Whole-genome sequencing and microarray analysis of ex vivo Plasmodium vivax reveal selective pressure on putative drug resistance genes

Abstract

Plasmodium vivax causes 25-40% of malaria cases worldwide, yet research on this human malaria parasite has been neglected. Nevertheless, the recent publication of the P. vivax reference genome now allows genomics and systems biology approaches to be applied to this pathogen. We show here that whole-genome analysis of the parasite can be achieved directly from ex vivo-isolated parasites, without the need for in vitro propagation. A single isolate of P. vivax obtained from a febrile patient with clinical malaria from Peru was subjected to whole-genome sequencing (30× coverage). This analysis revealed over 18,261 single-nucleotide polymorphisms (SNPs), 6,257 of which were further validated using a tiling microarray. Within core chromosomal genes we find that one SNP per every 985 bases of coding sequence distinguishes this recent Peruvian isolate, designated IQ07, from the reference Salvador I strain obtained in 1972. This full-genome sequence of an uncultured P. vivax isolate shows that the same regions with low numbers of aligned sequencing reads are also highly variable by genomic microarray analysis. Finally, we show that the genes containing the largest ratio of nonsynonymous-to-synonymous SNPs include two AP2 transcription factors and the P. vivax multidrug resistance-associated protein (PvMRP1), an ABC transporter shown to be associated with quinoline and antifolate tolerance in Plasmodium falciparum. This analysis provides a data set for comparative analysis with important potential for identifying markers for global parasite diversity and drug resistance mapping studies.

Host immune responses accelerate pathogen evolution

“…Thirty-six (97.3%) of the 37 single-nucleotide polymorphisms predicted by Illumina were confirmed by Sanger sequencing (Table 1). Similar results have been previously reported (Dharia et al, 2010;Wong et al, 2012).…”

“…High coverage of reads generated by the G2 Illumina resulted in a false-positive rate of only one per 9 200 000 bp. Although we obtained very high coverage per base position and negligible false-positive rates by Illumina sequencing (Dharia et al, 2010), we confirmed all the non-synonymous and frameshift mutation resulting in amino-acid substitutions by Sanger sequencing (Supplementary Table S2). Thirty-six (97.3%) of the 37 single-nucleotide polymorphisms predicted by Illumina were confirmed by Sanger sequencing (Table 1).…”

Selective sweep suggests transcriptional regulation may underlie Plasmodium vivax resilience to malaria control measures in Cambodia

“…Notably, a previous comparison of a 1990s era Peruvian P. vivax isolate to the reference Sal1 strain collected 35 y earlier found an increased dN/dS signal at two AP2-containing transcription factors, suggesting that evolutionary changes in these genes are not confined to P. vivax in Cambodia (47). Because this was a withinspecies comparison of nonsynonymous to synonymous SNP ratio (dN/dS), it is difficult to determine the time-scale (e.g., whether ancient or recent) associated with this selection (48).…”

High Degree of Plasmodium vivax Diversity in the Peruvian Amazon Demonstrated by Tandem Repeat Polymorphism Analysis

“…34 Nonetheless, the large-scale genomic structure underlying the population structure has yet to be determined. Whole genome analysis beyond the currently published data 35,36 will be required to further determine the genomic features underlying the population biology and evolution of P. vivax in Peru, which is of particular interest in light of the low malaria transmission intensity in this region. In this study, we have demonstrated that use of the TR markers in the hypoendemic malaria region of the Amazon effectively and efficiently distinguishes infecting strains of P. vivax.…”