Prevalence of Crimean-Congo Hemorrhagic Fever Virus in Healthy Population,Livestock and Ticks in Kosovo

Crimean-Congo hemorrhagic fever (CCHF) is an acute, tick borne disease often associated with hemorrhagic presentations and high case fatality rate. Kosovo is a highly endemic area for CCHF, with a significant case fatality rate. The aim of our study was to determine the prevalence of CCHF in Kosovo. We tested 1105 serum samples from healthy population in both endemic and non-endemic areas in the country. Our results revealed a seroprevalence of 4.0% (range 0–9.3%) which is comparable to the seroprevalence in other countries. We show that seroprevalence is correlated to the disease incidence in each studied municipality. We also tested 401 animal sera (353 cow, 30 sheep, 10 goat and 8 chicken) in four endemic municipalities in Kosovo. We detected specific antibodies in all animals except in chicken. Seroprevalence in cows is comparable to other endemic areas and correlates to the seroprevalence in humans. No CCHF RNA could be detected in 105 tick samples obtained in 2012 and 2013. Sequencing of CCHFV positive ticks from 2001 revealed that the virus is most closely related to viral strains that were detected in CCHF patients from Kosovo. Results suggest that mild CCHF cases are most probably underdiagnosed and consequently that the burden of disease is higher than reported. Our study provides key information for CCHF surveillance and raises awareness for possible imported cases in CCHF non-endemic countries.     

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.     

Epidemiology and Mutational Analysis of Global Strains of Crimean-Congo Haemorrhagic Fever Virus

Crimean-Congo hemorrhagic fever (CCHF) is a severe illness with high fatality.Cases are reported in several countries in Africa,Europe,the Middle East,and Asia.Phylogenetic analyses based on the virus S (nucleocapsid),M (glycoprotein),and L (polymerase) genome segments sequences indicate distinct geographic lineages exist but their specific genetic characteristics require elucidation.In this work we collected all full length S segment sequences and generated a phylogenetic tree based on the alignment of the...     

Epidemiology and Mutational Analysis of Global Strains of Crimean-Congo Haemorrhagic Fever Virus

Crimean-Congo hemorrhagic fever (CCHF) is a severe illness with high fatality.Cases are reported in several countries in Africa,Europe,the Middle East,and Asia.Phylogenetic analyses based on the virus S (nucleocapsid),M (glycoprotein),and L (polymerase) genome segments sequences indicate distinct geographic lineages exist but their specific genetic characteristics require elucidation.In this work we collected all full length S segment sequences and generated a phylogenetic tree based on the alignment of these 62 samples.We then analyzed the alignment using entries from AAIndex,the Amino Acid Index database,to identify amino acid mutations that performed significant changes in charge,pka,hydropathy and side chain volume.Finally,we mapped these changes back to the tree and alignment to identify correlated mutations or sites that characterized a specific lineage.Based on this analysis we are able to propose a number of sites that appear to be important for virus function and which would be good candidates for experimental mutational analysis studies.     

Structure, Function, and Evolution of the Crimean-Congo Hemorrhagic Fever Virus Nucleocapsid Protein

Crimean-Congo hemorrhagic fever virus (CCHFV) is an emerging tick-borne virus of the Bunyaviridae family that is responsible for a fatal human disease for which preventative or therapeutic measures do not exist. We solved the crystal structure of the CCHFV strain Baghdad-12 nucleocapsid protein (N), a potential therapeutic target, at a resolution of 2.1 Å. N comprises a large globular domain composed of both N- and C-terminal sequences, likely involved in RNA binding, and a protruding arm domain with a conserved DEVD caspase-3 cleavage site at its apex. Alignment of our structure with that of the recently reported N protein from strain YL04057 shows a close correspondence of all folds but significant transposition of the arm through a rotation of 180 degrees and a translation of 40 Å. These observations suggest a structural flexibility that may provide the basis for switching between alternative N protein conformations during important functions such as RNA binding and oligomerization. Our structure reveals surfaces likely involved in RNA binding and oligomerization, and functionally critical residues within these domains were identified using a minigenome system able to recapitulate CCHFV-specific RNA synthesis in cells. Caspase-3 cleaves the polypeptide chain at the exposed DEVD motif; however, the cleaved N protein remains an intact unit, likely due to the intimate association of N- and C-terminal fragments in the globular domain. Structural alignment with existing N proteins reveals that the closest CCHFV relative is not another bunyavirus but the arenavirus Lassa virus instead, suggesting that current segmented negative-strand RNA virus taxonomy may need revision.     

Antagonistic Antiviral Activity between IFN-Lambda and IFN-Alpha against Lethal Crimean-Congo Hemorrhagic Fever Virus In Vitro

MethodsHuman A549 and HuH7 cells were treated with increasing amounts of IFN-λ1, or IFN-α or a combination of them, infected with CCHF; the extent of virus yield inhibition and the induction of MxA and 2’-5’OAS mRNA was measured.

Results and Conclusions

Our study pointed out that type III IFN possess an antiviral activity against CCHFV, even if lower than type I IFN. Moreover, a clear antagonism between IFN-λ and IFN–α was observed in both cell lines (A549 and HuH7 cells), in terms of antiviral effect and activation of pivotal ISGs, i.e. MxA and 2’-5’OAS. Elucidating the interplay between type I and III IFNs will help to better understand innate defence mechanisms against viral infections and may provide novel scientific evidence for a more rational planning of available and future treatments, particularly against human diseases caused by high concern viruses.     

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.     

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.