Rescue from Cloned cDNAs and In Vivo Characterization of Recombinant Pathogenic Romero and Live-Attenuated Candid #1 Strains of Junin Virus, the Causative Agent of Argentine Hemorrhagic Fever Disease

Abstract

The New World arenavirus Junin virus (JUNV) is the causative agent of Argentine hemorrhagic fever (AHF), which is associated with high morbidity and significant mortality. Several pathogenic strains of JUNV have been documented, and a highly attenuated vaccine strain (Candid #1) was generated and used to vaccinate the human population at risk. The identification and functional characterization of viral genetic determinants associated with AHF and Candid #1 attenuation would contribute to the elucidation of the mechanisms contributing to AHF and the development of better vaccines and therapeutics. To this end, we used reverse genetics to rescue the pathogenic Romero and the attenuated Candid #1 strains of JUNV from cloned cDNAs. Both recombinant Candid #1 (rCandid #1) and Romero (rRomero) had the same growth properties and phenotypic features in cultured cells and in vivo as their corresponding parental viruses. Infection with rRomero caused 100% lethality in guinea pigs, whereas rCandid #1 infection was asymptomatic and provided protection against a lethal challenge with Romero. Notably, Romero and Candid #1 trans-acting proteins, L and NP, required for virus RNA replication and gene expression were exchangeable in a minigenome rescue assay. These findings support the feasibility of studies aimed at determining the contribution of each viral gene to JUNV pathogenesis and attenuation. In addition, we rescued Candid #1 viruses with three segments that efficiently expressed foreign genes introduced into their genomes. This finding opens the way for the development of a safe multivalent arenavirus vaccine.Arenaviruses are enveloped viruses with a bisegmented negative-strand (NS) RNA genome. Each genomic RNA segment, L (ca. 7.3 kb) or S (ca. 3.5 kb), uses an ambisense coding strategy to direct the synthesis of two polypeptides in opposite orientations and separated by a noncoding intergenic region (IGR) that acts as a transcription termination signal for the virus polymerase (21, 29). The S RNA encodes the viral glycoprotein precursor (GPC) and the nucleoprotein (NP). The GPC is posttranslationally cleaved by the cellular site 1 protease to yield the two glycoproteins GP1 and GP2, which, embedded in the lipid bilayer, form the viral spikes in the mature virion that are crucial for receptor recognition and cell entry. The L RNA encodes the viral RNA-dependent RNA polymerase (or L polymerase) and the small (ca. 11-kDa) RING finger protein Z that is the arenavirus counterpart of the M protein found in many other NS RNA viruses (24,28,30).

Inhibition of Arenavirus by A3, a Pyrimidine Biosynthesis Inhibitor

“…LCMV (Armstrong strain 53b), r3LCMV/CAT-GFP, r3LCMV/GFP-Gluc, live attenuated Candid#1 vaccine strain of JUNV, and r3Candid#1/CAT-GFP were produced by infecting either BHK-21 (LCMV) or Vero (Candid#1) cells at a multiplicity of infection (MOI) of 0.01 and harvesting tissue culture supernatants (TCS) at 72 h postinfection (h.p.i.) (33)(34)(35)(36).…”

Rescue of a Recombinant Machupo Virus from Cloned cDNAs and In Vivo Characterization in Interferon (αβ/γ) Receptor Double Knockout Mice

“…The rescue of rMACV was completed in a manner similar to that described previously by our laboratory (42). Briefly, equimolar amounts of the two full-segment MACV plasmids and the two expression plasmids were transfected into BHK21 cells.…”

“…To determine the sequences of the 5= and 3= 19-nucleotide terminal regions of the S and L segments, total RNA was isolated from Vero cells infected with MACV. RNA was treated with RNA 5= Tobacco acid pyrophosphatase (Epicentre) and ligated using T4 RNA ligase I as previously described (42). The ligated RNA was reverse transcribed utilizing the primers MACV_SsegR312 (5=-AGGGTGACTGACTGGAACTC-3=) and MACV_SsegF3129 (5=-GACA TGAGCCTATCCACTTC-3=) and the primers MACV_LsegR349 (5=-TGTG ATGGATGTCGGTAGTG-3=) and MACV_LsegF6917 (5=-AGGCGTGTGC TTCACAGGAC-3=) for the S and L segments, respectively.…”

“…The plasmids expressing MACV L and S segment MG were generated as described previously (42). Briefly, viral genes on the pPol-I-MACVSag and pPol-I-MACVLag template plasmids were replaced by the green fluorescent protein (GFP) and firefly Luciferase (fLuc) reporter genes (see Fig.…”

Endothelial Cell Permeability and Adherens Junction Disruption Induced by Junín Virus Infection

“…Recently, a study to rescue JUNV strains from cloned cDNAs suggests that Candid#1 does, in fact, replicate more slowly than Romero. 51 Of course, in this in vitro system, there are no immune responses; therefore, in vivo studies are needed to evaluate the effects of immune responses to Candid#1 replication and dissemination and more clearly elucidate the mechanism of attenuation. Although it might also call into question whether the observed endothelial response to JUNV infection is a general response to viral infection rather than a specific response to JUNV, the distinction, for the purposes of investigating pathogenesis, is irrelevant.…”