Innate immune signalling induced by Crimean-Congo haemorrhagic fever virus proteins in vitro
Crimean-Congo haemorrhagic fever orthonairovirus (CCHFV) is a tick-borne viral zoonosis associated with haemorrhagic fever in humans. The World Health Organisation identified CCHFV as a priority pathogen for research. The disease is widespread globally with regions of endemicity in Africa, Asia and eastern Europe; however, the emergence and re-emergence of disease in endemic and non-endemic regions is a cause for concern. Additionally, the lack of rapid point-of-care assays, a vaccine or therapeutic interventions approved for use in humans complicates the control and management of disease, which requires an improved understanding of the virus-host interactions. Viruses have co-evolved with their host/s. Dysregulation of the immune response is a common strategy utilised to evade immune detection and clearance. The innate immune response is a robust non-specific response to infection with the aim of limiting virus replication and spread while activating the adaptive immune response that mediates virus clearance and protection. In this study, innate immune modulation by selected non-structural CCHFV proteins, including the NSM protein and ovarian-tumour like (OTU) protease, and Hazara orthonairovirus (HAZV), a possible model for CCHFV, was investigated. The CCHFV NSM protein, encoded on the M-segment, was expressed in vitro to evaluate the ability of the protein to modulate innate immune signalling. In South Africa, isolates containing an M-segment that are genetically related to other South African isolates (non-reassortant) and isolates containing an M-segment that are genetically similar to Asian isolates (reassortant) have been identified. The CCHFV NSM proteins from both reassortant and non-reassortant CCHFV isolates were evaluated. Despite 92,88% amino acid sequence similarity between the reassortant and non-reassortant NSM protein, the non-reassortant NSM protein downregulated key innate immune markers, including DDX58 (RIG-1), IFNB1, IFNAR1 and STAT1, whereas the reassortant NSM protein upregulated DDX58 (RIG-I) and IFNB1 expression. The results indicate that the reassortant NSM protein induces innate immune signalling, whereas the non-reassortant NSM protein significantly downregulates innate immune signalling. The non-reassortant NSM protein may therefore potentially compromise virus recognition and the induction of an antiviral state. The CCHFV OTU protease, encoded on the L-segment, has deubiquitinating and deISGylating activity that interferes with the regulation of cellular processes, including innate immune signalling. Modulation of innate immune signalling at transcriptional level by the CCHFV OTU protease was determined by gene expression analysis. Expression of the CCHFV OTU protease resulted in downregulation of IFNB1, IFNAR1 and STAT1 expression. The results suggest that, in addition to interfering with innate immune signalling by deubiquitination and deISGylation, the CCHFV OTU protease also downregulates innate immune signalling at transcriptional level. The CCHFV OTU protease may therefore significantly compromise the host's innate immune response to infection and represents a potential target for the development of therapeutic interventions. HAZV, a closely related virus to CCHFV, has been proposed as a clinical surrogate for disease in type I IFN-receptor deficient mice. Innate immune signalling induced by HAZV and HAZV RNA was evaluated to determine whether HAZV modulates innate immune signalling. A significant downregulation in IFNB1 expression during infection was observed and a lack of upregulation of several key innate immune markers. The results suggest that HAZV has the ability to downregulate innate immune signalling without completely abolishing innate immune activation. Further investigation to determine the mechanism utilised by HAZV may provide insights into whether HAZV would be suitable as a surrogate for the screening of therapeutic interventions to overcome innate immune signalling modulation post-infection. HAZV provides a safer alternative to CCHFV since HAZV can be cultured and handled in a biosafety level (BSL)-2 facility. This study has identified targets for the development of therapeutic interventions and potential inclusion in vaccines. In vivo studies are required to determine the significance of the findings during infection.