1. Molecular and Serological Aspects of HBsAg-negative( Silent)HBV infections in North America The uncommon finding of """"""""silent"""""""" hepatitis B virus (HBV), characterized by the presence of HBV DNA in serum or liver tissue in the absence of hepatitis B surface antigen (HBsAg), anti-HBc and anti-HBs in serum, is thought to be caused in part by HBV mutants that produce a very low level of HBsAg, which escape detection by currently available serologic tests. This is an issue for blood transfusion safety. HCC and adjacent liver tissues from forty-three patients whose sera were HBsAg-negative, as well as three positive and four negative controls (a total of 87 DNAs) were further studied for HBV DNA using nested PCR, including 9 from various areas of the USA, 22 from Vancouver, Canada, and 12 from China. HBV DNA was detected by PCR in 5 of the 9 patients (55%)from HBsAg-negative patients from the USA, in 12 of the 22 from Canada (55%), and in 12/12 from Qidong, China (100%). Among all HCC patients from the USA and Canada who were HBsAg-negative, HBV DNA could be detected in 55%. Sequencing of the 241-bp S gene PCR product was performed in the patients from the USA and Canada; each was found to contain the expected S gene sequence with one or more nucleotide variations differing from the consensus sequence and from each other. Genotyping was determined using primers for type-specific sequences in the S gene. Genotype C was found to be the dominant type in these patients, both Oriental and non-Oriental from the US and Canada (70% in non-Oriental patients, 80% in Oriental patinets). Replication of HBV, as determined by the presence of replicative intermediates (covalently closed circular [CCC] HBV DNA)was found in 2/17(12%), indicating that active replication of HBV was present at least in these patients. PCR (bldg 29/rm 320). 2. Early Detection of Hepatitis B Virus Infection: Comparative Sensitivity of HBV DNA Detection by PCR and HBsAg Detection by a New Generation of Assays. Based on tests of the FDA HBsAg lot-release panel in the present study, newer HBsAg tests have sensitivity of 0.07-0.18 ng/ml, compared to 0.13-0.62 ng/ml for 4 licensed tests evaluated in this study. Tests on the WHO HBV DNA standard showed that the viral load at cut-off was 102-364 IU/ml for newer HBsAg tests, and 363-1069 for licensed HBsAg tests. In 90 samples collected during the exponential phase from 10 seroconversion panels, HBsAg tests detected 31-61% and single-sample PCR detected 82-99% of samples. In the pre-exponential phase, no sample was detected by licensed HBsAg tests, while single-sample PCR detected HBV at 30-80% of these samples. The newer HBsAg tests detected infections 2 weeks earlier and single-sample PCR detected HBV infections about one month earlier than currently available HBsAg tests. 4. Quantitation of HBV genome copies required to infect Chimpanzees, using Taqman PCR. Tabor et al. determined the infectious dose of HBV for chimpanzees in 1983. They used 3 different serotypes, serotypes adw, ayw, and adr. For quantitation of HBV inocula, we used a series of dilutions of HBV-containing plasmids as quantitative standards to create standard curves, and used WHO HBV-DNA as an additional reference standard. A WHO Working Standard (WHO-WS, containing 500 IU/ml)was included in each experiment as a run control. The mean genome equivalent for WHO-WS determined by Taqman in our study (of 11 tests values) was 3019 geq/ml. Validation of the Taqman PCR quantitation for HBV were performed, limits of detection and various influencing factors were defined, comparisons of different extraction methods and different designs of probes and primers were carried out. Sequencing of the segments of S and C gene of the 3 HBV inocula has been finished, and new probes and primers were designed to investigate the different levels of infectivity in greater detail.
