Results - Summary of the main objectives of the project

Objectives 1, 9 and 10 will be summarised during the second or third year as they contain long-term tasks that going to be fulfilled on the later stages of the project.

Objective 2: To develop and apply nucleic acid detection techniques without thermocycling, with special regards to Invader and NASBA methods (work performed within WP3 of the technical annex).

During this reporting period existing alignments for ASFV, CSFV and FMDV have been improved (Partner 2) and new ones have been made for NDV and HPAIV (Partner 5). An existing ASFV Invader assay have been further characterised and four new CSFV specific Invader assays designed and complementary RNA control cloned and transcribed. The CSFV Invader assays were manufactured by Third Wave Technologies, Madison, Wisconsin, USA. NASBA oligos and molecular beacons have been designed and purchased.

Main achievements: i, All necessary alignments have been done; ii, CSFV Invader assays and appropriate positive RNA controls are ready; iii, AIV/NDV NASBA assays are designed and are being tested.

Objective 3: To develop and evaluate novel nucleic acid detection methods (padlock probes and rapid readout formats) in order to elaborate a new generation of microarrays (work performed within WP4 of the technical annex).

A preliminary set of padlock probes has been designed and synthesized. The variability of the FMDV genome was found to be so extensive that only four general probes have been designed to recognize all 139 strains identified in WP4.1. An alternative strategy for the FMDV serotyping is under investigation. Nine padlock probes have been designed for SVDV, and 23 for VSV. All padlock probes can be amplified using the same PCR primer pair, and each is equipped with a unique tag-sequence that allows the amplification products to be sorted on an oligonucleotide array. A detected signal from a fluorescently labelled PCR primer will reveal what (if any) virus was present in the sample, and in the case of VSV, what serotype.

A preliminary protocol for ligation and amplification of the padlock probes described in WP4.2 has been established. The work performed in collaboration with subcontractor to Partner 1, OLINK AB. A new polymerase that specifically amplifies only circular probes, leaving the remaining linear probes unamplified has been introduced. This procedure allowed a substantial reduction in the number of subsequent PCR cycles, which are the main source of background in the assay. Thus, the sensitivity has been increased by a factor of approximately 100, compared to the previous protocol. The new protocol will be used in the analysis of cDNA prepared from FMDV, SVDV and VSV samples.

Objective 4 (and partly Objective 1): To develop novel real-time PCR assays for detection of OIE List A viruses (work performed within WP5 of the technical annex).

Primer and probe pairs for further development and application of PriProET have been selected. P3 have tested the PriProET technology in a one-step RT-PCR set-up. Real-time PCR detection of RNA viruses using the PriProET technology is dependent on preceding cDNA synthesis. By development of one-step PriProET RT-PCR assays for CSFV and FMDV the assay set-up is markedly simplified, which makes such assays more suited for automation and also for adaptation to portable PCR machines. Primers and probe for the detection of SVDV were designed by P1 and PriProET real-time PCR assay was developed and optimised.

A MGB probe assay was designed for AFSV as an alternative to the LUX primers given their poor performance. This assay is fully optimised and only requires further testing on alternative strains and specificity testing. An FMDV assay has been redesigned again using the Qiagen Quantiprobe software. This assay works well on DNA target and is currently being optimised for RNA. The design process for assays to detect CSFV is well underway. An FMDV and ASFV LUX primer set has been designed with the Invitrogen design software and is currently being tested. Initial optimisation on plasmid DNA shows low amplification and high background. The work is ongoing.

An AIV and a NDV detecting LUX assay has been developed and optimised.

Objective 5: To adapt the real-time PCR systems to portable PCR equipment, as further support for the “first line” diagnosis (work performed within WP5 of the technical annex).

P3 have searched the market for portable PCR machines suitable for first-line PCR detection of OIE List A viruses. Alternatives are the SmartCycler from Cepheid and the R.A.P.I.D. cycler from Idaho Technologies. Recently, the latter company also launched a new portable real-time PCR machine called RAZOR, which seems to be the lowest price.

Objective 6: To simplify antigen-antibody assays, with special regard to improvement of sensitivity and simplification of Ag-ELISA systems (work performed within WP6 of the technical annex).

Viruses considered in this objective include FMDV, serotype A, avian influenza viruses, types H5, H7 and swine influenza viruses. A set of MAbs cross-reactive with 4 to all 7 FMDV serotypes had been identified (MAbs originated from various fusions of splenocytes from mice immunised with different FMDV serotypes). MAbs specific for FMDV type SAT 1 and SAT 2 are available. Preliminary characterisation indicates that they include MAbs showing various patterns of reactivity, representative of different target epitopes. Production of MAbs to SAT 3 still needs to be enhanced. The characterisation of SAT 1 and SAT 2 neutralising MAbs will continue, through the selection and sequencing of neutralisation escape-mutants, that will allow the differentiation and the precise mapping of the target antigenic sites.

The approach pursued by P7 is the development of sandwich ELISA using MAbs as coating and conjugated antibody for FMDV antigen detection. The number of MAbs evaluated as catching and/or as conjugated antibody and the numbers of generated combinations (overall 186 different combinations). Results show that four FMDV serotypes can simultaneously detected by the various MAbs combinations. MAbs pan-reactive with the seven FMDV serotypes do not work as catching antibody, probably because the target epitopes are not exposed or because virus bound by these MAbs is not accessible to other MAbs. Due to this drawback, the possibility to develop a universal FMDV detection ELISA is decreasing; however, several MAbs combinations work efficiently for the detection of at least 4 types simultaneously, so that the objective to develop a pan-reactive ELISA could still be achieved by mixing MAbs selected to cover the complete spectrum of FMD viruses. Development of MAbs against FMDV serotypes SAT 1,2,3 and H5, H7 influenza viruses is ongoing. MAbs common to all A type AIV and MAbs specific for H5, H7, H3, H1 were identified. Among MAbs common to any influenza viruses, two MAbs against the NpA, with high titres and affinities, were selected to develop sandwich ELISAs suitable for the detection of all type A influenza viruses, originated from both avian and swine species. Each ELISA uses the same MAb as catching and conjugated antibody.

Objective 7: To develop simple dip-stick tests for the rapid field or abattoir-diagnosis of OIE List A diseases (work performed within WP7 of the technical annex).

Partner 6 has collected MAbs for FMDV, CSFV, SIV, NDV, HPAIV and VSV from Partner 7 and 9. Devices to FMDV and VSV has been put together and tested by Partner 6 and Partner 9 (FMDV, VSV) and Partner 7 (FMDV) with positive results. The MAb recognizing strain Indiana 1 have been used in a dip-stick which was evaluated with inactivated viral suspension and on live material at the location of Partner 9 by Partner 6. The results showed specific positive result on tested samples. The received specific to CSFV MAbs has been used to prepare devices with all possible combinations of membrane/latex MAbs. The combinations have been tested for any unspecific reaction in negative samples. No inactivated viral culture exists, thus Partner 6 has no ability to test these sticks that has to be evaluated by Partner 3 and 7.

Objective 8: To automate the virus-detection procedures, in order to accelerate and simplify the diagnosis (work performed within WP5 of the technical annex).

P9 has compared the ability two automated robots (BR9604, Qiagen and MagNA PURE LC, Roche) to extract nucleic acids from different biological matrices. A decimal titration series of FMDV was prepared by spiking the virus into different sample types. The relative amounts of FMDV in the prepared nucleic acid samples were determined using real-time RT-PCR. In general, results showed similar performance (identical limit of detection) of both robotic methods with all the lysis buffer conditions tested for FMDV samples in cell culture media and serum.

Objective 11: To initiate and maintain a global dissemination programme aimed at animal health services and authorities, including practitioners, field laboratories, abattoirs, central laboratories and policy makers (work performed within WP9 of the technical annex).

Contacts between Animal Health Staff in different countries of Central and South America (Costa Rica, Panama, Belice, Nicaragua and Argentina) as well as with OIRSA organization (Organismo Internacional Regional de Sanidad Agropecuaria) present in all Central America countries, Africa (Morocco and Algeria) Asia (China) and Middle East (Iran) was established. Several conversations have been taken place with the Research Center Borstel in Germany (Subcontractor to Partner 4) to establish the connection with other countries.

A workshop is planed to organize in China for the dissemination of knowledge emerging from the LAB-ON-SITE project. The time and the place of this meeting should be determined later after the establishing the information flow with Professor JM. Sánchez-Vizcaíno (P4). The objectives of the meeting are creation of regional cooperation, analysis of livestock diseases situation and improvement of integrated disease control measures through distribution and implementation of novel diagnostic and epidemiological tools.