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Vertical transmission from mother to child, the main route of chronic hepatitis B virus (HBV) infection in the East Asia, is considered one of the most important predictors for the response to antiviral therapies as well as its complications such as cirrhosis and hepatocellular carcinoma. Therefore, it is critical in both etiologic and prognostic aspects to confirm whether or not chronic HBV infection is acquired vertically. This study investigated whether mother-to-child infection could be proved by the phylogenetic analyses of HBV pre-S/S genes ever since several decades have elapsed in mother-child pairs with presumed vertical transmission. The pre-S and S regions of HBVs were compared and analyzed phylogenetically in a total of 36 adults (18 mother-child pairs) with chronic HBV infection. All of the isolates of HBV were genotype C and serotype adr. The divergence between mothers and offsprings was 0 to 1.5%. Phylogenetic trees revealed that 17 of 18 pairs (94%) with presumed vertical transmission were grouped into the same cluster. Vertical transmission from mother to child could be strongly suggested even in adults with a history of several decades of HBV infection using the phylogenetic analyses of pre-S and S genes. Graphical Abstract Keywords: Hepatitis B Virus, Multilocus Sequence Typing, Mutation, Infectious Disease Transmission, Vertical INTRODUCTION Chronic hepatitis B virus (HBV) infection is considered as a major risk factor of chronic liver diseases including cirrhosis and hepatocellular carcinoma (HCC). Parenteral infection during the adulthood is a main route of chronic HBV infection in western countries, whereas vertical (or perinatal) transmission from mother to child is thought of a critical one in the East Asia (1, 2). Of importance, the modes of infection, together with other factors such as viral titer, genotype, genetic mutations of HBV, age and gender of the host may determine the long-term clinical course of chronic HBV infection. The mode of vertical transmission, in particular, is one of the most important determinants for the therapeutic responsiveness to antiviral therapies, or the natural course in terms of hepatitis e antigen seroconversion and even the development of complications (3, 4, 5). Previous studies using a comparison of HBV DNA sequences and phylogenetic analyses have shown that HBV of chronically infected children originates mainly from their respective mothers or fathers (6, 7, 8). Although there has been hitherto a molecular evidence of vertical transmission of HBV in the pairs of mother-young child, it is improbable to identify exactly the mode of infection (especially vertical vs non-vertical) in real clinical settings because medical information from the adults for the source of HBV, relying on history taking alone, is highly obscure and there could be recall bias. There have been, so far, no reports about whether it was possible to demonstrate the molecular evidence of vertical transmission of HBV even after several decades of infection. Taken together, 'Vertical or non-vertical transmission' is so crucially informative that it enables clinicians to manage effectively the patients with chronic HBV infection and also foresee the prognosis or therapeutic responses. Therefore, this study was conducted to prove the vertical transmission by showing the molecular closeness of HBVs between adult patients with clinically presumed vertical transmission and their respective mothers with the phylogenetic analyses of pre-S/S genes of HBV. It must provide valuable information to design a further strategy of management if the mode of HBV infection can be disclosed even after long-term period of infection.