Daniel J. Park

Genomic surveillance elucidates Ebola virus origin and transmission during the 2014 outbreak

In its largest outbreak, Ebola virus disease is spreading through Guinea, Liberia, Sierra Leone, and Nigeria. We sequenced 99 Ebola virus genomes from 78 patients in Sierra Leone to ~2000× coverage. We observed a rapid accumulation of interhost and intrahost genetic variation, allowing us to characterize patterns of viral transmission over the initial weeks of the epidemic. This West African variant likely diverged from central African lineages around 2004, crossed from Guinea to Sierra Leone in May 2014, and has exhibited sustained human-to-human transmission subsequently, with no evidence of additional zoonotic sources. Because many of the mutations alter protein sequences and other biologically meaningful targets, they should be monitored for impact on diagnostics, vaccines, and therapies critical to outbreak response.

Identifying Recent Adaptations in Large-Scale Genomic Data

Although several hundred regions of the human genome harbor signals of positive natural selection, few of the relevant adaptive traits and variants have been elucidated. Using full-genome sequence variation from the 1000 Genomes (1000G) Project and the composite of multiple signals (CMS) test, we investigated 412 candidate signals and leveraged functional annotation, protein structure modeling, epigenetics, and association studies to identify and extensively annotate candidate causal variants. The resulting catalog provides a tractable list for experimental follow-up; it includes 35 high-scoring nonsynonymous variants, 59 variants associated with expression levels of a nearby coding gene or lincRNA, and numerous variants associated with susceptibility to infectious disease and other phenotypes. We experimentally characterized one candidate nonsynonymous variant in Toll-like receptor 5 (TLR5) and show that it leads to altered NF-κB signaling in response to bacterial flagellin. PAPERFLICK:

SNP Genotyping Defines Complex Gene-Flow Boundaries Among African Malaria Vector Mosquitoes

Signals of Mosquito Speciation Malaria in Africa is transmitted by the mosquito species complex Anopheles gambiae. Neafsey et al. (p. 514) made high-resolution single-nucleotide arrays to map genetic divergence among members of the species. Differentiation between populations was observed and evidence obtained for selective sweeps within populations. Most divergence occurred within inversion regions around the centrosome and in genes associated with development, pheromone signaling, and from the X chromosome. The analysis also revealed signals of sympatric speciation occurring within similar chromosomal regions in mosquitoes from different regions in Africa. Lawniczak et al. (p. 512) sequenced the genomes of two molecular forms (known as M and S) of A. gambiae, which have distinctive behavioral phenotypes and appear to be speciating. This effort resolves problems arising from the apparently chimeric nature of the reference genome and confirms the observed genome-wide divergences. This kind of analysis has the potential to contribute to control programs that can adapt to population shifts in mosquito behavior arising from the selective effects of the control measures themselves. Populations of African malaria vectors show signs of selective sweeps and ongoing speciation in their genomes. Mosquitoes in the Anopheles gambiae complex show rapid ecological and behavioral diversification, traits that promote malaria transmission and complicate vector control efforts. A high-density, genome-wide mosquito SNP-genotyping array allowed mapping of genomic differentiation between populations and species that exhibit varying levels of reproductive isolation. Regions near centromeres or within polymorphic inversions exhibited the greatest genetic divergence, but divergence was also observed elsewhere in the genomes. Signals of natural selection within populations were overrepresented among genomic regions that are differentiated between populations, implying that differentiation is often driven by population-specific selective events. Complex genomic differentiation among speciating vector mosquito populations implies that tools for genome-wide monitoring of population structure will prove useful for the advancement of malaria eradication.

Ebola Virus Epidemiology, Transmission, and Evolution during Seven Months in Sierra Leone

Summary The 2013–2015 Ebola virus disease (EVD) epidemic is caused by the Makona variant of Ebola virus (EBOV). Early in the epidemic, genome sequencing provided insights into virus evolution and transmission and offered important information for outbreak response. Here, we analyze sequences from 232 patients sampled over 7 months in Sierra Leone, along with 86 previously released genomes from earlier in the epidemic. We confirm sustained human-to-human transmission within Sierra Leone and find no evidence for import or export of EBOV across national borders after its initial introduction. Using high-depth replicate sequencing, we observe both host-to-host transmission and recurrent emergence of intrahost genetic variants. We trace the increasing impact of purifying selection in suppressing the accumulation of nonsynonymous mutations over time. Finally, we note changes in the mucin-like domain of EBOV glycoprotein that merit further investigation. These findings clarify the movement of EBOV within the region and describe viral evolution during prolonged human-to-human transmission.

Use of high-density tiling microarrays to identify mutations globally and elucidate mechanisms of drug resistance in Plasmodium falciparum.

BackgroundThe identification of genetic changes that confer drug resistance or other phenotypic changes in pathogens can help optimize treatment strategies, support the development of new therapeutic agents, and provide information about the likely function of genes. Elucidating mechanisms of phenotypic drug resistance can also assist in identifying the mode of action of uncharacterized but potent antimalarial compounds identified in high-throughput chemical screening campaigns against Plasmodium falciparum.ResultsHere we show that tiling microarrays can detect de novo a large proportion of the genetic changes that differentiate one genome from another. We show that we detect most single nucleotide polymorphisms or small insertion deletion events and all known copy number variations that distinguish three laboratory isolates using readily accessible methods. We used the approach to discover mutations that occur during the selection process after transfection. We also elucidated a mechanism by which parasites acquire resistance to the antimalarial fosmidomycin, which targets the parasite isoprenoid synthesis pathway. Our microarray-based approach allowed us to attribute in vitro derived fosmidomycin resistance to a copy number variation event in the pfdxr gene, which enables the parasite to overcome fosmidomycin-mediated inhibition of isoprenoid biosynthesis.ConclusionsWe show that newly emerged single nucleotide polymorphisms can readily be detected and that malaria parasites can rapidly acquire gene amplifications in response to in vitro drug pressure. The ability to define comprehensively genetic variability in P. falciparum with a single overnight hybridization creates new opportunities to study parasite evolution and improve the treatment and control of malaria.

Population genomic sequencing of Coccidioides fungi reveals recent hybridization and transposon control

We have sequenced the genomes of 18 isolates of the closely related human pathogenic fungi Coccidioides immitis and Coccidioides posadasii to more clearly elucidate population genomic structure, bringing the total number of sequenced genomes for each species to 10. Our data confirm earlier microsatellite-based findings that these species are genetically differentiated, but our population genomics approach reveals that hybridization and genetic introgression have recently occurred between the two species. The directionality of introgression is primarily from C. posadasii to C. immitis, and we find more than 800 genes exhibiting strong evidence of introgression in one or more sequenced isolates. We performed PCR-based sequencing of one region exhibiting introgression in 40 C. immitis isolates to confirm and better define the extent of gene flow between the species. We find more coding sequence than expected by chance in the introgressed regions, suggesting that natural selection may play a role in the observed genetic exchange. We find notable heterogeneity in repetitive sequence composition among the sequenced genomes and present the first detailed genome-wide profile of a repeat-induced point mutation (RIP) process distinctly different from what has been observed in Neurospora. We identify promiscuous HLA-I and HLA-II epitopes in both proteomes and discuss the possible implications of introgression and population genomic data for public health and vaccine candidate prioritization. This study highlights the importance of population genomic data for detecting subtle but potentially important phenomena such as introgression.

Genetic diversity and protective efficacy of the RTS,S/AS01 malaria vaccine

BACKGROUND The RTS,S/AS01 vaccine targets the circumsporozoite protein of Plasmodium falciparum and has partial protective efficacy against clinical and severe malaria disease in infants and children. We investigated whether the vaccine efficacy was specific to certain parasite genotypes at the circumsporozoite protein locus. METHODS We used polymerase chain reaction-based next-generation sequencing of DNA extracted from samples from 4985 participants to survey circumsporozoite protein polymorphisms. We evaluated the effect that polymorphic positions and haplotypic regions within the circumsporozoite protein had on vaccine efficacy against first episodes of clinical malaria within 1 year after vaccination. RESULTS In the per-protocol group of 4577 RTS,S/AS01-vaccinated participants and 2335 control-vaccinated participants who were 5 to 17 months of age, the 1-year cumulative vaccine efficacy was 50.3% (95% confidence interval [CI], 34.6 to 62.3) against clinical malaria in which parasites matched the vaccine in the entire circumsporozoite protein C-terminal (139 infections), as compared with 33.4% (95% CI, 29.3 to 37.2) against mismatched malaria (1951 infections) (P=0.04 for differential vaccine efficacy). The vaccine efficacy based on the hazard ratio was 62.7% (95% CI, 51.6 to 71.3) against matched infections versus 54.2% (95% CI, 49.9 to 58.1) against mismatched infections (P=0.06). In the group of infants 6 to 12 weeks of age, there was no evidence of differential allele-specific vaccine efficacy. CONCLUSIONS These results suggest that among children 5 to 17 months of age, the RTS,S vaccine has greater activity against malaria parasites with the matched circumsporozoite protein allele than against mismatched malaria. The overall vaccine efficacy in this age category will depend on the proportion of matched alleles in the local parasite population; in this trial, less than 10% of parasites had matched alleles. (Funded by the National Institutes of Health and others.).

Sequence-based association and selection scans identify drug resistance loci in the Plasmodium falciparum malaria parasite

Through rapid genetic adaptation and natural selection, the Plasmodium falciparum parasite—the deadliest of those that cause malaria—is able to develop resistance to antimalarial drugs, thwarting present efforts to control it. Genome-wide association studies (GWAS) provide a critical hypothesis-generating tool for understanding how this occurs. However, in P. falciparum, the limited amount of linkage disequilibrium hinders the power of traditional array-based GWAS. Here, we demonstrate the feasibility and power improvements gained by using whole-genome sequencing for association studies. We analyzed data from 45 Senegalese parasites and identified genetic changes associated with the parasites’ in vitro response to 12 different antimalarials. To further increase statistical power, we adapted a common test for natural selection, XP-EHH (cross-population extended haplotype homozygosity), and used it to identify genomic regions associated with resistance to drugs. Using this sequence-based approach and the combination of association and selection-based tests, we detected several loci associated with drug resistance. These loci included the previously known signals at pfcrt, dhfr, and pfmdr1, as well as many genes not previously implicated in drug-resistance roles, including genes in the ubiquitination pathway. Based on the success of the analysis presented in this study, and on the demonstrated shortcomings of array-based approaches, we argue for a complete transition to sequence-based GWAS for small, low linkage-disequilibrium genomes like that of P. falciparum.

Identification and functional validation of the novel antimalarial resistance locus PF10_0355 in Plasmodium falciparum.

The Plasmodium falciparum parasites ability to adapt to environmental pressures, such as the human immune system and antimalarial drugs, makes malaria an enduring burden to public health. Understanding the genetic basis of these adaptations is critical to intervening successfully against malaria. To that end, we created a high-density genotyping array that assays over 17,000 single nucleotide polymorphisms (∼1 SNP/kb), and applied it to 57 culture-adapted parasites from three continents. We characterized genome-wide genetic diversity within and between populations and identified numerous loci with signals of natural selection, suggesting their role in recent adaptation. In addition, we performed a genome-wide association study (GWAS), searching for loci correlated with resistance to thirteen antimalarials; we detected both known and novel resistance loci, including a new halofantrine resistance locus, PF10_0355. Through functional testing we demonstrated that PF10_0355 overexpression decreases sensitivity to halofantrine, mefloquine, and lumefantrine, but not to structurally unrelated antimalarials, and that increased gene copy number mediates resistance. Our GWAS and follow-on functional validation demonstrate the potential of genome-wide studies to elucidate functionally important loci in the malaria parasite genome.

Nomenclature- and database-compatible names for the two Ebola virus variants that emerged in Guinea and the Democratic Republic of the Congo in 2014.

In 2014, Ebola virus (EBOV) was identified as the etiological agent of a large and still expanding outbreak of Ebola virus disease (EVD) in West Africa and a much more confined EVD outbreak in Middle Africa. Epidemiological and evolutionary analyses confirmed that all cases of both outbreaks are connected to a single introduction each of EBOV into human populations and that both outbreaks are not directly connected. Coding-complete genomic sequence analyses of isolates revealed that the two outbreaks were caused by two novel EBOV variants, and initial clinical observations suggest that neither of them should be considered strains. Here we present consensus decisions on naming for both variants (West Africa: “Makona”, Middle Africa: “Lomela”) and provide database-compatible full, shortened, and abbreviated names that are in line with recently established filovirus sub-species nomenclatures.