The polymerase chain reaction (PCR) was used to investigate the presence of hepatitis B virus (HBV)-related DNA sequences in blood from three blood donors and two transfusion recipients who developed posttransfusion non-A, non-B hepatitis (NANBH). In the first case, the sole donor was positive for antibody to hepatitis B surface (HBs) and core (HBc) antigens and had elevated alanine aminotransferase (ALT) levels, while the recipient had no HBV serologic markers. Both the donor and the recipient had serologic markers of hepatitis C virus (HCV) and were found positive for HBV DNA and HCV RNA sequences by PCR. The second case involved two donors and one recipient. Serologic tests for conventional HBV markers were negative in all three individuals, but one of the donors had elevated ALT. HBV DNA sequences were detected by PCR in the serum of the recipient and of the donor with high ALT, but not in the serum of the donor with normal ALT. Anti-HCV was detected in the serum of the recipient and of the suspect donor but not in that of the donor with normal ALT. The sequences amplified in the S region and determined after cloning of PCR products for both donor-recipient pairs were indistinguishable from each other and identical to the sequence of the major HBV subtype of adw in the first case and ayw in the second case. Furthermore, for the second case, an identical single-point mutation was found in both the donor and the recipient. These data confirm the transmission of conserved HBV sequences together with HCV in posttransfusion NANBH.(ABSTRACT TRUNCATED AT 250 WORDS)

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Summary In order to define more accurately the initial events that take place during rabbit haemorrhagic disease virus (RHDV) infection, different organs of experimentally infected rabbits were analysed for the presence of the virus and correlated with histopathological observations. A total of 24 rabbits were intranasally inoculated with a viral suspension, and tissue samples were taken from the liver, spleen, kidney, lung, thymus, lymph node and tonsil at different intervals post‐inoculation (2, 4, 6, 12, 18, 24, 30, 36, 48, 50, 51, 70 and 72 h). Histopathological observations revealed the presence of the first significant lesions at 30 h post‐inoculation (p.i.) in the liver. Using an ELISA and a haemagglutination test (HAT), the virus was detected in the liver at 36 h p.i. The reverse transcriptase‐polymerase chain reaction (RT‐PCR) showed that the RHDV RNA was present as early as 18 h p.i. in the liver and spleen, whereas thymus, kidney, tonsil and lymph node were found to be positive after more than 36 h p.i. The lungs presented a variable positivity between 0 and 36 h p.i., but remained positive after this time.

The presence of hepatitis B surface protein (HBs) and hepatitis B core protein (HBc) was investigated, by flow cytometry, on the surface of peripheral mononuclear cells (PBMC) from cells of the following phenotype: CD3 (T lymphocytes), CD4 (T helper/ inducer), CD8 (T cytotoxic/suppressor), CD19 (B lymphocytes) and CD56 [natural killer (NK) cells] among eight patients suffering from chronic hepatitis B and five healthy HBV-negative subjects. This study demonstrated the presence of HBsAg and HBcAg on the lymphocyte surface for most of the patients. The mean percentage of labelled cells was 17% for HBsAg and 15% for HBcAg. Among the different lymphocyte subsets only B lymphocytes and the NK cells expressed HBsAg for 57% and 26% of cells, respectively. Similarly HBcAg was also detected among CD19 and CD56 cells only. Polymerase chain reaction (PCR) was used to search for the presence of hepatitis B virus (HBV) DNA and RNA in PBMC, using primers located in the S gene. HBV DNA was detected with variable intensity in the CD3, CD4, CD19 and CD56 subsets following their separation with a cell sorter. For HBV RNA the signal obtained after PCR and Southern blotting was higher for CD56 and CD19 cells than for CD3 cells and undetectable for CD4 cells. This study demonstrates that replication and transcription of the HBV can occur in CD19- and CD56-positive cells. Positive signals in CD3 cells may be due to contamination of this subpopulation by NK cells.

Polymerase chain reaction (PCR) with a reverse transcriptase step characterized a specific transcription activity in hepatitis B virus (HBV)-infected peripheral blood mononuclear cells (PBMC) in two patients (1 and 2) with chronic hepatitis positive for antibody to hepatitis B core antigen (HBc). Patient 1 was also coinfected with human hepatitis delta virus. A patient who cleared HBV replication after antiviral treatment with vidarabine served as negative control. HBV-specific RNA poly A sequences were detected by PCR in PBMC of patients 1 and 2 even without detectable HBV DNA (patient 2) as shown by dot blot and PCR assays. RNA sequences were found in both the nucleus and cytoplasm. The demonstration of HBV mRNA sequences within PBMC suggests the transcription of viral DNA, in agreement with the findings of HBV surface antigen in PBMC. The results in patient 1 demonstrated HBV mRNA sequences in leukocytes even without PCR-detectable HBV DNA sequences, likely due to ongoing hepatitis delta virus replication.