Biology subjects

Slonchak, A.

Publications and source records attributed to Slonchak, A..

9 recordsLinked to original sources

bioRxiv2026

Single-cell analysis of West Nile virus infection in brain organoids identified a serine synthesis enzyme as a determinant of viral neural tropism and a druggable antiviral target

The cellular and molecular determinants of human neuroinvasive West Nile virus (WNV) disease are currently poorly defined. Previous studies of WNV neural infection relied on rodent models and immortalised cell lines that diverge from human brain tissue. Herein, we established a reproducible human cerebral organoid model of WNV infection and profiled it by virus-inclusive single-cell RNA sequencing (scRNA-Seq) to obtain an understanding of WNV-host interactions in the context of human neural tissue. We found that WNV showed preferential tropism to progenitor cells, immature neurons, and astrocytes, while the mature neurons were largely resistant to infection. Astrocytes were identified as the principal source of interferon (IFN) and the main hub of IFN-signalling, whereas mature neurons expressed little viral RNA-sensing machinery or STAT1 and responded weakly to infection and to IFN. The differential gene expression analysis between WNV-permissive and nonpermissive cells demonstrated that virus-susceptible cells had high baseline expression of the serine synthesis and one-carbon metabolism genes PHGDH, SHMT2, and MTHFD2, which were not induced by infection. RNA-interference and pharmacological inhibition of phosphoglycerate dehydrogenase (PHGDH) restricted WNV replication, confirming the requirement of this gene for WNV infection. In addition, the FDA-approved drug disulfiram, known to inhibit PHGDH, also reduced viral replication in neural progenitors and cerebral organoids. Overall, these results provide the first comprehensive single-cell-level characterisation of WNV neural infection in human neural tissue. They identify de novo serine synthesis as a metabolic dependency of WNV in human neural cells that can be targeted as a host-directed antiviral therapy.

microbiology↗

bioRxiv2025

Omicron evolution drives increased ciliated cell tropism and dysfunction in nasal epithelia.

SARS-CoV-2 Omicron variants demonstrate greater replication efficiency in the upper respiratory tract than both ancestral virus and Delta variant, potentially due to stronger interactions with motile cilia in human nasal epithelial cells. Despite the emergence of numerous Omicron variants, there has been no comprehensive investigation comparing their interactions with ciliated cells. Here, we use differentiated primary human nasal epithelial cell (NEC) cultures to compare an ancestral isolate (QLD02) to Omicron variants BA.1, BA.5 and XBB. Transcriptomic analysis revealed that BA.5 and XBB infection, unlike BA.1 and QLD02, drives a distinct host response, defined by the profound downregulation of genes essential for cilium assembly and function at 48 hours post-infection. The BA.5/XBB signature was also characterized by the strong upregulation of pro-apoptotic and inflammatory pathways. Immunofluorescence of nucleocapsid-positive cells in NECs showed a significant 13-fold increase for BA.5 relative to QLD02. Additionally, BA.1 and BA.5 exhibited greater tropism for ciliated cells (7.6-fold for BA.1, 9-fold for BA.5) than QLD02. BA.5 and BA.1 infection were associated with elevated apoptosis in both infected and bystander cells, however, the total number of ciliated cells remained unchanged. Consistent with this, BA.5 infection significantly reduced DNAH5 outer dynein arm protein in the cilia, indicating dysfunction of cilia motility rather than overt cilia loss. Overall, we demonstrate a stepwise evolution at the nasal barrier: while enhanced ciliated cell tropism and elevated apoptosis are conserved features of Omicron infection, ciliary dysfunction, evident at both the transcriptional and protein level, is a more pronounced phenotype emerging with BA.5 and preserved in the XBB variant. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/689397v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@11b1b60org.highwire.dtl.DTLVardef@a25e0borg.highwire.dtl.DTLVardef@1cc3904org.highwire.dtl.DTLVardef@b35447_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Characterisation and phylogenetic analysis of Murray Valley encephalitis virus (MVEV) from the Ralph Doherty collection.

Murray Valley encephalitis virus (MVEV) is a zoonotic flavivirus endemic to Australia and Papua New Guinea. A recent outbreak of MVEV has prompted renewed concerns regarding the potential for MVEV to generate disease outbreaks. Currently, nine full length sequences of MVEV are publicly available, divided into four genotypes (G1-G4). Herein, we sequenced MVEV isolates from the Ralph Doherty Virus Collection, a virus bank with Australian field isolates dating between the 1950s-1980s, and determined their phylogenetic relationship with existing isolates to provide insights into virus evolution and genetic diversity. Additionally, we characterised isolates from different genotypes both in vitro using human neuronal cells, and in vivo using C57BL/6J mice, to provide additional insight into MVEV pathogenicity and establish models of MVEV disease that recapitulate MVEV human disease. We found 15 new full length sequences of MVEV, which primarily clustered into the dominant genotype, G1. Additionally, we show MVEV can be lethal and neuroinvasive in C57BL/6J mice, recapitulating histological lesions identified in human infection. Overall, our study contributes significant genomic sequences to the current MVEV database and establishes mouse models of disease and infection which can be used for mechanistic studies and evaluation of new interventions.

microbiology↗

bioRxiv2024

Passage of Zika virus in Rag1-deficient mice selects for unique envelope glycosylation motif mutants that show enhanced replication

N-linked glycosylation of flavivirus envelope proteins is widely viewed as a requirement for optimal folding, processing and/or transit of envelope proteins, and the assembling virons, through the endoplasmic reticulum (ER) and Golgi. Herein we show that serial passage of ZIKVNatal in Rag1-/- mice generated two unique envelope glycan-deficient mutants, ZIKV-V153D and ZIKV-N154D, that, surprisingly, produced titers [~]1 to 2.6 logs higher than the glycosylated parental ZIKVNatal in Vero E6 cells and human brain organoids. RNA-Seq of infected organoids suggested that this increased replication fitness was associated with upregulation of the unfolded protein response (UPR). Cell death, cellular viral RNA and viral protein levels were not significantly affected, arguing that these glycan mutants enjoyed faster ER/Golgi folding, processing, assembly, transit, and virion egress, assisted by an upregulated UPR. Thus, ZIKV envelope N-linked glycosylation is not essential for promoting envelope folding, assembly and transit through the ER/Golgi, as aspartic acid (D) substitutions in the glycosylation motif achieve this with significantly greater efficiency. V153D and N154D mutants have not been employed in flavivirus envelope glycosylation studies. Instead, mutants such as N154A have been used, which may impart unfavorable properties that have a greater impact than the loss of the glycan. ZIKV-V153D and -N154D may avoid this by preserving the surface negative charge provided by the glycan moiety in the parental ZIKVNatal. In Ifnar-/- mice ZIKV-V153D and -N154D showed faster viremia onsets, but reduced viremic periods, than the parental ZIKVNatal, consistent with the contention that these glycans have evolved to delay neutralizing antibody activity. IMPORTANCEStudies seeking to understand the role(s) of N-linked glycosylation of flavivirus envelope proteins often introduce amino acid substitutions that disrupt the glycosylation motif, which in ZIKV has the sequence 153VNDT156. Unfortunately, such substitutions, for instance N154A, may themselves impart unfavorable properties on envelope that have a greater impact than the loss of the glycan moiety. Herein we describe two unique glycosylation motif mutants, ZIKV-V153D and -N154D that were positively selected during passage of ZIKVNatal in Rag1-/- mice. These N154 glycan-deficient viruses produced viral titers up to [~]400 fold higher than the parental ZIKVNatal in Vero cells and in human brain organoids. Such glycans are thus clearly not a requirement for optimal folding and trafficking of ZIKV envelope through the endoplasmic reticulum/Golgi. These results provide new insights into the molecular mechanisms underpinning viral fitness in vitro and in vivo, and also have implications for virus-like-particle vaccine design and production.

microbiology↗

bioRxiv2023

A novel tamanavirus (Flaviviridae) of the European common frog (Rana temporaria) encodes a divergent class 1b XRN1-resistant RNA element.

Flavivirids are small, enveloped, positive-sense RNA viruses from the Flaviviridae family with genomes between [~]9-13kb. Metatranscriptomic analyses of metazoan organisms have revealed a diversity of flavivirus-like or flavivirid viral sequences in fish and marine invertebrate groups. To date, however, no flavivirus-like or flavivirid has been identified in amphibians. To remedy this, we investigated the virome of the European common frog (Rana temporaria) in the United Kingdom, utilising high-throughput sequencing at six catch locations. De novo assembly revealed a coding-complete virus contig of a novel flavivirid [~]11.2kb in length. The virus encodes a single open reading frame of 3456 amino acids and 5 and 3 untranslated regions (UTRs) of 227 and 666nt, respectively. We named this virus Rana tamanavirus (RaTV), as BLASTp analysis of the polyprotein showed the closest relationships to Tamana bat virus (TABV) and Cyclopterus lumpus virus from Pteronotus parnellii and Cyclopterus lumpus, respectively. Phylogenetic analysis of the RaTV polyprotein compared to Flavivirus and Flavivirus-like members indicated that RaTV was sufficiently divergent and basal to the vertebrate Tamanavirus clade. In addition to the Mitcham strain, partial but divergent RaTV, 95.64-97.39% pairwise nucleotide identity, were also obtained from the Poole and Deal samples, indicating that RaTV is widespread in UK frog samples. Bioinformatic analyses of putative secondary structures in the 3'-UTR of RaTV indicated a potential exoribonuclease-resistant RNA (xrRNA) structure identified in flaviviruses and TABV. To examine this biochemically, we conducted an in vitro XRN1 digestion assay showing that RaTV likely forms a divergent but functionally homologous XRN1-resistant xrRNA.

microbiology↗

bioRxiv2023

Choroid plexus defects in Down syndrome brain organoids enhance neurotropism of SARS-CoV-2

Why individuals with Down Syndrome (DS, trisomy 21) are particularly susceptible to SARS CoV-2 induced neuropathology remains largely unclear. Since the choroid plexus (CP) performs important barrier and immune-interface functions, secretes the cerebrospinal fluid and strongly expresses the ACE2 receptor and the chromosome 21 encoded TMPRSS2 protease, we hypothesized that the CP could play a role in establishing SARS-CoV-2 infection in the brain. To investigate the role of the choroid plexus in SARS-CoV-2 central nervous system infection in DS, we established a new type of brain organoid from DS and isogenic euploid control iPSC that consists of a core of appropriately patterned functional cortical neuronal cell types that is surrounded by a patent and functional choroid plexus (CPCOs). Remarkably, DS-CPCOs not only recapitulated abnormal features of DS cortical development but also revealed defects in ciliogenesis and epithelial cell polarity of the developing choroid plexus. We next demonstrate that the choroid plexus layer facilitates SARS-CoV-2 replication and infection of cortical neuronal cells, and that this is increased in DS-CPCOs. We further show that inhibition of TMPRSS2 and Furin activity inhibits SARS-CoV-2 replication in DS CPCOs to the level observed in euploid organoids. We conclude that CPCOs are a useful model for dissecting the role of the choroid plexus in euploid and DS forebrain development and enables screening for therapeutics that can inhibit SARS-CoV-2 induced neuro-pathogenesis.

neuroscience↗

bioRxiv2021

Structural analysis of 3'UTRs in insect flaviviruses reveals novel determinant of sfRNA biogenesis and provides new insights into flavivirus evolution

Insect-specific flaviviruses (ISFs) circulate in nature due to vertical transmission in mosquitoes and do not infect vertebrates. ISFs include two distinct lineages - classical ISFs (cISFs) that evolved independently and dual host associated ISFs (dISFs) that are proposed to diverge from mosquito-borne flaviviruses (MBFs). Compared to pathogenic flaviviruses, ISFs are relatively poorly studied, and their molecular biology remains largely unexplored. In this study we focused on the characterisation of ISF 3UTRs and their ability to produce subgenomic flaviviral RNAs - noncoding viral RNAs that are known as important determinants of transmission and replication of pathogenetic flaviviruses. We demonstrated that cISFs and dISFs produce sfRNAs by employing a highly conserved mechanism of resistance to degradation by the cellular 5-3 exoribonuclease XRN1. We determined the secondary structures of complete 3UTRs and experimentally identified structured RNA elements that resist degradation by XRN1 (xrRNAs) in divergent representatives of cISF and dISF clades. We discovered a novel class of xrRNAs in dISFs and identified structurally divergent xrRNA in Anopheles-associated cISFs. Phylogenetic analyses based on sequences and secondary structures of xrRNAs and complete 3UTRs reveal that xrRNAs of cISFs and MBFs/dISFs evolved from a common xrRNA ancestor similar to the xrRNA of Anopheles-associated cISFs. Additionally, we found that duplications of xrRNAs occurred independently in ISF and MBF clades. Using ISF mutants deficient in the production of sfRNAs, we found that individual sfRNAs of ISFs have redundant functions. We conclude that duplicated xrRNAs were selected in the evolution of flaviviruses to ensure that sfRNA is produced if one of the xrRNAs lose XRN1 resistance due to mutations or misfolding.

microbiology↗

bioRxiv2021

Zika virus noncoding RNA cooperates with the viral protein NS5 to inhibit STAT1 phosphorylation and facilitate viral pathogenesis

Zika virus (ZIKV) is a re-emerging pathogenic flavivirus, which causes microcephaly in infants and poses a continuing threat to public health. ZIKV, like all other flaviviruses, produces highly abundant noncoding RNA known as subgenomic flaviviral RNA (sfRNA). Herein we utilized wild-type and mutant ZIKV defective in production of sfRNA to elucidate for the first time how production of sfRNA affects all aspects of ZIKV pathogenesis. We found that in mouse pregnancy model of infection sfRNA is required for trans-placental dissemination of ZIKV and subsequent infection of fetal brain. Using human brain organoids, we showed that sfRNA promotes apoptosis of neural progenitor cells leading to profound cytopathicity and disintegration of organoids. We also found by transcriptome profiling and gene network analysis that in infected human placental cells sfRNA inhibits multiple antiviral pathways and promotes apoptosis with STAT1 identified as a key shared factor linking these two interconnected sfRNA activities. We further showed for the first time that sfRNA inhibits phosphorylation and nuclear translocation of STAT1 by a novel mechanism which involves binding to and stabilizing viral protein NS5. This allows accumulation of NS5 at the levels required for efficient inhibition of STAT1 phosphorylation. Thus, we elucidated the molecular mechanism by which ZIKV sfRNA exerts its functions in vertebrate hosts and discovered a co-operation between viral noncoding RNA and a viral protein as a novel strategy employed by viruses to counteract antiviral responses.

microbiology↗

bioRxiv2020

Systems vaccinology analysis of a recombinant vaccinia-based vector reveals diverse innate immune signatures at the injection site

Poxvirus systems have been extensively used as vaccine vectors. Herein a systems vaccinology analysis of intramuscular injection sites provides detailed insights into host innate immune responses, as well as expression of vector and recombinant immunogen genes, after vaccination with a new multiplication defective, vaccinia-based vector, Sementis Copenhagen Vector. Chikungunya and Zika virus immunogen mRNA and protein expression was associated with necrosing skeletal muscle cells surrounded by mixed cellular infiltrates. Adjuvant signatures at 12 hours post-vaccination were dominated by TLR3, 4 and 9, STING, MAVS, PKR and the inflammasome. Th1 cytokine signatures were dominated by IFN{gamma}, TNF and IL1{beta}, and chemokine signatures by CCL5 and CXCL12. Multiple signatures associated with dendritic cell stimulation were evident. By day seven, vaccine transcripts were absent, and cell death, neutrophil, macrophage and inflammation annotations had abated. No compelling arthritis signatures were identified. Such innate systems vaccinology approaches should inform refinements in poxvirus-based vector design.

systems biology↗