Research Project: Control Strategies to Prevent and Respond to Diseases Outbreaks Caused by Avian Influenza Viruses

Location: Exotic & Emerging Avian Viral Diseases Research

2024 Annual Report

Objectives
1. Characterize the ecology, epidemiology, and pathogenesis of emerging avian influenza viruses with a focus on the One-Health concept. 1.A. Characterize the pathogenesis of new and variant avian influenza virus (AIV) isolates and determine quantifiable species-specific transmission parameters of AIVs for modeling and outbreak preparedness. 1.B. Conduct the molecular characterization of new and variant AIVs including phylogenetics, and network analysis. 1.C. Examine novel or emerging viruses that may have an impact on poultry health or where poultry pathogens affect public health. 1.D. Assess the inter- and intra-species transmission dynamics of LPAI viruses, which will also contribute to investigating mechanisms and pathways of intra-host virus evolution. 1.E. Investigate determinants of virulence and mechanism behind increased pathology seen with some LPAI virus subtypes. 2. Elucidate the host-pathogen interactions of avian influenza virus infections. 2.A. Investigate virus-specific factors and viral molecular markers associated with infectivity, pathogenicity, and transmissibility of influenza viruses in avian species including virus tissue tropism and replication. 2.B. Investigate host-specific factors associated with the infectivity, pathogenicity, and transmissibility in different avian species of current and emerging influenza viruses including species, breed, age, and physiological state of the bird, and concomitant infections. 2.C. Characterize the innate and adaptive immune response to avian influenza virus infection in different avian models that are either susceptible, tolerant, or resistant to infection. 3. Develop intervention strategies to effectively control avian influenza viruses and contain disease outbreaks. 3.A. Improve virus control and recovery strategies by producing data on the environmental ecology of AIV. 3.B. Evaluate and improve existing and new diagnostic tests and testing strategies for avian influenza virus surveillance, detection, and recovery from disease outbreaks. 3.C.1. Evaluate existing or develop new vaccine platforms and strategies designed to rapidly control and prevent avian influenza virus outbreaks in the various components of poultry production. 3.C.2 Investigate the impact of immunosuppressive viruses on the efficacy of AIV vaccines in chickens. 3.D. Characterize the effect of vaccine induced immunity on virus evolution. 3.E. Utilize precision engineering of the chicken genome to develop genome edited poultry with increased resistance to avian influenza virus. 3.F. Identify correlates of vaccine protection in avian species, including different breeds and ages. 3.G. Determine mechanisms and immune-system-wide effects of vaccines with rapid onset of broadly protective immunity. 3.H. Determine the role of vaccines in driving escape mutations and how to prevent them.

Approach
These objectives include a combination of basic and applied research that will generate knowledge and help develop tools to improve our ability to prevent and control avian influenza virus (AIV). These research goals are highly interrelated and will be accomplished with similar tools and approaches (Figure 1); thus, experiments will often contribute to more than one objective. The first objective includes the characterization of new strains of AIV and other viruses, which constantly emerge in nature. The second objective complements the first with a more in-depth focus on the specific viral and host factors that contribute to host adaptation, transmission, and virulence. The third objective will improve current practical intervention strategies including diagnostics, vaccines, development of AIV resistant poultry, and will enhance our understanding of the ecology of AIV in poultry. For objectives 1 and 3: Utilizing sequencing in vitro and in vivo models, low pathogenic avian influenza virus (LPAIV) will be tested to identify how host range is determined and markers for virus pathogenicity identified. Multiple types of immune system models and reagents with in vitro models and in vivo models will be used to characterize the immune response to LPAIV viral infection and vaccines.

Progress Report
During FY2024 progress was made on all objectives and was directed to respond to the current H5 highly pathogenic avian influenza virus (HPAIV) outbreak in the U.S. This virus has affected more than 90 million domestic birds in 49 States and has spread to dairy cattle in the U.S. since it was first detected in wild birds in 2021. Under Objective 1, research continued to support the United States poultry and livestock industries from HPAIV and emerging viral disease threats. In late March 2024, highly pathogenic avian influenza virus (HPAIV) infections were discovered in dairy cows, soon it was determined that high quantities of virus were being excreted into milk. ARS researchers in Athens, Georgia, worked with the United States Food and Drug Administration (FDA) and the USDA Animal and Plant Health Inspection Service (APHIS), the USDA Food Safety Inspection Service (FSIS), NIH National Institute of Biomedical Imaging and Bioengineering (NIBIB), and ARS researchers in Wyndmoor, Pennsylvania, to confirm the safety of the food supply. Testing was conducted to confirm that standard pasteurization would inactivate the virus. In collaboration with the FDA testing was conducted with a continuous flow pasteurizer to accurately simulate real-world milk processing. It was found that continuous flow pasteurization using standard condition for high temperature short time (HTST) of 72°C for 15seconds is sufficient to inactivate high levels of virus in homogenized raw milk. Work continues to determine the parameters of HPAIV inactivation in milk products are different temperatures. To determine the risk associated with HPAIV in milk products, ARS researchers in Athens, Georgia, also collaborated with the FDA to evaluate the levels of virus in milk prior to and post processing. A survey was conducted on retail dairy products from around the United States in April and June 2024. Although the virus genetic material could be detected in fluid milk with PCR based methods, no infectious virus was found in any pasteurized products. Milk in bulk storage tanks was also tested. Although 57.4% of the samples were positive for virus genetic material, only about 14% were positive for infectious virus. Even when infectious virus was found, the quantity was much lower than what was predicted by PCR based methods. To help screen milk for HPAIV during routine microbiological quality control testing of milk, Lateral flow assays (LFAs) were optimized to milk testing in collaboration with NIH-NIBB. LFAs are rapid (10-15min) tests that don’t need to run in a lab. They are very specific, but less sensitive than PCR based methods. Processing of raw milk was optimized for testing by LFAs and eight different commercial kits were tested for evaluated for sensitivity in milk. The LFAs can be used as a rapid test for detection of HPAIV in milk which can be used to determine if the milk is safe or needs to be diverted or destroyed. ARS researchers in Athens, Georgia, collaborated with ARS researchers in Wyndmoor, Pennsylvania, to characterize how cooking temperatures inactivate infectious HPAIV. Hamburgers were artificially contaminated with a low pathogenic avian influenza virus (LPAIV) related to the virus in cattle. Hamburgers were cooked to rare, medium, and well-done temperatures (120, 145 and 160F respectively), then were tested for infectious virus. Infectious virus was only recovered from the hamburgers cooked at the lowest temperature, which is below the recommended temperature for ground beef. Work to confirm that LFAs for SARS-CoV-2 could be used with environmental samples from mink farms were completed. Three tests were evaluated and all three were shown to be robust against false negatives and false positives with material. It was also shown that SARS-CoV-2 did not remain infectious on mink pelts under routine pelt storage conditions. To determine the transmission risk of emergent avian metapneumovirus (aMPV) in poultry, ARS researchers in Athens, Georgia, collaborated with veterinarians to evaluate the possibility of vertical transmission by eggs in aMPV positive flocks. Eggs were received from aMPV-positive hens from four states. Shell and embryo tissues were tested virus using PCR based methods; the presence of virus could not be detected on the eggs or in the embryos. Because newly-hatched turkeys can be transported long distances, these results address concerns of interstate movement of poults spreading aMPV. Under Objective 1, ARS researchers in Auburn, Alabama, performed experiments to assess the inter- and intra-species transmission dynamics of LPAIV using strains isolated from different avian species. These were submitted to next generation sequencing (NGS) evaluation to obtain the complete genome sequence when needed and to evaluate changes after passage in embryonated chicken eggs. In addition, primary cell cultures were infected with LPAUV and followed for seven days to evaluate the infection. Different patterns of susceptibility and resistance to infection, were observed among strains and cell types. Studies were conducted to help elucidate the role of different bat species as hosts for SARS-CoV-2. A key determinant of infection is the attachment of the virus to the host receptor. An ARS researcher in Athens, Georgia, expressed the receptors from common vampire bat and pallid bat in an avian cell line to and showed that SARS-CoV-2 could bind to them. Virus replication in cells expressing bat receptors differed between virus variants tested. Under Objective 2 studies were conducted to characterize the immune systems of chickens and how they respond to AIV. Dendritic cells (DCs) are professional antigen-presenting cells and key components of the immune system, but their response to AIV is relatively unknown. In these studies, ARS scientists in Athens, Georgia produced chicken DCs to characterize the innate immune responses to AIV. A strong proinflammatory response and stimulation of the interferon pathway were observed early after infection. Microscopically, the DCs underwent morphological changes from classic elongated dendrites to a more general rounded shape that eventually lead to cell death with the presence of scattered cellular debris. Differences in onset of morphologic changes were observed between H5 and H7 subtypes of AIV. The elevated expression of cytokines from infected DCs may be indicative with the dysregulation of the immune response typically seen with AIV infections. Under Objective 3, several studies were also completed that addressed vaccination for HPAIV and modifications for surveillance that would be needed with vaccination for HPAIV. First, a study was conducted to evaluate inactivated vaccines in turkeys. Turkeys were vaccinated at three ages with an in-house vaccine closely matched to the current virus in the field in the United States. In addition to showing protection by vaccines and good antibody development. During this study serum samples were collected at several time points after infection to evaluate with a new test to identify whether turkeys were infected with HPAIV after vaccination. Because current tests can’t distinguish between vaccinated and infected animals, this new test would be needed for surveillance when vaccinating. ARS researchers in Athens, Georgia, in collaboration with the researchers at the University of Georgia have an H9N2 modified live influenza vaccine (MLV). Modified live virus vaccines, with their potential for mass application, offer an advantage over existing options. The MLV stimulated robust innate and humoral immune responses. In protection studies, vaccination of birds with MLV protected 100% birds from challenge. It was demonstrated that this novel technology creates a strategy for protecting poultry from avian influenza virus. Infection with HPAIV killed at least 21 California condors. ARS researchers in Athens, Georgia, collaborated with State and Federal groups to vaccinate endangered Condors for HPAI. Vaccine safety and antibody development was ensured by testing a related species, vultures, in rehab centers. Vaccine has been administered to over 140 condors by June 2024. A study was done to help elucidate how AIV changes during vaccination. The long-term goal is to help inform vaccine selection for antigens that will induce broader immunity that will improve protection and will extend the “lifespan” of the virus. The data show that the same virus can change in different ways to escape the same immune response. Therefore, anticipating antigenic drift with vaccine selection will need to take the whole landscape of antigenic changes into account. The use of clustered regularly interspaced short palindromic repeats (CRISPR) as an anti-viral approach to avian influenza control in poultry was determined to expand approaches to control AIV that are alternatives to vaccine usage. ARS researchers in Athens, Georgia, utilized transgenic methods to incorporate guides into the genome of avian cells that match the genetic sequence of avian influenza viruses. When expressed during virus infection, the guides target the genomic RNA of influenza virus for destruction. Results demonstrate significant reduction of infectious virus in cells expressing the CRISPR guides across a number of different subtypes supporting the concept for alternatives to vaccination. ARS researchers in Auburn, Alabama, established antibody panels to identify chicken immune cell markers, such as lymphocytes, B cells, dendritic cells, monocytes, macrophages, and others. They established a standardized assay for analysis of chicken immune cells that will be applied to characterizing the immune response and to optimize vaccines.

Accomplishments
1. Vaccination at day of age is effective against highly pathogenic avian influenza (HPAI) in chickens. Two vectored vaccines that can be administered at day of age, or in ovo, were evaluated by ARS researchers in Athens, Georgia, for efficacy against the current HPAI viruses in the US. These vaccines are also compatible with the modified poultry surveillance programs needed with vaccination. Both vaccines provided protection against death and disease and reduces virus excretion, which will limit virus spread. Because these vaccines can be given en masse by the in ovo route or in the hatchery they offer a practical initial vaccination that could help protect poultry from HPAI early in life.

2. Retail dairy products were shown to be free of infectious highly pathogenic avian influenza virus (HPAIV). Dairy cattle were shown to be infected with HPAIV for the first time and the virus was discovered in milk. Retail milk products were collected by collaborators with the US Food and Drug Administration then ARS researchers in Athens, Georgia, tested the milk for HPAIV. Although viral genetic material could be detected in about 20% of the products tested, no infectious virus was found. This confirms that the milk safety programs which includes pasteurization and diversion of poor-quality milk, have succeeded in keeping HPAIV out of the milk supply.

3. Continuous flow pasteurization inactivated high levels of highly pathogenic avian influenza virus (HPAIV) in milk. Dairy cows were first recognized to be infected with HPAIV in March 2024 and their milk contained high levels of virus that could be detected in raw milk in bulk storage tanks. ARS researchers in Athens, Georgia, worked with scientists from the US Food and Drug Administration (FDA) to closely simulate standard industry practices. It was demonstrated that the minimum pasteurization conditions required by the FDAs Pasteurized Milk Ordinance, 72C (161F) for 15 seconds is sufficient to inactivate at least 1 million infectious units of virus. This shows that if HPAIV were to be present in milk, pasteurization would render the virus non-infectious.

4. Avian influenza virus (AIV) is inactivated at recommended cooking temperatures in ground beef. ARS researchers in Athens, Georgia, and Wyndmoor Pennsylvania, collaborated to whether cooking hamburgers at three common target temperatures 120°F, 145°F, and 160°F would inactivate the virus. It was shown that the virus was destroyed and no longer infectious at the two highest temperatures, but infectious virus remained present at the lowest temperature which is below the recommended minimum cooking temperature for ground beef. This demonstrates that target cooking temperatures for bacteria will also inactivate AIV.

5. Recent highly pathogenic avian influenza viruses (HPAIV) from the US have a lower infectious dose in chickens and turkeys. The infectious dose of HPAIV can vary among viral strains so a recent US isolate was compared with an older United States isolate, and a related strain from Europe for infectivity in chickens and turkeys by ARS researchers in Athens, Georgia,. The most recent US isolate is the most infectious for chickens and the infectious dose remains very low for turkeys. This provides evidence that the current virus is spreading so easily to poultry, especially chickens, because of the low infectious dose. Biosecurity and other control measures will need to be adjusted to control the virus more effectively than in the past.

6. Bird study skin specimens are safe from highly pathogenic avian influenza virus (HPAIV) and Newcastle disease virus (NDV) for transfer a month after preparation. Study skins were prepared from chickens infected with HPAIV or NDV and the duration that the virus remained infectious was determined by ARS researchers in Athens, Georgia. Study skins are like a bird carcass that has been preserved by taxidermy but was made to study the species. The skins can have great historical values and are shared among researchers internationally. It was shown that neither virus remained infectious for month, therefore the skins can be safety shared and imported soon after preparation.

7. Transgenic methods enhance resistance against both high and low pathogenic avian influenza virus (AIV). In these studies, ARS researchers in Athens, Georgia, stably introduced a mouse AIV resistance gene into chicken cells to enhance the immune response against AIV. Following infection, titers of AIV were significantly decreased in cells expressing the mouse gene. In addition, considerably less cytopathic effect and virus staining was observed. These results demonstrate methods for establishing protection against multiple AIV subtypes. This work provides foundational studies for use of gene-editing to enhance innate disease resistance against AIV.

8. Comparative analysis of polymerase (PB2) gene of H5 avian influenza virus (AIV) isolated from birds and mammals demonstrates preferential differences in sequence based on species. AIVs do not normally infect mammals, however, recent outbreaks have demonstrated the ability to transmit to other species. In these studies, ARS researchers in Athens, Georgia, compare known genetic markers of adaption in the PB2 gene at position 627. It was demonstrated that recent isolates of H5 AIV from mammalian species have a higher conversion of glutamic acid to lysine compared to bird isolates. In addition, the >97% of all avian species demonstrated a glutamic acid at position 627 regardless of the H5 lineage examined. These studies broaden our understanding of AIV adaption in other species.

Review Publications
Kapczynski, D.R., Chrzastek, K., Shanmugasundaram, R., Zsak, A., Segovia, K., Sellers, H., Suarez, D.L. 2023. Efficacy of recombinant H5 vaccines delivered in ovo or day of age in commercial broilers against the 2015 U.S. H5N2 clade 2.3.4.4c highly pathogenic avian influenza virus. Virology Journal. 20(1):298. https://doi.org/10.1186/s12985-023-02254-1.
Roberts, E., Allen, C., Brennen, R., Swartz, A., Dines, B., Cigel, F., Killian, M.L., Crossley, B., Suarez, D.L., Torchetti, M., Watson, C., Slavinsky, S., Toohey-Kurth, K., Newbury, S. 2023. Discovery of Influenza A(H7N2) in a cat after admission to an animal shelter: A case report. Journal of Shelter Medicine and Community Animal Health. 2(1):1-8. https://doi.org/10.56771/jsmcah.v2.61.
Spackman, E., Suarez, D.L., Lee, C.W., Pantin Jackwood, M.J., Lee, S.A., Youk, S., Ibrahim, S. 2023. Efficacy of inactivated and RNA particle vaccines against a North American clade 2.3.4.4b H5 highly pathogenic avian influenza virus. Vaccine. 41(49):7369-7376. https://doi.org/10.1016/j.vaccine.2023.10.070.
Ghorbani, A., Ngunjiri, J.M., Rendon, G., Brooke, C.B., Kenney, S.P., Lee, C.W. 2023. Diversity and complexity of internally deleted viral genomes in influenza A virus subpopulations with enhanced interferon-inducing phenotypes. Viruses. 15(10):2107. https://doi.org/10.3390/v15102107.
Pantin Jackwood, M.J., Spackman, E., Leyson, C., Youk, S., Lee, S.A., Moon, L.M., Torchetti, M.K., Killian, M.L., Lenoch, J.B., Kapczynski, D.R., Swayne, D.E., Suarez, D.L. 2023. Pathogenicity in chickens and turkeys of a 2021 United States H5N1 highly pathogenic avian influenza clade 2.3.4.4b wild bird virus compared to two previous H5N8 clade 2.3.4.4 viruses. Viruses. 15(11):2273. https://doi.org/10.3390/v15112273.
Fasina, Y.O., Suarez, D.L., Ritter, G.D., Gerken, E.C., Farnell, Y.Z., Wolfenden, R., Hargis, B. 2024. Unraveling frontiers in poultry health (part 1) – Mitigating economically important viral and bacterial diseases in commercial chicken and turkey production. Poultry Science. 103(4). Article 103500. https://doi.org/10.1016/j.psj.2024.103500.
Youk, S., Torchetti, M.K., Lantz, K., Lenoch, J.B., Killian, M.L., Leyson, C., Bevins, S.N., Dilione, K., Ip, H.S., Stallknecht, D.E., Poulson, R.L., Suarez, D.L., Swayne, D.E., Pantin Jackwood, M.J. 2023. H5N1 highly pathogenic avian influenza clade 2.3.4.4b in wild and domestic birds: introductions into the United States and reassortments, December 2021-April 2022. Virology. 587. Article 109860. https://doi.org/10.1016/j.virol.2023.109860.
Briggs, K., Sweeney, R.P., Blehert, D.S., Spackman, E., Suarez, D.L., Kapczynski, D.R. 2023. SARS-CoV-2 utilization of ACE2 from different bat species allows for virus entry and replication in vitro. Virology. 586:122-129. https://doi.org/10.1016/j.virol.2023.07.002.
Briggs, K., Kapczynski, D.R. 2023. Comparative analysis of PB2 residue 627E/K/V in H5 subtypes of avian influenza viruses isolated from birds and mammals. Frontiers in Veterinary Science. https://doi.org/10.3389/fvets.2023.1250952.
Mo, J., Spackman, E., Swayne, D.E. 2023. Prediction of highly pathogenic avian influenza vaccine efficacy in chickens by comparison of in vitro and in vivo data: a meta-analysis and systematic review. Vaccine. 41(38):5507-5517. https://doi.org/10.1016/j.vaccine.2023.07.076.