Biology subjects

Dijkman, R.

Publications and source records attributed to Dijkman, R..

24 records · Page 2Linked to original sources

bioRxiv2020

Efficient inactivation of SARS-CoV-2 by WHO-recommended hand rub formulations and alcohols

The recent emergence of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing COVID-19 is a major burden for health care systems worldwide. It is important to address if the current infection control instructions based on active ingredients are sufficient. We therefore determined the virucidal activity of two alcohol-based hand rub solutions for hand disinfection recommended by the World Health Organization (WHO), as well as commercially available alcohols. Efficient SARS-CoV-2 inactivation was demonstrated for all tested alcohol-based disinfectants. These findings show the successful inactivation of SARS-CoV-2 for the first time and provide confidence in its use for the control of COVID-19. ImportanceThe current COVID-19 outbreak puts a huge burden on the worlds health care systems. Without effective therapeutics or vaccines being available, effective hygiene measure are of utmost importance to prevent viral spreading. It is therefore crucial to evaluate current infection control strategies against SARS-CoV-2. We show the inactivation of the novel coronavirus for the first time and endorse the importance of disinfectant-based hand hygiene to reduce SARS-CoV-2 transmission.

microbiology↗

bioRxiv2020

LY6E impairs coronavirus fusion and confers immune control of viral disease

Zoonotic coronaviruses (CoVs) are significant threats to global health, as exemplified by the recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)1. Host immune responses to CoV are complex and regulated in part through antiviral interferons. However, the interferon-stimulated gene products that inhibit CoV are not well characterized2. Here, we show that interferon-inducible lymphocyte antigen 6 complex, locus E (LY6E) potently restricts cellular infection by multiple CoVs, including SARS-CoV, SARS-CoV-2, and Middle East respiratory syndrome coronavirus (MERS-CoV). Mechanistic studies revealed that LY6E inhibits CoV entry into cells by interfering with spike protein-mediated membrane fusion. Importantly, mice lacking Ly6e in hematopoietic cells were highly susceptible to murine CoV infection. Exacerbated viral pathogenesis in Ly6e knockout mice was accompanied by loss of hepatic and splenic immune cells and reduction in global antiviral gene pathways. Accordingly, we found that Ly6e directly protects primary B cells and dendritic cells from murine CoV infection. Our results demonstrate that LY6E is a critical antiviral immune effector that controls CoV infection and pathogenesis. These findings advance our understanding of immune-mediated control of CoV in vitro and in vivo, knowledge that could help inform strategies to combat infection by emerging CoV.

microbiology↗

bioRxiv2020

Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform

Reverse genetics has been an indispensable tool revolutionising our insights into viral pathogenesis and vaccine development. Large RNA virus genomes, such as from Coronaviruses, are cumbersome to clone and to manipulate in E. coli hosts due to size and occasional instability1-3. Therefore, an alternative rapid and robust reverse genetics platform for RNA viruses would benefit the research community. Here we show the full functionality of a yeast-based synthetic genomics platform for the genetic reconstruction of diverse RNA viruses, including members of the Coronaviridae, Flaviviridae and Paramyxoviridae families. Viral subgenomic fragments were generated using viral isolates, cloned viral DNA, clinical samples, or synthetic DNA, and reassembled in one step in Saccharomyces cerevisiae using transformation associated recombination (TAR) cloning to maintain the genome as a yeast artificial chromosome (YAC). T7-RNA polymerase has been used to generate infectious RNA, which was then used to rescue viable virus. Based on this platform we have been able to engineer and resurrect chemically-synthetized clones of the recent epidemic SARS-CoV-24 in only a week after receipt of the synthetic DNA fragments. The technical advance we describe here allows to rapidly responding to emerging viruses as it enables the generation and functional characterization of evolving RNA virus variants - in real-time - during an outbreak.

microbiology↗

bioRxiv2019

Establishment of primary transgenic human airway epithelial cell cultures to study respiratory virus-host interactions

Primary human airway epithelial cell (hAEC) cultures represent a universal platform to propagate respiratory viruses and characterize their host interactions in authentic target cells. To further elucidate specific interactions between human respiratory viruses and important host factors in airway epithelium, it is important to make hAEC cultures amenable to genetic modification. However, the short and finite lifespan of primary cells in cell culture creates a bottleneck for the genetic modification of these cultures. In the current study, we show that the incorporation of the Rho-associated protein kinase (ROCK) inhibitor (Y-27632) during cell propagation extends the life span of primary human cells in vitro and thereby facilitates the incorporation of lentivirus-based expression systems. Using fluorescent reporters for FACS-based sorting, we generated homogenously fluorescent hAEC cultures that differentiate normally after lentiviral transduction. As proof-of-principle, we demonstrate that host gene expression can be modulated post-differentiation via inducible short hairpin (sh)RNA-mediated knockdown. Importantly, functional characterization of these transgenic hAEC cultures with exogenous poly(I:C), as a proxy for virus infection, demonstrates that such modifications do not influence the host innate immune response. Moreover, the propagation kinetics of both human coronavirus 229E (HCoV-229E) and human respiratory syncytial virus (RSV) were not affected. Combined, these results validate our newly established protocol for the genetic modification of hAEC cultures thereby unlocking a unique potential for detailed molecular characterization of virus - host interactions in human respiratory epithelium.

microbiology↗

bioRxiv2018

Determination of host cell proteins constituting the molecular microenvironment of coronavirus replicase complexes by proximity-labeling

Positive-sense RNA viruses hijack intracellular membranes that provide niches for viral RNA synthesis and a platform for interactions with host proteins. However, little is known about host factors at the interface between replicase complexes and the host cytoplasm. We engineered a biotin ligase into a coronaviral replication/transcription complex (RTC) and identified >500 host proteins constituting the RTC microenvironment. siRNA-silencing of each RTC-proximal host factor demonstrated importance of vesicular trafficking pathways, ubiquitin-dependent and autophagy-related processes, and translation initiation factors. Notably, detection of translation initiation factors at the RTC was instrumental to visualize and demonstrate active translation proximal to replication complexes of several coronaviruses.\n\nCollectively, we establish a spatial link between viral RNA synthesis and diverse host factors of unprecedented breadth. Our data may serve as a paradigm for other positive-strand RNA viruses and provide a starting point for a comprehensive analysis of critical virus-host interactions that represent targets for therapeutic intervention.

microbiology↗

bioRxiv2018

Assessment of the zoonotic potential of the ruminant-associated influenza D virus

Influenza viruses are notorious pathogens that frequently cross the species barrier with often severe consequences for both animal and human health. In 2011, a novel member of the Orthomyxoviridae family, Influenza D virus (IDV), was identified in the respiratory tract of pigs with influenza-like symptoms and subsequently also in cattle, a species that previously never was associated with influenza virus infection. Epidemiological surveys among livestock demonstrated that IDV is worldwide distributed among ruminants, but the most striking observation is the detection of IDV-directed antibodies among humans with occupational exposure to livestock. As a first step toward identifying the zoonotic potential of the newly emerging IDV we determined the replication kinetics and cell tropism at the primary site of replication using an in vitro respiratory epithelium model of humans. The inoculation of IDV on human airway epithelial cell (hAEC) cultures revealed efficient replication kinetics and apical progeny virus release of IDV at different body temperatures. Intriguingly, the replication characteristics of IDV revealed many similarities to the human-associated Influenza C virus, including the predominant cell tropism for ciliated cells. Moreover, analysis of the host response during IDV infection revealed only a pronounced upregulation of Type III interferon (IFN) transcripts. Nevertheless, viral progeny virus is replication competent and can be efficiently sub-passaged in hAEC cultures from different donors. Highlighting, that there is no intrinsic impairment of IDV replication within the human respiratory epithelium and might explain why IDV-directed antibodies can be detected among humans with occupational exposure to livestock.\n\nImportanceInfluenza viruses are notorious pathogens that frequently cross the species barrier with often severe consequences for both animal and human health. In 2011, a novel member of the Orthomyxoviridae family, Influenza D virus (IDV), was identified among pigs with influenza-like symptoms and subsequently also in cattle. IDV infections in humans have not yet been described, although IDV-directed antibodies have been found among people with occupational exposure to livestock. This observation suggests a possible spillover from livestock to humans. Using an in vitro human respiratory epithelium model we demonstrate there is no inherent restriction for IDV to replicate within the human respiratory epithelium and this might explain why IDV-directed antibodies are detected among humans with occupational exposure to livestock.

microbiology↗