Molecular approaches to validate disinfectants against human hepatitis B virus

Abstract

Disinfection is an important measure to prevent hepatitis B virus (HBV) transmission by instruments. However, virucidal testing of disinfectants against HBV is difficult, because no simple quantitative infectivity assay exists. Since molecular changes of viral epitopes and the genome may indicate virus inactivation, we measured the alteration of these constituents with 0.065% peracetic acid (PAA) for exposure times up to 1 h. Plasma of a chronic HBV carrier with 10(9) HBV genomes/ml served as viral source in the form of a 10% dilution or of a purified HB-antigen preparation. Alterations of HBV epitopes were analyzed with four monoclonal antibodies in an enzyme-linked immunosorbent assay. Changes of the HBV genomes were determined by the inability to amplify the target sequence with a quantitative real-time polymerase chain reaction, either of a short fragment (189 bp) or of the full-length (3,200 bp). The determination of the epitope and genome alteration was quantified as log10 reduction factor (RF) with the parallel line bioassay. Under a high protein load of 10% human plasma, PAA induced a HBV genome alteration of RF = 1.5 after an exposure time of 60 min. Similar RFs were seen with the four HB epitopes. Without protein load, the alteration of these epitopes amounted to a RF of more than 3.5 within 30 min. Such inhibition of PAA activity by protein load was also seen in the virucidal tests with parvovirus. Although the RF were higher in the virucidal tests, the time-dependent dose-response curves for the epitope and genome alteration and for the infectivity inactivation followed the same inactivation kinetics. The molecular alteration and disintegration epitope and genome test may therefore be suitable to measure antiviral activity of disinfectants against HBV.

Lipid-Mediated Introduction of Hepatitis B Virus Capsids into Nonsusceptible Cells Allows Highly Efficient Replication and Facilitates the Study of Early Infection Events

“…The pellet was resuspended in 50 l H 2 O. Ten microliters H 2 O containing the DNA of 2 ϫ 10 4 cells was subjected to PCR as described by Jursch et al (16).…”

“…From the 50-l eluate, 10 l was subjected to PCR as described by Jursch et al (16). As a standard, a geometric dilution series of extracted DNA from human HBV-containing plasma comprising 2.5 ϫ 10 5 to 2.5 ϫ 10 1 HBV genomes was used.…”

Basal Core Promoter and Precore Mutations in the Hepatitis B Virus Genome Enhance Replication Efficacy of Lamivudine-Resistant Mutants

“…Newly synthesized HBV virions released into supernatant were PEG precipitated 4 days after transfection and measured using a quantitative real-time PCR approach (28). Linearized HBV 1.0 wt plasmid was serially diluted as a standard, and a reliable standard curve was obtained by which reference serum from patients with acute HBV infection could be accurately quantified (data not shown).…”

Simple Technique for Internal Control of Real-Time Amplification Assays

“…The amplified ICOs are detected by the same probe that detects the amplified target. We successfully applied this technique to a newly designed LightCycler assay for rapid detection of MTBC and to three virus-specific PCR assays published by others (8,11 ). We have routinely used ICOs for several months without any major problems.…”

“…PCR amplification for the HBV assay was performed as described previously (8 ). For fluorescence acquisition during amplification, the temperature of the reaction was increased to 60°C.…”