RESEARCH ARTICLE Evolutionary analyses of the major variant surface antigen-encoding genes reveal population structure of Plasmodium falciparum within and between continents 1,2,3 1¤ 1,4 Gerry Tonkin-HillID , Shazia Ruybal-PesaÂntezID , Kathryn E. TiedjeID , 5 1,4 6 7,8 Virginie RougeronID , Michael F. DuffyID , Sedigheh Zakeri , Tepanata PumpaiboolID , 8,9 10,11 11 Pongchai HarnyuttanakornID , OraLee H. BranchID , Lastenia Ruiz-MesõÂaID , 1 5 2,12,13,14,15 a1111111111 Thomas S. Rask , Franck PrugnolleID , Anthony T. PapenfussID , Yao- 12,16 1,4 a1111111111 ban ChanID , Karen P. DayID * a1111111111 1 School of BioSciences, Bio21 Institute, The University of Melbourne, Melbourne, Australia, a1111111111 2 Bioinformatics Division, Walter and Eliza Hall Institute, Melbourne, Australia, 3 Parasites and Microbes, a1111111111 Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, United Kingdom, 4 Department of Microbiology and Immunology, Bio21 Institute and Peter Doherty Institute, The University of Melbourne, Melbourne, Australia, 5 Laboratoire MIVEGEC, Universite de Montpellier-CNRS-IRD, Montpellier, France, 6 Malaria and Vector Research Group (MVRG), Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran, 7 Biomedical Science, Graduate School, Chulalongkorn University, Bangkok, Thailand, 8 Malaria Research Programme, College of Public Health Science, Chulalongkorn University, Bangkok, OPEN ACCESS Thailand, 9 Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand, Citation: Tonkin-Hill G, Ruybal-PesaÂntez S, Tiedje 10 Concordia University, Portland, Oregon, United States of America, 11 Universidad Nacional de la KE, Rougeron V, Duffy MF, Zakeri S, et al. (2021) AmazonõÂa Peruana, Iquitos, PeruÂ, 12 School of Mathematics and Statistics, The University of Melbourne, Evolutionary analyses of the major variant surface Melbourne, Australia, 13 Peter MacCallum Cancer Centre, Victorian Comprehensive Cancer Centre, Melbourne, Australia, 14 Department of Medical Biology, The University of Melbourne, Melbourne, Australia, antigen-encoding genes reveal population structure 15 Sir Peter MacCallum Department of Oncology, The University of Melbourne, Melbourne, Australia, of Plasmodium falciparum within and between 16 Melbourne Integrative Genomics, The University of Melbourne, Melbourne, Australia continents. PLoS Genet 17(2): e1009269. https:// doi.org/10.1371/journal.pgen.1009269 ¤ Current address: Population Health and Immunity Division, Walter and Eliza Hall Institute, Melbourne, Australia; Department of Medical Biology and Bio21 Institute, The University of Melbourne, Melbourne, Editor: Carmen Buchrieser, Institut Pasteur, CNRS Australia; Burnet Institute, Melbourne, Australia UMR 3525, FRANCE * [email protected] Received: June 13, 2020 Accepted: November 10, 2020 Abstract Published: February 25, 2021 Peer Review History: PLOS recognizes the Malaria remains a major public health problem in many countries. Unlike influenza and HIV, benefits of transparency in the peer review where diversity in immunodominant surface antigens is understood geographically to inform process; therefore, we enable the publication of disease surveillance, relatively little is known about the global population structure of all of the content of peer review and author responses alongside final, published articles. The PfEMP1, the major variant surface antigen of the malaria parasite Plasmodium falciparum. editorial history of this article is available here: The complexity of the var multigene family that encodes PfEMP1 and that diversifies by https://doi.org/10.1371/journal.pgen.1009269 recombination, has so far precluded its use in malaria surveillance. Recent studies have Copyright: © 2021 Tonkin-Hill et al. This is an open demonstrated that cost-effective deep sequencing of the region of var genes encoding the access article distributed under the terms of the PfEMP1 DBLα domain and subsequent classification of within host sequences at 96% iden- Creative Commons Attribution License, which tity to define unique DBLα types, can reveal structure and strain dynamics within countries. permits unrestricted use, distribution, and reproduction in any medium, provided the original However, to date there has not been a comprehensive comparison of these DBLα types author and source are credited. between countries. By leveraging a bioinformatic approach (jumping hidden Markov model) Data Availability Statement: The sequences for designed specifically for the analysis of recombination within var genes and applying it to a this project have been deposited at DDBJ/ENA/ dataset of DBLα types from 10 countries, we are able to describe population structure of GenBank: PRJNA385207, PRJNA385208, PLOS Genetics | https://doi.org/10.1371/journal.pgen.1009269 February 25, 2021 1 / 26 PLOS GENETICS Population structure of P. falciparum within and between continents PRJNA630836, KY328840±KY341897, DBLα types at the global scale. The sensitivity of the approach allows for the comparison of KX845707±KX851405, KP219986- KP221189.The the global dataset to ape samples of Plasmodium Laverania species. Our analyses show relevant data used to produce the figures are deposited in https://github.com/gtonkinhill/global_ that the evolution of the parasite population emerging out of Africa underlies current patterns var_manuscript. of DBLα type diversity. Most importantly, we can distinguish geographic population structure Funding: This research was supported by the within Africa between Gabon and Ghana in West Africa and Uganda in East Africa. Our evo- National Institute of Allergy and Infectious Disease, lutionary findings have translational implications in the context of globalization. Firstly, DBLα National Institutes of Health [Grant number: R01- type diversity can provide a simple diagnostic framework for geographic surveillance of the AI084156 to K.P.D.] (https://www.niaid.nih.gov), rapidly evolving transmission dynamics of P. falciparum. It can also inform efforts to under- Fogarty International Center at the National Institutes of Health [Program on the Ecology and stand the presence or absence of global, regional and local population immunity to major Evolution of Infectious Diseases (EEID), Grant surface antigen variants. Additionally, we identify a number of highly conserved DBLα types number: R01-TW009670 to K.P.D.] (https://www. that are present globally that may be of biological significance and warrant further fic.nih.gov), and the National Institute of Allergy characterization. and Infectious Disease, National Institutes of Health [Grant number: R01-AI149779 to K.P.D.] (https:// www.niaid.nih.gov). Salary support was provided by R01-AI084156 to G.T-H, K.E.T, V.R, and T.S.R; R01-TW009670 to K.E.T; The University of Melbourne to K.E.T and M.F.D. S.R-P was Author summary supported by a Melbourne International Globalization has led to the spread of pathogens through increased human movement. Engagement Award from The University of Microbiologists track epidemics of these pathogens by cataloguing geographic diversity in Melbourne. The funders had no role in study design, data collection and analysis, decision to the genes that encode for variant surface antigens (VSA). Here, we developed a computa- publish, or preparation of the manuscript. tional approach to explore the evolution of specific DNA sequences of the major VSA gene of the human malaria parasite, Plasmodium falciparum. First, we tested the method Competing interests: The authors have declared that no competing interests exist. by comparing DNA sequences of these genes from P. falciparum to those of Plasmodium species that infect chimpanzees and gorillas. We showed that it could distinguish DNA signatures specific to each species. Next, we asked whether our method could detect geo- graphic signatures within these genes by analyzing a global collection of P. falciparum iso- lates from 23 locations in 10 countries. The important outcome of our work was the ability to identify geographic signatures specific to countries and continents that were consistent with the ªout of Africaº origin of P. falciparum. We can now identify malaria parasites from countries within Africa, South America, and Asia/Oceania using a diverse region of VSA genes without having to sequence and assemble whole parasite genomes. This methodology has potential applications in malaria surveillance to track parasites as they move around the world. Introduction Plasmodium falciparum continues to present a significant economic and public health burden globally. The pathogen is endemic across many resource poor countries such as those in tropi- cal Africa and parts of Asia [1] and re-emerging in Latin America [2]. Part of the pathogen's success in remaining endemic, while also highly prevalent in many regions, can be attributed to the extreme diversity of the major variant surface antigen of the blood stages, known as P. falciparum erythrocyte membrane protein 1 (PfEMP1). This molecule is encoded by the var multigene family [3] and each parasite possesses approximately 60 var genes [4,5]. Genome sequencing has shown that each parasite carries a different var gene repertoire [6]. Analysis of the population structure of the var genes encoding PfEMP1 is thus important for the control and prevention of the disease as well as for the design of any vaccine targeting PfEMP1 based on an understanding of variant-specific immunity [7]. PLOS Genetics | https://doi.org/10.1371/journal.pgen.1009269 February