Whole genome sequencing of Plasmodium vivax isolates reveals frequent sequence and structural polymorphisms in erythrocyte binding genes

Plasmodium vivax malaria is much less common in Africa than the rest of the world because the parasite relies primarily on the Duffy antigen/chemokine receptor (DARC) to invade human erythrocytes, and the majority of Africans are Duffy negative. Recently, there has been a dramatic increase in the reporting of P. vivax cases in Africa, with a high number of them being in Duffy negative individuals, potentially indicating P. vivax has evolved an alternative invasion mechanism that can overcome Duffy negativity. Here, we analyzed single nucleotide polymorphism (SNP) and copy number variation (CNV) in Whole Genome Sequence (WGS) data from 44 P. vivax samples isolated from symptomatic malaria patients in southwestern Ethiopia, where both Duffy positive and Duffy negative individuals are found. A total of 236,351 SNPs were detected, of which 21.9% was nonsynonymous and 78.1% was synonymous mutations. The largest number of SNPs were detected on chromosomes 9 (33,478 SNPs; 14% of total) and 10 (28,133 SNPs; 11.9%). There were particularly high levels of polymorphism in erythrocyte binding gene candidates including reticulocyte binding protein 2c (RBP2c), merozoite surface protein 1 (MSP1), and merozoite surface protein 3 (MSP3.5, MSP3.85 and MSP3.9). Thirteen genes related to immunogenicity and erythrocyte binding function were detected with significant signals of positive selection. Variation in gene copy number was also concentrated in genes involved in host-parasite interactions, including the expansion of the Duffy binding protein gene (PvDBP) on chromosome 6 and several PIR genes. Based on the phylogeny constructed from the whole genome sequences, the expansion of these genes was an independent process among the P. vivax lineages in Ethiopia. We further inferred transmission patterns of P. vivax infections among study sites and showed various levels of gene flow at a small geographical scale. The genomic features of P. vivax provided baseline data for future comparison with those in Duffy-negative individuals, and allowed us to develop a panel of informative Single Nucleotide Polymorphic markers diagnostic at a micro-geographical scale.