SARS-CoV-2 genomic surveillance identifies naturally occurring truncations of ORF7a that limit immune suppression (preprint)

Over 200,000 whole genome sequences of SARS-CoV-2 have been determined for viruses isolated from around the world. These sequences have been critical for understanding the spread and evolution of SARS-CoV-2. Using global phylogenomics, we show that mutations frequently occur in the C-terminal end of ORF7a. We have isolated one of these mutant viruses from a patient sample and used viral chal-lenge experiments to demonstrate that {Delta}115 mutation results in a growth defect. ORF7a has been implicated in immune modulation, and we show that the C-terminal truncation results in distinct changes in interferon stimulated gene expression. Collectively, this work indicates that ORF7a mutations occur frequently and that these changes affect viral mechanisms responsible for suppressing the immune response. HighlightsO_LIORF7a mutations are found in SARS-CoV-2 genomes isolated from around the globe. C_LIO_LIORF7a mutation results in a replication defect. C_LIO_LIAn ORF7a mutation limits viral suppression of the interferon response. C_LI

Effects of inactivation method on SARS-CoV-2 virion proteins and structure (preprint)

The risk posed by Severe Acute Respiratory Syndrome Coronavirus -2 (SARS-CoV-2) dictates that live-virus research is conducted in a biosafety level 3 (BSL3) facility. Working with SARS-CoV-2 at lower biosafety levels can expedite research yet requires the virus to be fully inactivated. In this study, we validated and compared two protocols for inactivating SARS-CoV-2: heat treatment and ultraviolet irradiation. The two methods were optimized to render the virus completely incapable of infection while limiting destructive effects of inactivation. We observed that 15 minutes of incubation at 65{degrees}C completely inactivates high titer viral stocks. Complete inactivation was also achieved with minimal amounts of UV power (70,000 J/cm2), which is 100-fold less power than comparable studies. Once validated, the two methods were then compared for viral RNA quantification, virion purification, and antibody recognition. We observed that UV irradiation resulted in a 2-log reduction of detectable genomes compared to heat inactivation. Protein yield following virion enrichment was equivalent for all inactivation conditions, but the resulting viral proteins and virions were negatively impacted by inactivation method and time. We outline the strengths and weaknesses of each method so that investigators might choose the one which best meets their research goals.

Intrinsic Signal Amplification by Type-III CRISPR-Cas Systems Provides a Sequence-Specific Viral Diagnostic (preprint)

To combat viral pandemics, there is an urgent need for inexpensive new technologies that enable fast, reliable, and scalable detection of viruses. Here we repurposed the type III CRISPR-Cas system for sensitive and sequence specific detection of SARS-CoV-2 in an assay that can be performed in one hour or less. RNA recognition by type III systems triggers Cas10-mediated polymerase activity, which simultaneously generates pyrophosphates, protons and cyclic oligonucleotides. We show that amplified products of the Cas10-polymerase are detectable using colorimetric or fluorometric readouts.