Effect of fetal calf serum on propagation of duck hepatitis A virus genotype 3 in duck embryo fibroblast cells

Abstract

Background Duck viral hepatitis (DVH) is a highly contagious viral disease affecting ducks. It can be caused by five agents, including duck hepatitis A virus genotypes 1 (DHAV-1), 2 (DHAV-2), and 3 (DHAV-3), as well as duck hepatitis virus 2 and duck hepatitis virus 3. Since 2007, DHAV-3 has been known to be the most prevalent in East and South Asia. So far, the information regarding the propagation of DHAV-3 in cultured cells is limited. In this study, we describe the comparative studies on the growth properties of DHAV-3 in primary duck embryo fibroblast (DEF) cells using two different strains: a virulent strain C-GY and an attenuated strain YDF120. The effect of fetal calf serum (FCS) and chick serum (CS) on DHAV-3 replication and the mechanism of the inhibitory effect conferred by FCS were also investigated. Results Following serial passages, both C-GY and YDF120 failed to produce cytopathic effect and plaques. The combined quantitative real-time PCR and indirect immunofluorescence staining methods showed that the two viruses could be propagated productively in DEF cells. Investigation of the viral growth kinetics revealed that the two viruses replicated in DEF cells with similar efficiencies, while the viral load of the virulent C-GY strain peaked more rapidly when compared with the attenuated YDF120 strain. Neutralization assay and time-of-drug-addition study indicated that FCS displayed inhibitory effect on DHAV-3 replication. Analysis on the mechanism of action of FCS against DHAV-3 demonstrated that the inhibitory effect was reflected at three steps of the DHAV-3 life cycle including adsorption, replication, and release. Conclusions Both virulent and attenuated DAHV-3 strains can establish noncytocidal, productive infections in DEF cells. The virulent strain replicates more rapidly than the attenuated strain in early infection period. FCS has an inhibitory effect on DHAV-3 replication, which may be attributed to action of a non-specific inhibitory factor present in FCS directly on the virus. These findings may provide new insights into the development of potential antiviral agents.

Fetal Calf Serum Exerts an Inhibitory Effect on Replication of Duck Hepatitis A Virus Genotype 1 in Duck Embryo Fibroblast Cells

“…Earlier work in our laboratory showed that the use of CS in maintenance medium is of help to the replication of DHAV-3 in DEF cells. Unlike DHAV-1, DHAV-3 cannot produce cytopathic changes in DEF cells [37]. Thus, the present observations further demonstrated that DHAV-1 and DHAV-3 infections lead to disparate effects on DEF cells.…”

“…At present, it is uncertain whether the effect of FCS on virus loads in supernatant is associated with FCS-mediated inhibitory impact on virus assembly and release. A recent work in our laboratory showed that FCS exerts an inhibitory effect on DHAV-3 through the same mechanisms [37], suggesting that an FCS-mediated inhibitory effect on DHAV replication in cell cultures shows no genotype specificity. Collectively, these data provide a promising strategy for the development of a potent inhibitor against DHAV by further biochemical analysis of FCS.…”

“…Confluent monolayers of DEF cells grown in 24-well plates were inoculated with virus, as described in Section 2.4, and 0.5 mL of 2% CS DMEM or 2% FCS DMEM was added. At 24 hpi, the cells were stained with chicken antiserum against DHAV-1 and fluorescein isothiocyanate (FITC)-conjugated goat anti-chicken IgY (KPL, Milford, MA, USA), as described previously [37]. The nuclei were stained with DAPI (Sigma-Aldrich, St Louis, MO, USA) for 5 min at room temperature.…”

“…The plaque phenotype of DHAV-1 was determined, as described previously [37], employing an agarose overlay containing DMEM, 2% FCS or 2% CS, 1% low-melt agar (Macgene, Beijing, China), 100 U/mL of penicillin, and 0.1 mg/mL streptomycin (designated 2% FCS agarose overlay and 2% CS agarose overlay, respectively). The viruses were individually diluted with DMEM in 10-fold steps to 10 −8 .…”

“…The effect of FCS on DHAV-1 was investigated following the protocol used in neutralization test [37], employing CS as a control. Briefly, three dilutions of serum were individually mixed with an equal volume of virus (2 × 10 5 RNA copies), to produce three serum concentrations (0.5%, 1%, and 2%).…”