Experimental models of ecological niches for African swine fever virus.

In this study, we investigated the possible biological factors affecting the survival of the African swine fever virus (ASFV) in the environment and their potential to influence the ecology of the ASFV. Specifically, we tested the survival and replication of ASFV in four phylogenetically distinct organisms: Paramecium caudatum, Dendrobaena alpine, Aedes aegypti andXeropicta derbentina using qReal-Time PCR and hemadsorbtion analysis. Levels of ASFV in earthworms (Dendrobaena alpina) and soil declined at similar rates, suggesting that earthworms likely have no influence on the ecology of the ASFV. Ciliates (Paramecium caudatum) significantly increase the rate of ASFV disappearance from the aquatic environment, probably using the virus as a food source. Mosquitoes (Aedes aegypti) do not provide significant support for the persistence of ASF virus in the environment, with no evidence for transmission to their offspring or pigs that ingested mosquitoes. ASFV persisted for much longer in air-breathing land snails (Xeropicta derbentina) than in the soil. Moreover, transcription of viral genes was maintained within the snail, although the question of full-fledged viral replication is still open. In addition, the active movements of snails suggests that they could play a role in the spread of the virus. The virus is likely to be localized in the intestines of snails as it is regularly excreted from their feces. These results highlight the importance of investigating invertebrates for understanding ASFV surviving, spreading and transmission in natural populations with zoonotic transmission potential.

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.

Serum Concentrations of Vascular Endothelial Growth Factor, Stromal Cell-Derived Factor, Nitric Oxide and Endothelial DNA Proliferation in Development of Microvascular Pathology in Acute African Swine Fever.

There is significant evidence that pathology of the microcirculation occurs in African swine fever (ASF); however, the mechanisms by which it develops are largely unknown. In the present experimental infection study, we show that an increase in vascular permeability in the initial stages of acute ASF is dependent on viraemia and elevation of the concentration of serum nitric oxide (NO). Macrophages activated by ASF virus (ASFV) are stimulated to produce NO and simultaneously to sensitize the endothelial cells through the action of vascular endothelial growth factor Β (VEGFΒ), which is followed by an increase in VEGF-mediated endothelial permeability. In the later stages of disease, the endothelial cells undergo DNA proliferation, which may additionally provoke capillary leakage, point haemorrhages and migration of blood cells into tissues. The possible mechanism of a shift in the cell cycle from the G1 to S and G2 stages could be a direct effect of ASFV. The terminal stages of disease are characterized by triggering of compensatory mechanisms such as stimulation of the synthesis of stromal cell-derived factor-1.

[Effect of human brain extracts on cultured neuroblastoma cells]. / Vliianie ékstraktov golovnogo mozga cheloveka na kletki kul'tury neiroblastomy.

OBJECTIVE: To identify the effect of brain extracts from people of different age groups on possible changes in cell physiology and behavior in vitro. MATERIAL AND METHODS: Human frontal cortical segments were obtained 12-24 hours after autopsy. Brain tissue extract was taken from young people who died at age of 22.5±2.7 years and old people at 80.9±3.0 years. SK-N-MC human neuroblastoma cells were cultured in a medium containing 50 mg/ml of brain tissue extracts; blood serum (50 mg/ml) from healthy people was used as a control. Procedures for cytophotometry of DNA and acidic proteins and polarized light microscopy were used. RESULTS: A short-term decrease in acidic protein levels in the nucleus and nucleolus was found to be affected by brain extracts from old people. There were higher cytoplasmic acidic protein levels. At the same time, the same indicators generally remained noticeably unchanged under the influence of brain extracts from young people. There were also simultaneous pronounced changes in cell morphology and behavior in vitro; namely, neuronal cell processes became shorter and their proliferative activity increased, which was not least a result of the unblocking of cells in the G2 phase under the influence of brain extracts from old people. CONCLUSION: The factors that accelerate cell proliferation in vitro accumulate in the human brain with age. Simultaneously with the acceleration of cell proliferation, there are changes in cell metabolic activity and morphology.

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.

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.

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.

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.

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.

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.

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.

New indices in morphometry of nuclear structure in the NIH 3T3 cell line.

We analyzed the connection of changes in nucleus ploidy with changes in nucleolar apparatus of NIH 3T3 cells. The quantity of nucleoli does not depend on the quantity of nucleolar DNA, but instead depends on euploidy: the majority of euploid cells have 1-3 nucleoli. The quantity of DNA in the nucleolus is correlated with the quantity of nucleolar DNA, and does not depend on ploidy changes. The nucleolar area has a tendency to increase in line with an increase in their numbers in the nucleus. The relationship of the quantity of DNA in the nucleolus with that of the nucleus is stable. During the process of increase in the number of nucleoli in a nucleus, there is a corresponding decrease in the quantity of DNA in each nucleolus, and there is likewise no increase in the sum of nucleolar DNA. The ratio of sums of the nucleolar perimeters to nuclear perimeter is a significant factor, which increases linearly along with an increase in the number of nucleoli in a nucleus.