Studies of Variola Virus Replication
in Cell Cultures and Chick Embryo Chorion-allantois Membrane
Elena Ryabchikova, Elena Sokunova, Julia
Kononova, Alexandr Guskov
State Research Center of Virology and Biotechnology "Vector",
Russian Ministry of Public Health, Koltsovo, Novosibirsk region, 633559.
is the causative agent of smallpox, one of the most severe human diseases.
Variola virus belongs to the Orthopoxvirus genus together with cowpox,
mouse pox, vaccinia and monkey pox viruses. Experimental studies with
living variola virus were stopped after the global eradication of smallpox,
and knowledge about the variola virus biology was "frozen". However
the global eradication of smallpox announced by WHO at the end of the
1970s, failed to eliminate the overall problem of orthopoxvirus infections.
In the meantime, epidemiological data demonstrate a high pathogenic
potential for orthopoxviruses. Sporadic cases of severe human diseases
caused by cowpox, buffalopox, and vaccinia viruses have been reported.
Monkeypox virus, the cause of recent infectious outbreaks in Africa,
is the most dangerous among presently circulating poxviruses [1-4].
On the other hand, the threat of use of variola virus by terrorists
calls for developing new, improved vaccines for smallpox prophylaxis.
There is a great need of renovation of knowledge about variola virus
biology in order to successfully development new vaccines and medicinal
Data about virus
interaction with cells and parameters of viral morphogenesis are among
the basic biological characteristics of any virus. Variola virus infection
in cells has been poorly examined; no detailed studies concerning features
of variola virus replication cycle and morphogenesis have been published.
Electron microscopy is the only single tool, which makes it possible
to visualize the viruses and their formation inside cells . Electron
microscopic characteristics of viral structure and replication are general
biological parameters and serves as features for virus classification.
The absence of comprehensive information about structural features of
variola virus reproduction is a gap in modern virology.
For these reasons,
we examined variola virus with a goal to describe the features of variola
virus reproduction in different cell cultures and in cells of chick
embryo chorion-allantois membrane using electron microscopy.
MATERIALS AND METHODS
(strains India-3, Congo-9 and Butler) derived from different geographic
zones were obtained from Russian National Collection of SRC VB "Vector"
and propagated in Vero cells to prepare viral inoculum.
cell cultures Vero and CV-1 (originated from green monkeys), Fl and
L-68 (originated from humans), and BHK-21 (originated from hamsters)
were infected with variola virus strains in multiplicity of 1-5 PFU/cell
and propagated for 24, 48 and 72 h. Each sample contained 1-1.5 x 106
cells. Infected cells were scraped down and fixed by 4% paraformaldehyde.
of 11 days of incubation were infected on chorion-allantois membrane
in a dose of 5-19 PFU and incubated during 48 and 72 days. Pocks were
dissected and fixes as cell cultures. All fixed samples were processes
for electron microscopy using routine technique and examined in Hitachi
RESULTS AND DISCUSSIONS
replication takes place in the cytoplasm of infected cells, as in other
orthopoxviruses. The replicating variola virus forms large inclusions
composed of unshaped viral material and viral particles. These inclusions
often are called as "viral factories". The initial stage of variola
virus replication is the appearance of viroplasm representing an accumulation
of the fibrillar and granular material. The next stage is assembly of
viral envelopes in viroplasm.
are seen on ultrathin sections as crescents and spheres in Fig. 1. The
variola virus envelope forms from viral proteins de novo and is not
a derivative of cellular membranes as are envelopes of influenza, vesicular
stomatitis, Marburg and Ebola, or other enveloped viruses. Completed
immature particles of variola virus are spherical in shape. The next
step of the virus morphogenesis is formation of nucleoid with a double-concave
oval (Fig. 2 insert). Mature particles of variola virus have brick-like
or rugby-ball-like shapes.
morphological parameters of assembly of different strains of variola
virus was not able to determine visible differences in morphogenesis
in Vero, CV-1, BHK-21, L-68 and FL cell cultures. Thus, origination
of cell culture or viral strain does not influence parameters of variola
virus morphogenesis. No visible differences have been found with morphogenesis
of other orthopoxviruses including vaccinia and cowpox viruses [6-8].
of variola virus in cell results in accumulation of mature virions (Fig.
2). The next step of the genetic "program" of virus is the infection
of other cells. This step requires emergence of viral particles from
the "parental" cell. One way is destruction of the cell, and most particles
of variola virus use this way. However, orthopoxviruses "found" additional
active exiting paths from the cell. They exploit the natural physiological
capacities of cells. Mature virions acquire an additional membrane envelope
derived from Golgi apparatus, and leave the cell like secretory granules.
This process was examined in details for vaccinia virus. "Enveloped"
virions differ from "non-enveloped" by antigenic and other biological
properties [9,10]. Examination of cell cultures infected with variola
virus revealed formation of "enveloped" particles. However, the number
of these particles were larger in Vero and CV-1 cells than in other
cultures. The process of "enveloping" itself is a rare occurrence in
prolonged cultures and may not be seen in each and every infected cell.
of variola virus is associated with the formation of unusual structures,
not observed in cells infected with other orthopoxviruses. There were:
curved membranes, tubular structures, polymorphous protein conglomerates,
dense aggregations of free nucleoids. Additional studies are needed
to establish a role for these structures. Probably they represent structures
forming in the case of alteration of variola virus reproduction.
infected cells of all examined cultures contained large amounts of mature
particles after 48 h of incubation. Many cells were destroyed and many
viral aggregations may be seen between the cells. Many virions were
bound with cellular debri. Samples of 72 h of incubation were mostly
composed of destroyed cells and cellular debri, and contained large
masses of virions associated with broken cells.
of the reproduction of India-3a, Congo-9 and Butler strains of variola
virus showed that ultrastructural parameters of their morphogenesis
are identical except for the number of forming "enveloped" virions.
membrane is the usual experimental system for examination of orthopoxviruses,
which induce formation of pocks visible by naked eyes. The Pocks are
a type of local damage of skin or mucous membranes [11,12]. Our previous
studies showed that morphology of the pocks, induced by vaccinia virus,
depends on viral genome . We performed comparative study of pocks
induced by India-3a, Congo-9 and Butler strains of variola virus on
chick embryo chorion-allantois membrane. A pock is in essence is a focus
of local damage of chorion epithelium. Underlying connective tissue
is also involved in formation of pock. Pocks are disk-like. A necrotic
zone develops in the center of the pock and forms a central cavity.
Size of the disk
depends mainly on proliferation of chorion epithelium. The largest pocks
were caused by India-3a strain, while Congo-9 and Butler strains caused
pocks of smaller sizes (2074+312, and 1428+256 micrometers, respectively).
Examination of pocks induced by variola virus strains on chick chorion-allantois
membrane revealed that parameters of viral morphogenesis are identical
to those observed in cell cultures.
total quantity of the viral progeny was much larger in the case of India-3a
strain. The "yield" of Congo-9 and Butler strains was the same. Pocks,
after 72 h of incubation, contain many infected cells filled with viral
particles, and infiltrating neutrophils (Fig. 4).
membrane represents a tissue system, and this makes possible to examine
tissue reactions to virus infection, including immune and inflammatory
reactions. A pock is the focus of damage induced by single virus particle,
so it is possible to compare features of virus reproduction, damage
and inflammation using pocks produced by different viral strains. Our
previous studies have been shown that the intensity of inflammation
is determined by intensity of tissue damage, vascular reaction and neutrophil
infiltration. In turn, the two first components completely depend on
virus reproduction . The present examination showed that the same
relationships operate in the case of variola virus infection. Neutrophil
infiltration of the pock initiates the defensive reaction of the organism
to virus replication. Three strains of variola virus cause very similar
infiltration. Sections of pocks showed the same number of neutrophils.
Lymphocytes also were present in pocks, but their number was very much
smaller than the number of neutrophils. The most interesting finding
was made in pocks induced by India-3a strain. Many neutrophils and lymphocytes
were apoptotic (Fig. 5). Apoptosis is a programmed death of cells, which
may be caused by various factors. Some viruses, for example, herpes
viruses, can induce apoptosis of immunocompetent cells . It should
be noted that apoptosis was observed only in uninfected immunocompetent
cells (neutrophils and lymphocytes). Apoptosis was not observed in infected
epithelium cells nor in cell cultures. The variola virus thereby kills
immunocompetent cells and provides more "comfortable housing" for its
replication. Thus, our studies showed the ability of variola virus to
induce apoptosis of neutrophils and lymphocytes in the zone of local
inflammation (in pocks) on chick chorion-allantois membrane. Most probably,
proteins encoding by viral genome are triggers of this apoptosis. Undoubtedly,
elimination of the cells of immune defense may be very important for
pathogenesis of variola virus infection. The next stage of the investigation
of this phenomenon should be examinations of animal infection.
features of the reproduction of variola virus strains (India-3a, Congo-9
and Butler) in prolonged cell cultures (Vero, CV-1, BHK-21, L-68 and
FL), and chick embryo chorion-allantois membrane have been studied.
Identical pattern of the virus morphogenesis was observed in all the
examined systems. Ability of variola virus to cause apoptosis of neutrophils
and lymphocytes infiltrating pocks has been shown.
- Arita, I., Jezek, Z., Khodakevich, L., Ruti, K. Am. J. Trop. Med.
Hyg. - 1985, 34, 781-789.
- Carra, L. and Dumbell, K.R. S. Afr. Med. J., 1987, , 846-848.
- Czerny, C.P., Eis-Hubinger, A.M., Mayr, A., Schneweis, K.E., and
Pfeiff, B. Zentralbl. Veterinarmed. (B), 1991, 38, 421-431.
- Jezek, Z., Marennikova, S.S., Mutumbo, M., Nakano, J.H., Paluku,
K.M., and Szczeniowski, M. J. Infect. Dis., 1986, 154, 551-555.
- Biel S.S., Gelderblom H.R. J. Clinical Virol. 1999. 13, 105-119.
- Moss B. Replication of poxviruses. In: Virology. V.3. Moscow, "Mir",
- Dales S. and Mosbach E. H. Virology. 1968. 35, 564-583
- Fenner F. Poxviruses. In: Fields Virology, Third Edition (ed.:
Fields B.N. et al) Lippincott-Raven Publishers, Philadelphia. 1996,
- Dunn W. J. Gen. Virol. 1998, 79, 877-887.
- Ichihashi Y. Virology. 1996, 217, 478-485.
- Marennikova, S.S. and Shchelkunov, S.N. Orthopoxviruses pathogenic
for humans. Moscow: KMK Scientific Press Ltd. Moscow, 1998.
- Buller R.M.L., Palumbo G.J. Microbiological Reviews. 1991, 55,
- Ryabchikova E.I., Streltsov V.V., Petrov V.S. Vopr. Virusol., 1990,
- Cuff S., Ruby J. Immunology and Cell Biology. 1996, 74, 527-537.
Variola virus, reproduction, cell cultures, chick embryo chorion allantois
Editor's Note: We thank Dr. Ryabchikova for this very important
and timely paper, which she presented at the CBMTS IV. Please see page
9 for information on Dr. Elena Ryabchikova's book on Ebola and Marburg
Fig. 1. Initial stages of variola virus morphogenesis
in cells of Vero culture Congo-9 and Butler strains). 1 - viroplasm;
2 - crescents and spherical virions; 3 - virion with dense material
Fig. 2. Cell of CV-1 culture containing many mature
particles of variola virus (India-3a strain). 1 - nucleus. Insert shows
virion at large magnification. Arrow shows nucleoid, thick arrow shows
Fig. 3. Pock on chick chorion-allantois membrane
induced by India-3a strain. 1- epithelium, 2 - connective tissue, 3
- blood vessel. Arrows show congested blood vessel near epithelium.
Thick arrow shows border of the pock.
Fig. 4. A part of pock on chick chorion-allantois
membrane induced by India-3a strain. 1 - infected epithelial cells;
2 - necrotic cells; 3 - neutrophil; 4 -lymphocyte; 5 - uninfected cells.
Viral particles are the small black spots in the infected epithelial
Fig. 5. Apoptotic cells in the central zone of
pock induced by India-3a strain of variola virus. Arrow shows nucleus
having typical for apoptosis appearance.