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.

The Third International Conference on Crimean -Congo Hemorrhagic Fever (CCHF) was held in Thessaloniki, Greece, September 19-21, 2023, bringing together a diverse group of international partners, including public health professionals, clinicians, ecologists, epidemiologists, immunologists, and virologists. The conference was attended by 118 participants representing 24 countries and the World Health Organization (WHO). Meeting sessions covered the epidemiology of CCHF in humans; Crimean -Congo hemorrhagic fever virus (CCHFV) in ticks; wild and domestic animal hosts; molecular virology; pathogenesis and animal models; immune response related to therapeutics; and CCHF prevention in humans. The concluding session focused on recent WHO recommendations regarding disease prevention, control strategies, and innovations against CCHFV outbreaks. This meeting report summarizes lectures by the invited speakers and highlights advances in the field.

Plasma membrane–located low-density lipoprotein receptor–related protein 1 (LRP1 or CD91) is a host factor that supports the early infection stages of a spectrum of RNA viruses. Viruses with an RNA genome are often the cause of zoonotic infections. In order to identify novel pro-viral host cell factors, we screened a haploid insertion–mutagenized mouse embryonic cell library for clones that are resistant to Rift Valley fever virus (RVFV). This screen returned the low-density lipoprotein receptor–related protein 1 (LRP1) as a top hit, a plasma membrane protein involved in a wide variety of cell activities. Inactivation of LRP1 in human cells reduced RVFV RNA levels already at the attachment and entry stages of infection. Moreover, the role of LRP1 in promoting RVFV infection was dependent on physiological levels of cholesterol and on endocytosis. In the human cell line HuH-7, LRP1 also promoted early infection stages of sandfly fever Sicilian virus and La Crosse virus, but had a minor effect on late infection by vesicular stomatitis virus, whereas encephalomyocarditis virus was entirely LRP1-independent. Moreover, siRNA experiments in human Calu-3 cells demonstrated that also SARS-CoV-2 infection benefitted from LRP1. Thus, we identified LRP1 as a host factor that supports infection by a spectrum of RNA viruses.

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.

The recent emergence of multiple SARS-CoV-2 variants has caused considerable concern due to reduced vaccine efficacy and escape from neutralizing antibody therapeutics. It is therefore paramount to develop therapeutic strategies that inhibit all known and future SARS-CoV-2 variants. Here we report that all SARS-CoV-2 variants analyzed, including variants of concern (VOC) Alpha, Beta, Gamma, and Delta, exhibit enhanced binding affinity to clinical grade and phase 2 tested recombinant human soluble ACE2 (APN01). Importantly, soluble ACE2 neutralized infection of VeroE6 cells and human lung epithelial cells by multiple VOC strains with markedly enhanced potency when compared to reference SARS-CoV-2 isolates. Effective inhibition of infections with SARS-CoV-2 variants was validated and confirmed in two independent laboratories. These data show that SARS-CoV-2 variants that have emerged around the world, including current VOC and several variants of interest, can be inhibited by soluble ACE2, providing proof of principle of a pan-SARS-CoV-2 therapeutic.

Crimean-Congo hemorrhagic fever virus (CCHFV) is the cause of severe hemorrhagic disease, which is often fatal. Present throughout Africa, Asia, and Southeast Europe, a diverse number of viral genotypes exist.

Viruses with an RNA genome are often the cause of zoonotic infections. In order to identify novel pro-viral host cell factors, we screened a haploid insertion-mutagenized mouse embryonic cell library for clones that rendered them resistant to the zoonotic Rift Valley fever virus (RVFV; family Phleboviridae , order Bunyavirales ). This screen returned the Low Density Lipoprotein Receptor-Related protein 1 (LRP1, or CD91) as top hit, a 600 kDa plasma membrane protein known to be involved in a wide variety of cell activities. Inactivation of LRP1 expression in human cells reduced RVFV RNA levels already at the attachment and entry stages of infection. Moreover, the role of LRP1 in promoting RVFV infection was dependent on physiological levels of cholesterol and on endocytosis. In the highly LRP1-positive human cell line HuH-7, LRP1 also promoted the early infection stages of Sandfly fever Sicilian virus (SFSV; family Phleboviridae , order Bunyavirales ), La Crosse virus (LACV; family Peribunyaviridae , order Bunyavirales ), had a minor effect on RNA levels during the late infection stages by vesicular stomatitis virus (VSV; family Rhabdoviridae , order Mononegavirales ), whereas infection by Encephalomyocarditis virus (EMCV, family Picornaviridae ) was entirely LRP1-independent. Moreover, siRNA experoments in human Calu-3 cells demonstrated that also SARS-CoV-2 infection benefitted from LRP1. Thus, we identified LRP1 as a host factor that supports infection by a spectrum of RNA viruses.

The recent emergence of the SARS-CoV-2 variant Omicron has caused considerable concern due to reduced vaccine efficacy and escape from neutralizing antibody therapeutics. Omicron is spreading rapidly around the globe and is suspected to account for most new COVID-19 cases in several countries, though the severity of Omicron-mediated disease is still under debate. It is therefore paramount to identify therapeutic strategies that inhibit the Omicron SARS-CoV-2 variant. Here we report using 3D structural modelling that Spike of Omicron can still associate with human ACE2. Sera collected after the second mRNA-vaccination did not exhibit a protective effect against Omicron while strongly neutralizing infection of VeroE6 cells with the reference Wuhan strain, confirming recent data by other groups on limited vaccine and convalescent sera neutralization efficacy against Omicron. Importantly, clinical grade recombinant human soluble ACE2, a drug candidate currently in clinical development, potently neutralized Omicron infection of VeroE6 cells with markedly enhanced potency when compared to reference SARS-CoV-2 isolates. These data show that SARS-CoV-2 variant Omicron can be readily inhibited by soluble ACE2, providing proof of principle of a viable and effective therapeutic approach against Omicron infections.

Crimean-Congo Hemorrhagic Fever virus (CCHFV; family Nairoviridae) is an extremely pathogenic member of the Bunyavirales order. Previous studies have shown that the N-terminal domain of the CCHFV polymerase (L) contains an ovarian tumor-type protease (OTU) domain with the capability to remove both ubiquitin and ISG15 molecules from proteins. The approximately 200 amino acids-long OTU domain, if ectopically expressed, can interfere with both the induction of antiviral type I interferons (IFN) as well as the IFN-stimulated signaling. A OTU protease mutant (C40A), by contrast, was inactive in that respect. However, the effect of the OTU protease activity in the context of the full-length L protein (approximately 4000 amino acids) is only poorly characterized, and recombinant CCHFV with the C40A mutation could not be rescued. Here, we employed transcriptionally active virus-like particles (tc-VLPs) to investigate the interaction between the L-embedded OTU protease and the IFN system. Our data show a cis requirement of the OTU protease for optimal CCHFV polymerase activity in human HuH-7 cells. The L-embedded OTU did not influence IFN signaling, the sensitivity to IFN, or IFN induction. Moreover, the attenuation of OTU C40A-mutated L could not be relieved by inactivating the IFN response, but after overexpression of conjugation-competent ISG15 the polymerase activity recovered to wild-type levels. Consequently, ISG15 was used to produce OTU-deficient tc-VLPs, a potential vaccine candidate. Our data thus indicate that in the context of full-length L the OTU domain is important for the regulation of CCHFV polymerase by ISG15.

There is currently no specific prophylaxis or vaccine against Crimean-Congo haemorrhagic fever virus (CCHFV). Crimean-Congo haemorrhagic fever (CCHF) is a severe febrile illness transmitted by Hyalomma ticks in endemic areas, handling of infected livestock or care of infected patients. We report here the successful protection against CCHFV-mediated disease in a non-human primate disease model. Cynomolgus macaques were vaccinated with a DNA-based vaccine using in vivo electroporation-assisted delivery. The vaccine contained two plasmids encoding the glycoprotein precursor (GPC) and the nucleoprotein (NP) of CCHFV. Animals received three vaccinations and we recorded potent antibody and T cell responses after vaccination. While all sham-vaccinated animals developed viraemia, high tissue viral loads and CCHF-induced disease, the NP + GPC vaccinated animals were significantly protected. In conclusion, this is evidence of a vaccine that can protect against CCHFV-induced disease in a non-human primate model. This supports clinical development of the vaccine to protect groups at risk for contracting the infection. A DNA-based vaccine elicits humoral and cellular immunity and provides protection against Crimean-Congo haemorrhagic fever virus-mediated disease in a non-human primate model.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne orthonairovirus that has become a serious threat to the public health. CCHFV has a single-stranded, tripartite RNA genome composed of L, M, and S segments. Cleavage of the M polyprotein precursor generates the two envelope glycoproteins (GPs) as well as three secreted nonstructural proteins GP38 and GP85 or GP160, representing GP38 only or GP38 linked to a mucin-like protein (MLD), and a double-membrane-spanning protein called NSm. Here, we examined the relevance of each M-segment non-structural proteins in virus assembly, egress and infectivity using a well-established CCHFV virus-like-particle system (tc-VLP). Deletion of MLD protein had no impact on infectivity although it reduced by 60% incorporation of GPs into particles. Additional deletion of GP38 abolished production of infectious tc-VLPs. The loss of infectivity was associated with impaired Gc maturation and exclusion from the Golgi, showing that Gn is not sufficient to target CCHFV GPs to the site of assembly. Consistent with this, efficient complementation was achieved in cells expressing MLD-GP38 in trans with increased levels of preGc to Gc conversion, co-targeting to the Golgi, resulting in particle incorporation and restored infectivity. Contrastingly, a MLD-GP38 variant retained in the ER allowed preGc cleavage but failed to rescue miss-localization or infectivity. NSm deletion, conversely, did not affect trafficking of Gc but interfered with Gc processing, particle formation and secretion. NSm expression affected N-glycosylation of different viral proteins most likely due to increased speed of trafficking through the secretory pathway. This highlights a potential role of NSm in overcoming Golgi retention and facilitating CCHFV egress. Thus, deletions of GP38 or NSm demonstrate their important role on CCHFV particle production and infectivity. GP85 is an essential viral factor for preGc cleavage, trafficking and Gc incorporation into particles, whereas NSm protein is involved in CCHFV assembly and virion secretion.

Background Many ruminant diseases of viral aetiology can be effectively prevented using appropriate vaccination measures. For diseases such as Rift Valley fever (RVF) the long inter-epizootic periods make routine vaccination programs unfeasible. Coupling RVF prophylaxis with seasonal vaccination programmes by means of multivalent vaccine platforms would help to reduce the risk of new RVF outbreaks. Methodology/Principal findings In this work we generated recombinant attenuated Rift Valley fever viruses (RVFVs) encoding in place of the virulence factor NSs either the VP2 capsid protein or a truncated form of the non-structural NS1 protein of bluetongue virus serotype 4 (BTV-4). The recombinant viruses were able to carry and express the heterologous BTV genes upon consecutive passages in cell cultures. In murine models, a single immunization was sufficient to protect mice upon RVFV challenge and to elicit a specific immune response against BTV-4 antigens that was fully protective after a BTV-4 boost. In sheep, a natural host for RVFV and BTV, both vaccines proved immunogenic although conferred only partial protection after a virulent BTV-4 reassortant Morocco strain challenge. Conclusions/Significance Though additional optimization will be needed to improve the efficacy data against BTV in sheep, our findings warrant further developments of attenuated RVFV as a dual vaccine platform carrying heterologous immune relevant antigens for ruminant diseases in RVF risk areas.

Toscana virus (TOSV) is an arthropod-borne human pathogen responsible for seasonal outbreaks of fever and meningoencephalitis in the Mediterranean basin. TOSV is a segmented negative-strand RNA virus (sNSV) that belongs to the genus phlebovirus (family Phenuiviridae, order Bunyavirales), encompassing other important human pathogens such as Rift Valley fever virus (RVFV). Here, we carried out a structural and functional characterization of the TOSV cap-snatching endonuclease, an N terminal domain of the viral polymerase (L protein) that provides capped 3'OH primers for transcription. We report TOSV endonuclease crystal structures in the apo form, in complex with a di-ketoacid inhibitor (DPBA) and in an intermediate state of inhibitor release, showing details on substrate binding and active site dynamics. The structure reveals substantial folding rearrangements absent in previously reported cap-snatching endonucleases. These include the relocation of the N terminus and the appearance of new structural motifs important for transcription and replication. The enzyme shows high activity rates comparable to other His+ cap-snatching endonucleases. Moreover, the activity is dependent on conserved residues involved in metal ion and substrate binding. Altogether, these results bring new light on the structure and function of cap-snatching endonucleases and pave the way for the development of specific and broad-spectrum antivirals.

Dear Editor,Crimean-Congo hemorrhagic fever (CCHF) is an emerging tick-borne viral disease widely distributed across countries of Africa, Southern Europe, the Middle East, and Asia1. CCHF is caused...

ABSTRACT Crimean-Congo hemorrhagic fever virus (CCHFV) is a bunyavirus causing severe hemorrhagic fever disease in humans, with high mortality rates. The requirement of a high-containment laboratory and the lack of an animal model hampered the study of the immune response and protection of vaccine candidates. Using the recently developed interferon alpha receptor knockout (IFNAR −/− ) mouse model, which replicates human disease, we investigated the immunogenicity and protection of two novel CCHFV vaccine candidates: a DNA vaccine encoding a ubiquitin-linked version of CCHFV Gc, Gn, and N and one using transcriptionally competent virus-like particles (tc-VLPs). In contrast to most studies that focus on neutralizing antibodies, we measured both humoral and cellular immune responses. We demonstrated a clear and 100% efficient preventive immunity against lethal CCHFV challenge with the DNA vaccine. Interestingly, there was no correlation with the neutralizing antibody titers alone, which were higher in the tc-VLP-vaccinated mice. However, the animals with a lower neutralizing titer, but a dominant cell-mediated Th1 response and a balanced Th2 response, resisted the CCHFV challenge. Moreover, we found that in challenged mice with a Th1 response (immunized by DNA/DNA and boosted by tc-VLPs), the immune response changed to Th2 at day 9 postchallenge. In addition, we were able to identify new linear B-cell epitope regions that are highly conserved between CCHFV strains. Altogether, our results suggest that a predominantly Th1-type immune response provides the most efficient protective immunity against CCHFV challenge. However, we cannot exclude the importance of the neutralizing antibodies as the surviving immunized mice exhibited substantial amounts of them. IMPORTANCE Crimean-Congo hemorrhagic fever virus (CCHFV) is responsible for hemorrhagic diseases in humans, with a high mortality rate. There is no FDA-approved vaccine, and there are still gaps in our knowledge of the immune responses to infection. The recently developed mouse models mimic human CCHF disease and are useful to study the immunogenicity and the protection by vaccine candidates. Our study shows that mice vaccinated with a specific DNA vaccine were fully protected. Importantly, we show that neutralizing antibodies are not sufficient for protection against CCHFV challenge but that an extra Th1-specific cellular response is required. Moreover, we describe the identification of five conserved B-cell epitopes, of which only one was previously known, that could be of great importance for the development of diagnostics tools and the improvement of vaccine candidates.

Purpose: Rift Valley fever virus (RVFV, family Bunyaviridae, genus Phlebovirus) is currently referenced by the WHO as one of the 8 emerging pathogens likely to cause serious outbreaks in the near future and for which few medical countermeasures exist. Transmitted by mosquitoes, RVFV causes large and devastating epidemics in Africa and Arabia, leading to hemorrhages and massive abortions in livestock ruminants, and hepatitis and hemorrhages in 10-20% of human hospitalized patients. While some RVFV proteins are known to interfere with apoptosis and antiviral signalling, a knowledge gap exists concerning the host cellular factors required for the virus cycle. Using a forward genetic screen, we aim to identify those host genes and pathways essential for RVFV entry, transcription and replication. Methods & Materials: Haploid organisms allow the study of gene knockouts, since recessive mutations will show a clear phenotype due to the absence of a second gene copy. Combined with the pluripotency of embryonic stem cells, the effect of the knockout on viral cycle can be assessed in any disease-relevant cell type. We used the recently developed mammalian haploid embryonic stem cells (mESCs) (Elling, 2011, Cell stem cell), which were mutagenized with a revertible genetrap vector, for infection with the RVFV MP-12 vaccine strain. Results: While wild-type cells massively died upon infection, a higher number of cells survived within the mutagenized cells. Surviving cells were submitted to next generation sequencing, and statistical analysis gave a list of 5 genes potentially involved in RVFV MP-12 cycle, but not essential for normal cell life. Those genes will be validated by infection of specific knockout mESCs clones (and their reverted version) by different strains of RVFV. Conclusion: Such an approach allows the identification with a great confidence of important host cofactors to RVFV, increasing our knowledge on the viral cycle, and giving access to several potential targets for antiviral design.

Crimean Congo hemorrhagic fever virus (CCHFV) is a highly virulent tick-borne pathogen that causes hemorrhagic fever in humans. The geographic range of human CCHF cases largely reflects the presence of ticks. However, highly similar CCHFV lineages occur in geographically distant regions. Tick-infested migratory birds have been suggested, but not confirmed, to contribute to the dispersal. Bats have recently been shown to carry nairoviruses distinct from CCHFV. In order to assess the presence of CCHFV in a wide range of bat species over a wide geographic range, we analyzed 1,135 sera from 16 different bat species collected in Congo, Gabon, Ghana, Germany, and Panama. Using a CCHFV glycoprotein-based indirect immunofluorescence test (IIFT), we identified reactive antibodies in 10.0% (114/1,135) of tested bats, pertaining to 12/16 tested species. Depending on the species, 3.6%–42.9% of cave-dwelling bats and 0.6%–7.1% of foliage-living bats were seropositive (two-tailed t-test, p = 0.0447 cave versus foliage). 11/30 IIFT-reactive sera from 10 different African bat species had neutralizing activity in a virus-like particle assay. Neutralization of full CCHFV was confirmed in 5 of 7 sera. Widespread infection of cave-dwelling bats may indicate a role for bats in the life cycle and geographic dispersal of CCHFV.

ABSTRACT Crimean-Congo hemorrhagic fever virus (CCHFV; genus Nairovirus) is an extremely pathogenic member of the Bunyaviridae family. Since handling of the virus requires a biosafety level 4 (BSL-4) facility, little is known about pathomechanisms and host interactions. Here, we describe the establishment of a transcriptionally competent virus-like particle (tc-VLP) system for CCHFV. Recombinant polymerase (L), nucleocapsid protein (N) and a reporter minigenome expressed in human HuH-7 cells resulted in formation of transcriptionally active nucleocapsids that could be packaged by coexpressed CCHFV glycoproteins into tc-VLPs. The tc-VLPs resembled authentic virus particles in their protein composition and neutralization sensitivity to anti-CCHFV antibodies and could recapitulate all steps of the viral replication cycle. Particle attachment, entry, and primary transcription were modeled by infection of naive cells. The subsequent steps of genome replication, secondary transcription, and particle assembly and release can be obtained upon passaging the tc-VLPs on cells expressing CCHFV structural proteins. The utility of the VLP system was demonstrated by showing that the endonuclease domain of L is located around amino acid D693, as was predicted in silico by B. Morin et al. (PLoS Pathog 6:e1001038, 2010, http://dx.doi.org/10.1371/journal.ppat.1001038). The tc-VLP system will greatly facilitate studies and diagnostics of CCHFV under non-BSL-4 conditions. IMPORTANCE Crimean-Congo hemorrhagic fever virus (CCHFV) is an extremely virulent pathogen of humans. Since the virus can be handled only at the highest biosafety level, research is restricted to a few specialized laboratories. We developed a plasmid-based system to produce virus-like particles with the ability to infect cells and transcribe a reporter genome. Due to the absence of viral genes, the virus-like particles are unable to spread or cause disease, thus allowing study of aspects of CCHFV biology under relaxed biosafety conditions.

Toscana virus is an emerging bunyavirus in Mediterranean Europe where it accounts for 80% of pediatric meningitis cases during the summer. The negative-strand ribonucleic acid (RNA) genome of the virus is wrapped around the virally encoded nucleoprotein N to form the ribonucleoprotein complex (RNP). We determined crystal structures of hexameric N alone (apo) and in complex with a nonameric single-stranded RNA. RNA is sequestered in a sequence-independent fashion in a deep groove inside the hexamer. At the junction between two adjacent copies of Ns, RNA binding induced an inter-subunit rotation, which opened the RNA-binding tunnel and created a new assembly interface at the outside of the hexamer. Based on these findings, we suggest a structural model for how binding of RNA to N promotes the formation of helical RNPs, which are a characteristic hallmark of many negative-strand RNA viruses.