Common marmoset experimentation and sample collection were approved by Southern Medical University (SMU) Animal Care and Use Committee (permit numbers: SYXK(Yue)2010-0056). All animal care and procedures (NFYYLASOP-037) were in accordance with national and institutional policies for animal health and well-being. All efforts were made to minimize suffering of animals.

Eight common marmosets (male, age 2–3 years and weight 250–350 g) were imported from Tianjin Medical University and individually fed in the Laboratory Animal Research Center of Nanfang Hospital, Guangzhou, China. HCV-CE1E2p7/GBV-B chimeric RNA and GBV-B RNA were prepared by in vitro transcription with a MEGAscript kit according to manufactory instruction (Ambion, Applied Biosystems, Austin, TX, USA). Marmosets (n = 8) were randomly separated into four animals for each group. An intrahepatic injection of 300 μg HCV/GBV-B chimeric RNA or GBV-B RNA were injected into animals’ livers at two points, respectively, as in a previously described method [13]. Blood samples were collected from marmosets via femoral vein at an interval of 2 weeks. At the end point of the experiment, liver tissues were collected specifically for this study from HCV chimera- or GBV-B-infected animals.

Viral RNA was extracted from sera of infected marmosets using the High Pure Viral Nucleic Acid kit (Roche Diagnostic GmbH, Mannheim, Germany). Viremic or hepatocytic RNA of HCV chimera- or GBV-B-infected marmosets was measured by RT-qPCR with primers targeting the GBV-B 5′ NCR as previously described [13].

Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the marmosets were measured in units per liter (U/L) by the commercial kits (Biosino Bio-technology and Science, Beijing, China) on a Hitachi 7170S fully automatic biochemical analyzer (Tokyo, Japan) [13].

Small sections of liver tissue from each animal’s liver were examined with hematoxylin and eosin (H&E) staining as described previously [13]. The necrosis and inflammation were graded on the scales 0–18 according to the modified histology activity index (HAI) system [14].

HCV and GBV-B peptides (Table S1) were selected by predicting peptide binding to Macaque MHC class I molecules from a website (http://www.mamu.liai.org/) accessed on 5 June 2014, which were commercially synthesized by the Chinese Peptide Company (Hangzhou, Zhejiang, China). HCV Chimera or GBV-B peptides (4 μg/mL each) were pooled in PBS. The specific IFN-γ-secretion T cell response of peripheral blood mononuclear cells (PBMCs) from marmosets was measured by stimulation with the pooled peptides in an enzyme-linked immunosorbent spot assay (ELISpot) as described previously [13]. Briefly, a pre-coated human gamma interferon ELISpot Plus plate (Mebtech AB, Nacka Strand, Sweden) was used in the assay. PBMCs were isolated from blood samples of marmosets and cultured in the pre-coated ELISpot plates at 3 × 10⁵ cells/well in 10% fetal calf serum, 100 IU/mL penicillin, and 100 IU/mL streptomycin 1640 medium containing pooled peptides and 100 U/mL interleukin-2 (IL-2). The negative and positive controls were 1640 medium or phytohemagglutinin (PHA), respectively. The result was normalized as the numbers of spot-forming cells (SFCs) per 10⁶ PBMCs.

Freshly separated marmosets’ PBMCs were examined for regulatory T (Treg) cells by incubating for 30 min at room temperature in 0.1% BSA PBS containing anti-CD4-PE (IgG2a, Cat: MHCD0404, clone: s3.5, Invitrogen, Carlsbad, CA, USA) for 5 μL/sample and anti-Foxp3-APC (Mouse IgG2a kapa, Clone: PCH101, eBioscience, San Diego, CA, USA) for 2 μL/sample. Propidium iodide was added after the final wash at 1 mg/mL to exclude dead cells in all experiments. Samples were analyzed on a Becton Dickinson (San Jose, CA, USA) FACSort apparatus. Ten thousand events were collected and analyzed using CellQuest software version 3.3 (Becton Dickinson, San Jose, CA, USA).

PBMCs (1 × 10⁶ per well) were stimulated for incubation for 5 days at 37 °C and 5% CO₂ with peptides, 1640 medium, and PHA controls, respectively. Afterwards, the IFN-γ in the supernatants of cell cultures were measured by the human IFN-γ ELISA kit (U-Cytech Biosciences, Utrecht, Netherland).

Total RNA of PBMCs were extracted using TRIzol method (Invitrogen) and reverse-transcripted using Reverse Transcription System according to the manufacturer’s instructions (Roche, Basel, Switzerland). Quantitative RT-PCR reactions for IL-10 and TGF-β mRNA transcripts were performed with SYBR Master Mix following the manufacturer’s protocol (Roche, Basel, Switzerland). Specific IL-10 primers were 5′-CTGCCTCACATGCTTCGAGA-3′ (forward) and 5′-TGGCAACCCAGGTAACCCTTA-3′ (reverse), and TGF-β primers were 5′- GGGTTCTCTTGGCTGTTACTG-3′ (forward) and 5′-TGTCTAAGAAAAGAGTTCCATTATC-3′ (reverse), respectively.

All experiments were repeated at least three times independently. The data were analyzed using the statistical package SPSS v. 19.0. The results were presented as the mean ± SD. Differences between groups were analyzed using Student’s t-test, and p-values < 0.05 were considered statistically significant.

Four marmosets, M37, M38, M43, and M45, were infected with HCV-CE1E2p7/GBV-B chimeric virus (HCV chimera), and four marmosets, M40, M41, M44, and M46, were infected with GBV-B, respectively. After intrahepatic injection of viral genomic RNA, 2 mL of blood samples were collected at an interval of two weeks from all marmosets. Viral replication was measured by detecting viremic RNA with RT-qPCR (Figure 1). Among the HCV chimera-infected group, marmosets M37, M38, and M43 were examined for 43 weeks, while M45 died 17 weeks after infection (Figure S1A). An overall pattern of viral replication exhibited a typical persistent infection with viral loads ranging between 7.41 (±2.37) × 10² and 2.95 (±0.48) × 10⁵ copies/mL (Figure 1A). In the GBV-B-infected group, marmosets M41, M44, and M46 were examined for 43 weeks, and M40 died 15 weeks after infection (Figure S1B), of which an overall pattern showed a self-limited infection with viral loads ranging between 24 (±2.41) and 1.34 (±0.49) × 10⁵ copies/mL and then the virus spontaneously cleared in 23 weeks (Figure 1B). The results suggested that HCV chimera-infected marmosets progressed to chronic infection, while GBV-B-infected marmosets resolved their infections naturally.

The serum ALT was detected within a normal range (<40 U/L) from both HCV chimera- and GBV-B-infected marmosets. The AST was progressively elevated over 40 U/L in the serum of HCV chimera-infected marmosets during the course of detection, while the AST was mostly stable below 40 U/L in the serum of GBV-B-infected marmosets (Figure 1 and Figure S1).

The histopathologic examination for liver tissues was conducted from HCV chimera- and GBV-B-infected marmosets at the end point of follow-up detection (Figure 2; Table S2). The viral load was measured for 10³–10⁴ copies/mg in liver tissue from HCV chimera-infected marmosets, but not from GBV-B-infected animals except for animal M40 that died at week 15 (Figure S2). The HCV chimera-infected marmosets presented typical viral hepatitis in the liver tissues, such as lymphocytic infiltration, ballooning, ground-glass changes, and eosinophilic changes, while the GBV-B-infected animals exhibited mild viral hepatitis in liver tissues (Figure 2A). The results suggested that HCV-CE1E2p7 chimera persistent infection caused more severe pathological changes than GBV-B resolved infection in marmosets (Figure 2B).

In order to characterize the specific cellular immune responses between HCV chimera- and GBV-B-infected marmosets, the PBMCs were freshly isolated from these two different virus infected animals, and the specific IFN-γ-secretion T cell responses were detected by ELISpot (Figure 3 and Figure S3). In four GBV-B-infected marmosets, the specific IFN-γ-secretion T cell response increased rapidly to the high level (>100 SFC/10⁶ PBMCs) at weeks 5–7 after infection and was then maintained at a relatively high level > 50 SFC/10⁶ PBMCs (Figure 3A,B). In HCV chimera-infected marmosets, the IFN-γ-secretion T cell response slowly increased to the plateau (<100 SFCs/10⁶ PBMCs) at weeks 13–19 after infection and was then maintained at a low level < 50 SFCs/10⁶ PBMCs (Figure 3C,D). The results suggested that HCV chimera-infected marmosets exhibited a delayed and lower specific T cell response, whereas GBV-B-infected marmosets showed a rapid and strong specific T cell response after the different virus infections.

Further, to investigate the differences in suppressive immune response between the two different virus infected marmosets, the Treg cell response was measured by flow cytometry with Foxp3+/CD4+ biomarker antibodies (Figure 4 and Figure S4). In HCV chimera-infected marmosets, the ratio of Treg cells was rapidly increased to approximately 4% in 3 weeks, 6% at the top in 9 weeks, and then maintained for 4–6% at a high level until 43 weeks among lymphocytes after virus infection. In GBV-B-infected marmosets, Treg cell response was not activated and the ratio of Treg cells was around 2% at a baseline level before or after virus infection. The results suggested that HCV chimera infection rapidly activated and amplified the suppressive T cell response, while GBV-B infection did not induce a Treg response in the marmosets.

To verify the activation of Treg cells by HCV chimera via its structural proteins, the specific Treg cell response was measured in vitro by stimulating the marmosets’ PBMCs with HCV structural protein peptides in comparison with GBV-B peptides, respectively (Figure 5 and Figure S5). From HCV chimera-infected marmosets (Figure S5A), the mean ratio of Treg cells was detected at 8.5% among PBMCs stimulated with HCV structural protein peptides, which were significantly higher than that at 4.6% in negative controls (Figure 5A; p < 0.001). From GBV-B-infected marmosets (Figure S5B), the mean ratio of Treg cells was detected at 2% among PBMCs stimulated with GBV-B structural protein peptides, which was not significantly different from negative controls (Figure 5A; p > 0.05). The ratio of Treg cells in HCV peptide-stimulated PBMCs was higher than that in GBV-B peptide-stimulated PBMCs from two different virus infected marmosets (Figure 5A; p < 0.001). The results suggested that HCV whole structural proteins could specifically activate Treg cells in PBMCs from HCV chimera-infected marmosets, whereas GBV-B whole structural proteins could not activate Treg cells in PBMCs from GBV-B-infected marmosets. At the mRNA level, IL-10 and TGF-β transcripts were higher in HCV peptide-stimulated PBMCs than those in GBV-B peptide-induced cells (Figure 5B,C; p < 0.001).

To investigate whether the low level of IFN-γ-secretion T cell response in HCV chimera-infected marmosets was inhibited by the early activated Treg cells, the PBMCs were stimulated correspondingly by the negative control (NC), HCV structural protein peptides plus PHA, and PHA only, respectively. The IFN-γ-secretion T cell response was measured as 17 ± 4 SFC (NC), 129 ± 15 SFC (HCV peptides + PHA), and 273 ± 47 SFC (PHA only) per million PBMCs, respectively (Figure S6A). Comparably, by stimulating PBMCs from GBV-B-infected marmosets with NC, GBV-B structural protein peptides plus PHA, and PHA only, IFN-γ-secretion T cell responses were measured as 16 ± 3 SFC, 250 ± 27 SFC, or 261 ± 33 SFC/10⁶ PBMCs, respectively (Figure S6B). The data showed that IFN-γ-secretion T cell response induced by HCV peptides plus PHA was lower than that induced by PHA only (Figure 5D; p < 0.01), while IFN-γ-secretion T cell response had no significant differences between GBV-B peptides plus PHA and PHA only inductions (Figure 5D; p > 0.05), suggesting that the HCV structural protein might suppress IFN-γ-secretion T cell response. By quantifying the secreted IFN-γ in supernatants of stimulated PBMCs from HCV chimera- or GBV-B-infected marmosets, IFN-γ was significantly decreased in simulation with HCV peptides plus PHA than that with PHA only (Figure 5E; p < 0.01). Overall, the data suggested that HCV structural proteins inhibited specific IFN-γ-secreting T cell responses via activation of Treg cells, but GBV-B did not activate Treg cells in the virus-infected marmosets.