_Archives

Vi rology

Arch Virol (1995) 140:1523-1539

© Springer-Verlag 1995 Printed in Austria

Quantitative molecular methods in virology

Brief Review M. Clementi1~, S. Menzo1, A. Manzinj, and P. BagnarellP

~Istituto di Microbiologia, UniversitAdi Ancona, Ancona, 2Dipartimento di Scienze Biomediche, Universit/l di Trieste, Trieste, Italy Accepted May 8, 1995 Summary. During the past few years, significant technical effort was made to develop molecular methods for the absolute quantitation of nucleic acids in biological samples. In virology, semi-quantitative and quantitative techniques of different principle, complexity, and reliability were designed, optimized, and applied in basic and clinical researches. The principal data obtained in successful pilot applications in vivo are reported in this paper and show the real usefulness of these methods to understand more details of the natural history of viral diseases and to monitor specific anti-viral treatments in real time. Theoretical considerations and practical applications indicate that the competitive polymerase chain reaction (cPCR) and competitive reverse-transcription PCR (cRTPCR) assay systems share several advantages over other quantitative molecular methodologies, thus suggesting that these techniques are the methods of choice for the absolute quantitation of viral nucleic acids present in low amounts in biological samples. Although minor obstacles to a wide use of these quantitative methods in clinical virology still remain, further technical evolution is possible, thus making the quantitative procedures easier and apt to routine applications. Introduction

A great deal of new informations has recently indicated that the direct and quantitative investigation of viral activity in vivo is important not only for a complete understanding of the pathogenic steps of most persistent viral infections, but also for a correct virological diagnosis, prognosis, and clinical management of infected patients. Analysis of viral activity by biological methods (including viral isolation in permissive cell culture systems) only approximates the real situation in vivo albeit performed on a quantitative basis. In fact, as in the case of human immunodeficiency virus type 1 (HIV- 1) infection [41, 66], in vitro propagation of a biologically non-homogeneous viral population increases the

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representation of the viruses with high replicative capacity in the particular cell system employed. On the other hand, several characteristics are required, in theory, in a molecular method adopted to evaluate viral activity and/or viral load in vivo and in vitro. These characteristics include: (i) high sensitivity and specificity (very low amounts of nucleic acids have to be detected in some viral infections or in some biological samples), (ii) technical flexibility (similar or identical procedures must be apt for the study of different viral infections and of samples of different origin), and (iii) adaptability to quantitative evaluation of the level of viral activity (absolute quantitation of the target sequence is often required for a precise monitoring of treated and untreated infected patients). Currently, polymerase chain reaction (PCR) is the method of choice for the detection of viral nucleic acids present in low amounts in biological samples, thus allowing a direct and sensitive approach to most acute and persistent viral infections. However, the qualitative features of PCR-based applications limited, in a first phase, the use of this molecular methodology to those conditions where the presence or the absence of a specific target sequence was to be assayed. Nonetheless, quantitative PCR-based strategies, amplification methods other than PCR, and novel hybridization strategies were also proposed in virology. In this review, quantitative molecular methodologies developed for and applied to the analysis of viral activity in persistent infections are analyzed, and virological data obtained are summarized. In particular, major aspects emerging from the quantitative, direct in vivo study of several persistent viral infections are discussed including HIV-1 infection, hepatitis B and hepatitis C virus (HBV; HCV) infections.

Quantitative polymerase chain reaction Common interferences to quantitative applications of PCR Development of PCR-amplification [86] rapidly introduced a real technical improvement in virological laboratories more than in any other biological and biomedical field. The sensitivity and specificity performances of PCR are to date unquestionably the best available for the purpose of detecting specific viral nucleic acids present in low amounts in biological samples. However, a commonly encountered drawback of PCR-amplification is the low tube-to-tube reproducibility of the amount of amplified product, and this may occur even under the most tightly controlled assay conditions. This variability may be dependent on different causes including machine performance, reaction conditions, presence ofinhibitors, differences in sample preparation and purification of nucleic acids, and degradation of templates. Due to the exponential features of PCR, minor differences in amplification efficiency give rise to very large differences in the final product yield. Under these conditions, although a linear relationship was documented between the input template and amplification product copy numbers [2, 74, 94], the unpredictable slope of this linear relationship hinders sample template quantitation by direct measurement of the amplification

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product using different methodologies [56, 101]. Similar problems affect PCRbased methods using limiting dilution analysis of samples or external standard reference curves. In these cases, although semi-quantitative results can undoubtedly be obtained, absolute quantitation of RNA or DNA species may be totally unreliable. Finally, co-amplification of two different templates in the same tube using two primer sets (and following correlation of PCR products) [50] is incorrect from a quantitative point of view due to the different amplification efficiencies of both template species.

The competitive polymerase chain reaction Presently, the most efficient, reproducible, and flexible approach to molecular quantitation using PCR is based on co-amplification of two similar template species (the wild-type sequence to be quantified and the reference template introduced at known amount) of equal or similar length and sharing the primer recognition~sites [34]. During PCR amplification, these similar templates compete for the same primer set (competitive PCR; cPCR) and, consequently, amplify at the same rate (independently of the number of cycles and of any predictable or unpredictable variable influencing PCR amplification) [27]. In this method, the final amount of amplified products can be calculated by different procedures [9, 27, 40, 68]; the ratio of these products precisely reflects the ratio of the initial concentrations of both template species [24, 25] (see below), and under these conditions, the results are completely independent of the number of cycles and the amount of DNA molecules generated by the reaction [27]. Several practical aspects of cPCR methods which provide the key for the development of reliable applications deserve careful attention; these aspects may be summarized as follows: (i) great diversity in sequence length between the wildtype sequence and the competitor template (RNA or DNA) may give rise to differences in amplification efficiency; for this principal reason, internal insertions or deletions have to be reduced to the minimum necessary to obtain discrete bands corresponding to the different PCR products after gel electrophoresis; (ii) although correct in theory, the use of competitors bearing a new restriction site [38, 48, 69] should be avoided since variable efficiency of enzymatic digestion may bias quantitative analysis of the reaction products (see below); (iii) quantitation of RNA targets is possible using cRT-PCR, while use of DNA competitors after reverse-transcription of the wild-type sequence [47, 105, 107] (assuming a theoretical efficiency of the reverse-transcription reaction) is methodologically incorrect and may be highly imprecise (in cRT-PCR, the wild-type sequence and competitor RNA are reverse-transcribed in the same tube, under identical reaction conditions, and with the same efficiency); (iv) use of random primers for reverse transcription is incorrect and should be avoided in cRT-PCR (in this reaction, identical RT-efficiency is ensured for both template species using the same primer); (v) not only concentration but also integrity of the competitor RNA used in the assay have to be carefully ascertained in cRT-PCR applications.

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Outlinesfor new competitor templates In cPCR and cRT-PCR, an initial and central aspect is the choice and construction of appropriate DNA or RNA competitors. Ideally, according to the general concept of competitive methods, the competitor templates (i) share identical primer recognition sites with the wild-type sequence to be quantified, (ii) have similar internal sequence, (iii) and similar (or identical) size. These characteristics ensure identical thermodynamics and amplification efficiency to both template species. Under these conditions, the well known function Y= Y' (1 + e)n (where Yis the product yield, Y' the starting sequence copy numbers, e the amplification efficiency, and n the number of cycles) is written for both templates amplified in cPCR as follows:

C'(I+e)"= C' W W'(l+e)"

W'

(where C and W are the product yield of competitor template and wild-type template, and C' and W' the initial amount of both template species, respectively); since e and n are identical in cPCR, the relative product ratio (C/W) directly depends on the initial concentration of both template species (C'/W') [24, 27]. Generally, different concentrations of competitors are challenged in different tubes against a constant amount of wild-type sample in order to cover the possible wild-type concentration range. Finally, the ratio of both values is plotted against the competitor copy numbers used in the assay. All this principally indicates that the choice of competitor templates requires the utmost care (Table 1A). Competitors with different length than that of the wild-type sequence were used extensively in cPCR. Competitor DNA templates may rapidly be generated by PCR-mediated insertion (or deletion) mutagenesis [27, 30, 44], quantified, and used directly avoiding cumbersome procedures of cloning in appropriate vectors; nevertheless, RNA competitors derived from transcription in vitro of mutated DNA fragments (cloned in appropriate expression vectors) are necessary for those applications aiming at quantifying viral mRNAs or cell-free genomes of RNA viruses (competitive RT-PCR; cRT-PCR) [10, 36, 62, 68, 78]. A commonly experienced advantage using competitors with different size than that of the wild-type template is that the amplified products can be recognized and quantified directly after gel electrophoresis and ethidium bromide staining. However, it was underlined [25] that great diversity in sequence length (higher that 10-15%) may give rise to significant differences in amplification efficiencies; for this principal reason, internal insertions or deletions have to be reduced to the minimum necessary for obtaining discrete bands of the PCR-products by gel electrophoresis. To minimize differences in amplification efficiency due to differences in size between the wild-type sequence and the competitor template, competitors bearing a novel restriction site have been proposed [38, 48, 69]. In this application, both template species have very close or identical size, and after PCR amplification, products are differentiated using digestion with the specific enzyme and finally quantified. However, this strategy

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Table 1. Characteristics of competitor templates (A) and strategies for detection and analysis of competitive amplification products (B) used in different cPCR- and cRT-PCR applications A. Types of RNA or DNA competitor templates

References

A. 1 Different size

internal deletion internal insertion

5, 44, 62, 68, 78, 79 27, 36, 61, 71

A.2 Similar or identical size

differentinternal sequence new restriction site

10, 82 38, 48, 69

B. Detection of cPCR (or cRT-PCR) products Gel electrophoresis

Hybridization

References ethidium bromide staining capillaryelectrophoresis

5, 6, 7, 27, 62, 65 68, 78, 79, 90 96

southern blotting liquid hybridization capture probes

106 82 10, 61,105

should be avoided because an additional step whose efficiency may vary unpredictably is introduced in the technique, and because precise quantitative analysis of digested amplification products may be difficult and imprecise. In some applications, competitors with similar or even identical size but different internal sequence may be necessary [10] as in the case where capture probes (and not gel electrophoresis) are used to separate the cPCR products. In these cases, careful attention and repeated testing are necessary to verify (not only theoretically, but also practically case-by-case) the relative influence of the internal modifications on amplification efficiency. Similarly, identity of the hybridization efficiency of the capture probes employed in the final steps of the procedure should carefully be ascertained. Of note, quantitation of competitor RNA or DNA deserves the utmost care. Precise evaluation of competitor concentration is achieved by gel electrophoresis, end-point dilution amplification performed on the basis of the spectrophotometric data, and final Poisson's analysis of the last dilution that gives positive scores after PCR or RT-PCR. The quantified competitor should also be challenged against a fixed amount of the wild-type template (previously quantified by end-point dilution and Poisson's distribution) by cPCR or cRT-PCR [68]. Possible obstacles to a correct use o f competitive PCR-based assays

At present, the effect of RNA purification on template recovery is the only step that remains uncontrolled in competitive PCR-based methods (as well as in all qualitative and quantitative molecular procedures). Quantitative methods allow direct evaluation of the mean loss of template copy numbers that follows any

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extraction procedure; this evaluation may easily be performed by first quantitation of viral specific nucleic acids after purification, reextraction, and second quantitative analysis [62, 68]. However, sample-to-sample diverging loss of nucleic acids (albeit minimal, lower than 5% in our experience) is possible using clinical specimens from different infected individuals [5]. More recently, a replication-competent mutant HIV-1 (25bp insert) was generated and used as an internal control in an application of the competitive strategy [71]; interestingly, using this mutant virus (which is added to the sample at known amount before RNA extraction) possible variability of RNA recovery during purification steps should be normalized, thus increasing, in theory, the accuracy of the competitive assay. An additional, important aspect of the optimization of quantitative methodologies used in diagnostic applications is the choice of the more appropriate clinical specimen; in a comparative quantitative analysis of serum and plasma samples of HCV infected patients, a highly variable loss of cell-free HCV genome molecules was observed in all serum samples [62], thus indicating that (although widely used in qualitative studies) serum specimens are totally inadequate for a correct quantitation of cell-free virus in HCV infection. Finally, general measures to prevent product contamination (in particular due to competitor products) are clearly necessary in competitive PCR-based methods, and they should be followed with the utmost care. Negative samples (for both template species, wild-type and competitor templates) and reagents controls are necessary in each run.

Analysis of competitive reactions According to the general concept ofcPCR, the final step requires quantitation of both amplification products (the wild-type and competitor template amplicons, respectively). Similarly to qualitative PCR-applications, quantitative cPCR and cRT-PCR assays allow amplification products to be visualized (and subsequently quantified) using different procedures (Table 1B): hybridization (liquid phase hybridization, Southern blotting, capture probes), gel electrophoresis and ethidium bromide staining, or capillary electrophoresis. Densitometric evaluation of ethidium bromide-stained gels makes the final step easier, faster and more direct than Southern hybridization or other procedures. Gels are generally scanned using a densitometer either directly (by positive fluorescent emission on the transilluminator) or after photography. The peak areas of both amplified products may be calculated, but, when deleted competitor or a competitor with a small insertion is used, the corresponding value has to be corrected for the different levels of ethidium bromide incorporation [68]. Preliminary data [96] suggest that capillary electrophoresis may be a suitable method for precise final analysis of cPCR products; with this technique, one-step evaluation and quantitation of the cPCR products without staining is possible. Other procedures (with or without capture of competitive amplification products by hybridization and colorimetric quantitation of the amplicons) are proposed

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commercially; generally, these methods (aiming at making simple the final detection of cPCR products) introduce unnecessary additional steps that may significantly hamper the reliability and reproducibility performances of the technique.

Other (PCR-based and non-PCR) quantitative molecular methods Semi-quantitative analysis of nucleic acids present in biological specimens is possible by conventional hybridization methods (Southern- and Northernblotting, dot-blot, and spot-hybridization). However, a major obstacle to a wide use of these techniques in basic and clinical virology is their low sensitivity in most cases. Recently, a novel hybridization procedure (named branched DNA; bDNA) was developed; in this technique, sensitivity was increased using a branched hybridization procedure for signal amplification [100]. More recently, this procedure was applied to the direct quantitation of cell-free HIV-1 and HCV genomic RNA molecules in serum samples. In these virological applications of bDNA, viral particles present in serum are incubated with lysis buffer and specific probes; the viral RNA-probe complex is then incubated in microplate wells in the presence of a second (solid-phase absorbed) probe. The immobilized target is hybridized by using multiple copies of probes labeled with alkaline phosphatase and then incubated with a chemiluminescent substrate. Finally, light emission is measured by a luminometer and compared to the data obtained using standard samples. Specific applications of the bDNA assay system were developed for studying HIV-1 [99] and HCV [19, 93] infected patients under therapy with specific anti-viral compounds. Independently of the accuracy of the absolute quantitation using this assay system (which still requires thorough evaluation since, in theory, small differences of hybridization efficiency may lead to gross final differences after the signal amplification procedure of bDNA), major limits ofbDNA are, at present, low sensitivity [59, 60] and poor flexibility in the analysis of different specific viral substrates. The combination of in situ PCR and flow cytometry analysis was principally proposed for a quantitative evaluation of the frequency of peripheral blood mononuclear cells (PBMCs) infected with HIV- 1 [80, 104]. Although very easy to perform, heavy doubts on the specificity performances of these and other in situ PCR applications still remain. In all these applications, a simple negative control including negative (uninfected) PBMCs and spikes of previously amplified extracellular, HIV-1 specific DNA molecules should be necessary; under these conditions, lack of intracellular signal should confirm the result's specificity. In the last few years, amplification procedures other than PCR were also developed [37, 55, 103]; one of these methodologies (named nucleic acid sequence-based amplification; NASBA) is carried out isothermally using three enzymes (avian mieloblastosis virus reverse transcriptase, T7RNA polymerase, and RNase H) and was recently applied to specifically detect HIV-1 RNA molecules in plasma samples and HIV-1 transcriptional activity [13]. More recently, a quantitative adaptation of this technique was commerciallyproposed

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using internal calibrators and evaluation of the relative amounts of amplificate products. Molecular analysis of viral activity in vivo

Qualitative or quantitative molecular methods? Recently, epidemiological, diagnostic, and pathogenic studies of different viral infections were carried out at the molecular level [4, 21, 31, 33, 39, 43, 46, 58, 75, 98]; in most cases, these studies supplied more complete and (probably) more precise data than those obtained by viral isolation or immunological methods. In particular, RT-PCR applications allowed specific viral transcripts to be detected in vivo by highly sensitive procedures [4, 70, 91]. However, insufficient information on the level of viral gene expression was generally provided by assays indicating the mere presence or absence of specific viral transcripts in infected cells. More recently, sharp molecular evidence suggested that the viral evolutionary potential is much greater than once thought in most persistent viral infections. It was observed that a virus infecting a host can no longer be considered a strain in many circumstances, but rather an heterogeneous population of genomically evolving viruses named quasispecies [16, 26, 28, 29, 54, 77, 95]. From this point of view, it was hypothesized that viruses (principally RNA, but also D N A viruses in some cases) are engaged in an evolutionary race against host factors during persistence [11, 15, 18, 22, 63, 72, 77, 88, 95]. This genetic variability and the following (possible) selection of biologically diverse viral variants means that most of the major viral properties may change with time, including regulatory functions [52] (and consequently viral gene expression and replication), and drug resistance [49, 57, 97]. Quantitative molecular methods and HIV-1 infection In HIV-1 infection, biological and molecular data underlined the importance of reliable quantitative analysis of viral activity and viral load directly in vivo. Results of PCR studies showed unambiguously that HIV-1 genomic R N A sequences in plasma samples [3], specific viral transcripts in peripheral CD4+ T-lymphocytes [4, 39, 91], and proviral D N A sequences [43, 92] are detectable in almost all HIV-1-infected individuals regardless of the presence of clinical symptoms and drop of CD4+ peripheral T-cells. Although the results did not exclude the possibility that a real latency may occur at the level of individual infected cells [85], these data indicated that, in HIV-1 infected patients, the virological counterpart of clinical progression from the asymptomatic state to clinical disease is not an on/off switch from latency to virus expression, but rather a slow increase in mean HIV-1 transcriptional activity rate and viral replication. Recently, we and others studied the HIV- 1 infection in vivo by using quantitative cRT-PCR methods [5, 6, 78] and obtained data on HIV- 1 viremia levels in different clinical conditions. The quantitative results obtained in these transsectional studies demonstrated that the molecular parameters of viral activity are significantly correlated with disease progression and fall of CD4+ T-lymphocyte

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levels [5, 78]. Interestingly, monitoring of HIV-1 load in untreated infected patients as a function of the natural history of this infection revealed that increasing viral load is significantly correlated to progressive immunosuppression and presence of clinical symptoms [6], thus suggesting quantitation of the HIV-1 activity levels may be the key for the precise understanding of the events related to clinical progression in these patients. Quantitative analysis of the very early phase (primary infection) of the HIV-1 infection was recently performed; preliminary data [6, 23] depicted the profile of viral load (drop of both viral genome copy numbers in plasma and specific HIV-1 transcripts in PBMCs) occurring at serocoversion in these patients and underlined the residual HIV-1 activity during the clinical latency. In particular, two pilot studies of long-term non-progressor patients were carried out using this competitive strategy [8, 76] and demonstrated that low (but persistent) HIV-1 viremia is present in these subjects (most of whom tested negative using viral isolation from plasma samples) at stable levels for several years; otherwise, increasing HIV-1 viremia levels were observed in normal or rapid progressors. More recently two pathogenic studies [42, 102] used quantitative molecular methods to evaluate the turnover of HIV-1 virions infected individuals, thus supplying further evidence of the importance of reliable quantitative methodologies in different virological applications. Quantitative molecular methods and HB V and HC V infections

A major aspect that calls for quantitative methods in virology emerges from the study of the persistent HBV infection. Quantitative PCR analysis of HBV viremia in samples from asymptomatic and symptomatic HBV infected patients was carried out extensively; significantly, these studies (in partial contrast with the data obtained using conventional hybridization procedures) indicated that a high proportion of these patients are viremic [33, 64] and suggested that quantitative methods for the precise evaluation of HBV DNA levels in serum samples [61] are important for the pathogenic investigation of chronic HBV liver disease. Similarly, it was observed that a correct approach to the natural history of HCV infection requires quantitative molecular methods for HCV RNA detection in tissues, or blood samples. Semi-quantitative PCR-based studies were initiallyemployed to study mother-to-infant transmission of the HCV infection [73], response to therapies with ~- and 13-interferon [12, 20, 32], the role of HCV in auto-immune extrahepatic diseases [1, 17], and the procedures adopted to eliminate viral particles from blood and blood-products [87]. More recently, different cRT-PCR assays were developed for quantifying HCV RNA molecules in biological samples [10, 36, 38, 47, 48, 53, 62, 69, 82]; these studies aimed at evaluating the levels of HCV viremia in infected patients with different (clinically and histologically defined) liver disease. According to the results obtained by these applications, several authors suggested that a direct correlation exists between cell-free HCV RNA levels in plasma samples and liver damage [36, 48, 69].

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Quantitative molecular methods and other viral infections To evaluate aspects of different acute and persistent viral infections, quantitative molecular strategies were developed and used during in vivo and in vitro researches. In particular, competitive RT-PCR methods were developed to determine hepatitis A virus (HAV) [35] and Coxsackievirus [65] R N A copy numbers in biological samples. Similarly, uterine cervical tissues from patients with human papillomavirus type 16 (HPV 16) infection were assayed by cPCR to determine HPV D N A copy numbers in different histological lesions [51]. Latency of herpes simplex virus type 1 (HSV-1) was investigated in vitro by cPCR and cRT-PCR [81]; in this study, both viral D N A copy numbers and latency-associated viral transcript molecules were quantified in rat brain ceils infected with attenuated HSV mutants. Furthermore, quantitative molecular methods were planned for studying infections with a different human herpesvirus in vivo, the human cytomegalovirus (HCMV). In fact, quantitation of HCMV D N A molecules in clinical samples is a general requirement for successful monitoring of HCMV infection in immunocompromised hosts and during therapies. Recently, different cPCR strategies were planned and used for quantification of HCMV D N A copy numbers in PBMCs ofimmunocompromised patients [90, 106]. Finally, a cPCR method for feline immunodeficiency virus (FIV) proviral D N A was recently optimized and used with the aim of evaluating the relative distribution of viral burden in the different tissues of infected hosts [79].

Molecular monitoring of specific anti-viral therapies Availability of quantitative methods for reliable analysis of viral load directly in vivo supplied a theoretical and practical basis for monitoring the efficacy of specific anti-viral compounds. During anti-viral treatments, quantitative techniques can supply information on both efficacy of therapies and selection of drugresistant viral variants in real time as documented in HIV-1 infection [7, 8, 67, 102]; the impact of this aspect might be dramatic in the near future in medical virology laboratories, and this is particularly true if an increasing number of specific antiviral compounds or new efficient strategies for the treatment of viral infections (such as gene therapy) will be introduced in clinical practice as expected. In HCV infection, a large proportion of patients are currently treated for several months with preparations of or- and 13-interferon (IFN) of different origin (natural or recombinant); in this case, a major question is whether the treatment can lead to virus eradication from infected hosts in the presence of sustained biochemical and histological improvement (so-called "long-term responders" to IFN). Conflicting results were recently reported using qualitative or semi-quantitative analysis of HCV viremia in patients under-anti-viral therapy; persistence of cellfree HCV R N A in blood was shown by some authors [14, 45, 89] whereas others report that long term response to IFN is unvaryingly associated with complete clearance of HCV R N A [83, 84]. This aspect should be clarified in the near future to evaluate precisely the real clinical efficacy of this very expensive therapy.

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The key role of quantitative virological methods in the near future Most of the aspects summarized here suggest that availability of quantitative molecular methods extends significantly the diagnostic potential of medical virology laboratories and allows pathogenicity studies to be carried out directly in vivo. Clearly, further technical improvement and optimization is necessary for a wide use of these methods since most of these techniques are presently cumbersome and can be used efficiently in experienced laboratories only. However, theoretical aspects and practical evidence recently indicated that appropriate molecular strategies may efficiently be used in virology as well as in all those biological applications where absolute quantitation of nucleic acids present in low amounts in biological samples is necessary.

Acknowledgements The authors' work in this field is supported by grants from the Italian "Ministero dell'Universit~ e della Ricerca Scientifica e Tecnologica" (MURST), "Consiglio Nazionale delle Ricerche" (C.N.R.), and "Ministero della Sanit/t" (VIII AIDS Project).

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