An unexplained increasing incidence of parapneumonic empyema (PPE) in pneumonia cases has been reported in recent years. The present study investigated the genetic and biological specifications of new isolates of Torque Teno Mini Virus (TTMV) detected in pleural effusion (PE) samples from children hospitalised for severe pneumonia with PPE.A pathogen discovery protocol was applied in undiagnosed PE samples and led us to the identification of 3 new isolates of TTMV (TTMV-LY). Isolated TTMV-LY genomes were transfected into A549 and HEK293T cells and viral replication was assessed by qPCR and full-length genome amplification. A549 cells were further infected with released TTMV-LY virions and the induced-innate immune response was measured by multiplex immunoassays.Genetic analyses of the 3 TTMV-LY genomes revealed a classic genomic organisation but a weak identity (<64%) with known sequences. We demonstrated here the in vitro replication of TTMV-LY in alveolar epithelial cells and the effective release of infectious viral particles. We also showed a selective production of inflammatory mediators in response to TTMV infection.This study reports the description of replicative TTMV-LY isolated from parapneumonic effusions of children hospitalised with PPE, suggesting the potential role of the virus in the pathogenesis of pneumonia.