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The number of electric vehicles on the road increases exponentially every year. Due to the quieter nature of these vehicles when operating at low speeds, there is significant concern that pedestrians and bicyclists will be at increased risk of vehicle collisions. This research explores the detectability of six electric vehicle acoustic additive sounds produced by two sound dispersion techniques: (1) using the factory approach versus (2) an exciter transducer-based system. Detectability was initially measured using on-road participant tests and was then replicated in a high-fidelity immersive reality lab. Results were analyzed through both mean detection distances and pedestrian probability of detection. This research aims to verify the lab environment in order to allow for a broader range of potential test scenarios, more repeatable tests, and faster test sessions. Along with pedestrian drive-by tests, supplemental experiments were conducted to evaluate stationary vehicle acoustics, 10 and 20 km/h drive by acoustics, and interior acoustics of each additive sound.

The number of electric vehicles on the road is increasing rapidly every year. Due to the decreased sound produced by these vehicles at low speeds, there is significant concern that pedestrians and bicyclists will be at increased risk of vehicle collisions. This is particularly true for those with vision impairment who cannot rely on visual cues to alert them of an approaching vehicle. This thesis explores pedestrian aural detectability of six electric vehicle additive sounds produced by two additive sound systems: a modified version of the factory equipped system and a prototype exciter transducer-based system. All additive sounds and systems were first evaluated for regulatory compliance at stationary, 10 km/h, and 20 km/h conditions and then pedestrian detectability was assessed using 16 blind folded participants and on-road drive by tests. Participant drive by tests were then replicated using 3D sound field recordings played in a high-fidelity immersive reality lab. Results were used to verify the accuracy of lab environment and its potential applicability to future testing. The exciter transducer acoustic warning system was found to created more uniform sound levels on the passenger and drivers' sides of the vehicle than the factory system but produced lower sound levels on the front side of the vehicle. Additive sound modulation rate was not determined to be a key differentiator in pedestrian detectability and low frequency emphasis sounds were found to have the highest level of pedestrian detectability. As expected, vehicle speed played a critical role in participant detection with the 20 km/h speed condition producing higher average detection distances. The immersive reality lab was found to not replicate on-road environment however a perceived linear offset was observed between the two environments. Master of Science The number of electric vehicles on the road increases every year due to growing consumer demand for clean and sustainable transportation. Due to the decreased sound produced by these vehicles at low speeds there is significant concern that pedestrians and bicyclists will be at increased risk of vehicle collisions. This is particularly true for those with vision impairment who cannot rely on visual cues to alert them of an approaching vehicle. This thesis explores pedestrians' ability to detect six electric vehicle additive sounds produced by two sound systems: a modified version of the factory equipped system and a prototype system designed to produce uniform sound around the vehicle. All sounds and systems were evaluated see if they met current regulations at stationary, 10 km/h, and 20 km/h conditions. Pedestrians' ability to detect the vehicle was assessed using 16 blind folded participants and on-road tests where participants were asked to press a button when they heard an approaching vehicle. Participant drive by tests were then replicated using recordings taken on the same section of road and played in a lab environment. Results were used to see if the lab environment matched the results seen on the road. The prototype system created more uniform sound levels on the passenger and drivers' sides of the vehicle than the factory system but consistently produced lower sound levels on the front side of the vehicle. Sound modulation rate was not determined to be a key differentiator in pedestrian detectability and low frequency emphasis sounds were found to be the most easily detected by pedestrians. As expected, vehicle speed played a critical role in participant detection with the 20 km/h speed condition producing higher detection distances. The lab environment was found to not replicate on-road environment however similar offsets and sound ordering was observed between the two environments. Further work will be needed assess and correct this disagreement.

Coronavirus disease 2019 (COVID-19) convalescents living in regions with low vaccination rates rely on post-infection immunity for protection against re-infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluate humoral and Tcell immunity against five variants of concern (VOCs) in mild-COVID-19 convalescents at 12months after infection with ancestral virus. In this cohort, ancestral, receptor-binding domain (RBD)-specific antibody and circulating memory B cell levels are conserved in most individuals, and yet serum neutralization against live B.1.1.529 (Omicron) is completely abrogated and significantly reduced for other VOCs. Likewise, ancestral SARS-CoV-2-specific memory Tcell frequencies are maintained in >50% of convalescents, but the cytokine response in these cells to mutated spike epitopes corresponding to B.1.1.529 and B.1.351 (Beta) VOCs were impaired. These results indicate that increased antigen variability in VOCs impairs humoral and spike-specific Tcell immunity post-infection, strongly suggesting that COVID-19 convalescents are vulnerable and at risk of re-infection with VOCs, thus stressing the importance of vaccination programs.

Additional file 10. Symptom questionnaire sent to patients to assess presence of long COVID associated symptoms.

Additional file 1: Figure S1: Stability of anti-Spike and anti-RBD antibody titres over time. (A-E) Anti-Spike and (F-J) anti-RBD IgG, IgG1, IgG3, IgM and IgA titres plotted as a function of time. End point titres are reported as log10 area under the curve (AUC). The blue line represents the line of best fit from a linear regression analysis. The shaded areas represent the 95% confidence interval. The P value shown is from the linear regression. Red dashed lines represent the mean AUC + 2 SD in healthy controls for each isotype.

Additional file 3: Figure S3: Adjusting for differences in immune cell populations. We repeated the differential expression analysis multiple times, each time adjusting for differences in the frequency of major immune cell populations among individuals. Each panel shows the enrichment of selected pathways (same as those shown in Fig. 4F) among genes identified as being significantly up-regulated in each analysis (FDR 1.25-fold). None = no immune cell population adjustment.

Additional file 11. Flow cytometry gating strategy.

Additional file 2: Figure S2: Expression of genes in two pathways identified as downregulated in COVID-19 convalescents and healthy controls. (A) Reactome pathway R-HSA-76002 ���Platelet activation, signaling and aggregation��� and (B) KEGG pathway hsa00190 ���Oxidative phosphorylation���. Oxidative phosphorylation genes are sub-divided into nuclear and mitochondrially encoded, with the same x axis order of samples in each panel. Only differentially expressed genes (FDR 1.25-fold) within each pathway are shown. (C) Serum CRP levels in samples collected from healthy controls (HC) and COVID-19 convalescent individuals at 12, 16 and 24 weeks post infection.

Immunosuppression by passive transfer of antibody has been extensively studied and has resulted in at least one practical human application. Human immunoglobulin G anti-Rho is now used widely in the prevention of Rh antibody production by Rh negative mothers. Although considerable knowledge has accrued concerning the mechanisms involved, many aspects of the role of passive antibody are still uncertain. The principal purpose of this was to determine if the low molecular weight subunit ( ? 160,000) of the IgM molecule is similar to the entire molecule (? 160,000 MW) of IgG and IgM (1,000,000) in regards to its capacity to cause immunosuppression. Rabbits immunized with human erythrocytes and injected with homologous immunoglobulins or their fractions were found to vary quantitatively in their ability to produce antibody. Homologous anti-human erythrocyte IgG exhibited the greatest capability to cause immunosuppression. Homologous, specific IgM appeared much less capable in suppression of antibody production. Homologous IgM low molecular weight fragments had little or no effect on antibody production.

The interferon-stimulated gene viperin has been shown to have antiviral activity against hepatitis C virus (HCV) in the context of the HCV replicon, although the molecular mechanisms responsible are not well understood. Here we demonstrate that viperin plays an integral part in the ability of interferon to limit replication of cell culture derived HCV (JFH-1) that accurately reflects the complete viral life cycle. Using confocal microscopy and Fluorescence Resonance Energy Transfer (FRET) analysis we demonstrate that viperin localizes and interacts with HCV NS5A at the lipid droplet interface. In addition viperin also associates with NS5A and the pro-viral cellular factor, VAP-A at the HCV replication complex. The ability of viperin to limit HCV replication was dependent on residues within the C-terminus as well as an N-terminal amphipathic helix. Removal of the amphipathic helix redirected viperin from the cytosolic face of the ER and the lipid droplet to a homogenous cytoplasmic distribution, coinciding with a loss of antiviral effect. C-terminal viperin mutants still localized to the lipid droplet interface and replication complexes but did not interact with NS5A proteins as determined by FRET analysis. In conclusion we propose that viperin interacts with NS5A and the host factor VAP-A to limit HCV replication at the replication complex. This highlights the complexity of host control of viral replication by interferon stimulated gene expression.

Fluctuations of the East-West component of the Earth's geomagnetic field were measured at a remote land site. The resulting data were transmitted by a solar powered telemetry system to the Naval Postgraduate School at Monterey, California, and the power spectra for the frequency range of .02 - 20 Hz calculated. The measurements, which covered a 4-month interval (July 20 - October 10, 1981), consistently show a minimum of activity in the interval 3 - 7 Hz. At frequencies below the minimum, in the range of .02 - 3 Hz , the typical monotonic decrease in background activity with frequency was observed. At 1 Hz an average power spectral density of 1X10" 2 nT2 /Hz was observed during the day and 3.1X10" 3 nT 2 /Hz at night a In contrast, at frequencies above the minimum, in the range 7-14 Hz, the activity is dominated by the first Schumann resonance. An evaluation of the East-West component spectra and concurrent underwater horizontal component measurements showed a 90% correlation with the underwater spectra. The underwater field strengths were normally 3-5 dB less than the strengths measured on land. Splitting of the first Schumann resonance peak into a doublet structure was observed in 10% of the land data. http://archive.org/details/powerspectrofgeo1094520645 Captain, United States Army Approved for public release; distribution is unlimited.

This report amplifies and refines some of the data alreadyissued covering the water resources of Volusia County, which were published asReport of Investigations No. 21. The work in the report was accomplished as acooperative program between the Department of Natural Resources, the U. S.Geological Survey and the Board of County Commissioners of Volusia County.Volusia County is almost totally dependent upon the water which falls upon thecounty and has a recharge area contained along the western portion and thecentral portions of the county. Excellent water is produced in the areal rechargeand it is anticipated that this data will expand the existing knowledge of thewater resources to permit the development of a great capacity for existingutilities and to offset and solve some of the problems now in the area. (PDF has 71 pages.)

Although endothelial cell (EC) infection is not widespread during dengue virus (DENV) infection in vivo, the endothelium is the site of the pathogenic effects seen in severe DENV disease. In this study, we investigated DENV infection of primary EC and defined factors that influence infection in this cell type. Consistent with in vivo findings where EC infection is infrequent, only 3%–15% of EC became productively DENV-2-infected in vitro. This low level infection could not be attributed to inhibition by heparin, EC donor variation, heterogeneity, or biological source. DENV-infection of EC was associated with induction of innate immune responses, including increased STAT1 protein, STAT1- phosphorylation, interferon (IFN)-β, OAS-1, IFIT-1/ISG56, and viperin mRNA. Antibody blocking of IFN-β inhibited the induction of OAS1, IFIT1/ISG56, and viperin while shRNA knockdown of viperin enhanced DENV-infection in EC. DENV-infection of EC resulted in increased activity of sphingosine kinase 1, a factor important in maintaining vascular integrity, and altered basal and stimulated changes in barrier integrity of DENV-infected EC monolayers. Thus, DENV productively infects only a small percentage of primary EC but this has a major influence on induction of IFN-β driven innate immune responses that can restrict infection while the EC themselves are functionally altered. These changes may have important consequences for the endothelium and are reflective of pathogenic changes associated with vascular leakage, as seen in DENV disease. Refereed/Peer-reviewed

Zika virus (ZIKV) infection has emerged as a global health threat and infection of pregnant women causes intrauterine growth restriction, spontaneous abortion and microcephaly in newborns. Here we show using biologically relevant cells of neural and placental origin that following ZIKV infection, there is attenuation of the cellular innate response characterised by reduced expression of IFN-β and associated interferon stimulated genes (ISGs). One such ISG is viperin that has well documented antiviral activity against a wide range of viruses. Expression of viperin in cultured cells resulted in significant impairment of ZIKV replication, while MEFs derived from CRISPR/Cas9 derived viperin−/− mice replicated ZIKV to higher titers compared to their WT counterparts. These results suggest that ZIKV can attenuate ISG expression to avoid the cellular antiviral innate response, thus allowing the virus to replicate unchecked. Moreover, we have identified that the ISG viperin has significant anti-ZIKV activity. Further understanding of how ZIKV perturbs the ISG response and the molecular mechanisms utilised by viperin to suppress ZIKV replication will aid in our understanding of ZIKV biology, pathogenesis and possible design of novel antiviral strategies.

Hepatitis C virus (HCV) NS5A protein is essential for HCV RNA replication and virus assembly. Here we report the identification of NS5A phosphorylation sites Ser-222, Ser-235 and Thr-348 during an infectious HCV replication cycle and demonstrate that Ser-235 phosphorylation is essential for HCV RNA replication. Confocal microscopy revealed that both phosphoablatant (S235A) and phosphomimetic (S235D) mutants redistribute NS5A to large juxta-nuclear foci that display altered colocalization with known replication complex components. Using electron microscopy (EM) we found that S235D alters virus-induced membrane rearrangements while EM using 'APEX2'-tagged viruses demonstrated S235D-mediated enrichment of NS5A in irregular membranous foci. Finally, using a customized siRNA screen of candidate NS5A kinases and subsequent analysis using a phospho-specific antibody, we show that phosphatidylinositol-4 kinase III alpha (PI4KIII alpha) is important for Ser-235 phosphorylation. We conclude that Ser-235 phosphorylation of NS5A is essential for HCV RNA replication and normal replication complex formation and is regulated by PI4KIII alpha. Refereed/Peer-reviewed

Sphingosine kinase (SK) 1 is a host kinase that enhances some viral infections. Here we investigated the ability of SK1 to modulate dengue virus (DENV) infection in vitro. Overexpression of SK1 did not alter DENV infection; however, targeting SK1 through chemical inhibition resulted in reduced DENV RNA and infectious virus release. DENV infection of SK1-/-murine embryonic fibroblasts (MEFs) resulted in inhibition of infection in an immortalized line (iMEF) but enhanced infection in primary MEFs (1ºMEFs). Global cellular gene expression profiles showed expected innate immune mRNA changes in DENV-infected WT but no induction of these responses in SK1-/-iMEFs. Reverse transciption PCR demonstrated a low-level induction of IFN-β and poor induction of mRNA for the interferon-stimulated genes (ISGs) viperin, IFIT1 and CXCL10 in DENV-infected SK1-/-compared with WT iMEFs. Similarly, reduced induction of ISGs was observed in SK1-/-1ºMEFs, even in the face of high-level DENV replication. In both iMEFs and 1ºMEFs, DENV infection induced production of IFN-β protein. Additionally, higher basal levels of antiviral factors (IRF7, CXCL10 and OAS1) were observed in uninfected SK1-/-iMEFs but not 1ºMEFs. This suggests that, in this single iMEF line, lack of SK1 upregulates the basal levels of factors that may protect cells against DENV infection. More importantly, regardless of the levels of DENV replication, all cells that lacked SK1 produced IFN-β but were refractory to induction of ISGs such as viperin, IFIT1 and CXCL10. Based on these findings, we propose new roles for SK1 in affecting innate responses that regulate susceptibility to DENV infection. Refereed/Peer-reviewed

Tumor necrosis factor alpha (TNF-α) has an antiviral role in some infections but in dengue virus (DENV) infection it is linked to severe pathology. We have previously shown that TNF-α stimulation cannot activate nuclear factor κB (NF-κB) to the fullest extent in DENV-2-infected cells. Here, we investigate further responses of DENV-2-infected cells to TNF-α, focussing particularly on cell death and pro-survival signals. TNF-α stimulation of productively DENV-2-infected monocyte-derived macrophages or HEK-293 cells induced caspase-3-mediated cell death. While TNF-α induced comparable degradation of the inhibitor of NF-κB alpha (IκB-α) and NF-κB activation in mock-infected and DENV-2-infected cells early in infection, later in infection and coinciding with TNF-α-induced cell death, TNF-α-stimulated IκB-α degradation and NF-κB activation was reduced. This was associated with reduced levels of sphingosine kinase-1 (SphK1) activity in DENV-2-infected cells; SphK1 being a known mediator of TNF-α-stimulated survival signals. Transfection experiments demonstrated inhibition of TNF-α-stimulated NF-κB activation by expression of DENV-2 capsid (CA) but enhancement by DENV-2 NS5 protein. DENV-2 CA alone, however, did not induce TNF-α-stimulated cell death or inhibit SphK1 activity. Thus, productively DENV-2-infected cells have compromised TNF-α-stimulated survival pathways and show enhanced susceptibility to TNF-α-stimulated cell death, suggesting a role for TNF-α in the killing of healthy productively DENV-2-infected cells. Additionally, the altered ability of TNF-α to activate NF-κB as infection progresses is reflected by the opposing actions of DENV-2 CA and NS5 proteins on TNF-α-stimulated NF-κB activation and could have important consequences for NF-κB-driven release of inflammatory cytokines.

The host protein viperin is an interferon stimulated gene (ISG) that is up-regulated during a number of viral infections. In this study we have shown that dengue virus type-2 (DENV-2) infection significantly induced viperin, co-incident with production of viral RNA and via a mechanism requiring retinoic acid-inducible gene I (RIG-I). Viperin did not inhibit DENV-2 entry but DENV-2 RNA and infectious virus release was inhibited in viperin expressing cells. Conversely, DENV-2 replicated to higher tires earlier in viperin shRNA expressing cells. The anti-DENV effect of viperin was mediated by residues within the C-terminal 17 amino acids of viperin and did not require the N-terminal residues, including the helix domain, leucine zipper and S-adenosylmethionine (SAM) motifs known to be involved in viperin intracellular membrane association. Viperin showed co-localisation with lipid droplet markers, and was co-localised and interacted with DENV-2 capsid (CA), NS3 and viral RNA. The ability of viperin to interact with DENV-2 NS3 was associated with its anti-viral activity, while co-localisation of viperin with lipid droplets was not. Thus, DENV-2 infection induces viperin which has anti-viral properties residing in the C-terminal region of the protein that act to restrict early DENV-2 RNA production/accumulation, potentially via interaction of viperin with DENV-2 NS3 and replication complexes. These anti-DENV-2 actions of viperin show both contrasts and similarities with other described anti-viral mechanisms of viperin action and highlight the diverse nature of this unique anti-viral host protein., Author Summary Viperin is a virally induced host protein that has been previously shown to have antiviral activity against a variety of viruses. Here we have demonstrated that viperin is also anti-viral against the medically significant arbovirus, dengue virus. Viperin was able to inhibit dengue virus at the level of viral replication, and cell lines unable to produce normal levels of viperin grew the virus to higher titres. These anti-dengue effects of viperin were mediated by amino acid residues in its C-terminus, and did not require structural domains of the N-terminal region as has been previously shown by us and others for the related virus, hepatitis C virus. Viperin was also demonstrated to co-localise and interact with the dengue capsid protein on the surface of lipid droplets, as well as with the NS3 protein and viral RNA. Viperin's association with NS3 was further demonstrated to be involved in its anti-dengue activities. The anti-viral activities of viperin presented in this manuscript show both similarities and contrasts with other described anti-viral mechanisms for the protein and highlight the diverse nature of this unique anti-viral host protein.