AU1949099A - Stealth virus nucleic acids and related methods - Google Patents

Description

WO 99/34019 PCT/US98/27744 Description Stealth Virus Nucleic Acids and Related Methods Technical Field The present invention primarily relates to diagnosis of infections that are caused by atypically structured cytopathic viruses. It provides viral isolates and sequence data on representative isolates. Methods are described for obtaining nucleic acid sequences contained within additional isolates of this molecularly heterogeneous grouping of cytopathic viruses and for using the sequence data for the design and implementation of sensitive virus detection methods, and for determining modes of virus transmission, choices of therapy, and likely mechanisms of induced cell damage. Sequence data are also useful in the classification, characterization and quantitation of individual viral isolates, and in determining other potential uses of isolated viruses, such as their use as vectors for the transfer of genetic information. Background Art This section provides an up-to-date overview of conventional viruses, their modes of detection, using culture, immunological and molecular probe based assays, and the utility of deriving nucleic acid sequence information on viral isolates. A. Viruses 1. Definition: Viruses are very small organism, which can grow and multiply, only inside of a cell. They require the contents of the cell to manufacture the various components of the virus. These components are; i) DNA or RNA nucleic acids, comprising a chain or polymer of nucleotides connected through a phohodiester bond from the 5' to 3' carbon atoms of the sugar moiety of the nucleotide. These are the genes of the virus. Viral particles contain either RNA or DNA but not both, whereas bacteria and cells have both RNA and DNA. The amount of DNA or RNA in a virus varies depending on the type of virus. Small viruses, e.g. papillomavirus have about 9,000 nucleotide molecules strung in a row. Big viruses, e.g. a herpesvirus can have 200,000 nucleotides. ii) A protein (or proteins) which associate with the DNA or RNA and provides some protection as the virus passes between cells. The proteins make up the capsid of the virus and are formed by the aggregation of normally soluble proteins into a shell-like structure iii) Some viruses also have an outer protective layer of proteins in a lipid envelope, and sometimes also between the capsid and the envelope (termed tegument proteins). Finally, some but not all viruses possess various enzymes involved directly or indirectly in the replication of the viral nucleic acids 2. Classification: Viruses are classified in several ways, for example. i) Whether the viral particles contain RNA or DNA. Some RNA viruses convert to DNA after they enter a cell and are called retroviruses. ii) Whether the genome is single (ss) or double stranded (ds). iii) For single stranded RNA viruses, the sequence may directly code for its protein (positive, -+ve, stranded), or WO 99/34019 2 PCT/US98/27744 consist of the opposite or negative, -ve, strand. iv) The genome may be linear or circular and may be in a single segment, or in multiple segments. iv) The genome size and overall shape of the virus and whether an envelope is present are also major criteria used for classification. v) Finally, the kinds of diseases produced can distinguish different viruses. Related viruses are successively grouped into species, genera. families and orders. 3. Viral Diseases: Viruses which cause disease are termed pathogenic. Examples of virus induced diseases include; influenza, the common cold, chickenpox, measles, mumps, rubella, hepatitis, infectious mononucleosis, polio, etc. Some viruses are thought to play a contributing role in causing certain types of cancers, such as papillomavirus in cervical cancer, and Epstein-Barr virus in Burkitt's lymphoma. Viruses are not widely regarded as a cause of lung cancers, breast cancers, gastrointestinal tumors, brain tumors and melanomas. A retrovirus called human immunodeficiency virus (HIV) damages the immune system and causes the Acquired Immunodeficiency Syndrome (AIDS). 4. Mechanism of Viral-Induced Diseases. Some viruses can directly damage cells by triggering a cellular destructive process termed apoptosis; and/or by usurping the cells metabolic resources; and/or by producing toxic components, all of these processes can interfere with the cells' normal functions. Cell damaging viruses will generally induce a morphological alteration in infected cells which is commonly referred to as a cytopathic effect (CPE). This change can be seen in infected tissues and under suitable cell culture conditions can be seen in cells grown in vitro in tissue cultures. Viruses can also cause cells to express viral and/or altered cellular components that may become targets for anti-cellular immunity. HIV directly attacks the immune system leaving the infected host vulnerable to secondary infections by other pathogens. Viruses that have been associated with cancers are thought to act mainly by causing mutations in critical cellular genes. 5. Distinction between Prions and Viruses. Certain types of cellular proteins have the property, similar to viral capsid proteins, that when present in a particular configuration, will complex with corresponding soluble protein resulting in the assembly of insoluble structures formed by induced aggregation of the molecules. Abnormally configured cellular protein can initiate a chain reaction leading to the progressive accumulation of protease-resistant material termed amyloid. Such proteins have been termed "prions" for "proteinaceous infectious particles". Certain humans and animals inherit genetically altered normal protein with an enhanced tendency to "spontaneously" generate the small quantity of the abnormally configured protein to begin the process. If other individuals simply become exposed to these proteins, e.g. by ingestion with food, disease can occur through the progressive conversion of the new host' s proteins, along with the potential of further transmission. Prion diseases include scrapie in sheep, bovine spongiform encephalopathy or mad cow disease in cows and both Creutzfeldt-Jakob disease and kuru in humans. A role for nucleic acids in prion disease transmission has been discounted by the scientific community largely because the protein itself appears capable of causing disease and, to date, no transmissible viral nucleic acids have been identified in association with these diseases.. B. Laboratory Diagnosis of Viral Infection: Immunological, tissue culture and molecular biological methods are used to diagnose viral WO 99/34019 PCT/US98/27744 3 infections and to ascertain the extent of viral damage and the quality and intensity of the body's immune response against the viral proteins. The antibody response to a viral infection normally starts with the relatively transient production of IgM followed by a long lasting production of IgG. For this reason, a specific IgM antibody response to viral components (termed antigens) can generally be taken as a sign of early infection. The serological detection of free viral antigen can precede the earliest serological response and this type of assay can be useful for certain infections, e.g. parvovirus B19, HIV, hepatitis B virus (HBV) and human herpesvirus-6 (HHV-6). Antigen assays can also be used to distinguish persisting infection, in spite of IgG response, from a cured infection. Again, this type of approach has been employed with HIV and HHV-6. The quality and intensity of the anti-viral cellular immune responses can be assessed using lymphocyte subset analyses, natural killer cell mediated cytotoxicity, and measurements of cytokine production including 1L2, IL4, interferon, etc. The composition of the viral proteins that act as targets for antibody and cellular immunity can be deduced from a knowledge of the nucleic acid sequences that comprise the virus genome. Tissue culture methods can provide conclusive evidence of active infection by cytopathic viruses, as well as provide the viral isolate for susceptibility studies to anti-viral agents. The appearance of the CPE and the susceptibility of various cell types to the CPE can be used as distinguishing criteria in the identification of different types of viruses. As noted above, several types of viruses induce cellular damage through the induction of apoptosis. This is seen morphologically as cellular shrinkage and progressive degeneration. Some viruses tend to cause cellular enlargement, rather than shrinkage. A cause of cellular swelling can be disruption of mitochondrial catabolism of lipids, yielding foamy vacuolated cells. Infected cells can fuse with each other yielding cell syncytia. In general, once a CPE has begun it will progress throughout the culture. With many viruses, however, only certain cell types will display the CPE. For example, human cytomegalovirus (HCMV) will grow in human but not in animal fibroblasts, and not very well in human epithelial cells. The major limitation of tissue culture techniques has been the apparent failure of routine viral cultures to detect the types of viruses to be described in more detail in this application. Molecular probes: Virus infection can also be inferred from the results of molecular probe based assays. The complimentarity between the nucleotide sequences of a molecular probe and a target nucleic acid results in binding of the probe to the target. Moreover, either through the addition of nucleic acid synthesizing enzymes (polymerases) or simple reliance on the viruses own replicating enzymes, a complex of probe with a target sequence can elicit a synthetic reaction such that the probe is extended from its 3' end with a sequence that is fully complimentary to the part of the target sequence adjacent to the site of initial binding by the probe. This feature has formed the basis of a recycling DNA synthetic technique termed the polymerase chain reaction (PCR). PCR refers to enzymatic amplification of a defined DNA sequence. It requires the following reactants: target DNA containing a known sequence to be amplified; oligonucleotide primers complimentary to the flanking regions, on opposing DNA strands, of the particular segment of double stranded DNA to be replicated; DNA polymerase enzyme; deoxynucleotide triphosphates (dNTP) and buffer. The PCR is performed in a thermal cycling machine. PCR proceeds by denaturing the double stranded DNA molecule by heat; and cooling in the presence of the oligonucleotide primers. Because of their high WO 99/34019 PCT/US98/27744 4 concentration and greater mobility in solution, the primers bind more rapidly to the target DNA than the slower reannealing process exhibited by the larger complimentary DNA strands. The primer DNA complex provides a substrate for DNA polymerase. In the presence of dNTP, the polymerase will extend the primers in a DNA synthesis reaction. Each newly synthesized strand will be complimentary to the template DNA and will acquire at its 3' end, the sequence complimentary to the other primer used in the PCR. On reheating, the newly formed hybrids will denature, thereby providing two additional template molecules during the next primer annealing step. With each successive cycle of heating, primer annealing and primer extension, there will be an exponential increase in the targeted segment of DNA. Eventually, the reaction will become rate limiting due mainly to competition between primer binding and reannealing of the greatly amplified single DNA molecules synthesized during the PCR. In a typical reaction, however, amplification in the order of 105 fold can be achieved in from 25-30 cycles. The specifically amplified PCR product will be of uniform size corresponding to the distance separating the 5' ends of the two primer binding sites on the opposing strands of the target segment of DNA. It can be identified by electrophoresis in agarose gels and further characterized by a hybridization reaction using a labeled probe reactive with the amplified sequence. For RNA viruses, an initial reverse transcriptase reaction can be performed to convert the RNA to a DNA sequence. The single stranded DNA is replicated to a double stranded molecule which is then amplified by PCR. Either PCR derived products or synthetic oligonucleotides can be used directly to determine the presence of a viral sequence in clinical material. Various approaches have been used to detect the binding (hybridization) of the probe to its corresponding viral sequence. The major limitation of molecular approaches, including PCR and direct probe based assays, to virus diagnosis, is the requirement to know at least some of the actual sequence of the virus. This issue can, however, be addressed by using conditions that favor the binding of probes that only partially match their target. PCR products generated in such low stringency reactions will reflect actual sequences of the bound target. By sequencing the PCR products, information can be obtained that will allow for the design of highly specific primer sets. C. Utility of Nucleic Acid Sequence Information Obtained on Viruses Sequence information on a virus can provide a wealth of useful information way beyond the design of specific PCR primers for diagnostic purposes. The sequences can predict the actual proteins that are encoded by the virus. Such proteins can be synthesized for use as antigens to assess anti-viral immune responses, and to evoke protective immune responses through immunizations. Antibodies that are formed through infection or by experimental immunization, can also be used to detect the specific virus. Protein data can help determine how a particular virus is able to mediate CPE in tissues and in tissue cultures. Relatively little efforts are directed towards sequencing of individual viral isolates from patients, because of the general assumption that all viruses within a particular category are essentially identical. As will be disclosed below, novel sequence information, especially on previously unrecognized viruses, can provide important new insights into basic molecular mechanisms of viral pathogenesis. In particular, it can help explain how some disease causing viruses have successfully evaded confrontation with the cellular immune system.

WO 99/34019 PCT/US98/27744 5 D. Viruses as a Potential Cause of Common Non-Inflammatory Diseases In spite of advances in understanding the molecular nature of viruses, and widespread availability of sophisticated viral diagnostic techniques, little regard has been given by others to the possible role of viruses in non-inflammatory illnesses affecting humans and animals. An appreciation, by the inventor, of the existence of previously unrecognized "stealth virus" infections has opened many opportunities to pursue important avenues of clinical and basic research. Although, the information provided in this disclosure is primarily directed towards methods of obtaining and utilizing nucleic acid sequence data from various stealth viral isolates, it is likely that the basic concepts will drastically change the approach that clinicians will take to human diseases. Areas of special concern include viral induced brain damage presenting as psychiatric symptoms in adults, learning and behavioral disorders in children and dementia in the elderly. As will be described, the issue of viruses as a cause of common cancers will need to be reopened. The significance of the work also extends to potential contamination of blood and other biological products, transmission of diseases between humans and between humans and animals, possible contamination of foods and other environmental sources. It disclosing the invention it is appropriate to begin with a general overview of stealth viruses, review the established culture methods for these viruses and then address the main topic of this application, which is obtaining and using nucleic acid sequence information on these viruses. Disclosure of Invention Stealth Adaptation of Viruses as a Means to Evade the Immune System. A major function of the cellular immune system is to recognize and respond to virus infections. A successful immune response can eradicate foreign viruses by destroying infected cells prior to the release of progeny viruses. Symptoms of an acute cytopathic virus infection commonly occur during the time period required to generate a primary cellular immune response, and can also be a byproduct of cellular immune damage inflicted on viral infected cells. Unusually severe infections can occur if the immune system is impaired, for example, as a result of immaturity, chemotherapy, coincident infection with human immunodeficiency virus (HIV), or specific genetic deficits in immune competence. Certain viruses may also interfere with immunological defenses through such mechanisms as downregulation of the expression of histocompatibility antigens, induction of immunosuppressive cvtokines and related virokines, and by remaining inactive, as in latent infections. Stealth adaptation was originally proposed by the inventor as a distinct process whereby a virus could remain actively cvtopathic in the absence of an accompanying inflammatory cellular immune response. Based on histological findings on brain biopsies, and on repeated isolations of atypically structured cytopathic viruses, it was suggested that these viruses lacked critical genes encoding effective targets for the cellular immune system. This original hypothesis is still foreign to most virologists who intuitively believe, either that all viruses possess ample targets for immune WO 99/34019 PCT/US98/27744 6 recognition and/or that viruses without such critical elements would be unable to replicate, cause cell damage or pass between individuals. Stealth viruses were defined as cytopathic viruses able to establish a persistent infection because of deletion and/or mutation of specific genes, which, if present, would code for immunogenic components able to evoke an effective anti-viral cellular immune response. The existence of stealth viruses is best understood within the framework of varying strategies used by viruses to evade immune elimination. Essentially, the basic postulates to explain stealth viruses are: i) That relatively few viral components serve as major immunologic targets (epitopes) for T cell recognition of infected cells; and ii) deletion and/or mutation of the genes coding these immunogenic epitopes can yield pathogenic viral variants (stealth viruses) that strategically avoid confrontation with the body's cellular immune defenses. The definition of stealth viruses does not follow the usual approach of defining specific molecular and morphological characteristics. Thus, as described in the "Background Art" section, the usual classification of viruses begins with the distinction between DNA and RNA genome. Other characteristics include single stranded or double stranded nucleic acid, polarity of single stranded RNA viruses, mode of viral replication, size and shape of the virion, and whether the virus is enveloped. The approach taken to initially define stealth viruses was based on the appearance of a fairly characteristic vacuolating, foamy cell appearing CPE in culture along with the exclusion of other known viruses. As the work progressed, several individual stealth viral isolates were defined in terms of more conventional criteria, including their electron micrographic appearance, electrophoretic pattern of isolated viral DNA, partial sequencing and probable origin. Even with the best characterized of these stealth virus isolates, however, the strict chemical and morphological classification schemes fail to account for the microheterogeneity and sub-genomic expression that is observed. Moreover, the precise chemical features of one isolate do not adequately encompass the broader concept of a diverse group of cytopathic viruses in which deletion and/or mutation of normally immunogenic components has occurred. Stealth adaptation is viewed as a mechanism to facilitate persistent infection by structurally losing the capacity to evoke an effective anti-viral cellular inflammatory response. Given the above considerations, stealth viruses do not originate from a single source. It is likely that stealth adaptation can occur with all of the presently known human herpesviruses and many of the additional cytopathic viruses known to affect humans and animals. Viral sources other than herpesviruses are certainly not excluded. In fact, as viruses downsize and simplify, their initial distinguishing characteristics tend to become less important compared to their common pathogenic capacity of overtaxing the metabolic resources of a cell. The application of DNA/RNA isolation and cloning techniques, as provided for in this disclosure, should continue to provide informative sequence data on various stealth viral isolates to help define their individual origins. Tissue Culture Based Screening Methods for the Detection of Stealth Viruses. Stealth viruses can most easily be screened for using basic tissue culture techniques. The viruses are identified by their capacity to induce a CPE in tissue cultures. It is advantageous to use a frozen-thawed extract of peripheral blood mononuclear cells added to normal human fibroblasts. The cells are monitored for CPE, and in particular, the formation of enlarged, rounded, vacuolated cells, that often form WO 99/34019 PCT/US98/27744 7 syncytia. An important observation is that the intensity of the CPE is enhanced by regularly replacing the medium in the tissue cultures, as a means to limit the accumulation of viral inhibiting components. The CPE can also be enhanced by the addition of 30% boiled supernatants of HCMV infected cultures, and/or by the inclusion of boiled or otherwise inactivated preparations of various viral vaccines, such as a commercial feline rhinotracheitis-calici-panleukopenia-chlamydia psittaci vaccine manufactured by BioCor inc. Omaha, Nebraska. As discussed below, it appears that extraneous viral and bacterial and cellular DNA can become incorporated into the viral replicative process and enhance stealth virus yields. Such additions, are contraindicated when the goal is to obtain primary sequence data on the clinical isolates. Stealth viruses can also be isolated from tissue biopsies, cerebrospinal fluid, semen, urine, throat swabs and feces. The routine successful culturing of stealth viruses has provided valuable insights into the wide range of illnesses that can fit the pattern of a persisting viral infection. While, the initial focus was on patients with neuropsychiatric illnesses, including the chronic fatigue syndrome (CFS), it became apparent that very severe, and even fatal neurological illnesses, were occurring in virus infected patients. The unique susceptibility of the brain to stealth virus infections, could be explained on the basis of spatial segregation of the various functions of the brain. Thus unlike other organs, limited damage to the brain can not be compensated for by heightened activity elsewhere. The induction of malignancy provides another example where damage to even a single cell can have dire consequences. Evidence for stealth viruses has been obtained in a number of patients with myelomas, melanomas, brain tumors, lymphomas and breast cancers (see below). Stealth viruses have also been cultured from animals, including symptomatic pets of patients with neuropsychiatric and oncogenic illnesses. The finding of atypical cytopathic viruses in patients with such a diverse array of clinical findings has unfortunately tended to impede rather than accelerate scientific acceptance of the basic culture findings. This is one reason for again emphasizing the potential value of actual sequence data on cultured virus isolates. The combination of tissue culture and molecular methods has provided informative sequence data on several stealth virus isolates. Moreover, recombinant clones resulting from these studies have provided a useful set of reagents to pursue stealth viral studies as well as other research and commercial endeavors. Obtaining Sequence Information on Stealth Viruses. Methods are in place such that once a stealth virus is cultured, one can readily proceed to defining its composition. As described in the "Modes for Carrying Out the Invention" this can be achieved using a variety of molecular methods, including PCR, primer induced nucleic acid synthesis, isolation of viral DNA and/or RNA. The nucleic acids can be sequenced and this information used to ascertain the potential biological properties of the viral isolate. Other approaches are also available including amino acid sequencing of protein products in culture supernatants and deriving, by reverse genetics, the probable nucleotide sequence of the coding gene. Cellular toxic products can also be isolated from cultures and their composition can be determined and related back to the genetic composition of the virus. Of these various approaches, the "Best Mode for Carrying Out the Invention" proceeds from culturing a stealth virus and determining its genetic sequence as described in the following section.

WO 99/34019 PCT/US98/27744 8 Modes for Carrying Out the Invention The culturing of the stealth viruses comprises inoculating a test tube containing human MRC 5 fibroblasts with a frozen-thawed aliquot of ficoll-paque isolated and washed mononuclear cells obtained from the equivalent of 4 mis of blood collected in an ACD (acid citrate dextrose) anti coagulated blood sample. The MRC-5 cells are maintained in 5 mis of X-Vivo 15 serum free medium (BioWhittaker, Walkersville Maryland, USA). The tubes are placed on a rotator at 12 revolutions per minute in a 37oC incubator. The cultures are observed daily for a CPE. A positive culture is defined as i) the loss of the following normal features of the cell monolayer. Normal characteristics include a monolayer consisting of an inconspicious sheet of elongated flat cells without noticeable intercellular gaps. The normal monolayer extends to the base of the test tube without evidence of cellular detachment. Similarly, the periphery of the normal monolayer should have < 5% of cells showing signs of rounding and/or enlargement. The monolayer should also not display areas of pigmentation or granularity. In addition to the loss of such normal features, a typical positive culture will show most of the following changes. The monolayer transforms to a rather coarse interwoven meshwork of readily apparent cells with intracellular retraction" evidence of detachment of cells, especially from the base and periphery, with scattered islands of reattached cells. Parts of the monolayer and the reattaching cells become foci of enlarged, rounded, or distinctly abnormal appearing cells, either alone or in apparent syncytia. Multiple areas of increased pigmentation and/or patches of residual cellular debris can be seen. An important feature of any questionable culture is the marked deterioration in the overall appearance of the culture and in individual cells 15-120 minutes following replacement of the old media with fresh media. Signs of cellular destruction, abundant lipid accumulations, extraneous particulate matter, and formation of granular, rod shaped and branching structures can be observed over several days. Typically, the positive cultures do not undergo the cell shrinkage and disappearance seen in control cultures. Moreover, the CPE can be transferred from a positive culture by using a frozen-thawed extract of centrifuged cells, detached by gentle vortexing a positive culture. Secondary and tertiary cultures will typically show a more subdued and focal changes. Frequent, even daily refeeding, especially with media enhanced with boiled supernatants of human cytomegaloviral or other herpesviruses and even positive stealth viruses, is sometimes a critical element in maintaining strongly positive cultures. Typically a 30% addition of boiled supernatant of a human cytomegalovirus culture, is used for this purpose. Additional confin-natory tests can include positive immunofluorescence of infected cells using selected human sera, wide range of permissive cell types, and exclusion of intact conventional viruses using specific antisera and molecular based assays. One aspect of the molecular based confirmatory assays is the generation of atypically sized PCR products using various primer sets based on sequences of known conventional viruses or on known sequences of previously cultured stealth viruses. Sequence information obtained on such atypically sized PCR products, including products generated from reverse transcribed RNA, can allow for the design of additional PCR primers tailored for the specific stealth virus being cultured. The following information provides an outline of the approach taken, and describes the results obtained in the molecular analysis of a "Stealth Adapted African Green Monkey Simian Cytomegalovirus" Stealth Virus. The virus was originally isolated using culture methods from a patient with chronic fatigue syndrome. An aliquot was deposited at the American type Culture Collection ATCC and given accession number VR2343. A culture was reestablished from a frozen aliquot of an early passage and grown on human fibroblasts, (BioWhittaker, Inc., Walkersville. MD.).

WO 99/34019 PCT/US98/27744 9 PCR Products: The cultures were used to generate PCR products. Several primer sets were initially used. A set of primers, designated SK43 and SK44, based on the tax genes of HTLV I and HTLV II, respectively, yielded 2 products of approximately 1,500 bases and a smaller product of 0.67 kb bases. DNA Extraction and Cloning. PCR products were isolated from agarose gels and ligated into a commercially available plasmid ( pBluescript, available from Stratagene, La Jolla, CA). Purified DNA was also extracted from the cultures, cut with a restriction enzyme, and cloned into pBluescript. Two independent experiments were performed on culture-derived DNA, yielding two sets of cloned DNA. An interesting observation was made during the work up phase for these studies. Restriction enzyme digestion of a conventionally structured virus is expected to yield equimolar amounts of contiguous fragments representing the entire genome. On agarose gel electrophoresis, the digestion products should be seen as multiple bands of essentially equal intensity. In contrast to these expected findings, electrophoresis of restriction enzyme digested DNA isolated from the stealth virus cultures yielded numerous bands of widely differing intensities. The uncut DNA migrated as a band of approximately 20 kilobases (kb), yet the aggregate size of the bands following digestion clearly exceeded 50 kb. The PCR generated products and the products of enzyme digestion were ligated into pBluescript plasmids and cloned. Clones and Sequencing. The PCR clones were designated 15-5-2, 15-5-4 and SK43/43. The 3B series of clones was obtained using EcoRl digestion of DNA extracted from the material, pelleted by ultracentrifugation, that was present in filtered supernatants of infected MRC-5 cells. The C1 6 series of clones was obtained using SacI digestion of agarose banded DNA extracted from the material, pelleted by ultracentrifugation, present in filtered supernatant of lysed virus-infected cells. The T3 and T7 promoter sites of the pBluescript vector were used in sequencing reactions to obtain partial sequence data from the ends of each of the inserts. Extended sequences of the inserts in selected clones were obtained using primers based on the T3 and T7 readouts. The sequencing services of the BioServe Biotechnology, Laurel, MD, U.S. Biochemical, Cleveland, OH, City of Hope Cancer Center, Durate CA, Midland Certified Reagent Company, Midland, TX; and Lark Technology, Houston, TX, were used with excellent correlation of the occasional duplicate and even triplicate testing of the same clones. The individual sequences were analyzed against GenBank entries using the gapped BlastN and unfiltered BlastX programs of the National Center for Biotechnology Information (NCBI) of the National Library of Medicine. A "p" value of < e -2.0 was used as a cutoff for a significant nucleotide or amino acid sequence homology. Results. The PCR product 15-5-4 was 15 kb bases in length and flanked by the SK44 primer. Sequencing of this product showed a statistical significant relatedness, by both BlastN analysis, to a portion of the human cytomegalovirus (HCMV) genome that codes a protein designated UL34. The 15-5-2 product of a similar size and was also flanked by the SK44 primer. Its internal sequence could not, however, be matched to any known sequence. The 0.67 kb product was flanked by the SK43 primer and when first examined had no significant matching with any GenBank entry. At least partial sequencing has also been obtained on over 300 clones from the virus infected cultures.

WO 99/34019 10 PCT/US98/27744 For many of the clones, the sequencing merely comprise a relatively short readout from the T3 and T7 promoter sites of the pBluescript plasmid. For selected clones, complete (C) sequencing has been obtained. As with the 15-5-4 PCR product, several of the clones contained sequences that could be statistically aligned by BlastN analysis to at least some portion of the protein coding regions of the HCMV genome. The BlastX program, which is based on the deduced amino acid sequences coded by a nucleotide sequence, identified many additional clones with significant partial sequence homology to various HCMV proteins. The matching was often incomplete with apparent gaps and occasional insertions in the sequences of the stealth virus clones. An overall indication of the HCMV proteins for which at least some portions could be aligned with a sequence contained in one or more of the stealth virus clones is shown in Table 1. Table 1. HCMV Proteins Identified by Homology Searching Using Sequences in the DNA Extracted From Cultures of Stealth Virus- I * ULI4 UL22 UL23 UL29 UL31 UL32 UL34 UL35 UL36 UL37 UL38 UL40 UL41 UL42 UL43 UL44 UL45 UL47 UL48 UL49 UL50 UL51 UL52 UL54UL56 UL57 UL69 UL70 UL71 UL72 UL75 UL76 UL77 UL78 UL84 UL85 UL86 UL87 UL88 UL89 UL93 UL95 UL97 UL98 UL102UL104UL105UL112UL122ULI23UL124 UL126ULI30OULI32UL141 UL144UL145US18 US21 US22 US23 US24 US25 US26 US27 US28 US29 US30 * Greater than 5 clones matched to the proteins that are underlined and ten or more clones matched to the proteins highlighted by bold print. Note: The genome of the fully sequenced, laboratory-adapted, ADI 169 strain of HCMV comprises 235,000 nucleotides base pairs. The virus has two linear segments, designated unique long (UL) and unique short (US). Each segment is flanked by relatively small regions of repetitive sequences. The potential proteins coded by the UL and US regions are designated numerically, and extend from UL1-132 and US 1-36. Additional potential open reading frames are present within the repeat regions that flank both the UL and US segments. Fresh clinical isolates of HCMV contain some additional protein coding sequences, designated UL 133-151, that are not present in the laboratory adapted AD169 strain. Sequence comparisons of animal and human herpesviruses indicate a greater conservatism of the central core sequences from UL30 to UL125, compared to the sequences prior to UL30 and beyond UL125. As shown in the above Table, the nucleotide and protein matching regions of the stealth virus are widely distributed throughout much of the central core region of the HCMV genome and in the later part of the US region. The matching sequences were not uniformly distributed through the central core region of the HCMV genome. For example of 300 clones, 10 or more clones matched to the UL36, UL52, UL86 and US28 regions of the HCMV genome, while other regions were not represented by any of the clones from which sequence data have so far been obtained.. The homologue of the UL83 gene of HCMV was not represented in any of the sequenced stealth virus WO 99/34019 PCT/US98/27744 11 clones. The UL83 lower matrix protein (pp65) which is the major target for cytotoxic T cell mediated immunity against HCMV (Wills MR, Carmichel AJ, Mynard K., et al. "The human cytotoxic T lymphocyte response to cytomegalovirus is dominated by structural protein pp65: frequency, specificity, and T-cell receptor usage ofpp65-specific CTL. J. Virol. 70: 7569-79, 1996). This finding is consistent with the underlying assumption concerning gene deletion as a means for stealth adapted viruses to bypass cellular immune defense mechanisms. Major deletions were also seen in portions of many of the proteins that were identified using the BlastX program. Moreover, many of the deduced amino acid sequences were not included in analyses based on putative open reading frames (data not shown).. To recapitulate the findings, the number of clones that matched to specific regions of the HCMV genome varied from a single clone to multiple clones. The regions of the HCMV genome for which > 5 clones contained statistically significant matching sequences by BlastX analysis are underlined in the Table. Regions of the HCMV genome in the vicinity of the UL36, UL52, UL86 and US28 gene product were each represented by 10 or more of the 300 analyzed clones. Noticeably absent were sequences of the major target for anti-HCMV cellular immunity UL83, whereas 6 clones were identified that matched to portions of the neighboring UL84. All of these clones extended forward towards the UL86 and UL87 gene products. In addition to apparent absence of major proteins, many of the proteins for which partial matching was seen, showed major deletions when compared with the corresponding protein in HCMV. Moreover, individual clones that matched to similar regions of the HCMV genome, often displayed minor sequence differences, indicative of an unstable viral genome. These differences occasionally disrupted the potential of a contiguous nucleotide sequences to provide a functional open reading frame. They also could account for the unequal distribution and surprisingly large number of products seen following restriction enzyme digestion of the DNA derived from the stealth virus cultures. Also of note, the aggregate lengths of the cloned stealth viral DNA sequences that match to non-overlapping regions of the HCMV genome exceeded 100 kb. Since, as mentioned above, the stealth virus DNA migrates in agarose gels with a size of only approximately 20 kb, it can be assumed that the genome consists of multiple fragments, rather than as an entire full length cytomegaloviral genome. Limited nucleotide and amino acid sequence data are available for rhesus cytomegalovirus (RhCMV). Several of these sequences corresponded to regions of the HCMV genome which matched to portions of the prototype stealth virus sequence. For these regions, it was possible to compare the relative relatedness of the stealth virus sequence with that of RhCMV and HCMV. This analysis showed a significantly higher homology of the stealth virus sequences to RhCMV compared to HCMV. A comparison of the p values seen when matching the nucleotide sequences using BlastN program and deduced amino acid sequences using BlastX program of the stealth virus clones against homologous regions of HCMV and RhCMV sequences are listed in Table 2.

WO 99/34019 PCT/US98/27744 12 Table 2. Comparison of Relative Homology of Sequences Present in Various Clones Derived From a Stealth Virus Culture to Corresponding Sequences in Human and Rhesus Cytomegaloviruses Analyzed Using BlastN and BlastX Programs Clone Region of BlastN Analysis BlastX Analysis Homology HCMV RhCMV HCMV RhCMV C16289.T3 UL31 NS 9e-21 2e-41 8e-53 C16292.T7 UL45 NS 5e-04 2e-26 le-33 3B51.T7 UL47 NS 6e-13 5e-25 6e-38 3B642.C UL48 NS NS NS 9e-26 C16169.T7 UL54 5e-07 4e-44 2e-42 2e-50 3B529.C UL71 5e-12 8e-23 5e-09 le-14 3B559.T3 UL76 NS 3e-15 4e- 11 3e-26 3B3.T7 UL87 NS e-104 2e-83 e-102 C16123.T7 UL89 5e-51 e- 113 7e-84 2e-89 C16145.T7 UL105 NS le-05 le-13 3e-16 3B546.C UL123 7e-09 le-71 8e-28 3e-61 C162113.T3 US21 NS NS 2e-61 8e-27 3B656.T7 US22 NS 7e-09 8e-28 3e-61 The higher homology to RhCMV compared to HCMV identified the stealth virus as not of HCMV origin. The variable statistical levels of the matching clearly distinguishes the stealth virus from RhCMV. Complete identity over a stretch of hundred or more bases will typically yield p values of< e-100. Very limited genetic information is currently available for other primate cytomegaloviruses. There are, however, several sequences in GenBank for African green monkey simian cytomegalovirus (SCMV). This provided an opportunity to directly compare a stealth virus sequence with several cytomegaloviruses. A much greater homology was seen with SCMV than for any other cytomegalovirus. Sequence data supporting the unequivocal origin of the stealth virus from is provided by comparing the sequence of the immediate-early gene transcriptional unit of SCMV with that of stealth virus clone 3B546 (Table 3). Several small gaps had to be placed in the stealth virus sequence for a proper alignment. The extraordinary high degree of homology between the stealth virus and regions of the SCMV genome, with p values often reaching zero, unequivocally identified the virus as a derivative of SCMV.

WO 99/34019 PCT/US98/27744 13 Table 3. Comparison of Sequence of Clone 3B546 Derived from a Stealth Virus With the Sequence of the Immediate Early IE 94 Transcriptional Unit of SCMV Stealth Virus Sequence SCMV Sequence Nucleotide Identity p Value 1 -421 8410 - 7984 408/428 0.0 408 -930 7986- 7464 504/525 0.0 929 -4193 7435 -4168 3085/3272 0.0 4185 -4360 3985 -3880 259/297 3e-95 4496 -4853 3707 -3347 510/536 0.0 5046- 5333. 2941 -2654 263/288 e-111 5394 -6026 2557 -1925 594/633 0.0 6043 7926 1893 - 11 1782/1884 0.0 The overall alignment of the two sequences required that variable sized gaps be present between the above listed matching segments. The size of the gaps for SCMV were larger than those for the stealth virus, consistent with other indications of genetic deletions. Most of the additional sequences deleted from the stealth virus were in non-coding areas, including introns, known to exist within the SCMV IE gene. Highly significant matching to two other regions of the SCMV genome was also present. The identification of an SCMV-derived stealth virus was not an isolated finding. Indeed, a young woman with a prior history of bi-polar psychiatric illness was admitted to hospital because of coma and seizures. Although her cerebrospinal fluid was essentially non-inflammatory, it grew out an atypical virus showing extensive molecular cross-reactivity in PCR and direct probe-based assays as did the viral isolate from the CFS patient. Yet by culture, the wide host range of permissive cells, and the formation of vacuolated foamy syncytia, clearly distinguished the stealth viruses from the standard Colburn laboratory strain of SCMV. Such differences should be reflected in the nucleotide sequences of the viruses. Consistent with this conclusion was the fact that while SCMV had clearly contributed multiple genetic sequences to the stealth virus, other regions of the virus could not be aligned to known herpesviral sequences. The virus DNA appeared to comprise multiple fragments with stretches of herpesviral-derived DNA, adjacent to sequences of uncertain origin. The virus also exhibited considerable sequence micro-heterogeneity. Similar stretches of the viral genome often showed multiple deletions, additions, substitutions and duplications. Sequence heterogeneity was confirmed in stretches of the genome in which genetic recombination had clearly occurred.

WO 99/34019 14 PCT/US98/27744 The possibility of cellular sequences contributing to portions of the prototype stealth virus genome was suggested by the significant partial homology (p < 0.05) of a portion of one of the viral DNA-derived clones to a cellular gene. With the ever increasing database of cellular sequences and the recent introduction of a "gapped" BlastN program, redoing the original analysis has provided many additional examples of cellular-derived sequences within the cloned DNA from the stealth virus culture. For example, the SK43/43 derived PCR product matches closely to a cellular gene in the Xp22 166-169 region of the human genome (GenBank accession AC005145) with a p value of 0.0. Other examples of clones from the SCMV-derived stealth virus culture containing cellular related sequences are provided in Table 4. Table 4. Regions of Homology of Stealth Virus Associated Sequences with Cellular Genes Stealth virus Region of clone Cellular gene(s) with Number of p value clone number matching to cell maximum homology matching sequence (GenBank accession nucleotides (No. of bases) (*reiterated sequence) C1629.T3 124-345 AA570230* 98/111 1 e-20 (512) C 1629.T7 25-76 Z97200* 50/52 I e-15 (410) Cl6261 .T3 511-534 N52914 26/27 5 e-04 (676) C16261.T7 97-119 AC002480 21/21 4 e-03 (536) C16242.T3 388-413 AL008718* 29/29 3 e-08 (620) C16242.T7 None detected (563) C 16145.T3 335-361 AA535872 27/27 1 e-05 (U27784) (561) C16145 T7 None detected** (U27938) (528) WO 99/34019 15 PCTIUS98/27744 * Reiterated sequence were present in multiple copies within the cellular gene. Most of the matching sequences were also repetitive with the BlastN program identifying somewhat similar sequences in multiple cellular genes. ** Partial homology to human CMV is present. Specifically, 81 of the first 95 bases match to human cytomegalovirus (GenBank accession number X17403) at position 153,273 to 153,367 (p=l e-12) The presence of rearranged, mutated, cellular sequences in stealth viral cultures is corroborated by the sequences present in various PCR products generated using the same set of SK43/SK44 primers in stealth virus cultures from three additional patients. Multiple distinct products were generated from each of the patients. Sequencing of the products showed variable patterns of incorporation of the different primers. Several of the products were identifiable on the basis of Blast analysis as being of cellular origin Table 5 Table 5 Matching to Cellular Sequences of Cloned PCR Products Amplified From Stealth Viral Cultures of Additional Patients Using the SK43/SK44 Primers Patient SK primer(s) Number of BLASTN homology to cellular gene Clone No. incorporated* bases GenBank accession, Nucleotide match, p value sequenced Patient LB LB.43 43/44 507 AA429618 175/189 1 e-66 AA429619 104/115 4 e-32 AA429619 89/90 4 e-22 Patient KE Clll3.T3 43 211 AA429618 162/176 5 e-57 C1113.T7 44 280 AA429619 37/38 2e-10 Cl1123.T3 44 207 None identified C 1123.T7 43 216 AF027598 63/69 1 e-16 C 132.T3 43 217 None identified C1132.T7 44 272 None identified C 142.T3 43 246 AC002377 48/53 2 e-09 C1142.T7 44 275 AC003105 31/31 3 e-08 C 1151.T3 43 228 Z82216 122/133 7 e-43 Cl151.T7 44 236 Z82250 127/144 5 e-25 WO 99/34019 PCT/US98/27744 16 C 1163.T3 43 131 None identified C1163.T7 44 216 None identified Patient TR C 131 1.T3 43 299 None identified C 1311.T7 44 227 None identified C1313.T3 43 205 AF027598 63/69 1 e-17 C1313.T7 44 171 None identified C 1322.T3 44 165 None identified C 1322.T7 43 89 AA429618 26/26 5 e-06 C1325.T3 44 182 U73168 100/105 3 e-38 C 1325.T7 43 158 None identified C1333.T3 44 169 Z47066 82/94 3 e-16 C1333.T7 43 161 None identified C 1334.T3 44 185 None identified C1334.T7 43 164 None identified C1335.T3 43 138 M32672 33/36 2 e-04 C1335.T7 44 236 AC003015 31/31 2 e-08 *Slight differences were occasionally noted in the primer sequences. Selection for minor variations in primer sequence has been previously noted in performing PCR assays with these primers. Additional primer sets that have been used to generate PCR products in various stealth virus cultures. Some of the more successful primers have been based on internal sequences of various clones obtained from the stealth virus culture. An initial indication of a potentially successful primer is based is heightened synthesis of RNA and/or DNA when the primer is added directly to an extract of a stealth virus culture. Synthesis is presumably being induced by a viral or activated cellular polymerase. Additional synthesis can be induced by the addition of DNA and RNA dependent polymerases. The importance of RNA dependent DNA polymerase was highlighted in a series of PCR assays performed on a stealth virus culture from a nurse with CFS. No products were seen using the regular PCR format, yet multiple distinct products were seen if an initial reverse transcriptase reaction was performed. This finding indicated that the template to which the primer was binding was RNA. The actual sequences used in this study were based on several clones that contained commonly WO 99/34019 17 PCT/US98/27744 occurring regions of homology to HCMV, and subsequently shown to be even more akin to sequences of RhCMV. The sequences are shown in Table 6. Table 6. Primers Based on Sequences of Cloned Regions of Stealth Virus-1 Used to Amplify Products by Reverse Transcription Followed by PCR From Positive Stealth Virus Culture From the Cerebrospinal Fluid of a Nurse With a Chronic Fatigue Syndrome Like Neurological Illness ...............................................---------------------------------------------------------------.. GenBank Plasmid Size of PCR product Primer sequence accession No. designation with stealth virus-I U27952 C 162114 279 bp 5'-AACCATGTCTGCCACATCG-3' 5'-CAATAGGATCTCTCGCGCAC-3' U27916 3B647 373 bp 5'-CGCTGTCGCTCTCTTCCTT-3' 5'-GAGCACGATACGGTGTTGC-3' U27888 3B412 374 bp 5'-CCTGTTGTCATCTTGTTCAGG-3' 5'-AATGTTCGACAGTCTGCGC-3' PCR assays were initially performed on DNA extracted from the patient's cultures with DNA from an SCMV culture serving as a positive control. Each of the primer sets tested gave negative results when tested on DNA extracted from the patient's positive culture. When the same primers were used in reverse transcriptase PCR (RT-PCR) assay on RNA extracted from patient's culture, clearly defined PCR products were generated. The PCR product generated with the primer set based on plasmid C 162114 was similar in size to the 278 base pair (bp) product expected for stealth virus- 1. This primer set yielded a similarly sized, but faint product with SCMV. The primer set based on plasmid B614 gives a similar size product with SCMV and stealth virus-I of 279 bp. A distinctly larger product than seen with SCMV was generated using the patient's culture (480 bp compared to 373 bp). Multiple additional faint bands were also generated. The primer set based on plasmid 3B412, generated two products (550 bp and a less intense band of 425 bp). Both of these products were considerably larger than the product seen with SCMV or predicted for stealth virus-1 (374 bp).

WO 99/34019 PCT/US98/27744 18 Renewed interest in pursuing cellular as well as viral sequences in stealth viruses was prompted by the realization that certain stealth viruses could potentially assimilate and transmit genes able to transform cells. A search was, therefore, made for any cellular sequences related to known oncogenes. Preliminary indications for this were found with sequences obtained from certain stealth virus cultures and confirmed by additional sequencing of the SCMV-derived prototype stealth virus. Specifically the sequence data show that parts of the virus has acquired several copies of a gene with a deduced amino acid sequence closely related to that of the oncogene termed melanoma growth stimulatory activity (MGSA/GRO-alpha) chemokine. Please note that this specific example was not included in the application for which priority is requested. The concept and prediction of this finding was, however, expressed. The 3B516 clone was fully sequenced during 1998. The ends of five partially sequenced clones matched by BlastX analysis to the MGSA/GRO alpha and to other closely related chemokine proteins. One of the clones, 3B516, was completely sequenced. It comprised 5,820 nucleotides. Except for matches to the other four clones from the stealth virus cultures (3B33, 3B624, 3B654 and 3B675), BlastN did not reveal significant homologies with current GenBank entries. By BlastX analysis, however, the nucleotide sequence from the left side of the clone could be translated into a string of proteins with highly significant homologies to the ULI41, UL144, and UL145 proteins respectively of the Toledo strain of HCMV. An apparent open reading frame (ORF) was situated slightly beyond the region coding for the UJL145 protein. The deduced amino acid sequence of this ORF failed to show a statistically significant match to any protein entries on GenBank. It did, however, show a weak partial matching to a human cadherin tumor suppressor protein. The right side of the clone contained three discrete regions, each of which could be translated into proteins that matched to MGSA/GRO-alpha chemokine. Two of the matches were statistically highly significant. Another potential open reading frame was situated between the region of clone 3B516 that showed a weak homology to the cadherin tumor suppressor protein and the region showing an identified, but statistically insignificant, homology to the MGSA/GRO-alpha chemokine. BlastX analysis matched this region to a human alpha chemokine that is only distantly related to the MGSA/GRO-alpha chemokine. The BlastX results indicating the best matching proteins for the various regions of clone 3B516, are summarized in Table 7. The Table also shows the percent identity and calculated "p" value for the HCMV matching sequences.

WO 99/34019 19 PCT/US98/27744 Table 7. Proteins that Most Closely Match to the Deduced Amino Acid Sequences Coded by Clone 3B516 Using BlastX Analysis. Nucleotide Matching Sequence Region Gene Accession # Identity p value 970-1,884 HCMV UL41 il 1167926 127/305 le-59 2,231-2.268 HCMV UL44 gi1167929 54/146 I 3e-18 3,122-3,361 HCMV UL145 gi1167930 52/80 1e-22 3,584-4,015 cadherin-related tumor suppressor d1014097 23/85 0.23 4,095-4,343 alpha chemokine human gi2580586 31/83 0.015 4,583-4,807 GRO human P09341 28/75 1.5 4,938-5.198 53/87 8e-23 5,469-5.795 45/109 5e-09 The deduced amino acid sequences of the potential ORF that comprised the 4 regions of clone 3B516 that matched to a cellular chemokine gene, are shown in Table 8. The sequences differ significantly from each other, and to varying degrees, from the amino acid sequence of the MGSA/GRO-alpha precursor protein. Also shown for comparison is the amino acid sequence of the closely related alpha chemokine, macrophage inflammatory protein-2-alpha (Gro-beta). The alignments for 3 of the sequences were obtained from the BlastX program. The amino acids that match to those of the MGSA/GRO-alpha precursor protein are underlined. The cystines involved in disulphide bond formation are indicated with an asterisk. Sequence 2 contained an apparent insert that separated two closely matching segments. This insert is shown beneath the site separating the matching segments. The initial codon for this insert was a stop codon indicated by the # symbol. The 4h sequence did not match to MGSA/GRO-alpha, but rather, as noted in Table 7, to a more distantly related human alpha chemokine. The sequence analysis shown in Tables 7 and Table 8, is consistent with a genetic recombination between a portion of the SCMV-derived stealth virus that encodes several cytomegalovirus related proteins and a region of the cellular genome that encodes a protein with chemokine activity. The data emphasize the value of obtaining sequence data on stealth virus isolates, and the potential that exists for stealth viruses to transmit potential oncogenes.

WO 9 9/34019 20 PCT/US98/27744 C, 2 > >) > > o z 0 > o > <( < I C L UVI UVI > < - : LI 2:: zI > >1 > > > >V to UI V) m) 01 0 1 D Z) 0 fl D, 0iOl 01 0 01 Y0 Y. Z U I u uI HY :) LI V WOLoI W O L >) U) '4: V) OH V) U) VH V) VD V) E- -n 0 0 0 0 0 > W ('4 '4 (> >5 >5 >) >)U WO 99/34019 PCT/US98/27744 21 It is unlikely that the chemokine genes were present in the original SCMV from which the stealth virus was derived. The HCMV Toledo strain in which the UL144 and ULl145 coding genes have been sequenced, continues with sequential genes that code for proteins designated UL146 to UL148. While the UL147 proteins of both the Toledo and Towne strains of HCMV do show weak and statistically insignificant (p value 0. 15), homology to an alpha chemokine ofquinea pig origin by BlastX analysis, the common sequences show very little overlap with those coded by the 3B516 clone. Moreover, this would not account for the multiple copies of the human chemokine related sequences in the stealth virus. Additional differences between the stealth virus and HCMV clinical isolates include the lack of ULl42 and UL143 genes. Further clarification of this important issue should be forthcoming from sequencing studies on SCMV isolates. The previously alluded to genetic instability of stealth virus-I genome may account for differences seen in the 4 regions of clone 3B516 that matched, by BlastX analysis, to the MGSA/GRO-alpha chemokine. One of the regions clearly has a major insert, while all have apparent deletions. Slightly less statistically significant amino acid matching of two of the regions occurred with macrophage inflammatory protein-2-alpha chemokine, also known as GRO-beta. This was not unexpected since, as shown in Table 8, there is a strong homology between this protein and the MGSA/GRO-alpha chemokine. Other members of the superfamily include macrophage inflammatory protein-2-beta (GRO-gamma), neutrophil activating peptide/1L8, platelet factor-4, beta thromboglobulin and interferon-inducible protein-10. The human MGSA/GRO-alpha chemokine gene has been more extensively studied than the other chemokines because of its possible role in the autocrine stimulation of melanoma cell growth. The 1,895 nucleotide MGSA/GRO-alpha gene comprises a 5' non-coding region containing NF kappaB, HMG(1)Y, IUR, and Spl binding sites. It has 4 exons and 3 introns. The first exon codes a signal peptide (nucleotide 130-229) while the second, third and fourth exons comprise the mature protein (nucleotides 330...451; 565...648, 1180-1195). A long non-coding 3' end extends from nucleotides 1196 to 1895. cDNA sequences of the MGSA/GRO-beta and MGSA/GRO-gamma genes show extensive homology with the mRNA sequence of the alpha gene. A pseudogene has also been identified with homology covering the 5' non-coding region, the first and second exon and the first intron of the MGSA/GRO-alpha gene. The absence of intron sequences in the assimilated MGSA/GRO-alpha related regions of clone 3B516 indicates that the putative recombination event occurred at the level of RNA, rather than at the level of genomic DNA. The fidelity of replication by reverse transcription is generally less than DNA dependent replication. This may help explain the greater divergence seen when comparing nucleotide sequences than when comparing amino acid sequences. At the same time, the apparent amino acid conservation, especially with regards to the capacity to form disulfide linkages (as indicated in Table 8), suggests that the proteins are providing some positive selective pressure. It has not been established, however, that any of the chemokine related coding sequences are being transcribed and translated into functional proteins. The patient from whom stealth virus-1 was isolated has not developed any malignancies. As discussed below, evidence for stealth viral infection has, however, been obtained from a number of WO 99/34019 PCT/US98/27744 22 cancer patients and from several animals with various tumors. Useful information should be forthcoming from sequencing studies on stealth virus isolates from these cancer patients. Positive Stealth Virus Cultures in Cancer Patients Multiple myeloma: Viral culture techniques were used to screen patients with multiple myeloma for stealth viruses. This inquiry was prompted by a woman who had previously tested positive, as had her son, informing me that her father had developed multiple myeloma. It turned out that he had been depressed for several years with many of the same symptoms experienced by his daughter and grandson. He tested strongly positive, as have over 100 additional patients. On careful clinical review, it is apparent that significant emotional and cognitive difficulties and sleep impairment, are remarkably common in patients with multiple myeloma. Breast cancer: Several patients have had either their blood or tumor sample tested using the viral culture technique. Again, it has been surprisingly easy to obtain positive, transmissible CPE, from these patients. Other cancers: The daughter of the multiple myeloma patient developed a uterine cancer. An medical office worker with a prior clinical history of Lyme disease developed a melanoma on her finger. She, as well as her ill husband, gave strong positive stealth virus cultures. Additional stealth virus culture positive patients have developed lymphomas, brain tumors, thyroid cancers, salivary gland tumors, etc. In addition to viral and cellular sequences, the data on clones generated from the prototype SCMV-derived stealth virus indicate that the viral replicative process can extend to the incorporation of genes of bacterial origin. Indeed, there is good evidence for bacterial-derived sequences in at least 8% of the DNA clones derived from stealth virus-I infected cells. As outlined above, the inserts in 180 clones of the 3B series and in 120 clones of the C 16 series have been partially or completely sequenced. Of the 300 clones, the majority have at least a portion of their overall sequence that is homologous, by BlastN and/or by BlastX analysis, to the HCMV and/or SCMV genome. Other regions of the same clones and numerous additional clones, contain sequences that do not correspond to HCMV and/or to the limited known regions of the SCMV genome. Of these non-matching clones, 24 contained sequences that by BlastX analysis, could be partially matched to different protein sequences of bacterial origin. An additional clone had a nucleotide sequence that was similar to a bacterial ribosomal gene complex. The bacterial nucleotide and protein sequences that most closely matched to the sequences contained in the various clones derived from the stealth virus-1 infected culture are summarized in Table 9. For many of the matches, additional bacterial sequences were also identified, usually comprising groups of functionally similar entities. Several clones contained non overlapping nucleotide sequences that, when translated by the BlastX program, could potentially encode amino acid sequences that corresponded to portions of quite distinct proteins, not known to be coded by any contiguous set of genes in bacterial genomes, and in some cases, seemingly derived from widely divergent bacteria. For several clones, the matching with a particular bacterial gene occurred in discrete segments separated by variable size gaps. The degree of identity and the corresponding statistical "p" values for each of the matching segments are shown in Table 9.

WO099/34019 23 PCT/US98/27744 800. '- j" P jLJ I E I L II.I JL. LU~E 1 2u~~1 .z LL 07 1=1z~ 2 U - ) 5 , U ol UV14 0 ~ 72 CL~~I -) C <0 73 F-4~ 0 u QI U~~ 0- -Oe Uo f-, r-r4 n C r-I 4 0 a. I- oU C I t-t In U- r u uI eII tII U -q IE 4 -t Z IA ei ~ ':*~L1 WO 99/34019 24 PCT/US98/27744 0I Iti SC 151 - 1-, 1I-. Ir-16, 1- - q .- I m o e I-,E I I- O~ z C- YI L I- rL 5 a. E Z I ui0 Infp N t -0 -t 0 _ n :2 0 - 14. 4 I4 Ir II II 4, I I n\,IC Io. f- ocr ~ WO 99/34019 25 PCTIUS98/27744 E z -J VJ r 0_ C4 Ir- 0 00 a,0 00 'n In 0 00 I w 0 r.

0 ~K ze f -N 0% ccq .. Ulu Ir WO 99/34019 PCTIUS98/27744 26 C) C) -J - 0 = C)

I

C) 0' C C) ~ 0 N = -~ C) C) ~ C) r- u F- C) 0 2 C) -' *0 = C) C) C) - C) . C) C) C C) C) - C~) 0 - 0 0 -~ '-~ C) 0 C) EN 7 ~ .r. C) 0 C) -t 0. ~ 0 0' ~ Cl) ~ C) EN F; o ~ U, o '-' ~ C) 0 C) EN 0 o ~ ~ 0 C) o C) F- E~C 0 -~ C) 0 ~ C) -t ~- 0 C) C.) F C) C) -J C) C) F.- o 0 o 0. C) o (~ C) ~ C) U C) = -. C) C) > ~ 0 ~ 0 ~ .2 U ~ ~J) ~. C) N 0 ~ 0 .~ .-,. C) 0 -. C) 0 C) U, -~ ~ 0 -. -t o ~ C) C) 'N C) C) ~- C C) C) ~. C) - ~ ', U, - C)C - C) C) . 0* - 0 a 0 ~ o ~ * C - C) C) 0 - C) U ~ - ~-' C) * F - - ~ o o - C) 0 * f- WO 99/34019 PCT/US98/27744 27 The data presented in Table 9, indicate the existence of bacterial-related sequences in cloned DNA extracted from the stealth virus-1 infected culture. The diverse range of bacterial species identified using the BlastN and BlastX programs, argues strongly against the bacterial DNA being a contaminant of the cloning process. The high levels of identity also exclude the possibility of diverged herpesviral sequences being mistaken for bacterial sequences. A flanking sequence in clone 3B313 matched to the human C-C chemokine receptor, which in turn matches to the G-protein coupled receptor coded by the US28 gene of HCMV. With this possible exception, the demonstration of a viral sequence followed by a bacterial sequence in the same clone has yet to be documented. This can clearly be inferred, however, from the information provided in the Footnote to Table 9. Specifically, near identity was observed between a stretch of clone 3B313 and a sequence contained in clone C16246 which aligned to the HCMV gene coding the US28 protein. Moreover, additional regions of clone C16246 show a strong homology with several HCMV coded genes. Similarly, the limited overlap between clones 3B513 and 3B525 is informative. Beyond the overlapping region, clone 3B525 shows highly significant matching to HCMV. The overall sequencing data are consistent with variable patterns of recombination between various sequences of viral, cellular and bacterial origins. It could be argued that the bacterial sequences have, in fact, incorporated viral sequences, rather than the reverse. When dealing with obligate intracellular microorganisms, the distinction between virus and bacteria is somewhat irrelevant. This is especially so when several of the functions ascribed to the bacteria, are in fact, mediated by bacterial plasmids and/or subgenomic insertion elements. The predominance of virus-related sequences, and the lack of bacterial structures seen on detailed electron microscopy of stealth virus-I cultures, strongly favor the notion of a virus with assimilated bacterial-related sequences. The term viteria is proposed for a eukaryotic virus which has incorporated genes of bacterial and/or bacterial plasmid origin. Many of the matching bacterial sequences correspond to genes involved in rather unique energy generating and metabolic conversion reactions. Particularly, noteworthy are sequences contained in genes that participate in the transport, activation and/or synthesis of uncommon metabolites. Given such a wide array of metabolic functions, it is conceivable that viteria could maintain a limited capacity to metabolize, and possibly even to replicate, outside the confines of a cell. Certain bacteria could derive a competitive advantage by being infected with a viteria that encode functionally useful genes. The improved metabolic performance of a viteria infected bacteria, (or an infected fungus), could facilitate transmission of the underlying virus infection. Conversely, some of the pathogenicity of stealth viruses for human and animal hosts, could be mediated by toxic byproducts of the various metabolic pathways encoded by the assimilated bacterial genes. Toxic products have been detected in blood, urine and cerebrospinal fluids of stealth virus infected patients, and also in the supernatants of stealth virus cultures (unpublished). Supemrnatants from mixed bacterial cultures, obtained from a stool sample of the patient from whom stealth virus- I was originally isolated, also induced a vacuolating CPE in cell culture (unpublished). In addition to one or more toxin, the supernatants contain a filterable cytopathic agent that can be passed in tissue culture. Molecular studies on this agent have yet to be performed.

WO 99/34019 28 PCT/US98/27744 The presence of bacterial-related gene sequences in the stealth virus-1 culture has relevance to diagnostic microbiology. Positive PCR based assays, using primers reactive with various bacterial sequences, have been reported in patients with chronic fatigue syndrome, Gulf war syndrome, chronic Lyme disease, Alzheimer's disease, multiple sclerosis, arteriosclerosis and other diseases. These reports may reflect the presence of limited bacterial-related nucleotide sequences contained within an essentially viral pathogen. Minor differences in the ribosomal sequences of bacterial species have also led investigators to use PCR to classify the postulated bacterial pathogens that were presumably being detected. The BlastX findings showed an even better matching of amino acid sequences than nucleotide sequences. Viteria encoded proteins might be expected, therefore, to evoke antibodies that could be misinterpreted as evidence for infection with conventional bacterial pathogens.

WO 99/34019 PCT/US98/27744 29 Industrial Applicability The detection of nucleic acids in stealth virus infected cultures, and in clinical materials derived from stealth virus infected patients and animals, has provided new insights into the molecular heterogeneity and potential derivation of stealth viruses from both viral, cellular and bacterial sequences. The utilization of molecular based probing and genetic sequencing expands upon the capacity of culture based techniques to detect stealth viruses and provides a method to distinguish between stealth virus isolates. Sequence determination of a given stealth virus can be useful in relating abnormality in specific gene expression and the clinical disease manifestations. For example, it is expected that the stealth viruses isolated from patients with multiple myeloma may have incorporated and lead to aberrant expression of a B cell growth promoting cytokine such as interleukin 6. This possibility can be determined using the techniques outlined in this application. The determination of stealth virus-isolate specific sequences can be used epidemiologically to trace disease transmission. This approach can be applied to the stealth virus epidemic currently being studied in patients in the Mohave Valley. PCR based assays on patients' blood and CSF samples using primers reactive with previously isolated prototype stealth virus were clearly useful in documenting infection in the patient who died with a cerebral vasculitis, the comatose patient with a prior history of a bi-polar illness and in the health care worker in whom probing of her stealth virus culture for atypical RNA sequences yielded positive PCR results. The application of this research extend well beyond the field of medical diagnostics. To address this area first, it has been extremely useful to perform stealth virus cultures in patients presenting with severe neurological illnesses of uncertain etiology. In previously published papers and in a clinical paper recently accepted for publications, the positive stealth virus findings have prompted clinicians to introduce anti-viral therapies, such as ganciciovir. An extensive outbreak of stealth virus associated illnesses was recorded within the Mohave Valley region of the United States. The illnesses in many of these patients began with a gastrointestinal disorder, possibly transmitted via stealth virus infected bacteria. Several adults have died with multi-system illnesses, including signs of progressive encephalopathy. An accepted publication describes a 7 year old boy from this region in whom two brain biopsies were performed. The biopsies showed marked vacuolization, not unlike that seen with brain diseases attributed to prions. Interestingly, for over 7 months, his illness was simply attributed to attention deficit, oppositional defiant behavior, etc. Even his parents, both of whom are physicians, failed for a long time to see signs of an organic illness. His MRI was grossly abnormal, yet his clinical neurological signs were initially quite minimal. He eventually became clinically severely ill, being unable to walk and suffering severe headaches. Yet the concept of a virus encephalopathy was still not considered by specialist neurologists, who favored a degenerative process. Following the successful culture of a stealth virus from brain and cerebrospinal fluid, the child was begun on anti viral therapy 9ganciclovir). his downhill clinical course began to stabilize within the second week of treatment. Multiple other patients, variously labeled as having chronic fatigue Gulf war syndrome, Lyme disease, amyelotrophic lateral sclerosis, schizophrenia, depression and dementia, have been begun on ganciciovir on the basis of a positive stealth virus culture. Ongoing studies have also focussed on patients with various malignancies, and on patients thought to have a prion associated illness.

WO 99/34019 30 PCT/US98/27744 The identification of a stealth virus infection will be aided by sequence information pertaining to the particular virus isolate. This will help remove subjective uncertainty that can result from always distinguishing a toxic effect from a viral cytopathic effect. It will help define the origins and composition of a particular isolate. It will help identify patterns of disease transmission, for example between family members and within a workplace environment. The possibility of disease transmission via blood and other biological products, and even in foods and from other environmental sources can be effectively explored when one has sequence data to help tag and quantitate the virus. Sequence data can also assist in understanding the replicative processes employed by various stealth virus isolates. While some viruses, may replicate primarily through a DNA dependent DNA polymerase, there is evidence for reverse transcription of RNA to DNA. This is relevent to the current model in which fragments of RNA are possibly being assembled on pieces of DNA forming a type of scaffold. Such speculations will be refined by knowledge of actual sequence data. More importantly, this model needs to be tested in terms of whether shorter pieces of nucleic acids could potentially disrupt the putative scaffold and lead to cessation of viral replication. The sequence data themselves are also useful since they relate to overall functions of the stealth viruses. This is seen particularly with the cellular and bacterial sequences that can apparently become assimilated into the viral replicative process. The sequence data can also define antigenic targets for potential antibody production, both as adjunct diagnostic agents and as a possible means to evoke in vivo protective antibodies that may prevent viruses entering the brain. While stealth adapted viruses are mainly being viewed as pathogenic agents, they may also find beneficial scientific and commercial uses as potent vectors for the transfer of genetic information. Both the viruses themselves and the various genes identified through sequencing efforts on stealth viruses, should find widespread applicability, especially if species barriers do not exist for at least some of the isolates. Finally, the discovery of stealth viruses and an understanding of their sequence composition has led to a series of publications, some of which have been summarized earlier. A listing of the major publication, including several that are "in press" is as follows: Martin W.J. Detection of viral related sequences in CFS patients using the polymerase chain reaction.in "The Clinical and Scientific Basis of Myalgic Encephalomyelitis Chronic Fatigue Syndrome." Byron M. Hyde Editor. Nightingdale Research Foundation Press. Ottawa Canada pp 278-283, 1992. Martin W.J. Viral infection in CFS patients. in "The Clinical and Scientific Basis of Myalgic Encephalomyelitis Chronic Fatigue Syndrome." Byron M. Hyde Editor. Nightingdale Research Foundation Press. Ottawa Canada pp 325-327, 1992. Martin WJ, Zeng LC, Ahmed K, Roy M Cytomegalovirus-related sequences in an atypical cytopathic virus repeatedly isolated from a patient with the chronic fatigue syndrome. Am. J. Path. 145: 441-452, 1994.

WO 99/34019 PCT/US98/27744 31 Martin W.J. Stealth viruses as neuropathogens. College of American Pathologist's publication "CAP Today" 8 67-70, 1994 Martin WJ. Stealth virus isolated from an autistic child. J. Autism Dev. Dis. 25:223-224,1995. Martin WJ, Ahmed KN, Zeng LC, Olsen J-C, Seward JG, Seehrai JS. African green monkey origin of the atypical cytopathic 'stealth virus' isolated from a patient with chronic fatigue syndrome. Clin. Diag. Virol. 4: 93-103, 1995. Martin WJ, Glass RT. Acute encephalopathy induced in cats with a stealth virus isolated from a patient with chronic fatigue syndrome. Pathobiology 63: 115-118, 1995. Martin WJ. Severe stealth virus encephalopathy following chronic fatigue syndrome-like illness: Clinical and histopathological features. Pathobiology 64:1-8, 1996. Martin WJ. Stealth viral encephalopathy: Report of a fatal case complicated by cerebral vasculitis. Pathobiology 64:59-63, 1996. Martin WJ. Simian cytomegalovirus-related stealth virus isolated from the cerebrospinal fluid of a patient with bipolar psychosis and acute encephalopathy. Pathobiology 64:64-66, 1996. Gollard RP, Mayr A, Rice DA, Martin WJ. Herpesvirus-related sequences in salivary gland tumors. J. Exp. Clin. Can. Res. 15: 1-4, 1996. Martin WJ. Genetic instability and fragmentation of a stealth viral genome. Pathobiology 64:9-17, 1996. Martin WJ, Anderson D: Stealth virus epidemic in the Mohave Valley. I Initial report of viral isolation. Pathobiology 1997; 65:51-56. Martin WJ: Detection of RNA Sequences in Cultures of a Stealth Virus Isolated from the Cerebrospinal Fluid of a Health Care Worker with Chronic Fatigue Syndrome. Pathobiology Martin WJ: Cellular sequences in stealth viruses. Pathobiology 1998;66:53-58 Martin WJ: Bacteria related sequences in a simian cytomegalovirus-derived stealth virus culture. Exp Mol Path (in press). Martin WJ: Melanoma Growth Stimulatory Activity (MGSA/GRO-alpha) Chemokine Genes Incorporated into an African Green Monkey Simian Cytomegalovirus (SCMV)-Derived Stealth Virus. Exp Mol Path (in press). Martin WJ Anderson D. Stealth virus epidemic in the Mohave Valley: Severe vacuolating encephalopathy in a child presenting with a behavioral disorder. Exp Mol Path (in press).

Claims (22)

1. A method of detecting and characterizing a stealth virus, comprising; i) the isolation of DNA or RNA from a sample suspected of containing a stealth virus, such as a culture of cells showing a viral cytopathic effect, ii) testing the reactivity of the isolated DNA or RNA with a molecular probe(s) that contain 18 or more contiguous nucleotides identical to the sequences previously identified in a stealth virus, and, optionally, iii) sequencing the isolated DNA or RNA molecules that react with the probe.

2. The method of claim 1 in which the sequence of the molecular probe corresponds to the sequence of a viral gene, including genes of cytomegaloviruses, and in particular those of African green monkey derived simian cytomegalovirus (SCMV).

3. The method of claim I in which the sequence of the molecular probe corresponds to the sequence of a cellular gene that might be expected to react with one or more cellular genes that have been incorporated into the genome of a stealth virus, and, as a result of this incorporation, is/are likely to show minor sequence variations when compared to the original cellular gene.

4. The method of claim 1 in which the sequence of the molecular probe corresponds to the sequences present in the stealth virus isolate that has been deposited with the ATCC, and assigned accession no. VR 2343.

5. The method of claim 1 in which the sequence of the molecular probe corresponds to the sequences present in the stealth virus isolated from patients in the Mohave Valley and deposited with the ATCC and assigned accession no. VR 2568.

6. The method of claim I in which the sequence of the molecular probe corresponds to the sequences present in the stealth viruses previously isolated from other patients or animals.

7. The method of claim I in which the sequence of the molecular probe corresponds to a sequence present in a stealth virus that is subsequently isolated and portions of its sequence determined using the methods described in this patent application

8. A kit containing a nucleic acid probe reactive with a viral gene that encodes a major antigen involved in eliciting an effective cellular immune response, such that the probe could be used to screen for stealth-adapted viruses in which this particular gene has been deleted

9. A kit containing a nucleic acid probe reactive with a stealth virus and capable of distinguishing a stealth virus infected cell from an uninfected cell.

10. The method of claim I in which the testing is for the purpose of diagnosing a stealth-virus associated disease. WO 99/34019 PCT/US98/27744 33

11. The method of claim 1 in which the testing is for the purpose of detecting a stealth virus in biological products.

12. The method of claim I in which the testing is for the purpose of detecting a stealth virus in foods.

13. The method of claim I in which the testing is for the purpose of detecting a stealth virus in the environment.

14. The method of claim 1 in which the testing is for quantitating the levels of a stealth virus for the purpose of evaluating agents for their inhibitory effect on stealth virus replication.

15. The method of claim I in which the testing is for quantitating the levels of a stealth virus for the purpose of evaluating agents for their ability to enhance the replication of stealth viruses.

16. The methods of claim I in which the testing is for purposes of determining the capacity of stealth viruses to recombine with, and to potentially alter the nucleic acid sequences of another virus.

17. The methods of claim I in which the testing is for the purpose of determining the capacity of stealth viruses to recombine with, and to potentially alter the nucleic acid sequences of a cell.

18. The method of claim I in which the testing is for the purpose of determining the capacity of stealth viruses to recombine with, and to potentially alter the nucleic acid sequences of a bacteria.

19. Nucleic acid molecules that contain nucleotide sequences identical over a contiguous region of at least 30 nucleotides to the nucleotides present within any of the sequences identified as SEQ Number I- , submitted together with this application.

20. Peptide and protein molecules that contain amino acid sequences identical over a contiguous region of at least 10 amino acids to the deduced amino acids coded by a stretch of nucleotide sequences present within any of the sequences identified as SEQ Number 1 submitted together with this application.

21. The stealth virus isolate that has been deposited with the ATCC, and assigned accession no. VR 2343.

22. The stealth virus isolate that has been deposited with the ATCC, and assigned accession no. VR 2568.