Molecular Characterization of African Swine Fever Viruses Circulating in Can Tho City, Vietnam

African swine fever (ASF) is a highly contagious and deadly viral disease in domestic and feral pigs. Since 2018, the disease has spread and caused large socioeconomic consequences to the pig industry in several Asian countries including China, Vietnam, and South Korea. This study aims to determine the genotype, serotype, and genetic variation of representative ASF viruses (ASFV) responsible for the outbreaks in 2019–2022 in Can Tho city, a central administrative province in the Mekong delta, Vietnam. For outbreak investigation, the presence of causative ASFVs was tested using conventional PCR targeting the B646L gene. Subsequently, the amplification and sequencing of the DNA fragments of the putative B646L gene encoding the major capsid protein p72, EP402R gene encoding the viral hemagglutinin CD2-like protein (CD2v), and intergenic region (IGR) between the l73R and I329L genes were performed for molecular characterization. Phylogenetic analyses based on B646L and EP402R genes confirmed that all ASFVs detected in Can Tho city belonged to genotype 2 and serotype 8. In addition, this study revealed that at least two variants of ASFVs, namely, IGR II and IGR III, based on the nucleotide variation of the IGR sequence, cocirculated, and caused outbreaks in Can Tho city. The molecular characterization study provides great significance for understanding the evolution of ASFVs and tracing possible sources of infection in Can Tho and Mekong delta.


Introduction
African swine fever (ASF) is a highly contagious disease in swine characterized by acute hemorrhagic fever and high mortality. Owing to the high transmissibility and serious socioeconomic consequences of the disease, the World Organization for Animal Health (OIE) classifed ASF as a list A notifable disease (https://www.woah.org/en/what-we-do/ animal-health-and-welfare/animal-diseases/old-classifcationof-diseases-notifable-to-the-oie-list-a/). ASF is caused by African swine fever virus (ASFV), which belongs to the Asfvirus genus, the only member of the Asfarviridae family [1]. Te ASFV genome is a linear double-stranded DNA of approximately 170-193 kbp encoding at least 160 open reading frames [2,3]. Currently, there are 24 genotypes and 8 serotypes that have been reported worldwide based on the B646L gene encoding the major capsid protein p72 and EP402R gene encoding the viral hemagglutinin CD2-like protein (CD2v), respectively [4][5][6]. Furthermore, diferentiation between close strains within each genotype can be determined by the variation of the nucleotide sequences of the partial intergenic region (IGR) located between the I73R and I329L genes [7].
In Vietnam, the frst ASF outbreak was reported at a backyard pig farm in the northern province in February 2019 [8]. Since then, serial outbreaks of ASF in domestic pigs have continuously been reported and have become endemic across the country [9]. Recently, several studies have been conducted to determine the genotypes and serotypes of ASFV circulating in Vietnam. All of these studies indicated that ASFVs causing outbreaks also belonged to genotype 2 and serotype 8 as in previous studies, suggesting that these are the most predominant serotype and genotype in Vietnam [9][10][11]. Furthermore, genetic characterization has been used previously to genetically diferentiate the variation between closely related ASFVs based on the analysis of the IGR between the I73R and I329L genes [7]. In Vietnam, IGR I and IGR II variants with three tandem repeat sequences (TRS) were initially reported in the northern areas in 2020 [12], and variant IGR III with four TRS was later found in at least four diferent provinces of Northern Vietnam during the 2019-2022 outbreaks [9], indicating that multiple variants of genotype II of ASFVs currently circulate in Vietnam.
In Southern Vietnam, limited molecular studies on ASFVs have been conducted. A few published studies showed that ASFVs circulating in this region also belonged to genotype 2 and serotype 8 [13,14]. However, these studies still provide limited information on the molecular properties and epidemiology of this virus. Terefore, further investigations are needed to provide more insights into the genetic characterization and variation of ASFVs. In this study, Can To city, which is considered an epicenter of ASF in the region due to its geographical and socioeconomic importance in the Mekong delta, has been selected as a representative study site for the molecular investigation of ASFVs in the Mekong delta. Tis study aims to determine the genotype, serotype, and genetic variations of representative ASFVs causing outbreaks in Can To city in 2019-2022 based on the sequences of B646L (p72) and EP402R (CD2v) genes and the TRSs between the I73R and I329L genes, respectively.

Study Areas and Sample Collection.
During 2019-2022, seven representative ASF outbreaks in domestic pigs reported by the provincial Can To Sub-Department of Animal Health were selected for this study. An outbreak was defned as at least one blood or tissue sample taken from afected pigs on a farm returning a positive result when tested with ASF real-time PCR. Tese seven outbreaks occurred in six (out of nine) administrative units of Can To that were selected for the purposes of this study, namely, including Binh Tuy, Cai Rang, Co Do, O Mon, Toi Lai, and Vinh Tanh ( Figure 1). Tissue samples including sets of lymph nodes and spleens and/or whole blood were collected from fatal and symptomatic pigs with ASF to detect causative ASFVs and molecular characterization. Te details of space-time and types of collected samples are shown in Table 1.

DNA Extraction and PCR Assay.
Total viral DNAs were extracted from the collected tissue samples or whole blood using the TopPURE ® Tissue Viral Extraction kit and Top-PURE ® Serum Viral Extraction kit (TBR, Vietnam), respectively, following the recommendation of the manufacturer. Genomic DNA was eluted in 35 μL of elution bufer of the kit and stored at −80°C until further use. Next, the DNA extracts from ASFV-infected samples were used for conventional PCR amplifcation with three pairs of primers of (i) P72-U/P72-D targeting the B646L gene [4], (ii) ASF_CD2v_ga3611-F/ASF_CD2v_ga4220-R targeting the EP402R gene [15], and (iii) ASF_IGR_I73R_F/ASF_I-GR_I73R_R targeting the fragment of IGR located in the I73R and I329L genes [14]. Te detail of the primer sequences used in this study is described in Table 2.
Te PCR amplifcation reactions were performed in a 22 μL volume, containing 11 μL MyTaq ™ master mix (Bioline, USA), 3 μL template DNA, 1 μL of 10 μM each primer (forward and reverse), and 6 μL ultrapure water. Te thermocycling condition for PCR detection of ASFV followed 40 cycles of denaturation at 98°C for 10 s and extension at 72°C for 60 s with a 7-min elongation at 72°C after an initial denaturation step at 95°C for 5 min. Te following thermal cycling program was used with a moderate modifcation in annealing temperature from 55°C to 58°C based on the melting temperature of the primers indicated in Table 2. Te products of PCR amplifcation were electrophoretically analyzed on a 1.5% agarose gel containing ethidium bromide (1 μg/mL) in 1 × Tris-acetate-EDTA (TAE) bufer before visualization and imaging using UV transillumination (BIO-RAD, USA).
Te DNA purifed products were sent to Genlab Co., Ltd., Vietnam, for Sanger sequencing. Sanger sequencing that used the ABI Prism BigDye ™ Terminator v1.1 cycle sequencing kit was conducted using an by ABI PRISM 3500 × l Genetic Analyzer. Te nucleotide sequences of seven ASFVs derived from three defned gene fragments were analyzed with BioEdit ® Sequence Alignment Editor version 7.1.9. Te basic local alignment search tool was used to verify the similarities of the sequences obtained in this study with reference sequences retrieved from the GenBank. Te Clustal W process in MEGA 7.0 was used for sequence alignment, and nucleotide positions after alignment were numbered according to the numbering scheme of Georgia 2007/1 (NC_044959.2) throughout the text. A maximumlikelihood tree for each B646L and EP402R gene sequence was constructed using MEGA 7.0 with a resampling process of 1000 replicates to determine the genotype and serotype of ASFVs [4,6,16].

Laboratory Confrmation of ASFV.
During the period of 2019-2022, seven representative outbreaks of ASF in domestic pigs were subjected to this molecular investigation. Tissue samples and/or whole blood collected from fatal and symptomatic pigs in six representative districts of Can To were tested for the presence of ASF viral DNA using conventional PCR. Diagnostic confrmation was conducted based on the ASFV-specifc primers, B646L gene encoding for protein p72, EP402R gene encoding for protein CD2v, and IGR between the I73R and I329L genes. Figure 2 shows the PCR results with evident amplicons of the expected sizes that confrmed the presence of ASFVs in all collected samples.

Genotyping ASFVs Using the Partial B646L (p72) Gene.
To determine the genotype and genetic relationship of the ASFVs in this study and other reference ASFVs previously deposited in the GenBank, a phylogenetic tree was constructed based on the partial sequences of the B646L (p72) gene. Te phylogenetic tree indicated that all Can To ASFVs were grouped into genotype 2, together with ASFVs previously detected in Vietnam and China ( Figure 3).

Serotyping ASFV Using the Partial EP402R (CD2v) Gene.
To determine the serotype of the representative ASFVs, another phylogenetic tree based on the partial sequence of the EP402R (CD2v) gene was constructed. Te result indicated that Can To ASFVs clustered into serotype 8 is identical to the serotype of ASFVs detected in Vietnam, China, and Korea (Figure 4(a)). Noteworthily, the sequence alignment of the EP402R gene detected an 18-bp nucleotide deletion of "CTACTACCCAATATCCCG" that results in six-amino-acid deletion "LLPNIP" in the CD2v of ASF/VN/    CanTo-OM/2021 (Figure 4(b)). Te short mutation sequence in the EP402R gene was confrmed as unique because there was no homology to other sequences previously deposited in the GenBank (data not shown).

Discussion
ASF has been endemic in Can To and it posed a major obstacle to the development of the pig industry in the region. A total of 2,377 ASF outbreaks have been reported across nine districts in this region in early 2019 [17]. To the best of our knowledge, this study is the frst report on the genetic characterization of representative ASFVs that caused outbreaks in Can To city in 2019-2022.
A phylogenetic tree based on the putative gene B646L (p72) was constructed to determine the genotype of ASFVs. Our result showed that all ASFVs in this study belonged to genotype 2 ( Figure 3). Tis result is in agreement with several previous studies that have reported that only ASFVs belonging to genotype 2 have been in circulation in Vietnam [10,11]. Tis fnding indicates that ASFV infections that were initially described only in Northern Vietnam (north central and the Red River delta regions) may have spread to the southern region through movement, trade of pigs, and pig products despite the ofcial control strategies in those regions. Since then, it has been confrmed that genotype 2 is the most prevalent genotype in the southern region since the molecular characterization of the circulating ASFVs originating from local outbreaks was serially grouped into genotype 2.
In addition, to determine the serotype of the studied viruses, further genetic analyses of seven ASFVs in Can To based on the phylogenetic tree of the EP402R (CD2v) gene were performed. Te EP402R phylogenetic analysis showed that the ASFVs detected in Can To together with other reference strains from Vietnam, China, and South Korea are a member of serogroup 8 (Figure 4(a)). However, the sequence alignment of the EP402R gene detected a mutation with the absence of an 18-bp nucleotide (six-amino-acid deletion "LLPNIP") in a single strain, ASF/VN/CanTo-OM/2021, out of the seven representative ASFVs (Figure 4(b)). Importantly, the short mutation sequence was confrmed as a unique mutant because there was no Although only ASFV genotype 2 and serotype 8 were identifed in Can To, our analysis of the TRS in the IGR indicates more diversity among these isolates. It has been reported that the analysis of the IGR between the I73R and I329L genes based on the TRS "GGAATATATA" has previously been used for distinguishing between closely related ASFVs [7]. Te current analysis of IGR that used seven samples collected during the 2019-2022 outbreaks revealed that there were two diferent variants, namely, IGR II and IGR III variants of ASFV genotype 2, circulating in Can To     K  P  L  P  S  I  P  L  L  P  N  I  P  P  L  domestic pig population. Te IGR II variants were the most prominent (6/7 strains) and were detected in pigs in six of the provinces tested, followed by IGR III (1/7 strains) ( Figure 5). Te cocirculating of IGR II and III detected in the current study suggests that the spread of ASFVs was most likely caused by transportation of infected pigs from northern to southern localities [9,12]. Interestingly, IGR III variants have never been detected in samples collected from Can To or any other regions in subsequent years. Terefore, it can be suggested that IGR variant II, which was initially found in northern provinces of Vietnam, spread further into almost all geographical locations of Vietnam before giving rise to variant III (in 2020-2022) when ASF occurred serially across the whole country before spreading into Can To city.

C A C T A C C T A G T A T C C C G C T A C T A C C C A A T A T C C C G C C A T T A T C T
Altogether, this study revealed that representative ASFVs causing outbreaks in Can To city, a central administrative region in the Mekong delta, belonged to genotype 2 and serotype 8, and at least two variants (based on the classifcation of TRS in the IGR) were detected in the area. Terefore, continuous surveillance and genetic characterization are needed to facilitate sufcient understanding of local ASFV dynamics, epidemiology, and incursion of ASFV in Can To, Vietnam.

Data Availability
Te data that support the fndings of this study are available upon request from the authors.

Conflicts of Interest
Te authors declare that they have no conficts of interest.