The discovery of an emerging viral disease, severe fever with thrombocytopenia syndrome (SFTS), caused by SFTS virus (SFTSV), has prompted the need to understand pathogenesis of SFTSV. We are unique in establishing an infectious model of SFTS in C57/BL6 mice, resulting in hallmark symptoms of thrombocytopenia and leukocytopenia. Viral RNA and histopathological changes were identified in the spleen, liver, and kidney. However, viral replication was only found in the spleen, which suggested the spleen to be the principle target organ of SFTSV. Moreover, the number of macrophages and platelets were largely increased in the spleen, and SFTSV colocalized with platelets in cytoplasm of macrophages in the red pulp of the spleen. In vitro cellular assays further revealed that SFTSV adhered to mouse platelets and facilitated the phagocytosis of platelets by mouse primary macrophages, which in combination with in vivo findings, suggests that SFTSV-induced thrombocytopenia is caused by clearance of circulating virus-bound platelets by splenic macrophages. Thus, this study has elucidated the pathogenic mechanisms of thrombocytopenia in a mouse model resembling human SFTS disease.animal model | phlebovirus | pathology | reduction of platelets | macrophage infection S evere fever with thrombocytopenia syndrome (SFTS) is a recently identified emerging viral infectious disease in China that is caused by a novel phlebovirus in the family Bunyaviridae, SFTS virus (SFTSV) (1). Clinical features of SFTS patients include abrupt high fever, thrombocytopenia, leukocytopenia, and gastrointestinal symptoms. Laboratory tests commonly show elevated serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), lactate dehydrogenase, creatine kinase, creatine kinase MB fraction, as well as elongated activated partial-thromboplastin time (1). These abnormally changed laboratory parameters are indicative of the pathological lesions that occur in multiple organs and altered homeostasis of the coagulation systems of SFTS patients. Pathological studies of SFTS are absent because patient tissues are rarely donated after death in rural areas of China. Therefore, the causes of illness and death, as well as pathological changes within organs, remain unclear. To systematically investigate the pathogenic mechanisms of SFTS and understand key symptoms, such as thrombocytopenia, infectious animal models for SFTSV are urgently needed. ResultsEstablishment of a SFTSV Pathogenic Mouse Model. In our initial study, to identify an infectious animal model that could mimic most clinical features during SFTSV infection, the susceptibilities of three commonly used laboratory rodent strains for phlebovirus (2-4), C57/BL6 mice, BalB/C mice, and Syrian hamsters, were examined. The SFTSV strain HB29 was inoculated at 10 5 TCID 50 (50% tissue culture infective dose) per mouse or 5 × 10 5 TCID 50 per hamster through four different routes of infection, including intravenous, intramuscular, intraperitoneal, and intracerebral...
The papain-like protease (PL pro ) of SARS-CoV-2 is a validated antiviral drug target. Through a fluorescence resonance energy transfer-based high-throughput screening and subsequent lead optimization, we identified several PL pro inhibitors including Jun9-72-2 and Jun9-75-4 with improved enzymatic inhibition and antiviral activity compared to GRL0617 , which was reported as a SARS-CoV PL pro inhibitor. Significantly, we developed a cell-based FlipGFP assay that can be applied to predict the cellular antiviral activity of PL pro inhibitors in the BSL-2 setting. X-ray crystal structure of PL pro in complex with GRL0617 showed that binding of GRL0617 to SARS-CoV-2 induced a conformational change in the BL2 loop to a more closed conformation. Molecular dynamics simulations showed that Jun9-72-2 and Jun9-75-4 engaged in more extensive interactions than GRL0617 . Overall, the PL pro inhibitors identified in this study represent promising candidates for further development as SARS-CoV-2 antivirals, and the FlipGFP-PL pro assay is a suitable surrogate for screening PL pro inhibitors in the BSL-2 setting.
Severe fever with thrombocytopenia syndrome bunyavirus is a newly discovered bunyavirus with high pathogenicity to human. The transmission model has been largely uncharacterized. Investigation on a cluster of severe fever with thrombocytopenia syndrome cases provided evidence of person-to-person transmission through blood contact to the index patient with high serum virus load.
The invitro antiviral activity of lactoferrin against common human coronaviruses and SARS-CoV-2 is mediated by targeting the heparan sulfate co-receptor, Emerging Microbes & Infections,
The main protease (M pro ) of SARS-CoV-2 is a validated antiviral drug target. Several M pro inhibitors have been reported with potent enzymatic inhibition and cellular antiviral activity, including GC376, boceprevir, calpain inhibitors II, and XII, with each containing a reactive warhead that covalently modifies the catalytic Cys145. Coupling structure-based drug design with the one-pot Ugi four-component reaction, we discovered one of the most potent noncovalent inhibitors, 23R (Jun8-76-3A) that is structurally distinct from the canonical M pro inhibitor GC376. Significantly, 23R is highly selective compared with covalent inhibitors such as GC376, especially toward host proteases. The cocrystal structure of SARS-CoV-2 M pro with 23R revealed a previously unexplored binding site located in between the S2 and S4 pockets. Overall, this study discovered 23R, one of the most potent and selective noncovalent SARS-CoV-2 M pro inhibitors reported to date, and a novel binding pocket in M pro that can be explored for inhibitor design.
The main protease (M pro ) is a validated antiviral drug target of SARS-CoV-2. A number of M pro inhibitors have now advanced to animal model study and human clinical trials. However, one issue yet to be addressed is the target selectivity over host proteases such as cathepsin L. In this study we describe the rational design of covalent SARS-CoV-2 M pro inhibitors with novel cysteine reactive warheads including dichloroacetamide, dibromoacetamide, tribromoacetamide, 2-bromo-2,2-dichloroacetamide, and 2-chloro-2,2-dibromoacetamide. The promising lead candidates Jun9-62-2R (dichloroacetamide) and Jun9-88-6R (tribromoacetamide) had not only potent enzymatic inhibition and antiviral activity but also significantly improved target specificity over caplain and cathepsins. Compared to GC-376 , these new compounds did not inhibit the host cysteine proteases including calpain I, cathepsin B, cathepsin K, cathepsin L, and caspase-3. To the best of our knowledge, they are among the most selective covalent M pro inhibitors reported thus far. The cocrystal structures of SARS-CoV-2 M pro with Jun9-62-2R and Jun9-57-3R reaffirmed our design hypothesis, showing that both compounds form a covalent adduct with the catalytic C145. Overall, these novel compounds represent valuable chemical probes for target validation and drug candidates for further development as SARS-CoV-2 antivirals.
Host restriction factors constitute a formidable barrier for viral replication to which many viruses have evolved counter-measures. Human SAMD9, a tumor suppressor and a restriction factor for poxviruses in cell lines, is antagonized by two classes of poxvirus proteins, represented by vaccinia virus (VACV) K1 and C7. A paralog of SAMD9, SAMD9L, is also encoded by some mammals, while only one of two paralogs is retained by others. Here, we show that SAMD9L functions similarly to SAMD9 as a restriction factor and that the two paralogs form a critical host barrier that poxviruses must overcome to establish infection. In mice, which naturally lack SAMD9, overcoming SAMD9L restriction with viral inhibitors is essential for poxvirus replication and pathogenesis. While a VACV deleted of both K1 and C7 (vK1L-C7L-) was restricted by mouse cells and highly attenuated in mice, its replication and virulence were completely restored in SAMD9L-/- mice. In humans, both SAMD9 and SAMD9L are poxvirus restriction factors, although the latter requires interferon induction in many cell types. While knockout of SAMD9 with Crispr-Cas9 was sufficient for abolishing the restriction for vK1L-C7L- in many human cells, knockout of both paralogs was required for abolishing the restriction in interferon-treated cells. Both paralogs are antagonized by VACV K1, C7 and C7 homologs from diverse mammalian poxviruses, but mouse SAMD9L is resistant to the C7 homolog encoded by a group of poxviruses with a narrow host range in ruminants, indicating that host species-specific difference in SAMD9/SAMD9L genes serves as a barrier for cross-species poxvirus transmission.
SARS-CoV-2 main protease (M pro ) is a cysteine protease that mediates the cleavage of viral polyproteins and is a validated antiviral drug target. M pro is highly conserved among all seven human coronaviruses, with certain M pro inhibitors having broad-spectrum antiviral activity. In this study, we designed two hybrid inhibitors UAWJ9-36-1 and UAWJ9-36-3 based on the superimposed X-ray crystal structures of SARS-CoV-2 M pro with GC-376, telaprevir, and boceprevir. Both UAWJ9-36-1 and UAWJ9-36-3 showed potent binding and enzymatic inhibition against the M pro ’s from SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-OC43, HCoV-NL63, HCoV-229E, and HCoV-HKU1. Cell-based Flip-GFP M pro assay results show that UAWJ9-36-1 and UAWJ9-36-3 inhibited the intracellular protease activity of SARS-CoV-2 M pro . In addition, UAWJ9-36-1 and UAWJ9-36-3 had potent antiviral activity against SARS-CoV-2, HCoV-OC43, HCoV-NL63, and HCoV-229E, with UAWJ9-36-3 being more potent than GC-376 in inhibiting SARS-CoV-2. Selectivity profiling revealed that UAWJ9-36-1 and UAWJ9-36-3 had an improved selectivity index over that of GC-376 against host cysteine proteases calpain I and cathepsin L, but not cathepsin K. The X-ray crystal structures of SARS-CoV-2 M pro with UAWJ9-36-1 and UAWJ9-36-3 were both solved at 1.9 Å, which validated our design hypothesis. Overall, hybrid inhibitors UAWJ9-36-1 and UAWJ9-36-3 are promising candidates to be further developed as broad-spectrum coronavirus antivirals.
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