African swine fever virus (Asfarviridae, Asfivirus) strains from the central regions of Russia, carrying variant 5 of the central variable region (CVR), are characterized by tandem duplication in the intergenic region MGF 360-13L – MGF 360-14L

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Introduction

African swine fever (ASF) is a contagious viral disease of pigs and wild boars that has spread widely throughout the world since 2007, causing significant economic damage. The lack of a reliable vaccine, as well as the identified trend towards a decrease in virulence and an increase in the infectivity of the ASF virus (Asfarviridae, Asfivirus, African swine fever virus) [1] make it difficult to control outbreaks of the disease. This points to the necessity for further in-depth study of the spatial and temporal circulation of the virus, changes in its genome, and the characteristics of genetic lines. In this regard, it seems important not only to classify the samples obtained according to known schemes, but also to perform their whole-genome analysis for the possible detection of previously undescribed regions that have potential phylogenetic significance.

To classify ASF virus strains of genotype II circulating in Eurasia, subgenotyping schemes based on a set of marker sites are currently used [2–4]. The central variable region (CVR) in the B602L gene is one such phylogenetic marker with regional resolution [2–5]. Since 2013, three CVR variants have been registered in the central regions of Russia: CVR-I, CVR-V, and CVR-VI [4, 6], but there are discrepancies between the frequency of occurrence of different CVR variants and the proportion of described whole-genome sequences carrying these variants. Since 2016, the frequency of detection of the CVR-V single nucleotide polymorphism (SNP) has increased [4, 6], but so far only one complete genome containing this SNP has been described [7]. It is of interest to analyze a larger number of whole-genome sequences carrying the CVR-V polymorphism to better understand the spread of the virus, its variability, and to identify additional markers of diversity.

The aim of the study is to perform whole-genome sequencing, subgenotyping, and bioinformatics analysis of previously unstudied ASF virus isolates obtained in the Lipetsk, Penza, and Tambov regions in 2016–2021, and to identify additional markers of diversity within the genetic lineage.

Materials and methods

The study analyzed ASF virus sequences from samples obtained during three ASF outbreaks in the Russian Federation in the Lipetsk (2016), Penza (2019), and Tambov (2021) regions. The obtained genomes were published in GenBank under numbers PX488473–PX488476.

DNA isolation. DNA was isolated from tissue samples of domestic pigs from pig farms. The presence of ASF virus in the samples had previously been confirmed by polymerase chain reaction (PCR) (ASF test system form 4, AmpliSens, Russia).

Viral DNA enrichment was not performed. The tissue samples were homogenized, then a 10% suspension was prepared with 800 μL of sterile saline. The samples were centrifuged at 200g for 1 min and the supernatant was used for extraction using the HiPure Blood DNA mini kit (Magen, China). DNA quality was confirmed by measurement with a nanophotometer (Implen); the A260/A280 ratio was in the range of 1.65–1.90. The concentration of the isolated DNA was measured using a Qubit fluorometer (Applied Biosystems, Thermo Fisher Scientific, USA) and the HS QuDye dsDNA assay kit (Lumiprobe, Russia). It was 8–10 ng/μL.

Sanger sequencing. Prior to whole-genome sequencing, several regions (the C-terminal region of the p72 protein, the central variable region in the B602L (CVR) gene, and the region located between the I73R and I329L genes) were sequenced using Sanger sequencing with an AB3500 genetic analyzer. For this purpose, total genomic DNA was extracted using the MagnoSorb kit (AmpliSens, Russia) on the KingFisher Flex system (Thermo Fisher Scientific, USA). DreamTaq PCR Master Mix (2X) (Thermo Fisher Scientific, USA) was used to amplify each primer pair. The ExoSAP-IT kit (Thermo Fisher Scientific, USA) was used to purify PCR reaction products, the BigDye Terminator v. 1.1 kit (Thermo Fisher Scientific, USA) was used for PCR analysis, and the BigDye XTerminator kit (Thermo Fisher Scientific, USA) was used for post-PCR purification.

DNA library preparation and high-throughput sequencing. For sample preparation, 200 ng of DNA was used with the MGIEasy FS DNA library preparation kit (MGI Tech, China) according to the manufacturer’s instructions. The resulting DNA library was sequenced using the DNBSEQ-G50RS high-throughput sequencing kit (FCL PE100, China) on a DNBSEQ-G50 instrument according to the manufacturer’s instructions.

Data analysis. Sanger sequencing data were analyzed using CLC Main Workbench 20 (Qiagen, Netherlands).

Whole-genome sequencing data were processed to remove adapters and low-quality reads. The processed pairs were then aligned to the Sus scrofa reference genome (GenBank accession number GCA_000003025.6) to remove host data using CLC Genomics Workbench 22 (Qiagen, Netherlands).

Unmatched reads were collected and filtered for quality using Trimmomatic v. 0.39 [8] to remove or trim the ends of low-quality reads. After that, read errors were corrected in SPAdes v. 4.2.0 [9]. Based on the corrected reads, genomes were assembled by aligning short reads to the Georgia 2007/1 reference sequence using the BWA-MEM v. 0.7.17-r1188 algorithm on the UGENE v. 52.1 platform [10]. To search for variations and create a consensus using the BWA-MEM method, the assembly was analyzed using the bcftools v. 1.22 package [11], after which the resulting consensus sequence was manually verified. To determine open reading frames (ORFs) using GATU software [12], the Georgia 2007/1 reference strain was used.

Multiple alignment of the obtained virus sequences with 67 genomes downloaded from GenBank (See Appendix 1 in the supplementary files of the article on the journal site: https://doi.org/10.36233/0507-4088-357-1) was performed using MAFFT v. 7.526 [13]. Phylogenetic analysis of the isolates was performed using Mega 11 [14] according to the recommended Tamura (T92) model with optional gamma distribution (G) parameters and consideration of invariant sites (I) selected based on the calculation of the Bayesian coefficient (BIC = 524,795.816) and the adjusted Akaike coefficient (AICc = 523,469.554). Phylogenetic trees were constructed using the Maximum Likelihood (ML) method with Bootstrap support of 1000 repetitions.

Molecular-epizootological maps were created in Evergis Online Map.

Authors confirm compliance with institutional and national standards for the use of laboratory animals in accordance with “Consensus author guidelines for animal use” (IAVES 23 July 2010). The research protocol was approved by the Ethics Committee of the institution Cherkizovo Research and Testing Center LLC (Protocol No. 1 dated 04 March 2025).

Results

As expected, preliminary Sanger sequencing of all isolates identified genotype II according to the generally accepted classification.

All isolates had a glutamic acid substitution for lysine at position 201 of the B602L protein and were therefore classified as CVR-V [6]. Also, all IGR I73R/I329L sequences obtained by Sanger method included a tandem repeat corresponding to variant II among the known IGR variants [15].

The lengths of the obtained genomes were 190,596 bp for ASFV_Lipezk-2016, 190,589 for ASFV_Penza-2019a, 190,596 for ASFV_Penza-2019b, and 190,592 for ASFV_Tambov-2021.

Cluster analysis was performed according to the supplemented scheme for subgenotyping ASF virus genotype II (modification of the subgenotype classification by C. Gallardo et al., 2023)¹ [2]. The presence of a specific SNP at position 27009 (MGF 360-10L III polymorphism) allowed the sequences to be assigned to the Georgia-2007 clade of the “Russia” genetic lineage according to the above scheme. To date, this combination of MGF 360-10L III and CVR-V has been found only once, in the ASFV/Ulyanovsk/19WB-5699 isolate [7]. The other known cases of CVR-V variant detection were not based on whole-genome sequencing, but on Sanger sequencing of CVR regions [4, 6].

Based on 67 complete ASF virus sequences downloaded from GenBank and the four genomes described above, a phylogenetic analysis was performed, the results of which are shown in Fig. 1. The MAD/01/1998 genotype strain isolated in Madagascar in 1998 was used as an outgroup. The results classify the isolates under consideration as belonging to the Georgia 2007 clade, which is characterized by close relatedness to the Georgia 2007/1 strain and relatively early isolation [16]. The highest homology was observed with the ASFV/Ulyanovsk/19WB-5699 isolate. The data obtained confirm the results of subgenotyping.

 

Fig. 1. Phylogenetic tree constructed based on 71 complete genome sequences of ASFV, genotype II.

The isolates covered by this research are labeled with ▀.

Рис. 1. Филогенетическое древо, построенное на основании 71 полногеномной последовательности вируса АЧС, II генотип.

Изоляты, полученные в данном исследовании, отмечены ▀.

 

Three insertions, one reading frame shift, and 22 SNPs relative to the reference sequence were found in the genomes described, of which five SNPs and two insertions are unique (Table). The polyC, polyG, and variation regions at the ends of the genome are considered unreliable and were therefore not taken into account.

 

Table. Variations in the complete genome sequences of the ASF virus of the studied isolates

Таблица. Вариации полногеномных последовательностей вируса АЧС исследованных изолятов

Position in Georgia-2007/1 Позиция в Georgia-2007/	Gene / intergenic region Ген / межгенная область	Georgia 2007/1	Lipezk-2016	Penza-2019a	Penza-2019b	Tambov-2021	Changes in amino acids Изменения в аминокислотах
2153*	MGF 360-1La	A	A	C	C	C	C > G
2161*	MGF 360-1La	A	A	GTAC	GTAC	GTAC	M > ST
12568	G ACD 00190	A	–	–	–	–	Reading frame shift Сдвиг рамки считывания
15320*	MGF 110-13La	A	A	G	G	G	–
27009	MGF 360-10L	A	G	G	G	G	–
28434*	MGF 360-11L	T	T	A	A	A	Q > H
32767–32768*	IGR MGF 360-13L – MGF 360-14L	–	CAGTCTATAAGA	CAGTCTATAAGA	CAGTCTATAAGA	CAGTCTATAAGA	–
39215	MGF 505-5R	G	A	A	A	A	–
41616*	IGR MGF 505-6R – MGF 505-7R	T	T	A	A	A	–
45002	MGF 505-9R	C	T	T	T	T	H > Y
62387	F1055L	G	A	A	A	A	–
63081*	F1055L	G	G	A	G	G	–
63729	F1055L	A	G	G	G	G	–
69509	EP1242L	G	A	A	A	A	–
90001	IGR C62L – C962R	T	T	С	С	C	–
98321*	B438L	T	C	T	T	T	–
102668	B602L	C	T	T	T	T	E > K
103309	IGR B602L – B385R	G	–	–	–	–	–
120769	CP2475L	C	T	T	T	T	–
143368	D1133L	G	G	A	A	A	P > S
156891	H240R	G	A	A	A	A	R > H
157890	R298L	C	T	T	T	T	–
158225	Q706L	G	G	A	A	A	A > V
167129	E199L	T	G	T	T	T	Q >H
167188	E199L	C	C	C	G	C	A > P
173408–173409	IGR I73R – I329L	–	GAATATATAG	GAATATATAG	GAATATATAG	GAATATATAG	–
182971	I9R	A	G	G	G	G	K > E

Note. * – the positions of previously undescribed polymorphisms are marked.

Примечание. * – позиции ранее не описанных полиморфизмов.

 

The greatest similarity of all isolates studied is observed with ASFV/Ulyanovsk/19WB-5699. These are 7 synonymous substitutions at positions 27009, 39215, 62387, 63729, 69509, 120769, and 157890, a deletion at position 103309 in the intergenic region B602L – B385R, as well as 4 non-synonymous SNPs leading to the replacement of histidine with tyrosine at position 465 of MGF 505-9R, glutamic acid to lysine at position 201 of the B602L gene, arginine to histidine at position 186 of the H240R gene, and lysine to glutamic acid in the I9R gene.

The close relationship between ASFV/Ulyanovsk/19/WB-5699 isolates from Penza and Tambov is also confirmed by transitions at position 90001 in IGR C62L – C962R, proline to serine substitution at position 457 of the D113L gene, and alanine to valine substitution at position 607 of the Q706L gene.

In this study, we discovered for the first time an insertion of 12 nucleotides CAGTCTATAAGA in IGR MGF 360-13L – MGF 360-14L in all sequenced samples, resulting in a tandem repeat (Fig. 2). Sanger sequencing of this region confirmed the presence of the insertion. Furthermore, based on multiple alignment data and the fact that tandem repeats are poorly mapped when assembled using standard methods, a similar repeat may be present instead of an adenine insertion at the same position in the ASFV/Ulyanovsk/19/WB-5699 isolate.

 

Fig. 2. Insertion in IGR MGF 360-13L – MGF 360-14L. Clade Georgia 2007.

The isolates covered by this research are labeled with *.

Рис. 2. Инсерция в IGR MGF 360-13L – MGF 360-14L. Клада Georgia 2007.

Изоляты, полученные в данном исследовании, отмечены *.

 

Unique polymorphisms at positions 2153 and 2161 in the ASFV_Penza-2019a, b and ASFV_Tambov-2021 samples lead to the replacement of cysteine with glycine at position 165 and methionine with threonine and serine at position 162 of the predicted MGF 360-1La protein, respectively. Synonymous SNPs at positions 15320 and 41616, as well as the replacement of glutamine with histidine at position 22 of MGF 360-11L, are also described for the first time for the outbreaks in Penza and Tambov.

Discussion

According to the methodological recommendations developed by the Federal Center for Animal Health (ARRIAH) (Russia)¹, the Georgia 2007 clade of the “Russia” genetic lineage includes those variants of the virus in which SNP A>G is present at position 27009 (the MGF 360-10L gene was proposed as a possible marker in [7], the Georgia 2007 clade was defined in [16]). These are isolates Kashino 04/13, ASFV/POL/2015/Podlaskie, ASFV/LT14/1490, ASFV/Ulyanovsk/19/WB-5699, and those described by us, ASFV_Lipezk-2016, ASFV_Penza-2019a, ASFV_Penza-2019b, and ASFV_Tambov-2021 (Fig. 3).

 

Fig. 3. Markers that allow grouping of isolates within the Russia lineage of the Georgia 2007 clade.

Sequences united by the presence of SNPs MGF 360-10L III; CVR-I, CVR-V and CVR-VI are shown; A – CAGTCTATAAGA insertion in IGR MGF 360-13L – MGF 360-14L; B – T>C substitution at position 90001 (IGR C62L – C962R); C – G>A transition at position 143368 (D1133L); D – G>A transition at position 158225 (Q706L). The isolates covered by this research are labeled with ▀.

Рис. 3. Маркеры, группирующие изоляты внутри линии «Россия» клады Georgia 2007.

Показаны последовательности, объединенные наличием ОНП MGF 360-10L III; CVR-I, CVR-V и CVR-VI; A – инсерция CAGTCTATAAGA в IGR MGF 360-13L – MGF 360-14L; B – замена T>C в позиции 90001 (IGR C62L – C962R); C – транзиция G>A в позиции 143368 (D1133L); D – транзиция G>A в позиции 158225 (Q706L). Изоляты, полученные в данном исследовании, отмечены ▀.

 

Within this lineage, several subgroups can be distinguished based on the CVR sequence. Since 2013, three CVR variants have been described in the central regions: CVR-I, CVR-V, and CVR-VI [4, 6]. CVR-I (identical to the reference sequence) is the most common. CVR-VI polymorphism (A>T substitution at position 459 of the B602L gene) was described for isolates from the Tver, Moscow, and Vladimir regions from 2013 to 2016 and is currently not detected. Although the prevalence of the CVR-V variant (C>T at position 601 of the B602L gene), described only for the central regions of Russia, has increased significantly over time [4], only one complete genome containing CVR-V and CVR-VI has been described so far: ASFV/Ulyanovsk/19WB-5699 and Kashino 04/13, respectively.

The ASFV/POL/2015/Podlaskie and ASFV/LT14/1490 strains, which are closely related and were isolated in Eastern Europe, have the CVR-I variant. However, they do not cluster with other strains with CVR-I circulating in Russia since 2013 and only carry the original CVR variant.

The other two subgroups identified in central Russia differ in terms of CVR-V and CVR-VI variants, which are not found among representatives of other genetic lines. It should be noted that the authors who described these polymorphisms and analyzed their distribution in Russia worked mainly with partial sequences of the B602L gene rather than complete genomes, describing 6 isolates containing CVR-VI and 26 isolates containing CVR-V [4, 6].

CVR-V, circulating in central Russia, the Volga region, European South, and part of the Urals between 2016 and 2022 [4, 6], has a non-synonymous substitution of glutamic acid for lysine at position 201 of the B602L protein. This variant was present in the ASFV/Ulyanovsk/19WB-5699 strain [7] and in all the sequences we isolated. The appearance of CVR-V and CVR-VI variants in isolates of the “Russia” lineage after the formation of MGF 360-10L-III, which characterizes this lineage, suggests that these variants were formed within this lineage and that all sequences with these polymorphisms belong to it.

To differentiate genomes with the CVR-V variant circulating in central regions of Russia, four sites with phylogenetic potential are proposed. The grouping of isolates using the proposed markers is shown in Fig. 3. The first site is an additional copy of the CAGTCTATAAGA sequence in IGR MGF 360-13L – MGF 360-14L, which we found and which leads to the formation of a tandem duplication. In the reference genome, there are four more similar sequences 9–12 nucleotides long on both sides of the duplicated region, one of which is identical to the insertion (Fig. 2). These regions probably arose as a result of duplication events and then changed independently. The ASF virus is characterized by variation in the number of tandem repeats both within genes and in non-coding regions [17]. In [18], a mechanism of chain slippage during replication, known for bacterial genomes [19], was proposed for the formation of indels in poxviruses. In this case, even if the repeat is not completely identical, the template and emerging strands shift, and duplication occurs. As a result, there are at least three copies on the new strand. On the other hand, there is an opinion that indels are significantly determined by homologous recombination events [20].

The CAGTCTATAAGA duplication is present (ASFV_Lipezk-2016, ASFV_Penza-2019a, ASFV_Penza-2019b, and ASFV_Tambov-2021), or is presumed to be present (ASFV/Ulyanovsk/19/WB-5699) in the analyzed strains of the Russia lineage of the CVR-V subgroup. In our opinion, this insertion may serve as a potential marker of diversity for a more detailed classification of ASF virus within the Russia genetic lineage. Sequencing of additional regions or whole-genome sequencing of previously selected and new ASF isolates could provide greater clarity on the extent of this insertion.

The following three sections are common transitions for four isolates: ASFV/Ulyanovsk/19/WB-5699, ASFV_Penza-2019a, ASFV_Penza-2019b, and ASFV_Tambov-2021, which are linked by the geographical proximity of their isolation sites in central Russia and a short time interval. These SNPs may be of phylogenetic significance and allow differentiation of sequences obtained since 2019 in central regions from earlier isolates of this lineage (Fig. 4).

 

Fig. 4. Polymorphisms that allow grouping of isolates in the Georgia 2007 clade sequences.

A – IGR C62L – C962R; B – partial sequence of the D1133L gene; C – partial sequence of the Q706L gene. The isolates covered by this research are labeled with – *.

Рис. 4. Полиморфизмы, группирующие изоляты в последовательностях клады Georgia 2007.

A – IGR C62L – C962R; В – частичная последовательность гена D1133L; С – частичная последовательность гена Q706L. Изоляты, полученные в данном исследовании, отмечены*.

 

The first of these is the T>C transition in IGR C62L – C962R (Fig. 4 A). The substitution is expected to be fairly stable and suitable as a marker site, as intergenic regions do not experience significant selection pressure.

The second is the replacement of proline with serine at position 457 of the D1133L protein, caused by a G>A transition (Fig. 4 B). It is not strictly conservative, since although the charge of the polar groups of serine in a neutral environment is compensated and equal to that of neutral proline, these amino acids have different hydrophobicity, which may slightly affect the protein structure. However, the detection of this mutation in several isolates suggests that it does not critically affect the normal function of D1133L. D1133L is an intermediate-late protein with nuclear and cytoplasmic localization during infection [21]. It has helicase activity and may also be involved in RNA metabolism and ribosomal function [22]. When D1133L is overexpressed, ASF virions replicate more efficiently in cells [23]. In vitro, it interacts with several host proteins—VIM and TRIM21, which enhance virus replication, and the translation elongation factor TUFM, which inhibits ASF virus replication in cell culture [22]. The polymorphism found may be a probable marker, but due to the possible influence of selection, it is not as reliable as a conservative replacement.

The third substitution, G>A transition, results in a fairly conservative substitution of alanine for valine at position 607 of the Q706L protein (Fig. 4 C), which should not lead to functional changes and should not be subject to increased selection pressure. This protein is an RNA helicase and is quite conservative, although it has a small variability between ASF virus genotypes from different regions [24]. Another SNP in this gene, characterizing isolates from the Bryansk region, is reported in [16]. The SNP we propose can also be considered a marker for the central regions of Russia since 2019.

The marker sites proposed for use allow differentiation of ASF virus strains from Central Russia carrying the CVR-V polymorphism, as well as strains that appeared after 2019, from earlier variants. The question of the prevalence of the new insertion in IGR MGF 360-13L – MGF 360-14L in relation to the occurrence of CVR-V can be clarified by whole-genome sequencing of a larger number of ASF virus isolates. A molecular-epizootological map showing the distribution of both confirmed and presumed Russia lineage isolates, with their marker sites indicated, is shown in Fig. 5. Fig. 6 shows the prevalence of ASF genotype II genetic lineages in Europe according to the above-mentioned methodological recommendations.

 

Fig. 5. Distribution of isolates of the Russia lineage.

● – variant CVR-I; ● – variant CVR-VI; ● – variant CVR-V, contains the CAGTCTATAAGA insertion in IGR MGF 360-13L – MGF 360-14L; ● – variant CVR-V, contains an insertion in IGR MGF 360-13L – MGF 360-14L, as well as transitions in IGR C62L – C962R and in genes D1133L and Q706L; ● – isolates carrying the CVR-V variant, presumably belonging to the Russia lineage.

Рис. 5. Распространение изолятов линии «Россия».

● – вариант CVR-I; ● – вариант CVR-VI; ● – вариант CVR-V, содержит инсерцию CAGTCTATAAGA в IGR MGF 360-13L – MGF 360-14L; ● – вариант CVR-V, содержит инсерцию в IGR MGF 360-13L – MGF 360-14L, а также транзиции в IGR C62L – C962R и в генах D1133L и Q706L; ● – изоляты, несущие вариант CVR-V, предположительно относящиеся к линии «Россия».

 

Fig. 6. Distribution of ASFV genotype II genetic lineages in Europe. Isolates presumably belonging to the Russia lineage are not listed.

● – Georgia 2007/Russia; ● – Georgia 2007/Unclassified lineages; ● – Eastern Europe/Poland; ● – Eastern Europe/Kyiv 2016; ● – Eastern Europe/Kaliningrad; ● – Europe/Italy; ● – Europe/Serbia; ● – Eastern Europe/Germany 2020; ● – Eastern Europe/Germany 2021; ● – Europe/Pskov 2021; ● – Europe/Central Russia 2021; ● – Europe/Bryansk 2021.

Рис. 6. Встречаемость генетических линий АЧС II генотипа в Европе. Изоляты, предположительно относящиеся к линии «Россия», не указаны.

● – Georgia 2007/Россия; ● – Georgia 2007/Неклассифицированные линии; ● – Восточная Европа/Польша; ● – Восточная Европа/Киев 2016; ● – Восточная Европа/Калининград; ● – Европа/Италия; ● – Европа/Сербия; ● – Восточная Европа/Германия 2020; ● – Восточная Европа/Германия 2021; ● – Европа/Псков 2021; ● – Европа/Центр России 2021; ● – Европа/Брянск 2021.

 

The isolates we studied also contain previously undescribed variations. A structure similar to polymorphisms 2153 and 2161 is observed in the paralog of the MGF 360-1L – MGF 360-21R gene, but no other substitutions characteristic of this region were found. This combination was also found in some incorrectly assembled sequences from GenBank, which we did not use for analysis. These two paralogs are among the most closely related in the ASF virus genome (69% identity) [17], which increases the likelihood of errors during genome assembly. The MGF-360-1L gene does not play a significant role in ASF virulence [25], therefore, these mutations should not lead to changes in the properties of the virus.

The Q>H substitution at position 22 observed in the MGF 360-11L protein of ASFV_Penza-2019a, b and ASFV_Tambov-2021 is non-conservative, which should affect the conformation of the protein molecule. The MGF 360-11L protein has an inhibitory effect on the production of type I interferon (IFN) and interleukins IL-1β and IL-6, inhibiting the activity of the IFN-β promoter and ISRE sequence, interfering with the normal functioning of the cellular immune response and limiting the synthesis of inflammatory factors [26].

The substitution of alanine for proline at position 85 of the E199L protein found in ASFV_Penza-2019b was also found in other isolates in Russia and other countries. However, as shown in [16], the polymorphism of the E199L gene is not significant as a marker of diversity, since its changes are associated not with geographical distribution, but with the adaptation of the virus to the host. This protein is necessary for the fusion event that leads to the penetration of the virus into the host cell [27] and also induces autophagy [28]. The same applies to the replacement of glutamine with histidine at position 104 of E199L in ASFV_Lipezk-2016.

The remaining unique polymorphisms detected do not lead to changes in the amino acid sequence, although they may also have potential phylogenetic significance in the future when describing new complete virus sequences isolated in central regions of Russia.

Conclusion

Based on the analysis, it was established that ASF virus isolates detected in the Lipetsk, Penza, and Tambov regions between 2016 and 2021 belong to the CVR-V variant of the Georgia 2007 clade of the Russia genetic lineage. A new probable marker of diversity was found in IGR MGF 360-13L – MGF 360-14L, and polymorphisms were proposed in IGR C62L – C962R and in the D1133L and Q706L genes, which have phylogenetic potential for differentiating ASF virus strains in the central regions of Russia.

 

¹ Chernyshev R.S., Sprygin A.S., Lavrentyev I.A., Igolkin A.S. Methodological recommendations for subgenotyping of ASF virus isolates of genotype II into topotypes and genetic lines. Vladimir; 2025, 14 p. (In Russ.).

About the authors

Daria A. Skorobagatko

Cherkizovo Research and Testing Center LLC

Author for correspondence.
Email: d.skorobagatko86@gmail.com
ORCID iD: 0000-0003-3239-9004

Candidate of Biological Sciences (Ph. D.), Specialist in Department of Genomics and Molecular Biology

Russian Federation, 108805, Moscow

Ekaterina S. Tolkova

Cherkizovo Research and Testing Center LLC

Email: katerina.tolkova@gmail.com
ORCID iD: 0000-0002-4770-3796

Main Specialist in Department of Genomics and Molecular Biology

Russian Federation, 108805, Moscow

Olga A. Shepeleva

Cherkizovo Research and Testing Center LLC

Email: o.shepeleva@cherkizovo.com
ORCID iD: 0000-0002-3366-8184

Head of the Department of Genomics and Molecular Biology

Russian Federation, 108805, Moscow

Sergey O. Shapovalov

Cherkizovo Research and Testing Center LLC

Email: s.shapovalov@cherkizovo.com
ORCID iD: 0000-0002-5630-5247

Doctor of Sciences in Biology, Head

Russian Federation, 108805, Moscow

References

Supplementary files