The role of gastropods in African swine fever virus ecology.

The spread of the African swine fever virus (ASF virus) genotype ii in the Eurasian region has been very successful and often inexplicable. The virus spreads rapidly and persists in areas with wild boar populations, but areas without feral pig populations are also affected. The virus has shown the ability to survive for a long time in the environment without a population of susceptible hosts, both pigs and Ornithodoros soft ticks. Published data indicated that ASF viruses persist significantly longer in an environment with some freshwater snails (especially Pomacea bridgesii, Tarebia granifera, Asolene spixii, Melanoides tuberculate, and Physa fontinalis), compared to freshwater without snails. Data obtained in this study suggest that gastropods theoretically can be the hosts of the ASF virus. Also, we have proven the possibility of long-term existence of an infectious virus when infected in vitro.

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.

Study of different heterocycles showing significant anti-severe acute respiratory syndrome 2 activity in vitro and in vivo.

Background and Aim: With the emergence of severe acute respiratory syndrome-related coronavirus (SARS-CoV-2), antiviral drug development has gained increased significance due to the high incidence and potentially severe complications of the resulting coronavirus infection. Heterocycle compounds, acting as antimetabolites of DNA and RNA monomers, rank among the most effective antiviral drugs. These compounds' antiviral effects on various SARS-CoV-2 isolates, as found in existing data collections, form the basis for further research. The aim of this study was to examine the possible antiviral effect of some originally synthesized heterocyclic compounds. Materials and Methods: The main methods were cell culturing, cytotoxicity assay, qRT-PCR assay, tissue and blood cells analysis, and micro-computed tomography (micro-CT) imaging. Results: In both in vitro and in vivo conditions, the elimination of SARS-Cov-2 occurred significantly earlier after administration of the compounds compared to the control group. In hamsters, the primary symptoms of coronavirus disease disappeared following administration of heterocycle compounds. Conclusion: Using delta and omicron strains of the SARS-CoV-2 virus, newly created heterocycle compound analogs dramatically reduced SARS-CoV-2 multiplication, resulting in a drop in viral RNA load in the supernatant under in vitro conditions. Improvements in pathological manifestations in the blood, bone marrow, and internal organs of hamsters demonstrated that heterocycle compounds inhibited SARS-CoV-2 replication both in vitro and in vivo.

Infection of Human Macrophage-Like Cells by African Swine Fever Virus.

BACKGROUND: The African swine fever (ASF) virus (ASFV) and ASF-like viral sequences were identified in human samples and sewage as well as in different water environments. Pigs regularly experience infections by the ASFV. The considerable stability of the virus in the environment suggests that there is ongoing and long-term contact between humans and the ASFV. However, humans exhibit resistance to the ASFV, and the decisive factor in developing infection in the body is most likely the reaction of target macrophages to the virus. Therefore, this study aimed to characterize the responses of human macrophages to the virus and explore the distinct features of the viral replication cycle within human macrophages. METHODS: The ASFV Armenia/07 strain was used in all experiments. In this study, quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine the ASFV gene expression; flow cytometry analysis was performed to evaluate the effects of the inactive and active ASFV (inASFV and aASFV) treatments on the phenotype of THP-1-derived macrophages (Mφ0) and inflammatory markers. Moreover, other methods such as cell viability and apoptosis assays, staining techniques, phagocytosis assay, lysosome-associated membrane protein (LAMP-1) cytometry, and cytokine detection were used during experiments. RESULTS: Our findings showed that the virus initiated replication by entering human macrophages. Subsequently, the virus shed its capsid and initiated the transcription of numerous viral genes, and at least some of these genes executed their functions. In THP-1-derived macrophages (Mφ0), the ASFV implemented several functions to suppress cell activity, although the timing of their implementation was slower compared with virus-sensitive porcine alveolar macrophages (PAMs). Additionally, the virus could not complete the entire replication cycle in human Mφ0, as indicated by the absence of viral factories and a decrease in infectious titers of the virus with each subsequent passage. Overall, the infection of Mφ0 with the ASFV caused significant alterations in their phenotype and functions, such as increased TLR2, TLR3, CD80, CD36, CD163, CXCR2, and surface LAMP-1 expression. Increased production of the tumor necrosis factor (TNF) and interleukin (IL)-10 and decreased production of interferon (IFN)-α were also observed. Taken together, the virus enters human THP-1-derived macrophages, starts transcription, and causes immunological responses by target cells but cannot complete the replicative cycle. CONCLUSION: These findings suggest that there may be molecular limitations within human macrophages that at least partially restrict the complete replication of the ASFV. Understanding the factors that hinder viral replication in Mφ0 can provide valuable insights into the host-virus interactions and the mechanisms underlying the resistance of human macrophages to the ASFV.

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.

The pattern of stability of African swine fever virus in leeches.

African swine fever virus (ASFV) can accumulate and survive in leeches for a long time. The reasons for the survival of ASFV in leeches are not entirely clear. Here, we elucidate the virus survival pathway in infected leeches. One of the questions reported previously is addressed in this article. How the virus concentration in the body of the leech is equal to or higher than in the water infected with ASFV? Examination of blood swallowed by leeches reveals that the blood cells retain their morphological characteristics for several weeks. It can explain the long-term persistence of the high levels of ASFV in the leeches that ingested ASFV-infected pig blood. qRT-PCR assay showed the transcription of ASFV genes in infected leeches. However, the infectious particles of the virus measured by HADU haven't increased. Quantitative studies of the ASFV revealed a high content of both viral genes and infectious particles in the skin of leeches compared with other body parts. Electron microscopy analysis revealed the ability of the ASFV to effectively bind to the skin surface of the leeches, which explained the high concentrations of ASFV in the leeches' skin. A significant difference in the transcriptional activity between early and late viral genes indicates that the virus entered the initial stage of replication, but for some reason failed to complete it, which is typical of abortive infections.

Comparative staining of Rhinolophus spp. white blood cells in blood smears.

Background and Aim: A drawback of studies on bat blood smears in the field is the lack of time for fixation because blood sampling using a non-lethal method often provides less time for fixation in smear preparations due to the small volume of blood collected. Usually, there is insufficient blood for another smear preparation, so it is necessary to use blood smears as rationally as possible, especially for rare bats. Many stains are used for staining peripheral blood smears, and they have advantages and disadvantages. This study aimed to examine commonly used stains for blood smears to select the best stain for staining peripheral blood smears in bats. Materials and Methods: In this study, 48 blood smears of Rhinolophus spp. bats were examined using several blood staining methods. Four methods that showed the best results were used in further experiments: Romanowsky-Giemsa, Pappenheim, hematoxylin-eosin, and eosin methylene blue. Results: Comparative analysis of different methods for staining bat blood smears revealed that the most convenient method for analyzing blood cells is Pappenheim method. Conclusion: Staining blood smears using Pappenheim method yield the least number of unsuccessful blood smear stains and are quite effective for the morphological analysis of blood cells.

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.

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.

Possibility of long-term survival of African swine fever virus in natural conditions.

BACKGROUND AND AIM: In modern scientific literature presents an understanding that African swine fever (ASF) ASF virus (ASFV) is remarkably stable in the environment, and carcasses of the pigs which were died after ASF, play a key role as ASFV reservoir. The aim of this study was to evaluate the possibility of the ASFV (different isolates) survival in bodies of dead animals, bones, remnants of bone marrow, residual organ matrix in natural conditions. MATERIALS AND METHODS: Skeletons of ASFV infected pigs which were died and left/abandoned in forests or buried in Armenia at diverse time points and locations had been excavated and examined for the presence of ASFV genome by real-time polymerase chain reaction (PCR) assay and for infection abilities through in vitro (hemadsorption test and infection in porcine lung macrophages) as well as by intramuscular infection in healthy pigs. RESULTS: Current exploration showed that in several samples (with different times of exposure) of excavated skeletons had been detected the presence of the virus gene (p72) using real-time PCR. However, in none of these porcine samples, infectious ASFV could be isolated. Data obtained by real-time PCR at frequent intervals indicated the presence of the virus gene (p72), especially within the case of the acute form of the disease. This can be explained by the highest levels of the virus during the latter case mentioned above. CONCLUSION: ASFV seems to be very sensitive to environmental temperature. The best place for ASFV long-term survival in the natural environment is bone marrow from intact big tubular bones (like femur or tibia) of buried carcasses. In artificial "graves," complete bones with not destructed bone marrow can preserve the virus gene (p72) for a very long time (more than 2 years). Infectious particles in underground conditions survive not so long: In complete bones with not affected bone marrow, possible presence of the virus for several months.