Assessment of the Zoonotic Potential of a Novel Bovine Influenza Virus

A novel orthomyxovirus was recently identified from pigs, with subsequent work suggesting the natural reservoir being bovine populations. The virus had genome characteristics most similar to influenza C viruses (ICV) but, due to the extent of sequence divergence, was proposed as a new genus, influenza D virus (IDV). Current literature on IDV has largely focused on the agricultural significance of the virus and provided evidence for the agricultural impact via observation of widespread prevalence and pathology in laboratory infected cattle. However, only one study, which identified 1.3% seroprevalence in a small cohort, has addressed the zoonotic potential of IDV to date, despite evidence that the virus can infect multiple mammalian species. Regardless of zoonotic potential, it is clear that IDV have distinct host ranges from ICV but the molecular markers responsible are not known. In this dissertation we assessed the zoonotic potential of D/swine/Oklahoma/1334/2011 (D/OK), a representative IDV, and conducted studies to investigate receptor binding specificity, temperature sensitivity of replication kinetics, and pH of inactivation, all factors known to affect influenza A virus (IAV) host range. In order to better address zoonotic potential of D/OK we independently verified the high seroprevalence of D/OK in cattle in the US and found evidence of D/OK circulation in this animal population since at least 2003. We also identified 1% seroprevalence in a cohort of older humans who lived in a rural community with likely exposure to cattle. This seropositivity rate was not, however, elevated compared to earlier studies in populations with low exposure to cattle suggesting that the responses measured were not specific. Further analysis of the seropositive sera indeed found that the IDV seroreactivity was most likely due to cross-reactivity of antibodies induced after prior ICV infection. Despite our inability to identify strong serologic support for zoonotic IDV infection, we did show that D/OK was able to replicate and transmit by direct contact in ferrets and that it replicated robustly in differentiated human respiratory cells, both of which are consistent with an ability to replicate in humans for IAV. We next explored possible mechanisms for the differences in host range of IDV, which has multiple host species, and ICV, which infects primarily humans. Characterization of the HEF proteins of D/OK and a representative ICV demonstrated that D/OK exhibits altered receptor binding specificity and replicates at higher temperatures than ICV although it does bind receptors present in the human respiratory tract. Using virus-like particles with mutant hemagglutinin-esterase fusion (HEF) proteins, we found that the differences in receptor binding of D/OK were at least partially attributable to residues F143, W201, and F256 that line the putative receptor binding pocket. Surprisingly, we also found that, unlike other orthomyxoviruses, the replication of D/OK was not affected by prior incubation at low pH, raising the possibility that its replication might be pH independent. Reassortment of orthomyxoviruses is a known mechanism of pandemic emergence of IAV and an informal proxy for genus distinction with viruses from distinct genera considered unable to successfully reassort. Contradictory to published data using conventional approaches we found that, using reverse genetics to force reassortment, D/OK genes could complement each of the corresponding genes from ICV and viable reassortants were produced. It is unclear, however, the biologic impact of these reassortments. The answer to this and other aspects of our work will require a resolution to the current US Governments pause on gain-of-function research. This observation does, however, bring into question the validity of classification of a new influenza genus despite IDV exhibiting the phylogenetic and antigenic divergence used to distinguish a novel genus. Together the evidence described in this study show that D/OK is widespread in cattle and has characteristics consistent with a zoonotic potential, although we were unable to find convincing evidence for such transmission in a small cohort of humans. We did find that D/OK has many features such as host range, receptor usage, sensitivity to pH, and optimal replication that are distinct from ICV and, we propose, supports its classification as a new genus with the orthomyxovirus family. Continued surveillance and investigation of host species barriers is necessary to further address the public health risk presented by this novel virus

Trapping a Highly Reactive Nonheme Iron Intermediate That Oxygenates Strong CH Bonds with Stereoretention

An unprecedentedly reactive iron species (2) has been generated by reaction of excess peracetic acid with a mononuclear iron complex [FeII(CF3SO3)2(PyNMe3)] (1) at cryogenic temperatures, and characterized spectroscopically. Compound 2 is kinetically competent for breaking strong C―H bonds of alkanes (BDE ≈ 100 kcal·mol−1) through a hydrogen-atom transfer mechanism, and the transformations proceed with stereoretention and regioselectively, responding to bond strength, as well as to steric and polar effects. Bimolecular reaction rates are at least an order of magnitude faster than those of the most reactive synthetic high-valent nonheme oxoiron species described to date. EPR studies in tandem with kinetic analysis show that the 490 nm chromophore of 2 is associated with two S = 1/2 species in rapid equilibrium. The minor component 2a (∼5% iron) has g-values at 2.20, 2.19, and 1.99 characteristic of a low-spin iron(III) center, and it is assigned as [FeIII(OOAc)(PyNMe3)]2+, also by comparison with the EPR parameters of the structurally characterized hydroxamate analogue [FeIII(tBuCON(H)O)(PyNMe3)]2+ (4). The major component 2b (∼40% iron, g-values = 2.07, 2.01, 1.95) has unusual EPR parameters, and it is proposed to be [FeV(O)(OAc)(PyNMe3)]2+, where the O―O bond in 2a has been broken. Consistent with this assignment, 2b undergoes exchange of its acetate ligand with CD3CO2D and very rapidly reacts with olefins to produce the corresponding cis-1,2-hydroxoacetate product. Therefore, this work constitutes the first example where a synthetic nonheme iron species responsible for stereospecific and site selective C―H hydroxylation is spectroscopically trapped, and its catalytic reactivity against C―H bonds can be directly interrogated by kinetic methods. The accumulated evidence indicates that 2 consists mainly of an extraordinarily reactive [FeV(O)(OAc)(PyNMe3)]2+ (2b) species capable of hydroxylating unactivated alkyl C―H bonds with stereoretention in a rapid and site-selective manner, and that exists in fast equilibrium with its [FeIII(OOAc)(PyNMe3)]2+ precursor

Association Between Double-Leg Squat and Single-Leg Squat Performance and Injury Incidence Among Incoming NCAA Division I Athletes: A Prospective Cohort Study

OBJECTIVES: Determine the association between performance on the double-leg squat (DLS) and single-leg squat (SLS) and prospective injury incidence in athletes. DESIGN: Prospective cohort study. SETTING: National Collegiate Athletic Association (NCAA) Division I university. PARTICIPANTS: 111 incoming NCAA Division I athletes from 10 varsity sports teams. MAIN OUTCOME MEASURES: Performance on the DLS and SLS were assessed as poor or non-poor. Lower extremity (LE) injury data for the following year were extracted from electronic medical records. Multivariate Poisson regression was used to compare the incidence of LE injuries in athletes with poor versus non-poor performance on the DLS and SLS. RESULTS: The final models for the DLS and SLS were adjusted for sex and LE injury history and yielded an incidence rate ratio (IRR) of 1.33 (95% CI: 0.80 2.22) for the DLS and 1.62 (95% CI: 0.98, 2.66) for the SLS when comparing poor to non-poor movers. CONCLUSIONS: Athletes with poor LE movement quality assessed on the DLS or SLS had greater incidence of LE injury than those with non-poor movement quality. This is the first study to demonstrate an association between performance on the double-leg squat and single-leg squat and injury risk in NCAA athletes

The iNanoBIT project: integration of Nano - and Biotechnology for Beta-cell and Islet Transplantation

Preclinical trials with porcine pancreatic islets have started and in recent years major progress has been made, particularly in the development of novel immunosuppressive regimens and genetically multi-modified donor pigs with an excellent potential to become a clinically relevant option for the treatment of type-1 diabetes (T1D). Ethical and regulatory frameworks for this new branch of medicine are currently being developed, including safety requirements. High resolution imaging of the function and faith of transplanted porcine xenoislets and beta-cells in large animals and patients for testing ATMPs is a currently unmet need for preclinical/clinical testing. The iNanoBIT EU H2020 project is aimed to apply nanotechnologies for imaging porcine pancreatic islet cellular transplants and induced pluripotent stem cell-derived beta-cells and subsequent regenerative processes in vivo in a porcine model. The project is developing novel highly sensitive nanotechnology-based imaging approaches allowing for monitoring of survival, engraftment, proliferation, function and whole body distribution of the cellular transplants in a preclinical porcine model with excellent translational potential to humans. We develop and validate the application of SPECT and optoacoustic imaging technologies in preclinical transgenic humanized type-1 diabetic pig model to observe transplanted porcine xenoislets and in vitro differentiated human beta-cells. The consortium of 5 SME and 3 Academic partners is progressing in generating new transgenic reporter pigs and human induced pluripotent cell lines for optoacoustic imaging and testing them in transplantable bioartificial islet devices. Novel nanomolecules have been generated and being tested for nuclear imaging of islets and beta-cells using a new, high-resolution SPECT imaging device for large animals. Overall, the combined multidisciplinary expertise of the project partners allows progress towards creating much needed technological tool-boxes for the xenotransplantation and ATMP field, and thus reinforce the European healthcare supply chain for regenerative medicinal products

Cold-Adapted Viral Attenuation (CAVA): Highly Temperature Sensitive Polioviruses as Novel Vaccine Strains for a Next Generation Inactivated Poliovirus Vaccine.

The poliovirus vaccine field is moving towards novel vaccination strategies. Withdrawal of the Oral Poliovirus Vaccine and implementation of the conventional Inactivated Poliovirus Vaccine (cIPV) is imminent. Moreover, replacement of the virulent poliovirus strains currently used for cIPV with attenuated strains is preferred. We generated Cold-Adapted Viral Attenuation (CAVA) poliovirus strains by serial passage at low temperature and subsequent genetic engineering, which contain the capsid sequences of cIPV strains combined with a set of mutations identified during cold-adaptation. These viruses displayed a highly temperature sensitive phenotype with no signs of productive infection at 37°C as visualized by electron microscopy. Furthermore, decreases in infectious titers, viral RNA, and protein levels were measured during infection at 37°C, suggesting a block in the viral replication cycle at RNA replication, protein translation, or earlier. However, at 30°C, they could be propagated to high titers (9.4-9.9 Log10TCID50/ml) on the PER.C6 cell culture platform. We identified 14 mutations in the IRES and non-structural regions, which in combination induced the temperature sensitive phenotype, also when transferred to the genomes of other wild-type and attenuated polioviruses. The temperature sensitivity translated to complete absence of neurovirulence in CD155 transgenic mice. Attenuation was also confirmed after extended in vitro passage at small scale using conditions (MOI, cell density, temperature) anticipated for vaccine production. The inability of CAVA strains to replicate at 37°C makes reversion to a neurovirulent phenotype in vivo highly unlikely, therefore, these strains can be considered safe for the manufacture of IPV. The CAVA strains were immunogenic in the Wistar rat potency model for cIPV, inducing high neutralizing antibody titers in a dose-dependent manner in response to D-antigen doses used for cIPV. In combination with the highly productive PER.C6 cell culture platform, the stably attenuated CAVA strains may serve as an attractive low-cost and (bio)safe option for the production of a novel next generation IPV

Management of Patients at Risk of Anaphylaxis during the Covid-19 Pandemic A Position Paper by the Medical Association of German Allergologists (AeDA), the German Society for Allergology and clinical Immunology (DGAKI), the Society for Pediatric Allergology and Environmental Medicine (GPA) and the German Allergy- and Asthma Association (DAAB)

Klimek L, Worm M, Lange L, et al. Management von Anaphylaxie-gefährdeten Patienten während der Covid-19-Pandemie Ein Positionspapier des Ärzteverbandes Deutscher Allergologen (AeDA)A, der Deutschen Gesellschaft für Allergologie und klinische Immunologie (DGAKI)B, der Gesellschaft für Pädiatrische Allergologie und Umweltmedizin (GPA)C und des Deutschen Allergie- und Asthmabundes (DAAB)D. Allergo Journal . 2020;29(7):16-26