The Glycoprotein Precursor Gene of Junin Virus Determines the Virulence of the Romero Strain and the Attenuation of the Candid #1 Strain in a Representative Animal Model of Argentine Hemorrhagic Fever

Abstract

The New World arenavirus Junin virus (JUNV) is the causative agent of Argentine hemorrhagic fever (AHF), a potentially deadly disease endemic to central regions of Argentina. The live-attenuated Candid #1 (Can) strain of JUNV is currently used to vaccinate the human population at risk. However, the mechanism of attenuation of this strain is still largely unknown. Therefore, the identification and functional characterization of viral genetic determinants dictating JUNV virulence or attenuation would significantly improve the understanding of the mechanisms underlying AHF and facilitate the development of novel, more effective, and safer vaccines. Here, we utilized a reverse genetics approach to generate recombinant JUNV ( A rgentine hemorrhagic fever (AHF), a severe human disease caused by the New World arenavirus Junin virus (JUNV), is clinically characterized by hemorrhagic manifestations and central nervous system (CNS) involvement (1). The incubation period of AHF is usually from 6 to 14 days, followed by the onset of fever with a flu-like syndrome that is considered to be the first day of clinical AHF. Based on the clinical symptoms, the severity of the neurological involvement, and disease outcome, patients are grouped into mild, moderate, and severe clinical forms (2). Patients with severe forms of AHF present with marked CNS manifestations, including areflexia, muscular hypotonia, ataxia, seizures, and coma. Fatal cases are commonly associated with a terminal shock syndrome; superimposed bacterial infections can also be observed (3). Leucopenia and thrombocytopenia are detected during the first and second week after the onset of symptoms. The most frequent hemorrhagic manifestations are petechiae in the mouth and the axillary region and bleeding of the gums. Less common manifestations are epistaxis, hematuria, metrorrhagia, hemoptysis, and gastrointestinal hemorrhages (4). The mortality rates can be up to 30% in cases without specific treatment.Guinea pigs infected with JUNV reproduce most of the symp-

Machupo Virus Expressing GPC of the Candid#1 Vaccine Strain of Junin Virus Is Highly Attenuated and Immunogenic

“…This is in line with the findings of previous studies performed by others and us, in which GPC was identified to be the major viral factor for the attenuation of the vaccine strain of JUNV (14,16,22). Consistent with our previous report, rMACV infection was lethal to IFN-␣␤/␥ R Ϫ/Ϫ mice (15,18).…”

“…It has been established that the Cd#1 GPC renders pathogenic JUNV avirulent in a mouse model and in guinea pigs (14,16). To determine if Cd#1 GPC also renders MACV fully attenuated in vivo, rMACV/ Cd#1-GPC was intraperitoneally inoculated into IFN-␣␤/␥ R Ϫ/Ϫ mice, which succumb to parental rMACV infection (18).…”

“…The importance of the F427I mutation in JUNV attenuation was also demonstrated in guinea pigs infected with JUNV. However, this mutation itself was apparently not sufficient to fully attenuate the virus; mild diseases and virus dissemination still occurred in these infected animals (16). In contrast, guinea pigs infected with a JUNV strain that expresses the whole GPC of Cd#1 origin did not develop symptoms and were protected against subsequent challenge with a virulent JUNV strain.…”

“…A plaque reduction neutralization test (PRNT) was performed on Vero cells as described previously (16). Briefly, 80 PFU of rMACV was The growth of rMACV/Cd#1-GPC was characterized in Vero cells (MOI ϭ 0.01).…”

Arenavirus Genome Rearrangement for the Development of Live Attenuated Vaccines

“…This strategy overcomes safety concerns affecting other live attenuated vaccine approaches currently licensed, which are based on a limited number of amino acid substitutions (67). Particularly relevant to arenaviruses, a single amino acid mutation (F427I) within GP2 of the pathogenic XJ13 strain of JUNV attenuated XJ13 neurovirulence in suckling mice (25,26) and was also highly attenuating in the guinea pig model of AHF although it was insufficient to prevent virus dissemination and mild clinical and pathological symptoms (68). These findings suggest that similar limited genetic changes may result in reversion of Candid#1 to a more virulent form, which raises safety concerns and further supports arguments for the development of arenavirus live attenuated vaccines based on alternative approaches involving more drastic changes to the viral genome, such as the rearrangement of the viral genome proposed in the present study.…”