Dengue is a major public health problem worldwide with distinct clinical manifestations: an acute presentation (dengue fever, DF) similar to other febrile illnesses (OFI) and a more severe, life-threatening form (severe dengue, SD). Due to nonspecific clinical presentation during the early phase of dengue infection, differentiating DF from OFI has remained a challenge, and current methods to determine severity of dengue remain poor early predictors. We present a prospective clinical cohort study conducted in Caracas, Venezuela from 2001-2005, designed to determine whether clinical and hematological parameters could distinguish DF from OFI, and identify early prognostic biomarkers of SD. From 204 enrolled suspected dengue patients, there were 111 confirmed dengue cases. Piecewise mixed effects regression and nonparametric statistics were used to analyze longitudinal records. Decreased serum albumin and fibrinogen along with increased D-dimer, thrombin-antithrombin complex, activated partial thromboplastin time and thrombin time were prognostic of SD on the day of defervescence. In the febrile phase, the day-to-day rates of change in serum albumin and fibrinogen concentration, along with platelet counts, were significantly decreased in dengue patients compared to OFI, while the day-to-day rates of change of lymphocytes (%) and thrombin time were increased. In dengue patients, the absolute lymphocytes to neutrophils ratio showed specific temporal increase, enabling classification of dengue patients entering the critical phase with an area under the ROC curve of 0.79. Secondary dengue patients had elongation of Thrombin time compared to primary cases while the D-dimer formation (fibrinolysis marker) remained always lower for secondary compared to primary cases. Based on partial analysis of 31 viral complete genomes, a high frequency of C-to-T transitions located at the third codon position was observed, suggesting deamination events with five major hot spots of amino acid polymorphic sites outside in non-structural proteins. No association of severe outcome was statistically significant for any of the five major polymorphic sites found. This study offers an improved understanding of dengue hemostasis and a novel way of approaching dengue diagnosis and disease prognosis using piecewise mixed effect regression modeling. It also suggests that a better discrimination of the day of disease can improve the diagnostic and prognostic classification power of clinical variables using ROC curve analysis. The piecewise mixed effect regression model corroborated key early clinical determinants of disease, and offers a time-series approach for future vaccine and pathogenesis clinical studies.

Human Parainfluenza viruses (HPIV) type 1 and 3 are important causes of respiratory tract infections in young children globally. HPIV infections do not confer complete protective immunity so reinfections occur throughout life. Since no effective vaccine is available for the two virus subtypes, comprehensive understanding of HPIV-1 and HPIV-3 genetic and epidemic features is important for diagnosis, prevention, and treatment of HPIV-1 and HPIV-3 infections. Relatively few whole genome sequences are available for both HPIV-1 and HPIV-3 viruses, so our study sought to provide whole genome sequences from multiple countries to further the understanding of the global diversity of HPIV at a whole-genome level. We collected HPIV-1 and HPIV-3 samples and isolates from Argentina, Australia, France, Mexico, South Africa, Switzerland, and USA from the years 2003-2011 and sequenced the genomes of 40 HPIV-1 and 75 HPIV-3 viruses with Sanger and next-generation sequencing with the Ion Torrent, Illumina, and 454 platforms. Phylogenetic analysis showed that the HPIV-1 genome is evolving at an estimated rate of 4.97 × 10-4 mutations/site/year (95% highest posterior density 4.55 × 10-4 to 5.38 × 10-4) and the HPIV-3 genome is evolving at a similar rate (3.59 × 10-4 mutations/site/year, 95% highest posterior density 3.26 × 10-4 to 3.94 × 10-4). There were multiple genetically distinct lineages of both HPIV-1 and 3 circulating on a global scale. Further surveillance and whole-genome sequencing are greatly needed to better understand the spatial dynamics of these important respiratory viruses in humans.

Following its introduction into New York State (NYS) in 1999, West Nile virus (WNV; Flavivirus, Flaviviridae) underwent a rapid expansion throughout the USA and into Canada and Latin America. WNV has been characterized as being evolutionarily stable, with weak geographic structure, a dominance of purifying selection and limited adaptive change. We analyzed all available full-genome WNV sequences, focusing on the 543 available sequences from NYS, which included 495 newly sequenced 2000-15 isolates. In addition, we analyzed deep-sequencing data from 317 of these isolates. While our data are generally in agreement with the limited pace of evolutionary change and broad geographic and temporal mixing identified in other studies, we have identified some important exceptions. Most notably, there are 14 codons which demonstrated evidence of positive selection as determined by multiple models, including some positions with evidence of selection in NYS exclusively. Coincident with increased WNV activity, genotypes possessing one or more of these mutations, designated NY01, NY07, and NY10, have increased in prevalence in recent years and displaced historic strains. In addition, we have found a geographical bias with many of these mutations, which suggests selective pressures and adaptations could be regional. Lastly, our deep-sequencing data suggest both increased overall diversity in avian tissue isolates relative to mosquito isolates and multiple non-synonymous minority variants that are both host-specific and retained over time and space. Together, these data provide novel insight into the evolutionary pressures on WNV and the need for continued genetic surveillance and characterization of emergent strains.

We report 26 complete genomes of Zika virus (ZIKV) isolated after passaging the Zika virus strain FLR in mosquito (C6/36) and mammalian (Vero) cell lines. The consensus ZIKV genomes we recovered show greater than 99% nucleotide identify with each other and with the FLR strain used as input.

The recent emergence of Zika virus (ZIKV) has been concentrated in the Caribbean, Southeastern United States, and South- and Central America; resulting in travel-based cases being reported around the globe. As multi-disciplinary collaborations are combatting the ZIKV outbreak, the need to validate the sequence of existing strains has become apparent. Here, we report high-quality sequence data for multiple ZIKV strains made publicly available through the National Institutes of Health- (NIH) funded biorepository, BEI Resources (www.beiresources.org). Next-generation sequencing, 3′ rapid amplification of cDNA ends (RACE), and viral genome annotation pipelines generated GenBank sequence records for 16 BEI Resources strains. Minor variants, consensus mutations, and consensus insertions/deletions were identified within the viral stocks using next-generation sequencing (NGS) and consensus changes were confirmed with Sanger sequencing. Bioinformatics analyses of the sequencing results confirm that the virus stocks available to the scientific research community through BEI Resources adequately represent the viral population diversity of ZIKV.

We report here the whole-genome sequence of 11 Zika virus (ZIKV) samples from six pediatric patients in Nicaragua. Serum samples were collected, and ZIKV was isolated in tissue culture. Both serum and virus isolates were sequenced. The consensus ZIKV genomes are greater than 99% identical to each other.

Chikungunya virus (CHIKV) has been detected sporadically since the 1950s and includes three distinct co-circulating genotypes. In late 2013, the Asian genotype of CHIKV was responsible for the Caribbean outbreak (CO) that rapidly became an epidemic throughout the Americas. There is a limited understanding of the molecular evolution of CHIKV in the Americas during this epidemic. We sequenced 185 complete CHIKV genomes collected mainly from Nicaragua in Central America and Florida in the United States during the 2014-2015 Caribbean/Americas epidemic. Our comprehensive phylogenetic analyses estimated the epidemic history of the Asian genotype and the recent Caribbean outbreak (CO) clade, revealed considerable genetic diversity within the CO clade, and described different epidemiological dynamics of CHIKV in the Americas. Specifically, we identified multiple introductions in both Nicaragua and Florida, with rapid local spread of viruses in Nicaragua but limited autochthonous transmission in Florida in the US. Our phylogenetic analysis also showed phylogeographic clustering of the CO clade. In addition, we identified the significant amino acid substitutions that were observed across the entire Asian genotype during its evolution and examined amino acid changes that were specific to the CO clade. Deep sequencing analysis identified specific minor variants present in clinical specimens below-consensus levels. Finally, we investigated the association between viral phylogeny and geographic/clinical metadata in Nicaragua. To date, this study represents the largest single collection of CHIKV complete genomes during the Caribbean/Americas epidemic and significantly expands our understanding of the emergence and evolution of CHIKV CO clade in the Americas.

Currently the National Center of Biotechnology Information (NCBI) assigns individual taxonomy identifiers to each distinct influenza virus isolate submitted to GenBank. To support this practice, individual flu isolates must be manually added to the NCBI taxonomy database and unique taxonomy identifiers generated. This added layer of manual processing is unique to influenza virus and prevents automatization of the flu sequence submission process. Here we outline a new NCBI policy that normalizes influenza virus taxonomy processing but maintains features supported by the previous approach. This change will reduce the amount of manual handling necessary for flu submissions and pave the way for increased automation of the submissions process. While this automation may disrupt some historic practices, it will better align influenza virus data processing with other viruses and ultimately lower the submission burden on data providers.

We report here the complete genome of a Dezidougou virus (DEZV) isolated from a passaged culture of the Zika virus strain DAK AR 41524. The consensus DEZV sequence we recovered shows 99% nucleotide similarity using BLASTN to a previously reported DEZV (accession no. JQ675604.1). The current sequence has additional repeat regions as well as a deleted repeat region, which we confirmed by Sanger sequencing, that were not present in the originally published sequence, JQ675604.1.

Culex quinquefasciatus (the southern house mosquito) is an important mosquito vector of viruses such as West Nile virus and St. Louis encephalitis virus, as well as of nematodes that cause lymphatic filariasis. C. quinquefasciatus is one species within the Culex pipiens species complex and can be found throughout tropical and temperate climates of the world. The ability of C. quinquefasciatus to take blood meals from birds, livestock, and humans contributes to its ability to vector pathogens between species. Here, we describe the genomic sequence of C. quinquefasciatus : Its repertoire of 18,883 protein-coding genes is 22% larger than that of Aedes aegypti and 52% larger than that of Anopheles gambiae with multiple gene-family expansions, including olfactory and gustatory receptors, salivary gland genes, and genes associated with xenobiotic detoxification.

BACKGROUND:In order to maintain genome information accurately and relevantly, original genome annotations need to be updated and evaluated regularly. Manual reannotation of genomes is important as it can significantly reduce the propagation of errors and consequently diminishes the time spent on mistaken research. For this reason, after five years from the initial submission of the Entamoeba histolytica draft genome publication, we have re-examined the original 23 Mb assembly and the annotation of the predicted genes.PRINCIPAL FINDINGS:The evaluation of the genomic sequence led to the identification of more than one hundred artifactual tandem duplications that were eliminated by re-assembling the genome. The reannotation was done using a combination of manual and automated genome analysis. The new 20 Mb assembly contains 1,496 scaffolds and 8,201 predicted genes, of which 60% are identical to the initial annotation and the remaining 40% underwent structural changes. Functional classification of 60% of the genes was modified based on recent sequence comparisons and new experimental data. We have assigned putative function to 3,788 proteins (46% of the predicted proteome) based on the annotation of predicted gene families, and have identified 58 protein families of five or more members that share no homology with known proteins and thus could be entamoeba specific. Genome analysis also revealed new features such as the presence of segmental duplications of up to 16 kb flanked by inverted repeats, and the tight association of some gene families with transposable elements.SIGNIFICANCE:This new genome annotation and analysis represents a more refined and accurate blueprint of the pathogen genome, and provides an upgraded tool as reference for the study of many important aspects of E. histolytica biology, such as genome evolution and pathogenesis.

We present the genome sequences of a new clinical isolate of the important human pathogen, Aspergillus fumigatus, A1163, and two closely related but rarely pathogenic species, Neosartorya fischeri NRRL181 and Aspergillus clavatus NRRL1. Comparative genomic analysis of A1163 with the recently sequenced A. fumigatus isolate Af293 has identified core, variable and up to 2% unique genes in each genome. While the core genes are 99.8% identical at the nucleotide level, identity for variable genes can be as low 40%. The most divergent loci appear to contain heterokaryon incompatibility (het) genes associated with fungal programmed cell death such as developmental regulator rosA. Cross-species comparison has revealed that 8.5%, 13.5% and 12.6%, respectively, of A. fumigatus, N. fischeri and A. clavatus genes are species-specific. These genes are significantly smaller in size than core genes, contain fewer exons and exhibit a subtelomeric bias. Most of them cluster together in 13 chromosomal islands, which are enriched for pseudogenes, transposons and other repetitive elements. At least 20% of A. fumigatus-specific genes appear to be functional and involved in carbohydrate and chitin catabolism, transport, detoxification, secondary metabolism and other functions that may facilitate the adaptation to heterogeneous environments such as soil or a mammalian host. Contrary to what was suggested previously, their origin cannot be attributed to horizontal gene transfer (HGT), but instead is likely to involve duplication, diversification and differential gene loss (DDL). The role of duplication in the origin of lineage-specific genes is further underlined by the discovery of genomic islands that seem to function as designated “gene dumps” and, perhaps, simultaneously, as “gene factories”.

The human malaria parasite Plasmodium vivax is responsible for 25–40% of the ∼515 million annual cases of malaria worldwide. Although seldom fatal, the parasite elicits severe and incapacitating clinical symptoms and often causes relapses months after a primary infection has cleared. Despite its importance as a major human pathogen, P. vivax is little studied because it cannot be propagated continuously in the laboratory except in non-human primates. We sequenced the genome of P. vivax to shed light on its distinctive biological features, and as a means to drive development of new drugs and vaccines. Here we describe the synteny and isochore structure of P. vivax chromosomes, and show that the parasite resembles other malaria parasites in gene content and metabolic potential, but possesses novel gene families and potential alternative invasion pathways not recognized previously. Completion of the P. vivax genome provides the scientific community with a valuable resource that can be used to advance investigation into this neglected species. Four distinct Plasmodium species are known to regularly infect humans: Plasmodium falciparum, P. vivax, P. malariae and P. ovale. The genome sequence of P. falciparum, the cause of the most severe type of human malaria, was completed in 2002 at the same time as the mosquito vector, Anopheles gambiae. In this week's Nature, which focuses on the malaria parasite, two further malaria genome sequences are described. First that of P. vivax, which contributes significant numbers to malaria incidence in humans, though in contrast to P. falciparum, the resulting disease is usually not fatal. The genome of this rather neglected species is presented together with a comparative analysis with the genomes of other Plasmodium species. Second, we publish the genome sequence of Plasmodium knowlesi. For long regarded as a monkey malaria parasite, it is increasingly becoming recognized as the fifth human-infecting Plasmodium species. In particular, it is prevalent in South East Asia where it is often misdiagnosed as another human malaria parasite P. malariae. As a model organism P. knowlesi stands out: not only is it a primate system, useful for work on vaccines, but it can be cultured in vitro and subjected to efficient transfection and gene knockouts. In a Review Article, Elizabeth Winzeler considers the progress made towards using the genome sequence to understand basic malaria parasite biology, and in particular the work on developing rational therapeutic approaches to combat P. falciparum infections. See also the Editorial. For a comprehensive collection of resources visit Nature's past malaria specials: Malaria killer blow ; Outlook on malaria ; Malaria web focus ; Malaria Insight ; Nature Medicine focus on malaria ; Focus on malaria

We present a draft sequence of the genome of Aedes aegypti , the primary vector for yellow fever and dengue fever, which at ∼1376 million base pairs is about 5 times the size of the genome of the malaria vector Anopheles gambiae . Nearly 50% of the Ae. aegypti genome consists of transposable elements. These contribute to a factor of ∼4 to 6 increase in average gene length and in sizes of intergenic regions relative to An. gambiae and Drosophila melanogaster . Nonetheless, chromosomal synteny is generally maintained among all three insects, although conservation of orthologous gene order is higher (by a factor of ∼2) between the mosquito species than between either of them and the fruit fly. An increase in genes encoding odorant binding, cytochrome P450, and cuticle domains relative to An. gambiae suggests that members of these protein families underpin some of the biological differences between the two mosquito species.

Parasitic nematodes that cause elephantiasis and river blindness threaten hundreds of millions of people in the developing world. We have sequenced the ∼90 megabase (Mb) genome of the human filarial parasite Brugia malayi and predict ∼11,500 protein coding genes in 71 Mb of robustly assembled sequence. Comparative analysis with the free-living, model nematode Caenorhabditis elegans revealed that, despite these genes having maintained little conservation of local synteny during ∼350 million years of evolution, they largely remain in linkage on chromosomal units. More than 100 conserved operons were identified. Analysis of the predicted proteome provides evidence for adaptations of B. malayi to niches in its human and vector hosts and insights into the molecular basis of a mutualistic relationship with its Wolbachia endosymbiont. These findings offer a foundation for rational drug design.

The availability of the genome sequences of multiple Aspergillus spp. presents the research community with an unprecedented opportunity for discovery. The genomes of Neosartorya fischeri and Aspergillus clavatus have been sequenced in order to extend our knowledge of Aspergillus fumigatus, the primary cause of invasive aspergillosis. Through comparative genomic analysis, we hope to elucidate both obvious and subtle differences between genomes, developing new hypotheses that can be tested in the laboratory. A preliminary examination of the genomes and their predicted proteomes reveals extensive conservation between protein sequences and significant synteny, or conserved gene order. Comparative genomic analysis at the level of these closely related aspergilli should provide important insight into the evolutionary forces at play and their effect on gene content, regulation and expression.

The ciliate Tetrahymena thermophila is a model organism for molecular and cellular biology. Like other ciliates, this species has separate germline and soma functions that are embodied by distinct nuclei within a single cell. The germline-like micronucleus (MIC) has its genome held in reserve for sexual reproduction. The soma-like macronucleus (MAC), which possesses a genome processed from that of the MIC, is the center of gene expression and does not directly contribute DNA to sexual progeny. We report here the shotgun sequencing, assembly, and analysis of the MAC genome of T. thermophila, which is approximately 104 Mb in length and composed of approximately 225 chromosomes. Overall, the gene set is robust, with more than 27,000 predicted protein-coding genes, 15,000 of which have strong matches to genes in other organisms. The functional diversity encoded by these genes is substantial and reflects the complexity of processes required for a free-living, predatory, single-celled organism. This is highlighted by the abundance of lineage-specific duplications of genes with predicted roles in sensing and responding to environmental conditions (e.g., kinases), using diverse resources (e.g., proteases and transporters), and generating structural complexity (e.g., kinesins and dyneins). In contrast to the other lineages of alveolates (apicomplexans and dinoflagellates), no compelling evidence could be found for plastid-derived genes in the genome. UGA, the only T. thermophila stop codon, is used in some genes to encode selenocysteine, thus making this organism the first known with the potential to translate all 64 codons in nuclear genes into amino acids. We present genomic evidence supporting the hypothesis that the excision of DNA from the MIC to generate the MAC specifically targets foreign DNA as a form of genome self-defense. The combination of the genome sequence, the functional diversity encoded therein, and the presence of some pathways missing from other model organisms makes T. thermophila an ideal model for functional genomic studies to address biological, biomedical, and biotechnological questions of fundamental importance.

Entamoeba histolytica is an intestinal parasite and the causative agent of amoebiasis, which is a significant source of morbidity and mortality in developing countries. Here we present the genome of E. histolytica, which reveals a variety of metabolic adaptations shared with two other amitochondrial protist pathogens: Giardia lamblia and Trichomonas vaginalis. These adaptations include reduction or elimination of most mitochondrial metabolic pathways and the use of oxidative stress enzymes generally associated with anaerobic prokaryotes. Phylogenomic analysis identifies evidence for lateral gene transfer of bacterial genes into the E. histolytica genome, the effects of which centre on expanding aspects of E. histolytica's metabolic repertoire. The presence of these genes and the potential for novel metabolic pathways in E. histolytica may allow for the development of new chemotherapeutic agents. The genome encodes a large number of novel receptor kinases and contains expansions of a variety of gene families, including those associated with virulence. Additional genome features include an abundance of tandemly repeated transfer-RNA-containing arrays, which may have a structural function in the genome. Analysis of the genome provides new insights into the workings and genome evolution of a major human pathogen.