Hepadnavirus detected in bile and liver samples from domestic pigs of commercial abattoirs

Abstract

Background: Preliminary studies showed the prevalence of a virus similar to human hepatitis B virus (HBV-like) in swine from farms in China and the molecular evidence of Hepadnavirus infection in domestic pigs herds in Brazil. In this study, we genetically characterize the swine Hepadnavirus strains in swine from slaughterhouses located in certified abattoirs from Rio de Janeiro State, Brazil and evaluate its hepatotropic potential.

Results: Bile and liver samples from swine were positive for partial genome amplification (ORF S and ORF C), direct sequencing and viral load quantification. Sequencing of the gene encoding the surface antigen allowed classification of Hepadnavirus into genotypes, similar to HBV genotype classification. Indirect immunofluorescence confirmed the presence of HBsAg antigen in liver tissue sections.

Conclusions: So far our data suggest that commercial swine house an HBV-like virus and this relevant finding should be considered in studies on the origin and viral evolution.

Figure 1

Phylogenetic analysis of swine Hepadnavirus strains and Hepadnaviridae family. The Bayesian phylogenetic tree was constructed by using partial nucleotide sequence of open reading frame S (566 nt) of HBV and related viruses. For each sequence used, the GenBank accession number and country of isolation are shown. Multiple nucleotide sequence alignment was analyzed by using the Markov Chain Monte Carlo method implemented in the program MrBayes version 3.1.2 under GTR + G nucleotide substitution model, selected using the jModeltest program. The tree was rooted at midpoint. Posterior probabilities are shown at the branch label. Newly described swine hepadnavirus sequences are indicated. Scale bar indicates evolutionary distance.

Figure 2

Phylogenetic analysis of swine Hepadnavirus strains and HBV from human samples. The Bayesian phylogenetic tree was constructed by using partial nucleotide sequence of open reading frame S (972 nt) of HBV. For each sequence used, the GenBank accession number and country of isolation are shown. Multiple nucleotide sequence alignment was analyzed by using the Markov Chain Monte Carlo method implemented in the program MrBayes version 3.1.2 under GTR + G nucleotide substitution model, selected using the jModeltest program. The tree was rooted at midpoint. Posterior probabilities are shown at the branch label. Newly described swine hepadnavirus sequences are indicated. Scale bar indicates evolutionary distance.

Figure 3

Detection of hepatitis B virus surface antigen in liver of swine by immunofluorescence staining. Different frozen sections of HBsAg-positive cells (A and B) from liver of pigs stained in green using Alexa Fluor 488-conjugated anti-mouse IgG as secondary antibody. Evans Blue (red) and DAPI (blue) counterstained all liver sections. Images were examined under a confocal immunofluorescent microscope (Original magnifications x40). Liver sections from negative Hepadnavirus-DNA animals were used as negative controls (C).