Analysis of avian Usutu virus infections in Germany from 2011 to 2018 with focus on dsRNA detection to demonstrate viral infections

Abstract

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.

Combining host and vector data informs emergence and potential impact of an Usutu virus outbreak in UK wild birds

“…Although, histopathological changes in other tissues were generally non-specific, immunolabelling demonstrated widespread virus antigen distribution in both bird species, which is similar to reports of USUV infection elsewhere 4 , 13 , 14 . Immunolabelling was disproportionately greater in the brain and heart in contrast to the minimal or absent histological changes observed in these organs: similar contrasting results of histological and immunohistochemical examinations of USUV-infected wild birds have previously been reported 12 . Although only brain and kidney samples were examined using USUV RT-PCR, our findings, together with earlier reports 4 , 14 , demonstrate that viral antigen can be detected in abundance in the heart and liver, suggesting that these organs could be useful for molecular diagnostic sampling.…”

“…The most prevalent and notable histological changes in the blackbirds and house sparrow with confirmed USUV infection were those in the liver and spleen, consisting of necrosis and lymphohistiocytic inflammation along with moderate to abundant virus antigen labelling. Whilst neurotropism resulting in brain necrosis and lymphohistiocytic inflammation has been reported in studies which examined large numbers of wild blackbirds with USUV infection in continental Europe 4 , 12 , we found minimal evidence of neural lesions in the five wild birds examined in this study. Although, histopathological changes in other tissues were generally non-specific, immunolabelling demonstrated widespread virus antigen distribution in both bird species, which is similar to reports of USUV infection elsewhere 4 , 13 , 14 .…”

Spread of West Nile Virus and Usutu Virus in the German Bird Population, 2019–2020

“…However, in 2019, only two dead birds with USUV lineage Africa 3 infections were detected in the same zoo in Berlin. In addition, it was recently announced that in a retrospective study, USUV of lineage Africa 2 was also detected in a Eurasian blackbird in Hannover, in the northwestern part of Germany in 2018 [59]. However, no further data are available for this retrospective study for this region and lineage in 2019 and 2020.…”

Expedited retrieval of high-quality Usutu virus genomes via Nanopore sequencing with and without target enrichment

“…Three are still regularly isolated to date: USUV lineages Europe 3 and Africa 3 are spread throughout Germany (Ziegler et al, 2022), while lineage Europe 2 is only present in the east (Michel et al, 2019;Santos et al, 2021;Ziegler et al, 2022). By contrast, USUV Africa 2 was only found in Germany in the past: in Berlin (2015,2017,2018), in Leipzig ( , 2018( ), and in Hannover (2018 (Ziegler et al, 2016;Michel et al, 2019;Santos et al, 2021;Störk et al, 2021). USUV Europe 5 was proposed as a novel putative lineage in Bonn in 2014 (Cadar et al, 2015) yet was never isolated again in subsequent years.…”

“…Even though partial USUV sequences are available from 2017 to 2020 ( Michel et al, 2019 ; Ziegler et al, 2022 ), whole-genome sequences of USUV isolates from birds in Germany are limited ( Becker et al, 2012 ; Ziegler et al, 2016 ; Störk et al, 2021 ). This makes it difficult to assess geographic and temporal dynamics of USUV in Germany.…”