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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

Location: Exotic and Emerging Avian Viral Diseases Research Unit

2010 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. Dates for latest lab inspection by APHIS: BSL-2 1/29/2008 and BSL-3AG 5/15/2007.


3.Progress Report
The Avian Influenza project has remained active nationally and internationally to meet the objectives and milestones of the research project. Major accomplishments have been achieved for all objectives. This has included:.
1)discovering that the pandemic H1N1 influenza has poor ability to infect poultry, however breeder turkeys can be infected through the reproductive tract during artificial insemination;.
2)improving sample processing methods for avian influenza virus (AIV) testing which drastically improves test sensitivity;.
3)Superior vaccine strains were identified for viruses which are currently circulating in poultry in Central America, improving control programs and reducing risk for U.S. poultry;.
4)numerous wild bird origin viruses have been evaluated for their ability to infect and cause disease in poultry, since these are the primary reservoir for poultry; it was found that turkeys were generally more susceptible to infection with wild bird origin viruses, but the ability to cause infection and disease varied greatly among the isolates;.
5)AIV normally does not cause disease and death in ducks, however the Asian H5N1 strain has gained this ability, it was discovered that young ducks are more susceptible to disease and death than older ducks from these viruses.

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, Department of State 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. Sample processing for avian influenza virus diagnostic test improved to increase test sentivitivty. Cloacal swab samples from poultry and wild birds are a common sample type for avian influenza virus detection, but the fecal material in these samples often contains substances that will inhibit the diagnostic test. ARS researchers in Athens, GA developed a new method of processing the specimens which effectively washes away the inihibtors. This results in a substantial increase in test sensitivity and therefore results in more accurate detection of avian influenza virus from poultry and wild birds.

2. Turkeys are highly susceptible to influenza viruses from numerous different species therefore can potentially mediate transmission between other species. Influenza viruses from numerous species of wild birds and domestic poultry were tested for their ability to infect domestic turkeys, chickens and ducks. ARS scientists in Athens, GA helped demonstrate that turkeys could be most easily infected in comparison to chickens and ducks as they were susceptible to the lowest doses. Turkeys were also susceptible to swine influenza which is frequently observed in the field. These data show that turkeys may be more susceptible for influenza from numerous avian and mammalian species, thus emphasizing the importance of disease control and that turkeys are highly susceptible hosts that have the potential to mediate infection between species.

3. Sixteen domestic origin low pathogenic avian influenza viruses from wild birds were shown to cause minimal clinical diseases in chickens or turkeys. Wild birds are the primary reservoir of avian influenza virus for chickens and turkeys, but it is unknown whether strains that cause severe disease are common or are the exception. Also it was not known whether there are differences between chickens and turkeys in susceptibilty to these viruses. ARS researchers in Athens, GA discovered that of the 16 isolates evaluated most caused no or only mild disease, but that more isolates caused disease in turkeys or that disease was more severe in turkeys than in chickens. These results show that these strains would be difficult to detect because of their mild nature and that turkeys appear to be more susceptible than chickens to avian influenza virus from wild birds.

4. Improvements to current diagnostics tests for a critical avian influenza virus subtype (H7) were completed. The H7 subtype is one of the two most important subtypes of avian influenza virus (the other being H5), because some strains have the ability to cause severe disease and even infection with mild strain and can have serious economic impact. Because the virus constantly changes, diagnostic tests which identify this subtype need to be periodically updated. An update of the currently used test was substantially improved so that it identified all American H7 strains was completed by ARS scientists in Athens, GA. This update, which enables the test to identify more H7 strains than ever before was implemented by the National Veterinary Services Laboratories as an official USDA test.

5. Development of an oral vaccine for avian influenza using yeast cells. Traditional vaccination methods for high pathogenicity avian influenza viruses require costly and time-consuming injection of individual birds, often multiple times, in order to provide adequate protection. The use of yeast as an expression system for influenza proteins can potentially be inexpensive and can be given in the feed. Yeast also provides high quality nutrition when added to the feed. ARS scientists in Athens, GA, successfully expressed the hemagglutinin protein from a subtype H5N1 highly pathogenic avian influenza virus (the protective protein in flu vaccines) on the surface of the yeast strain Pichia pastoris. Functionally, the protein retained its function in the yeast and oral vaccination of chickens produced antibodies which could block influenza infection. This study represents the first step in the development of a yeast-based vaccine for poultry for highly pathogenic strains of avian influenza virus that can be administered in feed.

6. The 2009 pandemic H1N1 influenza virus does not easily infect young poultry. When the pandemic H1N1 influenza virus emerged in the spring of 2009 it was unknown whether the virus could cause disease in poultry or whether poultry could act as reservoir for the virus. ARS scientists in Athens, GA determined that young chickens and turkeys could not be infected by respiratory route, however low levels of virus could be recovered from quail. This demonstrated that the virus is poorly adapted to poultry, therefore poultry would likely not serve as a reservoir and the virus has a minimal disease potential for young poultry.

7. Adult turkey hens are susceptible to 2009 pandemic H1N1 virus by reproductive tract insemination. In constrast to information about infection of young poultry with the human pandemic H1N1 virus, turkey breeder flocks world-wide became infected with the H1N1 pandeimc virus, presumably from infected humans as there was no epidemiological data for other birds as a source of infection. In order to understand how this occurred ARS scientists from Athens, GA exposed adult breeder age turkeys to the pandemic H1N1 by the respiratory route and through the reproductive tract by artificial insemination procedures. Only the turkeys that were exposed by artificial insemination methods were infected. These results suggest that the turkey breeders could have been infected by infected insemination crews and that biosecurity practices for artificial insemination, which is universally practiced by the turkey industry, need to be modified.

8. Superior H5 avian influenza virus strains were identified for vaccines for Central American poultry. Vaccination is widely used to control avian influenza virus in Latin America, however the virus continually changes so that it can evade the immune system, therefore vaccines that were produced with a virus from 1994 were no longer adequately effective. ARS scientists in Athens, GA tested the old vaccine and new strains for their ability to protect chickens against recent strains circulating in Central America. New strains that provided superior protection were identified. Vaccine manufacturers can utilize these new strains of virus to make improved vaccines for the control of avian influenza virus. Improved control of avian influenza virus in Central America reduces the risk of virus infecting US poultry.

9. The pathogenesis of the 2009 pandemic H1N1 virus was characterized in mice and was found to be milder than high pathogenicity avian influenza virus. The mouse is a widely used model system for studying influenza virus pathogenesis and has been used to look at numerous mammalian and avian influenza viruses, and thus contributes invaluable information about the transmission and biology of the virus in different species. ARS scientists from Athens, GA compared the pandemic H1N1 with several other influenza viruses including the Asian high pathogenicity H5N1 virus and found that the pandemic virus caused relatively mild damage in the lungs. Mouse models have been crucial for evaluating and understanding influenza strains from many species and can help assess their pandemic potential, evaluate vaccines and identify species specific characteristics.

10. The ecology and dissemination of high pathogenicity avian influenza viruses in Pakistan was characterized by completing genetic analysis on isolates from 1995 to 2004. High pathogenicity avian influenza viruses of the H7N3 subtypes have persisted in poultry in Pakistan despite vaccination and bio-security programs, therefore it has been unclear whether these are new introductions of virus or whether the virus is maintained within Pakistan with occasional transmission to poultry. ARS scientists from Athens, GA produced full genome sequence of these viruses and discovered that the viruses in Pakistan are distinct from other avian influenza viruses indicating that the virus is maintained in a reservoir within the country. They also found that the virus was genetically mixing with other influenza subtypes that were infecting poultry. Now that the source of the virus has been identified as being within the country and not new introductions from other regions, control programs can be developed to focus on local biosecurity. Additionally, this provides broader information on the ecology and dissemination of high pathogenicity avian influenza viruses which could be introduced into US poultry, which enhances US control programs.

11. A South American avian influenza virus (AIV) isolate with genes related to both the 2002 high pathogenicity virus in Chilean poultry and North American wild bird viruses was discovered. Prior to an outbreak of high pathogenicity AIV in poultry in Chile in 2002, no AIV had been reported in South America and little was known about AIV in wild birds in South America. However, ARS scientists from Athens, GA discovered an AIV in wild bird specimens collected in Bolivia in 2001. Genetic analysis of the virus revealed that some genes were related to the virsues from 2002 in Chile and some genes were related to wild bird viruses from North America. The source of the virus was a cinnamon teal (Anas cyanoptera), which is a non-migratory duck, indicating that AIV is found in South American wild birds.

12. Age at infection affects the pathogenicity of Asian highly pathogenic avian influenza H5N1 viruses (AIV) in ducks. A unique trait of the Asian H5N1 high pathogenicity influenza viruses is that some strains have developed the abililty to cause disease in waterfowl, such as ducks. ARS scientists from Athens, GA evaluated the ability of several of these strains to affect 2 and 5 week old ducks. Although the severity of disease and mortality was somewhat dependent on the strain used, the older ducks were much more likely to survive infection and experienced less severe disease. It was also found that disease correlates with the level of viral replication in tissues. This reveals a critical aspect of the Asian H5N1 AIV biology and can aid control programs by focusing on prevention of infection in young ducks.

13. Commercial vaccines have variable efficacy for protecting chickens and ducks against H5N1 highly pathogenic avian influenza (HPAI) viruses from Vietnam. Vaccination is the primary control method that has been employed for avian influenza viruses in Vietnam and there are numerous commercial vaccines, but little data is available about how well they work. ARS scientists in Athens, GA evaluated three commercial vaccines. The vaccines provided different levels of protection in chickens and ducks following infection with HPAI H5N1 and some were protected from mortality, but viral shedding occurred for at least 5 days post challenge depending on the vaccine, species and challenge virus used. Although the vaccines tested were effective in protecting against disease and mortality, updated and more efficacious vaccines are needed to maintain optimal protection.

14. The optimal detection methods for avian influenza virus (AIV) from wild birds depends on the prevalence of virus. Surveillance for AIV in wild birds is conducted worldwide and the detection methods employed depend on the resources of the labs conducting the surveillance. In order to determine the optimal methods for AIV detection in specimens from wild birds ARS scientists from Athens, GA compared numerous methods; cell culture, chicken eggs and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) (which detects the genetic material of the virus). When cost and virus recovery were taken into account it was shown that the most cost effective method depended on whether most of the specimens were from infected birds or not; the cheapest method, real-time RT-PCR was best when infection rates were low, but virus isolation in chickens eggs was better if infection rates were high.

15. H6N2 low pathogenicity viruses from poultry in CA and NY are adapted to chickens and not ducks making detection of infection difficult. H6N2 is a common subtype of low pathogenicity avian influenza virus seen in poultry and wild birds in North America. ARS scientists from Athens, GA evaluated several isolates from chickens and turkeys in CA and NY for their ability to cause disease in chickens and ducks. The viruses infected chickens more easily than ducks, but caused only minimal disease in either species. This indicates that these viruses have been circulating in chickens long enough to adapt to them and become less adapted to ducks. Since the disease was so mild and low amounts of virus was shed, yet were adequate to spread among chickens, there is a danger that these virsues could circulate without being easily detected.

16. Wild birds can disseminate avian influenza virus (AIV) widely throughout Asia. Samples were collected from wild birds throughout Mongolia from 2005-2007 for AIV testing. Since Mongolia has little poultry production any virus isolates were assumed to be disseminated by wild birds only. In addition to the Asian H5N1 high pathogenicity virus strain, ARS scietists in Athens, GA isolated 10 low pathogenicity AIVs from numerous species, and influenza was detected in specimens from two species for the first time (Luscinia svecica and Calandrella cheleensis). Genetic analysis revealed that there was variation among the viruses indicating that each year new AIVs are introduced into wild bird populations in Asia where they can mix with older viruses.


Review Publications
Spackman, E., Mccracken, K.G., Winker, K., Swayne, D.E. 2007. An avian influenza virus from waterfowl in South America contains genes from North American avian and equine lineages. Avian Diseases (Supplemental). 51:273-274.

Pantin Jackwood, M.J., Suarez, D.L., Spackman, E., Swayne, D.E. 2007. Age of infection affects the pathogenicity of Asian highly pathogenic avian influenza H5N1 viruses in ducks. Virus Research. 130:151-161.

Van Borm, S., Suarez, D.L., Boschmans, M., Ozhelvaci, O., Van Den Berg, T.P. 2010. Rapid detection of Eurasian and American H7 subtype influenza A viruses using a single TaqManMGB real-time RT-PCR. Avian Diseases. 54:632-638.

Adcock, N.J., Rice, E.W., Sivaganesan, M., Brown, J.D., Stallknecht, D.E., Swayne, D.E. 2009. The use of bacteriophages of the family Cystoviridae as surrogates for H5N1 highly pathogenic avian influenza viruses in persistence and inactivation studies. Journal of Environmental Science and Health, Part A. 44(13):1362-1366.

Thomas, C., Swayne, D.E. 2009. Thermal inactivation of H5N2 high pathogenicity avian influenza virus in dried egg white with 7.5% moisture. Journal of Food Protection. 72(9):1997-2000.

Dong, J., Matsuoka, Y., Maines, T.R., Swayne, D.E., O'Neill, E., Davis, C., Van-Hoven, N., Balish, A., Yu, H., Katz, J.M., Klimov, A., Cox, N., Li, D., Wang, Y., Guo, Y., Yang, W., Donis, R.O., Shu, Y. 2009. Development of a new candidate H5N1 avian influenza virus for pre-pandemic vaccination production. Influenza and Other Respiratory Viruses. 3:287-295.

Das, A., Spackman, E., Pantin Jackwood, M.J., Suarez, D.L. 2009. Removal of real-time reverse transcription polymerase chain (RT-PCR) inhibitors associated with cloacal swab samples and tissues for improved diagnosis of avian influenza virus by RT-PCR. Journal of Veterinary Diagnostic Investigation. 21:771-778.

Pfeiffer, J., Suarez, D.L., Sarmento, L., To, T., Nguyen, T., Pantin Jackwood, M.J. 2010. Efficacy of commercial vaccines in protecting chickens and ducks against H5N1 highly pathogenic avian influenza viruses from Vietnam. Avian Diseases. 54:262-271.

Faust, C., Stallknecht, D., Swayne, D.E., Brown, J. 2009. Filter-feeding bivalves can remove avian influenza viruses from water and reduce infectivity. Proceedings of the Royal Society of London: Biological Sciences. 276:3727-3735.

Tibbetts, C., Borsuk, L., Lichanska, A., Lorence, M., Weslowski, B., Schafer, K., Stenger, D., Lin, B., Malanowski, A., Wang, Z., Blaney, K., Long, N., Schnur, J., Metzgar, D., Myers, C., Faix, D., Russell, K., Brown, J., Saad, M., Thomas, C., Swayne, D.E. 2010. Simultaneous detection and identification of influenza virus types, subtypes and emergent variants using re-sequencing microarrays. PLOS One 5(2):e8995, doi:10.1371/journal.pone.0008995.

Pillai, S.S., Pantin Jackwood, M.J., Yassine, H., Saif, Y.M., Lee, C. 2010. The high susceptibility of turkeys to influenza viruses of different origins implies their importance as potential intermediate host. Avian Diseases. 54:522-526.

Moresco, K.A., Stallknecht, D., Swayne, D.E. 2010. Evaluation and attempted optimization of avian embryos and cell culture methods for efficient isolation and propagation of low pathogenicity avian influenza viruses. Avian Diseases. 54:622-626.

Lira, J., Moresco, K.A., Stallknecht, D., Swayne, D.E., Fisher, D.S. 2010. Single and combination diagnostic test efficiency and cost analysis for detection and isolation of avian influenza virus from wild bird cloacal swabs. Avian Diseases. 54:606-612.

Morales,Jr., A., Hilt, D.A., Williams, S.M., Pantin Jackwood, M.J., Suarez, D.L., Spackman, E., Stallknecht, D.E., Jackwood, M.W. 2009. Biological characterization of H4, H6, and H9 type low pathogenicity avian influenza viruses from wild birds in chickens and turkeys. Avian Diseases. 53:552-562.

Pedersen, J., Killian, M., Hines, N., Senne, D., Panigrahy, B., Ip, H., Spackman, E. 2010. Validation of a real-time reverse transcriptase-PCR assay for the detection of H7 avian influenza virus. Avian Diseases. 54:639-643.

Kwon, Y., Thomas, C., Swayne, D.E. 2010. Variability in pathobiology of South Korean H5N1 high-pathogenicity avian influenza virus infection for 5 species of migratory waterfowl. Veterinary Pathology. 47(3):495-506.

Jadhao, S., Suarez, D.L. 2010. New approach to delist highly pathogenic avian influenza viruses from BSL3+ select agents to BSL2 non-select status for diagnostics and vaccines. Avian Diseases. 54:302-306.

Arafa, A., Suarez, D.L., Aly, M.M., Hassan, M.K. 2010. Phylogenetic analysis of hemagglutinin and neuraminidase genes of highly pathogenic avian influenza H5N1 Egyptian strains isolated from 2006 to 2008 indicates heterogeneity with multiple distinct sublineages. Avian Diseases. 54:345-349.

Wasilenko, J.L., Sarmento, L., Spatz, S.J., Pantin Jackwood, M.J. 2010. Cell surface display of highly pathogenic avian influenza hemagglutinin on the surface of Pichia pastoris cells using alpha-agglutinin for production of oral vaccines. Biotechnology Progress. 26(2):542-547.

Jackwood, M., Suarez, D.L., Hilt, D., Pantin Jackwood, M.J., Woolcock, P., Cardona, C. 2010. Biologic characterization of chicken-derived H6N2 low pathogenic avian influenza viruses in chickens and ducks. Avian Diseases. 54:120-125.

Swayne, D.E., Pantin Jackwood, M.J., Kapczynski, D.R., Spackman, E., Suarez, D.L. 2009. Limited susceptibility of Japanese quail (Coturnix japonica) and resistance of other poultry species to the 2009 novel H1N1 influenza A virus. Emerging Infectious Diseases. 15(12):2061-2063.

Spackman, E., Swayne, D.E., Joly, D., Gilbert, M., Karesh, W., Suarez, D.L., Sodnomdarjaa, R., Cardona, C. 2009. Characterization of low pathogenicity avian influenza viruses isolated from wild birds in Mongolia 2005 through 2007. Virology Journal. 6:190-198.

Suguitan, A.L., Marino, M.P., Desai, P.D., Chen, L., Matsuoka, Y., Donis, R.O., Jin, H., Swayne, D.E., Kemble, G., Subbarao, K. 2009. The influence of the multi-basic cleavage site of the H5 hemagglutinin on the attenuation, immunogenicity and efficacy of a live attenuated influenza A H5N1 cold-adapted vaccine virus. Virology. 395:280-288.

Pantin Jackwood, M.J., Wasilenko, J.L., Spackman, E., Suarez, D.L., Swayne, D.E. 2010. Susceptibility of turkeys to pandemic H1N1 virus by reproductive tract insemination. Virology Journal. 7:27.

Eggert, D.L., Thomas, C., Spackman, E., Pritchard, N., R0jo, F., Bublot, M., Swayne, D.E. 2010. Characterization and efficacy determination of commercially available Central American H5N2 avian influenza vaccines for poultry. Vaccine. 28:4609-4615.

Abbas, M.A., Spackman, E., Swayne, D.E., Ahmed, Z., Sarmento, L., Siddique, N., Naeem, K., Hameed, A., Rehmani, S. 2010. Sequence and phylogenetic analysis of H7N3 avian influenza viruses isolated from poultry in Pakistan 1995-2004. Virology Journal. 7:137.

Cilloniz, C., Pantin Jackwood, M.J., Ni, C., Goodman, A.G., Peng, X., Proll, S.C., Carter, V.S., Rosenzweig, E.R., Szretter, K.J., Katz, J.M., Korth, M.J., Swayne, D.E., Tumpey, T.M., Katze, M.G. 2010. Lethal dissemination of H5N1 influenza virus is associated with dysregulation of inflammation, hematological function and lipoxin signaling in a mouse model of infection. Journal of Virology. 84(15):7613-7624.

Belser, J.A., Wadford, D.A., Pappas, C., Gustin, K.M., Maines, T.R., Pearce, M.B., Zeng, H., Swayne, D.E., Pantin Jackwood, M.J., Katz, J.M., Tumpey, T.M. 2010. Pathogenesis of pandemic influenza A (H1N1) and triple-reassortant swine influenza A (H1) viruses in mice. Journal of Virology. 84(9):4194-4203.

Avellaneda, G.E., Sylte, M.J., Lee, C., Suarez, D.L. 2010. A heterologous neuraminidase subtype strategy for the differentiation of infected and vaccinated animals (DIVA) for avian influenza virus using an alternative neuraminidase inhibition test. Avian Diseases. 54:272-277.

Spackman, E. 2008. Avian influenza virus RNA extraction from tissue and swab material. In: Spackman, E., editor. Avian Influenza Virus Methods. Totowa, NJ: Humana Press. p. 13-18.

Spackman, E. 2008. A brief introduction to avian influenza virus. In: Spackman, E., editor. Avian Influenza Virus Methods. Totowa, NJ: Humana Press. p. 1-6.

Liu, Y., Mundt, E., Mundt, A., Sylte, M.J., Suarez, D.L., Swayne, D.E., Garcia, M. 2010. Development and evaluation of an avian influenza, neuraminidase subtype 1, indirect enzyme-linked immunosorbent assay for poultry using the differentiation of infected from vaccinated animals control strategy. Avian Diseases. 54:613-621.

Last Modified: 7/9/2014
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