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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.IMPORTANCE MLB astroviruses are emerging viruses infecting humans. More studies are required to determine their exact epidemiology, but several reports have already identified them as the cause of unexpected clinical diseases, including severe neurologic diseases. Our study provides the first description of a cell culture system for the propagation of MLB astroviruses, enabling the study of their replicative cycle. Moreover, we demonstrated the unknown capacity of MLB astrovirus to establish persistent infections in cell culture. Whether these persistent infections are also established in vivo remains unknown, but the clinical consequences would be of high interest if persistence was confirmed in vivo Finally, our analysis of IFN expression provides some trails to understand the mechanism by which MLB astroviruses can cause persistent infections in the assayed cultures.

SUBMITTER: Vu DL

PROVIDER: S-EPMC6580956 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Human Astrovirus MLB Replication <i>In Vitro</i>: Persistence in Extraintestinal Cell Lines.

Vu Diem-Lan DL Bosch Albert A Pintó Rosa M RM Ribes Enric E Guix Susana S

Journal of virology 20190614 13

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 neurol ...[more]

PMID: 31019055

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Project description:Astrovirus VA1/HMO-C (VA1; mamastrovirus 9) is a recently discovered astrovirus genotype that is divergent from the classic human astroviruses (mamastrovirus 1). The gastrointestinal tract is presumed to be the primary site of infection and pathogenicity for astroviruses. However, VA1 has been independently detected in brain tissue of five cases of human encephalitis. Studies of the pathogenicity of VA1 are currently impossible because there are no reported cell culture systems or in vivo models that support VA1 infection. Here, we describe successful propagation of VA1 in multiple human cell lines. The initial inoculum, a filtered clinical stool sample from the index gastroenteritis case cluster that led to the discovery of VA1, was first passaged in Vero cells. Serial blind passage in Caco-2 cells yielded increasing copies of VA1 RNA, and multistep growth curves demonstrated a >100-fold increase in VA1 RNA 72 h after inoculation. The full-length genomic and subgenomic RNA strands were detected by Northern blotting, and crystalline lattices of viral particles of ?26-nm diameter were observed by electron microscopy in infected Caco-2 cells. Unlike other human astrovirus cell culture systems, which require addition of exogenous trypsin for continued propagation, VA1 could be propagated equally well with or without the addition of trypsin. Furthermore, VA1 was sensitive to the type I interferon (IFN-I) response, as VA1 RNA levels were reduced by pretreatment of Caco-2 cells with IFN-?1a. The ability to propagate VA1 in cell culture will facilitate studies of the neurotropism and neuropathogenesis of VA1.IMPORTANCE Astroviruses are an emerging cause of central nervous system infections in mammals, and astrovirus VA1/HMO-C is the most prevalent astrovirus in cases of human encephalitis. This virus has not been previously propagated, preventing elucidation of the biology of this virus. We describe the first cell culture system for VA1, a key step necessary for the study of its ability to cause disease.

Dataset Information

Human Astrovirus MLB Replication In Vitro: Persistence in Extraintestinal Cell Lines.

Publications

Human Astrovirus MLB Replication <i>In Vitro</i>: Persistence in Extraintestinal Cell Lines.

Similar Datasets

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