No abstract

Genome editing technology provides new possibilities for animal breeding and aid in understanding host-pathogen interactions. In poultry, retroviruses display one of the most difficult pathogens to control by conventional strategies such as vaccinations. Avian leukosis virus subgroup J (ALV-J) is an oncogenic, immunosuppressive retrovirus that causes myeloid leukosis and other tumors in chickens. Severe economic losses caused by ALV-J remain an unsolved problem in many parts of the world due to inefficient eradication strategies and lack of effective vaccines. ALV-J attachment and entry are mediated through the specific receptor, chicken Na + /H + exchanger type 1 (chNHE1). The non-conserved amino acid tryptophan 38 (W38) in chNHE1 is crucial for virus entry, making it a favorable target for the introduction of disease resistance. In this study, we obtained ALV-J-resistance in a commercial chicken line by precise deletion of chNHE1 W38, utilizing the CRISPR/Cas9-system in combination with homology directed repair. The genetic modification completely protected cells from infection with a subgroup J retrovirus. W38 deletion did neither have a negative effect on the development nor on the general health condition of the gene edited chickens. Overall, the generation of ALV-J-resistant birds by precise gene editing demonstrates the immense potential of this approach as an alternative disease control strategy in poultry.

B cells have first been described in chickens as antibody producing cells and were named after the Bursa of Fabricius, a unique organ supporting their development. Understanding different factors mediating the early migration of B cells into the bursa of Fabricius is crucial for the study of B cell biology. While CXCL12 (stromal derived factor 1) was found to play an important role in B lymphocyte trafficking in mammals, its role in the chicken is still unknown. Previous studies indicated that chicken CXCL12 and its receptor CXCR4 are simultaneously expressed during bursal development. In this study, we investigated whether the CXCR4/CXCL12 interaction mediates B cell migration in chicken embryo. We used the CRISPR/Cas9 system to induce a CXCR4 knockout in chicken B cells which led to chemotaxis inhibition toward CXCL12. This was confirmed by adoptive cell transfer and inhibition of the CXCR4/CXCL12 interaction by blocking with the small inhibitor AMD3100. In addition, we found that the chicken exhibits similarities to mice when it comes to CXCR4 being dependent on B cell receptor expression. B cells lacking the B cell receptor failed to migrate toward CXCL12 and showed no response upon CXCL12 stimulation. Overall, we demonstrated the significance of CXCR4/CXCL12 in chicken B cell development in vivo and the importance of the B cell receptor in CXCR4 dependent signaling.

With the exception of inactivated vaccines, all SARS-CoV-2 vaccines currently used for clinical application focus on the spike envelope glycoprotein as a virus-specific antigen. Compared to other SARS-CoV-2 genes, mutations in the spike protein gene are more rapidly selected and spread within the population, which carries the risk of impairing the efficacy of spike-based vaccines. It is unclear to what extent the loss of neutralizing antibody epitopes can be compensated by cellular immune responses, and whether the use of other SARS-CoV-2 antigens might cause a more diverse immune response and better long-term protection, particularly in light of the continued evolution towards new SARS-CoV-2 variants. To address this question, we explored immunogenicity and protective effects of adenoviral vectors encoding either the full-length spike protein (S), the nucleocapsid protein (N), the receptor binding domain (RBD) or a hybrid construct of RBD and the membrane protein (M) in a highly susceptible COVID-19 hamster model. All adenoviral vaccines provided life-saving protection against SARS-CoV-2-infection. The most efficient protection was achieved after exposure to full-length spike. However, the nucleocapsid protein, which triggered a robust T-cell response but did not facilitate the formation of neutralizing antibodies, controlled early virus replication efficiently and prevented severe pneumonia. Although the full-length spike protein is an excellent target for vaccines, it does not appear to be the only option for future vaccine design.

Targeted analysis of Coffea arabica and Coffea canephora green coffees (total sample size n = 57) confirmed 2-O-β-d-glucopyranosyl-carboxyatractyligenin (6) as the quantitatively dominating carboxyatractyligenin derivative. Its abundance in Arabicas (2425 ± 549 nmol/g, n = 48) exceeded that in Robustas (34 ± 12 nmol/g, n = 9) roughly by a factor of 70. Coffee processing involving heat (e.g., steam treatment and decaffeination) reduced concentrations of 6 and increased those of the decarboxylated derivative. The bioavailability of compound 6 in Caenorhabditis elegans was demonstrated by ultraperformance liquid chromatography-tandem mass spectrometry analysis of extracts prepared from nematode cultures incubated in a liquid medium containing 6. A toxicity assay performed to assess the impact of 6 in vivo showed a 20-fold higher median lethal dose (LD50 = 11.7 ± 1.2 mM) concentration compared to that of the known phytotoxic adenine-nucleotide transporters inhibitor carboxyatractyloside (2, LD50 = 0.61 ± 0.05 mM), whereas 1 mM 6 and 0.1 mM 2 were sufficient to decrease the survival of wild type C. elegans, already 10–20-fold lower doses reduced reproduction. Because the insulin/insulin-like growth factors signaling cascade (IIS) is a key regulator of life span and stress resistance, the impact of compound 6 on the survival of long-living daf-2 C. elegans was tested. As the susceptibility of these nematodes to 6 was as high as that in wild type, an impact on central metabolic processes independent of IIS was suggested. Analysis of the in vivo adenosine triphosphate (ATP) content of adult C. elegans revealed no changes after 1 and 24 h, but a 50% reduction after treatment with 1 mM 6 during the entire postembryonic development. These data speak for a developmental-stage-dependent modulation of the ATP pool by 6.