Development of reverse genetic tools to study Chapare and Machupo viruses

Abstract

Arenaviruses are highly pathogenic viruses that pose a serious public health threat. Chapare virus (CHAV) and Machupo virus (MACV), two New World arenaviruses, cause hemorrhagic fevers with case fatality rates of up to 45%. Research on therapeutic drug targets and vaccines for these viruses is limited because biosafety level 4 containment is required for handling them. In this study, we developed reverse genetics systems, including minigenomes and recombinant viruses, that will facilitate the study of these pathogens. The minigenome system is based on the S segment of CHAV or MACV genomes expressing the fluorescent reporter gene ZsGreen (ZsG). We also generated recombinant CHAV and MACV with and without the ZsG reporter gene. As a proof-of-concept study, we used both minigenomes and recombinant viruses to test the inhibitory effects of previously reported antiviral compounds. The new reverse genetics system described here will facilitate future therapeutic studies for these two life-threatening arenaviruses.

Keywords: Antiviral; Arenaviruses; Chapare virus; Hemorrhagic fever; Machupo virus; Reverse genetics.

Fig. 1.. Design and characterization of minigenomes.

(A) Schematics of Chapare and Machupo virus minigenomes and support plasmids (pC-N and pC-L). CHAV and MACV minigenomes contain the antigenome UTRs of the CHAV S segment and MACV S segment, respectively, in which the nucleocapsid protein (N) and glycoprotein precursor (GPC) coding sequences have been replaced with ZsGreen (ZsG). Plasmids expressing CHAV or MACV polymerase (L) and nucleoprotein (N) were provided in trans. (B) Replication of CHAV and MACV minigenomes with or without their respective support plasmids. Representative fluorescence images of Vero-E6 cells transfected with minigenomes of indicated viruses with (+L + N) or without support plasmids specific that minigenome. (C) Sequence similarity between CHAV and MACV genes and proteins. The table shows the percent nucleotide similarity between the L and S segments of CHAV and MACV along with the percentage similarity between the different proteins of CHAV and MACV. (D) Compatibility of CHAV and MACV proteins. After transfection with the indicated combinations of support plasmids and minigenomes, minigenome replication was measured using BioTek Cytation for ZsG-positive cells. Relative ZsG expression was measured with values normalized to non-transfected cells. All experiments were done in quadruplicate.

Fig. 2.. Design and characterization of recombinant viruses.

(A) Schematic representation of the genomes of recombinant CHAV and MACV with or without ZsG protein. Recombinant reporter viruses contain ZsG coding sequence fused to N via a porcine teschovirus-1 2A (P2A). (B) Immunofluorescence assay (IFA) was performed on Vero-E6 cells infected with different recombinant viruses using either anti-CHAV nucleoprotein antibody (for CHAV) or hyperimmune serum raised against South American arenaviruses (Guanarito, Machupo, and Sabiá viruses; for MACV). Representative IFA images (magnification 20 × ; EVOS microscope) of recombinant wild-type CHAV (rCHAV), recombinant reporter CHAV (rCHAV-ZsG), recombinant wild-type MACV (rMACV), and recombinant reporter (rMACV-ZsG). (C) Growth kinetics of different recombinant viruses in Vero-E6 cells.

Fig. 3.. Screening antivirals using reverse genetics tools.

(A) Screening using minigenome assays. Concentration-response curves in Huh-7 cells transfected with CHAV (CHAV-MG, blue circles) and MACV (MACV-MG, green triangles) minigenomes and their respective support plasmids treated with nucleoside analogs. Reductions in ZsG fluorescence in infected cells were measured, with values normalized to mock-treated cells (DMSO only). For each concentration of antivirals, cell viability was measured via CellTiter-Glo assay (purple), and values were normalized to those in mock-treated cells. Each point on the graph represents the mean, and error bars indicate standard deviations of quadruplicate wells. (B) Screening using recombinant viruses. Concentration-response curves in Huh-7 cells infected with rCHAV-ZsG (blue circles) or rMACV-ZsG (green triangles) after treatment with various antiviral compounds. For each concentration of antivirals, cell viability was measured via CellTiter-Glo assay (purple), and values were normalized to those in mock-treated cells. Each point on the graph represents mean and error bars indicate standard deviations of quadruplicate wells. (C) 50% effective inhibition concentration (EC₅₀), 50% cell cytotoxicity (CC₅₀), and selective index (SI) values for each compound against CHAV-MG, MACV-MG, rCHAV-ZsG, or rMACV-ZsG.