WO1999058638A2

WO1999058638A2 - Sequences of tt viruses for use in diagnosis, prevention and treatment of ttv infections

Info

Publication number: WO1999058638A2
Application number: PCT/EP1999/003183
Authority: WO
Inventors: Geert Maertens; Karen De Vreese
Original assignee: Innogenetics N.V.
Priority date: 1998-05-13
Filing date: 1999-05-10
Publication date: 1999-11-18
Also published as: AU4259299A; WO1999058638A3

Abstract

The present invention relates to a TTV polynucleic acid having a nucleotide sequence which is unique to at least TTV type 3, type 4, type 5 or type 6, TTV subtypes 2d, 3a, 4a, 5a, or 6a as well as to polypeptides or peptides encoded by the same. The present invention also relates to the use of such sequences or antigens for the detection, treatment or prevention of TTV infection.

Description

New sequences of TT viruses for use in diagnosis, prevention and treatment of TTV infections.

The present invention relates to new sequences of TT viruses for use in diagnosis, prevention and treatment of TTV infections

A novel virus named TT virus (TTV), associated with posttransfusion hepatitis of unknown etiology, has been identified recently (Nishizawa et al., 1997; Okamoto et al., 1998). In the current state of the art, the clinical significance of this agent remains to be further investigated.

By means of representational difference analysis, a viral clone (N22) of 500 nucleotides was isolated from serum of a patient TT with posttransfusion hepatitis of unknown etiology. Oligonucleotide primers were deduced from this clone for detection by polymerase chain reaction. Studies by Nishizawa et al. (1997) revealed the sequence to be associated with a DNA virus. Okamoto et al. (1998) further determined the complete genomic sequence of the prototype TTV, and demonstrated that the agent was a non-enveloped, single-stranded DNA virus which exhibited a genomic organization similar to that of parvoviruses (two open reading frames encoding 770 and 202 amino acids, respectively). Recently, an extensive study on the biophysical and molecular characteristics of TTV suggested, however, that TTV is a member of a new family of viruses, which was tentatively named the Circinoviridae (Mushahwar et al., 1999). Okamoto et al. (1998) further detected TTV DNA in 9 of 19 patients with fulminant hepatitis, and in 41 of 90 patients with chronic liver disease of unknown etiology. In 78 sera from blood donors and hepatitis patients, the analysis of a partial sequence spanning nucleotides 1902-2257 (fragment amplified from serum by PCR, primer sequences excluded) revealed two types of viruses which differed by about 30% in the nucleotide sequence (Okamoto et al., 1998). Seventy six belonged to group (G; type) 1 and two belonged to type 2. Type 1 consisted of 2 subtypes la and lb containing 52 and 24 isolates, respectively; the two type 2 isolates showed as much divergence as is observed between Gla and Gib and could therefore be classified into G2a and G2b (Okamoto et al., 1998).

So far, only data from Japan have been reported. It is therefore an aim of the present invention to provide new TTV nucleotide and amino acid sequences enabling the detection, prevention and treatment of TTV infection.

Another aim of the present invention is to provide new TTV nucleotide and amino acid sequences enabling the classification of infected biological samples into different groups.

Another aim of the present invention is to provide new TTV nucleotide and amino acid sequences enabling a better overall TTV detection rate.

Another aim of the present invention is to provide new TTV nucleotide and amino acid sequences useful for the design of TTV prophylactic or therapeutic vaccine compositions.

Another aim of the present invention is to provide antibodies specifically recognizing the new TTV amino acid sequences, for therapy or diagnosis.

All these aims have been met by the embodiments of the present invention.

The present invention relates more particularly to a TTV polynucleic acid having a nucleotide sequence which is unique to at least TTV type 3, type 4, type 5 or type 6, or TTV subtype 2d, subtype 3a, subtype 4a, subtype 5a or subtype 6a with said TTV sequences being classified as shown in Tables 2.2, 4, 6, 8, and 10, or Figures 1, 2, 3, 4, 5.

In the present invention, 36 serum samples from patients with hepatitis of unknown etiology, were analysed for the presence of TTV by PCR. Seventeen out of 36 (47.2%) samples were positive. Unexpectedly, sequence determination of nucleotides 1959-2180 revealed, in addition to 3 subtype lb isolates and one subtype 2b isolate, that the majority of samples belonged to previously unknown genotypes of TTV. Four samples belonged to a new genotype 3, and one sample belonged to another new genotype 4. All four genotype 3 isolates belonged to the same subtype 3a; the type 4 isolate was classified into subtype 4a. Furthermore, 4 isolates showed homology to genotype 2 but could not be classified as subtype 2a or subtype 2b. Two of these were classified into subtype 2c and the remaining two isolates were grouped into a new subtype 2d. Extensive testing of 507 more serum samples revealed the presence of even more new genotypes, i.e. genotype 5 and genotype 6. The subtypes corresponding to these genotypes were named subtype 5a and subtype 6a, respectively

The use of genotype-specific sequences of TTV genotypes and subtypes 2d, 3, 3a, 4, 4a, 5, 5a, 6, and 6a is thought to become important in the diagnosis, prevention, or treatment of TTV infection Furthermore, sequences common to TTV genotypes 1, la, lb, 2, 2a, 2b, 2c, 2d, 3, 3a, 4, 4a, 5, 5a, 6 and 6a may become instrumental in the detection of TTV genomic sequences by hybridization or amplification techniques The use of genotype-specific nucleotide or ammo acid sequences is thought to become important for diagnosis in a single assay or for discnmmation, in multiple or multi-parameter assays, of all vanant strains worldwide Similarly, prevention of certain types of TTV infection may require the synthesis of antigens from the given genotype of TTV, or may require the isolation or generation of human or humanized antibodies binding to the given genotype of TTV Also the design of drugs interfenng with the TTV replication cycle will need to be effective against the majonty of TTV genotypes, thus drug development will require the use of TTV proteins, which are active in the TTV replication cycle, isolated from strains belonging to different TTV genotypes

TTV types have been found to show more than 70%, preferably more than 75% similanty, while TTV subtypes have been found to show more than 85%, preferably more than 89% similanty

It should be clear that the classification into types and subtypes as given m the present application might still be susceptible to changes as new TTV sequences are being elucidated Therefore, current type 3 sequences might for instance be classified as type 4 and type 4 as type 3 sequences

The present invention more particularly relates to a polynucleic acid sequence encoding a TTV polyprotem compnsing in its amino acid sequence at least one of the type- or subtype specific ammo acid residues as indicated in Table 11

The present invention still more specifically relates to a polynucleic acid sequence as defined above having a sequence selected from any of SEQ ID NO 5, 7, 9, 11, 13, 15, 17, 27, 29, 31, 33, 35, 37, 39, 45, 47, 49, 51, or a part of said polynucleic acid which is unique to at least one of the TTV subtypes or types as defined above, and which contains at least one nucleotide diffenng from known TTV nucleotide sequences, or the complement thereof The term "polynucleic acid" refers to a single- stranded or double-stianded nucleic acid sequence which may contain at least 5 contiguous nucleotides common with the complete nucleotide sequence (e g at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 60, 75 or more contiguous nucleotides) A polynucleic acid which is up till about 100 nucleotides m length is often also referred to as an oligonucleotide A polynucleic acid may consist of deoxynbonucleotides or nbonucleotides, nucleotide analogues or modified nucleotides, or may have been adapted for therapeutic purposes A polynucleic acid may also compnse a double stranded cDNA clone which can be used for cloning purposes, or for in vivo therapy, or prophylaxis

The ohgonucleotides according to the present invention, used as pπmers or probes may also contain or consist of nucleotide analogous such as phosphorothioates (Matsukura et al , 1987), alkylphosphonates (Miller et al , 1979) or peptide nucleic acids (Nielsen et al , 1991, Nielsen et al , 1993) or may contain intercalating agents (Asselme et al , 1984)

As most other vanations or modifications introduced into the onginal DNA sequences of the invention these vanations will necessitate adaptions with respect to the conditions under which the oligonucleotide should be used to obtain the required specificity and sensitivity However, the eventual results will be essentially the same as those obtained with the unmodified ohgonucleotides

The introduction of these modifications may be advantageous m order to positivily influence charactenstics such as hybndization kinetics, reversibility of the hybnd-formation, biological stability of the oligonucleotide molecules, etc

The polynucleic acids of the invention may be compnsed m a composition of any kind Said composition may be for diagnostic, therapeutic or prophylactic use

The expression "sequences which are unique to TTV type or subtype" refers to sequences which are not shared by any other type or subtype of TTV, and can thus be used to uniquely detect that TTV type or subtype Sequence vanabi ty is demonstrated the present invention between the newly found TTV types and subtypes (see Figures 1 to 5) and the known TTV types and subtypes, and it is therefore from these regions of sequence vanabihty in particular that type- or subtypes-specific polynucleic acids, ohgonucleotides, polypeptides and peptides may be obtained The term type- or subtype-specific refers to the fact that a sequence is unique to that TTV type or subtype involved It is to be noted that the terms type and genotype as well as subtype and subgenotype are used interchangeably herein

The expression "nucleotides corresponding to" refers to nucleotides which are homologous or complementary to an indicated nucleotide sequence or region within a specific TTV sequence

The term "coding region" corresponds to the regιon(s) of the TTV genome that encode TTV proteins, including possible polyproteins In fact, it compnses the complete genome with the exception of the untranslated regιon(s)

Also included within the present invention are sequence vaπants of the polynucleic acids as selected from any of the nucleotide sequences as given in any of the above given SEQ ID numbers with said sequence vanants containing either deletions and/or insertions of one or more nucleotides, especially insertions or deletions of 1 or more codons, mamly at the extremities of ohgonucleotides (either 3' or 5'), or substitutions of some non-essential nucleotides (l e nucleotides not essential to discnmmate between different genotypes of TTV) by others (including modified nucleotides an/or inosine)

Particularly preferred vanant polynucleic acids of the present invention include also sequences which hybndise under stnngent conditions with any of the polynucleic acid sequences of the present invention Particularly, sequences which show a high degree of homology (similanty) to any of the polynucleic acids of the invention as descnbed above Particularly sequences which are at least 80%, 85%, 90%, 95% or more homologous to said polynucleic acid sequences of the invention Preferably said sequences will have less than 20%, 15%, 10%, or 5% vanation of the ongmal nucleotides of said polynucleic acid sequence

Polynucleic acid sequences according to the present invention which are similar to the sequences as represented by a SEQ ID NO can be charactenzed and isolated according to any of the techniques known in the art, such as amplification by means of sequence-specific pπmers, hybndization with sequence-specific probes under more or less stnngent conditions, sequence determination of the genetic information of TTV, serological screening methods or via the LiPA typing system.

Other prefeπed vanant polynucleic acids of the present invention include sequences which are redundant as a result of the degeneracy of the genetic code compared to any of the above-given polynucleic acids of the present invention. These vanant polynucleic acid sequences will thus encode the same amino acid sequence as the polynucleic acids they are deπved from

Sequence determination of the remaining parts of the sequences of the TTV isolates may be done by techniques known in the art.

The present invention also relates to an oligonucleotide pπmer compπsing part of a polynucleic acid as defined above, with said pπmer being able to act as pπmer for specifically sequencing or specifically amplifying the nucleic acid of a certain isolate belonging to the genotype from which the pπmer is deπved.

The term "pπmer" refers to a single stranded DNA oligonucleotide sequence capable of acting as a point of initiation for synthesis of a pπmer extension product which is complementary to the nucleic acid strand to be copied The length and the sequence of the pπmer must be such that they allow to prime the synthesis of the extension products Preferably the pπmer is about 5-50 nucleotides Specific length and sequence will depend on the complexity of the required DNA or RNA targets, as well as on the conditions of pnmer use such as temperature and ionic strength

The fact that amplification pπmers do not have to match exactly with coπespondmg template sequence to wanant proper amplification is amply documented in the literature (Kwok et al., 1990).

The amplification method used can be either polymerase chain reaction (PCR; Saiki et al., 1988), hgase chain reaction (LCR; Landgren et al, 1988; Wu & Wallace, 1989; Barany, 1991), nucleic acid sequence-based amplification (NASBA, Guatelh et al., 1990; Compton, 1991), transcπption-based amplification system (TAS; Kwoh et al., 1989), strand displacement amplification (SDA; Duck, 1990; Walker et al., 1992) or amplification by means of Qβ rep case (Lizardi et al., 1988; Lomeli et al , 1989) or any other suitable method to amplify nucleic acid molecules using pπmer extension Duπng amplification, the amplified products can be conveniently labelled either using labelled pπmers or by incorporating labelled nucleotides Labels may be isotopic (³²P, ³⁵S, etc.) or non-isotopic (biotin, digoxigemn, etc.). The amplification reaction is repeated between 20 and 70 times, advantageously between 25 and 45 times.

Any of a vaπety of sequencing reactions known in the art can be used to directly sequence the viral genetic information and determine the genotype by translating the sequence of the sample into the coπespondmg amino acid sequence, and compaπng this ammo acid sequence with the exemplary ammo acids listed in table 11 Exemplary sequencing reactions include those based on techniques developed by Sanger (Sanger et al, 1977, Proc. Natl. Acad. Sci. USA 74. 5463) or Maxam and Gilbert (Maxam and Gilbert, 1977, Proc. Natl Acad Sci. USA 74. 560). It is also contemplated that a vaπety of automated sequencing procedures may be utilized when performing the subject assays (Biotechmques 1995, 19" 448), including sequencing by mass spectrometry (see, for example. PCT publication WO 94/16101; Cohen et al 1996, Adv Chromatogr. 36: 127-162; and Gπffm et al. 1993, Appl. Biochem. Biotech. 38: 147-159). It will be evident to one skilled in the art that, for example the occurence of only two or three nucleic bases need to be determined m the sequencing reaction

The present invention also relates to an oligonucleotide probe compπsing part of a polynucleic acid as defined above, with said probe being able to act as a hybndization probe for specific detection and/or classification into types and/or subtypes of a TTV nucleic acid containing said nucleotide sequence, with said probe being possibly labelled or attached to a solid substrate

The term "probe" refers to single stranded sequence-specific ohgonucleotides which have a sequence which is complementary to the target sequence of the TTV genotype(s) to be detected

Preferably, these probes are about 5 to 50 nucleotides long, more preferably from about 10 to 25 nucleotides. The term "solid support" can refer to any substrate to which an oligonucleotide probe can be coupled, provided that it retains its hybndization charactenstics and provided that the background level of hybndization remains low Usually the solid substrate will be a microtiter plate, a membrane (e g nylon or nitrocellulose) or a microsphere (bead) Pπor to application to the membrane or fixation it may be convenient to modify the nucleic acid probe m order to facilitate fixation or improve the hybndization efficiency Such modifications may encompass homopolymer tailing, coupling with different reactive groups such as aliphatic groups, NH₂ groups, SH groups, carboxy c groups, or coupling with biotin or haptens

The present invention also relates to a diagnostic kit for use in determining the presence of a TTV isolate, said kit compπsing a primer as defined above

The present invention also relates to a diagnostic kit for use m determining the presence of a TTV isolate, said kit compπsing a probe as defined above

The present invention also relates to a diagnostic kit as defined above, wherein said probe(s) ιs(are) attached to a solid support

The present invention also relates to a diagnostic kit as defined above, wherein a range of said probes are attached to specific locations on a solid support

The present invention also relates to a diagnostic kit as defined above, wherein said solid support is a membrane stπp and said probes are coupled to the membrane in the form of parallel lines

The term "nucleic acid" can also be refeπed to as analyte strand and corresponds to a single- oi double-stranded nucleic acid molecule

The term "biological sample" refers to any biological sample (tissue or fluid) containing TTV nucleic acid sequences and refers more particularly to blood serum or plasma samples

The term ' universal TTV pπmer" refers to oligonucleotide sequences complementary to any of the conserved regions of the TTV genome.

The expression "appropπate" hybndization and washing conditions are to be understood as stnngent and are generally known in the art (e.g. Maniatis et al., Molecular Cloning- A Laboratory Manual, New York, Cold Spπng Harbor Laboratory, 1982)

However, according to the hybndization solution (SSC, SSPE, etc ), these probes should be hybπdized at their appropπate temperature in order to attain sufficient specificity.

The term "labelled" refers to the use of labelled nucleic acids. This may include the use of labelled nucleotides incorporated duπng the polymerase step of the amplification such as illustrated by Saiki et al (1989) or Bej et al. (1990) or labelled pπmers, or by any other method known to the person skilled in the art.

The process of the invention compπses the steps of contacting any of the probes as defined above, with one of the following elements: either a biological sample in which the nucleic acids are made available for hybndization, or the puπfied nucleic acids contained in the biological sample - or a single copy deπved from the puπfied nucleic acids, or an amplified copy deπved from the puπfied nucleic acrds, with said elements or with said probes being attached to a solid substrate

The expression "infernng the presence of one or more TTV genotypes present from the observed hybridization pattern" refers to the identification of the presence of TTV genomes m the sample by analyzing the pattern of binding of a panel of oligonucleotide probes. Single probes may provide useful information concerning the presence or absence of TTV genomes m a sample. On the other hand, the vaπation of the TTV genomes is dispersed in nature, so rarely is any one probe able to identify uniquely a specific TTV genome. Rather, the identity of an TTV genotype may be infened from the pattern of binding of a panel of oligonucleotide probes, which are specific for (different) segments of the different TTV genomes. Depending on the choice of these oligonucleotide probes, each known TTV genotype will conespond to a specific hybndization pattern upon use of a specific combination of probes Each TTV genotype will also be able to be discriminated from any other TTV genotype amplified with the same pπmers depending on the choice of the oligonucleotide probes Compaπson of the generated pattern of positively hybπdizing probes for a sample containing one or more unkown TTV sequences to a scheme of expected hybndization patterns, allows one to clearly infer the TTV genotypes present in said sample

The present invention also relates to a method for the detection of TTV nucleic acids present in a biological sample, compπsing (l) possibly extracting sample nucleic acid,

(n) amplifying the nucleic acid with at least one primer as defined above,

(in) detecting the amplified nucleic acids

Most of these probes target the most type- or subtype-specific regions of TTV genotypes, but some can be caused to hybridize to more than one TTV genotype

According to the hybndization solution (SSC, SSPE, etc ), these probes should be stπngently hybπdized at then appropπate temperature m order to attain sufficient specificity However, by slightly modifying the DNA probes, either by adding or deleting one or a few nucleotides at their extremities (either 3' or 5'), or substituting some non-essential nucleotides (I e nucleotides not essential to discπmmate between types) by others (including modified nucleotides or inosine) these probes or vanants thereof can be caused to hybπdize specifically at the same hybndization conditions (l e the same temperature and the same hybndization solution) Also changing the amount (concentration) of probe used may be beneficial to obtain more specific hybndization results It should be noted in this context, that probes of the same length, regardless of their GC content, will hybπdize specifically at approximately the same temperature in TMAC1 solutions (Jacobs et al , 1988)

Suitable assay methods for purposes of the present invention to detect hybπds formed between the oligonucleotide probes and the nucleic acid sequences in a sample may compπse any of the assay formats known m the art, such as the conventional dot-blot format, sandwich hybndization or reverse hybndization For example, the detection can be accomplished using a dot blot format, the unlabelled amplified sample being bound to a membrane, the membrane being incorporated with at least one labelled probe under suitable hybndization and wash conditions, and the presence of bound probe being monitored

An alternative and prefened method is a "reverse" dot-blot format, in which the amplified sequence contains a label. In this format, the unlabelled oligonucleotide probes are bound to a solid support and exposed to the labelled sample under appropnate stnngent hybndization and subsequent washing conditions. It is to be understood that also any other assay method which relies on the formation of a hybπd between the nucleic acids of the sample and the oligonucleotide probes according to the present invention may be used.

According to an advantageous embodrment, the process of detecting one or more TTV genotypes contained in a biological sample comprises the steps of contacting amplified TTV nucleic acid copies deπved from the biological sample, with oligonucleotide probes which have been immobilized as parallel lines on a solid support

According to this advantageous method, the probes are immobilized in a Line Probe Assay (LiPA) format. This is a reverse hybndization format (Saiki et al., 1989) using membrane stπps onto which several oligonucleotide probes (including negative or positive control ohgonucleotides) can be conveniently applied as parallel lines.

The invention thus also relates to a solid support, preferably a membrane stπp, carrying on its surface, one or more probes as defined above, coupled to the support in the form of parallel lines

The LiPA is a very rapid and user-fπendly hybndization test. Results can be read after 4 hours, after the start of the amplification. After amplification dunng which usually a non-isotopic label is incorporated in the amplified product, and alkaline denaturation, the amplified product is contacted with the probes on the membrane and the hybndization is earned out for about 1 to 1,5 h. The hybπdized polynucleic acid is subsequently detected. From the hybndization pattern generated, the TTV type can be deduced either visually, but preferably using dedicated software The LiPA format is completely compatible with commercially available scanning devices, thus rendenng automatic interpretation of the results very reliable. All those advantages make the LiPA format liable for the use of TTV detection in a routine setting The LiPA format should be particularly advantageous for detecting the presence of different TTV genotypes

The present invention also relates to a method for the detection of TTV nucleic acids present in a biological sample, compnsing

(I) possibly extracting sample nucleic acid,

(n) possibly amplifying the nucleic acid with at least one pπmer as defined above or with a universal TTV pπmer, (in) hybndizmg the nucleic acids of the biological sample, possibly under denatured conditions, at appropnate conditions with one or more probes as defined above, with said probes being possibly attached to a solid substrate, (IV) possibly washing at appropπate conditions,

(v) detecting the hybπds formed

The present invention also relates to a method for detecting the presence of one or more TTV genotypes present in a biological sample, compπsing

(l) possibly extracting sample nucleic acid,

(n) specifically amplifying the nucleic acid with at least one pπmer as defined above,

(in) detecting said amplified nucleic acids, (iv) infernng the presence of one or more genotypes of TTV present from the observed pattern of amplified fragments

The present invention also relates to a method for detecting the presence of one or more TTV genotypes present in a biological sample, compπsing (I) possibly extracting sample nucleic acid,

(n) possibly amplifying the nucleic acid with at least one pnmer as defined above or with a universal TTV pπmer,

(m) hybπdizing the nucleic acids of the biological sample, possibly under denatured conditions, at appropnate conditions with one or more probes as defined above, with said probes being possibly attached to a solid substrate,

(iv) possibly washing at appropriate conditions,

(v) detecting the hybπds formed, (vi) infernng the presence of one or more TTV genotypes present from the observed hybndization pattern.

The present invention also pertains to a method for detecting the presence of one or more TTV genotypes present in a biological sample, compπsing

(l) amplifying the nucleic acid with at least one pπmer as defined herein,

(n) contacting said amplified TTV nucleic acid copies, with oligonucleotide probes as defined herein, which have been immobilised as parallel lines on a solid support (in) infernng from the result of step (n) the presence of one or more TTV genotypes

More particular, the present invention pertains to a method as above, wherein said probes are immobilised in a LiPA format.

The present invention also pertains to a method for detecting the genotype of TTV nucleic acids as defined above, present in a biological sample, compnsing¹

(i) possibly extracting sample TTV nucleic acid,

(n) possibly amplifying the TTV nucleic acid,

(in) sequencing the TTV nucleic acid, (iv) mfernng from the result of step (in) the TTV genotype present in said sample

The expression "mfernng from the result of step (rrr) the TTV genotype present in said sample" refers to the identification of the presence of TTV genomes m the sample by companson of the obtained sequences with the exemplary TTV sequences as defined herein. Companson can be earned out by aligning these sequences by any of the methods well known m the art Depending on the sequence identity between the obtained sequences and the exemplary TTV sequences, the TTV genotype present in said sample is assigned.

The present invention also relates to a method as defined above, wherein said nucleic acids are labelled dunng or after amplification

The present invention also relates to a polypeptide having an ammo acid sequence encoded by a polynucleic acid as defined above, or a part thereof which is unique to at least one of the TTV subtypes or types as defined above, and which contains at least one ammo acid diffeπng from any of the known TTV types or subtypes amino acid sequences, or an analog thereof being substantially similar and biologically equivalent

The term 'polypeptide' refers to a polymer of ammo acids and does not refer to a specific length of the product; thus, peptides, o gopeptides, and protems are included within the definition of polypeptide. This term also does not refer to or exclude post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. Included within the definition are, for example, polypeptides containing one or more analogues of an ammo acid (including, for example, unnatural ammo acids, PNA, etc ), polypeptides with substituted linkages, as well as other modifications known m the art, both naturally occurnng and non-naturally occurnng

By "biologically equivalent" as used throughout the specification and claims, it is meant that the compositions are immunogenically equivalent to the proteins (polypeptides) or peptides of the invention as defined above and below

By "substantially homologous" as used throughout the ensuing specification and claims to descnbe proteins and peptides, it is meant a degree of homology m the amino acid sequence to the proteins or peptides of the invention. Preferably the degree of homology is in excess of 90, preferably in excess of 95, with a particularly prefened group of proteins being m excess of 99 homologous with the proteins or peptides of the invention

The term "analog" as used throughout the specification or claims to descπbe the proteins or peptides of the present invention, includes any protein or peptide having an amino acid residue sequence substantially identical to a sequence specifically shown herein in which one or more residues have been conservatively substituted with a biologically equivalent residue Examples of conservative substitutions include the substitution of one polar (hydrophobic) residue such as isoleucme, valine, leucme or methionme for another, the substitution of one polar (hydrophillic) residue for another such as between arginme and lysme, between glutamine and asparagine, between glycme and senne, the substitution of one basic residue such as lysme, arginme or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another. Examples of allowable mutations according to the present invention can be found in the Table as set out below

The phrase "conservative substitution" also includes the use of a chemically denvatized residue in place of a non-deπvatized residue provided that the resulting protein or peptide is biologically equivalent to the protein or peptide of the invention.

"Chemical derivative" refers to a protein or peptide having one or more residues chemically deπvatized by reaction of a functional side group Examples of such denvatized molecules, include but are not limited to, those molecules m which free amino groups have been deπvatized to form amine hydiochloπdes, p-toluene sulfonyl groups, carbobenzoxy groups, t- butyloxycarbonyl groups, chloracetyl groups or formyl groups. Free carboxyl groups may be deπvatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be deπvatized to form O-acyl or O-alkyl deπvatives. The imidazole nitrogen of histidme may be denvatized to form N-imbenzylhistidme. Also included as chemical denvatives are those proteins or peptides which contain one or more naturally-occurπng ammo acrd denvatives of the twenty standard amino acids For examples 4-hydroxyprolme may be substituted for prohne; 5 -hydroxyl ysine may be substituted for lysine; 3-methylhιstιdme may be substituted for histidme, homoseπne may be substituted for senne; and omithme may be substituted for lysine The proteins or peptides of the present invention also include any protein or peptide having one or more additions and/or deletions or residues relative to the sequence of a peptide whose sequence is shown herein, so long as the peptide is biologically equivalent to the proteins or peptides of the invention.

It is to be noted that, at the level of the amino acid sequence, at least one ammo acid difference (with respect to known TTV amino acid sequences) is sufficient to be part of the invention, which means that the polypeptides of the invention conespond to polynucleic acids having at least one nucleotide difference (with known TTV polynucleic acid sequences) involving an ammo acid difference in the encoded polyprotem.

The peptides according to the present invention contain preferably at least 3, preferably 4, 5 contiguous TTV amino acids, 6, 7 preferably however at least 8 contiguous TTV amino acids, at least 10 or at least 15 (for instance at least 9, 10, 11, 12, 13, 14, 15. 16, 17, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more amino acids).

TABLE

Amino acids Synonymous groups

Ser (S) Ser, Thr, Gly, Asn

Arg (R) Arg, His, Lys, Glu, Gin

Leu (L) Leu; He, Met, Phe, Val, Tyr

Pro (P) Pro, Ala, Thr, Gly

Thr (T) Thr, Pro, Ser, Ala, Gly, His, Gin

Ala (A) Ala, Pro, Gly, Thr

Val (V) Val, Met, He, Tyr, Phe, Leu, Val

Gly (G) Gly, Ala, Thr, Pro, Ser

He (I) He, Met, Leu, Phe, Val, He, Tyr

Phe (F) Phe, Met, Tyr, He, Leu, Tip, Val

Tyr (Y) Tyr, Phe, Trp, Met, He, Val, Leu

Cys (C) Cys, Ser, Thr, Met

His (H) His, Gin, Arg, Lys, Glu, Thr

Gin (Q) Gin, Glu, His, Lys, Asn, Thr, Arg

Asn (N) Asn, Asp, Ser, Gin

Lys (K) Lys, Arg, Glu, Gin, His

Asp (D) Asp, Asn, Glu, Gin

Glu (E) Glu, Gin, Asp, Lys, Asn, His, Arg

Met (M) Met, He, Leu, Phe, Val

Table Overview of the amino acid substitutions which could form the basis of analogs (muteins) as defined above

The polypeptides of the invention, and particularly the fragments, can be prepared by classical chemical synthesis.

The synthesis can be carried out in homogeneous solution or in solid phase.

For instance, the synthesis technique in homogeneous solution which can be used is the one descnbed by Houbenweyl in the book entitled "Methode der organischen chemie" (Method of organic chemistry) edited by E Wunsh, vol. 15-1 et LI THLEME. Stuttgart 1974

The polypeptides of the invention can also be prepared in solid phase according to for example the methods descnbed by Atherton and Shepard in their book entitled "Solid phase peptide synthesis" (IRL Press, Oxford, 1989).

The polypeptides according to this invention can be prepared by means of recombinant DNA techniques as for example descnbed by Mamatis et al., Molecular Cloning- A Laboratorv Manual, New York, Cold Spπng Harbor Laboratory, 1982)

The present invention also relates to a polypeptide as defined above compπsing in its ammo acid sequence at least one of the type or subtype specific ammo acid residues as indicated in Table 11, or a part of said polypeptide which is unique to at least one of the TTV subtypes or types as defined above, and which contains at least one ammo acid diffeπng from known TTV types or subtypes am o acid sequences, or an analog thereof being substantially similar and biologically equivalent to said polypeptide.

The present invention also relates to a polypeptide having an ammo acid sequence as represented in any of SEQ ID NO 6, 8, 10, 12, 14, 16, 18, 28, 30, 32, 34, 36, 38, 40, 46, 48, 50, 52, or a part thereof which is unique to at least one of the TTV subtypes or types as defined above, and which contains at least one ammo acid diffeπng from known TTV types or subtypes ammo acid sequences, or an analog thereof being substantially similar and biologically equivalent to said polypeptide.

The terms "epitope" and "antigemc determinant" mean an amino acid sequence that is lmmunoreactive. Generally an epitope consists of 4, and more usually 5, 6, 7, 8 or 9 contiguous amino acids However, it should also be clear that an epitope need not to be composed of a contiguous amino acid sequence. The lmmunoreactive sequence may be separated by a linker, which is not a functional part of the epitope. The linker does not need to be an amino acid sequence, but can be any molecule that allows the formation of the desired epitope The present invention also relates to a recombmant polypeptide encoded by a polynucleic acid as defined above, or a part thereof which is unique to at least one of the TTV subtypes or types as defined above, and which contains at least one amino acid diffenng from known TTV types or subtypes ammo acid sequences, or an analog thereof being substantially similar and biologically equivalent to said polypeptide

The present invention also relates to a method for production of a recombmant polypeptide as defined above, compπsmg transformation of an appropnate cellular host with a recombmant vector, in which a polynucleic acid or a part thereof as defined above has been inserted under the control of the appropnate regulatory elements, cultuπng said transformed cellular host under conditions enabling the expression of said insert, and, harvesting said polypeptide

The present invention also relates to a recombmant expression vector compπsing a polynucleic acid or a part thereof as defined above operably linked to prokaryotic, eukaryotic or viral transcπption and translation control elements

The term "vector" may compπse a plasmid, a cosmid, a phage, or a virus or a transgenic animal Particularly useful for vaccine development may be BCG or adenoviral vectors, as well as avipox recombmant viruses

The term 'recombinantly expressed' used withm the context of the present invention refers to the fact that the proteins of the present invention are produced by recombmant expression methods be it in prokaryotes, or lower or higher eukaryotes as discussed in detail below

The term 'lower eukaryote' refers to host cells such as yeast, fungi and the like Lower eukaryotes are generally (but not necessanly) unicellular Prefened lower eukaryotes are yeasts, particularly species with Saccharomyces, Schizosaccharomyces, Kluiveromyces, Pichia (e g Pichia pastons), Hansenula (e g Hansenula polymorpha), Schwamomyces, Schizosaccharomyces, Yarowia, Zygosaccharomyces and the like Saccharomyces cerevisiae, S carlsbergensis and K lactis are the most commonly used yeast hosts, and are convenient fungal hosts

The term 'prokaryotes' refers to hosts such as E.coh, Lactobacillus, Lactococcus, Salmonella, Streptococcus, Bacillus subtihs or Streptomyces Also these hosts are contemplated withm the present invention.

The term 'higher eukaryote' refers to host cells derived from higher animals, such as mammals, reptiles, insects, and the like. Presently prefened higher eukaryote host cells are denved from Chinese hamster (e.g. CHO), monkey (e.g COS and Vero cells), baby hamster kidney (BHK), pig kidney (PK15), rabbit kidney 13 cells (RK13), the human osteosarcoma cell line 143 B, the human cell line HeLa and human hepatoma cell lines like Hep G2. and insect cell lines (e.g Spodoptera frugiperda) The host cells may be provided m suspension or flask cultures, tissue cultures, organ cultures and the like Alternatively the host cells may also be transgenic animals

The term 'recombmant polynucleotide or nucleic acid' intends a polynucleotide or nucleic acid of genomic, cDNA, semisynthetic, or synthetic ongm which, by virtue of its oπgm or manipulation (1) is not associated with all or a portion of a polynucleotide with which it is associated in nature, (2) is linked to a polynucleotide other than that to which it is linked m nature, or (3) does not occur in nature

The term 'recombmant host cells', 'host cells', 'cells', 'cell lines', 'cell cultures', and other such terms denoting microorganisms or higher eukaryotic cell lines cultured as unicellular entities refer to cells which can be or have been, used as recipients for a recombmant vector or othei transfer polynucleotide, and include the progeny of the onginal cell which has been transfected It is understood that the progeny of a single parental cell may not necessaπly be completely identical in morphology or in genomic or total DNA complement as the onginal parent, due to natural, accidental, or deliberate mutation.

The term 'rephcon' is any genetic element, e.g., a plasmid, a chromosome, a vims, a cosmid, etc., that behaves as an autonomous unit of polynucleotide replication within a cell; i.e., capable of replication under its own control.

The term 'vector' is a rephcon further compnsing sequences providing replication and/or expression of a desired open reading frame

The term 'control sequence' refers to polynucleotide sequences which are necessary to effect the expression of coding sequences to which they are hgated The nature of such control sequences differs depending upon the host organism, in prokaryotes, such control sequences generally include promoter, nbosomal binding site, splicing sites and terminators, in eukaryotes, generally, such control sequences include promoters, splicing sites, terminators and, in some instances, enhancers The term 'control sequences' is intended to include, at a minimum, all components whose presence is necessary for expression, and may also include additional components whose presence is advantageous, for example, leader sequences which govern secretion

The term 'promoter' is a nucleotide sequence which is compπsed of consensus sequences which allow the binding of RNA polymerase to the DNA template m a manner such that mRNA production initiates at the normal transcπption initiation site for the adjacent structural gene

The expression 'operably linked' refers to a juxtaposition wherein the components so descnbed are in a relationship permitting them to function in their intended manner A control sequence 'operably linked' to a coding sequence is hgated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences

The segment of the TTV DNA encoding the desired sequence inserted into the vector sequence may be attached to a signal sequence Said signal sequence may be that from a non-TTV source, e g the IgG or tissue plasminogen activator (tpa) leader sequence for expression m mammalian cells, or the -mating factor sequence for expression into yeast cells, but particularly prefened constmcts according to the present invention contain signal sequences appeanng in the TTV genome before the respective start points of the proteins

Higher eukaryotes may be transformed with vectors, or may be infected with a recombmant virus, for example a recombmant vaccinia virus Techniques and vectors for the insertion of foreign DNA into vaccinia virus are well known m the art, and utilize, for example homologous recombination A wide vaπety of viral promoter sequences, possibly terminator sequences and poly(A)-addιtιon sequences, possibly enhancer sequences and possibly amplification sequences, all required for the mammalian expression, are available in the art. Vaccinia is particularly prefened since vaccinia halts the expression of host cell proteins. For vaccination of humans the avipox and Ankara Modified Vims (AMV) are particularly useful vectors

Also known are insect expression transfer vectors denved from baculovirus Auto rapha cahfornica nuclear polyhedrosis virus (AcNPV), which is a helper-independent viral expression vector. Expression vectors denved from this system usually use the strong viral polyhedπn gene promoter to dnve the expression of heterologous genes. Different vectors as well as methods for the introduction of heterologous DNA into the desired site of baculovirus are available to the man skilled in the art for baculovirus expression. Also different signals for posttranslational modification recognized by insect cells are known in the art

The present invention also relates to a host cell transformed with a recombmant vectoi as defined above

The present invention also relates to a method for detecting antibodies to TTV present in a biological sample, compnsmg:

(l) contacting the biological sample containing antibodies to be analysed for the presence of TTV with a polypeptide as defined above, (n) detecting the immunological complex formed between said antibodies and said polypeptide.

The immunoassay methods according to the present invention may utilize antigens from the different domains of the new and unique polypeptide sequences of the present invention that maintain linear (in case of peptides) and conformational epitopes (in case of polypeptides) recognized by antibodies in the sera from individuals infected with TTV. It is withm the scope of the invention to use for instance single or specific o gomenc antigens, dimenc antigens, as well as combinations of single or specific ohgomenc antigens. The TTV antigens of the present invention may be employed in virtually any assay format that employs a known antigen to detect antibodies. Of course, a format that denatures the TTV conformational epitope should be avoided or adapted A common feature of all of these assays is that the antigen is contacted with the body component suspected of containing TTV antibodies under conditions that permit the antigen to bind to any such antibody present in the component. Such conditions will typically be physiologic temperature, pH and ionic strenght using an excess of antigen The incubation of the antigen with the specimen is followed by detection of immune complexes compnsed of the antigen

Design of the lmmunoassays is subject to a great deal of vaπation, and many formats are known in the art. Protocols may, for example, use solid supports, or immunoprecipitation Most assays involve the use of labeled antibody or polypeptide; the labels may be, for example, enzymatic, fluorescent, chemilummescent, radioactive, or dye molecules Assays which amplify the signals from the immune complex are also known; examples of which are assays which utilize biotin and avidin or streptavidin, and enzyme-labeled and mediated lmmunoassays, such as ELISA assays

The immunoassay may be, without limitation, in a heterogeneous or in a homogeneous format, and of a standard or competitive type. In a heterogeneous format, the polypeptide is typically bound to a solid matnx or support to facilitate separation of the sample from the polypeptide after incubation Examples of solid supports that can be used are nitrocellulose (e g., in membrane or microtiter well form), polyvinyl chloπde (e.g., in sheets or microtiter wells), polystyrene latex (e.g., in beads or microtiter plates, polyvmyhdine fluoπde (known as Immunolon™), diazotized paper, nylon membranes, activated beads, and Protein A beads. For example. Dynatech Immunolon™ 1 or Immunolon 2 microtiter plates or 0.25 inch polystyrene beads (Precision Plastic Ball) can be used in the heterogeneous format. The solid support containing the antigemc polypeptides is typically washed after separating it from the test sample, and pπor to detection of bound antibodies. Both standard and competitive formats are know in the art

In a homogeneous format, the test sample is incubated with the combination of antigens in solution. For example, it may be under conditions that will precipitate any antigen-antibody complexes which are formed. Both standard and competitive formats for these assays are known in the art

In a standard format, the amount of TTV antibodies in the antibody-antigen complexes is directly monitored This may be accomplished by determining whether labeled anti-xenogeneic (e.g. anti- human) antibodies which recognize an epitope on anti-TTV antibodies will bind due to complex formation. In a competitive format, the amount of TTV antibodies in the sample is deduced by monitonng the competitive effect on the binding of a known amount of labeled antibody (or other competing ligand) in the complex.

Complexes formed compnsmg anti-TTV antibody (or in the case of competitive assays, the amount of competing antibody) are detected by any of a number of known techniques, depending on the format. For example, unlabeled TTV antibodies in the complex may be detected using a conjugate of anti-xenogeneic Ig complexed with a label (e.g. an enzyme label)

In an immunoprecipitation or agglutination assay format the reaction between the TTV antigens and the antibody forms a network that precipitates from the solution or suspension and forms a visible layer or film of precipitate. If no anti-TTV antibody is present in the test specimen, no visible precipitate is formed

There cunently exist three specific types of particle agglutination (PA) assays These assays are used for the detection of antibodies to vanous antigens when coated to a support. One type of this assay is the hemagglutmation assay using red blood cells (RBCs) that are sensitized by passively adsorbing antigen (or antibody) to the RBC. The addition of specific antigen antibodies present in the body component, if any, causes the RBCs coated with the puπfied antigen to agglutinate

To eliminate potential non-specific reactions in the hemagglutmation assay, two artificial earners may be used instead of RBC m the PA. The most common of these are latex particles However, gelatin particles may also be used The assays utilizing either of these earners are based on passive agglutination of the particles coated with purified antigens.

The TTV antigens of the present invention compπsed of conformational epitopes will typically be packaged in the form of a kit for use in these lmmunoassays. The kit will normally contain separate containers the native TTV antigen, control antibody formulations (positive and/or negative), labeled antibody when the assay format requires the same and signal generating reagents (e.g. enzyme substrate) if the label does not generate a signal directly. The native TTV antigen may be already bound to a solid matnx or separate with reagents for binding it to the matnx. Instructions (e.g. wntten, tape, CD-ROM, etc.) for canying out the assay usually will be included in the kit

In cases of a positive reactivity to the TTV antigen, it is preferable to repeat the immunoassay to lessen the possibility of false positives. For example, in the large scale screening of blood for the production of blood products (e.g. blood transfusion, plasma, Factor VIJT, lmmunoglobulm, etc.) 'screening' tests are typically formatted to increase sensitivity (to insure no contaminated blood passes) at the expense of specificity; i.e. the false-positive rate is increased. Thus, it is typical to only defer for further testing those donors who are 'repeatedly reactive'; i.e. positive in two or more runs of the immunoassay on the donated sample However, for confirmation of TTV-positivity, the 'confirmation' tests are typically formatted to increase specificity (to insure that no false-positive samples are confirmed) at the expense of sensitivity

The solid phase selected can include polymenc or glass beads, nitrocellulose, microparticles, microwells of a reaction tray, test tubes and magnetic beads The signal generating compound can include an enzyme, a luminescent compound, a chromogen, a radioactive element and a chemilummescent compound. Examples of enzymes include alkaline phosphatase, horseradish peroxidase and beta-galactosidase Examples of enhancer compounds include biotm, anti-biotin and avidin Examples of enhancer compounds binding members include biotin, anti-biotm and avidin. In order to block the effects of rheumatoid factor-like substances, the test sample is subjected to conditions sufficient to block the effect of rheumatoid factor-like substances. These conditions compπse contacting the test sample with a quantity of anti-human IgG to form a mixture, and incubating the mixture for a time and under conditions sufficient to form a reaction mixture product substantially free of rheumatoid factor-like substance

The present invention also relates to a method for TTV typing, compnsmg:

The present invention also relates to a diagnostic kit for use in detecting the presence of TTV, said kit compnsmg at least one polypeptide as defined above, with said polypeptide being possibly bound to a solid support.

The present invention also relates to a diagnostic kit for TTV typing, said kit comprising at least one polynucleic acid as defined above, with said polynucleic acid being possibly bound to a solid support.

The present invention also relates to a diagnostic kit as defined above, said kit comprising a range of polypeptides which are attached to specific locations on a solid substrate.

The present invention also relates to a diagnostic kit as defined above, wherein said solid support is a membrane strip and said polypeptides are coupled to the membrane in the form of parallel lines.

The present invention also relates to a pharmaceutical composition comprising at least one polypeptide as defined above and a suitable excipient, diluent or carrier.

The present invention also relates to a method of preventing TTV infection, comprising administering the pharmaceutical compositon as defined above to a mammal in effective amount to stimulate the production of protective antibody or protective T-cell response.

The present invention also relates to the use of a composition as defined above in a method for preventing TTV infection as defined above.

The present invention also relates to a vaccine for immunizing a mammal against TTV infection, comprising at least one polypeptide as defined above, in a pharmaceutically acceptable carrier.

Moreover, the present invention pertains to a vaccine for immunizing a mammal against TTV infection, comprising at least one TTV polynucleic acid as defined above, encoding a TTV polypeptide, in a pharmaceutically acceptable carrier. The term 'immunogenic' refers to the ability of a substance to cause a humoral and/or cellular response, whether alone or when linked to a earner, in the presence or absence of an adjuvant 'Neutralization' refers to an immune response that blocks the mfectivity, either partially or fully, of an infectious agent A 'vaccine' is an immunogenic composition capable of eliciting protection against TTV, whether partial or complete A vaccine may also be useful for treatment of an individual, in which case it is called a therapeutic vaccine

The term 'therapeutic' refers to a composition capable of treating TTV infection The term 'effective amount' refers to an amount of epitope-beaπng polypeptide sufficient to induce an immunogenic response in the individual to which it is administered, or to otherwise detectably immunoreact in its intended system (e g , immunoassay) Preferably, the effective amount is sufficient to effect treatment, as defined above The exact amount necessary will vary according to the application For vaccine applications or for the generation of polyclonal antiserum / antibodies, for example, the effective amount may vary depending on the species, age, and general condition of the individual, the seventy of the condition being treated, the particular polypeptide selected and its mode of administration, etc It is also believed that effective amounts will be found withm a relatively large, non-cntical range An appropnate effective amount can be readily determined using only routine experimentation Prefened ranges of proteins for prophylaxis of TTV disease are 0 Of to fOO μg/dose, preferably 0 1 to 50 μg/dose Several doses may be needed per individual in order to achieve a sufficient immune response and subsequent protection against TTV disease

Pharmaceutically acceptable earners include any earner that does not itself induce the production of antibodies harmful to the individual receiving the composition Suitable earners are typically large, slowly metabolized macromolecules such as proteins, polysacchandes, polylactic acids, polyglyco c acids, polymenc am o acrds, ammo acid copolymers, and inactive virus particles Such earners are well known to those of ordinary skill in the art

Prefened adjuvants to enhance effectiveness of the composition include, but are not limited to alumimm hydroxide (alum), N-acetyl-muramyl-L-threonyl-D-isoglutamme (thr-MDP) as found in U S Patent No 4,606,918, N-acetyl-normuramyl-L-alanyl-D-isoglutamme (nor-MDP), N- acetylmuramyl-L-alanyl-D-ιsoglutamιnyl-L-alanιne-2-(l'-2'-dιpalmιtoyl-sn-glycero-3-hydroxy- phosphoryloxy)-ethylamιne (MTP-PE) and RIBI, which contains three components extracted from bactena, monophosphoryl lipid A, trehalose dimycolate, and cell wall skeleton (MPL + TDM + CWS) in a 2% squalene/Tween 80 emulsion. Any of the 3 components MPL, TDM or CWS may also be used alone or combined 2 by 2. Additionally, adjuvants such as Strmulon (Cambπdge Bioscience, Worcester, MA) or SAF-1 (Syntex) may be used. Further, Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (LFA) may be used for non-human applications and research purposes.

The immunogenic compositions typically will contain pharmaceutically acceptable vehicles, such as water, saline, glycerol, ethanol, etc Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffenng substances, preservatives, and the like, may be included in such vehicles

Typically, the immunogenic compositions are prepared as mjectables, either as liquid solutions or suspensions, solid forms suitable for solution in, or suspension m, liquid vehicles pπor to injection may also be prepared The preparation also may be emulsified or encapsulated m liposomes for enhanced adjuvant effect The proteins may also be incorporated into Immune Stimulating Complexes together with saponins, for example Quil A (ISCOMS)

Immunogenic compositions used as vaccines compπse a 'sufficient amount' or 'an immunologically effective amount' of the proteins of the present invention, as well as any other of the above mentioned components, as needed. 'Immunologically effective amount', means that the administration of that amount to an individual, either in a single dose or as part of a seπes, is effective for treatment, as defined above. This amount vanes depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated (e.g. nonhuman pπmate, pπmate, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, the strain of infecting TTV, and othei relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine tnals. Usually, the amount will vary from 0.01 to 1000 μg/dose, more particularly from 0.1 to 100 μg/dose.

The present invention also relates to a vaccine as defined above, compnsmg at least one polypeptide as defined above, with said polypeptide being unique for at least one of the TTV subtypes as defined above The present invention also relates to a peptide conespondmg to an amino acid sequence encoded by at least one of the TTV polynucleic acids as defined above, with said peptide compnsmg an epitope being unique to at least one of the TTV subtypes or types as defined above, and with said peptide containing at least one ammo acid diffenng from any of the known TTV types or subtypes amino acid sequences, or an analog thereof being substantially similar and biologically equivalent

The present invention also relates to a method for detecting antibodies to TTV present in a biological sample, compπsing (I) contacting the biological sample to be analysed for the presence of TTV with a peptide as defined above, (π) detecting the immune complex formed between said antibodies and said peptide

The present invention also relares to a method for TTV typing, compπsing (l) contacting the biological sample to be analysed for the presence of TTV with a peptide as defined above, (n) detecting the immune complex formed between said antibodies and said peptide

The present invention also relates to a diagnostic kit for use in detecting the presence of TTV, said kit compnsmg at least one peptide as defined above, with said peptide being possibly bound to a solid support

The present invention also relates to a diagnostic kit for TTV typing, said kit compnsmg at least one peptide as defined above, with said peptide being possibly bound to a solid support

The present invention also relates to a diagnostic kit as defined above, said kit compnsmg a range of peptides which are attached to specific locations on a solid substrate

The present invention also relates to a diagnostic kit as defined above, wherein said solid support is a membrane stnp and said peptides are coupled to the membrane in the form of parallel lines

The present invention also relates to a pharmaceutical composition compnsmg at least one peptide as defined above and suitable excipient, diluent or earner The present invention also relates to a method of preventing TTV infection, compnsmg admmistenng the pharmaceutical composition as defined above to a mammal m effective amount to stimulate the production of protective antibody or protective T-cell response

The present invention further relates to the use of a composition as defined above in a method for preventing TTV infection as defined above

The present invention also relates to a vaccine for immunizing a mammal against TTV infection, compnsmg at least one peptide as defined above, in a pharmaceutically acceptable earner.

The present invention also relates to a vaccine as defined above, compnsmg at least one peptide as defined above, with said peptide being unique for at least one of the subtypes or types as defined above.

The present invention also relates to an antibody raised upon immunization with at least one polypeptide or peptide as defined above, with said antibody being specifically reactive with any of said polypeptides or peptides, and with said antibody being preferably a monoclonal antibody

The present invention also lelates to a method for detecting TTV antigens present m a biological sample, compnsmg:

(I) contacting said biological sample with an antibody as defined above,

(n) detecting the immune complexes formed between said TTV antigens and said antibody.

The present invention relates also to a method for TTV typing, compnsmg:

(l) contacting said biological sample with an antibody as defined above,

The present invention relates also to a diagnostic kit for use in detecting the presence of TTV, said kit comprising at least one antibody as defined above, with said antibody being possibly bound to a solid support

The present invention relates also to a diagnostic kit for TTV typing, said kit compπsing at least one antibody as defined above, with said antibody being possibly bound to a solid support

The present invention also relates to a diagnostic kit as defined above, said kit compnsmg a range of antibodies which are attached to specific locations on a solid substrate

The present invention also relates to a pharmaceutical composition compnsmg at least one antibody as defined above and a suitable excipient, diluent or earner

The present invention also relates to a method of preventing or treating TTV infection, compnsmg ddmimstenng the pharmaceutical composition as defined above to a mammal in effective amount

The present invention also relates to the use of a composition as defined above m a method for preventing or treating TTV infection as defined above

FIGURE LEGENDS

Figure 1 Alignment of the 222bp nucleotide sequences (nt 1959-2180) of the isolates of the newly identified types and subtypes of example 1 in comparison with known prototype isolates of types and subtypes. Hyphens denote identical nucleotides as TA278.

Figure 2 Alignment of the 222bp nucleotide sequences (nt 1959-2180) of the isolates of the newly identified types and subtypes of example 2 in comparison with known prototype isolates of types and subtypes. Hyphens denote identical nucleotides as TA278.

Figure 3 Alignment of the 222bp nucleotide sequences (nt 1959-2180) of the isolates of the newly identified types and subtypes of example 3 in comparison with known prototype isolates of types and subtypes. Hyphens denote identical nucleotides as TA278.

Figure 4 Alignment of the 222bp nucleotide sequences (nt 1959-2180) of the isolates of the newly identified types and subtypes of example 4 in comparison with known prototype isolates of types and subtypes. Hyphens denote identical nucleotides as TA278.

Figure 5 Alignment of the 222bp nucleotide sequences (nt 1959-2180) of the isolates of the newly identified types and subtypes of example 5 in comparison with known prototype isolates of types and subtypes. Hyphens denote identical nucleotides as TA278.

Figure 6 Alignment of the 74 aa amino acid sequences (aa 451-524 of ORFl) of the isolates of the newly identified types and subtypes of example 1 in comparison with known prototype isolates of types and subtypes. Hyphens denote identical amino acids as TA278. Figure 7

Alignment of the 74 aa ammo acid sequences (aa 451-524 of ORFl) of the isolates of the

identified types and subtypes of example 2 in companson with known prototype isolates of types and subtypes Hyphens denote identical ammo acids as TA278

Figure 8

Alignment of the 74 aa ammo acid sequences (aa 451-524 of ORFl) of the isolates of the newly identified types and subtypes of example 3 in companson with known prototype isolates of types and subtypes Hyphens denote identical amino acids as TA278

Figure 9

Alignment of the 74 aa ammo acid sequences (aa 451-524 of ORFl) of the isolates of the newh identified types and subtypes of example 4 in companson with known prototype isolates of types and subtypes Hyphens denote identical ammo acids as TA278

Figure 10

Alignment of the 74 aa ammo acid sequences (aa 451-524 of ORFl) of the isolates of the newly identified types and subtypes of example 5 in companson with known prototype isolates of types and subtypes Hyphens denote identical ammo acids as TA278

EXAMPLES

EXAMPLE 1

Serum samples

36 serum samples from patients with hepatitis of unknown etiology were analyzed for the presence of TTV The samples were previously found to be negative for hepatitis A, B and C markers Four samples (IG20254, IG20256, IG20266 and IG21425) were HGV positive

DNA isolation, PCR and sequencing

DNA was isolated from lOOμl serum as descnbed in Example 4 PCR was performed with the semi -nested pπmeiset NG59/63-NG61/63 (see Table 1) as descnbed m Okamoto et al (1998) The PCR consisted of 3 mm at 95°C, 35 cycles with denaturation at 94°C 1 mm, annealing at 50°C 1 mm, elongation at 72°C 1 mm and finally 10 mm at 72°C Surpnsingly, 17 out of 36 samples were positive These amplified products were sequenced using the pπmerset NG061/063 No amplification product could be obtained from samples IG20030, IG20033, IG20188, IG20189, IG20241, IG20249, IG20252, IG20253, IG20254, IG20255, IG20262, IG20266, IG20270, IG20281, IG21009, IG21268, IG21424, IG22425 and IGLT

Table 1 Sequence of the pπmers used in PCR and sequence analysis (Okamoto et al , 1998)

A TTV sequence was obtained for 14 out of 17 PCR fragments Withm the TTV-positive samples, only isolate IG20256 was co-mfected with HGV

Identification of new types and subtypes When Okamoto et al. (1998) compared a sequence of 356bp among 78 isolates, two genetic groups were found which differed by 30% (similarity of 70%). They were named Gl and G2. These genetic groups were divided into subgroups differing by 11 to 15% (similarity of 85-89%) in sequence from each other, and designated Gia and Gib as well as G2a and G2b.

The % similarity we observed with our samples compared to the described la, lb, 2a and 2b isolates as well as compared to each other are shown in Table 2.1. The samples that proved to belong tolb (IG20002, IG20256, IG27753) or 2b (IG20251, IG20296) are not included in this table. These samples showed a similarity of more than 90% in comparison with the already described subtype of lb (IG20002, IG20256, IG27753), and a similarity of more than 85% compared to 2b (IG20296), respectivily.

Table 2.1 clearly shows that 4 samples (IG20008, IG20260, IG21010 and IG21285) belong to a new type 3, and that they cluster in the same subtype 3a. One sample, IG21286, can be designated to another new genotype 4 and is classified into subtype 4a. Furthermore, 4 isolates showed homology to type 2. IG20272 and IG20010 clearly can be classified as new subtypes 2c, whereas IG21011 (and IG21012) belongs to a different subtype 2d.

For some of the samples, a number of indeterminate bases were obtained. However, when repeating the nested PCR with the same primer set, tagged with the SP6 and T7 sequence to facilitate sequence analysis, a very clear sequence emerged. In such cases, the sequence was quite different from the sequence which was obtained previously. For IG20251 (formerly classified as type 2d), such a newly obtained sequence classified as type 2b instead.

These fenomena can be explained by the presence of a mixed population of different TTV genotypes in the sample (coinfection).

Table 2.1

Table 2.2 gives an overview of the sequences belonging to newly identified TTV genotypes

Table 2.2

Figure 1 shows a nucleotide sequence alignment of the sequences obtained in the present example as listed m Table 2.2 Nucleotide vanations become clearly apparent in such an alignment and each of the vanations (conserved positions are indicated by a dash) may be used to diagnose, treat or prevent TTV infection according to the detailed descnption of the invention Figure 6 shows an ammo acid sequence alignment of the sequences obtained in the present example as listed in Table 2.2. Ammo acid vanations become clearly apparent m such an alignment and each of the vanations (conserved positions are indicated by a dash) may be used to diagnose, treat or prevent TTV infection according to the detailed descnption of the invention. EXAMPLE 2

Serum samples

202 semm samples from Brazilian blood donors (102 with ALT<65 IU/1, 100 with ALT>65 lU/l) were analyzed for the presence of TTV. The samples were previously found to be negative for hepatitis B and C, HIV, HTLV, Chagas and Syphilis.

77 samples from haemophiliacs were also tested. Five of those patients were PCR-positive for HCV, one of them was positive both for HCV and HGV. One sample originated from a Von Willebrand patient, another sample originated from a multitransfused patient.

DNA isolation, PCR and sequencing

DNA was isolated from fOOμl serum as in Example 4. PCR was performed with the semi-nested primerset NG59/63-NG61/63 (see Table 1) as described in Okamoto et al. (1998). The PCR consisted of 3 min at 95°C, 35 cycles with denaturation at 94°C 1 min, annealing at 50°C 1 min, elongation at 72°C 1 min and finally 10 min at 72°C. 6 out of the 77 haemophiliac samples (8%), 3 of the 202 blood donors (1.5%) and the Von Willebrand patient were positive. These amplified products were sequenced using the primerset NG061/063. A TTV sequence was obtained for the samples 1M24, lb210, 1M042, 1M287, 1 372, lb2540, lb2572 and lb3126.

Identification of new types and subtypes

When Okamoto et al. (1998) compared a sequence of 356bp among 78 isolates, two genetic groups were found which differed by 30% (similarity of 70%). They were named Gl and G2. These genetic groups were divided into subgroups differing by 11 to 15% (similarity of 85-89%) in sequence from each other, and designated Gfa and Gfb as well as G2a and G2b. The percentages of similarity we observed between the genotypes of the present invention and the described la, lb, 2a and 2b isolates are shown in Table 3. The sample that proved to belong to lb (lb 1287) is not included in this table. This sample showed a similarity of 95% with the already described subtype of lb.

Table 3 clearly shows that 1 sample (lb210) belongs to a new type 5a. Furthermore, 6 isolates showed homology to type 2. Ib3126, lb2540, 1 042 and lb2572 clearly can be classified into subtype 2c, whereas 372 and lbl24 belong to the other new subtype 2d. Table 3.

Table 3. continued

Table 4 gives an overview of the sequences belonging to newly identified TTV genotypes of example 2

Figure 2 shows a nucleotide sequence alignment of the sequences obtained in the present example as listed in Table 4 Nucleotide vanations become clearly apparent in such an alignment and each of the vanations (conserved positions are indicated by a dash) may be used to diagnose, treat or prevent TTV infection according to the detailed descnption of the invention Figuie 7 shows an ammo acid sequence alignment of the sequences obtained in the present example as listed m Table 4 Ammo acid vanations become clearly apparent in such an alignment and each of the vanations (conserved positions are indicated by a dash) may be used to diagnose, treat or prevent TTV infection according to the detailed descnption of the invention

EXAMPLE 3

Serum samples

23 serum samples from 22 patients with hepatitis of unknown ongin from Flanders, Belgium, were analyzed for the presence of TTV DNA isolation, PCR and sequencing

DNA was isolated from lOOμl serum as described in Example 4. PCR was performed with the semi-nested primerset NG59/63-NG61/63 (see Table 1) as described in Okamoto et al. (1998). The PCR consisted of 3 min at 95°C, 35 cycles with denaturation at 94°C f min, annealing at 50°C 1 min, elongation at 72°C 1 min and finally 10 min at 72°C. Six out of the 22 patients (27%) were positive. These amplified products were sequenced using the primerset NG061/063. A TTV sequence was obtained for all the samples.

Identification of new types and subtypes

When Okamoto et al. (1998) compared a sequence of 356bp among 78 isolates, two genetic groups were found which differed by 30% (similarity of 70%). They were named Gl and G2. These genetic groups were divided into subgroups differing by 11 to 15% (similarity of 85-89%) in sequence from each other, and designated Gla and Gib as well as G2a and G2b.

Percentages of similarity of the isolates are compared with the previously described genotypes la, lb, 2a, 2b and with genotypes 2c, 2d, 3a and 4a of the present invention in Table 5. The samples that proved to belong to la (gdg592), lb (gdg577) and 2b (gdg583) based on high similarities are not included in this table. gdg592 showed a similarity of 92% with the already described subtype of la, gdg577 is 95% similar to lb, and gdg583 has 95% similarity with 2b. Table 5 clearly shows that the other 4 samples (gdg566, gdg568, gdg569 and gdg575) belong to genotype 3a of the present invention. gdg566 and gdg568 originate from the same patient, with 1 month interval.

Table 6 gives an overview of the sequences belonging to newly identified TTV genotypes of example 3.

Table 6.

Figure 3 shows a nucleotide sequence alignment of the sequences obtained in the present example as listed in Table 6. Nucleotide variations become clearly apparent in such an alignment and each of the variations (conserved positions are indicated by a dash) may be used to diagnose, treat or prevent TTV infection according to the detailed description of the invention. Figure 8 shows an amino acid sequence alignment of the sequences obtained in the present example as listed in Table 6. Amino acid variations become clearly apparent in such an alignment and each of the variations (conserved positions are indicated by a dash) may be used to diagnose, treat or prevent TTV infection according to the detailed description of the invention.

EXAMPLE 4

Serum samples

200 serum samples from healthy Belgian blood donors were analyzed for the presence of TTV.

DNA isolation, PCR and sequencing

DNA was isolated from 100 μl serum using High Pure PCR Template Preparation kit (Boehnnger Mannheim). PCR was performed with the semi-nested pnmerset NG59/63-NG61/63 (see Table 1) as descnbed in Okamoto et al. (1998). The PCR consisted of 3 min at 95°C, 35 cycles with denaturation at 94°C 1 mm, annealing at 50°C 1 min, elongation at 72°C 1 mm and finally f 0 mm at 72°C. Thirteen samples were positive for TTV(6.5%). These amplified products were sequenced using the primerset NG061/063. A TTV sequence was obtained for 6 of the 13 samples.

Identification of new types and subtypes

When Okamoto et al. (1998) compared a sequence of 356bp among 78 isolates, two genetic groups were found which differed by 30% (similanty of 70%) They were named Gl and G2. These genetic groups were divided into subgroups diffenng by 11 to 15% (similanty of 85-89%) in sequence from each other, and designated Gla and Gib as well as G2a and G2b Percentages of similanty of the 13 sequences were compared with the previously published genotypes la, lb, 2a, 2b and genotypes 2c, 2d, 3a, 4a of the present invention in Table 7. The sample that belongs to lb (bde911) is not included m this table. This sample showed a similanty of 95% with the already descnbed subtype of lb.

Table 7 clearly shows that 2 isolates showed homology to type 2c (bde943 and bde953). The other three samples belong to type 3a. The classification of new TTV isolates from Belgium (examples 3 and 4) into the newly discovered genotype 2d, and 3a groups (example 1) clearly demonstrates the widespread prevalence of these TTV genotypes in Belgium. Table 7.

Table 8 gives an overview of the sequences belonging to newly identified TTV genotypes of example 4.

Table 8.

Figure 4 shows a nucleotide sequence alignment of the sequences obtained in the present example as listed in Table 8. Nucleotide variations become clearly apparent in such an alignment and each of the variations (conserved positions are indicated by a dash) may be used to diagnose, treat or prevent TTV infection according to the detailed description of the invention. Figure 9 shows an amino acid sequence alignment of the sequences obtained in the present example as listed in Table 8. Amino acid variations become clearly apparent in such an alignment and each of the variations (conserved positions are indicated by a dash) may be used to diagnose, treat or prevent TTV infection according to the detailed description of the invention.

EXAMPLE 5

Serum samples

Five serum samples from HCV-infected chimpanzees were analyzed for the presence of TTV.

DNA isolation, PCR and sequencing

DNA was isolated from lOOμl serum as descnbed in Example 4. PCR was performed with the semi-nested pnmerset NG59/63-NG61/63 (see Table 1) as descnbed in Okamoto et al (1998). The PCR consisted of 3 mm at 95°C, 35 cycles with denaturation at 94°C 1 min, annealing at 50°C 1 mm, elongation at 72°C 1 mm and finally 10 mm at 72°C. One chimpanzee was positive for TTV. This amplified product was sequenced using the pπmerset NG061/063.

Identification of new types and subtypes

Percentages of similanty with the previously known genotypes and with genotypes 2c, 2d, 3a, 4a, 5a of the present invention are shown in Table 9. About 50% homology is obtained which clearly indicates the segregation of CHIMl into a new TTV genotype 6, of which the particular subtype is designated 6a. It should be clear that TTV genotype nomenclature may still be adapted such that a cunent 6a may later be denoted 4a, 5a, 7a, 6b, or any other name.

Table 9.

^the deletions are considered as mismatches in this calculation

Table 10 gives an overview of the sequences belonging to newly identified TTV genotypes of example 5

Table 10.

Figure 5 shows a nucleotide sequence alignment of the sequence obtained in the present example as listed in Table 10. Nucleotide vanations become clearly apparent in such an alignment and each of the vanations (conserved positions are indicated by a dash) may be used to diagnose, treat or prevent TTV infection according to the detailed descnption of the invention. Figure 10 shows an amino acid sequence alignment of the sequence obtained in the present example as listed in Table 10. Ammo acid vanations become clearly apparent in such an alignment and each of the vanations (conserved positions are indicated by a dash) may be used to diagnose, treat or prevent TTV infection according to the detailed description of the invention.***

EXAMPLE 6

Based on the amino acid alignments from the previous examples, type-specific amino acids can be distinguished. Table 11 gives an overview of the amino acids occurring at each position of the analyzed sequence for the different types and subtypes.

Table 11.

AA nr. Typel Type 2 Type 3 Type 4 Type 5 Type 6

REFERENCES

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Bej A, Mahbubam M, Miller R, Di Cesare J, Haff L, Atlas R. (1990) Multiplex PCR amplification and immobilized capture probes for detection of bacteπal pathogens and indicators in water Mol Cell Probes 4:353-365.

Compton J (1991) Nucleic acid sequence-based amplification. Nature 350. 91-92.

Duck P (1990) Probe amplifier system based on chimeric cycling ohgonucleotides Bwtechniques 9. 142-147.

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Nielsen P, Egholm M, Berg R, Buchardt O (1993) Sequence specific inhibition of DNA restriction enzyme cleavage by PNA. Nucleic-Acids-Res. 21(2): 197-200. Nishizawa, T., Okamoto, H., Komshi, K., Yoshizawa, H., Miyakawa, Y. and Mayumi, M. (1997). A novel DNA virus (TTV) associated with elevated transammase levels in posttransfusion hepatitis of unknown etiology. Biochem.Biophys.Res.Comm. 241, 92-97.

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Claims

1 A TTV polynucleic acid having a nucleotide sequence which is unique to at least TTV type 3, type 4, type 5 or type 6 or TTV subtypes 2d, subtype 3a, subtype 4a, subtype 5a or subtype 6a with said TTV sequences being classified as shown m Table 2 2, 4, 6, 8, 10 or Figure 1, 2, 3, 4,

5

2 A polynucleic acid sequence encoding a TTV polyprotem compnsmg in its amino acid sequence at least one of the type- or subtype specific ammo acid residues as indicated in Table

11

3 A polynucleic acid sequence according to claim 1 or 2 having a sequence selected ftom any of SEQ ID NO 5, 7, 9, 11, 13, 15, 17, 27, 29, 31, 33, 35, 37, 39, 45, 47, 49, 51, or a part of said polynucleic acid which is unique to at least one of the TTV subtypes or types as defined in claim 1, and which contains at least one nucleotide diffe╧Çng from known TTV nucleotide sequences, or the complement thereof

4 An oligonucleotide pnmer compnsmg part of a polynucleic acid according to any of claims 1 to 3, with said pnmer being able to act as pnmer for specifically sequencing or specifically amplifying the nucleic acid of a certain isolate belonging to the genotype from which the pnmer is de╧Çved

5 An oligonucleotide probe compnsmg part of a polynucleic acid according to any of claims 1 to 3, with said probe being able to act as a hybndization probe for specific detection and/or classification into types and/or subtypes of a TTV nucleic acid containing said nucleotide sequence, with said probe being possibly labelled or attached to a solid substrate

6. A diagnostic kit for use in determining the presence of a TTV isolate, said kit compnsmg a pnmer according to claim 4

7. A diagnostic kit for use in determining the presence of a TTV isolate, said kit compnsmg a probe according to claim 5

8 A diagnostic kit according to claim 7, wherein said probe(s) is (are) attached to a solid support

9. A diagnostic kit according to claim 8, wherein a range of said probes are attached to specific locations on a solid support.

10. A diagnostic kit according to claim 8 or 9, wherein said solid support is a membrane strip and said probes are coupled to the membrane in the form of parallel lines.

11. One or more probes according to claim 5, coupled to a solid support, preferably a membrane strip, in the form of parallel lines.

12. A method for the detection of TTV nucleic acids present in a biological sample, comprising: (i) possibly extracting sample nucleic acid,

(ii) possibly amplifying the nucleic acid with at least one primer according to claim

4 or with a universal TTV primer, (iii) hybridizing the nucleic acids of the biological sample, possibly under denatured conditions, at appropriate conditions with one or more probes according to claim 5, with said probes being possibly attached to a solid substrate, (iv) possibly washing at appropriate conditions,

(v) detecting the hybrids formed.

13. A method for detecting the presence of one or more TTV genotypes present in a biological sample, comprising:

(i) possibly extracting sample nucleic acid,

(ii) specifically amplifying the nucleic acid with at least one primer according to claim 4,

(iii) detecting said amplified nucleic acids,

(iv) inferring the presence of one or more genotypes of TTV present from the observed pattern of amplified fragments.

14. A method for detecting the presence of one or more TTV genotypes present in a biological sample, comprising:

(i) possibly extracting sample nucleic acid,

4 or with a universal TTV primer, (in) hyb╧Çdizing the nucleic acids of the biological sample, possibly under denatured conditions, at approp╧Çate conditions with one or more probes according to claim 5, with said probes being possibly attached to a solid substrate,

(iv) possibly washing at approp╧Çate conditions, (v) detecting the hybnds formed,

(vi) mfernng the presence of one or more TTV genotypes present from the observed hybndization pattern.

15. A method for detecting the presence of TTV nucleic acids present in a biological sample, compnsmg

(l) possibly extracting sample nucleic acid,

(n) amplifying the nucleic acid with at least one p╧Çmer according to claim 4,

(in) detecting the amplified nucleic acids

16. A method for detecting the genotype of TTV nucleic acids according to any of claims 1 to

3 present in a biological sample, compnsmg:

(I) possibly extracting sample TTV nucleic acid,

(╧Ç) possibly amplifying the TTV nucleic acid,

17. A method for detecting the presence of one or more TTV genotypes present in a biological sample, compnsmg

(l) amplifying the nucleic acid with at least one pnmer according to claim 4, (u) contacting said amplified TTV nucleic acid copies, with oligonucleotide probes according to claim 5, which have been immobilised as parallel lines on a solid support (in) mfernng from the result of step (n) the presence of one or more TTV genotypes.

18. A method according to claim 17, wherein said probes are immobilised in a LiPA format

19. A method according to any of claims 12 to 18, wherein said nucleic acids are labelled dunng or after amplification

20. A diagnostic kit for TTV typing, said kit comp╧Çsing at least one polynucleic acid according to any of claims 1 to 5, with said polynucleic acid being possibly bound to a solid support

21. A polypeptide having an ammo acid sequence encoded by a polynucleic acid according to any of claims 1 to 3, or a part thereof which is unique to at least one of the TTV subtypes or types as defined in claims 1 to 3, and which contains at least one ammo acid diffenng from any of the known TTV types or subtypes ammo acid sequences, or an analog thereof being substantially similar and biologically equivalent.

22. A polypeptide according to any of claims 21, 23 or 24, which is recombinantly expressed

23. A polypeptide comp╧Çsing in its ammo acid sequence at least one of the type or subtype specific ammo acid residues as indicated in Table 11, or a part of said polypeptide which is unique to at least one of the TTV subtypes or types as defined in claim 1, and which contains at least one ammo acid diffenng from known TTV types or subtypes ammo acid sequences, or an analog thereof being substantially similar and biologically equivalent to said polypeptide.

24. A polypeptide having an ammo acid sequence as represented in any of SEQ ID NO 6, 8, 10, 12, 14, 16, 18, 28, 30, 32, 34, 36, 38, 40, 46, 48, 50, 52, or a part thereof which is unique to at least one of the TTV subtypes or types as defined in claim 1, and which contains at least one ammo acid differing from known TTV types or subtypes ammo acid sequences, or an analog thereof being substantially similar and biologically equivalent to said polypeptide.

25. A diagnostic kit for use in detecting the presence of TTV, said kit comp╧Çsing at least one polypeptide according to any of claims 21 to 24, with said polypeptide being possibly bound to a solid support.

26. A method for production of a recombmant polypeptide according to any of claims 21 to 24, comp╧Çsing: - transformation of an appropnate cellular host with a recombmant vector, m which a polynucleic acid or a part thereof according to any of claims 1 to 3 has been inserted under the control of the appropnate regulatory elements, cultu╧Çng said transformed cellular host under conditions enabling the expression of said insert, and, harvesting said polypeptide

27 A recombmant expression vector comp╧Çsing a polynucleic acid or a part thereof according to any of claims 1 to 3 operably linked to prokaryotic, eukaryotic or viral transcnption and translation control elements

28 A host cell transformed with a recombmant vector according to claim 27

29 A method for detecting antibodies to TTV present in a biological sample, compnsmg (I) contacting the biological sample containing antibodies to be analysed for the presence of TTV with a polypeptide according to any of claims 21 to 24, (n) detecting the immunological complex formed between said antibodies and said polypeptide

30 A method for TTV typing, compnsmg

(l) contacting the biological sample containing antibodies to be analysed for the presence of TTV with a polypeptide according to any of claims 21 to 24, (n) detecting the immunological complex formed between said antibodies and said polypeptide

31 A diagnostic kit for use in detecting the presence of TTV, said kit comp╧Çsing at least one polypeptide according to any of claims 21 to 24, with said polypeptide being possibly bound to a solid support

32 A diagnostic kit according to claim 31, compnsmg a range of polypeptides which are attached to specific locations on a solid support

33 A diagnostic kit according to claim 32, wherein said solid support is a membrane stnp and said polypeptides are coupled to the membrane in the form of parallel lines

34 A pharmaceutical composition compnsmg at least one polypeptide according to any of claims 21 to 24, and a suitable excipient, diluent or earner

35 A method of preventing TTV infection, compnsmg admmistenng the pharmaceutical compositon according to claim 34, to a mammal in effective amount to stimulate the production of protective antibody or protective T-cell response

36. Use of a composition according to claim 34, in a method for preventing TTV infection according to claim 35

37. A vaccine for immunizing a mammal against TTV infection, compnsmg at least one polypeptide according to any of claims 21 to 24, in a pharmaceutically acceptable earner

38 A vaccine for immunizing a mammal against TTV infection, compnsmg at least one TTV polynucleic acid according to any of claims 1 to 3, encoding a TTV polypeptide, in a pharmaceutically acceptable earner