J. gen. Virol. (197o), 7, 75-79
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75
D i r e c t Cell to Cell Transfer o f Bittner Virus
( A c c e p t e d 25 N o v e m b e r ~969)
Bittner virus consists of an internal component which arises in the cytoplasm and becomes
enveloped by budding through a cell membrane (Moore, 1962). During a study of this process
we obtained evidence that the virus could be transmitted directly from cell to cell and that
in some instances an unusual form of particle was involved. In this communication we
describe this unusual form and the engulfment of virus particles.
Pieces of spontaneous mammary tumours from C3H mice, originally obtained from the
Christie Institute, Manchester, were fixed in 3% glutaraldehyde in Sorensen's phosphate
buffer o f p H 7"2 for e hr at 4 °, post-fixed in I ~o osmium tetroxide for 2 hr at 4 ° and embedded
in Maraglas by standard techniques. Sections were stained by alcoholic uranyl acetate
followed by lead citrate.
Budding particles were found at cytoplasmic vacuoles and at the plasma membrane.
Particles which budded at the plasma membrane were not randomly distributed at the
periphery but tended to concentrate at free cell surfaces (Fig. I). Some particles, however,
budded where cell surfaces were in apposition, and these were frequently undergoing pinocytosis by the adjacent cell (Fig. 2 A, B, C). Only'immature B' particles have been observed
within pinocytic vesicles. Initiation of pinocytosis did not require the presence of a free
virus particle since vesicles began to form around particles before completion of the budding
process (Fig. 2D, E). Many empty pinocytic vesicles were seen (Fig. 2 F) while some appeared
to be engulfing part of the cytoplasm of an adjacent cell (Fig. 2G).
About 1% of all the particles budding at the plasma membrane had an elongated internal
component which extended from the tip to the base of the bud (Fig. 3 A). These unusual forms
had a diameter of 6o to 65 rim. with an internal component of diameter 5o to 55 nm. Some
were expanded at their distal end and may release normal 'immature B' particles. In contrast to the more usual form of budding particle these forms (stems) were found only where
the plasma membranes of adjacent cells were apposite. The tip of the stem was frequently
situated in a pinocytic vesicle forming in an adjacent cell (Fig. 3 B, C).
All the pinocytic vesicles involved in the processes described had membranes which were
thicker and denser than the original, normal plasma membrane (Fig. 2, 3). In those best
resolved the thickened membrane appeared as a central electron dense line with projections
which, though present on both sides, were more prominent on the convex surface (Fig. 2 D).
Enveloped mammalian viruses may enter a cell in a pinocytic vesicle or by fusion of the
virus envelope with the plasma membrane. Herpes virus (Morgan, Rose & Mednis, 1968) and
influenza virus (Morgan & Rose, I968 ) utilize both methods. There are, however, two
morphologically different types of pinocytic vesicle: those surrounded by a normal 'unit
membrane' structure and those (fuzzy vesicles) in which the membrane is thickened (Fawcett, 1966). Influenza (Morgan & Rose, I968) and Sendai viruses (Morgan & Howe, ~968)
are occasionally found within fuzzy vesicles whereas herpesviruses (Morgan et aI. I968) are
found in vesicles showing no thickening of the membranes. It is not known which method of
uptake is more efficient in initiating virus replication but, if the ceil to cell transmission of
virus which we describe is a biologically significant process, Bittner virus must be released
from fuzzy vesicles in a viable state. The process of direct cell to cell transfer could allow
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76
Short communications
spread of virus with little interference from i m m u n e processes. I f so, the observation that
stems are p r o d u c e d mainly, if n o t exclusively, where cell m e m b r a n e s are in close apposition
suggests that they are significant in dissemination of virus within a n animal.
Fig. I, Bittner virus in a mouse mammary tumour cell. Virus particles budding (B) at the free cell
surface or into small cytoplasmic vacuoles (V), Where adjacent cells are in apposition no budding
virus is seen. Both immature B (IB) and mature B (MB) forms are present.
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Fig, 2. A, B, C. Various stages of the engulfment of free virus in fuzzy vesicles. (D), (E) Virus partly
engulfed by vesicles before budding is complete. (F) Two apparently empty pinocytic vesicles.
(G) A pinocytic vesicle apparently engulfing part of the cytoplasm of an adjacent cell.
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78
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Fig. 3. (A) An unusual form of Bittner virus showing elongation of the internal component. The
normal double shell structure is represented by two parallel lines at each side of the stem. (B) A
Bittner virus stem. The enlarged tip is partly engulfed in a fuzzy vesicle. A stem cut obliquely is also
present (arrow). (C) A Bittner virus stem partly engulfed in a fuzzy vesicle.
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79
Our thanks are due to Professor K. B. Fraser for advice and encouragement and to
Miss P. Clyde for excellent technical assistance. The work was supported by the National
Fund for Research into Crippling Diseases.
Department of Microbiology
The Queen's University of Belfast
Grosvenor Road
Belfast
BTI2 6BN
F . W . GAY
J . K . CLARKE
EVELYN DERMOTT
REFERENCES
FAWCETT, D. W. (I966). The Cell, its Organelles and lnclusions : an Atlas of Fine Structure. W. B. Saunders Co.
Philadelphia.
MOORE, D. H. (1962). T h e milk agent. I n Tumors Induced by Viruses, p. II3. Ed. by A. J. D a l t o n a n d
F. H a g u e n a u . N e w Y o r k a n d L o n d o n : A c a d e m i c Press.
MORGAN, C. & HOWE, C. (I968). Structure a n d development of viruses as observed in the electron microscope.
IX. Entry of parainfluenza I (Sendal) virus. Journal of Virology 2, I I22.
MORGAN, C. & ROSE, H. M. (I968). Structure a n d development of viruses as observed in the electron microscope. VIII. Entry of i n f u e n z a virus. Journal of Virology 2, 925.
MORGAN, C., ROSE, H. U. & MEDNIS, B. (1968). Electron microscopy of herpes simplex virus. I. Entry. Journal
o f Virology 2, 507.
(Received 22 October I969)
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