RESUMEN
Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants.
RESUMEN
Nsp15 is a uridine specific endoribonuclease that coronaviruses employ to cleave viral RNA and evade host immune defense systems. Previous structures of Nsp15 from across Coronaviridae revealed that Nsp15 assembles into a homo-hexamer and has a conserved active site similar to RNase A. Beyond a preference for cleaving RNA 3 of uridines, it is unknown if Nsp15 has any additional substrate preferences. Here we used cryo-EM to capture structures of Nsp15 bound to RNA in pre- and post-cleavage states. The structures along with molecular dynamics and biochemical assays revealed critical residues involved in substrate specificity, nuclease activity, and oligomerization. Moreover, we determined how the sequence of the RNA substrate dictates cleavage and found that outside of polyU tracts, Nsp15 has a strong preference for purines 3 of the cleaved uridine. This work advances our understanding of how Nsp15 recognizes and processes viral RNA and will aid in the development of new anti-viral therapeutics.
RESUMEN
New therapeutics are urgently needed to inhibit SARS-CoV-2, the virus responsible for the on-going Covid-19 pandemic. Nsp15, a uridine-specific endoribonuclease found in all coronaviruses, processes viral RNA to evade detection by RNA-activated host defense systems, making it a promising drug target. Previous work with SARS-CoV-1 established that Nsp15 is active as a hexamer, yet how Nsp15 recognizes and processes viral RNA remains unknown. Here we report a series of cryo-EM reconstructions of SARS-CoV-2 Nsp15. The UTP-bound cryo-EM reconstruction at 3.36 [A] resolution provides molecular details into how critical residues within the Nsp15 active site recognize uridine and facilitate catalysis of the phosphodiester bond, whereas the apo-states reveal active site conformational heterogeneity. We further demonstrate the specificity and mechanism of nuclease activity by analyzing Nsp15 products using mass spectrometry. Collectively, these findings advance understanding of how Nsp15 processes viral RNA and provide a structural framework for the development of new therapeutics.
RESUMEN
OBJECTIVE@#To review the management experience of a consecutive series of patients presenting as acute surgical abdomen whom were ultimately diagnosed to have DF (Dengue fever)/DHF (Dengue hemorrhagic fever).@*METHODS@#Clinical data of all cases of apparent acute abdomen (AA) which were later confirmed as having DF/DHF reviewed by two surgical units from December 2012 to December 2013 were analyzed. Initially confirmed patients with DF/DHF who developed abdominal symptoms were not considered.@*RESULTS@#Out of the seventeen cases (7 males, age range 10-71 years) presented with fever and AA; appendicitis, cholecystitis, pancreatitis and non-specific peritonitis were suspected initially in 8, 5, 1 and 3 cases, respectively. Neutropenia or thrombocytopenia signifying DF/DHF occurred only in 11 patients at first evaluation thus six remained as surgical candidates beyond 24 h. One patient underwent appendicectomy with a prolonged hospital stay. DF was confirmed by serology in all patients, latest by fourth day of admission. One required blood product transfusion, 4 needed critical care treatment and there was 1 death.@*CONCLUSIONS@#DF/DHF misleads the clinicians when it presents as AA. Initial hematological and ultrasonographic findings may be equivocal creating a diagnostic and management dilemma. Vigilant clinical suspicion and early dengue serological assessment is advisable in equivocal cases of AAs with fever in dengue endemic areas, to confirm/exclude the infection in order to avoid unnecessary surgical morbidity in the presence of DF.
RESUMEN
Objective To review the management experience of a consecutive series of patients presenting as acute surgical abdomen whom were ultimately diagnosed to have DF (Dengue fever)/DHF (Dengue hemorrhagic fever). Methods Clinical data of all cases of apparent acute abdomen (AA) which were later confirmed as having DF/DHF reviewed by two surgical units from December 2012 to December 2013 were analyzed. Initially confirmed patients with DF/DHF who developed abdominal symptoms were not considered. Results Out of the seventeen cases (7 males, age range 10–71 years) presented with fever and AA; appendicitis, cholecystitis, pancreatitis and non-specific peritonitis were suspected initially in 8, 5, 1 and 3 cases, respectively. Neutropenia or thrombocytopenia signifying DF/DHF occurred only in 11 patients at first evaluation thus six remained as surgical candidates beyond 24 h. One patient underwent appendicectomy with a prolonged hospital stay. DF was confirmed by serology in all patients, latest by fourth day of admission. One required blood product transfusion, 4 needed critical care treatment and there was 1 death. Conclusions DF/DHF misleads the clinicians when it presents as AA. Initial hematological and ultrasonographic findings may be equivocal creating a diagnostic and management dilemma. Vigilant clinical suspicion and early dengue serological assessment is advisable in equivocal cases of AAs with fever in dengue endemic areas, to confirm/exclude the infection in order to avoid unnecessary surgical morbidity in the presence of DF.