Silver N-heterocyclic carbene complexes are potent uncompetitive inhibitors of the papain-like protease with antiviral activity against SARS-CoV-2

The ongoing SARS-CoV-2 pandemic has caused a high demand for novel innovative antiviral drug candidates. Despite promising results, metal complexes have been relatively unexplored as antiviral agents in general and in particular against SARS-CoV-2. Here we report on silver NHC complexes with chloride or iodide counter ligands that are potent inhibitors of the SARS-CoV-2 papain-like protease (PLpro) but inactive against 3C-like protease (3CL(pro)) as another SARS-CoV-2 protease. Mechanistic studies on a selected complex confirmed zinc removal from a zinc binding domain of PLpro as relevant factor of their activity. In addition, enzyme kinetic experiments revealed that the complex is an uncompetitive inhibitor and with this rare type of inhibition it offers great pharmacological advantages in terms selectivity. The silver NHC complexes with iodide ligands showed very low or absent host cell toxicity and triggered strong effects on viral replication in cells infected with SARS-CoV-2, making them promising future antiviral drug candidates.

Novel SARS-CoV-2 variants induce higher toxicity in cardiovascular cells

Objective: SARS-CoV-2 causes the coronavirus disease 2019 (COVID-19) and has spawned a global health crisis. Virus infection can lead to elevated markers of cardiac injury and inflammation associated with a higher risk of mortality. However, it is so far unclear whether cardiovascular damage is caused by direct virus infection or is mainly secondary due to inflammation. Recently, additional novel SARS-CoV-2 variants have emerged accounting for more than 70% of all cases in Germany. To what extend these variants differ from the original strain in their pathology remains to be elucidated. Here, we investigated the effect of the novel SARS-CoV-2 variants on cardiovascular cells. Results: To study whether cardiovascular cells are permissive for SARSCoV-2, we inoculated human iPS-derived cardiomyocytes and endothelial cells from five different origins, including umbilical vein endothelial cells, coronary artery endothelial cells (HCAEC), cardiac and lung microvascular endothelial cells, or pulmonary arterial cells, in vitro with SARS-CoV-2 isolates (G614 (original strain), B.1.1.7 (British variant), B.1.351 (South African variant) and P.1 (Brazilian variant)). While the original virus strain infected iPS-cardiomyocytes and induced cell toxicity 96h post infection (290±10 cells vs. 130±10 cells;p=0.00045), preliminary data suggest a more severe infection by the novel variants. To what extend the response to the novel variants differ from the original strain is currently investigated by phosphoproteom analysis. Of the five endothelial cells studied, only human coronary artery EC took up the original virus strain, without showing viral replication and cell toxicity. Spike protein was only detected in the perinuclear region and was co-localized with calnexin-positive endosomes, which was accompanied by elevated ER-stress marker genes, such as EDEM1 (1.5±0.2-fold change;p=0.04). Infection with the novel SARS-CoV-2 variants resulted in significant higher levels of viral spike compared to the current strain. Surprisingly, viral up-take was also seen in other endothelial cell types (e.g. HUVEC). Although no viral replication was observed (850±158 viral RNA copies at day 0 vs. 197±43 viral RNA copies at day 3;p=0.01), the British SARS-CoV-2 variant B.1.1.7 reduced endothelial cell numbers (0.63±0.03-fold change;p=0.0001). Conclusion: Endothelial cells and cardiomyocytes showed a distinct response to SARS-CoV-2. Whereas cardiomyocytes were permissively infected, endothelial cells took up the virus, but were resistant to viral replication. However, both cell types showed signs of increased toxicity induced by the British SARS-CoV-2 variant. These data suggest that cardiac complications observed in COVID-19 patients might at least in part be based on direct infection of cardiovascular cells. The more severe cytotoxic effects of the novel variants implicate that patients infected with the new variants should be even more closely monitored.

Detection and infectivity of SARS-CoV-2 in exhumated corpses.

Postmortem detection of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) after the exhumation of a corpse can become important, e.g. in the case of subsequent medical malpractice allegations. To date, data on possible detection periods [e.g. by reverse transcription polymerase chain reaction (RT-PCR)] or on the potential infectivity of the virus after an exhumation are rare. In the present study, these parameters were examined in two cases with a time span of approximately 4 months between day of death and exhumation. Using SARS-CoV-2 RT-PCR on swabs of both lungs and the oropharynx detection was possible with cycle threshold (Ct) values of about 30 despite signs of beginning decay. RT-PCR testing of perioral and perinasal swabs and swabs collected from the inside of the body bag, taken to estimate the risk of infection of those involved in the exhumation, was negative. Cell culture-based infectivity testing was negative for both, lung and oropharyngeal swabs. In one case, RT-PCR testing at the day of death of an oropharyngeal swab showed almost identical Ct values as postmortem testing of an oropharyngeal swab, impressively demonstrating the stability of viral RNA in the intact corpse. However, favorable climatic conditions in the grave have to be taken into account, as it was wintertime with constant low temperatures. Nevertheless, it was possible to demonstrate successful postmortem detection of SARS-CoV-2 infection following exhumation even after months in an earth grave.

Infectivity of deceased COVID-19 patients.

The duration of infectivity of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) in living patients has been demarcated. In contrast, a possible SARS-CoV-2 infectivity of corpses and subsequently its duration under post mortem circumstances remain to be elucidated. The aim of this study was to investigate the infectivity and its duration of deceased COVID-19 (coronavirus disease) patients. Four SARS-CoV-2 infected deceased patients were subjected to medicolegal autopsy. Post mortem intervals (PMI) of 1, 4, 9 and 17 days, respectively, were documented. During autopsy, swabs and organ samples were taken and examined by RT-qPCR (real-time reverse transcription-polymerase chain reaction) for the detection of SARS-CoV-2 ribonucleic acid (RNA). Determination of infectivity was performed by means of virus isolation in cell culture. In two cases, virus isolation was successful for swabs and tissue samples of the respiratory tract (PMI 4 and 17 days). The two infectious cases showed a shorter duration of COVID-19 until death than the two non-infectious cases (2 and 11 days, respectively, compared to > 19 days), which correlates with studies of living patients, in which infectivity could be narrowed to about 6 days before to 12 days after symptom onset. Most notably, infectivity was still present in one of the COVID-19 corpses after a post-mortem interval of 17 days and despite already visible signs of decomposition. To prevent SARS-CoV-2 infections in all professional groups involved in the handling and examination of COVID-19 corpses, adequate personal safety standards (reducing or avoiding aerosol formation and wearing FFP3 [filtering face piece class 3] masks) have to be enforced for routine procedures.