11 research outputs found
The DEVD motif of Crimean-Congo hemorrhagic fever virus nucleoprotein is essential for viral replication in tick cells
No full textThe DEVD motif of Crimean-Congo hemorrhagic fever virus nucleoprotein is essential for viral replication in tick cell
Recombinant Rift Valley fever viruses encoding bluetongue virus (BTV) antigens: Immunity and efficacy studies upon a BTV-4 challenge.
No full textBackgroundMany 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 findingsIn 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/significanceThough 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
ISG15 overexpression compensates the defect of Crimean-Congo hemorrhagic fever virus polymerase bearing a protease-inactive ovarian tumor domain.
No full textCrimean-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
Low-density lipoprotein receptor–related protein 1 (LRP1) as an auxiliary host factor for RNA viruses
No full textStructure of Crimean-Congo hemorrhagic fever virus nucleoprotein: superhelical homo-oligomers and the role of caspase-3 cleavage
No full textCrimean-Congo hemorrhagic fever, a severe hemorrhagic disease found throughout Africa, Europe, and Asia, is caused by the tick-borne Crimean-Congo hemorrhagic fever virus (CCHFV). CCHFV is a negative-sense single-stranded RNA (ssRNA) virus belonging to the Nairovirus genus of the Bunyaviridae family. Its genome of three single-stranded RNA segments is encapsidated by the nucleocapsid protein (CCHFV N) to form the ribonucleoprotein complex. This ribonucleoprotein complex is required during replication and transcription of the viral genomic RNA. Here, we present the crystal structures of the CCHFV N in two distinct forms, an oligomeric form comprised of double antiparallel superhelices and a monomeric form. The head-to-tail interaction of the stalk region of one CCHFV N subunit with the base of the globular body of the adjacent subunit stabilizes the helical organization of the oligomeric form of CCHFV N. It also masks the conserved caspase-3 cleavage site present at the tip of the stalk region from host cell caspase-3 interaction and cleavage. By incubation with primer-length ssRNAs, we also obtained the crystal structure of CCHFV N in its monomeric form, which is similar to a recently published structure. The conformational change of CCHFV N upon deoligomerization results in the exposure of the caspase-3 cleavage site and subjects CCHFV N to caspase-3 cleavage. Mutations of this cleavage site inhibit cleavage by caspase-3 and result in enhanced viral polymerase activity. Thus, cleavage of CCHFV N by host cell caspase-3 appears to be crucial for controlling viral RNA synthesis and represents an important host defense mechanism against CCHFV infection.link_to_OA_fulltex
The interplays between Crimean-Congo hemorrhagic fever virus (CCHFV) M segment-encoded accessory proteins and structural proteins promote virus assembly and infectivity
No full textInternational audienceCrimean-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
Genome-Wide Expression Profiling Deciphers Host Responses Altered during Dengue Shock Syndrome and Reveals the Role of Innate Immunity in Severe Dengue
No full textInternational audienceBackgroundDeciphering host responses contributing to dengue shock syndrome (DSS), the life-threatening form of acute viral dengue infections, is required to improve both the differential prognosis and the treatments provided to DSS patients, a challenge for clinicians.Methodology/Principal FindingsBased on a prospective study, we analyzed the genome-wide expression profiles of whole blood cells from 48 matched Cambodian children: 19 progressed to DSS while 16 and 13 presented respectively classical dengue fever (DF) or dengue hemorrhagic fever grades I/II (DHF). Using multi-way analysis of variance (ANOVA) and adjustment of p-values to control the False Discovery Rate (FDR<10%), we identified a signature of 2959 genes differentiating DSS patients from both DF and DHF, and showed a strong association of this DSS-gene signature with the dengue disease phenotype. Using a combined approach to analyse the molecular patterns associated with the DSS-gene signature, we provide an integrative overview of the transcriptional responses altered in DSS children. In particular, we show that the transcriptome of DSS children blood cells is characterized by a decreased abundance of transcripts related to T and NK lymphocyte responses and by an increased abundance of anti-inflammatory and repair/remodeling transcripts. We also show that unexpected pro-inflammatory gene patterns at the interface between innate immunity, inflammation and host lipid metabolism, known to play pathogenic roles in acute and chronic inflammatory diseases associated with systemic vascular dysfunction, are transcriptionnally active in the blood cells of DSS children.Conclusions/SignificanceWe provide a global while non exhaustive overview of the molecular mechanisms altered in of DSS children and suggest how they may interact to lead to final vascular homeostasis breakdown. We suggest that some mechanisms identified should be considered putative therapeutic targets or biomarkers of progression to DSS
Third International Conference on Crimean-Congo Hemorrhagic Fever in Thessaloniki, Greece, September 19-21, 2023.
No full textThe 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
Immunization with DNA Plasmids Coding for Crimean-Congo Hemorrhagic Fever Virus Capsid and Envelope Proteins and/or Virus-Like Particles Induces Protection and Survival in Challenged Mice
No full textCrimean-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 FDAapproved 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.Funding Agencies|European Commission under the Health Cooperation Work Programme [260427]; European Unions Horizon research and innovation program [732732]</p
Crimean-Congo haemorrhagic fever virus uses LDLR to bind and enter host cells
No full textClimate 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. Laboratory and clinical strains of Crimean-Congo haemorrhagic fever virus use LDLR to bind and enter host cells in blood vessel organoids and mice. Infection can also occur through ApoE, possibly present on virus particles
