Viral Subpopulation Screening Guides in Designing a High Interferon-Inducing Live Attenuated Influenza Vaccine by Targeting Rare Mutations in NS1 and PB2 Proteins.

Influenza A viruses continue to circulate among wild birds and poultry worldwide, posing constant pandemic threats to humans. Effective control of emerging influenza viruses requires new broadly protective vaccines. Live attenuated influenza vaccines with truncations in nonstructural protein 1 (NS1) have shown broad protective efficacies in birds and mammals, which correlate with the ability to induce elevated interferon responses in the vaccinated hosts. Given the extreme diversity of influenza virus populations, we asked if we could improve an NS1-truncated live attenuated influenza vaccine developed for poultry (PC4) by selecting viral subpopulations with enhanced interferon-inducing capacities. Here, we deconstructed a de novo population of PC4 through plaque isolation, created a large library of clones, and assessed their interferon-inducing phenotypes. While most of the clones displayed the parental interferon-inducing phenotype in cell culture, few clones showed enhanced interferon-inducing phenotypes in cell culture and chickens. The enhanced interferon-inducing phenotypes were linked to either a deletion in NS1 (NS1Δ76-86) or a substitution in polymerase basic 2 protein (PB2-D309N). The NS1Δ76-86 deletion disrupted the putative eukaryotic translation initiation factor 4GI-binding domain and promoted the synthesis of biologically active interferons. The PB2-D309N substitution enhanced the early transcription of interferon mRNA, revealing a novel role for the 309D residue in suppression of interferon responses. We combined these mutations to engineer a novel vaccine candidate that induced additive amounts of interferons and stimulated protective immunity in chickens. Therefore, viral subpopulation screening approaches can guide the design of live vaccines with strong immunostimulatory properties.IMPORTANCE Effectiveness of NS1-truncated live attenuated influenza vaccines relies heavily on their ability to induce elevated interferon responses in vaccinated hosts. Influenza viruses contain diverse particle subpopulations with distinct phenotypes. We show that live influenza vaccines can contain underappreciated subpopulations with enhanced interferon-inducing phenotypes. The genomic traits of such virus subpopulations can be used to further improve the efficacy of the current live vaccines.

Respiratory and Gut Microbiota in Commercial Turkey Flocks with Disparate Weight Gain Trajectories Display Differential Compositional Dynamics.

Communities of gut bacteria (microbiota) are known to play roles in resistance to pathogen infection and optimal weight gain in turkey flocks. However, knowledge of turkey respiratory microbiota and its link to gut microbiota is lacking. This study presents a 16S rRNA gene-based census of the turkey respiratory microbiota (nasal cavity and trachea) alongside gut microbiota (cecum and ileum) in two identical commercial Hybrid Converter turkey flocks raised in parallel under typical field commercial conditions. The flocks were housed in adjacent barns during the brood stage and in geographically separated farms during the grow-out stage. Several bacterial taxa, primarily Staphylococcus, that were acquired in the respiratory tract at the beginning of the brood stage persisted throughout the flock cycle. Late-emerging predominant taxa in the respiratory tract included Deinococcus and Corynebacterium Tracheal and nasal microbiota of turkeys were identifiably distinct from one another and from gut microbiota. Nevertheless, gut and respiratory microbiota changed in parallel over time and appeared to share many taxa. During the brood stage, the two flocks generally acquired similar gut and respiratory microbiota, and their average body weights were comparable. However, there were qualitative and quantitative differences in microbial profiles and body weight gain trajectories after the flocks were transferred to geographically separated grow-out farms. Lower weight gain corresponded to the emergence of Deinococcus and Ornithobacterium in the respiratory tract and Fusobacterium and Parasutterella in gut. This study provides an overview of turkey microbiota under field conditions and suggests several hypotheses concerning the respiratory microbiome.IMPORTANCE Turkey meat is an important source of animal protein, and the industry around its production contributes significantly to the agricultural economy. The microorganisms present in the gut of turkeys are known to impact bird health and flock performance. However, the respiratory microbiota in turkeys is entirely unexplored. This study has elucidated the microbiota of respiratory tracts of turkeys from two commercial flocks raised in parallel throughout a normal flock cycle. Further, the study suggests that bacteria originating in the gut or in poultry house environments influence respiratory communities; consequently, they induce poor performance, either directly or indirectly. Future attempts to develop microbiome-based interventions for turkey health should delimit the contributions of respiratory microbiota and aim to limit disturbances to those communities.

Farm Stage, Bird Age, and Body Site Dominantly Affect the Quantity, Taxonomic Composition, and Dynamics of Respiratory and Gut Microbiota of Commercial Layer Chickens.

The digestive and respiratory tracts of chickens are colonized by bacteria that are believed to play important roles in the overall health and performance of the birds. Most of the current research on the commensal bacteria (microbiota) of chickens has focused on broilers and gut microbiota, and less attention has been given to layers and respiratory microbiota. This research bias has left significant gaps in our knowledge of the layer microbiome. This study was conducted to define the core microbiota colonizing the upper respiratory tract (URT) and lower intestinal tract (LIT) in commercial layers under field conditions. One hundred eighty-one chickens were sampled from a flock of >80,000 birds at nine times to collect samples for 16S rRNA gene-based bacterial metabarcoding. Generally, the body site and age/farm stage had very dominant effects on the quantity, taxonomic composition, and dynamics of core bacteria. Remarkably, ileal and URT microbiota were compositionally more related to each other than to that from the cecum. Unique taxa dominated in each body site yet some taxa overlapped between URT and LIT sites, demonstrating a common core. The overlapping bacteria also contained various levels of several genera with well-recognized avian pathogens. Our findings suggest that significant interaction exists between gut and respiratory microbiota, including potential pathogens, in all stages of the farm sequence. The baseline data generated in this study can be useful for the development of effective microbiome-based interventions to enhance production performance and to prevent and control disease in commercial chicken layers.IMPORTANCE The poultry industry is faced with numerous challenges associated with infectious diseases and suboptimal performance of flocks. As microbiome research continues to grow, it is becoming clear that poultry health and production performance are partly influenced by nonpathogenic symbionts that occupy different habitats within the bird. This study has defined the baseline composition and overlaps between respiratory and gut bacteria in healthy, optimally performing chicken layers across all stages of the commercial farm sequence. Consequently, the study has set the groundwork for the development of interventions that seek to enhance production performance and to prevent and control infectious diseases through the modulation of gut and respiratory bacteria.

A Consistent and Predictable Commercial Broiler Chicken Bacterial Microbiota in Antibiotic-Free Production Displays Strong Correlations with Performance.

Defining the baseline bacterial microbiome is critical to understanding its relationship with health and disease. In broiler chickens, the core microbiome and its possible relationships with health and disease have been difficult to define, due to high variability between birds and flocks. Presented here are data from a large, comprehensive microbiota-based study in commercial broilers. The primary goals of this study included understanding what constitutes the core bacterial microbiota in the broiler gastrointestinal, respiratory, and barn environments; how these core players change across age, geography, and time; and which bacterial taxa correlate with enhanced bird performance in antibiotic-free flocks. Using 2,309 samples from 37 different commercial flocks within a vertically integrated broiler system and metadata from these and an additional 512 flocks within that system, the baseline bacterial microbiota was defined using 16S rRNA gene sequencing. The effects of age, sample type, flock, and successive flock cycles were compared, and results indicate a consistent, predictable, age-dependent bacterial microbiota, irrespective of flock. The tracheal bacterial microbiota of broilers was comprehensively defined, and Lactobacillus was the dominant bacterial taxon in the trachea. Numerous bacterial taxa were identified, which were strongly correlated with broiler chicken performance across multiple tissues. While many positively correlated taxa were identified, negatively associated potential pathogens were also identified in the absence of clinical disease, indicating that subclinical dynamics occur that impact performance. Overall, this work provides necessary baseline data for the development of effective antibiotic alternatives, such as probiotics, for sustainable poultry production.IMPORTANCE Multidrug-resistant bacterial pathogens are perhaps the greatest medical challenge we will face in the 21st century and beyond. Antibiotics are necessary in animal production to treat disease. As such, animal production is a contributor to the problem of antibiotic resistance. Efforts are underway to reduce antibiotic use in animal production. However, we are also challenged to feed the world's increasing population, and sustainable meat production is paramount to providing a safe and quality protein source for human consumption. In the absence of antibiotics, alternative approaches are needed to maintain health and prevent disease, and probiotics have great promise as one such approach. This work paves the way for the development of alternative approaches to raising poultry by increasing our understandings of what defines the poultry microbiome and of how it can potentially be modulated to improve animal health and performance.

Influenza A virus infection in turkeys induces respiratory and enteric bacterial dysbiosis correlating with cytokine gene expression.

Turkey respiratory and gut microbiota play important roles in promoting health and production performance. Loss of microbiota homeostasis due to pathogen infection can worsen the disease or predispose the bird to infection by other pathogens. While turkeys are highly susceptible to influenza viruses of different origins, the impact of influenza virus infection on turkey gut and respiratory microbiota has not been demonstrated. In this study, we investigated the relationships between low pathogenicity avian influenza (LPAI) virus replication, cytokine gene expression, and respiratory and gut microbiota disruption in specific-pathogen-free turkeys. Differential replication of two LPAI H5N2 viruses paralleled the levels of clinical signs and cytokine gene expression. During active virus shedding, there was significant increase of ileal and nasal bacterial contents, which inversely corresponded with bacterial species diversity. Spearman's correlation tests between bacterial abundance and local viral titers revealed that LPAI virus-induced dysbiosis was strongest in the nasal cavity followed by trachea, and weakest in the gut. Significant correlations were also observed between cytokine gene expression levels and relative abundances of several bacteria in tracheas of infected turkeys. For example, interferon γ/λ and interleukin-6 gene expression levels were correlated positively with Staphylococcus and Pseudomonas abundances, and negatively with Lactobacillus abundance. Overall, our data suggest a potential relationship where bacterial community diversity and enrichment or depletion of several bacterial genera in the gut and respiratory tract are dependent on the level of LPAI virus replication. Further work is needed to establish whether respiratory and enteric dysbiosis in LPAI virus-infected turkeys is a result of host immunological responses or other causes such as changes in nutritional uptake.

Protective immunity against influenza virus challenge by norovirus P particle-M2e and HA2-AtCYN vaccines in chickens.

Development of a broadly reactive influenza vaccine that can provide protection against emerging type A influenza viruses is a big challenge. We previously demonstrated that a vaccine displaying the extracellular domain of the matrix protein 2 (M2e) on the surface loops of norovirus P-particle (M2eP) can partially protect chickens against several subtypes of avian influenza viruses. In the current study, a chimeric vaccine containing a conserved peptide from the subunit 2 of hemagglutinin (HA) glycoprotein (HA2) and Arabidopsis thaliana cyanase protein (AtCYN) (HA2-AtCYN vaccine) was evaluated in 2-weeks-old chickens. Depending on the route of administration, the HA2-AtCYN vaccine was shown to induce various levels of HA2-specific IgA in tears as well as serum IgG, which were associated with partial protection of chickens against tracheal shedding of a low pathogenicity H5N2 challenge virus. Furthermore, intranasal administration with a combination of HA2-AtCYN and M2eP vaccines resulted in enhanced protection compared to each vaccine alone. Simultaneous intranasal administration of the vaccines did not interfere with secretory IgA induction by each vaccine. Additionally, significantly higher M2eP-specific proliferative responses were observed in peripheral blood mononuclear cells of all M2eP-vaccinated groups when compared with the mock-vaccinated group. Although tripling the number of M2e copies did not enhance the protective efficacy of the chimeric vaccine, it significantly reduced immunodominance of P-particle epitopes without affecting the robustness of M2e-specific immune responses. Taken together, our data suggests that mucosal immunization of chickens with combinations of mechanistically different cross-subtype-conserved vaccines has the potential to enhance the protective efficacy against influenza virus challenge.

Heterosubtypic protection against avian influenza virus by live attenuated and chimeric norovirus P-particle-M2e vaccines in chickens.

Avian influenza in poultry continues to be a great concern worldwide, and the currently licensed inactivated influenza vaccines are not effective against the novel strains of influenza virus that continue to emerge in the field. This warrants the development of more broadly protective influenza vaccines or vaccination regimens. Live attenuated influenza vaccines (LAIVs) and subunit vaccines derived from viral peptides, such as the highly conserved ectodomain of influenza virus matrix protein 2 (M2e), can offer a more broadly reactive immune response. In chickens, we previously showed that a chimeric norovirus P particle containing M2e (M2eP) could provide partial but broad immunity, when administered as a standalone vaccine, and also enhanced the protective efficacy of inactivated vaccine when used in a combination regimen. We also demonstrated that a naturally-selected NS1-truncated H7N3 LAIV (pc4-LAIV) was highly efficacious against antigenically distant heterologous H7N2 low pathogenicity avian influenza virus challenge, especially when used as the priming vaccine in a prime-boost vaccination regimen. In this study, we investigated the cross-subtype protective efficacy of pc4-LAIV in conjunction with M2eP using single vaccination, combined treatment, and prime-boost approaches. Chickens vaccinated with pc4-LAIV showed significant reduction of tracheal shedding of a low pathogenicity H5N2 challenge virus. This cross-subtype protective efficacy was further enhanced, during the initial stages of challenge virus replication, in chickens that received a vaccination regimen consisting of priming with pc4-LAIV at 1 day of age and boosting with M2eP. Further, H5N2-specific serum IgG and pc4-LAIV-specific hemagglutination-inhibition antibody titers were enhanced in LAIV-primed and M2eP boost-vaccinated chickens. Taken together, our data point to the need of further investigation into the benefits of combined and prime-boost vaccination schemes utilizing LAIV and epitope-based vaccines, to develop more broadly protective vaccination regimens.

Specific-pathogen-free Turkey model for reoviral arthritis.

Turkey arthritis reovirus (TARV) infections have been recognized since 2011 to cause disease and significant economic losses to the U.S. turkey industry. Reoviral arthritis has been reproduced in commercial-origin turkeys. However, determination of pathogenesis or vaccine efficacy in these turkeys can be complicated by enteric reovirus strains and other pathogens that ubiquitously exist at subclinical levels among commercial turkey flocks. In this study, turkeys from a specific-pathogen-free (SPF) flock were evaluated for use as a turkey reoviral arthritis model. One-day-old or 1-week-old poults were orally inoculated with TARV (O'Neil strain) and monitored for disease onset and progression. A gut isolate of turkey reovirus (MN1 strain) was also tested for comparison. Disease was observed only in TARV-infected birds. Features of reoviral arthritis in SPF turkeys included swelling of hock joints, tenosynovitis, distal tibiotarsal cartilage erosion, and gait defects (lameness). Moreover, TARV infection resulted in a significant depression of body weights during the early times post-infection. Age-dependent susceptibility to TARV infection was unclear. TARV was transmitted to all sentinel birds, which manifested high levels of tenosynovitis and tibiotarsal cartilage erosion. Simulation of stressful conditions by dexamethasone treatment did not affect the viral load or exacerbate the disease. Collectively, the clinical and pathological features of reoviral arthritis in the SPF turkey model generally resembled those induced in commercial turkeys under field and/or experimental conditions. The SPF turkey reoviral arthritis model will be instrumental in evaluation of TARV pathogenesis and reoviral vaccine efficacy.

Efficacy and synergy of live-attenuated and inactivated influenza vaccines in young chickens.

Outbreaks of novel highly pathogenic avian influenza viruses have been reported in poultry species in the United States since 2014. These outbreaks have proven the limitations of biosecurity control programs, and new tools are needed to reinforce the current avian influenza control arsenal. Some enzootic countries have implemented inactivated influenza vaccine (IIV) in their control programs, but there are serious concerns that a long-term use of IIV without eradication may result in the selection of novel antigenically divergent strains. A broadly protective vaccine is needed, such as live-attenuated influenza vaccine (LAIV). We showed in our previous studies that pc4-LAIV (a variant that encodes a C-terminally truncated NS1 protein) can provide significant protection against heterologous challenge virus in chickens vaccinated at 2-4 weeks of age through upregulation of innate and adaptive immune responses. The current study was conducted to compare the performances of pc4-LAIV and IIV in young chickens vaccinated at 1 day of age. A single dose of pc4-LAIV was able to induce stronger innate and mucosal IgA responses and protect young immunologically immature chickens better than a single dose of IIV. Most importantly, when 1-day-old chickens were intranasally primed with pc4-LAIV and subcutaneously boosted with IIV three weeks later, they showed a rapid, robust, and highly cross-reactive serum antibody response and a high level of mucosal IgA antibody response. This vaccination regimen warrants further optimization to increase its range of protection.