Download Infectious pancreatic necrosis virus induces apoptosis in vitro and in

Virology 342 (2005) 13 – 25
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Infectious pancreatic necrosis virus induces apoptosis in vitro and
in vivo independent of VP5 expression
Nina Santi a, Ane Sandtrø b, Hilde Sindre c, Haichen Song d, Jiann-Ruey Hong e, Beate Thu b,
Jen-Leih Wu f, Vikram N. Vakharia d, Øystein Evensen b,*
a
Section for Pathology, National Veterinary Institute, 0033 Oslo, Norway
Department of Basic Sciences and Aquatic Medicine, Norwegian School of Veterinary Science, PO Box 8146 Dep., N-0033 Oslo, Norway
c
Section for Virology, National Veterinary Institute, 0033 Oslo, Norway
d
Center for Biosystems Research, University of Maryland Biotechnology Institute and VA-MD Regional College of Veterinary Medicine,
University of Maryland, College Park, MD 20742, USA
e
Laboratory of Molecular Virology and Biotechnology, Institute of Biotechnology, National Cheng-Kung University, Tainan 701, Taiwan
f
Laboratory of Marine Molecular Biology and Biotechnology, Institute of Zoology, Academia Sinica, Taipei 115, Taiwan
b
Received 28 February 2005; returned to author for revision 25 April 2005; accepted 14 July 2005
Available online 26 August 2005
Abstract
Infectious pancreatic necrosis virus (IPNV), the causative agent of a highly infectious disease in salmonid fish, encodes a small nonstructural protein designated VP5. This protein contains Bcl-2 homologous domains and inhibits apoptosis when expressed in cell culture.
We have previously reported the generation of three VP5 mutants of IPNV – Sp serotype, using reverse genetics (Santi, N., Song, H.,
Vakharia, V.N., Evensen, Ø, 2005. Infectious pancreatic necrosis virus VP5 is dispensable for virulence and persistence. J. Virol. 79 (14),
9206 – 9216). The wild-type rNVI15 virus encodes a truncated 12-kDa VP5 protein, rNVI15-15K encodes a full-length 15-kDa VP5, whereas
rNVI15-DVP5 is deficient in VP5 expression. In the present report, the role of VP5 in apoptosis was assessed both in vitro and in vivo, using
the recombinant IPNV strains. Apoptosis was observed in hepatocytes of Atlantic salmon post-smolts challenged with all three VP5 mutant
viruses. Using a double-labeling technique to detect apoptotic cells and IPNV antigens, we found that viral antigen and apoptotic cells codistributed. In addition, numerous double-positive cells were seen. The recombinant viruses also induced apoptosis in infected cell cultures,
and the morphology and membrane integrity of infected cells at different time points was similar. In summary, these results indicate that
IPNV induces apoptosis in infected cell cultures and in fish, independent of VP5 expression. However, substitutions of putative functionally
important amino acids in the BH2 domain of VP5 of IPNV – Sp strains were identified, which might influence the anti-apoptosis effect of the
protein, and partly explain the apparent absence of this specific function.
D 2005 Elsevier Inc. All rights reserved.
Keywords: IPNV; VP5; Apoptosis; Bcl-2
Introduction
Infectious pancreatic necrosis virus (IPNV) is the
causative agent of an economically important disease in
farmed Atlantic salmon (Salmo salar L.), infectious
pancreatic necrosis (IPN). Outbreaks are typically seen at
fry start feeding, and shortly after sea transfer of smolts. As
* Corresponding author. Fax: +47 22597310.
E-mail address: [email protected] (Ø. Evensen).
0042-6822/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.virol.2005.07.028
indicated by the name, necrosis of exocrine pancreatic tissue
is the most common manifestation of the disease. In
addition, severe necrosis has been observed in the liver of
experimentally infected Atlantic salmon fry (Taksdal et al.,
1997; Santi et al., 2004). Following acute infection,
survivors continue to harbor virus in their bodies for long
periods, presumably in head kidney macrophages (Reno et
al., 1978; Mangunwiryo and Aguis, 1988; Johansen and
Sommer, 1995).
IPNV is a member of the genus Aquabirnavirus in the
family Birnaviridae. The non-enveloped icosahedral capsid
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N. Santi et al. / Virology 342 (2005) 13 – 25
has a diameter of 60 nm and contains two genome segments
(A and B) of double-stranded RNA (Dobos, 1995). The
smaller segment B encodes the virus polymerase, VP1
(Duncan et al., 1991). Segment A contains a large open
reading frame (ORF) encoding a 107-kDa polyprotein that
is processed into the major structural proteins VP2 and VP3
by the viral protease (VP4) (Macdonald and Dobos, 1981;
Duncan et al., 1987; Petit et al., 2000). A second ORF,
overlapping the N-terminal region of VP2, encodes the
small non-structural protein, VP5 (Håvardstein et al., 1990;
Magyar and Dobos, 1994).
Apoptosis is a genetically controlled process of cell
suicide in response to a variety of stimuli. Apoptosis is
considered a part of the innate immune response to virus
infection, limiting the time and cellular machinery available
for viral replication (Everett and McFadden, 1999). Apoptotic cell death in tissues typically involves scattered
individual cells, recognized by certain morphological
features, such as detachment from neighboring cells,
condensation and aggregation of chromatin along the inner
surface of the nuclear membrane, and segmentation of the
nucleus and the cell into ‘‘apoptotic bodies’’ that are rapidly
engulfed by adjacent cells or by macrophages (Kerr et al.,
1995). Infectious bursal disease virus (IBDV), another
member of the Birnaviridae family, induces apoptosis in
peripheral blood lymphocytes and in tissue from infected
chickens (Vasconcelos and Lam, 1994; Lam, 1997; Tanimura and Sharma, 1998). Apoptosis precedes necrosis in
cultured fish cells infected with IPNV (Hong et al., 1998),
but so far attempts to detect apoptosis in IPNV-infected fish
have been inconclusive (Eléouët et al., 2001; Imajoh et al.,
2005). IBDV also contains a small ORF that encodes a 17kDa non-structural protein called VP5 (Mundt et al., 1995).
VP5 of both IBDV and IPNV is dispensable for viral
replication in cell culture (Mundt et al., 1997; Weber et al.,
2001; Santi et al., 2005) but their function appears to be
different. Expression of IBDV-VP5 in cell culture leads to
accumulation of the protein in the cell membrane, alteration
in membrane permeability, and induction of apoptosis
(Lombardo et al., 2000; Yao and Vakharia, 2001). A
recombinant IBDV mutant, deficient in VP5 expression,
has decreased cytotoxic and apoptotic effect in cell culture
and does not induce lesions in the bursa of inoculated
chickens (Yao et al., 1998). IPNV-VP5, on the other hand,
contains Bcl-2 homologous domains, and stable expression
enhances cell viability following exposure to apoptotic
inducers (Hong et al., 2002). To date, there are no studies on
the role of this protein in IPNV-induced apoptosis using
strains deficient in VP5 expression.
We recently reported the recovery of three VP5 mutants
of IPNV by reverse genetics (Santi et al., 2005). The
recombinant rNVI15-DVP5 is a VP5 knockout mutant
virus, whereas rNVI15 encodes a truncated 12-kDa VP5,
and rNVI15-15K encodes the full-length 15-kDa protein.
The replication kinetics of the three viruses in cell culture is
similar, and they all cause IPN disease in challenged
Atlantic salmon post-smolts, resulting in a cumulative
mortality of 86%, 81%, and 81%, respectively (Santi et
al., 2005). Since both cellular and viral Bcl-2 homologues
have been associated with viral persistence (Levine et al.,
1993; Gangappa et al., 2002), we assessed the involvement
of VP5 in persistent IPNV infection. The results demonstrated that VP5 is not required for the establishment of a
carrier state. Thus, the physiological effect of VP5
expression was not apparent from in vivo studies of VP5
mutant strains. In the present study, we analyze apoptosis
induction in tissues during acute IPNV infection in Atlantic
salmon post-smolts and evaluate the effect of VP5
expression on the kinetics of IPNV-induced apoptosis in
cell culture using recombinant IPNVs of the Sp serotype.
Results
In vitro infection of cells
IPNV-infected cell cultures exhibit cytopathic effects
(CPE) as the cells die from virus infection. We wanted to
determine if VP5 expression influence morphological
changes in IPNV-infected cell cultures. CHSE-214 cells
were infected with multiplicity of infection (MOI) 1, using
three IPNV-VP5 mutant strains. Morphological changes
were observed by phase contrast microscopy at regular
intervals, and the monolayers were photographed at 0, 24,
32, and 36 h post-infection (p.i.). CPE was first detected 24
h p.i., affecting single cells or cells in small groups (Fig. 1).
A slight increase in the number of affected cells was
observed at 32 h p.i. At the 36-h time point, almost full
CPE was observed, as most cells had detached from the
monolayer. There was no apparent difference in the
morphology of monolayers infected with the VP5-deficient
strain compared to cells infected with the VP5-expressing
strains at any time point throughout the observation period.
This experiment was also performed at MOI = 0.1 and
MOI = 10, again demonstrating a synchronized development of CPE in CHSE-214 cells infected with the VP5
mutant IPNV strains (data not shown).
Alterations in membrane permeability
Studies of IBDV-VP5 have demonstrated accumulation
of the protein within the plasma membrane, resulting in cell
lysis by disruption of the plasma membrane (Lombardo et
al., 2000). To determine if IPNV-VP5 expression has any
effect on the membrane permeability of infected cells,
lactate dehydrogenase (LDH) activity in supernatants of cell
infected with rNVI15, rNVI15-15K, and rNVI-DVP5
viruses was measured and compared with mock-infected
cells (Fig. 2). For all three recombinant strains, a slight
decrease in LDH activity was detected at 16 and 20 h p.i.,
followed by a steady increase and reaching maximum
activity at 55 h. There was no statistical difference in LDH
N. Santi et al. / Virology 342 (2005) 13 – 25
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Fig. 1. Phase contrast micrographs of CHSE-214 cells infected with three recombinant IPNV – Sp VP5 mutants at an MOI = 1.00. Scale bar = 100 Am.
Fig. 2. Lactate dehydrogenase (LDH) activity in supernatants from CHSE-214 cells infected with three recombinant IPNV – Sp VP5 mutants at an MOI = 1.00.
The data shown are mean of three parallel samples, and error bars indicate SD. The values for control cells and lysed cells are represented by the mean of 24
independent samples; the range of absorbance values was 0.154 – 0.186 for control cells and 0.391 – 0.542 for lysed cells.
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activity between these three viruses at any time point during
the observation period.
Induction of apoptosis in vitro
Hong et al. (1998 and 2002) have previously shown that
apoptosis precedes necrosis in IPNV-infected CHSE-214
cells, and that VP5 overexpression enhances host viability
and delays induction of internucleosomal DNA cleavage
induced by IPNV. When CHSE-214 monolayers were
infected with the recombinant strains, the characteristic
apoptotic laddering of DNA started to occur at 16 –18 h p.i.
for all three viruses (Fig. 3A). The bands were weak at 16,
18, and 20 h, but from 24 h onwards strong DNA laddering
was observed (Fig. 3B). Similar results were obtained in
another independent experiment (data not shown). Thus, it
was not possible to detect delayed induction of internucleosomal cleavage by VP5-expressing strains compared to the
VP5-deficient strains.
Annexin V binds to phosphatidylserine exposed at the
external cell surface of apoptotic cells. Flow cytometric
analysis of annexin V/propidium-iodide-stained CHSE-214
cells was performed for quantitative measurement of
apoptosis induced by the three recombinant VP5 strains.
Results from this experiment indicated a slight antiapoptotic effect due to VP5 expression. The proportion of
apoptotic cells was similar at 0, 16, 20, and 32 h time
points (Fig. 4A). At the 24-h time point, there were more
apoptotic cells in cultures infected by rNVI15-DVP5 (11%)
compared to rNVI15 (7%) and rNVI15-15K (4%)
(Fig. 4A). The number of dead cells did not rise
considerably before the 32-h time point in any of the
infected cell cultures (Fig. 4B).
We previously reported a substitution of residue 221 in
VP2 (Ala to Thr) of IPNV isolates propagated in CHSE-214
cells (Santi et al., 2004). We recently discovered that strains
encoding Ala at this position have significantly reduced
infectivity to CHSE-214 cells compared to cell-cultureadapted strains (Song et al., 2005). The virus stocks used in
this study have the same low passage number in CHSE
cells, and nucleotide sequencing has shown that mutants are
present at a very low level (data not shown). However, an
uneven distribution of these mutants could influence the
outcome of the flow cytometric assay, and consequently it
was decided to repeat this experiment with rNVI15-15 and
rNVI15-DVP5 free of cell-culture-adapted mutants. In this
experiment, apoptosis was induced at the same time and at
comparable levels in cells infected with the VP5-encoding
and the VP5-deficient strain (Fig. 4C). The percentage of
dead cells was however significantly higher ( P > 0.05) in
cells infected with rNVI15-DVP5 compared to cells infected
with rNVI15-15K at 42, 48, and 54 h p.i. (Fig. 4D). A
relatively high number of dead cells were observed both for
virus infected and mock-infected cells at the 0-h time point,
this is probably related to rough handling of the cells during
harvest.
Induction of apoptosis in vivo
To investigate the role of apoptosis in the pathogenesis of
IPN, tissue sections of liver and pancreas from Atlantic
salmon post-smolts experimentally infected with rNVI15,
rNVI15-15K, and rNVI-DVP5 viruses were sampled for
histopathological examination. In liver samples harvested 7
days after infection, single cell death of hepatocytes
morphologically resembling apoptosis was detected in fish
challenged with all three recombinant viruses. After 9 days
and onwards, apoptotic cells were found scattered in the
liver parenchyma of most fish examined, except for control
fish (Fig. 5). Apoptosis was confirmed by in situ labeling of
DNA fragmentation using the TUNEL assay. Positive cells
were detected in the liver of fish infected with all these
viruses, but not in control fish (Fig. 6). Morphological
evidence of apoptosis was also detected in the exocrine
pancreas of infected cells; however, intensive viral replication followed by secondary necrosis and high enzymatic
activity in the exocrine pancreatic cells resulted in high level
of background staining in the TUNEL-labeled sections (data
not shown). In liver sections, the distribution of viral antigen
detected by immunohistochemistry seemed to overlap the
distribution of apoptotic cells (Fig. 7). This observation was
verified using double staining with TUNEL and IPNV
immunohistochemistry. Viral antigen and apoptotic cells codistributed, and numerous double-positive cells were
detected, although the darker stain of the TUNEL assay
(black color) seemed to mask some of the IPNV immunostaining (red color) (Fig. 8).
Sequence alignments of IPNV VP5
Results from the alignment of VP5 protein of rNVI1515K and rNVI15 viruses with cellular and viral Bcl-2
homologous proteins revealed structural differences in the
BH2 domain of VP5 compared to the E1S strain used by
Hong et al. (2002) (Fig. 9). Recombinant rNVI15 virus has a
premature stop codon at nucleotide 427 and encodes a
putative 12-kDa VP5, corresponding to a BH2 deletion
mutant. Several amino acid substitutions were detected in
the BH2 domain of rNVI15-15K-VP5 when compared to
IPNV-E1S. In particular, two arginine (Arg) residues (115
plus 121) of rNVI15-15K-VP5 have replaced two tryptophan (Trp) residues (129 plus 135) of the E1S-VP5 BH2
homology domain (Fig. 9).
Discussion
In this study, we demonstrate for the first time the
induction of apoptosis in hepatocytes of IPNV-infected
Atlantic salmon post-smolt. In a previous study of IPNVinfected rainbow trout (Oncorhynchus mykiss) fry, apoptotic
nuclei were not clearly demonstrated in the pancreas but
were widespread in the lamina propria of the intestine
N. Santi et al. / Virology 342 (2005) 13 – 25
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Fig. 3. Agarose gel electrophoresis of DNA isolated from CHSE-214 cells infected with three recombinant IPNV – Sp VP5 mutants at an MOI = 1.00. (A) Cells
were collected at 14, 16, 18, 20, and 24 h post-infection. (B) Cells were collected 20, 24, 28, 32, and 36 h post-infection. Lane M: 1 kb+ marker. Lane C:
uninfected control cells.
(Eléouët et al., 2001). Further, in a previous IPNV challenge
study with Atlantic salmon fry, diffuse necrosis of hepatic
and exocrine pancreatic tissues was found in diseased fish
(Santi et al., 2004). These lesions developed over short time
span, involving most of the liver and pancreas within a day
or two after onset of mortality. Minor lesions were detected
in the gastric glands, and in contrast to the necrosis found in
liver and pancreas, the affected cells in gastric glands often
displayed the morphological features of apoptotic cells
(Santi et al., 2004).
Host cell apoptosis is part of the innate immune response
to virus infection, serving to limit the progeny production by
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N. Santi et al. / Virology 342 (2005) 13 – 25
Fig. 4. CHSE-214 cells were mock infected or infected with various recombinant IPNV strains at an MOI = 1.00 and incubated at 15 -C before annexin V/
propidium iodide staining. (A) Percentage of apoptotic cells (Annexin-V-positive/propidium-iodide-negative cells) at different time points in cell cultures
infected with IPNV – Sp strains encoding the full-length VP5 protein (rNVI15-15K), a putative 12-kDa VP5 (rNVI15), and a VP5-deficient strain (rNVI15DVP5). (B) Percentage of dead cells (annexin V/propidium iodide double-positive cells) at corresponding time points. (C) Percentage of apoptotic cells at
different time points in CHSE-214 cells infected with virus stocks of one VP5-encoding (rNVI15-15K) and one VP5-deficient (rNVI15-DVP5) strain free of
cell-culture-adapted mutants. (D) Percentage of dead cells at corresponding time points. In panels A and C, the percentage of apoptotic cells is calculated from
the population of viable cells after the necrotic propidium iodide has been gated away. In panels B and D, the percentage of necrotic cells is calculated from the
total number of cells.
the virus. Double-stranded RNA (dsRNA) formed during
viral replication, in addition to being a potent inducer of
interferon (IFN), activates protein kinase R (PKR), leading
to phosphorylation of the eukaryotic translational initiation
factor 2 (eIF2a) and inhibition of protein synthesis. Further,
dsRNA also induces apoptosis through PKR activation, and
IFN sensitizes cells to this dsRNA-mediated apoptosis
(Barber, 2001; Clemens, 2003; Gil and Esteban, 2000;
Kibler et al., 1997). Evidence points to the activation of the
PKR pathway in IPNV-infected fish cells, as IPNV infection
in RTG-2 cells leads to increased eIF2a phosphorylation
(Garner et al., 2003). This suggests that apoptosis induction
in IPNV-infected cells could be linked to the activation of
PKR. However, IPNV induces non-typical apoptotic cell
morphology in CHSE-cells, characterized by the formation
of small holes in the membrane, associated with loss of
integrity, finally leading to post-apoptotic necrosis (Hong et
al., 1999). It is conceivable that in the battle between host
cell and virus, the cell, in an effort to limit the viral
replication, responds by induction of apoptosis. The virus,
on the other hand, will aim at producing as many progeny as
possible, with a final release of virus particles from the
infected cell through lysis/necrosis. The outcome for an
IPNV-infected cell might depend upon the host cell’s ability
N. Santi et al. / Virology 342 (2005) 13 – 25
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Fig. 5. HE staining of liver sections from Atlantic salmon post-smolts mock infected (A) or infected with IPNV strains rNVI15-15K (B), rNVI15 (C), or
rNVI15-DVP5 (D). Single rounded cells with condensed cytoplasm and chromatin aggregation morphologically resembling apoptotic cells (arrows) is seen in
the livers of IPNV-infected fish, but not in control fish. The livers sections shown were selected from representative fish sampled at 20 (A), 10 (B), 14 (C), and
13 (D) days p.i., respectively.
to mount an apoptotic response to the virus infection. If true,
this may provide an explanation for the differences observed
in the liver pathology of IPNV infected fry compared to
post-smolts. If the virus has the upper hand, it will replicate
at high rate, resulting in production of a high number of
progeny, causing cell lysis and necrosis at the end. Rapidly
developing diffuse liver necrosis is typically seen in IPNV
infected fry. When the balance is in favor of the host cell,
Fig. 6. TUNEL assay of liver sections from Atlantic salmon post-smolts mock infected (A) or infected with IPNV strains rNVI15-15K (B), rNVI15 (C), or
rNVI15-DVP5 (D). TUNEL-positive cells (black) are seen in the livers of infected fish, but not in control fish. Aggregation of chromatin, characteristic for
apoptotic cells, is observed (arrows). The livers sections shown were selected from representative fish sampled at 20 (A), 10 (B), 14 (C), and 13 (D) days p.i.,
respectively.
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N. Santi et al. / Virology 342 (2005) 13 – 25
Fig. 7. Immunohistochemistry of liver sections from Atlantic salmon post-smolts mock infected (A) or infected with IPNV strains rNVI15-15K (B), rNVI15
(C), or rNVI15-DVP5 (D). IPNV antigen-positive cells (red) are seen in the livers of infected cells, but not in control fish. Some of the IPNV-positive cells
resemble apoptotic cells (arrows). The livers sections shown were selected from representative fish sampled at 20 (A), 10 (B), 14 (C), and 13 (D) days p.i.,
respectively.
it will enter into apoptosis at an early stage of the
infection and limit the production and spread of virus
progeny. This is in line with the pathological changes
observed in post-smolt livers. Furthermore, interferon
sensitizes cells to the apoptosis-inducing effect of dsRNA;
consequently, the cells localized around the IPNV-infected
cell would respond quicker to the viral infection than the
cell initially infected. This is in accordance with our
observation that many TUNEL-positive cells encircle the
IPNV-positive cells in the liver tissues of post-smolts, and
Fig. 8. Dual labeling of liver sections from Atlantic salmon post-smolts mock infected (A) or infected with IPNV strains rNVI15-15K (B), rNVI15 (C), or
rNVI15-DVP5 (D) by TUNEL assay and IPNV immunohistochemistry. Viral antigen and apoptotic cells appear to co-distribute, and some double-labeled cells
are visible (arrows). The livers sections shown were selected from representative fish sampled at 20 (A), 10 (B), 14 (C), and 13 (D) days p.i., respectively.
N. Santi et al. / Virology 342 (2005) 13 – 25
21
Fig. 9. Sequence and structural homology of cellular and viral Bcl-2-related proteins. GenBank accession numbers for the protein sequences used are listed
below. Homo sapiens Bcl-2 (Human Bcl-2), P10415; Gallus gallus Bcl-2 (Chicken Bcl-2), BAA01978; H. sapiens Bcl-XL (Human Bcl-XL), CAA80661;
Danio rerio Bcl-XL (Zebrafish Bcl-XL), AAK81706; H. sapiens Mcl-1 (Human Mcl-1), BC017197; IPNV E1S NS protein, AAK71697; Epstein – Barr virus
BHRF1 (EBV Bhrf-1), QQBE4; human adenovirus type 9 E1B 19K (E1B19K), AAD16304; African swine fever virus LMV-5HL (ASFV 5-HL), AAA17034.
( ) Negative charge, (+) positive charge, (l) aliphatic, (t) tiny, (a) aromatic, (c) charged, (s) small, (p) polar, (h) hydrophobic, (b) big.
also in line with previous findings in IBDV-infected
chickens (Jungmann et al., 2001).
In persistently infected fish, IPNV is detected in
macrophages (Johansen and Sommer, 1995; Munro et
al., 2004). In a recent study of persistently infected
rainbow trout, IPNV-positive cells in leucocyte smears
from peripheral blood, head kidney, and spleen did not
have apoptotic nuclei (Imajoh et al., 2005). Assuming the
IPNV-positive cells to be macrophages, a possible
explanation for the lack of apoptotic morphology in these
cells is that differentiated macrophages are robust, longlived cells and resistant to numerous death stimuli
(Mangan et al., 1991; Munn et al., 1995; Perlman et al.,
1999). An alternative explanation for this observation is
that persistent infection of macrophages is associated with
specific inhibition of host-induced apoptosis.
Apoptosis was induced in hepatocytes of fish infected
with each of these three VP5 mutants. The absence of VP5
expression did not result in increased apoptosis frequency in
hepatocytes of fish infected with rNVI15-DVP5, as judged
by observation alone. An attempt to quantitatively measure
the level of apoptosis induction by the three VP5 variant
viruses was not pursued due to large individual variation in
the morphological appearances of livers in diseased fish.
Various assays were used in the studies of cell cultures
infected with the recombinant viruses. The results from the
LDH-assay demonstrated that, unlike VP5 of IBDV, IPNVVP5 expression did not have any detectable effect on cell
membrane permeability. However, the uptake of virus
apparently triggered a transient reduction in membrane
permeability, leading to a reduction in LDH leakage from
IPNV-infected cells, as compared to control cells at 16 and 20
h p.i. This is an interesting finding, as it indicates changes in
the membrane dynamics of the cell caused either by early
apoptotic responses in the host cell or by early replication of
the virus.
We did not observe differences in the morphological
appearance of cells infected with the VP5 mutant strains. No
delay in the induction of internucleosomal cleavage was
detected in cells infected by VP5-expressing strains
compared to the VP5-deficient strain, using the agarose
gel electrophoresis assay. In the first flow cytometry
experiment, Annexin-V-positive cells appeared at an earlier
time in cell cultures infected with the VP5-deficient IPNV
strains compared to the VP5-expressing strains, indicating a
slight anti-apoptotic effect of VP5 expression. However, this
was probably due to an unevenly distributed presence of
cell-culture-adapted mutants, as no difference could be
detected in apoptosis among cells infected with the VP5encoding and VP5-deficient strain in the second experiment.
The percentage of necrotic cells was still slightly higher in
cell cultures infected with the VP5 knockout mutant at the
later time points of the second experiment. The significance
of this finding is uncertain.
Using the annexin V/propidium iodide flow cytometric
assay, the percentage of apoptotic cells did not rise above
20% at any of the time points examined. It is however
important to keep in mind that, in contrast to cells expressing
IPNV antigen or being necrotic (propidium iodide positive)
cells, the apoptotic (annexin V positive/propidium iodide
negative) cells will not accumulate in infected cell cultures.
Cells are annexin V positive/propidium iodide negative only
for a short period, as the duration of the whole event of
apoptosis takes no more than a few hours. In the first paper
describing the annexin V/propidium iodide method for
detection of apoptotic cells, the maximum percentage of
apoptotic cells in relation to time elapsed was 24.5% after
irradiation and 8% after dexamethasone treatment (Vermes et
al., 1995). Parvovirus B19-induced apoptosis of hepatocytes
is shown to be infectious dose dependent, where an MOI of
10 leads to 10% apoptotic cells and an MOI of 1000 gives
27% apoptotic cells as measured by annexin V staining
(Poole et al., 2004).
In summary, no anti-apoptotic effect of VP5 was detected
for the IPNV serotype Sp strains studied. This is not in
accordance with the previously reported anti-apoptotic
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N. Santi et al. / Virology 342 (2005) 13 – 25
effect of VP5. Having said this, there are important
structural differences in the VP5 encoded by IPNV –Sp
strains compared to the E1S strain used in the study by
Hong and coworkers (Fig. 9). The BH2 domain of Bcl-2 is
required for inhibition of apoptosis and heterodimerization
with Bax (Hunter et al., 1996; Yin et al., 1994). Deletion of
the BH2 domain alone or both BH2 and BH1 domains
causes a loss of the anti-apoptosis function in the IPNV-E1S
VP5 (Hong et al., 2002). The putative 12-kDa VP5 encoded
by rNVI15 would correspond to a BH2 deletion mutant,
which could result in a complete or partial loss of antiapoptotic activity.
The carboxy-terminal part of the 15-kDa VP5 encoded
by rNVI15-15K is equivalent (in size) to the E1S-VP5.
However, in rNVI15-15K-VP5, two arginine (Arg) residues
(115 plus 121) have replaced two tryptophan (Trp) residues
(129 plus 135) of the E1S-VP5 BH2 homology domain
(Fig. 9). The first of these Trp residues corresponds to the
functionally important Trp 188 in Bcl-2, and replacement of
this residue with alanine (Ala) abrogates Bcl-2’s inhibition
of apoptosis (Yin et al., 1994). For Epstein –Barr virus
(EBV) Bcl-2 homolog BHRF1, it was shown that substitution of Trp-143 (corresponding to Trp-188 in Bcl-2)
with Ala leads to loss of the ability to confer protection
against cis-plantin-induced apoptosis (Khanim et al., 1997).
Thus, it cannot be excluded that the 15-kDa VP5 of rNVI1515K corresponds to a functional BH2 deletion mutant,
resulting in a reduction of or loss of the anti-apoptotic
function of E1S VP5. Furthermore, to the extent that the
anti-apoptotic activity of VP5 has any impact on the
virulence characteristics of IPNV, these observations could
explain the identical virulence characteristics of the three
VP5 variant viruses, as they may represent functionally
identical virus strains. Interestingly, an alignment of the
VP5-encoding sequence of twelve marine birnaviruses and
three IPNV strains (Zhang and Suzuki, 2004) shows that all
isolates encode Trp in VP5, which corresponds to the
functionally important Trp 188 in Bcl-2.
The data presented indicate a deficit in the anti-apoptotic
function of VP5 in the IPNV – Sp strains studied. Further
studies are needed to evaluate the importance of Trp residue
in the BH2 homologous domain for possible loss of
function.
strain rNVI15, recovered using reverse genetics (Santi et al.,
2005), is identical to the virulent field strain NVI-015
(GenBank accession nos. AY379740 and AY379741). It
encodes a putative 12-kDa VP5 due to a premature stop
codon at nucleotide position 427. Recombinant rNVI1515K virus encodes the full-length 15-kDa protein, whereas
rNVI15-DVP5 contains a mutation in the initiation codon of
VP5 gene, which ablates the expression of VP5. Viruses
were propagated by inoculation of the second (fish experiment) or third (cell culture experiments) passage supernatant
into CHSE-214 cells, followed by incubation at 15 -C.
When the cytopathic effect (CPE) was evident, cell culture
supernatants were harvested after a brief centrifugation.
Virus stocks free of cell-culture-adapted mutants were
prepared by inoculation of RTG-2 cells and confirmed by
sequencing as previously described (Song et al., 2005). For
fish challenge, the infectious titre was determined by end
point dilution on CHSE-214 cells grown in 96-well plates.
Fifty microliters of 10-fold dilutions (10 1 to 10 8) of cell
culture supernatants was used in six parallel wells per
dilution. The TCID50 was estimated by the method of
Kärber (1931). For in vitro assays, infectious titre was
determined by plaque assay. Briefly, monolayers of CHSE214 grown in 6-well plates were inoculated with 10-fold
dilutions (10 3 to 10 8) of cell culture supernatants. After 1
h incubation at 15 -C, the inoculum was removed and the
cells overlaid with 0.8% SeaPlaque Agarose (BioWhittaker)
in L-15 medium containing 5% FBS and 1% l-glutamine.
The cells were incubated for 3 days at 15 -C, and then fixed
using methanol and acetic acid (v/v 3:1). Overlays were
removed and the plaques were counted after staining the
monolayers with Giemsa (Sigma).
Observation of morphological changes in cell culture
CHSE-214 cells were grown in 24-well plates until 80%
confluence and infected with the three IPNV strains at a
multiplicity of infection (MOI) of 1.00 virus particles per
cell. Three parallel wells were examined for each IPNV
strain. After incubation at 15 -C, the cellular morphology
was observed using phase contrast microscopy (Olympus) at
0, 24, 32, and 36 h. Parallel experiments were performed at
MOI = 0.1 and MOI = 10.
Cytotoxicity and apoptosis assays
Materials and methods
Cells and viruses
Chinook salmon embryo cells (CHSE-214) were obtained
from the American Type Culture Collection (ATCC) and
maintained at 20 -C in Leibovitz’s L-15 medium (BioWhittaker) supplemented with 5% fetal bovine serum (FBS) and
25 Ag/ml gentamicin. Rainbow trout gonad cells (RTG-2,
ATCC CCL-55) were grown in L-15 medium supplemented
with 10% FBS at 20 -C. The recombinant IPNV serotype Sp
Leakage of cytoplasmatic contents due to damage of
the plasma membrane of infected cells was analyzed using
cytotoxicity detection kit (Roche). CHSE-214 cells were
seeded onto 96-well plates and grown until 80%
confluence and infected with rNVI15, rNVI15-15K, and
rNVI-DVP5 viruses at MOI = 1.00. After 0, 8, 16, 20, 24,
28, 32, 36, 40, 48, and 54 h of incubation, the
supernatants were collected and the lactate dehydrogenase
(LDH) activity was measured according to the manufacturer’s instructions.
N. Santi et al. / Virology 342 (2005) 13 – 25
DNA fragmentation assay was performed using apoptotic
DNA ladder kit (Roche) in accordance with the manufacturer’s instructions. Briefly, CHSE-214 cells were grown in
6-well plates and infected with rNVI15, rNVI15-15K, or
rNVI15-DVP5 viruses at MOI = 1.00. After 14, 16, 18, 20,
24, 28, 32, and 36 h of incubation, both detached and
adherent cells were collected, resuspended in binding/lysis
buffer, and incubated at room temperature for 10 min. DNA
was isolated using a column provided with the kit and eluted
in a total volume of 200 Al. After RNase A digestion (25 Ag/
ml) for 20 min at room temperature, DNA was dried and
dissolved in 30 Al dH2O. The samples were loaded onto a
1% agarose gel and electrophoresed at 50 V for 2 h,
followed by 70 V for 2 h. Gels were stained with ethidium
bromide and photographed under UV transillumination.
FITC – annexin V/PI double staining
The initial experiment was performed with CHSE-214
cells grown in 24-well plates and mock infected or
infected with rNVI15, rNVI15-15K, or rNVI15-DVP5
viruses (MOI = 1.00) and incubated at 15 -C for 0, 16,
20, 24, and 32 h. Both adherent and floating cells were
collected and washed with PBS before annexin V/propidium
iodide (PI) treatment, using the Annexin-V-FLUOS staining
kit in accordance with the manufacturer’s instructions
(Roche). Samples were analyzed on a FacsCalibur flow
cytometer (Becton Dickinson), and the WinMDI 2.8 software (J. Trotter) was used to determine the percentages of
viable, apoptotic, and necrotic cells. In short, the PI/annexin
V double-positive cells were gated and defined as necrotic
cells, and the remaining cell population was defined as viable
cells and investigated for the expression of annexin V. The
percentage of annexin V binding cells at the different time
point was then calculated. In the following experiment with
virus stocks free of cell-culture-adapted mutants, CHSE-214
cells grown in 24-well plates were mock infected or infected
with rNVI15-15K or rNVI15-DVP5 at MOI = 1. Due to the
low infectivity of non-attenuated viruses (Song et al., 2005),
cells were harvested after 0, 30, 36, 42, 48, and 54 h p.i.
Three parallel wells were sampled for each time point. Cells
were further analyzed as described above, except that the
staining reagents were supplied from a different manufacturer (annexin V – FITC apoptosis detection kit I, BD
Pharmingen).
Atlantic salmon post-smolt challenge
The challenge study with three VP5 mutant IPNVs was
described previously (Santi et al., 2005). The challenge was
conducted at VESO Vikan’s research facility in Namsos,
Norway, and approximately 2200 Atlantic salmon (S.
salar L.) parr (average weight 70 g) going through
smoltification were used in the study. Fish were challenged
by 1 h immersion with IPN virus at a concentration of 104
TCID50/ml. Fish in three parallel tanks were infected with
23
each of the three recombinant IPNV isolates, while fish in
two parallel tanks were given cell culture medium only and
kept as controls. One tank in each group was reserved for
sampling, from which three fish were collected each day and
organ specimens of pancreas and liver were fixed in 10%
phosphate-buffered formalin. Mortality was recorded on a
daily basis in the remaining tanks. The experiment was
terminated at 30 days post-challenge.
Immunohistochemistry and in situ detection of apoptotic
cells
The formalin-fixed tissue samples were embedded in
paraffin according to standard procedures. Tissue sections
were routinely stained with hematoxylin and eosin (HE) and
examined by light microscopy. Cells from liver samples
were observed specifically for morphological changes
characteristic for apoptosis, such as detachment from
neighboring cells, rounding, chromatin condensation, and
formation of apoptotic bodies. Selected liver samples with
representative changes (from minimum three fish in each
group) were examined by immunohistochemistry (IHC) and
terminal deoxynucleotidyl transferase mediated dUTP nickend-labeling (TUNEL) assay. These assays were performed
both independently and as double labeling on parallel liver
sections. IHC was performed as previously described (Santi
et al., 2004). Briefly, after blocking using bovine serum
albumin (BSA), the slides were incubated with a polyclonal
rabbit antiserum specific for IPNV at a dilution of 1:1000 for
30 min at room temperature, followed by the secondary
antibody (biotinylated anti-rabbit immunoglobulin diluted
1:300) for 30 min at room temperature. After incubation
with Streptavidin – biotin alkaline phosphatase (1:1000
dilution) for 1 h at 37 -C, staining was done with Fast
Red substrate (Sigma). Sections were counterstained with
hematoxylin and mounted in aqueous mounting medium
(Aquamount; BDH Laboratory). The TUNEL assay was
performed with the in situ cell death detection kit, POD
(Roche). Tissue sections were permeabilized by proteinase
K digestion (20 Ag/ml, 10 mM Tris –HCl, pH 7.4) for 20 min
at 37 -C. After washing with PBS, endogenous peroxidase
activity was blocked by incubation with 3% H2O2 in
methanol, before labeling of fragmented cellular DNA with
fluorescein-dUTP by the terminal deoxynucleotidyl transferase. Detection was carried out by incubation with antifluorescein antibody conjugated with horseradish peroxidase and diaminobenzidine staining with nickel/cobalt
enhancement. Sections were counterstained with hematoxylin. In double labeling, viral antigens were first detected by
IHC followed by identification of apoptotic cells using the
TUNEL assay.
Sequence alignments of IPNV VP5
The predicted amino acid sequences for the BH3, BH1,
and BH2 domain in VP5 protein of rNVI15 and rNVI15-
24
N. Santi et al. / Virology 342 (2005) 13 – 25
15K viruses were aligned to the corresponding domains
from several cellular Bcl-2 proteins, including human and
chicken Bcl-2, human and zebrafish Bcl-XL, and human
Mcl-1. They were also aligned to the proposed BH3, BH1,
and BH2 domains of viral Bcl-2 homologues proteins,
including the IPNV E1S strains, Epstein – Barr virus
BHRF1, human adenovirus type 9 E1B-19K, and African
swine fever virus LMV-5HL. The alignments were carried
out using the ClustalX program (Thompson et al., 1997).
Initial computer-assisted alignment of full-length proteins
were followed by several rounds of alignment of shorter
stretches of these proteins to arrive at an optimized
alignment, based on our knowledge of the structure of the
Bcl-2 proteins. The final alignment was printed with the aid
of the CHROMA software (Goodstadt and Ponting, 2001).
Acknowledgments
This study was supported by the Norwegian Research
Council, grant #134136/120 and #146790/120 to Ø.E.; by
the National Research Initiative of the USDA Cooperative
State Research, Education and Extension Service, grant
# 2001-35204-10065 to V.N.V; and by the Taiwan National
Science Council, grant # NSC-92-2313-B-001-011 to J.L.W.
The authors wish to thank Dan Torkehagen, Department
of Basic Sciences and Aquatic Medicine, Norwegian School
of Veterinary Science, for assistance in sampling of the postsmolts after challenge.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.virol.2005.
07.028.
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