Species' differences in cellular factors limit avian influenza A virus (IAV) zoonoses and human pandemics. The IAV polymerase, vPol, harbors evolutionary sites to overcome restriction and determines virulence. Here, we establish host ANP32A as a critical driver of selection, and identify host-specific ANP32A splicing landscapes that predict viral evolution. We find that avian species differentially express three ANP32A isoforms diverging in a vPolpromoting insert. ANP32As with shorter inserts interact poorly with vPol, are compromised in supporting avian-like IAV replication, and drive selection of mammalian-adaptive vPol sequences with distinct kinetics. By integrating selection data with multi-species ANP32A splice variant profiling, we develop a mathematical model to predict avian species potentially driving (swallow, magpie) or maintaining (goose, swan) mammalian-adaptive vPol signatures. Supporting these predictions, surveillance data confirm enrichment of several mammalian-adaptive vPol substitutions in magpie IAVs. Profiling host ANP32A splicing could enhance surveillance and eradication efforts against IAVs with pandemic potential.

Bovine respiratory disease complex (BRDC) is a multifactorial disease of cattle which presents as bacterial and viral pneumonia. The causative agents of BRDC work in synergy to suppress the host immune response and increase the colonisation of the lower respiratory tracts by pathogenic bacteria. Environmental stress and/or viral infection predispose cattle to secondary bacterial infections via suppression of key innate and adaptive immune mechanisms. This allows bacteria to descend the respiratory tract unchallenged. BRDC is the costliest disease among feedlot cattle, and whilst vaccines exist for individual pathogens, there is still a lack of evidence for the efficacy of these vaccines and uncertainty surrounding the optimum timing of delivery. This review outlines the immunosuppressive actions of the individual pathogens involved in BRDC and highlights the key issues in the development of vaccinations against them.

We demonstrate that novel bat HL17NL10 and HL18NL11 influenza virus NS1 proteins are effective interferon antagonists but do not block general host gene expression. Solving the RNA-binding domain structures revealed the canonical NS1 symmetrical homodimer, and RNA binding required conserved basic residues in this domain. Interferon antagonism was strictly dependent on RNA binding, and chimeric bat influenza viruses expressing NS1s defective in this activity were highly attenuated in interferon-competent cells but not in cells unable to establish antiviral immunity.T he complete genomes of two novel influenza A-like viruses (IAVs) were recently identified in Central and South American bat species and were provisionally designated the unique subtypes H17N10 and H18N11 due to their high sequence divergence from other IAVs (1, 2). Remarkably, the surface glycoproteins of bat IAVs (hemagglutinin [HA] and neuraminidase [NA]) lack canonical features normally associated with these proteins (3-6) and were recently proposed to be renamed HA-like (HL17 and HL18) and NA-like (NL10 and NL11) proteins (7). Although basic functions of the RNA replicative machinery appear largely conserved (1, 8-10), properties of other bat IAV proteins have yet to be determined. NS1 is a multifunctional virulence factor that acts as a major interferon (IFN) antagonist during IAV infection and has been associated with host range restriction (11-13). The mechanisms by which NS1 can pre-and posttranscriptionally inhibit cellular antiviral defenses are known to be highly strain specific (14-17). Here, we sought to characterize the structure and IFN-antagonistic functions of bat IAV NS1 proteins, which share only ϳ50% sequence identity with human and avian IAV NS1 proteins (1, 2, 18).Novel bat IAV NS1 proteins are IFN antagonists. A recent study revealed that transient expression of the HL17NL10 (HL17) NS1 protein is sufficient to antagonize the interferon-␤ (IFN-␤) response in human cells (18). Given that the HL17 and HL18NL11 (HL18) NS1 proteins differ by ϳ10%, we compared the abilities of these two IAV proteins to antagonize IFN-␤ promoter induction, along with the well-characterized NS1 from the PR8 strain.293T cells in 24-well plates were cotransfected with plasmids expressing V5-tagged glutathione S-transferase (GST), HL17 NS1, HL18 NS1, or PR8 NS1 proteins (1, 10, or 100 ng), together with an IFN-␤ promoter-dependent firefly luciferase (FF-Luc) expression plasmid (p125-FFLuc [25 ng]) and a constitutively active Renilla luciferase (Ren-Luc) expression plasmid (pRL-TK [25 ng]), which was used for normalization. Total plasmid DNA was kept constant with an empty expression vector. Twenty-four hours posttransfection, cells were infected with a defective interfering (DI) particle-rich stock of Sendai virus (SeV) for 16 h to stimulate the IFN-␤ promoter. High induction of FFLuc activity occurred in SeV-infected GST-expressing cells compared to mock-infected GST-expressing cells. However, this induction was antagonized in a dose-dependent manner in...

Pulsed electron–electron double resonance (PELDOR) is an electron paramagnetic resonance (EPR) spectroscopy technique for nanometer distance measurements between paramagnetic centers such as radicals. PELDOR has been recognized as a valuable tool to approach structural questions in biological systems. In this manuscript, we demonstrate the value of distance measurements for differentiating competing structural models on the dimerization of the effector domain (ED) of the non-structural protein 1 (NS1) of the influenza A virus. Our results show NS1 to be well amenable to nanometer distance measurements by EPR, yielding high quality data. In combination with mutants perturbing protein dimerization and in silico prediction based on crystal structures, we can exclude one of two potential dimerization interfaces. Furthermore, our results lead to a viable hypothesis of a NS1 ED:ED interface which is flexible through rotation around the vector interconnecting the two native cysteines. These results prove the high value of pulse EPR as a complementary method for structural biology.

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