WO2007030815A1

WO2007030815A1 - Methods for determining the pathogenicity of viral strains

Info

Publication number: WO2007030815A1
Application number: PCT/US2006/035258
Authority: WO
Inventors: Andrew J. Maniotis; Robert Folberg; Klara Valyi-Nagy; Tibor Valyi-Nagy
Original assignee: The Board Of Trustees Of The University Of Illinois
Priority date: 2005-09-09
Filing date: 2006-09-11
Publication date: 2007-03-15

Abstract

Methods and compositions are provided for rapidly detecting the presence and lytic potential of a pathogenic virus in a sample and for determining the pathogenicity of a viral strain to cells, as compared to merely detecting the presence of viruses in a sample. Assays determine the pathogenicity of viral strains based on the viral strains' ability to expose nuclease sites on the host cell's nuclear DNA (e.g., condensed chromatin in malignant cells) to a DNA nuclease.

Description

METHODS FOR DETERMINING THE PATHOGENICITY OF VIRAL

STRAINS

BACKGROUND

[0001] Methods and compositions for determining pathogenicity of viral strains in addition to detecting merely the presence of viral strains, are improvements in aiding decisions involving treatment plans and diagnosis. Detecting pathogenic viral strains from biological samples otherwise requires laborious methods and long periods of culturing.

[0002] Chromatin condensation states vary. For example, DNA in cancer cells is tightly packaged in protein complexes that contain disulfide bonds, making the DNA less susceptible to digestion by restriction enzymes, which are site-specific proteins that cut DNA at specific nucleotides. Detergent extraction assays, cell smear assays (a modified type of Pap smear), flow cytometry, touch preparations made from comparing human lesions with their margins in the normal tissue, and comparisons between isolated chromosome sets, all demonstrate that the more invasive the cancer cells, the more protection the cells' DNA has from restriction enzyme cleavage.

[0003] Enhanced DNA protection has been observed in many different types of cancer, suggesting that this may be a universal feature of malignant cells. Consequently, the fact that sequestration of DNA serves as a physical marker for malignancy may be broadly applicable in many if not all types of cancer detection, in contrast to current methods of cancer diagnostics which detect chemical markers that are highly variable from tumor to tumor and from patient to patient, or which are highly specific for certain types of tumors.

[0004] DNA in tumor cell genomes are more sequestered than the DNA from poorly invasive or normal cells. Assays measuring DNA sequestration, including the three- dimensional matrix chip assay, the constricting gel assay and therapeutic patch, have shown that the most malignant cells are the ones that are also the most resistant to most current forms of chemotherapy, including many chemotherapies that are designed to interfere with DNA metabolism. These assays are currently being employed to test new forms of therapeutic interventions. For example, current forms of cancer chemotherapy do not target, and will never target, the most malignant cells whose DNA is largely protected through a cytoarchitectural resistance mechanism deep within the malignant cell. [0005] Unraveling the mystery of DNA packaging or sequestration has also provided a new mechanistic paradigm to explain how eukaryotic genes are turned on or off, and how they are expressed or suppressed. Protein complexes and scaffolds, both outside and within the cell, can almost instantaneously affect the arrangement of DNA within the cell's nucleus. For example, tighter DNA packaging is triggered by the cell's cytoskeleton — the scaffolding of proteins that gives each cell its shape and keeps its internal components organized. Interestingly, proteins lurking in the area outside of the cell in the extracellular matrix environment also interact through the cell's cytoskeleton to provoke DNA sequestration. Exposure of even one edge of a cell to certain extracellular proteins can kick DNA sequestration into gear, a finding that has validated and provided a mechanistic basis for how the environment and cytoskeleton dictate cellular behaviors.

[0006] Condensed chromatin effects have other applications.

SUMMARY

[0007] Methods and compositions to determine the pathogenicity of viral strains are based on the ability of pathogenic viruses to uncover sequestered sites susceptible to nuclease degradation in cells with condensed chromatin characteristics.

[0008] Assays to determine the pathogenicity of viral strains based on the viral strains' ability to expose nuclease sites on chromatin, including condensed chromatin in malignant cells, are disclosed. Detecting pathogenic viruses and their pathogenicity in a sample, as compared to merely detecting the presence of viruses in a sample, is an useful tool in diagnosing viral infections.

[0009] Methods and compositions are provided not only for detecting the presence of a pathogenic virus in a sample and but also for determining the pathogenicity of a viral strain to cells. Methods for detecting the presence of a pathogenic virus in a sample include rapid infectivity and lysis assays (RILA). Methods include the steps of contacting cells, such as mammalian cells, with a sample including a viral strain, to produce a population of contacted, and presumptively infected, cells. The chromatin of the contacted/infected cells is then placed in contact with a DNA nuclease for a predetermined length of time, after which the cells are screened to determine what extent, if any, the nuclear DNA has been degraded. The amount of DNA present is determined through the use of a DNA binding fluorescent entity and the inference is made that that the viral strain is pathogenic based on the comparative degradation of DNA. The more pathogenic a viral strain, the more exposed the DNA in chromatin, and the more susceptible the DNA is to nuclease digestion (e.g., endonuclease degradation).

[00010] The steps of detecting and quantitating nuclear DNA includes contacting the cells with a DNA binding fluorescent moiety, removing the unbound moiety, and measuring fluorescence from the contacted cells to determine whether nuclear DNA is present. Any suitable method of DNA detection is within the scope of this disclosure. The methods also quantitate the DNA.

[00011] The DNA nuclease is generally an endonuclease, e.g., a restriction endonuclease that has a four base pair recognition sequence.

[00012] The rapid infectivity and lysis assay (RILA) detects viral species and predicts their pathogenic potential based on their ability to expose, rather than sequester, the

DNA of infected cells. [00013] Any viral strain that is capable of exposing nuclease sites on condensed chromatin is suitable for testing by RILA. For example, Herpes simplex viruses 1 and

2, cytomegalovirus (CMV) are suitable. [00014] RILA is also used to determine mutants resistant to drugs that are generated, for example, in patients undergoing antiviral therapy. [00015] A method for determining the pathogenicity of a viral strain from a biological sample includes the steps of:

(a) contacting a population cells with the viral strain to produce a population of contacted cells, wherein the population of cells include condensed chromatin;

(b) contacting the chromatin of the contacted cells with a DNA nuclease;

(c) detecting the presence of nuclear DNA of the contacted cells remaining after nuclease digestion; and

(d) inferring that the viral strain is pathogenic based on the digestion of nuclear DNA as compared to digestion of a control population of cells with condensed chromatin that was not contacted with the viral strain.

[00016] Pathogenicity of viral strains are determined in cells that have condensed chromatin, for example, malignant cells. The condensed chromatin includes sequestered restriction sites. Detecting the presence of nuclear DNA includes for example, contacting the cells with a nuclear DNA binding fluorescent moiety and measuring the fluorescence from the contacted cells to determine an amount of the nuclear DNA.

[00017] A DNA nuclease such as, for example, an endonuclease is used to detect the exposed nuclease sites. A suitable endonuclease is a restriction endonuclease that has a four base pair recognition sequence. A nuclease is selected from a group of nucleases for example, EcoRI, Saul, Pstl, Hindlll, Mspl, AIuI, DNase I, micrococcal nuclease, Sau3 AI, Dpnl, and Rsa I.

[00018] The cells used to determine the pathogenicity of viral strains, in an aspect, are permeablized with a detergent prior to being contacted with the restriction endonuclease.

[00019] The viral strain is in a concentration of about 0.001 plaque forming units

(PFU) per cell to 10 PFU/cell. [00020] The presence and amount of nuclear DNA of the contacted cells remaining after nuclease digestion is determined within about 3 hours after the population of the cells with the viral strain. [00021] A rapid infectivity lysis assay (RILA) to determine the pathogenic potential of a viral strain includes the steps of:

(a) contacting a population of cells with the viral strain, wherein the population of cells comprise nuclear DNA in a condensed chromatin;

(b) incubating the cells with a DNA nuclease; and

(c) detecting the presence of nuclear DNA remaining after nuclease digestion.

[00022] Pathogenicity determination assays described herein further include detecting lysis in the population of cells. The lysis in the population of cells is determined within about 2 days after contacting the population of the cells with the viral strain.

[00023] Pathogenicity determination assays described herein include cells, e.g., mammalian cells. [00024] Pathogenicity determination assays described herein are suitable for any virus capable of decondensing the chromatin and/or exposing the nuclease sites within the nuclear DNA. For example a suitable viral strain is a human virus. A suitable viral strain is herpes simplex virus (HSV) or cytomegalovirus (CMV). BRIEF DESCRIPTION OF THE DRAWINGS

[00025] FIG. 1 are photographs of nuclei of cells infected with a virus. Arrows in the 0.1 PFU/cell (Plaque-forming Units) picture point to the nuclei of cells that have been infected for 2 hours with 0.1 PFU/cell concentration of virus, whereas, the arrows in the 1.0 PFU/cell and the 10 PFU/cell point to cells still showing fluorescence whose DNA may not have been damaged, or they may indicate cells that have yet to completely exhibit the viral infection.

[00026] FIG. 2 shows infectivity level in a RILA assay. A low power (2OX, left) and high-power (63X, right) view of cell killing (any cell that is black) using the Trypan blue exclusion method. The arrow in the right photograph demonstrates the presence of a few living cells among all the dead (black) cells that take up the dye.

[00027] FIG. 3 shows that RILA assay detects that HSV-I is capable of decondensing chromatin and inducing cell lysis. [00028] FIG. 4 shows that heat inactivated HSV does not lead to chromatin decondensation and does not render it more sensitive to AIu I digestion when compared with mock infected cells (suggesting that heat treatment leads to inactivation of critical viral proteins needed for entry into host cells). UV-inactivated HSV does not lead to chromatin decondensation and does not render it more sensitive to Alul digestion when compared with mock infected cells (suggesting that de novo viral protein synthesis is required for chromatin decondensation). This suggests that the chromatin decondensation effect by Heipes simplex virus is a specific effect mediated by the virus and not due to random events caused during cell culture. DETAILED DESCRIPTION

[00029] Compositions and methods are described for rapidly detecting pathogenic viruses and their pathogenicity in a sample, as compared to merely detecting the presence of viruses in a sample. A method is provided for measuring the pathogenic potential of viruses. Even a small quantity of the virus in the sample is sufficient to determine the pathogenicity of the virus.

[00030] An assay is performed in vitro on cell cultures to measure the ability of a virus containing sample to induce exposure of a cell's chromatin to the enzymatic activity of nucleases. This enhanced exposure of the cells' nuclear DNA to the enzymatic activity of nucleases upon exposure and infection by a virus has been correlated with the pathogenicity (infectivity) of the virus as determined for example, by lysis of the cells. Correlations are standardized for each type of cells used in the assay, between uncovered DNA digestion sites and lysis.

[00031] In development of diagnostic assays for malignant cells, cells derived from inflammation reactions were concurrently tested because they were reported to confound other kinds of cancer diagnostic tests. In addition, human neurons exposed to high levels of amyloid protein, as in Alzheimer's disease, were tested as part of cancer diagnostics, because the amyloid protein was found to be highly expressed in the gene arrays of certain strains of melanoma cells. Herpes simplex virus was also tested, because it was reported that herpes virus causes DNA to become hypercondensed. Yet none of these pathological states mimicked the AIuI or Msp-I DNA sequestration observed in the DNA of malignant tumor cells, confirming the specificity for the sequestration reaction as being diagnostic for cancer. However, herpesvirus, but not mock-infected controls, was found to induce an exposure effect to the nuclease AIuI on DNA in the nuclei of infected cells at extremely low multiplicities of virus infection (MOI). Further tests showed that this reaction was followed by a lytic effect on 100% of the infected cells in monolayer cultures. Assays used to monitor chromatin susceptibility to nuclease activity are designated herein as "Rapid Infectivity and Lysis Assays (RILA)". The RILA is performed on samples from body fluids in less than 2 hours and detects very small quantities of virus (as little as 0.1 PFU/cell compared with a mock viral infection). The infectivity read-out after a 2 hour incubation with virus and a 1 hour incubation with AM demonstrates that the nuclear DNA has been exposed to the enzymatic activity of AIuI resulting in digestion of the DNA (as assayed by a simple fluorescent dye reaction that detects DNA; FIG. 1).

[00032] The RILA assay also measures the lytic potential of viruses within a day or two because at the same time it measures the extent of DNA exposure due to as few as 0.1 PFU/cell, it documents how many cells are killed by the infection (FIG. 2). The RILA assay provides a direct demonstration of the potential of any virus to expose cellular chromatin to the enzymatic activity of nucleases such as AIuI and Msp-I, and, at the same time, determine if the presence of virus is pathogenic to the cells harboring the infection. This characteristic exposure of chromatin and lysis in the RILA assay is different from all existing assays that are generally directed at identifying viruses through markers or surrogate markers thought to be associated with the virus itself, or by detecting host responses to a viral infection.

[00033] The RILA has the advantage of providing pathogenicity results rapidly.

Knowledge of pathogenic potential of a virus is highly desirable, because it is well established that many viral infections do no damage to the host's cells or cellular organelles and thus demand different treatment plans or do not require treatment. Accordingly, in spite of the ability of other assays to identify the presence of a particular virus type through the use of anti-viral specific antibodies, there is currently no way to subsequently determine if a particular viral infection is capable of affecting cellular DNA/chromatin. The ability to determine if a virus can infect a host cell and disturb host cell DNA/cliromatin structure and function, and/or cellular viability is particularly important in the context of therapeutic testing and resistance testing.

[00034] Certain antiviral therapies become ineffective because viruses mutate rapidly and develop resistance to a drug. However, the vast majority of mutations in a genetic code will typically generate defective organisms or viruses. This is especially true among genomes that only code for a few or up to a dozen or so proteins, such as most viruses. If any of the viral genes harbors a mistake or mutation, then the vast majority of the time, it will become non- viable, because all of its genes are required for replication and infectivity. Viruses harbor little, if any, extra DNA or RNA that isn't used during the viral life cycle to code an essential life-cycle function. Similarly, it is unlikely that organisms with genomes the size of viruses could mutate one of their 10 or so genes to evade a drug that was truly capable of targeting one of those 10 proteins. Tests (assays) described herein that rapidly determine if a tiny amount of virus from a patient's sera has pathogenic potential present a milestone in viral testing because the pathogenic property of a viral infection, if any, could be rapidly confirmed, or dismissed, within 3 hours using the RILA test.

[00035] A method for determining the presence and/or pathogenicity of a viral strain is provided. The method includes the steps of contacting a population of cells with a sample known to contain, or suspected of containing, a viral strain and determining if that contact resulted in modifying the cell's nuclear DNA/cliromatin. The method allows for the determination of whether the virus has induced a change in the cell's DNA/chromatin in a manner that makes the DNA/chromatin more susceptible to nucleases, relative to the nuclear DNA/cliromatin of cells not exposed to the virus. The cells are eukaryotic cells, for example, are mammalian cells including human cells. Suitable cells include primary cell cultures or established cell lines.

[00036] Malignant cells such as melanoma cells are suitable. However, because cells may vary in their response to viral infection, controls need to use the same cells, and quantitation is standardized for specific cell types. The cells contacted with the virus are typically cultured in vitro using standard techniques known to those skilled in the art. Cells are contacted with the virus at low viral titers, including titers as low as 10 PFU/cell, 5 PFU/cell, 1 PFU/cell, 0.5 PFU/cell or 0.1 PFU/cell. The cells and the virus are incubated together for a predetermined length of time under conditions suitable for attachment of the virus to the cells. After a predetermined length of time for exposure to the virus, the cells' DNA is contacted with a DNA nuclease. The cells are subjected to a wash to remove any non-bound material from said cells prior contacting the cells with the DNA nuclease.

[00037] The nuclear DNA/chromatin of the cells is contacted with a nuclease for a predetermined length of time. Those of ordinary skill in the art know how to test several times to standardize each set of experimental controls. The enzymatic activity of the nuclease is inhibited after the desired length of exposure of the cellular DNA/chromatin to the nuclease by the addition of a DNA binding fluorescent moiety such as ethidium bromide.

[00038] The length of time that the cellular DNA/chromatin is exposed to nuclease activity is dependent on the nuclease used, and the manner in which the nuclear DNA/chromatin is contacted with the nuclease. The cell nuclear DNA/chromatin may be contacted with the nuclease (in the form of a solution) after the cell membranes have been permeabilized. The cells are permeabilized using standard reagents and techniques known to those skilled in the art using organic solvents such as alcohols and acetone, detergents or other agents. For example, the cells are permeabilized by contacting the cells with a detergent such as Triton X-100. Alternatively, the cell membranes are mechanically disrupted to allow the solution containing the nuclease, access to the cell's nuclear DNA/chromatin. The cells are removed from the cell culture plate and allowed to dry on a solid support as a means of disrupting the cells membranes and allowing access of the nuclease to the cell's nuclear DNA/chromatin. A solution comprising the nuclease is then applied to the dried cells. [00039] The nuclease used to treat the viral contacted cells is selected from a group of any DNA nuclease including exonucleases and endonucleases. The nuclease may be an endonuclease, such as EcoRI, Saul, Pstl, Hindlll, Mspl, AIuI, Mspl, DNase I, or micrococcal nuclease. The nuclease is a restriction endonuclease that has a four base pair recognition site, for example, AIuI.

[00040] After the cells have been contacted with a solution containing a virus, and have been subsequently treated with a solution containing a nuclease, the cells are then analyzed to determine the extent the cell's nuclear DNA has been degraded. Any of the techniques known to those skilled in the art for detecting the presence of DNA can be used in accordance with this invention. The amount of intact DNA remaining after the nuclease treatment is measured by the use of a DNA binding fluorescent entity. The DNA binding fluorescent entity is one that specifically binds to double stranded DNA, such as the DNA intercalating agents ethidium bromide and theophyllinea. The amount of fluorescence emitted from the viral infected cells is compared to a control set of cells that were mock infected, exposed to the nuclease and then contacted with the DNA binding fluorescent entity. A detected decrease in fluorescence from the virus contacted cells relative to the control cells is indicative of enhanced DNA degredation and thus is indicative of the pathogenicity of the viral strain.

[00041] Accordingly, the use of RILA allows for the systematical testing a variety of different kinds of viruses, as well as allowing the determination of the pathogenicity of various strains of the same virus. In addition, in patients undergoing antiviral therapies, the procedures disclosed herein can be used to test whether a particular drug or therapeutic regiment has generated any " pathogenic mutants."

[00042] The following examples are intended only to further illustrate the invention and are not intended to limit the scope of the subject matter which is defined by the claims. EXAMPLES

[00043] The following examples are for illustrative purposes only and are not intended to limit the scope of the disclosure.

Example 1: Chromatin Organization Measured by AIuI Restriction Enzyme Changes with Malignancy. [00044] The ability of malignant cells to resist nuclease digestion of their condensed chromatin is exploited in the Rapid Infectivity and Lysis Assays (RILA). This example demonstrates the various conditions and steps that were practiced in determining chromatin organization of cancer cells exposed to nuclease treatments.

Cell Culture

[00045] Primary uveal melanoma cell lines of low (OCMl a) and high (M619) invasive potential, and a highly invasive metastatic uveal melanoma cell line (MUM2B) were used. Determination of the modal chromosome number revealed the OCMIa and M619 lines to be in the tetraploid range, while the MUM2B line was pseudo-diploid. These cell lines were also characterized by their differential ability to form laminin- rich patterns that appear to control melanoma morphogenesis. OCMIa uveal melanoma cells were a gift from Dr. June Kan-Mitchell, Karmanos Cancer Institute, Wayne State University, Detroit, MI, and UM54 normal uveal melanocytes were a gift from Dr. J. William Harbour, Washington University, St Louis, MO. Melanoma cells and uveal melanocytes were plated in EMEM (BioWhittaker, Inc., Walkersville, MD), and supplemented with heat inactivated 15% fetal bovine serum (Fisher, Ontario, Canada) without the addition of exogenous extracellular matrix molecules or growth factors.

[00046] Normal WI-38 human fibroblasts were obtained from the ATTC (Rockville,

MD), and were maintained in 10% serum in DMEM. Telomerase, large-T, and ras transfected fibroblasts were a gift from Dr. Igor Roninson (University of Illinois at Chicago). HTl 089 fibrosarcoma cells were obtained from the ATTC. No antibacterial or antifungal drugs were used in the maintenance of cell lines or in experiments, as their chronic use has been shown to interfere with the differentiation potential of other primary cell types. MCFlOA breast epithelial cells and MDA- MB231 breast carcinoma cells were obtained from the ATTC, and were maintained on DMEM plus heat inactivated calf serum. The isolation and phenotypes of HMT-3522 human mammary epithelial cells both nonmalignant (Sl) and their tumorigenic counterparts designated T4-2 were described previously. AU cell cultures were determined to be free of mycoplasma contamination using the GenProbe rapid detection system (Fisher, Itasca, IL). Restriction Enzyme Assays of Interphase Cells

[00047] Three assays were used to compare sequestration and exposure of AIuI binding sites in interphase cells of different invasive behaviors. In the permeabilized cell assay, cells in monolayer cultures were exposed to AIuI restriction enzyme after permeabilization with Triton X-100. In the cell smear assay, cells were mechanically dislodged from culture, applied to glass slides, allowed to air dry, and exposed to AIuI restriction enzyme, thus simulating a diagnostic cytological examination performed in a clinical setting (e.g., the examination of cells extracted by fine needle aspiration biopsy, brush biopsy, or scraping). In the flow cytometry assay, differential digestion of chromatin between cell lines by AM restriction enzyme over time was quantified, again using methodology that could be easily adapted to a clinical diagnostic setting by standardization techniques known to those of skill in the art.

Permeabilized Cell Assay

[00048] Cells grown as monolayer cultures in the absence of exogenous matrix molecules were exposed to 0.1% Triton X-100 (Sigma, St. Louis, MO) for 90 seconds followed by a rinse of DMEM. The preparation was exposed to AIuI restriction enzyme (0.5 μl in 40 μlDMEM; Promega, Madison, WI) for 60 minutes to 24 hours. The permeabilized cultures were then exposed to ethidium bromide (25 μl, 1 μg/ml; Sigma) and photographed with aLeica inverted microscope (Leica, Bannockburn, IL).

Cell Smear Assay

[00049] After mechanically dislodging cells grown under monolayer conditions, the slurry was suspended in IX PBS or serum-free DMEM, and a drop containing 15 μl of the suspension was placed onto a glass slide. The drops were allowed to evaporate over 30 minutes to 1 hour. AM restriction enzyme (0.5 μl in 40 μl) or Mspl restriction enzyme (0.5 μl in 40 μl DMEM; Promega) was applied to the dried cells, and the preparation was placed in a humidified 37°C chamber to optimize enzyme activity and minimize enzyme evaporation. Endonuclease digestions were terminated at pre-designated time points (30 minutes and hourly increments thereafter up to 24 hours) to determine the optimum digestion time that would allow for discrimination of differential chromatin digestion between cell lines. Ethidium bromide was added to terminate the digestion, and the preparation was photographed immediately. Touch preparations of normal human tissue and human tumor tissue were made and air dried. The preparation was incubated with AIuI restriction enzyme and the reaction was terminated with ethidium bromide at 5 and 24 hours.

[00050] To test the influence of different soluble ECM molecules on AIuI sensitivity, cells that had been growing under monolayer conditions were mechanically dislodged from flasks and was suspended in the slurry of dislodged cells in IX PBS or serum- free DMEM. Two rows of 15 μl drops of the suspension were applied to a glass slide, 3 drops per row. In some experiments as described below, soluble laminin, Matrigel, FBS, RGD-C, collagen Type I, bFGF, EGF (all reagents from Clontech, Palo Alto, CA), circularized RGD (RGD-C, a kind gift from Renata Pasquallini) were added to one of the two rows of drops. RGD-C is known to bind to integrin receptors. The test and control drops were then permitted to evaporate at room temperature for at least 1 hour, leaving "smears" of dried cells that had or had not been incubated with a test molecule. For each assay, cellular permeability was checked using the trypan blue exclusion method. The AIuI restriction enzyme (0.5 μl in 40 μl DMEM; Promega) was applied to each drop in a humidified 37° incubator for up to 24 hours. The buffers used in these assays did not contain either DTT or mercaptoethanol to avoid removing any proteins within the cytoplasm or nucleus that might sequester AM binding sites from enzyme digestion. Incubation with AIuI was terminated by adding ethidium bromide (Sigma; 250 μl, 100 ng/ml) to each drop per slide at 30 minutes and at hourly intervals thereafter up to 24 hours of incubation.

Scoring Results

[00051] In the cell smear assay and the permeabilized cell assay, samples were photographed with a Leica inverted microscope and were scored qualitatively from the micrographs as follows: 1) Nuclei in which no fluorescence was detected except for nucleoli were scored as complete digestion; 2) Nuclei in which fluorescence intensity was equal to that detected at time before application of restriction enzyme was scored as no digestion; and 3) Detection of non-nucleolar nuclear fluorescence that was less intense than detected before application of restriction enzyme was scored as partial digestion. Analysis was by standard statistical methods.

Flow Cytometry

[00052] To provide for a quantitative measure of DNA digestion, flow cytometry was performed. Cells from monolayer cultures of UM54 normal uveal melanocytes, poorly invasive OCMIa primary uveal melanoma, highly invasive M619 primary uveal melanoma, and highly invasive MUM2B metastatic uveal melanoma were suspended in DMEM and spun down at 1400 rpm for 5 minutes. The pellet was re- suspended in 0.1% Triton X-IOO, incubated for 1 minute at room temperature, and spun down again at 1400 rpm for 5 minutes. After re-suspension in DMEM, 0.5 μl of AM restriction enzyme in 40 μl of DMEM was added and the preparation was incubated at following time points: 0 (baseline), 1, 3, and 5 hours at 37°C. Propidium iodide (10 μl/ml; Molecular Probes, Eugene, OR) was added at the conclusion of each digestion period. The cells were analyzed in a FACS Calibur (BD Bioscience, San Jose, CA) equipped with a 488 laser, detectors for forward and side scatter, and 520, 575, and 675 nm detectors. 10,000 cells were counted and the results were analyzed with FACS dot-plots and histograms. Three AIuI digestions monitored by flow cytometry were performed for each of the three melanoma cell lines. The percentage of cells in M2-M4 (roughly corresponding to 10² to 10⁴ fluorescence intensity) was calculated and compared between cell lines at 5 hours of digestion with the t-test. The PI signal, representing stoichometrically the amount of labeled undigested DNA, was recorded immediately after cell permeabilization and labeling with PI and used as a baseline (0 time point), and was then measured for each cell line after 1, 3, and, 5 hours of exposure to AIuI restriction enzyme. CellQuest software (BD Bioscience) was used to generate overlays of the histograms for each cell line at each time point in the digestion experiment.

Isolated Chromosome Set Assay

[00053] The inventors have previously described a method by which the entire chromatin contents of nuclei can be extracted, producing a complete chromosome set for each cell thus manipulated (Maniotis, 1997). Chromatin sets were removed from cells that had not been pretreated with any agent. All chromosome extractions and digestions were carried out under isotonic culture conditions in DMEM at pH 7.4; similar results also were obtained using complete cell culture medium containing FBS.

[00054] Chromosome sets removed from metaphase cells of varying invasive potentials were studied under buffered media conditions that do not disturb the native structure of chromosomes. Cells from which chromosomes were to be removed were grown on small coverslips and placed in the center of a 35 -mm plastic dish lid containing 2 ml of gassed DMEM. This preparation was allowed to equilibrate at 37⁰C in a 5% CO₂ buffered incubator before micromanipulation. For optimal micromanipulation conditions, cells were grown to near confluence to produce strong attachments to each other and ECM needed to resist the pulling forces associated with rapid (within 1 second) micropuncture and removal of chromosomes without causing cell detachment or death of the manipulated cell. Chromosome sets were obtained either in the presence or absence of colchicine and cytochalasin-B. All chromosome set extractions and digestions were carried out under isotonic culture conditions in DMEM at pH 7.4; similar results also were obtained using complete cell culture medium containing serum.

[00055] Chromosome sets were extracted from living metaphase cells by rapidly piercing the cell directly on the side of a mitotic plate with a glass microneedle and then laterally drawing out the chromosomes through the hole on the cell surface created by the micropuncture using a Leitz micromanipulator (Leica Microsystems). In certain studies which used treatments that cause cell detachment (mercaptoethanol, DTT, and proteinase K), chromosome sets were removed from the cell with a pipette and placed on culture substrata. The chromosome sets were then deposited to pre- designated areas defined by scratching the Petri dish lid with a fine needle. Narishige micropipettes (Narishige Scientific Instruments, Tokyo, Japan) were pulled with a Sutter micropipette puller (Sutter Instrument Company, Novato, CA) adjusted to produce long barrels approximately 1 to 5 μm wide along a length of 40 to 100 μm (tip widths were consistently less than 0.5 μm).

[00056] In this isolated chromosome set assay, it is important to use restriction enzymes and proteases in the range of specific ion concentrations if they are to function efficiently. Buffered media conditions that preserve physiological ion concentrations were used to maintain the extent of chromatin compaction observed in living cells while providing the appropriate concentrations of NaCl and MgCl₂ to permit enzymes to work. The restriction enzymes were used in the absence of DTT or β-mercaptoethanol (which are typically included in commercially available kits used to digest DNA) to avoid interfering with DNA-associated proteins.

[00057] Restriction enzymes were dissolved in 2 ml of DMEM applied directly to 35 mm tissue culture dish lids on which the chromosome preparations were attached in the concentrations listed below. Changes in chromosome morphology were photographed by time-lapse video microscopy in real time. All microsurgical procedures were observed at 63x by phase and fluorescence microscopy with a Leica inverted microscope, and captured using a time-lapse video recorder (Sony) and Pinnacle Image software (Pinnacle Systems, Modesto, CA).

[00058] The following enzymes were tested: AM (5-50 units), Msp-I, (75 units,

Promega), DNase I (1-20 units/droplet; Promega,), micrococcal nuclease (1-10 units; Promega), EcoRI (5-50 units; Promega), HindIII (5-10 units; Promega), BamHI (5- 10 units; New England Biolabs, Beverly, MA), RNase A (1-100 units; Sigma), RNase I (1-100 units; Promega), EMBO (3 units; Promega), Sau-III (20 units; Promega), Pstl (30 units; Promega), XHO-I (40 units; Promega). Ethidium bromide (25 μl, 1 μg/ml; Sigma) was added to the medium after 1 to 3 hours of digestion with restriction enzyme and the preparations were photographed with a Leica inverted microscope.

[00059] Given that expression of many genes changes when cells become malignant or are placed in different microenvironments, a question was whether these changes were accompanied by global reorganization of chromatin. Sequestration or exposure of chromatin-sensitive sites to restriction enzymes was used to detect this reorganization. Alul-sensitive sites of nonmalignant cells were relatively more exposed compared to their malignant counterparts in cultured cells and human tumor samples. Changes in exposure and sequestration of Alul-sensitive sites in normal fibroblasts versus fibrosarcoma or those transfected with oncogenes, nonmalignant breast cells versus carcinomas and poorly metastatic versus highly invasive melanoma were shown to be independent of the cell cycle and may be influenced by proteins rich in disulfide bonds. Remarkably, regardless of degree of malignancy, Alul- sensitive sites became profoundly sequestered when cells were incubated with laminin, Matrigel, or a circular RGD peptide (RGD-C), but became exposed when cells were placed on collagen I or in serum-containing medium. Disruption of the actin cytoskeleton led to exposure, whereas disruption of microtubules or intermediate filaments exerted a sequestering effect. Thus, Alul-sensitive sites are more sequestered with increasing malignant behavior, but the sequestration and exposure of these sites is exquisitely sensitive to information conferred to the cell by molecules and biomechanical forces regulating cellular and tissue architecture. [00060] Example 2: Chromatin Organization Measured by AIuI Restriction

Enzyme Changes with Herpes Simplex Virus Type 1 (HSV-I) Infection. Currently available methods to detect (culture) infectious herpes simplex virus type 1 (HSV-I) and other viruses typically takes multiple days to weeks. The Rapid Infectivity and Lysis Assay (RILA) is intended for the rapid detection and quantification of infectious HSV-I and potentially other infectious viruses in body fluids within 1 to 2 days.

[00061 ] DNA in the malignant melanoma cell line MUM-2 is tightly packaged

(condensed), making it highly resistant to digestion by the restriction enzyme AM Experimental observations indicate that brief (2 hours) exposure of in vitro cultures of MUM-2 cells to infectious (but not to heat- or UV-inactivated) herpes simplex virus type 1 (HSV-I) leads to an increased susceptibility of MUM-2 DNA to digestion with AIuI (decondensation). The (RILA) assay is based on the measurement of changes in the sensitivity of MUM-2 DNA to AM digestion following exposure to biologic material (body fluids) and comparison of the detected changes to the effect of positive controls (HSV-I infection) and negative controls (mock infection). In vitro MUM-2 cultures are either exposed to the test body fluid, or known amounts of HSV-I (positive control) or sterile PBS (negative control) for 2 hours. This is followed by preparation of cell smears from the melanoma cultures, exposure of the smears to the AM restriction enzyme, ethidium bromide staining of DNA, microscopic visualization, photography and data analysis (cell smear assay).

[00062] Another set of MUM-2 cultures is either exposed to the test body fluid, HSV-I

(positive control) or PBS (negative control) for one hour and then incubated for up two weeks with tissue culture medium to detect growth of infectious virus by traditional culture methods.

[00063] Thus the RILA assay is intended for the rapid detection of infectious HSV-I in body fluids. Materials and Methods

[00064] The RILA assays were conducted as follows: MUM-2 cells were grown to approximately 70% confluency on 6-well tissue culture plates. Cell numbers per were counted. The tissue culture medium was then removed from the wells and the cells were exposed for 1 hour at 37 C to one of the following inocula: A. 0.5 ml of sterile PBS (mock infection, negative control) B. HSV-I with a calculated multiplicity of infection (MOI) ranging from 0.001 plaque forming units (PFU) per cell to 10 PFU/cell diluted in PBS to a final volume of 0.5 ml.

C. 0.5, 5 or 50 ul of test body fluid diluted to in PBS to a final volume of 0.5 ml. [00065] After 1 hour incubation, fresh tissue culture medium (1 ml) was added to each well and the cultures were either incubated for another hour at 37⁰C and then processed for the cell smear assay or further incubated in repeatedly refreshed culture medium for 2 weeks for traditional virus detection by culture.

Cell Smear Assay

[00066] Cultured and inoculated cells were washed twice in PBS and were then mechanically dislodged from the wells, pelleted and resuspended in IX PBS. A drop containing 15 ul of the cell suspension was then placed on a glass slide. The drops were allowed to evaporate over 30 minutes to 1 hour at room temperature.

[00067] AM restriction enzyme (Promega) (0.5 ul in 40 ul of DMEM) was applied to the dried cells, and the preparation was placed in a humidified 37⁰C chamber to optimize enzyme activity and minimize enzyme evaporation.

[00068] Endonuclease digestions were terminated at pre-designated time points (30 minutes and hourly increments thereafter up to 24 hours) to determine the optimum digestion time that allows for discrimination of differential chromatin digestion. Ethidium bromide was added to terminate the digestion and the preparation was photographed immediately using an inverted microscope and was scored qualitatively as follows:

1) Nuclei in which no fluorescence was detected except for nucleoli were scored as complete digestion;

2) Nuclei in which fluorescence intensity was equal to that detected at time before application of restriction enzyme was scored as no digestion (undigested);

3) Detection of non-nucleolar nuclear fluorescence that was less intense than detected before application of restriction enzyme was scored as partial digestion. [00069] The number of undigested, partially digested and completely digested nuclei was determined in each specimen (test specimens, positive controls, negative controls) by counting at least 3 times 100 nuclei in each specimen. The presence and quantity of infectious HSV-I in the test specimens was then determined by comparing the numbers of undigested, partially digested and completely digested nuclei following their inoculation relative to those detected in positive and negative controls. [00070] Results: The Rapid Infectivity and Lysis Assay can be performed on samples from body fluids in less than 2 hours and detects very small quantities of virus (as little as 0.1 PFU/cell compared with a mock viral infection). The read-out after a 2 hour incubation with virus and a 1 hour incubation with AIuI is the demonstration of digested DNA (as assayed by a simple fluorescent dye reaction that detects DNA see FIG. 1).

[00071] The RILA assay can also measure the lytic potential of viruses within a day or two because at the same time it measures the extent of DNA exposure due to as few as 0.1 PFU/cell, it documents how many cells are killed by the infection (FIG. 2).

[00072] One advantage that this assay provides is directly demonstrating the potential of any virus to expose cellular DNA to AIuI, and at the same time determining if the presence of virus is pathogenic to the cells harboring the infection. This ability stands in stark contrast to all existing assays that are principally directed at identifying viruses, typically through markers thought to be associated with the virus itself, or by detecting surrogate markers, or by detecting host responses to a viral infection. The RILA assay was repeated using various PFU concentrations using virology's most rigorous kinds of controls to demonstrate how the RILA assay detects that HSV-I is capable of decondensing chromatin, and inducing cell lysis. For example, HSV-I has been consistently observed to decondense chromatin, and with clarity of differential signal intensity (see FIG. 3A, High Magnification; and FIG. 3B, Low Magnification).

[00073] Considering results of mock infected, heat-inactivated, and UV-inactivated virus, the likely mechanism of decondensation involves the early steps of viral replication. Heat inactivated virus does not lead to chromatin decondensation (probably due to the heat denaturing the proteins needed for viral entry) (FIG. 4). UV-inactivated virus was also incapable of decondensing chromatin, indicating that novel viral protein synthesis from input viral DNA is required for chromatin decondensation and virion-associated stimuli (during virion attachment to the cell, virion entry into the cell, etc.) are not sufficient for this effect (FIG. 4). Mock infected controls continue to demonstrate that the decondensation effect of the virus on chromatin is not due to some trivial cause such as perturbation of the cultures while changing solutions. These overall effects support that the chromatin decondensation effect caused by Herpes Simplex virus is a specific effect mediated by the virus and not due to random events caused during cell culture. DOCUMENTS

Bojanowski, et al, J. Cellular Biøchem (1998), 69:127-142

Dayton, et al, Cell (1986), 44: 941-947

Maniotis et al, J. Cellular Biochem, (1997), VoI 65: 114-130.

Maniotis et al., Am. J. Pathol (2005), 166: 1187-1203

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Sodroski, et al.,. Nature (Lond.) (1986), 321: 412-417.

Claims

1. A method for determining the pathogenicity of a viral strain from a biological sample, the method comprising:

(a) contacting a population cells with the viral strain to produce a population of contacted cells, wherein the population of cells comprise condensed chromatin;

(b) contacting the chromatin of the contacted cells with a DNA nuclease;

2. The method of claim 1, wherein the cells are malignant cells.

3. The method of claim 1 , wherein the condensed chromatin comprises sequestered restriction sites.

4. The method of claim 1 , wherein the step of detecting the presence of nuclear DNA comprises contacting the cells with a nuclear DNA binding fluorescent moiety and measuring the fluorescence from the contacted cells to determine an amount of the nuclear DNA.

5. The method of claim 1, wherein the DNA nuclease is an endonuclease.

6. The method of claim 5, wherein the endonuclease is a restriction endonuclease that has a four base pair recognition sequence.

7. The method of claim 1, wherein the nuclease is selected from the group consisting of EcoRI, Saul, Pstl, Hindlll, Mspl, AIuI, DNase I, micrococcal nuclease, Sau3AI, Dpnl, and Rsa I.

8. The method of claim 6, wherein the cells are permeablized with a detergent prior to being contacted with the restriction endonuclease.

9. The method of claim 1 , wherein the viral strain is in a concentration of about 0.001 plaque forming units (PFU) per cell to 10 PFU/cell.

10. The method of claim 1 , wherein the presence of nuclear DNA of the contacted cells remaining after nuclease digestion is determined within 3 hours after the population of the cells with the viral strain.

11. A rapid infectivity lysis assay (RILA) to determine the pathogenic potential of a viral strain, the assay comprising:

(b) incubating the cells with a DNA nuclease; and

(c) detecting the presence of nuclear DNA remaining after nuclease digestion

12. The assay of claim 11 further comprising detecting lysis in the population of cells.

13. The assay of claim 11 , wherein the presence of the nuclear DNA after nuclease digestion is determined within 3 hours after contacting the population of the cells with the viral strain.

14. The assay of claim 12, wherein the lysis in the population of cells is determined within 2 days after contacting the population of the cells with the viral strain.

15. The assay of claim 11 , wherein the cells are mammalian cells.

16. The assay of claim 11 , wherein the viral strain is a human virus.

7. The assay of claim 11 , wherein the viral strain is herpes simplex virus (HSV).