Genetic characteristics of SARS-CoV-2 virus variants observed upon three waves of the COVID-19 pandemic in Ukraine between February 2021-January 2022.

The aim: of our study was to identify and characterize the SARS-CoV-2 variants in COVID-19 patients' samples collected from different regions of Ukraine to determine the relationship between SARS-CoV-2 phylogenetics and COVID-19 epidemiology. Patients and methods: Samples were collected from COVID-19 patients during 2021 and the beginning of 2022 (401 patients). The SARS-CoV-2 genotyping was performed by parallel whole genome sequencing. Results: The obtained SARS-CoV-2 genotypes showed that three waves of the COVID-19 pandemic in Ukraine were represented by three main variants of concern (VOC), named Alpha, Delta and Omicron; each VOC successfully replaced the earlier variant. The VOC Alpha strain was presented by one B.1.1.7 lineage, while VOC Delta showed a spectrum of 25 lineages that had different prevalence in 19 investigated regions of Ukraine. The VOC Omicron in the first half of the pandemic was represented by 13 lines that belonged to two different clades representing B.1 and B.2 Omicron strains. Each of the three epidemic waves (VOC Alpha, Delta, and Omicron) demonstrated their own course of disease, associated with genetic changes in the SARS-CoV-2 genome. The observed epidemiological features are associated with the genetic characteristics of the different VOCs, such as point mutations, deletions and insertions in the viral genome. A phylogenetic and transmission analysis showed the different mutation rates; there were multiple virus sources with a limited distribution between regions. Conclusions: The evolution of SARS-CoV-2 virus and high levels of morbidity due to COVID-19 are still registered in the world. Observed multiple virus sourses with the limited distribution between regions indicates the high efficiency of the anti-epidemic policy pursued by the Ministry of Health of Ukraine to prevent the spread of the epidemic, despite the low level of vaccination of the Ukrainian population.

The Natural Oligoribonucleotides Functionalized by D-Mannitol Affected Interactions of Hemagglutinin with Glycan Receptor Indicating Anti-Influenza Activity.

Hemagglutinin (HA), the class I influenza A virus protein is responsible for the attachment of virus particles to the cell by binding to glycan receptors, subsequent virion internalization, and cell entry. Consequently, the importance of HA makes it a primary target for the development of anti-influenza drugs. The natural oligoribonucleotides (ORNs) as well as their derivatives functionalized with D-mannitol (ORNs-D-M) possess anti-influenza properties in vitro and in vivo due to interaction with HA receptor sites. This activity suppresses the viral infection in host cells. In the present work, the complexes of ORNs and ORNs-D-M with HA protein were studied by agglutination assay, fluorescence spectroscopy, as well as molecular docking simulations. Acquired experimental data exhibited a decrease in HA titer by 32 times after incubation with the ORNs-D-M for 0.5-24 h. Quenching fluorescence intensity of the HA suggests that titration by ORNs and ORNs-D-M probably leads to changes in the HA structure. Detailed structural data were obtained with the molecular docking simulations performed for ORNs and ORNs-D-M ligands containing three and six oligoribonucleotides. The results reveal that a majority of the ORNs and ORNs-D-M bind in a non-specific way to the receptor-binding domain of the HA protein. The ligand's affinity to the hemagglutinin was estimated at the micromolar level. Presented experimental data confirmed that both natural ORNs and functionalized ORNs-D-M inhibit the interactions between HA and glycan receptors and demonstrate anti-influenza activity.

Complexes of Oligoribonucleotides with d-Mannitol Modulate the Innate Immune Response to Influenza A Virus H1N1 (A/FM/1/47) In Vivo.

Rapid replication of the influenza A virus and lung tissue damage caused by exaggerated pro-inflammatory host immune responses lead to numerous deaths. Therefore, novel therapeutic agents that have anti-influenza activities and attenuate excessive pro-inflammatory responses that are induced by an influenza virus infection are needed. Oligoribonucleotides-d-mannitol (ORNs-d-M) complexes possess both antiviral and anti-inflammatory activities. The current research was aimed at studying the ORNs-d-M effects on expression of innate immune genes in mice lungs during an influenza virus infection. Expression of genes was determined by RT-qPCR and Western blot assays. In the present studies, we found that the ORNs-d-M reduced the influenza-induced up-expression of Toll-like receptors (TLRs) (tlr3, tlr7, tlr8), nuclear factor NF-kB (nfkbia, nfnb1), cytokines (ifnε, ifnk, ifna2, ifnb1, ifnγ, il6, il1b, il12a, tnf), chemokines (ccl3, ccl4, сcl5, cxcl9, cxcl10, cxcl11), interferon-stimulated genes (ISGs) (oas1a, oas2, oas3, mx1), and pro-oxidation (nos2, xdh) genes. The ORNs-d-M inhibited the mRNA overexpression of tlr3, tlr7, and tlr8 induced by the influenza virus, which suggests that they impair the upregulation of NF-kB, cytokines, chemokines, ISGs, and pro-oxidation genes induced by the influenza virus by inhibiting activation of the TLR-3, TLR-7, and TLR-8 signaling pathways. By impairing activation of the TLR-3, TLR-7, and TLR-8 signaling pathways, the ORNs-d-M can modulate the innate immune response to an influenza virus infection.

Pre-Clinical and Clinical Efficiency of Complexes of Oligoribonucleotides with D-Mannitol against Respiratory Viruses.

Oligoribonucleotides-D-mannitol (ORNs-D-M) complexes possess antiviral, anti-inflammatory, and immunomodulatory actions. The aim of the present study was to evaluated an antiviral effect of ORNs-D-M against parainfluenza virus type 3 (PIV3); influenza CA709, PR834; avian influenza virus H5N2 (AIV) in vitro by a TCID50; hemadsorption and neuraminidase activity assays; and clinical efficiency of ORNs-D-M in patients with acute respiratory infections (ARIs) of various etiologies by PCR assay and AmpliSens test systems. It was observed that ORNs-D-M have an antiviral activity against the influenza CA709, PR834, PIV3, and AIV in vitro. The injectable dosage form of ORNs-D-M was shown to have a stronger antiviral effect compared to capsule form. It was also detected that the injectable form of ORNs-D-M significantly reduced the neuraminidase activity of influenza PR834. A complex treatment of patients with ORNs-D-M had a positive effect on the course of the disease, it accelerated patients' recovery. Treatment with ORNs-D-M caused eradication of adeno- and influenza viruses in patients with ARI. This drug contributed to significant decrease in duration of febrile period and cough. Comprehensive treatment with ORNs-D-M improved the disease clinical findings significantly. Collectively, these results suggested that ORNs-D-M may be used at co-infection with influenza and other respiratory viruses as a medical antiviral drug.

Complexes of Oligoribonucleotides with D-Mannitol Inhibit Hemagglutinin-Glycan Interaction and Suppress Influenza A Virus H1N1 (A/FM/1/47) Infectivity In Vitro.

The influenza virus hemagglutinin (HA) mediates both receptor (glycan) binding and membrane fusion for cell entry and has been the basis for subtyping influenza viruses. The oligoribonucleotides-d-mannitol (ORNs-d-M) complexes possess an anti-influenza activity in vitro and in vivo. In the present studies, we have found that ORNs-d-M interferes with hemagglutinin (HA)-glycan interaction and suppress viral infection in host cells. HA-glycan interactions were evaluated to indirectly quantify the amount of influenza virus titer by an agglutination assay. Influenza virus infectivity was determined by TCID50 assay. The direct virucidal action of the complexes was evaluated by both cytopathic effects (CPE) reduction assay and cell MTT assay. We found that ORNs-d-M hinders interaction between HA and glycan. These complexes decreased the infectivity of influenza virus and had a direct virucidal action. ORNs-d-M reduces influenza virus infectivity, affecting the HA-glycan interaction in vitro. By suppressing the influenza viral infection, the ORNs-d-M can have direct virucidal action.