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.
To improve the safety and efficacy of oral rotavirus vaccines, we developed an inactivated rotavirus vaccine (IRV) for parenteral administration. Since it remains unknown whether parenteral vaccination can induce mucosal immunity, we performed a comprehensive assessment of immune responses to IRV in mice with an adjuvant-free dissolving polymer MN patch or by alum-adjuvanted IM injection. We demonstrated that IRV induced the expression of the gut homing receptor LPAM-1 on T and B cells in spleen and mLN of vaccinated mice. MN patch IRV vaccination induced a slight Th1 phenotype while IM vaccination induced a balanced Th1/Th2 phenotype. In addition, a dose-sparing effect was seen for rotavirus-specific serum IgG and neutralizing activity for both vaccination routes. Our study is the first to show that parenterally administered IRV can induce mucosal immunity in the gut, in addition to strong serum antibody response, and is a promising candidate vaccine in achieving global immunization against rotavirus.
Cell types and mechanisms involved in type I interferon (IFN)‐mediated anti‐inflammatory effects are poorly understood. Upon injection of artificial double‐stranded RNA (poly(I:C)), we observed severe liver damage in type I IFN‐receptor (IFNAR) chain 1‐deficient mice, but not in wild‐type (WT) controls. Studying mice with conditional IFNAR ablations revealed that IFNAR triggering of myeloid cells is essential to protect mice from poly(I:C)‐induced liver damage. Accordingly, in poly(I:C)‐treated WT, but not IFNAR‐deficient mice, monocytic myeloid‐derived suppressor cells (MDSCs) were recruited to the liver. Comparing WT and IFNAR‐deficient mice with animals deficient for the IFNAR on myeloid cells only revealed a direct IFNAR‐dependent production of the anti‐inflammatory cytokine interleukin‐1 receptor antagonist (IL‐1RA) that could be assigned to liver‐infiltrating cells. Upon poly(I:C) treatment, IFNAR‐deficient mice displayed both a severe lack of IL‐1RA production and an increased production of proinflammatory IL‐1β, indicating a severely imbalanced cytokine milieu in the liver in absence of a functional type I IFN system. Depletion of IL‐1β or treatment with recombinant IL‐1RA both rescued IFNAR‐deficient mice from poly(I:C)‐induced liver damage, directly linking the deregulated IL‐1β and IL‐1RA production to liver pathology. Conclusion: Type I IFN signaling protects from severe liver damage by recruitment of monocytic MDSCs and maintaining a balance between IL‐1β and IL‐1RA production. (Hepatology 2014;59:1555‐1563)
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.