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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 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 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

1. Van Regenmortel M H V, Fauquet C M, Bishop D H L, Carstens E B, Estes M K, Lemon S M,

Maniloff J, Mayo M A, McGeoch D J, Pringle C R, and Wickner R B (2000) Virus .

Classification and Nomenclature of Viruses. Seventh Report of the International Committee on

Taxonomy of Viruses, pp. 395-480. (Academic Press, San Diego.)

2. Belloncik S and Mori H (1998) Cypoviruses. In: The Insect Viruses, ed. Miller L K and Ball LA, pp.

337-369. (Plenum Publishing Corporation, New York.)

3. Borsa J, Morash, B D, Sargent M D, Copps T P, Lievaart P A, and Szekely J G (1979) Two modes of

entry of reovirus particles into L cells. J. Gen. Virol. 45: 161-170.

4. Borsa J, Copps T P, Sargent M D, Long D G, and Chapman J D (1973) New intermediate subviral

particles in the in vitro uncoating of reovirus virions by chymotrypsin. J. Virol. 11: 552-564.

5. Kobayashi M (1972) Penetration of polyhedrosis viruses into the cultured migut cells of the silkworm,

Bombyx mori. J. Seric. Sci. Jpn. 41: 1-6.

6. Belloncik S, Berthiaume L, Alain R, and Arella M (1986) Replication of cytoplasmic polyhedrosis

virus (CPV) in cell culture: Electron microscopy study. J. Electron Microsc. (Jpn.) 35 (Suppl.):

3391-3392.

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7. Quiot J M and Belloncik S (1977) Characterisation d'une polyedrose cytoplasmique chez lepidoptere

Euxoa scandens, Riley (, Agrotinae): Etude in vivo et in vitro. Arch. Virol. 55: 145-153.

8. Arnott H J, Smith K M, and Fullilove S L (1968) Ultrastructure of a cytoplasmic polyhedrosis virus

affecting the monarch butterfly, Danaus plexippus. I. Development of virus and normal polyhedra in

the larva. J. Ultrastruct. Res. 24: 479-507.

9. Hatta T and Francki R I B (1982) Similarity in the structure of cytoplasmic polyhedrosis virus,

leafhopper A virus and Fuji disease virus particles. Intervirology 18: 203-208.

10. Zhang H, Zhang J, Yu X, Lu X, Zhang Q, Jakana J, Chen D H, Zhang X, and Zhou Z H (1999)

Visualization of protein-RNA interactions in cytoplasmic polyhedrosis virus. J. Virol. 73: 1624-1629.

11. Iwashita Y and Seki H (1973) The development of the cytoplasmic-polyhedrosis virus in the midgut of

the embyo and the young silkworm, Bombyx mori L. Bull. Coll. Agricult. Utsunomiya Univ. 8: 27-42.

12. Noguchi Y (1995) Newly isolated cytoplasmic polyhedrosis virus (CPV) derived from Dendrolimus

spectabilis CPV (DsCPV). J. Seric. Sci. Jpn 64: 493-497.

13. Noguchi Y and Yamaguchi K (1982) Development of disease in several species of Lepidopterous

subjected to cross-infection with cytoplasmic-polyhedrosis viruses. Jpn. J. appl. Entomol. Zool.

26: 281-287.

14. Sohi S S, Bird F T, and Hayashi Y (1971) Development of Malacosoma disstria cytoplasmic

polyhedrosis virus in Bombyx mori ovarian and tracheal tissue cultures. Proc. Ivth Int. Colloq. Insect

Pathol., College Park, Maryland pp. 340-351.

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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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