African swine fever (ASF) is a viral disease of swine that can lead to high mortality in domestic pigs and wild boar (Sus scrofa)
while being asymptomatic in wild suid reservoir hosts in Africa [1
]. ASF causes important economic damage due to mortality and production losses. The main economic influence of this virus comes from its radical control measures, as well as the ban on international trade of live animals and meat products [2
]. The causative agent of ASF is a large enveloped double-stranded DNA virus, the member of the family Asfarviridae
within the genus Asfivirus
. The length of the African swine fever virus (ASFV) genome can vary remarkably from 170 to 194 kbp, and the number of genes can vary from 150 to 167, depending on the isolate [4
]. The DNA molecule of the virus encodes between 151 and 167 open reading frames (ORFs) on both strands [1
]. The gain or loss of ORFs from the multigene families (MGF) encoded by the virus is the primary reason behind the differences in the virus’s genome length and gene number [5
]. A large number of highly variable multigene families in the genome of ASFV is one of the biggest challenges for understanding the functionality of the virus [5
Endemic in more than 20 sub-Saharan African countries [10
], as well as in Sardinia since the last century [11
], ASF arrived at a Black Sea harbor in Georgia in 2007 [12
]. The disease spread quickly west and northwards, reaching the European Union (EU) in 2014. Currently, ASFV is present in ten EU countries, including Lithuania, Poland, Latvia, Estonia, Romania, Bulgaria, Hungary, Belgium, Slovakia, and Greece, which was recently affected in 2020 [13
]. Outbreaks have also occurred in Asia since August 2018, when China declared the presence of ASFV in domestic pigs [14
]. The latter constitutes one of the most important jumps of the disease thus far. The continuous spread to other Asian countries, and recently to Oceania [15
], makes controlling the spread of the virus even more difficult. Early detection and coordinated countermeasures of ASFV are urgently needed; however, for such measures to be effective, information regarding the disease’s dynamics must be determined. Thus, molecular epidemiology has become an essential part of the epidemiological investigation.
The current genotyping of ASFV strains is based on partial nucleotide sequencing of the B646L
gene, which encodes the major capsid p72 protein [16
]. In total, 24 genotypes of ASFV have been determined worldwide [16
]. All genotypes are present in Africa, but only two of them (genotype I and II) have been found on other continents [16
]. In Europe, genotype I has circulated since 1978 on the Italian island of Sardinia. Genotype II began to circulate in large areas of eastern Europe in 2007 [3
], spreading consistently westwards and eastwards, affecting new countries and territories in Europe and Asia [23
]. Although the partial p72 sequencing has proved useful for placing the viruses within one of the defined 24 genotypes, a higher resolution is needed for an in-depth analysis. Several studies have described the suitability of the sequencing of tandem repeat sequences (TRSs) located in the central variable region (CVR) within the B602L
-gene to distinguish between closely related ASF isolates belonging to a single genotype [17
]. Until this study only one CVR variant (GII-CVR1, the “Georgia type variant”) was described in ASFV genotype II, characterized by the 10 amino acid TRS [23
] Nevertheless, this region was selected for a deeper molecular characterization of the selected Estonian ASFV isolates collected since September 2014, when the first case of ASF was diagnosed in a wild boar found dead in southern Estonia near the Latvian border. A few weeks later, an ASFV positive wild boar was found in the northeast of Estonia, near the border with the Russian Federation and 200 km away from the first case of ASF in the south [30
]. Based on further molecular and epidemiological analyses, this finding was not considered to be epidemiologically connected with the findings in the south [30
]. In the following three years, there was extensive spread of the ASFV in the wild boar population, and by the end of 2017, wild boar from 14 out of 15 counties were affected [33
]. The first domestic pig outbreak occurred in July 2015. In total, 27 outbreaks were reported in domestic pig farms, and 42,476 pigs were culled in outbreak farms during the period of 2015–2017 [33
Starting from the beginning of the ASF epidemics in 2014, virus strains have been collected systematically from both wild boar and domestic pigs to describe the genetic variability of ASFV strains circulating in Estonia and to investigate the distribution of genetic variants of the virus. A selection of the results from these investigations is presented in this paper.
The molecular surveillance of ASFV is an integral part of the disease intervention activities in affected countries. Most published studies use the molecular characterization of vp72 or/and variable regions containing an array of the tandem repeat sequences (TRS) for genotyping and subgrouping closely related isolates [16
]. Recent investigations of the genetic diversity of ASFV genotype II isolates have been based on sequencing the intergenic region (IGR) between the I73R and I329L genes [22
]. A study performed at the EURL (Valdeolmos, Spain) analyzed the nucleotide sequences of the IGR of 232 isolates collected in Estonia (2014–2018), but did not reveal any genetic modifications between them [23
]. All isolates had the same additional tandem-repeat sequence (TATATAGGAA) representative of the intergenic region (IGR) 2 variant [29
], and could not be separated into subgroups.
A higher phylogenetic resolution between closely related ASFV genotype isolates in Europe and in Africa was achieved by analyzing the amino-acid TRSs located in the CVR within the B602L
]. In this study, a comparative analysis of PCR CVR size fragments enabled the researchers to identify three different CVR variants of ASFV that have circulated in certain regions of Estonia since 2014. The selection of the samples was initially based on representing all affected areas. Nevertheless, the sample choice was mostly concentrated in the county of Tartu, where the first GII-CVR2 finding was detected, as well as in the two neighboring counties. The same strategy was used for samples originating from the county of Lääne, where the first GII-CVR1/SNP1 variant was detected. Therefore, the sampling scheme was not completely random, as the aforementioned areas were investigated more intensively. In total, 11.6% of all ASFV positive samples collected from wild boar were selected for further CVR sequence analyses. Sequencing of the 13 samples likely failed, due to a weak viral load or insufficient sample quality. The conventional PCR (cPCR) used in prior B602L
gene sequencing is less sensitive than the real-time PCR used in ASFV genome detection. ASFV positive samples with ct values over 34 are likely to fail in cPCR. Nevertheless, the method used in this study was slightly modified (see material and methods) for adaptation to our laboratory equipment and reagents to obtain optimal results. The real-time PCR method used in ASFV genome detection was also changed by adjusting its protocol to the PCR kit available in the laboratory. Despite the changes in the PCR reagents, the in-house validation of this method and excellent annual participation in interlaboratory comparison tests, organized by the EURL for ASF, confirm that this method is fit for its intended purpose.
CVR amplification using the cPCR of wild boar samples allowed us to identify, on July 2015, a size variation in the amplicon obtained from two wild boar samples collected in Tartumaa. Sequence analysis revealed that this difference in size was due to the deletion of three amino acid tetramer repeats compared to the GII-CVR1 “Georgia type” sequences circulating in Europe since 2007 [12
]. These isolates were classified as a new variant named the GII-CVR2 variant. Since the first identification of the GII-CVR2 variant, the number of investigations has noticeably increased in the region and in the two neighboring counties (Valga and Viljandi) to further clarify the spread of the novel variant strain. From the results obtained, we observed that the spread of the GII-CVR2 strain occurred over a relatively short span of time (from July 2015 to March 2016), and only in four municipalities of one county (Tartu)—where both GII-CVR variants co-circulated at the same time. Similar CVR size variation was previously described in Europe (in Sardinia), where genotype I predominates. The changes in the CVR region occurred a few decades after the virus entered Sardinia, placing the isolates into two clusters depending on their temporal distribution [48
]. Investigations in Estonia revealed that the amino acid deletion within genotype II occurred eight years after the virus entered into Georgia in 2007. This deletion could be related to a spontaneous mutation caused by the maintenance of ASFV within the wild boar population in the county of Tartu, since this phenomenon was not found in other affected areas in Estonia or in Europe [23
]. However, since new variants in the ASF genome are difficult to locate if the number of investigations is low, the introduction of GII-CVR2 to Estonia from other affected areas cannot be excluded completely.
Based on the results obtained, the geographical evolution indicates the extinction of new genetic variants over time. The animal trial conducted using one of these GII-CVR2 isolates (Est15/WB-Tartu14) classified the ASFV strain as moderately virulent [22
]. We, therefore, hypothesize that the disappearance of GII-CVR2 could be connected to the reduced virulence of the strain. However, this animal trial conducted with the Estonian GII-CVR2 (Est15/WB-Tartu14) isolate also included an Estonian GII-CVR1 strain (Est15/WB-Valga6) in parallel [22
]. Clinical signs of the disease observed in this study were acute, subacute, and chronic; such signs are usually connected to viral strains of moderate virulence [50
]. This was the first ASFV genotype II virus strain to show moderate virulence in Europe. Until this study, subacute and chronic causes of the disease, as well as moderate virulence, were only connected with genotype I in Europe [22
]. Irrespective of the variant strain used in the experiment, reduced virulence was observed [22
]. Therefore, the deletion of three amino acid tetramer repeats in the B602L
gene described in this study cannot be directly associated with reduced virulence, and the genes connected with moderately virulent strains need further research. We conclude that there is a possibility of domestic pigs and wild boar to develop chronic forms of this disease, a phenomenon common to ASFV strains with reduced virulence [22
]. Reduced virulence ASFV genotype II isolates were also described in two additional cases in Europe, while attenuated strains of the ASFV genotype II were found in Estonia [32
] and Latvia [55
]. By the time, genotype II ASFV circulating in Europe was known to be highly virulent and led to a 90–100% mortality in domestic pigs and European wild boar under experimental conditions [8
The GII-CVR1/SNP1 detected in the western and northern parts of Estonia is a non-synonymous mutation that alters the amino acid sequence, resulting in tyrosine coding instead of cysteine. The relevance of this change remains unknown and needs further investigation. Additional SNP variants within the genotype II CVR1 sequence have been reported in Poland and Lithuania, where GII-CVR1/SNP2 and GII-CVR1/SNP3 are circulating, respectively [23
]. However, compared to GII-CVR1/SNP1, the SNP variants detected in Poland and Lithuania are in a different position and do not alter the amino acid sequence [43
]. Using the whole genome sequencing approach, SNPs have also been described in other gene regions [24
], and are the most common types of genetic variations in the ASFV genome. Therefore, there is a possibility that SNP1 or other SNPs are also present in the southern and eastern areas of Estonia, but because positive ASF cases have decreased since the end of 2016 in the mentioned areas, these mutations may remain undetected.
Compared to GII-CVR2, the spread of the GII-CVR1/SNP1 strain in Estonia took a longer period of time (from November 2016 to December 2017) and covered a larger area (three counties). The epidemic of ASF was severe in the western part of Estonia at the time when GII-CVR1/SNP1 was identified in the county of Lääne. Therefore, the spread of this strain to neighboring counties was likely during this period. Due to the decreased wild boar population, the number of positive ASF cases in wild boar have decreased significantly since the end of 2017 [34
], which may explain the lack of GII-CVR1/SNP1 findings after this period.
Notably, GII-CVR1/SNP1 was also responsible for a domestic pig outbreak that occurred in the county of Pärnu in the middle of June 2017. A temporal investigation revealed the circulation of the GII-CVR1/SNP1 strain in the wild boar population in the same village only a few weeks before the outbreak. This finding agrees with the conclusions of another Estonian study that argued the presence of ASFV in wild boar populations is the main risk for domestic pig farms becoming infected [33
In conclusion, three changes were identified in the B602L
gene of ASFV isolated from the Estonian wild boar population, with the GII-CVR1 “Georgia type” variant being predominant. The GII-CVR2 variant remained only in a limited area and disappeared after the end of March 2016, whereas the GII-CVR1/SNP1 variant was no longer detected after December 2017. These data suggest that there is a predominant variant causing the outbreaks in Estonia, as no significant genetic changes have occurred in the regions evaluated over the five-year period. Although analysis of the CVR has been widely used to distinguish between closely related ASF isolates in Africa [27
] and Europe [29
], the low CVR genetic variability necessitates further research into alternative and more informative gene regions to clarify the relevant intra genotype relationships. Recent studies on the molecular evolution of genotype II EU strains revealed the presence of five different variants circulating in the EU when sequencing the IGR between the MGF505 9R and 10R genes [Gallardo personal communication 2019]. An extensive analysis of Estonian ASFV isolates using this genetic marker could help clarify questions regarding the epidemiology of ASF in Estonia.