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United States Department of Agriculture

Agricultural Research Service

Research Project: APPLICATION OF BIOLOGICAL AND MOLECULAR TECHNIQUES TO THE DIAGNOSIS AND CONTROL OF AVIAN INFLUENZA AND OTHER EMERGING POULTRY PATHOGENS
2009 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.


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
The Avian Influenza Research Project has remained active nationally and internationally to meet the objectives and milestones of the project. Major accomplishments have been achieved for all objectives. This has included:.
1)the identification of several possible virulence markers for chickens, also the 2009 emergent H1N1 influenza viruses were evaluated for their ability to replicate and cause disease in poultry;.
2)evaluation of different methods and technologies for vaccine seed strain selection, and performance of challenge studies to evaluate vaccine performance with recent isolates; .
3)a new reverse transcriptase polymerase chain reaction (RRT-PCR) test for the emergent 2009 H1N1 influenza lineage which can differentiate the new viruses from the classical swine H1N1 viruses was developed, also a critical update to the type A specific test to detect the 2009 lineage was completed; and.
4)phylogenetic analysis of wild bird isolates from the U.S. and abroad and analysis of isolates from poultry outbreaks abroad has continued and the data has been submitted to GenBank.

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 APHIS, the State Department, and U.N. Food and Agriculture Organization. These international projects have included vaccine efficacy studies and characterization of H5N1 viruses from the region.


4.Accomplishments
1. THE ROLE OF TURKEYS AS INTERMEDIATE HOSTS FOR INTRODUCING WILD BIRD AVIAN INFLUENZA VIRUSES TO CHICKENS. Because turkeys are more frequently reared in facilities where the turkeys can come in direct contact with wild birds than chickens, there have historically been more infections of turkeys with low pathogenicity avian influenza virus than chicken. It has also been postulated that turkeys may be more susceptible to viruses from wild birds and can serve as bridging species between ducks and chickens for unknown reasons. A study was performed to evaluate the susceptibility of turkeys, as well as quail, domestic ducks, and geese to infection with wild duck origin avian influenza virus and to compare it to that of chickens. With the duck origin virus the chickens were the least susceptible; turkeys, quail, domestic ducks and geese were more sensitive. This demonstrates how mixing of poultry species could facilitate cross-species spread of avian influenza virus.

2. THE REAL-TIME REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION (RT-PCR) FOR THE IDENTIFICATION OF THE AVIAN INFLUENZA VIRUS (AIV) H7 HEMAGGLUTININ SUBTYPE WAS UPDATED FOR CURRENT H7 VIRUSES. Assays to identify the most economically important stains of avian influenza are critical for timely control of outbreaks. Because AIV has such a high mutation rate, molecular detection assays must be continually monitored to assure that they are identifying with high sensitivity circulating strains of avian influenza. The currently available USDA test to identify the H7 subtype of avian influenza was updated to detect a broader range of H7 viruses that are found in the Western hemisphere. The test was evaluated for sensitivity, specificity and performance with clinical samples. The improved test has transferred to the National Veterinary Services Laboratories, (NVSL) Animal and Plant Health Inspection Services and is in use in the National Animal Health Laboratory Network (NAHLN) labs.

3. SPECIMENS COLLECTED FROM CROCODILES WERE TESTED FOR AVIAN INFLUENZA VIRUS. Crocodiles live in habitats which are frequented by waterfowl, the natural hosts of avian influenza virus and viable avian influenza virus can be recovered from unconcentrated pond water in areas in areas with high numbers of waterfowl. It is unknown if other species which share these habitats are susceptible to avian influenza virus, therefore, cloacal swab samples and serum samples were collected from farm reared crocodiles and were tested for evidence of influenza infection. Despite a sample that was weakly positive by a real-time polymerase chain reaction (PCR) test, no live virus was recovered and all antibody specimens were negative. This helps to add to our understanding of the ecology and host range of avian influenza virus.

4. WILD BIRDS FROM FOREST HABITATS IN CHINA WERE TESTED FOR AVIAN INFLUENZA VIRUS. Although it is well established that aquatic birds are reservoirs for avian influenza virus, the full natural host range among all avian species is not known. Because of the high prevalence of the H5N1 high pathogenicity viruses in Asia, China is an important location for sample collection to determine which birds can serve as host for this virus as well as for other low pathogenicity avian influenza virus. Samples were collected from land-habitat birds in southern China between 2004 and 2007. These samples were tested for avian influenza virus with polymerase chain reaction (PCR) tests and 2.3% were positive, indicating that species other than waterfowl serve as hosts for the virus. This data improves are understanding of what species may be important in the ecology of avian influenza virus.

5. CURRENT DIAGNOSTIC TESTS FOR CHICKENS AND TURKEYS WERE EVALUATED FOR PERFORMANCE WITH SPECIMENS FROM DOMESTIC DUCKS. Because ducks are natural hosts for avian influenza virus and can often be infected without showing any clinical signs, monitoring of commercially reared domestic ducks is critical for the poultry industry. Current diagnostic tests have only been evaluated and validated with specimens from chickens and turkeys, and since species of origin can affect diagnostic test performance it is necessary to evaluate avian influenza virus tests for use with duck specimens. It was shown than the virus detection assays, real-time reverse transcriptase polymerase chain reaction (RT-PCR) and virus isolation perform adequately with duck specimens, however some of the routine antibody detection tests for chickens and turkeys, do not perform adequately. The optimal virus detection and antibody detection methods for duck specimens from the currently available methods were determined thus providing the duck industry, the National Poultry Improvement Plan and veterinary diagnosticians with information to develop efficacious diagnostic programs for ducks.

6. CHARACTERIZATION OF H5N1 HIGH PATHOGENICITY AVIAN INFLUENZA VIRUS ISOLATES FROM NIGERIA. As the Asian origin H5N1 high pathogenicity avian influenza virus spreads into Africa it is important to characterize the isolates to establish their source and any possible changes in pathogenesis for poultry. Three virus, isolates from chickens in Nigeria, were sequenced for phylogenetic analysis and were tested for their pathogenicity in poultry. Sequencing revealed that the isolates were related to recent viruses from Europe and the Middle East. The viruses produced systemic and severe disease typical of this lineage of viruses based on pathogenesis studies with chickens. This helps to establish the dissemination of H5N1 high pathogenicity viruses in Africa and characterizes the biology of the virus over time and in new regions.

7. DETERMINATION OF THE SUSCEPTIBILITY OF HOUSE SPARROWS AND ROCK PIGEONS TO H5N1 HIGH PATHOGENICITY AVIAN INFLUENZA VIRUS. The current H5N1 high pathogenicity avian influenza viruses (HPAIV) are known to have a wide species range among poultry and aquatic birds, however little is known about the susceptibility of peri-domestic land-birds to this virus lineage. Pathogenesis, virus shed and minimum infective dose for a recent H5N1 HPAIV were evaluated for house sparrows (Passer domesticus) and rock pigeons (Columba livia). House sparrows were found to be highly susceptible to the virus, however rock pigeons were resistant to infection and only shed low titers of virus for short periods of time. This indicates that house sparrows could play a role in dissemination of the virus among birds including poultry, but rock pigeons are unlikely to be important for the ecology of this HPAIV lineage.

8. H5N1 HIGH PATHOGENICITY AVIAN INFLUENZA VIRUS CAUSES PNEUMONIA IN GUINEA PIGS. Guinea pigs have been suggested as a model to study influenza virus in humans, however, they have not been evaluated with avian influenza viruses, most importantly zoonotic strains such as the recent H5N1 high pathogenicity avian influenza virus (HPAIV) lineage. Guinea pigs were evaluated for their susceptibility to two virus isolates of the H5N1 HPAIV lineage. The guinea pigs could be infected, however disease was relatively mild and virus replication was restricted to the lungs. Microscopic evaluation of lung tissue revealed minimal lesions with one of the isolates, but the second isolate induced pneumonia. Disease was similar to that previously described with human H3N2 and H1N1, but less severe that what is seen in humans with the H5N1 HPAIV lineage viruses. This indicates that the guinea pig may serve as a model for human infection with some avian influenza viruses.

9. ROUTES OF TRANSMISSION OF H5N1 HIGH PATHOGENICITY VIRUSES IN MICE AND FERRETS. The replication and transmission of Asian-lineage high pathogenicity viruses in mammals is thought to occur primarily through the respiratory tract and not through the alimentary tract. Mice and ferrets were exposed to high pathogenicity avian influenza virus strains that are known to infect mammals by the digestive tract (where the mouth is bypassed) or through oral exposure. With the exception of one virus the animals were not infected when the mouth was bypassed, indicating that infection primarily occurs through the respiratory tract (through the connecting sinuses with oral exposure). This provides critical information on the mechanism of virus transmission from birds to mammals.

10. PHYLOGENETIC AND BIOLOGICAL CHARACTERIZATION OF H5N1 VIRUSES FROM VIETNAM. Vietnam is among the countries which have been most severely impacted by the high pathogenicity H5N1 avian influenza viruses. Development of successful control programs requires the characterization of viruses circulating in the field. Nineteen viruses, collected in Vietnam, were characterized genetically and biologically, which aids in vaccine selection and understanding the ecology of the virus. Successful control programs in areas which are affected by high pathogenicity avian influenza virus reduce the threat to the U.S. and provide valuable information on how the virus can be controlled. The sequencing and biological information helps to maintain an up-to-date knowledge of the most recent avian influenza isolates in the field.


6.Technology Transfer

Number of the New/Active MTAs (providing only)1

Review Publications
Aiki-Raji, C.O., Aguilar, P.V., Kwon, Y., Goetz, S.K., Suarez, D.L., Jethra, A.I., Nash, O., Adeyefa, C.O., Adu, F.D., Swayne, D.E., Basler, C.F. 2008. Phylogenetics and pathogenesis of early avian influenza viruses (H5N2), Nigeria. Emerging Infectious Diseases. 14(11):1753-1755.

Davis, L.M., Spackman, E. 2008. Do crocodilians get the flu? Looking for influenza A in captive crocodilians. Journal of Experimental Zoology. 309A:1-10.

Peterson, A.T., Bush, S.E., Spackman, E., Swayne, D.E. 2008. Influenza A virus infections in landbirds, People's Republic of China. Emerging Infectious Diseases. 14(10):1644-1646.

Spackman, E., Ip, H.S., Suarez, D.L., Slemons, R., Stallknecht, D. 2008. Analytical validation of a real-time reverse transcription polymerase chain reaction test for Pan-American lineage H7 subtype avian influenza viruses. Journal of Veterinary Diagnostic Investigation. 20:612-616.

Spackman, E., Pantin Jackwood, M.J., Swayne, D.E., Suarez, D.L. 2009. An evaluation of avian influenza diagnostic methods with domestic duck specimens. Avian Diseases. 53(2):276-280.

Swayne, D.E., Slemons, R.D. 2008. Using mean infectious dose of wild duck-and poultry-origin high and low pathogenicity avian influenza viruses as one measure of infectivity and adaptation to poultry. Avian Diseases. 52:455-460.

Pantin Jackwood, M.J., Rosenberger, S.S. 2008. Antigen detection systems. In: Dufour-Zavala, L., Swayne, D.E., Glisson, J.R., Pearson, J.E., Reed, W.M., Jackwood, M.J., Woolcock, P.R., editors. Isolation, Identification, and Characterization of Avian Pathogens. Fifth edition. Athens, GA: American Association of Avian Pathologists Inc. p. 233-240.

Brown, J.D., Stallknecht, D.E., Swayne, D.E. 2009. Infectious and lethal doses of H5N1 highly pathogenic avian influenza virus for house sparrows (Passer domesticus) and rock pigeons (Columbia livia). Journal of Veterinary Diagnostic Investigation. 21:437-445.

Thomas, C., Manin, T.B., Andriyasov, A.V., Swayne, D.E. 2008. Limited susceptibility and lack of systemic infection by an H3N2 swine influenza virus in intranasally inoculated chickens. Avian Diseases. 52(3):498-501.

Pfeiffer, J., Pantin Jackwood, M.J., To, T.L., Nguyen, T., Suarez, D.L. 2009. Phylogenetic and biological characterization of highly pathogenic H5N1 avian influenza viruses (Vietnam 2005) in chickens and ducks virus research. Virus Research. 142:108-120.

Last Modified: 12/20/2014
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