WO2008141400A1

WO2008141400A1 - A novel aetiologic agent of acute gastroenteritis (age), diagnostic methods and therapeutic treatment thereof

Info

Publication number: WO2008141400A1
Application number: PCT/AU2008/000746
Authority: WO
Inventors: Rodney Mark Ratcliff; Jane Louise Arthur
Original assignee: Medvet Science Pty Ltd
Priority date: 2007-05-24
Filing date: 2008-05-26
Publication date: 2008-11-27

Abstract

A novel aetiologic agent associated with acute gastroenteritis (AGE), referred to as Adelavirus, is disclosed. The invention particularly relates to an isolated single-stranded DNA virus referred to as Adelavirus, polynucleotide molecules encoding Adelavirus or portions thereof (eg the viral coat protein(s)), isolated protein(s) of the virus, and assays for the detection of the virus (eg for patient diagnosis).

Description

A NOVEL AETIOLOGIC AGENT OF ACUTE GASTROENTERITIS (AGE), DIAGNOSTIC METHODS AND THERAPEUTIC TREATMENT THEREOF

FIELD OF THE INVENTION The present invention relates to a novel aetiologic agent associated with acute gastroenteritis

(AGE), herein referred to as Adelavirus. More particularly, the invention relates to polynucleotide molecules encoding Adelavirus or portions thereof (eg the viral coat protein(s)), isolated protein(s) of the virus, and assays for the detection of the virus (eg for patient diagnosis). Further, the invention relates to the recombinant expression of Adelavirus protein(s) and antigenic fragments thereof, for eliciting antibodies and incorporation into a composition for use as a vaccine.

INCORPORATION BY REFERENCE

This patent application claims priority from: - AU 2007902783 titled "Novel virus" filed on 24 May 2007.

The entire content of this application is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

AGE is a common illness affecting all age groups and populations worldwide and therefore presents a significant public health concern. The average incidence of AGE in developed countries is one episode per person per year¹³ while in communities with inadequate sanitation, the incidence is greater. Globally, approximately three million deaths annually are estimated to be diarrhoea related¹³. Amongst young children and the elderly, the rate of illness is higher and the presentation of disease is more severe. However, for such a significant disease, present understanding of causality remains incomplete, although viral infection is attributable to the majority of reported AGE related cases.

Viruses such as rotavirus and adenovirus were first described over 30 years ago and have been implicated in diarrhoeal diseases; with rotavirus being the most common known cause of AGE. However, for many years, it was suspected that other viruses might also be responsible for AGE cases, but identification of such viruses were hampered by difficulties in, for example, viral culture and sensitivity limitations of available assays. More recently it has been found that the human calicivirus genus Norovirus (formerly Norwalk-like virus), is the most common cause of epidemic outbreaks of AGE (typically occurring in hospitals, aged care facilities, schools, military camps, and cruise ships) across all age groups⁵. Further, an association with AGE has now been established for a second human calicivirus genus, namely Sapovirus (formerly Sapporo-like virus or classic calicivirus), but these cases are less frequent than AGE cases caused by noroviruses, they cause symptoms that are less severe than both rotavirus and norovirus², and are mainly restricted to children that are less than 5 years of age. Another viral genus that has more recently been associated with AGE are astroviridiae. Human astroviruses have been reported to be particularly associated with AGE in children, with prevalence rates of 1.4% - 6.6% in Australia¹⁵, 8.6% in Thailand⁹, 6.5% in Mexico⁷, 4.9% in Spain⁸ and 5.9% in Japan¹⁷, although a greater frequency of illness (39%) has been reported in children less than 1 year old¹⁷, particularly in communities lacking adequate sanitation¹¹. Further, torovirus, picobirnavirus, picotrinavirus, pestivirus and coronavirus have all been implicated as potential causative viruses of AGE, although their true association with illness remains uncertain¹'⁴'⁶' ¹⁰- ¹⁶.

Despite this work to identify further causative agents of AGE, both in child and adult populations, the pathogen causing AGE remains unidentified in up to 70% of cases. Indeed, previous studies of AGE have found a "diagnostic void" of infection in which 59%¹⁹, 67%¹², 63%³, 40%¹⁸ and

46%¹⁴ of cases have been unattributable to any known bacterial or viral pathogens. Naturally, this is of considerable concern, since the diagnosis in many of these cases would allow a more complete clinical assessment of the patient thereby facilitating a proper assessment of the severity and likely progression of the illness, as well as recognition of the most appropriate available treatment. Further, there are considerable wider public health implications caused by the presence of a diagnostic void inasmuch that AGE cases that are unattributable to any known AGE-causing pathogen thwart attempts to identify the epidemiological association of such cases to sources or origins of illness which, in turn, prevents any opportunity to undertake intervening actions to limit spread of the illness (eg with an endemic outbreak) or prevent future illness (eg by identifying improvements to food handling practices).

The present applicant has previously undertaken a comprehensive prospective case control study of AGE in children presenting to hospital (unpublished) in an attempt to "close" the diagnostic void. This involved assessing whether the combination of a strict case definition (ie to prevent the inclusion of non-AGE cases) and the use of newly developed sensitive PCR assays for virus detection¹⁵, which had previously only been infrequently included in routine diagnostic screening for viruses (including norovirus genogroups 1 and 2, sapovirus, adenovirus and astrovirus), would substantially reduce, or even eliminate, the diagnostic void. However, despite some success in closing the diagnostic void, this study failed to identify a causal agent in 27% of cases. Subsequently, to determine whether unknown pathogens may be responsible for some or all of these AGE cases remaining undiagnosed, the present applicant identified, and has since characterised, a novel AGE associated virus, herein referred to as Adelavirus. Further to the clinical and public health benefits of diagnosing previously undiagnosed AGE cases, it is considered that the identification and characterisation of this previously unknown causative agent may also assist in the development of novel therapies and therapeutic agents for AGE prevention (eg vaccines and antiviral compounds and medicaments).

SUMMARY OF THE INVENTION Thus, in a first aspect, the present invention provides an isolated single-stranded DNA virus, characterised in that the virus comprises a viral DNA molecule comprising a nucleotide sequence that shows at least 73% sequence identity to the nucleotide sequence shown hereinafter as SEQ ID NO: 1.

In a second aspect, the present invention provides an isolated polynucleotide molecule comprising a nucleotide sequence that shows at least 73% sequence identity to the nucleotide sequence shown hereinafter as SEQ ID NO: 1.

The polynucleotide molecule of the second aspect encodes four viral proteins, namely a non- structural protein (NSl), a nucleoprotein (NPl), and two viral coat proteins (VPl and VP2).

In a third aspect, the present invention provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding a protein that comprises an amino acid sequence showing at least 75%, preferably at least 75.8%, more preferably at least 85%, even more preferably at least 95%, and still even more preferably at least 98% sequence identity to an amino acid sequence selected from the group consisting of the amino acid sequences shown hereinafter as SEQ ID NOS: 2 - 5 or a naturally occurring variant thereof.

The polynucleotide molecule of the second or third aspect may be used to express the encoded virus or an encoded protein by, for example, recombinant methodology involving cloning of the polynucleotide molecule into a suitable expression cassette or vector and thereafter introducing the expression cassette or vector into a suitable host cell.

Thus, in a fourth aspect, the present invention provides a cell transformed with the polynucleotide molecule of the second or third aspect. And, in a fifth aspect, the present invention provides an isolated protein comprising an amino acid sequence showing at least 70.3%, preferably at least 75%, more preferably at least 85%, even more preferably at least 95%, and still even more preferably at least 98% sequence identity to any one of the amino acid sequences hereinafter shown as SEQ ID NOS: 2 - 5 or a naturally occurring variant thereof.

The proteins of the invention can be employed in a variety of applications. For example, the proteins can be used as reagents, including control reagents, in various assays (including assays for antibodies against the virus of the present invention). Further, the proteins can be used to elicit antibodies against the virus of the present invention, and form the basis (ie immunogen) of vaccine compositions to protect a human subject against infection by said virus. Moreover, fragments of the proteins of the present invention, in particular antigenic fragments, can be used in such applications.

Accordingly, in a sixth aspect, the present invention provides an antigenic fragment of the protein of the fifth aspect.

In a seventh aspect, the present invention provides a virus-like particle (VLP) comprising a protein comprising an amino acid sequence showing at least 78.3% sequence identity to any one of the amino acid sequences shown hereinafter as SEQ ID NO: 4 and/or 5 or a naturally occurring variant thereof.

In an eighth aspect, the present invention provides a composition for eliciting an immune response in an animal, in particular an antibody response, said composition comprising one or more of: (i) an attenuated virus of the first aspect;

(ii) a deactivated preparation of the virus of the first aspect;

(iii) a polynucleotide molecule according to the third aspect comprising a nucleotide sequence encoding a protein comprising an amino acid sequence substantially corresponding to that shown hereinafter as SEQ ID NO: 4 or 5 or a naturally occurring variant thereof, or an antigenic fragment of said protein;

(iv) a transformed cell according to the fourth aspect expressing a protein comprising an amino acid sequence substantially corresponding to that shown hereinafter as SEQ ID NO: 4 or 5 or a naturally occurring variant thereof, or an antigenic fragment of said protein; (v) the protein of the fifth aspect; (vi) the antigenic fragment of the sixth aspect; and

(vii) the virus-like particle (VLP) of the seventh aspect.

The composition can be a vaccine composition.

In a ninth aspect, the present invention provides an isolated antibody or fragment thereof which specifically binds to the virus of the first aspect.

Preferably, the antibody or fragment thereof specifically binds to the protein of the fifth aspect, wherein said protein comprises an amino acid sequence substantially corresponding to that shown hereinafter as SEQ ID NO: 4 or 5 or a naturally occurring variant thereof.

In a tenth aspect, the present invention provides a method for the detection of the virus of the first aspect in a suitable sample from a subject, wherein said method comprises introducing to said sample a reagent that specifically binds to said virus or a viral protein thereof, and detecting any binding between said reagent and virus or viral protein thereof.

The reagent of the tenth aspect may, optionally, be an antibody or fragment thereof which specifically binds to said virus or a viral protein thereof.

Alternatively, the reagent of the tenth aspect may, optionally, comprises at least one polynucleotide for amplifying a target nucleotide(s) sequence specific to said virus, and said method comprises subjecting said sample to conditions for amplifying a target nucleotide(s) sequence specific to said virus, and detecting the generation of any amplification products.

Further, said reagent may comprises at least one polynucleotide which hybridises to said target nucleotide sequence(s) under high stringency conditions, wherein said method comprises detecting the presence of a target nucleotide(s) sequence specific to said virus.

The methods of the tenth aspect can be used to diagnose AGE cases that presently go undiagnosed. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 provides the nucleotide sequence (SEQ ID NO: 1) for the complete coding region of Adelavirus genogroup 1 strain Avl-153. It is considered that the nucleotide sequence may lack approximately 200 bases at the N- and C- terminal regions;

Figure 2 provides a nucleotide sequence alignment for Adelavirus genogroup 1 strain Avl-153, Adelavirus genogroup strain Av2-471, and partial sequence alignment of Adelavirus genogroup 2 strain Av2-93 with the closest related human bocavirus strains. Open reading frames (ORFs) for non-structural protein (NSl), nucleoprotein (NPl), viral proteins 1 and 2 (VPl and VP2) are indicated. Also indicated are the binding locations of nested primers for the Adelavirus molecular detection assays of the invention (primary reaction forward primers; Adelavirus GI outer forward, Adelavirus outer forward, Adelavirus real time forward, and reverse primers; Adelavirus GI outer reverse, Adelavirus outer reverse, Adelavirus real time reverse and specific RT reverse primers are indicated, and secondary reaction forward; Adelavirus GI inner forward, Adelavirus inner forward, and reverse, Adelavirus GI inner reverse, Adelavirus inner reverse, primers are indicated and the binding location of the molecular probes used in the real time assays (Adel/Boca probe is indicated);

Figure 3 provides an amino acid sequence alignment for Adelavirus genogroup 1 strain Avl-153, Adelavirus genogroup strain Av2-471, and partial sequence alignment of Adelavirus genogroup 2 strain Av2-93 with the closest related human bocavirus strains; (A) shows an alignment for nonstructural protein (NSl), (B) for nucleoprotein (NPl), (C) for viral protein 1 and viral protein 2 (VPl and VP2);

Figure 4 shows a dendrogram of genetic dissimilarity among unique strains of Adelavirus belonging to genogroups 1 and 2;

Figure 5 illustrates the primer structure and design for generating VPl and VP2 cDNA inserts for cloning into Gateway® and Baculodirect® expression vectors;

Figure 6 provides a schematic representation of the strategy for cloning VPl or VP2 cDNA inserts into a Gateway® vector;

Figure 7 provides a schematic representation of the strategy for cloning VPl or VP2 cDNA inserts into a Baculodirect® vector; and Figure 8 provides an electron microscopy (EM) image of virus-like particles comprising VP2 protein. The VLPs may be empty or packaged with cellular DNA.

DETAILED DESCRIPTION OF THE INVENTION

The present applicant has identified and characterised a novel, negative sense DNA virus, herein referred to as Adelavirus, which is associated with AGE. Preliminary studies have indicated that this virus has a clinically significant incidence rate with AGE cases presently going undiagnosed. Accordingly, the present applicant has identified and/or developed, inter alia, polynucleotide molecules encoding Adelavirus or portions thereof (eg the viral coat protein(s) and antigenic fragments thereof), isolated protein(s) of the virus, and assays for the detection of the virus (eg for patient diagnosis).

Thus, in a first aspect, the present invention provides an isolated single-stranded DNA virus, characterised in that the virus comprises a viral DNA molecule comprising a nucleotide sequence that shows at least 73% sequence identity to SEQ ID NO: 1.

The virus of the first aspect is characterised in that it comprises a viral DNA molecule comprising a nucleotide sequence that shows at least 75%, preferably at least 78.9%, more preferably at least 85%, even more preferably at least 95%, and still even more preferably at least 98% sequence identity to the nucleotide sequence shown as SEQ ID NO: 1.

Percentage values of nucleotide sequence identity given herein are calculated using the GenBank algorithm blastn.

Most preferably, the virus is characterised in that it comprises a viral DNA molecule comprising a nucleotide sequence substantially corresponding to the nucleotide sequence shown as SEQ ID NO: 1 or a naturally occurring variant thereof.

As used herein, the term "isolated", when used in relation to the virus, is to be understood as referring to a virus which is essentially free of viral particles of another species or strain, components thereof and/or other exogenous biological materials such as exogenous proteins, polypeptides, peptides and nucleic acids. As such, the isolated virus may be present in a preparation of identical viral particles. Such a preparation will comprise no more than 10% (by weight) of exogenous biological materials, and may be prepared by any of the methods well known to persons skilled in the art including affinity chromatography and sedimentation according to density.

The term "substantially corresponding" as used herein in relation to nucleotide sequences, is to be understood to encompass minor variations in the particular nucleotide sequence which due to degeneracy in the DNA code, do not result in a change to any encoded protein, polypeptide or peptide. Further, the term is to be understood as encompassing minor variations in the particular nucleotide sequence which may be required to enhance expression in a particular system but which do not result in any substantial alteration in the biological activity of any encoded protein, polypeptide or peptide.

The term "naturally occurring variant" as used herein in relation to a nucleotide sequence, is to be understood as referring to any nucleotide sequence that is derived from a naturally occurring variant strain of the virus. The naturally occurring variant sequence may, therefore, encode one or more amino acid substitutions, deletions and/or additions within any one or more of any encoded protein, polypeptide or peptide, but would generally vary from the particular nucleotide sequence by no more than 1% as determined by the GenBank algorithm blastn. Further, naturally occurring variant sequences may contain one or more silent nucleotide substitutions.

In a second aspect, the present invention provides an isolated polynucleotide molecule comprising a nucleotide sequence that shows at least 73% sequence identity to the nucleotide sequence shown as SEQ ID NO: 1.

The isolated polynucleotide molecule of the second aspect comprises a nucleotide sequence that shows at least 75%, preferably at least 78.9%, more preferably at least 85%, even more preferably at least 95%, and still even more preferably at least 98% sequence identity to the nucleotide sequence shown as SEQ ID NO: 1.

Most preferably, the isolated polynucleotide molecule comprises a nucleotide sequence substantially corresponding to the nucleotide sequence shown as SEQ ID NO: 1 or a naturally occurring variant thereof.

The term "isolated", as used herein in relation to a polynucleotide molecule, is to be understood as referring to a polynucleotide molecule which is essentially free of polynucleotide molecules of another species or strain, components thereof and/or other exogenous biological materials such as exogenous proteins, polypeptides and peptides. As such, the isolated polynucleotide molecules may be present in a preparation of identical polynucleotide molecules. Such a preparation will comprise no more than 10% (by weight) of polynucleotide molecules of another species or strain, components thereof and/or exogenous biological materials, and may be prepared by any of the methods well known to persons skilled in the art including ion-exchange chromatography, affinity chromatography and sedimentation according to density.

The polynucleotide molecule of the second aspect encodes four viral proteins, namely a nonstructural protein (NSl), a nucleoprotein (NPl), and two viral coat proteins (VPl and VP2). However, the present invention also extends to polynucleotide molecules comprising a nucleotide sequence encoding any combination of two or three of these viral proteins (eg a polynucleotide molecule encoding VPl and VP2) or, more preferably, any one of these viral proteins (eg a polynucleotide molecule encoding VPl alone).

Thus, in a third aspect, the present invention provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding a protein that comprises an amino acid sequence showing at least 75%, preferably 75.8%, more preferably at least 85%, even more preferably at least 95%, and still even more preferably at least 98% sequence identity to an amino acid sequence selected from the group consisting of those shown hereinafter as SEQ ID NOS: 2 - 5 or a naturally occurring variant thereof.

Percentage values of amino acid sequence identity given herein are calculated using the GenBank algorithm tblastx.

Most preferably, the isolated polynucleotide molecule of the third aspect comprises a nucleotide sequence encoding a protein that comprises an amino acid sequence substantially corresponding to any one of the amino acid sequences shown as SEQ ID NOS: 2 - 5 or a naturally occurring variant thereof.

The term "naturally occurring variant" as used herein in relation to an amino acid sequence, is to be understood as referring to any amino acid sequence that is derived from a naturally occurring protein variant of the virus. The naturally occurring variant sequence may, therefore, comprise one or more amino acid substitutions, deletions and/or additions, but would generally vary from the particular amino acid sequence by no more than 1% as determined by the GenBank algorithm tblastx. The term "substantially corresponding" as used herein in relation to an amino acid sequence, is to be understood as encompassing minor variations in the particular amino acid sequence(s) which do not result in any significant alteration of the biological activity of its derivative protein, polypeptide or peptide. These variations may include conservative amino acid substitutions. Exemplary conservative amino acid substitutions are provided in Table 1 below. Particular conservative amino acids envisaged are: G, A, V, I, L, M; D, E; N, Q; S, T; K, R, H; F, Y, W, H; and P, Nα-alkylamino acids.

Table 1 Exemplary conservative amino acid substitutions

Conservative Substitutions

Ala VaI*, Leu, He

Arg Lys*, GIn, Asn

Asn GIn*, His, Lys, Arg, Asp

Asp GIu*, Asn

Cys Ser

GIn Asn*, His, Lys,

GIu Asp*, γ-carboxyglutamic acid (GIa)

GIy Pro

His Asn, GIn, Lys, Arg*

He Leu*, VaI, Met, Ala, Phe, norleucine (NIe)

Leu NIe, He*, VaI, Met, Ala, Phe

Lys Arg*, GIn, Asn, ornithine (Orn)

Met Leu*, He, Phe, NIe

Phe Leu*, VaI, He, Ala

Pro GIy*, hydroxyproline (Hyp), Ser, Thr

Ser Thr

Thr Ser

Trp Tyr

Tyr Tφ, Phe*, Thr, Ser

VaI He, Leu*, Met, Phe, Ala, NIe

* indicates preferred conservative substitutions

The polynucleotide molecule of the second or third aspect may be used to express the encoded virus or protein by, for example, recombinant methodology involving cloning of the polynucleotide molecule into a suitable expression cassette or vector and thereafter introducing the expression cassette or vector into a suitable host cell. Suitable expression vectors may include functional sequences such as a multiple cloning site, detection tags (eg glutathione-S-transferase (GST) or green fluorescent protein (GFP)), tags for downstream purification (eg histidine tags (His)), or sequences encoding adjuvant molecules. Particularly preferred examples of suitable expression vectors include the Gateway® vector and the Baculodirect® vector.

In a fourth aspect, the present invention provides a cell transformed with the polynucleotide molecule of the second or third aspect.

The transformed cell may be selected from bacterial cells such as Escherichia coli, insect cells such Spodopterafrugiperda cells (particularly, Sf21 cells), and mammalian cells such as Vero cells (derived from primate kidney epithelial cells), HEK 293 cells (derived from human embryonic kidney cells), HeLa cells (derived from human epitheloid carcinoma cells), cell lines of human gastrointestinal origin such as Caco-2 (derived from colorectal adenocarcinoma cells), Kato-III and AGS cells (derived from gastric carcinoma cells), Int-407 (derived from small intestine cells) and HuTu-80 (derived from duodenal adenocarcinoma cells). The cell may be transformed using any of the methods well known to persons skilled in the art including direct uptake, transduction, or f-mating. The transformed polynucleotide molecule may be maintained in a non-integrated form (eg in a non-integrated plasmid expression vector), or alternatively, may be integrated into the genome of the transformed cell.

The transformed cell can be employed in a variety of applications that will be readily apparent to persons skilled in the art. For example, the transformed cell may be used for the culture of the virus (eg to provide a viral stock) or for the expression of viral protein(s). Further, the transformed cell may be applied to the investigation of viral gene expression and replication in cell culture, for the detection of protective antibodies (ie neutralising antibodies) to virus or viral particles, or for the identification of epitopes to elicit an immune response. Still further, the transformed cell may be used in challenge studies of whole organisms (eg an animal host), and in the screening of antiviral compounds for the treatment of viral infection.

Where the transformed cell is intended for the culture of the virus of the first aspect, the cell may be tested for its ability to support viral replication by assessing the capacity of the cell to bind complete virus or viral coat proteins. These binding studies will provide an indication of potential infectivity. Successful expression of the virus can be determined by, for example, Western immunoblot analysis for viral antigens, or electron microscopy performed on viral pellets purified by ultracentrifugation through 25% sucrose.

Where the transformed cell is intended for the expression of viral protein(s), the successful expression of the viral protein(s) can be determined by Western immunoblot analysis for viral antigens in a cell lysate or, otherwise, the cell supernatant where expression of the viral protein(s) has been in fusion with a suitable secretory signal peptide. Accordingly, the present invention provides for the recombinant expression of the proteins of the novel virus. The proteins can, however, be alternatively produced by isolation from a suitable virus-containing sample (eg a sample of a virus-expressing transformed cell culture or a sample obtained from a human subject infected with the virus of the present invention).

Thus, in a fifth aspect, the present invention provides an isolated protein comprising an amino acid sequence showing at least 70.3%, preferably 75%, more preferably at least 85%, even more preferably at least 95%, and still even more preferably at least 98% sequence identity to any one of the amino acid sequences shown as SEQ ID NOS: 2 - 5 or a naturally occurring variant thereof.

Most preferably, the isolated protein comprises an amino acid sequence substantially corresponding to any one of the amino acid sequences shown as SEQ ID NOS: 2 - 5 or a naturally occurring variant thereof.

The term "isolated" as used herein in relation to a protein, is to be understood as referring to a protein which is essentially free of other proteins and/or polypeptides and peptides, components thereof and/or other exogenous biological materials such as nucleic acids. As such, the isolated protein may be present in a preparation of identical proteins. Such a preparation will comprise no more than 10% (by weight) of other protein, polypeptides or peptides, components thereof and/or exogenous biological materials, and may be prepared by any of the methods well known to persons skilled in the art including ion-exchange chromatography, affinity chromatography and sedimentation according to density.

It is to be understood that the protein of the present invention may be a fusion protein (eg a fusion with Human serum albumin (HSA)).

The proteins of the invention can be employed in a variety of applications that will be readily appreciated by persons skilled in the art. For example, the proteins can be used to elicit antibodies against the virus of the present invention, and form the basis (ie immunogen) of vaccine compositions to protect a human subject against infection by said virus. Moreover, fragments of the proteins of the present invention, in particular antigenic fragments, can be used in such applications. Antigenic fragments can be identified by any of the methods well known to persons skilled in the art such as random fragmentation of the proteins and assaying the resultant fragments against antibodies to the virus of the present invention. Alternatively, antigenic fragments may be identified or designed following recognition of putative epitopes within the proteins (eg using epitope predicting algorithms).

Such fragments can be produced by recombinant methodology or may otherwise be synthesised using standard peptide synthesis techniques and apparatus. Typically, the fragments will be of a length in the range of 10 to 50, more preferably 15 to 30. amino acids in length.

The viral coat proteins °f 0 p^ r_\ 1 VP2) may also be assembled into virus-like particles (VLPs w

Thus, in a seventh aspect, virus-like particle (VLP) comprising a protein comprising an aminυ aciu sequence snowing at least 78.3% sequence identity to any one of the amino acid sequences shown as SEQ ID NO: 4 and/or 5 or a naturally occurring variant thereof.

VLPs can be expressed and purified by, for example, cloning a polynucleotide molecule encoding viral coat protein into an expression cassette wherein the cassette, optionally, comprises a purification tag. As VLPs are surface structural proteins they comprise viral surface antigens. Accordingly, purified VLPs are useful in many applications well known to persons skilled in the art including, for example, control reagents, in various assays (including diagnostic assays for antibodies against the virus of the present invention or in screening for therapeutic compounds useful in the treatment of Adelavirus infection). Further, VLPs may be useful in the development of vaccines or may themselves be used as an immunogen. Moreover, purified VLPs can be used to elicit antibodies against Adelavirus using standard methodologies. In an eighth aspect, the present invention provides a composition for eliciting an immune response in an animal, in particular an antibody response (ie humoral immune response), said composition comprising one or more of: (i) an attenuated virus of the first aspect;

(ii) a deactivated preparation of the virus of the first aspect (ie a viral preparation, such as a sonicated viral preparation, that is devoid of infective virus or which is otherwise unable to cause a viral infection of a cell typically prone to infection);

(iii) a polynucleotide molecule according to the third aspect comprising a nucleotide sequence encoding a protein comprising an amino acid sequence substantially corresponding to that shown as SEQ ID NO: 4 or 5 or a naturally occurring variant thereof, or an antigenic fragment of said protein;

(iv) a transformed cell according to the fourth aspect expressing a protein comprising an amino acid sequence substantially corresponding to that shown as SEQ ID NO: 4 or 5 or a naturally occurring variant thereof, or an antigenic fragment of said protein;

(v) the protein of the fifth aspect;

(vi) the antigenic fragment of the sixth aspect; and

(vii) a virus-like particle (VLP) of the seventh aspect.

Such a composition may be a vaccine composition for use in protecting a human subject against infection with the virus of the present invention. Accordingly, such a composition may further comprise a pharmaceutically-acceptable adjuvant (eg alum and complete or incomplete Freund's adjuvant). A particularly preferred vaccine composition according to the present invention is a subunit vaccine composition comprising one or both of the proteins comprising the amino acid sequence shown as SEQ ID NO: 4 and 5 (ie VPl and/or VP2 of the virus of the present invention). Another particularly preferred vaccine composition is a DNA vaccine comprising a polynucleotide molecule encoding one or both of the proteins comprising the amino acid sequence shown as SEQ ID NO: 4 and 5 (ie VPl and/or VP2 of the virus of the present invention).

Alternatively, the composition of the eighth aspect may be a composition for eliciting a humoral immune response in an animal such as a mouse, rabbit or sheep for the purposes of preparing polyclonal or monoclonal antibodies against the virus of the invention.

In a ninth aspect, the present invention provides an isolated antibody or fragment thereof which specifically binds to the virus of the first aspect. Preferably, the antibody or fragment thereof specifically binds to the protein of the fifth aspect, wherein said protein comprises an amino acid sequence substantially corresponding to that shown as SEQ ID NO: 4 or 5 or a naturally occurring variant thereof.

The antibody may be a polyclonal or monoclonal antibody.

The antibody fragment may be selected from fragments produced through enzymatic cleavage of an antibody such as Fab and F(ab')₂ fragments, and recombinant antibody fragments such as single chain Fv (scFv) fragments.

The antibody and fragment thereof of the present invention can be employed in methods for the detection of the virus of the present invention. Such methods can be used to diagnose AGE cases that presently go undiagnosed. In turn, this allows a more complete clinical assessment of the patient thereby facilitating a proper assessment of the severity and likely progression of the illness, as well as recognition of the most appropriate available treatment.

Thus, in a tenth aspect, the present invention provides a method for the detection of the virus of the first aspect in a suitable sample from a subject, wherein said method comprises introducing to said sample a reagent that specifically binds to said virus or a viral protein thereof, and detecting any binding between said reagent and virus or viral protein thereof.

Preferably, the antibody or fragment thereof used in the method is an antibody or fragment thereof which specifically binds to the protein of the fifth aspect, wherein said protein comprises an amino acid sequence substantially corresponding to that shown as SEQ ID NO: 4 or 5 or a naturally occurring variant thereof.

The method of the tenth aspect may be conducted in accordance with any of the antibody- or antibody fragment-based immunoassay formats well known to persons skilled in the art. Accordingly, the method may be conducted in accordance with standard ELISA formats, or where the sample is a tissue section, in situ immunofluorescence formats. The antibody or fragment thereof used in the method of the ninth aspect, may therefore be directly conjugated to a detectable label or may otherwise be detected via a secondary antibody or fragment thereof directly conjugated to a detectable label. Suitable detectable labels include chromophores, fluorophores (eg fluorescein or FITC), radiolabels (eg ¹²⁵I), and enzymes such as horseradish peroxidase. These labels can be used in methods and systems that are well known to persons skilled in the art, and which provide for the automation or partial automation of the detection of the label (eg by a microplate reader or by use of a flow cytometer).

The sample may be subjected to a pre-treatment, for example nuclease treatment to eliminate potentially cross-reactive exogenous nucleic acids, or heat treatment to ensure release of viral nucleic acid from viral particles present in the sample.

The amplification of the target nucleotide sequence(s) may be in accordance with any amplification methodology well known to persons skilled in the art. Accordingly, the amplification may be conducted in accordance with a polymerase chain reaction (PCR) methodology using, for example, the "traditional" two primers, or otherwise, one or more degenerate primers (eg DOP-PCR), nested primers (eg nested amplification or hanging drop nested amplification¹⁵), or a multiplex format.

The detection of any amplification products may be achieved by, for example, amplicon size fractionation by agarose gel electrophoresis, or fluorescent or calorimetric detection of amplicon specific probes or labelled primers. The latter may preferably be incorporated into real time assays, which are more conducive to routine diagnostic use. Particular examples include real-time assays incorporating Qiagen Quantitech reagent (Qiagen Inc., Valencia, CA, United States of America).

Preferably, the target nucleotide sequence(s) will be a nucleotide sequence that is at least 50 nucleotides in length, however typically the target nucleotide sequence will be between about 80 and 750 nucleotides in length, more preferably between 150 and 600 nucleotides in length. Particularly preferred primers for use in the method of the tenth aspect include; a forward primer comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS 6 - 12, and a reverse primer comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS 13 - 22.

As used herein, the term "high stringency conditions" refers to conditions under which polynucleotide or oligonucleotide molecules can be used to hybridise to similar nucleic acid molecules. Such standard high stringency conditions are disclosed, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, 1989). More particularly, the term high stringency conditions refers to conditions which permit hybridisation to nucleic acid molecules having at least about 85%, more preferably, at least 95% sequence identity. In particular embodiments, high stringency conditions for DNA:DNA hybrids include conditions comprising an ionic strength of 0.1X SSC (0.157 M Na⁺) and a temperature of between about 28⁰C and about 4O⁰C, more preferably, between about 35⁰C and about 45⁰C.

The detection of any hybridisation between the probe sequence(s) and the target nucleotide sequence(s) can be achieved by any of the methods well known to persons skilled in the art. In a particular embodiment, the method of the tenth aspect is conducted in accordance with a Southern blot or dot blot methodology, and as such, detection may be achieved by labelling the probe sequence(s) with a suitable detectable label such as those mentioned above, and detecting the label of bound (ie hybridised) probe sequence(s).

The sample used in the methods of the tenth aspect will typically be a faecal sample, or otherwise be prepared from a faecal sample, of the subject, however tissue sections (ie from biopsies) and vomitus, and samples from blood or urine, may also be suitable. Further, other samples could include samples of environmental substrates such as water, sludge and soil, samples from food products such as shellfish and cold meats, and samples prepared from food preparation surfaces (eg using swabs). The sample may be pre-treated by, for example, filtration, separation or extraction methods to partly or completely purify or isolate, for example, virus, viral proteins and/or viral nucleic acid molecules or fractions containing these components. Pre-treated samples may, optionally, also be treated with one or more nucleases to eliminate potentially cross-reactive exogenous nucleic acids.

The methods of the tenth aspect can be used to diagnose AGE cases that presently go undiagnosed.

The present invention further provides a kit for the detection of the virus of the first aspect in a suitable sample. As would be readily appreciated by persons skilled in the art, the components of such a kit will vary considerably depending upon the means by which the virus is to be detected. For example, if the kit is to be used in accordance with an immunoassay, the kit may minimally comprise an antibody or fragment thereof which specifically binds to the protein of the fifth aspect, wherein said protein comprises an amino acid sequence substantially corresponding to that shown as SEQ ID NO: 4 or 5 or a naturally occurring variant thereof. On the other hand, if the kit is to be used in accordance with a molecular assay, the kit may minimally comprise one or more primers for amplifying the target nucleotide sequence(s) specific to the virus.

Where the present invention resides in an isolated polynucleotide molecule comprising a nucleotide sequence showing sequence identity or, otherwise, substantially corresponding, to a given nucleotide sequence, it is to be understood that the invention extends to a polynucleotide sequence comprising a complementary nucleotide sequence thereto.

The present invention is hereinafter further described by way of the following, non-limiting examples and accompanying figures.

EXAMPLES

Example 1 Detection of a novel etiologic agent of AGE

Materials and Methods

Selection of faecal samples and screening for known infectious agents

A summer cluster of AGE consisting of 69 cases and 69 matched controls were selected, which was part of a larger case control study into the aetiology of paediatric AGE. The case definition of three or more loose stools within the last 24 hours, with or without vomiting, and a duration prior to presentation of less than 7 days was required for patient entry into the study. Age matched controls (within 2 months for patients less than 6 months of age, within 9 months for patients 6 months to 2 years of age, within 12 months for 2-5 year old patients and within 14 months for patients older than 5 years) were selected from patients without AGE symptoms presenting to hospital at a similar time (median 10 days, maximum 34 days). Control samples (stools) were screened for viruses only. The prevalence of bacteria and parasites in the AGE cases was known to be low, and samples from this AGE cluster were selected on a comparative prevalence of cases in which the initial testing failed to detect a causal agent (26 of 69; ie 38% compared with 27% for the larger case control study).

Preparation of clinical samples To recover viral nucleic acid from the samples for DOP-PCR, a 10% faecal suspension in sterile phosphate-buffered saline was centrifuged and filtered through a 0.2μm filter to remove solids and bacteria. Contaminating free nucleic acids were degraded and removed by nuclease digestion before the sample was ultracentrifuged to concentrate viral particles. Total nucleic acids (RNA and DNA) were extracted using the RNeasy Mini Kit according to the manufacturer's instructions (Qiagen Inc.).

POP- PCR amplification of nucleic acids

An aliquot for RNA extraction was predigested with DNase and reverse transcribed, and primed with random decamers to produce cDNA. An extract of a known adenovirus-positive sample was included in the batch as a control. DOP-PCR amplification²⁰ was performed on cDNA and DNA fractions using a single DOP primer and biphasic amplification cycles.

DOP-PCR amplification²⁰ was achieved using a single degenerate oligonucleotide primer to prime the amplification reaction. A four base anchor at the primer 3' "head" allows the primer to bind at approximately 250 bp intervals to both sense and antisense strands, but depending on the anchor sequence and target nucleic acid, binding will occur at variable intervals up to several kilobases apart. The random hexamer "core" assists in stabilising binding during the extension step. Amplification occurs in two cycling phases of the one amplification reaction. During the first, low temperature annealing and slow temperature ramping assist the DOP primers (ie annealing using 3' head and hexamer nucleotides only) to anneal and prime replication of all genomic sequences present. After several cycles of amplification, amplicons begin to form which have the primer Tag "tail" sequence (commonly 15 -18 bases) incorporated at their termini.

In this example, modification of the four base anchor and the tag of the published DOP primer²⁰ was required before satisfactory performance could be obtained with the adenovirus control. During the second cycling phase, the primer 5' Tag "tail" anneals to further nucleotide regions, increasing the robustness of binding despite mismatches occurring at the random hexamer "core". Thus, the second phase can incorporate higher annealing temperatures and faster temperature ramping. DOP-PCR typically produced a ladder of products up to several Kb when examined after gel electrophoresis.

The degree of amplification achieved was assessed by testing the pre- and post-DOP-PCR adenovirus genome copy number using an in-house semi-quantative Taqman real time adenovirus PCR assay. Batches not achieving a 10⁴-fold increase in copy number as measured by the control were repeated.

Cloning and sequencing of DOP-PCR products

Successfully DOP-PCR amplified product was "shot gun" cloned into Topo TA Cloning® (Invitrogen Corporation, Carlsbad, CA, United States of America) vector and plated onto selective media, according to the manufacturer's instructions. Clones containing an insert were further amplified using a specific PCR primed with standard M 13 primers (Invitrogen Corporation) targeting the vector M13 binding sites. The Ml 3 -specific PCR products were examined by agarose gel electrophoresis and twenty products varying in size from 200 to 800 bps were selected for sequencing. The selected products were purified (AMPure; Agencourt Bioscience Corporation, Beverly, MA, United States of America), sequenced using Big Dye 3 terminator chemistry (ABI), unincorporated dye removed using CleanSeq (Agencourt Bioscience Corporation) and the sequence determined using an ABI 3730 sequencer (Applied Biosystems Inc., Foster City, CA, United States of America).

Rules for sequence analysis

Approximately 20 colonies from each amplification were edited using Kodon (Applied Maths, Keistraat, Belgium) to remove vector and DOP primer sequence and then examined for nucleotide and putative amino acid sequence homology to sequences already present in a database of clone sequences (Kodon) and in GenBank (blastn and tblastx). Sequences were screened against GenBank using blastn (nucleotide) and tblastx (amino acid) homology searches for viral-Iike sequences (VLS).

VLS were classified according to the highest degree of homology suggested by comparison of the translated amino acid sequence with the GenBank database (tblastx). Sequences that yielded bit scores and protein homologies of > 50% were categorised according to the organism of highest homology. Sequences with low protein homology to the database (ie score <50 bits) were subdivided into low homology to virus of eukaryotes or non-viral (including bacteriophages) on the basis of the highest hit in GenBank.

High homology VLS included sequences with a GenBank blastn (nucleotide homology) or tblastx (translated amino acid homology; each frame in both orientations, 6 putative translated sequences) score of >50 bits and homology > 50%. Such sequences have sufficient nucleotide or inferred amino acid homology to recognise similarity to a known viral family.

Low homology VLS with recognisable amino acid homology included sequences with little nucleotide homology, and while the tblastx score was <50 bits the tblastx homology to viral sequences was >50% for shorter lengths of sequence up to 50 amino acids. These represent potential viral functional domains.

Results and Discussion

Homology score of unknown sequences

Using the sequence analysis rules described above on two adenovirus positive control faecal samples, the majority of the clones (10 out of 11, and 13 out of 13) tested from each sample were found to contain adenovirus sequences. When testing the 69 samples of the undiagnosed AGE infections, approximately 20 clones per case were sequenced. Of these (totalling 935 clones), 76% had high protein homology (score >50 bits and homology > 50%) to known or hypothetical proteins in GenBank. Amongst these, 53% contained sequences with high homology to bacterial proteins, 6% to bacteriophages and 9% to human or eukaryote proteins (Table 2). Sequences that were unambiguously of bacterial, human, animal, plant or bacteriophage origin were discarded from further consideration. Homology to previously recovered VLS was noted. A further 8% of the total clones had high homology to known viral sequences. These sequences came from 4 samples from 3 different subjects. Table 2 Sequences of DOP-PCR generated fragments as analysed by tblastx homology searches of GenBank

The first batch of DOP-PCR generated 19 clones derived from a "patient 153" faecal sample. All 19 clones contained sequences which were from an unknown virus, herein referred to as Adelavirus (AvI-153). A second batch of DOP-PCR generated from the same sample yielded another 19 clones containing Adelavirus DNA (from the 22 tested). A second faecal sample from this subject, taken three days after the first, was also DOP-PCR amplified and a further 24 Adelavirus clones obtained (from the 30 tested).

A second subject ("patient 93") was identified who by subsequent sequence analysis of a 520bp amplicon was later found to be shedding virus belonging to a second Adelavirus genogroup. The existence of a second distinct strain of Adelavirus (Av2-93) was able to confirm the initial characterisation of the virus isolated from patient 153 and therefore provide early and unequivocal evidence of the new virus.

Example 2 Adelavirus sequence characterisation

Methods and Materials

Adelavirus contiguous nucleotide sequence

The 19 clones generated from the first DOP-PCR amplification referred to in Example 1 ranged in size from approximately 400 - 800bp and discontinuously contained nearly 30% of the Adelavirus genomic sequence, based on alignment with the sequence of its closest relative, human bocavirus. Additional sequence was obtained by testing further colonies from the original and subsequent cloning, and then by designing primers to the cloned sequences to amplify and sequence product spanning several gaps between the cloned sequences.

Once all of the Adelavirus sequence fragments obtained by DOP-PCR and gap sequencing had been joined to form a single large contiguous sequence, a semi-DOP-PCR approach was used to "walk out" from the known sequence towards both termini.

Three primers homologous to the known sequence and designated as Pl, P2 and P3 were constructed to extend in the direction of the unknown sequence. PCR products were amplified using the most upstream specific primer (Pl) in combination with a non-specific DOP primer. A second round of "nested" amplification with the next most upstream primer (P2) in combination with a primer homologous to the DOP primer 5' tag (Tag) reduced non-specific amplification. "Nested" amplicons were sequenced directly, the reaction primed with the third and most downstream primer (P3) or cloned to segregate fragments as was done to sequence DOP amplified product. Primers PrI, Pr2 and Pr3 similarly facilitated the sequencing of the unknown sequence in the opposite orientation. This process was step-wise repeated with new primer triplets, progressively "walking" into the unknown sequence until the complete contiguous sequence was obtained. To confirm sequence identity, the Adelavirus genome sequence was regenerated using specific primers and amplified from the original faecal specimen.

Adelavirus translated sequence analysis

Sequences were assessed for their potential significance using a number of parameters. These included evidence of potential large open reading frames (ORF; generally one or no stop codons in at least one frame) using the ORF analysis module in Kodon (Applied Maths).

All 6 frames of potential encoding were examined for those in which there was evidence of at least one large ORF or no more than one stop codon. Such frames infer encoding sequence. The codon usage in these frames, which typically contain >70 codons, was assessed. The sequence was submitted to the codon usage comparison websites Graphical Codon Usage Analyser (http://www.cua.de) to assess bias in the VLS ORF codon usage frequencies toward that for procaryote (infers bacteriophage) or eukaryote (infers human virus). Results and Discussion

The complete contiguous sequence generated from the alignment of overlapping portions of sequence is provided in Figure 1. From the sequence analysis (Figure 1 ) and homology alignments to other members of the virus family shown in Figure 2, it is believed that the entire coding sequence has been identified. An identical Adelavirus sequence was obtained from the second faecal specimen from the same subject.

The complete sequence was aligned with the ten closest known related sequences as shown in Figure 2, showing 28% nucleotide variation from its closest relative, human bocavirus. Amino acid sequence alignment with known human bocavirus sequences confirmed the expected function of translated Adelavirus ORPs. As shown in Figure 3, the Adelavirus nucleotide sequence encodes four polypeptides corresponding to a non-structural protein (NSl, Figure 3A), a nucleoprotein (NP 1, Figure 3B) and two virus-like proteins (VPl and VP2, Figure 3C).

Example 3 Adelavirus molecular detection assay

Materials and Methods

Using the sequence information derived from Adelavirus (Av 1-153), primer sets were designed for use in a nested PCR¹⁵ targeting the predicted non-structural protein (NSl). The primary reaction was primed with primers having the nucleotide sequences shown as SEQ ID NO: 6 (outer forward primer) and SEQ ID NO: 13 (outer reverse primer), while the secondary reaction was primed with primers having the nucleotide sequences shown as SEQ ID NO: 7 (inner forward primer) and SEQ ID NO: 14 (inner reverse primer). Reactions of 25μl total volume were amplified at 94⁰C for 10 minutes (to activate AmpliTaq Gold; Applied Biosystems Inc.) followed by 40 cycles of: 94⁰C for 30 sees, 6O⁰C for 30 sees and 72⁰C for 90 sees. Reaction conditions were optimised using the Adelavirus controls described above for reference. The assay was validated by screening 76 cases and 64 control samples from a single summer cluster occurring in early 2001 wherein sequencing (in at least one direction) was performed on samples which were "positive" by agarose gel electrophoresis.

Since a certain level of "noise" was produced by the nested PCR, a more specific but "universal" (ie universal to all Adelavirus strains and genotypes) was sought. Accordingly, a single primer pair PCR detection assay and a further nested PCR detection assay were developed. The single reaction PCR assay utilised primers according to the nucleotide sequences shown as SEQ ID NO: 8 (outer forward primer) and SEQ ID NO: 15 (outer reverse primer) in a single reaction. To enhance sensitivity and specificity, this assay was nested with second round primers according to the nucleotide sequences shown as SEQ ID NO: 9 (inner forward primer) and SEQ ID NO: 16 (inner reverse primer). Assay conditions in both cases were maintained according to the methods described above, with the nested reaction incorporated into the single tube hanging-drop nested PCR described previously¹⁵. While some non-specific amplification occurred in both the single and nested PCR assays, the reaction conditions may be optimised by routine methods to eliminate, or at least, minimise, the amplification of non-specific products.

In addition, a universal real time detection assay was developed. The real time assay included primers according to the nucleotide sequences shown as SEQ ID NO: 10 (forward primer) and SEQ ID NO: 17 (reverse primer) and a probe according to the nucleotide sequence shown as SED ID NO: 23; with a reverse primer according to the nucleotide sequence shown as SEQ ID NO: 18 for the identification of Adelavirus genogroup 1 (Av 1 ), a reverse primer according to the nucleotide sequence shown as SEQ ID NO: 19 for the identification of Adelavirus genogroup 2 (Av2), and a reverse primer according to the nucleotide sequence shown as SEQ ID NO: 20 for the identification of bocavirus. Assay conditions were maintained as above except for a change in primer concentration to 15 picomoles and an increase in the number of amplification cycles to 60 cycles. As expected, this real time assay proved to be highly sensitive, and can be used in combination with any one of three real time PCR assays in which the reverse primer is replaced with a specific AvI, Av2 and bocavirus primer, respectively to ensure universality and to further characterise positives.

Results and Discussion

Twelve Adelavirus sequence confirmed positives were identified in the 76 cases (a prevalence rate of 17%), but only four in the 62 control samples (a prevalence rate of 6%) (p = 0.0211 Fisher's

Exact Probability Test). Thus, the Adelavirus was strongly associated with disease in this selected subset. Furthermore, in this period there was a total of 30 cases and 45 controls for which no viral agent had previously been detected. Of these, 8 cases (27%) and 2 controls (4%) were Adelavirus positive (p = 0.012 Fisher's Exact Probability Test). During the test period, Adelavirus was the second most common agent associated with AGE, exceeded only by norovirus-2 present in 21/76 cases and 2/64 controls (p <0.0001). Fifty five percent of the Adelavirus positive cases were hospitalised, a rate which was statistically equivalent to the hospitalisation rates associated with norovirus-2 and rotavirus for the same period.

Further testing of previously undiagnosed AGE specimens has confirmed the validity of the assay outside of the single test outbreak. Of the sequences tested, phylogenetic analysis revealed 24 unique viral sequences belonging to the novel Adelavirus viral species. A dendrogram illustrating the genetic diversity of members of the Adelavirus is shown in Figure 4.

The universal PCR detection assays were used to rescreen all of the case study samples to determine the prevalence of infection and disease association. The summer cluster of AGE patients showed 17/69 cases of Adelavirus infection in AGE patients (a prevalence of 24.6%) cases and 4/69 cases in controls (a prevalence of 5.9%) (p = 0.025). In all AGE cases from that year, 33/203 cases of Adelavirus were confirmed positive (a prevalence of 16%) and 16/203 controls showed Adelavirus infection (prevalence of 7.8%) (p = 0.014). Further, of all AGE related cases to date (ie from 2000 to 2007), 54/357 cases were positive for Adelavirus infection (a prevalence of 15%) and 23/288 control cases were Adelavirus positive (a prevalence of 8%) (p = 0.007).

To date, bocaviruses are present in 22/316 of all AGE related cases (a prevalence of 7%) and 20/327 control are positive for bocavirus (a prevalence of 6%), confirming that bocaviruses are not statistically associated with acute gastroenteritis. This, further, confirms the distinctness of Adelavirus from bocavirus.

While the Adelavirus strains detected in the outbreak specimens were largely homogeneous, based on an alignment of the 520 bp PCR-generated fragment (Figure 4), a number of variants and a strain from a second genotype were also revealed (strain Av2-93 and strain Av2-471; Figure 4). Of the analysed sequences, all belong to one of two genogroups (designated genogroups 1 and 2) and within each group, the sequences fall within 1% genetic dissimilarity of each other. Adelaviruses therefore contain a number of related variants and at least two different genotypes.

This was confirmed in further studies, in which the open reading frame sequences of 16 different isolates were aligned and phylogenetically compared (Figure 4). The NSl protein was conventionally expected to be the most conserved, and indeed, all but one on the isolates show a high degree of nucleotide conservation. The sequence from sample 208, designated Av 1-208 (GenBank, accession EU082214), was extended using the same primer sets as used to re-derive Avl-153, to recover 5156 nucleotides (99.9% similar to Avl-153) encompassing the complete encoding region, which was compared with that for Avl-153 (Table 3). The NSl region sequence in one sample, 93, was 17-6% variant from AvI strains (Figure 4) confirming a second genogroup of Adelaviruses.

Table 3 Comparison of open reading frames from Adelavirus Genogroup I (AvI),

Adelavirus Genogroup II (Av2) and Human bocavirus (HBoV stl)

*number of amino acids encoded by ORF

^Λpairwise percent dissimilarity at both the nucleotide (and amino acid level in brackets)

Total nucleotide pairwise dissimilarity between Adelavirus Genogroup I and Genogroup II was determined to be 21.1% dissimilarity. Notably, the nucleoprotein amino acid sequence consistently showed greater pairwise dissimilarity than the corresponding nucleotide sequences.

Example 4 Adelavirus serological detection assay

Materials and Methods

Synthesis of VPl and VP2 expression constructs

Analysis of Adelavirus sequence and alignment with the closest known relative, human bocavirus, indicated the presence of ORPs encoding potential viral capsid proteins (Figure 3C). The VPl ORF DNA was prepared for cloning by PCR amplification using primers having the nucleotide sequence shown as SEQ ID NO: 11 and SEQ ID NO: 21 and similarly, the VP2 ORF cDNA was prepared using primers having the nucleotide sequence shown as SEQ ID NO: 12 and SEQ ID NO: 21. Primers were constructed as shown in Figure 5 by combining Adelavirus- specific terminal sequences and the manufacturer's recommended attBl and α#B2 tags (Invitrogen Corporation). Since only the 5' ends of the VPl and VP2 ORFs differ, there was a dual use of the 3' end primer having the nucleotide sequence of SEQ ID NO: 21. To generate C-terminal fusion histidine (His)-tagged VPs, the terminal TAA stop codon was not included in the reverse primer as the integration of the endless ORF into the baculovirus DNA backbone allowed translation to continue on through a His-tag sequence to a termination codon encoded in the baculovirus genome. Therefore, a reverse primer having a nucleotide sequence as shown in SEQ ID NO: 22 was used in place of SEQ ID NO: 21 to generate His-tagged baculovirus clones.

The VPl and VP2 ORF cDNA fragments were recombined into pDONR™/Zeo Gateway® vector (Figure 6) as a precursor to the recombination of VP ORFs into Baculodirect® DNA (Figure 7) (Invitrogen Corporation) with and without C-terminal His tags. Cloning conditions were as specified in the manufacturer's instructions. His-tagged VPl and VP2 inserts were also generated using reverse primer SEQ ID NO: 22 which deletes the TAA stop codon from the end of the VPl and VP2 coding sequences. Loss of the stop codon results in translation continuing through into the baculovirus DNA to include a His tag.

The VPl and VP2 ORF cDNA fragments without C-terminal stop codons, can be cloned into the vector (Champion™ pET-DEST-42; Invitrogen Corporation) for expression of His-tagged VPl and VP2 in E.coli BL21(DE3)'. Expressed proteins can be purified using His-trapping technology (eg His-GraviTrap columns: GE Healthcare, Giles, United Kingdom) according to the manufacturer's instructions. This enables the production and purification of VPl and VP2 proteins for use in the generation of polyclonal antisera (which can be produced commercially in rabbits and guinea pigs) as potential alternatives to the antisera raised against the VLPs.

Results and Discussion

The four ORFs corresponding to VPl, VPl-minus stop codon, VP2 and VP2 minus stop codon were amplified and recombined into the Gateway® vector. Further, the VP ORFs were, in turn, successfully recombined from the Gateway® clones into the baculovirus DNA. Master stocks of all four (ie VPl, VPl -His, VP2 and VP2-His) recombinant baculoviruses were generated and His- tagged clones underwent two rounds of plaque purification. Non-His-tagged clones were subsequently plaque purified in three successive rounds of purification, and sequence identity was thereafter confirmed as corresponding to the viral sequence.

Expression of recombinant VLPs

Sf21 cells (Invitrogen Corporation) can be infected with varying ratios of VP-expressing baculoviruses to optimise particle production. Expressed VLPs without His-tags can be purified by gradient ultracentrifugation. On the other hand, expressed His-tagged VLPs can be purified using His-trapping technology (eg His-GraviTrap columns: GE Healthcare) according to the manufacturer's instructions. Purified VLPs can be used to generate polyclonal antisera in rabbits and guinea pigs.

To date, VLPs comprising VP2 (the major capsid protein) exhibiting the expected size and morphology have been expressed and visualised using electron microscopy (EM) (Figure 8). Briefly, the VP2-expressing baculovirus was cultured in SF21 cells for 72 hours, and the cells scraped from the culture vessel, pelleted by centrifugation, lysed with 1% Triton-xlOO, precipitated with PEG 8000 and collected after ultracentrifugation through a sucrose cushion at 145,000 G for 3 hours. The VLPs were negatively stained with 2% PTA and examined using EM. To date, VP 1 alone does not appear to produce VLPs.

Antigen capture assays

Antisera to Adelavirus VLPs, or VPs, can be diluted to optimised strength in a coating buffer (eg

Carbonate-bicarbonate buffer pH 9.6) and applied to microtitre assay plates to enable binding of VLP- (or VP-) targeted antibodies to the surface of assay wells. Prior to use, the assay wells will be pre-incubated with the assay (EIA) diluent (eg sodium casein in phosphate buffered saline with Tween20 and tritonX- 100). To test for the presence of Adelavirus particles or VPs (ie the target antigen), diagnostic samples (eg vomitus, faeces or blood) will be homogenised, centrifuged to sediment solids and mixed with EIA diluent, and thereafter applied to the antibody-coated assay wells. Captured antigen can be secondarily bound with guinea pig polyclonal antisera against Adelavirus (VLP or VP) in EIA diluent and then bound with enzyme-linked (eg horseradish peroxidase) rabbit anti-guinea pig antisera. Bound rabbit anti-guinea pig antibodies can be detected by the addition of horseradish peroxidase substrate (eg 3,3',5,5'-tetramethylbenzidine). The presence or absence of Adelavirus in the diagnostic sample will be assessed by comparison of assay well optical density to the established criteria and controls for the test. Sandwich ELISA assay

The purified recombinant antigens can also be used to produce rabbit polyclonal antisera for subsequent use in sandwich ELISA detection assays using anti-His-antibody coated plates (Qiagen, Inc.) according to the manufacturer's instructions. Briefly, anti-His-Antibody coated 96- well plates (Qiagen, Inc.) can be coated with either His-tagged recombinant VLP, VPl or VP2 diluted in phosphate buffered saline (PBS) to 1 μg/ml. Following 4 washes in PBS-Tween, 200 μl of serially diluted rabbit polyclonal antiserum can be applied to sets of wells for optimisation and, following a further 4 washes in PBS-Tween, one of either test specimen Av 1-153 or Av2-93 can be applied to a set of wells. Viral binding from test samples can be visualised by a reduction in alkaline phosphatase signal in comparison to control wells, following the application of anti- rabbit-alkaline phosphatase conjugate (Qiagen, Inc.). Wash buffers and binding conditions will be prepared and conducted according to the manufacturer's instructions (QIAexpress® Detection and Assay Handbook).

Antibody detection assay

The viral proteins (VP and VLPs) can be used to detect antibodies to Adelavirus in patient sera by coating assay well surfaces with protein to provide a substrate for the capture of antibodies. Bound human antibodies will be detected using enzyme-linked anti-human antisera from commercial sources followed by measurement of enzyme product (ie. colour or optical density change). The viral protein source most suitable for this assay will be assessed by comparative testing. Once antisera has been obtained, all proteins and corresponding antisera can be tested for optimum activity in the antibody and antigen capture assays. Secondary antibody systems for the detection of bound virus and bound human antibodies can be tested for sensitivity and specificity to Adelavirus to produce a sensitive and broadly reactive assays.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

All publications mentioned in this specification are herein incorporated by reference. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia or elsewhere before the priority date of each claim of this application.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

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20. Wells D, Bermudez MG. 2004. Whole genome amplification from single cells and minute DNA samples, p. 349-359. In Weissensteiner T, Griffin HG, Griffin A (ed.), PCR Technology, Current innovations, 2nd ed. CRC Press, Danvers, MA.

Claims

CLAIMS:

1. An isolated single-stranded DNA virus, characterised in that the virus comprises a viral DNA molecule comprising a nucleotide sequence that shows at least 73% sequence identity to the nucleotide sequence shown as SEQ ID NO: 1.

2. The virus of claim 1, wherein the virus comprises a viral DNA molecule comprising a nucleotide sequence that shows at least 78.9% sequence identity to the nucleotide sequence shown as SEQ ID NO: 1.

3. The virus of claim 1 , wherein the virus comprises a viral DNA molecule comprising a nucleotide sequence that shows at least 85% sequence identity to the nucleotide sequence shown as SEQ ID NO: 1.

4. The virus of claim 1, wherein the virus comprises a viral DNA molecule comprising a nucleotide sequence that shows at least 95% sequence identity to the nucleotide sequence shown as SEQ ID NO: 1.

5. An isolated polynucleotide molecule comprising a nucleotide sequence that shows at least 73% sequence identity to the nucleotide sequence shown as SEQ ID NO: 1.

6. The molecule of claim 5, wherein the virus comprises a viral DNA molecule comprising a nucleotide sequence that shows at least 78.9% sequence identity to the nucleotide sequence shown as SEQ ID NO: 1.

7. The molecule of claim 5, wherein the nucleotide sequence shows at least 85% sequence identity to the nucleotide sequence shown as SEQ ID NO: 1.

8. The molecule of claim 5, wherein the nucleotide sequence shows at least 95% sequence identity to the nucleotide sequence shown as SEQ ID NO: 1.

9. An isolated polynucleotide molecule comprising a nucleotide sequence encoding a protein that comprises an amino acid sequence showing at least 75% sequence identity to an amino acid sequence selected from the group consisting of the amino acid sequences shown as SEQ ID NOS: 2 - 5 or a naturally occurring variant thereof.

10. The molecule of claim 9, wherein the encoded protein comprises an amino acid sequence showing at least 75.8% sequence identity to an amino acid sequence selected from the group consisting of the amino acid sequences shown as SEQ ID NOS: 2 - 5 or a naturally occurring variant thereof.

11. The molecule of claim 9, wherein the encoded protein comprises an amino acid sequence showing at least 85% sequence identity to an amino acid sequence selected from the group consisting of the amino acid sequences shown as SEQ ID NOS: 2 - 5 or a naturally occurring variant thereof.

12. The molecule of claim 9, wherein the protein comprises an amino acid sequence showing at least 95% sequence identity to an amino acid sequence selected from the group consisting of the amino acid sequences shown as SEQ ID NOS: 2 - 5 or a naturally occurring variant thereof.

13. A cell transformed with the polynucleotide molecule of any one of claims 5 to 12.

14. An isolated protein comprising an amino acid sequence showing at least 70.3% sequence identity to any one of the amino acid sequences shown as SEQ ID NOS: 2 - 5 or a naturally occurring variant thereof.

15. The protein of claim 14 comprising an amino acid sequence showing at least 75% sequence identity to any one of the amino acid sequences shown as SEQ ID NOS: 2 - 5 or a naturally occurring variant thereof.

16. The protein of claim 14 comprising an amino acid sequence showing at least 85% sequence identity to any one of the amino acid sequences shown as SEQ ID NOS: 2 - 5 or a naturally occurring variant thereof.

17. The protein of claim 14 comprising an amino acid sequence showing at least 95% sequence identity to any one of the amino acid sequences shown as SEQ ID NOS: 2 - 5 or a naturally occurring variant thereof.

18. An antigenic fragment of the protein of any one of claims 14 to 17.

19. A virus-like particle (VLP) comprising a protein comprising an amino acid sequence showing at least 78.3% sequence identity to any one of the amino acid sequences shown as SEQ ID NO: 4 and/or 5 or a naturally occurring variant thereof.

20. A composition for eliciting an immune response in an animal, in particular an antibody response, said composition comprising one or more of:

(i) an attenuated virus of claim 1;

(ii) a deactivated preparation of the virus of claim 1;

(iii) a polynucleotide molecule according to claim 5 to 10, wherein the polynucleotide sequence comprises a nucleotide sequence encoding a protein comprising an amino acid sequence substantially corresponding to that shown as SEQ ID NO: 4 or 5 or a naturally occurring variant thereof, or an antigenic fragment of said protein;

(iv) a transformed cell according to claim 13 expressing a protein comprising an amino acid sequence substantially corresponding to that shown hereinbefore as SEQ ID NO: 4 or 5 or a naturally occurring variant thereof, or an antigenic fragment of said protein;

(v) the protein of any one of claims 14 to 17;

(vi) the antigenic fragment of claim 18; and

(vii) the virus-like particle (VLP) of claim 19.

21. The composition of claim 20, wherein the composition is a vaccine composition.

22. An isolated antibody or fragment thereof which specifically binds to the virus of any one of claims 1 to 4.

23. The antibody or fragment thereof of claim 22, which specifically binds to the protein of claim 11, wherein said protein comprises an amino acid sequence substantially corresponding to that shown as SEQ ID NO: 4 or 5 or a naturally occurring variant thereof.

24. A method for the detection of the virus of any one of claims 1 to 4 in a suitable sample from a subject, wherein said method comprises introducing to said sample a reagent that specifically binds to said virus or a viral protein thereof, and detecting any binding between said reagent and virus or viral protein thereof.

25. The method of claim 24, wherein said reagent is an antibody or fragment thereof which specifically binds to said virus or a viral protein thereof.

26. The method of claim 24, wherein said reagent comprises at least one polynucelotide for amplifying a target nucleotide(s) sequence specific to said virus, and said method comprises subjecting said sample to conditions for amplifying a target nucleotide(s) sequence specific to said virus, and detecting the generation of any amplification products.

27. The method of claim 24, wherein said reagent comprises at least one polynucleotide which hybridises to said target nucleotide sequence(s) under high stringency conditions, and said method comprises detecting the presence of a target nucleotide(s) sequence specific to said virus.