DNA sequences homologous to hepatitis C virus (HCV) in the extrachromosomal circular DNA in peripheral blood mononuclear cells of HCV-negative subjects.

OBJECTIVE: This study aimed to investigate DNA sequences that are substantially homologous to the corresponding RNA sequence sections of the hepatitis C virus (HCV). These DNA sequences are present in the whole DNA extracted from peripheral blood mononuclear cells (PBMCs) of HCV-negative subjects. We presumed that these experimentally proven 5'-noncoding region (5'-NCR) homologous DNA sequences could be contained in the extrachromosomal circular DNA (eccDNA) fraction as part of the whole cellular DNA. METHODS: Home-made polymerase chain reaction (PCR) with whole cellular and isolated eccDNA, nucleotide basic local alignment search tool (BLASTn) alignments, and tests for patterns of methylation in selected sequence sections were performed. RESULTS: The PCR tests revealed DNA sequences of up to 320 bp that broadly matched the corresponding sequence sections of known HCV genotypes. In contrast, BLASTn alignment searches of published HCV 5'-NCR sequences with human genome databases revealed only sequence segments of up to 36 bp of the 5'-NCR. The composition of these sequences shows missing base pairs, base pair mismatches as well as complete homology with HCV reference sequences. These short sequence sections are present in numerous copies on both the same and different chromosomes. The selected sequence region within the DNA sequences of the 5'-NCR revealed a broad diversity of individual patterns of methylation. CONCLUSIONS: The experimental results confirm our assumption that parts of the HCV 5'-NCR genomic RNA sequences are present at the DNA level in the eccDNA fraction of PBMCs. The tests for methylation patterns therein revealed individual methylomes which could represent an epigenetic feature. The respective sequence section might be subject to genetic regulation.

Overlooked: Extrachromosomal DNA and Their Possible Impact on Whole Genome Sequencing.

Extrachromosomal (ec) DNA in eukaryotic cells has been known for decades. The structures described range from linear double stranded (ds) DNA to circular dsDNA, distinct from mitochondrial (mt) DNA. The sizes of circular forms are described from some hundred base pairs (bp) up to more than 150 kbp. The number of molecules per cell ranges from several hundred to a thousand. Semi-quantitative determinations of circular dsDNA show proportions as high as several percentages of the total DNA per cell. These ecDNA fractions harbor sequences that are known to be present in chromosomal DNA (chrDNA) too. Sequencing projects on, for example the human genome, have to take into account the ecDNA sequences which are simultaneously ascertained; corrections cannot be performed retrospectively. Concerning the results of sequencings derived from extracted whole DNA: if the ecDNA fractions contained therein are not taken into account, erroneous conclusions at the chromosomal level may result.

Dilemma of concepts and strategies for the prevention of spread of HIV in relation to human behavior, law and human rights.

The new prevalence data regarding the estimated global number of human immunodeficiency virus positive (HIV+) cases, i.e., including people who are either aware or unaware of their HIV infection in 2010, lead many to wonder why the increase in incidence has reached today's unprecedented level and escalated within such a short time. This, in spite of prevention campaigns in countries affected by HIV/acquired immune deficiency syndrome (AIDS) with their urgent messages aimed at preventing HIV transmission by promoting changes in individual's behavior. This article analyzes the background of the prevention strategies, in particular their political, social and legal concepts in terms of human rights, and reveals traits of human behavior not considered thus far. A radical reappraisal is necessary, at social and legislative levels, as well as options additional to current concepts. When ethical issues come up, they become blamed for outmoded moralistic positions. However, ignoring the reality has led to dire consequences from prioritizing individual human rights over society's collective need to prevent the spread of HIV.

Effective inhibition of expression of hepatitis B virus genes by DNAzymes.

AIM: To evaluate the inhibitory effects of DNAzymes on the expressions of hepatitis B virus (HBV) s (HBsAg) and e (HBeAg) in 2.2.15 cells, and to explore the potential therapeutic effects of DNAzymes on replication of HBV genome. METHODS: DNAzymes DrzBS and DrzBC specific to HBV (ayw subtype) s gene ORF A157UG and e gene ORF A1816UG, were designed and synthesized. Inhibitory effects of DrzBS or DrzBC on the expressions of HBV s and e genes as well as HBV DNA levels in culture supernatants were observed in 2.2.15 cells. RESULTS: After being treated with DrzBS or DrzBC, the expression of HBV s or e genes in 2.2.15 cells was depressed dramatically. The maximum inhibition rate was 94.2% and 91.8% for DrzBS and DrzBC, respectively. The concentration for effective inhibition of both DrzBS and DrzBC was within 0.1-2.5 micromol/L, showing a dose-dependence. The efficiency of inhibiting HBsAg, HBeAg in 2.2.15 cells by DrzBS or DrzBC was higher than that of the same target genes by antisense oligonucleotides (ASON). The concentration for effective inhibition of DNAzymes was at least 10-fold lower compared with ASON controls. Neither inhibition on the replication of HBV DNA nor toxicity to 2.2.15 cells was observed. CONCLUSION: DrzBS and DrzBC can block the expression of HBV s- and e-genes in 2.2.15 cells and provide a specific and effective anti-HBV gene therapeutic means.

SARS-coronavirus replicates in mononuclear cells of peripheral blood (PBMCs) from SARS patients.

BACKGROUND: The etiologic agent of severe acute respiratory syndrome (SARS) is a recently identified, positive single-stranded RNA (ssRNA) coronavirus (SARS-CoV). Little is known about the dynamic changes of the viral replicative form in SARS cases. OBJECTIVES: Evaluate whether SARS-CoV can infect and replicate in peripheral blood mononuclear cells (PBMCs) of infected persons and reveal any dynamic changes to the virus during the course of the disease. STUDY DESIGN: Peripheral blood mononuclear cells collected from SARS cases infected by the same infectious source were tested for both negative-stranded RNA (minus-RNA, "replicative intermediates") and positive-stranded RNA (genomic RNA) of SARS-CoV during the course of hospitalization by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: SARS-CoV minus-RNA was detected in PBMCs from SARS patients. The viral replicative forms in PBMCs were detectable during a period of 6 days post-onset of the disease, while the plus-RNA were detectable for a longer period (8-12 days post-onset). CONCLUSIONS: SARS-coronavirus can infect and replicate within PBMCs of SARS patients, but viral replication in PBMCs seems subject to self-limitation.

Human DNA contains sequences homologous to the 5'-non-coding region of hepatits C virus: characterization with restriction endonucleases reveals individual varieties.

OBJECTIVE: To investigate a 272 base pair section of the 5'-non-coding region of genomic DNA from the peripheral blood monounuclear cells of healthy hepatitis virus C (HCV)-negative human subjects (not patients). METHODS: This sequence section bears interest because (1) it harbors several potential methylation (Cp-rich) sites, and (2) it represents the largest part of its internal ribosomal entry site. A pre-PCR digestion protocol was established making consistent use of four restriction endonucleases selected for certain features: SmaI, XmaCI, MspI, and HpaII are inhibited if methylation(s) are present at certain cytosines within their cutting sequences. RESULTS: The suspected HCV-specific sequence was found in the DNA of each subject tested. The pre-PCR digestion assay reveals individual differences in their pattern of methylation, which may be due to possible epigenetic phenomena. CONCLUSIONS: The results provide formal proof that these HCV-specific sequences are contained in the genomic or extra chromosomal target DNA, and probably belong to a new class of endogenous sequences.

Mutations in precore and core promoter region of HBV in patients with hepatic failure.

OBJECTIVE: To clarify the association of hepatitis B virus mutants in precore and core promoter regions in patients with hepatic failure and HBeAg state. METHODS: Precore and core promoter regions of 25 HBV isolates from the patients with hepatic failure were analyzed by polymerase chain reaction (PCR) direct sequencing approach. RESULTS: Precore G-to-A(1896) mutants were identified in 16 (64%) of the 25 isolates. The "hot spot" mutations at A-to-T(1762) and G-to-A(1764) were present together in 19 (76%) of the 25 isolates, while C-to-T(1653) and T-to-C(1753) existed in a mutually exclusive manner and more frequently in hepatic failure with liver cirrhosis group than in hepatic failure with chronic hepatitis group (100% vs 50%). Both A(1896) and T(1762)-A(1764) could be found frequently in HBeAg-positive subjects (77.8% and 88.9%), whereas T(1653)/C(1753) was more prevalent in anti-HBe-positive subjects than in HBeAg-positive subjects (93.8% vs 33.3%). CONCLUSIONS: The whole frequency of mutations in precore and core promoter gene will become more frequent as HBV infection is to be persistent. Mutation to T(1653)/C(1753) may be useful as a marker for hepatic failure. It requires further study whether the mixed infection of mutants and wilds will develop and affect the condition of HBeAg in serum along the progression of liver disease.

A high frequency of GBV-C/HGV coinfection in hepatitis C patients in Germany.

AIM:To detect infection rate of GBV-C/HGV in hepatitis C patients, to determine the methods of higher sensitivity and the primers of higher efficiency for GBV-C/HGV RNA detection and to study the dominant subtype and mutation of GBV-C/HGV.METHODS:Quantitative RT-PCR for detection pf HCV RNA concentration in serum samples, RT-nested PCR with two sets of primers for detection of GBV-C RNA, RT-PCR ELISA with two sets of primers for detection of HGV RNA, nucleotide sequence and putative amino acid sequence analysis.RESULTS:The positive rates of GBV-C RNA at the 5'-NCR and NS3 region in 211 serums amples from the patients with HCV infection were 31.8% and 22.8% respectively. The positive rates of HGV RNA at the 5'-NCR and NS5 region in the same samples were 47.9% and 31.8% respectively. The total positive rate of GBV-C/HGV RNA was as high as 55.5%. HCV copy numbers in the patients without GBV-C/HGV coinfection were statistically higher than that in the patients with GBV-C/HGV coinfection (P<0.01).Frequent mutation of nucleotide residue was present in the amplification products. Frameshift mutation was found in two samples with GBV-C NS3 region nucleotide sequences. All nucleotide sequences from amplification products showed higher homology to HGV genome than to GBV-C genome even though part of the sequences were amplified with GBV-C primers.CONCLUSION:A high frequency of GBV-C/HGV coinfection existed in the hepatitis C patients. RT-PCR ELISA was more sensitive than RT-nested PCR for detection of GBV-C/HGV RNA. The primers derived from the 5'-NCR was more efficient than those derived from the NS3 and NS5 regions. A reverse relationship was found to exist between HCV RNA concentration and GBV-C/HGV infection frequency. HGV was the dominant subtype of the virus in the local area. The major mutations of GBV-C/HGV genomes were random mutation of nucleotide residue.