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 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. 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.

e Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease characterized by high fever, thrombocytopenia, multiorgan dysfunction, and a high fatality rate between 12 and 30%. It is caused by SFTS virus (SFTSV), a novel Phlebovirus in family Bunyaviridae. Although the viral pathogenesis remains largely unknown, hemopoietic cells appear to be targeted by the virus. In this study we report that human monocytes were susceptible to SFTSV, which replicated efficiently, as shown by an immunofluorescence assay and real-time reverse transcription-PCR. We examined host responses in the infected cells and found that antiviral interferon (IFN) and IFN-inducible proteins were induced upon infection. However, our data also indicated that downregulation of key molecules such as mitochondrial antiviral signaling protein (MAVS) or weakened activation of interferon regulatory factor (IRF) and NF-B responses may contribute to a restricted innate immunity against the infection. NSs, the nonstructural protein encoded by the S segment, suppressed the beta interferon (IFN-␤) and NF-B promoter activities, although NF-B activation appears to facilitate SFTSV replication in human monocytes. NSs was found to be associated with TBK1 and may inhibit the activation of downstream IRF and NF-B signaling through this interaction. Interestingly, we demonstrated that the nucleoprotein (N), also encoded by the S segment, exhibited a suppressive effect on the activation of IFN-␤ and NF-B signaling as well. Infected monocytes, mainly intact and free of apoptosis, may likely be implicated in persistent viral infection, spreading the virus to the circulation and causing primary viremia. Our findings provide the first evidence in dissecting the host responses in monocytes and understanding viral pathogenesis in humans infected with a novel deadly Bunyavirus.

Waterfowl from northwestern Minnesota were sampled by cloacal swabbing for Avian Influenza Virus (AIV) from July – October in 2007 and 2008. AIV was detected in 222 (9.1%) of 2,441 ducks in 2007 and in 438 (17.9%) of 2,452 ducks in 2008. Prevalence of AIV peaked in late summer. We detected 27 AIV subtypes during 2007 and 31 during 2008. Ten hemagglutinin (HA) subtypes were detected each year (i.e., H1, 3–8, and 10–12 during 2007; H1-8, 10 and 11 during 2008). All neuraminidase (NA) subtypes were detected during each year of the study. Subtype diversity varied between years and increased with prevalence into September. Predominant subtypes during 2007 (comprising ≥5% of subtype diversity) included H1N1, H3N6, H3N8, H4N6, H7N3, H10N7, and H11N9. Predominant subtypes during 2008 included H3N6, H3N8, H4N6, H4N8, H6N1, and H10N7. Additionally, within each HA subtype, the same predominant HA/NA subtype combinations were detected each year and included H1N1, H3N8, H4N6, H5N2, H6N1, H7N3, H8N4, H10N7, and H11N9. The H2N3 and H12N5 viruses also predominated within the H2 and H12 subtypes, respectively, but only were detected during a single year (H2 and H12 viruses were not detected during 2007 and 2008, respectively). Mallards were the predominant species sampled (63.7% of the total), and 531 AIV were isolated from this species (80.5% of the total isolates). Mallard data collected during both years adequately described the observed temporal and spatial prevalence from the total sample and also adequately represented subtype diversity. Juvenile mallards also were adequate in describing the temporal and spatial prevalence of AIV as well as subtype diversity.

Viruses have adopted various mechanisms to evade or subvert host antiviral responses initiated by viral RNA or DNA through Toll-like receptor (TLR) (1, 2) and RIG-I-like receptor (RLR) (3-5) signaling pathways. Viral double-stranded RNA (dsRNA) can be sequestered by VP35 of Ebola virus (6) to avoid the activation of TLRs or RLRs. Viral proteins that contain the TIR domain can be interferon (IFN) antagonists such as A46R of vaccinia virus, which blocks upstream IFN signaling by directly targeting MyD88, MAL, TRIF, and TRAM, the TIR domain-containing adaptors (2). RLR sensors such as RIG-I and MDA5 and adaptor IPS1 are targeted by NS1 of influenza virus (7), V protein of paramyxoviruses (3), and the protease precursor protein 3ABC of hepatitis A virus (5). The adaptor protein TRIF of the TLR signaling pathway can be targeted for degradation by viral proteins containing protease activities, such as NS3/4A of flavivirus hepatitis C virus (HCV) (8) and 3C of picornavirus EV71 (9), which can also cleave transcription factor (TF) IRF7, compromising IFN induction. In addition, the N-terminal fragment of cleaved IRF7 interacts with and inhibits IRF3, another TF essential for IFN induction, thereby enhancing viral infection (10). Downstream TFs are also targeted by viral proteins, such as NS1 of West Nile virus, which inhibits TLR3 signaling by preventing IRF3 translocation into the nucleus (11). Between TLR/RLR sensors and downstream TFs, IKK complexes are also targeted by proteins encoded by HCV, vaccinia virus, and rabies virus (12-14), among others.Severe fever with thrombocytopenia syndrome (SFTS) is an emerging febrile illness caused by a novel phlebovirus, SFTS bunyavirus (SFTSV), in the family Bunyaviridae (15). SFTSV is a vector-borne zoonotic arbovirus transmitted to humans by tick bites, causing high fever, loss of white blood cells and platelets, and, in severe cases, multiorgan failure. It is a single-stranded negativesense RNA virus with three genomic segments, L, M, and S (15). The L segment encodes viral RNA polymerase, while the M segment encodes the two viral envelope glycoproteins, Gc and Gn. The S segment is an ambisense RNA of 1,744 bases encoding a nucleoprotein (NP) and a nonstructural protein (NSs). Our recent study demonstrated that NSs inhibits host IFN-␤ and NF-B responses, although the mechanism remains unclear (16). Here we report that NSs forms unique cytoplasmic inclusion bodies (IBs) in infected or transfected cells that appear to play a role in immune evasion through the interaction of NSs and the IKK complex. Our data indicate that SFTSV has adopted a novel host immune evasion strategy involving the sequestration of IKK complex components TBK1, IKKε, and IRF, which are critical for the activation of the IFN signaling pathway, into IBs. MATERIALS AND METHODSCells, viruses, and reagents. African green monkey kidney Vero, HeLa, human embryonic kidney 293T, and MDCK cells were grown in Dulbecco's modified Eagle's medium (DMEM; Gibco, Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovin...

When avian influenza viruses (AIVs) are transmitted from their reservoir hosts (wild waterfowl and shorebirds) to domestic bird species, they undergo genetic changes that have been linked to higher virulence and broader host range. Common genetic AIV modifications in viral proteins of poultry isolates are deletions in the stalk region of the neuraminidase (NA) and additions of glycosylation sites on the hemagglutinin (HA). Even though these NA deletion mutations occur in several AIV subtypes, they have not been analyzed comprehensively. In this study, 4,920 NA nucleotide sequences, 5,596 HA nucleotide and 4,702 HA amino acid sequences were analyzed to elucidate the widespread emergence of NA stalk deletions in gallinaceous hosts, the genetic polymorphism of the deletion patterns and association between the stalk deletions in NA and amino acid variants in HA. Forty-seven different NA stalk deletion patterns were identified in six NA subtypes, N1–N3 and N5–N7. An analysis that controlled for phylogenetic dependence due to shared ancestry showed that NA stalk deletions are statistically correlated with gallinaceous hosts and certain amino acid features on the HA protein. Those HA features included five glycosylation sites, one insertion and one deletion. The correlations between NA stalk deletions and HA features are HA-NA-subtype-specific. Our results demonstrate that stalk deletions in the NA proteins of AIV are relatively common. Understanding the NA stalk deletion and related HA features may be important for vaccine and drug development and could be useful in establishing effective early detection and warning systems for the poultry industry.

Studies of pathogen transmission typically overlook that wildlife hosts can include both migrant and resident populations when attempting to model circulation. Through the application of stable isotopes in flight feathers, we estimated the migration strategy of mallards (Anas platyrhynchos) occurring on California wintering grounds. Our study demonstrates that mallards- a principal host of avian influenza virus (AIV) in nature, contribute differently to virus gene flow depending on migration strategy. No difference in AIV prevalence was detected between resident (9.6%), intermediate-distance (9.6%) and long-distance migrants (7.4%). Viral diversity among the three groups was also comparable, possibly owing to viral pool mixing when birds converge at wetlands during winter. However, migrants and residents contributed differently to the virus gene pool at wintering wetlands. Migrants introduced virus from northern breeding grounds (Alaska and the NW Pacific Rim) into the wintering population, facilitating gene flow at continental scales, but circulation of imported virus appeared to be limited. In contrast, resident mallards acted as AIV reservoirs facilitating year-round circulation of limited subtypes (i.e. H5N2) at lower latitudes. This study supports a model of virus exchange in temperate regions driven by the convergence of wild birds with separate geographic origins and exposure histories.

Volatile organic compounds (VOCs) emanating from humans have the potential to revolutionize non-invasive diagnostics. Yet, little is known about how these compounds are generated by complex biological systems, and even less is known about how these compounds are reflective of a particular physiological state. In this proof-of-concept study, we examined VOCs produced directly at the cellular level from B lymphoblastoid cells upon infection with three live influenza virus subtypes: H9N2 (avian), H6N2 (avian), and H1N1 (human). Using a single cell line helped to alleviate some of the complexity and variability when studying VOC production by an entire organism, and it allowed us to discern marked differences in VOC production upon infection of the cells. The patterns of VOCs produced in response to infection were unique for each virus subtype, while several other non-specific VOCs were produced after infections with all three strains. Also, there was a specific time course of VOC release post infection. Among emitted VOCs, production of esters and other oxygenated compounds was particularly notable, and these may be attributed to increased oxidative stress resulting from infection. Elucidating VOC signatures that result from the host cells response to infection may yield an avenue for non-invasive diagnostics and therapy of influenza and other viral infections.

Severe fever with thrombocytopenia syndrome (SFTS) virus is an emerging bunyavirus that causes a hemorrhagic fever with a high mortality rate. The virus is likely tick-borne and replicates primarily in hemopoietic cells, which may lead to disregulation of proinflammatory cytokine induction and loss of leukocytes and platelets. The viral genome contains L, M, and S segments encoding a viral RNA polymerase, glycoproteins G(n) and G(c), nucleoprotein (NP), and a nonstructural S segment (NSs) protein. NSs protein is involved in the regulation of host innate immune responses and suppression of IFNβ-promoter activities. In this article, we demonstrate that NSs protein can form viroplasm-like structures (VLSs) in infected and transfected cells. NSs protein molecules interact with one another, interact with NP, and were associated with viral RNA in infected cells, suggesting that NSs protein may be involved in viral replication. Furthermore, we observed that NSs-formed VLS colocalized with lipid droplets and that inhibitors of fatty acid biosynthesis decreased VLS formation or viral replication in transfected and infected cells. Finally, we have demonstrated that viral dsRNAs were also localized in VLS in infected cells, suggesting that NSs-formed VLS may be implicated in the replication of SFTS bunyavirus. These findings identify a novel function of nonstructural NSs in SFTSV-infected cells where it is a scaffolding component in a VLS functioning as a virus replication factory. This function is in addition to the role of NSs protein in modulating host responses that will broaden our understanding of viral pathogenesis of phleboviruses.