Hepatitis B virus (HBV) is a major etiological agent for liver cirrhosis and hepatocellular carcinoma (1). The nature of the liver-specific HBV receptor(s) has been a longstanding puzzle in the field (2); this is partly attributable to the paucity of cell lines supporting productive HBV infection. Other than primary human hepatocytes (PHHs) and primary tupaia hepatocytes (PTHs), only the human liver progenitor cell line HepaRG could be infected with HBV after prolonged treatment with dimethyl sulfoxide (DMSO) (3). DMSO promotes the differentiation of HepaRG cell into foci of hepatocytes surrounded by biliary cells. Other human hepatoma cell lines such as HepG2 and Huh7 support HBV DNA replication and virion production upon transfection with cloned HBV genome but not after inoculation with HBV particles. HBV protein expression and genome replication are driven by several coterminal transcripts ranging from 0.7 to 3.5 kb (4). The subgenomic RNAs of 2.4 and 2.1 kb are responsible for the expression of three coterminal envelope proteins termed large (L), middle (M), and small (S), with the M protein having an extra preS2 domain than the S protein and the L protein having an extra preS1 domain than the M protein. In addition to their incorporation into virions, the envelope proteins, especially S and M, are secreted as capsid-free subviral particles that exceed virions by Ն1,000-fold. The large quantity of S protein associated with subviral particles is detected by enzyme-linked immunosorbent assay (ELISA) as hepatitis B surface antigen (HBsAg), which provides a sensitive serological marker of HBV infection. Another serological marker is hepatitis B e antigen (HBeAg), a secreted

Many Pseudomonas aeruginosa virulence traits that contribute to human infections are accepted as being associated with its environmental lifestyle. Therefore, identifying the molecular mechanisms that govern the lifestyle choice is of high significance. We previously reported that a mutation in suhB results in a decrease in swimming motility and increased biofilm formation compared to the wild-type strain. Yet, little is known about how this occurs. In this study, we demonstrated that SuhB inversely regulates motility and biofilm formation through the GacA-RsmY/Z-RsmA cascade. Mutations in gacA or the two small RNAs rsmY/rsmZ, or overproduction of the RsmA protein essentially rescued the motility defect of the suhB mutant. Additionally, we identified a c-di-GMP mediated mechanism for SuhB regulation of motility and biofilm formation. We showed that the ΔsuhB mutant displayed elevated levels of c-di-GMP, and the ΔsuhB motility and biofilm phenotypes could be switched by artificially decreasing c-di-GMP levels. Further experiments led to the identification of the diguanylate cyclase GcbA responsible for regulating the c-di-GMP concentration in ΔsuhB and hence the switch between planktonic and surface-associated growth. Together, our results demonstrate a novel mechanism for SuhB regulation of the lifestyle transition via the Gac/Rsm and c-di-GMP signaling networks in P. aeruginosa.

SUMMARY:The aim of this study was to analyze the molecular epidemiologic characteristics of Acinetobacter baumannii. A total of 398 isolates were collected in 7 regions of South China from January to June of 2012. Drug sensitivity was tested toward 15 commonly used antibiotics; thus, 146 multidrug-resistant strains (resistant to more than 7 drugs) were identified, representing 36.7z of all isolates. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used for molecular subtyping. According to the PFGE results (with a cutoff of 70z similarity for the DNA electrophoretic bands), 146 strains were subdivided into 15 clusters, with cluster A being the largest (33.6z, distributed in all districts except Jiaxing). Cluster B was also widespread and included 14.4z of all strains. In addition, MLST results revealed 11 sequence types (ST), with ST208 being the most prevalent, followed by ST191 and ST729. Furthermore, 4 novel alleles and 6 novel STs were identified. Our results showed that multi-drug-resistant A. baumannii in South China shares the origin with other widespread strains in other countries. The nosocomial infections caused by A. baumannii have been severe in South China. Continuous monitoring and judicious antibiotic use are required.

This study aimed to identify and characterize mutations in the hepatitis B virus (HBV) genome associated with advanced liver diseases. The 3.2-kb HBV genome of the C2 subgenotype was amplified from sera of 18 cirrhotic Korean patients with (10) or without (8) hepatocellular carcinoma (HCC), and two clones per patient were characterized by transient transfection experiments in human hepatoma cells. While A1762T/G1764A core promoter mutations were highly prevalent in both groups, the G1896A precore mutation to abolish hepatitis B e antigen (HBeAg) expression was more common in HCC clones (55% vs. 20%). High replication capacity was mostly found in HCC clones and associated with core promoter mutations, whereas more non-HCC clones harbored a nonfunctional core gene (34% vs. 8%). Large in-frame deletions in the preS region were found in 60% of HCC clones and 38% of non-HCC clones. They removed the first 11 residues of large envelope protein or impaired small envelope protein expression, or deleted a neutralizing epitope in the preS2 domain. Additional point mutations prevented middle envelope protein expression, or caused nonsense mutations in the preS or S region to truncate large and/or small envelope protein. Consequently, many clones were unable to express or secrete hepatitis B surface antigen (HBsAg). In conclusion, mutations associated with the advanced stage of chronic HBV infection are complex and diverse. Host immune pressure most likely selected for mutations in the HBV genome to abolish or reduce HBeAg or HBsAg production, to enhance genome replication, or to escape neutralizing antibodies. Some of these mutations may contribute to liver cirrhosis or HCC development.

Gene transfer mediated by mannosylated chitosan (MCS) is a safe and promising approach for gene and vaccine delivery. MCS nanoparticles based gene delivery system showed high in vivo delivery efficiency and elicited strong immune responses in mice. However, little knowledge about the cell binding, transfection efficiency and intracellular trafficking of MCS nanoparticles had been acquired. In this study, using gastrin-releasing peptide as a model plasmid (pGRP), the binding of MCS/pGRP nanoparticles to macrophages and the intracellular trafficking of MCS/pGRP nanoparticles in macrophages were investigated. MCS-mediated transfection efficiency in macrophages was also evaluated using pGL-3 as a reporter gene. The results showed that the binding and transfection efficiency of MCS nanoparticles in macrophages was higher than that of CS, which was attributed to the interaction between mannose ligands in MCS and mannose receptors on the surface of macrophages. Observation with a confocal laser scanning microscope indicated the cellular uptake of MCS/pGRP nanoparticles were more than that of CS/pGRP nanoparticles in macrophages. MCS/pGRP nanoparticles were taken up by macrophages and most of them were entrapped in endosomal/lysosomal compartments. After the nanoparticles escaping from endosomal/lysosomal compartments, naked pGRP entered the nucleus, and a few MCS might enter the nucleus in terms of nanoparticles. Overall, MCS has the potential to be an excellent macrophage-targeting gene delivery carrier.

Hepatitis B virus (HBV) has a 3.2 kb circular DNA genome. It employs four promoters in conjunction with a single polyadenylation signal to generate 3.5, 2.4, 2.1 and 0.7 kb co-terminal RNAs. The 3.5 kb RNA is subdivided into the precore RNA for e-antigen expression and pregenomic RNA for genome replication. When introduced to a genotype A clone, several core promoter mutations markedly enhanced HBV genome replication, but suppressed e-antigen expression through up-regulation of pregenomic RNA at the expense of precore RNA. In this study, we found such mutations also diminished envelope proteins and hepatitis B surface antigen, products of the 2.1 and 2.4 kb subgenomic RNAs. Indeed, Northern blot analysis revealed overall increase in 3.5 kb RNA, but reduction in all subgenomic RNAs. To validate transcriptional interference, we subcloned 1.1×, 0.7× and 0.6× HBV genome, respectively, to a vector with or without a cytomegalovirus (CMV) promoter at the 5' end, so as to produce the pregenomic RNA, 2.4 kb RNA, and 2.1 kb RNA in large excess or not at all. Parallel transfection of the three pairs of constructs into a human hepatoma cell line confirmed the ability of pregenomic RNA to suppress all subgenomic transcripts and established the ability of the 2.4 and 2.1 kb RNAs to suppress the 0.7 kb RNA. Consistent with our findings, pregenomic RNA of the related duck HBV has been reported to interfere with transcription of the subgenomic RNAs. Transcriptional interference might explain why HBV produces so little 0.7 kb RNA and HBx protein despite a strong X promoter.

Sodium taurocholate cotransporting polypeptide (NTCP) has been identified as a hepatitis B virus (HBV) receptor, and its overexpression in HepG2 cell lines leads to efficient secretion of hepatitis B e antigen (HBeAg) following challenge with a large dose of cell culture-derived HBV (cHBV) particles. However, NTCP-reconstituted HepG2 cells are inefficiently infected by patient serum-derived HBV (sHBV) and release very little hepatitis B surface antigen (HBsAg) following cHBV infection, unlike differentiated HepaRG cells, which are naturally susceptible to both cHBV and sHBV particles. Here, we investigated whether NTCP could explain the different behaviors of the two cell types. Endogenous NTCP protein from differentiated HepaRG cells was unglycosylated despite wild-type coding sequence. HepaRG cells stably transfected with an epitope-tagged NTCP expression construct displayed higher sHBV but not cHBV susceptibility than cells transfected with the null mutant. Tagged NTCP introduced to both HepG2 and HepaRG cells was glycosylated, with N5 and N11 being sites of N-linked glycosylation. Mutating N5, N11, or both did not alter cell surface availability of NTCP or its subcellular localization, with both the singly glycosylated and nonglycosylated forms still capable of mediating cHBV infection in HepG2 cells. In conclusion, nonglycosylated NTCP is expressed by differentiated HepaRG cells and capable of mediating cHBV infection in HepG2 cells, but it cannot explain differential susceptibility of HepaRG and HepG2/NTCP cells to cHBV versus sHBV infection and different HBsAg/HBeAg ratios following cHBV infection. The responsible host factor(s) remains to be identified. HBV can infect differentiated HepaRG cells and also HepG2 cells overexpressing NTCP, the currently accepted HBV receptor. However, HepG2/NTCP cells remain poorly susceptible to patient serum-derived HBV particles and release very little hepatitis B surface antigen following infection by cell culture-derived HBV. We found differentiated HepaRG cells expressed nonglycosylated NTCP despite a wild-type coding sequence. NTCP introduced to HepG2 cells was glycosylated at two N-linked glycosylation sites, but mutating either or both sites failed to prevent infection by cell culture-derived HBV or to confer susceptibility to serum-derived HBV. Overexpressing NTCP in HepRG cells did not increase infection by cell culture-derived HBV or distort the ratio between the two viral antigens. These findings suggest that host factors unique to HepaRG cells are required for efficient infection by serum-derived HBV, and factors other than NTCP contribute to balanced viral antigen production following infection by cell culture-derived HBV.

Chronic infection by hepatitis B virus (HBV) genotype C is associated with a prolonged replicative phase and an increased risk of liver cancer, compared with genotype B infection. We previously found lower replication capacity but more efficient virion secretion by genotype C than genotype B isolates. Virion secretion requires interaction between core particles and ENVELOPE proteins. In the present study, chimeric constructs between genotype B and genotype C clones were generated to identify the structural basis for differential virion secretion. In addition to dimeric constructs, we also employed 1.1mer constructs, where the cytomegalovirus (CMV) promoter drove pregenomic RNA transcription. Through transient transfection experiments in Huh7 cells, we found that exchanging the entire envelope gene or just its S region could enhance virion secretion by genotype B clones while diminishing virion secretion by genotype C. Site-directed mutagenesis established the contribution of genotype-specific divergence at codons 108 and 115 in the preS1 region, as well as codon 126 in the S region, to differential virion secretion. Surprisingly, exchanging the envelope gene or just its S region, but not the core gene or 3′ S region, could markedly increase intracellular replicative DNA for genotype C clones but diminish that for genotype B, although the underlying mechanism remains to be clarified.