A hepatitis B virus causes chronic infections in equids worldwide.

Preclinical testing of novel therapeutics for chronic hepatitis B (CHB) requires suitable animal models. Equids host homologs of hepatitis C virus (HCV). Because coinfections of hepatitis B virus (HBV) and HCV occur in humans, we screened 2,917 specimens from equids from five continents for HBV. We discovered a distinct HBV species (Equid HBV, EqHBV) in 3.2% of donkeys and zebras by PCR and antibodies against EqHBV in 5.4% of donkeys and zebras. Molecular, histopathological, and biochemical analyses revealed that infection patterns of EqHBV resembled those of HBV in humans, including hepatotropism, moderate liver damage, evolutionary stasis, and potential horizontal virus transmission. Naturally infected donkeys showed chronic infections resembling CHB with high viral loads of up to 2.6 × 109 mean copies per milliliter serum for >6 mo and weak antibody responses. Antibodies against Equid HCV were codetected in 26.5% of donkeys seropositive for EqHBV, corroborating susceptibility to both hepatitis viruses. Deltavirus pseudotypes carrying EqHBV surface proteins were unable to infect human cells via the HBV receptor NTCP (Na+/taurocholate cotransporting polypeptide), suggesting alternative viral entry mechanisms. Both HBV and EqHBV deltavirus pseudotypes infected primary horse hepatocytes in vitro, supporting a broad host range for EqHBV among equids and suggesting that horses might be suitable for EqHBV and HBV infections in vivo. Evolutionary analyses suggested that EqHBV originated in Africa several thousand years ago, commensurate with the domestication of donkeys. In sum, EqHBV naturally infects diverse equids and mimics HBV infection patterns. Equids provide a unique opportunity for preclinical testing of novel therapeutics for CHB and to investigate HBV/HCV interplay upon coinfection.

Splenic marginal zone lymphoma associated with hepatitis B virus infection, remission after viral treatment, and splenectomy: A case report and review of the literature.

We report the case of a patient diagnosed with a splenic marginal zone lymphoma with a simultaneous finding of hepatitis B virus infection, who responded to antiviral treatment and splenectomy. We highlighted this association described in the literature and its possible causal role, as well as the available therapeutic choices.

Evaluation of Hepatitis B Reactivation Among Patients With Chronic Myeloid Leukemia Treated With Tyrosine Kinase Inhibitors.

Hepatitis B reactivation (HBVr) in cancer patients is a well-established complication due to chemotherapy-induced immunosuppression. Studies have reported HBVr associated with immunosuppressive medications, such as rituximab, methotrexate, and high dose steroids. There are different risks for different types of chemotherapy with rituximab carrying one of the highest risks for hepatitis B reactivation. Tyrosine kinase inhibitors (TKIs) are the standard of care in patients with chronic myeloid leukemia (CML). The risk of HBVr in chronic myeloid leukemia has been reported in many studies, but to this date, there are no clear guidelines or recommendations regarding screening and monitoring of HBV in CML patients receiving TKIs. We conducted this review to identify the risk of HBVr in patients with CML who are treated with tyrosine kinase inhibitors. We recommend testing for HBV status in patients who are to be treated with TKIs and to consider giving prophylaxis in those who are positive for HBsAg at baseline. More studies are needed to assess the risk of reactivation in patients with Hepatitis B core antibody positive receiving TKIs. Currently, monitoring such patients for reactivation may be the best strategy.

Prevalence of hepatitis B and C virus co-infection in HIV positive patients attending a health institution in southeast Nigeria.

BACKGROUND: The health of people living with HIV/AIDS becomes progressively worse when co-infected with hepatitis B virus (HBV) and hepatitis C virus (HCV), resulting in shortened life span. The modes of transmission of HIV, HBV and HCV are similar. OBJECTIVE: To determine the prevalence of HBV and HCV co-infection in HIV patients. METHOD: This was a retrospective study of serology test results for hepatitis B surface antigen (HBsAg) and antibodies to HCV (anti-HCV) of HIV positive patients registered from 2008-2013 (6years) at the University of Nigeria Teaching Hospital. Adult patients with confirmed HIV seropositivity were included. Ethical approval was obtained and confidentiality of the patient information was maintained. Laboratory records were reviewed to obtain HBsAg, anti-HCV, and CD4 T-lymphocyte results. Prevalence was determined by the number of positive results over total number of patients tested. Chi-square test was used to determine relationships and p<0.05 was considered to be statistically significant. RESULTS: 4663 HIV patient records were included comprising 3024 (65%) females and 1639 (35%) males. Serology results showed 365/4663 (7.8%) tested HBsAg-positive only; 219/4663 (4.7%) tested anti-HCV-positive only; and 27/4663 (0.58%) tested both HBsAg and anti-HCV-positive. Correlation of age and sex were statistically significant with HBV and HCV (p<0.05) but not CD4 count (p>0.05). CONCLUSION: HBV co-infection was more prevalent than HCV, and triple infection was also observed. Screening for these viral infections in the HIV population is necessary for early identification to enable appropriate, holistic management of these patients.

Universal hepatitis B screening and management in patients with cancer who received immunosuppressive chemotherapy.

BACKGROUND: Clinical data to guide management of patients with cancer and hepatitis B virus (HBV) infection who are treated with immunosuppressive chemotherapy are lacking. The purpose of this study was to describe HBV+ rates in a population of patients with cancer and evaluate a risk-stratified management protocol for the prevention of HBV reactivation (HBVr). METHODS: This was a descriptive study conducted in an integrated healthcare delivery system. Patients with cancer and hepatitis B virus infection who received immunosuppressive chemotherapy between 1 January 2014 and 31 January 2016 were included. A risk-stratified management protocol that continued for six months after chemotherapy completion or 12 months after completion of B-cell targeted chemotherapy was assessed. Outcomes included the proportion of patients who were HBV+ and amongst patients who initiated immunosuppressive therapy, proportions who received hepatitis B virus monitoring or anti-hepatitis B virus prophylaxis, or experienced HBVr or hepatitis B virus-related complications. RESULTS: There were 2463 patients with cancer screened for hepatitis B virus with 114 (4.6%) HBV+ of whom 59 (51.8%) initiated chemotherapy. Included patients were primarily older, male, and white with gastrointestinal or hematologic cancers and initiated intermediate/low-risk cytotoxic chemotherapy. During follow-up, 41 (69.5%) received hepatitis B virus DNA monitoring and 17 (28.8%) initiated anti-hepatitis B virus prophylaxis. No HBVr was observed. ALT and AST abnormalities were common but mostly Grade 1 and primarily related to the patient's malignancy or medications. CONCLUSIONS: Universal hepatitis B virus screening coupled with a risk-stratified management strategy utilizing HBVr monitoring and anti-hepatitis B virus prophylaxis in HBV+ patients receiving immunosuppressive chemotherapy for cancer may prevent HBVr.

Altered oral microbiota in chronic hepatitis B patients with different tongue coatings.

AIM: To elucidate tongue coating microbiota and metabolic differences in chronic hepatitis B (CHB) patients with yellow or white tongue coatings. METHODS: Tongue coating samples were collected from 53 CHB patients (28 CHB yellow tongue coating patients and 25 CHB white tongue coating patients) and 22 healthy controls. Microbial DNA was extracted from the tongue samples, and the bacterial 16S ribosomal RNA gene V3 region was amplified from all samples and sequenced with the Ion Torrent PGM™ sequencing platform according to the standard protocols. The metabolites in the tongue coatings were evaluated using a liquid chromatography-mass spectrometry (LC-MS) platform. Statistical analyses were then performed. RESULTS: The relative compositions of the tongue coating microbiotas and metabolites in the CHB patients were significantly different from those of the healthy controls, but the tongue coating microbiota abundances and diversity levels were not significantly different. Compared with the CHB white tongue coating patients, the CHB yellow tongue coating patients had higher hepatitis B viral DNA (HBV-DNA) titers (median 21210 vs 500, respectively, P = 0.03) and a significantly lower level of Bacteroidetes (20.14% vs 27.93%, respectively, P = 0.013) and higher level of Proteobacteria (25.99% vs 18.17%, respectively, P = 0.045) in the microbial compositions at the phylum level. The inferred metagenomic pathways enriched in the CHB yellow tongue coating patients were mainly those involved in amino acid metabolism, which was consistent with the metabolic disorder. The abundances of bacteria from Bacteroidales at the order level were higher in the CHB white tongue coating patients (19.2% vs 27.22%, respectively, P = 0.011), whereas Neisseriales were enriched in the yellow tongue coating patients (21.85% vs 13.83%, respectively, P = 0.029). At the family level, the abundance of Neisseriaceae in the yellow tongue patients was positively correlated with the HBV-DNA level but negatively correlated with the S-adenosyl-L-methionine level. CONCLUSION: This research illustrates specific clinical features and bacterial structures in CHB patients with different tongue coatings, which facilitates understanding of the traditional tongue diagnosis.

Beneficial Effects of Bushen Formula Combined with Enticavir on Chronic Hepatitis B Patients with Suboptimal Response to Enticavir by Regulating B-Cell Differentiation.

BACKGROUND/AIMS: To investigate the clinical effects of the combination therapy with Bushen Formula (BSF) plus enticavir (ETV) on chronic hepatitis B (CHB) patients with suboptimal response to ETV and explore the regulatory mechanisms of BSF on B cells-mediated humoral immunity. METHODS: Sixty-four HBeAg-positive CHB patients with suboptimal response to ETV were enrolled, and were randomly assigned into control group (C-Group, placebo combined with ETV for 12 months) or treatment group (T-Group, BSF combined with ETV for 12 months). Serum samples from 57 treatment-naïve CHB patients and 15 healthy controls were collected. Serum HBV DNA levels were evaluated by real-time PCR. Characteristics of peripheral blood B-cell subtypes were analyzed by flow cytometry. Serum HBV markers and B cell-activating factor (BAFF) levels were detected by ELISA. Chinese medicine symptom complex score was evaluated and recorded. RESULTS: After treatment, the rates of patients with a reduction of HBsAg > 0.5 log10 IU/ml or 1.0 log10 IU/ml and the rates of HBeAg clearance in T-Group were all higher than those in C-group, with no significant intergroup difference. Only in T-Group, Chinese medicine symptom complex score and the frequency of total B cells were significantly decreased, and the frequencies of Bm1, CD24+CD27-switched B cells and plasma cells were markedly increased after treatment compared with those before treatment. Compared with healthy controls, serum BAFF levels in treatment-naïve CHB patients were increased, and there was a significant positive correlation between serum BAFF and HBsAg levels. However, serum BAFF levels did not differ after treatment in T-Group and C-Group. CONCLUSIONS: The combination therapy with BSF plus ETV promotes the reduction of HBsAg level and the clearance of HBeAg in CHB patients with partial response to ETV through regulating the differentiation of B-cell subsets.

Ancient hepatitis B viruses from the Bronze Age to the Medieval period.

Hepatitis B virus (HBV) is a major cause of human hepatitis. There is considerable uncertainty about the timescale of its evolution and its association with humans. Here we present 12 full or partial ancient HBV genomes that are between approximately 0.8 and 4.5 thousand years old. The ancient sequences group either within or in a sister relationship with extant human or other ape HBV clades. Generally, the genome properties follow those of modern HBV. The root of the HBV tree is projected to between 8.6 and 20.9 thousand years ago, and we estimate a substitution rate of 8.04 × 10-6-1.51 × 10-5 nucleotide substitutions per site per year. In several cases, the geographical locations of the ancient genotypes do not match present-day distributions. Genotypes that today are typical of Africa and Asia, and a subgenotype from India, are shown to have an early Eurasian presence. The geographical and temporal patterns that we observe in ancient and modern HBV genotypes are compatible with well-documented human migrations during the Bronze and Iron Ages1,2. We provide evidence for the creation of HBV genotype A via recombination, and for a long-term association of modern HBV genotypes with humans, including the discovery of a human genotype that is now extinct. These data expose a complexity of HBV evolution that is not evident when considering modern sequences alone.

Social-cultural, traditional beliefs, and health system barriers of hepatitis B screening among Hmong Americans: A case study.

BACKGROUND: The incidence of liver cancer in Hmong Americans is 5 times higher than that of non-Hispanic whites, and there is a low hepatitis B screening rate (24%) among Hmong adults compared with other Asian American populations. The purpose of this study was to examine the Hmong's perceptions on social-cultural determinants, traditional health beliefs, and health care system barriers that influenced community-based hepatitis B screening interventions. METHODS: A qualitative method was used, integrating a collective case study research design. In-depth interviews were used to collect data from 20 Hmong adults from the greater Sacramento area. A pattern matching analytic technique was used to analyze the data. The main core elements of Culture Care Theory were used to capture the key themes presented by the participants. RESULTS: Protecting a family's reputation; fear of doctors, medical procedures, and test results; lack of trust in medical doctors and medical care services; and using Hmong herbal medicines and practicing spiritual healing were identified as social-cultural and traditional health belief barriers to obtaining HBV screening. Health care costs, perceived discrimination, lack of transportation, linguistic discordance, and poor quality of care were identified as barriers to accessing high-quality health care services and obtaining hepatitis B screening. CONCLUSION: Providers, health policy makers, researchers, and community-based organizations will need to work together to develop intervention strategies to address the social-cultural factors, traditional health beliefs, and health care challenges that influence obtaining hepatitis B screening in the Hmong community. Cancer 2018;124:1576-82. © 2018 American Cancer Society.

Novel stable HBV producing cell line systems for expression and screening antiviral inhibitor of hepatitis B virus in human hepatoma cell line.

Chronic hepatitis B virus (HBV) infection is currently a major public health burden. Therefore, there is an urgent need for the development of novel antiviral inhibitors. The stable HBV-producing cell lines of genotype D are widely used to investigate the HBV life cycle and to evaluate antiviral agents. However, stable HBV-producing cell lines of different genotypes do not exist. To construct more convenient and efficient novel cell systems, stable cell lines of genotypes A, B, and C were established using a full-length HBV genome sequence isolated from chronic HBV patients in human hepatoma HepG2 cells. Novel HBV clones were identified and stable HBV-producing cell lines derived from these clones were constructed. HBV replication activities demonstrated time-dependent expression, and the novel cell lines were susceptible to several antiviral inhibitors with no cytotoxicity. Furthermore, infectious viruses were produced from these cell lines. In conclusion, we have established novel stable HBV-producing cell line systems of genotypes A, B, and C. These systems can provide valuable tools for screening antiviral agents and analyzing viral phenotypes in vitro.

Vitamin D deficiency and hepatitis viruses-associated liver diseases: A literature review.

The secosteroid hormone vitamin D has, in addition to its effects in bone metabolism also functions in the modulation of immune responses against infectious agents and in inhibiting tumorigenesis. Thus, deficiency of vitamin D is associated with several malignancies, but also with a plethora of infectious diseases. Among other communicable diseases, vitamin D deficiency is involved in the pathogenesis of chronic liver diseases caused by hepatitis B and C viruses (HBV, HCV) and high prevalence of vitamin D deficiency with serum levels below 20 mg/mL in patients with HBV and HCV infection are found worldwide. Several studies have assessed the effects of vitamin D supplementation on the sustained virological response (SVR) to interferon (IFN) plus ribavirin (RBV) therapy in HBV and HCV infection. In these studies, inconsistent results were reported. This review addresses general aspects of vitamin D deficiency and, in particular, the significance of vitamin D hypovitaminosis in the outcome of HBV- and HCV-related chronic liver diseases. Furthermore, current literature was reviewed in order to understand the effects of vitamin D supplementation in combination with IFN-based therapy on the virological response in HBV and HCV infected patients.

Effect of modified Xiaochaihu decoction­containing serum on HepG2.2.15 cells via the JAK2/STAT3 signaling pathway.

The present study aimed to investigate the possible mechanisms underlying the effect of modified Xiaochaihu decoction (mXCHD) in the treatment of chronic hepatitis B (CHB). Patients with CHB, in addition to liver stagnation and spleen deficiency syndrome were randomly assigned to receive either Chinese (mXCHD) or western (entecavir) treatment, with 30 cases in each group. Serum was collected following treatment with mXCHD or entecavir for 7 days. A healthy group of 30 individuals was also included. HepG2.2.15 cells were cultured in vitro and randomly divided into four groups: Healthy; entecavir­treated; 10% mXCHD­treated; and 20% mXCHD­treated. The HepG2.2.15 cells in the four groups were treated with either serum from the healthy volunteers, entecavir­containing serum, or mXCHD­containing serum at different concentrations (10 or 20%, respectively). Following treatment with the corresponding serum, cell proliferation was examined using an MTT assay, and the expression of hepatitis B surface antigen (HBsAg) in the cell supernatant was detected using an enzyme­linked immunosorbent assay. The mRNA and protein expression levels of Janus kinase (JAK)2 and signal transducer and activator of transcription (STAT)3 were measured using reverse transcription­quantitative polymerase chain reaction and western blot analyses, respectively. The results indicated that the most effective treatment for the promotion of HepG2.2.15 cell proliferation was a 20% concentration of mXCHD serum. The expression of HBsAg was significantly decreased in the groups treated with 10 and 20% mXCHD 48 h following intervention (P<0.01). The mRNA and protein expression levels of STAT3 in the 20% mXCHD serum group were significantly increased, compared with those in the healthy group (P<0.01 and P<0.05, respectively), whereas no significant difference was observed in the expression of JAK2 among the four groups. These results indicated that mXCHD suppressed the hepatitis B virus, and treatment of the cells with mXCHD­containing serum promoted HepG2.2.15 cell proliferation via modulating the expression of STAT3, which may contribute to the clinical efficacy of mXCHD against CHB.

Prevalence of Human Immunodeficiency Virus, Hepatitis C Virus, and Hepatitis B Virus Among Homeless and Nonhomeless United States Veterans.

BACKGROUND: Veterans are disproportionately affected by human immunodeficiency virus (HIV), hepatitis C virus (HCV), and hepatitis B virus (HBV). Homeless veterans are at particularly high risk for HIV, HCV, and HBV due to a variety of overlapping risk factors, including high rates of mental health disorders and substance use disorders. The prevalence of HIV, HCV, and HBV among homeless veterans nationally is currently unknown. This study describes national testing rates and prevalence of HIV, HCV, and HBV among homeless veterans. METHODS: Using data from the Department of Veterans Affairs (VA) Corporate Warehouse Data from 2015, we evaluated HIV, HCV, and HBV laboratory testing and infection confirmation rates and diagnoses on the Problem List for nonhomeless veterans and for veterans utilizing homeless services in 2015. RESULTS: Among 242740 homeless veterans in VA care in 2015, HIV, HCV, and HBV testing occurred in 63.8% (n = 154812), 78.1% (n = 189508), and 52.8% (n = 128262), respectively. The HIV population prevalence was 1.52% (3684/242740) among homeless veterans, compared with 0.44% (23797/5424685) among nonhomeless veterans. The HCV population prevalence among homeless veterans was 12.1% (29311/242740), compared with 2.7% (148079/5424685) among nonhomeless veterans, while the HBV population prevalence was 0.99% (2395/242740) for homeless veterans and 0.40% (21611/5424685) among nonhomeless veterans. CONCLUSIONS: To our knowledge this work represents the most comprehensive tested prevalence and population prevalence estimates of HIV, HCV, and HBV among homeless veterans nationally. The data demonstrate high prevalence of HIV, HCV, and HBV among homeless veterans, and reinforce the need for integrated healthcare services along with homeless programming.

Comparison of Detection Rate and Mutational Pattern of Drug-Resistant Mutations Between a Large Cohort of Genotype B and Genotype C Hepatitis B Virus-Infected Patients in North China.

The study aimed to investigate the association of prevalent genotypes in China (HBV/C and HBV/B) with HBV drug-resistant mutations. A total of 13,847 nucleos(t)ide analogue (NA)-treated patients with chronic HBV infection from North China were enrolled. HBV genotypes and resistant mutations were determined by direct sequencing and confirmed by clonal sequencing if necessary. HBV/B, HBV/C, and HBV/D occupied 14.3%, 84.9%, and 0.8% across the study population, respectively. NA usage had no significant difference between HBV/B- and HBV/C-infected patients. Lamivudine-resistant mutations were more frequently detected in HBV/C-infected patients, compared with HBV/B-infected patients (31.67% vs. 25.26%, p < 0.01). Adefovir- and entecavir-resistant mutation detection rates were similar, but the mutational pattern was different between the two genotypes. For adefovir-resistant mutations, HBV/C-infected patients had a higher detection rate of rtA181 V (HBV/C 5.29% vs. HBV/B 1.36%, p < 0.01) and a lower detection rate of rtN236T (2.70% vs. 6.54%, p < 0.01). For entecavir-resistant mutations, HBV/C-infected patients had a higher detection rate of rtM204 V/I+T184 substitution or S202G/C (3.66% vs. 2.16%, p < 0.01) and a lower detection rate of rtM204 V/I+M250 V/I/L substitution (0.67% vs. 1.46%, p < 0.01). Multidrug-resistant mutations (defined as coexistence of mutation to nucleoside and nucleotide analogues) were detected in 104 patients. HBV/C-infected patients had a higher detection rate of multidrug-resistant mutation than HBV/B-infected patients (0.83% vs. 0.35%, p < 0.05). The study for the first time clarified that HBV/C-infected patients had a higher risk to develop multidrug-resistant mutations, compared with HBV/B-infected patients; and HBV/C- and HBV/B-infected patients had different inclinations in the ETV-resistant mutational pattern.

[Spleen, liver and kidney-strengthening formula combined with polyethylene glycol interferon in treatment of chronic hepatitis B].

To observe the clinical efficacy of spleen, liver and kidney-strengthening formula combined with polyethylene glycol interferon in the treatment of HBeAg positive chronic hepatitis B(HP-HBV).One hundred and twenty-six patients with HP-HBV, who were treated in the hospital from June 2012 to December 2014, were selected and injected with polyethylene glycol interferon α-2a(or α-2b). The treatment course for the patients lasted for 24 weeks. Base on the level of HBV-DNA, patients are divided into response group and poor response group. According to random number table, the poor response group were randomized into control group and test group. Patients in the control group were injected with polyethylene glycol interferon α-2a(or α-2b), and patients in the test group were treated with spleen, liver and kidney-strengthening formula combined with polyethylene glycol interferon. Clinical efficacies of the 2 groups were observed, and changes in the level of HBeAg, ALT and HBV-DNA were observed before treatment and at the 24th week after treatment, and virological and serological response, biochemical responses, integral clinical symptoms and signs, adverse reactions were observed after 48 weeks of treatment.After 24 weeks of treatment, the response group was significantly better than the poor response group in HBeAg, ALT and the level of HBV-DNA(P<0.05). After 48 weeks of treatment, there was statistical significance in HBV-DNA negative conversion rate, HBeAg negative conversion rate between the 2 groups(P<0.05), and the test group was better in the two indicators. And the test group was significantly lower than the control group in clinical symptoms and signs score at the 48th week after treatment(P<0.05), with a significantly lower adverse reaction rate than the control group(P<0.05).Combination of spleen, liver and kidney-strengthening formula and polyethylene glycol interferon α-2a was effective and safe in the treatment of chronic hepatitis B, and so worth promoting in clinic.

Two-year treatment outcome of chronic hepatitis B infection treated with besifovir vs. entecavir: results from a multicentre study.

BACKGROUND & AIMS: We aimed to compare the viral suppression, safety and rate of drug resistance between besifovir (a new acyclic nucleotide analogue) and entecavir. METHODS: Treatment-naïve chronic hepatitis B patients receiving besifovir 90 mg (n=31), 150 mg (n=28) and entecavir 0.5 mg (n=30) were monitored for liver biochemistry, viral serology, HBV DNA levels, development of drug resistance mutations, and adverse events throughout 96 weeks of treatment. RESULTS: The mean decline of HBV DNA levels from baseline to week 96 were 5.29, 5.15, and 5.67 logs IU/ml for patients receiving besifovir 90 mg, 150 mg and entecavir 0.5 mg, respectively (p>0.05). Undetectable HBV DNA (<20 IU/ml) were achieved in 80.7%, 78.6%, and 80%; ALT normalization in 90.3%, 78.6%, and 93.3%; and loss of HBeAg in 20%, 21.4%, and 22.2% of patients respectively (all p>0.05). One patient receiving besifovir 90 mg had a virological breakthrough due to drug non-compliance. No patient developed drug resistance mutations. Ten patients had serious adverse events, which were not related to the study medications. The most common side effect related to besifovir was carnitine depletion. Carnitine supplements were prescribed to 83.9% and 100% of patients, who had low carnitine level for any one time during follow-up, receiving besifovir 90 mg and 150 mg respectively. No patient had increased creatinine>0.5 mg/dl from baseline. CONCLUSIONS: Besifovir had the same antiviral property as compared to entecavir over 96 weeks of treatment for chronic hepatitis B patients. Besifovir was well tolerated and also had a good clinical safety profile.

Rapid detection of hepatitis B virus variants associated with lamivudine and adefovir resistance by multiplex ligation-dependent probe amplification combined with real-time PCR.

Drug-resistant mutations of hepatitis B virus (HBV) are the major obstacles to successful therapy for chronic hepatitis B infection. Although there are many methods for detecting the antiviral drug-resistant mutations of HBV, their applications are restricted because of their shortcomings, such as low sensitivity, the time required, and the high cost. For this study, a multiplex ligation-dependent probe real-time PCR (MLP-RT-PCR) method was developed to simultaneously detect lamivudine (LAM)- and adefovir (ADV)-resistant HBV mutants (those with the mutations rtM204V/I, rtA181V/T, and rtN236T). The new method combined the high-throughput nature of multiplex ligation-dependent probe amplification (MLPA) with the rapid and sensitive detection of real-time PCR. In this report, MLP-RT-PCR was evaluated by detecting drug-resistant mutants in 116 patients with chronic hepatitis B infection. By MLP-RT-PCR analysis, LAM-resistant mutations were detected in 41 patients (35.3%), ADV-resistant mutations were detected in 17 patients (14.7%), and LAM- and-ADV-resistant mutations were detected in 5 patients (4.3%). Based on the results of MLP-RT-PCR, the mutations rtM204V, rtM204I, rtA181T, rtA181V, and rtN236T were 95.7% (111/116 patients), 98.3% (114/116 patients), 99.1% (115/116 patients), 98.3% (114/116 patients), and 99.1% (115/116 patients) concordant, respectively, with those of direct sequencing. The MLP-RT-PCR assay was more sensitive than direct sequencing for detecting mutations with low frequencies. Four samples containing the low-frequency (<10%) mutants were identified by MLP-RT-PCR and further confirmed by clonal sequencing. MLP-RT-PCR is a rapid and sensitive method that enables the detection of multidrug-resistant HBV mutations in clinical practice.

Integrated chip electrophoresis and magnetic particle isolation used for detection of hepatitis B virus oligonucleotides.

Rapid and sensitive detection is a key step in the effective and early response to the global hazard of various viral diseases. In this study, an integrated isolation of hepatitis B virus (HBV)-specific DNA fragment by magnetic nanoparticles (MNPs) and its immediate analysis by microchip CGE was performed. Microfluidic CE chip was used to accommodate the complete process of viral DNA isolation by MNPs including hybridization and thermal denaturation followed by CE separation. Beforehand, calibration curves of HBV fragments were constructed. For isolation by MNPs, specific streptavidin-biotin interaction was used to bind complementary HBV fragment to magnetic particles. After analysis of isolated HBV by regular MNPs method, innovative approach was performed. The commercial CE chip (Bio-rad) was successfully used to execute HBV fragment isolation. Detection using LIF with detection limit of 1 ng/mL was accomplished.

Screening and identification of compounds with antiviral activity against hepatitis B virus using a safe compound library and novel real-time immune-absorbance PCR-based high throughput system.

There are now seven nucleoside/tide analogues, along with interferon-α, that are approved by the FDA for the management of chronic hepatitis B virus (HBV) infection, a disease affecting hundreds of millions of people worldwide. These medications, however, are limited in usefulness, and significant side effects and the emergence of viral escape mutants make the development of novel and updated therapeutics a pressing need in the treatment of HBV. With this in mind, a library containing 2000 compounds already known to be safe in both humans and mice with known mechanisms of action in mammalian cells were tested for the possibility of either antiviral activity against HBV or selective toxicity in HBV producing cell lines. A modified real-time immune-absorbance-polymerase chain reaction (IA-PCR) assay was developed for this screen, utilizing cells that produce and secrete intact HBV virions. In this procedure, viral particles are first captured by an anti-HBs antibody immobilized on a plate. The viral load is subsequently assessed by real-time PCR directly on captured particles. Using this assay, eight compounds were shown to consistently reduce the amount of secreted HBV viral particles in the culture medium under conditions that had no detectable impact on cell viability. Two compounds, proparacaine and chlorophyllide, were shown to reduce HBV levels 4- to 6-fold with an IC50 of 1 and 1.5 µM, respectively, and were selected for further study. The identification of these compounds as promising antiviral drug candidates against HBV, despite a lack of previous recognition of HBV antiviral activity, supports the validity and utility of testing known compounds for "off-pathogen target" activity against HBV, and also validates this IA-PCR assay as an important tool for the detection of anti-viral activity against enveloped viruses.

Extraction and purification of hepatitis B virus-like M particles from a recombinant Saccharomyces cerevisiae strain using alumina powder.

A recombinant hepatitis B surface antigen (HBsAg) has been produced in the yeast Saccharomyces cerevisiae and used as a vaccine against hepatitis B virus (HBV) infection. The present study aimed to optimize the extraction of recombinant virus-like particles (rVLPs) to develop a simple purification procedure based on gel filtration and high performance size-exclusion chromatography. The findings showed that disruption of yeast cells with alumina powder increased the yield of the total proteins (290mg/l) and rVLPs (1mg/l) compared to the values for glass beads (171mg/l and 0.5mg/l), as estimated by quantitative ELISA. The purification of rVLPs was performed by two consecutive gel filtration chromatographic steps, namely Sephacryl S-200 followed by SEC-250 HPLC. The purified M protein was analyzed by atomic force microscopy and shown to assemble in particles that were able to recognize HBV antibodies in the sera of patients with chronic hepatitis B, providing evidence for their immunoreactivity.