Arndt, A. | Search bioRxiv

bioRxiv2025

Tandem split-GFP influenza A viruses for sensitive and accurate replication analyses

Reporter influenza A viruses (IAVs) are valuable tools for studying virus fitness, screening antivirals, and assessing host-virus interactions. However, the compact and segmented nature of the IAV genome presents major challenges for engineering genetically stable reporter viruses with minimal fitness defects. To address this, we developed a replication-competent IAV incorporating a tandem split-GFP reporter strategy. Specifically, we appended seven tandem repeats of the GFP11 peptide (GFP11x7) to the C-terminus of the PB2 or PA polymerase subunit. Viruses contained or lacked a self-cleaving 2A peptide between the viral and reporter genes to create polymerase fusion proteins or released GFP, respectively. These viruses are complemented in trans by cells expressing GFP1-10, allowing bright fluorescence with minimal disruption to viral function. The tandem GFP11 reporter viruses exhibit delayed replication kinetics, but are genetically stable over serial passages with a strong concordance between GFP signal and viral gene expression. We demonstrate their utility in high-throughput applications including fluorescence-based quantification of infection, neutralizing antibody titration, antiviral drug screening, and host factor identification, with results matching traditional assays but on an accelerated timeline. Furthermore, we engineered a battery of GFP1-10-expressing cell lines from multiple vertebrate species, illustrating the broad compatibility of this platform for comparative host studies. This system enables sensitive, scalable, and quantitative evaluation of influenza virus replication across diverse experimental contexts. The GFP11x7 reporter platform offers a versatile and robust tool for virology research and therapeutic screening, with potential for rapid adaptation to emerging IAV strains.

microbiology↗

bioRxiv2025

Why they occur here but not there - a case study of Great Spotted Woodpeckers in the city of Munich

O_LIMany animal species can occur in the urban environment. The factors determining whether a particular species can live in a particular place in the city are, however, often not known. Such knowledge is needed to be able to design a built environment that is suitable for species. C_LIO_LIThe Great Spotted Woodpecker, Dendrocopos major, breeds in self-made cavities in trees and regularly occurs in cities. Here, we used a number of approaches to elucidate the factors that influence the occurrence of woodpeckers in the build-up area of the city. C_LIO_LIWe studied the occurrence of D. major on 103 squares in Munich, Germany and linked their presence to a number of square features such as square size, position in the city, tree occurrences, and a number of other variables. We also observed the presence of woodpecker cavities and undertook behavioural observations to study tree use. C_LIO_LIWoodpeckers were observed on 60% of the squares. A random forest model showed that the number of humans on squares negatively affected woodpecker occurrence, while a number of tree-related variables, such as tree density, tree diversity, the number of old trees (>60 cm DBH), and the sum of DBH of trees on the square, positively affected occurrence. Some other variables were also important, such as the amount of green in the surroundings and the distance to the city centre. C_LIO_LINests were found only in trees with a DBH>20 cm, and there was a distinct preference for larger trees, whereby a range of species was accepted. Behavioural observations showed that woodpeckers used a wide variety of trees, including small ones. Particular species and trees with a DBH>20 cm are preferred. C_LIO_LIOur study shows that humans can design public places for the presence of the Great Spotted Woodpecker by providing a locally high abundance of trees of different species with total DBH close to 25m. Trees in the surroundings and the presence of softwood trees with >40 cm will make it more likely that woodpeckers will use such sites in the city. C_LI Statement on inclusionOur study brings both established scientists with permanent faculty positions with Ph.D.-students and students of the 2nd year of their B.Sc.-studies or 1st or 2nd year of their M.Sc.-studies for whom this work is the first experience in the publication of scientific results. Students were particularly encouraged to contribute to question and hypothesis development as well as analysis and interpretation.

ecology↗