Duck hepatitis B virus replicates in the yolk sac of developing embryos

Duck hepatitis B virus (DHBV) is the only member of the hepadnavirus family in which nearly 100% vertical transmission from carrier mother to embryo has been reported. Large quantities of maternally transmitted virus particles are present in the yolk prior to incubation of the eggs, and replicative forms of DHBV DNA are detectable in the liver at 6 days of incubation. Since the yolk sac is similar to the liver in its production of serum proteins, we examined the yolk sacs of developing embryos for signs of viral replication. We detected the supercoiled form of DHBV DNA, DHBV RNA transcripts similar to those in the virus-replicating liver, and DNA polymerase activity and viral DNA in corelike particles in extracts of yolk sac tissue of naturally infected eggs. DHBV core antigen was strongly stained in only the endodermal layer of the yolk sac by immunofluorescence. DHBV RNA was detectable in the yolk sac from 4 days of incubation until hatching, and a larger quantity of DHBV RNA was present in the yolk sac than in the liver during all the stages of embryogenesis. Our data indicate that DHBV replicates actively in the yolk sac from an earlier stage than that previously reported in studies of embryonic liver and that replication is limited to the endodermal cell layer, which is ontogenetically and functionally related to the liver. The yolk sac may support the vertical transmission of DHBV.