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Brief Report

Genetic Characterization of the Central Variable Region in African Swine Fever Virus Isolates in the Russian Federation from 2013 to 2017

1
Reference Laboratory for African Swine Fever Virus, FGBI “Federal Centre for Animal Health” (FGBI “ARRIAH”), 600901 Vladimir, Russia
2
Agricultural Research Council-Onderstepoort Veterinary Institute, 100 Old Soutpan Road, Onderstepoort 0110, South Africa
3
Department of Biotechnology, University of the Western Cape, Robert Sobukwe Road, Bellville 7535, South Africa
*
Author to whom correspondence should be addressed.
Pathogens 2022, 11(8), 919; https://doi.org/10.3390/pathogens11080919
Submission received: 27 July 2022 / Revised: 9 August 2022 / Accepted: 11 August 2022 / Published: 15 August 2022
(This article belongs to the Special Issue Emergence and Control of African Swine Fever)

Abstract

:
African swine fever virus (ASFV), classified as genotype II, was introduced into Georgia in 2007, and from there, it spread quickly and extensively across the Caucasus to Russia, Europe and Asia. The molecular epidemiology and evolution of these isolates are predominantly investigated by means of phylogenetic analysis based on complete genome sequences. Since this is a costly and time-consuming endeavor, short genomic regions containing informative polymorphisms are pursued and utilized instead. In this study, sequences of the central variable region (CVR) located within the B602L gene were determined for 55 ASFV isolates submitted from 526 active African swine fever (ASF) outbreaks occurring in 23 different regions across the Russian Federation (RF) between 2013 and 2017. The new sequences were compared to previously published data available from Genbank, representing isolates from Europe and Asia. The sequences clustered into six distinct groups. Isolates from Estonia clustered into groups 3 and 4, whilst sequences from the RF were divided into the remaining four groups. Two of these groups (5 and 6) exclusively contained isolates from the RF, while group 2 included isolates from Russia as well as Chechnya, Georgia, Armenia, Azerbaijan and Ukraine. In contrast, group 1 was the largest, containing sequences from the RF, Europe and Asia, and was represented by the sequence from the first isolate in Georgia in 2007. Based on these results, it is recommended that the CVR sequences contain significant informative polymorphisms to be used as a marker for investigating the epidemiology and spread of genotype II ASFVs circulating in the RF, Europe and Asia.

1. Introduction

African swine fever (ASF) is a fatal viral disease of domestic pigs and wild boars of all ages. The causative agent is the only known double-stranded DNA arbovirus, ASF virus (ASFV), which belongs to the Asfarviridae family in the genus Asfivirus. The genome ranges from 170 to 190 kilobase pairs (kbp) and encodes more than 150 open reading frames (ORFs), depending on the viral strain [1,2]. Traditionally, the disease has been confined to sub-Saharan Africa and the Italian island of Sardinia, but sporadic epidemics have affected a number of countries throughout the 20th century. However, in 2007, ASFV was first reported in Georgia, and from there, it spread to Armenia and Azerbaijan and subsequently into the Russian Federation (RF), Ukraine and Belarus [3]. In 2014, four European Union countries, Lithuania, Poland, Latvia, and Estonia, reported ASF prior to its subsequent spread to Belgium, Bulgaria, Czech Republic, Hungary, Romania and Slovakia [4]. In 2018, the disease spread to China, the world’s largest pig-producing country, resulting in devastating economic losses to the country [5]. In recent years, ASF has rapidly spread beyond China to neighboring countries, including Mongolia, Cambodia, North Korea, South Korea, the Philippines and India [6]. In July 2021, ASFV genotype II was reported in the Dominican Republic and Haiti, and additionally in Germany, Greece and Italy [7].
Currently, ASFV is classified into 24 genotypes based on sequence data from the C-terminal region of the open reading frame (ORF) B646L, encoding the major capsid protein p72 [8,9]. This gene region is frequently used to investigate the molecular epidemiology of ASF by determining and comparing the genotypes circulating in a region [9]. The additional differentiation of closely related viruses into sub-groups has been subsequently performed using the p54 locus (E183L), the central variable region (CVR) in the B602L gene [10], tandem repeat sequence (TRS) insertion in the intergenic regions (IGR) and multigene family (MGF) 505 9R/10R [11]. Additional genome markers, K145R, MGF 505-5R and O174L, have been used to differentiate isolates from Poland [12]. A novel 14 base pair (bp) TRS insertion of CAGTAGTGATTTTT was identified in the O174L gene of certain isolates from Poland [12]. Complete genome sequences from isolates in the RF recommended that MGF-360-10L, MGF-505-9R and I267L be included as additional genome markers, since they have the resolving capabilities of separating isolates from the RF, EU and China geographically into an eastern and western cluster [13]. The CVR region is frequently targeted for sequence analyses due to unique mutations capable of resolving phylogenies at a regional level, including clustering isolates into groups and sub-types [9,14]. Isolates from Africa were recently divided into various sub-types, based on informative polymorphisms observed within this gene region [15]. Despite the large number of ASFV genotype II outbreaks in Europe and Asia, variants in the CVR have only been described for isolates from Estonia, classifying these isolates into three uniquely distinctive CVR variant groups [16]. Based on the resolving power of this gene region in isolates from Africa and the polymorphisms observed in isolates from Estonia, it was hypothesized that this gene region could be used as a fast and cost-effective method to investigate the epidemiology, evolution and molecular relatedness of large numbers of isolates from the RF. The aim of this study was to characterize the CVR sequences of 55 ASFV isolates, each representing closely linked outbreaks from 23 different regions of the RF during 2013–2017, and subsequently determine the phylogenetic relationship between these isolates with ASFVs from Europe and Asia.

2. Materials and Methods

2.1. Ethics Statement

No animals were used during this study, but samples from clinically infected domestic pigs and wild boars were submitted for the laboratory confirmation of ASFV to the national reference laboratory at the Federal Center for Animal Health (FGBI “ARRIAH”) in Vladimir, Russia.

2.2. Isolates and Virus Identification

In this study, 55 ASFV PCR-positive samples were selected as representatives of the 526 outbreaks reported in 23 different regions of the RF at different times during the period 2013–2017. A brief summary of these isolates is provided in Table 1.
Blood or organ tissue samples were collected from domestic pigs (DPs) and either hunted or dead wild boars (WBs). These samples were refrigerated and shipped to FGBI ARRIAH within 24 h of collection. Viral DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, Germany) following the manufacturer’s recommendations, and the presence of ASFV nucleic acids was determined via real-time PCR according to recommendations of the OIE [17].

2.3. Sequence Alignment and Phylogenetic Analysis

A 233bp region of the CVR (B602L) gene was PCR-amplified as previously described using the primer pairs ORF9L-F (5′-AATGCGCTCAGGATCTGTTAAATCGG-3′) and ORF9L-R (5′-TCTTCATGCTCAAAGTGCGTATACCT-3′) [10,18]. These amplicons were submitted for Sanger sequencing at the FGBI “ARRIAH” institute using both primers incorporated during the generation of the amplicons. Both sequences were assembled to generate a consensus sequence representing the CVR gene of each isolate. Nucleotide sequences were aligned and compared to corresponding sequences from Genbank (Supplementary Table S1) using Bioedit v7.2.5 software (by Hall, T.A., CA, USA). The phylogenetic relatedness of these sequences was analyzed using Maximum Likelihood under General Time Reversal (GTR + GI = 4), with the consideration of all the sites to account for the gaps due to deletions in the analysis. The sequence of ASFV genotype I Liv13/33 was included as an outlier.

3. Results

The partial gene region of ORF B602L from 55 ASFV isolates, representing 526 outbreaks from 23 different regions within the RF during 2013–2017, were amplified and Sanger sequenced. The sequences were compared to data available from GenBank, representing additional genotype II ASFVs obtained from Europe and Asia. These sequences were aligned, and single-nucleotide polymorphisms (SNPs) were described using isolate Georgia-2007/1 (FR682468.2) as a reference. By comparing the new sequences with all the available data from Europe and Asia, the sequences were sub-divided into six distinct groups. These sub-divisions were based on five SNPs and one deletion and are subsequently described in detail (Figure 1, Figure 2 and Figure S1).
The first group (1) consisted of isolates that shared 100% sequence identity to Georgia 2007/1 (FR682468.2). This group included the majority of the new and old isolates from the RF as well as isolates from Europe and Asia with the exception of Estonia (Table 1, Supplementary Figure S1).
Sequences belonging to group 2 were characterized by two unique SNPs in contrast to the reference sequence Georgia 2007/1 (FR682468.2). A synonymous (C/T) SNP at position 480 and a non-synonymous (A/G) SNP at position 616 were described within these sequences (Supplementary Figure S1). The latter SNP resulted in threonine (T) exchange of an alanine (A) at position 206 of the complete B602L predicted protein (Supplementary Figure S2). This group contained 14 isolates from Eastern Europe, submitted between 2007 and 2014, with 12 sequences obtained from GenBank [19]. The sequences represent samples from Chechnya in 2007 (Che07; JX857524); Georgia in 2007 (Abk07; JX857523); Armenia in 2007 (Arm 07; JX857522); Azerbaijan in 2008 (Az08B; JX857530 and Az08D; JX857529); Ukraine in 2012 (Ukr12/Zapo; JX857535); and the RF between 2008 to 2016 (Oren08; JX857526, Ing08; JX857525, StPet09; JX857534; Kalmykia09; JX857533, Rostov 09; JX857532, Dagestan09; JX857531 as well as from this study: Krasnodar 07/15; ON098023 and Crimea 01\6 Martins; ON098024). Isolates from the RF belonging to this group were mainly submitted from the south-western regions of the RF (Figure 1).
Groups 3 and 4 were previously identified in a study performed by Vilem et al., 2020. Group 3 was characterized by a 35 bp deletion at position 481, resulting in an amino acid deletion of CASMCADTNVDT (Supplementary Figures S1 and S2). Group 4 contained a non-synonymous (A/G) SNP at position 506, which resulted in a cysteine (C) to tyrosine (Y) exchange at the predicted amino acid position 193 of the complete B602L protein. All sequences analyzed in both of these groups were obtained from Genbank, representing isolates unique to Estonia submitted between 2014 and 2017 [16] (Figure 2).
Group 5 had a non-synonymous (A/G) mutation at position 601, resulting in a lysine (K) exchange of glutamic acid (E) at the predicted amino acid position 201 of the B602L protein (Supplementary Figures S1 and S2). This SNP was observed in six isolates selected and sequenced in this study, representing outbreaks from the RF submitted to the laboratory in 2016 and 2017 (Sudogda-Vladimir 16-DP, Arkhangelsk 16-DP, Tambov 2016, Krasnodar 2016, Gorokhovets-Vladimir 17-WB/325 and Orel 17-WB/337) (Figure 2).
Lastly, group 6 was characterized by a single (A/T) SNP at position 459, identified in six isolates selected and analyzed in this study, representing samples submitted between 2013 and 2016 in Russia (Anino-Moscow 13-WB, Kashino-Tver 13-WB, Karamzino-Tver 13-WB, Shihobalovo-Vladimir 13-WB, Sobinka-Vladimir 15-WB and Sobinka-Vladimir 16-WB) (Figure 2). This is a synonymous SNP involving leucine (L) at amino acid position 153 (Supplementary Figures S1 and S2).
As represented by the phylogenetic tree, all isolates belonging to genotype II were clustered into six different groups, based on the mutations identified in the CVR of the virus genome. Group 1 constituted the largest number of isolates and shared 100% identity to the Georgia 2007/1 sequence. Isolates from the RF could be divided into four distinct groups: 1, 2, 5 and 6. Isolates from Azerbaijan, Georgia, Armenia and Ukraine clustered in group 2 along with samples from the RF (Figure 2). The latter had samples that were unique to groups 5 and 6 (Figure 2). Isolates from Estonia were sub-divided into groups 3 and 4, resulting in both groups being unique to this country (Figure 2).
Three of the five SNPs utilized during the demarcation of the six groups were non-synonymous, resulting in T206A exchange in 16 isolates and E201K exchange in 7 isolates submitted from the RF (Supplementary Figure S2). In addition, C169Y exchange was uniquely described for 24 isolates from Estonia in 2017 (Supplementary Figure S2) [16]. The isolates from Estonia in 2015 and 2016 were the only sequences that had a deletion of one of the tetramer-tandem repeats (CADT, NVDT and CASM) containing only seven of the eight tetramers (Supplementary Figure S2). The synonymous SNP in group six reduced the number of groups based on amino acid differentiation to five, compared to the six groups described based on nucleotide analysis (Supplementary Figures S1 and S2).

4. Discussion

Since the introduction of ASF into Georgia in 2007, the disease has been spreading in an unprecedented manner across Eurasia. Fear of ASF emergence in the territory, either in domestic pigs or in wild boar populations, exists in many countries currently still free from the disease [20]. From 2007 to 04.04.2022, about 62,351 outbreaks/cases of ASF have been reported in the territory of the RF, Europe, Asia and the Caribbean [7]. Of these, 2139 outbreaks were reported in the RF. Studies on the epidemiology of ASFVs in Europe indicated that wild boars and the products of affected pigs were the largest contributing factors pertaining to the spread of the disease in this region [21]. The subsequent monitoring of outbreaks and tracking of virus movements using genetic tools are therefore imperative in an efficient ASF control strategy. The gold standard in unraveling the relationship between ASFVs is based on the elucidation of a complete genome sequence of individual isolates and subsequent comparative analysis involving multiple ASFVs [22]. However, this procedure is time-consuming, labor-intensive and expensive [23]. Single genomic loci could provide a fast and cost-effective medium to resolve ASFV isolates based on differences in informative SNPs and size variations. Potential loci that could be used to resolve the molecular epidemiology of closely related genotype II ASFVs in Russia, Europe and Asia include I267L, MGF 505-5R and K145R and the CVR locus (B602L) [10,12,13,19]. Based on the analysis of O174L, three variants of ASFV were identified in Poland, whilst a single SNP in the K145R gene identified two additional variants of the virus [12]. Additionally, the IGR (I73R/I329L) verified the circulation of three variants [12]. Isolates in Vietnam clustered in a single group identical to Georgia 2007 based on the sequence analysis of the CVR, while the same isolates were divided into three groups based on their IGR sequences [6]. The CVR gene region is frequently applied to the intra-genotype differentiation of ASFVs circulating in African countries [14,15].
In this study, the partial B602L gene containing the CVR locus of 55 ASFVs, selected to represent outbreaks from different regions of the RF, was determined and compared to previously published sequence data from Europe and Asia. Based on these sequence analyses, samples from the RF were sub-divided into four unique clusters. In addition, ASFVs from Europe and Asia were divided into six distinct groups based on the same region (Supplementary Figure S1) [16]. This is due to the unique sequences previously described in Estonia between 2015 and 2017 [16].
The data generated in this study clustered the isolates into four groups that mirror the spatial or temporal origins of the isolates from the RF (Figure 1). This suggests that the outbreaks were highly clonal and that this marker could be used to track the origin and spread of viruses in future epidemiological studies. The identification of genetically highly related strains observed over multiple years within the same geographical location is indicative of the localized circulation of ASFV in possibly wild boar populations, which has been suggested by previous studies [24].
Interestingly, there were four exceptions to the observed spatiotemporal clustering of the defined groups. Two isolates from group 2, St. Petersburg in 2009 and Orenburg in 2008, as well as two isolates in group 5, Arkhangelsk in 2016 and Krasnodar in 2016, were submitted between 1300 and 1500 km from the nearest isolate belonging to the same cluster (Figure 1). This vast distances between outbreaks suggest possible transboundary incursions from outside the area of study, possibly related to movement of domestic pigs or pig-based products, rather than the localized wild boar transmission pathway. Additionally, group 2 included isolates from Armenia, Georgia, Azerbaijan and Ukraine, which further supports the hypothesis of the possible involvement of human actions in the spread of the disease across international borders [19].

5. Conclusions

This is the first study to differentiate isolates from the territory of the RF, based on CVR sequences. The sequences were clustered into four groups that mirrored the spatial and/or temporal distribution of the outbreaks represented by these isolates from the RF. Based on these results, representatives of outbreaks submitted from the same regions between 2018 and 2022 will be analyzed based on the CVR. The aim of these studies will be to identify any novel mutations and to evaluate if the variants identified within this study are still circulating in the same regions or have spread to new regions.

Supplementary Materials

The following supporting information can be downloaded at: www.mdpi.com/article/10.3390/pathogens11080919/s1, Figure S1: Alignment of CVR (B602L) nucleotide sequences of isolates representing the six groups; Figure S2: Alignment of the predicted partial amino acidic sequence of pB602L from representative isolates of each of six groups; Table S1: List of isolates obtained from Genbank used in this study to compare with new isolates from the RF.

Author Contributions

A.M., A.V.S., A.I. and A.S. (Alexander Sprygin) designed the study and revised the draft. A.M., A.V.S., F.I.K. and R.C. performed the experiments. A.S. (Andrey Shotin) and A.I. collected the samples. A.M., A.V.S. and A.S. drafted the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by grant no. 075-15-2021-1054 from the Ministry of Education and Science of Russia to implement objectives of the Federal Scientific and Technical Program for the Development of genetic technologies during 2019–2027.

Institutional Review Board Statement

No animals were sacrificed for this study.

Informed Consent Statement

Not applicable.

Data Availability Statement

The datasets presented in this study were submitted to the Genbank database (ON098019-ON098073). The names of the isolates and accession number(s) can be found in the article/Table 1.

Acknowledgments

The authors thank Zinyakov Nikolay for their excellent technical assistance.

Conflicts of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Abbreviations

ASFV: African swine fever virus; PCR, polymerase chain reaction; CVR, central variable region; kbp, kilobase pair; IGR, intergenic region; TRS, tandem repeat sequence; UPGMA, unweighted pair group method with arithmetic mean.

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Figure 1. Distribution of the ASFVs from the territory of the RF, EU and Asia based on the sub-division of sequences using the CVR locus. Sequences represent ASFV samples obtained between 2007 and 2020, from either DPs (circles) or WBs (squares). The sub-division of CVR sequences into six groups is graphically presented as indicated by the key provided in the figure.
Figure 1. Distribution of the ASFVs from the territory of the RF, EU and Asia based on the sub-division of sequences using the CVR locus. Sequences represent ASFV samples obtained between 2007 and 2020, from either DPs (circles) or WBs (squares). The sub-division of CVR sequences into six groups is graphically presented as indicated by the key provided in the figure.
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Figure 2. Maximum-likelihood phylogenetic tree based on the 281 bp partial sequence of the B602L gene (CVR) of the ASFV genome. Included in this analysis are the 55 sequences generated in this study from isolates within the RF, as well as sequences obtained from Genbank. Solid black circles are used to identify isolates from this study that belonged to groups other than group 1 (Georgia 2007/1); isolates that showed 100% identity to Georgia 2007/1 are not shown.
Figure 2. Maximum-likelihood phylogenetic tree based on the 281 bp partial sequence of the B602L gene (CVR) of the ASFV genome. Included in this analysis are the 55 sequences generated in this study from isolates within the RF, as well as sequences obtained from Genbank. Solid black circles are used to identify isolates from this study that belonged to groups other than group 1 (Georgia 2007/1); isolates that showed 100% identity to Georgia 2007/1 are not shown.
Pathogens 11 00919 g002
Table 1. Brief characteristics of samples used in this study, including the collection year and location as well as the GenBank accession number and the group each sequence is assigned to.
Table 1. Brief characteristics of samples used in this study, including the collection year and location as well as the GenBank accession number and the group each sequence is assigned to.
No.Collection DateHostIsolate NameRegionAccession Number of the CVR LocusGroup
Seq12016DPSudogda-Vladimir_16-DPVladimirON0980195
Seq22016DPArkhangelsk_16-DPArkhangelskON0980205
Seq32017WBGorokhovets-Vladimir_17-WB/325VladimirON0980215
Seq42017WBOrel_17-WB/337OrelON0980225
Seq52015DPKrasnodar_07/15KrasnodarON0980232
Seq62016DPCrimea_Martins_01/16-DPCrimeaON0980242
Seq72016WBSobinka-Vladimir_16-WBVladimirON0980256
Seq82013WBAnino-Moscow_13-WBMoscowON0980266
Seq92015WBSobinka-Vladimir_15-WBVladimirON0980276
Seq102013WBKashino-Tver_13-WBTverON0980286
Seq112013WBKaramzino-Tver_13-WBTverON0980296
Seq122013WBShihobalovo-Vladimir_13-WBVladimirON0980306
Seq132013DPBoguchary-Voronezh_13-DP/2051VoronezhON0980311
Seq142013DPVolgograd_13-DP/2078VolgogradON0980321
Seq152013DPVolgograd_13-DP/2059VolgogradON0980331
Seq162014DPVoronezh_Agro_14-DPVoronezhON0980341
Seq172014WBGrafskoe-Belgorod_14-WBBelgorodON0980351
Seq182014WBOdintsovo_02/14-Moscow_14-WBMoscowON0980361
Seq192014WBGribovo-Kaluga_14-WBKalugaON0980371
Seq202014WBVasilenki-Kaluga_08/14-WBKalugaON0980381
Seq212014DPAntonovo-Pskov_14-DPPskovON0980391
Seq222015WBRyazan_15-WBRyazanON0980401
Seq232015DPSaratov_01/15SaratovON0980411
Seq242015DPGuskhrustalny-Vladimir_15-DPVladimirON0980421
Seq252015WBRyazan(Autumn)_15-WBRyazanON0980431
Seq262015DPBolhovsky-Orel_15-DPOrelON0980441
Seq272015DPKurtnikovo-Moscow_15-DPMoscowON0980451
Seq282015WBSashino-Vladimir_15-WBVladimirON0980461
Seq292015DPSmolensk_15-DPSmolenskON0980471
Seq302015DPKursk_15-DPKurskON0980481
Seq312015DPSokolckie_Vol-Krasnodar_15-DPKrasnodarON0980491
Seq322015DPOrel_15-DPOrelON0980501
Seq332017DPKrasnodar_07/17KrasnodarON0980511
Seq342016DPSouth_Osetia_16/DP/2325South OssetiaON0980521
Seq352016WBRyazan_03/16RyazanON0980531
Seq362016WBRyazan_07/16-WBRyazanON0980541
Seq372016DPShatsky_-_Ryazan_16-DPRyazanON0980551
Seq382016DPOrel-Mtsensk_16-DPOrelON0980561
Seq392016DPKropotkin-Krasnodar_16-DPKrasnodarON0980571
Seq402016WBCrimea_16-WBCrimeaON0980581
Seq412016DPVrachovo-Moscow_16-DPMoscowON0980591
Seq422016DPPenza_16-DPPenzaON0980601
Seq432017DPKolchugino-Vladimir_17-DP/5662VladimirON0980611
Seq442017DPSobinka-Vladimir_17-DP/5660VladimirON0980621
Seq452017DPSobinka-_Vladimir_17-DP/328VladimirON0980631
Seq462017DPOmsk_17-DP/5665OmskON0980641
Seq472017WBVyaznikovski-Vladimir_17-WB/330VladimirON0980651
Seq482017DPIrkutsk_17-DP/447IrkutskON0980661
Seq492017DPSouth_Osetia_17-DP/2196South OsetiaON0980671
Seq502017WBCrimea_17-WB/470CrimeaON0980681
Seq512017DPKrasnoyarsk_10/2017KrasnoyarskON0980691
Seq522017DPOmsk_10/2017OmskON0980701
Seq532017DPBelgorod_10/17BelgorodON0980711
Seq542017WBBelgorod_12/17BelgorodON0980721
Seq552017DPKaliningrad_12/17KaliningradON0980731
DP = domestic pig; WB = wild boar.
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Mazloum, A.; Van Schalkwyk, A.; Chernyshev, R.; Shotin, A.; Korennoy, F.I.; Igolkin, A.; Sprygin, A. Genetic Characterization of the Central Variable Region in African Swine Fever Virus Isolates in the Russian Federation from 2013 to 2017. Pathogens 2022, 11, 919. https://doi.org/10.3390/pathogens11080919

AMA Style

Mazloum A, Van Schalkwyk A, Chernyshev R, Shotin A, Korennoy FI, Igolkin A, Sprygin A. Genetic Characterization of the Central Variable Region in African Swine Fever Virus Isolates in the Russian Federation from 2013 to 2017. Pathogens. 2022; 11(8):919. https://doi.org/10.3390/pathogens11080919

Chicago/Turabian Style

Mazloum, Ali, Antoinette Van Schalkwyk, Roman Chernyshev, Andrey Shotin, Fedor I. Korennoy, Alexey Igolkin, and Alexander Sprygin. 2022. "Genetic Characterization of the Central Variable Region in African Swine Fever Virus Isolates in the Russian Federation from 2013 to 2017" Pathogens 11, no. 8: 919. https://doi.org/10.3390/pathogens11080919

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