Defective interfering particles (DIPs) are incomplete viral genomes that modulate infection by competing with wild-type viruses and activating the innate immune response. Activation of the immune response leads to the production of cytokines and chemokines, including type I interferon (IFN), which restricts viral growth and may cause cell death. How DIPs interact with type I interferon (IFN) in spatially structured environments remains unclear. Focusing here on influenza A viruses, we developed a spatially explicit, stochastic model of in vitro viral infection that integrates virus and DIP replication, IFN signalling, and alternative dispersal modes. We find that: (1) our model captures the ring-like and patchy plaque morphologies observed experimentally; (2) IFN production peaks at an intermediate DIP ratio, reflecting a trade-off between early immune activation and sufficient co-infection; and (3) even a small fraction of long-range spread by virus and DIPs enables escape from the immune-based containment despite long-range IFN diffusion; this causes stronger antiviral responses but earlier peaks in virus egress at similar levels of cell loss. The model is available as an interactive platform: https://shiny-spatial-infection-app-production.up.railway.app/.

Highly pathogenic avian influenza viruses (HPAIVs) derive from H5 and H7 low pathogenic avian influenza viruses (LPAIVs). Although insertion of a furin-cleavable multibasic cleavage site (MBCS) in the hemagglutinin gene was identified decades ago as the genetic basis for the LPAIV-to-HPAIV transition, the mechanisms underlying the occurrence of insertion are unknown. Here, we show that transient H5 RNA structures, predicted to trap the influenza virus polymerase on purine-rich sequences, drive nucleotide insertions, providing empirical evidence of RNA structure involvement in MBCS acquisition. Introduction of H5-like sequences and structures into an H6 hemagglutinin resulted in MBCS-yielding insertions. Our results show that nucleotide insertions that underlie H5 HPAIV emergence result from an RNA structure–driven diversity-generating mechanism, which could also occur in other RNA viruses. High-pathogenicity influenza viruses have not only been devastating the planet’s wild and domestic bird populations, but they also represent a persistent threat of initiating a fatal human influenza pandemic. Funk et al. investigated how recombination of low-pathogenicity viruses is prone to incorporating sequences for the furin multibasic cleavage site in the hemagglutination gene, which promotes cell invasion by virus. The authors found that transient RNA structures in the virus replication machinery caused it to stutter on adenine-/uridine-rich sequences and allowed the nucleotide insertions that translate into the cleavage site. The authors suggest that these sorts of transient structures might also be present in other RNA viruses. —Caroline Ash

Fever during influenza A virus (IAV) infection is triggered by the innate immune response. Various factors contribute to this response, including IAV mini viral RNAs (mvRNA), which trigger RIG-I signaling when their replication and transcription are dysregulated by template loops (t-loops). It is presently not well understood whether the fever response to IAV infection affects subsequent viral replication and innate immune activation. Here, we show that IAV infection at temperatures that simulate fever leads to increased antiviral signaling in H1N1 and H3N2 infections. Mathematical modeling and experimental analyses reveal that differential IAV nucleoprotein and RNA polymerase production increase mvRNA and interferon production. Moreover, at the higher infection temperature, mvRNAs with dysregulating t-loops contribute most to the innate immune activation. We propose that fever during IAV infection can establish a positive feedback loop in which elevated aberrant RNA synthesis and innate immune activation can contribute to the dysregulation of cytokine production.

Cas13 is activated by the hybridization of a CRISPR RNA to a complementary single-stranded RNA protospacer in a target RNA. While Cas13 is not activated by double-stranded RNA in vitro, it robustly targets RNA in cellular environments where RNAs are highly structured. The mechanism by which Cas13 targets structured RNAs remains unknown. Here, we systematically probe the effects of secondary structure on Cas13. We find that secondary structure in the protospacer and 3' to it inhibits Cas13 activity and quantitatively explains the former effect through a strand displacement framework. We then harness strand displacement to generate an 'occluded' Cas13, which enhances mismatch discrimination up to 50-fold and enables sequence-agnostic mutation identification at low (<1%) allele frequencies. Using occluded Cas13, we identify human-adaptive mutations in SARS-CoV-2 and human and avian influenza A viruses, as well as oncogenic mutations in KRAS. Our work leverages improved mechanistic understanding of Cas13 to expand the scope of RNA diagnostics and enable structure-informed Cas13 approaches.

Influenza A virus (IAV) noncanonical RNAs are bound by retinoic acid-inducible gene I (RIG-I). However, innate immune activation is infrequent and it is not understood why noncanonical IAV RNAs activate RIG-I in a sequence- or RNA structure-dependent manner. We hypothesized that multiple events need to occur before IAV RNA synthesis activates RIG-I and investigated whether RIG-I activation is stimulated by the noncanonical or aberrant transcription of mini viral RNAs (mvRNA), an RNA that is overexpressed in highly pathogenic IAV infections. We find that mvRNAs can cause noncanonical transcription termination through a truncated 5' polyadenylation signal or a 5' transient RNA structure that interrupts polyadenylation. The resulting capped complementary RNAs stimulate the release of an mvRNA and complement RIG-I activation in trans. Overall, our findings indicate that sequential rounds of noncanonical or aberrant viral replication and transcription are needed for innate immune signaling by IAV RNA synthesis.

Background: Influenza A viruses (IAV) cause seasonal flu and occasional pandemics. In addition, the potential for the emergence of new strains presents unknown challenges for public health. Face masks and other personal protective equipment (PPE) can act as barriers that prevent the spread of these viruses. Metal ions embedded into PPE have been demonstrated to inactivate respiratory viruses, but the underlying mechanism of inactivation and potential for resistance is presently not well understood. Methods: In this study, we used hemagglutination assays to quantify the effect of zinc ions on IAV sialic acid receptor binding. We varied the zinc concentration, incubation time, incubation temperature, and passaged IAV in the presence of zinc ions to investigate if resistance to zinc ions could evolve. Results: We found that zinc ions impact the ability of IAV particles to hemagglutinate and observed inhibition within 1 min of exposure. Maximum inhibition was achieved within 1 h and sustained for at least 24 h in a concentration-dependent manner. Inhibition was also temperature-dependent, and optimal above room temperature. Serial passaging of IAV in the presence of zinc ions did not result in resistance. Conclusions: e conclude that zinc ions prevent IAV hemagglutination in a concentration and temperature-dependent manner for at least 24 h. Overall, these findings are in line with previous observations indicating that zinc-embedded materials can inactivate the IAV hemagglutinin and SARS-CoV-2 spike proteins, and they support work toward developing robust, passive, self-cleaning antiviral barriers in PPE.

Zoonotic viruses such as hantaviruses and influenza A viruses present a threat to humans and livestock. There is thus a need for methods that are rapid, sensitive, and relatively cheap to detect infections with these pathogens early. Here we use an amplification-free CRISPR-Cas13-based assay, which is simple, cheap and field-deployable, to detect the presence or absence of genomic hantavirus or influenza A virus RNA. In addition, we evaluate whether the use of multiple CRISPR RNAs (crRNAs) can improve the sensitivity of this amplification-free method. We demonstrate that for the hantaviruses Tula Virus (TULV) and Andes Virus (ANDV) a combination of two or three crRNAs provides the best sensitivity for detecting viral RNA, whereas for influenza virus RNA detection, additional crRNAs provide no benefit. We also show that the amplification-free method can be used to detect TULV and ANDV RNA in tissue culture infection samples and influenza A virus RNA in clinical nasopharyngeal swabs. In clinical samples, the Cas13 assay has an 85% agreement with RT-qPCR for identifying a positive sample. Overall, these findings indicate that amplification-free CRISPR-Cas13 detection of viral RNA has potential as a tool for rapidly detecting zoonotic virus infections.

Influenza A viruses (IAVs) typically cause a mild to moderate respiratory disease, whereas infections with pandemic and highly pathogenic avian IAV strains are frequently associated with high morbidity and death. Various noncanonical or aberrant transcription and replication products have been implicated in the effect of IAV infection on disease outcomes. While early research indicated that all these molecules may be defective, recent findings coupled with analyses of the structure of the IAV RNA polymerase suggest that the production of noncanonical RNAs is not solely driven by errors. Instead, their place in infection may be more nuanced. In this review, we discuss our current understanding of the molecular steps that underlie noncanonical transcription and replication and which molecular mysteries remain.

Influenza A virus encodes conserved promoter sequences. Using minimal replication assays-transfections with viral polymerase, nucleoprotein, and a genomic template-these sequences were identified as 13nt at the 5' end of the genomic RNA (U13) and 12nt at the 3' end (U12). Other than the fourth 3' nucleotide, the U12 and U13 sequences are identical between all eight RNA molecules of the segmented influenza A genome. However, individual segments can exhibit different dynamics during infection. Influenza NS2, which modulates transcription and replication differentially between genomic segments, may provide an explanation. Here, we assess how internal sequences of two genomic segments, HA and PB1, contribute to NS2-dependent replication and map such interactions down to individual nucleotides in PB1. We find that the expression of NS2 significantly alters sequence requirements for efficient replication beyond the identical U12 and U13 sequences, providing a potential mechanism for segment-specific replication dynamics across the influenza genome.

The influenza A virus nuclear export protein (NEP) is a multifunctional protein that is essential for the viral life cycle and has very high sequence conservation. However, since the open reading frame of NEP largely overlaps with that of another influenza viral protein, non-structural protein 1, it is difficult to infer the functional constraints of NEP based on sequence conservation analysis. In addition, the N-terminal of NEP is structurally disordered, which further complicates the understanding of its function. Here, we systematically measure the replication fitness effects of >1,800 mutations of NEP. Our results show that the N-terminal domain has high mutational tolerance. Additional experiments show that N-terminal domain mutations affect viral transcription and replication dynamics, host cellular responses, and mammalian adaptation of avian influenza virus. Overall, our study not only advances the functional understanding of NEP but also provides insights into its evolutionary constraints.

Influenza A virus (IAV) RNA synthesis produces full-length and deletion-containing RNA molecules, which include defective viral genomes (DVG) and mini viral RNAs (mvRNA). Sequencing approaches have shown that DVG and mvRNA species may be present during infection, and that they can vary in size, segment origin, and sequence. Moreover, a subset of aberrant RNA molecules can bind and activate host-pathogen receptor retinoic acid-inducible gene I (RIG-I), leading to innate immune signaling and the expression of type I and III interferons. Measuring the kinetics and distribution of these immunostimulatory aberrant RNA sequences is important for understanding their function in IAV infection. Here, we explored if IAV mvRNA molecules can be detected and quantified using amplification-free, CRISPR-LbuCas13a-based detection. We show that CRISPR-LbuCas13a can be used to measure the copy numbers of specific mvRNAs in samples from infected tissue culture cells. However, to efficiently detect mvRNAs in other samples, promiscuous CRISPR guide RNAs are required that activate LbuCas13a in the presence of multiple mvRNA sequences. One crRNA was able to detect full-length IAV segment 5 without amplification, allowing it to be used for general IAV infection detection nasopharyngeal swabs. Using CRISPR-LbuCas13a, we confirm that mvRNAs are present in ferret upper and lower respiratory tract tissue, as well as clinical nasopharyngeal swab extracts of hospitalized patients. Overall, CRISPR-LbuCas13a-based RNA detection is a useful tool for studying deletion-containing viral RNAs, and it complements existing amplification-based approaches.

During influenza A virus (IAV) infection, host pathogen receptor retinoic acid-inducible gene I (RIG-I) detects the partially complementary, 5'-triphosphorylated ends of the viral genome segments and non-canonical replication products. However, it has also been suggested that innate immune responses may be triggered by viral transcription. In this study, we investigated whether an immunostimulatory RNA is produced during IAV transcription. We show that the IAV RNA polymerase can read though the polyadenylation signal during transcription termination, generating a capped complementary RNA (ccRNA), which contains the 5' cap of an IAV mRNA and the 3' terminus of a cRNA instead of a poly(A) tail. ccRNAs are detectable and in both ribonucleoprotein reconstitution assays and IAV infections. Mutations that disrupt polyadenylation enhance ccRNA synthesis and increase RIG-I-dependent innate immune activation. Notably, while ccRNA itself is not immunostimulatory, it forms a RIG-I agonist by hybridizing with a complementary negative-sense viral RNA. These findings thus identify a novel non-canonical IAV RNA species and suggest an alternative mechanism for RIG-I activation during IAV infection.

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 the alignment of conserved RNA-dependent 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 genome 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 , 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 even short pieces of genetic, protein-coding information can be used to infer evolutionary relationships, potentially making metagenomic analyses more valuable.

Since the influenza pandemic in 1968, influenza A(H3N2) viruses have become endemic. In this state, H3N2 viruses continuously evolve to overcome immune pressure as a result of prior infection or vaccination, as is evident from the accumulation of mutations in the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). However, phylogenetic studies have also demonstrated ongoing evolution in the influenza A(H3N2) virus RNA polymerase complex genes. The RNA polymerase complex of seasonal influenza A(H3N2) viruses produces mRNA for viral protein synthesis and replicates the negative sense viral RNA genome (vRNA) through a positive sense complementary RNA intermediate (cRNA). Presently, the consequences and selection pressures driving the evolution of the polymerase complex remain largely unknown. Here, we characterize the RNA polymerase complex of seasonal influenza A(H3N2) viruses representative of nearly 50 years of influenza A(H3N2) virus evolution. The H3N2 polymerase complex is a reassortment of human and avian influenza virus genes. We show that since 1968, influenza A(H3N2) viruses have increased the transcriptional activity of the polymerase complex while retaining a close balance between mRNA, vRNA, and cRNA levels. Interestingly, the increased polymerase complex activity did not result in increased replicative ability on differentiated human airway epithelial (HAE) cells. We hypothesize that the evolutionary increase in polymerase complex activity of influenza A(H3N2) viruses may compensate for the reduced HA receptor binding and avidity that is the result of the antigenic evolution of influenza A(H3N2) viruses.

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 ( = 33), the median time to improvement was 8 days for the enisamium group and 13 days for the placebo group ( = 0.005). For patients treated within 10 days of the onset of COVID-19 symptoms ( = 154), the median time to improvement was 10 days for the enisamium group and 12 days for the placebo group ( = 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.

Highly pathogenic avian influenza viruses (HPAIVs) cause severe disease and high fatality in poultry. They emerge exclusively from H5 and H7 low pathogenic avian influenza viruses (LPAIVs). 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 transition, 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 sequencing 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.

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.

SUMMARYNegative and ambisense RNA viruses are the causative agents of important human diseases such as influenza, measles, Lassa fever, and Ebola hemorrhagic fever. The viral genome of these RNA viruses consists of one or more single-stranded RNA molecules that are encapsidated by viral nucleocapsid proteins to form a ribonucleoprotein complex (RNP). This RNP acts as protection, as a scaffold for RNA folding, and as the context for viral replication and transcription by a viral RNA polymerase. However, the roles of the viral nucleoproteins extend beyond these functions during the viral infection cycle. Recent advances in structural biology techniques and analysis methods have provided new insights into the formation, function, dynamics, and evolution of negative sense virus nucleocapsid proteins, as well as the role that they play in host innate immune responses against viral infection. In this review, we discuss the various roles of nucleocapsid proteins, both in the context of RNPs and in RNA-free states, as well as the open questions that remain.

RNA viruses are important human pathogens that cause seasonal epidemics and occasional pandemics. Examples are influenza A viruses (IAV) and coronaviruses (CoV). When emerging IAV and CoV spill over to humans, they adapt to evade immune responses and optimize their replication and spread in human cells. In IAV, adaptation occurs in all viral proteins, including the viral ribonucleoprotein (RNP) complex. RNPs consist of a copy of the viral RNA polymerase, a double-helical coil of nucleoprotein, and one of the eight segments of the IAV RNA genome. The RNA segments and their transcripts are partially structured to coordinate the packaging of the viral genome and modulate viral mRNA translation. In addition, RNA structures can affect the efficiency of viral RNA synthesis and the activation of host innate immune response. Here, we investigated if RNA structures that modulate IAV replication processivity, so-called template loops (t-loops), vary during the adaptation of pandemic and emerging IAV to humans. Using cell culture-based replication assays and sequence analyses, we find that the sensitivity of the IAV H3N2 RNA polymerase to t-loops increased between isolates from 1968 and 2017, whereas the total free energy of t-loops in the IAV H3N2 genome was reduced. This reduction is particularly prominent in the PB1 gene. In H1N1 IAV, we find two separate reductions in t-loop free energy, one following the 1918 pandemic and one following the 2009 pandemic. No destabilization of t-loops is observed in the influenza B virus genome, whereas analysis of SARS-CoV-2 isolates reveals destabilization of viral RNA structures. Overall, we propose that a loss of free energy in the RNA genome of emerging respiratory RNA viruses may contribute to the adaption of these viruses to the human population.

Mathematical models have played a crucial role in exploring and guiding pandemic responses. University campuses present a particularly well-documented case for institutional outbreaks, thereby providing a unique opportunity to understand detailed patterns of pathogen spread. Here, we present descriptive and modeling analyses of SARS-CoV-2 transmission on the Princeton University (PU) campus-this model was used throughout the pandemic to inform policy decisions and operational guidelines for the university campus. Epidemic patterns between the university campus and surrounding communities exhibit strong spatiotemporal correlations. Mathematical modeling analysis further suggests that the amount of on-campus transmission was likely limited during much of the wider pandemic until the end of 2021. Finally, we find that a superspreading event likely played a major role in driving the Omicron variant outbreak on the PU campus during the spring semester of the 2021-2022 academic year. Despite large numbers of cases on campus in this period, case levels in surrounding communities remained low, suggesting that there was little spillover transmission from campus to the local community.

Targeted protein degradation (TPD), as exemplified by proteolysis-targeting chimera (PROTAC), is an emerging drug discovery platform. PROTAC molecules, which typically contain a target protein ligand linked to an E3 ligase ligand, recruit a target protein to the E3 ligase to induce its ubiquitination and degradation. Here, we applied PROTAC approaches to develop broad-spectrum antivirals targeting key host factors for many viruses and virus-specific antivirals targeting unique viral proteins. For host-directed antivirals, we identified a small-molecule degrader, FM-74-103, that elicits selective degradation of human GSPT1, a translation termination factor. FM-74-103-mediated GSPT1 degradation inhibits both RNA and DNA viruses. Among virus-specific antivirals, we developed viral RNA oligonucleotide-based bifunctional molecules (Destroyers). As a proof of principle, RNA mimics of viral promoter sequences were used as heterobifunctional molecules to recruit and target influenza viral polymerase for degradation. This work highlights the broad utility of TPD to rationally design and develop next-generation antivirals.

The influenza A virus (IAV) RNA polymerase is an essential driver of IAV evolution. Mutations that the polymerase introduces into viral genome segments during replication are the ultimate source of genetic variation, including within the three subunits of the IAV polymerase (polymerase basic protein 2, polymerase basic protein 1, and polymerase acidic protein). Evolutionary analysis of the IAV polymerase is complicated, because changes in mutation rate, replication speed, and drug resistance involve epistatic interactions among its subunits. In order to study the evolution of the human seasonal H3N2 polymerase since the 1968 pandemic, we identified pairwise evolutionary relationships among ∼7000 H3N2 polymerase sequences using mutual information (MI), which measures the information gained about the identity of one residue when a second residue is known. To account for uneven sampling of viral sequences over time, we developed a weighted MI (wMI) metric and demonstrate that wMI outperforms raw MI through simulations using a well-sampled severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) dataset. We then constructed wMI networks of the H3N2 polymerase to extend the inherently pairwise wMI statistic to encompass relationships among larger groups of residues. We included hemagglutinin (HA) in the wMI network to distinguish between functional wMI relationships within the polymerase and those potentially due to hitch-hiking on antigenic changes in HA. The wMI networks reveal coevolutionary relationships among residues with roles in replication and encapsidation. Inclusion of HA highlighted polymerase-only subgraphs containing residues with roles in the enzymatic functions of the polymerase and host adaptability. This work provides insight into the factors that drive and constrain the rapid evolution of influenza viruses.

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.