Research Project: Intervention Strategies to Prevent and Control Disease Outbreaks Caused by Emerging Strains of Avian Influenza Viruses

Location: Exotic & Emerging Avian Viral Diseases Research

2021 Annual Report

Objectives
This in-house project has four general objectives, each of which is broken into sub-objectives: 1. Conduct studies to understand avian influenza viruses evolution and population dynamics, including the characterization of variant and emerging avian influenza viruses in live poultry markets and commercial production systems, and exploring the impact of variable host susceptibility on avian influenza virus persistence in different ecosystems. 1.1. Characterize new and variant avian influenza virus (AIV) isolates. 1.2. Investigate selection for AIV antigenic variation. 2. Elucidate the host-pathogen interactions of avian influenza virus infections, including determining the role of mutations at receptor binding sites on replication and pathogenesis, especially which mutations are important in changing host specificity, identifying molecular determinants of tissue tropism, and identifying molecular determinants of virulence in target animal species. 2.1. Identify genetic markers for AIV adaptation and/or increased virulence in different avian species. 2.2 Investigate host-specific factors associated with infectivity, pathogenicity and transmissibility of current and emerging AIV. 3. Conduct comparative immunology studies of avian species to determine variations in protective host defense mechanisms to avian influenza infections, including determining the innate and adaptive immune response to influenza virus infection in different avian species that are either susceptible, tolerant, or resistant to infection, and determining the contribution of host genetics on innate protection and other novel methods for disease resistance. 3.1. Identify innate defense mechanisms associated with disease resistance to AIV. 3.2. Characterize humoral responses to AIV and identify epitopes associated with adaptive immunity. 3.3. Improve resistance against AIV infections in poultry. 4. Develop intervention strategies to effectively control avian influenza viruses and contain disease outbreaks, including identifying risk factors in poultry production that favor transmission and spread of avian influenza viruses, improving existing diagnostic tests and testing strategies for avian influenza virus surveillance, detection, and recovery from disease outbreaks, developing new vaccine platforms designed to rapidly control and prevent avian influenza virus outbreaks in the various components of poultry production, and characterizing new or emerging poultry disease pathogens to evaluate potential impact on the U.S. poultry industry. 4.1. Maintain, update and improve diagnostic tests for avian influenza. 4.2. Evaluate vaccine strategies to better control and prevent avian influenza virus outbreaks.

Approach
These four 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. thus, experiments will often contribute to more than one objective. The first objective includes the characterization of new strains of AIV which constantly emerge in nature as well as the elucidation of how the virus changes under immune pressure using an experimental approach. 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 aims to improve our understanding of the avian immune response to AIV infection and vaccination in key poultry species. The fourth objective will improve current practical intervention strategies including diagnostics and vaccines.

Progress Report
This is the final report for this project, which will be replaced with a bridging project pending completion of research review. Two H7 highly pathogenic avian influenza virus (HPAIV) outbreaks occurred in poultry in the US during the course of this project: a H7N9 subtype outbreak in 2017, and a H7N3 outbreak in 2020. Research priorities were adjusted to provide data critical for the field response to aid industry and government responders. ARS researchers in Athens, Georgia, ensured the effectiveness of diagnostic tests used to identify the new strains, and evaluated the pathogenesis of the viruses in different avian hosts to inform surveillance. Advanced methods were used to elucidate the origin and evolution of the viruses and to determine genetic markers for host adaptation and virulence in different bird species. Vaccine trials were also conducted to identify the best vaccines should it be decided that vaccination would be necessary as part of the control efforts. Viruses from the two HPAIV U.S. outbreaks, as well as other Avian influenza viruses (AIV) isolates relevant to poultry, were characterized for their pathogenesis in several bird species. Each virus had unique infectivity, transmissibility, clinical signs, and shedding patterns that can impact how the viruses transmit and are diagnosed and controlled in the field. The pathobiology of the H7N9 low pathogenicity avian influenza virus (LPAIV) and HPAIV that caused the outbreak in chicken farms in Tennessee in 2017 was examined in commercial broiler breeders, the bird type most affected during the outbreak, and layer type chickens. Poor transmission with the LPAIV indicated that factors other than the type of bird were involved in the epidemiology of the outbreak. The pathogenicity in turkeys and chickens of the H7N3 LPAIV and HPAIV that caused the outbreak in turkeys in North and South Carolina in 2020 was evaluated and research findings confirmed that turkeys were much more susceptible to virus infection. Turkeys also shed virus in feces increasing the viral load in the environment. H5Nx HPAIVs of the Goose/Guangdong/96 lineage continue to circulate worldwide affecting both poultry and wild birds. One of these viruses caused the devastating poultry outbreak in the U.S. in 2015. The ARS researchers determined that the virus became more adapted and infectious to chickens as it spread in poultry, which likely complicated its eradication. In other studies, it was concluded that H5Nx HPAIV easily infected domestic ducks and geese, and that wild diving ducks, American black ducks, and lesser scaup, were also susceptible to infection, emphasizing the need of improved biosecurity to prevent contact of poultry with wild waterfowl. It was also discovered that broilers were more resistant to HPAIV infection than egg type chickens. This may explain the lack of affected broiler farms during the 2015 outbreaks. H2N2 LPAIV that has persisted in live bird markets (LBMs) in the Northeastern U.S. since 2014 was also studied. LBM H2N2 viruses were examined in chickens, Pekin ducks, and guinea fowl, birds commonly present in LBMs, with guinea fowl found to be the most susceptible to virus infection. Monitoring the biological characteristics of viruses from the LBM system is important because these viruses have the potential to be exposed to numerous hosts, including humans, which is conducive to crossing the species barrier and broadening host range. In addition, other novel emerging or exotic viruses that could pose a threat to poultry were evaluated. Because poultry are so widespread and have close and extended contact with humans and other mammals in many production systems including LBMs, susceptibility studies were conducted with SARS-CoV-2, and another human coronavirus (MERS-CoV), in five common poultry species and embryonating chicken eggs (ECE). Chickens, turkeys, ducks, quail, and white geese challenged with SARS-CoV-2 or MERS-CoV did not become infected and neither virus replicated in ECE, so it is unlikely that poultry will serve a role in the maintenance of either virus. Further work with a cell culture model system to predict host susceptibility is ongoing. Work was also conducted on adenovirus, Egg Drop Syndrome, which caused outbreaks in commercial layer for the first time in the U.S. The presence of the virus in the US has created a trade issue with some countries. Studies were conducted to examine the risk of spread from infected poultry through poultry products. It was determined that the virus was present for only short periods of time in broiler chickens and that spread of the virus through poultry meat was unlikely. Significant progress was made toward identifying genetic markers for adaptation and/or increased virulence of AIV in different avian species. Changes were identified in the 2015 H5N2 HPAIV proteins that contributed to the adaptation of this virus in chickens which allowed for increased transmission. The genetics of virulence of H5Nx HPAIV (H5N8) in waterfowl was also assessed. Multiple changes in the viral proteins were identified as the major factor contributing to mortality. Poultry outbreaks caused by H7 subtype 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 spill from the wild bird reservoir into poultry, next generation sequencing of numerous H7 viruses revealed that many wild bird AIV gene combinations were associated with HPAIV outbreaks. ARS researchers were able to determine common genetic changes that were introduced with poultry adaptation. 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. Progress was made toward disease resistance in poultry through genetic engineering. Utilizing the CRISPR/Cas system, genetic mutations in the germplasm in cell culture were introduced. Progress was also made identifying the regulatory molecules (promoters) used to drive expression of the introduced genes. As constant expression can be deleterious to the host, the chickens own natural systems for on/off expression was utilized. Results in cell culture have demonstrated significant reduction in virus replication. Current vaccines rely mostly on eliciting antibody responses against the viral hemagglutinin (HA) protein. However, this type of vaccine may not be long lasting and doesn’t prevent virus shedding after challenge. In collaboration with the University of Edinburgh and The University of Georgia, ARS researchers developed attenuated live virus H5 LPAIV vaccines based on mutations in the matrix gene demonstrating protection against four different HPAIV with minimal virus shedding. The development of a safe attenuated live virus vaccine allows for improved immunity over traditional and recombinant vaccines, and can be mass applied. Support to the National Veterinary Services Laboratories, APHIS continues with updated or improved real-time RT-PCR tests. A test for H2 LPAIV was developed to provide rapid confirmation to support the control of H2N2 LPAIV in the LBMs. Similar efforts were performed on H6N1 viruses from turkeys. An in-depth sequence analysis showed that a previously published H6 PCR test would be a good option for rapid confirmation of this lineage of virus. Efficacious vaccine strains for H7 AIVs present in North America were identified allowing for vaccines to be rapidly produced for poultry if needed. Progress was also made to characterize how immunity changes H7 subtype viruses. Like the influenza vaccine for humans, vaccines used in poultry need to be periodically updated to compensate for changes in the virus as it evades immunity. This process was characterized by using eggs instead of chickens showing that the virus changes can be monitored in real time. It was also determined that higher doses of vaccine can be used to maintain adequate protection until an updated vaccine can be produced. Vaccination for AIV is most often done with inactivated vaccines, but little data exists on the optimal adjuvants (chemicals added to improve vaccines) for use in chickens. Ten adjuvants for antibody response and protection against exposure to a virulent AIV strain were compared. It was determined that mineral oil based additives performed the best in chickens. An examination of the potential inhibition caused by chicken maternally derived antibodies (MDA) on the efficacy of virus vectored vaccines in AIV vaccination of broiler chickens was conducted. The MDA provides early protection from disease, but may interfere with active immunity in young chickens. MDA to AIV had minimal impact on the effectiveness of an AIV recombinant turkey herpesvirus vaccine in commercial broilers, however, MDA to AIV and/or Newcastle disease virus (NDV) can prevent development of protective immunity when using a spray- applied AIV recombinant NDV vaccine. This information is critical for developing effective vaccination programs to control avian influenza in poultry. To aid outbreak control and ensure that no viable virus remains after a fallow period or cleaning and disinfection, it was determined that the use of cotton gauze recovered the most virus in areas near the floor and was considered optimal for use at collection sites. The ability of LPAIV strains adapted to chickens to contaminate eggs laid by infected hens was determined, and although rare, virus could be detected on the surface of eggs, emphasizing that eggs should be sanitized prior to movement if they are coming from an infected flock. The project reaches it's 5 year term September 30, 2021.

Accomplishments
1. Identification of protective vaccines against contemporary North American H7 avian influenza viruses. In the past 5 years there have been 3 outbreaks of highly pathogenic H7 avian influenza in poultry in the United States (US). H7 avian influenza is also present in poultry in Mexico. ARS researchers in Athens, Georgia, tested several virus stains for use as vaccines against H7 viruses recently seen in North America and which would be expected to be similar to new H7 viruses that could emerge in the future. Two US-origin, non-virulent strains inducing a strong immune response in chickens were identified as good vaccine candidates. If an outbreak were to occur in the US and become widespread these virus strains could be rapidly utilized to produce a vaccine to protect poultry.

2. Selection and antigenic characterization of immune-escape mutants of H7N2 low pathogenic avian influenza virus. Like the influenza viruses affecting humans, avian influenza viruses change their genetic sequence frequently (mutations), so vaccines need to be changed regularly to match circulating strains to maintain adequate protection for poultry. If we understood the mutation process better, vaccination procedures, such as when and how much vaccine is needed, could be optimized. ARS researchers in Athens, Georgia, conducted a study to learn how avian influenza viruses mutate by simulating virus infection in vaccinated chickens by using eggs instead. They discovered that it took 20-30 generations for the virus to change, and that a vaccine could compensate for the changes if given at higher doses. This information provides guidelines to establish adequate vaccine doses, improving the vaccination process. An efficient and effective avian influenza vaccination for poultry will help save money and maintain a stable food supply when avian influenza outbreaks occur.

3. Identification of optimal sample collection for the detection of virus in the environment including in contamination in wire poultry cages. Before returning to normal business operations after an outbreak with avian influenza or any foreign animal disease, it must be confirmed that no virus remains in the farm environment. Better ways are needed to test wire chicken cages, which tend to tear sampling devices. Testing methods need to balance cost and effectiveness and only target the areas where virus can hide. ARS researchers in Athens, Georgia, conducted a study comparing several affordable and practical methods for sample collection for virus detection. The best method was to moisten 4-inch squares of cotton gauze and wipe areas of the coop that the chicken could touch. Using the most sensitive and efficient virus detection method helps to re-open farms after an outbreak by ensuring that poultry can be re-stocked safely, giving confidence to customers and trade partners that the threat of virus resurgence is minimized or even eliminated.

4. The susceptibility of five poultry species and chicken embryos to infection with SARS-CoV-2 and MERS-CoV. When SARS-CoV-2, the virus that causes COVID-19, first emerged the host range was unknown, and it was critical to determine which animal species could serve as reservoirs for humans and which species could be sickened by the virus. ARS researchers in Athens, Georgia, determined whether key poultry species, chickens, turkeys, Pekin ducks, Japanese quail, and Chinese domestic geese, could be infected with SARS-CoV-2. MERS-CoV, a similar but less widespread virus, was also tested. None of the poultry species showed any sign of being infected with either virus. Chicken eggs, a common laboratory system for making vaccines, were also tested and were not able to support infection with the virus. This established that poultry species cannot serve as sources of virus for humans or other animals and that SARS-CoV-2 and MERS-CoV will not harm poultry production.

5. Determining how the H5N2 highly pathogenic avian influenza virus adapted in chickens. Highly pathogenic avian influenza viruses of the H5Nx Goose/Guangdong lineage continue to circulate widely around the world affecting both poultry and wild birds. In the United States, a virus from this lineage caused a devastating outbreak in poultry in 2015. In time, the virus became more adapted to chickens, replicating and transmitting better in these birds. ARS researchers in Athens, Georgia, determined the genetic changes in the virus that contributed to this increase in host virus fitness by conducting bird experiments with viruses from early and later in the outbreak, and by analyzing the genetic sequence information of all the viruses detected during the outbreak. Genetic changes in four virus proteins, the PB1, NP, HA, and NA, were found to contribute to the adaptation of this virus in chickens. This knowledge is important for understanding how H5Nx HPAI viruses spreads to poultry farms.

6. Characterizing the avian influenza virus subtype H7N3 that caused the outbreak in turkeys in the United States in 2020. Highly pathogenic avian influenza viruses have devastating impact on the poultry industry. An outbreak of H7N3 low pathogenicity avian influenza virus of wild bird origin occurred in commercial turkeys in North and South Carolina in April 2020 and the virus subsequently changed to the highly pathogenic form. ARS scientists in Athens, Georgia, in collaboration with scientists from the University of Connecticut and the National Veterinary Services Laboratory, examined the genetic information of the viruses to determine the origin and evolution of the viruses, including the change in the hemagglutinin gene that resulted in the change to be a highly pathogenic virus. Another important change found in some of the viruses was a small deletion in the neuraminidase gene which has been commonly associated with increased adaptation of the virus to chickens and turkeys. Wild bird origin H7 avian influenza viruses have repeatedly spilled over from wild birds into poultry, and on multiple occasions have genetically changed into highly pathogenic avian influenza, highlighting the importance of avian influenza surveillance in wild birds and the continued vigilance, biosecurity, and surveillance in poultry.

7. Preparing for future human influenza pandemic viruses by producing vaccines against strains that are currently circulating in poultry. The highly pathogenic avian influenza viruses from the H5N1 A/goose/Guangdong/1996 lineage are widely found in poultry in several countries in Asia and Africa and have caused sporadic lethal infections in humans. Vaccines are important for virus control both for poultry and in prepandemic preparedness for humans. The sustained circulation of H5N1 Gs/GD lineage in the agricultural sector and some wild birds has led to the evolution and selection of distinct viral lineages involved in escape from vaccine protection. ARS researchers in Athens, Georgia, evaluated inactivated prepandemic vaccine strains, focusing on the genetic and antigenic diversity of field H5N1 viruses from the agricultural sector and assessing cross-protection in a chicken challenge model. Antigenic changes associated with virus escape from antibody neutralization in chickens were determined. Knowledge of these immunodominant regions is essential to proactively develop diagnostic tests, improve surveillance platforms to monitor avian influenza outbreaks, and design more efficient and broad-spectrum agricultural and human prepandemic vaccines.

8. Novel vaccines for avian influenza were developed and evaluated for protection against genetically diverse H5 high pathogenicity avian influenza viruses. The vaccines to the hemagglutinin protein were selected using an advanced computationally optimized broadly reactive antigen (COBRA) approach that considers a large number of high pathogenicity avian influenza viruses of H5 Goose/Guangdong lineage and makes a design that can potentially protects against all of them. The emergence of vaccine-resistant field viruses underscores the need for a broadly protective H5 influenza A vaccine. ARS researchers in Athens, Georgia, tested experimental herpesvirus of turkey (vHVT) vectored vaccines containing either a wild-type derived H5 inserts, or computer designed combination inserts. Protection was confirmed for all the tested vaccines, which provided clinical protection against virus challenge viruses and significantly decreased shedding of the virus. The wild-type-derived H5 vaccines elicited protection mostly against close antigenically viruses, while the COBRA-derived vaccines elicited antibody responses against antigenically diverse strains, which could be used to protect against future avian influenza variants.

9. The pathogenesis of H5Nx clade 2.3.4.4 highly pathogenic avian influenza virus in surf scoters, a large sea duck. In 2015, the United States experienced the worst foreign animal disease outbreak in our history, which was caused by avian influenza, and cost the economy an estimated $3 billion. It is believed that the virus was carried to North America by wild waterfowl. Many waterfowl species can migrate long distances and are known to be the natural hosts for avian influenza viruses. In order to expand our understanding of which birds can carry the virus, surf scoters, a large sea duck, ARS researchers in Athens, Georgia, tested them for their ability to become infected with H5Nx avian influenza viruses. Like many other ducks, surf scoters were able to become infected with the virus without becoming sick. This suggests that surf scoters, and possibly other sea ducks, could be involved in spreading avian influenza viruses over long distances.

10. Recombinant hemagglutinin proteins provided insight into binding of H5 subtype avian influenza viruses to cells of wild and domestic birds. H5 subtype Goose/Guangdong lineage highly pathogenic avian influenza viruses continue to cause devastating effects across wild and domestic bird populations. Influenza viruses use the hemagglutinin protein on the surface of the virus to attach to host cells and initiate infection. ARS researchers in Athens, Georgia, with collaborators at the University of Georgia, investigated differences in the intensity and distribution of cell binding of the hemagglutinin protein of a H5 highly pathogenic virus compared to a H5 low pathogenic avian influenza virus. They demonstrated that the highly pathogenic virus has higher tropism to tissues from a variety of bird species. These findings support why highly pathogenic viruses are more infectious and have a wider tissue distribution than low pathogenic viruses in experimental trials and natural disease outbreaks.

11. Detection of newly introduced Y280-lineage H9N2 avian influenza viruses in live bird markets in Korea. Live bird markets (LBMs) in Korea have been recognized as a reservoir and source of avian influenza viruses (AIVs); however, little is known about the role of LBMs in the epidemiology of AIVs in this country. H9N2 AIVs are a threat to poultry in many countries and have also caused infections in humans. ARS researchers in Athens, Georgia, with collaborators at the Konkuk University, South Korea, conducted in-depth sequence analysis on viruses isolated between 2006 and 2016. Results showed that there were three separate unique introductions of viruses into the LBMs of Korean domestic duck-origin and two wild aquatic bird-origin AIVs. This resulted in the generation of the five distinct types of H9N2 AIVs circulating in Korea. They also determined that the LBMs are where chickens became infected with the virus, with domestic ducks playing a major role in the transmission and evolution of the H9N2 viruses.

12. Avian influenza viruses may remain infectious for more than seven months in northern wetlands of North America. Avian influenza viruses maintained in wild bird hosts occasionally spillover to domestic poultry where they may cause clinical disease and ultimately lead to economically costly outbreaks. In rare instances, such viruses may spread to other domestic livestock or companion animals and/or infect humans, sometimes resulting in fatal outcomes. Thus, the maintenance of avian influenza viruses in the environment has important implications for economic interests and food security, as well as human and animal health. In collaboration with scientists from the U.S. Geological Survey, ARS scientists from Athens, Georgia, used a combination of field- and laboratory-based approaches to assess if influenza viruses shed by ducks could remain viable for extended periods in surface water within three wetland complexes of North America. The results of this study support surface waters of northern wetlands as a biologically important medium in which avian influenza viruses may be both transmitted and maintained, potentially serving as an environmental reservoir for infectious viruses during the overwintering period of migratory birds.

13. Molecular characterization of low pathogenicity avian influenza virus subtype H5N2 from the Dominican Republic. Low pathogenicity avian influenza virus (LPAIV) subtype H5N2 was detected in poultry in the Dominican Republic in 2007 and re-emerged in 2017. A similar virus has caused outbreaks in poultry in Mexico since 1993 and mutated into highly pathogenic avian influenza virus in 1994. A vaccination program against H5N2 in poultry was established in Mexico and the HPAIV was eradicated. However, LPAIV H5N2 persisted, and related viruses spread to neighboring countries. Since 2007, limited information on H5N2 LPAIVs and few genetic sequences have been reported. ARS researchers in Athens, Georgia, and collaborators from the Dominican Republic and the University of Connecticut, conducted whole-genome sequencing and phylogenetic analysis of nineteen H5N2 LPAIVs identified in the Dominican Republic during 2007–2019, showing the introduction of the virus from Mexico into poultry in the Dominican Republic, then divergence into three distinct genetic subgroups during 2007-2019. This information is important for understanding the epidemiology of the Mexican lineage H5N2 viruses, which continue to be a threat to the United States poultry industry.

14. Detection of H7N1 low pathogenicity avian influenza virus in poultry in the United States during 2018. High pathogenicity avian influenza viruses (HPAIV) have devastating impacts on the poultry industries. With infections in avian species, H5 and H7 low pathogenicity avian influenza viruses (LPAIV) can mutate to HPAIV. ARS researchers in Athens, Georgia, in collaboration with the University of Connecticut and the National Veterinary Service Laboratory in Ames, Iowa, reported three detections of H7N1 LPAIV from poultry in Missouri and Texas during February and March 2018. Detections from all three farms were made through routine premovement testing of either birds or eggs. Complete genome sequencing and comparative phylogenetic analysis suggest that the H7 LPAIV precursor viruses were circulating in wild birds in North America during the fall and winter of 2017 and spilled over into domestic poultry in Texas and Missouri independently during the spring of 2018. Wild bird origin H7 viruses have spilled over repeatedly to poultry. Given the potential to mutate into HPAIV, the present study shows how routine active surveillance in poultry and wild birds is important and necessary for monitoring and control of H5 and H7 viruses.

15. Vaccine protection against recent emergent antigenic variants of H5 avian influenza viruses in Bangladesh. H5N1 highly pathogenic avian influenza viruses have caused outbreaks in poultry in Bangladesh since 2007. ARS researchers in Athens, Georgia, with collaborators in Bangladesh, University of Connecticut and Food and Agricultural Organization reported good protection in chickens for a Chinese inactivated vaccine, and a recombinant herpesvirus of turkeys vaccine with an H5 insert, for protection against most recent field viruses in chickens. However, the emergence of antigenic variants of avian influenza viruses should be continuously monitored, and vaccines should be updated if field efficacy declines.

16. In March 2017, H7N9 highly pathogenic (HP) and low pathogenic (LP) avian influenza virus (AIV) were detected from poultry farms and backyard birds in several states in Southeast United States. Characterization of these viruses is important to understanding their ecology and pathogenesis. ARS researchers in Athens, Georgia, and in collaboration the National Veterinary Service Laboratory in Ames, Iowa, demonstrated virus replication and transmission of a duck LPAI isolate in chickens, with overt clinical signs of disease and shedding through both oral and cloacal routes. Genetic analysis identified numerous mutations in many gene segments and the receptor binding site in viruses recovered from chickens, indicating possible virus adaptation in the new host. Given the ability of these H7N9 viruses to readily jump between avian species, these studies demonstrate the potential of interspecies transmission on the evolution of the virus.

Review Publications
Sitaras, I., Spackman, E., De Jong, M.C., Parris, D.J. 2020. Selection and antigenic characterization of immune-escape mutants of H7N2 low pathogenic avian influenza virus using homologous polyclonal sera. Virus Research. 290:e198188. https://doi.org/10.1016/j.virusres.2020.198188.
Chung, H., Gomez, D.R., Vargas, J.M., Amador, B.L., Torchetti, M.K., Killian, M.L., Swayne, D.E., Lee, D. 2020. Low pathogenicity avian influenza (H5N2) viruses, Dominican Republic. Emerging Infectious Diseases. 26(12):3094-3096. https://doi.org/10.3201/eid2612.200268.
Criado, M.F., Sa-E-Silva, M., Lee, D., Salge, C.A., Spackman, E., Donis, R., Wan, X., Swayne, D.E. 2020. Cross-protection by inactivated H5 prepandemic vaccine seed strains against diverse Goose/Guangdong lineage H5N1 highly pathogenic avian influenza viruses. Journal of Virology. 94(24):e00720-20. https://doi.org/10.1128/JVI.00720-20.
Lee, D., Killian, M., Deliberto, T.J., Wan, X., Lei, L., Swayne, D.E., Torchetti, M. 2021. H7N1 low pathogenicity avian influenza virus in poultry in the United States during 2018. Avian Diseases. 65(1):59-62. https://doi.org/10.1637/aviandiseases-D-20-00088.
Youk, S., Leyson, C., Seibert, B., Perez, D., Jadhao, S., Suarez, D.L., Pantin Jackwood, M.J. 2021. Mutations in PB1, NP, HA, and NA contribute to increased virus fitness of H5N2 highly pathogenic avian influenza virus clade 2.3.4.4 in chickens. Journal of Virology. 95(5):e01675-20. https://doi.org/10.1128/JVI.01675-20.
Youk, S., Lee, D., Killian, M.L., Pantin Jackwood, M.J., Swayne, D.E., Torchetti, M.K. 2020. Highly pathogenic avian influenza A(H7N3) virus in poultry, United States, 2020. Emerging Infectious Diseases. 26(12):2966-2969. https://doi.org/10.3201/eid2612.202790.
Sims, L., Tripodi, A., Swayne, D.E. 2020. Spotlight on avian pathology: can we reduce the pandemic threat of H9N2 avian influenza to human and animal health?. Avian Pathology. 49(6):529-531. https://doi.org/10.1080/03079457.2020.1796139.
Suarez, D.L., Pantin Jackwood, M.J., Swayne, D.E., Lee, S.A., Deblois, S.M., Spackman, E. 2020. Lack of susceptibility to SARS-CoV-2 and MERS-CoV in poultry. Emerging Infectious Diseases. 26(12)3074-3076. https://doi.org/10.3201/eid2612.202989.
Bonney, P.J., Malladi, S., Ssematimba, A., Spackman, E., Torchetti, M., Culhane, M., Cardona, C.J. 2021. Estimating epidemiological parameters using diagnostic testing data from low pathogenicity avian influenza infected turkey houses. Scientific Reports. 11. Article 1602. https://doi.org/10.1038/s41598-021-81254-z.
Bertran, K., Kassa, A., Criado, M., Nunez, I.A., Lee, D., Killmaster, L.F., Sa E Silva, M., Ross, T.M., Mebatsion, T., Pritchard, N., Swayne, D.E. 2021. Efficacy of recombinant Marek’s disease virus vectored vaccines with computationally optimized broadly reactive antigen (COBRA) hemagglutinin insert against genetically diverse H5 high pathogenicity avian influenza viruses. Vaccine. 39(14):1933-1942. https://doi.org/10.1016/j.vaccine.2021.02.075.
Kapczynski,D.R., Segovia,K. 2020. Techniques for the Measurement of Cell Mediated Immune Responses to Avian Influenza Virus. In: Spackman, E. editor. Animal Influenza Virus Methods and Protocols. 3rd edition. New York, NY: Humana Press. p. 227-245. https://doi.org/10.1007/978-1-0716-0346-8_17.
Jerry, C., Stallknecht, D., Leyson, C., Berghaus, R., Jordan, B., Pantin Jackwood, M.J., Hitchener, G., França, M. 2020. Recombinant hemagglutinin glycoproteins provide insight into binding to host cells by H5 influenza viruses in wild and domestic birds. Virology Journal. 550:8-20. https://doi.org/10.1016/j.virol.2020.08.001.
Youk, S., Cho, A.Y., Lee, D., Sol, J., Kim, Y., Lee, S., Kim, T., Pantin Jackwood, M.J., Song, C. 2021. Detection of newly introduced Y280-lineage H9N2 avian influenza viruses in live bird markets in Korea. Transboundary and Emerging Diseases. 00:1-5. https://doi.org/10.1111/tbed.14014.
Lycett, S.J., Pohlmann, A., Staubach, C., Caliendo, V., Woolhouse, M., Beer, M., Kuiken, T., Swayne, D.E., Zoharia, S. 2020. Genesis and spread of multiple reassortants during the 2016/2017 H5 avian influenza epidemic in Eurasia. Proceedings of the National Academy of Sciences(PNAS). 117(34):20814-20825. https://doi.org/10.1073/pnas.2001813117.
Luczo, J., Prosser, D., Pantin Jackwood, M.J., Berlin, A., Spackman, E. 2020. The pathogenesis of H5Nx clade 2.3.4.4 group A highly pathogenic avian influenza virus in surf scoters (Melanitta perspicillata). BioMed Central (BMC) Veterinary Research. 16:351. https://doi.org/10.1186/s12917-020-02579-x.
Ramey, A.M., Reeves, A.B., Drexler, J., Ackerman, J., De La Crus, S., Lang, A., Leyson, C., Link, P., Prosser, D., Robertson, G., Wright, J., Youk, S., Spackman, E., Pantin Jackwood, M.J., Poulson, R.L., Stallknecht, D.E. 2020. Influenza A viruses remain infectious for more than seven months in northern wetlands of North America. Proceedings of the Royal Society B. 287(1934). Article 20200655. https://doi.org/10.1098/rspb.2020.1680.
Mo, J., Spackman, E., Stephens, C.B. 2020. Identification of optimal sample collection devices and sampling locations for the detection environmental viral contamination in wire poultry cages. Transboundary and Emerging Diseases. 00:1-7. https://doi.org/10.1111/tbed.13721.
Kwon, J., Lee, D., Criado, M., Killmaster, L.F., Ali, M., Giasuddin, M., Swayne, D.E. 2020. Genetic evolution and transmission dynamics of clade 2.3.2.1a highly pathogenic avian influenza A/H5N1 viruses in Bangladesh. Virus Evolution. 6(2):veaa046. https://doi.org/10.1093/ve/veaa046.
Kwon, J., Ferreira Criado, M., Killmaster, L.F., Ali, M.Z., Giasuddin, M., Samad, M.A., Karim, M.R., Brum, E., Hasan, M.Z., Lee, D., Spackman, E., Swayne, D.E. 2021. Efficacy of two vaccines against recent emergent antigenic variants of Clade 2.3.2.1a highly pathogenic avian influenza viruses in Bangladesh. Vaccine. 39(21):2824-2832. https://doi.org/10.1016/j.vaccine.2021.04.022.
Mo, J., Youk, S., Pantin Jackwood, M.J., Suarez, D.L., Lee, D., Killian, M., Bergeson, N.H., Spackman, E. 2021. The pathogenicity and transmission of live bird market H2N2 avian influenza viruses in chickens, Pekin ducks, and guinea fowl. Veterinary Microbiology. 260:109180. https://doi.org/10.1016/j.vetmic.2021.109180.