Human MxA protein inhibits the replication of Crimean-Congo hemorrhagic fever virus

Crimean-Congo hemorrhagic fever virus (CCHFV) belongs to the genus Nairovirus within the family Bunyaviridae and is the causative agent of severe hemorrhagic fever. Despite increasing knowledge about hemorrhagic fever viruses, the factors determining their pathogenicity are still poorly understood. The interferon-induced MxA protein has been shown to have an inhibitory effect on several members of the Bunyaviridae family, but the effect of MxA against CCHFV has not previously been studied. Here, we report that human MxA has antiviral activity against CCHFV. The yield of progeny virus in cells constitutively expressing MxA was reduced up to 1,000-fold compared with control cells, and accumulation of viral genomes was blocked. Confocal microscopy revealed that MxA colocalizes with the nucleocapsid protein (NP) of CCHFV in the perinuclear regions of infected cells. Furthermore, we found that MxA interacted with NP by using a coimmunoprecipitation assay. We also found that an amino acid substitution (E645R) within the C-terminal domain of MxA resulted in a loss of MxA antiviral activity and, concomitantly, in the capacity to interact with CCHFV NP. These results suggest that MxA, by interacting with a component of the nucleocapsid, prevents replication of CCHFV viral RNA and thereby inhibits the production of new infectious virus particles.     

Evaluation of the cell culture based and the mouse brain derived inactivated vaccines against Crimean-Congo hemorrhagic fever virus in transiently immune-suppressed (IS) mouse model

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus in the Nairoviridae family within the Bunyavirales order of viruses. Crimean-Congo hemorrhagic fever (CCHF) is the most widespread among tick-borne human viral diseases. It is endemic in many areas of Africa, Asia, the Middle East, in the Balkans, Russia and countries of the former Soviet Union. The confirmed CCHF cases were seen in Spain in 2016 to signify expansion of the virus into new geographical areas. CCHFV causes a viral human disease characterized by sudden onset of fever, headache, abdominal pain, nausea, hypotension, hemorrhage, and hepatic dysfunction with fatality rates up to 30%. Currently, there are no spesific treatments or licensed vaccines available for CCHFV. The absence of a susceptible animal model for CCHFV infection was severely hindered work on the development of vaccines. However, several animal models of CCHFV infection have been recently developed and used to assess vaccine efficacy. In this study, we have used the transiently immune-suppressed (IS) mouse model that MAb-5A3 was used to block IFN-I signaling in immune intact, wild-type mice at the time of CCHFV infection to evaluate the immune response and efficacy of the cell culture based and the mouse brain derived inactivated vaccines against CCHFV. Both vaccine preparations have provided complete protection but the cell culture based vaccine more effectively induced to CCFHV spesific antibodies and T cell responses. This is the first comparison of the cell culture based and the mouse brain derived vaccines for assessing the protective efficacy and the immunogenicity in the IS mouse CCHFV model.     

Crimean-Congo hemorrhagic fever

Crimean-Congo hemorrhagic fever (CCHF) is an acute infectious disease caused by CCHF virus (CCHFV), a member of the family Bunyaviridae, genus Nairovirus. The case fatality rate of CCHF ranges from 10-40%. Because CCHF is not present in Japan, many Japanese virologists and clinicians are not very familiar with this disease. However, there remains the possibility of an introduction of CCHFV or other hemorrhagic fever viruses into Japan from surrounding endemic areas. Development of diagnostic laboratory capacity for viral hemorrhagic fevers is necessary even in countries without these diseases. At the National Institute of Infectious Diseases, Tokyo, Japan, laboratory-based systems such as recombinant protein-based antibody detection, antigen-capture and pathological examination have been developed. In this review article, epidemiologic and clinical data on CCHF in the Xinjiang Uygur Autonomous Region, compiled through field investigations and diagnostic testing utilizing the aforementioned laboratory systems, are presented. CCHFV infections are closely associated with the environmental conditions, life styles, religion, occupation, and human economic activities. Based on these data, preventive measures for CCHFV infections are also discussed.     

Molecular Epidemiology of Crimean-Congo Hemorrhagic Fever Virus in Kosovo

Crimean-Congo hemorrhagic fever virus (CCHFV) is a zoonotic agent that causes severe, life-threatening disease, with a case fatality rate of 10–50%. It is the most widespread tick-borne virus in the world, with cases reported in Africa, Asia and Eastern Europe. CCHFV is a genetically diverse virus. Its genetic diversity is often correlated to its geographical origin. Genetic variability of CCHFV was determined within few endemic areas, however limited data is available for Kosovo. Furthermore, there is little information about the spatiotemporal genetic changes of CCHFV in endemic areas. Kosovo is an important endemic area for CCHFV. Cases were reported each year and the case-fatality rate is significantly higher compared to nearby regions. In this study, we wanted to examine the genetic variability of CCHFV obtained directly from CCHF-confirmed patients, hospitalized in Kosovo from 1991 to 2013. We sequenced partial S segment CCHFV nucleotide sequences from 89 patients. Our results show that several viral variants are present in Kosovo and that the genetic diversity is high in relation to the studied area. We also show that variants are mostly uniformly distributed throughout Kosovo and that limited evolutionary changes have occurred in 22 years. Our results also suggest the presence of a new distinct lineage within the European CCHF phylogenetic clade. Our study provide the largest number of CCHFV nucleotide sequences from patients in 22 year span in one endemic area.     

First international external quality assessment of molecular detection of Crimean-Congo hemorrhagic fever virus

Crimean-Congo hemorrhagic fever (CCHF) is a zoonosis caused by a Nairovirus of the family Bunyaviridae. Infection is transmitted to humans mostly by Hyalomma ticks and also by direct contact with the blood or tissues of infected humans or viremic livestock. Clinical features usually include a rapid progression characterized by hemorrhage, myalgia and fever, with a lethality rate up to 30%. CCHF is one of the most widely distributed viral hemorrhagic fevers and has been reported in Africa, the Middle East and Asia, as well as parts of Europe. There is no approved vaccine or specific treatment against CCHF virus (CCHFV) infections. In this context, an accurate diagnosis as well as a reliable surveillance of CCHFV infections is essential. Diagnostic techniques include virus culture, serology and molecular methods, which are now increasingly used. The European Network for the Diagnostics of "Imported" Viral Diseases organized the first international external quality assessment of CCHVF molecular diagnostics in 2011 to assess the efficiency and accurateness of CCHFV molecular methods applied by expert laboratories. A proficiency test panel of 15 samples was distributed to the participants including 10 different CCHFV preparations generated from infected cell cultures, a preparation of plasmid cloned with the nucleoprotein of CCHFV, two CCHFV RNA preparations and two negative controls. Forty-four laboratories worldwide participated in the EQA study and 53 data sets were received. Twenty data sets (38%) met all criteria with optimal performance, 10 (19%) with acceptable performance, while 23 (43%) reported results showing a need for improvement. Differences in performance depended on the method used, the type of strain tested, the concentration of the sample tested and the laboratory performing the test. These results indicate that there is still a need for improving testing conditions and standardizing protocols for the molecular detection of Crimean-Congo hemorrhagic fever virus.     

Induction of caspase activation and cleavage of the viral nucleocapsid protein in different cell types during Crimean-Congo hemorrhagic fever virus infection

Regulation of apoptosis during infection has been observed for several viral pathogens. Programmed cell death and regulation of apoptosis in response to a viral infection are important factors for host or virus survival. It is not known whether Crimean-Congo hemorrhagic fever virus (CCHFV) infection regulates the apoptosis process in vitro. This study for the first time suggests that CCHFV induces apoptosis, which may be dependent on caspase-3 activation. This study also shows that the coding sequence of the S segment of CCHFV contains a proteolytic cleavage site, DEVD, which is conserved in all CCHFV strains. By using different recombinant expression systems and site-directed mutagenesis, we demonstrated that this motif is subject to caspase cleavage. We also demonstrate that CCHFV nucleocapsid protein (NP) is cleaved into a 30-kDa fragment at the same time as caspase activity is induced during infection. Using caspase inhibitors and cells lacking caspase-3, we clearly demonstrate that the cleavage of NP is caspase-3-dependent. We also show that the inhibition of apoptosis induced progeny viral titers of ～80-90%. Thus, caspase-3-dependent cleavage of NP may represent a host defense mechanism against lytic CCHFV infection. Taken together, these data suggest that the most abundant protein of CCHFV, which has several essential functions such as protection of viral RNA and participation in various processes in the replication cycle, can be subjected to cleavage by host cell caspases.     

Crimean-Congo hemorrhagic fever virus activates endothelial cells

Crimean-Congo hemorrhagic fever virus (CCHFV) causes viral hemorrhagic fever with high case-fatality rates and is geographically widely distributed. Due to the requirement for a biosafety level 4 (BSL-4) laboratory and the lack of an animal model, knowledge of the viral pathogenesis is limited. Crimean-Congo hemorrhagic fever (CCHF) is characterized by hemorrhage and vascular permeability, indicating the involvement of endothelial cells (ECs). The interplay between ECs and CCHFV is therefore important for understanding the pathogenesis of CCHF. In a previous study, we found that CCHFV-infected monocyte-derived dendritic cells (moDCs) activated ECs; however, the direct effect of CCHFV on ECs was not investigated. Here, we report that ECs are activated upon infection, as demonstrated by upregulation of mRNA levels for E-selectin, vascular cell adhesion molecule 1 (VCAM1), and intercellular adhesion molecule 1 (ICAM1). Protein levels and cell surface expression of ICAM1 responded in a dose-dependent manner to increasing CCHFV titers with concomitant increase in leukocyte adhesion. Furthermore, we examined vascular endothelial (VE) cadherin in CCHFV-infected ECs by different approaches. Infected ECs released higher levels of interleukin 6 (IL-6) and IL-8; however, stimulation of resting ECs with supernatants derived from infected ECs did not result in increased ICAM1 expression. Interestingly, the moDC-mediated activation of ECs was abrogated by addition of neutralizing tumor necrosis factor alpha (TNF-α) antibody to moDC supernatants, thereby identifying this soluble mediator as the key cytokine causing EC activation. We conclude that CCHFV can exert both direct and indirect effects on ECs.     

Serial Expression of the Truncated Fragments of the Nucleocapsid Protein of CCHFV and Identification of the Epitope Region

The Crimean-congo hemorrhagic fever virus(CCHFV)is a geographically widespread fatal pathogen. Identification of the epitope regions of the virus is important for the diagnosis and epidemiological studies of CCHFV infections.In this study,expression vectors carrying series truncated fragments of the NP(nucleocapsid protein)gene from the S fragment of CCHFV strain YL04057 were constructed.The recombinant proteins were expressed in E.coli and purified for detection.The antigenic of the truncated fragments of ...     

Basolateral entry and release of Crimean-Congo hemorrhagic fever virus in polarized MDCK-1 cells

Crimean-Congo hemorrhagic fever virus (CCHFV) is an etiological agent of a disease with mortality rates in patients averaging 30%. The disease is characterized by fever, myalgia, and hemorrhage. Mechanisms underlying the hemorrhage have to our knowledge not been elucidated for CCHFV. Possibly, a direct or indirect viral effect on tight junctions (TJ) could cause the hemorrhage observed in patients, as TJ play a crucial role in vascular homeostasis and can cause leakage upon deregulation. Moreover, there is no knowledge regarding the site of entry and release of CCHFV in polarized epithelial cells. Such cells represent a barrier to virus dissemination within the host, and as a site of viral entry and release, they could play a key role in further spread. For the first time, we have shown preferential basolateral entry of CCHFV in Madin-Darby canine kidney 1 (MDCK-1) epithelial cells. Furthermore, we demonstrated basolateral release of CCHFV in polarized epithelial cells. Interestingly, by measuring transepithelial electrical resistance, we found no effect of CCHFV replication on the function of TJ in this study. Neither did we observe any difference in the localization of the TJ proteins ZO-1 and occludin in CCHFV-infected cells compared to mock-infected cells.     

Bayesian Phylogeography of Crimean-Congo Hemorrhagic Fever Virus in Europe

Crimean-Congo hemorrhagic fever (CCHF) is a zoonosis mainly transmitted by ticks that causes severe hemorrhagic fever and has a mortality rate of 5-60%. The first outbreak of CCHF occurred in the Crimean peninsula in 1944-45 and it has recently emerged in the Balkans and eastern Mediterranean. In order to reconstruct the origin and pathway of the worldwide dispersion of the virus at global and regional (eastern European) level, we investigated the phylogeography of the infection by analysing 121 publicly available CCHFV S gene sequences including two recently characterised Albanian isolates. The spatial and temporal phylogeny was reconstructed using a Bayesian Markov chain Monte Carlo approach, which estimated a mean evolutionary rate of 2.96 x 10^-4 (95%HPD=1.6 and 4.7 x 10^-4) substitutions/site/year for the analysed fragment. All of the isolates segregated into seven highly significant clades that correspond to the known geographical clades: in particular the two new isolates from northern Albania clustered significantly within the Europe 1 clade. Our phylogeographical reconstruction suggests that the global CCHFV clades originated about one thousand years ago from a common ancestor probably located in Africa. The virus then spread to Asia in the XV century and entered Europe on at least two occasions: the first in the early 1800s, when a still circulating but less or non-pathogenic virus emerged in Greece and Turkey, and the second in the early 1900s, when a pathogenic CCHFV strain began to spread in eastern Europe. The most probable location for the origin of this European clade 1 was Russia, but Turkey played a central role in spreading the virus throughout Europe. Given the close proximity of the infected areas, our data suggest that the movement of wild and domestic ungulates from endemic areas was probably the main cause of the dissemination of the virus in eastern Europe.     

Crimean-Congo Hemorrhagic Fever Virus Entry into Host Cells Occurs through the Multivesicular Body and Requires ESCRT Regulators

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne bunyavirus causing outbreaks of severe disease in humans, with a fatality rate approaching 30%. There are no widely accepted therapeutics available to prevent or treat the disease. CCHFV enters host cells through clathrin-mediated endocytosis and is subsequently transported to an acidified compartment where the fusion of virus envelope with cellular membranes takes place. To better understand the uptake pathway, we sought to identify host factors controlling CCHFV transport through the cell. We demonstrate that after passing through early endosomes in a Rab5-dependent manner, CCHFV is delivered to multivesicular bodies (MVBs). Virus particles localized to MVBs approximately 1 hour after infection and affected the distribution of the organelle within cells. Interestingly, blocking Rab7 activity had no effect on association of the virus with MVBs. Productive virus infection depended on phosphatidylinositol 3-kinase (PI3K) activity, which meditates the formation of functional MVBs. Silencing Tsg101, Vps24, Vps4B, or Alix/Aip1, components of the endosomal sorting complex required for transport (ESCRT) pathway controlling MVB biogenesis, inhibited infection of wild-type virus as well as a novel pseudotyped vesicular stomatitis virus (VSV) bearing CCHFV glycoprotein, supporting a role for the MVB pathway in CCHFV entry. We further demonstrate that blocking transport out of MVBs still allowed virus entry while preventing vesicular acidification, required for membrane fusion, trapped virions in the MVBs. These findings suggest that MVBs are necessary for infection and are the sites of virus-endosome membrane fusion.     

Recovery of Recombinant Crimean Congo Hemorrhagic Fever Virus Reveals a Function for Non-structural Glycoproteins Cleavage by Furin

Crimean Congo hemorrhagic fever virus (CCHFV) is a negative-strand RNA virus of the family Bunyaviridae (genus: Nairovirus). In humans, CCHFV causes fever, hemorrhage, severe thrombocytopenia, and high fatality. A major impediment in precisely determining the basis of CCHFV’s high pathogenicity has been the lack of methodology to produce recombinant CCHFV. We developed a reverse genetics system based on transfecting plasmids into BSR-T7/5 and Huh7 cells. In our system, bacteriophage T7 RNA polymerase produced complementary RNA copies of the viral S, M, and L segments that were encapsidated with the support, in trans, of CCHFV nucleoprotein and L polymerase. The system was optimized to systematically recover high yields of infectious CCHFV. Additionally, we tested the ability of the system to produce specifically designed CCHFV mutants. The M segment encodes a polyprotein that is processed by host proprotein convertases (PCs), including the site-1 protease (S1P) and furin-like PCs. S1P and furin cleavages are necessary for producing the non-structural glycoprotein GP38, while S1P cleavage yields structural Gn. We studied the role of furin cleavage by rescuing a recombinant CCHFV encoding a virus glycoprotein precursor lacking a functional furin cleavage motif (RSKR mutated to ASKA). The ASKA mutation blocked glycoprotein precursor’s maturation to GP38, and Gn precursor’s maturation to Gn was slightly diminished. Furin cleavage was not essential for replication, as blocking furin cleavage resulted only in transient reduction of CCHFV titers, suggesting that either GP38 and/or decreased Gn maturation accounted for the reduced virion production. Our data demonstrate that nairoviruses can be produced by reverse genetics, and the utility of our system uncovered a function for furin cleavage. This viral rescue system could be further used to study the CCHFV replication cycle and facilitate the development of efficacious vaccines to counter this biological and public health threat.     

Hydrogen inhalation reduced epithelial apoptosis in ventilator-induced lung injury via a mechanism involving nuclear factor-kappa B activation

Entry of enveloped viruses into cells is initiated by binding of their envelope glycoproteins (Envs) to cell surface-associated receptors. The Crimean-Congo hemorrhagic fever virus (CCHFV) has two Envs, Gn and Gc, with poorly understood role in binding to susceptible cells. We expressed codon optimized Gn and Gc, and identified independently folded soluble Env fragments, one of which (Gc residues 180–300) bound CCHFV susceptible cells supposedly by interacting with a putative receptor. This receptor binding domain (RBD) was used to identify its interacting partner by coimmunoprecipitation and mass spectrometry. Thus we identified the human cell surface nucleolin as a putative CCHFV entry factor. Nucleolin was expressed on all susceptible cells tested but not on the surface of cells resistant to CCHFV infection. Further studies are needed to explore the nucleolin function as a plausible CCHFV receptor and the molecular mechanisms of the Gc-nucleolin interactions. The identification of the CCHFV RBD and its binding partner could provide novel targets for therapy and tools for prevention as well as more complete understanding of the mechanisms of CCHFV entry and pathogenesis.     

Cryo-EM structure of glycoprotein C from Crimean-Congo hemorrhagic fever virus

Crimean-Congo hemorrhagic fever virus (CCHFV) is a causative agent of serious hemorrhagic diseases in humans with high mortality rates. CCHFV glycoprotein Gc plays critical roles in mediating virus-host membrane fusion and has been studied extensively as an immunogen. However, the molecular mechanisms involved in membrane fusion and Gc-specific antibody-antigen interactions remain unresolved largely because structural information of this glycoprotein is missing. We designed a trimeric protein including most of the ectodomain region of Gc from the prototype CCHFV strain, IbAr10200, which enabled the cryo-electron microscopy structure to be solved at a resolution of 2.8 ?. The structure confirms that CCHFV Gc is a class II fusion protein. Unexpectedly, structural comparisons with other solved Gc trimers in the postfusion conformation revealed that CCHFV Gc adopted hybrid architectural features of the fusion loops from hantaviruses and domain III from phenuiviruses, suggesting a complex evolutionary pathway among these bunyaviruses. Antigenic sites on CCHFV Gc that protective neutralizing antibodies target were mapped onto the CCHFV Gc structure, providing valuable information that improved our understanding of potential neutralization mechanisms of various antibodies.     

Ex vivo stability of the rodent-borne Hantaan virus in comparison to that of arthropod-borne members of the Bunyaviridae family

The possible effect of virus adaptation to different transmission routes on virus stability in the environment is not well known. In this study we have compared the stabilities of three viruses within the Bunyaviridae family: the rodent-borne Hantavirus Hantaan virus (HTNV), the sand fly-borne Phlebovirus sandfly fever Sicilian virus (SFSV), and the tick-borne Nairovirus Crimean-Congo hemorrhagic fever virus (CCHFV). These viruses differ in their transmission routes: SFSV and CCHFV are vector borne, whereas HTNV is spread directly between its hosts, and to humans, via the environment. We studied whether these viruses differed regarding stability when kept outside of the host. Viral survival was analyzed at different time points upon exposure to different temperatures (4 degrees C, 20 degrees C, and 37 degrees C) and drying at 20 degrees C. We observed clearly different stabilities under wet conditions, particularly at 4 degrees C, where infectious SFSV, HTNV, and CCHFV were detectable after 528, 96, and 15 days, respectively. All three viruses were equally sensitive to drying, as shown by drying on aluminum discs. Furthermore, HTNV and SFSV partially survived for 2 min in 30% ethanol, whereas CCHFV did not. Electron microscopy images of HTNV, SSFSV, and CCHFV stored at 37 degrees C until infectivity was lost still showed the occurrence of virions, but with abnormal shapes and densities compared to those of the nonincubated samples. In conclusion, our study points out important differences in ex vivo stability among viruses within the Bunyaviridae family.     

Serial expression of the truncated fragments of the nucleocapsid protein of CCHFV and identification of the epitope region

The Crimean-congo hemorrhagic fever virus (CCHFV) is a geographically widespread fatal pathogen. Identification of the epitope regions of the virus is important for the diagnosis and epidemiological studies of CCHFV infections. In this study, expression vectors carrying series truncated fragments of the NP (nucleocapsid protein) gene from the S fragment of CCHFV strain YL04057 were constructed. The recombinant proteins were expressed in E.coli and purified for detection. The antigenic of the truncated fragments of NP was detected with a polyclonal serum (rabbit) and 2 monoclonal (mAbs) (14B7 and 43E5) against CCHFV by Western-blot analyses. The results showed that the three expressed constructs, which all contained the region 235AA to 305AA could be detected by mAbs polyclonal serum. The results suggest that region 235-305 aa of NP is a highly antigenic region and is highly conserved in the NP protein.     

Crimean-Congo hemorrhagic fever virus glycoprotein processing by the endoprotease SKI-1/S1P is critical for virus infectivity

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 (>10⁶ PFU/ml), confirming that SKI-1/S1P processing is required for incorporation of viral glycoproteins. SKI-1/S1P may represent a promising antiviral target.     

The Non-structural Protein of Crimean-Congo Hemorrhagic Fever Virus Disrupts the Mitochondrial Membrane Potential and Induces Apoptosis

Viruses have developed distinct strategies to overcome the host defense system. Regulation of apoptosis in response to viral infection is important for virus survival and dissemination. Like other viruses, Crimean-Congo hemorrhagic fever virus (CCHFV) is known to regulate apoptosis. This study, for the first time, suggests that the non-structural protein NSs of CCHFV, a member of the genus Nairovirus, induces apoptosis. In this report, we demonstrated the expression of CCHFV NSs, which contains 150 amino acid residues, in CCHFV-infected cells. CCHFV NSs undergoes active degradation during infection. We further demonstrated that ectopic expression of CCHFV NSs induces apoptosis, as reflected by caspase-3/7 activity and cleaved poly(ADP-ribose) polymerase, in different cell lines that support CCHFV replication. Using specific inhibitors, we showed that CCHFV NSs induces apoptosis via both intrinsic and extrinsic pathways. The minimal active region of the CCHFV NSs protein was determined to be 93–140 amino acid residues. Using alanine scanning, we demonstrated that Leu-127 and Leu-135 are the key residues for NSs-induced apoptosis. Interestingly, CCHFV NSs co-localizes in mitochondria and also disrupts the mitochondrial membrane potential. We also demonstrated that Leu-127 and Leu-135 are important residues for disruption of the mitochondrial membrane potential by NSs. Therefore, these results indicate that the C terminus of CCHFV NSs triggers mitochondrial membrane permeabilization, leading to activation of caspases, which, ultimately, leads to apoptosis. Given that multiple factors contribute to apoptosis during CCHFV infection, further studies are needed to define the involvement of CCHFV NSs in regulating apoptosis in infected cells.