After this incubation, sections were washed in PBS and labeled with fluorescein-conjugated goat antimouse or -rabbit secondary Ab (1:100; Bio-Rad, France). Sections were stained with Evans blue, mounted, and finally examined with a Leica DM RXE confocal microscope (Leica Microsystems, Wetzlar, Germany). Sequences were edited using the SeqMan program in the LASERGENE package PS 341 (DNASTAR, Inc., Madison, WI). Sequences were thereafter aligned with the corresponding region in sequences retrieved from GenBank. Phylogenetic analysis was carried out with the ClustalX program package version 2. Phylogenetic trees were

constructed using neighbor joining in the ClustalX package. Genotypes and subgenotypes were determined by analysis of the amplified fragments of the S gene with sequences from previously genotyped and subgenotyped strains.[25] The deduced amino acid sequence of the S gene region was used to determine the serotype, which was assessed from the amino acids at codons 122, 127, and 160.[26] Assessment of possible recombination was investigated by using the software packages, Simmonic 2005 v1.6 and SimPlot v3.5.1, both implementing PHYLIP (Phylogeny Inference Package v3.68; J. Felsenstein, Department of Genome Sciences, University of Washington, Seattle, WA[27]). We investigated the

natural HBV infection www.selleckchem.com/products/17-AAG(Geldanamycin).html in sera samples from two macaque species, the M. sylvanus and M. fascicularis, belonging to the Cercopithecidae family. Two hundred and sixty serum samples from macaques were tested for HBV DNA by PCR (Table 1). Of the 120 Asian M. fascicularis sera and 20 Moroccan M. sylvanus sera, all were HBV negative. By contrast, 25.8% (31 of 120) Mauritius M. fascicularis sera showed HBV DNA positivity with a viral load ranging from 101 to 106 HBV DNA copies/mL (mean viral load: 8.62 × 103 viral genome equivalents [VGE]/mL). Viremia subsequently could be performed for 6 HBV DNA–positive macaques, and after an 8-month period, all 6 animals maintained viral DNA levels Oxalosuccinic acid between 101 and 103, peaking at 106 HBV

DNA copies/mL for 1 animal (Fig. 1). The majority of animals exhibited only modest viremia variations over 8 months of follow-up. In addition, each quantitative PCR for HBV DNA detection was performed in triplicate and exhibited only limited variations. Next, we analyzed liver biopsies from Mauritius Island M. fascicularis and demonstrated the presence of HBV DNA sequences in 21 of 50 (42%) analyzed samples (Table 1). In addition, HBsAg and HBcAg was investigated by immunostaining in liver tissue of 9 HBV DNA–positive Mauritius macaques and showed, for all these animals, 20%-30% of strongly stained hepatocytes (Fig. 2). Liver histological examination did not reveal any significant pathological changes (data not shown).