Cutavirus Infection in Large-Plaque Parapsoriasis, a Premalignant Condition of Mycosis Fungoides

Abstract

Background

Cutavirus (CuV) is associated with mycosis fungoides; however, the CuV status in parapsoriasis en plaques (PP), a premalignant inflammatory condition of mycosis fungoides, has not been fully delineated.

Methods

Fifty-five Japanese patients with chronic inflammatory skin diseases, including 13 patients with PP, were studied.

Results

CuV DNA was detected significantly more frequently in biopsies of the lesional skin from patients with PP (38%; 4 of 13) than in those from patients with other inflammatory skin diseases (2%; 1 of 42; P = .009). All CuV-positive PP cases were of the large-plaque parapsoriasis (LPP) subtype. The viral loads ranged from 83 450 to 2 164 170 copies/10³ cells. We recovered near–full-length CuV sequences from the CuV-positive LPP biopsies, all of which were of the Japanese/Asian genotype. The CuV genome appeared to be present within lymphoid cells infiltrating the epidermis and dermis. CuV NS1 and VP1 gene transcripts were also detected in the affected tissues.

Conclusions

The detection of high levels of CuV DNA with the expression of viral mRNA suggests a potential role for CuV in the pathogenesis of LPP, making it necessary to study further the impact of CuV, especially regarding the viral genotype, on the outcomes of patients with CuV-positive LPP.

Cutavirus (CuV) is a recently discovered human parvovirus with a single-stranded linear DNA genome of approximately 4.5 kb [1–3]. Using metagenomics, this virus was discovered initially in fecal specimens from Brazilian children with diarrhea [1]. Since then, our group and others have detected CuV DNA in biopsy specimens from patients with cutaneous T-cell lymphoma, especially mycosis fungoides (MF) [1, 4–6]. Moreover, we have shown the potential impact of CuV on the unfavorable outcome of patients with MF [6]. Although these findings suggest an association between CuV and MF, the pathogenetic relevance of CuV remains to be fully delineated.

Studies have also reported the detection of CuV DNA in 9 of 237 (3.8%) and 127 of 678 (18.7%) surface skin swabs collected from healthy individuals from Germany [7] and Japan [8], respectively. These findings suggest that CuV is found in the human skin virome in healthy individuals, although the prevalence may vary according to ethnicity.

MF is more often diagnosed in men and elderly persons than in women and young individuals [9, 10]. Etiologically, MF is believed to result from chronic antigenic stimulation with long-standing inflammation responses, leading to uncontrolled clonal expansion of T-helper cells in the skin [11, 12]. Many patients with MF present a long history of previous reactive inflammatory conditions such as parapsoriasis en plaques (PP) [13]. PP is a rare chronic papulosquamous dermatosis with asymptomatic recalcitrant patches that may persist for many years. This disorder is divided into 2 subtypes: small-plaque parapsoriasis (SPP) and large-plaque parapsoriasis (LPP) [14]. SPP is considered a benign disorder with no or minimal risk of malignant transformation [14, 15], whereas LPP can progress to overt MF in up to 35% of cases [16]. Because LPP and MF overlap considerably clinically and histopathologically, most dermatologists consider the 2 conditions to be different clinical stages of the same disease [14, 17].

If CuV is associated with the development of MF as a persistent antigenic T-cell stimulator in the skin, it may be assumed that this virus is also present at high levels in the premalignant condition of MF. A study reported the absence of CuV DNA in 8 skin biopsies of French patients with PP [1]. By contrast, 8 of 12 Finnish patients with PP had CuV DNA in biopsies from their lesional and healthy skin [18]. However, the 2 studies have stemmed from patients living in Europe. A worldwide survey including an Asian population will provide additional information on the geographical distribution of CuV in this premycotic dermatosis because Japanese/Asian-specific CuV strains, which are genetically distinct from the strains detected in the skin of individuals from Europe, are prevalent in the Asian populations [8].

Based on this background, we assessed the prevalence and loads of CuV DNA in lesion biopsies and skin swabs from Japanese patients with PP. We also analyzed the CuV genotype using the near–full-length CuV genome successfully recovered from the LPP skin. Furthermore, we performed RNAscope-in situ hybridization (ISH) and viral mRNA expression analysis in the CuV DNA-positive LPP tissues. We report not only a high prevalence of the high-load CuV DNA with the Japanese/Asian genotype, specifically in patients with LPP, but also the expression of the CuV RNA transcripts.

METHODS

Patients

The study cohort included a total of 55 Japanese patients (ie, persons of Japanese descent who resided in Japan) with chronic inflammatory skin diseases (Table 1), for whom formalin-fixed, paraffin-embedded (FFPE) biopsy specimens from lesional skin had been stored under good conditions and in sufficient quantity for the evaluation of CuV status. Among them, 13 patients with PP (5 with SPP and 8 with LPP) who were diagnosed based on the dermatological examination and histopathological findings of biopsy specimens at the Kochi University Hospital from 2010 to 2021 were enrolled in this study: 8 women and 5 men; age range 50–83 years, median 70 years. One or 2 experienced pathologists analyzed the biopsy specimens from each patient. SPP and LPP were categorized according to published criteria [19]. T-cell receptor gene rearrangement, which is thought to be a marker of malignant T-cell proliferation [20], was absent in all of the PP specimens tested (Table 2). None of the patients with PP had received UV phototherapy before the biopsy.

A total of 21 skin swabs were also collected from 7 (4 with SPP and 3 with LPP) of the enrolled 13 patients with PP: 1 swab from patch/plaque and 2 from healthy skin areas (trunk and forehead skin). Swab collection was performed by rubbing an area of about 50 cm² of the skin back and forth 5–10 times using sterile cotton swabs moistened with phosphate-buffered saline.

This study was approved by the Ethics Committee of the Kochi Medical School, Kochi University. Written informed consent was obtained from all participants who were followed on an outpatient basis. In the case of archived FFPE specimens, the Institutional Review Board waived the requirement for written informed consent from patients because the study was retrospective.

Detection and Quantification of CuV DNA

DNA extraction from FFPE and swab specimens and TaqMan-based real-time quantitative polymerase chain reaction (qPCR) were performed as described in our previous studies [6, 8]. Primers and probes were prepared to amplify the gene encoding the CuV viral capsid protein 1 (VP1) and VP2 at nucleotide positions 4245–4335 based on the CuV reference sequence (GenBank accession number, NC_039050; Supplementary Table 1) [5]. To prevent cross-contamination, microtome blades were changed between each embedded block. All runs included the negative DNA extraction controls (reagent blank controls that consist of reagents used for DNA extraction but contain no tissue) and molecular biology-grade water as no-template controls. Precautions were also taken to prevent contamination in the PCR assays, as described elsewhere [8]. The negative controls exhibited no amplification in any of the assays performed in this study. RNase P was used as a control for the quality of the DNA samples, and also as a control in the PCR reactions that were used for cell-count quantification. A standard curve for the VP1/VP2 PCR product was generated, from which we calculated the viral copy number [8]. All specimens were tested twice independently in a blind manner. Specimens with cycle threshold (Ct) values <40 (the average of 2 experimental results) were considered positive for CuV DNA according to the published criteria [21, 22]. The results are expressed as viral copies/10³ cells and viral copies/μg of DNA.

RNAscope-In Situ Hybridization

Colorimetric RNA ISH was performed on 5-μm sections of FFPE biopsy tissues using the RNAscope 2.5 HD Reagent Kit-Brown (Advanced Cell Diagnostic), according to the manufacturer's instructions [23]. Briefly, sections were treated with heat and protease and then hybridized with the probe cocktail that includes 20 pairs of probes (V-CuV-VP1-VP2-C1) targeting the region of the nonstructural protein 1 (NS1) gene, the middle open reading frame (ORF), and the VP1/VP2 genes in the Japanese/Asian type of CuV strain (nucleotide positions 1189–3931). Assays were done in duplicate. Probes targeting the human peptidylprolyl isomerase B (PPIB) and the bacterial dihydrodipicolinate reductase (DapB) were included as positive and negative technical controls, respectively. Positive staining was identified as brown punctate dots [1].

Immunohistochemistry

Immunohistochemistry was performed on FFPE sections as described elsewhere [24]. The tissue sections were incubated with antibodies against human CD3, CD4, and CD20 (Nichirei Bioscience).

Reverse Transcription PCR

Total RNA was extracted from the biopsies using the RNeasy FFPE kit (QIAGEN). The aliquots were treated with DNase to avoid the amplification of viral DNA and were reverse transcribed using SuperScript IV VILO Master Mix with ezDNase Enzyme (Thermo Fisher Scientific). The cDNAs were used for the subsequent PCR amplification as described [25]. The primer sequences are listed in Supplementary Table 1. The β-actin gene (ACTB) was amplified to confirm the presence of PCR-amplifiable cDNA.

CuV Sequence Analysis and Phylogenetic Analysis

The near–full-length CuV sequences and the partial VP1/VP2 sequences were amplified using PCR with different combinations of the primer sets listed in Supplementary Table 1. The PCR products were purified and sequenced directly as described [25]. Phylogenetic trees were constructed using the maximum-likelihood method. A nucleotide identity analysis was performed using the BLAST program with default parameters [26]. The CuV sequences obtained in this study were deposited in the GenBank database under accession numbers LC772178–LC772186.

Statistical Analysis

The statistical correlations with CuV positivity rates were analyzed using Fisher exact test. Significance was set at P < .05.

RESULTS

CuV DNA Detection and Quantification

We evaluated 55 biopsies obtained from patients with chronic inflammatory skin diseases for the presence and viral loads of CuV. The CuV DNA was found in 4 of 13 biopsies from PP (31%), a detection rate significantly higher than that from other types of inflammatory skin diseases (1 of 42; 2%; P = .009; Table 1). Notably, all CuV-positive PP patients (patient numbers PP3, PP8, PP9, and PP12) were of the LPP subtype. The viral loads ranged from 83 450 to 2 164 170 copies/10³ cells and 1409 to 71 541 copies/μg (Table 2). We also evaluated 40 swabs obtained from the lesional skin of patients with inflammatory skin diseases (Table 1). The CuV DNA was detected in 1 of 7 swabs from PP (14%) and 1 of 33 swabs from other types of inflammatory skin diseases (3%), which was not statistically significantly different (P = .323). This lack of statistical significance could be attributed to the fact that, except for 1 patient (PP8, with LPP), all patients with PP enrolled in this swab study were negative for CuV DNA in their biopsy specimens. Therefore, we assumed that the CuV DNA detection rate in the PP swabs was apparently low. Of note, patient PP8 had CuV DNA in his biopsy and swab from the lesional skin, which was analyzed blinded (Table 3). The CuV DNA was also detected in his 2 swabs collected from the nonlesional skin at different sites. The viral loads in the swabs from the nonlesional skin (15 966 copies/μg from forehead skin and 1042 copies/μg from trunk skin) were much lower than in the swab from the lesional skin (6 014 259 copies/μg).

Correlation Between CuV DNA Status and Clinical Characteristics

The demographics and CuV status of each patient with PP are listed in Table 2. The patient variables were compared between the 2 groups of patients stratified according to the CuV DNA status. No statistical difference in age was observed between the CuV DNA-positive and CuV DNA-negative groups (P = .063, Mann-Whitney nonparametric U test). The CuV DNA-positive group included significantly more men than the CuV DNA-negative group (P = .007, Fisher exact test).

The mean and median follow-up periods were 100 and 106 months for patients with SPP (range, 28–158) and 47 and 43 months for patients with LPP (range, 13–80), respectively (Table 2). None progressed to manifest MF during the follow-up time, irrespective of CuV DNA status.

RNAscope-ISH and CuV mRNA Expression

FFPE biopsy tissues from 6 patients with LPP (4 CuV DNA PCR-positive and, as controls, 2 CuV DNA PCR-negative tissues) were hybridized with the CuV RNAscope-ISH probes. Positive signals were observed in 4 of 4 LPP tissues that were positive for CuV DNA, but in none of the tissues negative for CuV DNA (Table 2). The CuV-positive signals were observed in the nucleus and/or cytoplasm of affected cells localized in the dermis and epidermis (Figure 1). In some cases, the strong signals formed small clusters. The positive signals were reproducibly detected in the same regions of the 5-μm sections sliced in sequential series in 2 independent experiments (data not shown). In addition, the CuV DNA-negative tissue controls, and positive and negative technical controls gave the expected results in this assay. Immunohistochemistry revealed that CD3⁺CD4⁺CD20^– lymphocytes mainly infiltrated the dermis, and epidermotropic CD4⁺ T cells were also observed. Based on the immunohistochemical findings, the CuV-positive signals appeared to exist primarily within T cells of the infiltrate, but the presence of some CuV-infected keratinocytes could not be excluded.

We also investigated mRNA expression of the CuV NS1 and VP1 genes by reverse transcription PCR (RT-PCR) in the biopsied tissues used for RNAscope-ISH (Table 2 and Figure 2). Among the 4 CuV DNA-positive specimens, 2 (PP8 and PP12) expressed the NS1 transcript, and 3 (PP8, PP9, and PP12) expressed the VP1 transcript. The expression levels of the genes were also determined by semiquantitative RT-PCR (Supplementary Figure 1). No correlation between viral mRNA expression levels and DNA load levels was observed in this assay.

CuV Genome Sequencing and Phylogenetic Analysis

All of the qPCR products obtained in this study were confirmed as CuV sequences by sequencing (Supplementary Figure 2). In the PCR assays of the FFPE extracts from LPP biopsy, we obtained 2 near–full-length CuV sequences with 4455 bp (nucleotide positions 2–4456; sequence numbers JPN-PP9 and JPN-PP12), 1 concatenated 1573-bp sequence (2513–3025 and 3397–4456; JPN-PP8), and 1 concatenated 1234-bp sequence (2852–3025 and 3397–4456; JPN-PP3). In the PCR assays of the swab extracts from patient PP8, we obtained 3 near–full-length CuV sequences, including 1 sequence from lesional skin (JPN-PP8-SW1) and 2 from nonlesional skin (JPN-PP8-SW2 from the forehead skin and JPN-PP8-SW3 from the trunk skin). We first performed a phylogenetic study using the near–full-length CuV sequences, including our 5 sequences obtained from the biopsies and skin swabs and the previously published CuV sequences from Europe and Japan [1, 8, 27] (Figure 3A). All of our sequences belonged to a Japanese clade (designated clade 1 [8]), which was distinct from a clade comprising sequences originating from European patients (clade 2). A phylogenetic tree, including the sequences of bufavirus and gray fox amdovirus used as outgroups, is shown in Supplementary Figure 3. We analyzed nucleotide identities among these near–full-length CuV sequences and confirmed that our sequences were relatively distinct from the European sequences of clade 2 (Supplementary Table 2). We next constructed a phylogenic tree based on the 1234-bp sequences of the CuV VP1/VP2 region, which included all 4 of our CuV sequences recovered from LPP biopsies (Figure 3B). This also showed that our sequences belong in clade 1.

Sequence analysis showed that the swab sequences of JPN-PP8-SW1 and JPN-PP8-SW2 from patient PP8 were identical, and JPN-PP8-SW3 had only a single nucleotide substitution: G→A at position 218 (data not shown). We next compared the JPN-PP8 biopsy sequence with these 3 swab sequences. We found several single-nucleotide substitutions with amino acid replacements in the VP1/VP2 region of JPN-PP8 (Figure 4). This shows that the CuV DNA sequences differed between viral strains on the skin surface and strain in the lesional tissue in this patient. The sequence analysis may also suggest the presence of multiple distinct CuV strains in the LPP tissue of patient PP8 (Figure 4).

We next conducted a phylogenetic analysis based on the short 207-bp CuV VP1/VP2 fragments to compare our CuV sequences from LPP biopsies with 5 CuV sequences from PP biopsies of patients from Finland (1 from LPP [AS11_230] and 4 from SPP patients [AS2_230, AS5_583, AS7_583, and AS9_230]) [18], which were available in GenBank as of 1 July 2023 (Figure 3C). Our sequences formed a phyletic Japanese group distinct from a clade comprising the sequences originating from Finland. Unexpectedly, the LPP sequence from Finland (AS11_230) was included in clade 1. Several nucleotide mismatches were found between the CuV sequences from Japan and Finland (Supplementary Figure 4). Nucleotide identity analysis also confirmed that the sequences from Japanese patients with LPP were closer to the AS11_230 sequence of clade 1 than to the other sequences of clade 2 from the Finnish patients with SPP (Supplementary Table 3).

DISCUSSION

The present study shows a possible link between CuV and LPP. Our study had several strengths. One advantage was that our data on CuV DNA loads in LPP biopsies could be compared with our previous data on those loads in a series of MF biopsies (n = 13) [6] because the 2 studies used the same methods to measure the viral loads. There was no overlap in the patients between the studies. Intriguingly, the CuV DNA loads in the LPP specimens (83 450–2 164 170 copies/10³ cells; median, 409 096) were significantly higher than those in the MF specimens (23–3922 copies/10³ cells; median, 141 [6]; P < .001; Supplementary Figure 5). This suggests that LPP skin provides a better habitat for CuV than MF tumors.

This study also provides the first data on near-complete CuV sequences (4455 bp) successfully recovered from LPP. Phylogenetic analysis clearly showed that all CuV sequences belonged to the Asian/Japanese clade, which we previously proposed for geographically related genetic classification of CuV [8]. To date, 5 shorter CuV DNA fragments with lengths of 216–567 bp derived from PP plaques of patients from Finland have been deposited in GenBank. Only 1 Finnish LPP sequence unexpectedly belonged to the Asian/Japanese clade (Figure 3C). In this context, whether or not the Asian/Japanese CuV genotype is more closely associated with LPP than the European clade needs to be investigated further.

Another notable finding in this study was that patient PP8, who carried CuV DNA in his biopsied tissue, also had CuV DNA in his 3 swabs obtained from plaque and healthy skin. These swab CuV sequences were almost identical, even though one was from lesional skin and the others from healthy skin. However, the CuV DNA load in the lesional skin swab was much higher than the viral loads in the healthy skin swabs (Table 3). Moreover, the CuV sequence obtained from the biopsied lesional tissue had many nucleotide alterations with amino acid replacement in the VP1/VP2 genes compared with the sequences obtained from swab specimens (Figure 4). These observations may suggest that disease-associated genetic alterations occurred during this patient's disease progression of LPP. Therefore, it should also be further investigated whether the potential variant CuV antigen might increase antigenic activity, leading to the persistent inflammatory reaction in LPP tissues.

We also present here data on the localization of CuV in LPP tissues. Although we could not identify the exact infected cell type, our findings suggested that CuV appeared to be present within T cells infiltrating the dermis and epidermis. Furthermore, we detected the transcripts of the CuV NS1 and VP1 genes in the LPP tissues, suggesting an active CuV replication. Previous studies showed that NS1 of B19 parvovirus (B19V) upregulates proinflammatory cytokines, such as interleukin 6 and tumor necrosis factor-α, and it is thought that B19V NS1 plays a role in the progression of some chronic and inflammatory diseases that have been linked to B19V infections [28–30]. Similar to B19V, CuV can persist after the primary infection in the skin [2, 8]. Therefore, the significance of the CuV mRNA expression in persistent inflammation observed in LPP is worth studying.

The present study had some limitations. The main limitation of this work is that our observations were made in a small patient cohort. In addition, this study involved a retrospective case series with data collection from records of patients at a single institution. The development of MF is mostly very slow. A study showed that the median period from the first skin symptoms to the development of histologically confirmed MF was 10 years in patients with SPP and 6 years in patients with LPP [16]. In the present study, none of our patients developed manifest MF during the median follow-up time of 106 and 43 months in patients with SPP and LPP, respectively. Thus the impact of CuV on the progression to MF from LPP could not be fully evaluated. Second, we could only assess the CuV status once at diagnosis; therefore, the dynamics of CuV status and viral load could not be evaluated during the clinical course. Lastly, we used FFPE-archived tissues to investigate the expression of CuV transcripts. Fresh frozen specimens may have an increased sensitivity to mRNA amplification by RT–PCR [31–33].

Taken together, this study shows not only the detection of CuV DNA with high viral load in LPP but also the expression of viral RNA transcripts, suggesting a potential role for CuV in the pathogenesis of LPP in a subset of patients. However, the higher viral load with a tendency to be found at the premalignant LPP stage before progression to overt MF suggests that CuV may be a trigger for inflammation rather than an oncogenic virus during carcinogenesis. Although the signals were strong, the small number of CuV-positive cells observed in the LPP tissues also argues against a direct oncogenic role for CuV. In this context, it is worth studying whether specific CuV antigens might induce immune responses that lead to uncontrolled clonal expansion and the accumulation of T-helper cells in the LPP skin and whether subsequent secondary genetic mutations might occur during tumor progression [34–36]. Previous studies detected viral DNA of human herpesvirus 8 and human polyomavirus 6 in the skin of patients with PP [37–39], but the pathogenetic relevance has not been demonstrated. Although the exact role for CuV in LPP remains uncertain, our present studies are expected to stimulate further worldwide studies to gain more insights into the pathogenicity of CuV and to evaluate the risk of coinfection with other viruses for the progression to MF. The impact of geographically related variant viral genotypes on the outcomes of patients with LPP is also worthy of study.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org/). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.

Notes

Author contributions. Y. H. contributed formal analysis, funding acquisition, investigation, methodology, visualization, and writing the original draft. K. N. contributed formal analysis and resources. T. H. contributed visualization. T. U. performed investigations. K. N. contributed resources. M. D. contributed conceptualization, formal analysis, funding acquisition, methodology, project administration, supervision, visualization, writing the original draft, and reviewing and editing.

Data availability. The data that support the findings of this study are available from the corresponding author upon reasonable request.

Financial support. This work was supported by the Japan Society for the Promotion of Science (grant number 22K08434); and the Kochi Shin-kin Medical Research Prize.

References

Author notes