Abstract
Polyomaviruses (PyVs) infect a wide range of animals and provoke wasting diseases, particularly in immunosuppressed hosts. Recently, a novel Rattus norvegicus polyomavirus 2 (RatPyV2) has been identified in a colony of X-linked severe combined immunodeficiency (X-SCID) rats in the United States. Here, we describe the first report of the RatPyV2 infection in an X-SCID rat colony in Japan. The affected rats exhibited adult-onset wasting. Histologically, we observed large basophilic intranuclear inclusion bodies within the hyperplastic or dysplastic epithelial cells in the salivary glands, Harderian glands, extraorbital lacrimal glands, and in respiratory and reproductive tissues. Among these organs, the parotid salivary, Harderian, and extraorbital lacrimal glands were most obviously affected. In particular, the parotid salivary glands were the most severely and diffusely affected and atrophic lesions were prominent even at 1 month of age, which suggested that the parotid salivary glands would be highly susceptible to RatPyV2 in X-SCID rats. RatPyV2 inclusion bodies were also detected in the tail of the epididymis and deferent duct. Such reproductive lesions developed significantly in the later stage of breeding age, and therefore may be associated with the reduced fecundity observed in the infected X-SCID rats. We also established a simple, rapid, and non-invasive diagnostic method based on the Amp-FTA method, using buccal swabs for the detection of RatPyV2 in immunodeficient rats. Our findings contribute to the early detection and diagnosis of RatPyV2 infections.
Keywords: Amp-FTA, intranuclear inclusion body, Rattus norvegicus polyomavirus 2, salivary gland, X-SCID rat
Polyomaviruses are a family of non-enveloped icosahedral DNA viruses that infect a variety of mammals, birds, marine fish, and insects. Most mammalian polyomaviruses cause latent subclinical infections [3, 10]. In humans, some immunosuppressed patients develop polyomavirus-associated diseases such as nephritis (BK virus), progressive multifocal leukoencephalopathy (JC virus), pneumonia (Washington University virus; WUPyV), and Merkel cell carcinoma (Merkel cell polyomavirus) [3, 5]. In laboratory mice, polyomaviruses cause tumor formation (Murine polyomavirus) and pneumonia (Murine pneumotropic virus) in immunodeficient mice or experimentally-inoculated newborn mice [5, 9, 13]. The prevalence of polyomavirus infection is considered to be quite low in contemporary mouse colonies [13].
In rats, Rattus norvegicus polyomavirus 1 (RnorPyV1), detected in feral Norway rats, was genetically characterized for the first time in 2015, although the infected rats were asymptomatic [6, 10]. Polyomavirus-associated diseases in rats were reported in athymic nude (NIH-Foxn1rnu) rats [7, 14, 16]. Infected nude rats developed a wasting disease with sialadenitis and/or pneumonia. Intranuclear inclusion bodies were present within the ductal and glandular epithelium in the salivary, Harderian, and laryngeal glands, and bronchi. Based on virus morphology and its immunoreactivity to the antibody to simian virus 40 (SV40), belonging to the Betapolyomaviriade genus [4], this virus was determined to be a polyomavirus [16]. Recently, a novel Rattus norvegicus polyomavirus 2 (RatPyV2) has been identified in a colony of X-linked severe combined immunodeficiency (X-SCID) rats lacking the interleukin-2 receptor gamma (Il2rg) gene in the United States [15]. Infected X-SCID rats showed adult-onset wasting, decreased fecundity, and/or dyspnea, and histopathological examination revealed inflammation, epithelial hyperplasia/dysplasia, and intranuclear inclusion bodies in multiple epithelial sites including the salivary glands, Harderian glands, and respiratory and reproductive tissues.
In 2015, a 6.5-month-old female X-SCID rat from a rat colony of Kyoto University exhibited severe dyspnea and wasting. This rat was euthanized, and its lungs were collected for histopathology and microbial examination for Pneumocystis carinii (P. carinii). The affected rat was negative for P. carinii in pathological, serological, and PCR examination. However, we detected large basophilic intranuclear inclusions in the lung epithelium, suggesting polyomavirus infection.
In the present study, we conducted further necropsy examination of both premature and aged X-SCID rats to reveal detailed pathology and highly susceptible target tissues. We also established a simple and rapid diagnostic method to detect RatPyV2 using buccal swab samples in immunodeficient rat strains.
MATERIALS AND METHODS
Animals
X-SCID and F344/NSlc (Japan SLC, Inc., Shizuoka, Japan) rats were maintained at the Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University. All animal experiments were approved by the Animal Research Committees of Kyoto University (MedKyo 17536) and were conducted according to the regulations on animal experimentation of Kyoto University.
Histopathology and transmission electron microscopy (TEM)
Rats were euthanized under isoflurane anesthesia. Tissues collected from 1- to 8.5-month-old X-SCID rats (n=23) were fixed in 10% neutral buffered formalin, and embedded in paraffin. The following tissues were sampled: liver, spleen, kidneys, heart, lungs, thymus, salivary glands, exorbital lacrimal glands, Harderian glands, testes, deferent ducts, epididymis, prostate glands, uterus, ovaries, and oviducts. Four µm-thick sections were cut and stained with hematoxylin and eosin (HE). Formalin-fixed tissues were also stored in 2.5% glutaraldehyde in 0.1 M phosphate buffer (PB; pH 7.4), post-fixed with 1% osmic tetraoxide at 4°C overnight, and embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate, and examined in a Hitachi H-7500 electron microscope (Hitachi, Tokyo, Japan).
Immunohistochemistry
For immunohistochemistry, paraffin-embedded tissue sections were dewaxed and pretreated in a microwave with Tris-EDTA buffer (pH 9.0) for 15 min. Sections were then incubated for 1 hr at room temperature with mouse anti-SV40 T antigen antibody (clone PAb416, 1:1,500; Millipore, Billerica, MA, U.S.A.). Horseradish peroxidase–conjugated polymer (Histofine Simple Stain MAX PO; Nichirei, Tokyo, Japan) was used as a secondary antibody. Signals were visualized with 3,3′-diaminobenzidine (DAB Substrate Kit; Nichirei).
PCR for RatPyV2 and sequence analysis
Genomic DNA was extracted from formalin-fixed paraffin-embedded (FFPE) sections (lung and salivary gland tissues) and feces, using NucleoSpin DNA FFPE XS and NucleoSpin DNA Stool (MACHEREY-NAGEL, Düren, Germany). PCR reactions (35 to 37 cycles) using primers for the β-actin and RatPyV2 virus protein 1 (VP1) were performed as described previously [15]. Sequencing of a larger region of VP1 (primer pair F14-R7, 432-bp [15]) was performed by Macrogen Japan Corp. (Kyoto, Japan).
For buccal swabs, direct PCR amplification by the Amp-FTA method was conducted [12]. Briefly, buccal swabs were collected with 4N6 FLOQSwabs (Copan, Brescia, Italy) and immediately smeared on the Whatman Indicating FTA Card (GE Healthcare, Little Chalfont, U.K.). Discs (1.5 mm diameter) were punched out from the FTA card by a standard ear punch and were directly used as templates (one disc for each reaction). PCR reactions (40 cycles) using primers for β-actin and RatPyV2 VP1 [15] were performed in Ampdirect Plus buffer (Shimadzu Corp., Kyoto, Japan). As a negative control, we used immunocompetent F344/NSlc strains that were negative for RatPyV2 serological assay, as conducted by IDEXX BioResearch (Columbia, MO, U.S.A.).
RESULTS
Clinical symptoms of RatPyV2 infected X-SCID rats
X-SCID rats exhibited adult-onset wasting and reduced fecundity, and affected rats showed high mortality after 6 months of age. Clinical symptoms included dyspnea, emaciation, low fecundity, and chromodacryorrhea that ranged from mild to severe (Supplemental Table 1). Although the fertility rate was 85.7% and the average number of pups per litter was 8.94, female X-SCID rats rarely had second or third litters, which indicated that the fecundity rate seemed to decrease after 3 or 4 months of age.
Pathology of RatPyV2 infection
At necropsy, mild to severe atrophy of the salivary glands (the parotid, submandibular, and sublingual glands), Harderian glands, and exorbital lacrimal glands was observed. The parotid salivary glands and Harderian glands were severely affected even at 1 to 2 months of age. In aged X-SCID rats with respiratory distress, lungs were mottled red or pink and showed varying degrees of red consolidation.
Histopathologically, multiple organs were affected; the salivary glands, Harderian glands, extraorbital lacrimal glands, and the respiratory system as well as the reproductive organs (Table 1). The most characteristic finding was basophilic or amphophilic large intranuclear inclusion bodies within the hyperplastic or dysplastic epithelial cells (Fig. 1). Inclusion bodies showed various appearances; dense homogeneous, glassy, or pale granular, with or without a halo. Infected epithelial cells could be detected by immunohistochemistry for anti-SV40 (PAb416) antibody as previously reported (Figs. 1G and 2B) [2, 15].
Table 1. Histopathologic lesions in RatPyV2 infected rats in this study.
| Age and number of rats (M: male, F: female) |
1 month of age | 2–3 months of age | 4–5 months of age | >6 months of age | |
|---|---|---|---|---|---|
| 3 (M:2, F:1) | 9 (M:7, F:2) | 6 (M:4, F:2) | 7 (M:3, F:4) | ||
| Parotid salivary glands | Intranuclear inclusion bodies | Presenta) | Presenta) | Presenta) | Presenta) |
| Epithelial hyperplasia and dysplasia | Diffuse, moderate | Diffuse, moderate to severe | Diffuse, severe | Diffuse, severe | |
| Glandular atrophy, loss, and necrosis | Diffuse, moderate | Diffuse, moderate to severe | Diffuse, severe | Diffuse, severe | |
| Mononuclear cell infiltration | Diffuse, moderate to severe | Diffuse, moderate to severe | Diffuse, moderate to severe | Diffuse, moderate to severe | |
| Fibrosis | Mild | Diffuse, moderate | Diffuse, moderate to severe | Diffuse, severe | |
| Submandibular glands | Intranuclear inclusion bodies | ― | Presenta) | Presenta) | Presenta) |
| Epithelial hyperplasia and dysplasia | ― | Multi-focal, mild | Multi-focal to locally extensive, moderate | Multi-focal to locally extensive, moderate | |
| Glandular atrophy, loss, and necrosis | ― | Multi-focal, mild | Multi-focal to locally extensive, moderate | Multi-focal to locally extensive, moderate | |
| Mononuclear cell infiltration | ― | Multi-focal, mild | Multi-focal to locally extensive, moderate | Multi-focal to locally extensive, moderate | |
| Fibrosis | ― | ― | Mild | Mild | |
| Sublingual glands | Intranuclear inclusion bodies | ― | Presenta) | Presenta) | Presenta) |
| Epithelial hyperplasia and dysplasia | ― | Focal, mild | Focal to multi-focal, mild | Focal to multi-focal, mild | |
| Glandular atrophy and loss | ― | ― | ― | ― | |
| Mononuclear cell infiltration | ― | ― | Focal to multi-focal, mild | Focal to multi-focal, mild | |
| Harderian glands | Intranuclear inclusion bodies | ― | Presenta) | Presenta) | Presenta) |
| Epithelial hyperplasia and dysplasia | ― | Multi-focal, moderate | Diffuse, moderate to severe | Diffuse, severe | |
| Glandular atrophy, loss, and necrosis | ― | Multi-focal, moderate | Diffuse, moderate to severe | Diffuse, severe | |
| Mononuclear cell infiltration | ― | Multi-focal, moderate | Diffuse, moderate to severe | Diffuse, moderate to severe | |
| Fibrosis | ― | Mild | Diffuse, moderate | Diffuse, severe | |
| Extraorbital lacrimal glands | Intranuclear inclusion bodies | ―a) | Presenta) | Presenta) | Presenta) |
| Mononuclear cell infiltration | Focal, mild to moderate | Focal to multi-focal, moderate | Multi-focal, moderate | Multi-focal, moderate to severe | |
| Glandular atrophy and loss | ― | Focal to multi-focal, mild to moderate | Multi-focal, moderate | Diffuse, moderate to severe | |
| Karyomegaly | ― | Presenta) | Presenta) | Presenta) | |
| Lung | Bronchiolar intranuclear inclusion bodies | Presenta) | Presenta) | Presenta) | Presenta) |
| Alveolar intranuclear inclusion bodies | ― | ― | ― | Presenta) | |
| Bronchiolar hyperplasia | Mild to moderate | Moderate | Moderate | Moderate | |
| Interstitial pneumonia | Mild to moderate | Mild to moderate | Moderate | Severe | |
| Prostate glands | Intranuclear inclusion bodies | ― | ― | Presenta) | Presenta) |
| Epithelial hyperplasia and dysplasia | ― | ― | Moderate | Severe | |
| Glandular atrophy and degeneration | ― | ― | Mild to moderate | Severe | |
| Deferent ducts | Intranuclear inclusion bodies | ― | ― | ― | Presenta) |
| Epididymis | Intranuclear inclusion bodies | ― | ― | ― | Presenta) |
| Karyomegaly | ― | ― | ― | Presenta) | |
| Uterus | Intranuclear inclusion bodies | ― | ― | ― | Presenta) |
| Epithelial hyperplasia and dysplasia | ― | ― | Mild to moderate | Moderate to severe | |
| Epithelial necrosis | ― | ― | ― | Moderate | |
―: Lesions were not detected. a) SV40 positive cells were detected by immunohistochemistry.
Fig. 1.
Histopathology of affected organs of X-SCID rats. Intranuclear inclusion bodies with (black arrowheads) or without (yellow arrowheads) a halo are evident. (A) Parotid salivary gland, 1 month of age; (B) Parotid salivary gland, 7 months of age; (C) Harderian gland, 2.5 months of age; (D) Harderian gland, 7 months of age; (E) Lung, 1 month of age; and (F) Pulmonary alveoli, 7 months of age. (G) Immunohistochemistry for SV40 T antigen (PAb416). Parotid salivary gland, 1 month of age. (H) Transmission electron microscopy image of lung. Numerous virus particles in the bronchial epithelial cell. The virus particles show icosahedral shape and are 40 to 50 nm in diameter. Bar=50 µm (A–C and E–G), 100 µm (D), and 100 nm (H).
Fig. 2.
Pathological analyses of the epididymis. (A) Histopathology of the 7-month-old-X-SCID rat. Intranuclear inclusion bodies (yellow arrowheads) and large atypical nuclei (arrows) are observed. (B) Immunohistochemistry for SV40 T antigen on the epididymis of a 7-month-old X-SCID rat. Intranuclear inclusion bodies are immunopositive for SV40 T antigen. Bar=50 µm.
In the salivary, Harderian, and extraorbital lacrimal glands, we observed severe loss, degeneration, or necrosis of acinar and ductal epithelia with mononuclear cell infiltration (Fig. 1A–D). The remaining epithelial components were hyperplastic, dysplastic, or disorganized. In advanced lesions, tissues were severely atrophied with areas of interstitial fibrosis and inflammation (Fig. 1B and 1D). There were many intranuclear inclusion bodies with halos (Fig. 1; black arrowheads) as well as full-type inclusion bodies in both acinar and ductal epithelial cells (Fig. 1; yellow arrowheads).
Among salivary glands, parotid salivary glands were the most severely and diffusely affected (Fig. 1Aand 1B). Diffuse atrophic lesions were prominent from at least 1 month of age (Fig. 1A). In the submandibular salivary glands, moderate multifocal to locally extensive lesions appeared starting at around 3 months of age. Meanwhile, the sublingual and lingual salivary glands, which consisted of mucinous glands, were rarely affected. In these tissues, mild focal to multifocal lesions were observed starting at 7 months of age (Table 1).
In the Harderian glands, epithelial hyperplasia and dysplasia, and glandular atrophy were observed from at least 2.5 months of age (Fig. 1C). After 6 months of age, the Harderian glands were almost completely replaced with fibrotic tissue and disorganized or dilated glands (Fig. 1D).
In the extraorbital lacrimal glands, a few SV40-positive cells were detected in focal inflammatory areas starting at 1 month of age, although intranuclear inclusion bodies were not observed. Karyomegaly appeared in inflammatory foci and glandular atrophic lesions from 2 months of age in both sexes. Focal or multi-focal lesions could be detected from around 3 months of age (Table 1).
In the lungs, bronchiolar hyperplasia with atypia and large basophilic intranuclear inclusion bodies (full type) were identified starting at 1 month of age (Fig. 1E). Inclusion bodies were mainly located at the apical side of respiratory epithelial cells. In affected aged rats, inclusion bodies as well as karyomegaly were also detected in pneumocytes (Fig. 1F; 7 months of age), and interstitial pneumonia was present. On TEM examination of lung tissue, numerous icosahedral virus particles, 40 to 50 nm in diameter, were detected in the nuclei of the bronchial epithelial cells (Fig. 1H).
The uterus and prostate gland were also affected as previously described [1, 14]. We also detected intranuclear inclusion bodies or karyomegaly in the epithelial cells of the deferent ducts and the tail of the epididymis (Fig. 2). These reproductive organs showed significant lesions from around 6 months of age, although any apparent inclusion bodies were not observed before about 4 months of age.
Detection of RatPyV2 by PCR
We detected RatPyV2 VP1 products in all DNA samples extracted from feces and FFPE tissues of X-SCID rats, but not from the immunocompetent F344 strains (Fig. 3A). The sequences of the 432-bp VP1 fragment showed a 99–100% match with those of RatPyV2 (GenBank accession No. KX574453 and KX808699). We also detected single nucleotide polymorphisms (SNPs) at nucleotide positions 2085 (A/G) and 2092 (A/C) that were identical with those previously reported by Rigatti et al. [14] (Fig. 3B). The Amp-FTA method using buccal swabs detected RatPyV2 VP1 products in X-SCID rats by this method starting at around 1 month of age (Fig. 4).
Fig. 3.
Detection of RatPyV2 DNA by PCR and sequence analysis. (A) PCR amplification from feces. RatPyV2 VP1 products (432 bp) are detected from X-SCID rats but not immunocompetent F344 rats. β-actin (191 bp) is used as an internal control. (B) Two SNPs at nucleotide position 2085 (A/G) and 2092 (A/C) are detected in the RatPyV2 VP1 region.
Fig. 4.
Detection of RatPyV2 DNA from buccal swabs by the Amp-FTA method. RatPyV2 VP1 products (432 bp) are detected even in 1-month-old X-SCID rats. β-actin (191 bp) is used as an internal control.
DISCUSSION
In this study, we confirmed that the histological lesions observed in the X-SCID rats in Kyoto University were similar to those reported previously and that the VP1 DNA sequences were identical with those found in the United States [15]. Moreover, PCR analyses revealed that all of the X-SCID rats examined (n=25) were positive for RatPyV2, which suggests that the prevalence of RatPyV2 in the X-SCID rat colony was significantly high. The X-SCID rats that were involved in the outbreak in the United States were obtained from Kyoto University in June 2015 [15]. Thus, these findings suggest that the RatPyV2-infected X-SCID rats were shipped to the U.S.A. rather than having become infected with RatPyV2 during or after shipping.
Previous pathological analyses demonstrated that epithelial hyperplasia and dysplasia of the salivary glands, Harderian glands, and lungs were characteristic in the RatPyV2-infected X-SCID rats. Glandular atrophy and loss in the salivary and Harderian glands were also characteristic [15]. These pathological characteristics were confirmed in the X-SCID rats used in this study. In addition, we found that the severity of the histological lesions varied among organs. Specifically, the parotid salivary glands and extraorbial lacrimal glands were affected as early as 1 month of age, and the Harderian gland lesions developed between 1 to 2 months of age. Therefore, we considered that the parotid salivary and extraorbital lacrimal glands were the most highly susceptible to RatPyV2 in X-SCID rats.
Intranuclear inclusion bodies were present in the salivary glands, lacrimal glands, and lung at 1 month of age, which strongly suggested that viral transmission occurred at least one week after the weaning. As intranuclear inclusion bodies were prominent in the epithelia of active mammary glands and the uterus [15], it is likely that X-SCID fetuses would be exposed to RatPyV2 in utero and that direct contact with oral, nasal, or respiratory secretions would largely contribute to RatPyV2 infection.
In laboratory rats, karyomegaly is often observed in the lacrimal gland. These lesions are predominantly found in male rats and are age-related [8]. Our histological study showed that karyomegaly was observed in both male and female rats starting at 2–3 months of age. Although it is necessary to distinguish these lesions from pseudoinclusions, it is likely that karyomegaly resulted from RatPyV2 infection.
Several PyVs have been reported to be involved in tumor development that is mediated by tumor antigens [5]. The RatPyV2 genome encodes the open reading frame for large T antigen [15]. Some PyV large T antigens, such as SV40 large T antigen, are oncoproteins that can induce neoplastic transformation in the host cell [1]. We observed no neoplastic lesions, but did observe hyperplastic and dysplastic changes with atypical or karyomegaly cells in the affected atrophic organs of the RatPyV2-infected X-SCID rats. We could not eliminate the possibility that these hyperplastic and dysplastic changes may be the result of RatPyV2 infection. Further investigation would clarify the involvement of RatPyV2 in hyperplastic and dysplastic cellular changes.
In the present study, we established a rapid, simple, and non-invasive diagnostic method based on the Amp-FTA method using buccal swabs [11]. This method could detect RatPyV2 infection in our X-SCID rat colony. FTA discs could be directly used as PCR templates and therefore the purification of DNA was not required. In addition, the buccal swabs were suitable starting materials, because the salivary glands were highly susceptible to RatPyV2. Indeed, this method allowed us to detect the RatPyV2 gene in X-SCID rats as early as 1 month of age. Thus, we recommend the Amp-FTA method using buccal swabs for the early detection of RatPyV2 in colonies of immunodeficient rats.
A serological survey for RatPyV2 among immunocompetent rats in North America revealed that 32% of samples were positive for the anti-RatPyV2 antibody, which suggested widespread RatPyV2 infection in laboratory rat populations [2]. At this time, a serological test for RatPyV2 is only available from IDEXX BioResearch in the United States. Therefore, we are planning to evaluate the utility of the Amp-FTA method using buccal swabs for detecting RatPyV2-infected immunocompetent rats.
In summary, we have been the first to detect the RatPyV2 infection in a colony of X-SCID rats in Japan. Our histological examination strongly suggested that the parotid salivary glands and extraorbital lacrimal glands were the most highly susceptible tissue to RatPyV2. The Amp-FTA method using buccal swabs is useful for the rapid antemortem diagnosis of RatPyV2 in immunodeficient rats.
Supplementary
Acknowledgments
We wish to thank the National Bio Resource Project–Rat in Japan (http://www.anim.med.kyoto-u.ac.jp/NBR/) for providing F344-Il2rgem7Kyo rats (NBRP Rat No. 0694). We are also grateful to K. Yamasaki, Y. Neoda, and K. Hagiwara for technical assistance in animal breeding and care.
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