All Publications


  • An optimized reverse genetics system suitable for efficient recovery of simian, human and murine-like rotaviruses. Journal of virology Sanchez-Tacuba, L., Feng, N., Meade, N. J., Mellits, K. H., Jais, P. H., Yasukawa, L. L., Resch, T. K., Jiang, B., Lopez, S., Ding, S., Greenberg, H. B. 2020

    Abstract

    An entirely plasmid-based reverse genetics (RG) system was recently developed for rotavirus (RV), opening new avenues for in-depth molecular dissection of RV biology, immunology, and pathogenesis. Several improvements to further optimize the RG efficiency have now been described. However, only a small number of individual RV strains have been recovered to date. None of the current methods have supported the recovery of murine RV, impeding the study of RV replication and pathogenesis in an in vivo suckling mouse model. Here, we describe useful modifications to the RG system that significantly improve rescue efficiency of multiple RV strains. In addition to the 11 RVA segment-specific (+)ssRNAs, a chimeric plasmid was transfected, from which the capping enzyme NP868R of African swine fever virus (ASFV) and the T7 RNA polymerase were expressed. Secondly, a genetically modified MA104 cell line was used in which several compounds of the innate immune were degraded. Using this RG system, we successfully recovered the simian RV RRV strain, the human RV CDC-9 strain, a reassortant between murine RV D6/2 and simian RV SA11 strains, and several reassortants and reporter RVs. All these recombinant RVs were rescued at a high efficiency (≥80% success rate) and could not be reliably rescued using several recently published RG strategies (<20%). This improved system represents an important tool and great potential for the rescue of other hard-to-recover RV strains such as low replicating attenuated vaccine candidates or low cell culture passage clinical isolates from humans or animals.IMPORTANCE Group A rotavirus (RV) remains as the single most important cause of severe acute gastroenteritis among infants and young children worldwide. An entirely plasmid-based reverse genetics (RG) system was recently developed opening new ways for in-depth molecular study of RV. Despite several improvements to further optimize the RG efficiency, it has been reported that current strategies do not enable the rescue of all cultivatable RV strains. Here, we described helpful modification to the current strategies and established a tractable RG system for the rescue of the simian RRV strain, the human CDC-9 strain and a murine-like RV strain, which is suitable for both in vitro and in vivo studies. This improved RV reverse genetics system will facilitate study of RV biology in both in vitro and in vivo systems that will facilitate the improved design of RV vaccines, better antiviral therapies and expression vectors.

    View details for DOI 10.1128/JVI.01294-20

    View details for PubMedID 32759316

  • Reverse genetics reveals a role of the rotavirus VP3 phosphodiesterase activity in inhibiting RNase L signaling and contributing to intestinal viral replication in vivo. Journal of virology Song, Y., Feng, N., Sanchez-Tacuba, L., Yasukawa, L. L., Ren, L., Silverman, R. H., Ding, S., Greenberg, H. B. 2020

    Abstract

    Our understanding of how rotavirus (RV) subverts host innate immune signaling has greatly increased over the past decade. However, the relative contribution of each virus-encoded innate immune antagonist has not been fully studied in the context of RV infection in vivo Here, we present both in vitro and in vivo evidence that the host IFN-inducible 2'-5'-oligoadenylate synthetase (OAS) and ribonuclease L (RNase L) pathway effectively suppresses the replication of heterologous RV strains. VP3 from homologous RVs relies on its 2'-5'-phosphodiesterase (PDE) domain to counteract RNase L mediated antiviral signaling. Using a RV reverse genetics system, we show that compared to the parental strain, VP3 PDE mutant RVs replicated at a lower level in the small intestine and shed less in the feces of wild-type mice and such defects were rescued in Rnasel -/- suckling mice. Collectively, these findings highlight an important role of VP3 in promoting viral replication and pathogenesis in vivo in addition to its well characterized function as the viral RNA capping enzyme.ImportanceRotaviruses are significant human pathogens that result in diarrhea, dehydration, and deaths in many children around the world. Rotavirus vaccines have suboptimal efficacy in low to middle income countries, where the burden of the diseases is the most severe. With the ultimate goal to improve current vaccines, we aim to better understand how rotavirus interacts with the host innate immune system in the small intestine. Here, we demonstrate that the interferon-activated RNase L signaling blocks rotavirus replication in a strain-specific manner. In addition, virus encoded VP3 antagonizes RNase L activity both in vitro and in vivo These studies highlight an ever-evolving arms race between antiviral factors and viral pathogens and provide a new means of targeted attenuation for the next-generation rotavirus vaccine design.

    View details for DOI 10.1128/JVI.01952-19

    View details for PubMedID 32051268

  • Retinoic Acid and Lymphotoxin Signaling Promote Differentiation of Human Intestinal M Cells. Gastroenterology Ding, S., Song, Y., Brulois, K. F., Pan, J., Co, J. Y., Ren, L., Feng, N., Yasukawa, L. L., Sánchez-Tacuba, L., Wosen, J. E., Mellins, E. D., Monack, D. M., Amieva, M. R., Kuo, C. J., Butcher, E. C., Greenberg, H. B. 2020

    Abstract

    Intestinal microfold (M) cells are a unique subset of intestinal epithelial cells in the Peyer's patches that regulate mucosal immunity, serving as portals for sampling and uptake of luminal antigens. The inability to efficiently develop human M cells in cell culture has impeded studies of the intestinal immune system. We aimed to identify signaling pathways required for differentiation of human M cells and establish a robust culture system using human ileum enteroids.We analyzed transcriptome data from mouse Peyer's Patches to identify cell populations in close proximity to M cells. We used the human enteroid system to determine which cytokines were required to induce M cell differentiation. We performed transcriptome, immunofluorescence, scanning electron microscope, and transcytosis experiments to validate the development of phenotypic and functional human M cells.A combination of retinoic acid and lymphotoxin induced differentiation of glycoprotein 2-positive human M cells, which lack apical microvilli structure. Upregulated expression of innate immune-related genes within M cells correlated with a lack of viral antigens after rotavirus infection. Human M cells, developed in the enteroid system, internalized and transported enteric viruses, such as rotavirus and reovirus, across the intestinal epithelium barrier in the enteroids.We identified signaling pathways required for differentiation of intestinal M cells, and used this information to create a robust culture method to develop human M cells with capacity for internalization and transport of viruses. Studies of this model might increase our understanding of antigen presentation and the systemic entry of enteric pathogens in the human intestine.

    View details for DOI 10.1053/j.gastro.2020.03.053

    View details for PubMedID 32247021