diseases then interest would be limited.

Prof. Mertens stated that sequencing representatives of the genera was more the remit of the project. Dr Rodriguez said that there were monoclonal antibodies to seadornaviruses and aquareoviruses for diagnostic purposes. Dr Rodriguez thought that the work they were doing with antibodies should be finished by the end of the year. Prof. Mertens commented that the production of monoclonal antibodies was a very useful approach and could be used for future studies.

Dr Attoui said that antibodies to coltivirus proteins and seadornaviruses had been made using baculovirus expressed proteins. These have already been used in Blots and ELISA=s and these techniques were currently undergoing validation. Blood samples from Greece and the Czech Republic had been tested for Eyach virus. He also said that collaboration with Thailand, China and other Asian countries had been done to check for antibodies to seadornaviruses.

Some preliminary structural studies have been started. A discussion was held concerning partners viewpoints on which systems were best for viral protein expression. Dr Attoui suggested Grass Carp Virus was a good candidate to start with because it grows to very high titre.

PCR=s for coltiviruses and seadornaviruses have already been validated and papers published. An AHS paper is published. Dr Rodriguez said that a paper on aquareoviruses is Acoming@. The orbiviruses need sequence data but this will be the focus ovedr the next 12 months. Sequence Data for EHDV will be obtained. Generally it was agreed that swapping and sharing data between partners has been good.

Dr Zientara described the problems of PCR and the possibility for contamination. He suggested that perhaps a common positive control could be used. Dr Attoui suggested Uracil Glycosidase as a treatment for primers, DNTP=s etc. However it was agreed that this doesnt help with the problem of laboratory mistakes. The OIE would recommend a gold standard test (ie VNT). It was agreed that we should work towards research, validation and acceptance of PCR as an acceptable test. A discussion ensued on a suitable positive control for PCR diagnostic assays. Dr Mertens asked Dr Zientara to prepare a brief document describing what was required as a positive control and this issue should be thought about and discussed at the next meeting. Abrief description of Dr Billinis use of β-Actin as a positive control should also be drawn up for discussion. Prof. Mertens then went on to describe RNA profiling and how this could be of great value for virus identification.

The Greek partners have already been using an ELISA for testing for AHS and EHD.

Work is ongoing on an EHD, Pallyam and EEV assay.

Seadornaviruses and Eyacu have been discussed earlier.

CTMV have been difficult to eastablish collaboration with.the Americans They generally wish to have reagents sent to them and then they do the work rather than actively collborate.

There has been a very low percentage of antibodies detected so far in animal bloods to Eyach Virus. As mentioned previously collaboration with the Czech Republic for samples from Eastern Europe where it was thought that conditions may be more favourable to virus spread. Ie there is greater proximity between animals and and people which may therefore mean Tick borne viruses.

Comments on workpackage 9 were dealt with earlier in the session and were not re-iterated at this time. Dr Rodriguez made the comment that it is simply a matter of gaining more isolates. The PCR needs to be up and running but the samples will be needed to validate the techniques properly. He stated that there is a clear way forward for this workpackage.

Work Package10. The website.

The other viruses (aquareoviruses etc.) Should now go up on the website. The site should also have more link as discussed previously and additionally have links to the partners websites. The dsRNA and ReoID (private) website will remain the same for the time being.

It was stated that a review which is being submitted by Dennis Bamford and others will reference the sites and so they should be kept up to date. Prof. Mertens also asked that if anyone should notice any errors on the sites then they should inform him. The EU is keen that the website should exist.Dr Zientara also made the comment that there was a European BTV website that there should be a link to.

The next meeting will be in six months time and it is proposed to hold a joint meeting with the epidemiology grant to which the commission will be invited. It is proposed that possibly it could be in Madeira but was probably too big for there. It was decided that possibly a meeting held in Greece with the vaccine project might be better. Prof. Mertens and Dr Mellor will discuss this.

Dr Zientara asked that the request to attend the meeting be made in June to help with the administration. Prof mertens agreed to this.

The annual meeting in 1 years time will probably be held in Corsica. Prof Mertens also said that it was intended that at the end of the grant there would be an international meeting and that a book would be published. Support from the commission and OIE will be sought to fund this. The likely time for such an event would be Spring 2005.