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. In the past 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). In addition, two unusual low pathogenic avian influenza viruses, H7N2 in cats and H2N2 in live bird markets in the Northeast U.S. were detected. Viruses from each of these outbreaks continued to be characterized for their pathogenesis and infectivity in several bird species. 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. In order to better characterize the wild avian species which could spread the H5 HPAI viruses, American Black Ducks (Anas rubripes), which are closely related to mallards, were challenged with the North American H5N2 and H5N8 index HPAI viruses. The ducks could be infected with low doses of both viruses, and shed virus for over 7 days. This suggests that Black Ducks could serve as efficient reservoirs for the H5 HPAI viruses. Most research efforts studying avian influenza virus (AIV) pathogenicity in waterfowl thus far have been directed toward dabbling ducks. In an effort to understand the possible role of diving ducks, the susceptibility and pathogenesis of the H7N8 and H7N9 HPAI viruses that caused the most recent outbreaks in the United States was investigated in Lesser Scaup, a type of diving duck. No mortality or clinical disease was observed with either virus, but ducks became infected and shed virus up to 14 days. These results demonstrate that Lesser Scaup are susceptible to HPAI viruses, and similar to dabbling duck species, they shed virus for long periods and don’t present with disease. The pathogenesis of the H7N9 low pathogenicity avian influenza virus (LPAI) virus was also investigated in commercial broiler breeders, the bird type mostly affected in this outbreak. The study showed sub-optimal adaptation for sustained transmission with the H7N9 LPAI isolate, indicating that factors other than the birds genetic background may explain the epidemiology of the outbreak. Greater susceptibility and easier transmission of the H7N9 HPAI virus were found which could favor the spread of HPAI over LPAI viruses during outbreaks. An H2N2 LPAI virus isolated from live bird markets in the Northeast United States was also evaluated. In 2014 the H2N2 LPAI virus was isolated from a variety of poultry species from live bird markets. This subtype of avian influenza had been repeatedly isolated from the market system since then and appears to be endemic. Although the virus is low pathogenic in poultry, there is concern about public health because the H2 subtype historically has infected humans. Sequence analysis and chicken transmission studies were performed to evaluate if the representative viruses from 2014 to 2018 are becoming more chicken adapted and represent an increased risk of spread to other species. Another unusual outbreak of avian influenza in New York City was detected in cats with clinical disease in the shelter system. The cats were infected with an H7N2 avian influenza virus that was most closely related to poultry viruses observed in 2000 from the live bird market system in the Northeast United States. Because of the similarity of the viruses seen in 2000, the virus was inoculated into chickens and ducks to evaluate how poultry-adapted these viruses were. Results showed the viruses were poorly adapted to both species and the feline virus was unlikely to have been derived from the live bird market system. 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 HPAI 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. The pathogenesis of a more recent European H5N8 HPAI virus, similar to the ones causing the outbreaks in poultry and wild birds in many countries in 2016-2017, showed that, as expected for a HPAI virus, chickens infected with the virus exhibited high mortality. However, different from previous H5N8 viruses, high mortality was also observed in mallards. Recombinant viruses are being generated to determine which genes and mutations are responsible for this increase in virulence in mallards. This could help explain why this virus has spread more widely and affected more avian species than the 2014-2015 H5N8 virus. Viral changes related to host adaptation were also determined for the Mexican H7N3 HPAI viruses. Outbreaks of HPAI virus subtype H7N3 have been occurring in commercial poultry in Mexico since 2012. Changes in pathogenicity and host adaptation of 2012, 2015, and 2016 viruses were evaluated in chickens and mallards. All three viruses caused high mortality when given to chickens. No mortality or clinical signs were observed with the 2012 and 2016 viruses in mallards but shedding of the 2016 virus was minimal and the virus didn’t transmit to contacts, indicating this virus was less adapted to ducks. Genetic differences were determined between the 2012 and 2016 H7N3 viruses, with some of these changes previously associated with changes in virulence or host adaptation. This shows that as the H7N3 HPAI viruses passaged in poultry for four years it decreased in adaptation to mallards remaining highly lethal to chickens. Immunology studies were conducted in avian species to determine variations in protective host defense mechanisms to avian influenza infections. Mallard ducks are widely recognized as reservoirs for LPAI viruses in nature and differences in prevalence of viral subtypes are likely influenced by flock immunity in these birds. Heterosubtypic immunity (HSI) refers to the ability of one subtype of Avian Influenza virus to protect against different subtype. Under experimental conditions, the protection of the HSI induced by infection with different LPAI viruses was assessed in mallards after challenge with a H5N8 HPAI virus. We demonstrate for the first time that previous exposure to LPAI viruses induced protective immunity and resulted in decreased or no infection of mallards following HPAI challenge. These findings provide important information on the contributions of HSI and implicate a potential role for cellular immunity in protection of birds previously exposed to LPAI viruses in the field. Also, a next generation sequencing method was developed to examine changes related to vaccine-induced immune pressure in genes from the 2014-15 H5 HPAI viruses. In these studies, virus adaptation to immune pressure was captured using Next Generation Sequence (NGS) analysis of swab samples taken at various time points after challenge of vaccinated animals. Overall, analysis of 50 samples containing complete viral genomes identified 198 amino acid (AA) mutations in either sham or vaccinated birds. These studies demonstrate that evolution of HPAI virus populations could be influenced by immune pressure and species change. Transgene molecules utilizing RNAi or CRISPR technologies for knock-in and knock-out capabilities were produced as first step in this process. A construct was selected for in ovo injection, and an avian cell line that restricts avian influenza virus replication was created. The efficacy of two vaccines licensed in the U.S. for their potential in controlling H5Nx clade 18.104.22.168 HPAI viruses in poultry was determined in domestic ducks. A commercial H5 vector vaccine based on RNA replicon system, which uses an alphavirus backbone to express the H5 gene from a hemagglutinin (HA) clade 22.214.171.124 H5N8 HPAI virus, and an inactivated H5 vaccine, also based on the same HA, were used to determine protection of Pekin ducks from challenge with a H5N2 HPAI virus from the 2015 U.S. outbreak. The vaccines were given either as single vaccination at 2 days of age or in a prime-boost strategy at 2 and 15 days of age. Both vaccines, regardless of the vaccination strategy used, were immunogenic in ducks and reduced or prevented virus shedding after challenge.
1. The effect of immunosuppressive viruses on avian influenza virus (AIV) vaccine efficacy. Infectious bursal disease virus (IBDV) is an immunosuppressive virus of chickens that is common in poultry worldwide. Immunosuppression caused by IBDV has been known to decrease the efficacy of vaccines in chickens, however it was not known if this was true for vaccine against AIV as well. ARS researchers at Athens, Georgia, showed that chickens infected with IBDV and then vaccinated for AIV were not as well protected as chickens that had not been infected with IBDV. The same number of birds died as those that were not vaccinated. The importance of this is that for vaccination for AIV to be successful, IBDV must be controlled as well. This also helps to explain why vaccinated chickens can still become sick and die from AIV in the field.
2. Thermal inactivation of avian influenza virus in poultry bedding as a method to decontaminate poultry houses. One of the biggest costs of recovery from an avian influenza virus (AIV) outbreak is decontaminating infected premises, especially organic material such as bedding made of wood shavings or sawdust. Chemical disinfection is too inefficient with this type of material, but heating the bedding along with the whole house is achievable in many situations. ARS researchers at Athens, Georgia, conducted studies testing different temperatures with used poultry bedding that was contaminated with AIV to determine how long the virus survived at each temperature. This data has been used to create guidelines for times and temperatures at which to heat a poultry house to inactivate AIV in the environment. This results in substantial cost savings because the litter does not have to be disposed of while infections, and poultry houses can be decontaminated more efficiently. In addition, the risk of virus spread is reduced because the decontamination process is more efficient that chemical methods.
3. Ten different adjuvants were evaluated for use in vaccines for chickens to protect against highly pathogenic avian influenza virus (HPAIV). Most vaccines, including those for HPAIV, contain material that enhances the immune response, called adjuvants. The best adjuvants vary by animal species and minimal data have been reported for chickens. Ten adjuvants were tested by ARS researchers at Athens, Georgia, in chickens for their ability to elicit a protective immune response to HPAIV. Commercial and experimental compounds including mineral oil, vegetable oil, calcium phosphate, and chitosan were tested. It was found that the mineral oil based adjuvants induced the best immune response. This will lead to better HPAIV vaccines and more efficient vaccination since lower doses can be administered.
4. Detection of airborne-transmissible highly pathogenic influenza virus during processing of infected poultry. Human infections with H5N1 highly pathogenic avian influenza (HPAI) virus have occurred following exposure to virus-infected poultry in live-poultry markets. ARS researchers at Athens, Georgia, detected infectious droplets and aerosols during laboratory-simulated processing of chickens infected with human- and avian- origin H5N1 viruses. In contrast, processing of infected ducks was less efficient in generating infectious airborne particles. Naïve chickens and ferrets exposed to the same air space as the processing of virus-infected chickens became infected, suggesting that the slaughter of infected chickens is an efficient source of airborne virus for avian and mammalian infections. The results support the tenet that airborne transmission of HPAI viruses can occur among poultry and from poultry to humans during home or live-poultry market slaughter of infected poultry.
5. Pathobiology of clade 126.96.36.199 H5Nx high pathogenicity avian influenza virus infections in minor gallinaceous poultry supports early backyard flock introductions in Western United States. In 2014-2015, the U.S. experienced an unprecedented outbreak of Eurasian clade 188.8.131.52 H5 highly pathogenic avian influenza (HPAI) virus. Initial cases affected mainly wild birds and mixed backyard poultry species, while outbreaks in 2015 affected mostly commercial chickens and turkeys. The pathogenesis, transmission, and intra-host evolutionary dynamics of initial Eurasian H5N8 and reassortant H5N2 clade 184.108.40.206 HPAI viruses in the U.S. were investigated by ARS researchers at Athens, Georgia, in minor gallinaceous poultry species (i.e. species for which the U.S. commercial industries are small): Japanese quail, Bobwhite quail, Pearl guinea fowl, Chukar partridges, and Ring-necked pheasants. High virus infectivity, sustained virus shedding with transmission to contact-exposed birds, alongside long incubation periods, could enable unrecognized dissemination and adaptation to other gallinaceous such as chickens and turkeys. The findings suggest that these gallinaceous poultry are permissive for infection and sustainable transmissibility with 2014 initial wild bird-adapted clade 220.127.116.11 virus, with potential acquisition of mutations leading to adaptation to other hosts. This information is critical in understanding the epidemiology of HPAI virus and its control.
6. The efficacy of recombinant turkey herpesvirus vaccines targeting the H5 of highly pathogenic avian influenza virus from the 2014/2015 North American outbreak. The highly pathogenic avian influenza virus outbreak in North America during 2014 and 2015 was extremely devastating to the U.S. poultry industry and federal government. The licensed poultry vaccines currently available for use in the U.S. during outbreaks were developed years ago and target older and different influenza virus lineages than the one responsible for the recent outbreak. By developing effective vaccines that target currently circulating strains of avian influenza virus, the USDA can be better prepared with a more effective vaccine if poultry vaccination is required for disease control and eradication in future outbreaks. Three vaccines were tested by ARS researchers at Athens, Georgia, in chickens for their effectiveness against the recent highly pathogenic avian influenza virus. Two of the test vaccines had mixed results while one vaccine was 100% effective for survival and significantly reduced viral shedding from infected chickens. Further studies of the most effective test vaccine are needed to determine its effectiveness against other lineages and strains of avian influenza.
7. Evolution, global spread, and pathogenicity of highly pathogenic avian influenza H5Nx clade 18.104.22.168. Unique virus strains of deadly H5 avian influenza (highly pathogenic avian influenza, HPAI) which originated in China (Goose/Guangdong lineage) have changed over time into four genetically distinct subgroups (A – D) within group 22.214.171.124, as shown by ARS researchers at Athens, Georgia. Since 2014, HPAI group 126.96.36.199 viruses have spread rapidly worldwide through migratory wild aquatic birds and changed through mixing genes with prevailing local mild (low pathogenicity avian influenza, LPAI) viruses. The Group A viruses caused bird outbreaks during 2014-2015, in Asia, Europe, and North America while Group B viruses caused bird outbreaks in Asia, Europe, and Africa during 2016-2017. Group C viruses originated in China have caused bird outbreaks in South Korea and Japan during winter season of 2016-17, and Group D H5N6 viruses have caused bird outbreaks in China and Vietnam. A wide range of bird species, including wild and domestic waterfowl, domestic poultry, and even zoo birds, have been infected with and/or transmitted the group 188.8.131.52 HPAI viruses. This information is important to understand the generation and dissemination of H5Nx HPAI viruses.
8. H5N6 clade 184.108.40.206 highly pathogenic avian influenza virus in Taiwan, 2017. Since 1996, Asian-origin H5 A/goose/Guangdong/1/1996 (Gs/GD) lineage of high pathogenicity avian influenza viruses (HPAIV) have caused outbreaks in Asia, Europe, Africa, and North America. In a collaborative study between ARS researchers in Athens, Georgia and the Animal Health Research Institute, Taiwan, an H5N6 avian influenza virus was detected in a domestic duck found dead in Taiwan during February 2017. The virus was HPAIV based of the amino acid sequence at the hemagglutinin cleavage site, PLRERRRKR/G, had high lethality in chickens on intravenous inoculation (IVPI= 3.0), and had mixing of internal virus genes (reassortment) from low pathogenicity avian influenza viruses (LPAIV) from Eurasia wild birds. Genetic analyses suggested that the virus belonged to clade 220.127.116.11 Group C, genotype C5, which has been found in China, Korea, and Japan during 2016-2017. The endemic situation of HPAIV in eastern Asia and continued evolution with mixing of genes from of a variety of LPAIV in Taiwan raises the possibility of spread of this HPAIV via migratory waterfowl into North America.
9. Spontaneous emergence of highly pathogenic avian Influenza (H7N9) virus in Tennessee, USA, 2017. The deadly form of avian influenza (highly pathogenic avian influenza) was detected in chickens in Tennessee during March 2017. Examination for other avian influenza viruses in the area and genetic analysis of such viruses by ARS researchers at Athens, Georgia, supports multiple independent introductions of a mild avian influenza virus (low pathogenicity avian influenza) from wild bird to farms at the border of Tennessee and Alabama. On one farm, the mild form changed to the deadly form by mutation and subsequent spread to a second farm. This information is important for understanding outbreaks of avian influenza in poultry.
10. Protection of commercial turkeys following inactivated or recombinant H5 vaccine application against the 2015 U.S. H5N2 clade 18.104.22.168 highly pathogenic avian influenza virus. During December 2014-June 2015, the U.S. experienced the worst high pathogenicity avian influenza (HPAI) outbreak event for the poultry industry. Three vaccines, developed based on updating existing registered vaccines or currently licensed technologies, were evaluated by ARS researchers at 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) was assessed in White Leghorn chickens against clade 22.214.171.124 H5N2 HPAI virus challenge. In Study 1, single (rHVT-H5) and prime-boost (rHVT-H5 + rgH5N1 or rHVT-H5 + RP-H5) vaccination strategies protected 3-week-old chickens with high levels of protective immunity and significantly reduced virus shedding. In Study 2, single vaccination with either rgH5N1 or RP-H5 vaccines provided clinical protection in adult chickens and significantly reduced virus shedding. In Study 3, 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 clade 126.96.36.199 H5Nx HPAI virus in young and adult layers.
11. Short- and long-term protective efficacy against clade 188.8.131.52 H5N2 highly pathogenic avian influenza virus following prime-boost vaccination in turkeys. In 2014-2015, H5N2 highly pathogenic avian influenza (HPAI) virus, clade 184.108.40.206, caused a devastating outbreak in poultry in the United States. Meat turkey and laying hen production systems in the Midwest were particularly affected, causing the largest animal health emergency in recent history in the U.S. In this study, ARS researchers at Athens, Georgia, and collaborators, examined the efficacy of 2 vaccines for reduction of virus shedding and clinical signs of disease in turkeys at 6 and 16 weeks of age challenged with a clade 220.127.116.11 H5N2 HPAI virus. Three different vaccine regimes were used. Vaccinated turkeys showed significantly reduced virus shedding and mortality compared to unvaccinated control birds. However, the timing between vaccination and challenge affected the protective efficacy of the vaccine regimes tested. The study highlights the importance of examining not only different vaccine platforms but also vaccination strategies to maximize protection of poultry against HPAIV.
12. Characterization of H9N2 avian influenza viruses from the Middle East. Next-generation sequencing (NGS) technologies are a valuable tool to monitor changes in viral genomes and determine the genetic heterogeneity of viruses. One advantage of the NGS platform is the possibility to sequence the genetic material in samples without any prior knowledge of the sequence contained within. ARS researchers at Athens, Georgia, applied NGS to poultry samples from Jordan to detect and directly sequence eleven H9N2LPAI viruses. Sequence analysis demonstrated a high degree of heterogeneity at specific locations in the hemagglutinin (HA) gene, which targets increased specificity to receptors found on avian or mammalian species. Moreover, additional amino acid changes corresponding with increased replication and virulence were identified among the viruses detected. It has been demonstrated that low pathogenic AI viruses can mutate to cross species barriers and replicate in mammals. Therefore, the detection and characterization of these LPAI viruses is critical for identifying emerging strains in poultry with zoonotic potential.
13. Mallard ducks are a primary reservoir for low pathogenic avian influenza viruses (AIV) in nature and flock immunity to these viruses influence the AIV subtypes isolated in these birds. Heterosubtypic immunity (HIS) refers to the ability of infection against one subtype of AIV to protect against different AIV subtype. However, the level of HSI induced following infection remains poorly understood. To examine this question we assessed the ability of HSI induced by infection with H3N8 AIV to increase resistance to infection from H4N6 or H6N2 AIV. ARS researchers at Athens, Georgia, and collaborators demonstrate that induced HSI resulted in increasing levels of challenge virus necessary to establish infection in mallards. In addition, the ability of HSI to increase resistance to infection was directly correlated to the genetic relatedness between the viruses. Thus, the closer the viruses were related, the more increased HSI was observed. These findings are important as they explain the dynamics of AIV subtype diversity in mallards.
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