УДК 616.988-092.18-06:616-018.1

   Chronically infected cell cultures are a long-term associations between a host cells and viruses, characterized by long coexistence of a virus and cell. The preservation of the viability of infected cells during persistence is caused by the interaction of various viral and cellular factors. During chronic viral infection a virus or cell or both are changed. The evolution occurs through selection of more resistant population of cells or less cytopathogenic virus [1]. Poliovirus can persistently infect some human cell lines in vitro. Arising in vitro conditions of a chronic virus infection by picornaviruses, and in particular by the poliomyelitis virus, is caused by the appearance of resistant cell clones [2]. In such cells a virus is replicated in lower titers compared to control cell without causing destructive changes. The point of interest is the study of proliferative potential changes in cancer cell cultures and degree of cells differentiation under the influence of chronic virus infection.
   Such parameters as the ploidy, size and the quantity of nuclei and nucleoli are a valuable diagnostic attributes of the transformed cells proliferation speed. [3,4,5].
   Materials and methods. Cells. In work we used the transformed continuous cell culture of a human larynx cancer - НЕр-2. Cells were cultivated in Eagle medium with glutamine and 10% bovine serum. A monolayer of the intact cells was used in 48 hours after the passage. Cells were resowed in dose 1×105 cell/ml. The cell lines were received from laboratory U 322 INSERM "Retrovirus et maladies associeees" Marseilles. The choice of НЕр-2 as a model is motivated by the facts from publications on susceptibility of these cells to the Sabin poliovirus strains.
   Virus. In work was used the standard trivalent Oral Polio Vaccine (OPV) (Polio Sabin^TM [oral] Poliomyelitis vaccine, live attenuated SB BIOLOGICALS Rixensart - BELGIUM).
   Chronic virus infection was received by one time infection of 48-hour monolayers of culture НЕр-2 by OPV. The multiplicity of infection was 0,000001 TCD₅₀ per cell. Infected cells were incubated at 36,5-37⁰C. Persistent infection was repeated 3 times and summarized data were presented. Viral titer was calculated by the method of Karber. As a control the parallel conducted passages of noninfected НЕр-2 culture were used. Their summarized data are given in the table.
   Quantitation of nuclear DNA. For the analysis of the received data, the cells preparations of НЕр-2 culture were fixed in 96⁰ ethyl alcohol for 30 minutes and stained in fresh Shiffs reactive, by Feulgen. The content of DNA in a nucleus and nucleolus was defined by means of computer-equipped microscope-photometer SMP 05 (OPTON). The television method was used on 575 nm waves. In each case 50 - 100 cells were measured.
   The DNA content is expressed in a "c" scale in which 1c is half (haploid) the mean nuclear DNA content of cells from a normal (non-pathological) diploid population in G₀/G₁ cell cycle phase. The DNA image cytometric measurements identified cell nucleuses as aneuploid if they deviate more than 10% from the 2c, 4c, 8c, 16 c, i.e. if they are outside 2c±0,2, 4c±0,4, 8c±0,8, 16c±1,6. The number of all cells in euploid regions of the DNA histogram rescaled by the mean corrective factor of the tissue type under investigation (1.8c - 2.2c; 3.6c - 4.4c; 7.2c - 8.8c; 14.4-17.6) also was calculated [6].
   Statistical analysis. For comparison of two groups the non-parametric U test analysis according to Mann-Whitney was applied. Also statistical analyses were performed with Student's t test in the SPSS version 8.0-computer software package (SPSS, Inc., Chicago, IL).
   Results and discussion. After the infection, during 2-3 passages of OPV in cell culture, cytopathogenic action of a virus was found as the degradation of the monolayer. However, destruction of a monolayer was not complete, and after the 3rd passage the cytopathogenic action of the virus was reduced. Received chronically infected culture was characterized by insular and slower growth (approximately twice), lack of capacity to form monolayer and constant allocation of a virus. During 2nd 3rd passages, cytopathogenic action of a virus was found. After the 3rd passage the reduction of cytopathogenic action of a virus was observed. The virus was found from the 1, 2, 3, 5, 9 passages in cell-free medium in titers 1,5-0,5 lg/ml. The highest titer was observed only once at 3-th passage (1,75). During the further passages the virus was found out only in cells after their destruction by freezing in low titres (0,25-0,75). A virus accumulated on sensitive intact cell culture НЕр-2. So, low viral titres may be explained as a result of decreased quantity of the sensitive cells in infected culture, and in first passages as results of low multiplicity of infection. Quantity of the cells in all passages after 2 was significantly less in comparison with control.
   So in the 1-st passage, we can observe the significant growth in the number of cells with 4 nucleoli, as well as the tendency to increase in the percentage of cells with 5 nucleoli (0,72±0,08). This happened first of all due to significant decrease in the number of cells with 1 nucleolus. From the 2-nd passage the number of 4 nucleolar cells decreases and at the same time the quantity of cells with 1 nucleolus increases. These processes continue and by the 5-th passage the number of cells with 4 nucleoli sharply decreases, cells with 5 nucleoli almost disappear (in following passages they percent vary from 0,07±0,01 up to 0,16±0,03). In comparison with the control and the 1-st passage the quantity of cells with 1 nucleoli significantly raised. From 6-th passage the quantity of nonnucleolar cells significantly increases, at practically the same parameters of cells of other types. By the 12-th passage all these parameters almost do not changes. Only the number of nonnucleolar cells in relation to the 6-th passage increases. In this passage in relation to the control a significant decrease in the number of 4 nucleolar cells and increase in the quantity of 1 nucleolar cells is observed. In all the passages significantly increased the number of dead cells, and by 12-th passage the quantity of mitoses significantly decreased. From the 6-th passage appeared significant number in comparison with the control of nonnucleolar cells, the number of which continued to increase, and by the 12-th passage it was significant not only in relation to the control, but also to the 6-th passage. At the same time the number of dead cells significantly increased and the quantity of mitoses decreases (except the 5-th passage). Significant changes in the quantity 2- and 3- nucleolar cells were not fixed in any passage in comparison with the control.