2007 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. BL-2 and BSL-3AG; 05/23/2003. 4.Accomplishments Cooking kills highly pathogenic avian influenza (HPAI) and Newcastle disease viruses in poultry. HPAI viruses can be present in the meat of infected poultry and a prior study demonstrated cooking was effective in killing an H5N1 HPAI virus. Two additional HPAI viruses (H5N2 Pennsylvania/83 and H5N2 Texas/04) and two Newcastle disease viruses (avirulent Ulster and virulent California/02 strains) were tested for thermal inactivation in naturally or artificially infected meat. Cooking at 70C or 73.9C (165F) were effective at killing the viruses in less than 1 minute. Therefore, proper cooking of poultry using the FSIS salmonella standards would be effective at killing both AI and Newcastle disease viruses. This accomplishment is in National Program 103 Animal Health (100%), Component 1 – Bio-defense research. Problem Statement 1A: Foreign Animal Disease. Lower transmission risk for H5N1 HPAI virus occurs through the digestive tract compared to respiratory system exposure. The H5N1 highly pathogenic avian influenza (HPAI) virus has been transmitted from poultry to humans causing infections, disease, and death. Various mammalian models have been used as comparative disease models for human influenza infections. Exposure of pigs, ferrets, guinea pigs, and mice through intranasal or intragastric routes to an H5N1 HPAI virus produced variable results for infection, disease, and death. Infection was more easily achieved through intranasal than intragastric inoculation in pigs and guinea pigs, but one H5N1 virus strain produced death in mice after either intranasal and intragastric inoculation. The most recent H5N1 strains grew better in pigs than the older strains, but did not produce death. These data suggest H5N1 strains vary in ability to infect different experimental mammalian models, but digestive tract exposure is less likely to produce infection than the respiratory exposure route. This accomplishment is in National Program 103 Animal Health (100%), Component 4 – Countermeasures to prevent and control respiratory diseases. Problem Statement 4C: Poultry Respiratory Diseases. Some highly pathogenic avian influenza viruses cause severe disease in several species of wild ducks in experimental infections. Since 2002, H5N1 HPAI viruses have caused mortality in numerous species of wild aquatic birds in Asia and Europe. In collaboration with Southeastern Cooperative Wildlife Disease Study (University of Georgia), five species of wild ducks were intranasally inoculated with an Asian strain of H5N1 HPAI virus. The wood duck was from 2-4 times more susceptible to infection than chickens, the latter are highly susceptible to the virus. Mallards (Anas platyrhynchos), northern pintails (Anas acuta), blue-wing teals (Anas crecca), and redheads (Aythya americana) less sensitive to infection, produced virus in low concentrations for short periods of time, and did not exhibit clinical signs. The data suggests that the wood duck would represent a sensitive indicator species for H5N1 HPAI should it enter North America. This accomplishment is in National Program 103 Animal Health (100%), Component 1 – Bio-defense research. Problem Statement 1B: Emerging diseases. An internal positive control was developed for rapid diagnostic test for avian influenza virus. The real-time RT-PCR (RRT-PCR) rapid diagnostic test for avian influenza has been adopted for routine use for poultry in the U.S., but this test was found to have a high false positive rate when testing wild birds because of PCR inhibitors in the fecal samples. The RRT-PCR test was modified to include an internal control that provides an independent measure of when PCR inhibitors are in a sample and alerts the user to when there is a problem with the test. The understanding that PCR inhibitors are a problem in some types of samples is an important step in reducing false negative samples and improving the specificity of rapid diagnostic tests, and the use of an internal control provides greater test assurance particularly for fecal or tissue samples. This accomplishment is in National Program 103 Animal Health (100%), Component 4 – Countermeasures to prevent and control respiratory diseases. Problem Statement 4C: Poultry Respiratory Diseases. The use of reverse genetics to make avian influenza viruses with a specific sequence has proven to be a valuable tool to examine how these viruses cause disease in poultry. From 1994 to 2006 low pathogenic H7N2 avian influenza viruses circulated in the live bird markets of the Northeast United States, and there was considerable concern that these viruses might change or mutate to the virulent form of the virus. Using a technique called reverse genetics, a representative H7N2 virus was changed in specific ways to try and understand the minimum number of changes needed for the virus to become virulent. The results of this study showed that the virus needed insertions of amino acids at a key site in the virus, the cleavage site, to become virulent, and only simple mutations at the cleavage site would not make it virulent. This study improved our understanding of how avian influenza viruses become virulent and will help us understand risk of low pathogenic viruses changing to the highly pathogenic form in the future. This accomplishment is in National Program 103 Animal Health (100%), Component 4 – Countermeasures to prevent and control respiratory diseases. Problem Statement 4C: Poultry Respiratory Diseases. The real-time RT-PCR (RRT-PCR) rapid diagnostic test for avian influenza virus was evaluated to allow better detection of Asian H5N1 highly pathogenic avian influenza virus. The original RRT-PCR test used to detect H5 avian influenza viruses was designed specifically to detect North American viruses, and although it did detect Asian H5N1 highly pathogenic avian influenza, it did so with lower sensitivity. The primers and probe were evaluated for changes that would improve the sensitivity of the test, and it was identified that the forward primer was found to be a problem for sensitivity. This primer was changed and better sensitivity was seen for Asian H5N1 viruses, and the use of both primers also allowed high sensitivity for North American viruses also. This study documents the importance of periodic review of molecular diagnostic tests to assure they are performing to a high level, and that changes are sometimes necessary to maintain that performance. This accomplishment is in National Program 103 Animal Health (100%), Component 1 – Bio-defense research. Problem Statement 1A: Foreign Animal Disease. Sequencing of avian influenza virus genomes following random amplification allows the sequencing of unusual or uncharacterized avian influenza viruses. Standard real-time PCR tests provides only minimal information on the virus genome, and PCR based genomic sequencing requires preliminary information about the virus’ hemagglutinin and neuraminidase subtypes to allow proper selection of the primers. We have developed a universal method for the quick and precise characterization of all types of avian influenza viruses that does not requires the use of specific PCR primers. This method provides a fast alternative to diagnose disease and to characterize viruses, and because it has the potential to detect mixed infections it may facilitate the understanding of disease symptoms in poultry. This accomplishment is in National Program 103 Animal Health (100%), Component 4 – Countermeasures to prevent and control respiratory diseases. Problem Statement 4C: Poultry Respiratory Diseases. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of new CRADAs and MTAs 3 Number of invention disclosures submitted 1 Number of non-peer reviewed presentations and proceedings 20 Review Publications Lvov, D.K., Prilipov, A.G., Shchelkanov, M.Y., Deryabin, P.G., Grebennikova, T.V., Fedyakina, I.T., Galegov, G.A., Shilov, A.A., Sadykova, G.K., Lyapina, O.V., Peiris, M., Suarez, D.L. 2006. Isolation of highly pathogenic avian influenza (HPAI) H5N1 strains from wild birds in the epizootic outbreak on the Ubsu-Nur Lake and their incorporation to the Russian Federation State collection of viruses. Voprosy Virusologii. 51:14-18. Lee, C., Lee, Y., Senne, D.A., Suarez, D.L. 2006. Pathogenic potential of North American H7N2 avian influenza virus: A mutagenesis study using reverse genetics. Virology. 353:388-395. Desheva, J.A., Lu, X.H., Rekstin, A.R., Rudenko, L.G., Swayne, D.E., Cox, N.J., Katz, J.M., Klimov, A.I. 2006. Characterization of an influenza a H5N2 reassortant as a candidate for live-attenuated and inactivated vaccines against highly pathogenic H5N1 viruses with pandemic potential. Vaccine. 24(47-48):6859-6866. Das, A., Spackman, E., Senne, D., Pedersen, J., Suarez, D.L. 2006. Development of an internal positive control for rapid diagnosis of avian influenza virus infections by real-time reverse-transcription-PCR with lyophilized reagents. Journal of Clinical Microbiology. 44(9):3065-3073. Scott, A., Zepeda, C., Garber, L., Smith, J., Swayne, D.E., Rohrer, A., Keller, J., Shimshony, A., Batho, H., Caporale, V., Giovannini, A. 2006. The concept of compartmentalisation. Reviews of the Scientific and Technical Office of International Epizootics. 25(3):873-879. Winker, K., Mccracken, K.G., Gibson, D.D., Pruett, C.L., Meier, R., Huettmann, F., Wege, M., Kulikova, I.V., Zhuravlev, Y.N., Perdue, M. ., Spackman, E., Suarez, D.L., Swayne, D.E. 2007. Movements of birds and avian influenza from Asia into Alaska. Emerging Infectious Diseases. 13(4):547-552. Brown, J.D., Swayne, D.E., Cooper, R. ., Burns, R.E., Stallknecht, D.E. 2007. Persistence of H5 and H7 avian influenza viruses in water. Avian Diseases. 51(Supplement):285-289. Brown, J.D., Stallknecht, D.E., Beck, J.R., Suarez, D.L., Swayne, D.E. 2006. Susceptibility of North American ducks and gulls to H5N1 highly pathogenic avian influenza viruses. Emerging Infectious Diseases. 12(11):1663-1670. Callison, S.A., Hilt, D.A., Boynton, T.O., Sample, B.F., Swayne, D.E., Jackwood, M.W. 2006. Development and evaluation of a real-time Taqman RT-PCR assay for the detection of infectious bronchitis virus from infected chickens. Journal of Virological Methods. 138(1-2):60-65. Bublot, M., Le Gros, F., Nieddu, D., Pritchard, N., Mickle, T.R., Swayne, D.E. 2007. Efficacy of two H5N9 inactivated vaccines against challenge with a recent H5N1 highly pathogenic avian influenza isolated from a chicken in Thailand. Avian Diseases. 51(Supplement):332-337. Bublot, M., Pritchard, N., Cruz, J.S., Mickle, T.R., Selleck, P., Swayne, D.E. 2007. Efficacy of a fowlpox-vectored avian influenza H5 vaccine against Asian H5N1 highly pathogenic avian influenza virus challenge. Avian Diseases. 51(Supplement):498-500. Lee, C.W., Lee, Y.J., Swayne, D.E., Senne, D., Linares, J., Suarez, D.L. 2007. Assessing potential pathogenicity of avian influenza virus: current and experimental system. Avian Diseases. 51:260-263. Thomas, C., Swayne, D.E. 2007. Thermal inactivation of H5N1 high pathogenicity avian influenza virus in naturally infected chicken meat. Journal of Food Protection. 70(3):674-680. Pletnev, A.G., Swayne, D.E., Speicher, J., Rumyantsev, A.A., Murphy, B.R. 2006. Chimeric West Nile/dengue virus vaccine candidate: Preclinical evaluation in mice, geese, and monkeys for safety and immunogenicity. Vaccine. 24(40-41):6392-6404. Callison, S.A., Riblet, S.M., Sun, S., Ikuta, N., Hiltl, D., Leiting, V., Kleven, S.H., Suarez, D.L., Garcia, M. 2006. Development and validation of a real-time Taqman polymerase chain reaction assay for the detection of Mycoplasma gallisepticum in naturally infected birds. Avian Diseases. 50:537-544. Tsukamoto, K., Imada, T., Tanimura, N., Okamatsu, M., Mase, M., Mizuhara, T., Swayne, D.E., Yamaguchi, S. 2007. Impact of different husbandry conditions on contact and airborne transmission of H5N1 highly pathogenic avian influenza virus to chickens. Avian Diseases. 51(1):129-132. Elvinger, F., Akey, B., Pierson, F., Porter-Spalding, B., Spackman, E., Suarez, D.L. 2007. Characteristics of diagnostic tests used in the 2002 low pathogenicity avian influenza H7N2 outbreak in Virginia. Journal of Veterinary Diagnostic Investigation. 19(4):341-348. Suarez, D.L., Das, A., Ellis, E.H. 2007. Review of rapid molecular diagnostic tools for avian influenza. Avian Diseases. 51:201-208. Tumpey, T.M., Maines, T.R., Van Hoeven, N., Glaser, L., Solorzano, A., Pappas, C., Cox, N.J., Swayne, D.E., Palese, P., Katz, J.M., Garcia-Sastre, A. 2007. A two-amino acid change in the hemagglutinin of the 1918 influenza virus abolishes transmission. Science. 315(5812):655-659. Swayne, D.E. 2007. Understanding the complex pathobiology of high pathogenicity avian influenza viruses in birds. Avian Diseases. 51 (Supplement):242-249. Afonso, C.L. 2007. Sequencing of avian influenza virus genomes following random amplification. Biotechniques. 43(2):188-192.