S evere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China, at the end of 2019. Researchers have identified close relatives to SARS-CoV-2 in bats (1) and pangolins (order Pholidota) (2,3). Whether the pandemic was initiated by direct transmission from bats or through an intermediate mammalian host is still under debate (4). During the 2002-2004 severe acute respiratory syndrome pandemic, researchers documented the causative virus in raccoon dogs (Nyctereutes procyonoides) in China, indicating that these animals might have been intermediate hosts for the virus (5). Fur producers in China own >14 million captive raccoon dogs, accounting for ≈99% of the global share of raccoon dogs (6) (Appendix Figure 1, panel A, https:// wwwnc.cdc.gov/EID/article/26/12/20-3733-App1. pdf). However, whether these animals are susceptible to SARS-CoV-2 is unknown. Using our established study design (7), we characterized susceptibility, viral shedding, transmission potential, serologic reactions, and pathologic lesions of raccoon dogs after experimental SARS-CoV-2 infection.

Serratia marcescens and several other bacteria produce the red-colored pigment prodigiosin which possesses bioactivities as an antimicrobial, anticancer, and immunosuppressive agent. Therefore, there is a great interest to produce this natural compound. Efforts aiming at its biotechnological production have so far largely focused on the original producer and opportunistic human pathogen S. marcescens. Here, we demonstrate efficient prodigiosin production in the heterologous host Pseudomonas putida. Random chromosomal integration of the 21 kb prodigiosin biosynthesis gene cluster of S. marcescens in P. putida KT2440 was employed to construct constitutive prodigiosin production strains. Standard cultivation parameters were optimized such that titers of 94 mg/L culture were obtained upon growth of P. putida at 20°C using rich medium under high aeration conditions. Subsequently, a novel, fast and effective protocol for prodigiosin extraction and purification was established enabling the straightforward isolation of prodigiosin from P. putida growth medium. In summary, we describe here a highly efficient method for the heterologous biosynthetic production of prodigiosin which may serve as a basis to produce large amounts of this bioactive natural compound and may provide a platform for further in-depth studies of prodiginine biosynthesis.

The evolution of eukaryotic organisms is often strongly influenced by microbial symbionts that confer novel traits to their hosts. Here we describe the intracellular Enterobacteriaceae symbiont of the invasive ant Cardiocondyla obscurior, 'Candidatus Westeberhardia cardiocondylae'. Upon metamorphosis, Westeberhardia is found in gut-associated bacteriomes that deteriorate following eclosion. Only queens maintain Westeberhardia in the ovarian nurse cells from where the symbionts are transmitted to late-stage oocytes during nurse cell depletion. Functional analyses of the streamlined genome of Westeberhardia (533 kb, 23.41% GC content) indicate that neither vitamins nor essential amino acids are provided for the host. However, the genome encodes for an almost complete shikimate pathway leading to 4-hydroxyphenylpyruvate, which could be converted into tyrosine by the host. Taken together with increasing titers of Westeberhardia during pupal stage, this suggests a contribution of Westeberhardia to cuticle formation. Despite a widespread occurrence of Westeberhardia across host populations, one ant lineage was found to be naturally symbiont-free, pointing to the loss of an otherwise prevalent endosymbiont. This study yields insights into a novel intracellular mutualist that could play a role in the invasive success of C. obscurior.

The deeply red-colored natural compound prodigiosin is a representative of the prodiginine alkaloid family, which possesses bioactivities as antimicrobial, antitumor, and antimalarial agents. Various bacteria including the opportunistic human pathogen Serratia marcescens and different members of the Streptomycetaceae and Pseudoalteromonadaceae produce prodiginines. In addition, these microbes generally accumulate many structurally related alkaloids making efficient prodiginine synthesis and purification difficult and expensive. Furthermore, it is known that structurally different natural prodiginine variants display differential bioactivities. In the herein described mutasynthesis approach, 13 different derivatives of prodigiosin were obtained utilizing the GRAS (generally recognized as safe) classified strain Pseudomonas putida KT2440. Genetic engineering of the prodigiosin pathway together with incorporation of synthetic intermediates thus resulted in the formation of a so far unprecedented structural diversity of new prodiginine derivatives in P. putida. Furthermore, the formed products allow reliable conclusions regarding the substrate specificity of PigC, the final condensing enzyme in the prodigiosin biosynthesis pathway of S. marcescens. The biological activity of prodigiosin toward modulation of autophagy was preserved in prodiginine derivatives. One prodiginine derivative displayed more potent autophagy inhibitory activity than the parent compound or the synthetic clinical candidate obatoclax.

Although conventional immunohistochemistry for neurotropic rabies virus (RABV) usually shows high preference for neurons, non-neuronal cells are also potential targets, and abortive astrocyte infection is considered a main trigger of innate immunity in the CNS. While in vitro studies indicated differences between field and less virulent lab-adapted RABVs, a systematic, quantitative comparison of astrocyte tropism in vivo is lacking. Here, solvent-based tissue clearing was used to measure RABV cell tropism in infected brains. Immunofluorescence analysis of 1 mm-thick tissue slices enabled 3D-segmentation and quantification of astrocyte and neuron infection frequencies.Comparison of three highly virulent field virus clones from fox, dog, and raccoon with three lab-adapted strains revealed remarkable differences in the ability to infect astrocytes in vivo. While all viruses and infection routes led to neuron infection frequencies between 7-19%, striking differences appeared for astrocytes. Whereas astrocyte infection by field viruses was detected independent of the inoculation route (8-27%), only one lab-adapted strain infected astrocytes route-dependently [0% after intramuscular (i.m.) and 13% after intracerebral (i.c.) inoculation]. Two lab-adapted vaccine viruses lacked astrocyte infection altogether (0%, i.c. and i.m.). This suggests a model in which the ability to establish productive astrocyte infection in vivo functionally distinguishes field and attenuated lab RABV strains.Cells 2020, 9, 412 2 of 20 M [4-10], and neuronal survival regulation by G [11][12][13][14]. Most pathogenicity studies, however, were performed on already attenuated virus backbones. Thus, differences in their ability to cause disease between highly virulent field virus isolates and lab-adapted, less pathogenic RABV strains are poorly understood. Moreover, it is unclear how molecular differences identified in virulent and attenuated viruses affect virus replication and spread in the infected animal and how the complex virus-host interplay eventually results in either disease or an abortive infection.In vivo, after infection of neurons, RABV spreads trans-synaptically from infected to connected neurons [15]. Retrograde axonal transport of RABV over long distances [16,17] along microtubules [18,19] is a key step in RABV neuroinvasion and is essential for infection of the central nervous system (CNS) through the peripheral nervous system. Co-internalization together with the neuronal p75NTR (tumor necrosis factor receptor superfamily member 16; TNFRSF16) receptor, subsequent retrograde axonal transport of RABV particles in endocytic vesicles, and post-replicative anterograde axonal transport of newly formed RABV have been visualized by live virus particle tracking in sensory neurons [20,21], emphasizing the capacity of hijacking neuron-specific machineries for long distance transport to synaptic membranes. However, internalization and axonal transport of lab-adapted viruses [20,21] together with the use of vaccine virus vectors for trans-syn...