Kinetics of hepatitis A virus replication in vivo and in vitro using negative-strand quantitative PCR

• Published in: European journal of clinical microbiology & infectious diseases 2009-10, Vol.28 (10), p.1167-1176
• Main Authors: de Paula, V. S, Perse, A. S, Amado, L. A, de Morais, L. M, de Lima, S. M. B, Tourinho, R. S, Gaspar, A. M. C, Pinto, M. A
• Format: Article
• Language: English
• Subjects: 5' Untranslated Regions; Animal models; Animals; Biological and medical sciences; Biomedical and Life Sciences; Biomedicine; Capsid Proteins; Cell culture; Cells, Cultured; Disease Models, Animal; DNA, Complementary - biosynthesis; Gene Amplification; Genomes; Hepatitis; Hepatitis A; Hepatitis A - virology; Hepatitis A virus; Hepatitis A virus - physiology; Hepatitis A Virus, Human - physiology; Human viral diseases; Humans; Immunization; Infections; Infectious diseases; Internal Medicine; Kinetics; Liver; Liver - virology; Medical Microbiology; Medical sciences; Pathogenesis; Peptide Fragments; Polymerase chain reaction; Polymerase Chain Reaction - methods; Polymerase Chain Reaction - standards; Primates; Proteins; Reverse Transcriptase Polymerase Chain Reaction - methods; RNA, Viral - genetics; Sensitivity and Specificity; Thermus thermophilus; Vaccines; Viral diseases; Viral hepatitis; Virus Replication - physiology; Viruses
• ISSN: 0934-9723; 1435-4373
• DOI: 10.1007/s10096-009-0759-8

The replication of hepatitis A virus (HAV) is via a complementary negative-strand RNA. Each negative strand may serve as a template for the synthesis of many positive strands. The aim of this study was to detect the intermediate replicative (negative strand) of HAV in order to monitor its replication in vitro and in vivo. Real-time polymerase chain reaction (PCR) was standardized to detect the intermediate replicative of HAV in cell culture and liver from non-human primates infected experimentally. HAV primers from the 5′ non-translated region and VP3 were used in the cDNA synthesis of negative-strand RNA. The negative strand was detected in the infected cell lines and liver by highly strand-specific rTth recombinant Thermus thermophilus DNA polymerase reverse transcription followed by quantitative PCR. The results indicate that the negative-strand HAV RNA can be detected in vivo and in vitro. This model is an approach for assessing the dynamic patterns of replication and should represent a valuable tool for the monitoring of HAV replications in cell cultures and for the evaluation of experimental infections in animal models.