A Universal Next-Generation Sequencing Protocol To Generate Noninfectious Barcoded cDNA Libraries from High-Containment RNA Viruses

ABSTRACT Several biosafety level 3 and/or 4 (BSL-3/4) pathogens are high-consequence, single-stranded RNA viruses, and their genomes, when introduced into permissive cells, are infectious. Moreover, many of these viruses are select agents (SAs), and their genomes are also considered SAs. For this reason, cDNAs and/or their derivatives must be tested to ensure the absence of infectious virus and/or viral RNA before transfer out of the BSL-3/4 and/or SA laboratory. This tremendously limits the capacity to conduct viral genomic research, particularly the application of next-generation sequencing (NGS). Here, we present a sequence-independent method to rapidly amplify viral genomic RNA while simultaneously abolishing both viral and genomic RNA infectivity across multiple single-stranded positive-sense RNA (ssRNA+) virus families. The process generates barcoded DNA amplicons that range in length from 300 to 1,000 bp, which cannot be used to rescue a virus and are stable to transport at room temperature. Our barcoding approach allows for up to 288 barcoded samples to be pooled into a single library and run across various NGS platforms without potential reconstitution of the viral genome. Our data demonstrate that this approach provides full-length genomic sequence information not only from high-titer virion preparations but it can also recover specific viral sequence from samples with limited starting material in the background of cellular RNA, and it can be used to identify pathogens from unknown samples. In summary, we describe a rapid, universal standard operating procedure that generates high-quality NGS libraries free of infectious virus and infectious viral RNA. IMPORTANCE This report establishes and validates a standard operating procedure (SOP) for select agents (SAs) and other biosafety level 3 and/or 4 (BSL-3/4) RNA viruses to rapidly generate noninfectious, barcoded cDNA amenable for next-generation sequencing (NGS). This eliminates the burden of testing all processed samples derived from high-consequence pathogens prior to transfer from high-containment laboratories to lower-containment facilities for sequencing. Our established protocol can be scaled up for high-throughput sequencing of hundreds of samples simultaneously, which can dramatically reduce the cost and effort required for NGS library construction. NGS data from this SOP can provide complete genome coverage from viral stocks and can also detect virus-specific reads from limited starting material. Our data suggest that the procedure can be implemented and easily validated by institutional biosafety committees across research laboratories

Analysis of the Aedes albopictus C6/36 genome provides insight into cell line utility for viral propagation

BACKGROUND: The 50-year-old Aedes albopictus C6/36 cell line is a resource for the detection, amplification, and analysis of mosquito-borne viruses including Zika, dengue, and chikungunya. The cell line is derived from an unknown number of larvae from an unspecified strain of Aedes albopictus mosquitoes. Toward improved utility of the cell line for research in virus transmission, we present an annotated assembly of the C6/36 genome. RESULTS: The C6/36 genome assembly has the largest contig N50 (3.3 Mbp) of any mosquito assembly, presents the sequences of both haplotypes for most of the diploid genome, reveals independent null mutations in both alleles of the Dicer locus, and indicates a male-specific genome. Gene annotation was computed with publicly available mosquito transcript sequences. Gene expression data from cell line RNA sequence identified enrichment of growth-related pathways and conspicuous deficiency in aquaporins and inward rectifier K+ channels. As a test of utility, RNA sequence data from Zika-infected cells were mapped to the C6/36 genome and transcriptome assemblies. Host subtraction reduced the data set by 89%, enabling faster characterization of nonhost reads. CONCLUSIONS: The C6/36 genome sequence and annotation should enable additional uses of the cell line to study arbovirus vector interactions and interventions aimed at restricting the spread of human disease

A draft genome sequence for the Ixodes scapularis cell line, ISE6 [version 1; referees: 2 approved]

Background: The tick cell line ISE6, derived from Ixodes scapularis, is commonly used for amplification and detection of arboviruses in environmental or clinical samples. Methods: To assist with sequence-based assays, we sequenced the ISE6 genome with single-molecule, long-read technology. Results: The draft assembly appears near complete based on gene content analysis, though it appears to lack some instances of repeats in this highly repetitive genome. The assembly appears to have separated the haplotypes at many loci. DNA short read pairs, used for validation only, mapped to the cell line assembly at a higher rate than they mapped to the Ixodes scapularis reference genome sequence. Conclusions: The assembly could be useful for filtering host genome sequence from sequence data obtained from cells infected with pathogens

The Ebola virus VP35 protein binds viral immunostimulatory and host RNAs identified through deep sequencing

<div><p>Ebola virus and Marburg virus are members of the <i>Filovirdae</i> family and causative agents of hemorrhagic fever with high fatality rates in humans. Filovirus virulence is partially attributed to the VP35 protein, a well-characterized inhibitor of the RIG-I-like receptor pathway that triggers the antiviral interferon (IFN) response. Prior work demonstrates the ability of VP35 to block potent RIG-I activators, such as Sendai virus (SeV), and this IFN-antagonist activity is directly correlated with its ability to bind RNA. Several structural studies demonstrate that VP35 binds short synthetic dsRNAs; yet, there are no data that identify viral immunostimulatory RNAs (isRNA) or host RNAs bound to VP35 in cells. Utilizing a SeV infection model, we demonstrate that both viral isRNA and host RNAs are bound to Ebola and Marburg VP35s in cells. By deep sequencing the purified VP35-bound RNA, we identified the SeV copy-back defective interfering (DI) RNA, previously identified as a robust RIG-I activator, as the isRNA bound by multiple filovirus VP35 proteins, including the VP35 protein from the West African outbreak strain (Makona EBOV). Moreover, RNAs isolated from a VP35 RNA-binding mutant were not immunostimulatory and did not include the SeV DI RNA. Strikingly, an analysis of host RNAs bound by wild-type, but not mutant, VP35 revealed that select host RNAs are preferentially bound by VP35 in cell culture. Taken together, these data support a model in which VP35 sequesters isRNA in virus-infected cells to avert RIG-I like receptor (RLR) activation.</p></div

Initial genome sequencing of the sugarcane CP 96-1252 complex hybrid [version 1; referees: 2 approved]

The CP 96-1252 cultivar of sugarcane is a complex hybrid of commercial importance. DNA was extracted from lab-grown leaf tissue and sequenced. The raw Illumina DNA sequencing results provide 101 Gbp of genome sequence reads. The dataset is available from https://www.ncbi.nlm.nih.gov/bioproject/PRJNA345486/

An analysis of host RNAs highlights transcripts bound by VP35.

<p>(A) Total number of sequencing reads from triplicate samples of wildtype and mutant VP35 proteins infected in the SeV infected groups. The pie charts depict the percentage of reads that map to the SeV DI genome and the percentage of reads that do not map to the SeV DI. (B) Multidimensional scaling (MDS) plot of triplicate samples from wild-type VP35 (black squares), wild-type VP35 infected with SeV (black circles), mutant VP35 (black triangle) and mutant VP35 infected with SeV (black diamond). Axes in the MDS plot (Leading logFC dim1 and Leading logFC dim2) are arbitrary, and the values on the axes are distance units. (C) Heat map showing the binding of the wild-type and mutant VP35 protein to human mRNA transcripts for all samples included in the analysis. Each row represents an experimental replicate, and each column represents a single transcript. Colors indicate relative abundance for each gene, where orange is low abundance and white is high abundance. (D) Sorting of the 62 most statistically significantly enriched mRNAs associated with VP35 (from the mock-infected wild-type VP35 samples). Y-axis denotes the p-value of each sample and X-axis denotes fold-change of transcript abundance between wild-type and mutant VP35.</p

VP35 proteins from Marburg virus and all five <i>Ebolavirus</i> species antagonize SeV induced promoter activity.

<p>The abilities of the VP35 proteins from Marburgvirus and the five species of Ebolavirus to antagonize the IFN response induced by virus infection were compared in the context of a luciferase reporter under the control of the IFN-β promoter. (A) 293T cells were transfected with increasing amounts (4 ng, 20 ng, or 100 ng) of pCAGGS-based plasmids expressing N-terminally FLAG-tagged filoviral proteins, an empty pCAGGS plasmid to transfect equal amounts between samples, and a plasmid expressing Renilla luciferase as a transfection control. The following day, cells were either mock-infected or infected with SeV to induce IFN-β promoter activity. The third day, cells were harvested and luciferase expression was measured. Error bars represent standard error of the mean of triplicates. MARV, Marburg Virus; EBOV, Ebola Virus (Mayinga); SUDV, Sudan Virus; BDBV, Bundibugyo Virus; RESTV, Reston Virus; TAFV, Taï Forest Virus. (B) Western blot analysis against the FLAG tag shows relative expression of filoviral proteins when 100 ng of each FLAG-tagged protein-expressing plasmid was transfected.</p

Mutations in VP35 important for dsRNA binding abrogate its ability to bind the immunostimulatory SeV DI.

<p>(A) Schematic of the EBOV VP35 protein containing a coiled-coil domain important for oligomerization in the N-terminal half of the protein and dsRNA-binding domain in the C-terminal half. Residues K309 and R312 were mutated to alanine to generate the EBOV VP35 RNA-binding mutant. (B) Protein staining of immunoprecipitated FLAG-tagged wild-type and mutant VP35 from which the RNA transfected in (C) and sequenced in (D) were recovered. (C) Immunostimulatory activity of RNA following immunoprecipitation of the pCAGGS empty vector (EV), wild-type EBOV VP35, and mutant EBOV VP35 in cells infected with SeV or mock-infected. (D) Next-generation sequencing and read mapping to the SeV genome. RNA associated with the pCAGGS empty vector, wild-type EBOV VP35, and mutant EBOV VP35 was purified and subjected to Illumina sequencing and the resulting reads were mapped to the SeV genome. The graph depicts nucleotide coverage (Y-axis) at each position of the SeV genome (X-axis).</p

Whole genome sequencing, variant analysis, phylogenetics, and deep sequencing of Zika virus strains

Abstract 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