AU2002306472A1 - Neurovirulent Virus (Cryptovirus) Within the Rubulavirus Genus and Uses Therefor - Google Patents

Description

A NOVEL VIRUS (CRYPTOVIRUS) WITHIN THE RUBULAVIRUS GENUS AND USES

THEREFOR

Throughout the application various publications are referenced in parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference in the application in order to more fully describe the state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the medical arts and particularly to the field of virology, and, more particularly, to a novel human Rubulavirus which has been designated as a "Cryptovirus" .

2. Discussion of the Related Art

The Rubulavirus genus of the Paramyxoviridae (see Fig. 1) are enveloped viruses characterized by a single minus-stranded RNA genome. There is substantial evidence that some rubulaviruses infect domestic animals and can cause neurological diseases in them These neuropathic rubulaviruses include Canine Parainfluenza Vims Type 2, Porcine Rubulaviais (aka La Piedad-Michoacan-Mexico Vims), and Menangle Virus. The strong sequence homologics amongst these viruses imply that each of these vimscs represent separate host-adapted species of a common ancestral Rubulavirus species. Canine Parainfluenza Vims Type 2 (which is also known as CPIV) is associated with infectious tracheobronchitis in dogs. This is a non-lethal disease (usually) of the respiratory tract (Appel and Binn, Canine Infectious Tracheobronchitis. In Vims Infections of Carnivores. Elsevier Science Publishing Co., New York, N.Y., pp 201-21 1, 1987). However, the vims has also been found to be associated with posterior paresis (Evermann et al. J.A. V.M.A. 177: 1132-1134. 1980), neurological dysfunction (Baumgartner et al. Infect. Immun. 3 L 1 177-1183,1981), encephalitis

(Evermann et al. Arch. Virol. 68: 165-172, 1981) and hydrocephalus (Baumgartner et al. Vet. Pathol. 19:79-92, 1982) in dogs.

Porcine Rubulavirus (which is also known as La Piedad-Michoacan-Mexico Vims or LPMV) is associated with Blue-Eye Disease (BED) of pigs. The symptoms of the disease include corneal opacity, extreme "nervousness" in young pigs and infertility in sows and boars (Ramirez-Mendoza et al, J. Comp. Pathol. 117:237-252. 1997). The vims persists in the central nervous system of pigs after recovery from BED (Wiman et al. 1998; Hjertner et al. 1998). This vims also shares more than

95% of its nucleotide sequence with Cryptovirus . Menangle vims is an emergent rubulavirus that was first identified in Australia in 1997 (Philbey et al., Emerging Infectious Diseases 4:269-271, 1998). This vims is associated with severe degeneration of the brain and spinal cord of stillborn piglets (Philbey et al. 1998). Neurons of these animals contained Menangle vims inclusion bodies (nucleocapsids). Serological studies have also found neutralizing antibodies to the vims in pigs, fruit bats and at least two human piggery workers.

A number of human rubulaviruses are known to cause illness in humans. These include: (1) mumps vims (causes human mumps); (2) human parainfluenza vims type 2 (aka HPIV-2; associated with relatively mild upper respiratory, flu-like illness; (3) human parainfluenza vims types 4A and 4B (aka HPIV-4A, HPIV-4B; also associated with relatively mild upper respiratory, flu-like illnesses). With mumps there is also evidence of nervous system involvement in a significant number of patients, although this is virtually never life threatening.

In contrast, there are no published studies clearly demonstrating that another Rubulavirus, Simian Vims 5 (SV5), causes disease either in humans or in experimentally-infected animals, and there has been at least one published study demonstrating that SV5 does not cause disease in experimentally-infected mice, even mice with severe combined immunodeficiency (SCID mice)

(Didcock et al., J. Virol. 73:3125-3133, 1999).

In 1978, a report described the isolation of an "infectious agent" from the bone marrow of patients with multiple sclerosis (MS). (Mitchell, DN el al, Isolation of an infectious agent from bone marrow of patients with mutiple sclerosis. Lancet ii:387-391 [1978]). Subsequent reports described five separate "human SV5 isolates" derived from different MS patients. (Goswami, KKA et al,

Does simian virus 5 infect humans? Journal of General Virology 65: 1295-1303 [1984]; Goswami, KKA et al. , Evidence for the persistence of paramyxoviruses in human bone marrows, Journal of General Virology 65: 1881-1888 [1985]; Randall, RE et al , Isolation and characterization of monoclonal antibodies to simian virus 5 and their use in revealing antigenic differences between human, canine and simian isolates, Journal of General Virology 68:2769-2780 [1987]).

Nevertheless, a causal link between SV5 and MS remained speculative.

In 1987, Goswami et al. reported that the cerebrospinal fluid (CSF) of some MS patients contained antibodies to SV5. (Goswami KKA et al, Antibodies against the paramyxovirus SV5 in the cerebrospinal fluids of some multiple sclerosis patients, Nature 327:244-247 [1987]). However, this report was controversial, since the results subsequently failed to be reproducible by other well respected paramyxovirologists. (Vandvik, B. and Norrby, E., Paramyxovirus SV5 and multiple sclerosis, Nature 338:769-771 [1989]; but see, Russell, WC and Randall, RE, Multiple sclerosis and paramyxovirus, Nature 340: 104 [1989]). Therefore, a clear causal link between SV5 and MS was not established in the art.

Multiple Sclerosis is a chronic degenerative central nervous system disease that most commonly affects young and early middle-aged adults (between 18 and 40 years of age). It is less commonly diagnosed in adolescents and even less so in children. Affecting 350,000 Americans, MS is one of the most frequent causes of neurologic disability except for traumatic injuries. (S.L. Hauser, Multiple Sclerosis and other demyelinating diseases In: Harrison 's Principles of Internal Medicine, 13th ed., K.J. Isselbacher et al. (eds.), McGraw-Hill, pp.2287-95 [1994]). The onset, progression and outcome of the disease are highly variable with patients manifesting one of several patterns of illness. For example, for reasons that are unclear, MS affects twice as many females as males. Although the individual components that comprise the diagnostic, clinical tableau of MS have long been delineated, their sequence and severity of presentation from case to case are subject to great variation. (Hallpike J.F., Adams C.W.M., and Tourtellotte W.W., Eds, 1983, Multiple Sclerosis: Pathology, Diagnosis and Management, Chapman & Hall, London; McAlpine E. et al, 1972, Multiple Sclerosis: A Reappraisal, Churchill Livingstone, Edinburgh; Rose A.S , 1972, Multiple Sclerosis: A Clinical

Interpretation. In Multiple Sclerosis: Immunology, Virology and Uitrastruclure Wolfgram F., Ellison G.W., Stevens J.G., and Andrews J.M., Eds., Academic Press, New York). It is fair to say that no two patients with MS are alike, and, consequently, there is contention as to what constitutes the stereotypic clinical history. Most commonly, MS first presents as a series of neurological attacks followed by complete or partial remissions where symptoms lessen only to return after some period of stability (relapsing- remitting MS). In other patients, the disease is characterized by a gradual decline with no clear remissions but sometimes with brief plateaus or minor relief of symptoms (primary-progressive MS). In still other patients, there can be a relapsing and remitting course of illness in the early stages followed by progressive decline (secondary-progressive MS).

In general, the primary manifestations of chronic progressive and chronic relapsing MS do not vary greatly. Evidence for an insidious disease (apathy, depression, fatigue, loss of weight, muscle pains) often can be uncovered from the patient's chart before the first neurological manifestations. Among the first signs in about 50% of all definite MS cases are limb weakness, numbness, or tingling (parathesias) in one or more limbs, the extremities, or around the trunk. There is often discordance between signs and symptoms. Adams and Victor (1997, Multiple sclerosis and allied demyelinative diseases. In Principles of Neurology. Adams R.D. and Victor M., Eds., McGraw Hill. New York) mention that "it is a common aphorism that the patient with MS presents with symptoms in one leg and signs (bilateral Babinski) in both." Another common initial sign is a short-lived episode of retrobulbar neuritis affecting one or both eyes. Many MS patients will display papilhtis (swelling of the optic nerve head), which depends on the proximity of the demyelinated plaque to the nerve head. There is considerable debate as to whether optic neuritis in a significant percentage of cases constitutes a separate disease or subclass of MS, but in about 50% of cases, the disease progresses to MS (Arnason et al., J. Neurol Sci. 22:419, 1974).

As diagnosis becomes established, a more regular group of clinical syndromes develops either progressively or in a remitting fashion. The majority of patients display a mixed or generalized type of disease involving optic nerves, brain stem, cerebellum, and spinal cord. About one third will exhibit a spinal form, and about 5% will display a cerebellar or pontobulbar-cerebellar form, and a similar percentage will have an amaurotic form. Adams and Victor (supra) estimate that at least 80% of their own clinical material comprised cerebrospinal and spinal forms of the disease.

Psychologic disturbances are frequently observed and can present as an inappropriate euphoric state, attributed by Adams and Victor (supra) probably to extensive white matter lesions in the frontal lobes. In a much higher percentage of MS patients depression and irritability are observed.

Until relatively recently, MS and epilepsy were considered discrete entities. The publication of numerous recent studies demonstrating an "overlap" between these disorders has corrected this misperception. Epidemiological and demographic studies conducted over the last decade have provided substantial evidence of concurrent epileptifomi symptomology in a significant proportion of MS patients. While the concurrence of epileptiform symptoms is markedly higher in early onset MS

(i.e. in children and adolescents), the overall prevalence of epilepsy in MS patients is many times higher than in the general population.

Human epilepsy is an enigmatic medical condition which, in fact, is not a specific disease - or even a single syndrome - but, rather a broad category of symptom complexes arising from any number of disordered brain functions that themselves can be secondary to a variety of pathologic processes. Today, a large number of clinical phenomena are recognized as epileptic seizures, some of which (e.g., myoclonic and atonic seizures) are currently poorly understood and could, in fact, reflect neuronal mechanisms that are somewhat different from the pathophysiologic processes traditionally considered to be "epileptic." Perhaps the best reflection of the enigmatic and complex nature of these illnesses is the simple fact that the etiology of the disease, in the overwhelming majority of the cases

(greater than 70%), is either "cryptogenic" (i.e., of obscure, indeterminate origin) or "idiopathic" (i.e., of unknown cause). Epilepsy is more than seizures Epileptics typically exhibit a spectrum of responses, from little or no seizure activity, through mild activity (petit mal or "absence" seizures), to recurrent and intractable grand mat seizures (the occurrence of which is often misunderstood by the lay public to be the defining symptom of all forms of epilepsy, see Epilepsy A Comprehensive Textbook, Engel, Jr J and Pedley, T A , Eds , Lippincott-Raven, 1997) The condition in its entirety is comprised of many facets, different for each individual, that contribute to disability and impaired quality of life While the physical spectrum of symptoms ranges from extremely subtle petit mal or "absence" seizures to profoundly disabling grand mal seizures, many patients experience other co-morbid processes (e g , memory loss, confusion, lethargy, sleep disturbances, and clinical depression) which can be equally disabling Treatment that focuses solely on seizures often does little to lessen disability This is perhaps best illustrated by the patient who, having undergone successful surgical resection of epileptogenic brain tissue, becomes seizure-free but remains socially isolated and unemployed, with little evidence of an improved life Therapeutic intervention can be optimal only when the multiple and interacting medical, psychological, and environmental factors that constitute epilepsy are addressed

Another epileptiform disease is Subacute Sclerosing Panencephalitis (SSPE), a rare and fatal degenerative central nervous system disease of children and adolescents (Sever and Zeman, editors, Measles Vims and Subacute Sclerosing Panencephalitis Neurology (Supplement I) 18 1-192, 1968, Payne and Baubhs, Per pectives in Virology 7 179-195, 1971, Johannes and Sever, Ann Rev Med 26 589-601, 1975, Meulen et at , Comp Virol 18 105-159, 1983, Dykcn, Neurol Clin 3 179-196,

1985) In its early stages it commonly presents as an affective or other behavioral disorder and progresses over a period of months to profound epileptiform neurological disease Its later stages are characterized by intractable seizures, decerebrate rigidity, coma and death At some time, virtually all SSPE patients are "misdiagnosed" with epilepsy Cases of SSPE have been described in both industrialized and developing countries throughout the world (Canal and Torck, ./ Neurol Sci 1 380-389, 1964, Pettay et al , J Inject Dis 124 439-444, 1971, Haddad et a! , Lancet 2 1025, 1974, Softer et al , Israeli J Med Sci 11 1-4, 1975, Naruszewicz-Lesiuk et al , Przeg Epidemiologiczna 23 1-8, 1979, Moodie et al , South African Med .1 58 964-967, 1980) The frequency of the disease varies greatly, ranging from 0 06 and 0 10 cases per million total population per year (cpmpy) in Britain (Dick, Brit Med J 3 359-360, 1973) and the United States (Jabbour et al , . I A M A 220 959-962, 1972) to 3 40 and 7 70 cpmpy in Israel (Soffer, et al , Israeli .7 Med Sci 1-4, 1975) and New Zealand (Baguley and Glasgow, Lancet 2 763-765, 1973) The factor(s) that are responsible for the ultimate etiopathogeneis of the disease are unclear

Numerous studies have shown that the central nervous system tissues of SSPE patients are persistently-infected with measles vims Substantial evidence indicates that the disease involves the recmdescence of a persistent measles vims infection acquired earlier in life Specific findings in

SSPE patients which support this hypothesis include (1) a history of childhood measles, (2) markedly elevated titers of measles virus-specific antibodies in serum, (3) the presence of measles virus-specific antibodies in cerebrospinal fluid, (4) the presence of measles vims antigens in CNS tissues demonstrated by specific immunofluorescence, (5) intracellular inclusions of paramyxoviral nucleocapsids in oligodcndroghal and neuronal cells, and (6) the isolation of infectious measles vims from brain and lymphatic tissues when co-cultivated with susceptible cells (Bouteille et al , Revue Neurologic U3 454-458, 1965, Connolly et al , Lancet 1 542-544, 1967, Lcgg, Brit Med J 3 350- 354, 1967, Payne et al , New Eng J Med 281585-589, 1969, Horta-Barbosa et al , Nature 221 974, 1969) Finally, and perhaps most convincingly, cpidemiological evidence suggests that vaccination against measles substantially reduces the risk of developing the disease (Modhn et al , Pediatrics

59 505-512, 1977, Halscy et al , Am J Epidemiol \ \ \ 415-424, 1980, Dyken et al , Morb Mortal Weekly Report 3J_. 585-588, 1982)

Despite these findings, there are a number of anomalies that have been observed which arc inconsistent with measles vims alone being the sole cause of the illness These include the following First, neurovirulence Clinical isolates of measles vims from patients with rubeola have not been shown to cause an SSPE-hke illness in experimentally infected animals Cell-associated SSPE- deπved strains, however, have been shown to cause such disease in ferrets, marmosets and monkeys (Katz et al , J Infect Dis \2\ 188-195, 1970, Thormar et al , J Infect Dis 121 678-685, 1973, Ueda et al , Biken Journal 8 179-181, 1975, Yamanouchi et al , Japan J Med Sci Biol 29177-186, 1976, Thormar et al , J Infect Dis 136 229-238, 1977, Albrecht et a , Science 195 64-66, 1977,

Thormar et al , J Exp Med 148 674-691, 1978, Ohuchi et al , Microbiol Immunol 25 887-893 [1981])

Second, distribution and morphology of vims inclusion bodies Measles vims antigens in infected cells form large coalescing lntracytoplasmic inclusions when examined by fluorescent antibody techniques When labeled with SSPE patient sera, vims antigens demonstrate distinctly different patterns in cell-associated SSPE-deπved vims strains and in experimentally-infected animal CNS tissues In such materials, intracellular inclusion bodies demonstrate a "peppery," particulate and/or "splattered" distribution (Doi et al , Japan J Med Sci Biol 25 321-333, 1972, Kimoto and Baba, Biken Journal 18 123-133, 1975, de Fehci et al , Annales Microbwlogie 126 523-538, 1975, O uc i et al , Microbiol Immunol 23 877-888, 1979)

Third, ultrastructural morphology of vims nucleocapsids In measles vims infected cells, cytoplasmic nucleocapsids are predominantly of a "fuzzy" or "granular" morphology (Tawara, Virus (Osaka) 14 85-88, 1965, Matsumoto, Bull Yamaguchi Med School 3 167-189, 1966, Nakai et al ,

Virology 38 50-67, 1969, Nakai and Imagawa, J Virol 3 187-197, 1969) In SSPE-deπved CNS tissues, both fuzzy and smooth nucleocapsids have been consistently observed (Oyanagi et al , J Virol 7 176-182, 1971, Dubois-Dalcq et al , Arch Neurol 31 355-364, 1974) Smooth cytoplasmic nucleocapsids have also been observed in most cell -associated SSPE-deπved cell lines (Doi et al , Japan J Med Sci Biol 25 321-333, 1972, Makino et al , Microbiol Immunol 2Λ 193-205, 1977,

Ueda et al , Biken Journal 18 113-122, 1975, Burnstein et al , Infect Immun 10 1378-1382, 1974, Mirchamsy et al , Intervirology 9 106-118, 1978, Schott et al , Revue Neurologie 135 653-664, 1979) with some containing only such stmctures (Doi et al , Japan J Med Sci Biol 25 321-333, 1972, Thormar et al , J Exp Med 148 674-691, 1978) Fourth, immunoreactivity of vims nucleocapsids While fuzzy nucleocapsid aggregates are labeled with HRP-conjugated measles virus-specific antibody experimentally raised in animals, smooth vims nucleocapsids arc not (Dubois-Dalcq et al , Lab Invest 30 241-250. 1974, Brown et al , Acta Neuropathologica 50 181-186, 1980) Most interestingly, both can readily be labeled with SSPE sera (Brown et al , Acta Neuropathologica 50 181-186, 1980) And fifth, epidemiology The least understood feature of the measles vims theory of SSPE aetiopathogenesis is the extremely low incidence of the disease Despite numerous investigations into the role of socioeconomic, demographic and genetic factors (Canal and Torck. J Neurol Sci \ 380- 389, 1964, Pettay et al , J Infect Dis YU 439-444, 1971, Haddad et al , Lancet 2 1025, 1974, Soffer et al , Israeli J Med Sci V\_ 1-4, 1975, Naruszewicz-Lesiuk et al , Przeg Epidemiologiczna 23 1-8, 1979, Moodie et al , South African Med J 58 964-967, 1980, Dick, Brit Med J 3 359-360, 1973,

Jabbour et al , J A M A 220 959-962, 1972, Baguley and Glasgow, Lancet 2 763-765, 1973, Modhn et al , Pediatrics 59 505-512, 1977, Halsey et al , Am J Epidemiol 111 415-424, 1980, Dyken et al , Morh Mortal Weekly Report 3J. 585-588, 1982), it has, until now, been completely unclear why SSPE is so rare when measles vims annually infects millions of children throughout the world

A much more common ldiopathic neurological and/or neuropsychiatπc disease, which affects more than a half million Americans, is chronic fatigue syndrome (CFS), which frequently involves concurrent epileptiform symptomology (P H Levine, What we know about chronic fatigue syndrome and its relevance to the practicing physician, Am J Med 105(3A) 100S-03S [1998]) Chronic fatigue syndrome is characterized by a sudden onset of persistent, debilitating fatigue and energy loss that lasts at least six months and cannot be attributed to other medical or psychiatric conditions, symptoms include headache, cognitive and behavioral impairment (e g , short-term memory loss), sore throat, pain in lymph nodes and joints, and low grade fever (M Terman et al ,

Chronic Fatigue Syndrome and Seasonal, Affective Disorder Comorbidity, Diagnostic Overlap, and Implications jor Treatment, Am J Med 105(3A) 115S-24S [1998]) Depression and related symptoms are also common, including sleep disorders, anxiety, and worsening of premenstmal symptoms or other gynecological complications (A L Komaroff and D Buchwald, Symptoms and signs of chronic jatigue syndrome, Rev Infect Dis 13 S8-S11 [1991], B L Harlow et al ,

Reproductive correlates oj ^'chronic fatigue .syndrome, Am J Med 105(3A) 94S-99S [1998]) Other physiologic abnormalities are also associated with CFS in many patients, including neurally-mediated hypotension, hypocortisolism, and immunologic dysregulation (P H Levine [1998]) A subgroup of CFS patients complain of exacerbated mood state, diminished ability to work and difficulty awakening during winter months, reminiscent of seasonal affective disorder (M Terman et al

[1998])

The etiology of CFS has been unknown, and the heterogeneity of CFS symptoms has precluded the use of any particular diagnostic laboratory test (P H Levine [1998]) Symptomatic parallels have been suggested between CFS and a number of other disease conditions, resulting from viral or bacterial infection, toxic exposure, orthostatic hypotension, and stress, but none of these has been shown to have a causal role in CFS (E g , I R Bell et al , Illness from low levels of environmental chemicals relevance to chronic fatigue syndrome and flbromyalgia, Am J Med 105(3A) 74S-82S [1998], R L Bruno et al , Parallels between post-polio fatigue and chronic fatigue syndrome a common pathophysiology? , Am J Med 105(3A) 66S-73S [1998], R Glaser and J K Kiecolt-Glaser, Stress-associated immune modulation relevance to viral inf ctions and chronic jatigue syndrome, Am J Med 105(3A) 35S-42S [1998], P C Rowe and H Calkins, Neurally mediated hypotension and chronic fatigue syndrome, Am J Med 105(3A) 15S-21S [1998], L A

Jason et al , Estimating the prevalence of chronic fatigue syndrome among nurses, Am J Med

105(3A) 91S-93S [1998]) Accordingly, there has been no known cause to which diagnosis and/or treatment of CSF could be directed Consequently, the diagnosis and treatment of CFS have continued to be directed to symptoms, rather than to an underlying treatable cause For example, the use of relaxin has been described for relaxing the involuntary muscles and thus relieve pain associated with CFS (S K Yue Method of treating myofascial pain syndrome with relaxin, U S Patent No 5,863,552)

There remains a need for an underlying causal factor for many ldiopathic neurological, neurodegenerative, neuropsychological and neuropsychiatric disorders and primary tracheobronchial and/or lymphadenopathy-associated diseases, to which diagnostic testing, research and development, including screening of potential new antiviral dmgs, and treatment can be directed This and other benefits of the present invention are described herein

SUMMARY OF THE INVENTION

The present invention is based on the discovery and isolation of a novel human vims that has been designated as a "Cryptovirus" , which falls within the genus Rubulavirus of the family

Paramyxoviπdae The genome of the isolated Cryptovirus of the present invention is a minus strand RNA having a nucleotide sequence entirely complementary to (SEQ ID NO 1)

The present invention relates to isolated nucleic acids that are Cryptovirus -specific The inventive Cryptovirus -specific nucleic acids encompass (A) nucleotide sequence of contiguous nucleotide positions 1-15246 of (SEQ ID NO 1), such as, but not limited, to plus strand RNAs (e g , mRNAs) and cDNAs, or (B) a nucleotide sequence complementary to contiguous nucleotide positions 1-15246 of (SEQ ID NO 1), such as, but not limited to minus strand RNAs (e g , genomic or cloned RNAs) and cDNAs, or (C) Cryptovirus -specific fragments of (A) or (B), such fragments being at least about five nucleotides long The present invention encompasses both RNAs and DNAs, and thus it is understood by the skilled artisan that the present invention encompasses nucleic acids, I e ,

RNAs, in which uracil residues ("U") replace the thymine residues ("T") in (SEQ ID NO 1) The inventive nucleic acids include useful Cryptovirus -specific probes and primers

Inventive nucleic acid constmcts, including cloning vectors and expression vectors, are provided that contain the inventive nucleic acid Such inventive recombinant vectors are contained in a host cell of the present invention

The present invention also relates to an isolated Cryptovirus protein encoded by a Cryptovirus -specific nucleic acid segment The inventive Cryptovirus proteins include isolated Cryptovirus nuclcocapsid and envelope proteins and chimeric proteins comprising a Cryptovirus protein moiety The invention relates to an isolated viπon or other viral particle that contains the inventive

Cryptovirus nucleic acid, such as a viral expression vector, or contains the inventive Cryptovirus protein, such as an inventive pseudotyped virion or an inventive isolated Cryptovirus virion or other Cryptovirus particle.

Inventive compositions of matter that include the inventive Cryptovirus nucleic acid, Cryptovirus protein, or isolated virions and other viral particles, together with a carrier, are also included in the present invention.

Moreover, the present invention provides a method of isolating a Cryptovirus virion. The inventive method involves culturing a plurality of peripheral blood mononuclear cells (PBMNCs) that have been obtained from a human having a Cryptovirus infection. The PBMNCs are cultured in an artificial aqueous medium that includes an agent that increases cellular guanylyl cyclase activity, such as but not limited to cyclic GMP. The PBMNCs are then co-cultured in with a plurality of mammalian amnion cells in fresh aqueous medium including the agent, and the co-culture is passaged one or more times Passaging is followed by co-cultivating a plurality of mammalian epithelial cells together with the PBMNCs and the mammalian amnion cells in fresh aqueous medium comprising the agent. This co-cultivation results in the production of Cryptovirus virions that are released into the aqueous medium. A supernatant of the aqueous medium is separated from the cells in the culture, to obtain the Cryptovirus virions, which are found in the supernatant. The inventive method facilitates the isolation from cellular material of Cryptovirus virions in great numbers. Virions isolated thereby can be further propagated by an inventive method of propagating a Cryptovirus, which the present invention provides. The inventive method of propagating a Cryptovirus involves exposing a plurality of mammalian epithelial cells to a plurality of cell-free Cryptovirus virions, thus isolated, and further cultivating the Cryptovirus virion-exposed mammalian epithelial cells in an artificial aqueous medium comprising an agent that increases the activity of cellular guanylyl cyclase. Thus, a mammalian epithelial cell acutely infected with Cryptovirus is provided, which inventive cell is produced by the method.

The present invention also relates to a method of producing a mammalian cell line nonproductively infected with Cryptovirus. The method involves co-culturing PBMNCs that have been obtained from a human having a Cryptovirus infection, with mammalian amnion cells (e.g., rodent or primate amnion cells), in an artificial aqueous medium comprising an agent that increases cellular guanylyl cyclase activity, such that the mammalian amnion cells become nonproductively infected by Cryptovirus. After passaging the nonproductively infected mammalian amnion cells with the peripheral blood mononuclear cells, the co-culture becomes a monoculture of the nonproductively infected mammalian amnion cells. The present invention also relates to a cell nonproductively infected with Cryptovirus, which cell is produced in accordance with the method.

Cryptovirus is associated with cryptogenic and idiopathic forms of human disease, e.g., epilepsy. Cryptovirus is also associated with other human neurological, neurodegenerative, and/or neuropsychiatric diseases where neural dysfunction and neuropathology are evident and where epileptiform symptomology is always concurrent (e.g. subacute sclerosing panencephalitis, SSPE) or is frequently concurrent (e.g., multiple sclerosis [MS] and chronic fatigue syndrome [CFS]). Thus, the inventive cell lines, viral particles and virions are particularly useful for screening potential antiviral agents to discover those that could be effective in treating mammals, including humans, infected with Cryptovirus.

In particular, useful in vitro methods of screening a potential antiviral therapeutic agent are provided. In accordance with the in vitro screening methods, the inventive Cryptovirus -infected cells are cultured, and then exposed to the potential antiviral therapeutic agent. If acutely infected mammalian epithelial cells are used, then the effect of the potential antiviral therapeutic agent on Cryptovirus replication and/or Cryptovirus virion assembly is measured (e.g., effect on Cryptovirus genomic replication, Cryptovirus transcription, and/or translation, i.e., protein synthesis, from Cryptovirus mRNAs, effect on numbers of Cryptovirus virions produced or completeness of Cryptovirus particles). Inhibition of Cryptovirus replication and/or Cryptovirus virion assembly, relative to a control not receiving the agent, indicates antiviral activity of the potential therapeutic agent. Alternatively, if nonproductively infected cells are used, measurement is made of the effect of the potential antiviral therapeutic agent on Cryptovirus replication, Cryptovirus genome replication, and/or Cryptovirus-specϊfic transcription. Inhibition of Cryptovirus replication, Cryptovirus genome replication, and/or Cry/^ov/ s'-specific transcription, relative to a control not receiving the agent, indicates antiviral activity of the potential therapeutic agent. These inventive methods are useful for identifying, screening, or isolating promising new antiviral dmgs. Once the potential of a chemical agent is identified by the inventive methods, then, further research can be done to ascertain its clinical usefulness. Thus, the inventive methods of screening a potential chemotherapeutic agent are of benefit in finding and developing pharmaceutical antiviral dmgs aimed at treating Cryplovirus- related conditions and other conditions associated with other viruses of the Mononegavirales. The present invention now also provides an animal model for the study of human diseases, for example a neurological, neurodegenerative, and/or neuropsychiatric disease (e.g., idiopathic epileptiform diseases, such as epilepsy, SSPE, MS, and CFS). The animal model involves a non- human mammal, which has been inoculated with an infectious cell-free Cryptovirus having a genome comprising a single stranded RNA complementary to (SEQ ID NO:l), or has been inoculated with a cell nonproductively-infected with the Cryptovirus. The inoculated non-human mammal of the animal model exhibits at least one symptom characteristic of a human disease after being thus inoculated, which was not previously exhibited by the non-human mammal before inoculation. The animal model is useful in an in vivo method of screening a potential antiviral therapeutic agent. The method involves administering the potential therapeutic agent to be screened, to the inventive animal model. Before administration of the potential therapeutic agent, the non-human mammal exhibits at least one symptom characteristic of a human disease. After administration of the potential therapeutic agent, the presence or absence of a beneficial antiviral effect is detected; the presence of a beneficial antiviral effect, in comparison to a control animal not receiving the agent, indicates activity of the potential therapeutic agent.

Employing an alternative embodiment of the inventive animal model, an in vivo method of screening a potential antiviral prophylactic agent is provided. The method involves administering a potential prophylactic agent to be screened to a non-human mammal, which does not have a symptom of a human disease, for example a neurological, neurodegenerative, and/or neuropsychiatric disease.

Then the animal is inoculated, as previously described, with an infectious cell-free Cryptovirus having a genome comprising a single stranded RNA complementary to (SEQ ID NO: l), or with a mammalian cell nonproductively-infected with the Cryptovirus. Subsequently, the presence or absence in the non-human mammal of a beneficial antiviral effect is detected, compared to a control not receiving the potential prophylactic agent. The subsequent presence of a beneficial antiviral effect in the inoculated non-human mammal indicates activity of the potential prophylactic agent.

The inventive nucleic acid constmcts, Cryptovirus proteins, and particles and virions are also particularly useful in producing Cryptovirus -specific antibodies, and in the production or manufacture of vaccines, which antibodies and vaccines are directed specifically against Cryptovirus proteins, such as the nucleocapsid or envelope proteins of Cryptovirus. These vaccines can include live attenuated vims; killed vims; recombinant chimeric viruses; proteins or other parts of vims; or one or more isolated or recombinantly expressed Cryptovirus proteins.

The present invention relates also to an isolated antibody that specifically binds a Cryptovirus protein and the use of the inventive antibody in manufacturing a medicament for the treatment of Cryptovirus infections. Also provided are compositions of matter comprising the antibody and a carrier. In other aspects, the invention is usefully directed to methods and assays, e g , for determining whether biological materials are contaminated with Cryptovirus or whether a mammal, including a human, is or has been infected with Cryptovirus

In particular, the invention provides methods of detecting the presence or absence of a Cryptovirus protein, Cryptovirus-specific RNA, or Cryptovirus -specific antibody in a sample of a biological material, such as serum

In the method of detecting Cryptovirus protein, the sample of the biological material is contacted with an inventive antibody that specifically binds a Cryptovirus protein, and if the presence of specific binding of the antibody to a constituent of the sample is detected, this indicates the presence of the Cryptovirus protein in the sample

Similarly, in the method of detecting Crypto virus -specific RNA in a sample of a biological material containing RNA, the sample is contacted with the inventive Cryptovirus-specific probe under at least moderately stringent hybridization conditions, and the formation of detectable hybridization products indicates the presence of the Cryptovirus RNA in the sample Alternatively, the sample containing RNA is subjected to an amplification of Cryptovirus- specific RNA in the sample, using at least one inventive Cryptovirus-specific primer in an amplification reaction mixture By detecting the presence or absence of Crypto virus -specific nucleic acid amplification products in the amplification reaction mixture, the presence or absence of Cryptovirus -specific RNA in the sample can be determined, with the presence of Cryptovirus-specific amplification products in the reaction mixture indicating the presence of the Cry/>/ov7π«-specιfιc

RNA in the sample

The present invention also provides a method of detecting the presence or absence of a Cryptovirus-specific antibody in a sample of an antibody-containing biological material, such as serum The method involves contacting the sample of biological material with the inventive protein, such as a Cryptovirus envelope protein, or alternatively, with the inventive virion or viral particle, under conditions allowing the formation of a specific protein-antibody complex, or antibody-bound vims complex, respectively Detection of the presence of such specific protein-antibody complexes, or antibody-bound vims complexes, indicates the presence of the Cr /?/ov/π«-specιfic antibody in the sample Inventive anti-Cryptovirus antibody detecting kits are also provided, which are useful for practicing the method

Thus, by practicing any of the foregoing inventive methods of detecting the presence or absence of a Cryptovirus protein, Cryptovirus -specific RNA, or Cryptovirus -specific antibody, with a sample of biological materials from a mammal, including a human, an inventive method of detecting or diagnosing a Cryptovirus infection in the mammal is provided, as indicated by the presence in the sample of Cryptovirus protein, Cryptovirus-specific RNA, or Cryptovirus-specific antibody. These diagnostic methods are valuable because, regardless of the therapeutic strategy, it is advantageous to begin therapy at the time of "primary" infection (i.e., the first exposure to the vi s) or as soon as possible thereafter (i.e., during development of the primary infection).

Other features, objects, and advantages of the invention will be apparent from the accompanying drawings and the detailed description of the preferred embodiments hereinbelow.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a chart showing the taxonomic assignment of the human Cryptovirus of the present invention to the genus Rubulavirus of the Paramyxoviridae.

Fig. 2 is a phylogenetic tree, modified from the version appearing in Collins et al.

(Chapter 41, page 1206, Parainfluenza Viruses, in Virology, 3^rd Ed., Fields, Knipe, and Howley, Eds.,

Lippincott-Raven, Philadelphia, 1996). The modified tree emphasizes the clustering of three Rubulavirus species (Porcine Rubulavirus; Canine Parainfluenza Vims Type 2; and the human

Cryptovirus of the present invention) as distinct from the prototype Rubulavirus Simian Vims 5.

Fig. 3 is a representation of the genetic maps of typical members of each genus of the family Paramyxoviridae. The gene size is drawn to scale. Vertical lines demark gene boundaries. The pneumovims L gene transcription overlaps that of the 22K (M2) gene and is thus shown in a staggered format. This overlap configuration is seen in human and animal vimses, but not in other pneumoviruses (Lamb and Kolakofsky, Chapter 40, page 1181, The Viruses and Their Replication, in Virology, supra).

Fig. 4 is a representation of revised Rubulavirus genetic maps, which distinguish Simian Vims 5 from a cluster of related vimses that demonstrate neurotropism and encode an additional 22 amino acid "tail" at the carboxy terminus of their fusion proteins (i.e., the "neurotropic species" of human Cryptovirus, Canine Parainfluenza Type 2, and Porcine Rubulavirus). The fusion (F) proteins of each neurotropic vims species are more closely related to each other than they are to the fusion protein of Simian Vims 5 (see Fig. 10).

Fig. 5 shows a schematic and comparative autoradiograms of the [³⁵S]-methionine labeled proteins of gradient-purified human Cryptovirus (Strain BBR) and Simian Virus 5 (NIH 2I005-2WR strain) following SDS-PAGE on 10% acrylamide Laemmli slab gels under reducing conditions. Fig 6 is a collage of matched sets of fluorescent photomicrographs taken of various SSPE- deπved nonproductively infected cell cultures following direct double labeling with rhodamine isothiocyanate-labeled goat anti-measles vims semm (Panels A, C, E, G and I) and rabbit anti- Cryptovirus sem , then followed with fluorescein isothiocyanate-labeled goat anti-rabbit IgG (Panels B, D, F, H and J) Panels A and B represent AV₃/SSPE/MV cells persistently-infected with

Cryptovirus and also infected with the Edmonston stram of measles vims before being passaged onto covershps for these linmunofluorcscent studies, Panels C and D represent the nonproductive SSPE- deπved cell line designated "Kitaken" (Ueda et al , Biken Journal J_8 179-181, 1975), Panels E and F represent the nonproductive SSPE-deπved cell line designated "Nugata" (Doi et al , Japan J Med Sci Biol 25 321-333, 1972), Panels G and H and I and J, respectively, represent the nonproductive

SSPE-deπved cell line designated "Biken" (Yamanouchi et al , Japan J Med Sc Biol 29 177-186, 1976. Ohuchi et al , Microbiol Immunol 25 887-983, 1981)

Fig 7 shows photographs of two male Colored mice, bom of the same litter, two months after neonatal (two days after birth) intracerebral inoculation with plaque-purified Cryptovirus (strain BBR, Fig 7A) or with the NIH 21005-2WR strain of Simian Vims 5 (SV5, Fig 7B)

Fig 8 shows photographs of two female Colored mice, bom of the same litter, three months after neonatal (two days after birth) intracerebral inoculation with plaque-purified Cryptovims (strain BBR, Fig 8A) and six months after neonatal (two days after birth) intracerebral inoculation with plaque-purified Cryptovims (Fig 8B) Fig 9 is a comparison of the FASTA formatted (I e , mRNA sense 5' to 3') sequence of human Cryptovirus Strain BBR (SEQ ID NO 1) and Simian Vims 5 Strain W3A (SEQ ID NO 2) The number of variations from (SEQ ID NO 1) in each line of (SEQ ID NO 2) is tallied in the right-hand margin The Cryptovirus-specific nucleotide positions that differ from the sequence of SV5 are in bold underlined type, if the difference is in a coding region, the relevant amino acid encoded is printed above the codon of the Cryptovirus nucleotide sequence, and if the different Cryptovirus nucleotide results in a codon encoding a different amino acid than the SV5 codon in the analogous position, an arrow leads from the SV5 amino acid to the different amino acid in the analogous Cryptovirus protein Boxed nucleotides indicate known SV5 and analogous Cryptovirus start or stop sites, as indicated Fig 10 is a comparison of Rubulavirus F Protein nucleotide (Fig 10A, comparison of the

FASTA formatted, l e , mRNA sense 5' to 3' sequence) and encoded amino acid (Fig 10B) sequences The first line (uppermost) represents an embodiment of the sequence of an inventive Cryptovirus F protein ("CV" [Strain BBR]), the second line represents Canine Parainfluenza Vims Type 2 ("CPV" [Strain TJ], see Ito et al , J Gen Virol 81 719-727, 2000), the third line represents Porcine Rubulavirus ("PR", Klenk and Klenk, Direct Submission to EMBL / GenBank Databases, September 2000, GenBank Accession AJ278916), the fourth line represents Simian Vims 5 ("W3A" [Strain W3A], Paterson et al , Proc Natl Acad Sci USA 81 6706-6710, 1984), and the fifth (bottom) line represents Simian Vims 5 ("WR" [Strain WR], Ito et al , J Virol 7J. 9855-9858, 1997) Ammo acids that are bold and underlined denote amino acids that differ from those in the analogous sequence of the Cryptovims F protein, and the tallies in the right margin are the number of differences for each sequence block Fig 11 demonstrates expression of Cryptovirus proteins Fig 11 is a photograph of an autoradiogram of gradient-purified [³⁵S]-methιonιne-labeled Cryptovirus virions produced in acutely- infected Vero cells after SDS-PAGE under reducing conditions The approximate molecular weights of the proteins indicated on the right side of Fig 12A were calculated by comparing their migrations to marker proteins of known molecular weight (Sigma Biochemicals) L = the largest nucleocapsid associated protein, the major component of the viπon-associated RNA dependent RNA polymerase,

HN = the hcmagglutinin protein, one of the envelope-associated glycoproteins, F₀ = the uncleaved fusion protein, a second envelope-associated glycoprotein, NP = the nucleocapsid protein, the major stmctural protein associated with the nucleocapsid, Fi = the larger fragment of the cleaved fusion protein, P = the nucleocapsid associated phosphoprotein, M = the viπon-associated matrix or membrane protein, V = a minor RNA binding protein thought to be a component of the viral polymerase, F = the smaller fragment of the cleaved fusion protein Note the SH protein (about 5 kD), a small envelope-associated protein, ran off the gel and is not shown Fig 12 shows photographs of autoradiograms of typical radioimmunoassay profiles (RIPs) obtained by the precipitation and SDS-PAGE separation of [³⁵SJ-methιonιne-labeled virus-specific proteins using the cerebrospinal fluids (CSFs) of a patient diagnosed with subacute sclerosing paneneccphahtis (Fig

12A) and the CSFs of six randomly-selected neurology/ neurosurgery patients who had CSF taken for microbiological screening (Fig 12B) Fig 12A shows the RIPs resulting from the precipitation of [³⁵S]-metluonιne-labeled CV-lc cells acutely-infected with the Edmonston strain of measles vims (Lane MV), identically-labeled CV-1_C cells acutely-infected with the BBR strain of Cryptovirus (Lane CV) or a mixture of both (Lane B) by the CSF of an 11 year male SSPE patient Lane V represents a SDS-PAGE profile of [³⁵S]-methιonιne-labeled gradient purified Cryptovirus virions (see also Fig 11) Fig 12B shows the RIPs resulting from the precipitation of proteins from [³⁵S]- methionine-labeled CV-lc cells acutely-infected with the BBR strain of Cryptovirus by the CSFs of six neurology/neurosurgery patients The patient whose RIP profile appears in Lane 2 was an adult male who had presented with ataxia, confusion and memory loss and had not been given a specific diagnosis The patient whose RIP profile appears in Lane 4 was an infant female who presented with hydrocephalus and intractable seizures and subsequently died in status epilepticus None of the CSFs from the patients in Fig 12B precipitated any of the envelope proteins of measles vims (data not shown) Fig 12A is the same as Fig 23 (described below), but is reduced to the same scale as Fig 12B for the purpose of comparison

Fig 13 shows a higher resolution autoradiogram of the radioimmunoassay profiles (RIPs) of the Cryptovirus -specific proteins precipitated from [³⁵S]-methιonιne-labeled CV-lc cells acutely- infected with the BBR strain of Cryptovirus by two CSF specimens (Fig 13 A) and a schematic showing the migration of the major corresponding stmctural proteins of gradient-purified virions of the BBR strain of Cryptovirus (Fig 13B Lane CV) and the NIH 21005-WR strain of SV5 (Fig 13B Lane SV5) The RIPs in Fig 13A represent CSF precipitates from patients assessed as Cryptovirus- negative (Lane "— ", I e not containing Cryptovirus-specific antibodies) and Cryptovirus -positive (Lane "+" , I c containing Cryptovirus-specific antibodies) Fig 13B is a schematic showing the near co-migration of the Fo and HN proteins of Cryptovirus and their separate migration in Simian Vims 5 (see also SDS-PAGE profiles in Fig 5)

Fig 14 shows an ELISA of matched semm and CSF specimens from four seropositive neurology/neurosurgery patients using gradient-purified Cryptovirus virions as the target Control sera were rabbit antisera generated against mock-infected CV-lc cells (column 1, "-") and hypeπmmunc rabbit antisera generated against gradient-purified Cryptovirus virions (column 2, "+") FN = infant female diagnosed with hydrocephalus and intractable seizures, SG = adult female diagnosed with idiopathic lntracranial hypertension, WK = male child diagnosed with acute viral meningitis, JK = adult male having an undetermined diagnosis Semm dilutions began at 1 20 (in the top rows) and proceeded by 2-fold serial dilution to the bottom CSF dilutions began at 1 2, at the top, before proceeding likewise Semm specimens were aliquoted from left to right while CSF specimens were aliquoted from right to left Note that although all of the patients had Cryptovirus- specific antibodies in their semm, only the patient with a seizure disorder (FN) had such antibodies in her CSF Fig 15 is a photograph of RIP assays using three sets of matched semm (S) and CSF (C) samples from patients diagnosed with Alzheimer's disease Fig 16 is a photograph of an autoradiogram following an RIP analysis using four CSF specimens from patients diagnosed with chronic fatigue syndrome (CFS). Lanes 1-3 were assessed as "Cryptovirus positive"; Lane 4 assessed as "Cryptovirus negative".

Fig. 17 is a photograph of an autoradiogram following an RIP analysis using CSF samples obtained as "Collection 1" (see hereinbelow). The positive CSF precipitate in Lane 2 was subsequently found to have been obtained from a 55 year-old adult male who presented with ataxia, memory loss, blackouts, seizures, diplopia, and headaches.

Fig. 18 is a photograph of an autoradiogram following a RIP analysis using CSF samples obtained as "Collection 2" (see hereinbelow). Fig 19 is a photograph of an autoradiogram following an RIP assay conducted with semm samples obtained from 5 MS patients (out of the 38 samples obtained).

Fig. 20 is a photograph of an autoradiogram following an RIP assay conducted with semm samples from an 25 additional MS patients (out of the 38 samples obtained).

Fig. 21 is a photograph of an autoradiogram following an RIP assay conducted with 16 CSF specimens obtained from 16 MS patients

Fig. 22 is a photograph of an autoradiogram obtained following creation of RIP profiles of the Cryptovirus NP protein (p63) precipitated from [³⁵S]-mcthionine-labeled AV₃/SSPE cells by the sera of six Australian SSPE patients (Lanes 1-6) and six control sera (Lanes 7-12; sera from pediatric patients without antibodies to the Cryptovirus major envelope proteins (F₀ and HN) Fig. 23 is a photograph of an autoradiogram of RIP profiles of measles vims-specific proteins or Cryptovirus-specific proteins precipitated from [³⁵S]-methionine-labeled measles vims-infected CV-lc cells (Lane MV), Cryptovirus-infected CV-1_C cells (Lane CV) or a mixture of both (Lane B) by CSF from an 1 1 year old male diagnosed with SSPE. Lane V = gradient-purified Cryptovirus virions from [³⁵S]-methionine-labeled Cryptovirus-infected CV-lc cells. L = the largest nucleocapsid associated protein, the major component of the virion-associated RNA dependent RNA polymerase;

HN = the hemagglutinin protein, one of the envelope-associated glycoprotcins; F₀ = the uncleaved fusion protein, a second envelope-associated glycoprotein; NP = the nucleocapsid protein, the major stmctural protein associated with the nucleocapsid; Fj = the larger fragment of the cleaved fusion protein; P = the nucleocapsid associated phosphoprotein; M = the virion-associated matrix or membrane protein; V = a minor RNA binding protein thought to be a component of the viral polymerase, F₂ = the smaller fragment of the cleaved fusion protein Note the SH protein (about 5 kD), a small envelope-associated protein, ran off the gel and is not shown

Fig 24 shows photomicrographs of Cryptovirus -infected neurons Fig 24 A demonstrates Cryptovirus -specific immunofluorescence in a single neuron in the brain of a Colored mouse inoculated when two days old with Cryptovirus Strain BBR (sacrificed 2 months post inoculation after presenting with seizures) Fig 24B demonstrates cytoplasmic immunofluorescence in a single neuron from the brain of a guinea pig presenting with a subacute encephalopathy after inoculation with the Nugata-l strain of SSPE-denved cell-associated vims (detected by an indirect fluorescent antibody technique using SSPE serum)(Doι et al , Japan J Med Sci Biol 25 321-333, 1972) Fig 25 shows photomicrographs of differential lmmunogold-labeling of the intracellular nucleocapsids of Cryptovirus and measles vims in persistently-infected AV₃/SSPE/MV cells Fig 25 A shows labeling of the about 15-nm to about 17-nm "smooth" and narrow nucleocapsids of Cryptovirus with 10-nm gold beads The etching technique used results in a loss of resolution of the fine structure of the smooth nucleocapsids making the herringbone pattern somewhat difficult to sec Fig 25 B shows labeling of the 25-nm "fuzzy" and wide nucleocapsids of measles vims with 15-nm gold beads Magnification is approximately 500,000X

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The novel vims described herein has been designated a Cryptovirus (CV) on the basis of its lnapparcnt, or "cryptic," cytopathology in cultured human cells and its slow and "encrypted" pathogenesis in experimentally infected animals Given the nucleotide sequence present in the vims, and its stmctural, biological, and pathogenetic characteristics, Cryptovirus fits best within the Rubulavirus genus of the family Paramyxoviridae (Fig 1) More specifically, this vims most closely resembles the viruses known as Canine Parainfluenza Vims Type 2 (which is also known as Canine Parainfluenza Vims, CPI and CP1V) and Porcine Rubulavirus (which is also known as La-Piedad- Michoacan-Mexico Vims and LPMV) (see Fig 2) The relationships between Cryptovirus and these two vimses can be seen in the relationships between their sequences and their stmctural, biological, and pathogenetic characteristics (sec Fig 3) CPIV shares more than 95% of its nucleotide sequence with Cryptovirus The extent of Menangle vims nucleotide sequence homology with Cryptovirus is presently unknown as the sequence of the Menangle vims genome has not yet been published There is also an apparent relationship between Cryptovirus and (1) simian parainfluenza vims type 5 (which is also known as simian vims 5 and SV5, see Fig 4, Fig 5, Fig 9, and Fig 10, here, there is a relationship between the sequences and stmctural and immunological properties of the vimses but little or no biological or pathogenetic similarity); (2) human mumps vims (here, there are certain stmctural, biological, and pathogenetic relationships); and (3) human measles vims (here again, there are certain stmctural, biological and pathogenetic relationships). These relationships help to classify Cryptovirus and to establish its novelty. In addition to having a role in idiopathic and cryptogenic forms of epilepsy or epileptiform disease, i.e , an illness, disorder, or condition having epileptiform symptomology (e.g., CFS, MS, SSPE), Cryptovirus is also implicated in a spectrum of idiopathic disorders of the central nervous system (CNS) that present with compulsive or iterative physical, behavioral, or psychological symptoms The manifestation of symptoms of these disorders as a consequence of Cryptovirus infection is exclusively subacute or slow in nature taking weeks, months, or even years to develop.

The spectmm of physical symptoms that have been presented by human patients that have been infected with Cryptovirus includes febrile response, opthalmological disorders (photosensitivity, blurred vision, nystagmus, loss of vision) parathesias, paralysis, tremor, myoclonus, and grand mal and petit mal (absence) seizures. The spectmm of behavioral or psychological symptoms that have been presented by patients includes repetitive movements and compulsive behaviors (characteristic of obsessive compulsive disorder), sleep disturbances, memory loss, and dysophoria, anorexia nervosa, autism, mental retardation, affective disorder, dysthymia (clinical depression), schizophrenia, and bipolar disorder.

While not essential features of the present invention, the portal of entry for Cryptovirus infection can be the oral mucosa of the throat (i e., the tracheo-bronchial epithelium), and the vims' incubation period can be of subacute duration (i.e., many days to weeks). Newly infected individuals can develop a febrile pharyngitis and lymphadenopathy of prolonged duration, not unlike infectious mononucleosis. Alternatively, it is thought that the portal of entry for the vims can also be transplacental, so that a mother carrying the vims can transmit it to her child in utero, and the child can subsequently develop a neurological, neurodegenerative, and/or neuropsychiatric disease or other developmental disorder (e.g., autism, cerebral palsy, hydrocephalus, birth defect, partial paralysis). Such a child is frequently diagnosed or labelled as "retarded". Indeed, the incidence of epilepsy and seizures are dramatically higher in the severely "mentally-retarded" (as much as 50-fold higher than the general population). The nucleotide sequence of the human Cryptovirus genome (15,246 contiguous nucleotides), in FASTA format (i.e., mRNA sense, 5' to 3'), is shown in Fig. 9 (SEQ ID NO:l). The actual genome of the vims is negative-stranded (antisense to mRNA), having a nucleotide sequence entirely complementary to (SEQ ID NO. l). Accordingly, the present invention encompasses an isolated human negative-stranded RNA vims that, in FASTA format (;. e. in positive-stranded, mRNA-sense, the reverse and complementary sequence to the actual genome), has the sequence of SEQ ID NO:l. In Fig. 9, nucleotides that vary from those of the W3A strain of Simian Vims 5 are highlighted and the number of variations in each line is tallied in the right margin. The FASTA formatted sequence of human Cryptovims Strain BBR was compared to Simian Vims 5 Strain W3A (SEQ ID NO:2; see Fig. 9). Comparisons between various Rubulavims F Protein amino acid sequences have also been made (Fig 10).

A Cryptovirus "particle" is an entire Cryptovirus virion, as well as encompassing particles which are intermediates in virion formation (e.g., nucleocapsids), or otherwise partial. Cryptovirus particles generally have one or more Cryptovirus proteins associated with the Cryptovirus-specific nucleic acid they contain. A preferred Cryptovirus particle or virion is Cryptovirus Strain BBR, which is deposited as ATCC Accession No.

The present invention also relates to a composition of matter comprising the inventive Cryptovims particle and a carrier. As used herein a "carrier" can be an organic or an inorganic carrier or excipient, such as water or an aqueous solution, or an emulsion such as an oil/water or water/oil emulsion, and various types of wetting agents. The active ingredient, such as the inventive viral particle, nucleic acid constaict, protein, or antibody, can optionally be compounded in a composition formulated, for example, with non-toxic, physiologically acceptable carriers for infusions, tablets, pellets, capsules, solutions, emulsions, suspensions, or in any other formulation suitable for its intended in vitro or in vivo use. Such carriers also include glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, com starch, keratin, colloidal silica, potato starch, urea, medium chain length triglycerides, dextrans, normal saline, phosphate buffered saline and other carriers suitable for use in manufacturing preparations, in solid, semisolid, or liquid form. In addition auxiliary, stabilizing, thickening and coloring agents and perfumes can be used as appropriate. Other example of suitable carriers are described hereinbelow, but any suitable carrier known in the art is intended.

In the inventive method of isolating the Cryptovirus virion, as described hereinabove, the PBMNCs that have been obtained from a human having a Cryptovirus infection are cultured in an artificial aqueous medium that includes, importantly, an agent that increases cellular guanylyl cyclase activity.

For purposes of the present invention, the artificial aqueous medium is made by adding the agent that increases cellular guanylyl cyclase activity to a known minimal cell culture medium, such as IMEMZO, MEM, HYQPF Vcro (Hyclone), or RPMI, buffered (e.g , with HEPES) to pH 6.8-7.8 and most preferably to pH 6.8-7.2. The agent operates to permit and facilitate the isolation and/or propagation of Cryptovims in accordance with the invention.

Optionally, fetal calf semm (about 2% v/v to about 10% v/v) is added to the medium. Antibiotics, such as penicillin or streptomycin, in conventional amounts, can also be added to the medium.

It is not essential to the present invention that cellular guanylyl cyclase activity actually be measured. In addition, the present invention is dependent neither upon any particular mechanism by which the agent may actually operate to increase cellular guanylyl cyclase activity (or not), nor upon any mechanism by which the agent operates to permit and/or facilitate the isolation and/or propagation of Cryptovims in accordance with the invention.

Useful examples of the agent that increases cellular guanylyl cyclase activity include most preferably guanosine 3',5'-cyclic monophosphate ("cyclic GMP") (free acid, or preferably, a pharmaceutically acceptable salt thereof, such as a sodium, potassium, magnesium, calcium, or ammonium salt, or the like), insulin (preferably human insulin), zinc dication (preferably provided in a chloride, sulfate, carbonate, bicarbonate, nitrate, acetate, or other pharmaceutically acceptable salt thereof), or a combination of any or all of these.

Preferably, the cyclic GMP is used in a concentration of about 0.05 to about 5 mM in the artificial aqueous medium. More preferably, the cyclic GMP concentration in the medium is about

0.5 to about 2.5 mM, and most preferably about 0.75 mM to about 1.25 mM. A concentration above about 5 mM cyclic GMP is not optimally conducive to cultivating, propagating, or isolating

Cryptovirus.

A preferred concentration range for insulin in the artificial aqueous medium is about 1 to about 10 mg/L, more preferably about 2 to about 6 mg/L, and most preferably about 3 to about 5 mg/L. A preferred concentration range for zinc dication in the artificial aqueous medium is equivalent to about 0.05 to about 0.25 mg/L of ZnS0₄-7H₂0, more preferably equivalent to about 0.10 to about 0 20 mg/L of ZnS0₄-7H₂0, or most preferably equivalent to about 0.13 to about 0.15 mg/L of ZnS0₄ 7H₂0.

Alternatively, in some embodiments, the agent that increases cellular guanylyl cyclase activity is nitric oxide or a nitric oxide donor. Nitric oxide gas is fully permeable across biological membranes. Inhalable nitric oxide gas can be administered to a mammalian subject by, for example, a mask in a controlled gas mixture as is known in the art. (E.g., Kieler- Jensen, N. et al, Inhaled nitric oxide in the evaluation of heart transplant candidates with elevated pulmonary vascular resistance, J Heart Lung Transplant 13(3) 366-75 [1994], Rajek, A et al , Inhaled nitric oxide reduces pulmonary vascular resistance more than prostaglandin E(l) during heart transplantation, Anesth Analg 90(3) 523-30 [2000], Sohna, A et al , A comparison of inhaled nitric oxide and milrinone for the treatment of pulmonary hypertension in adult cardiac surgery patients, J Cardiothorac Vase Anesth 14(1) 12-17 [2000], Fullerton, D A et al , Effective control of pulmonary vascular resistance with inhaled nitric oxide after cardiac operation, J Thorac Cardiovasc Surg 111(4) 753-62, discussion 762-3 11996]) The concentration in the gas mixture of nitric oxide (NO) is preferably about 1 to 100 ppm NO, more preferably about 4 to 80 ppm NO, and most preferably about 20 to 40 ppm NO The gas mixture also contains appropriate concentrations of oxygen and nitrogen and or other inert gases, such as carbon dioxide, helium or argon

Nitric oxide donors are compounds that produce NO-related physiological activity when applied to biological systems Thus, NO-donors can mimic an endogenous NO-rclated response or substitute for an endogenous NO deficiency The skilled artisan is aware that in biological systems there are at least three redox states of NO that can be released by various NO donors (NO⁺, NO⁰, or NO ), all of which are encompassed by the terms "nitric oxide" or "NO" for purposes of the present invention The redox state of NO makes a substantial difference to the NO donors reactivity towards other biomolecules, the profile of by-products, and the bioresponse (Feehsch, M , I he use oj nitric oxide donors in pharmacological studies, Naunyn-Schmiedebergs Arch Pharmacol 358 1 13-22 [1998]) Some classes of NO donors require enzymatic catalysis, while others produce NO non-enzymatically, some NO donors require reduction, for example by thiols, and some oxidation, m order to release NO

Preferred examples of nitric oxide donors include organic nitrate compounds, which are nitric acid esters of mono- and polyhydπc alcohols Typically, these have low water solubility, and stock solutions are prepared in ethanol or dimethyl sulfoxide (DMSO) Examples are glyceryl tπnitrate (GTN) or nitroglyceπn (NTG), pentaerythrityl tetranitrate (PETN), isosorbide dinitrate (ISDN), and isosorbide 5-mononιtrate (IS-5-N) Administration of organic nitrates can be done intravenously, intraperitoneal ly, intramuscularly, transdermally, or in the case of PETN, ISDN, NTG, and IS-5-N, orally

Other preferred examples of nitric oxide donors are S-mtrosothiol compounds, including S-nitroso-N-acetyl-D L-penicillamine (SNAP), S-nitrosoglutathione (SNOG), S-nitrosoalbumin,

S-mtrosocysteine S-nitrosothiol compounds arc particularly light-sensitive, but stock solutions kept on ice and m the dark are stable for several hours, and chelators such as EDTA can be added to stock solutions to enhance stability. Administration is preferably by an intravenous or intra-arterial delivery route.

Other preferred examples of nitric oxide donors include sydnonimine compounds, such as molsidomine (N-ethoxycarbonyl-3-morpholino-sydnonimine), linsidomine (SIN-1; 3-morpholino-sydnonimine or 3-morpholinylsydnoneimine or

5-amino-3morpholinyl-l,2,3-oxadiazolium, e.g., chloride salt), and pirsidomine (CAS 936). Stock solutions are typically prepared in DMSO or DMF, and are stable at 4°C to room temperature, if protected from light. Linsidomine is highly water soluble and stable in acidic solution in dcoxygenated distilled water, adjusted to about pH 5, for an entire day. At physiological pH, SIN-1 undergoes rapid non-enzymatic hydrolysis to the open ring form SIN-1 A, also a preferred nitric oxide donor, which is stable at pH 7.4 in the dark. Administration is preferably by an intravenous or intra-arterial delivery route.

Also useful as nitric oxide donors are iron nitrosyl compounds, such as sodium nitropmsside (SNP; sodium pentacyanonitrosyl ferrate(II)). Aqueous stock solutions are preferably made freshly in deoxygcnated water before use and kept in the dark; stability of stock solutions is enhanced at pH

3-5. Inclusion in the delivery buffer of a physiologically compatible thiol, such as glutathione, can enhance release of NO. SNP is administered by intravenous infusion, and the skilled practitioner is aware that long-term use is precluded by the release of five equivalents of toxic CN-pcr mole SNP as NO is released. A most preferred nitric oxide donor is chosen from among the so-called NONOate compounds. The NONOates are adducts of NO with nucleophilic residues (X^"), such as an amine or sulfite group, in which an NO dimer is bound to the nucleophilic residue via a nitrogen atom to form a functional group of the stmcture X[-N(0)NO]\ The NONOates typically release NO at predictable rates largely unaffected by biological reactants, and NO release is thought to be by acid-catalyzed dissociation with the regeneration of X^" and NO.

NONOates include most preferably diethylamine-NONOate (DEA/NO; N-Ethylethanamine: 1,1 -Diethyl-2-hydroxy-2-nitrosohydrazine (1 : 1) or l-[N,N-diethylamino]diazen-l-ium-l,2-diolate). Other preferred NONOates include diethylene triamine-NONOate(DETA/NO; 2,2'-Hydroxynitrosohydrazino]bis-ethanamine), sperminc-NONOate (SPER/NO; N-(4-[-l-(3-Aminopropyl)-2-hydroxy-2-nitrosohydrazino] butyl)- 1 ,3-propanediamine), propylamino-propylamine-NONOate (PAPA/NO;

3-(2-Hydroxy-2-nitroso-l-propylhydrazino)-l-propanamine or (Z)-l-[N-(3-aminopropyι)-N- (n-propyl)amιno]dιazen-l-mm-l,2-dιolate), MAHMA-NONOate (MAHMA/NO,

6-(2-Hydroxy- 1 -methyl-2- nιtrosohydrazιno)-N -methyl- 1 -hexanamine), dφropylenetπamine- NONOate (DPTA/NO, 3,3'- (Hydroxynιtrosohydrazιno)bιs-l-propanamιne), PIPERAZI/NO, proh-NONOate (PROLI/NO, l-([2-carbo\ylato]pyrrohdιn-l-yl)dιazen-l-ιum-l ,2-dιolate-methanol, e g , disodium salt), SULFO-NONOate (SULFO/NO, hydroxydiazenesulfomc acid 1 -oxide, e g , diammomum salt), the sulfite NONOate (SULFI/NO) and Angehs salt (OX1/NO)

Almost all NONOate compounds are highly soluble in water, and aqueous stock solutions are prepared in cold deoxygenated 1 to 10 mM NaOH (preferably about pH 12) just prior to use Alkaline stock solutions are stable for several hours if kept on ice in the dark The characteristic UV absorbance of NONOates can be used for spectrophotometπc quantification of NONOate in aqueous solutions NONOates are preferably administered intravenously or lntra-arteπally

Nitric oxide donors have different potencies (Ferraro, R et al , Comparative effects of several nitric oxide donors on intracellular cyclic GMP levels in bovine chromaffln cells correlation with nitric oxide production, Br J Pharmacol 127(3) 779-87 [1999]) For example, DEA NO is among the most potent nitric oxide donors, with a half-life of about 2 to 4 minutes, less potent are PAPA/NO (tι ₂ about 15 mιnutes),SPER/NO (t_)/2 about 34-40 minutes), even less potent are DETA/NO (t_υ2 about 20 hours) and SNAP (tm about 33 to 41 hours, although this can be shortened in the presence of a physiological reductant such as glutathione) SNP is also a potent NO donor (See, Ferrero et al [1999], Salom, J B et al , Relaxant effects of sodium nitroprusside and NONOates in rabbit basilary artery, Phaπnacol 57(2) 79-87 [1998], Salom, J B et al , Comparative relaxant effects of the NO donors sodium nitroprus side, DEA/NO and SPER/NO in rabbit carotid arteries, Gen Pharmacol 32(1) 75-79 [1999], Salom, J B et al , Relaxant effects of sodium nitroprus side and NONOates in goat middle cerebral artery delayed impairment by global ischemia-reperfusion, Nitric Oxide 3(1) 85-93 [1999], Kimura, M et al , Responses of human basilar and other isolated arteries to novel nitric oxide donors, J Cardiovac Pharmacol 32(5) 695-701 [1998]) Consequently, effective concentrations or doses of NONOates or other NO donors will vary, but can be determined by routine screening

Stock solutions of NO donors are preferably made up freshly before use (at the appropriate pH for each particular NO donor), chilled on ice, and protected from light (e g , by the use of darkened glass vials wrapped in aluminum foil), although organic nitrates can be stored for months to years if the vial is properly sealed Preferably, immediately before administration to the subject, final dilutions are prepared in pharmaceutically acceptable buffer and the final pH of the NO donor-containing buffer is checked for physiological suitability, especially when strongly acidic (e.g , hydrochloride salts) or alkaline (e.g., NONOates) stock solutions are used.

The product of NO exposure time and NO concentration largely determines the quality and magnitude of the biological response to exogcnously supplied NO. Short-lived NO donors, such as DEA/NO, are most preferably administered by continuous infusion rather than by bolus to avoid delivering only a short burst of NO.

In accordance with the invention, the artificial aqueous medium preferably, but not necessarily, further includes glutamine at a preferred concentration of about 0 5 to about 5 mM concentration. A more preferred concentration of glutamine in the medium is about 1 to about 3 mM. In the inventive methods of isolating a Cryptovirus virion and of producing a mammalian cell line nonproductively infected with Cryptovirus, the PBMNCs are co-cultured with mammalian amnion cells in the artificial aqueous medium, as described above

Examples of useful mammalian cells include, but arc not limited to, rodent, lagomorph, primate, ovine, bovine, canine, feline or porcine cells. In accordance with the present invention, one preferred embodiment is a primate cell, i e , a cell originating from a primate source. A primate is a member of the mammalian order Primates, including lemurs, tarsiers, monkeys (e.g., African Green Monkeys, colobus monkeys, and baboons), apes (e.g., chimpanzees, gorillas, orangutans, and gibbons), and humans.

An amnion cell is a cultured cell originally derived from an amniotic membrane or amniotic sac.

A preferred primate amnion cell is a human amnion cell, e.g., AV₃.

An example of the inventive cell nonproductively infected by the method is AV3/SSPE, which is deposited as ATCC Accession No. .

In these inventive methods, passaging of a co-culture of PBMNCs and mammalian amnion cells is done one or more times. Passaging of cultured cells into fresh culture medium (culture medium as described above), is typically done about twice per week. Preferably, at least about two to about 12 passages are done in accordance with the methods. Typically after about eight to twelve passages of the co-culture, virtually all mammalian amnion cells are nonproductively infected with Cryptovirus. Generally, within about two to about three passages, the PBMNCs have disappeared from the culture, leaving the mammalian amnion cells.

In accordance with the present invention, a mammalian epithelial cell is a cultured cell originally derived from a mammalian epithelial tissue. In one preferred embodiment, the mammalian epithehl cell is a rodent epithelial cell, such as baby hamster kidney (BHK) cells. In another preferred embodiment, the mammalian epithelial cell is a simian epithelial cell, for example a Vero or a CV-1 cell Most preferably the CV-1 cell is subline CV-1_C, which is deposited as ATCC Accession No. .

An example of a primate epithelial cell acutely infected with Cryptovirus, in accordance with the method of propagating a Cryptovirus is deposited as ATCC Accession No. . In addition to the sequence information provided herein to identify the inventive Cryptovirus particle, the inventive Cryptovirus and its viral subcomponents can be, and have been, characterized by numerous virological, biochemical, and molecular techniques, including the following, by way of example'

Plaque Titration Assay- Formation of macroscopically visible plaques on monolayers of mammalian epithelial cells (e.g , BHK, Vero or CV-lc) can be used to quantitate preparations of infectious Cryptovirus (Robbins et al, J. Infect. Disease 143:396-403. 1981).

Neutralization Titration Assay: Plaque formation can be inhibited by serial dilutions of clinical seaun specimens and Cryptovirus-specific antisera generated in rabbits (see e.g., Robbins et al, J. Infect. Disease 143:396-403, 1981). Neutralization titration assays are routinely used in medical virological research to demonstrate that a given patient has neutralizing antibodies to a particular viais. A neutralization assay can be used diagnostically for the presence or absence of neutralizing antibody to Cryptovirus. In a typical example of a neutralization assay, serial dilutions of a biological material, such as a sample of semm or CSF to be tested, are typically incubated for about one hour at 4°C with sufficient infectious vims to yield a net plating concentration of between about 100-200 plaque forming units of the vims per 0.2 mL of final diluent (including the diluted semm or CSF). After incubation, about 0.2 mL of the diluted virus-serum (or CSF) mixtures are then typically plated onto monolayers of susceptible cells (e.g. Vero or CV-1) and the cells are incubated at 37°C in a partial C0₂ atmosphere (e.g., 5% v/v) (typically, with redistribution of the inoculum every 15 minutes). At the end of the incubation period, inoculated monolayers are typically overlayed with sufficient volumes of a 2% (w/v) solution of carboxymethylcellulose in an artificial aqueous cell culture medium (e.g., IMEMZO medium, buffered at pH between about 6.8 and about 7.4, typically containing fetal calf seaim, and a suitable quantity of antibiotic, such as about 200 Units penicillin / mL and/or 100 μg streptomycin / mL) to last 10-12 days (i.e., enough volume so that the monolayers won't dry out). Optimally, the plates must not be moved during the incubation period. After 10-12 days, the overlay is aspirated and the cells are fixed with formalin fixative and stained with a protein stain (e.g , Giemsa). The number of plaques formed on each plate is then enumerated and the PRD50 calculated (PRD₅₀ = the Plaque Reduction Dilution; the dilution of seaim or CSF at which a 50% reduction in the number of plaques formed on controls [tubes containing viais and saline only] is observed).

Density Gradient Purification: Virions and intracellular nucleocapsids from productively- (e.g., Vero and CV-lc) and nonproductively-infected (e.g., AV₃/SSPE) cells can be purified on sucrose-potassium tartrate gradients (virions) and CsCl gradients (nucleocapsids) (see Robbins et al,

J. Infect. Disease 143:396-403, 1981; Rapp and Robbins, Intervirology 16: 160-167, 1981; Robbins and Rapp, Arch. Virol. 71 :85-91, 1982; and Robbins and Abbott-Smith, . Virol. Meth. 11 :253-257, 1985).

Electron Microscopy: Electron microscopy, such as transmission or scanning electron microscopy are useful for examining the inventive Cryptovirus virion. When examined by electron microscopy, the Cryptovirus has been shown to have a morphology and ultrastmcture consistent with other members of the Paramyxoviridae (i.e., enveloped pleomorphic virions, about 100 nm to about 120 nm in diameter, containing helical nucleocapsids). Intracellular inclusions of the vims in thin sections of productively- (e.g., Vero and CV-1_C) and nonproductively-infected cells (e.g., AV3/SSPE) have also been shown to be comprised of aggregates of filamentous structures with dimensions similar to the nucleocapsids of other members of the Paramyxoviridae (i.e., helical herringbone-like stmctures, about 15 to about 17 nm in diameter) (see Robbins et al, J. Infect. Disease 143:396-403. 1981 and Robbins and Rapp, Arch. Virol. 71:85-91, 1982).

Radioimmunoprecipilation (RIP) Assay: Extensive data has been generated by the comparative analysis of Cryptovirus-specific immunoprecipitates of [ Sj-methionine-labeled uninfected, nonproductively- and productively-infected mammalian cells by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; see below).

SDS-PAGE: Purified virions and cytoplasmic nucleocapsids of the vims have been analyzed by SDS-PAGE under reducing and non-reducing conditions (see Fig. 11 and Fig. 12), an autoradiogram of gradient-purified [³⁵S]-methionine-labeled Cryptovirus virions produced in acutely- infected Vero cells after SDS-PAGE under reducing conditions. The approximate molecular weights of the proteins indicated on the right side of Fig. 11 were calculated by comparing their migrations to marker proteins of known molecular weights (Sigma Chemical Co., St. Louis, MO). The SH protein, a small envelope-associated protein having a MW of about 5 kD, is not shown in Fig. 11, because it has mn off the gel.

Immuno-ultraslructural Analysis (Immunogold Analysis). The intracellular nucleocapsids of nonproductively and productively-infected mammalian cells are typically localized under the electron microscope using Cryptovirus -specific or Cryptovirus nucleocapsid-specific antibodies, for example hypenmmune rabbit antibodies and an indirect immunogold labeling technique (discussed hereinbelow)

The preceding are merely illustrative, and not an exhaustive list, of the known techniques typically useful for characterizing the isolated Cryptovirus virion of the present invention

Additional conventional techniques, or virological techniques yet to be discovered, can also be employed to further characterize the inventive Cryptovirus virion

Additional characteristics of Cryptovirus include the following

Latency and Persistence Cryptovirus latently and persistently infects human peripheral blood mononuclear cells (PBMNCs) No other member of the Paramyxoviridae has been shown to do this The evidence for infection includes (1) detection of Cryptovirus-specific proteins by an indirect lmmunofluorescent antibody technique in PBMNCs following in vitro cultivation and induction with mitogens and/or cyclic GMP, (2) recovery of the vims from PBMNCs by serial cocultivation with mammalian cells (see Robbins et al , J Infect Dis 143 396-403, 1981) and (3) the ability to repeatedly recover the vims from PBMNCs drawn from an SSPE patient over a period of 18 months

Defective Fusion Activity Cell fusion, which is a hallmark of the Paramyxoviridae, is either defective or extremely limited in experimental Cryptovirus infections in vitro (i e , in dysgenic nonproductive infections of human amnion cells (AV₃) and productive infections of monkey kidney cells (e g Vero and CV-1_C, see Robbins et al , J Infect Dis 143 396-403, 1981, and Robbins and

Rapp Arch Virol 71 85-91, 1982)

Restricted Expression in Latently-Infected Cells Cryptovirus-specific protein expression is dysgenic in experimental nonproductive latent infections of mammalian amnion cells (e g , human AV₃ cells) This restriction involves severely decreased expression, or non-expression, of the viais- encoded envelope proteins (F, HN and SH) (see Robbins and Rapp, Arch Virol 71 85-91, 1982)

B Cell Lymphotropism. Cryptovirus demonstrates a tropism for B cells, and can be harbored by such cells in situ This has been demonstrated by successfully infecting EBV-transformcd B cell lines from human donors with the viais (i e , by detecting the progressive formation of Cryptovirus- specific inclusion bodies in the cytoplasm of experimentally-infected EBV-transformed B cell lines by Cryptovirus -specific immunofluorescence) In contrast, Cryptovirus -specific proteins could not be detected in an experimentally-infected human T cell line, CCRF-CEM Accordingly, Cryptovirus can reside in B cells in infected individuals Neurotropism. Cryptovirus also demonstrates a clear tropism for neurons in mice following intracerebral inoculation of neonatal animals (as detected by Cryptovirus-specific immunofluorescence) It is less clear whether other nervous system tissues are infected While neurotropism, itself, may not be unique to Cryptovirus when compared and contrasted to other human members of the Paramyxoviridae (e.g., Measles Vims, Mumps Vims), some of the neuropathological consequences of Cryptovirus infection of CNS tissues (including neurons) appear to be (see below).

Hind Limb Atrophy and Paralysis. Hind limb paralysis and atrophy were seen in approximately 33% of Quackenbush mice intracerebrally-inoculated with Cryptovirus as newborns. In addition, hind limb atrophy and paralysis was observed in some of the offspring of adult female Quackenbush mice that had been inoculated with Cryptovirus as newborns but did not develop any overt symptomology. The frequency of the symptoms appearing in the latter situation was difficult to assess because the mothers tended to cannibalize the newborn animals that were bom with, or subsequently developed, such characteristics.

Subacute/Slow Encephalopathic and Epileptigenic Potential Approximately 30% of neonatal Colored mice that were inoculated with infectious Cryptovirus preparations went on to develop subacute/slow encephalopathic and/or epileptiform illness (the specific symptoms displayed by such animals are described below). The number of animals that actually developed encephalopathic and/or epileptiform illness was likely higher than 30%, because a number of previously asymptomatic animals were found dead in their cages in clonic postures (a symptom associated with death following or during intractable seizure). On at least two occasions, this occurred in animals after they had suffered from recurrent seizures the day before The animals that developed such illnesses were predominantly male (approximately 2: 1, male: female).

Slow Psychopalhogenic Potential Of the Colored mice that survived intracerebral inoculation with infectious Cryptovirus preparations as newborns and did not develop epileptiform illness as adolescent or young adult animals, approximately 30% went on to develop profound physical and behavioral abnormalities as adults. The abnormalities displayed by these animals arc described hereinbelow Sudden death was not seen in this group of animals. The animals which developed such symptoms were predominantly female (approximately 3: 1, female: male).

The preceding are merely illustrative, and are not an exhaustive list, of some of the observable properties of the inventive Cryptovirus particle.

The present invention also relates to isolated nucleic acids and isolated proteins that are "Cry/?/ov//7<.y-specific," i.e., unique to Cryptovirus. A Cryptovirus -specific nucleic acid segment or protein is determined by sequence similarity or homology to known sequences of bases or amino acids, respectively, for example other rubulavirus nucleic acid or protein sequences Base and amino acid sequence homology is determined by conducting a sequence similarity search of a genomics/proteomics data base, such as the GenBank database of the National Center for Biotechnology Information (NCBI, www ncbi nlm nih gov/BLAST/), using a computerized algorithm, such as PowerBLAST, QBLAST, PSI-BLAST, PHJ-BLAST, gapped or ungapped BLAST, or the "Align" program through the Baylor College of Medicine server (www hgsc bcm tmc edu/seq_data) (E g , Altchul, S F , et al , Gapped BLAST and PSI-BLAST a new generation of protein database search programs, Nucleic Acids Res 25(17) 3389-402 [1997], Zhang, J , & Madden, T L , PowerBLAST a new network BLAST application for interactive or automated sequence analysis and annotation, Genome Res 7(6) 649-56 [1997], Madden, T , et al , Applications of network BLAST server, Methods Enzymol 266 131-41 [1996], Altschul, S F , et al , Basic local alignment search tool, J Mol Biol 215(3) 403-10 [ 1990])

For purposes of the present invention the term "isolated" encompasses ^"'purified" Thus, an isolated nucleic acid, protein, viral particle, or antibody that is further purified to a greater level of homogeneity, is also "isolated "

For purposes of the present invention the term "nucleic acid" includes a polynucleotide, of any length, either polymeric ribonucleotidcs (RNA) or polymeric deoxyπbonucleotidcs (DNA), such as cDNA The term "isolated nucleic acid" refers to a Cryptovirus genomic RNA which is essentially free, I e . contains less than about 50%, preferably less than about 70%, and even more preferably less than about 90% of the polypeptides with which the Cryptovirus genome is naturally associated Alternatively, an "isolated" nucleic acid of the present invention is a Cryptovirus -specific "recombinant polynucleotide", which as used herein intends a polynucleotide of genomic RNA, sense RNA (I e , mRNA sense), cDNA, semisynthctic, or synthetic origin, which, by virtue of its origin or manipulation (1) is not associated with all or a portion of a polynucleotide with which it is associated in nature, (2) is linked to a polynucleotide other than that to which it is linked in nature, or (3) does not occur in nature The inventive nucleic acid can be in a sense or antisense orientation

As used herein, the "sense strand" of a nucleic acid contains the sequence that has sequence homology to that of mRNA The "anti-sense strand" contains a sequence which is complementary to that of the "sense strand " Inventive nucleic acids also include double- and single-stranded DNA and RNA Techniques for purifying viral polynucleotides from viral particles are known in the art, and include for example, dismption of the particle with a chaotropic agent, differential extraction and separation of the polynucleotide(s) and polypeptides by ion-exchange chromatography, affinity chromatography, and sedimentation according to density Inventive nucleic acids also encompass polynucleotides with known types of modifications, for example, labels, methylation, "caps", substitution of one or more of the naturally occurring nucleotides with a nucleotide analog, internucleotide modifications such as, for example, polynucleotides with uncharged linkages (e.g., methyl phosphonatcs, phosphotriesters, phosphoamidatcs, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc ), those containing pendant moieties, such as, for example proteins

(including for e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing dictators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide A "nucleic acid segment" is a polynucleotide subportion of a larger nucleic acid.

A nucleotide sequence complementary to an inventive Cry/?tov/n«-specific nucleotide sequence, as used herein, is one binding specifically or hybridizing with a Cryptovirus-specific nucleotide base sequence. The phrase "binding specifically" or "hybridizing" encompasses the ability of a polynucleotide sequence to recognize a complementary base sequence and to form double-helical segments therewith via the fonnation of hydrogen bonds between the complementary base pairs.

Thus, a complementary sequence includes, for example, an antisense sequence with respect to a sense sequence or coding sequence As known to those of skill in the art, the stability of hybrids is reflected in the melting temperature (T_m) of the hybrids. In general, the stability of a hybrid is a function of sodium ion concentration and temperature. Typically, the hybridization reaction is performed under conditions of relatively low stringency, followed by washes of varying, but higher, stringency.

Reference to hybridization stringency relates to such washing conditions.

As used herein, the phrase "moderately stringent hybridization" refers to conditions that permit targct-RNA or DNA to bind a complementary nucleic acid that has at least about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to the target RNA segment or DNA segment. Preferably, moderately stringent conditions are conditions approximately equivalent in stringency to hybridization in 50% formamide, 5 x Denhart's solution, 5 x SSPE, 0.2% SDS at 42°C, followed by washing in 0.2 x SSPE, 0.2% SDS, at 65°C. The phrase "high stringency hybridization" refers to conditions that permit hybridization of only those nucleic acid sequences that form stable hybrids in 0.018 M NaCl at 65°C (i.e., if a hybrid is not stable in 0.018 M NaCl at 65°C, it will not be stable under high stringency conditions, as contemplated herein). High stringency conditions can be provided, for example, by hybridization in 50% formamide, 5X Denhart's solution, 5X SSPE, 0.2% SDS at 42°C, followed by washing in 0.1 x

SSPE, and 0.1% SDS at 65°C.

The phrase "low stringency hybridization" typically refers to conditions equivalent to hybridization in 10% formamide, 5X Denhart's solution, 6 x SSPE, 0.2% SDS at 42°C, followed by washing in 1 x SSPE, 0.2% SDS, at 50°C. Denhart's solution and SSPE (see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press [1989]) are well known to those of skill in the art, as are other suitable hybridization buffers.

The inventive nucleic acids include a Cryptovirus-specific nucleic acid fragment at least about five contiguous nucleotides long, and up to 15245 contiguous nucleotides long, of SEQ ID NO: 1, or a complementary sequence. Thus, useful fragments include nucleic acid segments consisting of an open reading frame of the Cryptovims genome or a complementary sequence. An "open reading frame" (ORF) is a region of a polynucleotide sequence which encodes a polypeptide; this region may represent a portion of a coding sequence or an entire coding sequence.

A "coding sequence" is a polynucleotide sequence which is transcribed into mRNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus. A coding sequence can include, but is not limited to, mRNA, cDNA, and recombinant polynucleotide sequences.

Useful examples of the fragment include nucleic acid segments that encode Cryptovirus proteins, such as (i) contiguous nucleotide positions 152-1678 of (SEQ ID NO:l)(also designated

[SEQ ID NO-3]); (ii) contiguous nucleotide positions 1850-2515 of (SEQ ID NO: l)(also designated |SEQ ID NO.5]); (iii) contiguous nucleotide positions 1850-3023 of (SEQ ID NO: l)(also designated [SEQ ID NO:33J); (iv) contiguous nucleotide positions 1850-3023 of (SEQ ID NO: l) combined with a further insertion of two guanine (G) residues between nucleotide position 2339 of (SEQ ID NO: l) and nucleotide position 2340 of (SEQ ID NO: l)(the combined sequence including the "GG" insertion being designated [SEQ ID NO:7]); (v) contiguous nucleotide positions 3141-4271 of (SEQ ID NO: l)(also designated [SEQ ID NO:9j); (vi) contiguous nucleotide positions 4530-6182 of (SEQ ID NO: l)(also designated [SEQ ID NO 1 1J), (vii) contiguous nucleotide positions 4587-6182 of (SEQ ID NOT)(also designated [SEQ ID NO: 13]); (viii) contiguous nucleotide positions 4587-4835 of (SEQ ID NO: l)(also designated [SEQ ID NO: 15]); (ix) contiguous nucleotide positions 4836-6182 of (SEQ ID NO: l)(also designated [SEQ ID NO: 17]); (x) contiguous nucleotide positions 4272-6515 of (SEQ ID NO l)(also designated [SEQ ID NO 34J); (xi) contiguous nucleotide positions 6303-6434 of (SEQ ID NOT)(also designated [SEQ ID NO: 19]); (xii) contiguous nucleotide positions 6584-

8278 of (SEQ ID NO: l)(also designated [SEQ ID NO 21]); or (xiii) contiguous nucleotide positions 8414-15178 of (SEQ ID NO: l)(also designated [SEQ ID NO:23]). A nucleotide complementary to any of (SEQ ID NOS:3, 5, 7, 9, 1 1, 13, 15, 17, 19, 21, 23, 33, or 34), or a degenerate coding sequence of any of (SEQ ID NOS 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 33, or 34) is also encompassed by the nucleic acid fragment.

As used herein, the term "degenerate coding sequence", or interchangeably, "degenerate sequence", refers to a protein-encoding nucleic acid sequence that has at least one codon that differs in at least one nucleotide position from any reference nucleic acid, e.g., any of SEQ ID NO:3, 5, 7, 9, 1 1, 13, 15, 17, 19, 21, or 23, but which encodes the same amino acids as the reference nucleic acid. For example, codons specified by the triplets "UCU", "UCC", "UCA", and "UCG" are degenerate with respect to each other since all four of these codons encode the amino acid serine.

In some embodiments, the Cryptovirus-specific fragment is up to about 500 nucleotides long. In other embodiments, the fragment is up to about 50 nucleotides long. Other embodiments of the inventive nucleic acid fragment are about fifteen nucleotides to about 35 nucleotides long; for example, this is a preferred length for a Cryptovirus-specific primer of the present invention, which is a Cryptovirus-specific oligonucleotide for use in nucleic acid amplification reactions. Most preferably, the inventive Cryptovirus -specific primer is about 17 to about 22 nucleotides long, but primers as short as 7 contiguous nucleotides may be useful for some gene-specific sequences. (E.g., Vincent, J., et al, O gonucleonucleotides as short as 7-mers can be used for PCR amplification, DNA Cell Biol. 13(l):75-82 [1994])

The inventive probe is preferably about 7 to about 500 nucleotides long, most preferably about 15 to about 150 nucleotides long, and comprises, for at least part of its length, a Cryptovirus- specific nucleotide sequence at least 7 to 15 nucleotides long, such that the probe hybridizes to a Cryptovirus-specific single stranded nucleic acid under suitably stringent hybridization conditions. For example, probes comprising the inventive oligonucleotide primer sequences described herein can be labeled for use as probes for detecting or analyzing Cry/>tøv/π/.s'-specιfic nucleic acid, including nucleic acid amplification products. Non-limiting examples of the Cryptovirus-specific fragments useful as primers or probes include nucleic acids comprising contiguous nucleotide positions 1684-1701 of SEQ ID NO: l (designated SEQ ID NO:35); contiguous nucleotide positions 1700-1717 of SEQ ID NO: l (designated SEQ ID NO:36); contiguous nucleotide positions 4283-4300 of SEQ ID NO: l (designated SEQ ID NO:37); contiguous nucleotide positions 4299-4316 of SEQ ID NO. l

(designated SEQ ID NO:38); contiguous nucleotide positions 4285-4302 of SEQ ID NO:l (designated SEQ ID NO'39); contiguous nucleotide positions 4300-4317 of SEQ ID NO: l (designated SEQ ID NO:40); contiguous nucleotide positions 4518-4535 of SEQ ID NO: l (designated SEQ ID NO 41); contiguous nucleotide positions 4533-4550 of SEQ ID NO:l (designated SEQ ID NO:42); contiguous nucleotide positions 61 16-6133 of SEQ ID NO: l

(designated SEQ ID NO:44); contiguous nucleotide positions 6192-6209 of SEQ ID NO: l (designated SEQ ID NO:45); contiguous nucleotide positions 6191-6208 of SEQ ID NO: l (designated SEQ ID NO:43); contiguous nucleotide positions 7501-7518 of SEQ ID NO: l (designated SEQ ID NO:46); contiguous nucleotide positions 7517-7534 of SEQ ID NO: l (designated SEQ ID NO.47); or a nucleotide sequence complementary to any of the preceding SEQ

ID NOS:35-47. A polynucleotide particularly useful as a probe, especially for probing nucleic acids in samples of biological materials originating from a human or amplification products derived therefrom, is a nucleic acid comprising contiguous nucleotide positions 4292-4549 of SEQ ID NO:l (designated SEQ ID NO:48) or a complementary sequence For probing nucleic acids derived from a sample of biological material from a human, even a large nucleic acid segment of SEQ ID NO: l can be used, for example a nucleic acid comprising contiguous nucleotide positions 4272-6515 of SEQ ID NO: 1 (designated SEQ ID NO.34) or a complementary sequence.

The primer is capable of acting as a point of initiation of synthesis of a polynucleotide strand when placed under appropriate conditions. The primer will be completely or substantially complementary to a region of the polynucleotide strand to be copied. T us, under conditions conducive to hybridization, the primer will anneal to the complementary region of the analyte strand. Upon addition of suitable reactants, (e.g , a polymerase, nucleotide triphosphates, and the like), the primer is extended by the polymerizing agent to form a copy of the analyte strand The primer may be single-stranded, or alternatively maybe partially or fully double-stranded. The terms "analyte polynucleotide" and "analyte strand" refer to a single- or double- stranded nucleic acid molecule which is suspected of containing a Cryptovirus-specific target sequence, and which may be present in a sample of biological material. The inventive probe is a stmcture comprised of a polynucleotide which forms a hybrid staicture with a Cryptovirus-specific target sequence, due to complementarity of at least one nucleotide sequence in the probe with a sequence in the target region. The polynucleotide regions of probes may be composed of DNA, and/or RNA, and/or synthetic nucleotide analogs. "Target region" refers to a region of the nucleic acid which is to be amplified and/or detected The term "target sequence" refers to a Cryptovirus-specific sequence with which a probe or primer will form a stable hybrid under desired conditions.

Included within probes are "capture probes" and "label probes". Preferably the probe does not contain a sequence complementary to sequence(s) used to prime a nucleic acid amplification reaction.

The term "capture probe" as used herein refers to a polynucleotide comprised of a single- stranded polynucleotide coupled to a binding partner The single-stranded polynucleotide is comprised of a targeting polynucleotide sequence, which is complementary to a target sequence in a target region to be detected in the analyte polynucleotide. This complementary region is of sufficient length and complementarity to the target sequence to afford a duplex of stability which is sufficient to immobilize the analyte polynucleotide to a solid surface (via the binding partners). The binding partner is specific for a second binding partner; the second binding partner can be bound to the surface of a solid support, or may be linked indirectly via other staictures or binding partners to a solid support The term "binding partner" as used herein refers to a molecule capable of binding a ligand molecule with high specificity, as for example an antigen and an antibody specific therefor. In general, the specific binding partners must bind with sufficient affinity to immobilize the analyte copy/complementary strand duplex (in the case of capture probes) under the isolation conditions. Specific binding partners are known in the art, and include, for example, biotin and avidin or streptavidin, IgG and protein A, the numerous known receptor-ligand couples, and complementary polynucleotide strands. In the case of complementary polynucleotide binding partners, the partners are generally at least about 15 bases in length, and may be at least 40 bases in length; in addition, they have a content of Gs and Cs of at least about 25% and as much as about 75%. The polynucleotides may be composed of DNA, RNA, or synthetic nucleotide analogs. "Coupled" as used herein refers to attachment by covalent bonds or by strong non-covalent interactions (e.g., hydrophobic interactions, hydrogen bonds, etc.). Covalent bonds may be, for example, ester, ether, phosphoester, amide, peptide, imide, carbon-sulfur bonds, carbon-phosphoms bonds, and the like. A "support" refers to any solid or semi-solid surface to which a desired binding partner may be anchored. Suitable supports include glass, plastic, metal, polymer gels, and the like, and may take the form of beads, wells, dipsticks, membranes, and the like.

As used herein, the term "label probe" refers to an oligonucleotide which is comprised of a targeting polynucleotide sequence, which is complementary to a target sequence to be detected in the analyte polynucleotide. This complementary region is of sufficient length and complementarity to the target sequence to afford a duplex comprised of the "label probe" and the "target sequence" to be detected by the label The oligonucleotide is coupled to a label either directly, or indirectly via a set of ligand molecules with high specificity for each other. A "label" includes any atom or moiety which can be used to provide a detectable (preferably quantifiable) signal, and which can be attached by conventional means to a polynucleotide or to polypeptide, such as an antibody. The label can be used alone or in conjunction with additional reagents Such labels are themselves well-known in the art. The label can be a radioisotope, such as ¹⁴C, ³²P, ³⁵S, H, or ¹⁵0, which is detected with suitable radiation detection means. Alternatively, the label can be a fluorescent labeling agent that chemically binds to antibodies or antigens without denaturation to form a fluorochrome (dye) that is a useftil immunofluorcscent tracer. A description of lmmunofluorescent analytic techniques is found in DeLuca, "Immunofluorescence Analysis", in Antibody As a Tool, Marchalonis et al, eds , John Wiley & Sons, Ltd., pp. 189-231 (1982). As well, any of diverse fluorescent dyes can optionally be used to label probes or primers or amplification products for ease of detection and/or analysis. Useful fluorescent dyes include, but are not limited to, rhodamine, fluorcscein, SYBR Green I, YlO-PRO-1, thiazole orange, Hex (i.e., 6-carboxy- 2',4',7',4,7-hexachlorofluoroscein), pico green, edans, fluorescein, FAM (i.e., 6-carboxyfluorescein), or TET (i.e., 4,7,2',7'-tetrachloro-6-carboxyfluoroscein). (E.g., J. Skeidsvoll and P.M. Ueland, Analysis of double-stranded DNA by capillary electrophoresis with laser-induced fluorescence detection using the monomenc dye SYBR green I, Anal. Biochem. 231(20):359-65 [1995]; H.

Iwahana et al. , Multiple fluorescence-based PCR-SSCP analysis using internal fluorescent labeling of PCR products, Biotechniques 21(30 510-14, 516-19 | 1996|).

The inventive nucleic acid constmcts include recombinant cloning and expression vectors (including plasmids and viral expression vectors, such as retroviral or adenoviral vectors), that contain the inventive nucleic acid. A "vector" is a replicon in which another polynucleotide segment is attached, so as to bring about the replication and/or expression of the attached segment. A "replicon" is any genetic element, e.g., a plasmid, a chromosome, a vims, a cosmid, etc., that behaves as an autonomous unit of polynucleotide replication within a cell, i.e., being capable of replication under the replicon's own control. Inventive recombinant expression vectors contain one or more inventive nucleic acid segments and include at least a promoter region operatively linked to the inventive nucleic acid segment in a transcriptional unit. Preferred examples of inventive nucleic acid constructs are those that include one or more nucleic acid segments encoding a Cryptovirus protein, the Cryptovirus coding sequence(s) being thus suitably placed and operatively linked to suitable regulatory sequences within one or more transcriptional units within the constmct.

"Control" or "regulatory" sequences, elements or regions refers to polynucleotide sequences which arc necessary to effect the expression of coding sequences to which they are ligated The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and terminators; in cukaryotes, generally, such control sequences include promoters, terminators and, in some instances, enhancers. The term "control sequences" is intended to include, at a minimum, all components whose presence is necessary for expression, and may also include additional components whose presence is advantageous, for example, leader sequences. As used herein, "expression" refers to the process by which genes are transcribed into mRNA, which is in turn translated into peptides, polypeptides, or proteins. With respect to a recombinant expression vector, a promoter region refers to a segment of nucleic acid that controls transcription of a coding sequence to which it is operatively linked. The promoter region includes specific sequences that are sufficient for RNA polymerase recognition, binding and transcription initiation. In addition, the promoter region includes sequences that modulate this recognition, binding and transcription initiation activity of RNA polymerase. These sequences may be cis acting or may be responsive to trans acting factors. Promoters, depending upon the nature of the regulation, may be constitutive or regulated developmcntally or inducibly. Exemplary promoters contemplated for use in the practice of the present invention include the SV40 early promoter, the cytomegalovirus (CMV) promoter, the mouse mammary tumor vims (MMTV) stcroid-inducible promoter, Moloney murine leukemia viais (MMLV) promoter, and the like.

For optimal expression of foreign genes in mammalian cells (e.g., the Cryptovirus genes of the present invention), the expression vector may also require terminator sequences and poly A addition sequences; enhancer sequences which increase expression may also be included, and sequences which cause amplification of the gene may also be desirable Such sequences are known in the art.

As used herein, the term "operatively linked" refers to the functional relationship of nucleic acid with regulator)' (control or effector) nucleotide sequences, such as promoters, enhancers, transcriptional and translational stop sites, and other signal sequences. For example, operative linkage of DNA or RNA to a promoter refers to the physical and functional relationship between the DNA or RNA and the promoter such that the transcription of such DNA or RNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA or RNA, respectively. A regulatory sequence "operatively linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the regulatory sequences. Thus, for example, within a transcriptional unit, the promoter sequence, is located upstream (i e , 5' in relation thereto) from the coding sequence and the coding sequence, is 3' to the promoter, or alternatively is in a sequence of genes or open reading frames 3' to the promoter and expression is coordinately regulated thereby. Both the promoter and coding sequences are oriented in a 5' to 3' manner, such that transcription can take place in vitro in the presence of all essential enzymes, transcription factors, co-factors, activators, and reactants, under favorable physical conditions, e.g , suitable pH and temperature This does not mean that, in any particular cell, conditions will favor transcription. For example, transcription from a tissue-specific promoter is generally not favored in heterologous cell types from different tissues.

The inventive expression vector, comprising a Cryptovirus-specific nucleic acid, is used to transform a cell. "Transformation", as used herein, refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion, for example, direct uptake, transfection, transduction, f-mating, microparticle bombardment, or electroporation. The exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host genome. A "transformed" host cell refers to both the immediate cell that has undergone transformation and its progeny that maintain the originally exogenous polynucleotide.

Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.

Exemplary, eukaryotic expression vectors, include the cloned bovine papilloma vims genome, the cloned genomes of the murine retrovimses, and eukaryotic cassettes, such as the pSV-2 gpt system (described by Mulligan and Berg, 1979, Nature Vol. 277:108-114) the Okayama-Berg cloning system (Mol Cell Biol. Vol. 2: 161-170, 1982), pGAL4, pCI (e.g., pCI-neo), and the expression cloning vector described by Genetics Institute (Science Vol. 228:810-815, 1985), are available which provide substantial assurance of at least some expression of the protein of interest in the transformed mammalian cell. Preferred are vectors which contain regulatory elements that can be linked to the inventive nucleic acids, for transfection or transduction of mammalian cells Examples are cytomegalovims (CMV) promoter-based vectors such as pcDNAl (Invitrogen, San Diego, CA), MMTV promoter- based vectors such as pMAMNeo (Clontech, Palo Alto, CA) and pMSG (Phaπnacia, Piscataway, NJ), and SV40 promoter-based vectors such as pSVβ (Clontech, Palo Alto, CA) In one embodiment of the present invention, adenovirus-transferπn/polylysine-DNA (TfAdpl-DNA) vector complexes (Wagner et al , 1992, PNAS, USA, 89 6099-6103, Cunel et al , 1992, Hum Gene Therapy, 3 147- 154, Gao et al , 1993, Hum Gene Ther , 4 14-24) are employed to transduce mammalian cells with heterologous Cryptovirus -specific nucleic acid Any of the plasmid expression vectors described herein may be employed in a TfAdpl-DNA complex

In addition, expression vectors may contain appropriate packaging signals that enable the vector to be packaged by a number of viral virions, e g , retrovimses, such as human immune- deficiency vims, lentivimses, mumps vims, herpes vimses, adenovimses, resulting in the formation of a "viral vector " (See, e g , Anderson, W F , Gene therapy scores against cancer, Nat Med 6(8) 862- 63 [2000]) These viral vectors include, for example, Herpes simplex vims vectors (e g , Geller et al ,

1988, Science, 241 1667-1669), Vaccinia vims vectors (e g , Piccini et al , 1987, Meth in Enzymology, 153 545-563), Cytomegalovims vectors (Mocarski et al , in Viral Vectors, Y Gluzman and S H Hughes, Eds , Cold Spring Harbor Laboratory, Cold Spring Harbor, N Y , 1988, pp 78-84), Moloney murine leukemia vims vectors (Danos et al , 1980, PNAS, USA, 85 6469), adenovims vectors (e g , Logan et al , 1984, PNAS, USA, 81 3655-3659, Jones et al , 1979, Cell, 17 683-689,

Berkner, 1988, Biotechmques, 6 616-626, Cottcn et al , 1992, PNAS, USA, 89 6094-6098, Graham el al , 1991, Meth Mol Biol , 7 109-127), adeno-associated viais vectors, rctrovims vectors (see, e g , U S Patent 5,252,479, WIPO publications WO 92/07573, WO 90/06997, WO 89/05345, WO 92/05266 and WO 92/14829, U S Patent Nos 4,405,712 and 4,650,764, Shackleford et al , 1988, PNAS, USA, 85 9655-9659), and the like

A preferred viral vector is Moloney murine leukemia vims and the pseudotyped retroviral vector derived from Moloney vims called vesicular-stomahtis-viais-glycoprotein (VSV-G)-Moloney murine leukemia vims A most preferred viral vector is a pseudotyped (VSV-G) lentiviral vector derived from the HIV vims, which is used to transduce mammalian cells (Naldini, L , et al , In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector, Science 272 263-

267 [1996]) This gene delivery system employs retroviral particles generated by a three-plasmid expression system In this system a packaging constmct contains the human cytomegaloviais (hCMV) immediate early promoter, driving the expression of all viral proteins The constmct's design eliminates the cis-acting sequences cmcial for viral packaging, reverse transcription and integration of these transcripts. The second plasmid encodes a heterologous envelope protein (env), namely the G glycoprotein of the vesicular stomatitis vims (VSV-G). The third plasmid, the transducing vector (pHR'), contains cis-acting sequences of human immunodeficiency vims (HIV) required for packaging, reverse transcription and integration, as well as unique restriction sites for cloning heterologous complementary DNAs (cDNAs). For example, a genetic selection marker, such as green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), blue fluorescent protein, yellow fluorescent protein, a fluorescent phycobiliprotein, β-galactosidase, and/or a gene encoding another preselected product is cloned downstream of the hCMV promoter in the HR'vector, and is operatively linked so as to form a transcriptional unit. A VSV-G pseudotyped retroviral vector system is capable of infecting a wide variety of cells including cells from different species and of integrating into the genome. Some retroviaises, i e., lentivimses, such as HIV, have the ability to infect non-dividing cells. Lentiviaises have a limited capacity for heterologous DNA sequences, the size limit for this vector being 7-7.5 kilobases (Verma, I.M. and Somia, N., Gene Therapy - promises, problems and prospects, Nature 389:239-242 [1997]). In vivo experiments with lentivimses show that expression does not shut off like other retroviral vectors and that in vivo expression in brain, muscle, liver or pancreatic-islet cells, is sustained at least for over six months - the longest time tested so far (Verma and Somia [1997J; Anderson, WF , Human Gene Therapy, Nature (Suppl). 392:25-30 [1998]). All of the above vimses may require modification to render them non-pathogenic or less antigenic. Other known viral vector systems, however, are also useful within the confines of the invention.

A particularly useful expression vector which is useftil to express foreign cDNA and which may be used in vaccine preparation is Vaccinia vims. In this case, the heterologous cDNA is inserted into the Vaccinia genome. Techniques for the insertion of foreign cDNA into the vaccinia viais genome arc known in the art, and utilize, for example, homologous recombination. The insertion of the heterologous cDNA is generally into a gene which is non-essential in nature, for example, the thymidine kinase gene (tk), which also provides a selectable marker. Plasmid vectors that greatly facilitate the construction of recombinant vimses have been described (see, for example, Mackett et al. (1984) in "DNA Cloning" Vol II IRL Press, p. 191, Chakrabarti et al (1985), Mol. Cell Biol.

5:3403; Moss (1987) in "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, eds., p. 10). Expression of the desired polypeptides containing immunoreactive regions then occurs in cells and mammals that are infected and/or immunized with the live recombinant Vaccinia viais The inventive "nucleic acid constaict" also encompasses a constmct that is not contained in a vector, for example, a synthetic antisense oligonucleotide, such as a phosphorothioate oligodeoxynucleotide Synthetic antisense oligonucleotides, or other antisense chemical staictures designed to recognize and selectively bind to mRNA, are constaicted to be complementary to portions of the Cryptovirus coding strand, for example, to coding sequences shown in SEQ ID NO 3,

5, 7, 9, 1 1, 13, 15, 17, 19, 21, or 23 When taken up by a mammalian cell, the antisense oligonucleotide prevents translational expression of at least part of the Cryptovirus coding region, the inventive antisense oligonucleotide is useful to prevent expression of a Cryptovirus protein Antisense oligonucleotides inactivate target mRNA sequences by either binding thereto and inducing degradation of the mRNA by, for example, RNase I digestion, or inhibiting translation of mRNA target sequence by interfering with the binding of translation-regulating factors or πbosomes, or by inclusion of other chemical staictures, such as πbozyme sequences or reactive chemical groups which either degrade or chemically modify the target mRNA Gene-based therapy strategies employing antisense oligonucleotides are well known in the art (E g , Rait, A ct al , 3'-End conjugates of minimally phosphorothioate-protected oligonucleotides with 1 -O-hexadecylglycerol synthesis and anti-ras activity in radiation-resistant cells, Bioconjug Chem , 11(2) 153-60 [2000], Stenton, G R et al , Aerosolized syk antisense suppresses syk expression, mediator release from macrophages, and pulmonary inflammation. J Immunol . 164(7) 3790-7 [2000], Suzuki, J ct al . Antisense Bcl-x oligonucleotide induces apoptosis and prevents arterial neoinhmal formation in murine cardiac allografts, Cardiovas Res , 45(3) 783-7 [2000J, Kim, J W et al , Antisense oligodeoxynucleotide of glyceraldehyde-3-phosphate dehydrogena.se gene inhibits cell proliferation and induces apoptosis in human cervical carcinoma cell line, Antisense Nucleic Acid Drug Dev , 9(6) 507-13 [1999], Han, D C et al , Therapy with antisense TGF-belal ohgodeoxynucleotides reduces kidney weight and matrix mRNAs in diabetic mice, Am J Physiol Renal Physiol , 278(4) F628-F634 [ 2000], Scala, S et al , Adenovirus-mediated suppression of HMGI X) protein synthesis as potential therapy of human malignant neoplasms, Proc Natl Acad Sci USA , 97(8) 4256-4261 [2000], Artcaga, C L , el al , Tissue-targeted antisense c-fos retroviral vector inhibits established breast cancer xenografts in nude mice, Cancer Res , 56(5) 1098-1103 [1996], Muller, M et al , Antisense phosphorothioate oligodeoxynucleotide down-regulation of the insulin-like growth factor I receptor in ovarian cancer cells, Int J Cancer, 77(4) 567-71 [1998]; Engelhard, H H , Antisense Oligodeoxynucleotide

Technology Potential Use for the Treatment of Malignant Brain Tumors, Cancer Control, 5(2) 163- 170 [19981, Alvarez-Salas, L M et al . Growth inhibition of cervical tumor cells by antisense ohgodeoxynucleotides directed to the human papillomavirus type 16 E6 gene, Antisense Nucleic Acid Daig Dev , 9(5) 441-50 [1999], Im, S A , et al , Antiangiogenesis treatment for gliomas transfer oj antisense-vascular endothe al growth factor inhibits tumor growth in vivo, Cancer Res , 59(4) 895- 900 [1999], Maeshima, Y et al , Antisense oligonucleotides to proliferating cell nuclear antigen and Kι-67 inhibit human mesangial cell proliferation, J Am Soc Nephrol , 7(10) 2219-29 [1996], Chen, D S et al , Retroviral Vector-mediated transfer of an antisense cychn GI construct inhibits osteosarcoma tumor growth in nude mice, Hum Gene Ther, 8(14) 1667-74 [1997], Hirao, T et al , Antisense epidermal growth factor receptor delivered by adenoviral vector blocks tumor growth in human gastric cancer, Cancer Gene Ther 6(5) 423-7 [1999], Wang, X Y ct al , Antisense inhibition of protein kinase Calpha reverses the transformed phenotype in human lung carcinoma cells, Exp Cell Res , 250(1) 253-63 [1999], Sacco, M G et al , In vitro and in vivo antisense-mediated growth inhibition of a mammary adenocarcmoma from MMTV-neu transgenic mice. Gene Ther , 5(3), 388-93 [1998], Leonetti, C et al . And tumor effect of c-myc antisense phosphorothioate ohgodeoxynucleotides on human melanoma cells in vitro and in mice, J Natl Cancer Inst , 88(7) 419-29 [1996], Laird, A D et al , Inhibition of tumor growth in liver epithelial cells transfected w th a transforming growth factor alpha antisense gene, Cancer Res 54(15) 4224-32 (Aug 1, 1994),

Yazaki, T et al , Treatment of ghoblastoma U-87 by systemic administration of an antisense protein kinase C-alpha phosphorothioate oligodeoxynucleotide, Mol Pharmacol , 50(2) 236-42 [1996], Ho, P T et al , Antisense oligonucleotides as therapeutics for malignant diseases, Semm Oncol , 24(2) 187-202 [1997], Muller, M et al , Antisense phosphorothioate oligodeoxynucleotide down- regulation of the insulin-like growth factor I receptor in ovarian cancer cells, Int J. Cancer,

77(4) 567-71 [1998], Elez, R et al , Polo-like kinasel, a new target for antisense tumor therapy. Biochem Biophys Res Commun , 269(2) 352-6 [2000], Monia, B P et al., Antitumor activity of a phosphorothioate antisense oligodeoxynucleotide targeted against C-raf kinase, Nat Med , 2(6) 668- 75 [ 1996]) The present invention relates to an isolated Cryptovirus protein The term "protein" refers to a polymer of amino acids of any length, I e , a polypeptide, and does not refer to a specific length of the product, thus, "polypeptides", "peptides", and "o gopeptides", are included within the definition of "protein", and such terms arc used interchangeably herein with "protein" The term "protein" also includes post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like Included within the definition of "protein" are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural ammo acids, etc ), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. Methods of inserting analogs of amino acids into a peptide sequence are known in the art

The phrase "isolated Cryptovirus protein" refers to a cryptoviral protein which is substantially free, i e , contains less than about 50%, preferably less than about 70%, and even more preferably less than about 90%, of the cellular components and/or contaminants normally associated with a native in vivo environment. An isolated Cryptovirus protein of the present invention can also be further isolated from one or more other components of a Cryptovirus particle, for example, otlier Cryptovirus protein species, phospholipid components of the viral envelope, or the viral genome. In some useftil embodiments the inventive isolated Cryptovirus protein is purified to homogeneity by known virological and biochemical methods.

The inventive protein is encoded by a Cryptovirus-specific nucleic acid segment, which nucleic acid segment comprises'

(i) contiguous nucleotide positions 152-1678 of (SEQ ID NO: l)(also designated SEQ ID NO:3), or a degenerate coding sequence, which encodes the Cryptovirus nucleocapsid (NP) protein; (ii) contiguous nucleotide positions 1850-2515 of (SEQ ID NO: l)(also designated SEQ ID

NO:5), or a degenerate coding sequence, which encodes a Cryptovirus RNA binding (V) protein thought to be a component of the viral RNA-dependent RNA polymerase;

(in) contiguous nucleotide positions 1850-3023 of (SEQ ID NO: l)(also designated SEQ ID NO 33) combined with a ftirther insertion of two guanine (G) residues between nucleotide position 2339 of (SEQ ID NO: l) and nucleotide position 2340 of (SEQ ID NO: l)(the combined coding sequence including the "GG" insertion being designated [SEQ ID NO:7|), or a degenerate coding sequence thereof; this frameshift-causing insertion into the mRNA encoding the Cryptovirus nucleocapsid-associated phosphoprotein (P protein) occurs during processing of the mRNA and is not templated by the Cryptoviais minus stranded RNA genomic sequence; (iv) contiguous nucleotide positions 3141-4271 of (SEQ ID NO: l)(also designated SEQ ID

NO:9), or a degenerate coding sequence, which encodes the Cryptovirus virion-associated matrix or membrane (M) protein;

(v) contiguous nucleotide positions 4530-6182 of (SEQ ID NO: l)(also designated SEQ ID NO.11) or a degenerate coding sequence, which encodes the Cryptovirus (uncleaved) fusion (F) protein, which is a propeptide fonn of a major envelope-associated glycoprotein, and includes a 19- amino acid signal region at its amino terminus;

(vi) contiguous nucleotide positions 4587-6182 of (SEQ rD NO: l)(also designated SEQ ID NO 13), or a degenerate coding sequence, which encodes the Cryptovirus (uncleaved) fusion (F₀) protein, which is a propeptide form of a major envelope-associated glycoprotein, minus the 19-amino acid signal region at its amino terminus;

(vii) contiguous nucleotide positions 4587-4835 of (SEQ ID NO: l)(also designated SEQ ID NO: 15), or a degenerate coding sequence, which encodes the cleaved F₂ protein; (viii) contiguous nucleotide positions 4836-6182 of (SEQ ID NO: l)(also designated SEQ ID

NO: 17), or a degenerate coding sequence, which encodes the cleaved Fi protein, including a 22- amino acid carboxy terminal peptide segment that is thought to be important to Cryptovirus infectivity;

(ix) contiguous nucleotide positions 6303-6434 of (SEQ [D NO: l)(also designated SEQ ID NO: 19), or a degenerate coding sequence, which encodes the Cryptovirus SH protein, a small envelope-associated protein;

(x) contiguous nucleotide positions 6584-8278 of (SEQ ID NO:l)(also designated SEQ ID NO:21), or a degenerate coding sequence, which encodes the Cryptovirus hemagglutinin (HN) protein, another major envelope protein; or (xi) contiguous nucleotide positions 8414-15178 of (SEQ ID NO:l)(also designated SEQ ID

NO:23), or a degenerate coding sequence, which encodes the Cryptovirus largest nucleocapsid associated protein (L protein).

These are the amino acid sequences corresponding to the preceding inventive proteins in the same order: (i) NP has the following amino acid sequence (SEQ ID NO:4):

Met Ser Ser Val Leu Lys Ala Tyr Glu Arg Phe Thr Leu Thr Gin Glu

1 5 10 15

Leu Gin Asp Gin Ser Glu Glu Gly Thr lie Pro Pro Thr Thr Leu Lys 20 25 30 Pro Val lie Arg Val Phe Val Leu Thr Ser Asn Asn Pro Glu Leu Arg 35 40 45

Ser Arg Leu Leu Leu Phe Cys Leu Arg lie Val Leu Ser Asn Gly Ala

50 55 60

Arg Asp Ser His Arg Phe Gly Ala Leu Leu Thr Met Phe Ser Leu Pro 65 70 75 80

Ser Ala Thr Met Leu Asn His Val Lys Leu Ala Asp Gin Ser Pro Glu

85 90 95

Ala Asp lie Glu Arg Val Glu lie Asp Gly Phe Glu Glu Gly Ser Phe 100 105 110 Arg Leu lie Pro Asn Ala Arg Ser Gly Met Ser Arg Gly Glu lie Asn 115 120 125

Ala Tyr Ala Ala Leu Ala Glu Asp Leu Pro Asp Thr Leu Asn His Ala

130 135 140

Thr Pro Phe Val Asp Ser Glu Val Glu Gly Thr Ala Trp Asp Glu lie 145 150 155 160 Glu Thr Phe Leu Asp Met Cys Tyr Ser Val Leu Met Gin Ala Trp lie

165 170 175

Val Thr Cys Lys Cys Met Thr Ala Pro Asp Gin Pro Ala Ala Ser lie 180 185 190 Glu Lys Arg Leu Gin Lys Tyr Arg Gin Gin Gly Arg lie Asn Pro Arg 195 200 205

Tyr Leu Leu Gin Pro Glu Ala Arg Arg lie lie Gin Asn Val lie Arg

210 215 220

Lys Gly Met Val Val Arg His Phe Leu Thr Phe Glu Leu Gin Leu Ala 225 230 235 240

Arg Ala Gin Ser Leu Val Ser Asn Arg Tyr Tyr Ala Met Val Gly Asp

245 250 255

Val Gly Lys Tyr lie Glu Asn Cys Gly Met Gly Gly Phe Phe Leu Thr 260 265 270 Leu Lys Tyr Ala Leu Gly Thr Arg Trp Pro Thr Leu Ala Leu Ala Ala 275 280 285

Phe Ser Gly Glu Leu Thr Lys Leu Lys Ser Leu Met Ala Leu Tyr Gin

290 295 300

Thr Leu Gly Glu Gin Ala Arg Tyr Leu Ala Leu Leu Glu Ser Pro His 305 310 315 320

Leu Met Asp Phe Ala Ala Ala Asn Tyr Pro Leu Leu Tyr Ser Tyr Ala

325 330 335

Met Gly lie Gly Tyr Val Leu Asp Val Asn Met Arg Asn Tyr Ala Phe 340 345 350 Ser Arg Ser Tyr Met Asn Lys Thr Tyr Phe Gin Leu Gly Met Glu Thr 355 360 365

Ala Arg Lys Gin Gin Gly Ala Val Asp Met Arg Met Ala Glu Asp Leu

370 375 380

Gly Leu Thr Gin Ala Glu Arg Thr Glu Met Ala Asn Thr Leu Ala Lys 385 390 395 400

Leu Thr Thr Ala Asn Arg Gly Ala Asp Thr Arg Gly Gly Val Asn Pro

405 410 415

Phe Ser Ser Val Thr Gly Thr Thr Gin Met Pro Ala Ala Ala Thr Gly 420 425 430 Asp Thr Phe Glu Ser Tyr Met Ala Ala Asp Arg Leu Arg Gin Arg Tyr 435 440 445

Ala Asp Ala Gly Thr His Asp Asp Glu Met Pro Pro Leu Glu Glu Glu

450 455 460

Glu Glu Asp Asp Thr Ser Ala Gly Pro Arg Thr Glu Pro Thr Pro Glu 465 470 475 480

Gin Val Ala Leu Asp He Gin Ser Ala Ala Val Gly Ala Pro He His

485 490 495

Thr Asp Asp Leu Asn Ala Ala Leu Gly Asp Leu Asp He

500 505 //(SEQ ID NO 4),

(u) V has the following amino acid sequence (SEQ ID NO 6):

Met Asp Pro Thr Asp Leu Ser Phe Ser Pro Asp Glu He Asn Lys Leu

1 5 10 15

He Glu Thr Gly Leu Asn Thr Val Glu Tyr Phe Thr Ser Gin Gin Val 20 25 30 Thr Gly Thr Ser Ser Leu Gly Lys Asn Thr He Pro Pro Gly Val Thr

35 40 45

Gly Leu Leu Thr Asn Ala Ala Glu Ala Lys He Gin Glu Ser He Asn 50 55 60 His Gin Lys Gly Ser Val Gly Gly Gly Thr Asn Pro Lys Lys Pro Arg 65 70 75 80

Ser Lys He Ala He Val Pro Ala Asp Asp Lys Thr Val Pro Glu Lys

85 90 95

Pro He Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Pro Ser Thr Gin 100 105 110

Thr Val Leu Asp Leu Ser Gly Lys Thr Leu Pro Ser Gly Ser Tyr Lys

115 120 125

Gly Val Lys Leu Ala Lys Phe Gly Lys Glu Asn Leu Met Thr Arg Phe

130 135 140 He Glu Glu Pro Arg Glu Asn Pro He Ala Thr Ser Ser Pro He Asp

145 150 155 160

Phe Lys Arg Gly Arg Asp Thr Gly Gly Phe His Arg Arg Glu Tyr Ser

165 170 175

He Gly Trp Val Gly Asp Glu Val Lys Val Thr Glu Trp Cys Asn Pro 180 185 190

Ser Cys Ser Pro He Thr Ala Ala Ala Arg Arg Phe Lys Cys Thr Cys

195 200 205

His Gin Cys Pro Val Thr Cys Ser Glu Cys Glu Arg Asp Thr

210 215 220 //(SEQ ID N0:6);

(iii) P has the following amino acid sequence (SEQ ID NO:8):

Met Asp Pro Thr Asp Leu Ser Phe Ser Pro Asp Glu He Asn Lys Leu

1 5 10 15

He Glu Thr Gly Leu Asn Thr Val Glu Tyr Phe Thr Ser Gin Gin Val 20 25 30

Thr Gly Thr Ser Ser Leu Gly Lys Asn Thr He Pro Pro Gly Val Thr

35 40 45

Gly Leu Leu Thr Asn Ala Ala Glu Ala Lys He Gin Glu Ser He Asn 50 55 60 His Gin Lys Gly Ser Val Gly Gly Gly Thr Asn Pro Lys Lys Pro Arg 65 70 75 80

Ser Lys He Ala He Val Pro Ala Asp Asp Lys Thr Val Pro Glu Lys

85 90 95

Pro He Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Pro Ser Thr Gin 100 105 110

Thr Val Leu Asp Leu Ser Gly Lys Thr Leu Pro Ser Gly Ser Tyr Lys

115 120 125

Gly Val Lys Leu Ala Lys Phe Gly Lys Glu Asn Leu Met Thr Arg Phe

130 135 140 He Glu Glu Pro Arg Glu Asn Pro He Ala Thr Ser Ser Pro He Asp

145 150 155 160

Phe Lys Arg Gly Ala Glu He Pro Val Gly Ser He Glu Gly Ser Thr

165 170 175

Gin Ser Asp Gly Trp Glu Met Lys Ser Arg Ser Leu Ser Gly Ala He 180 185 190 His Pro Val Leu Gin Ser Pro Leu Gin Gin Gly Asp Leu Asn Ala Leu

195 200 205

Val Thr Asn Val Gin Ser Leu Ala Leu Asn Val Asn Glu He Leu Asn

210 215 220 Thr Val Arg Asn Leu Asp Ser Arg Met Asn Gin Leu Glu Thr Lys Val

225 230 235 240

Asp Arg He Leu Ser Ser Gin Ser Leu He Gin Thr He Lys Asn Asp

245 250 255

He He Gly Leu Lys Ala Gly Met Ala Thr Leu Glu Gly Met He Thr 260 265 270

Thr Val Lys He Met Asp Pro Gly Val Pro Ser Asn Val Thr Val Glu

275 280 285

Asp Val Arg Lys Lys Leu Ser Asn His Ala Val Val Val Pro Glu Ser

290 295 300 Phe Asn Asp Ser Phe Leu Thr Gin Ser Glu Asp Val He Ser Leu Asp

305 310 315 320

Glu Leu Ala Arg Pro Thr Ala Thr Ser Val Lys Lys He Val Arg Lys

325 330 335

Val Pro Pro Gin Lys Asp Leu Thr Gly Leu Lys He Thr Leu Glu Gin 340 345 350

Leu Ala Lys Asp Cys He Ser Lys Pro Lys Met Arg Glu Asp Tyr Leu

355 360 365

Leu Lys He Asn Gin Ala Ser Ser Glu Ala Gin Leu He Asp Leu Lys 370 375 380 Lys Ala lie He Arg Ser Ala He

385 390 //(SEQ ID NO:8);

(iv) M has the following amino acid sequence (SEQ ID NO: 10):

Met Pro Ser He Ser He Pro Ala Asp Pro Thr Asn Pro Arg Gin Ser 1 5 10 15

He Lys Ala Phe Pro He Val He Asn Ser Asp Gly Gly Glu Lys Gly

20 25 30

Arg Leu Val Lys Gin Leu Arg Thr Thr Tyr Leu Asn Asp Leu Asp Thr 35 40 45 His Glu Pro Leu Val Thr Phe Val Asn Thr Tyr Gly Phe He Tyr Glu 50 55 60

Gin Asn Arg Gly Asn Ala He Val Gly Glu Asp Gin Leu Gly Lys Lys 65 70 75 80

Arg Glu Ala Val Thr Ala Ala Met Val Thr Leu Gly Cys Gly Pro Asn 85 90 95

Leu Pro Ser Leu Gly Asn Val Leu Arg Gin Leu Ser Glu Phe Gin Val

100 105 110

He Val Arg Lys Thr Ser Ser Lys Ala Glu Glu Met Val Phe Glu He 115 120 125 Val Lys Tyr Pro Arg He Phe Arg Gly His Thr Leu He Gin Lys Gly 130 135 140

Leu Val Cys Val Ser Ala Glu Lys Phe Val Lys Ser Pro Gly Lys Val 145 150 155 160

Gin Ser Gly Met Asp Tyr Leu Phe He Pro Thr Phe Leu Ser Val Thr 165 170 175 Tyr Cys Pro Ala Ala He Lys Phe Gin Val Pro Gly Pro Met Leu Lys

180 185 190

Met Arg Ser Arg Tyr Thr Gin Ser Leu Gin Leu Glu Leu Met He Arg 195 200 205 He Leu Cys Lys Pro Asp Ser Pro Leu Met Lys Val His He Pro Asp 210 215 220

Lys Glu Gly Arg Gly Cys Leu Val Ser Val Trp Leu His Val Cys Asn 225 230 235 240

He Phe Lys Ser Gly Asn Lys Asn Gly Ser Glu Trp Gin Glu Tyr Trp 245 250 255

Met Arg Lys Cys Ala Asn Met Gin Leu Glu Val Ser He Ala Asp Met

260 265 270

Trp Gly Pro Thr He He He His Ala Arg Gly His He Pro Lys Ser 275 280 285 Ala Lys Leu Phe Phe Gly Lys Gly Gly Trp Ser Cys His Pro Leu His 290 295 300

Glu He Val Pro Ser Val Thr Lys Thr Leu Trp Ser Val Gly Cys Glu 305 310 315 320

He Thr Lys Ala Lys Ala He He Gin Glu Ser Ser He Ser Leu Leu 325 330 335

Val Glu Thr Thr Asp He He Ser Pro Lys Val Lys He Ser Ser Lys

340 345 350

His Arg Arg Phe Gly Lys Ser Asn Trp Gly Leu Phe Lys Lys Thr Lys 355 360 365 Ser Leu Pro Asn Leu Thr Glu Leu Glu

370 375 //(SEQ ID NO: 10);

(v) F has the following amino acid sequence (SEQ ID NO: 12):

Met Ser Thr He He Gin Ser Leu Val Val Ser Cys Leu Leu Ala Gly 1 5 10 15

Ala Gly Ser Leu Asp Pro Ala Ala Leu Met Gin He Gly Val He Pro

20 25 30

Thr Asn Val Arg Gin Leu Met Tyr Tyr Thr Glu Ala Ser Ser Ala Phe 35 40 45 He Val Val Lys Leu Met Pro Thr He Asp Ser Pro He Ser Gly Cys 50 55 60

Asn He Thr Ser He Ser Ser Tyr Asn Ala Thr Val Thr Lys Leu Leu 65 70 75 80

Gin Pro He Gly Glu Asn Leu Glu Thr He Arg Asn Gin Leu He Pro 85 90 95

Thr Arg Arg Arg Arg Arg Phe Ala Gly Val Val He Gly Leu Ala Ala

100 105 110

Leu Gly Val Ala Thr Ala Ala Gin Val Thr Ala Ala Val Ala Leu Val 115 120 125 Lys Ala Asn Glu Asn Thr Ala Ala He Leu Asn Leu Lys Asn Ala He 130 135 140

Gin Lys Thr Asn Ala Ala Val Ala Asp Val Val Gin Ala Thr Gin Ser 145 150 155 160

Leu Gly Thr Ala Val Gin Ala Val Gin Asp His He Asn Ser Val He 165 170 175 Ser Pro Ala He Thr Ala Ala Asn Cys Lys Ala Gin Asp Ala He He

180 185 190

Gly Ser He Leu Asn Leu Tyr Leu Thr Glu Leu Thr Thr He Phe His 195 200 205 Asn Gin He Thr Asn Pro Ala Leu Ser Pro He Thr He Gin Ala Leu 210 215 220

Arg He Leu Leu Gly Ser Thr Leu Pro Thr Val Val Glu Lys Ser Phe 225 230 235 240

Asn Thr Gin He Ser Ala Ala Glu Leu Leu Ser Ser Gly Leu Leu Thr 245 250 255

Gly Gin He Val Gly Leu Asp Leu Thr Tyr Met Gin Met Val He Lys

260 265 270

He Glu Leu Pro Thr Leu Thr Val Gin Pro Ala Thr Gin He He Asp 275 280 285 Leu Ala Thr He Ser Ala Phe He Asn Asn Gin Glu Val Met Ala Gin 290 295 300

Leu Pro Thr Arg Val He Val Thr Gly Ser Leu He Gin Ala Tyr Pro 305 310 315 320

Ala Ser Gin Cys Thr He Thr Pro Asn Thr Val Tyr Cys Arg Tyr Asn 325 330 335

Asp Ala Gin Val Leu Ser Asp Asp Thr Met Ala Cys Leu Gin Gly Asn

340 345 350

Leu Thr Arg Cys Thr Phe Ser Pro Val Val Gly Ser Phe Leu Thr Arg 355 360 365 Phe Val Leu Phe Asp Gly He Val Tyr Ala Asn Cys Arg Ser Met Leu 370 375 380

Cys Lys Cys Met Gin Pro Ala Ala Val He Leu Gin Pro Ser Ser Ser 385 390 395 400

Pro Val Thr Val He Asp Met His Lys Cys Val Ser Leu Gin Leu Asp 405 410 415

Asp Leu Arg Phe Thr He Thr Gin Leu Ala Asn Val Thr Tyr Asn Ser

420 425 430

Thr He Lys Leu Glu Thr Ser Gin He Leu Pro He Asp Pro Leu Asp 435 440 445 He Ser Gin Asn Leu Ala Ala Val Asn Lys Ser Leu Ser Asp Ala Leu 450 455 460

Gin His Leu Ala Gin Ser Asp Thr Tyr Leu Ser Ala He Thr Ser Ala 465 470 475 480

Thr Thr Thr Ser Val Leu Ser He He Ala He Cys Leu Gly Ser Leu 485 490 495

Gly Leu He Leu He He Leu Leu Ser Val Val Val Trp Lys Leu Leu

500 505 510

Thr He Val Ala Ala Asn Arg Asn Arg Met Glu Asn Phe Val Tyr His 515 520 525 Asn Ser Ala Phe His His Pro Arg Ser Asp Leu Ser Glu Lys Asn Gin 530 535 540

Pro Ala Thr Leu Gly Thr Arg 545 550 //(SEQ ID NO: 12); (vi) Fo has the following amino acid sequence (SEQ ID NO 14)

Leu Asp Pro Ala Ala Leu Met Gin He Gly Val He Pro Thr Asn Val

1 5 10 15

Arg Gin Leu Met Tyr Tyr Thr Glu Ala Ser Ser Ala Phe He Val Val 20 25 30

Lys Leu Met Pro Thr He Asp Ser Pro He Ser Gly Cys Asn He Thr

35 40 45

Ser He Ser Ser Tyr Asn Ala Thr Val Thr Lys Leu Leu Gin Pro He 50 55 60 Gly Glu Asn Leu Glu Thr He Arg Asn Gin Leu He Pro Thr Arg Arg 65 70 75 80

Arg Arg Arg Phe Ala Gly Val Val He Gly Leu Ala Ala Leu Gly Val

85 90 95

Ala Thr Ala Ala Gin Val Thr Ala Ala Val Ala Leu Val Lys Ala Asn 100 105 110

Glu Asn Thr Ala Ala He Leu Asn Leu Lys Asn Ala He Gin Lys Thr

115 120 125

Asn Ala Ala Val Ala Asp Val Val Gin Ala Thr Gin Ser Leu Gly Thr

130 135 140 Ala Val Gin Ala Val Gin Asp His He Asn Ser Val He Ser Pro Ala

145 150 155 160

He Thr Ala Ala Asn Cys Lys Ala Gin Asp Ala He He Gly Ser He

165 170 175

Leu Asn Leu Tyr Leu Thr Glu Leu Thr Thr He Phe His Asn Gin He 180 185 190

Thr Asn Pro Ala Leu Ser Pro He Thr He Gin Ala Leu Arg He Leu

195 200 205

Leu Gly Ser Thr Leu Pro Thr Val Val Glu Lys Ser Phe Asn Thr Gin

210 215 220 He Ser Ala Ala Glu Leu Leu Ser Ser Gly Leu Leu Thr Gly Gin He

225 230 235 240

Val Gly Leu Asp Leu Thr Tyr Met Gin Met Val He Lys He Glu Leu

245 250 255

Pro Thr Leu Thr Val Gin Pro Ala Thr Gin He He Asp Leu Ala Thr 260 265 270

He Ser Ala Phe He Asn Asn Gin Glu Val Met Ala Gin Leu Pro Thr

275 280 285

Arg Val He Val Thr Gly Ser Leu He Gin Ala Tyr Pro Ala Ser Gin

290 295 300 Cys Thr He Thr Pro Asn Thr Val Tyr Cys Arg Tyr Asn Asp Ala Gin

305 310 315 320

Val Leu Ser Asp Asp Thr Met Ala Cys Leu Gin Gly Asn Leu Thr Arg

325 330 335

Cys Thr Phe Ser Pro Val Val Gly Ser Phe Leu Thr Arg Phe Val Leu 340 345 350

Phe Asp Gly He Val Tyr Ala Asn Cys Arg Ser Met Leu Cys Lys Cys

355 360 365

Met Gin Pro Ala Ala Val He Leu Gin Pro Ser Ser Ser Pro Val Thr 370 375 380 Val He Asp Met His Lys Cys Val Ser Leu Gin Leu Asp Asp Leu Arg 385 390 395 400

Phe Thr He Thr Gin Leu Ala Asn Val Thr Tyr Asn Ser Thr He Lys

405 410 415

Leu Glu Thr Ser Gin He Leu Pro He Asp Pro Leu Asp He Ser Gin 420 425 430

Asn Leu Ala Ala Val Asn Lys Ser Leu Ser Asp Ala Leu Gin His Leu

435 440 445

Ala Gin Ser Asp Thr Tyr Leu Ser Ala He Thr Ser Ala Thr Thr Thr

450 455 460 Ser Val Leu Ser He He Ala He Cys Leu Gly Ser Leu Gly Leu He

465 470 475 480

Leu He He Leu Leu Ser Val Val Val Trp Lys Leu Leu Thr He Val

485 490 495

Ala Ala Asn Arg Asn Arg Met Glu Asn Phe Val Tyr His Asn Ser Ala 500 505 510

Phe His His Pro Arg Ser Asp Leu Ser Glu Lys Asn Gin Pro Ala Thr

515 520 525

Leu Gly Thr Arg

530 //(SEQ ID NO: 14);

(vii) F₂ has the folowing amino acid sequence (SEQ ID NO: 16):

Leu Asp Pro Ala Ala Leu Met Gin He Gly Val He Pro Thr Asn Val

1 5 10 15

Arg Gin Leu Met Tyr Tyr Thr Glu Ala Ser Ser Ala Phe He Val Val 20 25 30

Lys Leu Met Pro Thr He Asp Ser Pro He Ser Gly Cys Asn He Thr

35 40 45

Ser He Ser Ser Tyr Asn Ala Thr Val Thr Lys Leu Leu Gin Pro He 50 55 60 Gly Glu Asn Leu Glu Thr He Arg Asn Gin Leu He Pro Thr Arg Arg 65 70 75 80

Arg Arg Arg

//(SEQ ID NO:16);

(viii) Fi has the amino acid sequence (SEQ ID NO: 18);

Phe Ala Gly Val Val He Gly Leu Ala Ala Leu Gly Val Ala Thr Ala

1 5 10 15

Ala Gin Val Thr Ala Ala Val Ala Leu Val Lys Ala Asn Glu Asn Thr 20 25 30

Ala Ala He Leu Asn Leu Lys Asn Ala He Gin Lys Thr Asn Ala Ala

35 40 45

Val Ala Asp Val Val Gin Ala Thr Gin Ser Leu Gly Thr Ala Val Gin 50 55 60 Ala Val Gin Asp His He Asn Ser Val He Ser Pro Ala He Thr Ala 65 70 75 80

Ala Asn Cys Lys Ala Gin Asp Ala He He Gly Ser He Leu Asn Leu

85 90 95

Tyr Leu Thr Glu Leu Thr Thr He Phe His Asn Gin He Thr Asn Pro 100 105 110

Ala Leu Ser Pro He Thr He Gin Ala Leu Arg He Leu Leu Gly Ser

115 120 125

Thr Leu Pro Thr Val Val Glu Lys Ser Phe Asn Thr Gin He Ser Ala 130 135 140

Ala Glu Leu Leu Ser Ser Gly Leu Leu Thr Gly Gin He Val Gly Leu

145 150 155 160

Asp Leu Thr Tyr Met Gin Met Val He Lys He Glu Leu Pro Thr Leu

165 170 175 Thr Val Gin Pro Ala Thr Gin He He Asp Leu Ala Thr He Ser Ala

180 185 190

Phe He Asn Asn Gin Glu Val Met Ala Gin Leu Pro Thr Arg Val He

195 200 205

Val Thr Gly Ser Leu He Gin Ala Tyr Pro Ala Ser Gin Cys Thr He 210 215 220

Thr Pro Asn Thr Val Tyr Cys Arg Tyr Asn Asp Ala Gin Val Leu Ser

225 230 235 240

Asp Asp Thr Met Ala Cys Leu Gin Gly Asn Leu Thr Arg Cys Thr Phe

245 250 255 Ser Pro Val Val Gly Ser Phe Leu Thr Arg Phe Val Leu Phe Asp Gly

260 265 270

He Val Tyr Ala Asn Cys Arg Ser Met Leu Cys Lys Cys Met Gin Pro

275 280 285

Ala Ala Val He Leu Gin Pro Ser Ser Ser Pro Val Thr Val He Asp 290 295 300

Met His Lys Cys Val Ser Leu Gin Leu Asp Asp Leu Arg Phe Thr He

305 310 315 320

Thr Gin Leu Ala Asn Val Thr Tyr Asn Ser Thr He Lys Leu Glu Thr

325 330 335 Ser Gin He Leu Pro He Asp Pro Leu Asp He Ser Gin Asn Leu Ala

340 345 350

Ala Val Asn Lys Ser Leu Ser Asp Ala Leu Gin His Leu Ala Gin Ser

355 360 365

Asp Thr Tyr Leu Ser Ala He Thr Ser Ala Thr Thr Thr Ser Val Leu 370 375 380

Ser He He Ala He Cys Leu Gly Ser Leu Gly Leu He Leu He He

385 390 395 400

Leu Leu Ser Val Val Val Trp Lys Leu Leu Thr He Val Ala Ala Asn

405 410 415 Arg Asn Arg Met Glu Asn Phe Val Tyr His Asn Ser Ala Phe His His

420 425 430

Pro Arg Ser Asp Leu Ser Glu Lys Asn Gin Pro Ala Thr Leu Gly Thr

435 440 445

Arg //(SEQ ID NO: 18);

(ix) SH has the following amino acid sequence (SEQ ID NO:20):

Met Leu Pro Asp Pro Glu Asp Pro Glu Ser Lys Lys Ala Thr Arg Arg 1 5 10 15 Thr Gly Asn Leu He He Cys Phe Leu Phe He Phe Phe Leu Phe Val

20 25 30

Thr Leu He Val Pro Thr Leu Arg His Leu Leu Ser

35 40 //(SEQ ID NO 20),

(x) HN has the following amino acid sequence (SEQ ID NO 22)

Met He Ala Glu Asp Ala Pro Val Lys Gly Thr Cys Arg Val Leu Phe

1 5 10 15

Arg Thr Thr Thr Leu He Phe Leu Cys Thr Leu Leu Ala Leu Ser He 20 25 30

Ser He Leu Tyr Glu Ser Leu He Thr Gin Lys Gin He Met Ser Gin

35 40 45

Ala Gly Ser Thr Gly Ser Asn Ser Gly Leu Gly Gly He Thr Asp Leu 50 55 60 Leu Asn Asn He Leu Ser Val Ala Asn Gin He He Tyr Asn Ser Ala 65 70 75 80

Val Ala Leu Pro Leu Gin Leu Asp Thr Leu Glu Ser Thr Leu Leu Thr

85 90 95

Ala He Lys Ser Leu Gin Thr Ser Asp Lys Leu Glu Gin Asn Cys Ser 100 105 110

Trp Gly Ala Ala Leu He Asn Asp Asn Arg Tyr He Asn Gly He Asn

115 120 125

Gin Phe Tyr Phe Ser He Ala Glu Gly Arg Asn Leu Thr Leu Gly Pro

130 135 140 Leu Leu Asn He Pro Ser Phe He Pro Thr Ala Thr Thr Pro Glu Gly

145 150 155 160

Cys Thr Arg He Pro Ser Phe Ser Leu Thr Lys Thr His Trp Cys Tyr

165 170 175

Thr His Asn Val He Leu Asn Gly Cys Gin Asp His Val Ser Ser Asn 180 185 190

Gin Phe Val Ser Met Gly He He Glu Pro Thr Ser Ala Gly Phe Pro

195 200 205

Ser Phe Arg Thr Leu Lys Thr Leu Tyr Leu Ser Asp Gly Val Asn Arg

210 215 220 Lys Ser Cys Ser He Ser Thr Val Pro Gly Gly Cys Met Met Tyr Cys

225 230 235 240

Phe Val Ser Thr Gin Pro Glu Arg Asp Asp Tyr Phe Ser Thr Ala Pro

245 250 255

Pro Glu Gin Arg He He He Met Tyr Tyr Asn Asp Thr He Val Glu 260 265 270

Arg He He Asn Pro Pro Gly Val Leu Asp Val Trp Ala Thr Leu Asn

275 280 285

Pro Gly Thr Gly Ser Gly Val Tyr Tyr Leu Gly Trp Val Leu Phe Pro

290 295 300 He Tyr Gly Gly Val He Lys Asn Thr Ser Leu Trp Asn Asn Gin Ala

305 310 315 320

Asn Lys Tyr Phe He Pro Gin Met Val Ala Ala Leu Cys Ser Gin Asn

325 330 335

Gin Ala Thr Gin Val Gin Asn Ala Lys Ser Ser Tyr Tyr Ser Ser Trp 340 345 350 Phe Gly Asn Arg Met He Gin Ser Gly He Leu Ala Cys Pro Leu Gin

355 360 365

Gin Asp Leu Thr Asn Glu Cys Leu Val Leu Pro Phe Ser Asn Asp Gin

370 375 380 Val Leu Met Gly Ala Glu Gly Arg Leu Tyr Met Tyr Gly Asp Ser Val

385 390 395 400

Tyr Tyr Tyr Gin Arg Ser Asn Ser Trp Trp Pro Met Thr Met Leu Tyr

405 410 415

Lys Val Thr He Thr Phe Thr Asn Gly Gin Pro Ser Ala He Ser Ala 420 425 430

Gin Asn Val Pro Thr Gin Gin Val Pro Arg Pro Gly Thr Gly Asp Cys

435 440 445

Phe Ala Thr Asn Arg Cys Pro Gly Phe Cys Leu Thr Gly Val Tyr Ala

450 455 460 Asp Ala Trp Leu Leu Thr Asn Pro Ser Ser Thr Ser Thr Phe Gly Ser

465 470 475 480

Glu Ala Thr Phe Thr Gly Ser Tyr Leu Asn Ala Ala Thr Gin Arg He

485 490 495

Asn Pro Thr Met Tyr He Ala Asn Asn Thr Gin He He Ser Ser Gin 500 505 510

Gin Phe Gly Ser Ser Gly Gin Glu Ala Ala Tyr Gly His Thr Thr Cys

515 520 525

Phe Arg Asp Thr Gly Ser Val Met Val Tyr Cys He Tyr He He Glu 530 535 540 Leu Ser Ser Ser Leu Leu Gly Gin Phe Gin He Val Pro Phe He Arg 545 550 555 560

Gin Val Thr Leu Ser

565 //(SEQ ID NO:22);

and

(xi) L has the following amino acid sequence (SEQ ID NO:24):

Met Ala Gly Ser Arg Glu He Leu Leu Pro Glu Val His Leu Asn Ser

1 5 10 15

Pro He Val Lys His Lys Leu Tyr Tyr Tyr He Leu Leu Gly Asn Leu 20 25 30

Pro Asn Glu He Asp He Asp Asp Leu Gly Pro Leu His Asn Gin Asn

35 40 45

Trp Asn Gin He Ala His Glu Glu Ser Asn Leu Ala Gin Arg Leu Val 50 55 60 Asn Val Arg Asn Phe Leu He Thr His He Pro Asp Leu Arg Lys Gly 65 70 75 80

His Trp Gin Glu Tyr Val Asn Val He Leu Trp Pro Arg He Leu Pro

85 90 95

Leu He Pro Asp Phe Lys He Asn Asp Gin Leu Pro Leu Leu Lys Asn 100 105 110

Trp Asp Lys Leu Val Lys Glu Ser Cys Ser Val He Asn Ala Gly Thr

115 120 125

Ser Gin Cys He Gin Asn Leu Ser Tyr Gly Leu Thr Gly Arg Gly Asn 130 135 140 Leu Phe Thr Arg Ser Arg Glu Leu Ser Gly Asp Arg Arg Asp He Asp

145 150 155 160

Leu Lys Thr Val Val Ala Ala Trp His Asp Ser Asp Trp Lys Arg He

165 170 175 Ser Asp Phe Trp He Met He Lys Phe Gin Met Arg Gin Leu He Val

180 185 190

Arg Gin Thr Asp His Asn Asp Pro Asp Leu He Thr Tyr He Glu Asn

195 200 205

Arg Glu Gly He He He He Thr Pro Glu Leu Val Ala Leu Phe Asn 210 215 220

Thr Glu Asn His Thr Leu Thr Tyr Met Thr Phe Glu He Val Leu Met

225 230 235 240

Val Ser Asp Met Tyr Glu Gly Arg His Asn He Leu Ser Leu Cys Thr

245 250 255 Val Ser Thr Tyr Leu Asn Pro Leu Lys Lys Arg He Thr Tyr Leu Leu

260 265 270

Ser Leu Val Asp Asn Leu Ala Phe Gin He Gly Asp Ala Val Tyr Asn

275 280 285

He He Ala Leu Leu Glu Ser Phe Val Tyr Ala Gin Leu Gin Met Ser 290 295 300

Asp Pro He Pro Glu Leu Arg Gly Gin Phe His Ala Phe Val Cys Ser

305 310 315 320

Glu He Leu Asp Ala Leu Arg Gly Thr Asn Ser Phe Thr Gin Asp Glu

325 330 335 Leu Arg Thr Val Thr Thr Asn Leu He Ser Pro Phe Gin Asp Leu Thr

340 345 350

Pro Asp Leu Thr Ala Glu Leu Leu Cys He Met Arg Leu Trp Gly His

355 360 365

Pro Met Leu Thr Ala Ser Gin Ala Ala Gly Lys Val Arg Glu Ser Met 370 375 380

Cys Ala Gly Lys Val Leu Asp Phe Pro Thr He Met Lys Thr Leu Ala

385 390 395 400

Phe Phe His Thr He Leu He Asn Gly Tyr Arg Arg Lys His His Gly

405 410 415 Val Trp Pro Pro Leu Asn Leu Pro Gly Asn Ala Ser Lys Gly Leu Thr

420 425 430

Glu Leu Met Asn Asp Asn Thr Glu He Ser Tyr Glu Phe Thr Leu Lys

435 440 445

His Trp Lys Glu He Ser Leu He Lys Phe Lys Lys Cys Phe Asp Ala 450 455 460

Asp Ala Gly Glu Glu Leu Ser He Phe Met Lys Asp Lys Ala He Ser

465 470 475 480

Ala Pro Lys Gin Asp Trp Met Ser Val Phe Arg Arg Ser Leu He Lys

485 490 495 Gin Arg His Gin His His Gin Val Pro Leu Pro Asn Pro Phe Asn Arg

500 505 510

Arg Leu Leu Leu Asn Phe Leu Gly Asp Asp Lys Phe Asp Pro Asn Val

515 520 525

Glu Leu Gin Tyr Val Thr Ser Gly Glu Tyr Leu His Asp Asp Thr Phe 530 535 540

Cys Ala Ser Tyr Ser Leu Lys Glu Lys Glu He Lys Pro Asp Gly Arg 545 550 555 560

He Phe Ala Lys Leu Thr Lys Arg Met Arg Ser Cys Gin Val He Ala

565 570 575

Glu Ser Leu Leu Ala Asn His Ala Gly Lys Leu Met Lys Glu Asn Gly 580 585 590

Val Val Met Asn Gin Leu Ser Leu Thr Lys Ser Leu Leu Thr Met Ser

595 600 605

Gin He Gly He He Ser Glu Arg Ala Arg Lys Ser Thr Arg Asp Asn

610 615 620 He Asn Arg Pro Gly Phe Gin Asn He Gin Arg Asn Lys Ser His His

625 630 635 640

Ser Lys Gin Val Asn Gin Arg Asp Pro Ser Asp Asp Phe Glu Leu Ala

645 650 655

Ala Ser Phe Leu Thr Thr Asp Leu Lys Lys Tyr Cys Leu Gin Trp Arg 660 665 670

Tyr Gin Thr He He Pro Phe Ala Gin Ser Leu Asn Arg Met Tyr Gly

675 680 685

Tyr Pro His Leu Phe Glu Trp He His Leu Arg Leu Met Arg Ser Thr

690 695 700 Leu Tyr Val Gly Asp Pro Phe Asn Pro Pro Ala Asp Thr Ser Gin Phe

705 710 715 720

Asp Leu Asp Lys Val He Asn Gly Asp He Phe He Val Ser Pro Arg

725 730 735

Gly Gly He Glu Gly Leu Cys Gin Lys Ala Trp Thr Met He Ser He 740 745 750

Ser Val He He Leu Ser Ala Thr Glu Ser Gly Thr Arg Val Met Ser

755 760 765

Met Val Gin Gly Asp Asn Gin Ala He Ala Val Thr Thr Arg Val Pro

770 775 780 Arg Ser Leu Pro Thr Leu Glu Lys Lys Thr He Ala Phe Arg Ser Cys

785 790 795 800

Asn Leu Phe Phe Glu Arg Leu Lys Cys Asn Asn Phe Gly Leu Gly His

805 810 815

His Leu Lys Glu Gin Glu Thr He He Ser Ser His Phe Phe Val Tyr 820 825 830

Ser Lys Arg He Phe Tyr Gin Gly Arg He Leu Thr Gin Ala Leu Lys

835 840 845

Asn Ala Ser Lys Leu Cys Leu Thr Ala Asp Val Leu Gly Glu Cys Thr

850 855 860 Gin Ser Ser Cys Ser Asn Leu Ala Thr Thr Val Met Arg Leu Thr Glu

865 870 875 880

Asn Gly Val Glu Lys Asp He Cys Phe Tyr Leu Asn He Tyr Met Thr

885 890 895

He Lys Gin Leu Ser Tyr Asp He He Phe Pro Gin Val Ser He Pro 900 905 910

Gly Asp Gin He Thr Leu Glu Tyr He Asn Asn Pro His Leu Val Ser

915 920 925

Arg Leu Ala Leu Leu Pro Ser Gin Leu Gly Gly Leu Asn Tyr Leu Ser 930 935 940 Cys Ser Arg Leu Phe Asn Arg Asn He Gly Asp Pro Val Val Ser Ala 945 950 955 960 Val Ala Asp Leu Lys Arg Leu He Lys Ser Gly Cys Met Asp Tyr Trp

965 970 975

He Leu Tyr Asn Leu Leu Gly Arg Lys Pro Gly Asn Gly Ser Trp Ala 980 985 990 Thr Leu Ala Ala Asp Pro Tyr Ser He Asn He Glu Tyr Gin Tyr Pro 995 1000 1005

Pro Thr Thr Ala Leu Lys Arg His Thr Gin Gin Val Leu Met Glu Leu

1010 1015 1020

Ser Thr Asn Pro Met Leu Arg Gly He Phe Ser Asp Asn Ala Gin Ala 1025 1030 1035 1040

Glu Glu Asn Asn Leu Ala Arg Phe Leu Leu Asp Arg Glu Val He Phe

1045 1050 1055

Pro Arg Val Ala His He He He Glu Gin Thr Ser Val Gly Arg Arg 1060 1065 1070 Lys Gin He Gin Gly Tyr Leu Asp Ser Thr Arg Ser He Met Arg Lys 1075 1080 1085

Ser Leu Glu He Lys Pro Leu Ser Asn Arg Lys Leu Asn Glu He Leu

1090 1095 1100

Asp Tyr Asn He Asn Tyr Leu Ala Tyr Asn Leu Ala Leu Leu Lys Asn 1105 1110 1115 1120

Ala He Glu Pro Pro Thr Tyr Leu Lys Ala Met Thr Leu Glu Thr Cys

1125 1130 1135

Ser He Asp He Ala Arg Ser Leu Arg Lys Leu Ser Trp Ala Pro Leu 1140 1145 1150 Leu Gly Gly Arg Asn Leu Glu Gly Leu Glu Thr Pro Asp Pro He Glu 1155 1160 1165

He Thr Ala Gly Ala Leu He Val Gly Ser Gly Tyr Cys Glu Gin Cys

1170 1175 1180

Ala Ala Gly Asp Asn Arg Phe Thr Trp Phe Phe Leu Pro Ser Gly He 1185 1190 1195 1200

Glu He Gly Gly Asp Pro Arg Asp Asn Pro Pro He Arg Val Pro Tyr

1205 1210 1215

He Gly Ser Arg Thr Asp Glu Arg Arg Val Ala Ser Met Ala Tyr He 1220 1225 1230 Arg Gly Ala Ser Ser Ser Leu Lys Ala Val Leu Arg Leu Ala Gly Val 1235 1240 1245

Tyr He Trp Ala Phe Gly Asp Thr Leu Glu Asn Trp He Asp Ala Leu

1250 1255 1260

Asp Leu Ser His Thr Arg Val Asn He Thr Leu Glu Gin Leu Gin Ser 1265 1270 1275 1280

Leu Thr Pro Leu Pro Thr Ser Ala Asn Leu Thr His Arg Leu Asp Asp

1285 1290 1295

Gly Thr Thr Thr Leu Lys Phe Thr Pro Ala Ser Ser Tyr Thr Phe Ser 1300 1305 1310 Ser Phe Thr His He Ser Asn Asp Glu Gin Tyr Leu Thr He Asn Asp 1315 1320 1325

Lys Thr Ala Asp Ser Asn He He Tyr Gin Gin Leu Met He Thr Gly

1330 1335 1340

Leu Gly He Leu Glu Thr Trp Asn Asn Pro Pro He Asn Arg Thr Phe 1345 1350 1355 1360

Glu Glu Ser Thr Leu His Leu His Thr Gly Ala Ser Cys Cys Val Arg 1365 1370 1375

Pro Val Asp Ser Cys He He Ser Glu Ala Leu Thr Val Lys Pro His

1380 1385 1390

He Thr Val Pro Tyr Ser Asn Lys Phe Val Phe Asp Glu Asp Pro Leu 1395 1400 1405

Ser Glu Tyr Glu Thr Ala Lys Leu Glu Ser Leu Ser Phe Gin Ala Gin

1410 1415 1420

Leu Gly Asn He Asp Ala Val Asp Met Thr Gly Lys Leu Thr Leu Leu 1425 1430 1435 1440 Ser Gin Phe Thr Ala Arg Gin He He Asn Ala He Thr Gly Leu Asp

1445 1450 1455

Glu Ser Val Ser Leu Thr Asn Asp Ala He Val Ala Ser Asp Tyr Val

1460 1465 1470

Ser Asn Trp He Ser Glu Cys Met Tyr Thr Lys Leu Asp Glu Leu Phe 1475 1480 1485

Met Tyr Cys Gly Trp Glu Leu Leu Leu Glu Leu Ser Tyr Gin Met Tyr

1490 1495 1500

Tyr Leu Arg Val Val Gly Trp Ser Asn He Val Asp Tyr Ser Tyr Met 1505 1510 1515 1520 He Leu Arg Arg He Pro Gly Ala Ala Leu Asn Asn Leu Ala Ser Thr

1525 1530 1535

Leu Ser His Pro Lys Leu Phe Arg Arg Ala He Asn Leu Asp He Val

1540 1545 1550

Ala Pro Leu Asn Ala Pro His Phe Ala Ser Leu Asp Tyr He Lys Met 1555 1560 1565

Ser Met Asp Ala He Leu Trp Gly Cys Lys Arg Val He Asn Val Leu

1570 1575 1580

Ser Asn Gly Gly Asp Leu Glu Leu Val Val Thr Ser Glu Asp Ser Leu 1585 1590 1595 1600 He Leu Ser Asp Arg Ser Met Asn Leu He Ala Arg Lys Leu Thr Leu

1605 1610 1615

Leu Ser Leu He His His Asn Gly Leu Glu Leu Pro Lys He Lys Gly

1620 1625 1630

Phe Ser Pro Asp Glu Lys Cys Phe Ala Leu Thr Glu Phe Leu Arg Lys 1635 1640 1645

Val Val Asn Ser Gly Leu Ser Ser He Glu Asn Leu Ser Asn Phe Met

1650 1655 1660

Tyr Asn Val Glu Asn Pro Arg Leu Ala Ala Phe Ala Ser Asn Asn Tyr 1665 1670 1675 1680 Tyr Leu Thr Arg Lys Leu Leu Asn Ser He Arg Asp Thr Glu Ser Gly

1685 1690 1695

Gin Val Ala Val Thr Ser Tyr Tyr Glu Ser Leu Glu Tyr He Asp Ser

1700 1705 1710

Leu Lys Leu Thr Pro His Val Pro Gly Thr Ser Cys He Glu Asp Asp 1715 1720 1725

Ser Leu Cys Thr Asn Asp Tyr He He Trp He He Glu Ser Asn Ala

1730 1735 1740

Asn Leu Glu Lys Tyr Pro He Pro Asn Ser Pro Glu Asp Asp Ser Asn 1745 1750 1755 1760 Phe His Asn Phe Lys Leu Asn Ala Pro Ser His His Thr Leu Arg Pro

1765 1770 1775 Leu Gly Leu Ser Ser Thr Ala Trp Tyr Lys Gly He Ser Cys Cys Arg

1780 1785 1790

Tyr Leu Glu Arg Leu Lys Leu Pro Gin Gly Asp His Leu Tyr He Ala 1795 1800 1805 Glu Gly Ser Gly Ala Ser Met Thr He He Glu Tyr Leu Phe Pro Gly 1810 1815 1820

Arg Lys He Tyr Tyr Asn Ser Leu Phe Ser Ser Gly Asp Asn Pro Pro 1825 1830 1835 1840

Gin Arg Asn Tyr Ala Pro Met Pro Thr Gin Phe He Glu Ser Val Pro 1845 1850 1855

Tyr Lys Leu Trp Gin Ala His Thr Asp Gin Tyr Pro Glu He Phe Glu

1860 1865 1870

Asp Phe He Pro Leu Trp Asn Gly Asn Ala Ala Met Thr Asp He Gly 1875 1880 1885 Met Thr Ala Cys Val Glu Phe He He Asn Arg Val Gly Pro Arg Thr 1890 1895 1900

Cys Ser Leu Val His Val Asp Leu Glu Ser Ser Ala Ser Leu Asn Gin 1905 1910 1915 1920

Gin Cys Leu Ser Lys Pro He He Asn Ala He He Thr Ala Thr Thr 1925 1930 1935

Val Leu Cys Pro His Gly Val Leu He Leu Lys Tyr Ser Trp Leu Pro

1940 1945 1950

Phe Thr Arg Phe Ser Thr Leu He Thr Phe Leu Trp Cys Tyr Phe Glu 1955 1960 1965 Arg He Thr Val Leu Arg Ser Thr Tyr Ser Asp Pro Ala Asn His Glu 1970 1975 1980

Val Tyr Leu He Cys He Leu Ala Asn Asn Phe Ala Phe Gin Thr Val 1985 1990 1995 2000

Ser Gin Ala Thr Gly Met Ala Met Thr Leu Thr Asp Gin Gly Phe Thr 2005 2010 2015

Leu He Ser Pro Glu Arg He Asn Gin Tyr Trp Asp Gly His Leu Lys

2020 2025 2030

Gin Glu Arg He Val Ala Glu Ala He Asp Lys Val Val Leu Gly Glu 2035 2040 2045 Asn Ala Leu Phe Asn Ser Ser Asp Asn Glu Leu He Leu Lys Cys Gly 2050 2055 2060

Gly Thr Pro Asn Ala Arg Asn Leu He Asp He Glu Pro Val Ala Thr 2065 2070 2075 2080

Phe He Glu Phe Glu Gin Leu He Cys Thr Met Leu Thr Thr His Leu 2085 2090 2095

Lys Glu He He Asp He Thr Arg Ser Gly Thr Gin Asp Tyr Glu Ser

2100 2105 2110

Leu Leu Leu Thr Pro Tyr Asn Leu Gly Leu Leu Gly Lys He Ser Thr 2115 2120 2125 He Val Arg Leu Leu Thr Glu Arg He Leu Asn His Thr He Arg Asn 2130 2135 2140

Trp Leu He Leu Pro Pro Ser Leu Gin Met He Val Lys Gin Asp Leu 2145 2150 2155 2160

Glu Phe Gly He Phe Arg He Thr Ser He Leu Asn Ser Asp Arg Phe 2165 2170 2175

Leu Lys Leu Ser Pro Asn Arg Lys Tyr Leu He Thr Gin Leu Thr Ala 2180 2185 2190

Gly Tyr He Arg Lys Leu He Glu Gly Asp Cys Asn He Asp Leu Thr

2195 2200 2205

Arg Pro He Gin Lys Gin He Trp Lys Ala Leu Gly Cys Val Val Tyr 2210 2215 2220

Cys His Asp Pro Met Asp Gin Arg Glu Ser Thr Glu Phe He Asp He 2225 2230 2235 2240

Asn He Asn Glu Glu He Asp Arg Gly He Asp Gly Glu Glu He 2245 2250 2255 //(SEQ ID NO 24)

The inventive protein also encompasses a polypeptide having substantially the same amino acid sequence as (SEQ ID NO 4), (SEQ ID NO 6) (SEQ ID NO 8), (SEQ ID NO 10), (SEQ ID NO 12), (SEQ ID NO 14), (SEQ ID NO 16), (SEQ ID NO 18), (SEQ ID NO 20), (SEQ ID NO 22), or (SEQ ID NO 24) As employed herein, the term "substantially the same amino acid sequence" refers to amino acid sequences having at least about 80%, still more preferably about 90% ammo acid identity with respect to a reference amino acid sequence, with greater than about 95% amino acid sequence identity being especially preferred It is recognized, however, that polypeptide containing less than the described levels of sequence identity arising as splice variants or that are modified by conservative amino acid substitutions are also encompassed within the scope of the present invention

The degree of sequence homology is determined by conducting an amino acid sequence similarity search of a protein data base, such as the database of the National Center for Biotechnology Information (NCBI, www ncbi nlm nih gov/BLAST/), using a computerized algorithm, such as PowerBLAST, QBLAST, PSI-BLAST, PHI-BLAST, gapped or ungapped BLAST, or the - Align" program through the Baylor College of Medicine server (www hgsc bcm tmc edu/seq_data) (E g .

Altchul, S F , et al , Gapped BLAST and PSI-BLAST a new generation of protein database search programs, Nucleic Acids Res 25( 17) 3389-402 [1997], Zhang, J , & Madden, T L , PowerBLAST a new network BLAST application for interactive or automated sequence analysis and annotation, Genome Res 7(6) 649-56 [ 1997|, Madden, T L , el al , Applications of network BLAST server. Methods Enzymol 266 131-41 [1996], Altschul, S F , et al , Basic local alignment search tool. J

Mol Biol 215(3) 403-10 [1990])

Also encompassed by the term Cryptovirus protein, are biologically functional or active peptide analogs thereof The term peptide "analog" includes any polypeptide having an ammo acid residue sequence substantially identical to a sequence specifically shown herein in which one or more residues have been conservatively substituted with a functionally similar residue and which displays the ability to mimic the biological activity of a native Cryptovirus protein Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine. leucine or methionine for another, the substitution of one polar (hydrophihc) residue for another such as between argimne and lysine, between glutamine and asparagine. between glycine and serine, the substitution of one basic residue such as lysine. argimne or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another

The phrase "conservative substitution" also includes the use of a chemically derivatized residue in place of a non-denvatized residue, provided that such polypeptide displays the requisite biological activity "Chemical derivative" refers to a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group Such derivatized molecules include, for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups Free carboxyl groups may be derivatized to fonn salts, methyl and ethyl esters or other types of esters or hydrazidcs Free hydroxyl groups may be derivatized to form O-acyl or

O-alkyl derivatives The lmidazole nitrogen of histidine may be derivatized to form N-nn-benzylhistidine Also included as chemical derivatives are those peptides which contain one or more naturally occurring ammo acid derivatives of the twenty standard ammo acids For example, 4-hydroxyprohne may be substituted for proline, 5-hydroxylysιne may be substituted for lysine, 3-methylhιstιdιnc may be substituted for histidine, homoserme may be substituted for serine, and onnthine may be substituted for lysine The inventive polypeptide also includes any polypeptide having one or more additions and or deletions of residues, relative to the sequence of an inventive polypeptide whose sequence is shown herein, so long as the requisite biological activity is maintained The present invention also encompasses a variant of a Cryptovirus protein designated by

(SEQ ID NO 4), (SEQ ID NO 6) (SEQ ID NO 8), (SEQ ID NO 10), (SEQ ID NO 12), (SEQ ID NO 14), (SEQ ID NO 16), (SEQ ID NO 18), (SEQ ID NO 20), (SEQ ID NO 22), or (SEQ ID NO 24), a "variant" refers to a polypeptide in which the amino acid sequence of the designated Cryptovirus protein has been altered by the deletion, substitution, addition or rearrangement of one or more ammo acids in the sequence Methods by which variants occur (for example, by recombination) or are made (for example, by site directed mutagenesis) are known in the art

The Cryptovirus protein can also include one or more labels, which are known to those of skill in the art The inventive proteins are isolated or purified by a variety of known biochemical means, including, for example, by the recombinant expression systems described herein, precipitation, gel filtration, ion-exchange, reverse-phase and affinity chromatography, electrophoresis, and the like.

Other well-known methods are described in Dcutscher et al , Guide to Protein Purification: Methods in Enzymology Vol. 182, (Academic Press, [1990])

The isolated Cryptovirus protein of the present invention can also be chemically synthesized. For example, synthetic polypeptide can be produced using Applied Biosystems, Inc. Model 430A or 431 A automatic peptide synthesizer (Foster City, CA) employing the chemistry provided by the manufacturer and the amino acid sequences provided herein. Alternatively, the Cryptovirus protein can be isolated or purified from native cellular sources. Alternatively, the Cryptovirus protein can be isolated from the inventive chimeric proteins by the use of suitable proteases.

Alternatively, the Cryptovirus proteins can be recombinantly derived, for example, produced by eukaryotic or prokaryotic cells genetically modified to express Cryptovirus protein-encoding polynucleotides in accordance with the inventive technology as described herein. Recombinant methods are well known, as described, for example, in Sambrook et al , supra., 1989). An example of the means for preparing the inventive Cryptovirus protein is to express nucleic acids encoding the Cryptovirus protein of interest in a suitable host cell that contains the inventive expression vector, such as a bacterial cell, a yeast cell, an insect cell, an amphibian cell (i.e., oocyte), or a mammalian cell, using methods well known in the art, and recovering the expressed polypeptide, again using well-known methods

"Recombinant host cells", "host cells", "cells", "cell lines", "cell cultures", and other such terms denoting prokaryotic or eukaryotic cell lines cultured as unicellular or monolayer entities, and refer to cells which can be, or have been, used as recipients for a recombinant expression vector or other foreign nucleic acids, such as DNA or RNA, and include the progeny of the original cell which has been transfected. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation.

The present invention includes chimeric proteins. The term "chimeric protein" generally refers to a polypeptide comprising an amino acid sequence drawn from two or more individual proteins that are not naturally so linked In the present invention, "chimeric protein" is used to denote a polypeptide comprising a Crytovirus protein, or a tmncate or polypeptide variant thereof having an altered amino acid sequence, fused to a non-Cryptovirus protein or polypeptide moiety. Chimeric proteins are most conveniently produced by expression of a fused gene, which encodes a portion of one polypeptide at the 5' end and a portion of a different polypeptide at the 3' end, where the different portions are joined in one reading frame which may be expressed in a suitable host cell In some embodiments, the Cryptovirus protein is positioned at the carboxy terminus of the chimeric protein In other embodiments the Cryptovirus protein is positioned at the ammo terminus of the chimeric protein

The non-Cry ptovirus protein moiety, or "chimeric partner", of the inventive chimeric protein can be a functional enzyme fragment Suitable functional enzyme fragments are those polypeptides which exhibit a quantifiable activity when expressed ftised to the Cryptovirus protein Exemplary enzymes mclude, without limitation, β-galactosidase (β-gal), β-lactamase, horseradish peroxidase

(HRP), glucose oxidase (GO), human superoxide dismutase (hSOD), urease, and the like These enzymes are convenient because the amount of chimeric protein produced can be quantified by means of simple colorimetπc assays Alternatively, one may employ antigemc proteins or fragments, e g , human superoxide dismutase (hSOD), to permit simple detection and quantification of chimeric proteins using antibodies specific for the non-Cryptovirus protein chimeric partner In chimeric proteins, useful chimeric partners include ammo acid sequences that provide for secretion from a recombinant host, enhance the immunological reactivity of a Cryptovirus protein epιtope(s), or facilitate the coupling of the Cryptovirus polypeptide to a support or a vaccine carrier (See, e g , EPO Pub No 1 16,201, U S Pat No 4,722,840, EPO Pub No 259,149, U S Pat No 4,629,783) Embodiments of the inventive Cryptovirus protein arc useful as immunoreactive polypeptides, including use for the production of a Cryptovirus -specific antibody "Immunoreactive" refers to the ability of a polypeptide to bind immunologically to an antibody and/or to a lymphocyte antigen receptor due to antibody or receptor recognition of a specific epitope contained within the polypeptide, or the ability of a polypeptide to be immunogenic An "immunogenic" protein is a polypeptide that elicits a cellular and/or humoral immune response, whether alone or linked to a carrier in the presence or absence of an adjuvant The immunogenicity of various isolated Cryptovirus proteins, or Cryptovirus protein fragments, of interest is determined by routine screening Immunological reactivity may be determined by antibody binding, more particularly by the kinetics of antibody binding, and/or by competition in binding using as competιtor(s) a known polypeptιde(s) containing an epitope against which the antibody is directed The techniques for determining whether a polypeptide is immunologically reactive with an antibody are known in the art Particularly useful examples of immunogenic Cryptovirus proteins are the envelope proteins, l e , F, Fo, F₂, F_1; , HN, and SH proteins As used herein, "epitope" refers to an antigenic determinant of a polypeptide An epitope can comprise 3 amino acids in a spatial conformation which is unique to the epitope. Epitopes typically are mapped to comprise at least about five amino acids, more usually at least about 8 amino acids to about 10 amino acids, or more. Methods of determining the spatial conformation of amino acids are known in the art, and include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance.

Immunogenic Cryptovirus proteins are useful for producing or manufacturing vaccines. As described above, Cryptovirus belongs to the Paramyxoviridae family of vimses. Numerous vaccines have been developed for humans and domestic animals against vimses in this vims family. For example, there are effective vaccines against measles vims, mumps vims, canine distemper vims, canine parainfluenza vims type 2, and Newcastle's disease vims (of fowl). These viaises were amenable to the development of effective vaccines because they have a narrow species tropism (; e , they infect only one, or only a few, species), they exist as only one, or only one predominant, serotype (making a vaccine universally protective against the viais), they cause significant morbidity in their host (i.e., they are significant causes of human illness), and they are pandemic (i.e., there is a worldwide concern).

Cryptovirus is amenable to vaccine development for the same reasons other family members have proven so. There is evidence for cross- neutralizability of hyper immune rabbit antiseaim made against different sources of the vims, and very similar nucleotide sequences of the viais genome have been obtained from two sources of the viais (BBR strain and Niigata cell-associated strain).

In accordance with the invention, multivalent or monovalent vaccines can be prepared against one or more Cryptovirus proteins. In particular, vaccines are contemplated comprising one or more Cryptovirus proteins, such as, but not limited to, envelope proteins F, F₀, F₂, Fi, HN, and/or SH. Methods for manufacturing vaccines which contain an immunogenic polypeptide as an active ingredient, are known to one skilled in the art Typically, such vaccines are prepared as injectable compositions comprising the Cryptovirus protein(s), either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified, or the protein encapsulated in liposomes. The active immunogenic ingredients are typically mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients or carriers are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine. Examples of adjuvants which may be effective include but are not limited to: aluminum hydroxide, N-acetyl-muramyl-L-theronyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L- alanyl-D-isoglutamine (CGP 1 1637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D- isoglutaminyl-L-alanine-2-(l-2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalcne/Tvveen 80 emulsion. The effectiveness of a particular adjuvant can be determined by measuring the amount of antibodies directed against an immunogenic Cryptovirus protein resulting from administration of this protein in vaccines which are also comprised of the adjuvant. The proteins can be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, maleic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine,

2-ethylamino ethanol, histidine, procaine, and the like.

The vaccines arc conventionally administered parenterally by injection, for example, either subcutaneously or intramuscularly, but they can also be delivered intranasally, enterically, or by any other delivery route. Administration can be with or without adjuvants Additional formulations of the vaccine composition that are suitable for other non-injection modes of administration include suppositories and, in some cases, oral fonnulations. For suppositories, traditional binders and carriers can include, for example, polyalkylene glycols or triglyccrides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably l%-2%. Oral formulations include such normally employed excipients as, for example, phannaceutical grades of mannitol, lactose, starch, magnesium stearatc, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, caplets, sustained release formulations or powders and contain typically 10%-95% of active ingredient, preferably 25%-70%.

In addition to the above, it is also possible to manufacture live vaccines of attenuated microorganisms, e.g., weakened or avimlent vims particles, including mutated Cryptovirus particles, and other attenuated vims particles containing an inventive recombinant nucleic acid encoding one or more Cryptovirus proteins, or host cells that express recombinant Cryptovirus proteins encoded by inventive expression vectors, as described herein. Suitable attenuated microorganisms are known in the art and include, for example, vimses (e.g., vaccinia vims) as well as bacteria.

Alternatively, killed Cryptovirus particles or virions, or killed pseudotyped viral particles or virions bearing Cryptovirus envelope proteins, are useftil in the manufacture of a vaccine. Virions are killed for vaccine purposes by known methods.

The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effective. The quantity to be administered, which is generally in the range of about 5 μg to about 250 μig of antigen per dose, depends on the subject to be vaccinated, capacity of the subject's immune system to synthesize antibodies, and the degree of protection desired Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and can be particular to each vaccinated animal or human. Any suitable vertebrate animal can be vaccinated, particularly a member of a mammalian species, including rodents, lagomorphs, goats, pigs, cattle, sheep, and primates.

The vaccine may be given in a single dose schedule, or preferably in a multiple dose schedule. A multiple dose schedule is one in which a primary course of vaccination may be with 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and/or reinforce the immune response, for example, at 1-4 months for a second dose, and if needed, a subsequent dose(s) after several months. The dosage regimen will also, at least in part, be determined by the immunological characteristics and needs of the individual animal or human to be vaccinated and must be dependent upon the judgment of the practitioner.

In addition, the vaccine containing the Cryptovirus proteins described above, can be administered in conjunction with other immunoregulatory agents, for example, immunoglobulins.

Compositions of the present invention can be administered to individual animals or humans to generate polyclonal antibodies (purified or isolated from semm using conventional techniques) which can then be used in a number of applications. For example, the polyclonal antibodies can be used to passively immunize an animal or human, or as immunochemical reagents, as described hereinbelow.

The present invention is also directed to an isolated antibody that specifically binds a Cryptovirus protein, such as the Cryptovirus NP, V, P, M, F, F₀, F₂, Fi, SH, HN, or L proteins. The term "antibody" refers to a polypeptide or group of polypeptides which are comprised of at least one antibody binding domain. A "binding domain" is formed from the folding of variable domains of an antibody molecule to fonu three-dimensional binding spaces with an internal surface shape and charge distribution complementary to the features of an epitope of an antigen, which allows an immunological reaction with the antigen. An antibody binding domain can be formed from a heavy and/or a light chain domain (V_H and V_L, respectively), which form hypervanable loops which contribute to antigen binding. Typical vertebrate antibodies are tetramers or aggregates thereof, comprising light and heavy chains which are typically aggregated in a "Y" configuration and which may or may not have covalent linkages between the chains. In vertebrate antibodies, the amino acid sequences of all the chains of a particular antibody are homologous with the chains found in one antibody produced by the lymphocyte which produces that antibody in vivo, or in vitro (for example, in hybridomas).

An "isolated" antibody is an antibody, for instance polyclonal antibody, removed from the body of an animal or human that produced it. The inventive polyclonal antibody can be further purified from cellular material, e.g., in blood, lymph, or milk. A preferred embodiment is in the form of an antiscmm directed against one or more Cryptovirus proteins. Alternatively, an "isolated" antibody of the present invention includes antibodies the production of which involves a manipulation or human intervention, for example, monoclonal antibody or chimeric antibody. The inventive "antibody" includes any immunoglobulin, including IgG, IgM, IgA, IgD or

IgE, or antibody fragment that binds a specific Cryptovirus epitope. Such antibodies can also be produced by hybridoma, chemical synthesis or recombinant methods known in the art. (E.g., Sambrook et al, Molecular Cloning: A Laboratory Manual (2 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA [1989]); Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory [1988]). Both mti-Cryptovirus protein and anti-chimeric protein antibodies can be useful within the scope of the present invention. (See, e.g., Bahouth et al, Trends Phannacol. Sci. 12:338 [1991]; Ausubel et al, Current Protocols in Molecular Biology (John Wiley and Sons, NY [1989]). Examples of chimeric antibodies are discussed in U.S. Pat. Nos. 4,816,397 and 4,816,567. Flurorescent-labeled antibodies, enzyme-conjugated antibodies, or antibodies otherwise labeled for facility of detection, as known in the art, are also included within

"antibody."

"Antibody" also includes "chimeric antibody." Chimeric antibodies are antibodies in which the heavy and/or light chains are chimeric proteins. Typically the constant domain of the chains is from one particular species and/or class, and the variable domains are from a different species and/or class. Also included is any antibody in which either or both of the heavy or light chains are composed of combinations of sequences mimicking the sequences in antibodies of different sources, whether these sources be differing classes, or different species of origin, and whether or not the fusion point is at the variable/constant boundary. Thus, it is possible to produce antibodies in which neither the constant nor the variable region mimic known antibody sequences. It then becomes possible, for example, to constmct antibodies whose variable region has a higher specific affinity for a particular antigen, or whose constant region can elicit enhanced complement fixation, or to make other improvements in properties possessed by a particular constant region. Included also within the definition of "antibody" are Fab and F(ab^')₂ fragments of antibodies. The "Fab" region refers to those portions of the heavy and light chains which are roughly equivalent, or analogous, to the sequences which comprise the branch portion of the heavy and light chains, and which have been shown to exhibit immunological binding to a specified antigen, but which lack the effector F_c portion. A "Fab" fragment is an aggregate of one heavy and one light chain A F(ab')₂ fragment, which also lacks the effector F_c portion, is a tetramer containing the 2H and 2L chains, which arc capable of selectively reacting with a designated antigen or antigen family. Methods of producing "Fab" and F(ab')₂ fragments of antibodies are known within the art and include, for example, proteolysis, and synthesis by recombinant techniques. Thus, the inventive anti- Cryptovirus antibodies can also be Fab or F(ab^')₂ antibody fragments Also useful is a "single domain antibody" (dAb), which is an antibody which is comprised of a V_H domain, which reacts immunologically with a Cryptovirus antigen. A dAB does not contain a V_L domain, but may contain other antigen binding domains known to exist in antibodies, for example, the kappa and lambda domains. Methods for preparing dABs arc known in the art. (See, e g , Ward et al, Nature 341: 544 [1989]). Other preferred embodiments include altered antibodies such as humanized, CDR-graftcd or bifunctional, i e., divalent antibodies, all of which can also be produced by methods well known in the art. "Altered antibodies", which refers to antibodies in which the naturally occurring amino acid sequence in a vertebrate antibody has been varied. Utilizing recombinant DNA techniques, antibodies can be redesigned to obtain desired characteristics. The possible variations are many, and range from the changing of one or more amino acids to the complete redesign of a region, for example, the constant region. Changes in the constant region, in general, to attain desired cellular process characteristics, e.g , changes in complement fixation, interaction with membranes, and other effector functions. Changes in the variable region may be made to alter antigen binding characteristics. The antibody may also be engineered to aid the specific delivery of a molecule or substance to a specific cell or tissue site. The desired alterations may be made by known techniques in molecular biology, e.g., recombinant techniques, site directed mutagenesis, etc.

A preferred embodiment of the inventive antibody specifically binds a Cryptovirus envelope protein described hereinabove. Another preferred embodiment of the inventive antibody specifically binds a Cryptovirus nucleocapsid protein, but antibodies that specificaally bind any other Cryptovirus protein are also useful and preferred.

The inventive antibody can be used, inter alia, in diagnostic or assay methods and systems to detect Cryptovirus proteins present in a sample of biological material. With respect to the detection of such polypeptide, the antibodies can be used for in vitro diagnostic or assay methods, or in vivo imaging methods. Such antibodies can also be used for the immunoaffmity or affinity chromatography purification of the inventive Cryptovirus proteins.

The present invention includes an in vitro method of detecting the presence or absence of a Cryptovirus protein in a sample of a biological material. The method is particularly, but not exclusively, useful for testing clinical or experimental biological materials for diagnostic or pathology purposes.

The sample is contacted with the inventive antibody described herein; and known immunological procedures are employed for detecting specific binding of the antibody to a constituent of the sample. The presence of specific binding indicates the presence of the Cryptovirus protein in the sample.

Immunological procedures, useful for in vitro detection of target Cryptovirus proteins in a sample, include immunoassay systems that employ the inventive Cryptovirus protein-specific antibody in a detectable form.

Known protocols for such immunoassay techniques and systems are based, for example, upon competition, or direct reaction, or sandwich type assays. Such immunoassay techniques and systems include, for example, ELISA, immunoblotting, immunofluorescence assay (IFA), Pandex microfluorimetric assay, agglutination assays, flow cytometry, semm diagnostic assays and immunohistochemical staining procedures which are well known in the art.

An antibody can be made detectable by various means well known in the art. Assay systems that amplify the signals from a primary antibody-antigen complex are also known; examples of which are assays which utilize biotin and avidin, and enzyme-labeled and mediated immunoassays, such as ELISA assays. A detectable marker can be directly or indirectly attached to a primary or secondary antibody in the assay protocol. Useful markers include, for example, radionuclides, enzymes, fluorogens, chromogens and chemiluminescent labels. Embodiments can employ solid support matrices, or can involve immunoprecipitation. These same immunoassay techniques and systems can be employed in the inventive method of detecting the presence or absence of a Cry/>tov/rw.y-specific antibody in an antibody-containing biological material. Antibody-containing biological materials include, but are not limited to, whole blood and blood components, plasma, seaim, spinal fluid, lymph fluid, the external sections of the respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, white blood cells, and myelomas.

Processed antibody-containing fractions or dilutions of any of these are also considered antibody- containing biological materials.

One preferred embodiment of the method of detecting the presence or absence of a Cry/?tov/rw.y-specific antibody involves contacting the sample originating from an individual suspected of having a Cryptovirus infection with a viral virion or other viral particle containing one or more Cryptovims envelope proteins, as described herein, such that, if antibody selectively binding Cryptovirus antigen is present, an antibody-bound complex forms. Then any antibody-bound Cryptovirus antigen complexes thus formed are contacted with anti-human antibody-binding immunoglobulin or anti-human antibody-binding fragments thereof, for example, Fab and/or F(ab^')₂, fragments, and complexes of the immunoglobulin or the fragments thereof, arc allowed to form with the antibody-bound Cryptovirus complexes. The presence or absence of any complexes formed is detected, by any known immunodetection means as described herein. The presence of such complexes indicates the presence in the sample of antibody that selectively binds Cryptovirus antigen.

Another more preferred embodiment involves contacting the sample originating from an individual suspected of having a Cryptovirus infection with the inventive Cryptovirus envelope protein, such that, if antibody selectively binding Cryptovirus is present, an antibody-bound envelope protein complex forms. Any antibody-bound envelope protein complexes thus formed are then contacted with anti-human antibody-binding immunoglobulin or anti-human antibody-binding fragments, such as Fab and F(ab^')₂ fragments. The formation of complexes of the immunoglobulin or the Fab and F(ab') fragments thereof is allowed with the antibody-bound envelope protein complexes; and the presence or absence of any antibody-bound envelope protein complexes thus formed is detected. The presence of such complexes indicating the presence in the sample of antibody selectively binding Cryptovirus .

The terms "selective" or "specific" binding of antibody to Cryptovirus proteins or Cryptovirus antigens therein, includes asymmetric cross-reactive binding with closely related mbulavimses, such as SV5, but does not include non-specific binding to unrelated antigens or surfaces. The skilled artisan is aware of important controls that are preferably included in any immunoassay system for the determination of non-specific antibody binding Typically, for example in ELISA, a background level of non-specific binding is determined and used as a baseline.

"Complexed" means that a protein, such as an antibody, is a constituent or member of a complex, i.e., a mixture or adduct resulting from chemical binding or bonding between and/or among the other constituents. Chemical binding or bonding can have the nature of a covalent bond, ionic bond, hydrogen bond, hydrophobic bond, or any combination of these bonding types linking the constituents of the complex at any of their parts or moieties, of which a constituent can have one or a multiplicity of moieties of various sorts. Antibody-antigen binding is typically non-covalent. Not every constituent of a complex need be bound to every other constituent, but each constituent has at least one chemical bond with at least one other constituent of the complex. For example, a secondary antibody in the assay system may not be directly bound with the Cryptovirus antigen, or the viral particle or virion, yet it is "complexed" with it.

By way of further non-limiting illustration of a clinical diagnostic embodiment for detecting Cryptovirus infection in a human patient, a semm specimen from the patient is screened for Cryptovirus-specific antibodies to the major envelope proteins of the vims (Fo and HN) by ELISA, radio-immunoprecipitation, immunoblotting techniques or any other immunological technique (e.g., direct or indirect fluorescent antibody techniques, immunobeads, etc). Optionally, another blood sample is obtained from any seropositive individual. The viais can be detected in the PBMNCs of such samples by isolating the cells on a suitable gradient medium, culturing the cells in the presence of cyclic GMP and then either (1) screening the cells for intracellular Cryptovirus-specific inclusions with Cr /?/ov/Vz(.y-spccific fluorescent antibodies or (2) PCR amplification of induced PBMNC with Cry/?tov?/ -specifιc nucleotide primers

While Cry >/ vzrw.y-specific antibodies have been found in the semm of seropositive individuals, indicating current or former Cryptovirus infection, these antibodies arc not necessarily indicative of epileptiform or encephalopathic disease. Cryptovirus appears to infect and be carried in the PBMNCs of a significant proportion of individuals without necessarily causing encephalopathic disease. These individuals can overtly appear to be asymptomatic. The neuropathological (e.g., epileptiform, encephalopathic, and other neurological, neurodegenerative, and/or neuropsychiatric disease) potential of the vims only appears to become manifest in individuals in whom the viais has infected nervous system tissues. Consequently, only Cryptovirus-specific antibodies (i.e. those directed against the major envelope proteins of the viais, F and HN) found in the cerebrospinal fluid (CSF) are fully indicative of neurological, neurodegenerative, and or neuropsychiatric disease and then, they are virtually always indicative. By way of ftirther example, ten human patients who were previously diagnosed with chronic fatigue syndrome involving significant short-term memory loss, and for whom CSF samples could be obtained, were all found to have Cryptovirus-specific antibody in their CSF and electroencephalographic profiles consistent with a diagnosis of absence epilepsy (i.e., petit mal epilepsy). The present invention also relates to an ti-Cryptovirus antibody detecting kit, useful for testing or assaying a biological sample, in particular an antibody-containing biological material. Thus, the inventive kits are beneficial for screening clinical supplies of human blood, seaim, platelets, plasma, tissues and organs, to determine their safety for transfusion or transplantation purposes. Diagnostic applications of the inventive kits are also useful. The inventive kit is particularly useftil for practicing the inventive assay methods for detecting antibody that selectively binds Cryptovirus and its antigens and the inventive methods of detecting the presence or absence of a Cryptovirus-specific antibody. In some preferred embodiments, the kit contains an isolated Cryptovirus particle, comprising a genome having a nucleotide sequence entirely complementary to (SEQ ID NO:l). The kit also contains labeled anti- human antibody-binding antibody, preferably anti-human immunoglobulin or labeled anti-human antibody-binding antibody fragments, such as Fab and/or F(ab')₂ A preferred embodiment of the kit further contains a solid matrix for supporting the Cryptovirus particle(s). In a preferred embodiment, the Cryptovims particles in the anti-Cryptovirus antibody detecting kit are Cryptovirus virions, and in a preferred embodiment the Cryptovirus virions are produced from an acutely Cryptovirus-infected cell line, such as an acutely infected baby hamster kidney (BHK) cell-derived cell line, a Vero- dcrived cell line, or a CV-1-derived cell line (e.g., a CV-l_c-derived cell line, described hereinbelow, and deposited with ATCC as Accession No. ).

Alternatively, in a most preferred embodiment of the inventive wnti-Cryptovirus antibody detecting kit, which does not include Cryptovirus particles, the kit includes a plurality of one or more kinds of isolated Cryptovirus proteins and/or chimeric proteins comprising a Cryptovirus protein moiety, as described hereinabove. In some preferred embodiments, the Cryptovims protein or protein moiety is an envelope protein, as described herein. Such embodiments also contain labeled anti-human antibody-binding antibody, such as anti-human immunoglobulin or labeled anti-human antibody-binding antibody fragments, such as Fab and/or F(ab')₂ fragments. A preferred embodiment of such a kit further contains a solid matrix for supporting the Cryptovirus proteins.

Solid matrices, or supports, and methods for attaching or adsorbing viral particles and proteins to a solid matrix, are well known in the art. In accordance with the inventive kits, the term "solid matrix" includes any solid or semi-solid support or surface to which the viral particle or protein can be anchored or adhered Suitable matrices are made of glass, plastic, metal, polymer gels, and the like, and may take the form of beads, wells (e g., single- or multi-well seaim plates) slides, dipsticks, membranes, and the like.

As is known to the skilled artisan in using such kits, e g., for ELISA, R1A, or sandwich-type assays, the biological sample, optimally solubilized or suspended and in an optimized dilution, is contacted with the viral particles or proteins, typically supported on the surface of the solid matrix (e.g., well of a semm plate), and after appropriate washes and incubations, is further contacted with the labeled anti-human antibody-binding immunoglobulin or labeled anti-human antibody-binding fragments. After further washes, commercially available plate readers and/or other accessor}' detection equipment are typically employed in conjunction with the inventive kit, for detecting the formation of bound anti-human antibody complexes with human antibody that has bound to Cryptovirus particles or proteins.

Instructions for use are included in the kit. "Instructions for use" typically include a tangible expression describing the reagent concentration or at least one assay method parameter, such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions, incubations, washes, and the like, typically for an intended purpose, in particular the inventive assay methods as described herein.

Optionally, the kit also contains other useful components, such as, diluents, buffers, or other acceptable carriers, specimen containers, syringes, pipetting or measuring tools, paraphernalia for concentrating, sedimenting, or fractionating samples, or the inventive antibodies for use in controls.

The materials or components assembled in the kit can be provided to the practitioner stored in any convenient and suitable ways that preserve their operability and utility For example the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room, refrigerated or frozen temperatures. The components are typically contained in suitable packaging material(s). As employed herein, the phrase "packaging material" refers to one or more physical stmctures used to house the contents of the kit. The packaging material is constaictcd by well known methods, preferably to provide a sterile, contaminant-free environment.

The packaging materials employed in the kit are those customarily utilized in vims- and peptide-based systems. As used herein, the term "package" refers to a suitable solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding the individual kit components. Thus, for example, a package can be a glass vial used to contain suitable quantities of an inventive composition containing viral or peptide components The packaging material generally has an external label which indicates the contents, quantities, and/or purpose of the kit and/or its components

Thus the present invention provides immunoassay methods and kits, useftil for research, clinical diagnostics, and screening of blood, blood components or products, and tissue and organs intended for transfusion or transplantation These applications are of great value and utility, because strong evidence shows that peripheral blood mononuclear cells (PBMNCs) can harbor Cryptovirus and can subsequently infect and cause disease in a patient who receives such contaminated blood, blood products, tissues, or cells The inventive methods of detecting the presence or absence of a Cryptovirus -specific RNA m a sample of a biological material, described hercinabove, are also particularly useful for these and other purposes In accordance with the most preferred embodiments of the method, Cryptovirus RNAs are amplified by any of numerous known methods of amplifying nucleic acid segments, in the form of RNA or cDNA Before amplification, it is preferable, but not necessary, to extract or separate RNA from DNA in the sample and to amplify nucleic acids remaining in that fraction of the sample separated from the DNA The amplifications products, if any, are then analyzed to detect the presence of Cryptovirus-specific amplification products If Cryptovirus-specific amplification products are present, the findings are indicative of the presence of Cryptovirus RNA in the sample For greater confidence in the interpretation of negatives (l e , no detectable Cryptovirus -specific amplification products), analysis is optionally carried out following a control amplification of mRNAs specific for a cellular housekeeping gene, for example, a gene encoding β-actm, phosphofaictokinase (PFK), glyceraldehyde 3-phosphate dehydrogenase, or phosphoglycerate kinase

The RNAs in the sample, are amplified by a suitable amplification method For example, in a preferred embodiment, a reverse transcπptase-mediated polymerase chain reaction (RT-PCR) is employed to amplify Cryptovirus -specific nucleic acids Briefly, two enzymes are used in the amplification process, a reverse transcriptase to transcribe Cryptovirus -specific cDNA from a Cryptovirus-specific RNA template in the sample, a thermal resistant DNA polymerase (e g , Jaq polymerase), and Cryptovirus -specific primers to amplify the cDNA to produce Cryptovirus-specific amplification products The use of limited cycle PCR yields semi-quantitative results (E g , Gelfand et al , Reverse transcription with thermostable DNA polymerase-high temperature reverse transcription, U S Patent Nos 5,310,652, 5,322,770, Gelfand et al , Unconventional nucleotide substitution in temperature selective RT-PCR, U S Patent No 5,618,703) In another preferred embodiment of the inventive method, single enzyme RT-PCR is employed to amplify Cryptovirus-specific nucleic acids. Single enzymes now exist to perform both reverse transcription and polymerase functions, in a single reaction For example, the Perkin Elmer recombinant Thermus thermophilus (rTth) enzyme (Roche Molecular), or other similar enzymes, are commercially available. Cycling instmments such as the Perkin Elmer ABI Prism 7700, the so- called Light Cycler (Roche Molecular), and/or other similar instalments are useftil for carrying out RT-PCR. Optionally, single enzyme RT-PCR technology, for example, employing rTth enzyme, can be used in a PCR system.

By way of illustration only, RT-PCR-based testing is quite sensitive for detection of the vims. For example, a RT-PCR-priming technique has been used to confirm a detectable Cryptovirus carnage rate in PBMNCs nonproductively harboring the vims (without culturing, cyclic GMP induction, and passaging) on the order of 1 : 10⁵ PBMNC. In addition, numerous fragments of the Cryptovirus genome have been cloned from AV₃/SSPE cells using Cry/5/øv rw.y-specific primers and a RT-PCR-based amplification technique. Preferably, amplification and analysis are carried out in an automated fashion, with automated extraction of RNA from a sample, followed by PCR, and fluorescence detection of amplification products using probes, such as TaqMan or Molecular Beacon probes. Typically, the instrumentation includes software that provides quantitative analytical results during or directly following PCR without further amplification or analytical steps. In another preferred embodiment, transcription-mediated amplification (TMA) is employed to amplify Cryptovirus -specific nucleic acids. (E.g., K Kamisango et al, Quantitative detection of hepatitis B virus by transcription-mediated amplification and hybridization protection assay, J. Clin. Microbiol. 37(2):310-14 [1999]; M. Hirosc et al, New method to measure telomera.se activity by transcription-mediated amplification and hybridization protection assay, Clin Chem 44(12)2446-52 [19981). Rather than employing RT-PCR for the amplification of a cDNA, TMA uses a probe that recognizes a Cr y?tov/ «.y-specifιc (target sequence) RNA; in subsequent steps, from a promoter sequence built into the probe, an RNA polymerase repetitively transcribes a cDNA intermediate, in effect amplifying the original RNA transcripts and any new copies created, for a level of sensitivity approaching that of RT-PCR. The reaction takes place isothermally (one temperature), rather than cycling through different temperatures as in PCR

Other useful amplification methods include a reverse transcriptase-mediated ligase chain reaction (RT-LCR), which has utility similar to RT-PCR. RT-LCR relies on reverse transcriptase to generate cDNA from mRNA. then DNA ligase to join adjacent synthetic oligonucleotides after they have bound the target cDNA

Most preferably, amplification of a Cryptovirus -specific nucleic acid segment in the sample can be achieved using Cryptovirus -specific oligonucleotide primers of the present invention, as described herein

Optionally, high throughput analysis may be achieved by multiplexing techniques well known in the art, employing multiple primer sets, for example primers directed not only to Cryptovirus -specific nucleic acids, but to amplifying expression products of housekeeping genes (controls) or of other potential diagnostic markers, to yield additional diagnostic information (E g . Z Lin el al , Multiplex genotype determination at a large number of gene loci, Proc Natl Acad Sci

USA 93(6) 2582-87 [1996], Demetnou et al , Method and probe for detection of gene as sociated with liver neoplastic disease, U S Patent No 5.866,329)

Hybridization analysis is a preferred method of analyzing the amplification products to detect the presence or absence of Cryptovirus-specific nucleic acid amplification products, employing one or more Cryptovirus -specific probe(s) that, under suitable conditions of stringency, hybndιze(s) with single stranded Cryptovirus -specific nucleic acid amplification products comprising complementary nucleotide sequences The amplification products are typically deposited on a substrate, such as a cellulose or nitrocellulose membrane, and then hybridized with labeled Cryptovirus -specific probc(s). optionally after an electrophoresis Conventional dot blot, Southern, Northern, or fluorescence in situ (FISH) hybridization protocols, in liquid hybridization, hybridization protection assays, or other semi-quantitative or quantitative hybridization analysis methods arc usefully employed along with the Cryptovirus -specific probes of the present invention As is readily apparent to the skilled artisan, such analytical hybridization techniques and others (e g , Northern blotting), are useful in accordance with other embodiments of the inventive method of detecting the presence or absence of a Cryptovirus -specific RNA in a sample of a biological material that do not involve any amplification step(s) In these embodiments, the inventive Cryptovirus -specific probes are contacted directly with RNA in the sample to perform hybridization analysis

Alternatively, electrophoresis for analyzing or detecting amplification products is done rapidly and with high sensitivity by using any of various methods of conventional slab or capillary electrophoresis, with which the practitioner can optionally choose to employ any facilitating means of nucleic acid fragment detection, including, but not limited to, radionuclides, UV-absorbance or laser- induced fluorescence (K Keparnik et al , Fast detection of a (CA)18 microsatellite repeat in the IgE receptor gene by capillary electrophoresis with laser-induced fluorescence detection, Electrophoresis 19(2),249-55 [1998], H Inoue et al . Enhanced separation of DNA sequencing products by capillary electrophoresis using a stepwise gradient of electric field strength, J Chromatogr A 802(1) 179-84 [1998], N J Dovichi, DNA sequencing by capillary electrophoresis, Electrophoresis 18(12-13) 2393- 99 [1997], H Arakawa et al , Analysis of single-strand conformation polymorphisms by capillary electrophoresis with laser induced fluorescence detection, 1 Phaπ Biomed Anal 15(9-10) 1537-44

[1997], Y Baba, Analysis of disease-causing genes and DNA-based drugs by capillary electrophoresis Towards DNA diagnosis and gene therapy for human diseases, J Chromatgr B Biomed Appl 687(2) 271-302 [1996], K C Chan et al , High-speed electrophoretic separation of DNA fragments using a short capillary, J Chromatogr B Biomed Sci Appl 695(1) 13-15 [1997]) Any biological material can be sampled for the purpose of practicmg the inventive methods of detecting the presence or absence of a Cryptovirus -specific protein or Cryptovirus -specific RNA in a sample of a biological material Preferred biological materials for sampling include blood or seaim, lymphoid tissue nervous tissue, including brain tissue However, the biological material can be cerebrospinal fluid (CSF), lymph, plasma, feces, semen, prostatic fluid, tears, saliva, milk, gastric juice, mucus, synovial fluid, pleural effusion, peritoneal efftision, pencardial effusion, skin, vascular epithelium, oral epithelium, vaginal epithelium, cervical epithelium, uterine epithelium, intestinal epithelium, bronchial epithelium, esophageal epithelium, or mesothelium, or other biopsy sample of cellular material from any tissue Cellular material includes any sample containing mammalian cells, including samples of tissues, expressed tissue fluids, tissue wash or tissue πnsate fluids, blood cells (e g , peripheral blood mononuclear cells), tumors, organs, and also samples of in vitro cell culture constituents (including but not limited to conditioned medium resulting from the growth of cells in cell culture medium, putatively virally infected cells, recombinant cells, and cell components), or the like

Tissue samples that can be collected include, but are not limited to, cell-containing material from the brain, blood, spleen, lymph node, vasculaturc, kidney, pituitary, ureter, bladder, urethra, thyroid, parotid gland, submaxillary gland, sub ngual gland, bone, cartilage, lung, mediastinum, breast, uteais, ovary, testis, prostate, cervix uteri, endometπum, pancreas, liver, adrenal, esophagus, stomach, and/or intestine

The sample is alternatively derived from cultured mammalian cells, cell-free extracts, or other specimens indirectly derived from a mammalian subject's body, as well as from substances taken directly from a subject's body The samples can be stored before detection methods are applied (for example nucleic acid amplification and/or analysis, or lmmunochemical detection) by well known storage means that will preserve nucleic acids or proteins in a detectable and/or analyzable condition, such as quick freezing, or a controlled freezing regime, in the presence of a cryoprotectant, for example, dimethyl sulfoxide (DMSO), glycerol, or propanediol-sucrose Samples may also be pooled before or after storage for purposes of amplifying their Cryptovirus-specific nucleic acids for analysis and detection, or for purposes of detecting Cryptovirus protein The sample is optionally pre-treated by refrigerated or frozen storage overnight, by dilution, by phenol-chloroform extraction, or by other like means, to remove factors that may inhibit various amplification reactions that may be employed, such as heme-contaimng pigments or urinary factors (E g , J Mahony et al , Urine specimens from pregnant and non-pregnant women inhibitory to amplification of Chlamydia trachomatis nucleic acid by PCR, ligase chain reaction, and transcription-mediated amplification identification of urinary substances as sociated with inhibition and removal of inhibitory activity, J Clm Microbiol 36(11) 3122-26 [1998])

The present invention is also directed to an animal model for the study of human diseases, preferably, but not limited to, neurological, neurodegenerative, and or neuropsychiatric diseases The term "neurological diseases" refers to diseases of the nervous system, including neuropathies manifested in the central nervous system and/or the peripheral nervous system These include epileptiform diseases and non-epileptiform CNS diseases (e g Parkinsomsm) and peripheral nervous system dιsease(s) (e g amyotrophic lateral sclerosis or "Lou Gehπg's Disease " "Neurodegenerative" diseases involve wasting or paralytic neurological diseases, which typically present with tremor, weakness and atrophy, for example Lou Gehπg's Disease or Alzheimer's disease The terms "neuropsychiatric" and "neuropsychological" arc used interchangeably herein

"Neuropsychiatric diseases" are neurological diseases that also include behavioral symptoms that derive from the underlying ncurophysiological processes The animal model is a non-human mammal, such as, but not limited to, a rodent, a lagomorph, or a non-human primate

A rodent is any of the relatively small gnawing mammals of the order Rodentia , such as mice, rats, hamsters, guinea pigs, squirrels, mannots, beavers, gophers, voles, porcupines, and agoutis A lagomorph is any of various herbivorous mammals belonging to the order Lagomorpha, which includes rabbits, hares, and pikas

The animal is, or has been, inoculated with an infectious cell-free Cryptovirus virion of the present invention Alternatively, the animal is, or has been, artificially inoculated with a cell nonproductively-infected with Cryptovirus, such as AV₃/SSPE

Inoculation can be by a peripheral or an intracerebral delivery route For the study of neurological neurodegenerative, and/or neuropsychiatric diseases, intracerebral inoculation is preferred, although for diseases presenting with involvement of the peripheral nervous system, peripheral inoculation can also be useful

Intracerebral inoculation is by any suitable means, but preferably by direct injection into the brain, preferably into neural tissue, and most preferably by stereotactic injection means known in the art Alternatively, intracerebral inoculation with Cryptovirus or nonproductitively infected cells can be by lntraarteπal (e g , lntracarotid) or intravenous injection or infusion, in conjunction with at least transient dismption of the blood bram barrier by physical or chemical means, delivered simultaneously with the Cryptovirus or nonproductively infected cells

"Simultaneously" means that the physical or chemical means for disrupting the blood brain baaier are administered contemporaneously or concurrently with the Cryptovirus virions or nonproductively infected cells "Simultaneously" also encompasses disaipting means being administered within about one hour after the Cryptovirus or nonproductively infected cells are last administered, preferably within about 30 minutes after, and most preferably, being administered simultaneously with the Cryptovirus or nonproductively infected cells Alternatively. "simultaneously" means that the medicant is administered within about 30 minutes before, and preferably within about 15 minutes before the Cryptovirus or nonproductively infected cells are first administered

Physical disaiption of the blood brain barrier includes by means of "mechanical" injury or other physical trauma that breaches the blood brain barrier in at least one location of the brain's vasculature Chemical dismption includes by an agent that transiently permeabihzes the blood-brain baaier and allows the Cryptovirus to enter the brain from the blood stream via the brain microvasculaturc Such permeabilizing agents are known, for example, bradykinin and bradyki n analogs, and activators of calcium-dependent or ATP -dependent potassium channels (e g , B Malfroy-Camine, Method for increasing blood-brain barrier permeability by administering a bradykinin agonist of blood-brain barrier permeability, U S Patent No 5,1 12,596, J W Kozanch et al , Increasing blood brain barrier permeability with permeabihzer peptides, U S Patent No 5,268,164, Inamura, T et al , Bradykinin selectively opens blood-tumor barrier in experimental brain tumors, J Cereb Blood Flow Metab 14(5) 862-70 [1994], K L Black, Method for selective opening of abnormal brain tissue capillaries, U S Patent Nos 5,527,778 and 5,434,137, N G Rainov, Selective uptake of viral and monocrystalline particles delivered intra-arterially to experimental brain neoplasms, Hum Gene Ther 6(12) 1543-52 [1995], N G Rainov et a! , Long-term survival in a rodent brain tumor model by bradykinin-enhanced intra-arterial delivery of a therapeutic herpes simplex virus vector, Cancer Gene Ther 5(3) 158-62 [1998], F H Bamett et al . Selective delivery of herpes virus vectors to experimental brain tumors using RMP-7, Cancer Gene Ther 6(1) 14-20 [1999]. WO 01/54771 A2. and WO 01/54680 A2)

The inoculated non-human mammal exhibits at least one symptom characteristic of a human neurological, neurodegenerative, and/or neuropsychiatric disease after being thus inoculated, which was not previously exhibited by the non-human mammal before inoculation Such symptoms include subacute symptoms and more slowly developing symptoms

Generally, the subacute symptoms (developing from about 3 weeks to about 2 months post inoculation) associated with such experimental infections include (1) cachexia/anorexia (i e , wasting or diminution of body mass and size), (2) degenerative neurologic wasting or paralysis, (3) atrophy of lnnb(s), (4) hindhmb paralysis, (5) photosensitivity or repetitive blinking, (6) hypcractivity or hypcresthesia (e g . nervousness, agitation, racing, jumpiness, extreme sensitivity to touch and sound), (7) ataxia (i e , loss of balance, wobbly gait), (8) hypesthcsia, (9) withdrawal and isolation from other animals, closing of eyes, "hunched" posture, (10) stupor (i e , rigidity, semi-comatose, somnambulant motionlessness). (11) convulsions or seizures (i e , flaying of limbs, loss of consciousness, whirling, rolling and/or circling), (12) muscle spasms or myoclonus (e g , tremor, twitching of muscles, repetitive jerking of muscles), (13) corneal opacity (a clouding of the cornea) and (14) sudden death Individual animals can present with one or more of the preceding subacute symptoms, but are generally observed displaying a complex of two or more symptoms Subacute symptoms are more frequently observed in male animals compared to female animals More slowly developing symptoms (i e , those developing after about two months and sometimes not for about six months or more after inoculation) include (1) obesity, (2) hypesthesia (I e , decreased sensitivity to sensory stimuli), (3) extreme lethargy and prolonged sleeping, (4) hyperactivity or hypercsthesia (I e , increased sensitivity to sensor}' stimuli), (5) aggressiveness (e g , jumping or biting), (6) obsessive compulsive behavior (e g , excessive and prolonged washing of the face or continual scratching), (7) self-mutilation (the extreme end of obsessive compulsive washing or scratching where the skin is damaged), (8) still-born fetuses and deformities in newborn animals (usually paralysis or limb atrophy) born to experimentally-infected females, and (9) infanticide (cannibalism of numerous newborns or entire litters) Individual animals can present with one or more of the preceding more slowly developing symptoms, but are generally observed displaying a complex of two or more symptoms More slowly developing symptoms are more frequently observed in female animals compared to male animals

The inventive animal model is an excellent model system for the study of neurodegenerative, wasting or paralytic neurological diseases which typically present with tremor, weakness and atrophy The inventive animal model is also, in particular, an excellent model system for the study of idiopathic epileptiform diseases because the infected animals present with virtually the entire spectmm of symptoms associated with epileptiform illnesses in humans. At present, most existing animal models of epilepsy (e.g., induction of seizure by inoculation with the glutamate receptor agonist, kainite, or by partial suffocation) are contrived to produce seizures and the gross anatomical pathology associated with seizures without reference to the etiology of the actual symptomatic spectmm of the illnesses in humans While these models are useful in developing therapeutics for seizure activity, there is little or no evidence that they are relevant to the ultimate aetiopathogenesis of epileptiform illnesses in humans or the actual spectaim of symptoms which occur In contrast, the animal model of the present invention is a tally homologous animal model, that is, one in which the actual factors/symptoms associated with the disease in humans are extant and can be specifically targeted by both therapeutic and prophylactic strategies. Thus, the inventive animal models disclosed herein can be used to screen antiviral medications or medicaments, including anti-epileptic and anti-psychotropic medicaments, as well as to test vaccines and other prophylactic remedies and to determine how to best coordinate and optimize any and all treatment strategies.

Cryptovirus is mildly cytopathic in cell culture but causes profound neuropathological disease in experimentally-infected animals Any of the cytopathogenic and neuropathogenic traits of the vims can be used as markers in screens designed to identify and test potential antiviral therapeutic and/or prophylactic agents. Accordingly, the present invention features in vitro and n vivo methods of screening potential antiviral therapeutic agents and/or antiviral prophylactic agents, including immunoprophylactic agents. A "potential" antiviral therapeutic or prophylactic agent is an agent that has not yet been clinically confirmed (i c , in phase III clinical trials) to have antiviral properties effective against Cryptovirus. Agents that have not been tested clinically against Cryptovirus infections or have been tested clinically against Cryptovirus infections only with respect to phase 1 and phase II clinical trials are also encompassed by "potential" antiviral therapeutic and/or prophylactic agents for purposes of the present invention.

In accordance with the inventive in vitro methods of screening a potential therapeutic or prophylactic agent, either acutely- or productively-infected mammalian cell cultures (e.g., BHK, Vero, or CV-lc cells) or nonproductively infected carrier cultures (e.g., AV₃/SSPE cells) can be used to evaluate the potential antiviral agent. While the acutely infected (productive) cellular system is preferentially useful for screening agents targeted at the processing and assembly of Cryptovirus envelope glycoprotcins (e.g., protease inhibition of F₀ cleavage activation), the nonproductively infected cellular system (e.g., AV₃/SSPE cells) is preferred for screening for the efficacy of long-term treatment with transcriptional or other polymerase inhibitors (inhibiting the buildup of intracellular nucleocapsids and the eventual triggering of apoptotic cell death).

The inventive animal model is usefully employed in the in vivo method of screening a potential antiviral therapeutic agent. The method involves administering the potential therapeutic agent to be screened, to the inventive animal model after its inoculation with Cryptovirus, in accordance with the inventive animal model.

An alternative embodiment of the inventive animal model is employed in the in vivo method of screening a potential antiviral prophylactic agent. The method involves administering a potential prophylactic agent to be screened to a non-human mammal, which does not have a symptom of a human disease, such as but not limited to a neurological, neurodegenerative, and/or neuropsychiatric disease (e g , an epileptiform disease). Then the animal is inoculated, as described herein, with the infectious cell-free Cryptovirus or with the mammalian cell nonproductively-infected with the Cryptovirus The method is particularly, but not exclusively, useftil for identifying potential anti- epileptic or anti-psychotropic antiviral prophylactic agents.

Administration of the potential prophylactic agent or therapeutic agent is by any suitable delivery route, enteral (e.g., orally or by suppository) or parenteral (e.g., by injection, infusion, transmembrane, transdermal, or inhalation delivery route)

Examples of agents that can be evaluated, in accordance with the invention, include compounds or substances with known antiviral properties against viaises other than Cryptovirus, novel compounds or mixtures of compounds, such as cell, plant or animal extracts, with potential antiviral activity; and vaccines, as described hereinabove; or any combination of these.

Using the inventive in vivo method of screening, potential immunoprophylactic agents (i.e., vaccines which stimulate the immune system to respond to, attack or inhibit vims replication, assembly or any other process associated with vims reproduction and spread) are also amenable to testing because non-human mammals can be inoculated with a putative prophylactic agent or vaccine (as mentioned above) and then challenged with infectious Cryptovirus to assess its utility in preventing the development of Cryptovirus -associated diseases. The use of such agents discovered in accordance with the invention may ultimately be necessary to control and eradicate Cryptovirus- associated diseases in the human population much as measles and mumps vaccines have been used to bring these diseases under control in many countries

In addition to those named above, one of ordinary skill in the art will recognize numerous potential antiviral chemo- and molecular-therapeutic agents that could be analyzed or evaluated using the in vitro (cell culture) or in vivo methods of screening provided herein These potential antiviral therapeutic and/or prophylactic agents can include existing antiviral agents known to affect vimses other than Cryptovirus (e g , Ribaviπn™, which is also known as Virazole™) and new potential antiviral agents For example, molecular therapeutic agents ( g . anti-sense nucleotides and πbozymes) or protease inhibitors can also be tested using the inventive in vitro and/or in vivo methods of screening Agents that might inhibit the cleavage of the viral fusion protein (F₀) can be sought, and these could be particularly valuable, as there is evidence that cleavage of the fusion protein and its association with the viral hcmagglutinin/neuraminidasc protein (HN) are critical events in determining the pathogemcity of infections by other Paramyxovirdae (Yao et al , J Virol 21 650- 656, 1997) Further, these potential antiviral agents may be directed, for example, at Cryptovirus replication or assembly, or the expression or activity of Cryptovirus genes and proteins, such as. but not limited to, the Cryptovirus -encoded RNA-dependent RNA polymerase comprising the L protein and its companion P and V proteins The inventive screening methods also can be employed to develop broad-spectmm antiviral agents, effective against vimses other than Cryptovirus Immunotherapeutic agents, such as those that attack, or stimulate the immune system to attack, infected cells or vims particles (by, e g , passive antibody administration or introduction of Cryptovirus -specific monoclonal antibodies) are also amenable to testing because they can block or inhibit the assembly, release or ccll-to-ccll transfer of virions However, administration of these agents may be of only limited value in "curing" persistent and chronic Cryptovirus infections because the viais appears to survive in situ by shutting off production of its envelope proteins and going into a

"latent" or inapparent state, in which it appears to be undetectable by the immune system

Appropriate amounts of potential prophylactic or therapeutic agents vary and are determined by routine screening

In accordance with the inventive in vivo methods of screening a potential therapeutic agent or prophylactic agent, the agent is evaluated for an ability to induce, create, bring about, or result in a beneficial antiviral effect in the inventive animal model A "beneficial antiviral effect" includes the prevention of infection with Cryptovirus or a reduction in the duration or severity of at least one symptom associated with Cryptovirus infection, in the animal subjected to the assay, compared to tissues in control animals A "beneficial antiviral effect" also includes a prevention or reduction of cytopathic effect (CPE) in tissues sampled from the animal subjected to assay by the screening method Also encompassed by a "beneficial antiviral effect" is an inhibitory effect on Cryptovirus replication and/or Cryptovirus virion assembly (e g , inhibitory effect on Cryptovirus genomic replication, Cryptovirus transcription, and or translation, l e , protein synthesis, from Cryptovirus mRNAs, or a diminution in the numbers of Cryptovirus virions produced or a relative lack of completeness of Cryptovirus particles, compared to a suitable control), which effect is measured by known means in cells or tissues sampled from the animal subjected to the assay.

Appropriate controls for use in the screening methods will be self-evident to the skilled artisan. Such controls can include: (1) animals administered with the same potential prophylactic or therapeutic agent and challenged with sterile artificial aqueous culture medium alone or the culture medium containing a strain of SV5; (2) animals mock-treated with saline (or the same carriers used in delivering the potential prophylactic or therapeutic agent) and challenged with Cryptovirus; and (3) animals mock-treated with saline (or the same carriers used in delivering the potential prophylactic or therapeutic agent) and challenged with sterile artificial aqueous culture medium alone or the culture medium containing a strain of SV5.

The practice of the present invention will employ, unless otherwise indicated, conventional or other known techniques of biochemistry, molecular biology, microbiology, virology, recombinant nucleic acid technology, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, (e.g., Maniatis et al, Molecular Cloning: A Laboratory Manual,

Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA (1982); DNA Cloning, Vols. I and II (D. N Glover ed. 1985); Sambrook et al., Molecular Cloning: A Laboratory Manual (2 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA (1989); Davis et al, Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1986); or Methods in Enzymology: Guide to Molecular Cloning Techniques Vol.152, S. L. Berger and A. R.

Kimmerl Eds., Academic Press Inc., San Diego, USA (1987); Oligonucleotide Synthesis (M. J. Gait ed, 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Animal Cell Culture (R. I. Freshney ed. 1986); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In Enzymology (Academic Press, Inc.); Gene Transfer Vectors

For Mammalian Cells (J. H. Miller and M. P. Calos eds. 1987, Cold Spring Harbor Laboratory), Methods in Enzymology Vol. 154 and Vol. 155 (Wu and Grossman, and Wu, eds., respectively), Mayer and Walker, eds. (1987), Immunochemical Methods In Cell And Molecular Biology (Academic Press, London), Scopes, (1987), Protein Purification: Principles And Practice, Second Edition (Springer-Verlag, N.Y.), and Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell eds 1986).

The invention will now be described in greater detail by reference to the following non- limiting examples. EXAMPLES

Example 1. Detection of Cryptovirus in Infected Cells and Isolation and Purification of Cryptovirus Viral Particles.

In accordance with the present invention, cell-free Cryptovirus particles were recovered from cells of the buffy coat (peripheral blood mononuclear cells; PBMNC) obtained from the peripheral blood of patients with SSPE. The technique used was modified from that of Robbins et al. (J. Infect. Dis. 143:396-403, 1981). Modifications included the addition of cyclic GMP (to a final concentration of 1 mM) to the aqueous culture medium, in accordance with the present invention, which medium was added to the initial PBMNC cultures and the primary cocultivate with human amnion cells (AV₃) Results were ftirther optimized by using as the mammalian epithelial cell line in a co- cultivation step with the PBMNCs and amnion cells, a clonal subline of CV-1 cells (CV-1_C).

Successful isolation of the vims requires viable PBMNCs. Such PBMNCs were separated from other blood components by standard procedures on Ficoll-Hypaquc™ gradient media. After centrifugation, the buffy coat cells banded at the interface of the media and were removed with a sterile pipette. They were then gently washed by dilution in 50 volumes of RPMI cell culture media containing 1-2% fetal calf semm and centrifuged in a table-top refrigerated centrifuge (1000 rpm for 5 minutes) The pelleted PBMNCs were then diluted to 2 x 10⁵ cells per ml in RPMI media containing

10% fetal calf semm and 1 mM cyclic GMP (sodium salt) and incubated without disturbance at 37°C for 12-18 hours. Following this incubation, the cultures were seeded with sufficient AV₃ cells in Richtcr's Modified Minimal Essential Medium (IMEMZO) (supplemented with insulin, zinc and

HEPES buffer, 2 mM L-glutamine, 200 Units penicillin mL, 100 pg streptomycin/mL, 5-10% (v/v) fetal calf seaim, pH between 6.8 and 7.0) to yield a net cell concentration of 2 x 10⁵ cells per mL (for all cells in the culture) and were reincubated at 37°C. Once the cultures reached confluence (2-

3 days), the monolayer was chelated with a solution of 0.02% w/v EDTA in CMF-PBS (calcium and magnesium free phosphate buffered saline), the cells were dispersed, and passaged at 2 x 10 ⁵ cells

/mL in IMEMZO as before.

The cultures were then blindly passaged in the same way when confluent every 3-4 days

(roughly twice a week) for 2-3 weeks. After two weeks, a slide culture was prepared to examine the cells for the presence of C/7/>/ov/π<.y-specifϊc inclusions in the cytoplasm using a Cryptovirus -specific indirect fluorescent antibody technique (exposure to hyperimmune rabbit anti-Cryptovirus antisera followed by labeling with fluorescein-conjugated goat anti-rabbit IgG).

When 5-10% of the cells were positive for Cryptovirus-specific inclusions, the cultures were ready for co-cultivation with the permissive CV-1_C cells mentioned above This involved the 1 1 cocultivation of the passaged primary PBMNC/AV₃ cultures with CV-1_C cells in Richter's Modified Minimal Essential Medium diluted to yield a net concentration of 2 X 10⁵ cells/mL. These cultures were then monitored for the development of subtle cytopathic effects (CPE; stellation and rounding of cells or the formation of multinucleated cells containing three or more nuclei) over the ensuing 4-5 days. If no CPE developed before the cultures become confluent, they were passaged and monitored again. If no CPE developed after three such passages, the cultures were discarded.

Once CPE was observed, the whole culture was frozen at -70°C, thawed, and the cells were dispersed and dispensed into 1.0 mL aliquots. These aliquots represented the putative primary isolation of the vims. The vims was then plaque-purified by titration on monolayers of CV-1_C cells overlaid with a semi-solid solution of 1% w/v sodium carboxymcthylcellulose (NaCMC) containing

2% fetal calf semm, which was made up in Richter's Modified Minimal Essential Medium. The cultures were incubated at 37°C in a partial C0₂ atmosphere (5% v/v). Plaques formed in 8-12 days and were then picked and replaqued, as above. Once triply plaque-purified, the vims was aliquoted onto CV-1_C monolayers (in IMEMZO media containing 5% fetal calf semm and supplements listed above) in 25-75 cm² tissue culture flasks. Once sufficient CPE developed, involving half or more of the cultured cells, the whole cultures were frozen, thawed, and the lysate was dispersed, re-aliquoted and refrozcn at -70°C. Samples of the vims stock were then titrated for further use by the method of Robbins et al. (Robbins et al, J. Infect. Disease 143:396-403, 1981).

Density Gradient Purification: Virions and intracellular nucleocapsids isolated from productively- (Vero and CV-1_C) and nonproductively-infected (AV₃/SSPE) cells were further purified on sucrose-potassium tartrate gradients (virions) and CsCl gradients (nucleocapsids) by the method of Robbins el al. (Robbins et al, J. Infect. Disease 143:396-403, 1981 ; Rapp and Robbins, Intervirology 16: 160-167, 1981; Robbins and Rapp, Arch. Virol. 71:85-91, 1982; and Robbins and Abbott-Smith, J. Virol. Meth. 11:253-257, 1985).

Example 2. Cryptovirus Propagation and Virion Isolation and Purification.

Once isolated, cell-free vims stocks were grown in simian epithelial cell lines (e.g. Vero or CV-1 cells). The Cryptovirus isolates used in the studies described herein were triply-plaquc purified and grown in a clonal subline of CV-1 cells designated CV-lc. Optimal production of infectious vims occurred when using IMEMZO supplemented with insulin, zinc and HEPES buffer, 2 mM L- glutamine, 200 Units penicillin mL, 100 μg streptomycin/ml, 5-10% (v/v) fetal calf se m, at a pH between 6.8 and 7.0. The presence of insulin, and optionally zinc dication, in the tissue culture medium was useful in obtaining viable titers of infectious vims. Independent attempts to grow the vims in CV-1 cells using standard media (e.g. MEM) produced very poor results. Conversely, the expression of Cryptovirus proteins and the productivity of Cryptovirus infections in primate cell cultures was dramatically enhanced (50- to 100-fold) by addition of cyclic GMP (1 mM; sodium salt) to standard media (specifically MEM). The enhancement obtained was very similar to the enhancement of measles vims replication published earlier (Robbins, Intervirology 32:204-208, 1991).

Virion Isolation and Purification. Virions were isolated and further purified from the supernatant tissue culture medium of acutely infected CV-1 cells 72 hours after infection. The procedure employed involved the separation of the virus particles by differential centrifugation

The supernatant medium of infected cultures was decanted into a sterile plastic 50-mL Falcon centrifuge tube and clarified at 2000 rpm for 10 minutes. The supernatant was then transferred to an impact resistant glass centrifuge tube (Sorvall) and further clarified at 10,000 rpm for 10 minutes. All clarifications took place at 4° C in an RC2B Sorvall centrifuge The supernatant fluid from the second clarification step was layered over a 60% w/v sucrose cushion (in 10 mM Tris, 5 mM EDTA, pH 7.2) and centrifuged at approximately 130,000 x G in a Beckman SW-28 rotor at 4° C for 90 minutes in a Beckman L70 ultracentrifuge. Materials were collected from the tissue culture medium- sucrose interface, pooled, diluted with tissue culture medium and rcccntπfuged onto another 60% sucrose cushion as described above. The materials at the interface were again removed, diluted with tissue culture medium, and centrifuged at 35,000 rpm (280,000 x G) for 60 minutes through a 30% w/v over 60% w/v discontinuous (i.e. layered) sucrose gradient prepared in the Tris EDTA buffer described above) The virions were collection from the 30%'60% sucrose interface, diluted with cold Tris EDTA buffer and pelleted in a Beckman SW41 rotor at 41,000 rpm and 4° C for 60 minutes. Pelleted virions were resuspended in a variable amount of the cold Tris EDTA buffer and frozen at -

70° C until ftirther use. Total protein in each virion preparation was determined by the method of Lowry et al. (1951)

Example 3. Preparation of Antisera.

Antisera were raised in adult New Zealand White rabbits against sucrose-potassium tartrate gradient-purified virions of Cryptovirus, CsCl gradient-purified nucleocapsids (from infected CV-1_C cell cytoplasm), and against the major viral nucleocapsid protein, NP, eluted from polyacrylamide gels after SDS-PAGE. Rabbit antisera were also raised against the NIH 21005-2WR strain of SV5, and the Edmonston stram of measles vims.

Animals were inoculated by a pincushion technique which involved three series of three separate inoculations in each animal using a sterile 27 gauge needle and 1.0-mL syringe (one inoculation intradermally on the back; one inoculation intraperitoneally and one inoculation in a hind foot pad) The first series of inoculations were made using gradient purified and dialyzed virions

(100 μg of virions in 0.3 mL of a 10 mM Tris 5 mM EDTA solution) mixed 1: 1 with Freund's Complete Adjuvant and each inoculation contained approximately 200 μL of the virion adjuvant mixture. The second series of inoculations were made two weeks later in the same locations but on the opposite side of each animal and consisted of the same amount of virions mixed with Freund's Incomplete Adjuvant. The third series of inoculations were made two weeks later in the same locations as the first inoculations but using only virions (diluted in 0.6 mL of the Tris-EDTA solution described above). Blood was harvested by intracardiac exsanguination of the animals two weeks after the final series of inoculations. The harvested blood was centrifuged (2000 rpm for 10 minutes) and allowed to clot on ice. The upper semm component was harvested and adsorbed against the pelleted component (2000 rpm for 10 minutes) of saline-washed freeze-dried acetone :methanol- extracted monkey kidney tissue (4° C for 1 hour with agitation every 15 minutes). The adsorbed semm was harvested by centrifugation (2,000 for 10 minutes) and stored in 1.0-mL aliquots at -20° C).

All of the anti-Cryptovirus antisera were strongly reactive with the corresponding Cry/?/ov/rM.y-spccιfic materials from which they were generated when analyzed by (1) immunoprecipitation, (2) immunofluorescence, (3) immunoblotting, (4) ultrastmctural immunolabelling techniques (immunogold), and (5) in the case of antisera generated against gradient- purified viais particles, neutralization titration assays. All the hypenmmune vims-specific antisera that were generated in the rabbits had homologous neutralization titers in excess of 1280 and, usually, in excess of 2560 (reciprocal dilution of PRD₅₀).

All experimentally-generated antisera were adsorbed against saline washed, freeze-dried, acetone: methanol extracts of monkey kidney tissue or similar extracts of AV₃ cells and/or CV-lc cells While clinical sera were similarly adsorbed, CSF specimens were NOT preadsorbed due to the small volumes that were usually available and the requirement to retain aliquots for duplicate and parallel studies

The precipitating "titers" of the experimental sera raised against purified nucleocapsids and purified vimses were not specifically determined although, routinely, 5-10 μL were used in positive control immunoprecipitation reactions and 25 μL of positive control antisera (diluted 1 10 or 1 20) for positive controls in ELISA assays

Crypto virus -specific antisera were also produced in mice experimentally inoculated with gradient-purified infectious Cryptovirus virions These antisera were analyzed by immunoprecipitation. and were found to strongly precipitate all Cryptovirus envelope proteins

There was clear asymmetric cross-reactivity between the antisera raised against Cryptovirus virions and antiserum raised against virions of the NIH 2WR-21005 strain of SV5 The asymmetry observed in this regard was always such that the heterologous reactions (I e , Cryptovirus -specific antisera vs SV5 materials and SVS-specific antisemm vs Cryptovirus materials) were two- to four- fold weaker that the homologous reactions (l e , Cryptovirus -specific antisera vs Cryptovirus materials and SVS-specific antisera vs SV5 materials) Another antisemm, which was independently prepared against NIH 21005-2WR strain of SV5 and kindly provided by Dr Pumell Choppin, behaved in a similar asymmetric manner to the antisemm against SV5 described hereinabove

Such cross reactivity is not surprising Precisely the same sort of asymmetric cross-reactivity occurs when examining other paramyxovirus systems (e g , there is also a two to four fold asymmetric cross-reactivity between measles vims antibodies when reacted with the closely related vimses of canine distemper and rinderpest and vice versa) There was also limited (/ e , much weaker) cross- reactivity between Cryptovirus -specific antibodies and other paramyxoviaises (e g , measles viais)

Example 4 Characterization of isolated Cryptovirus

Cryptovirus Neurovirulence and Neurotropism As shown in Fig 24A, Cryptovirus demonstrated a tropism for neurons Intracranial inoculation of mice with infectious Cryptovirus or nonproductive virus-carrying cells (AV₃/SSPE) resulted in the subacutc/slow development of a spectmm of neuropathological conditions that had epileptiform, neurological and/or neuropsychological components These responses were similar to the "experimental SSPE" reported earlier in animals following inoculation with "cell-associated measles-like" vims such as Nngata, Kitaken and Biken (see Fig 24B, Doi et al , Japan J Med Sci Biol 25 321-333, 1972, Ueda et al , Biken Journal 18 179-181. 1975, Yamanouchi et al , Japan J Med Sci Biol 29 177-186, 1976, Ohuchi et al , Microbiol Immunol 25 887-983, 1981)

Cryptovirus Presence in Neurovirulent SSPE-denved Virus-carrying Cell Lines Four virus- carrying cell lines derived from patients with SSPE were tested by immunofluorescence for the presence of measles vims- and/or Cryptovirus specific antigens These were AV₃/SSPE/MV (an SSPE-denved cell line derived from PBMNC from an SSPE patient cocultivated with AV₃ cells which was experimentally-infected with Edmonston strain measles vims, Robbins, unpublished data) the nonproductive SSPE-denved cell line designated "Kitaken" (Ueda et al , Biken Journal J_8 179-

181, 1975), the nonproductive SSPE-denved cell line designated "Nugata" (Doi et al , Japan J Med S i Biol 25 321-333, 1972), and the nonproductive SSPE-denved cell line designated "Biken" (Yamanouchi et al , Japan J Med Sci Biol 29 177-186. 1976, Ohuchi et al , Microbiol Immunol 25 887-983, 1981) With the possible exception of the Nngata cell line, all of these virus-carrying cell lines expressed both measles virus-specific and Cryptovirus -specific antigens when examined by vims-specific immunofluorescent techniques (shown in Fig 6) Given that no cell-free clinical isolates of measles viais have ever been shown to cause SSPE- ke illnesses in experimentally- infected animals, the presence of Crytovirus m these cultures strongly suggests that the subacute/slow neuropathies seen in these animals are due to the presence of Cryptovirus in the cultures — not measles vims

Cryptovirus inclusion bodies (I c cytoplasmic nucleocapsids) displayed the same sort of "peppery" and or "splattered" distribution in both acutely-infected cells (CV-1_C) and nonproductively- and persistently-infected cells (AV₃/SSPE) as that previously described in CNS biopsy and autopsy materials from SSPE patients and in SSPE-denved nonproductive vims-carrying cell lines (e g , de Felici et al , Annales Microbiologie 126 523-538 [1975]. Makino et al ,

Microbiology and Immunology 21 193-205 [ 1977], Brown et al , Acta Neuropathologica 50 181 -186 119801) This is most clearly evident in Panels B, D, F, H and J of Fig 6 These characteristics were in sharp contrast to the discrete and "coalescing" distribution and morphology of intracellular measles viais inclusions bodies (sec Panels A, C, E and G of Fig 6)

Neutralization Titration A ssay Formation of macroscopically visible plaques on monolayers of mammalian cells (c g , BHK, Vero and CV-lc) can be used to quantitate preparations of infectious

Cryptovirus Plaque formation can be inhibited by serial dilutions of clinical seaim specimens and Cryptovirus-specific antisera generated in rabbits (see Robbins et al, J. Infect. Disease 143:396-403. 1981). Plaque titration assays were conducted to determine the PRD₅₀ of isolated Cryptovirus .

Briefly, ten-fold serial dilutions of semm or CSF specimens to be tested were incubated for one hour at 4°C with sufficient infectious vims to yield a net plating concentration of between 100-200 plaque forming units of the vims / 0 2 mL of final diluent (including the diluted seaim or

CSF). After incubation, 0.2 mL of the diluted vims-semm (or vims-CSF) mixtures was then plated onto monolayers of susceptible cells (e g. Vero or CV-1_C) and the cells were incubated at 37°C in a partial C0₂ atmosphere (5%v/v) (with redistribution of the inoculum every 15 minutes). At the end of the incubation period, inoculated monolayers were overlayed with sufficient volumes of a 2% (w/v) solution of carboxymethylcellulose (made up in IMEMZO medium containing 2% fetal calf seaim, insulin, zinc, and HEPES buffer, 2 mM L-glutamine, 200 Units penicillin / mL, 100 μg streptomycin / mL, pH between 6 8 and 7.0) to last 10-12 days (i.e., enough volume so that the monolayers won't dry out). The plates were not moved during the incubation period. After 10-12 days, the overlay was aspirated and the cells were fixed with formalin fixative and stained with a protein stain (e.g., Giemsa). The number of plaques fonned on each plate was then enumerated and the PRD₅₀ calculated.

In particular, cross neutralization assays involved the determination of the titer of antisera made against each species of viais which would neutralize 50% of the plaque forming units (PFUs) of each vims. Vims stocks of the BBR strain of Cryptovirus and the NIH 21005-2WR strain of SV5 were diluted in semm-free minimal essential medium (Eagle's MEM containing 2mM L-glutamine,

200 units of penicillin and 100 μg of streptomycin/ml with the pH adjusted to between 6.8 and 7.0 with NaHC0₃) to a titer of 1,000 PFUs per mL (resulting in 100 PFUs per well after dilution and plating). Antisemm raised in New Zealand White rabbits was serially-diluted in 10-fold increments in the same semm-free MEM. Aliquots (0.5 mL) of the diluted viais stocks were then mixed with 0 5 ml aliquots of each dilution of the antisera, gently mixed with a vortex and incubated on ice for one hour with gentle mixing every 15 minutes. Following this incubation period, the medium was aspirated from monolayers of CV-1 cells in 6-well cluster plates (NUNC), the monolayers were washed with warm saline, the saline was aspirated and 0.2 mL of each of the diluted antisera-viais incubates were plated onto two monolayers. The inoculated cluster plates were subsequently incubated at 37° C in a C0₂ incubator (containing 5% C0₂ v/v) for 1 hour with manual redistribution of the inocula every 15 minutes. Following this adsorption period, each well was overlayed with 10 0 mL of a semisolid overlay medium (1% w/v sodium carboxymethylcellulose in Eagle's MEM containing 2 mM L-glutamine, 200 units of penicillin and 100 μg of streptomycin/ml, 2% v/v fetal calf seaim with the pH adjusted to between 6 8 and 7 0 with NaHC0₃) and incubated for 10-12 days at 37° C in a C0₂ incubator (containing 5% C0₂ v/v) without being disturbed Following this incubation period, the overlay was aspirated, the monolayers were gently washed with warm saline, and then fixed in formalin fixative (3 7% by weight formaldehyde gas in saline) for 1 hour or longer Following fixation, the fixative was aspirated and the fixed monolayers were gently washed with distilled water and stained with 1-2 mL per well of Giemsa stain (0 5 gm Giemsa powder dissolved in 42 mL of warmed [55° C] glycerin, 42 mL of absolute methanol, filtered and diluted 1 5 with formalin fixative immediately before use) for 1 hour at room temperature The stain was subsequently decanted and the monolayers were washed under tap water and allowed to dry at room temperature Plaques on monolayers were illuminated on a light box, enumerated under a magnifying lens and recorded for each dilution, vims and antisera series The neutralization titer of each antisemm vims series was calculated to be the reciprocal of the dilution of antisera resulting in a 50% decrease in the number of plaques formed

The calculated neutralization titer for each crossed neutralization set (I e anti-Cryptovirus antisemm versus Cryptovirus and anti-Cryptovirus antisemm versus SV5, antι-SV5 antisemm versus

Cryptoviais and antι-SV5 antisera versus SV5) was consistently 2-4 fold less for the heterologous mixtures (I e anti-Crytovirus antisera versus SV5 and antι-SV5 antisera versus Cryptovirus) than for the homologous mixtures (anti-Cryptoviais antisera versus Cryptoviais and antι-SV5 antisera versus SV5) On no occasion did any heterologous mixture have less than a 2-fold difference when compared to the homologous pair (in three separate trials)

Cryptovirus Ultrastructural and Immunoultrastructural Characterization AV₃/SSPE/MV cells (AV₃/SSPE cells persistently and nonproductively infected with the Edmonston strain of measles vims), AV₃/SSPE cells, and CV-1_C cells acutely infected with the BBR strain of Cryptovirus were fixed in situ (on glass cover slips) in 2% formaldehyde and picric acid and 3% glutaraldehyde in

0 1 M cacodylate buffer, pH 7 2, for 15 minutes at room temperature Osmium tetroxide post-fixation was omitted for specimens that were to be treated with antibody (1 e which were prepared for immunoultrastmctural studies) Cover slips with fixed cells were washed in three changes of cacodylate buffer, dehydrated to 70% ethanol and embedded in LR White resin Resin was polymerized at 50°C for 24 hours Ultrathin sections were cut and mounted on uncoated nickel grids

Stained thin sections of CV-lc cells acutely-infected with the BBR strain of Cryptovirus and AV3/SSPE cells were examined by electron microscopy In infected CV-1_C cells, pleomorphic virion particles, 100-120 nm in diameter, were seen budding from the surface of acutely-infected cells and numerous accumulations of filamentous staictures (helical nucleocapsids, 15-17 nm in diameter) were observed in the cell cytoplasm (data not shown). Both the virions and nucleocapsids were similar to those described for other members of the Paramyxoviridae. While virions were not observed budding from the surfaces of AV₃/SSPE cells, inclusions of intracellular nucleocapsids were seen in abundance and these were identical to those seen in the acutely-infected cells.

The intracellular nucleocapsids of nonproductively and productively-infected mammalian cells can readily be localized under the electron microscope using Cry/?/ovzra.y-specific or Cryptovirus nucleocapsid-specific hypenmmune rabbit antibodies and an indirect immunogold labeling technique.

Immunolabelling was performed on sections of AV₃/SSPE/MV cells by floating sections mounted on nickel grids (processed as described without osmium tetroxide post-fixation) on drops of solution (see below) in a closed, humid chamber. Sections were etched according to the method of Ingram et al. (Parasitology Research 74:208-215, 1988). Non-specific labeling was reduced by incubation of the sections with 5% bovine semm albumin in modified Tris buffer (20 mM Tris, 0.5 M NaCl, 20 mM sodium azide and 0.05% Tween 20, pH 8.2) for 30 minutes at 37°C prior to immunolabeling The modified Tris buffer was used for all dilutions and washes.

In single labeling experiments, sections were incubated with rabbit antisera (anti-Edmonston measles vims or anti-BBR strain of Cryptoviais) diluted 1 :20 in modified Tris buffer for 2 hours at 37°C, washed in three changes of buffer, and incubated with a goat anti-rabbit IgG colloidal gold (10 or 15 nm particle size, 1 :20 dilution, 1 hour at 37°C) After washing with two changes of buffer, followed by two changes of distilled water, sections were lightly contrasted with 2% uranyl acetate and led citrate, and examined in a JEOL 1200EX transmission electron microscope.

In double labeling experiments, sections were immunolabeled on one face, as described for single labeling, using rabbit anti-Edmonston measles viais and 15 nm colloidal gold particles, and ensuring that the reverse face of the section was not contaminated by labeling solutions The labeled face was then coated with a thin film of Celloidin to reduce possible cross reaction of antibodies while the reverse face of the section was labeled. Immunolabeling of the reverse face of the sections was performed as described above, using the second antisemm (rabbit anti-BBR strain of Cryptovirus) and 10 nm colloidal gold particles. Examination of these double labeled sections allowed simultaneous comparison of labeling patterns of the two antisera.

The results of these studies were unequivocal and are shown in Fig. 25. The first labeling sequence (15 nm gold beads) labeled only the wider "fuzzy" measles viais nucleocapsids (as shown in Fig. 25 B), while the second labeling sequence (10 nm gold beads) labeled only the narrower smooth Cryptovirus nucleocapsids (as shown in Fig. 25A).

Example 5 Characterization of Isolated Cryptovirus Proteins Radioimmunoprecipitalion (RIP) Assay: Extensive data were generated by the comparative analysis of Cryptovirus-specific immunoprecipitates of [³⁵S]-methionine-labeled uninfected, nonproductively- and productively-infected human and primate cells by sodium dodccyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; see below)

CV-1_C or Vero cell monolayers were infected with the BBR strain of Cryptovims, the NIH 21005 -2WR strain of S V5 or the Edmonston strain of measles vims at a multiplicity of infection of 1 -

2 PFU/cell using procedures as described elsewhere (Robbins and Rapp, Virology 106:317-326, 1981)

Labeling was accomplished twenty-four hours after infection by the following procedure Tissue culture medium was removed from infected cell monolayers and the cells were washed with a seaim and methioninc-free Eagle's based MEM (starvation medium). The infected cultures were then supplemented with the starvation medium for 60-90 minutes and incubated at 37°C. Following the starvation period, cultures were labeled with the starvation medium containing 100 μCi/mL of [³⁵S]-methionine (Amersham). Labeling was carried out at 37°C in a 5% C0₂ atmosphere for 5-6 hours Immunoprecipitations were carried out according to the procedure of Lamb et al, Virology 91:60-78, 1978.

SDS-PAGE: Virions and immunoprecipitates were analyzed under denaturing and reducing conditions on 10% (or to detect the presence of very small peptide species [e.g., SH protein], 20%) polyacrylamide slab gels (Laemmli, 1970) After electrophoresis, gels were treated with a fluor solution (Amersham), were dried, and were then exposed to X-ray film.

Purified virions of the vims were analyzed by SDS-PAGE under reducing and non-reducing conditions (see Fig 11, an autoradiogram of gradient-purified [³⁵S]-methionine-labeled Cryptovirus virions produced in acutely-infected Vero cells after SDS-PAGE under reducing conditions). The approximate molecular weights of the proteins indicated on the right side of the figure were calculated by comparing their migrations to marker proteins of known molecular weights (Sigma

Chemical Co., St. Louis, MO). The annotations are defined in the brief description of the drawing. The SH protein, a small envelope-associated protein having a MW of about 5 kD, is not shown because it has mn off the gel (see below)

SH Protein Due apparently to the small size of the SH protein and its relatively low methionine content, the SH protein was difficult to detect in radio-labeled virion preparations of both the BBR strain of Cryptovirus and the NIH 21005-2WR strain of SV5 When unlabeled purified virion preparations of both vimses were ain on 20% polyacrylamide slab gels under denaturing and reducing conditions alongside of low molecular weight marker proteins (BioRad) and stained with a silver-staining technique (BioRad), a small protein with an M_r of approximately 5 kD, identified as the SH protein, was found in both Cryptovirus and SV5 virion preparations There was no detectable difference between the migration of the SH protein from the BBR strain of Cryptovirus or the NIH 21005-2WR strain of SV5

Fo and HN Co-migration Anomaly Although the major envelope proteins (F₀ and HN) of many Rubulavimses (e g , SV5) were readily discernible as separate bands on SDS-PAGE gels, the larger size of the Cryptovirus F₀ protein (i e , +22 ammo acids) resulted in a significantly slower rate of migration for this protein (M_r = 69 kD) As shown in Figs 13A-B, close examination of such gels enabled one to discern both proteins, albeit with some difficulty Figs 13A and 13B show photographs of migration patterns of the major Cryptovirus envelope proteins, F₀ and HN Fig 13A illustrates the observed near co-migration of the major Cryptovirus envelope proteins, F₀ and HN

Enlargement of the RIP from the CSF-positive patient (right lane) in Fig 13A shows the "bowed" or "crested" stmcture that resulted from the near co-migration of the F₀ and HN proteins of Cryptovirus A diagrammatic interpretation of the near co-migration of the F_n and HN proteins of Cryptovirus and the discrete migration of the analogous proteins of Simian vims 5 are shown in Fig 13B

Example 6 Experimental Infection of Mice Creation of an Animal Model

Infectious Cryptovirus stocks (prepared in CV-L cells) and live nonproductively-infected AV₃/SSPE cells were used to lntraccrebrally inoculate two strains of laboratory mice (Quackenbush and Colored, an outbred strain of C57 Black) Briefly, neonatal mice (1-2 days old) were inoculated by injection with 0 025 mL (phosphate buffered saline, pH 7 4) containing either 5 x 10⁴ PFU of cell-free Cryptovirus or 5 x 10³ nonproductively infected human amnion cells (AV₃/SSPE) Following inoculation, the neonatal mice were returned to their mothers who were provided food and water ad libitum Observations of inoculated mice were made daily Symptom of disease first appeared in affected animals after 21 days, and in others not until after more than 60 days Observed symptoms included cachexia, muscle spasms, tremors, compulsive behaviors (e g , extended periods of scratching, mbbing. or mnning in circles), hyperactivity/hyperesthesia, seizures and convulsions, and stupor These results demonstrated that intracerebral inoculation with Cryptovirus results in subacute central nervous system (CNS) disease Neurological, neurodegenerative, and/or neuropsychiatric disease presentation in mice is virtually indistinguishable from presentation in humans

Wlnle all of the inoculated animals developed antibodies to the nucleocapsid protein of the vims (NP), not all of them developed antibodies to the envelope proteins (F, HN, and SH) More than 90% of the mice inoculated with Cryptovirus virions developed antibodies to the envelope proteins but only 33% of those inoculated with AV₃/SSPE cells did so

Concurrently, while many of the animals inoculated with infectious Cryptovirus stocks developed profound neuropathological disease, fewer of the animals inoculated with nonproductively- infected AV₃/SSPE cells developed such illnesses, and there was a strong correlation between development of antibodies to the envelope proteins of the vims and the development of CNS symptoms This suggests that the development of CNS disease depends on the establishment of an acute or subacute CNS infection by the vims and the expression of all of the vims' stmctural proteins in some cells or tissues

More detailed examples follow

Quackenbush mice Two litters of 1-2 day old Quackenbush mice were lntracerebrally- moculatcd (in the right cerebral hemisphere) with 5 x 10⁴ PFUs of either Cryptovirus (strain BBR)(10 individuals) or measles vims (Edmonston stram)(8 individuals), were returned to their mothers and were periodically observed over a period of three months Two animals (one inoculated with Cryptovirus and one inoculated with measles vims) were found dead and partially consumed the next morning Their deaths were attributed to "needle trauma" and/or maternal cannibalism While none of the mice inoculated with measles vims developed any neurological, neurodegenerative, physiological or neuropsychiatric symptoms over the course of the study, two of the male mice inoculated with Cryptovirus developed atrophy and contralateral hindhmb paralysis (in their left hind legs) three to four weeks after inoculation A third (female) mouse was observed dragging its left hind leg (unatrophied) approximately four weeks after inoculation but was found killed and partially eaten a day later Wlnle 3 of 9 animals inoculated with Cryptovirus developed hind limb paralysis (33%), none of the animals showed overt signs of seizure, wasting or neurophychiatric symptoms over the course of the study. Hind limb paralysis was also seen in a number of the offspring of adult female Quackenbush mice that had been inoculated with Cryptovirus as newborns but that did not develop any overt symptomology. The frequency of this phenomenon was difficult to assess because the mothers tended to cannibalize the newborn animals that were bom with, or subsequently developed, such characteristics.

Colored mice. Three litters of 1-2 day old Colored mice (comprising 26 individuals) were observed daily following intracerebral inoculation with 5 x 10⁴ PFUs of Cryptovirus (strain BBR), Simian Vims 5 (NIH 21005-2WR strain) or measles vims (Edmonston strain) or mock-infected cells Half of each litter was inoculated with Cryptovirus (13 animals) while the other half was apportioned into three groups and inoculated with either Simian Vims 5 (6 animals), measles viais (4 animals) or mock-infected CV-lc lysate (3 animals). Each group was marked with phenol red stain on the upper skin of one foot to distinguish them (i.e. right front, left front, right rear, left rear) One animal inoculated with Cryptovirus died between 24 and 48 hours post-inoculation and this was attributed to "needle trauma/starvation" as it had stopped feeding (or was rejected) when it was returned to its mother. Between three and four weeks later, one male and one female animal were found dead in their cages in tonic-clonic posture — both having been inoculated with Cryptovirus. It was noted that both also appeared to be underweight when compared to their littermates. Two months after inoculation with Cryptovirus, a third mouse (male) was observed to have cachexia, anorectic wasting, tremors and seizures (Fig. 7A) Over the next month (approximately 11 weeks after inoculation), a fourth animal (also male) developed tremors and seizures although no wasting was observed (data not shown). A male littcrmate of the Cry/?/ovzr«.y-infected mouse shown in Fig 7A, which was inoculated with the NIH 21005-2WR strain of SV5, is shown in Fig. 7B. The same results were obtained when mice were inoculated with either the Edmonston strain of measles vims, mock-infected CV-lc cells, or homogenized AV₃/SSPE cells (i.e. all remained healthy and none developed neurological, neurodegenerative or neuropsychiatric symptoms; data not shown).

Over the ensuing six months (observed up to nine months post inoculation), a significant number of the remaining animals (4 of the remaining 8) inoculated with Cryptovirus developed physiological, neurological and/or neuropsychiatric symptoms. Such late onset animals presented with symptoms that were in marked contrast to the overt seizure disorders observed in the subacute onset animals (i.e. those that developed symptoms in first three months post inoculation). These symptoms were dominated by physiological, neuropsychiatric and behavioral disturbances rather than more overt neurological symptoms and included, marked weight gain, extreme aggressive/passive responses to stimuli, obsessive/compulsive behaviors, ataxia and tremor. Aggression was most frequently characterized in afflicted animals by physical agitation and a predisposition to biting when handled. Passive animals tended to cat excessively, gain weight, and sleep. Repetitive behavior was also sporadically observed and consisted primarily of endless pacing and/or facial washing so extreme as to result in the loss of fur on the head and neck and the development of abrasive wounds. One of the animals (a female shown in Fig. 8A) had abnormal cranial stmcture (microcephaly) and manifested a spectmm of physiological and behavioral symptoms at six months including obesity, tics and muscle twitching (along the back and left side) and obsessive/compulsive facial washing and scratching. Episodes of such obsessive/compulsive behavior were observed to last for an hour or more. A second female animal (shown in Fig. 8B) appeared overtly normal during the first five months post inoculation but between five and six months began displaying marked ataxia, tremors and aggression. While this animal maintained a normal body weight and appearance, it was prone to splaying its feet to maintain its balance when resting and stumbling when walking and biting and hissing/snarling when handled or disturbed. Neither animal shown in Fig 8 developed overt (grand mal) seizures, in contrast to the animal shown in Fig. 7A Two other animals (one male and one female) also developed mixtures of the slow onset symptomology (data not shown). Overt seizure activity was never observed in any of the late onset animals and none of the animals inoculated with SV5, measles vims or mock-infected cells developed any similar symptoms.

Of the 13 nconate animals inoculated with Cryptovirus, one died from needle trauma (sex uncertain), two died in tonic-clonic posture with signs of wasting (indicative of sudden death due to status epilepticus (one male; one female); two became wasted and developed grand mal seizures (both males); four developed a spectmm of slow onset neurological/neuropsychological symptoms (three females, one male); one committed infanticide after being bred (a female); and three never developed any symptoms (two females and one male). Removing the needle trauma death from the equation, 9 of 12 animals developed neurological, degenerative and/or neuropsychiatric symptoms

(75%). Removing the two clonic-posture deaths as well, 7 of 10 animals developed symptoms (70%). This was highly significant compared to the combined results for the control inoculated mice (7 of 10 mice inoculated with Cryptovirus presenting with neurological symptoms versus 0 of 13 mice in the control groups; P = 0.0005, 2-sided Fisher exact test), and was also significant compared to just the SV5 inoculated mice (7 of 10 versus 0 of 6; P = 0.01, 2-sided Fisher exact test) and even when compared to just the measles inoculated mice (7 of 10 versus 0 of 4; P = 0.035, one-sided Fisher exact test). Statistical significance was not quite reached compared to the mice inoculated with uninfected cell lysate, because of the small number of mice in this group (7 of 10 versus 0 of 3, P = 0.069, single-sided Fisher exact). However, when the two clonic-posture deaths were included even this small control group was significantly different (9 of 12 versus 0 of 3, P = 0.044, 2-sided Fisher exact). Thus, the disease(s) and symptoms resulting from Cryptovirus infection was profoundly significant.

Infanticide. Of the four Colored mice which were inoculated as neonates with Cryptovirus but did not develop subacute or slow onset symptoms over the first nine months of the study, three were female and were subsequently bred with uninfected males at nine months of age. While all of the offspring of two of the three resulting litters developed normally, all of the offspring in one of the litters (comprising 10 animals) were killed and wholly or partially cannibalized by their mother. Such infanticide did not occur in litters to females that had been inoculated with SV5, measles or mock- infected cells. In a separate study (see below), one of the females which was inoculated with live AV3/SSPE cells — but did not develop any overt neurological symptoms — developed late physiological and behavioral symptoms and also committed infanticide of its whole litter after being bred with an uninfected male.

Animal Model Employing Inoculation of Nonproductively Infected AV₃/SSPE cells. In a separate study, two litters of neonatal mice (18 animals) were inoculated (1-2 days after birth) with either live or homogenized AV₃/SSPE cells (six animals each) or live or homogenized AV₃ cells (three animals each). There were no needle trauma deaths. While none of the animals inoculated with the homogenized AV₃/SSPE cells, live AV₃ cells or homogenized AV₃ cells (12 animals) developed any subacute or slow onset symptoms whatsoever, one of the six animals inoculated with live AV₃/SSPE cells developed subacute degenerative and neurological symptoms (a male) and one developed slow onset symptoms (1 female). The animal that developed subacute symptomology began presenting with symptoms 24 days after intracerebral inoculation. Over a five day period, the symptoms presented included cachexia, wasting, hunched posture, repetitive chirping and clicking, hyperesthcsia, incontinence of urine, tremors, muscle spasms and coma. Overt seizures were not observed. It was sacrificed when coma developed. The animal that developed slow onset symptomology (a female) presented at five to six months (post-inoculation) with repetitive pacing, aggression and progressive obesity. The animal was bred at six months with an uninfected male. Seven days after delivering a litter of eight offspring, it killed the whole litter and partially consumed each individual. No seizures or other signs of overt neurological symptoms were observed. None of the other females which were inoculated with homogenized AV₃/SSPE cells, AV₃ cells or homogenized AV₃ cells and bred (six animals in total) committed infanticide. Example 7. Cryptovirus -Specific Antibodies Are Present In The Seaim And Cerebrospinal Fluid (CSF) Of Human Patients Diagnosed With Neurological. Neurodegenerative And/Or Neuropsychiatric Diseases. Evidence for the presence of Cr ptøvzrw.y-specifιc antibodies to the major envelope proteins of the vims (F₀ and HN) in the seaim and CSF of patients was determined by immunoprecipitation of [³⁵S]-methιonιne-labeled Cry/?tovzra.y-spccific proteins produced in acutely-infected CV-1_C cells. Figs. 12A and 12B arc photographs of autoradiograms, which serve as examples of RIP profiles of measles vims- or Cry/?/ovzπz.y-specιfic proteins precipitated from [³⁵S]-methionine-labeled acutely infected CV-1 cells by clinical CSF specimens followed by SDS-PAGE (reduced). In Fig. 12A, lane "V" contains gradient-purified Cryptovirus virions from acutely-infected, ³ [S]methionine- labeled CV-1_C cells (BBR Strain). Lane "MV" contains proteins precipitated by the CSF of an 11- year old male SSPE patient from radiolabled CV-1_C cells acutely-infected with measles vims (Edmonston Strain). Lane "B" contains proteins precipitated by the same CSF specimen from a 1 : 1 mixture of radiolabled CV-1_C cells acutely-infected with either measles vims or Cryptovirus. Lane

"CV contains proteins precipitated by the same CSF specimen from radio-labeled CV-1_C cells acutely-infected with Cryptovirus. In Fig. 12B are shown RIP profiles of the Cryptovirus-specific proteins precipitated by the CSFs of six randomly-selected neurology/neurosurgery patients who had CSF taken for diagnostic screening The patient whose sample appears in Lane 2 was an adult male who had presented with ataxia, confusion and memory loss (tentatively diagnosed with ataxic cerebellar syndrome) The patient whose sample appears in Lane 4 was an infant female who presented with hydrocephalus and intractable seizures and who subsequently died in status epilepticus

Example 8. Cryptovirus Is Implicated In The Aetiopathogenesis Of Disease in Patients Diagnosed with Idiopathic Human Neurological. Neurodegenerative, And/Or Neuropsychiatric Diseases.

Cryptovirus is implicated in the aetiopathogenesis of disease in patients diagnosed with idiopathic neurological, neurodegenerative, and/or neuropsychiatric diseases, including anorexia nervosa, multiple sclerosis (MS), epilepsy, subacute sclerosing panencephalitis (SSPE), autism, mental retardation, affective disorder, dysthymia (clinical depression), schizophrenia, obsessive compulsive disorder, manic depression (bipolar disorder), chronic fatigue syndrome (CFS), hydrocephalus, ataxic cerebellar syndrome and atypical viral meningitis. Most patients who had Cry/p/c»vzrw.y-specific antibodies in their CSF had been given multiple diagnoses. Thus, there is a correlation between the presence of Cry/>tovz^'ra.y-specific antibody to the major envelope proteins of the vims (F₀ and HN) in the CSF of neurology or neurosurgery patients and prior diagnosis of a condition with a significant "iterative" or compulsive component.

Although Cryptovirus seropositivity did not necessarily correlate to CSF positivity (i.e , the presence of antibody to the Cryptovirus F₀ and HN proteins in the CSF) or a diagnosis of any neuropathological condition, CSF positivity strongly correlated with a prior diagnosis of a significant disorder of the central nervous system These correlations were consistently found for patients with certain diagnoses (e.g., SSPE, MS, CFS, and certain forms of idiopathic epilepsy) and incidentally found for specimens from patients with other diagnoses (e.g , Alzheimer's Disease). Similar results were obtained for two CSF specimens analyzed by an immunblotting technique (data not shown), or for semm and CSF specimens analyzed by an enzyme-linked- lmmunosorbcnt-assay (ELISA; see Fig. 14), although these assays were performed on only a proportion of CSF specimens due to the limited volumes available in some samples.

Although some patients presented with some of the above-mentioned symptoms and did not have antibody to the vims in their CSF specimens, in no instances were Cryptovirus-specific antibodies found in the CSF of patients that did not present with many of the symptoms and who had not been diagnosed with a significant neuropathological or neuropsychological disorder.

In addition, seropositive individuals (i.e , those who have Cryptovirus-specific antibodies in their semm) harbor the vims in a nonproductive, inapparent but inducible state in their PBMNCs. While the presence of the viais in an individual patient's PBMNCs did not symmetrically correlate with the development of neuropathological disorder, these findings imply that the vims can gain entry into the CNS via a microvascular incident (i.e., leakage of Cryptovirus carrying PBMNCs into the CNS) or by immune system responses to other CNS stimuli (i.e., diapedisis of Cryptovirus- carrying lymphocytes into the CNS as part of an inflammatory response to another infection; a Trojan Horse phenomenon). Reference to a lack of symmetrical correlation means that, while all individuals whose PBMNCs were examined and had Cryptovirus-specific antibodies in their CSF carried the vims in those cells, not all individuals who were found to be carrying the vims in their PBMNCs were, at that time, suffering from any neurological, neurodegenerative, and/or neuropsychiatric disorder. The following examples reveal more detail.

(a) Alzheimer 's Disease. As shown in Fig 15, three matched sets of semm and CSF (provided by the National Neurological Research Specimen Bank (NNRSB) in Los Angeles, CA) were examined by RIP analysis for the ability to precipitate the Cryptovirus F₀ and HN proteins from radiolabeled acutely-infected CV-1_C cells. While all three had Cryptovirus-specific antibodies in their semm, only Patient 3 had these antibodies in his or her CSF. This implies that the illness Patient 3 was suffering, diagnosed as Alzheimer's disease, was complicated by concurrent Cryptovirus infection of the CNS tissues. Alternatively, Patient 3 could have been misdiagnosed, in which case he or she could actually be suffering from a Cryptovirus-related neuropathy.

Even though the sample size is small, it is interesting that all three of the Alzheimer's disease patients had been exposed to Cryptovirus and were probably carrying it in their lymphocytes. It appeared, unlikely, however, that Cryptovirus plays a role in the development of Alzheimer's disease, because Patients 1 and 2 did not appear to have the vims in their CNS tissues.

(b) Ataxic Cerebellar Syndrome, Atypical Viral Meningitis, Hydrocephalus, Idiopathic Parasthesia and Status Epilepticus. A blind screen (i.e., none of the diagnoses or medical histories pertaining to any of the specimens was provided prior to specimen screening) was conducted of 66 CSF specimens from neurology or neurosurgery patients who had CSF specimens taken for diagnostic screening by the Department of Clinical Microbiology at the Royal Brisbane and Royal Children's Hospitals, in Brisbane, Queensland, Australia. Of these CSF specimens, ten were Cry/?tovzπz.y-positive (see Fig. 17). One of the ten Cryptovirus -positive CSF specimens was identified as being from an adult male patient who had been diagnosed with ataxic cerebellar syndrome, (see Fig. 12B). Another of the ten Cryptovirus-positive CSF specimens was identified as being from an adult female patient who had been diagnosed with atypical viral meningitis (data not shown). A third positive CSF specimen came from a 55 year old male that had presented with ataxia, memory loss, blackouts, seizures, diploplia and headache and had been diagnosed with hydrocephalus, chronic fatigue syndrome and possible epilepsy; a fourth positive CSF specimen was from an adult male who had been diagnosed with idiopathic parasthesia; and a fifth positive CSF specimen was from a female infant who presented with clonic hand movements and intractable seizures and was diagnosed with hydrocephalus and status epilepticus (see also c and d, below). Diagnoses and symptoms of the remaining five Cry/?/ovz zz.y-positive CSF specimens were unavailable.

(c) Chronic Fatigue Syndrome (CFS). A number of adolescent and adult patients who presented with symptoms of CFS were subsequently found to have high titers of ti-Cryptovirus antibodies in their sera, demonstrating that primary infection with the vims can manifest as a chronic febrile tracheo-bronchial illness with associated chronic malaise and lymphadenopathy. This is not unlike infectious mononucleosis in presentation (i.e., a sore throat and persistent "glandular fever"). There was no evidence of acute encephalitic (or encephalopathic) disease in such patients or in any other patient found to have Cryptovirus-specific antibodies in his or her semm or CSF. "Acute" is taken here to mean presenting with rapid onset and symptoms within seven days. Fifty-six semm specimens from patients who had been diagnosed with CFS were provided for Cryptovirus screening by regional physicians (Brisbane and Southeast Queensland). Eleven matching CSF specimens were subsequently obtained. RIP analysis revealed that 54/56 (96.4%) of the seaim samples and 10/11 (90.9%) of the CSF specimens contained Cryptovirus-specific antibodies (Fig. 16). Including the patient who had been codiagnosed with hydrocephalus, epilepsy and CFS (sec

Fig. 17 and data for Epilepsy, below), a total of 12 CSF specimens from CFS patients were analyzed by RIP analysis and 1 1/12 (91.7%) had Cry/? ov/πz.y-specific antibodies in them.

Patients who had been diagnosed with CFS almost always had two, or more, concurrent diagnoses. These included: anorexia nervosa, MS, epilepsy, dysthymia (clinical depression), schizophrenia, and manic depression (bipolar disorder). For example, one adolescent girl who was co-diagnosed with both anorexia nervosa and chronic fatigue syndrome (CFS) had Cryptovirus- specific antibodies in her CSF. It is of note that the etiology of virtually all of these disorders is idiopathic.

Wlnle the symptoms presented by CFS patients cover a broad spectmm, the spectaim is, in fact, fairly discrete and representative of the illness. This is perhaps best illustrated by examination of the medical records of five patients, presented below:

Patient PR was an adult male, 55 years of age, who was suffering primarily from mental confusion, lethargy, memory loss, blurred vision, dysthymia, and petit mal seizures. EEG results were abnormal, which indicates epileptiform disease. Patient PR was ambulatory with progressively deteriorating CNS symptoms.

Patient DF was an adult male, 52 years of age, who was suffering primarily from mental confusion, lethargy, memory loss, dysthymia, and petit mal seizures. EEG results were abnormal, showing epileptiform responses in cortical and subcortical functions of the anterior hemispheres. Patient DF was ambulatory with progressively deteriorating CNS symptoms. Patient NB was an adult female, 36 years of age, who was suffering primarily from mental confusion, lethargy and extreme fatigue, memory loss, dysthymia, ataxia, blurred vision, and parathesias, and had a history of glandular fever, recurrent sore throats of prolonged duration, tremors, and petit mal seizures. NB's sister was diagnosed with anorexia and myoclonus. Patient NB was bedridden or partially ambulatory with progressively deteriorating CNS symptoms.

Patient KT was an adult female, 27 years of age, who was suffering primarily from mental confusion, loss of concentration, memory loss, anorexia, lethargy and extreme fatigue, and tremors, and had a history of recurrent febrile lymphadenopathy. Patient KT was stable but bedridden and only partially ambulatory.

Patient SS was an adult female, 23 years of age, who was suffering primarily from loss of concentration, memory loss, and lethargy, and had a history of dysthymia beginning at age 14 and EBV-negative glandular fever. Immediate family members (mother, father, two sisters, and one brother) were all seropositive. In addition, SS's mother had a 9 year history of dysthymia, and

Cryptovirus antigens were detected in her cultured PBMNC. Two years after sampling, Patient SS was stable and ambulatory.

(d) Epilepsy and Hydrocephalus. RIP analysis was used to determine the presence of Cryptovirus-specific antibodies in two clinical collections of CSF specimens. The first collection included 66 specimens that were selected at random from those submitted to the Department of Clinical Microbiology at Royal Brisbane Hospital in Brisbane, Queensland by physicians in the Department of Neurology and Neurosurgery (see b above). None of the diagnoses or medical histories pertaining to any of the specimens was provided prior to specimen screening. Fig. 17 illustrates the results of RIP assays conducted with CSF from this collection. The positive CSF precipitate in Lane 2 was subsequently found to have come from a 55-year old adult male (RW) who presented with ataxia, memory loss, blackouts, seizures, diploplia, and headaches. He was determined to have a hydrocephalic condition and underwent surgery to insert a ventricular shunt to alleviate the condition. He had been diagnosed with hydrocephalus, epilepsy and Chronic Fatigue Syndrome (CFS).

Ten of the 66 CSF specimens in Collection 1 were found to contain Cry/?tovzrzz.y-specιfic antibody (15%). Diagnoses were obtained for five of these patients and included: (1) hydrocephalus and intractable seizures in an infant female who subsequently died (see Fig. 12B), (2) ataxic cerebellar syndrome in an adult male (see Fig. 12B), (3) atypical viral meningitis in a female child, (4) parathesia in an adult male, and (5) hydrocephalus, epilepsy and CFS in the patient described in connection with Fig. 17 Diagnoses were obtained for only two of the 56 Cry/?/ovz^'πz.y-negative CSF specimens: one patient (WK, a male) was diagnosed with acute viral meningitis and one (SG, a female) was diagnosed with idiopathic intracranial hypertension.

The second collection (Collection 2) included 20 CSF specimens from children (<12 years old) that were collected by neurologists at Camperdown Children's Hospital in Sydney, New South

Wales, Australia. Again, none of the diagnoses or medical histories pertaining to any of the specimens was provided prior to specimen screening. However, in this collection a request had been made to include an undisclosed number of CSF specimens from children who had either presented with epileptiform illness or had been diagnosed with some form of idiopathic epilepsy. Fig. 18 illustrates the results of RIP assays conducted with CSF from Collection 2. The CSF precipitate analyzed in Lane 1 was from a newborn infant who developed intractable seizures and died in status epilepticus (Patient CT, below) and the precipitate analyzed in Lane 2 was from a child who had been given a diagnosis of Lennox-Gasteau/generalized epilepsy (Patient LB, below), respectively. The background noise in this autoradiogram was high as a result of the long-term exposure (30 days) required to see the bands.

Six of the 20 CSF specimens provided in the (biased) Collection 2 were found to have Cry/?tovz^'rw.y-specific antibodies, and it was subsequently learned that this screening had identified 6 of the 7 specimens from patients who had been diagnosed with epilepsy or other forms of epileptiform illness and had been included in the collection. The six Cryptovirus -positive CSF specimens came from the following patients: (1) CT, a neonate with intractable fits and seizures, who died in status epilepticus, (2) LB, who was diagnosed with Lennox-Gasteau epilepsy and generalized epilepsy, (3) BM, who was diagnosed with severe retardation and epilepsy, (4) FZ, a two-month old child with intractable seizures who died in status epilepticus, (5) CN, who had hydrocephalus, cerebral palsy, and epileptiform seizures, and (6) LD, who had primary infantile spasms. Hydrocephalus was codiagnosed in 3 of 8 patients diagnosed with epilepsy or other epileptiform illness.

Although one of the 14 Cry/?tovz^'π*.y-negative CSF specimens was obtained from a patient who had been diagnosed with epilepsy, diagnoses were not provided for the remaining specimens. They were simply characterized as pediatric neurology or neurosurgery specimens from asymptomatic patients (i.e., patients who had not presented with epileptiform symptoms or been diagnosed with epileptiform illness). (e) Multiple Sclerosis (MS). Clinical specimens from patients with MS comprise one of the largest groups of materials screened (38 semm samples and 30 CSF samples including 30 matched sets of each). Eight of the seaim samples came from MS patients in Brisbane, Queensland who had debilitating disease and were living in a nursing home ain by the National Multiple Sclerosis Society of Australia. No CSF specimens were acquired from these patients. The 30 matched sets of semm and CSF were provided by the National Neurological Research Specimen Bank (NNRSB) in Los Angeles.

Fig. 19 illustrates the results of RIP assays conducted with seaim samples of 5/30 MS patients provided by the NNRSB. The results obtained from an additional 25 semm specimens provided by the NNRSB are shown in Fig. 20, and the RIP results from 16/30 of the CSF specimens from MS patients provided by the NNRSB are shown in Fig. 21. RIPs performed using the remaining 8 specimens resulted in similar profiles (data not shown). As shown in Figs. 19-21, the results of these analyses demonstrated that all patients had high levels of Crypto virus -specific antibodies in their semm (100%) and 29/30 had Cry/?zovz>zz.y-specific antibodies in their CSF (96.7%).

(f) Subacute Sclerosing Panencephalitis (SSPE). The anomalies that have been observed which are inconsistent with measles vims alone being the sole cause of SSPE (see Discussion of Related Art) can be explained by the evidence that the aetiopathogenesis of SSPE involves dual infection of the CNS by measles and Cryptovirus (which was isolated from SSPE patients). Sera from SSPE patients were found to precipitate the major nucleocapsid protein of the viais

(NP, 63 kD) from nonproductively-infected AV₃/SSPE cells (see Fig. 22). Fig. 22 is a photograph of an autoradiogram obtained following creation of RIP profiles of the Cryptovirus NP protein (p63) precipitated from [³⁵S]-methionine-labeled AV₃/SSPE cells by the sera of six Australian SSPE patients (Lanes 1-6) and six control sera (Lanes 7-12; sera from pediatric patients without antibodies to the Cryptovirus major envelope proteins (F₀, HN).

Fig. 23 is a photograph of an autoradiogram of RIP profiles of measles vims-specific and Cryptovirus-specific proteins precipitated from [³⁵S]-methionine-labeled measles vims-infected CV- lc cells (Lane MV), Cryptovirus-infected CV-1 cells (Lane CV) or a mixture of both (Lane B) by CSF sampled from an 11-year old male diagnosed with SSPE. Lane V = gradient-purified Cryptovirus virions from [³⁵S]-mcthioninc-labeled Cryptovirus -infected CV-lc cells. Fig. 23 shows that CSF from this SSPE patient precipitated both Cryptovirus and measles viais proteins. The results of this assay demonstrate that SSPE CSF contains both measles viais-specific and Cr)7?/6>vz>z/.y-specifιc antibodies (i.e. antibody to the HN protein of measles vims (Lane MV) and the HN and F₀ proteins of Cryptovirus vims (Lane CV) and that both are present in nearly equal amounts This was unique, since none of the other CSFs samples that precipitated Cryptovirus proteins (e g , from MS. CFS, or epilepsy patients) also preciptated the measles viais HN protein

The RIP profile of Cryptovirus -specific proteins precipitated by this CSF specimen is typical of those produced by the "Cry/?zovzr«.y-posιtιve" CSFs of MS patients, CFS patients and idiopathic epilepsy patients tested to date (compare Figs 12A and 17) While there was considerable variation in the strength of the antibody response to the F₀ and HN proteins, there appeared to be little variation in the presence of antibody to one protein or the other There was, however, a variable response to the F, and F₂ proteins (z e , in many patients, such responses appeared to be absent) This paradox may relate to the proteolytic cleavage of the protein n situ and corresponding immune responses (i e , the Fo protein is efficiently cleaved by some patients, generating the F, and F₂ fragments and ultimately exposing their immune system to them), in other patients the protein may be cleaved less efficiently (or not at all) and, therefore, these patients do not generate as much of an antibody response to the fragments)

Example 9 Correlations Between Affected Human Patients and Experimentally-Infected Animals

Some of the examples herein highlight the epileptiform symptomology presented by many of the patients who have Cryptovirus -specific antibodies in their cerebrospinal fluid (CSF) or by mice experimentally-infected with the vims This association is strong, but not all patients with Cryptovirus-specific antibodies in their CSF present with overt seizures or convulsions There is instead a spectaim of responses, from little or no seizure activity, through mild activity (petit mal or "absence" seizures), to recurrent and intractable grand mal seizures (the occurrence of which is often misunderstood by the lay public to be the defining symptom of all forms of epilepsy, see Epilepsy A Comprehensive Textbook, Engel, Jr J and Pedley, T A , Eds , Lippincott-Raven, 1997) With regard to the development of symptoms and manifestations of human Cryptovirus- mfections, it is essential to be cognizant of the spatial ("where"), temporal ("when"), and quantum ("what else") factors involved (1) which cells, tissues, neural tracts and CNS staictures become infected by the vims, (2) the developmental state of those systems at the time of infection, and (3) the role of environmental and host factors in development and progress of the infection, respectively For example, the data presented here establish a strong correlation between the development of epileptiform symptomology and early CNS infection with the vims (i e , in infancy, early childhood or adolescence and in experimentally-infected neonatal mice), this correlation is less strong in adults (and adult mice who do not develop epileptiform symptoms) and the spectmm of CNS manifestations observed is much wider.

Generally, the characteristic most frequently and consistently presented by humans or animals that have been experimentally infected with the viais is the development of "iterative" or "compulsive" neuropathies and behaviors. This is most likely due to the selective loss of (or immunopathological damage done to) neurons (e.g. interneurons) or neuron tracts in different parts of the central nervous system (CNS) at different times or at different stages of CNS development.

When the medical records of patients who had Cryptovirus-specific antibodies in their CSF were examined, all of the patients had been diagnosed with one or more serious neurological disorders. These included, but were not limited to:

(1) subacute sclerosing panencephalitis (SSPE): 4 of 4 CSFs tested (100%), all adolescent patients);

(2) idiopathic / cryptogcnic epilepsy: 6 of 7 CSFs tested (85.7%), from infants and children presenting with seizures and diagnosed with idiopathic or cryptogenic forms of epilepsy; (3) multiple sclerosis (MS): 29 of 30 CSFs tested (96.7%) all adult specimens; and

(4) chronic fatigue syndrome (CFS) / clinical depression: 11 of 12 CSFs tested (91.7%), all adult specimens.

These results demonstrate a clear coaelation between Cry/?tovzrw.y-specifϊc antibodies in the CSF and a narrow spectaim of CNS diseases. Although the diseases listed above have been defined as representatives of discrete pathognomonic entities, there is in reality substantial overlap between the symptoms presented by these patients and their diagnoses. For example, virtually every patient eventually diagnosed with SSPE is initially diagnosed with epilepsy. Similarly, early stage MS is extremely similar in presentation to CFS, and clinical depression is a common characteristic of both. Not surprisingly, another name for CFS is "atypical multiple sclerosis" (in Bell, The Disease of a Thousand Names, Pollard Publications, Lyndonville, NY [1991]).

There is a strong correlation between (l) the age of those patients who had severe epileptiform illness (SSPE and epilepsy, the majority of whom are infants, children, or adolescents) and (2) the age of those patients who had more diffuse or subtle neurological dysfunction (e.g., MS and CFS patients who were all adults). Furthermore, SSPE, certain forms of idiopathic and cryptogenic epilepsy, and MS have many neuropathological characteristics in common. These include areas of discrete, focal or disseminated sclerosis (scar formation in CNS tissue), dysplastic lesions (either as the result of immunopathological processes or neuron tract loss), and perivascular cuffing of immune cells (evidence of inflammatory processes in the vicinity of lesions). Thus, each of these diseases could represent a different pathological "complex" of spatial, temporal, and quantum factors that have Cryptovirus infection of CNS tissues as a shared characteristic. With respect to SSPE, previous data have established (and the data here confirm) that measles vims is also involved in this illness. SSPE is caused by widespread CNS infection by both vimses (resulting in inflammatory and disseminated sclerosis across the white matter of the brain) while certain forms of idiopathic epilepsy represent early infection of the CNS by Cryptovirus alone (resulting in the loss of susceptible inteaieurons and neuron tracts and the development of discrete dysplastic lesions). MS, occurring almost exclusively in adults, represents the pathological outcome of late and focal Cryptovirus infection of the CNS - due to the restriction of Cryptovirus replication in fully differentiated CNS tissues and the effective partitioning of brain by the mature glial architecture.

In summary, intracranial inoculation of mice with the vims, or with cells nonproductively- infected with the vims, results in the subacute development of neuropathological diseases in a significant proportion of the animals. These diseases are closely akin to the spectaim of human neuropathies seen in patients with Cry/?tovzrw.y-specific antibodies in their cerebrospinal fluids. Further, although patients with Cryptovirus-positive CSF had been diagnosed with a spectmm of illnesses, there was a clear partitioning of patients into two groups (1) infants, children and adolescents with illnesses dominated by subacute epileptiform physical symptoms which were often life-threatening and (2) young adults and adults with slowly-developing chronic illnesses which presented with less pronounced physical symptoms but significant neuropsychological components which were usually not life-threatening.

These findings support the conclusion that Cryptovirus is responsible for a neurological spectrum disorder whose ultimate manifestation depends on (1) the age of the individual when they contract the primary infection, (2) the mechanism by which the vims gains entry into the CNS tissue, (3) the extent of the infection at that time, (4) the stage of development of the CNS when it becomes infected, (5) the part of the CNS which becomes infected, (6) genetic factors (e.g., immune system defects, neurological malformations, the presence for absence of viais receptors on CNS tissues, etc ) and (7) other environmental factors (e.g., the occurrence of head trauma, neurosurgery of any kind, prior or concurrent infection of CNS tissue by other agents, exposure to dmgs or toxic chemicals, etc.).

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims (1)

1. An isolated nucleic acid, comprising:

(A) contiguous nucleotide positions 1-15246 of (SEQ ID NO: l);

(B) a nucleotide sequence complementary to (A); or

(C) a Cry/?to vz^'rw.y-specific fragment of (A) or (B), at least about five nucleotides long.

2. The nucleic acid of Claim 1, wherein the Cry/?/ovz>zz.y-specific fragment of (C) comprises a nucleic acid segment selected from the group consisting of:

(i) contiguous nucleotide positions 152-1678 of (SEQ ID NO: l), a complementary sequence, or a degenerate coding sequence;

(ii) contiguous nucleotide positions 1850-2515 of (SEQ ID NO: l), a complementary sequence, or a degenerate coding sequence;

(iii) contiguous nucleotide positions 1850-3023 of (SEQ ID NO: l), a complementary sequence, or a degenerate coding sequence;

(iv) contiguous nucleotide positions 1850-3023 of (SEQ ID NO. l) combined with a further insertion of two guanine residues between nucleotide position 2339 of (SEQ ID NO: I) and nucleotide position 2340 of (SEQ ID NO: l), a complementary sequence, or a degenerate coding sequence;

(v) contiguous nucleotide positions 3141-4271 of (SEQ ID NO: l), a complementary sequence, or a degenerate coding sequence;

(vi) contiguous nucleotide positions 4530-6182 of (SEQ ID NO: l), a complementary sequence, or a degenerate sequence; (vii) contiguous nucleotide positions 4587-6182 of (SEQ ID NO: l), a complementary sequence, or a degenerate sequence;

(viii) contiguous nucleotide positions 4587-4835 of (SEQ ID NOT), a complementary sequence, or a degenerate sequence;

(ix) contiguous nucleotide positions 4836-6182 of (SEQ ID NOT), a complementary sequence, or a degenerate sequence;

(x) contiguous nucleotide positions 4272-6515 of (SEQ ID NO: l), a complementary sequence, or a degenerate coding sequence;

(xi) contiguous nucleotide positions 6303-6434 of (SEQ ID NOT), a complementary sequence, or a degenerate coding sequence;

(xii) contiguous nucleotide positions 6584-8278 of (SEQ ID NOT), a complementary sequence, or a degenerate coding sequence; and

(xiii) contiguous nucleotide positions 8414-15178 of (SEQ ID NOT), a complementary sequence, or a degenerate coding sequence.

3. The nucleic acid of Claim 1, comprising the C/ />tovz^'πz.y-specific fragment, said fragment being about seven nucleotides to about 500 nucleotides long.

4. The nucleic acid of Claim 3, wherein said fragment is about seven nucleotides to about 50 nucleotides long.

5. The nucleic acid of Claim 4, wherein said fragment is about fifteen nucleotides to about 35 nucleotides long.

6. The nucleic acid of Claim 1, wherein the nucleic acid is RNA.

7. The nucleic acid of Claim 1, wherein the nucleic acid is cDNA. A composition of matter, comprising the nucleic acid of Claim 1; and a caaier.

A nucleic acid constmct, comprising the nucleic acid of Claim 1.

10 Use of the nucleic acid of Claim 1 in manufacturing a vaccine.

Use of the nucleic acid constmct of Claim 9 in manufacturing a vaccine.

12. An expression vector, comprising the nucleic acid constmct of Claim 9.

13 A cloning vector, comprising the nucleic acid constmct of Claim 9

14. A host cell, comprising the expression vector of Claim 12 or the cloning vector of

Claim 13.

15. The host cell of Claim 14, wherein the cell is a mammalian cell.

16. An isolated Cryptovirus protein encoded by a nucleic acid segment comprising:

(A) contiguous nucleotide positions 152-1678 of (SEQ ID NOT) or a degenerate sequence;

(B) contiguous nucleotide positions 1850-2515 of (SEQ ID NOT) or a degenerate sequence;

(C) contiguous nucleotide positions 1850-3023 of (SEQ ID NOT) combined with a ftirther insertion of two guanine residues between nucleotide position 2339 of (SEQ ID NOT) and nucleotide position 2340 of (SEQ ID NO: l), or a degenerate sequence;

(D) contiguous nucleotide positions 3141 -4271 of (SEQ ID NOT) or a degenerate sequence;

(E) contiguous nucleotide positions 4530-6182 of (SEQ ID NOT) or a degenerate sequence; (F) contiguous nucleotide positions 4587-6182 of (SEQ ID NOT) or a degenerate sequence;

(G) contiguous nucleotide positions 4587-4835 of (SEQ ID NOT) or a degenerate sequence;

(H) contiguous nucleotide positions 4836-6182 of (SEQ ID NO: l) or a degenerate sequence;

(I) contiguous nucleotide positions 6303-6434 of (SEQ ID NOT) or a degenerate sequence,

(J) contiguous nucleotide positions 6584-8278 of (SEQ ID NOT) or a degenerate sequence; or

(K) contiguous nucleotide positions 8414-15178 of (SEQ ID NOT) or a degenerate sequence.

17. The protein of Claim 16, wherein the protein is a Cryptovirus envelope protein encoded by a nucleic acid segment comprising (E), (F), (G), (H), (I), or (J).

18. A composition of matter, comprising the protein of Claim 16; and a carrier.

19 A chimeric protein, comprising a Cryptovirus protein encoded by a nucleic acid segment comprising:

(A) contiguous nucleotide positions 152-1678 of (SEQ ID NOT) or a degenerate sequence;

(B) contiguous nucleotide positions 1850-2515 of (SEQ ID NO: l) or a degenerate sequence; (C) contiguous nucleotide positions 1850-3023 of (SEQ ID NO 1) combined with a further insertion of two guanine residues into the nucleotide sequence between nucleotide position 2339 of (SEQ ID NO 1) and nucleotide position 2340 of (SEQ ID NO 1), or a degenerate sequence,

(D) contiguous nucleotide positions 3141-4271 of (SEQ ID NO 1) or a degenerate sequence,

(E) contiguous nucleotide positions 4530-6182 of (SEQ ID NO 1) or a degenerate sequence,

(F) contiguous nucleotide positions 4587-6182 of (SEQ ID NO 1) or a degenerate sequence.

(G) contiguous nucleotide positions 4587-4835 of (SEQ ID NO 1) or a degenerate sequence,

(H) contiguous nucleotide positions 4836-6182 of (SEQ ID NO 1) or a degenerate sequence,

(I) contiguous nucleotide positions 6303-6434 of (SEQ ID NO 1) or a degenerate sequence,

(J) contiguous nucleotide positions 6584-8278 of (SEQ ID NO 1) or a degenerate sequence, or

(K) contiguous nucleotide positions 8414-15178 of (SEQ ID NO 1) or a degenerate sequence

20 The chimeric protein of Claim 19, wherein the Cryptovirus protein is a Cryptovirus envelope protein encoded by a nucleic acid segment comprising (E). (F), (G), (H), (I), or (J)

21 Use of the protein of Claim 16 or Claim 19 in producing a Cryptovirus-specific antibody 22 Use of the protein of Claim 16 or Claim 19 in producing a vaccine

23 An isolated antibody that specifically binds the protein of Claim 16

24 A composition of matter, comprising the antibody of Claim 23, and a carrier

25 The antibody of Claim 23. that specifically binds a Cryptovirus envelope protein encoded by

(A) contiguous nucleotide positions 4530-6182 of (SEQ ID NO 1) or a degenerate sequence

(B) contiguous nucleotide positions 4587-6182 of (SEQ ID NO 1) or a degenerate sequence,

(C) contiguous nucleotide positions 4587-4835 of (SEQ ID NO 1) or a degenerate sequence,

(D) contiguous nucleotide positions 4836-6182 of (SEQ ID NO 1) or a degenerate sequence,

(E) contiguous nucleotide positions 6303-6434 of (SEQ ID NO 1) or a degenerate sequence or

(F) contiguous nucleotide positions 6584-8278 of (SEQ ID NO 1) or a degenerate sequence

26 The antibody of Claim 23, wherein the antibody is polyclonal

27 The antibody of Claim 23, wherein the antibody is monoclonal

28 The antibody of Claim 23, wherein the antibody is chimeric 29 Use of the antibody of Claim 23 in manufacturing a medicament for the treatment of Cryptovirus infections

30 An isolated viral particle, comprising the nucleic acid of Claim 1

31 A composition of matter, comprising the virion of Claim 30, and a carrier

32 An isolated viral particle, comprising the protein of Claim 17

33 An isolated Cryptovirus particle, comprising a genome having a nucleotide sequence entirely complementary to (SEQ ID NO 1)

34 Use of the viral particle of Claim 32 in manufacturing a vaccine

35 Use of the Cryptovirus particle of Claim 33 in manufacturing a vaccine

36 The use of Claim 34 or 35, wherein the viral particle is an attenuated virion

37 The use of Claim 34 or 35, wherein the viral particle is a killed virion

38 An isolated Cryptovirus particle, wherein the Cryptovirus is Strain BBR

39 A probe, comprising the nucleic acid of Claim 3

40 A primer, comprising the nucleic acid of Claim 5

41 A method of detecting the presence or absence of a Cryptovirus protein in a sample of a biological material, comprising

contacting the sample of the biological material with the antibody of Claim 23, and

detecting specific binding of the antibody to a constituent of the sample, wherein the presence of specific binding indicates the presence of the Cryptovirus protein in the sample

42. A method of detecting the presence or absence of a Cry/?tøvzVιz.y-specific RNA in a sample of a biological material, comprising:

obtaining a sample of a biological material comprising RNA;

contacting the sample with the probe of Claim 39 under at least moderately stringent hybridization conditions, wherein the formation of detectable hybridization products indicates the presence of the Cryptovirus-specific RNA in the sample.

43. A method of detecting the presence or absence of a Czy/?zovz>z<.y-specific RNA in a sample of a biological material, comprising:

obtaining a sample of a biological material comprising RNA;

amplifying Cryptovirus-specific RNA in the sample using at least one primer of Claim 40 in an amplification reaction mixture;

then detecting the presence or absence of Cryptovirus-specific nucleic acid amplification products in the amplification reaction mixture, wherein the presence of the amplification products in the reaction mixture indicates the presence of the Cryptovirus RNA in the sample.

44. The method of any of Claims 41, 42, or 43, wherein the biological material is a cellular material.

45. The method of any of Claims 41, 42, or 43, wherein the biological material is blood or semm.

46. The method of any of Claims 41, 42, or 43, wherein the biological material is cerebrospinal fluid.

47. The method of any of Claims 41, 42, or 43, wherein the biological material is lymphoid tissue.

48. The method of any of Claims 41, 42, or 43, wherein the biological material is nervous tissue.

49. The method of Claim 48, wherein the nervous tissue is brain tissue.

50. A method of detecting the presence or absence of a Cry/j>/ovzr«.y-specific antibody in a sample of a biological material, comprising:

contacting the sample with the protein of Claim 16;

allowing the formation of a specific protein-antibody complex;

detecting the presence of the specific protein-antibody complex, wherein the presence of a specific protein-antibody complex indicates the presence of the Cry/?t6>vz zz.y-specific antibody in the sample.

1. A method of detecting the presence or absence of a Cr pzovz -spccific antibody in a sample of a biological material, comprising:

contacting the sample with the protein of Claim 19;

allowing the formation of a specific protein-antibody complex;

detecting the presence of the specific protein-antibody complex, wherein the presence of a specific protein-antibody complex indicates the presence of the Cry ?tøvz>w.y-specific antibody in the sample.

52. An assay method for detecting the presence or absence of an antibody that selectively binds Cryptovirus in a sample of an antibody-containing biological material originating from a human, comprising:

contacting the sample, the sample originating from an individual suspected of having a Cryptovirus infection, with the envelope protein of Claim 17, such that, if antibody selectively binding Cryptovirus is present, an antibody-bound envelope protein complex forms; contacting any antibody-bound envelope protein complexes thus formed with anti-human antibody-binding antibody, and allowing the formation of complexes of the antibody, with the antibody-bound envelope protein complexes, and

detecting the presence or absence of any antibody-bound envelope protein complexes thus formed, the presence of such complexes indicating the presence in the sample of antibody selectively binding Cryptovirus

53 An assay method for detecting the presence or absence of antibody that selectively binds Cryptovirus antigen in a sample of an antibody-containing biological material originating from a human the method comprising

contacting the sample, the sample originating from an individual suspected of having a Cryptovirus infection, with the viral particle of Claim 32, such that, if antibody selectively binding Cryptovirus antigen is present, an antibody-bound vims complex forms,

contacting any antibody-bound vims complexes thus formed with anti-human antibody- binding antibody, and allowing the formation of complexes of the anti-human antibody-binding antibody with the antibody-bound vims complexes, and

detecting the presence or absence of any complexes formed, the presence of such complexes indicating the presence in the sample of antibody selectively binding Cryptovirus antigen

54 A method of detecting a Cryptovirus infection in a mammal, comprising

obtaining a sample of a biological material from the mammal, and

performing the method of any of Claims 41. 42, 43, 50, 51, 52, or 53, using the sample, whereby detecting the presence of the Cryptovirus protein, Cryptovirus -specific RNA, and/or Cryptoviru y-spccific antibody in the sample indicates a Cryptovirus infection in the mammal

55 The method of Claim 54, wherein the mammal is a human

56. The method of Claim 54, wherein the human has a neurological, neurodegenerative, and/or neuropsychiatric disease.

57. The method of Claim 54, wherein the human has a primary tracheobronchial and/or lymphadenopathy-associated illness.

58. A cultured mammalian epithelial cell, wherein the cell is acutely infected with Cryptovirus and has the characteristics of a simian epithelial cell line deposited with ATCC as Accession No

59. A cultured mammalian amnion cell, wherein the cell is nonproductively infected with Cryptovirus and has the characteristics of a cell line AV₃/SSPE deposited with ATCC as Accession No

60. A method of isolating a Cryptovirus virion, comprising:

(a) culturing a plurality of peripheral blood mononuclear cells that have been obtained from a human having a Cryptovirus infection, in an artificial aqueous medium comprising an agent that increases cellular guanylyl cyclase activity:

(b) co-cultuπng the plurality of peripheral blood mononuclear cells with a plurality of mammalian amnion cells in fresh artificial aqueous medium comprising an agent that increases cellular guanylyl cyclase activity;

(c) passaging the peripheral blood mononuclear cells with the mammalian amnion cells in co- culture;

(d) co-cultivating a plurality of mammalian epithelial cells together with the peripheral blood mononuclear cells and the mammalian amnion cells in fresh artificial aqueous medium comprising an agent that increases cellular guanylyl cyclase activity; and

(e) separating a supernatant of the aqueous medium from the cells, to obtain a Cryptovirus virion in the supernatant.

61. A method of propagating a Cryptovirus, comprising:

(a) exposing a plurality of mammalian epithelial cells to a plurality of cell-free Cryptovirus virions, said Cryptovirus virions having been isolated by the method of Claim 60; and

(b) further cultivating the mammalian epithelial cells, thus virion-cxposed, in an artificial aqueous medium comprising an agent that increases the activity of cellular guanylyl cyclase

62 A method of producing a mammalian cell line nonproductively infected with

Cryptovirus, comprising:

(a) co-culturing peripheral blood mononuclear cells that have been obtained from a human having a Cryptovirus infection, with mammalian amnion cells, in an artificial aqueous medium comprising an agent that increases cellular guanylyl cyclase activity, such that the mammalian amnion cells become nonproductively infected by Cryptovirus; and

(b) passaging the nonproductively infected mammalian amnion cells with the peripheral blood mononuclear cells, whereby the co-culture becomes a monoculture of the nonproductively infected mammalian amnion cells.

63. The method of Claim 60 or Claim 62, wherein the mammalian amnion cells are human amnion cells.

64. The method of Claim 63, wherein the human amnion cells arc AV₃ cells.

65. The method of Claim 60 or Claim 61, wherein the mammalian epithelial cells are simian epithelial cells selected from the group consisting of Vero or CV-1 cells.

66. The method of Claim 65, wherein the CV-1 cells are sublinc CV-1

cells. 67 The method of Claims 60, 61, or 62, wherein the agent that increases cellular guanylyl cyclase activity is cyclic GMP, insulin, zinc dication, or a combination of any of these

68 The method of Claim 67. wherein the cyclic GMP is in a concentration of about 0 05 to about 5 mM in the artificial aqueous medium

69 The method of Claim 60, 61, or 62, wherein the agent that increases cellular guanylyl cyclase activity is nitric oxide or a mtπc oxide donor selected from the group consisting of organic nitrate compounds, iron nitrosyl compounds, S-nitrosothiol compounds, sydnonimine compounds, and NONOate compounds

70 The method of Claims 60, 61, or 62, wherein the aqueous medium ftirther comprises glutamine

71 A method of producing a mammalian epithelial cell line acutely infected with Cryptovirus, comprising the method of Claim 61

72 A mammalian epithelial cell acutely infected with Cryptovirus. said cell being produced by the method of Claim 61

73 A cell nonproductively infected with Cryptovirus, wherein said cell is produced in accordance with the method of Claim 62

74 An in vitro method of screening a potential antiviral therapeutic agent, comprising

(a) culturing the cell of Claims 58 or 72,

(b) exposing the cells to the potential antiviral therapeutic agent, and

(c) measuring the effect of the agent on Cryptovirus replication and or Cryptovirus virion assembly, wherein inhibition of Cryptovirus replication and/or Cryptovirus virion assembly relative to a control indicates antiviral activity of the potential therapeutic agent

75 An in vitro method of screening a potential antiviral therapeutic agent, comprising (a) culturing the cell of Claim 59 or Claim 73;

(b) exposing the cells to the potential antiviral therapeutic agent; and

(c) measuring the effect of the agent on Cryptovirus replication, Cryptovirus genome replication, and/or Cryptovirus-specific transcription, wherein inhibition of Cryptovirus replication,

Cryptovirus genome replication, and/or Cryptovirus-specific transcription, relative to a control, indicates antiviral activity of the potential therapeutic agent.

76 An animal model for the study of human diseases, comprising a non-human mammal, said non-human mammal having been artificially inoculated with an infectious cell-free Cryptovirus having a genome comprising a single stranded RNA complementary to (SEQ ID NOT), or having been inoculated with a cell nonproductively-infected with the Cryptovirus, whereby the non-human mammal exhibits at least one symptom characteristic of a human disease after being thus inoculated, said symptom not being previously exhibited by the non-human mammal.

77. The animal model of Claim 76, wherein the non-human mammal is a rodent or lagomorph.

78. The animal model of Claim 76, wherein the non-human mammal is a non-human primate.

79 The animal model of Claim 76, wherein the human disease is a neurological, neurodegenerative, and/or neuropsychiatric disease.

80 An in vivo method of screening a potential therapeutic agent, comprising:

(a) administering the potential therapeutic agent to be screened, to the animal model of Claim 76, wherein the non-human mammal exhibits, before administration of the potential therapeutic agent, at least one symptom characteristic of a human disease; and

(b) detecting the presence or absence of a beneficial antiviral effect of the potential therapeutic agent, wherein the presence of a beneficial antiviral effect indicates activity of the potential therapeutic agent.

81. An in vivo method of screening a potential prophylactic agent, comprising:

(a) administering the potential prophylactic agent to be screened, to a non-human mammal not previously having a symptom of a human disease;

(b) inoculating the non-human mammal with an infectious cell-free Cryptovirus having a genome comprising a single stranded RNA complementary to (SEQ ID NO: l), or with a mammalian cell nonproductively-infected with the Cryptovirus; and

(c) detecting the subsequent presence or absence in the non-human mammal of a beneficial antiviral effect, whereby the presence of a beneficial antiviral effect in the inoculated non-human mammal indicates activity of the potential prophylactic agent.

82 The method of Claim 81, wherein the potential prophylactic agent is an immunoprophylactic agent.

83. The method of Claim 80 or Claim 81, wherein the non-human mammal is a rodent or lagomorph.

84. The method of Claim 80 or Claim 81, wherein the non-human mammal is a non- human primate.

85. The method of Claim 80 or Claim 81, wherein the human disease is a neurological, neurodegenerative, and/or neuropsychiatric disease.

86. An anti-C ryptovirus antibody detecting kit, comprising:

the Cryptovirus particle of Claim 33; and

a labeled anti-human antibody-binding antibody. 87 The anti-Cryptovirus antibody detecting kit in accordance with Claim 86, further comprising a solid matrix for supporting said Cryptovirus particle

88 The anti-Cryptovirus antibody detecting kit in accordance with Claim 86, wherein the Cryptovirus particle is produced from a cell line deposited with ATCC as Accession No

89 An anti-Cryplovirus antibody detecting kit, comprising

the protein of Claim 16 or Claim 19, and

a labeled anti-human antibody-binding antibody

90 The anti-Cryptovirus antibody detecting kit in accordance with Claim 89, further comprising a solid matrix for supporting said protein

91 The nucleic acid of Claim 1, wherein the Cryptovirus-specific fragment of (C) comprises (SEQ ID NO 34), (SEQ ID NO 35), (SEQ ID NO 36), (SEQ ID NO 37), (SEQ ID NO 38). (SEQ ID NO 39). (SEQ ID NO 40). (SEQ ID NO 41), (SEQ ID NO 42). (SEQ ID NO 43). (SEQ ID NO 44). (SEQ ID NO 45), (SEQ ID NO 46), (SEQ ID NO 47). or (SEQ ID NO 48), or comprises a nucleotide sequence complementary to any of these