Stanley Qi, Postdoctoral Faculty Sponsor
Development of CRISPR as an Antiviral Strategy to Combat SARS-CoV-2 and Influenza.
The coronavirus disease 2019 (COVID-19) pandemic, caused by the SARS-CoV-2 virus, has highlighted the need for antiviral approaches that can target emerging viruses with no effective vaccines or pharmaceuticals. Here, we demonstrate a CRISPR-Cas13-based strategy, PAC-MAN (prophylactic antiviral CRISPR in human cells), for viral inhibition that can effectively degrade RNA from SARS-CoV-2 sequences and live influenza A virus (IAV) in human lung epithelial cells. We designed and screened CRISPR RNAs (crRNAs) targeting conserved viral regions and identified functional crRNAs targeting SARS-CoV-2. This approach effectively reduced H1N1 IAV load in respiratory epithelial cells. Our bioinformatic analysis showed that a group of only six crRNAs can target more than 90% of all coronaviruses. With the development of a safe and effective system for respiratory tract delivery, PAC-MAN has the potential to become an important pan-coronavirus inhibition strategy.
View details for DOI 10.1016/j.cell.2020.04.020
View details for PubMedID 32353252
- Cross-neutralization of SARS coronavirus-specific antibodies against bat SARS-like coronaviruses SCIENCE CHINA-LIFE SCIENCES 2017; 60 (12): 1399–1402
Discovery of a rich gene pool of bat SARS-related coronaviruses provides new insights into the origin of SARS coronavirus
2017; 13 (11): e1006698
A large number of SARS-related coronaviruses (SARSr-CoV) have been detected in horseshoe bats since 2005 in different areas of China. However, these bat SARSr-CoVs show sequence differences from SARS coronavirus (SARS-CoV) in different genes (S, ORF8, ORF3, etc) and are considered unlikely to represent the direct progenitor of SARS-CoV. Herein, we report the findings of our 5-year surveillance of SARSr-CoVs in a cave inhabited by multiple species of horseshoe bats in Yunnan Province, China. The full-length genomes of 11 newly discovered SARSr-CoV strains, together with our previous findings, reveals that the SARSr-CoVs circulating in this single location are highly diverse in the S gene, ORF3 and ORF8. Importantly, strains with high genetic similarity to SARS-CoV in the hypervariable N-terminal domain (NTD) and receptor-binding domain (RBD) of the S1 gene, the ORF3 and ORF8 region, respectively, were all discovered in this cave. In addition, we report the first discovery of bat SARSr-CoVs highly similar to human SARS-CoV in ORF3b and in the split ORF8a and 8b. Moreover, SARSr-CoV strains from this cave were more closely related to SARS-CoV in the non-structural protein genes ORF1a and 1b compared with those detected elsewhere. Recombination analysis shows evidence of frequent recombination events within the S gene and around the ORF8 between these SARSr-CoVs. We hypothesize that the direct progenitor of SARS-CoV may have originated after sequential recombination events between the precursors of these SARSr-CoVs. Cell entry studies demonstrated that three newly identified SARSr-CoVs with different S protein sequences are all able to use human ACE2 as the receptor, further exhibiting the close relationship between strains in this cave and SARS-CoV. This work provides new insights into the origin and evolution of SARS-CoV and highlights the necessity of preparedness for future emergence of SARS-like diseases.
View details for DOI 10.1371/journal.ppat.1006698
View details for Web of Science ID 000416888500016
View details for PubMedID 29190287
View details for PubMedCentralID PMC5708621
IFNAR2-dependent gene expression profile induced by IFN-alpha in Pteropus alecto bat cells and impact of IFNAR2 knockout on virus infection
2017; 12 (8): e0182866
Bats are important reservoirs of many viruses, which are capable of infecting the host without inducing obvious clinical diseases. Interferon and the downstream interferon regulated genes (IRGs) are known to act as the first line of defense against viral infections. Little is known about the transcriptional profile of genes being induced by interferon in bats and their role in controlling virus infection. In this study, we constructed IFNAR2 knockout bat cell lines using CRISPR technology and further characterized gene expression profiles induced by the most abundant IFN-α (IFN-α3). Firstly, we demonstrated that the CRISPR/Cas9 system is applicable for bat cells as this represents the first CRIPSR knockout cell line for bats. Our results showed the pleiotropic effect of IFN-α3 on the bat kidney cell line, PaKiT03. As expected, we confirmed that IFNAR2 is indispensable for IFN-a signaling pathway and plays an important role in antiviral immunity. Unexpectedly, we also identified novel IFNAR2-dependent IRGs which are enriched in pathways related to cancer. To our knowledge, this seems to be bat-specific as no such observation has been reported for other mammalian species. This study expands our knowledge about bat immunology and the cell line established can provide a powerful tool for future study into virus-bat interaction and cancer biology.
View details for DOI 10.1371/journal.pone.0182866
View details for Web of Science ID 000407196700075
View details for PubMedID 28793350
View details for PubMedCentralID PMC5549907
Bat Severe Acute Respiratory Syndrome-Like Coronavirus WIV1 Encodes an Extra Accessory Protein, ORFX, Involved in Modulation of the Host Immune Response
JOURNAL OF VIROLOGY
2016; 90 (14): 6573–82
Bats harbor severe acute respiratory syndrome (SARS)-like coronaviruses (SL-CoVs) from which the causative agent of the 2002-2003 SARS pandemic is thought to have originated. However, despite the fact that a large number of genetically diverse SL-CoV sequences have been detected in bats, only two strains (named WIV1 and WIV16) have been successfully cultured in vitro These two strains differ from SARS-CoV only in containing an extra open reading frame (ORF) (named ORFX), between ORF6 and ORF7, which has no homology to any known protein sequences. In this study, we constructed a full-length cDNA clone of SL-CoV WIV1 (rWIV1), an ORFX deletion mutant (rWIV1-ΔX), and a green fluorescent protein (GFP)-expressing mutant (rWIV1-GFP-ΔX). Northern blotting and fluorescence microscopy indicate that ORFX was expressed during WIV1 infection. A virus infection assay showed that rWIV1-ΔX replicated as efficiently as rWIV1 in Vero E6, Calu-3, and HeLa-hACE2 cells. Further study showed that ORFX could inhibit interferon production and activate NF-κB. Our results demonstrate for the first time that the unique ORFX in the WIV1 strain is a functional gene involving modulation of the host immune response but is not essential for in vitro viral replication.Bats harbor genetically diverse SARS-like coronaviruses (SL-CoVs), and some of them have the potential for interspecies transmission. A unique open reading frame (ORFX) was identified in the genomes of two recently isolated bat SL-CoV strains (WIV1 and -16). It will therefore be critical to clarify whether and how this protein contributes to virulence during viral infection. Here we revealed that the unique ORFX is a functional gene that is involved in the modulation of the host immune response but is not essential for in vitro viral replication. Our results provide important information for further exploration of the ORFX function in the future. Moreover, the reverse genetics system we constructed will be helpful for study of the pathogenesis of this group of viruses and to develop therapeutics for future control of emerging SARS-like infections.
View details for DOI 10.1128/JVI.03079-15
View details for Web of Science ID 000378658800033
View details for PubMedID 27170748
View details for PubMedCentralID PMC4936131