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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

2019 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
Outbreaks of H7N3 highly pathogenic avian influenza (HPAI) have occurred in poultry in Mexico since 2012 and vaccination has been used to control the disease. Mexican H7N3 HPAI viruses from 2015 and 2017 were isolated and fully sequenced. Phylogenetic analyses show divergence of all eight gene segments of the Mexican H7N3 HPAI virus from the original introduction into three genetic clusters by 2015 with no reassortment with other avian influenza viruses. We compared the sequences of the Mexican lineage H7N3 viruses and American ancestral wild bird avian influenza (AI) viruses to characterize the virus evolutionary dynamics and found that the nucleotide substitution rates in PB2, PB1, PA, HA, NP, and NS genes greatly increased once the virus was introduced into poultry with strong purifying selection. Continuous monitoring and molecular characterization of the H7N3 HPAI virus is important for better understanding of the virus evolutionary dynamics and further implementing measures to control the spread of the virus. Progress was made towards identifying genetic markers for avian influenza virus that are predictive of adaptation and/or increased virulence in different avian species. H5N2 highly pathogenic avian influenza (HPAI) virus emerged in the United States at the end of 2014 and caused severe poultry outbreaks during 2015. In previous infectivity and pathogenicity studies, poultry H5N2 isolates had shown increased adaptation to chickens and decreased adaptation in mallards when compared to the index H5N2 virus that was isolated from a wild duck. To establish the genetic basis of this change in virus adaptation, we generated H5N2 recombinant viruses using the index H5N2 virus and a chicken adapted isolate. These results indicate that mutations in the PB1, NP, HA and NA genes that occurred during the H5N2 HPAI virus circulation in poultry are associated with increased adaptation to chickens which allows for increased transmission and a lower dose required to establish infection. The goose Guangdong lineage of highly pathogenic avian influenza (HPAI) caused widespread outbreaks in the United States and Europe from 2014-2017. Although all the viruses shared some virulence characteristics in chickens, important differences were observed when testing a more recent European H5N8 HPAI virus. Different from previous H5N8 viruses, high mortality was observed in mallards, which was not seen with earlier related viruses, which may help explain why this virus has spread more widely and affected more avian species than the 2014-2015 H5N8 virus. Recombinant viruses were generated to determine which genes and mutations are responsible for the increase in adaptation and virulence in mallards. Experimental infection in mallards indicates a role of the M and NP genes in the increased virulence observed with the H5N8 HPAI virus. Influenza A virus (IAV) can produce virus particles of different size and shape including spherical virions of approximately 100 nm in diameter and filaments up to 30 µm long. The majority of non-laboratory adapted mammalian IAV strains are thought to be filamentous, however avian IAV budding morphology remains largely uncharacterized. To investigate this, 22 avian viruses representing the 11 major clades found from phylogenetic analysis of segment 7 were characterized for budding morphology. The majority of viruses produced filaments up to 10µm but a sizeable minority were non-filamentous. Budding phenotype did not correlate with any particular subtype of virus or clade. However, the filamentous phenotype was more common in duck viruses but less common in chicken viruses. Mutagenesis of closely related strains with differing budding morphologies identified positions 59, 169 and 234 as IAV strain-dependent determinants. Furthermore, statistical analysis of the galliforme and anseriforme strains characterized here coupled with bioinformatic analysis of all avian segment 7s identified amino acid specificity between the two at key epitopes. These results suggest that filamentous viruses are more common in ducks than chickens and the filamentous trait may be associated with enhanced shedding in nature. The pathogenesis of the H7N9 low pathogenic avian influenza (LPAI) and highly pathogenic avian influenza (HPAI) viruses that caused the outbreaks in poultry in the U.S. in 2017 and of a closely related wild duck origin H7N9 LPAI virus isolated 6 months previously in Wyoming was examined in mallards. Mallards infected with the LPAI viruses required moderate to high virus doses to infect the mallards and transmit to contacts. The HPAI virus had high infectivity and transmissibility but caused no clinical disease or mortality in the ducks. This should be taken in consideration when implementing biosecurity to control the spread of the virus in poultry. Immunology studies were conducted in mallard ducks to examine host defense mechanisms to highly pathogenic avian influenza virus (HPAIV) infections. Mallard ducks are widely recognized as major reservoirs for low pathogenic avian influenza (LPAIV) viruses in nature. In these studies, mallards were exposed to live LPAIV given at monthly intervals prior to challenge with H5N8 HPAIV clade 2.3.4.4. Our results demonstrate that in general mallards after H5N8 HPAIV challenge were resistant to mortality, including infection and shedding, when exposed to single or multiple subtypes of LPAIV prior to challenge. In addition, a positive correlation between the number of exposures to LPAIV with decreased levels of HPAIV shedding or seroconversion were demonstrated. Deep sequence analysis of virus populations found in swab samples identified changes in a number of different viral genes. Taken together these data suggests that young or immunologically-naive birds may pose the biggest risk to spread of the virus, disease, and genetic variants. The M2 ion channel of influenza A virus (IAV) is a single-pass type III membrane protein that plays important roles during both viral entry and egress. We have described a rare variant of the viral ion channel, M42, that can functionally replace M2 in the viral lifecycle. The objectives of these studies were to determine the biology that dictates the localization and expression of M2 and M42. Differential localization patterns of M2 and M42 were confirmed by quantification and co-localization to be a predominately Golgi-based localization of M42. This is in contrast to M2 which is found to be more widespread with a cytoplasmic/ plasma membrane-based localization. Viruses with altered M2 or M42 expression demonstrated decrease replication in both cell culture and in chicken embryos with changes in general virus morphology. This work defines a new functional motif in the M2 ectodomain that helps explain the functional constraints that underlay its conservation, and may play a role in protecting the HA during its initial progression from LP to HP forms. Current control strategies for poultry against avian influenza virus (AIV) is dependent on proper biosecurity and in some cases vaccine application, but there is considerable interest in enhancing the genetic resistance of poultry against infection of the virus. Efforts to introduce the mouse MX1 transgene molecules, a molecule shown to improve resistance of mice to avian influenza, into the chicken genome is being developed to allow for increased innate resistance to AIV. Transfection of avian cell lines were isolated that expressed the transgene. Infection studies were performed with H6N2, H5N2 and H7N2 AIV which demonstrated increased resistance to viral growth in cells expressing the Mx1 gene (a 10 to 100 fold reduction of virus). These results demonstrate that mouse Mx could be useful for enhanced protection of AI in birds. Vaccines and vaccination have emerged during the past three decades as essential tools in influenza control. In these studies we generated an attenuated live virus low pathogenic avian influenza H5N2 vaccine based on disruption of segment 7 expressing either M2 or M42 protein. The vaccines replicated in chickens and induced a protective immune response against two different highly pathogenic avian influenza challenges. The mutations resulted in virions with increased size and differential morphology compared to wild-type virus. Importantly, when applied to chickens the vaccine virus did not transmit to susceptible cohorts demonstrating a decreased ability to spread. The development of a safe live but attenuated vaccine allows for improved immune response over traditional killed vaccines and allows the possibility of mass administration of the vaccine. Recovery from outbreaks requires effective elimination of virus from the environment, which includes disposal of carcasses and litter and cleaning and disinfection of the premises. Several related projects are underway to help improve these processes to provide improved methods of virus elimination or improve methods to verify premises are free of infectious virus. Disposal of carcasses during an avian influenza virus (AIV) outbreak is a major problem because of the perception that they can contaminate the environment with virus. Studies are underway to determine how long AIV remains viable in chicken and turkey carcasses. Once the environment has been decontaminated the environment must be tested to ensure that infectious virus is absent. Studies are in progress to identify the most efficient sample collection device and the optimal surfaces for testing within different types of poultry houses. Very little data is available on the contamination of eggs laid by hens infected with AIV. Experiments are in progress to characterize egg contamination from hens infected with different strains of AIV.

Accomplishments
1. Improving efficiency of avian influenza virus (AIV) detection by improving the procedure for quantifying AIV. Isolating and quantifying avian influenza virus (AIV) is expensive and resource intensive because of the need to use chicken’s eggs to culture the virus. Chicken's eggs are the most sensitive culture system for AIV. To improve the efficiency of quantifying AIV, ARS researchers in Athens, Georgia, worked with statisticians to validate a streamlined procedure which utilizes 40 percent fewer eggs per test. Using fewer eggs will substantially decrease costs and allow for more efficient processing of samples.

2. Characterization of the susceptibility of avian influenza virus (AIV) in lesser scaup (Athya affinis). The ecology of AIV in many species of waterfowl has not been defined but is important to understand which species can spread the virus over long distances during migration. Lesser scaup were evaluated by ARS researchers in Athens, Georgia, for their ability to carry virulent H7 subtype AIV. The ducks could be infected with AIV at low doses and did not get sick, however they excreted high levels of virus. This suggests that Lesser scaup could carry virulent AIV during migration and contaminate the environment along their route. This information is important for understanding the epidemiology of AIV and the role of wild birds in disseminating the virus.

3. Determining the duration of avian influenza virus vaccinal immunity in chickens. Data on the duration of avian influenza virus (AIV) vaccination in long-term vaccination programs in chickens is very limited. ARS researchers in Athens, Georgia, worked with collaborators at the University of Delaware to determine the duration of immunity for different AIV vaccines and vaccination programs through 36 weeks in layer chickens. Protection varies among programs, but programs that were effective through 36 weeks of age were identified. Data on the immune response to the vaccines indicates that the immunity may last much longer, possibly through the lifespan of a layer chicken. Developing effective long-term vaccination programs for long-lived poultry will allow the implementation of the most effective vaccination programs, which will help control AIV.

4. Loss of fitness in mallards of Mexican H7N3 highly pathogenic avian influenza virus after extended circulation in chickens. Outbreaks of highly pathogenic avian influenza (HPAI) virus subtype H7N3 have been occurring in commercial chickens in Mexico since its first introduction in 2012. In order to determine changes in virus pathogenicity and adaptation in different avian species, ARS researchers in Athens, Georgia, examined H7N3 HPAI viruses from 2012, 2015, and 2016 in chickens and mallards and found that, as the Mexican H7N3 HPAI virus has passaged through large populations of chickens in a span of several years, it has retained its high pathogenicity for chickens but has decreased in fitness in mallards which could limit the potential spread of this HPAI virus by waterfowl. 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.

5. Age-dependent pathogenesis of clade 2.3.4.4A H5N2 highly pathogenic avian influenza virus in experimentally infected Broad Breasted White turkeys. Highly pathogenic avian influenza (HPAI) is a viral disease with devastating consequences to the poultry industry as it results in high morbidity, mortality and international trade restrictions. ARS researchers in Athens, Georgia, with collaborators at the University of Georgia, characterized age-related differences in terms of pathology in commercial white broad breasted turkeys inoculated with a H5N2 HPAI clade 2.3.4.4A virus from the largest HPAI poultry outbreak that affected the Unites States in 2014-2015. Turkeys infected at 6-weeks of age showed little clinical signs with rapid disease progression, reaching 100% mortality at 3 days post infection (dpi). In contrast, turkeys infected at 16-weeks of age developed ataxia and lethargy and reached 100% mortality by 5 dpi. These findings indicate that age is a determinant factor in the progression of the disease and delay of mortality during infection with H5N2 HPAI virus in turkeys.

6. Pathobiology of Tennessee 2017 H7N9 low and high pathogenicity avian influenza viruses in commercial broiler breeders and layer chickens. In March 2017, H7N9 highly pathogenic avian influenza (HPAI) virus was detected in broiler breeder farms in the state of Tennessee. Subsequent surveillance detected the low pathogenicity avian influenza (LPAI) virus precursor in multiple broiler breeder farms and backyard poultry in Tennessee and neighboring states. ARS researchers in Athens, Georgia, investigated the pathogenesis of the H7N9 LPAI virus in commercial broiler breeders, the bird type mostly affected in this outbreak and layer type chickens. The findings suggest sub-optimal adaptation for sustained transmission with the H7N9 LPAI isolate, indicating that factors other than the birds genetic background may explain the epidemiology of the outbreak. The HPAI isolate when examined in layer chickens was more infectious than the LPAI isolate. Greater susceptibility and easier transmission of the H7N9 HPAI virus are features of the highly pathogenic form of the virus that could favor the spread of HPAI over LPAI viruses during outbreaks. This information is critical in understanding the epidemiology of avian influenza virus and its control.

7. Efficacy of two licensed H5 vaccines against challenge with a 2015 United States H5N2 clade 2.3.4.4 highly pathogenic avian influenza virus in domestic ducks. Highly pathogenic avian influenza (HPAI) clade 2.3.4.4 viruses caused a major outbreak in poultry in the United States in 2015. Although the outbreak was controlled, vaccines were considered as an alternative control method and new vaccines were approved and purchased by the National Veterinary Stockpile for emergency use. ARS researchers in Athens, Georgia, evaluated the efficacy of two of these vaccines in protecting Pekin ducks against challenge with a H5N2 HPAI poultry isolate. A recombinant alphavirus-based vaccine and an inactivated adjuvanted reverse genetics vaccine were used to immunize the ducks. Both vaccines, regardless of the vaccination strategy used, were immunogenic in ducks and reduced or prevented disease and virus shedding after challenge. This information is important for the control of avian influenza in domestic ducks.

8. Highly pathogenic and low pathogenic avian H5 subtype viruses in wild birds in Ukraine. There have been three waves of highly pathogenic avian influenza (HPAI) outbreaks in commercial, backyard poultry and wild birds in Ukraine. In a collaborative study between ARS researchers in Athens, Georgia, and scientists in Ukraine, wild bird surveillance for avian influenza (AI) virus was conducted from 2006 to 2016 in Ukraine regions suspected of being intercontinental flyways. A total of 21,511 samples were collected from 105 species of wild birds representing 27 families and 11 orders. Ninety-five avian influenza (AI) viruses were isolated from wild birds with a total of 26 antigenic hemagglutinin (HA) and neuraminidase (NA) subtype combinations. The results demonstrate the great diversity of AI viruses in wild birds in Ukraine, as well as the importance of this region for studying the ecology of the virus.

9. Intercontinental spread of Asian-origin H7 avian influenza viruses by captive bird trade. Wild bird migration and illegal trade of infected poultry, eggs, and poultry products have been associated with the spread of avian influenza (AI) viruses. During 1992–1996, H7N1 and H7N8 low pathogenic AI viruses were identified from captive wild birds including Pekin robin, magpie robin, flycatcher sp., a parakeet, sun conure, painted conure, fairy bluebird, and common iora, kept in aviaries or quarantine stations in England, The Netherlands, Singapore and the United States. ARS researchers in Athens, Georgia, sequenced these H7 viruses isolated from quarantine facilities and aviaries and conducted a comparative phylogenetic analysis of complete genome sequences to elucidate patterns of virus spread. The analysis suggested that H7 viruses originated from a common source, even though they were obtained from birds in distant geographical regions. 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. Changes in the hemagglutinin viral protein resulted in escape of a 2015 Mexican H7N3 highly pathogenic avian influenza virus from vaccine-induced immunity. Since 2012, H7N3 highly pathogenic avian influenza (HPAI) has produced negative economic and animal welfare impacts on poultry in central Mexico. ARS researchers in Athens, Georgia, vaccinated chickens with two different recombinant fowlpox virus vaccines (rFPV-H7/3002 with 2015 H7 hemagglutinin [HA] gene insert, and rFPV-H7/2155 with a 2002 H7 HA gene insert). The chickens were then challenged three weeks later with a 2015 H7N3 HPAI virus. The rFPV-H7/3002 vaccine conferred 100% protection against mortality and morbidity, and significantly reduced virus shedding form the birds. In contrast, 100 percent of rFPV-H7/2155 vaccinated birds shed virus at higher titers and died within 4 days. One possible explanation for differences in vaccine efficacy is the antigenic drift between circulating viruses and vaccines. Molecular analysis demonstrated that the Mexican H7N3 strains have continued to rapidly evolve since 2012, and mutations in the HA antigenic sites, including increased glycosylation sites, accumulated in the recent circulating strains, which possibly altered the recognition of neutralizing antibodies from the older vaccine strain. These studies highlight the importance of frequent updating of vaccines seed strains for long-term effective control of HPAI viruses.

11. Transmission dynamics of highly pathogenic avian influenza virus subtype H5N2, clade 2.3.4.4, causing the outbreak in birds in North America from 2014–2015 were evaluated. Highly pathogenic avian influenza virus (HPAIV) H5N2 clade 2.3.4.4 emerged in North America at the end of 2014 and caused outbreaks affecting more than 50 million poultry in the United States before eradication in June 2015. ARS researchers in Athens, Georgia, investigated the underlying ecologic and epidemiologic processes associated with this viral spread by performing a comparative genomic study using 268 full-length genome virus sequences and information from the outbreak. The HPAIV H5N2 virus circulated in wild birds along the Pacific flyway before several spillover events resulting in transmission of the virus to commercial poultry farms. The analysis suggests that more than 3 separate introductions of HPAIV H5N2 into Midwest states occurred during March–June 2015; transmission to Midwest poultry farms from Pacific wild birds occurred approximately 2 months before detection. Once established in poultry, the virus rapidly spread between turkey and chicken farms in neighboring states. This information is critical in understanding the epidemiology of HPAI virus and its control.

12. A new clade 2.3.4.4b avian influenza subtype H5N6 virus was identified in wild birds in South Korea. In a collaborative study between ARS researchers in Athens, Georgia, and scientists in South Korea, a new reassortant avian influenza (H5N6) virus was isolated from feces of wild waterfowl in South Korea during 2017–18. Phylogenetic analysis suggested that reassortment occurred between clade 2.3.4.4b H5N8 virus and Eurasian low pathogenicity avian influenza viruses circulating in wild birds. This information is important for understanding the role of wild birds in the generation of new avian influenza viruses.

13. Maternal antibody inhibition of a virus vectored vaccine in avian influenza vaccination of broiler chickens. Maternally-derived antibodies (MDA) provide early protection from disease but may interfere with active immunity in young chickens. In countries with highly pathogenic avian influenza virus (HPAIV), broiler chickens typically have MDA to Newcastle disease virus (NDV) and HPAIV, and their impact on active immunity from recombinant vectored vaccines is unclear. ARS researchers in Athens, Georgia, assessed the effectiveness of a spray-applied recombinant NDV vaccine with H5 AIV insert (rNDV-H5) and a recombinant turkey herpesvirus (HVT) vaccine with H5 AIV insert (rHVT-H5) in commercial broilers with MDA to NDV alone or to NDV plus AIV to provide protection against a HPAIV challenge. The results demonstrate that MDA to AIV had minimal impact on the effectiveness of rHVT-H5, but MDA to AIV and/or NDV at the time of vaccination can prevent development of protective immunity from a rNDV-H5 vaccine. This information is critical for developing effective vaccination programs to control avian influenza in poultry.

14. A computationally designed H5 antigen shows immunological breadth of coverage and protects against drifting highly pathogenic avian influenza virus strains in chickens. Since the first identification of the H5N1 Goose/Guangdong lineage of highly pathogenic avian influenza viruses (HPAIV) in 1996, this virus has spread worldwide becoming endemic in domestic poultry. Sporadic transmission to humans has raised concerns of a potential pandemic and underscores the need for a broad cross protective influenza vaccine. In a collaborative study between ARS researchers in Athens, Georgia, and scientists in other institutions, a previously described methodology termed Computationally Optimized Broadly Reactive Antigen (COBRA), was tested to generate a novel hemagglutinin (HA) gene, termed COBRA-2 that was based on HA sequences from H5 viruses from 2005 to 2006. COBRA-2 HA virus-like particle vaccines were used to vaccinate chickens. The results demonstrated seroprotective antibody responses against genetically diverse clades and sub-clades of H5 HPAIV and protective efficacy against a recent drifted variant using this globular head-based design strategy. This information is important for improving vaccination to control highly pathogenic avian influenza.

15. Previous immunity conferred by avian influenza virus infection increases the infectious dose required to infect Mallard ducks with a subsequent avian influenza virus. Field and experimental studies have demonstrated that heterosubtypic immunity (HSI) is a potential driver of avian influenza virus (AIV) prevalence and subtype diversity in mallards. Prior infection with AIV can reduce viral shedding during subsequent reinfection with AIV that have genetically related hemagglutinins (HA). In a collaborative study between ARS researchers in Athens, Georgia, and scientists from the University of Georgia, the effect of HSI conferred by an H3N8 AIV infection was evaluated against increasing challenge doses of closely and distantly related AIV subtypes in mallards. The results showed that the infectious dose necessary to infect mallards with AIV can increase as a result of HSI and that this effect is most pronounced when the HA of the viruses are more genetically related. This information is important for understanding the epidemiology of AIV in wild waterfowl.

16. An inactivated influenza virus adjuvanted with a bispecific antibody complex targeting chicken CD40 and avian influenza virus M2e protein confers protection against highly pathogenic avian influenza. In a collaborative study between ARS researchers in Athens, Georgia, and scientists from other institutions, an immunization strategy using inactivated highly pathogenic avian influenza (HPAI) virions and a bispecific antibody complex (anti-CD40/M2e) was used to vaccinate chickens. Administration of a single vaccine dose yielded 56%–64% survival against challenge with H5N1 highly pathogenic avian influenza virus, and 100% protection was achieved upon boosting. These results represent a feasible strategy to effectively target whole inactivated influenza virus to chicken antigen presenting cells, regardless of the AIV clade and without phenotyping or purifying the virus from crude allantoic fluid. The data represent proof of principle for the unique prophylactic efficacy and versatility of a CD40-targeting adjuvation strategy that can in principle also be harnessed in other poultry vaccines.

U.S. DEPARTMENT OF AGRICULTURE

Exotic & Emerging Avian Viral Diseases Research: Athens, GA