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 (105 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.

Monitoring of Foodborne Hepatitis A virus Outbreaks in the Fresh Foods

“…This may be explained at least in part by non-viability of viral particles in the specimens that can be detect by RT-PCR was used for HAV detection. The results showed of the concentrated drainage water, strawberry and lettuce samples were positive for HAV-RNA by RT-PCR while were positive for HAV antigen by ELISA test with sensitive and specificity, after HAV propagated in cell line culture our result was in accordance with result obtained by Costa et al (2013).…”

“…HAV antigen has been detected in stool, cell culture and environmental samples by using radioimmunoassays and enzyme immunoassays (Hollinger & Emerson, 2001); ELISA in situ was performed to quantify the infectivity of HAV in cell culture (Costa et al, 2013). The main routine detection of HAV in food include the presence of inhibitory substances in the samples and the low concentration of virus recovered (Costa-Mattioli et al, 2002), Immunomagnetic separation (IMS) is one of the sample treatment methods that can address these limitations during using RT-PCR for detection of HAV in food (Costa-Mattioli et al, 2002 andAbd El Galil et al, 2004).…”

Study The prevalence of hepatitis A virus among patients of Sohag Governorate, Egypt

“…HAV antigen has been detected in stool, cell culture and environmental samples by using radio immuno assays and enzyme immunoassays (Hollinger & Emerson, 2001); ELISA in situ was performed to quantify the infectivity of HAV in cell culture (Costa et al, 2013). The main routine detection of HAV in food include the presence of inhibitory substances in the samples and the low concentration of virus recovered (Costa-Mattioli et al, 2002).…”

Inter- and Intra-Host Nucleotide Variations in Hepatitis A Virus in Culture and Clinical Samples Detected by Next-Generation Sequencing

“…HAV is a non-enveloped, RNA virus belonging to the family Picornaviridae, whose single-stranded genome of approximately 7.5 kilobases (kb) in length, contains a single open reading frame encoding a single polyprotein flanked by 5′ and 3′ untranslated regions, as well as a 3′ poly(A) tail [ 14 , 15 ]. Since its discovery in 1973 by Steven Feinstone [ 16 ], HAV has evolved through nucleotide mutations and recombination and is classified into three genogroups among which the nucleotide variation is more than 15% [ 17 ].…”