A new hepadnavirus in domestic dogs

Overall, a total of 40 out of 635 (6.3%) sera tested positive by quantitative screening (qPCR) and domestic dog hepadnavirus (DDH) DNA was found at low titer . The geometric mean value of DDH viremia in canine sera was 2.70 × 10² copies per ml (min range 1.36 × 10²—maximum 4.03 × 10⁴ DNA copies per mL). Of 40 dogs infected with DDH, we were able to retrieve information on hematological and serum biochemical parameters of 23 animals (Supplementary Table 1). Eleven dogs (47.8%) had either an increase in alanine transaminase and/or aspartate transaminase and 14 (60.8%) had an increase in alkaline phosphatase (Table 1). Ninety-six% (38/40) of the DDH-positive dogs were over 1 year old, 57% (23/40) were over 7 years old, and 70% (28/40) were male.

The complete genome of strain DDH 570/ITA (GenBank accession no. MZ201309) was 3184 bp long (Fig. 1). The virus showed 98.0% nucleotide (nt) identity at the whole genome level with Italian DCH strain ITA/2018/165-83 (GenBank accession no. MK117078) and 96.9% nt identity with the Australian DCH reference strain Sydney2016 (GenBank accession no.MH307930), respectively. For an additional DDH strain, 43/ITA, we were able to reconstruct a contig spanning approximately 80.7% (2572 nt) of the genome, including partial C and P genes and the complete S gene. Sequence analysis showed that the DDH 43/ITA strain was 97.7%, 97.8% and 98.7% nt identical to the C, P and S gene sequences of the DDH 570/ITA strain, respectively. . During the phylogenetic analysis of ORFs C, S and P, the two DDH strains were closely related to each other and mixed with other DCH strains (Fig. 2). These results suggest the possibility of free circulation of hepadnaviruses among domestic carnivores, rather than different viral species with a specific host range. A similar situation has been observed in equines, with similar hepadnaviruses circulating in zebras and donkeys^ten.

Phylogenetic trees based on three different genetic targets, (a) kernel (partial length), (b) polymerase (partial length) and (vs) surface (full length), hepadnaviruses extracted from the GenBank database. GenBank accession numbers are provided for reference strains. The trees were generated using the maximum likelihood method, the Hasegawa-Kishino-Yano model with gamma distribution and invariant sites, and bootstrapping up to 1000 replicates. Bootstrap values ​​> 70% are displayed. Italian DDH strains 43/ITA and 570/ITA (GenBank accession number MZ201309) are indicated by black bullets. White sucker hepadnavirus (NC_027922) was used as an outgroup. The scale bar indicates nt substitutions per site.

A limitation of this study was the fact that we only identified DDH in dog sera, while we did not test tissues or organs. Screening of liver biopsies from animals with liver disease could be helpful in gathering more accurate information about this virus. However, when tested in Western blot (WB), a subset (not = 20) of the DDH-positive (viremic) sera, a total of 13/20 (65.0%) reacted with the DCH core protein. Of these, 2 sera (15.4%, 2/13) were positive only for IgM, 3 samples (23.0%, 3/13) reacted only for IgG and 8 additional sera (61.5% , 8/13) showed reactivity for both IgM or IgG.

HBV-specific antibodies have already been reported in dogs in two studies dating from the 1980s^5.6. In a 1983 study in the United States, a total of 172 sera from 33 animal species were screened and HBV S antigen (HBsAg) and antibodies against protein C (anti-HBc) were not detected. . However, 48% (82/172 serum samples from 19 animal species) contained antibodies against Protein S (anti-HBs), including 75% of the tested dog sera, collected from inbred Maryland Beagles.⁵. Similar results were reported in a 1983 study in Taiwan. Anti-HBV antibodies to HBV were detected in 39/66 (59.1%) stray dog ​​sera, with older animals being more prone to HBV infection⁶. Dog sera tested negative for HBsAg and anti-HBc. Similarly, in a small sero-epidemiological survey in Iraq, 2020, 9% (7/78) of stray dogs had anti-HBs and serological positivity correlated with increased liver markers⁸.

In a 2019 study in Brazil, using an ELISA kit capable of detecting HBV markers (HBsAg and total anti-HBc), HBsAg was detected in 5.8% (11/189) of stray dogs and the Total anti-HBc was detected in 10% (19/189) of animals. Using two PCR assays with primers targeted to the preS/S1 genomic region and the HBV core gene, hepadnavirus DNA was identified in 10% of dogs. On partial sequencing (approximately 1 kb) of the S gene, the virus was 98.2% similar at the nt level to HBV. In addition, HBV-like virus DNA was detected in the sera of 5.9% (8/136) of pigs⁷. Notably, the Brazilian hepadnavirus sequence was far (61.2% nt) from the sequences of the DDH strains identified in our survey.

Interpretation of these data is difficult, given the diversity of the sampling in terms of number of animals and inclusion criteria, and the different diagnostic strategies. In all of the above studies, antigens and immunological reagents directed against HBV were used. Some authors have hypothesized that the presence of anti-HBs in animal sera could be due to nonspecific reactivity (false positivity), antigenic cross-reactivity with HBV-like viruses, or immunization against HBsAg disseminated in the environment.⁵. Interestingly, antigenic cross-reactivity was observed between DCH and HBV¹¹. Using a polyclonal serum specific for HBV basic protein, the DCH antigen was identified in feline tissues by immunostaining. Therefore, it is possible that the serological findings reported in previous studies in dogs are in fact due to cross-reactivity with HBV-like viruses, rather than exposure of dogs to HBV.

With the exception of canine adenovirus type 1, the causative agent of canine infectious hepatitis (Rubarth’s disease), few other viruses have been reported to target the liver of dogs.¹². Since hepadnaviruses are generally hepatotropic, it will be important to assess whether DDH also has the potential to impair liver functionality and damage liver tissue in dogs. In our study, altered liver markers were found in DDH-positive dogs (Table 1). Additionally, we hypothesized that a potentially immunosuppressive condition of dogs, infection with Leishmaniaspp., may be associated with DDH, as observed for immunosuppressive retroviruses in humans and cats¹³. However, only 12/40 DDH-positive dogs (30%) had specific antibodies for Leishmaniaagainst 170/635 (26.7%) of the entire serum collection.

Finally, since hepadnaviruses in humans and cats are capable of causing long-term infections^3.4 and this may amplify the possibility of transmission of the virus through blood and body fluids, it will be important to include DDH in diagnostic canine blood screening for dogs, to minimize potential risks, if any, to the canine health.