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. 2022 Jun 15;9:912224. doi: 10.3389/fvets.2022.912224

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

Kaichuang Shi 1,2,*,†, Huixin Liu 2,†, Yanwen Yin 1, Hongbin Si 2,*, Feng Long 1, Shuping Feng 1
PMCID: PMC9240437  PMID: 35782548

Abstract

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.

Keywords: African swine fever virus, B646L (p72) gene, E183L (p54) gene, EP402R (CD2v) gene, genotype, serogroup, phylogenetic analysis

Introduction

African swine fever virus (ASFV) is an enveloped double-stranded DNA virus of the genus Asfivirus in the family Asfarviridae (1). ASFV causes African swine fever (ASF), a devastating hemorrhagic disease infecting domestic pigs and wild boars, with high mortality rate up to 100% (2). ASF was first identified in Kenya in the 1920s, Europe in 1957, the Caucasus region and southern Russia in 2007 (3, 4), China in August 2018 (5), and other Asian countries, such as Mongolia, Korea, Vietnam, Laos, Cambodia, the Philippines, and Indonesia, since the end of 2018 (6, 7). Currently, the outbreaks of ASF are still ongoing in Africa, the trans-Caucasus region, eastern Europe, Russian Federation, Asia, and Latin America, which pose a huge challenge to the swine industry in these regions (8, 9). ASF has caused huge economic losses to the swine industry worldwide since the 1920s, and is a notifiable disease to the World Organization for Animal Health (OIE).

The ASFV genome varies by ~170–193 kb and encodes over 168 kinds of protein (10). Based on the partial B646L (p72) gene sequences, ASFV strains from different countries are currently classified into 24 genotypes (11, 12). Of all the 24 genotypes of ASFV found in Africa, only genotypes I and II have outbroken outside Africa until now (13, 14). Genotype I first occurred in Portugal in 1957, and then in other European countries, southern America, and Caribbean island countries (15). Genotype II entered Georgia from southern Africa in 2007 (16), later spread to eastern Europe and westward into western Europe arriving in Belgium in September 2018 (17), and introduced into Asia arriving in China in August 2018 (18). Since then, genotype II strains have spiraled out of control, spreading to multiple Chinese provinces and reaching other Asian countries, such as Vietnam, Cambodia, Laos, and Korea (8, 9).

Since China raises more than 50% of the world's pigs every year, it is very important to master the molecular characterization of ASFV strains circulating in China. ASF was first identified in China in August 2018 (5), where it rapidly spread to most provinces in China within a short time (19). To date, genotypes I and II ASFV strains have been reported in China (20, 21), and the wild-type and gene-deleted ASFV strains have been identified in several provinces in China (22, 23). However, the molecular characterization of ASFV strains circulating in China has been limited, thus the ASFV genotypes mapping in the country is incomplete until now. In this study, the tissue samples, which were collected in the field during the ASF outbreaks from 2019 to 2020 in Guangxi province, southern China, were tested for ASFV by real-time quantitative polymerase chain reaction (qPCR). The positive samples were randomly selected to amplify and sequence the C-terminal region of B646L gene encoding protein 72 (p72), the complete E183L gene encoding protein 54 (p54), the central variable region (CVR) of B602L gene, the full MGF505-2R gene, and the tandem repeat sequence (TRS) of intergenic region (IGR) between the I73R and I329L genes (I73R/I329L genes), and analyze the genetic characteristics and variations of ASFV strains circulating in Guangxi province. Furthermore, the serogroups of ASFV strains were determined by sequence analysis of the variable region of EP402R gene encoding protein CD2v (pCD2v). This is the first detailed report on the molecular characterization of the ASFV strains circulating in southern China.

Materials and Methods

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 phosphate-buffered 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 (24–29) with some modifications. The targeted genes were amplified by PCR using the Tks Gflex™ 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).

Table 1.

Primers used for evaluation and amplification of ASFV in this study.

Target Name Primer sequence (5′-3′) References
B646L (p72) p72-U GGCACAAGTTCGGACATGT (24)
p72-D GTACTGTAACGCAGCACAG
E183L (p54) p54-U CGAAGTGCATGTAATAAACGTC (25)
p54-D TGTAATTTCATTGCGCCACAAC
B602L (CVR) B602L-U AATGCGCTCAGGATCTGTTAAATCGG (26)
B602L-D TCTTCATGCTCAAAGTGCGTATACCT
MGF505-2R MGF505-2R-U GCAGAGGTATGATGTCCTTA (27)
MGF505-2R-D TTCCTGTTGAACAAGTATCT
I73R/I329L I73R/I329L-U CCATTTATCCCCCGCTTTGG (28)
I73R/I329L-D TCGTCATCCTGAGACAGCAG
EP402R (CD2v) CD2v-U YCTGTTGATTCCCCAACTATTACA (29)
CD2v-D ATGGCGGGATATTGGGTAGT
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Genomic Analysis

Nucleotide and amino acid alignments were carried out using the EditSeq and Megalign program in the DNAstar 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 (30–32). 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).

Results

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.

Amplification of the Targeted Genes of ASFV

A total of 66 positive samples were randomly selected for amplifying six targeted genes, including B646L (p72), E183L (p54), EP402R (CD2v), B602L (CVR), MGF505-2R and IGR between I73R/I329L genes. The results showed that the B646L (p72), E183L (p54), EP402R (CD2v), B602L (CVR), and IGR between I73R/I329L genes were amplified from all the 66 positive samples. However, the MGF505-2R gene fragments could be amplified from 58 of the 66 samples. The targeted fragments were purified, cloned and sequenced. The generated sequences of the 66 strains from Guangxi province have been deposited in GenBank under accession numbers OM986132-OM986197 for the B646L (p72) gene, OM986396-OM986461 for the E183L (p54) gene, OM986264-OM986329 for the EP402R (CD2v) gene, OM986198-OM986263 for the B602L (CVR) gene, OM986330-OM986395 for the IGR between I73R/I329L genes, and OM986074-OM986131 for the MGF505-2R gene (Table 2).

Table 2.

Summary of the 66 ASFV strains from domestic pigs in Guangxi province in this study.

No. Strain Date B646L E183L CVR profile TRS ER402R GenBank accession number
B646L (p72) E183L (p54) EP402R (CD2v) B602L (CVR) IGR between I73R/I329L MGF505-2R
1 China/GX/201901 Jan-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986132 OM986396 OM986264 OM986198 OM986330 OM986074
2 China/GX/201902 Jan-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986133 OM986397 OM986265 OM986199 OM986331 OM986075
3 China/GX/201903 Jan-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986134 OM986398 OM986266 OM986200 OM986332 OM986076
4 China/GX/201904 Mar-2019 II IIa BNDBND-NAA Tet-9a 8 OM986135 OM986399 OM986267 OM986201 OM986333 OM986077
5 China/GX/201905 May-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986136 OM986400 OM986268 OM986202 OM986334 OM986078
6 China/GX/201906 May-2019 II IIa —–(NK)—– (NK) 8 OM986137 OM986401 OM986269 OM986203 OM986335 OM986079
7 China/GX/201907 May-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986138 OM986402 OM986270 OM986204 OM986336 OM986080
8 China/GX/201908 May-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986139 OM986403 OM986271 OM986205 OM986337 OM986081
9 China/GX/201909 May-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986140 OM986404 OM986272 OM986206 OM986338 OM986082
10 China/GX/201910 May-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986141 OM986405 OM986273 OM986207 OM986339 OM986083
11 China/GX/201911 May-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986142 OM986406 OM986274 OM986208 OM986340 OM986084
12 China/GX/201912 Aug-2019 II IIa BNDBN—AA Tet-7 8 OM986143 OM986407 OM986275 OM986209 OM986341 OM986085
13 China/GX/201913 Aug-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986144 OM986408 OM986276 OM986210 OM986342 OM986086
14 China/GX/201914 Aug-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986145 OM986409 OM986277 OM986211 OM986343 OM986087
15 China/GX/201915 Aug-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986146 OM986410 OM986278 OM986212 OM986344 OM986088
16 China/GX/201916 Aug-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986147 OM986411 OM986279 OM986213 OM986345 OM986089
17 China/GX/201917 Aug-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986148 OM986412 OM986280 OM986214 OM986346 OM986090
18 China/GX/201918 Aug-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986149 OM986413 OM986281 OM986215 OM986347 OM986091
19 China/GX/201919 Aug-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986150 OM986414 OM986282 OM986216 OM986348 OM986092
20 China/GX/201920 Aug-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986151 OM986415 OM986283 OM986217 OM986349 OM986093
21 China/GX/201921 Sep-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986152 OM986416 OM986284 OM986218 OM986350 OM986094
22 China/GX/201922 Sep-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986153 OM986417 OM986285 OM986219 OM986351 OM986095
23 China/GX/201923 Sep-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986154 OM986418 OM986286 OM986220 OM986352 OM986096
24 China/GX/201924 Sep-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986155 OM986419 OM986287 OM986221 OM986353 OM986097
25 China/GX/201925 Sep-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986156 OM986420 OM986288 OM986222 OM986354 OM986098
26 China/GX/201926 Sep-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986157 OM986421 OM986289 OM986223 OM986355 OM986099
27 China/GX/201927 Oct-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986158 OM986422 OM986290 OM986224 OM986356 OM986100
28 China/GX/201928 Oct-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986159 OM986423 OM986291 OM986225 OM986357 OM986101
29 China/GX/201929 Oct-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986160 OM986424 OM986292 OM986226 OM986358 OM986102
30 China/GX/201930 Oct-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986161 OM986425 OM986293 OM986227 OM986359 OM986103
31 China/GX/201931 Oct-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986162 OM986426 OM986294 OM986228 OM986360 OM986104
32 China/GX/201932 Oct-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986163 OM986427 OM986295 OM986229 OM986361 OM986105
33 China/GX/201933 Oct-2019 II IIa BND-NDBNAA Tet-9b 8 OM986164 OM986428 OM986296 OM986230 OM986362 OM986106
34 China/GX/201934 Oct-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986165 OM986429 OM986297 OM986231 OM986363 OM986107
35 China/GX/201935 Oct-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986166 OM986430 OM986298 OM986232 OM986364 OM986108
36 China/GX/201936 Oct-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986167 OM986431 OM986299 OM986233 OM986365 OM986109
37 China/GX/201937 Oct-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986168 OM986432 OM986300 OM986234 OM986366 OM986110
38 China/GX/201938 Oct-2019 II IIa BNDBNDFNAA Tet-10b 8 OM986169 OM986433 OM986301 OM986235 OM986367 OM986111
39 China/GX/201939 Oct-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986170 OM986434 OM986302 OM986236 OM986368 OM986112
40 China/GX/201940 Oct-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986171 OM986435 OM986303 OM986237 OM986369 OM986113
41 China/GX/201941 Oct-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986172 OM986436 OM986304 OM986238 OM986370 OM986114
42 China/GX/201942 Oct-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986173 OM986437 OM986305 OM986239 OM986371 OM986115
43 China/GX/201943 Oct-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986174 OM986438 OM986306 OM986240 OM986372 OM986116
44 China/GX/201944 Oct-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986175 OM986439 OM986307 OM986241 OM986373 OM986117
45 China/GX/201945 Oct-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986176 OM986440 OM986308 OM986242 OM986374 OM986118
46 China/GX/201946 Oct-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986177 OM986441 OM986309 OM986243 OM986375 OM986119
47 China/GX/201947 Oct-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986178 OM986442 OM986310 OM986244 OM986376 OM986120
48 China/GX/201948 Nov-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986179 OM986443 OM986311 OM986245 OM986377 OM986121
49 China/GX/201949 Nov-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986180 OM986444 OM986312 OM986246 OM986378 OM986122
50 China/GX/201950 Nov-2019 II IIa BNDBNDBNAA Tet-10a 8 OM986181 OM986445 OM986313 OM986247 OM986379 OM986123
51 China/GX/202001 Jan-2020 II IIa BNDBNDBNAA Tet-10a 8 OM986182 OM986446 OM986314 OM986248 OM986380 OM986124
52 China/GX/202002 Jan-2020 I Ia BN——AA Tet-4 4 OM986183 OM986447 OM986315 OM986249 OM986381 /
53 China/GX/202003 Jan-2020 I Ia BNDBNDBNAA Tet-10a 4 OM986184 OM986448 OM986316 OM986250 OM986382 /
54 China/GX/202004 Jan-2020 I Ia BNDBNDBNAA Tet-10a 4 OM986185 OM986449 OM986317 OM986251 OM986383 /
55 China/GX/202005 Jan-2020 I Ia BNDBNDBNAA Tet-10a 4 OM986186 OM986450 OM986318 OM986252 OM986384 /
56 China/GX/202006 Jan-2020 II IIa BNDBNDBNAA Tet-10a 8 OM986187 OM986451 OM986319 OM986253 OM986385 OM986125
57 China/GX/202007 Jan-2020 I Ia BNDBNDBNAA Tet-10a 4 OM986188 OM986452 OM986320 OM986254 OM986386 /
58 China/GX/202008 Jan-2020 I Ia BNDBNDBNBA Tet-10c 4 OM986189 OM986453 OM986321 OM986255 OM986387 /
59 China/GX/202009 Jan-2020 I Ia BNDBNDBNAA Tet-10a 4 OM986190 OM986454 OM986322 OM986256 OM986388 /
60 China/GX/202010 Jan-2020 I Ia BNDBNDBNAA Tet-10a 4 OM986191 OM986455 OM986323 OM986257 OM986389 /
61 China/GX/202011 Feb-2020 II IIa BNDBNDBNAA Tet-10a 8 OM986192 OM986456 OM986324 OM986258 OM986390 OM986126
62 China/GX/202012 Sep-2020 II IIa BNDBNDBNAA Tet-10a 8 OM986193 OM986457 OM986325 OM986259 OM986391 OM986127
63 China/GX/202013 Nov-2020 II IIa BNDBNDBNAA Tet-10a 8 OM986194 OM986458 OM986326 OM986260 OM986392 OM986128
64 China/GX/202014 Nov-2020 II IIa BNDBNDBNAA Tet-10a 8 OM986195 OM986459 OM986327 OM986261 OM986393 OM986129
65 China/GX/202015 Dec-2020 II IIa BNDBNDBNAA Tet-10a 8 OM986196 OM986460 OM986328 OM986262 OM986394 OM986130
66 China/GX/202016 Dec-2020 II IIa BNDBNDBNAA Tet-10a 8 OM986197 OM986461 OM986329 OM986263 OM986395 OM986131
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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).

Figure 1.

Figure 1

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Nucleotide sequence alignment of the strains lacking the MGF505-2R gene. Eight clinical samples lacking the MGF505-2R gene by the commercial qPCR detection kit (Lijian, China) were further verified by sequence alignment, and confirmed to be lacking the partial MGF360-9R, whole MGF360-10R~MGF360-14R, MGF505-1R and MGF505-2R, and partial MGF505-3R genes with a total deletion of 10,134 bp.

Figure 2.

Figure 2

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Phylogenetic trees based on nucleotide sequences of the B646L (p72) (A) and E183L (p54) (B) genes. The evolutionary history was inferred by the maximum likelihood method based on the Kimura 2-parameter model [B646L (p72) gene] and the Kimura 2-parameter + G model [E183L (p54) gene]. The phylogeny was inferred following 1,000 bootstrap replications, and node values showed percentage bootstrap support. (A) Phylogenetic analysis of the B646L (p72) gene. The ASFV strains from Guangxi province were clustered into genotypes I and II. (B) Phylogenetic analysis of the E183L (p54) gene. The ASFV strains from Guangxi province were clustered into genotypes Ia and IIa. The strains from this study were highlighted with black squares (■) before the names.

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 from GenBank (Supplementary Table 1), could be clustered into 24 genotypes. The 66 strains from this study distributed in genotypes I and II, of which 8 strains belonged to genotype I and 58 strains belonged to genotype II (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 sub-genotype 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).

Figure 3.

Figure 3

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Phylogenetic tree based on nucleotide sequences of the ER402R (CD2v) gene. The evolutionary history was inferred by the maximum likelihood method based on the Tamura 3-parameter + G model. The phylogeny was inferred following 1,000 bootstrap replications, and node values showed percentage bootstrap support. The ASFV strains from Guangxi province were clustered into serogroups 4 and 8. The strains from this study were highlighted with black squares (■) before the names.

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.

Figure 4.

Figure 4

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Amino acid sequence alignment of the tetrameric tandem repeat sequences (TRSs) of the central variable region (CVR) of the B602L gene. The ASFV strains from Guangxi province generated 8 different TRS variants. Only one representative strain of each variant was presented in this figure. Codes were as follows and as previously described: (CAST, CVST, CTST, CASI = A) (CADT, CADI, CTDT, CAGT, CVDT = B) (GAST, GANT = C) (CASM = D) (CANT, CAAT = F) (CTNT = G) (RAST = H) (GTDT = J) (CTSP = K) (YTNT = L) (NEDT = M) (NVDT, NVGT, NVDI = N) (NANI, NADI, NASI = O) (SAST = S) (NVNT = T) (NIDT, NTDT = U) (NAGT, NAST, NAVT, NADT, NANT = V) (SADT, SVDT = W) (NTDI = X).

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).

Figure 5.

Figure 5

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Nucleotide sequence alignment of the intergenic region (IGR) between I73R/I329L genes. The ASFV strains from Guangxi province belonged to IGR II with one insertion of 10 nucleotides (GGAATATATA) and IGR III with two insertions of 10 nucleotides (GGAATATATA). All the strains from this study belonged to IGR II and only partial strains were presented in this figure.

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%.

Table 3.

Similarity analysis of the tandem repeat sequence (TRS) between I73R/I329L genes.

Strain Accession No. Date Origin Fragment/bp Similarity (%)
China/GX/202010 (genotype I)
China/GX/202010 OM986389 2020 China 385 –
NHV NC044943 1968 Spain 385 99.74
OURT 88/3 NC044957 2004 Portugal 385 99.74
China/Guangxi/2019/domestic pig MK670729 2019 China 356 91.37
Bel13/Grodno KJ620043 2013 Belarus 367 90.72
Ukr12/Zapo KJ620037 2012 Ukraine 366 90.72
VN/Pig/Hanoi/02 MW054561 2019 Vietnam 367 90.72
Indo/2020/Pig/West Java MT851947 2020 Indonesia 363 90.65
China/GX/201904 (genotype II)
China/GX/201904 OM986333 2019 China 366 –
VN/Pig/Hanoi/2019/01 MT332153 2020 Vietnam 366 100
Bel13/Grodno KJ620043 2014 Belarus 366 100
Ukr12/Zapo KJ620037 2014 Ukraine 366 100
VN/Pig/Hanoi/02 MW054561 2020 Vietnam 366 100
Indo/2020/Pig/West Java MT851947 2020 Indonesia 363 100
Voronezh 2016 KY385893 2016 Russia 362 100
MNG/7/BU / 2019 MT852023 2019 Mongolia 357 100
CN2018/01 MH735144 2018 China 346 100
Belgium/2018/Etalle MH998359 2018 Belgium 342 100
Tula06/2012 KP137625 2012 Russia 219 100
Pol20_36120_O52/20 MT951797 2020 Poland 217 100
China/Guangxi/2019/domestic pig MK670729 2019 China 356 97.19
ASFV-wbBS01 MK238345 2019 China 341 97.15
China/Jilin/2018/boar MK189457 2019 China 336 97.11
Arm07 KJ620028 2014 Armenia 356 97
VN/Pig/Hanoi/07 MW054562 2019 Vietnam 357 97
MAL/19/Karonga/4 MN755870 2019 Malawi 357 96.99
Tan_17_01 MK577991 2019 Tanzania 337 96.83
MAL/19/Karonga/2 MN755868 2019 Malawi 357 96.73
MAL/19/Karonga/1 MN755867 2019 Malawi 356 96.72
Irkutsk2017 KY982843 2017 Russia 355 96.69
4/Ol/02 KT718707 2017 Italy 345 90.23
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China/GX/202010 strain was used as the representative of genotype I, and China/GX/201904 strain was used as the representative of genotype II.

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 wild-type 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, 30–35, 48–52). 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, 52–55). 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 gene-deleted 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.

Funding

This study was supported by the Key Research and Development Program (AB21238003) and the Science and Technology Major Project (AA17204057) of Guangxi Science and Technology Bureau, China, and the Agricultural Science and Technology Program (Z201954, Z202031) of Guangxi Agricultural and Rural Bureau, China.

Conflict 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.

Publisher's Note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Acknowledgments

We thank to Guangxi Center for Animal Disease Control and Prevention (CADC) for providing all the clinical samples used in this study. Guangxi CADC was approved by the Ministry of Agriculture and Rural Affairs, China for collection and detection of ASFV in clinical samples (Approval Number: 2018-154-25).

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fvets.2022.912224/full#supplementary-material

Click here for additional data file. (36KB, DOCX)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

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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.

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