Helen Karlberg

Crimean–Congo hemorrhagic fever virus infection is lethal for adult type I interferon receptor-knockout mice

Crimean-Congo hemorrhagic fever virus (CCHFV) poses a great threat to public health due to its high mortality, transmission and geographical distribution. To date, there is no vaccine or specific treatment available and the knowledge regarding its pathogenesis is highly limited. Using a small-animal model system, this study showed that adult mice missing the type I interferon (IFN) receptor (IFNAR(-/-)) were susceptible to CCHFV and developed an acute disease with fatal outcome. In contrast, infection of wild-type mice (129 Sv/Ew) was asymptomatic. Viral RNA was found in all analysed organs of the infected mice, but the amount of CCHFV RNA was significantly higher in the IFNAR(-/-) mice than in the wild-type mice. Furthermore, the liver of IFNAR(-/-) mice was enlarged significantly, showing that IFN is important for limiting virus spread and protecting against liver damage in mice.

An Antibody against a Novel and Conserved Epitope in the Hemagglutinin 1 Subunit Neutralizes Numerous H5N1 Influenza Viruses

ABSTRACT The spread of the recently emerged, highly pathogenic H5N1 avian influenza virus has raised concern. Preclinical studies suggest that passive immunotherapy could be a new form of treatment for H5N1 virus infection. Here, a neutralizing monoclonal antibody (MAb) against the hemagglutinin (HA) of the influenza A/chicken/Hatay/2004 H5N1 virus, MAb 9F4, was generated and characterized. MAb 9F4 binds both the denatured and native forms of HA. It was shown to recognize the HA proteins of three heterologous strains of H5N1 viruses belonging to clades 1, 2.1, and 2.2, respectively. By use of lentiviral pseudotyped particles carrying HA on the surface, MAb 9F4 was shown to effectively neutralize the homologous strain, Hatay04, and another clade 1 strain, VN04, at a neutralization titer of 8 ng/ml. Furthermore, MAb 9F4 also neutralized two clade 2 viruses at a neutralizing titer of 40 ng/ml. The broad cross-neutralizing activity of MAb 9F4 was confirmed by its ability to neutralize live H5N1 viruses of clade 2.2.2. Epitope-mapping analysis revealed that MAb 9F4 binds a previously uncharacterized epitope below the globular head of the HA1 subunit. Consistently, this epitope is well conserved among the different clades of H5N1 viruses. MAb 9F4 does not block the interaction between HA and its receptor but prevents the pH-mediated conformational change of HA. MAb 9F4 was also found to be protective, both prophylactically and therapeutically, against a lethal viral challenge of mice. Taken together, our results showed that MAb 9F4 is a neutralizing MAb that binds a novel and well-conserved epitope in the HA1 subunit of H5N1 viruses.

Crimean-Congo hemorrhagic fever virus delays activation of the innate immune response.

As a first line of defence against virus infection, mammalian cells elicit an innate immune response, characterized by secretion of type I interferons and the up‐regulation of interferon stimulated genes. Many viruses down‐regulate the innate immune responses in order to enhance their virulence. Crimean‐Congo hemorrhagic fever virus (CCHFV), a Nairovirus of the family Bunyaviridae is the causative agent of severe hemorrhagic fever in humans with high mortality. Knowledge regarding the innate immune response against CCHFV is most limited. Interestingly, in this study it is shown that replicating CCHFV delays substantially the IFN response, possibly by interfering with the activation pathway of IRF‐3. In addition, it is demonstrated that CCHFV replication is almost insensitive to subsequent treatment with interferon‐α. Once the virus is replicating, virus replication is more or less insensitive to the antiviral effects induced by the interferon. By using an interferon bioassay, it is shown that infected cells secrete interferon relatively late after infection, that is, 48 hr post‐infection. In summary, the results suggest the presence of a virulence factor encoded by CCHFV that delays the host defence in order to allow rapid viral spread in the host. J. Med. Virol. 80:1397–1404, 2008.

Structure of Crimean-Congo Hemorrhagic Fever Virus Nucleoprotein: Superhelical Homo-Oligomers and the Role of Caspase-3 Cleavage

ABSTRACT Crimean-Congo hemorrhagic fever, a severe hemorrhagic disease found throughout Africa, Europe, and Asia, is caused by the tick-borne Crimean-Congo hemorrhagic fever virus (CCHFV). CCHFV is a negative-sense single-stranded RNA (ssRNA) virus belonging to the Nairovirus genus of the Bunyaviridae family. Its genome of three single-stranded RNA segments is encapsidated by the nucleocapsid protein (CCHFV N) to form the ribonucleoprotein complex. This ribonucleoprotein complex is required during replication and transcription of the viral genomic RNA. Here, we present the crystal structures of the CCHFV N in two distinct forms, an oligomeric form comprised of double antiparallel superhelices and a monomeric form. The head-to-tail interaction of the stalk region of one CCHFV N subunit with the base of the globular body of the adjacent subunit stabilizes the helical organization of the oligomeric form of CCHFV N. It also masks the conserved caspase-3 cleavage site present at the tip of the stalk region from host cell caspase-3 interaction and cleavage. By incubation with primer-length ssRNAs, we also obtained the crystal structure of CCHFV N in its monomeric form, which is similar to a recently published structure. The conformational change of CCHFV N upon deoligomerization results in the exposure of the caspase-3 cleavage site and subjects CCHFV N to caspase-3 cleavage. Mutations of this cleavage site inhibit cleavage by caspase-3 and result in enhanced viral polymerase activity. Thus, cleavage of CCHFV N by host cell caspase-3 appears to be crucial for controlling viral RNA synthesis and represents an important host defense mechanism against CCHFV infection.

Crimean-Congo hemorrhagic fever virus activates endothelial cells

ABSTRACT 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.

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.

Development and deployment of a rapid recombinase polymerase amplification Ebola virus detection assay in Guinea in 2015.

In the absence of a vaccine or specific treatments for Ebola virus disease (EVD), early identification of cases is crucial for the control of EVD epidemics. We evaluated a new extraction kit (SpeedXtract (SE), Qiagen) on sera and swabs in combination with an improved diagnostic reverse transcription recombinase polymerase amplification assay for the detection of Ebola virus (EBOV-RT-RPA). The performance of combined extraction and detection was best for swabs. Sensitivity and specificity of the combined SE and EBOV-RT-RPA were tested in a mobile laboratory consisting of a mobile glovebox and a Diagnostics-in-a-Suitcase powered by a battery and solar panel, deployed to Matoto Conakry, Guinea as part of the reinforced surveillance strategy in April 2015 to reach the goal of zero cases. The EBOV-RT-RPA was evaluated in comparison to two real-time PCR assays. Of 928 post-mortem swabs, 120 tested positive, and the combined SE and EBOV-RT-RPA yielded a sensitivity and specificity of 100% in reference to one real-time RT-PCR assay. Another widely used real-time RT-PCR was much less sensitive than expected. Results were provided very fast within 30 to 60 min, and the field deployment of the mobile laboratory helped improve burial management and community engagement.

Healthy individuals immune response to the Bulgarian Crimean-Congo hemorrhagic fever virus vaccine

Crimean-Congo hemorrhagic fever virus (CCHFV) poses a great threat to public health due to its high mortality and transmission rate and wide geographical distribution. There is currently no specific antiviral therapy for CCHF. This study provides the first in-depth analysis of the cellular and humoral immune response in healthy individuals following injection of inactivated Bulgarian vaccine, the only CCHFV vaccine available at present. Vaccinated individuals developed robust, anti-CCHFV-specific T-cell activity as measured by IFN-γ ELISpot assay. The frequency of IFN-γ secreting T-cells was 10-fold higher in individuals after vaccination with four doses than after one single dose. High levels of CCHFV antibodies were observed following the first dose, but repeated doses were required to achieve antibodies with neutralizing activity against CCHFV. However, the neutralizing activity in these groups was low.

Diagnostic Assays for Crimean-Congo Hemorrhagic Fever

On-site testing would diminish time, costs, and risks involved in handling of highly infectious materials.

Comparison of Antiviral Activity of Recombinant and Natural Interferons Against Crimean-Congo Hemorrhagic Fever Virus

As a first line of defence against a virus infection, mammalian cells elicit an innate immune response, characterized by secretion of type I interferons (IFN) and up-regulation of interferon stimulated genes (ISGs). We have previously included Crimean Congo Hemorrhagic Fever Virus (CCHFV) in the list of type I IFN-sensitive viruses. In this in vitro study, we have compared the antiviral activity of two recombinant IFN-alpha preparations (Roferon A and Intron A) with a natural IFN-alpha produced in human leukocytes (Multiferon). Our results clearly demonstrate that these commercially available IFNs have significant antiviral activities against CCHFV. However, we could show that Multiferon inhibits viral replication more efficiently than the two recombinant IFN alpha preparations.