Roman S. Chernyshev

In 2023, a non-haemadsorbing African swine fever virus (ASFV) of genotype II (isolate ASFV/Kirov/WB2023/1448) was recovered from a wild boar in the Kirov Region of Russia—the first such case during the current epidemic (2007–present). Whole-genome analysis of the non-haemadsorbing ASFV revealed a large deletion (~1,300 base pairs) in the EP153R and EP402R genes, and a smaller deletion (~50 base pairs) in the MGF 110-1 L gene. Phylogenetic analysis confirmed that ASFV/Kirov/WB2023/1448 belongs to the genetic lineage «II/EU1/RU7», which is typical for the European part of Russia. Following intramuscular inoculation of eight domestic pigs with isolate ASFV/Kirov/WB2023/1448 at a dose of 10 ³ TCID ₅₀ moderate virulence was observed, with the development of acute and subacute forms of ASF. Furthermore, the non-haemadsorbing virus was shown to be contagious, as two pigs maintained in direct contact with the inoculated animals developed ASF. The mortality rate among pigs infected via the intramuscular and contact routes was 60%. Two out of ten infected pigs recovered from ASF; persistence of the non-haemadsorbing virus was not detected, as complete elimination of the virus from the host organism was observed within 28 days post-infection. Upon challenge of the recovered pigs with the highly virulent haemadsorbing isolate ASFV/Kirov/WB2023/1447—which had been recovered from the same wild boar population as ASFV/Kirov/WB2023/1448—the animals exhibited no clinical signs of disease, viraemia, virus shedding, or virus dissemination in organs and tissues. To investigate the possible circulation of the non-haemadsorbing virus in wildlife, 122 field samples collected from hunted and found-dead wild boars within a single population in Kirov Region and adjacent regions of the Russian Federation during the period 2021–2023 were examined. Two hunted animals sampled in 2023 in the Republic of Mari El (bordering Kirov Region) were found to be seropositive with no detectable ASFV. The present study may indicate the emergence of natural mechanisms of virulence attenuation of ASFV genotype II in the European wild boar population over a relatively short period of pathogen circulation (15–16 years).

Introduction. The current state of pig industry in Russia is characterized by the dominance of pig farms of III–IV compartments and a reduction in livestock in private subsidiary and peasant farms against the background of the spread of infectious diseases, mainly caused by viruses of African and classical swine fever (ASFV and CSFV), and porcine reproductive and respiratory syndrome (PRRSV). The classical method of virus cultivation in vitro is still relevant for the diagnosis of diseases, isolation of relevant strains, studying their properties, attenuation, and obtaining vaccine raw materials. The aim of the study is to select of a universal biological model for in vitro replication of ASFV, CSFV, and PRRSV based on the analysis of permissiveness levels of various cell cultures (primary cell cultures, subcultures, and continuous cell lines). Materials and methods. The study utilized known virulent, vaccine, and attenuated strains of ASFV, CSFV, and PRRSV. Five primary cell cultures, two subcultures and eleven transferable cell lines / sublines were tested. The degree of tolerance was calculated based on the results of semi-quantitative estimates using the real-time PCR method for five consecutive passages. Results. For in vitro replication of both virulent and attenuated strains of ASFV, the optimal biological models were primary cell cultures of pBMC, PAM, PL, PS, ST, and PK. For the virulent strain of CSFV, the following were optimal: PAM, PL, ST, PK, PS, RRC, and PK-15 (ATCC). For the vaccine strain of CSFV, the optimal models were synovial membrane subcultures of lamb (MSO/ARRIAH) and kid (MSC/ARRIAH), as well as ST, PAM, PL, and PS. For virulent strains of PRRSV, both American and European types, the optimal model was the primary cell culture PAM. For the attenuated strain of American-type PRRSV, PAM, PL, and MA-104 were suitable, while for the attenuated strain of European-type PRRSV, PAM, PL, and Marc-145 were optimal. Conclusion. It has been established that some primary cell cultures, such as PAM and PL, with a predominance of mononuclear cells are universal biological models for the in vitro reproduction of virulent and attenuated strains of ASFV, CSFV, and PRRSV.

Development and validation of an alternative method for isolating ASFV from biological materials from domestic pigs and wild boar in primary cultures using pig testes.Pig testis cell culture can be used to isolate viruses if donors are not available, despite lower sensitivity (4.33±0.33–5.76±0.13 lg GAdE50/cm3) compared to pig spleen cells (5.0 ± 0.12–6.0±0.14 lg GAdE50/cm3). The developed method has 100 % specificity and 90.91 % diagnostic sensitivity when isolating the ASFV from both biological material from domestic pigs and wild boars.The studies conducted have made it possible to expand the arsenal of methods for virological diagnosis of ASF. Evaluation of an alternative cell culture suitable for isolation allowed us to establish the optimal inoculation concentration of cells, at which the high titer of the ASFV can reach 6.33±0.06 lg GAdE50/cm3. The validated method of ASFV isolation allows it to be used in veterinary laboratories when donors cannot be used.

Introduction. The identification of I/II genotype recombinant African swine fever virus (ASFV) in China (2021) and its subsequent introduction to Primorsky region of Russia (2023) is a new challenge in control against ASF. The potential recombinant virus circulation may negate efforts to develop vaccines against ASF, vaccines that are based on genotype II strains) do not provide protection against recombinant virus. The aim of the study was to develop qPCR for differentiation of genotype I and new recombinant ASFV variant in clinical samples from infected domestic pigs and wild boars. Materials and methods. Samples of viral culture suspensions and biological material from pigs, ASF- free and infected with the recombinant ASFV strain Primorsky 2023/DP-4560 or genotype II strains were used. Positive control sample and standard plasmid were obtained using complex molecular cloning techniques to construct circular and linearized forms of plasmid pJET1.2_IREC. Results. Primers and a TaqMan probe (modified with LNA, locked linear nucleic acid) were designed to the fragment of MGF 110-1L gene (93 bp). The specificity of RT-PCR was enhanced by increasing the annealing temperature to 65°C. Developed method had precision (repeatability (CV = 0.7–1.3%) and reproducibility (CV = 2.76–5.69%), 100% diagnostic specificity, 96% clinical sensitivity, and high linear dynamic range (R2 = 99.62) and efficiency (E = 95%). The limit of detection was 7 copies/µL (95% CI: 4–10 copies/µL) or 70 copies/per reaction. Conclusion. The monoplex RT-PCR has been developed to study the extent of circulation in Russia of atypical variants originating from East Asia.

Introduction. Classical swine fever (CSF) remains a critical challenge in global pig production. In the Russian Federation the last reported outbreak occurred in 2020 among wild boar populations, but the risk of re-emergence is sustained. To reduce the existing threats the targeted disease surveillance and control measures are needed to be improved. Objective. To analyze the current classical swine fever situation and the outcomes of epizootic monitoring in the Russian Federation, and to develop evidence-based proposals for its improvement. Materials and methods. This analysis draws upon laboratory test results from 2020 to 2024, as recorded in the “Vesta” electronic state information system (part of the FGIS “VetIS”); epidemiological data from the World Organization for Animal Health (WOAH); and the official “Guidelines for planning laboratory testing and sampling to improve classical swine fever surveillance in the Russian Federation”, developed and approved by the Federal Centre for Animal Health. Geospatial data were visualized using the MapChart platform, while statistical analyses were performed with Microsoft Excel. Results. Drawing on international experience in disease eradication and control, this study outlines a phased approach for the eradication of classical swine fever in the Russian Federation, with a view toward achieving official recognition of disease-free status from the World Organization for Animal Health. The proposed disease surveillance strategy is comprehensive and multifaceted, comprising: early detection measures, including immediate notification of suspected cases, syndromic analysis, and clinical examinations with necropsies; routine monitoring at key control points, such as ante-mortem and post-mortem inspections; and confirmatory procedures, consisting of strategic sampling, laboratory diagnostics, and surveillance in sentinel units. The study further explores the prospects for a strategic transition, including the zoning of Russian territory, the phased discontinuation of immunization with live (attenuated) vaccines, and the potential introduction of marker vaccines. Conclusion. The proposed approaches are fully aligned with international standards and are specifically designed to achieve classical swine fever freedom in the Russian Federation. The full implementation of the proposed measures will significantly strengthen classical swine fever control in Russia and, consequently, enhance the export potential of the domestic pork industry.

African swine fever (ASF) has been a persistent threat to Eurasian pig populations since its emergence in 2007. The disease has become endemic in numerous countries, including Poland, Germany, Romania, Hungary, Italy, the Philippines, and several others. Epidemiological data reveals that over 99% of outbreaks are attributed to a highly virulent hemadsorbing virus belonging to genotype II. Traditional genotyping methods, primarily relying on the B646L gene, have faced significant limitations in providing a comprehensive understanding of virus dissemination patterns. Previous attempts to identify a universal marker for tracking virus spread through analysis of the CVR locus of the B602L gene and the I73R/I329L locus failed to produce a coherent picture of the virus’s geographical distribution across Eurasia. To address these challenges, a comprehensive study was conducted involving the analysis of 250 ASFV isolates/strains from 25 countries across Europe and Asia between 2007 and 2024. This research led to the development of a novel sub-genotyping algorithm for ASFV genotype II. The study identified four topotypes: «CAU1», «EU1», «EU2», and «ASIA1». Within these topotypes, 31 genetic lineages were detected, each characterized by specific single-nucleotide polymorphisms (SNPs). Based on the comparison of two methods of sub-genotyping Eurasian ASFVs—the classification by Gallardo C. et al. (2023) based on genetic variations of 6 loci, and the proposed classification into topotypes and genetic lineages using whole-genomes—it was established that the multigenic approach has insufficient resolution. At the same time, significant differences were observed at the level of whole-genomes. The creation of a new spatiotemporal classification has significant applications in international surveillance of ASF outbreaks, local disease monitoring, and investigation of new infection cases.

Cell cultures are an essential tool for laboratory diagnosis of porcine viral infections. However, interpreting the results requires considering the species and tissue origin of cell lines as well as the specific virus replication characteristics (cytopathic effect). This guide discusses the development of techniques for the primary isolation of viruses from biological material and provides recommendations for culturing viruses in different cell types. According to the World Organization for Animal Health, laboratory diagnosis should aim to isolate the virus in cell culture. We have studied the evolution of virus isolation methods for various diseases affecting pigs, including African swine fever virus (ASFV), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (Aujeszky’s disease, PRV), rotaviruses (RV), teschoviruses (PTVs), swine pox virus (SwPV), swine influenza A virus (IAVs), parvovirus (PPV), coronaviruses, circoviruses (PCVs), diseases with vesicular syndrome, and others. During our analysis of the literature and our own experience, we found that the porcine kidney (PK-15) cell line is the most suitable for isolating most viral porcine pathogens. For ASFV and PRRSV, the porcine alveolar macrophages (PAMs) continue to remain the primary model for isolation. These findings can serve as a starting point for virological reference laboratories to select optimal conditions for cultivating, obtaining field isolates, and strain adaptation.

The onset of a new African swine fever pandemic prompted the development of alternative approaches to national and international surveillance, control, and disease management. One such approach involved the use of molecular epidemiology methods. Because of the high degree of genetic diversity of tandem repeats (TRs) in the I73R/I329L and MGF 505-9R/10R intergenic regions of ASFV genotype II isolates, these genome markers are used in epidemiological studies. In this study, Sanger sequencing was used to analyze the I73R/I329L and MGF 505-9R/10R intergenic regions of 129 Russian ASFV isolates from 2018–2022. The variants IGR-I (3.1%) and IGR-II (96.9%) of the intergenic region I73R/I329L, as well as MGF-I (95.3%), MGF-II (3.9%), and MGF-V (0.8%) of the non-translated fragment MGF 505-9R/10R, were found among the isolates. In addition, based on PCR and Sanger sequencing of the intergenic regions and the B646L fragment, a method for differentiating ASFV genotypes I and II from a recombinant variant circulating in China, Vietnam, and Eastern Russia is proposed. Our study confirms that the I73R/I329L and MGF 505-9R/10R intergenic regions can be used as markers for epidemiological surveillance of ASFV along the borders of the Russian Federation. However, their usefulness in the study of virus circulation might be limited by the tendency for identical TR insertions to occur spontaneously in unrelated isolates from geographically remote regions of Eurasia (e.g., IGR-III in Poland and China; IGR-IV in Poland and Vietnam; MGF-V in Lithuania and Volgograd Region of Russia).

Introduction. The Lower Volga region, including the Volgograd Oblast, remains one of Russia’s most rabies-affected areas to date. Data on the genetic diversity of rabies viruses (RABVs) currently circulating in the Volgograd Oblast are insufficient, making phylogenetic analysis of RABV isolates from this region a relevant scientific objective.Objective. The study aims to conduct a phylogenetic analysis of current RABV isolates recovered from animals in the Volgograd Oblast, based on the full-length nucleoprotein gene sequence.Materials and methods. Brain tissue samples from animals diagnosed with rabies were used. The obtained nucleotide sequences of the RABV nucleoprotein gene were analyzed using the Bayesian strict molecular clock method. The spatial distribution of RABV isolates was described using Natural Earth physical map.Results. Full-length nucleoprotein gene sequencing was performed for 13 RABV isolates collected in the Volgograd Oblast. Phylogenetic analysis revealed that the RABV population in this region comprises distinct genetic variants of genetic group C, formed at different times. Genetic relationship to the isolates from Kazakhstan, Ukraine, Moldova and Central/Southern Russia indicates intensive RABV circulation in Southern European Russia. Notably, distinct virus variants were detected on the left and right banks of the Volga River.Conclusion. All studied RABV isolates collected in the Volgograd Oblast belonged to genetic group C and exhibited high genetic diversity among variants.

Introduction. The most effective strategy to control African swine fever is to implement a set of anti-epizootic measures aimed at preventing introduction and spread of the disease pathogen. Currently, there is a wide range of commercially available disinfectants used at the facilities subject to veterinary control. Their effectiveness against African swine fever virus is unknown and is only confirmed by the manufacturers, who do not always provide substantiated evidence.Objective. The objective of the research is to test virucidal activity of various disinfectants against African swine fever pathogen in the laboratory.Materials and methods. Twelve samples of disinfectants with different chemical compositions were tested. The first in vitro assessment stage was carried out using suspension method, i.e. working solutions of the tested disinfectants in experimental concentrations and exposure times were added to the liquid-phase virus-containing material. During the second stage, swabs from concrete test plates contaminated with African swine fever virus were tested following treatment of surfaces with the working disinfectant solutions. Each stage was performed in two variants: without organic contamination and with its imitation (application of inactivated bovine serum on the test surface). The samples were tested using virus isolation in a sensitive porcine spleen cell culture. Results were assessed and interpreted in hemadsorption test. The disinfectant sample was considered to exhibit virucidal activity, if no reproduction of African swine fever virus was observed.Results. Nine out of twelve tested disinfectants demonstrated a virucidal effect against reference African swine fever virus Arm 07 strain (genotype II), when tested on test surfaces. Such results suggest the need to evaluate further the efficacy of various disinfectants against this pathogen.Conclusion. The fact that such disinfectant products that are incapable of inactivating African swine fever virus under the conditions specified in their instructions are potentially marketed underlines the need to improve regulatory framework in order to ensure effectiveness of general disease prevention and control measures.