Journal of General Virology (2012), 93, 2299–2309
DOI 10.1099/vir.0.042424-0
Assembly of the viroplasm by viral non-structural
protein Pns10 is essential for persistent infection of
rice ragged stunt virus in its insect vector
Dongsheng Jia,1 Nianmei Guo,1 Hongyan Chen,1 Fusamichi Akita,2
Lianhui Xie,1 Toshihiro Omura2 and Taiyun Wei1
1
Correspondence
Fujian Province Key Laboratory of Plant Virology, Institute of Plant Virology, Fujian Agriculture and
Forestry University, Fuzhou, Fujian 350002, PR China
Toshihiro Omura
[email protected]
2
National Agricultural Research Center, 3-1-1 Kannondai, Tsukuba, Ibaraki 305-8666, Japan
Taiyun Wei
[email protected]
Received 25 February 2012
Accepted 16 July 2012
Rice ragged stunt virus (RRSV), an oryzavirus, is transmitted by brown planthopper in a
persistent propagative manner. In this study, sequential infection of RRSV in the internal organs
of its insect vector after ingestion of virus was investigated by immunofluorescence microscopy.
RRSV was first detected in the epithelial cells of the midgut, from where it proceeded to the
visceral muscles surrounding the midgut, then throughout the visceral muscles of the midgut and
hindgut, and finally into the salivary glands. Viroplasms, the sites of virus replication and
assembly of progeny virions, were formed in the midgut epithelium, visceral muscles and salivary
glands of infected insects and contained the non-structural protein Pns10 of RRSV, which
appeared to be the major constituent of the viroplasms. Viroplasm-like structures formed in nonhost insect cells following expression of Pns10 in a baculovirus system, suggesting that the
viroplasms observed in RRSV-infected cells were composed basically of Pns10. RNA
interference induced by ingestion of dsRNA from the Pns10 gene of RRSV strongly inhibited
such viroplasm formation, preventing efficient virus infection and spread in its insect vectors.
These results show that Pns10 of RRSV is essential for viroplasm formation and virus replication
in the vector insect.
INTRODUCTION
Plant reoviruses are found in the genera Phytoreovirus,
Fijivirus and Oryzavirus in the family Reoviridae (Boccardo
& Milne, 1984). Rice ragged stunt virus (RRSV), an
oryzavirus (Hibino, 1996; Hibino et al., 1977), has spread
rapidly throughout southern China and Vietnam and causes
severe damage to rice (Hoang et al., 2011). For example,
since 2006, RRSV has spread widely in Fujian, Hainan,
Yunnan, Guangxi and Guangdong provinces in China.
RRSV has an icosahedral capsid, ~70 nm in diameter, to
which spikes are attached (Miyazaki et al., 2008). The RRSV
genome consists of ten dsRNA segments that encode at least
seven structural proteins, P1, P2, P3, P4A, P5, P8B and P9,
and three non-structural proteins, Pns6, Pns7 and Pns10
(Boccardo & Milne, 1984; Hagiwara et al., 1986; Upadhyaya
et al., 1996, 1997, 1998). Among the structural proteins
encoded by RRSV, P2, P3, P4A and P5 are a putative
guanylyltransferase, capsid shell protein, putative RNAdependent RNA polymerase and capping enzyme, respectively (Boccardo & Milne, 1984; Hagiwara et al., 1986;
A supplementary figure is available with the online version of this paper.
042424 G 2012 SGM
Supyani et al., 2007; Upadhyaya et al., 1998), P8 is a major
outer-capsid protein (Hagiwara et al., 1986) and P9 is a
spike protein involved in transmission via the insect vector
(Zhou et al., 1999). Among the non-structural proteins
encoded by RRSV, Pns6 functions as a viral RNA-silencing
suppressor and a viral movement protein (MP) (Wu et al.,
2010a, b), whilst Pns7 has been identified as an NTP-binding
protein (Spear et al., 2012; Upadhyaya et al., 1997). The
functions of the remaining proteins are unknown.
RRSV is transmitted by the brown planthopper (BPH),
Nilaparvata lugens (Stål), in a persistent propagative manner
(Hibino et al., 1977, 1979). As a persistent propagative plant
virus that is transmitted by an insect vector following
ingestion during feeding on diseased plants, RRSV must
enter the epithelial cells of the alimentary canal in its insect
vector, replicate and assemble progeny virions to move into
the salivary glands from which RRSV can be introduced into
a plant host during feeding (Hogenhout et al., 2008). Thus,
RRSV must enter insect vector cells to establish persistent
infection, that is, virus must replicate and accumulate
progeny virions in the body of BPHs. During RRSV
infection in BPHs, cytoplasmic inclusion bodies, known as
viroplasms, form in the salivary glands, gut and muscles in
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 22:13:29
Printed in Great Britain
2299
D. Jia and others
infected BPHs, as observed by electron microscopy (Hibino
et al., 1979). Virus replication and assembly of progeny
virions have been proposed to occur in viroplasms for plant
reoviruses (Boccardo & Milne, 1984). Thus, the formation of
a viroplasm for virus replication and assembly of progeny
virions may play a crucial role in the propagation of RRSV in
BPHs.
Viral non-structural proteins are essential for formation
of the viroplasm matrix of plant reoviruses, such as P9-1 of
three fijiviruses [rice black-streaked dwarf virus (RBSDV),
Southern rice black-streaked dwarf virus (SRBSDV) and
Mal de Rı́o Cuarto virus (MRCV)], Pns12 of the phytoreovirus rice dwarf virus (RDV) and Pns9 of rice gall dwarf
virus (RGDV), also a phytoreovirus (Akita et al., 2011,
2012; Jia et al., 2012; Maroniche et al., 2010; Wei et al.,
2006b). However, viral non-structural proteins involved in
formation of the viroplasms induced by oryzaviruses are
unknown.
RNA interference (RNAi), a conserved sequence-specific
gene-silencing mechanism induced by dsRNA (Fire et al.,
1998), has been developed into an important tool to
investigate the functional role of fijivirus replication
proteins in insect vectors (Jia et al., 2012). Ingestion of
dsRNA via membrane feeding is an effective method of
inducing RNAi in BPHs to knock down specific insect
genes (Chen et al., 2010; Li et al., 2011). Thus, dsRNAmediated gene silencing offers an opportunity for us to
investigate the functional roles of viral non-structural
proteins in the infection cycle of RRSV in BPHs.
In this study, using immunofluorescence microscopy and
an RNAi strategy, the functional roles were determined for
RRSV Pns10 in formation of the viroplasm and virus
infection in BPHs. Our results suggested that Pns10 of
RRSV is responsible for formation of the viroplasm matrix
in which the assembly of progeny virions occurs in BPHs.
RNAi induced by ingestion of dsRNA of the Pns10 gene
strongly inhibited such viroplasm formation, preventing
efficient virus infection and spread in BPHs. Our results
indicate that assembly of the viroplasm by Pns10 is
essential for persistent infection of RRSV in BPHs.
RESULTS
Infection route of RRSV in BPHs revealed by
confocal microscopy
To trace the infection route of RRSV within infected BPHs,
immunofluorescence microscopy was used to elucidate the
distribution of viral antigens in the body of BPHs after
ingestion of RRSV from diseased plants. In preliminary
tests, ~40 % of BPHs became infected after a latent period
of 9 days (Table 1). At 1, 3, 4, 6 and 9 days post-first access
to diseased plants (p.a.d.p.), internal organs from 50 BPHs
were dissected and processed for immunofluorescence
microscopy. The actin-specific dye phalloidin–rhodamine
was first used to stain the alimentary canal of BPHs. As in
other types of planthopper (Tsai & Perrier, 1996), the
alimentary canal of BPHs consists of the oesophagus,
anterior diverticulum, midgut and hindgut (Fig. 1a). The
midgut of BPHs consists of a single layer of epithelial cells,
with extensive microvilli on the lumenal side and basal
lamina on the outer side, surrounded by visceral muscle
tissues (Fig. 1a). Viral antigens were observed at 1 day
p.a.d.p. in the midgut lumen in ~50 % of BPHs examined
(Fig. 1b), suggesting that RRSV had travelled through the
oesophagus into the midgut lumen. At 3 days p.a.d.p.,
RRSV was mainly restricted to a few epithelial cells of the
midgut in ~30 % of the insects examined (Fig. 1c, Table 1).
At 4 days p.a.d.p., RRSV in the epithelial cells had
traversed the basal lamina and infected the visceral muscle
tissues encircling the midgut epithelium in ~28 % of BPHs
examined (Fig. 1d, Table 1). By 6 days p.a.d.p., RRSV had
spread to the oesophagus, anterior diverticulum, midgut,
hindgut and salivary glands in a higher proportion of BPHs
(~26 %; Fig. 1e, Table 1); RRSV was present in the visceral
muscle tissues of the entire midgut and hindgut but absent
in the epithelium (Fig. 1f–h). By 9 days p.a.d.p., the
presence of RRSV was extensive in the oesophagus,
anterior diverticulum, muscles of the midgut and hindgut,
and salivary glands in a high proportion of BPHs tested
(~40 %) (Fig. 1i, j, Table 1). At this time, RRSV was still
absent in the epithelium of the midgut and hindgut (Table
1). Taken together, these results indicated that RRSV first
accumulates in the midgut epithelium, procedes to the
Table 1. Occurrence of RRSV antigens and viroplasms of Pns10 in various organs/tissues of BPHs as detected by
immunofluorescence microscopy
Organ/tissue examined
Midgut epithelium
Visceral muscle (midgut)
Visceral muscle (hindgut)
Oesophagus
Anterior diverticulum
Salivary gland
2300
No. positive insects with viral antigens and viroplasms of Pns10 in different tissues (n550)
3 days p.a.d.p.
4 days p.a.d.p.
6 days p.a.d.p.
15
0
0
0
0
0
18
14
0
7
5
0
0
21
14
19
19
13
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 22:13:29
9 days p.a.d.p.
0
20
20
20
20
18
Journal of General Virology 93
Viroplasm essential for RRSV infection in vector
(a)
(b)
os
mg
ad
mg
me mv
gl
Lumen side
mv
mt
hg
mv
gl
vm
me
1 day
Muscle side
Lumen side Muscle side
(c) hg
gl
me
mv
vm
(d)
me
vm
vm
mg
mg
3 days
4 days
(e)
Lumen side
(f) (i)
(f) (ii)
Muscle side
mg
vm
ad
vm
os
(g)
(h)
vm (hg)
hg
vm (mg)
sg
6 days
(i)
(j)
sg
mg
sg
ad
os
9 days
sg
sg
Fig. 1. Infection route of RRSV in the insect vector. The internal organs of BPHs were stained for viral antigens with virus–FITC
(green) and for actin with phalloidin–rhodamine (red) and examined by confocal microscopy. (a) The alimentary canal of BPH.
Insets: single optical section of the lumen side (upper panel) and muscle side (lower panel) of the midgut. (b) At 1 day p.a.d.p.,
viral antigens accumulated in the midgut lumen. (c) By 3 days p.a.d.p., viral antigens were detected in a few epithelial cells of the
midgut. Inset: enlarged image of the boxed area. (d) At 4 days p.a.d.p., viral antigens accumulated in the epithelium and visceral
muscle of the midgut. The image is a projection of 12 optical sections taken at 0.5 mm intervals. Insets: single optical sections of
the lumen side (left panel) and muscle side (right panel) of the midgut. (e) By 6 days p.a.d.p., viral antigens had accumulated
throughout the digestive system. (f) Single optical sections of the lumen side (panel i) and muscle side (panel ii) of the midgut at
6 days p.a.d.p. (g, h) Viral antigens were detected in the visceral muscle of the midgut (g) and hindgut (h) at 6 days p.a.d.p. The
inset in (h) is an enlargement of the boxed area. (i, j) At 9 days p.a.d.p., viral antigens accumulated throughout the digestive
system. ad, Anterior diverticulum; mg, midgut; hg, hindgut; mt, Malpighian tubules; os, oesophagus; sg, salivary gland; gl, gut
lumen; mv, microvilli; me, midgut epithelium; vm, visceral muscle. Bars, 70 mm.
http://vir.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 22:13:29
2301
D. Jia and others
visceral muscles surrounding the midgut, spreads throughout the visceral muscles of the midgut and hindgut, and
finally spreads into the salivary glands.
RRSV Pns10 is sufficient to induce the formation
of viroplasm-like structures in non-host insect
cells
During infection of BPHs by RRSV, viroplasms, the putative
sites for virus replication and assembly of progeny virions,
are formed in the alimentary canal and salivary glands of
BPHs, as revealed by electron microscopy (Boccardo &
Milne, 1984; Hibino et al., 1977, 1979). Viral non-structural
proteins are essential for formation of the viroplasm matrix
of plant reoviruses (Akita et al., 2011, 2012; Maroniche et al.,
2010; Wei et al., 2006b). To identify which non-structural
protein encoded by RRSV had an inherent ability to form
the viroplasm matrix, a baculovirus system was used to
express each of the three non-structural proteins, Pns6, Pns7
and Pns10, fused to a 66His tag (Pns6–His and Pns7–His)
or Strep Tag II (Pns10–Strep). As seen by immunofluorescence microscopy, Pns6–His was associated exclusively with
the plasma membrane (Fig. 2a, panel i), corresponding to
previous evidence that Pns6 is a viral MP (Wu et al., 2010b).
Pns6–His
(a)
To determine whether Pns6 or Pns7 could be recruited into
viroplasm-like structures formed by Pns10, recombinant
baculoviruses that expressed Pns6–His or Pns7–His were
co-infected with recombinant baculoviruses expressing
Pns10–Strep in Sf9 cells. Co-infection led to redistribution
of Pns6–His into the viroplasm-like structures formed by
Pns10–Strep (Fig. 2b). By contrast, Pns7–His was not
observed in association with the viroplasm-like structures
formed by Pns10–Strep (data not shown). Rabbit anti66His tag polyclonal antibody and anti-Strep Tag II mAb
in non-infected cells were not observed to react with
cellular structures (data not shown). Thus, our findings
Pns7–His
(ii)
(i)
(b)
Pns7–His formed filament-like structures in the cytoplasm
or protruding from the plasma membrane (Fig. 2a, panel ii),
whilst Pns10–Strep aggregated to form punctate inclusions in the cytoplasm (Fig. 2a, panel iii), resembling the
viroplasm matrix in virus-infected cells (Hibino et al., 1979).
Our results indicated clearly that, among the three nonstructural proteins of RRSV, Pns10 alone was sufficient for
the formation of viroplasm-like structures in Spodoptera
frugiperda (Sf9) cells. Taken together, our results suggested
that Pns10 might self-aggregate to form the viroplasm
matrix in RRSV-infected host cells.
Pns10–Strep
Pns10–Strep
(iii)
Pns6–His
Merged
Fig. 2. RRSV Pns10 aggregates to form viroplasm-like inclusions in the absence of virus infection. Sf9 cells infected with
recombinant baculoviruses containing Pns6–His, Pns7–His or Pns10–Strep were fixed 3 days after infection and prepared for
immunofluorescence microscopy as described in Methods. (a) Pns6–His was associated with the plasma membrane (i), Pns7–
His formed filament-like structures (ii) and Pns10–Strep formed punctate structures (iii). (b) Pns6–His was associated with the
punctate structures formed by Pns10–Strep when co-infected with recombinant baculoviruses containing Pns6–His and
Pns10–Strep. Bars, 5 mm.
2302
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 22:13:29
Journal of General Virology 93
Viroplasm essential for RRSV infection in vector
suggested that Pns6 might be recruited to the viroplasm
through the association of Pns6 with Pns10.
RRSV Pns10 is the constituent of the viroplasm
matrix in the body of infected BPHs
To localize Pns10 in virus-infected cells, antibodies against
this protein were prepared. Western blot analysis using antiPns10 antibodies showed a 33 kDa protein (in accordance
with the expected size of the RRSV segment 10-encoded
protein) present in protein extracts from infected rice plants
(Fig. S1, available in JGV Online). No reaction was observed
with proteins from uninfected plants, confirming that
Pns10-specific antibodies were able to specifically recognize
the protein produced by RRSV infection.
(a)
Virus
To determine whether Pns10 plays a key role in formation
of the viroplasm, subcellular localization of Pns10 in the
body of infected BPHs was examined by immunofluorescence microscopy. Double labelling showed that viral
inclusions that stained with Pns10-specific IgG directly
conjugated to rhodamine (Pns10–rhodamine) co-localized
with punctate inclusions that stained with viral antigenspecific IgG directly conjugated to FITC (virus–FITC) in
the midgut epithelium at 3 days p.a.d.p., in the visceral
muscle tissues surrounding the midgut at 6 days p.a.d.p.
and in the salivary gland at 9 days p.a.d.p. (Fig. 3a, b, Table
1). These results indicated that RRSV Pns10 is a constituent
of viral inclusions where viral antigens accumulated.
To confirm whether the viral inclusions of Pns10 observed
by immunofluorescence microscopy were the viroplasm
Pns10
Actin
Merged + DIC
me
mg
(b)
Virus
Actin
Pns10
Merged + DIC
vm
mg
(c)
Virus
Pns10
Merged + DIC
sg
Fig. 3. Viroplasms containing Pns10 antigens co-localize with viral antigens in infected BPHs. The internal organs of BPHs
were stained for viral antigens with virus–FITC (green), for viroplasms with Pns10–rhodamine (red) or for actin with phalloidin–
Alexa Fluor 647 carboxylic acid (blue) and then examined by confocal microscopy. Viroplasms containing Pns10 antigens (red)
and viral antigens (green) were distributed in the midgut epithelium at 3 days p.a.d.p. (a), in the visceral muscle tissues
surrounding the midgut at 6 days p.a.d.p. (b) and in the salivary glands at 9 days p.a.d.p. (c). DIC, Differential interference
contrast; me, midgut epithelium; mg, midgut, sg, salivary gland; vm, visceral muscle. Bars, 70 mm.
http://vir.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 22:13:29
2303
D. Jia and others
where viral particles accumulate, immunoelectron microscopy was used to localize Pns10 in the salivary glands of
infected BPHs. As shown in Fig. 4(a), Pns10 antibodies
reacted specifically with the viroplasm matrix in salivary
glands, and the labelling was consistent with the results of
the immunofluorescence microscopy (Fig. 3c). Careful
analysis of the electron micrographs revealed that core-like
particles of ~50 nm in diameter were distributed within the
viroplasm matrix, whereas intact, double-layered viral
particles of ~70 nm in diameter accumulated at the
periphery of the viroplasm matrix (Fig. 4b). Moreover,
RRSV particles aggregated to form paracrystalline arrays at
the periphery of the viroplasm matrix (Fig. 4c). These results
confirmed that Pns10 is the constituent of the viroplasm
matrix induced by RRSV infection. Furthermore, our results
clearly indicated that the viroplasm is the putative site of
virus assembly in the body of infected BPHs.
(a)
VP
(b)
VP
Ingestion of dsRNA of the Pns10 gene strongly
inhibits virus infection in BPHs
An RNAi strategy was used next to investigate the
functional role of RRSV Pns10 in the virus replication
cycle in BPHs. Second-instar nymphs of BPHs were fed
0.5 mg dsRNA ml21 in 10 % sucrose by membrane feeding
for 1 day, allowed a 2-day acquisition on RRSV-infected
rice plants and then fed on fresh rice seedlings. RT-PCR
was used to determine the effects of dsRNA treatment on
the transcript levels of viral genes of the non-structural
protein Pns10 and the major outer-capsid protein P8 in
infected insects that received either dsRNA or sucrose diet
alone at 9 days p.a.d.p. (Upadhyaya et al., 1996, 1997). Our
results showed that ~40 % of BPHs (n5100, three
repetitions) that received dsRNA of the gfp gene (dsGFP)
contained transcripts for the Pns10 and P8 genes (Table 2).
By contrast, ~12 % (n5100, three repetitions) of BPHs that
received dsRNA of the Pns10 gene (dsPns10) contained
transcripts for the Pns10 and P8 genes (Table 2). The
number of positive samples found in BPHs that received
dsGFP did not differ significantly from the controls that
received the sucrose diet only (Table 2). These results
indicated that ingestion of dsPns10 using the membrane
feeding method can efficiently induce RNAi and inhibit
virus infection in BPHs.
To analyse in more detail the inhibition of virus infection
caused by ingestion of dsPns10 in the body of BPHs,
internal organs from 50 BPHs receiving dsRNA or sucrose
diet alone were dissected at 3, 6 and 9 days p.a.d.p. and
processed for immunofluorescence microscopy. Virus
infection was revealed by double labelling of internal
organs with virus–FITC and Pns10–rhodamine. At 3 days
p.a.d.p., Pns10 and viral antigens were restricted to a
limited number of epithelial cells of the midgut in 30 % of
BPHs receiving dsGFP but only in 10 % of those receiving
dsPns10 (Table 2). These results suggested that RNAi
induced by dsPns10 could significantly inhibit early RRSV
infection in the cells of the midgut of insect vectors. At
2304
(c)
VP
Fig. 4. RRSV Pns10 is the component of the viroplasm matrix. (a)
Immunogold labelling of RRSV Pns10 in the viroplasm matrix in
infected salivary glands. Salivary glands were immunostained using
Pns10-specific antibodies as the primary antibody, followed by
treatment with goat anti-rabbit antibodies conjugated to 15 nm
gold particles as the secondary antibody. The inset shows an
enlargement of the boxed area. Black arrows indicate virus
particles, whilst white arrows indicate gold particles. (b, c)
Morphogenesis of RRSV particles associated with the viroplasm
matrix in infected salivary glands. The inset in (b) shows an
enlargement of the boxed area. Black arrows indicate core-like
particles, whilst white arrows indicate intact viral particles. VP,
Viroplasm. Bars, 200 nm.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 22:13:29
Journal of General Virology 93
41
40
Diet control
38
38
40
37
dsGFP
http://vir.sgmjournals.org
*Second-instar nymphs of BPHs were fed with dsPns10 or dsGFP (0.5 mg ml21) or diet alone for 1 day, allowed a 2-day acquisition access period (AAP) on virus-infected rice plants and then fed on
uninfected rice seedling.
DVirus antigens and viroplasms of Pns10 were detected in the midgut epithelium (me), visceral muscle of the midgut (vm-mg), visceral muscle of the hindgut (vm-hg) and salivary glands (sg) of
BPHs by immunofluorescence microscopy.
0
3
3
0
10
18
0
11
17
0
6
7
0
18
20
0
21
19
10
dsPns10
13
12
3
6
9
3
6
9
3
6
9
5
6
0
15
0
0
16
0
0
0
4
6
0
13
20
0
14
19
sg
vm-hg
vm-mg
me
Days p.a.d.p.
III
II
I
No. of insects positive for virus and Pns10 antigens in different tissues (n550)D
No. of insects positive for Pns10 and P8 at 9 days p.a.d.p. (n5100)
Treatment*
Table 2. Ingestion of dsRNA of the Pns10 gene strongly inhibits virus infection, as revealed by the accumulation of Pns10 and viral antigens of RRSV in the body of BPHs
Viroplasm essential for RRSV infection in vector
6 days p.a.d.p., Pns10 and viral antigens were detected in
both visceral muscle tissues and salivary glands in 20 % of
BPHs receiving dsGFP but only in 6 % of BPHs receiving
dsPns10 (Table 2). Furthermore, RRSV was absent from
the midgut epithelium in BPHs receiving dsGFP, but virus
could be detected in 12 % of those receiving dsPns10
(Table 2). At 9 days p.a.d.p., Pns10 and viral antigens were
detected in the visceral muscles of 40 % of BPHs receiving
dsGFP and in the salivary glands of 36 % of BPHs receiving
dsGFP (Table 2). However, Pns10 and viral antigens were
only seen in ~12 % of the visceral muscle tissues and 6 % of
the salivary glands in BPHs receiving dsPns10 (Table 2).
No significant difference in the number of positive samples
was found between BPHs that received dsGFP and diet
alone (Table 2). Therefore, RNAi induced by dsPns10
inhibited efficient virus infection and spread in the body of
insect vectors.
DISCUSSION
RRSV Pns10 is responsible for formation of the
viroplasm matrix in virus-infected cells
Immunoelectron and immunofluorescence microscopy of
RRSV-infected cells showed that the non-structural protein
Pns10 of RRSV was a component of the viroplasm matrix
where core-like particles and viral particles accumulated
(Figs 3 and 4), confirming that viroplasms are the site of
virus assembly for plant reoviruses. Among the three nonstructural proteins encoded by RRSV, only expression of
Pns10 in Sf9 cells, a non-host of RRSV, resulted in formation
of viroplasm-like structures, whereas neither of the other
two non-structural proteins, Pns6 and Pns7, appeared to
form viroplasm-like structures in Sf9 cells (Fig. 2a). These
results suggested that Pns10 was the minimal viral factor
required for viroplasm formation during RRSV proliferation
and that formation of the viroplasm matrix was not specific
to host plant or insect vector cells and did not require hostspecific components. Our studies also showed that Pns10
could recruit Pns6 into the viroplasm-like structures formed
by Pns10 during co-expression of these two proteins in
Sf9 cells (Fig. 2b). Thus, Pns6 might be recruited to the
viroplasm through the association of Pns6 with Pns10
during virus infection. Our results suggested that RRSV
Pns6, which functions as a viral MP in plant hosts (Wu
et al., 2010b), is probably involved in virus replication.
Furthermore, the filament-like structures protruding from
the plasma membrane formed by RRSV Pns7 strongly
resembled those formed by P7-1 of SRBSDV and Pns10 of
RDV (Liu et al., 2011; Wei et al., 2006a). The efficient spread
of RDV among insect vector cells is dependent on the
formation of tubular structures induced by Pns10 (Wei et al.,
2006a). It will be interesting to examine whether RRSV Pns7
also forms the same kind of structures and plays a similar
role in the spread of virus among insect vector cells.
Non-structural proteins essential for formation of the
viroplasm matrix have common characteristics among
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 22:13:29
2305
D. Jia and others
viruses in the family Reoviridae. RRSV Pns10 contains the
amino acid sequence motifs typical of an ATPase protein
(Z.-X. Gong, unpublished data; http://www.doc88.com/p33746844520.html) and RNA-binding protein (Upadhyaya
et al., 1997), suggesting that Pns10 might have ATPase and
RNA-binding abilities. The viroplasm matrix protein P9-1
of RBSDV and MRCV can bind RNA (Akita et al., 2012;
Maroniche et al., 2010). In addition, P9-1 of MRCV has
ATPase activity (Maroniche et al., 2010). The RNA-binding
and NTPase activities of the viroplasm matrix protein
NSP2 of rotavirus in the family Reoviridae have been
studied in detail (Kumar et al., 2007; Vasquez-Del Carpio
et al., 2006). All these proteins are able to form viroplasmlike structures in non-host cells (Akita et al., 2012;
Fabbretti et al., 1999; Maroniche et al., 2010). The parallels
among Pns10 of RRSV, NSP2 of rotavirus and P9-1 of
MRCV and RBSDV suggest that these proteins might play
similar roles in formation of the viroplasm matrix during
virus replication cycles. It is interesting to note that
viroplasm matrix proteins such as rotavirus NSP2, RBSDV
P9-1 and RGDV Pns9 can form octameric structures (Akita
et al., 2011, 2012; Jiang et al., 2006); thus, RRSV Pns10 may
have a similar structure.
Assembly of the viroplasm by Pns10 accompanies
the sequential infection by RRSV of the internal
organs of BPHs
In our study of the sequential infection of RRSV in the
internal organs of BPHs, the accumulation of virus and the
formation of viroplasms were analysed by double labelling
the internal organs with virus–FITC and Pns10–rhodamine. As early as 1 day after the virus was ingested, masses
of viral particles had accumulated in the lumen or were
attached to the microvillar membrane of the midgut (Fig.
1b). However, only a limited number of viral particles in
the lumen had successfully crossed the microvilli into the
epithelial cells of the midgut (Fig. 1c, Table 1). The fact that
not all insects feeding on RRSV-infected rice plants became
infected could be due to the infection barrier posed by the
midgut. At an early stage of virus infection in the epithelial
cells of the midgut, even in a single virus-infected cell,
RRSV could initiate the formation of nascent viroplasms
(Fig. 3a), which serve as the sites for assembly of progeny
virions. It is interesting that most of the progeny RRSV
virions traversed the basal lamina of the midgut to infect
the visceral muscle tissues bordering the infected region,
rather than spreading extensively into the adjacent
epithelial cells of the midgut (Fig. 1d, Table 1). Subsequently, progeny virions spread to the visceral muscle
tissues surrounding the midgut and hindgut (Figs 1f–h and
3b, Table 1). RRSV might disseminate directly from the
visceral muscle tissues into the haemolymph and then into
the salivary glands. After a latency period of ~9 days, the
salivary glands were heavily infected, as shown by almost
complete distribution of virus particles and viroplasms
throughout the glands, which were proposed to support
the assembly of a large number of progeny virions (Figs 1i,
2306
j and 3c). Based on the preceding discussion, formation of
the viroplasm for assembly of progeny virions is probably
essential for the establishment of a persistent infection of
RRSV in insect vectors.
Our recent findings revealed a significantly different
infection route for RDV, a phytoreovirus, in its leafhopper
vector (Chen et al., 2011). RDV initially infected the
epithelial cells of the leafhopper filter chamber. Following
accumulation of progeny virions in these cells, most RDV
particles spread to the epithelial cells of other organs such
as the midgut. Furthermore, RDV may exploit nerves to
spread into the visceral muscle tissues surrounding the
anterior midgut, although we did not observe particles
associated with the neural tissues of BPHs (data not
shown).
RRSV Pns10 is essential for virus infection in
BPHs
Our results showed that RNAi induced by ingestion of
dsPns10 strongly inhibited efficient virus infection and
spread in the body of BPHs. Treatment with dsPns10
reduced the number of positive BPHs with early virus
infection in the epithelial cells of the midgut by ~67 %
(Table 2). In RRSV-positive BPHs that received dsPns10,
RRSV still could infect a limited number of the epithelial
cells of the midgut, but the subsequent spread of RRSV
from the initial infection site to additional tissues was
strongly inhibited (Table 2). Thus, RNAi induced by
dsPns10 treatment remained activated, even in RRSVpositive BPHs, which may have caused the slower virus
spread in the body of BPHs. RNAi induced by dsRNA is a
conserved sequence-specific gene-silencing mechanism
(Fire et al., 1998). Because the sequences of Pns10 and
other viral genes of RRSV showed no homology (data not
shown), we deduced that the RNAi response induced by
dsPns10 most probably inhibited expression of the Pns10
gene in BPHs. Due to the essential role of RRSV Pns10 in
biogenesis of the viroplasm matrix, we thus determined
that knockdown of Pns10 expression would block formation of the viroplasm necessary for virus replication and
assembly of progeny virions in the epithelial cells of the
midgut, preventing efficient virus infection and spread in
the body of BPHs. This conclusion agrees with our recent
finding for SRBSDV, a fijivirus, on the inhibition of
viroplasm assembly and virus replication in the body of
white-backed planthoppers by RNAi induced by dsRNA
targeting the viral gene for the viroplasm matrix protein
P9-1 (Jia et al., 2012). Similarly, short-interfering RNAs
against a viral gene for NSP2, the viroplasm matrix protein
of rotavirus, inhibited viroplasm formation, genome
replication, virion assembly and synthesis of the other
viral proteins (Silvestri et al., 2004). Recently, transgenic
rice plants, in which the expression of viroplasm matrix
proteins of plant reoviruses, including RBSDV, RDV and
RGDV, was silenced by RNAi, were shown to be strongly
resistant to virus infection (Shimizu et al., 2009, 2011,
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 22:13:29
Journal of General Virology 93
Viroplasm essential for RRSV infection in vector
2012). These results all support the hypothesis that the
viroplasm plays a pivotal role in replication of viruses in
the family Reoviridae. In this context, identification of RRSV
Pns10 as the driving force for viroplasm formation is a step
towards identifying suitable targets for pathogen-derived
resistance strategies to control disease. In this study, RNAi
induced by synthesized dsRNA could overcome the lack of a
reverse genetics system in RRSV and opens up new
opportunities to understand the biological activities of viral
proteins in replicative cycles in vivo.
METHODS
Antibody preparation. The preparation of RRSV antigen-specific
IgGs has been described previously (Takahashi et al., 1991). Rabbit
polyclonal antiserum against RRSV Pns10 was prepared as described
by Akita et al. (2012). IgG was isolated from specific polyclonal
antiserum using a protein A–Sepharose affinity column (Pierce).
RRSV antigen-specific IgGs were conjugated directly to FITC (virus–
FITC) and Pns10-specific IgGs were conjugated directly to rhodamine
(Pns10–rhodamine) according to the manufacturer’s instructions
(Invitrogen).
To determine the specificity of anti-Pns10 antibodies, total proteins
were extracted from 1 g RRSV-infected and healthy rice plants. The
proteins were separated by SDS-PAGE, transferred to a PVDF
membrane and detected on immunoblots using prepared antibodies,
as described previously (Spinelli et al., 2006).
Baculovirus expression of the non-structural proteins of RRSV.
A baculovirus system was used to express the three non-structural
proteins of RRSV, Pns6, Pns7 and Pns10, as described previously (Wei
et al., 2006b). Recombinant baculovirus vectors containing Pns6 and
Pns7 fused to a 66His tag (Pns6–His and Pns7–His) and Pns10 fused
to a Strep Tag II (Pns10–Strep) were introduced into DH10Bac
(Invitrogen) for transposition into the bacmid. Recombinant bacmids
were transfected into Sf9 cells in the presence of Cellfectin (Invitrogen)
according to the manufacturer’s instructions. Sf9 cells infected with
recombinant bacmids were incubated for 72 h, fixed in 4 %
paraformaldehyde and processed for analysis by immunofluorescence
microscopy, as described previously (Wei et al., 2006a, b). Cells infected
with recombinant baculoviruses containing Pns6–His or Pns7–His
were stained with rabbit anti-66His tag polyclonal antibody (Abcam)
and rhodamine-conjugated anti-rabbit IgG antibody (Sigma) as the
secondary antibody. Cells infected with recombinant baculoviruses
containing Pns10–Strep were stained with anti-Strep Tag II mAb (IBA)
and FITC-conjugated anti-mouse IgG (Sigma) as the secondary
antibody. Samples were examined under a Leica TCS SP5 inverted
confocal microscope, as described previously (Wei et al., 2006a, b).
Immunofluorescence staining of the internal organs of BPHs
after acquisition of virus. Second-instar nymphs of BPHs were
allowed a 2-day AAP on rice plants infected with RRSV. On different
days after the AAP, internal organs from BPHs were removed, fixed in
4 % paraformaldehyde and processed for immunofluorescence
microscopy, as described previously (Wei et al., 2006a, b). RRSV
particles were stained with virus–FITC. Viroplasms were stained with
Pns10–rhodamine. Actin was stained with phalloidin–rhodamine or
phalloidin–Alexa Fluor 647 carboxylic acid (Invitrogen). The particles
were then imaged by a Leica TCS SP5 inverted confocal microscope,
as described previously (Wei et al., 2006a, b).
Electron microscopy. The salivary glands of infected BPHs were
fixed, dehydrated and embedded, as described previously (Wei et al.,
http://vir.sgmjournals.org
2006a, b). Cell sections were then incubated with antibodies against
Pns10 and immunogold labelled with goat antibodies against rabbit
IgG that had been conjugated to 15 nm gold particles (Sigma) (Wei
et al., 2006a, b).
dsRNA production. A DNA fragment spanning a 793 bp segment of
the Pns10 gene of RRSV was amplified by PCR using forward primer
59-ATTCTCTAGAAGCTTAATACGACCACTATAGGGCGTGCAATTCCCGAACTTGT-39 and reverse primer 59-ATTCTCTAGAAGCTTAATACGACTCACTATAGGGACCAGACCAATGTCGCTTGAC-39,
both possessing a T7 promoter (shown in italic) at the 59 end. A DNA
fragment spanning a 717 bp segment of the gfp gene was amplified by
PCR using forward primer 59-ATTCTCTAGAAGCTTAATACGACTCACTATAGGGATGAGTA AAGGAGAAGAACTT-39 and reverse
primer 59-ATTCTCTAGAAGCTTAATACGACTCACTATAGGGTTATTTGTATAGTTCATCCATG-39, also with a T7 promoter (shown
in italic) at the 59 end. PCR products were used for dsRNA synthesis
according to the protocol for the T7 RiboMAX Express RNAi System
kit (Promega).
Examination of the effect of dsRNAs on virus infection in the
insect vectors. dsRNAs targeting viral genes were delivered to BPHs
using a membrane feeding approach (Chen et al., 2010). Briefly,
second-instar nymphs of BPHs were fed a diet of 0.5 mg dsRNA ml21
diluted in 10 % sucrose on a membrane for 1 day, allowed a 2-day
AAP on RRSV-infected rice plants and then fed on uninfected rice
seedlings. The transcript levels of viral genes for the targeted nonstructural protein Pns10 and major outer-capsid protein P8 were
determined by RT-PCR (Upadhyaya et al., 1996, 1997). In addition,
internal organs from BPHs receiving dsRNAs or diet alone were fixed
and processed for immunofluorescence microscopy, as described
elsewhere (Wei et al., 2006a, b).
ACKNOWLEDGEMENTS
This research was supported by the National Basic Research Program
of China (no. 2010CB126203), projects from the Ministry of
Education of China (nos 211082 and 20103515120007), the key
project of Department of Education of Fujian Province, China (no.
JA10097), the National Natural Science Foundation of China (nos
31130044, 31070130 and 30970135), the New Century of Excellent
Talents at Universities (no. NCET-09-0011) and the program for
Promotion of Basic Research Activities for Innovative Biosciences of
the Bio-oriented Technology Research Advancement Institution
(BRAIN) of Japan (to T. O.).
REFERENCES
Akita, F., Miyazaki, N., Hibino, H., Shimizu, T., Higashiura, A.,
Uehara-Ichiki, T., Sasaya, T., Tsukihara, T., Nakagawa, A. & other
authors (2011). Viroplasm matrix protein Pns9 from rice gall dwarf
virus forms an octameric cylindrical structure. J Gen Virol 92, 2214–
2221.
Akita, F., Higashiura, A., Shimizu, T., Pu, Y., Suzuki, M., UeharaIchiki, T., Sasaya, T., Kanamaru, S., Arisaka, F. & other authors
(2012). Crystallographic analysis reveals octamerization of viroplasm
matrix protein P9-1 of Rice black streaked dwarf virus. J Virol 86, 746–
756.
Boccardo, G. & Milne, R. G. (1984). Plant Reovirus Group. CMI/AAB
Descriptions of Plant Viruses, no. 294. Kew, UK: Commonwealth
Microbiology Institute/Association of Applied Biology.
Chen, J., Zhang, D., Yao, Q., Zhang, J., Dong, X., Tian, H., Chen, J. &
Zhang, W. (2010). Feeding-based RNA interference of a trehalose
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 22:13:29
2307
D. Jia and others
phosphate synthase gene in the brown planthopper, Nilaparvata
lugens. Insect Mol Biol 19, 777–786.
Chen, H., Chen, Q., Omura, T., Uehara-Ichiki, T. & Wei, T. (2011).
Sequential infection of Rice dwarf virus in the internal organs of its
insect vector after ingestion of virus. Virus Res 160, 389–394.
Fabbretti, E., Afrikanova, I., Vascotto, F. & Burrone, O. R. (1999).
Two non-structural rotavirus proteins, NSP2 and NSP5, form
viroplasm-like structures in vivo. J Gen Virol 80, 333–339.
Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E. &
Mello, C. C. (1998). Potent and specific genetic interference by
double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–
811.
Hagiwara, K., Minobe, Y., Nozu, Y., Hibino, H., Kimura, I. & Omura, T.
(1986). Component proteins and structures of rice ragged stunt virus.
Shimizu, T., Yoshii, M., Wei, T., Hirochika, H. & Omura, T. (2009).
Silencing by RNAi of the gene for Pns12, a viroplasm matrix protein
of Rice dwarf virus, results in strong resistance of transgenic rice plants
to the virus. Plant Biotechnol J 7, 24–32.
Shimizu, T., Nakazono-Nagaoka, E., Akita, F., Uehara-Ichiki, T.,
Omura, T. & Sasaya, T. (2011). Immunity to Rice black streaked dwarf
virus, a plant reovirus, can be achieved in rice plants by RNA silencing
against the gene for the viroplasm component protein. Virus Res 160,
400–403.
Shimizu, T., Nakazono-Nagaoka, E., Akita, F., Wei, T., Sasaya, T.,
Omura, T. & Uehara-Ichiki, T. (2012). Hairpin RNA derived from the
gene for Pns9, a viroplasm matrix protein of Rice gall dwarf virus,
confers strong resistance to virus infection in transgenic rice plants.
J Biotechnol 157, 421–427.
J Gen Virol 67, 1711–1715.
Silvestri, L. S., Taraporewala, Z. F. & Patton, J. T. (2004). Rotavirus
Hibino, H. (1996). Biology and epidemiology of rice viruses. Annu Rev
Phytopathol 34, 249–274.
replication: plus-sense templates for double-stranded RNA synthesis
are made in viroplasms. J Virol 78, 7763–7774.
Hibino, H., Roechan, M., Suoarisman, S. & Tantera, D. M. (1977). A
Spear, A., Sisterson, M. S. & Stenger, D. C. (2012). Reovirus genomes
virus disease of rice (kerdil hampa) transmitted by brown
planthopper, Nilaparvata lugens Stål, in Indonesia. Contr Centr Res
Inst Agric Bogor 35, 1–15.
Hibino, H., Saleh, N. & Roechan, M. (1979). Reovirus-like particles
from plant-feeding insects represent a newly discovered lineage within
the family Reoviridae. Virus Res 163, 503–511.
Spinelli, S., Campanacci, V., Blangy, S., Moineau, S., Tegoni, M. &
Cambillau, C. (2006). Modular structure of the receptor binding
associated with rice ragged stunt diseased rice and insect vector cells.
Ann Phytopath Soc Japan 45, 228–239.
proteins of Lactococcus lactis phages. The RBP structure of the
temperate phage TP901-1. J Biol Chem 281, 14256–14262.
Hoang, A. T., Zhang, H.-M., Yang, J., Chen, J.-P., Hébrard, E., Zhou,
G.-H., Vinh, V. N. & Cheng, J.-A. (2011). Identification, characteriza-
Supyani, S., Hillman, B. I. & Suzuki, N. (2007). Baculovirus expression of the 11 mycoreovirus-1 genome segments and identification
of the guanylyltransferase-encoding segment. J Gen Virol 88, 342–
350.
tion, and distribution of Southern rice black-streaked dwarf virus in
Vietnam. Plant Dis 95, 1063–1069.
Hogenhout, S. A., Ammar, D., Whitfield, A. E. & Redinbaugh, M. G.
(2008). Insect vector interactions with persistently transmitted
viruses. Annu Rev Phytopathol 46, 327–359.
Jia, D., Chen, H., Zheng, A., Chen, Q., Liu, Q., Xie, L., Wu, Z. & Wei, T.
(2012). Development of an insect vector cell culture and RNA
interference system to investigate the functional role of fijivirus
replication protein. J Virol 86, 5800–5807.
Jiang, X. F., Jayaram, H., Kumar, M., Ludtke, S. J., Estes, M. K. &
Prasad, B. V. V. (2006). Cryoelectron microscopy structures of
rotavirus NSP2–NSP5 and NSP2–RNA complexes: implications for
genome replication. J Virol 80, 10829–10835.
Kumar, M., Jayaram, H., Vasquez-Del Carpio, R., Jiang, X.,
Taraporewala, Z. F., Jacobson, R. H., Patton, J. T. & Prasad, B. V. V.
(2007). Crystallographic and biochemical analysis of rotavirus NSP2
with nucleotides reveals a nucleoside diphosphate kinase-like activity.
J Virol 81, 12272–12284.
Li, J., Chen, Q., Lin, Y., Jiang, T., Wu, G. & Hua, H. (2011). RNA
interference in Nilaparvata lugens (Homoptera: Delphacidae) based
on dsRNA ingestion. Pest Manag Sci 67, 852–859.
Liu, Y., Jia, D., Chen, H., Chen, Q., Xie, L., Wu, Z. & Wei, T. (2011). The
P7-1 protein of southern rice black-streaked dwarf virus, a fijivirus,
induces the formation of tubular structures in insect cells. Arch Virol
156, 1729–1736.
Maroniche, G. A., Mongelli, V. C., Peralta, A. V., Distéfano,
A. J., Llauger, G., Taboga, O. A., Hopp, E. H. & del Vas, M.
(2010). Functional and biochemical properties of Mal de Rı́o Cuarto
virus (Fijivirus, Reoviridae) P9-1 viroplasm protein show further
similarities to animal reovirus counterparts. Virus Res 152, 96–
103.
Takahashi, Y., Omura, T., Shohara, K. & Tsuchizaki, T. (1991).
Comparison of four serological methods for practical detection of ten
viruses of rice in plants and insects. Plant Dis 75, 458–461.
Tsai, J. & Perrier, J. L. (1996). Morphology of the digestive and
reproductive systems of Dalbulus maidis and Graminella nigrifrons
(Homoptera: Cicadellidae). Fla Entomol 79, 563–567.
Upadhyaya, N. M., Zinkowsky, E., Li, Z., Kositratana, W. &
Waterhouse, P. M. (1996). The Mr 43K major capsid protein of rice
ragged stunt oryzavirus is a post-translationally processed product of
a Mr 67,348 polypeptide encoded by genome segment 8. Arch Virol
141, 1689–1701.
Upadhyaya, N. M., Ramm, K., Gellatly, J. A., Li, Z., Kositratana, W. &
Waterhouse, P. M. (1997). Rice ragged stunt oryzavirus genome
segments S7 and S10 encode non-structural proteins of Mr
68,025 (Pns7) and Mr 32,364 (Pns10). Arch Virol 142, 1719–
1726.
Upadhyaya, N. M., Ramm, K., Gellatly, J. A., Li, Z., Kositratana, W. &
Waterhouse, P. M. (1998). Rice ragged stunt oryzavirus genome
segment S4 could encode an RNA dependent RNA polymerase
and a second protein of unknown function. Arch Virol 143, 1815–
1822.
Vasquez-Del Carpio, R., Gonzalez-Nilo, F. D., Riadi, G.,
Taraporewala, Z. F. & Patton, J. T. (2006). Histidine triad-like motif
of the rotavirus NSP2 octamer mediates both RTPase and NTPase
activities. J Mol Biol 362, 539–554.
Wei, T., Kikuchi, A., Moriyasu, Y., Suzuki, N., Shimizu, T., Hagiwara, K.,
Chen, H., Takahashi, M., Ichiki-Uehara, T. & Omura, T. (2006a). The
spread of Rice dwarf virus among cells of its insect vector exploits virusinduced tubular structures. J Virol 80, 8593–8602.
Miyazaki, N., Uehara-Ichiki, T., Xing, L., Bergman, L., Higashiura, A.,
Nakagawa, A., Omura, T. & Cheng, R. H. (2008). Structural evolution
Wei, T., Shimizu, T., Hagiwara, K., Kikuchi, A., Moriyasu, Y., Suzuki, N.,
Chen, H. & Omura, T. (2006b). Pns12 protein of Rice dwarf virus is
of Reoviridae revealed by Oryzavirus in acquiring the second capsid
shell. J Virol 82, 11344–11353.
essential for formation of viroplasms and nucleation of viral-assembly
complexes. J Gen Virol 87, 429–438.
2308
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 22:13:29
Journal of General Virology 93
Viroplasm essential for RRSV infection in vector
Wu, J., Du, Z., Wang, C., Cai, L., Hu, M., Lin, Q., Wu, Z., Li, Y. & Xie, L.
(2010a). Identification of Pns6, a putative movement protein of
cell-to-cell movement of Tobacco mosaic virus-based chimeric virus.
Virus Res 152, 176–179.
RRSV, as a silencing suppressor. Virol J 7, 335.
Wu, Z., Wu, J., Adkins, S., Xie, L. & Li, W. (2010b). Rice ragged stunt
Zhou, G. Y., Lu, X. B., Lu, H. J., Lei, J. L., Chen, S. X. & Gong, Z. X.
(1999). Rice ragged stunt oryzavirus: role of the viral spike protein in
virus segment S6-encoded nonstructural protein Pns6 complements
transmission by the insect vector. Ann Appl Biol 135, 573–578.
http://vir.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 22:13:29
2309