Background
African swine fever (ASF), a hemorrhagic contagious viral disease affecting domestic pigs and wild boars, is characterized by high mortality and has become a significant threat to the global pig industry [
1]. ASF is caused by African swine fever virus (ASFV), a large DNA virus and the only known member of the genus
Asfivirus within the
Asfarviridae virus family [
2]. The genome is a double-stranded DNA varying between 170 to 193 kbp in size containing 150 to 167 proteins coding open reading frames (depending on the virus strains) and a conserved central region of about 125 kb, while the ends are variable in size [
3]. The epidemiology is complex and embraces various patterns in endemic regions [
4,
5]. To date, neither therapy nor vaccine are available against the disease and therefore, the primary prevention methods rely on the application of strict biosecurity measures, early diagnosis and stamping out the infected pig herds [
6]. Based on the sequencing of the B646L gene, which encodes the capsid protein p72, 24 genotypes have been currently reported worldwide and all of them are known to circulate in Africa [
7,
8].
Previous studies have identified p72 genotype I, IX and XIV to be present in the Democratic Republic of Congo (DRC) [
9,
10]. In addition, our recent study has identified for the first time the presence of p72 genotype X in DRC [
11] in symptomatic domestic pigs during outbreaks. This emphasize the need for continued characterization of ASFV strains responsible for outbreaks to better understand the spread of the disease in DRC and to develop preventive measures to control the spread. To date, only two complete and fully annotated genomes of ASFV genotype X are available in the GenBank, both of which are from Kenyan virus strains [
12]. However, no ASFV complete genome sequencing study has been reported in DRC, an ASFV endemic country, limited a better understanding of molecular evolution and spread of this virus within the country. In this study, we report the first complete genome sequence of ASFV genotype X and serogroup 7 associated with ASF outbreaks in domestic pigs in the South Kivu province in eastern DRC in 2019. Analysis of this ASFV genome in comparison with the previously sequenced and available ASFV genomes provides new insights into genetic diversity of African strains and knowledge that may advance our understanding of host-virus interaction and pathogenicity.
Discussion
In this study, we generated the first complete genome sequence of an ASFV strain, the Uvira B53, directly from a spleen tissue sample of ASF symptomatic domestic pig sourced from Uvira district in South Kivu province, eastern DRC in 2019. Overall, the assembled 180,916 bp long genome sequence obtained was of a high-quality BLAST-match with the previously reported p72 genotype X sequences available in the GenBank. The Uvira B53 ASFV strain is genetically more closely related to the Kenyan strains genotype X than to other ASF viruses of different genotypes.
Compared to the two published genotype X which are all from Kenya, the Uvira B53 genome sequence contained a group of fully conserved genes and a group of divergent genes with SNPs and/or indels (insertions/deletions). In addition, a couple of ORFs found in the two Kenyan genotype X were absent in the Uvira B53 strain: DP96R coding for the UK protein [
25] and p285L with an unknown function. The absence of the DP96R gene has also been reported in the virulent Ken06.Bus strain genotype IX [
20] and the p285L gene was reported in the virulent Benin 97/1 strain [
21]. DP96R is among the known ASFV virulence factors [
27]. Based on this information, the absence of DP96R in Uvira B53 may suggest that the virulence of the DRC strain is different from that of the Kenyan strains genotype X. However, DP96R and p285L were found in the non-virulent strain OURT 88/3 [
21] as well as in the Malawian virulent strain Tengani 62 [
17]. Therefore, further study and information are needed to determine the role of these genes in Uvira B53 and improve our understanding of variable levels of pathogenicity between different ASFV strains.
Like in the two published genotype X, Kenya 1950 and Ken05/Tk1, the terminal inverted repeats were missing at both ends of the Uvira B53 genome sequence. This could be due to lack of enough ASFV DNA in the spleen DNA sample sequenced as we did not use any virus enrichment method (e.g. filtration, cell culture or ultracentrifugation) during viral DNA preparation; furthermore, it could also be due to the difficulty to sequence extensive homopolymer and ITR regions of the ASF virus genome. In addition, the Illumina sequencing platform used generates only short reads, which makes it difficult to effectively sequence and assemble repetitive regions as short repeats could collapse. The ASFV genome sequence represented 0.1% of the 42 Gbp total reads obtained, which corresponded to 42 Mbp of virus sequences. Assuming that the ASFV genome was about 200 kbp long, then the 42 Mbp reads will correspond to 210 × genome coverage. In support to this estimation, our two longest contigs could assemble into the 180,916 bp long genome with an average depth of 78.77 reads per nucleotide. Therefore, the amount of virus DNA used should not be the main cause of the observed sequencing limitation but rather the short length of reads, which could be addressed by using a sequencing platform that will generate long reads.
Several non-conserved divergent genes were identified to be affected by mutations including three proteins involved in putative signal peptide and transmembrane region (C262R, CP123L and E248R), one helicase superfamily II (F1055L), one AP endonuclease class II (E296R) and one RNA polymerase subunit 3 (H359L). However, the effects of these substitutions on protein function are unknown.
Several genetic variations including SNPs, indels (insertions/deletions) and complete loss of ORFs were observed within the MGF member genes between Uvira B53 and known genotype X strains, specifically in genes previously reported to be implicated in determining host range and virulence such as MGF 360, MGF 505 and MGF 110. Indels and loss of ORFs were among the major causes of the differences in length between these genomes as previously reported [
28]. They were observed in all the three strains genotype X analyzed but at higher magnitude in Uvira B53 than the two other strains, making the newly completed 180,916 bp long Uvira B53 genome 10 and 13 kbp shorter than the Kenya 1950 and Ken05/Tk1 genomes, respectively. The biological implications of these variations remain to be determined.
The high level of genetic conservation was observed in some ORFs of MGF 360 (9L, 10L, 13L, 15R and 16) and MGF 505 (3R, 4R and 10R) among Uvira B53 and Kenya 1950, a virulent strain [
20] whereas they were absent in the Ken05/Tk1, an avirulent strain [
20]. This feature may suggest difference and similarity in the Uvira B53 pathogenicity with Ken05/Tk1 and Kenya 1950, respectively. This hypothesis is in agreement with a previous study that demonstrated that some members of MGF 360 and MGF 505 such as MGF360-12L, -13L, and -14L and MGF505-1R, -2R, and -3R were associated with ASFV host range specificity, blocking of the host innate response, and virus virulence, as they were present in several virulent strains of ASFV while absent in the avirulent strains like OURT 88/3 and NHV [
29‐
31]. Further analysis of these differences should provide more insights into the molecular mechanisms underlying the virulence of Uvira B53 genotype X in pigs in South Kivu.
The comparative study based of the intergenic region between I73R and I329L genes showed the genome sequence of the Uvira B53 strain was closer to Kenya 1950 than Ken05/Tk1, as both the Uvira B53 and Kenya 1950 contained deletions compared to Ken05/Tk1. However, the Uvira B53 deletion was 33 bp longer than that of Kenya 1950. This observation corroborates our previous comparative analysis of this intergenic region which revealed this deletion in genotype X strains from symptomatic pigs in South Kivu, DRC [
11].
Analysis of phylogenetic relationship was performed using some representative sequence data sets retrieved from the GenBank. The analysis clustered the ASFV Uvira B53 together with genotype X strains reported in Kenya. This result is in accordance with our previous study reporting circulation of ASFV genotype X in symptomatic ASFV infected pigs from South Kivu province [
11]. Uvira B53 is different from genotype IX, I and XIV that have recently been reported in some provinces of DRC such as Bandundu, Equateur, Katanga, Kinshasa, Maniema and Province Orientale [
10]. They are sylvatic cycle-associated genotype that has been identified from ticks, warthog and domestic pigs in Kenya, Tanzania and Burundi [
5,
32‐
34]. The phylogenetic analysis of some of the divergent protein-coding gene products, particularly the I196L and I177L, matched with the phylogenetic relationship of the complete genome sequence that grouped the Uvira B53 with Kenya 1950 and Ken05/Tk1 genotype X strains. Two other gene products (EP153R and KP177R) were highly divergent in all the strains. A similar observation was reported by Chapman et al. [
18] where phylogenetic analyses of several divergent gene products did not match with one of the complete genome sequences. The divergence may probably be due to the occurrence of recombination events and antigenic diversity. Because of this, we suggest to be cautious when performing phylogenetic relationships between ASFV strains based on a small number of genes.
Several variations were identified in the C-type lectin-like protein (EP153R) in all the strains, making it one of the most divergent genes. Variations in this protein may alter the expression of the functional protein (C-type lectin) that may influence the immuno-modulatory functions as previously demonstrated [
34].
In addition to SNPs, variations in this region of CD2v (EP402R) were characterized by the presence of the tandem repeat amino acid sequence PPPKPY [
35,
37,
38] that was repeated 3–11 times among the strains analyzed. This CD2v variable region is identical in the Uganda (KM609361) strain of serogroup 7 and Uvira B53 with 5 tandem repeats. BLAST search also revealed that the Uganda (KM609361) strain’s CD2v antigen was the most closely related antigen to Uvira B53′s CD2v, displaying 99% amino acid identity. This finding implied that Uvira B53 strain belongs to the ASFV serogroup 7. However, Kenya 1950 found to be the closest genotype to Uvira B53 was shown to cluster with ASFV serogroup 6 based on C-type lectin protein sequence (35). Our study is the first report of a complete genome sequence of ASFV of the serogroup 7. In addition to the serogroups 1 and 2 already reported in DRC [
36], ASFV Uvira B53 genotype X and serogroup 7 represents the third ASFV serogroup to be found in DRC.
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