Endogenous G-quadruplex-forming RNAs inhibit the activity of SARS-CoV-2 RNA polymerase.

Replication of RNA viruses is catalysed by virus-specific polymerases, which can be targets of therapeutic strategies. In this study, we used a selection strategy to identify endogenous RNAs from a transcriptome library derived from lung cells that interact with the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2. Some of the selected RNAs weakened the activity of RdRp by forming G-quadruplexes. These results suggest that certain endogenous RNAs, which potentially form G-quadruplexes, can reduce the replication of viral RNAs.

Remdesivir does not affect mitochondrial DNA copy number or deletion mutation frequency in aged male rats: A short report.

Remdesivir is a leading therapy in patients with moderate to severe coronavirus 2 (SARS-CoV-2) infection; the majority of whom are older individuals. Remdesivir is a nucleoside analog that incorporates into nascent viral RNA, inhibiting RNA-directed RNA polymerases, including that of SARS-CoV-2. Less is known about remdesivir's effects on mitochondria, particularly in older adults where mitochondria are known to be dysfunctional. Furthermore, its effect on age-induced mitochondrial mutations and copy number has not been previously studied. We hypothesized that remdesivir adversely affects mtDNA copy number and deletion mutation frequency in aged rodents. To test this hypothesis, 30-month-old male F333BNF1 rats were treated with remdesivir for three months. To determine if remdesivir adversely affects mtDNA, we measured copy number and mtDNA deletion frequency in rat hearts, kidneys, and skeletal muscles using digital PCR. We found no effects from three months of remdesivir treatment on mtDNA copy number or deletion mutation frequency in 33-month-old rats. These data support the notion that remdesivir does not compromise mtDNA quality or quantity at old age in mammals. Future work should focus on examining additional tissues such as brain and liver, and extend testing to human clinical samples.

Low expression of EXOSC2 protects against clinical COVID-19 and impedes SARS-CoV-2 replication.

New therapeutic targets are a valuable resource for treatment of SARS-CoV-2 viral infection. Genome-wide association studies have identified risk loci associated with COVID-19, but many loci are associated with comorbidities and are not specific to host-virus interactions. Here, we identify and experimentally validate a link between reduced expression of EXOSC2 and reduced SARS-CoV-2 replication. EXOSC2 was one of the 332 host proteins examined, all of which interact directly with SARS-CoV-2 proteins. Aggregating COVID-19 genome-wide association studies statistics for gene-specific eQTLs revealed an association between increased expression of EXOSC2 and higher risk of clinical COVID-19. EXOSC2 interacts with Nsp8 which forms part of the viral RNA polymerase. EXOSC2 is a component of the RNA exosome, and here, LC-MS/MS analysis of protein pulldowns demonstrated interaction between the SARS-CoV-2 RNA polymerase and most of the human RNA exosome components. CRISPR/Cas9 introduction of nonsense mutations within EXOSC2 in Calu-3 cells reduced EXOSC2 protein expression and impeded SARS-CoV-2 replication without impacting cellular viability. Targeted depletion of EXOSC2 may be a safe and effective strategy to protect against clinical COVID-19.

Equivalence of the GeneXpert System and GeneXpert Omni System for tuberculosis and rifampicin resistance detection.

A laboratory validation study was conducted to assess the equivalence of Xpert MTB/RIF Ultra testing on the GeneXpert System and the GeneXpert Omni System ('Omni') for tuberculosis and rifampicin resistance. High concordance of the two devices was demonstrated for well-characterized clinical samples as well as control materials, with controls tested on Omni at normal and challenging environmental conditions (i.e. 35°C, 90% relative humidity). Equivalence of the Cts for all probes was also shown. Equivalence was demonstrated for the Omni and GeneXpert devices for tuberculosis and rifampicin resistance detection for a diverse range of clinical specimens and environmental conditions.

New Investigations with Lupane Type A-Ring Azepane Triterpenoids for Antimycobacterial Drug Candidate Design.

Twenty lupane type A-ring azepano-triterpenoids were synthesized from betulin and its related derivatives and their antitubercular activity against Mycobacterium tuberculosis, mono-resistant MTB strains, and nontuberculous strains Mycobacterium abscessus and Mycobacterium avium were investigated in the framework of AToMIc (Anti-mycobacterial Target or Mechanism Identification Contract) realized by the Division of Microbiology and Infectious Diseases, NIAID, National Institute of Health. Of all the tested triterpenoids, 17 compounds showed antitubercular activity and 6 compounds were highly active on the H37Rv wild strain (with MIC 0.5 µM for compound 7), out of which 4 derivatives also emerged as highly active compounds on the three mono-resistant MTB strains. Molecular docking corroborated with a machine learning drug-drug similarity algorithm revealed that azepano-triterpenoids have a rifampicin-like antitubercular activity, with compound 7 scoring the highest as a potential M. tuberculosis RNAP potential inhibitor. FIC testing demonstrated an additive effect of compound 7 when combined with rifampin, isoniazid and ethambutol. Most compounds were highly active against M. avium with compound 14 recording the same MIC value as the control rifampicin (0.0625 µM). The antitubercular ex vivo effectiveness of the tested compounds on THP-1 infected macrophages is correlated with their increased cell permeability. The tested triterpenoids also exhibit low cytotoxicity and do not induce antibacterial resistance in MTB strains.

A Methyltransferase-Defective Vesicular Stomatitis Virus-Based SARS-CoV-2 Vaccine Candidate Provides Complete Protection against SARS-CoV-2 Infection in Hamsters.

The current pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to dramatic economic and health burdens. Although the worldwide SARS-CoV-2 vaccination campaign has begun, exploration of other vaccine candidates is needed due to uncertainties with the current approved vaccines, such as durability of protection, cross-protection against variant strains, and costs of long-term production and storage. In this study, we developed a methyltransferase-defective recombinant vesicular stomatitis virus (mtdVSV)-based SARS-CoV-2 vaccine candidate. We generated mtdVSVs expressing SARS-CoV-2 full-length spike (S) protein, S1, or its receptor-binding domain (RBD). All of these recombinant viruses grew to high titers in mammalian cells despite high attenuation in cell culture. The SARS-CoV-2 S protein and its truncations were highly expressed by the mtdVSV vector. These mtdVSV-based vaccine candidates were completely attenuated in both immunocompetent and immunocompromised mice. Among these constructs, mtdVSV-S induced high levels of SARS-CoV-2-specific neutralizing antibodies (NAbs) and Th1-biased T-cell immune responses in mice. In Syrian golden hamsters, the serum levels of SARS-CoV-2-specific NAbs triggered by mtdVSV-S were higher than the levels of NAbs in convalescent plasma from recovered COVID-19 patients. In addition, hamsters immunized with mtdVSV-S were completely protected against SARS-CoV-2 replication in lung and nasal turbinate tissues, cytokine storm, and lung pathology. Collectively, our data demonstrate that mtdVSV expressing SARS-CoV-2 S protein is a safe and highly efficacious vaccine candidate against SARS-CoV-2 infection. IMPORTANCE Viral mRNA cap methyltransferase (MTase) is essential for mRNA stability, protein translation, and innate immune evasion. Thus, viral mRNA cap MTase activity is an excellent target for development of live attenuated or live vectored vaccine candidates. Here, we developed a panel of MTase-defective recombinant vesicular stomatitis virus (mtdVSV)-based SARS-CoV-2 vaccine candidates expressing full-length S, S1, or several versions of the RBD. These mtdVSV-based vaccine candidates grew to high titers in cell culture and were completely attenuated in both immunocompetent and immunocompromised mice. Among these vaccine candidates, mtdVSV-S induces high levels of SARS-CoV-2-specific neutralizing antibodies (Nabs) and Th1-biased immune responses in mice. Syrian golden hamsters immunized with mtdVSV-S triggered SARS-CoV-2-specific NAbs at higher levels than those in convalescent plasma from recovered COVID-19 patients. Furthermore, hamsters immunized with mtdVSV-S were completely protected against SARS-CoV-2 challenge. Thus, mtdVSV is a safe and highly effective vector to deliver SARS-CoV-2 vaccine.