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Department of Microbiology and Molecular Biology, Brigham Young University, 887 WIDB, Provo, UT 84602, USA
Correspondence
F. Brent Johnson
brent_johnson{at}byu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The autonomous parvoviruses, which include the genera Parvovirus, Erythrovirus, Amdovirus and Bocavirus, do not require a helper virus, but they require S phase for productive infection (Johnson, 1983; Lederman et al., 1983). BPV is classified in the genus Bocavirus (Fauquet et al., 2005). It is an interesting and unique model among the helper-independent parvoviruses because its capsid is composed of three structural proteins and it has a mid-ORF encoding a phosphorylated non-structural protein. Recently, a new human bocavirus was detected by PCR in nasopharyngeal specimens collected from children with lower respiratory tract infections (Ma et al., 2006).
During virus–substrate interactions, free BPV particles attach to sialic acid to mediate the haemagglutination (HA) reaction (Thacker & Johnson, 1998) and to bind to bovine cells in culture (Johnson et al., 2004). The erythrocyte receptor is the O-linked 
2,3-neuraminic acid on glycophorin A (Blackburn et al., 2005). The three known functional ORFs in the BPV genome encode the non-structural protein NS1 (large left ORF), the non-structural nuclear phosphoprotein NP1 (small mid-ORF) and the three structural proteins (Johnson & Hoggan, 1973), VP1, VP2 and VP3 (large right ORF) (Chen et al., 1986). After protein synthesis, viral proteins are translocated to the nucleus, where the assembly process is completed. Egress of the virus is presumably by degradation of the cell (Durham & Johnson, 1985) and rupture of the nuclear membrane (Johnson, 1983), but it has previously been unclear whether cell degradation followed apoptosis or necrosis.
Viruses are known to kill infected cells by inducing apoptosis or necrosis or other types of cell death. Currently, many subtleties of cell death pathways are under investigation and separate pathways are being recognized and given separate designations. The current study, however, was an initial investigation of BPV-induced cell death that included some of the standard markers for apoptosis and necrosis. Apoptosis is an active process of cell death that is tightly controlled. It is ‘programmed cell death’, an orderly series of biochemical events resulting in the purposeful removal of useless, unwanted or damaged cells. Cells undergoing apoptosis show characteristic morphological and biochemical changes, such as the appearance of membrane blebbing, inversions of phosphatidylserine molecules so that they become oriented on the outer surface of the plasma membrane, the appearance of caspases and the presence of a nucleosomal ladder due to non-random DNA fragmentation by caspase-activated DNase and other DNases (Hengartner, 2000; Yuan & Horvitz, 2004). Many viruses encode gene products that induce apoptosis, which may contribute directly to their cytopathogenic effects (Gaddy & Lyles, 2005; Kopecky et al., 2001; Selliah & Finkel, 2001; Shen & Shenk, 1995). On the other hand, there are viruses that inhibit apoptosis of the infected cells by the action of viral anti-apoptosis proteins (Koyama et al., 2000). These viruses prolong the life of the cell, presumably enhancing virus yield.
Necrosis, another form of cell death, involves a different manner of cell killing. Necrosis is called ‘accidental’ cell death, differentiating it from programmed death. It is the destructive, disorderly process that occurs when cells are exposed to serious physical or chemical insult or as a result of virus-induced cytolysis. Necrosis begins when the ability of the cell to maintain homeostasis is impaired, leading to an influx of water and extracellular electrolytes. Intracellular organelles, notably mitochondria, swell and the cell ruptures, liberating the cytoplasmic contents. The DNA is fragmented in a random fashion and appears as a smear on electrophoretic gels without the appearance of the DNA fragment laddering characteristic of apoptosis. Moreover, no caspase involvement has been shown in necrosis. Some viruses kill their host cells by inducing necrotic cell death. Detecting the release of cytoplasmic contents is one way of identifying cell necrosis.
Studies of how viruses induce death in cells have been reported for several members of the family Parvoviridae. Parvovirus H-1 causes cell death by a non-apoptotic process in permissive cells (Li et al., 2005; Raj et al., 2001) or by apoptosis in non-permissive cells (Ohshima et al., 1998), and has been studied as a possible agent for tumour therapy (Moehler et al., 2001, 2005). Rat parvovirus (RPV/UT) causes apoptotic cell death of thymic lymphoma cells (Ueno et al., 2001). Parvovirus B19 NS1 protein induces apoptosis in COS-7 cells (Hsu et al., 2004) and in erythroid lineage cells (Chisaka et al., 2002; Clark & Boyles, 1999; Moffatt et al., 1998; Yaegashi et al., 1999). Moreover, B19 caused apoptosis in hepatocytes (Poole et al., 2004). Furthermore, feline panleukopenia virus (FPLV) induces apoptosis in feline lymphoid cells (Ikeda et al., 1998) and apoptosis was detected in FPLV and canine parvovirus (CPV) enteritis in animals (Bauder et al., 2000). Aleutian mink disease virus (ADV) and mink enteritis virus induce apoptosis in permissive Crandell feline kidney cells (Best et al., 2002).
Here, we investigated whether BPV infection of embryonic bovine tracheal (EBTr) cells induced apoptotic cell death or necrotic cell death, the latter being characterized by membrane failure and the release of intracellular contents.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). EBTr cells (ATCC NBL-4), a diploid cell strain, were grown in Dulbecco's modified Eagle's medium (DMEM) containing 0.11 % sodium bicarbonate, 10 mM HEPES, 50 µg gentamicin ml–1 and either 5 % cosmic calf serum (HyClone) or Fetal Clone III serum (HyClone). Cosmic calf serum contains anti-BPV antibody; therefore, in cultures where virus infection was measured, Fetal Clone III serum was used instead of cosmic calf serum.
4,6-Diamidino-2-phenylindole (DAPI) staining.
EBTr cells were cultured on round coverslips (12 mm) in shell vials in DMEM containing 5 % Fetal Clone III serum. Various sets of five shell vials each were prepared for DAPI staining including BPV-infected cells and positive and negative controls. Staurosporine at a concentration of 5 µM was used as the positive control and was incubated with cells at 37 °C for 4 h. In the virus-infected cell set, BPV was added to cells at an m.o.i. of 0.27 and incubated at 37 °C for 48 h to ensure that most of the cells were infected. Negative controls consisted of EBTr cells with no virus or other treatment. The cells were then placed on microscope slides in drops of mounting fluid containing DAPI [phosphate-buffered glycerol (pH 9) containing 0.2 mg p-phenylenediamine ml–1 and 0.5 µg DAPI ml–1). Cells were analysed by fluorescence microscopy for nuclear morphology changes. Apoptotic cells on each coverslip were counted blind by two people, as were cells in all subsequent experiments. Cells that were positive for apoptosis were defined morphologically by nuclear shrinkage, chromatin fragmentation and membrane blebbing.
Detection of apoptosis by annexin V–FITC staining.
Cells were cultured on coverslips as above. Various sets comprising five shell vial cultures were prepared for annexin V staining to show membrane-associated phosphatidylserine inversion in apoptotic cells. The sets included BPV-infected cells, a cycloheximide (10 µg ml–1) positive control, a staurosporine (5 µM) positive control and negative controls. A culture set was inoculated with BPV (m.o.i.=0.27) and incubated as described above. Negative controls consisted of uninfected and untreated EBTr cells grown in DMEM containing 5 % Fetal Clone III serum. The staining reagents were obtained as a kit from BioVision and the manufacturer's instructions were followed. Unfixed cells were stained by adding annexin V–FITC reaction mixture (10 µl annexin V–FITC, 5 µl propidium iodide) and incubating at room temperature for 10 min in the dark. Cells were then washed with PBS and distilled water, placed on microscope slides and inspected microscopically for apoptosis-positive cells under UV illumination. In apoptosis-positive cells that had bound annexin V–FITC, the plasma membrane stained green, whilst negative cells remained unstained.
CaspGLOW fluorescein caspase staining.
The various sets of infected and control cells were cultured on coverslips as described above for annexin V staining. The principle of CaspGLOW fluorescein caspase staining is the irreversible attachment of the FITC-labelled caspase inhibitor z-VAD-fmk to a number of activated caspases such as caspases 1, 3, 4, 5, 6, 7, 8, 9, which can be seen as fluorescently labelled cells by fluorescence microscopy. Cells were labelled using the CaspGLOW reagent kit (BioVision) following the manufacturer's instructions. Following preparation of the culture sets, cells were stained by adding FITC–z-VAD-fmk reaction mixture (1 µl FITC-labelled z-VAD-fmk in 300 µl wash buffer) to each coverslip and incubating for 1 h at 37 °C. Cells were then examined by fluorescence microscopy. Caspase-positive cells stained bright green, whilst negative cells remained unstained.
DNA fragmentation as an indicator of apoptosis.
Four 75 cm2 flasks of cultured EBTr cells were grown to 70 % confluence. One culture served as a negative control, whilst another served as a positive control in which staurosporine (to 5 µM) was added. The cultures were incubated at 37 °C for 6 h. DMSO was added to the third culture as an additional negative control (the solvent control), whilst the culture in the fourth flask was infected with BPV (m.o.i. of 0.0066). The cultures were incubated until a complete cytopathic effect (CPE) was achieved in the infected culture. In this culture, most of the cells were freshly infected during the last replication cycle, providing reasonable comparison with the staurosporine control. The cells were then trypsinized and collected for DNA extraction. This process was carried out using the method of Herrmann et al. (1994). DNA samples prepared in parallel from the four cultures were electrophoresed in 1.5 % agarose gel at 100 V for 3–4 h until the tracking dye (brilliant blue) had run a distance of two-thirds of the gel. Included in each replicate run was a 100 bp DNA ladder control consisting of 15 blunt-ended fragments of between 100 and 1500 bp and an additional fragment at 2072 bp (obtained from Invitrogen). Gels were stained with 1x SYBR Green (Molecular Probes) for 20 min. Apoptotic cells present a DNA ladder on agarose gels (Herrmann et al., 1994).
Testing of cultures for virus-induced cell necrosis.
Experiments were carried out to determine the release of cell cytoplasmic contents as an indicator of cytolysis by necrosis. The markers assessed were cell-released infectious virus, viral HA and the cellular enzyme lactate dehydrogenase (LDH). In these experiments, EBTr cells were grown in monolayers to 90 % confluence, washed with serum-free medium and renewed with 5 % Fetal Clone III serum/DMEM. BPV was inoculated into one culture at an m.o.i. of 0.006 and another culture served as an uninfected negative control. The cultures were incubated at 37 °C until complete CPE (4+) was achieved. During this incubation period, 1 ml samples were taken from the medium every 6 h, centrifuged in a microfuge to remove any unattached cells from the samples and supernatants were collected. Infectivity assays on the supernatants were performed in flat-sided tissue culture tubes (Nunc), as described previously (Johnson et al., 2004), to detect infectious virus particles released into the medium of the cultures. Following incubation, cells were fixed with formaldehyde/ethanol/acetic acid and stained with an immunoperoxidase (IP) stain to identify cells positive for viral antigen (Luker et al., 1991). The stained cells were counted and infectious virus titres determined and notated as focus-forming units (f.f.u.) ml–1.
HA assays.
HA tests were performed in 96-well U-bottomed plates. Samples were diluted 1 : 10, 1 : 100 and 1 : 1000 in HA buffer (0.005 % gelatin, 0.1 % BSA in PBS, pH 7.0; Thacker & Johnson, 1998). Fifty microlitres of each diluted sample was mixed with 50 µl 0.5 % human type O red blood cell suspension and the plate was incubated at 4 °C overnight. Negative results were scored as red cell button formation, whereas positive results were scored as agglutinated cell sheets. To obtain a final titre, twofold dilutions between the tenfold steps were tested as needed.
LDH assays.
Medium supernatants collected from infected and control cells in the experiments designed to test for the release of cell cytoplasmic contents were tested for LDH activity. The assay reagents were purchased in kit form [Cytotoxicity Detection kit (LDH); Roche Diagnostics]. This test is a colorimetric assay for the quantification of cell death and cell lysis based on the measurement of LDH activity released from the cytosol of damaged cells into the supernatant. The amount of enzyme activity detected in the culture supernatant correlates with the proportion of lysed cells. The assays were conducted following the manufacturer's instructions, in flat-bottomed wells of 96-well plates. One hundred microlitres of each sample was added to 100 µl reaction reagent (prepared as a mixture of 250 µl catalyst, diaphorase/NAD+, with 11.25 ml dye solution containing iodotetrazolium chloride and sodium lactate). These reaction mixtures were incubated for 30 min at 15–25 °C in the dark. Following incubation, the absorbance of samples was measured at a wavelength of 490 nm as a measure of enzyme activity using an ELISA plate reader. The LDH released from bovine cells was readily detected by this method.
Detection of cell proliferation and cell death by monitoring mitochondrial dehydrogenases using the WST-1 reagent.
Cells were grown in 24-well plates. Cells in some wells were infected with virus at various m.o.i., whilst other wells served as uninfected controls. Cell proliferation and viability were measured using the cell proliferation reagent WST-1 (Roche). This is a colorimetric assay for the quantification of cell proliferation and cell viability, based on cleavage of the tetrazolium salt WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) to formazan by mitochondrial dehydrogenases in viable cells. Necrotic, dead cells do not catalyse this conversion. Viral CPE resulting in cell death can be detected by this method. At appropriate time intervals, 100 µl WST-1 (prepared according to the manufacturer's instructions) was added to the wells and incubated in the dark for 4 h at 36 °C for substrate conversion. Samples (200 µl) were removed from the wells, placed in flat-bottomed wells of a 96-well plate and the absorbance read on an ELISA plate reader at a wavelength of 450 nm. High absorbance readings were consistent with cell viability.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. The results indicated that the numbers of apoptosis-positive cells in the BPV-infected cultures and the negative controls were equivalent. As expected, the positive control (staurosporine-treated cells) responded with significantly larger numbers of apoptotic cells. The number of virus-positive cells (total number of antigen-positive cells) was also much larger than the number of apoptotic cells in any of the cultures, confirming a high level of infected cells, which would be potential candidates for virus-induced apoptosis.
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(a). The results indicated that the numbers of apoptosis-positive cells in the uninfected control cells and in the virus-infected cells were similar. As expected, the positive-control cultures had significantly larger numbers of apoptotic cells. The number of cells positive for virus antigen was also larger than the number of apoptotic cells in BPV-infected cells.
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. These results, together with two experimental replicates, demonstrated the failure of the virus to increase the numbers of apoptotic cells over the number seen in negative-control cells.
DNA fragmentation
In apoptotic cells, the DNA is cleaved non-randomly into fragments yielding DNA pieces that are multimers of about 180 bp consisting of nucleosomal units. They appear as DNA ladders on agarose gels. In contrast, in necrosis the cellular DNA is fragmented in a random fashion and appears as a smear on electrophoretic gels. The DNA of cells exposed to various treatments was extracted in parallel and separated by electrophoresis. Gel separation of the DNA isolated from infected cells and from positive- and negative-control cells is shown in Fig. 3. The negative-control cells and DMSO-treated cells had an intact DNA band (Fig. 3, lanes 2 and 3) with no laddering effect. Cells treated with staurosporine (positive control; Fig. 3, lane 5) exhibited a ladder of DNA fragments that were multimers separated at 180 bp intervals. In BPV-infected cells, the DNA was fragmented in a random fashion forming a smear with no evidence of laddering (Fig. 3, lane 4). A 100 bp size marker control ladder was included (Fig. 3, lane 1). Consistent results were obtained in replicate experiments. These results suggested a necrotic cell death pathway in virus-infected cells, rather than apoptosis.
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shows the results of titrations detecting the number of infectious units in the medium. In the negative-control medium, no infectious particles were detected, whilst in the BPV-infected culture, the number of virions increased with time as the CPE increased.
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. The data confirmed that, as the CPE increased, the titre of HA protein increased in the supernatant of the BPV-infected culture. The negative control did not show HA activity.
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Samples were collected from the medium of infected and control cultures at various time intervals and centrifuged to remove contaminating cells. LDH activity was determined and the results are shown in Fig. 6. LDH concentration in both culture supernatants was approximately the same at the beginning of the experiment. As increasing times p.i., viral CPE was detected in BPV-infected cells. At 42 h p.i., the concentration of the released enzyme increased. Significantly higher levels of LDH were detected in virus-infected cell supernatants than in the control supernatants, indicating necrotic cell damage in the infected cells.
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). Cultures in 24-well plates were infected with m.o.i. ranging from 0.0001 to 0.5 and observed for 4 days for CPE development. At this time, culture supernatant samples were tested for LDH activity (Fig. 7a). Enzyme activity rose significantly in cultures infected at an m.o.i. of 0.1 or 0.5. The highest levels of LDH correlated with cultures infected at the highest m.o.i. and this culture also exhibited the greatest level of CPE. A time-course experiment (Fig. 7b) demonstrated LDH release as early as 24 h p.i. with maximum activity noted at day 4, the end of the experiment. LDH release was also dependent on m.o.i. in this study. An additional set of controls that tested LDH release in cycloheximide-treated cultures (a second ‘negative’ control for necrosis) was included and showed LDH release in amounts indistinguishable from the untreated cell controls. The release of this enzyme from infected cells thus confirmed the necrotic cell death of BPV-infected EBTr cells.
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) showed loss of cell viability at an m.o.i. of 0.1 or 0.5. Cell death was consistent with increasing m.o.i. and correlated with CPE development. Loss of cell viability in virus-infected cells was tested over a 4 day time period (Fig. 8b). Loss of viability in infected cultures was discernable by day 4.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Cells undergoing apoptosis exhibit cardinal features such as changes in cellular and nuclear morphology, non-random DNA fragmentation, phosphatidylserine inversion on the plasma membrane, caspase activation and the formation of apoptotic bodies. In this study, several of these signs were examined to determine whether BPV-infected cells undergo apoptosis. As not all markers of apoptosis may be detected as positive in cells killed by apoptosis, several markers were assessed to see whether any of them would reveal evidence of apoptotic cell death. None of the findings suggested an apoptotic mechanism.
Cells undergoing necrosis release cytoplasmic contents due to bursting. The rationale for the approach employed in this study to assess cytotoxicity/necrosis was similar to that of Blanco et al. (2003), who reported cell killing by human immunodeficiency virus 1 protease by showing the release of intracellular proteins into the medium and the release of LDH. If BPV-infected cells are killed by necrosis, unassembled viral proteins, infectious virus particles and cellular materials should be present in the medium of cultured cells that are infected with the virus and should be released in a burst rather than slowly over a relatively long period of time, as would be expected if apoptotic degradation were the mechanism of virus egress. The number of infectious particles and HA titres in the medium increased rapidly with time p.i. and increased as CPE increased, indicating the release of infected-cell contents into the medium. The time course or kinetics of material release was consistent with asynchronous cell degradation. BPV matures by assembly in the nucleus. Thus, the finding of infectious, cell-released virus in the culture medium was suggestive of plasma membrane and nuclear membrane breakdown consistent with cell necrosis. Additionally, an LDH enzyme assay was used to detect cytolysis (Blanco et al., 2003). The enzyme is a constitutive enzyme present in the cytoplasm of all cell types. When the plasma membrane is damaged due to necrotic burst, LDH is released into the medium of cultured cells. In contrast, the cell membranes remain intact during apoptosis, preventing the release of LDH into the supernatant. The results of the LDH assay demonstrated enzyme release into the supernatant of BPV-infected cells, verifying that plasma membrane damage occurred in the virus-infected cells. LDH release from infected cells over time correlated with CPE development. The enzyme was released early into the medium of cultures infected at higher m.o.i., but not into cultures infected at low m.o.i. at early times p.i. before spread of virus within the culture. These observations confirmed cell bursts and death by necrosis.
Supporting evidence for necrosis, in addition to release of the HA antigen, assembled particles and LDH, was obtained in studies of cell viability, which tested for mitochondrial dehydrogenases. The reagent WST-1 was used to test for mitochondrial activity in cells. As cells lyse in the necrotic pathway, the mitochondria become non-functional. In apoptotic cells, mitochondrial activity remains intact and is only slowly reduced as the pathway progresses. Cells shown to be non-viable at early times p.i. by this test, as in our observations, are killed by necrosis, whereas apoptosis must be considered in cell populations killed over long periods of time and in which mitochondrial enzyme activity is slowly reduced. None of the markers for apoptosis were positive, confirming that cell death detected by WST-1 was mediated by necrosis. Thus, the data obtained in this study indicate that BPV-infected EBTr cells die by necrosis.
These findings do not eliminate the possibility that this virus may induce apoptosis in other host cells or even in EBTr cells under different conditions. For example, H-1, another parvovirus, kills QGY-7703 human hepatocellular carcinoma cells by a non-apoptotic process (Li et al., 2005), but it kills C6 rat glioblastoma cells by apoptosis (Ohshima et al., 1998). Several other parvoviruses, including minute virus of mouse (MVM), B19, RPV, FPLV, CPV and ADV, as noted earlier, induce apoptotic responses in host cells. It was therefore somewhat surprising that apoptosis was not detected in BPV-infected cells. We are currently assessing other cell types for BPV-induced apoptosis.
Future studies will examine the role of non-structural proteins in virus-induced cytotoxicity. The pleiotropic NS1 non-structural protein of parvoviruses plays numerous critical roles during virus replication. Among other functions, NS1 has been assigned the role of the virus effector of cytotoxicity in MVM-infected cells (Anouja et al., 1997; Legendre & Rommelaere, 1992). Some reports show that NS1 and NS2 act together to cause cytotoxicity in infected cells (Brandenburger et al., 1990; Legrand et al., 1993). Furthermore, NS2 is thought to act in the process of nuclear egress of progeny MVM particles (Eichwald et al., 2002). The parvovirus B19 replicates in erythroid precursor cells, but in human infections it can persist in multiple tissues and evidence suggests its involvement in a variety of diseases (Poole et al., 2004) where NS1 transcription is important in cell death. NS1 transfection has been shown to be cytotoxic (Caillet-Fauquet et al., 1990; Moffatt et al., 1998; Momoeda et al., 1994). Hepatocytes undergoing apoptosis in cases of acute fulminant liver failure are linked to B19 NS1 expression (Poole et al., 2004). In fact, NS1 is sufficient to induce apoptosis in liver-derived cells and does so through the initiation of intrinsic caspase pathways, probably caspase 3- and 9-dependent pathways (Poole et al., 2006). Thus, parvovirus infection in naturally non-permissive cells, in some cases, may result in cell damage by apoptosis mediated by the non-structural proteins. BPV NP1 is another candidate, in addition to NS1, for mediation of cytotoxicity and may enhance cytotoxic activity synergistically with NS1, since NS2 works together with NS1 in the H-1 system. Whether BPV NS1 or NP1 is a mediator of cell cytotoxicity will be examined in future studies.
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ACKNOWLEDGEMENTS |
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Received 7 February 2006;
accepted 19 May 2006.
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, Virus-infected cultures; 
, uninfected control cultures.
).
, Uninfected controls (m.o.i.=0);