Isolation and identification of a new porcine astrovirus 5 demonstrated that oxidative stress enhances porcine astrovirus replication.

Background: Porcine astrovirus (PAstV) poses a major risk to the pig industry by causing diarrhea in suckling piglets. Despite its global prevalence and five genotypes, the virus's pathogenic mechanism is not well understood due to difficulties in isolating and culturing it in vitro. Studying PAstV from clinical samples and its interaction with host cells is crucial for understanding its pathogenesis and developing antiviral treatments.

Methods: To isolate porcine astrovirus (PAstV) from clinical specimens, fecal samples from PAstV-positive pigs were collected in August 2018, inoculated into PK-15 cells, and subjected to three successive blind passages. The in vitro growth characteristics of the isolated strain were subsequently evaluated, and the morphology of the virus particles was examined through electron microscopy. The complete genome sequence of the isolated strain was determined, followed by sequence alignment, homology analysis, phylogenetic analysis, and recombination analysis. To investigate the induction of reactive oxygen species (ROS) production in PK-15 cells infected with the isolated strain, the cells were infected, and ROS production was quantified using the MitoSOX probe. Furthermore, the expression levels of the antioxidant factors Nrf2 and HO-1 were analyzed via Western blotting. Mitochondrial damage resulting from PAstV infection was observed using transmission electron microscopy, and the effect of PAstV infection on mitochondrial membrane potential was assessed using the JC-1 probe. Finally, the impact of ROS on PAstV replication was explored using IFA and RT-qPCR.

Results: In this study, a strain of PAstV was isolated from porcine fecal samples, demonstrating an ability to adapt effectively to PK-15 cells with a viral titer reaching up to 10^7.85 TCID₅₀/mL. Genetic evolution analysis classified the isolated strain as PAstV5, revealing high genetic homology with other representative PAstV5 strains. The isolated strain was designated as PAstV5-GX2. Sequence alignment identified 11 consecutive amino acid deletions at the 3' end of ORF1a in the PAstV5-GX2 strain, resulting in alterations to the three-dimensional structure of the nsp1a/4 protein. Further investigation indicated that PAstV infection in PK-15 cells enhances mitochondrial ROS production and diminishes the protein expression levels of the antioxidant molecules Nrf2 and HO-1. Concurrently, PAstV infection induces mitochondrial swelling, cristae rupture, and vacuolization, along with a reduction in mitochondrial membrane potential. Through the application of H₂O₂ and NAC to modulate cellular ROS levels, it was determined that ROS can facilitate viral replication.

Conclusions and relevance: Our study successfully isolated a novel strain of PAstV5 characterized by an 11-amino acid deletion in the nsp1a protein, leading to significant alterations in the three-dimensional structure of the nsp1a/4 protein. This strain was observed to induce the production of mitochondrial ROS, downregulate the expression of Nrf2 and HO-1, and cause mitochondrial damage. Furthermore, the generation of mitochondrial ROS was found to facilitate the replication of PAstV. These findings offer valuable insights into the genetic evolution and pathogenic mechanisms of PAstV.