Hand-foot-and-mouth disease (HFMD) caused by human enterovirus A71 (EV-A71) infection has been associated with severe neurological complications. With the lack of an internationally approved antiviral, coupled with a surge in outbreaks globally, EV-A71 has emerged as a neurotropic virus of high clinical importance. Andrographolide has many pharmacological effects including antiviral activity and its derivative, andrographolide sulfonate, has been used in China clinically to treat EV-A71 infections. This study sought to identify novel andrographolide derivatives as EV-A71 inhibitors and elucidate their antiviral mode of action. Using an immunofluorescence-based phenotypic screen, we identified novel EV-A71 inhibitors from a 344-compound library of andrographolide derivatives and validated them with viral plaque assays. Among these hits, ZAF-47, a quinolinoxy-andrographolide, was selected for downstream mechanistic studies. It was found that ZAF-47 acts on EV-A71 post-entry stages and inhibits EV-A71 protein expression. Subsequent luciferase studies confirm that ZAF-47 targets EV-A71 genome RNA replication specifically. Unsuccessful attempts in generating resistant mutants led us to believe a host factor is likely to be involved which coincide with the finding that ZAF-47 exhibits broad-spectrum antiviral activity against other enteroviruses (CV-A16, CV-A6, Echo7, CV-B5, CV-A24 and EV-D68). Furthermore, ZAF-46 and ZAF-47, hits from the screen, were derivatives of the same series containing quinolinoxy and olefin modifications, suggesting that an andrographolide scaffold mounted with these unique moieties could be a potential anti-EV-A71/HFMD strategy.

The midgut of insects is a crucial organ that enables them to obtain nutrients and resist pathogen invasion. In this study, the midgut cell clusters of the silkworm (Bombyx mori), a model species of lepidopteran insects, were analyzed using single-nucleus RNA sequencing technology (snRNA-seq) (PRJNA1280782) to understand the functional diversity of midgut tissues, identify the key cell clusters involved in the response to pathogen infection, and screen molecular targets for disease resistance. Twenty cell clusters (C0-C19) were obtained and annotated. The known marker genes, KEGG and GO pathway enrichment analysis were used to identify five types of cells: enterocyte cells (ECs, 54.4%), goblet cells (GCs, 25.9%), enteroendocrine cells (EEs, 10.6%), intestinal stem cells (ISCs, 2.17%), and muscle cells (MCs, 7.0%). Additional representative cell marker gene Cel of GCs was identified. We conducted snRNA-seq on the midgut tissues of B. mori infected with the fungal and viral pathogens Nosema bombycis (N.b) and Bombyx mori nucleopolyhedrovirus (BmNPV) at 24 h (early stage of infection) and 96 h (later stage of infection) to determine the immune response patterns of different cell clusters. The immune-related genes of ECs C2 and C16 were significantly altered after pathogen infection, making them important cell clusters for screening disease resistance target genes. The common susceptible cell clusters C4 and C13 between different pathogens were successfully identified, and related genes that could simultaneously affect N.b and BmNPV proliferation were screened in C2, C13 and C16. The results provide a basis for exploring potential common molecular target genes of various pathogens.