TRANSMISSION BOTTLENECKS AND WITHIN-HOST EVOLUTIONARY DYNAMICS OF INFLUENZA A VIRUS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1022827
• Grant No.: 2020-67015-31563
• Cumulative Award Amt.: $500,000.00
• Proposal No.: 2019-05932
• Project Start Date: Jun 1, 2020
• Project End Date: May 31, 2025
• Grant Year: 2020
• Program Code: [A1221]- Animal Health and Production and Animal Products: Animal Health and Disease

Recipient Organization
UNIVERSITY OF GEORGIA
200 D.W. BROOKS DR
ATHENS,GA 30602-5016

Performing Department
Population Health

Non Technical Summary
Influenza is one of the most important respiratory diseases of swine, caused by influenza A viruses (IAV). Although IAV infections in pigs generally result in mild respiratory signs, it can significantly impact production efficiency in swine herds due to its high prevalenceand lead to production losses estimated to be $3 to $10 per head. The epidemiology of swine IAV is complex, with many genetically different viruses co-circulating in pigs in the United States. Human-to-swine transmissions have been reported frequently in the past decades and directly contribute to this current diversity of swine viruses, complicating the control of the disease. One eventwas identified recently: a novel H3N2 virus of human origin became established in swine herds in the U.S. and is now one of the predominant viruses in circulation. Despite the importance of these human-to-swine spillover events, little is known about the circumstances under which human IAVs transmit and become adapted to swine. Typically, an influenza virus has to undergo specific molecular changes to effectively replicate and transmit in a new species. Such changes result from pressures imposed by the new host, creating bottlenecks that lead to the selection of viruses with advantageous characteristics. We hypothesize that these selective bottlenecks alter the virus's molecular characteristics early on during the adaptation between humans and swine and that these processes are dependent on specific host factors. The objectives of this project are to i) follow how a human IAV virus evolves genetically over time within an individual animal and between animals as it is transmitted in pigs; ii) evaluate if specific components of the animal's immune response impose pressure to result in virus evolution; iii) evaluate how genetic changes detected on the viral surface proteins affect the virus replication characteristics. We will evaluate the genetic changes of human-origin IAV during consecutive infections in pigs. Additionally, we will evaluate the virus genetic evolution under pressure from a swine-specific immune component. Any changes that are detected will be evaluated for its effect on virus replication and fitness. The results from this research could help filter swine surveillance data for viruses that have "adaptation signatures" and identify new spillover events that are likely to be sustained in pigs. Furthermore, this information can be used to improve biosecurity measures that could decrease the viral spread and prevent the establishment ofnovel viruses. Ultimately, this can help reduce the economic cost of IAV to producers and the swine industry.

Animal Health Component

25%

Research Effort Categories

Basic

75%

Applied

25%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3510	1040	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3510 - Swine, live animal;

Field Of Science
1040 - Molecular biology;

Keywords

adaptation

bottleneck

evolution

human

influenza a virus

mutation

swine

Goals / Objectives
The long-term goal of this proposal is to better understand the process of adaptation of influenza A viruses and how host factors shape virus evolution and transmission. We expect to determine the selective bottlenecks that happen during replication and transmission of human viruses in pigs and identify the molecular signatures that allow human influenza viruses to display expanded host range and become adapted to swine. Such detailed understanding will lead to novel strategies for influenza disease prevention and control in swine. This project is focused on two specific aims:Aim 1. Evaluate the within- and between-host evolution of human influenza viruses during adaptation to swine.Aim 2. Evaluate the changes in the hemagglutinin (HA) and neuraminidase (NA) proteins and the factors that may lead to these changes during the adaptation of human influenza viruses to pigs.

Project Methods
We will use a reassortant H3N2 human virus (2HA/NAVic11:6TRIG) previously demonstrated to be transmissible in pigs to assess virus evolution and adaptation during consecutive transmissions in swine. This virus will be compared to a wildtype swine-origin virus (OH04). Four-week-old seeder pigs (n=3) will be inoculated with a high dose of each virus and age-matched naïve pigs will be introduced consecutively in the same space when pigs are confirmed infected, for a total of seven serial transmissions. Nasal swabs will be collected, and viral RNA sequenced on a high-throughput Illumina MiSeq sequencing platform. Additionally, the two viruses above will be serially passaged in MDCK cells after incubation with surfactant protein D (SP-D), a collectin of the innate immune system with antiviral activity against IAVs, to determine if SP-D imposes selective pressure for virus adaptation to pigs. Viruses will be incubated with a previously determined concentration of recombinant porcine SP-D and then inoculated in confluent MDCK monolayers. Viruses will be serially passaged under the same conditions for a minimum of 10 times. Cell culture supernatant will be collected and viral RNA will be sequenced on a high-throughput Illumina MiSeq sequencing platform for each passage. To evaluate the evolutionary dynamics of the human seasonal HA and NA in the new host, the sequences of the input (inoculum or sample from previous animal passage) and output (tissue culture supernatant or animal samples) virus populations will be analyzed. Sequences will be curated, viral diversity quantified within and between hosts using consensus sequences. Variant calling analysis will be performed, nucleotide diversity will be estimated as nonsynonymous and synonymous sites, and Shannon entropy of each sample will be calculated. Bayesian phylodynamic methods will be applied to describe the population dynamics of IAV within and between hosts.Mutant viruses will be generated via reverse genetics with single or multiple amino acid substitutions representing all mutations (individual or in combination) that were fixed in the HA and/or NA after serial passages in pigs or cells. These mutant viruses will be tested for their growth kinetics in swine cells, for their susceptibility to SP-D using hemagglutination inhibition assay, for their binding specificity and avidity using a glycan-binding assay and a solid phase assay, and for their NA enzymatic activity using a neuraminidase fluorescence assay. All assays have been previously described and are routinely used. Reassortant viruses will be generated with different combinations of HA and NA segments (from mutants obtained above) on the 2HA/NAVic11:6TRIG internal genes. These reassortant viruses will be tested for the HA/NA balance by plotting the quantitative results from the solid-phase HA binding avidity assay and the NA-activity fluorescent assay.Results obtained throughout the life of the project will be presented as posters and/or oral presentations in national and international veterinary and scientific meetings, such as the American Association of Swine Veterinarians, the Conference of Research Workers in Animal Diseases, or the American Society of Virology, and published in peer-reviewed journals. At least one graduate student will be trained on the laboratory techniques planned for this project and is expected to graduate. One postdoctoral associate will be trained in the bioinformatics tools and analysis planned for this project. The following milestones have been set for the project:2020 - preparation of viral stocks and inoculum2020 - 2021 - Transmission in vivo study to evaluate virus diversity over time2021 - 2022 - In vitro studies to test the effect of porcine SP-D on selection pressure2020 - 2023 - Sequencing and analysis to determine diversity over time2021 - 2023 - Generation of mutants and reassortants for phenotypic characterization in vitro2022 - 2023 - HA and NA activity in vitro analysis2022 - 2023 - Virus replication characterization in vitro2021 - 2023 - Presentation at meetings and publications2024 - 2025 - graduate student thesis defense/graduationAt the end of each period, milestones will be evaluated with the following criteria: not initiated, ongoing, completed.

Progress 06/01/20 to 05/31/25

Outputs
Target Audience:The project targets researchers focused on influenza virus and swine diseases, veterinarians, veterinary diagnostic laboratories and technicians, swine farmers, vaccine companies. During the project period, our data was presented at the CRWAD, IPVS, ASV, CEIRR, Options Influenza, and CVM-UGA meetings. The presentations reached the national and international scientific community, veterinarians and personnel working in the swine industry, and veterinary students. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two PhD students, one Master student and two undergraduate students were trained during the reporting period in techniques such as animal experimentation, cell culture, molecular biology, sequencing, glycan analysis, and bioinformatics analysis. Two PhD students that previously worked on the project graduated and accepted postdoctoral positions at the United States Department of Agriculture-ARS and University of Georgia. The Master student is on track to graduate in the Spring 2026. How have the results been disseminated to communities of interest?The results were presented at the American Society for Virology Annual meeting in 2023 and 2024, the Center for Influenza Research and Response Annual meeting in 2023 and 2024, CRWAD in 2020, 2021, 2023, 2024, and 2025, the International Pig Veterinary Society Congress (IPVS) in 2022, the Steeve Giguere Science of Veterinary Medicine Symposium at University of Georgia in 2020 and 2022, and Swine in Biomedical Research Conference (SBRC) in 2024. The results have been published at Journal of Virology, Nature Communications Biology, and PLOS Pathogens. Another manuscript is under revision and will be re-submitted to PLOS Pathogens. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Specific Aim 1: To evaluate the within- and between-host evolution of human influenza viruses during adaptation to swine, we quantified genetic diversity during infection and following transmission in pigs. We performed consecutive serial transmissions in pigs with a recombinant virus containing the surface genes (HA/NA) from a human-origin influenza virus and a mutant virus containing a mutation (A138S) in the surface protein that was previously demonstrated to improve transmissibility in swine. The viruses were passaged through 5 consecutive transmissions between naïve pigs. Our results showed that the virus with human HA/NA produced a self-limiting infection, while the virus containing the A138S mutation was transmitted through all contacts. Overall, titers in nasal swabs and bronchoalveolar lavage fluid (BALF) samples of pigs exposed to the A138S mutant virus increased in each contact during the course of infection, leading to high titers at 6 days post contact (dpc). This virus infected the upper respiratory tract, both cranial lobes and the left caudal lobe while the human HA/NA virus was only found in nose and trachea samples. Flow cytometry of lung samples revealed a significant reduction of alveolar macrophages (AMs) in pigs infected with the mutant virus while the neutrophil population was increased. There was a distinct pattern of expression of a variety of pro-inflammatory cytokines such as IL-8, TNF-alpha, and IFN-beta which could partially explain the unique cell migration pattern into the lungs. After two serial passages in swine, the virus gained a second mutation, now in the NA segment (D113A), located in the low-affinity calcium-binding site. Specific Aim 2. To evaluate the changes in the hemagglutinin (HA) and neuraminidase (NA) proteins observed during transmission of human influenza viruses to pigs (HA A138S and NA D113A mutations), a series of in vitro assays were performed. The A138S did not visibly change the particle morphology and did not affect plaque formation compared to the human-origin HA. Replication in MDCK cells was not affected. The A138S mutation increased affinity for alpha2,6 receptors and increased thermal stability compared to the human-origin HA but did not reach the level of the swine-origin HA. The affinity of the mutant HA for different SA structures was analyzed using a glycan array system. Interestingly, the A138S mutation prevented binding to N-glycolylneuraminic acid (Neu5Gc) and reduced the pool of glycans supporting binding, exhibiting a preference for extended a2,6-liknked molecules with at least 3 LacNAc repeats. The D113A mutation in the NA increased replication, viral particle release, and aerosol infection of MDCK and porcine PK15 cells when low calcium concentrations were added to the media, but only when the A138S mutation was also present. D113A also increased NA enzymatic activity and thermostability when no calcium was present in the reaction buffer, showing a similar behavior as the swine-origin NA. The HA/NA balance was assessed using Bio-Layer Interferometry (BLI) by looking at the elution of the viruses from the sensor in presence of NA activity. This analysis revealed that presence of the D113A mutation in NA with A138S mutation in HA increased elution times, resembling the swine influenza virus control. OVERALL IMPACT: Overall, our study indicates that adaptation of human viruses to the swine host involves an increased affinity for the lower respiratory tract, alpha-2,6 receptors and selection for less sensitive NA proteins to calcium concentrations, suggesting a novel role of calcium in the host range of influenza viruses. Further, our findings highlight the role of glycan topology and receptor affinity in the adaptation of a human-origin HA to pigs.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Daniela Rajao. Interspecies transmission of influenza A viruses (FLUAV) at the swine-human interface: a model for FLUAV adaptation. (2024). In Swine in Biomedical Research 2024 Conference. Madison, Wisconsin, USA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Matias Cardenas, Brittany Seibert, Brianna Cowan, L. Claire Gay, Flavio Cargnin Faccin, C. Joaquin Caceres, Amy L. Vincent Baker, Tavis K. Anderson, Daniel R. Perez, Daniela S. Rajao. Immunization affects the evolutionary pathway of human-origin H3N2 influenza virus in pigs and selects for mutations in the HA receptor-binding site. (2024). In Options XII for the Control of Influenza. Brisbane, Australia.
• Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Matias Cardenas, Pradeep Chopra, Brittany Seibert, Brianna Cowan, L. Claire Gay, Flavio Cargnin Faccin, C. Joaquin Caceres, Geert-Jan Boons, Daniel R. Perez, Amy L. Baker, Tavis K. Anderson, Daniel S. Rajao. Adaptation of a human-origin H3N2 Influenza A virus to pigs restricts receptor specificity for extended glycans and enhances sialic acid binding. (2025). In Conference of Research Workers in Animal Diseases. Chicago, USA.
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Cardenas, M., Seibert, B., Cowan, B., Caceres, C. J., Gay, L. C., Cargnin Faccin, F., Perez, D., Anderson, T., Baker, A., Rajao, D. Modulation of human-to-swine influenza a virus adaptation by the neuraminidase low-affinity calcium-binding pocket. Commun Biol 7, 1230 (2024). https://doi.org/10.1038/s42003-024-06928-6

Progress 06/01/23 to 05/31/24

Outputs
Target Audience:The project targets researchers focused on influenza virus and swine diseases, veterinarians, veterinary diagnostic laboratories and technicians, swine farmers, vaccine companies. During this reporting period, our data was presented at the CRWAD, ASV, SBRC and CEIRR meetings. The presentations reached the scientific community, veterinarians and personnel working in the swine industry, and veterinary students. Changes/Problems:No changes for this reporting period. What opportunities for training and professional development has the project provided?Two PhD students were trained during the reporting period in techniques such as animal experimentation, cell culture, molecular biology, sequencing, glycan analysis, and bioinformatics analysis. One PhD student that previously worked on the project graduated and accepted a postdoctoral position at the United States Department of Agriculture-ARS. The other two students are on track to graduate in the Fall of 2024 and 2025. How have the results been disseminated to communities of interest?The results were presented at the American Society for Virology Annual meeting in 2023 and 2024, the Center for Influenza Research and Response Annual meeting in 2023, CRWAD 2024, and Swine in Biomedical Research Conference (SBRC) 2024. Part of the results have been published at Journal of Virology and PLOS Pathogens. Another manuscript is under review at Nature Communications Biology. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, our final, we will focus in finishing analyzing all results and manuscript submission.

Impacts
What was accomplished under these goals? Specific Aim 1: Evaluate the within- and between-host evolution of human influenza viruses during adaptation to swine. To quantify genetic diversity during infection and following transmission, we performed consecutive serial transmissions in pigs with a recombinant virus containing the surface genes from a human-origin FLUAV. We also tested a virus containing the same constellation, but also containing a mutation (A138S) in the surface protein that was previously demonstrated to improve transmissibility of the human HA/NA in swine. The viruses were passaged through 5 consecutive transmissions between naïve pigs. Our results showed that the virus with human HA/NA produced a self-limiting infection, while the virus containing the A138S mutation was transmitted through all contacts. This virus infected the upper respiratory tract, both cranial lobes and the left caudal lobe while the human HA/NA virus was only found in nose and trachea samples. Flow cytometry of lung samples revealed a significant reduction of alveolar macrophages (AMs) in pigs infected with the mutant virus while a MHCIIlow/CD163neg population was increased. There was a distinct pattern of expression of a variety of pro-inflammatory cytokines such as IL-8, TNF-alpha, and IFN-beta which could partially explain the unique cell migration pattern into the lungs. After two serial passages in swine, the virus gained a second mutation, now in the NA segment (D113A), located in the low-affinity calcium-binding site. Specific Aim 2. Evaluate the changes in the hemagglutinin (HA) and neuraminidase (NA) proteins and the factors that may lead to these changes during adaptation of human influenza viruses to pigs. To assess the impact of the two amino acid changes (HA A138S and NA D113A) on virus characteristics, a series of in vitro assays were performed. The A138S did not visibly change the particle morphology and did not affect plaque formation compared to the human-origin HA. Replication in MDCK cells was not affected. The A138S mutation increased affinity for alpha2,6 receptors and increased thermal stability compared to the human-origin HA, but did not reach the level of the swine-origin HA. The affinity of the mutant HA for different SA structures was analyzed using a glycan array system. Interestingly, the A138S mutation prevented binding to N-glycolylneuraminic acid (Neu5Gc) and reduced the pool of glycans supporting binding, exhibiting a preference for extended a2,6-liknked molecules with at least 3 LacNAc repeats. The D113A mutation in the NA increased replication, viral particle release, and aerosol infection of MDCK and PK15 cells when low calcium concentrations were added to the media, only when the A138S mutation was also present. D113A also increased NA enzymatic activity and thermostability when no calcium was present in the reaction buffer, showing a similar behavior as the swine-origin NA. The HA/NA balance was assessed using BLI by looking at the elution of the viruses from the sensor in presence of NA activity. This analysis revealed that presence of the D113A mutation in NA with A138S mutation in HA increased elution times, resembling the swine FLUAV control. OVERALL IMPACT: Overall, our study indicates that adaptation of human viruses to the swine host involves an increased affinity for the lower respiratory tract, alpha-2,6 receptors and selection for less sensitive NA proteins to calcium concentrations, suggesting a novel role of calcium in the host range of influenza viruses. Further, our findings highlight the role of glycan topology and receptor affinity in the adaptation of a human-origin HA to pigs.

Publications

• Type: Journal Articles Status: Published Year Published: 2024 Citation: Matias Cardenas, Brittany Seibert, Brianna Cowan, Ana Luiza S. Fraiha, Silvia Carnaccini, L. Claire Gay, Flavio Cargnin Faccin, C. Joaquin Caceres, Tavis K. Anderson, Amy L. Vincent Baker, Daniel R. Perez, Daniela S. Rajao. Amino acid 138 in the HA of a H3N2 subtype influenza A virus increases affinity for the lower respiratory tract and alveolar macrophages in pigs. PLoS Pathog 20(2):e1012026. https://doi.org/10.1371/journal.ppat.1012026
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Matias Cardenas, Sasha Compton, C. Joaquin Caceres, Adolfo Garcia-Sastre, Daniel R. Perez & Daniela S. Rajao. MHCII as an entry receptor of H3N2 influenza A viruses: implication for cross-species transmission at the human-swine interface. (2024). In CEIRR Annual Network Meeting. New York, NY.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Matias Cardenas, Brittany Seibert, Brianna Cowan, L. Claire Gay, Flavio Cargnin Faccin, C. Joaquin Caceres, Amy L. Vincent Baker, Tavis K. Anderson, Daniel R. Perez & Daniela S. Rajao. (2024). Structural features of the N2 neuraminidase low-affinity calcium-binding site modulate calcium binding and promote adaptation of human-origin H3N2 viruses to pigs. In 43nd American Society for Virology meeting. Columbus, OH.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Matias Cardenas, Brittany Seibert, Brianna Cowan, Ana Luiza Soares Fraiha, L. Claire Gay, Flavio Cargnin Faccin, C. Joaquin Caceres, Amy L. Vincent Baker, Tavis K. Anderson, Daniel R. Perez & Daniela S. Rajao. (2024). Adaptation of a human-origin H3 and N2 to pigs increases affinity for the lower respiratory tract and select for mutations increasing NA activity and stability under calcium-depleted conditions. In Conference of Research Workers in Animal Diseases (CRWAD). Chicago, IL.

Progress 06/01/22 to 05/31/23

Outputs
Target Audience:The project targets researchers focused on influenza virus and swine diseases, veterinarians, veterinary diagnostic laboratories and technicians, swine farmers, vaccine companies. During this reporting period, our data was presented at the CRWAD, IPVS, ASV, and CVM-UGA meetings. The presentations reached the scientific community, veterinarians and personnel working in the swine industry, and veterinary students. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three PhD students were trained during the reporting period in techniques such as animal experimentation, cell culture, molecular biology, sequencing, and bioinformatics analysis. One of the PhD students working on the project graduated and accepted a postdoctoral position at the United States Department of Agriculture-ARS. How have the results been disseminated to communities of interest?The preliminary results were presented at the International Pig Veterinary Society Congress (IPVS), at the Steeve Giguere Science of Veterinary Medicine Symposium, at the University of Georgia, at the CRWAD meeting (January 2023) in 2022, and at the annual meeting for the American Society for Virology in 2023. Part of the results have been published at Journal of Virology. What do you plan to do during the next reporting period to accomplish the goals?Specific Aim 1: Evaluate the within- and between-host evolution of human influenza viruses during adaptation to swine. We will continue to perform bioinformatic analysis of the samples collected and titrated from the transmission studies in swine, finish analyzing all results and submit for publication. Specific Aim 2. Evaluate the changes in the hemagglutinin (HA) and neuraminidase (NA) proteins and the factors that may lead to these changes during adaptation of human influenza viruses to pigs. We will continue to perform in vitro phenotypic characterization of mutants found in the in vivo experiments.

Impacts
What was accomplished under these goals? Specific Aim 1: Evaluate the within- and between-host evolution of human influenza viruses during adaptation to swine. To quantify genetic diversity during infection and following transmission, we performed consecutive serial transmissions in pigs with a recombinant virus containing the surface genes from a human-origin IAV. We also tested a virus containing the same constellation, but also containing a mutation (A138S) in the surface protein that was previously demonstrated to improve transmissibility of the human HA/NA in swine. The viruses were passaged through 5 consecutive transmissions between naïve pigs. Our results showed that the virus with human HA/NA produced a self-limiting infection, while the virus containing the A138S mutation was transmitted through all contacts. This virus infected the upper respiratory tract, both cranial lobes and the left caudal lobe while the human HA/NA virus was only found in nose and trachea samples. Flow cytometry of lung samples revealed a significant reduction of alveolar macrophages (AMs) in pigs infected with the mutant virus while a MHCIIlow/CD163neg population was increased. There was a distinct pattern of expression of a variety of pro-inflammatory cytokines such as IL-8, TNF-alpha, and IFN-beta which could partially explain the unique cell migration pattern into the lungs. After two serial passages in swine, the virus gained a second mutation, now in the NA segment (D113A), located in the low-affinity calcium-binding site. Specific Aim 2. Evaluate the changes in the hemagglutinin (HA) and neuraminidase (NA) proteins and the factors that may lead to these changes during adaptation of human influenza viruses to pigs. To assess the impact of the two amino acid changes (HA A138S and NA D113A) on virus characteristics, a series of in vitro assays were performed. The A138S did not visibly change the particle morphology and did not affect plaque formation compared to the human-origin HA. Replication in MDCK cells was not affected. The A138S mutation increased affinity for alpha2,6 receptors and increased thermal stability compared to the human-origin HA, but did not reach the level of the swine-origin HA. The D113A mutation in the NA increased replication, viral particle release, and aerosol infection of MDCK and PK15 cells when low calcium concentrations were added to the media, only when the A138S mutation was also present. D113A also increased NA enzymatic activity and thermostability when no calcium was present in the reaction buffer, showing a similar behavior as the swine-origin NA. OVERALL IMPACT: Overall, our study indicates that adaptation of human viruses to the swine host involves an increased affinity for the lower respiratory tract, alpha-2,6 receptors and selection for less sensitive NA proteins to calcium concentrations, suggesting a novel role of calcium in the host range of influenza viruses.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: Mo JS, Abente EJ, Cardenas Perez M, Sutton TC, Cowan B, Ferreri LM, Geiger G, Gauger PC, Perez DR, Vincent Baker AL, Rajao DS. Transmission of Human Influenza A Virus in Pigs Selects for Adaptive Mutations on the HA Gene. J Virol. 2022 Nov 23;96(22):e0148022. doi: 10.1128/jvi.01480-22. Epub 2022 Nov 1. PMID: 36317880; PMCID: PMC9682980.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Mo JS, Abente E, Sutton T, Ferreri L, Geiger G, Gauger P, Perez D, Vincent A, Rajao D. (2022). Transmission of human influenza A virus in pigs selects for mutations on the HA gene segment. In International Pig Veterinary Society Congress. Rio de Janeiro, Brazil.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Cardenas, Matias; Cowan, Brianna; Seibert, Brittany; Gay, Lindsey; Cargnin, Flavio; Caceres, Carlos Joaquin; Perez, Daniel; Vincent, Amy; Anderson, Tavis; Rajao, Daniela. (2022). Multiple transmissions of human influenza A virus in pigs leads to improved replication. In Conference of Research Workers in Animal Diseases (CRWAD). Chicago, IL.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Matias Cardenas, Brittany Seibert, Brianna Cowan, Ana Luiza Soares Fraiha, L. Claire Gay, Flavio Cargnin Faccin, C. Joaquin Caceres, Daniel R. Perez, Amy L. Vincent Baker, Tavis K. Anderson, Daniela S. Rajao. (2023). Adaptation of a human-origin influenza virus to the swine host increases affinity for the lower respiratory tract. In 42nd American Society for Virology meeting. Athens, GA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Cardenas, Matias; Cowan, Brianna; Seibert, Brittany; Gay, Lindsey; Cargnin, Flavio; Caceres, Carlos Joaquin; Perez, Daniel; Vincent, Amy; Anderson, Tavis; Rajao, Daniela. (2022). Multiple transmissions of human influenza A virus in pigs leads to improved replication in the respiratory tract. In Steeve Giguere Science of Veterinary Medicine Symposium, University of Georgia. Athens, GA.

Progress 06/01/21 to 05/31/22

Outputs
Target Audience:The project targets researchers focused on influenza virus and swine diseases, veterinarians, veterinary diagnostic laboratories and technicians, swine farmers, vaccine companies. During this reporting period, our data was presented at the CRWAD and the IPVS meetings. The presentations reached the scientific community in general and veterinary professionals and students. Changes/Problems:Due to the poor transmission observed for the recombinant virus originally planned for the swine studies, we were required to test different conditions than proposed. A second experiment was performed using a higher infection dose than used in the first experiment, in addition to comingling each group of contacts together with all previously infected/exposed pigs. Since this approach did not result in increased transmission, we switched the virus for the third experiment, consisting of a virus that only differed in a single mutation on the HA gene that had been previously detected after successful transmission of the human-origin recombinant in pigs. This virus resulted in sustained transmission until the last contact (#4), allowing for the viral population analysis to move forward. What opportunities for training and professional development has the project provided?Two PhD students and one Masters student were trained during the reporting period in techniques such as animal experimentation, cell culture, molecular biology, sequencing, and bioinformatics analysis. One of the PhD students working on the project recently graduated and accepted a postdoctoral position at the United States Department of Agriculture-ARS. How have the results been disseminated to communities of interest?The preliminary results were presented at the CRWAD meeting in 2021 and at the International Pig Veterinary Society Congress (IPVS) in 2022 by the PD. Part of the results have been submitted for publication at Journal of Virology (https://www.biorxiv.org/content/10.1101/2022.04.04.487085v1). What do you plan to do during the next reporting period to accomplish the goals?Specific Aim 1: Evaluate the within- and between-host evolution of human influenza viruses during adaptation to swine. In the first months of the next reporting period, we will perform sequencing and bioinformatic analysis of the samples collected and titrated from the transmission studies in swine. Specific Aim 2. Evaluate the changes in the hemagglutinin (HA) and neuraminidase (NA) proteins and the factors that may lead to these changes during adaptation of human influenza viruses to pigs. Cells will be immortalized and used to test effect of innate immune components on virus evolution.

Impacts
What was accomplished under these goals? Specific Aim 1: Evaluate the within- and between-host evolution of human influenza viruses during adaptation to swine. To quantify genetic diversity during infection and following transmission, we performed consecutive serial transmissions in pigs with a recombinant virus containing the surface genes from a human-origin IAV, compared to a swine IAV with the same internal gene constellation. Although the recombinant virus with human-origin HA/NA had been previously shown to replicate and transmit in pigs, transmission was only confirmed until contact 2 in our experiments (direct infection followed by two consecutive transmissions). This was confirmed in a separate experiment with modified conditions. Therefore, we generated a mutant virus containing a mutation that was found after transmission of the recombinant virus with human-origin HA/NA between pigs in a previous experiment. This mutant virus transmitted for four rounds of consecutive transmission. Overall, titers in nasal swabs of pigs exposed to the human-origin mutant virus increased in each contact during the course of infection, leading to high titers at 6 days post contact (dpc), similar to titers in directly infected pigs. Titers were not different from titers in pigs infected or exposed to the swine virus. However, titers in the nasal swabs of pigs exposed to the swine virus remained stable throughout the study, regardless of the contact number or dpc. Pigs exposed to human-origin mutant virus showed an increase in the viral titers in bronchoalveolar lavage fluid (BALF) samples with each contact, reaching titers similar to the swine virus in contact 4 pigs. Similar to nasal swab samples, viral titers in BALF remained stable in the swine virus contact pigs. Specific Aim 2. Evaluate the changes in the hemagglutinin (HA) and neuraminidase (NA) proteins and the factors that may lead to these changes during adaptation of human influenza viruses to pigs. Primary swine nasal, tracheal, and bronchial epithelial cells were freshly collected and isolated from 7-week-old male and female pigs. Cells will be immortalized and differentiated in air-liquid interface (ALI) for the studies in Aim 2. OVERALL IMPACT: None to date; project is ongoing and concludes May, 2023.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Rajao D, Perez D, Vincent A, Anderson T. (2021). Transmission bottlenecks during adaptation of human influenza A virus to pigs. In Conference of Research Workers in Animal Diseases. Chicago, USA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Mo JS, Abente E, Sutton T, Ferreri L, Geiger G, Gauger P, Perez D, Vincent A, Rajao D. (2022). Transmission of human influenza A virus in pigs selects for mutations on the HA gene segment. In International Pig Veterinary Society Congress. Rio de Janeiro, Brazil.
• Type: Journal Articles Status: Submitted Year Published: 2022 Citation: Mo JS, Abente E, Sutton T, Ferreri L, Geiger G, Gauger P, Perez D, Vincent Baker A, Rajao D. Transmission of human influenza A virus in pigs selects for adaptive mutations on the HA gene. https://www.biorxiv.org/content/10.1101/2022.04.04.487085v1.

Progress 06/01/20 to 05/31/21

Outputs
Target Audience:The project targets researchers focused on influenza virus and swine diseases, veterinarians, veterinary diagnostic laboratories and technicians, swine farmers, vaccine companies. During this reporting period, our data was presented at the CRWAD virtual meeting and the virtual UGA Giguère Science of Veterinary Medicine Symposium. The presentations reached the scientific community in general and veterinary professionals and students. Changes/Problems:Our original plan was to perform the baseline swine sustained transmission study in year 1 to provide sequencing information for the bioinformatics analysis that will follow. However, due to COVID-19 and consequent space restrictions for animal experiments, we encountered a delay. Since then, anew BSL2 animal facility was built at the University, and, with the approval of our National Program Leaders, we decided to wait for construction to finalize and perform the animal studies in the new facility, which will provide a better space for the logistics needed for the study.The animal studies are on track to begin in the coming weeks. What opportunities for training and professional development has the project provided?One PhD student was trained during the reporting period in techniques such as cell culture, sequencing, protein modeling, and bioinformatics analysis. How have the results been disseminated to communities of interest?The preliminary results were presented at the CRWAD virtual meeting in 2020 and in a research seminar at the College of veterinary Medicine at UGA, by a PhD student involved with initial studies. What do you plan to do during the next reporting period to accomplish the goals?In the first months of the next reporting period, we will perform a pilot study in swine to define infectious doses of the 2HA/NAVic11:6TRIG and control viruses, followed by the baseline transmission in vivo study to evaluate virus diversity over time that was originally planned for year 1. These studies are on track to start in the next month. Additionally, we will conduct in vitro studies to test the effect of innate immune components on virus evolution.

Impacts
What was accomplished under these goals? During the reporting period 6/01/2020 through 8/10/2020 the University was under essential research only mandate and continued with reduced occupancy restrictions until vaccination roll-out in early 2021. The report should consider these circumstances. Specific Aim 1: Evaluate the within- and between-host evolution of human influenza viruses during adaptation to swine. We will quantify genetic diversity during infection and following transmission by consecutive serial infections in pigs with human and swine IAV, and identify mutant viruses selected after transmission in pigs. We generated a recombinant virus containing the same internal gene constellation as the human-origin recombinant virus (termed 2HA/NAVic11:6TRIG) that will be tested in our experiments, to serve as a control and to focus our analysis on the evolution of the surface genes. The control virus was generated by reverse genetics, and contains the matrix gene from the 2009 pandemic H1N1 virus and remaining genes from a typical swine-origin H3N2 triple reassortant internal gene (TRIG) virus. Both viruses were grown in large stocks and baseline virus genetic diversity was determined by high throughput sequencing. Aim 2. Evaluate the changes in the hemagglutinin (HA) and neuraminidase (NA) proteins and the factors that may lead to these changes during adaptation of human influenza viruses to pigs. Virus population diversity will be evaluated in swine respiratory epithelial cells under different concentrations of surfactant protein D (SP-D), a component of the innate immune system that has antiviral activity against the HA. We established primary swine tracheal epithelial cells (STEC) that were successfully differentiated in air-liquid interface (ALI). Both viruses whose stocks were produced in Aim 1 were serially passaged in the STEC cells 10 times. Virus titers of the 2HA/NAVic11:6TRIG recovered from cell supernatants increased over time, stabilizing at passage 4, while titers for the control virus were high from the first passage. Preliminary variant analysis of passages 1, 3, 5, and 7 of 2HA/NAVic11:6TRIG revealed two mutations in the HA1 portion (N165Kand N216K) that were predominant in passage 5 and fixed (>99%)in passage 7. No mutations were fixed in the NA protein.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Mo JS, Ferreri L, Abente E, Sutton T, Perez D, Vincent A, Rajao D. (2020). Transmission of an influenza A virus with human seasonal H3 in pigs resulted in adaptive mutations in the HA gene. In Conference of Research Workers in Animal Diseases. Virtual.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Mo JS, Ferreri L, Abente E, Sutton T, Perez D, Vincent A, Rajao D. (2020). Transmission and Adaptation of an influenza H3N2 virus with human seasonal HA in pigs. In Gigu�re Science of Veterinary Medicine Symposium. Virtual.