Novel method for sub‐grouping of genotype II African swine fever viruses based on the intergenic region between the A179L and A137R genes

Abstract Background African swine fever (ASF) is a highly contagious and deadly viral disease affecting domestic and wild pigs of all ages. African swine fever virus (ASFV) has spread rapidly through Eastern and Southeastern Asia first appearing in Vietnam in 2019. Objectives Molecular typing of African swine fever virus (ASFV) in Vietnam has identified two principal variants circulating based on the sequencing of the intergenic region (IRG) between the I73R and I329L genes. Identification of additional genetic markers would enable higher resolution tracing of outbreaks within the country. Methods Sequence analysis suggested the IRG between the A179L and A137R genes may also exhibit variability, PCR primers were designed and samples from Vietnam were subject to Sanger sequencing. Results We developed a novel method for sub‐grouping of ASFV based on the IRG between the A179L and A137R genes of ASFV. Our results demonstrated that the finding of the insertion or deletion of an 11‐ nucleotide sequence (GATACAATTGT) between the A179L‐A137R genes. Conclusions The sub‐grouping method may provide useful insights into the evolution of genotype II ASFV as well as providing evidence of a relationship between geographically separated outbreaks.

F I G U R E 1 Alignment of the region between the A179L and A137R genes of the indicated p72 genotype I and II ASFV isolates. Alignment was prepared using MEGA7, and displayed using NCBI Multiple Sequence Alignment Viewer, Version 1.20.1. Numbers above the alignment show the positions relative to the Georgia 2007/1 isolate (FR682468.2), black bars indicate the GATACAATTGST repeats and the sequences from Hanoi/02 and Hanoi/07 are highlighted in red text (Rep. of), Timor-Leste, Indonesia, Papua New Guinea, India, Malaysia and Bhutan. ASF has also been reported on the Caribbean countries of Haiti and the Dominican Republic. From early February 2019, an ASF outbreak in Vietnam was reported officially, and within seven months, ASF had spread across the whole country. Approximately six million pigs on infected farms and households have been culled, indicating the socio-economic impact on the pig industry (Tran et al., 2021;Truong et al., 2020).
Our previous reports that the ASFV strains circulating in Vietnam belong to B646L(p72) genotype II and EP402R (CD2v) serotype 8 and that there are least two different variants based on the intergenic region located between the I73R and I329L genes (Tran et al., 2021).
Most investigations of ASFV genotypes have been identified based on sequencing of the B646L and E183L (p54) genes and/or analysis of the central variable region or intergenic region (IGR) between I73R and I329L genes (Gallardo et al., 2009;Gallardo et al., 2014). On the other hand, the recent research on the evolution of the MGF505 family, K145R, and O174L genes of ASFV strains revealed minor genetic diversity within these genes, indicating slow but consistent molecular evolution of ASFV strains in a different region (Malogolovkin et al., 2015;Mazur-Panasiuk et al., 2020). In this study, we developed a new method for ASFV sub-grouping analysis of genotype II viruses based on the intergenic region between the A179L and A137R genes. Additionally, the data obtained from this marker region is useful to provide the evidence of a relationship between geographically separated outbreaks.
We have shown that two ASFV strains, ASFV/VN/Pig/Hanoi/02 and ASFV/VN/Pig/Hanoi/07, obtained from the Thach That district of Hanoi city, belonged to B646L genotype II, EP402Rv serotype 8 and were closely related to strains circulating in China (Tran et al., 2021).
Based on the IGR located between the I73R and I329L genes, four different variants of ASFVs have been reported, including IGR I, II, III and IV variants. Since the introduction of ASFV into Vietnam most viruses circulating in the country, including ASFV/VN/Pig/Hanoi/02 strain, belong to the p72 genotype II and IGR II variant. However, ASFV/VN/Pig/Hanoi/07 strain was classified as p72 genotype II ASFV and IGR I variant (Tran et al., 2021).
An alignment and analysis of the nucleotide sequences of ASFV strains from B646L genotype I and II revealed the presence of one to three copies of an 11-nucleotide repeat (GATACAATTST) within the intergenic region between the A179L and A137R genes (Figure 1a, Supplementary Table S1). A pair of primers to amplify a 270-bp fragment was designed based on the reference sequence and the primer binding sites were as follows: forward primer, 5′-CCA TAG CGG CAC CCT ATA TT-3′; reverse primer, 5′-CCT CCT GGT CGA GTT TGG TA-3′. The optimal annealing temperature was 50 • C. PCR was performed, and the products were subjected to sequencing by the Sanger method. Lymph node samples from two dead pigs that the ASFV/VN/Pig/Hanoi/02 and ASFV/VN/Pig/Hanoi/07 viruses were originally isolated were used to investigate the A179L-A137R intergenic region. Our results showed that an 11-nucleotide deletion was found in ASFV/VN/Pig/Hanoi/07 isolate when compared to ASFV/VN/Pig/Hanoi/02 and other viruses isolated from Vietnam ( Figure 1a). Importantly, a similar deletion was identified in an ASFV isolate from Vietnam that we had previously sequenced, ASFV_Hanoi_2019. In addition, the deletion was still present in Hanoi/07 after three passages through porcine alveolar macrophages. Further analysis revealed that the A179L-A137R intergenic regions from forty-five other genotype II ASFV sequences from

CONFLICT OF INTEREST
There are no potential conflicts of interest

ETHICS STATEMENT
The study was conducted in compliance with the institutional rules for the care and use of laboratory animals and using a protocol approved by the Ministry of Agriculture and Rural Development (MARD) Vietnam (TCVN 8402:2010).