Abstract
BackgroundLa Crosse virus (LACV) causes pediatric encephalitis in the USA. LACV induces severe inflammation in the central nervous system, but the recruitment of inflammatory cells is poorly understood. A deeper understanding of LACV-induced neural pathology is needed in order to develop treatment options. However, there is a severe limitation of relevant human neuronal cell models of LACV infection.MethodsWe utilized human neural stem cell (hNSC)-derived neuron/astrocyte co-cultures to study LACV infection in disease-relevant primary cells. hNSCs were differentiated into neurons and astrocytes and infected with LACV. To characterize susceptibility and responses to infection, we measured viral titers and levels of viral RNA, performed immunofluorescence analysis to determine the cell types infected, performed apoptosis and cytotoxicity assays, and evaluated cellular responses to infection using qRT-PCR and Bioplex assays.ResultshNSC-derived neuron/astrocyte co-cultures were susceptible to LACV infection and displayed apoptotic responses as reported in previous in vitro and in vivo studies. Neurons and astrocytes are both targets of LACV infection, with neurons becoming the predominant target later in infection possibly due to astrocytic responses to IFN. Additionally, neuron/astrocyte co-cultures responded to LACV infection with strong proinflammatory cytokine, chemokine, as well as MMP-2, MMP-7, and TIMP-1 responses.ConclusionshNSC-derived neuron/astrocyte co-cultures reproduce key aspects of LACV infection in humans and mice and are useful models to study encephalitic viruses. Specifically, we show astrocytes to be susceptible to LACV infection and that neurons and astrocytes are important drivers of the inflammatory responses seen in LACV infection through the production of proinflammatory cytokines and chemokines.
Highlights
La Crosse virus (LACV) causes pediatric encephalitis in the USA
Our results indicate that both neurons and astrocytes are highly susceptible to LACV, and that LACV infection induces strong proinflammatory responses, which likely play a major role in the observed neuroinflammation and breakdown in the blood-brain barrier (BBB)
HNSCs were cultured as nonadherent neurospheres in DMEM/ F12 (Corning) media supplemented with epidermal growth factor (EGF) (20 ng/mL) (R&D Systems), fibroblast growth factor (FGF) (20 ng/mL) (R&D Systems), leukocyte inhibitory factor (LIF) (10 ng/mL) (Chemicon), heparin (5 μg/mL) (Sigma-Aldrich), and insulin (25 μg/ mL) (Sigma-Aldrich)
Summary
La Crosse virus (LACV) causes pediatric encephalitis in the USA. La Crosse virus (LACV), family Peribunyaviridae (genus Orthobunyavirus), is a leading cause of pediatric arboviral encephalitis in the USA [1]. LACV was responsible for 665 confirmed cases of encephalitis from 2003 to 2012, the true incidence of disease is thought to be underestimated [2]. Endemic areas of infection include the Midwest and Appalachian regions, with county-level incidence of 0.2–228 cases per 100,000 children under the age of 15, but LACV is becoming an important emerging pathogen of the Southern and Western United States [3]. The majority of cases present as mild febrile illness, but in a minority of cases, LACV causes severe neuroinvasive disease including encephalitis, meningitis, and meningoencephalitis [5]. While the disease is rarely (< 1%) fatal, neurological deficits such as epilepsy (in 10–28% of cases), reduced IQ, and attentiondeficit-hyperactivity disorder (ADHD) are not uncommon [4,5,6]
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