A simple and rapid Hepatitis A Virus (HAV) titration assay based on antibiotic resistance of infected cells: evaluation of the HAV neutralization potency of human immune globulin preparations
© Konduru et al; licensee BioMed Central Ltd. 2008
Received: 05 December 2008
Accepted: 18 December 2008
Published: 18 December 2008
Hepatitis A virus (HAV), the causative agent of acute hepatitis in humans, is an atypical Picornaviridae that grows poorly in cell culture. HAV titrations are laborious and time-consuming because the virus in general does not cause cytopathic effect and is detected by immunochemical or molecular probes. Simple HAV titration assays could be developed using currently available viral construct containing selectable markers.
We developed an antibiotic resistance titration assay (ARTA) based on the infection of human hepatoma cells with a wild type HAV construct containing a blasticidin (Bsd) resistance gene. Human hepatoma cells infected with the HAV-Bsd construct survived selection with 2 μg/ml of blasticidin whereas uninfected cells died within a few days. At 8 days postinfection, the color of the pH indicator phenol red in cell culture media correlated with the presence of HAV-Bsd-infected blasticidin-resistant cells: an orange-to-yellow color indicated the presence of growing cells whereas a pink-to-purple color indicated that the cells were dead. HAV-Bsd titers were determined by an endpoint dilution assay based on the color of the cell culture medium scoring orange-to-yellow wells as positive and pink-to-purple wells as negative for HAV. As a proof-of-concept, we used the ARTA to evaluate the HAV neutralization potency of two commercially available human immune globulin (IG) preparations and a WHO International Standard for anti-HAV. The three IG preparations contained comparable levels of anti-HAV antibodies that neutralized approximately 1.5 log of HAV-Bsd. Similar neutralization results were obtained in the absence of blasticidin by an endpoint dilution ELISA at 2 weeks postinfection.
The ARTA is a simple and rapid method to determine HAV titers without using HAV-specific probes. We determined the HAV neutralization potency of human IG preparations in 8 days by ARTA compared to the 14 days required by the endpoint dilution ELISA. The ARTA reduced the labour, time, and cost of HAV titrations making it suitable for high throughput screening of sera and antivirals, determination of anti-HAV antibodies in human immune globulin preparations, and research applications that involve the routine evaluation of HAV titers.
Hepatitis A Virus (HAV), a Picornaviridae that causes acute hepatitis in humans, is a significant public health problem in developing nations with approximately 1.4 million new infections per year . The virus is mainly transmitted via the fecal-oral route, either from person to person or by ingestion of contaminated food and water. Community wide outbreaks can result from the consumption of oysters and mussels harvested from contaminated waters, fresh produce from contaminated water-irrigated fields, and food prepared by infected handlers [2–4]. For example, a recent HAV outbreak originated from contaminated green onions resulted in over 600 infection cases and 3 deaths . Hepatitis A is an age-dependent disease, and children 6 year old and younger in general develop a subclinical form of the disease. Older children and adults develop a more severe form of hepatitis A, which in some rare instances can result in fulminant hepatitis. In developing countries, water- and food-borne HAV infections are common during childhood, which induces life-long immunity.
The overall incidence of HAV has decreased in recent years in the United States and Europe [6, 7] but the proportion of travel-related cases has increased in the United States. HAV vaccination and immune globulin (IG) are recommended for international travellers who plan to visit countries that are considered intermediate to high endemic zones for HAV infection [7, 8]. IG is recommended in addition to vaccination for elderly persons who are immunocompromised, have chronic liver disease, or have chronic medical conditions and are travelling to endemic zones. HAV vaccine does not prevent infection if administered three or more weeks post virus infection, but protection is conferred by administration of IG two weeks after exposure to the virus [9, 10]. It has recently been shown that both HAV vaccine and IG are similarly effective for post-exposure prophylaxis within 2 weeks of the exposure to HAV . IG preparations are derived from pools of plasma from human donors. Anti-HAV antibody levels vary among different lots of IG preparations . HAV vaccinated donors tend to have 10–50 fold lower anti-HAV titers than donors who were naturally infected with HAV .
HAV grows poorly in cell culture and in general does not induce cytopathic effect (CPE). Cytopathic strains of HAV have been isolated but CPE takes a long time to develop, the plaques are difficult to visualize, and CPE is dependent on the multiplicity of infection [14, 15]. Modified HAV plaque assays that detect HAV antigen in fixed cells have been developed but are time-consuming and laborious [15–17]. ELISA-based endpoint dilution assays to titrate HAV are simple to perform but require 2 weeks of incubation to detect antigen at the higher dilutions [18–23]. We reported previously that the insertion of a blasticidin resistance (Bsd) gene into the genome of wild type (wt) HAV allowed the selection of a cell line with enhanced susceptibility to wt HAV infection . In the present study, we used the HAV-Bsd construct to develop a rapid and simple titration assay based on the selection of blasticidin-resistant cells, and used this assay to evaluate the HAV neutralization potency of commercially available human IG preparations.
Titration of HAV by the antibiotic resistance titration assay (ARTA)
Evaluation of total anti-HAV antibodies in human IG preparations
Evaluation of anti-HAV neutralizing antibodies in the human IG preparations by ARTA
Comparison of the ARTA and ELISA methods to evaluate anti-HAV neutralizing antibodies levels in IG preparations
The ARTA method to titrate HAV is based on the antibiotic resistance of HAV-infected cells conferred by the blasticidin deaminase gene inserted into the virus genome. In the presence of blasticidin, HAV-Bsd-infected cells continue to metabolize and acidify the cell culture medium whereas uninfected cells die and the cell culture media turns pink to purple. At 8 days postinfection, there is a 100% correlation between the presence of surviving cells in the wells and the color of the cell culture medium, which obviates the use techniques of immunochemistry or molecular biology to detect the presence of the virus in the wells. Indeed, the HAV-Bsd titers determined by ARTA at 8 days post-infection and by ELISA at 2 weeks postinfection were almost identical (compare Figures 4 and 5) indicating that the ARTA and ELISA methods are equivalent. Since the Huh7-A-I cells are highly sensitive to low concentrations of blasticidin, the viral titers can also be determined at 5 days postinfection before the change in the color of the cell culture media becomes apparent by examination of the plates under the microscope (data not shown).
HAV is commonly titrated in monkey kidney or human fibroblast cell lines using attenuated strains of HAV that are highly adapted to grow in these cell cultures and contain a significant number of adapting mutations. For the ARTA, we selected a wild type based HAV vector and the human liver-derived Huh7-A-I cells to mimic as much as possible the HAV infection conditions found in vivo. Therefore, the ARTA described in this work is a better system to evaluate anti-HAV antibodies and screen for antivirals than other assays based on attenuated strains and non-hepatic cell substrates.
Human IG preparations are recommended for HAV pre- and post-exposure prophylaxis [11, 12]. Although anti-HAV specific antibodies in the IG preparations for human use are evaluated by immunochemistry, an HAV neutralization test can provide a more direct evaluation of the potency of the IG preparations. We used the ARTA to evaluate the HAV neutralization potency of two commercially available IG preparations and a WHO anti-HAV standard. These three IG preparations contained similar levels of anti-HAV antibodies and had comparable neutralization potencies. Our data showed that the ARTA is a simple, rapid, and robust method to assess the HAV neutralization potency of human IG preparations. The ARTA described in this work reduces significantly the time, effort, and cost of HAV titrations and could be exploited for high throughput applications such as HAV epidemiological studies, screening of antivirals, and the routine assessment of the HAV neutralization potency of IG preparations for human use.
The ARTA is a rapid and simple method to determine HAV titers by examining the color of the cell culture medium. HAV titers obtained by the ARTA and ELISA endpoint dilution assays are comparable. However, the ARTA requires shorter incubation times and no additional staining for viral antigens. We have successfully used the ARTA to evaluate the HAV neutralization potency of human IG preparations. The ARTA is an ideal assay to determine HAV titers for research purposes, and could also be used for large scale epidemiological studies, high throughput screening of antivirals, and the evaluation of anti-HAV antibodies in IG preparations for human use.
Cells and viruses
Huh7-A-I cells, a clone of human hepatoma Huh7 cells that support the stable growth of wt HAV in cell culture , were grown in Dulbeco's modified Eagle's medium supplemented with 10% fetal bovine serum (complete medium) at 37°C in a 5% CO2 incubator. The HAV-Bsd construct was derived from pHAV8Y-Bsd, which codes for the infectious cDNA of the wild type HM-175 strain of human HAV containing a A216V substitution at amino acid 216 of the 2B protein and a blasticidin resistance gene at the 2A-2B junction . HAV-Bsd was grown in Huh7-A-I cells in the presence of 2 μg/ml blasticidin, and viral stocks were prepared by washing infected cell cultures and subjecting the cells to 3 freeze-and-thaw cycles.
Stocks of commercially available liquid IG preparations, Immune Globulin (Human) and Immune Globulin Subcutaneous (Human), from two different Manufactures (Lab 1 and Lab 2) and the 1st WHO International Standard for Hepatitis A Immunoglobulin  containing 100 IU anti-HAV antibodies per ml were prepared as 16% IgG solutions. A stock of an anti-HAV antibody-negative human plasma donation prepared as a 1% IgG solution was used as negative control.
Determination of anti-HAV antibodies in immune globulin preparations
IG preparations were diluted in complete medium and levels of anti-HAV antibodies were determined by the HAVAB EIA test (Abbott Laboratories) according to the manufacturer's instructions. Samples with OD values above the cutoff value were considered nonreactive, while those with OD values less than or equal to the cutoff value were considered reactive for anti-HAV antibodies. Samples were analyzed in duplicates, and the means and standard deviations of the OD from each dilution were calculated and plotted.
Neutralization reactions in 0.3 ml of complete medium containing 1% IG preparations and 105 TCID50 HAV-Bsd were incubated rotating overnight at 4°C followed by 1 h at 37°C. The neutralization reactions were diluted in complete medium and titrated on 96-well plates containing confluent monolayers of Huh7-A-I cells.
HAV titer determination by ARTA
Four- or ten-fold dilutions of the neutralization reaction in complete medium were titrated on 96-well plates containing confluent monolayers of Huh7-A-I cells. Four or six replica wells were inoculated per dilution. Plates were washed three times with serum free medium followed by the addition of 0.2 ml complete medium containing 2 μg/ml blasticidin per well. Plates were placed in a CO2 incubator at 35°C. After 8 days, plates were observed under the microscope, and wells containing cells were scored as positive. Alternatively, the plates were examined by the naked eye to determine the color of the cell culture media in each well. Orange and yellow wells were considered positive and pink and purple wells were considered negative. Viral titers were calculated using the Reed and Muench method .
HAV titer determination by ELISA
The same neutralization reactions evaluated by ARTA were titrated in parallel by an endpoint dilution ELISA in 96-well plates containing Huh7-A-I cells but in the absence of blasticidin. Eight replica wells were inoculated per dilution, and plates were placed in a CO2 incubator at 35°C. Two weeks post-infection, HAV-Bsd titers were determined by ELISA . Briefly, cells were fixed with 90% methanol, and stained with a 1:4,000 dilution of anti-HAV monoclonal antibody K2-4F2 (Commonwealth Labs), and a 1:5,000 dilution of peroxidase-labeled goat anti-mouse secondary antibody. TMB one-component peroxidase substrate (KPL Inc) was added to the wells, and the colorimetric reaction was stopped with 1% H2SO4. An increase in absorbance of at least two folds above the uninfected negative control wells was considered positive. Viral titers were determined by the method of Reed and Muench .
Viral titers and standard deviations were calculated using the ID50 program developed by John L. Spouge (National Center for Biotechnology Information, NIH).
We thank K.V.K Mohan K.V.K., and the NIH Fellows Editorial Board for critical review of the manuscript. The work was supported by FDA intramural funds to MYY and GGK.
- Hepatitis A vaccines. World Health Organization Weekly Epidemiological Record 2000, 75: 38-44.Google Scholar
- Shieh YC, Khudyakov YE, Xia G, Ganova-Raeva LM, Khambaty FM, Woods JW, Veazey JE, Motes ML, Glatzer MB, Bialek SR, Fiore AE: Molecular confirmation of oysters as the vector for hepatitis A in a 2005 multistate outbreak. J Food Prot 2007, 70: 145-150.PubMedGoogle Scholar
- Hutin YJ, Pool V, Cramer EH, Nainan OV, Weth J, Williams IT, Goldstein ST, Gensheimer KF, Bell BP, Shapiro CN, et al.: A multistate, foodborne outbreak of hepatitis A. National Hepatitis A Investigation Team. N Engl J Med 1999, 340: 595-602. 10.1056/NEJM199902253400802View ArticlePubMedGoogle Scholar
- Niu MT, Polish LB, Robertson BH, Khanna BK, Woodruff BA, Shapiro CN, Miller MA, Smith JD, Gedrose JK, Alter MJ, et al.: Multistate outbreak of hepatitis A associated with frozen strawberries. J Infect Dis 1992, 166: 518-524.View ArticlePubMedGoogle Scholar
- Wheeler C, Vogt TM, Armstrong GL, Vaughan G, Weltman A, Nainan OV, Dato V, Xia G, Waller K, Amon J, et al.: An outbreak of hepatitis A associated with green onions. N Engl J Med 2005, 353: 890-897. 10.1056/NEJMoa050855View ArticlePubMedGoogle Scholar
- Mutsch M, Spicher VM, Gut C, Steffen R: Hepatitis A virus infections in travelers, 1988–2004. Clin Infect Dis 2006, 42: 490-497. 10.1086/499816View ArticlePubMedGoogle Scholar
- Hepatitis Surveillance Report No. 61. 2005 Atlanta, GA: U.S. Department of Health and Human Services. Centers for Disease Control and Prevention; 2006.Google Scholar
- Prevention of hepatitis A through active or passive immunization: recommendations of the Advisory Committee on Immunization Practices (ACIP) Morbid Mortal Wkly Rep (MMWR) 2007, 1080-1084.Google Scholar
- Mosley JW, Reisler DM, Brachott D, Roth D, Weiser J: Comparison of two lots of immune serum globulin for prophylaxis of infectious hepatitis. Am J Epidemiol 1968, 87: 539-550.PubMedGoogle Scholar
- Werzberger A, Mensch B, Kuter B, Brown L, Lewis J, Sitrin R, Miller W, Shouval D, Wiens B, Calandra G, et al.: A controlled trial of a formalin-inactivated hepatitis A vaccine in healthy children. N Engl J Med 1992, 327: 453-457.View ArticlePubMedGoogle Scholar
- Victor JC, Monto AS, Surdina TY, Suleimenova SZ, Vaughan G, Nainan OV, Favorov MO, Margolis HS, Bell BP: Hepatitis A vaccine versus immune globulin for postexposure prophylaxis. N Engl J Med 2007, 357: 1685-1694. 10.1056/NEJMoa070546View ArticlePubMedGoogle Scholar
- Winokur PL, Stapleton JT: Immunoglobulin prophylaxis for hepatitis A. Clin Infect Dis 1992, 14: 580-586.View ArticlePubMedGoogle Scholar
- Stapleton JT: Hepatitis A: biology, pathogenesis, epidemiology, clinical description, and diagnosis. In Viral Hepatitis: Diagnosis, Therapy and Prevention. Edited by: Spector S. Humana Press, Totowa, NJ; 1999:7-33.Google Scholar
- Cromeans T, Sobsey MD, Fields HA: Development of a plaque assay for a cytopathic, rapidly replicating isolate of hepatitis A virus. J Med Virol 1987, 22: 45-56. 10.1002/jmv.1890220107View ArticlePubMedGoogle Scholar
- Anderson DA: Cytopathology, plaque assay, and heat inactivation of hepatitis A virus strain HM175. J Med Virol 1987, 22: 35-44. 10.1002/jmv.1890220106View ArticlePubMedGoogle Scholar
- Lemon SM, Binn LN, Marchwicki RH: Radioimmunofocus assay for quantitation of hepatitis A virus in cell cultures. J Clin Microbiol 1983, 17: 834-839.PubMed CentralPubMedGoogle Scholar
- Counihan NA, Daniel LM, Chojnacki J, Anderson DA: Infrared fluorescent immunofocus assay (IR-FIFA) for the quantitation of non-cytopathic and minimally cytopathic viruses. J Virol Methods 2005, 133: 62-69. 10.1016/j.jviromet.2005.10.023View ArticlePubMedGoogle Scholar
- Lemon SM, Binn LN: Serum neutralizing antibody response to hepatitis A virus. J Infect Dis 1983, 148: 1033-1039.View ArticlePubMedGoogle Scholar
- Cohen JI, Rosenblum B, Ticehurst JR, Daemer RJ, Feinstone SM, Purcell RH: Complete nucleotide sequence of an attenuated hepatitis A virus: comparison with wild-type virus. Proc Natl Acad Sci USA 1987, 84: 2497-2501. 10.1073/pnas.84.8.2497PubMed CentralView ArticlePubMedGoogle Scholar
- Funkhouser AW, Purcell RH, D'Hondt E, Emerson SU: Attenuated hepatitis A virus: genetic determinants of adaptation to growth in MRC-5 cells. J Virol 1994, 68: 148-157.PubMed CentralPubMedGoogle Scholar
- Silberstein E, Dveksler G, Kaplan GG: Neutralization of hepatitis A virus (HAV) by an immunoadhesin containing the cysteine-rich region of HAV cellular receptor-1. J Virol 2001, 75: 717-725. 10.1128/JVI.75.2.717-725.2001PubMed CentralView ArticlePubMedGoogle Scholar
- Silberstein E, Xing L, Beek W, Lu J, Cheng H, Kaplan GG: Alteration of hepatitis A virus (HAV) particles by a soluble form of HAV cellular receptor 1 containing the immunoglobin-and mucin-like regions. J Virol 2003, 77: 8765-8774.PubMed CentralView ArticlePubMedGoogle Scholar
- Feigelstock DA, Thompson P, Kaplan GG: Growth of hepatitis A virus in a mouse liver cell line. J Virol 2005, 79: 2950-2955. 10.1128/JVI.79.5.2950-2955.2005PubMed CentralView ArticlePubMedGoogle Scholar
- Konduru K, Kaplan GG: Stable growth of wild-type hepatitis A virus in cell culture. J Virol 2006, 80: 1352-1360. 10.1128/JVI.80.3.1352-1360.2006PubMed CentralView ArticlePubMedGoogle Scholar
- Graff J, Emerson SU: Importance of amino acid 216 in nonstructural protein 2B for replication of hepatitis A virus in cell culture and in vivo. J Med Virol 2003, 71: 7-17. 10.1002/jmv.10457View ArticlePubMedGoogle Scholar
- Reed LJ, Muench H: A simple method of estimating fifty per cent end points. Am J Hyg 1938, 27: 493-497.Google Scholar
- Gerety RJ, Smallwood LA, Finlayson JS, Tabor E: Standardization of the antibody to hepatitis A virus (anti-HAV) content of immunoglobulin. Dev Biol Stand 1983, 54: 411-416.PubMedGoogle Scholar
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