Genomic surveillance of SARS-CoV-2 evolution by a centralised pipeline and weekly focused sequencing, Austria, January 2021 to March 2023.

BackgroundThe COVID-19 pandemic was largely driven by genetic mutations of SARS-CoV-2, leading in some instances to enhanced infectiousness of the virus or its capacity to evade the host immune system. To closely monitor SARS-CoV-2 evolution and resulting variants at genomic-level, an innovative pipeline termed SARSeq was developed in Austria.AimWe discuss technical aspects of the SARSeq pipeline, describe its performance and present noteworthy results it enabled during the pandemic in Austria.MethodsThe SARSeq pipeline was set up as a collaboration between private and public clinical diagnostic laboratories, a public health agency, and an academic institution. Representative SARS-CoV-2 positive specimens from each of the nine Austrian provinces were obtained from SARS-CoV-2 testing laboratories and processed centrally in an academic setting for S-gene sequencing and analysis.ResultsSARS-CoV-2 sequences from up to 2,880 cases weekly resulted in 222,784 characterised case samples in January 2021-March 2023. Consequently, Austria delivered the fourth densest genomic surveillance worldwide in a very resource-efficient manner. While most SARS-CoV-2 variants during the study showed comparable kinetic behaviour in all of Austria, some, like Beta, had a more focused spread. This highlighted multifaceted aspects of local population-level acquired immunity. The nationwide surveillance system enabled reliable nowcasting. Measured early growth kinetics of variants were predictive of later incidence peaks.ConclusionWith low automation, labour, and cost requirements, SARSeq is adaptable to monitor other pathogens and advantageous even for resource-limited countries. This multiplexed genomic surveillance system has potential as a rapid response tool for future emerging threats.

eIF4E1b is a non-canonical eIF4E protecting maternal dormant mRNAs.

Maternal mRNAs are essential for protein synthesis during oogenesis and early embryogenesis. To adapt translation to specific needs during development, maternal mRNAs are translationally repressed by shortening the polyA tails. While mRNA deadenylation is associated with decapping and degradation in somatic cells, maternal mRNAs with short polyA tails are stable. Here we report that the germline-specific eIF4E paralog, eIF4E1b, is essential for zebrafish oogenesis. eIF4E1b localizes to P-bodies in zebrafish embryos and binds to mRNAs with reported short or no polyA tails, including histone mRNAs. Loss of eIF4E1b results in reduced histone mRNA levels in early gonads, consistent with a role in mRNA storage. Using mouse and human eIF4E1Bs (in vitro) and zebrafish eIF4E1b (in vivo), we show that unlike canonical eIF4Es, eIF4E1b does not interact with eIF4G to initiate translation. Instead, eIF4E1b interacts with the translational repressor eIF4ENIF1, which is required for eIF4E1b localization to P-bodies. Our study is consistent with an important role of eIF4E1b in regulating mRNA dormancy and provides new insights into fundamental post-transcriptional regulatory principles governing early vertebrate development.

Selective Eradication of Colon Cancer Cells Harboring PI3K and/or MAPK Pathway Mutations in 3D Culture by Combined PI3K/AKT/mTOR Pathway and MEK Inhibition.

Colorectal cancer (CRC) is the second deadliest cancer in the world. Besides APC and p53 alterations, the PI3K/AKT/MTOR and MAPK pathway are most commonly mutated in CRC. So far, no treatment options targeting these pathways are available in routine clinics for CRC patients. We systematically analyzed the response of CRC cells to the combination of small molecular inhibitors targeting the PI3K and MAPK pathways. We used CRC cells in 2D, 3D spheroid, collagen gel cultures and freshly isolated organoids for drug response studies. Readout for drug response was spheroid or organoid growth, spheroid outgrowth, metabolic activity, Western blotting and immunofluorescence. We found profound tumor cell destruction under treatment with a combination of Torin 1 (inhibiting mTOR), MK2206 (targeting AKT) and selumetinib (inhibiting MEK) in 3D but not in 2D. Induction of cell death was due to apoptosis. Western blot analysis revealed efficient drug action. Gedatolisib, a dual PI3K/mTOR inhibitor, could replace Torin1/MK2206 with similar efficiency. The presence of PI3K and/or RAS-RAF-MAPK pathway mutations accounted for treatment responsiveness. Here, we identified a novel, efficient therapy, which induced proliferation stop and tumor cell destruction in vitro based on the genetic background. These preclinical findings show promise to further test this combi-treatment in vivo in mice and to potentially develop a mutation specific targeted therapy for CRC patients.

Multiplexed detection of SARS-CoV-2 and other respiratory infections in high throughput by SARSeq.

The COVID-19 pandemic has demonstrated the need for massively-parallel, cost-effective tests monitoring viral spread. Here we present SARSeq, saliva analysis by RNA sequencing, a method to detect SARS-CoV-2 and other respiratory viruses on tens of thousands of samples in parallel. SARSeq relies on next generation sequencing of multiple amplicons generated in a multiplexed RT-PCR reaction. Two-dimensional, unique dual indexing, using four indices per sample, enables unambiguous and scalable assignment of reads to individual samples. We calibrate SARSeq on SARS-CoV-2 synthetic RNA, virions, and hundreds of human samples of various types. Robustness and sensitivity were virtually identical to quantitative RT-PCR. Double-blinded benchmarking to gold standard quantitative-RT-PCR performed by human diagnostics laboratories confirms this high sensitivity. SARSeq can be used to detect Influenza A and B viruses and human rhinovirus in parallel, and can be expanded for detection of other pathogens. Thus, SARSeq is ideally suited for differential diagnostic of infections during a pandemic.