1

Pathogenicity and persistence of infection in red pandas (Ailurus fulgens)

C. Alex1, S. Kubiski1, L. Li2, Reza Saghedi2, R. Wack1, M. McCarthy1, E. Delwart2, P.

Pesavento1

1. University of California, Davis, Davis, California, USA (CA, SK, RW, MM, PP)

2. Blood Systems Research Institute, San Francisco, California, USA (LL, ED)

Corresponding author:

P. A. Pesavento, 4206 VM3A: Pathology, Microbiology, and Immunology, School of

Veterinary Medicine, UC Davis, 1 Shields Avenue, Davis, CA 95616, USA.

Email: [email protected] 2

ABSTRACT

Aleutian mink disease is the type species in the genus Amdoparvovirus, and in mink and other can cause either subclinical disease or fatal chronic immune stimulation and immune complex disease. The authors describe a novel amdoparvovirus in the endangered red panda ( Ailurus fulgens), discovered using viral metagenomics. The authors analyzed the prevalence, tissue distribution, and disease association by PCR, in situ hybridization, electron microscopy, and histology in a group of 6 red pandas from a single zoological collection. The study incorporates a fecal shedding survey and analysis of tissues from 4 necropsied animals over a 12-year span. The tentatively named red panda amdoparvovirus (RpAPV) was detected in the feces and/or tissues of all animals tested. At necropsy of 1 geriatric animal, infection was associated with pyogranulomatous peritonitis, pancreatitis, and myocarditis. Other animals had detectable low-level viral nucleic acid in lymph nodes and both oral and intestinal epithelium at the time of necropsy. Full-length genome sequences of RpAPV strains from 2 animals had 12% sequence divergence, demonstrating genetic diversity even among in-contact animals. RpAPV is a persistent infection in this cohort of red pandas, and has variable clinical expression.

KEYWORDS:

Ailurus fulgens; amdoparvovirus; electron microscopy; endangered species; in situ hybridization; metagenomics; red panda 3

Amdoparvovirus is one of eight genera currently recognized within the

(Cotmore SF, et al 2014 Archives Virol). The prototype virus, Aleutian mink disease virus (AMDV), was first discovered in 1952 as a fatal contaminant in vaccines for commercial mink, but has recently been reclassified as Carnivore amdoparvovirus 1 after recognition of an expansive host range, with natural or experimental infections observed in other mustelids (, badgers, weasels, otters) and many small carnivores (e.g. skunk, bobcat, dog, and cat) (Canuti et al., 2015; Canuti et al 2016;

Alexandersen 1985; Porter 1982; Mañas 2001; Fournier-Chambrillon 2004; Jepsen

2009; LaDouceur 2015; need ref for vaccine contaminant). AMDV was the sole member of the genus until recent discoveries of distinct amdoparvoviruses in the gray fox, the red fox, and the raccoon dog (Li EID 2011; Bodewes Virol J 2014; Shao XQ EID 2014).

In the mink, the most typical outcome of AMDV infection is subclinical disease, but a systemic disease syndrome characterized by chronic plasmacytosis, immune complex deposition, and progressive wasting is well-recognized (Bloom 1994). The sequelae of amdoparvovirus infection in other animals are less well characterized, but collective studies would at least indicate that, like AMDV in the mink, outcome of infection can range from innocuous to fatal. Recent studies using sensitive molecular detection methods have demonstrated that AMDV and the human parvovirus B19 are capable of long-term persistence (Santonja 2017, Molenaar-de Beck 2016, Nowalany-Kozielska

2015, Best 2005, Kaaden 1990). The variety in possible outcomes of infection is remarkable considering that these are among the smallest known , with a single- stranded DNA genome of ~2.5 kb. The significance of viral persistence by some parvoviruses is unknown. Among the confusing spectrum of suggestions from human 4 medicine include that persistence is innocuous, that they can reactivate and cause disease, and/or that reactivation of infection in cancer cells might contribute to host immunity and cancer surveillance (Santonja 2017, Molenaar-de Backer 2016,

Nowalany-Kozielska 2015, Best 2005, Kaaden 1990).

Red pandas (Ailurus fulgens) are native to central and eastern Asia, and are currently listed as endangered by the International Union for Conservation of Nature (IUCN) and as appendix 1 species according to the Convention on International Trade in

Endangered Species (CITES) (Glatston 2015). Approximately 200 red pandas are in human care in the United States, distributed among numerous zoological institutions and managed by an Association of Zoos and Aquariums (AZA) Species Survival Plan

(SSP) (Delaski 2015). Zoo placement and breeding programs have a key role in conservation efforts for the endangered red panda, part of which is maintaining vigilant surveillance for potential pathogens.

This report describes the discovery and characterization of a novel amdoparvovirus infecting a zoo cohort of red pandas. Tentatively named red panda amdoparvovirus

(RpAPV), the virus was discovered by viral metagenomics in tissues from two geriatric animals, one of which had systemic chronic inflammatory lesions, and one that died of neoplasia. To further characterize RpAPV and its association with disease, PCR testing was performed on serial fecal samples from four live red pandas that had been in contact with the index animal. Two of these in-contact animal subsequently died, and multiple tissues from the three necropsied animals (including the two in which RpAPV was discovered) were also tested by PCR and in situ hybridization (ISH). Archived tissues were also available from one red panda that originated from the same zoo and 5 died 12 years prior to the first case in this study; these were also tested by ISH. Taken together, results suggest that RpAPV has been present and ubiquitous within this group for at least ~1.5 years, and possibly much longer. Sites of persistence and shedding were determined, and infection in at least one animal correlated tightly with inflammation and tissue damage.

MATERIALS AND METHODS

Animals and sample collection

The five animals in this study were members of a cohort of red pandas (Ailurus fulgens fulgens) housed at the Sacramento Zoo. The cohort included three females and two males, ranging in age from 5 to 19 years during the time of the study. All were offspring of red pandas that had been in captivity at various zoological institutions dating back at least two generations, and all five animals were distantly related, sharing at least one common ancestor within the previous four generations. All five were acquired at least three years prior to this study and housed at the same institution. Animals 1, 2, and 3 were in direct contact, having shared enclosures and outdoor exhibit space. Animals 4 and 5 were added to the collection later and housed together in a separate enclosure.

The indoor housing allowed for potential direct or indirect contact among all five red pandas over a three-year period. Outdoor exhibit space was also exchanged between the groups at various times during their stay.

Three geriatric red pandas (cases 1, 2, and 3 – aged 19, 16, and 19 years, respectively) 6 died or were euthanized over a period of approximately 14 months between 2015 and

2016. In all three cases, complete routine necropsies were performed within 24 hours of death at the William R. Pritchard Veterinary Medical Teaching Hospital at the University of California, Davis. Tissue samples were fixed in 10% neutral buffered formalin, and select fresh tissues were stored at -80°C.

Beginning approximately eight months after the death of case 1, serial fecal samples were collected from each of the four remaining red pandas in the collection.

Opportunistic sampling spanned a period of six weeks, with samples collected from enclosure floors approximately once per week. Animals were housed individually at night, and fecal samples for each animal were collected from floors of their individual dens in the morning. Fecal samples were stored at -80°C until use. In total, between 6 and 11 fecal samples were collected for each individual.

Viral Discovery

Tissues from case 1 were pooled, frozen and thawed on dry ice thrice, approximately 2

X volume of PBS added and homogenized with a hand help rotor. After 10 min spin in a table-top microfuge (15000 g), supernatant (400 µl) was collected and filtered through a

0.45 µm filter (Millipore) to remove eukaryotic and bacterial cell-sized particles. The filtrates were treated with a mixture of DNases [Turbo DNase (Ambion), Baseline-ZERO

(Epicentre), benzonase (Novagen)] and RNase (Fermentas) at 37 °C for 90 min

(Victoria et al., 2009) to enrich for viral capsid-protected nucleic acids. Nucleic acids were then extracted using magnetic beads of the MagMAX Viral RNA Isolation kit 7

(Ambion) according to the manufacturer’s instructions. An Illumina MiSeq library was constructed using random RT-PCR followed by use of the Illumina Nextera kit and sequenced on an Ilumina MiSeq platform using 250 bases paired end. Sequence reads were de novo assembled using the Ensemble program and contigs and singletons then translated into hypothetical protein sequences, which were compared to all viral proteins in the NCBI virus RefSeq database using BLASTx.

RpAPV specific PCR

A conventional PCR assay was developed to screen feces from cases 2-5. The target was a 154-bp-sequence from the non-structural gene (NS-1) of the RpAPV genome.

The 25 μL reactions contained HotStarTaq Master Mix (Qiagen), 0.5 μM forward primer

RPAmdoNS_F2 (5’- CGCCAAAACCAACCGACCAA-3’) and reverse primer

RPAmdoNS_R2 (5’- AACACGCCCTTAGCTGTGCTT-3’) (Integrated DNA

Technologies), and approximately 100 ng of purified DNA. The reaction took place in an

MJ Research PCT-200 thermal cycler with an initial denaturation step at 95°C for 10 min, followed by 40 cycles of denaturation at 94°C for 30 seconds, annealing at 54°C for

1 minute, and elongation at 72°C for 30 seconds, with a final elongation step at 72°C for

10 min. Amplicons were evaluated by 2% agarose gel electrophoresis, and confirmed by Sanger sequencing (DNA sequencing facility, UC Davis).

Histopathology 8

Tissue samples from cases 1-3 were collected at necropsy, immersion-fixed in 10% neutral buffered formalin (pH 7.2) for at least 24 hours, then processed using standard histologic techniques. 4-µm-thick sections were cut and stained with hematoxylin and eosin for histologic, histochemical, and immunohistochemical evaluation.

In situ Hybridization

In conjunction with Advanced Cell Diagnostics (ACD) we designed an ISH diagnostic test comprised of thirty probe pairs (20-25 oligomer probes within VP1) that would hybridize to the capsid portion of the RpAPV-CA1 genome (ACD catalog No. 453991,

V-RPAmdoV-VP-custom). The ISH test was designed to recognize RpAPV DNA

(genome) and/or VP1 transcript. Chromogenic in situ hybridization was carried out according to the manufacturer’s instructions for nucleic acid detection (RNAscope 2.0

Brown Kit). FFPE slides were baked at 60°C for 1 hour, then deparaffinized with exposure to xylene twice, 10 minutes each time, followed by stirring in 100% ethanol twice and air-drying, then rehydration with dH2O for 2 minutes. “Pretreatment solution

1” was applied to the slides for 10 minutes at room temperature. The slides were then boiled in “pretreatment solution 2”, at 100°C for 15 minutes, followed by protease digestion in “pretreatment solution 3” for 30 minutes at 40°C to allow target accessibility.

Specific or control probes were applied and the slides were incubated at 40°C for 2 hours. Slides were washed twice with 1X wash buffer for 2 min at room temperature. A series of six signal amplification steps were performed per manufacturer’s instructions.

Incubation with DAB solution was performed at room temperature and the reaction was quenched with dH2O. Gill’s Hematoxylin was applied for 2 minutes, and slides were 9 then rinsed in water, counterstain and then water. Slides were passed through 100% ethanol, 70% ethanol, and twice in xylene before coverslipping with xylene-based mounting medium.

RESULTS

Clinical cases

Case 1 had systemic pyogranulomatous inflammation in multiple tissues, and, tests designed to rule in/out causative bacteria that performed on fresh or fixed tissues included Gomori Methenamine silver, Warthin-Starry silver, Periodic Acid Schiff, Brown and Brenn modified Gram stain, Giemsa, and Ziehl-Neelsen acid-fast stains, and immunohistochemistry for virus, canine coronavirus, and West Nile virus, all with negative results. Case 2 was euthanized for age- and cancer-related deterioration. Significant lesions included hepatic necrosis, intestinal and biliary carcinomas, and chronic cholangiohepatitis. Testing in this case included Periodic Acid

Schiff, Brown and Brenn modified Gram stain, Giemsa, Gomori Methenamine silver, and Fites acid-fast stains on various tissues, all with negative results. Cases 1 and 2 were submitted for (separate) viral metagenomic analyses.

Virus discovery

For case 1, colon, small intestine, spleen, kidney, and liver tissues were pooled and processed for viral metagenomics (Methods). 1.98 million reads were generated. 1.66%

(n=32946) of the total reads showed BLASTx E score <10-5 to amdoparvovirus proteins.

The entire protein coding sequence of the parvovirus genome was assembled and 10 deposited in GenBank (NC_031751.1) as red panda amdoparvovirus strain CA1

(RpADV-CA1). Also detected were 617 reads (0.033%) related to polyomaviruses. After using PCR and Sanger sequencing to fill gaps the complete polyomavirus genome could be assembled which was deposited in GenBank (KT878838.1) as red panda polyomavirus strain CA1 (RpPyV-CA1). In case 1, a low number of reads were also identified with closest similarity to anelloviruses (n=5) and papillomaviruses (n=6).

Feces and a tissue pool (lung, liver, tonsil) from case 2 were also analyzed separately by viral metagenomics. A small percentage of the reads derived from feces (n=24700 out of 12297030 or 0.02%) and a significant percentage from the tissue pool (n=524997 out of 8658526 or 6%) originated from RpAPV. Reads were assembled into a separate full coding sequence parvovirus genome (RpADV-CA2, KY564173). RpADV-CA2 is

93% identical at a nucleotide level to RpADV-CA1. No other viral sequences were detected in this case.

Phylogenetic analyses were performed to classify the red panda amdoparvovirus (Figs

1 and 2). The current criteria for the family Parvoviridae proposed by the International

Committee on the Taxonomy of Viruses (ICTV) requires that the NS1 of distinct virus species in the same Parvoviridae genus should be <85% identical at the amino acid sequence level (Cotmore 2014). It is important to note that the two full length RPAPVs reported here were 12% divergent at the NS1???? These animals were in-contact animals that died approximately 1 year apart. Skunks infected with variants of AMDV can also show a high degree of sequence diversity reaching as high as xx% between

AMDV NS1 sequences.

The taxonomy was revised in 2016 to accommodate a rapidly growing 11

number of newly discovered viruses in diverse animal species (1, 2). A new criterion for

creation of novel polyomavirus species is based on >15% difference in sequence

identity of the non-structural T antigen (T Ag) coding sequence compared to the most

closely related species. The red panda polyomavirus LT protein is closest to that of Mus musculus polyomavirus 2 (ALN69895.1) with 65% identity indicating that the RpPyV is the first representative of a new species in the Betapolyomavirus genus.

Detection of RpAPV in in-contact red pandas

A primer set was designed to amplify a 154 bp segment of the RpAPV NS1 gene that was 100% conserved between the CA1 and 2 isolates. To establish whether other pandas in the cohort were infected with RpAPV, serial fecal samples were collected from all four in-contact red pandas (cases 2-5) over a 6-week period, beginning eight months after the death of case 1. Two of these animals subsequently died (cases 2 and

3). The collection period ended four weeks prior to the death of case 2 and four months prior to the death of case 3. All four in-contact animals had detectable virus in their feces. In three of the four cases (cases 3-5), all fecal samples were positive. In case 2, virus was detected in all but two fecal samples (Supplemental table S1 – fecal PCR results; Supplement figure S1 – gel).

The same NS1 primer set was used to analyze tissue distribution of the virus in select frozen tissues of necropsied animals (cases 1-3). Size- and sequence-specific amplicons were detected in the spleen, liver, lung, small intestine, and kidney of case 1, in kidney, spleen, liver, lung of case 2, and kidney, liver, lung, and a mesenteric lymph node of case 3 (Supplemental Table S2 – PCR and ISH results table). 12

Histopathology and In-situ Hybridization

Three geriatric red pandas (cases 1-3) died or were euthanized within an approximately

1 year period of time. Causes of death or euthanasia were distinct in all three cases.

Complete histologic examinations were performed for all three animals, and full sets of tissues were probed by ISH designed to detect VP1 of RpAPV. Because RpAPV was tightly associated with lesions in case 1, a full histologic and ISH description is provided here.

In case 1, there were scattered aggregates and perivascular cuffs of disorganized lymphocytes, plasma cells, and histiocytes within the mesentery and under serosal surfaces of the urinary bladder and intestines (FIG 3a). In the intestines, inflammation multifocally extended into the longitudinal muscularis, where it was associated with degeneration and necrosis of individual myocytes or groups of myocytes. In the pancreas, inflammation was most dense around vessels of the interlobular septae, but multifocally extended into the parenchyma to surround and separate exocrine glands, with occasional necrosis of the affected glands (FIG3a). Similar chronic inflammation was scattered throughout the myocardium, with associated cardiomyocyte degeneration and necrosis. An enlarged mesenteric lymph node contained large aggregates of macrophages that regionally expanded the subcapsular sinuses and extended into the adjacent cortex (FIG4a). RpAPV-CA1 nucleic acid (punctate cytoplasmic and nuclear staining) was consistently detected by ISH within all regions of inflammation [FIG3a-c

(intestine, pancreas) and FIG4a-c (mesenteric lymph node)]. The hybridization was 13 interpreted to be in macrophages based on the cell size, shape, and location, but specific cell identity could not be assigned definitively.

For cases 2 and 3, pathology results are summarized in Supplemental table S3. Major histologic findings in case 2 included carcinoma (liver, small intestine) and chronic cholangiohepatitis. Case 3 died naturally with a variety of gross and histologic changes attributable to advanced age, with no definitive cause of death identified.

In case 2 sections of tongue and pharynx were available. There were segmental regions of individual cell apoptosis in the basal and spinous epithelial layers, associated with minimal or no inflammation. Rare individual epithelial cell nuclei had peripheralized chromatin with central, variably distinct, eosinophilic intranuclear inclusions [FIGS 5a

(tongue), 6a (pharynx)]. By in situ hybridization there was dense probe hybridization that localized to the inclusion-laden and apoptotic cells (FIGS 5b, 6b). Moderate numbers of cells in the jejunal carcinoma also exhibited dense hybridization in the submucosa (FIG

7).

As in case 1, there was positive hybridization in presumed dendritic cells in many germinal centers of the spleen and lymph nodes, with approximately 75% of germinal centers in examined sections displaying at least small amounts of hybridization (FIG 8).

In case 3, hybridization was seen only in rare scattered individual cells in the spleen, but sections of mesenteric lymph nodes showed positive probe hybridization in germinal centers.

In all three cases, sections of intestines also had scattered, individual ISH-positive cells in the lamina propria and submucosa (Supplemental FIG S2). These were most 14 frequent in sections of small intestine, but were rarely identified in the stomach and colon.

Archived tissues of another red panda from the same zoo were available. This animal died approximately 12 years prior to the death of case 1, with a mural colitis that was attributed to constipation. ISH in this case demonstrated positive hybridization in few mesenteric lymph node germinal centers, but not in inflammatory lesions in the GI tract.

DISCUSSION

The recent, rapid expansion of the genus Amdoparvovirus has been driven in large part by highly sensitive metagenomics techniques, as were used in the present study. Here, the discovery of a novel amdoparvovirus is combined with diagnostic methods (ISH,

PCR) that provide information about the natural history and pathogenic potential of

RpAPV in a zoo cohort of red pandas. The combined results, which include a 6-week fecal shedding survey and one 7-year old retrospective sample, demonstrate that

RpAPV is persistent and ubiquitous in this group. Among three red pandas that died during this study, RpAPV was present in lymphoid tissues, on mucosal gastrointestinal surfaces (presumed site of shedding) in two cases???, and in one case distribution overlapped directly with regions of inflammation, and infection was considered a significant factor in the animal’s death.

Amdoparvoviruses appear to be relatively widely distributed in small carnivore species, and undergo frequent species jumps among carnivores (AMDV in different species?).

The predicted evolutionary mutation rate for parvoviruses has been calculated, however 15 evolutionary pressure like a host species “jump,” may accelerate it so true evolutionary rate is difficult to predict. Recombination among parvoviruses is also rampant. Within the expanding Amdoparvovirus genus, there is remarkable sequence diversity. NS-1 protein identity comparisons among the mink isolates show up to16% divergence. By comparisons of either NS1 alone or the whole genomes of RpAPV CA1 and 2, which were derived from the same cohort of animals approximately a year apart, there is a

~12% divergence in sequence identity indicating that these two infections likely occurred from different sources. The closest genetic relatives of RpAPV at the whole genome level??? (skunk, Raccoon dog, Mink isolates) are between 11 and 17% divergent. Viral transfer from carnivorous mesopredators near the zoo is a reasonable source of amdoparvovirus infection. Further surveillance for RpAPV in other collections, and for regional amdoparvoviruses in free ranging mesopredator carnivore species, including sequence analyses would help to clarify the dynamics of parvoviral transmission among red pandas and other carnivores. The question of natural host ranges for RpAPVs is perhaps the most critical consideration in elucidating its pathogenic potential and conservation implications.

Evidence that this virus has successfully adapted to the red panda host arises directly from those animals that were infected without related clinical sequelae. PCR results from serial fecal samples across the healthy cohort demonstrated detectable shedding of viral nucleic acid from all in-contact red pandas. Moreover, the presence of virus in lymphoid tissue (lymph node and spleen) of all three necropsied animals suggests some degree of immunologic burden attributable to the virus. It is assumed in cases 2 and 3 that viral infection was incidental to the cause of death, but the combination of 16 histologic lesions and in situ hybridization would argue that viral replication was very active. In case 2, there were inclusions and viral nucleic acid in oral and intestinal mucosal cells, especially in the epithelium of the tongue, where there were a high number of infected cells seen by ISH along with apoptosis/cytolysis of individual and clusters of cells in the mucosa. In this same animal, an intestinal carcinoma contained many cells that had detectable viral nucleic acid. There is a sensible correlation of parvoviruses with infection of a rapidly dividing cell population, such as the neoplastic cells and inflammatory cells in this region, however the significance of that association here is entirely speculative. In human medicine, parvoviruses have alternately been proposed to contribute to cancer or to be an innate “screen” by cytolysis or clearance of neoplastic cells.

The association of RpAPV with fatal inflammatory lesions in case 1, in the face of evidence that in other animals this can be a clinically undetectable persistent infection is an enigma. The association is persuasive, since multiple tissues with inflammation had abundant virus present within lesions. The important histologic/ISH distinction between this case and the others is that in case 1 virus was present on serosal surfaces, within tissues (heart, pancreas, intestine) where it was central in regions of inflammation, within extracellular karyorrhectic debris, and in both the cytoplasm and nuclei of inflammatory cells. The localization in other cases was limited to the dendritic cells

(germinal centers) or epithelial and/or inflammatory cells of the gastrointestinal tract. All animals that died were geriatric. It is possible that the immunosuppression that accompanies old age, or potentially co-infections, were somehow unique in case 1. For example, the Red panda polyomavirus seen in case 1 was present within scattered 17 kidney tubular epithelium (data not shown). While the polyomavirus in this case was considered incidental to the cause of death because it was not overlapping in its distribution with RpAPV, its presence is consistent with viral reactivation post- immunosuppression.

The identification of a novel and potentially pathogenic amdoparvovirus in red pandas could have implications for conservation and captive management of the species. Initial studies have allowed some preliminary conclusions to be drawn regarding the nature and consequences of RpAPV infection.

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Figures 22

Figures 1-2. Phylogenetic analyses. 1. Predicted amino acid alignment of NS-1. 2.

Alignment of the whole genome sequences, demonstrating closest phylogenetic neighbors for RpAPV. Sequence comparisons were performed using the Clustal

Omega(1) plugin of Geneious version 9.1.8(2). Phylogenetic reconstruction of maximum likelihood (ML) trees was performed in Geneious using PhyML(3) plugin with

100 bootstrap replicates. (Sievers 2011, Kearse 2012, Markowitz 2012)

Figure 3. RpAPV infection, duodenum and pancreas, red panda, case 1. There is lymphoplasmacytic and histiocytic subserosal inflammation in the duodenum, with regional extension into the outer tunica muscularis, and a similar inflammatory infiltrate in the pancreas. Punctate ISH staining demonstrates RpAPV genome in infiltrating inflammatory cells. 3a – H&E; 3b – ISH; 3c – Control.

Figure 4. RpAPV infection, mesenteric lymph node, red panda, case 1. A dense aggregate of macrophages (CD204-positive, not shown) extends from the subcapsular sinus, regionally displacing or replacing the adjacent cortical architecture. Punctate ISH staining demonstrates RpAPV genome in the cytoplasm of infiltrating macrophages. 4a

– H&E; 4b – ISH; 4c – Control.

Figure 5. RpAPV infection, tongue, red panda, case 2. Multifocal, segmental regions of individual or clustered apoptotic/cytolytic cells in the lingual mucosa are associated with minimal inflammation. ISH demonstrates dense hybridization in the affected regions, and stong, specific staining in cytolytic or apoptotic cells. 5a – H&E; 5b – ISH.

Figure 6. RpAPV infection, pharyngeal mucosa, red panda, case 2. There are scattered, individual apoptotic/cytolytic cells in the mucosa, most prominently in the 23

basal and spinous layers. Several cells have nuclei with peripheralized chromatin and

central, eosinophilic inclusion bodies (arrows). 6a – H&E; 6b – ISH.

Figure 7. RpAPV infection and jejunal carcinoma, jejunum, red panda, case 2. There

are moderate numbers of strongly ISH-positive cells associated with the neoplasm,

most prominently in the lamina propria but possibly also involving the epithelial

population. 7a – H&E; 7b – ISH.

Figure 8. RpAPV infection, spleen, red panda, case 2. There is strong, positive ISH

hybridization in most lymphoid germinal centers. 8a – H&E; 8b – ISH.

Supplements

Figure S1. PCR gel

Figure S2. RpAPV infection, intestine, red panda, case 2. There are rare, scattered, individual, ISH-positive cells in the lamina propria and occasionally in the submucosa.

S2a – H&E; S2b – ISH

Table S1. Fecal PCR results

Table S2. Tissue PCR and ISH results

Table S3. Summary of histopathology findings