Molecular Genetics, Microbiology and Virology最新文献

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative viral pathogen in the coronavirus disease 2019 (COVID-19) pandemic, which prompted an immediate global response to the development of vaccines and antiviral therapeutics. Drug repurposing in antiviral therapeutics allows for the rapid advancement of existing clinical candidates and therapies into human clinical trials to be tested as COVID-19 therapies. Screening method for drug repurposing are required to prepare the next emerging infectious diseases. In the present study, surface plasmon resonance imaging (SPRi) technique was used to find drug repositioning candidates by taking SARS-CoV2 as an example. Preventing viral entry is an effective antiviral treatment strategy used early during symptom onset. SARS-CoV-2 enters angiotensin-converting enzyme 2 (ACE2)-expressing cells when the receptor-binding domain of the spike protein on the surface of SARS-CoV-2 binds to ACE2, followed by cleavage at two cut sites by transmembrane protease, serine 2 (TMPRSS2). Therefore, a molecule capable of inhibiting the protease activity of TMPRSS2 could be a valuable antiviral therapy. We evaluated whether it was possible to detect specific binding by measuring interactions of drugs and TMPRSS2. In a result, ulinastatin bound with recombinant TMPRSS2 immobilized on biochips pH-dependently. These results suggested that SPRi can be useful technique to discover drug repositioning candidates without in vitro assay, which contributes to the rapid screening.

The spike protein of SARS-CoV-2 plays an essential role in viral pathogenesis. It binds to human cells' angiotensin-converting enzyme 2 (ACE2) receptor through the receptor-binding domain (RBD), mediating virus entry into the human host cell. The genomic changes of SARS-CoV-2 can affect its pathogenic potential, making the development of treatments and vaccines more challenging. The virus accumulates mutations to evade immune response while preserving or enhancing the binding feature to ACE2. In this study, we aimed to identify mutations in the RBD region of the spike gene from SARS-CoV-2 RNA samples taken from infected patients. We use two-step RT-PCR (cDNA synthesis followed by separate PCR amplification) reactions to amplify the RBD sequence. We aligned the sequencing data with the reference RBD sequence of the Wuhan-Hu-1 (wild-type virus) to identify the different mutations. In addition, further bioinformatic analyses were performed to evaluate the impact of the mutation(s) on the spike protein, including the prediction of protein structure and its binding affinity to ACE2. Our results show that the substitutions Y421I, Q493H, S494P, and F497S may decrease the stability of the spike protein due to the different physicochemical properties of the substituted amino acids, affecting their interactions with other RBD amino acids. Moreover, molecular docking results indicate that Q493H and S494P substitutions cause an increased binding affinity of spike protein to ACE2. At the same time, Y421I and F497S substitutions decrease the binding potential of these two proteins. Based on our findings, new mutations can cause the emergence of dangerous strains so continuous monitoring of the virus and ongoing research is crucial to prevent the spread of new, potentially dangerous strains.

Abstract

A comparative analysis of the genome of nontoxigenic and toxigenic V. cholerae O1 El Tor strains identified in Russia in 2023 and their phylogenetic analysis were performed. We used V. cholerae O1 El Tor strains isolated from patients and from an aquatic environment in 2023 in Russia. Analysis of their sequenced genomes was performed with the UGENE v.45.1 and Bandage v.0.8.1 software. To carry out SNP analysis, nucleotide sequences of the complete genomes of 63 V. cholerae El Tor strains obtained by us (41 strains) and from the NCBI GenBank database (22 strains) were used. Conventional methods were used to assess the phenotypic characteristics of strains (susceptibility to antibiotics, hemolysin and protease production, and motility). Analysis of the sequenced complete genomes of V. cholerae El Tor strains isolated from patients and from an aquatic environment in 2023 in Russia showed their genetic diversity. Toxigenic strains belonged to different types of genovariants with different mutations in the genome of transposable elements, as well as in core genes associated with pathogenicity, the ability to spread epidemically, and drug resistance. The clinical strain M3208 had a full set of altered virulence genes (ctxB7, tcpA^CIRS101, and rtxA4) and epidemicity (VSP-II^Δ) characteristic of new variants with high pathogenic potential that were widespread during the third wave of the pandemic. In addition, the resulting mutations led to the emergence of resistance to nalidixic acid (Nal^r) and the loss of resistance to polymyxin B (Pol^s). The second toxigenic strain M3210, isolated from river water, differed from the strain M3208 in the composition of mutant genes, since it had only one altered gene (ctxB). The genotype of this strain (ctxB1tcpA^EltorrtxA1VSP-II) was identical to that of previously appearing genovariants with a lower level of virulence from the second wave of the pandemic, which persisted only in several endemic regions. By contrast, the nontoxigenic isolate M3209 from a patient with acute enteric disease was devoid of not only the CTXφ prophage but also other transposable elements carrying key pathogenicity and epidemic genes. Only the deleted VPI-2 pandemic island was present in its genome. This strain was shown to produce additional pathogenicity factors (hemolysin, soluble hemagglutinin/protease, and motility), which could lead to the development of gastroenteritis in infected individuals. The genomic diversity of V. cholerae El Tor strains introduced into Russia in 2023 was revealed. Toxigenic strains were shown to be different genovariants with altered epidemically important properties. Among them, the clinical toxigenic strain M3208 was classified as a new genovariant with high pathogenic potential based on its mutant genes.

Non-coding RNAs (ncRNAs) are RNA transcribed from DNA not including any encoded protein and they generally function as a regulator of gene expression in epigenetic terms. Long non-coding RNAs (lncRNAs) are a large class of RNA transcribe with lengths longer than 200 nucleotides, whereas microRNAs (miRNAs) are a small class of transcribed RNA molecules of length 21–25 nucleotides. Both classes play important roles in a variety of cellular processes. Dysregulation of lncRNAs and miRNAs contributes to many human diseases, particularly carcinogenesis. Although lncRNA and miRNA have been largely studied and reported in several malignancies, information is still limited on the regulation network of cellular ncRNAs and virus-associated tumors, including human papillomavirus (HPV) and Epstein–Barr virus (EBV). Here, we have reviewed the regulation and correlation of lncRNAs or miRNAs with either HPV or EBV in various kinds of cancers and cell lines to better elucidate and understand carcinogenesis and the viral stage of infection-associated cancer via an epigenetic alteration. Either cellular ncRNAs or viruses could be controlled by each other where associated with cancer development and progression; they could be useful as a therapeutic target in HPV and EBV-associated cancer.

Abstract

The review presents the results of investigations of the mechanisms of action of long-used and new antituberculosis drugs, and information about new chemical compounds as potential antituberculosis drugs. Data are given on mutations in a number of infectant genes associated with or leading to bacterial resistance to these drugs. The review comprises the results of 67 publications, systematic reviews, and electronic databases.

The present study was conducted to investigate the frequency of antibiotic resistance genes and mutations and expression level of GraSR and WalKR systems associated with vancomycin non-susceptibility in methicillin-resistant Staphylococcus aureus. In 2022, a total of 200 S. aureus isolates were investigated in terms of methicillin resistance and reduced susceptibility to vancomycin. In vitro antibiotic susceptibility testing of MRSA isolates and the frequency of 12 antibiotic resistance genes were assessed using disk diffusion and PCR technique, respectively. Amino acid alteration in two-component regulatory systems including GraSR and WalKR in VISA isolates were determined by PCR-sequencing, the expression of these systems was analysed by RT-qPCR whereas agr- and spa-typing were used for molecular typing of these isolates. Overall, 125 (62.5%) isolates were methicillin-resistant and 75% were multiple-antibiotic-resistant. Vancomycin was the most efficient antibiotic against the isolates and blaZ gene was shown to be the most common (96.66%) gene among MRSA. A highly diverse amino acid substitution was observed in WalKR and GraSR in VISA and VRSA strains. Interestingly, 100% of vancomycin non-susceptible strains had the D148Q substitution in the GraR and I59L was the most common change in GraS. VISA isolates showed higher walR expression levels than the mean level of their progenitor VSSA. Agr types 1 and 3 and spa types t030, t421, t037, t325, t084 and t005 were detected among VISA isolates. The present results indicate high frequency of antibiotic resistance genes in methicillin-resistant S. aureus isolates. Furthermore, our results suggest that GraS, WalK, and WalR are important in vancomycin non- susceptibility and also these findings support the need for future surveillance studies to better elucidate vancomycin resistance in MRSA strains.

Human respiratory syncytial virus (RSV) is an RNA virus of the family Pneumoviridae and is a common causative agent of acute respiratory infections. Infection with this virus is often severe and can result in fatalities and chronic complications, particularly in high-risk groups such as children under the age of 5 and elderly individuals over 65 years old. This review presents the latest data on the taxonomy, virion structure, and genome map of RSV, as well as its role in the etiology of acute respiratory diseases. We have analyzed over 100 publications on RSV genotyping in different parts of the world including Russia published between 2015 and 2023. The genetic composition of RSV isolates in Russia is similar to that in other regions of the world, which are predominantly represented by genotypes ON1 and BA9. The development of effective vaccines is crucial for successful disease control. The review summarizes global advances in the development of interventions against RSV, emphasizing promising treatments currently undergoing clinical trials and the ones that have recently been approved. Similar interventions (vaccines) are yet to be developed in Russia, but the similarities in genetic composition of RSV in Russia and worldwide offer hope for the rapid development of domestic vaccines against this infection.

In the intensively developing field of synthetic biology, synonymous gene recoding is a leading method, which is not without drawbacks in its use. There are natural limitations in the use of codons in genes. Computer analysis in 30 genes of viruses, bacteria, archaea, and human beings was used to study the nucleotide composition, the translational code of the proteins encoded by them, and the composition of dinucleotides, dicodons, and tricodons. To identify limitations in gene coding, sequences of quantitative indicators of codons (complementarity indices (CIs) and dimension indices) were used. For each gene, limitations (particular) in the nucleotide composition, the translational code, and the composition of dinucleotides and dicodons were identified. A general limitation in gene coding is that the difference in the CIs of neighboring tricodons read with a frame shift of one codon, as happens when mRNA is translated on ribosomes, was no more than 2 in the vast majority of tricodons. Since in genes each codon is included in three sequentially read tricodons, with the exception of the first and last three codons, the CI of each codon is associated with the CIs of the two preceding and two subsequent codons. This leads to the recognition of the existence of a continuum of the codon connectivity in genes based on their CI values.

Acute viral gastroenteritis may lead hospitalization and death in individuals with underlying health conditions such as immunosuppression. Identification of pathogen causing gastroenteritis is important for appropriate treatment. It is important to demonstrate the presence of the viral nucleic acid by real-time polymerase chain reaction (RT-PCR) in the stool specimens of the patients for the laboratory diagnosis of viral gastroenteritis. The aim of this study was to determine the frequency of viral gastroenteritis and evaluate the distribution and characteristics of viral agents among patients hospitalized with gastroenteritis at a university hospital from June 2019 to December 2022. A total of 340 stool samples from 265 patients with gastroenteritis submitted to the Virology Laboratory were included in the study. The gastrointestinal syndromic (GIS) panel was performed on stool samples of the patients. Of the 340 samples, 121 (35%) were positive. Viral agents were detected in 21 (17%) of these positive samples. One hundred and seventy three samples were sent from immunocompromised patients with transplants, human immunodeficiency virus (HIV) infection, and malignancies etc. Among these, 47 (27%) samples were positive by GIS panel. When comparing the norovirus frequency versus the other viral agents, norovirus was the most viral agent detected in samples from immunocompromised patients. Immunosuppression was found as a risk factor for norovirus gastroenteritis in this study. Early diagnosis of viral gastroenteritis in immunocompromised patients is crucial to reduce transmission. Therefore, the use of a GIS panel in these patients for the diagnosis of viral gastroenteritis should become common.

Infectious diseases remain a serious problem that requires constant improvement of effective methods of therapy and prevention. Traditional vaccination methods provide a durable immune response, but require time to develop antibodies. Passive immunization with the use of antibodies offers immediate protection but is limited by the short period of antibody circulation in the body. The recombinant adeno-associated viral vector (rAAV) has low immunogenicity and the ability for prolonged gene expression in host cells, which ensures the induction of persistent immune protection and overcomes the limitations of other methods. These properties make it a promising platform for passive immunization and long-term protection against infectious agents. The use of rAAVs, which carry genes for specific neutralizing antibodies to protect against viral diseases such as influenza, coronaviruses, and HIV, demonstrate high efficacy and safety in preclinical and clinical studies. The first phases of clinical trials of rAAV preparations for the prevention and treatment of HIV infection showed their safety and good tolerability. Also, the expression of monoclonal antibodies specific to bacterial exotoxins (enterotoxin A, cytotoxin B, botulinum toxin A, etc.) in rAAVs can be used to treat the corresponding bacterial infections (clostridiosis, botulism, etc.). This review examines current studies on the use of rAAVs as an innovative strategy for the delivery and expression of genes encoding neutralizing antibodies to provide long-term protection against infectious diseases. A review of the above studies highlights the significance and prospects of the use of rAAVs in the fight against infectious diseases, as well as indicating prospects for further research in this area.