Inhibition of SARS-CoV-2 Infections in Engineered Human Tissues Using Clinical-Grade Soluble Human ACE2.

We have previously provided the first genetic evidence that angiotensin converting enzyme 2 (ACE2) is the critical receptor for severe acute respiratory syndrome coronavirus (SARS-CoV), and ACE2 protects the lung from injury, providing a molecular explanation for the severe lung failure and death due to SARS-CoV infections. ACE2 has now also been identified as a key receptor for SARS-CoV-2 infections, and it has been proposed that inhibiting this interaction might be used in treating patients with COVID-19. However, it is not known whether human recombinant soluble ACE2 (hrsACE2) blocks growth of SARS-CoV-2. Here, we show that clinical grade hrsACE2 reduced SARS-CoV-2 recovery from Vero cells by a factor of 1,000-5,000. An equivalent mouse rsACE2 had no effect. We also show that SARS-CoV-2 can directly infect engineered human blood vessel organoids and human kidney organoids, which can be inhibited by hrsACE2. These data demonstrate that hrsACE2 can significantly block early stages of SARS-CoV-2 infections.

P53-Independent G1-Cell Cycle Arrest Increases SARS-CoV-2 RNA Replication.

While having already killed more than 7 million of people worldwide in 4 years, SARS-CoV-2, the etiological agent of COVID-19, is still circulating and evolving. Understanding the pathogenesis of the virus is of capital importance. It was shown that in vitro and in vivo infection with SARS-CoV-2 can lead to cell cycle arrest but the effect of the cell cycle arrest on the virus infection and the associated mechanisms are still unclear. By stopping cells in the G1 phase as well as targeting several pathways involved using inhibitors and small interfering RNAs, we were able to determine that the cell cycle arrest in the late G1 is beneficial for SARS-CoV-2 replication. This cell cycle arrest is independent of p53 but is dependent on the CDC25A-CDK2/cyclin E pathway. These data give a new understanding in SARS-CoV-2 pathogenesis and highlight some possible targets for the development of novel therapeutic approaches.

Crimean-Congo haemorrhagic fever virus uses LDLR to bind and enter host cells.

Climate change and population densities accelerated transmission of highly pathogenic viruses to humans, including the Crimean-Congo haemorrhagic fever virus (CCHFV). Here we report that the Low Density Lipoprotein Receptor (LDLR) is a critical receptor for CCHFV cell entry, playing a vital role in CCHFV infection in cell culture and blood vessel organoids. The interaction between CCHFV and LDLR is highly specific, with other members of the LDLR protein family failing to bind to or neutralize the virus. Biosensor experiments demonstrate that LDLR specifically binds the surface glycoproteins of CCHFV. Importantly, mice lacking LDLR exhibit a delay in CCHFV-induced disease. Furthermore, we identified the presence of Apolipoprotein E (ApoE) on CCHFV particles. Our findings highlight the essential role of LDLR in CCHFV infection, irrespective of ApoE presence, when the virus is produced in tick cells. This discovery holds profound implications for the development of future therapies against CCHFV.

Identification of CCZ1 as an essential lysosomal trafficking regulator in Marburg and Ebola virus infections.

Marburg and Ebola filoviruses are two of the deadliest infectious agents and several outbreaks have occurred in the last decades. Although several receptors and co-receptors have been reported for Ebola virus, key host factors remain to be elucidated. In this study, using a haploid cell screening platform, we identify the guanine nucleotide exchange factor CCZ1 as a key host factor in the early stage of filovirus replication. The critical role of CCZ1 for filovirus infections is validated in 3D primary human hepatocyte cultures and human blood-vessel organoids, both critical target sites for Ebola and Marburg virus tropism. Mechanistically, CCZ1 controls early to late endosomal trafficking of these viruses. In addition, we report that CCZ1 has a role in the endosomal trafficking of endocytosis-dependent SARS-CoV-2 infections, but not in infections by Lassa virus, which enters endo-lysosomal trafficking at the late endosome stage. Thus, we have identified an essential host pathway for filovirus infections in cell lines and engineered human target tissues. Inhibition of CCZ1 nearly completely abolishes Marburg and Ebola infections. Thus, targeting CCZ1 could potentially serve as a promising drug target for controlling infections caused by various viruses, such as SARS-CoV-2, Marburg, and Ebola.

Redirecting Imipramine against Bluetongue Virus Infection: Insights from a Genome-wide Haploid Screening Study.

Bluetongue virus (BTV), an arbovirus of ruminants, is a causative agent of numerous epidemics around the world. Due to the emergence of novel reassortant BTV strains and new outbreaks, there is an unmet need for efficacious antivirals. In this study, we used an improved haploid screening platform to identify the relevant host factors for BTV infection. Our screening tool identified and validated the host factor Niemann-Pick C1 (NPC1), a lysosomal membrane protein that is involved in lysosomal cholesterol transport, as a critical factor in BTV infection. This finding prompted us to investigate the possibility of testing imipramine, an antidepressant drug known to inhibit NPC1 function by interfering with intracellular cholesterol trafficking. In this study, we evaluated the sensitivity of BTV to imipramine using in vitro assays. Our results demonstrate that imipramine pretreatment inhibited in vitro replication and progeny release of BTV-4, BTV-8, and BTV-16. Collectively, our findings highlight the importance of NPC1 for BTV infection and recommend the reprofiling of imipramine as a potential antiviral drug against BTV.

Serological and molecular study of Crimean-Congo Hemorrhagic Fever Virus in cattle from selected districts in Uganda.

BACKGROUND: Crimean-Congo Hemorrhagic Fever (CCHF) is a severe tick-borne viral hemorrhagic disease caused by Crimean-Congo Hemorrhagic Fever Virus (CCHFV) that poses serious public health challenges in many parts of Africa, Europe and Asia. METHODS: We examined 500 cattle sera samples from five districts for CCHFV antibodies using in-house and commercially available (IDVet) ELISA, Immunofluorescent assay (IFA) and Real-time polymerase chain reaction (RT-PCR). RESULTS: 500 cattle (73.8 % females) were analyzed; CCHFV seropositivity was 12.6 % (n = 63) and 75.0 % (n = 375) with the in-house and IDVet ELISAs, respectively. Seropositivity was associated with geographical location, increasing age, being female, and having a higher tick burden. Twenty four out of the 37 (64.8 %) were seropositive for CCHFV using IFA and all were negative for virus on RT-PCR. The IFA results were more comparable to IDVet (κcoefficient = 0.88, p = <0.01) than to in-house (κcoefficient = 0.32, p = 0.02). CONCLUSIONS: Our study confirmed the presence and high prevalence of anti-CCHF antibodies in cattle based on three methods from all the five study districts, confirming presence and exposure of CCHFV. Given the zoonotic potential for CCHFV, we recommend a multidisciplinary public health surveillance and epidemiology of CCHFV in both animals and humans throughout the country.

Human soluble ACE2 improves the effect of remdesivir in SARS-CoV-2 infection.

There is a critical need for safe and effective drugs for COVID-19. Only remdesivir has received authorization for COVID-19 and has been shown to improve outcomes but not decrease mortality. However, the dose of remdesivir is limited by hepatic and kidney toxicity. ACE2 is the critical cell surface receptor for SARS-CoV-2. Here, we investigated additive effect of combination therapy using remdesivir with recombinant soluble ACE2 (high/low dose) on Vero E6 and kidney organoids, targeting two different modalities of SARS-CoV-2 life cycle: cell entry via its receptor ACE2 and intracellular viral RNA replication. This combination treatment markedly improved their therapeutic windows against SARS-CoV-2 in both models. By using single amino-acid resolution screening in haploid ES cells, we report a singular critical pathway required for remdesivir toxicity, namely, Adenylate Kinase 2. The data provided here demonstrate that combining two therapeutic modalities with different targets, common strategy in HIV treatment, exhibit strong additive effects at sub-toxic concentrations. Our data lay the groundwork for the study of combinatorial regimens in future COVID-19 clinical trials.