Sphingosine kinases promote Ebola virus infection and can be targeted to inhibit filoviruses, coronaviruses, and arenaviruses using late endocytic trafficking to enter cells

Entry of enveloped viruses in host cells requires the fusion of the viral and host cell membranes, a process that is facilitated by viral fusion proteins protruding from the viral envelope. For fusion, viral fusion proteins need to be triggered by host factors and for some viruses, such as Ebola virus (EBOV) and Lassa fever virus, this event occurs inside endosomes and/or lysosomes. Consequently, these late-penetrating viruses must be internalized and delivered to entry-conducive intracellular vesicles. Because endocytosis and vesicular trafficking are tightly regulated cellular processes, late penetrating viruses also depend on specific host factors, such as signaling molecules, for efficient viral delivery to the site of fusion, suggesting that these could be targeted for antiviral therapy. In this study, we investigated a role for sphingosine kinases (SKs) in viral entry and found that chemical inhibition of sphingosine kinase 1 (SK1) and/or SK2 and knockdown of SK1 or SK2, inhibited entry of EBOV into host cells. Mechanistically, inhibition of SK1 and/or SK2 prevented EBOV from reaching late-endosomes and lysosomes that are positive for the EBOV receptor, Niemann Pick C1 (NPC1). Furthermore, we present evidence that suggests the trafficking defect caused by SK1/2 inhibition occurs independently of S1P signaling through cell-surface S1PRs. Lastly, we found that chemical inhibition of SKs prevents entry of other late-penetrating viruses, including arenaviruses and coronaviruses, in addition to inhibiting infection by replication competent EBOV and SARS-CoV-2 in Huh7.5 cells. In sum, our results highlight an important role played by SKs in endocytic trafficking which can be targeted to inhibit entry of late-penetrating viruses. SK inhibitors could serve as a starting point for the development of broad-spectrum antiviral therapeutics.

Highly divergent white-tailed deer SARS-CoV-2 with potential deer-to-human transmission

Wildlife reservoirs of SARS-CoV-2 may enable viral adaptation and spillback from animals to humans. In North America, there is evidence of unsustained spillover of SARS-CoV-2 from humans to white-tailed deer (Odocoileus virginianus), but no evidence of transmission from deer to humans. Through a biosurveillance program in Ontario, Canada we identified a new and highly divergent lineage of SARS-CoV-2 in white-tailed deer. This lineage is the most divergent SARS-CoV-2 lineage identified to date, with 76 consensus mutations (including 37 previously associated with non-human animal hosts) and signatures of considerable evolution and transmission within wildlife. Phylogenetic analysis also revealed an epidemiologically linked human case. Together, our findings represent the first clear evidence of sustained evolution of SARS-CoV-2 in white-tailed deer and of deer-to-human transmission.

Identification of a SARS-CoV-2 host metalloproteinase-dependent entry pathway differentially used by SARS-CoV-2 and variants of concern Alpha, Delta, and Omicron

To infect cells, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) binds to angiotensin converting enzyme 2 (ACE2) via its spike glycoprotein (S), delivering its genome upon S-mediated membrane fusion. SARS-CoV-2 uses two distinct entry pathways: 1) a surface, serine protease-dependent or 2) an endosomal, cysteine protease-dependent pathway. In investigating serine protease-independent cell-cell fusion, we found that the matrix metalloproteinases (MMPs), MMP2/9, can activate SARS-CoV-2 S fusion activity, but not that of SARS-CoV-1. Importantly, metalloproteinase activation of SARS-CoV-2 S represents a third entry pathway in cells expressing high MMP levels. This route of entry required cleavage at the S1/S2 junction in viral producer cells and differential processing of variants of concern S dictated its usage. In addition, metalloproteinase inhibitors reduced replicative Alpha infection and abrogated syncytia formation. Finally, we found that the Omicron S exhibit reduced metalloproteinase-dependent fusion and viral entry. Taken together, we identified a MMP2/9-dependent mode of activation of SARS-CoV-2 S. As MMP2/9 are released during inflammation and severe COVID-19, they may play important roles in SARS-CoV-2 S-mediated cytopathic effects, tropism, and disease outcome.

Regional connectivity drove bidirectional transmission of SARS-CoV-2 in the Middle East during travel restrictions

Regional connectivity and land-based travel have been identified as important drivers of SARS-CoV-2 transmission. However, the generalizability of this finding is understudied outside of well-sampled, highly connected regions such as Europe. In this study, we investigated the relative contributions of regional and intercontinental connectivity to the source-sink dynamics of SARS-CoV-2 for Jordan and the wider Middle East. By integrating genomic, epidemiological and travel data we show that the source of introductions into Jordan was dynamic across 2020, shifting from intercontinental seeding from Europe in the early pandemic to more regional seeding for the period travel restrictions were in place. We show that land-based travel, particularly freight transport, drove introduction risk during the period of travel restrictions. Consistently, high regional connectivity and land-based travel also disproportionately drove Jordans export risk to other Middle Eastern countries. Our findings emphasize regional connectedness and land-based travel as drivers of viral transmission in the Middle East. This demonstrates that strategies aiming to stop or slow the spread of viral introductions (including new variants) with travel restrictions need to prioritize risk from land-based travel alongside intercontinental air travel to be effective. HighlightsO_LIRegional connectivity drove SARS-CoV-2 introduction risk in Jordan during the period travel restrictions were in place in genomic and travel data. C_LIO_LILand-based travel rather than air travel disproportionately drove introduction risk during travel restrictions. C_LIO_LIHigh regional connectivity disproportionately drove Jordans export risk, with significant contribution from land-based travel. C_LIO_LIRegional transmission dynamics were underestimated in genomic data due to unrepresentative sampling. C_LI

Arsenal of Nanobodies for Broad-Spectrum Countermeasures against Current and Future SARS-CoV-2 Variants of Concerns

Nanobodies offer several potential advantages over mAbs for the control of SARS-CoV-2. Their ability to access cryptic epitopes conserved across SARS-CoV-2 variants of concern (VoCs) and feasibility to engineer modular, multimeric designs, make these antibody fragments ideal candidates for developing broad-spectrum therapeutics against current and continually emerging SARS-CoV-2 VoCs. Here we describe a diverse collection of 37 anti-SARS-CoV-2 spike glycoprotein nanobodies extensively characterized as both monovalent and IgG Fc-fused bivalent modalities. The panel of nanobodies were shown to have high intrinsic affinity; high thermal, thermodynamic and aerosolization stability; broad subunit/domain specificity and cross-reactivity across many VoCs; wide-ranging epitopic and mechanistic diversity; high and broad in vitro neutralization potencies; and high neutralization efficacies in hamster models of SARS-CoV-2 infection, reducing viral burden by up to six orders of magnitude to below detectable levels. In vivo protection was demonstrated with anti-RBD and previously unreported anti-NTD and anti-S2 nanobodies. This collection of nanobodies provides a therapeutic toolbox from which various cocktails or multi-paratopic formats could be built to tackle current and future SARS-CoV-2 variants and SARS-related viruses. Furthermore, the high aerosol-ability of nanobodies provides the option for effective needle-free delivery through inhalation.

Relative Ratios of Human Seasonal Coronavirus Antibodies Predict the Efficiency of Cross-Neutralization of SARS-CoV-2 Spike Binding to ACE2

BackgroundAntibodies raised against human seasonal coronaviruses (sCoVs), which are responsible for the common cold, are known to cross-react with SARS-CoV-2 antigens. This prompts questions about their protective role against SARS-CoV-2 infections and COVID-19 severity. However, the relationship between sCoV exposure and SARS-CoV-2 correlates of protection are not clearly identified. MethodsWe performed a cross-sectional analysis of cross-reactivity and cross-neutralization to SARS-CoV-2 antigens (S-RBD, S-trimer, N) using pre-pandemic serum from four different groups: pediatrics and adolescents, persons 21 to 70 years of age, older than 70 years of age, and persons living with HCV or HIV. FindingsAntibody cross-reactivity to SARS-CoV-2 antigens varied between 1.6% and 15.3% depending on the cohort and the isotype-antigen pair analyzed. We also show a range of neutralizing activity (0-45%) in serum that interferes with SARS-CoV-2 spike attachment to ACE2. While the abundance of sCoV antibodies did not directly correlate with neutralization, we show that neutralizing activity is rather dependent on relative ratios of IgGs in sera directed to all four sCoV spike proteins. More specifically, we identified antibodies to NL63 and OC43 as being the most important predictors of neutralization. InterpretationOur data support that exposure to sCoVs triggers antibody responses that influence the efficiency of SARS-CoV-2 spike binding to ACE2, and may also impact COVID-19 disease severity through other latent variables. Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSThere is a growing body of evidence showing that within the population there are varying levels of pre-existing immunity to SARS-CoV-2 infection and possibly COVID-19 disease severity. This immunity is believed to be attributable to prior infection by four prevalent seasonal coronaviruses (sCoVs) responsible for the common cold. Pre-existing immunity can be assessed in part by antibodies directed to sCoVs that also cross-react to SARS-CoV-2 antigens. The SARS-CoV-2 spike and, more specifically, the receptor binding domain are the primary targets for neutralizing antibodies. It is unclear if cross-reactive antibodies to SARS-CoV-2 are neutralizing and are also responsible for the broad spectrum of COVID-19 disease severity, from asymptomatic to critical, observed in the infected population. Added-value of this studyHere we carried out a detailed analysis of sCoV prevalence in samples acquired before the pandemic from individuals of various age groups and in people living with HIV and HCV. We then analyzed the frequency of all the different types of antibodies that cross-react to three SARS-CoV-2 antigens. We found a high level of people with cross-reactive antibodies, surprisingly we also detected that some people have antibodies that block the SARS-CoV-2 spike from binding to its human receptor, ACE2. By using machine learning, we were able to accurate predict which individuals can neutralize SARS-CoV-2 spike-ACE2 interactions based on their relative ratios of antibodies against the four sCoVs. Implications of all the available evidenceWe demonstrate that it not absolute levels of sCoVs antibodies that are predictive of neutralization but the relative ratios to all four sCoVs, with NL63 being the most weighted for this prediction. Machine learning also highlighted the existence of latent variables that contribute to the neutralization and that may be related to the type of cellular immune response triggered by the infection to certain sCoVs. This study is one of the first to identify a functional relationship between prior-exposure to sCoV and the establishment of a certain degree of immunity to SARS-CoV-2 by way of a cross-reactive antibody response. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/21260079v3_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@e74392org.highwire.dtl.DTLVardef@1052bd5org.highwire.dtl.DTLVardef@80d88eorg.highwire.dtl.DTLVardef@10976cb_HPS_FORMAT_FIGEXP M_FIG C_FIG

Consistent and High-Frequency Identification of an Intra-Sample Genetic Variant of SARS-CoV-2 with Elevated Fusogenic Properties

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a virus that is continuously evolving. Although its RNA-dependent RNA polymerase exhibits some exonuclease proofreading activity, viral sequence diversity can be produced by replication errors and host factors. A diversity of genetic variants can be observed in the intra-host viral population structure of infected individuals. Most mutations will follow a neutral molecular evolution and wont make significant contributions to variations within and between infected hosts. Herein, we profiled the intra-sample genetic diversity of SARS-CoV-2 variants using high-throughput sequencing datasets from 15,289 infected individuals and infected cell lines. Most of the genetic variations observed, including C->U and G->U, were consistent with errors due to heat-induced DNA damage during sample processing and/or sequencing protocols. Despite high mutational background, we identified recurrent intra-variable positions in the samples analyzed, including several positions at the end of the gene encoding the viral Spike (S) protein. Strikingly, we observed a high-frequency C->A missense mutations resulting in the S protein lacking the last 20 amino acids (S{Delta}20). We found that this truncated S protein undergoes increased processing and increased syncytia formation, presumably due to escaping M protein retention in intracellular compartments. Our findings suggest the emergence of a high-frequency viral sublineage that is not horizontally transmitted but potentially involved in intra-host disease cytopathic effects. IMPORTANCEThe mutation rate and evolution of RNA viruses correlate with viral adaptation. While most mutations do not have significant contributions to viral molecular evolution, some are naturally selected and cause a genetic drift through positive selection. Many recent SARS-CoV-2 variants have been recently described and show phenotypic selection towards more infectious viruses. Our study describes another type of variant that does not contribute to inter-host heterogeneity but rather phenotypic selection toward variants that might have increased cytopathic effects. We identified that a C-terminal truncation of the Spike protein removes an important ER-retention signal, which consequently results in a Spike variant that easily travels through the Golgi toward the plasma membrane in a pre-activated conformation, leading to increased syncytia formation.