Biological systems are protected by innate immune mechanisms initiated by host sensors called pattern recognition receptors (‘PRRs’) that recognize specific “foreign” features of invading pathogens to initiate multiple downstream anti-pathogen cascades. PRRs that detect nucleic acid structures characteristic of viral infection (such as single- or double-stranded RNA or DNA) are among the innate responders that protect diverse cell types from viral pathogenesis (for review, see 
). How the cell handles viral double-stranded RNA (dsRNA) is of special interest because dsRNA is a necessary intermediate in the replication of RNA viruses. In addition to dsRNA that forms during replication of the virus genome, RNA duplexes can form due to self-complementarity in the virus genome, and in some instances, from sense-antisense transcription of overlapping genes.
Four of the most studied families of PRRs for dsRNA are: (a) cytoplasmic RNA helicases like Retinoic acid-inducible gene I & Melanoma differentiation-associated gene-5 (“RIG-I” & “Mda-5,” which trigger mitochondrial-localized antiviral pathways); (b) Protein Kinase R (“PKR,” which induces a translational arrest state in cells after sensing dsRNA); (c) 2′–5′ oligoadenylate synthetase (“OAS,” which stimulates the ssRNase activity of RNase L in response to dsRNA); and (d) Toll-like receptors (“TLRs,” which bind various forms of RNA or DNA). All of these PRRs trigger the Interferon (IFN) responses, and activate IFN-stimulated genes (ISGs) that establish an antiviral state in the infected cell (for review, see 
). The IFN signaling pathway is central to the detection of, and response to, virus infections in cells. Type I IFNs (IFN-α and IFN-β) make up one of the first lines of defense in the innate immune response to viruses by inducing antiviral ISGs, modulating the levels of specific host-encoded miRNAs 
, and in a feedback loop, that of PKR and OAS. Many viruses are also susceptible to treatment with Type I IFNs, and conversely, cells that have higher basal activity of ISGs seem to mount a more successful antiviral response, and are not targeted by viruses 
Dicer is another PRR that recognizes dsRNA, chopping it into smaller duplexes called siRNAs that are 19–27 nucleotides (nt) long 
. These siRNAs have a terminal 5′ mono-phosphate and a terminal 3′ hydroxyl on both strands, generally have 2 nt 3′ overhangs, and are fed into an RNA-induced silencing complex “RISC” (for review on Dicer and Argonautes, see 
). siRNA duplexes are unwound, and only one strand remains associated with RISC (the mechanism of unwinding and choice of strand is poorly understood; for review, see 
). One of the key components of RISC is a protein called Argonaute-2 (Ago-2), which belongs to the Argonaute family of proteins. Ago-2 is the only member of the family that has cleavage activity, and is the designated ‘slicer’ protein in RISC that mediates cleavage of mRNA in a sequence-directed manner by a process termed RNA interference, or ‘RNAi’ 
There is strong evidence for an antiviral role for RNAi in plant and invertebrate systems (for review, see 
). Viruses replicate most effectively in these systems in the absence of key elements of the RNAi pathway: either in cells lacking components of the RNAi machinery, or in the presence of virus-encoded suppressors of the silencing pathway (for review, see 
). As expected, virus-derived siRNAs (vsRNAs) can be detected in some plant and invertebrate systems that are capable of mounting a successful/partially successful RNAi response 
. A population of vsRNAs would be an expected component of any viral defense pathway that acted through an RNAi mechanism.
In mammalian cells, short duplex RNAs can effectively enter the RNAi pathway and function in sequence-specific silencing, while duplexes longer than 30 nt generally produce a more complex response including the induction of multiple non-specific pathways including the IFN response (for review, see 
). Indeed, RNA and DNA viruses have evolved a host of defense mechanisms to counteract the nonspecific signaling effects of dsRNA. For example, Adenovirus VA RNA sequesters PKR 
, while proteins from Vaccinia virus (E3L), Porcine Rotaviruses (NSP3), and Influenza A virus (NS1) sequester dsRNA and prevent stimulation of the IFN response 
. Viral proteins can also inhibit signaling downstream of dsRNA binding, as in the case of the HCV protease NS3/4A, which cleaves IPS-1 (the RIG-I/MDA-5 signaling partner) to consequently disrupt induction of IFN responses 
. Several of these dsRNA-binding proteins may also facilitate viral evasion of host immune responses by inhibiting RNAi 
. Additionally, some viruses make their genomes inaccessible to PRRs of various types including IFN effectors and the siRNA-programmed RISC complex (e.g. 
Viruses may also perturb another class of effectors involved in RNAi called micro-RNAs (miRNAs), which are a class of cellular small RNAs generated by Dicer from hairpin structures. Cellular miRNA profiles are frequently modulated upon infection by viruses, and this may contribute in some cases to infectivity and pathogenesis 
. Conversely, some viruses usurp the host miRNA machinery for processing miRNA-like structures encoded in the viral genome, potentially using these molecules for regulation of virus/host gene expression 
With so much potential for RNA-mediated cross talk between the IFN response, the RNAi pathway, and the virus itself, it has been difficult to demonstrate a precise role for the RNAi pathway in vertebrate antiviral defense. The difficulties in segregating IFN and RNAi functions have given rise to speculations that the antiviral role of RNAi may have been lost during evolution, or alternatively, that RNAi-based defense may only be harnessed by triggers such as short hairpins and siRNAs that do not stimulate the IFN pathway. There has been some attempt at demonstrating recognition of viral RNA by the RNAi machinery. For instance, in Vero cells (which lack IFNα/β), inhibition of RNAi by Dicer knockdown increases replication of an RNA virus, the Influenza A Virus 
. Additionally, there are cases where short virus-derived RNAs can be detected in vertebrate systems (e.g. from HDV 
by high-throughput sequencing, and the HCV replicon 
, by bulk analysis methods). However, it is still not clear how general the presence of such RNAs is, and whether these RNAs can participate in host defense mechanisms. To complicate this issue, many of the classically-studied virus-host systems have been chosen based on the ability of the virus to rapidly replicate and kill host cells; these experimental infection systems may artificially under-represent the capacity of vertebrate cells to protect themselves, hence biasing against systems where RNAi might have a significant role in host-virus interactions.
Here, we sought a broader survey of potential RNA-derived defenses in viral infection systems. Given no knowledge of which virus type might engage the RNAi machinery, and which cell types might efficiently use this machinery in defense, we cast a wide net in terms of both virus families and host cells. In this study, we describe small RNA populations from six different RNA viral pathogens, each in a variety of animal cell infection systems (including both immune-competent and immune-compromised hosts). Upon examining small RNA populations from ~150 samples with sample-specific DNA barcodes, we found viral-derived small RNAs (vsRNAs) from each virus, with vsRNA populations sensitive to both viral and host characteristics. A more detailed analysis of vsRNAs in two viral infection models (Hepatitis C Virus and Poliovirus) in various host types revealed that multiple distinct pools of vsRNAs may co-exist during infection: as single strands, as part of duplexes, and in complexes that may contain Argonautes. We also observed specific changes in cell-derived miRNA populations, providing a clear indication of host perturbation by the virus. The characterization of small RNA populations during RNA virus infections provides both an experimental entry point, and an indication of the complexity that will need to be addressed in understanding roles for small RNAs in host and viral processes.