We performed a survey on liver samples collected from 191 Iberian lynxes subjected to necroscopy in 2017–2023 throughout the Iberian Peninsula. Our screening also included 103 serum samples obtained from 100 lynxes affiliated with health programs in a 14-year time frame spanning 2010–2023. Both liver and serum samples were available for 7 lynxes. We obtained all samples from serum and tissue banks at the Center for Analysis and Diagnosis of Wildlife (Andalusia, Spain) and stored them at −80°C before shipment to the Department of Veterinary Medicine, University of Bari (Bari, Italy) for the analyses. We homogenized (10% wt/vol) liver tissues in Dulbecco modified Eagle’s medium and extracted viral DNA from the supernatant of the homogenates and from the serum by using the IndiSpin Pathogen Kit (Indical Bioscience GmbH, https://www.indical.com). We screened DNA extracts for the presence of DCH by using a quantitative PCR (10) and a qualitative PCR with panhepadnavirus primers targeting the polymerase ORF (11).

Our analyses revealed 1 (0.5%) of the 191 liver samples testing positive for viral DNA by qualitative PCR; none of the serum samples contained viral DNA. We traced the DCH-positive sample to a 7-year-old male Iberian lynx, raised in captivity in Andalusia in southern Spain (collection date March 2021). Samples for the animal included 4 serum samples collected over a 5-year period, 2016–2020; screening showed DCH DNA in only the fourth serum sample (collection date December 2020). We performed DNA enrichment for the DCH-positive liver sample (SPA/2022/Iberian lynx/296-23-81 strain) by using a rolling circle amplification technique with a TempliPhi 100 amplification kit (GE Healthcare, https://www.gehealthcare.com). We used the amplification product as a template for amplifying DCH genome fragments in PCR. A total of 500 ng of equimolar pooled PCR products made up the input for a library prepared using the Ligation Sequencing Kit V14 (Oxford Nanopore Technology, https://nanoporetech.com), according to manufacturer’s guidelines. We performed sequencing by using flongle flow cell R10.4.1 adapted in the MinION Mk1C platform (Oxford Nanopore Technology) for 24 hours.

Figure

Figure. Neighbor-joining phylogenetic tree based on the complete genome of domestic cat hepadnavirus (DCH) from a study of DCH infection in Iberian lynxes. We elaborated the tree by using the alignment...

We generated a complete DCH genome of the SPA/2022/Iberian lynx/296-23-81 strain (GenBank accession no. PP347721) measuring 3,184 bp in length. The Iberian lynx strain displayed 98.3% nucleotide identity to the Thailand strain CP87H_THA/2019 (GenBank accession no. MT506044) and <96.5% nucleotide identity to other DCH strains from Europe. On phylogenetic analysis, the strain SPA/2022/Iberian lynx/296-23-81 segregated with Thailand DCH strains within genotype A, into the distinct subtype A3, apart from other DCH strains from Europe, which segregated within either subtype A1 or subtype A2 (Figure).

We tested all serum samples at a dilution of 1:100 by using 2 in-house ELISA assays, one based on the recombinant core (DCHcAg) antigen and one based on the surface (DCHsAg) antigen (12,13), to evaluate the serologic response against DCH. The 4 serum samples collected at different points from the DCH-positive Iberian lynx reacted for DCHcAg IgG but not for DCHcAg IgM or DCHsAg IgG. Our testing detected viral DNA only in the last serum sample from the animal. That pattern is consistent with the status of HBV reactivation, characterized by a peak of viremia in persons with inactive infection, wherein the virus is barely detectable in the serum although replicating in the liver (3). Our analysis also revealed DCHcAg IgG in 32 (32.3%) of 99 serum samples, with the highest detection rate in adult free-ranging lynxes (7/16, 43.8%), and no DCHcAg IgM. Only 10 (31.2%) of the 32 serum samples with DCHcAg IgG had also DCHsAg IgG, suggesting clearance from the infection. In humans, HBVcAg IgG is persistent and indicative of exposure to HBV, regardless of the evolutive stage of the infection (14).

Our study results provide evidence for a wide circulation of DCH in the Iberian lynx population, with a seroprevalence rate (32.3%) higher than those observed in cats (25%) and dogs (10%) in Italy (12,13). We propose the need for additional studies to assess the effect of this virus on the health status of the Iberian lynx. Because cats are considered the primary reservoir of feline leukemia virus infection for the lynx population (9), it will be specifically important to investigate the role of domestic cats as a potential source of DCH infection for lynxes.

DCH appears to follow a pattern similar to that of HBV, presenting different types and subtypes based on nucleotide sequence diversity. In HBV, genotypes and subgenotypes might even play a crucial role in clinical outcomes, influencing disease evolution and drug resistance (15). In our study, the Iberian lynx DCH strain did not segregate phylogenetically with other EDCH strains from Europe detected in cats, raising questions as to the epidemiology of DCH and whether DCH subtype A3 exists in feline populations across the Iberian Peninsula or whether it is a hallmark of the Iberian lynx population. As more clinical and epidemiologic research on DCH unfolds, so might a greater understanding of whether different DCH types and subtypes exhibit phenotypic variations.

The viromes of closely related animal species, or even of species more distant in the evolutionary scale, are largely interconnected, with repeated events of interspecies transmissions and several examples of successful adaptation. Still, the patterns of infection and disease of viruses in a heterologous species remain unpredictable. The One Health model recommends intensifying the efforts in the study of animal pathogens to improve animal health and welfare and ensure animal conservation. This approach strongly applies to endangered animal species such as Iberian lynx.