Detection of replication-defective hepatitis A virus based on the correlation between real-time polymerase chain reaction and ELISA in situ results

Abstract

ELISA in situ can be used to titrate hepatitis A virus (HAV) particles and real-time polymerase chain reaction (RT-PCR) has been shown to be a fast method to quantify the HAV genome. Precise quantification of viral concentration is necessary to distinguish between infectious and non-infectious particles. The purpose of this study was to compare cell culture and RT-PCR quantification results and determine whether HAV genome quantification can be correlated with infectivity. For this purpose, three stocks of undiluted, five-fold diluted and 10-fold diluted HAV were prepared to inoculate cells in a 96-well plate. Monolayers were then incubated for seven, 10 and 14 days and the correlation between the ELISA in situ and RT-PCR results was evaluated. At 10 days post-incubation, the highest viral load was observed in all stocks of HAV via RT-PCR (10(5) copies/mL) (p = 0.0002), while ELISA revealed the highest quantity of particles after 14 days (optical density = 0.24, p < 0.001). At seven days post-infection, there was a significant statistical correlation between the results of the two methods, indicating equivalents titres of particles and HAV genome during this period of infection. The results reported here indicate that the duration of growth of HAV in cell culture must be taken into account to correlate genome quantification with infectivity.

Fig. 1. comparison between the numbers of viral particles analyzed by ELISA in situ and the number of amplified copies of the genome of hepa titis A virus analyzed by real-time (RT) polymerase chain reaction according to the days of incubation. dpi: days post-infection; EIA: ensyme immunoassay; OD: optical density; *: analysis of variance of RT values; **: ELISA values.

Fig. 2. correlation of the amounts of hepatitis A virus particles and RNA, estimated by tissue culture infective dose (TCID50) and cycle threshold (Ct) values of real-time polymerase chain reaction. dpi: days post-infection.