Visualization of viral DNA assembly using nonfluorescent staining.

We present an easy test for rapid visualization of viral DNA assemblies in infected cell cytoplasm. We selected the best stains for nuclear staining: Nile blue A, Bismarck brown, gallocyanin chrome alum, methyl green pyronin and azure II. None of the staining techniques is fluorescent, which facilitates their use in everyday experiments. Methyl green is most promising for routine detection of viral DNA assemblies in the cytoplasm; the procedure enables ready detection of viral DNA accumulation in the cytoplasm.

Third wave of African swine fever infection in Armenia: Virus demonstrates the reduction of pathogenicity.

AIM: First cases of clinically uncommon African swine fever (ASF), caused by virus genotype II are described in this article. These cases occurred in Armenia, Tavush region, Dilijan municipality in 2011. The aim of this study was to identify and describe the new pathogenic forms of ASF in Armenia. MATERIALS AND METHODS: The isolation and identification of ASF virus (ASFV) were carried out using conventional techniques. Clinical signs of infection were recorded daily. Gross anatomical pathology characteristics were observed during routine postmortem examinations. Blood and serum were obtained by puncture of the jugular vein using a vacutainer system. RESULTS: The presence of ASFV DNA in the spleens was confirmed by polymerase chain reaction. Sequenced sections of p72 showed phylogenetic identity to genotype 2. The pathology exhibits unusual manifestations of the main disease. The unusual form of ASF demonstrates characteristics of a subacute form of the disease, with the possibility of conversion to a chronic form. Decreased lethality, low level of hemorrhages, and absence of severe pancytopenia in smears from spleen, lymph nodes, and blood are common features of the new form of ASF. Unlike severe thrombocytopenia in the typical ASF, the unusual form exhibited moderate or minor decrease of this feature. Despite a moderate decrease in hemadsorption titers, the unusual pattern of the disease was characterized by viremia and the presence of the virus in the visceral organs, including the brain. CONCLUSION: Our data allow assuming that new nosological form of ASF (genotype II) may present as a transitional form of the disease with the possibility of chronization.

Hemophagocytic lymphohistiocytosis in acute African swine fever clinic.

INTRODUCTION: Hemophagocytic lymphohistiocytosis (HLH) usually has been defined as the combination of a proliferation of cytologically benign, actively phagocytic macrophages in bone marrow, spleen, lymph nodes, etc. in association with fever, cytopenia, splenomegaly, and hypertriglyceridemia. HLH is often triggered by viral infection. The aim of this study was to ascertain the features of HLH involvement in African swine fever virus (ASFV) (genotype II) pathogenesis. METHODS: The serum levels of macrophage colony-stimulating factor (MCSF) and granulocyte-macrophage colony-stimulating factor (GMCSF), as well as the histological constitution (for hemophagocytic macrophages detection) of various organs of pigs infected with ASFV genotype II were investigated. The diagnosis of HLH was made according to universally accepted human criteria. RESULTS: The association of fever, cytopenias, splenomegaly, and hemophagocytosis was present in 87.5% of the infected pigs (absence of hyperthermia in one of eight pigs). Marked hypertriglyceridemia was observed at 3-4days post infection. Previously it was shown that ASFV induced a significant decrease in the level of fibrinogen from day 5 till the end of experiment. Progression of the HLH coincided with a temporary increase in the serum levels of MCSF levels (early stage of disease) and GMCSF levels (2-3 pays post infection). CONCLUSIONS: Hemophagocytic syndrome should be suspected in ASFV (genotypeII) infected pigs.

Characterization of the atypical lymphocytes in African swine fever.

AIM: Atypical lymphocytes usually described as lymphocytes with altered shape, increased DNA amount, and larger size. For analysis of cause of genesis and source of atypical lymphocytes during African swine fever virus (ASFV) infection, bone marrow, peripheral blood, and in vitro model were investigated. MATERIALS AND METHODS: Atypical lymphocytes under the influence of ASFV were studied for morphologic, cytophotometric, and membrane surface marker characteristics and were used in vivo and in vitro models. RESULTS: This study indicated the increased size, high metabolic activity, and the presence of additional DNA amount in atypical lymphocytes caused by ASFV infection. Furthermore, in atypical lymphocytes, nuclear-cytoplasmic ratio usually decreased, compared to normal lymphocytes. In morphology, they looking like lymphocytes transformed into blasts by exposure to mitogens or antigens in vitro. They vary in morphologic detail, but most of them are CD2 positive. CONCLUSIONS: Our data suggest that atypical lymphocytes may represent an unusual and specific cellular response to ASFV infection.

[Influence of ionizing radiation on enzymatic activity and state of nucleus-nucleolar apparatus in rat hepatocytes].

The effects of a single exposure of rats to the whole-body roentgen irradiation at the doses of 3.5 Gy and 4.5 Gy on the activity of creatine kinase, purine nucleoside phosphorylase, alanine aminotransferase, aspartate aminotransferase, as well as on the state of the nuclear-nucleolar apparatus in rat hepatocytes on the 6th and 13th days after radiation exposure have been studied. Irradiation at the above doses induced changes in the levels of enzymatic activity of different values and different directions within the same time periods, as well as oscillating changes in this type of enzymatic activity over time. This demonstrates various radiosensitivity and adaptation abilities of these enzymatic activities. The changes in the enzymatic activity significantly correspond to the changes in the morphometric indices of nuclear-nucleolar apparatus of hepatocytes, as well as the distribution of hepatocytes within the ploidy classes: in particular, stabilization of the enzymatic activity on the 13th day after irradiation correlates with the increased transcriptional activity, which is detectable through the increased number of nucleoli per nucleus and the expanded space of a hepatocyte nucleus. The compensation mechanisms are likely to be targeted at the changes in the functional activity of surviving hepatocytes, rather than at the replacement of the damaged cells by the new ones.

[Dynamics of changes in the composition of leukocyte population of peripheral blood during the African swine fever].

The comparison of the composition of leukocytes of peripheral blood of healthy and Infected swine revealed the quantitative alterations, as well as the appearance of new cells in leukocyte population during African swine fever. It was determined that African swine fever virus induced mass-scale mortality of cells of peripheral blood, especially lymphocytes and neutrophiles. The number of the dead cells reaches 60% of the initial number of all cells at the end of infection. It was also revealed that the appearance of atypical lymphocytes and lymphoblasts was observed during viral Infections. Most of these cells are characterized by the presence of additional nucleus.

Interaction of Paramecium caudatum and Picornaviruses.

In our paper we have researched the relationship between picornaviruses (poliovirus, foot-and-mouth disease virus and encephalomyocarditis virus) and Ciliata (Paramecium caudatum). We show that the number of Paramecium in medium sharply increased during coincubation with picornaviruses within 2-5 days. This cannot be explained only by the fact that viruses were nutrient source for Paramecium because in case of inactivated viruses the number of infusorians in medium increased a little. At the same time the titer of viruses harshly decreased whereas in the control group, which is free of Paramecium, the fall of titer was little. Picornaviruses were eliminated from medium if only living Parameciums were present in medium. After 7-9 days of coincubation only a few number of viruses were liberated from destroyed Parameciums. These results will be especially useful for management of reservoirs of picornaviruses in water and prevention of diseases.

[The reaction of the neuroblastoma cells in the culture on the influence of tretionine and neurotoxine].

Effect of the tretionine (retinoid) and aluminum chloride (neurotoxin) on the growth and differentiation of neuroblastoma cells in culture after their introduction into the medium separately and in combination was studied. The introduction of these substances creates a new information field in the medium, which becomes apparent by the reactions of neuroblastoma found on the populational and cellular levels of its organization. The presence of tretionine stimulates proliferation and induces differentiation of the cells into astrocytes. Aluminum chloride inhibits cell proliferation and enhances the process of their destruction in the monolayer. The variety of the reactions of neuroblastoma cells to the presence of these substances in the medium indicates the existence and functioning of a mechanism that selects from the information introduced only the portion which may contribute to adaptation of neuroblastoma cells to the changed culture conditions.

[Pathology of lymphoid tissue cells infected by African swine fever virus in vitro].

The authors studied the pathology of bone marrow (BM) lymphoid cell from pigs infected by African swine fever virus (ASFV) in vitro. Monocytes were shown to be primarily afflicted in unstimulated BM culture. These cells disappeared completely 72 hours after infection. Just 24 hours following ASFV infection, there were atypical lymphocytes amounting to 12% of the general lymphoid population at hour 72 after inoculation.The area and perimeter of minor, middle, and large lymphocytes tended to reduce during both BM cell cultivation and inoculation. Lymphoblasts and monocytes were generally triploid in both the control and test groups, but among them there were diploid, triploid, and tetraploid cells. Cytophotometric assay revealed that the amount of nuclear DNA significantly increased in BM lymphoblasts and monocytes in the early stages of ASFV infection (within 24 hours). This effect was also rather pronounced in the lymphoblasts in the later stages (at hour 72).

Properties of cells with increased resistance to some picornaviruses.

The resistance to picornaviral infection cells of susceptible lines has similar changes in the phenotype. They have decreased number of nucleoli and increased percentage of euploidy. Also the percentage of euploid cells those were resistant to the picornaviral infection increased in all highly transformed cultures. In resistant cells of all cultures has been found reduction of DNA. RNA amount also decreased both in nucleus and in cytoplasm. All these data correlated with the increased euploidy of the resistant population. The resistant cells had a less transformed phenotype, and decreased proliferative activity. Decreased nucleolar status became apparent by reduction of absolute and relative nucleolar indices. Consequently the reduction of viral titer (viral titters reduction) in resistant cells could be the direct result of diminished activity of the RNA synthesis machinery. It is important to note that the cells lose resistance while another type of virus, even from the same family, infects the culture once.

Phenotypic and cytologic studies of lymphoid cells and monocytes in primary culture of porcine bone marrow during infection of African swine fever virus.

We have modeled in vitro infection of African swine fever virus (ASFV) in primary unstimulated cells of the porcine bone marrow and have studied the phenotypical changes in the population of porcine lymphoid cells by cytophotometry. Monocytes and large-sized lymphocytes completely vanished in 72 h of infection which is result of high sensitivity of those cells to ASFV. We describe DNA synthesis in monocytes at 24 h post infection. Cytophotometry of the uninfected cells revealed the few number of atypical lymphocytes and lymphoblasts after 72 h of cultivation; whereas in viral infected cultures, atypical cells appeared in large quantity (about 14%) with 24 h. Most of atypical lymphocytes and lymphoblasts had altered nucleus, and only a small number of atypical cells had additional nucleus. The cytophotometry of main and additional nuclei showed that DNA content didn't exceed diploid standard which indicates that the additional nuclei were consequence of fragmentation of nuclei in lymphocytes.

[The influence of solcoseryl on population and cellular parameters of HeLa and RD cultures].

Changes of population and cellular parameters of HeLa and RD cultures after introducing of solcoseryl in culture medium were studied by methods of scanning cytophotometry and cytomorphometry. Monolayer density, proliferation activity, the number of dead cells in a monolayer, the number of nucleoli in nuclei and distribution of cells in the populations by this parameter, RNA and DNA masses in nuclei and nucleoli, total volumes and surface areas of the nuclei and nucleoli were determined. It has been shown that solcoseryl differently affects the cultures both on population and on cellular levels of their organization. The results of multi-parametric analysis of the influence of solseryl on the cultures allow considering it as a biologically active compound with the features typical for cell and cell population growth regulating factors.

[Multiparametric study and cluster-analysis of the transformed cultures of human cells].

The cells of nepatocarcinoma (HEp-G2), adenocarcinoma of large intestine (Caco-2), embryonal kidney (HEK-293), neuroblastoma (SH-SY5Y), rabdomyosarcoma (RD), and larynx cancer (Hep-2) were studied by the methods of scanning cytophotometry, cytochemistry and cytomorphometry during 96 h of cultivation. The density of monolayers, proliferation activity, the number of dead cells, DNA content in the nuclei and distribution of the cells in the population by this parameter, total DNA content in the nucleoli (circumnucleolar chromatin), the number of nucleoli in the nuclei, distribution of cells by their number, the volume and area of the nucleus surface, total volume and area of the nucleoli surface were determined. The data obtained were used in the treelike cluster analysis of the cultures by Pierson correlation. As a result, the SH-SY5Y culture was put in a separate cluster, while Caco-2, HEp-G2, HEK-293, Hep-2 and RD cultures were placed in the tree of another cluster. The least transformed culture of neuroblastoma (SH-SY5Y) had no relationship with other cultures, which showed various rate of similarity. The cultured HEK-293, Hep-2 and RD appeared to be close to each other by all parameters. The parameters studied are of different significance for the formation of general pattern of the cell cultures. The greatest "weight" is carried by the parameters, which characterize the population as whole: the density of the monolayer, mitotic coefficient and the number of dead cells. They are followed by the content of DNA in the nuclei, the total area of the nucleoli surface, and ratios of DNA content in the nucleoli to DNA content in the nucleus and of total surface of the nucleoli to the surface of the nuclei. Other parameters are not so significant.

Comparative multiparametric analysis of HeLa and RD cell culture reactions to solcoseryl.

Reactions of continuous HeLa and RD cell cultures and their nuclear and nucleolar apparatus to addition of solcoseryl into the medium were studied. The monolayer density, proliferation activity, percentage of dead cells, RNA and DNA content in the nuclei and nucleoli, number of nucleoli in the nuclei, cell distribution in the population by the number of nucleoli in the nuclei, volume and complete surface area of the nuclei and nucleoli, and the nucleolar/nuclear ratio were evaluated. The cultures differently reacted to solcoseryl in the medium at the population and cellular levels of their organization. By the results of multiparametric analysis of the reactions of cells and their nuclear and nucleolar apparatus, solcoseryl can be referred to bioactive substances with characteristics of a factor regulating cell population growth.

Behavior of lymphoid cell population, cell nuclei and nucleoli in periodic disease and leukemia.

Similar behavior of lymphoid cells, their nuclei and nucleoli in periodic disease and leukemia attest to nonspecific reaction of the immune system to these diseases, but the intensity of this reaction and mechanisms of the population recovery are different. DNA hyperreplication plays an important role in this process: in periodic disease it is realized via gene amplification, which manifests by the formation of H2c nuclei and increase in the number of nucleoli, while in leukemia bone marrow lymphoblasts double the DNA content during S phase, maturate during G2 phase, and then divide. We called this mechanism "reserve lymphopoiesis" by analogy with reserve erythropoiesis discovered previously by us.

[Human large intestine adenocarcinoma cells (CaCo-2) in the process of cultivation].

Using cytomorphometry and cytophotometry cells of human large intestine adenocarcinoma (CaCo-2) were studied under condition of a 10 day cultivation. A reverse dependence was established between proliferative activity and monolayer density. The increase of the latter inhibits proliferation and promotes the formation of islets of polymorph cells. 2c-cells could be seen only at the beginning of culture growth; a larger part of cells polyploidized by cell blocking in G2-phase. These cells do not divide, which is testified by the absence of 2c-cells, but some part of 4c-cells start the next cycle, accumulates 8c-DNA and then divides, replenishing the 4c-cells population. In the process of cultivation, we observed an increase in the number and total volume of nucleoli in the nuclei, and a rise in DNA amount in the peri-nucleolar chromatin. The formation of numerous 4c-cells with multi-nucleolar nuclei may define an increase of functional activity of CaCo-2 culture as the whole, whereas the formation of separated groups of such cells in the monolayer may denote a possible initiation of their differentiation.

Comparison of impact of EMCV replication on the nuclear apparatus of NIH 3T3 and HEp-2 cells.

We have investigated differences between the actions of encephalomyocarditis virus (EMCV) on cytometric indices in cultured NIH 3T3 and HEp-2 cells, which are characterized by different levels of transformation. HEp-2 cells surviving 48 h after EMCV infection showed lower nuclear ploidy, reduced nuclear area, fewer nucleoli and a higher percentage of euploid cells. There was a significant increase of nucleolar/nuclear DNA 6-24 h after EMCV infection. However, EMCV had markedly different effects on NIH 3T3 cells: there was a consistent increase in population ploidy, but the average number of nucleoli and the number of euploid cells in the population remained constant. The nucleolar/nuclear DNA ratio was almost unchanged. These different viral effects might be explained by the contrasting levels of differentiation of the cultured cell lines. The number of nucleoli does not depend on the amount of nuclear DNA in either viral-infected or intact cells but on the euploidy-to-aneuploidy ratio. The ratio of the sums of the nucleolar perimeters to the nuclear perimeter increases linearly with the number of nucleoli per nucleus in both intact and virus-infected cells. In both cell lines, the amount of DNA per nucleolus decreases as the number of nucleoli increases.

Comparative analysis of effects of various types of double-stranded RNA on cultured human laryngeal cancer cells.

We compared the effects of Na+ and Ca2+ double-stranded RNA on cultured human laryngeal cancer cells by cytomorphometry and cytophotometry. Both agents inhibited proliferation and other cell functions, but to a different extent: Ca2+ double-stranded RNA was more active than Na+ double-stranded RNA.

[Nuclei and nucleolar organizing regions in chromosomes of lymphocytes on different stages of periodic disease]. / Povedenie iader i iadryshkoobrazuiushchikh raionov khromosom limfotsitov na raznykh stadiiakh razvitiia periodicheskoi bolezni.

By scanning cytomorphometry a cytological study was first performed on the behavior of nuclei and nucleolar organizing regions (NOR) in chromosomes of peripheral blood lymphocytes of healthy men and of patients with periodic disease (familial Mediterranean fever, FMF) on different stages of development, including its complication with amyloidosis. The volume and total surface of nuclei, the sum total volume and sum total surface of NOR, the mean number of NOR for one nucleus and distribution of nuclei according to NOR number were measured. It is shown that the parameters of nuclei and NOR for patients with FMF on all stages clearly and trustworthy differ from those for healthy men. They are sufficiently informative, can be successfully used in clinical practice and even serve as an early diagnostic test for amyloidosis complication.

Morphometry of nuclear and nucleolar structures in a CaCo-2 cell line.

The number of the nucleoli in a CaCo-2 cell nucleus does not generally depend on the quantity of DNA in the nucleus, but nucleolar DNA content is directly proportional to total nuclear DNA. However, in multinucleolar cells (three or more nucleoli), the nucleolar DNA content increases after 96 h incubation in culture without concomitant quantitative changes in nuclear DNA. The percentage of multinucleolar cells and the average number of nucleoli per nucleus increase with increasing incubation time. After 72 and 96 h in culture, multinucleolar cells show distinctive morphologies. The ratio of the sum of nucleolar perimeters to the nuclear perimeter increases linearly when the number of nucleoli in a nucleus increases, but there is no concomitant increase in total nucleolar area or DNA content, except in the 72 and 96 h populations. When the number of nucleoli in CaCo-2 cells increases after 48 and 60 h in culture, the amount of DNA per nucleolus decreases.