Usutu virus (USUV) is a zoonotic arbovirus causing avian mass mortalities. The first outbreak in North-Western Germany occurred in 2018. This retrospective analysis focused on combining virological and pathological findings in birds and immunohistochemistry. 25 common blackbirds, one great grey owl, and one kingfisher collected from 2011 to 2018 and positive for USUV by qRT-PCR were investigated. Macroscopically, most USUV infected birds showed splenomegaly and hepatomegaly. Histopathological lesions included necrosis and lymphohistiocytic inflammation within spleen, Bursa fabricii, liver, heart, brain, lung and intestine. Immunohistochemistry revealed USUV antigen positive cells in heart, spleen, pancreas, lung, brain, proventriculus/gizzard, Bursa fabricii, kidney, intestine, skeletal muscle, and liver. Analysis of viral genome allocated the virus to Europe 3 or Africa 2 lineage. This study investigated whether immunohistochemical detection of double-stranded ribonucleic acid (dsRNA) serves as an alternative tool to detect viral intermediates. Tissue samples of six animals with confirmed USUV infection by qRT-PCR but lacking viral antigen in liver and spleen, were further examined immunohistochemically. Two animals exhibited a positive signal for dsRNA. This could indicate either an early state of infection without sufficient formation of virus translation products, occurrence of another concurrent virus infection or endogenous dsRNA not related to infectious pathogens and should be investigated in more detail in future studies.

Rift Valley fever phlebovirus (RVFV, Phenuiviridae) is an emerging arbovirus that can cause potentially fatal disease in many host species including ruminants and humans. Thus, tools to detect this pathogen within tissue samples from routine diagnostic investigations or for research purposes are of major interest. This study compares the immunohistological usefulness of several mono- and polyclonal antibodies against RVFV epitopes in tissue samples derived from natural hosts of epidemiologic importance (sheep), potentially virus transmitting insect species (Culex quinquefasciatus, Aedes aegypti) as well as scientific infection models (mouse, Drosophila melanogaster, C6/36 cell pellet). While the nucleoprotein was the epitope most prominently detected in mammal and mosquito tissue samples, fruit fly tissues showed expression of glycoproteins only. Antibodies against non-structural proteins exhibited single cell reactions in salivary glands of mosquitoes and the C6/36 cell pellet. However, as single antibodies exhibited a cross reactivity of varying degree in non-infected specimens, a careful interpretation of positive reactions and consideration of adequate controls remains of critical importance. The results suggest that primary antibodies directed against viral nucleoproteins and glycoproteins can facilitate RVFV detection in mammals and insects, respectively, and therefore will allow RVFV detection for diagnostic and research purposes.

Insects play an important role in ecosystems. Changes in their abundance and biodiversity are of paramount interest, as there has not only been an alarming decline of insects important for ecosystem health throughout the past decades, but also an increase in insects detrimental for biomes. Furthermore, insects pose a threat to modern society as arbovirus-transmitting vectors. Therefore, detailed knowledge of insect staining characteristics could be beneficial as a basis for further studies, whether in the context of species conservation or control of insect pests. Thus, this study compared 14 histochemical stains for their usefulness in insects regarding nervous tissue, connective tissue components, mucins and polysaccharides, mineralization, and microorganisms. The study used formalin-fixed paraffin-embedded tissue sections of mammals ( Equus caballus) and 2 dipterans ( Culex pipiens biotype molestus, Drosophila melanogaster). Several histochemical stains were suitable for tissue assessment in insects and mammals, in particular for nervous tissue (Bielschowsky silver stain, luxol fast blue–cresyl violet) and polysaccharides (alcian blue, periodic acid–Schiff with and without diastase treatment, toluidine blue). Other stains proved useful for visualization of insect-specific organ characteristics such as Gomori’s reticulin stain for tracheoles in both dipteran species, Heidenhain’s azan for midgut-associated connective tissue, and von Kossa for mineral deposition in Malpighian tubules of C. pipiens biotype molestus. In summary, this study provides comparable insights into histochemical procedures in mammals and insects and their usefulness for histological assessment of C. pipiens biotype molestus and D. melanogaster.

Arthropod-borne diseases represent one of the greatest infection-related threats as a result of climate change and globalization. Repeatedly, arbovirus-infected mosquitoes show behavioral changes whose underlying mechanisms are still largely unknown, but might help to develop control strategies. However, in contrast to well-characterized insects such as fruit flies, little is known about neuroanatomy and neurotransmission in mosquitoes. To overcome this limitation, the study focuses on the immunohistochemical characterization of the nervous system of Culex pipiens biotype molestus in comparison to Drosophila melanogaster using 13 antibodies labeling nervous tissue, neurotransmitters or neurotransmitter-related enzymes. Antibodies directed against γ-aminobutyric acid, serotonin, tyrosine-hydroxylase and glutamine synthetase were suitable for investigations in Culex pipiens and Drosophila melanogaster, albeit species-specific spatial differences were observed. Likewise, similar staining results were achieved for neuronal glycoproteins, axons, dendrites and synaptic zones in both species. Interestingly, anti-phosphosynapsin and anti-gephyrin appear to represent novel markers for synapses and glial cells, respectively. In contrast, antibodies directed against acetylcholine, choline acetyltransferase, elav and repo failed to produce a signal in Culex pipiens comparable to that in Drosophila melanogaster. In summary, present results enable a detailed investigation of the nervous system of mosquitoes, facilitating further studies of behavioral mechanisms associated with arboviruses in the course of vector research.

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