Supplementary Table 1 from Distribution and Hepatocellular Carcinoma–Related Viral Properties of Hepatitis B Virus Genotypes in Mainland China: A Community-Based Study

Aim: To compare blood pressure (BP) and safety outcomes in patients with hypertension initiating bisoprolol, versus other β-blockers, angiotensin-converting enzyme inhibitors/angiotensin II receptor blockers, calcium channel blockers or diuretics. Materials & methods: New user cohort study. Patients initiating bisoprolol were matched with up to four patients, in each comparator cohort using propensity score. BP outcomes were compared using linear mixed models and safety outcomes using Cox proportional hazards. Results: Differences in average systolic and diastolic BP variation were ≤3 mmHg between bisoprolol versus the compared classes. No difference was observed in risk of diabetes, obesity or erectile dysfunction. An increased dyslipidemia risk was only observed versus diuretics (hazard ratio: 0.76; 98.75% CI: 0.58, 0.99). Conclusion: No differences in BP variation and safety outcomes.

Chikungunya virus (CHIKV) is an alphavirus of the Togaviridae family. After autoproteolytic cleavage, the CHIKV capsid protein (CP) is involved in RNA binding and assembly of the viral particle. The monomeric CP is approximately 30 kDa in size and is small enough for passive transport through nuclear pores. Some alphaviruses are found to harbor nuclear localization signals (NLS) and transport of these proteins between cellular compartments was shown to be energy dependent. The active nuclear import of cytoplasmic proteins is mediated by karyopherins and their export by exportins. As nuclear and cytoplasmic trafficking may play a role in the life cycle of CHIKV, we have sought to identify nuclear localization and nuclear export signals in CHIKV CP in a virus-free system.

Hepatitis B virus (HBV) is classified into 8 genotypes with distinct geographical distribution. Genotype D (HBV/D) has the widest distribution area and is comprised of 7 subgenotypes. Subgenotypes D1, D2 and D3 appear worldwide, while D4-D7 have a more restricted distribution. Within the Mediterranean area, HBV/D and subgenotype D3 are the most prevalent. The purpose of this study was to characterize the full genome of Serbian HBV/D3 isolates by comparison and phylogenetic analysis with HBV/D3 sequences (66 samples) found in GeneBank/DDBJ databases from different parts of the world. Isolates were obtained from three patients diagnosed with chronic hepatitis B (HBsAg+). All three isolates have two very rare nucleotide substitutions, A929T and T150A, which indicate the same ancestor. Phylogenetic analysis of HBV/D3 genome sequences throughout the world follows an ethno-geographical origin of isolates with rare exceptions, which could be explained by human travelling and migration. The geographically close but ethnically different Serbian and Italian isolates clustered in the same subnode, and on a common branch with strains from Northern Canada. To test the apparently close HBV phylogenetic relationship between completely separated patients from Serbia and Northern Canada we analyzed in depth a 440 bp region of the HBsAg from Canadian (n=73) and Serbian (n=70) isolates. The constructed parsimony tree revealed that strains from Serbia and Northern Canada fell along the same branch which indicates independent evolution within regions of each country. Considering that HBsAg sequence has limited variability for phylogenetic analyses, our hypothesis needs further confirmation with more HBV complete genome sequences.

Background Hepatitis E virus (HEV) is a non-enveloped plus-strand RNA virus that causes acute hepatitis. The capsid protein open reading frame 2 (ORF2) is known to induce endoplasmic reticulum stress in ORF2 expressing cells. Methodology/Principal Findings In this study we found that HEV ORF2 activates the expression of the pro-apoptotic gene C/EBP homologous protein (CHOP). ORF2 stimulates the CHOP promoter mainly through AARE (amino acid response elements) and to a minor extent the ERSE (endoplasmic reticulum stress response elements). Activating transcription factor 4 (ATF4) protein binds and activates the AARE regulatory sites of the CHOP promoter. ORF2 expression also leads to increased phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) that in turn initiates the translation of ATF4 mRNA. The pro-apoptotic gene CHOP is an important trigger to initiate endoplasmic reticulum stress induced apoptosis. However, the activation of CHOP by ORF2 in this study did not induce apoptosis, nor did BCL2-associated X protein (Bax) translocate to mitochondria. Microarray analysis revealed an ORF2 specific increased expression of chaperones Hsp72, Hsp70B', and co-chaperone Hsp40. Co-immunoprecipitation (Co-IP) and in silico molecular docking analysis suggests that HEV ORF2 interacts with Hsp72. In addition, Hsp72 shows nuclear accumulation in ORF2 expressing cells. Conclusions/Significance These data provide new insight into simultaneously occurring counter-acting effects of HEV ORF2 that may be part of a strategy to prevent host suicide before completion of the viral replication cycle.

Chronic hepatitis B virus (HBV) infection is the major risk for hepatocellular carcinomas (HCC). HBV X protein (HBx) and p53 tumor suppressor family interactions may be crucial for HCC induction. We compared p53 and p73 interactions with HBx in normal and HCC tumor cell lines differing in their p53 status. In the latter, HBx was pro-apoptotic but exhibited opposite effects in non-tumor cells. In these normal cells, p53 and p73 were retained in the cytoplasm. In hepatoma cells, however, HBx led to nuclear translocation of p53 and p73, followed by enhanced transactivation of p53-dependent promoters. The nuclear transfer of p53, but not of p73, was abrogated by protein kinase C inhibitor Gö6976. HBx overexpression in HCC cells led to strong p53 phosphorylation at Ser15, but not in non-tumor cells. Our results define ATM kinase as mediator for HBx-induced p53 phosphorylation. While HBx promotes cell death in p53/p73-positive hepatoma cells also in presence of increased levels of the oncogenic ΔTAp73 isoform, it significantly potentiates ΔTAp73-mediated proliferation and malignant transformation of fibroblasts. Our data suggest that prevention of apoptosis in normal cells by HBx through inhibition of pro-apoptotic p53 family members via direct interaction and coaction with anti-apoptotic ΔTAp73 seems to be the key element in the decision in favor of cell survival. The complex cell context-dependent interactions between p53 family members and HBx in the regulation of apoptosis may be essential in HBV-induced HCC and anticancer therapy.

AbstractIntroduction: Hepatitis B virus (HBV) genotypes, replication status, and mutations have been associated with the risk of hepatocellular carcinoma (HCC). Our aim was to study the distribution and HCC-related viral properties of HBV genotypes/subgenotypes in Mainland China.Methods: A multistage cluster probability sampling method was applied to select 81,775 participants between 1 and 59 years at 160 national disease surveillance points. We examined hepatitis B surface antigen, HBV genotypes and subgenotypes, hepatitis B e antigen, viral load, and mutations in the PreS and core promoter regions of HBV genome.Results: HBV subgenotypes B2 (27.3%), C1 (10.7%), and C2 (58.0%) were predominant. Genotype D (D1, 80.8%) was frequent in the Uygur. We identified a new subgenotype, C9, mainly in Tibetans. Compositions of subgenotypes B2 and C1 and genotype mixture increased from the North to Central South, which was consistently associated with the increasing prevalence of hepatitis B surface antigen. Hepatitis B e antigen positivity and viral loads were higher in the young with genotype B and declined more rapidly with increasing age than those with genotype C. In contrast to G1896A, PreS deletion, T31C, T1753V, and A1762T/G1764A were more frequent in subgenotype C2 than in subgenotype B2. A1762T/G1764A, T1753V, C1653T, and G1896A, except PreS deletion, consecutively increased with increasing age.Conclusion: HBV subgenotypes B2, C1, and C2 are endemic in Mainland China. HBV genotype C exhibits less replication activity in the young and harbors higher frequencies of the HCC-associated mutations than genotype B.Impact: These basic data could help evaluate the association of HBV variations with HCC. Cancer Epidemiol Biomarkers Prev; 19(3); 777–86

Background The alphavirus capsid is multifunctional and plays a key role in the viral life cycle. The nucleocapsid domain is released by the self-cleavage activity of the serine protease domain within the capsid. All alphaviruses analyzed to date show this autocatalytic cleavage. Here we have analyzed the sequence requirements for the cleavage activity of Chikungunya virus capsid protease of genus alphavirus. Results Amongst alphaviruses, the C-terminal amino acid tryptophan (W261) is conserved and found to be important for the cleavage. Mutating tryptophan to alanine (W261A) completely inactivated the protease. Other amino acids near W261 were not having any effect on the activity of this protease. However, serine protease inhibitor AEBSF did not inhibit the activity. Through error-prone PCR we found that isoleucine 227 is important for the effective activity. The loss of activity was analyzed further by molecular modelling and comparison of WT and mutant structures. It was found that lysine introduced at position 227 is spatially very close to the catalytic triad and may disrupt electrostatic interactions in the catalytic site and thus inactivate the enzyme. We are also examining other sequence requirements for this protease activity. Conclusions We analyzed various amino acid sequence requirements for the activity of ChikV capsid protease and found that amino acids outside the catalytic triads are important for the activity.

Die Übertragung von Erregern einer viralen Hepatitis infolge sexueller Aktivität ist ein häufiges Problem in der ärztlichen Praxis. Zur Diagnostik der viralen Hepatitis wird ein Stufenschema vorgeschlagen. Zum initialen Screening genügt die Untersuchung auf anti-HBc, HBsAg, anti-HAV und anti-HCV. Je nach Konstellation schließen sich dann weitere Untersuchungen zu HBV, HCV und HAV an. Bei fehlendem Nachweis im initialen Screening folgen weitere Untersuchungen zu HEV oder Epstein-Barr-Virus, Zytomegalievirus oder weiteren Agenzien.

Hepatitis B virus (HBV) infection is a global health problem associated with severe liver disorders. Viral load and HBV genotype affect the clinical outcome, guide antiviral therapy and provide long term prognosis for HBV infected patients. Various types of detection and quantitation assays are currently in use with a different effectiveness. The aim of this study was to develop a method that would provide simultaneous identification and quantitation of genotypes A and D in a single-tube reaction. Sera from infected patients were analyzed by a TaqMan based real time PCR. Optimized reagents were used for HBV DNA quantitation while the genotypes A and D were quantified separately by our design of the assay. Multiplex real time PCR was achieved and was specific for HBV genotypes A and D within a single-tube reaction. Simulation of mixed virus populations was identified reproducibly in vitro. Quantitation of these individual genotypes was exceptionally reliable, so much so that the sum of individual genotypes was equal to the total viral load determined in a separate reaction. Therefore, a straightforward, conceptually simple and reliable approach to issues involving HBV genotypes A and D is submitted. Identity and exact titer of these genotypes in the Caucasian population can now be determined easily.

Hepatitis B virus (HBV) genotypes and subgenotypes show distinct geographical prevalence. A genotyping analysis of 28 samples from asymptomatic HBV carriers from the Philippines gave a distribution of HBV genotypes as expected from a previous study: 54% B (15/28), C5 18% (5/28), 14% D (4/28), 7% A1 (2/28). In addition, 7% (2/28) of the samples showed a double infection with genotypes B and D. One of the isolates sequenced completely, ph105, did not group into one of the known subgenotypes after phylogenetic analysis. Ph105 formed a separate clade in genotype C. With a genome length of 3,215 nt. and a serological subtype adr, ph105 exhibited the main features of most genotype C strains. However, ph105 differed by 4.1–7.2% from HBV subgenotypes C1 to C5 when comparing the nucleotide sequence of whole genomes. With only 4.1% difference ph105 was most closely related to subgenotype C2. SimPlot analysis gave no indication for recombination with known HBV genotypes. Ph105 fulfils all criteria for a new subgenotype C6. J. Med. Virol. 81:983–987, 2009. © 2009 Wiley‐Liss, Inc.

Hepatitis B virus (HBV) genotypes (2, 4, 5, 7-9) and subgenotypes (11, 13, 14, 16-18) are important factors that influence the course of the disease and the outcome of treatment. Because of the clinical importance of the HBV classification, an accurate assignation of newly found subgenotypes to their respective clades and an unambiguous nomenclature are needed. A new HBV subgenotype should differ from previously established by at least 4%, and the segregation into a new clade should be supported by robust bootstrapping data (3). In the June 2009 issue of the Journal of Clinical Microbiology, a new C6 subgenotype of HBV found in the Papua province of Indonesia (C6 Papua-Indonesia) is described (15). However, a tentative C6 subgenotype found in the Philippines (C6 Philippines) has already been published contemporaneously (1). To keep up with an unambiguous system of HBV subgenotypes, it is important to analyze if C6 Papua-Indonesia and C6 Philippines are identical or map to different clades. Using methods described by Cavinta et al. (1) and published sequences from Utsumi et al. (15), we made a phylogenetic analysis using whole genomes that clearly showed that both C6 groups mapped to different clades (Fig. ​(Fig.11). FIG. 1. Phylogenetic tree of complete HBV genomes of genotypes A to H. An alignment of complete sequences was performed with ClustalW using the program DNASTAR. The alignment was further analyzed by bootstrapping with 500 replicates, using the neighborhood-joining ... C6 Papua-Indonesia and C6 Philippines differed by 5.1% from each other and diverged by at least 4.1% from the other HBV subgenotypes (Table ​(Table11). TABLE 1. Nucleotide distance between proposed subgenotypes C6 Philippines and C6 Papua-Indonesia of HBV and other reference genotype strains from the database Thus, both contemporaneously described subgenotypes (1, 15) fulfill all criteria for having new and separate subgenotypes. Because the designation C6 has been claimed for partial sequences found in the Papua province of Indonesia already (6), we suggest that the subgenotype C6 found in the Papua province of Indonesia shall keep the designation C6, whereas the tentative subgenotype from the Philippines shall be called C7. To avoid similar confusion in the future, we have suggested a procedure that would involve the International Committee for Taxonomy of Viruses to allot new genotype/subgenotype designations (10).

The transmission of hepatitis viruses as a result of sexual activity is a common problem in medical practice. A system of stages is proposed to diagnose viral hepatitis. Testing for anti-HBc, HBsAg, anti-HAV and anti-HCV is sufficient for initial screening. Depending on the circumstances, this may be followed by further tests for HBV, HCV and HAV. If initial screening provides no evidence, further tests for HEV, Epstein-Barr virus, cytomegalovirus or other agents are conducted.

(2) Sequencing of only parts of the HBV genomes led to the tentative suggestion of subgenotypes A4 and A5 [20] . In an article in Emerging Infectious Diseases sequencing whole genomes of tentative A4 isolates, Andernach et al. [21] now group this subgenotype into the clade of A5. (3) Based on whole genome sequencing two new subgenotypes have been called C6 in two contemporaneous publications. C6 has been identified in the Philippines [22] and Papua, Indonesia [23] . Isolates from these two publications map into two clades with a divergence of 5.1% and thus make up a new subgenotype C7 from the Philippines [Schaefer S, unpublished]. (4) A similar problem seems to arise with subgenotype D6. There are two concurrent publications that describe a new subgenotype D6 in Tunisia [24] and Papua, Indonesia [23] . (5) Regarding the delimitation of subgenotypes, these have been described as the major divisions within the phylogenetic trees in genotypes by Norder et al. [4] . Later on Kramvis et al. [14] defined subgenotypes as differing by at least 4% and supported this by robust bootstrapping data, although yielding largely overlapping results with the definition by Norder et al. [4] . This competing definition is partly contradictory to the original paper [4] , where 4 major clades of genotype D, designated D1–D4, were described with an overall divergence for some of these clearly below 4% [25] . However, these subgenotypes Hepatitis B virus (HBV) genotypes [1–6] and subgenotypes [7–12] have an impact on the course of disease and treatment decisions. In Germany, HBV genotyping is recommended before starting interferon therapy treatment of HBV infection [13] . Thus, an accurate and universal system of classification is of prime importance. Several largely overlapping definitions for HBV genotypes have been presented by leading experts in this field in the past [4, 14–17] . However, due to the lack of universally accepted rules, irregularities have accumulated within the last 20 years of HBV genotype research. Some of these are found below. (1) In a letter to the editor in this issue of Intervirology Ahn et al. (pp. 321–322) raise an important point. The assignation of subgenotypes C1 and C2 to clades is contradictory in the literature. As outlined by Ahn et al. there are two influential papers in which C1 following the nomenclature of Norder et al. [4] is C2 according to the classification of Huy et al. [18] and vice versa. However, regarding the figure in the letter, an uninitiated reader might get the impression that Huy et al. [18] also defined subgenotypes C3–C5. Also, only C1–C4 were described by Norder et al. [4] . C5 was described by Sakamoto et al. [19] in 2006. The reason for the discrepancies in nomenclature would have been more obvious if the references in the figure had been Huy et al. [18] and Norder et al. [4] , respectively. Published online: September 29, 2009

BACKGROUND AND AIMS:The major risk factors for acute hepatitis B (AHB) in China and the viral factors determining the progression from acute to chronic hepatitis B remain largely unknown.METHODS:Epidemiological studies within a population-based surveillance for AHB in adults were performed in Shanghai, China, including 294 patients, 588 matched controls and 572 family members of the patients.RESULTS:Invasive medical procedures, household contact with hepatitis B virus (HBV) carriers, body care and beauty treatments, and lack of HBV vaccination were independently associated with AHB. Among those risks, pedicure in bath centres emerged. Sixty-eight of 128 patients with AHB were genotyped including 33 with HBV B2 and 35 with HBV C2. Twenty-five (8.50%) of the 294 patients, including 20 with HBV C2 and 5 with HBV B2 (p = 0.013), progressed to chronic infection. Multivariate analysis showed that HBV C2 was independently associated with chronicification of AHB. Patients with HBV B2 were younger and there was a higher proportion of women than those with HBV C2. The prevalence of HBV B2 was higher in the patients than in neighbourhood chronic carriers. The chronic carriers with HBV B2 showed higher viral loads, higher hepatitis B e antigen (HBeAg) seropositivity, and with higher proportion in men than those with HBV C2, implying that sexual contact plays a role in the transmission of HBV B2. Phylogenetic analysis showed that HBV C2 was frequently involved in transmissions within households.CONCLUSIONS:Despite lower viral load and HBeAg status in the chronic carriers, HBV C2 was more prone to causing chronic infection than was HBV B2.

Hepatitis B virus (HBV) is a causative agent of chronic hepatitis and hepatocellular carcinoma. Recent findings demonstrating p73 and specifically N-terminally truncated p73 (Delta TAp73) accumulation in hepatocellular carcinoma suggest that p73 plays a role in the malignant phenotype. Here, we investigated the mechanism of HBV pregenomic core promoter/ enhancer II (cp/EII) regulation by full-length TAp73 and its oncogenic counterpart Delta TAp73. Ectopic and endogenous expression of TAp73 leads to a significant downregulation of cp/EII activity in p53-deficient hepatoma cell lines. In contrast, overexpression of Delta TAp73 results in significant cp/EII activation and increased HBV core (HBc) expression. TAp73-mediated repression of HBV transcription was substantially abolished by Delta TAp73. We show that both TAp73 and Delta TAp73 proteins directly bind to the Sp1 transcription factor, a key stimulator of HBV gene expression. However, only TAp73 abolishes Sp1 binding to cp/EII, whereas the Delta TAp73-Sp1 complex further persists on the DNA. The inhibitory effect of p53/p73 on HBc expression is associated with the inhibition of viral replication, while Delta TAp73 is not. These data strongly support the fact that the p73-isoform-related interaction with Sp1 is the underlying mechanism of the diverse outcome on HBc expression, suggesting a new mechanism by which oncogenic Delta TAp73 could enhance the carcinogenic process in liver cells.

There are eight genotypes A–H of hepatitis B virus (HBV). Most genotypes are further divided into subgenotypes. Genotypes and subgenotypes influence the natural course of infection and therapy. We analysed nine sera from HBV carriers from Peru. Using the small hepatitis B surface protein HBs, all samples could be grouped to genotype F. Sequencing of three complete Peruvian genomes showed that HBV from Peru belongs to subgenotype F1. Two of the genomes from HBeAg positive carriers coded surprisingly for a stop codon in the polymerase-ORF leading to a translational stop after 213 and 214 aa, respectively. The third isolate from an HBe Ag positive carrier had three deletions: aa 1–53 and aa 111–142 in preS. In addition nt. 2002–2087 in the HBc-ORF were deleted, leading to an HBc starting at aa 66.

The role of genotype mixture and subgenotypes remains controversial in determining the clinical outcome of chronic hepatitis B virus (HBV) infection. We aimed to determine their role on the development and the recurrence of hepatocellular carcinoma (HCC). HBV genotypes, serum viral load and hepatitis B e antigen (HBeAg) seroconversion were determined in 462 HCC patients, 234 chronic hepatitis patients and 425 asymptomatic carriers born in Eastern China. In the 462 HCC patients, 62 (13.4%), 337 (72.9%) and 49 (10.6%) had HBV subgenotype B2, C2 and genotype mixture, respectively. Genotype mixture in HCC patients and hepatitis patients was associated with higher viral load than HBV C2 (P = 0.012, P = 0.000) and more frequent than asymptomatic carriers (P = 0.005, P = 0.000). HBV C2 was more prevalent in HCC patients compared with controls. Proportion of HBV B2 in HCC patients decreased consecutively from <30 to 50-59 years group (P = 0.024). Age-related changes of HBeAg seroconversion were not consistent with serum viral load in HCC patients with HBV B2 and genotype mixture, quite in contrast to hepatitis patients. By multivariate regression analysis, age >or=40 years and serum viral load (>or=10 000 copies/ml) were independently associated with hepatocarcinogenesis, whereas age

There are eight genotypes of hepatitis B virus (HBV). Most genotypes can be further divided into subgenotypes. HBV genotypes influence the course of disease and treatment, and show geographic preferences. In Europe, A and D are the main genotypes found. Genotype A is more prevalent in northern Europe, where genotype D is mainly found in countries surrounding the Mediterranean Sea and in Eastern Europe. Subgenotype A2 is the dominant subgenotype in Europe, but the geographic prevalence of the four subgenotypes found in genotype D is not yet clear. On treatment with interferon HBV, genotype A, compared to genotype D, showsbetter virological response and in a large proportion of patients, even development of anti‐HBs. However, in the first year of treatment with lamivudine, higher rates of emergence of YMDD variants are observed in genotype A. This work summarizes the current knowledge on HBV genotypes in countries with low and intermediate HBV carriership from Europe.

Background: There are eight genotypes (A-H) and numerous subgenotypes of hepatitis B virus (HBV). The genotype has been shown to affect the course of HBV infection.Objectives: To develop an efficient genotyping and subgenotyping method for large-scale epidemiological surveys of HBV infection in countries with high prevalence of HBV B and C such as China.Study design: We designed genotype and subgenotype-specific primer pairs, and adjusted PCR conditions for a multiplex-PCR using common Taq polymerase to identify HBV genotypes A-F in one reaction and for the main subgenotypes B1/B2 and C1/C2 in another reaction.Results: We have developed a multiplex-PCR system, which specifically amplifies DNA of HBV genotypes and the corresponding main subgenotypes B and C from the sera of HBV patients. Our patients were infected with HBV of subgenotypes B2 (n = 18) C1 (n = 2) and C2 (n = 48). Eleven patients were doubly infected and three showed a triple infection with HBV A, B and C.Conclusions: The low-cost multiplex-PCR for identification of HBV genotypes A-F and main subgenotypes of HBV B and C, is rapid, reliable and sufficient for large-scale epidemiological surveys and clinical studies. (C) 2006 Elsevier B.V. All rights reserved.