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

Location: Exotic & Emerging Avian Viral Diseases Research

2023 Annual Report

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.

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
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 58 million domestic birds in 47 States in the U.S. since first detected in wild birds in 2021. Under Objective 1, examined the infectivity and transmissibility of one of the early H5N1 HPAIV U.S. isolates in chickens, turkeys, and mallards. It was demonstrated that turkeys were more susceptible to virus infection and transmitted the virus better than chickens. More importantly, the virus was more infectious to both chickens and turkeys than the H5N2 HPAIV that caused the major outbreak in poultry in the U.S. in 2014-2015. Mallards were shown to have the potential to be efficient reservoirs to amplify and disseminate the H5N1 HPAIVs because they excrete high amounts of virus and generally only have mild-to-moderate disease. These studies provide essential information on the epidemiology of these viruses in poultry and wild birds and data to inform models used to identify when and what type of samples to collect for optimal virus detection. Data has been shared with stakeholders and the United States Department of Agriculture-Animal and Plant Health Inspection Service (USDA-APHIS) for epidemiological modeling. Work with SARS-CoV-2 that was initiated with the emergence of the new virus continued in the context of an emerging virus affecting agriculture. Efforts were primarily directed to supporting USDA-APHIS and the U.S. Fish and Wildlife Service in determining animal susceptibility to the SARS-CoV-2 virus and its variants, Delta, Lambda, and Omicron. Multiple transgenic cell lines that represent eight different bat species, in addition to several domestic, peri-domestic, and wild animal species, were created. The cell lines were tested for whether they could be infected by SARS-CoV-2, which is an initial indication of whether the animal species could be infected with SARS-CoV-2. Based on this screening the animal species that may be susceptible to SARS-CoV-2 infection were identified and shared with stakeholders. Significant progress was also made in determining whether SARS-CoV-2 could contaminate mink pelts or could be spread in mink manure. It was shown that the virus does not remain infectious after the normal conditions for the early stages of processing mink pelts. Also, the virus does not remain infectious for more than 2-3 days at temperatures as low as 15°C (60°F). Finally, over the counter point-of-care COVID-19 test kits were evaluated for their ability to detect SARS-CoV-2 virus in materials from mink farms, so they could be utilized as non-invasive on-farm screenings test. It was demonstrated that the virus could be detected in droppings pans and other swabbing of the mink farm environment. Under Objective 2, several approaches to enhancing host resistance by improving the immune response to infection with HPAIV were evaluated in cell lines in vitro. First, new cell lines were developed with novel genetic changes (i.e., transgenic cell lines) that enhance the host’s innate immune system, which is the first line of defense against the virus. The cell lines were tested for their ability to reduce infection with H5 HPAIV, as well as low pathogenic AIVs. A significant reduction in viral growth and reduced cell destruction were observed. Second, using state of the art biotechnology, called CRISPR/Cas13, avian cells in culture were engineered to express genes that bind the influenza virus genetic material and inhibit virus replication. This also resulted in vastly reduced levels of virus in the cells. This serves as a proof of concept for alternative approaches to traditional vaccines for inducing protection and the studies provide an essential framework for the breeding and development of poultry with enhanced genetic resistance to AIV. Data were shared with stakeholders and commercial companies interested in developing avian influenza resistant birds. Under Objective 3, to develop intervention strategies to effectively control avian influenza viruses and contain disease outbreaks, numerous vaccine studies with H5N1 HPAIV were completed. First, challenge studies were conducted with U.S. licensed inactivated and vectored vaccines, along with inactivated and vectored vaccines produced in-house by ARS. Because the vaccines are based on different platforms, and have different antigens (the protein which induces immunity) they need to be tested for how well they protect against the current virus in the U.S. A total of seven vaccines were evaluated for protection in chickens: 1) a commercial inactivated reverse genetics (rg) generated H5N1 product with a 2014 H5 hemagglutinin (HA) antigen (rgH5N1); 2) a commercial alphavirus RNA particle (RP) vaccine with the 2022 H5 HA antigen; 3) an in-house inactivated rg vaccine with the 2022 H5 HA antigen (SEP-22-N1); 4) an in-house inactivated rg produced vaccine with the 2022 H5 HA antigen and a North American wild bird N9 neuraminidase (NA) (SEP-22-N9); 5) a commercial herpes virus of turkeys (HVT) vectored vaccine with an older Eurasian H5 HA antigen; 6) a commercial HVT vectored vaccine with a more recent H5 HA antigen; and 7) an in-house produced HVT vectored vaccine with a 2022 H5 HA. Each vaccine was tested to determine how well it reduced mortality, morbidity, and virus shed (which indicates how well the vaccine will reduce transmission) using a standard procedure. All vaccines were able to reduce morbidity and mortality. However, there were differences in quantities of virus shed by the oral and cloacal routes. The inactivated vaccines with the 2022 inserts reduced virus shed the most among vaccinated groups but all vaccines reduced shed compared to sham vaccinated controls. Vaccination of poultry for HPAI would require modifications to current surveillance programs for avian influenza virus (AIV) in U.S. poultry. There are several possible approaches, but a program based on antibody testing is preferred because it would fit into current diagnostic lab workflows and would be more economical than testing for virus by molecular methods. Therefore, an antibody-based method that would differentiate animals that have been vaccinated and infected from animals that have only been vaccinated (DIVA-VI) was evaluated. Serum samples were collected from chickens vaccinated with DIVA-VI compatible vaccines (SEP-22-N9, RP, all three HVT vectored vaccines) at different days post challenge to evaluate detection of antibodies to the challenge virus. The serum was tested for antibody to the N1 NA, a surface protein, of the challenge virus with a test called enzyme linked lectin assay (ELLA), or for antibody to the nucleoprotein (NP) of the challenge virus using a commercial test called enzyme linked immunosorbent assay (ELISA). Antibody could be detected as early as seven days post challenge by both tests. The ELLA detected antibody in more birds and the quantity of positive birds differed by vaccine but was >50% at all time points regardless of test or vaccine (data for the HVT vectored vaccines is still pending). Vaccinal immunity is not expected to last the lifetime of breeder and egg laying poultry. Therefore, a prime-boost strategy is needed to achieve the necessary longer duration of immunity. To determine whether order of vaccination affects the development of antibodies, a key correlate of protection, different prime-boost scenarios were tested. Because data with the RP vaccine are very limited because this is a new vaccine platform it was tested with a well characterized inactivated vaccine. Chickens were vaccinated with either an inactivated vaccine (SEP-22-N1) or the RP vaccine (prime), then three weeks later were vaccinated with the same vaccine or the other vaccine. The antibody response was quantified three weeks after the second vaccine and showed that the order of vaccination with an inactivated vaccine and RP vaccine did not affect the antibody response. Vaccines lose their efficacy over time against HPAI because the virus mutates, so we have been engaged in an international project organized by the United Nations Food and Agriculture Organization and World Organization for Animal Health, to monitor H5 HPAIV mutations that can affect vaccines. We are provided with materials to test viruses from North America against reference strains from around the world and the data are compiled and updated as needed so that vaccines can be compared to the viruses in poultry throughout the world. Similar work was completed for the current U.S. H5N1 HPAIV and the vaccines that are available in the U.S. as part of the initial vaccine testing by using serum from birds that were vaccinated with different H5N1 HPAIVs or commercially available vaccines in the U.S. It was shown that antibody to the current virus in the U.S. has reduced reactivity with the viruses from 2014-2015. However, based on the results of the vaccine studies above, it was not sufficient to eliminate vaccine efficacy. These results have been shared with the USDA-APHIS and industry stakeholders. Finally, a meta-study was conducted to evaluate ideal parameters for vaccines based on the current literature. Data from all available literature on inactivated vaccines and HVT vectored vaccines in chickens was used to establish: 1) how closely related a vaccine virus needed to be to the challenge virus to provide protection against mortality and to reduce virus shed 100-fold, and 2) what antibody titers to the challenge virus were needed to provide protection against mortality and reduce virus shed (i.e., define necessary antibody levels as a correlate of protection). For both vaccine types that were evaluated, mortality and virus shed could be reduced with antibody at the lowest detectable levels, and the vaccines would consistently provide protection if they were at least 95% related to the field virus.

1. Vaccines currently available in the U.S. protect chickens against recent strains of highly pathogenic avian influenza virus (HPAIV). Vaccination is being considered to help control HPAIV spread in poultry due to an ongoing outbreak of HPAI in the U.S. If vaccination is implemented, surveillance programs will need to be modified to identify vaccinated birds that have also been infected with virus (DIVA-VI). ARS researchers in Athens, Georgia, tested two U.S. licensed commercial vaccines and two in-house produced vaccines for their effectiveness against the current U.S. HPAIV’s and evaluated two tests for DIVA-VI. All four vaccines provided protection against death and disease but varied in how well they reduced virus shedding by chickens. Both DIVA-VI tests could identify infected birds seven days after infection, however there was variation in sensitivity among the vaccines and tests. This study has confirmed the efficacy of several vaccines and associated DIVA-VI tests that could be used in U.S. poultry. The data are critical for establishing vaccination and associated surveillance programs that will meet the disease control and regulatory needs of government and industry stakeholders.

2. The infectivity and transmissibility of a 2022 North American H5N1 highly pathogenic avian influenza virus (HPAIV) varies between chickens and turkeys. HPAIV’s of the clade goose/Guangdong/1996 H5 lineage remain a major threat to poultry because wild birds carry the virus and contaminate the environment. H5N1 HPAIV was first detected in December 2021 in South Carolina, U.S., and since then, then virus has infected a great number of wild and domestic birds. ARS researchers in Athens, Georgia, evaluated the pathobiology in chickens and turkeys of an early 2022 U.S. H5N1 isolate. Differences in clinical signs, mean death times, and virus transmissibility were found between chickens and turkeys. Although the amount of virus required to infect both species was low compared to similar clade viruses, turkeys transmitted the virus better than chickens because it took longer for them to get sick even when they were excreting virus, which increased the virus shedding period and facilitated transmission. These differences affect the epidemiology of the H5N1 HPAIV and will inform the development of species strategies to prevent, diagnose, and mitigate HPAI outbreaks.

3. Mallards are highly susceptible to, and shed high quantities of the 2022 North American H5N1 highly pathogenic avian influenza virus (HPAIV). HPAIV’s of the clade goose/Guangdong/1996 H5 lineage continue to be a problem in poultry and wild birds in much of the world. The recent incursion of a H5N1 clade HPAIV from this lineage into North America has resulted in widespread outbreaks in poultry and consistent detections of the virus across diverse families of wild birds and occasionally mammals. ARS researchers in Athens, Georgia, characterized the pathobiology of this virus in young mallards (Anas platyrhynchos), which are a primary reservoir of avian influenza viruses in the wild and are closely related to domestic ducks (i.e., Pekin ducks). The infectious dose was extremely low and 100% transmission was observed. No clinical signs were seen in most ducks; however, some of the observed clinical signs, such as neurological signs, would likely be fatal in the wild, but may not occur with older ducks. The mallards shed virus by both the oral and cloacal routes and 65% of the ducks where still shedding virus cloacally through 14 days post-exposure. Based on the high transmissibility, high virus shed titers, and mild-to-moderate disease, mallards could serve as efficient reservoirs to amplify and disseminate recent North American clade viruses.

4. The infectivity, transmissibility, and pathobiology of clade H5Nx highly pathogenic avian influenza viruses (HPAIV) is variable in chickens. Clade Eurasian lineage H5Nx highly pathogenic avian influenza viruses have become the dominant and caused global outbreaks since 2014. The clade viruses have evolved into eight hemagglutinin subgroups ( ARS researchers in Athens, Georgia, evaluated the infectivity (i.e., infectious dose), pathobiology, and transmissibility of seven clade viruses in chickens. The results showed that all the viruses caused high mortality, but the transmissibility of the viruses in chickens was variable. Changes in the pathogenicity and transmissibility of clade HPAIV’s warrant careful monitoring of the viruses to establish effective control strategies because of virus epidemiology could vary.

5. Zoonotic avian influenza transmission is reduced during household poultry slaughter using a behavior change tool for limited literacy audiences. Human infections in Egypt with highly pathogenic avian influenza (HPAI) likely due to airborne transmission of HPAI virus (HPAIV) during home slaughter of poultry predominately affect women and children, who are the primary caretakers of household poultry. ARS researchers in Athens, Georgia, developed a safe contained poultry slaughter procedure to reduce airborne HPAIV and zoonotic infections and simultaneously created an educational outreach tool for teaching the modified procedure. The tool designed for limited literacy audiences used two illustrated posters and handouts for teaching the safe contained poultry slaughter procedure. The posters were developed with advice of animal health professionals and then refined by target audience women's focus groups. The safe contained poultry slaughter procedure will help reduce human exposure to HPAIV and will subsequently reduce human infection with the virus.

6. Multivalent vectored vaccines are efficacious against viral infections in poultry. Vaccines are an essential tool for the control of viral infections in domestic birds. ARS researchers in Athens, Georgia, in collaboration with scientists at the University of Georgia generated recombinant vector herpesvirus of turkeys (vHVT) vaccines expressing computationally optimized broadly reactive antigens (a method called “COBRA”) of H5 avian influenza virus (AIV) alone in a herpes virus of turkeys (rHVT) vector (vHVT-AI) or in combination with virus protein 2of infectious bursal disease virus (IBDV) (vHVT-IBD-AI) or fusion protein of Newcastle disease virus (NDV) (vHVT-ND-AI). In vaccinated chickens, all three vaccines provided 90-100% clinical protection against three divergent clades of high pathogenicity avian influenza viruses (HPAIV), and significantly decreased both the number of birds shedding virus and the quantities of virus excreted by the oral route. The multivalent vHVT-IBD-AI and vHVT-ND-AI vaccines provided 100% clinical protection against IBDVs and NDV, respectively. These findings demonstrate that multivalent HVT vector vaccines were efficacious for simultaneous control of HPAIV and other viral infections which is improves the practicality of these vaccines.

7. Bat species are susceptible to infection by 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. SARS-CoV-2 is believed to have an animal origin and bats are a suspected natural host. A key determinant of the ability of the virus to cause infection is the attachment of the viral spike protein to the host receptor, angiotensin-converting enzyme 2 (ACE2). ARS researcher in Athens, Georgia, in collaboration with scientists at U.S. Geological Survey and U.S. Fish and Wildlife Service, developed avian cell lines that expressed the ACE2 protein of seven bat species to determine their ability to support attachment and replication of SARS-CoV-2 viruses. It was demonstrated that the ACE2 receptor of all seven species made the avian cells permissible to become infected with SARS-CoV-2. The level of virus replication differed among bat species and virus variants tested. These cell lines provide a practical in vitro method for screening animal species for potential susceptibility to current and emerging SARS-CoV-2 variants.

8. Development of a non-transmissible live attenuated avian influenza vaccine. Continued global outbreaks of H5 highly pathogenic avian influenza viruses (HPAIV) have been reported in poultry since the emergence of the Asian Goose/Guangdong lineage of H5N1 HPAIV. Consequently, vaccines have been deployed to protect poultry flocks in numerous countries. ARS researchers in Athens, Georgia, in collaboration with the researchers at the University of Edinburgh have created a novel, attenuated H5 influenza vaccine. The vaccine attenuates the virus through deletion of the M2 or M42 gene that is found on viral segment 7. Vaccines made with M2 and M42 deleted virus stimulated robust immune responses but did not transmit to co-housed naïve birds. In protection studies, vaccination of birds with M2 or M42 deleted vaccine viruses protected 100% birds from lethal challenge from numerous lineages of H5 HPAIV. This novel technology is a potential new platform for HPAIV vaccines.

Review Publications
Ghorbani, A., Ngunjiri, J.M., Abundo, M.C., Pantin Jackwood, M.J., Kenney, S.P., Lee, C.W. 2023. Development of in ovo-compatible NS1-truncated live attenuated influenza vaccines by modulation of hemagglutinin cleavage and polymerase acidic x frameshifting sites. Vaccine. 41(11):1848-1858.
Youk, S., Leyson, C., Killian, M.L., Torchetti, M.K., Lee, D., Suarez, D.L., Pantin Jackwood, M.J. 2022. Evolution of the North American lineage H7 avian influenza viruses in association with H7 virus's introduction to poultry. Journal of Virology. 96(14):e00278-22.
Clark, A.A., Eid, S., Hassan, M.K., Carter, K., Swayne, D.E. 2022. Reducing zoonotic avian influenza transmission at household poultry slaughter using a behavior change tool for limited literacy audiences. Zoonoses and Public Health. 69(8):956-965.
Chrzastek, K., Sellers, H.S., Kapczynski, D.R. 2023. A universal, single-primer amplification protocol to perform whole-genome sequencing of segmented dsRNA avian orthoreoviruses. Avian Diseases. 66(4):479-485.
Spackman, E., Swayne, D.E. 2022. Zoonotic potential of influenza A viruses of poultry and other avian species. Council for Agricultural Science and Technology Issue Paper. SP33:23-25.
Criado, M.F., Kassa, A., Bertran, K., Kwon, J., Sa E Silva, M., Killmaster, L.F., Ross, T.M., Mebatsion, T., Swayne, D.E. 2023. Efficacy of multivalent recombinant herpesvirus of turkey vaccines against high pathogenicity avian influenza, infectious bursal disease, and Newcastle disease viruses. Vaccine. 41(18):2893-2904.
Spackman, E., Pantin Jackwood, M.J., Lee, S.A., Prosser, D. 2023. The pathogenesis of a 2022 North American highly pathogenic clade H5N1 avian influenza in mallards (Anas platyrhynchos). Avian Pathology. 52(3):219-228.
Kwon, J., Bertran, K., Lee, D., Criado, M.F., Killmaster, L.F., Pantin Jackwood, M.J., Swayne, D.E. 2023. Diverse infectivity, transmissibility and pathobiology of clade H5Nx highly pathogenic avian influenza viruses in chickens. Emerging Microbes & Infections. 12:2218945.