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United States Department of Agriculture

Agricultural Research Service



2008 Annual Report

1a. Objectives (from AD-416)
The project plan for this CRIS project has four general objectives that are designed to increase our basic understanding of avian influenza virus (AIV) and to develop improved control measures. The specific objectives are listed below. 1. Identify determinants of virulence, tissue tropism and host range of AIV. 2. Develop vaccines that effectively stop outbreaks, allow differentiation from natural infection and can be administered in a cost effective manner. 3. Improve existing diagnostic tests and develop new diagnostic tests that are rapid, sensitive, and improve the detection of avian influenza. 4. Use molecular epidemiologic techniques and viral genomics to understand virus transmission and spread of AI outbreaks in poultry and wild birds. These objectives include a mixture of basic and applied research to give us the additional knowledge and tools to help control AI. This virus remains a difficult target because it can naturally infect a number of wild bird species, which provides numerous exposure opportunities for poultry and other domestic animal species. Additionally, avian influenza virus is highly variable because of both a high mutation rate and the ability to reassort gene segments, which has resulted in many unexpected phenotypes of the virus. The first objective includes the characterization of new or unusual avian influenza viruses in multiple species to provide improved risk assessment of isolates and increase our understanding of the pathogenesis of AIV. Sequence analysis and molecular epidemiology, objective 4, are also an important part of this characterization. The ultimate goal is to understand which specific amino acid changes are correlated to virulence, tissue tropism, and host range. The applied aspect of this project is the development of improved control tools for avian influenza in poultry, objectives 2 and 3. This includes the development and evaluation of new vaccines for use in poultry. Current vaccines, in general, provide solid protection from disease but have serious limitations in how they are applied and their ability to be used in a DIVA (differentiate infected from vaccinated animals) vaccination program. Many vaccine technologies are available that may be mass applied and are DIVA compatible, but more research needs to be done to determine if these technologies can be modified for avian influenza and are cost effective in the poultry industry. The remaining objective is to improve current diagnostic tests and develop new testing technologies. The continued development of existing technologies like real-time RT-PCR will provide increased performance or utility for testing different sample types, and to increase throughput and reliability of the test technology. Alternative technologies will also be explored with collaborative partners to achieve faster, more sensitive and economical testing. Additionally, improved serologic diagnostic tests and procedures will be examined, particularly as allied tests for DIVA vaccines.

1b. Approach (from AD-416)
A multidisciplinary approach will be used to study avian influenza virus (AIV) in poultry with particular emphasis on highly pathogenic avian influenza. The use of molecular biological techniques including RT-PCR, cloning, and sequencing will be used for molecular epidemiology, development of an influenza sequence database, and the use of reverse genetics to engineer influenza viruses to examine an individual viral genes role in virulence. For studying the pathogenesis of influenza, gross and clinical pathology, histology, immunohistochemistry, and quantitative RT-PCR will be used to examine the effect of infection with different viral strains and protection in vaccine trials. Cellular biology, immunology, and host genetics will be used to determine the role of host resistance to influenza infection. Improved diagnostic tests, emphasizing rapid detection, will be developed. Continued surveillance of wild bird isolates will continue with collaborators from several different institutions. These efforts should provide better control measures.

3. Progress Report
Avian Influenza Research has remained active nationally and internationally to meet the objectives and milestones of the research project. Major accomplishments have been achieved for all four objectives. This has included; 1) the increased use of reverse genetics technology to study the pathogenesis of avian influenza viruses, 2) the study of several new vaccine technologies for the control of avian influenza, 3) the phylogenetic analysis of wild bird isolates from the U.S. and outbreaks of avian influenza in the United States and abroad, 4) the development of new real-time reverse transcriptase polymerase chain reaction (RT-PCR) tests for several subtypes of avian influenza and improved ribonucleic acid (RNA) extraction procedures from meat samples. Collaborative work continues with a number of national and international partners to study avian influenza. University partners include but are not limited to the University of Georgia, The Ohio State University, the University of Delaware, the University of Alaska Fairbanks, Georgia Tech, Michigan State University, University of Connecticut, and Auburn University. Collaborative work with industry have included projects with Merial, Vaxin Inc, and American Egg Board. We also have received funding from several other government departments for special projects including the Homeland Security, and Centers for Disease control. We have been involved internationally with several major projects in Indonesia, Egypt, and Vietnam with additional funding from Animal and Plant Health Inspection Service (APHIS), the State Department, and United Nations Food and Agriculture Organization. These international projects have included vaccine efficacy studies and characterization of H5N1 viruses from the region. This progress addresses National Program 103 Animal Health, Component 1 Bio-defense Research Foreign and Emerging Animal Diseases and Component 4 Countermeasures to prevent and control respiratory diseases. The research addresses Agency Performance Measure 3.2.1 Provide scientific information to protect animals from pests, infectious diseases, and other disease causing entities that affect animal and human health and 3.2.3 Develop and transfer tools to the agricultural community, commercial partners, and Federal agencies to control or eradicate domestic and exotic diseases that affect animal and human health.

4. Accomplishments
1. Characterization of Low Pathogenicity H5N1 Avian Influenza Viruses from North America. Avian influenza viruses of many different antigenic subtypes (H1-H16) are found commonly in wild birds, but only the H5 and H7 subtypes are known to have the potential for being highly pathogenic in poultry. The H5N1 subtype is of particular importance because of the widespread outbreaks of highly pathogenic avian influenza in Europe, Asia, and Africa, and extensive surveillance of wild birds was conducted in the Americas to evaluate the chance of these highly pathogenic viruses entering the United States through wild birds. Several low pathogenic H5N1 viruses were isolated in wild birds in collaboration with Animal and Plant Health Inspection Service (APHIS) and United States Geological Survey (USGS), and these viruses were sequenced and shown to be of North American lineage that are separate from the highly pathogenic H5N1 viruses found in Europe, Asia, and Africa. The biologic and sequence characterization of these viruses continue to provide evidence that highly pathogenic H5N1 viruses have not traveled to the Americas in wild birds, and clearly documents that low pathogenic H5N1 viruses are normally found at a low prevalence level in the Americas. This study also included experimental animal studies in collaboration with The Ohio State University that showed that these viruses did not replicate well in poultry and pose only a small threat of introduction to our poultry populations. This accomplishment relates to National Program 103 Animal Health (100%), Component 1 – Bio-defense research. Problem Statement 1B: Emerging diseases.

2. Detection of H5N1 High Pathogenicity Avian Influenza Virus in Meat and Tracheal Samples from Experimentally Infected Chickens. Avian influenza virus replicates in the meat and tissues of infected poultry, and the potential for food borne infections of humans or other animals is a serious concern, but current methods for sampling flu for avian influenza are slow and cumbersome. A new procedure for extracting ribonucleic acid (RNA) from meat and other tissues was developed that allows for the rapid detection of avian influenza virus using the real-time reverse transcriptase polymerase chain reaction (RT-PCR) method. This new procedure allows for testing to be conducted in less than three hours on meat samples in a safe and cost effective approach, and the studies in experimental poultry show that samples can be detected in bird samples as early as 24 hours after infection. This new procedure has already been adopted by the Food Safety Inspection Service (FSIS) for testing of poultry meat to allow rapid testing of samples if a suspected or confirmed outbreak of avian influenza occurs in the United States. This modification of the existing real-time RT-PCR test provides a new application of the technology that provides assurance that poultry meat is safe for human consumption. This accomplishment relates to National Program 103 Animal Health (100%), Component 4 –Countermeasures to prevent and control respiratory diseases. Problem Statement 4C: Poultry Respiratory Diseases.

3. Predicting transmission of avian influenza viruses. Only a few avian influenza viruses (AIV) have been transmitted from wild bird reservoirs to poultry resulting in outbreaks, and predicting which AIV will cause an outbreak has not been possible. We developed an infectivity model which predicts transmissibility based on the intranasal bird mean infectious dose (BID50) test for chickens, turkeys, domestic ducks and geese, and Japanese quail. The quantity of avian influenza virus (AIV) needed to produce an infection in poultry is dependent upon both the bird species and virus strain, and most AIV that caused outbreaks had BID50 of 3log10 or less of virus, while low infectivity was observed for viruses with 4.7log10 or greater BID50. Chickens were not easily infected with most wild bird AIV, but domestic ducks and geese, Japanese quail and turkeys were easily infected and they could serve as key bridging species for waterfowl-origin AIV crossing into domestic poultry. Furthermore, these data suggest mixing of poultry species during rearing and using outdoor production systems is a major risk factor for transmission of AIVs from wild birds to domestic poultry. This accomplishment relates to National Program 103 Animal Health (100%), Component 1 – Bio-defense research. Problem Statement 1B: Emerging diseases.

4. Assessment of urban wild birds as reservoirs and transmission hosts for H5N1. A variety of wild bird species have been sporadically infected with and have died from infections with H5N1 high pathogenicity avian influenza viruses (HPAIV), but which species might be involved as urban and rural reservoirs and transmission hosts are unknown. In cooperative studies with University of Georgia, Southeastern Cooperative Wildlife Disease Study and Southeast Poultry Research Laboratory, infectivity and pathogenicity of three H5N1 HPAI viruses were determined for a variety of wild bird species. Swans, geese, gulls and house sparrows were highly susceptible to H5N1 HPAI virus and are good sentinels for detecting H5N1 HPAIV within an area since mortality in infected birds is high, while dabbling ducks (Genus Anas) were resistant to clinical disease and excreted low levels of virus. Pigeons were resistant to H5N1 HPAIV, and swans and geese were asymptomatic shedders for up to 5 days with a potential for short to intermediate transmission. These studies indicate that only some wild birds species could be involved with transmission of H5N1 HPAI viruses while others would be good sentinels for detection in wild bird populations. This accomplishment relates to National Program 103 Animal Health (100%), Component 1 – Bio-defense research. Problem Statement 1A: Foreign Animal Disease.

5. Transmission of H5N1 to mammals through infected meat. The H5N1 highly pathogenic avian influenza (HPAI) virus has been transmitted from poultry to humans and carnivorous mammals causing infections, disease and death, and transmission through consumption of infected poultry has been proposed as a potential but unproven risk factor. Three H5N1 HPAI viruses from Asia were examined for the ability to produce experimental infection in ferrets through consumption of infected meat or direct intragastric administration of minced infected meat. The three H5N1 viruses were transmitted through eating infected raw chicken meat, but the infections were predominately initiated in and affected the respiratory system. However, with one H5N1 systemic virus strain, A/Vietnam/1203/04, the digestive system was also concurrently affected and the site of initial replication was the small intestines, followed by spread to the liver and pancreas. Our results confirm that consumption of raw H5N1 HPAI virus infected products could initiate infection as has likely occurred naturally with tigers, leopards, domestic cats and dogs, but because humans consume almost exclusively cooked poultry product where cooking inactivates the virus, foods are unlikely to serve as vehicles of transmission of H5N1 HPAI virus to humans. This accomplishment relates to National Program 103 Animal Health (100%), Component 1 – Bio-defense research. Problem Statement 1A: Foreign Animal Disease.

6. The use of reverse genetics to determine the contribution of avian influenza (AI) viral genes in pathogenicity and host immune response. Avian influenza viruses can vary widely in their ability to cause disease in poultry, but few virulence characteristics have been genetically mapped in the virus. Reverse genetics techniques were used to study the role of the nucleoprotein (NP) and non-structured 1 (NS1) genes in the pathogenesis of H5N1 avian influenza and to determine how the chicken immune response to the virus is altered by changes in these genes. We identified one change at position 184 in the NP protein that resulted in greatly increased virus replication and spread in tissues, increased mortality, decreased mean death times and up-regulation of several host genes involved in the innate immune response as well as an increased level of nitric oxide production by the host in response to virus infection. A change in amino acid at position 148 in the NS1 protein had no effect on mortality of the chickens however up-regulated host interferon alpha gene expression levels in tissues. This study provides new insights into how avian influenza virus (AIV) causes disease, information that will be used in improving control methods. This accomplishment relates to National Program 103 Animal Health (100%), Component 1 – Bio-defense research. Problem Statement 1A: Foreign Animal Disease.

7. Detection of Avian Influenza Virus Using an Interferometric Biosensor. The rapid and sensitive diagnosis of avian influenza is critical for the cost effective control of outbreaks, but currently available tests that can be used in the field have low sensitivity and are unable to subtype avian influenza viruses. An antibody based detection system using a novel detection platform was developed that can both detect and subtype avian influenza from clinical samples. This new technology has the potential to provide rapid and sensitive testing of field samples for avian influenza viruses which can provide a more rapid diagnosis of outbreaks and a more rapid response. This accomplishment relates to National Program 103 Animal Health (100%), Component 4 – Countermeasures to prevent and control respiratory diseases. Problem Statement 4C: Poultry Respiratory Diseases.

8. Development and Bench Validation of Real Time RT-PCR Protocols for Rapid Detection of the Subtypes H6, H9 and H11 of Avian Influenza Viruses. Avian influenza virus has 16 distinct antigenic subtypes, but certain subtypes are responsible for most diseases outbreaks in poultry. A real-time reverse transcriptase polymerase chain reaction (RT-PCR) test for the rapid identification of H6, H9, and H11 subtypes of avian influenza were developed and bench validated. The rapid detection and identification of avian influenza viruses, particularly the H6 and H9 subtypes, provide additional tools to diagnose avian influenza outbreaks. The H6 and H9 subtypes are commonly found in other countries and rapid diagnostic tools are needed to rapidly diagnose if these subtypes infect poultry in the United States. This accomplishment relates to National Program 103 Animal Health (100%), Component 4 – Countermeasures to prevent and control respiratory diseases. Problem Statement 4C: Poultry Respiratory Diseases.

9. Pathogenicity and transmission study of the first United States parrot H5N2 virus of Mexican lineage in different poultry species. Avian influenza viruses are sporadically isolated from psittacine birds in quarantine stations, smuggled birds, or pet birds in the United States, and these viruses pose a potential risk of spread to our domestic poultry populations. In 2004 a H5N2 virus was isolated from a parrot in California, and previous sequence analysis showed it to be similar to a Mexican lineage H5N2 poultry virus. Further in vivo studies of the parrot isolate in chickens, ducks and turkeys showed that the virus, though did not cause any clinical signs, could replicate to high titers in these birds and efficiently transmit to contact control cage mates. This study documents the risk of psittacine birds harboring and likely carrying avian influenza viruses from other countries into the United States. This work emphasizes the risks and suggests enhanced surveillance of this avian population to prevent the introduction of foreign animal diseases into poultry. This accomplishment relates to National Program 103 Animal Health (100%), Component 1 – Bio-defense research. Problem Statement 1A: Foreign Animal Disease.

5. Significant Activities that Support Special Target Populations

Review Publications
Swayne, D.E., Senne, D.A., Suarez, D.L. 2008. Avian influenza. In: Dufour-Zavala, L., editor. Isolation, Identification, and Characterization of Avian Pathogens. Jacksonville, FL: American Association of Avian Pathologists. p. 128-134.

Swayne, D.E. 2008. Avian influenza. Foreign Animal Diseases, 7th Edition. St. Joseph, Missouri. U.S. Animal Health Association. p. 137-143.

Spackman, E., Suarez, D.L. 2008. Detection and identification of the H5 hemagglutinin subtype by real-time RT-PCR. In: Spackman, E., editor. Avian Influenza Virus Methods. Totowa, NJ: Humana press. p. 27-34.

Spackman, E., Suarez, D.L. 2008. Type-A influenza virus detection and quantitation by real-time RT-PCR. In: Spackman, E., editor. Avian Influenza Virus Methods. Totowa, NJ: Humana Press. p. 19-26.

Spackman, E., Suarez, D.L., Senne, D. 2008. Avian influenza diagnostics and surveillance methods. In: Swayne, D.E., editor. Avian Influenza Virus. Ames, IA: Blackwell. p. 299-308.

Das, A., Spackman, E., Thomas, C., Swayne, D.E., Suarez, D.L. 2008. Detection of H5N1 high pathogenicity avian influenza virus in meat and tracheal samples from experimentally infected chickens. Avian Diseases. 52:40-48.

Swayne, D.E., Halvorson, D.A. 2008. Influenza. In: Saif, Y.M., Fadly, A.M., Glisson, J.R., McDougald, L.R., Nolan, L.K., Swayne, D.E., editors. Diseases of Poultry. 12th edition. Ames, IA:Blackwell Publishing. p. 153-184.

Brown, J.D., Stallknecht, D.E., Valeika, S., Swayne, D.E. 2007. Susceptibility of wood ducks to H5N1 highly pathogenic avian influenza virus. Journal of Wildlife Diseases. 43(4):660-667.

Rice, E.W., Adcock, N.J., Sivaganesan, M., Brown, J.D., Stallknecht, D.D., Swayne, D.E. 2007. Chlorine inactivation of H5N1 highly pathogenic avian influenza virus. Emerging Infectious Diseases. 13(10):1568-1570.

Swayne, D.E., Suarez, D.L. 2007. Current developments in avian influenza vaccines including food safety aspects in vaccinated birds. Developments in Biologicals. 30:121-131.

Das, A., Suarez, D.L. 2007. Development and bench validation of real-time reverse transcription polymerase chain reaction protocols for rapid detection of the subtypes H6, H9, and H11 of avian influenza viruses in experimental samples. Journal of Veterinary Diagnostic Investigation. 19:625-634.

Pappas, C., Matsuoka, Y., Swayne, D.E., Donis, R.O. 2007. Development and evaluation of an influenza subtype H7N2 vaccine candidate for pandemic preparedness. Clinical and Vaccine Immunology. 14(11):1425-1432.

Swayne, D.E. 2008. High pathogenicity avian influenza in the Americas. In: Swayne, D.E., editor. Avian Influenza. Ames, Iowa: Blackwell Publishing. p. 191-216.

Swayne, D.E. 2008. Avian influenza control strategies. In: Swayne, D.E., editor. Avian Influenza. Ames, Iowa: Blackwell Publishing. p. 287-297.

Swayne, D.E. 2008. The global nature of avian influenza. In: Swayne, D.E., editor. Avian Influenza. Ames, Iowa: Blackwell Publishing. p. 123-143.

Spackman, E., Swayne, D.E., Suarez, D.L., Senne, D.A., Pedersen, J.C., Killian, M.L., Pasick, J., Handel, K., Pillai, S.P., Lee, C., Stallknecht, D., Slemons, R., Ip, H.S., Deliberto, T. 2007. Characterization of low- pathogenicity H5N1 avian influenza viruses from North America. Journal of Virology. 81(21):11612-11619.

Hanson, B.A., Luttrell, M.P., Goekjian, V.H., Niles, L., Swayne, D.E., Senne, D.A., Stallknecht, D.E. 2008. Is the occurrence of avian influenza virus in charadriiformes species and location dependent?. Journal of Wildlife Diseases. 44(2):351-361.

Swayne, D.E., Thomas, C. 2008. Trade and food safety aspects for avian influenza viruses. In: Swayne, D.E., editor. Avian Influenza. Ames, Iowa: Blackwell Publishing. p. 499-512.

Brown, J.D., Stallknecht, D.E., Swayne, D.E. 2008. Experimental infection of swans and geese with highly pathogenic avian influenza virus (H5N1) of Asian lineage. Emerging Infectious Diseases. 14(1):136-142.

Suarez, D.L. 2008. Influenza A virus. In: D.E. Swayne, editor. Avian Influenza. Amea, IA: Wylie-Blackwell. p. 3-22.

Yassine, H.M., Lee, C.W., Suarez, D.L., Saif, Y.M. 2008. Genetic and antigenic relatedness of H3 subtype influenza A viruses isolated from avian and mammalian species. Vaccine. 26:966-977.

Dugan, V.G., Chen, R., Spiro, D.J., Sengamalay, N., Zaborsky, J., Ghedin, E., Nolting, J., Swayne, D.E., Runstadler, J., Happ, G.M., Senne, D.A., Wang, R., Slemons, R.D., Holmes, E.C., Taubenberger, J.K. 2008. The evolutionary genetics and emergence of avian influenza viruses in wild birds. PLoS Pathogens [serial online]. 4(5):e1000076. Available:

Swayne, D.E., Pantin Jackwood, M.J. 2008. Pathobiology of avian influenza virus infections in birds and mammals. In: Swayne, D.E., editor. Avian Influenza. Ames, Iowa: Blackwell Publishing. p. 87-122.

Swayne, D.E. 2008. Epidemiology of avian influenza in agricultural and other man-made systems. In: Swayne, D.E., editor. Avian Influenza. Ames, Iowa: Blackwell Publishing. p. 59-85.

Pillai, S.S., Suarez, D.L., Pantin Jackwood, M.J., Lee, C. 2008. Pathogenicity and transmission studies of H5N2 parrot avian influenza virus of Mexican lineage in different poultry species. Veterinary Microbiology. 129:48-57.

Jadhao, S.J., Achenbach, J., Swayne, D.E., Donis, R., Cox, N., Matsuoka, Y. 2008. Development of Eurasian H7N7/PR8 high growth reassortant virus for clinical evaluation as an inactivated pandemic influenza vaccine. Vaccine. 26:1742-1750.

Thomas, C., King, D.J., Swayne, D.E. 2008. Thermal inactivation of avian influenza and Newcastle disease viruses in chicken meat. Journal of Food Protection. 71(6):1214-1222.

Winker, K., Spackman, E., Swayne, D.E. 2008. Rarity of influenza A virus in spring shorebirds, southern Alaska. Emerging Infectious Diseases. 14(8):1314-1316.

Wasilenko, J.L., Lee, C., Sarmento, L., Spackman, E., Kapczynski, D.R., Suarez, D.L., Pantin Jackwood, M.J. 2008. NP, PB1 and PB2 viral genes contribute to altered replication of H5N1 avian influenza viruses in chickens. Journal of Virology. 82(9):4544-4553.

Brown, J.D., Stallknecht, D.E., Swayne, D.E. 2008. Transmission of H5N1 high pathogenicity avian influenza virus to Herring gulls (Larus argentatus) through intranasal inoculation of virus and ingestion of virus-infected chicken meat. Avian Pathology. 37(4):393-397.

Lipatov, A.S., Kwon, Y., Sarmento, L., Lager, K.M., Spackman, E., Suarez, D.L., Swayne, D.E. 2008. Domestic pigs have low susceptibility to H5N1 highly pathogenic avian influenza viruses. Public Library of Science for Pathogens [serial online]. 4(7):e1000102. Available:

Sarmento, L., Pantin Jackwood, M.J., Kapczynski, D.R., Swayne, D.E., Afonso, C.L. 2008. Immediate early responses of avian tracheal epithelial cells to infection with highly pathogenic avian invluenza virus. Biologicals. 132:175-183.

Lee, C., Jung, K., Jadhao, S., Suarez, D.L. 2008. Evaluation of chicken-origin (DF-1) and quail-origin (QT-6) fibroblast cell lines for replication of avian influenza viruses. Journal of Virological Methods. 153:22-28.

Last Modified: 2/23/2016
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