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Letter
Entry of Bombyx mori cypovirus 1 into midgut cells in vivo
Yu-Rong Tan, Jing-Chen Sun, Xin-Ying Lu, De-Ming Su and Jing-Qiang Zhang*
Division of Structural Biology, State Key Laboratory for Biocontrol, College of Life Sciences, Zhongshan
University, Guangzhou 510275, China
* Corresponding author:
Prof. Jing-Qiang Zhang
Division of Structural Biology, State Key Laboratory for Biocontrol, College of Life Sciences, Zhongshan
University, #135 Xingang West Road, Guangzhou 510275, China
Telephone number: 86-20-84112286
Fax number: 86-20-84110108
E-mail address: [email protected]
Running title: Entry of BmCPV-1
Keywords: Bombyx mori cypovirus 1, in vivo, intact virion, direct penetration, electron microscopy
The total number of pages is nine and the total number of figures is three.
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Abstract
In vivo entry of Bombyx mori cypovirus 1 (BmCPV-1) into silkworm midgut cells was studied by electron
microscopy. Virions were observed adhering to the plasma membrane of microvilli of the columnar cells,
embedding in the membrane, and settling themselves intact inside the microvilli. These behaviors suggested
that intact BmCPV-1 virions enter columnar cells by means of direct penetration through the cell membrane.
In addition, goblet cells, muscle cells and the hemocoele were also involved in early events of the virus
infection. However, no replication of the virus had ever been detected in these invaded cells except for
columnar cells.
Keywords: Bombyx mori cypovirus 1, in vivo, intact virion, direct penetration, electron microscopy
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Cytoplasmic polyhedrosis viruses (cypoviruses, CPVs), the second major group of insect pathogens, belong
to the genus Cypovirus in the family Reoviridae [1] These viruses mainly infect midgut epithelial cells of
host larvae, and are characterized by the production of huge numbers of virion-occluded polyhedra in the
cytoplasm [2]. Viruses belonging to Reoviridae are nonenveloped, double-stranded RNA viruses with
segmented genomes. Two modes of virus entry, viropexis and direct penetration, have been proposed for
mammalian reovirus, a member of the Reoviridae family [3]. Direct penetration is the major mode of entry
for the intermediate subviral particles (ISVPs), which lose all of the outer capsid protein except for the
spike protein [4], while intact virions enter host cells primarily by viropexis. ISVPs induce leakage of
radioactive 51Cr from host cells and are in close proximity to regions of apparent discontinuity of the
plasma membrane. As for CPVs, the mode of virus entry into host cells is not finally resolved. According to
Kobayashi [5], the core material of BmCPV-1 is injected via the viral spikes into the cytoplasm of primary
cultured midgut cells. Whereas the study on Euxoa scandens CPV (EsCPV) showed that the virions enter
the cultured cells by viropexis [6-7]. We herein report the results of electron microscopic observation which
suggests direct penetration of intact BmCPV-1 virions into midgut cells of silkworm Bombyx mori.
Purified polyhedra of BmCPV-1 were generously donated by Prof. Xinyao Xu (South China Agriculture
University, Guangzhou, China). Second-instar silkworm larvae were fed with fresh mulberry leaves at
room temperature. Prior to infection, the leaves were smeared with a high concentration of polyhedron
preparation (2.3h107 polyhedra/ml) and air-dried. Newly molted second-instar larvae were fed with the
virus-contaminated leaves for 6 hours and then replaced with virus-free fresh leaves. Larvae fed only with
virus-free fresh leaves were set as the control.
For electron microscopy, the posterior half of the midguts from both infected and control larvae were
dissected at the following time intervals post infection (p.i.): 1 h, 2 h, 3 h, 8 h, 12 h, 24 h, and 48 h (the start
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of administration of virus-contaminated leaves is set as zero time). The midguts were fixed with 2.5%
glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 2 h, washed with phosphate buffer, and postfixed
with 1% osmium textroxide in phosphate buffer for 1 h. Then washed again with phosphate buffer,
dehydrated in graded series of ethanol, and embedded in Spurr low viscosity epoxy mixture. Ultrathin
sections (<60-90 nm) were obtained with an LKB-Nova Ultrotome, stained with uranyl acetate for 30 min,
and then with lead citrate for 20 min. Sections were examined under a JEOL electron microscope at 100
kV.
During the first 8 hours of virus infection, no formation of the virogenic stroma, the first morphological
sign of CPV replication perceivable by electron microscopy, was detected in the midgut cells. The virions
observed during this period should occur as the result of invasion and not as the result of replication. At 3 h
p.i., virions were observed outside and inside midgut cells. As seen in Fig. 1a, virions released from
polyhedra were detected in the midgut lumen, within the peritrophic membrane, and in association with
microvilli of columnar cells. Apparently the fibrous peritrophic membrane did not prevent the invasion of
virions. Virions adhered to the surface of microvilli, embedded in the plasma membrane, or settled
themselves inside the microvilli (Fig. 1b-d). The plasma membrane with embedded virion was
discontinuous only at the virus penetration site, and no obvious disruption of the membrane was observed.
Some virions were seen close to the proximal end of microvillus, or partially inside it, and some were
found free in the cytoplasm (Fig. 1d). It was noted that all these virions retained their integrity of an
icosahedron, and seemed not impaired by the entry. Since none of plasma membrane invaginations
containing virions, or cytoplasmic vesicles containing virions had been detected, we excluded the
possibility of virion internalization by means of viropexis. Therefore, we summarized the process of entry
of BmCPV-1 virions into columnar cells as follows: first the virions recognized and attached themselves to
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the plasma membrane of microvilli by viral spikes; then the virions continued to interact with the
membrane, resulting in a barely discernible membrane disruption at the site; and eventually, the virions
penetrated intact through the microvilli. Now the invading virions had settled in the cytoplasm of columnar
cells, and it is of great interest to know how they would behave later on.
In addition to columnar cells, goblet cells, muscle cells and the hemocoele were also involved in early
events of BmCPV-1 infection at 3h p.i.. Intact virions were seen in the goblet chamberˈand in the
cytoplasmic projections and cytoplasm of goblet cells (Fig. 2a). Occasionally, virions were caught
penetrating the membrane of cytoplasmic projections. These observations suggested that virions in the
midgut lumen could enter the goblet chamber through the apical opening, and penetrate the cytoplasm of
goblet cells through these cytoplasmic projections. Virions were also detected in midgut muscle cells
adjacent to the basal lamina and in the hemocoele (Fig. 2b). These observations indicated that some of the
virions that entered the cytoplasm of midgut epithelial cells continued to migrate toward the basal portion
of the cell, penetrated through the basal plasma membrane, and entered muscle cells or the hemocoele. Still,
we should not rule out the possibility that some virions might reach their destination through the
intercellular space between epithelial cells.
Unfortunately, no virions or virus-like particles were observed at 1 h, 2 h and 8 h p.i.. The reason may be
as following: (ν) It is very difficult to catch the transient image of virus entry which occurred within split
second, the image of BmCPV-1 entry was not observed except in 3 h samples; (ξ) In vivo, the entry of the
virus is asynchronous which makes our work tough and hazardous; (ο) During the eclipse phase of virus
infection no virus-like particles or virions could be found yet within the cells.
The virogenic stroma and progeny virions were first seen in infected columnar cells at 12h p.i.. The
mode of in vivo BmCPV-1 replication was the same as described previously for other CPV infections [8].
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The virus particles first appeared in the virogenic stroma with a micronet structure, and many of mature
virions were then occluded into crystalline protein forming polyhedra (Fig. 3a). Occasionally, empty virus
particles (i.e., viral capsids without the core) were produced, together with partially filled and mature virus
particles (Fig. 3b), suggesting that the viral capsid was produced and was subsequently filled with the core
material via the spike. In this work, we did not find any signs of virus replication in non-columnar cells of
the midgut.
The present study provides the first in vivo morphological evidence that intact BmCPV-1 virions enter B.
mori midgut cells by direct penetration, as was the case with ISVPs of reovirus [3]. Morphologically, CPV
virions are similar to ISVPs in that they lack the outer coat of reovirus except for the spike protein [9]. The
direct penetration of intact BmCPV-1 virions into the cell is in accordance with our previous study that the
transcriptase of BmCPV-1, a prerequisite for viral productive infection, is located at the capsid and retains
its position on the capsid when viral RNA has dropped out [10], providing a most effective way to ensure
the accurate delivery of all ten genes and enzymes to the cytoplasm for further initiation of virus
replication.
Commonly, midgut columnar cells are the target cells in CPV infection, and occasionally midgut goblet,
regenerative and basal granular cells could also be infected [11-12]. In the present study, we have
demonstrated that BmCPV-1 invasion also occurred in goblet cells, muscle cells and in the hemocoele
although no virus replication took place. There have been several reports that demonstrate CPV replication
in tracheal and muscle cells associated with the ovary or midgut [12-14]
In short, a new mode of virus entry is demonstrated for BmCPV-1 infection in vivo by electron
microscopy. The virions enter host cells by direct penetration of the plasma membrane. The finding
presents a morphological evidence for the virus entry and will facilitate a better understanding of CPV
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infection and replication.
Acknowledgments
We thank Dr. Debbie Rae and Dr. Z. Hong Zhou for advice in the preparation of the manuscript. This work
was supported by special funds of National Natural Science Foundation of China (No. 30070169).
References
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Figure legends
Fig. 1. Electron micrographs of B. mori midgut columnar cells infected with BmCPV-1, showing direct
entry of intact virions into cells at 3 h p.i.. (a) Virions (arrow) administrated per os were present in the
midgut lumen (L), in the peritrophic membrane (PM) and associated with the microvilli (Mv) of the
columnar cells. (b) Cross section of microvilli (Mv) showing different stages of virus entry. Intact virions
were seen to adhere to (blank arrow), penetrate (filled black arrow) and be inside (arrowhead) the microvilli.
Insert: higher magnification of a virion penetrating the membrane. (c) Longitudinal section of microvilli
showing the virion invasion (arrow) of microvilli (Mv). (d) Direct entry of intact virions (arrow) into the
columnar cells (CC) through the membrane of microvilli (Mv). Arrowheads show virions penetrating the
membrane. Triangle indicates the plasma membrane adjacent to the next columnar cell. All bars represent
100nm.
Fig. 2. Presence of perorally adminstrated BmCPV-1 virions in B. mori midgut cells other than columnar
cells at 3 h p.i.. (a) Virions (arrow) were detected in the goblet chamber (GCh), and in the cytoplasmic
projections (CP) and the cytoplasm of the goblet cell (GC). CC: a neighboring columnar cell. (b) Virions
(arrow) were distributed among myofibrils in the cytoplasm of midgut muscle cell (MC). Several virions
(arrow) occurred in the hemocoele (H). All bars represent 100nm.
Fig. 3. Presence of virogenic stroma and progeny virions of BmCPV-1 in B. mori columnar cells at 24 h p.i.
(a) and 48 h p.i. (b), respectively. (a) Progeny virions (arrow) and polyhedra (P) containing mature virions
occurred in or near the virogenic stroma (VS) within the cytoplasm. (b) Empty virus particles (blank arrow),
partially filled virus particles (filled black arrow), and mature virus particles (arrowhead) were seen in the
cytoplasm, suggesting that the viral capsid was produced and subsequently filled with the core material via
the spike. P: polyhedron. All bars present 100nm.
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