SUMMARY Chronic hepatitis B virus (HBV) infection is a complex clinical entity frequently associated with cirrhosis and hepatocellular carcinoma (HCC). The persistence of HBV genomes in the absence of detectable surface antigenemia is termed occult HBV infection. Mutations in the surface gene rendering HBsAg undetectable by commercial assays and inhibition of HBV by suppression of viral replication and viral proteins represent two fundamentally different mechanisms that lead to occult HBV infections. The molecular mechanisms underlying occult HBV infections, including recently identified mechanisms associated with the suppression of HBV replication and inhibition of HBV proteins, are reviewed in detail. The availability of highly sensitive molecular methods has led to increased detection of occult HBV infections in various clinical settings. The clinical relevance of occult HBV infection and the utility of appropriate diagnostic methods to detect occult HBV infection are discussed. The need for specific guidelines on the diagnosis and management of occult HBV infection is being increasingly recognized; the aspects of mechanistic studies that warrant further investigation are discussed in the final section.

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HBV genotypes differ in pathogenicity. In addition, genotype-specific differences in the regulation of transcription and virus replication exist in HBV, but the underlying mechanisms are unknown. Here, we show the presence of a G-quadruplex motif in the promoter of the preS2/S gene; this G-quadruplex is highly conserved only in HBV genotype B but not in other HBV genotypes. We demonstrate that this G-quadruplex motif forms a hybrid intramolecular G-quadruplex structure. Interestingly, mutations disrupting the G-quadruplex in HBV genotype B reduced the preS2/S promoter activity, leading to reduced hepatitis B surface antigen (HBsAg) levels. G-quadruplex ligands stabilized the G-quadruplex in genotype B and enhanced the preS2/S promoter activity. Furthermore, mutations disrupting the G-quadruplex in the full-length HBV genotype B constructs were associated with impaired virion secretion. In contrast to typical G-quadruplexes within promoters which are negative regulators of transcription the G-quadruplex in the preS2/S promoter of HBV represents an unconventional positive regulatory element. Our findings highlight (a) G-quadruplex mediated enhancement of transcription and virion secretion in HBV and (b) a yet unknown role for DNA secondary structures in complex genotype-specific regulatory mechanisms in virus genomes.

Background: G-quadruplexes are increasingly recognized as regulatory elements in human, animal, bacterial and plant genomes. The presence and function of G-quadruplexes are not well studied among herpesviruses; in particular, there are no systematic genome-wide analysis of these important secondary structures in herpesvirus genomes.Results: We performed genome-wide analysis of putative quadruplex sequences (PQS) in human herpesviruses. We found unusually high PQS densities among human herpesviruses. PQS are enriched in the repeat regions and regulatory regions of human herpesviruses. Interestingly, PQS densities are higher in regulatory regions of immediate early genes compared to early and late genes in most herpesviruses. In addition, the majority of genes functionally conserved across human herpesviruses contain one or more PQS within the regulatory regions. We also describe the existence of unique intramolecular PQS repeats or repetitive G-quadruplex motifs in herpesviruses. Functional studies confirm a role for G-quadruplexes in regulating the gene expression of human herpesviruses. Conclusion: The pervasiveness of PQS, their enrichment and conservation at specific genomic locations suggest that these structural entities may represent a novel class of functional elements in herpesviruses. Our findings provide the necessary framework for studies on the biological role of G-quadruplexes in herpesviruses.

Parvoviruses are rapidly evolving viruses that infect a wide range of hosts, including vertebrates and invertebrates. Extensive methylation of the parvovirus genome has been recently demonstrated. A global pattern of methylation of CpG dinucleotides is seen in vertebrate genomes, compared to "fractional" methylation patterns in invertebrate genomes. It remains unknown if the loss of CpG dinucleotides occurs in all viruses of a given DNA virus family that infect host species spanning across vertebrates and invertebrates. We investigated the link between the extent of CpG dinucleotide depletion among autonomous parvoviruses and the evolutionary lineage of the infected host. We demonstrate major differences in the relative abundance of CpG dinucleotides among autonomous parvoviruses which share similar genome organization and common ancestry, depending on the infected host species. Parvoviruses infecting vertebrate hosts had significantly lower relative abundance of CpG dinucleotides than parvoviruses infecting invertebrate hosts. The strong correlation of CpG dinucleotide depletion with the gain in TpG/CpA dinucleotides and the loss of TpA dinucleotides among parvoviruses suggests a major role for CpG methylation in the evolution of parvoviruses. Our data present evidence that links the relative abundance of CpG dinucleotides in parvoviruses to the methylation capabilities of the infected host. In sum, our findings support a novel perspective of host-driven evolution among autonomous parvoviruses.T he relative abundance of dinucleotides, particularly the CpG dinucleotide, has received much attention recently. The CpG dinucleotide content is normally expressed as a ratio of the actual (observed) number of CpG dinucleotides and the expected number of CpG dinucleotides (O/E ratio). The relative abundance of CpG dinucleotides varies greatly across viruses (1, 2). Depletion of CpG dinucleotides has been reported to occur in all small DNA viruses (Ͻ30 kb) infecting humans (2), while most large DNA viruses (Ͼ30 kb) infecting humans show little or no CpG dinucleotide depletion. Several possible reasons have been suggested to play a role in the depletion of CpG dinucleotides, including (i) lower transcription rate for CpG-containing codons (3), (ii) stimulation of Toll-like receptor 9-mediated innate immune response by unmethylated CpGs (4), and (iii) spontaneous deamination of methylated cytosines in CpG dinucleotides (5). Deamination of unmethylated cytosines results in C-to-U transitions which are amenable to mismatch repair mechanisms, whereas the deamination of 5-methylcytosine leads to C-to-T transitions that are often irreversible, resulting in high mutation rates in methylated CpGs and a depletion of CpG dinucleotides (6, 7).In most vertebrate genomes, the O/E ratio for CpG dinucleotides is Ͻ0.35, suggesting that vertebrate genomes have lost about two-thirds of CpG dinucleotides (5, 8). In contrast, invertebrate genomes show minor or no depletion of CpG dinucleotides (5). Early investigations on insects showed that...

BackgroundGlobally, the number of internet users has crossed the three-billion mark, while in India users grew over 17% in the first 6 months of 2015 to 354 million. This study presented a background on internet use and the existence of excessive internet use.AimTo study the extent of internet use in 11th and 12 grade students and the psychopathology, if any, associated with excessive internet use.Methods426 students who met the inclusion criteria were recruited from 11th and 12th grade classes from Kendriya Vidyalaya, New Delhi, India, and were assessed by Young’s Internet Addiction Test and the Strength and Difficulties Questionnaire.ResultsAmong the 426 students, the mean internet addiction total score was 36.63 (20.78), which indicated mild level of internet addiction. 1.41% (six students) was diagnosed as excessive internet users, while 30.28% and 23.94% were classified as moderate and mild internet users, respectively. The prevalence of internet addiction between gender was 58.22% in males and 41.78% in females. While both positive (prosocial) and negative (hyperactivity, emotional, conduct and peer problem) impacts of internet use were reported by students, in the current study excessive use of internet had a negative impact on students’ lives as compared with positive impact, which was statistically significant (p<0.0001).ConclusionExcessive internet use led to abnormal behaviours which cause negative consequences to users. Early diagnosis of risk factors related to excessive internet use, provides education about responsible use and supervision of students by family members.

Chronic HBV infection is a major cause of hepatocellular carcinoma (HCC). The association between hepatitis B “e” antigen (HBeAg) and HCC is well-established by epidemiological studies. Nonetheless, the biological role of HBeAg in HCC remains enigmatic. We investigate the role of HBeAg in HBV-related HCC. Our findings suggest that HBeAg enhances cell proliferation and accelerates progression from G0/G1 phase to the S phase of the cell cycle in Huh7 cells. Examination of host gene expression and miRNA expression profiles reveals a total of 21 host genes and 12 host miRNAs that were differentially regulated in cells expressing HBeAg. Importantly, HBeAg induced the expression of miR-106b, an oncogenic miRNA. Interestingly, HBeAg-expression results in a significant reduction in the expression of retinoblastoma (Rb) gene, an experimentally validated target of miR-106b. Inhibition of miR-106b significantly increased the expression of the Rb gene, resulting in reduced cell proliferation and slowing of cell cycle progression from the G0/G1 phase to S phase. These observations suggest that the up-regulation of miR-106b by HBeAg contributes to the pathogenesis of HBV-related HCC by down-regulating the Rb gene. Our results highlight a role for HBeAg in HCC and provide a novel perspective on the molecular mechanisms underlying HBV-related HCC.

Defective hepatitis B virus (dHBV) particles contain genomes corresponding to singly spliced HBV RNA. A limited number of studies show that dHBV is present in all chronically HBVinfected patients. Clinical studies have linked dHBV and dHBV gene products to high virus loads and liver damage. The replication characteristics of dHBV genomes remain poorly understood. We found that the splice donor/acceptor sites critical for the formation of dHBV genomes are conserved across HBV genotypes. We report a novel method to create dHBV constructs from corresponding wild-type (WT) HBV constructs. We assessed the transcriptional characteristics of the dHBV constructs with those of the corresponding WT construct using a cell culture model. Interestingly, dHBV constructs had higher pre-genomic RNA levels, transcription efficiency, HBV e antigen levels and intracellular HBV core antigen levels compared with the corresponding WT HBV constructs. Our findings highlight previously unrecognized fundamental molecular characteristics of dHBV genomes and their potential role in the pathogenesis of HBV infection.