Circulating Immune Cells Mediate a Systemic RNAi-Based Adaptive Antiviral Response in Drosophila.

Effective antiviral protection in multicellular organisms relies on both cell-autonomous and systemic immunity. Systemic immunity mediates the spread of antiviral signals from infection sites to distant uninfected tissues. In arthropods, RNA interference (RNAi) is responsible for antiviral defense. Here, we show that flies have a sophisticated systemic RNAi-based immunity mediated by macrophage-like haemocytes. Haemocytes take up dsRNA from infected cells and, through endogenous transposon reverse transcriptases, produce virus-derived complementary DNAs (vDNA). These vDNAs template de novo synthesis of secondary viral siRNAs (vsRNA), which are secreted in exosome-like vesicles. Strikingly, exosomes containing vsRNAs, purified from haemolymph of infected flies, confer passive protection against virus challenge in naive animals. Thus, similar to vertebrates, insects use immune cells to generate immunological memory in the form of stable vDNAs that generate systemic immunity, which is mediated by the vsRNA-containing exosomes.

Insular Microbiogeography: Three Pathogens as Exemplars.

Traditional taxonomy in biology assumes that life is organized in a simple tree. Attempts to classify microorganisms in this way in the genomics era led microbiologists to look for finite sets of 'core' genes that uniquely group taxa as clades in the tree. However, the diversity revealed by large-scale whole genome sequencing is calling into question the long-held model of a hierarchical tree of life, which leads to questioning of the definition of a species. Large-scale studies of microbial genome diversity reveal that the cumulative number of new genes discovered increases with the number of genomes studied as a power law and subsequently leads to the lack of evidence for a unique core genome within closely related organisms. Sampling 'enough' new genomes leads to the discovery of a replacement or alternative to any gene. This power law behaviour points to an underlying self-organizing critical process that may be guided by mutation and niche selection. Microbes in any particular niche exist within a local web of organism interdependence known as the microbiome. The same mechanism that underpins the macro-ecological scaling first observed by MacArthur and Wilson also applies to microbial communities. Recent metagenomic studies of a food microbiome demonstrate the diverse distribution of community members, but also genotypes for a single species within a more complex community. Collectively, these results suggest that traditional taxonomic classification of bacteria could be replaced with a quasispecies model. This model is commonly accepted in virology and better describes the diversity and dynamic exchange of genes that also hold true for bacteria. This model will enable microbiologists to conduct population-scale studies to describe microbial behaviour, as opposed to a single isolate as a representative.

RNA interference-mediated intrinsic antiviral immunity in invertebrates.

In invertebrates such as insects and nematodes, RNA interference (RNAi) provides RNA-based protection against viruses. This form of immunity restricts viral replication and dissemination from infected cells and viruses, in turn, have evolved evasion mechanisms or RNAi suppressors to counteract host defenses. Recent advances indicate that, in addition to RNAi, other related small RNA pathways contribute to antiviral functions in invertebrates. This has led to a deeper understanding of fundamental aspects of small RNA-based antiviral immunity in invertebrates and its contribution to viral spread and pathogenesis.

Predicting Epitope Candidates for SARS-CoV-2.

Epitopes are short amino acid sequences that define the antigen signature to which an antibody or T cell receptor binds. In light of the current pandemic, epitope analysis and prediction are paramount to improving serological testing and developing vaccines. In this paper, known epitope sequences from SARS-CoV, SARS-CoV-2, and other Coronaviridae were leveraged to identify additional antigen regions in 62K SARS-CoV-2 genomes. Additionally, we present epitope distribution across SARS-CoV-2 genomes, locate the most commonly found epitopes, and discuss where epitopes are located on proteins and how epitopes can be grouped into classes. The mutation density of different protein regions is presented using a big data approach. It was observed that there are 112 B cell and 279 T cell conserved epitopes between SARS-CoV-2 and SARS-CoV, with more diverse sequences found in Nucleoprotein and Spike glycoprotein.

Functional Genomics Platform, A Cloud-Based Platform for Studying Microbial Life at Scale.

The rapid growth in biological sequence data is revolutionizing our understanding of genotypic diversity and challenging conventional approaches to informatics. With the increasing availability of genomic data, traditional bioinformatic tools require substantial computational time and the creation of ever-larger indices each time a researcher seeks to gain insight from the data. To address these challenges, we pre-computed important relationships between biological entities spanning the Central Dogma of Molecular Biology and captured this information in a relational database. The database can be queried across hundreds of millions of entities and returns results in a fraction of the time required by traditional methods. In this paper, we describe Functional Genomics Platform (formerly known as OMXWare), a comprehensive database relating genotype to phenotype for bacterial life. Continually updated, the Functional Genomics Platform today contains data derived from 200,000 curated, self-consistently assembled genomes. The database stores functional data for over 68 million genes, 52 million proteins, and 239 million domains with associated biological activity annotations from Gene Ontology, KEGG, MetaCyc, and Reactome. The Functional Genomics Platform maps all of the many-to-many connections between each biological entity including the originating genome, gene, protein, and protein domain. Various microbial studies, from infectious disease to environmental health, can benefit from the rich data and connections. We describe the data selection, the pipeline to create and update the Functional Genomics Platform, and the developer tools (Python SDK and REST APIs)which allow researchers to efficiently study microbial life at scale.

Analysis and forecasting of global real time RT-PCR primers and probes for SARS-CoV-2.

Rapid tests for active SARS-CoV-2 infections rely on reverse transcription polymerase chain reaction (RT-PCR). RT-PCR uses reverse transcription of RNA into complementary DNA (cDNA) and amplification of specific DNA (primer and probe) targets using polymerase chain reaction (PCR). The technology makes rapid and specific identification of the virus possible based on sequence homology of nucleic acid sequence and is much faster than tissue culture or animal cell models. However the technique can lose sensitivity over time as the virus evolves and the target sequences diverge from the selective primer sequences. Different primer sequences have been adopted in different geographic regions. As we rely on these existing RT-PCR primers to track and manage the spread of the Coronavirus, it is imperative to understand how SARS-CoV-2 mutations, over time and geographically, diverge from existing primers used today. In this study, we analyze the performance of the SARS-CoV-2 primers in use today by measuring the number of mismatches between primer sequence and genome targets over time and spatially. We find that there is a growing number of mismatches, an increase by 2% per month, as well as a high specificity of virus based on geographic location.

Semi-Supervised Pipeline for Autonomous Annotation of SARS-CoV-2 Genomes.

SARS-CoV-2 genomic sequencing efforts have scaled dramatically to address the current global pandemic and aid public health. However, autonomous genome annotation of SARS-CoV-2 genes, proteins, and domains is not readily accomplished by existing methods and results in missing or incorrect sequences. To overcome this limitation, we developed a novel semi-supervised pipeline for automated gene, protein, and functional domain annotation of SARS-CoV-2 genomes that differentiates itself by not relying on the use of a single reference genome and by overcoming atypical genomic traits that challenge traditional bioinformatic methods. We analyzed an initial corpus of 66,000 SARS-CoV-2 genome sequences collected from labs across the world using our method and identified the comprehensive set of known proteins with 98.5% set membership accuracy and 99.1% accuracy in length prediction, compared to proteome references, including Replicase polyprotein 1ab (with its transcriptional slippage site). Compared to other published tools, such as Prokka (base) and VAPiD, we yielded a 6.4- and 1.8-fold increase in protein annotations. Our method generated 13,000,000 gene, protein, and domain sequences-some conserved across time and geography and others representing emerging variants. We observed 3362 non-redundant sequences per protein on average within this corpus and described key D614G and N501Y variants spatiotemporally in the initial genome corpus. For spike glycoprotein domains, we achieved greater than 97.9% sequence identity to references and characterized receptor binding domain variants. We further demonstrated the robustness and extensibility of our method on an additional 4000 variant diverse genomes containing all named variants of concern and interest as of August 2021. In this cohort, we successfully identified all keystone spike glycoprotein mutations in our predicted protein sequences with greater than 99% accuracy as well as demonstrating high accuracy of the protein and domain annotations. This work comprehensively presents the molecular targets to refine biomedical interventions for SARS-CoV-2 with a scalable, high-accuracy method to analyze newly sequenced infections as they arise.

Monitoring the microbiome for food safety and quality using deep shotgun sequencing.

In this work, we hypothesized that shifts in the food microbiome can be used as an indicator of unexpected contaminants or environmental changes. To test this hypothesis, we sequenced the total RNA of 31 high protein powder (HPP) samples of poultry meal pet food ingredients. We developed a microbiome analysis pipeline employing a key eukaryotic matrix filtering step that improved microbe detection specificity to >99.96% during in silico validation. The pipeline identified 119 microbial genera per HPP sample on average with 65 genera present in all samples. The most abundant of these were Bacteroides, Clostridium, Lactococcus, Aeromonas, and Citrobacter. We also observed shifts in the microbial community corresponding to ingredient composition differences. When comparing culture-based results for Salmonella with total RNA sequencing, we found that Salmonella growth did not correlate with multiple sequence analyses. We conclude that microbiome sequencing is useful to characterize complex food microbial communities, while additional work is required for predicting specific species' viability from total RNA sequencing.

Control of RNA viruses in mosquito cells through the acquisition of vDNA and endogenous viral elements.

Aedes aegypti transmit pathogenic arboviruses while the mosquito itself tolerates the infection. We examine a piRNA-based immunity that relies on the acquisition of viral derived cDNA (vDNA) and how this pathway discriminates between self and non-self. The piRNAs derived from these vDNAs are essential for virus control and Piwi4 has a central role in the pathway. Piwi4 binds preferentially to virus-derived piRNAs but not to transposon-targeting piRNAs. Analysis of episomal vDNA from infected cells reveals that vDNA molecules are acquired through a discriminatory process of reverse-transcription and recombination directed by endogenous retrotransposons. Using a high-resolution Ae. aegypti genomic sequence, we found that vDNAs integrated in the host genome as endogenous viral elements (EVEs), produce antisense piRNAs that are preferentially loaded onto Piwi4. Importantly, EVE-derived piRNAs are specifically loaded onto Piwi4 to inhibit virus replication. Thus, Ae. aegypti employs a sophisticated antiviral mechanism that promotes viral persistence and generates long-lasting adaptive immunity.

Food authentication from shotgun sequencing reads with an application on high protein powders.

Here we propose that using shotgun sequencing to examine food leads to accurate authentication of ingredients and detection of contaminants. To demonstrate this, we developed a bioinformatic pipeline, FASER (Food Authentication from SEquencing Reads), designed to resolve the relative composition of mixtures of eukaryotic species using RNA or DNA sequencing. Our comprehensive database includes >6000 plants and animals that may be present in food. FASER accurately identified eukaryotic species with 0.4% median absolute difference between observed and expected proportions on sequence data from various sources including sausage meat, plants, and fish. FASER was applied to 31 high protein powder raw factory ingredient total RNA samples. The samples mostly contained the expected source ingredient, chicken, while three samples unexpectedly contained pork and beef. Our results demonstrate that DNA/RNA sequencing of food ingredients, combined with a robust analysis, can be used to find contaminants and authenticate food ingredients in a single assay.

The Diversity, Structure, and Function of Heritable Adaptive Immunity Sequences in the Aedes aegypti Genome.

The Aedes aegypti mosquito transmits arboviruses, including dengue, chikungunya, and Zika virus. Understanding the mechanisms underlying mosquito immunity could provide new tools to control arbovirus spread. Insects exploit two different RNAi pathways to combat viral and transposon infection: short interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs) [1, 2]. Endogenous viral elements (EVEs) are sequences from non-retroviral viruses that are inserted into the mosquito genome and can act as templates for the production of piRNAs [3, 4]. EVEs therefore represent a record of past infections and a reservoir of potential immune memory [5]. The large-scale organization of EVEs has been difficult to resolve with short-read sequencing because they tend to integrate into repetitive regions of the genome. To define the diversity, organization, and function of EVEs, we took advantage of the contiguity associated with long-read sequencing to generate a high-quality assembly of the Ae. aegypti-derived Aag2 cell line genome, an important and widely used model system. We show EVEs are acquired through recombination with specific classes of long terminal repeat (LTR) retrotransposons and organize into large loci (>50 kbp) characterized by high LTR density. These EVE-containing loci have increased density of piRNAs compared to similar regions without EVEs. Furthermore, we detected EVE-derived piRNAs consistent with a targeted processing of persistently infecting virus genomes. We propose that comparisons of EVEs across mosquito populations may explain differences in vector competence, and further study of the structure and function of these elements in the genome of mosquitoes may lead to epidemiological interventions.

Cricket paralysis virus antagonizes Argonaute 2 to modulate antiviral defense in Drosophila.

Insect viruses have evolved strategies to control the host RNAi antiviral defense mechanism. In nature, Drosophila melanogaster C virus (DCV) infection causes low mortality and persistent infection, whereas the closely related cricket paralysis virus (CrPV) causes a lethal infection. We show that these viruses use different strategies to modulate the host RNAi defense machinery. The DCV RNAi suppressor (DCV-1A) binds to long double-stranded RNA and prevents processing by Dicer2. In contrast, the CrPV suppressor (CrPV-1A) interacts with the endonuclease Argonaute 2 (Ago2) and inhibits its activity without affecting the microRNA (miRNA)-Ago1-mediated silencing. We examined the link between viral RNAi suppressors and the outcome of infection using recombinant Sindbis viruses encoding either CrPV-1A or DCV-1A. Flies infected with Sindbis virus expressing CrPV-1A showed a marked increase in virus production, spread and mortality. In contrast, Sindbis pathogenesis was only modestly increased by expression of DCV- 1A. We conclude that RNAi suppressors function as virulence factors in insects and can target the Drosophila RNAi pathway at different points.

Harnessing endogenous miRNAs to control virus tissue tropism as a strategy for developing attenuated virus vaccines.

Live attenuated vaccines remain the safest, most cost-effective intervention against viral infections. Because live vaccine strains are generated empirically and the basis for attenuation is usually ill defined, many important viruses lack an efficient live vaccine. Here, we present a general strategy for the rational design of safe and effective live vaccines that harnesses the microRNA-based gene-silencing machinery to control viral replication. Using poliovirus as a model, we demonstrate that insertion of small miRNA homology sequences into a viral genome can restrict its tissue tropism, thereby preventing pathogenicity and yielding an attenuated viral strain. Poliovirus strains engineered to become targets of neuronal-specific miRNAs lost their ability to replicate in the central nervous system, leading to significant attenuation of neurovirulence in infected animals. Importantly, these viruses retained the ability to replicate in nonneuronal tissues. As a result, these engineered miRNA-regulated viruses elicited strong protective immunity in mice without producing disease.