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. 2021 Feb 27;13(3):376.
doi: 10.3390/v13030376.

Human Astroviruses: A Tale of Two Strains

Virginia Hargest  1 , Amy E Davis  1   2 , Shaoyuan Tan  1 , Valerie Cortez  1 , Stacey Schultz-Cherry  1
Affiliations

Human Astroviruses: A Tale of Two Strains

Virginia Hargest et al. Viruses. .

Abstract

Since the 1970s, eight closely related serotypes of classical human astroviruses (HAstV) have been associated with gastrointestinal illness worldwide. In the late 2000s, three genetically unique human astrovirus clades, VA1-VA3, VA2-VA4, and MLB, were described. While the exact disease associated with these clades remains to be defined, VA1 has been associated with central nervous system infections. The discovery that VA1 could be grown in cell culture, supports exciting new studies aimed at understanding viral pathogenesis. Given the association of VA1 with often lethal CNS infections, we tested its susceptibility to the antimicrobial drug, nitazoxanide (NTZ), which we showed could inhibit classical HAstV infections. Our studies demonstrate that NTZ inhibited VA1 replication in Caco2 cells even when added at 12 h post-infection, which is later than in HAstV-1 infection. These data led us to further probe VA1 replication kinetics and cellular responses to infection in Caco-2 cells in comparison to the well-studied HAstV-1 strain. Overall, our studies highlight that VA1 replicates more slowly than HAstV-1 and elicits significantly different cellular responses, including the inability to disrupt cellular junctions and barrier permeability.

Keywords: HAstV-1; VA1; barrier permeability; human astrovirus; nitazoxanide; viral replication.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Nitazoxanide effectively inhibits VA1 replication when added up to 12 h post-infection. (A) At the indicated times post-infection, nitazoxanide (NTZ; 2.5μM) was added to VA1-infected Caco-2 cells. At 24 hpi, cells were fixed and stained for the presence of VA1 capsid protein. (B) The percent of VA1 capsid positive cells for each NTZ treatment was quantitated and compared to vehicle alone (DMSO). All error bars indicate standard deviation of two combined, independent experiments performed in triplicate, and asterisks show statistical significance as measured by multiple t tests as follows: ***, p < 0.001.
Figure 2
Figure 2
VA1 replication is delayed in comparison to HAstV-1. Caco-2 cells were mock-infected or infected with HAstV-1 or VA1 (MOI of 2). At various time points, cells were stained for viral capsid and double-stranded RNA (dsRNA) by immunofluorescent staining. (A) Representative images, taken at 40× magnification on EVOS FL, of HAstV-1 and VA1 capsid production and dsRNA formation at 24 hpi. The percent of dsRNA positive (B) and capsid positive (C) cells at the indicated timepoints was quantitated.
Figure 3
Figure 3
ERK1/2 is activated by VA1 and required for productive replication. Lysates from HAstV-1 (A) and VA1 (B) infected cells taken at the indicated times post-infection were blotted and probed for pERK, ERK, and β-actin. (C) Bands were then quantified by densitometry and normalized to β-actin then compared to mock-infection. (D) Caco-2 cells were pre-incubated 10 μM U0126 vehicle alone (DMSO) and then infected with VA1 (MOI of 2). Percent infection was measured by immunofluorescent staining for VA1 capsid at 24 hpi. Error bars indicate standard deviations from two independent experiments performed in triplicate, and asterisks show statistical significance as measured by ordinary one-way ANOVA as follows: *, p < 0.05; **, p < 0.01; ***, p  < 0.001.
Figure 4
Figure 4
HAstV-1 and VA1 infection does not induce cytokines or cell death. A panel of 13 cytokines were analyzed from cell supernatants following 48 hpi for VA1 and serum-containing mock-infected wells and compared with supernatants from HAstV1 and serum free mock-infected wells 24 hpi. Levels of TNF-α, IL-12p70, IFN-β, IL-29, IL-10 and IFN-γ were all undetectable and are not shown. For the other 7 cytokines (A–G), the lower limit of detection is denoted by the dashed lines. Asterisks (*) denote statistical significance as measure by Mann Whitney U, with a cutoff value of p < 0.05. (H) Cell viability following VA1 and HAstV-1 infection at 24 hpi was measured using an ATP detection assay.
Figure 5
Figure 5
VA1 infection does not induce barrier permeability. (A) Representative images, taken at 60·magnification on Zeiss LSM 780 NLO, of Caco-2 monolayers infected with VA1 (MOI of 10) at 24 hpi show no disruption of occludin expression compared to mock-infected cells. Caco-2 monolayers grown on semi-permeable supports were infected with HAstV-1, VA1 at MOIs of 1 (B) and 10 (C), or mock-infected and transepithelial electrical resistance (TER) was measured from 0–24 hpi.
Figure 6
Figure 6
Alignment of classical HAstV-1 capsid structures with VA1 homology models. Crystal structure of the HAstV-1 (blue and green) aligned with homology models of VA1 capsid core (A) and spike (C). VA1 models (B,D) were marked with different colors based on its similarity with HAstV-1 reference: residues that are perfectly conserved (red), residues that are not perfectly conserved but that have a BLOSUM62 score higher than 0 (pink) and residues with a BLOSUM62 score lower than 0 or residues that are aligned to a gap in the reference (white).
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