Hepatitis C virus persistence after spontaneous or treatment-induced resolution of hepatitis C

Abstract

It is presumed that resolution of hepatitis C, as evidenced by normalization of liver function tests and disappearance of hepatitis C virus (HCV) RNA from serum, as determined by conventional laboratory assays, reflects virus eradication. In this study, we examined the expression of the HCV genome in the sera, peripheral blood mononuclear cells (PBMC), and, on some occasions, monocyte-derived dendritic cells (DC) long after resolution of hepatitis C by using a highly sensitive reverse transcription (RT)-PCR-nucleic acid hybridization (RT-PCR-NAH) assay. The samples obtained from 16 randomly selected patients (5 with spontaneous and 11 with treatment-induced resolution), monitored for up to 5 years, were studied by qualitative and semiquantitative RT-PCR-NAH and by real-time RT-PCR to detect the HCV RNA positive strand. The replicative HCV RNA negative strand was examined in PBMC after culture with a T-cell proliferation stimulating mitogen. The findings show that HCV RNA was carried in the convalescent-phase sera and/or PBMC in all 16 individuals investigated. Also, DC from six of seven patients were reactive for the HCV genome. Importantly, traces of the HCV RNA negative strand, suggesting progressing virus replication, were detected in the majority of mitogen-stimulated PBMC, including four samples collected 5 years after recovery. Sequencing of the HCV 5' untranslated region fragment revealed genotype 1b in four of nine individuals examined and genotypes 1a and 2a in three and two patients, respectively. These results imply that HCV RNA can persist at very low levels in the serum and peripheral lymphoid cells and that an intermediate replicative form of the HCV genome can persist in PBMC for many years after apparently complete spontaneous or antiviral therapy-induced resolution of chronic hepatitis C.

FIG. 1.

Effect of mitogen stimulation on the detection of HCV RNA in PBMC from individuals with resolved chronic hepatitis C. Peripheral lymphoid cells isolated from individuals with spontaneous (cases 1, 3, and 4) and therapeutically induced (case 7) resolution of chronic hepatitis C were treated (T) by culture with IL-2 and PHA for 72 h or were untreated (UT). Total RNA (3 μg) was transcribed to cDNA and amplified by nested PCR with HCV 5′-UTR primers, and the amplicons were detected by Southern blot hybridization. For contamination controls water was added instead of cDNA, amplified by direct and nested reactions (NW), and mock (M) treated as test RNA. Negative controls included untreated and treated PBMC from a healthy donor. The positive controls included RNA extracted from PBMC of a patient with clinically evident chronic hepatitis C (CH) and serial dilutions of a recombinant HCV UTR-E2 fragment as quantitative standards (vge). Positive signals showed the expected 244-bp oligonucleotide fragments. Numbers under the panel represent relative densitometric units (DU) given by hybridization signals.

FIG. 2.

Detection of HCV RNA in parallel serum and PBMC samples from individuals convalescent from chronic hepatitis C. Sera (S) and IL-2- and PHA-stimulated PBMC (P) from individuals who (a) resolved hepatitis spontaneously and were monitored for at least 12 months thereafter or (b) resolved hepatitis due to antiviral therapy and were monitored for 60 months after the apparently complete virological recovery were tested for HCV RNA by nested RT-PCR-NAH assay. Serum and PBMC from a healthy individual (Healthy) and water (DW and NW) and cDNA-free mock (M) samples were used as negative controls. Positive samples showed the expected 244-bp band.

FIG. 3.

Identification of HCV RNA in parallel samples of serum and PBMC by RT-PCR-NAH using both UTR and E2 region-specific primers. Total RNA was extracted from sera (S) and PBMC (P) from case 2, with self-limited acute infection, and from case 13, with therapeutically induced recovery from chronic hepatitis. Serum and PBMC from a healthy individual and a patient with clinically evident chronic hepatitis C (CH) served as negative and positive controls, respectively. Water (DW and NW) and cDNA-free mock (M) samples were included as contamination controls. Positive samples showed the expected 244-bp band for UTR and the 442-bp fragment for the E2 region by Southern blot hybridization analysis.

FIG. 4.

Nucleotide sequence alignment of the 5′-UTR fragments of HCV RNA amplified from serum or PBMC of individuals with apparently complete serological resolution of chronic hepatitis C following antiviral therapy. Samples from case 12 were obtained 15 (PBMC) or 30 months (serum) after sustained response to treatment. In case 16, the first sample was taken during symptomatic chronic hepatitis (Chronic) and the second 12 months after evidence of sustained clinical resolution of hepatitis. The sequences obtained were aligned with the prototype HCV genotype 1b (10). Nucleotides in the sequences from the patients' samples identical to those in the HCV genotype reference (top line) are shown as dashes, and differences are identified by letters.

FIG. 5.

Expression of the HCV RNA positive strand in monocyte-derived DC from individuals with long-term recovery from chronic hepatitis C. Cultured DC (C) and concentrates of their culture supernatant (S) were tested for HCV RNA by nested RT-PCR-NAH. Three micrograms of DC RNA and all RNA extracted from the pellet obtained after ultracentrifugation of 10 ml of DC culture supernatant were used for analysis. Water (DW and NW) instead of cDNA and mock (M) sample treated as a test RNA sample served as negative controls, while DC and their culture supernatant derived from a patient with chronic hepatitis C (CH) were used as positive controls. Hybridization signals showed 244-bp bands.

FIG. 6.

Detection of the HCV RNA negative strand in PBMC from individuals with clinical and apparently complete virological resolution of hepatitis C. Total RNA was extracted from IL-2- and PHA-stimulated PBMC isolated from individuals with self-limited (case 4) or therapeutically induced (cases 7, 11, 12, and 16) recovery from hepatitis and from control untreated (UT) and IL-2- and PHA-treated (T) PBMC obtained from a healthy donor and a patient with actively progressing chronic hepatitis C (CH). RNA was reverse transcribed and cDNA was amplified by PCR with HCV RNA negative strand-specific 5′-UTR primers. Serial dilutions of a synthetic HCV RNA negative strand (sHCV RNA neg) amplified in parallel were used as semiquantitative standards. Water (DW and NW) and mock (M) samples were used as negative and contamination controls. The positive signals (244-bp fragments) were visualized by hybridization to the recombinant HCV UTR-E2 probe. Values under the panel represent relative densitometric units (DU) given by positive hybridization signals.