Biology subjects

Chandran, K.

Publications and source records attributed to Chandran, K..

At least 37 records · Page 2Linked to original sources

bioRxiv2022

Divergent microbial structure still results in convergent microbial function during arrested anaerobic digestion of food waste at different hydraulic retention times

In this study, two arrested anaerobic digestion bioreactors fed with food waste operated under different hydraulic retention times (HRTs) exhibited long-term stable volatile fatty acid (VFA) production performance including similar total yields (p = .085) with propionic acid (PA) being the most abundant VFA. Meta-omics analysis revealed distinct microbial structures (p = .02) at the two HRTs while there were no differences in potential and extant functionality as indicated by the whole-genome and whole-transcriptome sequencing, respectively. The highest potential (relative abundance of DNA sequence reads) and extant (relative abundance of mRNA sequence reads) functionality corresponded with PA production compared to other acids. The most abundant genus Prevotella produced PA mainly through the acryloyl-CoA pathway. Based on our results, the mechanistic basis for the similar VFA production performance observed under the HRTs tested lies in the community-level redundancy in convergent acidification functions and pathways, rather than trends in community structure.

microbiology↗

bioRxiv2022

Two point mutations in protocadherin-1 disrupt Andes hantavirus recognition and afford protection against lethal infection

Andes virus and Sin Nombre virus are the etiologic agents of severe hantavirus cardiopulmonary syndrome (HCPS) in the Americas for which no FDA-approved countermeasures are available. Protocadherin-1 (PCDH1), a cadherin-superfamily protein recently identified as a critical host factor for ANDV and SNV, represents a new antiviral target; however, its precise role remains to be elucidated. Here, we used computational and experimental approaches to delineate the binding surface of the ANDV glycoprotein complex on PCDH1s first extracellular cadherin repeat domain. Strikingly, a single amino acid residue in this PCDH1 surface influenced the host species-specificity of SNV glycoprotein-PCDH1 interaction and cell entry. Mutation of this, and a neighboring residue, substantially protected Syrian hamsters from pulmonary disease and death caused by ANDV. We conclude that PCDH1 is a bona fide entry receptor for ANDV and SNV whose direct interaction with hantavirus glycoproteins could be targeted to develop new interventions against HCPS.

microbiology↗

bioRxiv2021

Reovirus infection is regulated by NPC1 and endosomal cholesterol homeostasis

Cholesterol homeostasis is required for the replication of many viruses, including Ebola virus, hepatitis C virus, and human immunodeficiency virus-1. Niemann-Pick C1 (NPC1) is an endosomal-lysosomal membrane protein involved in cholesterol trafficking from late endosomes and lysosomes to the endoplasmic reticulum. We identified NPC1 in CRISPR and RNA interference screens as a putative host factor for infection by mammalian orthoreovirus (reovirus). Following internalization via clathrin-mediated endocytosis, the reovirus outer capsid is proteolytically removed, the endosomal membrane is disrupted, and the viral core is released into the cytoplasm where viral transcription, genome replication, and assembly take place. We found that reovirus infection is significantly impaired in cells lacking NPC1, but infection is restored by treatment of cells with hydroxypropyl-{beta}-cyclodextrin, which binds and solubilizes cholesterol. Absence of NPC1 did not dampen infection by infectious subvirion particles, which are reovirus disassembly intermediates that bypass the endocytic pathway for infection of target cells. NPC1 is not required for reovirus attachment to the plasma membrane, internalization into cells, or uncoating within endosomes. Instead, NPC1 is required for delivery of transcriptionally active reovirus core particles into the cytoplasm. These findings suggest that cholesterol homeostasis, ensured by NPC1 transport activity, is required for reovirus penetration into the cytoplasm, pointing to a new function for NPC1 and cholesterol homeostasis in viral infection. Author summaryGenetic screens are useful strategies to identify host factors required for viral infection. NPC1 was identified in independent CRISPR and RNA interference screens as a putative host factor required for reovirus replication. We discovered that NPC1-mediated cholesterol transport is dispensable for reovirus attachment, internalization, and disassembly but required for penetration of the viral disassembly intermediate from late endosomes into the cytoplasm. These findings pinpoint an essential function for cholesterol in the entry of reovirus and raise the possibility that cholesterol homeostasis regulates the entry of other viruses that penetrate late endosomes to initiate replication.

cell biology↗

bioRxiv2021

Glycerol-driven Denitratation: Process Kinetics, Microbial Ecology, and Operational Controls

Denitratation, the selective reduction of nitrate to nitrite, is a novel process when coupled with anaerobic ammonium oxidation (anammox) could achieve resource-efficient biological nitrogen removal of ammonium- and nitrate-laden waste streams. Using a fundamentally-based, first principles approach, this study optimized a stoichiometrically-limited, glycerol-driven denitratation process and characterized mechanisms supporting nitrite accumulation with results that aligned with expectations. Glycerol supported selective nitrate reduction to nitrite and near-complete nitrate conversion, indicating its viability in a denitratation system. Glycerol-supported specific rates of nitrate reduction (135.3 mg-N/g-VSS/h) were at least one order of magnitude greater than specific rates of nitrite reduction (14.9 mg-N/g-VSS/h), potentially resulting in transient nitrite accumulation and indicating glycerols superiority over other organic carbon sources in denitratation systems. pH and ORP inflection points in nitrogen transformation assays corresponded to maximum nitrite accumulation, indicating operational setpoints to prevent further nitrite reduction. Denitratation conditions supported enrichment of Thauera sp. as the dominant genus. Stoichiometric limitation of influent organic carbon, coupled with differential nitrate and nitrite reduction kinetics, optimized operational controls, and a distinctively enriched microbial ecology, was identified as causal in glycerol-driven denitratation.

bioengineering↗

bioRxiv2021

Genotype-specific Features Reduce the Susceptibility of South American Yellow Fever Virus Strains to Vaccine-Induced Antibodies

The resurgence of yellow fever in South America has prompted mitigation through vaccination against the etiologic agent, yellow fever virus (YFV). Current vaccines are based on a virulent African isolate, and their capacity to induce neutralizing antibodies against the vaccine strain is widely used as a surrogate for protection. However, the sensitivity of genetically distinct South American strains to vaccine-induced antibodies is unknown. Here, we show that antiviral potency of the polyclonal antibody response in both U.S. and Brazilian vaccinees is attenuated against an emergent Brazilian strain. This reduction was attributable to genetic changes at two sites in the central domain II of the glycoprotein E, including the acquisition of an N-linked glycosylation site, which are unique to and shared among most South American YFV strains. Our findings call for a reevaluation of current approaches to YFV immunological surveillance in South America and suggest approaches for designing updated vaccines.

microbiology↗

bioRxiv2021

A combination of RBD and NTD neutralizing antibodies limits the generation of SARS-CoV-2 spike neutralization-escape mutants

Most known SARS-CoV-2 neutralizing antibodies (nAbs), including those approved by the FDA for emergency use, inhibit viral infection by targeting the receptor-binding domain (RBD) of the spike (S) protein. Variants of concern (VOC) carrying mutations in the RBD or other regions of S reduce the effectiveness of many nAbs and vaccines by evading neutralization. Therefore, therapies that are less susceptible to resistance are urgently needed. Here, we characterized the memory B-cell repertoire of COVID-19 convalescent donors and analyzed their RBD and non-RBD nAbs. We found that many of the non-RBD-targeting nAbs were specific to the N-terminal domain (NTD). Using neutralization assays with authentic SARS-CoV-2 and a recombinant vesicular stomatitis virus carrying SARS-CoV-2 S protein (rVSV-SARS2), we defined a panel of potent RBD and NTD nAbs. Next, we used a combination of neutralization-escape rVSV-SARS2 mutants and a yeast display library of RBD mutants to map their epitopes. The most potent RBD nAb competed with hACE2 binding and targeted an epitope that includes residue F490. The most potent NTD nAb epitope included Y145, K150 and W152. As seen with some of the natural VOC, the neutralization potencies of COVID-19 convalescent sera were reduced by 4-16-fold against rVSV-SARS2 bearing Y145D, K150E or W152R spike mutations. Moreover, we found that combining RBD and NTD nAbs modestly enhanced their neutralization potential. Notably, the same combination of RBD and NTD nAbs limited the development of neutralization-escape mutants in vitro, suggesting such a strategy may have higher efficacy and utility for mitigating the emergence of VOC. ImportanceThe US FDA has issued emergency use authorizations (EUAs) for multiple investigational monoclonal antibody (mAb) therapies for the treatment of mild to moderate COVID-19. These mAb therapeutics are solely targeting the receptor binding domain of the SARS-CoV-2 spike protein. However, the N-terminal domain of the spike protein also carries crucial neutralizing epitopes. Here, we show that key mutations in the N-terminal domain can reduce the neutralizing capacity of convalescent COVID-19 sera. We report that a combination of two neutralizing antibodies targeting the receptor binding and N-terminal domains may have higher efficacy and is beneficial to combat the emergence of virus variants.

microbiology↗

bioRxiv2021

MAVERICC: Efficient marker-free rescue of vaccinia virus recombinants by in vitro CRISPR-Cas9 engineering

Vaccinia virus (VACV)-based vectors are in extensive use as vaccines and cancer immunotherapies. VACV engineering has traditionally relied on homologous recombination between a parental viral genome and a transgene-bearing transfer plasmid, a highly inefficient process that necessitates the use of a selection or screening marker to isolate recombinants. Recent extensions of this approach have sought to enhance the recovery of transgene-bearing viruses through the use of CRISPR-Cas9 engineering to cleave the viral genome in infected cells. However, these methods do not completely eliminate the generation of WT viral progeny and thus continue to require multiple rounds of viral propagation and plaque purification. Here, we describe MAVERICC (marker-free vaccinia virus engineering of recombinants through in vitro CRISPR/Cas9 cleavage), a new strategy to engineer recombinant VACVs in a manner that overcomes current limitations. MAVERICC also leverages the CRISPR/Cas9 system but requires no markers and yields essentially pure preparations of the desired recombinants in a single step. We used this approach to rapidly introduce point mutations, insertions, and deletions at multiple locations in the VACV genome, both singly and in combination. The efficiency and versatility of MAVERICC make it an ideal choice for generating mutants and mutant libraries at arbitrarily selected locations in the viral genome to build complex VACV vectors, effect vector improvements, and facilitate the study of poxvirus biology. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/425447v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@17151cborg.highwire.dtl.DTLVardef@b0f480org.highwire.dtl.DTLVardef@184166corg.highwire.dtl.DTLVardef@dc177d_HPS_FORMAT_FIGEXP M_FIG C_FIG Overview of MAVERICC. Conceptual overview of the approach outlined in this manuscript. To make VACV recombinants, the parental virus is first purified and vDNA is isolated with phenol:chloroform extraction. This purified vDNA is then treated with Cas9 enzyme and sgRNAs that are directed to a specific locus in the VACV genome. The cleaved vDNA is then transfected into FWPV-infected BSC-40 cells along with a transfer amplicon containing an insertion or mutation of interest flanked by homologous sequences. Recombination is allowed to occur for 5-7 days, during which time the cleaved vDNA is healed by the transfer amplicon, thus editing the VACV genome, and packaged into infectious viral particles. Individual plaques are grown up and rVACVs are isolated after a single round of plaque purification. Image was created with Biorender.com.

microbiology↗

bioRxiv2021

Genetic depletion studies define receptor usage by virulent hantaviruses in human endothelial cells

Hantaviruses are a large group of RNA viruses that include known epidemic threats and other agents poised for emergence. Several rodent-borne hantaviruses cause zoonoses accompanied by severe illness and death. However, assessments of zoonotic risk and the development of countermeasures alike are challenged by our limited knowledge of the molecular mechanisms of hantavirus infection, including the identities of cell entry receptors and their roles in influencing viral host range and virulence. Previous work has implicated several cell-surface molecules, most notably {beta}3- and {beta}1-containing integrin heterodimers, decay-accelerating factor (DAF), and the cadherin superfamily protein protocadherin-1 (PCDH1), in hantavirus entry in endothelial cells, the major targets of viral infection in humans. Despite the fact that {beta}3/{beta}1 integrins have been presumed to be the major hantavirus entry receptors for over two decades, rigorous genetic evidence supporting their requirement, and that of DAF as an entry cofactor, is lacking. Here, we used CRISPR/Cas9 engineering to knock out four candidate hantaviral receptors, singly and in combination, in a human endothelial cell line that recapitulates the properties of primary microvascular endothelial cells. PCDH1 loss substantially reduced entry and infection by a subset of hantaviruses endemic to the Americas. In contrast, the loss of {beta}3 integrin, {beta}1 integrin, and/or DAF had little or no effect on entry by any of a large panel of hantaviruses tested. We conclude that the major host molecules necessary for endothelial cell entry by PCDH1-independent hantaviruses remain to be discovered.

microbiology↗

bioRxiv2020

Meta-azotomics of engineered wastewater treatment processes reveals differential contributions of established and novel models of N-cycling

The application of metagenomics and metatranscriptomics to field-scale engineered biological nitrogen removal (BNR) processes revealed a complex N-cycle network (the meta-azotome) therein in terms of microbial structure, potential and extant function. Autotrophic nitrification bore the imprint of well-documented Nitrosomonas and Nitrospira in most systems. However, in select BNR processes, complete ammonia oxidizing bacteria, comammox Nitrospira, unexpectedly contributed more substantially to ammonia oxidation than canonical ammonia oxidizing bacteria, based on metatranscriptomic profiling. Methylotrophic denitrification was distinctly active in methanol-fed reactors but not in glycerol-fed reactors. Interestingly, glycerol metabolism and N-reduction transcript signatures were uncoupled, possibly suggesting the role of other carbon sources in denitrification emanating from glycerol itself or from upstream process reactors. In sum, the meta-azotome of engineered BNR processes revealed both traditional and novel mechanisms of N-cycling. Similar interrogation approaches could potentially inform better design and optimization of wastewater treatment and engineered bioprocesses in general.

systems biology↗

bioRxiv2020

A glycoprotein mutation that emerged during the 2013-2016 Ebola virus epidemic alters proteolysis and accelerates membrane fusion

Genomic surveillance of viral isolates during the 2013-2016 Ebola virus epidemic in Western Africa--the largest and most devastating filovirus outbreak on record--revealed several novel mutations. The responsible strain, named Makona, carries an A to V substitution at position 82 in the glycoprotein (GP), which is associated with enhanced infectivity in vitro. Here, we investigated the mechanistic basis for this enhancement, as well as the interplay between A82V and a T to I substitution at residue 544 of GP, which also modulates infectivity in cell culture. We found that both 82V and 544I destabilize GP with the residue at 544 impacting overall stability, while 82V specifically destabilizes proteolytically cleaved GP. Both residues also promote faster kinetics of lipid mixing of the viral and host membranes in live cells, individually and in tandem, which correlates with faster times to fusion following co-localization with the viral receptor Niemann-Pick C1 (NPC1). Further, GPs bearing 82V are more sensitive to proteolysis by cathepsin L (CatL), a key host factor for viral entry. Intriguingly, CatL processed 82V variant GPs to a novel product of [~]12K size, which we hypothesize corresponds to a form of GP more fully primed for fusion than previously detected. We thus propose a model in which 82V promotes more efficient GP processing by CatL, leading to faster viral fusion kinetics and higher infectivity. ImportanceThe 2013-2016 outbreak of Ebola virus disease in West Africa demonstrated the potential for previously localized outbreaks to turn into regional, or even global, health emergencies. With over 28,000 cases and 11,000 confirmed deaths, this outbreak was over 50 times as large as any previously recorded. This outbreak also afforded the largest ever collection of Ebola virus genomic sequence data, allowing new insights into viral transmission and evolution. Viral mutants arising during the outbreak have attracted attention for their potentially altered patterns of infectivity in cell culture, with potential, if unclear, implications for increased viral spread and/or virulence. Here, we report on the properties of one such mutation in the viral glycoprotein, A82V, and its interplay with a previously described polymorphism at position 544. We show that mutations at both residues promote infection and fusion activation in cells, but that A82V additionally leads to increased infectivity under cathepsin-limited conditions, and the generation of a novel glycoprotein cleavage product.

microbiology↗

bioRxiv2020

The evolutionary history of ACE2 usage within the coronavirus subgenus Sarbecovirus

SARS-CoV-1 and SARS-CoV-2 are not phylogenetically closely related; however, both use the ACE2 receptor in humans for cell entry. This is not a universal sarbecovirus trait; for example, many known sarbecoviruses related to SARS-CoV-1 have two deletions in the receptor binding domain of the spike protein that render them incapable of using human ACE2. Here, we report three sequences of a novel sarbecovirus from Rwanda and Uganda which are phylogenetically intermediate to SARS-CoV-1 and SARS-CoV-2 and demonstrate via in vitro studies that they are also unable to utilize human ACE2. Furthermore, we show that the observed pattern of ACE2 usage among sarbecoviruses is best explained by recombination not of SARS-CoV-2, but of SARS-CoV-1 and its relatives. We show that the lineage that includes SARS-CoV-2 is most likely the ancestral ACE2-using lineage, and that recombination with at least one virus from this group conferred ACE2 usage to the lineage including SARS-CoV-1 at some time in the past. We argue that alternative scenarios such as convergent evolution are much less parsimonious; we show that biogeography and patterns of host tropism support the plausibility of a recombination scenario; and we propose a competitive release hypothesis to explain how this recombination event could have occurred and why it is evolutionarily advantageous. The findings provide important insights into the natural history of ACE2 usage for both SARS-CoV-1 and SARS-CoV-2, and a greater understanding of the evolutionary mechanisms that shape zoonotic potential of coronaviruses. This study also underscores the need for increased surveillance for sarbecoviruses in southwestern China, where most ACE2-using viruses have been found to date, as well as other regions such as Africa, where these viruses have only recently been discovered.

evolutionary biology↗

bioRxiv2020

A replication-competent vesicular stomatitis virus for studies of SARS-CoV-2 spike-mediated cell entry and its inhibition

There is an urgent need for vaccines and therapeutics to prevent and treat COVID-19. Rapid SARS-CoV-2 countermeasure development is contingent on the availability of robust, scalable, and readily deployable surrogate viral assays to screen antiviral humoral responses, and define correlates of immune protection, and to down-select candidate antivirals. Here, we describe a highly infectious recombinant vesicular stomatitis virus bearing the SARS-CoV-2 spike glycoprotein S as its sole entry glycoprotein that closely resembles the authentic agent in its entry-related properties. We show that the neutralizing activities of a large panel of COVID-19 convalescent sera can be assessed in high-throughput fluorescent reporter assay with rVSV-SARS-CoV-2 S and that neutralization of the rVSV and authentic SARS-CoV-2 by spike-specific antibodies in these antisera is highly correlated. Our findings underscore the utility of rVSV-SARS-CoV-2 S for the development of spike-specific vaccines and therapeutics and for mechanistic studies of viral entry and its inhibition.

microbiology↗

bioRxiv2020

Broad sarbecovirus neutralizing antibodies define a key site of vulnerability on the SARS-CoV-2 spike protein

Broadly protective vaccines against known and pre-emergent coronaviruses are urgently needed. Critical to their development is a deeper understanding of cross-neutralizing antibody responses induced by natural human coronavirus (HCoV) infections. Here, we mined the memory B cell repertoire of a convalescent SARS donor and identified 200 SARS-CoV-2 binding antibodies that target multiple conserved sites on the spike (S) protein. A large proportion of the antibodies display high levels of somatic hypermutation and cross-react with circulating HCoVs, suggesting recall of pre-existing memory B cells (MBCs) elicited by prior HCoV infections. Several antibodies potently cross-neutralize SARS-CoV, SARS-CoV-2, and the bat SARS-like virus WIV1 by blocking receptor attachment and inducing S1 shedding. These antibodies represent promising candidates for therapeutic intervention and reveal a new target for the rational design of pan-sarbecovirus vaccines.

immunology↗

bioRxiv2020

Direct intracellular visualization of Ebola virus-receptor interaction by in situ proximity ligation

Ebola virus (EBOV) entry into host cells comprises stepwise and extensive interactions of the sole viral surface glycoprotein GP with multiple host factors. During the intricate process, following virus uptake and trafficking to late endosomal/lysosomal compartments, GP is proteolytically processed to GPCL by the endosomal proteases cathepsin B and L unmasking GPs receptor-binding site. Engagement of GPCL with the universal filoviral intracellular receptor Niemann-Pick C1 (NPC1) eventually culminates in fusion between viral and cellular membranes, cytoplasmic escape of the viral nucleocapsid and subsequent infection. Mechanistic delineation of the indispensable GPCL:NPC1 binding step has been severely hampered by the unavailability of a robust cell-based assay assessing interaction of GPCL with full-length endosomal NPC1. Here, we describe a novel in situ assay to monitor GPCL:NPC1 engagement in intact, infected cells. Visualization of the subcellular localization of binding complexes is based on the principle of DNA-assisted, antibody-mediated proximity ligation. Virus-receptor binding monitored by proximity ligation was contingent on GPs proteolytic cleavage, and was sensitive to perturbations in the GPCL:NPC1 interface. Our assay also specifically decoupled detection of virus-receptor binding from steps post-receptor binding, such as membrane fusion and infection. Testing of multiple FDA-approved small molecule inhibitors revealed that drug treatments inhibited virus entry and GPCL:NPC1 recognition by distinctive mechanisms. Together, here we present a newly established proximity ligation assay, which will allow us to dissect cellular and viral requirements for filovirus-receptor binding, and to delineate the mechanisms of action of inhibitors on filovirus entry in a cell-based system. IMPORTANCEEbola virus causes episodic but increasingly frequent outbreaks of severe disease in Middle Africa, as shown by a currently ongoing outbreak in the Democratic Republic of Congo. Despite considerable effort, FDA-approved anti-filoviral therapeutics or targeted interventions are not available yet. Virus host-cell invasion represents an attractive target for antivirals; however our understanding of the inhibitory mechanisms of novel therapeutics is often hampered by fragmented knowledge of the filovirus-host molecular interactions required for viral infection. To help close this critical knowledge gap, here, we report an in situ assay to monitor binding of the EBOV glycoprotein to its receptor NPC1 in intact, infected cells. We demonstrate that our in situ assay based on proximity ligation represents a powerful tool to delineate receptor-viral glycoprotein interactions. Similar assays can be utilized to examine receptor interactions of diverse viral surface proteins whose studies have been hampered until now by the lack of robust in situ assays.

microbiology↗

bioRxiv2020

A virion-based assay for glycoprotein thermostability reveals key determinants of filovirus entry and its inhibition

Ebola virus (EBOV) entry into cells is mediated by its spike glycoprotein (GP). Following attachment and internalization, virions traffic to late endosomes where GP is cleaved by host cysteine proteases. Cleaved GP then binds its cellular receptor, Niemann-Pick C1. In response to an unknown cellular trigger, GP undergoes conformational rearrangements that drive fusion of viral and endosomal membranes . The temperature-dependent stability (thermostability) of the pre-fusion conformers of Class I viral fusion glycoproteins, including those of filovirus GPs, has provided insights into their propensity to undergo fusion-related rearrangements. However, previously described assays have relied on soluble glycoprotein ectodomains. Here, we developed a simple ELISA-based assay that uses the temperature-dependent loss of conformational epitopes to measure thermostability of GP embedded in viral membranes. The base and glycan cap subdomains of all filovirus GPs tested suffered a concerted loss of pre-fusion conformation at elevated temperatures, but did so at different temperature ranges, indicating virus-specific differences in thermostability. Despite these differences, all of these GPs displayed reduced thermostability upon cleavage to GPCL. Surprisingly, acid pH enhanced, rather than decreased, GP thermostability, suggesting it could enhance viral survival in hostile endo/lysosomal compartments. Finally, we confirmed and extended previous findings that some small-molecule inhibitors of filovirus entry destabilize EBOV GP and uncovered evidence that the most potent inhibitors act through multiple mechanisms. We establish the epitope-loss ELISA as a useful tool for studies of filovirus entry, engineering of GP variants with enhanced stability for use in vaccine development, and discovery of new stability-modulating antivirals. ImportanceThough a vaccine for Ebola virus has been approved by the FDA within the past year, no FDA-approved therapeutics are available to treat infections by Ebola virus or other filoviruses. The development of such countermeasures is challenged by our limited understanding of the mechanism by which Ebola virus enters cells, especially at the final step of membrane fusion. The sole surface-exposed viral protein, GP, mediates key steps in virus entry, including membrane fusion, and undergoes major structural rearrangements during this process. The stability of GP at elevated temperatures (thermostability) can provide insights into its capacity to undergo these structural rearrangements. Here, we describe a new assay that uses GP-specific antibodies to measure GP thermostability under a variety of conditions relevant to viral entry. We show that proteolytic cleavage and acid pH have significant effects on GP thermostability that shed light on their respective roles in viral entry. We also show that the assay can be used to study how small-molecule entry inhibitors affect GP stability. This work provides a simple and readily accessible assay to engineer forms of GP with enhanced stability that could be useful as part of an antiviral vaccine and to discover and improve drugs that act by modulating the stability of GP.

microbiology↗

bioRxiv2019

The generation of plasma cells and CD27-IgD- B cells during Hantavirus infection are associated with distinct pathological findings

Human hantavirus infections can cause hemorrhagic fever with renal syndrome (HFRS), major signs of the disease being thrombocytopenia and transient kidney dysfunction. By a comprehensive and longitudinal study of circulating B cells, we demonstrate that these two pathologies associate with distinct effects on the humoral immune system during HFRS. Low thrombocyte counts strongly associated with an abnormal frequency of plasmablasts in circulation, whereas kidney dysfunction was indicative of an accumulation of CD27- B cells and plasmablasts. Finally, we provide evidence that high levels of extracellular ATP in circulation during HFRS correlates with shedding of surface CD27 on B cells via a metallomatrix proteinase-8-mediated mechanism. Since extracellular ATP is known to regulate kidney function, our study reveals a link between kidney dysfunction and the generation of CD27-IgD- B cells, and a potential molecular target for treatment of the symptomatic phase of HFRS.

immunology↗

bioRxiv2019

Within-host dynamics of virulent viruses in bat reservoirs for emerging zoonotic disease

Bats host virulent zoonotic viruses without experiencing disease. A mechanistic understanding of the impact of bats virus hosting capacities, including uniquely constitutive immune pathways, on cellular-scale viral dynamics is needed to elucidate zoonotic emergence. We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data. Best fit models recapitulated expected immune phenotypes for representative cell lines, supporting robust antiviral defenses in bat cells that correlated with higher estimates for within-host viral propagation rates. In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host. Rapidly-replicating viruses that have evolved with bat immune systems will likely cause enhanced virulence following emergence into secondary hosts with immune systems that diverge from those unique to bats.

ecology↗

bioRxiv2018

Human, Nonhuman Primate, and Bat Cells Are Broadly Susceptible to Tibrovirus Particle Cell Entry

In 2012, the genome of a novel rhabdovirus, Bas-Congo virus, was discovered in the acute-phase serum of a Congolese patient with presumed viral hemorrhagic fever. In the absence of a replicating virus isolate, fulfilling Kochs postulates to determine whether Bas-Congo virus is indeed a human virus and/or pathogen has been impossible. However, experiments with vesiculoviral particles pseudotyped with Bas-Congo glycoprotein suggested that Bas-Congo virus particles can enter cells from multiple animals, including humans. In 2015, genomes of two related viruses, Ekpoma virus 1 and Ekpoma virus 2, were detected in human sera in Nigeria. Isolates could not be obtained. Phylogenetic analyses led to the classification of Bas-Congo virus, Ekpoma virus 1, and Ekpoma virus 2 in the same genus, Tibrovirus, together with five biting midge-borne rhabdoviruses (i.e., Beatrice Hill virus, Bivens Arm virus, Coastal Plains virus, Sweetwater Branch virus, and Tibrogargan virus) not known to infect humans. Using individual recombinant vesiculoviruses expressing the glycoproteins of all eight known tibroviruses and more than 75 cell lines representing different animal species, we demonstrate that the glycoproteins of all tibroviruses can mediate vesiculovirus particle entry into human, bat, nonhuman primate, cotton rat, boa constrictor, and Asian tiger mosquito cells. Using four of five isolated authentic tibroviruses (i.e., Bivens Arm virus, Coastal Plains virus, Sweetwater Branch virus, and Tibrogargan virus), our experiments indicate that many cell types may be partially resistant to tibrovirus replication after virion cell entry. Consequently, experimental data solely obtained from experiments using tibrovirus surrogate systems (e.g., vesiculoviral pseudotypes, recombinant vesiculoviruses) cannot be used to predict whether Bas-Congo virus, or any other tibrovirus, infects humans.

microbiology↗