HK1058804A - Sentinel virus ii - Google Patents

Description

Sentinel virus II

Technical Field

The present invention relates to the field of viruses, and more particularly, to hepatitis viruses.

Background

The strictly defined term "hepatitis" denotes an inflammation of the liver. Hepatitis is induced by a wide variety of chemical, viral and biological agents. However, the term hepatitis more generally denotes inflammation of the liver caused by viral infections, in particular infections with hepadnaviruses.

Viral hepatitis can be divided into two broad categories: acute and chronic. Acute viral hepatitis is characterized by jaundice, malaise, nausea and elevated liver enzymes in the blood. Although most cases of viral hepatitis resolve spontaneously, a proportion of acute hepatitis victims (generally less than about 10%) develop fulminant necrotizing hepatitis, a condition of very high morbidity and mortality. Interestingly, many cases of acute hepatitis are so mild that they are overlooked or eliminated as "flu". Chronic hepatitis poses a more significant public health problem and is the most common cause of liver transplantation in the united states. Chronic hepatitis is characterized by an exacerbation or "flare-up" with symptoms similar to acute hepatitis, as well as portal hypertension and cirrhosis (scarring of the liver) leading to liver failure. Because acute hepatitis infection can proceed unnoticed, many chronic hepatitis patients are not diagnosed until their disease has progressed too far; this limits the choice of treatment.

There are six different virus families called "hepatitis viruses" (A, B, C, D, E and G, F has been found to be an artifact (artifactual)). In developed countries, from the point of view of public health, those hepatitis viruses that establish chronic infections are widely regarded as the most important viruses. Among the hepatitis viruses, only hepatitis b virus and hepatitis c virus are known hepatitis viruses known to establish chronic infection associated with chronic hepatitis. However, HBV and HCV are not responsible for all cases of transfusion hepatitis. The terms "unexplained hepatitis" and "non-A-G" are used to denote transfusion hepatitis that cannot be attributed to known hepatitis viruses.

Hepatitis b, previously known as "transfusion hepatitis", is transmitted by the percutaneous, sexual, and vertical routes. Hepatitis B virus, which is a member of the Hepadnaviridae (Hepadnaviridae) family, causes both acute and chronic hepatitis. Hepatitis B Virus (HBV) has been well characterized and a wide variety of screening and diagnostic assays are generally available. In addition, a recombinant vaccine has been produced in the united states that is currently required by most school-age children.

Hepatitis c, previously known as "non-a, non-b hepatitis", is primarily transmitted by the transdermal route, although, like HBV, there is also a sexual route as well as a vertical route. Only a few acute Hepatitis C Virus (HCV) infections are clinically evident; this is problematic because this virus establishes chronic infection at a very high rate. In the united states, this combination makes chronic HCV infection the leading cause of liver transplantation.

The advent of screening assays for the detection of anti-HBV and/or HCV antibodies in donated blood significantly reduced the transmission of "transfusion hepatitis". However, 20-30% of the infectious blood donations are still in an undetected state. It is believed that the failure to detect these infectious samples is primarily due to the presence of one or more hepatitis viruses that have not yet been identified.

More recently, in addition to the six known hepatitis viruses, new viruses have been identified that are associated with hepatitis. The virus known as TTV was first identified by a panel of japan; the group identified genomic sequences from the virus using the differential expression analysis (RAD) technique (Nishizawa et al, 1997, biochem. Biophys. Res. common, 241 (1): 92-97). This virus, originally identified as a member of the Parvoviridae (Parvoviridae), is a relatively small virus with a buoyant density much lower than that of the Parvoviridae. Due to the circular single-stranded DNA genome of TTV, TTV has been proposed as a human-related prototypical member of the family of viruses known as the circoviridae (Circinoviridae) (Mushahwar et al, 1999, Proc. Natl. Acad. Sci. USA, 96 (6): 3177-.

Recently, Diasorin, Inc. has announced the isolation of a new hepatitis virus. Later, the virus called SEN-V was found to be very prevalent in healthy populations and was not limited to blood samples from hepatitis patients; and is therefore unlikely to be a hepatitis virus. They do not disclose the SEN-V polynucleotide sequence nor methods for isolating SEN-V.

Thus, there is a need in the art for compositions and methods for detecting non-A/non-G hepatitis, as well as for compositions and methods for preventing infection by non-A/non-G hepatitis and for compositions and methods for treating non-A/non-G hepatitis infection.

Disclosure of the invention

We have discovered a novel virus associated with non-A/non-G hepatitis of unknown cause. Various valuable inventions have been derived therefrom, which provide, for example:

1) a composition comprising an isolated SVII virus. Examples of isolated SVII viruses include the isolated viruses comprising the polynucleotide sequences of FIG. 1.

2) An isolated polynucleotide comprising: an isolated polynucleotide which selectively hybridizes to the nucleotide sequence of figure 1 and its complementary strand; an isolated polynucleotide encoding an isolated SVII protein or fragment thereof and the complementary strand thereof. The isolated polynucleotide may be an antisense polynucleotide.

3) A composition comprising an isolated SVII protein or fragment thereof.

4) A vaccine composition comprising an isolated SVII protein or fragment thereof. The vaccine composition may comprise a pharmaceutically acceptable excipient and/or an adjuvant.

5) An expression vector comprising an isolated polynucleotide encoding an SVII protein or fragment thereof.

6) An expression vector comprising an isolated polynucleotide; wherein transcription of the isolated polynucleotide results in the production of an SVII antisense polynucleotide.

7) Isolated polyclonal and monoclonal antibodies that bind to a SVII virus or a protein thereof.

8) A method for detecting SVII virus, said method comprising contacting a sample with an antibody that binds to SVII virus or a protein thereof, and detecting a complex of said antibody and SVII virus or a protein thereof.

9) A method for detecting SVII virus, said method comprising contacting a sample with a probe polynucleotide that selectively hybridizes to a SVII polynucleotide, and detecting hybridization of said probe to the SVII polynucleotide.

10) A method for detecting a SVII virus, the method comprising contacting a sample with a first primer polynucleotide that selectively hybridizes to a SVII polynucleotide and a second primer polynucleotide that hybridizes to the complement of the SVII polynucleotide, thereby performing primer extended DNA synthesis; and detecting the synthesis product.

Brief Description of Drawings

FIG. 1 shows the nucleotide sequence and conceptual open reading frame translation (using standard one-letter codes; k, m, r, s, y and w represent T/G, A/C, G/A, G/C, T/C, A/T, respectively) from a Sentinel Virus (Sentinel Virus) II (SVII) clone. The strand called "positive" is marked with a "+" and its opposite complement is marked with a "-". The numbering refers to the nucleotide sequence of the + strand. Frames P1, M1, and M2 are also shown. Read-chain and reading frames of M1 and M2 from right to left.

Detailed Description

We have discovered and isolated a novel hepatitis virus, designated sentinel virus II (SVII), associated with non-A-G hepatitis of unknown cause. The prototype virus comprises a DNA genome of at least about 371 bases. The genomic sequence from the prototype virus is shown in figure 1. Accordingly, the present invention provides isolated SVII.

In one aspect, the invention provides isolated polynucleotides comprising the SVII viral genome and fragments thereof. The polynucleotide may be DNA or RNA. Isolated nucleotide probes or primers for use in detecting SVII infection and/or SVII virus itself are also provided. The probes and/or primers can also be used in methods for identifying and isolating novel variants of SVII.

Another aspect of the invention provides isolated SVII viral proteins and/or fragments thereof and fusion proteins comprising SVII viral proteins or fragments thereof fused to heterologous (non-SVII) proteins. Chimeric polypeptides comprising at least two SVII epitopes are also included. In chimeric polypeptides of the invention comprising two epitopes from the same SVII protein, the amino acids inserted between the epitopes are substantially deleted or replaced with heterologous sequences. In another aspect, a chimeric polypeptide of the invention can comprise two epitopes from different SVII proteins, or comprise homologous epitopes from at least two viruses of the SVII family.

The present invention provides recombinant expression constructs comprising a polynucleotide sequence derived from an SVII virus open reading frame operably linked to a promoter operable in a prokaryotic or eukaryotic host cell. Expression vectors and recombinant host cells comprising the expression constructs are also provided.

Antibodies specific for epitopes of the SVII virus family are also provided. Including monoclonal antibodies and isolated polyclonal antibodies.

In another aspect, the invention provides assays and kits for performing the assays for detecting SVII infection and/or detecting SVII virus. The assays of the invention may be immunoassays using the polypeptides or antibodies of the invention, or nucleic acid-based assays using hybridization or amplification techniques with one or more polynucleotides of the invention.

In another aspect, the invention provides a vaccine for preventing and/or treating SVII infection. The vaccine may be a protein-based vaccine or may be a DNA-based vaccine. The protein-based vaccine comprises one or more SVII-derived polypeptides, optionally in combination with an adjuvant. A DNA-based vaccine comprises an isolated polynucleotide encoding a SVII polypeptide or polypeptide fragment operably linked to a promoter that is active in vivo in the recipient to be vaccinated (e.g., active in human cells if the recipient to be vaccinated is a human). General techniques

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology; such conventional techniques are within the skill of the art. Such techniques are fully described in the following documents, such as "Molecular Cloning: a Laboratory Manual "second edition (Sambrook et al, 1989)," Oligonucleotide Synthesis "(M.J. Gate edition, 1984)," Animal Cell Culture "(R.I. Freevine edition, 1987)," Methods in enzymology "(Academic Press, Inc.)," Handbook of Experimental immunology "(D.M.Weir & C.C. Black edition)," Gene Transfer vector for Mammalian Cell "(J.M.M.and M.P. Millos edition, 1987)," Current protocols in Molecular Biology "(F.M.sub.et al edition, 1987, and periodic update material)," PCR: the Polymerase Chain Reaction (Mullis et al, 1994), "Current Protocols in Immunology" (J.E.Coligan et al, 1991, and periodic updates of material), and "Immunology in Practice" (Johnstone and Thorpe, editors, 1996, Blackwell Science). Definition of

The terms "sentinel virus II" and "SVII" refer to a virus, virus type or virus class that can be transmitted in humans by transdermal exposure and is serologically distinct from Hepatitis A Virus (HAV), Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Hepatitis D Virus (HDV), Hepatitis E Virus (HEV), and Hepatitis G Virus (HGV). SVII comprises a genome having a major Open Reading Frame (ORF) that has at least about 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% amino acid complete sequence homology to the amino acid sequence of FIG. 1 and/or at least about 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% nucleotide complete sequence identity to the amino acid sequence of FIG. 1. In another aspect, an "SVII variant" can have at least about 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% complete nucleotide sequence identity to the sequence of FIG. 1 and encode an ORF that has at least about 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% complete amino acid homology to the amino acid sequence of FIG. 1 and/or at least about 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% complete amino acid sequence identity.

A "SVII polypeptide" or "SVII protein" is a polypeptide encoded by an ORF of the SVII virus genome. Typical SVII polypeptides are shown in the amino acid sequence shown in FIG. 1. Preferably, the SVII polypeptide is at least about 8,10, 12, 15, 20, 25, 30, 40, or 50 amino acids, and can be less than about 250, 200, 150, 134, 125, 110, 100, 90, 80, 70, 60, or 50 amino acids; wherein the upper and lower limits are independently selected, except that the upper limit is always greater than the lower limit.

A "variant SVII polypeptide" is a polypeptide that has at least about 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% amino acid sequence homology to the corresponding amino acid sequence in FIG. 1 and/or at least about 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% amino acid sequence identity to the corresponding portion of the amino acid sequence in FIG. 1. Preferably, the variant SVII polypeptide is at least about 8,10, 12, 15, 20, 25, 30, 40, or 50 amino acids and may be less than about 250, 200, 150, 134, 125, 110, 100, 90, 80, 70, 60, or 50 amino acids; wherein the upper and lower limits are independently selected, except that the upper limit is always greater than the lower limit.

A "SVII polynucleotide" is a polynucleotide having a sequence equivalent to the polynucleotide shown in FIG. 1 or a fragment thereof, or a complement thereof, or a polynucleotide encoding a SVII polypeptide, or a complement thereof. Preferably, the length of the SVII polynucleotide is at least about 15, 20, 25, 30, 35, 40, 50 or 60 nucleotides and less than about 371, 350, 300, 250, 200, 150, 125, 75, 50, 40 or 30 nucleotides; wherein the upper and lower limits are independently selected, except that the upper limit is always greater than the lower limit. A "complement" of a polynucleotide of interest is a polynucleotide that has the reverse complementarity of the reference polynucleotide according to Watson/Crick base pairing. Under suitable conditions, complementary polynucleotides are capable of hybridizing to the reference polynucleotide using Watson/Crick base pairing.

A "variant SVII polynucleotide" is a polynucleotide that encodes a variant SVII polypeptide or the complement thereof; or a polynucleotide that selectively hybridizes to a SVII polynucleotide or its complement, but does not fall within the definition of a SVII polynucleotide. In any known sequence, no variant SVII polynucleotide is found. Preferably, the variant SVII polynucleotide is at least about 15, 20, 25, 30, 35, 40, 50, or 60 nucleotides in length and less than about 400, 370, 367, 350, 300, 200, 150, 125, 100, 75, or 50 nucleotides in length; wherein the upper and lower limits are independently selected, except that the upper limit is always greater than the lower limit.

"amino acid sequence homology" and "amino acid sequence identity" refer to the percentage of amino acids that are homologous or identical when two sequences are compared. Such sequence alignments, and percentages of sequence homology or sequence identity, can be determined using software programs known in the art, such as those described in table 7.7.1, section 7.7.18 of Current Protocols in Molecular Biology (edited by f.m. ausubel et al, 1987) appendix 30. For sequence alignment, default parameters are preferred. For the purposes of the present invention, the sequence alignment program is BLASTP, using the following default parameters: database (databases) is non-redundant (non-redundant) (non-redundant GenBank CDS translation) + PDB + SwissProt + PIR + PRE), low complexity filtering (low complexity filtering) is ON, expected value (expect) is 10, matrix (matrix) is BLOSUM62 (gap existence penalty) 11, gap per residue (gap permeability) is 1, λ 0.85) and string size (word size) is 3. The sequence alignment may be performed with or without the introduction of gaps, and preferably with the introduction of gaps. The following Internet web site is pressed: http: // www.ncbi.nlm.nih.gov/cgi-bin/BLAST, details of the BLASTP implementation and these parameters described above are available.

"nucleotide sequence identity" refers to the percentage of identical nucleotide residues when two sequences are compared. Such sequence alignments, and percentages of sequence identity, can be determined using software programs known in the art, such as those described in table 7.7.1, section 7.7.18 of Current Protocols in molecular Biology (edited by f.m. ausubel et al, 1987), appendix 30. For sequence alignment, default parameters are preferred. For the purposes of the present invention, the sequence alignment program is BLASTN, using the following default parameters: database-non-redundant (all non-redundant GenBank) + EMBL + DDBJ + PDB sequences), low complexity filtering-ON, expected value-10, matrix-BLOSUM 62, gap existence penalty-5, gap extension penalty-2, mismatch penalty-3, match prize-1, and string size-11. The sequence alignment may be performed with or without the introduction of gaps, and preferably with the introduction of gaps. The following internet address of intenet (intenet) is given: http: // www.ncbi.nlm.nih.gov/cgi-bin/BLAST, details of the BLASTN implementation and these parameters described above are available.

A polynucleotide that "selectively hybridizes" to an SVII polynucleotide sequence is (i) a polynucleotide that hybridizes to an SVII polynucleotide sequence and not to a known viral polynucleotide sequence; or a polynucleotide that specifically initiates amplification of a SVII polynucleotide sequence without initiating amplification of a known viral polynucleotide sequence. Hybridization of selectively hybridizable polynucleotides can be accomplished, suitably under conditions of high stringency, moderate stringency, or low stringency (e.g., taking into account mismatches). Highly stringent conditions utilize a deterministic, lower T than the desired hybrid_m12-20 ℃ wash, while medium and low stringency hybridizations utilize a decisive, lower T than the hybrid_mWashing at 21-30 deg.C and 31-40 deg.C. According to T_m＝81.5-16.6(log₁₀[Na⁺]) +0.41 (% G + C) -0.63 (% formamide) -600/N, T for long-chain polynucleotide_mWhere N is the length of the selectively hybridizable polynucleotide under study; according to T_m＝81.5-16.6(log₁₀[Na⁺]) +0.41 (% G + C) -600/N, T for oligonucleotides of approximately from 70 nucleotides to 15 nucleotides in length can be obtained_m(ii) a According to T_m2(A + T) +4(G + C), T which gives short oligonucleotides of ≤ 14 nucleotides_m. Preferably in a Polymerase Chain Reaction (PCR) (e.g., 50mM KCl, 10mM Tris-HCl, pH 8.3 (at 20 deg.C), 1.5mM MgCl₂Optionally with 0.01% gelatin) and a modified form of Thermus aquaticus (T.aquaticus) DNA polymerase AmpliTaq Gold^TMPriming of the amplification was performed under standard conditions (PEbiosystems).

An "isolated" virus, viral structure (e.g., capsid), polynucleotide or polypeptide is one that has been at least partially purified away from contaminating components found in its normal environment. For example, an isolated virus is one that has been at least partially purified to remove blood, serum, or tissue proteins. In the case of an isolated viral polynucleotide, the polynucleotide is at least partially purified of viral proteins and/or other viral components, and may additionally be removed from its normal environment (e.g., the nucleotide sequences normally flanking the polynucleotide may be deleted).

When used herein, when the sequence of interest and the regulatory sequence are covalently linked in such a way as to place the expression or transcription of the sequence of interest under the influence or control of the regulatory sequence; the sequence of interest and the regulatory sequence are said to be "operably linked". The term "operably linked" refers to the orientation of polynucleotide elements in functional relationship. Operably linked means that the DNA sequences being linked are typically physically adjacent; and where necessary to join two protein coding regions, the DNA sequences are contiguous and in reading frame. However, since enhancers generally function when several kilobases apart from a promoter, intron sequences can be of variable length; thus, certain polynucleotide elements may be operably linked, but not contiguous. If translation of the sequence of interest into a functional protein is desired, then if induction of the promoter in the 5' regulatory sequence results in transcription of the sequence of interest, and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frameshift mutation, (2) interfere with the ability of the promoter region to direct transcription of the sequence of interest, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein; the two DNA sequences are said to be operably linked. Thus, if the promoter region is capable of effecting transcription of a sequence of interest such that the resulting transcript can be translated into a desired protein or polypeptide; the promoter region is operably linked to the DNA sequence. It should be noted that: the term "operably linked" includes an operable linkage in which the final protein coding region requires frameshifting or splicing to maintain a proper reading frame through the entire coding region of the protein (as seen, for example, in certain retroviral systems).

As used herein, the term "antibody" refers to an immunoglobulin molecule or a fragment of an immunoglobulin molecule that has the ability to specifically bind a particular antigen. Antibodies are well known to those of ordinary skill in the art of immunology. As used herein, the term "antibody" means not only an intact antibody molecule, but also a fragment of an antibody molecule that retains the ability to bind antigen. Such fragments are also well known in the art and are commonly used both in vitro and in vivo. In particular, as used herein, the term "antibody" means not only intact immunoglobulin molecules of any isotype (IgA, IgG, IgE, IgD, IgM), but also the well-known active (i.e., antigen-binding) fragments F (ab)₂、Fab、Fv、scFv、Fd、V_HAnd V_L. For antibody fragments, see: for example, "Immunochemistry in Practice" (edited by Johnstone and Thorpe, 1996, Blackwell Science), page 69. The term "antibody" additionally includes single chain antibodiesAntibodies, CDR-grafted antibodies, diabodies, chimeric antibodies, humanized antibodies, and Fab expression libraries. The term also includes fusion polypeptides comprising an antibody of the invention and another polypeptide or a portion of a polypeptide ("fusion partner"). Examples of fusion partners include modulators of biological responses, lymphokines, cytokines, and cell surface antigens. "antibody activity" refers to the ability of an antibody to bind a particular antigen in preference to other potential antigens via an antigen binding site located within the immunoglobulin variable region. The term "serologically distinct" describes polypeptides, proteins or viruses that are immunologically identifiable with specific antibodies with respect to other species of polypeptides, proteins or viruses by virtue of antigenic differences between the polypeptides, proteins or viruses and such other species.

As used herein, the term "comprising" and its cognate terms are used in their inclusive sense, i.e., to refer to the term "including" and its cognate terms. Isolated SVII viruses

Preferably, isolated SVII is prepared from plasma or serum derived from an individual infected with SVII. SVII virus can be isolated from plasma or serum using any technique known in the art; any of the techniques described include, but are not limited to, precipitation, isopycnic gradient centrifugation, especially on a preparative scale, and immunoseparation.

SVII viral particles can be isolated using precipitation techniques known in the art (e.g., ultracentrifugation). The SVII virus-containing material is treated to remove a substantial amount of debris and any cells (e.g., by filtration or medium/high speed centrifugation) and then ultracentrifuged to pellet the SVII virus. Preferably, the SVII virus-containing material is diluted, for example with tris buffered saline or with tris buffered saline containing EDTA, prior to precipitating the virus. For example, SVII virus can be precipitated by centrifugation at 200,000 Xg for 18 hours (e.g., centrifugation at 200,000 Xg in one SW41Ti rotor, dilution of SVII-containing serum with TEN buffer).

Fractionation of SVII by isopycnic gradient centrifugationThe analysis shows that: SVII has a value of-1.26 g/cm when determined by sucrose density gradient³(ii) a density of (d); whereas, in CsCl gradient, SVII has a value of-1.26-1.28 g/cm³The density of (c). Any gradient forming compound known in the art that will form a suitable gradient may be used, preferably one that will form about 1.2-1.35 grams per cubic centimeter (g/cm)³) Gradient forming compounds to perform isopycnic gradient centrifugation; sucrose and cesium chloride (CsCl) are preferred gradient forming compounds. Layering plasma or serum containing SVII on a sucrose gradient in a suitable centrifuge tube; alternatively, plasma or serum containing SVII is introduced into a suitable centrifuge tube as a homogeneous mixture in CsCl; then, it was centrifuged to equilibrium. By collecting fractions of the gradient of appropriate density, isolated SVII virus can be recovered.

The immunological separation techniques utilize an SVII-specific antibody in combination with any suitable separation medium known in the art. Preferred separation media include solid plastic matrices (e.g., as used in panning), chromatographic media (e.g., immunoaffinity chromatography), and magnetic particles (e.g., immunomagnetic separation). The SVII antibody is conjugated to a separation medium and the medium is contacted with a material containing SVII virus. Removing unbound material and washing away residual unbound material from the immuno-separation matrix; the bound SVII is then eluted, typically by using an elution buffer that varies in pH or has a high salt concentration.

Alternatively, isolated SVII viruses can be prepared using in vitro culture methods. A variety of such methods are known in the art and generally involve infecting a suitable host cell, preferably a liver cell line, with SVII; culturing the infected cells and collecting SVII viral particles from the culture medium or by lysing the cells. Density gradient separation or immuno-separation techniques can be used to further isolate the virus. Isolated SVII polynucleotides

By any of the methods known in the art, for example, by directly isolating viral DNA from viral particles, by directly isolating transcribed viral RNA that is part of the SVII life cycle, by using hybridization methods (i.e., identifying viral DNA in a DNA library prepared from viral DNA, or plasma or serum containing viruses), by using amplification methods (i.e., polymerase chain reaction of viral DNA, a library containing viral DNA, or DNA isolated from plasma or serum), or by direct synthesis; isolated SVII polynucleotides can be prepared. The polynucleotide sequences shown in FIG. 1 can be used to design probes or primers for use in hybridization and amplification methods, and to select sequences for synthesis. The probes or primers selected are preferably unique (e.g., not found in GenBank or other sequence databases).

Isolated genomic polynucleotides can be prepared by extraction of the isolated virions. The isolated virions can be subjected to any of the procedures known in the art for DNA extraction, such as guanidine hydrochloride extraction; optionally followed by further purification and/or concentration operations, such as agarose gel purification, phenol/chloroform extraction, or ethanol precipitation in the presence of salts.

The preparation of DNA libraries is well known in the art. DNA isolated from viral particles, or virus-containing plasma or serum, can be cloned into convenient library vectors using techniques commonly used in the art. Most commonly lambda phage-based library vectors are used to construct the library, although cosmid libraries and plasmid libraries are also commonly used. Plating phage-based libraries by infecting "lawn" of e.coli host cells; while cosmid libraries and plasmid libraries are generally transformed into plated cells. After plating, DNA from the library is transferred to a screening filter; and screened using SVII polynucleotide probes. Although other modified probes (e.g., digoxigenin or biotin-labeled) may be detected by using a modifying enzyme (e.g., alkaline phosphatase or luciferase) that binds to the labeled probe and acts on a chromogenic or other detectable substrate; however, it is preferred to modify the probe, typically by incorporating a radionuclideAcids (e.g. of³²P) to modify said probe so as to enable detection of hybridization. Purifying clones that hybridize to the SVII polynucleotide probe by one or more "rounds" of purification (e.g., repeating the plating and screening steps with progressively more purified clones); this is well known in the art. SVII DNA can be prepared by harvesting DNA from clones isolated in the screening step; and optionally further isolating SVII DNA from the library vector DNA by restriction enzyme digestion. Alternatively, cloned DNA isolated by screening can be used as a substrate to amplify SVII virus DNA using the Polymerase Chain Reaction (PCR) method. PCR primers may be designed based on SVII viral DNA, or more conveniently, PCR primers may be designed that hybridize to DNA sequences in the library vector that flank the site of insertion of the library DNA; this will be apparent to those skilled in the art.

SVII viral polynucleotides can also be isolated by amplification from a sample containing SVII DNA. Primers for amplification can be designed based on the sequences shown in FIG. 1; and the primers used for amplification are preferably designed so as to amplify SVII DNA without amplifying viral DNA from other viruses or genomic DNA from animals or prokaryotes. In addition, as is well known in the art, the primer sequences are selected to minimize any secondary structure within the molecule; the secondary structure greatly inhibits amplification and may even prevent amplification. Protocols for polymerase chain reaction amplification are well known in the art, as are protocols for other amplification methods such as ligase chain reaction. Following amplification, the SVII DNA may be further purified by size selection (e.g., gel electrophoresis) or chemical extraction (e.g., phenol/chloroform extraction) and/or concentrated by ethanol precipitation in the presence of salt.

SVII polynucleotides can also be chemically synthesized; although it is preferred that the length of the synthesized SVII polynucleotide is less than about 50-60 nucleotides, because the yield of polynucleotide synthesis decreases as the chain length increases. Methods for synthesizing polynucleotides are well known in the art and typically involve the iterative addition of nucleotides (or modified nucleotides) to the growing end of a synthesized polynucleotide. A wide variety of different systems are available in the art and will give the skilled person the choice of a particular method and chemistry.

SVII polynucleotides have a variety of uses, including: detecting SVII virus (which is useful in diagnosing SVII infection), producing SVII polypeptides, constructing SVII-based expression/transduction vectors, and as antisense oligonucleotides or for constructing antisense SVII vectors.

An antisense SVII polynucleotide is a SVII polynucleotide that is capable of selectively hybridizing to a segment of an mRNA molecule produced by the SVII genome. The antisense SVII polynucleotide can be any size of SVII polynucleotide, but is preferably less than about 200 nucleotides in length. In SVII-infected cells, the antisense SVII polynucleotide prevents expression of SVII proteins and/or replication of SVII virus. Thus, antisense SVII polynucleotides can be used to treat SVII infection and/or reduce the symptoms of SVII infection, including attenuating SVII viremia.

If the SVII antisense polynucleotide is chemically synthesized, it is preferably synthesized as a modified oligonucleotide to increase resistance to nucleases. Modified oligonucleotides comprising phosphoramidites (phosphoriodites) at the 5 ' end and the 3 ' end (Dagle et al, 1990, Nucl. acids Rs.18: 4751-4757) can be synthesized to introduce ethyl phosphonate analogues or methyl phosphonate analogues disclosed in U.S. Pat. No. 4,469,863, to introduce phosphorothioate (Stein et al, 1988, Nucl. acids Res.16: 3209-3221) or 2 ' -O-methyl ribonucleotides (Inove et al, 1987, Nucl. acids Res.15: 6131), or chimeric oligonucleotides as complex RNA-DNA analogues (Inove et al, 1987, FEBS Lett.215: 327).

The antisense SVII polynucleotides can be delivered to individuals infected with SVII virus as "naked DNA", typically by parenteral injection, preferably by intravenous injection or introduction into the portal vein, to take advantage of the uptake of naturally occurring oligonucleotides. Alternatively, the antisense SVII polynucleotide can be introduced into the target cell with the aid of a vector, such as a viral vector. The vector includes a promoter operably linked to a polynucleotide sequence that is operable in a host cell infected with SVII virus, and preferably in a human host cell, and transcription of the polynucleotide results in the production of SVII antisense polynucleotides. Preferred viral vectors include, but are not limited to, adeno-associated viral vectors known in the art. Preferably, the SVII antisense polynucleotide delivered by the viral vector is administered intravenously, and preferably, into the portal vein. Isolated SVII polypeptides

The SVII protein can include a complete ORF from SVII virus, one or more fusion proteins from SVII virus, a single protein from SVII virus, or a fragment thereof. Also included are "chimeric proteins" comprising two or more fragments of an SVII protein within the same protein. The fragments of SVII protein in the chimeric protein can be from the same SVII protein, or from different SVII proteins. Where the SVII protein fragments are derived from the same SVII protein, the amino acid sequences normally separating the fragments are either greatly deleted or replaced with an unrelated "spacer" sequence. Another chimeric protein encompassed by the present invention is a "superepitope" chimeric protein comprising a homologous form of at least one type of epitope from at least two different SVII viruses. For example, in a screening assay, a superepitope chimeric protein can be used to detect SVII virus infection species.

SVII polypeptides can be prepared by any method known in the art; the method comprises the following steps: purification from isolated virions, recombinant production, and chemical synthesis. Because of the relative difficulty in isolating large quantities of virus particles from natural sources, recombinant production and/or chemical synthesis are preferred methods for production.

Recombinant production of proteins is well known in the art. Typically, a polynucleotide sequence encoding a protein of the invention is cloned into an "expression vector" which is then introduced into a suitable host cell. Culturing said host cell under conditions suitable for expression of the protein; and collecting the recombinant protein. Although the expression construct will generally comprise a promoter/operator or promoter/enhancer operable in the host cell, and a selectable marker, to enable selection of cells containing the marker; the exact details of the expression construct will vary depending on the nature of the desired host and expression construct; this will be apparent to those skilled in the art. Preferably, the promoter/operator or promoter/enhancer is "controllable" in that a change in culture conditions will result in expression of the SVII protein (or fusion protein of SVII protein).

It should be noted that: for recombinant production, the SVII peptides can be combined into "fusion proteins". The fusion protein comprises a protein of interest (e.g., an SVII protein) linked to a fusion partner, and optionally includes a specific cleavage site between the protein of interest and the fusion partner, thereby allowing the two moieties to be separated. The fusion partner may be at the amino-terminus or the carboxy-terminus of the protein, although fusion proteins introduced as "inserts" within the coding region sequence into the protein of interest are also contemplated. As a screening tool, fusion proteins comprising an SVII protein insert may be particularly useful, for example, when introduced into a "phage display" system (e.g., in the case of inserting the SVII protein sequence into a lambda phage coat protein).

Useful fusion partners include: proteins that facilitate easy purification of the fusion protein (e.g., glutathione-S-transferase, oligohistidine, and certain sequences derived from myc oncogenes), proteins that increase the solubility of the fusion protein (e.g., DsbA from E.coli, disclosed in U.S. Pat. No. 5,629,172), or proteins that form a "linker" that allows the protein to bind to a substrate (e.g., a SVII protein can be attached to a substrate with a polyglycine having a terminal lysine for use in immunoassays).

In general, expression constructs are constructed by inserting a polynucleotide encoding a protein of the invention into an appropriate recombinant DNA expression vector using appropriate restriction enzymes. The site of the restriction enzyme may be a naturally occurring or synthetic site introduced by any method known in the art; such as site-directed mutagenesis, PCR or ligation of linkers/linkers to the polynucleotide. Alternatively, the polynucleotide may be a synthetic sequence designed to contain convenient restriction enzyme sites and/or to optimize codon usage for the intended host cell. The cleavage pattern of the restriction enzyme of the parent expression vector used will determine the particular endonuclease used. The restriction enzyme sites are selected so that the coding and control sequences are properly oriented for proper reading and expression of the protein in frame.

The polynucleotide may be inserted into any suitable expression vector. Expression vectors are available in several forms, including but not limited to: plasmids, cosmids, Yeast Artificial Chromosomes (YACs), and viral vectors. Typically, the expression vector will comprise an autonomous replication site that is active at least in the organism that propagated the vector, and often also in the recombinant host cell. The expression vector will also typically comprise a marker sequence capable of providing phenotypic selection in transformed cells, e.g., such as an antibiotic resistance gene (e.g., bla, tet)^R、neo^ROr hyg^R) Or a positive selection marker such as a gene that supplements auxotrophy (e.g., trp or DHFR) and/or a negative selection marker such as thymidine kinase of herpes simplex virus type 1. The expression vector will likewise include the necessary sequences for initiating and terminating transcription and translation (e.g., promoter, Shine-Dalgarno sequence, ribosome binding site, transcription termination site), and may optionally include sequences that regulate transcription (e.g., the SV40 enhancer or lac repressor), and may also include sequences that direct processing, such as an intron or polyadenylation site, if necessary.

Inserting the polynucleotide of the invention into an expression vector in the correct orientation and in the correct relationship to the transcriptional and translational control sequences of the expression vector, thereby providing transcription from the promoter and translation from the ribosome binding site; both of these should be functional in the host cell in which the protein is to be expressed. The transcription control sequences are preferably inducible (i.e.they can be adjusted by changing the culture conditions; the transcription control sequences are for example the lac operon of E.coli or the metallothionein promoter for mammalian cells). An example of such an expression vector is a plasmid described in U.S. patent No. 5,304,493 to Belagaje et al. The gene encoding A-C-B proinsulin described in the reference can be removed from plasmid pRB182 with the restriction enzymes NdeI and BamHI. The gene encoding the protein of the invention can be inserted into the backbone of the plasmid in a NdeI/BamHI restriction cassette.

For recombinant expression of the proteins of the invention, microbial hosts are generally preferred; and any of the microbial hosts commonly used may be used, including, for example, Escherichia coli (E.coli) of W3110 (prototroph, ATCC 27325), Bacillus subtilis, and other species of Enterobacteriaceae such as Salmonella typhimurium (Salmonella typhimurium) or Serratia marcescens (Pseudomonas marcescens), and various species of Pseudomonas. Alternatively, eukaryotic host cells may be used; such eukaryotic host cells include not only yeasts such as Saccharomyces cerevisiae (Saccharomyces cerevisiae), Schizosaccharomyces pombe (Schizosaccharomyces pombe), but also higher eukaryotes such as non-yeast fungal cells, plant cells, insect cells (e.g., Sf9) and mammalian cells (e.g., COS, CHO).

Introducing the completed expression construct into a recombinant host cell by any suitable method known in the art; said method being, for example, CaCl₂Transfection, Ca₂PO₄Transfection, viral transduction, lipid-mediated transfection, electroporation, gene gun transfection, and the like. After introduction of the expression construct, the recombinant host cell is typically cultured under suitable conditions that can be selected for the presence of the expression construct (e.g., in a bacterial host with an expression construct comprising bla, cultured in the presence of ampicillin); alternatively, any suitable method (e.g., fluorescence activated cell sorting) may be used depending on the expression of the proteinFACS; using an antibody specific for the SVII protein) to select recombinant host cells.

After selection and suitable isolation steps (e.g.restreak isolation or limiting dilution cloning), the recombinant host cells are cultured on a production scale (which may range from 500ml shake flasks to several hundred-grade fermenters for microbial host cells, or from T25 flasks up to several hundred-grade bioreactors for mammalian host cells; depending on the requirements of the practitioner) using any suitable technique known in the art. If the promoter/enhancer in the expression vector is inducible, expression of the protein is appropriately induced (e.g., by addition of an inducer, or by removal of a repressor from the culture medium) depending on the particular construct after the culture reaches the appropriate cell density; otherwise, the cells are grown until they reach an appropriate density for harvesting. The harvest of the recombinant proteins of the invention will depend on the exact nature of the recombinant host cell, the expression construct and the polynucleotide encoding the protein of the invention; this will be apparent to those skilled in the art. As for the expression construct for producing a secreted protein, the protein is generally recovered by removing the medium from the culture vessel; expression constructs that cause intracellular protein accumulation typically require recovery and lysis of the cells to release the expressed protein.

Proteins expressed with high levels of bacterial expression systems are characteristically aggregated in the form of microparticles or inclusion bodies containing high levels of the protein over-expressed. Solubilizing the protein aggregates, thereby achieving further purification and isolation of the desired protein product; for example, a strongly denaturing solution of, for example, guanidine hydrochloride, perhaps in conjunction with a reducing agent such as Dithiothreitol (DTT), is used. Recovering the solubilized protein in active form after the "refolding" reaction; in the "refolding" reaction, it is generally involved to reduce the concentration of denaturant and to add an oxidizing agent. Protocols generally recognized as suitable for protein refolding are well known in the art and are disclosed, for example, in U.S. patent nos. 4,511,502, 4,511,503 and 4,512,922.

Short (e.g., less than about 20 amino acid residues) SVII proteins can also be conveniently produced using synthetic chemistry, a method well known in the art. Because yield decreases over long peptide lengths, synthesis is a preferred method for producing peptides of about 15 amino acid residues or less.

In a vaccine for preventing and/or treating SVII infection, SVII polypeptides can be used. In a SVII vaccine, any SVII polypeptide or combination of SVII polypeptides can be used. A SVII chimeric polypeptide comprising a plurality of epitopes from a SVII protein, wherein the amino acids normally separating the epitopes are deleted, is a preferred SVII protein for use in vaccine formulations. Another preferred SVII protein for use in vaccines is a superepitope protein that comprises homologous epitopes from a plurality of SVII viruses that have been fused into a single protein.

The SVII vaccine is formulated according to methods known in the art. The vaccine is preferably a liquid formulation for parenteral administration. Vaccines can be formulated comprising pharmaceutically acceptable excipients known in the art, such as physiologically and pharmaceutically acceptable salts, buffers, preservatives, fillers, osmolytes, and the like, in the USP (U)_NITED S_TATES P_HARMACOPEIAThey can be identified in United States pharmaceutical Convention, Inc., Rockville, Md., 1995).

Vaccines based on SVII proteins can also be formulated with adjuvants. Adjuvants for use in SVII protein-based vaccines, including chemical adjuvants, cytokine adjuvants, and oil-in-water emulsions such as Freund's complete adjuvant and Freund's incomplete adjuvant; such as aluminum hydroxide (particularly aluminum hydroxide gel), alum, protamine, aluminum phosphate and calcium phosphate, and cytokine adjuvants including interleukin 1. beta., tumor necrosis factor alpha and granulocyte-macrophage colony stimulating factor (GM-CSF), such as described in U.S. Pat. No. 5,980,911.

Preferably, the SVII vaccine is delivered parenterally, more preferably by transdermal administration. Preferred routes of administration include not only intramuscular and subcutaneous injections, but also transdermal pneumatic administration (e.g., needle-free injection). The vaccine may be administered as a single dose, or may be administered in multiple administrations. Where multiple administrations are used, multiple administrations, preferably separated by at least one day, week or month, are preferred. SVII antibodies

Antibodies against SVII can be prepared using the isolated virions and/or SVII viral proteins provided by the invention. Not only isolated polyclonal antibodies but also monoclonal antibodies can be prepared.

Preferably, an isolated polyclonal antibody to the SVII protein is prepared by injecting an immunogenic "SVII immunogen" (e.g., an isolated SVII virion, SVII protein, SVII oligopeptide linked to a carrier, or SVII fusion protein) into an animal; the animal is preferably a mammal, such as a rodent (e.g., a mouse, rat, or rabbit), goat, cow, or horse. The first injection of the SVII immunogen is most commonly performed with an oil/water emulsion complete adjuvant such as Freund's complete adjuvant; the complete adjuvant comprises a non-specific activator of the immune system, thereby improving the immune response to the injected immunogen. The latter injection is generally performed with incomplete adjuvant (e.g. non-specific immunostimulant in an aqueous/oil emulsion). Alternatively, the SVII immunogen can be introduced adsorbed onto a solid substrate, or the SVII immunogen can be introduced as a pure solution. Harvesting the serum; and the presence of specific antibodies in the serum is determined using any convenient assay, most typically a simple immunoassay, such as an ELISA (enzyme-linked immunosorbent assay) using the SVII immunogen as the target and using species-specific anti-immunoglobulin secondary antibodies.

Monoclonal antibodies of the invention can be prepared using a number of different techniques. For hybridoma technology, the reader is generally referred to Harrow and Lane (1988) U.S. Pat. Nos. 4,491,632, 4,472,500, and 4,444,887, and Methods in Enzymology 73B: 3(1981). Conventional monoclonal antibody technology involves immortalization and cloning of antibody-producing cells recovered from an immunized animal (typically a mouse) as described in the preceding paragraph. The cells can be immortalized, for example, by fusion with a non-productive myeloma, infection with EB virus, or transformation with oncogene DNA. The treated cells are cloned and cultured, and clones producing antibodies of the desired specificity are selected. Specific assays are performed using techniques such as using the immune antigen as a detection reagent in an immunoassay using culture supernatants. A batch of monoclonal antibodies from a selected clone can then be purified from the large volume of culture supernatant or from ascites fluid from an appropriately treated host animal injected with the clone.

An alternative method for obtaining monoclonal antibodies involves contacting an immunocompetent cell or virion with a protein of the invention. In this respect, "immunocompetence" means that without further gene rearrangement, the cell or particle has expressed or is capable of expressing an antibody specific for the antigen; and can be selected from a mixture of cells by presentation of the antigen. Obtaining immunocompetent eukaryotic cells from an immunized mammalian donor; alternatively, eukaryotic cells can be harvested from an unimmunized donor and pre-stimulated by in vitro culture in the presence of an immunogen and an immunostimulatory growth factor. Cells of the desired specificity can be selected by contacting with the immunogen under culture conditions that result in proliferation of specific clones but not specific clones. Immunologically active phage can be constructed to express immunoglobulin variable region fragments on their surface. See: marks et al, New Engl.J.Med.335: 730, 1996; international patent application nos. 94/13804, 92/01047, and 90/02809; and McGuinness et al, Nature Biotechnol.14: 1149, 1996. For example, phage of the desired specificity can be selected by adsorption to an SVII immunogen associated with a solid phase; then, it was amplified in E.coli.

Antibodies can be purified from serum, cell supernatants, lysates, or ascites fluid using a combination of conventional biochemical separation techniques; such as ammonium sulfate precipitation, ion exchange chromatography on a weak anion exchange resin such as DEAE, hydroxylapatite chromatography and gel filtration chromatography. The antibodies of the invention may also be isolated using specific affinity techniques, such as affinity chromatography using SVII immunogen as the affinity moiety, alone or in conjunction with conventional biochemical isolation techniques.

It is desirable to screen or purify the resulting antibodies based not only on their ability to react with SVII viral proteins, but also on their low cross-reactivity with potential cross-reactants that are also present in the sample of diagnostic interest. If desired, the cross-reactant or an antigenic preparation derived from the serum of an individual who is negative for SVII infection may be used to adsorb unwanted activity from the polyclonal antiserum.

By preparing fragments and determining the ability of the antibodies to bind, the epitope bound by a particular antibody can be mapped. For example, a continuous 12 amino acid peptide covering the entire sequence of the immunogen and overlapping 8 residues is prepared. SPOTS from Genosys can be used according to the manufacturer's instructions^TMKit for preparing said peptide on a nylon membrane support using F-Moc chemistry. Then, the prepared membranes were covered with the antibody, washed, and these membranes were covered with alkaline phosphatase or horseradish peroxidase-conjugated anti-human IgG. The assay results are visualized by adding the appropriate substrate for the particular enzyme conjugate used. Positive staining indicates the antigen fragment recognized by the antibody. The fragment can then be used to obtain additional antibodies that recognize the epitope of interest. In a standard immunoassay, two antibodies recognizing the same epitope will compete for binding.

The antibodies of the invention can be used to detect and/or identify SVII viruses, and can also be used to isolate viral particles and/or viral proteins. Detection of SVII

The polynucleotides, proteins and antibodies of the invention may be used in methods and kits for detecting infection by SVII virus and for detecting SVII virus itself. Depending on the desired utility of the assay, a wide variety of formats can be devised for assays employing the polynucleotides, proteins and/or antibodies of the invention.

SVII virus genomic DNA can be detected using the polynucleotides of the invention. Detection of SVII genomic DNA in a blood sample indicates that: the sample is contaminated with SVII virus and the source of the sample is infected with SVII. A wide variety of different assays for detecting nucleotides are known, although all such assays typically require a hybridization step in which a primer or probe is hybridized to the DNA in the sample.

Using the determined portion of the isolated SVII genomic sequence as a basis, oligomers of about eight or more nucleotides that hybridize to the SVII genome can be prepared, either by excision or by synthesis. The native or derivatized probes for the SVII polynucleotides are of a length that allows detection of the unique viral sequences by hybridization. Generally, probes are sequences of a minimum of 6 to 8 nucleotides in length, preferably at least 10 to 12 nucleotides, and those of at least about 20 nucleotides may be most preferred. Depending on the utility of the assay desired (e.g., detection of a single SVII virus type for detection of all SVH viruses), the probe can be based on a region of the SVII genomic sequence that is conserved among SVII viruses or that is highly divergent among SVII viruses. Conventional, standard methods, including automated oligonucleotide synthesis, can be used to prepare these probes. The complement of any unique portion of the SVII genome will generally be satisfactory. Generally in terms of probes, complete complementarity is desired; although complete complementarity may not be necessary when increasing the length of the fragment.

Generally, a test sample to be analyzed, such as blood or serum, is treated in order to extract the nucleic acids contained therein. Nucleic acid samples are typically adsorbed to a solid support (e.g., nitrocellulose) for analysis (with or without preliminary size separation, e.g., by gel electrophoresis); although solution phase patterned analysis, such as that described in U.S. patent No. 4,868,105, may also be used.

Depending on the format of the assay and the detection system, the probes may or may not be directly labeled or otherwise modified, allowing subsequent detection based on the binding of the label. Suitable labels and methods of attaching labels to probes are known in the art and include, but are not limited to: radiolabels introduced by nick translation or kinase treatment (kinasing), modifications that allow subsequent binding of the label, e.g. biotinylation, and fluorescent and chemiluminescent labels that can be bound directly to the probe or via the modified probe.

In a basic nucleic acid hybridization assay, single-stranded nucleic acid of a sample is contacted with the probe under hybridization conditions of appropriate stringency and washing conditions, and the resulting duplexes are detected. Control of stringency is well known in the art and depends on variables such as salt concentration, probe length, formamide concentration, temperature, and the like. Hybridization and washing are preferably carried out under stringent conditions. Detection of bound probes is performed as required by the labeling/detection system used for the assay. For example, in the case where the probe is radiolabeled, binding of the probe is detected by autoradiography. Where the probe is modified to allow subsequent binding of the probe (e.g., by covalently linking biotin or digoxigenin to the probe, or by adding a poly-a tail to the probe), a label is linked to a modified binding moiety (e.g., streptavidin is linked to a detectable enzyme such as alkaline phosphatase, green fluorescent protein, or luciferase, or a fluorescent or other label is bound to the anti-digoxigenin antibody). Detection of the fluorescent probe is typically accomplished with a fluorometer, and the luminescent label can be detected with a luminometer or a photographic negative. Branched DNA techniques and other methods of enhancing the signal from the assay can be used (Urdea et al, 1989, Clin. chem.35 (8): 1571-1575; U.S. Pat. No. 5,849,481).

Other assays use probes as primers for amplifying SVII genomic DNA in a sample. Methods such as polymerase chain reaction, ligase chain reaction, Q-beta replicase, NASBA (Compton, 1991, Nature 350 (6313): 91-92), etc., can be used to generate large copies of partial or complete SVII genomic DNA present in a sample. Detection in such assays, typically by detection of amplification products of the expected size, is typically detected by gel electrophoresis and visual inspection of any bands present.

SVII virus can also be detected by detecting the presence of viral proteins in a sample using the antibodies of the invention. Any of a variety of immunoassay formats known in the art can be used in conjunction with the antibodies of the invention to detect SVII virus or viral proteins.

In its most basic type, an immunoassay for detecting SVII virus or SVII virus proteins in a sample detects complexes of SVII proteins with the antibodies of the invention. Although a preferred immunoassay format requires at least two antibodies of the invention, at least one antibody of the invention is required.

Many assay formats require that the sample or the SVII proteins from the sample be immobilized on a solid support. The connection can be accomplished by a variety of methods known in the art; most commonly it is adsorbed to a protein binding surface (e.g. polystyrene plate or nitrocellulose or PVA membrane) or bound to an antibody bound to a substrate. This second arrangement is used in "sandwich" immunoassays; and this second arrangement is preferred for the detection of SVII viral proteins.

After immobilizing the sample (or the SVII protein in the sample) on the substrate, a detection antibody is contacted with the sample and the presence of the detection antibody is detected. The detection antibody itself may be detectable due to the modification of the detection antibody with a dye or colored particle; the detection antibody may be modified such that a detection reagent will bind to the detection antibody, or may be modified with an enzyme that acts on a chromogenic substrate.

The exact details of detecting the detection antibody will, of course, depend on the detection system used. Directly detectable detection antibodies can be detected by, for example, simple examination, optical microscopy or colorimetry (for antibodies modified with colored particles such as latex microbeads or colloidal metals), radiometric analysis (for antibodies modified with a radioactive compound) or fluorometry or epifluorescence microscopy (for antibodies labeled with a fluorescent dye). Detection antibodies that have been modified to include the enzyme are typically detected by incubating the test sample in a solution containing the substrate that has become detectable upon processing by the enzyme, and detecting any processed substrate by a suitable method (e.g., colorimetric for chromogenic substrates, fluorimetric for fluorogenic substrates, etc.). Other detection antibodies may be modified to allow for "indirect" detection, wherein a second reagent bound to the modified detection antibody allows for detection of the bound detection antibody. The second agent is modified to be detectable (either directly when a dye is used or when a colored particle is used, or indirectly when an enzyme is used with a detectable substrate).

Examples Example 1: isolation of SVII viral DNA

A modified expression level differential analysis (RAD) method described by Lititsyn et al (1993, Science 259: 946-951) was used to isolate DNA clones containing SVII genomic DNA. This method utilizes a "driver" DNA source to enrich for amplificates that amplify sequences unique to the "tester" DNA source.

Serum from a patient with unexplained hepatitis, designated H101, was used as the source of "tester" DNA. DNA was extracted by proteinase K digestion followed by phenol and chloroform extraction. DNA isolated from 100. mu. l H101 serum was digested with 10 units of Sau3A I at 37 ℃ for 3 hours to completion. The enzyme was inactivated by incubation at 65 ℃ for 20 minutes.

By mixing 1nmol each of the oligonucleotide linkers R-Bgl-24 (5'-AGCACTCTCCAGCCTCTCACCGCA-3') and R-Bgl-12 (5'-GATCTGCGGTGA-3') with the digested DNA in T4 DNA ligase buffer (with ATP from New EnlandBlabs), denaturing the mixture by incubation at 55 ℃ for 2 minutes, annealing the linkers by gradually cooling the mixture to 10-15 ℃ during about 1 hour, then adding 800 units of T4 DNA ligase (New Enland Biolabs) and incubating at 12-16 ℃ overnight; and the linker is ligated to the digested DNA.

Preparing a tester amplicon by nested polymerase chain reaction; since one round of PCR does not produce a measurable amount of DNA. A portion of the ligation product was mixed with PCR buffer, dNTPs and an additional 250pmol of R-Bgl-24 oligonucleotide and covered with mineral oil. The R-Bgl-12 oligonucleotide was 'released' by incubating the mixture for 3 minutes at 72 ℃. The overhangs were filled in by adding 7.5 units of AMPLITAQ  Taq DNA polymerase (PEbiosystems) and incubating at 72 ℃ for an additional 5 minutes. The experimental amplicons were constructed by subjecting the mixture to 20 cycles of 1 minute at 95 ℃ and 3 minutes at 72 ℃ followed by a final extension step of 10 minutes at 72 ℃. In the second round of PCR, 15 cycles were performed under the same conditions as in the first round using 10. mu.l of the product of the first round PCR and the linker R-Bgl-25 (5'-ACTCTCCAGCCTCTCACCGCAGATC-3'). The product was then extracted with phenol/chloroform and precipitated with sodium acetate and isopropanol. The pellet was collected by centrifugation, and after removing the supernatant, it was air-dried and resuspended in TE (tris-EDTA) buffer. The R-Bgl-24 linker was removed by performing Sau3A I digestion essentially as described above, followed by inactivation of the enzyme at 65 ℃. Precipitating the digestion product with sodium acetate and ethanol in the presence of glycogen; the precipitate was collected by centrifugation, after removal of the supernatant, air-dried and resuspended in TE. The products were then separated on a 1% agarose gel electrophoresis in 1 × TAE; and the gel fraction corresponding to 150-1500 nucleotides was excised. The digested tester amplicons were purified from this Gel using the QIAGEN  Qiaex II Gel Extraction (QIAGEN  Qiaex II Gel Extraction) kit according to the manufacturer's instructions. Mu.g of the tester amplicon DNA was ligated to the J-Bgl-24 linker and the J-Bgl-12 linker (5'-ACCGACGTCGACTATCCATGAACA-3' and 5'-GATCTGTTCATG-3', respectively) essentially as described for the R-Bgl linker.

Driver amplicons were prepared from DNA extracted from pooled sera from 10 healthy donors essentially as described for the tester amplicon, except that no new linker was added after the second Sau3A I digestion.

Mixing the driver amplicon and the tester amplicon at a mass ratio of 100: 1; extracted with phenol/chloroform and precipitated with sodium acetate and ethanol. The pellet was collected by centrifugation, after removal of the supernatant, it was air-dried and resuspended in 4. mu.l of EE X3 buffer (30mM EPPS, pH 8.0, 3mM EDTA). The mixture was covered with mineral oil and hybridization was performed by denaturation at 98 ℃ for 5 minutes, addition of 1. mu.l of 5M NaCl, incubation at 98 ℃ for an additional 2 minutes, and then incubation at 65 ℃ for 20 hours.

The tester/driver hybridization mixture is amplified under conditions that selectively amplify only double-stranded tester DNA. A portion of the hybridization mixture was amplified for 10 cycles essentially as was done for amplification of J-Bgl ligated tester DNA, except that the extension cycle was performed at 70 ℃. The amplified product was collected, extracted with phenol/chloroform/isoamyl alcohol, and then precipitated with sodium acetate and isopropanol. The pellet was collected by centrifugation, and after removing the supernatant, it was air-dried and resuspended in TE. Removing single-stranded DNA by digestion with mung bean nuclease (New England Biolabs) at 30 ℃ for 30 minutes; followed by heat inactivation of the enzyme at 98 ℃ for 5 minutes. The digestion product was reamplified for 15 cycles in the presence of additional J-Bgl-24 oligonucleotides.

Collecting the amplified product, extracting with phenol/chloroform/isoamyl alcohol, and precipitating with sodium acetate and isopropanol; the precipitate was collected by centrifugation, washed with 70% ethanol, after removal of the supernatant, air-dried and resuspended in TE to form the first Difference Product (the first Difference Product) (DP 1).

The R-Bgl linker was changed to the J-Bgl linker essentially as described above by digesting DP1 with Sau3A I and replacing the J-Bgl linker with N-Bgl linkers (N-Bgl-12 and N-Bgl-24, 5'-GATCTTCCCTCG-3' and 5'-AGGCAACTGTGCTATCCGAGGGAA-3', respectively); hybridizing the N-linker DP1 to the driver amplicon at a mass ratio of 1: 800; and amplification/digestion/amplification was performed as described for DP1, except for extension during amplification at 72 ℃; thereby producing a Second difference product (the Second differential product) (DP 2).

By digesting DP2 with Sau3A I and replacing the N-Bgl linker with a J-Bgl linker; followed by 4 x 10⁵Driver to tester mass ratio of 1, hybridized to driver amplicon; and amplification/digestion/amplification as described for DP 1; thereby producing a Third Difference Product (the Third Difference Product) (DP3)

After three rounds of subtractive hybridization and selective amplification, clear bands were seen after gel electrophoresis when compared to the ` ill-defined fragmented ` band patterns of the original tester amplicons. DNA was isolated from each band using QIAGEN  gel extraction Kit (QIAGEN  Gelextraction Kit) (Cat. No.: 28704) according to the manufacturer's instructions; then, the DNA was ligated into TA plasmid 2.1(In Vitrogen Cat. K2000-01). The resulting plasmid library was then transformed into E.coli, and E.coli was plated. 30 colonies from each library were selected and sequenced using a PE Biosciences PCR sequencing kit according to the manufacturer's instructions. Sequences were compared against the GenBank database at the DNA and protein levels. Clones that showed no significant homology to the database were classified as "unknown". Each "unknown" was tested for the presence in human genomic DNA by PCR using primers designed from each "unknown" sequence. For any sequences present in human genomic DNA, the analysis is terminated. An unknown of 371 nucleotides, originally designated "clone 33" or "h101. c 33", was selected for further characterization. The nucleotide sequence of h101.c33 is shown in figure 1.

The nucleotide sequence of clone 33 was analyzed by translation conceptually in all six possible reading frames. A large Open Reading Frame (ORF) was identified: ORF 1. The amino acid sequence of ORF 1 is also shown in FIG. 1.

The non-human origin of clone 33 was confirmed by PCR. After amplification of human genomic DNA with clone 33 specific PCR primers, no amplified product was detected. Clone 33 was renamed to sentinel virus II or SVII because it was confirmed to be of non-human origin.

The presence of SVII in the serum of patient H101 was confirmed at two time points corresponding to peak ALT levels using PCR primers specific for SVII.Example 2: identification of physical Properties of SVII virions

SVII positive sera were fractionated by density gradient ultracentrifugation to determine the buoyant density of SVII virions.

On the surface of a continuous sucrose density gradient (20-65% sucrose, w/w), 500. mu.l of SVII positive serum sample spiked with HBV as a marker was spread. The sample was centrifuged at 39,000rpm at 6 ℃ for 15 hours using a Beckmann SW41Ti rotor. Fractions (500. mu.l) were collected by aspiration from the bottom of the centrifuge tube via a glass capillary tube connected to a silicone tube.

SVII in each fraction was analyzed using nested PCR. The first round used the primer pair 33.1/33.2 (5'-GGATTGACGACGACGACGAC-3', 5'-TGTCAAATACCCGCTCAGGA-3', respectively) and the second round used the primer pair 33.3/33.4 (5'-GACGACGACGACGACATTG-3' and 5'-CAAATACCCGCTCAGGAAGG-3', respectively). Similarly, two rounds of PCR were used to analyze HBV in each fraction; primers HBV1 and HBV 4(5 '-CATCTTCTTRTTGGTCTTCTGG-3' and 5'-CAAGGCAGGATAGCCACATTGTG-3', respectively) and primers HBV3 (5'-CCTATGGGAGTGGGCCTCAG-3') and HBV4 were used in the first round. At a value corresponding to 1.26g/cm³SVII virus was found in the fraction (2).

The buoyant density of SVII in CsCl gradient was also determined. Mu.l of an SVII-positive serum sample spiked with HBV as a marker and a homogeneous CsCl solution (density 4.2 g/cm) were placed in a suitable centrifuge tube³And a refractive index of 1.3645). The samples were centrifuged at 35,000rpm for 70 hours at 6 ℃ using Beckmann SW41Ti rotor heads. Fractions were collected by puncturing the sidewall near the bottom of the tube and collecting 500 μ Ι of fractions; and the fractions were analyzed as described for the sucrose gradient experiment. In the range corresponding to 1.26-1.28g/cm³In the fraction (b), SVII was found.Example 3: prevalence of SVII infection

More than 700 serum samples were analyzed by PCR for the presence of SVII. Dividing the sample into: (a) an "ultra-normal" donor (normal blood value, no hepatitis marker, and no less than 5 blood donations, no involvement of events related to blood transfusion); (b) "normal" blood donors (meeting blood donation criteria); (c) "non-eligible" donors (healthy individuals who are not eligible for blood donation in the current standard); and (d) "hepatitis" patients, classified as "hepatitis" patients of acute hepatitis, HBV chronic hepatitis, HCV chronic hepatitis, hepatitis of unknown cause (non A-E), and hepatitis of superinfection (HBV plus HCV or HBV plus HDV).

Samples were analyzed by nested PCR amplification of DNA extracted from serum samples using primer pair 33.1/33.2 and primer pair 33.3/33.4. SVII genomic DNA is not detected in serum samples from individuals who meet the blood donation screening criteria; and SVII genomic DNA was found to be prevalent only at very low levels in healthy individuals who did not meet the criteria for blood donation screening. However, in sera from patients with acute hepatitis, SVII was found to be highly prevalent; and SVII has also been found in sera from chronic hepatitis patients, especially chronic HCV patients and patients who are superinfected with more than one type of hepatitis virus. The results are summarized in table 1.

Table 1 group samples positive over normal 1000%, not qualified 960% 1722% hepatitis 37418%

Acute hepatitis 2463%

791% of chronic HBV hepatitis

Chronic HCV hepatitis 9829%

9423% of hepatitis with unknown cause

Chronic HBV/HCV/HDV hepatitis 794%

Sequence Listing <110> Liu, en-Kuei

Lewis，Samantha

Batz，Hans-Georg

Ramaswamy，Latha

Bohenzky，Roy

Lin，Yu-Huei

Montiel，Janine

Chen, Benjamin <120> sentinel virus II <130> RDID 0070<150> US 60/202271<151>2000-05-05<160>5<170> PatentIn version 3.0<210>1<211>371<212> DNA <213> sentinel virus II <400>1 gatcggaaa cgyttsgctc ggtgcatgca gaaggacggg wtgaaggcgg acgggattga 60cgacgacgac gacattgcga tgaaagatgg gaccgcygcgcgac gtccttggcg gggcggagcg 120cgagaaccaa gacgacgagg acgaggacgt ctacgcgcgc atccgtttcc ttcctgagcg 180ggtatttgac acctccgcat tgctgatcct gaagttctcg cttgcagacg ctgattcagc 240gccgcttcgt cgcacctgct ttggacgctg caaaccgcac ggctcggacc atcgtcagtt 300tcctgcttca gaggtgaatt tccgaccccg ttggactttg ctctctcttc tctctctacc 360cgacgacgat c 371< 371 >2<211>372<212> DNA <213> sentinel virus II <220> <221> mi <221> sc _ feature <222> (372.) sentinel < 372. (372) > 223> unknown: may be a, t, g, c, or there may be no nucleotide present at that position. <400>2gatcgtcgtc gggtagagag agaagagaga gcaaagtcca acggggtcgg aaattcacct 60ctgaagcagg aaactgacga tggtccgagc cgtgcggttt gcagcgtcca aagcaggtgc 120gacgaagcgg cgctgaatca gcgtctgcaa gcgagaactt caggatcagc aatgcggagg 180tgtcaaatac ccgctcagga aggaaacgga tgcgcgcgta gacgtcctcg tcctcgtcgt 240cttggttctc gcgctccgcc ccgccaagga cgtccggt cccatctttc atcgcaatgt 300cgtcgtcgtc gtcaatcccg tccgccttca scccgtcttctgcatgcac cgagcwaarc 360gtttcckgat cn 372<210>3<211>123<212> PRT <213> post virus II <220> <221> michcellaneous <222> (14) < 14 > which residue may be Met or Leu. <220> <221> Miscellaneous <222> (5) <223> the residue may be Phe or Leu. <400>3Ile Arg Lys Arg Xaa Ala Arg Cys Met Gln Lys Asp Gly Xaa Lys Ala 151015 Asp Gly Ile Asp Asp Asp Asp Asp Ile Ala Met Lys Asp Gly Thr Ala

20 25 30Asp Val Leu Gly Gly Ala Glu Arg Glu Asn Gln Asp Asp Glu Asp Glu

35 40 45Asp Val Tyr Ala Arg Ile Arg Phe Leu Pro Glu Arg Val Phe Asp Thr

50 55 60Ser Ala Leu Leu Ile Leu Lys Phe Ser Leu Ala Asp Ala Asp Ser Ala65 70 75 80Pro Leu Arg Arg Thr Cys Phe Gly Arg Cys Lys Pro His Gly Ser Asp

85 90 95His Arg Gln Phe Pro Ala Ser Glu Val Asn Phe Arg Pro Arg Trp Thr

100 105 110Leu Leu Ser Leu Leu Ser Leu Pro Asp Asp Asp

115120 <210>4<211>124<212> PRT <213> sentinel virus II <220> <221> Miscellaneous <222> (111) <223> the residue may be Ala or Pro. <220> <221> Miscellaneous <222> (119) <223> the residue may be Gln or His. <220> <221> Miscellaneous <222> (120) <223> the residue can be Ser or Asn. <220> <221> Miscellaneous <222> (123) <223> the residue may be Gly or a stop codon. <400>4Asp Arg Arg Arg Val Glu Arg Glu Glu Arg Ala Lys Ser Asn Gly Val 151015 Gly Asn Ser Pro Leu Lys Gln Glu Thr Asp Asp Gly Pro Ser Arg Ala

20 25 30Val Cys Ser Val Gln Ser Arg Cys Asp Glu Ala Ala Leu Asn Gln Arg

35 40 45Leu Gln Ala Arg Thr Ser Gly Ser Ala Met Arg Arg Cys Gln Ile Pro

50 55 60Ala Gln Glu Gly Asn Gly Cys Ala Arg Arg Arg Pro Arg Pro Arg Arg65 70 75 80Leu Gly Ser Arg Ala Pro Pro Arg Gln Gly Arg Xaa Arg Ser His Leu

85 90 95Ser Ser Gln Cys Arg Arg Arg Arg Gln Ser Arg Pro Pro Ser Xaa Arg

100 105 110Pro Ser Ala Cys Thr Glu Xaa Xaa Val Ser Xaa Ser

115120 <210>5<211>25<212> PRT <213> sentinel virus II <220> <221> Miscellaneous <222> (12) <223> the residue may be Ser or Thr. <220> <221> Miscellaneous <222> (24) <223> the residue may be Leu or Arg. <400>5Met Ser Ser Ser Ser Ser Ile Pro Ser Ala Phe Xaa Pro Ser Phe Cys 151015 Met His Arg Ala Lys Arg Phe Xaa Ile

20 25

Claims (13)

1. A composition comprising an isolated SVII virus.

2. The composition of claim 1, wherein the isolated SVII virus comprises one of the polynucleotide sequences shown in figure 1.

3. An isolated polynucleotide selected from the group consisting of:

an isolated polynucleotide which selectively hybridizes to a nucleotide sequence shown in figure 1;

a complement of an isolated polynucleotide that selectively hybridizes to one of the nucleotide sequences shown in figure 1;

an isolated polynucleotide encoding a SVII protein or a SVII protein fragment; and

a complement of an isolated polynucleotide encoding a SVII protein or SVII protein fragment.

4. The isolated polynucleotide of claim 3, wherein said isolated polynucleotide is an antisense polynucleotide.

5. A composition, comprising:

an isolated SVII protein or fragment thereof.

6. A vaccine composition comprising:

an isolated SVII protein or fragment thereof; and

a pharmaceutically acceptable excipient.

7. The vaccine composition of claim 6, further comprising an adjuvant.

8. An expression vector comprising an isolated polynucleotide encoding an SVII protein or SVII protein fragment.

9. An expression vector comprising an isolated polynucleotide; wherein transcription of the isolated polynucleotide results in the production of an SVII antisense polynucleotide.

10. An isolated polyclonal antiserum that specifically binds to a SVII virus or a protein thereof.

11. A monoclonal antibody that binds to SVII virus or a protein thereof.

12. A method of detecting SVII virus, the method comprising:

contacting the sample with an antibody that specifically binds to SVII virus or a protein thereof; and

detecting complexes of the antibody with SVII virus or proteins thereof.

13. A method for detecting SVII virus, the method comprising:

contacting the sample with a probe polynucleotide that selectively hybridizes to an SVII polynucleotide; and

detecting hybridization of the probe to an SVII polynucleotide.