1a. Objectives (from AD-416):
This in-house project has four general objectives, each of which is broken into sub-objectives: 1. Conduct studies to understand avian influenza viruses evolution and population dynamics, including the characterization of variant and emerging avian influenza viruses in live poultry markets and commercial production systems, and exploring the impact of variable host susceptibility on avian influenza virus persistence in different ecosystems. 1.1. Characterize new and variant avian influenza virus (AIV) isolates. 1.2. Investigate selection for AIV antigenic variation. 2. Elucidate the host-pathogen interactions of avian influenza virus infections, including determining the role of mutations at receptor binding sites on replication and pathogenesis, especially which mutations are important in changing host specificity, identifying molecular determinants of tissue tropism, and identifying molecular determinants of virulence in target animal species. 2.1. Identify genetic markers for AIV adaptation and/or increased virulence in different avian species. 2.2 Investigate host-specific factors associated with infectivity, pathogenicity and transmissibility of current and emerging AIV. 3. Conduct comparative immunology studies of avian species to determine variations in protective host defense mechanisms to avian influenza infections, including determining the innate and adaptive immune response to influenza virus infection in different avian species that are either susceptible, tolerant, or resistant to infection, and determining the contribution of host genetics on innate protection and other novel methods for disease resistance. 3.1. Identify innate defense mechanisms associated with disease resistance to AIV. 3.2. Characterize humoral responses to AIV and identify epitopes associated with adaptive immunity. 3.3. Improve resistance against AIV infections in poultry. 4. Develop intervention strategies to effectively control avian influenza viruses and contain disease outbreaks, including identifying risk factors in poultry production that favor transmission and spread of avian influenza viruses, improving existing diagnostic tests and testing strategies for avian influenza virus surveillance, detection, and recovery from disease outbreaks, developing new vaccine platforms designed to rapidly control and prevent avian influenza virus outbreaks in the various components of poultry production, and characterizing new or emerging poultry disease pathogens to evaluate potential impact on the U.S. poultry industry. 4.1. Maintain, update and improve diagnostic tests for avian influenza. 4.2. Evaluate vaccine strategies to better control and prevent avian influenza virus outbreaks.
1b. Approach (from AD-416):
These four objectives include a combination of basic and applied research that will generate knowledge and help develop tools to improve our ability to prevent and control avian influenza virus (AIV). These research goals are highly interrelated and will be accomplished with similar tools and approaches. thus, experiments will often contribute to more than one objective. The first objective includes the characterization of new strains of AIV which constantly emerge in nature as well as the elucidation of how the virus changes under immune pressure using an experimental approach. The second objective complements the first with a more in-depth focus on the specific viral and host factors that contribute to host adaptation, transmission and virulence. The third objective aims to improve our understanding of the avian immune response to AIV infection and vaccination in key poultry species. The fourth objective will improve current practical intervention strategies including diagnostics and vaccines.
3. Progress Report:
Progress was made on all four objectives and their subobjectives. In the past two years, three outbreaks of highly pathogenic avian influenza (HPAI) have occurred in the United States; these outbreaks were caused by the following subtypes of the virus: H5N2 and H5N8 (2014-15), H7N8 (2016) and H7N9 (2017). Under Objective 1, viruses from each of these outbreaks have been sequenced and characterized for their pathogenesis and infectivity in several bird species including chickens, turkeys, and wild waterfowl. It was found that each virus has unique pathobiological features in each experimentally inoculated species (infectivity, transmissibility, clinical signs, time course of disease onset, shedding patterns). These viral traits can impact how the virus transmits and is diagnosed in the field. Studies have been completed with the H5 highly pathogenic influenza viruses from 2014-2015 in diving ducks, species for which there is little information on their role in avian influenza virus ecology. Similar to dabbling ducks, diving ducks were susceptible to infection; however, the relatively low amount of virus shed by the ducks indicates that they would not be the most efficient species for maintaining and disseminating the virus. On the other hand, these H5 viruses easily infected domestic ducks and geese, emphasizing the need to improve active surveillance and biosecurity in domestic waterfowl to reduce direct and indirect contact between poultry and wild waterfowl in order to prevent avian influenza in poultry. It was also determined that H5N2 highly pathogenic avian influenza viruses as they circulated in poultry species, underwent adaptation becoming more infectious for poultry, which complicates the control of the virus. A separate H7N8 high pathogenicity avian influenza outbreak occurred on a single turkey flock in Indiana during January 2016. Surveillance testing did not identify any additional highly pathogenic avian influena (HPAI) outbreaks, but low pathogenic H7N8, the mild form of the virus, was detected in surrounding turkey flocks. Genetic testing determined the mild and deadly forms of H7N8 influenza were closely related, and the deadly form arose from the mild form on a single farm. Experimental challenge studies showed the viruses were highly infectious in chickens, turkeys and ducks. In 2017, an outbreak of H7N9 highly pathogenic influenza occurred in two broiler breeder farms in Tennessee. As with the H7N8 Indiana outbreak, a low pathogenic version of the virus was also detected by surveillance in farms in this and other three states. Viruses from this outbreak have been sequenced showing the virus was closely related to wild bird viruses. Pathogenicity studies show that the virus is highly infectious and well adapted to chickens. Under Objective 2, significant progress was made towards identifying genetic markers for avian influenza virus adaptation and/or increased virulence in different avian species. Full genome sequencing of the H5 highly pathogenic avian influenza viruses that caused the outbreaks in poultry in the United States in 2014-15, helped identify possibly genetic markers related to adaptation in turkeys and chickens. Host-specific factors associated with infectivity, pathogenicity and transmissibility of emerging avian influenza virus were also examined. Studies determined that although age is not a determinant factor in susceptibility of broilers for H5N2 high pathogenicity avian influenza virus, compared to egg type chickens, broilers were more resistant than the layer chicken. This apparent lower susceptibility of broilers may explain the lack of affected broiler farms during the 2015 United States outbreaks. Under objective 3, several studies have been conducted on immunity to avian influenza virus following vaccination. In addition, a next generation sequencing method was developed to examine changes related to vaccine-induced immune pressure in genes from the 2014-15 H5 highly pathogenic avian influenza viruses. Another subobjective is the development of transgenic chickens as a tool to help investigate the interaction of avian influenza virus (AIV) and the chicken immune system with a longer term goal of developing chickens resistant to avian influenza. Transgene molecules utilizing RNAi or CRISPR technologies for knock-in and knock-out capabilities were produced as first step in this process. Vaccination for AIV is most often done with inactivated vaccines, but little data exists on the optimal adjuvants (additives which improve vaccines) to use in chickens. Under Objective 4, ten adjuvants were compared for antibody response and protection against exposure to a virulent avian influenza virus strain. It was discovered that mineral oil based additives performed the best in chickens compared to plant and insect shell based additives. Accurate diagnostic tests are critical to detecting novel influenza variants in animal populations. To ensure accuracy, proficiency testing is conducted for laboratories that conduct surveillance for animal influenza and antibody for influenza in animals. An analysis of the results of five years of testing was completed and evaluated in 41 labs from 23 countries. The study demonstrated that although specific testing methods may differ among the laboratories, testing for influenza or antibody to influenza was accurate with an overall pass rate of 86 percent. In contrast, subtype detection was less accurate with accuracy below 50 percent for avian influenza virus.
1. The pathogenesis of the H7N8 low and highly pathogenic avian influenza viruses from the Indiana 2016 outbreak varies between chickens, turkeys and mallards. Avian influenza virus often naturally mutates from low to high virulence in chickens and turkeys, but it is not known why. ARS researchers in Athens, Georgia, compared two closely related natural strains of avian influenza virus that only differ in one specific genetic mutation that determines their pathotype (low versus high pathogenicity) in chickens, turkeys and mallards. In all three species it took less virus to cause infection with the more pathogenic virus suggesting that the mutation may be a way for the virus to transmit better among birds. Also, the more pathogenic form of the virus did not cause disease or death in the ducks like in the chickens and turkeys, meaning they could easily carry the virulent form for poultry while remaining healthy themselves. This information is critical in understanding the epidemiology of avian influenza and its control.
2. H5N2 highly pathogenic avian influenza viruses from the United States 2014-2015 outbreak have an unusually long pre-clinical period in turkeys. The most severe and costly foreign animal disease outbreak in United States history occurred in 2014-2015 and was caused by a highly pathogenic avian influenza virus. It was unclear why this virus spread so well and eventually caused the destruction of over 50 million chickens and turkeys. ARS researchers in Athens, Georgia, characterized the virus and discovered that it had the unusual ability to infect turkeys, and instead of causing disease in less than 2 days as many previous highly pathogenic viruses, it would not cause disease for as long as 5 days, however the turkeys were infectious during this time. This suggests that virus spread may have been enhanced because turkeys were infected but not showing clinical disease, which would allow a longer period of time before quarantine measures would have been taken facilitating spread.
3. Pathogenicity of H5 highly pathogenic avian influenza (H5N8 and H5N2) United States index viruses in domestic waterfowl. In late 2014, a H5N8 highly pathogenic avian influenza virus spread by migratory waterfowl into North America mixing with low pathogenicity viruses to produce a H5N2 virus. Since domestic waterfowl are common backyard poultry frequently in contact with wild waterfowl, ARS researchers in Athens, Georgia, investigated the pathogenicity of these viruses in domestic ducks and geese. The H5 highly pathogenic avian influenza viruses infected these species and easily transmitted to contact birds, with geese being more susceptible to infection and disease than ducks. Most of the birds did not show clinical signs but excreted virus for several days, representing a risk to other poultry species. These findings emphasize the need to implement and improve active surveillance in domestic waterfowl and increase biosecurity to reduce direct and indirect contact between poultry and wild waterfowl in order to prevent and control avian influenza in poultry.
4. Pathogenicity of H5 highly pathogenic avian influenza (H5N8 and H5N2) United States index viruses in diving ducks. Waterfowl are the natural hosts of avian influenza virus and disseminate the virus worldwide through migration. Historically, surveillance and research efforts for the virus in waterfowl have focused on dabbling ducks, but the role of diving ducks in avian influenza virus ecology has not been well characterized. ARS researchers in Athens, Georgia found that diving ducks (Lesser Scaups and Ruddy Ducks), similar to dabbling ducks, are susceptible to infection with this H5 highly pathogenic avian influenza virus lineage. A lack of clinical disease suggests that they could serve as reservoirs of the virus; however, the relatively low virus titers shed by the ducks and duration of virus being shed indicates that they would not be the most efficient species for maintaining and disseminating the virus. This information is critical in understanding the ecology of avian influenza in reservoir species.
5. Comparison of the pathogenicity and transmission of H5 and H7 highly pathogenic avian influenza viruses in mallards. Wild aquatic birds have been associated with the intercontinental spread of Asian H5 subtype highly pathogenic avian influenza viruses, but dispersion by wild waterfowl has not been implicated with spread of other highly pathogenic viruses. To better understand why the Asian H5 subtype viruses infect and transmit more efficiently in waterfowl than other highly pathogenic avian influenza viruses, ARS researchers in Athens, Georgia, inoculated mallard ducks with one of fourteen different H5 and H7 highly pathogenic avian influenza viruses from previous outbreaks in poultry. All virus-inoculated ducks and contact exposed ducks became infected and excreted moderate to high amount of viruses, with the exception of mallards resistant to two H5 viruses. Clinical signs were only observed in ducks infected with three of the viruses. This study highlights the possible role of wild waterfowl in the spread of any highly pathogenic avian influenza viruses.
6. Changes in adaptation of H5N2 highly pathogenic avian influenza H5 viruses in chickens and mallards. Highly pathogenic avian influenza viruses of the H5N2 subtype caused a severe outbreak in poultry in the United States during 2015, the virus transmitting easily among poultry farms. Since initially the H5N2 viruses spread by wild birds, the question was if the virus had changed (adapted) to better infect chickens and turkeys. ARS researchers in Athens, Georgia, investigated the infectivity, pathogenesis and transmission of poultry H5N2 isolates in chickens and mallards in comparison to the wild duck 2014 index H5N2 virus. Increased virus adaptation to chickens was observed with the poultry H5N2 viruses; however, these viruses still easily infected mallards. Genetic changes in the viruses were also determined, which helps in understanding how avian influenza viruses change to better infect and replicate in poultry species.
7. Age is not a determinant factor in susceptibility of broilers for H5N2 highly pathogenic avian influenza virus. In 2014-2015, the United States experienced an unprecedented outbreak of deadly or high pathogenicity avian influenza virus. This outbreak mainly affected commercial turkey and layer farms in the Midwest, but not broiler (meat chickens) farms. To assess the impact of genetic resistance of broilers and/or any age-related effects, ARS researchers in Athens, Georgia, investigated the ability of a H5N2 HPAI virus to infect and cause disease in commercial 5-week-old, 8-week-old, and adult broilers. The death rate and virus growth were not different between the three ages of broilers but compared to egg type chickens, broilers were more resistant than the layer breed. This apparent lower susceptibility of broilers may have accounted, at least partially, for the lack of affected broiler farms in the midwestern outbreaks.
8. Deep sequencing of H7N8 avian influenza viruses from surveillance zone supports H7N8 highly pathogenic avian influenza was limited to a single outbreak farm in Indiana during 2016. An outbreak of deadly H7N8 high pathogenicity avian influenza occurred in a single flock of turkeys in Indiana during January 2016. During surveillance testing in the Control Zone, eight cases of mild H7N8 influenza (low pathogenicity) were found in surrounding turkey flocks but no additional cases of the deadly highly pathogenic form of avian influenza. Genetic testing conducted by ARS researchers in Athens, Georgia, determined the mild and deadly forms of H7N8 influenza were closely related, and the deadly form likely arose from the mild form on a single farm.
9. Protection of White Leghorn chickens by United States emergency H5 vaccines against H5N2 high pathogenicity avian influenza virus. During December 2014-June 2015, the United States experienced the worst highly pathogenic avian influenza outbreak event for the poultry industry. Three vaccines, developed based on updating existing registered vaccines or currently licensed technologies, were evaluated by ARS researchers in Athens, Georgia, for possible use. The efficacy of a reverse genetics avian influenza inactivated vaccine (rgH5N1), a recombinant herpesvirus turkey vectored vaccine (rHVT-H5), and an RNA particle vaccine (RP-H5) in White Leghorn chickens against clade 22.214.171.124 H5N2 HPAI virus challenge was assessed. Single rHVT-H5 and prime-boost (rHVT-H5 + rgH5N1, or rHVT-H5 + RP-H5) vaccination strategies protected 3-week-old chickens and significantly reduced virus shedding. Single vaccination with either rgH5N1 or RP-H5 vaccines provided clinical protection in adult chickens and significantly reduced virus shedding. Double rgH5N1 vaccination protected adult chickens from clinical signs and mortality when challenged 20 weeks post-boost, with high levels of long-lasting protective immunity and significantly reduced virus shedding. These studies support the use of genetically related vaccines for emergency vaccination programs against H5 highly pathogenic avian influenza virus in young and adult layers.
10. Virus-like particle vaccines were developed for avian influenza H5/H7/H9 antigens derived from the H5N1, H7N3 and H9N2 AI subtypes. A virus-like particle vaccine was prepared by ARS researchers in Athens, Georgia, using baculovirus expression vector and their biochemical, functional and antigenic characteristics. The virus-like particle vaccine was further evaluated in chickens for safety, immunogenicity, and protective efficacy against different avian influenza viruses including H5N2, H7N3 and H9N2 subtypes. All vaccinated birds survived challenge with H5N2 and H7N3 highly pathogenic avian influenza viruses, while all controls died. For low pathogenic avian influenza H9N2 challenge, no mortality was observed following challenge; however, immune responses to the vaccine were detected. Thus, triple-subtype H5/H7/H9 virus-like particle vaccines induced protection against multiple AI challenges.
11. Mallard ducks constitute the main reservoir for low pathogenic avian influenza viruses in nature and differences in prevalence of viral subtypes are likely influenced by immunity in these birds. Heterosubtypic immunity refers to the ability to protect against different avian influenza virus subtypes. Under experimental conditions, the magnitude of the heterosubtypic immunity induced by infection with an H3N8 avian influenza virus against similar (H4N6) or less prevalent subtypes (H10N7 and H14N5) of avian influenza virus in mallards at different time intervals was assessed by ARS researchers in Athens, Georgia. Furthermore, the boosting effect of subsequent infections with different avian influenza virus subtypes in the heterosubtypic immunity was evaluated. It was demonstrated that inoculation with the H3N8 subtype induced heterosubtypic immunity, and that cross protection was related to the genetic relatedness between the avian influenza viruses tested. In addition, the magnitude of heterosubtypic immunity increased with multiple subsequent infections. These findings offer possible explanations to the dynamics of avian influenza virus subtype diversity in wild waterfowl.
12. The use of next generation sequencing for metagenomics analysis of clinical samples. One advantage of the next generation sequencing platform is the possibility to sequence the genetic material in samples without any prior knowledge of the sequence contained within. However, virus in clinical samples is typically available in limited concentrations thus enrichment for nucleic acids of interest is needed to increase the sensitivity of next generation sequencing technologies. ARS researchers in Athens, Georgia, developed a simplified, sequence-independent single-primer amplification (SISPA) technique in combination with the MiSeq Platform to target viral genome sequences representing negative- and positive-sense single-stranded RNA viruses belonging to different virus families. This method allowed the successful assembly of sequences into full or near full length avian influenza virus, infectious bronchitis virus, and Newcastle disease virus viral genomes. Moreover, analysis of the sequence data demonstrated that from a single clinical sample, a mapping assembly representing 98.1 percent of Newcastle disease virus and 98.8 percent of infectious bronchitis virus genomes could be produced. This application can be adaptable to other RNA viruses due to non-specific nature of the amplification technique.
13. Highly pathogenic avian influenza H5N6 virus isolated from a wild Mandarin Duck in South Korea. Highly pathogenic avian influenza viruses have caused significant economic losses in the poultry industries and represent a serious threat to public health. In this study, the first detection of H5N6 highly pathogenic avian influenza from a wild bird sampled in South Korea during the fall 2016 was examined by ARS researchers in Athens, Georgia and collaborators. This virus was genetically close to H5N6 viruses of China, Vietnam, Laos and Hong-Kong including those causing human infections. The detection of the H5N6 HPAIV clade 126.96.36.199 in a migratory bird species in South Korea raises a concern over the potential for broad geographic dissemination of zoonotic H5N6 HPAIV via wild birds outside of East Asia.
14. Major role for migratory wild birds in the global spread of highly pathogenic avian influenza A H5N8 viruses in 2014 and 2015. The deadly H5N8 avian influenza virus emerged in South Korea during 2014 and was spread by wild birds to Japan, North America and Europe during 2014. ARS researchers in Athens, Georgia, helped analyze the sequence data of a large number of highly pathogenic H5 avian influenza virus isolates that clearly showed that long-distance migratory birds spread this H5N8 virus. The H5 hemagglutinin gene from this outbreak was found to easily spread or reassort with different neuraminidase subtypes to create new hybrid viruses that are better at surviving in the wild.
15. Novel reassortant avian influenza (H5N8) virus detected in wild aquatic birds, Russia, 2016. A novel reassortant H5N8 high pathogenicity avian influenza virus HPAIV from wild water birds in Siberia during June 2016 was reported and studied by ARS researchers in Athens, Georgia and collaborators. Sequence analysis showed the viruses were related to poultry outbreaks in the region and that this area comprises important wild aquatic bird migration routes. The re-emergence of a novel virus in wild birds is of concern for dissemination of novel reassortant virus during the coming fall migration.
16. Reoccurrence of a H5N2 clade highly pathogenic avian influenza virus in wild birds, Alaska, 2016. A deadly form of influenza virus caused a major disease outbreak in poultry in the USA during 2014-2015. This highly pathogenic avian influenza virus was detected again by collaborators of ARS researchers in Athens, Georgia, in a wild mallard in Alaska during August 2016 which indicates the virus has not disappeared and raises concern that the virus might return to the lower 48 states during the fall wild waterfowl migration.
17. Sequence analysis of an avian influenza-like H7N2 low pathogenic virus isolated from cats in New York City. In December of 2016 a H7N2 low pathogenic avian influenza virus was detected in cats in an animal shelter in New York City, and veterinary officials worked to control the outbreak. ARS researchers in Athens, Georgia, in collaboration with other researchers analyzed the sequence of the influenza virus and determined the virus was highly similar to viruses found in poultry from live bird markets in New York City in 1999-2000 time frame. The source of the outbreak was not determined, but the analysis highlights the importance of surveillance to monitor for new or reemerging influenza viruses in animals.
18. Vaccines can reduce the amount of infectious bursal disease virus (IBVD) in meat samples of infected chickens. Infection with some infectious bursal disease virus pathotypes results in virus being present in chicken muscle tissue and the role of vaccination providing humoral immunity was examined as a mitigation strategy. Infectious bursal disease virus causes an important disease to the chicken industries and impacts trade in chicken meat. ARS researchers in Athens, Georgia, determined that viable infectious bursal disease virus could only be isolated from breast and/or thigh meat of broiler chickens infected by the two most virulent strains tested. In a second experiment, birds were vaccinated according to a commercially used vaccination program for IBDV, and challenged after vaccination to evaluate the role of vaccination in decreasing the presence of virus in the meat. The vaccination protocol used in the study has shown to be efficient in decreasing the already low presence of virus in chicken meat.
Sa E Silva, M., Moresco, K., Bertran, K., Jackwood, D., Swayne, D.E. 2016. Infection with some infectious bursal disease virus pathotypes produces virus in chicken muscle tissue and the role of humoral immunity as a mitigation strategy. Avian Diseases. 60(4):758-764.
Pantin Jackwood, M.J., Costa-Hurtado, M., Shepherd, E.M., Dejesus, E.G., Smith, D.M., Spackman, E., Kapczynski, D.R., Suarez, D.L., Stallknecht, D., Swayne, D.E. 2016. Pathogenicity and transmission of H5 and H7 highly pathogenic avian influenza viruses in mallards. Journal of Virology. 90(21):9967-9982.
Dejesus, E.G., Costa-Hurtado, M., Smith, D.M., Lee, D., Spackman, E., Kapczynski, D.R., Torchetti, M.K., Killian, M.L., Suarez, D.L., Swayne, D.E., Pantin Jackwood, M.J. 2016. Changes in adaptation of H5N2 highly pathogenic avian influenza H5 clade 188.8.131.52 viruses in chickens and mallards. Virology. 499:52-64. doi:10.1016/j.virol.2016.08.036.
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Goraichuk, I.V., Dimitrov, K.M., Sharma, P., Miller, P.J., Swayne, D.E., Suarez, D.L., Afonso, C.L. 2017. Complete genome sequences of four avian paramyxoviruses of serotype 10 isolated from Rockhopper Penguins on the Falkland Islands. Genome Announcements. 5:e000472-17. doi:org/10.1128/genomeA.00472-17.
Kapczynski, D.R., Tumpey, T.M., Hidajat, R., Zsak, A., Chrzastek, K., Tretyakova, I., Pushko, P. 2016. Vaccination with virus-like particles containing H5 antigens from three H5N1 clades protects chickens from H5N1 and H5N8 influenza viruses. Vaccine. 34(13):1575-1581. doi:10.1016/j.vaccine.2016.02.011.
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Muzyka, D., Pantin Jackwood, M.J., Spackman, E., Stegniy, B., Rula, O., Muzyka, N., Smith, D.M. 2016. Isolation and genetic characterization of avian influenza viruses isolated from wild birds in the Azov-Black Sea Region of Ukraine (2001–2012). Avian Diseases. 60(1):365-377. doi:10.1637/11114-050115-Reg.
Balzli, C., Lager, K., Vincent, A., Gauger, P., Brockmeier, S., Miller, L., Richt, J.A., Ma, W., Suarez, D., Swayne, D.E. 2016. Susceptibility of swine to H5 and H7 low pathogenic avian influenza viruses. Influenza and Other Respiratory Viruses. 10(4):346-352.
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Lee, D., Sharshov, K., Swayne, D.E., Kurskaya, O., Sobolev, I., Kabilov, M., Alekseev, A., Irza, V., Shestopalov, A. 2017. Novel H5N8 clade 18.104.22.168 highly pathogenic avian influenza virus in wild awuatic birds, Russia, 2016. Emerging Infectious Diseases. 23(2):358-360. doi: 10.3201/eid2302.161252.
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Brown, I.H., Abolnik, C., Garcia Garcia, J., McCullough, S., Swayne, D.E., Cattoli, G. 2016. High pathogenicity avian influenza outbreaks since 2008 except multi-continental panzootic of H5 Goose/Guangdong-lineage viruses. In: Swayne, D.E., editor. Animal Influenza. 2nd edition. Ames, IA: Wiley-Blackwell. p. 248-270.
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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.
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