Ultra-deep pyrosequencing detects conserved genomic sites and quantifies linkage of drug-resistant amino acid changes in the hepatitis B virus genome.
BACKGROUND
Selection of amino acid substitutions associated with resistance to nucleos(t)ide-analog (NA) therapy in the hepatitis B virus (HBV) reverse transcriptase (RT) and their combination in a single viral genome complicates treatment of chronic HBV infection and may affect the overlapping surface coding region. In this study, the variability of an overlapping polymerase-surface region, critical for NA resistance, is investigated before treatment and under antiviral therapy, with assessment of NA-resistant amino acid changes simultaneously occurring in the same genome (linkage analysis) and their influence on the surface coding region.


RESULTS
Serum samples obtained from chronic HBV-infected patients at pre-treatment and during sequential NA treatment with lamivudine, adefovir, and entecavir were analyzed by ultra-deep pyrosequencing (UDPS) using the GS-FLX platform (454 Life Sciences-Roche). The pre-treatment HBV quasispecies was not enriched with NA-resistant substitutions. The frequencies of this type of substitutions at pre-treatment did not predict the frequencies observed during lamivudine treatment. On linkage analysis of the RT region studied, NA-resistant HBV variants (except for rtA181T) were present in combinations of amino acid substitutions that increased in complexity after viral breakthrough to entecavir, at which time the combined variant rtL180M-S202G-M204V-V207I predominated. In the overlapping surface region, NA-resistant substitutions caused selection of stop codons in a significant percentage of sequences both at pre-treatment and during sequential treatment; the rtA181T substitution, related to sW172stop, predominated during treatment with lamivudine and adefovir. A highly conserved RT residue (rtL155), even more conserved than the essential residues in the RT catalytic motif YMDD, was identified in all samples.


CONCLUSIONS
UDPS methodology enabled quantification of HBV quasispecies variants, even those harboring complex combinations of amino acid changes. The high percentage of potentially defective genomes, especially in the surface region, suggests effective trans-complementation of these variants.
Biochemistry Department, Hospital Universitari Vall d'Hebron, Vall d'Hebron Institut de Recerca, Universitat Autònoma de Barcelona, Barcelona, Spain. frarodri@vhebron.net
Francisco
F
Rodriguez-Frías
David
D
Tabernero
Josep
J
Quer
Juan I
JI
Esteban
Israel
I
Ortega
Esteban
E
Domingo
Maria
M
Cubero
Sílvia
S
Camós
Carles
C
Ferrer-Costa
Alex
A
Sánchez
Rosendo
R
Jardí
Melanie
M
Schaper
Maria
M
Homs
Damir
D
Garcia-Cehic
Jaume
J
Guardia
Rafael
R
Esteban
Maria
M
Buti
D000328:Adult; D000368:Aged; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D017124:Conserved Sequence / Q000235:genetics*; D018432:Drug Resistance, Multiple / Q000235:genetics; D024882:Drug Resistance, Viral / Q000235:genetics*; D005260:Female; D008040:Genetic Linkage; D016679:Genome, Viral / Q000235:genetics; D023281:Genomics; D006515:Hepatitis B virus / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D019694:Hepatitis B, Chronic / Q000097:blood / Q000188:drug therapy / Q000821:virology; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D012194:RNA-Directed DNA Polymerase