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In vitro replication competence of a Hepatitis B genotype D/A recombinant virus: dissimilar biological behaviour regarding its parental genotypes Trinks J1,?,*, Sugiyama M2,3, Tanaka Y2, Kurbanov F2, Benetucci J4, Giménez E5, Weissenbacher M C1,6, Mizokami M3, Oubiña J R1 1Instituto de Microbiología y Parasitología Médica (IMPAM), Universidad de Buenos Aires (UBA) - Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires; 2Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Kawasumi, Mizuho, Nagoya; 3The Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, Kohnodai, Ichikawa, Japan; 4Fundación de Ayuda al Inmunodeficiente (FUNDAI), Buenos Aires; 5Hospital Zonal General de Agudos ´Dr. Isidoro G. Iriarte´, Quilmes; 6National Academy of Medicine, Buenos Aires, Argentina. ?Current address: Instituto de Ciencias Básicas y Medicina Experimental (ICBME), Hospital Italiano de Buenos Aires, Buenos Aires, Argentina. *e-mail: julietatrinks@yahoo.com.ar Information regarding the effects of HBV recombinant genomes on the clinical, prognostic and therapeutic aspects of the infection is still lacking. In a previous study, our group characterized the first HBV genotype D/A recombinant genome from the American continent, which exhibited a HBV/A2 DNA region (nucleotide positions 147 to 636 of the S gene) inserted in a backbone corresponding to HBV/D3. Hence, the aim of this study was to preliminary analyse the very early replication dynamics of the infection of this HBV D/A recombinant in comparison to its parental genotypes (HBV/D and HBV/A) and to a highly replicative genotype (HBV/C) in an in vitro experimental system. pUC19 plasmids deprived of promoters and carrying 1.24-fold the HBV genome of HBV/A2, D3, C and D/A recombinant were constructed. Huh7 cells were transfected with these plasmids and transfection efficiency was monitored by using a pTARGET®-GFP expression vector. At 24 and 72 hours post-transfection (hpt), ALT/AST levels, viral load (COBAS TaqMan HBV Test, Roche), HBsAg and HBeAg (Abbott) were determined in supernatants. Cells were lysed at 72 hpt and the density of core associated-HBV DNA was compared by Southern blot hybridization. Except for Southern Blot, all experiments were conducted twice for each clone. Student?s t-test was used to compare the means and standard deviations between any pair of samples. A p value <0.05 was considered statistically significant. All clones were able to replicate in Huh7 cells and secrete HBsAg and HBeAg. The in vitroreplication kinetics of the D/A recombinant differed from its parental genotypes, exhibiting: a) higher extracellular levels of HBV DNA; b) similar to A2 or higher than D3 (p<0.0001) secreted HBsAg; and c) higher than both genotypes (p<0.0001) HBeAg values. Southern blotting of intracellular core-associated HBV DNA confirmed that the level of the recombinant was higher than those from its parental genotypes. Transfection efficiency and cell viability ?monitored by ALT/AST levels- were similar for all clones (p>0.05). Negative controls processed in parallel confirmed the specificity of the above mentioned results. Since this recombinant was obtained from an injecting drug user -an increasing vulnerable community in Argentina-, these results imply an unprecedented call of singular relevance for the regional public health. Further in vivo studies are urgently needed to determine the pathogenicity of these replicative competent clones.