Int. J. Exp. Path. (I992), 73, i-8

Infection of Chang cells with hepatitis C virus using hepatic biopsy specimens from patients with chronic hepatitis (type C) Tsuneo Ozeki, Kazumasa Hikiji*, Keisuke Funakoshit, Masayoshi Tsutsumi*

and Akira Yoshida*

The 3 rd Department of Internal Medicine, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, *Genetic Research Laboratory, SRL, Inc., Tokyo, and tThe Department of Pathology, Kyushu Dental College, Kitakyushu, Japan Received for publication I 3 March i 9 9 i Accepted for publication I 4 May I 9 9 I

Summary. The presence of hepatitis C virus sequence was detected in liver tissue extracts by the polymerase chain reaction (PCR) method using primers of non-coding region in six out of eight cases with chronic hepatitis seropositive for Chiron's antibody. Subsequently, liver extracts from these cases were added to cell cultures of Chang cells for 3 days. The liver extracts of the six cases positive for PCR appeared to infect the Chang cells. Keywords: hepatitis C virus, hepatic biopsy, Chang cells Now that hepatitis (type C) can be diagnosed hepatitis and also seropositive for Chirons's by commercial assay for antibodies to Cioo antibody. protein of hepatitis C virus (Chiron's antibody), a number of new hepatitis C virus Materials and methods studies have been published (Alter et al. I989; Van der Poel et al. I990; Roggendorf Liver biopsies were taken from eight patients et al. I 989). The presence of hepatitis C virus with chronic hepatitis who were seropositive sequence in serum as revealed by the PCR for Chiron's antibody, a patient with chronic method has been reported in about 50% of hepatitis (type B) and one with primary patients seropositive for Chiron's antibody biliary cirrhosis (PBS). Half of each biopsy (Garson et al. I990). The presence of hepati- specimen was stored at - 70°C and the tis C virus sequence detected by the PCR remainder was fixed by formalin; sections method appeared to be a better predictor of were stained with haematoxylin and eosin infectivity than is the presence of Chiron's (HE) and with azan for histological examinantibody. So far it has not proved possible to ation and diagnosis. infect cultured cells with hepatitis C virus; if Chang cells were obtained from Dainippon this could be achieved, such infected cells Pharmaceutical Co. Ltd. (Osaka, Japan). would be useful for further study of hepatitis After pipetting off the medium, the flask in C virus infectivity. In this paper, we report which the Chang cells had been planted and successful infection of cultured cells with stored was washed twice with minimal extracts positive for PCR derived from liver essential medium (MEM) containing 0.02% biopsy specimens from patients with chronic ethylenediaminetetra-acetic acid (EDTA) in Correspondence: Dr Tsumeo Ozeki, The Third Department of Internal Medicine, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan. I

Tsuneo Ozeki et al. One of each pair of duplicate slides was a 3 7TC water bath for 30 s. A trypsin EDTA fixed in formalin for staining by the avidinsolution (trypsin (I:250) 0.5 g/l, EDTA. 4 biotin complex (ABC) method. The other Na 0.2 g/l, in Hanks buffer solution) was added to the flask and the remaining cells slide was fixed in cold acetone for staining by removed by repeated pipetting of the trypsin the immunofluorescence (IF) method. solution. The cell-containing solution was centrifuged at I000 r.p.m. for 3 min at room Reverse transcriptase and polymerase chain temperature, the supernatant pipetted off, reaction and the remaining cells suspended in 4 5 ml Eagle's MEM solution without FBS. The cells The methods for reverse transcriptase and PCR were modified according to Garson's were counted in a Fuchs-Rosenthal chamber and their number adjusted to method (Garson et al. 1990). 200 u1 of lysis 4x I05 cells/ml. These cells were subse- buffer was added to 200 [1 of liver extract and incubated at 3 70C for 40 min. After the quently used in the infection experiment. mixture had cooled to room temperature, it Liver biopsy specimens were homogenized with sea sand with a pestle in an agate was extracted with a phenol: chloroform mixture i: i (total 400 ,ul) and once with mortar. Each homogenate was suspended in chloroform. The supernatant was repeatedly I ml of Eagle's MEM solution containing IO% fetal bovine serum (FBS), I00 units of extracted with the same solvent. Finally, the penicillin G and ioo units of streptomycin supernatant was extracted with chloroform, 20 ,ug of glycogen was added and an ethanol and then centrfuged at 2 700 g at 40C for I 5 precipitate (3 vol of ethanol) was obtained by min. 0.5 ml of the supernatant was added to treatment at - 700C for an hour. The solu4 X IO5 Chang cells and incubated at 3 70C tion was then centrifuged at 12 000 g for i 5 for 30 min. The mixture was then centri- min. The precipitate thus obtained was fuged at i800 r.p.m. for I0 min. The washed twice with 95% ethanol and precipitated cells were suspended in 2 ml of vacuum dried. The dried precipitate was dissolved in 25 j1 Eagle's MEM solution containing IO% FBS, I00 units of penicillin G and I00 units of of diethyl pyrocarbonate treated water and i0o u1 sample RNA solution was incubated at streptomycin and adjusted to I X I05 cells/ slide (LAB-TEK Chamber slide). Duplicate 9o°C for 2 min to denature the RNA. The suspension were incubated at 3 70C in 5% solution was cooled on ice and i0o jl mixed CO2 and 95% air for 3 days. As controls, with io mmol/l dithiothreitol, i mmol/l duplicate cultures of Chang cells alone, with- deoxynucleotide triphosphate (dNTP), 20 units of RNase inhibitor (Takara, Japan), out the liver supernatants, were incubated in I00 ng of primer A (Table i) (region encodthe same way. 2

Structural protein

.1

51 Round 1

Round 2

Non-structural protein

293 base --------------

Primer A

Primer S1

178 base ----Primer A Primer S2 Hepatitis C genome and primer locations. Fig. i.

3,

Infection of Chang cells with hepatitis C

Scfe&es Library

3

Table i. Oligonucleotide sequence of primers

Primer S, (Forward outer) Primer S2

Sequence (5'-. 3')

Nucleotide positions

C G A C A C T C C A C C A T A G A T C A

3-22

G A G C C A T A G T G G T C T G C G G A

II8-137

G A C T A T C C C A C G A A C G C T A

276-295

(Forward inner) Primer A (Reverse)

ing non-coding region), 200 units of cloned Moloney murine leukaemia virus reverse transcriptase (Pharmacia, Sweden), I00 mmol/l tris-HCl pH 8.3, I50 mmol/l potassium chloride, 6 mmol/l magnesium chloride in a final volume of 20 jl and incubated at 3 70C for go min. The cDNA was stored at -20OC.

Round of the PCR was carried out in 50 jul solution containing 50 mmol/l potassium chloride, i.5 mmol/l magnesium chloride, o.oi% (w/v) gelatin, mmol/l tris-HCl pH 8.3, unit recombinant Taq DNA polymerase (Cetus, USA), 200 4umol/l of dNTP, 30 ng of outer primer (primer S, primer A) (Table i) and 5 jul of the cDNA sample. An initial 5 min denaturation at 940C was done and 35 cycles of 9 5C for 8o s, 5 5 C for min and 720C for min were carried out. This was followed by incubation for 7 min at 720C. The reaction mixture for Rounds of the PCR was made in the same manner as for Round with I/50 volume of the first PCR product in the inner primers (primer S2, primer A) (Table i). Thus the second Round was carried out with 2 5 cycles of 9 50C for 80 s, 5 5C for min, and 72°C for min, followed by 7 min extension at 720C. The products of both the first and second reactions were examined by electrophoresis on a V for 30 min. Bands 2% agarose gel, at were revealed by ethidium bromide staining and photographs were taken at 302 nm. The primers were prepared on an Applied Biosystems 38 I A DNA synthesizer with reference to the 5-terminal sequence of the hepatitis C virus genome (Okamoto et al. I990). i

io

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The immunofluorescence (IF) method (direct method) The cultured cells on glass slide were fixed by chilled acetone for I 0 and dried in air. Antihepatitis C virus (Anti-HCV) antibody (positive control in Chiron's Kit diluted 20 times with cold PBS, pH 7.2) was dropped onto the cells and they were washed three times with cold PBS, pH 7.2. Anti-human IgG conjugated fluorescein isothiocyanate (FITC) (Dako, Westburg) was dropped on the cells and they were incubated at 40C, overnight in the moisture chamber. The cells were washed three times with cold PBS pH 7.2, embedded in phosphate-buffered glycerin and assessed by fluorescence microscopy. s

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Staining for avidin-biotin complex Cultured Chang cells fixed with io% formalin were stained for HCV by the ABC method (ABC Kit, Dako, Accurate Chemical and Scientific Corporation, Westburg), using the primary antibody, anti-HCV antibody (positive control of Chiron's Kit, diluted with PBS 5 times) and the second antibody, Antihuman IgG raised in sheep. The positive cells were counted by light microscopy.

ioo

Histological examination Chronic hepatitis was diagnosed according to International Group classification (International Group 1977).

Tsuneo Ozeki et al.

4

Table 2. Summary of data for each case Infection on cultured cells

Histological Case I

2 3 4 5 6 7

8 9 Io

Age/Sex TT MO TH KF TS MS NA TA

MM TSe

75/F 64/F

55/M 58/M 28/M 30/M 64/F 42/M 48/F 54/F

Anti-C ioo + + +

exam

IF

ABC

+

CAH mod CAH mod CAH mod CAH mod CAH mod CAH mild CAH mild CAH mild CAH mild PBC(Stge2)

+ i + + + + -

+

-

+ + + + +

+ +

+ + + -

PCR

(HB%Ag(+))

-

-

-

+ + + + + i

IF (immunofluorescence method): +, staining cells more than io% ±, less than I0%; -, no staining cells. ABC (avidin-biotin complex method): +, Staining cells more than 20% (32 - 20%) ±, staining cells less than 20% and more than 5%; -, no staining

cells.

Results As shown in Table 2, I0 patients were examined. Eight patients were seropositive for Anti-Cioo. Histological examination showed three to have chronic active hepatitis (CAH)(mild) and five cases to have CAH (moderate). In the liver function test, cases and 7 had 8o-I20 alanine aminotransaminase (ALT) units in their serum and these elevated transaminase levels persisted despite treatment. The ALT activity in the other cases fell to less than 6o units after therapy. The presence of hepatitis virus C sequence in liver extracts, as indicated by the PCR method, was detected in six out of eight cases (Table 2). These cases had a single band related to 78 bp (Fig. 2). With the immunofluorescence method, more than o% of cells were stained in cases I, 4, 6 and 7 (Fig. 3). In cultured Chang cells, cells containing positive products to Anti-Cioo antibody (ABC method) were detected in all eight cases. By the ABC method, six cases had more than 20% of cells stained after 3 days

incubation (Fig. 4). However, the numbers of positive cells in cases 2 and 8 were scarcely seen. There were no positive cells in the control group without liver extracts. Discussion

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In the diagnosis of hepatitis (type C) using Chiron's Kit, the diagnosis rate for acute hepatitis (type C) is about 40%. In patients with chronic hepatitis (non A, non B type), the rate has been reported to be about 6o70%. More recently, Garson's group (Garson et al. I990) reported positive PCR results in about 6o% of patients with chronic hepatitis seropositive for Chiron's antibody. They also reported that of the six cases with acute hepatitis seropositive for Chiron's antibody, only one case, which was also positive for PCR, caused post-transfusion hepatitis. The presence of hepatitis virus C sequence in serum detected by PCR is therefore thought to be a good indicator of the infectivity of hepatitis C.

Infection of Chang cells with hepatitis C

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Fig. 2. PCR pattern of the Second Round product of HCV sequences in liver extracts of io patients. MW: molecular weight marker, 0 x 1 74 Hae III digestion. i TT 2 MM 5 TA 4 NA 3 MO 9 TSe 8 TH IO MS 6 TS 7 KF i I and I 2, Positive control, B, Negative control (no

template).

The PCR method has recently been considerably developed by the discovery of Taq polymerase (Saiki et a). I988). However, in the PCR method, the determination of primers is more important. The type C virus RNA is thought to consist of three portions, namely, structural protein, non-structural protein, and a non-coding region (Fig.' i). The essential characteristics of the primer are (i) forward and reverse primer are present in the structural protein region; (2) PCR takes place in more than a 2 Round system; (3) the region between forward and reverse primer is not variable. It is thus possible to alter the detection rate of PCR according to the selection of primers. As indicated above, the region of the primers of

the PCR method used by Garson et a]. ( I 990) was located in the non-structural protein. The region of the primers which we used in this study lies in the non-coding region, which is relatively non-variable. In liver biopsy extracts, the rate of detection of hepatitis C virus sequence by PCR may be somewhat higher than the rate previously reported for serum (Enomoto et a]. I990). Up till now it has not proved possible to infect cultured cells with type C virus. The present study used Chang liver cells although it is thought that Chang cells are HeLa cells and not liver cells. Nevertheless, with both immunofluorescence techniques and the ABC staining method, many cells were positively stained in six of eight cases

6

Tsuneo Ozeki et al.

Fig. 3. Immunofluorescence pattern of cultured cells (case I): cultured cells (Chang cells) fluorescing a light blue colour (250-290 nm) can be seen. a, Control; b, Case i.

seropositive for Chiron's antibody. In general, cases seropositive for Chiron's antibody may have the following characteristics: (i) cases with active type C virus infection; (2) cases with specific antibody to previous type C virus infection; and (3) cases with a

non-specific antibody. Cases negative for PCR-in liver extract may also differ; (i) there may be no type C virus in the liver extract or (2) in the PCR method the template of cDNA lying between the forward and reverse primer may be liable to variation. So the two cases which were seropositive for Chiron's antibody and negative for PCR could be explained in two ways; (i) though the non-coding region may be relatively stable, it could still undergo some minor change. In such a case, the liver extract may become negative for PCR. There could then be sufficient type C virus in the liver extracts to account infection. (2) Alternatively, there is no type C virus in the

liver extract; seropositivity for Chiron's antibody is thought to reveal previous exposure to type C virus.

Alternatively, since cases with PBC and type B hepatitis seronegative for Chiron's antibody and negative for PCR in liver extracts do not have type C virus, the cells may not have been infected. ABC staining using the positive controls in Chiron's Kit is thought to be partly nonspecific. Nevertheless, since the cells cultured without liver extract (as-controls) or with liver extracts of PBC and type B hepatitis showed no staining, it seems probable that the majority of the ABC stained cells observed were indeed infected with type C virus, even though the number of infected cells was small. In this study, the effects of 6 days incubation were also investigated in all cases. However, the infection rates were no higher than after 3 days incubation.

Infection of Chang cells with hepatitis C

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References ALTER H.J., PURCELL R.H. & SHIN J.W. (I989) Detection of antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-A, non-B hepatitis. N. Engi. J. Med. 321, 1494-1500. ENOMOTO N., TAKASE S., TAKADA A. & DATE T. (I990) Detection of hepatitis C virus genomes from patient's plasma using PCR method. Gastroenterol jpn. 25, 404-409. GARSON J.A., TEDDER R.S., BRIGGS M., TIJKE P., GLAZEBROOK J.A., TRUTE A., PARKER D., BARBARA J.A.J., CONTRERAS M. & ALOYSIIJS S. (I 990) Detection of hepatitis C viral sequences in blood donations by 'nested' polymerase chain reaction and prediction of infectivity. Lancet 335, I419-1422. INTERNATIONAL GROUP (I977) Acute and chronic

hepatitis revisited by an International Group, Lancet ii, 914-919. OKAMOTO H., OKADA S., SIJGIYAMA Y., YOTSUMOTO S., TANAKA T., YOSHIZAWA H., TSIJDA F., MIYAKAWA Y. & MAYUMI M. (I990) The 5'-terminal sequence of the hepatitis C virus genome. Jpn J. Exp. Med. 63, i67-I 77. ROGGENDORF M., DEINHARDT F., RASSHOFER R., FBERLE J., HOPF U., MOLLER B., ZACHOVOL R., PAPE G., SEHRAMM W. & ROMMEL F. (I989) Antibody to hepatitis C virus. SAIKI R.K., GELFAND D.H., STOFFEL S., SCHARE S.J., HIGUCHI R., HORN G.T., MIJLLIs K.B. & ERLICH H.A. (i 988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487-49 I. VAN DER POEL C.L., REESINK H.W., SCHAASBERG G.W. et al. ( I990) Infectivity of blood seropositive for hepatitis C virus antibodies. Lancet 335,

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Infection of Chang cells with hepatitis C virus using hepatic biopsy specimens from patients with chronic hepatitis (type C).

The presence of hepatitis C virus sequence was detected in liver tissue extracts by the polymerase chain reaction (PCR) method using primers of non-co...
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