Immunohistochemical Detection of Japanese Encephalitis Virus in AVIAN Myeloid Lineage Cells

Introduction: Between 1997 and 2000, birds (mainly blackbirds and song thrushes) were collected during mortality episodes occurring in a wetland area in Tuscany, where cases of West Nile virus (WNV) infection in horses were observed in 1998. Materials and Methods: Formalin-fixed tissue samples from 14 birds collected in 1997 and seven collected in 2000 that tested positive for Japanese encephalitis (JE) group antigens by immunohistochemistry (IHC), were sent to the Central Research Institute of Epidemiology in Moscow, to carry out molecular testing for flaviviruses. Results: None of the samples tested positive for WNV, but PCR amplicons of the JE virus NS5 gene and the JEV E gene were obtained from tissues of seven birds. Sequencing of 552 bp fragments of the JEV E gene (GenBank AF501311-AF501315) and of NS5 amplicons showed high similarity (99%) with JEV genotype III (Nakayama strain). IHC for JEV (pAb, PG 10004) was positive in 13 of 21 bone marrow samples, with stem cells positive also for CD11b (pAb, ab75476). Positive reactions were also present in neurons, microglial cells, kidney, spleen, liver, lung and gastrointestinal tract ganglia. Conclusions: This is the first description of JEV localization in myeloid lineage cells of avian bone marrow, a possible important reservoir for this virus.