An heteroduplex mobility analysis assay based on capillary electrophoresis for the study of HCV quasispecies.
The quasispecies nature of the hepatitis C virus (HCV) genome is central to the transmission, persistence and pathogenesis of the infection. Heteroduplex mobility analysis (HMA) is a simple and an inexpensive technique for the qualitative and quantitative analysis of genetic variation of viral quasispecies. An original HMA for the HVR1 region of HCV was developed, based on a semi-automated, non-radioactive capillary electrophoresis system, which allows the processing of large numbers of samples in short times, the accurate measure of mobility shifts and the quantitation of heteroduplexes. A set of 120 HVR1 clones of known sequence was used to develop the assay, which was tested on HVR1 sequences amplified directly from sera of 17 HCV-infected patients. HVR1 sequence divergence directly correlated with the heteroduplex mobility ratio (HMR) of hybrid molecules between six and 40 mismatches. Heteroduplexes between one and six mismatches were resolved, although HMRs were not proportional to base changes, likely due to an effect of type and position of the substitutions. The assay sensitivity was 1% of the total sample size. This assay may allow the application of quasispecies analysis to a wider range of clinical and basic investigations.
Rossi
Laura
L
ASAEV-Associazione Studio Avanzato Epatiti Virali, via G. Garibaldi 13, 24040, BG, Bonate Sotto, Italy. laura.asaev@tiscali.it <laura.asaev@tiscali.it>
Leveri
Michela
M
Marinelli
Daniela
D
Belli
Luca
L
Civardi
Emilio
E
Silini
Enrico M
EM
D000328:Adult; D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000627:therapeutic use; D001483:Base Sequence; D019075:Electrophoresis, Capillary; D005260:Female; D014644:Genetic Variation; D016174:Hepacivirus / Q000145:classification* / Q000235:genetics*; D019698:Hepatitis C, Chronic / Q000188:drug therapy / Q000821:virology; D020180:Heteroduplex Analysis / Q000295:instrumentation / Q000379:methods; D006801:Humans; D007372:Interferons / Q000627:therapeutic use; D016031:Liver Transplantation; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D012254:Ribavirin / Q000627:therapeutic use; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D014764:Viral Proteins / Q000235:genetics*
D000998:Antiviral Agents; C400562:HVR1 protein, Hepatitis C virus; D014764:Viral Proteins; D012254:Ribavirin; D007372:Interferons