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ReoID Phylogenetic sequence analysis and improved diagnostic assay systems for viruses of the family Reoviridae
Edited by Peter. P. C. Mertens and Houssam Attoui |
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Reo ID: Technical Annex
Quality of Life and Management of Living Resources
Phylogenetic sequence analysis and improved diagnostic assay systems for viruses of the family Reoviridae
Reo ID
Key action 2: Control of Infectious diseases
Proposal No: QLRT-1999-30143.
1. OBJECTIVES AND EXPECTED ACHIEVEMENTS The International Committee for the Taxonomy of Viruses (ICTV) has accepted RNA sequence comparisons and PCR based assays for identification of virus species within the Reoviridae. A PCR based assay has also been developed for the identification of the 9 serotypes of AHSV. Such methods have two significant advantages over serological techniques. Firstly sequences of new virus isolates can be compared to those of previously characterised viruses, without standardised reagents or assay systems. This would allow a laboratory working in isolation, to access the sequence data and identify a new virus isolate as either an established or new virus species. Secondly, PCR based assays do not require live virus and can be carried out on samples that have been stored or inactivated in ethanol, reducing and risk of contamination and disease. They are also extremely sensitive and rapid, making them ideal for use where the time involved in virus identification is important. However, conventional serological assays (for example by ELISA and competition ELISA), or virus serotype (serum neutralisation assays) are still vital parts of current diagnostic methods for identification of viruses. Development of techniques for the purification of virus particles, or expression and purification of viral antigens, will allow the production of high titre polyclonal antisera that is also highly specific for viral proteins. Such antisera can be used to improve the sensitivity and specificity of serological assays by replacing convalescent sera that are frequently in current use. Purified virus particles and proteins also represent the starting material for structural analyses, by electrophoresis, electron microscopy, crystallography or cryoelectron microscopy. The project will increase the available sequence data for the family Reoviridae, making it possible to identify the viruses directly from RNA sequences, without multiple serological assay systems and expensive standardised diagnostic reagents. This will be particularly valuable for the identification of new or emerging viruses. Full genome sequencing will be undertaken for representative isolates of virus genera that are not yet fully characterised (including Coltivirus, Seadornavirus and Aquareovirus) and some Orbivirus species (complete sequences already exist for BTV and AHSV). The derived amino acid sequence data will be used to identify functions of the proteins encoded by the different genome segments of these viruses. The project will also generate sequence data for the conserved genome segments (Pol and T2 genes) of those Orbivirus species that are currently either uncharacterised or unclassified. This information resource will allow new or unassigned orbivirus isolates to be identified as members of a known or a new species, simply from their RNA sequences. These new RNA sequence data will be made available through established sequence databases. New web sites will be constructed for each of the genera, for the collection and further dissemination of data concerning the molecular biology and structure/function of the different viral RNAs and proteins The project will develop improved serological and PCR based assay systems for detection and identification of different coltiviruses, seadornaviruses, aquareoviruses and individual Orbivirus species. By targeting the genome segment that encodes the major serotype specific protein of the orbiviruses BTV or EHDV, attempts will also be made to construct rapid PCR based assays (similar to those recently published for AHSV). Using such assays, studies will be made of the prevalence of coltiviruses, and seadornaviruses in tick populations within Europe and in ticks provided by collaborating laboratories from around the world. Assay systems will also be constructed for aquareoviruses to assess their distribution, prevalence and impact in commercial fisheries. Assay systems for different BTV serotypes and the different orbivirus genera will be subjected to validation exercises, to assess their sensitivity, reliability and level of discrimination. Provided these steps are successfully completed these assays will be made available to diagnostic or reference laboratories within the community. The project will also develop reference collections for the genera Orbivirus, Aquareovirus, Coltivirus, Seadornavirus (as well as new or currently unclassified viruses). These collections will contain different virus isolates, species and serotypes as well as reference antisera and cDNA clones of different genome segments, for further study. The project objectives are to generate RNA sequence data, serological reagents, improved assay systems; cDNA clones and reference collections for certain genera and unassigned viruses, of the family Reoviridae. The RNA sequence data will identify new or unassigned viruses from the Reoviridae. The new assay systems will be validated, then made available to survey the prevalence and impact of these viruses in the EU and elsewhere. Purified virus particles will be used for structural studies by electron microscopy, cryoelectron microscopy, and for crystallisation trials making use of existing collaborations with other structural laboratories. Dedicated web sites will be constructed to collect and disseminate information concerning the viruses. It is anticipated that because of similarities in the techniques used there will be frequent visits and informal exchanges of information and personnel, complemented by workshops and conferences involving all of the partners on a regular basis (6 to 12 month). Reference Collections: Isolates of seadornaviruses, coltiviruses, aquareoviruses, and orbiviruses, which are already being collected by the partners, will form the basis of reference collections for the different genera. Efforts will continue to add further virus isolates. The viruses will be adapted to cell culture and used for cDNA cloning, sequencing, particle purification and antibody production. Cell adapted strains, cDNAs, antibodies and antibody generating cells will be added to the collections, to provide a resource of diagnostic reagents and for further study. RNA sequence analyses: Advances in cDNA cloning and sequencing methods for dsRNAs involve improved RNA isolation methods, ligation of defined oligonucleotides to the RNA and use of high efficiency primers. With improvements in commercially available sequencing machines, it is now possible to clone and sequence a large proportion of the genome of a dsRNA virus in a matter of weeks. Full genome sequences will be generated for representative seadornaviruses, coltiviruses and aquareoviruses, as well as for more conserved Pol and T2 genes, from different species of Orbivirus. These data will be used for phylogenetic comparison of viruses within and between different genera of the Reoviridae. This will allow unassigned viruses and new isolates to be identified, or define an additional genera and species. Comparisons of the derived amino acid sequences to established databases will also help to identify protein functions.Improved assay systems: Purified Bluetongue virus particles can be used to generate high quality antisera, for virus species (ELISA) and serotype specific (neutralisation tests) diagnostic assays. These methods will be adapted, to improve serological testing systems for both BTV and other orbiviruses. Polyclonal or monoclonal antibodies will also be generated for members of the other genera under study, allowing development of serological assays for detection of virus specific antigens or antibodies. RNA sequence data will be used to design primers and PCR based assays for members of the genera under study and for different orbivirus species (serogroups). Using a method similar to that recently developed for AHSV, a PCR based assay will also be developed to identify the different BTV serotypes. Each of these assays will have to undergo testing and validation prior to being made available to diagnostic laboratories within the EU. Virus distribution and their impact: The assay systems will be used to assess the distribution, prevalence and impact of aquareoviruses, coltiviruses, seadornaviruses, using samples obtained via existing collaborative links. This aspect is therefore dependent on earlier reagent and assay development. Assays for BTV and related orbiviruses must be carried out in laboratories that are appropriately equipped and have permission. If they offer real improvements over current tests, it is anticipated that such assays will become part of standard diagnostic methods for detection of Orbivirus species or serotypes within the diagnostic laboratories in the community.Web sites for molecular biological data: Web sites will be established to disseminate data concerning the reference collections, virus structure and the molecular of these viruses. The sites developed will have a standard format and will be edited by one or more scientist(s) who has direct experience of the virus genus or species (for example partners in Reo ID). The different pages will be under central editorial control and will be updated at least every six months. 2.2 Description of the Workpackages Workpackage number: 1 Set up virus reference collections Start date : Month 1 Completion Date: Month 36 Partner Responsible: 1, 2, 4, Person months per partner: partner 1 (6 months), partner 2 (6 months), partner 4 (6 months) Total = 18 months Objectives The objectives of WP1 are to set up long term storage and documentation for reference collections of orbiviruses, coltiviruses, aquareoviruses and other currently unassigned members of the Reoviridae. This will include those virus isolates that are currently available, together with any new. These viruses, together with cDNA clones and virus specific antibody preparations derived, will provide a resource for comparison and identification of new virus isolates. Samples from the collections will be made available to scientists working in laboratories with appropriate facilities for their study. Methodology and Study Materials Dedicated cryo-storage facilities (liquid nitrogen storage or 80 C freezer) space will be purchased and established within the three laboratories involved. The orbivirus, seadornavirus / coltivirus and aquareovirus collections will be held at different laboratories (partner 1, partner 2 and partner 4 respectively) reflecting their primary research involvement. Unassigned reoviruses will be held in any of the three collections. The number of such virus isolates is relatively limited and the numbers stored will depend on availability. 72 species and 235 serotypes are currently recognised within the family Reoviridae, the majority of which (19 and 153) belong to the genus Orbivirus. Reference samples of each species together with samples of available serotypes will be stored. Virus isolates will be fully documented regarding source and passage history. Tissue culture adapted and plaque-cloned isolates will be developed, as dictated by the other areas of the project. These strains will also be documented and stored. Virus isolates will be obtained from the American Culture Collection, from authors of relevant research papers and through established collaborative links. cDNA clones of viral genome segments and preparations of virus specific polyclonal and monoclonal antibodies will also be fully documented and stored, providing a resource of characterised material (particularly where cDNAs have been sequenced) as a reference and for further manipulation, expression studies, or for use in diagnostic assays. Deliverables WP1 will provide source materials for the project as a whole and will act as a permanent reference collection for these viruses that can be used by the partner laboratories and other relevant research organisations. Collections will include cDNA clones and monoclonal or polyclonal antibodies that are specific for these viruses. In particular the antibody preparations can be used as diagnostic reagents. It is expected that within six months the initial collections of viruses and storage will be in place, although additional virus isolates, cDNA clones and antibodies will be added throughout the project, as they are developed and characterised. (D1, D2, D3 & D19) Milestones
Work Package Number 2: Genome sequence analyses for seadornaviruses and coltiviruses Start date : Month 1 Completion Date: Month 36 Partner Responsible: 2 Person months per partner: partner 2 (36 months), Total = 36 months Objectives This WP will generate full-length sequence data for the genome of representative strains from the genera Seadornavirus and Coltivirus, as well as data for conserved genome segments (Pol and T2 genes) of other strains, for RNA sequence comparisons. This will also generate cDNA clones of the genome segments analysed that will be added to the reference collection. The sequence data will be submitted to relevant databases. Phylogenetic comparisons, using new sequences and data obtained from data bases, will determine genetic relationships within the family Reoviridae and will help identify and assign new or currently unassigned viruses. Methodology and Study Materials Improved methods (recently developed) will be used for the purification of viral genomic dsRNA. The RNAs will be linked at their 3 termini, to defined oligonucleotides, then denatured and copied into cDNA using complimentary oligonucleotide primers. The cDNA will be cloned and sequenced using established technology. These methods offer significant advantages over more conventional techniques (poly A tailing and oligo dT primers), particularly in the rapid production of full-length cDNA clones from many segments of the genome. The methods are usable with genomic dsRNA from any member of the Reoviridae. Eyach virus (EYAV Fr-578), Banna virus and Kadipiro virus are currently available as prototypes for full genome sequencing. Conserved genome segments (Pol and T2 genes) from other isolates will be analysed for comparative / phylogenetic purposes. Deliverables The full genome sequence will be analysed from Banna virus and Kadipiro virus (genus Seadornavirus), as well as Eyach virus (EYAV) Fr-578 (a French isolate from the genus Coltivirus). These first full genome sequences for either genus will provide a standard to help identify other virus isolates from these genera. Data will also be generated for conserved segments (Pol and T2 genes) of other isolates, particularly from Europe. Data for complete genome segments will be submitted to relevant databases. The sequence data will be used to compare viruses and help identify the function of individual viral proteins. It is anticipated that our understanding of the genetic relationships within and between different genera of the Reoviridae will be significantly improved. This will result, for the first time, in an ability to reliably identify new or unassigned multi-segmented dsRNA viruses from RNA sequence data alone. (D4, D11, D18 & D19). Milestones
Workpackage Number: 3 Virus genome sequence analyses for aquareoviruses Start date : Month 1 Completion Date: Month 36 Partner Responsible: 4, 2 Person months per partner: partner 4 (36 months), partner 2 (2 months), Total = 38 months Objectives The objectives of this WP are to generate full genome sequence data for Aquareovirus A, together with data for conserved segments (Pol and T2 genes) of the other 5 species (Aquareovirus B to Aquareovirus F) and the 5 currently unassigned isolates. This will permit other Aquareovirus isolates to be identified by RNA sequence comparisons alone. The cDNA clones generated for those genome segments analysed will be added to the relevant reference collection. The sequence data will be submitted to relevant databases. Phylogenetic comparisons of the RNA sequences, will help to identify new or unassigned viruses and to determine genetic relationships to other viruses within the family Reoviridae. Methodology and Study Materials Improved methods (that have recently been developed) will be used for the purification of viral genomic dsRNA. The RNAs will be linked at their 3 termini, to defined oligonucleotides, then denatured and copied into cDNA using complimentary oligonucleotide primers. The cDNA will be cloned and sequenced using established technology. These methods offer significant advantages over conventional techniques (poly A tailing and oligo dT primers), particularly in the rapid production of full length cDNA clones from many segments of the genome. These methods are usable with the genomic dsRNA from any member of the Reoviridae. Striped bass reovirus (SBRV) a member of the species Aquareovirus A and the prototype strain for the genus, will be used to provide RNA for full genome sequencing. Isolates of the five other species and the 5 currently unassigned virus isolates will be used (if available) for analysis and phylogenetic comparisons of conserved genome segments (particularly Pol and T2 genes). Deliverables The full genome of striped bass reovirus (species Aquareovirus A) will be sequenced. These will provide the first full length sequence data for the genus. Data will also be generated for conserved genome segments (Pol and T2 genes) of the five other species within the genus. Data will also be generated to help identify the five currently unassigned isolates and any new isolates that become available, particularly those from within Europe. Complete data for individual genome segments will be submitted to a sequence database. Phylogenetic comparisons of these viruses will help identify the function of individual viral proteins. It is expected that the project will significantly improve in our understanding of the genetic relationships within and between different genera from the Reoviridae. This will result, for the first time, in an ability to reliably identify new or unassigned multi-segmented dsRNA viruses from their RNA sequence alone. (D16, D18 & D19). Milestones
Workpackage Number: 4 Genome sequence analyses of orbiviruses Start date : Month 1 Completion Date: Month 36 Partner Responsible: 1, 2, 3, 5 Person months per partner: partner 1 (36 months), partner 2 (6 months), partner 3 (6 months), partner 5 (6 months), Total = 54 months Objectives: This WP will generate additional sequence data (eg. for the conserved Pol and T2 genes) from uncharacterised species or unassigned viruses within the genus Orbivirus. Comparison of the RNA sequence data will clarify relationships between the established species and permit identification of unassigned or new virus isolates,. This WP will also analyse further the sequences of the serotype specific genome segment 2 of BTV and EHDV and the cell exit (NS3) gene of BTV. These studies will generate cDNA clones for each of these genome segments that will be added to the relevant reference collection. The sequence data will be added to relevant databases. Phylogenetic comparisons will also help determine genetic relationships within the family Reoviridae. Methodology and Study Materials Improved methods (that have recently been developed) will be used for the purification of viral genomic dsRNA. The RNAs will be linked at their 3 termini, to defined oligonucleotides, then denatured and copied into cDNA using complimentary oligonucleotide primers. The cDNA will be cloned and sequenced using established technology. These methods offer significant advantages over conventional techniques (poly A tailing and oligo dT primers), particularly in the rapid production of full length cDNA clones from many segments of the genome. These methods are usable with the genomic dsRNA from any member of the Reoviridae. The target viruses for these studies are the nine Orbivirus species for which no Pol or T2 gene sequences are currently available. These include Changuinola virus, Chenuda virus, Chobar gorge virus, Equine encephalosis virus, Ieri virus, Lebombo virus, Orungo virus, Umatila virus and Wad Medani virus. There are also fourteen unassigned orbiviruses, these include Andasibe virus (ANDV), Codajas virus (COJV), Ife virus (IFEV), Itupiranga virus (ITUV), Japanaut virus (JAPV), Kammavanpettai virus (KMPV), Lake Clarendon virus (LCV), Matucare Virus (MATV), Ndelle virus (NDEV), Peruvian horse virus (PHV), Peruvian rodent virus (PC21)(PRV), St Croix River virus (SCRV), Tembe virus (TMEV) and Tracambe virus (TRCV). Isolates of these viruses have already been obtained. Isolates of all of the serotypes of BTV and EHDV are also available. Deliverables The sequence of Pol or T2 genes of representative isolates of each Orbivirus species will be determined. These data will help assign new or previously unassigned orbivirus isolates to appropriate species, without serological assays. These data will also facilitate the design of serogroup specific primers for development of PCR based species specific assays. Data for complete genome segments will be submitted to sequence databases. Phylogenetic sequence comparisons will identify protein function for previously uncharacterised viruses. The project will significantly improve our understanding of genetic relationships between different Orbivirus and within the family Reoviridae. (D17, D18 & D19). Milestones and expected results
Workpackage number: 5 Virus purification, production and characterisation of antibodies for serological assays Start date : Month 1 Completion Date: Month 36 Partner Responsible: 1, 2, 4, 5 Person months per partner: partner 1 (14 months), partner 2 (6 months), partner 4 (4 months ), partner 5 (8 months), Total = 32 months Objectives The principal objective of this WP is to generate high quality antibody preparations for the development and improvement of diagnostic assays. Virus purification methods already exist for BTV and some closely related Orbivirus species. These methods will be applied to additional BTV serotypes and further developed for use with other Orbivirus species. Development of purification methods will also attempted with seadornaviruses, coltiviruses and aquareoviruses. This will permit production of antisera to these viruses, which will be evaluated for use in diagnostic assays. Monoclonal antibodies will also be generated for aquareovirus. It may also be possible to express individual viral proteins to generate antigen for antibody production. Any particles that are purified during the project would be subjected to an initial structural analysis. Methodology and Study Materials Purification methods have been developed for Bluetongue virus, providing a basis for recent structural analyses and the generation of serogroup and serotype specific antibodies. These antisera, which are used in diagnostic ELISA for BTV specific antigens and antibodies, also have high neutralising titres and are suitable for determination of virus serotype. These methods will be applied to additional BTV serotypes to improve current serotyping reagents and methods, and to other Orbivirus species (eg Equine encephalosis virus and Palyam virus) that pose an economic threat. Initial studies indicate that it may be possible to purify Seadornaviruses, since they can be grown to high titres in cell culture. This will provide material suitable for antibody production and structural analyses. However, coltiviruses only grow to low titre in currently available systems and more development work is needed. The major conserved core protein (T13) of Coltivirus will be expressed in either bacterial or eucaryotic systems (baculovirus) for the generation of serogroup specific antibodies. An attempt will also be made to purify aquareovirus particles for both structural analyses and antibody production. Monoclonal antibodies will also be generated against aquareovirus particles using established technology. The antibodies will be evaluated for use in diagnostic assays. Structural studies will include electrophoretic analyses of viral proteins, and electron microscopy. If sufficient material can be obtained, particles will be used in crystallisation trials and/or cryo electron microscopy, using of established links with virus structure laboratories. Deliverables The WP will generate additional serological reagents to increase the speed and reliability of testing for BTV serotypes, and Orbivirus species (Equine encephalosis and Palyam virus ). It will also generate particles or antigens of seadornaviruses and coltiviruses for production of antisera and assay development. Polyclonal and or monoclonal antibodies will be generated against aquareoviruses for development of diagnostic assays. Purified particles will be used in structural studies of these viruses by electrophoresis and EM. (D10, D12, D13, & D19). Milestones
Workpackage number: 6 Design and test PCR based assay systems Start date : Month 12 Completion Date: Month 36 Partner Responsible: 3, 1, 2, 4, 5 Person months per partner: partner 3 (36 months), partner 1 (6 months), partner 2 (2 months)
Objectives This WP will use previously published sequence data, and data generated during this project to design PCR based assay systems for the detection and identification of viruses from: the genera Coltivirus, Seadornavirus and Aquareovirus; different virus species within the genus Orbivirus; and different serotypes of BTV and EHDV. A PCR based assay system has recently been developed for different serotypes of AHSV, which will be evaluated further during the project. The assay systems will be evaluated for the sensitivity of detection and discrimination of the relevant virus strains using blind panels of samples and samples derived from animals in a field situation. Assays that provide a real improvement over current systems will be made available to relevant reference/ diagnostic laboratories. Methodology and Study Materials By sequence comparisons, regions of the viral genome segments that are highly conserved between isolates from the same genus will be identified and selected (coltivirus, seadornavirus, or aquareovirus) for example the Pol, T2, or T13 genes. Oligonucleotide primers will then be designed for use in PCR that specifically target these regions. Specificity will assessed by the size of the products generated, by probes or sequence analysis of the products. Nested PCR procedures may also be used. It is possible that as a consequence of species or sub group variations, it will be difficult to design a single set of primers that will work for all members of a single genus. In these circumstances multiple primer sets will be designed, which will identify members of the genus as members of separate sub groups (species). An essentially similar approach will be taken with the design of PCR based assays to detect and distinguish different orbivirus species (Pol, T2, or T13 genes) or different BTV and EHDV serotypes (VP2 gene). Deliverables The project will deliver PCR based assays for seadornaviruses, coltiviruses and aquareoviruses, to detect and distinguish these viruses from members of other genera within the family Reoviridae. The project will also develop PCR based assays that can be used to detect and distinguish the members of different Orbivirus species. The project will develop BTV and EHDV specific PCR based assays that can be used to detect and distinguish the different serotypes within these virus species. The PCR based AHSV serotype specific PCR that is already in existence will also be further evaluated. (D5, D6, D7, D8, D14, D20 & D21).
Milestones
Workpackage number : 7 Evaluate and validate diagnostic assays for orbiviruses Start date : Month 6 Completion Date: Month 36 Partner Responsible: 1, 3, 5 Person months per partner: partner 1 (12 months), partner 3 (6 months), partner 5 (10 months)
Objectives The objectives of this WP are to evaluate and validate the different test systems and reagents developed during the project for detection and identification of different orbivirus species (serogroups) and serotypes. Antisera generated against purified viral particles will be used to develop ELISA and competition ELISA that are specific for the antigens or antibodies respectively, of individual Orbivirus species. PCR assays developed during the project will also be evaluated for use in diagnostic test systems for different orbivirus species. High titre antisera raised against purified intact virus particles of different BTV serotypes will be evaluated for use in serotype specific, serum neutralisation tests. PCR will be designed to detect and distinguish genome segment 2 from different BTV and EHDV serotypes. These assays will be evaluated for routine use in the identification of the serotype of BTV or EHDV isolates. Methodology and Study Materials A vital aspect of any test system, is some measure of its specificity, sensitivity and reliability. The specificity and reliability of serological and PCR based assays and reagents will be tested against blind panels of reagents derived from the homologous virus, from related but distinct viruses, or which represent negative controls. The sensitivity of the assays will also be tested and compared to conventional assay systems (where available) using tissue and serum samples from the relevant host species. These tests will mimic the samples and storage conditions that would be experienced during routine testing. Although PCR based assays appear likely to be the most sensitive it should be noted that the three assay systems (PCR, ELISA and competition ELISA) all detect different virus specific molecules (nucleic acid, proteins/antigens and antibodies respectively). They are therefore all relevant for the testing different sample types. PCR based assays will be tested using purified RNA and samples derived from infected animals. This will help to identify appropriate methods of sample preparation and storage. PCR based assay systems for Orbivirus species, or serotypes, provide the option of further confirmation by sequence analyses of the cDNA products. This possibility relies to some extent on sequence data generated in this project. BTV specific antisera will evaluated in neutralisation assays to ensure that they have a high titre and are serotype specific. PCR based serotyping assays will be compared against neutralisation assays to confirm their relative specificity and sensitivity. Deliverables The project will deliver test systems for the orbiviruses, which have been evaluated for sensitivity and reliability. These assays will include: Orbivirus species specific ELISA for antibodies and antigens (eg. Equine encephalosis virus and Palyam virus); species specific PCR based assays; BTV serotype specific serum neutralisation tests (high titre antisera); and PCR based assays for different serotypes of AHSV (already developed), BTV, EHDV. These assay systems will be made available to relevant diagnostic and reference laboratories. (D5, D13, D14, D20 & D21). Milestones
Workpackage number: 8 Epidemiological studies of seadornaviruses / coltiviruses Start date : Month 18 Completion Date: Month 36 Partner Responsible: 2 Person months per partner: partner 2 (36 months), Total = 36 months Objectives Serological screening of sera from healthy populations (blood donors) will be performed using samples collected in western, eastern and south-eastern Europe. Molecular screening of known arthropod vectors will be undertaken using PCR, on samples also collected in western, eastern and south-eastern Europe. Tick and mosquito species originating from the same regions (mentioned above) plus parts of Africa will be screened by PCR and virus growth in cell culture to determine the identity and prevalence of virus vectors. Screening of human sera and cerebro-spinal fluids from Thai patients with undiagnosed encephalitis will be used to assess the involvement of seadornaviruses in human disease. Screening of a collection of paired human sera, from European patients bitten by ticks, will be used for coltiviruses Methodology and Study Materials Serum samples will be collected via an existing network that has been established in collaboration with the Blood Bank of Marseille. Groups of 500 sera collected from either French blood donors, from different sites across western Europe, or from sites in eastern Europe will be analysed using: I) Antigens prepared from infected cell cultures (CTFV). II) Synthetic peptides designed from the sequences of the proteins encoded by the 12th gene of CTFV and EYAV. Serum samples from Thailand (500 sera), Malaysia (500 sera) and Vietnam (500 sera) will also be tested for coltiviruses, using the same methods. These samples will also be analysed using antigens prepared from cell cultures infected with seadornaviruses (Banna virus and Kadipiro virus). Partner 2 already collaborates with the WHO Collaborative Centre on Rickettsia (Faculty of Medicine, University of Marseille). This Unit will provide large collections of tick vectors originating from Europe, Africa and Asia that will be tested for coltiviruses, using diagnostic tests developed by partner 2. Ticks and mosquitoes will be obtained from the sources described above (in collaboration with the Units of Prof. Raoult, Prof. Gonzalez and Dr Jaafar). These arthropods will be tested by PCR for the presence of viral genomes and by cell culture for infectious virus. This will detect and help identify the virus vectors. Human sera and cerebro-spinal fluids from 100 Thai patients with undiagnosed encephalitis will be tested for seadornaviruses, to assess their involvement in human disease. All samples included in the study have already been tested for Dengue virus, Japanese Encephalitis virus, enteroviruses and alphaviruses. Serum and CSF samples will also be tested by serological methods and PCR. Paired sera from 200 European patients who have been bitten by ticks and developed febrile illnesses but have tested negative for tick-transmitted organisms , will be tested for coltiviruses using serological methods. In cases where the tick involved can be obtained it will also be tested by PCR and virus isolation procedures. Deliverables All testing of European samples and Thai samples will be completed during the first year of the study. The testing of Malaysian, Vietnamese and African samples will be completed during years 2 and 3 of the project. (D22). Milestones
Workpackage number: 9 Epidemiological studies of Aquareoviruses Start date : Month 18 Completion Date: Month 36 Partner Responsible: 4 Person months per partner: partner 4 (34 months), Total = 34 months Objectives The project will design, test and validate two complementary diagnostic tools for aquareoviruses. a PCR based method using nucleotide sequence information corresponding to the six known Aquareovirus genotypes (species); and ii) an ELISA test based on the use of mAbs developed against each genotype. The project will also. Conduct an epidemiological survey to determine the presence of Aquareoviruses in samples from wild and Aquacultivated fish and mollusc species. Methodology and Study Materials A representative member of each of the six Aquareovirus genotypes (species) will be propagated in vitro, using established fish cell lines, to prepare virus stocks for further work. The sequence of the genome segment coding for the RNA polymerase of a representative member of each of the six genotypes / species, will be isolated and used to generate cDNAs that will be cloned and sequenced using standard protocols. Sequence comparisons will be carried out. The results of this study will be used to design of a series of PCR primers for the specific amplification of Aquareovirus sequences. Primers will be also designed to allow discrimination between different genogroups / species. The usefulness of the different primers will be experimentally tested under laboratory and field conditions. Purified virus stocks will be used to immunise Balb-C mice. Spleen cells from the immunised animals will be used to generate hybridoma cell lines, producing specific mAbs against the different genogroups. The specificity of these antibodies will be determined by immunofluorescence, Western blot, and ELISA. Appropriate mAbs will then be used to develop diagnostic ELISA for detection of aquareoviruses in field samples. The PCR and ELISA based diagnostic tests for aquareoviruses, that are developed during the project, will be used to carry out a preliminary epidemiological survey. This will assess the incidence, prevalence and impact of aquareoviruses in Europe. This survey will be conducted using samples from wild and aquacultivated fish and from mollusc species obtained from different European countries. Deliverables The complete nucleotide sequence for Pol and / or T2 genes will be determined for all Aquareovirus genotypes / species, allowing any subsequent isolates to be more rapidly identified from their RNA sequences alone. A PCR-based diagnostic test will be developed for the detection of Aquareoviruses. A reference collection will be established of virus strains and mAbs that are specific for each Aquareovirus species (genotype). Diagnostic ELISA will be developed for the detection and identification of aquareoviruses. These assay systems will be used to conduct a preliminary epidemiological survey of Europe for Aquareoviruses in both wild and aquacultivated species. ( D23). Milestones 9.1 Conduct an epidemiological survey for the prevalence and impact of aquareoviruses
Workpackage number: 10 Set up and maintain web sites Start date : Month 1 Completion Date: Month 36 Partner Responsible: 1, 2, 4 Person months per partner: partner 1 (2 months), partner 2 (2 months), partner 4 (2 months)
Objectives The objectives of this work package are to set up permanent web sites for the collection and dissemination of molecular biological data concerning the different viruses within the family Reoviridae. Initially sites will be developed for BTV (as the prototype orbivirus), Eyach virus (as a representative coltivirus), Banna virus and Kadipiro virus (as representative seadornaviruses) and for striped bass virus (as a representative aquareovirus). Other collaborating scientists (from outside the project) will be invited to set up additional linked sites for other genera and species of dsRNA viruses that will be linked.
Methodology and Study Materials The sites will also cite a number of the most recent and relevant references. There will be a single responsible editor for each site, who will regularly update the information it contains (every six months). The sites will contain data concerning genome and genome segment sizes and nomenclature, RNA coding and non-coding regions, accession numbers, protein size, function and structural roles. The sites will be constructed using Microsoft Internet Information server (IIS) soft-wear and Microsoft Front Page 98. A Compac Alpha, NT4 server is currently available for these sites.
Deliverables
Milestones
2.3 Project structure, planning and timetable Coordinator : Contact Person : Dr Peter P. C. Mertens Participant 1 The Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey, GU24 ONF UK
Phone +44(0) 1483 232441 Direct line and voice mail +44(0) 1483 231017 Fax +44(0) 1483 232448 e-mail peter.mertens@bbsrc.ac.uk ____________________________________________________________________ Participant 2 Contact Person : Dr. Xavier de Lamballerie Unite des Virus Emergents, Faculte de Medecine de Marseille, 27 Bd Jean Moulin F13005 Marseilles Cedex 05 France
telephone: +33 (4) 91 32 45 53 fax: +33 (4) 91 32 44 95 e-mail xndl-virophdm@lac2.gulliver.fr ____________________________________________________________________ Participant 3 Contact Person : Dr. StJ phan Zientara Agence Francaise de Securite Sanitaire des Aliments 22, Rue Pierre Curie, Boite Postal 67, Maisons Alfort Cedex 94703 France
elephone: +33 (1) 4977 1300 fax: +33 (1) 4368 9762 e-mail s.zientara@afssa.fr ____________________________________________________________________
Participant 4 Contact Person : Dr. Jose F. Rodriguez Departamento de Biologia Molecular y Celular Centro Nacional de Biotecnologia (C.S.I.C.) U.A.M. Cantoblanco, 28049 Madrid SPAIN
telephone +34 (91) 5854558 fax +34 (91) 585450 e-mail jfrodrig@cnb.uam.es ____________________________________________________________________
Participant 5 Contact Person : Dr. Maria Koubati-Artopiou Laboratory of Microbiology and Infectious Diseases, Faculty of Veterinary Medicine, AristotleUniversity, 54006 Thessaloniki, Greece.
telephone +30 (31) 999 924 fax +30 (31) 999 959 e-mail koumbati@vet.auth.gr
Nature of the deliverable: R = Report, P = Prototype, D = Demonstrator, O = Other. Dissemination levels: PU = Public, RE = Restricted to a group specified by the consortium (including the Commission Services). CO = Confidential, only for members of the consortium (including the Commission Services). Page
Co-ordinator / Participant 1 The Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey, GU24 ONF UK http://www.reoviridae.org/index.html peter.mertens@bbsrc.ac.uk Co-ordinator / Orbivirus group Leader: Dr Peter P. C. Mertens Team Members Mr J. N. Burroughs (Scientist) Dr Philip Strachan (Scientist) Post doctoral Scientist (to be appointed) Arbovirus Group Leader: Dr. Philip Mellor Mr Chris Hamblin (Scientist) Research Assistant (to be appointed) Division of Epidemiology Mr Nick Knowles Major objectives
Contractual Links to other Participants None Workplan The specific details of the materials and methods are specified in each of the work packages (see above) the person months by this participant are as follows. Workpackages: WP1 (6 months), WP4 (36 months), WP5 (14 Months), WP6 (6 months), WP7 (12) WP10 (2 months). Deliverables Title Delivery Month D1 Set up initial orbivirus reference collection and storage 12 D5 Test PCR based assay for AHSV serotypes 12 D9 First year report 12 D10 Purify viruses and carry out initial structural analyses 24 D12 Generate polyclonal and or monoclonal antibodies to viral proteins 24 D13 Develop serological based assays 24 D14 Design and test PCR based assay for orbivirus species 24 D15 2nd year report 24 D17 Generate cDNA clones and sequence data for representatives of orbivirus species 36 D18 Phylogenetic comparisons of RNA sequences 36 D19 Add viruses cDNA clones and antibodies to reference collections 36 D20 Design and test PCR based assay for EHDV serotypes 36 D21 Design and test PCR based assay for Bluetongue virus serotypes 36 D24 Set up and maintain web site for BTV molecular biology data 36 D29 Set up and maintain web site for molecular biology data of other dsRNA viruses 36 D30 Final report 36
Participant 2 : Unite des Virus Emergents, Faculte de Medecine de Marseille, 27 Bd Jean Moulin, F13005 Marseilles cedex 05, France. Xndl-virophdm@lac2.gulliver.fr
Scientific team : Team Leader Dr. Xavier de Lamballerie Dr. Houssam Attoui Post doctoral Scientist (to be appointed) Major objectives
Set up websites for the coltiviruses and seadornaviruses to disseminate current information on molecular biology / structure. Contractual Links to other Participants None Workplan The specific details of the materials and methods are specified in each of the work packages (see above ) the person months by this participant are as follows. Workpackages: WP1 (6 months), WP2 (36 months), WP3 (2 months), WP4 (6 months), WP5 (6 months), WP6 (2 months), WP8 (36) WP10 (2 months). Deliverables Title Delivery Month D2 Set up initial reference Seadornavirus / coltivirus collection and storage 12 D4 Generate cDNA clones and a full genome sequence for a representative coltivirus 12 D6 Design and test PCR based assay for coltivirus 12 D7 Design and test PCR based assay for seadornavirus 12 D9 First year report 12 D10 Purify viruses and carry out initial structural analyses 24 D11 Generate cDNA clones and a full genome sequence for a representative Seadornavirus 24 D12 Generate polyclonal and or monoclonal antibodies to viral proteins 24 D13 Develop serological based assays 24 D15 2nd year report 24 D17 Generate cDNA clones and sequence data for representatives of orbivirus species 36 D18 Phylogenetic comparisons of RNA sequences 36 D19 Add viruses cDNA clones and antibodies to reference collections 36 D22 Epidemiological survey for Seadornaviruses and coltiviruses 36 D26 Set up and maintain web site for Coltivirus molecular biology data 36 D27 Set up and maintain web site for Seadornavirus molecular biology data 36 D30 Final report 36
Participant 3 Agence Francaise de Securite Sanitaire des Aliments, 22 Rue Pierre Curie, Boite Postal 67, Maisons Alfort Cedex 94703, France s.zientara@afssa.fr
Scientific team: Team Leader Dr Zientara Stιphan, Veterinary Officer, DVM, PhD
Major objectives
Contractual Links to other Participants None Workplan The specific details of the materials and methods are specified in each of the work packages (see above) the person months by this participant are as follows. Workpackages: WP4 (6 months), WP6 (36 months), WP7 (6). Deliverables contributed to Deliverables Title Delivery Month D5 validate PCR based assay for AHSV serotypes 12 D9 First year report 12 D13 Develop serological based assays 24 D14 Design and test PCR based assay for orbivirus species 24 D15 2nd year report 24 D17 Generate cDNA clones and sequence data for orbiviruses (VP2, T2, Pol genes) 36 D18 Phylogenetic comparisons of RNA sequences 36 D20 Design and test PCR based assay for EHDV serotypes 36 D21 Design and test PCR based assay for BTV serotypes 36 D30 Final report 36
Participant 4 Departamento de Biologia Molecular y Celular Centro Nacional de Biotecnologia (C.S.I.C.) U.A.M. Cantoblanco, 28049 Madrid, SPAIN
Scientific team : Team Leader Dr. Jose F. Rodriguez, Assistant Professor. Dr. Eleuterio Lombardo. Postdoctoral Researcher. Dr. Dolores Gonzαlez. Research assistant. Post doctoral Scientist (to be appointed) Major objectives
Contractual Links to other Participants None Workplan The specific details of the materials and methods are specified in each of the work packages (see above) the person months by this participant are as follows. Workpackages: WP1 (6 months), WP3 (36 months), WP5 (4 months), WP6 (2 months), WP9 (34) WP10 (2 months). Deliverables Title Delivery Month D3 Set up initial Aquareovirus reference collection and storage 12 D8 Design and test PCR based assay for Aquareovirus 12 D9 First year report 12 D10 Purify viruses and carry out initial structural analyses 24 D12 Generate polyclonal and or monoclonal antibodies to viral proteins 24 D13 Develop serological based assays 24 D15 2nd year report 24 D16 Generate cDNA clones and a full genome sequence for a 36 representative aquareovirus. D18 Phylogenetic comparisons of RNA sequences 36 D19 Add viruses cDNA clones and antibodies to reference collections 36 D23 Epidemiological survey for incidence, prevalence and 36 impact of Aquareovirus D28 Set up and maintain web site for Aquareovirus molecular biology data 36 D30 Final report 36
Participant 5 Laboratory of Microbiology and Infectious Diseases, Faculty of Veterinary Medicine, Aristotle University, 54006 Thessaloniki, Greece. e-mail orestisp@vet.auth.gr
Scientific team: Team Leader -M. Koumbati (DVM, PhD) /Ass. Professor / Virology infectious diseases
Major objectives
Contractual Links to other Participants None Workplan The specific details of the materials and methods are specified in each of the work packages (see above ) the person months by this participant are as follows. Workpackages: WP4 (6 months), WP5 (8 months), WP6 (12 months), WP7 (10)
Deliverables contributed to: Deliverable Title Delivery Month D9 First year report 12 D13 Develop serological based assays 24 D14 design and test PCR based assay for orbivirus species 24 D15 2nd year report 24 D17 Generate cDNA clones and sequence data for representatives of orbivirus species 36 D18 Phylogenetic comparisons of RNA sequences 36 D19 Add viruses cDNA clones and antibodies to reference collections 36 D20 Design and test PCR based assay for EHDV serotypes 36 D21 Design and test PCR based assay for Bluetongue virus serotypes 36 D22 Epidemiological survey for Seadornaviruses and coltiviruses 36 D30 Final report 36
4. PROJECT MANAGEMENT AND COORDINATION The project co-ordinator is responsible for the overall project management. The co-ordinator will keep each participant informed of the work in progress, will call for meetings, will be responsible for ensuring that the achievement of tasks and with the assistance of participants will prepare and collate progress and final reports. A project management team will be assigned. This team, which will consist of one member of each of the participants (usually the team leader), will be responsible for specific work-packages and for achievement of the aims and milestones that are in these work-packages. At the start of the project a meeting will held to discuss necessary arrangements and to co-ordinate the priorities to be pursued within the different laboratories, collaboration between them, exchange visits of personnel and exchange of techniques and reagents. Additional co-ordination meetings of the whole partnership will be organised every six to twelve months throughout the project, to co-ordinate the organisation of the work, to evaluate the achieved milestones, exchange results and data and to prepare reports. At each project meeting the partners will present their work, and explain their progress towards the expected milestones. Short-term visits between partners will be actively promoted, to provide access for all groups to the different expertise of each participant and to ensure the exchange of improved technology, reagents and data. Each participant will supply a concise written report on the progress of their research, including specific information relating to progress towards and achievement of milestones, to the co-ordinator at least 14 days prior to each meeting. The co-ordinator will send copies of each report to all partners within 7 days of receipt. Together with a report concerning the meeting and the discussions held these reports from individual partners will form the basis of an annual appraisal of the project. Hard copies of these reports as well as electronic versions on disc will be kept by the co-ordinator. Progress and final reports will be sent to the European Commission . All decisions taken concerning the structure and future direction of the project will be decided on the basis of full discussion and a consensus between all partners. Scientific reports concerning the work of the partnership will be published whenever possible in refereed scientific journals. In addition as a central part of this project web sites will be established and maintained for the collection and dissemination of molecular biology data concerning the viruses under study and the work of the partnership. The management and planning of the project will, follow approximately the pattern shown below. Meetings will be arranged in Greece, France, Spain and the UK every six to twelve months. The co-ordinator will also make visits to each participant laboratory.
Reo ID: General Management and Planning
The time spans for each of the proposal Work Packages, broken down into a number of Tasks, together with timings of milestones for Years 1, 2, and 3 of the proposal are as set out in Figure 2. The relationships of the different tasks involved in these work-packages are show in Figure 1. A MidTerm Review Meeting, organised by the Co-ordinator, shall take place between representatives of all of the Participants and the Commission. The Commission may be represented by independent experts who shall be subject to confidentiality agreements. The meeting shall take place between the 18th and 24th month of the project. The co-ordinator shall agree with the commission the date, agenda and the participants for the meeting at least two months in advance of the meeting. At this time progress in each of the completed and of all under-way actions, will be assessed by delegates from each involved partner, in the light of the milestones. The track record of each partner, in respect of their individual areas of responsibility, will also be assessed. Shortfalls in achievement will be examined in
Figure 2: Participants Contribution and Timetable
Figure 3 : Timing of Tasks and Workpackages
5. Exploitation and Dissemination Activities The findings of this proposal will be forwarded to the Commission in the form of 6-monthly or annual progress reports and a final report drawing together all of the main conclusions and results. As appropriate the findings will also be presented at national and international scientific meetings, trade conventions, via the media and as scientific publications or popular articles. The project will also include as a major aim the establishment and maintenance of web sites for the collection and dissemination of molecular biology data concerning these viruses. It is intended that these sites will include data generated by the partners. The co-ordinator shall provide, by contract signature, a one page publishable summary of the project , which can be easily disseminated and distributed to the public. In addition, not later than the first report , the co-ordinator shall provide to the Commission a publishable poster targeted to a non specialist audience and summarising the main features of the entire project. During the course of the project further dissemination of the progress of the work will be encouraged by contribution to such of the Commissions own organs of publication as may be deemed appropriate to ensure that findings of likely practical benefit reach the end users (farmers, national veterinary services, legislators, government departments, international animal health organisations) with the minimum of delay. Contractors shall submit at, or before the end of the Project a technology implementation plan acceptable to the Commission. This plan should indicate all potential foreground rights and exploitation intentions. It shall include a summary of the project , the forecast of the intentions of each contractor and description of achievements regarding use of the knowledge (including timetable). The RNA and protein sequence data generated during the project will be freely shared between participating laboratories. This will be necessary for the collaboration on design, testing and validation of PCR based assay systems by the partnership. Reagents generated and techniques developed during the project will also be exchanged freely between partners. The assay systems developed for the different viruses under study will be used primarily within the laboratories of the partners, or any other laboratories that have facilities and are approved for handling, diagnosis and research concerning these viruses. However the number of such laboratories within the community is limited and widespread use of these assay systems is not anticipated at this stage. The partners include regional reference centres for orbiviruses and laboratories with established track records for diagnosis or research of the genera of viruses under study. The partners will adopt a consortium policy on the sharing of any benefits that result from the project.
6 . ETHICAL ASPECTS AND SAFETY PROVISION The majority of the work detailed in this project is carried out by experimentation in vitro. However, some aspects of the project require use of experimental animals (rabbits, guinea pigs and mice) for preparation of antibodies. 11-day-old embryonating hens eggs will also be used, if required, for the isolation of viral agents from field specimens. In such an event the British Governments 1986 Animal Scientific Procedures Act (ASPA), which is internationally recognised as being the strictest legislation of its type in the world, will be complied with in all details. The use of embryonating hens eggs for virus isolation has already been authorised by the local ethical review process of Partner 1 (IAH). The appropriate licences for animal handling and generation of antibodies have already been obtained. At IAH these include license number PPL 70/5010 (Production of polyclonal antibodies against viruses transmitted by Arthropods). Licence numbers for personnel at IAH include PIL 90/00435 (licence holder: Chris Hamblin) and PIL 90/00269 (licence holder: Philip Mellor). The work described in the project is specifically designed to alleviate or prevent several animal diseases of international significance. It will thereby significantly promote animal health and welfare. In so doing it will have a beneficial financial effect, particularly upon the small farmer in rural communities whose animals are often the most severely affected by disease, but it will also benefit all aspects of those industries dependent upon the livestock industry, including enhancing trade by the EU as a whole. Additionally, it will facilitate the implementation of more targeted disease control strategies with reduced, adverse environmental impact. Consequently, there are no philosophical practical, or ethical dilemmas involved in carrying out the work described here - the results of this study will be wholly beneficial.
The work outlined in this project (partner 2) will involve use of diagnostic assays of human serum samples for detection of virus specific antibodies or antigens, or for viral nucleic acid. These samples will be obtained and the studies will be carried out entirely under qualified medical supervision and will comply fully with the appropriate ethical medical and safety regulations of France. Specify if your project involves:
The co-ordinator shall implement the research project in full respect of the legal and ethical national requirements and code of practice. Wherever authorisations have to be obtained from national bodies, these authorisations shall be considered as documents relevant to the project under article 27 of Annex II of the contract. Copies of all relevant authorisations shall be submitted to the Commission prior to commencement of the research project.
Safety is a prime consideration at all five of the participating laboratories. All procedures initiated will conform with and be subjected to project licences granted under national agreed legal requirements. In addition all of the laboratories have on site safety regulation and work schedules which have to be followed. The work described does not involve the creation or release of any genetically modified organisms. Transport of viruses between laboratories will be in strict accordance with procedures previously agreed with the appropriate government agencies, with relevant permission and agreements obtained at the time of shipment. The orbiviruses that may be used or isolated from the field during the course of this project will be conveyed to or from the laboratories of Partner 1 (IAH) under MAFF approved procedures, and under the authority of the British Governments Animal Health Act (Importation of Animal Pathogens Order 1980, Licence No. AHZ/1309/A).
All envisaged virological work to be carried out in the high security laboratories that form part of the facilities of each of the different Partners. Those at IAH Pirbright are covered by the MAFF Specified pathogens Order 1998.
The Co-ordinator shall take all measures to assure that appropriate environmental safety provisions are fulfilled in the course of the project by all contractors, particularly those related to the deliberate release of genetically modified organisms. In addition the co-ordinator shall take all measures to assure all contractors that when dealing with biological material, strict safety procedures are in place in compliance with national and EU regulations on biosafety. All work must be carried out in compliance with national and EU regulations on safety.
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