7.3. Studies using L41, Vero, and HLE cells

The intranuclear cycle of virus reproduction is completed with its "maturation". Morphologically, it is expressed by covering the capsid with an envelope that further probably has a

Mass release of virus particles from the cell occurs from 15 to 18 h and is accompanied by the formation of numerous structures of a platy type. At late stages of the disease, various types of viral particles at different stages of formation get beyond the cell limits during its destruction.

The experiments were conducted in a liquid thermostat of ЗЦ-1125М type with the optical device shown in Figure 5 inside. Monolayers of cultured virus-free and herpes simplex virusinfected cells were the research targets. Two identical optical cuvettes were prepared for the study. There were two identical substrates in each cuvette, one with the cells and the other cellfree with nutrient solution. The experiments used HSV-1 infectious titre 4.5 lg TCD 50/ml (tissue cytopathic doses) in a dilution of 10-3. Speckle dynamics films of frequency 25 Hz lasting 20–40 s were recorded in the first experiments for 18–20 h at half-hour intervals. Cells of L41 line were the research target. Typical joint dependences ηðtÞ for the nutrient solution

Analysis of dependences of ηðtÞ as well as σuðtÞ for virus-free and virus-infected cells shows that they have features agreeing with some phases of virus development in cultured cells, but they are reproduced in about 50% of the cases. The result obtained was probably related to two considerations. First, while dependences ηðtÞ were being recorded, the initial frame, starting from the second film, did not correspond to the experiment start. Second, the optical wave path variation could have been caused by several factors with relaxation times of the same

Figure 20. Typical joint dependences ηðtÞ for the nutrient solution (1), virus-free cells (2), and virus-infected cells (3).

protective function.

134 Optical Interferometry

7.2. Speckle control procedures

and virus-free and virus-infected cells are shown in Figure 20.

The studies using the upgraded technique were conducted using three cell types: L41, Vero, and HLE-3. Dependences <sup>~</sup>Iðt<sup>Þ</sup> and <sup>η</sup>ðt<sup>Þ</sup> of the nutrient solution, virus-free and virus-infected cells were considerably different for all the cell types and were well reproduced in a qualitative sense.

Figure 21 shows typical dependences <sup>~</sup>Iðt<sup>Þ</sup> for the monolayer of HLE-3 cells.

Figure 21. A typical dependence of <sup>~</sup>Iðt<sup>Þ</sup> of the HLE-3 line: (1) for the nutrient solution, (2) cells with virus, (3) for the cells without virus.

It is seen from the dependence for virus-infected cells that in the first 3 h, value ~I diminishes considerably to a certain level. This time coincides with the time necessary for penetration of the viral material into the cell and then into its nucleus. Then in the next 5 h, relatively weak fluctuations of value ~I compared with the variations of ~I occurring in the virus-free cell take place. This time interval coincides with the time interval during which the cell produces proteins necessary for appearance of new viruses. Then relatively strong quasiperiodic variations of value ~I reappear. It is known that in this time interval, a capsule with new virions grows.

In Figures 22–24 typical dependences ηðtÞ for L41, Vero, and HLE-3 cells are shown.

Figure 22. Typical dependences ηðtÞ for L41 line: (1) nutrient solution, (2) cells without virus, (3) cells with virus.

Figure 23. Typical dependences ηðtÞ for Vero line: (1) nutrient solution, (2) cells without virus, (3) cells with virus.

Figure 24. Typical dependences ηðtÞ for HLE-3 sells: (1) nutrient solution, (2) cells without virus, (3) cells with virus.

Every picture presents three graphs corresponding to the nutrient solution, virus-free and virus-infected cells. It is seen from the pictures that dependences (1), (2), and (3) differ considerably in the numerical sense. Dependences (2) and (3) are nonstationary processes, but their forms are similar: first value η decreases rapidly, then its decrease slows down. Respectively, σ<sup>u</sup> found by Eq. (30) supposing that 〈x1〉 ¼ 〈x2〉 first rapidly grows, and then its growth slows down. We evaluated the multiple correlation coefficient of three masses ηðtÞ corresponding to one cell type, and also to different virus-free and virus-infected cell types. For the three masses, the coefficient was in the range from 0.82 to 0.96. This character of curves ηðtÞ was probably related with the fact that in the solution, the amount of nutrients gradually decreases and the concentration of harmful cell activity products increases.

Analysis of dependences ηðtÞ enabled us to conclude that the presence of the virus can be reliably detected by the curve difference for virus-free cells η1ðtÞ and for virus-infected cells η2ðtÞ 10 min from the experiment start. Twofold excess of the noise amplitude by difference η1−η<sup>2</sup> at fixed t was considered the reliability criterion.

### 7.4. Conclusions

In Figures 22–24 typical dependences ηðtÞ for L41, Vero, and HLE-3 cells are shown.

136 Optical Interferometry

Figure 22. Typical dependences ηðtÞ for L41 line: (1) nutrient solution, (2) cells without virus, (3) cells with virus.

Figure 23. Typical dependences ηðtÞ for Vero line: (1) nutrient solution, (2) cells without virus, (3) cells with virus.

The conducted experiments showed that recording of dependences <sup>~</sup>Iðt<sup>Þ</sup> and <sup>η</sup>ðt<sup>Þ</sup> in the image plane of a cell monolayer on a transparent substrate permits reliable recording of difference in the virus-free and virus-infected cell activity. The necessary conditions are the following:

