Download Medaka haploid embryonic stem cells are susceptible to Singapore

Journal of General Virology (2013), 94, 2352–2359
DOI 10.1099/vir.0.054460-0
Medaka haploid embryonic stem cells are
susceptible to Singapore grouper iridovirus as well
as to other viruses of aquaculture fish species
Yongming Yuan,1 Xiaohong Huang,2 Lei Zhang,1 Yi Zhu,1
Youhua Huang,2 Qiwei Qin2 and Yunhan Hong1
Correspondence
1
Yunhan Hong
2
[email protected]
Qiwei Qin
Department of Biological Sciences, National University of Singapore, Singapore
Key Laboratory of Marine Bio-resources Sustainable Utilization, South China Sea Institute of
Oceanology, Chinese Academy of Sciences, Guangzhou, PR China
[email protected]
Received 17 April 2013
Accepted 30 June 2013
Viral infection is a challenge in high-density aquaculture, as it leads to various diseases and
causes massive or even complete loss. The identification and disruption of host factors that
viruses utilize for infection offer a novel approach to generate viral-resistant seed stocks for costefficient and sustainable aquaculture. Genetic screening in haploid cell cultures represents an
ideal tool for host factor identification. We have recently generated haploid embryonic stem (ES)
cells in the laboratory fish medaka. Here, we report that HX1, one of the three established medaka
haploid ES cell lines, was susceptible to the viruses tested and is thus suitable for genetic
screening to identify host factors. HX1 cells displayed a cytopathic effect and massive death upon
inoculation with three highly infectious and notifiable fish viruses, namely Singapore grouper
iridovirus (SGIV), spring viremia of carp virus (SVCV) and red-spotted grouper nervous necrosis
virus (RGNNV). Reverse transcription-PCR and Western blot analyses revealed the expression of
virus genes. SGIV infection in HX1 cells elicited a host immune response and apoptosis. Viral
replication kinetics were determined from a virus growth curve, and electron microscopy revealed
propagation, assembly and release of infectious SGIV particles in HX1 cells. Our results
demonstrate that medaka haploid ES cells are susceptible to SGIV, as well as to SVCV and
RGNNV, offering a unique opportunity for the identification of host factors by genetic screening.
INTRODUCTION
Food demand is increasing steadily worldwide, and
innovative technologies are urgently needed to meet future
food demands. With an annual growth of 6.9 % from 1970
to 2007, aquaculture has been the fastest-growing animal
food-producing sector (FAO, 2008), now representing a
major global industry with total production exceeding 50
million t and estimated values of almost US$80 billion
(FAO, 2009). A major challenge in aquaculture is viral
infection, which brings about massive death in larval
production (Walker & Winton, 2010). Viral infection
becomes more serious in high-density aquaculture, because
it often causes massive or even complete loss. Little is known
about treatments for fish viral diseases. The development of
virus-controlling biotechnologies holds enormous potential
for cost-efficient and sustainable aquaculture to meet the
increasing demands for fish production.
The identification of host factors that viruses used for
infection is essential to elucidate the mechanisms by which
One supplementary figure, one table and two data figures are available
with the online version of this paper.
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these viruses cause diseases. Likewise, an understanding of
how viruses depend on host factors to complete the
infectious cycle may accelerate the development of antiviral
strategies. Most intriguingly, targeted disruption of genes
encoding host factors essential for viral infection would
represent a revolutionarily novel biotechnology to create
virus-resistant cells and even virus-resistant whole animals.
However, host factors for viral infection have remained
largely undetermined. One of the exceptions in aquatic
organism is the laminin receptor, which acts as a binding
protein for Taura syndrome virus infection in the whiteleg
shrimp and whose knockdown leads to organism lethality
(Stockwell, 2002).
Genetic screening is a powerful tool to identify genes
essential for diverse cellular processes through the production of genome-wide mutations with discernible phenotypes. The best example is the classical work pioneered by
Muller (1927), which induced numerous mutations and
led to the unbiased elucidation of genes crucial for
biological processes in Drosophila. The elusiveness of the
generation and recovery of biallelic mutations in diploid
cells is a major barrier to the application of this approach
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Viral susceptibility of medaka haploid ES cells
in vertebrate organisms. Haploid cells offer a powerful
system for genetic analyses of molecular events because any
recessive mutations of essential genes will show a clear
phenotype due to the absence of a second gene copy.
Carette et al. (2009) initially demonstrated the power of
haploid genetic screening in KBM7, a near-haploid human
chronic myeloid leukaemia cell line (Kotecki et al., 1999).
By generating a library of KBM7 mutant cells using a genetrap retrovirus and screening for mutant cells resistant to a
range of bacterial toxins and cytotoxic viruses, they
identified host genes required for cell death by the toxins
or viruses (Carette et al., 2009, 2011b). Specifically, human
cytidine monophosphate N-acetylneuraminic acid synthetase (CMAS) and solute carrier family 35 (UDP-galactose
transporter), member A2 (SLC35A2) were thus identified
as host factors essential for influenza infection (Carette
et al., 2009), and the cholesterol transporter Niemann–Pick
C1 as essential for Ebola virus entry (Carette et al., 2011b).
This near-haploid cell line has also been used to assign
human genes to phenotypes by global gene disruption
(Carette et al., 2011a).
We recently generated three genuine haploid embryonic
stem (ES) cell lines HX1–HX3 from the laboratory fish
medaka (Oryzias latipes) and demonstrated the applicability of these haploid ES cells for whole animal
production by semi-cloning (Yi et al., 2009). This was
followed by similar success in mouse haploid ES cell
derivation and semi-cloning (Leeb & Wutz, 2011; Li et al.,
2012; Yang et al., 2012). The availability of medaka
haploid ES cells offers a unique opportunity for haploid
genetic screening in fish.
So far, more than 25 virus species have been reported in
diverse fish species of importance in aquaculture. Nine are
listed by the Office of International Epizootic (OIE) as
highly infectious and notifiable viruses (http://www.oie.
int). These include Singapore grouper iridovirus (SGIV),
spring viremia of carp virus (SVCV) and viral nervous
necrosis virus (VNNV). SGIV is a highly infectious
pathogen that causes massive death in wild and farmed
groupers and many other marine teleosts (Qin et al., 2003),
and its genome is a circular dsDNA of 140 131 bp and
predicts 162 ORFs (Song et al., 2004). We reported recently
that SGIV induces paraptosis-like cell death in its natural
host species such as groupers but apoptosis in cells of nonhost species (Huang et al., 2011a). SVCV has an 11 kb
RNA genome and causes massive death by haemorrhaging
and necrosis of internal organs in many fresh water species
of the families Cyprinidae, Siluridae, Salmonidae, Esocidae,
Centrarchidae and Poeciliidae in many countries. VNNV is
non-enveloped, has a 4.9 kb RNA genome and causes a
highly destructive disease in larvae of more than 30 marine
fish species from 14 families of the order Perciformes. A
total of five isolates have been reported for VNNV, which
originate from the striped jack, barfin flounder, tiger
puffer, red-spotted grouper [red-spotted grouper nervous
necrosis virus (RGNNV)] and orange spotted grouper
(Epinephelus coioides). Susceptibility to RGNNV has been
http://vir.sgmjournals.org
reported for medaka fry (Furusawa et al., 2006) and
differentiated somatic cell lines (Adachi et al., 2010).
As a prelude for genetic screening for host factors that
pathogens use for infection in aquaculture-important fish
species, this study aimed to test the viral susceptibility of
medaka haploid ES cells. We showed that medaka haploid
ES cells are susceptible to SGIV, as well as to SVCV and
RGNNV. By gene expression profiles and light microscope,
we demonstrated that SGIV infection in HX1 cells was able
to elicit an immune response and apoptosis. Most
importantly, we revealed by growth curve analysis and
electron microscopy that HX1 cells are susceptible to SGIV
and produce infectious particles following virus replication. Our data establish medaka haploid ES cells as a
unique system for the identification of host factors by
genetic screening.
RESULTS
Viral susceptibility screening using a cytopathic
effect (CPE) assay
To screen the candidate viruses with potential infectivity
towards medaka stem cells, we tested nine viruses that are
classified as highly infectious and notifiable viruses by the
OIE. Our results indicated that three of the nine listed
viruses were able to cause a distinct CPE in the medaka
haploid ES cell line (Table 1). In SGIV-infected HX1 cells,
typical CPE was observed at 3 days post-inoculation (p.i.)
and became more evident at 4 days p.i., when control cells
retained their normal shape and grew into a full
monolayer, whereas the majority of SGIV-infected cells
lost attachment to the substrata and started to collapse or
lyse (Fig. 1a, a9). A similar CPE was found in the medaka
diploid ES cell line MES1 (Fig. 1b, b9) and the diploid
spermatogonial cell line SG3 (Fig. 1c, c9). CPE was also
observed in HX1 cells infected with SVCV (Fig. S1a,
available in JGV Online) and RGNNV (Fig. S1b).
Molecular analyses of viral RNA and protein
expression
Based on the above CPE screening, the potential
susceptibility of medaka ES cells to SGIV, SVCV and
RGNNV was verified further by reverse transcription-PCR
(RT-PCR) and Western blotting. RT-PCR analysis
revealed that HX1 cells at 4 days p.i. expressed viral
structural genes from SGIV, SVCV and RGNNV (Fig. 2a).
In SGIV-infected medaka cells of the HX1, MES1 and SG3
lines, Western blotting revealed the correlative expression
of viral protein ORF018 (NCBI Protein no. YP_164113.1)
and ORF158 (NCBI Protein no. YP_164253.1) (Fig. 2b).
The former encodes a highly conserved structural protein
identified in mature virions (Wang et al., 2008) and the
latter encodes an SGIV-specific histone H3-binding
protein (Tran et al., 2011). A time-course detection of
viral gene expression level in HX1 was performed with
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Y. Yuan and others
Table 1. Virus CPE screening in medaka haploid ES cells*
Class
Family
Virus name (abbreviation)
DNA virus
Iridoviridae
RNA virus
Herpesviridae
Rhabdoviridae
CPE
Infectious spleen and kidney necrosis iridovirus (ISKV)
Red sea bream iridovirus (RSIV)
Singapore grouper iridovirus (SGIV)
Koi herpes virus (KHV)
Infectious haematopoietic necrosis virus (IHNV)
Spring viraemia of carp virus (SVCV)
Viral haemorrhagic septicemia virus (VHSV)
Infectious pancreatic necrosis virus (IPNV)
Red-spotted grouper nervous necrosis virus (RGNNV)
Birnaviridae
Nodaviridae
No
No
Yes
No
No
Yes
No
No
Yes
*In the OIE list of highly infectious and notifiable viruses, 25 fish viruses have been reported. They belong to seven families: Iridoviridae (ten),
Herpesviridae (three), Nodaviridae (four), Rhabdoviridae (three), Birnaviridae (one), Orthomyxoviridae (one) and Reoviridae (three).
semi-quantitative RT-PCR. The transcription of immediate-early gene orf086 (Xia et al., 2009) could be detected
as early as 2 h p.i., whereas the RNA of early gene orf158
and late gene orf018 was not detectable until 4 and 8 h p.i.,
respectively (Fig. 2c). The above results demonstrated
SGIV entry and gene expression in medaka stem cells
including haploid HX1 cells.
Mock
(a)
4 days post SGIV infection
(a′)
HX1
*
*
*
*
(b)
(b′)
MES1
*
*
*
(c)
(c′)
SG3
*
*
Fig. 1. CPE of SGIV in medaka stem cell cultures. Haploid ES cell
line HX1 (a, a9), diploid ES cell line MES1 (b, b9) and diploid
spermatogonial stem cell line SG3 (c, c9) at ~60 % confluency
were mock infected (control) or infected with SGIV and analysed
microscopically at 4 days p.i. Control cells showed normal growth
into a full monolayer with a round cell phenotype. SGIV-infected
cells display typical CPE, as evidenced by loss of attachment and
shrinkage (arrows), as well as lysis and collapse into dead cell
debris (asterisks). Bars, 20 mm.
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Host response at the molecular level
It has been shown that SGIV induces apoptosis in cells of
non-host species such as fathead minnow but paraptosislike cell death in cells of natural host species such as
grouper spleen cells (Huang et al., 2011a). We wanted to
examine the host-cell response at the molecular level by
determining the time-course expression of a selected set of
cellular genes (Fig. 2c). Five medaka genes homologous to
those involved in the immune response were identified by
sequence comparison, which included interferon (ifn), mx,
stat1 and viperin (vpr) (Fig. 2c). Interestingly, induction of
RNA expression was found for all of these immuneresponsive genes as early as 2 h p.i. but was undetectable in
the mock-inoculated control (not shown). The four
immune-responsive genes fell into three groups according
to their induced expression patterns. The first group
included ifn and mx, which displayed a steadily increasing
or persistent high level of expression throughout the period
examined. The second was represented by stat1, whose
RNA level peaked at 2 h p.i. and decreased subsequently.
The third was vpr, which was upregulated at 2 h p.i. and
persisted at a comparably low level. The expression level of
retinoblastoma (rb) was stable both in mock and virusinfected cell. Concurrently, cell death-associated genes,
namely p53 and p21, and more importantly, caspase 3a
(cas3a), an apoptosis-specific gene, exhibited obvious
upregulation at 4 h p.i. compared with the mock-infected
control cells (Fig. 2c), strongly suggesting that SGIV
infection induces the apoptosis pathway in HX1 cells.
These data established that SGIV infection elicits molecular
processes characteristic of a cellular immune response and
apoptosis in medaka haploid ES cells.
Host response at the cellular level
We continued our examination into the cellular phenotype
change after SGIV infection. A hallmark of apoptotic cells
is DNA fragmentation, which at the early stage will form a
series of differently sized chromosomal DNA fragments.
Such fragments will form a series of ladders that differ in
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Journal of General Virology 94
(b)
SGIV –
HX1
+
MES1
–
+
b-Actin
ORF018
ORF158
Mock
SVCV
Mock
SGIV
Mock
M
(a)
RGNNV
Viral susceptibility of medaka haploid ES cells
SG3
–
+
Time after SGIV inoculation (h)
0 2 4 6 8 12 24 48 72
(c)
orf086
orf158
orf018
ifn
mx
stat1
vpr
p53 +
–
+
p21
–
+
cas3a –
+
rb –
actin +
–
size by ~200 bp upon agarose gel electrophoresis. We
clearly observed such a ladder in SGIV-infected HX1 cells
at 48 h p.i. (Fig. 3a). Concurrently, we also detected nuclear
fragmentation, as evidenced by the appearance of apoptotic
bodies in SGIV-infected HX1 cells at 48 h p.i. (Fig. 3b).
HX1 cells were further analysed by staining with FITC–
Annexin V, which served as sensitive probe to detect the
externalization of phospholipid phosphatidylserine in the
early stage of apoptosis, together with Hoechst 33342 and
propidium iodide (PI). Hoechst 33342 stains the nuclei of
both live and dead cells, whereas PI stains the nuclei of
dead cells only. Early apoptotic cells are positive for
annexin V staining but negative for PI staining. Late
apoptotic cells are positive for both annexin V and PI
staining. SGIV-induced apoptosis in HX1 cells was seen at
the early stage until 36 h p.i. and at the late stage until 72 h
p.i. (Fig. 3c). These results demonstrated that SGIV
infection induces apoptosis in medaka haploid ES cells.
Infectivity of SGIV produced in medaka haploid
ES cells
SGIV derived from HX1 and grouper spleen (GS) cells was
inoculated back into both cell lines (Fig. 4). HX1-derived
SGIV was comparable to the GS-derived counterpart in
producing CPE in both HX1 (Fig. 4b, c) and GS cells (Fig. 4e,
f). Furthermore, the resulting viral growth curve indicated
that SGIV from either HX1 or GS cells retained its infectivity
and could replicate in the host. In cultures of HX1 or GS cells,
viruses from the two sources gave similar growth curves and
titres. However, the titre of virus in HX1 cells increased more
slowly than in GS cells and was about tenfold lower than that
in GS cells at 72 days p.i. (Fig. 4 g, h). The P value from
Student’s t-test (n53) indicated there was no significant
difference between the two curves in the same host. Therefore,
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Fig. 2. Gene expression profile in virusinfected medaka stem cells. (a) RT-PCR
analysis of viral gene expression in HX1 cells
mock infected (”) or infected with the indicated virus at 4 days p.i. (b) Western blot
analysis of SGIV proteins in three medaka
stem cell lines mock infected (”) or infected
with SGIV at 4 days p.i. (c) RT-PCR analysis
of viral and host gene expression in HX1 cells
at the indicated times after SGIV inoculation
(+) or in mock-infected cells (”). The viral
genes orf086, orf158 and orf018 are shown in
plain text. The immune-responsive host genes
of interferon (ifn), mx, stat1 and viperin (vpr)
are shown in bold. Apoptosis-related host
genes of p53, p21 and caspase 3a (cas3a)
are underlined. Results are also shown for the
host retinoblastoma (rb) gene, and b-actin
(actin) served as a loading control. Negative
results for viral genes and immune-response
genes are not shown in the figure.
SGIV produced in medaka haploid ES cells resembles SGIV
produced in its natural host cells in infectivity.
Electron microscopy of SGIV propagation and
release
Electron microscopy of ultrathin sections revealed the
presence of numerous virus particles in the cytoplasm of
SGIV-infected HX1 cells (Fig. 5a, b). There were also fully
matured particles in the budding cytoplasm (Fig. 5a),
suggesting that they were in the process of release from the
cells. Virions were distributed throughout the cytoplasm.
At a higher magnification, three major stages of virus
assembly were clearly visible in the cytoplasm (Fig. 5b). At
stage I, four- and five-sided intermediates of SGIV
assembly were seen together with elongated tubular-like
materials, suggesting continuity of the assembly intermediate with cellular membrane compartments. At stage II,
empty hexagonal capsids were apparent. At stage III, the
assembly process was complete, leading to the production
of mature virus particles. Similar stages have been
described for African swine fever virus (Rouiller et al.,
1998). These results suggest that medaka haploid ES cells
allows SGIV to complete the infectious cycle.
DISCUSSION
In this study, we have demonstrated for the first time that
stem cell lines from a small freshwater fish like medaka
are susceptible to infection by SGIV and other viruses.
Specifically, we have shown in more detail that SGIV, a
large DNA virus, can efficiently infect even medaka haploid
ES cells. Here, four independent lines of evidence clearly
point to the viral susceptibility of medaka stem cells
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Y. Yuan and others
Hoechst 33342
Time after SGIV inoculation (h)
0
12
24
48
72
Hoechst 33342/MitoTracker
(b)
SGIV
Mock
(a)
FITC
Hoechst 33342
PI
nu
36 h p.i.
ni
72 h p.i.
nu
nu
including the haploid ES cell line HX1. First, CPE occurred
for all three medaka stem cell lines tested, including the
haploid HX1 line, and was similar to that observed in
groupers, the natural host, following SGIV infection.
Secondly, SGIV gene transcription and protein synthesis
took place in all three stem cell lines. Thirdly, electron
microscopy revealed SGIV propagation, maturation and
budding in HX1 cells. Finally, and most convincingly, we
showed that HX1-derived SGIV had infectivity not only in
GS cells as the natural host but also in medaka haploid ES
cells. These results clearly demonstrate medaka haploid ES
cells as the first potential system for genetic screening to
identify host factors essential for infection by these virus
species of importance in aquaculture.
Medaka live in freshwater habitats and are a member of
the order Beloniformes. Carp are freshwater species of the
order Cypriniformes. Groupers are marine species of the
order Perciformes. The separation between Cypriniformes
and Beloniformes plus Perciformes has been estimated to
be approximately 300 million years ago (Yamanoue et al.,
2006). Our finding that the medaka haploid ES cell line
HX1 is susceptible to infection by SVCV, SGIV and
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Merge
Mock
(c)
Fig. 3. SGIV-induced apoptosis in medaka
haploid ES cells. (a) Agarose gel showing DNA
fragmentation. High-molecular-mass chromosomal DNA from SGIV-inoculated HX1 cells at
the indicated times p.i. was separated on a
0.8 % agarose gel. Arrows depict the ladder
diagnostic of apoptotic DNA fragmentation and
degradation, which was most evident at 48 h
p.i. (b) Apoptotic phenotype of SGIV-infected
HX1 cells. At 48 h p.i., cells were stained for
nuclei with Hoechst 33342 (blue) and for
mitochondria with MitoTracker (red) before
analysis by fluorescence microscopy. (c) Live
cell staining with FITC–Annexin V (green),
Hoechst 33342 (blue) and PI (red, indicating
dead nuclei). Arrowheads indicate virus assembly sites. nu, nucleus; ni, nucleolus. Bars, 5 mm.
RGNNV, whose natural host species belong to Cypriniformes and Perciformes, underscores its potential use in
genetic screening for host factors crucial for viral infection
in many cyprinids and perches of importance in aquaculture. In this regard, the sequenced medaka genome
(Kasahara et al., 2007) will contribute greatly to the
identification of host factors and to investigation of the
host-cell response to viral infection.
Recently, we revealed that SGIV induces paraptosis in its
natural host species but apoptosis in non-host cells (Huang
et al., 2011a). This finding is in agreement with our
observation in this study that haploid ES cells of the HX1
line from medaka as a non-host species are capable of
apoptosis. SGIV-induced apoptosis was supported by four
independent lines of evidence. First, apoptosis-associated
genes, in particular caspase 3a, were induced by SGIV
infection. Secondly, chromosomal fragmentation generated
DNA ladders visible on gels. Thirdly, nuclear fragmentation led to the formation of apoptotic bodies. Lastly, cells
positive for annexin V, a marker of apoptosis, were
induced and increased in number during SGIV infection.
In addition, we revealed that SGIV also elicited an immune
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Journal of General Virology 94
Viral susceptibility of medaka haploid ES cells
HX1-derived SGIV
Mock
GS-derived SGIV
(b)
(c)
(d)
(e)
(f)
GS cells
HX1 cells
(a)
(h)
6
GS-derived SGIV
HX1-derived SGIV
5
HX1
4
_
Virus titre [log10(TCID50 ml 1)]
_
Virus titre [log10(TCID50 ml 1)]
(g)
7
GS-derived SGIV
HX1-derived SGIV
6
GS
5
0
2
6
12 18 24
Time (h p.i.)
36
48
72
0
2
6
12 18 24
Time (h p.i.)
36
48
72
Fig. 4. Infectivity and titre assays of SGIV derived from HX1 and GS cells. SGIV derived from HX1 or GS cells was inoculated
back into both cell lines to determine the virus infectivity and growth curves based on a virus titre test. (a–c) CPE of HX1 cells
mock infected or infected with SGIV from HX1 and GS cells as indicated. (d–f) CPE of GS cells mock infected or infected with
SGIV from HX1 and GS cells as indicated. Bars, 10 mm. (g, h) One-step virus growth curves of HX1 and GS cells infected by
SGIV. HXI (g) or GS (h) cells were inoculated with SGIV derived from HX1 and GS cells as indicated. Student’s t-test (n53)
was performed to identify significant differences between two groups (P,0.05). In the same cell strain, there was no significant
difference in titre of yield virus after infection with virus derived from HX1 or GS cells.
response in medaka haploid ES cells, as evidenced by the
apparent induction and upregulation of immune-responsive genes such as ifn and stat1. It has been reported that fish
virus-induced interferon exerts an antiviral function
through the Stat1 pathway (Yu et al., 2010). These results
suggest that medaka haploid ES cells resemble cells of the
natural host species at molecular and cellular levels in
response to SGIV infection, and thus provide a system for
understanding the mechanisms underlying viral infection
and antiviral strategies.
METHODS
Cell culture. The medaka cell lines were maintained at 28 uC in
medium ESM4 as described previously (Hong & Schartl, 2006; Yi
http://vir.sgmjournals.org
et al., 2010). These included the diploid ES cell line MES1 (Hong et
al., 1996), the diploid spermatogonial cell line SG3 (Hong et al., 2004)
and HX1, one of the three haploid ES cell lines (Yi et al., 2009). The
grouper embryonic cell line GP (Qin et al., 2003) and grouper spleen
cell line GS (Huang et al., 2011a) were maintained at 25 uC in Eagle’s
minimal essential medium containing 10 % FBS as described
elsewhere.
Virus preparation and challenge. Susceptibility to the nine virus
species list in Table 1 was tested independently in three laboratories. A
first test was performed as a custom service by the Agri-Food and
Veterinary Authority of Singapore (http://www.ava.gov.sg/), which
identified SGIV and SVCV as infectious agents in medaka haploid ES
cells on the basis of apparent CPE. We further examined these two
virus species and RGNNV. SGIV (strain A3/12/98) originally isolated
from diseased brown-spotted grouper (Epinephelus tauvina) was
propagated in GP cells as described previously (Qin et al., 2003).
RGNNV and SVCV were propagated in GB cells (Huang et al., 2011b)
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Y. Yuan and others
(a)
(b)
I
nu
II
I
III
Fig. 5. Electron microscopy of SGIV in medaka haploid ES cells. HX1 cells at 4 days after SGIV infection were subjected to
electron microscopy. (a) Intact cell at low magnification, showing enlargement of the nucleus (nu) occupying a large part of the
cytoplasm and SGIV particles in budding from the cytoplasm during secretion (arrows). (b) Higher magnification of the area
framed in (a), highlighting the early (I), mid- (II) and late (III) stages of SGIV assembly. Bars, 1 mm (a), 200 nm (b).
and CO cells (Teng et al., 2007). For instance, SGIV was inoculated
onto confluent GP cells at an m.o.i. of ~0.1. Following the appearance
of an apparent CPE, cells containing SGIV were harvested and
centrifuged at 3000 g for 10 min at 4 uC, and the cell debris together
with partial supernatant were collected and stored at 280 uC until
use. Medaka cell lines were similarly challenged with different viruses
at 70 % confluency for further analysis.
Virus replication kinetics assay. To determine whether medaka
haploid ES cells were able to propagate infectious virus comparable to
that produced in the natural host cells, SGIV particles derived from
the infected HX1 and GS cultures were inoculated back into the two
cell types to test the viral growth curve. In detail, cells of HX1 and GS
(16106 cells per well) were seeded into six-well plates and cultured
for 24 h. SGIV collected from HX1 and GS cells was inoculated back
onto seeded cells at an m.o.i. of 0.1. The infected cells together with
supernatant were harvested at the indicated time points and stored at
280 uC for titre assays. Virus titres were determined with serially
diluted GS cells to determine the TCID50 after 72 h of incubation.
RT-PCR analysis. RNA isolation from cell culture and RT-PCR
analyses were performed as described previously (Hong et al., 2004; Yi
et al., 2009). The GenBank accession numbers of target genes are
listed in Table S1, and the sequences of the genes stat1 and p21 were
identified using amino acid sequence alignment (Data S1 and S2).
RT-PCR primers were designed according to the relative sequence
(Table S1). PCR was performed in a 20 ml volume containing 10 ng
cDNA for 30 (for b-actin as a loading control) or 35 cycles (other
genes) of 95 uC for 30 s, 60 uC for 20 s and 72 uC for 1 min. The
primers used are listed in Table S1. PCR product (4 ml) was loaded
and analysed by 2 % agarose gel electrophoresis.
Genomic DNA isolation. HX1 cells were harvested by trypsinization
and lysed in lysis buffer [10 mM Tris/HCl (pH 8.0), 1 mM EDTA,
1 % SDS, 100 mg proteinase K ml21], and the lysate was incubated at
50 uC for 3 h. After phenol/chloroform extraction, the DNA was
precipitated by adding 2 vols ethanol at 220 uC overnight and then
centrifuged at 12 000 g for 15 min. The DNA pellets were resuspended in distilled water with RNase A (50 mg ml21) and then
analysed by 1.5 % agarose gel electrophoresis.
Western blot analysis. Western blot analysis was performed
essentially as described previously (Xu et al., 2005) using antibodies
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against the cellular b-actin (clone AC-74; Sigma) and SGIV proteins
ORF018 (Wang et al., 2008) and ORF158 (Tran et al., 2011).
Cell staining. HX1 cells grown in six-well plates at 60 % confluency
were mock infected or infected with SGIV. Live cells were stained
with MitoTracker Red (Invitrogen) plus Hoechst 33342 (1 mg ml21)
or with an FITC : Annexin V Apoptosis Detection kit II (BD
Pharmingen) plus nuclear dyes PI (1 mg ml21) and Hoechst 33342
(1 mg ml21). After staining, the cells were analysed by fluorescence
microscopy.
Light microscopy. Light microscopy analyses were carried out as
described previously (Hong et al., 2010; Yang et al., 2012; Yi et al.,
2009).
Electron microscopy. SGIV-infected HX1 cells were subjected to
electron microscopy as described previously (Huang et al., 2011a).
Briefly, cells at 36 h p.i. were fixed in 2.5 % glutaraldehyde overnight,
washed with PBS and post-fixed in 1 % osmium tetroxide for 1 h.
After dehydration in an increasing ethanol series, the cells were
embedded in Epon resin and ultrathin sections were cut at 200 nm
thickness with a UC6/FC6 ultramicrotome (Leica). Sections were
double stained with uranyl acetate and lead citrate. Samples on grids
were examined and documented at 120 kV on a JEM-1400 electron
microscopy (JEOL).
ACKNOWLEDGEMENTS
We thank J. Deng for fish breeding and C. M. Foong for laboratory
management. This work was supported by the National Research
Foundation Singapore (NRF-CRP7-2010-03) and the National Basic
Research Program of China (973 Program: 2012CB114402).
REFERENCES
Adachi, K., Sumiyoshi, K., Ariyasu, R., Yamashita, K., Zenke, K. &
Okinaka, Y. (2010). Susceptibilities of medaka (Oryzias latipes) cell
lines to a betanodavirus. Virol J 7, 150.
Carette, J. E., Guimaraes, C. P., Varadarajan, M., Park, A. S.,
Wuethrich, I., Godarova, A., Kotecki, M., Cochran, B. H., Spooner, E.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 07:54:07
Journal of General Virology 94
Viral susceptibility of medaka haploid ES cells
& other authors (2009). Haploid genetic screens in human cells
identify host factors used by pathogens. Science 326, 1231–1235.
Qin, Q. W., Chang, S. F., Ngoh-Lim, G. H., Gibson-Kueh, S., Shi, C. &
Lam, T. J. (2003). Characterization of a novel ranavirus isolated from
Carette, J. E., Guimaraes, C. P., Wuethrich, I., Blomen, V. A.,
Varadarajan, M., Sun, C., Bell, G., Yuan, B., Muellner, M. K. & other
authors (2011a). Global gene disruption in human cells to assign
grouper Epinephelus tauvina. Dis Aquat Organ 53, 1–9.
genes to phenotypes by deep sequencing. Nat Biotechnol 29, 542–546.
reticulum. J Virol 72, 2373–2387.
Carette, J. E., Raaben, M., Wong, A. C., Herbert, A. S., Obernosterer,
G., Mulherkar, N., Kuehne, A. I., Kranzusch, P. J., Griffin, A. M. &
other authors (2011b). Ebola virus entry requires the cholesterol
Song, W. J., Qin, Q. W., Qiu, J., Huang, C. H., Wang, F. & Hew, C. L.
(2004). Functional genomics analysis of Singapore grouper iridovirus:
Rouiller, I., Brookes, S. M., Hyatt, A. D., Windsor, M. & Wileman, T.
(1998). African swine fever virus is wrapped by the endoplasmic
transporter Niemann–Pick C1. Nature 477, 340–343.
complete sequence determination and proteomic analysis. J Virol 78,
12576–12590.
FAO (2008). State of World Fisheries and Aquaculture. Rome: Food
and Agricultural Organisation of the United Nations.
Stockwell, B. R. (2002). Chemical genetic screening approaches to
FAO (2009). Fishstat Plus. Rome: Food and Agricultural Organisation
Teng, Y., Liu, H., Lv, J. Q., Fan, W. H., Zhang, Q. Y. & Qin, Q. W. (2007).
of the United Nations.
Characterization of complete genome sequence of the spring viremia
of carp virus isolated from common carp (Cyprinus carpio) in China.
Arch Virol 152, 1457–1465.
Furusawa, R., Okinaka, Y. & Nakai, T. (2006). Betanodavirus infection
in the freshwater model fish medaka (Oryzias latipes). J Gen Virol 87,
2333–2339.
Hong, Y. & Schartl, M. (2006). Isolation and differentiation of medaka
neurobiology. Neuron 36, 559–562.
Tran, B. N., Chen, L., Liu, Y., Wu, J., Velázquez-Campoy, A.,
Sivaraman, J. & Hew, C. L. (2011). Novel histone H3 binding protein
Hong, Y., Winkler, C. & Schartl, M. (1996). Pluripotency and
ORF158L from the Singapore grouper iridovirus. J Virol 85, 9159–
9166.
differentiation of embryonic stem cell lines from the medakafish
(Oryzias latipes). Mech Dev 60, 33–44.
shrimp. Vet Res 41, 51.
embryonic stem cells. Methods Mol Biol 329, 3–16.
Hong, Y., Liu, T., Zhao, H., Xu, H., Wang, W., Liu, R., Chen, T., Deng, J.
& Gui, J. (2004). Establishment of a normal medakafish spermato-
gonial cell line capable of sperm production in vitro. Proc Natl Acad
Sci U S A 101, 8011–8016.
Hong, N., Li, M., Zeng, Z., Yi, M., Deng, J., Gui, J., Winkler, C., Schartl,
M. & Hong, Y. (2010). Accessibility of host cell lineages to medaka
Walker, P. J. & Winton, J. R. (2010). Emerging viral diseases of fish and
Wang, F., Bi, X., Chen, L. M. & Hew, C. L. (2008). ORF018R, a highly
abundant virion protein from Singapore grouper iridovirus, is
involved in serine/threonine phosphorylation and virion assembly.
J Gen Virol 89, 1169–1178.
stem cells depends on genetic background and irradiation of recipient
embryos. Cell Mol Life Sci 67, 1189–1202.
Xia, L., Cao, J., Huang, X. & Qin, Q. (2009). Characterization of
Singapore grouper iridovirus (SGIV) ORF086R, a putative homolog
of ICP18 involved in cell growth control and virus replication. Arch
Virol 154, 1409–1416.
Huang, X., Huang, Y., Ouyang, Z. & Qin, Q. (2011a). Establishment of
Xu, H., Gui, J. & Hong, Y. (2005). Differential expression of vasa RNA
a cell line from the brain of grouper (Epinephelus akaara) for
cytotoxicity testing and virus pathogenesis. Aquaculture 311, 65–73.
and protein during spermatogenesis and oogenesis in the gibel carp
(Carassius auratus gibelio), a bisexually and gynogenetically reproducing vertebrate. Dev Dyn 233, 872–882.
Huang, X., Huang, Y., Ouyang, Z., Xu, L., Yan, Y., Cui, H., Han, X. &
Qin, Q. (2011b). Singapore grouper iridovirus, a large DNA virus,
induces nonapoptotic cell death by a cell type dependent fashion and
evokes ERK signaling. Apoptosis 16, 831–845.
Yamanoue, Y., Miya, M., Inoue, J. G., Matsuura, K. & Nishida, M. (2006).
Kasahara, M., Naruse, K., Sasaki, S., Nakatani, Y., Qu, W., Ahsan, B.,
Yamada, T., Nagayasu, Y., Doi, K. & other authors (2007). The
The mitochondrial genome of spotted green pufferfish Tetraodon
nigroviridis (Teleostei: Tetraodontiformes) and divergence time
estimation among model organisms in fishes. Genes Genet Syst 81,
29–39.
medaka draft genome and insights into vertebrate genome evolution.
Nature 447, 714–719.
Yang, H., Shi, L., Wang, B. A., Liang, D., Zhong, C., Liu, W., Nie, Y., Liu,
J., Zhao, J. & other authors (2012). Generation of genetically
Kotecki, M., Reddy, P. S. & Cochran, B. H. (1999). Isolation and
modified mice by oocyte injection of androgenetic haploid embryonic
stem cells. Cell 149, 605–617.
characterization of a near-haploid human cell line. Exp Cell Res 252,
273–280.
Leeb, M. & Wutz, A. (2011). Derivation of haploid embryonic stem
Yi, M., Hong, N. & Hong, Y. (2009). Generation of medaka fish haploid
embryonic stem cells. Science 326, 430–433.
cells from mouse embryos. Nature 479, 131–134.
Yi, M., Hong, N. & Hong, Y. (2010). Derivation and characterization of
Li, W., Shuai, L., Wan, H., Dong, M., Wang, M., Sang, L., Feng, C., Luo,
G. Z., Li, T. & other authors (2012). Androgenetic haploid embryonic
haploid embryonic stem cell cultures in medaka fish. Nat Protoc 5,
1418–1430.
stem cells produce live transgenic mice. Nature 490, 407–411.
Muller, H. J. (1927). Artificial Transmutation of the Gene. Science 66,
Yu, F. F., Zhang, Y. B., Liu, T. K., Liu, Y., Sun, F., Jiang, J. & Gui, J. F.
(2010). Fish virus-induced interferon exerts antiviral function
84–87.
through Stat1 pathway. Mol Immunol 47, 2330–2341.
http://vir.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 07:54:07
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