Biology subjects

Matthews, D. A.

Publications and source records attributed to Matthews, D. A..

At least 19 recordsLinked to original sources

bioRxiv2025

Identification of a novel structural motif and overexpression of key transcripts elucidated in Adenovirus 10

Adenoviruses are widely used as vectors for subunit vaccines and oncolytic therapies. Efficient vectors must infect target cells and deliver therapeutic transgenes at high levels. Species D adenoviruses, such as human adenovirus type 10 (HAdV-D10), are promising candidates due to low seroprevalence in humans. Here, we present the cryo-electron microscopy structure of the HAdV-D10 capsid alongside transcriptomic profiling of infected cells to inform vector development. The fiber shaft, essential for cell entry, was resolved at 4.6 [A], revealing a previously uncharacterized umbrella motif. Viral transcript analysis using an ORF-centric pipeline uncovered key differences from HAdV-C5, including abundant expression of a transcript encoding a protein equivalent to mature protein VII. These findings highlight the importance of detailed vector characterization prior to clinical translation and support the advancement of HAdV-D10 as a next-generation platform for gene delivery and vaccine development.

microbiology↗

bioRxiv2025

Hypoxia inducible factors regulate Pneumovirus replication by enhancing innate immune sensing

The immune mechanisms responsible for protection and pathogenesis in pneumoviral infection are not well defined. We demonstrated that pharmacological activation of the hypoxic inducible factor (HIF) signalling axis using Daprodustat limited viral replication through enhanced immune signalling. Transcriptomic analysis revealed HIF augmented activation of innate immune response genes, including interferon-stimulated gene 15 (Isg15), in the lung and spleen of mice infected with pneumonia virus of mice (PVM). In human respiratory syncytial virus (hRSV) infected airway epithelial cells, Daprodustat inhibited viral replication and enhanced ISG15 expression in a HIF-dependent manner. Importantly, inhibition of type I interferon signalling or the RIG-I sensing pathway abrogated the antiviral activity of HIF. Moreover, Daprodustat increased interferon signalling in response to viral RNA, suggesting that HIF inhibits pneumovirus replication through enhancing viral RNA sensing. Mechanistically, Daprodustat reduced N6-methyladenosine modification of viral RNA through upregulation of RNA demethylases, promoting detection by innate immune sensors. This study highlights the intricate interplay between hypoxia and antiviral immunity and offers valuable insights into pneumovirus-host interactions and potential therapeutic interventions. SIGNIFICANCE STATEMENTThis study explores the role of the hypoxic inducible factor (HIF) signalling pathway in limiting pneumoviral infections, such as respiratory syncytial virus (hRSV) and pneumonia virus of mice (PVM). Using Daprodustat, a clinically approved activator of HIF signalling we demonstrated that stimulation of this pathway enhanced innate immune signalling and limited viral replication. Transcriptomic analysis showed that HIF promoted innate immune response genes in mice infected with PVM and human airway epithelial cells infected with hRSV. Importantly, inhibition of type I interferon signalling or the RIG-I sensing pathway abrogated the antiviral activity of HIF. We uncovered a role for HIF to regulate the levels of N6-methyladenosine modification of viral RNA transcripts, resulting in the increased activation of nucleic acid sensing.

microbiology↗

bioRxiv2025

A direct RNA-seq-based EBV Latency Transcriptome Offers Insights into the Biogenesis of EBV Gene Products.

Epstein-Barr virus (EBV) ubiquitously infects humans, establishing lifelong persistence in B cells. In vitro, EBV-infected B cells can establish a lymphoblastoid cell line (LCL). EBVs transcripts in LCLs (Latency III) produce six nuclear proteins (EBNAs), two latency membrane proteins (LMPs) and various microRNAs and putative long non-coding RNAs (BARTs). The BART and EBNA transcription units are characterised by extensive alternative splicing. We generated LCLs with B95-8 EBV-BACs, including one engineered with "barcodes" in the first and last repeat of internal repeat 1 (IR1), and analysed their EBV transcriptomes using long-read nanopore direct RNA-seq. Our pipeline ensures appropriate mapping of the W promoter (Wp) 5 exon, and corrects W1-W2 exon counts that misalign to IR1. This suggests that splicing across IR1 largely includes all W exons, and that Wp-derived transcripts more frequently encode the EBNA-LP start codon than Cp transcripts. Analysis identified a short variant of exon W2 and a novel polyA site before EBNA2, provided insights into BHRF1 miRNA processing and suggested co-ordination between polyA and splice site usage, although improved read depth and integrity are required to confirm this. The BAC region disrupts the integrity of BART transcripts through premature polyadenylation and cryptic splice sites in the hygromycin expression cassette. Finally, a few transcripts extended across established gene boundaries, running from EBNA, to BART to LMP2 gene regions, sometimes including novel exons between EBNA1 and the BART promoter. We have produced an EBV annotation based on these findings to help others better characterise EBV transcriptomes in future. Data SummaryScripts (and the shell scripts used to combine commands into the pipeline), and guidance in their usage are available on Github (github.com/robertewhite/ebv-transcriptomics-tools). All of the RNA-seq raw data and analyses relevant to this study are available from the EMBL Nucleotide Archive Study accession PRJEB83447 (read files ERR14129300-303), or from the authors website, ebv.org.uk. Processed and analysed data is presented in Excel format in Supplementary tables ST1-8, and the intermediate data processing conducted in excel is linked from the front page of ebv.org.uk, alongside the scripts, raw reads and updated B95-8-BAC and prototype EBV transcriptome annotation (gff3) files. Impact statementThis article showcases the potential of direct RNA-seq to characterise complex transcriptomes like Epstein-Barr virus, highlights specific challenges of interpreting RNA-seq data, and presents tools to solve specific challenges of mapping RNA-seq reads to EBV exons. Biologically, the data analysis identifies several new aspects of Epstein-Barr virus transcription, offering insights into miRNA proteins, and identifies idiosyncrasies of the transcriptome of the widely used B95-8 BAC that will inform studies using this system. Finally we provide an improved annotation for EBV RNA-seq studies.

microbiology↗

bioRxiv2024

SARS-CoV-2 infection enhancement by Amphotericin B: Implications for disease management

Severe coronavirus disease 2019 (COVID-19) patients who require hospitalisation are at high risk of invasive pulmonary mucormycosis. Amphotericin B (AmB), which is the first line therapy for invasive pulmonary mucormycosis, has been shown to promote or inhibit replication of a spectrum of viruses. In this study, we first predicted that AmB and Nystatin had strong interactions with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins using in silico screening, indicative of drugs with potential therapeutic activity against this virus. Subsequently, we investigated the impact of AmB, Nystatin, Natamycin, Fluconazole and Caspofungin on SARS-CoV-2 infection and replication in vitro. Results showed that AmB and Nystatin actually increased SARS-CoV-2 replication in Vero E6, Calu-3 and Huh7 cells. At optimal concentrations, AmB and Nystatin increase SARS-CoV-2 replication by up to 100- and 10-fold in Vero E6 and Calu-3 cells, respectively. The other antifungals tested had no impact on SARS-CoV-2 infection in vitro. Drug kinetic studies indicate that AmB enhances SARS-CoV-2 infection by promoting viral entry into cells. Additionally, knockdown of genes encoding for interferon-induced transmembrane (IFITM) proteins 1, 2, and 3 suggests AmB enhances SARS-CoV-2 cell entry by overcoming the antiviral effect of the IFITM3 protein. This study further elucidates the role of IFITM3 in viral entry and highlights the potential dangers of treating COVID-19 patients, with invasive pulmonary mucormycosis, using AmB. ImportanceAmB and Nystatin are common treatments for fungal infections but were predicted to strongly interact with SARS-CoV-2 proteins, indicating their potential modulation or inhibition against the virus. However, our tests revealed that these antifungals, in fact, enhance SARS-CoV-2 infection by facilitating viral entry into cells. The magnitude of enhancement could be up to 10- or 100-fold depending on cell lines used. These findings indicate that AmB and Nystatin have the potential to enhance disease when given to patients infected with SARS-CoV-2 and therefore should not be used for treatment of fungal infections in active COVID-19 cases.

microbiology↗

bioRxiv2023

Hypoxia inducible factors inhibit respiratory syncytial virus infection by modulation of nucleolin expression

Respiratory syncytial virus (RSV) is a global healthcare problem, causing respiratory illness in young children and elderly individuals. However, our knowledge of the host pathways that define susceptibility to RSV infection and disease severity is limited. Hypoxia inducible factors (HIFs) define cellular metabolic processes in response to low oxygen and are recognised to play a key role in regulating inflammatory responses in the lower respiratory tract. We demonstrate a role for hypoxia inducible factors (HIFs) to suppress RSV entry and RNA replication. We show that hypoxia and HIF prolyl-hydroxylase inhibitors significantly reduce expression of the RSV entry receptor nucleolin and inhibit viral driven cell-cell fusion. We identify a HIF regulated microRNA, miR-494, that regulates nucleolin expression. In RSV-infected mice, treatment with the clinically approved HIF prolyl-hydroxylase inhibitor, Daprodustat, reduced the level of infectious virus and infiltrating monocytes and neutrophils in the lung. This study highlights a role for HIF-signalling to limit multiple aspects of RSV infection and associated inflammation and informs future therapeutic approaches for treating this respiratory pathogen.

molecular biology↗

bioRxiv2023

Recombinant SARS-CoV-2 lacking initiating and internal methionine codons within ORF10 is attenuated in vivo

SARS-CoV-2 has been proposed to encode ORF10 as the 3 terminal gene in the viral genome. However, the potential role and even existence of a functional ORF10 product has been the subject of debate. There are significant structural features in the viral genomic RNA that could, by themselves, explain the retention of the ORF10 nucleotide sequences without the need for a functional protein product. To explore this question further we made two recombinant viruses, firstly a control virus (WT) based on the genome sequence of the original Wuhan isolate and with the inclusion of the early D614G mutation in the Spike protein. We also made a second virus, identical to WT except for two additional changes that replaced the initiating ORF10 start codon and an internal methionine codon for stop codons (ORF10KO). Here we show that the two viruses have apparently identical growth kinetics in a VeroE6 cell line that over expresses TMPRSS2 (VTN cells). However, in A549 cells over expressing ACE2 and TMPRSS2 (A549-AT cells) the ORF10KO virus appears to have a small growth rate advantage. Growth competition experiments were used whereby the two viruses were mixed, passaged in either VTN or A549-AT cells and the resulting output virus was sequenced. We found that in VTN cells the WT virus quickly dominated whereas in the A549-AT cells the ORF10KO virus dominated. We then used a hamster model of SARS-CoV-2 infection and determined that the ORF10KO virus has attenuated pathogenicity (as measured by weight loss). We found an almost 10-fold reduction in viral titre in the lower respiratory tract for ORF10KO vs WT. In contrast, the WT and ORF10KO viruses had similar titres in the upper respiratory tract. Sequencing of viral RNA in the lungs of hamsters infected with ORF10KO virus revealed that this virus frequently reverts to WT. Our data suggests that the retention of a functional ORF10 sequence is highly desirable for SARS-CoV-2 infection of hamsters and affects the viruss ability to propagate in the lower respiratory tract.

microbiology↗

bioRxiv2023

SARS-CoV-2 NSP12 associates with the TRiC complex and the P323L substitution is a host adaption.

SARS-CoV-2 emerged into the human population in late 2019 and human to human transmission has dominated the evolutionary landscape and driven the selection of different lineages. The first major change that resulted in increased transmission was the D614G substitution in the spike protein. This was accompanied by the P323L substitution in the viral RNA dependent RNA polymerase (RdRp) (NSP12). Together, with D614G these changes are the root of the predominant global SARS-CoV-2 landscape. Here, we found that NSP12 formed an interactome with cellular proteins. The functioning of NSP12 was dependent on the T-complex protein Ring Complex, a molecular chaperone. In contrast, there was differential association between NSP12 variants and components of a phosphatase complex (PP2/PP2A and STRN3). Virus expressing NSP12L323 was less sensitive to perturbations in PP2A and supports the paradigm that ongoing genotype to phenotype adaptation of SARS- CoV-2 in humans is not exclusively restricted to the spike protein.

microbiology↗

bioRxiv2022

The SARS-CoV-2 protein ORF3c is a mitochondrial modulator of innate immunity

The SARS-CoV-2 genome encodes a multitude of accessory proteins. Using comparative genomic approaches, an additional accessory protein, ORF3c, has been predicted to be encoded within the ORF3a sgmRNA. Expression of ORF3c during infection has been confirmed independently by ribosome profiling. Despite ORF3c also being present in the 2002-2003 SARS-CoV, its function has remained unexplored. Here we show that ORF3c localises to mitochondria during infection, where it inhibits innate immunity by restricting IFN-{beta} production, but not NF-{kappa}B activation or JAK-STAT signalling downstream of type I IFN stimulation. We find that ORF3c acts after stimulation with cytoplasmic RNA helicases RIG-I or MDA5 or adaptor protein MAVS, but not after TRIF, TBK1 or phospho-IRF3 stimulation. ORF3c co-immunoprecipitates with the antiviral proteins MAVS and PGAM5 and induces MAVS cleavage by caspase-3. Together, these data provide insight into an uncharacterised mechanism of innate immune evasion by this important human pathogen.

microbiology↗

bioRxiv2022

Development of SARS-CoV-2 replicons for the ancestral virus and variant of concern Delta for antiviral screening.

SARS-CoV-2 is the aetiologic agent of COVID-19 and the associated ongoing pandemic. As the pandemic has progressed, Variants of Concern (VOC) have emerged with lineage defining mutations. Using a SARS-CoV-2 reverse genetic system, based on transformation associated recombination in yeast, a series of replicons were produced for the ancestral Wuhan virus and the SARS-CoV-2 VOC Delta in which different combinations of the Spike, membrane, ORF6 and ORF7a coding sequences were replaced with sequences encoding the selectable marker puromycin N-acetyl transferase and reporter proteins (Renilla luciferase, mNeonGreen and mScarlet). Replicon RNAs were replication competent in African green monkey kidney (Vero E6) derived cells and a range of human cell lines, with a Vero E6 cell line expressing ACE2 and TMPRSS2 showing much higher transfection efficiency and overall levels of Renilla luciferase activity. The replicons could be used for transient gene expression studies, but cell populations that stably maintained the replicons could not be propagated. Replication of the transiently expressed replicon RNA genomes was sensitive to remedesivir, providing a system to dissect the mechanism of action of antiviral compounds.

microbiology↗

bioRxiv2022

Allostery in STAT3 Variant D170A is Mediated by a Rigid Core

Signal Transducer and Activator of Transcription 3 (STAT3) plays a crucial role in cancer development and thus is a viable target for cancer treatment. STAT3 functions as a dimer mediated by phosphorylation of the SRC-homology 2 (SH2) domain, a key target for therapeutic drugs. While great efforts have been employed towards the development of compounds that directly target the SH2 domain, no compound has yet been approved by the FDA due to a lack of specificity and pharmacologic efficacy. Studies have shown that allosteric regulation of SH2 via the coiled-coil domain (CCD) is an alternative drug design strategy. Several CCD effectors have been shown to modulate SH2 binding and affinity, and at the time of writing at least one drug candidate has entered phase I clinical trials. However, the mechanism for SH2 regulation via CCD is poorly understood. Here, we investigate structural and dynamic features of STAT3 and compare the wild type to the reduced function variant D170A in order to delineate mechanistic differences and propose allosteric pathways. Molecular dynamics simulations were employed to explore conformational space of STAT3 and the variant, followed by structural, conformation, and dynamic analysis. The trajectories explored show distinctive conformational changes in the SH2 domain for the D170A variant, indicating long range allosteric effects. Multiple analyses provide evidence for long range communication pathways between the two STAT3 domains, which seem to be mediated by a rigid core which connects the CCD and SH2 domains via the linker domain (LD) and transmits conformational changes through a network of short-range interactions. The proposed allosteric mechanism provides new insight into the understanding of intramolecular signaling in STAT3 and potential pharmaceutical control of STAT3 specificity and activity. Author SummaryIn all living organisms, the proliferation and survival of cells are regulated by various proteins. Signal Transducers and Activators of Transcription 3(STAT3) protein is one of the important proteins. However, the abnormal regulation of these proteins will lead to cancer cell. The constitutive activation of STAT3 has been linked to several types of solid tumors, leukemia, and lymphomas. Consequently, STAT3 proteins have been a key target for cancer therapy. SH2(SRC-homology 2) domain is the key interaction site, great efforts have been attributed to target SH2 domain, which specificity has been a major challenge in drug discovery. Research showing regulation of SH2 domain via CCD has opened a new path for drug discovery, however is challenged by poor understanding of the allosteric mechanism. Here, we show that CCD regulates SH2 conformation via a rigid backbone. The perturbations in CCD is transmitted through -helix to the rigid core that concert the movement of CCD and LD (Link domain), leading to structural changes in the SH2 domain. The present findings provide allosteric mechanism with atomistic details underlying the regulation of CCD to SH2 domain in STAT3 protein. Which allows informed drug design targeting CCD for desired downstream effect on SH2 domain and the overall STAT3 function.

biophysics↗

bioRxiv2021

Rapid selection of P323L in the SARS-CoV-2 polymerase (NSP12) in humans and non-human primate models and confers a large plaque phenotype

The mutational landscape of SARS-CoV-2 varies at both the dominant viral genome sequence and minor genomic variant population. An early change associated with transmissibility was the D614G substitution in the spike protein. This appeared to be accompanied by a P323L substitution in the viral polymerase (NSP12), but this latter change was not under strong selective pressure. Investigation of P323L/D614G changes in the human population showed rapid emergence during the containment phase and early surge phase of wave 1 in the UK. This rapid substitution was from minor genomic variants to become part of the dominant viral genome sequence. A rapid emergence of 323L but not 614G was observed in a non-human primate model of COVID-19 using a starting virus with P323 and D614 in the dominant genome sequence and 323L and 614G in the minor variant population. In cell culture, a recombinant virus with 323L in NSP12 had a larger plaque size than the same recombinant virus with P323. These data suggest that it may be possible to predict the emergence of a new variant based on tracking the distribution and frequency of minor variant genomes at a population level, rather than just focusing on providing information on the dominant viral genome sequence e.g., consensus level reporting. The ability to predict an emerging variant of SARS-CoV-2 in the global landscape may aid in the evaluation of medical countermeasures and non-pharmaceutical interventions.

microbiology↗

bioRxiv2021

Direct RNA sequencing of Respiratory Syncytial Virus infected human cells generates a detailed overview of RSV polycistronic mRNA and transcript abundance

To characterize species of viral mRNA transcripts generated during respiratory syncytial virus (RSV) infection, human fibroblast-like MRC5 lung cells were infected with subgroup A RSV for 6, 16 and 24 hours. Total RNA was harvested and polyadenylated mRNA was enriched and sequenced by direct RNA sequencing on an Oxford nanopore device. This yielded over 150,000 direct mRNA transcript reads which were mapped to the viral genome and analysed to determine relative mRNA levels of viral genes using our in-house ORF-centric pipeline. We were also able to examine frequencies with which polycistronic readthrough mRNAs were generated and to assess the length of the polyadenylated tails for each group of transcripts. We show that there is a general but non-linear decline in gene transcript abundance across the viral genome, as predicted by the model of RSV gene transcription. However, the decline in transcript abundance is not consistent. We show that the polyadenylate tails generated by the viral polymerase are similar in length to those generated by the host cells polyadenylation machinery and broadly declined in length for most transcripts as infection progressed. Finally, we observed that the steady state abundance of transcripts with very short polyadenylate tails is much less for N, SH and G transcripts compared to NS1, NS2, P, M, F and M2 which may reflect differences in mRNA stability and/or translation rates.

microbiology↗

bioRxiv2021

Nanopore ReCappable Sequencing maps SARS-CoV-2 5' capping sites and provides new insights into the structure of sgRNAs

The SARS-CoV-2 virus has a complex transcriptome characterised by multiple, nested sub genomic RNAs used to express structural and accessory proteins. Long-read sequencing technologies such as nanopore direct RNA sequencing can recover full-length transcripts, greatly simplifying the assembly of structurally complex RNAs. However, these techniques do not detect the 5' cap, thus preventing reliable identification and quantification of full-length, coding transcript models. Here we used Nanopore ReCappable Sequencing (NRCeq), a new technique that can identify capped full-length RNAs, to assemble a complete annotation of SARS-CoV-2 sgRNAs and annotate the location of capping sites across the viral genome. We obtained robust estimates of sgRNA expression across cell lines and viral isolates and identified novel canonical and non-canonical sgRNAs, including one that uses a previously un-annotated leader-to-body junction site. The data generated in this work constitute a useful resource for the scientific community and provide important insights into the mechanisms that regulate the transcription of SARS-CoV-2 sgRNAs.

genomics↗

bioRxiv2021

Structure and mechanism of SARS-CoV-2 Spike N679-V687 deletion variant elucidate cell-type specific evolution of viral fitness

As the global burden of SARS-CoV-2 infections escalates, so does the evolution of viral variants which is of particular concern due to their potential for increased transmissibility and pathology. In addition to this entrenched variant diversity in circulation, RNA viruses can also display genetic diversity within single infected hosts with co-existing viral variants evolving differently in distinct cell types. The BriS{Delta} variant, originally identified as a viral subpopulation by passaging SARS-CoV-2 isolate hCoV-19/England/02/2020, comprises in the spike glycoprotein an eight amino-acid deletion encompassing the furin recognition motif and S1/S2 cleavage site. Here, we elucidate the structure, function and molecular dynamics of this variant spike providing mechanistic insight into how the deletion correlates to viral cell tropism, ACE2 receptor binding and infectivity of this SARS-CoV-2 variant. Moreover, our study reveals long-range allosteric communication between functional regions within the spike that differ in wild-type and deletion variant. Our results support a view of SARS-CoV-2 probing multiple evolutionary trajectories in distinct cell types within the same infected host.

biochemistry↗

bioRxiv2021

Identification and quantification of SARS-CoV-2 leader subgenomic mRNA gene junctions in nasopharyngeal samples shows phasic transcription in animal models of COVID-19 and aberrant pattens in humans

IntroductionSARS-CoV-2 has a complex strategy for the transcription of viral subgenomic mRNAs (sgmRNAs), which are targets for nucleic acid diagnostics. Each of these sgRNAs has a unique 5 sequence, the leader-transcriptional regulatory sequence gene junction (leader-TRS-junction), that can be identified using sequencing. ResultsHigh resolution sequencing has been used to investigate the biology of SARS-CoV-2 and the host response in cell culture models and from clinical samples. LeTRS, a bioinformatics tool, was developed to identify leader-TRS-junctions and be used as a proxy to quantify sgmRNAs for understanding virus biology. This was tested on published datasets and clinical samples from patients and longitudinal samples from animal models with COVID-19. DiscussionLeTRS identified known leader-TRS-junctions and identified novel species that were common across different species. The data indicated multi-phasic abundance of sgmRNAs in two different animal models, with spikes in sgmRNA abundance reflected in human samples, and therefore has implications for transmission models and nucleic acid-based diagnostics.

microbiology↗

bioRxiv2020

Amplicon and metagenomic analysis of MERS-CoV and the microbiome in patients with severe Middle East respiratory syndrome (MERS)

Middle East Respiratory Syndrome coronavirus (MERS-CoV) is a zoonotic infection that emerged in the Middle East in 2012. Symptoms range from mild to severe and include both respiratory and gastrointestinal illnesses. The virus is mainly present in camel populations with occasional spill overs into humans. The severity of infection in humans is influenced by numerous factors and similar to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underlying health complications can play a major role. Currently, MERS-CoV and SARS-CoV-2 are co-incident in the Middle East and a rapid way is required of sequencing MERS-CoV to derive genotype information for molecular epidemiology. Additionally, complicating factors in MERS-CoV infections are co-infections that require clinical management. The ability to rapidly characterise these infections would be advantageous. To rapidly sequence MERS-CoV, we developed an amplicon-based approach coupled to Oxford Nanopore long read length sequencing. The advantage of this approach is that insertions and deletions can be identified - which are the major drivers of genotype change in coronaviruses. This and a metagenomic approach were evaluated on clinical samples from patients with MERS. The data illustrated that whole genome or near whole genome information on MERS-CoV could be rapidly obtained. This approach provided data on both consensus genomes and the presence of minor variants including deletion mutants. Whereas, the metagenomic analysis provided information of the background microbiome.

molecular biology↗

bioRxiv2020

Sequential infection with influenza A virus followed by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to more severe disease and encephalitis in a mouse model of COVID-19.

COVID-19 is a spectrum of clinical symptoms in humans caused by infection with SARS-CoV-2, a recently emerged coronavirus that rapidly caused a pandemic. Coalescence of this virus with seasonal respiratory viruses, particularly influenza virus is a global health concern. To investigate this, transgenic mice expressing the human ACE2 receptor driven by the epithelial cell cytokeratin-18 gene promoter (K18-hACE2) were first infected with IAV followed by SARS-CoV-2. The host response and effect on virus biology was compared to K18-hACE2 mice infected with IAV or SARS-CoV-2 only. Infection of mice with each individual virus resulted in a disease phenotype compared to control mice. Although SARS-CoV-2 RNA synthesis appeared significantly reduced in the sequentially infected mice, they exhibited more rapid weight loss, more severe lung damage and a prolongation of the innate response compared to singly infected or control mice. The sequential infection also exacerbated the extrapulmonary encephalitic manifestations associated with SARS-CoV-2 infection. Conversely, prior infection with a commercially available, multivalent live-attenuated influenza vaccine (Fluenz tetra) elicited the same reduction in SARS-CoV-2 RNA synthesis albeit without the associated increase in disease severity. This suggests that the innate immune response stimulated by infection with IAV is responsible for the observed inhibition of SARS-CoV-2, however, infection with attenuated, apathogenic influenza vaccine does not result in an aberrant immune response and enhanced disease severity. Taken together, the data suggest that the concept of twinfection is deleterious and mitigation steps should be instituted as part of a comprehensive public health response to the COVID-19 pandemic.

microbiology↗

bioRxiv2020

The furin cleavage site of SARS-CoV-2 spike protein is a key determinant for transmission due to enhanced replication in airway cells.

SARS-CoV-2 enters cells via its spike glycoprotein which must be cleaved sequentially at the S1/S2, then the S2 cleavage sites (CS) to mediate membrane fusion. SARS-CoV-2 has a unique polybasic insertion at the S1/S2 CS, which we demonstrate can be cleaved by furin. Using lentiviral pseudotypes and a cell-culture adapted SARS-CoV-2 virus with a S1/S2 deletion, we show that the polybasic insertion is selected for in lung cells and primary human airway epithelial cultures but selected against in Vero E6, a cell line used for passaging SARS-CoV-2. We find this selective advantage depends on expression of the cell surface protease, TMPRSS2, that allows virus entry independent of endosomes thus avoiding antiviral IFITM proteins. SARS-CoV-2 virus lacking the S1/S2 furin CS was shed to lower titres from infected ferrets and was not transmitted to cohoused sentinel animals. Thus, the polybasic CS is a key determinant for efficient SARS-CoV-2 transmission.

microbiology↗