In September and October 2023, we collected a total of 26 whole blood samples from pigs suspected to be infected with ASFV from family farms in 6 different northern provinces of Vietnam (Hai Duong, Bac Giang, Hanoi, Phu Tho, Tuyen Quang, and Thai Nguyen) (Appendix Figure 1). We tested the samples by using an ASFV-specific real-time PCR (Median Diagnostics Inc., https://mediandiagnostics.com), according to the kit instructions. Our results showed that all samples were positive for ASFV; cycle threshold values ranged from 16.58 to 32.13 (data not shown). We then tested the samples by using conventional PCR to amplify the C-terminal region of B646L (p72) and the full-length sequences of the E183L (p54) and EP402R (CD2v) genes, followed by Sanger sequencing (1st BASE, https://base-asia.com) (11). We assembled and analyzed the sequences by using Geneious Prime (Geneious, https://www.geneious.com), and we performed the alignment of nucleotide and amino acid sequences by using BioEdit version 7.7.1.0 (https://thalljiscience.github.io/page2.html).

Figure 1

Figure 1. Phylogenetic trees of ASFV, based on the sequences found in the p72 (A), p54 (B), and CD2v (C) regions, Vietnam, 2023. Black squares indicate rASFV I/II strain from Vietnam, red...

Our phylogenetic analysis using MEGAX (https://www.megasoftware.net) revealed that 6 of the 26 samples contained p72 sequences of genotype I, but the p54 sequence belonged to genotype II, and the CD2v sequence belonged to serotype VIII (Table). This finding was consistent with recent pandemic ASFV genotype II viruses (Figure 1), suggesting that those 6 whole blood samples possibly contained recombinant ASFV p72 viruses of genotypes I and II (rASFV I/II). The p72, p54, and CD2v sequences of the remaining viruses were like the viruses from the ASFV genotype II viruses reported in Vietnam (8). We inoculated the 6 whole blood samples onto primary porcine alveolar macrophages obtained from the lungs of 8–10-week-old healthy pigs, and we monitored the cultures daily for 5–7 days for hemadsorption. We obtained ASFV isolates positive for hemadsorption from all 6 samples (Appendix Figure 2).

Figure 2

Figure 2. Alignment of ASFV CVR signatures between rASFV genotype I/II strains from Vietnam and China, and genotype I strains from China, recovered from domestic swine in Vietnam and China, 2023. Numbers...

To gain further insight into the genomes of the 6 rASFV I/II isolates, we amplified 10 additional genes (MGF-505–1R, B119L [9GL], I177L, DP96R [UK], A238L, A137R, MGF 360–12L, I226R, B602L, and IGR [between I73R and I329L]) from all 6 isolates by using previously reported and newly designed primers, and we sequenced by using Sanger sequencing (Appendix Table 1) (12–14). All of the gene sequences we obtained were deposited into GenBank (accession nos.: p72, OR999183–88; p54, OR999177–82; CD2v, OR999147–52; B119L, OR999135–40; DP96R, OR999153–58; B602L, OR999141–46; I177L, OR999159–64; MGF 505–1R, OR999171–76; A238L, PP464965–70; A137R, PP464971–76; MGF 360–12L, PP464977–82; I226R, PP464983–88; and IGR, OR999165–70). Our genetic analysis showed that all target gene sequences of the 6 Vietnamese rASFV I/II isolates matched at the nucleotide level with the corresponding gene sequences of the 3 rASFV I/II strains (JS/LG/21, HeN/123014/22, and IM/DQDM/22) previously reported in China, with the exception of the central variable region (CVR) (Appendix Table 2). The nucleotide sequences of the CVR region of all 3 rASFV I/II strains from China showed a 96-nucleotide insertion compared with the genotype I strain pig/SD/DY-I/2021 isolated from China. The isolate VNUA/rASFV/TN1/23 from Thai Nguyen Province showed the same 96-nucleotide insertion in the CVR region (Appendix Figure 3, panel A). However, the unique 36-nucleotide deletion observed in the CVR region of the JS/LG/21 strain from China was not detected in any of the rASFV I/II strains from Vietnam (Appendix Figure 3, panel B). Four rASFV I/II isolates from Vietnam (VNUA/rASFV/HD1/23, VNUA/rASFV/BG1/23, VNUA/rASFV/Hanoi1/23, and VNUA/rASFV/TQ1/23) had an additional 12-nucleotide insertion (108 nucleotides in total) in the CVR region. The Vietnam isolate VNUA/rASFV/PT1/23 from Phu Tho Province had an additional 108-nucleotide insertion (204 nucleotides in total) in the CVR region (Appendix Figure 3, panel A). Those differences were reflected in the CVR profile of the viruses (Figure 2).

We report the discovery of 6 rASFV I/II strains from northern Vietnam. To characterize the virus strains, while attempting to sequence the whole genome of the 6 isolates, we rapidly sequenced 13 target genes of these 6 isolates and compared their nucleotide sequences with the corresponding sequences of rASFV I/II strains from China. All sequences, except for the CVR region, matched with the corresponding sequences of rASFV I/II strains from China. The observation of 3 different CVR variants in Vietnam indicates 3 possible independent introductions of rASFV I/II strains into Vietnam. Because ASFV p72 genotype I strains have not been reported in Vietnam, the rASFV I/II strains detected in Vietnam may have originated from China.

Two live-attenuated ASFV vaccines are now licensed in Vietnam. Both vaccines have been proven to protect young pigs against highly pathogenic ASFV p72 genotype II strains circulating in Vietnam. However, the rASFV I/II strains detected in China appear to be resistant to the live-attenuated ASFV p72 genotype II vaccine candidates (7). Therefore, the newly discovered rASFV I/II strains in Vietnam are likely resistant to the immunity induced by the current live ASFV vaccines and could replace the ASFV p72 genotype II strains circulating in the region. This possibility poses a major challenge for disease control and vaccine development and emphasizes the need for increased vigilance in the global control of ASFV.