Assessment of Inhibitors of Pathogenic Crimean-Congo Hemorrhagic Fever Virus Strains Using Virus-Like Particles.
ABSTRACT: Crimean-Congo hemorrhagic fever (CCHF) is an often lethal, acute inflammatory illness that affects a large geographic area. The disease is caused by infection with CCHF virus (CCHFV), a nairovirus from the Bunyaviridae family. Basic research on CCHFV has been severely hampered by biosafety requirements and lack of available strains and molecular tools. We report the development of a CCHF transcription- and entry-competent virus-like particle (tecVLP) system that can be used to study cell entry and viral transcription/replication over a broad dynamic range (~4 orders of magnitude). The tecVLPs are morphologically similar to authentic CCHFV. Incubation of immortalized and primary human cells with tecVLPs results in a strong reporter signal that is sensitive to treatment with neutralizing monoclonal antibodies and by small molecule inhibitors of CCHFV. We used glycoproteins and minigenomes from divergent CCHFV strains to generate tecVLPs, and in doing so, we identified a monoclonal antibody that can prevent cell entry of tecVLPs containing glycoproteins from 3 pathogenic CCHFV strains. In addition, our data suggest that different glycoprotein moieties confer different cellular entry efficiencies, and that glycoproteins from the commonly used strain IbAr10200 have up to 100-fold lower ability to enter primary human cells compared to glycoproteins from pathogenic CCHFV strains.
Project description:Despite the serious public health impact of Crimean-Congo hemorrhagic fever (CCHF), the efficacy of antivirals targeting the causative agent, CCHF virus (CCHFV), remains debatable. Neutralizing monoclonal antibodies (MAbs) targeting the CCHFV glycoprotein Gc have been reported to protect mice against challenge with the prototype CCHFV strain, IbAr10200. However, due to extensive sequence diversity of CCHFV glycoproteins, it is unknown whether these MAbs neutralize other CCHFV strains. We initially used a CCHF virus-like particle (VLP) system to generate 11 VLP moieties, each possessing a glycoprotein from a genetically diverse CCHFV strain isolated in either Africa, Asia, the Middle East, or southeastern Europe. We used these VLPs in biosafety level 2 conditions to efficiently screen MAb cross-neutralization potency. Of the 16 MAbs tested, 3 (8A1, 11E7, and 30F7) demonstrated cross-neutralization activity with most CCHF VLPs, with 8A1 neutralizing all VLPs tested. Although binding studies suggest that none of the MAbs compete for the same epitope, combining 11E7, 30F7, or both 11E7 and 30F7 with 8A1 had no additive effect on increasing neutralization in this system. To confirm our findings from the VLP system, the 3 MAbs capable of strain cross-neutralization were confirmed to effectively neutralize 5 diverse CCHFV strains in vitro. Passaging CCHFV strains in the presence of sub-neutralizing concentrations of MAbs did not generate escape mutants resistant to subsequent neutralization. This study demonstrates the utility of the VLP system for screening neutralizing MAbs against multiple CCHFV strains, and provides the first evidence that a single MAb can effectively neutralize a number of diverse CCHFV strains in vitro, which may lead to development of future CCHF therapeutics.
Project description:BACKGROUND: Crimean-Congo Hemorrhagic Fever virus (CCHFV), a member of the genus Nairovirus, family Bunyaviridae, is a tick-borne pathogen causing severe disease in humans. To better understand the CCHFV life cycle and explore potential intervention strategies, we studied the biosynthesis and intracellular targeting of the glycoproteins, which are encoded by the M genome segment. RESULTS: Following determination of the complete genome sequence of the CCHFV reference strain IbAr10200, we generated expression plasmids for the individual expression of the glycoproteins GN and GC, using CMV- and chicken beta-actin-driven promoters. The cellular localization of recombinantly expressed CCHFV glycoproteins was compared to authentic glycoproteins expressed during virus infection using indirect immunofluorescence assays, subcellular fractionation/western blot assays and confocal microscopy. To further elucidate potential intracellular targeting/retention signals of the two glycoproteins, GFP-fusion proteins containing different parts of the CCHFV glycoprotein were analyzed for their intracellular targeting. The N-terminal glycoprotein GN localized to the Golgi complex, a process mediated by retention/targeting signal(s) in the cytoplasmic domain and ectodomain of this protein. In contrast, the C-terminal glycoprotein GC remained in the endoplasmic reticulum but could be rescued into the Golgi complex by co-expression of GN. CONCLUSION: The data are consistent with the intracellular targeting of most bunyavirus glycoproteins and support the general model for assembly and budding of bunyavirus particles in the Golgi compartment.
Project description:BACKGROUND: Crimean-Congo hemorrhagic fever (CCHF), a tick-borne disease caused by Crimean-Congo hemorrhagic fever virus (CCHFV), is a member of the genus Nairovirus in the family Bunyaviridae. Recently, CCHFV has been reported as an important emerging infectious viral pathogen in Sudan. Sporadic cases and multiple CCHF outbreaks, associated with nosocomial chain of transmission, have been reported in the Kordufan region of Sudan. AIMS: To confirm CCHF in an index patient and attending physician in North Kordufan region, Sudan, and to provide some information on virus genetic lineages. METHODS: Antibody captured ELISA, reverse transcription PCR, partial S segment sequences of the virus and subsequent phylogenetic analysis were used to confirm the CCHFV infection and to determine the virus genetic lineages. RESULTS: CCHF was confirmed by monitoring specific IgM antibody and by detection of the viral genome using RT-PCR. Treatment with oral ribavirin, replacement with fluid therapy, blood transfusion and administration of platelets concentrate resulted in rapid improvement of the health condition of the female physician. Phylogenetic analysis of the partial S segment sequences of the 2 CCHFV indicates that both strains are identical and belong to Group III virus lineage, which includes viruses from Africa including, Sudan, Mauritania, South Africa and Nigeria. CONCLUSION: Further epidemiologic studies including, CCHFV complete genome analysis and implementation of improved surveillance are urgently needed to better predict and respond to CCHF outbreaks in the Kordufan region, Sudan.
Project description:Crimean-Congo hemorrhagic fever (CCHF) is a zoonotic disease caused by a nairovirus belonging to family Bunyaviridae. The CCHF virus (CCHFV) can be transmitted to humans by Hyalomma ticks as well as by direct contact with infected body fluids or tissues from viremic livestock or humans. Our aim was to set up a fast RT-qPCR for detection of the different CCHFV genotypes in clinical samples, including an inactivation step to make the sample handling possible in lower biosafety levels (BSL) than BSL-4. This method was evaluated against commercial reference assays and international External Quality Assessment (EQA) samples. The analytical limit of detection for the developed CCHFV-S RT-qPCR was 11 CCHFV genomes per reaction. After exclusion of four dubious samples, we studied 38 CCHFV-positive samples (using reference tests) of which 38 were found positive by CCHFV-S RT-qPCR, suggesting a sensitivity of 100%. CCHFV-S RT q-PCR detected all eight different CCHFV strains representing five different CCHFV genotypes. In conclusion, the CCHFV-S RT-qPCR described in this study was evaluated using various sources of CCHFV samples and shown to be an accurate tool to detect human CCHFV infection caused by different genotypes of the virus.
Project description:Crimean-Congo hemorrhagic fever (CCHF) is a widely distributed viral hemorrhagic fever characterized by rapid onset of flu-like symptoms often followed by hemorrhagic manifestations. CCHF virus (CCHFV), a bunyavirus in the Nairovirus genus, is capable of infecting a wide range of mammalian hosts in nature but so far only causes disease in humans. Recently, immunocompromised mice have been reported as CCHF disease models, but detailed characterization is lacking. Here, we closely followed infection and disease progression in CCHFV-infected interferon ?/? receptor knockout (IFNAR(-/-)) mice and age-matched wild-type (WT) mice. WT mice quickly clear CCHFV without developing any disease signs. In contrast, CCHFV infected IFNAR(-/-) mice develop an acute fulminant disease with high viral loads leading to organ pathology (liver and lymphoid tissues), marked proinflammatory host responses, severe thrombocytopenia, coagulopathy, and death. Disease progression closely mimics hallmarks of human CCHF disease, making IFNAR(-/-) mice an excellent choice to assess medical countermeasures.
Project description:Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne viral zoonotic disease caused by Crimean-Congo hemorrhagic fever virus (CCHFV), a member of the genus Nairovirus in the family Bunyaviridae. CCHF is typically asymptomatic in animals but can be highly fatal in humans approaching case fatality rate of approximately 30%. In the present investigation, a cross sectional study was conducted to determine the prevalence of CCHF and to identify the potential risk factors associated with CCHFV seropositivity among the one-humped camel (Camelus dromedaries) in Central Sudan.A total of 361 camels selected randomly from six localities were employed in the study. Sera sampled were tested for the presence of CCHFV-specific immunoglobulin G (IgG) antibodies using enzyme-linked immunosorbent assay (ELISA).CCHFV seropositivity was recorded in 77 out of 361 animals accounting for a prevalence rate of 21.3%. Age (OR = 3.6, CI = 1.72-7.79, p-value = 0.026); locality (OR = 5.85, CI = 1.81-18.83, p- value = 0.003), tick number (OR = 4.6, CI = 1.37-9.81, P-value 0.04); tick control (OR = 2.2, CI, 1.11-4.35, P-value = 0.023) and breed (OR = 6.60, CI = 2.38-18.36, P-value = 0.001) were recorded as potential risk factors for contracting CCHF.The prevalence of CCHF is significantly high among camels in Khartoum State, Sudan. Age, breed, locality and tick control are considered as potential risk factors for contracting CCHF. This study would be expected to reduce the impact on the livelihood of pastoral communities and ultimately avoid disease spread in human.
Project description:Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus of the genus Nairovirus within the family Bunyaviridae. Infection can result in general myalgia, fever, and headache with some patients developing hemorrhagic fever with mortality rates ranging from 5% to 30%. CCHFV has a wide geographic range that includes Africa, Asia, the Middle East, and Europe with nucleotide sequence variation approaching 20% across the three negative-sense RNA genome segments. While phylogenetic clustering generally aligns with geographic origin of individual strains, distribution can be wide due to tick/CCHFV dispersion via migrating birds. This sequence diversity negatively impacts existing molecular diagnostic assays, leading to false negative diagnostic results. Here, we updated a previously developed CCHFV real-time reverse transcription polymerase chain reaction (RT-PCR) assay to include strains not detected using that original assay. Deep sequencing of eight different CCHFV strains, including three that were not detectable using the original assay, identified sequence variants within this assay target region. New primers and probe based on the sequencing results and newly deposited sequences in GenBank greatly improved assay sensitivity and inclusivity with the exception of the genetically diverse strain AP92. For example, we observed a four log improvement in IbAr10200 detection with a new limit of detection of 256 PFU/mL. Subsequent comparison of this assay to another commonly used CCHFV real-time RT-PCR assay targeting a different region of the viral genome showed improved detection, and both assays could be used to mitigate CCHFV diversity for diagnostics. Overall, this work demonstrated the importance of continued viral sequencing efforts for robust diagnostic assay development.
Project description:Crimean-Congo hemorrhagic fever (CCHF) virus is the cause of an important tick-borne disease of humans throughout regions of Africa, Europe, and Asia. Like other members of the genus Nairovirus, family Bunyaviridae, the CCHF virus M genome RNA segment encodes the virus glycoproteins. Sequence analysis of the CCHF virus (Matin strain) M RNA segment revealed one major open reading frame that potentially encodes a precursor polyprotein 1,689 amino acids (aa) in length. Comparison of the deduced amino acid sequences of the M-encoded polyproteins of Nigerian, Pakistani, and Chinese CCHF virus strains revealed two distinct protein regions. The carboxyl-terminal 1,441 aa are relatively highly conserved (up to 8.4% identity difference), whereas the amino-terminal 243 to 248 aa are highly variable (up to 56.4% identity difference) and have mucin-like features, including a high serine, threonine, and proline content (up to 47.3%) and a potential for extensive O-glycosylation. Analysis of released virus revealed two major structural glycoproteins, G2 (37 kDa) and G1 (75 kDa). Virus protein analysis by various techniques, including pulse-chase analysis and/or reactivity with CCHF virus-specific polyclonal and antipeptide antibodies, demonstrated that the 140-kDa (which contains the mucin-like region) and 85-kDa nonstructural proteins are the precursors of the mature G2 and G1 proteins, respectively. The amino termini of the CCHF virus (Matin strain) G2 and G1 proteins were established by microsequencing to be equivalent to aa 525 and 1046, respectively, of the encoded polyprotein precursor. The tetrapeptides RRLL and RKPL are immediately upstream of the cleavage site for mature G2 and G1, respectively. These are completely conserved among the predicted polyprotein sequences of all the CCHF virus strains and closely resemble the tetrapeptides that represent the major cleavage recognition sites present in the glycoprotein precursors of arenaviruses, such as Lassa fever virus (RRLL) and Pichinde virus (RKLL). These results strongly suggest that CCHF viruses (and other members of the genus Nairovirus) likely utilize the subtilase SKI-1/S1P-like cellular proteases for the major glycoprotein precursor cleavage events, as has recently been demonstrated for the arenaviruses.
Project description: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.
Project description:BACKGROUND: The genus Nairovirus in the family Bunyaviridae contains 34 tick-borne viruses classified into seven serogroups. Hazara virus (HAZV) belongs to the Crimean-Congo hemorrhagic fever (CCHF) serogroup that also includes CCHF virus (CCHFV) a major pathogen for humans. HAZV is an interesting model to study CCHFV due to a close serological and phylogenetical relationship and a classification which allows handling in a BSL2 laboratory. Nairoviruses are characterized by a tripartite negative-sense single stranded RNA genome (named L, M and S segments) that encode the RNA polymerase, the Gn-Gc glycoproteins and the nucleoprotein (NP), respectively. Currently, there are neither vaccines nor effective therapies for the treatment of any bunyavirus infection in humans. In this study we report, for the first time, the use of RNA interference (RNAi) as an approach to inhibit nairovirus replication. RESULTS: Chemically synthesized siRNAs were designed to target the mRNA produced by the three genomic segments. We first demonstrated that the siRNAs targeting the NP mRNA displayed a stronger antiviral effect than those complementary to the L and M transcripts in A549 cells. We further characterized the two most efficient siRNAs showing, that the induced inhibition is specific and associated with a decrease in NP synthesis during HAZV infection. Furthermore, both siRNAs depicted an antiviral activity when used before and after HAZV infection. We next showed that HAZV was sensitive to ribavirin which is also known to inhibit CCHFV. Finally, we demonstrated the additive or synergistic antiviral effect of siRNAs used in combination with ribavirin. CONCLUSIONS: Our study highlights the interest of using RNAi (alone or in combination with ribavirin) to treat nairovirus infection. This approach has to be considered for the development of future antiviral compounds targeting CCHFV, the most pathogenic nairovirus.