WO1999034019A1 - Acides nucleiques discrets et procedes associes - Google Patents

Acides nucleiques discrets et procedes associes Download PDF

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WO1999034019A1
WO1999034019A1 PCT/US1998/027744 US9827744W WO9934019A1 WO 1999034019 A1 WO1999034019 A1 WO 1999034019A1 US 9827744 W US9827744 W US 9827744W WO 9934019 A1 WO9934019 A1 WO 9934019A1
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stealth
virus
viruses
sequences
sequence
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PCT/US1998/027744
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William John Martin
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William John Martin
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Priority to EP98964327A priority Critical patent/EP0977895A1/fr
Priority to NZ338040A priority patent/NZ338040A/en
Priority to AU19490/99A priority patent/AU1949099A/en
Priority to CA002282858A priority patent/CA2282858A1/fr
Publication of WO1999034019A1 publication Critical patent/WO1999034019A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • 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
  • 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
  • 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
  • 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)
  • the sequence may directly code for its protein (positive, -t-ve, stranded), or 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 c ⁇ te ⁇ a used for classification v)
  • the kinds of diseases produced can distinguish different viruses Related viruses are successively grouped into species, genera, families and orders
  • 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 (FUN) damages the immune system and causes the Acquired Immunodeficiency Syndrome (AIDS)
  • viruses can directly damage cells bv 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)
  • CPE cytopathic effect
  • 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
  • Immunological, tissue culture and molecular biological methods are used to diagnose viral 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
  • a specific IgM antibody response to viral components 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 IL2, 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
  • 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.
  • HCMV human cytomegalovirus
  • 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
  • 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
  • PCR proceeds by denaturing the double stranded DNA molecule by heat, and cooling in the presence of the oligonucleotide primers Because of their high 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.
  • 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.
  • 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.
  • 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.
  • novel sequence information 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.
  • 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.
  • HIV human immunodeficiency virus
  • Certain viruses may also interfere with immunological defenses through such mechanisms as downre ulation of the expression of histocompatibility antigens, induction of immunosuppressive cytokines and related virokines, and by remaining inactive, as in latent infections.
  • 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.
  • stealth viruses does not follow the usual approach of defining specific molecular and morphological characteristics
  • 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.
  • 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 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, Kansas.
  • various viral vaccines such as a commercial feline rhinotracheitis-calici-panleukopenia-chlamydia psittaci vaccine manufactured by BioCor inc. Omaha, Kansas.
  • 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.
  • Stealth viruses have also been cultured from animals, including symptomatic pets of patients with neuropsychiatric and oncogenic illnesses.
  • 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 is 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 37°C 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 ⁇
  • the virus was originally isolated using culture methods from a patient with chronic fatigue syndrome An aliquot was deposited at the Ame ⁇ can 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 ) 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
  • the PCR clones were designated 15-5-2, 15-5-4 and SK43/43
  • the 3B series of clones was obtained using EcoRI digestion of DNA extracted from the material, pelleted by ultracentrifugation, that was present in filtered supernatants of infected MRC-5 cells
  • the Cl 6 series of clones was obtained using Sad 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 pBluescnpt 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 PCR product 1 5-5-4 was 1 5 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 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
  • the sequencing merely comprise a relatively short readout from the T3 and T7 promoter sites of the pBluescnpt plasmid.
  • complete (C) sequencing has been obtained.
  • 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 genome of the fully sequenced, laboratory-adapted, AD 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 ULl-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 AD 169 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 UL 125.
  • 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 clones.
  • pp65 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 of pp65-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). .
  • 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.
  • RhCMV rhesus cytomegalovirus
  • 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 Table 2.
  • RhCMV RhCMV compared to HCMV origin.
  • the variable statistical levels of the matching clearly distinguishes the stealth virus from RhCMN. Complete identity over a stretch of hundred or more bases will typically yield p values of ⁇ e-100.
  • 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.
  • 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.
  • 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 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.
  • GenBank Plasmid Size of PCR product Primer sequence accession No. designation with stealth virus- 1
  • PCR assays were initially perfonned 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.
  • RT-PCR reverse transcriptase PCR
  • 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.
  • 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 .
  • MGSA/GRO-alpha melanoma growth stimulatory activity
  • ORF apparent open reading frame
  • 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.
  • 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 4 th sequence did not match to MGSA/GRO-alpha, but rather, as noted in Table 7, to a more distantly related human alpha- chemokine.
  • the previously alluded to genetic instability of stealth virus- 1 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/IL8, 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(I)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, 1 180- 1 195).
  • a long non-coding 3' end extends from nucleotides 1 196 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 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- 1 infected cells.
  • 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.
  • 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
  • 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.
  • 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 C 16246 which aligned to the HCMV gene coding the US28 protein. Moreover, additional regions of clone C 16246 show a strong homology with several HCMV coded genes.
  • 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.
  • the bacterial sequences have, in fact, incorporated viral sequences, rather than the reverse.
  • 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 term viteria is proposed for a eukaryotic virus which has incorporated genes of bacterial and/or bacterial plasmid origin.
  • 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.
  • 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).
  • Supernatants from mixed bacterial cultures obtained from a stool sample of the patient from whom stealth virus- 1 was originally isolated, also induced a vacuolating CPE in cell culture (unpublished).
  • the supernatants contain a filterable cytopathic agent that can be passed in tissue culture. Molecular studies on this agent have yet to be performed.
  • 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.
  • 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.
  • 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.
  • 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
  • sequence data themselves are also useful since they relate to overall functions of the stealth viruses This is seen particularly with the cellular and bacte ⁇ al sequences that can apparently become assimilated into the viral replicative process
  • 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
  • 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

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Abstract

Les virus discrets comprennent un groupement hétérogène du point de vue moléculaire, de virus cytopathologiques vacuolants, à structure atypique, induisant une infection systémique persistante, accompagnée souvent de manifestations neuropsychiatriques et éventuellement oncogènes et auto-immunes, en l'absence d'inflammation antivirale manifeste. L'apparition, la progression et les spectres d'activités caractéristiques de l'effet cytopathologique (CPE) distinguent les virus discrets des virus cytopathologiques humains classiques, dont les herpèsvirus, les entérovirus et les adénovirus. On peut utiliser la microscopie électronique, la sérologie et d'autres analyses moléculaires pour encore distinguer les virus discrets des virus classiques. Lesdits virus discrets peuvent assimiler divers gènes dérivés de cellules et dévirés de bactéries et éventuellement transférer lesdits gènes entre individus ou similaires, voire même entre différentes espèces. L'invention concerne des procédés de détection de virus discrets, au moyen de sondes moléculaires et de détermination de la composition d'isolats de virus discrets, en fonction des séquences nucléotidiques d'ADN et/ou d'ARN, détectables dans des cultures et dans des matériels cliniques ou autres, contenant un virus discret.
PCT/US1998/027744 1997-12-30 1998-12-30 Acides nucleiques discrets et procedes associes WO1999034019A1 (fr)

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EP98964327A EP0977895A1 (fr) 1997-12-30 1998-12-30 Acides nucleiques discrets et procedes associes
NZ338040A NZ338040A (en) 1997-12-30 1998-12-30 Stealth virus nucleic acids and related methods especially relating to cytopathic viruses
AU19490/99A AU1949099A (en) 1997-12-30 1998-12-30 Stealth virus nucleic acids and related methods
CA002282858A CA2282858A1 (fr) 1997-12-30 1998-12-30 Acides nucleiques discrets et procedes associes

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US118497A 1997-12-30 1997-12-30
US09/001,184 1997-12-30

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001059106A1 (fr) * 2000-02-08 2001-08-16 Takeda Chemical Industries, Ltd. Nouvelles proteines receptrices couplees a des proteines g et adn associes
WO2002002770A1 (fr) * 2000-06-30 2002-01-10 Takeda Chemical Industries, Ltd. Nouvelle proteine receptrice couplee a une proteine g et adn associe
US7074590B2 (en) 2000-06-23 2006-07-11 Maxygen, Inc. Chimeric promoters

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GOLLARD R P, ET AL.: "HERPESVIRUS-RELATED SEQUENCES IN SALIVARY GLAND TUMORS", JOURNAL OF EXPERIMENTAL AND CLINICAL CANCER RESEARCH., ROME., IT, vol. 15, no. 02, 1 January 1996 (1996-01-01), IT, pages 103 - 106, XP002918296, ISSN: 0392-9078 *
LOMBARDINI J B: "INHIBITION OF THE SYNTHESIS OF TAUROCHOLIC ACID BY STRUCTURAL ANALOGUES OF TAURINE", BIOCHEMICAL PHARMACOLOGY, ELSEVIER, US, vol. 26, 1 January 1977 (1977-01-01), US, pages 1175 - 1177, XP002908295, ISSN: 0006-2952, DOI: 10.1016/0006-2952(77)90064-8 *
RETTIG M B, ET AL.: "KAPOSI'S SARCOMA-ASSOCIATED HERPESVIRUS INFECTION OF BONE MARROW DENDRITIC CELLS FROM MULTIPLE MYELOMA PATIENTS", SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, US, vol. 276, 1 June 1997 (1997-06-01), US, pages 1851 - 1854, XP002918294, ISSN: 0036-8075, DOI: 10.1126/science.276.5320.1851 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001059106A1 (fr) * 2000-02-08 2001-08-16 Takeda Chemical Industries, Ltd. Nouvelles proteines receptrices couplees a des proteines g et adn associes
US7074590B2 (en) 2000-06-23 2006-07-11 Maxygen, Inc. Chimeric promoters
WO2002002770A1 (fr) * 2000-06-30 2002-01-10 Takeda Chemical Industries, Ltd. Nouvelle proteine receptrice couplee a une proteine g et adn associe

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AU1949099A (en) 1999-07-19
NZ338040A (en) 2002-07-26
CA2282858A1 (fr) 1999-07-08
EP0977895A1 (fr) 2000-02-09

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