WO1995027082A1 - Human neurological disease virus - Google Patents

Abstract

Two cell systems are disclosed for the production, detection and isolation of Human Neurological Disease Virus (HNDV) from patients with neurological disease. HNDV is maintained and reproduced in two interleukin-2 independent malignant lymphocyte cell lines, CM-1 and CM-2. Compositions are derived from the novel viral isolate, including the whole virus, proteins, polypeptides, polynucleotide sequences and antibodies to antigenic sites on the virus. These compositions are useful in a variety of techniques for the diagnosis and treatment of human encephalomyelitits such as multiple sclerosis. In addition, these compositions are useful in a variety of techniques for the detection of and vaccination against HNDV. Detection methods include immunoassays for both the virus and antibodies to the virus, and the use of polynucleotide probes to detect the viral genome. Vaccines include both wholly and partially inactivated viruses and subunit vaccines.

Description

  
 



   HUMAN NEUROLOGICAL DISEASE VIRUS
 This application is a continuation-in-part of U. S. serial number 08/221,380, filed on March 31,1994, and which is hereby incorporated by reference.



   FIELD OF THE INVENTION
 This invention relates to the detection and treatment of viral infection. More particularly, the invention relates to the diagnosis, vaccination and treatment of neurological diseases such as multiple sclerosis or encephalomyelitis, and compositions and methods therefor.



   BACKGROUND OF THE INVENTION
 Destruction of myelin happens in a wide variety of central nervous system (CNS) disorders. Although the causes for some demyelinating diseases have been found, the etiologic agent for the human prototype demyelinating disease, multiple sclerosis, remains unknown (see generally, Handbook of
Clinical Neurology, volume 9, edited by Vinken and Bruyn, 1971).



   Multiple sclerosis (MS) is a CNS disease with a highly variable course of clinical development. It was discovered and described in the 19th century. There are about 200,000 patients afflicted with multiple sclerosis in the
United States. The disease usually has its onset when a patient is between 20 to 40 years old. Common clinical manifestations of multiple sclerosis include reversible episodes of visual loss (optic neuritis), sensory disturbance or weakness in the trunk or limbs, vertigo, and bladder disturbance. In most patients, the disease follows a relapsing and remitting course. In the early stages of disease the remissions are usually associated with complete or nearly complete return of normal neurologic function, but with each remission there is less improvement and greater  neurological dysfunction. Females are affected about twice as commonly as males.



   The primary pathology of multiple sclerosis is confined to the central nervous system. The characteristic lesion of multiple sclerosis is the plaque of demyelination-a patch varying from a few cubic millimeters to many cubic centimeters in which the myelin sheath of nerve fibers is destroyed, leaving the axons relatively intact. The plaques are distributed asymmetrically throughout the brain and spinal cord, but there are certain sites of predilection which determine the clinical pattern of the disease. The important functional consequences of demyelination is block of electrical conduction in the nerve fibers which leads to many of the clinical features of the disease.



   McFarlin and McFarland, The New England Journal of
Medicine, vol. 307 (20): 1246-1251,1982, stated:
 There is no preventive measure or definitive
 treatment that will alter the course of multiple
 sclerosis. The marked clinical variations that can
 occur in the disease and the lack of specific
 diagnostic tests or indicators of disease activity
 have rendered it difficult to evaluate therapy.



   Haahr et al., Lancet, vol. 337: 863-864,1991, stated:
 Various viruses have been isolated from or detected
 in samples from patients with MS, but a reliable
 association has never been found. The clinical
 similarity between MS and tropical spastic
 paraparesis, a disease caused by HTLV-1, led to
 interest in the possibility that a retrovirus might
 be associated with MS, but there is no proof of such
 an association. Antibody assays, nucleic acid
 techniques, and enzyme methods, used to seek a
 retrovirus, have so far yielded ambiguous results.



   Perron et al., Lancet, vol. 337: 862-863,1991, stated:
 We have isolated from the CSF of a patient with MS a
 retrovirus antigenically different from any known
 human retrovirus. This virus proved difficult to
 characterize: it was poorly expressed in the
 original leptomeningeal cell-line (LM7) and has not
 been passed in any continuous cell-line.



   Perron et al., Research in Virology, vol. 143: 337-350, 1992, stated:  
 We succeeded in transferring the original CSF
 isolate into normal cultures of uninfected human
 leptomeningeal cells-called LM11-by co-culture
 with irradiated LM7 cells. This cell type is the
 only one which, until now could be infected and
 continuously produce LM7 virions in culture
 supernatants. This new culture-called LM711
 enabled the propagation of the LM7 virus, but the
 extracellular virion production remained as low as
 in original LM7 cells.



   McDonald, The Cambridge World History of Human
Disease, pp. 883-887, Cambridge University Press, 1993, not admitted to be prior art, stated:
 There is good evidence from family studies and
 epidemiology that an environmental trigger, probably
 infective, is required in the genetically
 susceptible individual. The most likely infective
 agent would be a virus, though interest in
 spirochetes has been revived recently on rather
 slender grounds.



   Isolation of a virus or infective agent associated with MS would allow for the development of in vitro diagnostic tests for detection and monitoring of MS. In addition, the isolation of a virus involved in the etiology of MS or other neurological diseases would facilitate the development of vaccines for treatment and prevention of these diseases.



  These and other needs are addressed by the present invention.



   SUMMARY OF THE INVENTION
 This invention provides for a purified Human
Neurological Disease Virus (HNDV). The HNDV virus of the invention is an RNA virus containing reverse transcriptase activity. The virus is a spherical virion particle with an average size of about 95-113 nm, as determined by electron microscopy. The virus is non-reactive with antibodies raised against HTLV-1, HIV-1 and EBV viruses and is also non-reactive with anti-HIV-1 p24 antibodies and anti-HIV-2/SIV p26 antibodies. The HNDV virus of the invention is specifically immunoreactive with antibodies raised to the HNDV virus present in CM-1 and CM-2 cell lines. The purified Human
Neurological Disease Virus can be obtained, for example, from the CM-1 cell line (ATCC Number CRL 11594) or the CM-2 cell line (ATCC Number CRL 11595).  



   The invention also provides for a cell line infected by the Human Neurological Disease Virus. These cell lines include human cell lines and T lymphocyte cell lines. For example, the infected cell line can be the CM-1 cell line or the CM-2 cell line.



   The invention also provides for methods for continuous production of the Human Neurological Disease Virus.



  In these methods, a cell can be infected with the virus. The cell has the capacity for continuous growth after the infection with the virus. The cell number is multiplied under conditions suitable for cell growth, and the virus produced by the cell is recovered. The method of infecting the cell can involve cocultivating the virus with the cell. The cell can be a T lymphocyte.



   The invention also provides for a method of producing a cell line containing an antigen of the Human
Neurological Disease Virus. This method involves infecting a cell with the virus, to yield a cell capable of continuous production of the virus, and thereafter multiplying said cell under conditions suitable for cell growth. The cell can be a
T lymphocyte.



   The invention also provides for methods for diagnosing viral infection in a susceptible host. These methods involve obtaining a biological specimen from the host and detecting the presence of Human Neurological Disease Virus or antibodies to the virus in the specimen as an indication of infection by the virus.



   The diagnostic methods also provide for methods of detecting antibodies reactive with the Human Neurological
Disease Virus in a biological specimen, which can be a human specimen. These methods involve contacting a composition containing the Human Neurological Disease Virus or an antigen obtained from the Human Neurological Disease Virus with a biological specimen, and then incubating the composition with the biological specimen to form an antibody: Human Neurological
Disease Virus antigen complex. The complex is then detected.



   The diagnostic methods also provide for methods of detecting a virus in a biological specimen, which can be a  human biological specimen. These methods involve contacting a binding agent having a binding affinity to the HNDV with the biological specimen and incubating the binding agent with the biological sample to form a binding agent: Human Neurological
Disease Virus complex, and then detecting the complex. The binding agent can be an antibody. The binding agent can also include a variety of other compounds capable of binding the
HNDV virus such as nucleic acid probes and PCR primers.



   The invention also provides for antibodies that are specifically immunoreactive to the Human Neurological Disease
Virus, and nucleic acid probes capable of selectively hybridizing to a nucleic acids obtained from the Human
Neurological Disease Virus.



   In addition, the invention provides for methods of detecting a viral nucleic acid obtained from the Human
Neurological Disease Virus in a biological specimen by contacting the biological specimen with a nucleic acid probe.



  The nucleic acid probe, which is capable of selectively hybridizing to the viral nucleic acid, is incubated with the biological specimen to form a hybrid of the nucleic acid probe with complementary nucleic acid sequences present in the biological specimen. The extent of hybridization of the nucleic acid probe to the complementary nucleic acid sequences is then determined.



   The invention also provides for a diagnostic kit for detecting antibodies immunoreactive to the HNDV virus. The kit has a container containing the Human Neurological Disease
Virus or an antigen obtained from the Human Neurological
Disease Virus and instructional material. Diagnostic kits for detecting the HNDV virus are also provided by the instant invention. These kits include a container containing an antibody specifically immunoreactive with the Human
Neurological Disease Virus and instructional material.



   Furthermore, this invention relates to compositions and methods for detection of and vaccination against a novel human T lymphocyte retrovirus designated Human Neurological
Disease Virus (HNDV). The compositions include the whole virus and portions thereof, particularly including  polypeptides which are cross-reactive with antibodies specific for determinant sites characteristic of the virus, such as those found on the major envelope and core proteins. The compositions further include antibodies capable of reacting with the virus and polynucleotides which are capable of duplexing with the virus genome. The compositions may be prepared by the methods disclosed in the detailed description of the invention and standard methods disclosed in Harlow and
Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, 1988, U.

   S. patents 5,118,602 (Pedersen et al.) and 4,520,113 (Gallo et al.), PCT publication WO 92/20787 "Stealth Virus Detection in the Chronic Fatigue Syndrome," (Martin), and PCT publication in Sri Lanka in 1989 on Japanese
Encephalitis Virus (Fournier et al.), incorporated by reference herein.



   Using the compositions of the present invention, the virus and viral infection may be detected by a variety of techniques, particularly immunoassays and techniques employing nucleotide probes such as those disclosed in the detailed description of the invention and in U. S. patents 5,118,602 (Pedersen et al.) and 4,520,113 (Gallo et al.), incorporated by reference herein. Immunoassays provide for the detection of the virus or antibody to the virus in a physiological specimen, particularly blood and lymph tissue. Nucleotide probes are used to detect the presence of the virus genome in a physiological specimen including, but not limited to, peripheral blood or cerebrospinal fluid (CSF). Vaccines may be prepared from the whole virus, either by partial or complete inactivation. Alternatively, subunit vaccines may be prepared from antigenic portions of the viral proteins.



   Finally, this invention is directed to the cloning of HNDV using routine molecular cloning techniques disclosed in J. Sambrook, E. F. Fritsch, and T. Maniatis, Molecular
Cloning: Laboratory Manual, 2 Ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, New York, 1989, incorporated by reference herein.



   Two cell systems are disclosed for the production, detection and isolation of Human Neurological Disease Virus  from patients with neurological disease. HNDV is maintained and reproduced in two interleukin-2 independent malignant lymphocyte cell lines, CM-1 and CM-2. Compositions are derived from the novel viral isolate, including the whole virus, proteins, polypeptides, polynucleotide sequences and antibodies to antigenic sites on the virus. These compositions are useful in a variety of techniques for the diagnosis and treatment of human encephalomyelitis such as multiple sclerosis. In addition, these compositions are useful in a variety of techniques for the detection of and vaccination against HNDV. Detection methods include immunoassays for both the virus and antibodies to the virus, and the use of polynucleotide probes to detect the viral genome.

   Vaccines include both wholly and partially inactivated viruses and subunit vaccines.



   BRIEF DESCRIPTION OF THE DRAWINGS
 Fig. 1. shows the growth curve of the CM-1 cells.



   Fig. 2A and 2B: Morphology of Virus Particles: Fig.



  2A: A 220, OOOX view of a particle budding from the cell surface. Note prominent spikes and dense core. Fig 2B:
Central portion of virus particle above enhanced contrast image. Note suggestion of conical shaped coiled nucleofilament in reverse image.



   Fig. 3A and 3B: Entry and Exit of Virus Particles into the Cell: Fig. 3A: Note viral particles on left in vacuoles which may be either intracellular or extracellular.



  In the middle at the bottom of the photo a viral core may be present beneath the membrane. Two-thirds up on the right border, a suggestion of a particle entering the cytoplasm is seen. Fig 3B: In upper left hand corner note viral particle attaching to thickened area of membrane which may be invaginating, possibly suggesting a coated pit. Both views are at 68,750X.



   Fig. 4: shows RT-activity distribution according to density after centrifugation in sucrose density gradients.  



   STATEMENT OF DEPOSIT
 Two cell lines related to the present invention which are denoted Chinese Malignant T-Cell Line No. 1 (CM-1) and Chinese Malignant T-Cell Line No. 2 (CM-2) have been deposited in the American Type Culture Collection on March 30, 1994, prior to the filing of the parent application. Cell line CM-1 has been assigned ATCC deposit number CRL 11594 and cell line CM-2 has been assigned ATCC deposit number CRL 11595.



   DEFINITIONS
 The term"nucleic acids", as used herein, refers to either DNA or RNA."Nucleic acid sequence"or"polynucleotide sequence"refers to a single-or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3'end. It includes both self-replicating plasmids, infectious polymers of DNA or RNA and nonfunctional DNA or
RNA.



   "Nucleic acid probes"may be DNA or RNA fragments.



  DNA fragments can be prepared, for example, by digesting plasmid DNA, or by use of PCR, or synthesized by either the phosphoramidite method described by Beaucage and Carruthers,
Tetrahedron Lett. 22: 1859-1862 (1981), or by the triester method according to Matteucci, et al., J. Am. Chem. Soc., 103: 3185 (1981), both incorporated herein by reference. A double stranded fragment may then be obtained, if desired, by annealing the chemically synthesized single strands together under appropriate conditions or by synthesizing the complementary strand using DNA polymerase with an appropriate primer sequence. Where a specific sequence for a nucleic acid probe is given, it is understood that the complementary strand is also identified and included. The complementary strand will work equally well in situations where the target is a double-stranded nucleic acid.



   The phrase"selectively hybridizing to"refers to a nucleic acid probe that hybridizes, duplexes or binds only to a particular target DNA or RNA sequence when the target sequences are present in a preparation of total cellular DNA  or RNA."Complementary"or"target"nucleic acid sequences refer to those nucleic acid sequences which selectively hybridize to a nucleic acid probe. Proper annealing conditions depend, for example, upon a probe's length, base composition, and the number of mismatches and their position on the probe, and must often be determined empirically. For discussions of nucleic acid probe design and annealing conditions, see, for example, Sambrook et al., Molecular
Cloning: A Laboratory Manual (2nd ed.), Vols. 1-3, Cold Spring
Harbor Laboratory, (1989) or Current Protocols in Molecular
Biology, F. Ausubel et al., ed.

   Greene Publishing and Wiley
Interscience, New York (1987).



   The phrase"isolated"or"substantially pure"when referring to nucleic acid sequences encoding HNDV viral proteins refers to isolated nucleic acids that do not encode proteins or peptides other than HNDV proteins or peptides.



   The phrase"expression cassette", refers to nucleotide sequences which are capable of affecting expression of a structural gene in hosts compatible with such sequences.



  Such cassettes include at least promoters and optionally, transcription termination signals. Additional factors necessary or helpful in effecting expression may also be used as described herein.



   The term"operably linked"as used herein refers to linkage of a promoter upstream from a DNA sequence such that the promoter mediates transcription of the DNA sequence.



   The term"vector", refers to viral expression systems, autonomous self-replicating circular DNA (plasmids), and includes both expression and nonexpression plasmids.



  Where a recombinant microorganism or cell culture is described as hosting an"expression vector,"this includes both extrachromosomal circular DNA and DNA that has been incorporated into the host chromosome (s). Where a vector is being maintained by a host cell, the vector may either be stably replicated by the cells during mitosis as an autonomous structure, or is incorporated within the host's genome.



   The term"plasmid"refers to an autonomous circular
DNA molecule capable of replication in a cell, and includes  both the expression and nonexpression types. Where a recombinant microorganism or cell culture is described as hosting an"expression plasmid", this includes both extrachromosomal circular DNA molecules and DNA that has been incorporated into the host chromosome (s). Where a plasmid is being maintained by a host cell, the plasmid is either being stably replicated by the cells during mitosis as an autonomous structure or is incorporated within the host's genome.



   The phrase"recombinant protein"or"recombinantly produced protein"refers to a peptide or protein produced using non-native cells that do not have an endogenous copy of
DNA able to express the protein. The cells produce the protein because they have been genetically altered by the introduction of the appropriate nucleic acid sequence. The recombinant protein will not be found in association with proteins and other subcellular components normally associated with the cells producing the protein.



   The phrase"specifically binds to an antibody"or "specifically immunoreactive with", when referring to a protein or peptide, refers to a binding reaction which is determinative of the presence of the HNDV virus of the invention in the presence of a heterogeneous population of proteins and other biologics including viruses other than the
HNDV virus. Thus, under designated immunoassay conditions, the specified antibodies bind to the HNDV viral antigens and do not bind in a significant amount to other antigens present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, antibodies raised to the HNDV virus of CM-1 or CM-2 cells described herein can be selected to obtain antibodies specifically immunoreactive with the HNDV viral proteins and not with other proteins.

   A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See
Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold  
Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.



   The phrase"substantially purified"or"isolated" when referring to a HNDV virus or a particular HNDV viral protein, means a chemical composition which is essentially free of cellular components such as cellular proteins and nucleic acids.



   The phrases"specific antisera"or"specific antibody"as used herein, when referring to an antisera or antibody for a particular virus mean an antisera or antibody that is immunoreactive with that particular virus and which is not immunoreactive with other viruses and with cellular material.



   The phrase"non-reactive"of"not immunoreactive"as used herein, in reference to the binding of the antibody to a specified virus means that antibody binding is not detected above a specified background level under standard immunoassay conditions. The specified background level is that which is statistically significant above the non-specific binding background levels in the immunoassay.



   "Biological specimen"as used herein refers to any sample obtained from a living organism or from an organism that has died. Examples of biological specimens include body fluids and tissue specimens.



   DETAILED DESCRIPTION
A. Human Neurological Disease Virus (HNDV) :
 This invention is directed to a purified novel human virus, Human Neurological Disease Virus (HNDV). This novel virus was first isolated as follows:
 An interleukin-2 independent malignant lymphoma cell line, CM-1 cell line, was established from the peripheral blood lymphocytes of a female Chinese patient with neurological disease. The peripheral blood lymphocytes of a second female Chinese patient with multiple sclerosis were transformed into a malignant T lymphocyte cell line, CM-2 cell  line, by the filtered supernatant of the CM-1 cells. Electron micrographs showed virions in the CM-1 and CM-2 cells that have the ultra-structure characteristics of a retrovirus. The virus in the CM-1 and CM-2 cells is an RNA virus and possesses reverse transcriptase activity.

   Serological assay, molecular hybridization and polymerase chain reaction excluded the existence of a known human virus.



   The terms"human neurological disease virus","HNDV" or"HNDV virus"as used herein refer to a group of novel related viruses that are associated with human neurological diseases. HNDV is an RNA virus that contains reverse transcriptase activity. When viewed by electron microscopy,
HNDV viral particles are spherical in shape and have an average size of about 95-113 nm. Electron micrographs show that the HNDV viral particle is a spherical structure with prominent projecting spikes, lacking a distinct double membrane and having an electron dense inner spherical core.



  (See Figures 2A, 2B, 3A, and 3B herein.)
 HNDV viruses are non-reactive to specific antisera for HTLV-I, HIV-I or EBV viruses. In particular, specific antisera reactive with the p24 gag and p26 proteins of HIV-1 is nonreactive with the HNDV virus. In addition, HNDV nucleic acids do not hybridize nucleic acid probes from the W fragment, the LMP gene and the EDNA-1 gene of EBV under routine hybridization conditions on Southern blots under stringent hybridization wash conditions of 65% C, 0.2x SSC and 0.1% SDS.



   The HNDV virus of the invention is described immunologically. In particular, HNDV viruses are selectively immunoreactive to antisera generated against a defined immunogen such as the HNDV virus strain isolated from CM-1 or
CM-2 cells (the CM-1/CM-2 HNDV virus). Immunoreactivity is determined in an immunoassay using a polyclonal antiserum which was raised to the CM-1/CM-2 HNDV virus. This antiserum is selected to have low crossreactivity against other viruses and any such crossreactivity is removed by immunoabsorbtion prior to use in the immunoassay.  



   In order to produce antisera for use in the immunoassay, the CM-1/CM-2 HNDV virus is purified from CM-1 or
CM-2 cells as described in Example 8 (A) herein. Rabbits or an inbred strain of mice such as BALB/c are immunized with the purified virus using a standard adjuvant, such as Freund's adjuvant, and a standard immunization protocol (see Harlow and
Lane, supra). Polyclonal sera are collected and titered against the immunogen in an immunoassay, for example, a solid phase ELISA assay with the immunogen immobilized on a solid support. Polyclonal antisera with a titer of 104 or greater are selected and tested for their cross-reactivity against other cell lines, including those known to be infected by known retroviruses, by using a standard ELISA immunoassay as described in Harlow and Lane, supra.

   In particular, the antisera is checked for crossreactivity against human peripheral blood lymphocytes, against the T lymphocyte cell lines, Molt4-8, H9, and Hut78. The antisera is also checked for cross-reactivity against the MT2 cell line which is known to be infected with HTLV-1. The above cell lines are all obtainable from the American Type Culture Collection,
Rockville, MD, USA.



   The ability of the above viruses and cell lines to bind to antisera directed to the CM-1/CM-2 HNDV virus immunogen is determined. The crossreactivity for other viruses is determined by comparing titers in ELISA assays using the CM-1/CM-2 HNDV virus or the other viruses or cell lines as the solid phase antigen. The antibody titers are determined for each virus or cell line. Those antisera that are less than 10% crossreactive with each of the other viruses and cell lines listed above are selected and pooled. (This means that the antibody titer is at least 10-fold lower for the other viruses and cell lines as compared to the CM-1/CM-2
HNDV virus.) The crossreacting antibodies are then removed from the pooled antisera by immunoabsorption with the abovelisted viruses and cell lines using standard techniques.

   (See
Harlow, et al., supra.)
 The immunoabsorbed and pooled antisera prepared as described above are then used as the capture antibody in a  competitive binding immunoassay procedure as described in
Harlow, et al., supra., to. compare an unknown virus preparation to the CM-1/CM-2 HNDV virus. In order to make this comparison, the preparation of the unknown virus competes with a labeled preparation of the CM-1/CM-2 HNDV virus for binding to the solid phase pooled antisera. The virus preparations are each assayed at a wide range of concentrations. The amount of each virus preparation required to inhibit 50% of the binding of the antisera to the labeled immunogen protein is determined.

   Those viruses that specifically bind to an antibody generated to the CM-1/CM-2
HNDV virus immunogen are those viruses where the amount of virus needed to inhibit 50% of the binding of the labeled virus to the solid phase antibody does not exceed an established amount. This amount is no more than 10 times the amount of unlabeled CM-1/CM-2 HNDV virus that is needed for 50% inhibition of the binding of the labeled virus preparation. Thus, the HNDV virus of the invention can be defined by immunological comparison to the specific strain of the HNDV virus obtained from CM-1 and CM-2 cells.



  B. Propagation and production of the HNDV virus:
 This invention is also directed to continuous production of HNDV and the antigens of HNDV, such as in a cell line, using methods disclosed in the specification and standard techniques as disclosed in U. S. patents 4,647,773 (Gallo et al.) and 4,520,113 (Gallo et al.), incorporated by reference herein.



   Live virus can be propagated in susceptible cell lines such as CM-1 or CM-2. (See Example 3, herein.)
Purified HNDV virus can be obtained from the HNDV-infected cell lines as described below.



  C. Purification of HNDV virus:
 The HNDV virus can be purified from infected cell lines such as CM-1 or CM-2 by a variety of different methods known to those of skill in the art. For instance, HNDV virus  can be purified from the supernatant or cell lysate of infected cells as described in Example 8 (A) herein.



   Purified HNDV viral proteins can be isolated from the purified HNDV virus using standard protein purification procedures known to those of skill in the art, including selective precipitation with such substances as ammonium sulfate, column chromatography, immunopurification methods, and others. See, for instance, R. Scopes, Protein
Purification: Principles and Practice, Springer-Verlag: New
York (1982), incorporated herein by reference.

 

   HNDV RNA can be isolated from the purified virus using standard procedures for nucleic acid purification. (See
Sambrook, et al., supra) DNA corresponding to the viral RNA sequences can be prepared by the use of reverse transcriptase.



  The viral DNA sequences can be digested with restriction enzymes and inserted into vectors for cloning, sequencing and for the production of recombinant HNDV proteins as described in Sambrook, et al., supra. DNA sequencing of the viral nucleic acids is performed by routine procedures as described in Sambrook, et al., supra.



  D. Production of recombinantlv produced HNDV viral proteins:
 Once nucleic acids encoding HDNV viral protein (s) have been isolated, one can express HNDV viral proteins in a variety of recombinantly engineered cells. It is expected that those of skill in the sequences, and promoters useful for regulation of the expression of polynucleotide sequences encoding HNDV viral proteins. To obtain high level expression of a cloned gene, such as those polynucleotide sequences encoding HNDV viral proteins, it is desirable to construct expression plasmids which contain, at the minimum, a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription/translation terminator.

   The expression vectors may also comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the plasmid in both eukaryotes and prokaryotes, i. e., shuttle vectors, and selection markers for both prokaryotic and eukaryotic systems.



  See Sambrook et al. Examples of expression of HNDV viral proteins in both prokaryotic and eukaryotic systems are described below.



   1. Expression in Prokaryotes
 A variety of procaryotic expression systems may be used to express HNDV proteins. Examples include E. coli,
Bacillus, Streptomyces, and the like. For example, HNDV viral proteins may be expressed in E. coli.



   It is essential to construct expression plasmids which contain, at the minimum, a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription/translation terminator.



  Examples of regulatory regions suitable for this purpose in E. coli are the promoter and operator region of the E. coli tryptophan biosynthetic pathway as described by Yanofsky, C., 1984, J. Bacteriol., 158: 1018-1024 and the leftward promoter of phage lambda (PX) as described by Herskowitz, I. and Hagen,
D., 1980, Ann. Rev. Genet., 14: 399-445. The inclusion of selection markers in DNA vectors transformed in E. coli is also useful. Examples of such markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol. See Sambrook et al. for details concerning selection markers for use in E. coli.  



   HNDV viral proteins produced by prokaryotic cells may not necessarily fold properly. During purification from
E. coli, the expressed protein may first be denatured and then renatured. This can be accomplished by solubilizing the bacterially produced proteins in a chaotropic agent such as guanidine HC1 and reducing all the cysteine residues with a reducing agent such as beta-mercaptoethanol. The protein is then renatured, either by slow dialysis or by gel filtration.



  See U. S. Patent No. 4,511,503.



   Detection of the expressed antigen is achieved by methods known in the art as radioimmunoassay, or Western blotting techniques or immunoprecipitation. Purification from
E. coli can be achieved following procedures described in U. S.



  Patent No. 4,511,503.



   2. Expression in Eukaryotes
 A variety of eukaryotic expression systems such as yeast, insect cell lines, bird, fish, and mammalian cells, are known to those of skill in the art. As explained briefly below, HNDV viral proteins can be expressed in these eukaryotic systems.



   Synthesis of heterologous proteins in yeast is well known. Methods in Yeast Genetics, Sherman, F., et al., Cold
Spring Harbor Laboratory, (1982) is a well recognized work describing the various methods available to produce the protein in yeast.



   Suitable vectors usually have expression control sequences, such as promoters, including 3-phosphoglycerate kinase or other glycolytic enzymes, and an origin of replication, termination sequences and the like as desired.



  For instance, suitable vectors are described in the literature (Botstein, et al., 1979, Gene, 8: 17-24; Broach, et al., 1979,
Gene, 8: 121-133).



   The sequences encoding HNDV viral proteins can also be ligated to various expression vectors for use in transforming cell cultures of, for instance, mammalian, insect, bird or fish origin. Illustrative of cell cultures useful for the production of the polypeptides are mammalian  cells. Mammalian cell systems often will be in the form of monolayers of cells although mammalian cell suspensions may also be used. A number of suitable host cell lines capable of expressing intact proteins have been developed in the art.



  Animal cells useful for production of HNDV viral proteins are available, for instance, from the American Type Culture
Collection Catalogue of Cell Lines and Hybridomas (7th edition, 1992). Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter (e. g., the CMV promoter, a HSV tk promoter or pgk (phosphoglycerate kinase) promoter), an enhancer (Queen et al. (1986) Immunol. Rev. 89: 49), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites (e. g., an SV40 large T Ag poly A addition site), and transcriptional terminator sequences.



   The host cells are competent or rendered competent for transformation by various means. There are several well-known methods of introducing DNA into animal cells.



  These include: calcium phosphate precipitation, fusion of the recipient cells with bacterial protoplasts containing the DNA, treatment of the recipient cells with liposomes containing the
DNA, DEAE dextran, electroporation and micro-injection of the
DNA directly into the cells.



   The transformed cells are cultured by means well known to those of skill in the art. See Biochemical Methods in Cell Culture and Virology, Kuchler, R. J., Dowden,
Hutchinson and Ross, Inc., (1977). The expressed polypeptides are isolated from cells grown as suspensions or as monolayers.



  The latter are recovered by well known mechanical, chemical or enzymatic means. Recombinantly produced polypeptides can be directly expressed or expressed as a fusion protein. The protein is then purified by a combination of cell lysis (e. g., sonication) and affinity chromatography. For fusion products, subsequent digestion of the fusion protein with an appropriate proteolytic enzyme releases the desired polypeptide.



   The HNDV viral polypeptides can be purified to substantial purity by standard techniques well known in the  art, including selective precipitation with such substances as ammonium sulfate, column chromatography, immunopurification methods, and others. See, for instance, R. Scopes, Protein
Purification: Principles and Practice, Springer-Verlag: New
York (1982), incorporated herein by reference.



  G. In Vitro Diagnostic Methods: Detection HNDV Nucleic Acids
 by Nucleic Acid Hybridization and Detection of HNDV virus
 and antibodies reactive to HNDV by Immunoassay Methods:
 1. Nucleic Acid Hybridization Assays
 A variety of methods for specific DNA and RNA measurement using nucleic acid hybridization techniques are known to those of skill in the art. (See Sambrook, et al. supra). For example, nucleic acids can be extracted from a biological sample and run on agarose slab gels in buffer and transferred to membranes. Hybridization is carried out using the nucleic acid probes.



   Nucleic acid probes are based on the nucleic acid sequence of the HNDV RNA. Nucleic acid probes can be prepared by a variety of methods known to those of skill in the art.



  For example, oligonucleotides for use as probes can chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage, S. L. and
Carruthers, M. H., 1981, Tetrahedron Lett., 22 (20): 1859-1862 using an automated synthesizer, as described in
Needham-VanDevanter, D. R., et al., 1984, Nucleic Acids Res., 12: 6159-6168. Purification of oligonucleotides is by either native acrylamide gel electrophoresis or by anion-exchange
HPLC as described in Pearson, J. D. and Regnier, F. E., 1983, J.



  Chrom., 255: 137-149. The sequence of the synthetic oligonucleotide can be verified using the chemical degradation method of Maxam, A. M. and Gilbert, W. 1980, in Grossman, L. and Moldave, D., eds. Academic Press, New York, Methods in
Enzymology, 65: 499-560.



   Nucleic acid probes can also be prepared from HNDV nucleic acids, for example, by nucleic acid amplification techniques such as PCR. In PCR techniques, oligonucleotide  primers complementary to the two 3'borders of the DNA region to be amplified are synthesized. The polymerase chain reaction is then carried out using the two primers. See PCR
Protocols: A Guide to Methods and Applications. (Innis, M.,
Gelfand, D., Sninsky, J. and White, T., eds.), Academic Press,
San Diego (1990). Primers can be selected to amplify the entire regions encoding full-length HNDV proteins or to amplify smaller DNA segments as desired.



   A variety of nucleic acid hybridization formats are known to those skilled in the art. For example, common formats include sandwich assays and competition or displacement assays. Hybridization techniques are generally described in Nucleic Acid Hybridization, A Practical Approach,
Ed. Hames, B. D. and Higgins, S. J., IRL Press, 1985; Gall and
Pardue (1969), Proc. Natl. Acad. Sci., U. S. A., 63: 378-383; and
John, Burnsteil and Jones (1969) Nature, 223: 582-587.



   For example, sandwich assays are commercially useful hybridization assays for detecting or isolating nucleic acid sequences. Such assays utilize a"capture"nucleic acid covalently immobilized to a solid support and a labeled "signal"nucleic acid in solution. The clinical sample will provide the target nucleic acid. The"capture"nucleic acid and"signal"nucleic acid probe hybridize with the target nucleic acid to form a"sandwich"hybridization complex. To be effective, the signal nucleic acid cannot hybridize with the capture nucleic acid.



   Typically, labeled signal nucleic acids are used to detect hybridization. Complementary nucleic acids or signal nucleic acids may be labeled by any one of several methods typically used to detect the presence of hybridized polynucleotides. The most common method of detection is the use of autoradiography with 3H, 125I, 35S, 14C, or 32P-labeled probes or the like. Other labels include ligands which bind to labeled antibodies, fluorophores, chemiluminescent agents, enzymes, and antibodies which can serve as specific binding pair members for a labeled ligand.



   Detection of a hybridization complex may require the binding of a signal generating complex to a duplex of target  and probe polynucleotides or nucleic acids. Typically, such binding occurs through ligand and anti-ligand interactions as between a ligand-conjugated probe and an anti-ligand conjugated with a signal.



   The label may also allow indirect detection of the hybridization complex. For example, where the label is a hapten or antigen, the sample can be detected by using antibodies. In these systems, a signal is generated by attaching fluorescent or enzyme molecules to the antibodies or, in some cases, by attachment to a radioactive label.



  (Tijssen, P.,"Practice and Theory of Enzyme Immunoassays,"
Laboratory Techniques in Biochemistry and Molecular Biology,
Burdon, R. H., van Knippenberg, P. H., Eds., Elsevier (1985), pp. 9-20.)
 As described above, a variety of labels may be employed, including those which have been described above for use in immunoassay, for example radionucleotides. Suitable labels may be bound to the probe by a variety of techniques.



  Commonly employed is nick translation with a-32P-dNTP terminal phosphate hydrolysis with alkaline phosphatase followed by 5'-end labeling with radioactive 32P employing X-32P-NTP and
T4 polynucleotide kinase or 3'-end labeling with an a-32P-dNTP and terminal deoxynucleotidyl transferase. Alternatively, nucleotides can be synthesized where one or more of the atoms present are replaced with a radioactive isotope, e. g., hydrogen with tritium. In addition, various linking groups can be employed. The terminal hydroxyl can be esterified with inorganic acids, e. g., 32p phosphate or 14C organic acids, or else esterified with bifunctional reagents to provide other reactive groups to which labels can be linked.



   The sensitivity of the hybridization assays may be enhanced through use of a nucleic acid amplification system which multiplies the target nucleic acid being detected.



  Examples of such systems include the polymerase chain reaction (PCR) system and the ligase chain reaction (LCR) system.



  Other methods recently described in the art are the nucleic acid sequence based amplification (NASBA''", Cangene,
Mississauga, Ontario) and Q Beta Replicase systems.  



   An alternative means for detecting HNDV is in situ hybridization. In situ hybridization assays are well known and are generally described in Angerer, et al., Methods
Enzymol., 152: 649-660 (1987). In an in situ hybridization assay, cells are fixed to a solid support, typically a glass slide. If DNA is to be probed, the cells are denatured with heat or alkali. The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of labeled probes specific to HNDV nucleic acid sequences. The probes are preferably labeled with radioisotopes or fluorescent reporters.



   As described above, diagnostic tests for detecting the presence of HNDV in biological samples may also be performed using polynucleotide probes. Such polynucleotide probes may be prepared based on the sequence of the viral genome. The length of the probe is not critical, but will usually comprise at least about 12 bases, more usually comprising at least about 16 bases, which are substantially complementary to a portion of the viral genome. The probe itself may be DNA, RNA, or their analogs, and the probe need not have perfect match with the HNDV genome. One or two mismatched pairs are acceptable for probes up to 20 bases in length and three to five mismatched pairs in probes from 20 to 35 bases. The probes may be prepared synthetically to include a detectable label.

   Usually, the synthetic sequences are multiplied in commonly available cloning vectors and suitable hosts or by PCR in order to obtain large quantities. The vectors may themselves be labeled for use as probes, or shorter fragments containing complementary strands may be excised and labeled. Methods for the preparation and utilization of nucleotide probes for diagnostic testing are described above and in Sambrook et al., supra.



   2. Production of antibodies and immunoassays for
 detection of HNDV:
 In addition to detecting HNDV virus by nucleic acid hybridization, one can also use immunoassays to detect the proteins. Immunoassays can be used to qualitatively or  quantitatively analyze for the proteins. A general overview of the applicable technology can be found in Harlow and Lane,
Antibodies: A Laboratory Manual, Cold Spring Harbor Pubs.,
N. Y. (1988), incorporated herein by reference.
 a. Antibody Production
 A number of immunogens may be used to produce antibodies specifically reactive with HNDV. For example, recombinant viral proteins produced as described above can be used as immunogens for the production of monoclonal or polyclonal antibodies. HNDV or the purified naturally occurring viral proteins may also be used either in pure or impure form.

   Synthetic peptides of a viral protein sequence can also be used as an immunogen.



   Recombinant viral proteins can be expressed in eukaryotic or prokaryotic cells as described above, and purified as generally described above. The product is then injected into an animal capable of producing antibodies.



  Either monoclonal or polyclonal antibodies may be generated, for subsequent use in immunoassays to measure the protein.



   Methods of production of polyclonal antibodies are known to those of skill in the art. In brief, an immunogen, preferably a purified protein, is mixed with an adjuvant and animals are immunized. The animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the HNDV viral protein.



  When appropriately high titers of antibody to the immunogen are obtained, blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the protein can be done if desired.



  (See Harlow and Lane, supra).



   Monoclonal antibodies may be obtained by various techniques familiar to those skilled in the art. Briefly, spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (See,
Kohler and Milstein, Eur. J. Immunol. 6: 511-519 (1976), incorporated herein by reference). Alternative methods of immortalization include transformation with Epstein Barr
Virus, oncogenes, or retroviruses, or other methods well known  in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host.

   Alternatively, one may isolate
DNA sequences which encode a monoclonal antibody or a binding fragment thereof by screening a DNA library from human B cells according to the general protocol outlined by Huse, et al.



  (1989) Science 246: 1275-1281.
 b. Immunoassays for detection of HNDV antigen:
 Viral antigens can be measured by a variety of immunoassay methods. For a review of immunological and immunoassay procedures in general, see Basic and Clinical
Immunology 7th Edition (D. Stites and A. Terr ed.) 1991.



  Moreover, the immunoassays of the present invention can be performed in any of several configurations, which are reviewed extensively in Enzyme Immunoassay, E. T. Maggio, ed., CRC
Press, Boca Raton, Florida (1980);"Practice and Theory of
Enzyme Immunoassays,"P. Tijssen, Laboratory Techniques in
Biochemistry and Molecular Biology, Elsevier Science
Publishers B. V. Amsterdam (1985); and, Harlow and Lane,
Antibodies, A Laboratory Manual, supra, each of which is incorporated herein by reference.



   Immunoassays for measurement of HNDV can be performed by a variety of methods known to those skilled in the art. In brief, immunoassays to measure the virus can be either competitive or noncompetitive binding assays. In competitive binding assays, the sample analyte competes with a labeled analyte for specific binding sites on a capture agent bound to a solid surface. Preferably the capture agent is an antibody specifically reactive with HNDV produced as described above. The concentration of labeled analyte bound to the capture agent is inversely proportional to the amount of free analyte present in the sample.



   In a competitive binding immunoassay, the HNDV viral antigens present in the sample compete with labeled protein for binding to a specific binding agent, for example, an  antibody specifically reactive with HNDV antigens. The binding agent may be bound to a solid surface to effect separation of bound labeled protein from the unbound labeled protein. Alternately, the competitive binding assay may be conducted in liquid phase and any of a variety of techniques known in the art may be used to separate the bound labeled protein from the unbound labeled protein. Following separation, the amount of bound labeled protein is determined.



  The amount of protein present in the sample is inversely proportional to the amount of labeled protein binding.



   Alternatively, a homogenous immunoassay may be performed in which a separation step is not needed. In these immunoassays, the label on the protein is altered by the binding of the protein to its specific binding agent. This alteration in the labeled protein results in a decrease or increase in the signal emitted by label, so that measurement of the label at the end of the immunoassay allows for detection or quantitation of the protein.



   HNDV can also be determined by a variety of noncompetitive immunoassay methods. For example, a two-site, solid phase sandwich immunoassay is used. In this type of assay, a binding agent for the protein, for example an antibody, is attached to a solid phase. A second protein binding agent, which may also be an antibody, and which binds the protein at a different site, is labeled. After binding at both sites on the protein has occurred, the unbound labeled binding agent is removed and the amount of labeled binding agent bound to the solid phase is measured. The amount of labeled binding agent bound is directly proportional to the amount of protein in the sample.



   Western blot analysis can also be done to determine the presence of HNDV viral antigens in a sample.



  Electrophoresis is carried out, for example, on a tissue sample suspected of containing the virus. Following electrophoresis to separate the viral proteins, and transfer of the proteins to a suitable solid support such as a nitrocellulose filter, the solid support is then incubated with an antibody reactive with the protein. This antibody may  be labeled, or alternatively it may be detected by subsequent incubation with a second labeled antibody that binds the primary antibody.



   The immunoassay formats described above employ labeled assay components. The label can be in a variety of forms. The label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. A wide variety of labels may be used. The component may be labeled by any one of several methods.



  Traditionally a radioactive label incorporating 3H, 125I, 35S, 14C, or 32p was used. Non-radioactive labels include ligands which bind to labeled antibodies, fluorophores, chemiluminescent agents, enzymes, and antibodies which can serve as specific binding pair members for a labeled ligand.



  The choice of label depends on sensitivity required, ease of conjugation with the compound, stability requirements, and available instrumentation. For a review of various labeling or signal producing systems which may be used, see U. S. Patent
No. 4,391,904, which is incorporated herein by reference.
 c. Immunoassays for detection of anti-HNDV
 antibodies:
 Antibodies reactive with a particular protein can also be measured by a variety of immunoassay methods. For a review of immunological and immunoassay procedures applicable to the measurement of antibodies by immunoassay techniques, see Basic and Clinical Immunology 7th Edition (D. Stites and
A. Terr ed.) supra, Enzyme Immunoassay, E. T. Maggio, ed., supra, and Harlow and Lane, Antibodies, A Laboratory Manual, supra.



   Radioimmunoassay techniques for detecting antibodies in a physiological specimen of susceptible patients include radiolabeled assays of the so-called blot technique, such as the Western Blot technique. In addition, anti-HNDV antibodies can be detected by the enzyme-linked immunoabsorbent assay (ELISA). Antibodies to HNDV can also be detected by an indirect immunofluorescence assay. This latter assay can use the infected T-cell as a starting material. Examples of an  
ELISA assay and an indirect immunofluorescence assay for detection of anti-HNDV antibodies are described below.



   In brief, immunoassays to measure antisera reactive with HNDA can be either competitive or noncompetitive binding assays. In competitive binding assays, the sample analyte competes with a labeled analyte for specific binding sites on a capture agent bound to a solid surface. Preferably, the capture agent is a purified or partially purified HNDV viral protein preparation produced as described above. Preparations of HNDV virus or cell line producing the virus can also be used a capture agent. Other sources of HNDV proteins, including isolated or partially purified naturally occurring proteins, can also be used. Noncompetitive assays are typically sandwich assays, in which the sample analyte is bound between two analyte-specific binding reagents. One of the binding agents is used as a capture agent and is bound to a solid surface.

   The second binding agent is labeled and is used to measure or detect the resultant complex by visual or instrument means. A number of combinations of capture agent and labeled binding agent can be used. A variety of different immunoassay formats, separation techniques and labels can be also be used as described above for the measurement of HNDV viral antigens.



   This invention also embraces kits for detecting the presence of HNDV in tissue or blood samples which comprise a container containing antibodies selectively immunoreactive to the protein and instructional material for performing the test. The kit may also contain other components such as HNDV proteins, controls, buffer solutions, and secondary antibodies. Kits for detecting antibodies to HNDV proteins comprise a container containing a HNDV protein or virus or a cell line containing the virus, instructional material and may comprise other materials such as secondary antibodies and labels as described herein.



   This invention further embraces diagnostic kits for detecting HNDV nucleic acids in tissue or blood samples which comprise nucleic probes as described herein and instructional material. The kit may also contain additional components such  as labeled compounds, as described herein, for identification of duplexed nucleic acids.



  H. Vaccines for treatment or prevention of HNDV Infection
 Compositions of the present invention are also useful in preparing vaccines for protection against HNDV infection. For example, the whole virus may be wholly or partially inactivated. Partial inactivation may be achieved by passage at elevated temperatures or by contact with mutagens, such as ultraviolet light, ethyl methanesulfonate, and the like. Complete inactivation may be achieved by contact with other agents, including formalin, phenol, a-lactopropionate, ultraviolet light, heat, psorlens, platinum complexes, ozone and other viricidal agents.



   The viral proteins and portions thereof may also be used in the preparation of subunit vaccines prepared by known techniques. Polypeptides displaying antigenic regions capable of eliciting protective immune response are selected and incorporated in an appropriate carrier. Alternatively, an antigenic portion of a viral protein or proteins may be incorporated into a larger protein by expression of fused proteins. The preparation of subunit vaccines for other viruses is described in various references, including Lerner et al., (1981) Proc. Natl. Acad. Sci. USA 78: 3403 and
Bhatanagar et al., (1982) Proc. Natl. Acad. Sci. USA 79: 4400.



  See also, U. S. Patents Nos. 4,565,697 (where a naturally-derived viral protein is incorporated into a vaccine composition); 4,528,217 and 4,575,495 (where synthetic peptides forming a portion of a viral protein are incorporated into a vaccine composition). Other methods for forming vaccines employing only a portion of the viral proteins are described in U. S. Patent Nos. 4,552,757; 4,552,758; and 4,593,002. The relevant portions of each of these cited references and patents are incorporated by reference herein.



   The vaccines prepared as described above may be administered in any conventional manner, including oronasally, subcutaneously, or intramuscularly, except that oronasal administration will usually not be employed with a partially  inactivated virus vaccine. Adjuvants will also find use with subcutaneous and intramuscular injection of completely inactivated vaccines to enhance the immune response.



   Vaccine compositions of the invention are administered to a patient to elicit a protective immune response against the polypeptide. Amounts effective for this use will depend on, e. g., the manner of administration, the weight and general state of health of the patient, and the judgment of the prescribing physician. Dosages, formulations and administration schedules may vary in these patients compared to normal individuals. In general, dosages range for the initial immunization from about 10 pg to about 1,000 mg of the HNDV vaccine for a 70 kg patient, followed by boosting dosages of from about 10 pg to about 1,000 mg of the HNDV vaccine, pursuant to a boosting regimen over weeks to months depending upon the patient's response and condition. 

   The patient's response can be measured, for example, by measuring the appearance of anti-HNDV antibodies present in the patient's blood at intervals after the initial immunization.



   EXAMPLES
Example 1: Establishment of the CM-1 Cell Line
 Case history of the patient whose cells were used to
 derive the CM-1 cell line:
 A novel virus designated Human Neurological Disease
Virus (HNDV) has been discovered and isolated from the cell line CM-1. The CM-1 cell line was derived from the peripheral blood of a female Chinese patient (Xiang-Ying Lan, Yi Zeng,
De-Xin Wang, Zi-Jing Feng, Mei-Hua Tang, Yuan Ji, Geng-Geng
Yu, and Kun Li, Chinese Journal of Virology, Vol 8: 187-190, 1992). For one month in January 1990, the patient had progressive disturbance of consciousness, numbness of limbs and incontinence of urine. She reacte illness. The impressive diagnosis was encephalitis or encephalopathy.



   The patient (then age 36) was admitted to the
Neurological Department of Beijing Friendship Hospital on
February 2,1990. The second day after admission to the hospital, the patient had a fever of 39.5-40.0 C. Her mental condition deteriorated progressively to somnolence, semi-coma and coma. Both of her eyes gazed to the left side. Her muscle tone increased in the left extremities. Both plantar reflexes were equivocal. However, blood, urine and stool of the patient were normal. CSF was clear with normal pressure.



  Chest X-ray showed some shadows consistent with pneumonia. CT scanning of her brain showed multiple small irregular low density foci.



   The patient's condition improved after treatment with antibiotics and Dexamethasone. She regained consciousness. She had clear but slow comprehension.



  However, the muscle tone of all extremities of the patient increased. Cogwheel rigidity of right upper extremity and tremor of both hands and chin were observed. The clinical diagnosis of this patient was encephalitis due to an unknown viral infection.



   This patient shares the profile and symptoms of a multiple sclerosis patient in that a typical multiple sclerosis patient is a female having the onset of multiple sclerosis between age 20-40 having paresis, sensory and visual disturbance, spasticity, sphincter dysfunction, and plaques of demyelination in the brain and spinal cord.



   As of March, 1994, the patient was still alive and showed no signs of a tumor. However, the patient showed degradation of intelligence and some motion difficulties.



   B) Derivation and growth of the CM-1 cell line:
 The CM-1 cell line was derived from the peripheral blood lymphocytes of the above described patient. Two weeks after her admission to the hospital, the patient was still in a state of semicoma although the patient's body temperature had returned to nearly normal. At that time, 5 ml venous  blood was collected and the lymphocytes were separated by
Ficoll-Conray gradient centrifugation, and cultured with
RPMI-1640 medium supplemented with 20% fetal calf serum (FCS) containing 1% penicillin, 1% streptomycin and 1% glutamine.



  The culture was incubated at 37 C in 5% C02 atmosphere. No growth factor was added. The cell line thus established was designated CM-1.



   On the second day, the CM-1 cells grew rapidly and actively. On the third day, the CM-1 cells were subcultured.



  Since then, they were subcultured twice a week for more than three years, and their growth is still vigorous. Some CM-1 cells have been preserved in liquid nitrogen. The rate of resuscitation of frozen CM-1 cells was more than 90%.



   The CM-1 cells grew on the wall of ware flasks and dissociated continuously into the suspension. The adhered cells were polymeric and aggregated to form clumps.



   After the CM-1 cell line was established, the blood of the patient in convalescent phase was collected.



  T lymphocytes were separated and cultured under the same condition as that in establishing the CM-1 cell line.



  However, no cell line was established from these
T lymphocytes.



   The growth and morphological characteristics of the
CM-1 cells were directly observed under an inverted optic microscope.



   To determine the CM-1 cell growth curve the suspension of newly passed CM-1 cells at a concentration of 5x104 cell/ml was distributed into 42 3 ml culture bottles.



  The culture was incubated at 37 C in 5% C02 atmosphere. Three bottles were taken out every 24 hours and the supernatant was aspirated out. The cells growing on the wall of bottles were treated with trypsin solution. The number of cells was counted. The procedure was carried out for 14 days without change of culture medium.



   The growth of CM-1 cells in culture follows the pattern depicted in Fig. 1. A log following seeding is followed by a period of exponential growth (log phase, from the 2nd day to the 7th day). The concentration of the CM-1  cells reached 2.55xl06/ml, which is more than 50 times the cell concentration at the start. The plateau phase began from the llth day, although the CM-1 cells could survive on the 14th day. They could be subcultured by changing to fresh medium.



  Example 2. Studies of the CM-1 Cell Surface Marker
 The CM-1 cell surface marker was studied by indirect immunofluorescence technique with 28 different anti-leukocyte monoclonal antibodies. Target cells were reacted with anti-leukocyte monoclonal antibodies at 37 C for 30 minutes.



  Samples of cell suspension were spotted on a slide, air dried, and fixed in acetone. Slides were then washed and reacted for 30 min at 37 C with a dilute solution of fluoresceinconjugated anti-human IgG. Slides were again washed and examined under a microscope for fluorescence.



   (1) Target cells: the CM-1 cells and subclones of the CM-1 cells (colony 5, colony 9, colony A-30 and suspended colony 3).



   (2) Anti-leukocyte monoclonal antibodies (MaCbs):
 a. Monoclonal antibodies against T lymphocyte CD1,
CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD27.
 b. Monoclonal antibodies against the activated lymphocyte, CD25, CD71, HLA-DR.
 c. Monoclonal antibodies against myelocyte CD11 (B),
CD13, CD15, CD33, CD15 (HI98).
 d. Monoclonal antibodies against B lymphocyte CD9,
CD19, CD20, CD21, CD22, CD10 (CALLA), SmIg.
 e. Monoclonal antibodies against other leukocyte
CD38, CD45, CD45R, HLA-1.



   Table 1 shows the result of surface marker detection assay. The numbers in Table 1 are percentage of fluorescent cells on a slide.  



   TABLE 1
Analysis of the Surface Markers of CM-1 Cells and Clonal Cell
Lines by Anti-Leukocyte Monoclonal Antibodies (McAbs)
 Kinds of cells
MaCbs
 Colony Suspended
 CM-1 Colony 5 Colony 9 A-30 Colony 3
CD1 0 NT NT NT NT
CD2 59 NT NT NT NT
CD3 25 90  < 5 46 90
CD4  > 90  > 90 NT 90  > 90
CD5  > 90  > 90  > 90  > 90  > 90
CD6 0 NT NT NT NT
CD7  > 90  > 90 90  > 90  > 90
CD8 0 NT NT NT NT
CD27 95 NT NT NT NT
CD25 0 NT NT NT NT
CD71 95  > 90 NT  > 90  > 90
HLA-DR 0 0 0 0 0
CD11 (B) 0 NT NT NT NT
CD13  < 5 NT NT NT NT
CD14 0 NT NT NT NT
CD33 0  < 10  < 5  < 12 0
CD15 (HI98) 34 4 16 17 10
CD10 (CALLA)  > 95  > 90  > 90  > 90  > 90
CD9  > 95 NT NT NT NT
CD19 0 0 0 0  < 5
CD20 0 NT NT NT NT
CD21 0 NT NT NT NT
CD22 0 NT NT NT NT
SmIg 0 NT NT NT NT
CD38 100 NT NT NT NT
CD45 100 NT NT NT NT
CD45R  < 10 NT NT NT NT
HLA-1  > 95 NT NT NT NT
 NT: not tested.



   The results of indirect immunofluorescence assay with 28 different anti-leucocyte monoclonal antibodies showed that characteristic T lymphocyte markers were present on the membrane of all CM-1 cells, 20-30% of which also showed myelocyte marker.



  Example 3. Establishment of the CM-2 Cell Line
 The suspension of newly passed CM-1 cells was frozen and thawed three times, and then was centrifugalized at 3,000  rpm for 10 minutes. The supernatant was passed through a 0.45 ssm filter.



   The peripheral blood from a 67-year-old woman with multiple sclerosis was collected and the lymphocytes were separated by Ficoll-Conray gradient centrifugation, and cultured at 37 C in 5% C02 atmosphere with RPMI-1640 medium supplemented with 20% FCS, 5 Mg phytohemagglutinin (PHA) and 20 U/ml IL-2 (1% penicillin G, 1% streptomycin and 1% glutamine). 1/10 volume of the filtered supernatant of the
CM-1 cells was added into the medium.



   After three weeks, clones of the cells were visible to the naked eye. The cells could be subcultured and a continuous cell line was established and designated CM-2 (Chinese malignant T lymphocyte cell line-2). The characteristics and morphology of the CM-2 cells were the same as those of the CM-1 cells. The control lymphocytes without the filtered supernatant of the cultured CM-1 cells were all broken and dead three weeks later.



   In summary, two malignant T lymphocyte cell lines (CM-1 and CM-2) were established from the peripheral blood lymphocytes of the above described patient. Both CM-1 and
CM-2 proliferated rapidly and actively. The number of cells increased by 50 times in 14 days. Furthermore, CM-1 and CM-2 cells were able to grow spontaneously in vitro without the addition of any exogenous growth factors. Without wishing to be bound by theory, the CM-1 and CM-2 cells appeared to produce growth factors themselves.



  Example 4. Tumorigenesis of the CM-1 and CM-2 Cells in
 Nude Mice
 The tumorigenicity of CM-1 and CM-2 cells was determined in nude mice.



   The suspensions of newly passed CM-1 and CM-2 cells were separately collected and centrifugalized at 2,000 rpm for 10 min. The pellets were resuspended in a small volume of medium. 2x107 CM-1 and CM-2 cells were separately transplanted into nude mice by subcutaneous inoculation. The mice were reared and preserved.  



   About 10 weeks after the CM-1 cells were transplanted into nude mice by subcutaneous inoculation, a tumor grew to a size of 5x7x3 mm in the nude mice. Histopathological section showed that the tumor was a typical malignant lymphoma.



   When cultured cells of CM-2 were transplanted into nude mice by subcutaneous inoculation, tumors appeared in 75% of the mice 4-5 weeks later, and grew in size to 5x6x4 mm about 10 weeks later. Histopathological section showed that the tumor caused by CM-2 cells was also a typical malignant lymphoma.



   The tumors were taken out of the mice and subcutaneously transplanted into new nude mice. Tumors grew in all of the mice 7-8 weeks later. The growth period of the tumor was shortened. As of March of 1994, a tumor of a nude mouse has been subcultured for six generations, which is more than one year.



   In summary, when the CM-1 and CM-2 cells were transplanted into nude mice by subcutaneous inoculation, the nude mice developed tumors that were typical malignant lymphoma, as proved by histopathological dissection.



  Example 5. Electron Microscony Observation of Virus
 Cells from cultures CM-1, CM-2, and CM-3 were examined by electron microscopy. CM-3 is a cell line obtained by limiting dilution cloning of CM-1 cells. All cultures were stimulated with PHA for 48 hours prior to harvesting. Cells were pelleted and washed in phosphate buffered saline, and then transferred to solvent safe 1.8 ml Eppendorf tubes, and pelleted again. Fresh glutaraldehyde fixative was added to cell pellets, and the cells resuspended in fixative. After pelleting in fixative, cells were fixed in epoxy, and sectioned blocks were transferred to grids for viewing at final magnification of from 36,000x to 220,000x.



   Virus particles were seen in all lines examined (CM1,
CM2, CM3). Estimates of particle size based upon measurements at 68,750x were consistent with an average size of 109 nm, measurements made on photographs taken at 86,625x produced an average size measurement of 99 nm, with a range from 90-110  nm, and measurements taken on two well-formed virions at 220,000x were consistent with a size of 113 nm (see Figures 2A and 2B). Thus, this data indicates a virus with an average size of 95-113 nm. Concomitant measurement of EBV particles produced a size of 143 nm, consistent with results reported in the literature (data not shown).



   As in Figure 2A, unequivocal budding from the cell surface was seen. In addition, on some views particles appeared to be budding into spaces which could either represent intracellular vacuoles, or invaginations in the surface membrane (see Figure 3A). No intracellular or intranuclear inclusions were seen. Some suggestion of attachment of particles in sites which might represent coated pits was seen (see Figure 3B), and a few electron dense structures seen immediately below the cell surface membrane might have represented viral core assemblies (see Figure 3B), though this is far from certain.



   The morphology of the particles was consistent between the different cell lines. Particles examined at 86,625x and 220,000x display a spherical structure with prominent projecting spikes, without a distinct double membrane such as seen with herpes viruses, but with an electron dense inner spherical core or shell, and an angular (possibly icosahedral) core (see Figure 2A). Computerized image enhancement suggests that coiled nucleocapsid filament (s) may be present within the core of some particles (see Figure 2B).



  Example 6. Identification of Virus
 A. Serolovical Assays
 The CM-1 cells were tested as target cells for antibodies against known viruses by indirect immunofluorescence technique with standard antisera against these viruses.



   0.1 ml of cells (CM-2 cells, MT-2 cells carrying
HTLV-1, MT-4 cells infected by HIV-1 and B9-58 cells carrying
EBV) were separately placed on a microscope slide, fixed by cold acetone at 4 C for 15 min, and dried in air. 0.1 ml of diluted serum or cerebrospinal fluid (CSF) of the CM-1 donor  was added on the slide, incubated at 37 C for 45 minutes, and washed three times in the washing solution phosphate-buffered saline (PBS, pH 7.4). 0.05 ml fluorescein-labeled mouse anti-human IgG diluted in 0.01% Even's blue was added, incubated at 37 C for 30 min, and washed three times. The slide was observed under an Olympus fluorescence microscope.



   The serum and cerebrospinal fluid were tested for
HIV-1 antibody by Western blot method with the standard kit (Bio-Rad).



   (1) The immunological reactions between the serum or cerebrospinal fluid of CM-1 donor and the antigens of the above-mentioned viruses tested by Western Blot or immunofluorescence assay were all negative. This result showed that the patient was not infected by HTLV-1, HIV-1 or
EBV.



   (2) The assay for the virus antigens in the CM-1 cells by immunofluorescence assay showed that the virus in the
CM-1 cells did not possess the antigenicity of HTLV-1, HIV-1 or EBV.



   (3) There was no immunological reaction between the
CM-2 cells and the serum of its donor. However, the CM-2 cells reacted with the serum of the CM-1 donor, suggesting that the antigen in the CM-2 cells came from the filtered supernatant of the cultured CM-1 cells.



   Antigen capture assays for HIV-1 P24 and HIV-2/SIV P26 were also performed on supernatants obtained from CM1 and CM2 cell lines. The assays were performed with standard kits obtained from Coulter Corporation, Miami, Florida, USA. The results indicated that these cells are not producing HIV/SIV gag protein and are not infected by these viruses.



   B. Test for Viral Genes in the CM-1 Cells
 The suspension of subcultured CM-1 cells and the whole blood cells of the CM-1 cell donor in convalescent phase were collected and centrifugalized. The cell DNA was extracted by phenol-chloroform method (see Sambrook et al., supra).  



   (1) Molecular Hybridization Test
 The cell DNA was digested and cleaved by Hind III and tested for genes of known viruses with Southern-blot method.



  Probes included W-fragment, LMP gene and EBNA-1 gene of EBV
DNA. The probes were labeled by 32P-dCTP with nicktranslation method.



   The molecular hybridization test showed that the
W-fragment, LMP gene and EBNA-1 gene of EBV did not hybridize to the cell DNA. This result excludes the existence of EBV genes in the CM-1 cells.



   (2) Test for HIV-1 and HTLV-1 Genes with polymerase-chain-reaction (PCR)
 The concentration of CM-1 cell DNA was 0.1 cc mg/ml, the primer for HIV-1 was gag-pol, and the primer for HTLV-1 was gag-env.



   After an initial 5-min denaturation at 94 C, 30 cycles at 95 C for 1 min, 50 C for 1 min, 72 C for 1 min were performed (Perkin, Automated Thermal Cycler), followed by a 7-min extension at 72 C. The PCR products were analyzed by electrophoresis on a 4% agarose gel and the bands were visualized by ethidium bromide staining.



   The primers from HIV-1 and HTLV-1 did not amplify any fragment from the CM-1 DNA. The results of PCR confirmed that the virus in the CM-1 cells is different than HIV-1 or HTLV-1.



  Example 7. Identifying the Virus in the CM-1 Cells as an
 RNA Virus
 A. Purification of the Virus Nucleic Acid from the
 CM-1 Cells
 The subcultured CM-1 cells activated by 5 pg/ml PHA and 25 ng/ml TPA for 48-72 hours were collected and centrifugalized at 1,400 rpm and 4 C for 10 minutes to remove cell debris. This supernatant was centrifugalized at 27,000 rpm, 4 C for 3 hours in a Sorvall ultracentrifuge. The pellets were resuspended in TNE buffer to about 1/200 of the volume at beginning. The virus containing buffer was extracted by phenol and chloroform (1: 1) twice. The virus nucleic acid was precipitated by ethanol and 3 M Na3OAc.  



   B. Test for RNA Virus
 RNase A was used to digest the virus nucleic acid at 37 C for 1 hour. RNase A digest analyzed on 1% agarose gel was performed.



   Lane 1 contained molecular size marker sized from 400 bp to 12 kb.



   Lane 2 contained 10 pg of the virus nucleic acid untreated by RNase A.



   Lane 3 contained 10 pg of the virus nucleic acid digested by 10 pg of RNase A for 1 hour at 37 C.



   The result showed that the HDNV virus nucleic acid is sensitive to RNase A.



  Example 8. Purification of the Virus and Test for RT Activity
 A. Purification of the Virus
 The subcultured CM-2 cells activated by 5 pg/ml PHA and 25 ng/ml TPA for 48-72 hours were collected and centrifugalized at 3,000 rpm and 4 C for 20 min. The supernatant was placed in a dialyzer and was concentrated by
PEG (22,000) at 4 C, then was centrifugalized at 10,000 rpm for 30 min to remove cell debris. The pellets of cells were resuspended with a small amount of TNE buffer. The suspension was frozen and thawed three times and centrifugalized at 10,000 rpm for 30 min. This supernatant was combined with the concentrated supernatant and passed through a 0.45 pm filter.



  The filtered solution was centrifugalized at 35,000 rpm, 4 C for 2 hours in TFT 70.38 rotor of the Kontron T-2080 ultracentrifuge. The pellets were suspended in TNE buffer to about 1/200 of the volume at beginning. 0.4 ml of the concentrated preparations were layered on top of a 20-60% sucrose gradient and centrifugalized at 50,000 rpm, 4 C for 16 hours in the Kontron TST 60.4 rotor. The gradients were collected dropwise (0.5 ml per fraction) and the density of fractions were determined. Clumps of viruses purified from the CM-2 cells. were visualized by electron microscopy from the peak fractions collected from the sucrose gradient.  



   B. Test for Reverse Transcriptase Activitv
 50 l of each fraction was added into round bottom wells of a 12-well plate. 50 yl of disruption buffer (100 mmol/L Tris-HC1 (pH 8.0), 300 mmol/L KCl, 10 mmol/L DTT and 0.1% Triton X-100) was added into each well with the sample.



  The mixtures were incubated at 37 C for 15 min. 25 yl of reaction mixture was added into each well. The mixtures were incubated at 37 C for 20 hours. The components of the reaction mixture are 50 mmol/L Tris-HC1 (pH 8.0), 150 mmol/L
KCL, 12 mmol/L MgCl2,5mmol/L DTT, 0.05% Triton X-100,50 pg/ml Poly (rA): oligo (dT) 15 and 10 uCi/ml 3H-TTP. The reaction was stopped by adding 0.1 ml of ice-cold 10% TCA and the reactants were placed at 4 C for 20 min. The products of the reaction were collected with glass-fibre filters, and washed by 2 ml of ice-cold 10% TCA twice and 2 ml of ice-cold 95% ethanol once. The incorporated radioactivity of the filters in the solution of liquid scintillation was measured with a liquid scintillation counter (Beckman LS-5000 TA).



   All fractions obtained by ultracentrifugation in sucrose density gradients were assayed for RT activity with poly (A): oligo (dT) 15 as template-primers. The results showed that RT activity appeared in tube 5 whose density was 1.16 g/ml (Fig. 4). Thus, RT activity was detected in purified virus.



   RT activity was also determined by a PCR-linked RT assay known as product enhanced RT assay (PERT). (See Pyra H.,
Boni J., and Schupbach J., Proc Natl Acad Sci 91: 1544-1548.



  1994, for a description of the PERT assay.) This assay essentially detects the conversion of template RNA into DNA by
PCR and is highly sensitive and simple to perform on large numbers of samples. 10-6 units of AMV RT can be reproducibly detected by this procedure in our laboratory. The assay is performed by lysing samples of supernatant in RT lysis buffer (composed of 1.5 N KCL, 0.9% triton X-100,0.05% BSA) followed by the addition of bacteriophage MS2 template RNA which has been previously annealed to the appropriate primer. The bacteriophage MS2 is an RNA virus which has no DNA intermediate and therefore problems associated contaminating  
DNA template in the RNA PCR reaction are avoided. The assay was then performed as described in Pyra, et al., supra.



   Supernatant obtained from uninfected T-cell lines molt 4/8, H9 and Jurkat either unstimulated or stimulated with PMA was negative for RT production by PERT assay. In contrast, supernatant obtained from the chronically infected cell line
CM1 was positive for RT production by PERT assay either before or after stimulation with PMA. The level of RT produced by
CM1 cells was comparable to RT produced by a cell line chronically infected with HIV-2, however the ability of this assay to yield quantitative data has not been established.



   Particles from CM1 supernatant were also purified on sucrose gradients. Fractions were then analyzed for density on a refractometer and for the presence of RT activity by PERT assay. The results indicated that RT activity was present at density between 1.15-1.16 g/mi on the sucrose gradient. While not wishing to be bound by theory, this density is consistent with the density of known retroviruses.



   The results of serological assay, probe hybridization and PCR showed that the virus in the CM-1 and CM-2 cells is different than other known human retroviruses or other human non-retroviruses. Furthermore as is demonstrated above, this virus is capable of transforming T lymphocytes in vitro.



  Example 9. In Vitro Detection of Antibodies to HNDV in
 Specimens of Patients with Neurological Diseases:
 The detection of antibodies in patient specimens by
ELISA, western blot, and indirect immunofluorescence is described below.



   A) Detection of antibodies to HNDV by ELISA:
 The ELISA assay described below involves reacting a lysate of HNDV with a test specimen taken from a patient. The mixture is incubated with a peroxidase labeled antibody in wells. Any well positive for the presence of HNDV antibodies forms a detectable and measurable color product.



   The wells of a 96-well plate are coated overnight with a lysate of HNDV at 0.5 yg protein per well in 100 yl 50 mM sodium bicarbonate buffer, pH 9.6. The wells are washed with  water and incubated for 20 minutes with 100 1 of 5% bovine serum albumin in phosphate buffered saline (PBS). After washing, 100 gl of 20% normal goat serum in PBS are added to each well, followed by 5 or 10 gl of the test specimen, and allowed to react for 2 hours at room temperature. The wells are washed three times with 0.5% Tween-20 in PBS in order to remove unbound antibodies and incubated for 1 hour at room temperature with peroxidase labeled goat anti-human IgG at a dilution of 1: 2000 in 1% normal goat serum in PBS. Goat anti-human IgG is a second antibody that binds with the antibody antigen complex formed in positive wells.

   The wells are successively washed 4 times with 0.05% Tween 20 in PBS and 4 times with PBS to remove unbound goat antibody and reacted with 100 gl of the substrate mixture containing 0.05% orthophenylene diamine and 0.005% hydrogen peroxide in phosphatecitrate buffer, pH 5. This substrate mixture detects the peroxidase label and forms a color product. The reactions are stopped by the addition of 50 yl of (NH4) 2SO4 and the color yield measured using an ELISA reader which quantifies the color reading. Assays are performed in duplicate; absorbance readings greater than three times the average of 4 normal negative control readings are taken as positive.



   B) Detection of antibodies to HNDV by western blot:
 As indicated above, antibodies to HNDV may also be detected in a physiological specimen of susceptible patients by means of the Western Blot technique. In this procedure,
HNDV is lysed and electrophoretically fractionated on a polyacrylamide slab gel. Protein bands on the gel are then electrophoretically transferred to a nitrocellulose sheet according to the procedure of Towbin et al., Proc. Natl. Acad.



  Sci. USA, 76: 4350 (1979). The sheet is incubated at 37 C for 2 hr. with 5% bovine serum albumin in 10 mM Tris-HCl, pH 7.5 containing 0.9% NaCl and cut into 0.5 cm strips. Each strip is incubated for 2 hr. at 37 C and 2 hours at room temperature in a screw cap tube containing 2.5 ml of buffer 1 (20 mM
Tris-HCl, pH 7.5,1 mM EDTA, 0.2 M NaCl, 0.3% Triton X-100 and 2 mg/ml bovine serum albumin and 0.2 mg/ml of human antibody  fractions, Fab). Test specimen are then added to individual tubes containing the strips and incubation continued for 1 hour at room temperature and overnight in the cold. The strips are washed three times with solution containing 0.5% sodium deoxycholate, 0.1 M NaCl, 0.5% Triton X-100,1 mM phenylmethylsulfonyl fluoride and 10 mM sodium phosphate, pH 7.5.

   The strips are incubated for 1 hour at room temperature with 2.4 ml of buffer 1 and 0.1 ml of normal goat serum.



  *Affinity purified and 125I-labeled goat anti-human immunoglobulin (mu chain and Fc fragment) (1.25X106 cpm) are added to the reaction mixture and the incubation continues for 30 min. at room temperature. The strips are washed as described, dried, mounted and exposed to X-ray film. The appearance of bands on the X-ray film corresponding to the migration position of viral proteins is indicative of the presence of antibodies to the virus.



   C) Indirect Immunofluorescence Assay for Antibodies to
 HNDV
 The electron micrographs described above showed that the CM-2 cells carried more virions than the CM-1 cells. In addition, serological assays showed that the CM-2 cells had more antigen to the serum of the CM-1 cell donor than the CM-1 cells. Thus, CM-2 cells are preferentially used as the antigen of the virus in an immunofluorescence assay.



   Infected CM-2 cells are washed with phosphate buffered saline (PBS) and resuspended in PBS at 106 cells/ml.



  Approximately 50 samples of cell suspension are spotted on a slide, air dried, and fixed in acetone for 10 min. at room temperature. Slides are stored at-20 C until ready for use.



  20 ml of the test specimen diluted 1: 10 in PBS are added to the fixed cells and incubated for 1 hr. at 37 C. Slides are washed and reacted for 30 min. at room temperature with a dilute solution of fluorescein-conjugated goat anti-human IgG.



  Slides are again washed and examined under a microscope for fluorescence. A negative control uses uninfected parental cells. The above describes a fixed cell system in which the  antibody-antigen reaction is sought for both inside and outside the cell.



   For a live cell immunofluorescence assay all the above reactions are done in a tube instead of on a slide, but without chemical fixation of the cells. After reaction with the fluorescein conjugated antihuman antibody, the cells are added to the slide and examined under a microscope for antibody-antigen reaction on the surface of the cell.



   D. Test kits for ELISA. western blot and indirect
 immunofluorescent assays
 HNDV test kits are constructed for detecting antibodies using several different techniques for detection.



  One test kit for antibodies detection comprises a compartmented enclosure containing a plurality of wells, plates which are coated prior to use with HNDV and ELISA materials for enzyme detection consisting of normal goat serum and peroxidase, labeled goat antihuman IgG, and a color change indicator consisting of orthophenylene diamine and hydrogen peroxide in phosphate citrate buffer.

 

   A test kit for detecting antibodies using the Western
Blot technique comprises a container containing a nitrocellulose sheet and a polyacrylamide slab gel in the presence of sodium dodecylsulfate, and additionally surfactants as well as pH modifiers and bovine serum albumin and human Fab. Additionally this Western Blot analysis container also contains a supply of dilute normal goat serum and 125I labeled goat antihuman immunoglobulin and a source of
HNDV.



   An HNDV specific test kit for detecting antibodies using the indirect immunofluorescence assay comprises a compartmental container, human test serum containing HNDV, phosphate buffered saline, and a fluorescein-conjugated goat antiserum IgG.



   Other embodiments of this invention are disclosed in the claims.
  

Claims

WHAT IS-CLAIMED IS: 1. An isolated Human Neurological Disease Virus, wherein said virus has the following characteristics: a) is an RNA virus containing reverse transcriptase activity; b) is a spherical virion particle with an average size of about 95-113 nm, as determined by electron microscopy; c) is non-reactive with antibodies raised against HTLV-1, HIV-1 and EBV viruses; d) is non-reactive with anti-HIV-1 p24 antibodies and anti-HIV-2/SIV p26 antibodies; e) is specifically immunoreactive with antibodies raised to the HNDV virus present in CM-1 and CM-2 cell lines.

2. The purified Human Neurological Disease Virus of claim 1 wherein said virus is obtained from the CM-1 cell line (ATCC Number CRL 11594).

3. The purified Human Neurological Disease Virus of claim 1 wherein said virus is obtained from the CM-2 cell line (ATCC Number CRL 11595).

4. A cell line infected by the Human Neurological Disease Virus of claim 1.

5. The cell line of claim 4 wherein said cell line is a human cell line.

6. The cell line of claim 5 wherein said cell line is a T lymphocyte cell line.

7. The cell line of claim 5 wherein said cell line is CM-1. (ATCC Number CRL 11594).

8. The cell line of claim 5 wherein said cell line is CM-2 (ATCC Number CRL 11595).

9. A method for continuous production of the Human Neurological Disease Virus of claim 1, comprising the steps of: a) infecting a cell with said virus, said cell having the capacity for continuous growth after infection with said virus; b) multiplying said cell under conditions suitable for cell growth; and recovering said virus produced by said cell.

10. The method of claim 9 wherein said infecting comprises cocultivating said virus with said cell to produce a cell line and recovering said cell line.

11. The method of claim 9 wherein said cell is a T lymphocyte.

12. A method of producing a cell line containing an antigen of the Human Neurological Disease Virus of claim 1, comprising the steps of: infecting a cell with said virus, wherein said cell is capable of continuous production of said virus; and thereafter multiplying said cell under conditions suitable for cell growth.

13. The method of claim 12 wherein said cell is a T lymphocyte.

14. A method for diagnosing viral infection in a susceptible host, comprising the steps of: a) obtaining a biological specimen from said host; and b) detecting the presence of the Human Neurological Disease Virus of claim 1 or antibodies to said virus in said specimen as an indication of infection by said virus.

. 15. A method for detecting antibodies reactive with the Human Neurological Disease Virus of claim 1 in a biological specimen comprising the steps of: a) contacting a composition containing the Human Neurological Disease Virus of claim 1 or an antigen obtained from said Human Neurological Disease Virus with said biological specimen; b) incubating said composition with said biological specimen to form an antibody: Human Neurological Disease Virus antigen complex; and c) detecting said complex.

16. The method of claim 15 wherein said biological specimen is human.

17. A method of detecting a virus in a biological specimen comprising the steps of: a) contacting a binding agent having a binding affinity to the Human Neurological Disease Virus of claim 1 with said biological specimen; b) incubating said binding agent with said biological sample to form a binding agent: Human Neurological Disease Virus complex; and c) detecting said complex.

18. The method of claim 17 wherein said biological specimen is human.

19. The method of claim 17 wherein said binding agent is an antibody.

20. An antibody that is specifically immunoreactive to the Human Neurological Disease Virus of claim 1.

21. A nucleic acid probe capable of selectively hybridizing to a nucleic acid obtained from the Human Neurological Disease Virus of claim 1.

22. A method of detecting a viral nucleic acid obtained from the Human Neurological Disease Virus of claim 1 in a biological specimen comprising: a) contacting said biological specimen with a nucleic acid probe capable of selectively hybridizing to said nucleic acid; b) incubating said nucleic acid probe with the biological specimen to form a hybrid of the nucleic acid probe with complementary nucleic acid sequences present in the biological specimen; and c) determining the extent of hybridization of the nucleic acid probe to the complementary nucleic acid sequences.

23. The method of claim 22 wherein said biological specimen is human.

24. A diagnostic kit for detecting antibodies immunoreactive a virus, comprising a container containing the Human Neurological Disease Virus of claim 1 or an antigen obtained from said Human Neurological Disease Virus and instructional material.

25. A diagnostic kit for detecting a virus comprising a container containing an antibody specifically immunoreactive with the Human Neurological Disease Virus of claim 1 and instructional material.