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Introduction: For four years, SARS-CoV-2, the etiological agent of COVID-19, has been circulating among humans. By the end of the second year, an absence of immunologically naive individuals was observed, attributable to extensive immunization efforts and natural viral exposure. This study focuses on delineating the molecular and biological patterns that facilitate the persistence of SARS-CoV-2, thereby informing predictions on the epidemiological trajectory of COVID-19 toward refining pandemic countermeasures. The aim of this study was to describe the molecular biological patterns identified that contribute to the persistence of the virus in the human population., Materials and Methods: For over three years since the beginning of the COVID-19 pandemic, molecular genetic monitoring of SARS-CoV-2 has been conducted, which included the collection of nasopharyngeal swabs from infected individuals, assessment of viral load, and subsequent whole-genome sequencing., Results: We discerned dominant genetic lineages correlated with rising disease incidence. We scrutinized amino acid substitutions across SARS-CoV-2 proteins and quantified viral loads in swab samples from patients with emerging COVID-19 variants. Our findings suggest a model of viral persistence characterized by 1) periodic serotype shifts causing substantial diminutions in serum virus-neutralizing activity (> 10-fold), 2) serotype-specific accrual of point mutations in the receptor-binding domain (RBD) to modestly circumvent neutralizing antibodies and enhance receptor affinity, and 3) a gradually increasing amount of virus being shed in mucosal surfaces within a single serotype., Conclusion: This model aptly accounts for the dynamics of COVID-19 incidence in Moscow. For a comprehensive understanding of these dynamics, acquiring population-level data on immune tension and antibody neutralization relative to genetic lineage compositions is essential.

The spread of COVID-19 continues, expressed by periodic wave-like increases in morbidity and mortality. The reason for the periodic increases in morbidity is the emergence and spread of novel genetic variants of SARS-CoV-2. A decrease in the efficacy of monoclonal antibodies (mAbs) has been reported, especially against Omicron subvariants. There have been reports of a decrease in the efficacy of specific antiviral drugs as a result of mutations in the genes of non-structural proteins. This indicates the urgent need for practical healthcare to constantly monitor pathogen variability and its effect on the efficacy of preventive and therapeutic drugs. As part of this study, we report the results of the continuous monitoring of COVID-19 in Moscow using genetic and virological methods. As a result of this monitoring, we determined the dominant genetic variants and identified the variants that are most widespread, not only in Moscow, but also in other countries. A collection of viruses from more than 500 SARS-CoV-2 isolates has been obtained and characterized. The genetic lines XBB.1.9.1, XBB.1.9.3, XBB.1.5, XBB.1.16, XBB.2.4, BQ.1.1.45, CH.1.1, and CL.1, representing the greatest concern, were identified among the dominant variants. We studied the in vitro efficacy of mAbs Tixagevimab + Cilgavimab (Evusheld), Sotrovimab, Regdanvimab, Casirivimab + Imdevimab (Ronapreve), and Bebtelovimab, as well as the specific antiviral drugs Remdesivir, Molnupiravir, and Nirmatrelvir, against these genetic lines. At the current stage of the COVID-19 pandemic, the use of mAbs developed against early SARS-CoV-2 variants has little prospect. Specific antiviral drugs retain their activity, but further monitoring is needed to assess the risk of their efficacy being reduced and adjust recommendations for their use.

Vaccination against COVID-19 has occurred in Russia for more than two years. According to the Russian official clinical guidelines to maintain tense immunity in the conditions of the ongoing COVID-19 pandemic, it is necessary to use booster immunization six months after primary vaccination or a previous COVID-19 contraction. It is especially important to ensure the maintenance of protective immunity in the elderly, who are at risk of severe courses of COVID-19. Meanwhile, the immunological effectiveness of the booster doses has not been sufficiently substantiated. To investigate the immunogenicity of Sputnik V within the recommended revaccination regimen and evaluate the effectiveness of booster doses, we conducted this study on 3983 samples obtained from individuals previously vaccinated with Sputnik V in Moscow. We analyzed the level of antibodies in BAU/mL three times: (i) six months after primary immunization immediately before the booster (RV), (ii) 3 weeks after the introduction of the first component of the booster (RV1), and (iii) 3 weeks after the introduction of the second component of the booster (RV2). Six months after the primary vaccination with Sputnik V, 95.5% of patients maintained a positive level of IgG antibodies to the receptor-binding domain (RBD) of SARS-CoV-2. The degree of increase in the specific virus-neutralizing antibodies level after revaccination increased with a decrease in their initial level just before the booster dose application. In the group of people with the level of antibodies up to 100 BAU/mL six months after the vaccination, a more than eightfold increase (p < 0.001, Wilcoxon criterion with Bonferroni adjustment) in the level of specific antibodies was observed (Me = 8.84 (IQR: 3.63−30.61)). A significant increase in the IgG level after receiving both the first and the second booster doses occurred at the initial titer level up to 300 BAU/ mL (p < 0.001) in those who did not contract COVID-19 in the past and up to 100 BAU/mL (p < 0.001) in those who were previously infected with SARS-CoV-2. A significant increase in the antibody level after the first dose of the booster was noted for people who had up to 500 BAU/mL (p < 0.05), regardless of the previous COVID-19 infection. Thus, revaccination is most effective in individuals with an antibody level below 500 BAU/mL, regardless of the vaccinee age and COVID-19 contraction. For the first time, it has been shown that a single booster dose of the Sputnik vaccine is sufficient to form a protective immunity in most vaccinees regardless of age and preexisting antibody level., Competing Interests: The authors declare no conflict of interest.

An important goal of COVID-19 surveillance is to detect outbreaks using modern molecular epidemiology techniques based on methods to decode the full genome of the virus, since rapidly evolving RNA viruses, which include SARS-CoV-2, are constantly accumulating changes in their genomes. In addition to using these changes to identify the different virus lines spreading in the population, the availability of sequence information is very important. It will allow the identification of altered variants that may be more transmissible, cause more severe forms of disease, or be undetectable by existing diagnostic test systems. The global scientific community is particularly interested in changes in the spike protein (S-protein, Spike) because they are responsible for binding and penetration into the host cell, lead to false-negative results in diagnostic tests, and affect transmission rates, health outcomes, therapeutic interventions, and vaccine efficacy.Genomic surveillance uses next-generation sequencing (NGS) applications and makes data on the full genome of the virus available. These methods offer new means to detect variants that differ phenotypically or antigenically. This approach promotes earlier prediction as well as effective strategies to mitigate and contain outbreaks of SARS-CoV-2 and other new viruses long before they spread worldwide.Today, molecular typing of strains is playing an increasingly important role in this process, as it makes it possible to identify samples that share a common molecular «fingerprint».

In mid-2021, the Delta strain of SARS-CoV-2 caused the third wave of the COVID-19 pandemic. Huge efforts have been devoted to studying the effect of its mutations on the effectiveness of neutralizing antibodies. Much less attention was paid to the individual features of the presentation of its peptides by molecules of the major histocompatibility complex class I (MCHC-I). In this study, the correlation of the HLA-I genotype of patients under the age of 60 years with the severity of COVID-19 caused by the two most common variants of the SARS-CoV-2 Delta strain in the summer of 2021: AY.122 and B.1.617.2 was studied. Analysis of the severity of the course of COVID-19 revealed a more severe course of the disease caused by the AY.122 variant. Comparison of the mutation profile of the two most common variants of the Delta strain showed that that the G8R mutation in the NS8 protein makes the greatest contribution to the ability of MHC-I to present viral peptides. Given that the NS8 protein is able to suppress the maturation of MHC-I molecules, the appearance of a mutation in one of its immunogenic epitopes could make a significant contribution to the prevalence of the AY.122 variant in the Russian population., (© 2022. Pleiades Publishing, Ltd.)

Numerous studies showed that diabetes mellitus (DM) increases the risk of death from COVID-19 by five times. It is generally accepted that the high lethality of COVID-19 against the background of DM is due to the main complications of this disease: micro- and macroangiopathies, as well as heart and kidney failure. In addition, it was shown that acute respiratory viral infection increases the production of interferon gamma, increases muscle resistance to insulin, and modulates the activity of effector CD8+ T cells. The ability of CD8+ T cells to recognize SARS-CoV-2-infected cells depends not only on humoral factors but also on individual genetic characteristics, including the individual set of major histocompatibility complex class I (MHC-I) molecules. In this study, the relationship of the MHC-I genotype of patients with DM aged less than 60 years with the outcome of COVID-19 was studied using a sample of 222 patients. It was shown that lethal outcomes of COVID-19 in patients with DM are associated with the low affinity of the interaction of an individual set of MHC-I molecules with SARS-CoV-2 peptides., (© 2022. Pleiades Publishing, Ltd.)

Findings collected over two and a half years of the COVID-19 pandemic demonstrated that the level immunity resulting from vaccination and infection is insufficient to stop the circulation of new genetic variants. The short-term decline in morbidity was followed by a steady increase. The early identification of new genetic lineages that will require vaccine adaptation in the future is an important research target. In this study, we summarised data on the variability of genetic line composition throughout the COVID-19 pandemic in Moscow, Russia, and evaluated the virological and epidemiological features of dominant variants in the context of selected vaccine prophylaxes. The prevalence of the Omicron variant highlighted the low effectiveness of the existing immune layer in preventing infection, which points to the necessity of optimising the antigens used in vaccines in Moscow. Logistic growth curves showing the rate at which the new variant displaces the previously dominant variants may serve as early indicators for selecting candidates for updated vaccines, along with estimates of efficacy, reduced viral neutralising activity against the new strains, and viral load in previously vaccinated patients.

WHO has declared the outbreak of monkeypox as a public health emergency of international concern. In less than three months, monkeypox was detected in more than 30 000 people and spread to more than 80 countries around the world. It is believed that the immunity formed to smallpox vaccine can protect from monkeypox infection with high efficiency. The widespread use of Vaccinia virus has not been carried out since the 1980s, which raises the question of the level of residual immunity among the population and the identification of groups requiring priority vaccination. We conducted a cross-sectional serological study of remaining immunity among Moscow residents. To do this, a collection of blood serum samples of age group over 30 years old was formed, an in-house ELISA test system was developed, and a virus neutralization protocol was set up. Serum samples were examined for the presence of IgG antibodies against Vaccinia virus ( n =2908), as well as for the ability to neutralize plaque formation with a Vaccinia virus MNIIVP-10 strain ( n =299). The results indicate the presence of neutralizing antibody titer of 1/20 or more in 33.3 to 53.2% of people older than 45 years. Among people 30-45 years old who probably have not been vaccinated, the proportion with virus neutralizing antibodies ranged from 3.2 to 6.7%. Despite the higher level of antibodies in age group older than 66 years, the proportion of positive samples in this group was slightly lower than in people aged 46-65 years. The results indicate the priority of vaccination in groups younger than 45, and possibly older than 66 years to ensure the protection of the population in case of spread of monkeypox among Moscow residents. The herd immunity level needed to stop the circulation of the virus should be at least 50.25 - 65.28%., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gushchin, Ogarkova, Dolzhikova, Zubkova, Grigoriev, Pochtovyi, Iliukhina, Ozharovskaia, Kuznetsova, Kustova, Shelkov, Zrelkin, Odintsova, Grousova, Kan, Davtyan, Siniavin, Belyaeva, Botikov, Bessonova, Vasilchenko, Vasina, Kleymenov, Slutskiy, Tkachuk, Burgasova, Loginova, Rozhdestvensky, Shcheblyakov, Tsibin, Komarov, Zlobin, Borisevich, Naroditsky, Logunov and Gintsburg.)

K(+) channels distinguish K(+) from Na(+) in the selectivity filter, which consists of four ion-binding sites (S1-S4, extracellular to intracellular) that are built mainly using the carbonyl oxygens from the protein backbone. In addition to ionic discrimination, the selectivity filter regulates the flow of ions across the membrane in a gating process referred to as C-type inactivation. A characteristic of C-type inactivation is a dependence on the permeant ion, but the mechanism by which permeant ions modulate C-type inactivation is not known. To investigate, we used amide-to-ester substitutions in the protein backbone of the selectivity filter to alter ion binding at specific sites and determined the effects on inactivation. The amide-to-ester substitutions in the protein backbone were introduced using protein semisynthesis or in vivo nonsense suppression approaches. We show that an ester substitution at the S1 site in the KcsA channel does not affect inactivation whereas ester substitutions at the S2 and S3 sites dramatically reduce inactivation. We determined the structure of the KcsA S2 ester mutant and found that the ester substitution eliminates K(+) binding at the S2 site. We also show that an ester substitution at the S2 site in the KvAP channel has a similar effect of slowing inactivation. Our results link C-type inactivation to ion occupancy at the S2 site. Furthermore, they suggest that the differences in inactivation of K(+) channels in K(+) compared with Rb(+) are due to different ion occupancies at the S2 site.

C-type inactivation of K(+) channels plays a key role in modulating cellular excitability. During C-type inactivation, the selectivity filter of a K(+) channel changes conformation from a conductive to a nonconductive state. Crystal structures of the KcsA channel determined at low K(+) or in the open state revealed a constricted conformation of the selectivity filter, which was proposed to represent the C-type inactivated state. However, structural studies on other K(+) channels do not support the constricted conformation as the C-type inactivated state. In this study, we address whether the constricted conformation of the selectivity filter is in fact the C-type inactivated state. The constricted conformation can be blocked by substituting the first conserved glycine in the selectivity filter with the unnatural amino acid d-Alanine. Protein semisynthesis was used to introduce d-Alanine into the selectivity filters of the KcsA channel and the voltage-gated K(+) channel KvAP. For semisynthesis of the KvAP channel, we developed a modular approach in which chemical synthesis is limited to the selectivity filter whereas the rest of the protein is obtained by recombinant means. Using the semisynthetic KcsA and KvAP channels, we show that blocking the constricted conformation of the selectivity filter does not prevent inactivation, which suggests that the constricted conformation is not the C-type inactivated state.

Potassium channels conduct K(+) ions selectively and at very high rates. Central to the function of K(+) channels is a structural unit called the selectivity filter. In the selectivity filter, a row of four K(+) binding sites are created using mainly the backbone carbonyl oxygen atoms. Due to the involvement of the protein backbone, site-directed mutagenesis is of limited utility in investigating the selectivity filter. In order to overcome this limitation, we have developed a semisynthetic approach, which permits the use of chemical synthesis to manipulate the selectivity filter. In this chapter, we describe the protocols that we have developed for the semisynthesis of the K(+) channel, KcsA. We anticipate that the protocols described in this chapter will also be applicable for the semisynthesis of other integral membrane proteins of interest.

Chemical synthesis is a powerful method for precise modification of the structural and electronic properties of proteins. The difficulties in the synthesis and purification of peptides containing transmembrane segments have presented obstacles to the chemical synthesis of integral membrane proteins. Here, we present a modular strategy for the semisynthesis of integral membrane proteins in which solid-phase peptide synthesis is limited to the region of interest, while the rest of the protein is obtained by recombinant means. This modular strategy considerably simplifies the synthesis and purification steps that have previously hindered the chemical synthesis of integral membrane proteins. We develop a SUMO fusion and proteolysis approach for obtaining the N-terminal cysteine containing membrane-spanning peptides required for the semisynthesis. We demonstrate the feasibility of the modular approach by the semisynthesis of full-length KcsA K(+) channels in which only regions of interest, such as the selectivity filter or the pore helix, are obtained by chemical synthesis. The modular approach is used to investigate the hydrogen bond interactions of a tryptophan residue in the pore helix, tryptophan 68, by substituting it with the isosteric analogue, beta-(3-benzothienyl)-l-alanine (3BT). A functional analysis of the 3BT mutant channels indicates that the K(+) conduction and selectivity of the 3BT mutant channels are similar to those of the wild type, but the mutant channels show a 3-fold increase in Rb(+) conduction. These results suggest that the hydrogen bond interactions of tryptophan 68 are essential for optimizing the selectivity filter for K(+) conduction over Rb(+) conduction.

The ability to selectively conduct K(+) ions is central to the function of K(+) channels. Selection for K(+) and rejection of Na(+) takes place in a conserved structural element referred to as the selectivity filter. The selectivity filter consists of four K(+)-specific ion binding sites that are created using predominantly the backbone carbonyl oxygen atoms. Due to the involvement of the protein backbone, experimental manipulation of the ion binding sites in the selectivity filter is not possible using traditional site directed mutagenesis. The limited suitability of the site-directed mutagenesis for studies on the selectivity filter has motivated the development of a semisynthesis approach, which enables the use of chemical synthesis to manipulate the selectivity filter. In this chapter, we describe the protocols that are presently used in our laboratory for the semisynthesis of the bacterial K(+) channel, KcsA. We show the introduction of a spectroscopic probe into the KcsA channel using semisynthesis. We also review previous applications of semisynthesis in investigations of K(+) channels. While the protocols described in this chapter are for the KcsA K(+) channel, we anticipate that similar protocols will also be applicable for the semisynthesis of other integral membrane proteins.

We studied the current rectification properties and selectivity of class 1 porin (PorA) from Neisseria meningitidis (strain H44/76 Delta 3 Delta 4) reconstituted in planar lipid membranes varying salt concentrations and pH. PorA channel shows voltage gating with a characteristic time remarkably longer than other porins. Its current-voltage asymmetry, evaluated as the current rectification ratio, changes nonmonotonically with salt concentration. Interestingly, it reaches its maximum value at physiological concentration. Porin selectivity, quantified by reversal potential measurements, is also significantly asymmetric. Depending on the direction of the salt gradient, the channel becomes more or less selective (10:1 vs. 5:1 Na(+)/Cl(-)). Besides, the reversal potential measurements suggest that porin inserts directionally following the concentration gradient. Measurements over a wide range of pH show that although PorA is strongly cation selective at pH >6, its selectivity gradually changes to anionic in an acidic medium (pH < 4). We show that a continuum electrodiffusion model quantitatively accounts for conductance and reversal potential measurements at positive and negative applied voltages.

The mitochondrial channel, VDAC, regulates metabolite flux across the outer membrane. The open conformation has a higher conductance and anionic selectivity, whereas closed states prefer cations and exclude metabolites. In this study five mutations were introduced into mouse VDAC2 to neutralize the voltage sensor. Inserted into planar membranes, mutant channels lack voltage gating, have a lower conductance, demonstrate cationic selectivity, and, surprisingly, are still permeable to ATP. The estimated ATP flux through the mutant is comparable to that for wild-type VDAC2. The outer membranes of mitochondria containing the mutant are permeable to NADH and ADP/ATP. Both experiments support the counterintuitive conclusion that converting a channel from an anionic to a cationic preference does not substantially influence the flux of negatively charged metabolites. This finding supports our previous proposal that ATP translocation through VDAC is facilitated by a set of specific interactions between ATP and the channel wall.

VDAC, a major protein of the mitochondrial outer membrane, forms voltage-dependent, anion-selective channels permeable to most metabolites. Although multiple isoforms of VDAC have been found in different organisms, only one isoform (porin/DVDAC) has been previously reported for Drosophila melanogaster. We have examined the physiological properties of three other Drosophila proteins (CG17137, CG17139, and CG17140) whose primary sequences have significant homology to DVDAC. A comparison of their hydropathy profiles (beta-pattern) with known VDAC sequences indicates the same fundamental folding pattern but with major insertions and deletions. The ability of these proteins to form channels was tested on planar membranes and liposomes. Channel activity was observed with varying degrees of similarity to VDAC. Two of these proteins (CG17137 and CG17140) produced channels with anionic selectivity in the open state. Sometimes channels exhibited closure and voltage gating, but for CG17140 this occurred at much higher voltages than is typical for VDAC. CG17139 was not able to form channels. DVDAC and CG17137 were able to rescue the temperature-sensitive conditional-lethal phenotype of VDAC-deficient yeast, whereas CG17139 and CG17140 demonstrated no complementation. Similar structure and channel formation indicate that VDAC-like proteins are part of the larger VDAC family but the modifications are indicative of specialized functions.