GB2412731A

GB2412731A - method for diagnosing avian influenza virus comprisisng detecting NS1 specific antibodies

Info

Publication number: GB2412731A
Application number: GB0407526A
Authority: GB
Inventors: Ilaria Capua; Stefano Marangon; Giovanni Cattoli
Original assignee: Individual
Current assignee: Individual
Priority date: 2004-04-02
Filing date: 2004-04-02
Publication date: 2005-10-05
Also published as: GB0407526D0; WO2005095974A1

Abstract

A method for diagnosing avian influenza virus infection in a bird, the method comprising the following steps of providing an antibody sample from a bird to be tested and determining whether the sample comprises antibodies to non-structural protein 1 (NS1) of an avian influenza virus, the presence of anti-NS1 antibodies being indicative of infection of the bird with avian influenza virus. Preferably, the method comprises contacting the sample with a non-structural protein 1 (NS1) of an avian influenza virus, and determining whether antibodies in the sample to be tested bind thereto, for example using an indirect ELISA or immunofluorescence assay. The invention further provides a kit for performing the methods of the invention.

Description

24 1273 1

ASSAY

The present application relates to an assay for detecting infection of birds with avian influenza virus. In particular, the invention provides an assay which is able to differentiate between infected birds and birds which have been vaccinated with inactive virus particles.

Background art

The general structure and replicative mechanism of influenza viruses has long been known in the art. Three types of influenza, termed A, B and C, have been described and are distinguished by the antigenicity of their nucleoproteins. While all are responsible for infections of the upper respiratory tract, types A and B are the major cause of national epidemics and world-wide pandemics (Dimmock and Primrose, 1994).

so Avian influenza vinus (AIV) is a type A virus and a common pathogen of commercially-important domestic and wild birds, such as chickens, turkeys, ducks, geese, quails, ostrich, emus, and exotic birds such as parrots, cockatoos and cockatiels. In common with other influenza viruses, AIV possesses a sense RNA genome with eight genes encoding eight structural proteins that comprise the viral particle and two non- structural proteins not found in the mature virion.

The severity of AIV infection depends upon the pathogenicity of the infectious agent, the avian species infected and the age, sex and health of so the host. Symptoms include sneezing, coughing, huddling, cuffed feathers, oedema of the head and face, nervous disorder, diarrhoea and sinusitis, and disease is usually accompanied by decreased egg production.

s Although many infections are sub-clinical, certain virus subtypes are highly virulent and can cause devastating epidemics within populations of commercially-valuable domestic birds, such as poultry. For example, the "Foreign Animal Disease Report", USDA 1994 reported that in 1983- 1984 an outbreak of a highly pathogenic AIV strain resulted in the death lo of 17 million birds in Pennsylvania, Maryland, New Jersey and Virginia at a cost of over $500 million (US Patent Application No. 2002187162).

Despite its importance, the spread of avian influenza virus is generally only controlled by restricting trade and movement of birds to those that Is have not been exposed to virus. AIV surveillance and eradication programmer rely upon diagnostic assays that are capable of detecting serum antibodies specific for the virus and these allow identification of potential carriers of influenza and hosts with pre-symptomatic or subclinical infections.

I'resently, imtnunological surveillance prevents the use of available inactivated vaccines as immunisation generates individuals that are scrologically positive for virus and current diagnostic tests are unable to differentiate these birds from those harbouring live virus. Consequently Is vaccination programs that could protect domestic stocks have not been adopted, and broad export bans have been imposed on countries using standard vaccines.

Accordingly, alternative vaccines or diagnostic tests that would allow so hnmunised individuals to be examined for the presence of replicating virus are strongly desired. Recently, birds vaccinated with a specific inactivated strain possessing antigenic properties distinct from those of the field virus enabled serological identification of infected and uninfected vaccinated birds (Capua, 2002). While successful, this approach requires isolation and analysis of the field virus prior to application of a suitable vaccine and consequently may be of limited practical use. However, to date a diagnostic test capable of identifying viral infection in birds previously immunised with existing influenza vaccines has not been described.

Differential diagnostic tests have been described for hepatitis C virus (HCV) infection (Inoue et al., 1992) and foot and mouth disease virus (FMDV) infection (Nietzert et al., 1991) based on expression of viral nonstructural proteins. As these HCV and FMDV proteins are absent from mature viral particles and only expressed during productive infection, antibodies specific for these non-structural proteins arise in hosts exposed to live virus but not in those imnunised with inactivated virus, providing a diagnostic marker of disease.

so Influenza virus RNA segment 8 encodes two overlapping proteins, NS1 and NS2, that were originally termed 'non-structural' (Dimmock and Primrose, 1994). NSI contributes to viral pathogenesis, primarily by disarming the host cell interferon defence system (Krug et al., 2003).

NS2 performs a regulatory role during virus-specific RNA synthesis :5 (Bullido et al., 2001), but is now also known to be a minor structural component in some influenza virus types (Yasuda et al., 1993).

Several documents disclose efforts to develop assays for mammalian influenza infections based on the differential presence of non-structural so proteins in infected or uninfected vaccinated mammalian hosts. In marine and equine influenza, non-structural proteins are expressed during viral replication but are not present in the mature virion and can therefore be used as diagnostic markers of actively replicating virus (Birch-Machin et al., 1997; Ozaki et al., 2001).

Birch-Machin et al. (1997) describes the detection of antibodies specific for a non-structural protein, NS1, of equine influenza virus in vaccinated uninfected and vaccinated infected horses. This report indicated that anti- NS 1 antibodies were present in serum samples of animals experimentally lo infected with influenza virus, but not in unchallenged animals or those vaccinated with inactive virus particles. While the suggestion is that the differential detection of anti-NS1 antibodies may fonn the basis of a diagnostic assay for identifying live equine influenza in immunised horses, methodology for such an assay is not disclosed.

Ozaki et al. (2001) describes the detection of anti-NSI antibodies in experimentally infected mice and horses using an Enzyme Linked Immunosorbent Assay (ELISA) method. This report demonstrated the utility of antibodies specific for NS 1 as differential diagnostic markers for so murine and equine influenza infections and disclosed a method suitable for identifying infection by these viruses. Similarly, European Patent No. 0726316 disclosed an ELISA method using equine anti-NS I antibodies as a diagnostic marker of equine influenza infection in vaccinated animals.

Despite these disclosures, there are currently no reports of the presence of anti-NS1 antibodies in animals other than mammals, and there is no evidence to suggest that the diagnostic assays developed for horses and mice can be applied to other animals. Furthermore, it is currently unclear whether the NS1 protein represents a suitable differential diagnostic marker for influenza infections in animals other than horses or mice Hence, these exists a need for improved assays for detecting infection of birds with avian influenza virus which allow differentiation of infected and uninfected vaccinated animals.

Summary of the invention

A first aspect of the present invention provides a method for diagnosing avian influenza virus infection in a bird, the method comprising the lo following steps: (a) providing an antibody sample from a bird to be tested; and (b) determining whether the sample comprises antibodies to non- structural protein 1 (NS1) of an avian influenza virus the presence of anti-NSI antibodies being indicative of infection of the bird with avian influenza virus.

so It will be appreciated by persons skilled in the art that the method of the invention may be used to diagnose AIV in any species of bird, including both wild birds (such as wildfowl) and domesticated species (such as poultry). Preferably, the bird to be tested is selected from the group consisting of chickens, turkeys, ducks, geese, quails, ostrich, emus and exotic birds such as parrots, cockatoos and cockatiels. More preferably, the bird to be tested is a chicken or turkey.

In a preferred embodiment, the bird to be tested has previously been vaccinated prior to testing.

The method of the invention may be performed using any antibody sample from the bird to be tested. Advantageously, however, the antibody sample is a serum sample. Alternatively, the antibody sample may be collected from egg-yolk.

Advantageously, the antibody sample is diluted prior to testing. Optimal dilution levels may be determined by methods known in the art, such as checkboard titration. Briefly, serial dilution of the antigen and of the positive and negative reference sera are prepared and tested. The lo combination offering the best performances (i.e. highest signal for the positive reference serum associated with the lowest signal with the negative reference serum, lowest background) is selected and used to validate the test. It will be appreciated by persons skilled in the art that such a checkboard titration should be performed on every new batch of 1 5 reagents.

In a preferred embodiment of the first aspect of the invention, step (b) comprises contacting the antibody sample with a non-structural protein 1 (NSI) of an avian influenza virus, and determining whether antibodies in the sample to be tested bind thereto. Fragments and/or variants of NSI proteins which exhibit substantially the same antigenicity as complete NSI can also be used in this step. The antigenicity of such NS1 fragments and variants may be tested using methods well known in the art, e.g immunoaffinity and/or immunoprecipitation techniques.

It will be further appreciated by persons skilled in the art that the NS1 protein used in step (b) may be derived from an avian influenza virus of any strain, using known techniques (for example, see Porter et al., 1980, Proc. Natl. Acad. Sci. USA 77:5074-8).

Conveniently, the NS1 protein is from an avian influenza virus strain selected from the group consisting of A/ty/Italy/4426/00 (H7N1) LPAI, Alty/ltaly/7 159/02 (H7N3) LPAI and A/avian/NL/2586/03 HPAI.

Preferably, the NS I protein is from avian influenza virus strain A/ty/Italy/4426/00 (H7NI) LPAI.

For example, the NS1 protein may consist of or comprise the amino acid sequence of SEQ ID NO: 1, or a fragment thereof which exhibits lo substantially the same antigenic properties as a protein of SEQ ID NO: 1: DCYLWHIRKII.SMRDMVVDAPFDDRLRE<DQKALKGRCSTLGLDLRVATMEGKVED ILKSETDENLKIAIAS STAPRYITDMSIEEIS REWYMI.MPRQKVTGGLMVKMDQAIMDK

RITLKANFSVLFDQLETLVSLRAFTEDGAIVAEISPIPSMPGHSTEDVKNAIGILIGGLEW

I 5 NDNSIRASENIQRFAWGIRDENGGPPLPPKQKRMARRVESEVRDQMAHCMQKYTDKD

KQLTDNILASIATLTSE

iSEQ ID NO: 11 Such a polypeptide may be encoded by a nucleic acid molecule of SEQ ID NO: 2.

GATAACCTCGTriCA(iGTAGATTGrTATCTATG GCACATAAG AAAGATACTCAGTA TGAGAGACATGGTGGTAGA'l'(iCTCCCT'rTGATCiACAGGCTCCGAAGACACCAAAAG GcA r rAAAGGGAAGA rGcAGcAcAcTTGGAc rcGATcTA^GAGTGGcTAcAATGG AGGGGAAAAAGA'I'CGTTGAGGACA'l'CCTGAA(iAGTGAAACAGATGAAAACCTCAA AATAGCCA'l'TGCTTCCAG'rACTGCTCCTCGGTATATCACTGATA'r(iAG CATAGAGG AGATAAGCCGAGAATGGTACATGC"l'GATGC'C'[AGGCAGAAAG'rAACTGGAGGCCT TATGGTGAAAATGGACCAAGCCATAATG GA'I'AAGAGAATTACCCTTAAAG CTAATT TCTCAGTCCTArrTGA'rCAACTAGAAACATTAGTCTCTCTGAGGGCATTCACAGAAG ATGGTGCAATTGTG (i CTGAAATATCTCCCAT'f CCC'I'CCATGCCA(iGACATTCTACAG AGGATGTCAAAAATGCAA'rTGGAA'fCCTCA'l'CGGTGGACTTGAATGGAA'rGATAAC TCAATTCGAGCG'l'CTGAAAATATACAGAGATTCGC'l'TGGGGAA'rCCG'l'(iATGAGAA TG(iGGGACCTCCACTCCC'l'CCAAAGCAGAAACGCTAAATGGC(iAGACGAGTTGA(i'r CAGAAG m GAAGAGATCAGATGGCTCAfTGCTGAATGCAGAAATATACTGACAAA GACTGAAAACAGc m GAACAGATAACArRcrTGCAAGCATTGCAACTCTTACrTG AAGTTGAGAGTGA - 3' [SEQ ID NO. 2] Alternatively, the NS 1 protein is from avian influenza virus strain A/ty/Italy/7159/02 (H7N3) LPAI.

For example, the NS1 protein may consist of or comprise the amino acid lo sequence of SEQ ID NO: 3, or a fragment thereof which exhibits substantially the same antigenic properties as a protein of SEQ ID NO: 3:

VVS SFQVDCFLWH VRKRFADQELGDAPFLDRLRRDQKSLRGRGSTLGLDIETATRAGK

QIVERILEEESDEALKMTITS VPASRYLTDMTLEEMSRDWFMLMPKQKVAGSLCIRMD

QAIMDKNIKLKANFS VIFDRLETLILLRAFTEEGAIVGEISPLPSLPGlITDEDVKNAIGVLI

GGLEWNVNTVRVSETLQRFAWRSSNEDGRPPLPPKQKARTIESEV

[SEQ ID NO: 31 Such a polypeptide may be encoded by a nucleic acid molecule of SEQ ID NO: 4.

ACGTAGTGTCAAGCTTTCAGGTAGACTGC'm ClrTGGCACGTCCGCAAACGA m G CAGACCAAGAACTGGG'l'GATGCCCCATTCCrTGACCGGCtrCGCCGAGATCAGAAG TCCCTAAGAGGAAGGGGCAGCAC'FC'rTGG'rC'rGGACATCGAGACAGCTACTCGTGC GGGAAAGCAGATAGTGGAGCGGATTC'I'GGAGGAAGAATCTGATGAGGCACTTAAA ATGACTATTACT'I'CAGTGCCGGC'I'TCACGCTACCTAACTGACATGACTC'I'TGAAGAA ATGTCAAGGGACTGGI"fCATGC'l'CATGCCCAAGCAGAAAGTG(iCAGGTTCCCTTrG CATCAGAATGGACCAGGCAATAATGGATAAGAACATCAAAT'l'CiAAAGCAAACrTC AGTGT(, AT'rT'[TGACCG(iCTGGAAACCC'l'AATAC'l'AC'I TAGAGCTTTC'ACAGAAGA AGGAGCAAl"rGTGGGA(iAAA'l'CTCACCATTACCl"l'CTCTCCAGGACATACTGA'l'G AGGATGTCAAAAATGCAA'l'TG(iGGTCC'l'CATCGGAGGACl-"r(iAATGGAATGATAAC ACAGTTCGAGTCTCTGAAACTCTACAGAGATTCGCrl'(iGAGAAGCAGTAA'rGAGGA

TGGGAGACCTCCACTCCCTCCAAAACAGAAACGGAAAATGGCGAGAACAATTGAG

TCAGAAGTrTGAAGAAATAAGATGGTTGATTGAAGAAGTGCGACACAGATTGAAG ATTACAGAGAACAGCTTCGAACAGATAAC('TTTATGCAAGCCTTA(AACTATTGC'I' TGAAGTGGAGCAAGAGATAAGAA- 3' [SEQ ID NO. 4J In a further embodiment, the NSI protein is from avian influenza virus strain A/avian/NL/2586/03 HPAI.

o For example, the NS1 protein may consist of or comprise the amino acid sequence of SEQ ID NO: 5, or a fragment thereof which exhibits substantially the same antigenic properties as a protein of SEQ ID NO: 5: GIRDKNIMDSNAFSFQVDCFLWI IVRKRFADQEI.GDAPFLDRLRRDQKSI.RGRGSTLGL

DIETATRAGKQIVERILEEESDALKMTIASVLASRYLTDMTLEEMSRDWFMLMPKQK

VAGSLCIRMDQAIMDKNIILKANFSVIFDRLETLILLRAFTEEGAIVGEISPLPSLPGHTDE

DVKNAIGVLIGGLEWNDNTVRVSE'I'LQRFAWRSSN:EDGRPPLPPKQKRKMARTIESEV iSEQ ID NO: 5] Such a polypeptide may be encoded by a nucleic acid molecule of SEQ ID NO: 6.

CGGGATCCG'rGACAAAAACA'[AATGGA'l'TCCAACG(2'l'TTCAGCTT'rCAGGTA GACT GCTTTCIl'GGCATGTCCGCAAACGAT]"fGCAGACCAAGAACT(iGGTGATGCCCCA rTCCrTGACCGG CTTCGCCGAGATCAGAA ATCCCTA AGAGGAAGAGG CAG CACTCT TGGTCTGGACATCGAGACAGCTAC'I'(2GTGCGGGAAAGCAGATAGTGGAGCGGATTC TGGAGGAAGAATCTGA'fGAGGCA(2TTAAAATGACTATTGCrTCAG'l'(,CTGGCT'l'(2A CGCTACCTAACTGACATGACTCTTGAAGAAA'l'G'l'CAAGGGACTGGrl'CATGCTCA'r

GCCCAAGCAGAAAGTGGCAGGTTCCCTTTGCATCAGAATGGACCAGGCAATAATGG

ATAAAAACATCATATTGAAGGCAAACTJ'CAG'l'GTGATrffl'GACCGGCTGGAAACC CTAATA CTA CTTA (i A G CTrl'CA CA G AA(i AA GGAG ('A ATTGTG G G A GAAAT(21'CA C C ATTACCTTCTCTTCCAGGACA'I'ACTGATGAGGATG'I'CAAAAATGCAATTGGGGTCCT

CATCGGAGGACTTGAATGGAATGATAACACAGTTCGAGTCTCTGAAACTCTACAGA

GATTCGC'GGAGAAGCAGTAATGAGGATGGGAGACCTCCACTCCCTCCAAAGCAG

AAACGGAAAATGGCGAGAACAATTGAGTCAGAAGTTTGAAGAGATAAGATGGCTG

ATTGAAGAAGTGCGACATAGGTTGAAGATTACAGAGAACAGCTTTGAACAGATTAC

GTrrATGCAAGCClrACAACTATTGCTTGAAGTAGAGCAAG-3' [SEQ ID NO. 6] to a preferred embodiment, the NS 1 protein is a recombinant NS 1 protein. Methods for the production of recombinant proteins are well lo known in the art (for example, see Sambrook & Russell, 2001, Molecular Cloning: A Laboratory Manual, 3r edition, CSHL Press).

In brief, DNA encoding the NS1 protein (such as SEQ ID NOS: 2, 4 and 6) is inserted into an expression vector, such as a baculovirus expression Is vector, in proper orientation and correct reading frame for expression. If necessary, the DNA may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognised by the desired host, although such controls are generally available in the expression vector. Thus, the DNA insert may be operatively linked to an so appropriate promoter. Suitable promoters will be known to the skilled artisan. The expression constructs will desirably also contain sites for transcription initiation and termination, and in the transcribed region, a ribosome binding site for translation (e.g. see WO 98/16643).

The vector is then introduced into the host through standard techniques.

Generally, not all of the hosts will be transformed by the vector and it will therefore be necessary to select for transformed host cells. One selection technique involves incorporating into the expression vector a DNA sequence marker, with any necessary control elements, that codes for a so selectable trait in the transformed cell. Such markers include dihydrofolate reductase, G4 18 or neomycin resistance for eukaryotic cell culture.

Alternatively, the gene for such a selectable trait can be on another vector, which is used to co-transform the desired host cell.

The cells are then cultured under conditions which permit expression of the NS1 protein.

Preferably, the NSI protein is derived from transformed insect cells.

Advantageously, the transformed insect cells are Trichoplusia ni cells.

lo It will be appreciated by persons skilled in the art that the NSI protein may be provided in one of a number of suitable forms. For example, the NS I protein antigen may be provided in the form of transformed insect cells (this form is particularly suitable for detection of antibody-antigen binding by immunofluorescence assay), crude cell Iysate (this form is Is particularly suitable for detection of antibody-antigen binding by ELISA) or in a purified protein preparation. Methods of preparing such purified protein preparations are well known in the art.

Preferably, the NS1 protein is present in a cell. Conveniently, the NSI so protein is present in a cell lysate.

In a further preferred embodiment of the first aspect of the invention the NS 1 protein is immobilised on a surface, such as a surface of a multiwell plate. Methods of immobilization of a protein to a surface are well known in the art. For example, the protein may simply be exposed to the surface and allowed to adhere to it.

In step (b), the detection of binding of anti-NS 1 antibodies to NS I protein may be measured by methods known in the art, for example: (a) ELISA (enzyme-linked immunosorbent assay) ELISAs are perhaps the most widespread method of detecting antibody-antigen binding, and typically involve the use of antibody s enzyme conjugates which give a coloured reaction product. Enzymes such as horseradish peroxidase and phosphatase have been widely employed in such conjugates. A way of amplifying the phosphatase reaction is to use NADP as a substrate to generate NAD which now acts as a coenzyme for a second enzyme system. Pyrophosphatase lo from E. cold provides a good conjugate because the enzyme is not present in tissues, is stable and gives a good reaction colour. Chemi luminescent systems based on enzymes such as luciferase can also be used.

is Conjugation with the vitamin biotin is also frequently used since this can readily be detected by its reaction with enzyme-linked avidin or streptavidin to which it binds with great specificity and affinity.

(b) Radioimmunoassay The antibody content of a serum can also be assessed by the ability to bind to antigen which has been in and immobilised by physical absorption to a plastic tube or micro-agglutination tray with multiple wells; the bound immunoglobin may then be estimated by addition of Is a labelled anti-I" raised for anther species. For example, serum is added to a microwell coated with antigen, the antibodies will bind to the plastic and remaining serum proteins can be readily washed away.

Bound antibody can be estimated by addition of '25I-labelled purified rabbit anti IgG; after rinsing out excess unbound reagent, the radioactivity of the tube will be a measure of the antibody content of the serum.

(c) Immunofluorescence assay s Immunofluorescence assays work on a similar principle to both ELISA and radioimmunoassays, except that binding of the secondary antibody o the target anti-NS1 antibody is measured by fluorescence.

Typically, the secondary antibody is conjugated to a fluorophore, lo such as fluorescein isothiocyanate (FITC).

In a preferred embodiment of the method of the first aspect of the invention, step (b) is carried out by indirect ELISA.

Is In an alternative preferred embodiment of the method of the invention, step (b) is carried out by indirect IFA.

It will be appreciated by persons skilled in the art that step (b) should preferably by carried out under conditions that permit the specific so association of anti-NSI antibodies with NS1 protein. Thus, conditions should be sufficiently stringent to prevent host antibodies not raised against AIV NS1 epitopes from binding NS1 while permitting binding of anti-NSI antibodies. The method must allow the resulting NS1 proteinantibody complex to be identified, preferably by physically isolating the :5 complex from unbound antibody.

A second aspect of the invention provides a method of monitoring the spread of avian influenza virus infection in a population of birds comprising selecting a bird from the population to be tested and determining whether the bird is infected with avian influenza virus using a method according to the first aspect of the invention.

A third aspect of the invention provides a method of preventing the s spread of avian influenza virus infection in a population of birds comprising selecting a bird from the population to be tested, determining whether the bird is infected with avian influenza virus using a method according to the first aspect of the invention, and destroying the bird if it is found to be infected. Preferably, the method further comprises lo destroying birds which have been exposed to the infected bird.

A fourth aspect of the invention provides a kit of parts for performing a method according to the first aspect of the invention comprising a nonstructural protein 1 (NS1) of an avian influenza virus and means for Is detecting the binding of antibodies thereto.

Preferably, the NS 1 protein is from an avian influenza virus strain selected from the group consisting group consisting of A/ty/Italy/4426/00 (H7Nl) LPAI, A/ty/Italy/7159/02 (H7N3) LPAI and A/avian/NL/2586/03 so HPAI. More preferably, the NSI protein is from avian influenza virus strain A/ty/Italy/4426/00 (H7N1) LPAI.

Most preferably, the NS1 protein consists of or comprises the amino acid sequence of SEQ ID NO: 1, 3 or 5, or a fragment thereof which exhibits Is substantially the same antigenic properties as a polypeptide of SEQ ID NO: 1, 3 or 5, respectively.

Conveniently, the NS1 protein is a recombinant the NS1 protein.

Advantageously, the NS1 protein is derived from transformed insect cells, such as Trichoplusia ni cells.

In a preferred embodiment, the NS1 protein is provided in the form of a s cell, for example in a transformed insect cell (or culture of such cells).

Alternatively, the NSI protein may be provided in the form of a cell Iysate. As a further alternative, the NS1 protein may be provided in a purified form (such as a freeze-dried preparation or solution).

lo In a preferred embodiment of the fourth aspect of the invention, the kit further comprises a surface upon which the NS1 protein is immobilised or means for producing such as surface. Thus, in one embodiment the kits of the invention may comprise a surface to which NSI protein may be adhered and cells capable of expressing a NS1 protein (such as those Is identified above). Prior to using the kits, the cells are cultured in contact with the surface so as to deposit the NS 1 protein on said surface.

Conveniently, the surface is a surface of a multi-well plate.

so In a further preferred embodiment of the fourth aspect of the invention, the kit further comprises a negative control, i.e. a sample which does not contain anti-NS 1 antibodies. Preferably, the kit further comprises a positive control, i.e. a sample which does contain anti-NS1 antibodies.

as In a further preferred embodiment of the fourth aspect of the invention, the kit is for use with an El,ISA.

Thus, the kit may further comprise a secondary antibody with specificity for antibodies from the bird to be tested. For example, where the kit is to so be used for diagnosis of AIV infection of chickens, the secondary l antibody is an anti-chicken IgG antibody. Likewise, where the kit is to be used for diagnosis of AIV infection of turkeys' the secondary antibody is an anti-hurkey IgG antibody.

s Preferably, the secondary antibody is conjugated to horseradish peroxidase. More preferably, the kit further comprises a horseradish peroxidase substrate, such as hydrogen peroxide.

Advantageously, the kit further comprises 2,2'-azino-di-3-ethyl- io benzthiazoline sulfonic acid (ARTS).

In a further preferred embodiment of the fourth aspect of the invention, the kit further comprises a blocking agent, such as defatted milk powder.

Preferably, the defatted milk powder is dissolved in phosphate-buffered saline.

The kit of the invention may further comprise a washing agent, which agent may in hum comprise a detergent such as polyoxyethylene sorbitan monolaurate polysorbate 20 (Tween-20). Preferably, the detergent is so dissolved in phosphate-buffered saline.

Advantageously, the kit further comprises a stop reagent.

In an alternative preferred embodiment of the fourth aspect of the invention, the kit is for use with an indirect immunofluorescence assay (iIFA). Preferably, such a kit further comprising a secondary antibody with specificity for antibodies from the bird to be tested, such as an anti- chicken IgG antibody or anti-turkey IgG antibody.

Advantageously, the secondary antibody is conjugated to fluorescein isothiocyanate (FITC).

Preferred, non-limiting aspects of the invention will now be described s with reference to the following examples.

EXAMPLES

Materials and Methods Cloning of the avian influenza virus NSI gene Viral RNA from avian influenza virus strain H7N1 4426/V00 LPAI was extracted using standard methods. The gene encoding non-structural Is protein NS1 was amplified by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) using the following oligonucleotide primers: (s) 5'-CGGGATCCGTGACAAAAACATAATGGATTCCAAC-3' [SEQIDNO.7] (a/s) 5'-CGGGATCCTCATTAAATAAGCTGAAACGAGAAAG-3' [SEQ ID NO. 8] These primers are specific for the NS1 open reading frame (ORF) and Is include terminal BamNl restriction sites (underlined sequence) for cloning. Reactions were performed using a 'One Step RT-PCR protocol' composed of the following conditions: Pre-RT step: 60 C incubation 10 minutes so RT step: 42 C incubation 60 minutes Initial Denaturation 95 C incubation 10 minutes cycles as follows: Denaturation: 94 C incubation 1 minute s Annealing: 55 C incubation 1 minute Elongation: 72 C incubation 2 minutes Final elongation: 72 C incubation 10 minutes lo The resulting 870bp PCR product was subjected to DNA sequencing to confirm amplification of a eDNA encoding AIV NS 1.

Expression arid purification of recombinant 4IVNSl protein The AIV NS1 gene was expressed using the Bae-to-Bae) Baeulovirus expression system (Invitrogen) according to manufacturer's instructions.

The NS1 eDNA was BamHI digested and ligated into BamHI-digested pHTe histidine-tagged vector using standard methods. Escherichia cold strain DHlOBae cells were transformed with the pHTc-NS1 plasmid, and the resulting recombinant NS 1 -containing Baemid was isolated and transfeeted into Trichoplusia ni insect cells (I-lighFive cells, Invitrogen) using standard methods. Recombinant baculovirus particles were collected from cell cultures after 72 hours and titred by plaque assay.

Indirect ELISA assay (iELIS>l) IIighliveCR) cells were seeded on 75em2 flasks at a concentration of 0.9- o 1.0 x 106 eells/ml in Express-five SFM culture medium supplemented with 1% antibiotic solution (PBS stock solution: penicillin G 10,000 Ul/ml; streptomycin 10mg/ml; amphotericin B 5,000 Ul/ml; gentamycin mg/ml), incubated at 28 C for I hour and then infected with recombinant NSI- containing bacmid at multiplicity of infection (MOT) 2.

Infected cells expressing the recombinant NSI protein were washed in PBS after 48-72 hour incubation at 28 C and collected in Iml/flask sonication buffer (150mM NaCI; 2mM Pefabloc; 50mM Tris-HCI pH7.5). After three freeze-thaw cycles at -20 C, cells were sonicated and lo cell Iysates diluted in carbonate buffer (pH9.6) for ELISA assays. Optimal dilution of antigen and sera was derived by checkboard titration.

A volume of 501/well diluted antigen was adsorbed overnight at 4 C in 96 well microtitre plates (NUNC Polysorp), washed three times with TPBS (0.05% Tween 20 v/v in PBS solution, pE-I7.3) and stored at 4 C.

Prior to use, adsorbed plates were blocked for I hour at room temperature in blocking buffer (5% non-fat dried milk in TPBS) and washed three times in TPBS.

o Serum samples to be tested were stored at -20 C and pre-diluted 1:80 in 1% (w/v) non-fat dried milk in TPBS. Indirect ELTSA assays were performed according to the following protocol: 1. 501 of pre-diluted test serum dispensed into duplicate wells of a 96 wCll microtitre test plate containing adsorbed NSI antigen. 501 of pre- diluted reference-positive and reference-negative serum dispensed in triplicate into each microtitre test plate.

2. Microtitre plates incubated at 37 C for 45 minutes.

3. Wells subjected to three five-minute washes with 1001 TPT-3S.

4. 501 of pre-titred anti-chicken IgG-HRP conjugated antibody dispensed into each well.

5. Microtitre plates incubated at 37 C for 45 minutes.

6. Wells subjected to three five-minute washes with 1 00,ul T PBS.

7. 100!11 of ARTS solution dispensed into each well.

8. Absorbance of microtitre plate wells read at Optical Density (OD) 405mn using a spectrophotometer.

Results Cloning of a cDNA encoding the AIVNS1 protein The nuclcotide sequence of the cDNA encoding non-structural protein I (NS1) from A/ty/Italy/4426/00 (H7NI) LPAI is as follows: GATAACC'I'CGTTTCAGGTAGATTGTTATCTATGGCACATAAGAAAGATACTCAG'I'A TGAGAGACATGGTGGTAGATGCTCCCTTTGAT(iACAGGCTCCGAAGAGACCAAAAG GCAT'l'AAAGGGAAGATGCAGCACACTTGGACTCGATC'rAAGAGTGGCTACAATGG

AGGGGAAAAAGATCGTTGAGGACATCCTGAAGAGTGAAACAGATGAAAACCTCAA

AATAGCCA'l'TGCTTCCAGTAC'fGCTCC'l'CGGTATA'l'CACTGAl'ATGAGCATAGAGG A(iATAAGCCGAGAATGGTACATGCTGA'FGCCTAGGCAGAAAGTAACTGGAGGCCT TATGGTGAAAATGGACCAAGCCATAATGGATAAGAGAAT'l'ACCCTTAAAGCTAAT'r TCTCAGT('CTAT] "rGATCAA('TAGAAACATTAGTCTCTCTGAGGGCA'rTCACAGAAG ATGGTGCAATTGTGGCTGAAATATCTCCCATTCCCTCCA'FGCCAGGACATTC'l'ACAG AGGATGTCAAAAATGCAATTGGAATCCTCATCGGTGGACTTGAA'I'GGAATGATAAC TCAATTCGAGCGTCTGAAAATATACAGAGATTCGCl-fGGGGAATCCGTGATGAGAA TO GGG GACCTCCA CTCCCTCCAA AGCA GA AACG CTAAATGG CGAGA CGA GTTGAG'r CAGAAGT'ITGAAGAGATCAGATGGCTCATTGCTGAATGCAGAAATATACTGACAAA GACTGAAAACAGCT'l"l'GAACAGATAACA'rTC'rTGCAAGCATTGCAACTC'lTACT'rG AAGTTGAGAGTGA-3' [SEQ ID NO. 2]

DCYLWH IRKILSMRDMVVDAPFDDRLRRDQKALKGRCSTLGLDLRVATMEGKKIVED

ILKSE'rDENLKIAIASSTAPRYITDMSIEEISREWYMLMPRQKVTGGLMVKMDQAIMDK RITLKANFSVI. FDQLETLVSLRAFTEDGAIVAEISPIPSMPGHSTEDVKNAIGILIGGLEW NDNSIRASENIQRFAWGIENGGPPI.PPKQMAVESEVQMAHCMQKYTDw

KQLTDNILASIATLTSE

[SEQ ID NO. 1] Analysis of recombinant AIVNSI protein lo The identity of the recombinant protein purified from baculovirus was confirmed by Western blot analysis, using standard methods (for example, sce Sambrook J. Fritsch EF & Maniatis T. 1989, Molecular Cloning. A laboratory manual, Cold Spring Harbor Laboratory Press, USA).

Detection of chicken and turkey anti-NS1 antibodies using iELISA The gold standard test is an Agar Gel Immunodiffusion (AGID) assay (see EU Directive 92/40).

Validation of the iELISA test was performed on 998 sera samples from chicken and turkey that had previously been assessed using the AGID technique and divided into positive sera (reference number 434) and negative sera (reference number 564).

Methodology was as described above; the mean OD values for each test serum was determined and the percentage of positivity (PP) referred to the positive controls was fixed. The cut-off value was determined by a TG- ROC curve using the AGID test as gold standard. This cut-off was so used to calculate the relative sensitivity and specificity of the test.

Agreement between the iELISA and ACID was assessed using the kappa (K) value and benchmarks suggested by Everitt (1989): K value Degree of agreement >0.81 Almost perfect agreement.

0.61-0.81 Substantial agreement.

0.41-0.60 Moderate agreement.

0.21-0.40 Fair agreement.

lo 0.00-0.20 Slight agreement.

0.00 Poor agreement.

Results obtained from this preliminary validation were: Is Obtained K value 0.647 Relative sensitivity 76.97% Relative specificity 87.23% Cutoff value 51.57 so Thus, the obtained K value indicates substantial agreement of the iELISA with the ACID technique.

Discussion as Present serological assays are unable to differentiate birds vaccinated against, and infected with, avian influenza virus from birds vaccinated against, but not infected with, avian influenza virus.

The discovery by the present inventors that anti-NSI antibodies are only so present in birds infected with replicating virus present invention provides an assay capable of differentiating birds vaccinated against, and infected with, avian influenza virus from birds vaccinated against, but not infected with, avian influenza virus. Thus, the present invention uses recombinant AIV NSI protein to detect antibodies specific for the presence of live s virus in birds.

Validation of the assay of the present invention indicates substantial agreement with the current gold standard ACID test. Thus, the present invention provides an alternative assay to current serologic methods for lo examining AIV infections. The following are example applications of the present invention and additional uses may exist for this invention that will be obvious to a man skilled in the art: 1. The status of field exposure of a given flock may be identified is during a vaccination campaign against any subtype of AIV, thereby allowing birds vaccinated against but not infected with AIV to be exported and birds vaccinated against and infected with AIV to be culled.

2. The spread of infection in a vaccinated population may be evaluated, allowing appropriate corrective measures to be taken.

3. AIV prevalence in populations of poultry may be monitored, enabling the early identification of infections in vaccinated or unvaccinated flocks.

It will be appreciated that the usefulness of the NS 1 protein as a diagnostic marker of AIV infection depends upon the duration of the host antibody response following infection. A response that remains long so after productive infection has ended would obviously limit its use diagnostically. Results in equine influenza virus infections indicate that levels of circulating anti-NSI antibody decline rapidly after infection, as is the case for anti-haemagglutinin antibodies (Hannant et al., 1988; European Patent Application No 0 726 316). s

References 1. Birch-Machin I, Rowan A, Pick J. Mumford J. Binns M. (1997) Expression of the nonstructural protein NS1 of equine influenza A 0 virus: detection of anti-NS 1 antibody in post infection equine sera, Journal of Virological Methods 65:255-263 2. Bullido R. Gomez-Puertas P. Saiz MJ, Portela A. (2001) A virus NEP (NS2 protein) downregulates RNA synthesis of model Is template RNAs, Journal of Virology 75:4912-7.

3. Capua et al. (2002) The Veterinary Record 150:223 4. Capua, I. (2003) Development of a DIVA (Differentiating Infected so from Vaccinated Animals) strategy using a vaccine containing a heterologous neuraminidase for the control of avian influenza., Asian Pathology 32:47-55 5. Dimmock, NJ, Primrose, SB. (1994) Introduction to Modern Is Virology, Fourth Edition, Blackwell Publishing.

6. Everitt (1989) Statistical methods for medical investigation.

Oxford University Press.

7. Hannant, D, Mumford, JA, Jessett, DM (1988) Duration of circulating antibody and immunity following infection with equine influen%avirus, Veterinary Record 122: 125-128.

8. Inoue, Y. Suzuki, R. Matsuura, Y. Harada, S. Chiba, J. Watanabe, Y. Saito, I, Miyamura, T. (1992) Expression of the amino-terminal half of the NSI region of the hepatitis C virus genome and detection of an antibody to the expressed protein in patients with liver disease, Journal of General Virology 73:2151 -2154.

9. Krug RM, Yuan W. Noah DL, Latham AG. (2003) Warfare between human influenza viruses and human cells: the roles of the viral NS 1 protein, Virology 309: 181 -9.

1 O. Nietzert, E, Beck, E, De Mello, PA, Comes, I, Bergmann, IF (1991) Expression of the apEthovirus RNA polymerase gene in Escherichia cold and its use with other bioengineered nonstructural antigens in detection of late persistent infections, Virology l 84:799-804.

11. Ozaki 1l, Sugiura T. Sugita S. Imagawa H. Kida Il. (2001) Detection of antibodies to the nonstructural protein (NS 1) of influenza A virus allows distinction between vaccinated and infected horses, Veterinary Microbiology 82:111-119.

12. Yasuda J. Nakada S. Kato A, Toyoda T. Ishihama A. (1993) Assembly of influenza virus: association of the NS2 protein with virion matrix, Virology 196:249-55.

Claims

1. A method for diagnosing avian influenza virus infection in a bird, the method comprising the following steps: (a) providing an antibody sample from a bird to be tested; and (b) determining whether the sample comprises antibodies to non structural protein 1 (NS 1) of an avian influenza virus the presence of anti-NS I antibodies being indicative of infection of the bird with avian influenza virus.

2. A method according to Chin I wherein the bird to be tested is selected from the group consisting chickens, turkeys, ducks, geese, quails, ostrich, emus and exotic birds.

3. A method according to Claim 1 or 2 wherein the bird has been vaccinated prior to testing.

4. A method according to Claim 2 wherein the bird to be tested is a chicken or a turkey.

5. A method according to any one of Claims I to 4 wherein the antibody sample is a serum sample or an egg yolk sample.

6. A method according to Claim 5 wherein the sample is diluted prior to testing.

7. A method according to any one of Claims 1 to 6 wherein step (b) comprises contacting the sample with a non-structural protein 1 (NS 1) of an avian inflecnza virus, and determining whether antibodies in the sample to be tested bind thereto.

8. A method according to Claim 7 wherein the NSI protein is from an avian influenza virus strain selected from the group consisting of A/ty/ltaly/4426/00 (H7N1) LPAI, A/ty/ltaly/7159/02 (H7N3) LPAI and A/avian/NL/2586/03 HPAI.

9. A method according to Claim 7 wherein the NSI protein is from avian influenza virus strain A/ty/Italy/4426/00 (H7NI) LPAI.

10. A method according to Claim 7 wherein the NS1 protein consists Is of or comprises the amino acid sequence of SEQ ID NO: 1, or a fragmcut thereof which exhibits substantially the same antigenic properties as a protein of SEQ ID NO: 1.

11. A method according to Claim 7 wherein the NS1 protein is from so avian influenza virus strain A/ty/ltaly/7159/02 (H7N3) LPAI.

12. A method according to Claim 7 wherein the NS1 protein consists of or comprises the amino acid sequence of SEQ ID NO: 3, or a fragment thereof which exhibits substantially the same antigenic :5 properties as a protein of SEQ ID NO: 3.

13. A method according to Claim 7 wherein the NS1 protein is from avian influenza virus strain A/avian/NL/2586/03 HPAI.

14. A method according to Claim 7 wherein the NS1 protein consists of or comprises the amino acid sequence of SEQ ID NO: 5, or a fragment thereof which exhibits substantially the same antigenic properties as a protein of SEQ lD NO: 5.

15. A method according to any one Claims 7 to 14 wherein the NS1 protein is a recombinant NS I protein.

16. A method according to Claim 15 wherein the NS 1 protein is lo derived from transformed insect cells.

17. A method according to Claim 16 wherein the insect cells are Trichoplusia hi cells.

s

18. A method according to any one of Claims 7 to 17 wherein the NS 1 protein is present in a cell.

19. A method according to any one of Claims 7 to 17 wherein the NS I protein is present in a cell Iysate.

20. A method according to any one of Claims 7 to 17 wherein the NSI protein is immobilised on a surface.

21. A method according to Claim 20 wherein the surface is a surface as of a multiwell plate.

22. A method according to any one of Claims 1 to 21 wherein step (b) is carried out by indirect ELISA.

23. A method according to any one of Claims 1 to 21 wherein step (b) is carried out by indirect IFA.

24. A method of monitoring the spread of avian influenza virus s infection in a population of birds comprising selecting a bird from the population to be tested and determining whether the bird is infected with avian influenza virus using a method according to any one of Claims 1 to 23.

lo

25. A method of preventing the spread of avian influenza virus infection in a population of birds comprising selecting a bird from the population to be tested, determining whether the bird is infected with avian influenza virus using a method according to any one of Claims 1 to 23, and destroying the bird if it is found to be infected.

26. A method according to Claim 25 further comprising destroying birds which have been exposed to the infected bird.

so

27. A kit of parts for performing an assay according to any one of Claims 1 to 26 comprising a non-structural protein 1 (NS1) of an avian influenza virus and means for detecting the binding of antibodies thereto.

:5

28. A kit according to Claim 27 wherein the NS1 protein is from an avian influenza virus strain selected from the group consisting of A/ty/Ttaly/4426/00 (H7N1) LPAI, A/ty/Italy/7159/02 (H7N3) LPAI and A/avian/NL/2586/03 HPAI.

29. A kit according to Claim 27 wherein the NS1 protein is from avian influenza virus strain A/ty/Italy/4426/00 (H7Nl) LPAI.

30. A kit according to Claim 27 wherein the NS 1 protein consists of or comprises the amino acid sequence of SEQ ID NO: 1, or a fragment thereof which exhibits substantially the same antigenic properties as a protein of SEQ lD NO: 1.

31. A kit according to Claim 27 wherein the NS1 protein is from avian lo influenza virus strain A/ty/Italy/7159/02 (H7N3) LPAI.

32. A kit according to Claim 27 wherein the NS I protein consists of or comprises the amino acid sequence of SEQ ID NO: 3, or a fragment thereof which exhibits substantially the same antigenic Is properties as a protein of SEQ ID NO: 3.

33. A kit according to Claim 27 wherein the NS1 protein is from avian influenza virus strain A/avian/NL/2586/03 HPAI.

so

34. A kit according to Claim 27 wherein the NS 1 protein consists of or comprises the amino acid sequence of SEQ ID NO: 5, or a fragment thereof which exhibits substantially the same antigenic properties as a protein of SEQ ID NO: 5.

35. A kit according to any one Claims 27 to 34 wherein the NS1 protein is a recombinant NS1 protein.

36. A kit according to Claim 35 wherein the NSl protein is derived from transformed insect cells.

37. A kit according to Claim 36 wherein the insect cells are Trichoplusia ni cells.

38. A kit according to any one of Claims 27 to 37 wherein the NS1 protein is present in a cell.

39. A kit according to any one of Claims 27 to 37 wherein the NS1 protein is present in a cell Iysate.

lo

40. A kit according to any one of Claims 27 to 39 further comprising a surface upon which the NS1 protein is immobilized or means for producing such as surface.

41. A kit according to Claim 40 wherein the surface is a surface of a multi-well plate.

42. A kit according to any one of Claims 27 to 41 further comprising a negative control.

43. A kit according to any one of Claims 27 to 42 further comprising a positive control.

44. A kit according to any one of Claims 27 to 43 for use with an ELISA.

45. A kit according to Claim 44 further comprising a secondary antibody with specificity for antibodies from the bird to be tested.

46. A kit according to Claim 45 wherein the secondary antibody is so anti- chicken IgG antibody.

47. A kit according to Claim 45 wherein the secondary antibody is antiturkey IgG antibody.

48. A kit according to any one of Claims 45 to 47 wherein the secondary antibody is conjugated to horseradish pcroxidase.

49. A kit according to Claim 48 further comprising a horseradish peroxidase substrate.

50. A kit according to Claim 49 wherein the horseradish peroxidase substrate is hydrogen peroxide.

51. A kit according to any one of Claims 44 to 50 further comprising 2,2'-azino-di-3-ethylbenzthiazoline sulfonic acid (ARTS).

52. A kit according to any one of Claims 44 to 51 further comprising a blocking agent.

53. A kit according to Claim 52 wherein the blocking agent comprises defatted milk powder.

54. A kit according to Claim 53 wherein the dcfatted milk powder is dissolved in phosphate-buffered saline.

55. A kit according to any one of Claims 44 to 54 further comprising a washing agent.

56. A kit according to Claim 55 wherein the washing agent comprises so a detergent.

57. A kit according to Claim 56 wherein the detergent is polyoxyethylene sorbitan monolaurate polysorbate 20.

58. A kit according to Claim 56 wherein the detergent is dissolved in phosphate-buffered saline.

59. A kit according to any one of Claims 44 to 58 further comprising a stop reagent.

60. A kit according to any one of Claims 27 to 43 for use with an indirect immunofluorescence assay (iIFA).

61. A kit according to Claim 60 further comprising a secondary is antibody with specificity for antibodies from the bird to be tested.

62. A kit according to Claim 61 wherein the secondary antibody is antichicken IgG antibody.

fi3. A kit according to Claim 61 wherein the secondary antibody is antiturkey IgG antibody.

64. A kit according to any one of Claims 61 to 63 wherein the secondary antibody is conjugated to fluorescein isothiocyanate.

65. A method for diagnosing avian influenza virus infection in a bird substantially as herein described with reference to the description.

66. A method of monitoring the spread of avian influenza virus infection in a population of birds substantially as herein described

with reference to the description.

67. A method of preventing the spread of avian influenza virus infection in a population of birds substantially as herein described

with reference to the description.

68. A kit of parts for performing an assay according to any one of lo Claims I to 27 substantially as herein described with reference to

the description.