Insect viruses have evolved strategies to control the host RNAi antiviral defense mechanism. In nature Drosophila C Virus (DCV) infection causes low mortality and persistent infection, whereas the closely related Cricket Paralysis Virus (CrPV) causes a lethal infection. We show these viruses use different strategies to modulate the host RNAi defense machinery. The DCV RNAi suppressor (DCV-1A) binds to long double-stranded RNA (dsRNA) and prevents processing by Dicer2. In contrast, the CrPV suppressor (CrPV-1A) interacts with the endonuclease Ago2 and inhibits its activity, without affecting the miRNA-Ago1 mediated silencing. The link between viral RNAi suppressors and the outcome of infection was examined using recombinant Sindbis viruses encoding either CrPV-1A or DCV-1A. Flies infected with Sindbis virus expressing CrPV-1A showed a dramatic 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.

SUMMARY 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 RNA (siRNAs) and PIWI-interacting RNAs (piRNAs) [1, 2]. Endogenous viral elements (EVEs) are sequences from non-retroviral RNA viruses that are inserted into the mosquito genome and 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 LTR retrotransposons and organize into large loci (>50kbp) 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.

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, confers passive protection against virus challenge in naïve 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.

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 non-neuronal tissues. As a result, these engineered miRNA-regulated viruses elicited strong protective immunity in mice without producing disease.

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.

18The Aedes aegypti mosquito is a major vector for arboviruses including 19 dengue, chikungunya and Zika virus. Combating the spread of these viruses 20 requires a more complete understanding of the mosquito immune system. 21 Recent studies have implicated genomic endogenous viral elements (EVEs) 22 derived from non-retroviral RNA viruses in insect immunity. Because these 23 elements are inserted into repetitive regions of the mosquito genome, their large-24 scale structure and organization with respect to other genomic elements has 25 been difficult to resolve with short-read sequencing. To better define the origin, 26 diversity and biological role of EVEs, we employed single-molecule, real-time 27 sequencing technology to generate a high quality, long-read assembly of the Ae. 28 aegypti-derived Aag2 cell line genome. We leverage the quality and contiguity of 29 this assembly to characterize the diversity and genomic context of EVEs in the 30 genome of this important model system. We find that EVEs in the Aag2 genome 31 are acquired through recombination by LTR retrotransposons, and organize into 32 larger loci (>50kbp) characterized by high LTR density. These EVE containing 33 loci are associated with increased transcription factor binding sight density and 34 increased production of anti-genomic piRNAs. We also detected piRNA 35 processing corresponding to on-going viral infection. This global view of EVEs 36 and piRNA responses demonstrates the ubiquity and diversity of these heritable 37

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.

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.