WO2010011440A2

WO2010011440A2 - Diagnosis and treatment of lipogenic adenovirus infection associated with adipose tissue hypertrophy

Info

Publication number: WO2010011440A2
Application number: PCT/US2009/047531
Authority: WO
Inventors: Richard L. Atkinson
Original assignee: Obetech Llc
Priority date: 2008-06-16
Filing date: 2009-06-16
Publication date: 2010-01-28
Also published as: WO2010011440A3

Abstract

Lipogenic adenoviruses may serve as a biomarker for the identification of the development of abnormal adipose tissue hypertrophy. A diagnostic test may be used to identify abnormal adipose tissue hypertrophy in subjects associated with lipogenic adenovirus infection.

Description

DIAGNOSIS AND TREATMENT OF LIPOGENIC ADENOVIRUS INFECTIONASSOCIATED WITH ADIPOSE TISSUE HYPERTROPHY

CROSS-REFERENCES TO RELATED APPLICATIONS[0001] This application claims priority to and the benefit under 35 U.S. C. § 119(e) to U.S. Provisional Application Serial No. 61/061,870, filed on June 16, 2008, the disclosure of which is expressly incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Field of the Invention

[0002] This invention relates generally to lipogenic adenoviruses that act as biomarkers for abnormal adipose tissue hypertrophy and more particularly, as a biomarker for adipose tissue hypertrophy in HIV/ AIDS or Madelung's disease patients. The invention also relates to nucleic acid molecules, proteins, and antibodies useful for detecting the presence of lipogenic adenoviruses.

Related Art

[0003] HIV/ AIDS and Madelung's disease is associated with increased, non-cancerous tumors, and excess adipose tissue deposits. However, there is a poor understanding of the cause(s) of these diseases, thereby preventing effective treatment.

[0004] It would be a major advance in the field to demonstrate that lipogenic adenovirus infection is associated with adipose tissue hypertrophy in HIV/ AIDS patients. Moreover, a diagnostic tests for lipogenic adenovirus infection in HIV/ AIDS patients can be used to help healthcare providers determine the development of adipose tissue hypertrophy. In particular, these methods may allow for the onset of adipose tissue hypertrophy to be predicted with greater certainty and an effective course of therapy to be implemented.

BRIEF SUMMARY OF THE INVENTION

[0005] This invention provides a method for predicting the presence of or predisposition to abnormal adipose tissue hypertrophy by screening for the presence of lipogenic adenoviruses. The invention may be implemented in a number of ways.

[0006] According to one aspect of the invention, a method of predicting the presence of or predisposition to abnormal adipose tissue hypertrophy in a subject may include assaying a sample from the subject to determine whether the subject is infected with an lipogenic adenovirus, and correlating the presence or absence of lipogenic adenovirus based upon whether the subject is infected with an lipogenic adenovirus, where the presence of lipogenic adenovirus infection correlates with a predisposition to or the presence of abnormal adipose tissue hypertrophy.

[0007] The assaying step may include screening for antibodies reactive to the lipogenic adenovirus. The antibodies may be reactive to one or more peptides such as SEQ ID NO.: 1, SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15, SEQ ID NO.: 16, SEQ ID NO.: 17, and SEQ ID NO.: 18. The screening may be performed by employing a serum neutralization assay or ELISA.

[0008] The assaying step may include screening for lipogenic adenovirus nucleic acids. The nucleic acids may encode a hexon protein. The hexon protein may comprise SEQ ID No.: 29. The nucleic acids may encode the fiber coat protein. The nucleic acids encoding the fiber coat protein may comprise SEQ ID No.: 30. The nucleic acid sequences comprising SEQ ID NO.: 19, SEQ ID NO.: 20, SEQ ID NO.:21, SEQ ID NO.:22, SEQ ID NO.:23, SEQ ID NO.:24, and SEQ ID NO.:25 may be employed to screen for lipogenic adenovirus specific nucleic acids.

[0009] The lipogenic adenoviruses may include adenovirus type 5, adenovirus type 36, and adenovirus type 37. The sample may include a biological sample, body fluid, a tissue sample, an organ sample, feces, blood, saliva, and any combination thereof. The subject may be a human. The human may be afflicted with HIV/ AIDS or Madelung's disease. The subject may be a non-human animal.

[0010] The method may further include screening for the presence of a biomarker associated with a lipogenic adenovirus infection in the sample. The biomarker may include fatty acid synthetase (FAS), peroxisome proliferator-activated receptor (PPAR) family proteins, CCAAT/enhancer-binding proteins (C/EBP), adipose tissue differentiation dtermination-dependent factor 1 (ADD-l)/sterol response element-binding protein (SREBP- 1), glycerol-3-phosphdehydrogenase, and lipoprotein lipase. [0011] The method may also include modifying a drug regimen to a less adipogenic drug regimen as a result of the correlation, administering a vaccine effective against lipogenic adenovirus to the subject to prevent lipogenic adenovirus infection, or administering a compound that blocks the effects of a lipogenic adenovirus to produce abnormal adipose tissue hypertrophy.

[0012] Additional features, advantages, and embodiments of the invention may be set forth or apparent from consideration of the following detailed description, and claims. Moreover, it is to be understood that both the foregoing summary of the invention and the following detailed description are exemplary and intended to provide further explanation without limiting the scope of the invention as claimed.

DETAILED DESCRIPTION OF THE INVENTION

[0013] It is understood that the invention is not limited to the particular methodology, protocols, and reagents, etc., described herein, as these may vary as the skilled artisan will recognize. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention. It also is be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include the plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a cell" is a reference to one or more cells and equivalents thereof known to those skilled in the art.

[0014] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which the invention pertains. The embodiments of the invention and the various features and advantageous details thereof are explained more fully with reference to the non-limiting embodiments and examples that are described and/or illustrated in the accompanying drawings and detailed in the following description. It should be noted that the features illustrated in the drawings are not necessarily drawn to scale, and features of one embodiment may be employed with other embodiments as the skilled artisan would recognize, even if not explicitly stated herein. Descriptions of well-known components and processing techniques may be omitted so as to not unnecessarily obscure the embodiments of the invention. The examples used herein are intended merely to facilitate an understanding of ways in which the invention may be practiced and to further enable those of skill in the art to practice the embodiments of the invention. Accordingly, the examples and embodiments herein should not be construed as limiting the scope of the invention, which is defined solely by the appended claims and applicable law.

[0015] Accordingly, provided immediately below is a "Definition" section, where certain terms related to the invention are defined specifically for clarity, but all of the definitions are consistent with how a skilled artisan would understand these terms. Particular methods, devices, and materials are described, although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention. All references referred to herein are incorporated by reference herein in their entirety.

[0016] Ad-2 is adenovirus type 2

[0017] Ad-5 is adenovirus type 5

[0018] Ad-31 is adenovirus type 31

[0019] Ad-36 is adenovirus type 36

[0020] Ad-37 is adenovirus type 37

[0021] PPAR is peroxisome proliferator activated receptors

[0022] CEBP is CCAAT-enhancer binding protein

[0023] HIV is human immune deficiency virus or human immunodeficiency virus

[0024] AIDS is acquired immunodeficiency syndrome

[0025] NGNA is N-glycolylneuraminic acid

[0026] "Lipogenic adenovirus," as used herein, generally refers to adenoviruses that are capable of stimulating increase lipid production in cells, tissues, and/or organs by facilitating expression of lipogenic enzymes, which in turn produce excess fatty acids and promote fat storage. The lipogenic adenoviruses of the invention may include Ad-5, Ad-36, and Ad-37.

[0027] "Abnormal adipose tissue hypertrophy," as used herein, generally refers to an abnormal localized increase in adipose tissue distinguished from simple obesity. This may be a lipoma, a deposition of adipose tissue that is inappropriate for the anatomical location, or an infiltration of an normal gland or area with adipose tissue (e.g., infiltration of parotid glands with adipocytes in HIV+/ AIDS patients, "buffalo hump" subcutaneous adipose deposition on the upper back, or intra-thoracic deposition of adipose tissue). [0028] A "biological sample," as used herein, generally refers to a sample of tissue or fluid from a human or animal including, but not limited to plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal and genitourinary tracts, tears, saliva, blood cells, tumors, organs, tissue and sample of in vitro cell culture constituents.

[0029] "Organ," as used herein, generally refers to a tissue that performs a specific function or group of functions within an organism. An exemplary list of organs includes lungs, heart, blood vessels, blood, salivary glands, esophagus, stomach, liver, gallbladder, pancreas, intestines, rectum, anus, endocrine glands such as hypothalamus, pituitary or pituitary gland, pineal body or pineal gland, thyroid, parathyroids, adrenals, skin, hair, nails, lymph, lymph nodes, tonsils, adenoids, thymus, spleen, muscles, brain, spinal cord, peripheral nerves, nerves, sex organs such as ovaries, fallopian tubes, uterus, vagina, mammary glands, testes, vas deferens, seminal vesicles, prostate, and penis, pharynx, larynx, trachea, bronchi, diaphragm, bones, cartilage, ligaments, tendons, kidneys, ureters, bladder, and urethra.

[0030] "Organ system," as used herein, generally refers to a group of related organs. Organ systems include, without limitation, circulatory system, digestive system, endocrine system, integumentary system, lymphatic system, muscular system, nervous system, reproductive system, respiratory system, skeletal system, and urinary system.

[0031] An "isolated" or "substantially pure," nucleic acid (e.g., DNA, RNA, or a mixed polymer) for example, is one which is substantially separated from other cellular components which naturally accompany a native human or animal sequence or protein, e.g., ribosomes, polymerases, many other human or animal genome sequences and proteins. The term embraces a nucleic acid sequence or protein which has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biologically synthesized.

[0032] The term "antibody," as used herein generally refers to antibodies, digestion fragments, specified portions and variants thereof, including antibody mimetics or comprising portions of antibodies that mimic the structure and/or function of an antibody or specified fragment or portion thereof, including single chain antibodies and fragments thereof. The invention encompasses antibodies and antibody fragments capable of binding to a biological molecule (such as an antigen or receptor), such as the fiber coat protein of lipogenic adenoviruses, and specifically, Ad-36, or portions thereof. [0033] The term "nucleic acid sequence," as used herein generally includes an oligonucleotide, nucleotide, or polynucleotide, and fragments thereof. The term is not limited by length and is generic to linear polymers of polydeoxyribonucleotides (containing 2-deoxy- D-ribose), polyribonucleotides (containing D-ribose), and any other N-glycoside of a purine or pyrimidine base, or modified purine or pyrimidine bases. These terms include double- and single-stranded DNA, as well as double- and single-stranded RNA.

[0034] A nucleic acid, polynucleotide or oligonucleotide can comprise, for example, phosphodiester linkages or modified linkages including, but not limited to phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfone linkages, and combinations of such linkages.

[0035] A nucleic acid, polynucleotide or oligonucleotide can comprise the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil) and/or bases other than the five biologically occurring bases. For example, a polynucleotide of the invention can contain one or more modified, non-standard, or derivatized base moieties, including, but not limited to, N⁶-methyl-adenine, N⁶-tert-butyl-benzyl-adenine, imidazole, substituted imidazoles, 5- fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, A- acetylcytosine, 5 -(carboxyhydroxymethyl)uracil, 5 -carboxymethylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6- isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, uracil-5- oxyacetic acidmethylester, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, 2,6- diaminopurine, and 5-propynyl pyrimidine. Other examples of modified, non-standard, or derivatized base moieties may be found in U.S. Patent Nos. 6,001,611; 5,955,589; 5,844,106; 5,789,562; 5,750,343; 5,728,525; and 5,679,785. [0036] Furthermore, a nucleic acid, polynucleotide or oligonucleotide can comprise one or more modified sugar moieties including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and a hexose.

[0037] The term "fragment," as used herein generally includes any portion of a heterologous peptide or nucleic acid sequence. Heterologous peptide fragments retain at least one structural or functional characteristic of the subject heterologous polypeptides. Nucleic acid sequence fragments are greater than about 60 nucleotides in length, and most preferably includes fragments that are at least about 100 nucleotides, at least about 1000 nucleotides, and at least about 10,000 nucleotides in length.

[0038] The term "PCR," as used herein, generally refers to a method for amplifying, detecting, or quantifying a specific region of an analyte. One skilled in the art appreciates that there are several variations on the basic PCR technique such as allele-specific PCR, assembly PCR or polymerase cycling assembly (PCA), colony PCR, helicase-dependent amplification, hot start PCR, intersequence-specific (ISSR) PCR, inverse PCR, ligation- mediated PCR, methylation-specific PCR, multiplex ligation dependent probe amplification, multiplex PCR, nested PCR, overlap-extension PCR, quantitative PCR, quantitative real-time PCR, RT-PCR, thermal asymmetric interlaces (TAIL) PCR, touchdown PCR, and PAN- AC. Additionally, one skilled in the art would understand how to practice these variations on the basic PCR technique.

[0039] "Biomarker," as used herein, generally refers to an organic biomolecule that is differentially present in a sample taken from a subject of one phenotypic status (e.g., adipose tissue hypertrophy) as compared with another phenotypic status (e.g., no adipose tissue hypertrophy). A biomarker is differentially present between different phenotypic statuses if the mean or median expression level of the biomarker in the different groups is calculated to be statistically significant. Common tests for statistical significance include, among others, t- test, ANOVA, Kruskal-Wallis, Wilcoxon, Mann- Whitney and odds ratio. Biomarkers, alone or in combination, provide measures of relative risk that a subject belongs to one phenotypic status or another. As such, they are useful as markers for disease (diagnostics), therapeutic effectiveness of a drug (theranostics), and for drug toxicity. The differential presence of a biomarker may include, for example, an expression level of at least 10%, 20%, 50%, 100%, 200%, 500% or more in the lipogenic adenovirus infected cell compared with the expression level of the biomarker endogenously expressed in the normal cell. [0040] "Subject," as used herein, includes individuals who require intervention or manipulation due to the presence of lipogenic adenovirus infection. Furthermore, the term "subject" includes non-human animals and humans.

[0041] The invention relates to compositions and methods for predicting a predisposition to or the presence of abnormal adipose tissue hypertrophy. The methods of the invention include the detection of a lipogenic adenovirus alone or in combination with the detection of specific biomarkers that are selectively expressed with lipogenic adenovirus infection. The lipogenic adenovirus and biomarkers may be detected by any method known in the art, including, but not limited to nucleic acid and/or protein detection techniques, PCR, and antibody-based methods (including but not limited to immuno-cytochemistry).

[0042] Lipogenic adenoviruses may include adenoviruses that are capable of stimulating increase lipid production in cells, tissues, and/or organs by facilitating expression of lipogenic enzymes, which in turn produce excess fatty acids and promote fat storage. The lipogenic adenoviruses of the invention may include Ad-5, Ad-36, and Ad-37, for example. In some embodiments, the lipogenic adenoviruses may be responsible for producing abnormal adipose tissue deposits in subjects afflicted with Madelung's disease, in subjects infected with HIV, and in subjects having non-specific abnormal adipose tissue deposits, for example.

[0043] In a specific embodiment, the invention relates to compositions and methods for predicting a predisposition to or the presence of abnormal adipose tissue hypertrophy in patients afflicted with Madelung's disease. Madelung's disease is a rare disease characterized by abnormal fat deposits (i.e., abnormal adipose tissue hypertrophy), mainly in the upper body. The abnormal fat deposits of three patients with Madelung's disease was obtained and screened for the presence of lipogenic adenovirus by performing a nested PCR assay. The data showed the presence of Ad-36 DNA in abnormal adipose tissue of the three patients.

[0044] In a specific embodiment, the invention relates to compositions and methods for predicting a predisposition to or the presence of abnormal adipose tissue hypertrophy in HIV/ AIDS patients. The occurrence of fatty deposits in HIV/ AIDS patients upon initiation of anti-HIV agents is well known, especially after starting a regimen of protease inhibitors. Studies have estimated that deposits of abnormal adipose tissue occur in greater than about 80% of HIV/ AIDS patients. Typically, abnormal adipose tissue hypertrophy in HIV/ AIDS patients is characterized by non-cancerous lipomas and deposits of excess adipose tissue, particularly around the upper body and neck, along with areas of atrophy of the adipose tissue. Despite the prevalence of abnormal adipose tissue hypertrophy among HIV/ AIDS patient, the etiology of abnormal adipose tissue hypertrophy in HIV/AIDS patients has been unclear.

[0045] A study was conducted that showed a majority of HIV/ AIDS patients having abnormal adipose tissue hypertrophy, as described in the examples below, were found to be infected with lipogenic adenovirus. Tissue samples were taken from HIV/ AIDS patients of excess abnormal adipose tissue deposition in the neck and trunk areas. The samples were screened by performing a nested PCR assay and the results demonstrated that a majority of AIDS/HIV patients were positive for lipogenic adenovirus. Accordingly, there is a correlation between infection with lipogenic adenovirus and a predisposition to or the development of adipose tissue hypertrophy in HIV/AIDS patients.

[0046] In a specific embodiment, the invention relates to compositions and methods for predicting a predisposition to or the presence of abnormal adipose tissue hypertrophy in patients having non-specific abnormal adipose tissue deposits. Deposition of abnormal adipose tissue occurs sporadically in individuals and no specific diagnosis can be given. A study was conducted that examined a subject having abnormal fat deposits in the chest and abdominal cavity. The fat deposits were so severe that they interfered with respiration and surgical debulking of the fat was necessary to save the subject's life. Samples of the fat were assayed by nested PCR and the results demonstrated the presence of Ad-36 DNA in the fat samples extracted from the subject.

[0047] While not intending to be limited to a particular mechanism, in some embodiments, the molecular mechanism of lipogenic adenovirus infection is the stimulation of lipogenic enzymes that increase fat deposition in adipose tissues and cause differentiation of adult stem cells in adipose tissue to adipocytes. Specific lipogenic enzymes may be over-expressed or expressed in the cell, such as fatty acid synthase (FAS), glycerol-3-phosphodehydrogenase, lipoprotein lipase (LPL), SREBP-I, SCDl, CPT 1, PPAR-gamma, and L-type pyruvate kinase. These lipogenic enzymes may be responsible for the formation of excess fatty acids and promote fat storage within the cells of multiple organs.

[0048] In a specific embodiment of the invention, an interaction may occur between lipogenic adenovirus infection and other factors that stimulate adipogenesis, such as anti-HIV drugs (e.g., protease inhibitors). Addition of NGNA (a compound known to have anti-HIV activity) to 3T3-L1 cells (a mouse stem cell line) causes increased expression of fatty acid synthase as compared to control. FAS is the critical enzyme responsible for converting intracellular glucose into fatty acids. Higher levels of FAS mRNA produce greater amounts of FAS, which produces greater amounts of fatty acids within the cell. When 3T3-L1 cells are exposed to the combination of Ad-36 and NGNA, greater deposition of lipid occurs in those cells than without the NGNA. These data demonstrate that it is the combination of Ad- 36 and the anti-HIV drugs that cause an increased deposition of fat producing the abnormal fat deposits seen in some HIV+/ AIDS patients.

Biomarkers

[0049] A specific embodiment of the invention is directed to biomarkers that are characteristic of lipogenic adenovirus infection. The biomarkers of lipogenic adenovirus infection include lipogenic enzymes such as FAS, PPAR family proteins, C/EBP, ADD- 1/SREBP-l, glycerol-3-phosphdehydrogenase, and lipoprotein lipase. Depending on the cell type, following lipogenic adenovirus infection, lipogenic enzymes may by expressed in cells not normally expressing the specific lipogenic enzyme(s) or over-expressed in cells that normally express the specific lipogenic enzyme(s).

[0050] For example, FAS is normally expressed in adult cells such as epithelial cells of the duodenum and stomach, hemopoietic cells, appendix, ganglion cells of alimentary tract, hepatocytes, mast cells, seminal vesicle, umbrella cells of urinary bladder, adrenal zona fasciculate cells, adipocytes, anterior pituitary cells, basket cells of cerebellum, cerebral cortical neurons, deciduas, decidualized stromal cells of endometrium, epithelial cells of apocrine gland, duct and acinus of breast, prostate, and sebaceous gland, letein cells, and Type II alveolar cells of lung. Additionally, FAS is also expressed in fetal cells such as anterior pituitary cells, chondrocytes of tracheobronchial wall, endothelium of blood vessels and heart, epithelial cells of bronchus, esphagogastrointestinal tract, lung, pancreas, prostate, thyroid, tongue, trachea, proximal tubules of kidney, fibroblasts, nodal lymphocytes, neuroblasts in adrenal medulla, thymocytes, striated myocytes of tongue, epithelial cells of salivary glands and tracheobronchial glands, hemopoietic cells, heptocytes, Lanhans cells of chorionic villi, osteoblasts, perivertebral fibroblastic cells, Schwann cells of sympathetic ganglion and Auerbach plexus, subcapsular cells of adrenal, adipocytes, Leidig cells of testis, mast cells, uroepithelium of urinary tract, and adrenocortical cells of upper layer. Therefore, a baseline value of FAS expression can be determined in these cells and then compared with an over-expression value in cells infected with lipogenic adenovirus.

[0051] In another embodiment of the invention, a biomarker profile following lipogenic adenovirus infection may be used for determining therapeutic efficacy or toxicity of a compound. If the compound has a pharmaceutical impact on the subject, organ or cell following lipogenic adenovirus infection, the phenotype (e.g., the pattern or profile) of the biomarkers changes towards a non-lipogenic adenovirus infection profile. For example, FAS and lipoprotein lipase expression are increased following lipogenic adenovirus infection. Therefore, one can follow the course of the amounts of these biomarkers in the subject, organ, or cell during the course of treatment. Accordingly, this method involves measuring one or more biomarkers following lipogenic adenovirus infection. Methods for measuring the specific biomarkers are a matter of routine experimentation and are known by those of skill in the art.

Immunoanalytical Detection Methods

[0052] Antibodies reactive to lipogenic adenoviruses and biomarker proteins may be employed for the detection of lipogenic adenovirus and biomarker proteins in a subject. Exemplary screening immunoanalytical techniques include without limitation, standard virus neutralization assay techniques or enzyme immunoassay techniques well known in the art. The standard virus neutralization assay may be used to identify the presence of antibodies reactive to a lipogenic adenovirus in a biological sample such as a serum sample obtained from a subject. A standard virus neutralization assay is described in the specific examples, below.

[0053] Techniques for raising and purifying antibodies against these lipogenic adenoviruses or fragments thereof (e.g., fiber protein, hexon protein, or fragments thereof), or other proteins (or fragments thereof) from these viruses for use in these immunoassay techniques may be prepared by conventional techniques are well known in the art. In a specific embodiment of the invention, antibodies will bind lipogenic adenovirus virus or lipogenic adenovirus proteins from solution as well as react with these proteins on Western or immunoblots or polyacrylamide gels. In a specific embodiment, the following fiber protein sequences, in Table 1 below, may be employed to generate antibodies reactive to lipogenic adenoviruses or may be used to detect lipogenic adenovirus antibodies (e.g., in a serum neutralization assay): Table 1

Amino Acid Amino Acid End Sequence Start Position Position

11 17 FNPVYPY (SEQ ID NO.: 1)

24 35 NIPFLTPPFVSS (SEQ ID NO.: 2)

41 55 FPPGVLSLKLADPIA (SEQ ID NO.: 3)

57 73 ANGNVSLKVGGGLTVEQ (SEQ ID NO.: 4 )

75 88 SGKLSVDTKAPLQV (SEQ ID NO.: 5)

113 121 AGHGLAVVT (SEQ ID NO.: 6)

126 138 SLPSLVGTLWLT (SEQ ID NO.: 7)

189 195 PSPNCKV (SEQ ID NO.: 8)

201 232 SKLTLALTKCGSQILATVSLLWTGKYAIISD (SEQ ID NO.: 9 )

235 254 NPKQFSIKLLFNDKGVLLSD (SEQ ID NO.: 10)

275 281 YKEAVGF (SEQ ID NO.: 11)

316 329 LGGEVYQPGFIWK (SEQ ID NO.: 12 )

336 342 ANCAYSI (SEQ ID NO.: 13 )

348 359 WGKVYKDPIPYD (SEQ ID NO.: 14 )

VETARDSKLT (SEQ ID NO.: 15)

LGGEVYQPGFIWK (SEQ ID NO.: 16)

WGKVYKDPIPYD (SEQ ID NO.: 17)

GTGSSAHG (SEQ ID NO.: 18)

[0054] In another specific embodiment, antibodies will detect the presence of lipogenic adenovirus or lipogenic adenovirus proteins in a biological sample, using immunocytochemical techniques. Specific embodiments relating to methods for detecting lipogenic adenovirus or lipogenic adenovirus proteins include methods well known in the art such as enzyme linked immunosorbent assays (ELISA), radioimmunoassay (RIA), immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA), including sandwich assays using monoclonal and/or polyclonal antibodies.

Nucleic Acid Detection Methods

[0055] The lipogenic adenoviruses and biomarkers may be detected by nucleic acid detection techniques, such as PCR and non-PCR techniques. The nucleic acid probe hybridization assay techniques used in these methods of the invention will be standard techniques (optionally after amplification of DNA or RNA extracted from a sample of blood, other body fluid, feces, tissue or organ) using nucleic acid probes (and primers if amplification is employed) made available by the lipogenic adenoviruses identified and made available by the invention. The sequences of nucleic acids characteristic of the lipogenic adenoviruses can be determined by standard techniques once the viruses are conventionally isolated, and probes and primers that are specific for the viruses and that provide the basis for nucleic acid probes and primers that can be used in nucleic acid based assays for the viruses are prepared using conventional techniques on the basis of the sequences. [0056] Initially, screening involves amplification of the relevant lipogenic adenovirus sequences. In a specific embodiment of the invention, the screening method involves a non- PCR based strategy. Such screening methods include two-step label amplification methodologies that are well known in the art. Both PCR and non-PCR based screening strategies can detect target sequences with a high level of sensitivity.

[0057] One embodiment of the invention relates to target amplification. Here, the target nucleic acid sequence is amplified with polymerase. One specific method using polymerase- driven amplification is the polymerase chain reaction (PCR). The polymerase chain reaction and other polymerase-driven amplification assays can achieve over a million- fold increase in copy number through the use of polymerase-driven amplification cycles. Once amplified, the resulting nucleic acid can be sequenced or used as a substrate for DNA probes.

[0058] Quantitative amplification methods (e.g., quantitative PCR or quantitative linear amplification) can be used to quantify the amount of target nucleic acids. Methods of quantitative amplification are disclosed in, e.g., U.S. Patent Nos. 6,180,349; 6,033,854; and 5,972,602, as well as in, e.g., Gibson et al., Genome Research 6:995-1001 (1996); DeGraves, et al, Biotechniques 34(l):106-10, 112-5 (2003); Deiman B, et al., Mol Biotechnol 20(2): 163-79 (2002). Amplifications may be monitored in "real time."

[0059] In general, quantitative amplification is based on the monitoring of the signal (e.g., fluorescence of a probe) representing copies of the template in cycles of an amplification (e.g., PCR) reaction. In the initial cycles of the PCR, a very low signal is observed because the quantity of the amplicon formed does not support a measurable signal output from the assay. After the initial cycles, as the amount of formed amplicon increases, the signal intensity increases to a measurable level and reaches a plateau in later cycles when the PCR enters into a non-logarithmic phase. Through a plot of the signal intensity versus the cycle number, the specific cycle at which a measurable signal is obtained from the PCR reaction can be deduced and used to back-calculate the quantity of the target before the start of the PCR. The number of the specific cycles that is determined by this method is typically referred to as the cycle threshold (Ct). Exemplary methods are described in, e.g., Heid et al Genome Methods 6:986-94 (1996) with reference to hydrolysis probes.

[0060] One method for detection of amplification products is the 5 '-3' exonuclease "hydrolysis" PCR assay (also referred to as the TaqMan™ assay) (U.S. Pat. Nos. 5,210,015 and 5,487,972; Holland et al, Proc. Natl Acad. Sci. USA 88: 7276-7280 (1991); Lee et al., Nucleic Acids Res. 21 : 3761-3766 (1993)). This assay detects the accumulation of a specific PCR product by hybridization and cleavage of a doubly labeled fluorogenic probe (the "TaqMan™" probe) during the amplification reaction. The fluorogenic probe consists of an oligonucleotide labeled with both a fluorescent reporter dye and a quencher dye. During PCR, this probe is cleaved by the 5'-exonuclease activity of DNA polymerase if, and only if, it hybridizes to the segment being amplified. Cleavage of the probe generates an increase in the fluorescence intensity of the reporter dye.

[0061] Another method of detecting amplification products that relies on the use of energy transfer is the "beacon probe" method described by Tyagi and Kramer {Nature Biotech. 14:303-309 (1996)), which is also the subject of U.S. Patent Nos. 5,119,801 and 5,312,728. This method employs oligonucleotide hybridization probes that can form hairpin structures. On one end of the hybridization probe (either the 5' or 3' end), there is a donor fluorophore, and on the other end, an acceptor moiety. In the case of the Tyagi and Kramer method, this acceptor moiety is a quencher, that is, the acceptor absorbs energy released by the donor, but then does not itself fluoresce. Thus, when the beacon is in the open conformation, the fluorescence of the donor fluorophore is detectable, whereas when the beacon is in the hairpin (closed) conformation, the fluorescence of the donor fluorophore is quenched. When employed in PCR, the molecular beacon probe, which hybridizes to one of the strands of the PCR product, is in the open conformation and fluorescence is detected, and the probes that remain unhybridized will not fluoresce (Tyagi and Kramer, Nature Biotechnol. 14: 303-306 (1996)). As a result, the amount of fluorescence will increase as the amount of PCR product increases, and thus may be used as a measure of the progress of the PCR. Those of skill in the art will recognize that other methods of quantitative amplification are also available.

[0062] Various other techniques for performing quantitative amplification of a nucleic acid are also known. For example, some methodologies employ one or more probe oligonucleotides that are structured such that a change in fluorescence is generated when the oligonucleotide(s) is hybridized to a target nucleic acid. For example, one such method involves a dual fluorophore approach that exploits fluorescence resonance energy transfer (FRET), e.g., LightCycler™ hybridization probes, where two oligo probes anneal to the amplicon. The oligonucleotides are designed to hybridize in a head-to-tail orientation with the fluorophores separated at a distance that is compatible with efficient energy transfer. Other examples of labeled oligonucleotides that are structured to emit a signal when bound to a nucleic acid or incorporated into an extension product include: Scorpions™ probes (e.g., Whitcombe et al., Nature Biotechnology 17:804-807, 1999, and U.S. Pat. No. 6,326,145), Sunrise™ (or Amplifluor™) probes (e.g., Nazarenko et al., Nuc. Acids Res. 25:2516-2521, 1997, and U.S. Pat. No. 6,117,635), and probes that form a secondary structure that results in reduced signal without a quencher and that emits increased signal when hybridized to a target (e.g., Lux probes™).

[0063] In other embodiments, intercalating agents that produce a signal when intercalated in double stranded DNA may be used. Exemplary agents include SYBR GREEN™ and SYBR GOLD™. Since these agents are not template-specific, it is assumed that the signal is generated based on template-specific amplification. This can be confirmed by monitoring signal as a function of temperature because melting point of template sequences will generally be much higher than, for example, primer-dimers, etc.

[0064] In a specific embodiment, the following primers may be employed for PCR amplification of the nucleic acid sequence encoding the Ad-36 hexon protein:

SEQ ID NO.: 19: 5'-ggtggacaaccacccactac-3' (Forward primer) SEQ ID NO.:20: 5'- tggacaaccacccactacaa-3 ' (Forward primer)

SEQ ID NO.:21 : 5'- cagcggagcttgtatcttcc-3 ' (Reverse primer)

[0065] In a another specific embodiment, nested PCR may be used to detect Ad-36 DNA in biological samples. Four primers were designed to unique regions of the Ad-36 fiber protein gene for use in a nested PCR assay for detection of viral DNA, which are as follows: outer forward primer (5'-gtctggaaaactgagtgtggata) (SEQ ID No.: 22), outer reverse primer ( 5'- atccaaaatcaaatgtaatagagt) (SEQ ID No.: 23), inner forward primer (5'-ttaactggaaaaggaataggta) (SEQ ID No.: 24), inner reverse primer (5'- ggtgttgttggttggcttaggata) (SEQ ID No.: 25).

[0066] In a specific embodiment, the following primers may be employed for detecting Ad- 36 fiber protein using SYBRgreen method:

Forward: 5'-taagccaaccaacaacacca-3' (SEQ ID NO.: 32) Reverse: 5'-tgcacaattggcatcagttt-3' (SEQ ID NO.: 33) Forward: 5'-ggccatggtttagcagttgt-3' (SEQ ID NO.: 34) Reverse: 5'-gtccaaagggtgcgtgtatc-3' (SEQ ID NO.: 35) Forward: 5'-ttaactggaaaaggaataggta-3' (SEQ ID NO.: 36)

Reverse: 5'-ggtgttgttggttggcttaggata-3' (SEQ ID NO.: 37)

[0067] In another specific embodiment, the following primers may be employed for detecting Ad-36 fiber protein using the Taqman method:

Forward: 5'-caatgggaatgtctcactcaaggt-3' (SEQ ID NO.: 38)

Reverse: 5'-agggtgccttagtatccacact-3' (SEQ ID NO.: 39)

5'-cagttttccagactgttgttct-3' (SEQ ID NO.: 40)

[0068] When the probes are used to detect the presence of the lipogenic adenovirus nucleic acid sequences or biomarker nucleic acid sequences, nucleic acid may be first isolated from the biological sample. The sample nucleic acid may be prepared in various ways known in the art, to facilitate detection of the target sequence, e.g., denaturation, restriction digestion, electrophoresis or dot blotting. The targeted region of the analyte nucleic acid usually must be at least partially single-stranded to form hybrids with the targeting sequence of the probe. If the sequence is naturally single-stranded, denaturation will not be required. However, if the sequence is double-stranded, the sequence will probably need to be denatured. Denaturation can be carried out by various techniques well known in the art.

[0069] Analyte nucleic acid and probe are incubated under conditions which promote stable hybrid formation of the target sequence in the analyte. The region of the probes which is used to bind to the analyte can be made completely complementary to the targeted region of the lipogenic adenovirus of interest, and in particular the Ad-36 fiber coat protein or hexon protein. For example, the following probes may be used to detect the nucleic acid encoding Ad-36 fiber coat protein:

SEQ ID NO: 26: 5'-agttgaaacagcaagagactcaaag-3' SEQ ID NO: 27: 5'-ggtactggatcaagtgcacatggag-3' SEQ ID NO: 28: 5 '-ttgaaacagcaagagactcaaagctaac-3'

[0070] High stringency conditions may be desirable in order to prevent false positives. However, conditions of high stringency are used only if the probes are complementary to regions of the lipogenic adenovirus. The stringency of hybridization is determined by a number of factors during hybridization and during the washing procedure, including temperature, ionic strength, base composition, probe length, and concentration of formamide. [0071] Detection, if any, of the resulting hybrid is usually accomplished by the use of labeled probes. Alternatively, however, the probe may be unlabeled, but may be detectable by specific binding with a ligand which is labeled, either directly or indirectly. Suitable labels, and method for labeling probes and ligands are well known in the art, and include, for example, radioactive labels which may be incorporated by known methods (e.g., nick translation, random priming or kinasing), biotin, fluorescent groups, chemiluminescent groups (e.g., dioxetanes) enzymes, antibodies, gold nanoparticles and the like. Variations of this basic scheme are known in the art, and include those variations that facilitate separation of the hybrids to be detected from extraneous materials and/or that amplify the signal from the labeled moiety.

[0072] As noted above, non-PCR based screening assays are also contemplated by this invention. This procedure hybridizes a nucleic acid probe (or analog such as a methyl phosphonate backbone replacing the normal phosphodiester) to the low level DNA target. This probe may have an enzyme covalently linked to the probe, such that the covalent linkage does not interfere with the specificity of the hybridization. The enzyme-probe-conjugate- target nucleic acid complex can then be isolated away from the free probe conjugate and a substrate is added for enzyme detection. Enzymatic activity is observed as a change in color development or luminescent output resulting in about a 10³ to about a 10⁶ increase in sensitivity.

[0073] Two-step label amplification methodologies are known in the art. These assays work on the principle that a small ligand (such as digioxigenin, biotin, or the like) is attached to a nucleic acid probe capable of specific binding the adenovirus sequence region of interest. In one example, the small ligand attached to the nucleic acid probe is specifically recognized by an antibody-enzyme conjugate. In one embodiment of this example, digioexigenin is attached to the nucleic acid probe. Hybridization is detected by an antibody-alkaline phosphatase conjugate which turns over a chemiluminescent substrate. In a second example, the small ligand is recognized by a second ligand-enzyme conjugate that is capable of specifically complexing to the first ligand. A well known embodiment of this example is the biotin-avidin type interactions.

[0074] It is also contemplated within the scope of this invention that the nucleic acid probe assays of this invention will employ a cocktail of nucleic acid probes capable of detecting various species of adenoviruses. Thus, in one example to detect the presence of Ad-36, Ad- 37 and/or Ad-5, for example, in a biological sample, more than one probe complementary of the targeted regions of interest in the various types of adenovirus may be employed.

[0075] As the skilled will understand, more than one strain of lipogenic adenovirus may be tested for simultaneously in an immunological or nucleic acid-based assay method for testing for virus in accordance with the invention and kits may be assembled to facilitate carrying out the methods for a particular virus or a plurality of them.

Correlating

[0076] As discussed herein, the presence of or predisposition to abnormal adipose tissue hypertrophy is correlated with the presence of lipogenic adenovirus infection. Thus, in some embodiments, the presence of lipogenic adenovirus markers is sufficient for one to determine that an individual likely has, or has a predisposition to, abnormal adipose tissue hypertrophy. In some embodiments, the quantity of a particular lipogenic virus marker (e.g., a virus nucleic acid or protein, an antibody specific for such proteins, or a biomarker for lipogenic virus infection as described herein) is correlated with the presence or absence of lipogenic adenovirus infection. For example, in some embodiments, the presence lipogenic adenovirus infection may be compared to a threshold value that distinguishes between the presence or absence of lipogenic virus infection. It is understood that a threshold value will likely vary depending on the assays used to measure lipogenic adenovirus infection, but it is also understood that it is a relatively simple matter to determine a threshold value or range by measuring the presence of lipogenic adenovirus in diseased and normal samples using the particular desired assay and then determining a value that distinguishes at least a majority of the abnormal adipose tissue hypertrophy samples from a majority of non-adipose tissue hypertrophy samples.

[0077] Moreover, in making a correlation based on presence of lipogenic adenovirus biomarkers, such as the expression or over-expression of lipogenic enzymes, the presence of a lipogenic adenovirus biomarker may be compared to a threshold value that distinguished between one diagnosis, determination of a predisposition, risk assessment, etc., to another. The threshold value or range may be determined by measuring the presence of lipogenic adenovirus biomarker in diseased and normal samples using the particular desired assay and then determining a value that distinguished at least a majority of the abnormal adipose tissue hypertrophy from a majority of non-adipose tissue hypertrophy samples. [0078] A manual comparison can be made or a computer can compare and analyze the values to detect disease, assess the risk of contracting disease, determining a predisposition to disease, stage disease, diagnose disease, monitor, or aid in the selection of treatment for a person with disease.

[0079] In some embodiments the threshold value is set such that there is at least 10, 20, 30, 40, 50, 60, 70, 80% or more sensitivity and at least 70% specificity with regard to detecting abnormal adipose tissue hypertrophy. In some embodiments, the methods comprise recording a diagnosis, prognosis, risk assessment or classification, based on lipogenic adenovirus status determined from an individual. Any type of recordation is contemplated, including electronic recordation, e.g., by a computer.

Computer Program

[0080] The correlations for the methods described herein can involve computer-based calculations and tools. The tools are advantageously provided in the form of computer programs that are executable by a general purpose computer system (referred to herein as a "host computer") of conventional design. The host computer may be configured with many different hardware components and can be made in many dimensions and styles (e.g., desktop PC, laptop, tablet PC, handheld computer, server, workstation, mainframe). Standard components, such as monitors, keyboards, disk drives, CD and/or DVD drives, and the like, may be included. Where the host computer is attached to a network, the connections may be provided via any suitable transport media (e.g., wired, optical, and/or wireless media) and any suitable communication protocol (e.g., TCP/IP); the host computer may include suitable networking hardware (e.g., modem, Ethernet card, WiFi card). The host computer may implement any of a variety of operating systems, including UNIX, Linux, Microsoft Windows, MacOS, or any other operating system.

[0081] Computer code for implementing aspects of the invention may be written in a variety of languages, including PERL, C, C++, Java, JavaScript, VBScript, AWK, or any other scripting or programming language that can be executed on the host computer or that can be compiled to execute on the host computer. Code may also be written or distributed in low level languages such as assembler languages or machine languages.

[0082] The host computer system advantageously provides an interface via which the user controls operation of the tools. In the examples described herein, software tools are implemented as scripts (e.g., using PERL), execution of which can be initiated by a user from a standard command line interface of an operating system such as Linux or UNIX. Those skilled in the art will appreciate that commands can be adapted to the operating system as appropriate. In other embodiments, a graphical user interface may be provided, allowing the user to control operations using a pointing device. Thus, the present invention is not limited to any particular user interface.

[0083] Scripts or programs incorporating various features of the invention may be encoded on various computer readable media for storage and/or transmission. Examples of suitable media include magnetic disk or tape, optical storage media such as compact disk (CD) or DVD (digital versatile disk), flash memory, and carrier signals adapted for transmission via wired, optical, and/or wireless networks conforming to a variety of protocols, including the Internet.

Treatments

[0084] Subsequent to a determination of whether a subject has been infected with a lipogenic adenovirus, the subject may need specialized treatment. For example, subjects who are negative for lipogenic adenovirus may be prescribed vaccines or other agents that will prevent infection. In a specific aspect, subject who are HIV/ AIDS positive but lipogenic adenovirus negative may be administered a vaccine to prevent future infection.

[0085] The vaccines of the invention effective to prevent infection of lipogenic adenovirus in a subject and may be made by conventional methods known in the art. The vaccines include an immunogenic component that is live, inactive virus, killed virus, antigenic peptide generated from the fiber coat protein or hexon protein, or an epitope-comprising segment thereof. An anti-lipogenic adenovirus vaccine of the invention, where the active ingredient is nucleic acid, will also be a standard preparation for vaccines of that type. With vaccines of this type, the nucleic acid is not the immunogen but is expressed in vivo after administration of the vaccine as a peptide or protein which in turn is immunogenic. Vaccines of this type will be administered by techniques known in the art for such vaccines (e.g., intramuscular injection). Dosing will also be according to procedures known in the art to cause and maintain protective immunity against viral obesity in the vaccinated individual. [0086] The vaccine compositions may include carriers, excipients, adjuvants, buffers, antimicrobials, preservatives and the like as well understood in the art. Thus, in addition to the active ingredient, the vaccines will have suitable compositions, usually aqueous buffers, such as phosphate-buffered saline or the like, in which the active ingredient will be suspended along with, optionally, any of various immune-system stimulating adjuvants used in human vaccine preparations, antimicrobial compositions, and other compositions to stabilize the preparations. All compositions included with the vaccine preparation will be suitable for administration to humans. The vaccine preparation may be stored in lyophilized form and then combined with solution soon before administration. For oral administration, the vaccine composition may be in solution, tablet or pill form. The concentration of active component in solution with which it is administered typically will be between about 1 ng and about 1 mg/ml. The anti-lipogenic adenovirus vaccines of the invention will be administered intranasally, orally, or by injection intravenously, intramuscularly, subcutaneously or peritoneally.

[0087] Appropriate dosing of the anti-obesity vaccine is well within the skill of medical practitioners and will depend on a number of factors including the age of the subject being treated, the urgency of the subject's developing protective immunity, the status of the subject's immune system, and other factors known to those of skill in the art. The vaccine typically will be administered in several steps in order to cause and maintain protective immunity against lipogenic adenoviurs in the subject being vaccinated. Thus, after the primary vaccination, there may be between one and about ten booster vaccinations separated by periods between about 1 week and 10 years.

[0088] In another embodiment of the invention, subjects who are positive for lipogenic adenovirus infection may consider stopping or changing a specific regimen, such as drug or dietary regimen. For example, HIV/ AIDS patients may consider stopping treatment regimens that are associated with fat gain, such as anti-HIV treatments with protease inhibitors. Alternatively, HIV/ AIDS patients may consider changing anti-HIV treatments to treatments not associated with fat gain.

[0089] In a further embodiment, compounds may be administered to subjects positive for lipogenic adenovirus that that target the lipogenic enzymes that increase or promote fat deposition in adipose tissue or prevent the differentiation of adult stem cells to adipocytes. Moreover, antiviral effective against lipogenic adenoviruses may be used in all patients with abnormal adipose tissue hypertrophy to start such agents to block the virus from making more adipose tissue and potentially reducing the adipose tissue present. [0090] Without further elaboration, it is believed that one skilled in the art using the preceding description can utilize the invention to the fullest extent. The following examples are illustrative only, and not limiting of the disclosure in any way whatsoever.

EXAMPLES

Specific Example 1 :

[0091] Adipose tissue samples from 5 HIV patients were obtained and assayed for Ad-36 DNA by nested polymerase chain assay (PCR) using primers made to unique DNA sequences in the Ad-36 fiber protein genome. The HIV/ AIDS patients presented with lipodystrophy in that all patients have excess adipose tissue. 5 of the 5 samples, or 100%, had Ad-36 DNA in the adipose tissue. An agarose PCR gel demonstrated the presence of Ad-36 DNA in the adipose tissue of HIV infected patients. As noted above, the 5 samples were assayed in duplicate.

Specific Example 2:

[0092] The occurrence of abnormal fat deposits in HIV+ patients upon initiation of anti- HIV agents is well known, particularly after starting protease inhibitors. The etiology of these deposits previously has been unknown. Fifty two individuals who were positive for HIV and who had abnormal fat deposits were evaluated by obtaining biopsies of adipose tissue at the time of liposuction for removal of the deposits. The adipose tissue was tested for Ad-36 DNA by quantitative polymerase chain reaction assay (PCR) using proprietary specific primers and probe for Ad-36 fiber protein DNA and an ABI Plus One PCR apparatus. Of the 52 HIV+ individuals, 58% were positive for Ad-36 DNA. These data show that a majority of abnormal fat deposits in HIV+/AIDS patients are due to Ad-36.

Specific Example 3 :

[0093] A virus neutralization assay (serum neutralization assay) was used to assay serum for antibody reactive with lipogenic adenovirus in serum of test subjects. First, serum was thawed and heat-inactivated for about 30 minutes at 56⁰C. The assay was carried out in standard 96-well microtiter plates. Serial two fold dilutions (1 :2 to 1 : 1024) were made with the medium that is the A549 growth medium described in Example 3 but lacks the fetal calf serum and sodium bicarbonate. 50 microliters of each dilution was added in duplicate to the wells of the plate. 50 microliters of virus suspension (100 TCID50) was then added to each well. (TCID50 was calculated by serially diluting viral stock solution and inoculating A549 cells with the dilutions to determine the reciprocal of the highest dilution of virus which causes CPE in 50% of the material inoculated.) The plates were then incubated at 37⁰C for 1 hour. Then 100 microliters of A549 cell suspension, containing approximately 20,000 cells, was added to each well and the plate was further incubated at 37⁰C for 12 days. Crystal violet-ethanol was then added to each of the wells to fix and stain the cells and the plates were examined macroscopically for CPE. The highest serum dilution with no CPE is the titer. Controls used in the procedure were wells with no virus and wells with virus but no serum. A back titration was carried out to confirm that appropriate virus dilutions were used. Positive control was antisera to chicken adenovirus and human adenovirus. Presence of CPE with the virus and no CPE in the presence of serum was considered an indication of effective neutralization of the virus with antibody in serum, such that the serum was considered to have antibody against the virus. A titer of 1 :8 or greater was considered positive.

Specific Example 4:

[0094] The DNA sequence encoding Ad-36 (SEQ ID No.: 29) is as follows:

5'taacgaagaggctgtgaatttaatatttccagaatctatgattcttcaggctgacatagccagtgaagccatagttactcctctacatact cccactctgcctcccatacctgaattggaggaggatgaagaaatagacctccggtgctacgaggaaggttttcctcccagcgattcaga ggacgaacagggtgagcagcagatggctctaatctctgatttagcttgtgtgattgtggaggaacaagttgtgattgaaaaatctaccga gccagtacaaggctgtaggaactgccagtatcaccgggataagtccggagacccgaacgcttcctgcgctctgtgttacatgaaatct actttcagctttatttacagtaagtggagtgaatgtgagagaggctgagtgcttaacacataactgtaatgcttgaacagctgtgctaagtg tggtttatttttgttactaggtccggtgtcagaggatgagtcatcaccctcagaagaagaccacccgtctccccctgagctgtcaggcga aacgcccctgcaagtgcacagacccaccccagtcagagccagtggcgagaggcgagcagctgtagaaaaaattgaggacttgttac atgacatgggtggggatgaacctttggacctgagcttgaaacgccccaggaactaggcgcagctgcgcttagtcatgtgtaaataaag ttgtacaataaaagtatatgtgacgcatgcaaggtgtggtttatgactcatgggcggggcttagtcctatataagtggcaacacctgggc acttgggcacagaccttcagggagttcctgatggatgtgtggactatccttgcagactttagcaagacacgccggcttgtagaggatag ttcagacgggtgctccgggttctggagacactggtttggaactcctctatctcgcctggtgtatacagttaagaaggattataaagagga atttgaaaatctttttgctgactgctctggtctgctagattctctgaatcttggccaccagtcccttttccaggaaagggtactccacagcctt gatttttccagcccagggcgcactacagccggggttgcttttgtggtttttctggttgacaaatggagccaggacacccaactgagcag gggctacatcctggacttcgcagccatgcacctgtggagggcctggatcaggcagcggggacagagaatcttgaactactggcttct acagccagcagctccgggtcttcttcgtctacacagacaaacatccatgttggaggaagaaatgaggcaggccatggacgagaaccc gaggagcggcctggaccctccgtcggaagaggagctggattgaatcaggtatccagcctgtacccagagcttagcaaggtgctgac atccatggccaggggagtgaagagggagaggagcgatgggggtaataccgggatgatgaccgagctgactgccagtctgatgaat cggaagcgcccagagcgccttacctggtacgagctacagcaggagtgcagggatgagataggcctgatgcaggataaatatggcct ggagcagataaaaacccattggttgaacccagatgaggattgggaggaggctattaagaagtatgccaagatagccctgcgcccaga ttgcaagtacatagtgaccaagaccgtgaatatcagacatgcctgctacatctcggggaacggggcagaggtggtcatcgataccctg gacaaggccgccttcaggtgttgcatgatgggaatgagagcaggagtgatgaatatgaattccatgatcttcatgaacattaagttcaat ggagagaagtttaatggggtgctgttcatggccaacagccacatgaccctgcatggctgcagcttcttcggtttcaacaacatgtgcgc cgaggtctggggagctgctaagatcaggggatgtaagttttatggctgctggatgggcgtggtcggaagacccaagagcgagatgtc tgtgaagcagtgtgtgtttgagaaatgctacctgggagtctctaccgagggcaatgctagagtgagacactgctcttccatggagacgg gctgcttctgcctggtgaagggcacggcctctctgaagcataatatggtgaagggctgcacggatgagcgcatgtacaacatgctgac ctgcgattcgggggtctgccatatcctgaagaacatccatgtgacctcccaccccagaaagaagtggccagtgtttgagaataacctgc tgatcaagtgccatatgcacctgggtgccagaaggggcaccttccagccgtaccagtgcaactttagccagaccaagctgctgttgga gaacgatgccttctccagggtgaacctgaacggcatctttgacatggatgtctcggtgtacaagatcctgagatacgatgagaccaagt ccagggtgcgcgcttgcgagtgcgggggcagacacaccaggatgcaaccagtggccctggatgtgacagaggagctgagaccag accacctggtgatggcctgtaccgggaccgagttcagctccagtggggaggacacagattagaggtaggttttgagtagtgggcgtg gctaaggtgagtataaaggcggtgtcttacgagggtctttttgcttttctgcagacatcatgaacgggaccggcggggccttcgaaggg gggctttttagcccttatttgacaacccgcctgccgggatgggccggagttcgtcagaatgtgatgggatctacggtggatgggcgccc agtgcttccagcaaattcctcgaccatgacctacgcgaccgtggggagctcgtcgctcgacagcaccgccgcagccgcggcagccg cagccgccatgacagcgacgagactggcctcgagctacatgcccagcagcagcagtagcccytctgtgcccagttccatcatcgcc gaggagaaactgctggccctgctggcagagctggaagccctgagccgccagctggccgccctgacccagcaggtgtccgagctcc gcgagcaacagcagcagcaaaataaatgattcaataaacacagattctgattcaaacagcaaagcatctttattatttattttttcgcgcgc ggtaggccctggtccacctctcccgatcattgagagtgcggtggattttttccaggacccggtagaggtgggattggatgttgaggtac atgggcatgagcccgtcccgggggtggaggtagcaccactgcatggcctcgtgctctggggtcgtgttgtagatgatccagtcatagc aggggcgctgggcgtggtgctggatgatgtccttaaggaggagactgatggccacggggagccccttggtgtaggtgttggcgaag cggttgagctgggagggatgcatgcggggggagatgatgtgcagtttggcctggatcttgaggttggcaatgttgccgcccagatccc gcctggggttcatgttgtgcaggaccaccaggacggtgtagcccgtgcacttggggaacttatcatgcaacttggaagggaatgcgtg gaagaatttggagacgcccttgtgcccgcccaggttttccatgcactcatccatgatgatggcgatgggcccgtgggctgcggctttgg caaagacgtttctggggtcagagacatcgtaattatgctcctgggtgagatcatcataagacattttaatgaatttggggcggagggtgc cagattgggggacgatagttccctcgggccccggggcaaagttcccctcacagatctgcatctcccaggctttcatctcggaggggg ggatcatgtccacctgcggggcgatgaaaaaaacggtttccggggcgggggtgatgagctgcgaggagagcaggtttctcaacagc tgggacttgccgcacccggtcgggccgtagatgaccccgatgacgggttgcaggtggtagttcaaggacatgcagctgccgtcgtcc cggaggaggggggccacctcgttgagcatgtctctgacttggaggttttcccggacgagctcgccgaggaggcggtccccgcccag cgagaggagctcttgcagggaagcaaagtttttcaggggcttgagtccgtcggccatgggcatcttggcgagggtctgcgagaggag ctccaggcggtcccagagctcggtgacgtgctctacggcatctcgatccagcagacttcctcgtttcgggggttgggacgactgcgac tgtagggcacgagacgatgggcgtccagcgcggccagcgtcatgtccttccagggtctcagggtccgcgtgagtgtggtctccgtca cggtgaaggggtgggccccgggctgggcgcttgcaagggtgcgcttgagactcatcctgctggtgctgaaacgggcacggtcttcg ccctgcgcgtcggcgagatagcagttgaccatgagctcgtagttgagggcctcggcggcgtggcccttggcgcggagcttgcccttg gaagagcgcccgcaggcgggacagagaagggattgcagggcgtagagcttgggtgcgagaaaaacggactcgggggcgaagg cgtccgctccgcagtgggcgcagacggtctcgcactcgacgagccaggtgagctcgggctgctcggggtcaaaaaccagttttccc ccgttctttttgatgcgcttcttacctcgcgtctccatgagtctgtgtccgcgctcggtgacaaacaggctgtcggtgtccccgtagacgg acttgatgggcctgtcctgcagggacgtcccgcggtcctcctcgtagagaaactcggaccactctgagacaaaggcgcgcgtccacg ccaagacaaaggaggccacgtgcgaggggtagcggtcgttgtccaccagggggtccaccttttccaccgtgtgcagacacatgtcc ccctcctccgcatccaagaaggtgattggcttgtaggtgtaggccacgtgaccgggggtccccgacgggggggtataaaagggggc gggtctgtgctcgtcctcactctcttccgcgtcgctgtccacgagcgccagctgttggggtaggtattccctctctagagcgggcatgac ctcggcactcaggttgtcagtttctagaaacgaggaggatttgatgttggcctgccctgccgcgatgctttttaggagactttcatccatct ggtcagaaaagacaatttttttattgtcaagcttggtggcaaaggagccatagagggcgttggatagaagcttggcgatggatctcatgg tctgatttttgtcacggtcggcgcgctccttggccgcgatgtttagctggacatactcgcgcgcgacgcacttccattcggggaagacg gtggtgcgctcgtcgggcacgatcctgacgcgccagccgcggttatgcagggtgaccaggtccacgctggtggccacctcgccgc gcaggggctcgttggtccagcagagtctgccgcccttgcgcgagcagaacgggggcagcacatcaagcagatgctcgtcaggggg gtccgcatcgatggtgaagatgcccggacagagttccttgtcaaaataatcgatttttgaggatgcatcatccaaggccatctgccactc gcgggcggccagcgctcgctcgtaggggttgaggggcggaccccagggcatgggatgcgtgagggcggaggcgtacatgccgc agatgtcatacacatagatgggctccgagaggatgccgatgtaggtgggataacagcgccccccgcggatgctggcgcgcacgtag tcatacaactcgtgcgagggggccaagaaggcggggccgagattggtgcgctggggctgctcggcgcggaagacgatctggcga aagatggcgtgcgagtttgaggagatggtgggccgttggaagatgttaaagtgggcgtgaggcaggcggaccgagtcgcggatga agtgcgcgtaggagtcttgcagcttggcgacgagctcggcggtgacgaggacgtccatggcacagtagtccagcgtttcgcggatg atgtcataacccgcctctcctttcttctcccacagctcgcggttgagggcgtactcctcgtcatccttccagtactcccggagcgggaatc ctcgatcgtccgcacggtaagagcccagcatgtagaaatggttcacggccttgtagggacagcagcccttctccacggggagggcgt aagcttgagcggccttgcggagcgaggtgtgcgtcagggcgaaggtgtctctgaccatgactttcaagaactggtacttgaaatccga gtcgtcgcagccgccgtgctcccagagctcgaaatcggtgcgcttcttcgagagggggttaggcagagcgaaagtgacgtcattgaa gagaatcttgcctgcccgcggcatgaaattgcgggtgatgcggaaagggcccgggacggaggctcggttgttgatgacctgggcgg cgagcacgatctcgtcgaagccgttgatgttgtgcccgacgatgtagagttccatgaatcgcgggcggcctttgatgtgcggcagctttt tgagctcctcgtaggtgaggtcctcggggcattgcagtccgtgctgctcgagcgcccactcctggagatgtgggttggcttgcatgaa ggaagcccagagctcgcgggccatgagggtctggagctcgtcgcgaaagagacggaactgctggcccacggccatcttttcgggt gtgacgcagtagaaggtgagggggtcccgctcccagcgatcccagcgtaagcgcacggcgagatcgcgagcgagggcgaccag ctcggggtccccggagaatttcatgaccagcatgaaggggacgagctgcttgccgaaggaccccatccaggtgtaggtttctacatcg taggtgacaaagagccgctccgtgcgaggatgagagccgattgggaagaactggatttcctgccaccagttggacgagtggctgttg atgtgatgaaagtagaaatcccgccggcgaaccgagcactcgtgctgatgcttgtaaaagcgtccgcagtactcgcagcgctgcacg ggctgtacctcatccacgagatacacagcgcgtcccttgaggaggaacttcaggagtggcggccctggctggtggttttcatgttcgcc tgcgtgggactcaccctggggctcctcgaggacggagaggctgacgagcccgcgcgggagccaggtccagatctcggcgcggcg ggggcggagagcgaagacgagggcgcgcagttgggagctgtccatggtgtcgcggagatccaggtccgggggcagggttctgag gttgacctcgtagaagcgggtgagggcgtgcttgagatgcagatggtacttgatttctacgggtgagttggtggccgtgtccacgcatt gcatgagcccgtagctgcgcggggccacgaccgtgccgcggtgcgcttttagaagcggtgtcgcggacgcgctcccggcggcag cggcggttccggccccgcgggcaggggcggcagaggcacgtcggcgtggcgctcgggcaggtcccggtgctgcgccctgagag cgctggcgtgcgcgacgacgcggcggttgacatcctggatctgccgcctctgcgtgaagaccacgggccccgtgactttgaacctga aagacagttcaacagaatcaatctcggcgtcattgacggcggcctgacgcaggatctcttgcacgtcgcccgagttgtcctggtaggc gatctcggacatgaactgctcgatctcctcctcctggagatcgccgcggcccgcgcgctccacggtggcggcgaggtcattcgagat gcgacccattagctgcgagaaggcgcccaggccgctctcgttccagacgcggctgtagaccacgtccccgtcggcgtcgcgcgcg cgcatgaccacctgcgcgaggttgagctccacgtgccgcgtgaagacggcgtagttgcgcaggcgctggaagaggtagttgagggt ggtggcgatgtgctcggtgacgaagaagtacatgatccagcggcgcaggggcatctcgctgatgtcgccgatggcctccagcctttc catggcctcgtagaaatccacggcgaagttgaaaaactgggcgttgcgggccgagaccgtgagctcgtcttccaggagccggatga gctcggcgatggtggcgcgcacctcgcgctcgaaatccccggggacctcctcctcttcctcttcttccatgacgacctcttcttctatttct tcctctgggggcggtggtggtggcggggcccgacgacgacggcgacgcaccgggagacggtcgacgaagcgctcgatcatctcc ccgcggcggcgacgcatggtttcggtgacggcgcgaccccgttcgcgaggacgcagcgtgaagacgccgccggtcatctcccggt aatggggcgggtccccgttgggcagcgatagggcgctgacgatgcatcttatcaattgcggtgtaggggacgtgagcgcgtcgagat cgaccggatcggagaatctttcgaggaaagcgtctagccaatcgcagtcgcaaggtaagctcaaacacgtagcagccctgtggacg ctgttagaattgcggttgctgatgatgtaattgaagtaggcgtttttaaggcggcggatggtggcgaggaggaccaggtccttgggtcc cgcttgctggatgcggagccgctcggccatgccccaggcctggccctgacaccggctcaggttcttgtagtagtcatgcatgagccttt caatgtcatcactggcggaggcggagtcttccatgcgggtgaccccgacgcccctgagcggctgcacgagcgccaggtcggcgac gacgcgctcggcgaggatggcctgttgcacgcgggtgagggtgtcctggaagtcatccatgtcgacgaagcggtggtaggccccg gtgttgatggtgtaggtgcagttggccatgagcgaccagttgacggtctgcaggccgggttgcacgacctcggagtacctgagccgc gagaaggcgcgcgagtcgaagacgtagtcgttgcaggtgcgcacgaggtactggtagccgactaggaagtgcggcggcggctgg cggtagagcggccagcgctgggtggccggcgcgcccggggccaggtcctcgagcatgaggcggtggtagccgtagaggtagcg ggacatccaggtgatgccggcggcggtggtggaggcgcgcgggaactcgcggacgcggttccagatgttgcgcagcggcaggaa ataatccatggtcggcacggtctggccggtgagacgcgcgcagtcattgacgctctagaggcaaaaacgaaagcggttgagcgggc tcttcctccgtagcctggcggaacgcaaacgggttaggccgcgtgtgtaccccggttcgagtcccctcgaatcaggctggagccgcg actaacgtggtattggcactcccgtctcgacccgagcccgatagccgccaggatacggcggagagccctttttgccgaccgagtggg gtcgctagacttgaaagcggccgaaaaccccgccgggtagtggctcgcgcccgtagtctggagaagcatcgccagggttgagtcgc ggcagaacccggttcgcggacggccgcggcgagcgggacttggtcaccccgccgatttaaagacccacagccagccgacttctcc agttacgggagcgagcccccttttttctttttgccagatgcatcccgtcctgcgccaaatgcgtcccacccccccggcgaccaccgcga ccgcggccgtaacaggcgccggcgctagccagccacagacagagatggacttggaagagggcgaagggctggcgagactggg ggcgccgtccccggagcgacacccccgcgtgcagctgcagaaggacgtgcgcccggcgtacgtgcctgcgcagaacctgttcag ggaccgcagcggggaggagcccgaggagatgcgcgactgccggtttcgggcgggcagggagctgcgcgagggcctggaccgc cagcgcgtgctgcgcgacgaggatttcgagccgaacgagcagacggggatcagccccgcgcgcgcgcacgtggcggcggccaa cctggtgacggcctacgagcagacggtgaagcaggagcgcaacttccaaaagagtttcaacaaccacgtgcgcacgctgatagcgc gcgaggaggtggccctgggcctgatgcacctgtgggacctggcggaggccatcgtgcagaacccggacagcaagcctctgacgg cgcagctgttcctggtggtgcagcacagcagggacaacgaggcgttcagggaggcgctgctgaacatcgccgagcccgagggtcg ctggctgctggagctgatcaacatcttgcagagcatcgtagtgcaggagcgcagcctgagcctggccgagaaggtggcggcgatca actactcggtgctgagcctgggcaagttttacgcgcgcaagatttacaagacgccgtacgtgcccatagacaaggaggtgaagatag acagcttttacatgcgcatggcgctcaaggtgctgacgctgagcgacgacctgggcgtgtatcgcaacgaccgcatccacaaggccg tgagcacgagccggcggcgcgagctgagcgaccgcgagctgatgctgagcctgcgccgggcgctggtagggggcgccgccgg cggcgaggagtcctacttcgacatgggggcggacctgcattggcagccgagccggcgcgccttggaggccgcctacggtccagag gacttggatgaggatgaggaagaggaggaggatgcacccgttgcggggtactgacgcctccgtgatgtgtttttagatgtcccagcaa gccccggaccccgccataagggcggcgctgcaaagccagccgtccggtctagcatcggacgactgggaggccgcgatgcaacgc atcatggccctgacgacccgcaaccccgagtcctttagacaacagccgcaggccaacagactctcggccattctggaggcggtggtc ccctctcggaccaaccccacgcacgagaaggtgctggcgatcgtgaacgcgctggcggagaacaaggccatccgtcccgacgag gccgggctggtgtacaacgccctgctggagcgcgtgggccgctacaacagcacgaacgtgcagtccaacctggaccggctggtga cggacgtgcgcgaggccgtggcgcagcgcgagcggttcaagaacgagggcctgggctcgctggtggcgctgaacgccttcctgg cgacgcagccggcgaacgtgccgcgagggcaggacgattacaccaactttatcagcgcgctgcggctgatggtgaccgaggttcc ccagagcgaggtgtaccagtcgggcccggactactttttccagacaagccggcagggcctgcagacggtgaacctgagtcaggcttt caagaacctgcgcgggctgtggggcgtgcaggcgcccgtgggcgaccggtcgacggtgagcagcttgctgacgcccaactcgcg gctgctgctgctgctgatcgcgcccttcaccgacagcggcagcgtgaaccgcaactcgtacctgggccacctgctgacgctgtaccg cgaggccataggccaggcgcaggtggacgagcagaccttccaggagatcacgagcgtgagccgcgcgctggggcagaacgaca ccgacagtctgagggccaccctgaactttttgctgacaaatagacagcagaagatcccggcgcagtacgcactgtcggccgaggag gaaaggatcctgagatatgtgcagcagagcgtagggctgttcctgatgcaggagggtgccacccccagcgccgcgctggacatgac cgcgcgcaacatggaacctagcatgtacgccgccaaccggccgttcatcaataagctgatggactacctgcaccgcgcggcggcca tgaacacggactactttacaaacgccatattgaacccgcactggcttccgccgccggggttctacacgggcgagtacgacatgcccg accccaacgacgggttcctgtgggacgacgtggacagcgcggtgttctcgccgacctttcaaaagcgccaggaggcgccgccgag cgagggcgcggtggggaggagcccctttcctagcttagggagtttgcatagcttgccgggctcggtgaacagcggcagggtgagcc ggccgcgcttgctgggcgaggacgaatacctgaacgactcgctgctgcagccgccgcgggtcaagaacgccatggccaataacgg gatagagagtctggtggacaaactgaaccgctggaagacctacgctcaggaccatagggagcctgcgcccgcgccgcggcgaca gcgccacgaccggcagcggggcctggtgtgggacgacgaggactcggccgacgatagcagcgtgttggacttgggcgggagcg gtggggccaacccgttcgcgcatctgcagcccagactggggcggcggatgttttgaatgcaaaataaaactcaccaaggccatagcg tgcgttctcttccttgttagagatgaggcgtgcggtggtgtcttcctctcctcctccctcgtacgagagcgtgatggcgcaggcgaccct ggaggttccgtttgtgcctccgcggtatatggctcctacggagggcagaaacagcattcgttactcggagctggctccgcagtacgac accactcgcgtgtacttggtggacaacaagtcggcggacatcgcttccctgaactaccaaaacgaccacagcaacttcctgaccacg gtggtgcagaacaacgatttcacccccgccgaggccagcacgcagacgataaattttgacgagcggtcgcggtggggcggtgatct gaagaccattctgcacaccaacatgcccaatgtgaacgagtacatgttcaccagcaagtttaaggcgcgggtgatggtggctagaaag catcccaaagatgtagatgccagtgatttaagcaaggatatcttagagtataagtggtttgagtttaccctgcccgagggcaacttttccg agaccatgaccatagacctgatgaacaacgccatcttggaaaactacttgcaagtggggcggcagaatggcgtgctggagagcgata tcggagtcaagtttgacagcaggaatttcagactgggctgggacccggtgaccaagctggtgatgccaggggtctacacctacgagg ccttccacccggacgtggtgctactgccgggctgcggggtggacttcaccgagagccgcctgagcaacctcctgggcattcgcaag aagcaaccttttcaagagggcttcagaatcatgtatgaggatctagaagggggtaacatccccgctctcctggataccaaaaaatatctg gatagcaagaaggaacttgaggatgctgccaaggaagctgcaaagcaacagggagatggtgctgtcactagaggcgatacccacct cactgtagctcaagaaaaagcagctgaaaaggagctagtgatcgtaccaattgaaaaggatgagagcaacagaagttacaacctgat caaggacacccatgacaccctgtaccgaagctggtacctgtcctatacctacggggaccccgagaagggggtgcagtcgtggacgc tgctcaccaccccggacgtcacctgcggcgcggagcaagtctactggtcgctgccggacctcatgcaagaccccgtcaccttccgct ctacccagcaagtcagcaactaccccgtggtcggcgccgagctcatgcccttccgcgccaagagcttttacaacgacctcgccgtcta ctcccagctcatccgcagctacacctccctcacccacgtcttcaaccgcttccccgacaaccagatcctctgccgcccgcccgcgccc accatcaccaccgtcagtgaaaacgtgcctgctctcacagatcacgggacgcttccgctgcgcagcagtatccgcggagtccagcga gtgaccgtcactgacgcccgtcgccgcacctgtccctacgtctacaaggccctgggcatagtcgcgccgcgcgtgctctccagtcgc accttctaaaaaatgtctattctcatctcgcccagcaataacaccggctggggtcttactaggcccagcaccatgtacggaggagccaa gaagcgctcccagcagcaccccgtccgcgtccgcggtcacttccgcgctccctggggagcttacaagcgggggcgcactgccacc gccgccgccgtgcgcaccaccgtcgacgacgtcatcgactcggtggtcgccgacgcgcgcaactacacccccgccccctccaccg tggacgcggtcatcgacagcgtggtggccgacgcgcgcgactatgccagacgcaagagccggcggcgacggatcgccaggcgc caccggagcacgcccgccatgcgcgccgcccgggctctgctgcgccgcgccagacgcacgggccgccgggccatgatgcgagc cgcgcgccgcgccgccactgcaccccccgcaggcaggactcgcagacgagcggccgccgccgctgccgcggccatctctagcat gaccagacccaggcgcggaaacgtgtactgggtgcgcgactccgtcacgggcgtgcgcgtgcccgtgcgcacccgtcctcctcgt ccctgatctaatgcttgtgtcctcccccgcaagcgacgatgtcaaagcgcaaaatcaaggaggagatgctccaggtcgtcgccccgg agatttacggacccccggaccagaaaccccgcaaaatcaagcgggttaaaaaaaaggatgaggtggacgagggggcagtagagtt tgtgcgcgagttcgctccgcggcggcgcgtaaattggaaggggcgcagggtgcagcgcgtgttgcggcccggcacggcggtggt gttcacgcccggcgagcggtcctcggtcaggagcaagcgtagctatgacgaggtgtacggcgacgacgacatcctggaccaggcg gcggagcgggcgggcgagttcgcctacgggaagcggtcgcgcgaagaggagctgatctcgctgccgctggacgaaagcaaccc cacgccgagcctgaagcccgtgaccctgcagcaggtgctgccccaggcggtgctgctgccgagccgcggggtcaagcgcgagg gcgagagcatgtacccgaccatgcagatcatggtgcccaagcgccggcgcgtggaggacgtgctggacaccgtgaaaatggatgt ggagcccgaggtcaaggtgcgccccatcaagcaggtggcgccgggcctgggcgtgcagaccgtggacattcagatccccaccga catggatgtcgacaaaaaaccctcgaccagcatcgaggtgcagaccgacccctggctcccagcctccaccgctaccgtctccactttt accgccgccacggctaccgagcctcccaggaggcgaagatggggcgccgccagccggctgatgcccaactacgtgttgcatcctt ccatcatcccgacgccgggctaccgcggcacccggtactacgccagccgcaggcgcccagccgccaaacgccgccgccgcayt gccacccgccgccgtmtggcccccgcccgcgtgcgccgcgtaaccacgcgccggggccgctcgytcgttctgcccaccgtgcgc taccaccccagcatcctttaatccgtgtgctgtgatactgttgcagagagatggctctcacttgccgcctgcgcatccccgtcccgaatta ccgaggaagatcccgccgcaggagaggcatggcaggcagcggcctgaaccgccgccggcggcgggccatgcgcaggcgcctg agtggcgggtttctgcccgcgctcatccccataatcgccgcggccatcggcacgatcccgggcatagcttccgttgcgctgcaggcgt cgcagcgccgttgatgtgcgaataaagcctctttagactctgacacacctggtcctgtatatttttagaatggaagacatcaattttgcgtc cctggctccgcggcacggcacgcggccgttcatgggcacctggaacgagatcggcaccagccagctgaacgggggcgccttcaat tggagcagtgtctggagcgggcttaaaaatttcggctcgacgctccggacctatgggaacaaggcctggaatagtagcacggggca gttgttaagggaaaagctcaaagaccagaacttccagcagaaggtggtggacgggctggcctcgggcattaacggggtggtggaca tcgcgaaccaggccgtgcagcgcgagataaacagccgcctggacccgcggccgcccacggtggtggagatggaagatgcaactc ttccgccgcccaaaggcgagaagcggccgcggcccgacgcggaggagacgatcctgcaggtggacgagccgccctcgtacgag gaggccgtcaaggccggcatgcccaccacgcgcatcatcgcgccgctggccacgggtgtaatgaaacccgccacccttgacctgc ctccaccacccacgcccgctccaccgaaggcagctccggttgtgcaggcccctccggtggcgaccgccgtgcgccgcgtccccgc ccgccgccaggcccagaactggcagagcacgctgcacagtatcgtgggcctgggagtgaaaagtctgaagcgccgccgatgctatt gagagaggaaagaggacactaaagggagagcttaacttgtatgtgccttaccgccagagaacgcgcgaagatggccaccccctcg atgatgccgcagtgggcgtacatgcacatcgccgggcaggacgcctcggagtacctgagcccgggtctggtgcagtttgcccgcgc caccgacacgtacttcagcctgggcaacaagtttaggaaccccacggtggccccgacccatgatgtgaccacggaccggtcccagc gtctgacgctgcgcttcgtgcccgtggatcgcgaggacaccacgtactcgtacaaggcgcgcttcactctggccgtgggcgacaacc gggtgctagacatggccagcacgtactttgacatccgcggcgtcctggaccgcggtcccagcttcaaaccctactcgggcacggctt acaacagtttggcccccaagggcgcccccaactccagtcagtggactgacaaagaacggcaaaatggtggacaaccacccactaca aaagatgttacaaaaacattcggagtagcagccaggggagggcttcatattactgataaaggactacaaataggagaagatgaaaata acgaggatggtgaagaagagatatatgcagacaaaactttccagccagaacctcaagtaggagaggaaaactggcaagatactgat gttttctatggcggcagagcgcttaaaaaggaaaccaaaatgaaaccatgctatggctcttttgccagacctaccaatgaaaaaggagg tcaagctaaatttttaaatggcgaaaacggtcaaccttctaaagatcaagatattacattagctttctttgatcttaaacaaaatgacactgg aactactcaaaaccagccagatgttgtcatgtacactgaaaatgtgtatctggaaaccccagacacccatgtggtgtacaaacctggca aggaagatacaagctccgctgctaaccttacacaacagtccatgcccaacaggcccaactacattggtttcagggacaactttgtggg gctcatgtattacaacagcactggcaacatgggtgtgctggctggtcaggcctctcagttgaatgctgtggttgacttgcaagacagaaa caccgagctgtcttatcagctcttgctagattctctgggtgacagaaccagatactttagcatgtggaattctgcggtggacagctatgat ccagatgtcaggatcattgagaatcacggtgttgaagatgagcttccaaattattgcttcccactggatggatctggcagcaataccgca tatcaaggtgttaaatatgaaaacggagctggcaatggaagctggaaagtagatggcgaagttgcttctcagaatcagatcgccaagg gtaatctgtatgccatggagataaaccttcaggccaacctgtggaagagttttctgtactcgaacgtggcgctgtatctaccagactccta caagtacacgccggccaacatcacgctgcccaccaacaccaacacctacgagtacatgaacggccgcgtggtggcaccctcgctg gtggatgcctatgtcaacatcggtgcccgctggtcgctggaccccatggacaacgtcaaccccttcaaccaccaccgcaacgcgggt ctgcgctaccgctccatgcttctgggcaacggccgctacgtgcccttccacatccaagtgccccaaaagttctttgccatcaagaacct gctcctgcttcccggttcctacacctacgagtggaacttccgcaaggatgtcaacatgatcctgcaaagttccctcggcaacgacctgc gcgtcgacggcgcctccgtccgcttcgacagcgtcaacctctatgccaccttcttccccatggcgcacaacaccgcctccacccttga agccatgctgcgcaacgacaccaacgaccagtccttcaacgactacctctcggccgccaacatgctctacccaatcccggccaaggc caccaacgtgcccatctccatcccctcgcgcaactgggccgccttccgcggctggagtttcacccggctcaagaccaaggaaactcc ctccctcggctcgggtttcgacccctactttgtctactcgggctccattccctacctcgacggaaccttctacctcaaccacaccttcaag aaggtctccatcatgttcgactcctcggtcagctggcccggcaacgaccggctgctcacgccgaacgagttcgagatcaagcgcagc gtcgacggggagggctacaacgtggcccaatgcaacatgactaaggactggttcctcgtccagatgctctctcattacaacattggcta ccagggcttctacgtgcctgacggttacaaggaccgcatgtactccttcttccgcaacttccagcccatgagcaggcaggtggtcgatg agatcaactacaaggactacaaggccgtcaccctgcccttccagcacaacaactcgggcttcaccggctacctcgcacccaccatgc gtcaggggcagccataccccgccaacttcccctacccgctcatcggccagacagccgtgccctccgtcacccagaaaaagttcctct gcgacagggtcatgtggcgcatccccttctccagcaacttcatgtccatgggcgccctcaccgacctgggtcagaacatgctctacgc caactcggcccacgcgctcgacatgaccttcgaggtggaccccatggatgagcccaccctcctctatcttctcttcgaagttttcgacgt ggtcagagtgcaccagccgcaccgcggcgtcatcgaggccgtctacctgcgcacgcccttctccgccggaaacgccaccacataa gcatgagcggctccagcgaacgagagctcgcggccatcgtgcgcgacctgggctgcgggccctactttttgggaacccacgacaa gcgcttccctggcttcctcgccggcgacaagctggcctgcgccatcgtcaacacggccggccgcgagaccggaggcgtgcactgg ctcgccttcggctggaacccgcgctcgcgcacctgctacatgttcgacccctttgggttctcggaccgccggctcaagcagatttacag cttcgagtacgaggccatgctgcgccgcagcgccctggcctcctcgcccgaccgctgtctcagcctcgagcagtccacccagaccgt gcaggggcccgactccgccgcctgcggacttttctgttgcatgttcttgcatgccttcgtgcactggcccgaccgacccatggacggg aaccccaccatgaacttgctgacgggggtgcccaacggcatgctacaatcgccacaggtgctgcccaccctccggcgcaaccagga ggagctctaccgcttcctcgcgcgccactccccttactttcgatcccaccgcgccgccatcgaacacgccaccgcttttgacaaaatga aacaactgcgtgtatctcaataaacagcacttttattttacatgcactggagtatatgcaagttatttaaaagtcgaaggggttctcgcgctc gtcgttgtgcgccgcgctggggagggccacgttgcggtactggtacttgggaagccacttgaactcggggatcaccagtttgggaac cggaatctcggggaaggtctcgctccacatgcgccggctcatctgcagggcgcccagcatgtccggggcggagatcttgaaatcgc agttgggaccggtgctctgcgcgcgcgagttgcggtacacggggttgcagcactggaacaccatcagactggggtacttcacactgg ccagcacgctcttgtcggtgatctgatccttgtcaaggtcctcggcgttgctcaggccaaacggggtcatcttgcacagctggcggccc aggaagggcacgctctgaggcttgtggttacactcgcagtgaatgggcattagcatcatccccgcgccgcgctgcatattcgggtaga gggccttgacaaaggccgagatctgtttgaaagcttgctgggccttggctccctcgctgaaaaacagcccgcagctcttcccgctgaa ctggttattcccgcaaccggcatcctgcacgcagcagcgcgcgtcatggctggtcagttgcaccacgctccgtccccagcggttctgg gtcaccttggccttgctgggttgctccttcagcgcgcgctgtccgttctcgctggtcacatccatctccaccacgtggtctttgtggatcat caccgttccatgcagacacttgagctggccttccacctcggtgcagccgtgatcccacagggcgcatccggtgcactcccagttcttat gcgcgatcccgctgtggctgaagatgtaaccttgcaacaggcgacccatgacggtgctaaaggctttctgggtggtgaaggtcagttg cagaccgcgggcctcctcgttcatccaggtctggcacatcttctggaagatctcggtctgctctggcatgagcttgtaagcatcgcgca ggccgctgtcgacgcggtagcgttccatcagcacgttcatggtatccatgcccttctcccaggacgagaccagaggcagactcaggg ggttgcgcacgttcaggacaccgggggtcgcgggctcgacgatgcgttttccgtccttgccttccttcaacagaaccggaggctggct gaatcccactcccacgatcacggcgtcttcctggggcatctcttcgtctgggtctacctttgtcacatgcttggtctttctggcttgcttctttt ttggagggctgtccacggggaccacgtcctcctccgaagacccggagcccacccgctgatactttcggcgcttggtgggcagagga ggtggcggcgaggggctcctctcctgctccggcggatagcgcgccgacccgtggccccggggcggartggcctctcgctccatga accggcgcacgtcctgactgccgccggccattgtttcctaggggaagatggaggagcagccgcgtaagcaggagcaggaggagg acttaaccacccacgagcaacccaaaatcgagcaggacctgggcttcgaagagccggctcgtctagaacccccacaggatgaaca ggagcacgagcaagacgcaggccaggaggagaccgacgctgggctcgagcatggctacctgggaggagaggaggatgtgctgc tgaaacacctgcagcgccagtccctcatcctccgggacgccctggccgaccggagcgaaacccccctcagcgtcgaggagctgtgt cgggcctacgagctcaacntcttctcgccgcgcgtgccccccaaacgccagcccaacggcacctgcgagcccaacccgcgtctca acttctatcccgtctttgcggtccccgaggcccttgccacctatcacatctttttcaagaaccaaaagatccccgtctcctgccgcgccaa ccgcacccgcgccgacgcgctcctcgctctggggcccggcgcgcgcatacctgatatcgcttccctggaagaggtgcccaagatctt cgaagggctcggtcgggacgagacgcgcgcggcgaacgctctgaaagaaacagcagaggaagagggtcacactagcgccctgg tagagttggaaggcgacaacgccaggctggtcgtgctcaagcgcagcgtcgagctcacccacttcgcctaccccgccgttaacctcc cgcccaaggtcatgcgtcgcatcatggatcagcttatcatgccccacatcgaggccatcgatgagacccaagagcagcgccccgag gacgcccggcccgtggtcagcgacgagatgctcgcgcgctggctcgggacccgcgacccccaggctttggaacagcggcgcaag ctgatgctggccgtagtcctggtcaccctcgagctcgaatgcatgcgccgcttcttctgcgaccccgagaccctgcgcaaggtcgagg agaccctgcactacactttcagacacggtttcgtcaggcaagcctgcaagatctccaacgtggagctgaccaacctggtctcctgcctg gggatcctgcatgagaaccgcctggggcagacagtgctccactctaccctgaagggcgaggcacggcgggactatgtccgcgact gcgtctttctctttctatgccacacatggcaagcagccatgggcgtgtggcagcagtgtctcgaggacgagaacctgaaggagctgga caagcttcttgctagaaaccttaaaaagttgtggacgggcttcgacgagcgcaccgtcgcctcggacctggccgagatcgttttccccg agcgcctgaggcatacgctgaaaggcgggctgcccgacttcatgagccagagcatgttgcaaaactaccgcactttcattctcgagcg ctcgggtatcctgcccgccacctgcaacgccttcccctccgactttgtcccgctgagctaccgcgagtgtcccccgccgctgtggagc cactgctacctcttgcagctggctaactacatctcctaccactcggacgtgatcgaggacgtgagcggcgaggggctgctcgagtgcc actgccgctgcaacctgtgctccccgcaccgctccctggtctgcaacccccagctcctgagcgagacccaggtcatcggtaccttcga gctgcaaggtccggagaagtccaccgctccgctgaaactcacgccggggttgtggacttccgcgtacctgcgcaaatttgtacccga agactaccacgcccatgagataaagttcttcgaggaccaatcgcgtccgcagcacgcggatctcacggcctgcgtcatcacccaggg cgcgatcctcgcccaattgcatgccatccaaaaatcccgccaagagtttcttctgaaaaagggtagaggggtctacctggacccccag acgggcgaggtgctcaacccgggtctcccccagcatgccgaggaagaagcaggagccgctagtggaggagatggaagaagaatg ggacagccaggcagaggaggacgaatgggaggaggagacagaggaggaagaattggaagaggtggaagaggagcaggcaac agagcagcccgtcgccgcaccatccgcgccggcagccccggcggtcacggatacaacctccgctccggtcaagcctcctcgtaga tgggatcgagtgaagggtgacggtaagcacaagcggcagggctaccgatcatggagggcccacaaagccgcgatcatcgcctgct tgcaagactgcggggggaacatcgctttcgcccgccgctacctgctcttccaccgcggggtgaacatcccccgcaacgtgttgcatta ctaccgtcaccttcacagctaagaaaaagcaagtaagaggagtcgccggaggaggcctgaggatcgcggcgaacgagccctcgac caccagggagctgaggaaccggatcttccccactctttatgccatttttcagcagagtcgaggtcagcagcaagagctcaaagtaaaa aatcggtctctgcgctcgctcacccgcagttgcttgtaccacaaaaacgaagatcagctgcagcgcactctcgaagacgccgaggctc tgttccacaagtactgcgcgctcactcttaaagactaaggcgcgcccacccggaaaaaaggcgggaattacctcatcgccaccatga gcaaggagattcccaccccttacatgtggagctatcagccccagatgggcctggccgcgggcgcctcccaggactactccacccgc atgaactggctcagtgccggcccctcgatgatctcacgggtcaacggggtccgtaaccatcgaaaccagatattgttggagcaggcg gcggtcacatccacgcccagggcaaagctcaacccgcgtaattggccctccaccctggtgtatcaggaaatccccgggccgactac cgtactacttccgcgtgacgcactggccgaagtccgcatgactaactcaggtgtccagctggccggcggcgcttcccggtgcccgct ccgcccacaatcgggtataaaaaccctgatgatccgaggcagaggcacacagctcaacgacgagttggtgagctcttcgatcggtct gcgaccggacggagtgttccaactagccggagccgggagatcatccttcactcccaaccaggcctacctgaccttgcagagcagctc ttcggagcctcgctccggaggcatcggaaccctccagttcgtggaggagtttgtgccctcggtctacttcaaccccttctcgggatcgc caggcctctacccggacgagttcataccgaacttcgacgcagtgagagaagcggtggacggctacgactgaatgtcccatggtgact cggctgagctcgctcggttgaggcatctggaccactgccgccgcctgcgctgcttcgcccgggagagctgcggactcatctactttga gctgcccgaggagcaccccaacggccctgcacacggagtacggatcaccgtagagggcaccgccgagtctcacctggtcaggttc ttcacccagcaacccttcctggtcgagcgggaccggggcgccaccacctacaccgtctactgcatctgtcctaccccaaagttgcatg agaatttttgctgtactctttgtggtgagtttaataaaagctgaactaagaacctactttggaatcccttgtcgtcatcaaatccacaagacc atcaacttcacctttgaggaacaggtgaactttacctgcaagccacacaagaagtacgtcacctggttttaccagaacactactctagca gtagccaacacctgctcgaacgacggtgttcttcttccaaacaatctcaccagtggactaactttctcagtgaaaagggcaaagctaatt cttcatcgccctattgtagaaggaacttaccagtgtcagagcggaccttgcttccacagtttcactttggtgaacgttaccggcagcagca cagtcgctccagaaactaaccttctttctgatactaacactcctaaaaccggaggtgagctctgggttccctctctgacagaggggggta gtcatattgaagcggtcgggtatttgattttaggggtggtcctgggtgggtgcatagcggtgctatattaccttccttgctgggtcgaaatc agggtatttatctgctgggtcagacattgtggggaggaaccatgaaggggctcttgctgattatcctttccctggttgggggtttactggc ctgccacgaacagccacgatgtaacatcaccacaggcaatgagaggaacgactgctctgtagtgatcaaatgcgagcaccagtgtcc tctcaacattacattcaagaataagaccatgggaaatgtatgggtgggattctggcaaccaggagatgagcagaactacacggtcacta tccatggtagcgatggaaatcacactttcggtttcaaattcatttttgaagtcatgtgtgatatcacactgcatgtggctagacttcatggctt gtggccccctaccaaggagaacatggttgggttttctttggcttttgtgatcatggcctgtgcaatgtcaggtctgctggtaggggctcta gtgtggttcctgaagcgcaagcccaggtacggaaatgaggagaaggaaaaattgctataaatctttttctcttcgcagaaccatgaatac tttgaccagtgtcgtgctgctctctcttttagttattaatgtggaatgtgccgatcctattctagttagtgtagattggggaaaaaatcttacatt agagggtcctaaagaaacaccagttgaatggtggggtggaagaaacatacaacaactgtgcatagggaatcaaaccaaacataaag agctaagtcacagatgtaatgtccagaacataactttactgtttgtaaatactagttttaatggagactactttgggtttaaaaatgataacag cggtatgaaacattataaagtcacagttataccccctaaaccctccactcggaaacctctttctcctccacactatgtaaacgcaactatg gggcaaaacctaacattagtggggcctgcaaacattccagttacttggcttagtgaatatggcacgttgtgtgagggcaaaaaaattttg cacaaagnaattaaatcacacctgtaacgaacagaacctcacgttactgtttgttaatatgacacacaacggaccatattttggctttgac aaatacaacattgatagagagcagtatgaggtttctattattagtttgtttaaagttggcgctggacagaagaaaattgggaaaggacaga aaaaggaggaaaagacaaaaccaaactctagtgatttgggacaaagacaatccagaccaaagaaaaaagatattgttgaagaggtcc aaatcaaaacaggagaaaatcgaacccttgttggtccacctggaaaagttgattggattaaactttccagtggaaacaataatgttcttaa gttgtgtaatggcgacaagtatattaaacacacatgtgatggtcaaaatttaacattaattaatgtgactagaatttatgacggaacttattat ggttctagcaatgatggctcaagtcattacaaagttaccatctatgaattacacaaagttaataaaactaaatctatgcttaagccatacact acaaaaagaactacagtgaatgcaacagatgacagtgctcacaaaattgctttgcagcaggaaaataatgggcaaacagaaaatgatc aagaatcaaaaattccatctgctactgtggcaatcgtggtgggagtgattgcgggcttcataactataatcattgtcattctgtgctacatct gctgccgcaagcgtcccagggcatacaataatatggtagacccactactcagcttctcttactgagactcagtcactttcatttcagaacc atgaaggctttcacagcttgcgttctgattagcataattacacttagtttagcagcacctaaaccagaagtatatacacaagttaatgtcact aggggtgggaatgctacactagatggaccatttaacaataacacatggacaagatatcatgatgatgggagaaaaaacggatggatg aatatttgtaaatggtcagacccatcatacacatgtcatagtaatggaagccttagtatttttgctttcaacattagttcaggtaaatataaagt tcaaagttacactaacagttataatggattagatggttatgaaaaacttgaagttaaaatgtttaatctaacagtaattgagcctccaaccac tagagcacccaccacagttaggacaactaaggaaacaacacagcctaccactgtacccactacacatccaaccaccacagtcagtac aactattgagaccactactcatactacacagctagacacaacagtgcagaatactactttactgattgaatttttactaagagggaatgaa agtactactgatcagacagaggctacctcaagtgccttcagcagtactgcaaatttaacttcgcttgcttggactaatgaaaccggagtat cattgatgcatggccagccttactcaggtttggatattcaaattacttttctggttgtctgtgggatctttattcttgtggttcttctgtactttgtc tgctgcaaagccagagagaaatctagtaggcccatctacaggccagtaatcggggaacctcagcctctccaagtggaagggggtcta aggaatcttctcttctctttttcagtatggtgatcagccatgattcctaggttcttcctatttaacatcctcttctgtctcttcaacatctgcgctg ccttcgcggccgtctcgcacgcctcgcccgactgtctcgggcccttccccacctacctcctctttgccctgctcacctgcacctgcgtct gcagcattgtctgcctggtcgtcaccttcctgcagctcatcgactggtgctgcgcgcgttacaattatctccaccacagtcccgaataca gggacaagaacgtagccagaatcttaaggctcatctgaccatgcagactctgctgatactgctatccctcctctcccctgcccttgctga ctgtaaatttgcggacatatggaatttcttagactgttatcaagagaaaatggatatgccttcctattacttggtgattgtgggtgtagtcatg gtctgctcctgcactttctttgctatcatgatctacccctgttttgatctcggctggaactctgttgaggcattcacatacacactagaaagca gttcactagcctccacgccgccacccacaccgcctccccgcagaaatcagttccccctgattcagtacttagaagagccccctccccg gcccccttccactgttagctactttcacataaccggcggcgatgactgaccaccacctggacctcgagatggacggccaggcctccga gcagcgcatcctgcaactgcgcgtccgtcagcagcaggagcgggccgccaaggagctcctcgatgccatcaacatccaccagtgc aagaagggcatcttctgcctggtcaaacaggcaaagatcacctacgagctcgtgtccaacggcaaacagcatcgcctcacctatgag atgccccagcagaagcagaagttcacctgcatggtgggcgtcaaccccatagtcatcacccagcagtcgggcgagaccagcggctg catccactgctcctgcgaaagccccgagtgcatctactccctcctcaagaccctttgcggacttcgcgacctcctccccatgaactgatt gattaaagcccagaaaccaatcaaacccccttccccatcaccccaaataacaatcattggaaataatcattcaataaagatcacttacttg aaatctgaaagtatgtctctggtgtagttgttcagcagcacctcggtaccctcctcccagctctggtactccagtccccggcgggcggc gaacttcctccacaccttgaaagggatgtcaaattcctggtccacaattttcattgtcttccctctcagatgtcaaagaggctccgggtgga agatgacttcaaccccgtctacccctatggctacgcgcggaatcagaatatccccttcctcactcccccctttgtctcctccgatggattc caaaacttcccccctggggtcctgtcactcaaactggctgatccaatcgccatcgccaatgggaatgtctcactcaaggtgggagggg gactcactgtagaacaacagtctggaaaactgagtgtggatactaaggcacccttgcaagttgcaaatgacaacaaattggagctatctt atgatgatccatttaaggtagagaataacaaacttggaattaaagctggccatggtttagcagttgtaactaaagaaaacacaagtcttcc tagtctagttggaacacttgtagttttaactggaaaaggaataggtactggatcaagtgcacatggaggaactattgatgtaagacttggt gaaggaggtgggttatcatttgatgaaaaaggagacttagtagcttgggacaaaaaaaatgatacacgcaccctttggacaacacctgt ccttctccaaattgcaaagttgaaacagcaagagactcaaagctaaccttagcacttacaaaatgtggtagtcaaattttggccactgtat ctttacttgttgttacgggcaaatatgctattataagtgacacagtcaacccaaagcagttctctattaagttactgtttaatgacaagggtgt tttgttaagkgactcaaatcttgatgggacatattggaactatagaagcaacaataacaacataggcactccttataaagaggctgttggt tttatgccaagcacaacagcttatcctaagccaaccaacaacaccagcacagatccggataaaaaagtgagtcaaggtaaaaataaaa ttgtaagcaatatttatcttggaggagaggtatatcaaccaggatttattgttgttaaatttaatcaggaaactgatgccaattgtgcatactct attacatttgattttggatggggtaaggtgtataaggatcctataccatatgatacctcttcttttactttctcatatatcgctcaagaatgaaag accaataaacgtgtttttcattgaaaattttcatgtatctttattgatttttacaccagcacgggtagtcagtctcccaccaccagcccatttca cagtgtaaacaattctctcagcacgggtggccttaaataggggaatgttctgattagtgcgggaactggatttagtgtctataatccacac agtttcctggcgagccaaacggggatcggtgattgagatgaagccgtcctctgaaaagtcttccaagcgggcctcacagtccaaggtc acagtctggtggaatgagaagaacgcacagattcatactcggaaaacaggatgggtctgtgcctctccatcagcgccctcaacagtct ctgccgccggggctcggtgcggctgctgcagatgggatcgggatcgcaagtctctctgactatgatccccacagccttcagcatcagt ctcctggtgcgtcgggcacagcacctcatcctgatctcgctcatgttctcacagtaagtgcagcacataatcaccatgttattcagcagcc cataattcagggtgctccagccaaagctcatgttggggatgatggaacccacgtgaccatcataccaaatgcggcagtatatcaggtg cctgcccctcatgaacacactgcccatatacatgatctctttgggcatgtttctgttcacaatctggcggtaccaggggaagcgctggttg aacatgcacccgtaaatgactctcctgaaccacacggccagcarggtgcctcccgcccggcactgcagggagcccggggacgaac agtggcaatgcaggatccagcgntcgnacccgctcaccatctgagctctcaccaagtccagggtagcggggcacaggcacactga catacatctttttaaaatttttatttcctctggggtcaggatcatatcccaggggactggaaactcttggagcagggtaaagccagcagca catggtaatccacggacagaacttacattatgatattcagcatgatcacaatcgggcagcagggggtgttgttcagtcagtgaggccct ggtctcctcctcagatcgtggtaaacgggccctgcggtatggatgatggcggagcgaggtcgattgttcctcggtgctcattgtagtgc accctcttgcgtaccttgtcgtacttctgccagcagaaagtggcccgggaacagcagatacccctcctccgtccgtcctttcgctgctgc cgctcagtcatccaactgaagtacatccattcccgaaggttctggagaagttcctctgcatctgatgaaacaaaaagcccgtccatgcg aattcccctcatcacatcagccaggactctgtaggccatccccatccagttaatgctgccttgtctatcattcagagggggcggtggcag gattggaagaaccattatttttttactccaaacggtcgcgcagcaatataaaattcaggtcacggaggtggcacctctctcctccactgttt tggtggaaacagacagccaaatcaaaaattatgcgattctcaaggtgctctactgtggcttccagcagaggctccacacgtacatccag aaacaacagcacattaaaagcgggcccgccatcctgctcmtcaatcatcatattacagtcctgaaccatccccaggtaattttcgtttttc cagccttgaattatcgstacaa-3 '

Specific Example 5 : [0095] The nucleic acid encoding the Ad-36 hexon protein (SEQ ID NO.: 30) is as follows:

5'atggccaccc cctcgatgat gccgcagtgg gcgtacatgc acatcgccgg gcaggacgcc tcggagtacc tgagcccggg tctggtgcag tttgcccgcg ccaccgacac gtacttcagc ctgggcaaca agtttaggaa ccccacggtg gccccgaccc atgatgtgac cacggaccgg tcccagcgtc tgacgctgcg cttcgtgccc gtggatcgcg aggacaccac gtactcgtac aaggcgcgct tcactctggc cgtgggcgac aaccgggtgc tagacatggc cagcacgtac tttgacatcc gcggcgtcct ggaccgcggt cccagcttca aaccctactc gggcacggct tacaacagtt tggcccccaa gggcgccccc aactccagtc agtggactga caaagaacgg caaaatggtg gacaaccacc cactacaaaa gatgttacaa aaacattcgg agtagcagcc aggggagggc ttcatattac tgataaagga ctacaaatag gagaagatga aaataacgag gatggtgaag aagagatata tgcagacaaa actttccagc cagaacctca agtaggagag gaaaactggc aagatactga tgttttctat ggcggcagag cgcttaaaaa ggaaaccaaa atgaaaccat gctatggctc ttttgccaga cctaccaatg aaaaaggagg tcaagctaaa tttttaaatg gcgaaaacgg tcaaccttct aaagatcaag atattacatt agctttcttt gatcttaaac aaaatgacac tggaactact caaaaccagc cagatgttgt catgtacact gaaaatgtgt atctggaaac cccagacacc catgtggtgt acaaacctgg caaggaagat acaagctccg ctgctaacct tacacaacag tccatgccca acaggcccaa ctacattggt ttcagggaca actttgtggg gctcatgtat tacaacagca ctggcaacat gggtgtgctg gctggtcagg cctctcagtt gaatgctgtg gttgacttgc aagacagaaa caccgagctg tcatatcagc tcttgctaga ttctctgggt gacagaacca gatactttag catgtggaat tctacggtgg acagctatga tccagatgtc aggatcattg agaatcacgg tgttgaagat gagcttccaa attattgctt cccactggat ggatctggca gcaataccgc atatcaaggt gttaaatatg aaaacggagc tggcaatgga agctggaaag tagatggcga agttgcttct cagaatcaga tcgccaaggg taatctgtat gccatggaga taaaccttca ggccaacctg tggaagagtt ttctgtactc gaacgtggcg ctgtatctac cagactccta caagtacacg ccggccaaca tcacgctgcc caccaacacc aacacctacg agtacatgaa cggccgcgtg gtggcaccct cgctggtgga tgcctatgtc aacatcggtg cccgctggtc gctggacccc atggacaacg tcaacccctt caaccaccac cgcaacgcgg gtctgcgcta ccgctccatg cttctgggca acggccgcta cgtgcccttc cacatccaag tgccccaaaa gttctttgcc atcaagaacc tgctcctgct tcccggttcc tacacctacg agtggaactt ccgcaaggat gtcaacatga tcctgcaaag ttccctcggc aacgacctgc gcgtcgacgg cgcctccgtc cgcttcgaca gcgtcaacct ctatgccacc ttattcccca tggcgcgcaa caccgcctcc acccttgaag ccatgctgcg caacgacacc aacgaccagt ccttcaacga ctacctctcg gccgccaaca tgctctaccc aatcccggcc aaggccacca acgtgcccat ctccatcccc tcgcgcaact gggccgcctt ccgcggctgg agtttcaccc ggctcaagac caaggaaact ccctccctcg gctcgggttt cgacccctac tttgtctact cgggctccat tccctacctc gacggaacct tctacctcaa ccacaccttc aagaaggtct ccatcatgtt cgactcctcg gtcagctggc ccggcaacga ccggctgctc acgccgaacg agttcgagat caagcgcagc gtcgacgggg agggctacaa cgtggcccaa tgcaacatga ctaaggactg gttcctcgtc cagatgctct ctcattacaa cattggctac cagggcttct acgtgcctga gggttacaag gaccgcatgt actccttctt ccgcaacttc cagcccatga gcaggcaggt ggtcgatgag atcaactaca aggactacaa ggccgtcacc ctgcccttcc agcacaacaa ctcgggcttc accggctacc tcgcacccac catgcgtcag gggcagccat accccgccaa cttcccctac ccgctcatcg gccagacagc cgtgccctcc gtcacccaga aaaagttcct ctgcgacagg gtcatgtggc gcatcccctt ctccagcaac ttcatgtcca tgggcgccct caccgacctg ggtcagaaca tgctctacgc caactcggcc cacgcgctcg acatgacctt cgaggtggac cccatggatg agcccaccct cctctatctt ctcttcgaag ttttcgacgt ggtcagagtg aacgccacca cataa-3 '

Specific Example 6:

[0096] The nucleic acid encoding the Ad-36 fiber coat protein (SEQ ID NO.: 31) is as follows:

5'atgtcaaagaggctccgggtggaagatgacttcaaccccgtctacccctatggctacgcgcggaatcagaatatccccttcctcact cccccctttgtctcctccgatggattccaaaacttcccccctggggtcctgtcactcaaactggctgatccatgtctcactcaaggtggga gggggactcactgtagaacaacagtctggaaaactgagtgtggatactaaggcacccttgcaagttgcaaatgacaacaaattggagc tatcttatgatgatccatttaaggtagagaataacaaacttggaattaaagctggccatggtttagcagttgtaactaaagaaaacacaagt cttcctagtctagttggaacacttgtagttttaactggaaaaggaataggtactggatcaagtgcacatggaggaactattgatgtaagac ttggtgaaggaggtgggtatcatttgatgaaaaaggagacttagtagcttgggacaaaaaaaatgatacacg caccctttggacaacacctgatccttctccaaattgcaaagttgaaacagcaagagactcaaagctaaccttagcacttacaaaatgtgg tagtcaaattttggccactgtatctttacttgttgttacgggcaaatatgctattataagtgacacagtcaacccaaagcagttctctattaagt tactgtttaatgacaagggtgttttgttaagtgactcaaatcttgatgggacatattggaactatagaagcaacaataacaacataggcact ccttataaagaggctgttggttttatgccaagcacaacagcttatcctaagccaaccaacaacaccagcacagatccggataaaaaagt gagtcaaggtaaaaataaaattgtaagcaatatatcttggaggagaggtatatcaaccaggatttattgttgttaaatttaatcaggaaact gatgccaattgtgcatactctattacatttgatttggatggggtaaggtgtataaggatcctataccatatgatacctcttctactttctcatata tcgctcaagaatga-3 '

[0097] The examples given above are merely illustrative and are not meant to be an exhaustive list of all possible embodiments, applications or modifications of the invention. Thus, various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in cellular and molecular biology, chemistry, or in the relevant fields are intended to be within the scope of the appended claims. [0098] The disclosures of all references and publications cited above are expressly incorporated by reference in their entireties to the same extent as if each were incorporated by reference individually.

Claims

WHAT IS CLAIMED IS:

1. A method of determining the presence of or a predisposition to abnormal adipose tissue hypertrophy in a subject, comprising the steps of: assaying a sample from the subject to determine whether the subject is infected with an lipogenic adenovirus; and correlating the presence or absence of lipogenic adenovirus to the presence of, or predisposition to, abnormal adipose tissue hypertrophy, thereby predicting the presence of, or predisposition to, abnormal adipose tissue hypertrophy in the subject.

2. The method of claim 1 , wherein said step of assaying the sample comprises the step of screening for antibodies reactive to the lipogenic adenovirus in the sample.

3. The method of claim 2, wherein the antibodies in said screening step are reactive to one or more peptides encoded by the peptide sequences selected from the group consisting of SEQ ID NO.: 1, SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15, SEQ ID NO.: 16, SEQ ID NO.: 17, and SEQ ID NO.: 18.

4. The method of claim 2, wherein said screening step is performed by using a method selected from the group consisting of serum neutralization assay and ELISA.

5 . The method of claim 1 , wherein said step of assaying the sample comprises the step of screening for lipogenic adenovirus nucleic acids.

6. The method of claim 5, wherein the nucleic acids are nucleic acids encoding a hexon protein.

7 . The method of claim 6, wherein the hexon protein comprises SEQ ID

No. 29.

8. The method of claim 5, wherein the nucleic acids are nucleic acids encoding the fiber coat protein.

9. The method of claim 8, wherein the nucleic acid sequence comprises SEQ ID No.: 30.

10. The method of claim 5, wherein the nucleic acids in said screening step are detected using one or more nucleic acids selected from the group consisting of SEQ ID NO.: 19, SEQ ID NO.: 20, SEQ ID NO.:21, SEQ ID NO.:22, SEQ ID NO.:23, SEQ ID NO.:24, and SEQ ID NO.:25.

11. The method of claim 1 , wherein the lipogenic adenovirus is one or more lipogenic adenoviruses selected from the group consisting of adenovirus type 5, adenovirus type 36, and adenovirus type 37.

12. The method of claim 1, wherein the sample is selected from the group consisting of a biological sample, body fluid, a tissue sample, an organ sample, feces, blood, saliva, and any combination thereof.

13. The method of claim 1 , wherein the subject is a human.

14. The method of claim 13, wherein the human is afflicted with HIV/ AIDS or Madelung's disease.

15. The method of claim 1 , wherein the subject is a non-human animal.

16. The method of claim 1 , further comprising the step of screening for the presence of a biomarker associated with a lipogenic adenovirus infection in the sample.

17. The method of claim 16, wherein the biomarker is at least one biomarker selected from the group consisting of fatty acid synthetase (FAS), peroxisome proliferator-activated receptor (PPAR) family proteins, CCAAT/enhancer-binding proteins (C/EBP), adipose tissue differentiation dtermination-dependent factor 1 (ADD-l)/sterol response element-binding protein (SREBP-I), glycerol-3-phosphdehydrogenase, and lipoprotein lipase.

18. The method of claim 1, further comprising the step of modifying a drug regimen to a less adipogenic drug regimen as a result of the correlation.

19. The method of claim 1, further comprising the step of administering a vaccine effective against lipogenic adenovirus to the subject to prevent lipogenic adenovirus infection.

20. The method of claim 1, further comprising the step of administering a compound that blocks the effects of a lipogenic adenovirus to produce abnormal adipose tissue hypertrophy.