WO2010004251A1

WO2010004251A1 - Dna methylomes

Info

Publication number: WO2010004251A1
Application number: PCT/GB2009/001487
Authority: WO
Inventors: Manel Esteller
Original assignee: Oncomethylome Sciences Sa; Spencer, Matthew, Peter
Priority date: 2008-06-16
Filing date: 2009-06-16
Publication date: 2010-01-14

Abstract

Methods and kits for diagnosing and/or monitoring the progression of or otherwise staging a disease caused by an infection by a double stranded DNA virus in a test sample obtained from a subject comprise determining the methylation status of the viral genome. The presence of hypermethylation of the viral genome indicates a positive diagnosis of the disease and/or an increased level of methylation of the viral genome indicates the progression of the disease to a more advanced form. Relevant viruses includes, HPV18, HBV and EBV. Suitable diseases linked to viral infection include cancers such as anogenital, hepatic and nasopharyngeal cancers and lymphomas. The methylomes of HPV16, HPV18, EBV and HBV are provided.

Description

DNA METHYLOMES

Field of the invention The invention relates to diseases linked to viral infections such as cancers. The invention relates inter alia to viruses, especially DNA viruses and more particularly to double stranded DNA viruses such as HPV, HBV and EBV. Methods and kits are provided for diagnosing, staging or otherwise monitoring the progress of such diseases. These methods and kits generally involve determining the methylation status of at least a portion of a DNA virus genome.

Background to the invention

Epigenetics encompasses a large number of mechanisms underlying embryonic development, differentiation, and cell identity including DNA methylation and histone modifications, and is increasingly recognized as being involved in human diseases such as cancer and imprinting disorders, among others (1-5).

In contrast to the human genome, human epigenomes vary between tissues, among individuals, and between healthy and disease states. Under these circumstances, distinct epigenomes might explain why the same genotypes generate different phenotypes as occurs in the Agouti mice(6), cloned animals(7), and monozygotic twins(8). Determining the complete DNA methylome entails the description of all the methylated nucleotides in an organism. The gold standard technique for analyzing the methylation state of individual cytosines is bisulfite sequencing in which unmethylated cytosines are converted to uracils and read as thymines, while methylated cytosines are protected from conversion. Bisulfite sequencing yields precise nucleotide resolution data, but to date this method have been limited to relatively small genome coverage (9-11). Alternative approaches involve the isolation of methylated fractions of the genome by methylation-sensitive restriction (12), or immunoprecipitation with a methylcytosinespecific antibody (13-15), combined with hybridization to genomic microarrays. This is exemplified by the recent DNA methylation analyses of the Arabidopsis genome (16,17). Worlwide, it has been estimated that viral infection are etiologically linked to 15% of cancer cases, accounting for nearly 1.5 million new cases and 1,000,000 deaths annually20,21. Three well-studied oncogenic viruses are the human papilloma virus (HPV) (Papillomaviridae; Alphapapillomavirus; Human papillomavirus), the hepatitis B virus (HBV) (Hepadnaviridae; Orthohepadnavirus, Hepatitis B virus) and the Epstein-Barr Virus (EBV) (Herpesviridae; Gammaherpesvirinae; Lymphocryptovirus; Human herpesvirus 4). All of these are double-stranded DNA viruses, that are not only involved in the development of infectious diseases (such as infectious mononucleosis and hepatitis), but also in the formation of cervical, liver and nasopharyngeal tumors and lymphomas, respectively (20,21).

Description of the invention

In a first aspect, the invention provides a method of diagnosing and/or monitoring the progression of or otherwise staging a disease caused by an infection by a virus, in particular a DNA virus and especially a double stranded DNA virus in a test sample obtained from a subject comprising determining the methylation status of the viral genome wherein the presence of hypermethylation of the viral genome indicates a positive diagnosis of the disease and/or an increased level of methylation of the viral genome indicates the progression of the disease to a more advanced form.

These methods of the invention are most preferably ex vivo or in vitro methods carried out on a test sample. In certain embodiments the method may also include the step of obtaining the sample. The methods can be used to diagnose any disease caused by, or resulting from, infection by a virus, in particular a DNA virus and especially a double stranded DNA virus . By "double stranded DNA virus" is meant a virus comprising a DNA genome, at least part of which is, or may be, double stranded. Specific examples relevant to the present invention include HPV, such as HPV-16 and HPV-18, HBV and EBV. Such viruses are involved in a number of disease conditions including infectious and non-infectious disease and the invention may be applied to both - individually or in combination.

HBV infections increases the risk of conditions reflecting an early stage of carcinogenesis, such as active hepatitis and cirrhosis and also full blown cancer in particular, hepatocarcinoma (HCC). Thus, certain aspects of the invention are particularly applicable to liver disease and especially diagnosing and monitoring progression or treatment of liver neoplasias.

EBV infection is surprisingly frequent in B-lymphocyte cells and can manifest itself as reactive lymphadenitis and infectious mononucleosis (IM). Others conditions relevant in accordance with certain embodiments of the invention include cancer and in particular lymphoid and epithelial malignancies such as nasopharyngeal carcinoma, non-Hodgkin's lymphoma - including diffuse large B-cell lymphoma and peripheral T-cell lymphoma, Hodgkin's lymphoma and Burkitt's lymphoma. Thus any of these diseases may be monitored, diagnosed etc according to the invention where the methylation status of the EBV genome is detected.

HPV infections contribute to a number of conditions, ranging from warts and verrucas to more serious diseases such as cancer. The methods of the invention may, therefore, be used in a range of applications. In certain embodiments, the disease which is diagnosed, staged or otherwise monitored comprises, consists essentially of or consists of cancer. HPV infections may contribute to the incidence of a range of cancers. Thus, in specific embodiments where methylation of HPV genomes is detected, the cancer is selected from anogenital cancers or skin cancers. The cancer may be cervical cancer, skin cancer or head and neck cancer for example. The HPV may be HPV18 in certain embodiments.

In specific embodiments, the methods of the invention are utilised in order to stage the disease into one of several categories of varying severity. Thus the progression of the disease may be monitored having regard to the (relative) methylation status of the viral genome in the sample. The methods may detect progression from benign proliferative conditions to tumourigenic conditions. The categories may be, for example virus carrier, pre-malignancy or primary tumour. "Virus carrier" indicates an asymptomatic subject infected with the virus. By "asymptomatic" is meant that clinical symptoms are not displayed by the subject. "Pre-malignancy" indicates subjects who are suffering from pre-tumourigenic lesions, or neoplasias. "Primary tumour" indicates the presence of a tumourigenic lesion. These categories are linked to the methylation status of the viral genome. Generally, hypomethylation indicates a viral carrier and hypermethylation indicates a primary tumour. An intermediate methylation status between hypomethylation and hypermethylation may indicate pre-malignancy. Hypomethylation and Hypermethylation are well known terms in the art to describe (relatively) under and over methylated regions of the genome respectively.

In specific embodiments, where the cancer is cervical cancer, the pre-malignancy may comprise, consist essentially of or consist of cervical intraepithelial neoplasia. Also where the cancer is cervical cancer, the primary tumour may comprise, consist essentially of or consist of squamous cell carcinoma or adenocarcinoma. The presence of hypermethylation of the HPV genome such as the HPV18 genome may be used as an indication of a positive diagnosis of squamous cell carcinoma. In a related embodiment, the presence of hypermethylation of the HPV in particular HPV18 genome indicates a positive diagnosis of squamous cell carcinoma and the presence of less methylation/relative hypomethylation indicates the presence of cervical intraepithelial neoplasia. Thus, in one aspect the invention provides a method of diagnosing squamous cell carcinoma in a test sample obtained from a subject comprising determining the methylation status of a HPV and in particular HPV-18 genome wherein the presence of hypermethylation of the HPV-18 genome indicates a positive diagnosis of squamous cell carcinoma. The invention also provides a method of distinguishing squamous cell carcinoma and cervical intraepithelial neoplasia in a test sample obtained from a subject comprising determining the methylation status of a HPV and in particular HPV -18 genome wherein the presence of hypermethylation of the HPV and in particular HPV -18 genome indicates the presence of squamous cell carcinoma, whereas hypomethylation of the HPV and in particular HPV -18 genome indicates the present of cervical intraepithelial neoplasia.

In certain embodiments, the methods specifically exclude a method of distinguishing squamous cell carcinoma and cervical intraepithelial neoplasia in a test sample obtained from a subject comprising determining the methylation status of a HPV-16 genome wherein the presence of hypermethylation of the HPV-16 genome indicates the presence of squamous cell carcinoma, whereas hypomethylation of the HPV-16 genome indicates the present of cervical intraepithelial neoplasia.

In further specific embodiments, the methods of the invention may exclude a method of diagnosing and/or monitoring the progression of or otherwise staging a disease caused by a human papillomavirus (HPV) infection in a test sample obtained from a subject comprising determining the methylation status of a HPV genome wherein the presence of hypermethylation of the HPV genome indicates a positive diagnosis of the disease and/or an increased level of methylation of the HPV genome indicates the progression of the disease to a more advanced form. Specifically, the HPV is HPV16.

In further embodiments, the cancer is liver cancer. Here, the virus may be HBV. The pre-malignancy may comprise, consist essentially of or consist of chronic active hepatitis and/or hepatic cirrhosis. Also where the cancer is liver cancer, the primary tumour may comprise, consist essentially of or consist of a hepatocarcinoma. The presence of hypermethylation of the HBV genome may be used as an indication of a positive diagnosis of hepatocarcinoma. In a related embodiment, the presence of hypermethylation of the HBV genome indicates a positive diagnosis of hepatocarcinoma and the presence of less methylation/relative hypomethylation indicates the presence of chronic active hepatitis and/or hepatic cirrhosis. Thus, in one aspect the invention provides a method of diagnosing hepatocarcinoma in a test sample obtained from a subject comprising determining the methylation status of a HBV genome wherein the presence of hypermethylation of the HBV genome indicates a positive diagnosis of hepatocarcinoma. The invention also provides a method of distinguishing hepatocarcinoma and chronic active hepatitis and/or hepatic cirrhosis in a test sample obtained from a subject comprising determining the methylation status of a HBV genome wherein the presence of hypermethylation of the HBV genome indicates the presence of hepatocarcinoma, whereas (relative) hypomethylation of the HBV genome indicates the present of chronic active hepatitis and/or hepatic cirrhosis.

In specific embodiments, the cancer is a lymphoid and/or epithelial malignancy. Here, the virus may be EBV. The pre-malignancy may comprise, consist essentially of or consist of a benign condition. Examples include reactive lymphadenitis and infectious mononucleosis. Also where the cancer is a lymphoid or epithelical malignancy, the primary tumour may comprise, consist essentially of or consist of nasopharyngeal carcinoma, non-Hodgkin's lymphoma - including diffuse large B-cell lymphoma and peripheral T-cell lymphoma, Hodgkin's lymphoma and/or Burkitt's lymphoma. The presence of hypermethylation of the EBV genome may be used as an indication of a positive diagnosis of a lymphoid and/or epithelial malignancy. In a related embodiment, the presence of hypermethylation of the EBV genome indicates a positive diagnosis of lymphoid and/or epithelial malignancy and the presence of less methylation/relative hypomethylation indicates the presence of a benign condition. Thus, in one aspect the invention provides a method of diagnosing a lymphoid and/or epithelial malignancy in a test sample obtained from a subject comprising determining the methylation status of an EBV genome wherein the presence of hypermethylation of the EBV genome indicates a positive diagnosis of a lymphoid and/or epithelial malignancy. The invention also provides a method of distinguishing a lymphoid and/or epithelial malignancy and a benign condition such as reactive lymphadenitis and/or infectious mononucleosis in a test sample obtained from a subject comprising determining the methylation status of an EBV genome wherein the presence of hypermethylation of the EBV genome indicates the presence of a lymphoid and/or epithelial malignancy, whereas (relative) hypomethylation of the EBV genome indicates the present of a benign condition such as reactive lymphadenitis and/or infectious mononucleosis. As aforementioned, the lymphoid and/or epithelial malignancy may be selected from nasopharyngeal carcinoma, non-Hodgkin's lymphoma - including diffuse large B-cell lymphoma and peripheral T-cell lymphoma, Hodgkin's lymphoma and Burkitt's lymphoma.

The "test sample" may comprise, consist essentially of or consist of any suitable tissue sample or body fluid. A suitable test sample is thus one representative of the presence of the viral genome and in which the methylation status may be determined. Preferably, the test sample is obtained from a human subject. The type of sample which is appropriate can be readily determined by the nature of the disease to be diagnosed, staged or otherwise monitored according to the methods of the invention. Thus, for example, if the disease to be investigated is liver cancer, the sample may be a suitable liver sample. Suitable samples may be obtained by any method. For example, a suitable cervical sample for diagnosing cervical cancer may be obtained by a cervical scraping, from cervical smears and/or vaginal excretions.

Other DNA-containing samples which may be used in the methods of the invention include samples for diagnostic, prognostic, or personalised medicinal uses provided that they contain the viral genome (or at least a suitable portion thereof) for investigation of its methylation status as an indication of the disease. These samples may be obtained from surgical samples, such as biopsies or fine needle aspirates or from cytological samples, cervical conization or hysterectomy, from paraffin embedded tissues, from frozen tumor tissue samples, from fresh tumor tissue samples or from a fresh or frozen body fluid, for example. Non-limiting examples include whole blood or parts/fractions thereof, bone marrow, cerebrospinal fluid, peritoneal fluid, pleural fluid, lymph fluid, serum, plasma, urine, chyle, ejaculate, sputum, nipple aspirate, saliva, swabs specimens, colon wash specimens and brush specimens. The tissues and body fluids can be collected using any suitable method. Many such methods are well known in the art. Assessment of a paraffin-embedded specimen can be performed directly or on a tissue section.

"Diagnosis" is defined herein to include screening for a disease or pre-indication of a disease, identifying a disease or pre-indication of a disease, monitoring the staging and the state and progression of the disease, checking for recurrence of disease following treatment and monitoring the success of a particular treatment. The methods of the invention may also have prognostic value, and this is included within the definition of the term "diagnosis". The prognostic value of the methods of the invention may be used as a marker of potential susceptibility to the disease associated with a viral infection or as a marker for progression of the disease, for example from pre-malignancy to primary tumour, or from carrier to pre-malignancy for example. Thus patients at risk may be identified before the disease has a chance to manifest itself in terms of symptoms identifiable in the patient.

The methods of the invention may be carried out on purified or unpurified DNA- containing samples. However, in certain embodiments, DNA is isolated/extracted/purified from the sample. Any suitable DNA isolation technique may be utilised. Examples of purification techniques may be found in standard texts such as Molecular Cloning - A Laboratory Manual (Third Edition), Sambrook and Russell (see in particular Appendix 8 and Chapter 5 therein). In one embodiment, purification involves alcohol precipitation of DNA. Suitable alcohols include ethanol and isopropanol. Suitable purification techniques also include salt-based precipitation methods. Thus, in specific embodiments the DNA purification technique comprises use of a high concentration of salt to precipitate contaminants. The salt may comprise, consist essentially of or consist of potassium acetate and/or ammonium acetate for example. The method may further include steps of removal of contaminants which have been precipitated, followed by recovery of DNA through alcohol precipitation. In alternative embodiments, the DNA purification technique is based upon use of organic solvents to extract contaminants from cell lysates. Thus, in certain embodiments, the method comprises use of phenol, chloroform and isoamyl alcohol to extract the DNA. Suitable conditions are employed to ensure that the contaminants are separated into the organic phase and that DNA remains in the aqueous phase. Alternatively, appropriate filters may be utilised in the methods of the invention to isolate DNA from a sample, in particular to achieve separation of DNA from contaminants. Such filters are known in the art and commercially available.

In specific embodiments of these purification techniques, extracted DNA is recovered through alcohol precipitation, such as ethanol or isopropanol precipitation.

Amplification of DNA (using PCR) from natural sources is often inhibited by co-purified contaminants and various methods adopted for DNA extraction from environmental samples are available and provide an alternative for isolating DNA from test samples. Appropriate commercially available kits for isolating DNA from a test sample may thus be employed in the methods of the invention.

The methods of the invention may also, as appropriate, incorporate quantification of isolated/extracted/purified DNA in the sample. Quantification of the DNA in the sample may be achieved using any suitable means. Quantitation of nucleic acids may, for example, be based upon use of a spectrophotometer, a fluorometer or a UV transilluminator. Examples of suitable techniques are described in standard texts such as Molecular Cloning - A Laboratory Manual (Third Edition), Sambrook and Russell (see in particular Appendix 8 therein). In specific embodiments, kits such as the Picogreen® dsDNA quantitation kit available from Molecular Probes, Invitrogen may be employed to quantify the DNA.

Determining the methylation status of a (double stranded DNA) viral genome may be achieved by any suitable means. As discussed herein, the present invention provides the methylomes of, HPV-18, HBV and EBV for the first time. Thus, in certain embodiments, the methylation status of the entire viral genome is determined. By this is meant that a global assessment of viral genome methylation levels is carried out. Every CpG found in the known viral genome may be investigated. Equally, every CpG island in the viral genome, as can be identified using known software for example, may be investigated (see for example The CpGPLOT software program (http://bioinfo.hku.hk/cgi- bin) and the CpG Island Searcher software (see http://cpgislands.usc.edu/ and Takai D, Jones PA. The CpG island searcher: a new WWW resource. In Silico Biol. 3, 235-240 (2003)). This may be the HPV-16, HBV, HPV-18 or EBV methylomes in certain embodiments or may exclude HPV16 in other embodiments.

In specific embodiments, the methylation status of alM 10 CpG residues in the 7904 nucleotide HPV16 genome is determined. The primers of table 1 and SEQ ID NOs 1 to 42 define the regions of interest to be investigated within the HPV16 genome. Thus, the methods of the invention may comprise, consist essentially of or consist of determination of the methylation status of a plurality, up to all (110 CpG residues for the HPV16 genome) in individual increments, CpG residues contained within the nucleotide sequences which can be sequenced using the primers of table 1 and set forth as SEQ ID NOs 1 to 42, as defined herein. Reference can also be made to Fig. 3 and 7. Thus, in one embodiment the methylation patterns presented in Fig. 3 and/or 7 are utilised as a reference in order to facilitate the diagnosis. Other suitable controls are described herein. In alternative embodiments, the methylation status of at least 5, 10, 15, 20, 25, 30, 35 etc up to all (in individual increments) of the methylation sites may be investigated. In further embodiments, the methods of the invention comprise, consist essentially of or consist of determining the methylation status of the L2 gene of the HPV genome and/or determining the methylation status of the E2 binding sites in the upstream regulatory region. Again, this may be carried out in respect of the HPV-16 genome in a particular embodiment. Suitable primers defining the regions of the L2 gene which may be investigated are shown in table 1 (under the heading "MSP") and in SEQ ID NOs 43 to 46. Other regions of the genome may be investigated as required. Overall investigation of the entire genome may provide the most informative results.

In specific embodiments, the methylation status of all 168 CpG residues in the 7857 nucleotide HPV18 genome is determined. The primers of table 2 and SEQ ID NOs 53 to 107 define the regions of interest to be investigated within the HPV18 genome. Thus, the methods of the invention may comprise, consist essentially of or consist of determination of the methylation status of a plurality, up to all (168 CpG residues for the HPV18 genome) in individual increments, CpG residues contained within the nucleotide sequences which can be sequenced using the primers of table 2 and set forth as SEQ ID NOs 53 to 107, as defined herein. Reference can also be made to Fig. 8. Thus, in one embodiment the methylation patterns presented in Fig. 8 are utilised as a reference in order to facilitate the diagnosis. Other suitable controls are described herein. In alternative embodiments, the methylation status of at least 5, 10, 15, 20, 25, 30, 35 etc up to all (in individual increments) of the methylation sites may be investigated. In further embodiments, the methods of the invention comprise, consist essentially of or consist of determining the methylation status of the E2 gene of the HPV18 genome Suitable primers defining the regions of the E2 gene which may be investigated are shown in table 2 (under the heading "MSP") and in SEQ ID NOs 109 to 112. Other regions of the genome may be investigated as required. Overall investigation of the entire genome may provide the most informative results.

In specific embodiments, the methylation status of all CpG residues in the 3215 nucleotide HBV genome is determined. The primers of table 3 and SEQ ID NOs 119 to 140 define the regions of interest to be investigated within the HBV genome. Thus, the methods of the invention may comprise, consist essentially of or consist of determination of the methylation status of a plurality, up to all, CpG residues contained within the nucleotide sequences which can be sequenced using the primers of table 3 and set forth as SEQ ID NOs 119 to 140, as defined herein. Reference can also be made to Fig. 9. Thus, in one embodiment the methylation patterns presented in Fig. 9 are utilised as a reference in order to facilitate the diagnosis. Other suitable controls are described herein. In alternative embodiments, the methylation status of at least 5, 10, 15, 20, 25, 30, 35 etc up to all (in individual increments) of the methylation sites may be investigated. In further embodiments, the methods of the invention comprise, consist essentially of or consist of determining the methylation status of the HBVgp4 and/or HBVgp2 genes of the HBV genome. Suitable primers defining the regions of the HBVgp2 gene which may be investigated are shown in table 3 (under the heading "MSP") and in SEQ ID NOs 141 to 144. Other regions of the genome may be investigated as required. Overall investigation of the entire genome may provide the most informative results. In specific embodiments, the methylation status around all transcription start sites in the 171 ,823 nucleotide EBV genome is determined. This can be achieved, for example, by bisulphite sequencing of amplicons produced from the viral genome and which incorporate the transcription start sites for production of the 94 EBV proteins and optionally also the two structural RNAs, EBER1 and EBER2. This allows investigation of up to 95% of the CpG islands predicted to be contained within the EBV genome. The primers of table 4 and set forth as SEQ ID NOs 151 to 330 define the regions of interest to be investigated within the EBV genome. These primers allow the methylation status of 1 ,344 CpG residues to be determined. Thus, the methods of the invention may comprise, consist essentially of or consist of determination of the methylation status of a plurality, up to all (transcriptionally relevant - 1 ,344 CpG residues for the EBV genome) in individual increments, CpG residues contained within the nucleotide sequences which can be sequenced using the primers of table 4 and set forth as SEQ ID NOs 151 to 330, as defined herein. Reference can also be made to Fig. 4. Thus, in one embodiment the methylation patterns presented in Fig. 4 are utilised as a reference in order to facilitate the diagnosis. Other suitable controls are described herein. In alternative embodiments, the methylation status of at least 5, 10, 15, 20, 25, 30, 35 etc up to all (in individual increments) of the methylation sites may be investigated. In further embodiments, the methods of the invention comprise, consist essentially of or consist of determining the methylation status of the Wp and/or Cp genes of the EBV genome. Suitable primers defining the regions of the Wp and Cp genes which may be investigated are shown in table 4 (under the heading "MSP") and in SEQ ID NOs 331 to 334 for the Cp gene and SEQ ID NOs 335 to 338 for the Wp gene. Other regions of the genome may be investigated as required. Overall investigation of the entire genome may provide the most informative results.

Various methylation assay procedures are known in the art, and can be used in conjunction with the present invention. In specific embodiments, the methylation status is determined using a technique selected from bisulphite sequencing, methylation specific PCR (MSP), microarray-based techniques, real-time amplification techniques and COBRA, either alone or in combination. Certain techniques for analysis of DNA methylation rely on the inability of methylation-sensitive enzymes to cleave methylated cytosine residues. Others rely upon treatment of DNA samples with a reagent such as sodium bisulphite which converts unmethylated cytosine to a changed nucleotide which displays different base pairing properies to cytosine, such as uracil, while methylated cytosines are maintained (Furuichi et al., 1970). This conversion results in a change in the sequence of the original DNA which can then be detected through one or more of a plurality of techniques. Any suitable technique may be employed in the methods of the invention.

Specific examples of techniques useful in the methods of the invention comprise, consist essentially of or consist of: sequencing, methylation-specific PCR (MSP), melting curve methylation-specific PCR (McMS-PCR), MLPA with or without bisulfite treatment, QAMA (Zeschnigk et al, 2004), MSRE-PCR (Melnikov et al, 2005), MethyLight (Eads et al., 2000), ConLight-MSP (Rand et al., 2002), bisulfite conversion-specific methylation- specific PCR (BS-MSP)(Sasaki et al., 2003), COBRA (which relies upon use of restriction enzymes to reveal methylation dependent sequence differences in PCR products of sodium bisulfite - treated DNA), methylation-sensitive single-nucleotide primer extension conformation (MS-SNuPE), methylation-sensitive single-strand conformation analysis (MS-SSCA), Melting curve combined bisulfite restriction analysis (McCOBRA)(Akey et al., 2002), PyroMethA, HeavyMethyl (Cottrell et al. 2004), MALDI- TOF, MassARRAY, Quantitative analysis of methylated alleles (QAMA), enzymatic regional methylation assay (ERMA), QBSUPT, MethylQuant, Quantitative PCR sequencing and oligonucleotide-based microarray systems, Pyrosequencing, Meth-DOP- PCR. A review of some useful techniques is provided in Nucleic acids research, 1998, Vol. 26, No. 10, 2255-2264, Nature Reviews, 2003, Vol.3, 253-266; Oral Oncology, 2006, Vol. 42, 5-13, which references are incorporated herein in their entirety. Any of these techniques may be utilised in accordance with the present invention, as appropriate.

Additional techniques useful in the methods of the invention include those which utilize the ability of the methyl binding domain (MBD) of the MeCP2 protein to selectively bind to methylated DNA sequences (Cross et al, 1994; Shiraishi et al, 1999). Alternatively, the MBD may be obtained from MBP, MBP2, MBP4 or poly-MBD (Jorgensen et al., 2006). In one method, restriction exonuclease digested genomic DNA is loaded onto expressed His-tagged methyl-CpG binding domain that is immobilized to a solid matrix and used for preparative column chromatography to isolate highly methylated DNA sequences. Such methylated DNA enrichment-step may supplement the methods of the invention. Several other methods for detecting methylated CpG islands are well known in the art and include amongst others methylated-CpG island recovery assay (MIRA). Any of these methods may be employed in the present invention where desired.

In specific embodiments, the methylation status of the viral genome is determined using methylation specific PCR (MSP), or an equivalent amplification technique. The MSP technique will be familiar to one of skill in the art. In the MSP approach, DNA may be amplified using primer pairs designed to distinguish methylated from unmethylated DNA by taking advantage of sequence differences as a result of sodium-bisulphite treatment (Herman et al.,1996; and WO 97/46705).

A specific example of the MSP technique is designated real-time quantitative MSP (QMSP), which permits reliable quantification of methylated DNA in real time. These methods are generally based on the continuous optical monitoring of an amplification procedure and utilise fluorescently labelled reagents whose incorporation in a product can be quantified and whose quantification is indicative of copy number of that sequence in the template. One such reagent is a fluorescent dye, called SYBR Green I that preferentially binds double-stranded DNA and whose fluorescence is greatly enhanced by binding of double-stranded DNA. Alternatively, labelled primers and/or labelled probes can be used. They represent a specific application of the well known and commercially available real-time amplification techniques such as hydrolytic probes (TAQMAN®), hairpin probes (MOLECULAR BEACONS®), hairpin primers (AMPLIFLUOR®), hairpin probes integrated into primers (SCORPION®), oligonucleotide blockers and primers incorporating complementary sequences of DNAzymes (DzyNA®), specific interatction between two modified nucleotides (PlexorTM) etc as described in more detail herein. Often, these real-time methods are used with the polymerase chain reaction (PCR).

An alternative to MSP is the so-called HeavyMethyl technique. This method is described in detail in WO 02/072880 for example. In this method, the primers used in the amplification do not need to be methylation specific (although they can also serve this function if desired). Instead, non-extendable oligonucleotide blockers provide for the ability to discriminate between methylated and unmethylated DNA following bisulphite treatment instead of the primers themselves. The blockers bind to bisulphite-treated DNA in a methylation-specific manner, and their binding sites may optionally overlap the primer binding sites. When the blocker is bound, the primer cannot bind and/or direct complete amplification and therefore an amplification product is not generated. The HeavyMethyl technique can be used in combination with real-time or end-point detection, as required, in the methods of the invention.

Thus, in certain embodiments, the methylation status of the viral genome is determined by methylation specific PCR/amplification and/or by HeavyMethyl, which may be a realtime or end point version thereof. In specific embodiments, the real time PCR/amplification involves use of hairpin primers (Amplifluor)/hairpin probes (Molecular Beacons)/hydrolytic probes (Taqman)/FRET probe pairs (Lightcycler)/primers incorporating a hairpin probe (Scorpion)/primers incorporating complementary sequences of DNAzymes that cleave a reporter substrate included in the reaction mixture (DzyNA®)/fluorescent dyes (SYBR Green etc.)/oligonucleotide blockers/the specific interaction between two modified nucleotides (Plexor). For Scorpion type primers, the primer and/or the probe may distinguish between methylated and unmethylated DNA following bisulphite treatment as required.

Real-Time PCR detects the accumulation of amplicon during the reaction. Real-time methods do not need to be utilised, however. Many applications do not require quantification and real-time PCR is used principally as a tool to obtain convenient results presentation and storage, and at the same time to avoid post-PCR handling. Analyses can be performed only to determine whether the target DNA is present in the sample or not. End point verification is carried out after the amplification reaction has finished. This knowledge can be used (in a medical diagnostic laboratory for example), in the methods of the invention. In the majority of such cases, the quantification of DNA template is not very important. Amplification products may simply be run on a suitable gel, such as an agarose gel, to determine if the products of the expected size are present. This may involve use of ethidium bromide staining and visualisation of the DNA bands under a UV illuminator for example. Alternatively, fluorescence or other energy transfer can be measured to determine the presence of the methylated DNA. The end- point PCR fluorescence detection technique can use the same approaches as widely used for Real Time PCR: examples include the TaqMan assay, Molecular Beacons, Scorpion, Amplifluor etc as discussed in detail above. As an example, «Gene» detector allows the measurement of fluorescence directly in PCR tubes.

In real-time embodiments, quantitation may be on an absolute basis, or may be relative to a constitutively methylated DNA standard, or may be relative to an unmethylated DNA standard. Methylation status may be determined by using the ratio between the signal of the marker under investigation and the signal of a reference gene where methylation status is known (such as β-actin for example), or by using the ratio between the methylated marker and the sum of the methylated and the non-methylated marker. Alternatively, absolute copy number of the methylated marker gene can be determined. Suitable reference genes for the present invention include beta-actin, glyceraldehyde-3- phosphate dehydrogenase (GAPDH), ribosomal RNA genes such as 18S ribosomal RNA and RNA polymerase Il gene (Radonic A. et al., Biochem Biophys Res Commun. 2004 Jan 23;313(4):856-62).

In certain embodiments, each clinical sample is measured in duplicate and for both Ct values (cycles at which the amplification curves crossed the threshold value, set automatically by the relevant software) copy numbers are calculated. The average of both copy numbers (for each gene) is used for the result classification. To quantify the final results for each sample two standard curves are used, one for either the reference gene (β-actin or the non-methylated marker) and one for the methylated version of the marker. The results of all clinical samples (when m-Gene was detectable) are expressed as 1000 times the ratio of "copies m-Gene'7" copies β-actin" or "copies m-Gene'7"copies u-Gene+m-Gene" and then classified accordingly (methylated, non-methylated or invalid) (u=unmethylated; m=methylated).

In certain embodiments, primers useful in determining the methylation status of the HPV genome (by MSP) are provided. These primers may comprise, consist essentially of or consist of the nucleotide sequences set forth as SEQ ID NOs 43 to 46. These primers are useful in determining the methylation status of the HPV-16 L2 gene and, in addition to being useful in the methods of the invention, form separate aspects of the invention. The invention thus provides a primer for use in methylation specific PCR (MSP) to determine the methylation status of a HPV-16 genome selected from the primers comprising the nucleotide sequences set forth as SEQ ID NO: 43 to 46 and functional derivatives thereof which retain functionality in MSP. Effectively, the methods of the invention may involve determining the methylation status in regions of the HPV16 genome defined by the sequence between (and including) the primer binding sites. Further characteristics of these primers are summarized in the detailed description (experimental part) below. Thus, the methods of the invention may comprise, consist essentially of or consist of determination of the methylation status of a plurality, up to all (110 CpG residues for the HPV16 genome) in individual increments, CpG residues in the nucleotide sequences which can be amplified using the primers of table 1 and/or as set forth in SEQ ID NOs 43 to 46, as defined herein.

In certain embodiments, primers useful in determining the methylation status of the HPV18 genome (by MSP) are provided. These primers comprise, consist essentially of or consist of the nucleotide sequences set forth as SEQ ID NOs 109 to 112. These primers are useful in determining the methylation status of the HPV-18 E2 gene and, in addition to being useful in the methods of the invention, form separate aspects of the invention. The invention thus provides a primer for use in methylation specific PCR (MSP) to determine the methylation status of a HPV-18 genome selected from the primers comprising the nucleotide sequences set forth as SEQ ID NO: 109 to 112 and functional derivatives thereof which retain functionality in MSP. Effectively, the methods of the invention may involve determining the methylation status in regions of the HPV18 genome defined by the sequence between (and including) the primer binding sites. Further characteristics of these primers are summarized in the detailed description (experimental part) below. Thus, the methods of the invention may comprise, consist essentially of or consist of determination of the methylation status of a plurality, up to all in individual increments, CpG residues in the nucleotide sequences which can be amplified using the primers of table 2 and SEQ ID NOs 109 to 112, as defined herein.

In certain embodiments, primers useful in determining the methylation status of the HBV genome (by MSP) are provided. These primers comprise, consist essentially of or consist of the nucleotide sequences set forth as SEQ ID NOs 141 to 144. These primers are useful in determining the methylation status of the HBVgp2 gene and, in addition to being useful in the methods of the invention, form separate aspects of the invention. The invention thus provides a primer for use in methylation specific PCR (MSP) to determine the methylation status of a HBV genome selected from the primers comprising the nucleotide sequences set forth as SEQ ID NOS: 141 to 144 and functional derivatives thereof which retain functionality in MSP. Effectively, the methods of the invention may involve determining the methylation status in regions of the HBV genome defined by the sequence between (and including) the primer binding sites. Further characteristics of these primers are summarized in the detailed description (experimental part) below. Thus, the methods of the invention may comprise, consist essentially of or consist of determination of the methylation status of a plurality, up to all in individual increments, CpG residues in the nucleotide sequences which can be amplified using the primers of table 3 an/or as set forth in SEQ ID NOs 141 to 144, as defined herein.

In certain embodiments, primers useful in determining the methylation status of the EBV genome (by MSP) are provided. These primers comprise, consist essentially of or consist of the nucleotide sequences set forth as SEQ ID NOs 331 to 334 for determining the methylation status of the Cp gene and SEQ ID NOs 335 to 338 for determining the methylation status of the Wp gene. These primers are useful in determining the methylation status of the EBV Cp and Wp genes respectively and, in addition to being useful in the methods of the invention, form separate aspects of the invention. The invention thus provides a primer for use in methylation specific PCR (MSP) to determine the methylation status of an EBV genome selected from the primers comprising the nucleotide sequences set forth as SEQ ID NOs 331 to 334 for the Cp gene and SEQ ID NOs 335 to 338 for the Wp gene and functional derivatives thereof which retain functionality in MSP. Effectively, the methods of the invention may involve determining the methylation status in regions of the EBV genome defined by the sequence between (and including) the primer binding sites. Further characteristics of these primers are summarized in the detailed description (experimental part) below. Thus, the methods of the invention may comprise, consist essentially of or consist of determination of the methylation status of a plurality, up to all in individual increments, CpG residues in the nucleotide sequences which can be amplified using the primers of table 4 and/or as set forth in SEQ ID NOs 331 to 334 for the Cp gene and/or as set forth in SEQ ID NOs 335 to 338 for the Wp gene, as defined herein.

It is noted that variants of these primer sequences may be utilised in the present invention. In particular, additional sequence specific flanking sequences may be added, for example to improve binding specificity, as required. Variant sequences may have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% nucleotide sequence identity with the nucleotide sequences of the primers set forth above (and in the appropriate portions of the tables). The primers may incorporate synthetic nucleotide analogues as appropriate or may be RNA or PNA based for example, or mixtures thereof. Primers may be labelled with suitable probes or combined with probes to allow real-time and/or end point detection. Such probes, and in certain embodiments the primers themselves, may be fluorescently labelled to facilitate detection. A range of alternative fluorescent donor and acceptor moieties/FRET pairs may be utilised as appropriate. In addition to being labelled with the fluorescent donor and acceptor moieties, the primers (or probes as appropriate) may include modified oligonucleotides and other appending groups and labels provided that the functionality as a primer in the methods of the invention is not compromised. Similarly alternative fluorescent donor and acceptor moieties/FRET pairs may be utilised as appropriate. Molecules that are commonly used in FRET include fluorescein, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5'-dichloro-6- carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N, N₁N', N¹- tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'- dimethylaminophenylazo) benzoic acid (DABCYL), and 5-(2'-aminoethyl) aminonaphthalene-1 -sulfonic acid (EDANS). Whether a fluorophore is a donor or an acceptor is defined by its excitation and emission spectra, and the fluorophore with which it is paired. For example, FAM is most efficiently excited by light with a wavelength of 488 nm, and emits light with a spectrum of 500 to 650 nm, and an emission maximum of 525 nm. FAM is a suitable donor fluorophore for use with JOE, TAMRA, and ROX (all of which have their excitation maximum at 514 nm).

Thus, in certain embodiments, a donor moiety and acceptor moiety are selected from 5- carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5^l-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 5-(2'-aminoethyl)aminonapthalene-1 -sulfonic acid (EDANS), anthranilamide, coumarin, terbium chelate derivatives, Malachite green, Reactive Red 4, DABCYL, tetramethyl rhodamine, pyrene butyrate, eosine nitrotyrosine, ethidium, and Texas Red. In a further embodiment, a donor moiety is selected from fluorescein, 5-carboxyfluorescein (FAM)₁ rhodamine, 5-(2'-aminoethyl)aminonapthalene- 1 -sulfonic acid (EDANS), anthranilamide, coumarin, terbium chelate derivatives, Malachite green, and Reactive Red 4, and an acceptor moiety is selected from DABCYL, rhodamine, tetramethyl rhodamine, pyrene butyrate, eosine nitrotyrosine, ethidium, and Texas Red. In specific embodiments, the donor moiety is fluorescein or a derivative thereof, and the acceptor moiety is DABCYL. The fluorescein derivative may comprise, consist essentially of or consist of 6-carboxy fluorescein.

For all aspects and embodiments of the invention, the primers and/or probes where utilised may be labelled with donor and acceptor moieties as required during chemical synthesis of the primers or the label may be attached following synthesis using any suitable method. Many such methods are available and well characterised in the art.

In further embodiments of the methods of the invention, bisulphite sequencing is utilised in order to determine the methylation status of the viral genome. Bisulphite sequencing may be particularly useful in embodiments where the methylation status of the whole viral genome or at least the majority and up to all CpG islands, or the majority and up to all active transcription sites, is determined. By "whole viral genome" is meant determination of the methylation status of selected CpG residues throughout the viral genome. This may be carried out at regular intervals in particular at predicted CpG islands and/or around transcription start sites. Suitable residues to investigate may be determined by known methods such as through use of computer software to predict and identify CpG islands. This "global" approach provides particularly sensitive results. The HPV-18 methylome is shown in Fig. 8, the HBV methylome is shown in Fig. 9 and the EBV methylome is shown in Fig. 10 (the HPV-16 methylome is shown in Figures 3 and 7), and indicates the global approach which may be utilised. Sequencing primers may be designed for use in sequencing through the important CpG islands in the viral genome. Thus, primers may be designed in both the sense and antisense orientation to direct sequencing across the relevant regions of the viral genome, in particular HBV, HPV-18 or EBV viral genome, as discussed.

Suitable primers for use in the methods of the invention are set forth in the tables. In particular for HBV, the primers may comprise, consist essentially of or consist of the nucleotide sequences set forth as SEQ ID NO: 119 to 140 (and in table 9). Thus, the invention provides a primer for use in bisulphite sequencing of a HBV genome selected from the primers comprising the nucleotide sequences set forth as SEQ ID NO: 119 to 140 (and in table 9) and functional derivatives thereof which retain functionality in bisulphite sequencing. All appropriate combinations of these primers may be utilised as required. These primers are useful in determining the methylation status of the HBV genome and, in addition to being useful in the methods of the invention, form separate aspects of the invention. Effectively, the methods of the invention may involve determining the methylation status in regions of the HBV genome defined by the sequence between (and including) the primer binding sites. Thus, the methods of the invention may involve determination of the methylation status of a plurality, up to all, CpG residues in the nucleotide sequences which can be sequenced using the primers of table 9 and/or as set forth in SEQ ID NOs 119 to 140, as defined herein.

Further suitable primers for use in the methods of the invention are set forth as SEQ ID NO: 151 to 330 (and in table 10). Thus, the invention provides a primer for use in bisulphite sequencing of an EBV genome selected from the primers comprising the nucleotide sequences set forth as SEQ ID NO: 151 to 330 and functional derivatives thereof which retain functionality in bisulphite sequencing. All appropriate combinations of these primers may be utilised as required. These primers are useful in determining the methylation status of the EBV genome (in particular around the transcription start sites) and, in addition to being useful in the methods of the invention, form separate aspects of the invention. Effectively, the methods of the invention may involve determining the methylation status in regions of the EBV genome defined by the sequence between (and including) the primer binding sites. Thus, the methods of the invention may involve determination of the methylation status of a plurality, up to all, CpG residues in the nucleotide sequences which can be sequenced using the primers of table 10 and/or as set forth in SEQ ID NOs 151 to 330, as defined herein.

Additional suitable primers for use in the methods of the invention are set forth as SEQ ID NO: 53 to 108 (and in table 2). Thus, the invention provides a primer for use in bisulphite sequencing of an HPV18 genome selected from the primers comprising the nucleotide sequences set forth as SEQ ID NO: 53 to 108 and functional derivatives thereof which retain functionality in bisulphite sequencing. All appropriate combinations of these primers may be utilised as required. These primers are useful in determining the methylation status of the HPV-18 genome and, in addition to being useful in the methods of the invention, form separate aspects of the invention. Effectively, the methods of the invention may involve determining the methylation status in regions of the HPV18 genome defined by the sequence between (and including) the primer binding sites. Thus, the methods of the invention may involve determination of the methylation status of a plurality, up to all, CpG residues in the nucleotide sequences which can be sequenced using the primers of table 2 and/or as set forth in SEQ ID NOs 53 to 108, as defined herein.

Primers which may find use in the methods of the invention are set forth as SEQ ID NO: 1 to 42 (and in table 1). Thus, the invention provides a primer for use in bisulphite sequencing of a HPV genome selected from the primers comprising the nucleotide sequences set forth as SEQ ID NO: 1 to 42 and functional derivatives thereof which retain functionality in bisulphite sequencing. All appropriate combinations of these primers may be utilised as required. These primers are useful in determining the methylation status of the HPV-16 genome and, in addition to being useful in the methods of the invention, may form separate aspects of the invention. Effectively, the methods of the invention may involve determining the methylation status in regions of the HPV16 genome defined by the sequence between (and including) the primer binding sites. Thus, the methods of the invention may involve determination of the methylation status of a plurality, up to all, CpG residues in the nucleotide sequences which can be sequenced using the primers of table 1 and/or as set forth in SEQ ID NOs 1 to 42, as defined herein.

Further characteristics of these primers are summarized in the detailed description (experimental part) below. It is noted that variants of these sequences may be utilised in the present invention. In particular, additional sequence specific flanking sequences may be added, for example to improve binding specificity, as required. Variant sequences preferably have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% nucleotide sequence identity with the nucleotide sequences of the primers set forth above (and in the relevant tables). The primers may incorporate synthetic nucleotide analogues as appropriate or may be RNA or PNA based for example, or mixtures thereof. Primers may be fluorescently labelled to facilitate detection. Other nucleic acid amplification techniques, in addition to PCR (which includes real-time versions thereof and variants such as nested PCR), may also be utilised in the methods of the invention, as appropriate, to detect the methylation status of the viral genome. Such amplification techniques are well known in the art, and include methods such as NASBA (Compton, 1991) , 3SR (Fahy et al., 1991 ) and Transcription Mediated Amplification (TMA). Other suitable amplification methods include the ligase chain reaction (LCR) (Barringer et al, 1990), selective amplification of target polynucleotide sequences (US Patent No. 6,410,276), consensus sequence primed polymerase chain reaction (US Patent No 4,437,975), arbitrarily primed polymerase chain reaction (WO 90/06995), invader technology, strand displacement technlology, and nick displacement amplification (WO 2004/067726). This list is not intended to be exhaustive; any nucleic acid amplification technique may be used provided the appropriate nucleic acid product is specifically amplified. Thus, these amplification techniques may be tied in to MSP and/or HeavyMethyl and/or bisulphite sequencing techniques for example.

As discussed above, sequence variation that reflects the methylation status at CpG dinucleotides in the original genomic DNA offers different approaches to primer design. Both primer types may be utilised in the methods of the invention. Firstly, primers may be designed that themselves do not cover any potential sites of DNA methylation. Sequence variations at sites of differential methylation are located between the two primers. Such primers are used in bisulphite genomic sequencing, COBRA and Ms- SnuPE for example. Secondly, primers may be designed that anneal specifically with either the methylated or unmethylated version of the converted sequence. If there is a sufficient region of complementarity, e.g., 12, 15, 18, or 20 nucleotides, to the target, then the primer may also contain additional nucleotide residues that do not interfere with hybridization but may be useful for other manipulations. Examples of such other residues may be sites for restriction endonuclease cleavage, for ligand binding or for factor binding or linkers or repeats. The oligonucleotide primers may or may not be such that they are specific for modified methylated residues.

One way to distinguish between modified and unmodified DNA is to hybridize oligonucleotide primers which specifically bind to one form or the other of the DNA. After hybridization, an amplification reaction can be performed and amplification products assayed. The presence of an amplification product indicates that a sample hybridized to the primer. The specificity of the primer indicates whether the DNA had been modified or not, which in turn indicates whether the DNA had been methylated or not.

Another way to distinguish between modified and unmodified DNA is to use oligonucleotide probes which may also be specific for certain products. Such probes may be hybridized directly to modified DNA or to amplification products of modified DNA. Oligonucleotide probes can be labelled using any detection system known in the art. These include but are not limited to fluorescent moieties, radioisotope labelled moieties, bioluminescent moieties, luminescent moieties, chemiluminescent moieties, enzymes, substrates, receptors, or ligands.

As discussed above, in the MSP technique, amplification is achieved with the use of primers specific for the sequence of the gene whose methylation status is to be assessed. In order to provide specificity for the nucleic acid molecules, primer binding sites corresponding to a suitable region of the sequence may be selected. The skilled reader will appreciate that the nucleic acid molecules may also include sequences other than primer binding sites which are required for detection of the methylation status of the gene, for example RNA Polymerase binding sites or promoter sequences may be required for isothermal amplification technologies, such as NASBA, 3SR and TMA.

When determining methylation status, it may be beneficial to include suitable controls. This may be particularly useful in applications of the methods of the invention in which staging or progression of a particular disease is the aim. Suitable controls may also be advantageous in order to ensure the methods of the invention are working correctly and reliably. Thus, in one embodiment the determined methylation status (of the viral genome) is compared to a control. In one embodiment the control is a sample taken from the same subject at an earlier time point. This allows monitoring of disease progression in a subject. It may also allow the effectiveness of a given treatment to be determined - if the methylation status does not progress to a more methylated level, or in fact decreases, it may be determined that the treatment is having the desired effect. This may be tied in to the treatment methods of the invention as described in further detail. Thus, the basic correlation of the invention is that an increased level of methylation of the viral genome (up to and including the entire methylome) indicates the progression of the disease to a more advanced form. Additionally or alternatively, the control may comprise, consist essentially of or consist of one or more reference samples representing the methylation status of the viral genome at a defined stage of the disease. Thus, a whole collection of suitable control samples taken from subjects at various stages of the disease may be utilised as a reference point. Note, the samples themselves need not be available when the methods of the invention are carried out. The results of determining the methylation status of the viral genome are all that is required to compare with the output from the methods of the invention. Thus, it can be readily envisaged that a large databank of information can be accumulated and used as a point of reference to facilitate interpretation of results achieved using the methods of the invention. As discussed above, in certain embodiments the methylation patterns presented in any of figures 3, 7, 8, 9 and 10, in particular figures 8 (HPV18), 9 (HBV) and 10 (EBV) are utilised as a reference in order to facilitate the diagnosis. The methylation of the test sample can be appropriately presented and then compared against these references to reach a conclusion on the level of infection/disease progression.

Other suitable controls may include assessing the methylation status of a gene known to be methylated in the sample. This experiment acts as a positive control to ensure that false negative results are not obtained (i.e. a conclusion of a lack of methylation is made even though the viral genome may, in fact, be methylated). The gene may be one which is known to be methylated in the sample under investigation or it may have been artificially methylated, for example by using a suitable methyltransferase enzyme, such as Sssl methyltransferase.

Additionally or alternatively, suitable negative controls may be employed with the methods of the invention. Here, suitable controls may include assessing the methylation status of a gene known to be unmethylated or carrying out an amplification in the absence of DNA (for example by using a water only sample). This experiment acts as a negative control to ensure that false positive results are not obtained (i.e. a conclusion of methylation is made even though the viral genome may, in fact, be unmethylated). The gene may be one which is known to be unmethylated in the sample under investigation or it may have been artificially demethylated, for example by using a suitable DNA methyltransferase inhibitor. As mentioned above, the basic correlation of the invention is that an increased level of methylation of the viral genome indicates the progression of the disease to a more advanced form. Accordingly, the invention provides a method of monitoring the progression of a disease caused by a (double stranded DNA) viral infection in response to a treatment directed against the disease in a test sample obtained from a subject comprising determining the methylation status of a/the viral genome before and following treatment of the subject wherein the presence of decreased methylation of the viral genome following treatment indicates a positive effect of the treatment on the disease in terms of successfully inhibiting disease progression. The presence of equal methylation before and following treatment may indicate that the treatment has been successful in preventing progression of the disease. The presence of increased methylation following treatment may indicate that the treatment has been unsuccessful in preventing progression of the disease (and that therefore alternative treatments should be explored). For the avoidance of doubt, it is hereby stated that all embodiments and applications of the (other) methods of the invention (including disclaimers) apply mutatis mutandis to this particular aspect of the invention and are not repeated for reasons of conciseness.

The application of the methods of the present invention to extremely small amounts of abnormally-methylated DNA, that are released for example into test samples comprised of fluids may require the generation and amplification of a DNA library before testing for methylation of any specific gene. Suitable methods on whole genome amplification and libraries generation for such amplification (e.g. Methylplex and Enzyplex technology, Rubicon Genomics) are described in US2003/0143599, WO2004/081225 and WO2004/081183 for example. In addition, WO2005/090507 describes library generation/amplification methods that require either bisulfite conversion or non-bisulfite based application. Bisulfite treatment may occur before or after library construction and may require the use of adaptors resistant to bisulfite conversion. Meth-DOP-PCR (Di Vinci et al, 2006), a modified degenerate oligonucleotide-primed PCR amplification (DOP-PCR) that is combined with MSP, provides another suitable method for specific detection of methylation in small amounts of DNA. Improved management of patient care may require these existing methods and techniques to supplement the methods of the invention. As mentioned above, determining the methylation status at a number of locations in the HBV genome may be advantageous. Thus, in certain embodiments, the methods of the invention are utilised in order to determine the methylation status of at least the HBVgpi (or P)/HBVgp2 (or S)/HBVgp3 (or X)/HBVgp4 (or C) gene. In specific embodiments, the methylation status of the entire HBV genome is determined. Moreover, in order to improve the sensitivity of the methods of the invention the methods may comprise detecting an epigenetic change in a panel of genes comprising at least two, three or four of the genes, and optionally also including or selected from additional regions in the HBV genome. The level of methylation at each point of investigation contributes to the output of the methods of the invention in terms of diagnosing, staging or monitoring the progression of the disease. Thus, the methods of the invention generally include investigation of a significant number of cytosine nucleotides (in the context of CpG dinucleotide pairs) within the HBV genome. As aforementioned, the methods of the invention may involve determination of the methylation status of a plurality, up to all, CpG residues in the nucleotide sequences which can be sequenced or amplified using the primers of tables 3 and/or as set forth in SEQ ID NOs 119 to 140 (bisulphite sequencing) and SEQ ID NOS 141 to 144 (MSP), as defined herein. Even within each of the genes or regions of the genome, as discussed herein, which may be investigated, it is possible to investigate the methylation status of multiple cytosine residues. Individual primers and probes used in the methods of the invention may investigate the methylation status of a plurality of cytosine residues in each case through appropriate primer and probe design, as would be immediately apparent to the skilled person. For example, the primers and probes may be designed to overlap 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 etc cytosine residues and thus investigate the methylation status of multiple sites in a single reaction.

As mentioned above, determining the methylation status at a number of locations in the EBV genome may be advantageous. Thus, in certain embodiments, the methods of the invention are utilised in order to determine the methylation status of at least the BNRF1 EBER1 , EBER2, BCRF1 , Cp, BWRF1/1 , Wp, BWRF1/2, BWRF1/3, BWRF1/4, BWRF1/5, BWRF1/6, BWRF1/7, BWRF1/8, BHLF1 , BHRF1-mirBHRF1 , BFLF2 BFLF1-BFRF1A, BFRF1 , BFRF2, BFRF3, Fp, Qp, BPLF1 , BORF1-BOLF1 , BORF2, BaRFI , BMRF1 , BMRF2, BSLF2/BMLF1 , BSLF1-BSRF1 , BLRF1 , BLRF2-BLLF3, BLLF2, BLLF1, BZLF2, BZLF1 , BRRF1 , BRRF2-BRLF1 (Rta), BKRF2, BKRF3, BKRF4, BBRF1 , BBLF4, BBRF2, BBRF3-BBLF2/BBLF3 BBLF1 , BGLF5, BGLF4, BGLF3/5, BGLF3-BGRF1/BDRF1 , BGLF2, BGLF1, BDLF4, BDLF3/5, BDLF3, BDLF2, BDLF1, BcRFI , BcLFI , BTRF1 , BXLF2, BXRF1 , BVRF1-BXLF1 , BVRF2, BVLF1 , BdRFI , RPMS1-BILF2, LF3, LF2, LF1 , BILF1-miR-BART2 A73, BALF5, BALF4, BARFO, BALF3, BALF2, BARF1-BALF1 , LMP-2A, BNLF2b, BNLF2a, and LMP-2B - LMP-1 gene. In specific embodiments, the methylation status of the entire EBV genome, or all transcriptionally active regions of the genome is determined. Moreover, in order to improve the sensitivity of the methods of the invention the methods may comprise detecting an epigenetic change in a panel of genes comprising at least two, three, four, five, six etc. up to all of the genes, and optionally also including or selected from additional regions of the EBV genome. The level of methylation at each point of investigation contributes to the output of the methods of the invention in terms of diagnosing, staging or monitoring the progression of the disease. Thus, the methods of the invention generally include investigation of a significant number of cytosine nucleotides (in the context of CpG dinucleotide pairs) within the EBV genome. As aforementioned, the methods of the invention may involve determination of the methylation status of a plurality, up to all, CpG residues in the nucleotide sequences which can be sequenced or amplified using the primers of table 4 and/or as set forth in SEQ ID NOs 151 to 330 (bisulphite sequencing) and SEQ ID NOs 331 to 338 (MSP), as defined herein. Even within each of the genes or regions of the genome, as discussed herein, which may be investigated, it is possible to investigate the methylation status of multiple cytosine residues, individual primers and probes used in the methods of the invention may investigate the methylation status of a plurality of cytosine residues in each case through appropriate primer and probe design, as would be immediately apparent to the skilled person. For example, the primers and probes may be designed to overlap 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 etc cytosine residues and thus investigate the methylation status of multiple sites in a single reaction.

Determining the methylation status at a number of locations in the HPV18 genome may likewise be advantageous. Thus, in one embodiment, the methods of the invention are utilised in order to determine the methylation status of at least the E7/E1/E2/E4/E5/L2/L1 gene. In certain embodiments, the methylation status of the entire HPV-18 genome is determined. Moreover, in order to improve the sensitivity of the methods of the invention the methods may comprise detecting an epigenetic change in a panel of genes comprising at least two, three, four, five, six etc. up to all of the genes, and optionally also including or selected from additional regions such as the Upstream Regulatory Region (URR). The level of methylation at each point of investigation contributes to the output of the methods of the invention in terms of diagnosing, staging or monitoring the progression of the disease. Thus, the methods of the invention generally include investigation of a significant number of cytosine nucleotides (in the context of CpG dinucleotide pairs) within the HPV18 genome. As aforementioned, the methods of the invention may involve determination of the methylation status of a plurality, up to all, CpG residues in the nucleotide sequences which can be sequenced or amplified using the primers of table 2 and/or as set forth in SEQ ID NOs 53 to 108 (bisulphite sequencing) and SEQ ID NOs 109 to 112 (MSP), as defined herein. Even within each of the genes or regions of the genome, as discussed herein, which may be investigated, it is possible to investigate the methylation status of multiple cytosine residues. Individual primers and probes used in the methods of the invention may investigate the methylation status of a plurality of cytosine residues in each case through appropriate primer and probe design, as would be immediately apparent to the skilled person. For example, the primers and probes may be designed to overlap 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 etc cytosine residues and thus investigate the methylation status of multiple sites in a single reaction.

As mentioned above, determining the methylation status at a number of locations in the HPV genome may be advantageous. Thus, in one embodiment, the methods of the invention are utilised in order to determine the methylation status of at least the E7/E1/E2/E4/E5/L2/L1 gene. In specific embodiments, the methylation status of the entire HPV-16 genome is determined. Moreover, in order to improve the sensitivity of the methods of the invention the methods may comprise detecting an epigenetic change in a panel of genes comprising at least two, three, four, five, six etc. up to all of the genes, and optionally also including or selected from additional regions such as the Upstream Regulatory Region (URR). The level of methylation at each point of investigation contributes to the output of the methods of the invention in terms of diagnosing, staging or monitoring the progression of the disease. Thus, the methods of the invention generally include investigation of a significant number of cytosine nucleotides (in the context of CpG dinucleotide pairs) within the HPV genome. As aforementioned, the methods of the invention may involve determination of the methylation status of a plurality, up to all, CpG residues in the nucleotide sequences which can be sequenced or amplified using the primers of table 1 and SEQ ID NOs 1 to 42 (bisulphite sequencing) and 43 to 46 (MSP primers), as defined herein. Even within each of the genes or regions of the genome, as discussed herein, which may be investigated, it is possible to investigate the methylation status of multiple cytosine residues. Individual primers and probes used in the methods of the invention may investigate the methylation status of a plurality of cytosine residues in each case through appropriate primer and probe design, as would be immediately apparent to the skilled person. For example, the primers and probes may be designed to overlap 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 etc cytosine residues and thus investigate the methylation status of multiple sites in a single reaction.

Since DNA methylation, particularly in the promoter or other regulatory regions of a gene generally corresponds to a decrease in expression of the corresponding gene, the methods of the invention may employ determination of gene expression levels as an indication of the methylation status of the viral genome. As the disease progresses and methylation levels increase gene expression may decrease in corresponding fashion. Thus, the invention provides a method of diagnosing and/or monitoring the progression of or otherwise staging a disease caused an infection by a virus, in particular a double stranded DNA virus in a test sample obtained from a subject comprising determining the methylation status of a viral genome wherein the presence of hypermethylation of the viral genome indicates a positive diagnosis of the disease and/or an increased level of methylation of the viral genome indicates the progression of the disease to a more advanced form and wherein levels of expression of one or more viral genes are measured as an indication of the methylation status of the viral genome.

In specific embodiments, the methods of the invention include determining the methylation status of a HPV genome by determining whether E6 and/or E7 is overexpressed. Such overexpression may be considered surprising in light of the general correlation between methylation and decreased gene expression. However, as discussed in detail herein, methylation of E2 binding sites in the Upstream Regulatory Region (URR) of the HPV16 genome are associated with high levels of expression of E6 and E7 oncoproteins. Overexpression of E6 and E7 appears to be due to the fact that the E2 viral protein cannot bind to methylated E2-binding sites. This also indicates that determining the methylation status of the E2-binding sites in the URR of the HPV16 genome is useful in the methods of the invention. Likewise, methods for determining whether E2 has bound to its binding sites within the URR may also be employed in the methods of the invention as an indication of the methylation status of the HPV genome. Any suitable method of determining protein binding may be employed. In a specific embodiment, chromatin immunoprecipitation techniques are used to investigate whether the E2 protein has bound and thus whether the E2-binding sites are methylated. Accordingly, the invention provides a method of diagnosing and/or monitoring the progression of or otherwise staging a disease caused by a human papillomavirus (HPV) infection in a test sample obtained from a subject comprising determining the expression levels of E6 and/or E7 wherein the presence of overexpression of E6 and/or E7 indicates a positive diagnosis of the disease and/or an increased level of expression of E6 and/or E7 indicates the progression of the disease to a more advanced form. These aspects of the invention are particularly applicable to HPV-16.

In certain embodiments relating to HBV infection, expression levels of C and/or S viral proteins may be monitored as an indication of the methylation status of the corresponding genes and HBV genome. As discussed herein, methylation of the HBVgp4 (encoding the C protein) and HBVgp2 (encoding the S viral protein) is associated with a lack of expression. This may be measured at either the RNA or protein level as discussed herein. Expression can be restored through use of a DNA demethylating agent as described in further detail herein. Accordingly, the invention provides a method of diagnosing and/or monitoring the progression of or otherwise staging a disease caused by a HBV infection in a test sample obtained from a subject comprising determining the expression levels of C and/or S viral proteins wherein the presence of reduced expression of C and/or S viral proteins indicates a positive diagnosis of the disease and/or a decreased level of expression of C and/or S viral proteins indicates the progression of the disease to a more advanced form. Relevant disease conditions to HBV infection, such as liver disease and cancer, are discussed herein, which discussion applies mutatis mutandis (lower levels of expression correlate with increased methylation as the infection/disease progresses).

In certain embodiments relating to EBV infection, expression levels of proteins encoded by the EBNA2, BRLF1 and/or BHRF1 genes may be monitored as an indication of the methylation status of the corresponding genes. As discussed herein, methylation of the EBNA2, BRLF1 and BHRF1 genes and the UTR of the BHFR1 gene is associated with a lack of expression of the gene, or microRNA in the latter case (miR-BHFRF1-1). This may be measured at either the RNA, or protein level for genes such as EBNA2, BRLF1 and BHRF1 genes, as discussed herein. Expression can be restored through use of a DNA demethylating agent such as 5-aza-2'-deoxycytidine as described in further detail herein. Accordingly, the invention provides a method of diagnosing and/or monitoring the progression of or otherwise staging a disease caused by an EBV infection in a test sample obtained from a subject comprising determining the expression levels of EBNA2, BRLF1 and/or BHRF1 genes and/or miR-BHFRF1-1 wherein the presence of reduced expression of EBNA2, BRLF1 and/or BHRF1 genes and/or miR-BHFRF1-1 indicates a positive diagnosis of the disease and/or a decreased level of expression of EBNA2, BRLF1 and/or BHRF1 genes and/or miR-BHFRF1-1 indicates the progression of the disease to a more advanced form. Relevant disease conditions to EBV infection, such as lymphoid and epithelial malignancies, are discussed herein, which discussion applies mutatis mutandis (lower levels of expression correlate with increased methylation as disease progresses).

The methods of the invention may also incorporate investigating whether there are genomic deletions in the region of the E1/E2 genes of HPV16. Deletion in this region may cause loss of E2 expression resulting in decreased repression of E6 and E7 expression by E2. E6 and E7 are oncoproteins and thus detecting the deletion may be a reliable indicator of HPV16 associated tumourigenesis, in particular cervical tumourigenesis.

Gene expression may be determined at the RNA or protein level, as would be readily appreciated by one skilled in the art. "Overexpression" indicates an increase in expression relative to the level of expression in which there is no or normal levels of methylation of the viral genome, (for example at the E2 binding sites in the of the HPV 16 genome). With respect to E6 and/or E7, overexpression means increased expression as compared to the level of expression when the E2-binding sites in the URR are unmethylated. Overexpression may be determined by assessing protein activity, rather than by directly looking at expression levels themselves. Changes in the level of expression may, as necessary, be measured in order to determine if it is statistically significant in the sample. This helps to provide a reliable test for the methods of the invention. Any method for determining whether the expression levels are significantly altered may be utilised. Such methods are well known in the art and routinely employed. For example, statistical analyses may be performed. One example involves an analysis of variance test. Typical P values for use in such a method would be P values of < 0.05 or 0.01 or 0.001 when determining whether the relative expression or activity is statistically significant. A change in expression may be deemed significant if there is at least a 10% change for example. The test may be made more selective by making the change at least 15%, 20%, 25%, 30%, 35%, 40% or 50%, for example, in order to be considered statistically significant.

As discussed in respect of the methods involving direct determination of methylation levels in the viral genome, levels of expression or activity may be determined with reference to a control sample. Thus, for example, expression of E6 and/or E7 in the test sample may be measured and compared to control samples in which expression levels of E6 and/or E7 were determined and where there was no, partial or complete methylation of the E2-binding sites respectively.

Suitable additional controls may also be included to ensure that the test is working properly, such as measuring levels of expression or activity of a suitable reference gene in both test and control samples. Suitable reference genes for the present invention include beta-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal RNA genes such as 18S ribosomal RNA and RNA polymerase Il gene (Radonic A. et al., Biochem Biophys Res Commun. 2004 Jan 23;313(4):856-62).

Expression of a nucleic acid can be measured at the RNA level or at the protein level. Cells in test samples can be lysed and the mRNA levels in the lysates, or in the RNA purified or semi-purified from the lysates, determined. Alternatively, methods can be used on un-lysed tissues or cell suspensions. Suitable methods for determining expression at the RNA level are well known in the art and described herein.

Methods employing nucleic acid probe hybridization to the relevant transcript(s), such as the E6 and/or E7 transcripts may be employed for measuring the presence and/or level of the respective mRNA. Such methods are well known in the art and include use of nucleic acid probe arrays (microarray technology) and Northern blots. Advances in genomic technologies now permit the simultaneous analysis of thousands of genes, although many are based on the same concept of specific probe-target hybridization. Sequencing-based methods are an alternative. These methods started with the use of expressed sequence tags (ESTs), and now include methods based on short tags, such as serial analysis of gene expression (SAGE) and massively parallel signature sequencing (MPSS). Differential display techniques provide yet another means of analyzing gene expression; this family of techniques is based on random amplification of cDNA fragments generated by restriction digestion, and bands that differ between two tissues identify cDNAs of interest.

In one embodiment, the levels of gene expression are determined using reverse transcriptase polymerase chain reaction (RT-PCR). RT-PCR is a well known technique in the art which relies upon the enzyme reverse transcriptase to reverse transcribe mRNA to form cDNA, which can then be amplified in a standard PCR reaction.

Protocols and kits for carrying out RT-PCR are well known to those of skill in the art and are commercially available.

The RT-PCR can be carried out in a non-quantitative manner. End-point RT-PCR measures changes in expression levels using three different methods: relative, competitive and comparative. These traditional methods are well known in the art. Alternatively, RT-PCR is carried out in a real time and/or in a quantitative manner. Real time quantitative RT-PCR has been thoroughly described in the literature (see Gibson et al for an early example of the technique) and a variety of techniques are possible. Examples include use of hydrolytic probes (Taqman), hairpin probes (Molecular

Beacons), FRET probe pairs (LightCycler (Roche)), hairpin probes attached to primers (Scorpion), hairpin primers (Plexor and Amplifluor), DzyNA and oligonucleotide blocker systems. All of these systems are commercially available and well characterised, and may allow multiplexing (that is, the determination of expression of multiple genes in a single sample).

Appropriate RT-PCR primers are exemplified in the tables herein. In certain embodiments, primers useful in determining the methylation status of the HBV, EBV, HPV18 or HPV16 genome (by RT-PCR) are provided. These primers comprise, consist essentially of or consist of the nucleotide sequences set forth as SEQ ID NOs 145 to 150 for the S, C and X genes respectively of HBV, SEQ ID NOs 343 to 350 for the EBNA2, BRLF1 , BHRF1 and EBER1 genes respectively of EBV, SEQ ID NOs 113 to 118 for the E6 and E7 genes respectively of HPV18 and SEQ ID NOs 47 to 50 for the E6 and E7 genes respectively of HPV16. These primers are useful in determining the methylation status of the relevant genes of the relevant viruses by looking at corresponding mRNA expression and, in addition to being useful in the methods of the invention, form separate aspects of the invention. The invention thus provides a primer for use in RT-PCR to determine the expression level, related to the methylation status of an HBV, EBV, HPV18 or HPV16 genome selected from the primers comprising the nucleotide sequences set forth as SEQ ID NOs 145 to 150 for the S, C and X genes respectively of HBV, SEQ ID NOs 343 to 350 for the EBNA2, BRLF1 , BHRF1 and EBER1 genes respectively of EBV, SEQ ID NOs 113 to 118 for the E6 and E7 genes respectively of HPV18 and SEQ ID NOs 47 to 50 for the E6 and E7 genes respectively of HPV16 and functional derivatives thereof which retain functionality in RT-PCR. Effectively, these particular methods of the invention may involve determining the methylation status in regions of the viral genome defined by the sequence between (and including) the primer binding sites by looking at gene expression. Further characteristics of these primers are summarized in the detailed description (experimental part) below. Thus, the methods of the invention may comprise, consist essentially of or consist of determination of the expression levels of genes which can be amplified using the primers comprising the nucleotide sequences set forth as SEQ ID NOs 145 to 150 for the S, C and X genes respectively of HBV, SEQ ID NOs 343 to 350 for the EBNA2, BRLF1 , BHRF1 and EBER1 genes respectively of EBV, SEQ ID NOs 113 to 118 for the E6 and E7 genes respectively of HPV18 and SEQ ID NOs 47 to 50 for the E6 and E7 genes respectively of HPV16 and functional derivatives thereof which retain functionality in RT-PCR, as defined herein. Variants are also included within the scope of the invention, as defined herein, which definition applies mutatis mutandis.

TAQMAN was one of the earliest available real-time PCR techniques and relies upon a probe which binds between the upstream and downstream primer binding sites in a PCR reaction. A TAQMAN probe contains a 5¹ fluorophore and a 3¹ quencher moiety. Thus, when bound to its binding site on the DNA the probe does not fluoresce due to the presence of the quencher in close proximity to the fluorophore. During amplification, the 5' - 3' exonuclease activity of a suitable polymerase such as Taq digests the probe if it is bound to the strand being amplified. This digestion of the probe causes displacement of the fluorophore. Release of the fluorophore means that it is no longer in close proximity to the quencher moiety and this therefore allows the fluorophore to fluoresce. The resulting fluorescence may be measured and is in direct proportion to the amount of target sequence that is being amplified. These probes are sometimes generically referred to as hydrolytic probes.

In the Molecular Beacons system, the probe is again designed to bind between the primer binding sites. However, here the probe is a hairpin shaped probe. The hairpin in the probe when not bound to its target sequence means that a fluorophore attached to one end of the probe and a quencher attached to the other end of the probe are brought into close proximity and therefore internal quenching occurs. Only when the target sequence for the probe is formed during the PCR amplification does the probe unfold and bind to this sequence. The loop portion of the probe acts as the probe itself, while the stem is formed by complimentary arm sequences (to respective ends of which are attached the fluorophore and quencher moiety). When the beacon probe detects its target, it undergoes a conformational change forcing the stem apart and this separates the fluorophore and quencher. This causes the energy transfer to the quencher to be disrupted and therefore restores fluorescence.

During the denaturation step, the Molecular Beacons assume a random-coil configuration and fluoresce. As the temperature is lowered to allow annealing of the primers, stem hybrids form rapidly, preventing fluorescence. However, at the annealing temperature, Molecular Beacons also bind to the amplicons, undergo conformational reorganisation, leading to fluorescence. When the temperature is raised to allow primer extension, the Molecular Beacons dissociate from their targets and do not interfere with polymerisation. A new hybridisation takes place in the annealing step of every cycle, and the intensity of the resulting fluorescence indicates the amount of accumulated amplicon.

Scorpions primers are based upon the same principles as Molecular Beacons. However, here, the probe is bound to, and forms an integral part of, an amplification primer. The probe has a blocking group at its 5¹ end to prevent amplification through the probe sequence. After one round of amplification has been directed by this primer, the target sequence for the probe is produced and to this the probe binds. Thus, the name "scorpion" arises from the fact that the probe as part of an amplification product internally hybridises to its target sequence thus forming a tail type structure. Probe-target binding is kinetically favoured over intrastrand secondary structures. Scorpions primers were first described in the paper "Detection of PCR products using self-probing amplicons and fluorescence" (Nature Biotechnology. 17, p804-807 (1999)).

In similar fashion to Scorpions primers, Amplifluor primers rely upon incorporation of a Molecular Beacon type probe into a primer. Again, the hairpin structure of the probe forms part of an amplification primer itself. However, in contrast to Scorpions type primers, there is no block at the 5' end of the probe in order to prevent it being amplified and forming part of an amplification product. Accordingly, the primer binds to a template strand and directs synthesis of the complementary strand. The primer therefore becomes part of the amplification product in the first round of amplification. When the complimentary strand is synthesised amplification occurs through the hairpin structure. This separates the fluorophore and quencher molecules, thus leading to generation of florescence as amplification proceeds.

DzyNA primers incorporate the complementary/antisense sequence of a 10-23 nucleotide DNAzyme. During amplification, amplicons are produced that contain active (sense) copies of DNAzymes that cleave a reporter substrate included in the reaction mixture. The accumulation of amplicons during PCR/amplification can be monitored in real time by changes in fluorescence produced by separation of fluorophore and quencher dye molecules incorporated into opposite sides of a DNAzyme cleavage site within the reporter substrate. The DNAzyme and reporter substrate sequences can be generic and hence can be adapted for use with primer sets targeting various genes or transcripts (Todd et al., Clinical Chemistry 46:5, 625-630 (2000)).

The Plexor™ qPCR and qRT-PCR Systems take advantage of the specific interaction between two modified nucleotides to achieve quantitative PCR analysis. One of the PCR primers contains a fluorescent label adjacent to an iso-dC residue at the 5' terminus. The second PCR primer is unlabeled. The reaction mix includes deoxynucleotides and iso- dGTP modified with the quencher dabcyl. Dabcyl-iso-dGTP is preferentially incorporated at the position complementary to the iso-dC residue. The incorporation of the dabcyl-iso- dGTP at this position results in quenching of the fluorescent dye on the complementary strand and a reduction in fluorescence, which allows quantitation during amplification. For these multiplex reactions, a primer pair with a different fluorophore is used for each target sequence.

Real time quantitative techniques produce a fluorescent read-out that can be continuously monitored. Fluorescence signals are generated by dyes that are specific to double stranded DNA, like SYBR Green, or by sequence-specific fluorescently-labeled oligonucleotide primers or probes. Each of the primers or probes can be labelled with a different fluorophore to allow specific detection. These real time quantitative techniques are advantageous because they keep the reaction in a "single tube". This means there is no need for downstream analysis in order to obtain results, leading to more rapidly obtained results. Furthermore, keeping the reaction in a "single tube" environment reduces the risk of cross contamination and allows a quantitative output from the methods of the invention. This may be particularly important in a clinical setting for the present invention.

It should be noted that whilst PCR is a preferred amplification method, to include variants on the basic technique such as nested PCR, equivalents may also be included within the scope of the invention. Examples include without limitation isothermal amplification techniques such as NASBA, 3SR, TMA and triamplification, all of which are well known in the art and commercially available. Other suitable amplification methods without limitation include the ligase chain reaction (LCR) (Barringer et al, 1990), MLPA, selective amplification of target polynucleotide sequences (US Patent No. 6,410,276), consensus sequence primed polymerase chain reaction (US Patent No 4,437,975), invader technology (Third Wave Technologies, Madison, Wl), strand displacement technology, arbitrarily primed polymerase chain reaction (WO90/06995) and nick displacement amplification (WO2004/067726).

Suitable methods for determining expression, of the relevant viral proteins, at the protein level are also well known to one of skill in the art. Examples include western blots, immunohistochemical staining and immunolocalization, immunofluorescence, enzyme- linked immunosorbent assay (ELISA), immunoprecipitation assays, complement fixation assay, agglutination reactions, radioimmunoassay, flow cytometry, mass spectrophotometry, and equilibrium dialysis. These methods generally depend upon a reagent specific for identification of the appropriate gene product. Any suitable reagent may be utilised such as lectins, receptors, nucleic acids, antibodies etc. The reagent is preferably an antibody and may comprise monoclonal or polyclonal antibodies. Fragments and derivatized antibodies may also be utilised, to include without limitation Fab fragments, ScFv, single domain antibodies, nano-antibodies, heavy chain antibodies, aptamers etc. which retain gene product binding function. Any detection method may be employed in accordance with the invention. The nature of the reagent is not limited except that it must be capable of specifically identifying the appropriate (viral) gene product.

Measurement of expression of a gene or protein on its own does not necessarily conclusively indicate that the silencing is epigenetic, as the mechanism of silencing could be genetic, for example, by somatic mutation. Accordingly, in one embodiment, the methods of the invention incorporate an appropriate re-expression assay which is designed to reverse epigenetic silencing. Appropriate treatment of the sample using a demethylating agent, such as a DNA-methyltransferase (DMT) inhibitor may reverse epigenetic silencing of the relevant gene, or in the case of E6 and/or E7, E2-binding sites. Suitable reagents include, but are not limited to, DAC (5'-deazacytidine), TSA or any other treatment affecting epigenetic mechanisms present in cell lines. Typically, expression is reactivated or reversed upon treatment with such reagents, indicating that the silencing is epigenetic.

The methods of the invention may be applied to any virus which contributes to a disease condition and wherein the progression of the disease is linked to increased methylation of the viral genome. In specific embodiments, the methods and kits of the invention are applied to DNA viruses, in particular double stranded DNA viruses such as HBV and EBV. In further embodiments the methods of the invention are applied to HPV, which for example comprises, consists essentially of or consists of HPV16. In alternative or complementary embodiments, the HPV comprises, consists essentially of, or consists of HPV-18.

Methods of Treatment/Pharmaceutical Compositions etc. The invention relies upon the observation that methylation of viral genomes increases as an indication of progression of virus-associated disease to more advanced or aggressive forms. This observation opens up a number of therapeutic options, either for directing treatment or for providing specific treatments.

In the first instance, determining the methylation status of the viral genome may be used to direct therapy by providing an indication of the relative progression of the condition. Thus, for example, where the genome is unmethylated or (relatively) hypomethylated at the investigated locations, this may provide an indication that the disease is either not present or is less advanced and may, therefore, not require aggressive treatment for example by surgery, radiation treatment, immunotherapy or use of chemo-therapentics. Alternatively, detection of hypermethylation within the genome of the dsDNA virus, as discussed above, may provide an indication that the infection-associated disease has progressed to an advanced stage. Here, the decision taken may be to adopt a more aggressive treatment strategy to include for example surgery, radiotherapy, immunotherapy, chemotherapy etc. Therapies may include use of antiviral, cytotoxic, epigenetic modifying, gene therapy or immunomodulating agents for example.

Thus, the invention provides a method for selecting a suitable treatment of an infection by a double stranded DNA virus (and/or disease caused by the infection) comprising (in a sample obtained from a subject) the determination of the methylation status of the viral genome wherein the presence of hypermethylation of the viral genome results in selection of a suitable therapeutic intervention. The therapeutic intervention may be selected from surgery, including surgical resection, radiotherapy, chemoptherapy etc as discussed above. If an epigenetic modifying agent is selected this may be a suitable DNA demethylating agent as discussed herein.

The presence of hypomethylation or a lower level of methylation (as appropriate depending upon the genome and region under investigation) results in selection of an alternative treatment and/or no treatment. One treatment which may be selected is use of a DNA methylating agent. The DNA methylating agent may drive the virus into a silenced state, by inducing hypermethylation of the genome, and thus prevent the virus contributing to progression of, or initiation of, a disease caused by the virus. Thus, the methylation status of the viral genome can direct either an attempt to accelerate methylation of the genome to prevent disease progression through silencing of the virus, or, in certain embodiments on attempt to alleviate viral genome methylation to facilitate immune recognition by the host. This shows the importance of the discovered correlation between methylation of the viral genome and the mechanism of disease formation and progression, in terms of being able to direct therapy.

The invention thus also provides a method of treating an infection by a double stranded DNA virus (and thus a disease associated with the infection) in a subject in need thereof comprising administering a therapeutically effective amount of a DNA methylating agent to the subject to silence the virus. The subject has been selected for this treatment of the basis of the methods of the invention which correlate hypermethylation of the viral genome with disease progression to advanced forms. Thus, these treatment methods are applied to a specific targeted population where the viral genome is found to be present but is unmethylated or hypomethylated. The natural progression of the infection to more methylated forms may be pre-empted and prevented by forcing hypermethylation within the viral genome, thus silencing the viral genome without the opportunity for the associated initiation and/or progression of disease.

Any DNA methylating agent capable of increasing methylation of the viral genome may be employed. For example, DNA methyl transferases may be utilised or chemical agents such as nitrosoureas, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyll - N-nitrosourea (MNU) , dimethyl sulphate (DMS), methyl methanesulphonate (MMS) and s-Adenosine Methionine (SAM) may be employed.]

The invention similarly provides a DNA methylating agent for use in treating an infection by a double stranded DNA virus/disease associated with or caused by the infection wherein the subject carries an unmethylated double stranded DNA viral genome. Also provided is use of a DNA methylating agrent in the manufacture of a medicament for treating an infection by a dsDNA virus/disease associated with or caused by the infection wherein the subject carries an unmethylated or hypomethylated viral genome.

In a further aspect, the invention provides a method of treating an infection by a double stranded DNA virus in a subject (in need of such treatment) comprising administering a therapeutically effective amount of a DNA demethylating agent in order to prevent progression of the infection (by decreasing methylation of the viral genome or by preventing increased methylation of the viral genome). The invention similarly provides a method of treating a disease caused by an infection by a double stranded DNA virus comprising administering a therapeutically effective amount of a DNA demethylating agent in order to prevent progression of the disease to a more aggressive form (by decreasing methylation of the viral genome or by preventing increased methylation of the viral genome). Thus, these methods of the invention take account of the observed increase in methylation of the viral genome as the disease progresses and aim to assist in treatment by alleviation of hypermethylated forms of the viral genomes. As has been shown in the experimental section, use of DNA demethylating agents can alleviate hypermethylation of viral genomes, which may assist in stimulating a patient/host (immune) response against the virus. It is hypothesised that increased methylation of the viral genome leads to disease progression through evasion of host immune recognition.

These treatment methods generally include a selection of the subject for treatment according to the methods of the invention. The treatment may be applied to a target population where the viral genome is highly methylated or hypermethylated.

The methods of treatment may advantageously be combined with treatment using other chemotherapeutic agents in certain embodiments. Thus, the invention also provides a pharmaceutical composition comprising a DNA demethylating agent or DNA methylating agent, as appropriate, together with a chemotherapeutic agent. The nature of the chemotherapeutic agent may be determined by the virus infection to be treated and the disease condition resulting from the viral infection. The link between specific viruses and specific primary tumours is discussed herein, which discussion applies mutatis mutandis to these aspects. The selection of a DNA methylating or DNA methylating agent may be based upon use of the methods of the invention to determine whether a hypermethylated or hypomethlyated viral genome is present.

All embodiments of the diagnostic methods of the invention apply mutatis mutandis to the method of treatment aspects. Thus, treatment may be of suitable disease conditions linked to HBV, HPV or EBV infections as described in detail herein. The methods may also be coined as a DNA demethylating agent for use in preventing progression of, or treating, an infection by a double stranded DNA virus or a disease caused by or associated with the viral infection. It may similarly be coined as the use of a DNA demethylating agent in the manufacture of a medicament for preventing progression of, or treating, an infection by a double stranded DNA virus or a disease caused by or associated with the viral infectio. The subject carries a hypermethylated viral genome indicating the infection/disease is at a progressed/advanced stage (which may be detected according to the methods of the invention).

The methods of treatment of the invention are generally applied to human subjects/patients but may also have veterinary applications. Generally, the subject/patient will be one who has a viral infection; for example as identified using the methods according to the present invention. Thus, the diagnostic and pharmacogenetic methods of the invention may assist in identifying a specific patient population to which the treatment methods of the invention may be applied.

Pharmacogenetic methods of the invention

As expounded in the experimental section below, it has been shown that increased methylation of double stranded DNA viral genomes correlates with progression of virus associated diseases to more aggressive forms. The effect of methylation is believed to mask the virus from the host immune response. Relieving this methylation may, therefore, help in the treatment of appropriate infections and resultant disease. However, the silencing of viral genes caused by methylation may also be taken advantage of. Detection of a hypomethylated genome may allow treatment by forcing silencing of the virus using suitable methylating agents. This may prevent the disease manifesting itself or progressing in the patient. Thus, the invention provides for the use of viral methylation status as a pharmacogenetic indicator (for the use of DNA demethylating or methylating agents dependent upon the level of methylation).

Accordingly, the invention provides a method for predicting the probability of successful treatment of a viral infection and/or a disease caused by a viral infection with a DNA demethylating agent comprising, in a sample obtained from a subject, determining the methylation status of the viral genome, wherein methylation and in particular hypermethylation of the viral genome is indicative of a high or increased probability of successful treatment (compared to the situation where methylation levels are lower).

Similarly, the invention also provides a method of selecting a suitable treatment regimen for a viral infection and/or a disease caused by a viral infection comprising, in a sample obtained from a subject, determining the methylation status of the viral genome, wherein methylation and in particular hypermethylation of the viral genome indicates that treatment using a DNA demethylating agent is suitable.

Relative hypomethylation of the viral genome may indicate that treatment using a DNA demethylating agent is unsuitable - at that particular stage of the infection. Instead, as discussed above, detection of a hypomethylated genome may allow treatment by forcing silencing of the virus using suitable methylating agents. This may prevent the disease manifesting itself or progressing in the patient.

Accordingly, the invention provides a method for predicting the probability of successful treatment of a viral infection and/or a disease caused by a viral infection with a DNA methylating agent comprising, in a sample obtained from a subject, determining the methylation status of the viral genome, wherein low levels of methylation and in particular hypomethylation of the viral genome is indicative of a high or increased probability of successful treatment (compared to the situation where methylation levels are higher).

Similarly, the invention also provides a method of selecting a suitable treatment regimen for a viral infection and/or a disease caused by a viral infection comprising, in a sample obtained from a subject, determining the methylation status of the viral genome, wherein low levels of methylation and in particular hypomethylation of the viral genome indicates that treatment using a DNA methylating agent is suitable.

The pharmacogenetic methods of the invention may incorporate any and all of the aspects described in respect of the diagnostic and therapeutic methods described above, as appropriate. In particular, the diagnostic methods of the invention may be carried out as a prelude to, or as an integral part of, the pharmacogenetic methods of the invention.

Thus, for example, the description of suitable methods for determining methylation status of the viral genomes, suitable test samples, subjects and specific double stranded DNA viruses and associated disease conditions (in particular types of cancer) which may be monitored all apply mutatis mutandis to these aspects of the invention and are not repeated here simply for reasons of conciseness. Different pre-malignancies and primary tumours are linked to specific viral infections as discussed herein (for HBV, EBV, HPV18 etc).

For all of the relevant methods (pharmacogenetic methods, treatment regimen methods and methods of treatment) of the invention, the DNA demethylating agent may be any agent capable of demethylating at least a portion of the relevant viral genome. DNA demethylating agents may comprise, consist essentially of or consist of DNA methyltransferase inhibitors. The DNA methyltransferase inhibitor may be any suitable inhibitor of DNA methyltransferase which is suitable for decreasing levels of methylation of the viral genome.

The DNA methyltransferase inhibitor may, in certain embodiments, be one which reduces expression of DNMT genes, such as suitable antisense molecules, or siRNA molecules which mediate RNAi for example. The design of a suitable siRNA molecule is within the capability of the skilled person and suitable molecules can be made to order by commercial entities (such as Qiagen and Ambion). In embodiments, the DNA methyltransferase gene is (human) DNMT1 and/or DNMT3b. As discussed in the experimental section, double depletion of DNMT1 and DNMT3b using an appropriate short interference RNA (siRNA) can be used to induce hypomethylation events in the viral genomes of dsDNA based viruses.

Alternatively, the agent may be a direct inhibitor of DNMTs. Examples include modified nucleotides such as phosphorothioate modified oligonucleotides (Fig 6 of Villar-Garea, A. and Esteller, M. DNA demethylating agents and chromatin-remodelling drugs: which, how and why? Current Drug Metabolism, 2003, 4, 11-31) and nucleosides and nucleotides such as cytidine analogues. Suitable examples of cytidine analogues include 5-azacytidine, 5-aza-2'-deoxycytidine, 5-fluouro-2'-deoxycytidine, pseudoisocytidine, 5,6-dihydro-5-azacytidine, 1 -β-D-arabinofuranosyl-5-azacytosine (known as fazabarine) (see figure 4 of Villar-Garea, A. and Esteller, M. DNA demethylating agents and chromatin-remodelling drugs: which, how and why? Current Drug Metabolism, 2003, 4, 11-31). In specific emboidiments the inhibitor comprises, cosists essentially of or consists of 5-aza-2'-deoxycytidine.

In other embodiments, the DNA methyltransferase inhibitor comprises Decitabine (Supergen). Additional DNMT inhibitors include S-Adenosyl-Methionine (SAM) related compounds like ethyl group donors such as L-ethionine and non-alkylating agents such as S- adenosyl-homocysteine (SAH), sinefungin, (S)-6-methyl-6-deaminosine fungin, 6- deaminosinefungin, N4-adenosyl-N4-methyl-2,4-diaminobutanoic acid, 5'-methylthio-5'- deoxyadenosine (MTA)and 5'-amino-5'-deoxyadenosine (Villar-Garea, A. and Esteller, M. DNA demethylating agents and chromatin-remodelling drugs: which, how and why? Current Drug Metabolism, 2003, 4, 11-31).

Further agents which may alter DNA methylation and which may, therefore, be useful in the present compositions include organohalogenated compounds such as chloroform etc, procianamide, intercalating agents such as mitomycin C, 4-aminobiphenyl etc, inorganic salts of arsenic and selenium and antibiotics such as kanamycin, hygromycin and cefotaxim (Villar-Garea, A. and Esteller, M. DNA demethylating agents and chromatin-remodelling drugs: which, how and why? Current Drug Metabolism, 2003, 4, 11-31). Useful DNMT inhibitors in the present invention may comprise, consists essentially of or consists of 5-azacytidine and/or zebulaine.

The DNA demethylating agent may additionally or alternatively comprise a histone deacetylase inhibitor. Such an agent may complement the effect of the DNA methyltransferase inhibitor, or may be used separately to induce demethylation of the viral genome. In specific embodiments, the histone deacetylase (HDAC) inhibitor comprises or is selected from at least one of trichostatin A (TSA), suberoyl hydroxamic acid (SBHA), 6-(3-chlorophenylureido)caproic hydroxamic acid (3-CI-UCHA), m- carboxycinnamic acid bishydroxylamide (CBHA), suberoylanilide hydroxamic acid (SAHA), azelaic bishydroxamic acid (ABHA), pyroxamide, scriptaid, aromatic sulfonamides bearing a hydroxamic acid group, oxamflatin, trapoxin, cyclic-hydroxamic- acid containing peptides, FR901228, MS-275, MGCD0103 (Methylgene), short-chain fatty acids and N-acetyldinaline .

It should be noted that, for the purposes of the present invention, the designation of a particular DNA demethylating or methylating agent is considered to encompass all pharmaceutically acceptable forms of the active compound which are useful as DNA demethylating or methylating agents. Thus, stereoisomers, enantiomers, salts, esters etc are all encompassed within the scope of the invention as appropriate.

Thus, in a pharmaceutical composition incorporating a suitable DNA demethylating or methylating agent (optionally together with a chemotherapeutic agent), compositions may include pharmaceutically acceptable carriers including, for example, non-toxic salts, sterile water or the like. A suitable buffer may also be present allowing the compositions to be lyophilized and stored in sterile conditions prior to reconstitution by the addition of sterile water for subsequent administration. The carrier may also contain other pharmaceutically acceptable excipients for modifying other conditions such as pH, osmolarity, viscosity, sterility, lipophilicity, somobility or the like. Pharmaceutical compositions which permit sustained or delayed release following administration may also be used.

Suitable pharmaceutical compositions for use in the treatment methods or medical uses of the invention may be used together with other standard chemotherapeutic treatments which target tumour cells directly, as discussed above. Non limiting examples include paclitaxel, cyclaphosphomide and 5-fluoro-uracil (5-FU) and pharmaceutically acceptable derivatives thereof including salts, etc. Additionally, or alternatively, the pharmaceutical compositions of the invention may incorporate a DNA demethylating agent together with a suitable anti-viral agent. The anti-viral agent may be specific for HBV, HPV (16 or 18) or EBV in certain embodiments. Examples of anti-viral agents include cytokines, in particular interferons and nucleoside antimetabolite drugs that interfere with duplication of viral genetic material, such as ribavirin.

The therapeutic agent may, for example, be encapsulated and/or combined with suitable carriers in solid dosage forms for oral administration which would be well known to those of skill in the art or alternatively with suitable carriers for administration in an aerosol spray. Examples of oral dosage forms include tablets, capsules and liquids.

Alternatively, the therapeutic agent may be administered parenterally. Specific examples include intradermal injection, subcutaneous injection (which may advantageously give slower absorption of the therapeutic agent), intramuscular injection (which can provide more rapid absorption), intravenous delivery (meaning the drug does not need to be absorbed into the blood stream from elsewhere), sublingual delivery (for example by dissolving of a tablet under the tongue or by a sublingual spray), rectal delivery, vaginal delivery, topical delivery, transdermal delivery and inhalation, as appropriate.

Furthermore, as would be appreciated by the skilled practitioner, the specific dosage regime may be calculated according to the body surface area of the patient or the volume of body space to be occupied, dependent on the particular route of administration to be used. The amount of the composition actually administered will, however, be determined by a medical practitioner based on the circumstances pertaining to the disorder to be treated, such as the severity of the symptoms, the age, weight and response of the individual, f Kits

The invention also provides kits which may be used in order to carry out the methods of the invention. The kits may incorporate any of the preferred features mentioned in connection with the various methods (and uses) of the invention herein. Accordingly, there is provided a kit for use in the methods of the invention comprising, consisting essentially of or consisting of a primer pair selected from the primers of tables 1 to 4 and in particular tables 2 to 4. These primers are described in greater detail herein. Suitable pairs may be selected from primers for use in bisulphite sequencing of a double stranded DNA viral genome selected from the primers comprising the nucleotide sequences set forth as SEQ ID NO: 53 to 108 for HPV18, SEQ ID NO: 119 to 140 for HBV and SEQ ID NOL: 151 to 330 for EBV and functional derivatives thereof which retain functionality in bisulphite sequencing and primers for use in methylation specific PCR (MSP) to determine the methylation status of a double stranded DNA viral genome selected from the primers comprising the nucleotide sequences set forth as SEQ ID NO: 109 to 112 for the E2 gene of HPV18, SEQ ID NO: 141 to 144 for the HBVgp2 gene of HBV, SEQ ID NO: 331 to 334 for the Cp gene of EBV and SEQ ID NO: 335 to 338 for the Wp gene of EBV and functional derivatives thereof which retain functionality in MSP.

The kit may also comprise, consist essentially of or consist of a reagent for converting unmethylated cytosine residues to a changed nucleotide which displays different base pairing properies to cytosine, such as uracil, but which does not convert methylated cytosine residues. The reagent may be a chemical or an enzyme for example. In certain embodiments, the reagent comprises, consists essentially of or consists of a bisulphite reagent. In more specific embodiments, the reagent comprises, consists essentially of or consists of sodium bisulphite. An enzyme such as a cytidine deaminase may be used in the methods and kits of the invention to mediate conversion as appropriate (e.g. see Bransteitter et al. PNAS USA (2003) Apr 1 ; 100(7): 4102-4107).

In a further embodiment, the means for determining the methylation status of the viral genome, that is to say the primers included in the kit, enable the detection to be carried out in a single reaction. Multiplexing is made possible for example through use of appropriate fluorophores having separable emission spectra. TaqMan probes, Molecular Beacons, Scorpions, etc., to include all suitable techniques and as discussed in greater detail above, allow multiple markers to be measured in the same sample (multiplex PCR), since fluorescent dyes with different emission spectra may be attached to the different probes. Accordingly, suitably labelled probes and primers are encompassed by the kits of the invention.

Many suitable reagents for methylation detection are known in the art, and are discussed herein (which discussion applies here mutatis mutandis). In certain embodiments, the kit also includes means for carrying out the methylation detection in single tube format, which may optionally be in real time. Means for carrying out embodiments such as methylation specific PCR/amplification or HeavyMethyl in real time may comprise hairpin primers (Amplifluor), hairpin probes (Molecular Beacons), hydrolytic probes (Taqman), FRET probe pairs (Lightcycler), primers incorporating a hairpin probe (Scorpion), fluorescent dyes (SYBR Green etc.), DzyNA primers or oligonucleotide blockers. All appropriate combinations are envisaged within the scope of the invention. The end-point PCR fluorescence detection technique can use the same approaches as widely used for Real Time PCR - TaqMan assay, Molecular Beacons, Scorpion etc. as discussed in greater detail herein. Accordingly, the kits of the invention may include means for carrying out the end-point methylation detection, such as methylation specific PCR or HeavyMethyl. The means for carrying out end-point methylation specific

PCR/amplification may comprise primers and/or probes as explained for PCR in Realtime.

In the real-time and end-point detection embodiments, the probes for detection of amplification products may simply be used to monitor progress of the amplification reaction in real-time and/or they may also have a role in determining the methylation status of the HPV genome themselves. Thus, the probes may be designed in much the same fashion as the primers to take advantage of sequence differences following treatment with a suitable reagent such as sodium bisulphite dependent upon the methylation status of the appropriate cytosine residues (found in CpG dinucleotides).

The probes may comprise any suitable probe type for real-time detection of amplification products as discussed above. Notably, however, with the AMPLIFLUOR and SCORPION embodiments, the probes are an integral part of the primers which are utilised. The probes are typically fluorescently labelled, although other label types may be utilised as appropriate (such as mass labels or radioisotope labels). These probes are also suitable for end-point detection.

In certain embodiments, the kit may also incorporate means for processing the test sample. Depending upon the sample to be analyzed this may include reagents such as a homogenization buffer. The means for processing a sample may further or alternatively comprise reagents for extraction/isolation/concentration/purification of DNA. Suitable reagents are known in the art and may comprise, consist essentially of or consist of alcohols such as ethanol and isopropanol for precipitation of DNA. Salt-based precipitation may require high concentrations of salts to precipitate contaminants. The salt may comprise, consist essentially of or consist of potassium acetate and/or ammonium acetate for example. Organic solvents may also be included in the kits to extract contaminants from cell lysates. Thus, in certain embodiments, the means for processing the sample comprise, consist essentially of or consist of phenol, chloroform and isoamyl alcohol to extract the DNA. Suitable combinations of reagents are envisaged as appropriate.

The kits may incorporate reagents for quantification of DNA such as those found in the Picogreen® dsDNA quantitation kit available from Molecular Probes, Invitrogen.

The kits may, in certain embodiments, also include means for obtaining a sample. For example, where the kits are used in the diagnosis, monitoring and staging of cervical cancer, the kits of the invention may also contain means for removing cervical cells from a patient for analysis. For example a spatula, such as an Ayre's spatula and/or a brush, such as an endocervical brush may be incorporated in the kits (or used in the methods) of the invention in order to obtain a cervical sample.

Sensitivity of detection, staging and monitoring may conceivably be improved by increasing the quantity of DNA in the sample. Accordingly, in one embodiment the means for processing a sample comprises, consists essentially of or consists of primers for directing amplification of viral DNA in the sample. Any suitable primers which amplify the viral genome, or relevant portions thereof, may be utilised. Preferably, the primers do not discriminate between methylated and unmethylated DNA (i.e. the primer binding sites lies outside of the CpG islands) thus providing a general increase in the amount of the relevant viral DNA prior to determining whether the methylated form of the viral genome is present in the sample. In a further embodiment, the primers are designed specifically for the viral genome such that there is minimal sequence overlap, or sequence homology, with endogenous DNA from the subject under test (the infective viral genome is considered exogenous to the genomic DNA, even if it has integrated). This means that the viral genome will be selectively amplified, thus improving the sensitivity of the methods of the invention. The aim is to prevent non-specific amplification of genomic DNA from the subject which may influence the results. Such considerations are also applicable to the methods of the invention and may therefore also apply to the primer design as discussed above.

Preferably, the homology is less than about 5%, less than about 10%, less than about 12.5 %, less than about 15%, less than about 20%, less than about 30%, less than about 40%, 50%, 60%, 70% or 80% sequence identity with the corresponding nucleotide sequence from the genomic DNA of the subject under test. In specific embodiments, there is no sequence identity with the corresponding nucleotide sequence from the DNA of the subject under test over approximately 10, 20, 30, 40 or 50 contiguous nucleotides. In another embodiment, there is less than about 10% or less than about 12.5 %, 15%, 20%, 30%, 40%, 50% or 60% sequence identity over approximately 10, 20, 30, 40 or 50 contiguous nucleotides with the corresponding nucleotide sequence from the endogenous DNA of the subject under test.

As discussed with respect to the methods of the invention, suitable controls may be utilised in order to act as quality control for the methods. Accordingly, in certain embodiments, the kit of the invention further comprises, consists essentially of or consists of one or more control nucleic acid molecules of which the methylation status is known. These (one or more) control nucleic acid molecules may include both nucleic acids which are known to be, or treated so as to be, methylated and/or nucleic acid molecules which are known to be, or treated so as to be, unmethylated. One example of a suitable internal reference gene, which is generally unmethylated, but may be treated so as to be methylated, is beta-actin.

Furthermore, the kit of the invention may further comprise, consist essentially of or consist of primers for the amplification of the control nucleic acid. These primers may be designed according to the control nucleic acid selected. Suitable probes and/or oligonucleotide blockers for use in determining the methylation status of the control nucleic acid molecules may also be incorporated into the kits of the invention. The probes may comprise any suitable probe type for real-time detection of amplification products. The discussion provided above applies mutatis mutandis.

The kits of the invention may additionally include suitable buffers and other reagents for carrying out the claimed methods of the invention. Thus, the discussion provided in respect of the methods of the invention as to the requirements for determination of the methylation status of the various viral genomes apply mutatis mutandis here.

In certain embodiments, the kit of the invention further comprises, consists essentially of, or consists of nucleic acid amplification buffers. The kit may also additionally comprise, consist essentially of or consist of enzymes to catalyze nucleic acid amplification. Thus, the kit may also additionally comprise, consist essentially of or consist of a suitable polymerase for nucleic acid amplification. Examples include those from both family A and family B type polymerases, such as Taq, Pfu, Vent etc.

The various components of the kit may be packaged separately in separate compartments or may, for example be stored together where appropriate.

The kit may also incorporate suitable instructions for use, which may be printed on a separate sheet or incorporated into the kit packaging for example. For example, data may be provided to indicate typical levels of methylation which are expected for each stage of disease, based upon a number of previous results which have been accumulated and assessed. The results obtained using the kits may then be compared with this data to provide a diagnosis or indication of the stage or progression of the disease. The data may be provided in any suitable format - such as in software or printed form. For example, the methylation patterns presented in figures 1 to 4 may be presented and utilised as a reference in order to facilitate the diagnosis depending upon the virus concerned.

Alternative kits may include the RT-PCR primers of the invention, in the form of one or more primer pairs for determining methylation status at the level of gene expression of the appropriate genes. Additional components of such an RT-PCR kit would be known to the skilled person and may be selected from a reverse transcriptase to produce cDNA from RNA, buffers, RNase inhibitors etc. Real-time and end point versions of the kit may incorporate suitable detection systems, which may be fluorescence based for example. Reference can be made to the discussion above, which discussion applies mutatis mutandis to the RT-PCR applications.

Brief description of the figures Figure 1. The DNA methylome of HPV16.

FIG.1A. Unsupervised clustering analysis of the HPV16 DNA methylome in asymptomatic carriers of the virus, premalignant lesions (CIN), primary tumors (SCCs) and cancer cell lines (CaSki, SiHA, CCL-879 and CCL- 866). Black, dark grey and grey indicate methylated, unmethylated and deleted sequences, respectively. The HPV16 genome is shown on top.

FIG.1 B. Example of bisulfite genomic sequencing analysis of multiple clones for the HPV16 genome. Black and white squares indicate methylated and unmethylated CpG dinucleotides. Grey squares indicate deleted genome sequences.

FlG.1C. Bisulfite sequencing showing how the mostly unmethylated HPV16 DNA methylomes from pre-immortal keratynocytes undergo hypermethylation in immortalized cells.

FIG.1 D. Confocal studies of fluorescence in situ hybridization for HPV16 and 5- methylcytosine DNA staining demonstrate the colocalization of the integrated viral genome in a human chromosome and the DNA methylation signal in CaSki cells.

FIG.1 E. The use of a DNA demethylating agent in CaSki cells causes DNA hypomethylation of the HPV16 genome.

FIG.1 F. The expression of HPV16 E6 and E7 oncoproteins is associated with a loss of binding of the E2 repressor to the URR methylated sites. Shown is the expression of E2 and the methylated status of the URR binding site in CaSki.

FIG. 1G. DNA demethylating treatment reduces E6 and E7 expression in western-blot experiments

FIG. 1 H. DNA demethylating treatment reduces E6 and E7 expression in qRT-PCT experiments FIG. 11. DNA demethylating treatment reduces E6 and E7 expression in. association with a recruitment of the E2 protein to the demethylated URR E2 binding site, shown by q- ChIP.

FIG. U Methylation-specific PCR analysis of the HPV16 L2 sequence in cervical tumorigenesis. The presence of a band under the U or M lanes indicate unmethylated or methylated sequences. In vitro methylated DNA (IVD) is shown as positive control.

Figure 2. The DNA methylome of HPV18.

FIG. 2A Unsupervised clustering analysis of the HPV18 DNA methylome in asymptomatic carriers of the virus, primary tumors (SCCs) and cancer cell lines (HeLa and C4I). Black, dark grey and grey indicate methylated, unmethylated and deleted sequences, respectively. The HPV18 genome is shown on top.

FIG. 2B Example of bisulfite genomic sequencing analysis of multiple clones for the HPV18 genome. Black and white squares indicate methylated and unmethylated CpG dinucleotides. Grey squares indicate deleted genome sequences.

FIG. 2C Bisulfite sequencing showing how the mostly unmethylated HPV18 DNA methylome from pre-immortal keratynocytes undergoes hypermethylation in immortalized cells. Dark grey and grey indicate methylated and unmethylated sequences, respectively.

FIG 2D. Depletion of DNMT1 and DNMT3b by short interference RNA in HeLa cells causes a DNA hypomethylation of the HPV18 genome.

FIG 2E. The use of a DNA demethylating agent (5- aza-2'-deoxycytidine) does not change the expression of the HPV18 E6 and E7 oncoproteins in HeLa cells in association with an unmethylated URR E2 binding site, as shown by Conventional RT- PCRs. FIG. 2F. The use of a DNA demethylating agent (5- aza-2'-deoxycytidine) does not change the expression of the HPV18 E6 and E7 oncoproteins in HeLa cells in association with an unmethylated URR E2 binding site, as shown by q-RT-PCRs.

FIG. 2G Expression of E2 demonstrates an unmethylated URR E2 binding site.

FIG. 2H. Bisulfite sequencing also demonstrates an unmethylated URR E2 binding site.

FIG 2I. Methylation specific PCR analysis of HPV16 E2 sequence in cervical tumorigenesis. The presence of a band under the U or M lanes indicate unmethylated or methylated sequences. In vitro methylated DNA (IVD) is shown as positive control

Figure 3. The DNA methylome of HBV.

FIG 3A. Unsupervised clustering analysis of the HBV DNA methylome in carriers of the virus with premalignant lesions such as cirrhosis and hepatitis, primary liver tumors (HCC primary) and liver cancer cell lines (HCC cell line). Black, dark grey and grey indicate methylated, unmethylated and deleted sequences, respectively. The HBV genome is shown on top.

FIG 3B. Example of bisulfite genomic sequencing analysis of multiple clones for the HBV genome. Black and white squares indicate methylated and unmethylated CpG dinucleotides. Grey squares indicate deleted genome sequences.

FIG 3C. DNA methylation-associated silencing of the HBVgp4 (HBV-C) and HBVgp2

(HBV-S) genes in the hepatic carcinoma cell line HA22T and reactivation upon the use of a DNA demethylating agent (5-aza-2'- deoxycytidine). The HBVgp3 remains unmethylated and expressed, shown in RT-PCR experiments. The hepatoblastoma cell line HepG2, negative for the HBV virus, is used as negative control.

FIG. 3D. . DNA methylation-associated silencing of the HBV surface antigen (HBsAg) encoding gene in the hepatic carcinoma cell line HA22T and reactivation upon the use of a DNA demethylating agent (5-aza-2'- deoxycytidine), as shown in western-blot experiments. The hepatoblastoma cell line HepG2, negative for the HBV virus, is used as negative control.

FIG. 3E. Bisulfite sequencing results demonstrating the effect of demethylating agents and DNMT inhibition on methylation levels.

FIG 3F. Methylation-specific PCR analysis of the HBVgp2 sequence in liver tumorigenesis. The presence of a band under the U or M lanes indicate unmethylated or methylated sequences. In vitro methylated DNA (IVD) is shown as positive control.

Figure 4. The DNA methylome of EBV.

FIG 4A. Unsupervised clustering analysis of the EBV DNA methylome in the wild EBV, the B95-8 type EBV, lymphoblastoid cell line (BL2126), benign lesions (reactive lymphadenitis, RL, and infectious mononucleosis, IM), Post-transplant lymphoproliferative Disorder (PTLD), primary lymphomas (non- Hodgkin Lymphoma, NHL, Hodgkin Lymphoma, HL, Burkitt's Lymphoma, BL), lymphoma cell lines (Rael, Akata, Raji, AhM , Farage, Namalwa) and nasopharyngeal primary carcinoma (NPC) and cancer cell line (C666.1). Dark grey and grey indicate methylated and unmethylated CpG dinucleotides encompassing the corresponding 5'- end transcription start sites, respectively. The EBV genome is shown on top.

FIG 4B. Methylation-specific PCR analysis of the EBV FP, WP and CP 5'-end CpG islands in lymphoblastic and Burkitt's lymphoma cell lines. The presence of a band under the U or M lanes indicate unmethylated or methylated sequences. In vitro methylated DNA (IVD) is shown as positive control.

FIG 4C. Colocalization of the EBV viral genome determined by FISH and the 5- methylcytosine DNA staining in Akata cells..

FIG 4D 5'-CpG island methylation-associated silencing of the EBNA2, BRLF1 and BHRF1- H2H3 genes and the microRNA miRBHRFH and release of silencing upon the use of a DNA demethylating agent (5-aza-2'-deoxycytidine), shown in RT-PCR results. FIG. 4E. 5'-CpG island methylation-associated silencing of the EBNA2 gene and release of silencing upon the use of a DNA demethylating agent (5-aza-2'-deoxycytidine), shown in a western blot.

FIG 4F. 5'-CpG island methylation-associated silencing of the EBNA2, BRLF1 and

BHRF1- H2H3 genes and the microRNA miRBHRFH and release of silencing upon the use of a DNA demethylating agent (5-aza-2'-deoxycytidine), shown via bisulfite genomic sequencing

FIG 4G. 5'-CpG island methylation-associated silencing of the microRNA miRBHRF1.1 and release of silencing upon the use of a DNA demethylating agent (5-aza-2'- deoxycytidine), shown in q-RT-PCR.

FIG 4H EBV-immortalized ICF lymphocytes (DNMT3b defective) demonstrate a hypomethylated EBV genome in comparison to the one observed in DNMT3b-proficient EBV-transformed lymphocytes (LCL). Dark grey, and grey indicate methylated and unmethylated CpG dinucleotides encompassing the corresponding 5'-end transcription start sites, respectively.

FIG 41. The induction of the EBV lytic cycle in a lymphoma cell line (AKATA) upon addition to the media of anti-lgG caused a massive DNA hypomethylating event in the EBV-transcription start sites. Red, and green indicate methylated and unmethylated CpG dinucleotides encompassing the corresponding 5'- end transcription start sites, respectively. Below, example of western-blot for the EBNA2 protein showing the restoration of expression upon the demethylation of the corresponding CpG island.

Figure 5. FISH hybridization with the HPV16 probe labeled in red (shown as light spots in greyscale version and indicated by arrows in certain panels) in different cervical cancer cell lines. CaSki, SiHa and HeIa cells present high-number, low-number and none copies of the integrated HP16 genome, respectively.

Figure 6. FISH hybridization with the HBV probe labeled in red (shown as light spots in greyscale version and indicated by arrows in certain panels) in different hepatic carcinoma cell lines. HA22T, SNU354 and ALEX demonstrate integrated HBV genome. The hepatoblastoma cell line HepG2 is used as negative control.

Figure 7. FISH hybridization with the EBV probe labeled in red (shown as light spots in greyscale version and indicated by arrows in certain panels) in different lymphoma cell lines. Raji, Akata and Namalwa present high, intermediate and low-number copies of the EBV genome, respectively. The lymphoma cell line Ramos is used as negative control.

Figure 8. The induction of the EBV lytic cycle in a lymphoma cell line (AKATA) upon addition of anti-lgG to the media caused a major change in the EBV DNA methylome, in just 48 hours, characterized by massive hypomethylating events at the transcription start sites present in the EBV virus. This wave of DNA hypomethylation was accompanied by the restoration of gene expression of the previously methylated genes, which matched our expression microarray data [Yuan J, Cahir-McFarland E, Zhao B, Kieff E. Virus and cell RNAs expressed during Epstein-Barr virus replication. J Virol. 80, 2548-65 (2006)] . Red and green blocks indicate methylated and unmethylated.transcription start sites (TSS), respectively, a, DNA hypomethylating events associated with gene reactivation, b, Unmethylated TSSs with decreased expression, c, Upregulated TSSs.

The DNA methylome of HPV16

There are over 100 different papilloma family members and, in humans, these are responsible for a variety of benign proliferations. However, infection with two specific high-risk HPVs, HPV-16 and HPV-18, is associated with approximately 90% of uterine cervical cancers, more than 50% of other anogenital tumors and a small percentage of head and neck tumors (25).

HPV particles consist of circular DNA molecules, about =8,000 base pairs (bp) long, wrapped into a protein shell that is composed of two molecules (L1 and L2). The genome has the coding capacity for these two proteins and at least six so-called early proteins (E 1 , E2, E4-E7) that are necessary for the replication of the viral DNA and for the assembly of newly produced virus particles within the infected cells. Both sets of genes are separated by an upstream regulatory region (URR) of about 1000 bp that does not code for proteins but contains cis-elements required for regulation of gene expression, replication of the genome and its packaging into virus particles (20,21 ,25). Two of the viral proteins, E6 and E7, are essential for HPV-mediated cellular transformation (26). These interfere with many cellular processes, overriding signalling pathways and cell- cycle control by altering the function of basal transcription factors p53 and Rb, among other target proteins (26).

The natural history of the disease is intriguing, since only a minority of cervical tissues infected with HPV inevitable progress to cancer. Given that around 291 million women throughtout the world are carriers of HPV DNA (27), the incidence of cervical cancer is relatively low (25). Thus, the development of cervical cancer occurs in a few women who cannot resolve their infection and who maintain persistent active infection for years or decades following initial exposure. However, the molecular reasons why the infection is controlled or instead progresses to subsequent stages of tumorigenesisare largely unknown.

The complete HPV DNA methylomes obtained in our study might provide important clues to help us understand the described process. First, we sequenced the whole DNA methylome of the HPV16 virus (110 CpGs in 7904 nucleotides) in a collection of human cervical samples corresponding to the different progressive stages of the disease: asymptomatic carriers (n=5), premalignant disease (the so called Cervical Intraepithelial Neoplasia, CIN) (n=8) and the primary cervical carcinomas (n=5). We have also completed the HPV16 DNA methylome of four established cervical cancer cell lines (CaSki, SiHa, CCL-866 and CLL-879). We discovered that the HPV16 DNA methylome undergoes a progressive increase in its DNA methylation content from tissue of womenwho are carriers of the virus but without any symptom of clinical disease, through early pre-tumorigenic lesions, to full-blown primary cervical carcinomas (Figure 1A). In this sequence of events, the HPV16 DNA methylomes of the invasive cervical cancer cell lines CaSki and SiHa are the ones demonstrating the higher DNA methylation levels. We developed an unsupervised clustering analysis for the eighteen HPV16 DNA methylomes obtained that also distinguished the four different biological groups: carriers, premalignancies, primary tumors and cancer cell lines (Figure 1A). Examples of the bisulfite genomic sequencing analysis of multiple clones of the HPV16 virus genome are shown in Figure 1 B. To examine the HPV16 DNA methylomes please go to: http://ubio.bioinfo.cnio.es/Methylyzer/main/index.html (password: Methyl 502lyzer).

The dynamic changes experienced by the viral DNA methylome during the tumorigenic process were highlighted when we studied cultured primary human foreskin keratinocytes transfected with the entire genome of HPV1628. The HPV16 DNA methylome from the pre-immortal keratynocytes was almost completely unmethylated, whilst the immortal descendant cells featured a densely methylated viral genome (Figure 1C). Most importantly, genetic or nucleotidic changes in the HPV16 genome were not observed. This observation was a universal feature of our studies: because our DNA methylation analysis by bisulfite genomic sequencing also allows putative nucleotide changes (such as point mutations, insertions or deletions) to be identified, we were able to assess the contribution of these genetic changes in the virus itself to the progression of the disease. We did not observe any particular viral nucleotide change associated with the natural history of cervical tumorigenesis. To navigate through all the viral DNA genomes please go to: http://ubio.bioinfo.cnio.es/Methylyzer/main/index.html (password: Methyl 502lyzer).

The HPV16 genome does not code for any gene of the DNA methylation machinery, such as DNA methyltransferases (DNMTs). Thus, the viral genome is methylated by the host human cellular DNMTs. The integration of HPV16 in the human genome occurs in most cervical tumors and a significant proportion of premalignat lesions, and so it is reasonable to suggest that human DNMTs might recognize these sequences (1-5). Indeed, in our study, we have confirmed by FISH analysis that the HPV16 genome is integrated within the host cell DNA in CaSki and SiHa cells (see METHODS). Most importantly, confocal studies demonstrated the colocalization of the integrated viral genome and 5-methylcytosine DNA staining (Figure 1 D). The use of a DNA demethylating agent (5-aza-2'-deoxycytidine) in CaSki cells did indeed cause DNA hypomethylation of the HPV16 genome (Figure 1E).

Interestingly, the HPV16 genome is not merely a passive spectator of the process but might actively participate by recruiting DNMTs using the viral oncoprotein E7(29). The HPV16 DNA methylomes described here also reflect the proposed expression patterns of the virus in cervical tumorigenesis, which are characterized by a significant overexpression of E6 and E7 in the carcinomas(20,21 ,25,26). Our sequencing effort shows that, for a subset of cases spanning the range of stages, there are genomic viral regions that are deleted, particularly the E1/E2 region. The protein E2 inhibits cell proliferation by regulating the viral upstream regulatory region (URR), repressing the E6 and E7 oncoproteins, and, ultimately, causing cell cycle arrest at G2(30). Thus, the genomic loss of E2 could contribute to the progression of the disease. However, the HPV16 DNA methylomes described here show that there are many cases where there are not any ruptures at the E2 locus, and, accordingly, E2 is expressed (Figure 1 F). We observed instead methylation of the E2-binding sites at the URR region (Figure 1A and 1 F) that was associated with a high level of expression of the oncoproteins E6 and E7 (Figure 1 F). Most importantly, we showed by chromatin immunoprecipitation (ChIP) that the E2 viral protein could not bind to the methylated E2-binding sites at the URR region (Figure 1 F), and thus led to E6 and E7 overexpression. Finally, the induction of hypomethylation events in the E2-binding sites at the URR region by a DNA demethylating agent (5-aza-2'-deoxycytidine) induced the recruitment of E2 to its URR binding sites and a marked reduction of E6 and E7 expression (Figure 1 F).

We went one step further to confirm these results in a large collection (n=87) of human primary samples from the different stages of cervical carcinogenesis using the technique of methylation-specific PCR for the L2 region of the virus (Figure 1G). We observed the progressive presence of hypermethylation at the L2 locus in tumorigenesis: 0% (0 of 10) in asymptomatic carriers, 29% (5 of 17) in stage I of intraepithelial neoplasia (CIN I), 37% (16 of 43) in stage Il and III of intraepithelial neoplasia (CIN N-III), and 94% (16 of 17) in primary cervical carcinoma. Overall, the DNA methylomes of HPV16 outlined are evidence of the existence of an adaptive DNA methylation pattern of the viral genome, whereby there was an increased methylated- CpG content during the progression of the disease arising from the crosstalk between the viral and the host genomes.

The DNA methylome of HPV18 HPV16 is the most prevalent high-risk papilloma virus in the general population and is responsible for approximately 50% of cervical cancers, but HPV18 occupies second place in both instances. HPV16 and HP18 are representative of two different HPV species (alphaθ and alpha7, respectively), but share a global genomic structure that codes for almost identical proteins (20,21 ,25,26). Most importantly, a highly similar pattern was encountered when we obtained the complete DNA methylomes of the HPV18 virus. The bisulfite genomic sequencing of the entire HPV18 viral genome (168 CpGs in 7857 nucleotides) obtained from asymptomatic carriers of the virus demonstrated a low level of methylation, whilst an increased methylated genome was observed in primary cervical carcinomas (Figure 2A). The unsupervised clustering analysis was also able to distinguish infected tissues without clinical relevance from the full-blown tumors on the basis of the HPV18 DNA methylomes (Figure 2A). Examples of the bisulfite genomic sequencing of multiple clones are shown in Figure 2B. The HPV18 DNA methylomes may be examined at: http://ubio.bioinfo.cnio.es/Methylyzer/main/index.html (password: Methyl 502lyzer).

We did not only analyze the static nature of these DNA methylomes at given stages, but, as we did with HPV16, also we obtained the HPV18 DNA methylomes present during the immortalization crisis that keratynocyes transfected with the entire HPV18 genome undergo28. The HPV18 DNA methylome from the preimmortal keratynocytes was mostly unmethylated, whilst the immortal cells presented a densely methylated viral genome (Figure 2C). Most importantly, no genetic or nucleotidic changes in the HPV18 genome were observed.

The HPV18 genome does not encode for any DNMTs, thus it is believed to "use" the host enzymes. Short interference RNA depletion of DNMT1 and DNMT3b did indeed induce hypomethylation events in the HPV18 positive cervical cancer cell line HeLa (Figure 2D). Although HPV16 and HPV18 share many features in their DNA methylomes, one interesting distinction occurs for the viral oncoprotein expression standpoint. The levels of E6 and E7 expression in HPV18-infected cervical cancer cell lines, such as HeLa and C4I, were not modified by the treatment with a DNA demethylating agent (Figure 2E). These findings fits perfectly well with the observation that the E2 binding sites at the URR region of HPV18 in these cells were not methylated (Figure 2A and 2E) and thus the E2-mediated control of E6 and E7 expression could not be released by the DNA hypomethylating drug.

Finally, to extend our analysis to a larger collection (n=32) of human primary samples from the different stages of HPV18-mediated cervical carcinogenesis, we used the technique of methylation-specific PCR for the E2 region of the virus (Figure 2F). We confirmed the progressive presence of hypermethylation at the E2 locus in tumorigenesis: 0% (0 of 6) in asymptomatic carriers, 14% (1 of 7) in stage I of cervical intraepithelial neoplasia (CIN I), 67% (6 of 9) in stage Il and III of cervical intraepithelial neoplasia (CIN ll-lll), and 90% (9 of 10) in primary cervical carcinoma.

The DNA methylome of HBV

The third genome whose complete DNA methylome we obtained is that of the Hepatitis B Virus (HBV). The HBV infectious virion has an outer envelope, formed by the hepatitis B surface antigen (HBsAg) in a lipid bilayer. This encloses the nucleocapsid core, within which lies the viral genome (21 ,31). The latter is a relaxed circular, partially double- stranded DNA molecule of 3,215 bp in length, and contains four partially overlapping open reading frames. These code for the envelope glycoproteins (L, M and S HBsAgs), the precore/core (precursor of the soluble hepatitis B e antigen, HBeAg, and the core protein), the X protein (HBx) and the polymerase, which acts as reverse transcriptase and also has DNA polymerase activity (21 ,31). It is interesting that while HBV is a DNA virus, it replicates through an RNA intermediate and thus requires the described active viral reverse transcriptase polymerase enzyme.

HBV infects more than 2,000 million people worldwide, 400 million of which are chronically infected and are at high risk for the development of active hepatitis, cirrhosis and hepatocarcinoma (HCC)(21 ,31). The HBV carriers with chronic liver disease have 100-fold greater risk of developing HCC, which is the third leading cause of cancer death in the world(21 ,31). 90% of HBV-associated liver cancers show integration of the HBV genome within the human genome(21 ,31). Two major HBV-specific mechanisms contribute to the development of HCC: the viral genome integration in the human genome causes cis-effects that inactivate tumor suppressor genes and activate oncogenes; and the expression of trans-activating factors derived from the HBV genome, such as the HBx protein and the PreS2 activators(32), which disrupt the signal transduction pathways and alter the gene expression of the host cell(21 ,31).

We completed the HBV DNA methylomes (n=32) from different stages of the liver tumorigenesis, from chronic active hepatitis (CAH) and hepatic cirrhosis (HC) to primary hepatocarcinomas (HCCs) and a well characterized panel of hepatic cancer cell lines. The results are in concordance with those observed for HPV16 and HPV18: the HBV genome is almost completely unmethylated in the early stages of carcinogenesis, such as hepatitis and cirrhosis, whilst it becomes more methylated in the established liver tumors, both in patients and in cultured cancer cell lines (Figure 3A). Unsupervised clustering analysis was also able to classify these liver samples as pre-malignant or malignant stages on the basis of their HBV DNA methylomes (Figure 3A). The HBV DNA methylomes may be examined at: http://ubio.bioinfo.cnio.es/Methylyzer/main/index.html (password: Methyl 502lyzer). Examples of the bisulfite genomic sequencing of multiple clones are shown in Figure 3B.

Most importantly, the presence of DNA methylation at the HBVgp4 and HBVgp2 genes, which respectively code for the C and S viral proteins, is associated with their lack of expression (Figure 3C), whilst release of transcriptional silencing can be achieved by the use of a DNA demethylating agent (Figure 3C). Our sequencing data also shows that most of the HBV genomes, although more methylated than the premalignant lesions, retained the HBVgp3 gene that codes for the X protein (HB-X) in an unmethylated state (Figure 3C). HB-X was transcribed in all the studied liver cancer cell lines (Figure 3C). Interestingly, the HB-X protein might regulate DNMT activity(33). As with the HPVs, the HBV genome does not code for any DNMTs, and thus should "use" the host enzymes. In this regard, double depletion of DNMT1 and DNMT3b by short interference RNA in HA22T cells did indeed cause DNA hypomethylation of the HBV genome (Figure 3D).

Our extensive bisulfite genomic sequencing effort also established that the integrated HBV genome undergo significant deletions, as previously reported(21 ,31), that are more common in CAH and HC than in liver tumors. We developed a FISH analysis for the HBV virus to demonstrate further its presence in the liver tumors (Supplementary Information). Finally, to expand our study of the HBV DNA methylome and to confirm its dynamic nature we tested a larger collection (n=35) of human primary samples from the different stages of HBV-mediated liver carcinogenesis, using the technique of methylation-specific PCR for the HBVgp2 gene of the virus (Figure 3E). We have confirmed the progressive presence of hypermethylation at the HBVgp2 locus in the hepatic tumorigenesis: 0% (0 of 5) in HC, 11 % (1 of 9) in CAH, and 52% (11 of 21) in primary liver tumors. Thus, these data suggest that DNA methylation of the HBV genome may "mark" those cells that progress towards tumorigenesis, as was implied by the analysis of the HPV16 and HPV18 DNA methylomes The DNA methylome of EBV

The final extensive DNA methylation of a viral genome we analyzed in the present study is that of the Epstein-Barr Virus (EBV), which is several orders of magnitude larger than those considered above (171 ,823 bp). EBV was discovered more than 40 years ago examining cells cultured from Burkitt's lymphoma, a childhood tumour that is common in sub-Saharan Africa. However, instead of showing a restricted distribution, EBV has been found to be widespread in all human populations. 90% of the world's adult population is infected by the virus, and it persists in the vast majority of individuals as a lifelong, asymptomatic infection of the B-lymphocyte cells21 ,34-36, but can occasionally evolve to an infectious mononucleosis (IM). In immunocompromised humans, such as AIDS and post-transplant patients, EBV infection is implicated in the etiology of several different lymphoid and epithelial malignancies, such as nasopharyngeal carcinoma(21 ,34-36).

There are two alternative states of EBV infection: lytic and latent. Virions are only produced in lytic infection and its genome remains as an episome in the host cell(21 ,34- 36). EBV has the ability to transform resting B cells into permanent infected lymphoblastoid cell lines that constitutively express a limited set of viral products, which are called latent proteins. These oncogenic genes are the nuclear antigens EBNAs 1 , 2, 3A₁ 3B, 3C and LP; and the latent membrane proteins LMPs 1 , 2A and 2B. Other gene products are the immediate early genes (which are considered the switch between latent and lytic cycle), the early genes (for example, enzymes influencing the host cell nucleotide metabolism and DNA synthesis) and the late gene products (for example the virion structural proteins)(21 , 34-36). The large EBV genome, of almost 172 Kb, prevented us from deriving the entire EBV DNA methylome using the same technology we had employed for the HPV16, HPV 18 and HBV genomes. Instead, we undertook the most extensive DNA methylation analysis of the EBV genome to date using bisulfite genomic sequencing analysis of multiple clones for seventy-seven amplicons that contains the transcription start sites that code for the ninety-four EBV proteins and the two structural RNAs, EBER1 and EBER2. The complete set of EBV transcription start sites studied included 95% (72 of 76) CpG islands(37) and covered 1 ,344 CpGs of the EBV genome. Overall, twenty-two EBV DNA methylomes were obtained from a collection of lymphoid samples that included benign proliferating cells, such as reactive lymphadenitis and infectious mononucleosis, non-tumorigenic lymphoblastoid cell lines, post-transplant lymphoproliferative disorder and EBV-associated lymphomas from primary tissues and cell lines, such as non-Hodgkin's lymphoma (diffuse large B-cell lymphoma and peripheral T-cell lymphoma), Hodgkin's lymphoma and Burkitt's lymphoma. In addition, nasopharyngeal tumors were also included. We observed a similar EBV DNA methylation profile to that observed for HPV16, HPV18 and HBV. Since the EBV genome does not code for any DNA methyltransferase, the EBV DNA obtained from free viral particles is devoid of DNA methylation (Figure 4A), however, the EBV genome present in human cells corresponding to benign diseases, such as reactive lymphadenitis and infectious mononucleosis showed the incipient presence of methylated EBV transcription start sites (Figure 4A). The same occasional hypermethylation was observed in nontumorigenic human B cell-derived lymphoblastoid cell lines (Figure 4A). Remarkably, this pattern was significantly changed in cancer cells infected with EBV, where the majority of lymphoma and nasopharyngeal samples from primary tumors and cell lines had a large number of hypermethylated EBV transcription start sites (Figure 4A). Unsupervised clustering analysis was able to distinguish free virus DNA, EBV-related benign conditions and EBV-associated human lymphomas and epithelial carcinomas on the basis of the EBV DNA methylome (Figure 4A).

These results were confirmed by methylation-specific PCR in a second set of EBV- positive non-tumorigenic (n=16) vs EBV-lymphoma (n=21) cell lines: lymphoblastoid cell lines did not present promoter hypermethylation of Wp and Cp genes, whilst Burkitt's lymphoma cell lines had 43% and 23% hypermethylation frequencies, respectively

(Figure 4B). Confocal studies demonstrated the colocalization of the EBV viral genome determined by FISH and the 5-methylcytosine DNA staining in Burkitt's lymphoma cells (Figure 4C). The EBV DNA methylomes may be examined at: http://ubio.bioinfo.cnio.es/Methylyzer/main/index.html (password: Methyl 502lyzer).

Interestingly, of the seventy-seven transcription start site regions that originate the entire EBV mRNA transcriptome, four amplicons "escaped" the DNA methylation mark: the two structural RNAs EBER1 and EBER2 and the Qp and BZLF1 sequences (Figure 4A). Methylation-specific PCR analyses for the Qp sequence confirmed the absence of DNA methylation from the additional set of both lymphoblastoid (n=16) and Burkitt's lymphoma (n=21) cell lines (Figure 4B). RT-PCR expression confirmed that the always unmethylated EBER1 was expressed in all the analyzed samples (Figure 4D). On the other hand, the presence of a hypermethylated EBV transcription start site in the cancer cells was associated with the corresponding transcriptional silencing of the neighboring gene. We demonstrated this close association between a methylated transcription start site and its loss of transcription at the single gene level, using RTPCRs and Western- blots (Figure 4D) for the EBNA2, BRLF1 and BHRF1 genes, and at global level, matching the obtained EBV methylomes with our previous study of the EBV transcriptome by expression microarrays (see METHODS)(38). Notably, as it has also been shown in human cells(39,40), the presence of DNA methylation in a microRNA locus was also associated with its loss of expression. This was the case for miR- BHFRF1-1 , located in the 5'-untranslated region of the BHFR1 gene41 , which was hypermethylated in Akata cells (Figure 4D). The link between DNA methylation and silencing was further reinforced by the use of the DNA demethylating agent 5-aza-2'- deoxycytidine, which was able to restore the expression of the EBV methylation inactivated genes and of mirR-BHFRF1-1 (Figure 4D).

The EBV genome does not code for any known DNMT and, thus, could be a target of the human DNMTs. In the case of EBV, we have a research avenue that it has been provided by nature itself. Some individuals are born with a germline defect in DNMT3b (Immunodeficiency, Centromere Instability, Facial Anomalies syndrome, ICF, OMIM #242860)(42,43) and lymphocytes immortalized with EBV from these patients have been widely used to understand the DNA methylation machinery(44,45). The analysis of DNA methylation of EBV-immortalized ICF lymphocytes revealed a more hypomethylated EBV genome than that observed in DNMT3b-proficient EBV transformed lymphocytes (Figure 4E), which is evidence of the role of DNMT3b in establishing the EBV DNA methylome. Interestingly, in a similar fashion to what occurs with HPV16, HPV18 and EBV, there may be a cross-talk between the EBV-viral proteins and the DNA methyltransferases of the host human cell. The viral LMP1 protein induces the expression and activity of human DNMTs(46), but it is not known, whether induces hypermethylation of the EBV genome. Finally, as we showed for the other three viral double-strand DNA methylomes, the EBV DNA methylome was highly adaptable to the biological and environmental circumstances surrounding the host cell. The induction of the EBV lytic cycle in a lymphoma cell line (AKATA) upon addition of anti-lgG to the media38 caused a major change in the EBV DNA methylome, in just 48 hours, characterized by massive hypomethylating events at the transcription start sites present in the EBV virus (Figure 4F). This wave of DNA hypomethylation was accompanied by the restoration of gene expression of the previously methylated genes, which matched our expression microarray data(38) (see METHODS), and as is confirmed here by Western Blot (Figure 4F). Thus, the dynamic nature of the epigenome, exemplified here by the changing EBV DNA methylome, was further highlighted.

Our analysis provide for the first time the complete DNA methylomes of living organisms, the three double-stranded DNA viruses HPV16, HPV18 and HBV, in addition to the DNA methylation status of all the transcription start sites of another double-stranded DNA virus, EBV. These DNA methylomes have been studied in more than 200 human samples infected with these viruses at different stages of the disease and this large- scale sequencing effort has revealed the dynamic nature of the epigenome, at least at the level of DNA methylation. One of the main findings of our study is the progressive increase in the DNA methylation content in these viruses from asymptomatic carriers, through benign lesions and premalignant disease, to full-blown human tumors. Several explanations for these findings come to mind, and they should be the focus of future multidisciplinary research in the field, but one particularly interesting possibility is that DNA methylation might be a cloacking device to camouflage the virus from our immune system(47,48). A DNA methylation-associated blockade of viral antigen presentation could be used to evade immune control. Most interestingly, the use of clinically approved DNA demethylating agents for the treatment of hematological malignancies, in addition to restore the expression of epigenetically silenced tumor suppressor genes of the host cell(1-5), might also reactivate the immune response, thereby enhancing its therapeutic benefits. The DNA methylomes presented here could be a good starting point for biomedical researchers who wish to address these questions, or to understand how the viral proteins themselves are able to use our own DNMTs to favor the establishment of persistent infection. These issues go beyond basic research and might have a great impact in public health, since many millions of people in the world are carriers of these viruses. Above all, the DNA methylomes obtained here, although owned by very small organisms, could be an excellent trampoline for launching ultra-deep sequencing projects aimed at the complete description of the human DNA methylome, in a similar way to which microbial genomes stimulated the race to derive the human genome. Our viral DNA methylomes, in this regard, could be an excellent proof of principle for the successful completion of ongoing human epigenome projects.

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METHODS

Cell lines and primary tissues

The human cell lines used in this study were obtained from the American Type Culture Collection (Rockland, MD), the German Collection of Microorganisms and Cell Cultures (DMSZ, Braunschweig, Germany) and the Coriell Institute for Medical Research (Camden, NJ). Cell lines were maintained in the appropriate medium and treated with 5- aza-2-deoxycytidine at a concentration of 1 μmol/L for 3 days to achieve DNA demethylation. Cell lines of primary human foreskin keratinocytes transfected with HPV16 or HPV18 were obtained as previously described (Steenbergen et al., 1996). The EBV lytic cycle was induced in Akata cells upon addition of anti-lgG to the media as previously described (Yuan et al., 2006). Primary tissues from normal, premalignant, and tumoral samples were all obtained at the time of the clinically indicated procedures.

Western blot analysis

To study the levels of viral proteins in the cell lines used in the study, cells were lysed with RSB buffer [10 mmol/L Tris (pH 7.5), 10 mmol/L NaCI, 3 mmol/L MgCI2] in the presence of 1% NP40 (17) and resuspended in SDS-PAGE loading buffer. Equal amounts of proteins were separated on 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were then hybridized with antibodies against HPV16 E6 (N-17, sc-1584 1 :200), HPV16 E7 (ED17, sc-6981 1 :200), HPV16 E2 (TVG261 , ab17185: 1 :250), Hepatitis B Virus X antigen antibody (3F6-G10, ab 235, 1 :250), Hepatitis B Virus Surface Antigen (Ad/Ay) antibody (86C, ab20758, 1 :200) (Abeam, Cambridge, UK) and HBV HBcAg (C1-5, sc-23945 1 :200), EBNA2 (DakoCytomation M7004 1 : 100). The proteins were detected with the Enhanced

Chemiluminescence Plus Western Blotting Detection System (Amersham Biosciences, Piscataway, NJ).

Analysis of CpG methylation status DNA methylation patterns in the CpG islands were determined by bisulfite-mediated conversion of unmethylated, but not methylated, cytosines to uracil as previously described (Clark et al., 1995; Herman et al., 1996). Bisulfite genomic sequencing of multiple clones was performed following the detailed protocol described at http://www.epigenomenoe. net/researchtools/protocol.php?protid=34. In brief, we first modified the DNA with sodium bisulfite as described (Herman et al., 1996) and then used Methyl Primer Express® software (Applied Biosystems) to design bisulfite-modified DNA- specific oligonucleotides spanning the whole genomes of the viruses HPV16, HPV18, and HBV viruses, and all the transcription start sites of the EBV virus. We next amplified the promoter regions under standard PCR conditions, cloned the PCR products into pGEM®-TEasy Vector (Promega), randomly selected at least eight colonies per sample, and performed sequencing using an ABI Prism 3130XL Applied Biosystems DNA sequencer. Finally, we analyzed the methylation of every CpG using Sequencing Analysis 5.2 Software from Applied Biosystems. Both strands were sequenced. For primary samples we also developed a second analysis using methylation-specific PCR (MSP) (Herman et al., 1996). Placental DNA treated in vitro with Sss I methyltransferase was used as a positive control for all methylated sequences. Complete Tables with all the primers used are provided herein. To view all the viral DNA methylomes please go to http://ubio.bioinfo.cnio.es/Methylyzer/main/index.html (password: Methyl 502lyzer).

Quantitative chromatin immunoprecipitation (qChlP) assays

To investigate the presence of E2 protein at the E2-binding sites of the URR region of the HPV16 genome, chromatin immunoprecipitation (ChIP) assays were done as described previously (Ballestar et al., 2003) using the E2 antibody (TVG261 , ab17185). Cell lysates were sonicated for 20 min with 30 s on-and-off cycles at the high setting of a Bioruptor (Diagenode, Liege, Belgium) to produce chromatin fragments of 0.5 kb on average. At least three independent ChIP experiments were done for each cell line. PCR amplification was done in 20 μL with specific primers encompassing the URR region. The sensitivity of PCR amplification was evaluated in serial dilutions of total DNA collected after sonication (input fraction). PCR conditions for the ChIP assay are available on request. Negative controls (no antibody) and input were included for each PCR experiment; and each experiment was carried out in triplicate. For the real-time PCR reactions, we used the SYBR Green PCR master mix (Applied Biosystems, Foster City, CA) and a 7900HT Fast Real-Time PCR system (Applied Biosystems, Foster City, CA). Standard curves were calculated from serial dilutions (100-0.1 ng) of input genomic DNA. To evaluate the relative enrichment of target sequences after ChIP, we calculated the ratios of the signals in the immunoprecipitated DNA versus input DNA (relative enrichment over input).

Semiquantitative reverse-transcription PCR expression analysis We reverse transcribed total RNA (2 μg) treated with DNase I (Ambion, Austin, TX) using oligo(dT) primer with Superscript Il reverse transcriptase (Life Technologies, Gaithersburg, MD). We used 100 ng cDNA for PCR amplification and amplified all of the genes with multiple (20-35) cycles to determine the appropriate conditions for obtaining semiquantitative differences in their expression levels. Reverse transcription-PCR (RTPCR) primers were designed between different exons to avoid any amplification of DNA. PCRs were done simultaneously with two sets of primers, with glyceraldehyde-3- phosphate dehydrogenase as an internal control to ensure cDNA quality and loading accuracy.

Viral gene expression by quantitative RT-PCR

Total RNA was isolated from Caski, SiHa and HeLa cells by Trizol (Invitrogen, San

Diego, CA) extraction according to the manufacturer^'s instructions. Then, we transcribed total RNA (2 μg) treated with TURBO DNA-free (Ambion, Austin, TX), using oligo(dT) primer with Superscript Il reverse transcriptase (Life Technologies, Gaithersburg, MD). The gene specific primer sequences are available in the Primer Tables. The real-time PCR reactions typically contained sense and antisense gene specific primers (350 nM), 7 ul of SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) and 2 μl of diluted cDNAs (10 ng/μl) in a total volume of 14 μl. The reaction was done in a ABI 7900 HT. Glyceraldehyde-3-phosphate dehydrogenase expression was used as an internal control to ensure cDNA quality and loading accuracy.

miR-BHRF1-1 detection bv quantitative RT-PCR Total RNA was isolated from Akata cells and Akata cells treated with 5-aza-2^'- deoxycytidine (1 μmol/L for 72 h) by Trizol (Invitrogen, San Diego, CA), with extraction according to the manufacturer's instructions. Trace amounts of unwanted DNA were eliminated using TURBO DNA-free, from Ambion (Austin, TX). We then measured miRNA expression using quantitative-real-time reverse transcription PCR (qRT-PCR) using the Ncode SYBR Green ER miRNA qRTPCR Kit, from Invitrogen (San Diego, CA). Briefly, 1 ug of RNA was polyadenylated following the manufacturer's instructions and the reverse transcription was then performed to obtain first-strand cDNA. The gene- specific primer sequence was identical to the entire mature miRNA sequence (see Table). The real-time PCR reactions typically contained gene-specific primer (20OnM), universal qPCR primer (200 nM), 0.24 ul of ROX Reference Dye, 6 μl of Platinum SYBR Green qPCR SuperMix-UDG and 1.2 μl of diluted cDNAs (1 :5) in a total volume of 12 μl. The reaction was done in a ABI 7900 HT. hsa-miR-16 expression was used to normalize ebv-miR-BHRF1-1 expression levels.

Short interference RNA (siRNA) experiments

Short interference RNA experiments were undertaken as previously described (Ropero et al., 2006). DNMT1- and DNMT3b-specific siRNAs were designed and synthesized by Qiagen. siRNA for E7 (HPV16) and X (HBV) were provided by Genelink. Scramble siRNA was also purchased by Qiagen and used as a control. For adherent cells, transfection was carried out using oligofectamine (Invitrogen) according to the manufacturer's specifications. Suspension cells were transfected by electroporating 107 cells in 0.8 ml PBS with 10 μg of siRNA at 250 V and 975 μF. The cells were harvested at specific times for total RNA extraction, and the target genes were assessed by RTPCR.

Flourescent in situ hybridization (FISH)

Cell cultures were exposed to colchicine (0.5 μg/ml) for 4 h at 37⁰C₁ and harvested routinely. Metaphases were prepared from the immortalized cell lines following a conventional cytogenetic protocol: Cells were pelleted, gently resuspended in 2-3 ml 75 mM KCI prewarmed to 37⁰C, and incubated for 15 min at 37⁰C. Cells were pelleted and initially resuspended in a residual volume of 100 μL of freshly made fixation solution (3:1 v/v methanokacetic acid). Up to 3 ml_ of fresh fix was added dropwise while the tubes were vortexed at low speed. Two more fixation washes in the same fixation solution were done. To prepare spreads, 100 μL of cells were dropped onto a precleaned microscope slide, which was dried slowly in a fume hood and left overnight to age the chromosomes naturally. Bacterial artificial chromosome (BAC) clones containing the entire genomic region of the viruses were used to generate the FISH probes. BAC for HBV16 was kindly provided by Dr. Pulivarthi H. Rao (Texas Children's Cancer Center, Baylor College of Medicine, 6621 Fannin Street, MC 3-3320, Houston TX, USA) (Harris et al., 2003). BAC for HBV was kindly provided by Dr. Tian-Hua Huang (Research Center of Reproductive Medicine, Shantou University Medical College, Shantou 515041, Guangdong Province, China) (Huang et al., 2005). Dr. Teru Kanda (Center for Virus Vector Development, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815, Japan) kindly provided us with BAG for EBV virus (Kanda et al., 2004). In brief, all BACs were labeled directly by nick translation according to the manufacturer's specifications using the CGH Nick Translation Reagent Kit with Spectrum Red dUTP (Abbot Molecular, Inc., Des Plaines, IL, USA). The probes were blocked with Cot-1 Human DNA (Roche Diagnostic Corporation, Indianapolis, IN, USA) to suppress repetitive sequences. The probe was denatured at 96°C for 5 min and hybridized overnight at 37⁰C in a humid chamber. After post-hybridization washes, the cell metaphases were counterstained with 4',6-diamidino- 2-phenylindole (DAPI II) and included in "VECTASHIELD" mounting medium kit (Vector Laboratories, Inc., Burlingame, CA, USA) for chromatin counterstaining and fluorescent protection before microscopy. Cell images were captured using a CCD camera (Photometries SenSys) installed on an Olympus BX microscope connected to a computer running the analysis system CytoVision v. 3.1. Software package (Applied Imaging Corporation, San Jose, CA, USA). For 5mC immunodetection after FISH, cells were treated with 0.5N CIH for 30 min at 37⁰C, and neutralized in Tris-Borate-EDTA buffer for 5 min at RT. Treated samples were blocked in PBS-0.5% BSA for 15 min at RT and incubated with mouse monoclonal antibodies recognizing 5-methylcytidine (a gift from A. Nivelau). Primary antibodies were detected using Cy2-labeled secondary antibodies. Confocal optical sections were obtained using a Leica TCS SP2 microscope (Leica Microsystems) equipped with krypton and argon lasers. Images were acquired with Leica LCS Lite software, and processed with the publicly available GNU Image Manipulation Program.

Computational analysis

We developed a new Java software package, in the form of a web server, called 'Methylyzer'. It is able to analyze DNA methylation data obtained from sequences previously modified with bisulfite, and to publish them on the internet. Data obtained from the experiments (multiple sequence alignment files) were uploaded to and analyzed by Methylyzer. Original, new, and lost CpG sites are located and a methylation value is assigned. Mutations (replacements, insertions, and deletions) are also identified. To navigate through the genomes of these viruses we set up a genome browser (GBrowse) (Stein et al., 2002) that lets the user jump directly to Methylyzer and visualize the annotations. A set of tracks, which are very easy to activate, shows the different types of information, such as 'DNA methylation sequence¹, 'Genetic sequence', Transcription start site¹, 'Gene', Εxon', etc. By clicking, for example, over an annotated 'DNA methylation sequence' one can view the methylation data for the available samples of a particular amplicon. The samples are represented by a band colored according to the average level of DNA methylation. It is possible to click on these bands and the accompanying description, and this will take the user to another web page with detailed information for each CpG site of that sample. The information can be seen in the form of two types of image: those of the overall bisulfite genomic sequence and those of the bisulfite genomic sequence of multiple clones. By selecting all or a subset of the samples, Methylyzer builds images of methylation profiles that allow the direct comparison of the methylation over the different samples. The data matrix containing the CpGs methylation profiles for HPV16, HPV18, HBV, and EBV samples were obtained from Methylyzer. Unsupervised hierarchical clustering (UPGMA) analyses were carried out applying the City Block Metric (Manhattan) and Single Linkage methods.

References 1). Steenbergen RD, Walboomers JM, Meijer CJ, van der Raaij-Helmer EM, Parker JN, Chow LT, Broker TR, Snijders PJ. Transition of human papillomavirus type 16 and 18 transfected human foreskin keratinocytes towards immortality: activation of telomerase and allele losses at 3p, 10p, 11q and/or 18q. Oncogene. 13, 1249-1257 (1996). 2). Yuan J, Cahir-McFarland E, Zhao B, Kieff E. Virus and cell RNAs expressed during Epstein-Barr virus replication. J Virol. 80, 2548-65 (2006).

3). Clark SJ, Harrison J, Paul CL, Frommer M. High sensitivity mapping of methylated cytosines. Nucleic Acids Res. 22, 2990-2997 (2004).

4). Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A. 93, 9821-9826 (1996).

5). Ballestar E, Paz MF, VaIIe L, Wei S, Fraga MF, Espada J, Cigudosa JC, Huang TH, Esteller M. Methyl-CpG binding proteins identify novel sites of epigenetic inactivation in human cancer. EMBO J. 22, 6335-6345 (2003). 6). Ropero S, Fraga MF, Ballestar E₁ Hamelin R, Yamamoto H, Boix-Chornet M, Caballero R, Alaminos M, Setien F, Paz MF, Herranz M, Palacios J, Arango D, Orntoft TF, Aaltonen LA, Schwartz S Jr, Esteller M. A truncating mutation of HDAC2 in human cancers confers resistance to histone deacetylase inhibition. Nat Genet. 38, 566-569 (2006).

7). Harris CP, Lu XY, Narayan G, Singh B, Murty W, Rao PH. Comprehensive molecular cytogenetic characterization of cervical cancer cell lines. Genes Chromosomes Cancer. 36, 233-241 (2003). 8). Tian-Hua Huang, Qing-Jian Zhang, Qing-Dong Xie, Li-Ping Zeng, Xi-Fan Zeng. Presence and integration of HBV DNA in mouse oocytes. World J Gastroenterol 11 , 2869-2873 (2005).

9). Kanda T, Yajima M, Ahsan N, Tanaka M, Takada K. Production of high-titer Epstein- Barr virus recombinants derived from Akata cells by using a bacterial artificial chromosome system. J Virol. 78, 7004-15 (2004). 10). Stein LD, Mungall C, Shu S, Caudy M, Mangone M, Day A, Nickerson E, Stajich JE, Harris TW, Arva A, Lewis S. The generic genome browser: a building block for a model organism system database. Genome Res. 12, 1599-1610 (2002).

Table 1. Primers used for Human Papilloma Virus 16 assays. BS, bisulfite genomic sequencing; MSP, methylation specific PCR; RT, reverse transcription PCR; ChIP, chromatin immunoprecipitation.

Primers Forward 5' -> 3¹ Reverse 5* -> 3'

BS

BS-HPV16-1 TAATAATΓTATGTATAAAATTMGGG ATCCAAATATCTTTACTTTTCTT

BS-HPV16-2 TG7TAAAAGTTATTGTGTTTTΘAAG ATCCA.ACTAAACCATCTATTTCAT

BS-HPV16-3 ATGAAATAGATGGTTTAGTTGG TCATCTAATATAACATCCCCTATT

BS-HPV16-4 ATAGGGGATGTTATATTAGATG AATATATCTTTCACTAACACCC

BS-HPV16-5 TTAGTAATGTAAAGGTAGTAATGTTAG TTCCCCATMACATACTMAC

BS-HPV16-6 GTGTTTTTAATGTGTATGATG TACCTATATTAACTACACCATATAAT

BS-HPV16-7a TGTTGGTATAGATTTTAGGTGG CAACCMTATTAACACCACTTAA

BS~HPV1δ-7b ATAAGGTTAGAGAAATGGGATT AACCCTCTACCACMTTACTAAT

BS-HPV16-8 TAGTGGAAGTGTAGTTTGATGG CACTATCCACTAAATCTCTATACMCA

BS-HPV16-9a AAGTTGTTGTATAGAGATTTAGTGGA TAAACAMCACACAAAAACACA

BS-HPV16-9b TTTTGTGTG i I i I i GTGTGTTT AMCACCTAMCRCAAAAACTA

BS-HPV16-10a TAGTAG l I I I I GYGTTTAGGTG TTCAACAATMTTTTACCTTCAAC

BS-HPV16-1Cb GGTrGAAGGTAAAATTATTGTTG ACACCMCATCMTMAACTMTTT

BS-HPV16-11 AGTAATTAGTAGTATATTTATATTAGGG ATCATAATMTAATATACCTTAACACC

BS-HPV16-12 GGATTATATGATATTTATGTAGATGAT ATCCAACTACAMTMTCTAMTATTC

BS-HPV16-13 TAMGTATTAGGATTATMTATAGGG TCTATTATCCACACCTACATTTA

BS~HPV1δ-14a GGTTTTGGTGTTATGGATTTTA TTMTTACCCCMCAAATACCA

BS-HPV16-14b AMGGTTTTGΘGTTTATTGTAA ATTCCMTCCTCCAAAATMTA

BS-HPV16-15a AAAGGAAAAG I i I i I I GTAGATTT TAMTMCCACMCACMTTMTAM

BS-HPV16-15b AGGATTGAAGGTTAMTTAAMTT MCACATTTTATACCAAAAAACA

BS-HPV16-16 GTTGTATG I 1 1 1 1 1 GGTATAAMT ATTACTTATAAATATATAACCCMMTC

MSP

MSP-HPV16-4M TGCGATATAMCGTTTTGTAAMC MTATACCCMTACGTCCGC MSP-HPV16-4U TAATGTGATATAMTGTTTTGTAAMT AMTATACCCAATACATCCACCT

RT*

RT-HPV16-E6 ACTGCGACGTGAGGTGTATTA TGGMTCTTTGC I I 1 I I GTCC RT-HPV16-E7 CCGΘACAGAGCCCATTACMT ACGTGTGTGCTTTGTACGCAC

ChIP

ChIP-HPVI 6-E2 TTGC ATATTTGGC ATMG GTTT ACTMCCGGTTTCGGTTCAA

*de Boer, MA, Jordanαva, ES., Kenter, GG., Peters, AA., Cower, WE., Trimbos, JB. & Fleuren, GJ. High human papillomavirus oncogene niRNA expression and not viral DNA load is associated wiift poor prognosis in cervical cancer patients. CTm Cancer Res. 13, 13-2-8 (2007).

"Lamarcq, L., Deeds, J., Ginzinger, D., Peπy, J., Padmanabha, S. & Smith-McCune, K. Measurements of human papillomavirus transcripts by real time quantitative reverse transciipϋαn-polymerase chain reaction in samples collected for cervical cancer screening. J Mo! Diagn. 4, 97-102 (2002) Table 2. Primers used for Human Papilloma Virus 18 assays. BS, bisulfite genomic sequencing; MSP, methylation specific PCR; RT, reverse transcription PCR.

Primers Forward 5' -> 3' Reverse S' -> Z'

BS

BS-HPV18-0 TAATTGTAGTATATAAAAAAGGGAGT TACACAAATCAAATAACTTATAAAATC

BS-HPV18-1 GGTGTATATAAAAGATGTGAGAAATAT AXAAAATATACTATCTCTATACACCAC

BS-HPV18-2 TTTGTGTATGGAGATATATTGGA ACTCTAAATACAATACAATATCTTACAA

BS-HPV18-3 AGTATGTATGGATTTAAGGTAATATTG ATTACTTACTACTAAAATACACACCAC

BS-HPV18-4 GTGGTGTGTATTTTAGTAGTAAGTAAT CCTATACTATCTCTAACTCTACCTATTC

BS-HPV18-5 GAATA6GTAGA6TTAGAGATAGTATAGG CACTTCAAAACAACCATAAC

BS-BPV18-6 GTTGGAGGTGGATATAGAGTTAA ACTATTATTACCCTCTATACCCC

BS-HPV18-7 GGGGGTATAGAGGGTAATAATA TAACAACTATTAATCTACTCTTACCAC

BS-HPV18-8 GGGTTATAGTTATATTTGGAGT.AAATT TATCCACTAAAATATATCTCCCA

BS-HPV18-9 ATGGGAGATATATTTGAGTGGA ACATCATCTAACATAACCACCT

BS-HPV18-10.1 TTTATTAGTTATTTTTGGTTGGAA AAACCATATCCTTTCAAAAAAAC

BS-HPV18-10.2 TTTTTTGAAAGGATATGΘTTTA .AAAACCTTATAAMCCATTTAC

BS-HPV18-11 AMGTTATTGAATTGTAAATGGTTT AC.AATCCCCTATAACTTACACAA

BS-HPV18-12 TGATTTATGTAGTATGGGATAGTG AACAAATCCACAATACTACTTCTC

BS-HPV18-13 GAGAAGTAGTATTGTΘGATTTGTT TCATTACCTACACCTATCCAAT

BS-HPV18-14 I I I I I GTGTATGTATGTATGTGTGT ACATAAACAATAAAAAACAAAATACA

BS-HPV18-15 I I I I I IATG I 1 1 I l I GTAI I I I I GT CCCCTATACCACTACCAATAC

BS-HPV18-16 TTTGATGTTGTTTTTAAGGTGG TAAAACCCACAAATTCAATAACC

BS-HPV18-17 AGTGGTTATTGAATTTGTGGGT CTACCACCTCCCCAATTTATAA

BS-HPV18-18a AAGTTTTATAAATTGGGGAGGT CCATAAAATCTAAATCAAAAACATC

BS-HPVIS-fSb AGTGATGTTTTTGATTTAGATTTT AAAAATAAACCCTAAGACCTATTT

BS-HPV1S-19a TAGGTGTTAGGGTTTAI I 1 1 IATTATG CACATCCCAAAAAAAAATTAA

BS-HPV18-1913 TTTTAATTTTTTTTTGGGATGTG ACCACCTACAAAAACCCTAAAA

BS-HPV18-20 TTTGTAGGTGGTGGTAATAAGTAGGA CCCAATAACAAAAACACAACCC

BS-HPV18-21 TTGGGTTGTGTTΠTGTTATTG CACAATATCACCCATAATACCTAC

BS-HPVI 8-22 TAGAGGATTGGAATTTTGGTGTTTT ACACAAAACATACAAACACAACAATAA

BS-HPV18-23 GTATGATTGTATTGTATGGTATGTATG ACAAACATAAACCAAAAACAAC

BS-HPV18-24 TTTTGGTTTATGTTTGTGGTT TATAATATATACTACCCAACCTATTTC

MSP

MSP-HPV18-3M AGTATCGTGTTCGTG6GTATC ACAATACTACTTCTCCGCGAAT MSP-HPV18-3U TTTAGTATTGTGTTTGTGGGTATT ACAATACTACTTCTCCACAAATCCA

RT

RT-HPV18-FULL CAGAGGTATTTGAATTTGCATT AATCTATACATTTATGGCATGC

RT-HPV18-E6* AACTTACAGAGGTGCCTGCG TAGTGCCCAGCTATGTTGTG

RT-HPV18-E7 GACTCAGAGGAAGAAAACGAT GTGACGTTGTGGTTCGGCT

^•de Boer, IWA., Jordanova, ES., Keπter, GG., Peters, AA., Cocver, WE., Tπmbos, JB. & Reuren, GJ. High human papillomavirus oncogene mRNA expression and not viral DNA load is associated with poor prognosis in cervical cancer patients. CHn Cancer Res. 13, 132-8 (2007).

"Lamarcq, L, Deeds, J., Ginanger, D., Perry, J., Padmanabha, S. & Smith-McCuπe, K. Measurements of human papillomavirus transcripts by real time quantitative reverse transcripSon-pcIymerase chain reaction in samples collected for cervical cancer screening. J Mo! Diagn. 4, 97-102 (2002) Table 3. Primers used for HBV assays. BS, bisulfite genomic sequencing; MSP, methylation specific PCR; RT, reverse transcription PCR; ChIP, chromatin immunoprecipitation.

Primers Forward 5' -> 3' Reverse 5' -> 3'

BS

BS-HBV-1 TTTTAGAGTGAGGGGTTTATATT TMATACTCCCCCTAAAMATTA

BS-HBV-2 GGAi 1 1 1 i I I IAAI f Ti I TAGGGG CTAAMCCCACTCCCATAAAM

BS-HBV-3 GTAAGAI I I I I ATGGGAGTGGGT ACAACAAMCCCAAAAMCC

BS-HBV-4 MGAATTGTGGG I 1 1 I I I GGGT TMCMCACAACCTAACMCCA

BS-HBV-5 TATGGTTGTTAGGTTGTGTTGT ACCAATTTATACCTACAACCTC

BS-HBV-5.1 TGGTTGTTAGGTTGTGTTGTTA TAAAMCCCAMCRACCC

BS-HBV-5.2 TYGTTTGTGi 1 1 I 1 1 I ATTTGT CCMTTTATACCTACMCCTCC

BS-HBV-6 AAGTTTTTAAGTTGTGTTTTGGG CAAMACAATTTCTCTTCCAM

BS-HBV-7 TTATTTGGGTGGGAAGTMTTT AATTTCCCACCTTATAAATCCA

BS-HBV-8 TGGGI I 1 1 I I GATAGTTAATGM AAAAACCAACCTCCCATACTAT

BS-HBV-9 AGAGTTATAGTATGGGAGGTT CCACTACATAACCTAAMATMCT

MSP

MSF-HBV-1M TCGGMTAGTAMTTTTGTTTC CCGCCTATMCACGMCA MSP-HBV-2U GTTTTGGMTAGTAMTTTTGTTTT CCCACCTATMCACAAACMA

RT*

RT-HBV-S TTGGGGTGGAGCCCTCAGGCT MTGGCACTAGTAMCTGAG RT-HBV-C GCCTTAGAGTCTCCTGAGCA GTCCMGGAATACTMC RT-HBV-X GCTAGGCTGTGCTGCCMCTG GGGGAGTCCGCGTAAAGAGAGG

'Pollicino T., Squadrito, G., Cererszia, G., Cacciola, I.. Raffa_r G., Craxi A., Farinati, F., Missale, G., Smedfle, A., Tiribelli, C. Villa, E. & Raϊmαndo, G. Hepatlis B vims maintains its pra-oncogenic properties in the case of occult HBV infection.Gastroenterology 126, 102-110 (2G04).

Table 4. Primers used for EBV assays. BS, bisulfite genomic sequencing; MSP, methylation specific PCR; RT, reverse transcription PCR; ChIP, chromatin immunoprecipitation.

Primers Forward 5' -> 3' Reverse 5' -> 3'

BS

EBV-A73 GGATATΠGTAGGATTAGGTTAGTTT TCCCATCCCAACMTATATAT

EBV-BALF1 AGGGTTGGTAAAGGTAGGTΓTT CTCCTCTCCTAMCCCMTTTA

EBV-BALF2 GTTTAGGGTGGTGTTGTGTTA ACACCCTACATMTACCCAMA

EBV-BALF3 GGATTTΠTGGAGGGAGTGT ATAACCTCCAACTCRACCMC

EBV-BALF4 GAGYGATTGGATAGAGATTT CRAATCATCTCTCATTTAAMA

EBV-BALF5 AGTAATATGTTTTTGGTGAGGG TCAMC I H I I CMAAMCCTT

EBV-BARFO AI I I I I IATTTGGAGGTGTAGG ACCMAMACATTCCTMCTTC

EBV-BarF1 GAGTTGGGTTTG.AAGATTATTATGT CMACAMACCCTCATMTCAC

EBV-BARF1 AGGGTTGGTAAAGGTAGGTTTT CTCCTCTCCTAMCCCMTTTA

EBV-BBLF1 AGGTTTGGAATTTTAAAGATGT CTTACCACCCCCACTATMC

EBV-BBLF2/BBLF3 GGGGTTTTAGGGAGTTΠTA TCAATTAMCATTCACCTTTTAAA

EBV-BBLF4 GGTAGATGTTGGTGGTTAGTAGGTT TAACCAAAATCCCTATCCTCCT

EBV-BBRF1 AGTAGAGl i l l ! I GGGTTTGGT CTTCCTACCCTΓTTCMCCTATA

EBV-BBRF2 TTTGATTGAATTTTGGGAA.A CTTACTTTTTACATMTCMACTC

EBV-BBRF3 GGGGTΓTTAGGGAGTTTTTA TCAATTAMCATTCACCTTTTMA

EBV-BcLPI GTGTATGTGATGATTTTGGGTA ATCAMTAATTAMCACRACCCT

EBV-BCRFI GAGAAl I H I l I GTGTTTGGTT TCCACACTCAAATACCAMTM

EBV-BcRFI TGTTTGNTGGTMTTTTTGATTA CCCTCTTACCTTATATCATMCC

EBV-BDLF1 GTTTGTAGTGATTGTTGGTATTG CCAMAATAAAATACMCCMTC

EBV-BDLF2 GGTTTGGTTTTGTTGGATAATT TMTACCCCCTTTATCTTMCCM

EBV-BDLF3.5 AGAGTTTTAGTTGGTTGATGG TTATCAAAAAAAAAACCCTACC

EBV-BDLF3 TTTAAGGATGTGTTGGTTTTTA TCCAMTAAMCTTATCTCACAM

EBV-BDLF4 AGAAAGTTGGTGGTTTGGTTA ACCTTMTCTMCATTCCACCC

EBV-BdRFI TTGMGTATTTTAGTGATTTGG CAAACTATTACTCCTTCTCCTAC

EBV-BFLF1 AGATGTAGTGATTTAGGAGGTTT AAATCCTCCATCTTTAMATTC

EBV-BFLF2 TTAGGTTAAGGGTTTGGTTGTA CCAATTCCTTMTMCATCCAC

EBV-BFRF11 AAGGATAAGGTGGGTTAGTAAGTTT ACCCMAATCACACAAMATM

EBV-BFRF1A AGATGTAGTGATTTAGGAGGTTT AMTCCTCCATCTTTAMATTC

EBV-BFRF2 GTTGGGATTTGGATAAI I M I F CAAAMCATCAAMACAAMCC

EBV-BFRF3 TAGAGGTAGAATTGTTATTTGGTGA CCCMCCACACTAACTCTCTAC

EBV-BGLF1 3GGAGTTTGAATAAGTTTAGQG AMCTAAAAACCACATCCACC

EBV-BGLF2 GG.AAAGTTGTTGGGTG.AAAGTTATTA CTCAACACCCAAAMCACTCCT

EBV-BΘLF3.5 TGGMGGGTGAGGATATAGTTT TAATCCCTCCCAMTCTATCAC

EBV-BGLF3 ATTTGGATTTGGATTGGGGTGT CCMTACCMTTCCAMCACTAC

EBV-BGLF4 GGGGTGTTTTAGAGTTTTTG ACAATCACCTTCCCCAAAAAAA

EBV-BGLF5 TTTTAAAGGTGGTGTTGTTGGAG TCATCTCCTCCATAAAATCCTC

EBV-BGRF1/BDRF1 ATTTGGATTTGGATTGGGGTGT CCAATACCMTTCCAAACACTAC

EBV-BHLF1 TATGGAGGGGATTTTTTTGAG CCCAAACCRMCCCTMT

EBV-BHRF1 TGGTTGTTTTTGGGATGTATTA TCTTATCAACCTCTTCAMCOC

EBV-BILF1 TTGTTATTATTATYGGGAMGG ATCATATTMCCACCAAMTCC

EBV-BILF2 GGTTMGGGTAGTTGTTTATTT ATCTCTTACCTCACATAAAAM EBV-BKRF2 AGGGTGATTGTGTGTAGTTTTG ACCCAAWACCCCATAWAAAM

EBV-BKRF3 TTTTAATATGGGGTAATTGGGT AAAAAACAMWTTCTCCTWCA

EBV-BKRF4 AAGMGTATTTGGTTTTGATTTTT TCAMCTCATCAATATCACTCA

EBV-BLLF1 GGTGGGTATTTTTTGTTTTTTT WAAAAMACCMAATCTTCACC

EBV-BLLF2 TAGGTGGTGAAAATATAATATAGGTG AAAWMCCWTCCACCTAWAM

EBV-BLLF3 AGGTTTTTATGGTGATGATTTA CMAAAMAMTACAMCMCC

EBV-BLRF1 GGGTTAGG I I I I I I GGTAAAGA CACCATAAMACCTATCCACAC

EBV-BLRF2 AGGTTTTTATGGTGATGATTTA CAMAAAAAMTACAMCMCC

EBV-BMRF1 GGTGGAGGTAGAGATTGTTTTT ATCACAMCMCMCAAAMCC

EBV-BMRF2 TATAAGTTTTTGAGTGTGGGG ACTCTCCCAAATAMAATCCTM

EBV-BNLF2a GAGGTAGGAATATTTGTTGTTGA TAAAAATCCAMCAAMCACAA

EBV-BNLF2& GGTATTTTGTGTTTTGTTTGGA MWCTTTAWAACACCCCCAA

EBV-BNRF1 TrTTTGTTTGTTTGGATTTTTA ATAWTACCTCTCCATCWACCC

EBV-BOLF1 GTAWAGTATYGGWGGGWAM RTAWCCTCMCCMCCCAC

EBV-BORF1 GTATTAGTATYGGTTGGGTTAAA RTAWCCTCMCCAACCCAC

EBV-BORF2 GTTTGGGGATATTTAGTGTTATT TMCCWMCCWTAACTCATCA

EBV-BPLF1 GTATTTGTAAGGTTTTGGTGGA WCCTACCRTTACCCAMA

EBV-BRLF1 GAAAGAGTGATATTTTGTTTTGTG ACCATACMTACAMTAAATWCTCTT

EBV-BRRF1 GTWGGATGAAGAATTTGTTAG AACTCCTAAMTCAAAAAACAAC

EBV-BRRF2 TGTTGTGTTTTGTATGGTATTTT CCTCCAMAAAAATMCATCW

EBV-BSLF1 AGTTTGGGAGATAGMGGTTAT AAAAAAAAAAACCTCCTCC

EBV-BSLF2/BMLF1 TATGATAGAGGGATATTTGGGT ATCWAACTAACCTGAACCCTATW

EBV-BSRF1 AGTTTGGGAGATAΘAAGGTTAT AAAAMAMAACCTCCTCC

EBV-BTRF1 GTTTGTTTGA I I I I 1 1 IAAGAGMG CATWTCCCCAMCMTC

EBV-BVLF1 GWAGTAGTTTGGTTAGGGATAG RAMCATCCRACAMTAWCCAC

EBV-BVRF1 WGTGGTAGTTWGTAGTΠTA CCAAAACTAAAMCTCTACCT

EBV-BVRF2 GGl 1 1 1 I GTGATAWAGGT CTCAAAI I i i I ACACCTATC

EBV-BWRF1 GGGGGGTAGAGATAGGTAGG AAAAAAAMCAAMCCMCCC

EBV-BXLF1 WGTGGTAGWWGTAGWWA CCMAACTAAAMCTCTACCT

EBV-BXLF2 GGGGGATATTATAGTWGGATG CTCTAAAAMTCCACAAACACAA

EBV-BXRF1 TAGTGGGTGGGTATWGGTAΘ TCCCMCTCWMAAAAAAACM

EBV-BZLF1 TGTGGATAGATGGAWT6AGW MATCCATCATCWCMCAAAA

EBV-BZLF2 GΘWAAGTTTAAGTAATTGTTG C.AMAATMMCMTMCAMT

EBV-Cp TAGAMWAGWGAGAGGWAGTGT ATAAMCCWAATCCCCCC

EBV-EBER1 GGGAAATGAGGGWAGTATAGGT TMAAMACMCCACAMCACC

EBV-EBER2 GWWGATWAAA I 1 1 1 IGTWTAGGA TACCCTTCTCCCAMAMAWA

EBV-Fp GGGTAYGAAATATGGTGTATGT MCACTCCCTCAATAATCACC

EBV-LF1 H t I ITGGTWATGWGt H I f G AATCCTCTMTCCCTATCTCCA

EBV-LF2 WWGGGGGWTATAWWAGG AAMAAATCTCRTACWCCACA

EBV-LF3 AGGATMTGGAATWTATGGAT ACMTACCWAAAATWACTCAAAAA

EVB-LMP-1 AAGGTWAGGGMGAGGAGAGG CAWCCCACMCWACCCCC

EBV-LMP-2A TGWATAGWGWGGWTGGAGA ACCAWTCTAAAAACCCCATM

EVB-LMP-2B AAGGTWAGGGMGAGGAGAGG CAWCCCACAACWACCCCC EBV-miR-BART-2 TTGTTATTATTATYGGQAAAGG ATCATATTAACCACCAAAATCC

EBV-miR-BHRF1 TGGTTGTTTTTGGGATGTATTA TCTTATCMCCTCTTCAAACCC

EBV-Qp TTAGTTTGATTMGGGTGAGGT CAACTACCCAAAATACCAAAAT

EBV-RPMS1 GGTTAAGGGTAGTTGTTTATTT ATCTCTTACCTCACATAAAAAA

EBV-Wp AGTTTTAGGGAGGGGGATTATT TTAAAATCCACTTACCTCTAACCC

EBV-Wp2 AATTTTTGGTAGTGATTTGGAT AAAATAAAAMCCCCCTCTTAC

MSP

MSP-EBV-Qp-m ACGTTTTATTTGGGAGGAGC CAAAACGTAATTAATCCCGC

MSP-EBV-Cp-U ATAATGTTTTATTTGGGAGGAGT ACAAAACATMTTMTCCCACCC

MSP-EBV-Wp-m I t I I I j AGTTTAGCGCGTTTAC AACΘCTCTAATACGACCAAA

MSP-EBV-Wp-U GGTTA I I I I I I I AGTTTAGTGTGTTTAT CCTAACACTCTAATACAACCAAA

MSP-EBV-Qp-m TTGTTTGGTCGTTAGATGGC TATATTACCCGCCATCCGAT

MSP-EBV-Qp-U ATGTTGTTTGGTTGTTAGATGGT TATATTACCCACCATCCAATAAC

RT*

RT-EBV-EBNA2 AGAGGAGGTGGTAAGCGGTTC TGACGGGTTTCCMGACTATCC RT-EBV-BRLF1 ACCATACAGGACACAACACCT ΘATGTTGAGCGTGGCCATTAG

RT-EBV-BHRF1 GTCAAΘΘTTTCGTCTGΪGTG TTCTCTTGCTGCTAGCTCCA RT-EBV-EBER1 AAAACATGCGGACCACCAGC AGGACCTACGCTGCCCTAGA

Tierney, RJ., Steven, N., Young, LS. & RicWnsαn AB . EpsteiivBanr virus latency in blood mononuclear cells: analysis of viral gene transcription during primary infection and in {he carrier state. J Wra/ 68, 7374-85 (1994).

*FeltDn-Edkins,ZA.., Kαndrashσv, A., Karaii, D., Fairtey, JA₁ Dawson, C.W., Arraod, J.R., Young, LS. & WhSe, RJ.

Epsfein-Barr virus induces cellular transcription factors to allow active expression of EBER genes by RhiA polymerase

III. J BM Ctiem.281, 33871-βO {2006)

^*Luo, B., Wang, Y., Wang, X.F., Liang, H., Yan, L.P., Huang, B.H. & Zhao, P. Expression of Epstein-Barr virus genes in

EBV-associated gastric carcinomas. World J Gastroenterol. 11 , 629-33 (2005).

"Oudejans, J.J., van den Bru!e, A.J., Jiwa, N.M., de Bruin, P.C., Ossenkoppele, G.J., van der VaIk, P., Walboomers,

J.M. & Meijer, CJ. BHRFI , the Epsiein-Barr virus (EBV) homologue of the BCL-2 protooπcogene, is transcribed in

EBV-associated B-ceϋ lymohomas and in reactive lymphocytes. Blood 86, 1893-902 (1995).

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. Moreover, all embodiments described herein are considered to be broadly applicable and combinable with any and all other consistent embodiments, as appropriate. Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties.

Claims

1. A method of diagnosing and/or monitoring the progression of or otherwise staging a disease caused by an infection by a double stranded DNA virus in a test sample obtained from a subject comprising determining the methylation status of a double stranded DNA viral genome wherein the presence of hypermethylation of the double stranded DNA viral genome indicates a positive diagnosis of the disease and/or an increased level of methylation of the double stranded DNA viral genome indicates the progression of the disease to a more advanced form.

2. The method of any preceding claim wherein the disease comprises cancer.

3. The method of any preceding claim which is utilised in order to stage the disease as one of virus carrier, pre-malignancy or primary tumour depending upon the methylation status.

4. The method of claim 3 wherein hypomethylation of the viral genome indicates a virus carrier and/or pre-malignancy and hypermethylation indicates a primary tumour.

5. The method of claim 3 wherein an intermediate methylation status between hypomethylation and hypermethylation indicates pre-malignancy.

6. The method of any preceding claim wherein the methylation status of all transcription start site regions of the viral genome are determined.

7 The method of any preceding claim wherein the methylation status of the entire viral genome is determined.

8. The method of any preceding claim which comprises determination of the methylation status of a plurality of CpG residues in the nucleotide sequences which can be sequenced using the primers comprising the nucleotide sequences set forth as SEQ ID NOs 53 to 107 for Human Papilloma Virus 18 (HPV 18), SEQ ID NOs 119 to 140 for Hepatitis B Virus (HBV) and/or SEQ ID Nos 151 to 330 for Epstein Barr Virus (EBV) and/or amplified using the primers comprising the nucleotide sequences as set forth as SEQ ID NOs 109 to 112 for HPV18, SEQ ID NOs 141 to 144 for HBV and/or SEQ ID NO'S 331 to 338 for EBV.

9. The method of claim 2 and claims 3 to 8 when dependent from claim 2 wherein the cancer is liver cancer.

10 The method of claim 9 wherein the virus is HBV.

11. The method of claim 9 or 10 wherein the pre-malignancy comprises chronic active hepatitis and/or hepatic cirrhosis.

12. The method of any of claims 9 to 11 wherein the primary tumour comprises hepatocarcinoma.

13. The method of any of claims 9 to 12 wherein the presence of hypermethylation of the HBV genome indicates a positive diagnosis of hepatocarcinoma.

14. The method of any of claims 9 to 12 wherein the presence of hypermethylation of the HBV genome indicates a positive diagnosis of hepatocarcinoma and the presence of less methylation/relative hypomethylation indicates the presence of chronic active hepatitis and/or hepatic cirrhosis.

15. The method of any of claims 10 to 14 wherein determining the methylation status of the HBV genome comprises determining the methylation status of the HBVgp2 and/or H BVg p4 gene.

16. The method of any of claims 1 to 8 wherein the virus is EBV.

17. The method of claim 16 wherein the pre-malignancy comprises a benign condition such as reactive lymphadenitis and infectious mononucleosis.

18. The method of claim 16 or 17 wherein the primary tumour comprises a lymphoid and/or epithelial malignancy.

19. The method of any of claims 16 to 18 wherein the presence of hypermethylation of the EBV genome (in particular around the transcription start sites) indicates a positive diagnosis of lymphoid and/or epithelial malignancy.

20. The method of any of claims 16 to 18 wherein the presence of hypermethylation of the EBV genome indicates a positive diagnosis of lymphoid and/or epithelial malignancy and the presence of less methylation/relative hypomethylation indicates the presence of a benign condition.

21. The method of any of claims 16 to 20 wherein determining the methylation status of the EBV genome comprises determining the methylation status of the Wp and/or Cp gene and/or the miR-BHFRF1-1 microRNA locus.

22. The method of any of claims 1 to 8 wherein the virus is a HPV.

23 The method of claim 22 wherein the HPV is HPV18.

24. The method of claim 22 or 23 wherein the cancer is cervical cancer.

25. The method of any of claims 22 to 24 wherein the pre-malignancy comprises cervical intraepithelial neoplasia.

26. The method of any of claims 22 to 25 wherein the primary tumour comprises squamous cell carcinoma.

27. The method of any of claims 22 to 26 wherein the presence of hypermethylation of the HPV genome indicates a positive diagnosis of squamous cell carcinoma.

28. The method of any of claims 22 to 26 wherein the presence of hypermethylation of the HPV genome indicates a positive diagnosis of squamous cell carcinoma and the presence of less methylation/relative hypomethylation indicates the presence of cervical intraepithelial neoplasia.

29. The method of any of claims 22 to 28 wherein determining the methylation status of the HPV genome comprises determining the methylation status of the L2 gene.

30. A method of treating an infection by a double stranded DNA virus and/or disease caused by an infection by a double stranded DNA virus in a subject comprising administering a therapeutically effective amount of a DNA demethylating agent to the subject in order to treat or prevent progression of the infection, wherein the subject has been selected for treatment on the basis of carrying a hypermethylated viral genome.

31. A method of treating an infection by a double stranded DNA virus and/or disease caused by an infection by a double stranded DNA virus in a subject comprising administering a therapeutically effective amount of a DNA methylating agent to the subject in order to treat or prevent progression of the infection, wherein the subject has been selected for treatment on the basis of carrying a hypomethylated viral genome.

32. The method of claim 30 or 31 wherein the virus is selected from HBV, EBV and HPV.

33. The method of claim 32 wherein the HPV is HPV16 or 18.

34. The method of claim 30 wherein the disease is a primary tumour and is selected from a hepatocarcinoma (HBV), lymphoma (EBV), epithelial malignancy (EBV) or an anogenital or head and neck tumour (HPV16 or 18).

35. A method for predicting the probability of successful treatment of an infection by a double stranded DNA virus and/or a disease caused by an infection by a double stranded DNA virus, with a DNA demethylating agent comprising, in a sample obtained from a subject, determining the methylation status of the viral genome, wherein methylation and in particular hypermethylation of the viral genome is indicative of a high or increased probability of successful treatment.

36. A method for predicting the probability of successful treatment of an infection by a double stranded DNA virus and/or a disease caused by an infection by a double stranded DNA virus, with a DNA methylating agent comprising, in a sample obtained from a subject, determining the methylation status of the viral genome, wherein hypomethylation of the viral genome is indicative of a high or increased probability of successful treatment.

37. A method of selecting a suitable treatment regimen for an infection by a double stranded DNA virus and/or a disease caused by an infection by a double stranded DNA virus comprising, in a sample obtained from a subject, determining the methylation status of the viral genome, wherein methylation and in particular hypermethylation of the viral genome indicates that an aggressive therapeutic treatment is suitable.

38. The method of claim 37 wherein the treatment comprises surgery, radiation and/or chemotherapy, including use of DNA demethylating agents.

39. A method of selecting a suitable treatment regimen for an infection by a double stranded DNA virus and/or a disease caused by an infection by a double stranded DNA virus comprising, in a sample obtained from a subject, determining the methylation status of the viral genome, wherein hypomethylation of the viral genome indicates that treatment using a DNA methylating agent may be suitable.

40. A pharmaceutical composition useful in treating an infection caused by a double stranded DNA virus incorporating a DNA demethylating agent or DNA methylating agent and a chemotherapeutic agent and/or an antiviral agent for simultaneous, sequential or separate administration.

41. A primer for use in bisulphite sequencing of a double stranded DNA viral genome selected from the primers comprising the nucleotide sequences set forth as SEQ ID NO: 53 to 108 for HPV18, SEQ ID NO: 119 to 140 for HBV and SEQ ID NO: 151 to 330 for EBV and functional derivatives thereof which retain functionality in bisulphite sequencing.

42. A primer for use in methylation specific PCR (MSP) to determine the methylation status of a double stranded DNA viral genome selected from the primers comprising the nucleotide sequences set forth as SEQ ID NO: 109 to 112 for the E2 gene of HPV18 SEQ ID NO: 141 to 144 for the HBVgp2 gene of HBV SEQ ID NO: 331 to 334 for the Cp gene of EBV

SEQ ID NO: 335 to 338 for the Wp gene of EBV and functional derivatives thereof which retain functionality in MSP.

43. A kit for use in the method of any one of claims 1 to 39 comprising a primer pair selected from the primers claimed in claim 41 or 42.

44. The kit of claim 43 which further comprises a reagent for converting unmethylated cytosine residues to a changed nucleotide which displays different base pairing properies to cytosine, such as uracil but which does not convert methylated cytosine residues.

45. The method of any of claims 1 to 39 which utilises one or more primers as claimed in claim 41 or 42 or a kit as claimed in claim 43 or 44.