Protective neutralizing antibodies from human survivors of Crimean-Congo hemorrhagic fever.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a World Health Organization priority pathogen. CCHFV infections cause a highly lethal hemorrhagic fever for which specific treatments and vaccines are urgently needed. Here, we characterize the human immune response to natural CCHFV infection to identify potent neutralizing monoclonal antibodies (nAbs) targeting the viral glycoprotein. Competition experiments showed that these nAbs bind six distinct antigenic sites in the Gc subunit. These sites were further delineated through mutagenesis and mapped onto a prefusion model of Gc. Pairwise screening identified combinations of non-competing nAbs that afford synergistic neutralization. Further enhancements in neutralization breadth and potency were attained by physically linking variable domains of synergistic nAb pairs through bispecific antibody (bsAb) engineering. Although multiple nAbs protected mice from lethal CCHFV challenge in pre- or post-exposure prophylactic settings, only a single bsAb, DVD-121-801, afforded therapeutic protection. DVD-121-801 is a promising candidate suitable for clinical development as a CCHFV therapeutic.

ICTV Virus Taxonomy Profile: Nairoviridae 2024.

Nairoviridae is a family for negative-sense RNA viruses with genomes of about 17.2-21.1 kb. These viruses are maintained in and/or transmitted by arthropods among birds, reptiles and mammals. Norwaviruses and orthonairoviruses can cause febrile illness in humans. Several orthonairoviruses can infect mammals, causing mild, severe and sometimes, fatal diseases. Nairovirids produce enveloped virions containing two or three single-stranded RNA segments with open reading frames that encode a nucleoprotein (N), sometimes a glycoprotein precursor (GPC), and a large (L) protein containing an RNA-directed RNA polymerase (RdRP) domain. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) report on the family Nairoviridae, which is available at www.ictv.global/report/nairoviridae.

Bunyavirales: Scientific Gaps and Prototype Pathogens for a Large and Diverse Group of Zoonotic Viruses.

Research directed at select prototype pathogens is part of the approach put forth by the National Institute of Allergy and Infectious Disease (NIAID) to prepare for future pandemics caused by emerging viruses. We were tasked with identifying suitable prototypes for four virus families of the Bunyavirales order (Phenuiviridae, Peribunyaviridae, Nairoviridae, and Hantaviridae). This is a challenge due to the breadth and diversity of these viral groups. While there are many differences among the Bunyavirales, they generally have complex ecological life cycles, segmented genomes, and cause a range of human clinical outcomes from mild to severe and even death. Here, we delineate potential prototype species that encompass the breadth of clinical outcomes of a given family, have existing reverse genetics tools or animal disease models, and can be amenable to a platform approach to vaccine testing. Suggested prototype pathogens outlined here can serve as a starting point for further discussions.

Pathogenesis of Kasokero virus in experimentally infected Egyptian rousette bats (Rousettus aegyptiacus).

Egyptian rousette bats (ERBs; Rousettus aegyptiacus; family Pteropodidae) are associated with a growing number of bunyaviruses of public health importance, including Kasokero virus (KASV), which was first identified as a zoonosis in Uganda in 1977. In this study, formalin-fixed paraffin-embedded tissues from a previous experiment in which KASV infection was confirmed in 18 experimentally infected ERBs were used for an in-depth analysis using histopathology, in situ hybridization (ISH) for detection of viral RNA, immunohistochemistry (IHC) to assess the mononuclear phagocyte system response, and quantitative digital image analysis to investigate virus clearance from the liver and spleen within a spatial context. Significant gross and histological lesions were limited to the liver, where KASV-infected bats developed mild to moderate, acute viral hepatitis, which was first observed at 3 days postinfection (DPI), peaked at 6 DPI, and was resolved by 20 DPI. A subset of bats had glycogen depletion (n = 10) and hepatic necrosis (n = 3), rarely with intralesional bacteria (n = 1). Virus replication was confirmed by ISH in the liver, spleen, lymph nodes, and tongue. In the liver, KASV replicated in the cytoplasm of hepatocytes, to a lesser extent in mononuclear phagocytes, and rarely in presumptive endothelial cells. Most KASV RNA, as detected by ISH, was cleared from the spleen and liver by 6 DPI. It is concluded that ERBs have effective mechanisms to respond to this virus, clearing it without evidence of clinical disease.

Lack of Evidence for Crimean-Congo Hemorrhagic Fever Virus in Ticks Collected from Animals, Corsica, France.

In Corsica, France, 9.1% of livestock serum samples collected during 2014-2016 were found to have antibodies against Crimean-Congo hemorrhagic fever virus (CCHFV), an emerging tickborne zoonotic disease. We tested 8,051 ticks for CCHFV RNA and Nairovirus RNA. The results indicate that Corsica is not a hotspot for CCHFV.

Virus-Derived DNA Forms Mediate the Persistent Infection of Tick Cells by Hazara Virus and Crimean-Congo Hemorrhagic Fever Virus.

Crimean-Congo hemorrhagic fever (CCHF) is a severe disease of humans caused by CCHF virus (CCHFV), a biosafety level (BSL)-4 pathogen. Ticks of the genus Hyalomma are the viral reservoir, and they represent the main vector transmitting the virus to its hosts during blood feeding. We have previously shown that CCHFV can persistently infect Hyalomma-derived tick cell lines. However, the mechanism allowing the establishment of persistent viral infections in ticks is still unknown. Hazara virus (HAZV) can be used as a BSL-2 model virus instead of CCHFV to study virus/vector interactions. To investigate the mechanism behind the establishment of a persistent infection, we developed an in vitro model with Hyalomma-derived tick cell lines and HAZV. As expected, HAZV, like CCHFV, persistently infects tick cells without any sign of cytopathic effect, and the infected cells can be cultured for more than 3 years. Most interestingly, we demonstrated the presence of short viral-derived DNA forms (vDNAs) after HAZV infection. Furthermore, we demonstrated that the antiretroviral drug azidothymine triphosphate could inhibit the production of vDNAs, suggesting that vDNAs are produced by an endogenous retrotranscriptase activity in tick cells. Moreover, we collected evidence that vDNAs are continuously synthesized, thereby downregulating viral replication to promote cell survival. Finally, vDNAs were also detected in CCHFV-infected tick cells. In conclusion, vDNA synthesis might represent a strategy to control the replication of RNA viruses in ticks allowing their persistent infection. IMPORTANCE Crimean-Congo hemorrhagic fever (CCHF) is an emerging tick-borne viral disease caused by CCHF virus (CCHFV). Ticks of the genus Hyalomma can be persistently infected with CCHFV representing the viral reservoir, and the main vector for viral transmission. Here we showed that tick cells infected with Hazara virus, a nonpathogenic model virus closely related to CCHFV, contained short viral-derived DNA forms (vDNAs) produced by endogenous retrotranscriptase activity. vDNAs are transitory molecules requiring viral RNA replication for their continuous synthesis. Interestingly, vDNA synthesis seemed to be correlated with downregulation of viral replication and promotion of tick cell viability. We also detected vDNAs in CCHFV-infected tick cells suggesting that they could represent a key element in the cell response to nairovirus infection and might represent a more general mechanism of innate immunity against RNA viral infection.

Structure and Characterization of Crimean-Congo Hemorrhagic Fever Virus GP38.

Crimean-Congo hemorrhagic fever virus (CCHFV) is the causative agent of the most widespread tick-borne viral infection in humans. CCHFV encodes a secreted glycoprotein (GP38) of unknown function that is the target of a protective antibody. Here, we present the crystal structure of GP38 at a resolution of 2.5 Å, which revealed a novel fold primarily consisting of a 3-helix bundle and a ß-sandwich. Sequence alignment and homology modeling showed distant homology between GP38 and the ectodomain of Gn (a structural glycoprotein in CCHFV), suggestive of a gene duplication event. Analysis of convalescent-phase sera showed high titers of GP38 antibodies indicating immunogenicity in humans during natural CCHFV infection. The only protective antibody for CCHFV in an adult mouse model reported to date, 13G8, bound GP38 with subnanomolar affinity and protected against heterologous CCHFV challenge in a STAT1-knockout mouse model. Our data strongly suggest that GP38 should be evaluated as a vaccine antigen and that its structure provides a foundation to investigate functions of this protein in the viral life cycle.IMPORTANCE Crimean-Congo hemorrhagic fever virus (CCHFV) is a priority pathogen that poses a high risk to public health. Due to the high morbidity and mortality rates associated with CCHFV infection, there is an urgent need to develop medical countermeasures for disease prevention and treatment. CCHFV GP38, a secreted glycoprotein of unknown function unique to the Nairoviridae family, was recently shown to be the target of a protective antibody against CCHFV. Here, we present the crystal structure of GP38, which revealed a novel fold with distant homology to another CCHFV glycoprotein that is suggestive of a gene duplication event. We also demonstrate that antibody 13G8 protects STAT1-knockout mice against heterologous CCHFV challenge using a clinical isolate from regions where CCHFV is endemic. Collectively, these data advance our understanding of GP38 structure and antigenicity and should facilitate future studies investigating its function.

ICTV Virus Taxonomy Profile: Nairoviridae.

Members of the family Nairoviridae produce enveloped virions with three single-stranded RNA segments comprising 17.1 to 22.8 kb in total. These viruses are maintained in arthropods and transmitted by ticks to mammals or birds. Crimean-Congo hemorrhagic fever virus is tick-borne and is endemic in most of Asia, Africa, Southern and Eastern Europe whereas Nairobi sheep disease virus, which is also tick-borne, causes lethal haemorrhagic gastroenteritis in small ruminants in Africa and India. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Nairoviridae, which is available at ictv.global/report/nairoviridae.

A Minigenome Study of Hazara Nairovirus Genomic Promoters.

Hazara nairovirus (HAZV) is a trisegmented RNA virus most closely related to Crimean-Congo hemorrhagic fever virus (CCHFV) in the order Bunyavirales The terminal roughly 20 nucleotides (nt) of its genome ends are highly complementary, similar to those of other segmented negative-strand RNA viruses (sNSV), and act as promoters for RNA synthesis. These promoters contain two elements: the extreme termini of both strands (promoter element 1 [PE1]) are conserved and virus specific and are found bound to separate sites on the polymerase surface in crystal structures of promoter-polymerase complexes. The following sequences (PE2) are segment specific, with the potential to form double-stranded RNA (dsRNA), and the latter aspect is also important for promoter activity. Nairovirus genome promoters differ from those of peribunyaviruses and arenaviruses in that they contain a short single-stranded region between the two regions of complementarity. Using a HAZV minigenome system, we found the single-stranded nature of this region, as well as the potential of the following sequence to form dsRNA, is essential for reporter gene expression. Most unexpectedly, the sequence of the PE2 dsRNA appears to be equally important for promoter activity. These differences in sNSV PE2 promoter elements are discussed in light of our current understanding of the initiation of RNA synthesis.IMPORTANCE A minigenome system for HAZV, closely related to CCHFV, was used to study its genome replication. HAZV genome ends, like those of other sNSV, such as peribunyaviruses and arenaviruses, are highly complementary and serve as promoters for genome synthesis. These promoters are composed of two elements: the extreme termini of both 3' and 5' strands that are initially bound to separate sites on the polymerase surface in a sequence-specific fashion and the following sequences with the potential to anneal but whose sequence is not important. Nairovirus promoters differ from the other sNSV cited in that they contain a short single-stranded RNA (ssRNA) region between the two elements. The single-stranded nature of this region is an essential element of the promoter, whereas its sequence is unimportant. The sequence of the following complementary region is unexpectedly also important, a possible rare example of sequence-specific dsRNA recognition.

Small Interfering RNAs Are Highly Effective Inhibitors of Crimean-Congo Hemorrhagic Fever Virus Replication In Vitro.

Crimean-Congo hemorrhagic fever virus (CCHFV) is one of the prioritized diseases of the World Health Organization, considering its potential to create a public health emergency and, more importantly, the absence of efficacious drugs and/or vaccines for treatment. The highly pathogenic characteristic of CCHFV restricts research to BSL-4 laboratories, which complicates effective research and developmental strategies. In consideration of antiviral therapies, RNA interference can be used to suppress viral replication by targeting viral genes. RNA interference uses small interfering RNAs (siRNAs) to silence genes. The aim of our study was to design and test siRNAs in vitro that inhibit CCHFV replication and can serve as a basis for further antiviral therapies. A549 cells were infected with CCHFV after transfection with the siRNAs. Following 72 h, nucleic acid from the supernatant was extracted for RT Droplet Digital PCR analysis. Among the investigated siRNAs we identified effective candidates against all three segments of the CCHF genome. Consequently, blocking any segment of CCHFV leads to changes in the virus copy number that indicates an antiviral effect of the siRNAs. In summary, we demonstrated the ability of specific siRNAs to inhibit CCHFV replication in vitro. This promising result can be integrated into future anti-CCHFV therapy developments.

Biochemical and structural studies reveal differences and commonalities among cap-snatching endonucleases from segmented negative-strand RNA viruses.

Viruses rely on many host cell processes, including the cellular transcription machinery. Segmented negative-strand RNA viruses (sNSV) in particular cannot synthesize the 5'-cap structure for their mRNA but cleave off cellular caps and use the resulting oligonucleotides as primers for their transcription. This cap-snatching mechanism, involving a viral cap-binding site and RNA endonuclease, is both virus-specific and essential for viral proliferation and therefore represents an attractive drug target. Here, we present biochemical and structural results on the putative cap-snatching endonuclease of Crimean-Congo hemorrhagic fever virus (CCHFV), a highly pathogenic bunyavirus belonging to the Nairoviridae family, and of two additional nairoviruses, Erve virus (EREV) and Nairobi sheep disease virus (NSDV). Our findings are presented in the context of other cap-snatching endonucleases, such as the enzymatically active endonuclease from Rift Valley fever virus (RVFV), from Arenaviridae and Bunyavirales, belonging to the His- and His+ endonucleases, respectively, according to the absence or presence of a metal ion-coordinating histidine in the active site. Mutational and metal-binding experiments revealed the presence of only acidic metal-coordinating residues in the active site of the CCHFV domain and a unique active-site conformation that was intermediate between those of His+ and His- endonucleases. On the basis of small-angle X-ray scattering (SAXS) and homology modeling results, we propose a protein topology for the CCHFV domain that, despite its larger size, has a structure overall similar to those of related endonucleases. These results suggest structural and functional conservation of the cap-snatching mechanism among sNSVs.

Hazara nairovirus elicits differential induction of apoptosis and nucleocapsid protein cleavage in mammalian and tick cells.

The Nairoviridae family within the Bunyavirales order comprise tick-borne segmented negative-sense RNA viruses that cause serious disease in a broad range of mammals, yet cause a latent and lifelong infection in tick hosts. An important member of this family is Crimean-Congo haemorrhagic fever virus (CCHFV), which is responsible for serious human disease that results in case fatality rates of up to 30 %, and which exhibits the most geographically broad distribution of any tick-borne virus. Here, we explored differences in the cellular response of both mammalian and tick cells to nairovirus infection using Hazara virus (HAZV), which is a close relative of CCHFV within the CCHFV serogroup. We show that HAZV infection of human-derived SW13 cells led to induction of apoptosis, evidenced by activation of cellular caspases 3, 7 and 9. This was followed by cleavage of the classical apoptosis marker poly ADP-ribose polymerase, as well as cellular genome fragmentation. In addition, we show that the HAZV nucleocapsid (N) protein was abundantly cleaved by caspase 3 in these mammalian cells at a conserved DQVD motif exposed at the tip of its arm domain, and that cleaved HAZV-N was subsequently packaged into nascent virions. However, in stark contrast, we show for the first time that nairovirus infection of cells of the tick vector failed to induce apoptosis, as evidenced by undetectable levels of cleaved caspases and lack of cleaved HAZV-N. Our findings reveal that nairoviruses elicit diametrically opposed cellular responses in mammalian and tick cells, which may influence the infection outcome in the respective hosts.

Is the brain spared in Crimean-Congo haemorrhagic fever? An MR-SWI study to reveal CNS involvement.

OBJECTIVES: The aim of this prospective study is to investigate the central nervous system involvement in Crimean-Congo haemorrhagic fever (CCHF) with magnetic resonance imaging (MRI) in conjunction with clinical and laboratory findings. METHODS: Between July 2015 and August 2016, 36 patients with CCHF were undergone brain MRI including SWI. Two MRIs, one at the time of admission and the second in the convalescent period, were performed for each patient in order to see if there is any sign of central nervous system (CNS) involvement, especially in terms of intracranial haemorrhage or viral encephalitis. Clinical severity scoring was also done and laboratory findings were noted in order to correlate with clinical and imaging findings. RESULTS: None of the 36 patients showed any MRI findings of an acute intracranial event during the course of the disease. There was a significant difference between mild cases and moderate cases in terms of some laboratory parameters (p < 0.05). CONCLUSIONS: Although CCHF is a highly lethal disease which involves multiple organs and systems, CNS involvement seems to be extremely rare in mild and moderate cases. KEY POINTS: • MRI is the imaging method of choice to diagnose microbleeds and encephalitis • Although CCHF causes multisystem bleeding, intracranial haemorrhage seems to be very rare • CNS complications are uncommon, even in the setting of suggestive symptoms • Death usually results from extracranial bleeding and multiorgan failure • Severity scoring is associated with some laboratory abnormalities in CCHF.

Phylogenetic Characterization of Crimean-Congo Hemorrhagic Fever Virus, Spain.

Two cases of Crimean-Congo hemorrhagic fever were reported in Spain during 2016. We obtained the virus from a patient sample and characterized its full genomic sequence. Phylogenetic analysis indicated that the virus corresponds to the African genotype III, which includes viruses previously found in West and South Africa.

Novel clades of tick-borne pathogenic nairoviruses in Europe.

Members of the Orthonairovirus genus (family Nairoviridae) include many tick-borne viruses of significant human and animal health impact, with several recently-documented pathogenic viruses lacking sufficient epidemiological information. We screened 215 adult ticks of seven species collected in Bulgaria, Georgia, Latvia and Poland for orthonairoviruses, followed by nanopore sequencing (NS) for genome characterization. Initial generic amplification revealed Sulina virus (SULV, Orthonairovirus sulinaense), for which an updated amplification assay was used, revealing an overall prevalence of 2.7% in Ixodes ricinus ticks from Latvia. Three complete and additional partial SULV genomes were generated, that consistently formed a separate, distinct clade with further intragroup divergence in the maximum likelihood analyses. Comparisons with previously described viruses from Romania exhibited similar genome topologies, albeit with divergent motifs and cleavage sites on the glycoprotein precursor. Preliminary evidence of recombination involving the S segment was documented, in addition to variations in predicted viral glycoproteins. Generic screening further identified Tacheng tick virus 1 (TCTV1, Orthonairovirus tachengense), with documented human infections, in Dermacentor reticulatus ticks from Poland, with a prevalence of 0.9%. Subsequent NS and assembly provided the first complete TCTV1 genome outside of China, where it was originally described. Phylogenetic analysis of virus genome segments revealed TCTV1-Poland as a discrete taxon within the TCTV1 cluster in the Orthonairovirus genus, representing a geographically segregated clade. Comparable genome topology with TCTV1 from China was observed, aside from minor variations in the M segment. Similar to SULV, TCTV1 exhibited several mismatches on previously described screening primer binding sites, likely to prevent amplification. These findings indicate presence of novel TCTV1 and SULV clades in Eastern Europe, confirming the expansion of orthonairoviruses with pathogenic potential.

A wide distribution of Beiji nairoviruses and related viruses in Ixodes ticks in Japan.

Beiji nairovirus (BJNV), in the family Nairoviridae, the order Bunyavirales, was recently reported as a causative agent of an emerging tick-borne zoonotic infection in China. This study investigated the prevalence of BJNV in ticks in Japan. Screening of over 2,000 ticks from multiple regions revealed a widespread distribution of BJNV and BJNV-related viruses in Japan, particularly in the northern island, and in other high altitude areas with exclusive occurrence of Ixodes ticks. Phylogenetic analysis identified three distinct groups of nairoviruses in ticks in Japan: BJNV, Yichun nairovirus (YCNV) and a newly identified Mikuni nairovirus (MKNV). BJNV and YCNV variants identified in ticks in Japan exhibited high nucleotide sequence identities to those in China and Russia with evidence of non-monophyletic evolution among BJNVs, suggesting multiple cross-border transmission events of BJNV between the Eurasian continent and Japan. Whole genome sequencing of BJNV and MKNV revealed a unique GA-rich region in the S segment, the significance of which remains to be determined. In conclusion, the present study has shown a wide distribution and diversity of BJNV-related nairoviruses in Ixodes ticks in Japan and has identified unique genomic structures. The findings demonstrate the significance of BJNV as well as related viruses in Japan and highlight the necessity of monitoring emerging nairovirus infections and their potential risks to public health.

Evaluation of two inoculation routes of an adenovirus-mediated viral protein inhibitor in a Crimean-Congo hemorrhagic fever mouse model.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne nairovirus with a wide geographic spread that can cause severe and lethal disease. No specific medical countermeasures are approved to combat this illness. The CCHFV L protein contains an ovarian tumor (OTU) domain with a cysteine protease thought to modulate cellular immune responses by removing ubiquitin and ISG15 post-translational modifications from host and viral proteins. Viral deubiquitinases like CCHFV OTU are attractive drug targets, as blocking their activity may enhance cellular immune responses to infection, and potentially inhibit viral replication itself. We previously demonstrated that the engineered ubiquitin variant CC4 is a potent inhibitor of CCHFV replication in vitro. A major challenge of the therapeutic use of small protein inhibitors such as CC4 is their requirement for intracellular delivery, e.g., by viral vectors. In this study, we examined the feasibility of in vivo CC4 delivery by a replication-deficient recombinant adenovirus (Ad-CC4) in a lethal CCHFV mouse model. Since the liver is a primary target of CCHFV infection, we aimed to optimize delivery to this organ by comparing intravenous (tail vein) and intraperitoneal injection of Ad-CC4. While tail vein injection is a traditional route for adenovirus delivery, in our hands intraperitoneal injection resulted in higher and more widespread levels of adenovirus genome in tissues, including, as intended, the liver. However, despite promising in vitro results, neither route of in vivo CC4 treatment resulted in protection from a lethal CCHFV infection.

The current pathogenicity and potential risk evaluation of Crimean-Congo hemorrhagic fever virus to cause mysterious "Disease X"-An updated literature review.

Background and Aims: Crimean-Congo hemorrhagic fever (CCHF) is a severe and potentially lethal illness. Tick bites of the Hyalomma genus are the primary source of transmission of CCHF to humans. The virus responsible for CCHF is the CCHF virus (CCHFV). It is a single-stranded negative sensed RNA virus. The virus belongs to the Orthonairoviridae genus within the Nairoviridae family. It occurs in an extensive geographical area spanning the Middle East, western China, southern Asia, southeastern Europe, and much of Africa. The current study aimed to evaluate the pathogenicity and potential risk of CCHFV to cause a public health emergency of international concern. Methods: We searched updated relevant information from PubMed, Google Scholar, and Scopus databases using Crimean-Congo hemorrhagic fever, tick-borne virus, and Nairovirus as keywords. Results: The case fatality rate (CFR) varies by region. It can be more than 30% in some cases. Three segments in the genome of CCHFV (L, M, and S) are different in size and function. It is unknown whether the pathogenicity of CCHFV varied based on the genomic diversity. CCHFV can be transmitted through tick bites, handling of infected ticks, contact with infected humans, contaminated body fluids, and so on. A wide range of severity is associated with CCHF, ranging from a moderate fever with no apparent cause to increased vascular permeability, failure of several organs, bleeding, and shock. Hospitals with high-level isolation units should be the first choice for treating CCHF patients. Individual safety equipment is crucial in healthcare to prevent the spread of the virus. In the farm environment, using integrated pest management techniques, minimizing activity in tick-infested regions, and dressing appropriately in long sleeves and pants will help to reduce the risk of CCHFV infection via tick bites. Conclusion: There are no approved vaccinations or therapeutics for CCHF except supportive therapeutic approaches. Therefore, scientists recommend early ribavirin therapy for cases of high-risk exposures.

Exploring the Potential of Iminosugars as Antivirals for Crimean-Congo Haemorrhagic Fever Virus, Using the Surrogate Hazara Virus: Liquid-Chromatography-Based Mapping of Viral N-Glycosylation and In Vitro Antiviral Assays.

Crimean-Congo haemorrhagic fever virus (CCHFV) is a pathogen of increasing public health concern, being a widely distributed arbovirus and the causative agent of the potentially fatal Crimean-Congo haemorrhagic fever. Hazara virus (HAZV) is a genetically and serologically related virus that has been proposed as a surrogate for antiviral and vaccine testing for CCHFV. Glycosylation analysis of HAZV has been limited; first, we confirmed for the first time the occupation of two N-glycosylation sites in the HAZV glycoprotein. Despite this, there was no apparent antiviral efficacy of a panel of iminosugars against HAZV, as determined by quantification of the total secretion and infectious virus titres produced following infection of SW13 and Vero cells. This lack of efficacy was not due to an inability of deoxynojirimycin (DNJ)-derivative iminosugars to access and inhibit endoplasmic reticulum α-glucosidases, as demonstrated by free oligosaccharide analysis in uninfected and infected SW13 and uninfected Vero cells. Even so, iminosugars may yet have potential as antivirals for CCHFV since the positions and importance of N-linked glycans may differ between the viruses, a hypothesis requiring further evaluation.

Design and evaluation of neutralizing and fusion inhibitory peptides to Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever (CCHF) is a medically relevant tick-borne viral disease caused by the Bunyavirus, Crimean-Congo hemorrhagic fever virus (CCHFV). CCHFV is endemic to Asia, the Middle East, South-eastern Europe, and Africa and is transmitted in enzootic cycles among ticks, mammals, and birds. Human infections are mostly subclinical or limited to mild febrile illness. Severe disease may develop, resulting in multi-organ failure, hemorrhagic manifestations, and case-fatality rates up to 30%. Despite the widespread distribution and life-threatening potential, no treatments have been approved for CCHF. Antiviral inhibitory peptides, which antagonize viral entry, are licensed for clinical use in certain viral infections and have been experimentally designed against human pathogenic bunyaviruses, with in vitro and in vivo efficacies. We designed inhibitory peptides against CCHFV with and without conjugation to various polyethylene glycol and sterol groups. These additions have been shown to enhance both cellular uptake and antiviral activity. Peptides were evaluated against pseudotyped and wild-type CCHFV via neutralization tests, Nairovirus fusion assays, and cytotoxicity profiling. Four peptides neutralized CCHFV with two of these peptides shown to inhibit viral fusion. This work represents the development of experimental countermeasures for CCHF, describes a nairovirus immunofluorescence fusion assay, and illustrates the utility of pseudotyped CCHFV for the screening of entry antagonists at low containment settings for CCHF.