For workplaces which cannot operate as telework or remotely, there is a critical need for routine occupational SARS-CoV-2 diagnostic testing. Although diagnostic tests including the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel (CDC Diagnostic Panel) (EUA200001) were made available early in the pandemic, resource scarcity and high demand for reagents and equipment necessitated priority of symptomatic patients. There is a clearly defined need for flexible testing methodologies and strategies with rapid turnaround of results for (1) symptomatic, (2) asymptomatic with high-risk exposures and (3) asymptomatic populations without preexisting conditions for routine screening to address the needs of an on-site work force. We developed a distinct SARS-CoV-2 diagnostic assay based on the original CDC Diagnostic Panel (EUA200001), yet, with minimum overlap for currently employed reagents to eliminate direct competition for limited resources. As the pandemic progressed with testing loads increasing, we modified the assay to include 5-sample pooling and amplicon target multiplexing. Analytical sensitivity of the pooled and multiplexed assays was rigorously tested with contrived positive samples in realistic patient backgrounds. Assay performance was determined with clinical samples previously assessed with an FDA authorized assay. Throughout the pandemic we successfully tested symptomatic, known contact and travelers within our occupational population with a ~ 24–48-h turnaround time to limit the spread of COVID-19 in the workplace. Our singleplex assay had a detection limit of 31.25 copies per reaction. The three-color multiplexed assay maintained similar sensitivity to the singleplex assay, while tripling the throughput. The pooling assay further increased the throughput to five-fold the singleplex assay, albeit with a subtle loss of sensitivity. We subsequently developed a hybrid ‘multiplex-pooled’ strategy to testing to address the need for both rapid analysis of samples from personnel at high risk of COVID infection and routine screening. Herein, our SARS-CoV-2 assays specifically address the needs of occupational healthcare for both rapid analysis of personnel at high-risk of infection and routine screening that is essential for controlling COVID-19 disease transmission. In addition to SARS-CoV-2 and COVID-19, this work demonstrates successful flexible assays developments and deployments with implications for emerging highly transmissible diseases and future pandemics.

Purpose: Mycoplasma capricolum subsp. capripneumoniae (Mccp) causes a severe, usually fatal disease in goats known as Contagious Caprine Pleuropneumonia (CCPP). CCPP is listed by OIE as a notifiable animal diseases, causing economic losses in terms of high morbidity and mortality. Thus far, very limited information is available on the molecular characterization of the unique Mccp strains prevalent in Pakistan. The study was aimed to isolate Mccp local strain for the development of diagnostics and vaccines. Methods: Samples were collected during November 2017-December 2018 at Northern areas of Pakistan from 10 goat flocks each in Gilgit-Baltistan, Chitral, Swat, Buner, and Hazara. 900 sam-ples were collected; nasal swabs (n = 400), tracheal swabs (n = 150) from naturally infected goats showing clinical signs of CCPP, and lungs tissue (n = 200), pleural fluid (n = 150) from goats at necropsy. Results: The clinical signs recorded were mucopurulent nasal discharges, cough, abdominal respiration and hyperthermia. The post-mortem revealed, pulmonary consolidation, fibrinous pleuropneumonia, and accumulation pleural fluid. The fried egg like growth was observed on agar in 16 (4%), 11 (7.3%), 38 (19%), and 24 (16%) nasal swab, tracheal swabs, lungs and pleural fluid samples, respectively. PCR targeting 16S rRNA gene revealed isolates, belongs to Mycoplasma mycoides cluster, in 72 (8%) samples. Forty one (4.5%) isolates were Mccp by specie specific PCR generating an amplicon of 316 bp. Conclusions: We successfully isolated local strain of Mccp for the first time in Pakistan. This Mccp strain could be further utilized for the development of diagnostics and control measures against Mccp infection in goats. Copyright 2020, Taiwan Society of Microbiology. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/).

Porphyrins are vital pigments involved in biological energy transduction processes. Their abilities to absorb light, then convert it to energy, have raised the interest of using porphyrin nanoparticles as photosensitizers in photodynamic therapy. A recent study showed that self- assembled porphyrin-silica composite nanoparticles can selectively destroy tumor cells, but detection of the cellular uptake of porphyrin-silica composite nanoparticles was limited to imaging microscopy. Here we developed a novel method to rapidly identify porphyrin-silica composite nanoparticles using Atmospheric Solids Analysis Probe-Mass Spectrometry (ASAP-MS). ASAP-MS can directly analyze complex mixtures without the need for sample preparation. Porphyrin-silica composite nanoparticles were vaporized using heated nitrogen desolvation gas, and their thermo-profiles were examined to identify distinct mass- to-charge (M/Z) signatures. HeLa cells were incubated in growth media containing the nanoparticles, and after sufficient washing to remove residual nanoparticles, the cell suspension was loaded onto the end of ASAP glass capillary probe. Upon heating, HeLa cells were degraded and porphyrin-silica composite nanoparticles were released. Vaporized nanoparticles were ionized and detected by MS. The cellular uptake of porphyrin-silica composite nanoparticles was identified using this ASAP-MS method.

The envelope (E) protein of Dengue virus rearranges to a trimeric hairpin to mediate fusion of the viral and target membranes, which is essential for infectivity. Insertion of E into the target membrane serves to anchor E and possibly also to disrupt local order within the membrane. Both aspects are likely to be affected by the depth of insertion, orientation of the trimer with respect to the membrane normal, and the interactions that form between trimer and membrane. In the present work, we resolved the depth of insertion, the tilt angle, and the fundamental interactions for the soluble portion of Dengue E trimers (sE) associated with planar lipid bilayer membranes of various combinations of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and cholesterol (CHOL) by neutron reflectivity (NR) and by molecular dynamics (MD) simulations. The results show that the tip of E containing the fusion loop (FL) is located at the interface of the headgroups and acyl chains of the outer leaflet of the lipid bilayers, in good agreement with prior predictions. The results also indicate that E tilts with respect to the membrane normal upon insertion, promoted by either the anionic lipid POPG or CHOL. The simulations show that tilting of the protein correlates with hydrogen bond formation between lysines and arginines located on the sides of the trimer close to the tip (K246, K247, and R73) and nearby lipid headgroups. These hydrogen bonds provide a major contribution to the membrane anchoring and may help to destabilize the target membrane.

We describe a new method to measure the activation energy for unbinding (enthalpy ΔH*u and free energy ΔG*u) of a strongly-bound membrane-associated protein from a lipid membrane. It is based on measuring the rate of release of a liposome-bound protein during centrifugation on a sucrose gradient as a function of time and temperature. The method is used to determine ΔH*u and ΔG*u for the soluble dengue virus envelope protein (sE) strongly bound to 80:20 POPC:POPG liposomes at pH5.5. ΔH*u is determined from the Arrhenius equation whereas ΔG*u is determined by fitting the data to a model based on mean first passage time for escape from a potential well. The binding free energy ΔGb of sE was also measured at the same pH for the initial, predominantly reversible, phase of binding to a 70:30 PC:PG lipid bilayer. The unbinding free energy (20±3kcal/mol, 20% PG) was found to be roughly three times the binding energy per monomer, (7.8±0.3kcal/mol for 30% PG, or est. 7.0kcal/mol for 20% PG). This is consistent with data showing that free sE is a monomer at pH5.5, but assembles into trimers after associating with membranes. This new method to determine unbinding energies should be useful to understand better the complex interactions of integral monotopic proteins and strongly-bound peripheral membrane proteins with lipid membranes.

Since the introduction of micro total analytical systems (μTASs), significant advances have been made toward development of lab-on-a-chip platforms capable of performing complex biological assays that can revolutionize public health, among other applications. However, use of these platforms in low-resource environments (e.g. developing countries) has yet to be realized as the majority of technologies used to control microfluidic flow rely on off-device hardware with non-negligible size, cost, power requirements and skill/training to operate. In this paper we describe a magnetic-adhesive based valve that is simple to construct and operate, and can be used to control fluid flow and store reagents within a microfluidic device. The design consists of a port connecting two chambers on different planes in the device that is closed by a neodymium disk magnet seated on a thin ring of adhesive. Bringing an external magnet into contact with the outer surface of the device unseats and displaces the valve magnet from the adhesive ring, exposing the port. Using this configuration, we demonstrate on-device reagent storage and on-demand transport and reaction of contents between chambers. This design requires no power or external instrumentation to operate, is extremely low cost ($0.20 materials cost per valve), can be used by individuals with no technical training, and requires only a hand-held magnet to actuate. Additionally, valve actuation does not compromise the integrity of the completely sealed microfluidic device, increasing safety for the operator when toxic or harmful substances are contained within. This valve concept has the potential to simplify design of μTASs, facilitating development of lab-on-a-chip systems that may be practical for use in point-of-care and low-resource settings.

Despite significant progress in development of bioanalytical devices cost, complexity, access to reagents and lack of infrastructure have prevented use of these technologies in resource-limited regions. To provide a sustainable tool in the global effort to combat infectious diseases the diagnostic device must be low cost, simple to operate and read, robust, and have sensitivity and specificity comparable to laboratory analysis. In this mini-review we describe recent work using laser machined plastic laminates to produce diagnostic devices that are capable of a wide variety of bioanalytical measurements and show great promise towards future use in low-resource environments.

Pathogenic membrane-enveloped viruses cause devastating infectious diseases and pose major biodefense threats. For these viruses, fusion of the viral membrane with cellular membranes is essential for viral replication and infectivity. For flaviviruses, alphaviruses, and many arenaviruses fusion occurs within endosomes and is driven by pH-dependent conformational changes of a dedicated protein, called E in flaviviruses. The present work focuses on the dependence of fusion on membrane composition for the flavivirus Dengue (DEN). Recently it has been reported that full fusion only occurs with membranes containing anionic lipids, i.e. late endosomes.1 The origin of this strong dependence on lipid composition is currently unknown. Regarding cholesterol, different effects have been reported. One group reported that cholesterol is not absolutely required but increases the efficiency of fusion of the flavivirus tick-borne encephalitis (TBE) with liposomes.2 Another group reported that an excess of cholesterol blocks fusion of DEN in mammalian cells.3 Others reported that fusion of DEN in insect cells was independent of cholesterol.4 To provide insight into the membrane dependence of fusion for DEN we used several biophysical techniques (neutron reflectivity, quartz crystal microbalance, density gradient centrifugation and liposome coflotation analysis) to study the depth of insertion of E into lipid membranes, the influence of E binding on membrane curvature, and the anchoring energy of E in the membrane as a function of membrane composition. Important effects were discovered for both negatively-charged lipids and cholesterol. (1) Zaitseva et al PLoS Pathogens 2010, 6, e1001131. (2) Stiasny et al Journal of Virology 2003, 77, 7856. (3) Lee, et al Journal of Virology 2008, 82, 6470. (4) Umashankar et al Journal of Virology 2008, 82, 9245.

Dengue virus is a devastating human pathogen responsible for millions of infections each year. No antiviral therapies for Dengue currently exist, making effective treatment of the virus challenging. Dengue is taken into the cell through endocytosis. Low-pH mediated structural rearrangements of the envelope protein E leads to the formation of fusogenic E trimers that facilitate membrane fusion with late endosomes. The fusion mechanism is not fully understood, but is a key target for inhibiting the viral infection pathway. An important aspect of fusion is the dependence on endosomal membrane composition, and in particular, the requirement of anionic lipids. This study aims to characterize the biophysical reasons for this dependence. The work includes experimental studies and molecular simulations of the interactions of E with lipid membranes. These approaches revealed the structure of E bound to lipid membranes including the depth of its insertion into the membrane and the average angle with respect to the membrane, the fundamental interactions involved, the dependence of adsorption and anchoring energy on membrane composition, the membrane curvature induced upon insertion, and the correlation of the above with fusion efficiency of virus like particles (VLPs) with liposomes. As a part of this work we developed a new biophysical technique to measure the energy for pulling E out of a membrane, and distinguished anchoring (pull-out) and binding energies for this nonequilibrium system. We also developed a modeling approach combining molecular and continuum approaches to provide the first theoretical estimate of the binding energy. Taken together, this work lays the foundation for developing a systematic fundamental understanding of fusion in enveloped viruses that has been elusive to date.

463-Pos Board B232 Single Neuronal Snare Complexes Zipper in Three Distinct Stages Ying Gao, Sylvain Zorman, Gregory Gundersen, Zhiqun Xi, Lu Ma, George Sirinakis, James E. Rothman, Yongli Zhang. Yale University, New Haven, CT, USA. SNARE proteins drive membrane fusion by assembling into a four-helix bundle in a zippering process. Here we used optical tweezers to observe in real time a long-sought SNARE assembly intermediate in which only the membranedistal N-terminal half of the bundle is assembled. Our finding supports the zippering hypothesis, but suggests that zippering proceeds through three sequential binary switches, not continuously, in the Nand C-terminal halves of the bundle and the linker domain. The half-zippered intermediate was stabilized by externally applied force which mimicked the repulsion between apposed membranes being forced to fuse. This intermediate then rapidly and forcefully zippered, delivering free energy of 36 kBT to mediate fusion.