Brief Communication/Ku rzmitteilu ng G. G. Fr6sner, F. Deinhardt, R. Scheid, V. Gauss-Miiller, N. Holmes, V. Messelberger, G. Siegl, J. J. Alexander
Propagation of Human Hepatitis A Virus in a Hepatoma Cell Line Summary: Hepatitis A virus (HAV) was isolated directly from human feces and propagated serially in an HBsAg producing human hepatoma cell line. No cytopathic effect was observed in the tissue culture and no detectable amounts of HAV were present in the tissue culture supernatant fluid. However, increasing amounts of hepatitis A antigen (HAAg) were detected by radioimmunoassay in the cell extracts obtained by freezing and thawing of cells. Specificity of the HAAg determination was shown by neutralization with convalescent sera of marmosets experimentally infected with the MS-1 strain of hepatitis A and by the absence of this neutralization with preinoculation sera. HAAg was first detected after four weeks in the cell extract of infected cultures after inoculation of 102-t04 tissue culture infectious doses of HAV from second passage. Zusammenfassung: Zfichtung yon Hepatitis A Virus in einer menschlichen Hepatomazellinie. Hepatitis A Virus (HAV) konnte in einer HBsAg produzierenden menschlichen Hepatomazellinie direkt aus dem menschlichen Stuhl isoliert und passagiert werden. Dabei wurde weder ein zytopathischer Effekt noch das Auftreten von HAV im Gewebekulturtiberstand beobachtet. Zunehmende Mengen von Hepatitis A Antigen (HAAg) waren jedoch in Zellextrakten, die durch Frieren und Tauen aus den Zellen gewonnen worden waren, mit einem Radioimmunassay nachzuweisen. Die Spezifit~it dieses Nachweises wurde dutch Neutralisation des HAAg mit Rekonvaleszentenseren von experimentell mit dem MS-1 Stamm des HAV infizierten Krallenaffen gezeigt und durch das Fehlen dieser Neutralisation mit Seren, die vor der Inokulation gewonnen worden waren. HAAg konnte nach der Inokulation von 102-104 ftir die Gewebekultur infekti6sen Dosen des HAV der zweiten Gewebekulturpassage erstmals nach vier Wochen im Zellextrakt der infizierten Kulturen gefunden werden.
Introduction Until recently the only available experimental systems for showing infectivity of specimens for hepatitis A depended on the inoculation of marmoset monkeys and chimpanzees (for review see Deinhardt, 1976 [1]), and human volunteers (for review see Zuckerman, 1975 [2]). This situation changed when Provost and Hilleman reported in 1979 (3) the propagation of hepatitis A virus derived from infected livers after many passages in marmosets, in primary marmoset liver and fetal rhesus kidney (FRhK6) cell cultures. However, neither type of'cell culture is easy to maintain in the laboratory: primary liver cell cultures cannot be passaged and the FRhK6 cultures have a maximum population doubling of only twelve. Also it was not clear whether hepatitis A virus (HAV) could be isolated directly from human materials without previous adaptation in marmosets. In this paper the isolation and serial propagation of H A V directly from human feces in an easily maintained, permanent hepatoma cell line carrying the genome of hepatitis B virus (HBV) is described.
Materials and Methods Source of hepatitis A virus inoculum: A 5 % stool suspension of patient MBB collected five days before onset of hepatitis A, which occurred 23 days after the patient had returned from vacation in the Mediterranean (fever > 39° C, fatigue), was the initial inoculum. The HAV etiology of the disease was proved by development of antibodies to HAV (anti-HAV) in the previously anti-HAV negative patient, and by the detection of anti-HAV of the IgM class during the acute stage but not during the late convalescent stage of the disease. This stool specimen and several others from the same patient contained large amounts of HAAg (detected by radioimmunoassay) and typical 27-28 nm particles. Hepatoma cell culture: A hepatoma cell line (4), carrying four copies of the total genome of HBV per cell (IV. S. Robinson, personal communication) but expressing only the hepatitis B surface antigen (HBsAg), was used. The line was obtained at passage 54, and our experiments were performed during passage 90 to 100. Cells were cultivated in 50 cm3 flasks (Nunc, Wiesbaden, FRG) using Eagle's minimal essential medium (MEM) containing 100 U/ml penicillin and 100 gg/ml streptomycin. In addition, growth medium contained 10% fetal calf serum (North American Biological Association, Miami/USA, Lot 017981), and maintenance medium used after infection of the cultures contained 0.5-5% fetal calf serum. Medium was changed twice weekly. Trypsine and EDTA (Titriplex III; Merck, Darmstadt, FRG) were used to split the cells. Virus propagation: A 5% stool suspension diluted 1:16 with serum-free medium was used for initial inoculation, Virus was adsorbed by adding 16 ml of the diluted suspension to a flask with a dense cell layer for four hours at 37° C. After that, 0.75 ml of fetal calf serum were added and the flasks incubated at 32° C. The virus was passed in cell culture by inoculating fresh cultures with various dilutions of crude cell extracts of the previous passage. Cell extracts were prepared by washing the infected cell culture with serum-free medium, freezing and thawing the cells three times, resuspending in 1-3 ml serumfree medium, and centrifuging for five minutes at 10.000 g. The supernatant was inoculated in the original strength or in various dilutions, and is referred to as "cell extract" throughout the paper. Detection of hepatitis A virus antigen (HAAg): HAAg was determined in a solid phase radioimmunoassay (5) in 0.2 ml of tissue culture supernatant or in 0.2 ml of cell extracts (see above). Neutralization of HAAg in the solid phase radioimmunoassay by convalescent sera of experimentally infected
Received: 17 October 1979 Dr. G. G. Fr6sner, Prof. F: Deinhardt, Dr. R. Scheid, Dr. V. Gauss-Miiller, Dr. N. Holmes, V. Messelberger, Max von Pettenkofer-Institut, University of Munich, Pettenkoferstr. 9a, D-8000 Munich 2, West Germany; Dr. G. Siegl, Institut fiir Hygiene und Medizinische Mikrobiologic, University of Bern, Bern, Switzerland; Dr. J. J. Alexander, National Institute for Virology, Sandringham, South Africa. This study was supported by grants Fr. 400/6 and De 26l/t of the Deutsche Forschungsgemeinschaft, grant IA 2-5/176295 of the Bundesministerium fiir Jugend, Familie und Gesundheit and grant 3.945-0.78 of the Schweizer Nationalfonds.
Infection 7 (1979) Nr. 6
303
G. G. Fr6sner et al.: Hepatitis A Virus marmosets and absence of neutralization by preinoculation sera was used for determination of the specificity of the reaction.
Results
Propagation .of hepatitis A virus: The results are summarized in Table 1. No cytopathic effect was seen during incubation at 32 ° C of the inoculated hepatoma cells, nor was H A A g detected at any time in the medium. However, despite repeated medium changes and dilution of the origi-
Table t: Detection of hepatitis A virus antigen by solid phase radioimmunoassay in extracts of infected hepatoma cells from serial passages. Passage number 1
HAAg specific cpm of inoculum Total dilution of culture supernatant through repeated renewals of culture fluids** Total dilution of original inoculum based on dilution of cell extracts used for the next passage Day of harvest of infected cells HAAg specific cpm in harvested cell extracts
2
93 100
3
3907* 78*
10-5
1 0 - 1 5 10-29,**
0
10-1
10- 7.48***
45
49
20
39 070 235 665 42 750
* Calculated from test result of undiluted cell extract. ** Each change of culture fluid was considered to be a dilution of 1:10. *** Refers to accumulated dilution through the 3 passages. This is a minimal estimate.
20 000
cpm
j
nal viral inoculum by the passage of dilutions of the cell extracts, the amount of H A A g detectable by radioimmunoassay increased. Titration of hepatitis A virus after second passage: Cell cultures were inoculated with 1 ml of cell extract of the second passage diluted 10 -1, 10 -3, 10 -5 and 10 -7. After seven weeks of incubation H A A g was determined in the cell extracts. In three of four experiments performed with inoculum of different tissue culture flasks after the second passage, H A A g was detected up to a dilution of 10 -3 but not at a dilution of 10 -s (see Figure 1, fight side). In one experiment (not shown here) a low amount of H A A g was also detected at 10 -5 .
Growth curve of hepatitis A virus after second tissue culture passage: Cell extracts which contained between 102 and 104 tissue culture infective doses (TCDs0) were inoculated in 14 new flasks. The cell extracts of two flasks were tested for H A A g after 14 hours, and a further two flasks were tested after 1, 2, 3, 4, 5 and 7 weeks. H A A g was not detected during the first three weeks of cultivation. It was just detectable after four weeks and was present in high concentration after five and seven weeks (see Figure 1, left side). Specificity of the HAAg determination: To establish specificity of the detected H A A g , 0.1 ml of an extract of cells obtained at seven weeks in the growth curve experiment was diluted 1:10 and mixed with 0.1 ml of preinfection or convalescent sera of two marmosets inoculated with the MS-1 strain of hepatitis A (6, 7),and with the preinfection and convalescent serum of a patient (MRM) who contracted hepatitis A after accidental inoculation of the German GBM hepatitis A isolate (5). All three convalescent sera, but not the three preinfection sera, completely blocked H A A g reactivity in the solid phase radioimmunoassay (see Table 2).
x 57 220
7
15 000 -
10 OOO
Figure 1: Growth curve (left) and 5000
~l 1456 (Input)
controls)
/~o
1000
sis k._..~~r 1
304
(nontnfected
/ 1728
2
3
4
Infection 7 (1979) Nr. 6
5
(~ after
weeks inoculation
lO-1 lo~ lo-5 lo-7 dilution of inoculum
virus titration (right) of HAV obtained after second passage in hepatoma cells. Amount of HAAg was determined by standard solid phase radioimmunoassays (see Materials and Methods). Cpm = amount of 1251 anti-HAV bound by 0.2 ml of cell extracts.
G. G. Fr6sner et al.: Hepatitis A Virus Table 2: Neutralization of HAAg present in cell extract obtained 7 weeks after inoculation, with convalescent but not with preinoculation sera from two marmosets inoculated with the MS-I strain of hepatitis A and from a patient with hepatitis A. Sera used Marmoset number H 307/74-EQ-2
Marmoset number H 284/69-BH-7 Patient MMR
Cpm measured in the HAAg test preinoculation serum convalescent serum
3253 590 --I-
extracts of infected cells
preinoculation serum convalescent serum
3945 591
preinfection serum convalescent serum
2177 575
Pool of anti-HAV negative sera Pool of anti-HAV negative sera
Discussion The finding of Provost and Hillema~ (3) that primary marmoset liver cells and fetal rhesus kidney cells in culture are susceptible to H A V was a major advance in efforts to propagate this virus. However, the limited life span of these cell cultures brought the authors to conclude that these systems "cannot provide the means for substituting cell cultures for animal propagation of the virus". In our report the susceptibility to H A V of a continuously growing hepatoma cell line is described. If further experiments show the susceptibility of this system to be comparable to that of marmosets, this system may be used in future to detect and titrate infectious hepatitis A virus present in clinical specimens. This system may also be used to produce the H A A g needed for serological tests and to further characterise HAV. It is obvious that it can not be used for vaccine preparation because the virus is propagated in a tumour cell line. In contrast to Provost and Hilteman, who used a strain of H A V adapted by passage through marmosets, our report indicates that H A V can be isolated directly from human stool without previous adaptation of the virus to an animal. In all systems, i. e. in primary marmoset liver cells and RhK6 cells, and in hepatoma cells, H A V replicated without cytopathic effects, and no virus was detected in the medium. However, in this study H A A g was detected by radioimmunoassay in the cell extracts and by indirect immunofluorescence in a granular form in the cytoplasm (but not in the n u c l e u s ) o f undamaged cells in longterm
2084 extract of noninfected cells
601
cultures of the hepatoma cells (Deinhardt, F., Scheid, R., Gauss-MiiUer, V., Fr6sner, G., unpublished data). The growth rate of H A V was different in the experiments of Provost and Hilleman as compared to our own. The slower development of H A A g in our system may be caused by the incubation temperature of 32 ° C and by the lower passage level of the virus. These two possibilities are being examined.
Literature 1. Deinhardt, F.: Hepatitis in primates. Adv. Virus Res. 20 (1976) 113-157.
2. Zuckerman, A. J.: Human viral hepatitis. Hepatitis associated antigen and viruses. North-Holland Publishing Co., Amsterdam/Oxford 1975. 3. Provost, P. J., Hilleman, M. R.: Propagation of human hepatitis A virus in cell culture in vitro. Proc. Soc. Exp. Biol. Med. 160 (1979) 213-221. 4. Alexander, J., Macnab, G., Saunders, R.: Studies of in vitro production of hepatitis B surface antigen by a human hepatoma cell line. Perspectives in Virol. 10 (1978) 103-120. 5. Frfsner, G. G., Overby, L. R., Flehmig, B., Gerth, H.-J., Haas, H., Decker, R. H., Ling, Ch.-M., Zuckerman, A. J., Fr6sner, H.-R.: Seroepidemiological investigation of patients and family contacts in an epidemic of hepatitis A. J. Med. Virol. 1 (1977) 163-173.
6. Deinhardt, F., Peterson, D., Cross, G., Wolfe, L., Holmes, A. W.: Hepatitis in marmosets, Am. J. Med. Sci. 270 (1975) 73--80.
7. Holmes, A. W., Wolfe, L. G., Rosenblate, H., Deinhardt, F.: Hepatitis in marmosets; induction of disease with coded specimens. Science 165 (1969) 816-817.
Infection 7 (1979) Nr. 6
;305
Propagation of Human Hepatitis A Virus in a Hepatoma Cell Line Summary: Hepatitis A virus (HAV) was isolated directly from human feces and propagated serially in an HBsAg producing human hepatoma cell line. No cytopathic effect was observed in the tissue culture and no detectable amounts of HAV were present in the tissue culture supernatant fluid. However, increasing amounts of hepatitis A antigen (HAAg) were detected by radioimmunoassay in the cell extracts obtained by freezing and thawing of cells. Specificity of the HAAg determination was shown by neutralization with convalescent sera of marmosets experimentally infected with the MS-1 strain of hepatitis A and by the absence of this neutralization with preinoculation sera. HAAg was first detected after four weeks in the cell extract of infected cultures after inoculation of 102-t04 tissue culture infectious doses of HAV from second passage. Zusammenfassung: Zfichtung yon Hepatitis A Virus in einer menschlichen Hepatomazellinie. Hepatitis A Virus (HAV) konnte in einer HBsAg produzierenden menschlichen Hepatomazellinie direkt aus dem menschlichen Stuhl isoliert und passagiert werden. Dabei wurde weder ein zytopathischer Effekt noch das Auftreten von HAV im Gewebekulturtiberstand beobachtet. Zunehmende Mengen von Hepatitis A Antigen (HAAg) waren jedoch in Zellextrakten, die durch Frieren und Tauen aus den Zellen gewonnen worden waren, mit einem Radioimmunassay nachzuweisen. Die Spezifit~it dieses Nachweises wurde dutch Neutralisation des HAAg mit Rekonvaleszentenseren von experimentell mit dem MS-1 Stamm des HAV infizierten Krallenaffen gezeigt und durch das Fehlen dieser Neutralisation mit Seren, die vor der Inokulation gewonnen worden waren. HAAg konnte nach der Inokulation von 102-104 ftir die Gewebekultur infekti6sen Dosen des HAV der zweiten Gewebekulturpassage erstmals nach vier Wochen im Zellextrakt der infizierten Kulturen gefunden werden.
Introduction Until recently the only available experimental systems for showing infectivity of specimens for hepatitis A depended on the inoculation of marmoset monkeys and chimpanzees (for review see Deinhardt, 1976 [1]), and human volunteers (for review see Zuckerman, 1975 [2]). This situation changed when Provost and Hilleman reported in 1979 (3) the propagation of hepatitis A virus derived from infected livers after many passages in marmosets, in primary marmoset liver and fetal rhesus kidney (FRhK6) cell cultures. However, neither type of'cell culture is easy to maintain in the laboratory: primary liver cell cultures cannot be passaged and the FRhK6 cultures have a maximum population doubling of only twelve. Also it was not clear whether hepatitis A virus (HAV) could be isolated directly from human materials without previous adaptation in marmosets. In this paper the isolation and serial propagation of H A V directly from human feces in an easily maintained, permanent hepatoma cell line carrying the genome of hepatitis B virus (HBV) is described.
Materials and Methods Source of hepatitis A virus inoculum: A 5 % stool suspension of patient MBB collected five days before onset of hepatitis A, which occurred 23 days after the patient had returned from vacation in the Mediterranean (fever > 39° C, fatigue), was the initial inoculum. The HAV etiology of the disease was proved by development of antibodies to HAV (anti-HAV) in the previously anti-HAV negative patient, and by the detection of anti-HAV of the IgM class during the acute stage but not during the late convalescent stage of the disease. This stool specimen and several others from the same patient contained large amounts of HAAg (detected by radioimmunoassay) and typical 27-28 nm particles. Hepatoma cell culture: A hepatoma cell line (4), carrying four copies of the total genome of HBV per cell (IV. S. Robinson, personal communication) but expressing only the hepatitis B surface antigen (HBsAg), was used. The line was obtained at passage 54, and our experiments were performed during passage 90 to 100. Cells were cultivated in 50 cm3 flasks (Nunc, Wiesbaden, FRG) using Eagle's minimal essential medium (MEM) containing 100 U/ml penicillin and 100 gg/ml streptomycin. In addition, growth medium contained 10% fetal calf serum (North American Biological Association, Miami/USA, Lot 017981), and maintenance medium used after infection of the cultures contained 0.5-5% fetal calf serum. Medium was changed twice weekly. Trypsine and EDTA (Titriplex III; Merck, Darmstadt, FRG) were used to split the cells. Virus propagation: A 5% stool suspension diluted 1:16 with serum-free medium was used for initial inoculation, Virus was adsorbed by adding 16 ml of the diluted suspension to a flask with a dense cell layer for four hours at 37° C. After that, 0.75 ml of fetal calf serum were added and the flasks incubated at 32° C. The virus was passed in cell culture by inoculating fresh cultures with various dilutions of crude cell extracts of the previous passage. Cell extracts were prepared by washing the infected cell culture with serum-free medium, freezing and thawing the cells three times, resuspending in 1-3 ml serumfree medium, and centrifuging for five minutes at 10.000 g. The supernatant was inoculated in the original strength or in various dilutions, and is referred to as "cell extract" throughout the paper. Detection of hepatitis A virus antigen (HAAg): HAAg was determined in a solid phase radioimmunoassay (5) in 0.2 ml of tissue culture supernatant or in 0.2 ml of cell extracts (see above). Neutralization of HAAg in the solid phase radioimmunoassay by convalescent sera of experimentally infected
Received: 17 October 1979 Dr. G. G. Fr6sner, Prof. F: Deinhardt, Dr. R. Scheid, Dr. V. Gauss-Miiller, Dr. N. Holmes, V. Messelberger, Max von Pettenkofer-Institut, University of Munich, Pettenkoferstr. 9a, D-8000 Munich 2, West Germany; Dr. G. Siegl, Institut fiir Hygiene und Medizinische Mikrobiologic, University of Bern, Bern, Switzerland; Dr. J. J. Alexander, National Institute for Virology, Sandringham, South Africa. This study was supported by grants Fr. 400/6 and De 26l/t of the Deutsche Forschungsgemeinschaft, grant IA 2-5/176295 of the Bundesministerium fiir Jugend, Familie und Gesundheit and grant 3.945-0.78 of the Schweizer Nationalfonds.
Infection 7 (1979) Nr. 6
303
G. G. Fr6sner et al.: Hepatitis A Virus marmosets and absence of neutralization by preinoculation sera was used for determination of the specificity of the reaction.
Results
Propagation .of hepatitis A virus: The results are summarized in Table 1. No cytopathic effect was seen during incubation at 32 ° C of the inoculated hepatoma cells, nor was H A A g detected at any time in the medium. However, despite repeated medium changes and dilution of the origi-
Table t: Detection of hepatitis A virus antigen by solid phase radioimmunoassay in extracts of infected hepatoma cells from serial passages. Passage number 1
HAAg specific cpm of inoculum Total dilution of culture supernatant through repeated renewals of culture fluids** Total dilution of original inoculum based on dilution of cell extracts used for the next passage Day of harvest of infected cells HAAg specific cpm in harvested cell extracts
2
93 100
3
3907* 78*
10-5
1 0 - 1 5 10-29,**
0
10-1
10- 7.48***
45
49
20
39 070 235 665 42 750
* Calculated from test result of undiluted cell extract. ** Each change of culture fluid was considered to be a dilution of 1:10. *** Refers to accumulated dilution through the 3 passages. This is a minimal estimate.
20 000
cpm
j
nal viral inoculum by the passage of dilutions of the cell extracts, the amount of H A A g detectable by radioimmunoassay increased. Titration of hepatitis A virus after second passage: Cell cultures were inoculated with 1 ml of cell extract of the second passage diluted 10 -1, 10 -3, 10 -5 and 10 -7. After seven weeks of incubation H A A g was determined in the cell extracts. In three of four experiments performed with inoculum of different tissue culture flasks after the second passage, H A A g was detected up to a dilution of 10 -3 but not at a dilution of 10 -s (see Figure 1, fight side). In one experiment (not shown here) a low amount of H A A g was also detected at 10 -5 .
Growth curve of hepatitis A virus after second tissue culture passage: Cell extracts which contained between 102 and 104 tissue culture infective doses (TCDs0) were inoculated in 14 new flasks. The cell extracts of two flasks were tested for H A A g after 14 hours, and a further two flasks were tested after 1, 2, 3, 4, 5 and 7 weeks. H A A g was not detected during the first three weeks of cultivation. It was just detectable after four weeks and was present in high concentration after five and seven weeks (see Figure 1, left side). Specificity of the HAAg determination: To establish specificity of the detected H A A g , 0.1 ml of an extract of cells obtained at seven weeks in the growth curve experiment was diluted 1:10 and mixed with 0.1 ml of preinfection or convalescent sera of two marmosets inoculated with the MS-1 strain of hepatitis A (6, 7),and with the preinfection and convalescent serum of a patient (MRM) who contracted hepatitis A after accidental inoculation of the German GBM hepatitis A isolate (5). All three convalescent sera, but not the three preinfection sera, completely blocked H A A g reactivity in the solid phase radioimmunoassay (see Table 2).
x 57 220
7
15 000 -
10 OOO
Figure 1: Growth curve (left) and 5000
~l 1456 (Input)
controls)
/~o
1000
sis k._..~~r 1
304
(nontnfected
/ 1728
2
3
4
Infection 7 (1979) Nr. 6
5
(~ after
weeks inoculation
lO-1 lo~ lo-5 lo-7 dilution of inoculum
virus titration (right) of HAV obtained after second passage in hepatoma cells. Amount of HAAg was determined by standard solid phase radioimmunoassays (see Materials and Methods). Cpm = amount of 1251 anti-HAV bound by 0.2 ml of cell extracts.
G. G. Fr6sner et al.: Hepatitis A Virus Table 2: Neutralization of HAAg present in cell extract obtained 7 weeks after inoculation, with convalescent but not with preinoculation sera from two marmosets inoculated with the MS-I strain of hepatitis A and from a patient with hepatitis A. Sera used Marmoset number H 307/74-EQ-2
Marmoset number H 284/69-BH-7 Patient MMR
Cpm measured in the HAAg test preinoculation serum convalescent serum
3253 590 --I-
extracts of infected cells
preinoculation serum convalescent serum
3945 591
preinfection serum convalescent serum
2177 575
Pool of anti-HAV negative sera Pool of anti-HAV negative sera
Discussion The finding of Provost and Hillema~ (3) that primary marmoset liver cells and fetal rhesus kidney cells in culture are susceptible to H A V was a major advance in efforts to propagate this virus. However, the limited life span of these cell cultures brought the authors to conclude that these systems "cannot provide the means for substituting cell cultures for animal propagation of the virus". In our report the susceptibility to H A V of a continuously growing hepatoma cell line is described. If further experiments show the susceptibility of this system to be comparable to that of marmosets, this system may be used in future to detect and titrate infectious hepatitis A virus present in clinical specimens. This system may also be used to produce the H A A g needed for serological tests and to further characterise HAV. It is obvious that it can not be used for vaccine preparation because the virus is propagated in a tumour cell line. In contrast to Provost and Hilteman, who used a strain of H A V adapted by passage through marmosets, our report indicates that H A V can be isolated directly from human stool without previous adaptation of the virus to an animal. In all systems, i. e. in primary marmoset liver cells and RhK6 cells, and in hepatoma cells, H A V replicated without cytopathic effects, and no virus was detected in the medium. However, in this study H A A g was detected by radioimmunoassay in the cell extracts and by indirect immunofluorescence in a granular form in the cytoplasm (but not in the n u c l e u s ) o f undamaged cells in longterm
2084 extract of noninfected cells
601
cultures of the hepatoma cells (Deinhardt, F., Scheid, R., Gauss-MiiUer, V., Fr6sner, G., unpublished data). The growth rate of H A V was different in the experiments of Provost and Hilleman as compared to our own. The slower development of H A A g in our system may be caused by the incubation temperature of 32 ° C and by the lower passage level of the virus. These two possibilities are being examined.
Literature 1. Deinhardt, F.: Hepatitis in primates. Adv. Virus Res. 20 (1976) 113-157.
2. Zuckerman, A. J.: Human viral hepatitis. Hepatitis associated antigen and viruses. North-Holland Publishing Co., Amsterdam/Oxford 1975. 3. Provost, P. J., Hilleman, M. R.: Propagation of human hepatitis A virus in cell culture in vitro. Proc. Soc. Exp. Biol. Med. 160 (1979) 213-221. 4. Alexander, J., Macnab, G., Saunders, R.: Studies of in vitro production of hepatitis B surface antigen by a human hepatoma cell line. Perspectives in Virol. 10 (1978) 103-120. 5. Frfsner, G. G., Overby, L. R., Flehmig, B., Gerth, H.-J., Haas, H., Decker, R. H., Ling, Ch.-M., Zuckerman, A. J., Fr6sner, H.-R.: Seroepidemiological investigation of patients and family contacts in an epidemic of hepatitis A. J. Med. Virol. 1 (1977) 163-173.
6. Deinhardt, F., Peterson, D., Cross, G., Wolfe, L., Holmes, A. W.: Hepatitis in marmosets, Am. J. Med. Sci. 270 (1975) 73--80.
7. Holmes, A. W., Wolfe, L. G., Rosenblate, H., Deinhardt, F.: Hepatitis in marmosets; induction of disease with coded specimens. Science 165 (1969) 816-817.
Infection 7 (1979) Nr. 6
;305
