Small interfering RNAs are highly effective inhibitors regarding Crimean-Congo hemorrhagic fever virus replication in vitro

Crimean-Congo hemorrhagic fever virus (CCHFV) is one of the prioritized diseases of World Health Organization, considering its potential to create a public health emergency and more importantly, the absence of efficacious drugs and/or vaccines regarding treatment. The highly lethal nature characteristic to 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 siRNAs 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 hours, nucleic acid from the supernatant was extracted for Droplet Digital PCR analysis. Among the investigated siRNAs we identified four effective candidates against all three segments of CCHF genome: one for the S and M segments, whilst two for the L segment. Consequently, blocking any segment of CCHFV leads to changes in the virus copy number that indicates an antiviral effect of the siRNAs in vitro. The most active siRNAs were demonstrated a specific inhibitory effect against CCHFV in a dose-dependent manner. In summary, we demonstrated the ability of specific siRNAs to inhibit CCHFV replication in vitro. This promising result can be used in future anti-CCHFV therapy developments.


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Crimean-Congo hemorrhagic fever virus (CCHFV) categorically belongs to the Orthonairovirus 37 genus, the Nairoviridae family in the Bunyavirales order. CCHFV is causing a mild to severe 38 hemorrhagic disease in humans, with fatality rates from 5% up to 30% [1].

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Emerging infectious diseases (EIDs) are growing threats to animal and human health. CCHFV 45 is a tick-borne pathogen that causes an increasing number of severe infections and presents over a 46 wide geographic range, including areas in South-Eastern Europe, Western and Central Asia, the Middle East and Africa as well [1]. This virus is transmitted primarily by ticks, but the spectrum of 48 natural hosts for CCHFV includes a wide variety of domestic and wild animals [3].
There are neither vaccines nor effective antiviral therapies for the treatment of CCHFV infections 50 in humans to date [4]. There is a growing need for advantaged research and development activities 51 for such pathogens as CCHFV, since there is a constantly growing geographic and epidemiologic 52 burden of the disease and BSL-4 capacity is limited throughout the world, which can safely handle 53 such research.

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Among antiviral therapies, RNA interference (RNAi) can be used to suppress viral replication 55 by targeting either viral-or host genes that are needed for viral replication. Since its discovery in 1998 56 [5], it has revolutionized the mechanism of gene silencing and improved our understanding of the 57 endogenous mechanism of gene regulation to enhance the use of new tools for antiviral research.

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Silencing viral genes such as viral polymerases, master regulators of viral gene transcription and viral 59 genes that act early in the viral life cycle, may suppress viral replication more effectively than 60 targeting late or accessory viral genes. Moreover, RNAi could target viral proteins and pathways, 61 which are unique to the viral life cycle and it has become possible to interfere with viral infections 62 and replication without unacceptable host cell toxicity [6]. Accordingly, the major advantage of RNA 63 interference is its target specificity. In recent years, many viruses have been successfully targeted by 64 RNA interference such as human immunodeficiency virus (HIV) [7,8]

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CCHFV viral stock was titrated using the TCID50 method with the immunofluorescence assay.

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Briefly, serial 10-fold dilutions of CCHFV supernatant were inoculated (100 µl) on 60% confluent 107 A549 cells (30000 cells/well) in 48-well plates. Viral adsorption was allowed for 1 hour at 37°C. After 108 washing cells with PBS three times, cells were incubated for 3 days at 37°C in DMEM supplemented 109 with 2% FBS. The fixation and the immunofluorescence assay were performed as previously   (Table   121 1). Sequences were subjected to a BLAST search against GenBank to minimize off-target effects. All

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The use of cytotoxicity tests was important to find out the concentration at which siRNAs do not 149 cause cell death but their concentration is high enough to inhibit virus replication.

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A549 cells were seeded in 96-well plates at a density of 2x10 4 cells/well to achieve 60-70% confluent 153 cell monolayers on the day after in a humidified incubator at 37°C with 5% CO2.

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Cells were transfected in triplicate biological replicates with siRNAs in the following final

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The quantitative real-time TaqMan based assay was carried out using a One-step RT-PCR kit 179 (Qiagen) in the Light Cycler 2.0 system (Roche

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In our study, 15 siRNAs were designed and synthesized to test the inhibitory activity on CCHFV 221 replication and target the mRNAs produced by S, M and L segments. We analyzed the high inhibitory 222 effect of some S (siS2), M (siM1) and L (siL3, siL4) segment-specific siRNAs. We experienced that 223 siRNAs inhibited CCHFV replication in different efficiency and dose-dependent manner.  (Table 3).

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The siRNAs treatment could cause visual cytopathogenic effects (CPEs) and affect viral growth, 228 therefore we performed light microscopic observation to evaluate cell growth and viability. Firstly,

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we had to find the appropriate siRNA concentration that is effective in inhibiting CCHFV replication

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Based on the ddPCR results, a high and significant copy number decrease in the case of some 267 siRNAs (siS2, siM1, siL3 and siL4) was detected. As shown in Figure 1

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In our study, among siRNAs that were designed against the S segment, siS2 has inhibited effectively 354 (93%) CCHFV copy number. Our study is in agreement with previous works [15,27] that targeting 355 the S segment by siRNAs can produce an effective inhibitory impact. In consequence, using the S 356 segment as the target for silencing virus replication has proven to be an option for future therapeutics.

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In case of CCHFV, the largest of the three segments termed the L segment, encodes an RNA-372 dependent RNA polymerase (RdRp) that is characterized by several conserved functional regions [2].

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Moreover, next to nucleoprotein, L protein drives the processes of transcription and replication that 374 occur in the cytoplasm during the viral replication cycle. Thus, targeting this segment is likely to be 375 an exact strategy. In our study, a remarkable copy number decrease (by 92%) was caused by siL4.

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Taken together, our results provide further support for the use of RNA interference-based 377 technique in the development of antiviral drugs against CCHFV infections. Moreover, to our 378 knowledge, this is the first study that used designed siRNAs against CCHFV replication in vitro and 379 the first study to provide RNAi solution to all three genomic segments of a nairovirus. Currently,