RAPID IDENTIFICATION FOR SEROTYPING OF AFRICAN SWINE FEVER VIRUS BASED ON THE SHORT FRAGMENT OF THE EP402R GENE ENCODING FOR CD2-LIKE PROTEIN

The first confirmed case of African swine fever (ASF) in Vietnam was officially reported in February 2019. To date, the ASF virus (ASFV) has been detected in all 63 cities/provinces in Vietnam. In order to get a better understanding of the potential role of the EP402R gene in a grouping of ASFV serotypes, thirty ASFV sequences of EP402R genes (accession numbers: MN711757-86) from North Central Coast of Vietnam and 68 well-known references of serotype groups from previous studies were further analyzed. Interestingly, we found that a short fragment of 90 nucleotides was very typical for 8 serological groups of ASFVs. A primer set was designed to amplicon the short fragment of 90 nucleotides using the Primer3 program to establish a simplified method for the serotyping of ASFV. Our results indicated that phylogenetic analysis of the short fragment (90 nucleotides) of the EP402R gene is a very specific and useful method for ASFV serotyping when compared to the previous method using a long fragment (816 nucleotides) of this gene and well-known serotype references based on haemadsorption inhibition (HAI) assay.


INTRODUCTION
Since the first ASF outbreak in China in 2018, the disease has spread to some countries in Asia and the Pacific, including Mongolia, Vietnam, Cambodia, Korea, Laos, Myanmar, Philippines, Timor-Leste, Papua New Guinea and Indonesia. The first case of ASF outbreak in Vietnam was officially reported in February 2019, and to date, the disease has spread in 63/63 provinces/cities of Vietnam. Approximately six million pigs have been culled in infected farms, suggesting the risk of spreading of this virus. Many active measures were conducted by Vietnamese Government to inhibit the rapid spread of ASF virus. In a previous study, we have indicated that ASFVs isolated from 30 clinical samples in North Central Coast of Vietnam belonged to genotype II, serotype 8 and contained additional tandem repeat sequences between the I73R and the I329L protein genes [1]. All sequences have been deposited to GenBank with accession numbers as MN711727-56 for p72 genes, MN711757-86 for EP402R genes and MN733925-54 for TRS genes. Similarly, the data obtained from an ASFV isolated in the Red River Delta of Vietnam also indicated that this field strain belongs to genotype II and serotype 8 [2], suggesting that ASFVs circulating in Vietnam are very close to Eastern European and Chinese strains. Additionally, recent reports suggested that the updates of research methods based on current databases of ASFV sequences are very important for ASFV diagnosis, genotyping and serotyping [3,4].
To date, at least eight ASFV serogroups have been reported and some previous studies have suggested that ASF protective immunity appears to be serotype-specific. Additionally, it has been indicated that serotyping of ASFV is very important for vaccine design and development. Currently, two methods have been developed for grouping of ASFV serotypes, including HAI (haemadsorption inhibition) assay and long fragment PCR based on sequencing method developed by Sanna [5]. However, HAI assay has shown a disadvantage in which this method may not apply in the cases of non-haemadsorbing ASFV viruses, suggesting that PCR based on sequencing method is a good candidate for serotyping of ASFV [6,7]. ASFV serotyping method based on a long fragment (816 nucleotides) of EP402R gene has been first described in 2017 [5]. The primer set for this PCR sequencing method was designed based on genotype I sequences and some previous studies have indicated their successes when using this system for ASFV serotyping [5,8,9]. In order to get a better understanding of the potential role of EP402R gene in grouping of ASFV serotypes, thirty sequences of EP402R genes (accession numbers: MN711757-86) were further analyzed. Interestingly, we found that a short fragment of 90 nucleotides was very typical for 8 serological groups of ASFVs. Thus, in this study, we established a simplified method for ASFV serotyping based on the short fragment of 90 nucleotides of EP402R gene coding for CD2-like protein.

Virus isolation
The porcine alveolar macrophages (PAM) cultures used for the isolation of the virus were derived from domestic pigs that were tested negative for ASFV upon diagnosis with conventional PCR as previously described [3,10], and according to the recommendation by the World Organization for Animal Health [10]. Briefly, these cells were obtained and resuspended in Dulbecco's modified Eagle's medium (DMEM, Thermo Fisher Scientific) supplemented with streptomycin (Sigma-Aldrich), ampicillin (Sigma-Aldrich) and 5% fetal calf serum (FCS, Sigma-Aldrich) to a final concentration of 1 x 10 6 cells/mL. Six-well culture plate (Thermo Scientific), containing 2 mL of the cell suspension, were inoculated with 50 µL of a clarified 10 % spleen or lymph node suspension. After three days of incubation at 37 o C, in an atmosphere with 5% CO 2 , the first passage virus was harvested by freezing and thawing. For the second passage, 50 µL of this virus harvest was added to 2 mL fresh PAMs, then incubated and harvested as described for the first passage. For the third passage, 50 µL of this virus harvest was added to 2 mL fresh PAMs, then incubated at 37 o C, in an atmosphere with 5% CO 2 . After one-day incubation, a 20-µl preparation of 1% homologous red blood cells (RBC) in buffered saline was added to each well. The plates were examined for HAD and cytopathic effects over a 4-day period in PAM cells according to the recommendation by World Organization for Animal Health [10].

Conventional PCR method
The DNA of ASFV were extracted from infected pig-organ samples using PureLink Viral RNA/DNA Mini Kit (Invitrogen, Carlsbad, CA, USA) as given by the manufacturer's instructions. Conventional PCR were performed to amplify the partial gene CD2v (CP204L) gene from Vietnam ASFV strains using the specific primer set CD2-2F and 2R, to amplify a 816-bp fragment of EP402R gene coding for the cytoplasmic portion of CD2-like protein as described previously [5,11]. In the second conventional PCR, we designed a set of primers to amplify of short fragment CDS of CD2v gene,

Genomic characterization
Amplification products were electrophoresed on a 1.5% agarose gel against a 100 bp DNA leader marker (Thermo Scientific) and visualized by UV irradiation and ethidium bromide staining (Sigma-Aldrich, Louis, MO, USA). Amplicons of the correct size were excised from the agarose gel and purified using the QIAQuick gel extraction kit (QIAgen, Valencia, CA, USA) according to the manufacturer's specifications for sequencing (1st BASE, Selangor, Malaysia). The percentage of identities and similarity scores of the viral gene sequences were calculated using DNASTAR Lasergene (DNASTAR, Inc., Madison, USA). The gene identities and similarities of two ASFV strains from Vietnam were compared with the reference sequences of ASFV strains from the National Center for Biotechnology Information (NCBI).

RESULTS AND DISCUSSION
It has been indicated that CD2v protein of ASFV is required for detecting the HAD phenotype and mediating the HAI serological groups [7]. Additionally, the analysis of CD2v sequence is simple method for ASFV serotyping [6]. The first description of CD2v serogroups of ASFV based on 816 bq long fragment is reported in 2017 by Sanna [5] and this method has been applied successfully for ASFV serotyping [9]. In this study, two ASFV isolates from North Central Coast of Vietnam were used for viral isolation in combination with HAD test and a primer set to amplify the short fragment of CD2v gene was designed using Primer3 software (named as CV2v R1 and CD2v F1). Additionally, PCR primers developed by Sanna [5] with slight modification based on our previous results and GenBank databases for genotypes II were employed to establish a new analytic method for serotyping of ASFVs (Figure 1). Two field ASFV isolates, ASFV/VN/Pig/NA/1299 and ASFV/VN/Pig/TH/1238, were used to examine these primer sets. The HAD positive results and the success to amplify the short and long fragments of EP402R gene of ASFV/VN/Pig/NA/1299 (accession numbers MN711770) and ASFV/VN/Pig/TH/1238 (accession numbers MN711768) isolates by two primer sets were shown in Figure 2A and B.
It is demonstrated that serotyping based on EP402R gene sequences showed to be a useful and convenient tool when compared to hemagglutination inhibition (HAI) assay [5]. Some previous works indicated that the consideration when performing a vaccine design and development should be given to the fact that viruses within a serogroup provide cross-protection from challenge with viruses of the same serogroup. A recent study has found that ASFV isolates, clustered to serogroups 1, 2, or 4, were recognized as genotype I group based on p72 gene sequences. The alignment of short fragments of 90 nucleotide sequences from 30 ASFV sequences in North Central region of Vietnam (accession numbers MN711757-86) and 68 reference sequences of well-known serogroups was shown in Figure 3. Interestingly, it is clear that a short fragment of EP402R genes is very typical for 8 serotype groups of ASFVs. Phylogenetic analyses of nucleotide sequences of EP402R gene encoding for CD2-like protein was further constructed using the neighbor-joining method with a bootstrap value of 1,000 in the MEGA7 program [12]. The results in Figure 4A and 4B indicated an agreement exists between short fragment analysis ( Figure 4B) and the previous method based on 816 nucleotide long sequences ( Figure 4A) in which the two methods shares 22 nucleotides in the downstream sequences of 90 nucleotides short fragment. Additionally, ASFV serotyping based on 90 nucleotide short fragments is clearer and more specific when compared to long fragment analysis as seen in Figure  4. In previous studies, a good correlation for ASFV serotyping between phylogenetic analysis of EP402R gene coding for CD2-like protein and HAI serogrouping were observed and the information of ASFV serotypes is very necessary for vaccine design and development [6]. HAI assay is the golden method for ASFV serotyping. However, this method required a live virus, reference serum and may not apply in the cases of non-haemadsorbing ASF viruses. Additionally, ASFV antibodies may appear late or at low titre during infection, affecting directly to this assay [13][14][15][16]. Thus PCR based on sequencing method of EP402R gene encoding for CD2-like protein is good candidate and simple method for serotyping of ASFV [6]. Our results suggested that analysis of short fragments of EP402R gene is good tool for serotyping of ASFV strains circulating in Vietnam.