Biology subjects

Graham, M.

Publications and source records attributed to Graham, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Measles Whole Genome Sequencing by an Illumina Tiled Amplification Method

Measles virus remains a significant global health threat, and despite the availability of an effective vaccine, measles cases continue to increase worldwide in recent years. Genomic surveillance has become an essential tool for monitoring virus circulation and investigating outbreaks. Here, we describe a wet-laboratory method for whole-genome sequencing of measles virus using a tiled amplicon approach and Illumina sequencing technology. A previously published Oxford Nanopore-based tiled primer scheme was adapted to include both circulating measles genotypes and for use on the Illumina platform. Two Illumina library preparation kits, Illumina DNA Prep (IDP) and Nextera XT (XT), were evaluated for performance. The IDP kit demonstrated more complete genomes and consistent genome coverage compared with XT. Using quantified reference genomes, the limit of detection was determined to be 10,000 genome copies for genotype B3 and D8. Sequence accuracy was evaluated using previously characterized clinical samples and showed high concordance. This method provides a reliable and sensitive approach for measles virus whole-genome sequencing using Illumina platforms and is suitable for genomic surveillance applications.

genomics↗

bioRxiv2026

Multi-omics links microbial dysbiosis, systemic inflammation and metabolomic disruptions to SNAE risk in treated HIV

Serious non-AIDS events (SNAEs), including non-AIDS malignancies, cardiovascular disease, and hepatic complications, remain major causes of mortality in treated HIV infection. These outcomes are driven by persistent immune activation, systemic inflammation, and metabolic dysfunction despite effective viral suppression with antiretroviral therapy (ART). To investigate mechanisms underlying SNAE pathogenesis, we performed a cross-site multi-omic analysis integrating plasma proteins, plasma metabolites, and mucosal microbiomes (ileum and rectum) in 82 ART-treated people with HIV (PWH) and 10 people without HIV (PWoH) from the United States and Mexico. Geography was the dominant source of variation, particularly across lipid classes. However, individuals at high risk for SNAEs, defined by low CD4 T cell counts and low CD4/CD8 ratios, shared a consistent signature of systemic inflammation, mitochondrial dysfunction, and microbial dysbiosis including elevated plasma IL-6, {omega}-oxidation products (adipic and suberic acids), and depletion of short-chain fatty acid-producing commensals in the gut mucosa, including Akkermansia muciniphila, Bacteroides uniformis, and Ruminococcus. Notably, A. muciniphila abundance correlated with lower IL-6 levels, fewer HIV RNA-producing cells in lymph nodes, and higher CD4/CD8 ratios. Together, these findings identify a shared inflammatory and metabolic phenotype in PWH and implicate A. muciniphila as a potential microbiome-based target to mitigate immune activation and SNAE risk in treated HIV.

immunology↗

bioRxiv2025

Targeted disruption of phage liquid crystalline droplets abolishes antibiotic tolerance of bacterial biofilms

All bacterial biofilms contain an extracellular matrix rich in filamentous molecules 1 that self-associate 2-4, conferring emergent properties to bacteria 5, including antibiotic tolerance 6. Pseudomonas aeruginosa is a human pathogen that forms biofilms in diverse infectious settings 7,8, where the upregulation of a filamentous bacteriophage Pf4, has been shown to be a key virulence factor that protects bacteria from antibiotics 9,10. Here, we modelled biophysical characteristics of biofilm-linked liquid crystalline droplets formed by Pf4, which predicted that sub-stoichiometric phage binders had the ability to disrupt liquid crystals by changing the surface properties of the phage. We tested this prediction by developing nanobodies targeting the outer surface of the Pf4 phage, which disrupted in vitro reconstituted droplets, promoted antibiotic diffusion into bacteria, disrupted P. aeruginosa biofilm formation under a variety of conditions, and abolished antibiotic tolerance of biofilms. The inhibition strategy illustrated in this study could be extended to biofilms of other pathogenic bacteria, where filamentous molecules are pervasive in the extracellular matrix. Furthermore, our findings exemplify how targeting a biophysical mechanism, rather than a defined biochemical target, is a promising avenue for intervention, with the potential of applying this concept to other disease-related contexts.

microbiology↗

bioRxiv2025

The Golgi Rim is a Precise Tetraplex of Golgin Proteins that Can Self-Assemble into Filamentous Bands

Golgin proteins have long been suspected to be organizers of the Golgi stack. Using three-dimensional super-resolution microscopy, we comprehensively localize the human golgin family at the rim of the Golgi apparatus at 10-20 nm resolution in situ. Unexpectedly, we find that the golgins are precisely organized into a tetraplex with four discrete layers, each containing a specific set of rim golgins. We observe no golgins inside the stack between its membrane-bound cisternae. Biochemically characterizing most of the golgins as isolated proteins, we find that they form anti-parallel dimers and further self-assemble into bands of multi-micron-long filaments. Based on our findings, we propose an "outside-in" physical model, the Golgin Organizer Hypothesis, in which the Golgi stack of cisternae and its overall ribbon morphology directly result from bending circumferential bands of rim golgin filaments onto a membrane surface, explaining stack formation without the need for special "stacking proteins."

cell biology↗

bioRxiv2025

Three-dimensional quantitative characterization of Coxiella burnetii infection using focused ion beam-scanning electron microscopy

Coxiella burnetii is a highly virulent intracellular pathogen that causes acute and chronic Q fever in humans. The bacterium utilizes a Dot/Icm type IV secretion system (T4SS) to translocate over 100 effectors into host cells to facilitate biogenesis of Coxiella-containing vacuoles (CCVs), which are specialized lysosome-derived organelles that support bacterial replication. During replication in CCVs, C. burnetii undergoes a unique biphasic developmental cycle defined by two distinct cellular forms: the infectious and metabolically dormant small cell variant (SCV) that converts to a replicative large cell variant (LCV). This developmental cycle was believed to be intimately coupled with biogenesis of CCVs, but the underlying mechanisms remained unclear. Here, we combine advanced focused ion beam-scanning electron microscopy (FIB-SEM) with machine learning-based data analyses to visualize host cells infected by C. burnetii. We reveal significant pleiomorphism of both the bacteria and CCVs in three dimensions. We show that this technology can be leveraged to characterize CCV biogenesis defects displayed by C. burnetii mutants that are unable to generate the large CCV displayed by wild-type C. burnetii. Analysis of HeLa cells infected with a cig2::Tn mutant confirmed that this mutant creates vacuoles that have a defect in homotypic fusion but that the proportions of SCV to LCV in vacuoles in which this mutant resides are nearly identical to CCVs containing wild-type C. burnetii, which indicates the vacuole biogenesis defect displayed by the cig2::Tn mutant did not impact the developmental cycle intracellularly. Collectively, this study provides unprecedented three-dimensional images of the complex intracellular lifestyle of C. burnetii. This imaging technology also provides unique insights into the biphasic developmental cycle and will be a powerful approach to dissect CCV biogenesis defects displayed by mutant C. burnetii.

microbiology↗

bioRxiv2024

Fitness compatibility and dengue virus inhibition in a Bangladeshi strain of Aedes aegypti infected with the Wolbachia strain wAlbB

Dengue cases in Bangladesh have surged in recent years. The existing insecticide-based control program, implemented in parts of the country, is challenged by issues of insufficient household coverage and high levels of insecticide resistance in the primary dengue virus (DENV) vector, Aedes aegypti. A more sustainable, effective alternative could be the implementation of a Wolbachia-mediated disease management strategy. Infecting mosquitoes with Wolbachia can change their reproductive compatibility and their ability to transmit DENV. These new phenotypes can be exploited to suppress or replace wild-type Ae. aegypti populations. Such strategies require the development of well-characterised Wolbachia-infected strains with biological characteristics that are comparable with local mosquitoes. We created and characterised a Wolbachia-infected Ae. aegypti strain with a Dhaka wild-type genetic background, and compared its reproductive compatibility, maternal inheritance, fitness, and virus-blocking ability to the parental strains (Dhaka wild-type and wAlbB2-F4). The new Ae. aegypti strain wAlbB2-Dhaka demonstrated complete cytoplasmic incompatibility with the wild-type strain and complete maternal transmission, retaining levels of pyrethroid resistance of the Dhaka wild-type (70% survival to 10 times the dose of permethrin expected to kill susceptible mosquitoes). No significant fitness costs were detected during laboratory comparisons of fecundity, fertility, survival, mating competitiveness, or desiccation tolerance. Compared to the wild-type strain, wAlbB2-Dhaka mosquitoes had a significantly reduced number of DENV genome copies in the bodies (44.4%, p = 0.0034); two-fold reduction in dissemination to legs and wings (47.6%, p < 0.0001); and >13-fold reduction of DENV in saliva expectorates (proxy of transmission potential) (92.7%, p < 0.0001) 14 days after ingesting dengue-infected blood. Our work indicates that the wAlbB2-Dhaka strain could be used for Ae. aegypti suppression or replacement strategies for dengue management in Bangladesh. Author summaryBangladesh is currently grappling with a series of severe dengue outbreaks, resulting in more than 300 thousand infections, 1700 fatalities, and a significant influx of patients requiring hospitalisation in 2023. These outbreaks coincide with the emergence of high levels of insecticide resistance in the Ae. aegypti population, which is contributing to the failure of conventional, insecticidal control strategies. In search of more sustainable, effective dengue control strategies, approaches using the endosymbiotic bacteria Wolbachia that render mosquitoes resistant to arbovirus infection are being trialled in a number of countries. We transinfected Dhaka wild-type Ae. aegypti with the Wolbachia strain wAlbB2 by backcrossing it with a Wolbachia wAlbB2-infected Ae. aegypti strain and assessed the new strains suitability for field release. We compared the fitness and virus-blocking ability of the backcrossed strain with its parental strains. We demonstrated that the new strain, wAlbB2-Dhaka, is as fit as the Dhaka wild-type strain and blocked [~]92% of dengue virus transmission. This strain has great potential for Wolbachia-mediated dengue management strategies in Bangladesh.

zoology↗

bioRxiv2024

Structure of the virulence-associated Neisseria meningitidis filamentous bacteriophage MDA{varphi}

Neisseria meningitidis is a human commensal bacterium that can opportunistically invade the bloodstream and cross the blood-brain barrier, where it can cause septicaemia and meningitis. These diseases, if left untreated, can be lethal within hours. Hyperinvasive N. meningitidis strains often express a genomically encoded filamentous bacteriophage called MDA{Phi}, which promotes colonisation of mucosal host surfaces to facilitate invasion. How this phage is organised and how it promotes biofilm formation and infection at the molecular level is unclear. Here, we present an electron cryomicroscopy structure of the MDA phage, showing that MDA{Phi} is a class I filamentous inovirus, with the major capsid protein arranged within the phage as a highly curved and densely packed -helix. Comparison with other filamentous bacteriophages offers clues about inoviral genome encapsidation mechanisms, providing a framework for understanding the evolutionary diversity of inoviruses. A disordered, N-terminal segment in the major capsid protein presents hydrophobic patches on the surface of assembled phage particles, which, together with electron cryotomography data of phage bundles, furnishes a structural rationale for phage-phage interactions that were seen previously in an epithelium adhesion infection model of N. meningitidis. Taken together, our results shed light on the structure, organisation, and higher order assembly of a biomedically relevant phage encoded in the genome of a human pathogen. Molecular insights gleaned from this study increase our understanding of phage evolution, phage-mediated bacterial adhesion and pathogenicity.

microbiology↗

bioRxiv2024

Improved Zika virus plaque assay using Vero/TMPRSS2 cell line

Plaque assay is the gold standard for the quantification of viable cytopathic viruses like Zika virus (ZIKV). Some strains of ZIKV produce plaques that are very difficult to accurately visualize and count on the commonly used Vero cell line. From data generated in our lab, we became curious if Vero/TMPRSS2 cells may be a better alternative, therefore we compared the plaque forming units (PFU) of two strains of ZIKV on Vero/TMPRSS2 cells to those produced by Vero cells. We also compared the virus stock titer generated on Vero/TMPRSS2 cells to that generated by the Vero cell line. Although Vero cells generated higher quantity of ZIKV stocks, Vero/TMPRSS2 cells produced plaques with significantly improved morphology and visibility and may therefore be a better alternative to use for performing plaque assays for strains of ZIKV that are more difficult to titer on regular Vero cells.

microbiology↗

bioRxiv2024

Long term co-circulation of multiple arboviruses in southeast Australia revealed by xeno-monitoring of mosquitoes and metatranscriptomics

Arbovirus surveillance of wild-caught mosquitoes is an affordable and sensitive means of monitoring virus transmission dynamics at various spatial-temporal scales, and emergence and re-emergence during epidemic and interepidemic periods. A variety of molecular diagnostics for arbovirus screening of mosquitoes (known as xeno-monitoring) are available, but most provide limited information about virus diversity. PCR-based screening coupled with metatranscriptomics is an increasingly affordable and sensitive pipeline for integrating complete viral genome sequencing into surveillance programs. This enables large-scale, high-throughput arbovirus screening from diverse samples. We collected mosquitoes in CO2-baited light traps from five urban parks in Brisbane from March 2021 to May 2022. Mosquito pools of [≤]200 specimens were screened for alphaviruses and flaviviruses using virus genus-specific primers and reverse transcription quantitative PCR (qRT-PCR). A subset of virus-positive samples was then processed using a mosquito-specific ribosomal RNA depletion method and then sequenced on the Illumina NextSeq. Overall, 54,670 mosquitoes, representing 26 species were screened in 382 pools. Thirty detections of arboviruses were made in 28 pools. Twenty of these positive pools were further characterised using meta-transcriptomics generating 18 full-length genomes. These full-length sequences belonged to four medically relevant arboviruses: Barmah Forest, Ross River, Sindbis-like and Stratford viruses. Phylogenetic and evolutionary analyses revealed the evolutionary progression of arbovirus lineages over the last 100 years, highlighting long-distance dispersal across the Australian continent and continuous circulation characterised by constant turnover of virus lineages.

genomics↗

bioRxiv2023

Proportionality-based association metrics in count compositional data

MotivationCompositional data comprise vectors that describe the constituent parts of a whole. Data arising from various -omics platforms such as 16S and RNA-sequencing are compositional in nature. However, correlations between features on raw counts have no meaningful interpretation. Metrics of proportionality were formulated to address this problem. However, there is an inherent bias that arises when calculating these metrics empirically on count-based measures due to variability in read depths. ResultsWe quantify the bias introduced by empirically calculating proportionality-based association metrics in count data. Additionally, we propose a means of estimating these metrics within a logit-normal multinomial model in pursuit of more accurate estimates. The model-based estimates are shown to outperform empirical estimates in simulated data, and are additionally applied to a mouse embryonic stem-cell single-cell sequencing dataset as well as a pediatric-onset multiple sclerosis metagenomic dataset. Availability and ImplementationAn R package is available at https://CRAN.R-project.org/package=countprop. Supplementary informationSupplementary data are available at Bioinformatics online.

bioinformatics↗

bioRxiv2023

Central in vivo mechanisms by which C. difficile's proline reductase drives efficient metabolism, growth, and toxin production

Clostridioides difficile (CD) is a sporulating and toxin-producing nosocomial pathogen that opportunistically infects the gut, particularly in patients with depleted microbiota after antibiotic exposure. Metabolically, CD rapidly generates energy and substrates for growth from Stickland fermentations of amino acids, with proline being a preferred reductive substrate. To investigate the in vivo effects of reductive proline metabolism on C. difficiles virulence in an enriched gut nutrient environment, we evaluated wild-type and isogenic{Delta} prdB strains of ATCC43255 on pathogen behaviors and host outcomes in highly susceptible gnotobiotic mice. Mice infected with the{Delta} prdB mutant demonstrated extended survival via delayed colonization, growth and toxin production but ultimately succumbed to disease. In vivo transcriptomic analyses demonstrated how the absence of proline reductase activity more broadly disrupted the pathogens metabolism including failure to recruit oxidative Stickland pathways, ornithine transformations to alanine, and additional pathways generating growth-promoting substrates, contributing to delayed growth, sporulation, and toxin production. Our findings illustrate the central role for proline reductase metabolism to support early stages of C. difficile colonization and subsequent impact on the pathogens ability to rapidly expand and cause disease.

microbiology↗

bioRxiv2022

Biophysical basis of phage liquid crystalline droplet-mediated antibiotic tolerance in pathogenic bacteria

Inoviruses are abundant filamentous phages infecting numerous prokaryotic phyla, where they can symbiotically promote host fitness and increase bacterial virulence. Due to their unique properties, inoviruses have also been utilised in biotechnology for phage display and as models for studying phase behaviour of colloidal rods. Inoviral phages secreted by bacteria can self-assemble into liquid crystalline droplets that protect bacterial cells in biofilms from antibiotics, however, factors governing the formation of such droplets and the mechanism of antibiotic protection are poorly understood. Here, we investigate the structural, biophysical, and protective properties of liquid crystalline droplets formed by Pseudomonas aeruginosa and Escherichia coli inoviral phages. We report a cryo-EM structure of the capsid from the highly studied E. coli fd phage, revealing distinct biochemical properties of fd compared to Pf4 phage from P. aeruginosa. We show that fd and Pf4 form liquid crystalline droplets with diverse morphologies governed by the underlying phage particle geometry and biophysics, rather than their surface biochemical properties. Finally, we show that these morphologically diverse droplets made of either phage can protect rod-shaped bacteria from antibiotic treatment, despite differing modes of association with cells. This study advances our understanding of phage assembly into liquid crystalline droplets, and provides insights into how filamentous molecules protect bacteria from extraneous molecules under crowding conditions, which are found in biofilms or on infected host tissues.

microbiology↗

bioRxiv2022

Correction of susceptibility distortion in EPI: a semi-supervised approach with deep learning.

Echo planar imaging (EPI) is the most common approach for acquiring diffusion and functional MRI data due to its high temporal resolution. However, this comes at the cost of higher sensitivity to susceptibility-induced B0 field inhomogeneities around air/tissue inter-faces. This leads to severe geometric distortions along the phase encoding direction (PED). To correct this distortion, the standard approach involves an analogous acquisition using an opposite PED leading to images with inverted distortions and then non-linear image registration, with a transformation model constrained along the PED, to estimate the voxelwise shift that undistorts the image pair and generates a distortion-free image. With conventional image registration approaches, this type of processing is computationally intensive. Recent advances in unsupervised deep learning-based approaches to image registration have been proposed to drastically reduce the computational cost of this task. However, they rely on maximizing an intensity-based similarity measure, known to be suboptimal surrogate measures of image alignment. To address this limitation, we propose a semi-supervised deep learning algorithm that directly leverages ground truth spatial transformations during training. Simulated and real data experiments demonstrate improvement to distortion field recovery compared to the unsupervised approach, improvement image similarity compared to supervised approach and precision similar to TOPUP but with much faster processing.

neuroscience↗

bioRxiv2022

Clostridium scindens colonization of gnotobiotic mice promotes a chronic unresolving infection with Clostridioides difficile

The commensal Clostridium scindens has been regarded as a promising bacteriotherapeutic against Clostridioides difficile infection due to its ability to consume host factors that can promote C. difficile growth, and its production of the antimicrobial compound 1-acetyl-{beta}-carboline. We investigated C. scindens protective effects against C. difficile using defined colonization studies in gnotobiotic mice. Mice infected with C. difficile develop lethal infection within 48 hours. In contrast, 88% of mice pre-colonized with C. scindens survived acute infection with delayed C. difficile colonization, lower biomass, and toxin B levels at 24 hours after infection. However, two weeks post-challenge, surviving mice showed comparable levels of cecal C. difficile vegetative and spore biomass and toxin B, as seen during acute infection. After two weeks, co-colonized mice exhibited mucosal colonic hyperplasia with focal pseudomembranes, modeling a chronic and recurrent infection state. Our findings illustrate how the commensal microbiota can modulate host and pathogen interactions leading to chonic C. difficile carriage and infection.

microbiology↗

bioRxiv2022

Wolbachia wAlbB inhibit dengue and Zika infection in the mosquito Aedes aegypti with an Australian background

Biological control of mosquito vectors using the insect-specific bacteria Wolbachia is an emerging strategy for the management of human arboviral diseases. We recently described the development of a strain of Ae. aegypti infected with the Wolbachia strain wAlbB (referred to as the wAlbB2-F4 strain) through simple backcrossing of wild type Australian mosquitoes with a wAlbB infected Ae. aegypti strain from the USA. Field releases of male wAlbB2-F4 mosquitoes resulted in the successful suppression of a wild population of mosquitoes in the trial sites by exploiting the strains Wolbachia-induced cytoplasmic incompatibility. We now demonstrate that the strain is resistant to infection by dengue and Zika viruses and is genetically similar to endemic Queensland populations. There was a fourfold reduction in the proportion of wAlbB2-F4 mosquitoes that became infected following a blood meal containing dengue 2 virus (16.7%) compared to wild type mosquitoes (69.2%) and a 6-7 fold reduction in the proportion of wAlbB2-F4 mosquitoes producing virus in saliva following a blood meal containing an epidemic strain of Zika virus (8.7% in comparison to 58.3% in wild type mosquitoes). Restriction-site Associated DNA (RAD) sequencing revealed that wAlbB2-F4 mosquitoes have > 98% Australian ancestry, confirming the successful introduction of the wAlbB2 infection into the Australian genomic background through backcrossing. Genotypic and phenotypic analyses showed the wAlbB2-F4 strain retains the insecticide susceptibility phenotype and genotype of the Australian mosquitoes. We demonstrate that the Wolbachia wAlbB2-F4, in addition to being suitable for suppression programs, can be effective in population replacement programs given its high inhibition of virus infection in mosquitoes. The ease at which a target mosquito population can be transfected with wAlbB2, while retaining genotypes and phenotypes of the target population, shows the robustness of this strain as a biocontrol agent against the Ae. aegypti mosquito itself as well as the pathogens it transmits. IMPORTANCEEpidemics of arthopod-borne virus (arbovirus) diseases affect millions of people and are becoming more frequent and widespread. A successful strategy to control these diseases is by infecting mosquito populations with benign, insect-specific Wolbachia bacteria that render mosquitoes refractory to infection with pathogenic arboviruses. Here we show that a strain of the major mosquito vector Ae. aegypti that was infected with Wolbachia following a simple back-cross mating procedure is refractory to infection with dengue and Zika viruses. Importantly, the genetic background of the strain is equivalent to the target population, which is important for persistence of the strain and regulatory approval.

microbiology↗

bioRxiv2021

Boosting of SARS-CoV-2 immunity in nonhuman primates using an oral rhabdoviral vaccine

An orally active vaccine capable of boosting SARS-CoV-2 immune responses in previously infected or vaccinated individuals would help efforts to achieve and sustain herd immunity. Unlike mRNA-loaded lipid nanoparticles and recombinant replication-defective adenoviruses, replicating vesicular stomatitis viruses with SARS-CoV-2 spike glycoproteins (VSV-SARS2) were poorly immunogenic after intramuscular administration in clinical trials. Here, by G protein trans-complementation, we generated VSV-SARS2(+G) virions with expanded target cell tropism. Compared to parental VSV-SARS2, G-supplemented viruses were orally active in virus-naive and vaccine-primed cynomolgus macaques, powerfully boosting SARS-CoV-2 neutralizing antibody titers. Clinical testing of this oral VSV-SARS2(+G) vaccine is planned.

immunology↗

bioRxiv2020

CRISpy-pop: a web tool for designing CRISPR/Cas9-driven genetic modifications in diverse populations

CRISPR/Cas9 is a powerful tool for editing genomes, but design decisions are generally made with respect to a single reference genome. With population genomic data becoming available for an increasing number of model organisms, researchers are interested in manipulating multiple strains and lines. CRISpy-pop is a web application that generates and filters guide RNA sequences for CRISPR/Cas9 genome editing for diverse yeast and bacterial strains. The current implementation designs and predicts the activity of guide RNAs against more than 1000 Saccharomyces cerevisiae genomes, including 167 strains frequently used in bioenergy research. Zymomonas mobilis, an increasingly popular bacterial bioenergy research model, is also supported. CRISpy-pop is available as a web application (https://CRISpy-pop.glbrc.org/) with an intuitive graphical user interface. CRISpy-pop also cross-references the human genome to allow users to avoid the selection of sgRNAs with potential biosafety concerns. Additionally, CRISpy-pop predicts the strain coverage of each guide RNA within the supported strain sets, which aids in functional population genetic studies. Finally, we validate how CRISpy-pop can accurately predict the activity of guide RNAs across strains using population genomic data.

genetics↗

bioRxiv2020

Optimized pseudotyping conditions for the SARS-COV2 Spike glycoprotein

The SARS-COV2 Spike glycoprotein is solely responsible for binding to the host cell receptor and facilitating fusion between the viral and host membranes. The ability to generate viral particles pseudotyped with SARS-COV2 Spike is useful for many types of studies, such as characterization of neutralizing antibodies or development of fusion-inhibiting small molecules. Here we characterized the use of a codon-optimized SARS-COV2 Spike glycoprotein for the generation of pseudotyped HIV-1, MLV, and VSV particles. The full-length Spike protein functioned inefficiently with all three systems but was enhanced over 10-fold by deleting the last 19 amino acids of the cytoplasmic tail of Spike. Infection of 293FT target cells was only possible if the cells were engineered to stably express the human ACE-2 receptor, but stably introducing an additional copy of this receptor did not further enhance susceptibility. Stable introduction of the Spike activating protease TMPRSS2 further enhanced susceptibility to infection by 5-10 fold. Substitution of the signal peptide of the Spike protein with an optimal signal peptide did not enhance or reduce infectious particle production. However, modification of a single amino acid in the furin cleavage site of Spike (R682Q) enhanced infectious particle production another 10-fold. With all enhancing elements combined, the titer of pseudotyped particles reached almost 106 infectious particles/ml. Finally, HIV-1 particles pseudotyped with SARS-COV2 Spike was successfully used to detect neutralizing antibodies in plasma from COVID-19 patients, but not plasma from uninfected individuals. ImportanceWhen working with pathogenic viruses, it is useful to have rapid quantitative tests for viral infectivity that can be performed without strict biocontainment restrictions. A common way of accomplishing this is to generate viral pseudoparticles that contain the surface glycoprotein from the pathogenic virus incorporated into a replication-defective viral particle that contains a sensitive reporter system. These pseudoparticles enter cells using the glycoprotein from the pathogenic virus leading to a readout for infection. Conditions that block entry of the pathogenic virus, such as neutralizing antibodies, will also block entry of the viral pseudoparticles. However, viral glycoproteins often are not readily suited for generating pseudoparticles. Here we describe a series of modifications that result in the production of relatively high titer SARS-COV2 pseudoparticles that are suitable for detection of neutralizing antibodies from COVID-19 patients.

microbiology↗