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SECTION III: SCHEMATIC DESCRIPTION OF THE PROJECT

This text (one page maximum) should provide an updated description of the project, written in a way that can be understood by non-specialists in the field.  It should include only information that can be published.

Abstracts

Peter Mertens: Web sites and Reference collection  ICTV database

  The Web sites for dsRNA viruses and for the EU grants have proved to be a considerable success.  The dsRNA virus Protein/RNA tables are currently being updated and will be included in the handouts for the forthcoming dsRNA Virus Symposium in Tuscany (October 2003),

The username and password have been removed from the ReoID pages to allow greater and easier access, particularly to the list of viruses held in the reference collection at Pirbright. 

(see http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/viruses-at-iah.htm)

Tables of additional primers have recently included on the website, which can be used to identify genome segment 2 from individual BTV serotypes and to distinguish genome segment 2 from field or vaccine strains of the European BTV serotypes .

  (See http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/rt-pcr-primers.htm )

The Phylogenetic trees currently displayed on the website are in the process of being updated  (See: http://www.reoviridae.org/dsRNA_virus_proteins/orbivirus-phylogenetic-trees.htm)

The ICTV and The international sequence databases have recognized that a significant proportion of the sequence data currently available is from nucleic acids of insufficiently well defined origins.   In order to counteract these problems ICTV has set up a 'database' * to hold information concerning individual virus isolates.    Each virus entered will be given a unique identification number that can then be used permanently to identify it or search for information.   The new database will hold isolation data and provide links automatically to sequence data, publications, Reference collections  and any other sources of information  that are available via the web.

(see (http://www.ictvdb.iacr.ac.uk/Tutorial/tt_virin.htm   (may not work in Netscape). 

The ICTV has agreed  to press authors and publishers of the major Scientific Journals)  to include the unique reference number for all viruses included in publications.  This may well lead to the submission of virus data becoming a normal pre-publication requirement, in much the same way as getting accession numbers is for sequence data.   We have already started to create links between the ReoID orbivirus collection data and ICTVdb, which we plan to expand over the final year of the project.  We anticipate this will add to the value of the reference collection and will provide a better link to publications/sequences and other useful data for individual isolates.

( *Note:  P. Mertens is now a member of the ICTV database Sub Committee)

Sushila Maan* Karam Singh*,  Alan Samuel, Shujing Rao, Peter Mertens:   Recent work at IAH Pirbright will be described, concerning sequencing of BTV genome segment 2  and 6

The bluetongue virus has two outer capsid proteins, VP2 and VP5, encoded by genome segment 2 and segment 6 respectively. VP2 is the major neutralisation antigen and the most variable of the virion structural proteins, which is reflected by sequence variation in genome segment 2.   The absence of sequence data for segment 2 of many BTV serotypes, has previously made it impossible to design primers for RT-PCR based serotyping assays.   Full length sequences for genome segment 2 of representative isolates of all 24 BTV serotypes are now available (see Abstracts by S. Maan et al), as well as sequence data for multiple European isolates of BTV 1, 2, 4, 9 and 16.  This has facilitated the design of  'serotype-specific' primers, to identify isolates of different serotypes in less than 24 hrs.   However, the efficiency of the initial serotype specific primers for types 1 & 2 was variable with isolates from different geographic regions.  Additional primers were therefore designed and tested. Primers were also designed to differentiate field and vaccine strains of the European BTV serotypes. The results from these assays will be presented.

The complete nucleotide sequence of genome segment 6 from all 24 BTV serotypes was also analyzed, providing information on sequence variation and its correlation with virus serotype.  Differences between the vaccine and field isolates of BTV were also examined and may be sufficient to design RT-PCR primers to distinguish them. Similar methods are being used as the basis for serotype specific RT-PCR assays, to improve the speed and reliability of BTV serotype determination. The sequences generated will be added to those already available for the segments of different BTV serotypes in the international databases and listed at:  www.reoviridae.org/dsRNA_virus_proteins/btv_sequences.htm.

·      Joint first authors  Presenting Author is underlined

TITLE: S10 SEGMENT SEQUENCE ANALYSIS OF  GREEK BTV  FIELD STRAINS

Nikolakaki S. V, Nomikou K, Koumbati M.

Sequence analysis of the NS3/NS3A gene of Greek isolates from the 1979-2001 epizootics will be presented.

The sequences of the S10 gene of BTV field strains of serotypes BTV1, BTV4, BTV9 and BTV 16, were determined to define the molecular epidemiology of BTV infection in Greece.

Phylogenetic analysis segregated the Greek BT viruses into two monophyletic groups.

The first group, consisting of strains of serotype 4, presented high percentage of homology (98-100%) irrespective of the area and the year of isolation and were close to the Tunisian and the Corsican strains. The second group consisting of strains of serotype 1, 9 and 16, on the other hand, showed 97-99% homology and were closer to the Chinese, Australian and South African strains.

From the above results it may be concluded that two different groups of BTV strains seemed to coexist in Greece during the epizootics of 1979-2001.

NUCLEOTIDE SEQUENCE ANALYSIS OF GENOME SEGMENT 10 FROM BLUETONGUE VIRUSES ISOLATED IN THE CURRENT EUROPEAN OUTBREAK

O’Hara, R.S., Rao, S., and Mertens, P.P.C.,

Institute For Animal Health, Pirbright Laboratory,

Ash Road, Woking, Surrey, GU24 0NF(UK)

Seasonal outbreaks of Bluetongue disease have occurred across southern and eastern Europe since 1998. Five serotypes (1, 2, 4, 9 and 16) have recently been reported in Europe and the virus has extended its range northwards, into areas never previously affected by the disease, which do not support populations of C.imicola (the principal southern European and north African vector). This indicates that the virus is being transmitted by an alternative (new) vector (possibly C. pulicaris or C. obsoletus), which are widely distributed across north Western Europe. Virus isolates (of different serotypes) were obtained from several European countries. Viral dsRNA was extracted and its RNA electropherotype analysed by SDS-PAGE. Segment 10 was amplified by RT-PCR, before sequencing using a Beckman capillary sequencer. Full-length sequences were aligned and compared to existing sequences (held on the EMBL) database using the BioEdit sequence alignment editor, ClustalX and TreeView.  Segment 10 sequences from current European field strains were found to cluster within only 2 groups, which vary (between groups) by 15 – 20%. These differences are independent of serotype, but showed some correlation with geographic origin.   Initial analyses also indicate some correlation with the insect vector species involved in transmission. Comparisons of genome segment 2 and 10 from European isolates, provide evidence for genome segment reassortment in the field.

Presenting Author is underlined
 

cDNA Synthesis, Cloning and Sequencing of the Complete Genomes of Previously Uncharacterized or Unassigned Orbiviruses

Shujing Rao , Adam  Meyer, Alan Samuel, Houssam Attoui, Peter P. C Mertens,

Institute For Animal Health, Pirbright Laboratory, Ash Road, Woking, Surrey, GU24 0NF(UK)

The dsRNA genomes of fourteen previously unsequenced and / or unassigned orbiviruses were converted into cDNAs (using ligation of defined oligonucleotides to the 3' termini) and cloned into pGEM-T Easy vector for sequencing and future expression studies. This has permitted the rapid analyses of the entire genomes of these viruses.  The viruses studied include Peruvian horse sickness virus, Chobar Gorge virus, Tilligerry virus, Umatilla virus, Itupiranga virus, Eubenangee virus, Orungo virus, Andasibe virus, Tracambe virus, Changuinola virus, Codajas virus, Lebombo virus, Ieri virus, and Tembe virus.  The results of initial sequencing studies have revealed the relationship of each isolate to other (established) Orbivirus species and have indicated the function of the virus proteins.    These data form a basis for assignment of these viruses to established or new virus species.  The sequence data will be made available via the international sequence databases and via the dsRNA virus web site at: www.reoviridae.org/dsRNA_virus_proteins/ forming a resource for subsequent identification of other new orbivirus isolates. Consequently, it will be possible to identify different orbivirus species unambiguously by comparison to 'reference' sequence data obtained over the web, without the need for expensive (and frequently unavailable) serological reagents.   These data will also support the design of oligonucleotides primers for use in diagnostic RT-PCR based assays.

The viruses analysed were generously provided by Bob Shope. The authors also wish to thank the Instituto Evandro Chagas as the original source of isolates of Itupiranga, Tracambe, and Codajas viruses.

New viruses within genera Seadornavirus and Orbivirus isolated from mosquitoes obtained from China
and
Diagnostic assays for viruses of genera Coltivirus and Seadornavirus

Houssam Attoui

Two viruses with dsRNA segmented genome profiles were isolated from mosquitoes in China.

The first virus was isolated from Aedes dorsalis mosquitoes and contained 12 segments of dsRNA. Sequence analysis has shown that this virus is a new species of genus Seadornavirus. This virus was designated “Liao ning virus”. Amino acid identities between homologous genes of either banna virus or kadipiro virus ranged between 18 and 42%. The genome of LNV had higher G+C content that those of BAV or KDV. The conserved terminal sequences of this virus differed from those of either BAV or  KDV

The other virus was isolated from Culex tritaeniorhynchus mosquitoes and contained 10 segments of dsRNA. Sequence analysis has shown that this virus is a new species of genus Orbivirus. This virus was designated “Yunnan orbivirus”. Amino acid identities between homologous genes of either Banna virus or St Croix river virus ranged between 20 and 49%.

The consereved terminal sequence of this virus were found similar to those of other orbiviruses. The T2 protein of this virus is the VP2 as it is the case for tick borne orbiviruses (BRDV and SCRV).

Diagnostic assays based on expressed recombinant proteins were validated on human and immunised animal samples for Colorado tick fever, Eyach and Banna viruses.

An example of data submission to the ICTV database 

(DRAFT Copy)

00.060.0.02.003.02.109.001. Bluetongue virus type 2 ITL02/07

Isolation Details

Isolate designation: ITL02/07.

Isolation date: 18 July; 2002.

Location: S. Angelo di Brolo, Sicily, Italy.

Source of isolate: Sheep.

Virus was isolated by Ms Giuseppina Alimena, IZS Palermo, N.A., Palermo, Sicily, Italy.

Involved in the submission of the isolate was also Dr. Annalisa Guercio; IZS Palermo.

Classification

This is a description of an invertebrate, or vertebrate virus at the isolate level.

ICTVdB Virus Code: 00.060.0.02.003.02.109.001. Virus accession number: 3EAF0E9A.

Biocontainment Level

Health authorities recommend to handle this virus at the biocontainment level BSL-3.

Name, Synonyms and Lineage

Acronym(s): BTV-2/ITL02/07. Virus is assigned to the serotype and species 00.060.0.02.003. Bluetongue virus 00.060.0.02.003.02. serotype 2; genus 00.060.0.02. Orbivirus; family 00.060 Reoviridae; not assigned to an order.

Depositories and Collections

The isolate has been deposited and is available at an institution at Orbivirus Collection, Pirbright Laboratory, Institute for Animal Health and is listed in the catalogue with the reference number ITL02/07.

Morphology

Virions have a simple construction and consist of a capsid. Capsid/nucleocapsid is round. The capsid.

Nucleic Acid

The genome is segmented; consists of ten segments of linear double-stranded RNA. The complete genome is 19216 nucleotides long <Genome size is estimated from electrophoretic migration pattern>. The negative-sense or non-coding strand (complementary to the viral mRNA) is full-length. The 5'–terminal sequence has conserved regions. The 3'–terminus has conserved nucleotide sequences; of 6 nucleotides in length.

Biological Properties

Natural Host Range

Viral hosts belong to the Domain Eucarya.

Domain Eucarya

Kingdom Animalia.

Kingdom Animalia

Phylum Chordata.

Phylum Arthropoda

Subphylum Vertebrata.

Phylum Chordata, Subphylum Vertebrata

Class Mammalia.

Class Mammalia

Order Artiodactyla;                              
Family Bovidae:; Subfamily Caprinae; virus infects Genus Ovis aries (sheep).

Pathology

The virus can be detected best in spleen.

Geographical Distribution

The virus is known to occur in Mediterranean regions; viral host lives under aerobic conditions; viral host lives in the atmosphere. The viral host is found in a undisturbed environment yet with signs of human disturbance; an agricultural environment. The virus occurs in Italy.

Data Sources and Contributions

Virus description on the Web

 http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/viruses-at-iah.htm - ITL02/07. For further information on virus please contact the Study Group (SG) chair (see at ICTV web page).

Contributor

Data have been submitted online to ICTVdB on 29–04–2003; by                                     
Peter P. C. Mertens                             
Institute for Animal Health, Department of Molecular Biology                                         
Ash Road Pirbright                               
Woking; Surrey; GU24 0NF                  
UK                                                      
Tel: + +44 1483 231017.                        
Fax: + +44 1483 232448.                       
Email: peter.mertens@bbsrc.ac.uk.

This is a description from ICTVdB: the Universal Virus Database of the International Committee on Taxonomy of Viruses.

Copyright © 2002. International Committee on Taxonomy of Viruses. All rights reserved.

Last updated: 26 May 2003

Phylogenetic sequence analysis and improved diagnostic assay systems for viruses of the family Reoviridae

(Contract number QLK2-CT-2000-00143)

 MINUTES OF THE 4th  COORDINATION MEETING

(Seville, Spain, 10-11th July 2003)

Participants  

            Maria  Koubati-Artopiou                       University of Thessaloniki                      Partner 5

            Kiki  Nomikou                                        “                                                           “

            Stephan Zientara                                    AFSSA Maisons Alfort                        Partner 3

            Emmanuel Briard                                      “                                                           “

            Corrinne                                                   “                                                           “

            Houssam Attoui                                      University of Marseille              Partner 2

            Paqco Rodriguez                                    Madrid                                                 Partner 4

            Shujing Rao                                            IAH Pirbright                                        Partner 1

            Alan Samuel                                             “                                                           “

            Peter Mertens                                           “                                                           “           Co-ordinator

Welcome  (Peter Mertens)

             Professor Mertens welcomed the participants and thanked Paco Rodriguez   for organising the venue  in Seville.  Apologies for absence were recieved from Dr Rachel O’Hara and Dr Philip Mellor (IAH Pirbright) and Dr Xavier De Lamballeries ( University of Marseille).  

 Financial matters

            Professor Mertens reported that the scientific report for year 2 had been received by Isabel Minguez in Brussels and that she was currently reviewing it. As soon as any news is received concerning  its acceptance Professor Mertens promised to forward this to the ReoID partnership

Professor Mertens reported that all partners had received the initial tranche of 40% funding, but only Marseille (partner 2 ) had been paid the second amount of money (end of year 1) which had been paid by the administration of Partner 1 even though these funds have not yet been received from Brussels.

 The reasons that funding for the start of year 2 (25%) and year 3 (20%) have not yet been released by the Commission is that satisfactory financial reports have not yet been received for year 1 and year 2 from either Maisons Alfort or the University of Marseille.  As a result none of the funding for any partner for year 1 and 2 has been received. The financial reports from Partners 4 and 5 have been received by IAH (Mike Baker) and are acceptable.  The delay in funding is therefore due to lack of reports from partners 2 and 3 only  (University of Marseille and AFSSA Maisons Alfort).  

            Professor Mertens Urged both Dr Zientara and Dr Attoui to contact their respective financial representatives to ensure that these financial reports were delivered to IAH as soon as possible.  They both reported that they had already received several requests for the correct documents from Mike Baker (financial officer at IAH) and agreed to contact their finance departments with these requests  as a matter of urgency.  However they also reported that since they were not in control of their financial departments it might be also necessary for Professor Mertens as Co-ordinator to send demands for these statements to the financial officers and senior management of both organisations, reporting that these oversights were now becoming a serious embarrassment and might in his opinion affect future funding from the EU.        

Professor Mertens reported that he had requested a 10 month extension to the project (with no additional funding) to allow each of the partners to spend the funds that had been awarded to them.   Isabel Minguez has agreed to work on this request over the coming week and it is hoped that a reply will be received very soon.

If the application is successful it  would mean that the proposed annual meeting in May 2004, will not be the final meeting, which will then be required in late 2004/early 2005.

Professor Mertens also reported plans to have a joint final meeting between the three current  EU grants co-ordinated from IAH Pirbright  (the ReoID grant, The BTV vaccine grant and The culicoides Risk assessment grant)  This meeting is planned for early/mid 2005 and Peter Mertens & Philip Mellor will discuss provision of some support funding for this as an international meeting with the commission.  Archives of Virology has already agreed to publish proceedings of such a meeting as a book (supplement to the Journal) similar to that published for an AHSV meeting held in Morocco after the EU AHSV grants. 

Scientific Reports

1.                  Peter Mertens, Web sites, Reference collections & the ICTV database

Professor Mertens reported that he has been invited to present a plenary lecture to the dsRNA virus Symposium in Tuscany September 2003, concerning the molecular epidemiology of BTV in Europe.   He suggested that it would be appropriate to use this opportunity to report the major scientific observations concerning BTV that have been derived from the ReoID grant and would therefore like to use data from all partners (with appropriate acknowledgements) for this report.  He would also report the construction of sequence databases for segments 2, 6 and 10 from different serotypes.   The partners agreed with these proposals and would supply any relevant data required.

Pages from the dsRNA virus website (providing details on viral proteins/RNAs) will be included in an information book to be published for the Tuscany meeting. 

Professor Mertens reported that tables of primers for the amplification, detection and identification of BTV genome segment 2 from European vaccine and field strains (BTV 1, 2, 4, 9 and 16)   have been added to the ReoID web pages and are therefore, widely available.  It was agreed that these pages will be edited and circulated to the different partners for addition of other primers (developed by other members of the partnership) and comment on primer efficiency/discrimination etc.  The revised tables will then be posted on the web site.  

Professor Mertens reported his intention to add agarose genome profiles for representative species of the family Reoviridae, to the website, thereby providing an additional aid to virus identification.  It was agreed by the partnership that this would be valuable and where possible they would provide relevant images.    

Professor Mertens reported that the Israeli Veterinary Services had greed to send recent Israeli isolates of BTV to the ReoID reference collection at IAH Pirbright.  Initial communications suggest that this will include 20-30 isolates made over the last 3-4 years.   Negotiations are also underway with senior management at CSIRO, Geelong in Australia to provide multiple isolates of different Australian orbiviruses, including particularly, a complete collection of Australian EHDV isolates.  These will be use for sequencing studies similar to those already being conducted for BTV.  It is anticipated that this will provide a data base for the identification and molecular epidemiology of EHDV.  In view of partner 3’s specific interest in EHDV, these studies will be conducted primarily by partner 1 but in association with  partner 3. 

It was also reported that after the successful analysis of the Peruvian horse sickness virus (PHSV) genome, there is considerable interest from South American Colleagues in continued collaboration with IAH Pirbright and other members of the partnership.   In order to support such links Professor Mertens will explore funding opportunities to allow South American colleagues to visit and work at Pirbright (e.g. Wellcome travelling fellowship grants).   It is hoped that Anna  Hurtado will be able to come to Pirbright, bringing a collection of uncharacterised South American orbiviruses with her for sequencing and expression studies.   This would provide a valuable continuation of the work and useful additional isolates/data for the reference collection/sequence databases.

ICTV database:         Professor Mertens provided a brief report on the development of a virus isolate database by ICTV, after attending the ICTV database subcommittee meeting in St Louis (Professor Mertens is now a member of this committee). It becomes clear that both ICTV and EMBL are concerned that much of the sequence data that is currently available is derived from nucleic acids of poorly or inadequately defined origins, reducing the overall value of these data.   Initial estimates suggest that 65% of available sequences are inadequately identified.  As a result ICTV have set up databases to record and identify individual virus isolates.  This will be available via the internet and will include links (automatically updated) to other sources of information (sequences publications, reference collections etc).    

            Each Virus strain will be given a unique reference number to identify it and act as a identification mechanism in other data sources (e.g sequence databases publications etc).  Since each virus strain data entry will be refereed by the relevant ICTV study group it may be considered as a refereed publication in its own right.    

 Home base:    http://ictvdb.bio2.edu/

USA site:        http://www.ncbi.nlm.nih.gov/ICTVdb/

UK site:          http://www.ictvdb.iacr.ac.uk/

 Contributors will need to register and obtain a password but are then free to add data on individual virus isolates.

            Professor Mertens reported ICTV’s intention to ask both the sequence databases and the editors of Virology journals to make incorporation of the ICTVdb number a requirement for submission or publication of data concerning any new virus isolate (in much the same way as accession numbers are usually required before publication of sequences).  It is anticipated that this will lead to much greater uptake and acceptance of the ICTVdb system for virus strain identification.  Professor Mertens reported that he would add data for each isolate in the ReoID reference collection and obtain relevant strain identification numbers.    

Dr Rodriguez asked Prof. Mertens  if entering details of a virus isolate onto the ICTV data base  would commit the author/contributor to giving that isolate to other people  (in a manner similar to that implied by publication in refereed journals) .  Prof. Mertens replied that these issues had not yet been discussed at ICTVdb but decisions would have to be made in the near future.  He said that he would bring this matter up at the next ICTV db meeting.

Dr Rodriguez also asked whether, (in consideration of bio-terrorism) it was ‘safe’ to put sequence data for these viruses in the public domain.   In reply Prof Mertens pointed out that such  sequence data were already available for many important pathogens via the international databases and the matter was one for consideration by international agencies rather than by the ReoID partnership alone.   It is currently normal practice to submit such data to the international sequence databases. 

Prof. Mertens also reported that a colleague at Pirbright had questioned whether data concerning virus isolates and reference collections should  be put on open websites.  However it is evident that containment laboratories such as Pirbright must have sources of relevant virus pathogens.  The details of which specific strain are held are of secondary consideration and do not therefore represent a significant additional risk.   Indeed the availability of these data may contribute significantly to disease control programmes, which is clearly desirable .

Alan Samuel Suhila Maan, Karam Singh, Shujing Rao Rachel O Hara, & Peter Mertens,     Phylogenetic Analysis of Segment 2 & 6 of BTV.

In subsequent discussions:  Professor Mertens reported that the trees that had been shown for all 24 BTV serotypes and for multiple individual isolates of BTV 1, 2, 4, 9 and 16 had been placed or would appear on the ReoID website in the near future. 

 The data presented by Dr Samuel was discussed and it was concluded that from comparisons of the genome segment 2 sequences, the Turkish BTV-4 vaccine is only distantly related to recent BTV-4 field isolates from Greece.  It is therefore considered unlikely that the Turkish vaccine strain and vaccination campaign were responsible for these outbreaks.    However, it was also shown  that at least in genome segments 2 and 6 the South African and Turkish vaccine strains are very closely related.      

Coffee 11:15am

Presentation Maria 

Segment 10 data, 2x Monophyletic group Some were related to Far Eastern strains whilst others were more related to strains from the Middle East.

There was a brief discussion on who has been sequencing segment 10 and Maria said that the Italians (Dr Savinni) have been sequencing the S10 of an Israeli strain. Dr Zientara said that the S2 nd S10 were identical to the Corsican strain. 

Presentation . Prof. Mertens S10 analysis.

Discussion Kiki made some corrections to the distribution and incidence of BTV types in Europe over the last 5 years.  Kiki said that she had tested some 5000 sera, collected from all over Greece, for EHDV and AHS and that they were all negative.

Kiki also mentioned that  Turkey are vaccinating against Rinderpest  PPR (Clarification of this matter should be sought from Dr Anderson and Prof. Barrett at Pirbright.

PAGE analysis has revealed additional bands in several virus isolates, including the South African vaccine strains.  This highlights the fact that these isolates / vaccines are not cloned and confirms that mixed virus populations can exist (Quite often) in cell culture grown viruses. 

There was some discussion concerning the generation of two distinct sequences for segment 10 of the Lesbos BTV-1 isolate.   Maria and Kiki reported that the commercial sequencers they used for this isolate had reported that the cDNA contained more than one sequence.   This suggests that Rachel’s observation of two segment 10 sequences may be based on a mixed virus population in the original isolate.   Because of the relative importance of segment 10 (possible involvement in use of new vector species) it was agreed that Partner 1 (Rachel) would design primers to distinguish the two versions of segment 10 and test original isolate material  to be supplied by partner 5 for the presence of each version of seg 10, helping to establish the presence (or not) of a mixed virus population.   It was agreed to share these data and publish the segment 10 analyses for the Greek isolates as a joint publication.        Maria agreed to write an initial draft to be sent to Peter for correction and addition of extra data.  

Presentation Dr Shujing Rao

Discussion

Houssam reported that the cDNA synthesis method developed by Shujing was also entirely effective with ssRNA virus genomes, making it widely applicable and valuable.

Lunch

Presentation Dr Stephan Zientara

Discussion

Stephan reported that the French group had also developed type specific primers for the European BTV strains.  During validation tests of these primers (using viral RNA which was supplied by Pirbright) some genome segment 2s from heterologous BTV isolates were  amplified in a non-type specific manner.   These data suggest the presence of more than one serotype in several isolates, which may either represent genuinely mixed virus populations, or laboratory contamination.  Clearly this matter needs to be resolved.    Dr Zientara will liase with partner 1, to identify which RNA’s may be Mixtures/contaminated. The sequence data that he has obtained for some of these RNA’s can then be compared to the sequences held on the Pirbright BTV Sequence database. The RNA’s should also be checked with Partner 1’s serotype specific primers to see if the same phenomena are apparent.  

Dr Zientara reported detection of EHDV in Reunion Island.   This virus had been sent to Pirbright for serological identification.   Although a positive match had been found with an isolate from Australia the serotype remains unresolved.   Stephan and Peter agreed that such problems could be resolved by development of an EHDV sequence database for genome segment 2 coupled with RT-PCR and sequencing of such new virus isolates.  Peter reported that the new Ph.D student (Simon Anthony) at IAH Pirbright would be working specifically on these problems.  

It was agreed that Partners 1 and 3 would pool sequence data for genome segment 2 of the European BTV isolates, so that they could be published jointly.  Initially Partner 1 agreed to prepare a manuscript for BTV-2 sequences, which will be sent to partner 3 for addition al data and comments prior to joint publication.

Presentation Dr Housam Attoui

Peter congratulated Houssam on the amount of work/progress that he had achieved.   Peter confirmed that Unan orbivirus has similar terminal sequences to Peruvian Horse Sickness Virus, suggesting the possibility of a close relationship.  As a result it was agreed that Alan or Shujing would send sequences for PHSV to Housam for comparisons.  If it is found that there are high levels of similarity, this may indicate that PHSV and YOV are isolates of the same virus species.  In this case it was agreed that these data will be submitted as a joint publication. 

In order to speed up sequencing studies Partner 1 will send cDNA clones for the larger Ndelle virus genome segments and for the Indian Sheep Orthoreovirus  segments to Houssam for sequencing.  This will   lead to additional joint publications.

Presentation Dr Paco Rodriguez

Due to financial problems only the acquisition of strains continues. Therefore Dr Rodriguez said that he has no further progress to report within the work package. Prof. Mertens stated that if a ten month extension occurs and the money gets to Paco quickly then perhaps a break in progress on the work package would be more acceptable.

A discussion was held on where additional funding could be sought to continue the project and to keep the collaborative links together.

Kiki suggested that perhaps money could be sought for the validation of the diagnostic tests that have been devised.

Work packages over Year 3

As a final discussion It was agreed that work was generally progressing well but that some additional focusing on epidemiological survey work for different viruses in Europe would be required in the later stages of the project.  However everything appears to cutrrently be on target, provided the second and third blocks of finance can be got to partners very soon.  Professor Mertens promised to deal with this as a matter of priority on his return to Pirbright.

There was some discussion of the time and venue for the next meeting.  It was agreed that this should be in May 2004 in Corsica.  Dr Zientara and Dr Attoui agreed to help organize the dates and venue.