Screening for genetic heterogeneity in the interferon sensitivity determining region of the hepatitis C virus genome by polymerase chain reaction with melting curve analysis.
BACKGROUND
Although mutations in the interferon (IFN) sensitivity determining region (ISDR) of hepatitis C virus (HCV) have been reported to be useful as a predictive viral factor for IFN therapy in patients infected with HCV-1b, such laboratory research has not been favorably translated into the clinic. To promote such translation, we attempted the establishment of a rapid and simple polymerase chain reaction (PCR) combined with melting curve analysis (MCA) to screen for mutations in the ISDR and for the monitoring of HCV quasispecies.


METHODS
A PCR-MCA protocol was established using in-house primers and hybridization probes designed according to the results of direct sequencing of 34 HCV-1b samples. Then, the performance of PCR-MCA was verified by comparing with mutation profiles obtained by direct sequencing and sequencing after cloning.


RESULTS
The MCA assay revealed that melting temperature (Tm) was inversely correlated with the number of nucleotide (nt) and amino acid substitutions in the ISDR deduced on the basis of the results of direct sequencing. A boundary Tm of 58.0 degrees C allowed us to discriminate HCV genomes into two groups: one with a Tm >58.0 degrees C had no or a low number of nt substitutions, while the other genomes with a Tm <58.0 degrees C had a high number of nt substitutions, corresponding to wild-type in the former and mutant-type in the latter in respect of a clinical setting for IFN therapy. Moreover, this MCA assay provided precise discrimination of Tm between clones, reflecting the degree of the genetic complexity of HCV genomes.


CONCLUSIONS
This study indicates that the MCA assay is useful to rapidly and simply screen the mutational status of the ISDR of HCV, as well as in using the ISDR as one of the targets for discriminating the genetic complexity of HCV genomes. The MCA assay could also be applicable as a convenient and useful screen of the genetic heterogeneity of clones relating to HCV quasispecies.
Sasaki
Daisuke
D
Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, Sakamoto, Nagasaki City, Japan.
Sugahara
Kazuyuki
K
Inokuchi
Naoko
N
Yanagihara
Katunori
K
Hasegawa
Hiroo
H
Mori
Sayaka
S
Yamada
Yasuaki
Y
Kamihira
Shimeru
S
D000328:Adult; D000368:Aged; D005260:Female; D016679:Genome, Viral; D016174:Hepacivirus / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D019698:Hepatitis C, Chronic / Q000821:virology; D006801:Humans; D007372:Interferons / Q000494:pharmacology*; D008297:Male; D008875:Middle Aged; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics* / Q000302:isolation & purification; D044366:Transition Temperature
D012367:RNA, Viral; D007372:Interferons