Cell and Organism Technologies for Assessment of the SARS-CoV-2 Infectivity in Fluid Environment.

Under conditions of COVID-19 pandemic, considerable amounts of SARS-CoV-2 contained in household, municipal, and medical wastewaters inevitably reach natural water bodies. Possible preservation of virus infectivity in liquid environment is of a paramount epidemiological importance. Experiments demonstrated that SARS-CoV-2 is resistant to multiple freezing/thawing cycles and retains its infectivity in tap and river water for up to 2 days at 20°C and 7 days at 4°C. In natural milk, its viability is preserved in a refrigerator for 6 days. The exposure of aquarium fish to the virus-containing water fails to cause any infection.

Development of a Purified Viral Preparation for Studies of COVID-19 (SARS-CoV-2) Biology.

Different methods for producing bulk quantities of concentrated and purified SARS-CoV-2 for the use as antigens and for the research into COVID-19 biology were tested.

Role of Ebola Virus vp24 Protein in Inhibition of Interferonogenesis.

The effects of recombinant analog of natural Ebola virus protein vp24 in configurations virulent (vp24-ad) and avirulent (vp24-w) for guinea pigs on interferonogenesis were studied in vivo and in vitro. Amino acid differences were determined by His186 substitution in avirulent (nonlethal) configuration for Tyr in the virulent (lethal) one. Recombinant analogs vp24-w and vp24-ad inhibited interferonogenesis in vivo and in vitro. Inhibition by the two protein configurations was virtually the same.

A vesicular stomatitis pseudovirus expressing the surface glycoproteins of influenza A virus.

Pseudotyped viruses bearing the glycoprotein(s) of a donor virus over the nucleocapsid core of a surrogate virus are widely used as safe substitutes for infectious virus in virology studies. Retroviral particles pseudotyped with influenza A virus glycoproteins have been used recently for the study of influenza hemagglutinin and neuraminidase-dependent processes. Here, we report the development of vesicular-stomatitis-virus-based pseudotypes bearing the glycoproteins of influenza A virus. We show that pseudotypes bearing the hemagglutinin and neuraminidase of H5N1 influenza A virus mimic the wild-type virus in neutralization assays and sensitivity to entry inhibitors. We demonstrate the requirement of NA for the infectivity of pseudotypes and show that viruses obtained with different NA proteins are significantly different in their transduction activities. Inhibition studies with oseltamivir carboxylate show that neuraminidase activity is required for pseudovirus production, but not for the infection of target cells with H5N1-VSV pseudovirus. The HA-NA-VSV pseudoviruses have high transduction titers and better stability than the previously reported retroviral pseudotypes and can replace live influenza virus in the development of neutralization assays, screening of potential antivirals, and the study of different HA/NA reassortants.

[Study of the functional role of mutation in the guinea pig-adapted Ebola virus genome on a Drosophila melanogaster model].

Ebola virus virulence in guinea pigs, which appears through virus adaptation to this animal host, correlates with substitutions in the gene encoding vp24 protein. In particular, the substitution His-->Tyr186 was found when obtaining strain 8 ms. An attempt was made to clarify the functional role of this substitution in a transgenic fruit fly model. Using the drosophila transformation technique provided transgenic strains that contained genomic insertions of wild-type Ebola virus vp24 gene and the mutant gene with the His-->Tyr substitution at the above position. Thus, the drosophila strains carrying the sequences encoding for the vp24 proteins of Ebola virus Zaire and 8 ms in pUAST vector were obtained. This makes it possible to study the expression of transgenic constructs in various D. melanogaster organs and tissues.

[Antigenic differences in wild-type and guinea pig-adapted Ebola virus strains].

The splenocytes isolated from the mice immunized with wild-type or guinea pig-adapted Ebola virus strains were used to obtain hybridoma collections. Investigation of the monoclonal antibodies (mAb) obtained to one of the strains to another revealed antigenic interstrain differences in nucleoprotein and VP40. It is interesting that the differences were found in the hydridoma collection obtained against the wild-type strain. The mAbs produced by hydridomas to the adapted strain were found to equally well the antigens of both strains.

[Evaluation of Ebola virus reproduction in adult ICR white mice].

The investigators studied the ability of adult ICR mice (a laboratory model that was most approximated to the wildtype populations of mice) to maintain Ebola virus (EV) reproduction in the organism. The adult ICR mice inoculated with EV during 23 passages were shown to maintain viral reproduction in the liver. The elevated levels of platelets and the early generation of fibrin and fibrinogen degradation products suggested there were hemostatic changes that did not, however, progress to severe coagulopathy. The animals were in appearance apparently, other than adynamia observed on days 5-7. Thus, the susceptibility of the adult ICR mice to EV is characterized by their ability to maintain virus reproduction in the liver without evident signs of the infection. This pattern of susceptibility in the mice shows a possible role of this rodent species in the transmissive cycle of EV.

[Recombinant full-size human antibody to Ebola virus].

A full-size human antibody to Ebola virus was constructed by joining genes encoding the constant domains of the heavy and light chains of human immunoglobulin with the corresponding DNA fragments encoding variable domains of the single-chain antibody 4D1 specific to Ebola virus, which was chosen from a combinatorial phage display library of single-strand human antibodies. Two expression plasmids. pCH1 and pCL1, containing the artificial genes encoding the light and heavy chains of human immunoglobulin, respectively, were constructed. Their cotransfection into the human embryonic kidney cell line HEK293T provided the production of a full-size recombinant human antibody. The affinity constant for the antibody was estimated by solid-phase enzyme-linked immunoassay to be 7.7 x 10(7) +/- 1.5 x 10(7) M(-1). Like the parent single-chain antibody 4DI, the resulting antibody bound the nucleoprotein of Ebola virus and did not interact with the proteins of Marburg virus.

[Molecular mechanisms of Ebola virus reproduction].

The review presents recent data on the molecular mechanisms of the stages of an Ebola virus replication cycle, on the interaction of viral and cellular components at each stage, as well as on the mechanisms responsible for he realization of viral genetic information in the infected cell.

[Development of a method for rapid detection of Ebola virus antibodies and antigen].

Despite the wide spectrum of reliable methods for identifying Ebola virus, their performance requires highly-skilled personnel, specialized laboratories, complicated equipment, and much time. Therefore, there is a need for a method that allows a physician or a medical attendant to identify the causative agent in field or bedside tests without special equipment as soon as possible. The immunoassay involving nitrocellulose membrane immuno-filtration, by using a fixed antigen (antibodies) or their immunosols, is a tried-and-true method. The time of the analysis is 7-15 min.

N-Heterocyclic borneol derivatives as inhibitors of Marburg virus glycoprotein-mediated VSIV pseudotype entry.

There is currently no approved antiviral therapy for treatment of Marburg virus disease (MVD). Although filovirus infection outbreaks are quite rare, the high mortality rates in such outbreaks make the development of anti-filoviral drugs an important goal of medical chemistry and virology. Here, we performed screening of a large library of natural derivatives for their virus entry inhibition activity using pseudotype systems. The bornyl ester derivatives containing saturated N-heterocycles exhibited the highest antiviral activity. It is supposed that compounds with specific inhibitory activity toward MarV-GP-dependent virus entry will inhibit the rVSIV-ΔG-MarV-GP pseudotype much more efficiently than the control rVSIV-ΔG-G pseudotype. At the same time, the compounds similarly inhibiting both pseudotypes will likely affect rVSIV capsid replication or the cellular mechanisms common to the entry of both viruses. Borneol itself is not active against both pseudotypes and is nontoxic, whereas its derivatives have varying toxicity and antiviral activity. Among low-toxic borneol derivatives, six compounds turned out to be relatively specific inhibitors of MarV-GP-mediated infection (SC > 10). Of them, compound 6 containing a methylpiperidine moiety exhibited the highest virus-specific activity. Notably, the virus-specific activity of this compound is twice as high as that of the reference.

[The properties of Ebola virus proteins].

The paper describes the structure and functions of Ebola virus properties. It also presents information on the role of structural (NP, VP40, VP35, GP, VP30, VP24, and L) and secreted (sGP, delta-peptide, GP1, GP(1,2delta), ssGP) proteins in the viral replication cycle and in the pathogenesis of Ebola hemorrhagic fever.

[Hematological and immunological parameters during Ebola virus passages in guinea-pigs].

The trend in hematological and immunological parameters during Ebola virus passages in guinea-pigs indicated that pathophysiological changes occurred just during the second passage and further became stronger. The increase of some parameters and their correlation with the occurrence of fatal outcomes allowed the authors to reveal the most significant changes as increased juvenile platelets, whole blood virus appearance, higher echinocytes, a rise in the pro mil of blast cells and megakaryocytes in the bone marrow, and decreased neutrophilic phagocytic activity. Viral acquisition of the properties of lethality to guinea-pigs depends on the fine mechanisms responsible for viral interaction with host cells, which may lead to viral genetic changes during passages.

Adapted Lethality: What We Can Learn from Guinea Pig-Adapted Ebola Virus Infection Model.

Establishment of small animal models of Ebola virus (EBOV) infection is important both for the study of genetic determinants involved in the complex pathology of EBOV disease and for the preliminary screening of antivirals, production of therapeutic heterologic immunoglobulins, and experimental vaccine development. Since the wild-type EBOV is avirulent in rodents, the adaptation series of passages in these animals are required for the virulence/lethality to emerge in these models. Here, we provide an overview of our several adaptation series in guinea pigs, which resulted in the establishment of guinea pig-adapted EBOV (GPA-EBOV) variants different in their characteristics, while uniformly lethal for the infected animals, and compare the virologic, genetic, pathomorphologic, and immunologic findings with those obtained in the adaptation experiments of the other research groups.

[Human recombinant antibodies to Ebola virus: preparation and characteristics].

Human recombinant antibodies against a purified Ebola virus (EV) lysate were selected from a combinatorial library of scFv-antibodies using the phage display technique. Nine unique antibodies were identified after sequencing the Vh- and Vl-genes encoding the selected antibodies. Solid-phase enzyme immunoassay (EIA) indicated that these antibodies were able to bind both inactivated and native EV. Immunoblotting showed that 6 antibodies identified nucleoprotein (NP), one antibody did VP24 and another antibody did VP40. One of the selected antibodies reacted with two EP proteins: VP24 and VP40. Solid-phase EIA demonstrated cross-reactivity with Marburg virus (MAR) and defined VP24 MAR as a target protein for the antibody.

Immune and pathophysiological processes in baboons experimentally infected with Ebola virus adapted to guinea pigs.

The dynamics of pathophysiological and immunological parameters monitored in monkeys Papio hamadryas infected with the guinea pig-adapted Ebola virus strain demonstrated that this viral strain preserved its virulence for monkeys and caused the disease with characteristic features similar to those caused by non-adapted Ebola virus. However, certain previously unknown patterns have been observed: (1) prolongation of the febrile period by two days; (2) extended period was characterized by stability of serum biochemical parameters; (3) marked vacuolization of the neutrophil cytoplasm; (4) appearance of juvenile lymphocytes on day 3 and by the end of the disease; and (5) a considerable increase in the spontaneous mononuclear proliferation (along with a decrease in the mitogen-induced proliferation) during the terminal stage of infection. The severity of pathological coagulation was found to correlate with the activity of serum cytokines IFN-alpha and TNF-alpha: their activities increased about 250- and 100-fold, respectively. There was significant alteration in the activity of natural killer cells, that dropped by the time of animal death.

Suppressive effect of Ebola virus on T cell proliferation in vitro is provided by a 125-kDa GP viral protein.

Ebola virus (EV), an extremely infectious pathogen, causes severe hemorrhagic fever in humans and nonhuman primates. The disease pattern includes damage of parenchymal cells of vital organs in association with hemostatic and immune disorders. Vaccination with the inactivated virions does not provide an effective immune protection against the disease. The inadequate immune response may be directly caused by the virus, and, hence, it may presumably be crucial in the pathogenic process and prophylactic treatment of Ebola infection. The suggested immunosuppressive properties of EV were examined in this study. We have demonstrated that the whole heat-inactivated virions can dose-dependently suppress human lymphocyte mitogen-stimulated proliferation in vitro. In further analyses, we identified the viral protein responsible for the suppressive effect, and we showed that it was provided by a protein corresponding to a 125-kDa envelope glycoprotein (GP-125). The protein alone inhibited lymphocyte proliferation, whereas the other viral proteins were without significant effect on blastogenesis. To determine the immunosuppressive properties of different portions of GP-125, deletion mutants of GP were designed based on predicted localisation of antigen sites. They were expressed as recombinant proteins and studied in proliferation assays. We identified a 40-amino acid sequence at the N-terminus of GP-125 that exerted a suppressive effect on blastogenesis.

Elaboration of laboratory strains of Ebola virus and study of pathophysiological reactions of animals inoculated with these strains.

Selective passages in animals and cell cultures were used to produce a set of Ebola virus (EBO) laboratory strains with changed virulence for some animal genera. Comparative study of the genomes of wild-type EBO and selected variants formed the basis for studying the molecular causes of EBO virulence. Investigation of pathophysiological reactions of the animals inoculated with these strains allowed some key factors in Ebola fever pathogenesis to be determined.

Inactivation of Ebola virus with a surfactant nanoemulsion.

Hemorrhagic fever caused by Ebola virus (EBO) is a highly contagious infection. This necessitates that the contaminated instruments, clothes, and hospital premises must be completely disinfected. Nanoemulsions are a new form of disinfectant composed of detergents and vegetable oil suspended in water. The antiviral activity of nanoemulsion ATB has been investigated against EBO. The nanoemulsion was tested against two preparations of EBO (strain Zaire) obtained from Vero cell culture fluid (EBO-zc) and from blood of infected monkeys (EBO-zb). The nanoemulsion ATB was virucidal against both preparations of EBO, inactivating the purified virus within 20 min even when diluted 1:100 with the growth medium. Inactivation of the virus in tissue preparations was also complete, but required 1:10 dilutions with media or higher. After treatment with ATB (10 and 1% concentrations), no EBO was apparent even after two passages in Vero cell culture. These data indicate that the nanoemulsion is an effective disinfectant for EBO. Because of the excellent biocompatibility of nanoemulsions, studies are planned to determine whether the nanoemulsion-killed virus is suitable for developing a vaccine against EBO.

The growth of human HIV-1 infected U937 cells in immune-deprived mice.

We report in vivo growth of human promonocytic cells infected with HIV-1 presented in new mouse model. Cloned U937 cells chronically infected with HIV-1 were grafted in (CBA*C57B1/6)F1 mice deprived of immunity by thymectomia and total body irradiation with subsequent marrow reconstitution. Nine weeks after cell inoculation, HIV-1-positive cells were found only in mice that received an additional single dose of cyclophosphamide (100 mg/kg bw) prior to transplantation, whereas, in mice without further immune deprivation, the complete elimination of cells bearing viral antigen occurred already on the seventh day after transplantation. The approach described may be suitable for in vivo development of antiviral drugs against latent infection in macrophage-like cells which represent a serious problem in therapy of AIDS in humans.