Accumulated experience and future prospects of <i>in vivo</i> hepatitis B virus research

Nowadays, an estimated more than 300 million people live with hepatitis B virus (HBV) infection globally. One of the main goals of the World Health Organization (WHO) is to eliminate viral hepatitis by the year 2030. The study of the pathogenic and immunologic properties of HBV, as well as therapeutic substances and treatment regimens, is significantly complicated by the insufficient number of susceptible biological test subjects (animal models) and the lack of zoonotic reservoirs of the virus. In this regard, researching the properties of HBV and related hepadnaviruses provides invaluable material for understanding the biology of the pathogen and the developing methods of prevention and control of this chronic infectious disease, leading to severe hepatopathies (cirrhosis and hepatocellular carcinoma). Furthermore, prolonged HBV viremia leads to depletion of the immune system, reducing resistance against pathogens of other infections, especially those with a chronic course and socially determined spread. The aim of this research is to evaluate existing animal models of HBV infection in the context of pathogenesis, immunologic and pathomorphological features. For the first time, the hypothesis of the possible use of certain models for the research of HBV-associated socially significant infections is considered from the point of view of the development of pathomorphological features. To complete this review, we analyzed the information about the features of HBV infection models in vivo, published over the last 25 years in open sources (Web of Science, PubMed, Scopus, ScienceDirect, Springer). The main criteria for literature selection were the type of infecting agent, the observed immunologic features of the course of the infectious process and the availability of a description of the pathomorphological features in model organisms

Anatomical and physiological aspects of the HIV infection pathogenesis in animal models

Understanding the entire pathogenesis of HIV infection, from penetration at the gates of infection to the induction of severe immunodeficiency, is an essential tool for the development of new treatment methods. Less than 40 years of research into the mechanisms of HIV infection that lead to the development of acquired immunodeficiency syndrome have accumulated a huge amount of information, but HIV's own unique variability identifies new whitespaces. Despite the constant improvement of the protocols of antiretroviral therapy and the success of its use, it has not yet been possible to stop the spread of HIV infection. The development of new protocols and the testing of new groups of antiretroviral drugs is possible, first of all, due to the improvement of animal models of the HIV infection pathogenesis. Their relevance, undoubtedly increases, but still depends on specific research tasks, since none of the in vivo models can comprehensively simulate the mechanism of the infection pathology in humans which leads to multi-organ damage. The aim of the review was to provide up-to-date information on known animal models of HIV infection, focusing on the method of their infection and anatomical, physiological and pathological features

Sequence analysis of the non-coding control region of John Cunningham virus isolates from patients with multiple sclerosis treated with natalizumab

Introduction. The John Cunningham virus (JCPyV) causes a fatal demyelinating disease of the central nervous system known as progressive multifocal leukoencephalopathy (PML). In healthy people, the JCPyV non-coding control region (NCCR) is not rearranged, while NCCRs in immunocompromised patients are characterized by frequent rearrangements and can be associated with PML development. Therefore, patients treated with natalizumab, which decreases the migration of leukocytes and monocytes through the blood-brain barrier to inflammatory foci, are at increased risk of developing PML. The purpose of the study was to analyze NCCR sequences of JCPyV isolates from patients with multiple sclerosis (MS) treated with natalizumab. Materials and methods. A total of 26 blood plasma samples and 8 cerebrospinal fluid samples were analyzed using nested PCR to study the JCPyV NCCR structure in Russian MS patients treated with natalizumab. The NCCRs present in the samples were cloned and sequenced by Sanger sequencing. All the JCPyV NCCR sequences were compared with the archetype sequence and mapped. The NCCR sequences were also examined for presence of putative transcription factor binding sites. Results. A total of 48 NCCR sequences were found. The analysis showed that up to 55% of NCCRs were identified as rearranged NCCRs, while the other were archetype-like NCCRs. All the sequences can be divided into 6 types with one dominant rearrangement pattern. This rearranged NCCR was also found in a patient with the confirmed PML diagnosis and a poor prognosis. All the rearranged NCCRs were characterized by the presence of additional transcription factor binding sites. Conclusion. The study has helped identify previously unknown NCCR patterns typical of MS patients treated with natalizumab in Russia, thus confirming the need for the further research on NCCR rearrangements in MS patients undergoing natalizumab treatment to gain better understanding of the origin of neurovirulent JCPyV variants

Studying the genetic diversity of the varicella-zoster virus in selected regions of the Russian Federation using high-throughput sequencing

Introduction. Varicella-zoster virus (VZV), the causative agent of the disease of the same name and herpes zoster, is phylogenetically divided into 8 clades, the distribution of which is characterized by geographic reference to certain regions of the world. For most countries, VZV clades circulating in their territories have been identified, however, such information is almost unavailable for Russia. The purpose of the study is to develop an effective method for VZV typing using high-throughput sequencing technologies to identify the prevalence of various VZV clades in Moscow, Moscow Region, and Stavropol Territory. Materials and methods. To genotype VZV, it is enough to refer to 7 nucleotide positions. Their unique combinations can be used to assign the virus to one of the clades. Short sections of nucleotide sequences of open reading frames were obtained using a developed set of primers. Results. A VZV genotyping technique has been developed and optimized. Using this technique, primary data on the distribution of VZV clades in the studied regions have been obtained. Thus, it has been established that in Moscow and a number of other regions, the 1st, 3rd, and 5th clades of VZV are predominantly distributed. Conclusion. The developed technique, including a primer panel and a genotyping algorithm, allows VZV typing in a short time while reducing specimen preparation costs and simultaneously increasing the number of specimens in one sequencing cycle. The results obtained using this assay allow us to assume that in Moscow, Moscow Region, Stavropol Territory, VZV, clades 1, 3, and 5 are the most represented ones. To confirm this hypothesis, it is necessary to include a larger number of clinical specimens in subsequent studies, including from other regions of the country

The Russian database of HIV antiretroviral drug resistance

The development of sequencing technologies and bioinformatic analysis made it possible to conduct molecular and epidemiological studies, in which nucleotide sequences of the human immunodeficiency virus (HIV) are used as information added to the patient profile. From a practical perspective, studies of prevalence of HIV drug resistance (HIVDR) are of the highest significance. To promote such studies, different countries use databases that serve as repositories of genetic and epidemiological information. The Russian HIVDR database (https://hivresist.ru/) was created in 2009. Nevertheless, it was characterized by limited applicability for a long time. Since 2021, after the regulatory documents had been revised and updated, the entry of HIVDR research results into the Russian HIVDR database has been mandatory. Therefore, the priority attention has been given to upgrading the database and improving its functional capabilities. Different methods have been developed to enter clinical, epidemiological and genetic data. At the time of this study, the Russian database HIVDR contained 10,626 unique records about patients and 13,126 nucleotide sequences deposited by 10 institutions. The following functions have been provided for data analysis: quality control of the epidemiological and clinical information about a patient, quality control of nucleotide sequences, contamination check, subtyping, detection of DR mutations, identification of viral tropism and generation of standardized reports. The efforts toward further development of the Russian HIVDR database will be focused on designing tools for detection and analysis of molecular clusters, adaptation to routine application for epidemiological surveillance of HIV infection

Production of Bst polymerase for diagnosis of different infections using loop-mediated isothermal amplification

Introduction. The large fragment of DNA polymerase I from Geobacillus stearothermophilus GIM1.543 (Bst DNA polymerase) possesses 5'-3' DNA polymerase activity, 5'-3' displacement activity and high processivity. These properties make it possible to use Bst DNA polymerase in loop-mediated isothermal amplification (LAMP), which provides highly specific amplification of the target sequence and is used for rapid detection of agents causing human infectious diseases. The purpose of the study was to produce a recombinant Bst polymerase enzyme in the bacterial expression system and to assess its properties for LAMP-based diagnostics of infectious diseases. Materials and methods. Expression constructs carrying the Bst polymerase gene were obtained using genetic engineering techniques. Different Escherichia coli strains were used for protein expression. Metal-chelate and gel filtration chromatography techniques were used for protein purification. Catalytic characteristics of the enzyme were assessed in loop-mediated isothermal amplification reactions using AmpliSens SARS-CoV-2-IT, AmpliSens IAV-IT and AmpliSens IBV-IT diagnostic systems designed for high-quality detection of SARS-CoV-2, influenza A virus (IAV) and influenza B virus (IBV) RNA, respectively. Results. The offered protocol for production, extraction and purification of recombinant Bst polymerase makes it possible to produce the enzyme in the bacterial expression system using E. coli cells in a soluble form and reaching the yield up to 20% of the total cell mass. In LAMP reactions, the obtained enzyme demonstrates activity comparable with that of the commercial enzyme Bst 2.0 (NEB). Conclusion. Considering the fast purification and production of the enzyme, the obtained recombinant Bst polymerase can be used in LAMP-based diagnostic kits

Results of screening for antibodies to varicella-zoster virus in healthcare workers of a multidisciplinary hospital in Moscow

Introduction. Given the unfavorable epidemic situation with chickenpox and shingles in Russia, there is a high risk of virus introduction and spread in healthcare settings, including among medical staff who are not immune to varicella zoster virus (VZV). The objective of this study is to assess the immunity of employees of a multidisciplinary hospital in Moscow to VZV. Materials and methods. A selective screening study was carried out. Venous blood serum samples were taken from 1546 hospital employees as material for detection of IgG antibodies to VZV antigens using a commercial solid-phase enzyme immunoassay (ELISA) test system "Vecto VZV-IgG". All employees were questioned to obtain information about their infectious and vaccine history in relation to VZV. Results and discussion. Screening for antibodies to VZV in the hospital workers revealed that 6.3% of those workers are not immune to VZV. The proportion of seronegative individuals was the highest (12.6 ± 2.4%) in the age group of 29 years and younger. VZV seronegative healthcare workers were found in various departments, but the presence of non-immune individuals among the staff of the obstetrics and gynecology departments (6.5%) is of epidemiologic concern. The results of the survey showed that documented data on infection and vaccination history cannot be used to assess the protection of healthcare workers against VZV infection. Conclusion. The results of serologic screening for antibodies to VZV made it possible to identify a significant number of susceptible employees of the multidisciplinary hospital. In order to prevent the formation of multiple epidemic foci of varicella in medical organizations, it is advisable to include anti-VZV testing of medical staff in the state prevention programs with subsequent vaccination of non-immune individuals

SARS, SARS again, and MERS. Review of animal models of human respiratory syndromes caused by coronavirus infections

Since the beginning of the 21th century, major outbreaks of human respiratory syndromes caused by coronavirus infections have caused more than million deaths on the planet. Despite the fact that the first wave of the coronavirus infection took place back in 2002, even now there is not any adequate animal model that would meet the needs of the scientific community for reproducing the pathogenesis, clinical manifestations, immunogenicity, development and testing of preventive and therapeutic compounds specific to Severe Acute Respiratory Syndrome, Middle East Respiratory Syndrome, and Coronavirus Disease 2019 (COVID-19).The purpose of the study is to provide relevant information on known animal models of human respiratory syndromes caused by coronavirus infections and to focus the reader's attention on their adequacy, which consists in the most accurate imitation of clinical signs and pathomorphological changes

Characterization of Streptococcus pneumoniae strains causing invasive infections using whole-genome sequencing

Purpose: antigenic and genetic characterization of Streptococcus pneumoniae strains isolated from patients with invasive forms of pneumococcal infection using whole-genome sequencing.Materials and Methods. The study was performed on 46 S. pneumoniae strains isolated during the PEHASus multicenter studies in 2015-2018. Sequencing was performed using Illumina protocols and equipment. The SPAdes, SeroBA, PneumoCaT software were used for data processing, as well as BIGSdb software (PubMLST.org).Results and Discussion. Whole-genome sequences of strains were obtained; the information was entered into the PubMLST database (id: 51080-51125). Ten (21%) strains were found to have serotype 3. Five (11%) strains belonged to serotype 19F and five to serogroup 6; two of them belonged to serotype 6A; one strain had 6B and 1 had 6BE serotype; 1 strain showed discordant result (6A or 6BE). Serotype 15B was identified in 3 (6.5%) strains. Serotypes 7F, 8, 9V, 14, 22F, 23F and 28A were identified in two strains each; serotypes 1, 4, 9N, 10C, 12F, 18C, 35F, 37 and 38 were found once. The proportion of strains with serotypes included in PCV13 and PPV23 vaccines was 65% and 80%, respectively. 36 sequence types were found in strains; out of them, 6 sequence types were found for the first time. A dominant sequence type or clone complexes could not be identified using multilocus sequence typing except for serotype 3 strains. The inability to identify clonal complexes is in congruence with the previously obtained data on the absence of S. pneumoniae clones associated with pneumococcal meningitis in Russia.Conclusion. The information about serotypes of S. pneumoniae causing invasive infections together with epidemiologic data about strain sources and vaccination allows us to evaluate the effectiveness of pneumococcal vaccines and provide information for improving the PCR-based routine serotyping

Epidemiological significance of detection of SARS-CoV-2 RNA among different groups of population of Moscow and Moscow Region during the COVID-19 outbreak

The Central Research Institute of Epidemiology of Rospotrebnadzor presents priority data obtained from the largescale population PCR-based study of the changes in the rates of circulation of SARS-CoV-2 among relatively healthy residents of Moscow and Moscow Region