Genetic Elements Involved in Zika Virus Neuropathogenesis

Zikavirus (ZIKV) is a mosquito-borne flavivirus(Fig. 1) that is closely related to Japanese encephalitis, West Nile, yellow fever, and dengue viruses. ZIKV was first discovered in Uganda in 1947, but it was not until recent outbreaks, such as through Micronesia in 2007 and through Brazil in 2015 (Fig. 2), that it has been found to be associated with neurological diseases such as Guillain-Barrésyndrome and microcephaly

Generation of Polyclonal Rabbit Antisera Specific to the Zika Virus Capsid Protein

Zika virus (ZIKV), a mosquito-borne flavivirus, is an emerging zoonotic pathogen closely related to Japanese encephalitis virus, West Nile virus, dengue virus, and yellow fever virus. Although ZIKV infection generally produces only mild symptoms in some infected individuals, it has recently been associated with a growing number of neurological diseases, including Guillain-Barré syndrome in ZIKV-infected adults and microcephaly in infants born to ZIKV-infected women. Like all flaviviruses, ZIKV has a plus-strand RNA genome encoding ten functional proteins (designated C, prM, E, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). Of these ten, the C (capsid) protein is an essential structural protein required for the formation of infectious viral particles. In order to produce the antiserum specifically recognizing the ZIKV C protein in this study, we expressed and purified the ZIKV C protein as a glutathione-S-transferase (GST) fusion protein in E. coli. The ZIKV C protein-coding region was PCR-amplified using the genomic RNA of ZIKV PRVABC-59, and the amplicons were cloned into the pGEX-4T-1 E. coli expression vector. GST-C fusion proteins were purified using a glutathione sepharose column. Subsequently, the GST-C fusion proteins were used for immunization with rabbits. Western blot analysis using the ZIKV-infected Vero cell lysates were performed to examine the reactivity of the antisera to the ZIKV C protein. Thus, this study provides a useful reagent for the diagnosis and understanding of the viral morphogenesis in the ZIKV-infected cells

Development, Characterization, and Application of Two Reporter-Expressing Recombinant Zika Viruses

Zika virus (ZIKV), a mosquito-borne transplacentally transmissible flavivirus, is an enveloped virus with an ~10.8 kb plus-strand RNA genome that can cause neurological disease. To facilitate the identification of potential antivirals, we developed two reporter-expressing ZIKVs, each capable of expressing an enhanced green fluorescent protein or an improved luminescent NanoLuc luciferase. First, a full-length functional ZIKV cDNA clone was engineered as a bacterial artificial chromosome, with each reporter gene under the cap-independent translational control of a cardiovirus-derived internal ribosome entry site inserted downstream of the single open reading frame of the viral genome. Two reporter-expressing ZIKVs were then generated by transfection of ZIKV-susceptible BHK-21 cells with infectious RNAs derived by in vitro run-off transcription from the respective cDNAs. As compared to the parental virus, the two reporter-expressing ZIKVs grew to lower titers with slower growth kinetics and formed smaller foci; however, they displayed a genome-wide viral protein expression profile identical to that of the parental virus, except for two previously unrecognized larger forms of the C and NS1 proteins. We then used the NanoLuc-expressing ZIKV to assess the in vitro antiviral activity of three inhibitors (T-705, NITD-008, and ribavirin). Altogether, our reporter-expressing ZIKVs represent an excellent molecular tool for the discovery of novel antivirals

Vegetation Cover Analysis of Hazardous Waste Sites in Utah and Arizona Using Hyperspectral Remote Sensing

This study investigated the usability of hyperspectral remote sensing for characterizing vegetation at hazardous waste sites. The specific objectives of this study were to: (1) estimate leaf-area-index (LAI) of the vegetation using three different methods (i.e., vegetation indices, red-edge positioning (REP), and machine learning regression trees), and (2) map the vegetation cover using machine learning decision trees based on either the scaled reflectance data or mixture tuned matched filtering (MTMF)-derived metrics and vegetation indices. HyMap airborne data (126 bands at 2.3 x 2.3 m spatial resolution), collected over the U. S. Department of Energy uranium processing sites near Monticello, Utah and Monument Valley, Arizona, were used. Grass and shrub species were mixed on an engineered disposal cell cover at the Monticello site while shrub species were dominant in the phytoremediation plantings at the Monument Valley site. Regression trees resulted in the best calibration performance of LAI estimation (R-2 > 0.80. The use of REPs failed to accurately predict LAI (R-2 < 0.2). The use of the MTMF-derived metrics (matched filter scores and infeasibility) and a range of vegetation indices in decision trees improved the vegetation mapping when compared to the decision tree classification using just the scaled reflectance. Results suggest that hyperspectral imagery are useful for characterizing biophysical characteristics (LAI) and vegetation cover on capped hazardous waste sites. However, it is believed that the vegetation mapping would benefit from the use of higher spatial resolution hyperspectral data due to the small size of many of the vegetation patches (<1 m) found on the sites.open111

Direct Reprogramming of Cardiac Fibroblasts to Repair the Injured Heart

Coronary heart disease is a leading cause of mortality and morbidity. Those that survive acute myocardial infarction are at significant risk of subsequent heart failure due to fibrotic remodelling of the infarcted myocardium. By applying knowledge from the study of embryonic cardiovascular development, modern medicine offers hope for treatment of this condition through regeneration of the myocardium by direct reprogramming of fibrotic scar tissue. Here, we will review mechanisms of cell fate specification leading to the generation of cardiovascular cell types in the embryo and use this as a framework in which to understand direct reprogramming. Driving expression of a network of transcription factors, micro RNA or small molecule epigenetic modifiers can reverse epigenetic silencing, reverting differentiated cells to a state of induced pluripotency. The pluripotent state can be bypassed by direct reprogramming in which one differentiated cell type can be transdifferentiated into another. Transdifferentiating cardiac fibroblasts to cardiomyocytes requires a network of transcription factors similar to that observed in embryonic multipotent cardiac progenitors. There is some flexibility in the composition of this network. These studies raise the possibility that the failing heart could one day be regenerated by directly reprogramming cardiac fibroblasts within post-infarct scar tissue

Functional Genomics and Immunologic Tools: The Impact of Viral and Host Genetic Variations on the Outcome of Zika Virus Infection

Zika virus (ZIKV) causes no-to-mild symptoms or severe neurological disorders. To investigate the importance of viral and host genetic variations in determining ZIKV infection outcomes, we created three full-length infectious cDNA clones as bacterial artificial chromosomes for each of three spatiotemporally distinct and genetically divergent ZIKVs: MR-766 (Uganda, 1947), P6-740 (Malaysia, 1966), and PRVABC-59 (Puerto Rico, 2015). Using the three molecularly cloned ZIKVs, together with 13 ZIKV region-specific polyclonal antibodies covering nearly the entire viral protein-coding region, we made three conceptual advances: (i) We created a comprehensive genome-wide portrait of ZIKV gene products and their related species, with several previously undescribed gene products identified in the case of all three molecularly cloned ZIKVs. (ii) We found that ZIKV has a broad cell tropism in vitro, being capable of establishing productive infection in 16 of 17 animal cell lines from 12 different species, although its growth kinetics varied depending on both the specific virus strain and host cell line. More importantly, we identified one ZIKV-non-susceptible bovine cell line that has a block in viral entry but fully supports the subsequent post-entry steps. (iii) We showed that in mice, the three molecularly cloned ZIKVs differ in their neuropathogenicity, depending on the particular combination of viral and host genetic backgrounds, as well as in the presence or absence of type I/II interferon signaling. Overall, our findings demonstrate the impact of viral and host genetic variations on the replication kinetics and neuropathogenicity of ZIKV and provide multiple avenues for developing and testing medical countermeasures against ZIKV

Model Channel Ion Currents in NaCl - SPC/E Solution with Applied-Field Molecular Dynamics

Using periodic boundary conditions and a constant applied field, we have simulated current flow through an 8.125 Angstrom internal diameter, rigid, atomistic channel with polar walls in a rigid membrane using explicit ions and SPC/E water. Channel and bath currents were computed from ten 10-ns trajectories for each of 10 different conditions of concentration and applied voltage. An electric field was applied uniformly throughout the system to all mobile atoms. On average, the resultant net electric field falls primarily across the membrane channel, as expected for two conductive baths separated by a membrane capacitance. The channel is rarely occupied by more than one ion. Current-voltage relations are concentration-dependent and superlinear at high concentrations.Comment: Accepted for publication in Biophysical Journa

FluTE, a Publicly Available Stochastic Influenza Epidemic Simulation Model

Mathematical and computer models of epidemics have contributed to our understanding of the spread of infectious disease and the measures needed to contain or mitigate them. To help prepare for future influenza seasonal epidemics or pandemics, we developed a new stochastic model of the spread of influenza across a large population. Individuals in this model have realistic social contact networks, and transmission and infections are based on the current state of knowledge of the natural history of influenza. The model has been calibrated so that outcomes are consistent with the 1957/1958 Asian A(H2N2) and 2009 pandemic A(H1N1) influenza viruses. We present examples of how this model can be used to study the dynamics of influenza epidemics in the United States and simulate how to mitigate or delay them using pharmaceutical interventions and social distancing measures. Computer simulation models play an essential role in informing public policy and evaluating pandemic preparedness plans. We have made the source code of this model publicly available to encourage its use and further development

MAGE-A cancer/testis antigens inhibit MDM2 ubiquitylation function and promote increased levels of MDM4

Melanoma antigen A (MAGE-A) proteins comprise a structurally and biochemically similar sub-family of Cancer/Testis antigens that are expressed in many cancer types and are thought to contribute actively to malignancy. MAGE-A proteins are established regulators of certain cancer-associated transcription factors, including p53, and are activators of several RING finger-dependent ubiquitin E3 ligases. Here, we show that MAGE-A2 associates with MDM2, a ubiquitin E3 ligase that mediates ubiquitylation of more than 20 substrates including mainly p53, MDM2 itself, and MDM4, a potent p53 inhibitor and MDM2 partner that is structurally related to MDM2. We find that MAGE-A2 interacts with MDM2 via the N-terminal p53-binding pocket and the RING finger domain of MDM2 that is required for homo/hetero-dimerization and for E2 ligase interaction. Consistent with these data, we show that MAGE-A2 is a potent inhibitor of the E3 ubiquitin ligase activity of MDM2, yet it does not have any significant effect on p53 turnover mediated by MDM2. Strikingly, however, increased MAGE-A2 expression leads to reduced ubiquitylation and increased levels of MDM4. Similarly, silencing of endogenous MAGE-A expression diminishes MDM4 levels in a manner that can be rescued by the proteasomal inhibitor, bortezomid, and permits increased MDM2/MDM4 association. These data suggest that MAGE-A proteins can: (i) uncouple the ubiquitin ligase and degradation functions of MDM2; (ii) act as potent inhibitors of E3 ligase function; and (iii) regulate the turnover of MDM4. We also find an association between the presence of MAGE-A and increased MDM4 levels in primary breast cancer, suggesting that MAGE-A-dependent control of MDM4 levels has relevance to cancer clinically