Lysosomal enzyme trafficking factor LYSET enables nutritional usage of extracellular proteins.

Mammalian cells can generate amino acids through macropinocytosis and lysosomal breakdown of extracellular proteins, which is exploited by cancer cells to grow in nutrient-poor tumors. Through genetic screens in defined nutrient conditions, we characterized LYSET, a transmembrane protein (TMEM251) selectively required when cells consume extracellular proteins. LYSET was found to associate in the Golgi with GlcNAc-1-phosphotransferase, which targets catabolic enzymes to lysosomes through mannose-6-phosphate modification. Without LYSET, GlcNAc-1-phosphotransferase was unstable because of a hydrophilic transmembrane domain. Consequently, LYSET-deficient cells were depleted of lysosomal enzymes and impaired in turnover of macropinocytic and autophagic cargoes. Thus, LYSET represents a core component of the lysosomal enzyme trafficking pathway, underlies the pathomechanism for hereditary lysosomal storage disorders, and may represent a target to suppress metabolic adaptations in cancer.

Agent-based simulations for protecting nursing homes with prevention and vaccination strategies.

Due to its high lethality among older people, the safety of nursing homes has been of central importance during the COVID-19 pandemic. With test procedures and vaccines becoming available at scale, nursing homes might relax prohibitory measures while controlling the spread of infections. By control we mean that each index case infects less than one other person on average. Here, we develop an agent-based epidemiological model for the spread of SARS-CoV-2 calibrated to Austrian nursing homes to identify optimal prevention strategies. We find that the effectiveness of mitigation testing depends critically on test turnover time (time until test result), the detection threshold of tests and mitigation testing frequencies. Under realistic conditions and in absence of vaccinations, we find that mitigation testing of employees only might be sufficient to control outbreaks if tests have low turnover times and detection thresholds. If vaccines that are 60% effective against high viral load and transmission are available, control is achieved if 80% or more of the residents are vaccinated, even without mitigation testing and if residents are allowed to have visitors. Since these results strongly depend on vaccine efficacy against infection, retention of testing infrastructures, regular testing and sequencing of virus genomes is advised to enable early identification of new variants of concern.

A Rapid, Highly Sensitive and Open-Access SARS-CoV-2 Detection Assay for Laboratory and Home Testing.

RT-qPCR-based diagnostic tests play important roles in combating virus-caused pandemics such as Covid-19. However, their dependence on sophisticated equipment and the associated costs often limits their widespread use. Loop-mediated isothermal amplification after reverse transcription (RT-LAMP) is an alternative nucleic acid detection method that overcomes these limitations. Here, we present a rapid, robust, and sensitive RT-LAMP-based SARS-CoV-2 detection assay. Our 40-min procedure bypasses the RNA isolation step, is insensitive to carryover contamination, and uses a colorimetric readout that enables robust SARS-CoV-2 detection from various sample types. Based on this assay, we have increased sensitivity and scalability by adding a nucleic acid enrichment step (Bead-LAMP), developed a version for home testing (HomeDip-LAMP), and identified open-source RT-LAMP enzymes that can be produced in any molecular biology laboratory. On a dedicated website, rtlamp.org (DOI: 10.5281/zenodo.6033689), we provide detailed protocols and videos. Our optimized, general-purpose RT-LAMP assay is an important step toward population-scale SARS-CoV-2 testing.

Results of WICOVIR Gargle Pool PCR Testing in German Schools Based on the First 100,000 Tests.

Background: Opening schools and keeping children safe from SARS-CoV-2 infections at the same time is urgently needed to protect children from direct and indirect consequences of the COVID-19 pandemic. To achieve this goal, a safe, efficient, and cost-effective SARS-CoV-2 testing system for schools in addition to standard hygiene measures is necessary. Methods: We implemented the screening WICOVIR concept for schools in the southeast of Germany, which is based on gargling at home, pooling of samples in schools, and assessment of SARS-CoV-2 by pool rRT-PCR, performed decentralized in numerous participating laboratories. Depooling was performed if pools were positive, and results were transmitted with software specifically developed for the project within a day. Here, we report the results after the first 13 weeks in the project. Findings: We developed and implemented the proof-of-concept test system within a pilot phase of 7 weeks based on almost 17,000 participants. After 6 weeks in the main phase of the project, we performed >100,000 tests in total, analyzed in 7,896 pools, identifying 19 cases in >100 participating schools. On average, positive children showed an individual CT value of 31 when identified in the pools. Up to 30 samples were pooled (mean 13) in general, based on school classes and attached school staff. All three participating laboratories detected positive samples reliably with their previously established rRT-PCR standard protocols. When self-administered antigen tests were performed concomitantly in positive cases, only one of these eight tests was positive, and when antigen tests performed after positive pool rRT-PCR results were already known were included, 3 out of 11 truly positive tests were also identified by antigen testing. After 3 weeks of repetitive WICOVIR testing twice weekly, the detection rate of positive children in that cohort decreased significantly from 0.042 to 0.012 (p = 0.008). Interpretation: Repeated gargle pool rRT-PCR testing can be implemented quickly in schools. It is an effective, valid, and well-received test system for schools, superior to antigen tests in sensitivity, acceptance, and costs.

Prevalence of RT-qPCR-detected SARS-CoV-2 infection at schools: First results from the Austrian School-SARS-CoV-2 prospective cohort study.

BACKGROUND: The role of schools in the SARS-CoV-2 pandemic is much debated. We aimed to quantify reliably the prevalence of SARS-CoV-2 infections at schools detected with reverse-transcription quantitative polymerase-chain-reaction (RT-qPCR). METHODS: This nationwide prospective cohort study monitors a representative sample of pupils (grade 1-8) and teachers at Austrian schools throughout the school year 2020/2021. We repeatedly test participants for SARS-CoV-2 infection using a gargling solution and RT-qPCR. We herein report on the first two rounds of examinations. We used mixed-effects logistic regression to estimate odds ratios and robust 95% confidence intervals (95% CI). FINDINGS: We analysed data on 10,734 participants from 245 schools (9465 pupils, 1269 teachers). Prevalence of SARS-CoV-2 infection increased from 0·39% at round 1 (95% CI 028-0·55%, 28 September-22 October 2020) to 1·39% at round 2 (95% CI 1·04-1·85%, 10-16 November). Odds ratios for SARS-CoV-2 infection were 2·26 (95% CI 1·25-4·12, P = 0·007) in regions with >500 vs. ≤500 inhabitants/km2, 1·67 (95% CI 1·42-1·97, P<0·001) per two-fold higher regional 7-day community incidence, and 2·78 (95% CI 1·73-4·48, P<0·001) in pupils at schools with high/very high vs. low/moderate social deprivation. Associations of regional community incidence and social deprivation persisted in a multivariable adjusted model. Prevalence did not differ by average number of pupils per class nor between age groups, sexes, pupils vs. teachers, or primary (grade 1-4) vs. secondary schools (grade 5-8). INTERPRETATION: This monitoring study in Austrian schools revealed SARS-CoV-2 infection in 0·39%-1·39% of participants and identified associations of regional community incidence and social deprivation with higher prevalence. FUNDING: BMBWF Austria.

Sensitivity and specificity of the antigen-based anterior nasal self-testing programme for detecting SARS-CoV-2 infection in schools, Austria, March 2021.

This study evaluates the performance of the antigen-based anterior nasal screening programme implemented in all Austrian schools to detect SARS-CoV-2 infections. We combined nationwide antigen-based screening data obtained in March 2021 from 5,370 schools (Grade 1-8) with an RT-qPCR-based prospective cohort study comprising a representative sample of 244 schools. Considering a range of assumptions, only a subset of infected individuals are detected with the programme (low to moderate sensitivity) and non-infected individuals mainly tested negative (very high specificity).

Functional interrogation of a SARS-CoV-2 host protein interactome identifies unique and shared coronavirus host factors.

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has devastated the global economy and claimed more than 1.7 million lives, presenting an urgent global health crisis. To identify host factors required for infection by SARS-CoV-2 and seasonal coronaviruses, we designed a focused high-coverage CRISPR-Cas9 library targeting 332 members of a recently published SARS-CoV-2 protein interactome. We leveraged the compact nature of this library to systematically screen SARS-CoV-2 at two physiologically relevant temperatures along with three related coronaviruses (human coronavirus 229E [HCoV-229E], HCoV-NL63, and HCoV-OC43), allowing us to probe this interactome at a much higher resolution than genome-scale studies. This approach yielded several insights, including potential virus-specific differences in Rab GTPase requirements and glycosylphosphatidylinositol (GPI) anchor biosynthesis, as well as identification of multiple pan-coronavirus factors involved in cholesterol homeostasis. This coronavirus essentiality catalog could inform ongoing drug development efforts aimed at intercepting and treating coronavirus disease 2019 (COVID-19) and help prepare for future coronavirus outbreaks.