Rabies is an acute disease of warm-blooded animals that affects the central nervous system. All domestic and wild animal species, as well as human beings are susceptible to the disease [1-5].

The virus reproduction cycle includes its adsorption onto the cell surface, penetration into the cell, uncoating, transcription, translation, replication, assembly and budding.

Primary transcription does not require structural protein synthesis. There are 2 types of rabies virus transcription: 1) at first, leader RNA is transcribed with genomic RNA, then 5 monocistronic mRNAs are sequentially transcribed, from which N, P, M, L proteins are translated in the cytoplasm, and G gene mRNA is translated in polysomes associated with the Golgi apparatus membrane; 2) transcription of antigenomic (positive-sense) RNAs, which serve as templates for the synthesis of new generations of negative-sense genomic RNAs, with genomic RNA. Unlike primary transcription, genomic RNA replication by the described mechanism can occur only simultaneously with translation and synthesis of structural proteins, especially N and P ones, necessary for RNA encapsidation by nucleocapsid structures. L, N, and P proteins mainly control RNA transcription and replication, whereas M protein occupies an intermediate position between the nucleocapsid and the virion shell and participates in RNA condensation. Besides, M protein plays a key role in RNA synthesis regulation and budding of the virus [6, 7]. At the final stage of assembly and budding, nucleocapsids “put on” the viral envelope represented by G protein integrated into the cell membrane. Glycoprotein determines the neurovirulence and neuroinvasiveness of rabies virus.

The place of viral particle formation depends on the virus and host cells. For representatives of the genus Lyssavirus of the family Rhabdoviridae, to which rabies virus belongs, the synthesis and assembly of nucleocapsids occur in the cytoplasm, and budding occurs through the plasma membrane of the cell, from which the virion acquires an additional lipid layer.

Rabies virus does not cause characteristic cytopathic changes in cells. All transcription and replication events occur in the cytoplasm inside Negri bodies. These structures are typical for rabies and, thus, can be used as a pathognomonic symptom of the infection [8].

Flow cytometry is a modern technology that ensures rapid, high-quality and multiparametric analysis of cells. It is widely used in such fields of medicine as immunology, pharmacology, cytology, oncology, hematology, genetics, infectology [9-15].

Flow cytometry makes it possible to collect a variety of data: to determine DNA and RNA content in a cell, the total number of proteins and the number of specific proteins recognized by monoclonal antibodies, to investigate cell metabolism, to study the transport of calcium ions and the kinetics of enzymatic reactions [15-20].

Each cell has its life cycle from the moment of its formation by mother cell division to mitosis or death. It is called a cell cycle. The cell cycle consists of two periods: 1) cell growth period (interphase); 2) cell division period called M phase (from the Greek word “mitos”, meaning “thread”). In its turn, each of the said periods has several phases. Usually, the interphase takes at least 90% of the time of the entire cell cycle. Most of cell components are synthesized throughout the interphase, and this makes it difficult to identify individual stages within it.

The interphase is divided into G1, S and G2 subphases. A period within the interphase, during which cell nucleus DNA is replicated, is called “S phase” (from the word “synthesis”). It should be noted that not only DNA replication, but also the basic biosynthesis of structural and functional proteins of the cell occurs during the interphase (mainly in S phase). A period between M phase and the beginning of S phase is called G1 phase (from the word “gap”), and a period between the end of S phase and subsequent M phase is called G2 phase [21].

The use of flow cytometers in the studies made it possible to identify important data on the reproduction cycle phases of cells, as well as to obtain the results that allowed for the estimation of duration and predominance of the corresponding cell cycle G1, S, G2 + M phases at different stages of cultivation [18][19][20][22][23]. In the light of the above, it can be concluded that the nature of changes in the cell cycle during virus replication in a cell is important for understanding the process of virus reproduction, but is understudied.

The aim of the study was to examine the dynamics of DNA transformation in BHK-21/SUSP/ARRIAH cell culture during rabies virus reproduction using flow cytometry.

Cell line. Suspension continuous culture of newborn Syrian hamster kidney cells BHK-21/SUSP/ARRIAH was used in the work [24]. The cells were grown in metal bioreactors with a working capacity of up to 1,800 dm3 in accordance with the “Master formula record for production of vaccine against foot-and-mouth disease of different types” approved by the Director of the Federal Centre for Animal Health.

Rabies virus. Production “ARRIAH” strain of rabies virus was used to infect the cells.

Nutrient medium used to grow cells. Eagle’s medium supplemented with 5% of fetal bovine serum (Serana, Germany) and 0.25% blood protein hydrolysate (Russia) was used.

Rabies virus cultivation. Rabies virus reproduction was carried out during 48 hours in 0.5 dm3 flasks using Minisart® RC25 Syringe Filters 17764-ACK Ø 25 mm (Sartorius, Germany).

Growth rate was calculated as the ratio of the final (after 48 hours) and initial cell concentrations within one passage.

Cell infection with rabies virus. Suspension BHK-21/SUSP/ARRIAH cell culture was inoculated with culture rabies virus at a dose of 0.1 CCID50/cell. Samples containing infected cells were designated as test samples; cells not inoculated with the virus served as control and were cultivated under similar conditions.

The identification of cell cycle stages was carried out using an AccuriTM C6 flow cytometer and a BD CycletestTM kit for working with a cytometer, as well as a reagent kit for quantitative DNA content analysis in cells C6 Flow Cytometer Fluid Kit (Becton Dickinson and Company, USA).

The determination of rabies virus infectivity titre was carried out in accordance with “Methodical guidelines for indirect determination of infectivity titre of culture rabies virus “ARRIAH” strain in vaccine production seed with real-time reverse transcription polymerase chain reaction (real-time RT-PCR)”1.

Samples were collected every 3 hours throughout the entire time of rabies virus reproduction. The concentration of BHK-21/SUSP/ARRIAH cells in the suspension was determined using a Goryaev chamber for counting blood cells, dA0.000.851, compliant with TU 64-1-816-84. The cell suspension in a volume of 1 cm3 was supplemented with an equal volume of 0.2% trypan blue solution, thoroughly mixed and used to fill the chamber. The number of cells in 1 cm3 of the suspension was calculated using the formula [21]:

where X is the number of cells in 1 cm3; A is the total number of cells in the chamber; B is the suspension dilution.

The counting was performed using a microscope at 10× magnification.

Statistical data processing. Numerical data were statistically processed with generally accepted methods of variation statistics, using a personal computer and Microsoft Excel software.

The virus reproduction dynamics was assessed based on the following indicators:

– dependence of the virus infectivity titre on reproduction time;

– changes in apoptosis and debris;

– changes in cell cycle stages (G1, S, G2 + M).

During cultivation, the hydrogen ion concentration (pH) of the suspension was checked every 3 hours.

Figure 1 shows the dynamics of rabies virus infectivity during its reproduction in BHK-21/SUSP/ARRIAH cell culture.

It was found that the virus infectivity titre increased from (3.2 ± 0.2) lg CCID50/cm3 at the time of inoculation to (7.63 ± 0.3) lg CCID50/cm3 after 48 hours of reproduction, with the most intensive increase having been observed within the first 24 hours (up to 6.76 CCID50/cm3).

Within 48 hours, the cell concentration changed from 0.5 to 1.9 million/cm3, i.e. increased by a factor of 3.8 (Fig. 2).

At the next stage, experiments were conducted to study changes in rabies virus infectivity titre in BHK-21/SUSP/ARRIAH cells with different initial concentrations: (0.77 ± 0.1); (3.0 ± 0.2) and (1.1 ± 0.1) million/cm3. It was found that the accumulation of complete viral particles in the infected cells with different concentrations occurred within the first 12 hours of reproduction (Fig. 3). Subsequently, the virus accumulated 1.1–1.2 times more intensively in the culture with initial cell concentration of (0.77 ± 0.10) million/cm3. This is probably due to the limited amount of nutrients for the reproduction and growth of cells with a higher concentration.

Tests were carried out to study changes in cell cycle phases during rabies virus cultivation in suspension BHK-21/SUSP/ARRIAH cells. Under standard optimal conditions, intact populations are distributed according to cell cycle phases as follows: G1 – 30–75%, G2 + M – 2–18%, S – 2–33%, polyploids – up to 4% and debris – 1–20% [23].

It was found that the dynamics of changes in the apoptotic cell population in the control and test samples was similar within 36 hours of cultivation (a small time shift of 3 hours between the test and control samples was observed after 9 and 12 hours of reproduction). The proportion of apoptotic infected cells was 28–42% higher than that of apoptotic control cells 36 hours after the start of cultivation (Fig. 4).

After 9 hours, the proportion of cells undergoing G1 phase increased by 11.7% in the test samples, whereas it decreased by 16.6% in the control samples. Subsequently, the number of G1 phase cells in the control and test samples changed in the same way: a 40% decrease was observed after 15–18 hours, it was followed by a 45–46% growth jump, then again a 39–40% decrease and an increase were observed. After 33 hours of reproduction and till the end of cultivation, the proportion of infected cells undergoing G1 phase was significantly higher (by 12–21%) as compared with the control cells.

The comparison of the number of cells undergoing apoptosis + debris and G1 phase revealed a certain synchronicity between these phases: after 9 hours of the virus cultivation, G1 phase increased by 10–12%, at the same time apoptosis decreased by 10–11%, then a decrease in the percentage of G1 phase cells and an increase in the proportion of cells in apoptosis and debris were observed. A more pronounced “mirroring” of these cell cycle phases was observed after 24 hours of cultivation. A similar pattern was seen in the study of the cell cycle during foot-and-mouth disease virus reproduction [22].

The diploid phase of the cell cycle (G1), during which the synthesis of mRNA, structural proteins and other cell components began, prevailed in the cell cycle of intact BHK-21/SUSP/ARRIAH population. This phase accounted for 30 to 75% of cells, depending on the cultivation conditions, which is reflected in earlier studies [23].

It is known that during G1 phase, cells start to grow in size, mRNA and enzymes required for subsequent DNA replication are activated [21].

G1 phase сells in the suspension culture were maintained at 37–70% throughout the entire virus reproduction time, i.e. cells infected with the virus served as a source of energy and material for the synthesis of viral components, as well as for the assembly of complete virions.

The transition from diploid (G1) phase to synthetic (S) phase is one of the check points of the cell cycle. Depending on the amount of nutrients and energy, as well as on external factors of cultivation, the cell “decides” whether to enter the cell cycle or to go into a non-dividing quiescent state known as G0 phase that leads to apoptosis. The main event of S phase is DNA replication, which has its specific features [21].

In our tests, the percentage of S phase cells in the test and control samples was the same during the first day of the virus reproduction, with sharp jump-like 3.4- and 2.4-fold increases having been observed after 15 and 24 hours, respectively (Fig. 5). After 24 hours, the infected and control cells began to demonstrate differences, which gradually increased from 8 to 137% by the end of reproduction. It can be assumed that the cells undergoing the said phase also participated in the virus reproduction.

In G2 phase, the last of three successive phases of the cell cycle interphase, and M phase, a tetraploid population was formed. In the intact and rabies virus infected cells of BHK-21/SUSP/ARRIAH line, the phase of preparation for mitosis and mitosis itself accounted for 2 to 20% of the entire population. After 30 hours of reproduction, the proportion of test sample cells undergoing these phases began to decrease by 17–28% as compared with the control cells (Fig. 6). This was probably due to the fact that the overall biosynthesis and functions of the host cell were inhibited as a result of the biosynthesis of rabies virus components; therefore, the percentage of G2 + M phase cells decreased.

The number of polyploid cells in the test samples was approximately equal during 45 hours, with a sharp 2.0–2.5-fold increase having been observed only within the last hours of the virus reproduction. In the control cells, the population ploidy was abrupt: 2.06-, 2.7-, 1.56- and 3.18-fold increases were observed after 9, 21, 42 and 48 hours, respectively. It can be assumed that polyploid cells were partially resistant to the virus. This fact has already been noted earlier in the study of foot-and-mouth disease virus reproduction in BHK-21 cells [22].

The DNA histograms of BHK-21/SUSP/ARRIAH cells during rabies virus reproduction 24 hours after the start of infection and after 42 hours of cultivation (Fig. 7) summarize the graphs shown in Figures 4–6.

The phases of the cell cycle during rabies virus reproduction in the host cell were examined, the results obtained allowed for the estimation of duration and predominance of the corresponding cell cycle G1, S, G2 + M phases at different stages of cultivation.

When studying the dynamics of changes in the DNA of BHK-21/SUSP/ARRIAH cells infected with rabies virus, it was found that after 24 hours of the virus cultivation, the proportion of cells in G1 phase increased as compared with the control cells (by 12–21%). Since mRNA, structural proteins, other cell components that are also necessary for rabies virus virion assembly are synthesized during G1 phase, the growth of cells in this phase of the cycle can be considered expectable.

It was found that since the main event of S phase is DNA replication, a linear decrease of S phase during rabies virus cultivation after 24 hours of reproduction may also be indicative of the cell switch-over from cell DNA replication to viral RNA synthesis.

It was revealed that G2 phase and M phase (postsynthetic, or premitotic phase, and mitosis itself) of the test sample cell life cycle were relatively decreased (by 17–28%) as compared with the control cells, i.e. the infected cells spent less time in the division stage than the uninfected ones. This was probably due to the fact that the overall biosynthesis and functions of the host cell were inhibited as a result of the biosynthesis of rabies virus components.

The polyploid cells formed during the cell and virus cultivation were found to be partially resistant to rabies virus and probably had the least sensitivity.

It was established that BHK-21/SUSP/ARRIAH cell switch-over to the synthesis of complete rabies virus particles occurred after 24 hours of reproduction, as indicated by changes in the host cell cycle phases, as well as by the slowing down of the cell population growth.

1 Doronin M. I., Mikhalishin D. V., Borisov A. V., Balashov A. N., Mudrak N. S., Zakharov V. M. Methodical guidelines for indirect determination of infectivity titre of culture rabies virus “ARRIAH” strain in vaccine production seed with real-time reverse transcription polymerase chain reaction (real-time RT-PCR): approved by FGBI “ARRIAH” 23.12.2021 No. 66–21. Vladimir; 2021. 59 p.