Problems of Virology

Herpes simplex virus type 1 (HSV-1), newly named as Simplexvirus humanalpha1 is one of the most common pathogens in the human population, which can cause severe disease, often with fatal outcomes. Diagnostic methods currently in use are specific and sensitive, but time-consuming, require expensive laboratory equipment and highly qualified personnel. Existing therapeutic agents have a number of significant drawbacks. To successfully treat and prevent the spread of the infection, new rapid, easy-to-use, and highly sensitive diagnostic tools and effective therapeutic agents are required. One approach to achieve this goal is CRISPR-based technology.

This review analyzes information obtained from a literature search in the Scopus, Web of Science and MedLine databases on the topics «HSV-1, structure, distribution, life cycle», «new methods for molecular diagnosis of HSV-1-infection», «classification of CRISPR-Cas systems», «nucleic acid amplification methods», «CRISPR-Cas effector proteins», «application of CRISPR-Cas systems in molecular diagnostics of HSV-1-infection», «application of CRISPR-Cas systems in therapy of HSV-1-infection». New approaches of CRISPR using effector proteins Cas12 and Cas13 in the diagnosis of HSV-1 infections are reviewed. The article discusses the progress in the development of CRISPR-Cas-based therapies against HSV-1-infection in vitro and in vivo. CRISPR gene therapy in vivo has a great clinical potential, but its safety and efficacy require further investigation. An analysis of the available data suggests that CRISPR-based technologies offer promising prospects for expanding the arsenal of diagnostic tools and antiviral drugs in the context of current and future outbreaks of viral diseases.

Introduction. Nef provides high level of HIV-1 replication due to synergy of its multiple functions and is an important factor in the pathogenesis of HIV infection. Nef is considered as a target for development of therapeutic agents. Mutations of drug resistance to dolutegravir can occur in Nef protein. Natural amino acid substitutions in Nef protein have been associated with the degree of progression of HIV infection, development of neurodegenerative and cardiovascular diseases in patients.

The aim of the study is to investigate Nef genetic diversity in HIV-1 variants circulating in Russia and in Moscow region.

Materials and methods. Total 216 Nef sequences obtained from whole blood samples of patients and 77 sequences downloaded from the Los Alamos International Database were analyzed. Consensus sequences of Nef sub-subtype A6, subtype B, CRF02_AG, CRF63_02A6, CRF133_A6B, and the reference sequence NL4-3 were compared. Genetic diversity of Nef sub-subtype A6 (Nef-A6) in patients with different stages of the disease was assessed. The presence of dolutegravir-associated drug resistance mutations in the Nef protein in HIV-1 variants circulating in Russia was also investigated.

Results. Differences in the spatial structures in consensus sequences of the studied HIV-1 variants were determined. It was shown that the conservatism of Nef-A6 in groups of patients with later stages of the disease was significantly higher. No mutations of drug resistance to dolutegravir were detected.

Conclusion. The differences in Nef sequences of HIV-1 variants circulating in Russia could affect the functional properties of the protein and could be taken into account in creating Nef-based therapies in the future. Obtained results indicate that there is no risk of resistance to dolutegravir associated with – mutations in the Nef protein. It outlines possible directions for further research into the genetic diversity of Nef.

Introduction. Differentiation of vaccine strains and field isolates of bovine type 1 herpesvirus Varicellovirus bovinealpha1 is an urgent task to improve the quality of diagnosis of infectious bovine rhinotracheitis (IBR). There are several approaches to solve this problem. The most successful methods are those proposed by R.W. Fulton and S.K. Chothe in 2013 and 2018, respectively.

The aim of the study is to test the methods proposed by R.W. Fulton and S.K. Chothe to study the possibility of their optimization and implementation in routine laboratory diagnostics of IBR in Russia.

Materials and methods. 4 vaccine strains and 6 field isolates of the IBR virus were studied using PCR-based algorithms by R.W. Fulton and S.K. Chothe to determine the presence of single nucleotide substitutions at 11 control points of the virus genome in comparison with the nucleotide sequence of the reference strain Cooper JX898220.

Results. Both methods confirmed that the domestic strains of the IBR virus used for the production of inactivated vaccines originate from field isolates of the virus. Both the reference and modern epizootic isolates obtained by us and our colleagues at different times in Russia are epizootic strains that have no direct connection with the large-scale use of foreign vaccines, including live ones, both among our own indigenous livestock and among animals imported from abroad. None of the methods we tested allows us to distinguish between Varicellovirus bovinealpha1 and Varicellovirus bovinealpha5.

Conclusion. The methods proposed by R.W. Fulton (2013) and S.K. Chothe (2018) can be used to differentiate vaccine strains and field isolates of IBR virus only after our recommended preliminary differentiation of BoHV of types 1 and 5.

Introduction. The Kurkino and Sochi viruses of species Orthohantavirus dobravaense (ODOB) are among the pathogens that cause hemorrhagic fever with renal syndrome in the European part of Russia. However, the current literature provides limited data on the distribution of genetic variants of the ODOB in Russia.

The aim is to identify ODOB in several regions of Volga, Central and Ural Federal districts of the Russian Federation and analyze their genome.

Materials and methods. Total RNA was isolated from lung tissue samples of striped field mouse (Apodemus agrarius) and yellow-necked mouse (A. flavicollis) captured in a number of areas of the Volga Federal District and neighboring regions in 2015–2023. Orthohantavirus RNA was detected by RT-PCR using specific primers to ODOB. The PCR amplification products were separated in agarose gel, purified, and subjected to Sanger sequencing. Comparative and phylogenetic analyses were performed for the sequenced genome fragments.

Results. Orthohantavirus RNA was detected in one sample of A. flavicollis from the Ulyanovsk region. Based on the analysis of the nucleotide sequences of the sequenced PCR products, it was found that the highest values of similarity were obtained when comparing the identified strain with the reference Kurkino virus from the Tula region. The data from the phylogenetic analysis of the sequenced fragment of the S and M segments allowed us to establish that the identified isolate is closely related to the Kurkino virus found earlier in A. agrarius in the Tula region. Thus, the detected isolate was identified as variant of Kurkino virus, which is also widespread in the central regions of Russia, Wetern Siberia and close related to genome variants that are distributed in Central Europian countries.

Conclusions. It is proved for the first time that: 1) the range of Orthohantavirus dobravaense (Kurkino virus) extends to a part of the territory of the Volga Federal District; 2) Orthohantavirus dobravaense (Kurkino virus) and Orthohantavirus puumalaense (Puumala virus) are co-circulating in the Ulyanovsk region.