new mutation rtA181C (+rtL180M+rtM204V) etv resistance

Abstract
Background: Current data suggest that entecavir (ETV) resistance is caused by mutation at positions rt184, rt202, or rt250 in conjunction with mutation at position rt204 of HBV reverse-transcriptase domain. Here, we studied a novel HBV strain with mutations rtL180M+A181C+M204V selected in an ETV-refractory genotype C HBV-infected patient. Methods: HBV reverse-transcriptase gene was amplified by nested PCR and cloned into pGEM-Teasy vector for sequence analysis. Replication-competent wild-type (WT) and mutant HBV genomic amplicons were constructed and transfected into HepG2 cells. The transfected cells were cultured in the presence or absence of serially-diluted nucleos(t)ide analogs. Viral replication capacity was assessed by measuring secreted HBV DNA using quantitative PCR and validated by measuring intracellular HBV replicative intermediates using Southern blotting. Results: As shown in figure, this 43-year-old patient successively developed LAM- and ADV-resistance after 36 months of LAM and 14 months of ADV sequential therapies. Switching-to ETV suppressed virus to an undetectable level and normalized his ALT level. However, the patient developed virologic and biochemical breakthrough in 29 months. An HBV stain with novel mutational pattern rtL180M+A181C+M204V was detected and soon overwhelmed in virus pool. Compared with WT HBV, rtL180M+A181C+M204V mutant decreased >1000-fold susceptibility to LAM and 85.6 ± 10.8-fold susceptibility to ETV in vitro. By contrast, the mutant remained susceptible to ADV and TDF. In addition, another HBV strain harboring rtA181C (rtL180M+A181C+S202G+M204V) was detected in a patient who developed virologic breakthrough during rescue ETV therapy for LAM and ADV sequential treatment failure. Conclusion: HBV strain harboring mutation rtA181C in junction with rtL180M+rtM204V could be selected during ETV treatment and might confer resistance to ETV.

[Yasin76]

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