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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

2016 Annual Report

1a. Objectives (from AD-416):
1. Characterize variant and emerging avian influenza viruses in live poultry markets and commercial production systems. 2. Elucidate the host-pathogen interactions of avian influenza virus infections, including determining host-virus factors that influence infection outcome in different poultry hosts. 3. Develop intervention strategies to effectively control avian influenza and stop disease outbreaks, including identifying conserved B- and T-cell epitopes within and between virus subtype to target in new vaccine platforms or improvement of existing vaccines, and characterizing the humoral and cellular immune response to wild-type infection, and compare it to attenuated and inactivated vaccines to identify correlates of protection. 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. Experimental challenge in poultry and wild bird species will be conducted. 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. Transmission in chickens and pekin ducks will be evaluated. 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 the life of this project substantial data were generated and numerous goals were accomplished. In addition to the planned research objectives there were four major avian influenza virus (AIV) events to which we responded (2012 H7N3 highly pathogenic (HP) AIV in Mexico, 2013 H7N9 low pathogenicity (LP) AIV in China which infects humans, 2014-2015 H5 HP AIV in the U.S., and 2016 H7N8 HP AIV in the U.S.). We are only able to highlight a few of the most important achievements of this project over five years. Selected accomplishments from the planned research objectives include: Outbreak response. Responses to each outbreak were multi-disciplined and were coordinated with the field response and with collaborators when appropriate. First, a HP strain of AIV emerged in Mexico in 2012. Working with the Mexican government scientists, we conducted studies to elucidate the pathogenesis of the virus in chickens and to identify the best vaccines to aid in the control of the outbreak and to reduce the risk of being introduced into the United States. The second outbreak event was an outbreak of H7N9 subtype LP AIV in China. Although LP AIV outbreaks in domestic poultry are not uncommon, this strain caused (and still does cause) human infections with a case fatality rate near 60%, was found widely in China, and was seen as a potential human pandemic influenza virus threat. Our laboratory provided a rapid risk assessment for the role of different poultry species to carry and transmit the virus into humans. Key findings were that quail and chickens were the most probable disease vector and that the virus replicated primarily in the respiratory tract which helped guide surveillance procedures in poultry. Additionally, the PCR H7 subtype test was redesigned to allow for a more sensitive and accurate detection of the virus in the United States if the virus spread to the United States. As a follow-up, more advanced methods have been used to elucidate genetic markers for host specificity among different bird species. H5 and H7 HP AIV outbreaks occurred in the U.S. during the course of this project. During each outbreak research priorities were adjusted so that data critical for the field response could be generated and provided to the industry and government responders. Data included ensuring that current diagnostic tests were adequate, which lead to the development of a specific test for identifying the H5 strain and using genetic data from isolates to trace the origin and movement of the virus during the H5 outbreak. In addition the pathobiology of the H5 virus was evaluated in chickens, turkeys, quail, pheasants, partridges, guinea fowl, domestic and l wild duck species, domestic geese and other waterfowl. The pathogenesis studies revealed that the H5 virus had an unusually delayed clinical presentation in turkeys and chickens which may have contributed to the spread of the virus. It was confirmed that domestic and wild waterfowl could carry and transmit the virus easily with minimal clinical disease. In contrast the H7N8 HP AIV strain had a much more typical clinical presentation in chickens and turkeys. Finally, many vaccine trials were conducted with each strain to identify the best available vaccines in case vaccination was to be used as part of the control efforts. A reverse genetics vaccine was developed in the laboratory and transferred to a commercial company, and it was selected as one vaccine that was purchased for use in the APHIS Veterinary Stockpile. Diagnostics. During each outbreak the current diagnostic tests were evaluated for their performance with the outbreak isolates. In three of the events additional tests that were specific for the outbreak strains were developed and deployed to stakeholders. In addition to the modifications described for outbreak related tests, several other improvements were made for AIV diagnostics in poultry. First, the performance of rapid on-farm tests (lateral-flow devices) was characterized to show how soon post infection the virus can be detected from chickens and it was also shown that virus could be detected by these methods from chickens that appear healthy. Further the methods for collecting AIV samples were further refined to identity the best swab type (a flocked swab), the optimal media, and optimal transport conditions. Vaccines. Vaccine work with chickens, turkeys and ducks included evaluating commercially available vaccines for efficacy against newly emerging strains of AIV during each of the major events. Data were produced to identify the best strains to use as vaccines against these outbreak viruses. This included conducting potency and efficacy testing and characterizing the genetics of the outbreak viruses. Using reverse genetics vaccine technology, a representative H5 virus was modified from HP to LP to allow it to be used as killed vaccine. This virus was transferred to a commercial company for commercial production. Data from the work with H5 vaccines was used to inform the preparation of H5 AIV vaccines for the US veterinary stockpile. Other testing included the evaluation of commercial products recently licensed in U.S. Work was also completed on elucidating a key factor in AIV vaccine failure: maternal antibody. Maternal antibody to avian influenza virus suppresses the immune response to viral-vectored vaccines to avian influenza virus. Avian influenza is an important poultry pathogen that is often controlled through the use of vaccines abroad, but maternal antibody that comes from the egg yolk can potentially suppress the immune response to vaccination during the early life of the chick. Using a passive antibody model system to mimic maternal antibodies, two different viral-vectored vaccines were tested with or without maternal antibody and it was shown even for this type of vaccine that maternal antibody will at least partially suppress the immune response to this type of vaccine. It is important to get an early response to vaccines for poultry disease because the production life of chickens is so short, and this study shows that the new generation of vaccines does not resolve the vaccine suppression issue. Pathobiology. Substantial progress was made in identifying the genes involved in virulence for the “goose/Guangdong/1996” lineage of H5N1 HP AIV infection in ducks and the effect of duck age and species on clinical disease and response to vaccination. This group of HPAIVs is unique in their ability to cause disease in ducks. The study showed how age and breed of duck affects how well 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. Virus inactivation. Several accomplishments focused on HPAIV 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 (i.e. HPAIV), few eggs are laid which could contain the virus, however this research shows that the virus may be able to 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 cooking times and temperatures were evaluated which could inactivate the virus, which would allow for liquid egg products to be safety shipped, thus maintaining business operations and trade. Wild bird monitoring. Monitoring of wild bird specimens for AIV has been an ongoing effort since 1998. Samples from Asia and the continental US have been collected by collaborators yearly and tested. Some of the most important data are the detection of the H5 HP AIV involved in the outbreak in poultry in the U.S. in 2014-2015 in wild ducks in California during the outbreak. This helps to demonstrate that apparently healthy wild waterfowl were a reservoir for these viruses and that they can disseminate the viruses over long distances. This report serves as the final reprot for this project.

4. Accomplishments
1. How the immune system sees different strains of avian influenza virus (AIV) (called the antigenic structure) was characterized for the H7 subtype (one of the 2 most important subtypes in poultry), strain in North America. The antigenic structure of 93 AIV isolates of the H7 subtype were compared and mapped to inform vaccine development. Scientists in Athens, Georgia, were part of the team that found that the diversity in structure was more limited than expected. The importance of this work is that the limited antigenic variation observed allows for the development of fewer vaccines that will still provide good protection for the circulating virulent strains.

2. Surveillance for viruses in wild birds that are related to avian influenza virus (AIV) strains that caused the 2014-2015 outbreak in wild waterfowl was conducted. Wild waterfowl are the natural hosts of AIV and can harbor strains that cause severe disease in chickens and turkeys without becoming sick themselves. To determine whether the virulent strains that caused the H5 HP outbreak in poultry in the U.S. in 2014-2015 originated directly from wild birds, samples from wild waterfowl were collected by collaborators in several locations in the Pacific flyway and tested at SEPRL. This showed that the virus was present in relatively high levels in wild waterfowl at nearly the same time that outbreaks occurred in domestic poultry. Additional genetic analysis of the wild bird viruses provided additional epidemiologic data to support the role of wild birds as the source of the poultry outbreasks.

3. Characterization of avian influenza viruses from wild birds from Ukraine. Wild bird surveillance for avian influenza virus (AIV) was conducted from 2001 to 2012 in the Azov-Black Sea region of Ukraine, which is considered part of the transcontinental wild bird migration routes from northern Asia and Europe to the Mediterranean, Africa, and southwest Asia. From the 6281 samples collected, 69 AIVs belonging to 15 of the 16 known hemagglutinin (HA) subtypes were isolated. Sequencing and phylogenetic analysis of the HA genes revealed epidemiological connections between the Azov-Black Sea region and Europe, Russia, Mongolia, and Southeast Asia. Serology conducted on serum and egg yolk samples also demonstrated previous exposure of many wild bird species to different AIVs. The results demonstrate the great genetic diversity of AIVs in wild birds in the Azov-Black Sea region as well as the importance of this region for monitoring and studying the ecology of influenza viruses. This information furthers our understanding of the ecology of AIV in wild bird species.

4. Respiratory protection for researchers working with avian influenza viruses. Positive air purifying respirators (PAPRs) are common personal protective equipment used in working with zoonotic avian influenza viruses and failures are rare. Two incidences of failures, a tear in a breathing tube and separation of a breathing tube from a fitting joint, were reported. Although no exposure to avian influenza was likely to have occurred occurred, these incidents prompted additional research to reduce this time of accident from happening in the future. Improvements to PAPRs included the addition of breathing tube covers, reinforcement of breathing tube and fitting joints, and notification of the manufacturer of PAPR issues. These incidents demonstrate the need for continuous evaluation and updating of biosafety procedures and equipment.

5. Breed differences between layer- and meat-type chickens determines severity of disease from very virulent infectious bursal disease virus (vvIBDV) infections. Previous studies found more severe vvIBDV diseases in specific-pathogen-free (SPF) egg-type chickens than in conventional broiler chickens, but it is unclear if this resistance of broilers was genetic only or was influenced by other concurrent floral factors. We determined that SPF egg-type chickens were more susceptible to disease and lesions produced by vvIBDV infection than SPF broiler chickens. This study provides important information on impact of chicken breed on susceptibility to vvIBDV.

6. Pigs are susceptible to low pathogenic avian influenza viruses (LPAIV). Pigs are proposed as mixing vessel for development of human transmissible influenza viruses but their susceptibility to LPAIV are unknown. In this study pigs were inoculated with ten different H5 or H7 LPAIV and were shown to be susceptible to infection but did not develop clinical disease. Mild lung lesions were identified but no LPAIV transmitted to contact pigs. LPAI strains from various bird populations within the United States are capable of infecting pigs. Thus the subclinical nature of the infections demonstrates potential role in human-zoonotic LPAIV dynamics over long periods of time.

7. Long-term circulation of H5 high pathogenicity avian influenza virus (HPAIV) in Asian wild bird populations. H5 HPAIV of clade HPAI viruses caused major disease outbreaks in wild birds and poultry in Asian and North America during 2014-15. These HPAIV have evolved into multiple genetic subgroups during the wild bird breeding season and have had a long-term circulation in wild bird populations. In South Korea the subtype icA3 viruses was reintroduced by migratory waterfowl, but this virus was genetically distinct from the H5N8 HPAIV circulating on poultry farms. This demonstrates the long-term circulation and need for continued surveillance for HPAIV in Eurasian wild birds.

8. New H9N2 low pathogenic avian influenza viruses (LPAIV) were identified in Pakistan. H9N2 LPAIV commonly causes disease in poultry across North Africa, the Middle East, and Asia. We report four new H9N2 LPAIV in Pakistan during 2012 and 2015 which belonged to Middle East B genetic group of G1 lineage. These viruses retained genetic features for the potential to be infectious in mammals, including attachment to human associated sialic acids. Continued active surveillance in poultry and mammals is needed to monitor the spread and understand the potential for zoonotic infection by these H9N2 LPAIV.

9. Reassortment of H5 high pathogenicity avian influenza viruses (HPAIV) occurred in North America during the fall 2014. Asian HPAIV (H5N8) viruses spread into North America in 2014 likely during the autumn bird migration. Complete genome sequencing and phylogenetic analysis of 32 H5 viruses determined the H5N2 reassortant viruses emerged in November 2014, and H5N1 and H5N8 reassortant viruses emerged in December 2014 through reassortment with North American low-pathogenicity avian influenza viruses. These studies indicate the reassortment of H5N8 HPAIV and North American lineage LPAIV occurred very quickly after H5N8 HPAIV was introduced in North America.

10. Initial H5 high pathogenicity avian influenza viruses (HPAI) in USA during 2014 were poorly adapted to chickens. In 2014-2015, the U.S. experienced an unprecedented outbreak of Eurasian clade H5 HPAIV, initially affecting mainly wild birds and a few backyard and commercial poultry premises. Studies indicated these first H5 HPAIV were poorly chicken adapted, and had delayed appearance of lesions, longer mean death times, and reduced replication in endothelial cells as compared to historical Eurasian H5N1 HPAIV. However, after multi-generational passage in poultry they became more infectious and transmissible for chicken.

5. Significant Activities that Support Special Target Populations:

Review Publications
Lee, D., Fusaro, A., Song, C., Suarez, D.L., Swayne, D.E. 2016. Poultry vaccination directed evolution of H9N2 low pathogenicity avian influenza viruses in Korea. Journal of Virology. 488(2016):225-231. doi: 10.1016/j.virol.2015.11.023.

Kwon, J., Lee, D., Swayne, D.E., Yuk, S., Tseren-Ochir, E., Noh, J., Hong, W., Jeong, J., Jeong, S., Gwon, G., Song, C. 2016. Highly pathogenic avian influenza A(H5N8) viruses reintroduced into South Korea by migratory waterfowl, 2014–2015. Emerging Infectious Diseases. 22(3):507-510.

Pantin Jackwood, M.J., Kapczynski, D.R., Dejesus, E., Costa-Hurtado, M., Dauphin, G., Tripodi, A., Dunn, J.R., Swayne, D.E. 2016. Efficacy of a recombinant turkey herpesvirus H5 vaccine against challenge with H5N1 clades 1.1.2 and highly pathogenic avian influenza viruses in domestic ducks (Anas platyrhynchos domesticus). Avian Diseases. 60:22-23.

Balzli, C.L., Kozlovac, J.P., Swayne, D.E. 2015. Improvements in powered air purifying respirator protection in an ABSL-3E facility. Applied Biosafety. 20(4):175-178.

Spackman, E. 2016. Avian influenza virus. In: Liu, D. editor. Molecular Detection of Animal Viral Pathogens. Baca Raton, FL: CRC Press. p.377-382.

Costa-Hurtado, M., Afonso, C.L., Miller, P.J., Shepherd, E.M., Cha, R., Smith, D.M., Spackman, E., Kapczynski, D.R., Suarez, D.L., Swayne, D.E., Pantin Jackwood, M.J. 2015. Previous infection with virulent strains of Newcastle disease virus reduces highly pathogenic avian influenza virus replication, disease, and mortality in chickens. Veterinary Research. 46:97. doi: 10.1186/s13567-015-0237-5.

Reeves, A.B., Poulson, R.L., Muzyka, D., Ogawa, H., Imai, K., Bui, V., Hall, J.S., Pantin Jackwood, M.J., Stallknecht, D.E., Ramey, A.M. 2016. Limited evidence of intercontinental dispersal of avian paramyxovirus serotype 4 by migratory birds. Infection, Genetics and Evolution. 40:104-108.

Bertran, K., Swayne, D.E., Pantin Jackwood, M.J., Kapczynski, D.R., Spackman, E., Suarez, D.L. 2016. Lack of chicken adaptation of newly emergent Eurasian H5N8 and reassortant H5N2 high pathogenicity avian influenza viruses in the U.S. is consistent with restricted poultry outbreaks in the Pacific flyway during 2014-2015. Virology. 494:190-197.

Lee, D., Bahl, J., Torchetti, M.K., Killian, M.L., Ip, H.S., Swayne, D.E. 2016. Highly pathogenic avian influenza virus and generation of novel reassortants, United States, 2014-2015. Emerging Infectious Diseases. 22(7):1283-1285.

Sa E Silva, M., Rissi, D.R., Swayne, D.E. 2016. Very virulent infectious bursal disease virus produces more severe disease and lesions in specific pathogen free (SPF) Leghorn than in SPF broiler chickens. Avian Diseases. 60(1):63-66. doi: 10.1637/11230-070615-ResNote.1.

Lee, D., Swayne, D.E., Sharma, P., Rehmani, S.F., Wajid, A., Suarez, D.L., Afonso, C.L. 2016. H9N2 low pathogenic avian influenza in Pakistan (2012-2015). Veterinary Record. 3:e000171. doi:10.1136/vetreco-2016-000171.