NCBI's Virus Discovery Hackathon:Engaging Research Communities to Identify Cloud Infrastructure Requirements

A wealth of viral data sits untapped in publicly available metagenomic data sets when it might be extracted to create a usable index for the virological research community. We hypothesized that work of this complexity and scale could be done in a hackathon setting. Ten teams comprised of over 40 participants from six countries, assembled to create a crowd-sourced set of analysis and processing pipelines for a complex biological data set in a three-day event on the San Diego State University campus starting 9 January 2019. Prior to the hackathon, 141,676 metagenomic data sets from the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) were pre-assembled into contiguous assemblies (contigs) by NCBI staff. During the hackathon, a subset consisting of 2953 SRA data sets (approximately 55 million contigs) was selected, which were further filtered for a minimal length of 1 kb. This resulted in 4.2 million (Mio) contigs, which were aligned using BLAST against all known virus genomes, phylogenetically clustered and assigned metadata. Out of the 4.2 Mio contigs, 360,000 contigs were labeled with domains and an additional subset containing 4400 contigs was screened for virus or virus-like genes. The work yielded valuable insights into both SRA data and the cloud infrastructure required to support such efforts, revealing analysis bottlenecks and possible workarounds thereof. Mainly: (i) Conservative assemblies of SRA data improves initial analysis steps; (ii) existing bioinformatic software with weak multithreading/multicore support can be elevated by wrapper scripts to use all cores within a computing node; (iii) redesigning existing bioinformatic algorithms for a cloud infrastructure to facilitate its use for a wider audience; and (iv) a cloud infrastructure allows a diverse group of researchers to collaborate effectively. The scientific findings will be extended during a follow-up event. Here, we present the applied workflows, initial results, and lessons learned from the hackathon

Inherited chromosomally integrated HHV-6 demonstrates tissue-specific RNA expression in vivo

Thesis (Master's)--University of Washington, 2019Human herpesvirus-6A and 6B (HHV-6A, HHV-6B) are highly prevalent viruses unique in that they, besides Epstein-Barr virus, are the only human herpesviruses capable of chromosomal integration. When a chromosomal integration event happens in a germ cell the resulting progeny will have one copy of the virus in all of their cells, referred to as inherited chromosomally integrated human herpesvirus-6 (iciHHV-6). Active HHV-6 infections in those who are iciHHV-6 positive confound current DNA based PCR assays, highlighting a necessity for mRNA-based assays. As the transcriptional state of iciHHV-6 has not been defined in vivo, it is unclear which HHV-6 genes best determine actively infected iciHHV-6 individuals. Here, we screened DNA- Seq and RNA-Seq data for 650 individuals available through the Genotype-Tissue Expression (GTEx) project and identified 2 iciHHV-6A and 4 iciHHV-6B positive candidates. When corresponding tissue-specific gene expression signatures were analyzed, the HHV-6 genes U90 and U100 were expressed in the brain, testis, breast, adrenal gland, lungs, salivary gland, esophagus, skeletal muscle, colon, tibial nerve and artery, adipose tissue, heart, skin, and thyroid. Additionally, high levels of HHV-6 gene expression were detected in the brain, testis, esophagus, and adrenal gland. We also analyzed data from the Synapse Mount Sinai Brain Bank and found similar brain tissue expression in the 4 iciHHV-6 positive samples. We found no expression of U38, a viral polymerase subunit found to be expressed in HHV-6 active infections, in GTEx whole blood RNAseq samples. However, using an RT-qPCR assay specific to the HHV-6A and -6B U38 genes, we saw that U38 was expressed in some iciHHV-6 positive PBMC samples submitted for iciHHV-6 testing

Mutant KRAS regulates transposable element RNA and innate immunity via KRAB zinc-finger genes.

RAS genes are the most frequently mutated oncogenes in cancer, yet the effects of oncogenic RAS signaling on the noncoding transcriptome remain unclear. We analyzed the transcriptomes of human airway and bronchial epithelial cells transformed with mutant KRAS to define the landscape of KRAS-regulated noncoding RNAs. We find that oncogenic KRAS signaling upregulates noncoding transcripts throughout the genome, many of which arise from transposable elements (TEs). These TE RNAs exhibit differential expression, are preferentially released in extracellular vesicles, and are regulated by KRAB zinc-finger (KZNF) genes, which are broadly downregulated in mutant KRAS cells and lung adenocarcinomas in vivo. Moreover, mutant KRAS induces an intrinsic IFN-stimulated gene (ISG) signature that is often seen across many different cancers. Our results indicate that mutant KRAS remodels the repetitive noncoding transcriptome, demonstrating the broad scope of intracellular and extracellular RNAs regulated by this oncogenic signaling pathway

In vivo antiviral host transcriptional response to SARS-CoV-2 by viral load, sex, and age.

Despite limited genomic diversity, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has shown a wide range of clinical manifestations in different patient populations. The mechanisms behind these host differences are still unclear. Here, we examined host response gene expression across infection status, viral load, age, and sex among shotgun RNA sequencing profiles of nasopharyngeal (NP) swabs from 430 individuals with PCR-confirmed SARS-CoV-2 and 54 negative controls. SARS-CoV-2 induced a strong antiviral response with up-regulation of antiviral factors such as OAS1-3 and IFIT1-3 and T helper type 1 (Th1) chemokines CXCL9/10/11, as well as a reduction in transcription of ribosomal proteins. SARS-CoV-2 culture in human airway epithelial (HAE) cultures replicated the in vivo antiviral host response 7 days post infection, with no induction of interferon-stimulated genes after 3 days. Patient-matched longitudinal specimens (mean elapsed time = 6.3 days) demonstrated reduction in interferon-induced transcription, recovery of transcription of ribosomal proteins, and initiation of wound healing and humoral immune responses. Expression of interferon-responsive genes, including ACE2, increased as a function of viral load, while transcripts for B cell-specific proteins and neutrophil chemokines were elevated in patients with lower viral load. Older individuals had reduced expression of the Th1 chemokines CXCL9/10/11 and their cognate receptor CXCR3, as well as CD8A and granzyme B, suggesting deficiencies in trafficking and/or function of cytotoxic T cells and natural killer (NK) cells. Relative to females, males had reduced B cell-specific and NK cell-specific transcripts and an increase in inhibitors of nuclear factor kappa-B (NF-κB) signaling, possibly inappropriately throttling antiviral responses. Collectively, our data demonstrate that host responses to SARS-CoV-2 are dependent on viral load and infection time course, with observed differences due to age and sex that may contribute to disease severity

Molecular Features of the Measles Virus Viral Fusion Complex That Favor Infection and Spread in the Brain

International audienceMeasles virus (MeV) infection can cause serious complications in immunocompromised individuals, including measles inclusion body encephalitis (MIBE). In some cases, MeV persistence and subacute sclerosing panencephalitis (SSPE), another severe central nervous system (CNS) complication, develop even in the face of a systemic immune response

Comparative genomic, transcriptomic, and proteomic reannotation of human herpesvirus 6

Background: Human herpesvirus-6A and -6B (HHV-6) are betaherpesviruses that reach > 90% seroprevalence in the adult population. Unique among human herpesviruses, HHV-6 can integrate into the subtelomeric regions of human chromosomes; when this occurs in germ line cells it causes a condition called inherited chromosomally integrated HHV-6 (iciHHV-6). Only two complete genomes are available for replicating HHV-6B, leading to numerous conflicting annotations and little known about the global genomic diversity of this ubiquitous virus. Results: Using a custom capture panel for HHV-6B, we report complete genomes from 61 isolates of HHV-6B from active infections (20 from Japan, 35 from New York state, and 6 from Uganda), and 64 strains of iciHHV-6B (mostly from North America). HHV-6B sequence clustered by geography and illustrated extensive recombination. Multiple iciHHV-6B sequences from unrelated individuals across the United States were found to be completely identical, consistent with a founder effect. Several iciHHV-6B strains clustered with strains from recent active pediatric infection. Combining our genomic analysis with the first RNA-Seq and shotgun proteomics studies of HHV-6B, we completely reannotated the HHV-6B genome, altering annotations for more than 10% of existing genes, with multiple instances of novel splicing and genes that hitherto had gone unannotated. Conclusion: Our results are consistent with a model of intermittent de novo integration of HHV-6B into host germline cells during active infection with a large contribution of founder effect in iciHHV-6B. Our data provide a significant advance in the genomic annotation of HHV-6B, which will contribute to the detection, diversity, and control of this virus.Other UBCNon UBCReviewedFacult

Additional file 5: of Comparative genomic, transcriptomic, and proteomic reannotation of human herpesvirus 6

Figure S4. Gel image of silver stain of HHV-6B Z29 lysate in SupT1 cells or serum-free supernatant run on 4-12% TrisHCl gel in MES buffer. (PDF 1335 kb

Additional file 8: of Comparative genomic, transcriptomic, and proteomic reannotation of human herpesvirus 6

Table S4. HHV-6 Peptides Identified by Shotgun Proteomics. Mass spectrometry database search results are shown for HHV6 peptides identified using Protein Prospector v 5.19.1 described in Materials and Methods. The table reports the best matched peptide spectra. Provided are the mass to charge ratio (m/z), charge (z), mass error in ppm, the peptide sequence with previous and next amino acids in the sequence, variable modification, the fraction and retention time as spectrum identifiers. The start and end sequence numbers are given, along with Protein Prospector peptide score and peptide expectation value. (XLSX 88 kb

Additional file 3: of Comparative genomic, transcriptomic, and proteomic reannotation of human herpesvirus 6

Figure S2. Phylogenetic tree of HHV-6B complete U90/91 and U94/100 loci. HHV-6B genomes were aligned using MAFFT, curated for sequence outside of repeat regions, and phylogenetic trees were constructed using MrBayes along the 6 kb U90/91 (A), and 10 kb U94-100 (B) regions. HHV6-6B NY310 was used as an outgroup. Samples are colored and labeled for origin based on New York (green), Japan (blue), or iciHHV6-B from HSCT recipients or their donors in Seattle (black), as well as whether two genomes were recovered from first-degree relatives (red). Location images purchased from Adobe Stock. (ZIP 656 kb

Additional file 4: of Comparative genomic, transcriptomic, and proteomic reannotation of human herpesvirus 6

Figure S3. Non-contiguous gel images of silver stain of HHV-6B Z29 lysates in SupT1 cells or serum-free supernatant run on 10-20% TrisHCl gels in MOPS buffer. (PDF 3011 kb