Rice dwarf virus is engulfed into and released via
vesicular compartments in cultured insect vector
Taiyun Wei, Hiroyuki Hibino and Toshihiro Omura
National Agricultural Research Center, 3-1-1 Kannondai, Tsukuba, Ibaraki 305-8666, Japan
Received 12 March 2008
Accepted 24 June 2008
Vector insect cells infected with Rice dwarf virus had vesicular compartments containing viral
particles located adjacent to the viroplasm when examined by transmission electron and confocal
microscopy. Such compartments were often at the periphery of infected cells. Inhibitors of
vesicular transport, brefeldin A and monensin, and an inhibitor of myosin motor activity,
butanedione monoxime, abolished the formation of such vesicles and prevented the release of
viral particles from infected cells without significant effects on virus multiplication. Furthermore,
the actin-depolymerizing drug, cytochalasin D, inhibited the formation of actin filaments without
significantly interfering with formation of vesicular compartments and the release of viruses from
treated cells. These results together revealed intracellular vesicular compartments as a mode for
viral transport in and release from insect vector cells infected with a plant-infecting reovirus.
In vector cells grown in monolayers (VCMs), Rice dwarf
virus (RDV), a phytoreovirus, multiplies and spreads from
primarily infected cells to neighbouring cells (Wei et al.,
2006a) in addition to spreading via mature, free viral
particles. Infection via free viral particles was protected by
the addition of neutralizing antibodies to the cell culture
medium. As part of integrated studies on RDV prolifera-
tion in vector insects, we have focused our study on the
accumulation of the virus in cells of the insect vector and
on the subsequent release of the virus.
In electron micrographs of thin sections of insect tissues
infected with plant-pathogenic reoviruses, viral particles
were sequestered in spherical vesicular compartments
(Fukushi et al., 1962; Shikata & Maramorosch, 1965;
Shikata, 1969; Vidano, 1970; Omura et al., 1985). However,
the biological significance of these inclusions in RDV
infection has not been clarified because, for the most part,
the tissues examined were in the late stage of infection
which made it difficult to gather details on the formation
of these compartments. Similar vesicular compartments
appear to play a role in the release of viral particles from
cultured cells infected with animal viruses, such as severe
acute respiratory syndrome coronavirus (Ng et al., 2003)
and human immunodeficiency virus (HIV) (Nydegger
et al., 2003).
A method for the continuous culture of cells from the
leafhopper vector Nephotettix cincticeps, consisting of
VCMs, has allowed the study of infection with RDV
because such infection results in asymptomatic but
persistent infection (Peterson & Nuss, 1985; Kimura,
1986). VCMs have been used to reveal fundamental aspects
of viral activity at the cellular level, which suggests details
of the transmission, multiplication and cytopathology of
RDV in vector insects (Wei et al., 2006a, b, c, 2007, 2008).
In the present study with this system, we investigated the
role of vesicular compartments in the transport of RDV
and its release from infected VCMs by confocal and
electron microscopy, and the use of drugs that inhibit
We examined the intracellular distribution of RDV during
the late stages of viral infection when viral particles would
be released from the cells. VCMs grown on a coverslip
(15 mm in diameter) were inoculated with RDV at an
m.o.i. of 10 and fixed 36 h post-inoculation (p.i.) for
transmission electron microscopy as described previously
(Omura et al., 1998). In these figures, RDV particles,
confirmed by immunoelectron microscopy in our earlier
study (Wei et al., 2006a), were easily distinguished by their
spherical appearance and diameter (70 nm), and they were
not found in uninoculated controls (Fig. 1). Further, the
viroplasm, the site of viral replication and assembly,
reacted specifically with Pns12-specific antibodies as
reported earlier (Wei et al., 2006b, c), was easily recognized
by its characteristic appearance (Fig. 1a). As reported in
our earlier studies (Wei et al., 2006c), viral particles
distributed at the periphery of the viroplasm (Fig. 1a). We
sometimes observed viral particles in vesicular compart-
ments other than the viroplasm in the cytoplasm of RDV-
infected VCMs (Fig. 1a). These compartments varied in
size and often reached a diameter of 2 mm. Occasionally,
we observed viral particles in vesicular compartments at the
periphery of infected cells (Fig. 1b, c). Furthermore, viral
Journal of General Virology (2008), 89, 2915–2920
2008/002063G2008 SGMPrinted in Great Britain2915
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T. Wei, H. Hibino and T. Omura
2920 Journal of General Virology 89