Biology subjects

VanInsberghe, D.

Publications and source records attributed to VanInsberghe, D..

12 recordsLinked to original sources

bioRxiv2026

Viral dispersal drives changes in population composition

In an acute viral infection, a small seeding population expands rapidly over several orders of magnitude, then declines. This vast population growth is accompanied by spatial expansion as infection spreads to new anatomical sites within the host. Evolutionary theory predicts that spatially expanding populations experience more extreme stochastic losses of genetic diversity than spatially unstructured populations. To test this prediction in the context of viral dynamics, we evaluated the impact of spatial expansion on viral population genetic structure and assessed how modulators of viral dispersal affected this structure. To this end, we developed a means of implementing the spatial expansion of influenza A virus in cell culture and applied it to a viral population carrying a diverse genetic barcode. Across this simplified cellular landscape, we found that spatial expansion produces a patchwork of distinct viral subpopulations. We then evaluated the consequences for population structure of two modulators of viral dispersal: extracellular viscosity and a common polymorphism in the viral attachment protein. Both physical constraint and viral phenotype altered the extent of genetic bottlenecking and spatial heterogeneity that developed during expansion, indicating that environmental features and viral traits that modulate dispersal also modulate viral population composition, diversity and evolution.

microbiology↗

bioRxiv2026

Within-host antigenic selection of influenza A virus dominates over stochasticity but is limited by fitness tradeoffs and timing of the immune response

Despite antigenic evolution at the global scale, positive selection of influenza virus antigenic variants is not readily observed within hosts. Here, we tested the extent to which fitness tradeoffs, the timing of immune pressure, and stochastic effects impede antigenic selection within pre-immune hosts. We used genetically barcoded influenza A/Texas/50/2012 (H3N2) viruses (Tx/12) in a guinea pig model to probe these dynamics. Positive selection of an antigenic variant was reliant on a high strength of immune pressure acting early in infection. However, when fitness tradeoffs of the antigenic change were lessened, a lower strength and later introduction of immune pressure favored the antigenic variant. In all conditions, barcode dynamics revealed moderate stochastic effects. Our results suggest that stochastic evolution does not impede selection during acute influenza virus infection. The rarity of antigenic escape may instead stem from low mutational supply, fitness tradeoffs, and the intrinsic delay between infection and antibody recall.

microbiology↗

bioRxiv2026

Comparative analysis of flavivirus sfRNA dynamics and secondary structure

The accumulation of subgenomic flavivirus RNAs (sfRNAs) modulates viral fitness and pathogenicity in culture and in vivo. These noncoding RNAs are produced by incomplete digestion of the flavivirus genome by the cellular 5-3 exoribonuclease (XRN1). Diverse flaviviruses have conserved RNA structural elements (RSEs) that map to their 3-untranslated region (3-UTR): Xrn-resistant RNA structures, dumbbell structures, and a 3-stem loop (3SL). Despite the importance of the 3-UTR RSEs for flavivirus replication, the structural dynamics of sfRNA during flavivirus infection are understudied. Here, we use digital droplet PCR to quantify sfRNA levels during infection for a panel of mosquito-borne flaviviruses (MbFV) including dengue virus serotypes 1 (DENV1), 2 (DENV2), and 4 (DENV4), and Zika virus (ZIKV). We then used SHAPE-MaP on XRN1-digested, in vitro-transcribed sfRNAs from each virus to determine their secondary structures compared to the corresponding sfRNAs obtained from flavivirus-infected A549 cells. Results seen in-cell and in vitro were largely similar; however, motifs within the dumbbell, the small hairpin (sHP) directly upstream of the 3-SL, and 3-SL regions showed significant differences in the extent of nucleotide reactivity. These differences were consistent among the four flaviviruses examined and may indicate regions of sfRNA that are shielded by interaction with proteins or other nucleic acids during infection. However, strong protection indicative of sustained interaction was not apparent. Our findings suggest that sfRNA interactions with viral and host factors within the cell are few, occur via base-paired regions, or are highly transient. ImportanceFlaviviruses are highly prevalent human pathogens. The flavivirus genome contains RNA structural elements (RSEs), including those encoded in the 3-UTR, that are necessary for viral replication. Subgenomic flavivirus RNAs (sfRNAs) are produced by incomplete digestion of flavivirus genomic RNA due to the cellular exoribonuclease XRN1 encountering 3-UTR RSEs that promote its stalling and disassociation. Viruses unable to produce sfRNAs are highly attenuated, underlining their biological importance. sfRNA secondary structure has been investigated previously but little information is available on sfRNA secondary structure dynamics in infected cells. By comparing SHAPE-MaP reactivities in vitro and in cells, we determined that previously inferred structures are likely maintained within infected cells. We also identified differences in the extent of SHAPE reactivity between in vitro and in-cell environments that were common to multiple mosquito-borne flaviviruses. These differences suggest that sfRNAs may engage in transient interactions within the cell that may be important for their function.

microbiology↗

bioRxiv2026

Conserved stem-loops of the SARS-CoV-2 5'-UTR activate OAS1

The innate immune system relies on pathogen recognition receptors, such as the 2,5-oligoadenylate synthetase (OAS) proteins, to detect pathogen-associated molecular patterns like viral double-stranded (ds)RNA. A specific splicing variant of OAS1 (OAS1-p46) has been implicated in initiating an immune response that leads to decreased disease severity during SARS-CoV-2 infection. OAS1-p46 has a C-terminal lipid modification motif that allows for anchoring of the protein to intracellular membranes and thus potential colocalization with immunogenic viral RNA regions such as the SARS-CoV-2 5-untranslated region (5-UTR). Here, we show that OAS proteins can detect the 5-structured elements (5-SE)-comprising the 5-UTR and three additional stem-loop structures-and activate the cellular RNase L pathway. Through systematic 3-end truncations of the 5-SE, we show that the smallest 5-SE fragment capable of potently activating OAS1 is the conserved and highly structured SL1-4b region, containing the first four stem-loop structures and their intervening linking sequences (SL1-4), as well as an unstructured region ("SL4b") to the 3-side of SL4. Analyses of OAS1 activation and RNA secondary structure probing using selective 2-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) of additional SL1-4b RNA variants suggests a model in which SL4 acts as the primary OAS1 interaction site, while the unstructured SL4b region and two other stem-loops (SL1 and SL3) are necessary for optimal presentation of this region for OAS1 activation. Our findings reveal a structurally complex viral RNA region that potently activates OAS1, underscoring the potential complexity of RNAs that can strongly activate this innate immune sensor.

biochemistry↗

bioRxiv2026

Within-host adaptive evolution is limited by genetic drift in experimental human influenza A virus infections

Selection of advantageous mutations drives the emergence of dominant variants during seasonal influenza epidemics. However, within-host detection of such variants remains rare, limiting our understanding of how selection operates at the scale of individual hosts. In this study, we used a controlled human infection model to examine the within-host evolutionary dynamics in thirteen participants intranasally infected with a seasonal H3N2 influenza A virus. Although this clinical trial is ongoing, our work represents a pre-planned, interim, exploratory analysis. Results in this system were contrasted with those observed in a ferret model of infection. The inoculum, used in both humans and ferrets, carried standing diversity that enabled evaluation of variant trajectories during infection. Although the dynamics were variable among participants, in humans, the minor variants in the PA and NP gene segments tended to increase in frequency as infection progressed. Variant dynamics were more consistent among ferrets but showed differences from humans in the fate of the minor NP allele. Based on these observations, we fit a population genetic model to longitudinal measurements of variant frequencies. Estimates of variant selection coefficients and effective viral population sizes indicated that in humans the two minor variants had a selective advantage over the major variants, but genetic drift was strong, limiting the efficiency of selection. In ferrets, the PA minor variant also was estimated to have a selective advantage, while the NP minor variant was estimated to have a selective disadvantage. Moreover, effective viral population sizes were estimated to be considerably higher in ferrets than in humans, indicating that genetic drift was weaker in ferrets. Our analyses reveal differing selective environments acting on influenza viruses in human and ferret hosts and indicate that selection at the within-host level is weakened by genetic drift.

microbiology↗

bioRxiv2025

Viral lineage and mode of exposure modulate within host spatial dynamics of influenza A viruses

The upper and lower respiratory tracts (URT and LRT) present distinct environments for influenza A virus (IAV) replication. Their differential features have major implications for viral evolutionary dynamics, transmission potential, and pathogenesis. To investigate the implications of differential viral replication in the URT and LRT, we assessed dispersal of IAVs throughout the guinea pig respiratory system. Guinea pigs were inoculated intranasally with a 300 L volume to deliver inoculum to both the URT and LRT. Two strains were used to represent the circulating seasonal IAV lineages: influenza A/TX/50/2012 (H3N2) and influenza A/CA/07/2009 (H1N1) virus. The inclusion of a diverse genetic barcode enabled high-resolution tracing of viral dispersal for the H1N1 virus. While infectious virus was consistently detected in the URT, the H1N1 virus could be detected in LRT while the H3N2 virus could not. To determine whether replication of the H1N1 virus in the LRT extends to other modes of infection, virus distribution was evaluated following infection via aerosol exposure or transmission. Infectious virus in lung homogenates was observed in both cases, confirming the LRT tropism of the H1N1 virus. Sequencing genetic barcodes revealed that diversity was largely maintained in nasal samples and trachea but contracted upon dispersal to the lungs. This loss of diversity was associated with increased distance to and branching from the major airways, implicating long distance dispersal through the airways in imposing within-host population bottlenecks. These data underline the implications for within-host viral dynamics of the distinct environments of the upper and lower respiratory tracts. ImportanceThe upper (URT) and lower (LRT) respiratory tracts create different conditions for influenza A virus (IAV) spread and evolution. We studied how the virus moves through guinea pigs airways after infection with H3N2 or H1N1 strains of IAV. Whether delivered intranasally, by aerosol or by transmission, the H1N1 virus replicated in the nasal cavity, trachea, and lungs. By contrast, the H3N2 virus stayed mostly in the nasal cavity. Genetic barcodes were used to track how the H1N1 virus moved and changed. The populations replicating in the nasal cavity and trachea maintained high diversity but those sampled from the lungs showed low diversity. This bottlenecking effect was stronger for viral populations present deeper in the lungs. These findings show that the different environments of the URT and LRT strongly shape how influenza spreads and evolves inside a host.

microbiology↗

bioRxiv2025

Virion aggregation shapes infection dynamics and evolutionary potential

Viral spread is classically thought to be mediated by single viral particles. However, viruses often disseminate in groups - as aggregates, inside membranous vesicles, or as clusters associated with bacterial components or other complex surfaces. The implications of collective dispersal for viral infectivity and evolution remain incompletely defined. Here, we used mammalian orthoreovirus to evaluate the impact of aggregation on the propagation of infection and the generation of viral diversity through reassortment. Aggregation of free virions was induced by manipulating pH and ionic conditions. This treatment promoted coordinated delivery of viruses to cells, increasing the number of virions per infected cell and number of virions per occupied endosome at early times of infection. Likely due to a consolidation of infectious units, aggregation concomitantly reduced the overall infectivity of the viral population and progeny virus yields. When viral populations comprised two genetically distinct viruses, aggregation increased the frequency of mixed infection and of genetic exchange through reassortment. Thus, the formation of collective infectious units lowers the replicative potential of mammalian orthoreovirus populations but increases viral evolutionary potential by promoting genetic diversification. ImportanceA deeper understanding of processes shaping viral evolution will advance our ability to anticipate viral emergence, escape from immune responses and resistance to therapeutics. Although much is known about how genetic variation fuels viral evolution, how modes of viral spread influence the generation and structure of genetic variation remains poorly characterized. Here we examine how the collective dissemination of viruses modulates early infection dynamics and viral diversity. We find that, although infection in groups reduces the number of independently infected cells, it results in a more genetically diverse progeny population, an outcome that may enhance evolutionary potential.

microbiology↗

bioRxiv2023

Ventilation does not affect influenza virus transmission efficiency in a ferret playpen setup

Sustained community spread of influenza viruses relies on efficient person-to-person transmission. Current experimental transmission systems do not mimic environmental conditions (e.g., air exchange rates, flow patterns), host behaviors or exposure durations relevant to real-world settings. Therefore, results from these traditional systems may not be representative of influenza virus transmission in humans. To address this pitfall, we developed a modified, more realistic transmission setup and used it to investigate the impact of ventilation rates on transmission in a close-range, play-based scenario. In this setup, four immunologically naive recipient ferrets were exposed to a donor ferret infected with a genetically barcoded 2009 H1N1 virus (H1N1pdm09) for four hours. The ferrets interacted in a shared space that included toys, similar to a child care setting. The transmission efficiency was determined under conditions of low and high ventilation rates; air exchange rates of [~] 1.3 hr-1 and 23 hr-1, respectively. Despite the large difference in ventilation rate, transmission efficiency was the same, 50% in two independent replicate studies. The presence of infectious virus or viral RNA on surfaces and in air throughout the exposure area was similar regardless of ventilation rate. While high viral genetic diversity in donor ferret nasal washes was maintained during infection, recipient ferret nasal washes displayed low diversity, revealing a narrow transmission bottleneck regardless of ventilation rate. Our findings indicate that in exposures characterized by frequent close-range, play-based interactions and the presence of fomites, ventilation does not significantly impact transmission efficiency. SignificanceImproved ventilation in building has the potential to reduce transmission of respiratory viruses, but its effect in different settings is not well understood. We developed a novel system to study influenza virus transmission in the ferret animal model in an environment that mimics a child care center. We demonstrate that increased ventilation is not effective at disrupting transmission in this setting, suggesting that transmission occurs mainly at close-range or via fomites. Multiple interventions are needed to reduce the spread of influenza virus in this type of setting.

microbiology↗

bioRxiv2023

Viral expansion after transfer is a primary driver of influenza A virus transmission bottlenecks

For many viruses, narrow bottlenecks acting during transmission sharply reduce genetic diversity in a recipient host relative to the donor. Since genetic diversity represents adaptive potential, such losses of diversity are thought to limit the opportunity for viral populations to undergo antigenic change and other adaptive processes. Thus, a detailed picture of evolutionary dynamics during transmission is critical to understanding the forces driving viral evolution at an epidemiologic scale. To advance this understanding, we used a barcoded virus library and a guinea pig model of transmission to decipher where in the transmission process influenza A virus populations lose diversity. In inoculated guinea pigs, we show that a high level of viral barcode diversity is maintained. Within-host continuity in the barcodes detected across time furthermore indicates that stochastic effects are not pronounced within the inoculated hosts. Importantly, in both aerosol-exposed and direct contact animals, we observed many barcodes at the earliest time point(s) positive for infectious virus, indicating robust transfer of diversity through the environment. This high viral diversity is short-lived, however, with a sharp decline seen 1-2 days after initiation of infection. Although major losses of diversity at transmission are well described for influenza A virus, our data indicate that events that occur following viral transfer and during the earliest stages of natural infection have a central role in this process. This finding suggests that host factors, such as immune effectors, may have greater opportunity to impose selection during influenza A virus transmission than previously recognized.

microbiology↗

bioRxiv2023

Genetic drift and purifying selection shape within-host influenza A virus populations during natural swine infections

Patterns of within-host influenza A virus (IAV) diversity and evolution have been described in natural human infections, but these patterns remain poorly characterized in non-human hosts. Elucidating these dynamics is important to better understand IAV biology and the evolutionary processes that govern spillover into humans. Here, we sampled an IAV outbreak in pigs during a week-long county fair to characterize viral diversity and evolution in this important reservoir host. Nasal wipes were collected on a daily basis from all pigs present at the fair, yielding up to 421 samples per day. Subtyping of PCR-positive samples revealed the co-circulation of H1N1 and H3N2 subtype IAVs. PCR-positive samples with robust Ct values were deep-sequenced, yielding 506 sequenced samples from a total of 253 pigs. Based on higher-depth re-sequenced data from a subset of these initially sequenced samples (260 samples from 168 pigs), we characterized patterns of within-host IAV genetic diversity and evolution. We find that IAV genetic diversity in single-subtype infected pigs is low, with the majority of intra-host single nucleotide variants (iSNVs) present at frequencies of <10%. The ratio of the number of nonsynonymous to the number of synonymous iSNVs is significantly lower than under the neutral expectation, indicating that purifying selection shapes patterns of within-host viral diversity in swine. The dynamic turnover of iSNVs and their pronounced frequency changes further indicate that genetic drift also plays an important role in shaping IAV populations within pigs. Taken together, our results highlight similarities in patterns of IAV genetic diversity and evolution between humans and swine, including the role of stochastic processes in shaping within-host IAV dynamics.

evolutionary biology↗

bioRxiv2021

Rapid evolutionary turnover of mobile genetic elements drives microbial resistance to viruses

Although it is generally accepted that viruses (phages) drive bacterial evolution, how these dynamics play out in the wild remains poorly understood. Here we show that the arms race between phages and their hosts is mediated by large and highly diverse mobile genetic elements. These phage-defense elements display exceedingly fast evolutionary turnover, resulting in differential phage susceptibility among clonal bacterial strains while phage receptors remain invariant. Protection afforded by multiple elements is cumulative, and a single bacterial genome can harbor as many as 18 putative phage-defense elements. Overall, elements account for 90% of the flexible genome amongst closely related strains. The rapid turnover of these elements demonstrates that phage resistance is unlinked from other genomic features and that resistance to phage therapy might be as easily acquired as antibiotic resistance.

microbiology↗

bioRxiv2020

Identification of SARS-CoV-2 recombinant genomes

Viral recombination can generate novel genotypes with unique phenotypic characteristics, including transmissibility and virulence. Although the capacity for recombination among betacoronaviruses is well documented, there is limited evidence of recombination between SARS-CoV-2 strains. By identifying the mutations that primarily determine SARS-CoV-2 clade structure, we developed a lightweight approach for detecting recombinant genomes. Among the over 537,000 genomes queried, we detect 1175 putative recombinants that contain multiple mutational markers from distinct clades. Additional phylogenetic analysis and the observed co-circulation of predicted parent clades in the geographic regions of exposure further support the feasibility of recombination in these detected cases. An analysis of these detected cases did not reveal any evidence for recombination hotspots in the SARS-CoV-2 genome. Although most recombinant genotypes were detected a limited number of times, at least two recombinants are now widely transmitted. Recombinant genomes were also found to contain substitutions of concern for elevated transmissibility and lower vaccine efficacy, including D614G, N501Y, E484K, and L452R. Adjusting for an unequal probability of detecting recombinants derived from different parent clades, and for geographic variation in clade abundance, we estimate that at most 5% of circulating viruses in the USA and UK are recombinant. While the phenotypic characterization of detected recombinants was beyond the scope of our analysis, the identification of transmitted recombinants involving substitutions of concern underscores the need to sustain efforts to monitor the emergence of new genotypes generated through recombination.

evolutionary biology↗