ABSTRACT
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.
Subject(s)
Coronavirus Infections , COVID-19ABSTRACT
Background: COVID-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. Methods: We 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. Findings: On 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, with a maximum pool size of 64, with only 7.3 tests on the average, we can distinguish between prevalences of 1% and 5% with a probability of detection of 95% and probability of false alarm of 4%. Interpretation: The 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.
Subject(s)
COVID-19ABSTRACT
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.