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. 2019 Jul 9;10(4):e01455-19.
doi: 10.1128/mBio.01455-19.

Differential In Vitro Infection of Neural Cells by Astroviruses

Andrew B Janowski  1 , Robyn S Klein  2   3   4 , David Wang  5   6
Affiliations

Differential In Vitro Infection of Neural Cells by Astroviruses

Andrew B Janowski et al. mBio. .

Abstract

Recent advances in unbiased pathogen discovery have implicated astroviruses as pathogens of the central nervous system (CNS) of mammals, including humans. However, the capacity of astroviruses to be cultured in CNS-derived cells in vitro has not been reported to date. Both astrovirus VA1/HMO-C (VA1; mamastrovirus 9) and classic human astrovirus 4 (HAstV4; mamastrovirus 1) have been previously detected from cases of human encephalitis. We tested the ability of primary human neurons, primary human astrocytes, and other immortalized human nervous system cell lines (SK-N-SH, U87 MG, and SW-1088) to support infection and replication of these two astrovirus genotypes. Primary astrocytes and SK-N-SH cells supported the full viral life cycle of VA1 with a >100-fold increase in viral RNA levels during a multistep growth curve, detection of viral capsid, and a >100-fold increase in viral titer. Primary astrocytes were permissive with respect to HAstV4 infection and replication but did not yield infectious virus, suggesting abortive infection. Similarly, abortive infection of VA1 was observed in SW-1088 and U87 MG cells. Elevated expression of the chemokine CXCL10 was detected in VA1-infected primary astrocytes and SK-N-SH cells, suggesting that VA1 infection can induce a proinflammatory host response. These findings establish an in vitro cell culture model that is essential for investigation of the basic biology of astroviruses and their neuropathogenic potential.IMPORTANCE Encephalitis remains a diagnostic conundrum in humans as over 50% of cases are managed without the identification of an etiology. Astroviruses have been detected from the central nervous system of mammals in association with disease, suggesting that this family of RNA viruses could be responsible for cases of some neurological diseases that are currently without an ascribed etiology. However, there are significant barriers to understanding astrovirus infection as the capacity of these viruses to replicate in nervous system cells in vitro has not been determined. We describe primary and immortalized cultured cells of the nervous system that support infection by astroviruses. These results further corroborate the role of astroviruses in causing neurological diseases and will serve as an essential model to interrogate the neuropathogenesis of astrovirus infection.

Keywords: astrovirus; astrovirus VA1; cell culture; encephalitis; virology.

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Figures

FIG 1
FIG 1
Multistep growth curves of (a) VA1 and (b) HAstV4 gRNA of primary astrocytes, primary neurons, and immortalized cell lines, including SK-N-SH, SW-1088, U87 MG, and Caco-2 cells. Each data point is normalized to the gRNA copy number present at 1 h postinoculation for each cell line. Geometric means are plotted with error bars representing 1 geometric standard deviation. The horizontal dotted line represents the relative gRNA copy number at 1 h postinoculation.
FIG 2
FIG 2
Quantification of infectious titers over time of VA1 (left panel) and HAstV4 (right panel) by a TCID50 assay in cell lines that had >10-fold increases in gRNA levels after inoculation. Geometric means are plotted with error bars representing 1 geometric standard deviation. The horizontal dotted line represents the limit of detection of the assay. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
FIG 3
FIG 3
Single-step growth curve of VA1 (MOI of 3) in primary astrocytes and immortalized cell lines, including SK-N-SH, SW-1088, U87 MG, and Caco-2 cells. Data points are normalized to the gRNA copy number that was present 1 h after inoculation for each cell line. Geometric means with error bars representing 1 geometric standard deviation are plotted. The horizontal dotted line represents the relative gRNA copy number at 1 h postinoculation.
FIG 4
FIG 4
Detection of VA1 capsid in infected cells by immunofluorescence. Cells were incubated with polyclonal antibody to the capsid and labeled by staining with a secondary antibody conjugated with a green fluorescent dye. Merged fluorescence images are shown with counterstaining of nuclei (performed with DAPI). White scale bars represent 10 μm.
FIG 5
FIG 5
Quantification of sgRNA in single-step growth curves of VA1 (MOI of 3) from the cellular fraction of each cell line. Each plotted data point represents the geometric mean normalized to the copy number present at 1 h postinoculation with error bars representing 1 geometric standard deviation. The dotted horizontal line represents relative sgRNA present at 1 h postinoculation. The maximum P value for each cell line comparison at 24, 36, or 48 h postinoculation is depicted. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
FIG 6
FIG 6
Cytokine expression in mock-infected or infected cell lines at 24 h and 48 h postinoculation. Geometric mean cytokine concentrations are plotted in picograms per milliliter with error bars representing 1 geometric standard deviation. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
FIG 7
FIG 7
CXCL10 or MCP-1 mRNA expression in mock-infected or infected cell lines 24 h and 48 h postinoculation. Relative fold changes were calculated via the 2−ΔΔCT method. Geometric means are plotted with error bars representing 1 geometric standard deviation. The horizontal dotted line represents relative baseline mRNA expression of mock-infected cells. ND, not detected. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
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