Optimized quantification of intra-host viral diversity in SARS-CoV-2 and influenza virus sequence data.

High error rates of viral RNA-dependent RNA polymerases lead to diverse intra-host viral populations during infection. Errors made during replication that are not strongly deleterious to the virus can lead to the generation of minority variants. However, accurate detection of minority variants in viral sequence data is complicated by errors introduced during sample preparation and data analysis. We used synthetic RNA controls and simulated data to test seven variant-calling tools across a range of allele frequencies and simulated coverages. We show that choice of variant caller and use of replicate sequencing have the most significant impact on single-nucleotide variant (SNV) discovery and demonstrate how both allele frequency and coverage thresholds impact both false discovery and false-negative rates. When replicates are not available, using a combination of multiple callers with more stringent cutoffs is recommended. We use these parameters to find minority variants in sequencing data from SARS-CoV-2 clinical specimens and provide guidance for studies of intra-host viral diversity using either single replicate data or data from technical replicates. Our study provides a framework for rigorous assessment of technical factors that impact SNV identification in viral samples and establishes heuristics that will inform and improve future studies of intra-host variation, viral diversity, and viral evolution. IMPORTANCE When viruses replicate inside a host cell, the virus replication machinery makes mistakes. Over time, these mistakes create mutations that result in a diverse population of viruses inside the host. Mutations that are neither lethal to the virus nor strongly beneficial can lead to minority variants that are minor members of the virus population. However, preparing samples for sequencing can also introduce errors that resemble minority variants, resulting in the inclusion of false-positive data if not filtered correctly. In this study, we aimed to determine the best methods for identification and quantification of these minority variants by testing the performance of seven commonly used variant-calling tools. We used simulated and synthetic data to test their performance against a true set of variants and then used these studies to inform variant identification in data from SARS-CoV-2 clinical specimens. Together, analyses of our data provide extensive guidance for future studies of viral diversity and evolution.

Optimized Quantification of Intrahost Viral Diversity in SARS-CoV-2 and Influenza Virus Sequence Data.

High error rates of viral RNA-dependent RNA polymerases lead to diverse intra-host viral populations during infection. Errors made during replication that are not strongly deleterious to the virus can lead to the generation of minority variants. However, accurate detection of minority variants in viral sequence data is complicated by errors introduced during sample preparation and data analysis. We used synthetic RNA controls and simulated data to test seven variant calling tools across a range of allele frequencies and simulated coverages. We show that choice of variant caller, and use of replicate sequencing have the most significant impact on single nucleotide variant (SNV) discovery and demonstrate how both allele frequency and coverage thresholds impact both false discovery and false negative rates. We use these parameters to find minority variants in sequencing data from SARS-CoV-2 clinical specimens and provide guidance for studies of intrahost viral diversity using either single replicate data or data from technical replicates. Our study provides a framework for rigorous assessment of technical factors that impact SNV identification in viral samples and establishes heuristics that will inform and improve future studies of intrahost variation, viral diversity, and viral evolution. IMPORTANCE: When viruses replicate inside a host, the virus replication machinery makes mistakes. Over time, these mistakes create mutations that result in a diverse population of viruses inside the host. Mutations that are neither lethal to the virus, nor strongly beneficial, can lead to minority variants that are minor members of the virus population. However, preparing samples for sequencing can also introduce errors that resemble minority variants, resulting in inclusion of false positive data if not filtered correctly. In this study, we aimed to determine the best methods for identification and quantification of these minority variants by testing the performance of seven commonly used variant calling tools. We used simulated and synthetic data to test their performance against a true set of variants, and then used these studies to inform variant identification in data from clinical SARS-CoV-2 clinical specimens. Together, analyses of our data provide extensive guidance for future studies of viral diversity and evolution.

Initial Mapping of the New York City Wastewater Virome.

Bacteriophages are abundant members of all microbiomes studied to date, influencing microbial communities through interactions with their bacterial hosts. Despite their functional importance and ubiquity, phages have been underexplored in urban environments compared to their bacterial counterparts. We profiled the viral communities in New York City (NYC) wastewater using metagenomic data collected in November 2014 from 14 wastewater treatment plants. We show that phages accounted for the largest viral component of the sewage samples and that specific virus communities were associated with local environmental conditions within boroughs. The vast majority of the virus sequences had no homology matches in public databases, forming an average of 1,700 unique virus clusters (putative genera). These new clusters contribute to elucidating the overwhelming proportion of data that frequently goes unidentified in viral metagenomic studies. We assigned potential hosts to these phages, which appear to infect a wide range of bacterial genera, often outside their presumed host. We determined that infection networks form a modular-nested pattern, indicating that phages include a range of host specificities, from generalists to specialists, with most interactions organized into distinct groups. We identified genes in viral contigs involved in carbon and sulfur cycling, suggesting functional importance of viruses in circulating pathways and gene functions in the wastewater environment. In addition, we identified virophage genes as well as a nearly complete novel virophage genome. These findings provide an understanding of phage abundance and diversity in NYC wastewater, previously uncharacterized, and further examine geographic patterns of phage-host association in urban environments.IMPORTANCE Wastewater is a rich source of microbial life and contains bacteria, viruses, and other microbes found in human waste as well as environmental runoff sources. As part of an effort to characterize the New York City wastewater metagenome, we profiled the viral community of sewage samples across all five boroughs of NYC and found that local sampling sites have unique sets of viruses. We focused on bacteriophages, or viruses of bacteria, to understand how they may influence the microbial ecology of this system. We identified several new clusters of phages and successfully associated them with bacterial hosts, providing insight into virus-host interactions in urban wastewater. This study provides a first look into the viral communities present across the wastewater system in NYC and points to their functional importance in this environment.

The administration of intranasal live attenuated influenza vaccine induces changes in the nasal microbiota and nasal epithelium gene expression profiles.

BACKGROUND: Viral infections such as influenza have been shown to predispose hosts to increased colonization of the respiratory tract by pathogenic bacteria and secondary bacterial pneumonia. To examine how viral infections and host antiviral immune responses alter the upper respiratory microbiota, we analyzed nasal bacterial composition by 16S ribosomal RNA (rRNA) gene sequencing in healthy adults at baseline and at 1 to 2 weeks and 4 to 6 weeks following instillation of live attenuated influenza vaccine or intranasal sterile saline. A subset of these samples was submitted for microarray host gene expression profiling. RESULTS: We found that live attenuated influenza vaccination led to significant changes in microbial community structure, diversity, and core taxonomic membership as well as increases in the relative abundances of Staphylococcus and Bacteroides genera (both p < 0.05). Hypergeometric testing for the enrichment of gene ontology terms in the vaccinated group reflected a robust up-regulation of type I and type II interferon-stimulated genes in the vaccinated group relative to controls. Translational murine studies showed that poly I:C administration did in fact permit greater nasal Staphylococcus aureus persistence, a response absent in interferon alpha/beta receptor deficient mice. CONCLUSIONS: Collectively, our findings demonstrate that although the human nasal bacterial community is heterogeneous and typically individually robust, activation of a type I interferon (IFN)-mediated antiviral response may foster the disproportionate emergence of potentially pathogenic species such as S. aureus. TRIAL REGISTRATION: This study was registered with Clinicaltrials.gov on 11/3/15, NCT02597647 .

Filarial and Wolbachia genomics.

Filarial nematode parasites, the causative agents for a spectrum of acute and chronic diseases including lymphatic filariasis and river blindness, threaten the well-being and livelihood of hundreds of millions of people in the developing regions of the world. The 2007 publication on a draft assembly of the 95-Mb genome of the human filarial parasite Brugia malayi- representing the first helminth parasite genome to be sequenced - has been followed in rapid succession by projects that have resulted in the genome sequencing of six additional filarial species, seven nonfilarial nematode parasites of animals and nearly 30 plant parasitic and free-living species. Parallel to the genomic sequencing, transcriptomic and proteomic projects have facilitated genome annotation, expanded our understanding of stage-associated gene expression and provided a first look at the role of epigenetic regulation of filarial genomes through microRNAs. The expansion in filarial genomics will also provide a significant enrichment in our knowledge of the diversity and variability in the genomes of the endosymbiotic bacterium Wolbachia leading to a better understanding of the genetic principles that govern filarial-Wolbachia mutualism. The goal here is to provide an overview of the trends and advances in filarial and Wolbachia genomics.

Secretory and endocytic pathways converge in a dynamic endosomal system in a primitive protozoan.

Leishmania are a group of primitive eukaryotic trypanosomatid protozoa that are apically polarized with a flagellum at their anterior end. Surrounding the base of the flagellum is the flagellar reservoir that constitutes the site for endocytosis and exocytosis in these organisms. In the present study, we define a novel multivesicular tubular compartment involved in the intracellular trafficking of macromolecules in Leishmania. This dynamic structure appears to subtend the flagellar reservoir and extends towards the posterior end of the cell. Functional domains of several surface-expressed proteins, such as the gp63 glycosyl phosphatidyl inositol anchor and the 3'nucleotidase/nuclease transmembrane domain were fused to green fluorescent protein. These chimeric proteins were found to traffic through the secretory pathway and, while reaching their intended destinations, also accumulated within the intracellular tubular compartment. Using various compounds that are efficient fluid-phase markers used to track endocytosis in higher eukaryotes, we showed that this tubular compartment constitutes an important station in the endocytic pathway of these cells. Based on our functional observations of its role in the trafficking of expressed proteins and endocytosed markers, this compartment appears to have properties similar to endosomes of higher eukaryotes.

Dissection of the functional domains of the Leishmania surface membrane 3'-nucleotidase/nuclease, a unique member of the class I nuclease family.

Class I nucleases are a family of enzymes that specifically hydrolyze single-stranded nucleic acids. Recently, we characterized the gene encoding a new member of this family, the 3'-nucleotidase/nuclease (Ld3'NT/NU) of the parasitic protozoan Leishmania donovani. The Ld3'NT/NU is unique as it is the only class I nuclease that is a cell surface membrane-anchored protein. Currently, we used a homologous episomal expression system to dissect the functional domains of the Ld3'NT/NU. Our results showed that its N-terminal signal peptide targeted this protein into the endoplasmic reticulum. Using Ld3'NT/NU-green fluorescent protein chimeras, we showed that the C-terminal domain of the Ld3'NT/NU functioned to anchor this protein into the parasite cell surface membrane. Further, removal of the Ld3'NT/NU C-terminal domain resulted in its release/secretion as a fully active enzyme. Moreover, deletion of its single N-linked glycosylation site showed that such glycosylation was not required for the enzymatic functions of the Ld3'NT/NU. Thus, using the fidelity of a homologous expression system, we have defined some of the functional domains of this unique member of the class I nuclease family.

Inducible expression of suicide genes in Leishmania donovani amastigotes.

This study tests the feasibility of using the A2 gene regulatory system to create a Leishmania cell line in which attenuation is developmentally regulated when the parasite differentiates from promastigotes to amastigotes. The Leishmania donovani- inducible A2 gene regulatory system was used to differentially express in amastigotes two potential suicide genes: a truncated version of the L. donovani 3'-nucleotidase/nuclease expressed in the cytoplasm and the herpes simplex virus thymidine kinase gene. These genes were inserted between A2 noncoding regulatory sequences for up-regulation of expression in amastigotes. The accumulation of toxic products affected L. donovani cell replication and viability both in vitro and in vivo. The inducible expression of toxic gene products represents a valuable tool for the development of safe and effective vaccines.

A2rel: a constitutively expressed Leishmania gene linked to an amastigote-stage-specific gene.

The A2-A2rel gene copies are arranged in tandem arrays on a 850 kb chromosome in Leishmania donovani. Contrary to A2 mRNA which displays amastigote-stage-specific expression, A2rel gene expression is constitutive throughout the L. donovani life cycle. The A2rel sequence was found to be conserved in all Leishmania species tested, while the A2 sequence is specific to L. donovani and L. mexicana. The A2rel full length cDNA is of 2.3 kb and it contains one open reading frame coding for a putative protein of 436 amino acids.

Antibody response against a Leishmania donovani amastigote-stage-specific protein in patients with visceral leishmaniasis.

The antibody response against an amastigote-specific protein (A2) from Leishmania donovani was investigated. Sera from patients with trypanosomiasis and various forms of leishmaniasis were screened for anti-A2 antibodies. Sera from patients infected only with L. donovani or Leishmania mexicana specifically recognized the A2 recombinant protein. These results were consistent with karyotype analyses which revealed that the A2 gene is conserved in L. donovani and L. mexicana strains. The potential of this antigen in diagnosis was further explored by screening a series of sera obtained from patients in regions of the Sudan and India where L. donovani is endemic. The prevalence of anti-A2 antibodies was determined by Western blotting for all samples. Enzyme-linked immunosorbent assay (ELISA) and an immunoprecipitation assay were also performed on some of the samples. Anti-A2 antibodies were detected by ELISA in 82 and 60% of the samples from individuals with active visceral leishmaniasis (kala-azar) from the Sudan and India, respectively, while the immunoprecipitation assay detected the antibodies in 92% of the samples from India. These data suggest that the A2 protein may be a useful diagnostic antigen for visceral leishmaniasis.

Identification and overexpression of the A2 amastigote-specific protein in Leishmania donovani.

Leishmania protozoa must adapt rapidly to widely different environments and thus exist as promastigotes in their sandfly host and as amastigotes in their mammalian host. Promastigote differentiation into amastigotes is accompanied by both morphological and biological changes. The molecular mechanisms regulating the differentiation and survival of the different life cycle stages are poorly understood. We have therefore undertaken to identify and characterize amastigote-specific genes and their corresponding products based on the rationale that such products may be involved in the survival in the mammalian host. Previous studies in our laboratory have revealed that the A2 gene family-derived transcripts are abundant in L. donovani amastigotes but are barely detectable in promastigotes. In the present study, we have raised polyclonal and monoclonal antibodies against a recombinant A2 protein synthesized in Escherichia coli. These antibodies have been used to identify a family of A2 proteins ranging from 45 kDa to about 100 kDa which are specifically detected in L. donovani cells when they are cultured in 37 degrees C, and pH 4.5 (conditions which mimic the macrophage phagolysosome) but not in promastigotes cultured at 26 degrees C and pH 7.4. A2 protein therefore represents a unique amastigote-specific protein marker for L. donovani. It is also demonstrated that it was possible to overexpress the A2 protein specifically in amastigote-like cells using a plasmid construct containing the A2 coding and non-coding sequences. These advances set the foundation for defining the biological function of the A2 protein and other genes when specifically expressed in amastigotes.