Biology subjects

Radoshitzky, S.

Publications and source records attributed to Radoshitzky, S..

3 recordsLinked to original sources

bioRxiv2020

Molecular Detection of SARS-CoV-2 in Formalin Fixed Paraffin Embedded Specimens

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of human coronavirus disease 2019 (COVID-19), emerged in Wuhan, China in December 2019. The virus rapidly spread globally, resulting in a public-health crisis including more than one million cases and tens of thousands of deaths. Here, we describe the identification and evaluation of commercially available reagents and assays for the molecular detection of SARS-CoV-2 in infected formalin fixed paraffin embedded (FFPE) cell pellets. We identified a suitable rabbit polyclonal anti-SARS-CoV spike protein antibody and a mouse monoclonal anti-SARS-CoV nucleocapsid protein (NP) antibody for cross detection of the respective SARS-CoV-2 proteins by immunohistochemistry (IHC) and immunofluorescence assay (IFA). Next, we established RNAscope in situ hybridization (ISH) to detect SARS-CoV-2 RNA. Furthermore, we established a multiplex fluorescence ISH (mFISH) to detect positive-sense SARS-CoV-2 RNA and negative-sense SARS-CoV-2 RNA (a replicative intermediate indicating viral replication). Finally, we developed a dual staining assay using IHC and ISH to detect SARS-CoV-2 antigen and RNA in the same FFPE section. These reagents and assays will accelerate COVID-19 pathogenesis studies in humans and in COVID-19 animal models.

microbiology↗

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↗

bioRxiv2017

DDX3 is exploited by Arenaviruses to suppress type I interferons and favor their replication

Several arenaviruses cause hemorrhagic fever (HF) diseases that are associated with high morbidity and mortality in humans. Accordingly, HF arenaviruses have been listed as top-priority emerging diseases for which countermeasures are urgently needed. Because arenavirus nucleoprotein (NP) plays critical roles in both virus multiplication and immune-evasion, we used an unbiased proteomic approach to identify NP-interacting proteins in human cells. DDX3, a DEAD-box ATP-dependent-RNA-helicase, interacted with NP in both NP-transfected and virus-infected cells. Importantly, DDX3 deficiency compromised the propagation of both Old and New World arenaviruses, including the HF arenaviruses Lassa and Junin viruses. The DDX3 role in promoting arenavirus multiplication correlated with both a previously un-recognized DDX3 contribution to type I interferon suppression in arenavirus infected cells and a positive effect of DDX3 on viral RNA synthesis. Our results uncover novel mechanisms used by arenavirus to exploit the host machinery and subvert immunity, singling out DDX3 as a potential host target for developing new therapies against highly pathogenic arenaviruses.\n\nAUTHOR SUMMARYArenaviruses include severe clinical pathogens causing hemorrhagic fevers and have been recently incorporated by the World Health Organization in a list of critical emerging diseases for which additional research and identification of clinical targets is urgently required. A better understanding of how viral proteins interact with host cellular factors to favor arenavirus multiplication can illuminate novel pipelines on therapeutic strategies. Here we demonstrated that the ATP-dependent RNA helicase DDX3 interacted with the arenavirus nucleoprotein, which displays fundamental functions in different steps of the viral-cycle. Our work also revealed an unexpected new biology on the role that DDX3 might play during viral infections. In sharp contrast to previous studies showing DDX3 enhancement of IFN-I induction, we demonstrated that DDX3 suppressed IFN-I production at late time points after arenavirus infection, contributing to a DDX3 pro-viral effect. We also showed that early after infection, DDX3 pro-viral role was IFN-I independent and was mediated by DDX3 facilitation of viral RNA synthesis without affecting RNA translation. Altogether, our study established DDX3 as a critical host interacting partner of the arenavirus nucleoprotein and demonstrated two previously unrecognized DDX3-dependent strategies by which these deadly viruses exploit the host cellular machinery and suppress immunity.

microbiology↗