Altered RSV Epidemiology and Genetic Diversity Following the COVID-19 Pandemic

Respiratory Syncytial Virus (RSV) is a leading cause of acute respiratory tract infection, with greatest impact on infants, immunocompromised individuals, and older adults. RSV prevalence decreased substantially following the implementation of non-pharmaceutical interventions to mitigate the COVID-19 pandemic but later rebounded with initially abnormal seasonality. The biological and epidemiological factors underlying this altered behavior remain poorly defined. In this retrospective cohort study, we examined RSV epidemiology, clinical severity, and genetic diversity in the years surrounding the COVID-19 pandemic. We found that changes in RSV diagnostic platforms drove increased detections in outpatient settings after 2020 and that hospitalized adults with RSV-A were at higher risk of needing intensive care than those with RSV-B. While the population structure of RSV-A remained unchanged, the population structure of RSV-B shifted in geographically distinct clusters. Mutations in the antigenic regions of the fusion protein suggest convergent evolution with potential implications for vaccine and therapeutic development.


INTRODUCTION
The implementation of non-pharmaceutical interventions (NPIs) to limit the spread of COVID-19, such as masking, school closures, and social distancing, had dramatic impacts on the prevalence of other respiratory pathogens, including in uenza viruses and respiratory syncytial virus (RSV). 1 According to the Centers for Disease Control and Prevention (CDC), percent positivity in RSV diagnostic testing failed to reach even 1% during the 2020-2021 season, compared to an average of 12-16% peak positivity in years prior. 2 However, the easing of NPI enforcement in the United States (US) in the fall of 2021 was accompanied by an early and broadened RSV season in 2021-2022 3 followed by a particularly severe season in 2022-2023.The unanticipated spike in cases and hospitalizations overwhelmed many pediatric hospital systems, highlighting the need for improved RSV-speci c therapeutic options and preparedness. 4V is one of the leading causes of acute respiratory tract infections worldwide, with particularly high disease impact on pediatric populations, older adults, and immunocompromised individuals. 5Most children are initially infected with RSV before the age of 2, but rapidly waning immunity allows for regular reinfection throughout adulthood. 6,7While most often resulting in mild respiratory illness in adults, several risk factors for more severe disease have been identi ed, including older age, immune dysfunction, and comorbid conditions such as chronic obstructive pulmonary disease and congestive heart failure. 8Of the two major circulating subtypes of RSV, RSV-A has been more often associated with a higher risk of severe disease than RSV-B, though these studies have been largely limited to pediatric populations. 9, 10 Nonetheless, a lack of widespread testing for RSV, especially in adults, suggests that the true incidence and burden of this virus remains underestimated. 11ntil recently, there have been few speci c preventative measures to prevent RSV transmission, with the monoclonal antibody palivizumab available for use in selected pediatric populations.However, in 2023 the Food and Drug Administration (FDA) approved two rst-in-class RSV vaccines, one that is adjuvanted (Arexvy) and one bivalent, unadjuvanted vaccine (Abrysvo), which reduced the risk of RSV-lower respiratory tract infection (LRTI) in older adults by 94.1% 12 and in infants from vaccinated pregnant individuals by 81.8% 13 after the rst season of observation, respectively.Likewise, the monoclonal antibody nirsevimab received FDA approval in July 2023 with a reduction of medically attended lower respiratory tract infection from 5.0-1.2% for children under 2 years of age. 14While trials of these and other vaccines and monoclonal antibodies, most children and adults between 2 years of age and 60 have no current options approved to prevent RSV.
The use of these recently approved agents and the many ongoing clinical trials have increased the call for expanded genomic surveillance of RSV populations.While whole genome sequencing pipelines to monitor SARS-CoV-2 evolution proved to be critical to informing monoclonal antibody usage during the COVID-19 pandemic, RSV sequencing has remained much more limited. 15While some diagnostic platforms distinguish between RSV subtypes, 16 genotyping efforts are largely reliant on external surveillance/research teams and are typically limited to the sequencing of the attachment glycoprotein (G), which has the most variable open reading frame (ORF).This strategy precludes the detection of mutations in other parts of the genome, including those that arise in the viral fusion (F) protein, the main target of most vaccine and therapeutic strategies.However, efforts to standardize and expand global surveillance are currently underway by the World Health Organization Global RSV Surveillance Project (WHO-GRSVS) 17 .Previous lack of prioritization in RSV genomic surveillance has had direct consequences on effective therapeutic design.For example, monoclonal antibody suptavumab failed to meet its endpoints in phase 3 clinical trial due to the unrecognized high frequency of naturally occurring escape mutations (F: S173L & F: L172Q) in the US at the time of study 18 .Current genomic surveillance efforts have identi ed increased genetic diversity in RSV following the COVID-19 pandemic, particularly in RSV-B, but it is unclear how these mutations might impact the e cacy of future therapeutics.Monitoring emerging resistance to nirsevimab has thus far been limited to computational modeling 19 and in vitro assays 20 , with analyzed mutations limited to those that were prevalent before 2020 thus far.
Here, we present a retrospective cohort study that examined RSV epidemiology, clinical severity, and genetic diversity in Chicago, Illinois, US, in the years surrounding the COVID-19 pandemic.Our objective in this study was three-fold: (1) To elucidate shifts in RSV epidemiological behavior during and after implementation of NPIs; (2) To test for associations between viral subtype and clinical outcomes in adult populations; and (3) To assess viral genetic diversity over time and its likely impact on upcoming treatment and vaccine e cacy.

Alterations in RSV Epidemiology in the United States following the COVID-19 Pandemic
To assess how RSV epidemiological trends changed in the US before and after the COVID-19 pandemic, we calculated 3-week rolling averages of diagnostic tests administered, RSV detections, and percent positivity from the National Respiratory and Enteric Virus Surveillance System (NREVSS) from July 2010 to April 2023 for all participating US laboratories (Fig. 1A).Diagnostic testing for RSV through the 2019-2020 season followed a regular seasonal pattern with the total number of tests administered peaking near 40,000 tests per three-week rolling average in later years.Detections and percent positivity rate followed similar seasonal trends, with most years peaking around 15 to 20% positivity during December or January.Testing increased as expected early in the 2020-2021 season, but detections and percent positivity remained near zero.Hereafter, the number of tests administered remained high year-round with additional seasonal testing during the winter months pushing peak testing to over 140,000 tests per three weeks in early 2023.This increased testing helped capture the broad off-season peak during the summer and fall of 2021, followed by an early and dramatic peak in detections the following season in November of 2022.Despite the altered seasonality and testing patterns, percent positivity remained in line with historic trends, peaking near 15 to 20%.Likewise, regional trends where the single-center study took place recapitulated the aberrant RSV behavior observed in national data (Supplementary Fig. 1).
We then leveraged hospitalization data from the RSV-Hospitalization Surveillance Network (RSV-NET) 21 to determine whether increased viral detections coincided with increased hospital admission rates.
Hospitalization data for patients with RSV were obtained from 12 states (California, Colorado, Connecticut, Georgia, Maryland, Michigan, Minnesota, New Mexico, New York, Oregon, Tennessee, and Utah) participating in the Emerging Infections Program 22 or the In uenza Hospitalization Surveillance Program between July 2017 and April 2023. 23Irrespective of age, admission rates were below 1 case per 100,000 in the 2020-2021 season, reemphasizing the lack of RSV circulation at that time (Fig. 1B).
Infants 0 to 4 years of age displayed a broadened and off-season peak in hospitalizations in 2021 followed by a heightened peak in October 2022, approximately 3-4 times higher than in prior years.These trends are less apparent in other age groups, which showed a more typical seasonality, particularly for older adults aged 65 and older.Similarly, while hospitalizations increased for other age groups in the 2022-2023 season, the magnitude of the increase was not proportional to what was seen for infants.Taken together, these data con rm alterations in RSV epidemiological patterns following the COVID-19 pandemic but suggest these trends were largely driven by infants as opposed to older adults.

Shifts in RSV Testing Platforms Resulted in an Incidental Increase in Outpatient Detections
National RSV-associated hospitalizations did not increase proportionately with cases in the 2022-2023 season, suggesting an increase in less severe case detections.We hypothesize that the less severe case detection trend will likewise be observed within our single-center study.To test this hypothesis, we performed a single-center retrospective cohort study of patients who tested positive for RSV at a Northwestern Medicine (NM) a liated hospital or clinic between August 1st, 2009, and March 1st, 2023.clinical data collated through NM's centralized repository system identi ed 8,508 unique patient encounters over this timeframe, including 3,416 adult and 5,092 pediatric patients (Supplementary Fig. 2).Most NM patient encounters were geographically concentrated in the greater Chicago area (Cook County and the surrounding suburbs) (Fig. 2A).Within Cook County, encounters were aggregated in the north and far north regions of the city with lower representation in the southwest and far southwest regions.To verify that RSV behavior in Chicago was representative of broader regional and national trends, we obtained 3-week rolling averages of diagnostic tests administered, RSV detections, and percent positivity from the Chicago Department of Public Health (CDPH) from September 29th, 2019, to January 22nd, 2023 (Fig. 2B).As expected, these data con rmed aberrant seasonality and testing patterns citywide following the COVID-19 pandemic with increased detections in the 2022-2023 season.
We assessed patient outcomes (i.e., outpatient, hospitalized, ICU admission, and death) and diagnostic testing platform information for the 3,416 adult patient encounters from our clinical dataset.Adult patient encounters increased gradually from the 2009-2010 season through the 2019-2020 season (Fig. 2C).
These encounters were predominantly inpatients (> 75%) until the 2015-2016 season when encounters became more balanced at around 50% for both outpatient and inpatient encounters (Fig. 2D).After only a single observed encounter in the 2020-2021 season during the COVID-19 pandemic, cases rebounded to near-normal levels in 2021-2022 before doubling in the 2022-2023 season.These latter seasons had increased outpatient encounters, with over 75% outpatient proportion during the 2022-2023 surge.
Notably, outcomes among inpatients varied only slightly during the study period, with a consistent 25-40% of inpatients requiring intensive care or dying each year (Fig. 2D, bottom panel).These data suggest that most of the infected adult patients were detected in the outpatient setting.
Historically, many adult RSV cases in the NM system have been detected using either a broad, multiplexed, PCR-based respiratory panel or a similar PCR-based assay for the detection of RSV and in uenza viruses (Fig. 2E).After the start of the COVID-19 pandemic, however, platforms became predominated by multiplex tests for a range of viruses, including SARS-CoV-2 and RSV, or a rapid triplex PCR assay for SARS-CoV-2, RSV, and in uenza viruses; the former was generally utilized for hospitalized and high-risk adults while the latter was generally used in the outpatient setting. 24The adoption of these platforms, particularly the rapid triplex assay, closely mirrors the increase in outpatient detections (Supplementary Fig. 3).Altogether, our patient and diagnostic data suggest that the transition to multiplexed platforms for the detection of SARS-CoV-2 led to an incidental increase in RSV detections, particularly among patients not requiring inpatient care.As such, shifts in diagnostic testing platforms must be considered as an additional factor driving case counts in addition to virological and immunological behavior.

RSV Subtype A Infection is Associated with Increased Risk of ICU Admission in Hospitalized Adults
When controlling for clinical and demographic confounders, RSV-A has historically been associated with increased severity and worse patient outcomes compared to RSV-B. 5 , 24 , 9 , 10 , However, most of these studies were limited to pediatric populations before the COVID-19 pandemic.To determine whether subtype is associated with clinical outcomes in our adult cohort, we subsampled our dataset to only include adult encounters with RSV subtyping information.Of the 3,3416 adult encounters, only 886 were tested on a diagnostic platform that could discern between subtypes, with no typing data available after 2017.To recover subtyping information, we collected 551 residual diagnostic nasopharyngeal swabs from RSV-positive patients between December 18, 2017, and January 1, 2023 (Supplementary Fig. 2).RNA was extracted from each specimen and the RSV subtype was determined using an in-house quantitative reverse transcription PCR (qRT-PCR) assay.Merging these data with our clinical dataset, we recovered typing information for 1,437 patients, including outpatient (n = 495) and inpatient (n = 942) adults.
RSV-A and RSV-B were found to co-circulate each season, with year-to-year shifts in predominance (Fig. 3A).Following the COVID-19 pandemic, subtype predominance uctuated more dramatically with the 2021-2022 season having a 91.9% RSV-B predominance, and the 2022-2023 season having an 80.8% RSV-A predominance.Inpatient adults with typing information [RSV-A (n = 415) or RSV-B (n = 527)] were highly representative of our overall inpatient cohort (n = 1,565) (Table 1).The age distribution was similar among inpatients with RSV-A or RSV-B, skewing towards 60 years and older (Fig. 3B).Comorbidities were likewise similarly distributed among inpatients with different RSV subtypes, though patients with RSV-B reported slightly lower frequencies overall (Fig. 3C).We applied a Kaplan Meier survival model to observe the probability of discharge among hospitalized patients and found no statistical difference (p-value = 0.620) between subtypes with a 50% discharge probability within 5 days from hospital admission for both A and B infections (Fig. 3D).
To determine whether an association exists between subtype and patient outcome, we used multivariable logistic regression to model subtype infection (i.e., RSV-A versus RSV-B) while controlling for race, ethnicity, sex, age at admission, the month of patient admission, comorbidity sum, hospitalization length of stay, intensive care unit (ICU) admission, and RSV-associated death.This model indicated that ICU admission [adjusted Odds Ratio (aOR) 0.68, 95% con dence interval (CI) 0.49-0.94]and comorbidity sum (aOR 0.92, 95% CI 0.85-0.99)were signi cantly associated with RSV-A infection (the reference subtype) as compared to RSV-B (Fig. 3E, Supplementary Fig. 4).Likewise, we observed an overall increased proportion of ICU admissions in our RSV-A infected cohort (32.8%) in comparison to the RSV-B cohort (27.1%) (Fig. 3F).These same trends were observed when excluding inpatient encounters after 2020, suggesting that these associations were not confounded by COVID-19 pandemic-associated factors (Supplementary Fig. 4).Altogether, our modeling data are indicative of more severe outcomes in hospitalized adults with RSV-A compared to RSV-B and corroborate previous reports in pediatric populations.

Shift in RSV-B Population Structure Following the COVID-19 Pandemic
The RSV resurgence superseding the low incidence in the 2020-2021 season has been reported to be from previously existing RSV-A lineages that now predominate in the US. 25 To determine if the genetic diversity of RSV in Chicago changed throughout the COVID-19 pandemic, we expanded the use of the 551 residual diagnostic nasopharyngeal swabs used for typing for viral whole genome sequencing (WGS).Speci cally, viral genomic material from the collected isolates was extracted, reverse transcribed, ampli ed using a tiled fragment approach spanning the whole genome, and sequenced using the Illumina platform.In total, we obtained 218 whole genome sequences (110 subtype A and 108 subtype B) with a minimum of 90% coverage across the genome.Sequences were given a clade designation using both Nextstrain and G clades nomenclature 26 via Nextclade and were deposited in the National Center for Biotechnology Information (NCBI) GenBank (Supplementary Table 1).Finally, maximum-likelihood (ML) phylogenetic trees were generated for all RSV-A and RSV-B sequences.
Between 2017 and 2023, the RSV-A and RSV-B populations in Chicago were exclusively represented by existing clades GA2.3.5 and GB5.0.5a, respectively (Fig. 4A & B).The RSV-A population structure remained largely unchanged after 2020, showing evidence of multiple lineage expansions (Fig. 4A), consistent with prior reports from Arizona 27 , Washington State 28 , and Massachusetts 25 .Conversely, RSV-B exhibited monophyletic clustering with strong support (100% using Shimodaira-Hasegawa approximate likelihood-ratio test [aLRT] and 100% with bootstrap) after 2020 (Fig. 4B), suggestive of population bottlenecking followed by either genetic drift or positive selection.The de ning mutations for this monophyletic cluster were mainly in glycoprotein (I252T, I268T, S275P, P214S, P221L, T310I, and S100G) and the fusion protein (S190N, S211N, S389P).To better determine whether this cluster is speci c to the Chicagoland area, we pulled 340 publicly available RSV-A and 113 publicly available RSV-B whole genome sequences from the US and repeated our phylogenetic analysis.Again, RSV-A sequences after 2020 were broadly representative of GA.2.3.5 diversity in the US before that year (Fig. 4C).However, RSV-B sequences after 2020 formed a monophyletic cluster composed of our sequences alongside those from Washington and Massachusetts (Fig. 4D).Taken together, these results suggest a potential founder effect in the RSV-B population during the COVID-19 pandemic, which resulted in a more homogenous population structure in the US post-pandemic.

Convergent Evolution of Mutations in the RSV-B Fusion Protein Antigenic Sites
To determine whether this monophyletic cluster was solely identi ed in the US, we generated a temporal ML phylogenetic analysis using all publicly available RSV-B whole genome sequences collected between 1957 and 2023 (n = 723) (Fig. 5A).This analysis revealed genetically and geographically distinct clusters of RSV-B GB5.0.5a isolates arising in the 2021-2022 season (i.e., unique clusters were observed in Australia, France, Japan, the US, etc.).Similar temporal ML analyses of unique and complete RSV-B G (n = 983) and F (n = 2,067) sequences showed the monophyletic divergent lineage in RSV-B after 2020 (Supplementary Fig. 5).
Subsequently, we used a Bayesian approach to characterize the global population dynamics of RSV-B and compared it with RSV-A (Supplementary Fig. 6) to identify the possible origin and the extent of the expansion of the detected circulating monophyletic lineage in the US.Overall, the population dynamics observed using this approach depicted a temporal evolution of the effective population size for both RSV-A and RSV-B concordant with the epidemiological patterns previously described.More importantly, these analyses con rmed strong statistical support for a monophyletic origin of most of the currently circulating RSV-B viruses in the US with a most likely local origin [ancestral US location probability > 0.99].Using this approach, we also estimated a time to the most recent common ancestor (TMRCA) for this cluster to be June 9, 2018 [95% Highest Posterior Density (HPD) interval: November 23, 2017 -December 28, 2018] with the next MRCA for this cluster also being identi ed close to 2 NM sequences sampled between March and April 2018 that constituted a minority lineage at that time.This is indicative of possible selective processes involved in the population bottleneck observed due to the unlikeliness of random drift to a minority lineage instead of within the most prevalent lineages.Although most of the sequences that constituted this cluster were collected in the US, 4 sequences from Austria from NCBI collected during 2022 also clustered within this lineage, indicating ongoing expansion outside the US.On the other hand, RSV-A showed the appearance of multiple unrelated lineages after the period of the SARS-CoV-2 pandemic discarding any possible sampling bias leading to the identi cation of the dominant monophyletic US cluster in RSV-B.
To further characterize the mutations in the F ORF that de ne currently circulating isolates and test their possible selective advantage, we generated an ancestral sequence of the most recent common ancestor (MRCA).When comparing the MRCA sequence to all publicly available RSV-B sequences available after 2020 (n = 225), we observed an enrichment of mutations on the heptad-rich A domain (HRA), which is necessary for F protein conformational change and viral-host membrane fusion (Fig. 5B, left). 29When stratifying these data by country, three mutations in the HRA domain (K191R, I206M, and Q209R) were particularly enriched, predominating in 12 of the 15 countries with available sequencing data after 2020 (Fig. 5B, right).While these mutations were all in the majority before 2020, several minority mutations also arose in multiple countries.For example, S190N and S211N, also in the HRA domain, arose to predominance in the US, Austria, and Canada, despite being present in fewer than 2% of sequences before 2020.Not all mutations were localized to the HRA domain, with, for example, P312H and S389P in Domain I which arose in several countries.Notably, most of these mutations lie either in or are anking antigenic sites Ø, I, and V (Fig. 5C).
Given the position of several of these mutations in or near antigenic regions, we next sought to determine if any of these mutations were under positive selection.Each RSV-B ORF was analyzed independently for episodic diversifying selection using the Mixed Effects Model of Evolution (MEME) method using all available sequence data from 1957 to 2023.Most ORFs showed no evidence of positive selection over this time frame, including NS1, NS2, N, M, P, and SH.M2-1 (T188), M2-2 (S26, H50), and L (S69, S102, N500, I1989) each had positions showing evidence of episodic selection, but these mutations remained low frequency and none are currently circulating (Supplementary Fig. 7).However, both F (Fig. 5D, left) and G (Supplementary Fig. 7) showed high levels of episodic positive selection at positions 7 and 20, respectively.The T250I mutation in G is identi ed to undergo diversifying selection and is a de ning mutation in the monophyletic RSV-B cluster.However, no sites undergoing positive selection in F are fully represented in the cluster, with the F12L mutation only being represented in less than 5% of that cluster.
As expected, S173L, a known resistance mutation against Suptavumab in antigenic region V, is now xed in the RSV-B population.Expanding this analysis to include pervasive positive selection using the Fast Unconstrained Bayesian AppRoximation (FUBAR) method, two sites in the F signal peptide (I5 and F12) are additionally identi ed (Fig. 5D).If we constrain these analyses to clade GB5.0.5a in the last 5 years, only three mutations are identi ed to still be under episodic (D548) and pervasive (I5 and F12) positive selection (Fig. 5D, right).While the predominant mutations arising after 2020 are not found to be under statistically signi cant positive selection, several have high mean posterior difference values between nonsynonymous and synonymous mutations.
Lastly, we quanti ed the mutational frequency within the binding sites for the monoclonal antibody therapeutics nirsevimab (Fig. 5E), suptavumab, clesrovimab, and palivizumab (Supplementary Fig. 8) before and after 2020 (Fig. 5E).As expected, current RSV-B isolates contain a high frequency of mutations across the Suptavumab binding site both before and after 2020, with treatment-resistant mutations (i.e., S173L) observed in all isolates.However, several mutations in the Nirsevimab binding site, including I206M, Q209R, and S211N, have substantially increased in frequency since 2020 with minor increases observed at other positions (i.e., K68N).Overall, mutations in the Clesrovimab and Palivizumab binding sites remain low.

DISCUSSION
In this single-center study, we leveraged a 14-year clinical dataset and a matched biobank of residual diagnostic specimens to assess changes in RSV epidemiology, clinical severity, and genetic diversity in the years surrounding the COVID-19 pandemic.We demonstrated that changes in diagnostic testing, particularly multiplexing of SARS-CoV-2 and RSV diagnostics, were associated with increased outpatient detections, a factor that might contribute to the increased case counts and altered seasonality observed in recent seasons.We further found that RSV-A was associated with a higher risk of ICU admission among hospitalized adults, consistent with what has been previously reported in pediatric populations.Finally, we observed a shift in the RSV-B population structure to geographically distinct monophyletic clusters in 2021-2022, likely a result of population bottlenecking during the widespread use of NPIs to mitigate the spread of COVID-19.Despite the independent emergence of differing RSV-B clusters, the convergent evolution of several mutations in the antigenic regions of F suggests potential alterations to therapeutic e cacy pro les that should be considered as new treatment options progress through clinical trials.
Data from within our hospital system re ected local and national trends in RSV surveillance with a surge of detections in the 2022-2023 season.Reduced pathogen exposure on account of the widespread use of NPIs during the COVID-19 pandemic is theorized to have produced an "immunity debt" 30 with a larger susceptible population resulting in larger surges of infection.Increased transmission and skewed age distributions in the 2021-2023 RSV seasons support this association between increased incidence and waned population immunity in infants.Alternatively, adults likely retained immunity from prior RSV exposures despite a decreased probability of exposure during the COVID-19 pandemic.Consequently, data from RSV-NET showed a coordinated increase in pediatric (0-4 years) hospitalizations and detections, adult hospitalizations did not increase to the same extent 31 and overall percent positivity remained comparable to prior seasons.According to our data, this can be partially explained by an increase in outpatient detections, which is strongly associated with a shift in the diagnostic testing platforms being used in our hospital system.The increased use of rapid, pan-viral molecular diagnostics platforms may increase the number of incidental detections that must be considered when interpreting epidemiological trends.This observed increase in molecular diagnostic usage could likewise be attributed to shifts in practice and societal norms regarding testing.Namely, changes in institutional/medical practices to order diagnostics, widespread availability of triplex testing, and behavioral shifts to preferentially seek COVID-19 testing during the pandemic are examples of societal factors that potentially contribute to increased RSV detection in recent years.
Prior studies in pediatric populations have shown that RSV-A infections have more severe clinical manifestations and worse patient outcomes compared to RSV-B. 10,32While fewer studies have been done in adult populations, they likewise have indicated higher RSV-A virulence as measured by clinical severity scores. 33Following the resurgence of RSV post-2020, several multi-center studies observed increased infant hospitalization rates with unchanged clinical severity trends 34,35 , though no such contemporary studies have yet been reported in adults.Our data support the theory that worse clinical outcomes among hospitalized adults occur with RSV-A infection compared to RSV-B.While our dataset post-2020 is too sparse for a stand-alone model, our regression analyses illustrate that the inclusion or exclusion of adult inpatients sampled after 2020 does not contribute to associated risks of outcome and subtype infection.This suggests there is no change in relative severity between RSV-A and RSV-B following the COVID-19 pandemic, but larger, multi-center studies with more patients are still required.
Our phylogenetic analyses revealed a shift in RSV-B population structure post-2020, with multiple monophyletic clusters arising in distinct geographic locations in the 2021-2022 season.The isolates in Chicago appear closely related to those previously reported in Washington state 28 and Massachusetts 25 , suggestive of a US-speci c cluster.This pattern of divergence is most consistent with a genetic bottleneck event, likely resulting from the NPIs introduced in 2020 to stop the spread of SARS-CoV-2.However, RSV-A global sequences exhibited polyphyletic clustering in 2022, suggesting that the transmission patterns between these two subtypes differ despite their co-circulation in the 2019-2020 season.This is further supported by the lack of monophyletic clustering of RSV-A even in regions where this subtype was dominant after viral reemergence in 2021. 36Alternately, it is possible that the bottlenecking exacerbated an already ongoing transition in the RSV-B population.Viral populations with a mildly bene cial tness advantage that are expanding in a low-competing environment may become rapidly predominant.The seemingly independent selection of similar mutations in F domains, several of which existed to some degree before the pandemic, suggest some convergent evolution at these sites, though the impact of these changes on viral entry, transmission, and immune evasion remains to be elucidated.
The RSV sequences reported here add to a growing body of publicly available RSV genomic data, which will improve our ability to monitor for shifts in viral evolution and emerging resistance mutations.This is particularly critical as a slate of new interventions gain FDA approval or begin to go through clinical trials.
Prior literature on nirsevimab neutralization escape mutations showed low prevalence K68N (0.32%) and S211N (1.14%) and a global increase in Q209R (68.25%) and I206M (68.9%) between 2015 and 2021. 37,38Although neutralization potency is reduced marginally by I206M (5.0-fold), Q209R (0.5-fold), and S211N (1.2-fold), the K68N mutation exhibits 29.9-fold reduced susceptibility. 37The prevalence of each of these mutations has increased notably, especially S211N, which is now observed in 82% of sequences.Perhaps even more concerning, circulating RSV-B K68N mutations after May 2020 increased from < 1% prevalence to 1.8% in Australia and the United States, suggestive of a growing population of strongly resistant isolates.Fortunately, other monoclonal antibody epitopes for either FDA-approved (palivizumab), investigational, or unapproved therapeutics (clesrovimab and suptavumab) do not exhibit a large shift in variation.However, our data show the emergence of several other mutations in the HRD A domain anking antigenic sites Ø and V, which may cause issues for other antibodies targeting these sites.Altogether, the shift in RSV-B population structure and the emergence of nirsevimab-escape mutants strongly argue for further surveillance, particularly in regions committed to rolling out monoclonal antibody treatments in the 2023 RSV season.
Several limitations exist within our study design and analysis despite its contemporary insights into RSV epidemiology.For example, single-center cohort studies run the risk of not being representative of results in other medical institutions because of sampling methods, inclusion criteria, and model design.Despite this, the epidemiological data in Chicago appears indicative of US trends.Similarly, the incorporated clinical metadata was ltered to a smaller subset to focus on inpatient adults for our models.
Consequently, removing outpatients underpowers our modeling of the association with adult clinical severity and RSV subtype post-2020.Again, this observed shift in hospitalization-to-outpatient ratios shows the impact of diagnostic shifts on association studies.Lastly, the scarcity of publicly available genomes results in inherent under-sampling bias, impacting the elucidation of phylogenetic and dynamic patterns.
Overall, this study characterized the molecular epidemiology of RSV in the Chicagoland area spanning the COVID-19 pandemic.Speci cally, we sought to: (1) elucidate shifts in RSV epidemiological behavior during and after implementation of NPIs; (2) test for associations between viral subtype and clinical outcomes in adult populations; and (3) assess viral genetic diversity over time and its likely impact on upcoming treatment and vaccine e cacy.This multifaceted work demonstrates 1) the strengths that lie in utilizing epidemiology to inform clinical care, public health interventions, and molecular virology, and 2) the existing gaps in molecular surveillance in under-sample regions that impede our understanding of RSV evolution.Our ndings also highlight the merit of currently developing virological techniques for using primer and probe set from the World Health Organization Strategy for Global RSV Surveillance Project based on the In uenza Platform. 17Primers for RSV polymerase [L] (Forward: 5'-AATACAGCCAAATCTAACCAACTTTACA-3' and Reverse: 5'-GCCAAGGAAGCATGCAATAAA-3') used to target both RSV-A and RSV-B, whereas probes (RSV-A: 5'-TGCTATTGTGCACTAAAG-3' & RSV-B: 5'-CACTATTCCTTACTAAAGATGTC-3') distinguish between genotype.
cDNA Synthesis and Viral Genome Ampli cation cDNA synthesis was performed using SuperScript IV First-Strand Synthesis Kit using RSV-speci c primers. 40Due to amplicon dropout in the 2022-2023 RSV season, a Twist Pan-viral panel (Twist Library Preparation EF Kit 2.0 and Twist Comprehensive Viral Research Panel) for the enrichment of RSV was used for subsequent degenerate design (Forward: 5'-TATAGGCATGCACCYCCYTAT-3' and Reverse: 5'-ACGAGAAAAAAAGTGTYAAAAACT-3' ) per manufacturer's instruction.Direct ampli cation of RSV genome cDNA was performed in separate PCR reactions to generate ~ 4000 base pair fragments that span the genome.PCR ampli cation of the generated cDNA was performed using the Phusion Hot Start Flex system from New England Biolabs.Genomic ampli cation was con rmed via agarose gel electrophoresis, followed by reaction pooling and reaction cleanup using MAGwise Paramagnetic Beads.
After veri cation of genome ampli cation, the four amplicon sets were pooled before sequencing library preparation.

Sequencing Library Preparation and Whole Genome Sequencing
Two separate approaches to library preparation for sequencing on the Illumina MiSeq platform were used during this study, speci cally seqWell's plexWell and expressPlex library preparation.For the seqwell protocol, up to 384 ng of DNA was used for the total Viral genome sequencing reads were utilized to generate consensus sequences using the HAplotype and PHylodynamics pipeline (HAPHPIPE) for viral assembly, population genetics, and phylodynamics (https://github.com/gwcbi/haphpipe). 41 This pipeline consists of the following: (1) trimming sequencing reads to remove both adapters and low-quality sequences using Trimmomatic v1.0] and (2) three-step assembly re nement.The generated consensus reads were then annotated using Viral Genome ORF Reader (VIGOR). 42ylogenetic Analyses Generated consensus sequences and open reading frames were aligned using MAFFT v7.490, followed by inspection and removal of low coverage sequences using Seaview v. 5.0.4 43 46 , assuming an asymmetric transition model with separated rate parameters for each possible transition.MCMC was run for over 100 million steps with a burn-in of 20%.Parameters were sampled every 5,000 steps and trees were sampled every 10,000 steps.For both RSV-A and RSV-B phylogenies, the maximum clade credibility (MCC) trees were obtained from the tree posterior distribution using TreeAnnotator v2.7.6 after 20% burn-in.
Structural Modeling of the B5.0.5aF Protein A model of GB5.0.5aFusion pre-fusion conformation protein used to map contemporary mutations was built using SwissModel 47 using the PDB le of RSV strain B18537 Prefusion-stabilized glycoprotein F Variant DS-Cav1 (PDB: 6QOS.1.a)with as a template.ViralVar 48 was then utilized to visualize the mutation frequencies of isolates sampled after 2020.PyMOL v. 2.4.1 was used to visualize and annotate the following in RSV-B Fusion: (1) antigenic sites (2) predominant mutations appearing after 2020, and (3) mutations at binding sites of monoclonal antibodies nirsevimab, suptavumab, palivizumab, and clesrovimab.

Statistical Analyses
Tables Table 1 is available in the Supplementary Files section.
Figures   Figure 5

Figure 3 RSV
Figure 3 44d MEGA11 v. 0.1.IQ-Tree's v. 2.2.0 ModelFinder function44was used for the selection of nucleotide substitution model best-Both global (RSV-A = 860, RSV-B = 613) and USA(RSV-A = 340, RSV-B = 113) used in phylogenetic analysis from NCBI Genbank with sample dates up to May 2020 were scanned for high coverage > 90%.Due to the low number of publicly available genome sequences after May 2020, all genomes were obtained from the Nextstrain RSV-A and RSV-B databases.ORF analysis was done by extracting cds information using VIGOR v4.1.20200702.which was then used for subsequent phylogenetic and selection analysis.Bayesian time-scaled phylogenetic analysis after ltering outliers that did not t the root-to-tip correlation performed with TreeTime (1970 to 2022) and removing alignment positions with poor alignment quality using the default options in Gblocks implemented in Seaview v5.0.4.BEAST 2 v2.7.6 45 was used to estimate each subtypes' rate of evolution and identify the location and time of the most recent common ancestors (MRCA).BEAST 2 priors were introduced with BEAUTI v2.7.6 including a strict molecular clock model with a lognormal distribution of the evolutionary rate, with a prior for the mean of 4.42 × 10 − 4 substitutions per site per year for RSV-A and 4.22 × 10 − 4 for RSV-B, and a standard deviation of 0.2 for both after optimization with preliminary runs.We assumed a GTR substitution model with invariant sites, as the best-tted model obtained with ModelFinder, and a Coalescence Bayesian Skyline to model the population size changes through time.Markov chain Monte Carlo (MCMC) runs of at least 100 million states with sampling every 5,000 steps were computed. Th tted for each alignment and subsequent inference of Maximum Likelihood (ML) phylogeny.Assessment of tree topology was done ultrafast bootstrap (UFboot) and Shimodaira-Hasegawa approximate likelihood-ratio test (SH-aLRT) each with 1000 replications.Ancestral sequence reconstruction was performed using TreeTime v. 0.8.5.Likewise, TreeTime was used to estimate time-scaled phylogenies, estimate the evolutionary rate, and calculate the root-to-tip correlation by incorporating sequence sampling dates.