Entry Sites of Venezuelan and Western Equine Encephalitis Viruses in the Mouse Central Nervous System following Peripheral Infection

Abstract

Venezuelan and western equine encephalitis viruses (VEEV and WEEV; Alphavirus; Togaviridae) are mosquito-borne pathogens causing central nervous system (CNS) disease in humans and equids. Adult CD-1 mice also develop CNS disease after infection with VEEV and WEEV. Adult CD-1 mice infected by the intranasal (i.n.) route, showed that VEEV and WEEV enter the brain through olfactory sensory neurons (OSNs). In this study, we injected the mouse footpad with recombinant WEEV (McMillan) or VEEV (subtype IC strain 3908) expressing firefly luciferase (fLUC) to simulate mosquito infection and examined alphavirus entry in the CNS. Luciferase expression served as a marker of infection detected as bioluminescence (BLM) by in vivo and ex vivo imaging. BLM imaging detected WEEV and VEEV at 12 h postinoculation (hpi) at the injection site (footpad) and as early as 72 hpi in the brain. BLM from WEEV.McM-fLUC and VEEV.3908-fLUC injections was initially detected in the brain's circumventricular organs (CVOs). No BLM activity was detected in the olfactory neuroepithelium or OSNs. Mice were also injected in the footpad with WEEV.McM expressing DsRed (Discosoma sp.) and imaged by confocal fluorescence microscopy. DsRed imaging supported our BLM findings by detecting WEEV in the CVOs prior to spreading along the neuronal axis to other brain regions. Taken together, these findings support our hypothesis that peripherally injected alphaviruses enter the CNS by hematogenous seeding of the CVOs followed by centripetal spread along the neuronal axis. IMPORTANCEVEEV and WEEV are mosquito-borne viruses causing sporadic epidemics in the Americas. Both viruses are associated with CNS disease in horses, humans, and mouse infection models. In this study, we injected VEEV or WEEV, engineered to express bioluminescent or fluorescent reporters (fLUC and DsRed, respectively), into the footpads of outbred CD-1 mice to simulate transmission by a mosquito. Reporter expression serves as detectable bioluminescent and fluorescent markers of VEEV and WEEV replication and infection. Bioluminescence imaging, histological examination, and confocal fluorescence microscopy were used to identify early entry sites of these alphaviruses in the CNS. We observed that specific areas of the brain (circumventricular organs [CVOs]) consistently showed the earliest signs of infection with VEEV and WEEV. Histological examination supported VEEV and WEEV entering the brain of mice at specific sites where the blood-brain barrier is naturally absent. E astern, Venezuelan, and western equine encephalitis viruses (EEEV, VEEV, and WEEV; Alphavirus; Togaviridae) are mosquito-borne viruses in the Americas causing central nervous system (CNS) disease in humans and equids (1). EEEV, VEEV, and WEEV are maintained in nature via transmission cycles between vertebrate hosts and specific mosquito species (2). Historically, these alphaviruses have caused sporadic epizootics and epidemics in horses and humans, respectively (1, 3). Outbreaks have led to significant rates of morbidity...

Virus entry and replication in the brain precedes blood-brain barrier disruption during intranasal alphavirus infection

“…Others have reported that VEEV can move in a retrograde manner up olfactory neurons crossing the cribriform plate and infecting glomeruli in the olfactory bulb, which could seed infection of the cortex and ultimately the hindbrain. Alternatively, virus could enter the brain directly during epithelial disruption at the choroid plexus or by crossing the BBB after moving via lymphatic drainage into the circulation (Phillips et al, 2016). We used in vivo 2P imaging of wild type and Ifit1 −/− mice to analyze the spatiotemporal distribution of virus following intranasal infection.…”

Direct and indirect pro-inflammatory cytokine response resulting from TC-83 infection of glial cells

“…The viral supernatants were checked using plaque assays. For the plaque assay, Vero cells were plated in 12-well plates at a density of 2.5 x 10 [5] cells/well and cultured in DMEM at 37.1°C, 5% CO 2 . After a 1h adsorption with a serially diluted virus solution, wells were covered with Eagle’s Minimum Essential Medium (Quality Biological 115–073-101) (without phenol red, supplemented with 10% FBS, non-essential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, 20 U/ml of penicillin and 20 μg/ml of streptomycin) containing 0.6% agarose (Life Technologies).…”

Venezuelan and western equine encephalitis virus E1 liposome antigen nucleic acid complexes protect mice from lethal challenge with multiple alphaviruses

“…EEEV-FL93-NLuc plasmid was obtained from Dr. William Klimstra (The University of Pittsburgh) and rescued as described (Sun et al, 2014). WEEV-McM-FLuc was constructed and generated as previously described (Phillips et al, 2016). All recombinant viruses were used without further passage after rescue.…”