Characterization of liver histopathology in a transgenic mouse model expressing genotype 1a hepatitis C virus core and envelope proteins 1 and 2

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Characterization of liver histopathology in atransgenic mouse model expressing genotype1a hepatitis C virus core and envelope proteins1 and 2

Turaya Naas,1,2 Masoud Ghorbani,1 Ikuri Alvarez-Maya,1

Michael Lapner,1 Rashmi Kothary,3 Yves De Repentigny,3

Susantha Gomes,4 Lorne Babiuk,5 Antonio Giulivi,6 Catalina Soare,1,2

Ali Azizi1,2 and Francisco Diaz-Mitoma1,2

Correspondence

Francisco Diaz-Mitoma

[email protected]

1Division of Virology, Children’s Hospital of Eastern Ontario, 401 Smyth Road, Ottawa, ON,Canada, K1H 8L1

2Department of Microbiology Immunology and Biochemistry, University of Ottawa, Ottawa, ON,Canada, K1N 6N5

3Ottawa Health Research Institute, Molecular Medicine Program, Ottawa, ON, Canada,K1H 8L6

4Department of Veterinary Pathology, Western College of Veterinary Medicine,University of Saskatchewan, Saskatoon, SK, Canada, S7N 5B4

5Department of Veterinary Microbiology, Veterinary Infectious Disease Organization,University of Saskatchewan, Saskatoon, SK, Canada, S7N 5E3

6Division of Blood Borne Pathogens, Health Canada, Ottawa, ON, Canada, K1A 0L2

Received 13 February 2005

Accepted 11 April 2005

Hepatitis C virus (HCV) is a major cause of chronic hepatitis and hepatocellular carcinoma

worldwide. The purpose of this study was to determine how the HCV structural proteins affect the

dynamic structural and functional properties of hepatocytes and measure the extra-hepatic

manifestations induced by these viral proteins. A transgenic mouse model was established by

expressing core, E1 and E2 proteins downstream of a CMV promoter. HCV RNA was detected

using RT-PCR in transgenic mouse model tissues, such as liver, kidney, spleen and heart.

Expression of the transgene was analysed by real-time PCR to quantify viral RNA in different

tissues at different ages. Immunofluorescence analysis revealed the expression of core, E1 and E2

proteins predominantly in hepatocytes. Lower levels of protein expression were detected in

spleen and kidneys. HCV RNA and viral protein expression increased in the liver with age.

Histological analysis of liver cells demonstrated steatosis in transgenic mice older than 3 months,

which was more progressed with age. Electron microscopy analysis revealed alterations

in nuclei, mitochondria and endoplasmic reticulum. HCV structural proteins induce a severe

hepatopathy in the transgenic mouse model. These mice became more prone to liver and

lymphoid tumour development and hepatocellular carcinoma. In this model, the extra-hepatic

effects of HCV, which included swelling of renal tubular cells, were mild. It is likely that the HCV

structural proteins mediate some of the histological alterations in hepatocytes by interfering

with lipid transport and liver metabolism.

INTRODUCTION

Hepatitis C virus (HCV) is a major cause of chronic humanliver disease. Approximately 170 million people worldwideare infected with HCV (Cohen, 1999). HCV infection oftenprogresses to chronic hepatitis, liver cirrhosis and hepato-cellular carcinoma (HCC). Although death from fulminant

hepatitis C is rare, it accounts for the deaths of at least8000–10 000 Americans each year (Alter, 1997).

HCV belongs to the family Flaviviridae and has a posi-tive single-stranded RNA genome that encodes a 3000 aapolyprotein precursor. The structural proteins core, E1and E2, which form viral particles, are located at the

0008-0969 G 2005 SGM Printed in Great Britain 2185

Journal of General Virology (2005), 86, 2185–2196 DOI 10.1099/vir.0.80969-0


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amino-terminal end of the precursor protein. Downstreamare 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 arenot completely understood. Following an incubation periodof 3–12 weeks, HCV clinical manifestations are often mild.However, as many as 85 % of the infected individualsdevelop a chronic infection, frequently with severe long-term liver pathology (Hoofnagle, 1997). HCV infection isalso associated with the development of extra-hepatic mani-festations, such as type II mixed cryoglobulinemia, glomeru-lonephritis and B-cell non-Hodgkin’s lymphoma (Chanet al., 2001; Gasparotto et al., 2002; Ishikawa et al., 2003).Chronic HCV infection has been treated with interferon-abut less than 20 % of patients achieve a sustained responsewith this drug. Combination therapy of interferon-a anda nucleoside analogue, ribavirin, has increased the rate ofsustained response to more than 30 % (Gretch et al., 1996;Davis et al., 1998; Poynard et al., 1998). The broad geneticvariability of the virus genome has resulted in six identifiedgenotypes with more than 50 subtypes (Simmonds et al.,1993). Also, in individuals infected with HCV, the virusbecomes a quasispecies, a population of closely relatedvariants (Bukh et al., 1995; Kato, 2000). Thus, the under-standing of the biology of the infection, the diagnosis andthe development of a vaccine against HCV is complex.

Little is known about the mechanism of HCV pathogenesis.However, both immune-mediated mechanisms and directviral cytotoxicity have been suggested as factors in thepathology associated with hepatitis C (Chisari, 1997). Thelack of an appropriate viral culture system and a smallanimal model that can support virus replication has ham-pered detailed analysis of HCV pathogenesis and vaccinedevelopment (Chisari, 1997). Combined research effortswith different small animal models will facilitate detailedanalysis of HCV in vivo and clarify HCV pathogenesis.

A previous study has shown that HCV proteins are notdirectly cytopathic on liver cells and that the host immuneresponse plays an important role in hepatitis C infection(Wakita et al., 1998). In contrast, other studies have pro-vided evidence that HCV structural proteins play a directrole in the development of liver steatosis, and increase therisk of liver cancer in transgenic mice (Lerat et al., 2002).These transgenic mouse models utilize organ-specific pro-moters, which preferentially express viral proteins in theliver. In the present study, a transgenic mouse model thatexpressed the HCV structural proteins core, E1 and E2under the control of a universal CMV promoter wasdeveloped. The model shared several features of humanHCV infection, such as severe hepatopathy characterizedby the development of steatosis as well as liver and lym-phoid tumours. This animal model could be used to studypathogenesis and novel treatment modalities for HCVinfections and liver cancer.

METHODS

Construction of expression vector. Total RNA was extractedfrom the plasma of a patient infected with HCV genotype 1a. TheRNA was used as a template to amplify core, E1 and E2 genes. TheHCV fragment containing core, E1 and truncated E2 genes, encod-ing for amino acid residues 1–683 (2049 nt), was constructed byRT-PCR using the forward primer 59-ACCATGAGCACGAAT-CCTAAACCTC-39 and the reverse primer 59-TGGTAGGGTTGTG-AAGGAACACG-39. The amplified fragment was cloned into theEcoRI sites of pCR 2.1 vector using the TOPO-TA cloning kit(Invitrogen). The nucleotide sequence was verified by DNA sequen-cing using the University of Ottawa DNA sequencing facility. Thecore, E1 and E2 fragments were subsequently subcloned into pVAX(Invitrogen) downstream of a cytomegalovirus (CMV) promoter(Fig. 1a). The pVAX-HCV (core, E1, E2) was linearized by MluIrestriction enzyme digestion.

Production of transgenic mice. The linearized plasmid contain-ing HCV genes encoding core, E1 and E2 proteins was micro-injected into the pro-nuclei of fertilized eggs from a B6C3F1 murinestrain (Charles River Laboratories). The injected eggs were surgicallyimplanted into the oviducts of pseudopregnant mothers. Foundermice harbouring HCV fragments were identified as transgenic viaPCR using DNA isolated from tail biopsies according to the DNeasyTissues kit protocol (Qiagen). Five founder mice that demonstrateda high copy number of the transgene were used to establish trans-genic lines. Transgenic mice were maintained by breeding with non-transgenic mice to develop a heterozygous colony. All mice were fedordinary chows and were maintained in a specific, pathogen-freestate and received standard care following guidelines establishedin the animal care facility at the Faculty of Medicine, University ofOttawa.

RT-PCR. Total RNA was extracted from different mouse tissues(liver, spleen, kidney, brain, lung and heart) using the RNeasy minikit (Qiagen). Reverse transcription of total RNA was performed withMuLV reverse transcriptase (Applied Biosystems) and random hex-amers (Applied Biosystems) to generate cDNA, according to themanufacturer’s instructions. The cDNA was amplified by PCR usingthe 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 and18 months. Mice were anaesthetized and then perfused with normalsaline followed by 4 % paraformaldehyde. Liver, kidney, heart,spleen, lung, salivary glands and brain tissues were removed surgi-cally and placed in 4 % paraformaldehyde overnight at 4 uC. Apiece of tissue was placed onto a plastic mould, covered with tissueembedding medium and then frozen in isopentane on dry ice.Frozen tissue sections of 5 mm thickness were cut using a cryostatand placed onto lysine-coated slides. Frozen tissue sections wereincubated with blocking buffer (5 % normal goat serum and 0?1 %Triton X-100 in PBS) for 1 h at room temperature. Rabbit anti-coreE1, E2 polyclonal antibody (prepared in our laboratory accordingto the University of Ottawa animal care facility protocols for anti-body production) or anti-core monoclonal antibody (Biogenesis)were applied at dilutions of 1 : 50 and 1 : 500, respectively, for 1 h atroom 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 withVectashield mounting medium (Vector). In addition to using tissuesfrom non-transgenic littermates as negative controls, we also per-formed parallel immunostaining experiments using pre-immunizedrabbit serum, as well as secondary anti-rabbit FITC-labelled antibodyalone.

2186 Journal of General Virology 86

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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 uC using 1 mg RNA per

reaction in a 20 ml reaction volume. In order to stop this reaction,

the cDNA samples were incubated for 15 min at 72 uC. The real-

time PCR was performed in special optical tubes in 36-well micro-

titre 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, 59-ATGTG-

TCCGTCGTGGATCTGA-39 and 59-TTGAAGTCGCAGGAGAC-

AACCT-39, respectively. The HCV core gene was analysed using

the following oligonucleotide primers (300 nM): forward 59-

ACCATGAGCAATCCTAAACCTC-39 and reverse 59-GCAACAAG-

TAAACTCCACCAACGA-39. 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 ml and

no cDNA was added to the negative control. For each amplifica-

tion using real-time PCR, the protocol included 15 min at 95 uCand 40 cycles of 15 s at 94 uC, 30 s at 66 uC and 30 s at 72 uC. All

PCR experiments were performed with hot start and were run in

1 aa 683 aa

5' 3'Core E1 E2

P (A)n

ErpVAX

Fig. 1. Structure and expression of HCV core, E1 and E2 transgene. (a) Schematic representation of pVAX plasmidcontaining HCV core, E1 and E2 genes. The HCV cDNA fragment containing core, E1 and truncated E2 genes encoded forHCV polyprotein (aa 1–683, 2049 nt) was inserted downstream of CMV promoter. (b) Detection of HCV RNA in differenttissues of transgenic mice by RT-PCR. This was performed using oligonucleotides that amplify a 1?2 kb fragment of core, E1and E2. Lanes 2, 3, 4, 5, 6 and 7 are positive for viral RNA in tissues of a transgenic mouse corresponding to liver, spleen,heart, kidney, brain and lung, respectively, lane 8 is the RNA extracted from the liver of a non-transgenic mouse used as anegative control. Lane 1 is the molecular size marker, 1 kb DNA ladder. (c–d) Immunofluorescent staining of CHO cellstransfected with pVAX core, E1 and E2 vector. (c) CHO cells transfected with pVAX alone showing no expression of protein.(d) CHO cells transfected with pVAX containing HCV core, E1 and E2 showing expression of proteins in the cytoplasm of thehepatocytes (arrows). Rabbit polyclonal antibodies to core, E1 and E2 was used as primary antibody. FITC-conjugatedantibody was used as a secondary antibody (magnification 640). Bars, 50 mm.

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Liver changes in an HCV transgenic model


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triplicate. The iCycler software (Bio-Rad) detected the thresholdcycle (CT) for each amplicon. Normalization was performed usingthe 22DDC

T method (Livak & Schmittgen, 2001). Experimentalcontrols including non-reverse-transcribed RNA samples wereperformed.

Histological staining. Mouse tissues were fixed in 4 % paraformal-dehyde and embedded in paraffin. Sections of 5 mm thickness werestained with haematoxylin and eosin, Sudan black B or Masson’strichrome staining according to standard methods used in theDepartment of Pathology and Laboratory Medicine at the Faculty ofMedicine, University of Ottawa. Tissue examination was performedby one of us (S. G.) and interpreted according to standard histo-pathological morphology. To determine the extent of steatosis asemi-quantitative method was used, in which 1+ represented lipiddroplets in sporadic hepatocytes; 2+ represented moderate steatosis,in which hepatocytes with lipid droplets were seen clearly in morethan two of five fields examined; 3+ represented moderate to severesteatosis, in which lipid droplets were extensively distributed inmost areas of the liver; 4+ very severe steatosis, in which steato-sis was observed everywhere in the liver. Characterization of thetumours was performed according to standard morphological histol-ogy. For example, the diagnosis of lymphosarcoma in the liver andnodes was made based on the undifferentiated nature of the cellmorphology, which resembled early embryonic cells; the presenceof many mitotic figures and the invasiveness represented by a lack oftumour capsule, tumour size and invasion of nodes and liver. Inaddition, a large portion of normal hepatic tissue was replaced bytumour cells. In contrast, the diagnosis of adenoma was based oncell morphology, including the well differentiated nature of cellswith few mitotic figures and the lack of invasion of extra-hepatictissues.

Electron microscopy. The electron microscopy study was doneaccording to the standard procedures at the electron microscopyfacility at the Children’s Hospital of Eastern Ontario in Ottawa.Approximately 0?25 mm thick liver tissue slices were post-fixedbriefly in 2 % osmium tetroxide in 0?1 M cacodylate buffer, dehy-drated in graded ethanol and embedded in Spur epoxy resin.Ultrathin 60–70 nm sections were cut using a Leica Ultracut Rultra-microtome, mounted on 200 mesh copper grids and stainedwith 10 % uranyl acetate in 50 % methanol, followed by Reynold’slead citrate. Grids were examined and micro-graphed using a JEOL1010 transmission electron microscope at 60 KV.

RESULTS

In vitro expression of the transgene

An HCV expression vector containing the sequencesencoding the core, E1 and E2 proteins was constructedfrom a PCR product obtained from the serum of a patientinfected with genotype 1a HCV. The amplified fragment wasinserted into the pVAX vector containing a CMV promoter(Fig. 1a). To measure the HCV protein expression in vitro,Chinese hamster ovary (CHO) cells were transiently trans-fected with pVAX core, E1 and E2, and then cells wereanalysed by immunolabelling with anti-core monoclonalantibody or with anti-core, E1 and E2 rabbit polyclonalantibody using immunofluoresence analysis. Recombinantviral proteins were present mainly in the cytoplasm, and noprotein expression was detected in the nuclei (Fig. 1c, d).Cell culture of the transfected CHO cells expressing the HCV

recombinant proteins demonstrated no apparent morpho-logical cellular changes.

Generation of transgenic mice

Five transgenic founder (F0) mice were used to establish fivetransgenic lines. Transgenic mice were analysed for HCVtransgene expression by PCR using genomic DNA extractedfrom the tails. Founder mice were crossed with B6C3F1 miceto expand the mouse colony, and ensure the integratedtransgene was successfully transmitted to the offspring. F1(first generation) and F2 (second generation) heterozygousmice 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 tran-script in all tissues including, liver, kidney, spleen, heart,lung and brain (Fig. 1b), but the viral proteins were mainlyexpressed in the liver (Fig. 2a, b) demonstrating selectiveexpression in certain tissues. Viral protein detection wasnot observed in either the heart or the brain. Spleen cells,as well as epithelial cells in the renal microtubules of thekidney, demonstrated relatively low levels of viral proteinexpression. The liver demonstrated the highest level ofviral protein expression with a centrilobular distribution ofgroups of cells expressing the viral proteins. Tissues fromnon-transgenic littermates did not show HCV proteinexpression. When fetal tissue was analysed by immuno-fluorescence to detect the viral proteins, the fluorescentsignal was restricted to the salivary glands (Fig. 2e, f).However, salivary glands of adult mice did not express HCVproteins (data not shown). In addition, there was no detec-table viral protein expression in fetal livers (data not shown).There was no difference in the levels of protein expressionbetween the five lines. Core, E1 and E2 protein expressionwas tested in all tissues using both rabbit anti-core, E1, E2polyclonal and anti-core monoclonal antibodies.

Viral proteins accumulate in hepatocytes withage

The number of hepatocytes expressing HCV proteinswas directly associated with the age of transgenic animals.Hepatocytes expressing the HCV proteins were detectedonly in mice 2–3 months of age or older (Fig. 2a, b). Thesehepatocytes were scattered around the central veins ina centrilobular pattern. Fluorescent cells appeared con-tiguous, forming patches of fluorescent hepatocytes withincreasing age. At 12–18 months of age, almost all cells inthe liver expressed the viral proteins (Fig. 2c, d).

Viral RNA is increased with age in transgenicmice

For further confirmation of the increased expression ofthe viral transgene in older mice, a comparison betweenfour groups of transgenic mice (aged 4 and 18 months)was performed using real-time RT-PCR. Increased expres-sion of the HCV core RNA transcript was observed in older


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Fig. 2. Immunofluorescence staining of HCV structural proteins in frozen formalin-fixed transgenic mouse tissues. (a) Liver section ofa 2-month-old transgenic mouse showing the expression of HCV structural proteins in hepatocyte cytoplasm (arrows). (b) Liversection from a 2-month-old non-transgenic mouse showing no evidence of HCV structural proteins. Propidium iodide used as acounter stain. (c) Liver of 1-year-old transgenic mouse showing an increase in the number of hepatocytes expressing HCV structuralproteins in the cytoplasm (arrows). Cells without green fluorescence in the middle of the section are not expressing the protein.(d) Liver section of a 1-year-old non-transgenic mouse showing no expression of HCV structural proteins. (e–f) Immunofluorescencedetection of HCV structural proteins in frozen formalin-fixed sections of salivary glands of a transgenic mouse fetus stained with arabbit polyclonal antibody to HCV core, E1 and E2 proteins; (e) is lowmagnification (620), (f) high magnification (640). Proteins aredetected in both the epithelial cell layer – arranged aswedges forming an acinus around a central lumen – and in themyoepithelial celllayer surrounding them. Rabbit polyclonal antibody against core, E1 and E2was used as primary antibody. FITC-conjugated antibodywas used as a secondary antibody. For panels (a–d), magnification 640. Bars, 50 mm.




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mice (Table 1). There was a 1?9-, 274- and 776-fold increasein HCV transcript concentration at 6, 14 and 18 months,respectively, when compared with 4-month-old mice.

Differential viral RNA expression in transgenicmouse tissues

The viral RNA expression varied not only according to age,but also according to individual tissues (Table 2). The HCVcore, E1 and E2 expression was higher in the liver than inany other tissues examined by real-time PCR. Hepatic viralRNA expression was increased more than 68-fold over thebrain. In contrast, spleen and kidneys showed approxi-mately the same level of expression of viral RNA.

Histopathological observations in transgenicmice

Post-mortem examination of transgenic mice demonstratedthat the gross morphology of organs was normal, with theexception of the livers, which showed hepatomegaly andyellow discolouration. Transgenic mice (n=4) had a ratio ofliver to body mass of 6?1±1?6 % while the non-transgeniccounterpart (n=5) demonstrated a ratio of 4?1±0?6 %

(P<0?01). To determine if the expression and accumula-tion of HCV proteins induced morphological alterationin hepatocytes, paraffin-embedded liver sections werestained with haematoxylin and eosin. Transgenic hepato-cytes showed several morphological alterations includ-ing increased cell volume and chromatin disorganization.Normal hepatocytes showed a polygonal shape that was welldelineated and had arranged hepatic plates. In contrast,transgenic hepatocytes were round and swollen to aboutthree times the size of normal cells. There was a loss ofhepatic cell borders and a loss of hepatic plate arrangementin transgenic hepatocytes as well as non-uniform staining inthe nuclei (Fig. 3a, b). Cytoplasmic vacuolation character-istic of steatosis was prominent in hepatocytes of trans-genic mice at 3 months of age or older (Fig. 3b). Apercentage of 45?8 transgenic mice that developed steatosiswere male, while 54?2 % were female (Table 3). Both micro-vesicular and macrovesicular steatosis were observed. Therewas a centrilobular distribution of macrovesicular steatosis,while the microvesicular pattern was more generally distri-buted in the liver. Sudan black staining confirmed that theobserved cytoplasmic vacuolation in hepatocytes were infact lipid deposits in transgenic mice (Fig. 3c, d). The extent

Table 1. Relative expression of RNA in the livers of transgenic mice of different ages detected by real-time RT-PCR

Age

(months)

Mouse

no.

HCV

core CT

GAPDH

CT

DCT (average core CT”

average GAPDH CT)

DDCT (average DCTx”

average DCTxo)

Normalized core relative

to 4 months (2”DDCT)

18 4912 30?5 21?3

4924 30?0 20?1

4908 31?4 22?5

Average 30?6 21?3 9?3 9?6 776

14 2574 27?6 16?6

2575 28?2 16?6

2576 30?3 20?5

Average 28?7 17?9 10?8 8?1 274

6 0003 35?9 18?1

0021 36?5 19?6

0022 37?4 18?3

Average 36?6 18?6 18?0 0?9 1?9

4 0136 38?2 18?7

0137 37?8 19?5

Average 38?0 19?1 18?9 0 1

Table 2. Relative expression of RNA in various tissues detected by real-time RT-PCR

Transgenic

mouse tissue

HCV

core CT

GAPDH

CT

DCT (average core CT”

average GAPDH CT)

DDCT (average DCTx”

average DC liver)

Normalized core relative

to liver (2”DDCT)

Liver 25?4 13?00 12?40 0 1?00

Spleen 28?5 14?35 14?15 1?75 0?30

Kidney 25?4 11?20 14?20 1?80 0?29

Heart 26?5 10?45 16?05 3?65 0?08

Lung 29?6 11?45 18?15 5?25 0?03

Brain 30?9 12?40 18?50 6?10 0?01


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of the steatosis became more severe in older mice, whichresulted in involvement of more than 90 % of hepatocytes atthe age of 12 months. There was minimal or no inflamma-tory cell infiltration in liver sections of transgenic mice.Trichrome staining demonstrated deposits of collagen inportal areas and the collapse of lobular architecture of theliver in older mice (12–18 month-old) (Fig. 4f). Histologicalabnormalities were also found in the kidneys of trans-genic mice. Microtubular and glomerular epithelial cellswere swollen and demonstrated some cytoplasmic vacuola-tion (data not shown).

Evidence of lymphoproliferative disorders andtumours in transgenic mice

Several mice developed nodular hyperplasia in liver, spleenand lymph nodes, as well as development of lymphosarcomain the liver. Out of 109 transgenic mice investigated, 17(15?59 %) transgenic mice developed various tumours. Ofthese mice, 12 (70?5 %) were male suggesting that tumour

formation occurred more readily in males compared withfemales (Table 3). Mice developing lymphoproliferativedisorders were 18 months of age or older. Transgenic micedeveloped different hepatic tumours (Fig. 4), includingadenomas (29 %) (data not shown), hemangiomas (17?6 %)(Fig. 4a–c), lymphoid tumours (29 %) (Fig. 4d) and HCC(23 %) (Fig. 4e). All corresponding tissues from non-transgenic littermates did not demonstrate tumours orany evidence of lympoproliferative disorders.

Subcellular abnormalities in transgenichepatocytes

Electron microscopy demonstrated several abnormalitiesin the intracellular organelles of hepatocytes in transgenicmice (Fig. 5a, b). A loss of the rough endoplasmic reticulumpattern was observed as well as swelling of mitochondriaand loss of mitochondrial cristae. Additionally, the nucleushad lost the normal organization of chromatin, which

Fig. 3. Hepatic steatosis in transgenic mice. (a) Haematoxylin and eosin-stained paraffin-embedded liver section showingnormal liver histology in a 1-year-old non-transgenic mouse. (b) One-year-old transgenic liver section showing bothmacrovesicular (green arrows) and microvesicular (yellow arrows) steatosis. (c–d) Hepatic steatosis in transgenic mice asdetected by Sudan black B staining. (c) Four-month-old non-transgenic mouse liver cryosections (5 mm) showing no blackstained lipid droplets. (d) Four-month-old transgenic mouse liver section showing black stained lipid droplets indicated byarrows. The nucleus of the cell is stained red. Magnification 620. Bars, 50 mm.




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Fig. 4. Liver tumours in transgenic mice. (a–b) Haematoxylin and eosin-stained paraffin-embedded liver section showinghemangioma in a murine transgenic liver. Lesion appears consistent with early endothelial cell proliferation and peliosis –proliferating endothelial cells (arrows) aggressively invading hepatic sinusoids in the focal area – but there is more focal angiopathyrather than neoplasm, characteristic of benign hemangioma. (a) Shows the edge of the hemangioma. (b) Shows the centre of thehemangioma (magnification640). (c) Photomicrograph of a liver tumour in HCV-transgenic mouse stained with Masson’s trichromestain showing blue stained collagen trapped between hepatocytes. Red blood cells are shown in light brown colour (magnification660). (d) Haematoxylin and eosin staining of a liver section demonstrating infiltration by a lymphoid tumour. This mouse alsodeveloped tumours in the spleen and in cervical lymph nodes as well (magnification 620). (e) Trichrome staining of a transgenicliver section demonstrating development of HCC. Polymorphic nuclei and invasion of steatotic liver by anaplastic cells. Cells arearranged in macrotrabecular patterns – where trabeculae are eight or more cells thick – differentiating it from adenoma. Note theground glass appearance of tumour cells indicating protein synthesis (magnification620). (f) Trichrome staining of a liver section inan 18-month-old transgenic mouse. Capsular fibrosis due to disappearance of hepatocytes and approximation of the remainingreticulin fibres demonstrating collapse of the lobular structure, collagen deposition and inflammatory infiltrates. It involves largelobular areas and multiple lobules, producing approximation of portal fields together, which can still be recognized with a stain forelastic fibres (magnification 620). Bars, 50 mm.


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appears uncondensed and uneven in contrast to a normalnucleus.

DISCUSSION

The structural proteins core, E1 and E2 of HCV wereexpressed in a transgenic mouse model using a universal

promoter to elucidate the effects of HCV on hepatic andextra-hepatic tissues. Initially, the vector was transfectedinto CHO cells, in order to confirm authentic expression ofthe viral proteins and a high level of expression from thisvector. The HCV transgenic mice expressed RNA encodingthe structural proteins in all tissues examined. However, inadult mice viral proteins were expressed selectively in the

Table 3. Steatosis and tumour formation in the five transgenic lines investigated in the study

3+, Moderate to severe; 2+, moderate; 1+, mild; M, male; F, female; Tg+, transgenic; Tg2, non-transgenic.

Transgenic line Transgenic Total no. mice Gender No. M/F mice Steatosis Tumour formation

3+ 2+ 1+ Positive Negative

Line 1-1 Tg+ 53 M 26 13 6 7 6 20

F 27 10 12 5 2 25

Tg2 38 M 15 0 0 2 0 15

F 23 0 0 1 0 23

Line 2-1 Tg+ 6 M 3 3 0 0 1 2

F 3 2 1 0 0 3

Tg2 15 M 9 0 0 0 0 9

F 6 0 0 1 0 6

Line 3-1 Tg+ 27 M 9 7 2 0 1 8

F 18 12 6 0 0 18

Tg2 3 M 3 0 0 0 0 3

F 0 0 0 0 0 0

Line 4-1 Tg+ 5 M 0 0 0 0 0 0

F 5 3 2 0 2 3

Tg2 8 M 3 0 0 0 0 3

F 5 0 1 0 0 5

Line 5-1 Tg+ 18 M 12 10 2 0 4 8

F 6 4 2 0 1 5

Tg2 12 M 8 0 0 0 0 8

F 4 0 0 0 0 4

Fig. 5. Electron micrograph of a normal (a) and transgenic mouse liver (b). In the transgenic, extensive abnormalities areobserved in the hepatocyte organelles, with abnormal mitochondrial shape, swelling and loss of cristae as well as chromatindisorganization and lipid droplets in the cytoplasm (magnification 67500). L, Lipid; M, mitochondria; N, nucleus; GC, Golgicomplex.




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liver, but detected at low concentrations in both the kidneyand the spleen. It has been shown that chronic infectionwith HCV can lead to the immune complex syndromes ofcryoglobulinemia and membranoproliferative glomerulo-nephritis. Also hepatitis C is a complicating factor amongpatients with end-stage renal disease and renal transplants(Morales, 2004). The differential expression of viral proteinsin murine tissues suggests that viral protein expression maybe controlled via translational mechanisms. Immuno-histochemistry suggested that the level of viral proteins inthe liver increased with age. This was confirmed by real-timeRT-PCR, which was performed in transgenic mice livers ofdifferent age groups and in various tissues.

Our study showed that fetal transgenic mice expressedviral protein only in salivary glands. This expression wasnot observed in adult transgenic mice. Several studies haveshown that HCV can replicate in several tissues other thanthe liver, such as lymphocytes and salivary glands (Koikeet al., 1997; Toussirot et al., 2002; Ishikawa et al., 2003). Thismay have important implications for the pathogenesis ofHCV infections. There appears to be a strong association ofsalivary gland lesions, lymphocytic capillaritis and lympho-cytic adenitis with chronic hepatitis C. HCV infection oflymphocytes may be related to HCV-associated lymphomaand/or autoimmune disorders. Similarly, salivary glandinfection may be associated with Sjogren’s syndrome(Haddad et al., 1992; Toussirot et al., 2002).

The HCV structural proteins caused profound structuralabnormalities in the liver. Progressive steatosis, mitochon-drial swelling and nuclear abnormalities became moresevere with age. This hepatopathy was more widespread inthe livers of older mice. Eventually, there was increasingfibrosis and liver cancer in older mice. Thus, this transgenicmodel of HCV represents many of the liver abnormalitiesobserved in human infections. Moriya et al. (1997) des-cribed an HCV transgenic mouse model that expressed onlythe core protein. Their results showed that mice, 16 monthsof age, developed hepatic tumours, first appearing asadenomas with fat droplets in the cytoplasm. Addi-tionally, they demonstrated neoplasia development in anadenoma, a ‘nodule-in-nodule’ formation. The authorsconcluded that HCV core protein may have a primary rolein HCC development in transgenic mice and suggestedthat steatosis may develop as early as 3 months of age.Similarly, our transgenic mouse model developed steatosisas early as 3 months and developed adenoma and carcinomaafter 1 year of age. In contrast to the HCV core transgenic,our transgenic model expressed the three HCV structuralcore, E1 and E2 proteins.

The three HCV structural proteins are important for theviral life cycle, specifically in the entry and formation ofthe viral capsid (Santolini et al., 1994; Penin, 2003). Thesignificance of including core, E1 and E2 as a polyprotein inthis mouse model allows for the analysis of the impact ofthese three proteins as a complex in the liver over the lifespan of the transgenic mice. While younger transgenic mice

have no detectable HCV proteins, at 3 months of age anincreasing number of hepatocytes expressing these proteinswere observed around central veins. Viral protein expres-sion and steatosis appeared in a similar time frame. Trans-genic mice did not develop steatosis until they were at least3 months of age with both severe microvesicular and macro-vesicular steatosis eventually developing. The accumulationof lipids in hepatocytes is indicative of a disturbance in thelipid metabolism that may be caused by the accumulation ofviral proteins and it is likely related to defects in mito-chondrial and peroxisomal fatty acid oxidation and bio-synthesis (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 abnormalitieswith increasing age (Fig. 3a and b). Previous studies havedemonstrated steatosis is associated with a decreased anti-oxidant effect in the ageing liver. Similarly, a study performedby Lerat et al. (2002) also indicated that steatosis increasedwith age. However, their mouse model rarely developedsteatosis before 10 months of age.

A cross-sectional study by Kumar et al. (2002) suggested thatthe HCV genotype 3 but not genotype 1 was cytopathicand induced hepatic steatosis in HCV-infected patients.However, our mouse model expressing HCV genotype 1acaused severe steatosis, which demonstrates that this geno-type is also involved in the development of this abnor-mality. Both Rubbia-Brandt et al. (2000) and Adinolfi et al.(2001) suggested that steatosis in patients infected withHCV genotype 1 is not of viral origin. However, the modeldescribed in this study suggested that HCV genotype 1a mayinduce steatosis, and it is of viral origin. Steatosis causedby obesity was ruled out in this study, as the transgenicmice were not obese. More compelling evidence includedthat the non-transgenic littermates did not show steatosis.

In contrast to earlier studies by Lerat et al. (2002) usingan albumin liver-specific promoter and Moriya et al. (1997)using an exogenous promoter, the results in the presentstudy reflect the activity of a CMV promoter that has auniversal transcriptional regulator. Thus, protein expressionwas illustrated in extra-hepatic tissues. After establishingthe presence of HCV in older mice, fetal transgenic micewere analysed. In fetal mice, HCV structural proteins weredetected only in the salivary glands (Fig. 2). Koike et al.(1995) confirmed this in another study. In contrast to Koikeet al. (1995), there was no sialadenitis present in our trans-genic mouse model. However, Koike et al. (1995) developedmice that expressed only E1 and E2. In addition, the expres-sion of HCV proteins in the salivary glands of older miceseemed to be lost. The unstable expression of HCV proteinsin extra-hepatic tissues may explain the lack of sialadenitis inour model. Therefore, our transgenic mouse model may notbe as useful for characterizing extra-hepatic sites of patho-genesis associated with persistent HCV infection.

Several mice older than 18 months of age demonstratedlymphoproliferative disorders with nodular hyperplasiaand lymphoma developing in the liver. A recently described


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transgenic mouse model, expressing HCV core, developedlymphomas and liver adenomas at 20 months of age orolder (Ishikawa et al., 2003). Similarly, HCV patients maydevelop two types of lymphoproliferative disorders, cryo-globulinemia and non-Hodgkin’s B-cell lymphomas inthe liver (Chan et al., 2001; Gasparotto et al., 2002). Clonalexpansion of B-cells has been detected in bone marrow,blood and liver of HCV-infected patients. One studysuggests that HCV E2 could play a pathogenic role bystimulating a strong humoral response and the clonalexpansion of B-cells, resulting in lymphoproliferative dis-orders (Gasparotto et al., 2002). However, our model istolerant to the transgenes and chronic lymphocyte stimula-tion may therefore originate by other mechanisms. It islikely that the core protein may be involved in oncogenicityand lymphoproliferation. However, the study of this modelmay be useful to define the pathogenesis of HCV-associatedlymphoproliferative disorders.

Recent publications have illustrated that HCV confersoncogenic 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). Inaddition, HCC tumorigenesis in patients infected with HCVis correlated with steatosis (Lemon et al., 2000; Ohata et al.,2003). Transgenic mice with steatosis displayed dysplasticgrowth of cells evolving into tumour formation. Further-more, transgenic mice showed deposition of collagen andprogressive fibrosis (Kato et al., 2003). These transgenicmice expressing core with a liver-specific serum amyloid Pregulator did not demonstrate HCC. Therefore, the othertwo structural proteins of HCV (E1 and E2) used in thisstudy may be a requisite for HCC development. Althoughthe underlying mechanism of HCC progression in HCVtransgenic mice remains to be understood, our transgenicmice are acceptable models to elucidate this role.

Murine models are critical for understanding HCV patho-genesis and to explore the molecular interface betweenHCV, the liver and other tissues. Recently, transplantationof healthy human hepatocytes that carry a plasminogenactivator transgene (Alb-uPA) into SCID mice was carriedout (Mercer et al., 2001). This model may be useful in acutestudies of antiviral activity against HCV. In summary,expression of core, E1 and E2 in the liver of transgenic micemay contribute to the development of liver disease includ-ing steatosis, fibrosis and HCC. Further studies are requiredin order to explain the role of the structural proteins inmitochondrial injury and more generally in lipid metabo-lism. The transgenic mice presented in this study are suitablemodels to study the pathogenesis of HCV.

ACKNOWLEDGEMENTS

The authors thank Rita Frost for her help in preparing samples andassistance with RNA extraction. We also thank Helina Tadesse, KatrinaGee and Sue Aucoin for critical review. This work was supported bygrants from Health Canada and the Canadian Institutes for HealthResearch.

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Characterization of liver histopathology in a transgenic mouse model expressing genotype 1a hepatitis C virus core and envelope proteins 1 and 2