Biology subjects

Wider, M.

Publications and source records attributed to Wider, M..

7 recordsLinked to original sources

bioRxiv2022

The spike gene is a major determinant for the SARS-CoV-2 Omicron-BA.1 phenotype

Variant of concern (VOC) Omicron-BA1 has achieved global predominance in early 2022. Therefore, surveillance and comprehensive characterization of Omicron-BA.1 in advanced primary cell culture systems and multiple animal models is urgently needed. Here, we characterized Omicron-BA.1 and recombinant Omicron-BA.1 spike gene mutants in comparison with VOC Delta in well-differentiated primary human nasal and bronchial epithelial cells in vitro, followed by in vivo fitness characterization in naive hamsters, ferrets and hACE2-expressing mice, and in immunized hACE2-mice. We demonstrate a spike-mediated enhancement of early replication of Omicron-BA.1 in nasal epithelial cultures, but limited replication in bronchial epithelial cultures. In Syrian hamsters, Delta showed dominance over Omicron-BA.1 and in ferrets, Omicron-BA.1 infection was abortive. In mice expressing the authentic hACE2-receptor, Delta and a Delta spike clone also showed dominance over Omicron-BA.1 and an Omicron-BA.1 spike clone, respectively. Interestingly, in naive K18-hACE2 mice, we observed Delta spike-mediated increased replication and pathogenicity and Omicron-BA.1 spike-mediated reduced replication and pathogenicity, suggesting that the spike gene is a major determinant of both Delta and Omicron-BA.1 replication and pathogenicity. Finally, the Omicron-BA.1 spike clone was less well controlled by mRNA-vaccination in K18-hACE2-mice and became more competitive compared to the progenitor and Delta spike clones, suggesting that spike gene-mediated immune evasion is another important factor that led to Omicron-BA.1 dominance.

microbiology↗

bioRxiv2021

Enhanced fitness of SARS-CoV-2 variant of concern B.1.1.7, but not B.1.351, in animal models

Emerging variants of concern (VOCs) drive the SARS-CoV-2 pandemic. We assessed VOC B.1.1.7, now prevalent in several countries, and VOC B.1.351, representing the greatest threat to populations with immunity to the early SARS-CoV-2 progenitors. B.1.1.7 showed a clear fitness advantage over the progenitor variant (wt-S614G) in ferrets and two mouse models, where the substitutions in the spike glycoprotein were major drivers for fitness advantage. In the "superspreader" hamster model, B.1.1.7 and wt-S614G had comparable fitness, whereas B.1.351 was outcompeted. The VOCs had similar replication kinetics as compared to wt-S614G in human airway epithelial cultures. Our study highlights the importance of using multiple models for complete fitness characterization of VOCs and demonstrates adaptation of B.1.1.7 towards increased upper respiratory tract replication and enhanced transmission in vivo. Summary sentenceB.1.1.7 VOC outcompetes progenitor SARS-CoV-2 in upper respiratory tract replication competition in vivo.

microbiology↗

bioRxiv2021

Establishment of a reverse genetic system from a bovine derived Influenza D virus isolate.

The ruminant-associated Influenza D virus (IDV) has a broad host tropism and was shown to have zoonotic potential. To identify and characterize molecular viral determinants influencing the host spectrum of IDV, a reverse genetic system is required. For this, we first performed 5' and 3' rapid amplification of cDNA ends (RACE) of all seven genomic segments, followed by assessment of the 5' and 3' NCR activity prior to constructing the viral genomic segments of a contemporary Swiss bovine IDV isolate (D/CN286) into the bidirectional pHW-2000 vector. The bidirectional plasmids were transfected in HRT-18G cells followed by viral rescue on the same cell type. Analysis of the segment specific 5' and 3' non-coding regions (NCR) highlighted that the terminal 3' end of all segments harbours an Uracil instead of a Cytosine nucleotide, similar to other Influenza viruses. Subsequent analysis on the functionality of the 5' and 3' NCR in a minireplicon assay revealed that these sequences were functional and that the variable sequence length of the 5' and 3' NCR influences reporter gene expression. Thereafter, we evaluated the replication efficiency of the reverse genetic clone on conventional cell lines of human, swine and bovine origin by use of an in vitro model recapitulating the natural replicate site of IDV in bovine and swine. This revealed that the reverse genetic clone D/CN286 replicates efficiently in all cell culture models. Combined, these results demonstrate the successful establishment of a reverse genetic system from a contemporary bovine IDV isolate that can be used for future identification and characterization of viral determinants influencing the broad host tropism of IDV.

microbiology↗

bioRxiv2020

Susceptibility of well-differentiated airway epithelial cell cultures from domestic and wildlife animals to SARS-CoV-2

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread globally, and the number of cases continues to rise all over the world. Besides humans, the zoonotic origin, as well as intermediate and potential spillback host reservoirs of SARS-CoV-2 are unknown. To circumvent ethical and experimental constraints, and more importantly, to reduce and refine animal experimentation, we employed our airway epithelial cell (AEC) culture repository composed of various domesticated and wildlife animal species to assess their susceptibility to SARS-CoV-2. In this study, we inoculated well-differentiated animal AEC cultures of monkey, cat, ferret, dog, rabbit, pig, cattle, goat, llama, camel, and two neotropical bat species with SARS-CoV-2. We observed that SARS-CoV-2 only replicated efficiently in monkey and cat AEC culture models. Whole-genome sequencing of progeny virus revealed no obvious signs of nucleotide transitions required for SARS-CoV-2 to productively infect monkey and cat epithelial airway cells. Our findings, together with the previously reported human-to-animal spillover events warrants close surveillance to understand the potential role of cats, monkeys, and closely related species as spillback reservoirs for SARS-CoV-2.

microbiology↗

bioRxiv2020

Identification of five antiviral compounds from the Pandemic Response Box targeting SARS-CoV-2

With currently over 4 million confirmed cases worldwide, including more than 300000 deaths, the current Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic has a major impact on the economy and health care system. Currently, a limited amount of prophylactic or therapeutic intervention options are available against SARS-CoV-2. In this study, we screened 400 compounds from the antimicrobial Pandemic Response Box library for inhibiting properties against SARS-CoV-2. We identified sixteen compounds that potently inhibited SARS-CoV-2 replication, of which five compounds displayed equal or even higher antiviral activity compared to Remdesivir. These results show that five compounds should be further investigated for their mode of action, safety and efficacy against SARS-CoV-2. HighlightsO_LI400 compounds from the pandemic response box were tested for antiviral activity against SARS-CoV-2. C_LIO_LI5 compounds had an equal or higher antiviral efficacy towards SARS-CoV-2, compared to the nucleoside analogue Remdesivir. C_LI

microbiology↗

bioRxiv2020

Disparate temperature-dependent virus - host dynamics for SARS-CoV-2 and SARS-CoV in the human respiratory epithelium

Since its emergence in December 2019, SARS-CoV-2 has spread globally and become a major public health burden. Despite its close phylogenetic relationship to SARS-CoV, SARS-CoV-2 exhibits increased human-to-human transmission dynamics, likely due to efficient early replication in the upper respiratory epithelium of infected individuals. Since different temperatures encountered in the human respiratory tract have been shown to affect the replication kinetics of several viruses, as well as host immune response dynamics, we investigated the impact of temperatures during SARS-CoV-2 and SARS-CoV infection in the human airway epithelial cell culture model. SARS-CoV-2, in contrast to SARS-CoV, replicated more efficiently at temperatures encountered in the upper respiratory tract, and displayed higher sensitivity to type I and type III IFNs. Time-resolved transcriptome analysis highlighted a temperature-dependent and virus-specific induction of the IFN-mediated antiviral response. These data reflect clinical features of SARS-CoV-2 and SARS-CoV, as well as their associated transmission efficiencies, and provide crucial insight on pivotal virus - host interaction dynamics.

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↗