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.

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. 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). 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). 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.

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.

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.

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.

Emerging viral infections continue to challenge global health and highlight the importance of understanding host-virus interactions that determine infection and disease pathogenesis. This thesis investigates host factors that regulate viral entry, intracellular trafficking, and replication, with a primary focus on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and additional studies on filoviruses and Crimean-Congo hemorrhagic fever virus (CCHFV). Using a haploid genetic screening approach, human organoid models, and mechanistic analysis, the work examines host-dependent pathways that influence viral infection and validate potential antiviral strategies.The initial studies explored therapeutic targeting of viral entry. Human recombinant soluble angiotensin-converting enzyme 2 (hrsACE2) inhibited SARS-CoV-2 infection in cell cultures and in human blood vessel and kidney organoids, demonstrating that receptor-decoy strategies can block viral entry in physiologically relevant models. Combination treatment with hrsACE2 and the RNA-dependent RNA polymerase inhibitor remdesivir further reduced viral RNA levels and infectious virus production, supporting the benefit of targeting multiple stages of the viral life cycle.To identify host pathways required for viral entry, haploid genetic screening was performed. CCZ1, a regulator of endosomal maturation, was identified as a host factor required for viruses that utilize endosomal entry. Loss of CCZ1 disrupted the endosomal pathway, resulting in strong inhibition of Marburg and Ebola virus infection and reduced endocytosis-dependent SARS-CoV-2 entry.At the level of viral attachment, a chemically mutagenized haploid screen identified the low-density lipoprotein receptor (LDLR) as an entry receptor for CCHFV. LDLR directly interacted with the viral glycoprotein Gc, and soluble LDLR inhibited infection in a dose-dependent manner. LDLR-dependent infection was confirmed in human vascular organoids and supported by in vivo observations, demonstrating receptor-mediated entry.Finally, a chemically mutagenized haploid screen identified MED13 and DDX60 as host factors influencing SARS-CoV-2 infection. Functional analysis indicated roles for transcriptional regulation and interferon-associated pathways in modulating viral replication.Together, these studies characterize host-dependent mechanisms that influence viral entry and replication and demonstrate how genetic screening and human relevant infection models can identify host determinants of viral susceptibility. Understanding host-virus interactions like these can inform the development of antiviral strategies and are vital for preparedness for emerging viral diseases in the years to come.List of scientific papersThis thesis is based on the following papers, which are referred to in the text by their Roman numerals.I. Monteil V, Kwon H, Prado P, Hagelkrüys A, Wimmer RA, Stahl M, et al. Inhibition of SARS-CoV-2 infections in engineered human tissues using clinical-grade soluble human ACE2. Cell. 2020; 181(4):905–913.e7. https://doi.org/10.1016/j.cell.2020.04.004II. Monteil V, Dyczynski M, Lauschke VM, Kwon H, Wirnsberger G, Youhanna S, et al. Human soluble ACE2 improves the effect of remdesivir in SARS-CoV-2 infection. EMBO Molecular Medicine. 2021; 13(1): e13426. https://doi.org/10.15252/emmm.202013426III. Monteil V, Kwon H, John L, Salata C, Jonsson G, Vorrink SU, Appelberg S, Youhanna S, Dyczynski M, Leopoldi A, Leeb N, Volz J, Hagelkruys A, Kellner MJ, Devignot S, Michlits G, Foong-Sobis M, Weber F, Lauschke VM, Horn M, Feldmann H, Elling U, Penninger JM, Mirazimi A. Identification of CCZ1 as an essential lysosomal trafficking regulator in Marburg and Ebola virus infections. Nature Communications. 2023; 14:6785. https://doi.org/10.1038/s41467-023-42526-6IV. Monteil VM, Wright SC, Dyczynski M, Kellner MJ, Appelberg S, Platzer SW, Ibrahim A, Kwon H, Pittarokoilis I, Mirandola M, Michlits G, Devignot S, Elder E, Abdurahman S, Bereczky S, Bagci B, Youhanna S, Aastrup T, Lauschke VM, Salata C, Elaldi N, Weber F, Montserrat N, Hawman DW, Feldmann H, Horn M, Penninger JM, Mirazimi A. Crimean–Congo haemorrhagic fever virus uses LDLR to bind and enter host cells. Nature Microbiology. 2024; 9:1499–1512. https://doi.org/10.1038/s41564-024-01672-3V. Kwon H, Tim Wai ST, Michlits G, John L, Kellner MJ, Horn M, Weber F, Penninger JM, Monteil V, Mirazimi A. Identification of MED13 and DDX60 as important proviral host factors for SARS-CoV-2 infection using a haploid screening. Manuscript submitted to Journal of General Virology. 2026. [Submitted]