Pilot-scale adenovirus seed production through concurrent virus release and concentration by hollow fiber filtration

Recovery of recombinant adenoviruses from infected mammalian cell cultures often requires multiple unit operations such as cell lysis for virus release, microfiltration for clarification, and ultrafiltration for concentration. While development of these multiple unit operations is relatively straightforward, implementation under aseptic conditions in a closed system can be challenging for the production of virus seed at industrial scales. In this study, we have developed a simple, single-step, scaleable process to effectively recover adenoviruses from infected PER.C6 cell cultures for the production of concentrated adenovirus seeds under aseptic conditions. Specifically, hollow fiber tangential flow filtration technology was applied to maximize cell lysis of infected cultures for virus release while simultaneously concentrating the virus to an appropriate level of volume reduction. Hollow fiber filters with small lumen diameter of 0.5 mm were chosen to maximize the wall shear for a highly effective cell lysis and virus release. Cell lysis and virus release were shown to correlate with the exposure time in the hollow fiber cartridge: the shear zone. In most cases, a virus recovery yield of more than 80% and a 15- to 20-fold concentration (or up to 95% volume reduction) was achieved in less than 2 h of processing time. The virus seeds prepared using this process at lab scale and at 300-L scale without clarification have been successfully tested for sterility and potency and used for subsequent infection with consistent virus productivity. This process should enable rapid production of adenovirus seeds with minimal unit operations and high efficiency recovery for adenovirus production at 1000-L scale.     

Viral probe into the events of cellular (in vitro) aging

Viral infection by vesicular stomatitis virus (VSV) as well as cellular protection against it were studied in cultured human diploid fibroblast cells (WI38 strain) of varying senescence (passage) level. Full protection against viral infection can be achieved by pretreatment of cells with an interferon inducer (complex of polycytidylate and polyinosinate) or by pretreatment with concentrates of interferon itself. The late passages (over 45) require higher concentration of both agents than the medium passages (25–45); a complete failure of protective mechanisms occurs only close to cellular death. Furthermore, effects of confluency and of duration of induction impulse were studied. WI38 cells are sensitive to VSV infection through their entire life span; however, during the last passage before their death by senescence, VSV replication is significantly decreased. It is concluded that even in relatively senescent cells (a) protection mechanisms which were not used previously can be activated, (b) synthesizing machinery necessary for efficient support of viral replication is sustained, and (c) release of lysosomal enzymes ending the viral replication does not begin sooner than with cells of earlier passage level.     

Phage formation in Staphylococcus muscae cultures. X. The relationship between virus synthesis,the release of bacterial ribonucleic acid,virus liberation,and cellular lysis

1. Under a variety of conditions in which cells are infected with one or a few virus particles and the host cells are killed, but no infective particles or virus material is formed as indicated by plaque count, one-step growth curve, or protein or desoxyribonucleic determinations, the cells neither lyse nor release ribonucleic acid into the medium. 2. The "killing" effect of S. muscae phage is separate from its lytic property. 3. The release of ribonucleic acid into the medium is not simply due to the killing of the cell by the virus, and ribonucleic acid is never found in the medium unless virus material is synthesized. 4. Infected cells of S. muscae synthesizing virus release ribonucleic acid into the medium before cellular lysis begins and before any virus is liberated. 5. The higher the phage yield the more ribonucleic acid is released into the medium before any virus is released. 6. Phage may be released from one strain of Staphylococcus muscae without cellular lysis, although bacterial lysis begins shortly after the virus is released. In another strain, infected under similar conditions, virus liberation occurs simultaneously with cellular lysis. 7. The viruses liberated from both bacterial strains appear to be the same in so far as they cannot be distinguished by serological tests, have the same plaque type and plaque size, and need the same amino acids added to the medium in order to grow. Furthermore, the virus liberated from one strain can infect and multiply in the other strain and vice versa. 8. It is suggested that virus synthesis, in S. muscae cells infected with one or a few phage particles, leads to a disturbance of the normal cellular metabolism, resulting in lysis of the host cell.     

Adenovirus Death Protein (ADP) Is Required for Lytic Infection of Human Lymphocytes

The adenovirus death protein (ADP) is expressed at late times during a lytic infection of species C adenoviruses. ADP promotes the release of progeny virus by accelerating the lysis and death of the host cell. Since some human lymphocytes survive while maintaining a persistent infection with species C adenovirus, we compared ADP expression in these cells with ADP expression in lymphocytes that proceed with a lytic infection. Levels of ADP were low in KE37 and BJAB cells, which support a persistent infection. In contrast, levels of ADP mRNA and protein were higher in Jurkat cells, which proceed with a lytic infection. Epithelial cells infected with an ADP-overexpressing virus died more quickly than epithelial cells infected with an ADP-deleted virus. However, KE37, and BJAB cells remained viable after infection with the ADP-overexpressing virus. Although the levels of ADP mRNA increased in KE37 and BJAB cells infected with the ADP-overexpressing virus, the fraction of cells with detectable ADP was unchanged, suggesting that the control of ADP expression differs between epithelial and lymphocytic cells. When infected with an ADP-deleted adenovirus, Jurkat cells survived and maintained viral DNA for greater than 1 month. These findings are consistent with the notion that the level of ADP expression determines whether lymphocytic cells proceed with a lytic or a persistent adenovirus infection.     

Immediate-early gene expression is sufficient for induction of natural killer cell-mediated lysis of herpes simplex virus type 1-infected fibroblasts.

Herpes simplex virus type 1 (HSV-1)-infected human fibroblast (HSV-FS) targets are susceptible to lysis by natural killer (NK) cells, whereas uninfected FS are resistant to lysis. Studies were undertaken to determine the mechanism of this preferential susceptibility. HSV-FS were not intrinsically less stable than FS, as determined by a 51Cr release assay under hypotonic shock in the presence of rat granule cytolysin and by sensitivity to anti-human leukocyte antigen class I antibody plus complement. Single-cell assays in agarose demonstrated that although similar numbers of large granular lymphocytes bound to the HSV-FS and FS targets, the conjugates with HSV-FS were lysed at a much higher frequency than those with FS. These results suggested that both targets are bound by the NK cells but only the HSV-FS were able to trigger lysis. The requirement for active virus expression was demonstrated by failure of emetine-treated HSV-FS targets or targets infected with UV-inactivated HSV to be lysed by NK effectors. To evaluate the role of viral glycoproteins in conferring susceptibility to lysis, Fab were prepared from HSV-1-seropositive sera; these Fab were unable to block lysis of the HSV-FS. Furthermore, incubation in phosphonoacetic acid failed to reduce NK(HSV-FS) activity despite sharp reductions in viral glycoprotein synthesis. Finally, targets infected with tsLB2 at the nonpermissive temperature were lysed as well as or better than targets infected with wild-type virus, indicating that HSV immediate-early gene product expression is sufficient for conferring susceptibility to lysis. We conclude that expression of nonstructural viral proteins or virally induced cellular gene products early in the course of infection rather than structural glycoproteins is required for NK lysis of HSV-FS targets.     

Role of the pneumococcal autolysin (murein hydrolase) in the release of progeny bacteriophage and in the bacteriophage-induced lysis of the host cells.

The pneumococcal bacteriophage Dp-1 seems to require the activity of the N-acetylmuramic acid-L-alanine amidase of the host bacterium for the liberation of phage progeny into the medium. This conclusion is based on a series of observations indicating that the exit of progeny phage particles is prevented by conditions that specifically inhibit the activity of the pneumococcal autolysin. These inhibitory conditions are as follows: (i) growth of the bacteria on ethanolamine-containing medium; (ii) growth of the cells at pH values that inhibit penicillin-induced lysis of pneumococcal cultures and lysis in the stationary phase of growth; (iii) addition of trypsin or the autolysin-inhibitory pneumococcal Forssman antigen (lipoteichoric acid) to the growth medium before lysis; (iv) infection of an autolysin-defective pneumococcal mutant at a multiplicity of infection less than 10 (treatment of such infected mutant bacteria with wild-type autolysin from without can liberate the entrapped progeny phage particles); (v) release of phage particles and culture lysis can also be inhibited by the addition of chloramphenicol to infected cultures just before the time at which lysis would normally occur. Bacteria infected with Dp-1 under conditions nonpermissive for culture lysis and phage release secrete into the growth medium a substantial portion of their cellular Forssman antigen in the form of a macromolecular complex that has autolysin-inhibitory activity. We suggest that a phage product may trigger the bacterial autolysin by a mechanism similar to that operating during treatment of pneumococci with penicillin (Tomasz and Waks, 1975).     

Peptidoglycan Loss During Hen Egg White Lysozyme-Inorganic Salt Lysis of Streptococcus mutans

Streptococcus mutans BHT was grown in Todd-Hewitt dialysate medium containing N-acetyl[¹⁴C]glucosamine for 6 to 11 generations. After treatment with cold and hot trichloroacetic acid and trypsin, 52 to 65% of the radioactivity remained present in insoluble peptidoglycan-containing residues. Hen egg white lysozyme or mutanolysin treatment of the peptidoglycan residues resulted in the release of 80 and 97%, respectively, of the ¹⁴C label to the supernatant fraction. Hydrochloric acid hydrolysates of such supernatants showed that essentially all of the radioactivity present in insoluble peptidoglycan fractions was present in compounds that comigrated on paper chromatography with glucosamine (~60%) or muramic acid (~30%). Treatment of whole cells with low and high concentrations of lysozyme alone resulted in losses of 45 and 70% of the insoluble peptidoglycan, respectively, yet release of deoxyribonucleic acid from cells was not detected. Sequential addition of appropriate concentrations of selected inorganic salts after lysozyme treatment did result in the liberation of deoxyribonucleic acid. Deoxyribonucleic acid release was correlated with a further release of peptidoglycan from the insoluble fraction. However, the total amount of peptidoglycan lost effected by the low concentration of lysozyme and NaSCN (lysis) was significantly less than the amount of peptidoglycan hydrolyzed by high concentrations of lysozyme alone (no lysis), suggesting that the overall amount of peptidoglycan lost did not correlate well with cellular lysis. The total amount of insoluble peptidoglycan lost at the highest salt concentrations tested was found to be greater than could be accounted for by lysozyme-sensitive linkages of the peptidoglycan, possibly implicating autolysins. The results obtained suggested that hydrolysis of peptidoglycan bonds in topologically localized, but strategically important, sites was a more significant factor in the sequence that results in loss of cellular integrity (lysis).     

Injury-induced release of basic fibroblast growth factor from bovine aortic endothelium

Although the basic fibroblast growth factor (bFGF) gene lacks a traditional consensus signal peptide domain indicative for secretion, many cell types have receptors for bFGF. Since endothelium is a rich source of cell-associated bFGF, we asked under what conditions could bFGF be released or secreted from confluent cultures of bovine aortic endothelial (BAE) cells. The level of bFGF in BAE cell lysates was compared with the level of heparin-releasable bFGF in intact BAE cell monolayers, intact cells with exposed extracellular matrix (nonlytic matrices), and extracellular matrices prepared by cell lysis (lytic matrices). Less than 10% of total cell-associated bFGF was released from intact cell monolayers and nonlytic matrices. In contrast, the levels of bFGF released from lytic matrices depended upon the conditions used to prepare the matrices. Cell lysis at neutral pH generated matrices that released the highest bFGF levels (approximately 50% of total cell-associated bFGF). These matrices were heavily contaminated by histones, indicating the cellular release and adsorption of intracellular proteins to the matrix. Matrices prepared by BAE cell exposure to basic pH (100 mM NH4OH) contained low bFGF content and minor histone contamination. These latter matrices were chosen to study bFGF sequestration, under physiological conditions, into the extracellular matrix of confluent BAE cell cultures. Incubation with endotoxin, an agent acutely toxic to BAE cells, resulted in cellular release and adsorption of endogenous bFGF to cells and matrices, accompanied by histone deposition in the matrices. These results suggested that one mechanism for bFGF release from BAE cell monolayers was passive release induced by severe cell injury and/or cell lysis with secondary adsorption to the matrix.     

Cytomegalovirus Replication in Cells Pretreated with 5-Iodo-2′-Deoxyuridine

The replication of human cytomegalovirus (CMV) in cells pretreated with 5-iodo-2′-deoxyuridine (IUdR) was studied. Pretreatment of cells with IUdR enhanced several parameters of virus replication. Virus grown in drug-treated cells exhibited a shorter eclipse period and the cells produced more infectious virus sooner than did untreated cells. There was an approximate fivefold increase in virus yield per cell in the drug-treated samples when compared to control cultures. The time required for plaque development was shortened by 6 days in drug-treated cultures. Pretreatment of cells with IUdR also increased plaquing efficiency of the virus by approximately 10-fold. The enhancement of virus replication by IUdR was further demonstrated by varying the multiplicity of infection. In a 7-day period there was a 100-fold increase in sensitivity of the cultures for virus detection when the cells had been previously exposed to IUdR. The data presented indicate the possibility that IUdR interferes with the production of a cellular product inhibitory for CMV replication.     

In vitro generation of cytotoxic lymphocytes against radiation and radiation leukemia virus-induced tumors

Summary Cytotoxic T lymphocytes (CTL) to syngeneic radiation- or radiation leukemia virus (RadLV)-induced tumors were generated in vitro in mixed lymphocytetumor cultures (MLTC) using splenocytes of mice primed in vivo with inactivated tumor cells. Effective sensitization was obtained with virus-producer cell lines, while cells of a virus-nonproducer line did not sensitize.The CTL could lyse syngeneic, but not allogeneic, tumor cells of established lines producing C-type virus and therefore expressing membrane-associated viral antigenicity.Susceptibility of primary leukemias to cell-mediated lysis could not be tested due to a very high spontaneous ⁵¹ Cr release shortly after labeling. In a cold target competition assay, however, the RadLV-induced, but not the X-radiation-induced primary tumor cells inhibited the cytotoxic reactivity. This inhibition was correlated with the level of viral antigen expression on the inhibiting cells, which was high in the RadLV-induced and low in the radiation-induced primary tumors.These results suggest that antitumor CTL generated under conventional MLTC conditions are largely stimulated by and directed at virus-related antigens not necessarily associated with the malignant state of the cell.     

Hyperthermia and the generation and activity of murine influenza-immune cytotoxic T cells in vitro.

The rate of generation of murine secondary influenza virus-immune cytotoxic T cells in vitro is enhanced under limiting dilution conditions at hyperthermal temperatures (39 versus 37 degrees C). Increased mean values of cytotoxic activity were observed in the presence as well as absence of exogenous helper factors. Elevated cytotoxic activity at 39 degrees C was observed after day 3 to day 5 of culture. The number of autoreactive cytotoxic cells observed was not greater at 39 degrees C than at 37 degrees C. Elevated temperature did not influence target cell lysis or release of isotopes from killed target cells. The results are discussed with a view to the role of fever in augmenting the cellular immune response responsible for the host defense against primary viral infection.     

Isolation and characterization of heat-resistant (HR) mutants of Newcastle disease virus

Heat-resistant (HR) mutants (MR 70 and HR 74) of Newcastle disease virus (NDV) which exhibited significantly higher thermostability in their infectivity than wild-type virus were isolated and characterized. They differ from each other in their plaque morphology; HR 70 produces small turbid plaques, whereas those of HR 74 are large and clear. Cytopathogenicity of these mutants is much lower than that of the wild-type virus in cultured cells such as CEF, LLCMK2 and HeLa cells. Moreover, these HR mutants exhibited extended mean embryo survival times. Synthesis of cellular RNA's and proteins in cells infected with HR mutants was not significantly reduced under conditions in which synthesis of these macromolecules was strongly reduced in cells infected with wild-type virus. No significant differences were observed between HR mutants and wild-type virus in their other phenotypic characteristics such as the capacity for interferon production, growth characteristics at a low multiplicity of infection, and cleavage of viral glycoproteins in infected cells. From these findings, it was suggested that the inhibitory effect of virus infection on cellular macromolecular synthesis is a possible determinant of cytopathogenicity of NDV.     

Population dynamics of a continuous fermentation of recombinant Saccharomyces cerevisiae using flow cytometry

The plasmid instability of genetically modified microorganisms during prolonged bioreactor operations is one of the major problems to be overcome in the production of recombinant proteins. The use of flow cytometry to monitor a fermentation process with recombinant cells in a CSTR is reported here. This technique has been applied to determine the fraction of plasmid-bearing cells (P+) of a recombinant Saccharomyces cerevisiae strain harboring the EXG1 gene in a continuous stirred tank bioreactor with a working volume of 2 L. The different levels in the expression of the EXG1 gene, which encodes the enzyme exo-beta-glucanase, were used to determine the P+ fraction. Other parameters such as viability, cellular protein, cell size and structure were also monitored using flow cytometry. This technique has two main advantages over the conventional method of determining the P+ fraction (plating in selective and non-selective solid media): (a) it takes a very short period of time to obtain a measurement that provides multiple parametric information; and (b) it is more representative of the bioreactor cell population since it can analyze thousands of cells in the same sample. A continuous operation (432 h) with the recombinant strain in a CSTR was carried out to test the application of this technique. Measurements of cellular exo-beta-glucanase activity and cellular protein content closely correlates to the measured fraction of plasmid-containing cells in the population. Moreover, the standard deviation of the fraction of P+ cells determined using this technique was very low (about 2%). Recombinant protein production also increased the size of the yeast cells, whereas the recombinant cells also had a more complex internal structure than the non-recombinant host strain.     

Free fatty acids are produced in and secreted from target cells very early in cytotoxic T lymphocyte-mediated killing

Conjugation of CTL with their cognate targets elicits a number of early changes within the target cell that are thought to play an important role in the lytic mechanism. We now report that at times earlier than 5 min after conjugation with allospecific CTL, free fatty acids (FFA) are produced in and then secreted from alloantigen-bearing target cells. Using murine CTL clones with different alloantigen specificities, stimulation of FFA production from target cells was found to be Ag specific. FFA production does not appear to be specific for any particular FFA species. Indeed, a wide spectrum of cis unsaturated as well as saturated FFA are produced. FFA production is well correlated with, and specific for, CTL-mediated target cell lysis. Other means of perturbing or lysing target cells, including freeze/thaw disruption, detergent solubilization, or increasing membrane permeabilization with ionomycin, do not stimulate FFA production. In particular, FFA production is not stimulated by treatment with pore-forming granules under conditions that cause more than 90% target cell lysis. These results suggest that FFA production plays an important role in CTL-mediated lysis because stimulation of FFA release specifically requires an event that is CTL induced, occurs very early after conjugation, and is strongly correlated with CTL-mediated lysis.     

The Simian virus 40 late viral protein VP4 disrupts the nuclear envelope for viral release

Simian virus 40 (SV40) appears to initiate cell lysis by expressing the late viral protein VP4 at the end of infection to aid in virus dissemination. To investigate the contribution of VP4 to cell lysis, VP4 was expressed in mammalian cells where it was predominantly observed along the nuclear periphery. The integrity of the nuclear envelope was compromised in these cells, resulting in the mislocalization of a soluble nuclear marker. Using assays that involved the cellular expression of VP4 or the treatment of cells with purified VP4, we found that the central hydrophobic domain and a proximal C-terminal nuclear localization signal of VP4 were required for (i) cytolysis associated with prolonged expression; (ii) nuclear envelope accumulation; and (iii) disruption of the nuclear, red blood cell, or host cell membranes. Furthermore, a conserved proline within the hydrophobic domain was required for membrane perforation, suggesting that this residue was crucial for VP4 cytolytic activity. These results indicate that VP4 forms pores in the nuclear membrane leading to lysis and virus release.     

Lysis of Streptococcus mutans by hen egg white lysozyme and inorganic sodium salts.

Streptococcus mutans BHT was grown in a synthetic medium containing radioactive thymidine to monitor deoxyribonucleic acid release. Kinetic experiments demonstrated that although lysozyme alone could not liberate deoxyribonucleic acid, cellular deoxyribonucleic acid was liberated from lysozyme-treated cells by addition of low concentrations of inorganic sodium salts. When the salts were tested for their ability to dislodge cell-bound tritiated lysozyme, the extent of the initial release of enzyme by individual anions correlated with the anion potency for deoxyribonucleic acid liberation (SCN- greater than ClO4- greater than I- greater than Br- greater than NO3- greater than Cl- greater than F-), although the total amount of lysozyme dislodged did not correspond directly with cell lysis. Differences in the effectiveness of anions (SCN-, HCO3-, Cl- and F-) in potentiating cell lysis could be enhanced or minimized by varying the lysozyme, anion, and bacterial cell concentrations. As the anion concentration was increased for each enzyme concentration and cell concentration, the lysis increased, in some cases markedly, until maximum levels of released deoxyribonucleic acid were attained. The maximum levels of lysis of SCN- and HCO3- were similar and were greater than those for Cl- and F-. In addition, the maximum levels were observed to increase for each of the anions as the concentration of lysozyme increased.     

Inhibition of Cellular DNA Synthesis in Cells Infected with Infectious Pancreatic Necrosis Virus

In asynchronous RTG-2 cell cultures infected with infectious pancreatic necrosis (IPN) virus, inhibition of cellular DNA synthesis, but not protein synthesis, was detected 5 to 6 h postinfection and was 80 to 90% complete by 7 to 8 h. Inhibition of DNA synthesis was largely abolished by UV irradiation of the virus. Sedimentation analyses of phenol-extracted DNA indicated that native cellular DNA was not degraded during infection. Sedimentation on alkaline sucrose gradients of DNA from cells pulsed with radioactive thymidine for varying periods indicated that elongation of nascent DNA chains proceeded normally in infected cells. These and previous results suggest that IPN virus infection results in a reduction of the number of chromosomal sites active in DNA synthesis but does not affect the rate of polymerization at active sites. Cells synchronized with excess thymidine and hydroxyurea and infected with virus at the time of release from the block demonstrated an inhibition of DNA synthesis 3 h postinfection. Cells infected 4 h prior to release continued to synthesize normal amounts of DNA for 1 to 2 h after release. These results indicated that DNA synthesis in early synthetic phase is relatively insensitive to inhibition by IPN virus.     

Cell killing by HIV-1 protease

The human immunodeficiency virus protease (HIV-1 PR) was expressed both in the yeast Saccharomyces cerevisiae and in mammalian cells. Inducible expression of HIV-1 PR arrested yeast growth, which was followed by cell lysis. The lytic phenotype included loss of plasma membrane integrity and cell wall breakage leading to the release of cell content to the medium. Given that neither poliovirus 2A protease nor 2BC protein, both being highly toxic for S. cerevisiae, were able to produce similar effects, it seems that this lytic phenotype is specific of HIV-1 PR. Drastic alterations in membrane permeability preceded the lysis in yeast expressing HIV-1 PR. Cell killing and lysis provoked by HIV-1 PR were also observed in mammalian cells. Thus, COS7 cells expressing the protease showed increased plasma membrane permeability and underwent lysis by necrosis with no signs of apoptosis. Strikingly, the morphological alterations induced by HIV-1 PR in yeast and mammalian cells were similar in many aspects. To our knowledge, this is the first report of a viral protein with such an activity. These findings contribute to the present knowledge on HIV-1-induced cytopathogenesis.     

Phage formation in Staphylococcus muscae cultures; nucleic acid synthesis during virus formation

1. The total nucleic acid synthesized by normal and by infected S. muscae suspensions is approximately the same. This is true for either lag phase cells or log phase cells. 2. The amount of nucleic acid synthesized per cell in normal cultures increases during the lag period and remains fairly constant during log growth. 3. The amount of nucleic acid synthesized per cell by infected cells increases during the whole course of the infection. 4. Infected cells synthesize less RNA and more DNA than normal cells. The ratio of RNA/DNA is larger in lag phase cells than in log phase cells. 5. Normal cells release neither ribonucleic acid nor desoxyribonucleic acid into the medium. 6. Infected cells release both ribonucleic acid and desoxyribonucleic acid into the medium. The time and extent of release depend upon the physiological state of the cells. 7. Infected lag phase cells may or may not show an increased RNA content. They release RNA, but not DNA, into the medium well before observable cellular lysis and before any virus is liberated. At virus liberation, the cell RNA content falls to a value below that initially present, while DNA, which increased during infection falls to approximately the original value. 8. Infected log cells show a continuous loss of cell RNA and a loss of DNA a short time after infection. At the time of virus liberation the cell RNA value is well below that initially present and the cells begin to lyse.     

Virus reactivation: a panoramic view in human infections

Viruses are obligate intracellular parasites, relying to a major extent on the host cell for replication. An active replication of the viral genome results in a lytic infection characterized by the release of new progeny virus particles, often upon the lysis of the host cell. Another mode of virus infection is the latent phase, where the virus is 'quiescent' (a state in which the virus is not replicating). A combination of these stages, where virus replication involves stages of both silent and productive infection without rapidly killing or even producing excessive damage to the host cells, falls under the umbrella of a persistent infection. Reactivation is the process by which a latent virus switches to a lytic phase of replication. Reactivation may be provoked by a combination of external and/or internal cellular stimuli. Understanding this mechanism is essential in developing future therapeutic agents against viral infection and subsequent disease. This article examines the published literature and current knowledge regarding the viral and cellular proteins that may play a role in viral reactivation. The focus of the article is on those viruses known to cause latent infections, which include herpes simplex virus, varicella zoster virus, Epstein-Barr virus, human cytomegalovirus, human herpesvirus 6, human herpesvirus 7, Kaposi's sarcoma-associated herpesvirus, JC virus, BK virus, parvovirus and adenovirus.