Hepatitis B virus (HBV) is a partially double-stranded DNA virus from the Orthohepadnavirus genus and a member of the Hepadnaviridae family. HBV remains a major threat to global public health, affecting hundreds of millions of people and leading to high rates of chronic liver disease, cirrhosis, HBV-related cancer, and death. As of 2025, approximately 254 million people worldwide have chronic hepatitis B infection (Yan et al., 2025). China has a significant burden of HBV infection. According to a recent estimate, approximately 75 million people in China are infected with HBV, accounting for nearly one-third of the 254 million HBV-infected individuals worldwide. This high prevalence was attributed to the use of contaminated injections and blood transfusions between 1970 and 1992. With the introduction of the hepatitis B vaccine, the use of immunoglobulins, the advancement of medical technology, and increased awareness of HBsAg, the prevalence of HBsAg has decreased significantly from 9.7% in the early 1990s to 5.86% in the general Chinese population. Mother-to-child transmission has also decreased significantly, with a transmission rate of 0.23% in China. Among children aged 1–4, the HBsAg seroprevalence is currently only 0.3%, approaching the WHO’s 2030 elimination targets (Yan et al., 2025).

The evolutionary origins of HBV remain poorly understood, and no definitive zoonotic reservoir has been established. Phylogenetic studies suggest that human HBV infections have existed for approximately 15,000 years. In contrast, studies of endogenous avian hepadnavirus elements integrated into host genomes suggest a far deeper evolutionary history, extending back at least 40 million years (Locarnini et al., 2013). Hepadnaviruses infect a variety of mammals, including humans and certain primates (chimpanzees, gorillas, and gibbons) (Bonvicino et al., 2014; Lanford et al., 1998; Norder et al., 1996), several rodent species (including marmots, ground squirrels, and Arctic ground squirrels in the Americas) (Cohen et al., 1988; Seeger et al., 1984; Testut et al., 1996), and bats, which have been confirmed as natural hosts for numerous zoonotic pathogens (Lei et al., 2019; N’Dilimabaka, 2024; Nie et al., 2018). Although most animal hepadnaviruses exhibit strong host restriction, they serve as valuable models for understanding viruses’ origins and evolutionary dynamics. Notably, some animal HBV strains share a high genetic similarity with human HBV, suggesting that cross-species transmission and subsequent adaptation may have played a role in the emergence of human HBV infection. This evidence supports the hypothesis that human HBV may have originated from an ancestral zoonotic transmission (Norder et al., 1996).

Bat hepadnaviruses display notably greater genetic diversity than those found in other hosts. Multiple strains can coexist in a single bat species, and a single viral lineage may be distributed across diverse bat species, reflecting a long evolutionary history. Hepadnaviruses identified in New World bats share antigenic relatedness with HBV and can infect human liver cells (Drexler et al., 2013). Together with genetically diverse hepadnaviruses from New World rodents and non-human primates, these viruses exhibit a phylogenetic pattern where New World lineages often occupy basal positions. Rather than directly indicating a geographic origin, this pattern is more parsimoniously explained by the long-term preservation of early viral diversity under geographic isolation – a view supported by recent metagenomic studies revealing substantial hepadnavirus diversity in the New World (Ben et al., 2025). This pattern is compatible with the hypothesis that long-term geographic isolation may have preserved ancient viral diversity in the New World. Multiple host-switching events between bats and primates suggest that bats may be the source of the ancestral hepadnavirus in primates (Rasche et al., 2016). The frequent contact between bats and humans warrants continued vigilance and highlights the need for sustained virological surveillance.

Hepatitis D virus is a single-stranded circular RNA virus that lacks an independent envelope. It must rely on the HBsAg protein encoded by HBV to assemble into complete viral particles to achieve effective replication and transmission (Pan et al., 2023). Co-infection with both viruses often leads to more severe liver pathology and significantly increases the risk of severe hepatitis and hepatocellular carcinoma. Historically, this co-infection pattern has been considered exclusive to humans. However, recent studies have identified HDV-like viruses in various wildlife species. Their extensive genetic diversity suggests that the evolutionary history of HDV may be more complex than previously recognized.

We conducted a molecular epidemiological survey of BtHBV and BtHDV in bats from Yunnan Province, a key biogeographic region in China, to elucidate the genetic diversity, evolutionary patterns, and co-infection status of bat hepadnaviruses and deltaviruses.

Hepatitis B viruses and HDV infections collectively represent a substantial global public health burden, accounting for a significant proportion of liver-related morbidity and mortality worldwide. HBV is the primary causative agent of chronic hepatitis, cirrhosis, and hepatocellular carcinoma, whereas HDV relies exclusively on HBV-derived surface antigens for its replication cycle and transmission. Clinical evidence firmly establishes that HBV/HDV co-infection is associated with accelerated progression to severe hepatic complications and significantly higher liver-related mortality rates than HBV monoinfection (Cho et al., 2023).

Our findings provide important empirical data from southwestern China in this context. The high prevalence of BtHBV (36.86%) detected in Yunnan bats, particularly exceeding 40% in H. pomona, strongly suggests that this species may be a key reservoir sustaining enzootic transmission.

Phylogenetic and pairwise identity analyses revealed that all Yunnan BtHBV strains belong to the bat-specific evolutionary Lineage V. Within this lineage, distinct subclades were formed by the characterized strains BtHBV7/Nujiang71/Hpo/CHN, BtHBV7/Nujiang166/Hpo/CHN, and BtHBV7/Lincang10/Rfe/CHN. A phylogenetic analysis based on the amino acid sequences of the HBV polymerase (P) protein consistently supported this clustering pattern (Supplementary Figure 1). The pairwise nucleotide identities among these representative strains ranged from 81.84% to 82.55%. Within Lineage V, the Yunnan strains, while sharing high nucleotide identity (80.46%–94.13%) with strains reported from other Chinese provinces, such as Guizhou and Zhejiang, formed distinct, well-supported subclades (Drexler et al., 2013; He et al., 2013; Nie et al., 2018; Yang et al., 2018). A critical finding from our expanded phylogenetic analysis was the distant relationship between these bat viruses and other mammalian Orthohepadnaviruses. The entire Lineage V clade showed substantially lower sequence identities compared with viruses of other mammalian orders: identities ranged from 58.50% to 60.69% with primate viruses (Lineage II) and approximately 34.00% with rodent viruses (Lineage III). A gradient of relatedness was evident when compared to other bat virus lineages: identities remained high with other Asian bat viruses (Lineage IV, 80.18%–81.99%), but were markedly lower with bat viruses from geographically distant regions, such as the Americas (Lineage I, 54.31%–55.25%) (Drexler et al., 2013).

However, these bat viruses are only distantly related to other mammalian Orthohepadnaviruses. For instance, the recently reported rodent strain (C_AA050303.1) (Ben et al., 2025), which is positioned on a deep, basal branch of the phylogenetic tree and clearly separate from the bat virus clusters identified in this study, also exhibits a distant evolutionary relationship with the latter. The substantially lower sequence identity between Lineage V and viruses of other mammalian orders underscores a long history of independent evolution within bats, aligning with the hypothesis that bats harbored ancestral hepadnaviruses prior to their diversification in other mammalian orders, a finding that is further highlighted by the intermediate identity with geographically isolated bat viruses (Lineage I) and underscores the combined roles of evolutionary time and geographic separation in shaping current viral diversity. The formation of phylogenetically distinct subclades among the Yunnan strains–despite their high overall identity with strains from other provinces–further supports a pattern of regionalized diversification, hinting at multiple independent local transmission chains rather than widespread cross-regional spread.

The current understanding of HDV remains limited and is predominantly based on human studies. As a satellite virus, the HDV depends on HBV co-infection to complete its replication cycle and establish infection in humans (Khalfi et al., 2023). Clinical evidence indicates that HBV/HDV co-infection significantly increases the risk of fulminant hepatitis and hepatocellular carcinoma compared to HBV monoinfection (Chiou et al., 2022; Gerber et al., 2021; Khalfi et al., 2023). At least three distinct BtHDVs have been documented in bat populations in previous studies. PCR-based screening of the corresponding samples failed to detect typical hepatoviruses in these bats. In vitro experiments have confirmed that hepatitis viral proteins can efficiently support HDV replication and virion assembly (Bergner et al., 2021; Pan et al., 2023). Although experimental HDV infection has been achieved in woodchucks carrying woodchuck hepatitis virus, natural co-circulation of HBV and HDV has not been documented in wildlife (Freitas et al., 2012; Negro et al., 1989). Consequently, HDV remains the only human RNA virus without a confirmed natural reservoir in other animal species. Establishing the auxiliary virus status of HDV-like agents in wildlife is essential for clarifying their evolutionary origins, host spectrum, and zoonotic potential, thereby advancing our understanding of their ecology and pathogenesis.

In this study, HDV RNA was detected in a subset of samples that were positive for BtHBV. Despite a low detection rate (1.14%), with all positive cases restricted to H. pomona, this finding strongly implicates this bat species as a potential reservoir of HDV. Nevertheless, whether HBV functions as a helper virus for HDV in wildlife remains unclear.

In addition, we also successfully obtained a near-complete genome sequence of a bat HDV strain (designated BtHDV-YNNJ49). Comprehensive phylogenetic and pairwise identity analyses revealed that BtHDV-YNNJ49 forms a robust, distinct clade with a previously reported bat HDV from Peru (Bergner et al., 2021) yet exhibits substantial sequence divergence (37.50% nucleotide difference, ∼31.80% amino acid difference). This pattern defines BtHDV-YNNJ49 as a highly divergent member within a unique bat-associated deltavirus lineage. The extent of genetic divergence highlights its distinctiveness and contributes to the known diversity of deltaviruses. Collectively, these findings provide new genomic data relevant to understanding the evolutionary history and ecological range of HDV-related viruses.

Taken together, this study and existing evidence underscore that bats are a crucial natural reservoir for hepadnaviruses. Their substantial viral diversity provides key insights into the long-term evolution of these viruses. Furthermore, our detection of a highly divergent bat HDV lineage reinforces that the ecology of deltaviruses in wildlife constitutes a critical dimension in assessing viral diversity and potential zoonotic pathways.

In summary, this study confirms that bats in Yunnan harbor a diverse community of Orthohepadnaviruses belonging to the bat-specific Lineage V (BtHBV 7) and documents the co-circulation of a highly divergent bat HDV lineage. These findings substantially expand the known genetic spectrum of both virus families in wildlife. They underscore that the evolutionary history of hepadnaviruses and deltaviruses in bats is complex and merits further investigation. Future research should focus on expanded surveillance to map the geographic and host distribution of these lineages, and on evolutionary analyses to elucidate their deep-time diversification and ecological dynamics. This work provides a foundation for such studies and reinforces the importance of sustained virological surveillance in wildlife for understanding viral diversity and emerging disease risks.