Ultra-deep pyrosequencing of hepatitis B virus quasispecies from nucleoside and nucleotide reverse-transcriptase inhibitor (NRTI)-treated patients and NRTI-naive patients.
The dynamics of emerging nucleoside and nucleotide reverse-transcriptase inhibitor (NRTI) resistance in hepatitis B virus (HBV) are not well understood because standard dideoxynucleotide direct polymerase chain reaction (PCR) sequencing assays detect drug-resistance mutations only after they have become dominant. To obtain insight into NRTI resistance, we used a new sequencing technology to characterize the spectrum of low-prevalence NRTI-resistance mutations in HBV obtained from 20 plasma samples from 11 NRTI-treated patients and 17 plasma samples from 17 NRTI-naive patients, by using standard direct PCR sequencing and ultra-deep pyrosequencing (UDPS). UDPS detected drug-resistance mutations that were not detected by PCR in 10 samples from 5 NRTI-treated patients, including the lamivudine-resistance mutation V173L (in 5 samples), the entecavir-resistance mutations T184S (in 2 samples) and S202G (in 1 sample), the adefovir-resistance mutation N236T (in 1 sample), and the lamivudine and adefovir-resistance mutations V173L, L180M, A181T, and M204V (in 1 sample). G-to-A hypermutation mediated by the apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like family of cytidine deaminases was estimated to be present in 0.6% of reverse-transcriptase genes. Genotype A coinfection was detected by UDPS in each of 3 patients in whom genotype G virus was detected by direct PCR sequencing. UDPS detected low-prevalence HBV variants with NRTI-resistance mutations, G-to-A hypermutation, and low-level dual genotype infection with a sensitivity not previously possible.
Margeridon-Thermet
Severine
S
Department of Medicine, Stanford University, Stanford, CA, USA.
Shulman
Nancy S
NS
Ahmed
Aijaz
A
Shahriar
Rajin
R
Liu
Tommy
T
Wang
Chunlin
C
Holmes
Susan P
SP
Babrzadeh
Farbod
F
Gharizadeh
Baback
B
Hanczaruk
Bozena
B
Simen
Birgitte B
BB
Egholm
Michael
M
Shafer
Robert W
RW
D000225:Adenine / Q000031:analogs & derivatives / Q000627:therapeutic use; D000998:Antiviral Agents / Q000627:therapeutic use; D001483:Base Sequence; D017931:DNA Primers; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D024882:Drug Resistance, Viral; D014644:Genetic Variation; D005838:Genotype; D015658:HIV Infections / Q000150:complications / Q000188:drug therapy; D006509:Hepatitis B / Q000150:complications / Q000188:drug therapy / Q000821:virology*; D006515:Hepatitis B virus / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D019259:Lamivudine / Q000627:therapeutic use; D009154:Mutation; D063065:Organophosphonates / Q000627:therapeutic use; D016133:Polymerase Chain Reaction; D012194:RNA-Directed DNA Polymerase; D018894:Reverse Transcriptase Inhibitors / Q000627:therapeutic use*
D000998:Antiviral Agents; D017931:DNA Primers; D004279:DNA, Viral; D063065:Organophosphonates; D018894:Reverse Transcriptase Inhibitors; D019259:Lamivudine; C053001:adefovir; D012194:RNA-Directed DNA Polymerase; D000225:Adenine