3, 4 Moreover, Tupaia belangeri (a small mammal related to primates) has been successfully inoculated with human HBV and can develop a transient acute infection. However, the infection is rapidly resolved because of seroconversion to antibody to hepatitis B e antigen and antibody to hepatitis B surface antigen.5 The development of a new animal model susceptible to HBV infection that is closer to humans would be a very useful tool for anti-HBV
drug evaluation and for the improvement of anti-HBV therapies. In this respect, monkey species are very attractive, and chimpanzees were the first animals described for their ability to develop an acute HBV infection after the inoculation of sera from selleck products HBV-infected patients.6, 7 However, as this species is now protected, chimpanzees no longer represent an appropriate model for an antiviral research program. Some other great apes such as gibbons8 and baboons9 have demonstrated their susceptibility to HBV infection. More recently, we have suggested that macaques could represent a useful new primate model for selleck the study of HBV because we have demonstrated that HBV can successfully
replicate in Macaca sylvanus intrahepatically inoculated with an HBV DNA plasmid construct.10 The aims of our work were to confirm in vitro that human HBV can replicate in liver macaque cells and to demonstrate the relevance of macaque models for antiviral therapy evaluations. cccDNA, covalently closed circular DNA; CsCl, cesium chloride; DSL-DNA, double-stranded linear DNA; HBV, hepatitis B MCE virus; HBsAg, hepatitis B surface antigen; IFN, interferon; INSERM UMR-S, Institut National de la Santé et de la Recherche Médicale Unité Mixte de Recherche en Santé; LAM, lamivudine; ODN, oligodeoxynucleotide; PBMC, peripheral blood mononuclear cell; pgRNA, pregenomic RNA; PMH, primary macaque hepatocyte; PT, post-transduction; RC-DNA, relaxed circular DNA; TLR, Toll-like
receptor. Primary hepatocytes were isolated from the livers of cynomolgus macaques (Macaca fascicularis) as previously described.11 Primary macaque hepatocytes (PMHs) were next maintained in William’s medium (Invitrogen) supplemented with 10% fetal calf serum (Perbio), 50 U/mL penicillin/streptomycin (Invitrogen), 2 mM GlutaMAX (Invitrogen), 5 μg/mL bovine insulin, 5 × 10−5 M hydrocortisone hemisuccinate (Roche Diagnostics, Boehringer Mannheim), and 1.8% dimethyl sulfoxide (Sigma). Macaque livers were kind gifts from INSERM UMR-S 864 (Bron, France) and commissariat à l’Energic atomique (CEA) (Paris, France). PBMCs were separated by centrifugation on Lymphoprep (Abcys, France). Freshly isolated PBMCs were cultured at 5 × 106 cells/mL in Roswell Park Memorial Institute 1640 medium (Life Technologies, France) supplemented with 10% fetal calf serum (Hyclone, VWR, France), 2 mM L-glutamine (Invitrogen), and antibiotics (penicillin/streptomycin; Life Technologies, France).