Gastrointestinal effects associated with Coronavirus Disease 2019 (COVID-19) are highly variable for reasons that are not understood. In this study, we used intestinal organoid-derived cultures ...differentiated from primary human specimens as a model to examine interindividual variability. Infection of intestinal organoids derived from different donors with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) resulted in orders of magnitude differences in virus replication in small intestinal and colonic organoid-derived monolayers. Susceptibility to infection correlated with angiotensin I converting enzyme 2 (ACE2) expression level and was independent of donor demographic or clinical features. ACE2 transcript levels in cell culture matched the amount of ACE2 in primary tissue, indicating that this feature of the intestinal epithelium is retained in the organoids. Longitudinal transcriptomics of organoid-derived monolayers identified a delayed yet robust interferon signature, the magnitude of which corresponded to the degree of SARS-CoV-2 infection. Interestingly, virus with the Omicron variant spike (S) protein infected the organoids with the highest infectivity, suggesting increased tropism of the virus for intestinal tissue. These results suggest that heterogeneity in SARS-CoV-2 replication in intestinal tissues results from differences in ACE2 levels, which may underlie variable patient outcomes.
Methylobacterium extorquens strains are the best-studied methylotrophic model system, and their metabolism of single carbon compounds has been studied for over 50 years. Here we develop a new system ...for high-throughput batch culture of M. extorquens in microtiter plates by jointly optimizing the properties of the organism, the growth media and the culturing system. After removing cellulose synthase genes in M. extorquens strains AM1 and PA1 to prevent biofilm formation, we found that currently available lab automation equipment, integrated and managed by open source software, makes possible reliable estimates of the exponential growth rate. Using this system, we developed an optimized growth medium for M. extorquens using response surface methodologies. We found that media that used EDTA as a metal chelator inhibited growth and led to inconsistent culture conditions. In contrast, the new medium we developed with a PIPES buffer and metals chelated by citrate allowed for fast and more consistent growth rates. This new Methylobacterium PIPES ('MP') medium was also robust to large deviations in its component ingredients which avoided batch effects from experiments that used media prepared at different times. MP medium allows for faster and more consistent growth than other media used for M. extorquens.
Despite mounting evidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) engagement with immune cells, most express little, if any, of the canonical receptor of SARS-CoV-2, ...angiotensin-converting enzyme 2 (ACE2). Here, using a myeloid cell receptor-focused ectopic expression screen, we identified several C-type lectins (DC-SIGN, L-SIGN, LSECtin, ASGR1, and CLEC10A) and Tweety family member 2 (TTYH2) as glycan-dependent binding partners of the SARS-CoV-2 spike. Except for TTYH2, these molecules primarily interacted with spike via regions outside of the receptor-binding domain. Single-cell RNA sequencing analysis of pulmonary cells from individuals with coronavirus disease 2019 (COVID-19) indicated predominant expression of these molecules on myeloid cells. Although these receptors do not support active replication of SARS-CoV-2, their engagement with the virus induced robust proinflammatory responses in myeloid cells that correlated with COVID-19 severity. We also generated a bispecific anti-spike nanobody that not only blocked ACE2-mediated infection but also the myeloid receptor-mediated proinflammatory responses. Our findings suggest that SARS-CoV-2-myeloid receptor interactions promote immune hyperactivation, which represents potential targets for COVID-19 therapy.
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•C-type lectins and TTYH2 are myeloid cell-interacting partners of SARS-CoV-2 spike•C-type lectins interact with spike largely through regions outside of the RBD•Myeloid receptors promote SARS-CoV-2 proinflammatory responses but not infection•A bispecific nanobody blocked SARS-CoV-2 infection and inflammatory responses
Most immune cells express little, if any, of the canonical SARS-CoV-2 receptor, ACE2. Lu et al. report that C-type lectins and TTYH2 act as SARS-CoV-2 myeloid cell-interacting partners that trigger immune hyperactivation but not infection. These findings raise the possibility that these virus-myeloid cell interactions are directly involved in COVID-19 immunopathogenesis and could be targeted for COVID-19 therapy.
Historically, the evolution of bats has been analyzed using a small number of genetic loci for many species or many genetic loci for a few species. Here we present a phylogeny of 18 bat species, each ...of which is represented in 1,107 orthologous gene alignments used to build the tree. We generated a transcriptome sequence of Hypsignathus monstrosus, the African hammer-headed bat, and additional transcriptome sequence for Rousettus aegyptiacus, the Egyptian fruit bat. We then combined these data with existing genomic and transcriptomic data from 16 other bat species. In the analysis of such datasets, there is no clear consensus on the most reliable computational methods for the curation of quality multiple sequence alignments since these public datasets represent multiple investigators and methods, including different source materials (chromosomal DNA or expressed RNA). Here we lay out a systematic analysis of parameters and produce an advanced pipeline for curating orthologous gene alignments from combined transcriptomic and genomic data, including a software package: the Mismatching Isoform eXon Remover (MIXR). Using this method, we created alignments of 11,677 bat genes, 1,107 of which contain orthologs from all 18 species. Using the orthologous gene alignments created, we assessed bat phylogeny and also performed a holistic analysis of positive selection acting in bat genomes. We found that 181 genes have been subject to positive natural selection. This list is dominated by genes involved in immune responses and genes involved in the production of collagens.
Epidemic RNA viruses seem to arise year after year leading to countless infections and devastating disease. SARS-CoV-2 is the most recent of these viruses, but there will undoubtedly be more to come. ...While effective SARS-CoV-2 vaccines are being deployed, one approach that is still missing is effective antivirals that can be used at the onset of infections and therefore prevent pandemics. Here, we screened FDA-approved compounds against SARS-CoV-2. We found that atovaquone, a pyrimidine biosynthesis inhibitor, is able to reduce SARS-CoV-2 infection in human lung cells. In addition, we found that berberine chloride, a plant-based compound used in holistic medicine, was able to inhibit SARS-CoV-2 infection in cells through direct interaction with the virion. Taken together, these studies highlight potential avenues of antiviral development to block emerging viruses. Such proactive approaches, conducted well before the next pandemic, will be essential to have drugs ready for when the next emerging virus hits.
Understanding antibody responses to SARS-CoV-2 is indispensable for the development of containment measures to overcome the current COVID-19 pandemic. Recent studies showed that serum from ...convalescent patients can display variable neutralization capacities. Still, it remains unclear whether there are specific signatures that can be used to predict neutralization. Here, we performed a detailed analysis of sera from a cohort of 101 recovered healthcare workers and we addressed their SARS-CoV-2 antibody response by ELISA against SARS-CoV-2 Spike receptor binding domain and nucleoprotein. Both ELISA methods detected sustained levels of serum IgG against both antigens. Yet, the majority of individuals from our cohort generated antibodies with low neutralization capacity and only 6% showed high neutralizing titers against both authentic SARS-CoV-2 virus and the Spike pseudotyped virus. Interestingly, higher neutralizing sera correlate with detection of -IgG, IgM and IgA antibodies against both antigens, while individuals with positive IgG alone showed poor neutralization response. These results suggest that having a broader repertoire of antibodies may contribute to more potent SARS-CoV-2 neutralization. Altogether, our work provides a cross sectional snapshot of the SARS-CoV-2 neutralizing antibody response in recovered healthcare workers and provides preliminary evidence that possessing multiple antibody isotypes can play an important role in predicting SARS-CoV-2 neutralization.
Biological factors that influence the host range and spillover of Ebola virus (EBOV) and other filoviruses remain enigmatic. While filoviruses infect diverse mammalian cell lines, we report that ...cells from African straw-colored fruit bats (Eidolon helvum) are refractory to EBOV infection. This could be explained by a single amino acid change in the filovirus receptor, NPC1, which greatly reduces the affinity of EBOV-NPC1 interaction. We found signatures of positive selection in bat NPC1 concentrated at the virus-receptor interface, with the strongest signal at the same residue that controls EBOV infection in Eidolon helvum cells. Our work identifies NPC1 as a genetic determinant of filovirus susceptibility in bats, and suggests that some NPC1 variations reflect host adaptations to reduce filovirus replication and virulence. A single viral mutation afforded escape from receptor control, revealing a pathway for compensatory viral evolution and a potential avenue for expansion of filovirus host range in nature.
In an increasingly interconnected world, the exposure and subsequent spread of emergent viruses has become inevitable. This is particularly true for
(
) mosquito-vectored viruses, whose range has ...increased over the past decade from tropical to temperate regions. However, it is unclear if all populations of
mosquitoes in temperate New York City are able to successfully replicate and transmit arboviruses. To answer this question, we reared
mosquitoes living in a temperate climate from three locations in New York City. We first sequenced the salivary antiviral protein D7 from individual mosquitoes in each population and found single nucleotide variants that are both shared and unique for each
population. We then fed each population chikungunya virus (CHIKV) via an artificial blood meal. All three mosquito populations could be infected with CHIKV, yet viral titers differed between populations at 7 days post infection. Moreover, we found that these mosquitoes could transmit CHIKV to mice, and that virus RNA reached the saliva as early as two days post infection. Upon sequencing of the saliva CHIKV genomic RNA, we found mutations at sites correlated with increased transmission and virulence. These studies show that NYC
populations can be infected with and transmit CHIKV, CHIKV is able to evolve in these mosquitoes, and that host salivary factors display population-specific diversity. Taken together, these studies highlight the need to study how distinct mosquito populations control viral infections, both at the virus and host level.
The mechanisms underlying Zika virus (ZIKV)-related microcephaly and other neurodevelopment defects remain poorly understood. Here, we describe the derivation and characterization, including ...single-cell RNA-seq, of neocortical and spinal cord neuroepithelial stem (NES) cells to model early human neurodevelopment and ZIKV-related neuropathogenesis. By analyzing human NES cells, organotypic fetal brain slices, and a ZIKV-infected micrencephalic brain, we show that ZIKV infects both neocortical and spinal NES cells as well as their fetal homolog, radial glial cells (RGCs), causing disrupted mitoses, supernumerary centrosomes, structural disorganization, and cell death. ZIKV infection of NES cells and RGCs causes centrosomal depletion and mitochondrial sequestration of phospho-TBK1 during mitosis. We also found that nucleoside analogs inhibit ZIKV replication in NES cells, protecting them from ZIKV-induced pTBK1 relocalization and cell death. We established a model system of human neural stem cells to reveal cellular and molecular mechanisms underlying neurodevelopmental defects associated with ZIKV infection and its potential treatment.
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•Derivation of human neocortical and spinal cord neuroepithelial stem (NES) cells•Zika virus (ZIKV) infects NES cells and radial glia, impairing mitosis and survival•ZIKV induces mitochondrial sequestration of centrosomal phospho-TBK1•Nucleoside analogs inhibit ZIKV replication, protecting NES cells from cell death
Onorati et al. establish neuroepithelial stem (NES) cells as a model for studying human neurodevelopment and ZIKV-induced microcephaly. Together with analyses in human brain slices and microcephalic human fetal tissue, they find that ZIKV predominantly infects NES and radial glial cells, reveal a pivotal role for pTBK1, and find that nucleoside analogs inhibit ZIKV replication, protecting NES cells from cell death.
Methylobacterium extorquens strains are the best-studied methylotrophic model system, and their metabolism of single carbon compounds has been studied for over 50 years. Here we develop a new system ...for high-throughput batch culture of M. extorquens in microtiter plates by jointly optimizing the properties of the organism, the growth media and the culturing system. After removing cellulose synthase genes in M. extorquens strains AM1 and PA1 to prevent biofilm formation, we found that currently available lab automation equipment, integrated and managed by open source software, makes possible reliable estimates of the exponential growth rate. Using this system, we developed an optimized growth medium for M. extorquens using response surface methodologies. We found that media that used EDTA as a metal chelator inhibited growth and led to inconsistent culture conditions. In contrast, the new medium we developed with a PIPES buffer and metals chelated by citrate allowed for fast and more consistent growth rates. This new Methylobacterium PIPES ('MP') medium was also robust to large deviations in its component ingredients which avoided batch effects from experiments that used media prepared at different times. MP medium allows for faster and more consistent growth than other media used for M. extorquens.
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