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. RUSSIA

INTRODUCTION
          Variola virus 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 preparations.
          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 [5]. 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
          Variola virus (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.
          Monolayers of 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.
          Chick embryos 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 H-600 microscope.

RESULTS AND DISCUSSIONS
          Variola virus 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.
          Developing envelopes 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.
          Comparison of 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].
          Reproduction 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.
          Reproduction 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.
          Variola virus 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.
          So, examination 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.
          Chick chorion-allantois 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 [3]. 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. (Fig. 3).
          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.
          However, the 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).
          Chorion-allantois 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 [13]. 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 [14]. 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.

SUMMARY
          Ultrastructural 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.

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KEY WORDS
Variola virus, reproduction, cell cultures, chick embryo chorion allantois membrane.

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 Viruses.

FIGURES

Fig.1a           Fig.1b

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 of nucleoid.

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 virus envelope.

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 cell.

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.