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.
BACKGROUND
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.


RESULTS
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 microg/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.


CONCLUSIONS
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.
Konduru
Krishnamurthy
K
Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, 29 Lincoln Drive, Bethesda, Maryland 20892, USA. Krishnamurthy.konduru@fda.hhs.gov
Virata-Theimer
Maria Luisa
ML
Yu
Mei-Ying W
MY
Kaplan
Gerardo G
GG
D000914:Antibodies, Viral / Q000032:analysis* / Q000276:immunology; D045744:Cell Line, Tumor; D004352:Drug Resistance, Microbial; D006506:Hepatitis A / Q000175:diagnosis / Q000276:immunology / Q000821:virology*; D030041:Hepatitis A virus / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D007136:Immunoglobulins / Q000032:analysis* / Q000276:immunology; D009500:Neutralization Tests / Q000379:methods; D014773:Virology / Q000379:methods*; D014776:Virus Cultivation / Q000379:methods
D000914:Antibodies, Viral; D007136:Immunoglobulins