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Crimean-Congo hemorrhagic fever virus (CCHFV; family Nairoviridae ) is a tick-borne pathogen that frequently causes lethal disease in humans. CCHFV has a wide geographic distribution, and cases have been reported in Africa, Asia, the Middle East, and Europe. Availability of a safe and efficacious vaccine is critical for restricting outbreaks and preventing disease in endemic countries. We previously developed a virus-like replicon particle (VRP) vaccine that provides complete protection against homologous and heterologous lethal CCHFV challenge in mice after a single dose. However, the immune responses induced by this vaccine are not well characterized, and correlates of protection remain unknown. Here we comprehensively characterized the kinetics of cell-mediated and humoral immune responses in VRP-vaccinated mice, and demonstrate that they predominantly target the nucleoprotein (NP). NP antibodies are not associated with protection through neutralizing activity, but VRP vaccination results in NP antibodies possessing Fc-mediated antibody effector functions, such as complement activation (ADCD) and antibody-mediated cellular phagocytosis (ADCP). This suggests that Fc-mediated effector functions may contribute to this vaccine's efficacy., Competing Interests: Provisional US Patent Application No. 62/780,098. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Scholte, Karaaslan, O’Neal, Sorvillo, Genzer, Welch, Coleman-McCray, Spengler, Kainulainen, Montgomery, Pegan, Bergeron and Spiropoulou.)

Crimean-Congo haemorrhagic fever virus (CCHFV) is a tick-borne virus causing Crimean-Congo haemorrhagic fever (CCHF), a disease reported to have a high fatality rate in numerous countries. The virus is geographically widespread due to its vector, and numerous wild and domestic animals can develop asymptomatic infection. Serological and limited molecular evidence of CCHFV has previously been reported in Camelus dromedarius (the dromedary, or one-humped camel) in the United Arab Emirates (UAE). In this study, 238 camel samples were screened for CCHFV RNA where 16 camel samples were positive for CCHFV by RT-PCR. Analysis of full-length CCHFV genome sequences revealed a novel lineage in camels from the UAE, and potential reassortment of the M segment of the genome.

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., Competing Interests: The authors have declared that no competing interests exist.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus capable of causing a severe hemorrhagic fever disease in humans. There are currently no licensed vaccines to prevent CCHFV-associated disease. We developed a DNA vaccine expressing the M-segment glycoprotein precursor gene of CCHFV and assessed its immunogenicity and protective efficacy in two lethal mouse models of disease: type I interferon receptor knockout (IFNAR-/-) mice; and a novel transiently immune suppressed (IS) mouse model. Vaccination of mice by muscle electroporation of the M-segment DNA vaccine elicited strong antigen-specific humoral immune responses with neutralizing titers after three vaccinations in both IFNAR-/- and IS mouse models. To compare the protective efficacy of the vaccine in the two models, groups of vaccinated mice (7-10 per group) were intraperitoneally (IP) challenged with a lethal dose of CCHFV strain IbAr 10200. Weight loss was markedly reduced in CCHFV DNA-vaccinated mice as compared to controls. Furthermore, whereas all vector-control vaccinated mice succumbed to disease by day 5, the DNA vaccine protected >60% of the animals from lethal disease. Mice from both models developed comparable levels of antibodies, but the IS mice had a more balanced Th1/Th2 response to vaccination. There were no statistical differences in the protective efficacies of the vaccine in the two models. Our results provide the first comparison of these two mouse models for assessing a vaccine against CCHFV and offer supportive data indicating that a DNA vaccine expressing the glycoprotein genes of CCHFV elicits protective immunity against CCHFV.

Unlabelled: 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., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus (genus Nairovirus, family Bunyaviridae) associated with high case fatality disease outbreaks in regions of Africa, Europe, and Asia. The CCHFV genome consists of three negative-strand RNA segments, S, M, and L. The unusually large virus L polymerase protein and the need for biosafety level 4 (BSL-4) containment conditions for work with infectious virus have hampered the study of CCHFV replication. The L protein has an ovarian tumor (OTU) protease domain located in the N terminus, which has led to speculation that the protein may be autoproteolytically cleaved to generate the active virus L polymerase and additional functions. We report the successful development of efficient CCHFV helper virus-independent S, M, and L segment minigenome systems for analysis of virus RNA and protein features involved in replication. The virus RNA segment S, M, and L untranslated regions were found to be similar in support of replication of the respective minigenomes. In addition, the OTU domain located in the N terminus of the expressed virus L protein was shown to be a functional protease. However, no evidence of L protein autoproteolytic processing was found, and the OTU protease activity was dispensable for virus RNA replication. Finally, physiologically relevant doses of ribavirin inhibited CCHFV minigenome replication. These results demonstrated the utility of the minigenome system for use in BSL-2 laboratory settings to analyze CCHFV biology and in antiviral drug discovery programs for this important public health and bioterrorism threat.

Crimean-Congo hemorrhagic fever virus (CCHFV) causes severe human disease. The CCHFV medium RNA encodes a polyprotein which is proteolytically processed to yield the glycoprotein precursors PreGn and PreGc, followed by structural glycoproteins Gn and Gc. Subtilisin kexin isozyme-1/site-1 protease (SKI-1/S1P) plays a central role in Gn processing. Here we show that CCHFV-infected cells deficient in SKI-1/S1P produce no infectious virus, although PreGn and PreGc accumulated normally in the Golgi apparatus, the site of virus assembly. Only nucleoprotein-containing particles which lacked virus glycoproteins (Gn/Gc or PreGn/PreGc) were secreted. Complementation of SKI-1/S1P-deficient cells with a SKI-1/S1P expression vector restored release of infectious virus (>10(6) PFU/ml), confirming that SKI-1/S1P processing is required for incorporation of viral glycoproteins. SKI-1/S1P may represent a promising antiviral target.