Virological and pathological characterization of an outbreak of domestic cat hepatitis B virus infection in a cat shelter in Japan

Abstract

Domestic cat hepatitis B virus (DCHBV) infections are associated with chronic hepatitis and hepatic cancer in cats, similar to human HBV. We previously demonstrated that the positivity rate for DCHBV in Japan is less than 1% among tested samples, suggesting lower viral dissemination in Japan than in other countries. Furthermore, the transmission dynamics of DCHBV remain poorly understood. In this study, we document the first recorded outbreak of DCHBV in a cat shelter in Japan, which had an unprecedentedly high prevalence of 26.59% (46/173 cats). We investigated potential transmission routes via quantitative polymerase chain reaction analysis of matched blood, oral, and rectal swab samples. Blood and oral viral loads were significantly correlated, with viral DNA detected in both oral (40.54%) and rectal (19.44%) cavities of viremic cats. Phylogenetic analysis of the polymerase gene retrieved from deceased cats yielded >99.2% sequence identity, indicating the circulation of a single strain. Pathological assessments, including histopathology, immunohistochemistry, and RNA in situ hybridization, confirmed viral presence in hepatocytes and biliary epithelium, linking the outbreak with severe hepatic lesions, including cholangitis, fibrosis, and cholangiocarcinoma. Our findings indicate that the transmission route of DCHBV may be more complex than initially anticipated, underscoring the importance of understanding the mode of transmission to effectively control DCHBV infection, particularly in multi-cat facilities.

INTRODUCTION

Orthohepadnaviruses (Family Hepadnaviridae), which include hepatitis B virus (HBV), are known agents of severe liver diseases such as hepatitis, cirrhosis, and hepatocellular carcinomas in humans, apes, woodchucks, and birds [4, 5, 12, 16, 30]. Domestic cat HBV (DCHBV) was first identified in a domestic cat in 2018, marking the first reported case of hepadnavirus infection in a companion animal [2]. The recent detection of closely relative hepadnaviral DNA in canine serum [8] indicates the zoonotic potential of DCHBV.

DCHBV is a small DNA virus measuring 42–50 nm in diameter. Its genome comprises circular, partially double-stranded DNA approximately 3.2 kb long. As with other hepadnaviruses, the genome encodes four overlapping open reading frames that encode polymerase (L), surface (S), core (C), and X proteins [2, 18]. DCHBV has been identified in Brazil, Hong Kong, Italy, Japan, Malaysia, Taiwan, Thailand, Turkey, the United Kingdom, and the United States [1,2,3, 6, 8, 13, 15, 23, 27, 29, 31, 32].

The prevalence of DCHBV varies by country at 6.5% in Australia, 3.08% in the United Kingdom, 0.78% in Japan, and 14.9% in Malaysia [2, 3, 13, 31]. In Italy, the highest reported prevalence among several studies was 10.8% [10, 15, 25]. Also, antibody assay targeting the DCHBV core protein revealed that 25% of the cats tested were positive in the country [10]. A case in Thailand has the highest positivity rate at 18.5% [22]. Thus, DCHBV prevalence in Japan is lower than those in other countries.

DCHBV shares clinical features with human HBV and is often associated with immunosuppressive conditions in cats infected with feline immunodeficiency virus (FIV) or feline leukemia virus (FeLV) [2, 3, 23, 31]. The pathogenicity of DCHBV may be associated with chronic hepatitis and hepatocellular carcinoma. Although inconclusive, a recent study revealed the genomic integration of DCHBV in feline hepatocellular carcinoma, indicating a link between DCHBV and hepatocarcinogenesis via insertional mutagenesis, similar to HBV [7]. Furthermore, we recently found an association between DCHBV and feline cholangiocarcinoma [9].

The HBV transmission route can be determined by testing for HBV DNA in urine, saliva, tears, and sweat in patients with chronic infection [11, 14]. Like HBV, DCHBV has also been detected in ear, nasal, oral, and rectal swabs [35]. These observations indicate the potential horizontal transmission of HBV and DCHBV.

The present study investigated the first DCHBV outbreak, which occurred in a cat shelter in Japan. Our molecular testing demonstrated that the DCHBV positive rate was 26.59% (46 of 173 cats) at the shelter. Our pathological analysis found that several cats subsequently developed cholangitis and cholangiocarcinoma and led to death. Although the horizontal transmission of DCHBV requires further confirmation, its detection in oral and rectal swabs and phylogenetic analysis of circulating viral strains provides evidence supporting this possibility.

MATERIALS AND METHODS

Ethical approval

All procedures for the collection of feline samples were approved by the Animal Care and Use Committee of the University of Miyazaki (Approval No. 2025-052) and conducted in accordance with the University of Miyazaki experimentation regulations and relevant institutional guidelines.

Detection of DCHBV in cat blood

Blood samples (n=173) were collected from cats in a cat shelter. The samples were preserved in EDTA tubes containing 1 mL DNA/RNA Shield (Cat# R1100-250, Zymo Research, Irvine, CA, USA). We performed a duplex real-time polymerase chain reaction (PCR) assay to detect DCHBV in the DNA samples using the One Step Easy Direct RT-qPCR Kit (Non-treatment) (Cat# RC805A, TaKaRa, Kusatsu, Japan). The primers/probe for DCHBV were forward primer 5′-ACTCACCAACTTCCTGTCCT-3′, reverse primer 5′-CTTCCAATCCAGGAGAACCAAC-3′, and probe /56-FAM/ATCATTTAC/Zen/CTCTTGCTCCTGGCG/3IABkFQ/ (Integrated DNA Technologies [IDT], Inc., Coralville, IA, USA) [26]. The primers/probes for the feline gene Actb were forward primer 5′-TCTCGATCTGTGCAGGGTATTA-3′, reverse primer 5′-AGACCGGCAAGACAGAAATG-3′, and probe /5HEX/TGGCAAGAG/ZEN/TCCTGAACCAGTTGT/3IABkFQ/ (IDT) [26]. A 2-μL aliquot of each blood sample, prediluted with DNase/RNase-free water (1:4) was analyzed using the QuantStudio 3 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). The PCR conditions were 90°C for 3 min, followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec.

Detection of FIV and FeLV in blood samples

Testing for FIV and FeLV was performed by analyzing blood samples using a triplex real-time reverse transcription PCR assay with the PrimeDirect^® Probe RT-qPCR Mix (Cat# RR650A, TaKaRa). The primers/probe used in the analysis are summarized in Supplementary Table 1. A 2-μL aliquot of each blood sample, pre-diluted with DNase/RNase-free water (1:4), was used analyzed using the QuantStudio 5 Real-Time PCR System (Applied Biosystems). The PCR conditions were 90°C for 3 min, 60°C for 5 min, followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec.

Detection of DCHBV in oral and rectal swabs

Oral and rectal swabs were subsequently collected from cats that tested positive for DCHBV in blood. DNA samples were extracted using NucleoSpin^® VET (Cat# U0842C, TaKaRa) according to the manufacturer’s instructions. Samples were tested for DCHBV using the same protocol described above.

Quantification of DCHBV genome levels genome levels

Blood, oral, and rectal swabs that tested positive for DCHBV during the blood screening were subsequently analyzed by qPCR for viral quantification. The assay, kit, and primers/probe used were the same as those described above for DCHBV detection. A plasmid containing the DCHBV genome, as identified using the primers and probe used in this study, was used as the DNA standard. Plasmid copy numbers were calculated based on plasmid size and concentration. Correlation plots of Ct values and viral loads were generated using GraphPad Prism Version 10.4.1 (GraphPad Software, San Diego, CA, USA). In the analysis of the correlation between Ct values and viral loads, DCHBV samples that were undetermined were assigned a Ct value of 40.

Sequencing of the DCHBV genome

Liver tissues were collected at necropsy from deceased cats. The polymerase DCHBV genome collected from the liver was amplified using PrimeSTAR^® Max DNA Polymerase Ver. 2 (Cat# R047B, TaKaRa) according to the company’s instructions. The liver samples were homogenized by BioMasher (Cat# 893064, Nippi, Tokyo, Japan) according to the manufacturer’s instructions. Genomic DNA was extracted using the DNeasy Blood & Tissue Kit (250) (Cat# 69506, QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Before amplifying the polymerase DCHBV genome, DCHBV detection was performed as described above. The primers used were DCHBV-4F (5′- ACTCTCAAACAGGGAACATTCGT-3′), and DCHBV-2R (5′- GTCTAGATTGTGACGAGGGAAAAAC-3′) [31]. The PCR conditions were 40 cycles at 98°C for 10 sec, 55°C for 5 sec, and 68°C for 30 sec, followed by 68°C for 7 min. The amplicons were visualized in 0.6% agarose gel and extracted using the QIAquick Gel Extraction Kit (Cat# 28706, QIAGEN) according to the manufacturer’s instructions. We then sequenced the polymerase genes using six primer pairs, as shown in Supplementary Table 2. The sequences of the amplicons were determined using a SupreDye v3.1 Cycle Sequencing Kit (Cat# 063001, M&S TechnoSystems, Osaka, Japan) according to the manufacturer’s instructions. We used a gel filtration cartridge (Cat# 42453, M&S TechnoSystems) to purify the amplicons for sequencing. The samples were analyzed using the Spectrum Compact CE System (Promega, Madison, WI, USA).

The sequence assembly was performed using 4Peaks (Nucleobytes, Aalsmeer, The Netherlands) and Microsoft Word 2019 (Microsoft, Redmond, WA, USA). The sequence of the strain identified in this study was aligned with domestic cat hepadnavirus Japan/KT116/2021 (LC668427.1) and Sydney2016 (MH307930.1) using MEGA X (MEGA Software version 11.0.13).

Phylogenetic analysis of the DCHBV polymerase genome

A phylogenetic tree of Hepadnaviridae polymerase sequences obtained from GenBank was constructed using the MUSCLE algorithm in MEGA X. The analysis included aligned amino acid sequences extracted from the livers of DCHBV-positive deceased cats and those retrieved from public databases. Evolutionary analyses were performed using the maximum likelihood and neighbor-joining methods with the Jones–Taylor–Thornton matrix-based model.

Sequence alignment and pairwise identity calculation for DCHBV polymerase

Pairwise amino acid sequence identities were calculated using MEGA X. Sequences were aligned using the MUSCLE algorithm, and pairwise distances were computed using the p-distance model with complete gap deletion. Because MEGA expresses p-distance as the proportion of nucleotide differences between each pair of sequences, the percentage identity was subsequently calculated as (1 −p-distance) × 100. The alignment was manually checked and edited to minimize artificial gaps and ensure accurate comparisons.

Histopathological examination

Liver samples from eight cats confirmed positive for DCHBV by PCR were fixed in 10% neutral-buffered formalin, routinely processed, embedded in paraffin, sectioned at 3 µm thickness, and stained with hematoxylin and eosin (HE) and direct fast scarlet for amyloid detection.

Immunohistochemical staining (IHC)

Liver tissues from the five cases subjected to histopathological examination were analyzed by immunohistochemistry using a primary antibody specific for the DCHBV core protein core protein (57–75) [9] at a 1:2,000 dilution. The Histofine Simple Stain MAX-PO (MULTI) reagent (Cat# 424154, Nichirei Biosciences, Tokyo, Japan) was used as the secondary antibody. The incubation conditions were 60 min at 37°C for the primary antibody and 30 min at 37°C for the secondary antibody. After deparaffinization and rehydration, antigen retrieval was performed by heat treatment at 105°C for 10 min in pH 9.0 antigen activation buffer. Endogenous peroxidase activity was blocked with 3% H₂O₂ in methanol, followed by blocking with Blocking One (Cat# 03953-95, Nacalai Tesque, Kyoto, Japan). Color development was achieved using 3,3′-diaminobenzidine solution prepared in Tris buffer adjusted to pH 7.6 (D5637-50G, Sigma-Aldrich, St. Louis, MO, USA). Counterstaining was performed with hematoxylin (Cat# 9131-4P, Sakura Finetek Japan Co., Tokyo, Japan) for 15 sec, followed by rinsing, dehydration through a graded ethanol series, clearing in xylene, and mounting on a coverslip. Negative controls were performed by replacing the primary antibodies with PBS.

RNA in situ hybridization

Liver tissues from three cases subjected to histopathological examination were provided. RNA in situ hybridization (RNA-ISH) was performed using RNAscope (Cat# 322300, Advanced Cell Diagnostics, Hayward, CA, USA) according to the manufacturer’s instructions. Gene expression was localized within formalin-fixed, paraffin-embedded tissues. Tissue sections were deparaffinized and treated with Pretreat 1 reagent (hydrogen peroxide) at room temperature for 10 min. This was followed by treatment with Pretreat 2 reagent (Target Retrieval Buffer for antigen retrieval) at 105°C for 10 min and Pretreat 3 reagent (Protease Plus) at 40°C for 15 min. Sections were then hybridized at 40°C for 2 hr with the hepadnavirus probe (Cat# 575811, Advanced Cell Diagnostics), a positive control probe Fc-PPIB (Cat# 455011, Advanced Cell Diagnostics), and a negative control probe DapB (Cat# 310043, Advanced Cell Diagnostics). After hybridization, signals were amplified and visualized using the RNA ISH 2.5 HD Detection Kit–BROWN (Cat# 322310, Advanced Cell Diagnostics). Positive signals were indicated by brown punctate dots in the cytoplasm or nucleus.

Statistical analysis

Logistic regression was used to analyze the association between DCHBV status and the background of each cat. The response variable was DCHBV status (positive or negative), and the independent variables were birth year, sex, FIV status, and FeLV status.

RESULTS

Clinical course of a DCHBV-positive cat in a shelter

At a cat shelter in Japan, a spayed domestic cat of unknown age and positive for FIV presented with a chief complaint of loss of appetite and hematuria (Day 0). Ulcerative stomatitis localized to the molar region and rupture of the anal sac were noted on physical examination. Hematological and biochemical analyses revealed mild anemia and a slight decrease in albumin (2.2 g/dL). Initial treatment consisted of methylprednisolone acetate (Depo-Medrol) administered subcutaneously.

Re-evaluation of the cat on Day 39 revealed weight loss, progression of anemia with 4.23 × 10⁶/μL red blood cell count (RBC), 6.4 g/dL hemoglobin (HGB), 19.7% hematocrit (Hct), and hypoalbuminemia (1.9 g/dL), and elevated serum ammonia (94 μg/dL). Abdominal ultrasonography revealed gallstones and increased echogenicity of the gallbladder wall. The cat was administered orally with 2.5 mg prednisolone, 10 mg orbifloxacin, 25 mg ursodeoxycholic acid, and 10 mg timepidium bromide WP.

On Day 47, the cat showed further progression of anemia with RBC count of 3.60 × 10⁶/μL, HGB of 5.6 g/dL, Hct of 17.6%, and hypoalbuminemia. An irregular mass-like shadow in the liver with a small amount of ascites was observed on ultrasound. Since hepatic bleeding was suspected, an exploratory laparotomy and biopsy were performed on Day 51. During exploratory laparotomy, whitish discoloration of the entire lateral and medial segments of the left hepatic and quadrate lobes were observed, while the right hepatic lobes presented with the patchy lateral and medial segments, as well as severe fibrosis, neoplastic changes, and lesions resembling cirrhosis. A partial resection of the left lateral lobe was performed for biopsy. Pathological analysis of the collected specimen conducted by a pathology service company revealed intrahepatic cholangiocarcinoma with lymphatic metastasis.

Furthermore, blood and ascites samples were sent to a commercial laboratory for viral infection testing. While the ascites sample was negative for feline infectious peritonitis virus, the blood sample tested positive for DCHBV.

Prevalence of DCHBV in the cat shelter

Given the DCHBV infection identified in the cat, the rest of the cats in the shelter were subsequently tested. Blood samples were analyzed by qPCR analyses. In this test, amplification of Actb was used as an internal control to verify sample quality and PCR efficiency. Efficient amplification of Actb was confirmed in all blood samples. Subsequently, 46 out of 173 cats (26.59%) were DCHBV-positive. Furthermore, the FIV and FeLV infection statuses of the cats at the shelter were determined by qRT-PCR analyses. Supplementary Tables 3A and 3B summarizes the sex, birth year, and FIV/FeLV infection status of DCHBV-positive and DCHBV-negative samples, respectively.

DCHBV testing using oral and rectal swabs

We found that 26.59% of blood samples were positive for DCHBV (Supplementary Tables 3A and 3B), indicating horizontal transmission of the infection in the shelter. Understanding the transmission route is essential to control DCHBV infection. To assess the presence of DCHBV in the oral and rectal cavities of cats that were DCHBV-positive on blood screening, we collected swab samples and performed qPCR analyses on those samples. Samples with Ct values <35 were defined as positive, whereas those with Ct values ≥35 were defined as negative. The Ct cutoff (<35) was selected based on the assay’s analytical sensitivity, which is commonly used in real-time PCR studies. Among the 37 cats with DCHBV-positive blood samples, 15 were positive (40.54%), 7 were negative (Ct values ≥35), and 16 were negative (undetermined) in oral swabs. Among the 36 cats with DCHBV-positive blood samples, 7 were positive (19.44%), 9 were negative (Ct values ≥35), and 20 were negative (undetermined) in rectal swabs. A positive correlation was observed between Ct values in blood and in oral swabs (Fig. 1A) but none between blood and rectal swabs (Fig. 1B). Notably, a strong positive correlation was observed between oral and rectal swabs (Fig. 1C). Similarly, viral quantification revealed a strong positive correlation between blood and oral swabs (Supplementary Fig. 1A). However, no significant correlation was observed for viral loads between blood and rectal swabs (Supplementary Fig. 1B) or those between oral and rectal swabs (Supplementary Fig. 1C).

Fig. 1.

Correlation diagrams of Ct values of domestic cat hepatitis B virus in blood, oral swabs, and rectal swabs. Ct values for blood, oral swabs, and rectal swabs were analyzed by real-time polymerase chain reaction. Correlation diagrams of Ct values between blood and oral swabs (A), between blood and rectal swabs (B), and between oral swabs and rectal swabs (C). R-square and P values are indicated on the bottom right of each diagram.

Genetic Characteristics of the Identified DCHBV

As of August 12, 2025, 32 cats had died at the shelter. Of the 31 liver samples collected, 27 were positive for DCHBV. Compared with the initial infection status based on blood screening for DCHBV in March 2025, 13 cats remained positive, 4 remained negative, 13 became positive, and 1 became negative, indicating the spread of infections in the shelter.

For genetic characterization, we amplified the polymerase region of DCHBV from positive DNA samples. Phylogenetic trees of the polymerase genome were constructed from the resulting sequences (Fig. 2). The DCHBV strain identified in the present study showed a maximum sequence identity of 99% and a minimum sequence identity of 98.6% with Japan/KT116/2021 (LC668427.1), whereas it showed a maximum sequence identity of 98.2% and a minimum sequence identity of 97.7% with Sydney2016 (MH307930.1), a reference strain of DCHBV [2]. In DCHBV strains isolated from the cat shelter, the maximum sequence identity was 100%, and the minimum was 99.2% with each other.

Fig. 2.

Phylogenetic tree of domestic cat hepatitis B virus (DCHBV) strains isolated from the cat shelter. DCHBV sequences from the livers of deceased cats were used to construct a phylogenetic tree, which included Japan/KT116/2021 (LC668427.1) and Sydney2016 (MH307930.1). The viral strains obtained in this study are indicated by the red bracket. Sample names correspond to specimen numbers as listed in Supplementary Table 2 and the year/month/date of death.

Gross pathological findings

Multiple white foci measuring approximately 1–5 mm in diameter were observed on the liver surface in many individuals (Fig. 3A and 3B). Other gross findings included evidence of hemorrhage, such as petechial hemorrhages, fibrin deposition on the liver surface, adherence of blood clots, and a distinct lobular architecture. The findings observed in each individual are summarized in Table 1.

Fig. 3.

Gross pathological findings in the liver with domestic cat hepatitis B virus infection in cats. (A) White spots (white arrows), petechiae (black arrows), and distinct lobular architecture were noted in Case No. 8. (B) White spots (white arrows), petechiae (black arrows), and distinct lobular architecture were observed in Case No. 3, formalin-fixed tissue.

Table 1. Gross pathological findings in the liver.

No.	White spots	Hemorrhage	Adherent blood clot	Fibrin deposition	Distinct lobular architecture
1	+	+	+	+	+
2	+	+	+	-	-
3	+	+	+	-	+
4	+	+	+	-	+
5	+	+	+	-	-
6	+	-	-	-	+
7	+	-	-	+	+
8	+	+	-	-	+

Pathological examinations

Portal areas presented with infiltration of inflammatory cells predominantly composed of lymphocytes, as well as fibrosis and bile duct proliferation (Fig. 4A). Some cases also showed neutrophilic infiltration around and within the bile ducts (Fig. 4B). Hepatocytes exhibited vacuolar degeneration, deposition of brown pigments, necrosis, and infiltration of inflammatory cells in the surrounding areas (Fig. 4C). In Case No. 4 (Table 2), positive amyloid deposition, as indicated by direct fast scarlet staining, was noted in the space of Disse or blood vessels walls. Six cases showed basophilic substance in the space of Disse (Fig. 4D). Hemorrhage and fibrin deposition were observed in interstitial tissue in multiple cases (Fig. 4E). In one case, a tumor-like proliferation of biliary epithelial cells was identified (Fig. 4F). The findings for each case are summarized in Table 2.

Fig. 4.

Histopathological figures of livers positive for domestic cat hepatitis B virus infection (A) Infiltration of lymphocytes and plasma cells and proliferation of bile ducts within the portal tract in Case No. 2. Hematoxylin and eosin staining (HE), scale bar=60 μm. (B) Infiltration of neutrophils in the bile ducts and surrounding area (Suppurative cholangitis) in Case No. 2, HE, scale bar=30 μm (C) Vacuolation of hepatocytes and infiltration of neutrophils in sinusoids were observed in Case No. 4. HE, scale bar=30 μm. (D) Weakly basophilic substance observed in the space of Case No. 1. HE, scale bar=30 μm. (E) Severe hemorrhage in the hepatic parenchyma in Case No. 4. HE, scale bar=100 μm. (F) Neoplastic cells of cholangiocarcinoma proliferate in the hepatic parenchyma in Case No. 3. HE, scale bar=100 μm.

Table 2. Histopathological findings in the liver.

No.	Portal tract				Hepatocytes
	Infiltration of lymphocytes and plasma cell	Cholangitis	Bile duct proliferation	Fibrosis	Vacuolation	Necorsis	Bile pigmentation
1	+	+1	+	+	+	+	+
2	+	+1	+	+	+	+	+
3	+	+1	+	+	+	+	+
4	+	+1	+	+	+	+	+
5	+	+2	+	+	+	+	+
6	+	-	+	+	+	-	+
7	+	-	+	+	+	+	+
8	+	-	-	+	+	-	+

1, Suppurative cholangitis; 2, Lymphocytic cholangitis.

IHC revealed DCHBV-positive signals in the cytoplasm of hepatocytes and biliary epithelial cells (Fig. 5A and 5B). On RNA-ISH, DCHBV-positive signals were observed in the nuclei of hepatocytes (Fig. 5C). In the negative controls for IHC and ISH, no positive signals were observed (Fig. 5A and 5C).

Fig. 5.

Immunohistochemistry (IHC) and RNA in situ hybridization (RNA-ISH) of liver samples from cats positive for domestic cat hepatitis B virus infection. (A) Hepatocytes were positive for domestic cat hepatitis B virus (DCHBV) core antigen in Case No. 8 (A1). Hepatocytes from an uninfected cat were negative for DCHBV core antigen (A2). IHC, scale bar=30 μm. (B) Biliary epithelial cells are weakly positive for DCHBV core antigen (arrows) in Case No. 8. IHC, scale bar=30 μm. (C) Positive RNA-ISH signals were observed in the nuclei of hepatocytes (arrows) in Case No. 8. The inset shows a higher-magnification image of positive cells (C1). Hepatocytes from an uninfected cat were negative for the DCHBV-specific probe (C2). RNA-ISH, scale bar=30 μm.

Logistic regression analysis

The associations between DCHBV status and birth year, sex, FIV status, and FeLV status (Supplementary Tables 3A and 3B) were evaluated by multivariate logistic regression analysis. However, no significant independent variables were identified in the analysis.

DISCUSSION

In this study, 46 of 173 cats at a shelter in Japan tested positive for DCHBV, yielding a 26.59% positive rate. As previously noted, the prevalence of DCHBV in Japan was lower than in other countries. Thus far, the highest prevalence rate reported worldwide was in Thailand at 18.5% [22]. To the best of our knowledge, this is the first confirmed outbreak of DCHBV in a cat shelter. The World Health Organization (WHO) defines a disease outbreak as the occurrence of more cases than would normally be expected in a defined community, geographical area, or season. Because the prevalence of DCHBV observed in this cat shelter (26.59%) greatly exceeded the previously reported prevalence in Japan (0.78%), this situation could reasonably be defined as an outbreak.

In analyzing the transmission route of DCHBV, viral DNA of DCHBV was detected in oral and rectal swabs from cats that tested positive for DCHBV on blood screening. Among the cats that tested positive in blood, 40.54% were positive on oral swabs, while 19.44% were positive on rectal swabs. We set a cutoff value of Ct 35 for the viral load correlation analysis. Since we sought to include values above this threshold in our analysis, we based our correlation plot on Ct values. According to Fig. 1A, the Ct values between blood and oral swabs were positively correlated. Thus, higher blood viral loads are associated with higher viral DNA levels in the oral cavity. However, no correlation was observed between the Ct values obtained from blood samples and those from rectal swabs. The absence of a correlation suggests the presence of extrahepatic tissue tropism of DCHBV and the possible involvement of an alternative receptor with which DCHBV can combine. Further studies are needed to clarify the transmission route of DCHBV and its in vivo kinetics in infected cats. All individuals who tested positive in oral swabs were also positive in rectal swabs, with a strong correlation observed between Ct values of the two sample types (Fig. 1B). These findings indicate that a higher viral genetic load in the oral cavity may be associated with a higher likelihood of viral genes in the rectum. Therefore, cats with high viremia for DCHBV are more likely to test positive for DCHBV on oral and rectal swabs.

These results aligned with those of a previous study in Thailand [35]. Due to the numerous samples of DCHBV-positive blood, oral swabs, and rectal swabs collected in this study, we could more precisely predict the relationship between viral loads in blood and those in oral and rectal swabs. In the previous study above, DCHBV was detected in oral and rectal swabs from cats at 1.0 × 10⁷ copies/mL. In contrast, the lowest viral load in blood among individuals who tested positive in both oral and rectal swabs was 790,000 copies/mL, which was lower than that in the previous study. The study demonstrated that viral DNA could be detected in oral and rectal swabs, even when the viral load in blood is below 1.0 × 10⁷ copies/mL. However, some cats tested negative for DCHBV on oral and rectal swabs, even when viral loads in blood exceeded 790,000 copies, suggesting that the correlation between blood viral load and oral and rectal swabs needs further investigation.

Considering the transmission route, a previous study demonstrated that children chronically infected with HBV contain detectable [14]. Furthermore, when specimens were intravenously inoculated into human hepatocyte-transplanted chimeric mice, the mice subsequently tested positive for HBV DNA, indicating the presence of infectious HBV in tears. Since viral isolation of DCHBV has not been achieved in this study, the presence of infectious virus in the oral cavity or rectum cannot be determined at this time. Therefore, we cannot conclude that the oral cavity and feces are infectious when viral DNA is detected in oral and rectal swabs. This is the major limitation of this study and awaits further analysis. However, considering the circumstances within the shelter, where all cats were neutered or spayed, thereby ruling out the occurrence of any blood-shedding aggressive behavior, alternative routes of transmission, such as saliva-mediated or fecal–oral transmission remain plausible. Further studies are needed to clarify these possibilities.

Based on the DCHBV testing of liver samples from deceased cats, a substantial number of individuals were found to have seroconverted to positive compared with the results of the initial blood-based DCHBV testing in March 2025. Even after August 12, 2025, additional liver samples from deceased cats had converted to positive on DCHBV testing. Taken together, these findings indicate that DCHBV infection likely continued to spread within the shelter. To support this, sequencing of the viral genomes derived from DCHBV in the cat shelter demonstrated that DCHBV identified from multiple deceased cats exhibited 99.2–100% sequence identity. Therefore, we conclude that a single strain of DCHBV has been spreading in the cat shelter.

Previous histopathologic findings reported in the livers of cats infected with DCHBV include hepatocellular vacuolar degeneration and inflammatory cell infiltration within the portal areas. Owing to these findings observed in the present case, it is concluded that hepatic injury associated with DCHBV infection had occurred in this cat as well [21, 34]. HBV infection has been associated with cryoglobulinemic vasculitis and hemorrhage in humans [19, 24]. Cryoglobulinemia causes systemic vasculitis that primarily affects small vessels. Reports on HBV-associated cryoglobulinemia indicate that approximately 50% of affected patients have chronic hepatitis and 30% have liver cirrhosis [20]. Although the cause of the hemorrhage in the present case remains unknown, the close association between DCHBV and HBV may suggest that viral infection could be a contributing factor to hemorrhage.

We here identified one cat that presented with cholangiocarcinoma, similar to our recent study [9]. Proposed mechanisms for HBV-induced cholangiocarcinoma include carcinogenesis promoted by chronic hepatitis, inactivation of tumor suppressor genes by HBx, and abnormal differentiation or transformation of cholangiocytes; however, the exact cause remains unclear. Previous reports indicate that HBV infection of hepatocytes promotes the development of cholangiocarcinoma [17, 28, 33]. Although the DCHBV–cholangiocarcinoma association remains unclear, the virus may have exerted some influence. Further studies are needed to clarify this possibility

We analyzed the risk factors associated with DCHBV acquisition. Thus, no remarkable independent factors associated with DCHBV infection were identified owing to the few cases involving FIV or FeLV infection, as well as the limited information available regarding cats. Future epidemiological studies collecting more detailed information potentially related to the infection are warranted to identify the risk factors for DCHBV.

Based on findings from HBV studies, for preventing the spread of DCHBV infection, it is crucial to avoid reusing injection needles and to spay and neuter in order to reduce contact with body fluids and blood associated with mating and fighting. In addition, given the outbreak of DCHBV and the suggested potential for horizontal transmission within shelters, DCHBV testing should be carried out when introducing new cats into shelters or multicat households, as well as prior to adoption, similar to current control measures for FIV and FeLV.

In conclusion, the observation of outbreaks within the cat shelter, detection of DCHBV in oral and rectal swabs, and phylogenetic analysis of circulating viral strains strongly indicate the potential for horizontal transmission in DCHBV infection. The findings underscore the importance of implementing effective infection control measures in multicat facilities. However, further studies are needed for verification.

CONFLICTS OF INTEREST

The authors declare no conflict of interest.