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Sequencing Studies Lay Foundation for More Extensive Research on Widespread Malaria Parasite

NEW YORK (GenomeWeb News) – In a pair of studies online yesterday in Nature Genetics, two international research teams described the genome sequencing strategies they're using to study the human malaria parasite Plasmodium vivax and a closely related monkey malaria species, P. cynomolgi.

For one of the studies, researchers from the US and India did genome sequencing on P. vivax parasites from three continents, demonstrating that the genetic diversity in this relatively widespread species surpasses that found in isolates of the human malaria parasite, P. falciparum, the most common human malaria parasite in Africa.

In a second study, an American and Japanese team sequenced three strains of a monkey malaria parasite called P. cynomolgi, which infects Asian Old World monkeys. A comparison between these genomes, the P. vivax reference sequence, and sequences for another monkey malaria species verified the researchers' suspicion that P. cynomolgi and P. vivax are genomically very similar — supporting the notion that P. cynomolgi might serve as a useful stand-in for studying the more difficult to culture P. vivax parasite.

"[P. cynomolgi] is actually an ideal model system to study Plasmodium vivax," New York University genomics and systems biology researcher Jane Carlton, told GenomeWeb Daily News. "The reason for that is that you can't grow P. vivax in the lab, you can't grow it in cultures. It's a really difficult malaria parasite to deal with."

Carlton is senior author on one of the studies and co-senior author on the other.

Roughly half of the world's population lives in places where the P. vivax parasite poses a malaria threat, she and her co-authors noted. But difficulties in culturing P. vivax have made it trickier to study in the lab than its largely tropical counterpart P. falciparum, which has been the subject of several genetic and genomic studies.

Carlton was part of a team that published the first genome sequence for P. vivax in Nature in 2008. And at least one team successfully generated sequence and tiling array data using P. vivax DNA circulating in the bloodstream of an infected human host from Peru — information that that group used to track down candidate drug resistance genes in the parasite. Even so, it remains far less characterized than P. falciparum.

"Because Plasmodium vivax is such an intractable parasite, genomics is really the way forward," Carlton said. "We can learn so much by interrogating the genome."

To that end, she and her co-authors used Illumina's GAIIx and HiSeq 2000 platforms to sequence four P. vivax strains to between 35 and 87-fold average coverage.

The strains selected for the study, which originated in northwest Africa, South America, South Asia, and East Asia and had been adapted to grow in monkeys, were obtained through a large repository available to members of the malaria research community known as the Malaria Research and Reference Reagent Resource Center.

In addition to their P. vivax sequencing efforts, the team also sequenced four P. falciparum strains from the same parts of the world to allow for direct comparisons between the two species.

"What we found was that the P. vivax genomes were much easier to sequence and assemble compared to the P. falciparum genomes," Carlton noted. "And a lot of that is because the P. falciparum genomes are incredibly AT-rich."

Their comparison between genome sequences for each strain, meanwhile, revealed genetic diversity in the P. vivax strains that outpaced that found in P. falciparum — variability that is suspected of contributing to the complicated drug resistance patterns described for P. vivax, which is resistant to certain drugs in some parts of the world but not others.

These differences in diversity were not limited to SNPs, NYU's Carlton explained, but also included polymorphisms in microsatellite regions.

"The [P. vivax] diversity is genome wide and much more significant than we first thought, which is very worrisome," she said, explaining that such findings may have implications for programs aimed at eradicating malaria, such as the initiative spearheaded by the Bill and Melinda Gates Foundation.

Though such efforts have largely focused on P. falciparum, Carlton and her colleagues argue that the extent of the genetic diversity found in P. vivax may pose a particular challenge to eradication efforts.

In the accompanying Nature Genetics study, meanwhile, researchers used Illumina, Roche 454, and Sanger sequencing methods to sequence the genome a P. cynomolgi strain originating in Malaysia to an average depth of around 161-fold each. Two more P. cynomolgi strains, one from Malaysia and another from Cambodia, were also sequenced for the study.

They then compared these with the existing P. vivax reference sequence and to sequences for P. knowlesi, a parasite found in Southeast Asia that naturally infects monkeys but is sometimes transmitted to humans in areas where they encounter mosquitoes carrying the parasite.

That analysis uncovered SNPs, copy number variants, and microsatellite markers in the newly sequenced P. cytomolgi genome. It also pointed to sets of shared CNVs in the monkey malaria parasites that affect multi-gene families such as those mediating interactions with the host immune system.

Down the road, Carlton explained that some of the SNPs and microsatellite markers identified in the P. cynomolgi sequences could prove useful for tracking the parasites in Asian monkey populations.

And given the similarities between P. cynomolgi and P. vivax, researchers are keen to find ways of using the monkey parasite as a model for the tricky-to-culture human malaria culprit.

"We have generated a genetic map of P. cynomolgi, the sister species to P. vivax, so we can now push forward in creating a robust model system to study P. vivax," Osaka University malaria researcher Kazuyuki Tanabe, co-senior author on the P. cynomolgi study, said in a statement.

While they did not directly compare these P. cynomolgi genomes with the four P. vivax genomes sequenced for the newly published study, Carlton noted that that comparison is currently underway.

In addition, she and her colleagues have embarked on a seven-year project known as the "Center for the Study of Complex Malaria in India", which includes a large P. vivax malaria sequencing effort being done in partnership with India's National Institute of Malaria Research.

The center is funded through the US National Institutes of Health's International Centers of Excellence in Malaria Research program, an initiative that supports 10 malaria research centers in Asia, Africa, Melanesia, and South America.

The India-based team currently plans to sequence as many as 50 P. vivax isolates from patient samples collected in different parts of that country. Together with collaborators at the Broad Institute, Carlton noted that the group is in the process of developing a hybrid capture method based on probes targeting the P. vivax genome to help enrich for parasite DNA from patient blood samples.

"We're starting to develop the technologies to do direct [P. vivax DNA] sequencing from patients," she explained. "It's very challenging, but this really is the starting point — the P. vivax and P. cynomolgi papers — for us to really be able to delve into that sequencing of clinical isolates."