Kathie A. Mihindukulasuriya

Evaluation of 16s rDNA-based community profiling for human microbiome research

The Human Microbiome Project will establish a reference data set for analysis of the microbiome of healthy adults by surveying multiple body sites from 300 people and generating data from over 12,000 samples. To characterize these samples, the participating sequencing centers evaluated and adopted 16S rDNA community profiling protocols for ABI 3730 and 454 FLX Titanium sequencing. In the course of establishing protocols, we examined the performance and error characteristics of each technology, and the relationship of sequence error to the utility of 16S rDNA regions for classification- and OTU-based analysis of community structure. The data production protocols used for this work are those used by the participating centers to produce 16S rDNA sequence for the Human Microbiome Project. Thus, these results can be informative for interpreting the large body of clinical 16S rDNA data produced for this project.

Biogeography of the ecosystems of the healthy human body

BackgroundCharacterizing the biogeography of the microbiome of healthy humans is essential for understanding microbial associated diseases. Previous studies mainly focused on a single body habitat from a limited set of subjects. Here, we analyzed one of the largest microbiome datasets to date and generated a biogeographical map that annotates the biodiversity, spatial relationships, and temporal stability of 22 habitats from 279 healthy humans.ResultsWe identified 929 genera from more than 24 million 16S rRNA gene sequences of 22 habitats, and we provide a baseline of inter-subject variation for healthy adults. The oral habitat has the most stable microbiota with the highest alpha diversity, while the skin and vaginal microbiota are less stable and show lower alpha diversity. The level of biodiversity in one habitat is independent of the biodiversity of other habitats in the same individual. The abundances of a given genus at a body site in which it dominates do not correlate with the abundances at body sites where it is not dominant. Additionally, we observed the human microbiota exhibit both cosmopolitan and endemic features. Finally, comparing datasets of different projects revealed a project-based clustering pattern, emphasizing the significance of standardization of metagenomic studies.ConclusionsThe data presented here extend the definition of the human microbiome by providing a more complete and accurate picture of human microbiome biogeography, addressing questions best answered by a large dataset of subjects and body sites that are deeply sampled by sequencing.

Sequence Analysis of the Human Virome in Febrile and Afebrile Children

Unexplained fever (UF) is a common problem in children under 3 years old. Although virus infection is suspected to be the cause of most of these fevers, a comprehensive analysis of viruses in samples from children with fever and healthy controls is important for establishing a relationship between viruses and UF. We used unbiased, deep sequencing to analyze 176 nasopharyngeal swabs (NP) and plasma samples from children with UF and afebrile controls, generating an average of 4.6 million sequences per sample. An analysis pipeline was developed to detect viral sequences, which resulted in the identification of sequences from 25 viral genera. These genera included expected pathogens, such as adenoviruses, enteroviruses, and roseoloviruses, plus viruses with unknown pathogenicity. Viruses that were unexpected in NP and plasma samples, such as the astrovirus MLB-2, were also detected. Sequencing allowed identification of virus subtype for some viruses, including roseoloviruses. Highly sensitive PCR assays detected low levels of viruses that were not detected in approximately 5 million sequences, but greater sequencing depth improved sensitivity. On average NP and plasma samples from febrile children contained 1.5- to 5-fold more viral sequences, respectively, than samples from afebrile children. Samples from febrile children contained a broader range of viral genera and contained multiple viral genera more frequently than samples from children without fever. Differences between febrile and afebrile groups were most striking in the plasma samples, where detection of viral sequence may be associated with a disseminated infection. These data indicate that virus infection is associated with UF. Further studies are important in order to establish the range of viral pathogens associated with fever and to understand of the role of viral infection in fever. Ultimately these studies may improve the medical treatment of children with UF by helping avoid antibiotic therapy for children with viral infections.

Identification of a Novel Coronavirus from a Beluga Whale by Using a Panviral Microarray

ABSTRACT The emergence of viruses such as severe acute respiratory syndrome coronavirus and Nipah virus has underscored the role of animal reservoirs in human disease and the need for reservoir surveillance. Here, we used a panviral DNA microarray to investigate the death of a captive beluga whale in an aquatic park. A highly divergent coronavirus, tentatively named coronavirus SW1, was identified in liver tissue from the deceased whale. Subsequently, the entire genome of SW1 was sequenced, yielding a genome of 31,686 nucleotides. Phylogenetic analysis revealed SW1 to be a novel virus distantly related to but most similar to group III coronaviruses.

Metagenomic analysis of double-stranded DNA viruses in healthy adults

BackgroundThe Human Microbiome Project (HMP) was undertaken with the goal of defining microbial communities in and on the bodies of healthy individuals using high-throughput metagenomic sequencing analysis. The viruses present in these microbial communities, the `human virome,’ are an important aspect of the human microbiome that is particularly understudied in the absence of overt disease. We analyzed eukaryotic double-stranded DNA (dsDNA) viruses, together with dsDNA replicative intermediates of single-stranded DNA viruses, in metagenomic sequence data generated by the HMP. We studied 706 samples from 102 subjects were studied, with each subject sampled at up to five major body habitats: nose, skin, mouth, vagina, and stool. Fifty-one individuals had samples taken at two or three time points 30 to 359 days apart from at least one of the body habitats.ResultsWe detected an average of 5.5 viral genera in each individual. At least one virus was detected in 92% of the individuals sampled. These viruses included herpesviruses, papillomaviruses, polyomaviruses, adenoviruses, anelloviruses, parvoviruses, and circoviruses. Each individual had a distinct viral profile, demonstrating the high interpersonal diversity of the virome. Some components of the virome were stable over time.ConclusionsThis study is the first to use high-throughput DNA sequencing to describe the diversity of eukaryotic dsDNA viruses in a large cohort of normal individuals who were sampled at multiple body sites. Our results show that the human virome is a complex component of the microbial flora. Some viruses establish long-term infections that may be associated with increased risk or possibly with protection from disease. A better understanding of the composition and dynamics of the virome may hold important keys to human health.

Novel Bacterial Taxa in the Human Microbiome

The human gut harbors thousands of bacterial taxa. A profusion of metagenomic sequence data has been generated from human stool samples in the last few years, raising the question of whether more taxa remain to be identified. We assessed metagenomic data generated by the Human Microbiome Project Consortium to determine if novel taxa remain to be discovered in stool samples from healthy individuals. To do this, we established a rigorous bioinformatics pipeline that uses sequence data from multiple platforms (Illumina GAIIX and Roche 454 FLX Titanium) and approaches (whole-genome shotgun and 16S rDNA amplicons) to validate novel taxa. We applied this approach to stool samples from 11 healthy subjects collected as part of the Human Microbiome Project. We discovered several low-abundance, novel bacterial taxa, which span three major phyla in the bacterial tree of life. We determined that these taxa are present in a larger set of Human Microbiome Project subjects and are found in two sampling sites (Houston and St. Louis). We show that the number of false-positive novel sequences (primarily chimeric sequences) would have been two orders of magnitude higher than the true number of novel taxa without validation using multiple datasets, highlighting the importance of establishing rigorous standards for the identification of novel taxa in metagenomic data. The majority of novel sequences are related to the recently discovered genus Barnesiella, further encouraging efforts to characterize the members of this genus and to study their roles in the microbial communities of the gut. A better understanding of the effects of less-abundant bacteria is important as we seek to understand the complex gut microbiome in healthy individuals and link changes in the microbiome to disease.

Quaranfil, Johnston Atoll, and Lake Chad Viruses Are Novel Members of the Family Orthomyxoviridae

ABSTRACT Arboviral infections are an important cause of emerging infections due to the movements of humans, animals, and hematophagous arthropods. Quaranfil virus (QRFV) is an unclassified arbovirus originally isolated from children with mild febrile illness in Quaranfil, Egypt, in 1953. It has subsequently been isolated in multiple geographic areas from ticks and birds. We used high-throughput sequencing to classify QRFV as a novel orthomyxovirus. The genome of this virus is comprised of multiple RNA segments; five were completely sequenced. Proteins with limited amino acid similarity to conserved domains in polymerase (PA, PB1, and PB2) and hemagglutinin (HA) genes from known orthomyxoviruses were predicted to be present in four of the segments. The fifth sequenced segment shared no detectable similarity to any protein and is of uncertain function. The end-terminal sequences of QRFV are conserved between segments and are different from those of the known orthomyxovirus genera. QRFV is known to cross-react serologically with two other unclassified viruses, Johnston Atoll virus (JAV) and Lake Chad virus (LKCV). The complete open reading frames of PB1 and HA were sequenced for JAV, while a fragment of PB1 of LKCV was identified by mass sequencing. QRFV and JAV PB1 and HA shared 80% and 70% amino acid identity to each other, respectively; the LKCV PB1 fragment shared 83% amino acid identity with the corresponding region of QRFV PB1. Based on phylogenetic analyses, virion ultrastructural features, and the unique end-terminal sequences identified, we propose that QRFV, JAV, and LKCV comprise a novel genus of the family Orthomyxoviridae.

Nyamanini and midway viruses define a novel taxon of RNA viruses in the order Mononegavirales

ABSTRACT Here, we report the sequencing and classification of Nyamanini virus (NYMV) and Midway virus (MIDWV), two antigenically related viruses that were first isolated in 1957 and 1966, respectively. Although these viruses have been cultured multiple times from cattle egrets, seabirds, and their ticks, efforts to classify them taxonomically using conventional serological and electron microscopic approaches have failed completely. We used a random shotgun sequencing strategy to define the genomes of NYMV and MIDWV. Contigs of 11,631 and 11,752 nucleotides, representing the complete genome of NYMV and the near-complete genome of MIDWV, respectively, were assembled. Each virus genome was predicted to carry six open reading frames (ORFs). BLAST analysis indicated that only two of the ORF proteins of each virus, the putative nucleocapsid and polymerase, had detectable sequence similarity to known viral proteins. Phylogenetic analysis of these ORF proteins demonstrated that the closest relatives of NYNV and MIDWV are negative-stranded-RNA viruses in the order Mononegavirales. On the basis of their very limited sequence similarity to known viruses, we propose that NYMV and MIDWV define a novel genus, Nyavirus, in this order.

Exploration of bacterial community classes in major human habitats

BackgroundDetermining bacterial abundance variation is the first step in understanding bacterial similarity between individuals. Categorization of bacterial communities into groups or community classes is the subsequent step in describing microbial distribution based on abundance patterns. Here, we present an analysis of the groupings of bacterial communities in stool, nasal, skin, vaginal and oral habitats in a healthy cohort of 236 subjects from the Human Microbiome Project.ResultsWe identify distinct community group patterns in the anterior nares, four skin sites, and vagina at the genus level. We also confirm three enterotypes previously identified in stools. We identify two clusters with low silhouette values in most oral sites, in which bacterial communities are more homogeneous. Subjects sharing a community class in one habitat do not necessarily share a community class in another, except in the three vaginal sites and the symmetric habitats of the left and right retroauricular creases. Demographic factors, including gender, age, and ethnicity, significantly influence community composition in several habitats. Community classes in the vagina, retroauricular crease and stool are stable over approximately 200 days.ConclusionThe community composition, association of demographic factors with community classes, and demonstration of community stability deepen our understanding of the variability and dynamics of human microbiomes. This also has significant implications for experimental designs that seek microbial correlations with clinical phenotypes.

Nyamiviridae: Proposal for a new family in the order Mononegavirales

Nyamanini virus (NYMV) and Midway virus (MIDWV) are unclassified tick-borne agents that infect land birds and seabirds, respectively. The recent molecular characterization of both viruses confirmed their already known close serological relationship and revealed them to be nonsegmented, single- and negative-stranded RNA viruses that are clearly related to, but quite distinct from, members of the order Mononegavirales (bornaviruses, filoviruses, paramyxoviruses, and rhabdoviruses). A third agent, soybean cyst nematode virus 1 (SbCNV-1, previously named soybean cyst nematode nyavirus), was recently found to be an additional member of this new virus group. Here, we review the current knowledge about all three viruses and propose classifying them as members of a new mononegaviral family, Nyamiviridae.