Parsing the role of NSP1 in SARS-CoV-2 infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 19 (COVID-19) pandemic. Despite its urgency, we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis and its ability to antagonize innate immune responses. SARS-CoV-2 leads to shutoff of cellular protein synthesis and over-expression of nsp1, a central shutoff factor in coronaviruses, inhibits cellular gene translation. However, the diverse molecular mechanisms nsp1 employs as well as its functional importance in infection are still unresolved. By overexpressing various nsp1 mutants and generating a SARS-CoV-2 mutant in which nsp1 does not bind ribosomes, we untangle the effects of nsp1. We uncover that nsp1, through inhibition of translation and induction of mRNA degradation, is the main driver of host shutoff during SARS-CoV-2 infection. Furthermore, we find the propagation of nsp1 mutant virus is inhibited specifically in cells with intact interferon (IFN) response as well as in-vivo, in infected hamsters, and this attenuation is associated with stronger induction of type I IFN response. This illustrates that nsp1 shutoff activity has an essential role mainly in counteracting the IFN response. Overall, our results reveal the multifaceted approach nsp1 uses to shut off cellular protein synthesis and uncover the central role it plays in SARS-CoV-2 pathogenesis, explicitly through blockage of the IFN response.

Betulinic acid mitigates oxidative stress-mediated apoptosis and enhances longevity in the yeast Saccharomyces cerevisiae model.

Betulinic acid (BA), a pentacyclic triterpenoid found in certain plant species, has been reported to have several health benefits including antioxidant and anti-apoptotic properties. However, the mechanism by which BA confers these properties is currently unknown. Saccharomyces cerevisiae, a budding yeast with a short life cycle and conserved cellular mechanism with high homology to humans, was used as a model for determining the role of BA in aging and programmed cell death (PCD). Treatment with hydrogen peroxide (H2O2) exhibited significantly increased (30-35%) survivability of antioxidant (sod1Δ, sod2Δ, cta1Δ, ctt1Δ, and tsa1Δ) and anti-apoptotic (pep4Δ and fis1Δ) mutant strains when cells were pretreated with BA (30 µM) as demonstrated in spot and CFU (Colony forming units) assays. Measurement of intracellular oxidation level using the ROS-specific dye H2DCF-DA showed that all tested BA-pretreated mutants exhibited decreased ROS than the control when exposed to H2O2. Similarly, when mutant strains were pretreated with BA and then exposed to H2O2, there was reduced lipid peroxidation as revealed by the reduced malondialdehyde content. Furthermore, BA-pretreated mutant cells showed significantly lower apoptotic activity by decreasing DNA/nuclear fragmentation and chromatin condensation under H2O2-induced stress as determined by DAPI and acridine orange/ethidium bromide staining. In addition, BA treatment also extended the life span of antioxidant and anti-apoptotic mutants by ∼10-25% by scavenging ROS and preventing apoptotic cell death. Our overall results suggest that BA extends the chronological life span of mutant strains lacking antioxidant and anti-apoptotic genes by lowering the impact of oxidative stress, ROS levels, and apoptotic activity. These properties of BA could be further explored for its use as a valuable nutraceutical.

Pooling RT-PCR or NGS samples has the potential to cost-effectively generate estimates of COVID-19 prevalence in resource limited environments

BackgroundCOVID-19 originated in China and has quickly spread worldwide causing a pandemic. Countries need rapid data on the prevalence of the virus in communities to enable rapid containment. However, the equipment, human and laboratory resources required for conducting individual RT-PCR is prohibitive. One technique to reduce the number of tests required is the pooling of samples for analysis by RT-PCR prior to testing. MethodsWe conducted a mathematical analysis of pooling strategies for infection rate classification using group testing and for the identification of individuals by testing pooled clusters of samples. FindingsOn the basis of the proposed pooled testing strategy we calculate the probability of false alarm, the probability of detection, and the average number of tests required as a function of the pool size. We find that when the sample size is 256, using a maximum pool size of 64, with only 7.3 tests on average, we can distinguish between prevalences of 1% and 5% with a probability of detection of 95% and probability of false alarm of 4%. InterpretationThe pooling of RT-PCR samples is a cost-effective technique for providing much-needed course-grained data on the prevalence of COVID-19. This is a powerful tool in providing countries with information that can facilitate a response to the pandemic that is evidence-based and saves the most lives possible with the resources available. FundingBill & Melinda Gates Foundation Authors contributionsRL and KRN conceived the study. IF, KT, KRN, SB and RL all contributed to the writing of the manuscript and AH and JJ provided comments. KRN and AH conducted the analysis and designed the figures. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSThe pooling of RT-PCR samples has been shown to be effective in screening for HIV, Chlamydia, Malaria, and influenza, among other pathogens in human health. In agriculture, this method has been used to assess the prevalence of many pathogens, including Dichelobacter nodosus, which causes footrot in sheep, postweaning multisystemic wasting syndrome, and antibiotic resistance in swine feces, in addition to the identification of coronaviruses in multiple bat species. In relation to the current pandemic, researchers in multiple countries have begun to employ this technique to investigate samples for COVID-19. Added value of this studyGiven recent interest in this topic, this study provides a mathematical analysis of infection rate classification using group testing and calculates the probability of false alarm, the probability of detection, and the average number of tests required as a function of the pool size. In addition the identification of individuals by pooled cluster testing is evaluated. Implications of all the available evidenceThis research suggests the pooling of RT-PCR samples for testing can provide a cheap and effective way of gathering much needed data on the prevalence of COVID-19 and identifying infected individuals in the community, where it may be infeasible to carry out a high number of tests. This will enable countries to use stretched resources in the most appropriate way possible, providing valuable data that can inform an evidence-based response to the pandemic.

Isolation and characterization of SARS-CoV-2 from the first US COVID-19 patient

The etiologic agent of the outbreak of pneumonia in Wuhan China was identified as severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) in January, 2020. The first US patient was diagnosed by the State of Washington and the US Centers for Disease Control and Prevention on January 20, 2020. We isolated virus from nasopharyngeal and oropharyngeal specimens, and characterized the viral sequence, replication properties, and cell culture tropism. We found that the virus replicates to high titer in Vero-CCL81 cells and Vero E6 cells in the absence of trypsin. We also deposited the virus into two virus repositories, making it broadly available to the public health and research communities. We hope that open access to this important reagent will expedite development of medical countermeasures. Article SummaryScientists have isolated virus from the first US COVID-19 patient. The isolation and reagents described here will serve as the US reference strain used in research, drug discovery and vaccine testing.

Sinister Splenic Artery Pseudoaneursym: A Rare Case of Unidentified Aetiology.

Splenic Artery Pseudoaneurysms (SAP) are very rare. Giant SAPs are those which are more than 5 cm in diameter and are rarer. SAPs are usually caused by pancreatitis, trauma, surgery or other iatrogenic interventions, vasculitis, local infective or inflammatory processes. We report the successful surgical management of a giant SAP of unidentified aetiology. This case report highlights the significance that this entity may present atypically and hence, early recognition and aggressive management may be life saving.