We studied infection of NHA with the virulent strain of JUNV and showed that human astrocytes are highly susceptible to infection. Susceptibility of NHA to JUNV infection indicates the possibility that astrocytes might be a source of virus production in the brain of patients with AHF. Additionally, we analyzed the transcriptional response of NHA to JUNV infection early and late post challenge. To our knowledge, this is the first report of JUNV-induced response in human astrocytes, and the first study using mRNA profiling upon infection of human cells. Using transcriptional profiling that was partly confirmed by qRT-PCR and cytokine/chemokine/growth factor assay, we demonstrated the impact of virus infection on both immune/inflammatory response/IFN signaling and apoptosis. Amplification of pro-inflammatory cascade with time and pro-apoptotic response of NHA to JUNV infection are indicative of neurodegenerative dysfunctional reactive astrogliosis [43, 44]. In response to neurological injury initial early astrocyte activation and reactive astrogliosis (astrocyte hypertrophy, proliferation, migration, acute mild inflammation) represent CNS physiological local response to limit the lesions, repair the initial damage, regulate homeostasis, prevent neurodegeneration and stimulate neurogenesis [45–47]. Indicative of astrocyte proliferation M phase Cell cycle function was increased in Romero-infected NHA at 24 h p.i. (Additional file 2: Table S2). On the other hand, massive/prolong brain injury or astrocyte stress lead to amplification of a microglia-astrocyte crosstalk and uncontrolled release of pro-inflammatory cytokines, chemokines and reactive oxygen species that has been associated with chronic neuroinflammation and neurodegeneration [45, 46, 48–50].
Multiple anti- and pro-inflammatory cytokines (i.e. IFN-α, IFN-β, IFN-γ, TNF-α, IL-1β, and IL-6), chemokines [i.e. stromal cell derived factor-1 alpha (SDF-1α, CXCL12), MCP-1, MIP-1α, RANTES and IP-10] and growth factors are produced by activated astroglia in response to an injury or pathogen . Although, both neurogenesis and neurodegeneration has been associated with the factors produced by reactive astrocytes ; in our study transcriptom and cytokine/chemokine/growth factor profiles suggest the later response to JUNV infection. A link between chronic overproduction of type I IFNs and neurodegenerative diseases has been well established . Likewise, we observed amplification with time of type I IFN signaling in response to JUNV infection. Moreover, in NHA we found elevated production/expression of pro-inflammatory mediators [type I cytokines (IL-12, INF-γ), proinflammatory cytokines (IL-6, TNF-α), chemoattractants and chemokines (IL-8, IL-10, MCP-1, RANTES)] but not anti-inflammatory cytokines (IL-4, IL-5, IL-10, IL-13, IL-17) upon JUNV infection. However, at 24 h p.i. we detected an elevated expression of SDF-1a, a key chemokine responsible for recruitment, proliferation and survival of neuronal precursor cells, and an elevated expression and production of FGF2 (BFGF) and VEGF at 24 and 96 h p.i. respectively (Figure 1, 3A) .
Supportive of exacerbated pro-inflammatory astrogliosis, Inflammatory and Neurological disease functions were uniformly increased at both time points in the microarray profile of JUNV-infected NHA. Among the others, neurological diseases such as relapsing-remitting multiple sclerosis and progressive motor neuropathy were listed. Pathogenic role of astrocytes in neurodegenerative disease including Amyotrophic lateral sclerosis has been documented [46, 48, 50].
The transcriptional profile of Romero JUNV-infected NHA indicated an apoptosis induction late in infection. Correspondingly, earlier we demonstrated IFN-independent RIG-I enhanced apoptosis induction in human and non-human primate transformed cells in response to virulent (Romero) and vaccine (Candid #1) strains of JUNV. Apoptosis induction in those cells was confirmed through quantification of phosphatidylserine translocation, Caspase 3 activation, Poly (ADP-ribose) polymerase (PARP) cleavage and/or chromosomal DNA fragmentation . In agreement, activation of RIG-I/IRF3 pathway was evident in our microarray profile.
Importance of three branches of Death Receptor Signaling pathway CD95 (Fas)/CD95L (Fas L), TNF-TNFR1, and TNF-related apoptosis-inducing ligand (TRAIL) and it receptors has been described for cell death induction in the brain. Fas/FasL and TRAIL receptors are expressed by glia and neurons and responsible for triggering neuronal and astrocyte apoptosis; while Fas/FasL and TNF-TNFR1 pathways has been implicated in T-cell apoptosis induction [42, 52, 53]. In agreement with that, CASP6, CFLAR (FLIP), FADD, IKBKB, TNFSF10 (TRAIL), and TRADD transcripts were uniformly up-regulated at 96 h p.i. in Romero-infected NHA.
Complement components produced by glia interact with the apoptotic signals on the cell surface leading to complement receptor-dependent phagocytosis/clearance of apoptotic cells [42, 54]. Accordingly, C3, C1R, C1S, CFH, CFI and SERPING1 encoding transcripts were up-regulated at 96 h p.i. in NHA infected with Romero JUNV. Detrimental inflammatory as well as neuroprotective role of complement activation has been reported .
The recent study  reported transcriptional changes of reactive astrocytes purified from mouse brains after ischemic stroke or LPS-induced neuroinflammation. Interestingly, the transcriptional profile of Romero-infected NHA in our study was similar to the detrimental pro-inflammatory molecular phenotype of reactive astrocytes induced by LPS, but not to the beneficial or protective response of reactive astrocytes in the ischemia model. Similar to our data, antigen presentation, complement, and IFN pathways were induced in the LPS reactive astrocytes. In contrast, increased metabolic activity, cell-cycle genes, and transcription factors categories were more prominent in reactive astrocytes from transient ischemia model .
In several previous studies of chronic encephalitis in newborn rats and mice induced by JUNV intracerebral inoculation or in vitro JUNV-infected rat and mouse astrocytes, a prominent astrocyte reaction to infection was reported [55–64]. In contrast to JUNV infection of human astrocytes, a protective role of JUNV-stimulated astrogliosis dependent on the increased expression of inducible nitric oxide synthase (iNOS) and not associated with the induction of apoptosis was reported in those studies [58, 63]. This discrepancy could be attributed to the different mechanisms of JUNV interaction with cells of resistant or permissive hosts.
Although limited, autopsy findings of patients with AHF and pathology reports of AHF/LNS animal models suggest gliosis/astrogliosis and neuronal damage as part of the disease pathogenesis [17, 19, 20, 26, 27]. Our observation of neurodegenerative dysfunctional reactive astrogliosis in response to virulent strain of JUNV infection is in agreement with the human and animal model pathology. Additionally, production of proinflammatory cytokines (IL-6, TNF-α) and chemoattractant IL-8 by Romero-infected NHA reflects systemic cytokine profile of severely ill AHF patients [8, 9]. Moreover, the transcriptional changes of multiple genes involved in IFN signaling detected in the JUNV-infected astrocytes partially corresponded to the in vivo transcriptional changes of blood mononuclear cells of Rhesus macaques infected with a virulent strain of LCMV , furthermore suggesting the relevance of in vitro profile observed in our study to the in vivo response to JUNV.
In conclusion, our study clearly demonstrates for the first time the high susceptibility of human astrocytes to infection with hemorrhagic arenavirus. Additionally, our mRNA profile of JUNV-infected human astrocytes confirmed by qRT-PCR and cytokine/chemokine/growth factor assay suggests neurodegenerative dysfunctional reactive astrogliosis as a cellular response to this infection. Taken together, our results suggest potential role of astroglial cells in AHF pathogenesis, particularly pathogenesis of neurological symptoms and LNS.