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Research Project: Intervention Strategies to Control and Prevent Disease Outbreaks Caused by Avian Influenza and Other Emerging Poultry Pathogens

Location: Exotic & Emerging Avian Viral Diseases Research

2014 Annual Report

1a. Objectives (from AD-416):
1. Characterize variant and emerging avian influenza viruses in live poultry markets and commercial production systems. 2. Identify genetic and biological determinants of virulence, tissue tropism and host range of avian influenza virus. 3. Identify genetic and biological determinants of avian influenza virus susceptibility and resistance in avian species. 4. Improve existing diagnostic tests and testing strategies for avian influenza virus surveillance, detection, and recovery from disease outbreaks. 5. Develop new vaccine platforms designed to control and prevent avian influenza virus outbreaks.

1b. Approach (from AD-416):
These objectives include a combination of basic and applied research to give us the knowledge and tools to effectively control avian influenza virus (AIV). The first objective focuses on characterizing new and emerging strains of AIV, initially by genome sequencing and analysis, then by pathogenesis and transmission studies, and finally by antigenic characterization. The second objective will elucidate specific viral factors involved in pathogenesis and virulence at a molecular level including utilizaiton of variant isolates initially characterized under objective 1. The third objective will investigate the viral factors involved in transmission and host adaptation of AIV among avian species with reverse genetics and pathogensis studies. Under objective 4 diagnostic tests will be improved by characterizing novel isolates to assure specificity and by adapting novel technologies to improve sentivity and specificity. New vaccines will be developed and evaluated through a variety of approaches including antigenic and molecular characterization for objective 5.

3. Progress Report:
During FY2014 substantial progress was made for all milestones and sequencing of wild bird isolates from Alaska was completed. Progress has been made on all objectives of the project. Also during FY2014 the novel H7N9 strain from China, which has been responsible for fatal human infections, was a primary focus. The animal reservoir of the virus infection in humans is still unclear, although epidemiological data suggests poultry are involved. The role of different avian species was investigated further. This new flu strain did not cause disease in any of the birds tested and it was found that chickens are relatively difficult to infect, which makes them less likely to be a primary reservoir. Quail were the most susceptible, but are not a dominant species in areas affected by the virus. Additionally, natural variants of the virus were tested to determine if there were differences among the strains in their ability to infect poultry. Finally, diagnostic tests for use in poultry by U.S. veterinary labs, which are specific for this novel virus, were completed. Additional FY2014 accomplishments which include rapid tests (lateral-flow devices) for bird flu are available commercially, however data on their performance with the most virulent forms of the virus are lacking. Work was completed which provides performance data for these tests by evaluating how well they detected the virus from chickens at 12 hour intervals post exposure. The performance data will be used to develop response plans for using these devices with bird flu by showing how soon post exposure the virus can be detected and it was shown that virus could be detected from healthy birds. Several accomplishments focused on bird flu in chicken eggs and egg products. First, several strains were investigated for how they enter the egg, and it was shown that the virus targets the reproductive organs of the hen, therefore the virus can be passed into eggs. Since chickens die rapidly once infected with the deadly forms of bird flu, few eggs are laid which could contain the virus, however this shows that the virus can enter the egg. The main concern for this is that the virus can be spread by the movement of eggs to other poultry if the infection is not recognized until after the eggs are moved. Since cooking rapidly inactivates the virus, as shown by another element of the project that developed pasteurization standards for eggs and egg products, food safety is less of a concern than animal health. In this part of the project, specific cook times and temperatures were evaluated which could inactivate the virus; this would allow for liquid egg products to be safely shipped, thus maintaining business operations and trade. The H5N1 deadly bird flu was the subject of several studies. One showed that the virus could survive in water under simulated pond conditions for the same time as the non-deadly form of the virus. This is important because the environmental stability is related to how the virus can spread in nature. Another study showed how age and breed of duck affects how the virus grows, which will allow for the design of more efficient surveillance and control programs. It was also shown that a new strain of the deadly H5N1 viruses could cause disease in several species of migratory waterfowl, but may not kill them. Therefore these species, ruddy shelducks and bar-headed geese, could serve as short or medium distance carriers of the virus.

4. Accomplishments
1. The role of poultry in the spread of a novel flu strain (H7N9) and development of tests for the new strain. A new flu strain emerged in people in China in February, 2013. The source of the virus was thought to be animal markets, but the critical species involved needed to be identified. ARS researchers in Athens, Georgia, were able to show that the virus does infect several types of birds without causing any disease. Furthermore, since chickens and turkeys may be infected, the current tests for bird flu in the U.S. were modified to ensure that the virus could be accurately detected. This work demonstrated that some bird species could be involved in the life cycle of the novel influenza virus and diagnostic tests were implemented so we can confidently check our own birds, including poultry, for the virus.

2. Pasteurization standards for inactivation of avian influenza and Newcastle disease (ND) viruses in egg products were developed. Four million cases (38,623 metric tons) of liquid egg products are exported from the U.S. annually and the presence of avian influenza or ND can restrict trade of egg products. To address this trade issue, pasteurization standards were developed for five commonly exported egg products against low pathogenicity bird flu and high pathogenicity bird flu and non and highly virulent ND viruses. For sugared, fortified, plain, salted egg yolk and homogenized whole eggs, the established pasteurization temperatures and times to inactivate salmonella would also inactivate 100,000 units of bird flu virus or Newcastle virus at most temperatures and times of treatment. However, for the salted and sugared egg yolk products, an additional 0.65 and 1.6 minutes of treatment, respectively, at 63.3°C was necessary to inactivate 100,000 of virulent Newcastle virus. These standards will allow continued exports of pasteurized U.S.A. egg products during outbreaks of avian influenza and ND.

3. Virulent bird flu virus can be found in the reproductive tract of infected chickens. Bird flu is present in eggs laid by infected hens before they die, but where the virus comes from within the body is unknown. Chickens were inoculated with three different strains of bird flu. All three viruses produced lesions in the ovaries and oviducts 36 to 72 hours after inoculation and virus was present in cells of the ovary that surround the egg yolk, cells of the oviduct that secrete the albumin of the egg white and in the blood vessels of both tissues. This study explains why egg yolk and albumin are contaminated with virulent bird flu when laid by infected hens.

4. Wild ducks in Japan may have been exposed to bird flu. The ecology of bird flu is not completely understood, particularly which wild birds may be able to disseminate the virus. ARS researchers in Athens, Georgia, detected signs that wild pintail ducks in Japan had been exposed to H5 bird flu. A high proportion of the ducks tested (61%) were positive. This shows that bird flu may be common in pintail ducks which migrate between China and Japan and helps to elucidate which wild species are infected in the wild during some part of their migration which informs surveillance programs in what species to target.

5. The effect of species, breed and route of exposure was evaluated with deadly H5N1 bird flu viruses for their effect on domestic ducks. Domestic ducks are implicated in the spreading of H5N1I viruses. Therefore, the successful control of H5N1 in ducks is important for the eradication of the disease in poultry. ARS researchers in Athens, Georgia, showed that domestic ducks are susceptible to H5N1 virus infection by different routes of exposure, but the presentation of the disease varies by virus strain and duck species. This information helps support the planning and implementation of surveillance and control measures in countries with large domestic duck populations.

6. Disease caused by H5N1 deadly bird flu in wild waterfowl. Since 2005, different families of H5N1 have caused infections, disease and death in numerous species of wild waterfowl in Eurasia and Africa, but we do not know how the newest family (called clade 2.3.2) affects wild waterfowl. Ruddy shelducks (Tadorna ferruginea) were severely affected and died, but bar-headed geese (Anser indicus) were not as sick and did not die. ARS researchers in Athens, Georgia, demonstrated this new clade of H5N1 bird flu can cause infections and severe disease in some migratory waterfowl, therefore they may serve as short to medium distance disseminators of H5N1 bird flu.

7. Transmission of virulent bird flu to ferrets by eating infected meat. Cats, dogs, tigers and other meat eating mammals have naturally become infected with H5N1 bird flu after consuming infected chicken meat. In studies using a ferret model, animals were infected with different bird flu strains through ingestion of infected chicken meat. However, the dose of virus needed to infect ferrets through consumption of infected meat was much higher than if exposed by the respiratory route. In addition, H5N1 produced higher titers in the meat and more easily infects ferrets than the H7 flu. These findings from ARS researchers in Athens, Georgia, indicate that carnivorous mammals can be infected by eating H5N1 virulent bird flu infected meat but that infection is more likely to occur after breathing contaminated air.

8. The length of survival of deadly bird flu viruses in water was determined. Lakes and other natural bodies of water play a critical role in maintaining and spreading the mild forms of avian influenza viruses in wild waterfowl, but a similar role of these bodies of water for deadly H5N1 is unknown. Using an established laboratory model system, 11 strains of deadly H5N1 were examined for survivability under different simulated environmental conditions. The deadly H5N1 strains responded similarly to mild forms in different water temperatures and levels of salt, with all viruses being most stable at colder temperatures and fresh to brackish salinities. Therefore, ARS researchers in Athens, Georgia, showed that H5N1 has similar stabilities in water as mild bird flu, and suggests there has been no loss in environmental survival in the deadly form.

9. Collaborative approach to identify and correct errors in the influenza virus sequence database and efforts to correct them. Over 300,000 influenza gene sequences have been made available to the public sequence databases, but there are many errors in the sequence database that makes analysis of the data much harder. ARS researchers in Athens, Georgia, in collaboration with a local high school science class at Athens Academy, taught students basic skills to analyze influenza sequence data that allowed them to identify and classify common sequence errors present in the public databases. The students then contacted the submitters of the sequence data in an effort to have them corrected. Over 250 different influenza sequences were corrected as part of this program, and these corrections benefit the entire influenza community because the higher quality data provides better analytical results for their research.

Review Publications
Pantin Jackwood, M.J., Miller, P.J., Spackman, E., Swayne, D.E., Susta, L., Costa-Hurtado, M., Suarez, D.L. 2014. Role of poultry in the spread of novel H7N9 influenza virus in China. Journal of Virology. 88(10):5381-5390. DOI: 10.1128/JVI.03689-13.

Ramey, A., Spackman, E., Yeh, J., Fujita, G., Konishi, K., Uchida, K., Reed, J., Wilcox, B., Brown, J., Stallknecht, D. 2013. Antibodies to H5 subtype avian influenza virus and Japanese encephalitis virus in northern pintails (Anas acuta) sampled in Japan. Japanese Journal of Veterinary Research. 61(3):117-123.

Lebarbenchon, C., Pantin Jackwood, M.J., Kistler, W., Luttrell, P., Spackman, E., Stallknecht, D., Brown, J. 2012. Evaluation of a commercial enzyme-linked immunosorbent assay for detection of antibodies against the H5 subtype of Influenza A virus in waterfowl. Influenza and Other Respiratory Viruses. 7(6):1237-1240. DOI:10.1111/irv.12070.

Pantin Jackwood, M.J., Swayne, D.E., Smith, D.M., Shepherd, E.M. 2013. Effect of species, breed and route of virus inoculation on the pathogenicity of H5N1 highly pathogenic influenza (HPAI) viruses in domestic ducks. Veterinary Research. 44(1):62. DOI: 10.1186/1297-9716-44-62.

Awe, O.O., Ali, A., Elaish, M., Ibrahim, M., Murgia, M., Pantin Jackwood, M.J., Saif, Y.M., Lee, C. 2013. Effect of coronavirus infection on reproductive performance of turkey hens. Avian Diseases. 57:650-656.

Chmielewski, R.A., Beck, J.R., Swayne, D.E. 2013. Evaluation of the U.S. Department of Agriculture's egg pasteurization processes on the inactivation of high pathogenicity avian influenza virus and velogenic Newcastle disease virus in processed egg products. Journal of Food Protection. 76(4):640-645. DOI: 10.4315/0362-028X.JFP-12-369.

Chmielewski, R.A., Beck, J.R., Juneja, V.K., Swayne, D.E. 2013. Inactivation of low pathogenicity notifiable avian influenza virus and lentogenic Newcastle disease virus following pasteurization in liquid egg products. LWT - Food Science and Technology. 52(1):27-30.

Sa E Silva, M., Rissi, D.R., Pantin Jackwood, M.J., Swayne, D.E. 2013. High pathogenicity avian influenza virus in the reproductive tract of chickens. Veterinary Pathology. 50(6):956-960. DOI: 10.1177/0300985813490755.

Nemeth, N.M., Brown, J.D., Stallknecht, D.E., Howerth, E.W., Newman, S.H., Swayne, D.E. 2013. Experimental infection of bar-headed geese (Anser indicus) and ruddy shelducks (Tadorna ferruginea) with a clade 2.3.2 H5N1 highly pathogenic avian influenza virus. Veterinary Pathology. 50(6):961-970.

Bertran, K., Swayne, D.E. 2014. High doses of highly pathogenic avian influenza virus in chicken meat are required to infect ferrets. Veterinary Research. 45:60. DOI:10.1186/1297-9716-45-60. Available:

Swayne, D.E., Spackman, E., Pantin Jackwood, M.J. 2013. Success factors for avian influenza vaccine use in poultry and potential impact at the wild bird-agricultural interface. EcoHealth. 11:94-108. DOI:10.1007/s10393-013-0861-3.

Pepin, K., Spackman, E., Brown, J., Pabilonia, K., Garber, L., Weaver, T., Kennedy, D., Patyk, K., Huyvaert, K., Miller, R., Franklin, A., Pedersen, K., Bogich, T., Rohani, P., Shriner, S., Webb, C., Riley, S. 2014. Using quantitative disease dynamics as a tool for guiding response to avian influenza in poultry in the United States of America. Preventive Veterinary Medicine. 113(4):376-397. DOI: 10.1016/j.prevetmed.2013.11.011.

Maughan, M., Dougherty, L., Preskenis, L., Ladman, B., Gelb, J., Spackman, E., Keeler, C. 2013. Transcriptional analysis of the innate immune response of ducks to different species-of-origin low pathogenic H7 avian influenza viruses. Virology Journal. 10:94. DOI: 10.1186/1743-422X-10-94.

Sharshov, K., Sivay, M., Liu, D., Pantin Jackwood, M.J., Marchenko, V., Durymanov, A., Alekseev, A., Damdindorj, T., Gao, G.F., Swayne, D.E., Shestopalov, A. 2014. Molecular characterization and phylogenetics of a reassortant H13N8 influenza virus isolated from gulls in Mongolia. Virus Genes. DOI: 10.1007/s11262-014-1083-7.

Sivay, M.V., Sharshov, K.A., Pantin-Jackwood, M.J., Muzyka, V.V., Shestopalov, A.M. 2014. Avian influenza virus with Hemagglutinin-Neuraminidase combination H8N8, isolated in Russia. Genome Announcements. 2(3):e00545-14. DOI: 10.1128/genomeA.00545-14.

Suarez, D.L., Chester, N., Hatfield, J. 2014. Sequencing artifacts in the type A influenza databases and attempts to correct them. Influenza and Other Respiratory Viruses. 8(4):499-505. DOI: 10.1111/irv.12239