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.

Figure 1

Macroscopic findings in naturally Usutu virus infected birds. (A) Marked splenomegaly (asterisk) was present in a blackbird (Supplementary Table S1, #8). (B) Moderate to marked hepatomegaly (asterisk) was shown in a blackbird (Supplementary Table S1, #10). (C) Severe hemorrhagic enteritis characterized by dark red to black, coagulated blood in the intestinal lumen was detected in a blackbird (arrowheads; Supplementary Table S1, #8).

Figure 2

Histopathologic findings in naturally Usutu virus infected birds. (A) The Spleen of a great grey owl (Supplementary Table S1, #1) showed multifocal coagulative necrosis (asterisk) and lymphoid depletion (arrows; scale bar: 50 µm; HE). (B) The Bursa fabricii of a blackbird (Supplementary Table S1, #3) displayed marked subacute lymphocytolysis (asterisks) in follicular centers and additional histiocytic infiltrations (scale bar: 100 µm; HE). (C) The liver of a blackbird (Supplementary Table S1, #4) presented severe coagulation necrosis (asterisk) accompanied by mild infiltration of lymphocytes and hemosiderin-laden macrophages (scale bar: 50 µm; HE). (D) The heart of a blackbird (Supplementary Table S1, #3) exhibited mild to moderate, multifocal, predominantly interstitial, lymphohistiocytic myocarditis (arrows; scale bar: 50 µm; HE). (E) The cerebellum of a blackbird (Supplementary Table S1, #3) revealed mild, multifocal, perivascularly accentuated, lymphohistiocytic infiltrations (arrows; scale bar: 100 µm; HE). (F) The lung of a blackbird (Supplementary Table S1, #9) showed moderate, peribronchial, lymphohistiocytic inflammation (asterisk; scale bar: 50 µm; HE).

Figure 3

Immunohistochemical findings in naturally Usutu virus (USUV) infected birds. (A) USUV antigen was detected in the cytoplasm of a high numbers of cells, mostly macrophages, in both red and depleted white pulp in the spleen of a great grey owl (Supplementary Table S1, #1; scale bar: 50 µm; U433). (B) The Bursa fabricii of a blackbird (Supplementary Table S1, #2) showed low to moderate amounts of viral antigen, frequently located in the periphery of follicles (scale bar: 50 µm; U433). (C) The liver of a great grey owl (Supplementary Table S1, #1) presented moderate amounts of viral antigen multifocally in the cytoplasm of necrotic and intactly appearing hepatocytes as well as in Kupffer cells (scale bar: 50 µm; U433). (D) The cerebellum of a blackbird (Supplementary Table S1, #7) showed high amounts of viral antigen predominantly within Purkinje cells and glial cells (scale bar: 50 µm; U433). (E) In the cerebrum of a blackbird (Supplementary Table S1, #7) high numbers of unaltered neurons contained USUV antigen (scale bar: 50 µm; U433). (F) The lung of a great grey owl (Supplementary Table S1, #1) yielded low to moderate amounts of viral antigen in the interstitium and multifocally within lumina of small and large blood vessels (scale bar: 50 µm; U433).

Figure 4

Immunohistochemical detection of Caspase 3 positive apoptotic cells in naturally Usutu virus (USUV) infected birds. (A) The spleen of an USUV infected blackbird (Supplementary Table S1, #3) presented mildly to moderately increased numbers of apoptotic cells (scale bar: 50 µm; Caspase 3). (B) The spleen of an USUV negative control blackbird (Supplementary Table S1, #29) showed less numbers of apoptotic cells. Due to the poor state of preservation, non-specific background staining was increased (scale bar: 50 µm; Caspase 3). (C) The Bursa fabricii of an USUV infected blackbird (Supplementary Table S1, #3) displayed a moderately increased number of apoptotic cells in follicles (scale bar: 200 µm; Caspase 3). (D) In the Bursa fabricii of an USUV negative blackbird (Supplementary Table S1, #29) only small to moderate numbers of apoptotic cells were detected, mostly located in the center of follicles (scale bar: 100 µm; Caspase 3).

Figure 5

Comparison of distribution of Usutu virus (USUV) specific antigen and double-stranded ribonucleic acid (dsRNA) expression in the spleen between two animals with qRT-PCR confirmed USUV infection with (A-D) and without (E–H) immunohistochemical USUV antigen detection. (A) Moderate amounts of USUV antigen-labeled cells in the spleen of an animal (Table 2, #1) with USUV confirmed infection by qRT-PCR. (B) In contrast, only few cells labeled positive for dsRNA by applying the 9D5 antibody (arrowheads). Inset: 9D5-labeled cells at higher magnification. A moderate number of cells showed positive reaction with dsRNA-specific antibodies K1 (C, arrowheads) and J2 (D, arrowheads) similar to the USUV antigen distribution with respect to both, amount and distribution of immunopositive cells. Inset: K1- and J2-labeled cells at higher magnification. (E) Despite lack of viral antigen the spleen of a blackbird with qRT-PCR-confirmed USUV infection (Table 2, #7) was tested positive for dsRNA by the application of antibodies directed against dsRNA. Although, the amount of immunopositive cells varied substantially: Detection level of dsRNA by using the 9D5 antibody (F, arrowheads) remained low in comparison to K1 (G, arrowhead) and J2 (H, arrowhead) antibody, respectively. Insets: 9D5-, K1- and J2-labeled cells at higher magnification. (scale bar: 50 µm; immunohistochemistry).

Figure 6

Maximum likelihood phylogeny of USUV sequences. Phylogenetic tree was constructed using a subset (n = 31) of published USUV sequences from NCBI Genbank representing USUV strains from lineages Africa 2–3 and Europe 1–4. Maximum likelihood phylogeny was performed using nearly complete genomes (> 10,203 bp) with a GTR + G model of nucleotide substitution. The tree was generated using bootstrap support of 1000 replications. Bootstrap values are depicted on the major nodes. The tip labels represent the NCBI Genbank accession numbers of respective strains and the scale bar is proportional to the number of nucleotide substitutions per site. The novel sequences of the strains “HAN/SN/2018” and “HAN/TM/2018” that were generated via NGS in this study are marked with an asterisk and were obtained from a great grey owl and a black bird, respectively. The pictogram of the owl was created with BioRender.com. The pictogram of the blackbird is licensed under CC BY-SA 3.0 (https://creativecommons.org/licenses/by-sa/3.0/legalcode) and originally created by Andreas Plank and can be found under the following link: https://commons.wikimedia.org/wiki/File:Blackbird_Turdus_merula_female_silhouette.svg.