Construction and functional identification of a hepatitis B virus S protein small hairpin RNA recombinant adenovirus.

Hepatitis B virus S protein (HBs) plays an important role in hepatocellular carcinoma progression. However, to date, no direct and effective methods exist to research the function of HBs. Here, we combined the technology of RNA interference with recombinant adenovirus, constructed a recombinant adenovirus-expressing small hairpin RNA of HBs, and infected HepG2.2.15 cells. Then, reverse transcription-polymerase chain reaction (RT-PCR), quantitative real-time PCR, enzyme-linked immunosorbent assay, and Western blot analysis were performed to verify the interference effects. As a result, a recombinant adenovirus was successfully constructed and effectively packaged in AD293 cells, and it significantly inhibited HBs mRNA and protein expression in vitro. Our study may provide a novel tool to study HBs function.

An improved reverse dot hybridization for simple and rapid detection of adefovir dipivoxil-resistant hepatitis B virus.

Early detection of adefovir dipivoxil-resistant mutants during long-term treatment of chronic hepatitis B virus (HBV) infection with this drug is of great clinical importance. We developed an improved reverse dot hybridization test for simple and rapid detection of the rtA181V/T and rtN236T mutations associated with adefovir dipivoxil resistance in chronic hepatitis B patients. Probes were designed for genotypes B, C, and D of this resistance characteristic; a total of 70 clinical samples were analyzed with this improved reverse dot hybridization assay. Its usefulness was validated by comparing with sequencing data. Discordant results were confirmed by subclone sequencing. This reverse dot hybridization assay was sufficiently sensitive to detect 10(3) copies/mL; it also detected adefovir dipivoxil-resistant mutant strains when they comprised more than 5% of a mixed virus population. This reverse dot hybridization array correctly identified adefovir dipivoxil-resistant mutants; it had high concordance (98.5%) with direct sequencing data. There was no clear relationship between the HBV genotype and the development of adefovir dipivoxil-resistant mutants. This reverse dot hybridization assay proved to be simple and rapid for detection of rtA181V/T and rtN236T mutations associated with resistance to adefovir dipivoxil.

Molecular, cellular and functional characterizations of a novel ICAM-like molecule of the immunoglobulin superfamily from Leishmania mexicana amazonensis.

A molecule with two immunoglobulin (Ig) domains cloned from Leishmania mexicana amazonensis was characterized to have a sequence homology to the Ig domains of an ICAM-like molecule telencephalin, cloned from the brain of mammals, as well as to the variable domains of human immunoglobulin lambda light chain. The molecule therefore appears to be an ICAM-like molecule as well as a member of the immunoglobulin superfamily. We thus named it ICAM-L for Leishmania ICAM. The gene was coamplified with the ribonucleotide reductase M(2) subunit gene responsible for hydroxyurea resistance from hydroxyurea (Hu)-resistant Leishmania variants. As expected, an increase of the ICAM-L protein as well as an increase of the specific ICAM-L transcript of 2.1 kb was detected in the Hu-resistant variants with increasing doses of the drug used for resistance selection. Structurally, ICAM-L is more similar to the secretory adhesive molecules, such as 1Bgp and the link protein of the immunoglobulin superfamily, in that it lacks a transmembrane region and a GPI anchor sequence. Although ICAM-L was mainly localized in the nucleus of the parasite by confocal microscopy, however, detailed studies by electron microscopy and FACS analysis indicated that the protein was also localized on the surface of the parasite. The surface localization of the protein was furthered strengthened by the observations that anti-ICAM-L or ICAM-L itself can significantly block the binding of the parasite to macrophages. The blocking of the attachment of parasite to macrophages may indicate that ICAM-L functions as an intercellular adhesive molecule.