Tinker, tailor, antiviral: RNA virus inhibition by induced recombination.

Nucleotide analogs can help to combat RNA virus growth by stalling the viral RNA polymerase or by introducing lethal mutations into the viral genome. Janissen and Woodman et al. have used single-molecule, sequencing, and virological methods to reveal that antiviral T-1106 provides a third mechanism of counterattack: inducing recombination.

Evolution of highly pathogenic H5N1 influenza A virus in the central nervous system of ferrets.

Central nervous system (CNS) disease is the most common extra-respiratory tract complication of influenza A virus infections in humans. Remarkably, zoonotic highly pathogenic avian influenza (HPAI) H5N1 virus infections are more often associated with CNS disease than infections with seasonal influenza viruses. Evolution of avian influenza viruses has been extensively studied in the context of respiratory infections, but evolutionary processes in CNS infections remain poorly understood. We have previously observed that the ability of HPAI A/Indonesia/5/2005 (H5N1) virus to replicate in and spread throughout the CNS varies widely between individual ferrets. Based on these observations, we sought to understand the impact of entrance into and replication within the CNS on the evolutionary dynamics of virus populations. First, we identified and characterized three substitutions-PB1 E177G and A652T and NP I119M - detected in the CNS of a ferret infected with influenza A/Indonesia/5/2005 (H5N1) virus that developed a severe meningo-encephalitis. We found that some of these substitutions, individually or collectively, resulted in increased polymerase activity in vitro. Nevertheless, in vivo, the virus bearing the CNS-associated mutations retained its capacity to infect the CNS but showed reduced dispersion to other anatomical sites. Analyses of viral diversity in the nasal turbinate and olfactory bulb revealed the lack of a genetic bottleneck acting on virus populations accessing the CNS via this route. Furthermore, virus populations bearing the CNS-associated mutations showed signs of positive selection in the brainstem. These features of dispersion to the CNS are consistent with the action of selective processes, underlining the potential for H5N1 viruses to adapt to the CNS.

Deep mutational scanning reveals the functional constraints and evolutionary potential of the influenza A virus PB1 protein.

IMPORTANCE: The influenza virus polymerase is important for adaptation to new hosts and, as a determinant of mutation rate, for the process of adaptation itself. We performed a deep mutational scan of the polymerase basic 1 (PB1) protein to gain insights into the structural and functional constraints on the influenza RNA-dependent RNA polymerase. We find that PB1 is highly constrained at specific sites that are only moderately predicted by the global structure or larger domain. We identified a number of beneficial mutations, many of which have been shown to be functionally important or observed in influenza virus' natural evolution. Overall, our atlas of PB1 mutations and their fitness impacts serves as an important resource for future studies of influenza replication and evolution.

The Host Factor ANP32A Is Required for Influenza A Virus vRNA and cRNA Synthesis.

Influenza A viruses are negative-sense RNA viruses that rely on their own viral replication machinery to replicate and transcribe their segmented single-stranded RNA genome. The viral ribonucleoprotein complexes in which viral RNA is replicated consist of a nucleoprotein scaffold around which the RNA genome is wound, and a heterotrimeric RNA-dependent RNA polymerase that catalyzes viral replication. The RNA polymerase copies the viral RNA (vRNA) via a replicative intermediate, called the cRNA, and subsequently uses this cRNA to make more vRNA copies. To ensure that new cRNA and vRNA molecules are associated with ribonucleoproteins in which they can be amplified, the active RNA polymerase recruits a second polymerase to encapsidate the cRNA or vRNA. Host factor ANP32A has been shown to be essential for viral replication and to facilitate the formation of a dimer between viral RNA polymerases. Differences between mammalian and avian ANP32A proteins are sufficient to restrict viral replication. It has been proposed that ANP32A is only required for the synthesis of vRNA molecules from cRNA but not vice versa. However, this view does not match recent molecular evidence. Here we use minigenome assays, virus infections, and viral promoter mutations to demonstrate that ANP32A is essential for both vRNA and cRNA synthesis. Moreover, we show that ANP32A is not only needed for the actively replicating polymerase, but not for the polymerase that is encapsidating nascent viral RNA products. Overall, these results provide new insights into influenza A virus replication and host adaptation. IMPORTANCE Zoonotic avian influenza A viruses pose a constant threat to global health, and they have the potential to cause pandemics. Species variations in host factor ANP32A play a key role in supporting the activity of avian influenza A virus RNA polymerases in mammalian hosts. Here we show that ANP32A acts at two stages in the influenza A virus replication cycle, supporting recent structural experiments, in line with its essential role. Understanding how ANP32A supports viral RNA polymerase activity and how it supports avian polymerase function in mammalian hosts is important for understanding influenza A virus replication and the development of antiviral strategies against influenza A viruses.

The influenza virus RNA polymerase as an innate immune agonist and antagonist.

Influenza A viruses cause a mild-to-severe respiratory disease that affects millions of people each year. One of the many determinants of disease outcome is the innate immune response to the viral infection. While antiviral responses are essential for viral clearance, excessive innate immune activation promotes lung damage and disease. The influenza A virus RNA polymerase is one of viral proteins that affect innate immune activation during infection, but the mechanisms behind this activity are not well understood. In this review, we discuss how the viral RNA polymerase can both activate and suppress innate immune responses by either producing immunostimulatory RNA species or directly targeting the components of the innate immune signalling pathway, respectively. Furthermore, we provide a comprehensive overview of the polymerase residues, and their mutations, associated with changes in innate immune activation, and discuss their putative effects on polymerase function based on recent advances in our understanding of the influenza A virus RNA polymerase structure.

Enisamium Reduces Influenza Virus Shedding and Improves Patient Recovery by Inhibiting Viral RNA Polymerase Activity.

Infections with respiratory viruses constitute a huge burden on our health and economy. Antivirals against some respiratory viruses are available, but further options are urgently needed. Enisamium iodide (laboratory code FAV00A, trade name Amizon) is an antiviral, marketed in countries of the Commonwealth of Independent States for the treatment of viral respiratory infections, but its clinical efficacy and mode of action are not well understood. In this study, we investigated the efficacy of enisamium in patients aged between 18 and 60 years with confirmed influenza virus and other viral respiratory infections. Enisamium treatment resulted in reduced influenza virus shedding (at day 3, 71.2% in the enisamium group tested negative versus 25.0% in placebo group [P < 0.0001]), faster patient recovery (at day 14, 93.9% in the enisamium group had recovered versus 32.5% in placebo group [P < 0.0001]), and reduced disease symptoms (from 9.6 ± 0.7 to 4.6 ± 0.9 score points in enisamium group versus 9.7 ± 1.1 to 5.6 ± 1.1 score points in placebo group [P < 0.0001]) compared to those in the placebo group. Using mass spectrometry, and cell-based and cell-free viral RNA synthesis assays, we identified a hydroxylated metabolite of enisamium, VR17-04. VR17-04 is capable of inhibiting influenza virus RNA synthesis and is present in plasma of patients treated with enisamium. VR17-04 inhibits the activity of the influenza virus RNA polymerase more potently than its parent compound. Overall, these results suggest that enisamium is metabolized in humans to an inhibitor of the influenza virus RNA polymerase that reduces viral shedding and improves patient recovery in influenza patients. (This study has been registered at ClinicalTrials.gov under identifier NCT04682444.).

Real-time analysis of single influenza virus replication complexes reveals large promoter-dependent differences in initiation dynamics.

The viral RNA (vRNA) genome of influenza viruses is replicated by the RNA-dependent RNA polymerase (RNAP) via a complementary RNA (cRNA) intermediate. The vRNA promoter can adopt multiple conformations when bound by the RNAP. However, the dynamics, determinants, and biological role of these conformations are unknown; further, little is known about cRNA promoter conformations. To probe the RNA conformations adopted during initial replication, we monitored single, surface-immobilized vRNA and cRNA initiation complexes in real-time. Our results show that, while the 3' terminus of the vRNA promoter exists in dynamic equilibrium between pre-initiation and initiation conformations, the cRNA promoter exhibited very limited dynamics. Two residues in the proximal 3' region of the cRNA promoter (residues absent in the vRNA promoter) allowed the cRNA template strand to reach further into the active site, limiting promoter dynamics. Our results highlight promoter-dependent differences in influenza initiation mechanisms, and advance our understanding of virus replication.

The mechanism of resistance to favipiravir in influenza.

Favipiravir is a broad-spectrum antiviral that has shown promise in treatment of influenza virus infections. While emergence of resistance has been observed for many antiinfluenza drugs, to date, clinical trials and laboratory studies of favipiravir have not yielded resistant viruses. Here we show evolution of resistance to favipiravir in the pandemic H1N1 influenza A virus in a laboratory setting. We found that two mutations were required for robust resistance to favipiravir. We demonstrate that a K229R mutation in motif F of the PB1 subunit of the influenza virus RNA-dependent RNA polymerase (RdRP) confers resistance to favipiravir in vitro and in cell culture. This mutation has a cost to viral fitness, but fitness can be restored by a P653L mutation in the PA subunit of the polymerase. K229R also conferred favipiravir resistance to RNA polymerases of other influenza A virus strains, and its location within a highly conserved structural feature of the RdRP suggests that other RNA viruses might also acquire resistance through mutations in motif F. The mutations identified here could be used to screen influenza virus-infected patients treated with favipiravir for the emergence of resistance.

Single-Cell Virus Sequencing of Influenza Infections That Trigger Innate Immunity.

Influenza virus-infected cells vary widely in their expression of viral genes and only occasionally activate innate immunity. Here, we develop a new method to assess how the genetic variation in viral populations contributes to this heterogeneity. We do this by determining the transcriptome and full-length sequences of all viral genes in single cells infected with a nominally "pure" stock of influenza virus. Most cells are infected by virions with defects, some of which increase the frequency of innate-immune activation. These immunostimulatory defects are diverse and include mutations that perturb the function of the viral polymerase protein PB1, large internal deletions in viral genes, and failure to express the virus's interferon antagonist NS1. However, immune activation remains stochastic in cells infected by virions with these defects and occasionally is triggered even by virions that express unmutated copies of all genes. Our work shows that the diverse spectrum of defects in influenza virus populations contributes to-but does not completely explain-the heterogeneity in viral gene expression and immune activation in single infected cells.IMPORTANCE Because influenza virus has a high mutation rate, many cells are infected by mutated virions. But so far, it has been impossible to fully characterize the sequence of the virion infecting any given cell, since conventional techniques such as flow cytometry and single-cell transcriptome sequencing (scRNA-seq) only detect if a protein or transcript is present, not its sequence. Here we develop a new approach that uses long-read PacBio sequencing to determine the sequences of virions infecting single cells. We show that viral genetic variation explains some but not all of the cell-to-cell variability in viral gene expression and innate immune induction. Overall, our study provides the first complete picture of how viral mutations affect the course of infection in single cells.

A Mechanism for Priming and Realignment during Influenza A Virus Replication.

The influenza A virus genome consists of eight segments of single-stranded RNA. These segments are replicated and transcribed by a viral RNA-dependent RNA polymerase (RdRp) that is made up of the influenza virus proteins PB1, PB2, and PA. To copy the viral RNA (vRNA) genome segments and the cRNA segments, the replicative intermediate of viral replication, the RdRp must use two promoters and two different de novo initiation mechanisms. On the vRNA promoter, the RdRp initiates on the 3' terminus, while on the cRNA promoter, the RdRp initiates internally and subsequently realigns the nascent vRNA product to ensure that the template is copied in full. In particular, the latter process, which is also used by other RNA viruses, is not understood. Here we provide mechanistic insight into priming and realignment during influenza virus replication and show that it is controlled by the priming loop and a helix-loop-helix motif of the PB1 subunit of the RdRp. Overall, these observations advance our understanding of how the influenza A virus initiates viral replication and amplifies the genome correctly.IMPORTANCE Influenza A viruses cause severe disease in humans and are considered a major threat to our economy and health. The viruses replicate and transcribe their genome by using an enzyme called the RNA polymerases. To ensure that the genome is amplified faithfully and that abundant viral mRNAs are made for viral protein synthesis, the RNA polymerase must work correctly. In this report, we provide insight into the mechanism that the RNA polymerase employs to ensure that the viral genome is copied correctly.

Initiation, Elongation, and Realignment during Influenza Virus mRNA Synthesis.

The RNA-dependent RNA polymerase (RdRp) of the influenza A virus replicates and transcribes the viral genome segments in the nucleus of the host cell. To transcribe these viral genome segments, the RdRp "snatches" capped RNA oligonucleotides from nascent host cell mRNAs and aligns these primers to the ultimate or penultimate nucleotide of the segments for the initiation of viral mRNA synthesis. It has been proposed that this initiation process is not processive and that the RdRp uses a prime-realign mechanism during transcription. Here we provide in vitro evidence for the existence of this transcriptional prime-realign mechanism but show that it functions efficiently only for primers that are short or cannot stably base pair with the template. In addition, we demonstrate that transcriptional elongation is dependent on the priming loop of the PB1 subunit of the RdRp. We propose that the prime-realign mechanism may be used to rescue abortive transcription initiation events or cope with sequence variation among primers. Overall, these observations advance our mechanistic understanding of how influenza A virus initiates transcription correctly and efficiently.IMPORTANCE Influenza A virus causes severe disease in humans and is considered a major global health threat. The virus replicates and transcribes its genome by using an enzyme called the RNA polymerase. To ensure that the genome is amplified faithfully and abundant viral mRNAs are made for viral protein synthesis, the viral RNA polymerase must transcribe the viral genome efficiently. In this report, we characterize a structure inside the polymerase that contributes to the efficiency of viral mRNA synthesis.

Role of the PB2 627 Domain in Influenza A Virus Polymerase Function.

The RNA genome of influenza A viruses is transcribed and replicated by the viral RNA-dependent RNA polymerase, composed of the subunits PA, PB1, and PB2. High-resolution structural data revealed that the polymerase assembles into a central polymerase core and several auxiliary highly flexible, protruding domains. The auxiliary PB2 cap-binding and the PA endonuclease domains are both involved in cap snatching, but the role of the auxiliary PB2 627 domain, implicated in host range restriction of influenza A viruses, is still poorly understood. In this study, we used structure-guided truncations of the PB2 subunit to show that a PB2 subunit lacking the 627 domain accumulates in the cell nucleus and assembles into a heterotrimeric polymerase with PB1 and PA. Furthermore, we showed that a recombinant viral polymerase lacking the PB2 627 domain is able to carry out cap snatching, cap-dependent transcription initiation, and cap-independent ApG dinucleotide extension in vitro, indicating that the PB2 627 domain of the influenza virus RNA polymerase is not involved in core catalytic functions of the polymerase. However, in a cellular context, the 627 domain is essential for both transcription and replication. In particular, we showed that the PB2 627 domain is essential for the accumulation of the cRNA replicative intermediate in infected cells. Together, these results further our understanding of the role of the PB2 627 domain in transcription and replication of the influenza virus RNA genome.IMPORTANCE Influenza A viruses are a major global health threat, not only causing disease in both humans and birds but also placing significant strains on economies worldwide. Avian influenza A virus polymerases typically do not function efficiently in mammalian hosts and require adaptive mutations to restore polymerase activity. These adaptations include mutations in the 627 domain of the PB2 subunit of the viral polymerase, but it still remains to be established how these mutations enable host adaptation on a molecular level. In this report, we characterize the role of the 627 domain in polymerase function and offer insights into the replication mechanism of influenza A viruses.

Single-molecule FRET reveals the pre-initiation and initiation conformations of influenza virus promoter RNA.

Influenza viruses have a segmented viral RNA (vRNA) genome, which is replicated by the viral RNA-dependent RNA polymerase (RNAP). Replication initiates on the vRNA 3' terminus, producing a complementary RNA (cRNA) intermediate, which serves as a template for the synthesis of new vRNA. RNAP structures show the 3' terminus of the vRNA template in a pre-initiation state, bound on the surface of the RNAP rather than in the active site; no information is available on 3' cRNA binding. Here, we have used single-molecule Förster resonance energy transfer (smFRET) to probe the viral RNA conformations that occur during RNAP binding and initial replication. We show that even in the absence of nucleotides, the RNAP-bound 3' termini of both vRNA and cRNA exist in two conformations, corresponding to the pre-initiation state and an initiation conformation in which the 3' terminus of the viral RNA is in the RNAP active site. Nucleotide addition stabilises the 3' vRNA in the active site and results in unwinding of the duplexed region of the promoter. Our data provide insights into the dynamic motions of RNA that occur during initial influenza replication and has implications for our understanding of the replication mechanisms of similar pathogenic viruses.

Host restriction of influenza virus polymerase activity by PB2 627E is diminished on short viral templates in a nucleoprotein-independent manner.

Most avian influenza viruses do not replicate efficiently in human cells. This is partly due to the low activity of the RNA polymerase of avian influenza viruses in mammalian cells. Nevertheless, this impediment can be overcome through an E→K adaptive mutation at residue 627 of the PB2 subunit of the polymerase. Accordingly, viral ribonucleoprotein (RNP) reconstitution assays show that a viral polymerase containing PB2 627E has impaired activity in mammalian cells compared to a viral polymerase that contains PB2 627K, characteristic of mammalian-adapted influenza viruses. In contrast, purified viral polymerases containing either PB2 627E or PB2 627K show comparable levels of activity in transcription assays that require no RNP assembly. We sought to reconcile these conflicting observations by using an NP-independent cell-based transcription/replication assay to assess viral polymerase activity. We found that PB2 627E polymerase restriction in mammalian cells is independent of NP expression but is dependent on the length of the viral RNA template. In addition, restriction of PB2 627E polymerase was overcome by mutations specific to the viral RNA template promoter sequence. Consequently, we propose that PB2 627E affects recruitment of the viral RNA promoter by the viral polymerase in mammalian cells.

Common and unique features of viral RNA-dependent polymerases.

Eukaryotes and bacteria can be infected with a wide variety of RNA viruses. On average, these pathogens share little sequence similarity and use different replication and transcription strategies. Nevertheless, the members of nearly all RNA virus families depend on the activity of a virally encoded RNA-dependent polymerase for the condensation of nucleotide triphosphates. This review provides an overview of our current understanding of the viral RNA-dependent polymerase structure and the biochemistry and biophysics that is involved in replicating and transcribing the genetic material of RNA viruses.

Uncoupling of influenza A virus transcription and replication through mutation of the unpaired adenosine in the viral RNA promoter.

Transcription and replication of the influenza A virus RNA genome are mediated by the viral RNA polymerase from a promoter consisting of the partially base-paired 3' and 5' termini of viral genome segments. Here we show that transcription and replication can be uncoupled by mutation of an unpaired adenosine in the 5' strand of the promoter. This residue is important for transcription but not replication by being essential for the cap-binding activity of the RNA polymerase.

The SARS-coronavirus nsp7+nsp8 complex is a unique multimeric RNA polymerase capable of both de novo initiation and primer extension.

Uniquely among RNA viruses, replication of the ~30-kb SARS-coronavirus genome is believed to involve two RNA-dependent RNA polymerase (RdRp) activities. The first is primer-dependent and associated with the 106-kDa non-structural protein 12 (nsp12), whereas the second is catalysed by the 22-kDa nsp8. This latter enzyme is capable of de novo initiation and has been proposed to operate as a primase. Interestingly, this protein has only been crystallized together with the 10-kDa nsp7, forming a hexadecameric, dsRNA-encircling ring structure [i.e. nsp(7+8), consisting of 8 copies of both nsps]. To better understand the implications of these structural characteristics for nsp8-driven RNA synthesis, we studied the prerequisites for the formation of the nsp(7+8) complex and its polymerase activity. We found that in particular the exposure of nsp8's natural N-terminal residue was paramount for both the protein's ability to associate with nsp7 and for boosting its RdRp activity. Moreover, this 'improved' recombinant nsp8 was capable of extending primed RNA templates, a property that had gone unnoticed thus far. The latter activity is, however, ~20-fold weaker than that of the primer-dependent nsp12-RdRp at equal monomer concentrations. Finally, site-directed mutagenesis of conserved D/ExD/E motifs was employed to identify residues crucial for nsp(7+8) RdRp activity.

Using structure prediction of negative sense RNA virus nucleoproteins to assess evolutionary relationships.

Negative sense RNA viruses (NSV) include some of the most detrimental human pathogens, including the influenza, Ebola and measles viruses. NSV genomes consist of one or multiple single-stranded RNA molecules that are encapsidated into one or more ribonucleoprotein (RNP) complexes. These RNPs consist of viral RNA, a viral RNA polymerase, and many copies of the viral nucleoprotein (NP). Current evolutionary relationships within the NSV phylum are based on alignment of conserved RNA-directed RNA polymerase (RdRp) domain amino acid sequences. However, the RdRp domain-based phylogeny does not address whether NP, the other core protein in the NSV genome, evolved along the same trajectory or whether several RdRp-NP pairs evolved through convergent evolution in the segmented and non-segmented NSV genomes architectures. Addressing how NP and the RdRp domain evolved may help us better understand NSV diversity. Since NP sequences are too short to infer robust phylogenetic relationships, we here used experimentally-obtained and AlphaFold 2.0-predicted NP structures to probe whether evolutionary relationships can be estimated using NSV NP sequences. Following flexible structure alignments of modeled structures, we find that the structural homology of the NSV NPs reveals phylogenetic clusters that are consistent with RdRp-based clustering. In addition, we were able to assign viruses for which RdRp sequences are currently missing to phylogenetic clusters based on the available NP sequence. Both our RdRp-based and NP-based relationships deviate from the current NSV classification of the segmented Naedrevirales, which cluster with the other segmented NSVs in our analysis. Overall, our results suggest that the NSV RdRp and NP genes largely evolved along similar trajectories and that even short pieces of genetic, protein-coding information can be used to infer evolutionary relationships, potentially making metagenomic analyses more valuable.

RNA Polymerase Inhibitor Enisamium for Treatment of Moderate COVID-19 Patients: A Randomized, Placebo-Controlled, Multicenter, Double-Blind Phase 3 Clinical Trial.

Enisamium is an orally available therapeutic that inhibits influenza A virus and SARS-CoV-2 replication. We evaluated the clinical efficacy of enisamium treatment combined with standard care in adult, hospitalized patients with moderate COVID-19 requiring external oxygen. Hospitalized patients with laboratory-confirmed SARS-CoV-2 infection were randomly assigned to receive either enisamium (500 mg per dose, four times a day) or a placebo. The primary outcome was an improvement of at least two points on an eight-point severity rating (SR) scale within 29 days of randomization. We initially set out to study the effect of enisamium on patients with a baseline SR of 4 or 5. However, because the study was started early in the COVID-19 pandemic, and COVID-19 had been insufficiently studied at the start of our study, an interim analysis was performed alongside a conditional power analysis in order to ensure patient safety and assess whether the treatment was likely to be beneficial for one or both groups. Following this analysis, a beneficial effect was observed for patients with an SR of 4 only, i.e., patients with moderate COVID-19 requiring supplementary oxygen. The study was continued for these COVID-19 patients. Overall, a total of 592 patients were enrolled and randomized between May 2020 and March 2021. Patients with a baseline SR of 4 were divided into two groups: 142 (49.8%) were assigned to the enisamium group and 143 (50.2%) to the placebo group. An analysis of the population showed that if patients were treated within 4 days of the onset of COVID-19 symptoms (n = 33), the median time to improvement was 8 days for the enisamium group and 13 days for the placebo group (p = 0.005). For patients treated within 10 days of the onset of COVID-19 symptoms (n = 154), the median time to improvement was 10 days for the enisamium group and 12 days for the placebo group (p = 0.002). Our findings suggest that enisamium is safe to use with COVID-19 patients, and that the observed clinical benefit of enisamium is worth reporting and studying in detail.

Transient RNA structures underlie highly pathogenic avian influenza virus genesis.

Highly pathogenic avian influenza viruses (HPAIVs) cause severe disease and high fatality in poultry1. They emerge exclusively from H5 and H7 low pathogenic avian influenza viruses (LPAIVs)2. Although insertion of a furin-cleavable multibasic cleavage site (MBCS) in the hemagglutinin gene was identified decades ago as the genetic basis for LPAIV-to-HPAIV transition3,4, the exact mechanisms underlying said insertion have remained unknown. Here we used an innovative combination of bioinformatic models to predict RNA structures forming around the influenza virus RNA polymerase during replication, and circular sequencing5 to reliably detect nucleotide insertions. We show that transient H5 hemagglutinin RNA structures predicted to trap the polymerase on purine-rich sequences drive nucleotide insertions characteristic of MBCSs, providing the first strong empirical evidence of RNA structure involvement in MBCS acquisition. Insertion frequencies at the H5 cleavage site were strongly affected by substitutions in flanking genomic regions altering predicted transient RNA structures. Introduction of H5-like cleavage site sequences and structures into an H6 hemagglutinin resulted in MBCS-yielding insertions never observed before in H6 viruses. Our results demonstrate that nucleotide insertions that underlie H5 HPAIV emergence result from a previously unknown RNA-structure-driven diversity-generating mechanism, which could be shared with other RNA viruses.