Two divergent VAR2CSA lineages with distinct evolutionary signatures circulate in Plasmodium falciparum from Thailand.

VAR2CSA is the primary target for vaccines against placental malaria; however, its extensive polymorphism hinders vaccine development. While most studies focus on partial fragments, full-length sequence data from clinical isolates remain scarce. We determined the complete var2csa coding sequences (9.1-9.9 kb) of 31 symptomatic Plasmodium falciparum isolates from five endemic provinces in Thailand using long-range PCR and Sanger sequencing. Our analysis identified 24 distinct haplotypes with high diversity (h ± S.D. = 0.97 ± 0.02). Phylogenetic inference revealed two major lineages circulating in Thailand: a dominant 3D7-type and a distinct Dd2-type. The Dd2-type lineage, comprising seven isolates, is characterized by an extended ID4 region (645 bp) and extreme sequence divergence at the 3' end of the gene. Nucleotide diversity (π) was significantly higher in the 3' region (DBL6ε to ATS) compared to the 5' portion (NTS to DBL5ε) (π = 0.4253 vs. 0.1033, p < 0.0001). This disparity is driven by a deep evolutionary split between the 3D7 and Dd2 lineages, which exhibit pronounced nucleotide divergence (Dxy = 0.506 and Da = 0.444) from the DBL6ε domain through the ATS. Conversely, the 5' region showed more frequent intragenic recombination and shorter phylogenetic branching. These findings demonstrate that two deeply divergent VAR2CSA lineages with distinct evolutionary signatures co-circulate in Thailand. Understanding this bipartite variation is essential for developing a vaccine capable of providing broad protection against diverse global P. falciparum populations.