The virucidal action of solvent tributyl phosphate and detergent sodium cholate used in the production of immunoglobulin for inactivation of viruses with lipid envelope was studied on the model of duck hepatitis B virus. PCR analysis revealed no significant decrease in duck hepatitis B virus DNA concentrations after treatment with solvent/detergent. At the same time, in vivo experiments showed that treatment of duck hepatitis B virus with tributyl phosphate (concentration >0.15%) and sodium cholate (concentration >0.1%) at 37°C for 6 h or longer completely inactivated this model virus added to immunoglobulin solution in concentration 5 log ID50. Duck hepatitis B virus appears to be one of the most acceptable model viruses for validation of virus inactivating technologies in manufacturing human plasma preparations.

Viruses commonly utilize the cellular trafficking machinery of polarized cells to effect viral export. Hepatocytes are polarized in vivo, but most in vitro hepatocyte models are either nonpolarized or have morphology unsuitable for the study of viral export. Here, we investigate the mechanisms of trafficking and export for the hepadnaviruses hepatitis B virus (HBV) and duck hepatitis B virus (DHBV) in polarized hepatocyte-derived cell lines and primary duck hepatocytes. DHBV export, but not replication, was dependent on the development of hepatocyte polarity, with export significantly abrogated over time as primary hepatocytes lost polarity. Using Transwell cultures of polarized N6 cells and adenovirus-based transduction, we observed that export of both HBV and DHBV was vectorially regulated and predominantly basolateral. Monitoring of polarized N6 cells and nonpolarized C11 cells during persistent, long-term DHBV infection demonstrated that newly synthesized sphingolipid and virus displayed significant colocalization and fluorescence resonance energy transfer, implying cotransportation from the Golgi complex to the plasma membrane. Notably, 15% of virus was released apically from polarized cells, corresponding to secretion into the bile duct in vivo, also in association with sphingolipids. We conclude that DHBV and, probably, HBV are reliant upon hepatocyte polarity to be efficiently exported and this export is in association with sphingolipid structures, possibly lipid rafts. This study provides novel insights regarding the mechanisms of hepadnavirus trafficking in hepatocytes, with potential relevance to pathogenesis and immune tolerance.

Hepatitis B-virucidal testing of biocides in quantitative suspension tests using duck hepatitis B virus (DHBV) requires primary duck embryonic hepatocytes for viral propagation. To improve the test system and availability of these cells, commercial culture plates with different growth surfaces were tested for cell cultivation and different approaches for cryopreservation of hepatocyte suspension were examined. After 12 days of culture, the largest amounts of hepatocytes were grown in CellBIND and TTP plates and CellBIND surface showed the lowest tendency of monolayer detachment nearly comparable with collagen 1-coated CELLCOAT plates. For cryopreservation of hepatocyte suspension, the use of growth medium supplemented with fetal calf serum (FCS) and dimethyl sulfoxide (ME(2)SO), FCS supplemented with ME(2)SO or cryosafe-1 as cryoprotective agents provided the highest rates of surviving cells after thawing. The freezing-thawing process did not significantly reduce the susceptibility of hepatocytes to infection with DHBV. In conclusion, plates without collagen 1 such as CellBIND are recommended for cultivation of primary duck embryonic hepatocytes in infectivity experiments of DHBV for virucidal testing of biocides. The use of cryopreserved hepatocytes is possible when freshly isolated cells from the liver of duck embryos are not available.

Mother-infant transmission of hepatitis B virus (HBV) accounts for up to 30% of worldwide chronic infections. The mechanism and high-risk period of HBV transmission from mother to infant are unknown. Although largely prevented by neonatal vaccination, significant transmission continues to occur in high-risk populations. It is unclear whether HBV can traverse an intact epithelial barrier to infect a new host. Transplacental transmission of a number of viruses relies on transcytotic pathways across placental cells. We wished to determine whether infectious HBV can traverse a polarized trophoblast monolayer. We used a human placenta-derived cell line, BeWo, cultured on membranes as polarized monolayers, to model the maternal-fetal barrier. We assessed the effects of placental maturity and maternal immunoglobulin on viral transport. Intracellular viral trafficking pathways were investigated by confocal microscopy. Free HBV (and infectious duck hepatitis B virus) transcytosed across trophoblastic cells at a rate of 5% in 30 min. Viral transport occurred in microtubule-dependent endosomal vesicles. Additionally, confocal microscopy showed that the internalized virus traverses a monensin-sensitive endosomal compartment. Differentiation of the cytotrophoblasts to syncytiotrophoblasts resulted in a 25% reduction in viral transcytosis, suggesting that placental maturity may protect the fetus. Virus translocation was also reduced in the presence of HBV immunoglobulin. We show for the first time that transcytosis of infectious hepadnavirus can occur across a trophoblastic barrier early in gestation, with the risk of transmission being reduced by placental maturity and specific maternal antibody. This study suggests a mechanism by which mother-infant transmission may occur.

The use of a surrogate virus, namely duck hepatitis B virus (DHBV), has been recommended for testing the virucidal activity of chemical biocides against hepatitis B virus. To date, however, this model has not been recognized as a standard test in European countries, as its laboratory use is associated with considerable difficulties. As previous studies have demonstrated, several alternative procedures may improve the validation of DHBV infection in a cell culture system. Using indirect immunofluorescent antigen staining and the light cycler real-time polymerase chain reaction (PCR) technique, the virucidal activity of peracetic acid (PAA), povidone-iodine (PVP-I) and formaldehyde was tested against DHBV obtained from congenitally infected ducks or prepared from the transfected hepatoma D2 cell line. The results demonstrated that inactivation of DHBV from the D2 cell line was achieved with lower concentrations of the biocides and within shorter exposure time intervals. These lower concentration-exposure time values for DHBV from D2 cells in comparison with DHBV from infected ducks indicated a higher sensitivity of the virus derived from D2 cells. In addition, concentrations of PAA and PVP-I that significantly inactivated DHBV in suspension tests were not able to destroy the viral genome. In conclusion, DHBV from congenitally infected ducks should be used for virucidal testing of chemical biocides against DHBV; DHBV prepared from D2 cells is unsuitable due to its higher sensitivity to biocides. Indirect immunofluorescent staining allows reliable detection of DHBV infectivity, whereas the hepadnavirucidal effect can be evaluated by quantitative PCR.

Human adenoviruses are model viruses for testing the virucidal efficacy of disinfectants. However, a recent study has shown that the chemical sensitivity of adenovirus serotypes varies significantly, possibly due to the composition of the viral capsid and/or the resistance of nucleic acids. We have investigated the effect of molecular changes in the viral genome of the human adenovirus subgenera C and D. A common oligonucleotide fragment within the hexon gene was amplified by polymerase chain reaction (PCR) after exposure to liposomal povidone-iodine (PVP-I), peracetic acid (PAA), and formaldehyde. The findings were compared with infectivity in cell cultures. PVP-I (0.125%) destroyed the infectivity of most serotypes within 60 min. However, PCR revealed no destruction of the adenoviral genome in most serotypes, even after exposure to higher PVP-I concentrations. PAA (0.5%) failed to inactivate the hexon gene of adenovirus types 22 and 44. Furthermore, the hexon gene of adenovirus type 22 was not altered by 0.7% formaldehyde. In conclusion, the genomes of human adenoviruses show considerably more chemical resistance than the complete viral particle. The molecular resistance of individual serotypes also varies. However, there was no clear correlation between the differences in the effect of disinfectants on infectivity of the complete viral particle and destruction of the viral genome.

Hepatitis B virus (HBV) is an important virus used in disinfection procedures for blood spillage. However, validation of HBV inactivation is difficult, since there are no feasible infectivity assays. In some countries, the duck HBV (DHBV) is recognized as a suitable model for testing antiviral activity of chemical biocides against HBV. Currently, DHBV-infected ducks are required for preparation of the test virus as well as eggs from DHBV-free flocks for testing DHBV infectivity. To improve the practicality of the system, we suggested to use commercially available embryonated duck eggs for preparation of DHBV-susceptible hepatocyte cultures and to exclude infected hepatocytes by pre-screening with qualitative detection of DHBV DNA using polymerase chain reaction (PCR). A standardized DHBV test virus was prepared from the DHBV DNA-transfected hepatoma cell line D2, which contained 10(11)DHBV DNA molecules per mL detected by light cycler real-time PCR. Infection of cell cultures was most efficient 4 days after plating. The best identification of infected cultures was possible 6 days after infection with immunofluorescence using an antiserum against DHBV surface antigen.

The virucidal efficacy of monopercitric acid (MPCA) was evaluated against the enveloped vaccinia virus as well as the nonenveloped adenovirus type 2 and poliovirus type 1. The results were compared with that obtained with peracetic acid (PAA).

In the virucidal suspension test without and with protein burden, all viruses were inactivated by 0.5% MPCA within 0.5 min or by 0.1% MPCA within 5 min as measured by a >10(4)-fold reduction in virus titres. For MPCA, there was a better virucidal efficacy than for PAA which inactivated all viruses included in the test within 15-30 min at a concentration of 0.2%.

The high virucidal activity, short exposure times, and nontoxic by-products seem to make MPCA suitable as disinfectant for medical use and should warrant further investigation.

Since important agents of viral nosocomial infections like hepatitis B and C viruses and norovirus do not replicate sufficiently in cell culture systems, disinfectants with suspected efficacy against these viruses must be evaluated by different methods. Besides molecular approaches and indirect tests, the use of surrogate viruses with similar biophysical properties and genomic structure allows the assessment of virucidal efficacy of chemical disinfectants in quantitative suspension tests. Furthermore, insights into the survival of these viruses in the environment are possible. In recent years, duck hepatitis B virus and bovine viral diarrhoea virus have been tested as surrogates for hepatitis B and C viruses. Feline calicivirus serves as a surrogate for the group of norovirus. By including these viruses in inactivation experiments, valuable data from suspension tests can be derived on the virucidal efficacy of chemical disinfectants. Even in vivo tests using fingerpads of adult volunteers can be performed with these animal viruses without risk of infection. In contrast to in vitro examinations, the results of these tests allow use recommendations of chemical disinfectants for outbreak situations and daily routine disinfection.