With the noticeable exception of ribavirin recommended by the World Health Organization (WHO) to treat CCHFV infection , there is no specific medical therapy. Therefore, there is a need for the development of novel antiviral strategies against nairovirus infections.
In this work, for the first time, we evaluated the antiviral activity of siRNAs targeting the L (polymerase), M (glycoproteins) and S (nucleoprotein) transcripts of HAZV, a non pathogenic nairovirus in humans, which is considered as a surrogate CCHFV model.
We observed that siRNAs complementary to the mRNAs encoded by the L and M genomic segments had a lower effect than those targeting the S segment. Interestingly, three studies performed in arthropod cells showed that the nucleoprotein gene of orthobunyaviruses is a RNAi prim target. We demonstrated the efficient inhibition of HAZV in tick cells via RNAi induced by a Semliki Forest replicon expressing the S segment whereas the L segment had no effect . In earlier studies, Billecocq et al. and Powers et al. observed a similar phenomenom in mosquito cells infected by Rift Valley Fever virus  and La Crosse virus , respectively. Furthermore, in mammalian cells La Crosse virus replication was successfully decreased by siRNAs targeting the S segment and similarly to our results, the L and M siRNAs had a much weaker effect . Finally, orthobunyavirus Akabane replication was inhibited up to 99% by siRNAs directed against highly conserved regions of the nucleoprotein gene .
As other RNA viruses, nairovirus present a high mutation rate which might contribute to their escape from siRNAs inhibition. The emergence of viral mutants is lowered when using siRNAs targeting the most conserved viral sequences. Among the three genomic segments, the S segment is the most conserved within the Nairovirus genus because of its lowest mutation rate [49, 50]. This observation indicates that siRNAs directed against the S segment are potentially interesting anti-nairovirus molecules.
The nucleoprotein plays a crucial role in the regulation of viral transcription and replication. It associates with genomic RNA and serves as a template for the polymerase to initiate both steps during virus cycle. It is assumed that cytoplasmic NP concentration is important for the transition from transcription to replication  and therefore, a decrease in NP production should contribute to virus inhibition, as observed in our study.
Among all tested siRNAs, only three (siS1, siS2 and siS3) exhibited a strong inhibition of HAZV replication (up to 90%). The unequal siRNAs efficiency is in agreement with previous works [51–53]. The interaction of proteins with the viral transcripts or the presence of secondary structures in these mRNAs may interfere the proper recognition by the homologous siRNAs [54, 55], explaining the variability of siRNAs efficacy. Interestingly, the combination of siS1, siS2 and siS3 used at a concentration of 33 nM (100 nM siRNA final concentration) induced ~ 90% virus inhibition which is similar to the effect observed for each individual siRNAs (data not shown, figure 1A). The absence of additive or synergistic antiviral effect observed here was also reported in other studies. It was hypothesized that the competition of siRNAs for RISC might explain this lack of effect [56, 57].
In our study, the two most active siRNAs, siS1 and siS2, demonstrated a specific inhibitory effect against HAZV in a dose-dependent manner. These two siRNAs did not induce IFN production or cell toxicity. We showed that treatment with each siRNAs correlates with the reduction of nucleoprotein expression level. A concentration as low as 0.1 nM for both siRNAs was sufficient to reduce significantly HAZV replication. A similar antiviral effect at such concentrations had already been described in a previous study with Epstein-Barr virus . We observed that the siRNA stability lasted at least up to 72 hrs. The prolonged stability of siRNAs with a relatively long half life (from 3 to 8 days after transfection) was also shown in experiments with vaccinia virus , monkeypox virus , food and mouth disease virus  and HIV-1 . We also demonstrated that the antiviral activity of siRNAs was effective when carried out after infection. Taken together, the results are encouraging for the use of siRNA as future prophylactic and therapeutic treatments of nairovirus infection.
Since its discovery , RNAi technology has been assessed in several clinical trials. Among these, the most developed assays include the treatment of HIV-1  and RSV . siRNAs were successfully used against viral diseases in numerous animal models including infections affecting the liver: hepatitits C virus [64, 65], hepatitits B virus [31, 33, 34, 66, 67] or Hepatitis E virus . These data suggest that a similar strategy may be considered to treat patients infected with CCHFV which has an important hepatotropism.
We demonstrated in vitro the inhibitory activity of ribavirin against HAZV in a concentration dependent manner. This guanosine analogue acts through several mechanisms including the replication of viral nucleic acid. Ribavirin and siRNAs having distinct modes of action and different target, it was tempting to use them in combination to improve the efficacy of each separate treatment. We reported here the additive or synergistic effect of siRNAs in combination with ribavirin without increasing the cellular toxicity. Our results highlight the interest of a combined therapy. Such a therapy is known to reduce the drug toxicity by decreasing the active dose  and preventing the antiviral compound resistance [70, 71]. Ribavirin is a potent inhibitor of several viruses in vitro  and in vivo . Thus, it should be interesting to associate ribavirin and siRNAs to treat CCHFV infections.