Previous Usutu virus exposure partially protects magpies (Pica pica) against west Nile virus disease but does not prevent horizontal transmission

viruses Article Previous Usutu Virus Exposure Partially Protects Magpies (Pica pica) against West Nile Virus Disease But Does Not Prevent Horizontal Transmission Estela Escribano-Romero 1,† , Nereida Jiménez de Oya 1,† , María-Cruz Camacho 2,†, Ana-Belén Blázquez 1, Miguel A. Martín-Acebes 1, Maria A. Risalde 3,4 , Laura Muriel 3, Juan-Carlos Saiz 1and Ursula Höfle 2,*   Citation: Escribano-Romero, E.; Jiménez de Oya, N.; Camacho, M.-C.; Blázquez, A.-B.; Martín-Acebes, M.A.; Risalde, M.A.; Muriel, L.; Saiz, J.-C.; Höfle, U. Previous Usutu Virus Exposure Partially Protects Magpies (Pica pica) against West Nile Virus Disease But Does Not Prevent Horizontal Transmission. Viruses 2021,13, 1409. https:// doi.org/10.3390/v13071409 Academic Editors: Kateri Bertran, MartíCortey and Miria F. Criado Received: 7 June 2021 Accepted: 14 July 2021 Published: 20 July 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Department of Biotechnology, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA-CSIC), 28040 Madrid, Spain; [email protected] (E.E.-R.); [email protected] (N.J.d.O.); [email protected] (A.-B.B.); [email protected] (M.A.M.-A.); [email protected] (J.-C.S.) 2Health and Biotechnology Research Group SaBio, National Institute for Game Rearch IREC (University of Castilla—La Mancha UCLM-National Research Council CSIC-Regional Government of Castilla—La Mancha JCCM), 13005 Ciudad Real, Spain; mrcr[email protected] 3Departamento de Anatomía y Anatomía Patológica Comparadas, Facultad de Veterinaria, Agrifood Excellence International Campus (ceiA3), Universidad de Córdoba (UCO), 14014 Córdoba, Spain; [email protected] (M.A.R.); lauramurielcantarer[email protected] (L.M.) 4Infectious Diseases Unit, Hospital Universitario Reina Sofía de Córdoba, Instituto Maimonides de Investigación Biomédica de Córdoba (IMIBIC), University of Córdoba, 14004 Córdoba, Spain *Correspondence: [email protected] † These authors contributed equally to this work. Abstract: The mosquito-borne flaviviruses USUV and WNV are known to co-circulate in large parts of Europe. Both are a public health concern, and USUV has been the cause of epizootics in both wild and domestic birds, and neurological cases in humans in Europe. Here, we explore the susceptibility of magpies to experimental USUV infection, and how previous exposure to USUV would affect infection with WNV. None of the magpies exposed to USUV showed clinical signs, viremia, or detectable neutralizing antibodies. After challenge with a neurovirulent WNV strain, neither viremia, viral titer of WNV in vascular feathers, nor neutralizing antibody titers of previously USUV-exposed magpies differed significantly with respect to magpies that had not previously been exposed to USUV. However, 75% (6/8) of the USUV-exposed birds survived, while only 22.2% (2/9) of those not previously exposed to USUV survived. WNV antigen labeling by immunohistochemistry in tissues was less evident and more restricted in magpies exposed to USUV prior to challenge with WNV. Our data indicate that previous exposure to USUV partially protects magpies against a lethal challenge with WNV, while it does not prevent viremia and direct transmission, although the mechanism is unclear. These results are relevant for flavivirus ecology and contention. Keywords: avian host; Flaviviruses; co-infection; cross-protection; Usutu virus; West Nile virus; magpie 1. Introduction Usutu virus (USUV) is a mosquito-transmitted flavivirus that belongs to the Japanese encephalitis virus serocomplex [ 1 ]. After its first identification in South Africa in 1959 [ 2 ], the virus was detected in Europe in 2001 in an outbreak in birds in Austria [ 3 ], although retrospective identification in tissues from blackbird mortalities in Italy suggests it has been circulating in Europe since at least 1996 [ 4 ]. The virus has now spread throughout Europe, causing high bird mortality [ 5 ] and some human cases [ 6 – 10 ]. USUV seropositivity has been reported in more than 58 bird species belonging to 26 families and 13 orders, Passeriformes being the most affected [ 5 , 11 ]. In 2016, several countries in Western Europe reported the largest epizootic of the USUV registered so far in Europe, which caused a massive mortality of birds [ 5 ]. However, scarce experimental data on USUV infection in avian species are available [12–14]. Viruses 2021,13, 1409. https://doi.org/10.3390/v13071409 https://www.mdpi.com/journal/viruses Viruses 2021,13, 1409 2 of 15 At least two other mosquito borne-flaviviruses co-circulate with USUV in different regions of Europe. While the Bagaza virus (BAGV) has so far only affected birds [ 15 ], West Nile virus (WNV) is highly neurovirulent in humans [ 5 , 16 ]. During the last ten years, WNV outbreaks among birds and horses, and a worrying increase in human cases, with up to 2671 confirmed infections and 213 deaths, have been reported in the continent [17]. The magpie, one of the most abundant corvids in Europe [ 18 ], has recently been shown to be highly susceptible to WNV infection and a possible source for virus transmission [ 19 ], and the mortality of magpies due to lineage 2 WNV has been reported in Greece [ 20 ]. However, there are scarce data on the susceptibility of magpies to USUV infection. Freeliving, apparently healthy, and actively collected magpies have been reported positive for USUV RNA in a region with co-circulation of USUV and WNV [ 21 ], and the seropositivity of magpies against USUV has been detected occasionally in large-scale serosurveys in France, Italy, and Germany [ 22 – 26 ]. To date, there is a lack of understanding of the interaction between USUV and WNV in avian hosts where the circulation of both viruses overlaps [ 11 ]. With this background, we experimentally addressed the susceptibility of magpies to USUV infection and their subsequent response to a lethal challenge with WNV. 2. Materials and Methods 2.1. Experimental Design Magpies were captured between April and June 2018 in hunting estates in southcentral Spain under permit 346760/2018 of the regional government of the autonomic Community of Castilla-La Mancha (Spain), as part of a specific pest control program in the area. The presence of flavivirus (USUV and WNV)-specific neutralizing antibodies (NAbs) was tested using a plaque reduction neutralization test (PRNT) on Vero cells using twofold serial dilutions of heat-inactivated serum, as described in [ 27 ]. All birds were also tested for Flavivirus RNA in pooled oral and cloacal swabs and in their feather follicles [15]. A final group of 24 juvenile (less than one year old) magpies negative for USUV and WNV Nabs and RNA was transported to our biosafety level 3 (BSL-3) facilities, where they were housed in 2 separate boxes (Figure 1) in flight cages (12 birds/cage), equipped as described [ 19 ]. After one week of adaptation, animals were weighed and bled via the jugular vein for pre-inoculation serology. In the USUV box, one group of magpies (n = 9) was subcutaneously inoculated in the neck with 5 × 10 3 plaque-forming units (pfu)/bird of USUV strain SAAR-1776 (GenBank accession no. KU760915.1 [ 28 ]), diluted in 200 µ L Eagle Minimum Essential Medium (EMEM, BioWhittaker, Lonza, Verviers, Belgium), and 3 cagemates were similarly sham-inoculated with medium alone and served as contact controls. On day 18 post-USUV infection, eight out of the nine USUV-infected birds were challenged with 5 × 10 3 pfu/bird of WNV lineage 1 strain (GenBank accession no. KC407666; [ 29 ]). The remaining USUV-infected magpie and the three remaining USUV contact control birds were again sham-inoculated with medium alone. In the vehicle group (n = 12), housed in a separate cage (vehicle box), all magpies were inoculated with medium alone. On day 18, 9 birds were infected with WNV, and 3 were again inoculated with medium (contact control magpies) (Figure 1). Both viral inocula were back-titrated to confirm the injected dose. Food and water were provided ad libitum throughout the experiment. The magpies were monitored daily for clinical signs, and birds showing severe clinical signs were anesthetized with isoflurane and euthanized by intravenous injection of an overdose of sodium pentobarbital (Dolethal, Vetoquinol, Madrid, Spain), as were all surviving animals at the end of the experiment (37 days post infection (d.p.i.)). Viruses 2021,13, 1409 3 of 15 Viruses 2021, 13, x FOR PEER REVIEW 3 of 16 Figure 1. Experimental design. Representation of the USUV and WNV infection, sampling schedule, and distribution of the magpies in the experimental boxes. 2.2. Sampling At 0, 4, 7, 10, and 14 d.p.i. with USUV and at 4, 7, 10, 14, and 19 d.p.i. with WNV, all surviving animals were weighed and sampled (blood and feathers) as previously described [19]. Blood samples were allowed to clot overnight at 4 °C. Serum and all tissue and feather samples were stored at −80 °C until analysis. Growing feathers with pulp (vascular feathers) were collected from the dead and euthanized birds and placed into 0.5 mL of EMEM medium. Samples of brain, heart, and kidney were also collected into sterile containers and frozen at −80 °C until the analysis. In addition, cerebrum, cerebellum, medulla oblongata, heart, lung, spleen, liver, kidney, caecal tonsils, and duodenum samples were collected from deceased birds during a post mortem examination and were fixed in 10% neutral buffered formalin for histopathologic examination. 2.3. Immunological and Viral Assays Neutralizing antibodies for either virus (USUV and WNV) were analyzed using a plaque reduction neutralization test (PRNT) [30]. Titers were calculated as the reciprocal of the serum dilution, and diluted by at least 1:20, which reduced the plaque formation ≥90% (PRNT90). Sera were considered specific when only one of the viruses was neutralized or the titers were ≥4 times higher for one of them. Collected sera and vascular feathers were also tested for flavivirus (USUV and WNV) infectivity by plaque assay on Vero cell culture, as previously reported [31,32]. Figure 1. Experimental design. Representation of the USUV and WNV infection, sampling schedule, and distribution of the magpies in the experimental boxes. 2.2. Sampling At 0, 4, 7, 10, and 14 d.p.i. with USUV and at 4, 7, 10, 14, and 19 d.p.i. with WNV, all surviving animals were weighed and sampled (blood and feathers) as previously described [ 19 ]. Blood samples were allowed to clot overnight at 4 ◦ C. Serum and all tissue and feather samples were stored at − 80 ◦ C until analysis. Growing feathers with pulp (vascular feathers) were collected from the dead and euthanized birds and placed into 0.5 mL of EMEM medium. Samples of brain, heart, and kidney were also collected into sterile containers and frozen at − 80 ◦ C until the analysis. In addition, cerebrum, cerebellum, medulla oblongata, heart, lung, spleen, liver, kidney, caecal tonsils, and duodenum samples were collected from deceased birds during a post mortem examination and were fixed in 10% neutral buffered formalin for histopathologic examination. 2.3. Immunological and Viral Assays Neutralizing antibodies for either virus (USUV and WNV) were analyzed using a plaque reduction neutralization test (PRNT) [ 30 ]. Titers were calculated as the reciprocal of the serum dilution, and diluted by at least 1:20, which reduced the plaque formation ≥ 90% (PRNT90). Sera were considered specific when only one of the viruses was neutralized or the titers were ≥ 4 times higher for one of them. Collected sera and vascular feathers were also tested for flavivirus (USUV and WNV) infectivity by plaque assay on Vero cell culture, as previously reported [31,32]. Viruses 2021,13, 1409 4 of 15 The presence of WNV RNA was analyzed in the serum, homogenized tissue, and processed feather samples, as previously described [ 19 ], and RT-qPCR was performed using specific primers of the USUV 30non-coding region [33]. 2.4. Histopathology Formalin-fixed tissue samples were trimmed, embedded in paraffin, and processed to obtain 4 µ m sections that were stained with hematoxylin and eosin. These were independently examined by 2 investigators (U.H. and MC.C.) to determine the presence of USUV or WNV infection-associated lesions, respectively. 2.5. Immunohistochemistry (IHC) Sections of the formalin-fixed paraffin-embedded tissue samples were routinely processed for IHC using the avidin–biotin–peroxidase complex (ABC) method described by Gamino et al. [ 15 ], with some modifications. Briefly, endogenous peroxidase activity was exhausted by incubation with 0.3% hydrogen peroxide in methanol for 30 min at room temperature (RT). The sections were incubated with 0.2% proteinase K (Sigma-Aldrich, St. Louis, MO, USA) in 0.05 M Tris-buffered saline (TBS; pH 7.6) and treated in a microwave oven at 37 ◦ C for 8 min for antigen retrieval; after pretreatment, the sections were covered with 20% normal goat serum (Vector Laboratories, Burlingame, CA, USA) in 0.01 M phosphate-buffered saline (PBS) at RT for 30 min. For WNV and USUV antigen detection, a polyclonal antibody against the envelope protein E (BioReliance, Product 81–015, Rockville, MD, USA) was used in a 1:1000 dilution at 4 ◦ C overnight. Following this, the sections were incubated for 30 min at RT with biotinylated goat anti-rabbit IgG secondary Ab (Vector Laboratories, Burlingame, CA, USA) diluted 1:200 in TBS containing 10% normal goat serum. All tissue sections were finally treated with ABC complex (Vectastain ABC Elite Kit; Vector Laboratories Inc., Burlingame, CA, USA) for 1 h at RT, then rinsed in TBS and incubated in chromogen solution (NovaRED Substrate Kit; Vector Laboratories Inc., Burlingame, CA, USA). Finally, the slides were counterstained with Harris hematoxylin. Tissue sections of the magpies that tested positive for WNV by RT-qPCR served as positive controls. Negative controls included the substitution of the primary antibody by 10% normal goat serum and a negative rabbit antibody (product 81-015; BioReliance, Rockville, MD, USA), as well as tissue sections of non-infected (WNV/USUV RT-qPCR negative) magpies. To evaluate the number of immunolabeled cells on tissue sections, cell counts were carried out by three observers (M.-C.C., M.A.R., and L.M.) in 20 randomly chosen fields, of 0.2 mm 2 whenever possible, and they were blinded to the group that was being analyzed. The results were given as the number of positive cells per 0.2 mm 2 . Cellular identification was based on the morphologic features, location, and size of the cells. 2.6. Statistical Analyses Statistical analyses were performed using Graph Pad Prism for Windows, version 6 (Graph Pad Software, Inc., San Diego, CA, USA, 2005). Kaplan–Meier survival curves were analyzed by a log-rank test. Two-way analysis of variance (ANOVA) with Bonferroni’s correction for multiple comparisons was used to compare the proportion of change in body weight of the animals in the two groups throughout the experiment. An unpaired t-test was used to compare viremia and immunolabeled cells between the groups infected with the two viruses used. Statistically significant differences are indicated by asterisks (*) (p< 0.05). 3. Results None of the magpies inoculated with 5 × 10 3 PFU of USUV died (Figure 2), showed apparent signs of disease, or showed significant weight loss (Figure 3). No viremia, USUV genome, or USUV neutralizing antibodies were detected in their sera at any time point Viruses 2021,13, 1409 5 of 15 post-USUV analysis, 4, 7, 10, and 14 days post-infection. Similarly, none of the feathers collected at the same time points had USUV RNA. Viruses 2021, 13, x FOR PEER REVIEW 5 of 16 post-USUV analysis, 4, 7, 10, and 14 days post-infection. Similarly, none of the feathers collected at the same time points had USUV RNA. Figure 2. USUV exposure conferred protection against lethal WNV challenge. Survival rates in magpies infected with USUV (5 × 103 pfu/bird) or sham-inoculated (vehicle) and challenged 18 d.p.i. with 5 × 103 pfu/bird of WNV NY99. The asterisk represents a statistically significant difference between both groups (* p < 0.05). Upon WNV challenge, a significantly higher survival rate (n = 6/8, 75%, p = 0.0218) was recorded in USUV-exposed magpies than in the unexposed vehicle birds (n = 2/9, 22%) (Figure 2). Figure 2. USUV exposure conferred protection against lethal WNV challenge. Survival rates in magpies infected with USUV (5 × 10 3 pfu/bird) or sham-inoculated (vehicle) and challenged 18 d.p.i. with 5 × 10 3 pfu/bird of WNV NY99. The asterisk represents a statistically significant difference between both groups (* p< 0.05). Upon WNV challenge, a significantly higher survival rate (n = 6/8, 75%, p= 0.0218) was recorded in USUV-exposed magpies than in the unexposed vehicle birds (n = 2/9, 22%) (Figure 2). Viruses 2021, 13, x FOR PEER REVIEW 5 of 16 post-USUV analysis, 4, 7, 10, and 14 days post-infection. Similarly, none of the feathers collected at the same time points had USUV RNA. Figure 2. USUV exposure conferred protection against lethal WNV challenge. Survival rates in magpies infected with USUV (5 × 103 pfu/bird) or sham-inoculated (vehicle) and challenged 18 d.p.i. with 5 × 103 pfu/bird of WNV NY99. The asterisk represents a statistically significant difference between both groups (* p < 0.05). Upon WNV challenge, a significantly higher survival rate (n = 6/8, 75%, p = 0.0218) was recorded in USUV-exposed magpies than in the unexposed vehicle birds (n = 2/9, 22%) (Figure 2). Figure 3. Proportion of change in body weight over time of sham-inoculated and USUV-infected magpies challenged with WNV. Significant differences are marked with an asterisk. Notably, none of the surviving USUV-infected magpies challenged with WNV showed any signs of disease. In contrast, as previously reported [ 19 ], clinical signs of disease were observed in magpies infected with WNV alone that died, including lethargy, ruffled feathers, ataxia, inability to fly, and leg paralysis. WNV viremia was detected 4 days post-WNV infection in 75% (6/8) of the previously USUV-exposed birds and 78% (7/9) of the vehicle birds, respectively (Figure 4A). A ten- Viruses 2021,13, 1409 6 of 15 dency for the mean viremia to be lower in previously USUV-exposed magpies was evident but was not statistically significant. Only one animal in each group was viremic 7 days post-WNV infection. Similarly, infectious WNV was detected in the vascular feathers of birds from both groups from 4 to 10 d.p.i. (Figure 4B). WNV-specific neutralizing antibodies were recorded from 4 d.p.i. in both groups of animals until the end of the experiment (Figure 4C). These antibodies also had a neutralizing capacity against USUV but with titers that were >4 times lower than those against WNV, indicating that neutralization was due to cross-reactivity (Supplementary Figure S1). Viruses 2021, 13, x FOR PEER REVIEW 7 of 16 Figure 4. Infectious virus in sera and vascular feathers and neutralizing antibodies, developed by experimentally WNVinfected magpies. Infectious virus titers in sera (A) and vascular feathers (B) and neutralizing antibodies (C). Circles and triangles represent sham-inoculated vehicle birds and USUV-infected birds, respectively. Dotted lines represent the limit of detection of the assays. B C A Figure 4. Infectious virus in sera and vascular feathers and neutralizing antibodies, developed by experimentally WNV-infected magpies. Infectious virus titers in sera ( A ) and vascular feathers ( B ) and neutralizing antibodies ( C ). Circles and triangles represent sham-inoculated vehicle birds and USUV-infected birds, respectively. Dotted lines represent the limit of detection of the assays. Viruses 2021,13, 1409 7 of 15 No mortality was recorded in any of the seven contact magpies housed with WNVchallenged cage-mates, but four (three from the USUV and one from the vehicle group) were viremic 7 days after WNV infection of their cage-mates (Figure 5A), including the one previously infected with USUV. Two of them were still viremic three days later (day 10 p.i. of their cage-mates). Viral titers in these contact birds were lower and detected 3 days later than in their experimentally infected cage-mates. Infectious virus was also recovered from the vascular feathers of these magpies from 7 days post-challenge of their cage-mates, thus with a delay similar to that of viremia (Figure 5B). Contact-infected birds also developed specific WNV-NAbs after 10 d.p.i. of their cage-mates (Figure 5C). Thus, in all of the birds infected by contact, the variables analyzed (viremia, infectious virus in vascular feathers, and NAbs) showed a delay with respect to the experimentally infected magpies. Macroscopic lesions observed during post mortem examination of magpies that had died from WNV infection in either group included generalized congestion, especially marked in the brain; swollen kidneys; and enlarged liver and spleen. Microscopical lesions were more severe in the animals that died between 7 and 10 d.p.i., and very similar between the two previously USUV-exposed and the USUV-unexposed (vehicle) magpies, although slightly less extensive in the former. The most severely affected tissues were the spleen, liver, kidney, heart, brain, and intestine. In general, the main microscopic findings were the presence of vascular changes, such as congestion and hemorrhages, as well as inflammatory infiltrates, cellular degeneration, and necrosis. In the central nervous system, the most predominant lesions were vasculitis, gliosis, neuronal necrosis, and mild-moderate satellitosis, compatible with a moderate multifocal acute non-purulent encephalitis ( Figure 6A ). Lesions in the heart were dominated by an acute mild to moderate myocarditis characterized by the degeneration of myocardiocytes and a diffuse mild lymphohistiocytic infiltrate, as well as swelling of the endothelial cells. In the liver and kidneys, the most important lesions were an acute moderate multifocal hepatitis, acute multifocal interstitial and tubulonephritis characterized by a moderate multifocal mononuclear inflammatory infiltrate and associated multifocal coagulative necrosis in hepatocytes and renal tubular epithelial cells (Figure 6B–D). A brownish pigment (likely hemosiderin) was observed in the cytoplasm of the Kupffer cells in the liver and was also especially abundant in the spleen (Figure 6E). In addition, lymphocyte depletion, necrotic foci of lymphoid cells, and severe hemosiderosis were present in the spleen. In the duodenum, an acute mild enteritis was present that was characterized by a diffuse moderate lymphohistiocytic inflammatory infiltrate in the lamina propria and submucosa, leading to a thickening of the villi and mild to moderate crypt hyperplasia (Figure 6F). The antigen distribution in the tissues studied was assessed by IHC, and the antigen labeling in USUV-exposed magpies that died after WNV infection appeared to be considerably less intense and restricted to significantly fewer tissues than in the vehicle magpies that had succumbed to WNV infection (Chi-square, df 50, 1, z = 7.071, p< 0.0001, Figures 7 and 8). As an example, the WNV antigen was completely absent in the hearts and kidneys of USUV-exposed magpies, two of the organs highly positive in the vehicle birds (Figure 8). The duodenum was the only tissue in which WNV antigen staining was more intense in the previously USUV-exposed magpies, although the difference was not significant. Viruses 2021,13, 1409 8 of 15 Viruses 2021, 13, x FOR PEER REVIEW 8 of 16 Figure 5. WNV contact transmission. Infectious virus titers in sera (A) and vascular feathers (B), and neutralizing antibody titers (C) in WNV contact-infected magpies. Circles and triangles represent animals housed with sham-inoculated vehicle birds and USUV-infected birds, respectively. Dotted lines represent the limit of detection of the assays. A B C Figure 5. WNV contact transmission. Infectious virus titers in sera ( A ) and vascular feathers ( B ), and neutralizing antibody titers ( C ) in WNV contact-infected magpies. Circles and triangles represent animals housed with sham-inoculated vehicle birds and USUV-infected birds, respectively. Dotted lines represent the limit of detection of the assays. Viruses 2021,13, 1409 9 of 15 Viruses 2021, 13, x FOR PEER REVIEW 9 of 16 Macroscopic lesions observed during post mortem examination of magpies that had died from WNV infection in either group included generalized congestion, especially marked in the brain; swollen kidneys; and enlarged liver and spleen. Microscopical lesions were more severe in the animals that died between 7 and 10 d.p.i., and very similar between the two previously USUV-exposed and the USUV-unexposed (vehicle) magpies, although slightly less extensive in the former. The most severely affected tissues were the spleen, liver, kidney, heart, brain, and intestine. In general, the main microscopic findings were the presence of vascular changes, such as congestion and hemorrhages, as well as inflammatory infiltrates, cellular degeneration, and necrosis. In the central nervous system, the most predominant lesions were vasculitis, gliosis, neuronal necrosis, and mildmoderate satellitosis, compatible with a moderate multifocal acute non-purulent encephalitis (Figure 6A). Lesions in the heart were dominated by an acute mild to moderate myocarditis characterized by the degeneration of myocardiocytes and a diffuse mild lymphohistiocytic infiltrate, as well as swelling of the endothelial cells. In the liver and kidneys, the most important lesions were an acute moderate multifocal hepatitis, acute multifocal interstitial and tubulo-nephritis characterized by a moderate multifocal mononuclear inflammatory infiltrate and associated multifocal coagulative necrosis in hepatocytes and renal tubular epithelial cells (Figure 6B–D). A brownish pigment (likely hemosiderin) was observed in the cytoplasm of the Kupffer cells in the liver and was also especially abundant in the spleen (Figure 6E). In addition, lymphocyte depletion, necrotic foci of lymphoid cells, and severe hemosiderosis were present in the spleen. In the duodenum, an acute mild enteritis was present that was characterized by a diffuse moderate lymphohistiocytic inflammatory infiltrate in the lamina propria and submucosa, leading to a thickening of the villi and mild to moderate crypt hyperplasia (Figure 6F). Figure 6. Histopathologic lesions in magpies experimentally infected with West Nile virus. ( A ) Diffuse moderate gliosis and satellitosis (arrowheads) in cerebrum. Multifocal mononuclear inflammatory infiltrate in the liver ( B ) and kidney ( C ) (arrowheads). ( D ) Focally extensive degeneration and necrosis of myocardiocytes (arrowheads). ( E ) Massive hemosiderosis (arrowheads) and lymphocytic necrosis (arrow) in spleen. ( F ) Mononuclear inflammatory infiltrate (asterisk) in lamina propria of duodenum. Hematoxylin and eosin staining; bars = 50 µm. Viruses 2021, 13, x FOR PEER REVIEW 10 of 16 Figure 6. Histopathologic lesions in magpies experimentally infected with West Nile virus. (A) Diffuse moderate gliosis and satellitosis (arrowheads) in cerebrum. Multifocal mononuclear inflammatory infiltrate in the liver (B) and kidney (C) (arrowheads). (D) Focally extensive degeneration and necrosis of myocardiocytes (arrowheads). (E) Massive hemosiderosis (arrowheads) and lymphocytic necrosis (arrow) in spleen. (F) Mononuclear inflammatory infiltrate (asterisk) in lamina propria of duodenum. Hematoxylin and eosin staining; bars = 50 μm. The antigen distribution in the tissues studied was assessed by IHC, and the antigen labeling in USUV-exposed magpies that died after WNV infection appeared to be considerably less intense and restricted to significantly fewer tissues than in the vehicle magpies that had succumbed to WNV infection (Chi-square, df 50, 1, z = 7.071, p < 0.0001, Figures 7 and 8). As an example, the WNV antigen was completely absent in the hearts and kidneys of USUV-exposed magpies, two of the organs highly positive in the vehicle birds (Figure 8). The duodenum was the only tissue in which WNV antigen staining was more intense in the previously USUV-exposed magpies, although the difference was not significant. Figure 7. Viral antigen in cells of different tissues of fatally WNV-infected magpies. No. of antigenpositive cells/0.2 mm2 tissue section detected by immunohistochemistry using a polyclonal WNV antibody, in infected magpies from the vehicle group (n = 7) and the previously USUV-exposed, group (n = 2) that succumbed to the infection. Figure 7. Viral antigen in cells of different tissues of fatally WNV-infected magpies. No. of antigenpositive cells/0.2 mm 2 tissue section detected by immunohistochemistry using a polyclonal WNV antibody, in infected magpies from the vehicle group (n = 7) and the previously USUV-exposed, group (n = 2) that succumbed to the infection.