Postnatal persistent infection with classical Swine Fever virus and its immunological implications.

It is well established that trans-placental transmission of classical swine fever virus (CSFV) during mid-gestation can lead to persistently infected offspring. The aim of the present study was to evaluate the ability of CSFV to induce viral persistence upon early postnatal infection. Two litters of 10 piglets each were infected intranasally on the day of birth with low and moderate virulence CSFV isolates, respectively. During six weeks after postnatal infection, most of the piglets remained clinically healthy, despite persistent high virus titres in the serum. Importantly, these animals were unable to mount any detectable humoral and cellular immune response. At necropsy, the most prominent gross pathological lesion was a severe thymus atrophy. Four weeks after infection, PBMCs from the persistently infected seronegative piglets were unresponsive to both, specific CSFV and non-specific PHA stimulation in terms of IFN-γ-producing cells. These results suggested the development of a state of immunosuppression in these postnatally persistently infected pigs. However, IL-10 was undetectable in the sera of the persistently infected animals. Interestingly, CSFV-stimulated PBMCs from the persistently infected piglets produced IL-10. Nevertheless, despite the addition of the anti-IL-10 antibody in the PBMC culture from persistently infected piglets, the response of the IFN-γ producing cells was not restored. Therefore, other factors than IL-10 may be involved in the general suppression of the T-cell responses upon CSFV and mitogen activation. Interestingly, bone marrow immature granulocytes were increased and targeted by the virus in persistently infected piglets. Taken together, we provided the first data demonstrating the feasibility of CSFV in generating a postnatal persistent disease, which has not been shown for other members of the Pestivirus genus yet. Since serological methods are routinely used in CSFV surveillance, persistently infected pigs might go unnoticed. In addition to the epidemiological and economic significance of persistent CSFV infection, this model could be useful for understanding the mechanisms of viral persistence.

Cell cycle reactivation of cochlear progenitor cells in neonatal FUCCI mice by a GSK3 small molecule inhibitor.

Due to the lack of regenerative capacity of the mammalian auditory epithelium, sensory hair cell loss results in permanent hearing deficit. Nevertheless, a population of tissue resident stem/progenitor cells has been recently described. Identification of methods to trigger their activity could lead to exploitation of their potential therapeutically. Here we validate the use of transgenic mice reporting cell cycle progression (FUCCI), and stemness (Lgr5-GFP), as a valuable tool to identify regulators of cell cycle re-entry of supporting cells within the auditory epithelium. The small molecule compound CHIR99021 was used to inhibit GSK3 activity. This led to a significant increase in the fraction of proliferating sphere-forming cells, labeled by the FUCCI markers and in the percentage of Lgr5-GFP + cells, as well as a selective increase in the fraction of S-G2-M cells in the Lgr5 + population. Using whole mount cultures of the organ of Corti we detected a statistically significant increment in the fraction of proliferating Sox2 supporting cells after CHIR99021 treatment, but only rarely appearance of novel MyoVIIa+/Edu + hair cells. In conclusion, these tools provide a robust mean to identify novel regulators of auditory organ regeneration and to clarify the contribution of stem cell activity.

Intravital and whole-organ imaging reveals capture of melanoma-derived antigen by lymph node subcapsular macrophages leading to widespread deposition on follicular dendritic cells.

Aberrant antigens expressed by tumor cells, such as in melanoma, are often associated with humoral immune responses, which may in turn influence tumor progression. Despite recent data showing the central role of adaptive immune responses on cancer spread or control, it remains poorly understood where and how tumor-derived antigen (TDA) induces a humoral immune response in tumor-bearing hosts. Based on our observation of TDA accumulation in B cell areas of lymph nodes (LNs) from melanoma patients, we developed a pre-metastatic B16.F10 melanoma model expressing a fluorescent fusion protein, tandem dimer tomato, as a surrogate TDA. Using intravital two-photon microscopy (2PM) and whole-mount 3D LN imaging of tumor-draining LNs in immunocompetent mice, we report an unexpectedly widespread accumulation of TDA on follicular dendritic cells (FDCs), which were dynamically scanned by circulating B cells. Furthermore, 2PM imaging identified macrophages located in the subcapsular sinus of tumor-draining LNs to capture subcellular TDA-containing particles arriving in afferent lymph. As a consequence, depletion of macrophages or genetic ablation of B cells and FDCs resulted in dramatically reduced TDA capture in tumor-draining LNs. In sum, we identified a major pathway for the induction of humoral responses in a melanoma model, which may be exploitable to manipulate anti-TDA antibody production during cancer immunotherapy.

The Honey Bee Pathosphere of Mongolia: European Viruses in Central Asia

Parasites and pathogens are apparent key factors for the detrimental health of managed European honey bee subspecies, Apis mellifera. Apicultural trade is arguably the main factor for the almost global distribution of most honey bee diseases, thereby increasing chances for multiple infestations/infections of regions, apiaries, colonies and even individual bees. This imposes difficulties to evaluate the effects of pathogens in isolation, thereby creating demand to survey remote areas. Here, we conducted the first comprehensive survey for 14 honey bee pathogens in Mongolia (N = 3 regions, N = 9 locations, N = 151 colonies), where honey bee colonies depend on humans to overwinter. In Mongolia, honey bees, Apis spp., are not native and colonies of European A. mellifera subspecies have been introduced ~60 years ago. Despite the high detection power and large sample size across Mongolian regions with beekeeping, the mite Acarapis woodi, the bacteria Melissococcus plutonius and Paenibacillus larvae, the microsporidian Nosema apis, Acute bee paralysis virus, Kashmir bee virus, Israeli acute paralysis virus and Lake Sinai virus strain 2 were not detected, suggesting that they are either very rare or absent. The mite Varroa destructor, Nosema ceranae and four viruses (Sacbrood virus, Black queen cell virus, Deformed wing virus (DWV) and Chronic bee paralysis virus) were found with different prevalence. Despite the positive correlation between the prevalence of V. destructor mites and DWV, some areas had only mites, but not DWV, which is most likely due to the exceptional isolation of apiaries (up to 600 km). Phylogenetic analyses of the detected viruses reveal their clustering and European origin, thereby supporting the role of trade for pathogen spread and the isolation of Mongolia from South-Asian countries. In conclusion, this survey reveals the distinctive honey bee pathosphere of Mongolia, which offers opportunities for exciting future research.