This study was approved by the Institutional Review Board. TABLE 1 Correlation of serum HBV DNA, HDV RNA, and HBsAg levels and age with disease courses Detection of selleck chemicals Abiraterone original and novel dominant HDV strains and determination of variant-specific restriction sites. The original and novel dominant HDV quasispecies before (early time point) and after (late time point) serum ALT elevation were cloned from the sera of CHD patients. Detection of dominant viral strain RNA by reverse transcription (RT)-PCR and PCR cloning were performed as previously reported (32). More than 10 colonies from each time point of each case were randomly selected, and their sequences were analyzed with an ABI 373A sequencer (Perkin-Elmer Cetus Corp., Norwalk, CT).

The sequences of the original and novel dominant HDV quasispecies were compared, and restriction enzymes that were able to differentiate these quasispecies were determined by large-scale screening as described in the following section. Identification and functional analysis of HDV RNA quasispecies. In order to have equal chances to detect all of the HDV quasispecies, the primers for RT-PCR were synthesized on the basis of the most conserved parts of the HDAg sequences of the original and novel dominant quasispecies. Primer 120 (5��-ATG CCA TGC CGA CCC GAA GAG GAA-3��) and primer 214 (5��-CTC AGG GGA GGG TTC TCC GAC A-3��) were synthesized according to the conserved region of the hepatitis delta antigen coding region.

The amplified PCR products derived from HDV-infected serum obtained at different time points were ligated into plasmid vector pCR2 (Original TA Cloning Kit; Invitrogen Corporation, Carlsbad, CA) and then transformed into competent Escherichia coli strain DH5�� (Gibco BRL, Life Technologies, Gaithersburg, MD) according to the manufacturer’s instructions (32). More than 100 HDV colonies from each case at each time point were randomly selected and subjected to restriction fragment length polymorphism (RFLP) differentiation based on quasispecies-specific restriction enzyme digestion. The determination of quasispecies-specific restriction enzymes was based on comparisons of the nucleotide sequences of dominant HDV species obtained at the pre- and post-ALT elevation stages. For patient I (infected with HDV genotype 1), the 446-bp PCR products amplified with primers 214 and 120 were cut with restriction enzymes BlpI and HpyCH4V (TGCA), respectively.

The BlpI-cut PCR products of the original dominant species generated 200- and 246-bp fragments, while the PCR products of the novel dominant species could not be cut by restriction enzyme BlpI. Using the same method, the PCR products cut by HpyCH4V showed inverse results. For patient II (infected with HDV genotype 2), the PCR products cut by restriction enzyme FokI also gave two different RFLP Anacetrapib patterns.