Abstract
Background and AimsAlthough the advent of ultra-deep sequencing technology allows for the analysis of heretofore-undetectable minor viral mutants, a limited amount of information is currently available regarding the clinical implications of hepatitis B virus (HBV) genomic heterogeneity.MethodsTo characterize the HBV genetic heterogeneity in association with anti-viral therapy, we performed ultra-deep sequencing of full-genome HBV in the liver and serum of 19 patients with chronic viral infection, including 14 therapy-naïve and 5 nucleos(t)ide analogue(NA)-treated cases.ResultsMost genomic changes observed in viral variants were single base substitutions and were widely distributed throughout the HBV genome. Four of eight (50%) chronic therapy-naïve HBeAg-negative patients showed a relatively low prevalence of the G1896A pre-core (pre-C) mutant in the liver tissues, suggesting that other mutations were involved in their HBeAg seroconversion. Interestingly, liver tissues in 4 of 5 (80%) of the chronic NA-treated anti-HBe-positive cases had extremely low levels of the G1896A pre-C mutant (0.0%, 0.0%, 0.1%, and 1.1%), suggesting the high sensitivity of the G1896A pre-C mutant to NA. Moreover, various abundances of clones resistant to NA were common in both the liver and serum of treatment-naïve patients, and the proportion of M204VI mutants resistant to lamivudine and entecavir expanded in response to entecavir treatment in the serum of 35.7% (5/14) of patients, suggesting the putative risk of developing drug resistance to NA.ConclusionOur findings illustrate the strong advantage of deep sequencing on viral genome as a tool for dissecting the pathophysiology of HBV infection.
Highlights
Hepatitis B virus (HBV) is a non-cytopathic DNA virus that infects approximately 350 million people worldwide and is a main cause of liver-related morbidity and mortality [1,2,3]
We determined the polymerase chain reaction (PCR)-amplified hepatitis B virus (HBV) sequences derived from the expression plasmid using high-fidelity Taq polymerase to take the PCR-induced errors as well as sequencing errors into consideration
Viral sequences determined by the conventional Sanger method were used as reference sequences for aligning the amplicons obtained by ultra-deep sequencing
Summary
Hepatitis B virus (HBV) is a non-cytopathic DNA virus that infects approximately 350 million people worldwide and is a main cause of liver-related morbidity and mortality [1,2,3]. A highly characteristic nature of HBV infection is the remarkable genetic heterogeneity at the inter- and intra- patient level. The latter case of variability as a population of closely-related but nonidentical genomes is referred to as viral quasispecies [5,6]. Acute infection with the G1896A pre-C mutant represents a high risk for fulminant hepatic failure [10,11] These facts clearly illustrate the clinical implications of certain viral mutation, increasing evidence strongly suggests that the viral genetic heterogeneity is more complicated than previously thought [12,13]. The advent of ultra-deep sequencing technology allows for the analysis of heretoforeundetectable minor viral mutants, a limited amount of information is currently available regarding the clinical implications of hepatitis B virus (HBV) genomic heterogeneity
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