Biology subjects

Fitzsimmons, W. J.

Publications and source records attributed to Fitzsimmons, W. J..

10 recordsLinked to original sources

bioRxiv2026

H5N5 virus transmission is driven by repeated cross-Atlantic introductions and transmission among high latitude seabirds

In November 2025, the first global H5N5 infection was reported in a person in Washington State. Previously, these viruses had only been detected in seabirds and shorebirds near the Atlantic Ocean, suggesting potentially distinct ecology from dominant H5N1 strains. Using 354 global H5N5 genomes, we show that H5N5 viruses have been carried by high latitude seabirds and shorebirds across the Atlantic at least 9 times, with introductions more frequent in the fall and spring. We reconstruct a plausible path of cross-continental movement via Arctic or sub-Arctic regions, followed by a year of cryptic circulation prior to human spillover. These data show that H5N5 viruses have undergone far more intercontinental movements than previously recognized, and exhibit host and geographic patterns that are fundamentally distinct from dominant H5N1 lineages. The distinct host ecology, geography, and movement, paired with the capacity to infect a person, suggest that enhanced surveillance may be warranted.

evolutionary biology↗

bioRxiv2025

Influenza virus evolution and defective genome formation are shaped by host genotype and sex

Viral evolution during initial pandemic waves favors mutations that enhance replication and transmission over antigenic escape. Host genotype and sex strongly shape this early adaptation, yet their individual and combined effects remain unclear. We experimentally adapted influenza A virus to male and female BALB/c and C57BL/6 mice, generating 28 independent lineages, and employed a novel "rolling sphere" approach to identify mutational hotspots in three-dimensional protein structures. In BALB/c mice, adaptation favored nonsynonymous substitutions linked to increased virulence, including a hemagglutinin variant exclusively fixed in female lineages. It also revealed the first demonstration of sex-dependent selection shaping a viral protein interface. In female-adapted viruses, substitutions disrupting a key NS1 dimerization motif converged on a single residue, while in male-adapted viruses, they were dispersed across the same interface. Conversely, adaptation to C57BL/6 resulted in fewer substitutions but promoted defective viral genome formation, leading to reduced cytopathic effect and attenuated virulence. This provides the first in vivo evidence that host genotype alone can modulate defective viral genome formation. Our results offer critical insights into host-pathogen interactions and reveal that selective pressures imposed by specific genotype-sex combinations can increase virulence across host genotypes, enabling new epidemiological modeling and disease control strategies.

evolutionary biology↗

bioRxiv2024

In depth sequencing of a serially sampled household cohort reveals the within-host dynamics of Omicron SARS-CoV-2 and rare selection of novel spike variants

SARS-CoV-2 has undergone repeated and rapid evolution to circumvent host immunity. However, outside of prolonged infections in immunocompromised hosts, within-host positive selection has rarely been detected. The low diversity within-hosts and strong genetic linkage among genomic sites make accurately detecting positive selection difficult. Longitudinal sampling is a powerful method for detecting selection that has seldom been used for SARS-CoV-2. Here we combine longitudinal sampling with replicate sequencing to increase the accuracy of and lower the threshold for variant calling. We sequenced 577 specimens from 105 individuals from a household cohort primarily during the BA.1/BA.2 variant period. There was extremely low diversity and a low rate of divergence. Specimens had 0-12 intrahost single nucleotide variants (iSNV) at >0.5% frequency, and the majority of the iSNV were at frequencies <2%. Within-host dynamics were dominated by genetic drift and purifying selection. Positive selection was rare but highly concentrated in spike. Two individuals with BA.1 infections had S:371F, a lineage defining substitution for BA.2. A Wright Fisher Approximate Bayesian Computational model identified positive selection at 14 loci with 7 in spike, including S:448 and S:339. We also detected significant genetic hitchhiking between synonymous changes and nonsynonymous iSNV under selection. The detectable immune-mediated selection may be caused by the relatively narrow antibody repertoire in individuals during the early Omicron phase of the SARS-CoV-2 pandemic. As both the virus and population immunity evolve, understanding the corresponding shifts in SARS-CoV-2 within-host dynamics will be important.

microbiology↗

bioRxiv2024

Influenza A virus within-host evolution and positive selection in a densely sampled household cohort over three seasons

While influenza A virus (IAV) antigenic drift has been documented globally, in experimental animal infections, and in immunocompromised hosts, positive selection has generally not been detected in acute infections. This is likely due to challenges in distinguishing selected rare mutations from sequencing error, a reliance on cross-sectional sampling, and/or the lack of formal tests of selection for individual sites. Here, we sequenced IAV populations from 346 serial, daily nasal swabs from 143 individuals collected over three influenza seasons in a household cohort. Viruses were sequenced in duplicate, and intrahost single nucleotide variants (iSNV) were identified at a 0.5% frequency threshold. Within-host populations were subject to purifying selection with >75% mutations present at <2% frequency. Children (0-5 years) had marginally higher within-host evolutionary rates than adolescents (6-18 years) and adults (>18 years, 4.4x10-6 vs. 9.42x10-7 and 3.45x10-6, p <0.001). Forty-five iSNV had evidence of parallel evolution, but were not overrepresented in HA and NA. Several increased from minority to consensus level, with strong linkage among iSNV across segments. A Wright Fisher Approximate Bayesian Computational model identified positive selection at 23/256 loci (9%) in A(H3N2) specimens and 19/176 loci (11%) in A(H1N1)pdm09 specimens, and these were infrequently found in circulation. Overall, we found that within-host IAV populations were subject to purifying selection and genetic drift, with only subtle differences across seasons, subtypes, and age strata. Positive selection was rare and inconsistently detected.

microbiology↗

bioRxiv2024

Surveillance of H5 HPAI in Michigan using retail milk

An outbreak of highly pathogenic avian influenza virus (HPAI) H5N1 in dairy cattle in the United States has spread to at least 136 dairy herds in 11 states. Due to a lack of testing, it has been difficult to determine the number of farms affected and the extent of intrastate spread. We report a pilot study of real time RT-PCR (rRT-PCR) detection of influenza A virus (IAV) in milk purchased from local markets across Michigan. Two out of 13 samples were positive for IAV nucleic acid, suggestive of HPAI H5N1 infection in the originating herds. This small study of a convenience sample suggests that milk-based surveillance might be useful if applied systematically either at dairy processors or point of sale.

microbiology↗

bioRxiv2023

A rapid and flexible microneutralization assay for serological assessment of influenza viruses

BackgroundSerological responses from influenza vaccination or infection are typically measured by Hemagglutinin Inhibition (HAI) or Microneutralization (MN). Both methods are limited in feasibility, standardization, and generalizability. We describe a luciferase MN (LMN) assay that combines the advantages of the conventional MN assay with the ease of the HAI assay MethodsSera were obtained from the HIVE study. Plasmid transfection was used to generate recombinant influenza viruses expressing the hemagglutinin and neuraminidase of test strains, all other viral proteins from A/WSN33, and a NanoLuc reporter. Serum neutralization of luciferase-expressing targets was quantified as a reduction in Relative Light Unit (RLU) emission from infected cells. Neutralization titers were measured for cell- and egg-adapted versions of A/H3N2/Hong Kong/4801/2014 and A/H3N2/Singapore/INFIMH-16-0019/2016 and compared to HAI titers against egg-grown antigens. Results333 sera were collected from 259 participants between May 2016 and July 2018. Sampled participants ranged from 7 to 68 years of age and >80% were vaccinated against influenza. HAI and LMN titers were correlated for A/H3N2/Hong Kong/4801/2014 ({rho}=0.45, p[≤]0.01) and A/H3N2/Singapore/INFIMH-16-0019/2016 ({rho}=0.75, p[≤]0.01). LMN titers were lower for cell strains compared to egg strains (A/H3N2/Hong Kong/4801/2014 mean log2 fold change = -2.66, p[≤]0.01 and A/H3N2/Singapore/INFIMH-16-0019/2016 mean log2 fold change= -3.15, p[≤]0.01). ConclusionsThe LMN assay was feasible using limited sample volumes and was able to differentiate small antigenic differences, specifically between egg-adapted and cell-derived strains. The correspondence of these results with the commonly-used HAI confirms the potential utility of this assay for high-throughput studies of correlates of protection and vaccine response.

microbiology↗

bioRxiv2021

Temporal dynamics of SARS-CoV-2 mutation accumulation within and across infected hosts

Analysis of SARS-CoV-2 genetic diversity within infected hosts can provide insight into the generation and spread of new viral variants and may enable high resolution inference of transmission chains. However, little is known about temporal aspects of SARS-CoV-2 intrahost diversity and the extent to which shared diversity reflects convergent evolution as opposed to transmission linkage. Here we use high depth of coverage sequencing to identify within-host genetic variants in 325 specimens from hospitalized COVID-19 patients and infected employees at a single medical center. We validated our variant calling by sequencing defined RNA mixtures and identified a viral load threshold that minimizes false positives. By leveraging clinical metadata, we found that intrahost diversity is low and does not vary by time from symptom onset. This suggests that variants will only rarely rise to appreciable frequency prior to transmission. Although there was generally little shared variation across the sequenced cohort, we identified intrahost variants shared across individuals who were unlikely to be related by transmission. These variants did not precede a rise in frequency in global consensus genomes, suggesting that intrahost variants may have limited utility for predicting future lineages. These results provide important context for sequence-based inference in SARS-CoV-2 evolution and epidemiology.

microbiology↗

bioRxiv2020

The early evolution of oral poliovirus vaccine is shaped by strong positive selection and tight transmission bottlenecks

The evolution of circulating vaccine-derived polioviruses (cVDPV) from components of the live-attenuated oral poliovirus vaccine (OPV) presents a major challenge to global polio eradication. This process has largely been characterized by consensus sequencing of isolates collected from routine surveillance, and little is known about the early evolution of OPV within vaccinated hosts. These early events are critical steps in the progression of OPV to cVDPV. Here, we use whole genome, high depth of coverage sequencing to define the evolutionary trajectories of monovalent type 2 OPV in a cluster-randomized trial of polio vaccines in Matlab, Bangladesh. By sequencing 416 longitudinal samples from 219 mOPV2 recipients and 81 samples from 52 household contacts, we were able to examine the extent of convergent evolution in vaccine recipients and track the amount of viral diversity transmitted to new hosts. Using time-series data from a synchronized point of vaccine administration, we identify strong positive selection of reversion mutations at three known attenuating sites within two months post-vaccination. Beyond these three recognized "gate-keeper" mutations, we identify 19 mutations that exhibit significant parallelism across vaccine recipients, providing evidence for early positive selection not previously detected by phylogenetic inference. An analysis of shared genetic variants in samples from vaccinated individuals and their household contacts suggests a tight effective bottleneck during transmission. The absence of positively selected variants among household contacts across the cohort suggests that this tight bottleneck limits the transmission of these early adaptive mutations. Together, our results highlight the distinct evolutionary dynamics of live attenuated virus vaccines and have important implications for the success of novel OPV2 and other next generation approaches. SignificanceThe emergence of circulating vaccine-derived polioviruses (cVDPV) through evolution of the oral polio vaccine (OPV) poses a significant obstacle to global eradication. Understanding the genetic changes in OPV that occur as it evolves and transmits in populations is important for preventing future cVDPV outbreaks. Little is known about the early events in VDPV evolution and the selective forces that drive them. We used high depth-of-coverage genome sequencing to assess the within-host evolutionary dynamics of monovalent type 2 OPV in a vaccine trial in Matlab, Bangladesh. We leverage longitudinal sampling from vaccine recipients and household contacts to identify mutations that arise in parallel across individuals and estimate the size of the transmission bottleneck. We find evidence for strong positive selection on key sites in the capsid and the 5 noncoding region, many of which have not been previously identified. Our results also suggest that narrow transmission bottlenecks can constrain the spread of mutations selected within individuals. These results provide important insights into how OPV variants spread in populations and are highly relevant for ongoing poliovirus surveillance and the design of improved polio vaccines.

microbiology↗

bioRxiv2019

Influenza B viruses exhibit lower within-host diversity than influenza A viruses in human hosts

Influenza B virus undergoes seasonal antigenic drift more slowly than influenza A, but the reasons for this difference are unclear. While the evolutionary dynamics of influenza viruses play out globally, they are fundamentally driven by mutation, reassortment, drift, and selection within individual hosts. These processes have recently been described for influenza A virus, but little is known about the evolutionary dynamics of influenza B virus (IBV) at the level of individual infections and transmission events. Here we define the within-host evolutionary dynamics of influenza B virus by sequencing virus populations from naturally-infected individuals enrolled in a prospective, community-based cohort over 8176 person-seasons of observation. Through analysis of high depth-of-coverage sequencing data from samples from 91 individuals with influenza B, we find that influenza B virus accumulates lower genetic diversity than previously observed for influenza A virus during acute infections. Consistent with studies of influenza A viruses, the within-host evolution of influenza B viruses is characterized by purifying selection and the general absence of widespread positive selection of within-host variants. Analysis of shared genetic diversity across 15 sequence-validated transmission pairs suggests that IBV experiences a tight transmission bottleneck similar to that of influenza A virus. These patterns of local-scale evolution are consistent with influenza B virus slower global evolutionary rate.\n\nImportanceThe evolution of influenza virus is a significant public health problem and necessitates the annual evaluation of influenza vaccine formulation to keep pace with viral escape from herd immunity. Influenza B virus is a serious health concern for children, in particular, yet remains understudied compared to influenza A virus. Influenza B virus evolves more slowly than influenza A, but the factors underlying this are not completely understood. We studied how the within-host diversity of influenza B virus relates to its global evolution by sequencing viruses from a community-based cohort. We found that influenza B virus populations have lower within-host genetic diversity than influenza A virus and experience a tight genetic bottleneck during transmission. Our work provides insights into the varying dynamics of influenza viruses in human infection.

microbiology↗

bioRxiv2018

Antibodies against egg- and cell-grown influenza A(H3N2) viruses in adults hospitalized during the 2017-2018 season

BackgroundThe 2017-2018 US influenza season was severe with low vaccine effectiveness. Circulating A(H3N2) viruses from multiple genetic groups were antigenically similar to cell-grown vaccine strains. However, most influenza vaccines are egg-propagated.\n\nMethodsSerum was collected shortly after illness onset from 15 PCR confirmed A(H3N2) infected cases and 15 uninfected (controls) hospitalized adults enrolled in an influenza vaccine effectiveness study.\n\nGeometric mean titers against egg- and cell-grown A/Hong Kong/4801/2014 A(H3N2) vaccine strains and representative circulating viruses (including A/Washington/16/2017) were determined by microneutralization (MN) assays. Independent effects of strain-specific titers on susceptibility were estimated by logistic regression.\n\nResultsMN titers against egg-A/Hong Kong were significantly higher among those who were vaccinated (MN GMT: 173 vs 41; P = 0.01). However, antibody titers to cell-grown viruses were much lower in all individuals (P>0.05) regardless of vaccination. In unadjusted models, a 2-fold increase in MN titers against egg-A/Hong Kong was not significantly protective against infection (29% reduction; p=0.09), but a similar increase in cell-A/Washington titer (3C.2a2) was protective (60% reduction; p=0.02). A similar increase in egg-A/Hong Kong titer was not significantly associated with odds of infection when adjusting for MN titers against A/Washington (15% reduction; P=0.61). A 54% reduction of odds of infection was observed with a 2-fold increase in A/Washington (not significant; P=0.07), adjusted for egg-A/Hong Kong titer.\n\nConclusionAlthough individuals vaccinated in 2017-2018 had high antibody titers against the egg-adapted vaccine strain, antibody responses to cell-grown circulating viruses may not be sufficient to provide protection, likely due to egg-adaptation in the vaccine.

epidemiology↗