The pathogenic arenaviruses are genetically diverse, globally distributed, and capable of causing human illness ranging from encephalitis to severe and often lethal HF. Divided into New World and Old World lineages, these rodent-borne viruses are most often transmitted to humans by direct contact or aerosol exposure to infectious rodent excreta, or, in some cases, via a chain of human-to-human transmissions within hospital settings. The overall public health impact can range from only a few cases (e.g., Sabia or Chapare viruses) to over 100,000 cases per year (e.g., LASV) 
. LUJV is the most recently discovered pathogenic arenavirus, identified in 2008 after a high fatality (80%) cluster of cases among primary, secondary, and tertiary contacts with the index patient 
A number of factors mark LUJV as a unique arenavirus. Although the data from the 2008 outbreak are limited, the high case fatality was striking compared with most other arenavirus outbreaks that typically are associated with case fatalities of 10–40% 
. Phylogenetically, LUJV is distinct from all previously identified arenaviruses, forming a unique lineage more closely related to the Old World than New World arenaviruses, but with over 40% divergence from LASV at the nucleotide level 
. The unique genomic sequence and high antigenic diversity compared to other Old World and New World arenaviruses greatly complicated its initial diagnosis by molecular (RT-PCR) or serology (IgM/IgG) techniques 
. Unfortunately, due to these and other factors, the virus later named LUJV was only identified as the specific cause of the 2008 outbreak several weeks after the last fatal case of Lujo HF.
To assess the in vivo
characteristics of LUJV, we began with the traditional newborn and weanling outbred mouse models of arenavirus infection 
. These experiments further demonstrated the unique virulence properties of LUJV compared to New World (JUNV-XJ13) and Old World (LASV-Josiah) prototype arenaviruses. Both JUNV and LASV are highly virulent in newborn and weanling mice, respectively, causing lethality in an age-dependent pattern 
. In previous experiments, JUNV dosages as low as 1.0×101
FFU caused uniform lethality after intracranial inoculation into 2-day-old mice (data not shown). Similar dosages of LASV were lethal in 14-day-old mice (data not shown). However, LUJV was non-lethal in mice regardless of the route of inoculation (intracranial, subcutaneous, or intra-peritoneal), mouse age at inoculation (2 or 14 days old), or viral dose (ranging up to the maximum dose tested, 2.0×103
FFU; Fig. S1
, and data not shown).
In marked contrast with the mouse results, LUJV caused severe, rapidly progressive, and uniformly lethal and hemorrhagic disease in strain 13/N guinea pigs. In this model, we observed an apparent incubation period of 5 to 6 days from the time of inoculation to the first clinical signs of illness (fever and weight loss). Over the next 24 to 48 h, the animals began to display signs of progressive illness (bilateral ocular discharge, continued fever, weight loss, and dehydration) until they were found dead or humanely euthanized when moribund. By day 5 PI, significant hematological changes began to occur, including hypoproteinemia, thrombocytopenia, and lymphopenia (). Interestingly, although we saw consistent elevation in key serum transaminase enzyme (aspartate transferase, alkaline phosphatase, and alanine transferase) activities, these were neither dramatic nor statistically significant and are typical of the generally poor release of tissue transaminases in guinea pigs in the face of tissue damage or necrosis. Subjective comparisons of clinical illness severity, and gross and histologic pathology between guinea pigs infected with LUJV or LASV suggests that LUJV caused a more profound illness (greater and more rapid weight loss, and frank hemorrhage and congestion in gastrointestinal organs, bladder, lymph nodes, and abdominal cavity), tissue damage (hepatic and myocardial necrosis), and hallmarks that may be consistent with disseminated intravascular coagulation (DIC) than LASV in preliminary experiments completed in our laboratory and as reported in 
( and , and data not shown).
Previous studies of human pathogenic New World (JUNV, Guanarito (GTOV), Machupo (MACV)) and Old World (LASV) arenaviruses using rodents and other small animal models failed to demonstrate clear consistent signs of HF (reviewed in 
). LUJV infection of guinea pigs, however, resulted in severe infarction, fibrin deposition, and hemorrhage in multiple organs, suggesting DIC. The progressive reductions in platelet numbers (9, 12, and 14 days PI) are consistent with the consumption of platelets at sites of localized virus inflammation and tissue destruction, or, in the most extreme cases, may be consistent with DIC at later time points PI. Anecdotally, the animal with severe frank hemorrhage into the abdominal cavity developed profound thrombocytopenia (99×103
). Unfortunately, no assays to determine coagulation factor parameters (i.e., activated partial thromboplastin time (APTT) or prothrombin time (PT)) were completed to directly assess the influence of these coagulation pathways on the observed coagulopathy. It is clear that further work is essential to definitively characterize the underlying mechanisms of these observations before firm conclusions can be drawn. For these types of studies this guinea pig model may provide a potentially useful alternative to non-human primate models for studying basic pathogenesis of a bona fide human pathogenic arenavirus causing severe coagulopathy and HF.
The rapid rise and magnitude of LUJV-specific antigen deposition, histologic pathology, and RNA titers in tissues, blood, urine, and abdominal fluid were surprising. Within 48 h of infection, the virus already disseminated to the liver, spleen, kidneys, and lungs, and was rapidly replicating up to 1.8×105 TCID50 eq/g of liver. Interestingly, this high-titer replication continued for another 48–72 h before the onset of illness, indicated by increased body temperature and weight loss 5–6 days PI. Tissue viral loads remained extremely high throughout the course of the disease, with death occurring 11–16 days PI.
Like other arenaviruses, LUJV broadly modulates host immune responses during infection. The molecular basis of the seemingly high LUJV virulence in humans has not been characterized. However, recent work using reverse genetics-derived recombinant viruses indicates that LUJV has unique promoter elements that influence the expression of the viral NP and glycoprotein, and an unusually long intergenic region sequence on the viral L segment, which influences expression of the Z protein (Bergeron et al., in-review). Both LASV and lymphocytic choriomeningitis virus NP and Z proteins have immunomodulatory properties and function as potent antagonists of host cell antiviral responses (
and reviewed in 
). These and other yet unrecognized molecular motifs may augment the apparently enhanced virulence of LUJV by increasing viral replication or interfering with immunoregulatory mechanisms within the host.
The ability of LUJV to influence the immune response is illustrated in guinea pigs by the finding that, despite the very high viral loads by day 5 PI, pro-inflammatory cytokine/chemokine genes, such as IL-1b and RANTES, were downregulated 2–4-fold early during infection (). In contrast, as early as day 5 PI, potent mediators of macrophage and neutrophil activation and inflammation (IL-8, MCP-1) and pro-inflammatory molecules (IL-12p40, and IFNg) were transcriptionally upregulated (10 to >100-fold) compared to mock inoculated animals, especially in liver and kidney tissues (, and Table S1
). Potentially important and robust induction of the broad immunomodulatory mediator IL-10 was also detected in lungs by day 9 PI, and may signal attempts to limit and control pro-inflammatory activity, allowing for further virus replication.
Overall, the pattern of gene induction and histological evidence from our study allow for the speculation that, in early stages of infection, the animals mounted a pro-inflammatory and innate immune response (presumably involving macrophages, neutrophils, NK-cells (i.e., Kurloff cells in the guinea pig), and/or NKT-cells) in multiple tissues. This was followed by a predominantly Th1 response dominated by IFNg, MCP-1, and IL-12p40, which likely stimulated activation and enhanced function presumably of NK cells, CD4+ TH
1 cells, and/or CD8+ cytotoxic lymphocytes, resulting in a strong bias towards cell mediated immunity. Since LUJV is not highly cytopathic in cell culture, these responses may have been more deleterious than helpful to the host, due to immune cell-mediated destruction of vital organs. This hypothesis is consistent with recent work describing enhanced LASV pathogenesis due to deleterious T-cell mediated activation and stimulation of monocytes/macrophages, leading to tissue destruction in humanized HHD mice 
. Regardless of the immune mechanisms stimulated by LUJV infection, these responses were insufficient to control virus replication and dissemination throughout the host, and to prevent eventual lethality.
Similarly to LASV infection in humans and non-human primate animal models, LUJV does not appear, at least at the mRNA transcriptional level, to elucidate an end-stage cytokine storm as seen in fatal hemorrhagic cases of infection with Ebola, Marburg, or Rift Valley fever viruses 
. Although IFNu-producing cells (presumably natural killer cells, NKT-cells, and/or macrophages) are clearly activated, the increase in mRNA encoding the TNFa, iNOS, and IL-1b genes was not significant even in terminal cases. Among the limited genes analyzed in this study, we speculate that complete dysregulation of the host immune response is not responsible for the dramatic vascular permeability changes and coagulopathy observed in the guinea pig model of LUJV HF. Although our results are suggestive, more definitive studies of the guinea pig immune response are necessary before drawing distinct conclusions regarding the roles of inflammatory mediators, immune effector cells, and viral virulence factors in the pathogenesis of LUJV HF in this animal model.
The dramatic severity of the clinical illness, short survival times, high tissue viral loads, and the mRNA gene expression patterns in visceral organs further highlight the unique pathogenic characteristics of LUJV compared with other studies of LASV or JUNV 
. Our in vivo
data, taken together with recent insights into the unique genomic elements regulating viral replication in cell culture, stress the importance of further work to elucidate the possibly novel pathogenic mechanisms employed by LUJV to cause HF in both humans and guinea pigs. Direct comparisons of the precise pathogenic mechanisms utilized by LUJV and other pathogenic arenaviruses are underway, and may reveal insights into the apparent enhanced virulence of LUJV in humans and guinea pigs, and provide broader understanding of arenavirus HF. Regardless of the exact mechanisms, LUJV is clearly highly pathogenic, easily transmitted in health care settings, and a potential health threat in southern Africa. Establishing a robust and reliable animal model of severe and lethal LUJV HF is a critical first step for further investigations to increase our understanding of LUJV and for developing anti-viral therapeutics or experimental vaccines for this new and unique threat to human health.