Single-cell image-based genetic screens systematically identify regulators of Ebola virus subcellular infection dynamics.

Ebola virus (EBOV) is a high-consequence filovirus that gives rise to frequent epidemics with high case fatality rates and few therapeutic options. Here, we applied image-based screening of a genome-wide CRISPR library to systematically identify host cell regulators of Ebola virus infection in 39,085,093 million single cells. Measuring viral RNA and protein levels together with their localization in cells identified over 998 related host factors and provided detailed information about the role of each gene across the virus replication cycle. We trained a deep learning model on single-cell images to associate each host factor with predicted replication steps, and confirmed the predicted relationship for select host factors. Among the findings, we showed that the mitochondrial complex III subunit UQCRB is a post-entry regulator of Ebola virus RNA replication, and demonstrated that UQCRB inhibition with a small molecule reduced overall Ebola virus infection with an IC50 of 5 µM. Using a random forest model, we also identified perturbations that reduced infection by disrupting the equilibrium between viral RNA and protein. One such protein, STRAP, is a spliceosome-associated factor that was found to be closely associated with VP35, a viral protein required for RNA processing. Loss of STRAP expression resulted in a reduction in full-length viral genome production and subsequent production of non-infectious virus particles. Overall, the data produced in this genome-wide high-content single-cell screen and secondary screens in additional cell lines and related filoviruses (MARV and SUDV) revealed new insights about the role of host factors in virus replication and potential new targets for therapeutic intervention.

A Flexible, Quantitative Plasmonic-Fluor Lateral Flow Assay for the Rapid Detection of Orthoebolavirus zairense and Orthoebolavirus sudanense.

Filoviruses comprise a family of single-stranded, negative-sense RNA viruses with a significant impact on human health. Given the risk for disease outbreaks, as highlighted by the recent outbreaks across Africa, there is an unmet need for flexible diagnostic technologies that can be deployed in resource-limited settings. Herein, we highlight the use of plasmonic-fluor lateral flow assays (PF-LFA) for the rapid, quantitative detection of an Ebolavirus-secreted glycoprotein, a marker for infection. Plasmonic fluors are a class of ultrabright reporter molecules that combine engineered nanorods with conventional fluorophores, resulting in improved analytical sensitivity. We have developed a PF-LFA for Orthoebolavirus zairense (EBOV) and Orthoebolavirus sudanense (SUDV) that provides estimated limits of detection as low as 0.446 and 0.641 ng/mL, respectively. Furthermore, our assay highlights a high degree of specificity between the two viral species while also maintaining a turnaround time as short as 30 min. To highlight the utility of our PF-LFA, we demonstrate the detection of EBOV infection in non-human primates. Our PF-LFA represents an enormous step forward in the development of a robust, field-deployable assay for filoviruses.

Serological assays for differentiating natural COVID-19 infection from vaccine induced immunity.

BACKGROUND: Natural SARS-CoV-2 infection may elicit antibodies to a range of viral proteins including non-structural protein ORF8. RNA, adenovirus vectored and sub-unit vaccines expressing SARS-CoV-2 spike would be only expected to elicit S-antibodies and antibodies to distinct domains of nucleocapsid (N) protein may reliably differentiate infection from vaccine-elicited antibody. However, inactivated whole virus vaccines may potentially elicit antibody to wider range of viral proteins, including N protein. We hypothesized that antibody to ORF8 protein will discriminate natural infection from vaccination irrespective of vaccine type. METHODS: We optimized and validated the anti-ORF8 and anti-N C-terminal domain (NCTD) ELISA assays using sera from pre-pandemic, RT-PCR confirmed natural infection sera and BNT162b2 (BNT) or CoronaVac vaccinees. We then applied these optimized assays to a cohort of blood donor sera collected in April-July 2022 with known vaccination and self-reported infection status. RESULTS: We optimized cut-off values for the anti-ORF8 and anti-N-CTD IgG ELISA assays using receiver-operating-characteristic (ROC) curves. The sensitivity of the anti-ORF8 and anti-N-CTD ELISA for detecting past infection was 83.2% and 99.3%, respectively. Specificity of anti-ORF8 ELISA was 96.8 % vs. the pre-pandemic cohort or 93% considering the pre-pandemic and vaccine cohorts together. The anti-N-CTD ELISA specificity of 98.9% in the pre-pandemic cohort, 93% in BNT vaccinated and only 4 % in CoronaVac vaccinated cohorts. Anti-N-CTD antibody was longer-lived than anti-ORF8 antibody after natural infection. CONCLUSIONS: Anti-N-CTD antibody assays provide good discrimination between natural infection and vaccination in BNT162b2 vaccinated individuals. Anti-ORF8 antibody can help discriminate infection from vaccination in either type of vaccine and help estimate infection attack rates (IAR) in communities.

Host entry factors of Rift Valley Fever Virus infection.

Rift Valley Fever Virus (RVFV) is a negative sense segmented RNA virus that can cause severe hemorrhagic fever. The tri-segmented virus genome encodes for six (6) multifunctional proteins that engage host factors at a variety of different stages in the replication cycle. The S segment encodes nucleoprotein (N) and nonstructural protein S (NSs), the M segment encodes viral glycoproteins Gn and Gc as well as nonstructural protein M (NSm) and the L segment encodes the viral polymerase (L). Viral glycoproteins Gn and Gc are responsible for entry by binding to a number of host factors. Our recent studies identified a scavenger receptor, LDL receptor related protein 1 (Lrp1), as a potential pro-viral host factor for RVFV and related viruses, including Oropouche virus (OROV) infection. Coincidentally, several recent studies identified other LDL family proteins as viral entry factors and receptors for other viral families. Collectively, these observations suggest that highly conserved LDL family proteins may play a significant role in facilitating entry of viruses from several distinct families. Given the significant roles of viral and host factors during infection, characterization of these interactions is critical for therapeutic targeting with neutralizing antibodies and vaccines.

Disruption of Ebola NP0VP35 Inclusion Body-like Structures reduce Viral Infection.

Viral inclusion bodies (IBs) are potential sites of viral replication and assembly. How viral IBs form remains poorly defined. Here we describe a combined biophysical and cellular approach to identify the components necessary for IB formation during Ebola virus (EBOV) infection. We find that the eNP0VP35 complex containing Ebola nucleoprotein (eNP) and viral protein 35 (eVP35), the functional equivalents of nucleoprotein (N) and phosphoprotein (P) in non-segmented negative strand viruses (NNSVs), phase separates to form inclusion bodies. Phase separation of eNP0VP35 is reversible and modulated by ionic strength. The multivalency of eVP35, and not eNP, is also critical for phase separation. Furthermore, overexpression of an eVP35 peptide disrupts eNP0VP35 complex formation, leading to reduced frequency of IB formation and limited viral infection. Together, our results show that upon EBOV infection, the eNP0VP35 complex forms the minimum unit to drive IB formation and viral replication.

Lrp1 is essential for lethal Rift Valley fever hepatic disease in mice.

Rift Valley fever virus (RVFV) is an emerging arbovirus found in Africa. While RVFV is pantropic and infects many cells and tissues, viral replication and necrosis within the liver play a critical role in mediating severe disease. The low-density lipoprotein receptor-related protein 1 (Lrp1) is a recently identified host factor for cellular entry and infection by RVFV. The biological significance of Lrp1, including its role in hepatic disease in vivo, however, remains to be determined. Because Lrp1 has a high expression level in hepatocytes, we developed a mouse model in which Lrp1 is specifically deleted in hepatocytes to test how the absence of liver Lrp1 expression affects RVF pathogenesis. Mice lacking Lrp1 expression in hepatocytes showed minimal RVFV replication in the liver, longer time to death, and altered clinical signs toward neurological disease. In contrast, RVFV infection levels in other tissues showed no difference between the two genotypes. Therefore, Lrp1 is essential for RVF hepatic disease in mice.

Biochemical and HDX Mass Spectral Characterization of the SARS-CoV-2 Nsp1 Protein.

A major challenge in defining the pathophysiology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is to better understand virally encoded multifunctional proteins and their interactions with host factors. Among the many proteins encoded by the positive-sense, single-stranded RNA genome, nonstructural protein 1 (Nsp1) stands out due to its impact on several stages of the viral replication cycle. Nsp1 is the major virulence factor that inhibits mRNA translation. Nsp1 also promotes host mRNA cleavage to modulate host and viral protein expression and to suppress host immune functions. To better define how this multifunctional protein can facilitate distinct functions, we characterize SARS-CoV-2 Nsp1 by using a combination of biophysical techniques, including light scattering, circular dichroism, hydrogen/deuterium exchange mass spectrometry (HDX-MS), and temperature-dependent HDX-MS. Our results reveal that the SARS-CoV-2 Nsp1 N- and C-terminus are unstructured in solution, and in the absence of other proteins, the C-terminus has an increased propensity to adopt a helical conformation. In addition, our data indicate that a short helix exists near the C-terminus and adjoins the region that binds the ribosome. Together, these findings provide insights into the dynamic nature of Nsp1 that impacts its functions during infection. Furthermore, our results will inform efforts to understand SARS-CoV-2 infection and antiviral development.

Ebolavirus Species-Specific Interferon Antagonism Mediated by VP24.

Members of the Ebolavirus genus demonstrate a marked differences in pathogenicity in humans with Ebola (EBOV) being the most pathogenic, Bundibugyo (BDBV) less pathogenic, and Reston (RESTV) is not known to cause a disease in humans. The VP24 protein encoded by members of the Ebolavirus genus blocks type I interferon (IFN-I) signaling through interaction with host karyopherin alpha nuclear transporters, potentially contributing to virulence. Previously, we demonstrated that BDBV VP24 (bVP24) binds with lower affinities to karyopherin alpha proteins relative to EBOV VP24 (eVP24), and this correlated with a reduced inhibition in IFN-I signaling. We hypothesized that modification of eVP24-karyopherin alpha interface to make it similar to bVP24 would attenuate the ability to antagonize IFN-I response. We generated a panel of recombinant EBOVs containing single or combinations of point mutations in the eVP24-karyopherin alpha interface. Most of the viruses appeared to be attenuated in both IFN-I-competent 769-P and IFN-I-deficient Vero-E6 cells in the presence of IFNs. However, the R140A mutant grew at reduced levels even in the absence of IFNs in both cell lines, as well as in U3A STAT1 knockout cells. Both the R140A mutation and its combination with the N135A mutation greatly reduced the amounts of viral genomic RNA and mRNA suggesting that these mutations attenuate the virus in an IFN-I-independent attenuation. Additionally, we found that unlike eVP24, bVP24 does not inhibit interferon lambda 1 (IFN-λ1), interferon beta (IFN-ß), and ISG15, which potentially explains the lower pathogenicity of BDBV relative to EBOV. Thus, the VP24 residues binding karyopherin alpha attenuates the virus by IFN-I-dependent and independent mechanisms.

Real-world COVID-19 vaccine effectiveness against the Omicron BA.2 variant in a SARS-CoV-2 infection-naive population.

The SARS-CoV-2 Omicron variant has demonstrated enhanced transmissibility and escape of vaccine-derived immunity. Although first-generation vaccines remain effective against severe disease and death, robust evidence on vaccine effectiveness (VE) against all Omicron infections, irrespective of symptoms, remains sparse. We used a community-wide serosurvey with 5,310 subjects to estimate how vaccination histories modulated risk of infection in infection-naive Hong Kong during a large wave of Omicron BA.2 epidemic in January-July 2022. We estimated that Omicron infected 45% (41-48%) of the local population. Three and four doses of BNT162b2 or CoronaVac were effective against Omicron infection 7 days after vaccination (VE of 48% (95% credible interval 34-64%) and 69% (46-98%) for three and four doses of BNT162b2, respectively; VE of 30% (1-66%) and 56% (6-97%) for three and four doses of CoronaVac, respectively). At 100 days after immunization, VE waned to 26% (7-41%) and 35% (10-71%) for three and four doses of BNT162b2, and to 6% (0-29%) and 11% (0-54%) for three and four doses of CoronaVac. The rapid waning of VE against infection conferred by first-generation vaccines and an increasingly complex viral evolutionary landscape highlight the necessity for rapidly deploying updated vaccines followed by vigilant monitoring of VE.

Viral Targeting of Importin Alpha-Mediated Nuclear Import to Block Innate Immunity.

Cellular nucleocytoplasmic trafficking is mediated by the importin family of nuclear transport proteins. The well-characterized importin alpha (IMPA) and importin beta (IMPB) nuclear import pathway plays a crucial role in the innate immune response to viral infection by mediating the nuclear import of transcription factors such as IRF3, NFκB, and STAT1. The nuclear transport of these transcription factors ultimately leads to the upregulation of a wide range of antiviral genes, including IFN and IFN-stimulated genes (ISGs). To replicate efficiently in cells, viruses have developed mechanisms to block these signaling pathways. One strategy to evade host innate immune responses involves blocking the nuclear import of host antiviral transcription factors. By binding IMPA proteins, these viral proteins prevent the nuclear transport of key transcription factors and suppress the induction of antiviral gene expression. In this review, we describe examples of proteins encoded by viruses from several different families that utilize such a competitive inhibition strategy to suppress the induction of antiviral gene expression.

Ubiquitin variants potently inhibit SARS-CoV-2 PLpro and viral replication via a novel site distal to the protease active site.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has made it clear that combating coronavirus outbreaks benefits from a combination of vaccines and therapeutics. A promising drug target common to all coronaviruses-including SARS-CoV, MERS-CoV, and SARS-CoV-2-is the papain-like protease (PLpro). PLpro cleaves part of the viral replicase polyproteins into non-structural protein subunits, which are essential to the viral replication cycle. Additionally, PLpro can cleave both ubiquitin and the ubiquitin-like protein ISG15 from host cell substrates as a mechanism to evade innate immune responses during infection. These roles make PLpro an attractive antiviral drug target. Here we demonstrate that ubiquitin variants (UbVs) can be selected from a phage-displayed library and used to specifically and potently block SARS-CoV-2 PLpro activity. A crystal structure of SARS-CoV-2 PLpro in complex with a representative UbV reveals a dimeric UbV bound to PLpro at a site distal to the catalytic site. Yet, the UbV inhibits the essential cleavage activities of the protease in vitro and in cells, and it reduces viral replication in cell culture by almost five orders of magnitude.

Rift Valley Fever Virus Infects the Posterior Segment of the Eye and Induces Inflammation in a Rat Model of Ocular Disease.

People infected with the mosquito-borne Rift Valley fever virus (RVFV) can suffer from eye-related problems resulting in ongoing vision issues or even permanent blindness. Despite ocular disease being the most frequently reported severe outcome, it is vastly understudied compared to other disease outcomes caused by RVFV. Ocular manifestations of RVFV include blurred vision, uveitis, and retinitis. When an infected individual develops macular or paramacular lesions, there is a 50% chance of permanent vision loss in one or both eyes. The cause of blinding ocular pathology remains unknown in part due to the lack of a tractable animal model. Using 3 relevant exposure routes, both subcutaneous (SC) and aerosol inoculation of Sprague Dawley rats led to RVFV infection of the eye. Surprisingly, direct inoculation of the conjunctiva did not result in successful ocular infection. The posterior segment of the eye, including the optic nerve, choroid, ciliary body, and retina, were all positive for RVFV antigen in SC-infected rats, and live virus was isolated from the eyes. Proinflammatory cytokines and increased leukocyte counts were also found in the eyes of infected rats. Additionally, human ocular cell lines were permissive for Lrp1-dependent RVFV infection. This study experimentally defines viral tropism of RVFV in the posterior segment of the rat eye and characterizes virally-mediated ocular inflammation, providing a foundation for evaluation of vaccines and therapeutics to protect against adverse ocular outcomes. IMPORTANCE Rift Valley fever virus (RVFV) infection leads to eye damage in humans in up to 10% of reported cases. Permanent blindness occurs in 50% of individuals with significant retinal scarring. Despite the prevalence and severity of this outcome, very little is known about the mechanisms of pathogenesis. We addressed this gap by developing a rodent model of ocular disease. Subcutaneous infection of Sprague Dawley rats resulted in infection of the uvea, retina, and optic nerve along with the induction of inflammation within the posterior eye. Infection of human ocular cells induced inflammatory responses and required host entry factors for RVFV infection similar to rodents. This work provides evidence of how RVFV infects the eye, and this information can be applied to help mitigate the devastating outcomes of RVF ocular disease through vaccines or treatments.

Detection of biomarkers for filoviral infection with a silicon photonic resonator platform.

This protocol describes the use of silicon photonic microring resonator sensors for detection of Ebola virus (EBOV) and Sudan virus (SUDV) soluble glycoprotein (sGP). This protocol encompasses biosensor functionalization of silicon microring resonator chips, detection of protein biomarkers in sera, preparing calibration standards for analytical validation, and quantification of the results from these experiments. This protocol is readily adaptable toward other analytes, including cytokines, chemokines, nucleic acids, and viruses. For complete details on the use and execution of this protocol, please refer to Qavi et al. (2022).

Oropouche orthobunyavirus infection is mediated by the cellular host factor Lrp1.

Oropouche orthobunyavirus (OROV; Peribunyaviridae) is a mosquito-transmitted virus that causes widespread human febrile illness in South America, with occasional progression to neurologic effects. Host factors mediating the cellular entry of OROV are undefined. Here, we show that OROV uses the host protein low-density lipoprotein-related protein 1 (Lrp1) for efficient cellular infection. Cells from evolutionarily distinct species lacking Lrp1 were less permissive to OROV infection than cells with Lrp1. Treatment of cells with either the high-affinity Lrp1 ligand receptor-associated protein (RAP) or recombinant ectodomain truncations of Lrp1 significantly reduced OROV infection. In addition, chimeric vesicular stomatitis virus (VSV) expressing OROV glycoproteins (VSV-OROV) bound to the Lrp1 ectodomain in vitro. Furthermore, we demonstrate the biological relevance of the OROV-Lrp1 interaction in a proof-of-concept mouse study in which treatment of mice with RAP at the time of infection reduced tissue viral load and promoted survival from an otherwise lethal infection. These results with OROV, along with the recent finding of Lrp1 as an entry factor for Rift Valley fever virus, highlight the broader significance of Lrp1 in cellular infection by diverse bunyaviruses. Shared strategies for entry, such as the critical function of Lrp1 defined here, provide a foundation for the development of pan-bunyaviral therapeutics.

Plasmonic Fluor-Enhanced Antigen Arrays for High-Throughput, Serological Studies of SARS-CoV-2.

Serological testing for acute infection or prior exposure is critical for patient management and coordination of public health decisions during outbreaks. Current methods have several limitations, including variable performance, relatively low analytical and clinical sensitivity, and poor detection due to antigenic drift. Serological methods for SARS-CoV-2 detection for the ongoing COVID-19 pandemic suffer from several of these limitations and serves as a reminder of the critical need for new technologies. Here, we describe the use of ultrabright fluorescent reagents, Plasmonic Fluors, coupled with antigen arrays that address a subset of these limitations. We demonstrate its application using patient samples in SARS-CoV-2 serological assays. In our multiplexed assay, SARS-CoV-2 antigens were spotted into 48-plex arrays within a single well of a 96-well plate and used to evaluate remnant laboratory samples of SARS-CoV-2 positive patients. Signal-readout was performed with Auragent Bioscience's Empower microplate reader, and microarray analysis software. Sample volumes of 1 µL were used. High sensitivity of the Plasmonic Fluors combined with the array format enabled us to profile patient serological response to eight distinct SARS-CoV-2 antigens and evaluate responses to IgG, IgM, and IgA. Sensitivities for SARS-CoV-2 antigens during the symptomatic state ranged between 72.5 and 95.0%, specificity between 62.5 and 100%, and the resulting area under the curve values between 0.76 and 0.97. Together, these results highlight the increased sensitivity for low sample volumes and multiplex capability. These characteristics make Plasmonic Fluor-enhanced antigen arrays an attractive technology for serological studies for the COVID-19 pandemic and beyond.

Monoclonal antibodies binding data for SARS-CoV-2 proteins.

SARS-CoV-2 pandemic opens up the curiosity of understanding the coronavirus. This demand for the development of the regent, which can be used for academic and therapeutic applications. The present data provide the biochemical characterization of synthetically developed monoclonal antibodies for the SARS-CoV-2 proteins. The antibodies from phage-displayed antibody libraries were selected with the SARS-CoV-2 proteins immobilized in microwell plates. The clones which bind to the antigen in Fab-phage ELISA were selected, and a two-point competitive phage ELISA was performed. Antibodies binding kinetic of IgGs for SARS-CoV2 proteins further carried with B.L.I. Systematic analysis of binding with different control proteins and purified SARS-CoV-2 ensured the robustness of the antibodies.

The intrinsically disordered protein TgIST from Toxoplasma gondii inhibits STAT1 signaling by blocking cofactor recruitment.

Signal transducer and activator of transcription (STAT) proteins communicate from cell-surface receptors to drive transcription of immune response genes. The parasite Toxoplasma gondii blocks STAT1-mediated gene expression by secreting the intrinsically disordered protein TgIST that traffics to the host nucleus, binds phosphorylated STAT1 dimers, and occupies nascent transcription sites that unexpectedly remain silenced. Here we define a core region within internal repeats of TgIST that is necessary and sufficient to block STAT1-mediated gene expression. Cellular, biochemical, mutational, and structural data demonstrate that the repeat region of TgIST adopts a helical conformation upon binding to STAT1 dimers. The binding interface is defined by a groove formed from two loops in the STAT1 SH2 domains that reorient during dimerization. TgIST binding to this newly exposed site at the STAT1 dimer interface alters its conformation and prevents the recruitment of co-transcriptional activators, thus defining the mechanism of blocked transcription.

Rapid detection of an Ebola biomarker with optical microring resonators.

Ebola virus (EBOV) is a highly infectious pathogen, with a case mortality rate as high as 89%. Rapid therapeutic treatments and supportive measures can drastically improve patient outcome; however, the symptoms of EBOV disease (EVD) lack specificity from other endemic diseases. Given the high mortality and significant symptom overlap, there is a critical need for sensitive, rapid diagnostics for EVD. Facile diagnosis of EVD remains a challenge. Here, we describe a rapid and sensitive diagnostic for EVD through microring resonator sensors in conjunction with a unique biomarker of EBOV infection, soluble glycoprotein (sGP). Microring resonator sensors detected sGP in under 40 min with a limit of detection (LOD) as low as 1.00 ng/mL in serum. Furthermore, we validated our assay with the detection of sGP in serum from EBOV-infected non-human primates. Our results demonstrate the utility of a high-sensitivity diagnostic platform for detection of sGP for diagnosis of EVD.