The fluorescence-based real-time reverse transcription PCR (RT-PCR) is widely used for the quantification of steady-state mRNA levels and is a critical tool for basic research, molecular medicine and biotechnology. Assays are easy to perform, capable of high throughput, and can combine high sensitivity with reliable specificity. The technology is evolving rapidly with the introduction of new enzymes, chemistries and instrumentation. However, while real-time RT-PCR addresses many of the difficulties inherent in conventional RT-PCR, it has become increasingly clear that it engenders new problems that require urgent attention. Therefore, in addition to providing a snapshot of the state-of-the-art in real-time RT-PCR, this review has an additional aim: it will describe and discuss critically some of the problems associated with interpreting results that are numerical and lend themselves to statistical analysis, yet whose accuracy is significantly affected by reagent and operator variability.

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Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems.

Since the discovery of interferons (IFNs), considerable progress has been made in describing the nature of the cytokines themselves, the signalling components that direct the cell response and their antiviral activities. Gene targeting studies have distinguished four main effector pathways of the IFN-mediated antiviral response: the Mx GTPase pathway, the 2',5'-oligoadenylate-synthetase-directed ribonuclease L pathway, the protein kinase R pathway and the ISG15 ubiquitin-like pathway. As discussed in this Review, these effector pathways individually block viral transcription, degrade viral RNA, inhibit translation and modify protein function to control all steps of viral replication. Ongoing research continues to expose additional activities for these effector proteins and has revealed unanticipated functions of the antiviral response.

Interferon-inducible antiviral effectors

Folk wisdom has long suggested that stressful events take a toll on health. The field of psychoneuroimmunology (PNI) is now providing key mechanistic evidence about the ways in which stressors--and the negative emotions that they generate--can be translated into physiological changes. PNI researchers have used animal and human models to learn how the immune system communicates bidirectionally with the central nervous and endocrine systems and how these interactions impact on health.

Stress-induced immune dysfunction: implications for health.

The use of the polymerase chain reaction (PCR) in molecular diagnostics has increased to the point where it is now accepted as the gold standard for detecting nucleic acids from a number of origins and it has become an essential tool in the research laboratory. Real-time PCR has engendered wider acceptance of the PCR due to its improved rapidity, sensitivity, reproducibility and the reduced risk of carry-over contamination. There are currently five main chemistries used for the detection of PCR product during real-time PCR. These are the DNA binding fluorophores, the 5' endonuclease, adjacent linear and hairpin oligoprobes and the self-fluorescing amplicons, which are described in detail. We also discuss factors that have restricted the development of multiplex real-time PCR as well as the role of real-time PCR in quantitating nucleic acids. Both amplification hardware and the fluorogenic detection chemistries have evolved rapidly as the understanding of real-time PCR has developed and this review aims to update the scientist on the current state of the art. We describe the background, advantages and limitations of real-time PCR and we review the literature as it applies to virus detection in the routine and research laboratory in order to focus on one of the many areas in which the application of real-time PCR has provided significant methodological benefits and improved patient outcomes. However, the technology discussed has been applied to other areas of microbiology as well as studies of gene expression and genetic disease.

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Real-time PCR in virology

The action of interferons (IFNs) on virus-infected cells and surrounding tissues elicits an antiviral state that is characterized by the expression and antiviral activity of IFN-stimulated genes. In turn, viruses encode mechanisms to counteract the host response and support efficient viral replication, thereby minimizing the therapeutic antiviral power of IFNs. In this review, we discuss the interplay between the IFN system and four medically important and challenging viruses -- influenza, hepatitis C, herpes simplex and vaccinia -- to highlight the diversity of viral strategies. Understanding the complex network of cellular antiviral processes and virus-host interactions should aid in identifying new and common targets for the therapeutic intervention of virus infection. This effort must take advantage of the recent developments in functional genomics, bioinformatics and other emerging technologies.

Viruses and interferon: a fight for supremacy

Following ocular infection, herpes simplex virus type 1 (HSV-1) establishes latency in trigeminal ganglion (TG) neurons. Using reverse transcription-PCR, cytokine gene expression was analyzed in the TGs of mice infected with HSV-1. IL-2, TNF-alpha, IFN-gamma, IL-10, and RANTES mRNAs were readily detected in TGs taken from mice 7 days postinoculation (PI). Likewise, IL-2, IL-6, IL-10, and IFN-gamma protein were detected by ELISA of TG homogenates. Between 5 and 45 days PI, IL-10, IFN-gamma, TNF-alpha, and RANTES mRNAs were detected in nearly 100% of latently infected TGs (latent infection was confirmed by reverse transcription-PCR detection of HSV-1 latency-associated transcripts). T cell-associated cytokine and chemokine mRNAs (IL-2, IL-10, IFN-gamma, and RANTES) were still detected in the majority of latently infected TG samples taken between 60 and 135 days PI. In contrast, these cytokine mRNA species were rarely detected in uninfected TGs. Measurement of serum Abs to HSV-1 at different times revealed that anti-HSV-1 Ab concentrations approached a plateau in mice by 30 days PI but remained at high levels 67 and 125 days PI. Although there was molecular evidence of an ongoing immune response to HSV-1 in latently infected TG, histologic analysis indicated that very few mononuclear cells remained in the ganglion 60 days PI. Collectively, the results suggest that residual lymphocytes encounter viral Ag during HSV-1 latency with sufficient frequency to remain activated. The paradox of a persistent immune response against a latent infection is discussed.

Persistent cytokine expression in trigeminal ganglion latently infected with herpes simplex virus type 1.

Relative to wild-type herpes simplex virus type 1 (HSV-1), ICP0-null mutant viruses reactivate inefficiently from explanted, latently infected mouse trigeminal ganglia (TG), indicating that ICP0 is not essential for reactivation but plays a central role in enhancing the efficiency of reactivation. The validity of these findings has been questioned, however, because the replication of ICP0-null mutants is impaired in animal models during the establishment of latency, such that fewer mutant genomes than wild-type genomes are present in latently infected mouse TG. Therefore, the reduced number of mutant viral genomes available to reactivate, rather than mutations in the ICP0 gene per se, may be responsible for the reduced reactivation efficiency of ICP0-null mutants. We have recently demonstrated that optimization of the size of the ICP0 mutant virus inoculum and transient immunosuppression of mutant-infected mice with cyclophosphamide can be used to establish wild-type levels of ICP0-null mutant genomes in latently infected TG (W. P. Halford and P. A. Schaffer, J. Virol. 74:5957-5967, 2000). Using this procedure to equalize mutant and wild-type genome numbers, the goal of the present study was to determine if, relative to wild-type virus, the absence of ICP0 function in two ICP0-null mutants, n212 and 7134, affects reactivation efficiency from (i) explants of latently infected TG and (ii) primary cultures of latently infected TG cells. Although equivalent numbers of viral genomes were present in TG of mice latently infected with either wild-type or mutant viruses, reactivation of n212 and 7134 from heat-stressed TG explants was inefficient (31 and 37% reactivation, respectively) relative to reactivation of wild-type virus (KOS) (95%). Similarly, n212 and 7134 reactivated inefficiently from primary cultures of dissociated TG cells plated directly after removal from the mouse (7 and 4% reactivation, respectively), relative to KOS (60% reactivation). The efficiency and kinetics of reactivation of KOS, n212, and 7134 from cultured TG cells (treated with acyclovir to facilitate the establishment of latency) in response to heat stress or superinfection with a nonreplicating HSV-1 ICP4(-) mutant, n12, were compared. Whereas heat stress induced reactivation of KOS from 69% of latently infected TG cell cultures, reactivation of n212 and 7134 was detected in only 1 and 7% of cultures, respectively. In contrast, superinfection with the ICP4(-) virus, which expresses high levels of ICP0, resulted in the production of infectious virus in nearly 100% of cultures latently infected with KOS, n212, or 7134 within 72 h. Thus, although latent mutant viral genome loads were equivalent to that of wild-type virus, in the absence of ICP0, n212 and 7134 reactivated inefficiently from latently infected TG cells during culture establishment and following heat stress. Collectively, these findings demonstrate that ICP0 is required to induce efficient reactivation of HSV-1 from neuronal latency.

ICP0 Is Required for Efficient Reactivation of Herpes Simplex Virus Type 1 from Neuronal Latency

Green fluorescent protein (GFP) has gained widespread use as a tool to visualize spatial and temporal patterns of gene expression in vivo. However, it is not generally accepted that GFP can also be used as a quantitative reporter of gene expression. We report that GFP is a reliable reporter of gene expression in individual eukaryotic cells when fluorescence is measured by flow cytometry. Two pieces of evidence support this conclusion: GFP fluorescence increases in direct proportion to the GFP gene copy number delivered to cells by a replication-defective adenovirus vector, Ad.CMV-GFP, and the intensity of GFP fluorescence is directly proportional to GFP mRNA abundance in cells. This conclusion is further supported by the fact that the induction of GFP gene expression from two inducible promoters (i.e., the TRE and ICP0 promoters) is readily detected by flow cytometric measurement of GFP fluorescent intensity. Collectively, the results presented herein indicate that GFP fluorescence is a reliable and quantitative reporter of underlying differences in gene expression.

Green fluorescent protein is a quantitative reporter of gene expression in individual eukaryotic cells.

In vivo evidence suggests that T-cell-derived gamma interferon (IFN-γ) can directly inhibit the replication of herpes simplex virus type 1 (HSV-1). However, IFN-γ is a weak inhibitor of HSV-1 replication in vitro. We have found that IFN-γ synergizes with the innate IFNs (IFN-α and -β) to potently inhibit HSV-1 replication in vitro and in vivo. Treatment of Vero cells with either IFN-β or IFN-γ inhibits HSV-1 replication by

Alpha/Beta Interferon and Gamma Interferon Synergize To Inhibit the Replication of Herpes Simplex Virus Type 1

Primary cultures of trigeminal ganglion (TG) cells from herpes simplex virus type 1 (HSV-1) latently infected mice were used to study reactivation. Expression of HSV-1 latency-associated transcripts was noted in TG cell cultures. Infectious virus appeared in 75% of culture supernatants within 120 h after heat stress. Likewise, HSV-1 lytic-phase mRNA and proteins were detectable 24 h after heat stress. HSV-1 antigen first appeared in neurons after heat stress, indicating the neurons were the source of reactivation. The effect of heat stress duration on reactivation was determined. Reactivation occurred in 0, 40, or 67% of cultures after a 1-, 2-, or 3-h heat stress, respectively. However, 72-kDa heat shock protein expression was induced regardless of heat stress duration. Thus, reactivation was not a direct result of inducing the heat shock response. The capacities of several drugs to induce reactivation were also evaluated. While neither epinephrine, forskolin, nor a membrane-permeable cyclic AMP analog induced reactivation, dexamethasone did so in a dose-dependent manner. Furthermore, dexamethasone pretreatment enhanced the kinetics of heat stress-induced reactivation from TG cells. Collectively, the results indicate that TG cell cultures mimic important aspects of in vivo latency and reactivation. Therefore, this model may be useful for studying signalling pathways that lead to HSV-1 reactivation.

William P. Halford

Papers

Persistent cytokine expression in trigeminal ganglion latently infected with herpes simplex virus type 1.

ICP0 Is Required for Efficient Reactivation of Herpes Simplex Virus Type 1 from Neuronal Latency

Green fluorescent protein is a quantitative reporter of gene expression in individual eukaryotic cells.

Alpha/Beta Interferon and Gamma Interferon Synergize To Inhibit the Replication of Herpes Simplex Virus Type 1

Mechanisms of herpes simplex virus type 1 reactivation.