Monitoring of early events of experimental woodchuck hepatitis infection: Studies of peripheral blood mononuclear cells by cytofluorometry and PCR

Abstract

Abstract:The peripheral blood mononuclear cells (PBMC) of woodchucks experimentally infected by woodchuck hepatitis virus (WHV) were examined simultaneously for the presence of membrane associated WHV antigens by cytofluorometry, and for WHV DNA and RNA sequences by the polymerase chain reaction (PCR). Four woodchucks were inoculated: two with a well-defined infectious inoculum and two with an inoculum obtained from an animal at the late incubation phase, which was positive for WHV DNA by PCR but still devoid of WHV markers. Infection was demonstrated in all four inoculated woodchucks by the appearance at different times of WHV DNA and WHV antigens in both leucocytes and serum. WHV DNA was first detected by PCR either in the serum (two cases) or in leucocytes (two cases). The mean percentage of cells positive for membrane associated WHsAg or WHcAg detected by cytofluorometry were 37% + 25 and 17% _+ 15 respectively. After 8 weeks, all inoculated animals were WHsAg positive in serum. These data suggest that PBMC are involved in the early events of hepadnavirus infection. They also show that sera which are positive by PCR for WHV DNA may transmit viral infection even while still seronegative for WHV markers and for WHV DNA by dot blot.

Hepatitis B virus persistence after recovery from acute viral hepatitis.

“…Identification of HBV specific RNA in the PBMC of our patients is consistent with previous reports that hepadnavirus DNA, RNA and protein are detectable in human and woodchuck PBMC even in the absence of serologic evidence of ongoing virus infection (11, 13,16,[28][29][30][31][32][33][34][35][36]. Since infectious WHV is produced by mitogen-stimulated PBMC from chronically infected woodchucks (37,38) it is possible that these human cells may also be able to support HBV replication and, thus, serve as a reservoir for cell-to-cell transmission of the virus despite the presence of neutralizing anti-HBs.…”

“…The cells were homogenized and solubilized according to the acid guanidinum thiocyanate-phenol-chloroform method ( 14). The final RNA was treated with RNAase-free DNAase I (2 U/reaction) (Promega Corp., Madison, WI) for 30 min at 37°C and then with RNAase H ( 1.5 U/reaction) (Bethesda Research Laboratories, Gaithersburg, MD) for 30 min at 37°C to eliminate possible viral RNA/DNA hybrids resistant to DNAase digestion (15,16). The enzymes were subsequently inactivated by incubation at 95°C for 5 min and the RNA stored at -70°C.…”