Aug 31, 1987 - These data led to the conclusion that the observed cytopathology was caused by hepatitis A virus. The isolation and propagation of hepatitis A ...
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1987, p. 2967-2971
Vol. 53, No. 12
0099-2240/87/122967-05$02.00/0 Copyright © 1987, American Society for Microbiology
Production of Cytopathology in FRhK-4 Cells by BS-C-1-Passaged Hepatitis A Virus ABIDELFATAH M. NASSERt AND THEODORE G. METCALF* Department of Virology and Epidemiology, Baylor College of Medicine, Houston, Texas 77030 Received 1 May 1987/Accepted 31 August 1987
Cytopathic effects were produced in fetal rhesus monkey kidney (FRhK-4) cells 7 days postinfection by a serially BS-C-1-passaged strain of hepatitis A virus. Typical enterovirus cytopathology was produced by the HM-175 strain after 15 passages at 7-day intervals in BS-C-1 cells. No cytopathic effects were obtained after neutralization of virus with human anti-hepatitis A virus immunoglobulin G. Normal human serum had no effect on development of cytopathology. Maximum antigen and cDNA probe-based hybridization activity were associated with a CsCI gradient fraction having a density of 1.34 g/cm3. Large quantities of 27- to 30-nm virions typical of hepatitis A virus were associated with the same fraction. These data led to the conclusion that the observed cytopathology was caused by hepatitis A virus. passage in primate cultures at the time of receipt. Persistently infected AGMK cultures were used for virus propagation as previously described (20). Virus was recovered from cell lysates by chloroform extraction and purified by CsCl density gradient centrifugation. Fractions with a density of 1.34 g/cm3 were pooled and dialyzed for 16 h against 0.01 M phosphate-buffered saline (PBS) (pH 7.2) at 4°C. Mouse anti-HAV monoclonal antibody in the form of an ascites fluid was kindly supplied by Emilio Emini (Merck Sharp & Dohme Research Laboratories, West Point, Pa.). After clarification by centrifugation for 10 min at 14,000 x g, immunoglobulins were precipitated at 4°C with an equal volume of saturated ammonium sulfate, and mouse antiHAV immunoglobulin G (IgG) was purified by column chromatography on protein A-Sepharose (Sigma Chemical Co., St. Louis, Mo.) by the method of Emini et al. (6). As previously reported, this monoclonal antibody possessed high affinity for purified HAV (14). Human polyclonal antiHAV serum obtained from Blaine Hollinger (Baylor College of Medicine, Houston, Tex.) was precipitated with 50% ammonium sulfate, and the human anti-HAV IgG was purified by ion-exchange column chromatography on DEAEcellulose (7). Human anti-HAV IgG was labeled with 1251 by the
The isolation and propagation of hepatitis A virus (HAV) in cell culture was first reported by Provost and Hilleman (17). Since that report, HAV has been cultivated in primary and continuous African green monkey kidney (AGMK) cultures (2, 4, 20), fetal rhesus monkey kidney (FRhK-4) cells (2, 8, 23), human embryonic fibroblasts (10), and a hepatoma cell line that produces the hepatitis B surface antigen (9). In all these cultures HAV replicated without causing cytopathic effect (CPE). Adaptation of an HAV isolate to rapid growth in a FRhK-4 subline culture, Frp/3, followed by production of CPE in this culture was recently reported (22). The rapidly growing HAV replicated in human diploid fibroblast, Vero, and RC-37 cells, but did not produce CPE in any of these cultures. The CPE in the Frp/3 subline was attributed solely to HAV. During the course of studies seeking to increase HAV quantities produced while simultaneously decreasing the culture time required, we observed development of CPE in FRhK-4 cells. The following note describes the production of an enterovirus-type CPE by the HM-175 strain of HAV serially passaged in BS-C-1 cells. FRhK-4 cells obtained from the American Type Culture Collection at passage 30 were grown in Eagle minimal essential medium supplemented with 0.73% L-15 medium containing L-glutamine, 0.1 mM each of the nonessential amino acids, 0.1% NaHCO3, 0.4% HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer, 0.5% glucose, 0.004% each of penicillin, streptomycin, and gentamicin, and 10% heat-inactivated fetal bovine serum (FBS). Maintenance medium was the same except for 2% FBS. Cells were grown at 37°C and split at a ratio of 1:2 every 7 days. BS-C-1 cells, also obtained from the American Type Culture Collection at passage 43, were grown and maintained in the same media as described for FRhK-4 cells. The cell culture-adapted HM-175 strain of HAV used in the study was obtained from Robert Purcell (National Institutes of Health, Bethesda, Md.). The HM-175 stool isolate had been passaged six times in marmosets and was in its 16th
lodogen (1,3,4,6-tetrachloro-3a,6a-diphenyl glycoluril;
Pierce Chemical Co., Rockford, Ill.) method (19). lodination was performed by adding 35 RI1 of PBS, 5 ,ul of 1251 (0.5 mCi; Amersham Corp., Arlington Heights, Ill.), and 10 pu1 of anti-HAV IgG (1 mg/ml) to a tube containing a layer of Iodogen formed by evaporation of 50 ,ul of chloroform containing 2 ,ug of lodogen. The reaction was stopped after 2 min by transferring the iodination mixture to a new tube containing 0.5 ml of 0.01 M PBS. Free 125I was separated from bound 1251 on a PD-10 column (Pharmacia, Inc., Piscataway, N.J.). Detection of HAV antigen was made by solid-phase radioimmunoassay (SPRIA) by the method of Hollinger et al. (11). A 75-pI portion of anti-HAV monoclonal antibody (1:200 dilution in 0.1 M NaHCO3, pH 9) was added to each well of a polyvinyl microtiter plate (Dynatech Laboratories, Inc., Alexandria, Va.). The plate was incubated for 1 h at 37°C and then washed twice with 2% FBS in 0.01 M PBS. Next, 200 p.1 of 10% FBS in 0.01 M PBS was added to each well, and the plate was incubated for 30 min at room temperature. The wells were washed three times with
* Corresponding author. t Present address: Department of Environmental Sciences and Engineering, School of Public Health, University of North Carolina, Chapel Hill, NC 27514.
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2% FBS, after which 75-,ul portions of the HAV-containing sample diluted in 0.01 M PBS were added to each well. The plate was incubated for 16 h at room temperature. The wells were rinsed three times with 2% FBS, and 75 [lI of 1251_ labeled human anti-HAV IgG was added. The antibody was diluted with 10% FBS in 0.01 M PBS, and 75 ,ul with 200,000 cpm was added to each well. After 2 h of incubation at 37°C, the plate was washed four times with 0.01 M PBS containing 0.01% Tween 20. The radioactivity in each well was counted in a gamma counter. A positive-to-negative control ratio of 2.1 or greater was considered to be positive. Quantitative enumeration of HAV in BS-C-1 and AGMK cells was made by radioimmunofocus assay by the method of Lemon et al. (13). Duplicate tests were made with 0.25-ml inocula on 4- to 5-day-old monolayers in 60-mm petri dishes (Miles Scientific, Div. Miles Laboratories, Inc., Naperville, Ill.). After a 2-h incubation at 37°C, 5 ml of agarose overlay was added (0.5% Seakem agarose-minimal essential medium-L-15-based medium), and petri dishes were incubated at 36°C under 5% CO2 tension. After addition of a second agarose overlay at 7 days, the overlays were removed at 14 days postinfection (p.i.), and the monolayers were fixed for 5 min at -20°C with absolute ethanol. A 2-ml sample of 125I-labeled human anti-HAV IgG (106 cpm/ml) diluted in 10% FBS in 0.01 M PBS was added to each monolayer, and the petri dishes were incubated at 37°C for 3 h. The monolayers were washed six times, each time with 4 ml of 0.01 M PBS, and air dried, and the bottoms of the petri dishes were cut out. The monolayers were exposed to Kodak XAR film with intensifier screens at -70°C for 3 days, and after development of the film, the radioimmunofocus units were counted. Dot-blot hybridization analysis with an HAV cDNA probe was used to screen for the presence of HAV RNA as previously described (12). Phenol-chloroform-isoamyl alcohol (25:24:1, by volume)-extracted and absolute ethanolprecipitated nucleic acid was spotted on Zetabind membrane filters (AMF Cuno, Meriden, Conn.) with a 96-well microfiltration manifold (Schleicher & Schuell, Inc., Keene, N.H.). The filters were baked in vacuo for 2 h at 80°C and prehybridized for 2 h at 42°C in a prehybridization solution (4x SSC [lx SSC is 0.015 M NaCI plus 0.015 M sodium
citrate]-2.6x Denhardt solution [5]-0.1% sodium dodecycl sulfate-1.8 mM EDTA-80 pg of tRNA per ml-50% formamide). Filters were hybridized for 12 h at 42°C in the same solution with the addition of 2 x 106 cpm of 32P-labeled HAV cDNA per ml. Labeling by nick translation (18) with 32P-labeled dTTP (Amersham Corp.) resulted in routine specific activities of 1 x 108 to 2 x 108 cpm/,ug of cDNA. The filters were washed at 42°C (three times, 15 min each) in 2x SSC containing 0.2% sodium dodecyl sulfate and then in 2x SSC (three times, 15 min each). The filters were air dried and autoradiographed by exposure to XAR film for 18 h at -70°C with intensifier screens. Physical particle counts were performed by the pseudoreplication technique (21). Briefly, a 50-,u portion of the virus sample was spread evenly onto agar disks (2% Difco Noble agar in 0.85% saline) and allowed to dry. A 50-pI portion of Parlodion (0.75% in amyl acetate) was spread over the surface, drained, and dried. The Parlodion film was stripped from the agar disk and stained with 1% ammonium molybdate. The virus particle count was obtained with an RCA EMU 3F electron microscope (EM). Neutralization tests were performed by preparing serial 10-fold dilutions of HAV in serum-free minimal essential medium-L-15 medium and mixing 0.5-ml samples of each
APPL. ENVIRON. MICROBIOL.
dilution with an equal volume of a 1:10 dilution of human anti-HAV IgG or normal human serum (NHS). The mixtures were incubated for 1 h at 37°C, and duplicate 0.3-ml samples of each mixture were inoculated onto a 25-cm2 flask of confluent FRhK-4 cells. After a 1-h incubation at 37°C, 10 ml of maintenance medium was added to each flask. The cultures were incubated at 37°C and observed daily for development of CPE. BS-C-1 cultures were infected with HAV from a seventh passage, persistently infected AGMK culture. Passages in BS-C-1 cultures were made at 7-day intervals. HAV was detected intracellularly and in supernatant culture medium. The ratio of cell-associated virus versus virus released to culture medium was 80 to 20%, respectively, as determined by SPRIA. After 15 passages without production of CPE, the virus yield obtained from one 850-cm2 roller bottle was 1010 physical particles. Passage 15 virus was purified by CsCl density gradient centrifugation, and 1 ml of purified virus containing 107 radioimmunofocus-forming units was used to infect a confluent monolayer of FRhK-4 cells (multiplicity of infection, 10). CPE with virtually complete destruction of a monolayer was observed at 7 days p.i. An enterovirus-type CPE was produced, with cells showing an initial glistening appearance followed by rounding up and detachment from the surface of the flask. The CPE was produced by inocula from each of seven serial passages of HAV-containing culture medium. Fresh FRhK-4 monolayers were infected with 1 ml of culture medium from infected FRhK-4 cells at 7 days p.i. To establish that HAV was responsible for inducing the observed CPE, neutralization tests were performed with human anti-HAV IgG and NHS (control). The tests were made with 0.5-ml volumes of undiluted virus suspension harvested from 7- to 8-day-old FRhK-4 cultures showing CPE. Virus titers determined by quantal assay with FRhK-4 cultures were approximately 2 x 105 50% tissue culture infectious dose per ml. Figure 1 shows an uninfected monolayer of FRhK-4 cells (A) and an infected FRhK-4 monolayer at 8 days p.i. (B). No CPE was observed in cultures inoculated with HAV after incubation with human anti-HAV IgG (Fig. 1C). In contrast, CPE developed within 7 days p.i. in those cultures receiving HAV preincubated with NHS (Fig. 1D). Whereas high titers of HAV antigen were detected by SPRIA in cell lysates from cultures that developed CPE, no HAV antigen was found in cell lysates from CPE-negative cultures. To confirm the neutralization results, we partially purified HAV from infected FRhK-4 cell lysates by CsCl density gradient centrifugation (Fig. 2). Each fraction was tested for its density (refractometer reading), HAV antigenic activity (SPRIA), and RNA content (hybridization). The major peak of HAV antigen was detected in fraction 10, which corresponded to a density of 1.34 g/cm3 (Fig. 2A). Dot-blot hybridization gave the strongest signal in fraction 10, agreeing with the SPRIA results (Fig. 2C). A sample of fraction 10 that was evaluated by EM showed approximately 108 HAVlike virions 27 to 30 nm in diameter (Fig. 2B). These data, together with the results of the neutralization test, provide strong evidence that the agent causing the CPE in the FRhK-4 cells was HAV. AGMK passage 1 cells and passage 60 BS-C-1 cells infected with an HAV inoculum that had been passaged five times in FRhK-4 cells produced no CPE 15 days p.i. in either culture. Lysates of both AGMK and BS-C-1 infected cells were positive by SPRIA, showing that virus replication had occurred. These results indicated that CPE produced by the serially passaged HAV was limited to FRhK-4 cells; it was not produced either in the BS-C-1
VOL. 53, 1987
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FIG. 1. CPE in FRhK-4 cells as a result of HAV infection. (A) Uninfected FRhK-4 cell monolayer. (B) HAV-infected FRhK-4 cells. (C) FRhK-4 cells inoculated with neutralized HAV. (D) FRhK-4 cells inoculated with HAV preincubated with NHS.
culture in which the virus had been serially passaged or in AGMK cells. The development of CPE in FRhK-4 cells by HAV strain HM-175 adapted to growth in BS-C-1 cells was considered to be the result of large quantities of HAV and the presence of permissive host cells. Four kinds of evidence supportive of HAV as the sole cause of the CPE were collected. The CPE could be prevented by neutralization of HAV by human anti-HAV IgG but not with NHS. Occurrence of HAV antigen in FRhK-4 cells infected with nonneutralized HAV and the absence of antigen in FRhK-4 cells inoculated with neutralized HAV confirmed the specificity of the neutralization tests. The major peak of HAV antigenic activity occurred in CsCl gradient fraction 10 for which a density of 1.34 g/cm3 had been determined. Hybridization results showed the strongest signal to be associated with fraction 10. This same fraction also was shown to contain numerous 27to 30-nm virus particles with a morphology typical of HAV. The FRhK-4 cell line has often been found to be infected with polyomavirus (15). This conceivably might represent a situation in which CPE could be produced by the combined action of polyomavirus and HAV but not by HAV alone. Although the FRhK-4 culture used throughout the study had
been previously determined to be free of mycoplasma and polyomavirus, EM examination for 45-nm polyomavirus particles was made. No virus larger than 27 to 30 nm was detected in either FRhK-4 or BS-C-1 cells. Replication of HAV in FRhK-4, BS-C-1, and AGMK cultures was compared with a CsCl-purified inoculum prepared from an FRhK-4 culture showing 3+ CPE at 7 days p.i. (multiplicity of infection, 1.8). Replication was evaluated by EM, production of CPE, and SPRIA. All three criteria indicated maximum virus yields at 7 days p.i. in FRhK-4, compared with .14 days in AGMK or BS-C-1 as indicated by EM and SPRIA. Physical particles of HAV produced at the time of maximum yields in 25-cm2 cultures of approximately similar cell densities averaged 6.3 x 107 for FRhK-4, 9.2 x 106 for BS-C-1, and 7.4 x 106 for AGMK. A previous explanation for the production of CPE by a rapidly growing strain of HAV was based on a picornavirustype inhibition of host cell synthetic activities that eventually resulted in cell death (22). In studies in which CPE was observed in FRhK-4 cells or the Frp/3 subline, but not in BS-C-1 or other cultures, an explanation offered emphasized the crucial role of a host cell in determining the development of a picornavirus-type induced CPE (16, 22).
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APPL. ENVIRON. MICROBIOL.
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somewhat different. Only our data show that the virus particles have a density of 1.34 g/cm3 in CsCl and a diameter of 27 nm, properties identical to HAV. In addition, only our data prove by genomic hybridization test (cDNA) that the cytopathic agent is HAV. passage and conditions of culture are
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z
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40
LITERATURE CITED
E
0
0.
5 4c I
30 C 0
20
a
10
U
U).I
4
5
6 7 8 9 1011121314152122
Gradient Fraction Number
c Hybridization Autoradiograms FIG. 2. Characterization of HAV from infected FRhK-4 monolayers showing CPE. (A) Positive-to-negative (P/N) ratio for HAV antigen obtained by SPRIA and CsCI density gradient determined by refractometer reading. (B) Electron micrograph of material from fraction 10 showing 27-nm HAV particles. (C) Dot-blot hybridization made on a sample of each fraction from the same CsCl gradient. The strongest hybridization signal was detected in fraction 10.
We did not look at host cell intracellular activities, but EM studies indicated that the observed CPE in FRhK-4 cultures could have reflected an adverse host cell response to a rapid accumulation of virus products resulting from the greatly increased numbers of infective virions produced. We found persistently infected AGMK and BS-C-1 cultures to contain as much as 33% defective virions, whereas FRhK-4 cultures did not exceed 4% defective particles for the much greater numbers of physical particles produced. The data showed that about seven times more HAV, with eight times more infectious virions, was produced in FRhK-4 cultures in one-half or less time than that required for the lesser yields obtained in BS-C-1 cultures. We thank Joseph L. Melnick for his continued encouragement and support. The contributions made by Ed Calomeni in preparing and analyzing electron micrographs are gratefully acknowledged. This work was supported by the Texas A&M University Sea Grant Program (supported by the National Oceanic and Atmospheric Administration Office of Sea Grant, Department of Commerce) and by Public Health Service grant RR-05425 from the National Institutes of Health.
ADDENDUM
Two articles on the production of CPE by rapidly replicating isolates of HAV strain HM-175 were published in the Journal of Medical Virology after submission of this manuscript for publication (1, 3). We find our results, arrived at independently and without knowledge of the other studies, in agreement with the conclusions of those authors that rapidly replicating isolates of the cell culture-adapted HM-175 strain will produce demonstrable CPE. Our data offer additional and different evidence, however, that the CPE was caused by HAV per se. In particular, our history of
1. Anderson, D. A. 1987. Cytopathology, plaque assay, and heat inactivation of hepatitis A virus strain' HM-175. J. Med. Virol.
22:35-4.
2. Binn, L. N., S. M. Lemon, R. H. Marchwicki, R. R. Redfield, N. L. Gates, and W. H. Bapcroft. 1984.- Primary isolation and serial passage of hepatitis A virus strains in primate cell cultures. J. Clin. Microbiol. 20:28-g13. 3. Cromeans, T., M. D. Sobsey, and H. A. Fields. 1987. Develop-
ment of a plaque assay for a cytopathic, rapidly replicating isolate of hepatitis A virus. J. Med. Virol. 22:45-56. 4. Daemer, R. J., S. M. Feinstone, I. D. Gust, and R. H. Purcell. 1981. Propagation of human hepatitis A virus' in African green monkey kidney cell culture: primary isolation and serial passage. Infect. Immun. 32:388-393. 5. Denhardt, D. T. 1966. A membrane-filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23:641-646.
6. Emini, E. A., B. A. Jameson, A. J. Lewis, G. R. Larsen, and E. Wimmer. 1982. Poliovirus neutralization epitopes: analysis and localization with neutralizing monoclonal antibodies. J. Virol.
43:997-1005. 7. Fahey, J. L., and E. W. Terry. 1978. Ion exchange chromatography and gel filtration, p. 8.1-8.16. In D. M. Weir (ed.),
8.
9.
10. 11.
Handbook of experimental immunology, 3rd ed., vol. 1: Immunochemistry. Blackwell Scientific Publications, Ltd., Oxford. Flehmig, B. 1980. Hepatitis A virus in cell culture. I. Propagation of different hepatitis A virus isolates in a fetal rhesus monkey kidney cell line (Frhk-4). Med. Microbiol. Immunol. 168:239-248. Frosner, G. G., F. Deinhardt, R. Scheid, V. Gauss-Muller, N. Holmes, V. Messelberger, G. Siegl, and J. J. Alexander. 1979. Propagation of human hepatitis A virus in a hepatoma cell line. Infection 7:303-306. Gauss-Muller, V., G. G. Frosner, and F. Deinhardt. 1981. Propagation of hepatitis A virus in human embryo fibroblasts. J. Med. Virol. 7:233-239. Hollinger, F. B., D. W. Bradley, J. E. Maynard, G. R. Dreesman, and J. L. Melnick. 1975. Detection of hepatitis A viral
antigen by radioimmunoassay. J. Immunol. 115:1464-1466. 12. Jiang, X., M. K. Estes, T. G. Metcalf, and J. L. Melnick. 1986. Detection of hepatitis A virus in seeded estuarine samples by hybridization with cDNA probes. Appl. Environ. Microbiol. 52:711-717. 13. Lemon, S. L., L. N. Binn, and R. H. Marchwicki. 1983. Radioimmunofocus assay for quantitation of hepatitis A virus in cell cultures. J. Clin. Microbiol. 17:834-839.
14. Nasser, A. M., and T. G. Metcalf. 1987. An A-ELISA to detect hepatitis A virus in estuarine samples. Appl. Environ. Microbiol. 53:1192-1195. 15. Parry, J. V., J. E. Richmond, and S. D. Gardner. 1983. Polyomavirus in fetal rhesus monkey kidney cell lines used to grow hepatitis A virus. Lancet i:994. 16. Plagemann, P. G. W., and H. W. Swim. 1966. Replication of mengovirus. I. Effect on synthesis of macromolecules by host cell. J. Bacteriol. 91:2317-2326. 17. Provost, P. J., and M. R. Hilleman. 1979. Propagation of human hepatitis A virus in cell culture in vitro. Proc. Soc. Exp. Biol.
Med. 160:213-221. 18. Rigby, P. W., M. Dieckmann, C. Rhodes, and P. Berg. 1977. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113:237-251. 19. Salacinski, P. R. P., C. McLean, J. E. C. Sykes, V. V. Clement-
VOL. 53, 1987 Jones, and P. J. Lowry. 1981. lodination of proteins, glycoproteins, and peptides using a solid-phase oxidizing agent, 1,3,4,6tetrachloro-3a,6ct-diphenyl glycoluril (lodogen). Anal. Biochem. 117:136-146. 20. Simmonds, R. S., G. Szucs, T. G. Metcalf, and J. L. Melnick. 1985. Persistently infected cultures as a source of hepatitis A virus. Appl. Environ. Microbiol. 49:749-755. 21. Smith, K. O., and J. L. Melnick. 1962. Electron microscopic counting of virus particles by sedimentation on aluminized grids. J. Immunol. 89:279-284.
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22. Venuti, A., C. Di Russo, N. del Grosso, A.-M. Patti, F. Ruggeri, P. R. De Stasio, M. G. Martiniello, P. Pagnotti, A. M. Degener, M. Midulla, A. Pana, and R. Perez-Bercoff. 1985. Isolation and molecular cloning of a fast-growing strain of human hepatitis A virus from its double-stranded replicative form. J. Virol. 56:579588. 23. Wheeler, C. M., H. A. Fields, C. A. Schable, W. J. Meinke, and J. E. Maynard. 1986. Adsorption, purification, and growth characteristics of hepatitis A virus strain HAS-15 propagated in fetal rhesus monkey kidney cells. J. Clin. Microbiol. 23:434 440.
