Inhibition of Hazara nairovirus replication by small interfering RNAs. Part 2

The nairovirus are spherical enveloped particles of 100 nm in diameter. Their genome consists of three segments of single-stranded RNA of negative polarity designated S (Small), M (Medium) and L (Large) RNA segments. These three segments encode the nucleocapsid protein (NP), the envelope glycoproteins (Gn and Gc) and an RNA-dependent RNA polymerase (L), respectively. During the viral cycle, NP and L drive the processes of transcription (mRNA synthesis) and replication (synthesis of genomic RNA) that occur in the cytoplasm. Thus, targeting these proteins is likely an accurate strategy to inhibit the viral replication.

Currently, there are neither vaccines nor effective therapies to treat bunyavirus infection in humans. Ribavirin, however, has been shown to inhibit CCHFV replication in Vero cells, reducing the mean time to death in infant mice and partially protecting infected STAT-1 KO mice. Several studies reported the efficiency of oral or intravenous use of ribavirin to treat CCHFV infection cases but to date, no double-blind trial had been carried out. Therefore, it is important to initiate research programs aimed towards the development of new medical countermeasures against CCHFV.

Since its discovery in 1998, RNA interference (RNAi) has been successfully applied as a technology to inhibit gene expression. Small interfering RNAs (siRNAs), the mediators of RNAi, are a class of double-stranded RNA molecules (20-25 nucleotides in length) that interfere with translation by inducing sequence-specific degradation of homologous mRNA. Recently, several RNAi-based applications for gene silencing have been developed to target pathogenic human viruses causing acute or chronic infections including HIV-1, influenza virus, respiratory syncytial virus, hepatitis B and C viruses, as well as Marburg and Ebola filoviruses [38-40].

In this report, we tested various chemically synthesized siRNAs for their ability to inhibit HAZV replication in cell culture. We demonstrated that siRNAs targeting the NP mRNA depicted a stronger antiviral effect than those designed to inhibit the L and M segment encoded mRNAs. siRNAs were efficient when transfected in cells before or after HAZV infection and their use in combination with ribavirin induced a synergistic or an additive antiviral effect, according to the dose of ribavirin used. Thus, our study highlights the potential of RNAi in the antiviral treatment of nairovirus infection.

Cell lines and viruses

A549 cells (human lung carcinoma cell line, ATCC CCL-185) and Vero E6 cells (African green monkey kidney, ATCC CRL-1586) were grown in F12K medium and Dulbecco’s modified eagle medium DMEM (Gibco, Invitrogen Corporation, Paisley, United Kingdom), respectively, supplemented with 10% heat-inactivated fetal calf serum (FCS; Invitrogen, Sao Paulo, Brazil) and maintained at 37°C in a 5% CO2 atmosphere. BHK21 cells (baby hamster kidney, ATCC CCL10) were cultured in Glasgow minimum essential medium GMEM (Gibco, Invitrogen Corporation) with 10% FCS, 10% tryptose phosphate (Sigma-Aldrich, St Quentin-Fallavier, France) and 50 mM HEPES (Gibco, Invitrogen Corporation) at 37°C in a 5% CO2 atmosphere.

The HAZV strain JC280 used in these experiments was produced by infecting 90% confluent BHK21 cells at a multiplicity of infection (MOI) of 0.001. Virus was titrated using a focus-forming assay in Vero E6 cells as described below. Viral stocks usually reached 106-107 foci forming units per ml (ffu/ml).