Animal influenza Diagnostics - Europa

Animal influenza – Diagnostics

Anette Bøtner

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


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


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

Diagnostic methods for influenza in other animals species

• Tools as applied for Avian/Swine

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



• 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



• 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


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


DAY2


Possible research subjects based on previous initiatives

DAY2


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.


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.

Documents

Animal influenza Diagnostics - Europa