Isolation of Diverse Simian Arteriviruses Causing Hemorrhagic Disease.

Genetically diverse simian arteriviruses (simarteriviruses) naturally infect geographically and phylogenetically diverse monkeys, and cross-species transmission and emergence are of considerable concern. Characterization of most simarteriviruses beyond sequence analysis has not been possible because the viruses fail to propagate in the laboratory. We attempted to isolate 4 simarteriviruses, Kibale red colobus virus 1, Pebjah virus, simian hemorrhagic fever virus, and Southwest baboon virus 1, by inoculating an immortalized grivet cell line (known to replicate simian hemorrhagic fever virus), primary macaque cells, macrophages derived from macaque induced pluripotent stem cells, and mice engrafted with macaque CD34+-enriched hematopoietic stem cells. The combined effort resulted in successful virus isolation; however, no single approach was successful for all 4 simarteriviruses. We describe several approaches that might be used to isolate additional simarteriviruses for phenotypic characterization. Our results will expedite laboratory studies of simarteriviruses to elucidate virus-host interactions, assess zoonotic risk, and develop medical countermeasures.

Generation of the NeoThy mouse model for human immune system studies.

Humanized mouse models, created via transplantation of human hematopoietic tissues into immune-deficient mice, support a number of research applications, including transplantation immunology, virology and oncology studies. As an alternative to the bone marrow, liver, thymus humanized mouse, which uses fetal tissues for generating a chimeric human immune system, the NeoThy humanized mouse uses nonfetal tissue sources. Specifically, the NeoThy model incorporates hematopoietic stem and progenitor cells from umbilical cord blood (UCB) as well as thymus tissue that is typically discarded as medical waste during neonatal cardiac surgeries. Compared with fetal thymus tissue, the abundant quantity of neonatal thymus tissue offers the opportunity to prepare over 1,000 NeoThy mice from an individual thymus donor. Here we describe a protocol for processing of the neonatal tissues (thymus and UCB) and hematopoietic stem and progenitor cell separation, human leukocyte antigen typing and matching of allogenic thymus and UCB tissues, creation of NeoThy mice, assessment of human immune cell reconstitution and all experimental steps from planning and design to data analysis. This entire protocol takes a total of ~19 h to complete, with steps broken up into multiple sessions of 4 h or less that can be paused and completed over multiple days. The protocol can be completed, after practice, by individuals with intermediate laboratory and animal handling skills, enabling researchers to make effective use of this promising in vivo model of human immune function.

Robust engraftment of fetal nonhuman primate CD34-positive cells in immune-deficient mice.

Nonhuman primates (NHPs) represent one of the most important models for preclinical studies of novel biomedical interventions. In contrast with small animal models, however, widespread utilization of NHPs is restricted by cost, logistics, and availability. Therefore, we sought to develop a translational primatized mouse model, akin to a humanized mouse, to allow for high-throughput in vivo experimentation leveraged to inform large animal immunology-based studies. We found that adult rhesus macaque mobilized blood (AMb) CD34+-enriched hematopoietic stem and progenitor cells (HSPCs) engrafted at low but persistent levels in immune-deficient mice harboring transgenes for human (NHP cross-reactive) GM-CSF and IL3, but did not in mice with wild-type murine cytokines lacking NHP cross-reactivity. To enhance engraftment, fetal liver-derived HSPCs were selected as the infusion product based on an increased CD34hi fraction compared with AMb and bone marrow. Coupled with cotransplantation of rhesus fetal thymic fragments beneath the mouse kidney capsule, fetal liver-derived HSPC infusion in cytokine-transgenic mice yielded robust multilineage lymphohematopoietic engraftment. The emergent immune system recapitulated that of the fetal monkey, with similar relative frequencies of lymphocyte, granulocyte, and monocyte subsets within the thymic, secondary lymphoid, and peripheral compartments. Importantly, while exhibiting a predominantly naïve phenotype, in vitro functional assays demonstrated robust cellular activation in response to nonspecific and allogenic stimuli. This primatized mouse represents a viable and translatable model for the study of hematopoietic stem cell physiology, immune development, and functional immunology in NHPs. Summary Sentence: Engraftment of rhesus macaque hematopoietic tissues in immune-deficient mice yields a robust BLT/NeoThy-type primatized mouse model for studying nonhuman primate hematopoiesis and immune function in vivo.