Pilot survey of expressed sequence tags (ESTs) from the asexual blood stages of Plasmodium vivax in human patients

Abstract Background Plasmodium vivax is the most widely distributed human malaria, responsible for 70–80 million clinical cases each year and large socio-economical burdens for countries such as Brazil where it is the most prevalent species. Unfortunately, due to the impossibility of growing this parasite in continuous in vitro culture, research on P. vivax remains largely neglected. Methods A pilot survey of expressed sequence tags (ESTs) from the asexual blood stages of P. vivax was performed. To do so, 1,184 clones from a cDNA library constructed with parasites obtained from 10 different human patients in the Brazilian Amazon were sequenced. Sequences were automatedly processed to remove contaminants and low quality reads. A total of 806 sequences with an average length of 586 bp met such criteria and their clustering revealed 666 distinct events. The consensus sequence of each cluster and the unique sequences of the singlets were used in similarity searches against different databases that included P. vivax, Plasmodium falciparum, Plasmodium yoelii, Plasmodium knowlesi, Apicomplexa and the GenBank non-redundant database. An E-value of <10-30 was used to define a significant database match. ESTs were manually assigned a gene ontology (GO) terminology Results A total of 769 ESTs could be assigned a putative identity based upon sequence similarity to known proteins in GenBank. Moreover, 292 ESTs were annotated and a GO terminology was assigned to 164 of them. Conclusion These are the first ESTs reported for P. vivax and, as such, they represent a valuable resource to assist in the annotation of the P. vivax genome currently being sequenced. Moreover, since the GC-content of the P. vivax genome is strikingly different from that of P. falciparum, these ESTs will help in the validation of gene predictions for P. vivax and to create a gene index of this malaria parasite.

We would like to thank the anonymous reviewer 1 for the criticisms and suggestions that significantly increased the quality of this manuscript. To Professor Michael Lanzer for his initial support to this project. To Marcio M. Yamamoto for technical support with the cDNA library and to Luciana Terumi Nagao, Fernando Tadashi G. Matsunaga and Paulo H. Ahagon for other technical assistance. We are also grateful to Marcio K. Oikawa for putting the initial supplementary information into the malaria server at USP. Sequence data from rodent and monkey malaria parasites were produced by the Pathogen Sequencing Group at the Sanger Institute. Sequencing of the Plasmodium vivax Salvador I strain from TIGR was accomplished with support from the National Institute of Allergy and Infectious Diseases/Department of Defense (NIAID/DoD). This work was supported by Fundação de Amparo à Pesquisa do Estado de Sâo Paulo (FAPESP, ID 01/094010), Malaria Research and Reference Reagent Resource Center (MR4), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, ID 304651/907).

We would like to thank the anonymous reviewer 1 for the criticisms and suggestions that significantly increased the quality of this manuscript. To Professor Michael Lanzer for his initial support to this project. To Marcio M. Yamamoto for technical support with the cDNA library and to Luciana Terumi Nagao, Fernando Tadashi G. Matsunaga and Paulo H. Ahagon for other technical assistance. We are also grateful to Marcio K. Oikawa for putting the initial supplementary information into the malaria server at USP. Sequence data from rodent and monkey malaria parasites were produced by the Pathogen Sequencing Group at the Sanger Institute. Sequencing of the Plasmodium vivax Salvador I strain from TIGR was accomplished with support from the National Institute of Allergy and Infectious Diseases/Department of Defense (NIAID/DoD). This work was supported by "Fundação de Amparo à Pesquisa do Estado de Sâo Paulo (FAPESP, ID 01/09401-0), Malaria Research and Reference Reagent Resource Center (MR4), and "Conselho Nacional de Desenvolvimento Científico e Tecnológico" (CNPq, ID 304651/90-7).