Reframing wildlife disease management problems with decision analysis.

Contemporary wildlife disease management is complex because managers need to respond to a wide range of stakeholders, multiple uncertainties, and difficult trade-offs that characterize the interconnected challenges of today. Despite general acknowledgment of these complexities, managing wildlife disease tends to be framed as a scientific problem, in which the major challenge is lack of knowledge. The complex and multifactorial process of decision-making is collapsed into a scientific endeavor to reduce uncertainty. As a result, contemporary decision-making may be oversimplified, rely on simple heuristics, and fail to account for the broader legal, social, and economic context in which the decisions are made. Concurrently, scientific research on wildlife disease may be distant from this decision context, resulting in information that may not be directly relevant to the pertinent management questions. We propose reframing wildlife disease management challenges as decision problems and addressing them with decision analytical tools to divide the complex problems into more cognitively manageable elements. In particular, structured decision-making has the potential to improve the quality, rigor, and transparency of decisions about wildlife disease in a variety of systems. Examples of management of severe acute respiratory syndrome coronavirus 2, white-nose syndrome, avian influenza, and chytridiomycosis illustrate the most common impediments to decision-making, including competing objectives, risks, prediction uncertainty, and limited resources.

Replanteamiento del manejo de problemas por enfermedades de fauna mediante el análisis de decisiones Resumen El manejo actual de las enfermedades de la fauna es complejo debido a que los gestores necesitan responder a una amplia gama de actores, varias incertidumbres y compensaciones difíciles que caracterizan los retos interconectados del día de hoy. A pesar de que en general se reconocen estas complejidades, el manejo de las enfermedades tiende a plantearse como un problema científico en el que el principal obstáculo es la falta de conocimiento. El proceso complejo y multifactorial de la toma decisiones está colapsado dentro de un esfuerzo científico para reducir la incertidumbre. Como resultado de esto, las decisiones contemporáneas pueden estar simplificadas en exceso, depender de métodos heurísticos simples y no considerar el contexto legal, social y económico más amplio en el que se toman las decisiones. De manera paralela, las investigaciones científicas sobre las enfermedades de la fauna pueden estar lejos de este contexto de decisiones, lo que deriva en información que puede no ser directamente relevante para las preguntas pertinentes de manejo. Proponemos replantear los obstáculos para el manejo de enfermedades de fauna como problemas de decisión y abordarlos con herramientas analíticas de decisión para dividir los problemas complejos en elementos más manejables de manera cognitiva. En particular, las decisiones estructuradas tienen el potencial de mejorar la calidad, el rigor y la transparencia de las decisiones sobre las enfermedades de la fauna en una variedad de sistemas. Ejemplos como el manejo del coronavirus del síndrome de respiración agudo tipo 2, el síndrome de nariz blanca, la influenza aviar y la quitridiomicosis ilustran los impedimentos más comunes para la toma de decisiones, incluyendo los objetivos en competencia, riesgos, incertidumbre en las predicciones y recursos limitados.

Phage Therapy in the Management of Urinary Tract Infections: A Comprehensive Systematic Review.

Urinary tract infections (UTIs) are a problem worldwide, affecting almost half a billion people each year. Increasing antibiotic resistance and limited therapeutic options have led to the exploration of alternative therapies for UTIs, including bacteriophage (phage) therapy. This systematic review aims at evaluating the efficacy of phage therapy in treating UTIs. We employed a comprehensive search strategy for any language, any animal, and any publication date. A total of 55 in vivo and clinical studies were included. Of the studies, 22% were published in a non-English language, 32.7% were before the year 1996, and the rest were after 2005. The results of this review suggest that phage therapy for UTIs can be effective; more than 72% of the included articles reported microbiological and clinical improvements. On the other hand, only 5 randomized controlled trials have been completed, and case reports and case series information were frequently incomplete for analysis. Overall, this comprehensive systematic review identifies preliminary evidence supporting the potential of phage therapy as a safe and viable option for the treatment of UTIs.

Bayesian forecasting of disease spread with little or no local data.

Rapid and targeted management actions are a prerequisite to efficiently mitigate disease outbreaks. Targeted actions, however, require accurate spatial information on disease occurrence and spread. Frequently, targeted management actions are guided by non-statistical approaches that define the affected area by a pre-determined distance surrounding a small number of disease detections. As an alternative, we present a long-recognized but underutilized Bayesian technique that uses limited local data and informative priors to make statistically valid predictions and forecasts about disease occurrence and spread. As a case study, we use limited local data that were available after the detection of chronic wasting disease in Michigan, U.S. along with information rich priors obtained from a previous study in a neighboring state. Using these limited local data and informative priors, we generate statistically valid predictions of disease occurrence and spread for the Michigan study area. This Bayesian technique is conceptually and computationally simple, relies on little to no local data, and is competitive with non-statistical distance-based metrics in all performance evaluations. Bayesian modeling has added benefits because it allows practitioners to generate immediate forecasts of future disease conditions and provides a principled framework to incorporate new data as they accumulate. We contend that the Bayesian technique offers broad-scale benefits and opportunities to make statistical inference across a diversity of data-deficient systems, not limited to disease.

Conformational plasticity of the HIV-1 gp41 immunodominant region is recognized by multiple non-neutralizing antibodies.

The early humoral immune response to acute HIV-1 infection is largely non-neutralizing. The principal target of these antibodies is the primary immunodominant region (PID) on the gp41 fusion protein. The PID is a highly conserved 15-residue region displayed on the surface of HIV-1 virions. In this study, we analyzed the humoral determinants of HIV-1 gp41 PID binding using biophysical, structural, and computational methods. In complex with a patient-derived near-germline antibody fragment, the PID motif adopts an elongated random coil, whereas the PID bound to affinity-matured Fab adopts a strand-turn-helix conformation. Molecular dynamics simulations showed that the PID is structurally plastic suggesting that the PID can form an ensemble of structural states recognized by various non-neutralizing antibodies, facilitating HIV-1 immunodominance observed in acute and chronic HIV-1 infections. An improved understanding of how the HIV-1 gp41 PID misdirects the early humoral response should guide the development of an effective HIV-1 vaccine.

Risks posed by SARS-CoV-2 to North American bats during winter fieldwork.

The virus that causes COVID-19 likely evolved in a mammalian host, possibly Old-World bats, before adapting to humans, raising the question of whether reverse zoonotic transmission to bats is possible. Wildlife management agencies in North America are concerned that the activities they authorize could lead to transmission of SARS-CoV-2 to bats from humans. A rapid risk assessment conducted in April 2020 suggested that there was a small but significant possibility that SARS-CoV-2 could be transmitted from humans to bats during summer fieldwork, absent precautions. Subsequent challenge studies in a laboratory setting have shed new information on these risks, as has more detailed information on human epidemiology and transmission. This inquiry focuses on the risk to bats from winter fieldwork, specifically surveys of winter roosts and handling of bats to test for white-nose syndrome or other research needs. We use an aerosol transmission model, with parameter estimates both from the literature and from formal expert judgment, to estimate the risk to three species of North American bats, as a function of several factors. We find that risks of transmission are lower than in the previous assessment and are notably affected by chamber volume and local prevalence of COVID-19. Use of facemasks with high filtration efficiency or a negative COVID-19 test before field surveys can reduce zoonotic risk by 65 to 88%.

Snapshot of an influenza virus glycoprotein fusion intermediate.

Enveloped virus entry requires the fusion of cellular and viral membranes, a process directed by their viral fusion glycoproteins. Our current knowledge of this process has been shaped by structural studies of the pre- and post-fusion conformations of these viral fusogens. These structural snapshots have revealed the start and end states necessary for fusion, but the dynamics of the intermediate conformations have remained unclear. Using the influenza C virus hemagglutinin-esterase-fusion glycoprotein as a model, we report the structural and biophysical characterization of a trapped intermediate. Crystallographic studies revealed a structural reorganization of the C terminus to create a second chain reversal region, resulting in the N and C termini being positioned in opposing directions. Intrinsic tryptophan fluorescence and bimane-induced quenching measurements suggest intermediate formation is mediated by conserved hydrophobic residues. Our study reveals a late-stage extended intermediate structural event. This work adds to our understanding of virus cell fusion.

Optimization of peptidic HIV-1 fusion inhibitor T20 by phage display.

The HIV fusion inhibitor T20 has been approved to treat those living with HIV/AIDS, but treatment gives rise to resistant viruses. Using combinatorial phage-displayed libraries, we applied a saturation scan approach to dissect the entire T20 sequence for binding to a prefusogenic five-helix bundle (5HB) mimetic of HIV-1 gp41. Our data set compares all possible amino acid substitutions at all positions, and affords a complete view of the complex molecular interactions governing the binding of T20 to 5HB. The scan of T20 revealed that 12 of its 36 positions were conserved for 5HB binding, which cluster into three epitopes: hydrophobic epitopes at the ends and a central dyad of hydrophilic residues. The scan also revealed that the T20 sequence was highly adaptable to mutations at most positions, demonstrating a striking structural plasticity that allows multiple amino acid substitutions at contact points to adapt to conformational changes, and also at noncontact points to fine-tune the interface. Based on the scan result and structural knowledge of the gp41 fusion intermediate, a library was designed with tailored diversity at particular positions of T20 and was used to derive a variant (T20v1) that was found to be a highly effective inhibitor of infection by multiple HIV-1 variants, including a common T20-escape mutant. These findings show that the plasticity of the T20 functional sequence space can be exploited to develop variants that overcome resistance of HIV-1 variants to T20 itself, and demonstrate the utility of saturation scanning for rapid epitope mapping and protein engineering.

Tyrosyl phosphorylation of KRAS stalls GTPase cycle via alteration of switch I and II conformation.

Deregulation of the RAS GTPase cycle due to mutations in the three RAS genes is commonly associated with cancer development. Protein tyrosine phosphatase SHP2 promotes RAF-to-MAPK signaling pathway and is an essential factor in RAS-driven oncogenesis. Despite the emergence of SHP2 inhibitors for the treatment of cancers harbouring mutant KRAS, the mechanism underlying SHP2 activation of KRAS signaling remains unclear. Here we report tyrosyl-phosphorylation of endogenous RAS and demonstrate that KRAS phosphorylation via Src on Tyr32 and Tyr64 alters the conformation of switch I and II regions, which stalls multiple steps of the GTPase cycle and impairs binding to effectors. In contrast, SHP2 dephosphorylates KRAS, a process that is required to maintain dynamic canonical KRAS GTPase cycle. Notably, Src- and SHP2-mediated regulation of KRAS activity extends to oncogenic KRAS and the inhibition of SHP2 disrupts the phosphorylation cycle, shifting the equilibrium of the GTPase cycle towards the stalled 'dark state'.

Structure of the infectious salmon anemia virus receptor complex illustrates a unique binding strategy for attachment.

Orthomyxoviruses are an important family of RNA viruses, which include the various influenza viruses. Despite global efforts to eradicate orthomyxoviral pathogens, these infections remain pervasive. One such orthomyxovirus, infectious salmon anemia virus (ISAV), spreads easily throughout farmed and wild salmonids, constituting a significant economic burden. ISAV entry requires the interplay of the virion-attached hemagglutinin-esterase and fusion glycoproteins. Preventing infections will rely on improved understanding of ISAV entry. Here, we present the crystal structures of ISAV hemagglutinin-esterase unbound and complexed with receptor. Several distinctive features observed in ISAV HE are not seen in any other viral glycoprotein. The structures reveal a unique mode of receptor binding that is dependent on the oligomeric assembly of hemagglutinin-esterase. Importantly, ISAV hemagglutinin-esterase receptor engagement does not initiate conformational rearrangements, suggesting a distinct viral entry mechanism. This work improves our understanding of ISAV pathogenesis and expands our knowledge on the overall diversity of viral glycoprotein-mediated entry mechanisms. Finally, it provides an atomic-resolution model of the primary neutralizing antigen critical for vaccine development.

New structural and functional insight into the regulation of Ras.

Mutations in RAS and various components of the Ras signaling pathways are among the most common causative genetic alterations in human cancers, accounting up to 25% of lung cancers and over 90% of pancreatic cancers. Ras is a small GTPase that functions as a 'molecular switch' in a number of signaling pathways that regulate vital eukaryotic cellular functions. Despite our comprehensive understanding of the molecular mechanisms governing the activity of Ras, the clinical outcome of various pharmacologic anti-cancer strategies designed to directly inactivate Ras have been less than satisfactory. In this review, the more recently uncovered mode of regulation of Ras involving non-receptor tyrosine kinase and phosphatase, which have long been suspected of contributing to the oncogenic potential of Ras, will be discussed in the context of both function and structure.

Electrostatic Architecture of the Infectious Salmon Anemia Virus (ISAV) Core Fusion Protein Illustrates a Carboxyl-Carboxylate pH Sensor.

Segment 5, ORF 1 of the infectious salmon anemia virus (ISAV) genome, encodes for the ISAV F protein, which is responsible for viral-host endosomal membrane fusion during a productive ISAV infection. The entry machinery of ISAV is composed of a complex of the ISAV F and ISAV hemagglutinin esterase (HE) proteins in an unknown stoichiometry prior to receptor engagement by ISAV HE. Following binding of the receptor to ISAV HE, dissociation of the ISAV F protein from HE, and subsequent endocytosis, the ISAV F protein resolves into a fusion-competent oligomeric state. Here, we present a 2.1 Å crystal structure of the fusion core of the ISAV F protein determined at low pH. This structure has allowed us to unambiguously demonstrate that the ISAV entry machinery exhibits typical class I viral fusion protein architecture. Furthermore, we have determined stabilizing factors that accommodate the pH-dependent mode of ISAV transmission, and our structure has allowed the identification of a central coil that is conserved across numerous and varied post-fusion viral glycoprotein structures. We then discuss a mechanistic model of ISAV fusion that parallels the paramyxoviral class I fusion strategy wherein attachment and fusion are relegated to separate proteins in a similar fashion to ISAV fusion.

Src promotes GTPase activity of Ras via tyrosine 32 phosphorylation.

Mutations in Ras GTPase and various other components of the Ras signaling pathways are among the most common genetic alterations in human cancers and also have been identified in several familial developmental syndromes. Over the past few decades it has become clear that the activity or the oncogenic potential of Ras is dependent on the nonreceptor tyrosine kinase Src to promote the Ras/Raf/MAPK pathway essential for proliferation, differentiation, and survival of eukaryotic cells. However, no direct relationship between Ras and Src has been established. We show here that Src binds to and phosphorylates GTP-, but not GDP-, loaded Ras on a conserved Y32 residue within the switch I region in vitro and that in vivo, Ras-Y32 phosphorylation markedly reduces the binding to effector Raf and concomitantly increases binding to GTPase-activating proteins and the rate of GTP hydrolysis. These results suggest that, in the context of predetermined crystallographic structures, Ras-Y32 serves as an Src-dependent keystone regulatory residue that modulates Ras GTPase activity and ensures unidirectionality to the Ras GTPase cycle.

Crystal structures of beta- and gammaretrovirus fusion proteins reveal a role for electrostatic stapling in viral entry.

Membrane fusion is a key step in the life cycle of all envelope viruses, but this process is energetically unfavorable; the transmembrane fusion subunit (TM) of the virion-attached glycoprotein actively catalyzes the membrane merger process. Retroviral glycoproteins are the prototypical system to study pH-independent viral entry. In this study, we determined crystal structures of extramembrane regions of the TMs from Mason-Pfizer monkey virus (MPMV) and xenotropic murine leukemia virus-related virus (XMRV) at 1.7-Å and 2.2-Å resolution, respectively. The structures are comprised of a trimer of hairpins that is characteristic of class I viral fusion proteins and now completes a structural library of retroviral fusion proteins. Our results allowed us to identify a series of intra- and interchain electrostatic interactions in the heptad repeat and chain reversal regions. Mutagenesis reveals that charge-neutralizing salt bridge mutations significantly destabilize the postfusion six-helix bundle and abrogate retroviral infection, demonstrating that electrostatic stapling of the fusion subunit is essential for viral entry. Our data indicate that salt bridges are a major stabilizing force on the MPMV and XMRV retroviral TMs and likely provide the key energetics for viral and host membrane fusion.

A convenient and general expression platform for the production of secreted proteins from human cells.

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) (1,2), baculovirus-infected insect (S. frugiperda or T. ni) cells (3), and cell-free in vitro translation systems (2,4) have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins (5-9). However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories. Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein (5,10). In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.