Human astrovirus MLB replication in vitro: persistence in extraintestinal cell lines

Abstract

MLB astroviruses were identified 10 years ago in feces from children with gastroenteritis of unknown etiology and have been unexpectedly detected in severe cases of meningitis/encephalitis, febrile illness of unknown etiology, and respiratory syndromes. The aim of this study was to establish a cell culture system supporting MLB astrovirus replication. We used two clinical strains to infect several cell lines, an MLB1 strain from a gastroenteritis case, and an MLB2 strain associated with a neurologic infection. Efforts to propagate the viruses in the Caco-2 cell line were unsuccessful. In contrast, we identified two human nonintestinal cell lines, Huh-7 and A549, permissive for both genotypes. After serial passages in the Huh-7.5 cell line, the adapted strains were able to establish persistent infections in the Huh-7.5, Huh-7AI, and A549 cell lines, with high viral loads (up to 10 log10 genome copies/ml) detected by quantitative reverse transcription-PCR (RT-qPCR) in the culture supernatant. Immunofluorescence assays demonstrated infection in about 10% of cells in persistently infected cultures. Electron microscopy revealed particles of 32 to 33 nm in diameter after negative staining of cell supernatants and capsid arrays in ultrathin sections with a particularly high production in Huh-7.5 cells. Interferon (IFN) expression by infected cells and effect of exogenous IFN varied depending on the type of infection and the cell line. The availability of a cell culture system to propagate MLB astroviruses represents a key step to better understand their replicative cycle, as well as a source of viruses to conduct a wide variety of basic virologic studies