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At least 91 records · Page 5
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Improved detection of low-frequency within-host variants from deep sequencing: A case study with human papillomavirus

Abstract High-coverage sequencing allows the study of variants occurring at low frequencies within samples, but is susceptible to false-positives caused by sequencing error. Ion Torrent has a very low single nucleotide variant (SNV) error rate and has been employed for the majority of human papillomavirus (HPV) whole genome sequences. However, benchmarking of intrahost SNVs (iSNVs) has been challenging, partly due to limitations imposed by the HPV life cycle. We address this problem by deep sequencing three replicates for each of 31 samples of HPV type 18 (HPV18). Errors, defined as iSNVs observed in only one of three replicates, are dominated by C→T (G→A) changes, independently of trinucleotide context. True iSNVs, defined as those observed in all three replicates, instead show a more diverse SNV type distribution, with particularly elevated C→T rates in CCG context (CCG→CTG; CGG→CAG) and C→A rates in ACG context (ACG→AAG; CGT→CTT). Characterization of true iSNVs allowed us to develop two methods for detecting true variants: (1) VCFgenie, a dynamic binomial filtering tool which uses each variant’s allele count and coverage instead of fixed frequency cut-offs; and (2) a machine learning binary classifier which trains eXtreme Gradient Boosting models on variant features such as quality and trinucleotide context. Each approach outperforms fixed-cut-off filtering of iSNVs, and performance is enhanced when both are used together. Our results provide improved methods for identifying true iSNVs in within-host applications across sequencing platforms, specifically using HPV18 as a case study.

Virology↗
DOE OSTI2025

Identifying impacts of contact tracing on HIV epidemiological inference from phylogenetic data

Abstract Robust sampling methods are foundational to inferences using phylogenies. Yet the impact of using contact tracing, a type of non-uniform sampling used in public health applications such as infectious disease outbreak investigations, has not been investigated in the molecular epidemiology field. To understand how contact tracing influences a recovered phylogeny, we developed a new simulation tool called SEEPS (Sequence Evolution and Epidemiological Process Simulator) that allows for the simulation of contact tracing and the resulting transmission tree, pathogen phylogeny, and corresponding virus genetic sequences. Importantly, SEEPS takes within-host evolution into account when generating pathogen phylogenies and sequences from transmission histories. Using SEEPS, we demonstrate that contact tracing can significantly impact the structure of the resulting tree, as described by popular tree statistics. Contact tracing generates phylogenies that are less balanced than the underlying transmission process, less representative of the larger epidemiological process, and affects the internal/external branch length ratios that characterize specific epidemiological scenarios. We also examined real data from a 2007–2008 Swedish HIV-1 outbreak and the broader 1998–2010 European HIV-1 epidemic to highlight the differences in contact tracing and expected phylogenies. Aided by SEEPS, we show that the data collection of the Swedish outbreak was strongly influenced by contact tracing even after downsampling, while the broader European Union epidemic showed little evidence of universal contact tracing, agreeing with the known epidemiological information about sampling and spread. Overall, our results highlight the importance of including possible non-uniform sampling schemes when examining phylogenetic trees. For that, SEEPS serves as a useful tool to evaluate such impacts, thereby facilitating better phylogenetic inferences of the characteristics of a disease outbreak. SEEPS is available at https://github.com/MolEvolEpid/SEEPS.

Virology↗
DOE OSTI2025

Clarifying Terminology in Microbial Ecology: A Call for Precision in Scientific Communication

The rapid evolution of microbiology as a field of research has led to the introduction of new terminology and the adaptation of existing terms. However, inconsistencies in the use of these terms, including variations across different scientific disciplines, can lead to confusion and miscommunication within the scientific community. This article discusses the importance of precise terminology in microbiome research, highlighting examples where terms have been misused or redefined without clear justification. We also present a list of frequently used terms in microbial ecology along with their specific definitions. We argue that the misuse of terminology can hinder scientific progress by creating ambiguity and misunderstanding. To address this, we propose a set of guidelines for the consistent use of key terms and provide clear definitions for some of the most commonly misused or newly introduced terms in the field. The definitions provided herein will also function as a guide for young researchers new to the field of microbial ecology. Accurate and consistent use of terminology is crucial for effective communication and collaboration in microbiology research. By adhering to standardised definitions, researchers can ensure that their work is clearly communicated and contributes meaningfully to the progress of science.

definitions↗
DOE OSTI2024

Coinfection of Influenza A and B and Human OC43 Coronavirus in Normal Human Bronchial Epithelial Cells

Influenza viruses and seasonal coronaviruses are pathogens transmitted via an airborne route that can cause respiratory diseases in humans that have similar symptoms such as fever, cough, and pneumonia. These two viruses can infect similar human tissues, such as the respiratory tract and nasal, bronchial, and alveolar epithelial cells. Influenza virus and seasonal coronavirus coinfections are poorly understood. Here, we coinfected normal human bronchial epithelial (NHBE) cells with influenza A/California/04/09 (IAV) or B/Victoria/504/2000 (IBV) strains and the seasonal human beta-coronavirus OC43 and evaluated viral replication capacities. We also examined changes in the expression of various cytokines/chemokines by qPCR and Luminex assay. We observed that the replication of IAV and IBV was not affected by coinfection with OC43. However, coinfection reduced OC43 titers (~3-fold) compared with infection with OC43 alone. Select cytokine/chemokine expression was increased in coinfected cells compared with all single infections with greater differences seen between coinfected cells and cells infected with OC43 alone compared with IAV- or IBV-infected cells. In addition, IL-8 and IL-1RA showed the highest expression among a panel of 22 cytokines by Luminex. As the rate of influenza and seasonal coronavirus coinfection continue to increase, our findings may help set guidelines for the treatments of the individuals coinfected with both viruses.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Impact of Baloxavir Resistance-Associated Substitutions on Influenza Virus Growth and Drug Susceptibility

Baloxavir marboxil (baloxavir) is a recently FDA-approved influenza virus polymerase acidic (PA) endonuclease inhibitor. Several PA substitutions have been demonstrated to confer reduced susceptibility to baloxavir; however, their impacts on measurements of antiviral drug susceptibility and replication capacity when present as a fraction of the viral population have not been established. We generated recombinant A/California/04/09 (H1N1)-like viruses (IAV) with PA I38L, I38T, or E199D substitutions and B/Victoria/504/2000-like virus (IBV) with PA I38T. These substitutions reduced baloxavir susceptibility by 15.3-, 72.3-, 5.4-, and 54.5-fold, respectively, when tested in normal human bronchial epithelial (NHBE) cells. We then assessed the replication kinetics, polymerase activity, and baloxavir susceptibility of the wild-type:mutant (WT:MUT) virus mixtures in NHBE cells. The percentage of MUT relative to WT virus necessary to detect reduced baloxavir susceptibility in phenotypic assays ranged from 10% (IBV I38T) to 92% (IAV E199D). While I38T did not alter IAV replication kinetics or polymerase activity, IAV PA I38L and E199D MUTs and the IBV PA I38T MUT exhibited reduced replication levels and significantly altered polymerase activity. Differences in replication were detectable when the MUTs comprised ≥90%, ≥90%, or ≥75% of the population, respectively. Here, droplet digital PCR (ddPCR) and next-generation sequencing (NGS) analyses showed that WT viruses generally outcompeted the respective MUTs after multiple replication cycles and serial passaging in NHBE cells when initial mixtures contained ≥50% of the WT viruses; however, we also identified potential compensatory substitutions (IAV PA D394N and IBV PA E329G) that emerged and appeared to improve the replication capacity of baloxavir-resistant virus in cell culture.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Evolution of the North American Lineage H7 Avian Influenza Viruses in Association with H7 Virus’s Introduction to Poultry

Wild bird-origin H7 subtype avian influenza viruses are a constant threat to commercial poultry, both directly by the disease they cause and indirectly through trade restrictions that can be imposed when the virus is detected in poultry. It is important to understand the genetic basis of why the North American lineage H7 viruses have repeatedly crossed the species barrier from wild birds to poultry.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Molecular Modeling Predicts Novel Antibody Escape Mutations in the Respiratory Syncytial Virus Fusion Glycoprotein

Respiratory syncytial virus (RSV) causes severe disease in young infants, particularly those with heart or lung diseases or born prematurely. Because no vaccine is currently available, monoclonal antibodies are used to prevent severe RSV disease in high-risk infants. While it is known that RSV evolves to avoid recognition by antibodies, screening tools that can predict which changes to the virus may lead to antibody resistance are greatly needed.

molecular modeling↗
DOE OSTI2022

Deletion of the H108R Gene Reduces Virulence of the Pandemic Eurasia Strain of African Swine Fever Virus with Surviving Animals Being Protected against Virulent Challenge

African swine fever virus (ASFV) is the etiological agent of African swine fever (ASF), a devastating disease affecting domestic and wild swine and currently causing a global pandemic, severely affecting swine production. Here, we demonstrate that the deletion of the previously uncharacterized ASFV gene, H108R from the highly virulent ASFV-Georgia2007 (ASFV-G) genome strain, reduces virulence in domestic swine. ASFV-G-DH108R, a recombinant virus with the H108R gene deleted, was used to evaluate the involvement of the H108R gene for ASFV replication and virulence in swine. ASFV-G-DH108R showed a delayed replication in swine macrophage cultures. A group of five pigs, intramuscularly inoculated with 102 HAD50 of ASFV-G-DH108R, was observed over a 28-day period and compared with a similar group of animals inoculated with similar doses of the parental virulent virus. While all animals inoculated with ASFV-G developed an acute fatal disease, ASFV-G-DH108R inoculated animals, with the exception of one animal showing a protracted but fatal form of the disease, all survived the infection, remaining clinically healthy during the observational period. The surviving animals presented protracted viremias with lower virus titers compared with those of animals inoculated with the parental virus, and all of them developed a strong virus-specific antibody response. Importantly, all animals surviving ASFV-G-DAH108R infection were protected when challenged with the virulent parental strain, ASFV-G. This report constitutes the first evidence that the H108R gene is involved in ASFV virulence in swine and that the deletion of this gene may be used as a tool to increase the attenuation of currently experimental vaccines to improve their safety profiles.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Evaluation of the Deletion of MGF110-5L-6L on Swine Virulence from the Pandemic Strain of African Swine Fever Virus and Use as a DIVA Marker in Vaccine Candidate ASFV-G-ΔI177L

African swine fever virus (ASFV) is responsible for an ongoing pandemic that is affecting central Europe, Asia, and recently the Dominican Republic, the first report of the disease in the Western Hemisphere in over 40 years. ASFV is a large, complex virus with a double-stranded DNA (dsDNA) genome that carries more than 150 genes, most of which have not been studied. Here, we assessed the role of the MGF110-5L-6L gene during virus replication in cell cultures and experimental infection in swine. A recombinant virus with MGF110-5L-6L deleted (ASFV-G-ΔMGF110-5L-6L) was developed using the highly virulent ASFV Georgia (ASFV-G) isolate as a template. ASFV-G-DMGF110-5L-6L replicates in swine macrophage cultures as efficiently as the parental virus ASFV-G, indicating that the MGF110-5L-6L gene is nonessential for virus replication. Similarly, domestic pigs inoculated with ASFV-G-ΔMGF110-5L-6L presented with a clinical disease undistinguishable from that caused by the parental ASFV-G, confirming that the MGF110-5L-6L gene is not involved in producing disease in swine. Sera from animals inoculated with an efficacious vaccine candidate, ASFV-G-ΔMGF, strongly recognized the protein encoded by the MGF110-5L-6L gene as a potential target for the development of an antigenic marker differentiation of infected from vaccinated animals (DIVA) vaccine. To test this hypothesis, the MGF110-5L-6L gene was deleted from the highly efficacious ASFV vaccine candidate ASFV-G-ΔI177L, generating the recombinant ASFV-G-DI177L/DMGF110-5L-6L. Animals inoculated with ASFV-G-ΔI177L/DMGF110-5L-6L developed an ASFV-specific antibody response detected by enzyme-linked immunosorbent assay (ELISA). The sera strongly recognized ASFV p30 expressed in eukaryotic cells but did not recognize ASFV MGF110-5L6L protein, demonstrating that deletion of the MGF110-5L-6L gene can enable DIVA capabilities in preexisting vaccine candidates.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Evolution and Antigenic Advancement of N2 Neuraminidase of Swine Influenza A Viruses Circulating in the United States following Two Separate Introductions from Human Seasonal Viruses

Two separate introductions of human seasonal N2 neuraminidase genes were sustained in U.S. swine since 1998 (N2-98) and 2002 (N2-02). Herein, we characterized the antigenic evolution of the N2 of swine influenza A virus (IAV) across 2 decades following each introduction. The N2-98 and N2-02 expanded in genetic diversity, with two statistically supported monophyletic clades within each lineage. To assess antigenic drift in swine N2 following the human-to-swine spillover events, we generated a panel of swine N2 antisera against representative N2 and quantified the antigenic distance between wild-type viruses using enzyme-linked lectin assay and antigenic cartography. The antigenic distance between swine and human N2 was smallest between human N2 circulating at the time of each introduction and the archetypal swine N2. However, sustained circulation and evolution in swine of the two N2 lineages resulted in significant antigenic drift, and the N2-98 and N2-02 swine N2 lineages were antigenically distinct. Although intralineage antigenic diversity was observed, the magnitude of antigenic drift did not consistently correlate with the observed genetic differences. These data represent the first quantification of the antigenic diversity of neuraminidase of IAV in swine and demonstrated significant antigenic drift from contemporary human seasonal strains as well as antigenic variation among N2 detected in swine. These data suggest that antigenic mismatch may occur between circulating swine IAV and vaccine strains. Consequently, consideration of the diversity of N2 in swine IAV for vaccine selection may likely result in more effective control and aid public health initiatives for pandemic preparedness.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Modeling of hepatitis B virus infection spread in primary human hepatocytes

ABSTRACT Chronic hepatitis B virus (HBV) infection poses a significant global health threat, causing severe liver diseases including cirrhosis and hepatocellular carcinoma. We characterized HBV DNA kinetics in primary human hepatocytes (PHHs) over 32 days post-inoculation (p.i.) and modified ourin-vivoagent-based modeling (ABM) to gain insights into the HBV lifecycle and spreadin vitro. Parallel PHH cultures were mock-treated or treated with HBV entry inhibitor Myr-preS1 (6.25 µg/mL) was initiated 24 h p.i. In untreated PHH, three viral DNA kinetic patterns were identified: (i) an initial decline, followed by (ii) rapid amplification and (iii) slower amplification/accumulation. In the presence of Myr-preS1, viral DNA and infected cell numbers in phase 3 were effectively blocked, with minimal to no increase. This suggests that phase 2 represents viral amplification in initially infected cells, while phase 3 corresponds to viral spread to naïve cells. The ABM reproduced well the HBV kinetic patterns observed and predicted that the viral eclipse phase lasts between 18 and 38 h. After the eclipse phase, the viral production rate increased over time, starting with a slow production cycle of 1 virion per day, which gradually accelerated to 1 virion per hour after 3 days. Approximately 4 days later, virion production reached a steady state production rate of 4 virions/h. The estimated median efficacy of Myr-preS1 in blocking HBV spread was 91% (range: 90–92%). The HBV kinetics and the predicted estimates of the HBV eclipse phase duration and HBV production cycles in PHH are similar to those predicted in uPA/SCID mice with human livers. IMPORTANCE While primary human hepatocytes (PHHs) are the most physiologically relevant culture system for studying HBV infectionin vitro, a comprehensive understanding of HBV infection kinetics and spread in PHH is lacking. In this study, we characterize HBV viral kinetics and modify ourin vivoagent-based modeling (ABM) to provide quantitative insights into the HBV production cycle and viral spread in PHH. The ABM provides an estimate of the HBV eclipse phase duration, HBV production cycles, and Myr-preS1 efficacy in blocking HBV spread in PHH. The results resemble those predicted in uPA/SCID mice with human livers, demonstrating that estimated HBV infection kinetic parameters in PHHin vitromirror those observed in thein vivoHBV infection chimeric mouse model.

Virology↗
DOE OSTI2022

Antibody Recognition of CD4-Induced Open HIV-1 Env Trimers

Human immunodeficiency virus type 1 (HIV-1) envelope (Env), a heterotrimer of gp120-gp41 subunits, mediates fusion of the viral and host cell membranes after interactions with the host receptor CD4 and a coreceptor. CD4 binding induces rearrangements in Env trimer, resulting in a CD4-induced (CD4i) open Env conformation. Structural studies of antibodies isolated from infected donors have defined antibody-Env interactions, with one class of antibodies specifically recognizing the CD4i open Env conformation. In this study, we characterized a group of monoclonal antibodies isolated from HIV-1 infected donors (V2i MAbs) that displayed characteristics of CD4i antibodies. Binding experiments demonstrated that the V2i MAbs preferentially recognize CD4-bound open Env trimers. Structural characterizations of V2i MAb-Env-CD4 trimer complexes using single-particle cryo-electron microscopy showed recognition by V2i MAbs using different angles of approach to the gp120 V1V2 domain and the β2/β3 strands on a CD4i open conformation Env with no direct interactions of the MAbs with CD4. We also characterized CG10, a CD4i antibody that was raised in mice immunized with a gp120-CD4 complex, bound to an Env trimer plus CD4. CG10 exhibited characteristics similar to those of the V2i antibodies, i.e., recognition of the open Env conformation, but showed direct contacts to both CD4 and gp120. Structural comparisons of these and previously characterized CD4i antibody interactions with Env provide a suggested mechanism for how these antibodies are elicited during HIV-1 infection.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Deletion of the A137R Gene from the Pandemic Strain of African Swine Fever Virus Attenuates the Strain and Offers Protection against the Virulent Pandemic Virus

African swine fever virus (ASFV) is causing a devastating pandemic in domestic and wild swine within an extended geographical area from Central Europe to East Asia, resulting in economic losses for the regional swine industry. There are no commercial vaccines; therefore, disease control relies on identification and culling of infected animals. We report here that the deletion of the ASFV gene A137R from the highly virulent ASFV-Georgia2010 (ASFV-G) isolate induces a significant attenuation of virus virulence in swine. A recombinant virus lacking the A137R gene, ASFV-G-DA137R, was developed to assess the role of this gene in ASFV virulence in domestic swine. Animals inoculated intramuscularly with 102 50% hemadsorption doses (HAD50) of ASFV-G-DA137R remained clinically healthy during the 28-day observational period. All animals inoculated with ASFV-G-DA137R had medium to high viremia titers and developed a strong virus-specific antibody response. Importantly, all ASFV-G-DA137Rinoculated animals were protected when challenged with the virulent parental strain ASFV-G. No evidence of replication of challenge virus was observed in the ASFV-GDA137R-inoculated animals. Therefore, ASFV-G-DA137R is a novel potential live attenuated vaccine candidate and one of the few experimental vaccine strains reported to induce protection against the highly virulent ASFV Georgia virus that is the cause of the current Eurasian pandemic.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Antigenic Distance between North American Swine and Human Seasonal H3N2 Influenza A Viruses as an Indication of Zoonotic Risk to Humans

Human-to-swine transmission of influenza A virus (IAV) repeatedly occurs, leading to sustained transmission and increased diversity in swine; human seasonal H3N2 introductions occurred in the 1990s and 2010s and were maintained in North American swine. Swine H3N2 strains were subsequently associated with zoonotic infections, highlighting the need to understand the risk of endemic swine IAV to humans. We quantified antigenic distances between swine H3N2 and human seasonal vaccine strains from 1973 to 2014 using a panel of monovalent antisera raised in pigs in hemagglutination inhibition (HI) assays. Swine H3N2 lineages retained the closest antigenic similarity to human vaccine strains from the decade of incursion. Swine lineages from the 1990s were antigenically more similar to human vaccine strains of the mid-1990s but had substantial distance from recent human vaccine strains. In contrast, lineages from the 2010s were closer to human vaccine strains from 2011 and 2014 and the most antigenically distant from human vaccine strains prior to 2007. HI assays using ferret antisera demonstrated that swine lineages from the 1990s and 2010s had significant fold reductions compared to the homologous HI titer of the nearest pandemic preparedness candidate vaccine virus (CVV) or seasonal vaccine strain. The assessment of postinfection and postvaccination human serum cohorts demonstrated limited cross-reactivity to swine H3N2 from the 1990s, especially in older adults born before the 1970s. We identified swine strains to which humans are likely to lack population immunity or are not protected against by a current human seasonal vaccine or CVV to use in prioritizing future human CVV strain selection.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Detection of Airborne Influenza A and SARS-CoV-2 Virus Shedding following Ocular Inoculation of Ferrets

Despite reports of confirmed human infection following ocular exposure with both influenza A virus (IAV) and SARS-CoV-2, the dynamics of virus spread throughout oculonasal tissues and the relative capacity of virus transmission following ocular inoculation remain poorly understood. Furthermore, the impact of exposure route on subsequent release of airborne viral particles into the air has not been examined previously. To assess this, ferrets were inoculated by the ocular route with A(H1N1)pdm09 and A(H7N9) IAVs and two SARS-CoV-2 (early pandemic Washington/1 and Delta variant) viruses. Virus replication was assessed in both respiratory and ocular specimens, and transmission was evaluated in direct contact or respiratory droplet settings. Viral RNA in aerosols shed by inoculated ferrets was quantified with a two-stage cyclone aerosol sampler (National Institute for Occupational Safety and Health [NIOSH]). All IAV and SARS-CoV-2 viruses mounted a productive and transmissible infection in ferrets following ocular inoculation, with peak viral titers and release of virus-laden aerosols from ferrets indistinguishable from those from ferrets inoculated by previously characterized intranasal inoculation methods. Viral RNA was detected in ferret conjunctival washes from all viruses examined, though infectious virus in this specimen was recovered only following IAV inoculation. Low-dose ocular-only aerosol exposure or inhalation aerosol exposure of ferrets to IAV similarly led to productive infection of ferrets and shedding of aerosolized virus. Viral evolution during infection was comparable between all inoculation routes examined. Furthermore, these data support that both IAV and SARS-CoV-2 can establish a high-titer mammalian infection following ocular exposure that is associated with rapid detection of virus-laden aerosols shed by inoculated animals.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Deletion of E184L, a Putative DIVA Target from the Pandemic Strain of African Swine Fever Virus, Produces a Reduction in Virulence and Protection against Virulent Challenge

African swine fever (ASF) is currently causing a major pandemic affecting the swine industry and protein availability from Central Europe to East and South Asia. No commercial vaccines are available, making disease control dependent on the elimination of affected animals. Here, we show that the deletion of the African swine fever virus (ASFV) E184L gene from the highly virulent ASFV Georgia 2010 (ASFV-G) isolate produces a reduction in virus virulence during the infection in swine. Of domestic pigs intramuscularly inoculated with a recombinant virus lacking the E184L gene (ASFV-GDE184L), 40% experienced a significantly (5 days) delayed presentation of clinical disease and, overall, had a 60% rate of survival compared to animals inoculated with the virulent parental ASFV-G. Importantly, all animals surviving ASFV-G-DE184L infection developed a strong antibody response and were protected when challenged with ASFV-G. As expected, a pool of sera from ASFV-G-DE184L-inoculated animals lacked any detectable antibody response to peptides partially representing the E184L protein, while sera from animals inoculated with an efficacious vaccine candidate, ASFV-GDMGF, strongly recognize the same set of peptides. These results support the potential use of the E184L deletion for the development of vaccines able to differentiate infected from vaccinated animals (DIVA). Therefore, it is shown here that the E184L gene is a novel ASFV determinant of virulence that can potentially be used to increase safety in preexisting vaccine candidates, as well as to provide them with DIVA capabilities. To our knowledge, E184L is the first ASFV gene product experimentally shown to be a functional DIVA antigenic marker.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Transmission of Human Influenza A Virus in Pigs Selects for Adaptive Mutations on the HA Gene

Influenza A viruses (FLUAV) cause respiratory diseases in many host species, including humans and pigs. The spillover of FLUAV between swine and humans has been a concern for both public health and the swine industry. With the emergence of the triple reassortant internal gene (TRIG) constellation, establishment of human-origin FLUAVs in pigs has become more common, leading to increased viral diversity. However, little is known about the adaptation processes that are needed for a human-origin FLUAV to transmit and become established in pigs. Here, we generated a reassortant FLUAV (VIC11pTRIG) containing surface gene segments from a human FLUAV strain and internal gene segments from the 2009 pandemic and TRIG FLUAV lineages and demonstrated that it can replicate and transmit in pigs. Sequencing and variant analysis identified three mutants that emerged during replication in pigs, which were mapped near the receptor binding site of the hemagglutinin (HA). The variants replicated more efficiently in differentiated swine tracheal cells compared to the virus containing the wildtype human-origin HA, and one of them was present in all contact pigs. These results show that variants are selected quickly after replication of human-origin HA in pigs, leading to improved fitness in the swine host, likely contributing to transmission.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Utility of Human In Vitro Data in Risk Assessments of Influenza A Virus Using the Ferret Model

Both in vitro and in vivo models are employed for assessing the pandemic potential of novel and emerging influenza A viruses in laboratory settings, but systematic examinations of how well viral titer measurements obtained in vitro align with results from in vivo experimentation are not frequently performed. We show that certain viral titer measurements following infection of a human bronchial epithelial cell line are positively correlated with viral titers in specimens collected from virus-inoculated ferrets and employ mathematical modeling to identify commonalities between viral infection progression between both models.

60 APPLIED LIFE SCIENCES↗