Pathology and Clinics of Naturally Occurring Low-Virulence Variants of African Swine Fever Emerged in Domestic Pigs in the South Caucasus.

Shortly after the establishment of African swine fever virus (ASFV) genotype II in 2007, cases of acute fatal infection were observed. However, after several years of circulation in the Eurasian region, the clinical signs of the disease changed. Currently, this disease can occur acutely, subclinically, chronically, or asymptomatically. Cases of the complete recovery of infected pigs, and the disappearance of ASFV from their tissues and secretions have been described. This form of the disease first appeared in Armenia at the end of 2011. This virus was described and identified as the Dilijan2011IMB strain. The goal of our research was to study the main features of clinical, pathological, immunological, virological, and genetic parameters involved in the development of new forms of African swine fever (ASF). Chronic ASF was characterized with low titers of the virus and a decrease in the intensity of hemadsorption. Additionally, a reduced intensity in clinical symptoms and pathoanatomical results was noted. The absolute, but not the relative number of immune cells changes; the neutropenia (in bone marrow and spleen), lymphopenia (in bone marrow), lymphocytosis (only in spleen), lymphoid cell depletion (in bone marrow), and pancytopenia (in bone marrow) observed in the chronic form of ASF were less pronounced compared to in the acute form. When comparing the late stage of chronic ASF to the acute form, the key cytological indicators in the spleen, lymph nodes, and blood were less severe in the chronic stage. Bone marrow failure in the chronic form, expressed in a pronounced decrease in all cell types, generally coincided with the data in the acute form of ASF. The same data were obtained after assessing serum TNF-alpha levels. Thus, we can conclude that the chronic form of ASF occurs due to a less pronounced immune response, as well as a decrease in virus titers in the blood and tissues of infected pigs.

African Swine Fever Virus Manipulates the Cell Cycle of G0-Infected Cells to Access Cellular Nucleotides.

African swine fever virus manipulates the cell cycle of infected G0 cells by inducing its progression via unblocking cells from the G0 to S phase and then arresting them in the G2 phase. DNA synthesis in infected alveolar macrophages starts at 10-12 h post infection. DNA synthesis in the nuclei of G0 cells is preceded by the activation of the viral genes K196R, A240L, E165R, F334L, F778R, and R298L involved in the synthesis of nucleotides and the regulation of the cell cycle. The activation of these genes in actively replicating cells begins later and is less pronounced. The subsequent cell cycle arrest at the G2 phase is also due to the cessation of the synthesis of cellular factors that control the progression of the cell cycle-cyclins. This data describes the manipulation of the cell cycle by the virus to gain access to the nucleotides synthesized by the cell. The genes affecting the cell cycle simply remain disabled until the beginning of cellular DNA synthesis (8-9 hpi). The genes responsible for the synthesis of nucleotides are turned on later in the presence of nucleotides and their transcriptional activity is lower than that during virus replication in an environment without nucleotides.

The Effect of Low-Energy Laser-Driven Ultrashort Pulsed Electron Beam Irradiation on Erythropoiesis and Oxidative Stress in Rats.

Anemia is a commonly observed consequence of whole-body exposure to a dose of X-ray or gamma irradiation of the order of the mean lethal dose in mammals, and it is an important factor for the determination of the survival of animals. The aim of this study was to unravel the effect of laser-driven ultrashort pulsed electron beam (UPEB) irradiation on the process of erythropoiesis and the redox state in the organism. Wistar rats were exposed to laser-driven UPEB irradiation, after which the level of oxidative stress and the activities of different antioxidant enzymes, as well as blood smears, bone marrow imprints and sections, erythroblastic islets, hemoglobin and hematocrit, hepatic iron, DNA, and erythropoietin levels, were assessed on the 1st, 3rd, 7th, 14th, and 28th days after irradiation. Despite the fact that laser-driven UPEB irradiation requires quite low doses and repetition rates to achieve the LD50 in rats, our findings suggest that whole-body exposure with this new type of irradiation causes relatively mild anemia in rats, with subsequent fast recovery up to the 28th day. Moreover, this novel type of irradiation causes highly intense processes of oxidative stress, which, despite being relatively extinguished, did not reach the physiologically stable level even at the 28th day after irradiation due to the violations in the antioxidant system of the organism.

New composition of tungsten has a broad range of antiviral activity.

The water-based combination of two inorganic chemical compounds such as sodium tungstate dihydrate-Na2WO4 × 2H2O and Aluminum sulfate octadecahydrate-Al2 (SO4) 3 × 18H2O that we have conditionally named 'Vomifal' has a broad antiviral activity in various DNA and RNA viruses, including Human Herpes Virus (HHV), African Swine Fever Virus (ASFV), Vaccinia Virus (VV), Hepatitis C Virus (HCV), Foot and Mouth Disease Virus (FMDV), Influenza A virus (A/Aichi/2/68 (H3N2)). In vitro and In vivo assays in several tissue cultures as well as in laboratory animals, conformed 'Vomifal' has a very low toxicity and the antiviral properties partially are due to its ability to induce gamma-IFN. Based on the results obtained, we can assume the presence of at least two mechanisms of the antiviral action of the studied drug. First or early stage - an unknown mechanism, possibly related to the effect on cellular receptors. Second or late stage - main antiviral properties probably associated with an interferonogenic effect.

Severe Acute Respiratory Syndrome Coronavirus-2 Delta Variant Study In Vitro and Vivo.

At the end of 2019, an outbreak of a new severe acute respiratory syndrome caused by a coronavirus occurred in Wuhan, China, after which the virus spread around the world. Here, we have described the adaptive capacity and pathogenesis of the SARS-CoV-2 Delta variant, which is widespread in Armenia, in vitro and vivo on Syrian hamsters. We have studied the changes in the SARS-CoV-2genome using viral RNA sequencing during virus adaptation in vitro and in vivo. Our findings revealed that SARS-CoV-2 in Syrian hamsters causes a short-term pulmonary form of the disease, the first symptoms appear within 48 h after infection, reach 5-7 days after infection, and begin to disappear by 7-9 days after infection. The virus induces pathogenesis in the blood and bone marrow, which generally corresponds to the manifestation of the inflammatory process. The pulmonary form of the disease passes faster than changes in blood cells and bone marrow. Our data show that hamster organs do not undergo significant pathological changes in the Delta variant of SARS-CoV-2 infection.

Low-Energy Laser-Driven Ultrashort Pulsed Electron Beam Irradiation-Induced Immune Response in Rats.

The development of new laser-driven electron linear accelerators, providing unique ultrashort pulsed electron beams (UPEBs) with low repetition rates, opens new opportunities for radiotherapy and new fronts for radiobiological research in general. Considering the growing interest in the application of UPEBs in radiation biology and medicine, the aim of this study was to reveal the changes in immune system in response to low-energy laser-driven UPEB whole-body irradiation in rodents. Forty male albino Wistar rats were exposed to laser-driven UPEB irradiation, after which different immunological parameters were studied on the 1st, 3rd, 7th, 14th, and 28th day after irradiation. According to the results, this type of irradiation induces alterations in the rat immune system, particularly by increasing the production of pro- and anti-inflammatory cytokines and elevating the DNA damage rate. Moreover, such an immune response reaches its maximal levels on the third day after laser-driven UPEB whole-body irradiation, showing partial recovery on subsequent days with a total recovery on the 28th day. The results of this study provide valuable insight into the effect of laser-driven UPEB whole-body irradiation on the immune system of the animals and support further animal experiments on the role of this novel type of irradiation.

Patterns of alveolar macrophage activation upon attenuated and virulent African swine fever viruses in vitro.

The pattern of porcine alveolar macrophage (AM) activation upon classical stimuli of two strains of African swine fever (ASF) viruses, an attenuated ASFV-BA71V and virulent ASFV-Georgia2007 were investigated. In an in vitro experiment ASFV-Georgia2007-infected AM showed M1 polarization pattern different from the one induced by classical stimuli. Altered morphology, appearance of binuclear cells, decreased synthesis of IFN-alpha as well as IFN-epsilon was observed compared with attenuated ASFV-BA71V, and decreased synthesis of IFN-omega compared with intact cells. However, CD68 level did not significantly differ between alveolar macrophage populations infected by ASFV-Georgia2007 and control group, while both LPS/IFN-gamma stimulation and non-pathogenic ASFV-BA71V virus increased the level of CD68 soluble receptor. AM infection with ASFV-Georgia2007 resulted in remarkable DNA proliferation whereas LPS/IFN-gamma and ASFV-BA71V induced less expressed DNA proliferation in activated cells. The higher value of nitric oxide was obvious in the cells infected with ASFV-BA71V, compared to ASFV-Georgia2007 and LPS/IFN-gamma activated cells. In conclusion, pattern of activation of alveolar macrophages induced by ASFV-Georgia2007 virus differs from the one expressed in LPS/IFN-gamma- and ASFV-BA71V-activated cells. ASFV-BA71V and LPS/IFN-gamma share similar antiviral response of porcine AM. Therefore we assume that wild type virulent ASFV can partially down regulate antiviral response of AM and conclude that evolutionary decrease of virulence in ASFV is related to alterations of control of the host cell antiviral response.

Immune cell pathology in rabbit hemorrhagic disease.

AIM: The aim of this research was to study the effect of rabbit hemorrhagic disease virus (RHDV) on the host immune response by examining the cellular composition/pathology of lymphoid organs and serum levels of tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ). MATERIALS AND METHODS: Nine adult rabbits were inoculated with 1 ml of 10% infected liver homogenate, and three rabbits served as controls. The rabbit hemorrhagic disease (RHD)-induced animals were studied on 3 consecutive days post-infection. Diagnosis of RHD was made through routine hemagglutination tests and the polymerase chain reaction. Blood smears and tissue samples from bone marrow (BM), spleen, lymph nodes, and liver were analyzed for cell composition and cytopathology. Serum levels of TNF-α and IFN-γ were measured by enzyme-linked immunosorbent assay. RESULTS: RHD showed a decreased absolute cell count of blood as well as lymph nodes, spleen, and BM cell populations with marked left shift. This was seen as a progressive rise in immature and blast cells. Quantitative cellular changes were accompanied by an increase in specific inflammatory cytokines. Immunocytopathological alterations were evidenced by: Vacuolized, hyperactivated tissue macrophages, finding of Döhle bodies in neutrophils, and activated lymphocytes with increased nuclear-cytoplasmic ratio. Cytoplasmic eosinophilic viral inclusions found in tissue (liver, spleen, and BM) macrophages were shown for the 1st time in RHD. Megakaryocytic emperipolesis was a common feature of RHD. CONCLUSION: These studies suggest that RHDV induces pathology in leukocytes due to hyperactivation with left shift (toward immature stages of the different cell lineages). Macrophages are increased in number and show an expressed cytopathic effect often accompanied by viral eosinophilic cytoplasmic inclusions. They also developed a secretory activation (increased levels of pro-inflammatory cytokines).

Evaluation of Viral Genome Copies Within Viral Factories on Different DNA Viruses.

This article describes a simple method of measuring the number of viral genomes within viral factories. For this purpose, we use three DNA viruses replicating in the cytoplasm of the infected cells: wild-type African swine fever virus (ASFV)-Georgia 2007, culture-adapted type ASFV-BA71V, and Vaccinia virus (VV). The measurements are conducted in three steps. In the first step, after DNA staining, we evaluate Integrated Optical Density (IOD) of total DNA for each viral factory. The second step involves the calculations of the mass of DNA in the viral factories in picograms (pg). And, in the third step, by dividing the mass of DNA within viral factory by the weight of a single viral genome, we obtain the number of viral genomes within the factory.

Pathomorphology of the brain in the acute form of African swine fever

The brains of 10 infected pigs were examined for histopathology and presence of African swine fever virus (ASFV) DNA ASFV infection induces inflamed meninges, cerebral edema and vascular thrombosis, as well as subdural hematomas. Slight tension in the dura mater, flattening of the gyri and narrowing of the sulci were also observed at four days post infection (dpi). Enlarged perivascular spaces were detected for most vessels of the brain after three to four dpi. Considerable lymphocytic infiltration of the brain tissue was observed at the terminal stage of disease. ASFV was present in all investigated areas of brain beginning from three to four dpi. The isolated virus do not differ from the used in present study Georgia 2007 strain.

Evidence of hemolysis in pigs infected with highly virulent African swine fever virus.

AIM: The research was conducted to understand more profoundly the pathogenetic aspects of the acute form of the African swine fever (ASF). MATERIALS AND METHODS: A total of 10 pigs were inoculated with ASF virus (ASFV) (genotype II) in the study of the red blood cells (RBCs), blood and urine biochemistry in the dynamics of disease. RESULTS: The major hematological differences observed in ASFV infected pigs were that the mean corpuscular volume, mean corpuscular hemoglobin, and hematocrits were significantly decreased compared to controls, and the levels of erythropoietin were significantly increased. Also were detected the trends of decrease in RBC count at terminal stages of ASF. Analysis of blood biochemistry revealed that during ASF development, besides bilirubinemia significantly elevated levels of lactate dehydrogenase, and aspartate aminotransferase were detected. Analysis of urine biochemistry revealed the presence of bilirubinuria, proteinuria during ASF development. Proteinuria, especially at late stages of the disease reflects a severe kidney damage possible glomerulonefritis. CONCLUSION: The results of this study indicate the characteristics of developing hemolytic anemia observed in acute ASF (genotype II).

Polyploidization on SK-N-MC human neuroblastoma cells infected with herpes simplex virus 1.

Polyploidization is one of the most dramatic changes occurring within cell genome owing to various reasons including under many viral infections. We examined the impact of herpes simplex virus-1 (HSV-1) on SK-N-MC human neuroblastoma cell line. The infected cells were followed from 6 hours up to 96 hours post infection (hpi). A large number of polyploid cells with giant nuclei was observed under the influence of HSV-1 at 24 hpi with the DNA content of 32c to 64c or more, in comparison with control SK-N-MC cells that were characterized by relatively moderate values of ploidy, i.e. 8с to 16с (where 1c is the haploid amount of nuclear DNA found in normal diploid populations in G0/G1). After 48-96 hpi, the population of polyploid cells with giant nuclei decreased to the benchmark level. The SK-NMC cells infected with HSV-1 for 24 hours were stained with gallocyanine and monitored for cytological features. The infected cells underwent virus induced cellcell and nuclei fusion with the formation of dense nuclei syncytium. The metabolic activity of HSV-1 infected cells was higher in both nuclei and nucleoli when compared to control cells.

A study of lymphoid organs and serum proinflammatory cytokines in pigs infected with African swine fever virus genotype II.

African swine fever virus (ASFV), the causative agent of one of the most important viral diseases of domestic pigs for which no vaccine is available, causes immune system disorders in infected animals. In this study, the serum levels of proinflammatory cytokines, as well as the histological and cellular constitution of lymphoid organs of pigs infected with ASFV genotype II were investigated. The results showed a high degree of lymphocyte depletion in the lymphoid organs, particularly in the spleen and lymph nodes, where ASFV infection led to a twofold decrease in the number of lymphocytes on the final day of infection. Additionally, ASFV-infected pigs had atypical forms of lymphocytes found in all lymphoid organs. In contrast to lymphocytes, the number of immature immune cells, particularly myelocytes, increased dramatically and reached a maximum on day 7 postinfection. The serum levels of TNF-α, IL-1ß, IL-6, and IL-8 were evaluated. Proinflammatory cytokines showed increased levels after ASFV infection, with peak values at 7 days postinfection, and this highlights their role in the pathogenesis of ASFV. In conclusion, this study showed that ASFV genotype II, like other highly virulent strains, causes severe pathological changes in the immune system of pigs.

Evaluation of hemostaseological status of pigs experimentally infected with African swine fever virus.

African swine fever is a highly contagious hemorrhagic disease of pigs caused by African swine fever virus (ASFV). Hemorrhages are the most frequently reported lesions in acute and subacute forms of ASF. Hemorrhagic lesions are accompanied by impaired hemostasis, which includes thrombocytopenia and changes in the coagulation system. In the present study, experimental infection was conducted to elucidate whether a highly virulent ASFV genotype II circulating in the Trans-Caucasus and Eastern Europe affects the hemostasis of infected pigs. Platelet count changes and platelet size, as well as coagulation parameters were evaluated upon experimental infection. In contrast to other ASFV strains, ASFV genotype II showed a significant decrease in the number of platelets from 3rd dpi onwards. Furthermore, a decrease in platelet size was observed throughout the entire period of experiment. A significant increase in the number of platelet aggregates was observed from the beginning of infection. Unlike other ASFV strains, ASFV genotype II induced a slight shortening of an activated partial thromboplastin time (aPTT) throughout the experiment. Thrombin time (TT) was prolonged from day 5 onwards, whereas no changes in prothrombin time (PT) were found upon infection. The level of d-dimers was permanently higher than in control with a peak on day 3 post-infection. ASFV induced a significant decrease in the level of fibrinogen from day 5 till the end of experiment. Thus, it can be concluded that ASFV genotype II isolated in Armenia affects the hemostasis of infected pigs and causes changes that differ from that of other ASFV strains described previously.

Pathology of porcine peripheral white blood cells during infection with African swine fever virus.

BACKGROUND: African swine fever virus (ASFV) is the causative agent of African swine fever (ASF) that is the significant disease of domestic pigs. Several studies showed that ASFV can influence on porcine blood cells in vitro. Thus, we asked ourselves whether ASFV infection results in changes in porcine blood cells in vivo. A series of experiments were performed in order to investigate the effects of ASFV infection on porcine peripheral white blood cells. Nine pigs were inoculated by intramuscular injection with 104 50% hemadsorbing doses of virus (genotype II) distributed in Armenia and Georgia. The total number of fifteen cell types was calculated during experimental infection. RESULTS: Although band-to-segmented neutrophils ratio became much higher (3.5) in infected pigs than in control group (0.3), marked neutropenia and lymphopenia were detected from 2 to 3 days post-infection. In addition to band neutrophils, the high number of other immature white blood cells, such as metamyelocytes, was observed during the course of infection. From the beginning of infection, atypical lymphocytes, with altered nuclear shape, arose and became 15% of total cells in the final phase of infection. Image scanning cytometry revealed hyperdiploid DNA content in atypical lymphocytes only from 5 days post-infection, indicating that DNA synthesis in pathological lymphocytes occurred in the later stages of infection. CONCLUSION: From this study, it can be concluded that ASFV infection leads to serious changes in composition of white blood cells. Particularly, acute ASFV infection in vivo is accompanied with the emergence of immature cells and atypical lymphocytes in the host blood. The mechanisms underlying atypical cell formation remain to be elucidated.