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.
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 was made on all four objectives and their subobjectives. In March 2020, an outbreak of H7N3 low pathogenicity avian influenza (LPAI) occurred on 9 turkey farms in North Carolina and a farm in South Carolina (SC). On a second turkey farm in SC a H7N3 highly pathogenic avian influenza (HPAI) virus was detected. Immediate depopulation was performed on affected premises for a total of 361,000 birds. Under Objective 1, phylogenetic analyses were conducted to trace the origin and evolution of the 2020 H7N3 viruses in collaboration with scientists at the University of Connecticut and the National Veterinary Services Laboratories, APHIS, USDA, Ames, Iowa. Results showed that the viruses were closely related to North American lineage wild bird avian influenza (AI) viruses and that a single source of H7N3 LPAI virus was introduced to the turkey farms. The viruses were genetically distinct from recent H7 HPAI viruses including the Mexican H7N3, the 2016 Indiana H7N8, and 2017 Tennessee H7N9 viruses. With several recent incursions, this H7 Hemagglutinin (HA) clade represents a repetitive threat to domestic poultry. A manuscript with the results of the study has been submitted for publication. In addition, pathogenesis studies conducted in chickens and turkeys with these 2020 H7N3 viruses showed they had unique pathobiological features in each species which impacts how the virus transmits and is diagnosed in the field. There is little data on the ability of LPAI virus strains that are highly adapted to chickens to contaminate eggs laid by infected hens. Therefore, chicken hens in production were exposed to H9N2 or H5N2 LPAI strains known to be adapted to chickens and which cause moderate to severe disease. Although contamination on eggs was rare, virus could be detected on the surface of some eggs, emphasizing that eggs should be sanitized prior to movement if they are coming from a flock infected by these strains. In addition to AI virus, we evaluate other novel emerging or exotic viruses that could pose a credible threat to poultry. Coronaviruses (CoV) of animals periodically transmit to humans, as recently occurred with SARS-CoV-2. Because poultry are so widespread and have close contact with humans in many production systems, susceptibility studies were conducted with SARS-CoV-2, and another human coronavirus (MERS-CoV), in five common poultry species. We also tested embryonating chicken eggs (ECE) because of their importance as a laboratory host system and vaccine production. Chickens, turkeys, ducks, quail and white geese did not become infected with either virus and neither virus replicated in ECE. Based on this, poultry are unlikely to serve a role in the maintenance of either virus. This information has been conveyed to poultry stakeholders and a paper is under review for publication. Detailed characterization was conducted of clinical samples recovered during the 2017 U.S. H7N9 HPAI outbreak. Because introduction of LPAI virus from waterfowl to poultry is a known mechanism for evolution of HPAI viruses, we genetically characterized the original H7N9-positive clinical specimens recovered from backyard ducks. Sequence analysis determined quasi-species genomes in the duck virus samples. Inoculation of chickens with a duck LPAI virus resulted in infection and transmission. This work indicates that H7N9 viruses coming from backyard ducks were genetically diverse and rapidly adapted to chickens, which may have contributed to its spread in the region during the outbreak. Under Objective 2, significant progress was made towards identifying genetic markers for adaptation and/or increased virulence of AI viruses in different avian species. H5Nx HPAI viruses of the A/goose/Guangdong/1/96 lineage continue to circulate worldwide affecting both poultry and wild birds. These viruses constantly change which affects their ability to infect and cause disease in different avian hosts. A H5N2 virus from this lineage caused the severe poultry outbreak in the United States in 2015. In previous studies we showed that H5N2 viruses isolated in the later stages of the outbreak were more pathogenic in chickens than the earlier viruses. We found that mutations in the PB1, NP, HA, and NA virus proteins were associated with this increase in virulence and determined that HPAI viruses as they circulated in poultry species, underwent adaptation becoming more infectious, which complicates the control of the virus. We also identified genetic determinants that lead to severe disease outcomes in ducks infected with H5N8 HPAI viruses. Wild waterfowl play a major role in the spread of H5Nx HPAI viruses across geographical areas. Two distinct groups of viruses caused intercontinental H5N8 outbreaks in 2014-2015 and 2016-2017. We found that viruses from 2014 caused mild disease in mallards in contrast with 2016 viruses which caused severe disease and mortality. We assessed the genetic determinants associated with virulence of H5N8 in mallards and found that changes in the PB2, NP, and M viral proteins were major contributors. Pleomorphic virus particles are a characteristic influenza A virus, which can produce spherical or filamentous virions. Avian influenza virus budding morphology remains largely uncharacterized. To investigate this, we studied the budding morphology of a panel of AI viruses finding that the majority were filamentous. This phenotype did not correlate with any particular subtype of virus or passage history. However, the filamentous phenotype was more common in duck viruses than chicken isolates. Under Objective 3, high resolution T-cell profiling of the avian adaptive immune response to AI virus was conducted. The ability to detect virus-specific T-cell responses in birds is a critical gap in understanding immunity and an impediment to developing better vaccines. T-cell receptors (TCRs), located on the surface of T cells, recognize and bind influenza specific peptides presented on the surface of infected cells. We developed a PCR assay to allow detection of the TCR repertoire of CD4+ and CD8+ T-cells following AI virus infection. This information helps in understanding how the avian immune system responds to AI virus infection and contributes to the development of immunoassays and vaccines. Current control of AI is through biosecurity and in some cases vaccination. Chickens lack several innate immune response genes found in other species that inhibit AI virus replication, including myxovirus-resistance gene (Mx), which appears to be a key host regulator of virus replication that can be manipulated to defeat the virus. We developed an avian cell line expressing the mouse Mx gene. Infection study demonstrated up to 99% reduction of AI virus replication in vitro. Results from these studies will be used to develop transgenic birds with increased resistance to AI virus. Under Objective 4, in order to develop strategies to effectively control AI viruses, we determined the best method for environmental testing of the virus after an outbreak. After outbreaks of AI virus or other viruses, premises are tested to ensure that no viable virus remains after a fallow period or cleaning and disinfection. There is not much data on how to approach sample collection, so methods to detect AI and other viruses in poultry houses or wire cages (e.g. layer cages) were developed. Sample collection devices were compared for virus recovery, and to identify the sites where sample collection should be targeted, different areas of the poultry house or caging areas were tested. Cotton gauze was shown to recover the most virus and areas near the floor were optimal for sample collection.
1. Risk factors determined in the spread of high pathogenicity avian influenza during the outbreak in 2015 in the U.S. The incidence and economic impact of avian influenza in poultry remains high worldwide. From December 2014 through mid-June 2015 an H5 subtype highly pathogenic avian influenza (HPAI) virus that originated in Asia and spread to North America by migratory birds, caused the worst animal disease outbreak in U.S. history. Approximately 49.6 million commercial birds died or had to be euthanized. The eradication effort cost more than $1 billion and the impact to the US economy was over $3.2 billion. ARS researchers in Athens, Georgia, with collaborators at the University of Georgia and the University of Connecticut, found that the HPAI outbreak among poultry farms in the Midwestern United States was influenced by agricultural and geographic factors. After initial introduction of the HPAI virus into the poultry industry, no further introductions, such as from a wild bird reservoir or long-distance movement, were necessary to explain the continuation of the outbreak from March to June 2015. Additionally, evidence suggests that closeness of farms increased the chances of viral movement between two locations. While many theories could explain the transmission of virus among poultry farms, road density was found to be an important factor of virus movement, and human-based transportation plays a key role. This information is critical in understanding the epidemiology of HPAI viruses and developing methods for prevention and control of the disease.
2. Contamination of eggs with avian influenza virus laid by infected hens. Effective recovery from an avian influenza (AI) outbreak requires reliable knowledge of the risk for transmitting the virus on or in eggs so the risk can be mitigated. However, data on AI virus contamination from eggs is almost exclusively from field reports where the timeframe of infection is not clearly known. To understand the extent that eggs laid by infected hens could be contaminated with AI virus, ARS researchers in Athens, Georgia, infected hens in production with either low pathogenicity (LP) or highly pathogenic (HP) AI viruses and their eggs were collected and tested for the presence of virus on the eggshell or in the egg contents. The study showed that eggs could be contaminated with either LP or HP AI virus, but the levels of virus and numbers of infected eggs were much higher with the highly pathogenic avian influenza (HPAI) virus. This data shows that eggs need to be considered a fomite that could potentially spread AI unless sanitization measures are employed.
3. Rapid evolution of Mexican H7N3 highly pathogenic avian influenza viruses in poultry. Highly pathogenic avian influenza (HPAI) virus subtype H7N3 has been circulating in poultry in Mexico since 2012 and vaccination has been used to control the disease. However, the virus has not been eradicated and continues to be a threat to U.S. poultry. ARS researchers in Athens, Georgia, fully sequenced Mexican H7N3 HPAI viruses from 2015-2017. Phylogenetic analyses showed divergence of all eight virus gene segments into three genetic clusters by 2015, with differences in the hemagglutinin (HA) genes when compared to the index virus from 2012. Differences in the HA protein can result in antigenic differences which can affect the efficacy of vaccines. To understand the evolution of the virus, comparison of the sequences of the Mexican H7N3 HPAI viruses and American ancestral wild bird avian influenza (AI) viruses showed that that several virus genes (PB2, PB1, PA, HA, NP, and NS) greatly changed once the virus was introduced into poultry from the wild bird reservoir. These changes can increase the infectivity and transmissibility of the virus in poultry and render vaccines inefficient. Continuous monitoring and molecular characterization of the H7N3 HPAI virus is important for better understanding of the virus evolutionary dynamics and further improve control measures including vaccination.
4. Protection of chickens by recombinant fowlpox vector vaccine with an updated H5 insert against Mexican H5N2 avian influenza viruses. Despite decades of vaccination, surveillance, and biosecurity measures, H5N2 avian influenza (AI) virus infections continue in Mexico and other neighboring countries and pose a threat to the U.S. poultry industry. ARS researchers in Athens, Georgia, in collaboration with industry partners, analyzed the genome sequences of circulating H5N2 field viruses and the vaccines used to date. The analyses showed that the vaccine should be updated to protect against newly circulating H5N2 AI viruses in Mexico. A new recombinant fowlpox vectored vaccine was developed and tested in chickens. This new vaccine provided 100% protection from Mexican H5N2 low and highly pathogenicity AI viruses. The results confirm the efficacy of the new rFPVH5/2016 against antigenic drift of low pathogenic avian influenza (LPAI) virus in Mexico and suggest that this vaccine would be a good candidate, likely as a primer in a prime-boost vaccination program.
5. Pathogenicity and genomic changes of a 2016 H5N8 highly pathogenic avian influenza virus (Clade 126.96.36.199) in experimentally infected mallards and chickens. Highly pathogenic avian influenza H5N8 clade 188.8.131.52 viruses caused worldwide outbreaks in poultry and unusually high mortality in wild birds in 2016-2017. ARS researchers in Athens, Georgia, examined the pathogenicity of one of these viruses in mallards to determine why it was more virulent than previous clade 184.108.40.206 viruses. The virus caused severe disease and high mortality and transmitted to contact ducks. In chickens, as expected, the virus caused high mortality but only when given at a high dose. Viral genome sequences obtained from infected chickens had a higher number of changes than viral sequences obtained from infected mallards, consistent with what was found when viruses infect a host that it is not well adapted to. This information on changes in virus host adaptation is important for understanding the epidemiology of avian influenza viruses and the role that wild waterfowl may play in disseminating viruses adapted to terrestrial poultry.
6. Pathobiology and innate immune responses of gallinaceous poultry to subtype H5Nx highly pathogenic avian influenza virus infection. In 2014-2015, highly pathogenic avian influenza (HPAI) subtype H5 viruses caused a devastating outbreak in the U.S. Backyard flocks of mixed poultry and large commercial chicken and turkey operations were severely affected. Approximately 49.6 million commercial birds died or had to be euthanized. The eradication effort cost more than $1 billion and the impact to the U.S. economy was over $3.2 billion. To better understand differences in disease presentation among gallinaceous poultry, ARS researchers in Athens, Georgia, examined the pathobiology of the first U.S. H5Nx isolates in chickens, Japanese quail, Bobwhite quail, Pearl guinea fowl, Chukar partridges, and Ring-necked pheasants. The HPAI virus infections caused necrosis in many tissues in severely ill birds. In general, lesions and virus distribution in tissues were similar regardless of virus and species; however, only Pearl guinea fowl showed widespread virus replication in vascular endothelial cells. The HPAI virus infection was modulated in Japanese quail because of innate immune responses. The expression of IFN-gamma and IL-10 in Japanese quail, and IL-6 in chickens, were upregulated in later clinical stages, indicating differences in innate immune responses to the virus. Greater susceptibility and easier transmission of the H5Nx HPAI virus are features that could favor the spread of HPAI during outbreaks. This information is critical in understanding the epidemiology of avian influenza virus and its control.
7. American black ducks (Anas rubripes) can be asymptomatic carriers of highly pathogenic avian influenza virus. Ducks are a natural host for low pathogenic avian influenza virus, but there is not much data on whether highly pathogenic avian influenza (HPAI) virus, the most virulent form of avian influenza for chickens and turkeys, can be carried by all duck species. To determine if American black ducks can carry HPAI virus, ARS researchers in Athens, Georgia, exposed ducks to the virus, tested them for virus excretion and observed for disease. Like many dabbling duck species, American black ducks, could carry the virus and excrete large quantities without becoming sick. This demonstrates that American black ducks can serve as reservoirs of HPAI virus and may be able to disseminate the virus through migration, therefore contact of this species with poultry needs to be controlled.
8. Domestic ducks play a major role in the maintenance and spread of H5N8 highly pathogenic avian influenza (HPAI) viruses in South Korea. During 2014-2016 H5N8 HPAI viruses (HPAIV) induced the longest outbreak in South Korea and caused severe economic loss in poultry. ARS researchers in Athens, Georgia, with collaborators in the U.S. and South Korea, demonstrated that H5N8 HPAIV most likely transmitted from wild waterfowl to domestic ducks, and then maintained in domestic ducks followed by dispersal of HPAIV from domestic ducks to chickens, suggesting the domestic duck population plays a central role in the maintenance, amplification and spread of wild HPAIV to terrestrial poultry in Korea.
9. Live bird markets as evolutionary epicenters of H9N2 low pathogenicity avian influenza viruses in Korea. Live bird markets (LBMs) in Korea have been recognized as a reservoir, amplifier, and source of avian influenza (AI) viruses; however, little was known about the role of LBMs in the epidemiology of AI viruses in Korea until recently. Through 10 years of surveillance (2006-2016), H9N2 viruses in Korean LBMs were isolated and sequenced. To understand how H9N2 evolves and spreads in Korea, ARS researchers in Athens, Georgia, and collaborators In South Korea, conducted phylogenetic analysis that suggested that three separate introductions of progenitor virus gene pools, Korean domestic duck-origin and two wild aquatic bird-origin AI viruses, contributed to the generation of the five genotypes of H9N2 viruses in Korea. The results of the study suggest that the LBMs are where chickens become infected with the virus, with domestic ducks playing a major role in the transmission and evolution of the H9N2 viruses. Three increases in the genetic diversity of H9N2 viruses were observed and coincided with transitions in host species and the locations (domestic farm, LBM, slaughterhouse, and wild aquatic bird habitat) where the viruses were isolated, accompanying genetic reassortment. Following the introduction of a wild aquatic bird-origin AI virus in 2008, six genes of the Korean lineage H9N2 virus were replaced with genes originating from wild aquatic birds, and viruses with this new genotype became predominant in Korean LBMs. These results support the continued need for regulation of the captive wild bird trade to reduce the risk of introduction and dissemination of AI viruses throughout the world.
10. Eliminating chicken testing for human prepandemic influenza vaccine seed strains. A shortened time period is needed for delivery of human vaccines to prepare for an influenza pandemic. One critical need was an accelerated process to approve prepandemic candidate vaccine viruses (CVV) for use in vaccine manufacture. In a global collaborative study between public health and animal health institutes, ARS researchers in Athens, Georgia, provided 15 years of biosafety evidence in support of eliminating mandatory chicken safety testing requirements of avian influenza vaccine seed strains, thus expediting the transfer of vaccine candidates to manufacturing by nearly three weeks.
11. Assessment of 220.127.116.11 H5Nx high pathogenicity avian influenza viruses as potential pandemic viruses. The H5N1 highly pathogenic avian influenza (HPAI) viruses from Eurasia have caused sporadic human infections since 1997 in Asia and 2005 in Africa. A new group of these viruses called Clade 18.104.22.168 have been distributed via long distance migratory birds onto several continents and through poultry trade among neighboring countries. To date, these viruses have caused only sporadic human infections and lack the ability for transmission between humans. However, some 22.214.171.124 viruses transmit between ferrets in direct contact and have molecular signatures related to mammalian adaptation. ARS researchers in Athens, Georgia, with their global collaborators have called for surveillance of influenza viruses in domestic and wild birds to be expanded and fully integrated with laboratory and field- based risk assessment to allow for development and updated veterinary and public health countermeasures to reduce the threats of zoonotic and pandemic influenza.
12. H9N2 avian influenza in Pakistan. In 2017, H9N2 avian influenza virus were isolated in Pakistan as part of a surveillance program in live poultry markets. ARS researchers in Athens, Georgia, in a collaborative study conducted genetic analysis of a H9N2 virus and showed that the virus belonged to the G1 lineage of H9N2 viruses. In addition, this isolate possessed mammalian host-specific mutations which could possibly favor interspecies transmission, suggesting that Pakistani H9N2 viruses are still potentially infectious for mammals.
13. Detection of H9N2 avian influenza viruses in live poultry markets of Pakistan and their association with human infections. The ecology and epidemiology of influenza A viruses is highly complex, involving both birds and mammals and impacting human health. In a collaboration with St. Jude’s Children’s Hospital and Lahore University in Pakistan, ARS researchers in Athens, Georgia, identified H9N2 avian influenza antibodies in poultry workers in association with detection of H9N2 avian influenza viruses in chickens in live poultry markets of Pakistan. Live poultry markets are a source of avian influenza virus exposure to humans and for development of zoonotic infections.
14. First detection of H9N2 avian influenza in Kenya. As part of surveillance study of wild birds, ARS researchers in Athens, Georgia, and their collaborators collected samples birds in live bird markets, and backyard and commercial poultry, and screened these samples for avian influenza. Several H9N2 viruses were isolated from poultry from live bird markets, which is the first confirmed detection of avian influenza from poultry in Kenya. The viruses were sequenced and were shown to be genetically related to viruses H9N2 G1 lineage viruses from Uganda, a contiguous neighbor, and several North African countries. These viruses are a risk both to veterinary health and public health and deserve additional scrutiny in Kenya and the region.
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