Inoculation of HCV causes acute hepatitis and transient viremia in tupaias.
To begin this study, two distinct but related inocula were chosen for infection of tupaias. Serum from a chronic hepatitis patient (designated HCR6) was chosen for its defined genotype (genotype 1b), and genetic heterogeneity was ascertained by the process of cloning consensus cDNA. The infectivity of this serum was also experimentally defined in chimpanzees; a 50% chimpanzee infectious dose was estimated at 3.7 × 104 50% chimpanzee infectious doses/ml. Furthermore, the consensus genomic sequence of HCV was cloned from the serum (pHCR6; 9,611 bases; GenBank AY045702.1). For the second inoculum (referred to as RCV), clonal viral particles were reconstituted as described in Materials and Methods. This inoculum was expected to be free of neutralizing antibodies and thus was considered potentially more infectious than patient sera. In the case of RCV infection, genetic diversification of viral RNA, also known as quasispecies, can be regarded as a direct indication of de novo synthesis of progenitor virus in vivo.
Either patient serum or cDNA-derived RCV was inoculated into tupaias (Table , group I). Two animals (one female and one male) were tested against each inoculum. Age-matched animals were bred as infection-free controls.
All experimental infections are described in Materials and Methods and Table . Prior to experimental infection, the normal serum ALT level in tupaias was measured at 22.3 IU/liter (n = 23).
Inoculation with patient serum HCR6 caused rapid fluctuations in the serum ALT concentrations, from two- to fivefold, in both inoculated tupaias, suggesting acute hepatitis in vivo (Fig. ). Correlative quantitative RTD-PCR revealed HCV viremia soon after serum inoculation in Tup.5, which continued to show transient viremia long term. The appearance of viremia sometimes coincided with a steep elevation in the serum ALT (Fig. ). Conversely, HCV RNA was not detected in the serum of Tup.6 up to 60 weeks postinoculation and only twice thereafter. Acute-phase ALT elevations (3 to 4 weeks postinoculation) in Tup.6 might represent tight control of HCV infection by the host immune system (Fig. ).
FIG. 1. Course of infection with patient serum HCR6 and RCV. (A) The results of quantitative RTD-PCR for HCV RNA and serum ALT concentrations were combined and plotted to show the course of infection in Tup.5. The bars and the ordinates on the left represent (more ...)
Distinct results were obtained for the two animals (Tup.4 and Tup.8) inoculated with RCV. Both animals displayed sustained viremia up to 10 weeks postinoculation (Fig. ), indicating persistent HCV infection and inability to eradicate the virus. Viremia was detected intermittently throughout the course of infection, sometimes accompanying the elevation of serum ALT. Humoral immune responses in Tup.5 and Tup.6 (see Fig. S1A in the supplemental material) and Tup.4 and Tup.6 (see Fig. S1B in the supplemental material) were indicated.
We performed RTD-PCR to confirm whether HCV could replicate in the tupaias' livers (Tup.4, Tup.5, Tup.6, and Tup.8) and obtained the following results (Fig. ): 310 ± 117 copies/μg total RNA in Tup.5, 80 ± 11 copies/μg in Tup.6, 199 ± 77 copies/μg in Tup.4, and 292 ± 48 copies/μg in Tup.8. In contrast, HCV RNA was not detected in the liver of the mock-infected animal (Tup.15).
HCV RNA was also not detected in samples from either preinoculation or age-matched, infection-free control tupaias (Table , group III), nor were significant elevations in serum ALT observed for any of the three infection-free controls (data not shown).
HCV causes chronic hepatitis in tupaia liver, leading to fibrosis and cirrhosis.
Serum ALT and circulating HCV RNA levels in primary infected tupaias (Table , group I) were monitored for 3 years postinoculation. As described above, the magnitudes of serum ALT fluctuations varied substantially among infected animals (Fig. ). Tupaia livers were examined for histological lesions in order to elucidate if HCV caused chronic hepatitis. Liver biopsies via abdominal incisions were performed at 2 years postinoculation. All animals were sacrificed at 3 years postinoculation (4.5 years for uninfected animals). H&E staining of liver specimens from HCV-infected tupaias showed infiltrating lymphocytes within sinusoids and around portal areas, indicating chronic hepatitis in the tupaia livers (Fig. ). Infiltrating lymphocytes were also observed in limiting plates, indicating ongoing inflammation (Fig. ). Furthermore, a comparison of liver samples at 2 and 3 years postinoculation revealed that the hepatitis had worsened with time in all HCV-infected tupaias (Fig. and Table ).
FIG. 2. Micrographs of liver specimens stained with H&E. Liver tissue from HCR6-inoculated tupaias (A to D) and RCV-inoculated tupaias (E to H) was obtained at 2 and 3 years postinoculation (pi). (I and J) Liver specimens from uninfected animals age matched (more ...)
Fibrosis and cirrhosis were also examined. Mild fibrosis was seen in Tup.6, while severe fibrosis was seen in Tup.8. Cirrhosis was histologically investigated in all animals (Table ). There was no significant difference between groups I and III at 94 weeks postinfection (P = 0.194), but at 144 weeks postinfection, a slight difference was observed (P = 0.059; SPSS 12.0). Macroscopic observation of the liver biopsy specimens (taken 2 years postinoculation) indicated liver cirrhosis in Tup.8 (Fig. ) compared with Tup.15 (uninfected control) (Fig. ), while silver staining of histology samples revealed fibrosis and cirrhotic nodules (Fig. ). Macroscopic observation upon sacrifice (3 years postinoculation) indicated that liver cirrhosis in Tup.8 had worsened (Fig. ). In contrast, age-matched infection-free negative control tupaias displayed none of these pathologies (Fig. ).
FIG. 3. Macro- and microscopic features of tupaia liver. (A) Infection-free control tupaia (Tup.15; 92 weeks). (B) RCV-infected animal displaying liver cirrhosis (Tup.8; 84 weeks postinoculation). (C) RCV-infected animal with massive surface nodules (Tup.8; 144 (more ...)
Progressive lipid degeneration was noted in infected tupaias throughout the course of infection (Fig. ). In particular, Tup.5 displayed microvesicular lipid droplets in the first biopsy specimens (at 2 years), which developed into macrovesicular droplets and foamy degeneration in biopsy specimens at 3 years (Fig. ). Liver specimens from other infected animals displayed intracellular micro- and macrovesicular lipid droplets in hepatocytes at 3 years postinoculation (Fig. ). These anomalies were not present in liver specimens from infection-free control animals (Fig. ).
FIG. 4. Sudan IV-stained liver specimens exhibiting fatty liver degeneration. Cryosections of liver stained by Sudan IV as described in Materials and Methods show fatty liver degeneration. The left and right columns display biopsy specimens of infected animals (more ...) Transmission of viral-RNA-positive serum to naive animals reproduces acute hepatitis and viremia.
To confirm virion regeneration in vivo, and to exclude the possibility of false-positive serum HCV RNA results due to amplification of the original inocula, HCV RNA-positive sera from primary inoculated tupaias were used to inoculate naive tupaias. Three different sera were tested in this passage experiment, with two naive tupaias used as recipient animals for each trial (see Materials and Methods) (Table , group II).
In the first reinfection experiment, serum from Tup.5 (originally infected with patient serum HCR6) was collected at 5 weeks postinoculation and used to infect two naive animals. The recipient animals showed intermittent viremia over the subsequent 3 months (Fig. ). In the second and third cases of reinfection, sera from Tup.8 at 10 weeks postinoculation and from Tup.4 at 8 weeks postinoculation also induced viremia in the naive inoculated animals, similar to the first reinfection experiment (Fig. ). Furthermore, the PCR titers of the recipient tupaias were significantly greater than the inoculation titers (102 genome equivalents/animal) (Table ). For Tup.11, serum from 4 weeks postinoculation contained almost 104 genome equivalents/ml of HCV RNA (Fig. ). In addition, significant increases in serum ALT accompanied detection of serum HCV RNA. These results indicate that HCV RNA-positive sera from group I actually contained infectious virion particles. They also suggest that reconstituted HCV particles made from cDNA are infectious in tupaias.
FIG. 5. Results of a reinfection experiment. (A) Quantitative RTD-PCR for HCV RNA and serum ALT levels are shown. Two naive animals were inoculated with tupaia serum (using serum taken at 5 weeks postinoculation from Tup.5, originally inoculated with patient (more ...)
We amplified a portion of the NS5A sequence, which is known as the interferon sensitivity determining region, by reverse transcription-PCR as described in the supplemental material. Each PCR product was subcloned and sequenced to compare the encoded amino acid sequences. For the purposes of this study, animals were inoculated with a molecular clonal virus consisting of a unique viral sequence of cDNA. The interferon sensitivity determining region sequences recovered from an animal infected with clonal inoculum (Tup.8 at 103 weeks postinoculation) were found to be heterogeneous, with a few amino acid substitutions (K2212M for 2/10 cases, L2232P for 1/10 cases, and L2253S for 6/10 cases) (see Fig. S2E in the supplemental material). Interestingly, the codon for amino acid 2224 encodes valine, but it was found to be variant for alanine and valine in sequences from the original patient serum (HCR6). Tupaias infected with patient serum also exhibited variability at position 2224; valine occupancy was rare, as was seen in the original HCR6 population (see Fig. S2B and C in the supplemental material). On the other hand, this position was occupied solely by valine for sequences recovered from Tup.8 (see Fig. S2E in the supplemental material), indicating that genetic variations shown for Tup.8 originated from the pHCR6 cDNA sequence. Taken together, quasispecies detection of circulating virus represents further evidence demonstrating intrinsic replication of HCV in tupaias despite low levels and infrequent detection of viremia.