Domestic cat hepadnavirus detection in blood and tissue samples of cats with lymphoma

Abstract

Domestic cat hepadnavirus (DCH), a relative hepatitis B virus (HBV) in human, has been recently identified in cats; however, association of DCH infection with lymphoma in cats is not investigated. To determine the association between DCH infection and feline lymphoma, seven hundred and seventeen cats included 131 cats with lymphoma (68 blood and 63 tumor samples) and 586 (526 blood and 60 lymph node samples) cats without lymphoma. DCH DNA was investigated in blood and formalin-fixed paraffin-embedded (FFPE) tissues by quantitative polymerase chain reaction (qPCR). The FFPE lymphoma tissues were immunohistochemically subtyped, and the localization of DCH in lymphoma sections was investigated using in situ hybridization (ISH). Feline retroviral infection was investigated in the DCH-positive cases. DCH DNA was detected in 16.18% (11/68) (p = 0.002; odds ratio [OR], 5.15; 95% confidence interval [CI], 2.33–11.36) of blood and 9.52% (6/63) (p = 0.028; OR, 13.68; 95% CI, 0.75–248.36) of neoplastic samples obtained from lymphoma cats, whereas only 3.61% (19/526) of blood obtained from non-lymphoma cats was positive for DCH detection. Within the DCH-positive lymphoma, in 3/6 cats, feline leukemia virus was co-detected, and in 6/6 were B-cell lymphoma (p > 0.9; OR, 1.93; 95% CI, 0.09–37.89) and were multicentric form (p = 0.008; OR, 1.327; 95% CI, 0.06–31.18). DCH was found in the CD79-positive pleomorphic cells. Cats with lymphoma were more likely to be positive for DCH than cats without lymphoma, and infection associated with lymphoma development needs further investigations.

Keywords:

• B-cell lymphoma
• domestic cat hepadnavirus
• feline
• hepatitis virus
• localization

Introduction

Feline lymphoma, a hematopoietic cancer, is considered the most common neoplastic disease in domestic cats worldwide (Dorn et al. 1968; Louwerens et al. 2005). The occurrence of feline lymphoma is associated with various factors, including host variables that are typically present in crossbreed and middle-aged cats (Louwerens et al. 2005; Sato et al. 2014) and environmental factors such as exposure to tobacco, radon, and pesticides (Bertone et al. 2002; Gavazza et al. 2008; Ha et al. 2017). In addition to these modifiable risk factors, viral infections have been recognized as common risk factors for development of lymphoma not only in humans but also in felines (Louwerens et al. 2005). Feline retroviruses, including feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV), have been thought to be the most common etiological agent-induced feline lymphomas (Louwerens et al. 2005; Beatty 2014). Retroviral infective status in lymphoma cats is also considered a risk factor associated with disease progression (Fabrizio et al. 2014; Santagostino et al. 2015; Economu et al. 2021). Previously, up to 80% of lymphoma cats were found to be FeLV antigenic positive (Cotter et al. 1975; Francis et al. 1979; Hardy et al. 1980; Hartmann 2012). However, the FeLV positivity rate has been declining in a recent year contrary to the rising trend in feline lymphoma prevalence (Louwerens et al. 2005; Taylor et al. 2009; Economu et al. 2021). This decline in prevalence of FeLV infection in lymphoma cases indicates a shift in tumor causation in recent years or there are other undetectable underlying causes (Hartmann 2012; Schlecht-Louf et al. 2014; Studer et al. 2019). Although the lymphotropic nature of feline retroviruses has shed some light on the virus–lymphoma link (Hardy et al. 1980; Hartmann 2012), little is known about the underlying mechanistic cause or the impact of indirect mechanisms in the development of feline lymphoma mediated by non-lymphotropic viruses.

Domestic cat hepadnavirus (DCH), a novel virus that belongs to the Hepadnaviridae family, which includes the human hepatitis B virus (HBV), woodchuck hepatitis virus, and a variety of bat hepatitis viruses, was first discovered in a leukemia cat in 2016 (Aghazadeh et al. 2018; Lanave et al. 2019; Piewbang et al. 2020). Similar to the HBV as a prototype member of this viral family, DCH strongly prefers hepatocytes for infection, resulting in associations with hepatobiliary diseases and development of hepatocellular carcinoma (HCC) in chronically infected cases (Pesavento et al. 2019; Piewbang et al. 2020). Since the initial discovery of DCH, investigations of this virus in association with hepatopathy in cats have been conducted, resulting in evidence indicating that the virus is associated with an increase in liver enzymes (Lanave et al. 2019; Piewbang et al. 2022a). Cellular localization of DCH is mainly found within hepatocytes, where it is associated with pathological lesions, including interface hepatitis and HCC (Pesavento et al. 2019; Piewbang et al. 2020, 2022a). These findings support the association of DCH infection with hepatic diseases, reminiscent of the disease association found in HBV infection in humans (Mendy et al. 2010; Lamontagne et al. 2016; Levrero and Zucman-Rossi 2016; Lin and Zhang 2017). Regarding this, other HBV-associated diseases found in infected patients might be present in DCH-infected cats, which requires further studies. Extrahepatic HBV infection has been reported (Dienstag 1981; Mason et al. 1993; Kappus and Sterling 2013) and was found to be associated with other various clinical diseases where the virus was either localized or caused by chronic inflammatory reactions. Similar to HBV infection, DCH antigens can also be found in extrahepatic organs, displaying the cellular diversity of DCH tropism (Piewbang et al. 2020). Neither the roles of extrahepatic infection of DCH nor related diseases associated with chronic infection have been investigated; determination of diseases associated with chronic inflammation in tissues where DCH is chronically localized is, therefore, necessary. Apart from association with development of HCC, HBV has been found in cases of non-Hodgkin lymphoma (NHL) (Wang et al. 2007, 2012; Deng et al. 2015; Lemaitre et al. 2018; Li et al. 2018). Whether or not HBV is associated with the lymphoma is still controversial, several studies have found that the HBV antigen in sera was more prevalently detected in B-cell NHL cases (Wang et al. 2007; Fwu et al. 2011). In addition, the HBV antigen has been detected in lymphoma tissues, indicating that HBV might have some roles in association with lymphoma (Wang et al. 2012, 2018; Li et al. 2020). Because the hepatotropic nature that mimics HBV was found in DCH-infected cats, other direct or indirect roles of DCH as an oncogenic agent of lymphoma should be investigated.

The first study of DCH that reported that the virus was recovered from lymphoma tissue obtained from a cat (Aghazadeh et al. 2018), together with information of HBV in patients with NHL, prompted us to investigate the presence of DCH in lymphoma cases. In this study, we investigated the presence of DCH DNA in the blood obtained from cats with and without lymphoma. We also extended our investigation by postulating that DCH infection might exhibit extrahepatic behavior similar to what has been described in HBV infection. To elaborate on this speculation, we performed the qPCR and ISH to detect DCH infection in tumor tissues of different lymphoma subtypes, which were differentiated by immunohistochemistry (IHC). Tissues from benign lymphatic diseases were included in this investigation. Coinfection of other feline retroviruses was also investigated.

Materials and methods

Samples

In this study, we divided the research investigation into 2 parts: 1) detection of DCH DNA in the blood obtained from lymphoma and non-lymphoma cats and 2) investigation of DCH DNA in lymphoma tissues. First, 594 ethylenediaminetetraacetic acid (EDTA) blood included 68 cats with lymphoma and 526 cats without lymphoma that were collected for any medical purposes and were submitted for routine hematology at the Faculty of Veterinary Science, Chulalongkorn University, and private veterinary laboratories from 2019 to 2021. The EDTA blood of the lymphoma group was classified based on the historical data presenting that this blood was obtained from cats presenting tumors that were cytologically or histologically diagnosed as lymphomas, and the control group was the EDTA blood obtained from cats with or without a nonspecific medical condition and with no presence of any tumor mass when sampling. Owners were informed and asked to provide informed consent before collection of the cat samples.

Second, 123 retrospective formalin-fixed, paraffin-embedded (FFPE) tissues including 63 histologically diagnosed lymphoma and 60 lymph node samples obtained from cats that presented histological benign lymphatic diseases were examined in this study. The FFPE samples were obtained from the archives at the Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University, and private laboratories in Bangkok, Thailand, during 2020–2022. This study was conducted in compliance with the ARRIVE guidelines and regulations and with the Chulalongkorn University Animal Care and Use Committee (No. 2131004). Other case signalments including sex and age were included for further analysis.

Lymphoma subtype classification

The obtained FFPE samples were subjected to immunophenotyping using CD3, CD79, and Pax5 for subtype classification according to the Revised European American Lymphoma/World Health Organization (REAL/WHO) guidelines (Valli et al. 2011). Briefly, the FFPE samples were 4-µm sectioned, deparaffinized, and subsequently subjected to antigen retrieval using 10 mM citrate buffer (pH 6) incubation in a water bath at 95 °C for 20 min. The endogenous peroxidase enzyme was blocked by immersion in 0.3% (v/v) H₂O₂ for 30 min at room temperature. Nonspecific blocking was performed by incubation with 5% bovine serum albumin in a humidified chamber at 37 °C for 20 min. Subsequently, the sections were incubated with each primary antibody following the concentration and procedures described in a previous study (Sirivisoot et al. 2022). After incubation and triple washing, the slides were then incubated with a secondary antibody for 30 min using EnVision system mouse/rabbit horseradish peroxidase (Dako, Glostrup, Denmark) for CD3, CD79, and Pax5. Diaminobenzidine (Dako, Glostrup, Denmark) was used as the detection system for CD79 and Pax5, and the Vector VIP Substrate Kit (Vector Laboratories, Inc., CA, USA) was used for CD3. The lymphoma tissue samples were categorized based on their anatomic forms, histologically prognostic grading, and histological pattern according to the US National Cancer Institute working formulation and WHO classifications (Valli et al. 2000, 2011).

Viral nucleic acid extraction, quantification, and reverse transcription

EDTA blood samples were vigorously vortexed and centrifuged, and subsequently, about 200 µl of the supernatant was collected for nucleic acid extraction. The total viral nucleic acids were extracted using the IndiSpin Pathogen Kit (Indical Bioscience GmbH, Leipzig, Germany) following the manufacturer’s recommendation. The FFPE tissue samples were serially shaved to 75–100 µm thickness in total, collected in 1.5-ml Eppendorf tubes, and subsequently deparaffinized using xylene and alcohols. The deparaffinized tissues were incubated with 10 mg/ml proteinase K at 95 °C until dissolved. Total DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The quantity and quality of the extracted nucleic acids were measured using a spectrophotometer (Nanodrop Lite Spectrophotometer, Thermo Scientific, MA, USA). The extracted nucleic acids were subjected to determine the quality of extraction process using conventional PCR targeting the GADPH gene following previously described protocols (Piewbang et al. 2021). The complementary DNA (cDNA) was constructed using the Omniscript® Reverse Transcription Kit (Qiagen GmbH, Hilden, Germany) as described previously (Piewbang et al. 2017). The DNA and cDNA samples were then kept at −20 °C until used.

Detection of feline retroviruses in samples obtained from lymphoma cats

Because feline retroviruses are recognized as etiological agents in lymphoma development, nucleic acids extracted from the EDTA blood and constructed cDNA from the FFPE tissues obtained from lymphoma cats were examined to investigate the presence of feline retroviral genomes, including viral DNA (provirus) and RNA, respectively. The DNA and cDNA were used as a template for FIV and FeLV detection using SYBR-based qPCRs. Briefly, a total of 2 µl of DNA/cDNA was mixed with qPCR master mix using the KAPA SYBR Fast qPCR Master Mix (2X) Universal kit (KAPABIOSYSTEMS, Sigma-Aldrich, Cape Town, South Africa). Primers targeting the LTR U3 (Tandon et al. 2005) and gag (Weaver et al. 2005) genes of FeLV and FIV, respectively, were applied. The amplifications were processed on a Rotor-Gene Q real-time PCR cycler (Qiagen GmbH, Hilden, Germany) with thermal cycling conditions and settings as previously described (Tandon et al. 2005; Weaver et al. 2005). The extracted nucleic acid samples obtained from the FIV-positive lymph nodes of infected cats and FeLV plasmid (Lacharoje et al. 2021) served as positive controls. A non-template tube was used as a negative control.

Real-time PCR of DCH DNA

The presence of DCH DNA in the blood and FFPE samples obtained from diseased and control groups was investigated by qPCR targeting the DCH core gene using the previously described protocols (Piewbang et al. 2020, 2022a). Briefly, 2 μl of the extracted DNA sample was mixed with the 23-μl master mix of the KAPA SYBR Fast qPCR Master Mix (2X) Universal kit (KAPABIOSYSTEMS, Sigma-Aldrich, Cape Town, South Africa) that contained 200 nM of DCH-specific primer (Lanave et al. 2019). The viral loads of DCH in each tested sample were calculated on the basis of the standard curve, which was determined from 10-fold serial dilutions of the constructed DCH plasmid obtained from a previous study (Piewbang et al. 2020). Amplification present at a Ct value over 38 was considered negative, and a non-template tube was used as a negative control. All samples were run in triplicate.

In situ hybridization

DCH in lymphoma sections was determined by ISH to indicate viral DNA localization in DCH qPCR-positive cases. A DNA probe covering 290 bp of the DCH genomic portion between the polymerase and core genes was constructed using a PCR DIG Synthesis Kit (Roche Diagnostics, Basel, Switzerland) according to the manufacturer’s procedures. Cycling conditions and chemicals for probe synthesis were described in previous publications (Piewbang et al. 2022a). The success of probe synthesis was determined by size resolution on 1% (w/v) agarose gel. Chromogenic ISH was performed with the protocols as per previous descriptions. Briefly, the FFPE samples were 4-µm sectioned and placed on positively charged slides. After deparaffinization, the sections were pretreated with 10 µg/ml proteinase K enzyme at 37 °C for 10 min and post-fixed with 0.4% (v/v) cold formaldehyde for 5 min. After prehybridization with buffer containing 50% (v/v) deionized formamide in 4X saline-sodium citrate (SSC), the sections were incubated with hybridization buffer that contained 5 ng/ml DCH-DIG probe per slide at 55 °C overnight in a formamide-containing humidified slide incubator. The sections were then stringent washed with graded SSC solutions and blocked for nonspecific blocking using 5% (w/v) bovine serum albumin. The DCH-DIG probe binding to the DCH gene in the tissue was coupled with a secondary antibody against the DIG using anti-DIG-AP Fab Fragments (Roche, Basel, Switzerland) (1:200 in 1X blocking solution) for 60 min at room temperature. After triple washing, the PermaRed AP chromogen (Diagnostics Biosystem, CA, USA) was used as the detection system. The slides were then counterstained with hematoxylin, cover-slipped, and mounted. Red-dot precipitates in cellular morphology were considered positive. For negative controls, the lymphoma sections were incubated with DIG probe-targeted feline bocavirus-3 (FBoV-3). The lymphoma sections incubated with 1% (w/v) DNAse enzyme prior to hybridization with the DCH-DIG probe and the lymph node sections obtained from DCH-negative cats’ incubation with the DCH-DIG probe were used as additional negative controls. The DCH-positive liver section derived from a previous study (Piewbang et al. 2020) incubated with the DCH-DIG probe was used as a positive control. The intensity of the DCH-specific signals presented in the lymphoma cells of each case was subsequently scored as follows: no hybridization signals, rare hybridization signals, common hybridization signals, and widespread staining (Piewbang et al. 2022a). The intensity scores were used for further analysis.

Dual labeling

To confirm the presence of DCH DNA in lymphoma, dual chromogenic ISH for DCH DNA and IHC targeting CD79 that is specific to the B-cell lineage were performed following modified protocols (Chaiyasak et al. 2022; Piewbang et al. 2022b). IHC targeting CD79 was sequentially performed following ISH targeting DCH DNA. Briefly, the DCH qPCR-positive FFPE lymphoma tissues were cut to 4-µm thickness and placed on positively charged slides. The IHC was performed following the ISH procedures described above with the exception of counterstaining. After developing the colour for ISH (red precipitates), the sections were immersed in 0.3% (v/v) H₂O₂ for 15 min at room temperature to get rid of the remaining alkaline phosphatase activity and subsequently subjected to IHC targeting CD79 according to the protocols described above. After incubation with the CD79 primary antibody and triple washing with 1X phosphate saline buffer, the slides were incubated with an immune-alkaline-phosphatase polymer-conjugated anti-mouse/rabbit antibody (Histofine® simple stain AP [MULTI], Nichirei, Japan) as a secondary antibody. The immunohistochemical signals were visualized using StayGreen AP/Plus (Abcam, MA, USA). DCH-negative B-cell lymphoma section incubation with the FBoV-3 ISH probe was used as a negative control.

Statistical analysis

Descriptive statistics were expressed as means and standard deviations, and percentages were used for discrete data. Statistical analysis was performed using Fisher’s exact test to determine the association of DCH DNA with lymphoma cases or different lymphoma subclasses. Furthermore, the association the cases showing positive DCH DNA detection and the presence of retroviruses was also determined using Fisher’s exact test. Anatomical forms of lymphoma in association with the presence of DCH were statistically analysed using the Chi-square test. The differences were considered significant at p < 0.05. The associations between viral copy number and ISH staining signals were analysed using the Kruskal-Wallis test. The differences were considered significant at p < 0.05. Odds ratios were also calculated to indicate the strength of association between DCH and lymphoma. All statistical analyses were performed using GraphPad Prism version 9.2.0 (GraphPad Software, CA, USA).

Results

Sample population and information

The 594 EDTA blood samples included 68 lymphoma cats and 526 control cats, with 220 (37.04%) males and 374 (62.96%) females. Their ages ranged from 1 to 14 years (mean = 6.57 years, median = 6 years). Regarding breed, most cats were domestic short hair cats (n = 391, 95.82%), followed by Persian (n = 174, 29.30%), Scottish fold (n = 23, 3.87%), and others (n = 6, 1.02%). Within the 63 lymphoma cases, 56 (88.89%) and 7 (11.11%) were immunohistologically differentiated to B-cell and T-cell lymphomas, respectively (Figure 1A–D). Among them, multicentric lymphoma was the most predominant anatomic form (49.21%, 31/63). Details regarding the investigated samples are included in the Tables 1 and 2. Of the 60 samples obtained from cats with benign lymphatic diseases, 42 (70.00%) and 18 (30.00%) were diagnosed as reactive lymph nodes and normal histological lymph nodes, respectively. Regarding the detection of retroviral viruses in lymphoma cases, 61.76% (42/68) and 55.56% (35/63) of EDTA blood and FFPE tissue samples, respectively, were positive for FeLV.

Figure 1. Histopathological features of DCH infection in lymphoma tissue of cats. (A, E-J) Sections from case no. 3. (A) The lymphoid follicles are diffusely expanded by large neoplastic lymphocytes separating coarse fibrovascular stroma. Neoplastic lymphocytes have moderate amounts of cytoplasm with distinct cellular borders. The nuclei are 2 times larger than red blood cells, round, and finely stippled, containing a prominent nucleolus (square box is indicated for the inset). (B-D) DCH qPCR-positive lymphoma section of case no.6. (B) Diffuse, generalized cytoplasmic IHC staining for CD79. (C) Prominent nuclear IHC staining for pax-5 in multiple neoplastic cells. (D) Cytoplasmic IHC staining for CD3 in some single cells in lymphoma tissue. (E) In situ hybridization (ISH) for DCH DNA staining (red precipitates) in lymphoma tissue revealed intensely intranuclear staining in diffuse round cells (square box is indicated for the inset). (F) Prominent nuclear staining for DCH hybridization in large, pleomorphic round cells. No DCH labeling was observed within the small round cells. (G) Dual DCH ISH (red precipitates) and CD79 IHC (green color) revealed marked, diffuse staining in the nucleus and cytoplasm of neoplastic cells, respectively. (H) Higher magnification reveals intense DCH labeling (red precipitates) in the nucleus of the cells that were cytoplasmically stained with CD79 IHC (green color). (I) Negative control using unrelated probe. No hybridization signals were present in the DCH qPCR-positive lymphoma section that was incubated with the FBoV-3 ISH probe. (J) Negative control using DNase-treated section. No hybridization was present in the DCH qPCR-positive lymphoma section treated with DNase prior to incubation with the DCH probe. (K) Negative control for dual ISH/IHC labeling. Cytoplasmic IHC CD79 staining of the DCH qPCR-positive lymphoma section incubated with the FBoV-3 probe. Bars indicate 180 µm for A, B, D and E; 80 µm for F; 120 µm for C, G, I, and J; and 20 µm for H and K.

Table 1. Detection of DCH DNA in blood and lymph node tissues of investigated cats.

Samples	Sample characteristics		Lymphoma		Non-lymphoma		P- value (Odd ratio, 95% CI)
			No. of cases (A)^a %		No. of cases	%
EDTA Blood	Total case	N = 594					0.002 (5.1496, 2.33–11.36)
	DCH qPCR	No. of cases	68 (42)	100	526	100
		Positive	11 (5)	16.18	19	3.61
		Negative	57 (37)	83.82	507	96.39
FFPE Tissue	Total case	N = 123					0.028 (13.678, 0.75–248.36)
	DCH qPCR	No. of cases	63 (35)	100	60	100
		Positive	6 (3)	9.52	0	0
		Negative	57 (32)	90.48	60	100

A = number of cases that were co-detected with feline retroviruses. ^aThere was no statistical significance between DCH detection and retrovirus status in lymphoma cases (p = 0.3).

Table 2. Morphological characteristics of lymphoma cats and DCH DNA detection in paraffin-embedded lymphoma tissues.

Lymphoma classification	Number of tested cases (n)	DCH qPCR		P-value (Odd ratio, 95% CI)
		Positive	Negative
Subtypes	B-cell (56)	6	50	P >0.9 (1.93, 0.09–37.89)
	T-cell (7)	0	7
Anatomical form	Multicentric (31)	6	24	P = 0.008 (1.327, 0.06–31.18)
	Others (32)	0	32

DCH DNA in lymphoma-derived samples

The EDTA blood samples revealed 5.10% (30/594) positive DCH DNA detection. Significantly, the DCH DNA detection was higher in blood samples obtained from cats with lymphoma (16.18%, 11/68) than from control cats (3.61%, 19/526) (p = 0.002) (Table 1). In extracted lymphoma samples, DCH DNA was detected in 9.52% (6/63), but it was not detected in samples obtained from lymph nodes showing lymphoid follicular hyperplasia and normal lymph nodes. Similar to the results obtained from blood sample investigation, the positive rate of DCH DNA in the lymphoma tissue samples was significantly higher in the lymphoma cases than in the non-lymphoma group (p = 0.028). A significant association between DCH detection and lymphoma was observed in both study groups, revealing odds ratios of 5.1496 (95% CI, 2.33–11.36) and 13.678 (95% CI, 0.75–248.36) for the EDTA blood and lymph node tissue groups, respectively (Table 1). Within 17 DCH-positive lymphoma cats (including EDTA blood and FFPE tissue samples), FeLV was co-detected in 8 samples (p = 0.305). Details regarding DCH detection in lymphoma cases are provided in Table 1. Of the DCH-positive FFPE lymphoma cases, all were B-cell lymphomas, and all were in multicentric form. Regarding anatomical forms, multicentric lymphoma was a significant form in DCH detection compared with the other forms (p = 0.008) (Table 2). However, there was no statistically significant difference in association of DCH DNA with B-cell and T-cell lymphomas (p > 0.9). Most DCH-positive cases were low-grade lymphomas (4/6, 66.67%), and only 2 cases were characterized as high-grade lymphomas. Details regarding histological assessment according to WHO classification are provided in Table 3.

Table 3. Histological characteristics of DCH-positive feline lymphoma cases.

Case no.	Lymphoma subtype/anatomical form	Histological Grading	WHO classification	DCH loads (copies/ml)	ISH score^a	ISH staining pattern	Feline retroviral status^b
1	B-cell/Multicentric	Low grade	Peripheral B cell	2.35 × 10^4	+	Focal, dot-like pattern	Negative
2	B-cell/Multicentric	High grade	Diffuse large B-cell	2.82 × 10^4	+	Focal, dot-like pattern	FeLV
3	B-cell/Multicentric	Low grade	B-cell small lymphocytic	3.84 × 10^6	+++	Multifocal, multiple dot-like pattern	Negative
4	B-cell/Multicentric	Low grade	Follicle center	1.80 × 10^7	+++	Diffuse, intensity	FeLV
5	B-cell/Multicentric	High grade	Diffuse large B-cell	1.28 × 10^6	++	Patchy, multiple dot-like pattern	Negative
6	B-cell/Multicentric	Low grade	Follicle center	1.11 × 10^6	++	Focally extensive, multiple dot-like pattern	FeLV

^aThere is no significant correlation between DCH loads and DCH staining pattern (p = 0.067).

^bFeLV: feline leukemia virus.

DCH DNA loads and localization in lymphoma tissues

To obtain the DCH loads in blood and lymphoma tissues, qPCR targeting the DCH core gene was performed. The DCH loads in lymphoma cases ranged from 2.98 × 10³ to 7.65 × 10⁸ copies/ml in the blood samples and 2.35 × 10⁴ to 1.80 × 10⁷ copies/ml in the lymphoma tissues. There were no overlapping samples between blood and lymphoma tissues obtained from the same cats. Localization of DCH in lymphoma tissue was further determined by ISH. ISH was positive in all 6 DCH lymphoma cases, with different ISH staining intensities (Figure 1E); however, this did not correspond to the DCH loads presented in the qPCR results (p = 0.06) (Table 3). DCH DNA was mostly detected in pleomorphic lymphoma cells but not in small lymphocyte cells (Figure 1F). The hybridization patterns varied from patchy to diffuse staining, which covered the entire tumor mass in some cases. In cases with lower DCH loads, the ISH staining pattern was limited to the focal area of the tumor mass, and the hybridization signals scattered within the nucleus of malignant cells. No ISH signals were observed in the negative controls (Figure 1I and J). The ISH/IHC dual labeling indicated that the DCH DNA was localized within the nucleus of the pleomorphic cells (red precipitates) that were positive for IHC against CD79 within the cytoplasm (green colour) (Figure 1G and H). No reaction signal was presented within negative control sections for dual labeling (Figure 1K).

Discussion

The detection of DCH in feline samples leads to the question of the impact of this virus on feline health. Apart from hepatotropic nature of the hepadnavirus, extrahepatic manifestations of the hepadnavirus, such as lymphoproliferative disorders caused by HBV infection, have been reported, and the prevalence of HBV has been found to be higher in humans with NHL (Wang et al. 2007; Fwu et al. 2011; Wang et al. 2012; Deng et al. 2015; Lemaitre et al. 2018; Li et al. 2020). These led us to question the association of DCH with feline lymphoma. To the best of our knowledge, this study is the first to describe the presence of DCH in feline lymphoma cases by examining the differences in presence of DCH in blood and tissues obtained from cats with and without lymphoma. We also extended the analysis of DCH to different lymphoma subtypes and anatomical forms; in both, DCH DNA was confirmed using qPCR. DCH DNA localization in pleomorphic lymphoma cells may suggest the role of DCH in feline lymphoma development that needs further investigations.

In the present study, DCH infection was found to be significantly prevalent with lymphoma in cats. This study’s findings align with current knowledge on HBV in association with lymphomas in humans (Wang et al. 2007; Deng et al. 2015; Lemaitre et al. 2018; Li et al. 2018); however, the exact roles of this virus in development of NHL in humans or lymphoma in cats are not yet understood (Marcucci et al. 2012; Wang et al. 2012, 2018). Recent studies hypothesized that HBV invades the lymph node, involving local immune system alterations, and could result in NHL development (Wang et al. 2012; Deng et al. 2015; Wang et al. 2018). Furthermore, chronic immunogenic stimulation caused by chronic HBV infection could drive the development of NHL due to lymphomagenesis initiation and promotion (Deng et al. 2015, 2017). Because both HBV and DCH are members of the Hepadnaviridae family (Aghazadeh et al. 2018; Piewbang et al. 2020; Capozza et al. 2021), and chronic DCH infection has been reported in recent literature, the pathogenic role of DCH in lymphoma development could, therefore, be similar to that postulated in HBV infection. A better understanding of the molecular mechanisms underlying DCH-driven lymphomagenesis, whether acute or chronic DCH infection, will provide a clear idea on the association of this virus with lymphoma development. On the other hand, HBV reactivation due to chemotherapy and immunotherapy in lymphoma patients has been described (Kelling et al. 2018; Cao et al. 2021). Thus, findings of prevalent DCH in feline lymphoma cases in this study may be due to the immunosuppressive effect of tumor and/or chemotherapy–which could cause reactivation of DCH viremia or increase risk of infection. In addition, coinfection with retroviruses could enhance the susceptibility of the host for DCH infection. However, there were no overlapping samples between blood and lymphoma tissues obtained from the same cats and no information about lymphoma treatments, as an exclusion criterion, in the study groups. Thus, a definitive conclusion of the DCH in association with development of feline lymphomas could not be drawn based on this current study. Furthermore, a lower detection rate of the DCH DNA in non-lymphoma cases may be due to a latency stage of infection as found in the HBV infection in humans. Therefore, determination of negative DCH DNA detection in the non-lymphoma cases should be cautiously interpreted and this may not reflect the definitive number of infected cases. In addition, it should be reminded that molecular investigations may underestimate DCH prevalence and that the absence of DCH DNA does not indicate that the cat is uninfected.

Studies have reported that HBV infection is more frequently found in cases of B-cell lymphoma (Fwu et al. 2011; Deng et al. 2015; Cao et al. 2021). Similarly, we found that DCH was exclusively present in B-cell lymphoma; however, we found no statistically significant differences in DCH infection between lymphoma subtypes. Because of unequal proportions among investigated groups, with lower numbers in the T-cell-derived group, determination based on the results obtained in this study should be interpreted cautiously. Because B-cell lymphoma is a common subtype of feline lymphoma (Valli et al. 2000; Musciano et al. 2020), and this retrospective study included samples submitted during a single period, investigation of DCH in larger numbers in multiple periods must be performed for better understanding. In this study, the multicentric form of feline lymphoma was the most common anatomical form. This is inconsistent with previous studies in Australia (Gabor et al. 1998) and Japan (Sato et al. 2014) that indicated that alimentary lymphoma was the most common anatomical form. This discrepancy could result from the different study groups and study periods. Despite multicentric lymphoma being the most common form found in retroviral-related lymphoma (Rojko et al. 1989; Weiss et al. 2010; Cristo et al. 2019), DCH DNA has also significantly been found to be associated with this form compared with other forms. The findings of DCH DNA within the B-cell lymphomas that were confirmed by dual labeling may suggest the possibility of DCH association with multicentric B-cell lymphoma development, as presented in HBV-related NHL (Lemaitre et al. 2018; Li et al. 2020; Cao et al. 2021), or they could be co-incidental findings.

Although lymphoma development is multifactorial, virus-associated tumorigenesis, has been commonly reported not only in humans (Wang et al. 2007; Fwu et al. 2011; Marcucci et al. 2012; Wang et al. 2012; Deng et al. 2015; Lemaitre et al. 2018; Li et al. 2018, 2020; Cao et al. 2021), but also in felines (Louwerens et al. 2005; Hartmann 2012; Beatty 2014; Esau 2017). Even though we quantified the DCH in lymphoma tissues and identified the DCH localization in malignant B-cells, the role of DCH in development of feline lymphoma could not be determined. Although we tested about 700 samples, only a relatively small subset was from cats with lymphoma. Further research aiming to determine the presence of DCH DNA in samples obtained from a case-controlled study of retroviral-free lymphoma tissues is warranted to support our hypothesis.

In conclusion, the presence of DCH was more prevalent in feline lymphoma. Strikingly, this finding was in accordance with the finding of HBV with NHL, supporting some pathophysiological mechanisms similar to HBV infection. Identification of the DCH localization in malignant B-cell cells lymphoma may possess evidence for further study regarding the infection associated with development of this neoplastic disease. Studies aimed at investigating the mechanisms of DCH-mediated malignant B-cell transformation and the overlap between DCH-mediated processes in increasing the risk of feline lymphoma development would garner great attention.

Author contributions

C.P. and S.T. designed the study. C.P., S.W.W., J.S., and S. S. collected the samples and performed the experiments. C.P., S.W.W., J.S., S.S., and A.R. analyses. C.P. wrote the first draft of the manuscript and S.T. finalized the manuscript. All authors approved the manuscript.

Ethical approval

The authors confirm that the ethical policies of the journal, as noted on the journal’s author guidelines page, have been adhere to. This study was approved by Chulalongkorn University Animal Care and Use Committee (No. No. 2131004). Authors confirm that this study is reported in accordance with ARRIVE guidelines.

Disclosure statement

No potential conflict of interest was reported by the authors.

Data availability statement

All data generated or analysed during this study are included in this published article.

Additional information

Funding

C.P. and S.S. were supported by the Ratchadapisek Somphot Fund for Postdoctoral Fellowship, Chulalongkorn University. S.W.W. was granted by The Second Century Fund (C2F), Chulalongkorn University for Doctoral Scholarship. J.S. was supported by the 100th Anniversary Chulalongkorn University Fund for Doctoral Scholarship. S.T. was partly supported by National Research Council of Thailand (NRCT): R. Thanawongnuwech NRCT Senior Scholar 2022 #N42A650553. This Research is funded by National Research Council of Thailand (NRCT) (N41A640175) (to C.P.).