Short Communication Long-term monitoring drug resistance by ultra-deep pyrosequencing in a chronic hepatitis B virus (HBV)-infected patient exposed to several unsuccessful therapy schemes M. Sede a,b,1 , D. Ojeda a,1 , L. Cassino a,b , G. Westergaard c , M. Vazquez c , S. Benetti d , F. Fay d , H. Tanno e J. Quarleri a,b,⇑ a Centro Nacional de Referencia para el SIDA, Departamento de Microbiología, Facultad de Medicina, Universidad de Buenos Aires, Argentina b Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina c Instituto de Agrobiotecnología Rosario (INDEAR), Santa Fe, Argentina d Centro de Diagnóstico Médico de Alta Complejidad (CIBIC), Rosario, Argentina e Servicio de Gastroenterología y Hepatología del Hospital Provincial de Centenario, Rosario, Argentina article info Article history: Received 17 October 2011 Revised 29 February 2012 Accepted 6 March 2012 Available online xxxx Keywords: HBV UDPS Resistance NRTI abstract The aim of this study was to analyze the spectrum and dynamics of low-prevalent HBV mutations in the reverse transcriptase (rt) and S antigen by ultra-deep pyrosequencing (UDPS). Samples were obtained from a chronically infected patient who was followed throughout a thirteen-year period. This technology enabled simultaneous analysis of 4084 clonally amplified fragments from the patient allowing detecting low prevalent (<1%) mutations during the follow-up. At baseline, HBV sequences were predominately wild-type. Under sequential HBV monotherapies including lamivudine, adefovir and entecavir, a high fre- quency of rtM204I mutation was detected initially as unique and then coexisting with rtM204V. Both mutations were statistically associated with rtA200V and rtV207I, respectively. Once the entecavir and tenofovir combined therapy was started, polymerase and consequently envelope gene mutations appeared at several positions at a higher frequency than before, including the entecavir resistance-asso- ciated mutation rtT184L. Ó 2012 Elsevier B.V. All rights reserved. Hepatitis B virus (HBV) is a circular partially double stranded DNA virus containing a reverse-transcriptase (rt) enzyme. HBV replicates via an RNA intermediate that is responsible for the gen- eration of several related viral variants called quasispecies, favor- ing the emergence of HBV drug resistance (Locarnini and Zoulim, 2010). HBV resistance to antiviral therapy can lead to HBV treat- ment failure and progression of liver disease. In general, this is gradually acquired through the selection of pre-existing variants with polymerase resistance-conferring mutations and the accumu- lation of new amino acid substitutions (Durantel, 2010). As the reading frames of the envelope and polymerase genes overlap, sev- eral of such resistance mutations could simultaneously alter the antigenicity of HBsAg inducing an ‘‘immune escape’’ (Chotiyaputta and Lok, 2009; Sheldon and Soriano, 2008; Sloan et al., 2008; Torresi et al., 2002). The estimated mutation rate of HBV is >2 Â 10 À4 base substitu- tions/site/year. This is about 100 times higher than that of other DNA viruses, while about 1000 times lower than that of RNA viruses (Chu and Lok, 2002). The emergence of drug resistance oc- curs more slowly for HBV than for HIV-1 and HCV, two other viruses that exist as quasispecies (Lai et al., 2003). This fact could be explained by the combination of incomplete inhibition of virus replication by some NRTIs, slow turnover of covalently closed cir- cular DNA in chronically infected hepatocytes and constraints on HBV evolution imposed by its overlapping reading frames, in addi- tion to the host immune response (Soriano et al., 2008). Direct sequencing and reverse hybridization (line probe assay [LiPA])-based methods are techniques commonly used for detect- ing HBV drug resistance mutations. Nevertheless, mutations must be present in viral quasispecies with a prevalence P20% to be de- tected by sequencing, and only known mutations are reported by LiPA. The aim of the present study was to analyze the spectrum and dynamics of low-prevalent HBV mutations at both the reverse transcriptase (rt) and S gene by massively parallel ultradeep pyrosequencing (UDPS) in a patient who did not respond to a 13- year antiviral therapy with different NRTIs. This technology is based on a massively parallel PCR amplification and pyrosequencing 0166-3542/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.antiviral.2012.03.003 ⇑ Corresponding author at: Centro Nacional de Referencia para el SIDA, Departa- mento de Microbiología, Facultad de Medicina, Universidad de Buenos Aires, Argentina. Tel.: +54 11 4508 3689/3671; fax: +54 11 4508 3705. E-mail address: quarleri@fmed.uba.ar (J. Quarleri). 1 These authors contributed equally to this work. Antiviral Research xxx (2012) xxx–xxx Contents lists available at SciVerse ScienceDirect Antiviral Research journal homepage: www.elsevier.com/locate/antiviral Please cite this article in press as: Sede, M., et al. Long-term monitoring drug resistance by ultra-deep pyrosequencing in a chronic hepatitis B virus (HBV)- infected patient exposed to several unsuccessful therapy schemes. Antiviral Res. (2012), http://dx.doi.org/10.1016/j.antiviral.2012.03.003