Development and characterization of a reverse genetics system for the lineage II Chicava strain of Machupo virus in a guinea pig model

Abstract

Background: Machupo virus (MACV) is a New World mammarenavirus (hereafter referred to as "arenavirus") and the etiologic agent of Bolivian hemorrhagic fever (BHF). No vaccine or antiviral therapy exists for BHF, which causes up to 35% mortality in humans. New World arenaviruses evolve separately in different locations. To date, up to eight lineages of MACV have been identified in Bolivia. While the prototype MACV Carvallo strain belongs to lineage I discovered in the Memore Province in the 1960s, the MACV lineage II strains have become the dominantly-circulating lineage in the same province since 1993.

Methods: We report the development of a reverse genetics system for the MACV lineage II Chicava strain, using a pRF42 plasmid encoding the L and S segment genomic RNA under the transcriptional control of a murine DNA-dependent RNA polymerase I promoter sequence. Rescue of the recombinant MACV Chicava strain (rMACV-Chicava) was accomplished by expression of the L protein and nucleoprotein genes of the MACV Carvallo strain in trans in transfected baby hamster kidney (BHK-21) cells. We characterized the multiplication kinetics of rMACV-Chicava in African green monkey kidney epithelial Vero cells, followed by determining the virulence phenotype in outbred Hartley guinea pigs.

Principal findings: We demonstrated that the multiplication kinetics in Vero cells, virulence phenotype in guinea pigs, and neutralizing antibody titers are indistinguishable between rMACV-Chicava and the wild-type parental virus.

Conclusion and significance: We conclude that rMACV-Chicava provides a useful model system to investigate the emergence of MACV lineage II strains and the guinea pig model has utility for the development of candidate vaccines and therapeutic antibodies for BHF.

Fig 1. Rescue of MACV Chicava.

(A) Schematic of plasmids used for rescue of rCHICV. Two plasmids containing the full-length MACV Chicava S and L segments including the 5’ and 3’ untranslated regions (UTR) and intergenic regions (IGR) downstream of a murine Pol-I promoter (mPol-I.p) were transfected alongside two expression plasmids encoding the MACV Carvallo NP and L genes downstream of a chicken β-actin promoter (chicken β-actin.p). The silent A525G single-nucleotide substitution was added in the L segment as a marker for recombinant virus. (B) Multiplication kinetics of rescued MACV Chicava compared to the wild-type isolate in Vero cells at a MOI of 0.01. The mean of three replicates is shown, with error bars displaying standard deviation.

Fig 2. Wild-type and recombinant MACV produce similar disease in 6–8 week-old Hartley guinea pigs.

(A) Survival rate of guinea pigs (n = 5 per group) challenged with 10⁴ pfu of the indicated MACV strains. (B) Mean body weight change compared to baseline weight. (C) Mean body temperature. The dotted line indicates the clinical definition of fever (39.5°C) for a 6–8-week-old guinea pig. Pink asterisk (*) indicates a broken transponder that prevented the collection of further body temperature readings in one surviving animal (#49).

Fig 3. Wild-type and recombinant MACV cause inflammation in the liver and brain.

Representative H&E-stained images of (A) liver and (B) brain tissues collected at the time of euthanasia. Images are representative of animals that succumbed to challenge. Black arrows indicate areas of (A) focal hepatic inflammation or (B) perivascular cuffs.

Fig 4. Guinea pigs infected with recombinant MACV Chicava produce a heterologous neutralizing antibody response.

(A) Individual serum PRNT₅₀ titers against wtMACV Carvallo or (B) rMACV Carvallo GPC_{ΔN83/N166/F438I}. Box plots extend from the 25^th to 75^th percentiles, with the center line indicating the median. Whiskers represent the minimum and maximum values. The dotted line indicates the lower limit of detection (LOD), i.e., 1:30 PRNT₅₀ titer. Samples with no neutralizing activity detected were plotted at 1:15 PRNT₅₀ titer (50% LOD).