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The lack of a small-animal model has hampered the analysis of hepatitis C virus (HCV) pathogenesis. The tupaia (Tupaia belangeri), a tree shrew, has shown susceptibility to HCV infection and has been considered a possible candidate for a small experimental model of HCV infection. However, a longitudinal analysis of HCV-infected tupaias has yet to be described. Here, we provide an analysis of HCV pathogenesis during the course of infection in tupaias over a 3-year period. The animals were inoculated with hepatitis C patient serum HCR6 or viral particles reconstituted from full-length cDNA. In either case, inoculation caused mild hepatitis and intermittent viremia during the acute phase of infection. Histological analysis of infected livers revealed that HCV caused chronic hepatitis that worsened in a time-dependent manner. Liver steatosis, cirrhotic nodules, and accompanying tumorigenesis were also detected. To examine whether infectious virus particles were produced in tupaia livers, naive animals were inoculated with sera from HCV-infected tupaias, which had been confirmed positive for HCV RNA. As a result, the recipient animals also displayed mild hepatitis and intermittent viremia. Quasispecies were also observed in the NS5A region, signaling phylogenic lineage from the original inoculating sequence. Taken together, these data suggest that the tupaia is a practical animal model for experimental studies of HCV infection.
Hepatitis C virus (HCV) is a small enveloped virus that causes chronic hepatitis worldwide (32). HCV belongs to the genus Hepacivirus of the family Flaviviridae. Its genome comprises 9.6 kb of single-stranded RNA of positive polarity flanked by highly conserved untranslated regions at both the 5′ and 3′ ends (4, 27, 29). The 5′ untranslated region harbors an internal ribosomal entry site (29) that initiates translation of a single open reading frame encoding a large polyprotein comprising about 3,010 amino acids (35). The encoded polyprotein is co- and posttranslationally processed into 10 individual viral proteins (15).
In most cases of human infection, HCV is highly potent and establishes lifelong persistent infection, which progressively leads to chronic hepatitis, liver steatosis, cirrhosis, and hepatocellular carcinoma (9, 16, 21). The most effective therapy for treatment of HCV infection is administration of pegylated interferon combined with ribavirin. However, the combination therapy is an arduous regimen for patients; furthermore, HCV genotype 1b does not respond efficiently (19). The prevailing scientific opinion is that a more viable option than interferon treatment is needed.
The chimpanzee is the only validated animal model for in vivo studies of HCV infection, and it is capable of reproducing most aspects of human infection (5, 18, 23, 28, 35, 36). The chimpanzee is also the only validated animal for testing the authenticity and infectivity of cloned viral sequences (8, 14, 35, 36). However, chimpanzees are relatively rare and expensive experimental subjects. Cross-species transmission from infected chimpanzees to other nonhuman primates has been tested but has proven unsuccessful for all species evaluated (1).
The tupaia (Tupaia belangeri), a tree shrew, is a small nonprimate mammal indigenous to certain areas of Southeast Asia (6). It is susceptible to infection with a wide range of human-pathogenic viruses, including hepatitis B viruses (13, 20, 31), and appears to be permissive for HCV infection (33, 34). In an initial report, approximately one-third of inoculated animals exhibited acute, transient infection, although none developed the high-titer sustained viremia characteristic of infection in humans and chimpanzees (33). The short duration of follow-up precluded any observation of liver pathology. In addition to the putative in vivo model, cultured primary hepatocytes from tupaias can be infected with HCV, leading to de novo synthesis of HCV RNA (37). These reports strongly support tupaias as a valid model for experimental studies of HCV infection. However, longitudinal analyses evaluating the clinical development and pathology of HCV-infected tupaias have yet to be examined. In the present study, we describe the clinical development and pathology of HCV-infected tupaias over an approximately 3-year time course.
Table Table11 summarizes the tupaias used in this study. Tupaias born in laboratory captivity were obtained from the Laboratory Animal Center at the Kunming Institute of Zoology (Chinese Academy of Sciences). Tupaias were imported with permission from the Convention on International Trade in Endangered Species of Wild Fauna and Flora (7), quarantined for medical inspection, and housed individually in standard rat cages supplied with filtered air. The animals were fed a daily regimen of eggs, fruit, and the CMS-1 commercial diet for marmosets (CLEA, Japan). Their appetites and feces were carefully monitored. Animal care and experimental handling conformed to study guidelines established by the Subcommittee on Laboratory Animal Care at the Tokyo Metropolitan Institute of Science.
HCV genotype 1b serum, designated HCR6, was obtained from a patient with chronic active hepatitis C. The infectious titer of HCR6 was determined in chimpanzee and Molt4 cells and denoted plasma K (HCR6) by Shimizu et al. (24). The HCR6 serum exhibited a PCR titer of 6 × 106 genome equivalents/ml and an infectious titer of 3.7 × 104 50% chimpanzee infectious doses/ml. Serum aliquots were frozen at −80°C until they were used.
As described previously, pHCR6 (genotype 1b; 9,611 nucleotides; GenBank accession no. AY045720) is a plasmid carrying HCV genomic cDNA cloned from HCR6 serum (30). pHCR6Rz was designed for precisely trimmed RNA expression, with the entire genomic region of pHCR6Rz recloned under the control of the T7 promoter and the 5′ and 3′ distal ends flanked by hammerhead- and hepatitis D virus ribozyme-encoding sequences, respectively (22, 25).
For molecular reconstitution of HCV particles, pHCR6Rz was transfected into IMY-N9 cells as described previously (12). Briefly, semiconfluent IMY-N9 cells in 100-mm plastic dishes were transfected with 15 μg of plasmid using 40 μl of cationic lipids (DMRIE-C reagent; Life Technology) in accordance with the manufacturer's instructions. Five hours after transfection, the cells were infected with AdexCAT7 (2) (kindly provided by Y. Matsuura) at a multiplicity of infection of 20. After infection, the culture medium was replaced with Hepato-STIM (Becton Dickinson). The culture supernatants were collected at 24 h postinfection and stored at −80°C.
Animals were infected at 6 months of age. The anesthetic agent, ketamine hydrochloride, was administered intramuscularly at 50 mg/kg body weight prior to virus inoculation and bleeding of the tupaias. The inocula were introduced intravenously at 6 × 105 genome equivalents/animal for patient serum HCR6 and 1 × 107 genome equivalents/animal for reconstituted virions derived from the pHCR6Rz inoculation. Blood samples were drawn from infected and control animals pre- and postinfection. Briefly, the animals were bled weekly for 20 weeks and biweekly thereafter. At each time point, 0.5 ml of blood was drawn from the thigh vein; the sera were separated, aliquoted, and stored for subsequent assays.
Reinfection experiments were performed by transmission of HCV RNA-positive serum from group I (Table (Table1)1) to naive animals.
Serum alanine aminotransferase (ALT) concentrations were determined using a Transnase Nissui kit (Nissui Pharmaceutical Co.), standardized, and displayed as IU/liter.
Serum samples (100 μl) were tested for circulating HCV RNA in vivo using quantitative real-time detection (RTD)-PCR (TaqMan). RNA was extracted from the sera and livers of sacrificed animals using the acid guanidium-phenol chloroform method with tRNA as a carrier (3). Two tupaias (Tup.5 and Tup.6) were inoculated with patient serum HCR6. Another two animals (Tup.4 and Tup.8) were inoculated with reconstituted viral particles (RCV). Tup.15 served as a mock-infected control. Liver specimens (3- to 4-mm2 blocks) from these tupaias were homogenized with 1.5 ml of 5 M guanidine thiocyanate using a polytron-type homogenizer (Ultra-Turrax T25; IKA Laborteehnik, Staufen, Germany). RNA was then reextracted with 4 M guanidine thiocyanate.
RNA samples were subjected to RTD-PCR on an ABI 7700 sequence detector (Applied Biosystems) as described previously (26). The extracted RNA was dissolved in 200 μl of diethyl pyrocarbonate-treated water containing 10 mM dithiothreitol and 200 units/ml RNase inhibitor in a siliconized tube. RTD-PCR was performed using 1 μg of total RNA, one set of PCR primers, and a probe for a location within the 5′ noncoding region using the EZ rTth RNA PCR kit (Perkin Elmer) and the ABI Prism 7700 sequence detector system. A standard curve was constructed using a 10-fold dilution series of in vitro-transcribed and previously titrated synthetic HCV RNA.
Consequently, the quantities represented by genome equivalents correspond to an absolute standard curve (26). All quantitative RTD-PCR assays were performed using duplicate samples, with both negative control serum and HCV-positive serum included. The control sera were diluted before use and were estimated to contain low copy numbers of HCV RNA (100 genome equivalents/ml serum). Samples were deemed positive for HCV RNA if both duplicates yielded PCR-amplified product. Averages of the two estimated values are shown in the figures.
Tissue samples were carefully collected from anesthetized animals by abdominal incision, fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Silver and Sudan IV (Wako Pure Chemical Industries, Ltd.) staining were also carried out to visualize fiber generation and lipid degeneration, respectively. All histological staining was performed in accordance with conventional procedures. The histological status was determined using the modified hepatitis activity index scoring system, which grades necrosis and inflammation on a scale of 0 to 18 (periportal inflammation and necrosis, 0 to 10; lobular inflammation and necrosis, 0 to 4; portal inflammation, 0 to 4) (11). Fibrosis was scored using the Ishak fibrosis scale of 0 to 6 (0, no fibrosis; 1 or 2, portal fibrosis; 3 or 4, bridging fibrosis; and 5 or 6, cirrhosis). The values in each group (Table (Table2)2) represent the averages of the scores in five visual fields.
The statistical significance of differences between controls and HCV-infected animals was analyzed with the nonparametric Mann-Whitney U test. All comparisons were two tailed. The statistical analysis was conducted with SPSS 12.0 software (SPSS Inc., Chicago, IL).
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 (Table1,1, 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 Table1.1. 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. 1A and B). 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. (Fig.1A).1A). 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.1B1B).
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. 1C and D), 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. (Fig.1E):1E): 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 (Table1,1, group III), nor were significant elevations in serum ALT observed for any of the three infection-free controls (data not shown).
Serum ALT and circulating HCV RNA levels in primary infected tupaias (Table (Table1,1, group I) were monitored for 3 years postinoculation. As described above, the magnitudes of serum ALT fluctuations varied substantially among infected animals (Fig. 1A, B, C, and D). 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. 2B, D, and H). Infiltrating lymphocytes were also observed in limiting plates, indicating ongoing inflammation (Fig. 2G and H). 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. 2A to H and Table Table22).
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 (Table2).2). 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. (Fig.3B)3B) compared with Tup.15 (uninfected control) (Fig. (Fig.3A),3A), while silver staining of histology samples revealed fibrosis and cirrhotic nodules (Fig. 3E and F). Macroscopic observation upon sacrifice (3 years postinoculation) indicated that liver cirrhosis in Tup.8 had worsened (Fig. (Fig.3C).3C). In contrast, age-matched infection-free negative control tupaias displayed none of these pathologies (Fig. 3A, D, and G).
Progressive lipid degeneration was noted in infected tupaias throughout the course of infection (Fig. (Fig.4).4). 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. 4C and D). Liver specimens from other infected animals displayed intracellular micro- and macrovesicular lipid droplets in hepatocytes at 3 years postinoculation (Fig. 4F, H, and J). These anomalies were not present in liver specimens from infection-free control animals (Fig. 4A and B).
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 (Table1,1, 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. (Fig.5A).5A). 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. 5B and C). Furthermore, the PCR titers of the recipient tupaias were significantly greater than the inoculation titers (102 genome equivalents/animal) (Table (Table1).1). For Tup.11, serum from 4 weeks postinoculation contained almost 104 genome equivalents/ml of HCV RNA (Fig. (Fig.5B).5B). 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.
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.
In the present study, we described persistent HCV infection in tupaias. Long-term follow-up was performed and revealed histological progression of HCV-related liver disorders in infected tupaias, including steatosis, fibrosis, and cirrhosis, in addition to acute and chronic hepatitis. HCV genomic RNA was detected in animal sera intermittently throughout the entire course of infection. However, HCV RNA was detected in the liver upon sacrifice (3 years postinoculation). Furthermore, HCV RNA in serum contained genomic variants that had diverged from the inoculated virus (see Fig. S1 and S2 in the supplemental material). These data strongly indicate an established persistent infection in the tupaias studied. All animals exhibited HCV viremia soon after inoculation, yet the viremia was intermittent and accompanied by relatively low RTD-PCR titers compared with equivalent human and chimpanzee infections. The discrepancy between humans and tupaias might be due to host-dependent differences in replication efficiency. Over the course of HCV infection in these tupaias, serum ALT profiles indicated repeated liver injury, probably due to host immune responses mediated by agents such as cytotoxic T lymphocytes rather than direct viral cytopathic effects.
In cases of tupaia infection, experimental inoculations rarely led to sustained viremia, which for most human cases lasts for the entire course of infection. Even the course of infection appeared transient and self-resolved. It seems likely that HCV replication is less compatible with the tupaia host environment. This possibility was substantiated by a previous report by Xu et al. (34), where tissue-cultured virions of cloned genotype 1b, referred to as HCVcc in the paper, could not cause chronic infection with sustained viremia in tupaias. Although HCVcc actually infected most of the inoculated tupaias (83%; 10/12), chronic infection was seen for only a fraction of them (20%; 2/10). In this study, we also tried to detect a humoral response to HCV core antigen. We found that tupaia sera were HCV positive for antibodies only at occasional time points, observable as intermittent steep responses (data not shown). Overall, sustained seroconversion was not seen in this study, probably because HCV propagation in vivo was so limited or well controlled by host immunity. Given that models of HCV propagation are severely limited, the most important and interesting finding of this study is the successful detection of HCV RNA in livers of infected tupaias 3 years after inoculation, indicating that HCV persists in tupaias. Although the limited propagation of HCV in tupaias is a drawback of this model at the present time, the isolation of tupaia-adapted HCV may be feasible by performing multiple infection passages. This possibility is supported by both quasispecies development and successful reinfection.
The chimpanzee is the animal species most closely related to humans, and as a model, it has contributed significantly to our understanding of HCV infection and pathogenesis. However, reproducing HCV pathogenesis in humans or chimpanzees can take as long as 10 to 20 years. The chronically infected tupaias in the present study developed complicated liver disorders in a much shorter time. Using tupaias, with their relatively short life span (3 to 5 years in the laboratory), as a model of HCV infection, we can evaluate HCV pathogenesis and correlate senescence and duration of infection.
The recent development of a primary human hepatocyte xenograft-uPA/SCID mouse model opened up opportunities to test putative antivirals against HCV replication in vivo (10, 17). In this innovative model, human hepatocytes, which are transplanted into the lobe of a mouse liver, can support HCV replication effectively. As a result, the level of circulating HCV RNA is comparable to that of a human patient. However, this mouse model is immunodeficient, and thus, it lacks the interplay between host immunity and viral infection. Therefore, it does not provide a suitable platform for characterizing immune responses to HCV infection.
HCV infection in tupaias represents an important model of HCV infection, particularly for the study of key determinants controlling virus propagation in vivo. The pathogenesis of HCV infection can be substantially different among humans, chimpanzees, and tupaias, and the mechanisms governing these differences are of great interest. Comparative studies of HCV infection in these different species will help us to understand the basic mechanisms of persistent infection.
We thank Masahiro Shuda for helpful assistance and Etsuko Endo for creating the figures. We also thank the staffs of the Departments of Microbiology and Cell Biology and Mitsugu Takahashi for breeding the tupaias.
This study was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan; the Program for Promotion of Fundamental Studies in Health Sciences of the Pharmaceuticals and Medical Devices Agency of Japan; and the Ministry of Health, Labor and Welfare of Japan.
Published ahead of print on 21 October 2009.
†Supplemental material for this article may be found at http://jvi.asm.org/.