1 Division of Virology, Children's Hospital of Eastern Ontario, 401 Smyth Road, Ottawa, ON, Canada, K1H 8L1
2 Department of Microbiology Immunology and Biochemistry, University of Ottawa, Ottawa, ON, Canada, K1N 6N5
3 Ottawa Health Research Institute, Molecular Medicine Program, Ottawa, ON, Canada, K1H 8L6
4 Department of Veterinary Pathology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, Canada, S7N 5B4
5 Department of Veterinary Microbiology, Veterinary Infectious Disease Organization, University of Saskatchewan, Saskatoon, SK, Canada, S7N 5E3
6 Division of Blood Borne Pathogens, Health Canada, Ottawa, ON, Canada, K1A 0L2
Correspondence
Francisco Diaz-Mitoma
diaz{at}exchange.cheo.on.ca
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ABSTRACT |
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INTRODUCTION |
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HCV belongs to the family Flaviviridae and has a positive single-stranded RNA genome that encodes a 3000 aa polyprotein precursor. The structural proteins core, E1 and E2, which form viral particles, are located at the amino-terminal end of the precursor protein. Downstream are non-structural proteins P7, NS2, NS3, NS4A, NS4B, NS5A and NS5B, which are involved in virus replication (Choo et al., 1991; Grakoui et al., 1993
; Bartenschlager et al., 1994
; Lin et al., 1994
; Lo et al., 1996
).
The exact pathological processes of HCV infection are not completely understood. Following an incubation period of 312 weeks, HCV clinical manifestations are often mild. However, as many as 85 % of the infected individuals develop a chronic infection, frequently with severe long-term liver pathology (Hoofnagle, 1997). HCV infection is also associated with the development of extra-hepatic manifestations, such as type II mixed cryoglobulinemia, glomerulonephritis and B-cell non-Hodgkin's lymphoma (Chan et al., 2001
; Gasparotto et al., 2002
; Ishikawa et al., 2003
). Chronic HCV infection has been treated with interferon-
but less than 20 % of patients achieve a sustained response with this drug. Combination therapy of interferon-
and a nucleoside analogue, ribavirin, has increased the rate of sustained response to more than 30 % (Gretch et al., 1996
; Davis et al., 1998
; Poynard et al., 1998
). The broad genetic variability of the virus genome has resulted in six identified genotypes with more than 50 subtypes (Simmonds et al., 1993
). Also, in individuals infected with HCV, the virus becomes a quasispecies, a population of closely related variants (Bukh et al., 1995
; Kato, 2000
). Thus, the understanding of the biology of the infection, the diagnosis and the development of a vaccine against HCV is complex.
Little is known about the mechanism of HCV pathogenesis. However, both immune-mediated mechanisms and direct viral cytotoxicity have been suggested as factors in the pathology associated with hepatitis C (Chisari, 1997). The lack of an appropriate viral culture system and a small animal model that can support virus replication has hampered detailed analysis of HCV pathogenesis and vaccine development (Chisari, 1997
). Combined research efforts with different small animal models will facilitate detailed analysis of HCV in vivo and clarify HCV pathogenesis.
A previous study has shown that HCV proteins are not directly cytopathic on liver cells and that the host immune response plays an important role in hepatitis C infection (Wakita et al., 1998). In contrast, other studies have provided evidence that HCV structural proteins play a direct role in the development of liver steatosis, and increase the risk of liver cancer in transgenic mice (Lerat et al., 2002
). These transgenic mouse models utilize organ-specific promoters, which preferentially express viral proteins in the liver. In the present study, a transgenic mouse model that expressed the HCV structural proteins core, E1 and E2 under the control of a universal CMV promoter was developed. The model shared several features of human HCV infection, such as severe hepatopathy characterized by the development of steatosis as well as liver and lymphoid tumours. This animal model could be used to study pathogenesis and novel treatment modalities for HCV infections and liver cancer.
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METHODS |
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RT-PCR.
Total RNA was extracted from different mouse tissues (liver, spleen, kidney, brain, lung and heart) using the RNeasy mini kit (Qiagen). Reverse transcription of total RNA was performed with MuLV reverse transcriptase (Applied Biosystems) and random hexamers (Applied Biosystems) to generate cDNA, according to the manufacturer's instructions. The cDNA was amplified by PCR using the primers described above. RNA extract from the liver of a non-transgenic littermate was used as a negative control.
Immunofluorescence analysis.
Transgenic mice and non-transgenic littermates were sacrificed between the ages of 3 and 18 months. Mice were anaesthetized and then perfused with normal saline followed by 4 % paraformaldehyde. Liver, kidney, heart, spleen, lung, salivary glands and brain tissues were removed surgically and placed in 4 % paraformaldehyde overnight at 4 °C. A piece of tissue was placed onto a plastic mould, covered with tissue embedding medium and then frozen in isopentane on dry ice. Frozen tissue sections of 5 µm thickness were cut using a cryostat and placed onto lysine-coated slides. Frozen tissue sections were incubated with blocking buffer (5 % normal goat serum and 0·1 % Triton X-100 in PBS) for 1 h at room temperature. Rabbit anti-core E1, E2 polyclonal antibody (prepared in our laboratory according to the University of Ottawa animal care facility protocols for antibody production) or anti-core monoclonal antibody (Biogenesis) were applied at dilutions of 1 : 50 and 1 : 500, respectively, for 1 h at room temperature. After washing with PBS, FITC-conjugated anti-rabbit IgG (Sigma) or FITC-conjugated anti-mouse IgG (Sigma) were incubated at a dilution of 1 : 200 for 1 h at room temperature. After washing with PBS, the tissue sections were mounted with Vectashield mounting medium (Vector). In addition to using tissues from non-transgenic littermates as negative controls, we also performed parallel immunostaining experiments using pre-immunized rabbit serum, as well as secondary anti-rabbit FITC-labelled antibody alone.
Real-time RT-PCR assay.
Total RNA was extracted from mouse liver using the RNeasy mini kit (Qiagen). There were four groups of three mice (n=3) of different ages. Each mouse liver was tested in triplicate by real-time RT-PCR. For the reverse transcription reaction, the RNA was reverse transcribed using random hexamers (Applied Biosystems) and MuLV reverse transcriptase (Applied Biosystems), following the manufacturer's instructions. Reverse transcription was performed for 1 h at 37 °C using 1 µg RNA per reaction in a 20 µl reaction volume. In order to stop this reaction, the cDNA samples were incubated for 15 min at 72 °C. The real-time PCR was performed in special optical tubes in 36-well microtitre plates (Perkin-Elmer/Applied Biosystems) with an iCycler (Bio-Rad). Fluorescent signals were generated using the Quantitect SYBR Green PCR kit (Qiagen). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control gene with the following sense and antisense primer sequences, 5'-ATGTGTCCGTCGTGGATCTGA-3' and 5'-TTGAAGTCGCAGGAGACAACCT-3', respectively. The HCV core gene was analysed using the following oligonucleotide primers (300 nM): forward 5'-ACCATGAGCAATCCTAAACCTC-3' and reverse 5'-GCAACAAGTAAACTCCACCAACGA-3'. The core and GAPDH genes were amplified using the primers mentioned above and a cDNA template from both transgenic and non-transgenic animals. Target samples were added in individual reactions to a total volume of 50 µl and no cDNA was added to the negative control. For each amplification using real-time PCR, the protocol included 15 min at 95 °C and 40 cycles of 15 s at 94 °C, 30 s at 66 °C and 30 s at 72 °C. All PCR experiments were performed with hot start and were run in triplicate. The iCycler software (Bio-Rad) detected the threshold cycle (CT) for each amplicon. Normalization was performed using the 2CT method (Livak & Schmittgen, 2001
). Experimental controls including non-reverse-transcribed RNA samples were performed.
Histological staining.
Mouse tissues were fixed in 4 % paraformaldehyde and embedded in paraffin. Sections of 5 µm thickness were stained with haematoxylin and eosin, Sudan black B or Masson's trichrome staining according to standard methods used in the Department of Pathology and Laboratory Medicine at the Faculty of Medicine, University of Ottawa. Tissue examination was performed by one of us (S. G.) and interpreted according to standard histopathological morphology. To determine the extent of steatosis a semi-quantitative method was used, in which 1+ represented lipid droplets in sporadic hepatocytes; 2+ represented moderate steatosis, in which hepatocytes with lipid droplets were seen clearly in more than two of five fields examined; 3+ represented moderate to severe steatosis, in which lipid droplets were extensively distributed in most areas of the liver; 4+ very severe steatosis, in which steatosis was observed everywhere in the liver. Characterization of the tumours was performed according to standard morphological histology. For example, the diagnosis of lymphosarcoma in the liver and nodes was made based on the undifferentiated nature of the cell morphology, which resembled early embryonic cells; the presence of many mitotic figures and the invasiveness represented by a lack of tumour capsule, tumour size and invasion of nodes and liver. In addition, a large portion of normal hepatic tissue was replaced by tumour cells. In contrast, the diagnosis of adenoma was based on cell morphology, including the well differentiated nature of cells with few mitotic figures and the lack of invasion of extra-hepatic tissues.
Electron microscopy.
The electron microscopy study was done according to the standard procedures at the electron microscopy facility at the Children's Hospital of Eastern Ontario in Ottawa. Approximately 0·25 mm thick liver tissue slices were post-fixed briefly in 2 % osmium tetroxide in 0·1 M cacodylate buffer, dehydrated in graded ethanol and embedded in Spur epoxy resin. Ultrathin 6070 nm sections were cut using a Leica Ultracut R ultra-microtome, mounted on 200 mesh copper grids and stained with 10 % uranyl acetate in 50 % methanol, followed by Reynold's lead citrate. Grids were examined and micro-graphed using a JEOL 1010 transmission electron microscope at 60 KV.
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RESULTS |
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Generation of transgenic mice
Five transgenic founder (F0) mice were used to establish five transgenic lines. Transgenic mice were analysed for HCV transgene expression by PCR using genomic DNA extracted from the tails. Founder mice were crossed with B6C3F1 mice to expand the mouse colony, and ensure the integrated transgene was successfully transmitted to the offspring. F1 (first generation) and F2 (second generation) heterozygous mice from the five lines were used as models in this study.
Expression of the core, E1 and E2 transgenes
Transgenic mice expressed the HCV core, E1 and E2 transcript in all tissues including, liver, kidney, spleen, heart, lung and brain (Fig. 1b), but the viral proteins were mainly expressed in the liver (Fig. 2a, b
) demonstrating selective expression in certain tissues. Viral protein detection was not observed in either the heart or the brain. Spleen cells, as well as epithelial cells in the renal microtubules of the kidney, demonstrated relatively low levels of viral protein expression. The liver demonstrated the highest level of viral protein expression with a centrilobular distribution of groups of cells expressing the viral proteins. Tissues from non-transgenic littermates did not show HCV protein expression. When fetal tissue was analysed by immunofluorescence to detect the viral proteins, the fluorescent signal was restricted to the salivary glands (Fig. 2e, f
). However, salivary glands of adult mice did not express HCV proteins (data not shown). In addition, there was no detectable viral protein expression in fetal livers (data not shown). There was no difference in the levels of protein expression between the five lines. Core, E1 and E2 protein expression was tested in all tissues using both rabbit anti-core, E1, E2 polyclonal and anti-core monoclonal antibodies.
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Viral RNA is increased with age in transgenic mice
For further confirmation of the increased expression of the viral transgene in older mice, a comparison between four groups of transgenic mice (aged 4 and 18 months) was performed using real-time RT-PCR. Increased expression of the HCV core RNA transcript was observed in older mice (Table 1). There was a 1·9-, 274- and 776-fold increase in HCV transcript concentration at 6, 14 and 18 months, respectively, when compared with 4-month-old mice.
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Subcellular abnormalities in transgenic hepatocytes
Electron microscopy demonstrated several abnormalities in the intracellular organelles of hepatocytes in transgenic mice (Fig. 5a, b). A loss of the rough endoplasmic reticulum pattern was observed as well as swelling of mitochondria and loss of mitochondrial cristae. Additionally, the nucleus had lost the normal organization of chromatin, which appears uncondensed and uneven in contrast to a normal nucleus.
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DISCUSSION |
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Our study showed that fetal transgenic mice expressed viral protein only in salivary glands. This expression was not observed in adult transgenic mice. Several studies have shown that HCV can replicate in several tissues other than the liver, such as lymphocytes and salivary glands (Koike et al., 1997; Toussirot et al., 2002
; Ishikawa et al., 2003
). This may have important implications for the pathogenesis of HCV infections. There appears to be a strong association of salivary gland lesions, lymphocytic capillaritis and lymphocytic adenitis with chronic hepatitis C. HCV infection of lymphocytes may be related to HCV-associated lymphoma and/or autoimmune disorders. Similarly, salivary gland infection may be associated with Sjögren's syndrome (Haddad et al., 1992
; Toussirot et al., 2002
).
The HCV structural proteins caused profound structural abnormalities in the liver. Progressive steatosis, mitochondrial swelling and nuclear abnormalities became more severe with age. This hepatopathy was more widespread in the livers of older mice. Eventually, there was increasing fibrosis and liver cancer in older mice. Thus, this transgenic model of HCV represents many of the liver abnormalities observed in human infections. Moriya et al. (1997) described an HCV transgenic mouse model that expressed only the core protein. Their results showed that mice, 16 months of age, developed hepatic tumours, first appearing as adenomas with fat droplets in the cytoplasm. Additionally, they demonstrated neoplasia development in an adenoma, a nodule-in-nodule formation. The authors concluded that HCV core protein may have a primary role in HCC development in transgenic mice and suggested that steatosis may develop as early as 3 months of age. Similarly, our transgenic mouse model developed steatosis as early as 3 months and developed adenoma and carcinoma after 1 year of age. In contrast to the HCV core transgenic, our transgenic model expressed the three HCV structural core, E1 and E2 proteins.
The three HCV structural proteins are important for the viral life cycle, specifically in the entry and formation of the viral capsid (Santolini et al., 1994; Penin, 2003
). The significance of including core, E1 and E2 as a polyprotein in this mouse model allows for the analysis of the impact of these three proteins as a complex in the liver over the life span of the transgenic mice. While younger transgenic mice have no detectable HCV proteins, at 3 months of age an increasing number of hepatocytes expressing these proteins were observed around central veins. Viral protein expression and steatosis appeared in a similar time frame. Transgenic mice did not develop steatosis until they were at least 3 months of age with both severe microvesicular and macrovesicular steatosis eventually developing. The accumulation of lipids in hepatocytes is indicative of a disturbance in the lipid metabolism that may be caused by the accumulation of viral proteins and it is likely related to defects in mitochondrial and peroxisomal fatty acid oxidation and biosynthesis (Sabile et al., 1999
; Hope & McLauchlan, 2000
; Moriya et al., 2001
; Perlemuter et al., 2002
; Lerat et al., 2002
). Our results demonstrate aggravation of liver abnormalities with increasing age (Fig. 3a and b
). Previous studies have demonstrated steatosis is associated with a decreased antioxidant effect in the ageing liver. Similarly, a study performed by Lerat et al. (2002)
also indicated that steatosis increased with age. However, their mouse model rarely developed steatosis before 10 months of age.
A cross-sectional study by Kumar et al. (2002) suggested that the HCV genotype 3 but not genotype 1 was cytopathic and induced hepatic steatosis in HCV-infected patients. However, our mouse model expressing HCV genotype 1a caused severe steatosis, which demonstrates that this genotype is also involved in the development of this abnormality. Both Rubbia-Brandt et al. (2000)
and Adinolfi et al. (2001)
suggested that steatosis in patients infected with HCV genotype 1 is not of viral origin. However, the model described in this study suggested that HCV genotype 1a may induce steatosis, and it is of viral origin. Steatosis caused by obesity was ruled out in this study, as the transgenic mice were not obese. More compelling evidence included that the non-transgenic littermates did not show steatosis.
In contrast to earlier studies by Lerat et al. (2002) using an albumin liver-specific promoter and Moriya et al. (1997)
using an exogenous promoter, the results in the present study reflect the activity of a CMV promoter that has a universal transcriptional regulator. Thus, protein expression was illustrated in extra-hepatic tissues. After establishing the presence of HCV in older mice, fetal transgenic mice were analysed. In fetal mice, HCV structural proteins were detected only in the salivary glands (Fig. 2
). Koike et al. (1995)
confirmed this in another study. In contrast to Koike et al. (1995)
, there was no sialadenitis present in our transgenic mouse model. However, Koike et al. (1995)
developed mice that expressed only E1 and E2. In addition, the expression of HCV proteins in the salivary glands of older mice seemed to be lost. The unstable expression of HCV proteins in extra-hepatic tissues may explain the lack of sialadenitis in our model. Therefore, our transgenic mouse model may not be as useful for characterizing extra-hepatic sites of pathogenesis associated with persistent HCV infection.
Several mice older than 18 months of age demonstrated lymphoproliferative disorders with nodular hyperplasia and lymphoma developing in the liver. A recently described transgenic mouse model, expressing HCV core, developed lymphomas and liver adenomas at 20 months of age or older (Ishikawa et al., 2003). Similarly, HCV patients may develop two types of lymphoproliferative disorders, cryoglobulinemia and non-Hodgkin's B-cell lymphomas in the liver (Chan et al., 2001
; Gasparotto et al., 2002
). Clonal expansion of B-cells has been detected in bone marrow, blood and liver of HCV-infected patients. One study suggests that HCV E2 could play a pathogenic role by stimulating a strong humoral response and the clonal expansion of B-cells, resulting in lymphoproliferative disorders (Gasparotto et al., 2002
). However, our model is tolerant to the transgenes and chronic lymphocyte stimulation may therefore originate by other mechanisms. It is likely that the core protein may be involved in oncogenicity and lymphoproliferation. However, the study of this model may be useful to define the pathogenesis of HCV-associated lymphoproliferative disorders.
Recent publications have illustrated that HCV confers oncogenic potential to hepatocytes (Ikeda et al., 1993; Takano et al., 1995
; Chiba et al., 1996
; Silini et al., 1996
; Bruno et al., 1997
; Shibata et al., 1998
; Ueno et al., 2001
). In addition, HCC tumorigenesis in patients infected with HCV is correlated with steatosis (Lemon et al., 2000
; Ohata et al., 2003
). Transgenic mice with steatosis displayed dysplastic growth of cells evolving into tumour formation. Furthermore, transgenic mice showed deposition of collagen and progressive fibrosis (Kato et al., 2003
). These transgenic mice expressing core with a liver-specific serum amyloid P regulator did not demonstrate HCC. Therefore, the other two structural proteins of HCV (E1 and E2) used in this study may be a requisite for HCC development. Although the underlying mechanism of HCC progression in HCV transgenic mice remains to be understood, our transgenic mice are acceptable models to elucidate this role.
Murine models are critical for understanding HCV pathogenesis and to explore the molecular interface between HCV, the liver and other tissues. Recently, transplantation of healthy human hepatocytes that carry a plasminogen activator transgene (Alb-uPA) into SCID mice was carried out (Mercer et al., 2001). This model may be useful in acute studies of antiviral activity against HCV. In summary, expression of core, E1 and E2 in the liver of transgenic mice may contribute to the development of liver disease including steatosis, fibrosis and HCC. Further studies are required in order to explain the role of the structural proteins in mitochondrial injury and more generally in lipid metabolism. The transgenic mice presented in this study are suitable models to study the pathogenesis of HCV.
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ACKNOWLEDGEMENTS |
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Received 13 February 2005;
accepted 11 April 2005.