Upload
others
View
1
Download
0
Embed Size (px)
Citation preview
2 DTU Vet, Technical University of Denmark
Overview EU-funded projects
Project Species covered by the project Area covered by the project
Human Pig Poultry Other species Diagnostics
ANIHWA No No Yes No High
AIV VACC DIAGNOSIS No No Yes No High
AVIFLU No No Yes No High
ConFluTech No Yes Yes Yes Medium
EPIZONE Yes Yes Yes Yes Low
ESNIP2 No Yes No No High
ESNIP3 No Yes No No High
FLUAID No Yes Yes No High
FLU-LAB-NET No No Yes Yes High
FLUTEST No No Yes Yes High
FLUTRAIN No Yes Yes Yes High
INN-FLU No No No Yes Low
LAB-ON-SITE Yes Yes Yes Yes High
NEW-FLUBIRD No No No Yes Medium
3 DTU Vet, Technical University of Denmark
AIV VACC DIAGNOSIS (Dec 2006 – Nov 2010)
Development and validation of a diagnostic marker test (antibodies) for poultry that can be used in conjunction with marker or conventional vaccines.
AVIFLU (Oct 2002 – Sept 2006)
Development and validation of rapid diagnostic tests for poultry (AI NP ELISA and H5, H7 and M RRT-PCR)
LAB-ON-SITE (Nov 2004 – Jan 2008)
Development and validation of molecular diagnostic methods and improved ELISAs for both antigen and antibody detection for both AI and SIV
ESNIP2 (Jan 2006 – Dec 2008) and ESNIP3 (Nov 2010 – Oct 2013)
Harmonization of diagnostic tests (HI, NT, RT-PCR)
Standard protocols and reagents disseminated for virus detection, isolation and subtyping. Ring trial performed to confirm detection of all relevant SIV subtypes
4 DTU Vet, Technical University of Denmark
FLUAID (Jan 2006 – Oct 2010)
Development and validation of a diagnostic tests (anti-N ELISAs) for poultry that could be used in association with N-heterologous vaccines in a DIVA strategy framework.
The project also confirmed that the recommended molecular diagnostic procedures for AI diagnosis (EURL) could be transferred to portable PCR instruments that can be employed at the penside.
FLUTEST (Feb 2007 – Jan 2011)
The project developed and evaluated a range of novel molecular based tests for the rapid detection of AI and a range of ELISAs for detection of antibodies (including DIVA, use of egg yolk)
FLUTRAIN (Mar 2007 – May 2011)
The project aimed at training and transfer of AI diagnostics and disease management systems to developing countries.
Genetic characterization of influenza A viruses collected from avian species, sequences deposited in public databases
State of the art on animal influenza diagnostics
Ian Brown Animal and Plant Health Agency, Weybridge, UK
EURL for AI; OIE/FAO ref lab for AI/SI; OFFLU partner
Diagnostic methods for influenza in poultry
• Based upon EU AI Diagnostic Manual(Decision 2006/437/EC) reviewed annually
– Closely relates to OIE Terrestrial manual for Diagnostic Tests
• Test requirements
– Reliable and fit for purpose; DSp, DSe known
– Robustly validated
– Cost effective, ease of use
– Readily harmonised and can be applied different lab
settings
– In scope for QA schemes
Diagnostic methods for influenza in poultry
• EU AI Diagnostic Manual
– Covers wide range of scenarios from suspicion to
freedom testing
• Report Case: minimum sampling per epi
group/sample type
– 5-10 carcases
– 20 oropharyngeal swabs; 20 cloacal swabs; 20 bloods
• ‘Source and spread’ investigations
– Sampling modified according to virus/epidemiology
• Application to wild bird investigations
Laboratory diagnostic strategy
• Primary outbreak with NAI
– PCR, Virus isolation, HI, NI, IVPI, gene sequencing
• Secondary outbreaks
– PCR screening, limited VI, gene sequencing inc molecular epidemiology, virus pathotyping
• Surveillance
– Virological and serological, host related
Detection of
influenza A virus
Step
2
Step 3
Classical pathway
Virus isolation
(embryonated fowl
eggs)
Molecular
pathway
M-gene PCR
Serological
Characterisation
HI (H5, H7)
Step 1
Specific identification of
avian influenza virus
HA-gene PCR
(H5, H7)
Animal experiment
(IVPI)
Sequencing of
HA-cleavage site
Positive Negative
Pathotyping
LPAI HPAI
Figure 1: Schematic overview of diagnostic steps for confirmation of AI
Newly applied technologies/tools
• Molecular pathway – Real time PCR’s in use from FP5/6
– Microarray applications
– Genomics – ie NGS
• Classical pathway – Alternative substrates to eggs
• Serology – Range of ELISA’s
– Ability to define antigenic properties with cartography
Diagnostic methods for influenza in swine
• Sample types/frames – Typical mammalian tropism
• Molecular tools – Pan PCR (realtime) – Subtype specific – Full genome : Sanger or NGS
• Classical – Virus culture in cells or eggs – Conventional typing HI/NI
• Serology – Pan ELISA; Subtype specific – HI subtype specific
Where are we in harmonization of diagnostic methods, sharing of data, data analysis … at a global
level but in particular within the EU?
• Harmonisation
– EU Avian: high – manual; annual proficiency amongst EU NRLs; standard reagents; ongoing fitness for purpose coordinated through EURL
– Global Avian: OFFLU – OIE manual; standards; proficiency tests; limitations to one size fits all
– EU Swine: recommended protocols; harmonised and proficiency tested in ESNIP3 (finished Oct 2013) ;no ongoing activity
– Global swine: OFFLU – OIE manual; standards; proficiency tests; limitations to one size fits all
– EU/global: Other species no systems other than equine sector – Cross host utility exploited
Where are we in harmonization of diagnostic methods, sharing of data, data analysis … at a global
level but in particular within the EU?
• Data analyses/sharing
– EU Avian: network comms via web (FlulabNet maintained by EURL after FP6 project finished); partner to partner; analyses timely; EU reports/annual meetings of network; rapid virus sharing; emergency mode responses; sequence deposition ie GISAID; publications in scientific literature; EFSA, ECDC
– Global Avian: OFFLU sub-group range of technical and review activities; Close interaction with public health ie VCM, H5 evolution WG, sequence deposition, virus and reagent sharing
Where are we in harmonization of diagnostic methods, sharing of data, data analysis … at a global
level but in particular within the EU?
• Data analyses/sharing
– EU swine: no coordinated structure – institute level; ESNIP3: sequence deposition ie GISAID; publications in scientific literature; EFSA, ECDC
– Global Swine: OFFLU sub-group range of technical and review activities; Close interaction with public health; sequence deposition and genomic analyses, virus and reagent sharing; antigenic cartography (global maps)
New developments in diagnostic methods
• What is in the pipeline?
• Molecular pathway – Faster Genomics – inc analyses, bioinformatics – Microarray applications?
• Classical pathway – Alternative substrates to eggs – Review of in-vivo methods
• Penside – ??
• Serology – Enhanced/expanded antigenic cartography – Use of arrays
17 DTU Vet, Technical University of Denmark
Knowledge and data gaps based on previous initiatives
• Integrated and multiplexed rapid molecular tests (both penside and high-throughput lab tests) to detect and characterize all influenza viruses timely and cost effectively.
• Better serological tests to determine the subtype specificity of antibodies (i.e. to identify what subtype an animal has been infected with) and to determine antigenic characteristics of influenza viruses for improved evaluation of cross reaction/protection between viruses/vaccines.
• Improve virus recovery methods relating to sample sources, quality and virus isolation
• Integration of diagnostic methodologies with surveillance (real time data exchange)
• Integration of diagnostics with bioinformatic approaches to assist decision making
19 DTU Vet, Technical University of Denmark
Possible research subjects based on previous initiatives
DAY2
20 DTU Vet, Technical University of Denmark
Short-term applied research priorities:
• Optimize and validate a range of diagnostic tools and approaches to allow for improved serological and viral diversity surveillance, in particular for wild birds.
• Develop an engineered cell system permissive for the replication of influenza A viruses as an alternative to the currently used culture systems of embryonated chicken eggs or current cell culture systems
• Develop sample preservation substrates which are suitable for a wide range of diagnostic sample types (faeces, tissues, blood, oral and nasal fluids, serum, environmental) and which do not rely on maintaining the cold chain.
• Develop reliable diagnostic tools for rapid virus and antibody subtyping as an alternative to the current Haemagglutination Inhibition (HI) test.
• Validate RT-PCR test(s) to OIE standards as fit for purpose for international movement of horses.
Priority basic research:
• Measure cross species infection, especially subclinical infection.
21 DTU Vet, Technical University of Denmark
Additional research topics that are complementary to those listed above are selected from the list presented in Appendix, as possible research topics identified by other initiatives: • More sensitive pen-side tests or commercial test kits that can be used in
the field are needed, ideally with simultaneous identification of the most important HA and NA subtypes.
• Continue to monitor current molecular diagnostic tests for sensitivity and specificity with novel influenza isolates.
• There is a need to develop a rapid and cost effective full genome sequencing method (e.g. with next generation methods).
• Develop a rapid molecular test that is not easily affected by genetic mutations and able to detect all influenza viruses.
• Development of better serological tests to determine the subtype specificity of antibodies (i.e. to identify what subtype an animal has been infected with).
• Development of tests for better characterization of the antigenic differences among animal influenza isolates (critical for updating vaccines and evaluating vaccine-induced protection).
• Development and optimization of test sensitivity and specificity, in relation to virus subtype identification by molecular methods e.g. RT-PCR.
• Development of companion DIVA “differentiating infected from vaccinated animals” diagnostic tests that are validated for routine use.
• Further development of test methodologies for detection of reassortment.