New and emerging technologies: Improved laboratory
and on-site detection of OIE List A viruses in animals and
Time of action: LAB-ON-SITE started in November
2004 and is scheduled to end in January 2008
EU budget (funding): € 1.5 million
Without reliable molecular methods it is impossible or extremely
difficult to identify viral strains and to determine their
genetic variability. Novel, powerful molecular methods are
required, in order to track the spread of diseases and to
implement effective control measures. Accurate diagnosis is
the first, and arguably most important, step in controlling
epidemic diseases in animals.
But the value of even the best diagnostic techniques is limited
unless results can be obtained quickly. Highly pathogenic
viruses can spread through entire flocks in a matter of days,
so it is essential that diagnoses are made early, in time
for preventative and protective strategies to be put in place.
The LAB-ON-SITE project combines the expertise of three universities,
five veterinary research institutes and a Swedish biotechnology
company. Together these partners will develop, test and distribute
a range of new, improved diagnostic systems for virus detection.
These will include onsite systems that will allow breeders,
government agents and other people on the ‘front line’
to obtain immediate results. The group will also develop laboratory-based
procedures that can be used to confirm the quick-and-easy
pen-side approaches. The partners are focusing on nine diseases
including foot-and-mouth disease, classical swine fever, highly
pathogenic avian influenza and swine influenza.
For avian influenza the diagnostic tests will make use of
several basic technologies. PCR assays can amplify specific
viral sequences to detectable levels in just a few hours.
The idea is then to build portable PCR devices that can be
used for on-site testing. The project will also look at other
novel nucleic-acid detection technologies, some developed
in prior EU projects, that can detect viral sequences without
the need for the repetitive heating and cooling of samples
that is necessary with PCR.
ELISA assays, meanwhile, use antibodies to detect specific
viral proteins and will be used in laboratories for the universal
detection of influenza virus and for the specific identification
of H5 and H7 subtypes. The project is also developing a dipstick
technology for rapid pen-side detection of viruses.
The work of LAB-ON-SITE complements the investigations of
the extended AVIFLU project and it is hoped that the two will
coordinate their work together. The combination of improved
and standardized diagnostics and AVIFLU's insights into avian
influenza transmission should strengthen both Europe's position
in animal welfare in general, and its protection from the
immediate risk of avian influenza in particular.
Status (January 2006)
The work program is focused on new and emerging technologies
specifically the development, validation and dissemination
of robust, specific and sensitive diagnostic tests for nine
OIE List A diseases.
Establishment of a sample collection: A
virtual sample bank has been established containing 283 samples
of viruses used in the project. This database will evolve
and expand during the lifetime of the project to record details
of material that is available to the partners for the validation
of the assays generated by the other LOS work packages.
Application of rapid, simplified PCR techniques:
Genomic sequences of Highly pathogen avian influenza
virus (HPAIV, M-gene), Newcastle disease virus (NDV, F-gene),
African horse sickness virus (AHSV, VP7-gene) and Bluetongue
virus (BTV, NS1-gene) were collected from the Genbank. Alignments
were performed and primers/probes for rapid detection assays
(real-time PCR, capillary-based PCR) were selected and tested
on virus isolates and clinically related pathogens. The work
on setting the conditions for AHSV real-time PCR is ongoing.
Application of nucleic acid detection techniques
without thermocycling: Two assays are used to
fulfill the task: Invader and NASBA. An ASFV Invader assay
has been partly evaluated on clinical material from pigs infected
with the ASFV strain E75. Four CSFV specific Invader assays
have been designed against the Ns5b and 3’noncoding
regions, and manufactured by Third Wave Technologies. Optimisation
of the assays is ongoing. Oligonucleotides for the NASBA detection
assays against HPAIV and NDV were selected by using the Beacon
Designer and Lasergene softwares. The best candidates have
been purchased. The primers and probes have been designed
for the subsequent NASBA assays against HPAIV and NDV.
Development of novel nucleic acids detection
methods, including padlock probes and rapid readout formats:
The cornerstone of this approach lies in the
ability of padlock probes in conjunction with tag microarray
to simultaneously detect hundreds of viruses. A preliminary
set of padlock probes for the detection of Foot-and-mouth
disease virus (FMDV), Vesicular stomatitis virus (VSV) and
Swine vesicular disease virus (SVDV) has been designed and
synthesized. A protocol has been established for ligation
and amplification of the padlock probes. All padlock probes
can be amplified using the same PCR primer pair, and each
is equipped with a unique tagsequence 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. The method
has capacity even for the determination of various serotypes
of the viruses.
Detection of OIE List A viruses with real-time
PCR assays, including portable PCR and automated procedures:
Primers and probes for the detection of HPAIV, ASFV, CSFV,
NDV, SVDV, and FMDV using real-time PCR assays are designed.
Among the systems used are PriProET (Primer-Probe Energy Transfer)
assay, MGB (Minor Groove Binding) probes and LUX (Light Upon
extension) primers. 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. At the same time
two-step SVDV PriProET real-time was developed, and optimised.
The manuscript was recently submitted for publication to the
Archives of Virology.
Light Upon Extension (LUX) quantitative real-time PCR assays
were developed to detect HPAIV and NDV. Several primers and
two dyes (JOE and FAM) were tested in various combinations
and a reaction mixture was established allowing effective
virus detection. The assays proved to be specific that can
be conveniently confirmed by melting point analysis upon amplification.
An MGB probe assay was designed for AFSV as an alternative
to the LUX primers. This assay is fully optimised and only
requires further testing on alternative strains and specificity
testing. An FMDV MGB assay has also been designed. This assay
works well on DNA target and is currently being optimised
for RNA. The design process for assays to detect CSFV is well
The ability of two automated robots (BR9604, Qiagen and MagNA
PURE LC, Roche) to extract nucleic acids from different biological
matrices were compared. A Qiagen Biorobot 3000 for automated
extraction of nucleic acids from various biological materials
was installed and calibrated as well.
Development and optimisation of simple and sensitive
monoclonal antibody-based antigenantibody assays: Sandwich
ELISA using MAbs as coating and conjugated antibody detection
was developed. MAbs combinations for the specific typing of
six out of seven FMDV serotypes were evaluated. Competitive
ELISAs for the specific detection of antibodies to four FMDV
types (O, A, C and Asia 1) was developed and evaluated as
Development of rapid methods for the on-site
detection of viral antigens (Dip-stick tests):
Selection of MAbs and preparation of dip-stick devices was
performed on FMDV and VSV Indiana. The best MAb candidates
were selected as they react with all or most of the different
FMDV strains in capture ELISA. Dip-stick devices have been
put together and the best combinations of Mabs have been chosen.
The work is continued using three promising MAbs, which are
going to be tested on FMDV field samples. The work is ongoing
with AHSV, BTV, Classical swine fever virus (CSFV), Swine
influenza virus (SIV), NDV, HPAIV and VSV New Jersey.
Transfer of knowledge to EU member and candidate
countries, as well as to the third countries.
The involvement of nine partner laboratories, one third party
and two subcontractors, representing diagnostic institutes,
universities, one SME and international organizations of the
United Nations (IAEA, FAO) provide guarantee that the gained
knowledge will be directly applied in routine diagnostic laboratories,
in undergraduate and postgraduate teaching, in industries,
as well as in national and in international networks of biotechnology
and health sciences. Contact with Animal Health Staff in different
countries of Central and South America - Costa Rica, Panama,
Belize, 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.
All these countries will be included in LAB-ON-SITE information
Prof. Sándor Belák
Department of Virology
The National Veterinary Institute (SVA)
Ulls väg 2B
SE-751 89 Uppsala
List of partners (listed countrywise)
SE – National Veterinary Institute, Uppsala
UK – Queen's University of Belfast, Virology Laboratory,
DK – Danish Institute for Food and Veterinary Research,
ES – Universidad Complutense de Madrid, Facultad de
HU – Veterinary Institute of Debrecen, Debrecen
SE – SVANOVA Biotech AB, Uppsala
IT – Istituto Zooprofilattico Sperimentale della Lombardia
e dell´Emilia Romagna, Brescia
BE – Ghent University, Merelbeke
UK – Institute for Animal Health, Pirbright