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.     

Actin-Dependent Receptor Colocalization Required for Human Immunodeficiency Virus Entry into Host Cells

Human immunodeficiency virus (HIV) envelope binds CD4 and a chemokine receptor in sequence, releasing hydrophobic viral gp41 residues into the target membrane. HIV entry required actin-dependent concentration of coreceptors, which could be disrupted by cytochalasin D (CytoD) without an effect on cell viability or mitosis. Pretreatment of peripheral blood mononuclear cells, but not virus, inhibited entry and infection. Immunofluorescent confocal microscopy of activated cells revealed CD4 and CXCR4 in nonoverlapping patterns. Addition of gp120 caused polarized cocapping of both molecules with subsequent pseudopod formation, while CytoD pretreatment blocked these membrane changes completely.     

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.     

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.     

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.     

Fine Epitope Mapping of the Central Immunodominant Region of Nucleoprotein from Crimean-Congo Hemorrhagic Fever Virus (CCHFV)

Crimean-Congo hemorrhagic fever (CCHF), a severe viral disease known to have occurred in over 30 countries and distinct regions, is caused by the tick-borne CCHF virus (CCHFV). Nucleocapsid protein (NP), which is encoded by the S gene, is the primary antigen detectable in infected cells. The goal of the present study was to map the minimal motifs of B-cell epitopes (BCEs) on NP. Five precise BCEs (E1, ²⁴⁷FDEAKK²⁵²; E2a, ²⁵⁴VEAL²⁵⁷; E2b, ²⁵⁸NGYLNKH²⁶⁴; E3, ²⁶⁷EVDKA²⁷¹; and E4, ²⁷⁴DSMITN²⁷⁹) identified through the use of rabbit antiserum, and one BCE (E5, ²⁵⁸NGYL²⁶¹) recognized using a mouse monoclonal antibody, were confirmed to be within the central region of NP and were partially represented among the predicted epitopes. Notably, the five BCEs identified using the rabbit sera were able to react with positive serum mixtures from five sheep which had been infected naturally with CCHFV. The multiple sequence alignment (MSA) revealed high conservation of the identified BCEs among ten CCHFV strains from different areas. Interestingly, the identified BCEs with only one residue variation can apparently be recognized by the positive sera of sheep naturally infected with CCHFV. Computer-generated three-dimensional structural models indicated that all the antigenic motifs are located on the surface of the NP stalk domain. This report represents the first identification and mapping of the minimal BCEs of CCHFV-NP along with an analysis of their primary and structural properties. Our identification of the minimal linear BCEs of CCHFV-NP may provide fundamental data for developing rapid diagnostic reagents and illuminating the pathogenic mechanism of CCHFV.     

Heparan Sulfate Facilitates Rift Valley Fever Virus Entry into the Cell

Rift Valley fever virus (RVFV), an emerging arthropod-borne pathogen, has a broad host and cell tropism. Here we report that the glycosaminoglycan heparan sulfate, abundantly present on the surface of most animal cells, is required for efficient entry of RVFV. Entry was significantly reduced by preincubating the virus inoculum with highly sulfated heparin, by enzymatic removal of heparan sulfate from cells and in cells genetically deficient in heparan sulfate synthesis.     

Vascular Endothelial Growth Factor A Promotes Vaccinia Virus Entry into Host Cells via Activation of the Akt Pathway

Vaccinia virus (VV) is an enveloped DNA virus from the poxvirus family and has played a crucial role in the eradication of smallpox. It continues to be used in immunotherapy for the prevention of infectious diseases and treatment of cancer. However, the mechanisms of poxvirus entry, the host factors that affect viral virulence, and the reasons for its natural tropism for tumor cells are incompletely understood. By studying the effect of hypoxia on VV infection, we found that vascular endothelial growth factor A (VEGF-A) augments oncolytic VV cytotoxicity. VEGF derived from tumor cells acts to increase VV internalization, resulting in increased replication and cytotoxicity in an AKT-dependent manner in both tumor cells and normal respiratory epithelial cells. Overexpression of VEGF also enhances VV infection within tumor tissue in vivo after systemic delivery. These results highlight the importance of VEGF expression in VV infection and have potential implications for the design of new strategies to prevent poxvirus infection and the development of future generations of oncolytic VV in combination with conventional or biological therapies.     

A Polarized Cell Model for Chikungunya Virus Infection: Entry and Egress of Virus Occurs at the Apical Domain of Polarized Cells

Chikungunya virus (CHIKV) has resulted in several outbreaks in the past six decades. The clinical symptoms of Chikungunya infection include fever, skin rash, arthralgia, and an increasing incidence of encephalitis. The re-emergence of CHIKV with more severe pathogenesis highlights its potential threat on our human health. In this study, polarized HBMEC, polarized Vero C1008 and non-polarized Vero cells grown on cell culture inserts were infected with CHIKV apically or basolaterally. Plaque assays, viral binding assays and immunofluorescence assays demonstrated apical entry and release of CHIKV in polarized HBMEC and Vero C1008. Drug treatment studies were performed to elucidate both host cell and viral factors involved in the sorting and release of CHIKV at the apical domain of polarized cells. Disruption of host cell myosin II, microtubule and microfilament networks did not disrupt the polarized release of CHIKV. However, treatment with tunicamycin resulted in a bi-directional release of CHIKV, suggesting that N-glycans of CHIKV envelope glycoproteins could serve as apical sorting signals.     

Genetic monitoring of the Crimean-Congo Hemorrhagic Fever virus in Kazakhstan and Tajikistan in 2001-2003

Blood specimens obtained from 32 CCHF patients were tested for the presence of CCHF virus markers. In addition, 3210 ticks of the genera Hyalomma asiaticum, Hyalomma anatolicum, and Dermacentor niveus were examined to identify the CCHF virus antigen and RNA. This material was obtained during the 2001-2003 local outbreaks of CCHF in Kazakhstan and Tajikistan. The nucleotide sequence in the region 983-1282 of S segment of the CCHF virus for 12 wild type strains was determined. The phylogenetic relationships among the established biovariants of CCHF virus, and also between these biovariants and those from other regions of the world were identified. We were the first to demonstrate the presence of an African-like genotype of CCHF virus in the territory of Kazakhstan. The conclusion was made that two genotypes of CCHF virus were in circulation in Kazakhstan. It was also demonstrated that CCHF virus, circulating in the territories of Kazakhstan and Tajikistan, was genetically heterogeneous.     

Differential Responses of Disease-Resistant and Disease-Susceptible Primate Macrophages and Myeloid Dendritic Cells to Simian Hemorrhagic Fever Virus Infection

Simian hemorrhagic fever virus (SHFV) causes a fatal hemorrhagic fever in macaques but an asymptomatic, persistent infection in baboons. To investigate factors contributing to this differential infection outcome, the targets of SHFV infection, macrophages (MΦs) and myeloid dendritic cells (mDCs), were differentiated from macaque and baboon peripheral blood monocytes and used to compare viral replication and cell responses. SHFV replicated in >90% of macaque MΦs but in only ∼10% of baboon MΦs. Although SHFV infected ∼50% of macaque and baboon mDCs, virus replication was efficient in macaque but not in baboon mDCs. Both types of macaque cultures produced higher virus yields than baboon cultures. A more efficient type I interferon response and the production of proinflammatory cytokines, including interleukin-1β (IL-1β), IL-6, IL-12/23(p40), tumor necrosis factor alpha (TNF-α), and macrophage inflammatory protein 1α (MIP-1α), in response to SHFV infection were observed in macaque but not baboon cultures, suggesting less efficient counteraction of these responses by viral proteins in macaque cells. Baboon cultures produced higher levels of IL-10 than macaque cultures both prior to and after SHFV infection. In baboon but not macaque cell cultures, SHFV infection upregulated IL-10R1, a subunit of the IL-10 receptor (IL-10R), and also SOCS3, a negative regulator of proinflammatory cytokine production. Incubation of macaque cultures with human IL-10 before and/or after SHFV infection decreased production of IL-6, IL-1β, and MIP-1α but not TNF-α, suggesting a role for IL-10 in suppressing SHFV-induced proinflammatory cytokine production in macaques.     

Circulation of Crimean-Congo Hemorrhagic Fever Virus in the Former Yugoslav Republic of Macedonia Revealed by Screening of Cattle Sera Using a Novel Enzyme-linked Immunosorbent Assay

BackgroundThere are only few assays available for the detection of Crimean-Congo Hemorrhagic Fever Virus (CCHFV)-specific antibodies in animals, and data about diagnostic sensitivity and specificity are incompletely documented for most of these tests. This is unfortunate since CCHFV antibodies in animals can be used as indicator for virus circulation in a geographic area and therewith potential risk of human exposure. This paper therefore reports on a novel ELISA for the detection of CCHFV-specific antibodies in cattle and on its application for testing ruminant sera from the Former Yugoslav Republic of Macedonia.ConclusionThis article describes a fully validated, highly sensitive and specific ELISA for the detection of CCHFV-specific IgG antibodies in cattle. Using this assay, CCHFV-specific antibodies were detected for the first time in cattle in the Former Yugoslav Republic of Macedonia, giving evidence for an active circulation of this virus in the country. Supporting this conclusion, the occurrence of the main vector of CCHFV was demonstrated in the present work for the first time in Former Yugoslav Republic of Macedonia.     

感染流行性出血热病毒的正常人骨髓细胞膜脂和酶生物活性的研究

采用疏水性荧光探针ＤＰＨ（１,６,－苯基－１,３;５－已三烯）标记感染了流行性出血热病毒的正常人骨髓细胞的细胞膜,根据荧光偏振值的变化推导出膜脂流动性,同时通过细胞组化染色定量方法,测定细胞内酸性磷酸酶（ＡＣＰ）和琥珀酸脱氢酶（ＳＤＨ）的活性。动态观察１、６、２４、７２ｈ培养后的淋巴细胞、单核细胞、中性粒细胞,发现感染后细胞膜脂的流动性明显降低,并随培养时间的延长而明显,和正常对照组比较有显著性差异（Ｐ＜０．０５～０．０１）。通过组化染色和显微分光光度计单细胞扫描定量认为：感染细胞在培养开始的７２ｈ内ＡＣＰ、ＳＤＨ升高,１２０ｈ后明显下降,和正常相比亦有显著差异（Ｐ＜０．０５）,这与病毒侵犯和细胞机能的受损程度有关。