ANNEX III
COST Technical Committee “Agriculture and Biotechnology”
COST Action 857
Apicomplexan Biology in the Post-Genomic Era
PROGRESS REPORT Period: from November 2003 to December 2004 12.11.03 - 31.12.04
This Report is prepared by the Management Committee of the Action and presented to the relevant Technical Committee. The report is a "cumulative" report, i.e. it is updated annually and covers the period beginning from the start date of the Action.
1. Action Identification Data COST Action 857
Apicomplexan Biology in the Post-Genomic Era
TC Recommendation:13/06/2002 CSO Approval: 18/12/2002 Start date(1): 12/11/2003 Duration: 60 months Extension: End date: 11/11/2008
First MC meeting: 12/11/2003 Last MC meeting: Final Report: Evaluation Report(2): TC Evaluation:
Number of signatories: 13 Signatories and date of signature: Austria 930/04/2004 Belgium Bulgaria Croatia Cyprus
Czech Rep.9 14/04/2004 Denmark 9Not shown on web site Estonia Finland France 905/05/2003 Germany 917/12/2002
Greece Hungary Iceland Ireland Israel Italy 906/08/2003 Yugoslavia Former Yugoslav Rep.of Macedonia Latvia Lithuania Luxembourg Malta Netherlands 907/05/2004 Norway 908/10/2004
Poland Portugal 918/12/2002 Romania Slovakia Slovenia
Spain 917/12/2002 Sweden 927/05/2004 Switzerland 918/12/2002 Turkey United Kingdom 917/12/2002
Institutes of non-COST countries: Area: Action Web site: http://www.iah.bbsrc.ac.uk/Eimeria/COST%20857.htm Chairperson: Dr Fiona Tomley Tel.:+44 1635 577276 Institute for Animal Health Fax:+44 1635 577263 Compton, Newbury, Berkshire E-Mail:
[email protected] RG20 7NN UK TC Rapporteur: Henrik ANDERSEN,
[email protected] External Evaluator: Title, name, affiliation, country External Evaluator: Title, name, affiliation, country (1) Date of the first MC meeting. (2) When the report is received by TC Secretariat
2. OBJECTIVES The main objective is to understand the fundamental biology of apicomplexan parasites and the way they interact with the infected hosts, through the exploitation of genome sequence data and the application of post-genomics. The major areas are • Comparative genomics and bioinformatics • Gene expression • Host-Parasite interactions such as invasion, formation of a replication-competent vacuole, parasite modulation of host cell function and survival, regulation of stage-differentiation • Parasite biodiversity and population genetics The driving force is the urgent need to identify new drug targets and vaccine candidates to allow the development of novel, sustainable control strategies against diseases caused by apicomplexan parasites. The benefits will include:• Comparative analyses of apicomplexan genomes to give detailed knowledge of the classes of genes that are conserved across the phylum or restricted to certain genera. • Analyses of apicomplexan gene expression, leading to the definition of genes that are differentially regulated during parasite development or upon interaction with the host, or differentially expressed between defined parasite populations. • The application of proteomics to apicomplexan parasites and comparison of proteomes to define proteins that are conserved across the phylum, restricted to specific parasites or localised to specialised sub-cellular organelles and compartments. • The development of common approaches for processing and displaying post-genomic data to ensure compatibility between different research groups and thus facilitate rapid data exchange. • The development of common approaches towards understanding apicomplexan gene function, utilising the most amenable and appropriate experimental systems. • The identification of apicomplexan-derived proteins capable of interacting/interfering with normal host cell function (for example, signal-transduction pathways) • The elucidation of key parasite processes and metabolic pathways. • The identification of potential drug targets and vaccine candidates. 3. TECHNICAL DESCRIPTION AND IMPLEMENTATION To meet the scientific objectives and accomplish the research tasks of this Action it is clear that close collaboration and interaction between participants will be essential. Thus it is agreed that co-ordination of research tasks will be channelled primarily through Working Groups (WGs), which will hold specialised meetings for participants to discuss their latest data, formulate research plans and establish close collaborations. Short-term scientific missions (STSMs) are also seen as a very important part of the Action, together with the organisation of training workshops and networking opportunities for young scientists working on a variety of different apicomplexan parasites. The Action is organised according to COST400/94 ‘Rules and procedures for implementing COST actions’. It is managed by a Management Committee (MC), which convened for the first time in November 2003 and elected a chairperson and vice-chairperson. At this meeting, the MC also identified 2 co-chairpersons for each of 6 WGs: • WG1: Functional genomics and bioinformatics • WG2: Gene expression
• WG3: Motility, cytoskeleton and invasion biology • WG4: Intracellular survival and host cell modulation • WG5: Biodiversity and population genetics • WG6: Development of novel targets for disease control The WG chairpersons are responsible for ensuring that the goals of each WG are achieved and that there is appropriate interaction between the Groups. WG chairpersons have been asked to report back formally to the MC via the Action Chairperson once each year and to channel requests for holding WG meetings through the Action Chairperson. All WG meetings are open to all members of the Action and it was agreed by the MC that all working documents (abstracts of papers presented, minutes, conclusions, recommendations etc) should be made available to all members of the Action through a web site and by e-mail. A temporary web page was immediately set up for the Action, with the expectation that this would be improved and expanded during 2005. At the 2nd meeting of the MC in May 2004, a committee was elected to deal specifically with the processing of STSM applications. The chair of this committee deals directly with the COST office on all matters associated with STSMs. All other paperwork and communication with the COST office is through the Action Chairperson. 4. PARTICIPATION AND COORDINATION 4.1 Management Committee Chairperson: Dr Fiona TOMLEY , Institute for Animal Health, Compton, Newbury, Berkshire RG20 7NN, UK. Tl: +44 1635 577276 Fax: +44 1635 577263
[email protected] Vice Chairperson: Dr Catherine BRAUN-BRETON, UMR CNRS 5539, Bâtiment 24, CC 107, Place Eugène Bataillon, 34095 Montpellier Cedex 05, FRANCE, Tl:+33 4 67 14 34 55 Fax:+33 4 67 14 42 86
[email protected] Members AUSTRIA CZECH REPUBLIC DENMARK
Renate EDELHOFER Universität Wien Tomas MIKUS Biopharm, Jilove U Prahy
Heidi ENEMARK Danish Inst. for Food and Veterinary Copenhagen V FRANCE Pierre PERY Centre INRA, Jouy-en-Josas GERMANY Uwe GROSS Universität Göttingen ITALY Furio SPANO Istituto Superiore di Sanità, Roma NETHERLANDS Andrew WATERS Leiden University Medical Center NORWAY Lucy Robertson The Norwegian School of Veterinary Science, Oslo PORTUGAL Henrique CONDINHO DA SILVEIRA Instituto de Higiene e Medicina Tropical, Lisbon SPAIN Luis Miguel ORTEGA MORA
Henrik Vedel NIELSEN Statens Serum Institut, Copenhagen
Klaus LINGELBACH Universität Marburg Bruno ARCA Universitya di Napoli ‘Federico II’ Dick SCHAAP Intervet International bv, Boxmeer
Helder ESPINGUINHA CORTES Universidad de Evora
Emilio DEL CACHO
SWEDEN
SWITZERLAND UK
Universidad Complutense de Madrid Antonio BARRAGAN Karolinska Institutet, Stockholm Andrew HEMPHILL Institute of Parasitology, Bern Tony HOLDER National Institute for Medical Research,London
University of Zaragoza, Jens MATTSSON SVA (National Veterinary Institute of Sweden), Uppsala, Adrian HEHL Institut für Parasitologie, Zurich
4.2 Participating Institutions AUSTRIA CZECH REPUBLIC DENMARK FRANCE
GERMANY
ITALY
NETHERLANDS
NORWAY PORTUGAL
SPAIN
SWEDEN
Universitat Wien. Biopharm Research Insitute of Biopharmacy and Vet. Drugs, Prague. Institute of Parasitology, Ceske Budejovice. Danish Institute for Food and Veterinary Research, Copenhagen. Statens Serum Institut, Copenhagen. CNRS-UJF, Grenoble. Inserm U511, University Paris 6. Institut Cochin, Paris. Institut National de la Recherche Agronomique, Jouy-en-Josas. Institut National de la Recherche Agronomique, Nouzilly. Institut National de la Recherche Agronomique, Nantes. Institut Pasteur, Paris. Laboratoire Parasitologie-Mycologie, UFR Medecine, Reims. University Limoges. University Montpellier 2. Bernard Nocht Institute for Tropical Medicine, Hamburg. Justus Liebig University Giessen. Philipps’ University Marburg. Technische Unversitat Dresden. University of Heidelberg. University of Gottingen. University of Hannover. Istituto Superiore di Sanita, Roma. Kenton srl, Pomenzia. National Institute for Infectious Diseases, Roma. Universitya di Napoli. Intervet International, Boxmeer. Leiden University Medical Center. Utrecht University Norwegian School of Veterinary Science, Oslo. Faculdade de Medicina Veterinaria, Lisboa. Instituto Gulbenkian de Ciencia, Oeiras. Instituto de Higiene e medicina Tropical, Lisboa. Instituto de Investigacao Cientifica Tropical, Lisboa. Instituto Nacional de Engenharia e Tecnologia Industrial, Lisboa. Instituto Nacional de Saude DrRicardo Jorge, Porto. Unive Evora. Labratorios Hipra SA, Girona. Universidad Complutense de Madrid. Zaragoza University. Karolinska Institutet, Stockholm. National Veterinary Institute of Sweden, Uppsala. Swedish University of Agricultural Sciences, Uppsala.
SWITZERLAND
UK
Swiss Tropical Institute, Basel. Universitaet Basel. University of Bern. University of Geneva. University of Zurich. Cryptosporidium Reference Unit, Singleton Hospital, Swansea GKT Guy’s Hospital, London Imperial College, London Institute for Animal Health, Newbury London School of Hygiene and Tropical Medicine National Institute of Medical Research, London St George’s Hospital Medical School, London University of Dundee University of Edinburgh University of Glasgow University of Liverpool University of Leeds University of Strathclyde Wellcome Centre for Molecular Parasitology, Glasgow Wellcome Trust Sanger Institute, Hinxton.
4.3 Meetings of the Management Committee 12th November, 2003 3rd May 2004
Brussels Lisboa
Minutes of these meetings are included in Annex I 4.4 Meetings of the Working Groups 3-6th May, 2004 8-9th September, 2004 12-15th September, 2994 29th September, 2004 8th-9th November, 2004
All WGs (Action annual workshop) Lisbon, Portugal WG2, Liverpool, UK All WGs (PhD workshop) Brussels, Belgium WG1/3, London, UK WG5, Rome, Italy
Reports of these meetings are included in Annex II 4.5 Short-term scientific missions Five were approved during 2004 Bruno Douradinha (Portugese) dates: 25 September 2004 to 09 October 2004 (final report accepted) host: Joanne Thompson, Ashworth Laboratories, School of Biology, University of Edinburgh, The King's Buildings, West Mains Road, Edinburgh EH9 3JT, United Kingdom topics : role of the Plasmodium berghei cysteine-rich modular (CRM) protein family, during the hepatic phase of Malaria Charlotte Egan (English) dates: 6th to 26th September 2004 host: Dr. Antonio Barragan at the Karolinska Institute, Stockholm, Sweden topics: To determine the outcome of the absence of TcRγδ+ IEL on the invasion and
dissemination of Toxoplasma gondii in vivo Regina Lizundia (Spanish) dates: November 2004 to February 2005 host: Dr Dirk Werling, Royal Veterinary College, Hawkshead Lane. Hatfield, AL9 7TA. UK topics: Role of TGF in transformation of bovine B cells by Theileria Marco Lalle (Italian) dates: January 9th to February 9th 2005 host: Dr Adrian Hehl University of Zürich, Institute of Parasitology, Winterthurerstrasse 266a, CH 8057 Zürich, Switzerland topics: Heterologous expression of the Cryptosporidium parvum Cpa135 protein in the protozoan Giardia duodenalis Lorenza Putignani (Italian) dates: 17 January to 13 February 2005 host: Jonathan Wastling, Department of Pre-Clinical Veterinary Science, Faculty of Veterinary Science, University of Liverpool, PO Box 147, Liverpool, L69 3BX UK topics: Proteomic analysis of the mitochondrion-like organelle of Cryptosporidium parvum: an approach to highlight central metabolic pathways of the parasite
5. RESULTS In this first year of the Action, the main activities have been in bringing together research groups who specialise in work on the biology of a wide range of apicomplexan parasites and in developing the overall structure and focus of the Action. At the first workshop in Lisbon in May 2004, over 130 scientists attended and the work of nearly all of these was represented in the ~90 oral and poster presentations. At smaller working group meetings held during this first get together, it was possible to have in-depth discussions on all the major topics of the Action, to evaluate our relative strengths and weaknesses in each area and to draw together plans for how to tackle the main research objectives and to prioritise areas that required the most input. Notes of these working group meetings are included in Annex II. The major outcomes were: • A clear need to address the issue that a very large number of apicomplexan genes remain totally uncharacterised. Establishment of cross-genus bioinformatics approaches to allow scientists working on different parasites to easily cross-check/shortlist genes that are of interest for functional studies would be enormously beneficial • A need to prioritise lists of candidate genes (for functional studies) to manageable numbers. This should be directly related to advances in relevant functional genomics technologies. Thus for instance, advances in protocols for conditional mutagenesis would allow larger numbers of genes to be systematically analysed • Training in bioinformatics is required to get more value from genome(s) • There is a clear need to establish common networks for exchanging and comparing data on gene expression between different laboratories. Cross-genus bioinformatics is essential, as are cross- genus wet lab collaborations. • Theme-based meetings were popular amongst several working groups and will help to focus attention on key topics throughout the next year. • There is a need to establish a catalogue of parasites held, research topics, special methods etc. Uwe Gross (MC member and co-chair WG4) offered to co-ordinate the organisation of this.
• •
• •
It is important to ensure that evolutionary studies are well covered. There is an urgent need to develop good diagnostic tools for various apicomplexan genera and in particular to ensure that the COST Action platform be used to develop tools, standardise techniques and consult with experts in other fields and countries about the best ways to move forward. Selection strategies used to identify and validate new targets must be decided Differences between children and adults in their responses to drugs and vaccines and to efficacy (disease profiles, immunological status, toxicological risk…) should be prioritised.
We were very pleased with the number of younger scientists (PhD students and post-docs) who attended the 1st annual workshop in Lisboa and who are involved in working groups and STSMs. We also organised a specialised PhD training workshop in September in Brussels that included 16 students (14 COST funded and 2 HHMI funded). This was a great success (report is included in Annex II) and will be repeated in 2005. 6. DISSEMINATION OF RESULTS 6.1 Publications and Reports Abstract books were published for the 1st Annual Workshop in Lisbon and for the WG2 meeting in September that was held jointly with COST 854. For all other meetings, programmes and abstracts were circulated electronically to the whole COST 857 membership and lodged on the Action website. All programmes and collections of abstracts are included in Annexes III-V. 6.2 Conferences and Workshops 3-6th May, 2004 12-15th September, 2004
1st Action Annual Workshop, Lisboa, Portugal 1st Action PhD student Workshop, Brussels, Belgium
Reports from these meetings are included in Annex II 6.3 Web site A web site for the action was established at the beginning of 2004 for the purposes of (1) advertising WG and whole Action workshops,(2) publicising STSMs (3) publishing abstracts/meetings programmes and (4) providing links to COST documents. This web is house temporarily at http://www.iah.bbsrc.ac.uk/Eimeria/COST%20857.htm and is currently of very crude design. During 2005 a more sophisticated and informative site will be designed and developed. 6.4 Scientific and Technical Co-operation The WG2 workshop held in Liverpool in September 2004 was organised jointly with COST Action 854 as there are common interests between these two groups on gene expression and proteomics. The WG1/3 workshop held in London in September 2004 formed part of a local Apicomplexan meeting in London and included the participation of individuals with broader interests than the COST Action. The PhD training workshop, held in Brussels in September 2004 included the participation of two Mexican students funded through the Howard Hughes Medical Institute studentship programme. WG6 has established a link with COST Action B22 on anti-parasitic drugs and will form some joint activities during 2005. At the level of individual co-operation, a number of new collaborations are being started and will be built upon during 2005.
7.
ECONOMIC DIMENSION In 2004, ~250 person years were dedicated to the activities of the Action in its broadest sense (ie ~250 persons were employed on projects pertaining to the biology of apicomplexan parasites within the countries that are signed up to COST 857).
Funds received from the COST budget MC meeting, Brussels
Nov 12th 2003
Reimbursements
7098.3€
MC and Annual Workshop, Lisboa
May 3-6th 2004
Reimbursments
40883.84€
Grant to organiser
6363.05€
Abstract books
840.00€
WG2 meeting, Liverpool
Sept 8-9th 2004
Reimbursements
6077.35€
PhD Training workshop, Bruxelles
Sept12-15th 2004
Reimbursements
10096.26€
WG1/3 meeting, London
Sept 29th 2004
Reimbursements
6189.56€
WG5 meeting, Roma
Nov 10-12th 2004
Reimbursements
9332€
STSM (Dourahdinha)
1368€
Total
88248.36€
Apicomplexan Biology in the post-genomic era
COST- European Cooperation in the field of Scientific and Technical Research
COST 857 Annual Report 2004 ANNEX I MINUTES OF MC MEETINGS Meeting
Page
Minutes of the 1st MC meeting, Brussels, November 2003
2-5
Draft minutes of the 2nd MC meeting, Lisbon, May 2004.
6-7
1
Approved minutes of the first meeting of the Management Committee of COST Action 857 “Apicomplexan Biology in the post-genomic era” held on 12th November, 2003 at 10:30, Brussels. Present: John Williams (JW), Gema Alvarez (GA), Henrique Silveira (HS), Helder Cortes (HC), Pierre Pery (PP), Catherine Braun-Breton (CBB), Uwe Gross (UG), Furio Spano (FS), Tomas Mikus (TM), Heidi Enemark (HE), Fiona Tomley (FT). Emilio Del Cacho (EDC), Andy Waters (AW), Andrew Hemphill (AH) and Adrian Hehl (AH) arrived later. I.
Part I – Adminstrative matters
1. On behalf of COST, JW welcomed all participants to this Management Committee (MC) meeting and explained the format of the agenda. FT volunteered to take the minutes for today. 2. The agenda was formally adopted 3. Delegates introduced themselves and gave a brief summary of their scientific interests and their reasons for participating in COST 857. 4. Status of the Action. Currently there are 7 countries fully signed up to COST 857 (Spain, Portugal, UK, France, Germany, Switzerland, Italy) and 4 more are in the process of signing up (The Netherlands, Denmark, Czech Republic and Sweden). JW emphasised the importance of maximising the number of countries participating and stated that Actions under the Agricultural Technical Committee were averaging around 20 participants. Because of the inclusion of malarial parasites within this Action, it might be of interest to ask the Medical Technical Committee to nominate two delegates to our Management Committee. 5. General Information. JW went through this in some detail, particularly because some rules for administration of COST Actions have recently changed as a result of changes in COST itself. In particular, the Commission will no longer provide COST with secretarial support and from the 1st Jan 2004, COST (20-25 personnel) will occupy new offices at 19, Avenue Louise under the umbrella of the European Science Foundation (ESF). It was anticipated that between 70 and 90 million Euro will be made available by contract to ESF for financing COST Action activities during 2004. Information is available on the COST web site (http://cost.cordis.lu/src/documents.cfm) and delegates should look to this for details, as some documents are still being updated due to the recent administrative changes. However, the main points covered were: (i)
Re-imbursement. A new form is now in use and claims can be paid either to individuals or to their Institutes. The process of reimbursement will be much more rapid than before, as the COST office will process claims direct. Rules are laid down in the COST-office document “Rules for reimbursement of expenses for experts eligible for reimbursement”
(ii)
MC meetings are obligatory and must be held at least once per year, though this can be alongside a workshop/conference (see below). COST will reimburse a maximum of 2 delegates for up to 3 MC meetings per year of the 35 COST member and cooperating countries having signed up for the Action. Occasionally an outside expert may be invited and reimbursed.
(iii)
Non-COST countries or organisations can join the Action, either temporarily or permanently, though this must be validated by COST
(iv)
Working group meetings are typically small and focused, and meet independently of the MC usually in a lab – they are usually low cost and participants are reimbursed according the same rules as for the MC meetings.
2
(v)
Workshops/conferences. These are larger gatherings, typically once a year and may involve some or all of the working groups. Members of the Technical Committee should be invited to these, especially the Chair of the Evaluation Panel. COST does not pay for the whole of these meetings, but makes a contribution up to 50% of the total cost. The remainder is made up by contributions from the Host Institute (this includes the cost of secretarial, and professional time, overheads etc.) and from sponsorship, registration fees etc.
(vi)
Annual Grant. A specific budget has not yet been allocated and will be based on the number of countries signed up. It is likely to be between 60,000 and 80,000 Euro for 2004. Once the Action gets going there will be a weighting factor based on past activity. Each year, the allocation will be reviewed at the 6-8 month stage and funds may be re-distributed between Actions according to demand. It is likely that the COST office, which does not charge any administration costs, will hold funds on behalf of the Action. However, it is possible that COST will move to a TIST system, where annual money will be delivered at the beginning of each year and held by an Institute participating in the Action.
(vii)
Short term scientific missions (STSM). There has been a change to the way these will be administered and the COST office will now deal directly with individuals following approval of their applicaton by an MC evaluation committee. Individuals will be reimbursed up to 2000 Euro for a stay of one month and pro-rata for shorter times. The MC will need to set up an evaluation committee and decide what proportion of the budget should be allocated to STSM.
6. Internal rules of procedure for the Management Committee of COST 857. The following amendments to Annex II of the document were unanimously agreed:Article 7 : ‘tacitly’ was added to the end of the final sentence Article 8 : ‘two-thirds’ was changed to ‘one-half’ Article 9 : ‘twice’ was changed to ‘once’ Artile 13 : ‘EN’ was inserted as the main working language of the MC 7. Election of chair and vice-chair. Fiona Tomley (Chair, nominated NL, seconded FR) and Catherine Braun-Breton (Vice-Chair, nominated DE, seconded PT) were elected unanimously Break for Lunch. II.
Part II Organisation of COST 857
1. Expansion of the COST 857 mailing list. It was agreed that all members of the MC would attempt to increase the list of contacts, both within their own countries and, where possible, in countries not yet signed up for the Action. FT provided hard copies of current e-mail list at the meeting and will e-mail electronic copies to all MC members next week. AW will draw up a letter explaining the purpose and potential benefits of this Action and e-mail this to MC members next week. Please send names and e-mail addresses of potential new contacts to FT by 20th Nov so they can be incorporated into central list. 2. Working Group Chairpeople. It was agreed that each Working Group should have two co-chairs to guide and co-ordinate its activities and that these would be appointed for 1 year in the first instance, to allow for the possibility of rotating responsibilities to other participants throughout the Action. The following co-chairs were suggested:
3
WG1 Functional Genomics and Bioinformatics
Matt Berriman (UK); Robert Menard (FR)
WG2 Gene Expression
Andrew Hemphill (CH); Adrian Hehl (CH)
WG3 Motility, Cytoskeleton and Invasion Biology
Dominique Soldati (CH); Jean-Francois Dubremetz (FR)
WG4 Intracellular Survival and Host Cell Modification
Uwe Gross (DE); Klaus Lingelbach (DE)
WG5 Biodiversity and Population Genetics
Jonathan Wastling (UK); Susan Pedraza (ES)
WG6 Development of Novel Targets for Disease Control
Martin Shirley (UK); Jean-Christophe Barale (FR)
FT and CBB will contact each of these and confirm if they are willing 3. First Conference/Workshop of COST 857. After discussion, it was agreed that the Action should begin with a relatively large meeting at which the scientific sessions will be organised around the six working groups. It is hoped that the meeting would attract 70-80 scientists, and the MC agreed in principle to use funds up to ~ 35,000 Euro. It was agreed that for this first meeting, attendance would be limited to European laboratories in order to provide a good review of the level of activity within Europe on the topics covered by the Action. FT will ask WG chairs to suggest 2 or 3 names of potential speakers for their sessions (NB, some of these should be MC members or WG chairs as we will be able to offer reimbursement of funding to only ~35-40 people in total). The remainder of presentations will be selected from submitted abstracts. All partipants will be asked to bring a poster. Given that there are potentially 22 MC members plus 7 non-MC WG chairs that may wish to attend the meeting, we may be able to fund only 11 additional speakers. It was agreed that the meeting should be of 2.5-days/3 nights duration and should be held, if possible, during the second week of May. FT will approach Matt Berriman at the Sanger Institute UK, to see if the meeting could be held there and HS will investigate the possibility of hosting the meeting in Portugal. A decision on the venue should be made within the next month. The following preliminary timetable was agreed: 30th November 2003
1st call
6th January 2004
2nd call (preliminary programme/ keynote speakers)
9th February 2004
Final deadline for receipt of abstracts
st
1 March 2004
Names of participants to COST office
4. COST 857 Web Site This will be important for communicating information from the working groups to the membership of COST 857 and would also be extremely useful for housing mailing lists, dealing with registration for meetings and submitting abstracts. FT will investigate setting up and designing a site and will contact Sanger if necessary for help. AW will investigate whether the software used for registrations at the Woods Hole Molecular Parasitology meeting could be made available to us. 5. Short term scientific missions. It was agreed that these will most likely be in demand once the first meeting of COST 857 has been organised and people realise that these are a possibility. Election of an evaluation committee should wait until it is clear that the working group chairs have accepted their roles, and should consist of 3-5 people, drawn from MC and WGs. It was decided to advertise deadlines for applications for STSMs, rather than to have an open submission policy, with the first deadlines being June and October 2004. The amount to be spent on STSMs was not decided but will be considered at the next MC meeting and will depend on the number and quality of the applications.
4
There being no more business, the meeting closed at ~16:00. The MC will next meet in May, during the proposed workshop/conference
5
DRAFT Minutes of the 2nd MC meeting of COST 857 held on Monday May 3rd at 15:30 at the Hotel Real Palacio, Lisbon Present: Henrique Silveira (HS), Helder Cortes (HC), Heide Enemark (HE), Catherine Braun-Breton (CBB), Uwe Gross (UG), Jean-François Dubremetz (JFD) Furio Spano (FS), Tomas Mikus (TM), Heidi Enemark (HE), Fiona Tomley (FT), Andrew Hemphill (AH), Adrian Hehl (AH), Jens Mattsson (JM), Bouktje Stol (BS) Klaus Lingelbach (KL) arrived later. 1. FT welcomed all participants to this Management Committee (MC) meeting and explained the format of the agenda. 2. apologies had been received from Emilio del Cacho, Pierre Pery, Bruno Arca, Petr Bedrnik, Renate Edelhofer, Henrik Neilsen and Andy Waters. Delegates present at the meeting introduced themselves after which JM and CBB volunteered to take the minutes for today. 3. The minutes from the 1st MC meeting were accepted 4. The meeting formally accepted that those countries still in the process of signing up fro COST 857 (The Netherlands, Austria, Denmark, Norway and Sweden) can actively take part in the action as well as be part of the MC. FT pointed out that we would all keep on adding names to the email in order to enlarge our action. 5. FT has been trying to contact scientists in Ireland, Poland and Greece. FT emphasised the importance of maximising the number of countries participating and asked members of the MC to try to suggest or approach other countries as well. It is also important to try to integrate more countries from the Eastern Europe. 6. The first annual workshop and our expectations were discussed. The organising committee had been overwhelmed by abstract and in the end a total of 87 abstracts will be presented. 8 parasite genera are represented with a strong emphasis on Plasmodium. The number of persons attending the meeting will be 135 with a total of 28 from Portugal. Once again the MC expressed its thanks to the local organisers. 7. Discussions on the format on the workshop were also held and it was agreed that we should have an open mind and evaluate this years meeting in order to do necessary adjustments for next year. 8. The following arrangements will be organised through COST 857 during the rest of the year: A retreat for PhD students, in Brussels, September 13-15th incorporating members of all WGs. MC has agreed to allow a total of 20 reimbursements (16 PhD students/4 PIs). It was emphasized that information about the retreat must be distributed among PhD students within the COST Action network. A flyer has been circulated to everyone on the mailing list and FT will highlight the advertisement during her introduction this evening. A specialised proteomics workshop organised by Working Group 2 will be held in conjunction with annual COST 854 workshop in Liverpool, September 8-9th. The workshop will be focused on analysing subcellular compartments and will only allow a small number of people to participate (16 max). One full day seems necessary to make the best of it. Data sharing is going to be of high concern: people attending this workshop should think about how best to establish a COST 857 data base. A specialised transfection meeting will be held at the end of September in London together with the Happycomplexan group, organised by Working Groups 1 and 3. The aim will be to have about 10-12 labs represented at the meeting. A list has been drawn up but the organisers should also contact Franz Petry (Mainz) about transfections in Cryptosporidium. 9. A panel for the evaluation of short-term scientific missions (STSM) was appointed: Henrique Silveira, Jean-François Dubremetz, Furio Spano, and Catherine BraunBreton. CBB will be coordinating the panel. The deadlines for applications will formally be 1/1,1/4, 1/7, and 1/10. Depending on the amount of work involved, a slightly more flexible schedule might be adapted in the future. The STSM budget is
6
centralised and therefore most of the administration is taken care of by the COST office. The total budget for all COST actions are 2 000 000 Euro for 2004. Following application, it is expected that it will take two months before getting the grant (1 month for the evaluation procedure and 1 month requested for the COST office administration). A flyer should be prepared and circulated to the membership of COST. 10. The action has an unofficial web-site at http://www.iah.bbsrc.ac.uk/Eimeria/COST%20857.htm. The Institute of Animal Health (IAH), Compton, UK, currently hosts the homepage and it has a "functional but crude" design. No money is available through the COST office for web-page construction/design but BS said this will be discussed soon and the situation may change in the future. It was decided to leave the site at IAH for the moment. 11. At the end of the 1st year of the Action (November 2004) we will need to produce a written annual report. In part it will be the duty of representatives from each country to collect information about activities related to COST 857. The format of the report can vary and a definitive decision will be put on hold. The up coming year will certainly provide us with a clearer profile for forthcoming reports. FT will send a reminder note to MC and Chairs of working groups in September. 12. Meetings organised by the WGs will be vital to the health and success of the COST action and time is allocated during this meeting for all WGs to meet. Already, WG4 are planning to have have a meeting in Spring 2005 in Marburg, Germany in connection with the DFG workshop "Life in a cell". KL will be the organiser. Other WGs will hopefully be able to present their plans for 2005 on Thursday at the debriefing together with KL, UG, HS and FT. Discussions on next years annual meeting resulted in a plea for volunteers to step forward. The meeting will probably be held in May as this is a relatively ‘quiet’ time of year and will likely be organised again around the 6 working group themes. In 2006 the annual workshop may be held in conjunction with ICOPA in Glasgow. (FT) will be the main organiser. In 2007 the annual workshop might be held in Montpellier (JFD, CBB). 13. Nick Smith at UTS in Australia is currently setting up research network down under and has approached COST 857 for possible collaboration. The MC approved and decided to evaluate these contacts as the Australian Network develops. 14. MC meetings will be held once a year. The next MC meeting will be held next year at the annual Workshop. (NOTE added later – next year’s annual workshop will be organised by our Swiss colleagues – date and venue to be decided) There being no other business, the meeting closed at 16:45
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Apicomplexan Biology in the post-genomic era
COST- European Cooperation in the field of Scientific and Technical Research
COST 857 Annual Report 2004 ANNEX II REPORTS Report:
Page
Reporter
Report on the 1st Annual Workshop of COST Action 857 and WG meetings, May 2004
2-5
Fiona Tomley, Chair COST 857
Financial Report for 1st Annual Workshop
6
Henrique Silveira, MC National member and local organiser of 1st Annual Workshop
Report on WG2 (Gene Expression) meeting, September 2004
7-8
Andrew Hemphill, MC National member and co-chair WG2
Report on WG5 (BioDiversity and Population Genetics) meeting, November 2004.
9-12
Jonathan Wastling and Susan Pedraza, co-chairs WG5
Report on 1st PhD Students’ Workshop September, 2004.
13-14
Report on Activities of COST 857 in the Czech Republic
15
Dominique Soldati, co-chair WG3 and organiser of PhD Students’ workshop. Tomas Mikus, MC National member
1
Report on the 1st Annual Workshop of COST Action 857, Hotel Real Palacio, Lisbon, Portugal, May 3-5th, 2004 PARTICIPATION The workshop was attended by 135 scientists from 11 countries. There were a large number of participants from UK and France, which reflects the strength of these two nations in 35 research into Apicomplexan parasites. There 30 was also a good local participation, including 25 many PhD students and younger scientists. 20 We are very happy with the level of interest 15 in the Action and in the future we expect to 10 attract participants from more nations – as well as the 11 listed in Fig1, Austria and 5 Norway are in the process of signing the MOI 0 and researchers in other countries have been CH CZ DE DK ES FR IT NL PT SW UK approached. Fig 1 – National Attendance at Workshop MAIN SCIENTIFIC SESSIONS 25 20 15 10 5 0 WG1
WG2
WG3
WG4
WG5
WG6
87 scientific papers were delivered either orally or as posters. The main part of the meeting was organised around the themes of our 6 working groups and provided an excellent opportunity for us to determine the relative strengths of European research within each of these priority areas. There was a good balance of presentations across the 6 areas (Fig 2)
Fig 2 – Number of presentations per working group The quality of science was impressive and the breadth of work was spread across 8 different parasitic genera covering human, zoonotic and veterinary pathogens. There was a bias towards Plasmodium, partly because of the local research strength in this parasite (Fig 3).
40 35 30 25 20 15 10 5
N eo sp or a Pl as m od iu m Th ei le ria To xo pl as m a
M ix tu re
Ei m er ia
Ba be si a Be sn C oi ry tia pt os po rid iu m
0
An abstract book containing all presentations was published for the meeting and a copy is included as appendix 1 to this report.
Fig 3 – Number of presentations per parasitic genus MANAGEMENT COMMITTEE MEETING A full meeting of the Action MC took place at which the priorities for discussion were the planning of meetings over the next 12 months and the establishment of an evaluation committee for short term scientific missions. The full minutes of the MC are included as appendix 2 to this report.
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WORKING GROUP MEETINGS Meetings of all six working groups took place during the Workshop. The main aim of these was to bring interested parties together and to discuss the priorities for Action and WG activities over the next 12 month period. The chairs of each WG reported back to a subcommittee of the MC as follows: WG1 - Genomics and Bioinformatics (Chairs Matt Berriman/Robert Menard) ~15 people attended this WG and the major outcomes were: •
There is a clear need to address the issue that a very large number of apicomplexan genes remain totally uncharacterised. Establishment of cross-genus bioinformatics approaches to allow scientists working on different parasites to easily crosscheck/shortlist genes that are of interest for functional studies would be enormously beneficial
•
Approaches to prioritise lists of candidate genes (for functional studies) to manageable numbers should be directly related to advances in relevant functional genomics technologies. Thus for instance, advances in protocols for conditional mutagenesis would allow larger numbers of genes to be systematically analysed
•
Training in bioinformatics is required to get more value from the genome(s)
After discussions, it was agreed that the most practical way forwards in the first instance will be to organise bioinformatics training courses that cover all aspects of comparative genomics. It was agreed that to start this process, a 2 day general training in bioinformatics workshop should be held at the Sanger Institute in 2005. Thereafter, there would be more value in providing theme-driven workshops aimed at providing training in key areas of interest amongst groups of collaborators. Interested parties should contact the WG1 chairs and/or other bioinformatics groups to assess specific requirements. Suggested themes included parasite motility, signalling, cell cycle control. A full list should be established and priorities set by members of COST via e-mail circulation. WG2 - Gene expression (Chairs Adrian Hehl/Andrew Hemphill) 12 people attended this WG and the major outcomes were: •
There is a clear need to establish common networks for exchanging and comparing data on gene expression between different laboratories. Cross-genus bioinformatics is essential
•
Training in bioinformatics is required
Again, the most practical way forward will be to organise bioinformatics training courses and this will proceed in collaboration with WG1, as outlined above. In the first instance there is a meeting already planned for comparative proteomics, based on the analysis of subcellular proteomes of various secretory organelles/membrane fractions. This will take place in September 2004, in Liverpool UK WG3 - Cytoskeleton, motility, invasion (Chairs Jean-Francois Dubremetz/Dominique Soldati) 12 people attended this WG and the major outcomes were: •
There is a strong requirement for establishment of bioinformatics networks and databases to enhance comparative genomics/proteomics so that these can be applied to the study of molecules important in motility and invasion (cytoskeletal components, secretory organelles, surface proteins) It is envisaged that there will be strong links between WG3 and other working groups especially WGs 1, 2 and 4.
3
•
Theme-based meetings will be the top priority of the working group and some topics that should be developed through this WG are o Exocytosis and motility/invasion o Signalling in motility o Proteases
The group will be actively involved in the WG2 led meeting on subcellular proteomes, which will take place in September in Liverpool UK. The working group is also leading two other activities – a PhD training workshop that will be in Brussels and a transfection workshop (with WG1) which will be in London, both again in September 2004. WG4 - Intracellular survival and host cell modification (Chairs Klaus Lingelbach/Uwe Gross) 18 people attended this meeting and the major outcomes were: •
There is a need to establish a catalogue of parasites held, research topics, special methods etc. Uwe Gross offered to co-ordinate the organisation of this
•
Theme based workshops should have a high priority within the working group and suggestions for future activities included o A meeting on Toxoplasma and GC models, to be organised by Antony Barragan o A meeting on congenital toxoplasmosis, to be organised by Uwe Gross
In the first instance A COST working group 4 meeting will be held as a satellite to a larger meeting entitled ‘Life inside cells’ to be organised by Klaus Lingelbach in Marburg, Germany during March or April 2005. The precise content of this meeting will be decided by e-mail circulation to the membership. WG5 – Biodiversity and Epidemiology (Chairs Susana Pedraza/Jonathan Wastling) 11 people attended this meeting and the major outcomes were: •
There is a strong recognition that WG5 has direct relevance to other WGs, most particularly WG6 and that there should be good co-ordination of the activities of these two working groups
•
It is important to ensure that evolutionary studies are well covered within the WG
•
There is an urgent need to develop good diagnostic tools for various apicomplexan genera and in particular to ensure that the COST Action platform be used to develop tools, standardise techniques and consult with experts in other fields and countries about the best ways to move forward.
In the first instance, it is suggested that the WG holds its first scientific meeting in November, the precise content of which should be determined by e-mail circulation to the membership. WG6 – Drugs and Vaccines (Jean-Christophe Barale/Martin Shirley [absent]) ~12 people attended this working group meeting and the major outcomes were: •
Expectations are different for those working on drugs versus those working on vaccines, but there are some common features, most certainly:
4
o
Selection strategies used to identify and validate new targets
o
Differences between children and adults in their responses to drugs and vaccines and to efficacy (disease profiles, immunological status, toxicological risk…)
o
Problems concerning biological delivery of drugs/vaccines
o
Interactions between treatment and the immunological status of the patient
For those working in the drug development area, it is clearly important to increase their interactions with chemists and pharmacologists and it would be very valuable to invite such experts to a COST meeting or to organise a working group meeting based on chemicopharmacotherpay topics (eg how to define a lead compound, what about pseudopeptidic and combinatorial chemistry approaches, costs of development, toxicology studies. It was suggested that a valuable approach would be to organise a joing meeting with the EU COST Action B22 (Drug development for parasitic diseases) For those working on vaccines, it is important to ensure interaction with immunologists so again it was suggested that the COST Action should consider inviting some fundamental immunologist to its meetings and/or clinical immunologists with expertise about existing vaccines and the way they are working. A topic of interest identified was ‘adjuvants’ – do they have to be natural, synthetic, and which should be used for stimulating particular arms of the immune response. Report filed by Fiona Tomley, Chair COST 857
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Financial Report for 1st Annual Workshop (filed by Henrique Silveira) for the period from: 03-05-2004 to 06-05-2004 Meeting Title: COST 857 meeting - Apicomplexan Biology in the Post-Genomic Era Currency: euro Description
Supplier name
Room and technical equipment rental Room and technical equipment rental Room and technical equipment rental Coffee breaks and light refreshments Printing + Photocopying -Programe (preliminary, definitive) application form, draft report Printing + Photocopying -Programe (preliminary, definitive) application form, draft report Printing + Photocopying -Programe (preliminary, definitive) application form, draft report Personnel - staff involvement
Hotel Real Palacio
Phone, fax, mail - announcements, letters of confirmation Phone, fax, mail - announcements, letters of confirmation
CTT
Description Abstract Books
number of invoice 10125
Date of invoice 27-May
Amount
1223
30-Apr
84.07
1,877.25
Transportes Francisco Alberto, Lda. Transportes Francisco Alberto, Lda. Hotel Real Palacio
1242
5-May
84.07
10125
27-May
2,447.00
Papelaria Fernandes
46109
26-Apr
268.24
Albano R.N.Alves
13606
16-Mar
135.00
1785
5-Apr
150.00
307
23-Apr
867.42
5000427820
31-Mar
150.00
41757486
27-Apr
300.00
MicroDrive Celeste Figueiredo
Vodafone
Supplier name Leandro Branco, Lda.
number of invoice 984
Total
6,363.05
Date of invoice 23-Apr
Amount
Total
840.00 840.00
Date:31-05-2004
Date: 31-052004
Name of person in charge of the work: Henrique Silveira
Name of duly authorised responsible Financial Officer: Carla Ribeiro Brás
Signature of person in charge of the work:
Signature of duly authorised responsible Financial Officer:
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Report on WG2 (Gene Expression) meeting, September 2004 WG2 held a working group meeting on “Analysis of organellar proteomes” at Leahurst, Faculty of Veterinary Medicine, University of Liverpool, England, on Sept.8 / 9. This meeting was held in conjunction with the Annual Conference of COST854 (Protozoal reproduction losses in farm ruminants, Sept.6-9). This conference was organized by Sandy Trees and and Diana Williams (University of Liverpool), and our warmest thanks go to the organizers which allowed us to “parasitize their conference”. There is a considerable overlap of interests for proteomics technology in both COST actions. As a consequence, a large number of COST854 participants stayed on for the extra two half days. Overall, the presentations were as follows. September 8 started out with John Boothroyd (Stanford, USA) on genome approaches to the analysis of virulence and intracellular development of Toxoplasma. He pointed out new microarray techniques, which allow to study RNA synthesis, rather than abundance, and how the growing body of sequence data has been used to analyze the genomic pattern of polymorphisms and the evolution of virulence in nature. This presentation was followed by short talks on a number of topics relevant to both COST actions. Of particular interest was the update on the Neospora caninum genome sequencing project presented by Jonathan Wastling (University of Liverpool). September 9 was completely dedicated to proteomics and its applications in different species. Three excellent speakers started the session. First, Bob Sinden (Imperial College London), provided an exellent talk on the proteomic analysis of Plasmodium, and gave specific examples how in different life cycle stages proteomic analyses has somehow seemingly contradicted traditional views about stages-specific protein expression. Secondly, John Hyde (University of Manchester) gave a talk on how quantitative proteomics can be applied for Plasmodium falciparum. He presented a methodology which was applied to quantitatively assess protein expression levels upon treatments of the parasite or stage conversion, and which can be applied to essentially every one of the approximately 5300 proteins expressed by P. falciparum. The third speaker was Peter Brophy (University of Wales), who gave us a glimpse of the worm’s view on proteomics. He outlined how the group has focussed on the application of proteomics for elucidating the nature of those proteins important for the establishment of nematode infections in rodent models, and he highlighted the difficulties in applying this technology in organisms which are not genome-verified. Keynote speakers were followed by individual 15 minute presentations. Laetitia Vincensini (University of Montpellier, France) presented an excellent talk on the proteomic and functional analysis of the P. falciparum Maurer’s clefts, a membrane compartment in the infected erythrocyte cytoplasm, involved in provision of enzymatic activities necessary for secretion. For this, parasite-derived proteins were metabolically labelled with deuterated lysine and analysed by MS-MS. Julius Nyalwidhe (University of Marburg, Germany) presented an interesting strategy for the specific labelling of soluble proteins contained within the parasitophorous vacuole in P. falciparum infected erythrocytes, based on the selective permeabilization of the parasitophorous vacuole membrane by the pore forming enzyme streptolysin O. These selectively labelled proteins were analyzed by high throughput MALDI-TOF. Last but not least, Liz Bromley (Institute for Animal Health, Compton, England) provided us with new and exciting data on the proteome of apical organelles, micronemes and rhoptries, of Eimeria tenella. The recent sequencing of the respective genome has facilitated studies on their respective proteomes. The data on Eimeria is highly relevant to other apicomplexa for which those organelles are less abundant and purification protocols are less straightforward.
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In conclusion, this WG meeting provided a broad overview and exciting information on the state of the art technologies and applications of proteomics in apicomplexan and other parasites. It demonstrated how the sequencing of genomes and provision of expressed sequence tag databases has facilitated the studies on the expression of distinct proteins during development, and how genomics now allows us to determine the repertoire of proteins within distinct subcellular fractions or isolated organelles, through the application of proteomic technologies. These advances are highly relevant for further elucidating the nature and molecular basis of host-parasite interactions and the development of suitable tools for treatment or prevention of respective diseases. It also leaves us with a rather grim perspective for those parasites or parasitic systems which have not yet entered the genomic era. In terms of achieving rapid progress in the field of host-parasite interactions, they are likely to be left behind in the long run. Report filed by Andrew Hemphill, MC member and co-chair of WG2
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Report on Activities of COST 857 – Working Group 5 BIODIVERSITY AND POPULATION GENETICS OF THE APICOMPLEXA Working Group co-ordinators Dr JM Wastling (United Kingdom) Dr S Pedraza (Spain) Overview The objectives of this working group are to exploit recent developments in genomics to further our knowledge of the biodiversity and population genetics of the Apicomplexa. Full genome sequence information is now available for nearly all of the important apicomplexan parasites: a resource that can be exploited to develop highly discriminatory tools for studying genetic diversity, population structures and for field applications including disease tracking. These studies are not just of academic interest, but have important implications for public health (Plasmodium spp.and the zoonotic Apicomplexa Toxoplasma gondii, Cryptosporidium parvum and Babesia spp.) and also animal health (T. gondii, C. parvum, Eimeria spp., Babesia, Besnoitia besnoiti and Neospora caninum). One of the features of such studies is that they rely on large sampling areas, co-operation between agencies such as public health institutions, veterinary establishments and the academic research community. Moreover, they require resources outside the scope of a single agency, and if a European perspective is to be acquired, beyond the scope of a single country. Working Group 5 is thus ideally placed to act as a catalyst to enable researchers from the above disciplines to work together towards common scientific goals by enabling an exchange of resources (such as biological samples), standardisation of tools and wider access to epidemiological field data. For some of the less common apicomplexan parasites such as Besnoitia besnoiti Working Group 5 provides the opportunity for sample collection over wide geographical area thus potentially providing a critical body of data that would not otherwise be available. Working Group 5 met briefly at the inaugural meeting of COST 857 in Lisbon in May 2004 to plan activities for the coming year. A decision was taken to meet together for a workshop in the Autumn. This workshop took place in at the Institute Maria SS. Bambina, Rome on 8th and 9th November 2004. The meeting proceedings and outcomes are summarised below. Attendance and workshop structure The workshop was attended by representatives of academic, public health and veterinary research establishments drawn from the United Kingdom, Ireland, Sweden, Norway, Denmark, Italy, France, Spain and Portugal. COST 857 provided twelve reimbursements. In addition to the invited experts the workshop was attended by local Italian public health researchers who also provided generous hospitality and superb organisational assistance. A full list of the invited attendees is appended. Scientific Proceedings Day 1 of the workshop consisted of formal presentations aimed at setting the background for the discussion on day 2. Day 2 was a mixture of data presentation, questions and general discussion. The workshop concluded with an agreement on objectives which the Working Group felt could form a focus for the group and which had achievable and defined outcomes. A summary of a selection of the presentations and discussion is given below: Dr Susana Pedraza (Spain): Molecular epidemiology of cryptosporidiosis in farm animals: study of transmission routes and impact of farm practices for the human population.
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Dr Pedraza presented: • • • •
A description of a proposed epidemiological study in Spain to run for 3-5 years. The use of ribosomal DNA sequencing for species identification The use of microsatellite markers developed by other groups for sub-species identification Farm studies will be carried out to test genotypes over time and seasons.
The following problems were highlighted: • Method relies heavily upon reliable DNA extraction • Environmental and faecal samples require very different handling and it may be difficult to perform exactly the same techniques on both or at least to have directly comparable results. A more general point made was that Cryptosporidium taxonomy and classification needs to be agreed upon and then to remain as stable as possible. Daniel Ajzenberg (France): Genetic diversity, clonality and sexuality in Toxoplasma reconsidered by microsatellite analysis of unusual isolates Earlier studies by several other groups have shown that despite the worldwide distribution, wide host range and the presence of a sexual cycle in felids, Toxoplasma has a clonal population structure with low genetic diversity. Tibayrenc, Howe and Sibley found 15 genotypes out of a possible 1728 genotypes using 106 Toxoplasma isolates. Therefore, there was an over-representation of identical genotypes. However, recent work by Ajzenberg and Darde has suggested that recombination is occurring and that there had previously been a lack of samples/ information from the sylvatic cycle. A substantial set of unusual isolates in terms of their geographical origin was subjected to multilocus analysis using a multiplex microsatellite system. The 5 markers are located on different chromosomes. Interestingly there was a predominance of recombinant lineages in the unusual isolates (even if the genetic distance was remaining low at
