No viral antigen or nucleic acid was detected in the draining popliteal lymph node of the contralateral leg

No viral antigen or nucleic acid was detected in the draining popliteal lymph node of the contralateral leg. in osteoblasts, skeletal muscle myocytes, and in fibroblasts along fascial sheaths. The severity and extent of infection in peripheral tissues peaked at day 1 PI. In the neural phase of infection, virus was first detected in the brain on day 1 PI, with rapid interneuronal spread of infection leading to death by day 4 PI. EEE virus appeared to be directly cytopathic for neurons. The very rapid onset and apparently random and widely dispersed infection in the CNS, with concurrent sparing of olfactory neuroepithelium, strongly suggests that invasion of the CNS by EEE occurs by a vascular route, rather than via peripheral nerves or the olfactory neuroepithelium. Our finding that metaphyseal osteoblasts are an early site of amplifying viral replication may explain the higher-titer viremias and higher incidence of neuroinvasion and fulminant encephalitis seen in the young, and may also explain why mature animals become refractory to encephalitis after peripheral inoculation with EEE virus. Eastern equine encephalitis (EEE) virus is a single-stranded RNA virus in the genus (family mosquitoes rarely feed on mammalian hosts, other bridging mosquito species actually transmit the virus from infected birds to horses, and occasionally, to humans. Most EEE infections in humans are inapparent or produce a low-grade fever followed by malaise, arthralgia, and myalgia. However, in some cases, EEE virus crosses the blood-brain barrier and causes a severe, and often fatal, acute encephalitis, which kills 50 to 75% of infected humans and leaves many survivors with serious neurological sequelae.1,3 It has long been recognized that EEE infection in children tends to have a more rapid onset and to be more severe. Goldfield et al reported that one in eight young children developed fulminant encephalitis and only 1 1 in 23 had an inapparent infection.4 Similarly, EEE tends to be more severe in elderly patients.5 Histologically, infection in the human central nervous system (CNS) is characterized by a diffuse meningoencephalitis with widespread neuronal necrosis, perivascular cuffing with polymorphonuclear neutrophils and mononuclear cells, and vasculitis with vessel occlusion.6 The neuropathology of virulent EEE has been investigated in several CID16020046 species,7C9 including experimentally infected laboratory mice.10C13 However, time-course studies that apply hybridization (ISH) and immunohistochemistry (IHC) to track the progression of viral infection in extraneural tissues at early time points have not been reported. The purpose of this study was to investigate the early events in the pathogenesis of EEE virus infection in mice because the early viral cell targets and the interactions between viral replication, virus-specific immune responses, and nonspecific defenses in CID16020046 the pathogenesis of virulent EEE are still not well defined. It is likely that the initial events after infections play a critical role in determining the overall severity and eventual outcome of disease. A better understanding of the early cell targets of EEE virus may contribute to our understanding of virus pathogenesis and to CID16020046 CID16020046 the development of more effective vaccines and viral therapeutics. Materials and Methods Virus Strain The pathogenic EEE virus strain, FL91C4679, was originally isolated from mosquitoes collected in Florida in 1991 and had been passaged once in suckling mice, three times in Vero cells, and twice in BHK-21 cells.14 We propagated virus on BHK-21 cell monolayers maintained in Eagles CID16020046 minimal essential medium (EMEM) containing 10% fetal bovine serum (FBS). Viral titers in mouse blood samples and viral stocks were measured by plaque assay in Vero cells maintained in EMEM containing Ebf1 5% FBS. Mice Specific pathogen-free, 5-week-old C57BL/6 female mice (National Cancer Institute, Frederick, MD) were maintained in a biosafety level 3 (BSL-3) facility and were housed in microisolator cages and provided water and standard mouse chow hybridization methods. Briefly, lightly anesthetized mice (= 24) (Metofane; Pitman-Moore, Mundelein, IL) were inoculated in the subcutis of the right footpad with 105 plaque-forming units (PFU) of EEE virus (FL91C4679) in 0.025 ml of Hanks balanced salt solution (HBSS) diluent. Negative control mice (= 12) were inoculated with HBSS diluent alone. At 12 hours and 1, 2, and 4 days post-inoculation (PI), four randomly selected virus-infected mice and two negative control mice were bled from the retroorbital sinus for virus isolation and then killed by inhalation of CO2. After death, the mice were immediately perfused via the left ventricle with 20 ml of 10%.