ABSTRACT
Summary (Abstract)O_ST_ABSBackgroundC_ST_ABSFecal shedding of SARS-CoV-2 has raised concerns about transmission through fecal microbiota transplantation (FMT) procedures. While many tests have been authorized for diagnosis of COVID-19 using respiratory samples, no fully validated stool tests for detection of SARS-CoV-2 are currently available. We sought to adapt and validate an available test specifically for detection of SARS-CoV-2 in human stool. MethodsStool samples were spiked with inactivated SAR-CoV-2 virus for development and validation of the assay. A modified version of the CDC rRT-PCR SARS-CoV-2 test was used for detection of virus. Analytical sensitivity, assay reproducibility, and sample stability under a variety of storage conditions were assessed. We also performed the assay on stool samples collected from known COVID positive individuals. FindingsThe lower limit of detection (LoD) of the assay was found to be 3000 viral RNA copies per gram of original stool sample, with 100% detection across 20 replicates assessed at this concentration. Samples were relatively stable in all buffers tested at both 4{degrees}C and ambient temperature, with the exception of storage in STAR buffer at ambient temperature. Assay sensitivity was slightly diminished in low-copy-number samples after a single freeze-thaw cycle at -80{degrees}C. Thirty contrived SARS-CoV-2 samples were tested by a second laboratory and were correctly identified as positive or negative in at least one of two rounds of testing. Additionally, we detected SARS-CoV-2 RNA in the stool of known COVID-19 positive individuals using this method. InterpretationThis is a sensitive, reproducible, and validated assay for detection of SARS-CoV-2 RNA in human stool with potential uses in FMT donor screening, sewage monitoring, and further research into the impact of fecal shedding on the epidemiology of this pandemic. FundingNational Institute for Allergy and Infectious Diseases, NIH. Center for Biologics Evaluation and Research, FDA. Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSSince the onset of the COVID-19 pandemic, multiple studies have documented shedding of SARS-CoV-2 RNA in feces and considered the potential for fecal-oral transmission of this virus. This potential risk led to the U.S. Food and Drug Administration issuing a safety alert that contained the recommendation that no stool donated after December 1, 2019 be used for manufacture of Fecal Microbiota for Transplantation (FMT) products in the United States until such a time as sufficient screening procedures could be put in place to mitigate this risk. Added value of this studyHere, we report the development and validation of an assay specifically meant for the detection of SARS-CoV-2 RNA in the stool of healthy individuals. While studies have reported detection of viral RNA in stool previously, this is the first publication of a validated assay designed for this purpose. Implications of all the available evidenceThe work presented here provides a validated SARS-CoV-2 stool assay with potential application to FMT donor screening protocols, sewage monitoring protocols, as well as research studies assessing the role of stool shedding and transmission on the epidemiology of COVID-19.
ABSTRACT
Biochemical and structural analyses suggest that SARS-CoV-2 is well-adapted to infecting human and the presence of four residues (PRRA) at the S1/S2 site within the Spike protein may lead to unexpected tissue or host tropism. Here we report that SARS-CoV-2 efficiently utilized ACE2 of 9 species except mouse to infect 293T cells. Similarly, pseudoviruses bearing spike protein derived from either the bat raTG13 or pangolin GX, two closely related animal coronaviruses, utilized ACE2 of a diverse range of animal species to gain entry. Removal of PRRA from SARS-CoV-2 Spike displayed distinct effects on pseudoviral entry into different cell types. Strikingly, insertion of PRRA into the raTG13 Spike selectively abrogated the usage of horseshoe bat and pangolin ACE2 but conferred usage of mouse ACE2 by the relevant pseudovirus to enter cells. Together, our findings identified a previously unrecognized effect of the PRRA insert on SARS-CoV-2 and raTG13 spike proteins.
ABSTRACT
In this study, we aimed to evaluate the stability of SARS-CoV-2 under four different heat conditions (37, 42, 56, 60 {degrees}C) and report that the virus is stable at 37 {degrees}C for at least 24 hours. Heating at 56 {degrees}C for 30 minutes, however, effectively inactivated the virus while preserved the stability of viral RNA in both human sera and sputum samples. These findings provide critical information regarding the biology of the virus as well as a practical way to inactivate infectious virus that is potentially found in clinical specimens.
ABSTRACT
The global pandemic of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or 2019-nCoV) has prompted multiple clinical trials to jumpstart search for anti-SARS-CoV-2 therapies from existing drugs, including those with reported in vitro efficacies as well as those ones that are not known to inhibit SARS-CoV-2, such as ritonavir/lopinavir and favilavir. Here we report that after screening 19 antiviral drugs that are either in clinical trials or with proposed activity against SARS-CoV-2, remdesivir was the most effective. Chloroquine only effectively protected virus-induced cytopathic effect at around 30 {micro}M with a therapeutic index of 1.5. Our findings also suggest that velpatasvir, ledipasvir, ritonavir, litonavir, lopinavir, favilavir, sofosbuvir, danoprevir, and pocapavir do not have direct antiviral effect.
