Molecular Characterization of African Swine Fever Virus From 2019-2020 Outbreaks in Guangxi Province, Southern China

African swine fever virus (ASFV) causes contagious hemorrhagic disease of pigs with high morbidity and mortality. To identify the molecular characteristics of ASFV strains circulating in Guangxi province, southern China, a total of 336 tissue samples collected from 336 domestic pigs that died as a result of severe hemorrhagic disease during 2019–2020 were tested for ASFV. Furthermore, 66 ASFV strains were genetically characterized by sequence analysis of the C-terminal region of B646L (p72) gene, the complete E183L (p54) gene, the variable region of EP402R (CD2v) gene, the central variable region (CVR) of B602L gene, the full MGF505-2R gene, and the tandem repeat sequence (TRS) within intergenic region (IGR) between the I73R and I329L (I73R/I329L) genes. Phylogenetic analysis revealed that the ASFV strains from Guangxi province belonged to genotypes I and II based on the B646L (p72) and E183L (p54) genes, and there were eight different tetrameric TRS variants based on the CVR of B602L gene. Phylogenetic analysis of the EP402R (CD2v) gene revealed that these ASFV strains belonged to serogroups 4 and 8. Eight of the 66 strains belonged to genotype I and serogroup 4, and showed deletion of whole MGF505-2R gene. The sequence analysis of the IGR between the I73R/I329L genes showed that IGR II and III variants were co-circulating in Guangxi province. The results indicated that ASFV strains circulating in Guangxi province during 2019–2020 outbreaks showed high genetic diversity, of which genotypes I and II, as well as serogroups 4 and 8, were simultaneously circulating in Guangxi province, and there existed wild-type and naturally gene-deleted strains in the field. This is the first detailed report on the molecular characterization of the ASFV strains circulating in southern China, and serogroup 4 in China.


Sample Collection and Detection of ASFV Genome
From January 2019 to December 2020, a total of 336 tissue samples from 336 domestic pigs were collected from 86 different pig farms in 14 regions of Guangxi province, southern China in this study. The pig farms with high fever, hemorrhagic pig herds suspected of ASF were selected, and three to five dead pigs form each farm were selected randomly to collect clinical samples. The tissue samples, including lung, liver, spleen and lymph nodes from each dead pig, were collected, transported immediately at ≤4 • C to the laboratory, and stored at −70 • C until used. The tissue samples were homogenized (10%, W/V) in phosphatebuffered saline (PBS, pH7.2) using a blender (Tianlong, Xi'an, China), followed by shaking with small glass beads for 5 min, and then freeze-thawed three times, centrifuged at 12, 000 × g at 4 • C for 5 min. Total DNA was extracted from the supernatants using MiniBEST RNA/DNA Extraction Kit (TaKaRa, Beijing, China) and confirmed the presence of ASFV by a real-time qPCR targeting the B646L (p72) gene by the following specific primers and probe: ASFV-U: GGCGTATAAAAAGTCCAGGAAATTC, ASFV-D: TTCGGCGAGCGCTTTATC, ASFV-P: Texas Red-TCACCAAATCCTTTTGCGATGCAAGCT-BHQ2.
The parameters of real-time qPCR were as follows: 10 µl of Premix Ex Taq (TaKaRa, Beijing, China), 0.4 µl of each of ASFV primers (20 pmol/µl), 0.5 µl of ASFV probe (20 pmol/µl), 2 µl of total DNA as templates and distilled water to a total volume of 20 µl. The amplification parameters were as follows: 95 • C for 1 min; and then 40 cycles of 95 • C for 5 s, 59 • C for 34 s. The fluorescent signals were determined at the end of each cycle. The ASFV positive samples were randomly selected for genetic analysis.

Amplification and Sequencing of the Targeted Genes
The positive samples were randomly selected for amplifying the C-terminal region of the B646L (p72) gene, the E183L (p54) gene, the CVR of the B602L gene, the EP402R (CD2v) gene, the full MGF505-2R gene, and the TRS within IGR between the I73R/I329L genes, using six different pairs of primers ( Table 1) according to the previous reports (24)(25)(26)(27)(28)(29) with some modifications. The targeted genes were amplified by PCR using the Tks Gflex TM DNA Polymerase kit (TaKaRa, Beijing, China), and the products were electrophoresed in a 1.2% agarose gel and visualized using an imaging system (UVITEC, France). The amplified fragments were purified and cloned into a pMD18-T vector (TaKaRa, Beijing, China), transferred into E. coli DH5α competent cells (TaKaRa, Beijing, China) and sequenced with an ABI 3730XL sequencer (ABI, Los Angeles, CA, United States).

Genomic Analysis
Nucleotide and amino acid alignments were carried out using the EditSeq and Megalign program in the DNAstar

Target
Name CD2v-D ATGGCGGGATATTGGGTAGT package (DNAstar, Madison, WI, United States). All the reference sequences of ASFV were downloaded from the GenBank in the National Center for Biotechnology Information (NCBI) (Supplementary Tables S1-S3). Phylogenetic analysis based on the B646L (p72) gene and the E183L (p54) gene nucleotide sequences, respectively, were conducted using MEGA X, and the genotypes of ASFV strains were determined as previously described (30)(31)(32). The serogroups of the obtained ASFV strains were achieved by phylogenetic analysis of the EP402R (CD2v) gene sequences using MEGA X as previously described (33,34). The maximum likelihood (ML) and the best-fit model methods were used to compare the differences between the two typing methods and analyze gene sequences. Phylogeny was inferred following 1,000 bootstrap replications. The nucleotides of the CVR of the B602L gene and the TRS within the IGR between I73R/I329L genes were first translated into amino acids and searched by similarity analysis according to the ASFV amino acid sequences using BLASTp (v2.2.2.29). The CVR amino acid tetramers were matched with those previously reported codes (31,34,35).

Detection of ASFV Genome in Clinical Samples
Of all 336 clinical samples evaluated by real-time qPCR using primers ASFV-U/D and probe ASFV-P, 192 samples were positive for ASFV, with a positive rate of 57.14% (192/336). Sixty-six samples were randomly selected from these 192 positive samples and further confirmed by real-time qPCR using a commercial ASFV qPCR detection kit (Lijian, China) which targeted the B646L (p72), EP402R (CD2v) and MGF505-2R genes, before they were used for gene amplification, sequencing and analysis. The results showed that all the 66 samples were positive for ASFV based on the B646L (p72) and EP402R (CD2v) genes, of which eight samples lacked MGF505-2R gene. Then, these samples were further amplified and sequenced to verify the deletion of MGF505-2R gene.

Sequence Alignment of the MGF505-2R Gene
The evaluation of ASFV positive samples using a commercial qPCR detection kit (Lijian, China) suggested that eight samples lacked MGF505-2R gene. To verify this result, the MGF505-2R genes of all the 66 samples were amplified, sequenced and aligned. The results confirmed the deletion of the MGF505-2R gene in eight samples (Figure 1). The further analysis showed that these 8 strains belonged to genotype I based on the phylogenetic analysis of the B646L (p72) gene (Figure 2A) and serogroup 4 based on the phylogenetic analysis of the EP402R (CD2v) gene ( Figure 2B).

Phylogenetic Analysis of the B646L (p72) Gene
The B646L (p72) gene was amplified, sequenced and analyzed. Phylogenetic analysis of B646L (p72) nucleotide sequences revealed that all ASFV strains, including the 66 strains obtained in this study ( Table 2) and the reference strains downloaded  (Figure 2A).

Phylogenetic Analysis of the E183L (p54) Gene
The E183L (p54) gene was amplified, sequenced and analyzed. Since all the 66 strains from Guangxi province belonged to genotypes I and II based on phylogenetic analysis of the B646L (p72) gene, we wanted to know whether these strains also belonged to genotypes I and II based on phylogenetic analysis of the E183L (p54) gene. The result revealed that all the 66 strains from this study ( Table 2) and the reference strains downloaded from GenBank (Supplementary Table 2) could be clustered into different genotypes, and further into different sub-genotypes. Of all the 66 strains from this study, 8 strains belonged to subgenotype Ia, and the other 58 strains belonged to sub-genotype IIa which could be further divided into different branches ( Figure 2B).

Phylogenetic Analysis of the EP402R (CD2v) Gene
The EP402R (CD2v) gene was amplified, sequenced and analyzed. Phylogenetic analysis of EP402R (CD2v) nucleotide sequences revealed that all ASFV strains, including the 66 strains from this study ( Table 2) and the reference strains downloaded from GenBank (Supplementary Table 3), could be clustered into eight serogroups. Of the 66 strains from this study, eight strains of genotype I belonged to serogroup four, and the other 58 strains of genotype II belonged to serogroup eight (Figure 3).

Analysis of the CVR of the B602L Gene
The CVR of the B602L gene is a highly variable region and is often used for intragenotypic resolution of viruses belonging to the same B646L (p72) and E183L (p54) genotypes. Therefore, the CVR of the B602L gene of Guangxi ASFV strains was amplified, sequenced and analyzed. The results showed that the 66 strains from this study had different nucleotide lengths from 68 to 178 bp. The nucleotides were transformed into amino acids and analyzed. The sequencing analysis based on amino acids obtained eight different TRS variants from the circulating strains in Guangxi province ( Table 2). Of the eight TRS variants, three variants (Tet-4, Tet-10a, and Tet-10c) belonged to genotype I, and six variants (NK, Tet-7, Tet-9a, Tet-9b, Tet-10a, and Tet-10b) belonged to genotype II, of which Tet-10a was found in both genotypes I and II. The tetrameric TRS of BNDBNDBNAA (Tet-10a) was the most dominant variant in this study. Sequence alignment revealed that four new tetrameric TRSs were found due to the mutation of a single amino acid (Figure 4), indicating the presence of single nucleotide polymorphism (SNP) in CVR variants. The similarity analysis of the tetrameric TRSs on NCBI database showed that, except for the sequence of BNDBNDBNAA (Tet-10a), the other seven TRS variants did not find 100% identical sequence. Furthermore, it was found that the CVR of B602L gene of the 66 strains was not affected by the region of source, the time of collection and the genotype of the strains.

Analysis of the IGR Between the I73R/I329L Genes
The IGR between the I73R/I329L genes was amplified, sequenced and characterized by the presence of TRSs. The results showed that all the 66 strains from Guangxi province in this study belonged to IGR II variant with an TRS insertion of the sequence GGAATATATA compared to the Georgia 2007/1 (GenBank accession no. FR682468.1), while one previously reported strain (China/Guangxi/2019/domestic pig, GenBank accession no. MK670729) from Guangxi province (36) belonged to IGR III variant with two TRS insertions, indicating that two types of variants (IGR II and IGR III variants) were co-circulating in Guangxi province during 2019-2020 outbreaks (Figure 5). China/GX/202010 strain (GenBank accession no. OM986389) of genotype I and China/GX/201904 strain (GenBank accession no. OM986333) of genotype II from Guangxi province were selected as the representative strains for sequence similarity retrieval by BLAST software in the NCBI database ( Table 3). The genotype II strain had 100% identity of the IGR between I73R/I329L genes with some strains from Eurasian countries,  including Belarus, Ukraine, Russia, Belgium and Poland in Europe, and Mongolia, Vietnam, Indonesia and China in Asia. In addition, the IGR between I73R/I329L genes of the genotype I strain could not found a strain with 100% similarity in NCBI database, and the highest similar strains were the Portuguese strain OURT88/3 and the Spanish strain NHV with a similarity of 99.74%.

DISCUSSION
ASF was first reported in China on August 3, 2018 (5), and then rapidly spread to most provinces in China within a short time (19). According to a report from the Ministry of Agriculture and Rural Affairs of China, by August 2019, ASF had caused a loss of 190.9 million pigs, including 29.4 million sows with a 40.5% decrease in China (37), showing that ASF had caused huge losses to the pig industry within a very short time. It has been reported that genotypes I and II of ASFV were found to be simultaneously epidemic in the pig herds in China (20,21), and the wild-type ASFV strains with different virulence and the naturally gene-deleted ASFV strains with decreased virulence were identified in several provinces in China (22,23), indicating that many different ASFV strains with high genetic diversity were circulating in China. Therefore, the molecular characterization of ASFV strains circulating in China, which has not been reported in detail until now, is necessary to further study.
The tissue samples from dead pigs collected from 86 pig farms during 2019-2020 in Guangxi province, southern China, were tested for ASFV, and a high positive rate of 57.14% (192/336) was found, indicating that ASF occurred seriously in these pig farms. To further analyze the molecular characterization of the circulating ASFVs, 66 positive samples were randomly selected for gene sequencing and analysis. These samples were also tested by a commercial ASFV qPCR detection kit (Lijian, China), and 8 ASFV strains lacked MGF505-2R gene. These situations were further confirmed by the sequence alignment. Recently, there were reports on ASFV strains with different levels of virulence causing a broad range of clinical symptoms in susceptible animals. Per-acute and acute ASFV infections might lead to mortality rate up to 100% in naïve domestic pig populations (5,38,39), while pigs infected with low virulence strains could develop resistance to ASFV leading to an increased number of chronic or subclinical infections (20,22,40,41). Some researchers have reported that the gene-deleted ASFV strains showed decreased virulence and low pathogenicity, such as the non-pathogenic strain OURT88/3 in Portugal lacking the MGF505-2R gene (42), and the attenuated strain BA71 in Spain losing its EP402R (CD2v) gene (43). The naturally gene-deleted ASFV strains with decreased virulence has been reported in several provinces in eastern China (22,23), and the man-made gene-deleted strains with decreased virulence have also been reported in China (44). Of the 66 strains in this study, eight strains lacked MGF505-2R gene. Therefore, the wildtype and the gene-deleted ASFV strains were simultaneously circulating during 2019-2020 in Guangxi province, southern  China, and their harm to the pig industry needs to be further evaluated.
ASF was first reported in Kenya in the 1920s, introduced into Georgia in 2007 and has subsequently spread to Russia, eastern Europe, Asia and Latin America with devastating socioeconomic consequences (3,8,9,45,46). To date, two of the 24 currently described ASFV genotypes defined by the B646L (p72) gene, namely genotypes I and II, have been reported outside Africa, with genotype II being responsible for the ongoing ASF pandemic (13,14). In this study, the positive clinical samples, collected during 2019-2020 outbreaks from Guangxi province, were randomly selected for gene sequence analysis. The B646L (p72) and E183L (p54) gene sequences were used to determine the genotype of the viruses by phylogenetic analysis, while the EP402R (CD2v) gene sequence was analyzed to determine the serogroup. Phylogenetic analysis of the B646L (p72) gene revealed that all 66 strains from Guangxi province belonged to genotypes I (8/66) and II (58/66), and phylogenetic analysis of the E183L (p54) gene revealed that all 66 strains from Guangxi province belonged to genotypes Ia (8/66) and IIa (58/66), indicating that the genotyping by the B646L (p72) and E183L (p54) nucleotide sequences generated similar genotypes for the circulating ASFV strains in Guangxi province. Phylogenetic analysis of the EP402R (CD2v) gene revealed that the strains from Guangxi province could be clustered into two serogroups, serogroups 4 (8/66) and 8 (58/66). Traditionally, genotyping of ASFV strains depended on the phylogenetic analysis of the B646L (p72) and E183L (p54) gene sequences, and the analysis of the E183L (p54) gene sequences could improve the identification of each genotype (30,32,33,47,48). However, some scholars proposed that the B646L (p72) gene could not accurately define the serogroup of ASFV, distinguish viruses with different virulence, or predict the effectiveness of specific ASFV vaccines, so they advocated to determine the serogroup of ASFVs based on the phylogenetic analysis of the EP402R (CD2v) gene (34,49,50). Therefore, it is recommended that genotyping should be combined with serotyping for epidemiological investigation of ASFV. Many reports have used the genetic characteristics of the CVR in the B602L gene for genotyping and subgrouping closely related ASFV strains (25,(30)(31)(32)(33)(34)(35)(48)(49)(50)(51)(52). In this study, the ASFV strains circulating in Guangxi province from 2019 to 2020 presented a multi-complex profile with a high degree of heterogeneity that could be clustered into 8 different TRS variants, and three variants belonged to genotype I and six variants belonged to genotype II ( Table 2). To better track the source of these strains, the similarity analysis between these 8 TRS variants and the strains from GenBank database showed that only the TRS variant BNDBNDBNAA had 100% similar sequences, and the other seven variants could not find a completely consistent sequence. In addition, the situation that the TRSs within CVR of some genotype I strains was identical to those of the genotype II strains was also reported previously (50). The results demonstrated that the CVR in the B602L gene is a hypervariable genetic marker for high-resolution discrimination of viruses that are identical based on their B646L (p72) and E183L (p54) genotypes.
The CVR of the B602L gene has been widely used to distinguish closely related ASFVs, but the relatively low CVR genetic variability necessitates further research on alternative and more informative gene regions, so the sequence analysis of the IGR between I73R/I329L genes is used to determine the relationship within the relevant genotypes and origin of circulating ASFV strains (28,29,32,34,36,49,(52)(53)(54)(55). According to the results in this study, the IGR II and IGR III variants were simultaneously circulating in Guangxi province ( Figure 5). Furthermore, the circulating genotype II strains in Guangxi province had 100% identity with some strains from Eurasian countries ( Table 3). In addition, the IGR between I73R/I329L genes of the circulating genotype I strains in Guangxi province did not find a 100% similar sequence on NCBI database, and the most similar strain was the Portuguese OURT88/3 strain and Spanish NHV strain with 99.74% similarity. Therefore, Chinese ASFV strains, including the strains form Guangxi province, might be derived from Europe, and more genome sequences of ASFV strains circulating in pig herds will help precisely trace the origin of ASFV strains in China.
In conclusion, this study first reported that ASFV strains of two genotypes (genotypes I and II based on the B646L (p72) and E183L (p54) genes) and two serogroups (serogroups 4 and 8 based on the EP402R (CD2v) gene) were simultaneously circulating during 2019-2020 outbreaks in Guangxi province, southern China. There existed wild-type and naturally genedeleted ASFV strains in the field. These strains generated eight different CVR variants within the B602L gene and belonged to IGR II and III variants between I73R/I329L genes. To our knowledge, this is the first detailed report on the molecular characterization of ASFV strains circulating in southern China, and serogroup 4 ASFV strains in China.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material.

AUTHOR CONTRIBUTIONS
KS contributed to study design, laboratory supervision, and manuscript writing and editing. HL contributed to experiments, data analysis, and manuscript drafting. YY, FL, and SF contributed to sample collection and data analysis. HS contributed to study design and manuscript editing. All authors have read and approved the submitted manuscript.