NOVEL DIAGNOSTIC STANDARDS FOR VIRUS DETECTION AND QUANTIFICATION

Abstract: The invention provides replication incompetent viral particles that are engineered to contain one or more primer binding sites that are homologous to similar sites found in one or more target wild type viral genomes. Accordingly, the invention also provides methods of using the replication incompetent viral particles as controls for the detection and/or quantification of wild type viruses.

Description: NOVEL DIAGNOSTIC STANDARDS FOR VIRUS DETECTION AND QUANTIFICATION FIELD OF THE INVENTION The invention relates to the use of standards as controls for determining the presence or absence of a wild type virus in a biological specimen. BACKGROUND Detection and quantification of viral pathogens in subjects is important for determining infection rates, and assessing the severity of infection and response to anti-viral treatment. Accurate and sensitive methods for detecting the presence of viruses (such as HIV and HCV) in biological specimens are therefore important tools in modern medicine. Assays that detect viral nucleic acid depend on such technologies as the reverse transcriptase polymerase chain reaction (RT-PCR) (Mulder, 1994.J. Clin. Microbiol, 32:292-300), isothermal amplification (NASBA) (Compton, 1994, Nature 350:91-92), and production of branched chain DNA (Pachl et al., 1995, J. AIDS and Hum. Retrovir., 8:446-454). The sensitivity of these technologies depends upon the quality of the sample that is tested. If a significant span of time elapses between collecting and processing the sample, nucleases in the sample can degrade the RNA of interest, and interfere with the accuracy of the assay. (Damen et al., 1998, J. of Virological Methods 72:175-184). This is a particular problem when a viral standard, such as a naked RNA standard, is added to the sample prior to analysis, to help determine the quantity of virus present in the specimen. The viral standard may also be degraded by nucleases, which interferes with accurate quantitation of the viral load. Presently, RT-PCR is the most sensitive method for detecting or quantifying viral load in plasma. The four general steps involved in RT-PCR analysis of biological samples are sample preparation, reverse transcription, amplification, and detection. The amplification step uses short nucleotide primers, which bracket a target DNA sequence that is unique to the virus, to repetitively replicate the viral sequence. One form of quantitative PCR uses two sets of primers, one of which is specific for the target sequence, and the second of which is specific for a control sequence (such as an endogenous gene) that is expressed at consistent levels (a housekeeping gene). The PCR reaction can be performed in the presence of both sets of primers, and the products electorphoretically separated. The quantity of the target sequence is then compared to the quantity of the control sequence, to determine which sequence is present in greater quantities. This method is especially useful when the exact concentration of the target sequence is not needed. Another form of quantitative analysis is competitive PCR, which permits precise quantitation of target gene expression, or viral load. Primers used in the reaction hybridize to both the target sequence and a control sequence, and the control and the target sequences compete for primer binding (Gilliland et al., 1990, Proc. Natl. Acad. Sci. USA SI r :2725-729). The control used in this method is distinguishable from the target sequence by either being a different length than the target sequence, or by containing different restriction endonuclease recognition sites. Known amounts of the control are added to a constant amount of test sample, all of the samples are then subjected to the same PCR protocol, and the resulting products are electrophoretically separated. The products derived from the control can then be used to make a standard curve, and the concentration of the target sequence can be determined by comparing the products derived from its amplification to the standard curve. The accuracy of competitive PCR is contingent upon the sample quality, as well as upon the accuracy of the standard curve. Therefore, an ideal control for use in competitive PCR protocols would be one that can be safely added to the sample as soon as the sample is collected. This ensures that the sample and the control degrade at the same rate. SUMMARY The invention provides internal controls for viral amplification assays by taking advantage of the discovery that replication incompetent viral particles can be engineered to contain one or more primer binding sites that are homologous to similar sites found in one or more target wild type viral genomes. Furthermore, the region between the primer binding sites (genetically tagged sequences) in the replication incompetent viral particle is engineered such that it is distinguishable from that of the wild type virus. These engineered replication incompetent viral particles are used as controls during the detection of target wild type viruses in biological specimens, and are particularly useful in quantitative PCR to determine viral loads. Furthermore, they are particularly useful because they are biochemically and physically similar to the wild type virus. When used as a control, the replication incompetent viral particles are added to a biological specimen, for example at the time of collection, and the nucleic acid is then isolated. Subsequently, an amplification reaction is performed in which primers are used to amplify both the replication incompetent viral nucleic acid sequence and the target wild type viral nucleic acid sequence. The amplification products can then be separated and detected and/or quantified. Simply detecting the presence of the wild type viral nucleic acid sequence is useful for tracking the spread of a virus through a given population, which is important for designing an appropriate prevention scheme. Similarly, quantifying the viral load in a specific biological specimen can be useful on an individual basis to determine whether someone is responding to treatment, and what specific clinical steps should be taken to help improve the prognosis for a specific subject. The target wild type viral nucleic acid sequence can be any known form of nucleic acid, and can be retroviral in origin. In cases where the target viral nucleic acid sequence is RNA, the amplification process is preceded by a reverse transcription reaction. The wild type viral nucleic acid can be isolated from any biological specimen that may contain viral nucleic acid, such as blood or hepatic cells. The replication incompetent viral particles are engineered by using a vector, termed a standard vector, which can be derived from the intended target virus, or from a completely unrelated nucleic acid sequence. The standard vector is distinguished from the wild type viral nucleic acid sequence by one or more detectable characteristics, such as length or inclusion of one or more restriction endonuclease recognition sites. These distinguishing characteristics allow for the individual detection of the amplification products. The detection of products from the simultaneous amplification of the genetically tagged sequence and that of the target wild type virus can be accomplished using various means, such as by incorporating labels or post-amplification processing. The invention also provides a cell line that produces replication incompetent viral particles. An example of the genetically tagged sequence contained within a replication incompetent viral particle is shown in Seq. I.D. No. 10. The invention also provides a method of determining the relative quantity of target wild type viral nucleic acid in a sample. This method involves adding different known concentrations of the replication incompetent viral particles to samples containing a constant amount of the biological specimen. The samples are then subjected to an amplification process, and the products are separated. Once separated the products derived from the replication incompetent viral nucleic acid sequence can be used to create a standard curve. Subsequently, the products from the wild type nucleic acid sequence can be compared to the standard curve to determine the concentration of the target viral nucleic acid within the original biological specimen. The replication incompetent viral particles that are used as standards in the present invention avoid bio-safety concerns that would be raised by the use of replication competent viral particles as internal standards in PCR reactions. The PCR target sequence can also be inserted into the control without any concern for the effect on viral morphogenesis, because replication competence need not be preserved. Viral particles can also be constructed that contain multiple PCR target sequences from different viruses or genes in a single piece of RNA, for example by combining the PCR targets of HCV and HIV in one particle. Multiple control sequences can be placed in the retroviral particle, so that the same standard can be used as a control in the PCR amplification of many diverse viral targets. The present invention also avoids the difficulty of developing viral cell culture systems that would be needed to produce intact human viruses, such as HCV. Furthermore, the replication incompetent viral particles of the present invention allow for increased quality assurance. These particles can be used to determine sample integrity by adding the particles to a sample just after the sample is collected. Used in this way, the particle allows for the detection of false negative results. The particles can also be used to monitor the quality of the processing of the biological specimens. This can be achieved by adding the particles at various stages of the purification process. SEQUENCE LISTING Seq. ID. Nos. 1 and 2 show the nucleic acid sequences of the Xho I-Eco Rl adapters. Seq. ID. No. 3 shows the inserted sequence in clone NCC 16- A3. Seq. ID. Nos. 4 and 5 show the primers used to amplify the HCV clone. Seq. ID. Nos. 6 and 7 show the primers used to identify clones producing HCV virus particles. Seq. ID. Nos. 8 and 9 show the primers used to detect helper viral particles (RCR). Seq. ID. No. 10 shows the nucleic acid sequence of the amplification region containing the genetically tagged sequence. DETAILED DESCRIPTION I. Definitions Packaging cell line: Cell lines that contain the DNA encoding the structural proteins necessary for viral packaging, but not to package or transmit the RNAs encoding these functions. Infective: A virus or vector is "infective" when it transduces a cell, replicates, and (without the benefit of any complementary virus or vector) spreads progeny vectors or viruses of the same type as the original transducing virus or vector to other cells in an organism or cell culture, where the progeny vectors or viruses have the same ability to reproduce and spread throughout the organism or cell culture. Thus, for example, a nucleic acid encoding an HIV particle is not infective if the nucleic acid cannot be packaged by the HIV particle (e.g. if the HIV particle lacks an HIV packaging site), even though the nucleic acid can be used to transfect and transform a cell. Similarly, an HIV nucleic acid packaged by an HIV particle is not infective if it does not encode the HIV particle that it is packaged in. All known retroviruses share features of the replicative cycle, including packaging of viral RNA into virions, entry into target cells, reverse transcription of viral RNA to form the DNA provirus, and stable integration of the provirus into the target cell genome. Replication competent proviruses contain, at a minimum, regulatory long terminal repeats (LTRs) and the gag, pro, pol and env genes which encode core proteins, a protease, reverse transcriptase/RNAse H/integrase and envelope glycoproteins, respectively. Replication incompetent viruses lack one or more of these structural features. Genetically tagged sequence: A sequence that has been altered such that it can be differentiated from the wild type sequence that is being detected. The genetically tagged sequence is characterized by the presence of primer binding sites which have at least about 80% sequence identity to the comparable sequence contained in the target wild type sequence. Nucleic acid: A deoxyribonucleotide or ribonucleotide polymer in either single or double stranded form, which unless otherwise limited encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. Polynucleotide: A linear nucleotide sequence, including sequences of greater than about 100 nucleotide bases in length. Isolating: The term "isolating" or "purifying" generally refers to preparing a compound to be tested or analyzed in a manner suitable for the test or analysis. The terms are intended to encompass crude cell lysates and extracts containing nucleic acid, as well as substantially purified nucleic acid. The term co-isolating encompasses the meaning of isolating, and also refers to the isolation of more than one compound from a sample substantially simultaneously, and particularly refers to the isolation of more than one nucleic acid species. Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame. Promoter: A promoter is an array of nucleic acid control sequences that direct transcription of a nucleic acid. A promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription. ORF (open reading frame): a series of nucleotide triplets (codons) coding for amino acids without any termination codons. These sequences are usually translatable into a peptide. Recombinant: A recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Sequence identity: The similarity between two nucleic acid sequences, or two amino acid sequences is expressed in terms of the level of sequence identity shared between the sequences. Sequence identity is typically expressed in terms of percentage identity; the higher the percentage, the more similar the two sequences are. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman (1981) Adv. Appl. Math. 2: 482; Needleman & Wunsch (1970) J. Mol. Biol. 48: 443; Pearson & Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444; Higgins & Sharp (1988) Gene, 73: 237-244; Higgins & Sharp (1989) CABIOS 5: 151-153; Corpet et al. (1988) Nuc. Acids Res. 16, 10881-90; Huang et al. (1992) Computer Appls. in the Biosciences 8, 155-65; and Pearson et al. (1994) Meth. Mol. Bio. 24, 307-31. Altschul et al. (1990) /. Mol. Biol. 215:403-410, presents a detailed consideration of sequence alignment methods and homology calculations. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al. (1990) J. Mol. Biol. 215:403-410) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. It can be accessed at http//www.ncbi.nlm.nih.gov/BLAST/. A description of how to determine sequence identity using this program is available at http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html. For comparisons of nucleic acid sequences of less than about 150 nucleic acids, the Blast 2 sequences function is employed using the default 0 BLOSUM62 matrix set to default parameters, (OPEN GAP 5, extension gap 2). Nucleic acid sequences with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 45 % , at least 50 % , at least 70 % , at least 80 % , at least 85 % , at least 90 % , or at least 95% sequence identity. Probes and primers: A probe comprises an isolated nucleic acid attached to a detectable label or reporter molecule. Primers are short nucleic acids, preferably DNA or RNA oligonucleotides 10 nucleotides or more in length. Primers may be annealed to a complementary target DNA or RNA strand by nucleic acid hybridization to form a hybrid between the primer and the target sequence, and then extended along the target nucleic acid sequence by a DNA polymerase enzyme or a reverse transcriptase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods known in the art. One of skill in the art will appreciate that the specificity of a particular probe or primer increases with its length. Thus, for example, a primer comprising 20 consecutive nucleotides will anneal to a target with a higher specificity than a corresponding primer of only 15 nucleotides. Thus, in order to obtain greater specificity, probes and primers may be selected that comprise 15, 20, 25, 30, 35, 40, 50 or more consecutive nucleotides. Label: A label is a detectable moiety that is incorporated into the product of the amplification reaction. Examples of such detectable moieties are radioactive molecules, fluorescent molecules and dyes. In some cases the moiety itself will not be detectable, but it will provide at target for a secondary reaction. Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in the host cell, such as an origin of replication. A vector may also include one or more therapeutic genes and/or selectable marker genes and other genetic elements known in the art. A vector can transduce, transform or infect a cell, thereby causing the cell to express nucleic acids and/or proteins other than those native to the cell. A vector optionally includes materials to aid in achieving entry of the nucleic acid into the cell, such as a viral particle, liposome, protein coating or the like. Packaging cell line: A packaging cell line lacks the nucleic acid sequences necessary for packaging viral RNA. However, the cell line does express the viral RNA that encodes proteins necessary for the production of the capsid. That is, packaging cell line viral RNAs are not themselves encapsidated in the resulting viral particles for which they encode. Therefore, the resulting viral particles are replication incompetent and not infective. The packaging cell line optionally includes all of the components necessary for production of viral particles, or optionally includes a subset of the components necessary for viral proteins for packaging. Transfer vector: A vector that shuttles a transgene. Transduced and Transformed: A virus or vector "transduces" a cell when it transfers nucleic acid into the cell. A cell is "transformed" by a nucleic acid transduced into the cell when the DNA becomes stably replicated by the cell, either by incorporation of the nucleic acid into the cellular genome, or by episomal replication. As used herein, the term transformation encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration. Amplify (Amplification): This term refers to the process of increasing the number of nucleic acid sequences which are complementary to the region between two primer binding sites. This is typically accomplished through the use of PCR or NASBA techniques. Mammal: This term includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects. Animal: Living multicellular vertebrate organisms, a category which includes, for example, mammals and birds. Transgene: An exogenous gene supplied by a vector. Hybridization: DNA molecules and nucleotide sequences which are derived from the disclosed DNA molecules as described above may also be defined as DNA sequences which hybridize under stringent conditions to the DNA sequences disclosed, or fragments thereof. Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method of choice and the composition and length of the hybridizing DNA used. Generally, the temperature of hybridization and the ionic strength (especially the Na + concentration) of the hybridization buffer will determine the stringency of hybridization. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed by Sambrook et al. (1989) In Molecular Cloning: A laboratory Manual, Cold Spring Harbor, New York, of which chapters 9 and 11, herein incorporated by reference. By way of illustration only, a hybridization experiment may be performed by hybridization of a DNA molecule (for example, a variant of the cDNA) to a target DNA molecule which has been electrophoresed in an agarose gel and transferred to a nitrocellulose membrane by Southern blotting (Southern, 1975, J. Mol. Biol. 98:503), a technique well known in the art and described in Sambrook et al. (1989). Hybridization with a target probe labeled, for example, with [ 32 P]-dCTP is generally carried out in a solution of high ionic strength such as 6xSSC at a temperature that is 20- 25 °C below the melting temperature, T m , described below. For such Southern hybridization experiments where the target DNA molecule on the Southern blot contains 10 ng of DNA or more, hybridization is typically carried out for 6-8 hours using 1-2 ng/ml radiolabeled probe (of specific activity equal to 10 9 CPM/μg or greater). Following hybridization, the nitrocellulose filter is washed to remove background hybridization. The washing conditions should be as stringent as possible to remove background hybridization but to retain a specific hybridization signal. The term T m represents the temperature above which, under the prevailing ionic conditions, the radiolabeled probe molecule will not hybridize to its target DNA molecule. The T m of such a hybrid molecule may be estimated from the following equation (Bolton and McCarthy, 1962, Proc. Natl. Acad. Sci. USA 48:1390): T m = 81.5°C + 16.6(log 10 [Na + ]) + 0.41(%G+C) - 0.63(% formamide) - (600//) Where / = the length of the hybrid in base pairs. This equation is valid for concentrations of Na + in the range of 0.01 M to 0.4 M, and it is less accurate for calculations of T m in solutions of higher [Na + ] . The equation is also primarily valid for DNA's whose G+C content is in the range of 30% to 75%, and it applies to hybrids greater than 100 nucleotides in length (the behavior of oligonucleotide probes is described in detail in Ch. 11 of Sambrook et al., 1989). The T m of double-stranded DNA decreases by 1-1.5°C with every 1% decrease in homology (Bonner et al., 1973). For purposes of the present invention, "stringent conditions" encompass conditions under which hybridization will only occur if there is less than 25% mismatch between the hybridization probe and the target sequence. "Stringent conditions" may be broken down into particular levels of stringency for more precise definition. Thus, as used herein, "moderate stringency" conditions are those under which DNA molecules with more than 25% sequence variation (also termed "mismatch") will not hybridize; conditions of "medium stringency" are those under which DNA molecules with more than 15% mismatch will not hybridize, and conditions of "high stringency" are those under which DNA sequences with more than 10% mismatch will not hybridize. Conditions of "very high stringency" are those under which DNA sequences with more than 6% mismatch will not hybridize. Substantial Homology: When comparing the nucleic acid sequence of a primer to the target region for which the primer is intended to hybridize, the sequences of the primer and the target region should be substantially homologous. This term is defined functionally to include primer sequences that contain enough complementarity to allow for the specific amplification of the region between two primer binding sites. Specific amplification infers that after amplification a distinct product of the size of the intended amplification region will be detectable. The standard vector: A standard vector is a nucleotide construct that is used as a control, and contains the genetically tagged sequence and primer binding sites that are necessary for amplifying the genetically tagged sequence. The standard vector also contains the nucleotide sequences required for efficient packaging of its RNA genome (including the genetically tagged sequence) and may be made from a variety of proviral clones, such as, for example, HIV-2/ST (Kumar et al., 1991, J. Virol. 64:890-901; Arya et al., 1993, J. Acquir. Immune. Defic. Syndr. 6:1205-1211; Arya et al., 1993, 9:839-48). The standard vector can also be created through the modification of a number of different viral genomes. Furthermore, these modifications can be made using standard genetic engineering techniques which are well known in the art and further described below. Implementation of the Invention The invention involves using replication incompetent viral particles as positive controls in assays that detect viral contaminants. The replication incompetent viral nucleic acid sequences can be derived from a variety of wild type viral genomes, for example from HCV or HIV-1. Furthermore, the replication incompetent viral particles can be engineered to contain multiple primer binding sites so that a single replication incompetent viral particle can be used to detect multiple target viruses. O. The Creation of Replication Incompetent Viral Particles For the purposes of the present invention, any non-infectious viral particle that contains a nucleic acid sequence which can be engineered to incorporate primer binding sites will work. However, to date the most readily available method of generating replication incompetent viral particles is through the use of packaging cell lines and vectors derived from retroviruses. The method of engineering such packaging cell lines and vectors is described below, and following these teachings other systems which divide viral genomes into packaging components and transfer components can be generated. Another such non-retroviral system is the adenoviral system described in U.S. Patent No. 5,882,877 issued to Gregory et al. A. Retroviral Vectors The retroviral life cycle has been extensively studied and involves five basic steps. These steps are: attachment to the cell membrane; formation of DNA from the viral RNA genome; insertion of the viral DNA into the host cell genome; transcription and translation of viral structural proteins; and the transcription of the viral RNA genome which is then packaged into new virus particles. These processes have been adapted to carry exogenous nucleic acid sequences into host cells, where they will be incorporated into the host cell genome. However, the use of such viruses for this purpose introduces the possibility of infecting the host with a potentially harmful viral strain. The threat of introducing a harmful viral strain into a host has been minimized by the creation of packaging cell lines and associated transfer vectors. A packaging cell line is a cell line that has been engineered to produce various viral structural genes, such as gag, pol, and env, that are not encoded by the associated transfer vector. The transfer vector contains other viral proteins and regulatory sequences which, when transformed into the packaging cell line, yields replication incompetent viral particles. Even with the single division of viral genetic material between the packaging cell line and the transfer vector, occasional viral particles may be produced that will be replication competent. Recombination events can lead to the production of replication competent viral particles that potentially can cause disease. To overcome this problem, variations have been developed that divide the viral genetic material into multiple transfer vectors. This increases the number of recombination events that are necessary for a replication competent viral particle to be produced. In the present invention, the standard vector is roughly analogous to the transfer vector described above. The standard vector is similar to the transfer vector in that it contains the necessary nucleic acid signal sequence to package the viral nucleic acid sequence in the replication incompetent viral particles. However, unlike the transfer vector described above, the standard vector contains only the nucleic acid sequences for packaging the genetically tagged sequence into the viral capsid. However, a standard vector can contain the additional nucleic acid sequences necessary for transforming a host cell, if desired. Examples of packaging cell lines and transfer vectors can be found in US Patent No. 5,866,411 issued to Pedersen, et al., US Patent No 5,858,744 issued to Baum et al., and US Patent No. 5,766,945 issued to Miller. B. Other Replication Incompetent Virus Particles Given the strategy for making the packaging and target packageable nucleic acids described above, the construction of various other replication incompetent viral particles is now possible. Cloning methodologies to accomplish these ends, and sequencing methods to verify the sequence of nucleic acids, are well known in the art. Examples of appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises, are found in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, volume 152, Academic Press; Sambrook et al., 1989, Molecular Cloning, and Ausbel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., 1994. Further examples of systems that generate replication incompetent viral particles are described in U.S. Patent No. 5,882,877 issued to Gregory et al. The replication incompetent viral particles can be engineered to contain either dsDNA, ssDNA, dsRNA, ssRNA or mixtures thereof. Typically, the choice of which type of replication incompetent virus to use will depend upon the nature of the target viral nucleic acid. For example, if the target viral nucleic acid sequence is ssRNA, then the replication incompetent viral particle will also contain ssRNA. Furthermore, the target wild type viral nucleic acid sequence can be retroviral in origin, for example derived from HTLV-1, HTLV-2, HIV-1 and HIV-2. The nucleic acid compositions of this invention, whether RNA, cDNA, genomic DNA, or a hybrid of the various combinations, are isolated from biological sources or synthesized in vitro. The nucleic acids of the invention are present in transformed or transfected whole cells, in transformed or transfected cell lysates, or in a partially purified or substantially pure form. It is also possible to generate other alterations in a given nucleic acid construct. Such well-known methods include site-directed mutagenesis, PCR amplification using degenerate oligonucleotides, exposure of cells containing the nucleic acid to mutagenic agents or radiation, chemical synthesis of a desired oligonucleotide, and other well known techniques. See Gilman and Smith, 1979, Gene 8:81-97; Roberts et al., 1987, Nature 328:731-734; and Sambrook, Innis, and Ausbel, Id. One of ordinary skill in the art can select a desired nucleic acid of the invention based upon the sequences provided, and upon knowledge in the art regarding the production of replication incompetent viral particles. Finally, most modifications to nucleic acids are evaluated by routine screening techniques in suitable assays for the desired characteristics. For instance, changes in the immunological character of encoded polypeptides can be detected by an appropriate immunological assay. Modifications of other properties, such as nucleic acid hybridization to a complementary nucleic acid and amplification efficiency, are all standard techniques. m. Construction of Various Standard Vectors for use in Detecting the Presence of One or More Target Wild Type Viruses A. Using a Control Vector which Contains Regions of Homology to a Target Wild Type Viral Nucleic Acid Sequence The standard vector can be created by using the target wild type viral nucleic acid sequence as the starting point (as described in Example 2 below). The wild type viral sequence must first be isolated, reverse transcribed and amplified such that it is in the form of a double stranded DNA sequence. This sequence can then be cloned into an appropriate vector for subsequent manipulation. Manipulation of the sequence may involve removing some, if not all, of the genes that encode the capsid proteins. The remaining vector sequence is then modified such that, upon subsequent amplification by PCR primers, it is distinguishable from the original target wild type viral nucleic acid sequence. The vector can be made distinguishable from the target wild type viral nucleic acid sequence by linearizing the vector at a point between the two primer binding sites and then inserting or deleting one or more nucleic acids. The inserted nucleic acids, may for example, provide a new restriction site which can be recognized by a restriction endonuclease. B. Using a Control Vector which is not Homologous to a Target Wild Type Virus The standard vector can also be created by modifying a vector that is not homologous to the target wild type viral nucleic acid sequence. This can be done by selecting a vector which is derived from a different virus and incorporating primer binding sites into it. These sites, however, should be substantially homologous to primer binding sites found in the target wild type viral nucleic acid sequence. Amplification of the standard vector will result in a product which contains the primer binding sites on the 5' and 3' termini, and a region in between the primer sites that is unrelated to the target wild type viral nucleic acid sequence. Similarly, the amplification of the target wild type viral nucleic acid sequence will result in a sequence that has 5' and 3' ends that are the same as that of the primer binding sequences of the standard vector. C. Creating a Control Vector which is Capable of Being used to Detect the Presence of Multiple Wild Type Viruses A standard vector can also be created so that it can be used to detect multiple different target wild type nucleic acid sequences. This can be done by incorporating a series of primer binding sites into a single standard vector, such that the primers are substantially homologous to a variety of different wild type viruses. To perform a test for a variety of viruses, a biological specimen can be taken from a subject and allocated into several different vessels. The primers specific for a particular target wild type viral nucleic acid sequence can then be added to one vessel, and primers specific for another target wild type nucleic acid sequence can be added to another vessel and so on. These vessels can then be amplified using standard PCR techniques, and the presence of a particular virus can be detected. Optimally, the primer pairs will be such that they can be used in the same reaction (multiplexed) without causing PCR artifacts. The development of primer sets that can be used in a single reaction requires that each primer pair retain specificity. Testing to ensure that specificity is maintained can be performed by the following steps: 1) using one primer pair and amplifying; 2) confirming the size of the fragment; 3) using two primer pairs and amplifying; and 4) confirming that two distinct products are generated. Furthermore, if multiple primer pairs are used, the genetically tagged sequences are designed such that the resulting amplification products can be distinguished from each other, as well as from the target wild type viral nucleic acid sequences. IV. Addition of Replication Incompetent Viral Particles to the Biological Specimen The replication incompetent viral particle can be added immediately after the collection of the biological specimen, or at any time up until the time when the nucleic acid is isolated from the specimen. The biological specimen can be derived from a variety of sources, such as blood, cerebrospinal fluid, saliva, lymphatic tissue, seminal tissue, vaginal tissue, serum, plasma, lymphoid cells lymphocyte cells, B cells, T cells, monocytes, polymorphonuclear cells, macrophages, epithelial cells, nasopharyngeal epithelium, upper respiratory tract epithelium, labial epithelium, tumor cells, respiratory secretions, nasopharyngeal secretions, brain tissue, virus vesicle tissue wart tissue feces, urine, pleural and pericardial fluid, milk, salivary gland tissue, negri body tissue, and/or hepatic cells. V. Methods of Detecting PCR Products The genetically tagged sequences differ from the target wild type nucleic acid sequences by at least one detectable characteristic, but the characteristic does not affect the ability of the sequence to be amplified using the same primers. Genetically tagged sequences that are a different size than the region amplified from the target wild type viral nucleic acid sequence, or which contain a unique restriction cleavage site, have proved to be convenient. When detecting the presence of a wild type viral nucleic acid sequence which is DNA, the replication incompetent virus contains DNA, and when detecting wild type nucleic acid sequences which are RNA, it preferably is an RNA. The genetically tagged sequences generally differ from the target wild type nucleic acid sequence by 1 % to 20% of their lengths and will often differ by at least by 1 nucleotide. When the assay is intended to determine an actual quantity of the viral load (DNA or RNA), the quantity may be stated in the form of weight (mg, μg, ng, pg), or may be given as the number of copies of a certain nucleic acid molecule. Quantitation following amplification may be effected in different ways. Generally, multiple dilutions of the replication incompetent viral particles are placed in samples containing the same amount of the biological specimen. The nucleic acid is subsequently isolated from the specimen and subjected to an amplification procedure. The product from the replication incompetent viral nucleic acid sequence is then used to create standard curve. The product from the amplification of the target wild type viral nucleic acid sequence can then be compared to the standard curve, and the viral load determined. A. Florescent Tags Detection methods which are automatic, and which combine the separation and quantitation steps, have proved to be particularly suitable. A particular embodiment of the method according to the invention involves deteπnining the amounts of the amplified nucleic acid by using a nucleic acid detection device, preferably a fluorescence-sensitive nucleic acid detection device. Examples of such nucleic acid detection devices are automatic DNA sequencers with laser-induced fluorescence measuring means (e.g. Gene Scanner.RTM.373A of Applied Biosystems), HPLC or capillary electrophoresis system devices. With these devices it is possible to separate nucleic acid molecules which differ in length by one base pair. A particular advantage of the Gene Scanner. RTM. is that it is possible to differentiate between different fluorescent dyes in a single lane. This allows for the simultaneous processing of several samples in one lane on a gel. This method can analyze a plurality of PCR products, labeled with different fluorescent dyes, in a single lane (multiplex-PCR). When simultaneously detecting two different nucleic acids, e.g., in one sample, expenditures and costs are reduced. When using the method according to the invention in a routine operation, this is of particular advantage, e.g., when a blood sample is to be tested for multiple target viral nucleic acid sequences. B. Identification of Amplification Products using Various Dyes In addition to the fluorescent dyes discussed above, the amplification products can also be visualized and quantified through the use of various dyes. For example, the products can be separated out on an agarose gel, and then the gel can be exposed to a solution containing ethidium bromide. The ethidium bromide will then intercalate into the nucleic acid products, and the nucleic acid products can subsequently be visualized by exposure to ultraviolate light. Furthermore, the relative amount of nucleic acid in each product can be determined by photographing the gel and subsequently scanning the image into a graphic file. The graphic file can then be analyzed to determine the relative intensity of the bands appearing in the photographic image. C. Identification of Amplification Products using Radiolabeled Nucleic Acids Another convenient method of detecting the amplification products is by incorporating radiolabeled nucleic acid moieties into either the primers and/or into the reaction itself. For example, a primer can be subjected to treatment with a kinase in the presence of radiolabeled ATP. Used in this way, the terminal phosphate of the ATP molecule will be 32 P, and this radiolabeled phosphate will then be transferred by the kinase to the primer. Subsequently, the primer will be used in an amplification procedure, and it will become incorporated into the resulting products. The resulting products can then be electrophoresed separated on a gel and the level of radioactivity within the resulting bands can be quantified. Similarly, radiolabeled mononucleotides can be used directly in the amplification process. For example, α- 32 P dATP can be added directly to the reaction and the radiolabled mono-nucleotide will then be incorporated into the product of the reaction. Subsequently, the reaction products can be separated out and quantified using a phosphoimager, or by simply excising the bands and placing them in a scintillation counter. Radiolabeled probes can also be used to identify the products of the amplification reaction. Used in this way the radiolabeled probes are allowed to hybridize with the amplification products, as described above. EXAMPLES 1. Collection of Biological Specimens The HCV positive plasma sample used for molecular cloning, site-directed mutagenesis, and construction of virus particles was from patient H (H77 isolate) with chronic post-transfusion non-A, non-B hepatitis (Ogata et al., 1991, Proc. Natl. Acad. Sci. USA 88:3392-3396.). The viral nucleic acid was isolated by extraction of the subject's plasma with guanidimum thiocyanate, followed by centrifugation in 5.7 M cesium chloride solution (Sambrook, J. et al., 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Purified RNA was dissolved in diethylpyrocarbonate (DEPC)-treated water containing 2 mM Tris-HCl, pH 8.0, 0.2 mM EDTA, 5 mM dithiothreitol (DTT), and 200 units/ml RNase inhibitor (PE Applied Biosystems, Foster City, CA), and stored at -70°C before use. Three different HCV positive patient plasma samples and five HCV negative plasma samples from individual blood donors were also included in the study. 2. Cloning and Construction of an HCV control vector To construct HCV recombinant DNA containing the 5' untranslated region and core protein gene (nt 18-965), the complementary DNA (cDNA) to this region was synthesized by using avian myeloblastosis virus (AMV) reverse transcriptase (Promega, Madison, WI) using a 5'- phosphorylated primer C2 (see Table 1 below). The cDNA was amplified by the polymerase chain reaction using 5'-phosρhorylated primer pair C2 and NAF1 (see Table 1). The 948-bp PCR fragment was purified from 1 % agarose gel, blunt-ended by T4 DNA polymerase, and inserted into vector pT7T3 19U (Pharmacia Biotech Inc., Piscataway, NJ) at Sma I site using the standard molecular cloning procedures (Sambrook, J. et al., 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The recombinant clone HCV-NCC16 was identified by restriction enzyme digestion analysis and in vitro transcription analysis to demonstrate that the transcript synthesized in vitro by T7 RNA polymerase contained the sense strand of HCV sequence. This recombinant DNA was subsequently used for construction of a mutated sequence in the 5' noncoding region of the HCV genome. The potential target site for mutagenesis in the HCV 5' noncoding region was selected in a 24-base long sequence (nt 263-286) previously used as a probe for detection of HCV PCR products (Young, K. K. et al., 1993, J. Gin. Microbiol. 31: 882-886). Insertion of an exogenous sequence in this area disrupts the continuity of sequence complementary to the probe, therefore it can be used for differential detection between a wild type viral nucleic acid sequence and the standard vector sequences. HCV-NCC16 plasmid DNA was digested with Nru I (at nt 272) and dephosphorylated at its 5 '-ends by alkaline phosphatase. The resulting DNA was purified and ligated with Xho I - Eco RI adapter (Stratagene, La Jolla, CA): 5ΗO-TCGAGGAATTC-3' (Seq. ID. No. 1), 3'- CCTTAAG-p5' (Seq. ID. No. 2) by T4 DNA ligase for 24 hours at 5°C, followed by incubation with T4 DNA polymerase and T4 polynucleotide kinase in the presence of dNTP and ATP at 25°C for 30 minutes, and further incubated at 5°C for another 16 hours. One tenth of the ligation mixture was used to transform E. coli DH5α competent cells (Gibco BRL, Grand Island, NY). The recombinant clones were first screened by digestion of plasmid DNA's with Eco RI, then confirmed by DNA sequencing. The inserted sequence from one of the recombinant clones, HCV-NCC16-A3, is 36-bp long: 5'-GAATTCCTCGAGGAATTCGAATTCCTCGAGGAATTC-3' (Seq. ID. No. 3). Plasmid DNA from this clone was isolated from a large culture and purified by cesium chloride-ethidium bromide density centrifugation. The DNA was digested with restriction endonucleases Bam HI and Sac I to release a 1,000-bp fragment with 4-base overhangs on both ends, which contained a 948-bp HCV sequence, a 36-bp the inserted sequence, and a 16-bp vector sequence. The 1,000-bp DNA fragment was isolated from 1.0% agarose gel, purified by using a QIAEX II agarose gel extraction kit (QIAGEN, Valencia, CA), and blunt-ended by T4 DNA polymerase. This DNA fragment was cloned into retroviral vector pXTl (Stratagene) at a Bgl II site. Bgl II-digested pXTl DNA was blunt-ended by the Klenow fragment of E. coli DNA polymerase I, then mixed with the insert, T4 DNA ligase, ATP and DNA ligation buffer, and incubated at 5°C for 65 hours. One tenth of the DNA ligation mixture was used to transform E. coli DH5α cells. HCV retroviral recombinant clones were identified by 1,000-bp increment of plasmid DNA's size, and restriction enzyme digestion analysis to verify the orientation of HCV insert being in line with the retroviral transcriptional unit in vector DNA. One of the clones, pXT-HCV-NCC-D8, was selected for subsequent construction of viral particles in retrovirus packaging cells. 3. Generating Replication Incompetent Viral Particles The following method was used to construct murine amphotropic retrovirus particles containing the standard vector for use as an internal standard for the detection and quantification of HCV. Retrovirus packaging cell line PA317 (Miller, A. D. et al., 1986, Mol. Cell. Biol. 6: 2895- 2902) was obtained from American Type Culture Collection (ATCC, Manassas, VA), and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 4.5 g/L D- glucose, 2 mM L-glutamine, 20 μg/ml gentamicin, and 10% fetal bovine serum (complete DMEM). To transfect PA317 cells with pXT-HCV-NCC-D8 plasmid DNA, 12 μg of this plasmid DNA was mixed with 300 μl Opti-MEM I medium (Gibco BRL), and combined with a mixture of 15 μl Lipofectamine (Gibco BRL) and 300 μl Opti-MEM I. The mixture was incubated at 25 °C for 30 minutes. This incubation allowed the DNA-liposome complexes to form. The culture medium was removed from PA317 cells which were at 65%-70% confluence in 25-cm 2 flasks. The cells were then rinsed once with 8 ml Opti-MEM I, overlayed with 2.4 ml Opti-MEM I plus the DNA-liposome complexes, and incubated for 3 hours at 37°C in a C0 2 incubator. Subsequently, the cells were fed with 5 ml complete DMEM, and allowed to grow for another 18 hours. The transfected cells were then trypsinized, diluted to 40ml with complete DMEM plus 500 μg/ml antibiotic G418 (Gibcol BRL) for selection, and seeded into two 96- well plates with 200 μl each well. The plates were incubated for 6 days, then the media were refreshed and incubated for another 8 days. The G418-resistant colonies were marked under an inverted microscope, and subcultured into 12-well plates with 2 ml medium each well. When the cells in 12-well plates reached 90%-100% confluency, the culture supematants were collected and cleared by filtration through 0.45 μm filter units (Millipore, Bedford, MA). The cells were processed for storage in a liquid nitrogen freezer. To identify cell clones which produced virus particles containing HCV sequences, the RNA from the filtered culture supernatant was isolated and subjected to the combined reverse transcription and polymerase chain reaction (RT-PCR) using the primer pair NF5 and NR5 (see Table 1) specific for the HCV 5' noncoding region. The PCR products were analyzed on 5% polyacrylamide gel for detection of a 338-bp band. Control samples that did not contain reverse transcriptase were also run in order to eliminate the possible detection of proviral DNA. Cells from several tentatively identified positive clones were revived, grown successively from a 12-well plate, to a 6-well plate, to 25-cm 2 flasks in complete DMEM plus 500 μg/ml G418. When the cells reached 80% confluency in 25-cm 2 flasks, the culture media were replaced with complete DMEM without G418. After 24 hours incubation, the culture fluids were collected and filtered through 0.45 μm filter units. These filtered culture fluids were used to determine the viral titers by RT-PCR, and verify whether replication-competent retroviruses (RCR) were present. One of the clones, D8-54, which produced the highest virus titer and was free of any detectable RCR, was selected for further expansion to 500 ml - 1,000 ml cultures. The virus titer was determined by RT-PCR, colony forming units in NIH 3T3 cells, infectious units in mouse cell line P815, and particle counts by electron microscopy. Each batch of virus preparations was also verified for the absence of RCR with an assay sensitivity of detecting 0.2 infectious units per ml. 4. Screening for Helper Viral Particles Replication-competent retroviruses (RCR or helper viruses) are a potential biohazard, and the presence of such viral particles in the preparations of replication incompetent standards can eventually lead to erroneous particle counts by electron microscopy. Therefore, RCR analysis for each lot of replication incompetent standard may be performed to avoid these problems. The RCR testing was carried out according to the previously described methods (Cornetta, K. et al., 1991, Human Gene Ther. 2: 5-14) with some modifications. In brief, NIH 3T3 cells at 50% -60% confluency in a 25-cm 2 flask were infected with 6 ml filtered virus supernatant in the presence of 8 μg/ml Polybrene (Sigma, St. Louis, MO) for 5-16 hours, then the virus fluids were removed and replaced with 8 ml complete DMEM plus 4 μg/ml Polybrene. After 36-48 hours incubation, the cells were trypsinized and subcultured at 1 : 10 dilution in 8 ml complete DMEM plus 4 μg/ml Polybrene. When the cells reached 100% confluency (3-4 days), the culture fluids were collected and filtered through 0.45-μm pore-sized membrane filters, then the filtrates were saved as the second passage of virus supematants for helper virus detection by RT-PCR. The cells were further subcultured at 1 :20 dilution in 8 ml of the above-described media, and grown until 100% confluency (4-5 days). The culture supematants and the cells from the third passage were processed for helper virus detection by RT-PCR. The reaction conditions of RT-PCR for detecting helper virus were similar to those of HCV except that the primers were replaced by ENV1 and ENV3 (see Table 1) and the extension time at 72 °C in the thermal cycles was 45 seconds. The PCR target is 411-bp fragment from the envelope gene encoding gp70 protein of amphotropic virus (Morgan, R. A. et al., 1990, 1: 135-149). 5. Detecting Target Wild Type Viral Nucleic Acid in a Biological Sample The 50 μl reaction contained 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl 2 , 50mM KC1, 0.001% (w/v) gelatin, 200 μM each of dATP, dCTP, dGTP, and dTTP, 0.4 μM each of primers NF5 and NR5, 1 μM random hexanucleotides, 1 mM DTT, 4 units of RNase inhibitors, 2.5 units of Moloney murine leukemia virus (M-MuLV) reverse transcriptase, 1 unit of AmpliTaq DNA polymerase, and a 25 μl RNA sample. Most of the RT-PCR reagents, PE9600 DNA thermocycler, and the thin-walled MicroAmp tubes were obtained from PE Applied Biosystems. The RT reaction was carried out at 42°C for 20 minutes followed by 2 minutes incubation at 94°C to denature RNA-DNA heteroduplexes and inactivate the reverse transcriptase. PCR amplification was performed for 32-45 cycles with denaturation at 94°C for 20 seconds, primer annealing at 56°C for 45 seconds with 1 second increments per cycle, and extension at 72°C for 15 seconds. A final step of incubation for 5 minutes each at 56°C and 72°C was also included after completion of the thermal cycling. The PCR products were analyzed by 5% polyacrylamide gel electrophoresis, followed by ethidium bromide staining or by using Gene Analyzer 310 (PE Applied Biosystems) when the unlabeled NF5 was replaced by fluorescent-labeled NF5. TABLE 1. Primers for construction of the HCV internal standard and for subsequent use in screening assays. Primer' Sequence Position b NAFl (+) 5'-GGCGACACTCCACCATGAATCAC-3' 18-40 (Seq. ID. No.4) C2 (-) 5'-AGGGCAATCATTGGTGACATG-3' 945-965 (Seq. ID. No.5) NF5 (+) 5'-GTGAGGAACTACTGTCTTCACGCAG-3' 47-71 (Seq. ID. No.6) NR5 (-) 5'-TGCTCATGGTGCACGGTCTACGAGA-3' 324-348 (Seq. ID. No.7) ENV1 (+) 5'-ACCTGGAGAGTCACCAACCTG-3' 6491-6511 (Seq. ID. No.8) ENV3 (-) 5'-TACTTTGGAGAGGTCGTAGC-3' 6882-6901 (Seq. ID. No.9) a (+) and (-), sense and antisense, respectively. b Coordinates for primers NAFl, C2, NF5, and NR5 are from infectious HCV clone (GenBank accession no. AF009606), and coordinates for primers EN VI and ENV3 are from murine amphotropic leukemia virus (GenBank accession no. AF010170). Having illustrated and described the principles of the invention in multiple embodiments and examples, it should be apparent to those skilled in the art that the invention can be modified in arrangement and detail without departing from such principles. We claim all modifications coming within the spirit and scope of the following claims.

Claims: What is claimed is: 1. A method for detecting a target wild type viral nucleic acid sequence present in a biological specimen derived from a subject, the method comprising: adding to an aliquot of the sample, as an internal control, one or more replication incompetent viral particles comprising a nucleic acid sequence which contains at least two primer binding sites that have nucleic acid sequences that are substantially homologous to the target wild type viral nucleic acid sequence and are capable of amplifying a genetically tagged sequence contained within the replication incompetent viral nucleic acid sequence; co-purifying the target wild type nucleic acid sequence and the replication incompetent viral nucleic acid sequence from the sample; amplifying the target wild type viral nucleic acid sequence and the replication incompetent viral nucleic acid sequence using one or more primers specific for the two primer binding sites that have nucleic acid sequences which are substantially homologous to the target wild type viral nucleic acid sequence; and detecting the target wild type viral nucleic acid sequence and the replication incompetent viral nucleic acid sequence from the sample. 2. The method of claim 1 wherein the target wild type viral nucleic acid sequence is selected from the group consisting of ssRNA, dsRNA, ssDNA, dsDNA and mixtures thereof. 3. The method of claim 1 wherein the target wild type nucleic acid sequence is retroviral RNA. 4. The method of claim 1 wherein the target wild type nucleic acid sequence is DNA. 5. The method of claim 3 wherein the retroviral RNA is derived from a vims selected from the group consisting of HTLV-I, HTLV-2, HIV-1 and HIV-2. 6. The method of claim 1 wherein the biological specimen is obtained from a mammal. 7. The method of claim 1 wherein the amplifying comprises reverse transcription. 8. The method of claim 1 wherein the target wild typ nucleic acid is derived from HCV. 9. The method of claim 1 wherein the biological specimen is selected from the group consisting of: blood, cerebrospinal fluid, saliva, lymphatic tissue, seminal tissue, vaginal tissue, serum, plasma, lymphoid cells lymphocyte cells, B cells, T cells, monocytes, polymorphonuclear cells, macrophages, epithelial cells, nasopharyngeal epithelium, upper respiratory tract epithelium, labial epithelium, tumor cells, respiratory secretions, nasopharyngeal secretions, brain tissue, vims vesicle tissue wart tissue feces, urine, pleural and pericardial fluid, milk, salivary gland tissue, negri body tissue, and hepatic cells. 10. The method of claim 1 wherein the biological specimen comprises blood. 11. The method of claim 1 wherein the biological specimen comprises plasma. 12. The method of claim 1 wherein the replication incompetent viral particle is derived from a retrovirus. 13. The method of claim 1 further comprising quantitating the target wild type viral nucleic acid sequence and the replication incompetent viral nucleic acid sequence. 14. The method of claim 13 wherein the probe is labeled. 15. The method of claim 1 further comprising quantifying the target wild type nucleic acid sequence by separating the amplified target wild type viral nucleic acid sequence from the amplified genetically tagged sequence. 16. The method of claim 15 wherein separating consists essentially of electrophoresis of the amplified target wild type nucleic acid sequence and the amplified genetically tagged sequence. 17. The method of claim 1 wherein a product from amplifying the genetically tagged sequence is shorter than a product from amplifying the target wild type viral nucleic acid sequence. 18. The method of claim 1 wherein a product from amplifying the genetically tagged sequence is longer than a product from amplifying the target wild type viral nucleic acid sequence. 19. A replication incompetent retrovirus, comprising an genetically tagged sequence having an inserted genetic tag sequence, the genetic tag sequence provided by mutating a highly conserved, non-coding region of a corresponding target wild type retroviral nucleic acid sequence, wherein the genetic tag sequence is of a sufficient length that the genetic tag sequence can be quantitated after amplification with a corresponding target wild type sequence. 20. The method of claim 19 wherein the replication incompetent retrovirus comprises multiple genetically tagged sequences which are amplified by primers that are substantially homologous to more than one target wild type viral nucleic acid sequence. 21. The method of claim 1, wherein amplifying the genetically tagged sequence and the target wild type viral nucleic acid sequence is accomplished using a method selected from the group consisting of PCR or NASBA. 22. The genetically tagged replication incompetent vims of claim 19 wherein the amplification region comprises a nucleic acid sequence corresponding to: SEQ ID No. 10. 23. The method of claim 13 wherein quantitating the target wild type nucleic acid and the replication incompetent viral nucleic aid sequence is accomplished by hybridizing at least one hybridization probe with the amplified target wild type nucleic acid sequence and the genetically tagged sequence, wherein the hybridization probe is specific for at least one or both of the amplified target wild type nucleic acid sequence and the amplified genetically tagged sequence. 24. The method of claim 13, wherein quantitating comprises a method selected from the group consisting of labeled nucleotide incorporation, absorption spectroscopy, nucleic acid hybridization, antibody detection and restriction analysis. 25. A replication incompetent viral nucleic acid sequence containing multiple primer binding sites, comprising primer sites that are substantially homologous to a variety of wild type viral nucleic acid sequences. 26. A replication incompetent viral particle comprising the replication incompetent viral nucleic acid sequence of claim 25.

Inventors: SHIH JAMES W, WANG RICHARD YUAN-HU, SHIH, JAMES, W., WANG, RICHARD, YUAN-HU
Applicants: SHIH JAMES W, US HEALTH, WANG RICHARD YUAN HU, SHIH, JAMES, W., THE GOVERNMENT OF THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY, DEPARTMENT OF HEALTH & HUMAN SERVICES, WANG, RICHARD, YUAN-HU
Application year: 2000
Main classification: C12Q1
IPC codes: C12Q1