Detection of viruses present in cells

Abstract

Detection of viruses present in cells The present invention provides 1. A method for identifying viral DNA integrated into the genome of at least one mammalian test cell, comprising: (a) determining the presence or absence of viral DNA in a DNA sample obtained from a mammalian test cell; (b) determining the methylation status of at least one CpG site in the viral DNA of a DNA sample obtained from a mammalian test cell; and the presence of viral DNA after step (a) and a hypermethylated state in step (b) indicates that the viral DNA has been integrated into the mammalian test cell and that the virus is inactive, respectively; the presence of viral DNA after step (a) and a baseline methylation state or a hypomethylated state in step (b) indicates that the viral DNA has integrated into the mammalian test cell and that the virus is active, respectively; the absence of viral DNA after step (a) indicates that the viral DNA has not integrated into the mammalian test cell; Methods performed on arrays Regarding.

Description

[Technical Field]

[0001] The present invention provides The present invention relates to a method for detecting the presence or absence of a virus and / or its promoter in a cell, as well as viral activity. In particular, the method quantitatively measures the DNA methylation level at CpG sites in viral DNA in a cell. [Background technology]

[0002] Viral infections cause various human diseases, including cancer. Upon infecting a host cell, a virus hijacks the cellular machinery to replicate and produce progeny virus particles. To facilitate long-term intracellular persistence, some viruses integrate their genomic DNA into the host genome. This results in various effects, including gene disruption, chromosomal instability, genetic mutations, and tumorigenesis. Such integrated viral DNA is called a provirus. Proviruses passively replicate along with the host genome and are passed down through generations. Integration of viral DNA into the host genome can lead to latent or productive infection.

[0003] In a productive infection, the provirus transcribes to generate new virus particles and mRNA, thereby spreading the infection to other cells. In a latent infection, the provirus is not transcribing, but becomes activated and starts transcribing in response to changes in the host's environmental conditions. This hijacks the host cell's protein synthesis machinery, allowing further virus replication and destruction of the host cell.

[0004] Therefore, it is necessary not only to check the presence or absence of viral DNA in host cells, but also to predict viral activity within host cells and determine the severity of the virus's presence. Furthermore, checking for viral DNA integration is essential as part of the quality control of cell line evaluation, vaccine development, patient samples, etc.

[0005] Viral vectors are widely used to express transgenes for gene therapy, cell line development, and therapeutic protein production. Viral vectors are engineered by replacing a set of viral genes responsible for viral replication and virulence with a transgene cassette. Gene delivery using viral vectors results in high and long-term transgene expression. Viral promoters and enhancers are the main DNA regulatory elements of viral vectors, determining the transgene expression level in cells. Viral vector integration sites are susceptible to transcriptional regulation by epigenetic mechanisms such as histone modifications and DNA methylation. For example, the DNA methylation status of viral vectors is an important factor for protein production and expression stability in recipient cells. Increased DNA methylation at CpG sites in viral promoters leads to transgene silencing at the transcriptional level. For example, variations in protein productivity in CHO cells have been linked to DNA methylation-mediated regulation of the cytomegalovirus major immediate-early enhancer (CMV) promoter and the simian vacuolating virus 40 (SV40) promoter, the promoters most frequently used for recombinant protein production in CHO cells. Summary of the Invention [Problem to be solved by the invention]

[0006] However, as mentioned above, the presence of viral DNA in a host cell does not indicate that the host cell is seriously compromised or infected with a virus. Current methods used to test for the presence or integration of viral DNA in a host cell lack the ability to determine the impact of the presence of the virus on the survival of the host cell. Therefore, there is a need in the art for a technique that can quantitatively and qualitatively determine not only the presence of a virus in a host cell, but also the impact of DNA integration on the host cell. [Means for solving the problem]

[0007] Description of the Invention The present invention solves the above-mentioned problems by providing a DNA-based method for determining the DNA methylation level of CpG sites in viral DNA and / or viral promoters, which can be used to predict not only the activity of a virus in a host cell but also the expression of a transgene in the cell. In particular, the method according to any of the aspects of the present invention may be performed on a DNA array. Thus, the method according to any of the aspects of the present invention can provide an efficient, inexpensive, and accurate means for determining not only the presence of a virus in a cell but also the activity of the virus in the cell.

[0008] According to one aspect of the present invention, 1. A method for identifying viral DNA integrated into the genome of at least one mammalian test cell, comprising: (a) determining the presence or absence of viral DNA in a DNA sample obtained from a mammalian test cell; (b) determining the methylation status of at least one CpG site in the viral DNA of a DNA sample obtained from a mammalian test cell; and the presence of viral DNA after step (a) and a hypermethylated state in step (b) indicates that the viral DNA has been integrated into the mammalian test cell and that the virus is inactive, respectively; the presence of viral DNA after step (a) and a baseline methylation state or a hypomethylated state in step (b) indicates that the viral DNA has integrated into the mammalian test cell and that the virus is active, respectively; the absence of viral DNA after step (a) indicates that the viral DNA has not integrated into the mammalian test cell; Methods performed on arrays is provided.

[0009] The term "viral DNA integration" is used interchangeably with "integrated viral DNA" or "genomically integrated viral DNA" and refers to the integration of the entire genome or a portion of the genome of at least one virus into a host cell, particularly an animal cell, more specifically a mammalian cell. Viral DNA integration is a non-instantaneous means of viral insertion because the viral DNA is genomically integrated into the host cell, i.e., a mammalian cell. Viral DNA integration is a potential diagnostic and prognostic marker. In particular, viral DNA integration is a unique enzymatic process in which double-stranded linear viral DNA is inserted into the host genome, catalyzed by a virus-encoded integrase. However, many of these viruses remain dormant within infected cells despite their DNA being integrated into the chromosomal DNA of the host cell. Some viruses present within host cells can cause malignant transformation of the cell and ultimately produce tumors. Viruses from the Polyomaviridae family, such as Simian Virus 40 (SV40), as well as viruses from the Retroviridae and Herpesviridae families, are examples of viruses that can be associated with malignant tumors (causing death or disease) within host cells. Viral DNA can be transformed from viral RNA.

[0010] As used herein, the term "mammalian cells" refers to cells derived from any animal in the order Mammalia, including cells from mice, rats, monkeys, guinea pigs, dogs, minipigs, humans, cattle, sheep, pigs, goats, horses, donkeys, mules, hamsters, and the like. Mammalian cells may also include established cell lines or immortalized cell lines. For example, a mammalian cell according to any embodiment of the present invention may be a Chinese hamster ovary (CHO) cell line, which refers to an immortalized CHO cell line derived from Cricetulus griseus. In particular, the CHO cell line may be selected from the group comprising only CHO-K1 (ATCC), CHO-DG44 (Thermo Fisher Scientific), CHO-DXB11 (ATCC), ExpiCHO-S™ cells (Thermo Fisher Scientific), FreeStyle™ CHO-S™ cells (Thermo Fisher Scientific), CHO1-15 [subscript 500] (ATCC), Agarabi CHO (ATCC), and CHOK1SV cells containing all variants (e.g. POTELLIGENT®, Lonza, Slough, UK), CHOK1SV GS-KO (glutamine synthetase knockout) cells containing all variants (e.g. XCEED™, Lonza, Slough, UK). Mammalian cells can be derived from baby hamster kidney fibroblasts (BHK (ATCC CCL-10)) or Vero cells (ATCC CCL-81). Examples of human cells include human embryonic kidney (HEK) cells, such as HEK293 (ATCC CRL-1573), HEK 293T (ATCC CRL-3216), HeLa cells (ATCC CCL-2), NS0 cells (ECACC 85110503), or Sp2 / 0 cells (ATCC CRL-1581). Mammalian cells according to any embodiment of the invention can include mammalian cell cultures, which can be either adherent or suspension cultures.

[0011] According to any one of the aspects of the present invention, the first step includes: (a) determining the presence or absence of viral DNA in a DNA sample obtained from a mammalian test cell; Includes:

[0012] The presence or absence of viral DNA in a DNA sample can be determined by measuring the viral titer in the DNA from a biological sample, i.e., the mammalian cells being tested. As used herein, "viral titer" refers to the amount of virus in a given volume, typically expressed as the number of viral particles, transducing units, or infectious particles per milliliter (mL).

[0013] The presence or absence of viral DNA in a DNA sample obtained from a mammalian test cell can be determined using a DNA array according to any of the embodiments of the present invention. A DNA array according to any of the embodiments of the present invention can include probes that bind to all regions specific to viral DNA (i.e., CpG sites and non-CpG sites). Binding of a probe specific for at least one viral CpG site or non-CpG site to the DNA test sample indicates the presence of viral DNA and / or the presence of viral DNA integrated into the DNA of the mammalian test cell. More specifically, an array according to any of the embodiments of the present invention includes probes specific for non-CpG sites that can detect the presence of viral DNA integrated into the genome of the DNA sample being tested.

[0014] The presence of specific CpG and non-CpG sites in step (a) indicates a virus strain or a mutant thereof. As used herein, the term "mutant" in reference to a virus refers to a virus strain that has changed into a different version from the original virus, possibly due to mutation. The term "mutant" refers to a form of a virus strain that deviates from what exists in nature or what is considered wild-type. A mutant may have a sequence that is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% similar to that of a wild-type strain.

[0015] The second step according to any of the aspects of the present invention comprises: (b) determining the methylation status of at least one CpG site in the viral DNA of a DNA sample obtained from a mammalian test cell; Includes:

[0016] If the presence and integration of viral DNA, or viral DNA integrated into mammalian DNA, is confirmed in step (a), the methylation status of at least one CpG of the integrated viral DNA may be determined from DNA of the mammalian test cell.

[0017] As used herein, "CpG site" or "methylation site" refers to a nucleotide in a nucleic acid (DNA or RNA) that is susceptible to methylation, either by naturally occurring events in vivo or by events induced in vitro to chemically methylate the nucleotide. Some of these sites may be hypermethylated or hypomethylated in cells.

[0018] As used herein, the term "methylated nucleic acid molecule" refers to a nucleic acid molecule in which one or more nucleotides are methylated.

[0019] As used herein, the term "methylated nucleotide" or "methylated nucleotide base" refers to the presence of a methyl group on a nucleotide base, which is not present in typical nucleotide bases that are commonly recognized. For example, cytosine, in its normal form, does not contain a methyl group in its pyrimidine ring, but 5-methylcytosine contains a methyl group at the 5-position of the pyrimidine ring. Therefore, cytosine in its normal form is not considered a methylated nucleotide, and 5-methylcytosine is considered a methylated nucleotide. As another example, thymine may contain a methyl group at the 5-position of its pyrimidine ring, but for purposes of this specification, thymine is not considered a methylated nucleotide when present in DNA. Typical nucleotide bases in DNA are thymine, adenine, cytosine, and guanine. Typical bases in RNA are uracil, adenine, cytosine, and guanine. Correspondingly, a "methylation site" refers to a site where methylation may occur in a nucleic acid region of a target gene. For example, the site containing CpG is a methylation site, and cytosine may or may not be methylated.In particular, the term "methylated nucleotide" refers to the nucleotide that has a methyl group attached to the nucleotide position that is susceptible to methylation.These methylated nucleotides usually exist in nature, and up to now, it is considered that the methylated cytosine that mainly exists in dinucleotide CpG, and also exists in CpNpG sequence and CpNpN sequence is the most common.In principle, other nucleotides that exist in nature can also be methylated, but they are not considered in all aspects of the present invention.

[0020] In this specification, "CpG island" refers to a segment of DNA sequence that has a functionally or structurally deviant CpG density.For example, Yamada et al. list the measurement criteria of CpG island as follows: length is at least 400 nucleotides, GC content is more than 50%, and OCF / ECF ratio is more than 0.6 (Yamada et al., 2004, Genome Research, 14, 247-266).In addition, some researchers define CpG island more loosely, as length is at least 200 nucleotides, GC content is more than 50%, and OCF / ECF ratio is more than 0.6 (Takai et al., 2002, Proc.Natl.Acad.Sci.USA, 99, 3740-3745). In the present invention, the terms "methylation profile," "methylation pattern," "methylation status," or "methylation state" are used to describe the state, status, or condition of methylation of a genomic sequence, and refer to the methylation characteristics of a DNA segment at a specific genomic locus. These characteristics include, but are not limited to, whether any cytosine (C) residues within the DNA sequence are methylated, the location of methylated C residues, the proportion of methylated C residues in a specific residue region, and inter-allele differences in methylation due to, for example, differences in the origin of the alleles.

[0021] The term "methylation state" refers to the state of a particular methylation site (i.e., methylated vs. unmethylated), meaning that the residue or methylation site is methylated or unmethylated. Based on the methylation state of one or more methylation sites, a methylation profile can be determined.

[0022] The term "methylation level" refers to the level of a particular methylation site and ranges from 0 (unmethylated) to 1 (fully methylated). Therefore, a methylation profile can be determined based on the methylation levels of one or more methylation sites. Thus, the term "methylation profile" or "methylation pattern" refers to the relative or absolute concentration of methylated or unmethylated Cs at a particular residue region in a biological sample. For example, if a cytosine (C) residue that is not normally methylated in a DNA sequence is more methylated in the sample, it may be referred to as "hypermethylated." On the other hand, if a cytosine (C) residue that is normally methylated in a DNA sequence is less methylated, it may be referred to as "hypomethylated." Similarly, if a cytosine (C) residue in a DNA sequence (e.g., a sample nucleic acid) is more methylated compared to another sequence (e.g., compared to a normal nucleic acid) from a different region or a different individual, the sequence is considered to be hypermethylated compared to the other sequence. Alternatively, if a cytosine (C) residue in a DNA sequence is hypomethylated compared to another sequence from a different region or a different individual, the sequence is considered to be hypomethylated relative to other sequences. These sequences are said to be "differentially methylated." For example, if the methylation status differs between inflamed and non-inflamed tissues, the sequence is considered to be "differentially methylated." Measuring the level of differential methylation can be performed by various methods known to those skilled in the art. One method includes, but is not limited to, determining the methylation level of each CpG site under investigation by bisulfite sequencing. "Bisulfite treatment" of genomic DNA, used interchangeably with the term "bisulfite conversion," refers to the treatment of genomic DNA with a deaminating agent such as bisulfite (a salt of hydrogen sulfite), which can be used to treat all DNA, regardless of whether it is methylated or not. In particular, the term "bisulfite," as used herein, encompasses all suitable types of bisulfite salts, such as sodium bisulfite, or other chemicals that can chemically convert cytosine (C) to uracil (U) without chemically modifying methylated cytosine, and thus can be used to differentially modify DNA sequences based on the methylation status of the DNA. See, e.g., U.S. Patent Application Publication No. 2010 / 0112595. As used herein, "reagents that 'differentially modify' methylated or unmethylated DNA include all reagents that modify methylated and / or unmethylated DNA in a process that results in products that are distinguishable from methylated and unmethylated DNA, thereby allowing the methylation status of DNA to be distinguished. Such processes include, but are not limited to, chemical reactions (e.g., bisulfite conversion of C to U) and enzymatic treatments (e.g., cleavage by methylation-dependent endonucleases). Thus, an enzyme that preferentially cleaves or digests methylated DNA is an enzyme that can cleave or digest DNA molecules with much higher efficiency when the DNA is methylated. On the other hand, an enzyme that preferentially cleaves or digests unmethylated DNA exhibits significantly higher efficiency when the DNA is unmethylated.

[0023] Therefore, before carrying out step (a) according to any aspect of the present invention, the genomic DNA contained in / obtained or extracted from the cells is first bisulfite treated.

[0024] Instead of bisulfite treatment, alternative methods available in the art may be used. Those skilled in the art will understand which other methods should be used. In one example, TET-assisted pyridine borane sequencing (TAPS) is used to detect 5mC and 5hmC (Yibin Liu et al., Nature Biotechnology, 37:424-429 (2019)).

[0025] As used herein, the term "genomic material" refers to nucleic acid molecules or fragments of the genome of an animal according to any aspect of the present invention. In particular, such nucleic acid molecules or fragments are DNA, RNA, or hybrids thereof, and most preferably are molecules of the DNA genome of a subject or group of subjects.

[0026] As used herein, the term "biological sample" may be selected from the group consisting of muscle, organ tissue, milk, blood, brain, sperm, and other tissues or samples that provide genomic DNA for use in the methods according to any aspect of the present invention. In particular, a biological sample may include any biological material obtained from a subject, including DNA, and may be liquid, solid, or both, such as tissue or bone, or body fluids such as blood, lymph, etc. In particular, a biological sample useful in the present invention may include biological cells or fragments thereof.

[0027] As used herein, the term "DNA sample" refers to DNA extracted from mammalian cells according to any embodiment of the present invention using methods known in the art. In particular, the DNA used for the test is purified high-molecular-weight genomic fragments ranging in size from 50 kb to 150 kb to detect the presence or absence of viral DNA integrated into the genome of the DNA under test. Further washing steps remove low-molecular-weight DNA fragments representing episomal DNA, virion DNA, and cccDNA.

[0028] The term "test" used herein in conjunction with the term mammal or mammalian cell refers to a mammal or cell to be introduced into an array according to any embodiment of the present invention and that serves as the basis for the analytical application of the present invention to determine whether viral DNA has integrated into the genome of the test cell and whether the virus is active. Accordingly, a "test profile" refers to a profile obtained or generated for or in connection with an (individual) subject or group of subjects to be tested according to the present invention. Similarly, the term "sample" used according to any embodiment of the present invention refers to an entity that can be the subject of the methods of the present invention. Conversely, the term "reference" or "control" refers to an entity, most likely predetermined, that is used for comparison with a test subject. In particular, DNA extracted from a test mammalian cell to be subjected to the methods of the present invention may be used as a sample to first determine the presence or absence of virus in the cell, and then determine the DNA methylation profile of the test cell to determine whether viral DNA has integrated into the mammalian cell and whether the virus is active in the test cell. An additional step of comparing the methylation profile of the test cell with a control may be included. "Control" refers to a mammalian cell, presumably of the same taxonomic group, species, or cell line, whose characteristics as described above are already known and whose methylation status is already known and used as a reference.

[0029] The DNA methylation profile of steps (a) and (b) according to any embodiment of the present invention is determined using an array. In particular, a bead array is used. The array according to any embodiment of the present invention is advantageous because it not only allows early detection of viral DNA integrated into host mammalian cells, but also helps determine the effectiveness of viral vectors in cells to be used for heterologous protein production. In particular, the method according to any embodiment of the present invention can be used to better understand the genomic stability of mammalian cell lines, allowing better control over manufacturing / process development / product development / scale-up / validation processes, thereby assisting in the selection of mammalian cell lines more suitable for industrial applications.

[0030] Arrays enable a high-throughput, robust method for determining semi-quantitative or quantitative DNA methylation information using small samples of extracted DNA of interest. These custom-designed arrays may use Illumina iScan and Infinium platform technology, or their equivalents, which allows for the placement of, for example, 100,000 beads covalently attached to DNA methylation probes on each chip. Each probe represents a single CpG methylation site at the end of the probe sequence. Before hybridization on the array chip, the DNA sample undergoes bisulfite conversion, amplification, fragmentation, precipitation, and resuspension steps. Once the DNA hybridizes to the beads at each CpG site on the chip, methylation changes at each site can be specifically detected through single-nucleotide extension. The array method is particularly advantageous because it is simple and the array results are accurate and highly reproducible.

[0031] Furthermore, compared to conventional sequencing, which can take weeks to generate data, array technology has a much faster turnaround time. The volume and complexity of the data generated is lower compared to sequencing, which also reduces the computational load. This reduces the computational time required to obtain interpretable results from experimental groups. Overall, microarray technology is approximately 10 times faster and 10 times cheaper than conventional sequencing, yet it can quantify the methylation levels of specific CpG sites.

[0032] As used herein, the term "array" refers to an intentionally created collection of probe molecules, which can be prepared synthetically or biosynthetically. The probe molecules within an array can be identical or different from one another. Arrays can take a variety of forms, including libraries of soluble molecules, libraries of compounds bound to resin beads, silica chips, or other solid supports.

[0033] In particular, arrays provide a convenient platform for simultaneously analyzing a large number of CpG sites, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 500, 1,000, 5,000, 10,000, 100,000, or more sites or loci. In particular, arrays contain multiple different probe molecules that can be attached to a substrate or spatially distinguished within the array. Examples of arrays that can be used in accordance with any embodiment of the present invention include slide arrays, silicon wafer arrays, liquid arrays, bead arrays, and the like. In one example, the array technology used in accordance with any embodiment of the present invention combines a compact array platform with a high degree of assay multiplexing and scalable automation for sample handling and data processing. In particular, arrays according to any embodiment of the present invention can be arrays of arrays (also called composite arrays) having multiple individual arrays configured to process multiple samples simultaneously. Examples of composite arrays and the technology behind them are disclosed at least in U.S. Patent No. 6,429,027 and U.S. Patent Application Publication No. 2002 / 0102578. The substrate of a composite array may include multiple individual array locations. Each array location has multiple probes, and each array location is physically separated from other assay locations on the same substrate, thereby preventing fluid contacting one array location from contacting other array locations. Each array location may have multiple different probe molecules attached directly to the substrate or attached to the substrate via rigid particles within wells (also referred to herein as intra-well beads).

[0034] In one example, the array substrate can be a fiber optic bundle or array bundle, such as those described in U.S. Patent Nos. 6,023,540, 6,200,737, and / or 6,327,410. The fiber optic bundle or array bundle can have probes attached to it either directly to the fibers or via beads. One of skill in the art would readily be able to determine the most suitable substrate for an array according to any embodiment of the present invention. WO 2004 / 110246 further discloses other substrates that can be used for arrays according to any embodiment of the present invention, as well as methods for attaching beads to the substrate.

[0035] In one example, the surface of the substrate can be physically modified to allow probe attachment or to form array locations.For example, the surface of the substrate can be modified to include chemically modified sites that are useful for covalently or non-covalently attaching probe molecules or particles with probe molecules attached thereto.Probes can be attached using various methods known in the art, such as inkjet printing, spotting, photolithography synthesis, or mask-based printing.WO 2004 / 110246 discloses these techniques in more detail.

[0036] In one example, an array according to any embodiment of the invention can be a bead array, where the beads are attached to a solid support, such as those commercially available from Illumina, Inc. (San Diego, Calif.). Useful bead arrays according to any embodiment of the invention can also be in a fluidic format, such as the fluidic system of a flow cytometer or similar device. Commercially available fluidic formats for identifying beads include those used in Luminex's XMAP™ technology and Lynx Therapeutics' MPSS™ method.

[0037] As used herein, the terms "solid support," "support," and "substrate" are used interchangeably and refer to a material or group of materials having a rigid or semi-rigid surface or surfaces. In many instances, at least one surface of the solid support is substantially flat, although in some instances it may be desirable to physically separate synthesis regions for different compounds, e.g., by wells, raised areas, pins, etched trenches, etc.

[0038] Arrays or microarrays according to any embodiment of the invention may be very high density arrays, e.g., about 10,000,000 probes / cm 2 ~approximately 2,000,000,000 probes / cm 2 , or approximately 100,000,000 probes / cm 2 ~approximately 1,000,000,000 probes / cm 2 In any aspect of the invention, high density arrays are particularly useful for including a large number of CpG sites on the array.

[0039] Arrays according to any aspect of the invention can be used to analyze or assess multiple loci simultaneously or sequentially, as desired. In one example, multiple different probe molecules can be attached to a substrate or spatially separated within the array. Each probe is typically specific to a particular locus and can be used to distinguish the methylation status of that locus.

[0040] The term "probe molecule" or "probe", as used interchangeably herein, refers to a surface-immobilized molecule that can be recognized by a specific target. The probes used in the array can be specific to the methylated alleles of CpG sites, the unmethylated alleles of CpG sites, or both, or can be specific to the methylated alleles of non-CpG sites, the unmethylated alleles of non-CpG sites, or both.

[0041] The term "target" as used herein refers to a molecule that has affinity for a particular probe molecule. Targets may be naturally occurring or artificial molecules. They may be used in their unmodified state or as aggregates. Targets may be covalently or non-covalently bound to a binding member, either directly or via a specific binding substance. Examples of targets that can be used according to any embodiment of the present invention include methylated and unmethylated CpG sites. Targets are sometimes referred to in the art as antiprobes. Although the term "target" is used herein, it does not imply any difference in meaning.

[0042] As used herein, the term "complementary" refers to hybridization or base pairing between nucleotides or nucleic acids, such as between the two strands of a double-stranded DNA molecule or between an oligonucleotide primer and a primer-binding site on a single-stranded nucleic acid to be sequenced or amplified. Complementary nucleotides are typically A and T (or A and U) or C and G. Two single-stranded RNA or DNA molecules are said to be complementary when the nucleotides of one strand are optimally aligned and compared, and pair with at least about 80%, usually at least about 90%-95%, and more preferably about 98%-100% of the nucleotides of the other strand, with appropriate nucleotide insertions or deletions. Fully complementary refers to 100% complementarity throughout the entire length of the sequence. For example, a 25-base probe being fully complementary to a target means that all 25 bases of the probe are complementary to the contiguous 25-base sequence of the target, with no mismatches between the probe and the target throughout the entire length of the probe.

[0043] The presence of viral DNA after step (a) according to any embodiment of the present invention and the hypermethylated state in step (b) indicate that the viral DNA has integrated into the mammalian test cell and that the virus is inactive, respectively. Additionally, the presence of viral DNA after step (a) and a baseline methylation state or a hypomethylated state in step (b) indicate that the viral DNA has integrated into the mammalian test cells and that the virus is active, respectively. Additionally, the absence of viral DNA after step (a) indicates that the viral DNA has not integrated into the mammalian test cells.

[0044] The term "hypermethylation" refers to an average methylation state corresponding to an increased abundance of 5-mCyt found at one or more CpG dinucleotides within a DNA sequence of a test DNA sample compared to the amount of 5-mCyt found at the corresponding CpG dinucleotides in a normal control DNA sample.

[0045] The term "hypomethylation" refers to an average methylation state corresponding to a decreased abundance of 5-mCyt found at one or more CpG dinucleotides within a DNA sequence of a test DNA sample compared to the amount of 5-mCyt found at the corresponding CpG dinucleotides in a normal control DNA sample.

[0046] A virus is considered active when it is not only integrated into a host mammalian cell but also capable of replication and protein production. Some viruses integrate their genomic DNA into the host genome to promote long-term viral persistence within the cell. This results in various effects, such as gene disruption, chromosomal instability, genetic mutations, and tumorigenesis. Such viral DNA integrated into the host genome is called a provirus. Proviruses passively replicate along with the host genome and are passed down through generations. The integrated viral DNA can cause either latent or productive infection. In productive infection, proviral transcription generates new virus particles, which then produce mRNA and infect other cells. As used herein, "latent infection" refers to a state in which the provirus is not transcribing but is activated and initiates transcription in response to changes in the host's environmental conditions. This results in the host cell's protein synthesis machinery being hijacked, leading to the production of additional virus and the destruction of the host cell. Therefore, detecting viral DNA integration or integrated viral DNA is essential as part of quality control in cell line evaluation, vaccine development, patient samples, and so on. The opposite is true for inactive viruses.

[0047] Viral promoters are rich in CpG sites and prone to DNA methylation, which represses protein expression, so studying the DNA methylation of viruses and their promoters can accurately determine whether the virus is integrated and, if so, its activity within the DNA.

[0048] As used herein, the terms "promoter" or "gene promoter" are used interchangeably with the terms "regulatory region" or "regulatory sequence" and refer to the respective contiguous gene DNA sequence, or a contiguous portion thereof, extending from 1.5 kb upstream to 1.5 kb downstream from the transcription start site (TSS). In particular, "regulatory region" refers to the respective contiguous gene DNA sequence extending from 1.5 kb upstream to 0.5 kb downstream from the TSS. In some instances, "regulatory region" refers to the respective contiguous gene DNA sequence extending from 1.5 kb upstream to the downstream end of a CpG island that overlaps with the region 1.5 kb upstream to 1.5 kb downstream from the TSS (and therefore, in such cases, may extend further than 1.5 kb downstream), and a contiguous portion thereof. Alterations in DNA methylation on gene promoters responsible for protein glycosylation can lead to improved protein quality. Protein glycosylation is an important quality characteristic that regulates the efficacy, stability, and half-life of therapeutic proteins. Due to regulatory concerns, obtaining a consistent glycoform profile is desirable in protein production. Therefore, DNA methylation can serve as a tool to determine viral DNA integrated into mammalian host cells.

[0049] According to another aspect of the present invention, 1. A method for identifying viral DNA integrated into the genome of at least one mammalian test cell, comprising: (a) determining the presence or absence of one or more preselected CpG and / or non-CpG sites of viral DNA in a DNA sample obtained from a mammalian test cell; and / or (b) determining a test methylation profile of preselected CpG and / or non-CpG sites within a DNA sample obtained from the mammalian test cell; and the presence of one or more preselected CpG sites and / or non-CpG sites after step (a) and a hypermethylated state in step (b), respectively, indicates that viral DNA has integrated into the mammalian test cell but that the virus is inactive; the presence of one or more preselected CpG sites and / or non-CpG sites after step (a) and a baseline methylation state or a hypomethylated state indicate that the viral DNA has integrated into the mammalian test cell and that the virus is active, respectively; the absence of one or more preselected CpG sites and / or non-CpG sites after step (a) indicates that the viral DNA has not integrated into the mammalian test cell; Methods performed on DNA arrays is provided.

[0050] Herein, the term "preselected sites" is used interchangeably with "preselected CpG and non-CpG sites" and refers to sites selected from genes or regions considered during training of the method that exhibit the highest degree of methylation variation, meet certain quality criteria, such as a minimum sequence coverage of 5x or greater, and have five or more eligible CpG sites. Furthermore, genes with average methylation levels below 0.1 or above 0.9 can be excluded due to their limited dynamic range. A "reference methylation profile" can be defined based on multiple training samples using multivariate statistical methods such as principal component analysis and multidimensional scaling.

[0051] According to any one of the aspects of the present invention, there is an additional step of: (c) comparing the methylation status of the CpG sites obtained in step (b) with the methylation status of control mammalian cells into which the viral DNA has been integrated. may exist. Step (c) is optional because step (b) of measuring the methylation status is sufficient to determine hypomethylation or hypermethylation of CpG sites, providing information about virus and protein production without further comparison in step (c).

[0052] According to a further aspect of the present invention, 1. A DNA array-based method for identifying viral DNA integrated into at least one mammalian test cell, comprising: (a) determining the presence of at least one viral CpG site and / or non-CpG site in a DNA sample obtained from a mammalian test cell; (b) determining the methylation status of a plurality of CpG and / or non-CpG sites within a DNA sample obtained from the mammalian test cell; (c) comparing the methylation status of the CpG and / or non-CpG sites obtained from step (b) with the methylation status of mammalian control cells into which the viral DNA has been integrated; and the presence of CpG and / or non-CpG sites after step (a) and a hypermethylated state in step (c) indicate that the viral DNA has integrated into the mammalian test cell but that the virus is inactive; the presence of CpG sites and / or non-CpG sites and a baseline methylation state or a hypomethylated state after step (a) indicate that the viral DNA has integrated into the mammalian test cell and that the virus is active, respectively; the absence of CpG and / or non-CpG sites after step (a) indicates that viral DNA has not integrated into the mammalian test cell. is provided.

[0053] A method according to any aspect of the present invention comprises: (ii) contacting a DNA sample from a test cell with a DNA array Includes:

[0054] A method according to any aspect of the present invention comprises: (i) a step of bisulfite-modifying the DNA sample prior to step (a) Further includes:

[0055] As used herein, the term "contacting" refers to directly contacting a genomic material sample with a DNA array, e.g., a genomic material sample is DNA extracted from a biological sample derived from a test mammal, and directly contacting the DNA array with the probes.

[0056] In a method according to any of the aspects of the present invention, the mammalian cells are a CHO cell line and, as an additional step, (c) determining the methylation status of CpG and / or non-CpG sites within the viral DNA from step (b); (i) the methylation status of a first CHO control cell, wherein the viral DNA has been integrated into the genome of the control cell and the first CHO control cell has optimal heterologous protein productivity; and / or (ii) the methylation status of a second CHO control cell, in which viral DNA has been integrated into the genome of the control cell and which does not have optimal heterologous protein productivity; and comparing the a significant similarity of the test methylation state of the CpG site with the first CHO control cell indicates that the test CHO cell has optimal heterologous protein productivity, or a significant similarity of the test methylation state of the CpG site with the second CHO control cell indicates that the test CHO cell does not have optimal heterologous protein productivity; A difference between the test methylation state of the CpG site and the first CHO control cell indicates that the test CHO cell does not have optimal heterologous protein productivity, or a difference between the test methylation state of the CpG site and the second CHO control cell indicates that the test CHO cell has optimal heterologous protein productivity.

[0057] As used herein, the term "heterologous protein production" refers to the production of a protein that is not endogenous to a cell. This refers to the expression of a gene or part of a gene, particularly the expression of a transgene in a host CHO cell that does not naturally express the transgene. Assays commonly used to quantify heterologous protein production include enzyme-linked immunosorbent assay (ELISA), chromatography, and bioprocess analyzers. As used herein, the term "host cell" refers to a cell line for expressing a heterologous protein. For example, CHO cells are a primary host for the production of various therapeutic proteins.

[0058] As used herein, the term "therapeutic protein" refers to a genetically engineered version of a naturally occurring human protein. Examples of therapeutic proteins include therapeutic antibodies, anticoagulants, blood factors, bone morphogenetic proteins, artificial protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, etc.

[0059] As used herein, the term "optimal heterologous protein production" refers to CHO cells capable of high-level protein production, particularly in industrial or large-scale production of recombinant proteins. The protein in this case is typically a functional protein that is not naturally present in wild-type CHO cells. In particular, for optimal heterologous protein production, the CHO cell line minimizes the metabolic burden and toxic effects on the cells. More specifically, "optimal heterologous protein production" refers to high-level protein production in which the CHO cell line not only produces a high yield of the target protein, but also maintains constant protein production throughout the production period (i.e., a long-term culture period), resulting in a constant and maintained quality of the produced protein. In particular, for a CHO cell according to any embodiment of the present invention to be capable of "optimal heterologous protein production," the cell must exhibit at least one or more of the following target phenotypes: phenotypic homogeneity, protein productivity, and protein quality. More specifically, for "optimal heterologous protein production," CHO cells may have phenotypic homogeneity and protein productivity, or phenotypic homogeneity and protein quality, or protein productivity and protein quality, or phenotypic homogeneity, protein productivity and protein quality, and good protein titer. Optimal heterologous protein productivity can only be achieved if the cells (i.e., CHO cells) are stable and have high cell viability.

[0060] As used herein, the term "phenotypic homogeneity" refers to a state in which all cells in a cell population exhibit the same phenotype under certain conditions.

[0061] As used herein, the term "protein productivity" refers to a measure of protein production per viable cell at a single titer point. It is calculated by dividing the titer (mg) by the viable cell density (VCD or cells / mL), with the final measurement expressed as protein per cell (mg / cell).

[0062] The term "protein quality" refers to post-translational modifications of proteins that determine their efficacy and function. These modifications generally include phosphorylation, glycosylation, ubiquitination, methylation, acetylation, and protein folding. For example, protein glycosylation is an important quality characteristic that regulates the efficacy, stability, and half-life of therapeutic proteins. Protein quality can be determined using immunoprecipitation techniques, biochemical assays, mass spectrometry (MS), and other methods.

[0063] As used herein, "cell viability" refers to the ability of cells to survive and undergo cell proliferation. Cell viability is a measure of the proportion of living cells in a cell population. Cell proliferation refers to an increase in cell number due to cell division. Assays commonly used to test cell viability include the BrdU cell proliferation assay, the MTT cell proliferation assay, the trypan blue cell counting method, and the ATP cell viability assay.

[0064] As used herein, "cell exhaustion" refers to a state in which cells have lost the ability to perform metabolic activities such as heterologous protein production. Cell exhaustion can be determined by metabolite detection assays.

[0065] As used herein, "significantly similar," particularly in the context of comparing methylation profiles (e.g., comparing a test profile (derived from a test subject) to a reference profile), refers to a similarity observed by statistical means (i.e., using bioinformatics) and / or visual observation. Significant similarity is observed, for example, when a test profile overlaps with a reference profile defined by multiple training samples through multivariate statistical methods such as principal component analysis or multidimensional scaling. In particular, a test profile is significantly similar to a given reference profile if more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, or more than 95% of its methylation pattern / profile overlaps with the methylation pattern / profile of the reference profile. The significance of the similarity decreases when the test profile is similar to multiple reference profiles, such as two, three, or all of the reference profiles.

[0066] As used herein, the term "predetermined reference profile" refers to a typical or standard methylation profile of the genomic material of a CHO cell line, having particular characteristics that depend on the context in which the term is used. In one example, in a method for determining a CHO cell line that exhibits at least one phenotypic parameter according to any embodiment of the present invention that confers optimal heterologous protein production potential on the cell line, the term "predetermined reference profile" refers to a typical or standard methylation profile of the genomic material of a CHO cell line that exhibits one or more phenotypic parameters selected from the group consisting of optimal glucose consumption, optimal growth rate, optimal lactate production, and optimal ammonia accumulation only. The predetermined reference profile can be obtained from one or more reference CHO cell lines that each express one or more phenotypic parameters.

[0067] The method according to this aspect of the present invention may use the methylation profile of a CHO cell line with optimal heterologous protein production potential, because this cell line may exhibit cell viability, adaptability, low cell exhaustion, and favorable metabolic indicators. In particular, the method according to this aspect of the present invention provides a prognostic methylation profile of an ideal parent cell line before introduction of a transgene. This methylation profile may be used as a control.

[0068] Cells according to any aspect of the present invention may be obtained from a biological sample selected from the group including saliva, blood, brain, sperm, and other tissues or samples that provide genomic DNA only.

[0069] The virus according to any aspect of the invention may belong to the Herpesviridae and / or Polyomaviridae families, in particular the Herpesviridae virus is selected from the group consisting of only Simplexvirus, Mardivirus, Varicellovirus, Lymphocryptovirus, Cytomegalovirus, Muromegalovirus, Rosellovirus, Lymphocryptovirus and Rhadinovirus, and the Polyomaviridae virus is selected from the group consisting of only Alphapolyomavirus, Betapolyomavirus, Deltapolyomavirus, Epsilonpolyomavirus, Gammapolyomavirus and Zetapolyomavirus. The virus belonging to the Herpesviridae family is selected from the group consisting of Gallid alphaherpesvirus 2, human herpesvirus 1, human herpesvirus 2, bovine herpesvirus 1, bovine herpesvirus 5, human herpesvirus 3, porcine herpesvirus 1, human herpesvirus 5, murine herpesvirus 1, human herpesvirus 6A, human herpesvirus 6B, human herpesvirus 7, human herpesvirus 4, Kaposi's sarcoma-associated herpesvirus, and murine herpesvirus 4 only. The virus belonging to the Polyomaviridae family is selected from the group consisting only of human polyomavirus 5, human polyomavirus 8, human polyomavirus 9, human polyomavirus 12, human polyomavirus 13, human polyomavirus 1, human polyomavirus 2, human polyomavirus 3, human polyomavirus 4, human polyomavirus 6, human polyomavirus 7, human polyomavirus 10, human polyomavirus 11, and human polyomavirus 14. In particular, the virus may be human herpesvirus 5 (also known as human cytomegalovirus), murine herpesvirus 1 (also known as murine cytomegalovirus), or simian virus 40.

[0070] According to a further aspect of the present invention, (a) The presence of integrated viral DNA in mammalian cells; (b) the methylation status of at least one CpG site in the virus; The present invention provides the use of DNA arrays to determine the viral status of a virus. The mammalian cells may be an immortalized cell line of mammalian origin. According to yet another aspect of the present invention, (a) The presence of integrated viral DNA in mammalian cells; (b) the methylation status of at least one CpG site in the virus; and thereby provide a DNA array for determining the viral status. [Example]

[0071] The above describes preferred embodiments, and those skilled in the art will recognize that changes or modifications may be made in design, configuration, or operation without departing from the scope of the claims. For example, these modifications are intended to be included within the scope of the claims.

[0072] Experimental Example 1 Detection of viral promoters in CHO cells CHO cells contain the cytomegalovirus major immediate-early enhancer (CMV) promoter and the simian vacuolating virus 40 (SV40) promoter, which are the most frequently used promoters for recombinant protein production in CHO cells. CHO DG44 (Thermo Fisher Scientific) is a parent CHO cell line that does not contain any viral promoters integrated into its genome. The Humira 431 (A*STAR Bioprocessing Technology Institute) cell line is a genetically engineered cell line derived from CHO DG44 cells that expresses biosimilar Humira from a modified CMV promoter.

[0073] DNA extraction RNAse treatment was performed according to the manufacturer's instructions, and DNA was extracted using the PureLink Genomic DNA Isolation Minikit (Invitrogen). DNA quantity was measured using the PicoGreen assay, and DNA quality was assessed using a NanoDrop (Thermo Scientific) to ensure an A260 / A280 ratio of 1.8 or less. A small sample was then analyzed by automated electrophoresis on a TapeStation (Agilent) to confirm that each sample contained high-molecular-weight DNA.

[0074] Bisulfite treatment and BeadChip analysis Genomic DNA samples were bisulfite converted using the EZ DNA Methylation-Gold™ kit (Zymo Research), and methylation levels were then quantified using a customized Methylation BeadChip kit (Illumina), which allows for the quantitative analysis of over 50,000 methylation sites across the genome at single-nucleotide resolution.

[0075] Data Processing: Customized chip array data processing was performed in R version 4.1.2 using Sesame version 1.14.2. DNA methylation levels at each site were calculated as methylation beta values. Beta values ​​were defined as methylation signal / (methylation signal + unmethylation signal) and calculated using the getBetas function. Normalized beta values ​​were generated and quality control was performed using the SeSAMe pipeline (Zhou et al., 2018). Low-intensity detection (based on p-values) was called and determined using pOOBAH. Background subtraction was performed based on normal-exponential deconvolution using the out-of-band probe noob (Triche et al., 2013), with additional bleed-through subtraction performed as needed.

[0076] The proportion of viral probes that met the p-value from the data indicated the presence and type of virus integrated into the CHO genome.

[0077] result Three CHO DG44 samples and three CHO Humira431 samples were analyzed by TALOS. The CHO Humira431 cell line contained the CMV vector, but the CHO DG44 did not. The results indicated that the CMV-targeted probes could detect only the CHO Humira431 cell line. A total of 538 CMV probes were available in TALOS1. Table 1 shows the number of viral probes that were successfully detected. Table 1: Number of CMV probes successfully detected in three CHO DG44 samples and three CHO Humira431 samples

[0078] [Table 1]

[0079] Experimental Example 2 Quantification of viral promoter methylation levels within the CHO genome Variation in protein production in CHO cells is associated with DNA methylation-mediated regulation of the cytomegalovirus major immediate-early enhancer (CMV) promoter and the simian vacuolating virus 40 (SV40) promoter, which are the promoters most frequently used for recombinant protein production in CHO cells. Increased DNA methylation of CpG sites in viral promoters results in transcriptional repression of transgenes.

[0080] DNA extraction, bisulfite treatment, BeadChip analysis, quality control, data processing, and differential methylation analysis were performed as outlined in Example 1.

[0081] Analysis of the DNA methylation level of CpG sites in the viral promoter can predict transgene expression in CHO cells.

[0082] Three CHO DG44 samples and three CHO Humira431 samples were analyzed by TALOS. The CHO Humira431 cell line contained the CMV vector, whereas the CHO DG44 cell line did not. Table 2 lists all CMV-specific probes available for CMV detection and the beta values ​​for the CHO Humira431 samples.

[0083] Table 2: Beta values ​​of the CMV probe in three CHO Humira 431 samples [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6]

Claims

1. 1. A method for identifying viral DNA integrated into the genome of at least one mammalian test cell, comprising: (a) determining the presence or absence of viral DNA in a DNA sample obtained from said mammalian test cells; (b) determining the methylation status of at least one CpG site in the viral DNA of the DNA sample obtained from the mammalian test cell; and the presence of the viral DNA after step (a) and its hypermethylated state in step (b) indicates that the viral DNA has integrated into the mammalian test cell and that the virus is inactive, respectively; the presence of the viral DNA after step (a) and a baseline methylation state or a hypomethylated state in step (b) indicates that the viral DNA has integrated into the mammalian test cell and that the virus is active, respectively; the absence of said viral DNA after step (a) indicates that said viral DNA has not integrated into said mammalian test cell; The method is performed on an array.

2. The method of claim 1 , wherein the presence of specific CpG and non-CpG sites in step (a) is indicative of the virus strain or a mutant thereof.

3. 1. A method for identifying viral DNA integrated into the genome of at least one mammalian test cell, comprising: (a) determining the presence or absence of one or more preselected CpG and / or non-CpG sites of said viral DNA in a DNA sample obtained from said mammalian test cell; and / or (b) determining a test methylation profile of the preselected CpG and / or non-CpG sites within the DNA sample obtained from the mammalian test cell; and the presence of one or more preselected CpG and / or non-CpG sites after step (a) and a hypermethylated state in step (b), respectively, indicates that the viral DNA has integrated into the mammalian test cell but that the virus is inactive; the presence of one or more preselected CpG and / or non-CpG sites and a baseline methylation state or a hypomethylated state after step (a) indicates that the viral DNA has integrated into the mammalian test cell and that the virus is active, respectively; the absence of one or more preselected CpG and / or non-CpG sites after step (a) indicates that the viral DNA has not integrated into the mammalian test cell; The method is carried out on a DNA array.

4. The method according to any one of claims 1 to 3, wherein the mammal is a mouse, rat, guinea pig, dog, minipig, human, cow, sheep, pig, goat, horse, donkey, mule, or hamster.

5. The method according to any one of claims 1 to 4, wherein the mammalian cell is an immortalized cell line derived from the mammal.

6. 6. The method of claim 5, wherein the immortalized cell line is selected from the group consisting of only CHO, BHK, Vero, HEK293, HEK293T, HeLa cells, NS0 cells, Sp2 / 0 cells, and derivatives thereof.

7. The method according to any one of claims 1 to 6, wherein the mammalian cells are CHO cell lines.

8. As an additional process, (c) determining the methylation status of the CpG sites in the viral DNA from step (b) by: (i) the methylation status of a first CHO control cell, wherein the viral DNA has been integrated into the genome of the control and the first CHO control cell has optimal heterologous protein productivity; (ii) the methylation status of a second CHO control cell, wherein the viral DNA has been integrated into the genome of the control and the second CHO control cell does not have optimal heterologous protein productivity. and comparing the a significant similarity between the test methylation status of the CpG site and the first CHO control cell indicates that the test CHO cell has optimal heterologous protein productivity, or a significant similarity between the test methylation status of the CpG site and the second CHO control cell indicates that the test CHO cell does not have optimal heterologous protein productivity; 8. The method of claim 7, wherein a difference between the test methylation state of the CpG site and the first CHO control cell indicates that the test CHO cell does not have optimal heterologous protein productivity, or a difference between the test methylation state of the CpG site and the second CHO control cell indicates that the test CHO cell has optimal heterologous protein productivity.

9. The method according to any one of claims 1 to 8, wherein the integrated viral DNA is derived from at least one virus of the Herpesviridae and / or Polyomaviridae genus.

10. The virus of the Herpesviridae genus is selected from the group consisting of simplex virus, mardi virus, varicellovirus, lymphocryptovirus, cytomegalovirus, muromegalovirus, roserovirus, lymphocryptovirus and rhadinovirus, and 10. The method of claim 9, wherein the Polyomaviridae virus is selected from the group consisting of only alpha polyomaviruses, beta polyomaviruses, delta polyomaviruses, epsilon polyomaviruses, gamma polyomaviruses and zeta polyomaviruses.

11. the virus of the Herpesviridae genus is selected from the group consisting of Gallid alphaherpesvirus, Human herpesvirus 1, Human herpesvirus 2, Bovine herpesvirus 1, Bovine herpesvirus 5, Human herpesvirus 3, Porcine herpesvirus 1, Human herpesvirus 5, Murine herpesvirus 1, Human herpesvirus 6A, Human herpesvirus 6B, Human herpesvirus 7, Human herpesvirus 4, Kaposi's sarcoma-associated herpesvirus, Murine herpesvirus 4 only; and 11. The method of claim 9 or claim 10, wherein the Polyomaviridae virus is selected from the group consisting of only human polyomavirus 5, human polyomavirus 8, human polyomavirus 9, human polyomavirus 12, human polyomavirus 13, human polyomavirus 1, human polyomavirus 2, human polyomavirus 3, human polyomavirus 4, human polyomavirus 6, human polyomavirus 7, human polyomavirus 10, human polyomavirus 11, and human polyomavirus 14.

12. 12. The method of any one of claims 1 to 11, wherein the virus is human herpesvirus 5 (also known as human cytomegalovirus), murine herpesvirus 1 (also known as murine cytomegalovirus), or simian virus 40.

13. (a) the presence of integrated viral DNA in mammalian cells; (b) the methylation status of at least one CpG site of the virus; and 13. Use of a DNA bead array in the method of any one of claims 1 to 12 for determining the viral status of a virus.

14. The use according to claim 13, wherein the mammalian cell is an immortalized cell line derived from the mammal.

15. (a) the presence of integrated viral DNA in mammalian cells; (b) the methylation status of at least one CpG site of the virus; and A DNA bead array for use in the method according to any one of claims 1 to 12 for determining the viral status of a virus.

Images