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

Location: Exotic & Emerging Avian Viral Diseases Research

2022 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
Several objectives were accomplished in response to the current H5 highly pathogenic avian influenza virus (HPAIV) outbreak in the U.S. HPAIVs have devastating impact on the poultry industry. Beginning in November and December of 2021, H5N1 HPAI clade 2.3.4.4b viruses were detected in wild birds in Canada and in February of 2022 in the US and were most closely related to viruses in northern Europe from Spring 2021. These viruses have been circulating in Europe causing outbreaks in poultry and wild birds in many countries. Once in the U.S., the virus spread in the wild bird population and spilled over to backyard birds and poultry. As of June 2022, HPAI has been confirmed in 186 commercial flocks and 191 backyard flocks across 34 states, and there have been 1635 wild bird detections. ARS researchers in Athens, Georgia, in collaboration with APHIS scientists from the National Veterinary Services Laboratories, Ames, Iowa, and the Wildlife Services National Wildlife Disease Program, Fort Collins, Colorado, examined the genetic information of the viruses to determine the origin and evolution of the viruses. Analysis conducted with full genome sequences from more than 1,369 viruses, corroborated virus spread via wild bird circulation, with most detections in poultry premises and non-poultry flocks consistent with independent wild bird introductions. After an initial introduction, minimal farm to farm spread for a limited number of outbreaks was also found. Additionally, the original virus recombined with several different North American origin avian influenza (AI) viruses, and these H5N1 reassortants continue to predominate among wild bird detections and have been identified in commercial and backyard flocks across several states. A representative HPAIV isolate was characterized by experimental challenge of virus in chickens and turkeys that demonstrated that turkeys were more susceptible to virus infection and transmitted the virus better than chickens. These studies provide essential information on the epidemiology of HPAIV in poultry, and data to inform models used to identify what type of samples and when to collect them for optimal virus detection during an outbreak. Data was shared with stakeholders and the United States Department of Agriculture-Animal and Plant Health Inspection Service (USDA-APHIS) for epidemiological modeling and to help responders recognize the clinical signs in the field. (Objectives 1 and 2). Diagnostic tests were updated to include internal positive controls and sample collection paradigms were evaluated to ensure best practices in the field. An initial antigen characterization of the US HPAIV viruses was also conducted to help determine how closely related the virus is to HPAIV strains from the U.S. in 2015 and to determine how closely matched they are to available vaccines. Specific reference serum was produced and shared with USDA-APHIS to support diagnostic efforts (Objective 3). In addition to avian influenza virus (AIV), we worked with SARS-CoV-2 in the context of an emerging virus affecting agriculture. Efforts were primarily directed to support the mink industry and included determining the thermal stability of SARS-CoV-2 virus in mink products (pelts) and farm waste (manure). Commercial point-of-care COVID-19 antigen tests were evaluated with environmental samples from mink farms for use as a non-invasive, rapid test for infected herds. In addition, a cell-culture based assay was developed to predict species susceptibility based on expression of the ACE2 protein from different species. This model strongly correlated with animal challenge studies to predict susceptibility. Also, under Objective 2, significant progress was made towards identifying genetic markers for adaptation and/or increased virulence of AI viruses in different avian species. Poultry outbreaks caused by H7 subtype low pathogenicity avian influenza viruses (LPAIVs) have occurred several times in North America in the last 20 years, and on six occasions the virus mutated to HPAIV. To understand the genetic basis of why these H7 subtype viruses repeatedly spread from the wild bird reservoir into poultry, next generation sequencing of numerous H7 viruses was conducted and revealed that many wild bird AIV gene combinations were associated with HPAIV outbreaks. Common genetic changes were identified once the viruses were introduced into poultry. Within-host virus dynamics contributing to the evolution of AIVs was also examined. Using samples from studies conducted in chickens, turkeys, and ducks, the variation of virus found within the host was explored. The results suggest that the maintenance of within-host virus diversity is an indication of better adaptation of a virus to a particular host. Objective 3, in order to develop strategies to effectively control AI viruses, we completed the evaluation of environmental sample collection from poultry farms using surrogate viruses. Sample collection devices were tested and pre-moistened 4”x4” cotton gauze was found to be the most sensitive collection device for numerous surfaces. Sites on the farm was compared for virus detection to identify targets for testing and cleaning/disinfection. Areas that were near bird level where dust accumulated and areas where caretakers touched surfaces were the most contaminated. To help support vaccination strategies a meta-analysis on vaccine protection on poultry was conducted. A thorough search of the literature was conducted and studies on HPAIV efficacy in chickens or turkeys with comparable methods were selected. Data were extracted and odds-ratios for protection against mortality and virus shed were calculated for: type of vaccine, strength of antibody response, relationship between the vaccine and challenge virus, and age at vaccination. Analysis are being completed but indicate a complex relationship among the metrics evaluated. Also, we completed studies with recombinant herpesvirus of turkey vaccines with multiple inserts from poultry pathogens that demonstrated efficacy against H5 avian influenza, Marek’s disease, and additional poultry viral diseases, and an application method that fits into day-old or in ovo vaccination program of chickens in the field. Support to the National Veterinary Services Laboratories, APHIS continues with updated or improved real-time RT-PCR tests.

Accomplishments
1. Optimal methods identified for virus detection in poultry farms. Uniform methods for environmental sample collection that would verify cleaning and disinfection of infected premises have not been developed. ARS researchers in Athens, Georgia, in collaboration with scientists from the University of Georgia, conducted studies in several housing types using surrogate viruses for avian influenza virus to identify the best sample collection devices and areas from which to collect samples in poultry housing. Cotton gauze pads that were pre-moistened with transport media recovered the most virus from the most samples compared to the 3 other devices (swabs). The most virus was detected from areas where dust settled near bird level, and sites heavily touched by personnel were the best places for virus detection. The results have been communicated to stakeholders and have informed testing during 2022 highly pathogenic avian influenza (HPAIV) outbreak.

2. The pathobiology of H7N3 low and high pathogenicity avian influenza viruses from the United States outbreak in 2020 differs between turkeys and chickens. An outbreak caused by H7N3 low pathogenicity avian influenza virus (LPAIV) occurred in commercial turkey farms in the states of North Carolina (NC) and South Carolina (SC), United States, in March of 2020. Subsequently, H7N3 high pathogenicity avian influenza virus (HPAIV) was detected on a farm in SC. The pathogenicity of the H7N3 HPAIV and two LPAIV isolates were studied in turkeys and chickens by ARS researchers in Athens, Georgia. High infectivity and transmissibility were observed with both the LPAIVs and with the HPAIV in turkeys. In contrast, the dose to infect chickens was higher than for turkeys and no transmission was observed. These results show clear differences in the pathobiology of AIVs in turkeys and chickens and corroborate the high susceptibility of turkeys to both LPAIV and HPAIV infections. These types of studies provide essential information on the epidemiology of AIVs in poultry, and inform models used to identify the type of samples and the time to collect them for optimal virus detection during an outbreak.

3. Mexican lineage H5N2 low pathogenic avian influenza viruses continue to change and are well adapted to chickens. The Mexican lineage H5N2 low pathogenic avian influenza viruses (LPAIVs) were first detected in 1994 and mutated to highly pathogenic avian influenza viruses (HPAIVs) in 1994-1995 causing widespread outbreaks in poultry. By using vaccination and other control measures, the HPAIVs were eradicated but the LPAIVs continued circulating in Mexico and spread to several other countries. ARS researchers in Athens, Georgia, analyzed the full genome sequences of 49 H5N2 LPAIVs detected from 1994 to 2019. Genetic changes and reassortments between H5N2 viruses and with previously unidentified avian influenza viruses were identified. Increased infectivity and transmission were observed with a 2011 H5N2 LPAIV in chickens compared to a 1994 virus also demonstrated improved adaptation of the virus to chickens. The genetic changes that occur as this lineage of H5N2 LPAIVs continues circulating in poultry is concerning not only because of the effect of these changes on vaccination efficacy, but also because of the potential of the viruses to mutate to HPAIV.

4. Low pathogenicity H7N3 avian influenza viruses have higher within-host genetic diversity than a closely related high pathogenicity H7N3 virus in infected turkeys and chickens. Within-host viral diversity offers a view into the early stages of viral evolution occurring after a virus infects a host. ARS researchers in Athens, Georgia, examined within-host viral diversity in turkeys and chickens experimentally infected with closely related H7N3 avian influenza viruses (AIVs). Consistent with the high mutation rates of AIVs, an abundance of intra-host changes was observed in all samples collected. Furthermore, a small number of common changes were observed between turkeys and chickens, or between directly inoculated and contact-exposed birds. Notably, the LPAIVs have significantly higher number and diversity of changes than the HPAIV in both turkeys and chickens. These findings highlight the dynamics of AIV populations within-host and the potential impact of genetic changes on AIV virus populations and evolution.

5. Multiple gene segments are associated with enhanced virulence of H5N8 highly pathogenic avian influenza virus in mallards. Highly pathogenic avian influenza viruses (HPAIVs) from the H5Nx Goose/Guangdong/96 lineage continue to cause outbreaks in domestic and wild bird populations around the world. Epidemiological evidence has shown that wild waterfowl play a major role in the spread of these viruses. ARS researchers in Athens, Georgia, demonstrated that three viral gene segments (PB2, NP, and M) are associated with enhanced virulence of H5N8 HPAIVs in mallards. Phylogenetic analyses established that gene segments related to the more virulent 2016 H5N8 virus have persisted in the contemporary H5Nx HPAIV gene pool until 2020. These findings advance our knowledge on the pathobiology of HPAIVs in waterfowl and has potential implications in the ecology and epidemiology of H5Nx HPAIV in wild bird populations.

6. Age associated changes in recombinant H5 highly pathogenic and low pathogenic avian influenza hemagglutinin tissue binding in domestic poultry species. The 2014 outbreak of highly pathogenic avian influenza (HPAIV), known as clade 2.3.4.4c, led to the culling of millions of commercial chickens and turkeys and death of many wild bird species. ARS researchers in Athens, Georgia, discovered in this outbreak, older chickens and turkeys were more commonly infected and succumbed to clinical disease compared to younger aged birds such chicken broilers. Differences in H5 Hemagglutinin (HA) tissue binding was evaluated across age groups in chickens, ducks (Mallard, Pekin, Muscovy) and turkeys. Age-related differences in HA binding of the LPAIV and HPAIV demonstrated in this study may partially, but not completely, explain differences in host susceptibility to infection observed during avian influenza outbreaks and in experimental infection studies.

7. Avian influenza viruses replicate differently in gulls and mallards. Wild aquatic birds are natural reservoirs of low pathogenicity avian influenza viruses (LPAIVs). Laughing gulls inoculated with four gull-origin LPAIVs had a predominate respiratory infection. ARS researchers in Athens, Georgia, discovered that by contrast, mallards inoculated with two mallard-origin LPAIVs became infected and had similar virus amounts in respiratory and fecal samples. The trend toward mostly respiratory shedding in gulls suggest a greater role of direct bird transmission in maintenance, whereas mallards shedding suggests importance of fecal-oral transmission through water contamination. This information is important for understanding the epidemiology of avian influenza viruses in the wild bird reservoir.

8. Virus adaption following experimental infection of chickens with a domestic duck low pathogenic avian influenza isolate from the 2017 USA H7N9 outbreak identifies mutations in multiple gene segments. In March 2017, highly pathogenic (HP) and low pathogenic (LP) avian influenza virus (AIV) subtype H7N9 were detected in poultry farms and backyard birds in several states in the southeast United States. Because interspecies transmission is a known mechanism for evolution of AIVs, ARS researchers in Athens, Georgia, sought to characterize infection and transmission of a domestic duck-origin H7N9 LPAIV in chickens and genetically characterize the viruses replicating in the chickens. Chickens became infected, with overt clinical signs of disease and shedding through both respiratory and fecal routes, and next generation sequencing (NGS) analysis identified numerous mutations in the polymerase genes (i.e., PA, PB1, and PB2) and the hemagglutinin (HA) receptor binding site in viruses recovered from the chickens, indicating possible virus adaptation in the new host. This work demonstrates that the H7N9 viruses could readily jump between avian species, which may have contributed to the evolution of the virus and its spread in the region.

9. Transmission dynamics of low pathogenicity avian influenza (H2N2) viruses in live bird markets of the Northeast United States of America, 2013–2019. Live bird market (LBM) surveillance was conducted in the Northeast United States (US) to monitor for the presence of avian influenza viruses (AIV) in domestic poultry and market environments. 384 H2N2 low pathogenicity AIVs (LPAIV) were isolated by ARS researchers in Athens, Georgia, from LBM system of New York, Connecticut, Rhode Island, New Jersey, Pennsylvania, and Maryland during 2013–2019 by the National Veterinary Services Laboratories and sequenced. Comparative analysis showed that a wild-bird-origin H2N2 virus may have been first introduced into the LBMs in Pennsylvania and independently evolved since March 2012 followed by spread to LBMs in New York City during late 2012–early 2013. LBMs in New York state played a key role in the maintenance and dissemination of the virus to LBMs in the Northeast US. The frequent detections in the domestic ducks and market environment with viral transmissions between birds and environment possibly led to viral adaptation and circulation in domestic gallinaceous poultry in LBMs, suggesting significant roles of domestic ducks and contaminated LBM environment as reservoirs in maintenance and dissemination of H2N2 LPAIV.

10. Patent HA-specific influenza virus attenuated vaccine comprising mutations in segment 7 and uses therefor. Patent No. US 11,214,799 B2. Date of Patent Jan. 4, 2022. Continued global outbreaks of H5Nx highly pathogenic avian influenza viruses (HPAIV) have been reported in poultry since the emergence of the Asian Goose/Guangdong lineage of HPAIV H5N1. Consequently, vaccines have been developed and employed to protect commercial and non-commercial poultry flocks. However, constant changes in virus immunogenetics makes vaccine candidates that prevent morbidity, mortality, reduce shedding, and prevent transmission a moving target. A novel, attenuated H5 influenza vaccine, has been developed by ARS researchers in Athens, Georgia. The virus vaccines (M2 and M42) infected chickens efficiently and stimulated robust immune responses; however, unlike the wild type virus, they did not transmit to naïve-contact birds but did protect 100% of birds from lethal challenges with H5 HPAIV strains from North American and Goose/Guangdong lineages, and significantly reduced virus shedding. Taken together, these studies demonstrate a strategy for developing a non-transmittable but highly protective live attenuated virus for AIV.

11. Development of an in vitro model for animal species susceptibility to SARS-CoV-2. The SARS-CoV-2 virus has caused a worldwide pandemic because of the virus’s ability to transmit efficiently human-to-human. A key determinant of infection is the attachment of the viral spike protein to the host receptor know as angiotensin-converting enzyme 2 (ACE2). Because of the presumed zoonotic origin of SARS-CoV-2, there is no practical way to assess the susceptibility of every animal species to SARS-CoV-2 by direct challenge studies. Therefore, ARS researchers in Athens, Georgia, developed a unique and sensitive cell-line culture that can be modified using two key host receptors (ACE2 and another called TMPRSS2) and serve as a model to test the potential infection and transmission of the SARS-CoV-2 in a wide range of hosts. Their resulting models showed that SARS-CoV-2 can replicate in cat, Golden hamster, and goat species, but not pig or horse, which correlated with the results of reported challenge studies. The virus also grew in cells expressing genes from the bat species but not as well. The development of this cell culture model allows for more efficient testing of the potential susceptibility of many different animal species for SARS-CoV-2 and emerging variant viruses.

Review Publications
Youk, S., Leyson, C., Parris, D., Kariithi, H., Suarez, D.L., Pantin Jackwood, M.J. 2022. Phylogenetic analysis, molecular changes, and adaptation to chickens of Mexican lineage H5N2 low-pathogenic avian influenza viruses from 1994 to 2019. Transboundary and Emerging Diseases. https://doi.org/10.1111/tbed.14476.
Chrzastek, K., Kraberger, S., Schmidlin, K., Fontenele, R.S., Kulkarni, A., Chappell, L., Dufor-Zavala, L., Kapczynski, D.R., Varsani, A. 2021. Diverse single-stranded DNA viruses identified in chicken buccal swabs. Microorganisms. 9(12):2602. https://doi.org/10.3390/microorganisms9122602.
Mo, J., Stephens, C.B., Jordan, B., Ritz, C., Swayne, D.E., Spackman, E. 2022. Optimizing sample collection methods for detection of respiratory viruses in poultry housing environments. Transboundary and Emerging Diseases. https://doi.org/10.1111/tbed.14547.
Leyson, C.M., Criado, M.F., Youk, S., Pantin Jackwood, M.J. 2022. Low pathogenicity H7N3 avian influenza viruses have higher within-host genetic diversity than a closely related high pathogenicity H7N3 virus in infected turkeys and chickens. Viruses. 14(3):554. https://doi.org/10.3390/v14030554.
Kapczynski, D.R., Sweeney, R.P., Spackman, E., Pantin Jackwood, M.J., Suarez, D.L. 2022. Development of an in vitro model for animal species susceptibility to SARS-CoV-2 replication based on expression of ACE2 and TMPRSS2 in avian cells. Virology. 569:1-12. https://doi.org/10.1016/j.virol.2022.01.014.
Chrzastek, K., Segovia, K., Torchetti, M., Killian, M.L., Pantin Jackwood, M.J., Kapczynski, D.R. 2021. Virus adaption following experimental infection of chickens with a domestic duck low pathogenic avian influenza isolate from the 2017 USA H7N9 outbreak identifies polymorphic mutations in multiple gene segments. Viruses. 13(6):1166. https://doi.org/10.3390/v13061166.
Criado, M.F., Leyson, C.M., Youk, S., Deblois, S.M., Olivier, T.L., Killian, M.L., Torchetti, M.L., Parris, D.J., Spackman, E., Kapczynski, D.R., Suarez, D.L., Swayne, D.E., Pantin Jackwood, M.J. 2021. The pathobiology of H7N3 low and high pathogenicity avian influenza viruses from the United States outbreak in 2020 differs between turkeys and chickens. Viruses. 13(9):1851. https://doi.org/10.3390/v13091851.
Spackman, E., Pantin Jackwood, M.J., Sitaras, I., Stephens, C.B., Suarez, D.L. 2021. Identification of efficacious vaccines against contemporary North American H7 avian influenza viruses. Avian Diseases. 65:113–121. https://doi.org/10.1637/aviandiseases-D-20-00109.
Leyson, C.M., Youk, S., Ferreira, H.L., Suarez, D.L., Pantin Jackwood, M.J. 2021. Multiple gene segments are associated with enhanced virulence of clade 2.3.4.4 H5N8 highly pathogenic avian influenza virus in mallards. Journal of Virology. 95(18):e00955-21. https://doi.org/10.1128/JVI.00955-21.
Jerry, C., Stallknecht, D.E., Leyson, C., Berghaus, R., Jordan, B., Pantin Jackwood, M.J., Franca, M.S. 2021. Age-associated changes in recombinant H5 highly pathogenic and low pathogenic avian influenza hemagglutinin tissue binding in domestic poultry species. Animals. 11(8):2223. https://doi.org/10.3390/ani11082223.
Criado, M.F., Moresco, K.A., Stallknecht, D.E., Swayne, D.E. 2021. Low-pathogenicity influenza viruses replicate differently in laughing gulls and mallards. Influenza and Other Respiratory Viruses. 15(6):701-706. https://doi.org/10.1111/irv.12878.
Kapczynski, D.R. 2021. Foreword-Avian Immunology 3rd Edition. In: Kaspers, B., Schat, K.A., Gobel, T.W., Vervelde, L., editors. Avian Immunology. 3rd edition. London, England: Academic Press p.xix-xx. https://doi.org/10.1016/C2018-0-00454-5.
Chung, D.H., Torchetti, M.K., Killian, M.L., Swayne, D.E., Lee, D. 2022. Transmission dynamics of low pathogenicity avian influenza (H2N2) viruses in live bird markets of the Northeast United States of America, 2013-2019. Virus Evolution. 8(1). Article veac009. https://doi.org/10.1093/ve/veac009.