Comparative analysis of the gut microbiota of wild adult rats from nine district areas in Hainan, China.

BACKGROUND: Wild rats have the potential to hold zoonotic infectious agents that can spread to humans and cause disease. AIM: To better understand the composition of gut bacterial communities in rats is essential for preventing and treating such diseases. As a tropical island located in the south of China, Hainan province has abundant rat species. Here, we examined the gut bacterial composition in wild adult rats from Hainan province. METHODS: Fresh fecal samples were collected from 162 wild adult rats, including three species (Rattus norvegicus, Leopoldamys edwardsi, and Rattus losea), from nine regions of Hainan province between 2017-2018. RESULTS: We analyzed the composition of gut microbiota using the 16S rRNA gene amplicon sequencing. We identified 4903 bacterial operational taxonomic units (30 phyla, 175 families, and 498 genera), which vary between samples of different rat species in various habitats at various times of the year. In general, Firmicutes were the most abundant phyla, followed by Bacteroidetes (15.55%), Proteobacteria (6.13%), and Actinobacteria (4.02%). The genus Lactobacillus (20.08%), unidentified_Clostridiales (5.16%), Romboutsia (4.33%), unidentified_Ruminococcaceae (3.83%), Bacteroides (3.66%), Helicobacter (2.40%) and Streptococcus (2.37%) were dominant. CONCLUSION: The composition and abundance of the gut microbial communities varied between rat species and locations. This work provides fundamental information to identify microbial communities useful for disease control in Hainan province.

Influence of the basal core promoter and precore mutation on replication of hepatitis B virus and antiviral susceptibility of different genotypes.

Mutations in the basal core promoter (BCP) and precore (PC) regions of the hepatitis B virus (HBV) are more common in genotypes B and C than in genotype A, suggesting that these mutations might affect replication competency depending on genotype. The purpose of the study was to investigate the influence of these mutations on the capacity of HBV for replication and antiviral drug susceptibility according to genotype. Genotypes A, B, and C of HBV strains with a BCP mutation, PC mutation, or BCP + PC mutation were made by site-directed mutagenesis. Replication competency of each construct and susceptibility to nucleos(t) ide analogues were tested in an Huh7 cell line. In genotype A, the BCP and BCP + PC mutations increased the viral replication around 6.5 times compared with the wild type, and the PC mutation alone similarly increased the viral replication around three times. In genotypes B and C, all three mutant types increased viral replication to a similar extent, regardless of mutation pattern. Interestingly, the BCP mutation appeared to have a greater effect on viral replication in genotype A than in genotypes B and C. This finding was unexpected because the BCP mutation is more common in HBV genotypes B and C. Moreover, the BCP, PC, and BCP + PC mutations decreased the sensitivity of HBV to antiviral agents to various degrees (2- to 10-fold) regardless of genotype. In conclusion, BCP and PC mutations increased viral replication regardless of HBV genotype and decreased in vitro antiviral susceptibility to the nucleos(t) ide analogues.

Different mechanism of selection of adefovir-resistant mutant viruses during adefovir monotherapy in patients with lamivudine-resistant chronic hepatitis B.

BACKGROUND: Adefovir (ADV) resistance is more frequent in lamivudine (LMV)-resistant chronic hepatitis B (CHB) patients than in nucleos(t)ide analogue-naïve patients. The majority of LMV-resistant mutants harbor the rtM204V/I mutation, while a minor fraction harbor the rtA181V/T mutation. We aimed to elucidate the mechanism of the high rate of ADV resistance in LMV-resistant patients during ADV therapy. METHODS: We performed a clonal analysis of HBV reverse transcriptase in treatment-naïve (n = 3) and LMV-resistant patients before ADV therapy (n = 14). Dynamic changes in the viral population (n = 9) during ADV therapy were also analyzed. RESULTS: Before ADV therapy, rtA181V/T was observed in 30 of 680 clones (4.4%) from 7 patients with LMV resistance under dominant rt204V/I mutation and in one of 150 clones in treatment-naïve patients. The rtA181V/T mutation was more frequently found in clones from LMV-resistant patients than in treatment-naïve patients (p = 0.029). The rtN236T mutation was not observed in any clone. During ADV therapy, most rtM204V/I mutants were replaced by wild type in all 3 patients without the rtA181V/T mutation and in one patient with the rtA181V/T mutation. Subsequently, wild type was replaced by the rtN236T and/or rtA181V/T mutant. In patients with the rtA181V/T mutation (n = 6), the rtA181V/T mutant overtook the rtM204V/I mutant in 3 of 4 patients with ADV resistance. In 2 patients without ADV resistance, most of the viral population was replaced by wild type by the last follow-up. CONCLUSION: The high rate of ADV resistance in patients with LMV-resistance might be attributable to preexisting rtA181V/T mutant virus.

Response to adefovir depends on mutation patterns in precore region, basal core promoter and reverse transcriptase, and on-treatment responses in Lamivudine-resistant chronic hepatitis B patients.

OBJECTIVE: In vitro studies showed that mutations in the basal core promoter (BCP) or precore (PC) region restore the replication inefficiency of the lamivudine-resistant mutant. The aim of this study was to clarify the effect of molecular characteristics on the antiviral response to adefovir in patients with lamivudine-resistant chronic hepatitis B (CHB). METHODS: Sixty-six lamivudine-resistant patients who were treated with adefovir monotherapy were studied. Sequences of BCP, PC region and reverse transcriptase were determined before adefovir therapy. In patients with virologic breakthrough, reverse transcriptase sequencing was performed. RESULTS: The cumulative probabilities of virologic response were 23.3, 46, 52.7 and 59.5% at years 1, 2, 3 and 4, respectively. PC mutation, the absence of compensatory mutations (rtL80I/V or rtV173L), and a decrease in serum hepatitis B virus (HBV) DNA by 3 log or greater at 6 months were independent predictors of virologic response. The cumulative probabilities of virologic breakthrough were 0, 12.9, 30.7 and 44.5% at years 1, 2, 3 and 4, respectively. BCP mutation and a less than 3 log decrease in serum HBV DNA at 6 months were 2 independent risk factors for virologic breakthrough. CONCLUSION: Response to adefovir depends on mutation patterns in the BCP, PC region and reverse transcriptase, and on-treatment decreases in serum HBV DNA in lamivudine-resistant CHB patients.

Molecular characteristics and functional analysis of full-length hepatitis B virus quasispecies from a patient with chronic hepatitis B virus infection.

It has been hypothesized that naturally occurring mutations in HBV may play a role in the pathogenesis of HBV-related disease. We determined the molecular characteristics of naturally occurring HBV isolates and performed a functional analysis of full-length hepatitis B virus quasispecies from a patient with chronic hepatitis B. The 10 HBV clones isolated were identified as HBV genotype B4 and subtype adw. In most clones, amino acid substitutions and nucleotide changes occurred in a specific region of the core protein, X protein and in the core promoter. In the core protein, cI3L, cL60M, and cI97L were detected in 8 of 10 clones and cP130T was detected in all 10 clones. In the X protein, xI127M was detected in 5 clones. In the basal core promoter, the A1762T/G1764A mutation was found only in 1 clone. An 11-bp nucleotide insertion between nucleotides 1772 and 1773, was found in 2 clones. Six clones that were replication-competent exhibited variation in the level of replicating capacity in vitro even though the average genetic distance between the HBV clones was only 0.5% (range: 0.3-0.7%). Among the replication-competent 6 clones, 5 clones showed higher replication competency compared with clone B9 (reference clone used in this study) in Huh7 cells. However, 4 clones showed lower replication competency compared with clone B9 in HepG2 cells. In conclusion, the HBV virus exhibits genetic variation in the form of quasispecies with different mutation patterns, and these quasispecies may be recognized by distinct viral replication patterns even in patients with subtle genetic mutation.

New scoring system for predicting relapse after lamivudine-induced hepatitis B e-antigen loss in chronic hepatitis B patients.

AIM: In endemic areas of hepatitis B virus (HBV) infection, lamivudine-induced hepatitis B e-antigen (HBeAg) loss is not durable. We developed a scoring system to predict the relapse after lamivudine-induced HBeAg loss. METHODS: Of 93 HBeAg-positive chronic hepatitis B patients receiving lamivudine therapy, we studied the 30 patients who achieved HBeAg loss. Qualitative polymerase chain reaction assays for HBV DNA were performed after HBeAg loss; if results were negative on two consecutive occasions, lamivudine treatment was stopped. Serum HBV DNA levels at the time of HBeAg loss were determined using stored serum by the Cobas Amplicor HBV Monitor Kit. RESULTS: Cumulative relapse rates were 44%, 55.8% and 59.8%, after 1, 2 and 5 years, respectively. Univariate analysis showed that the risk factors for relapse were age (>/= 30 years), HBV DNA level (>/= 60 IU/mL) at the time of HBeAg loss and liver cirrhosis. Independent risk factors for relapse were age (odds ratio [OR], 4.3; 95% confidence interval [CI], 1.2-17.6) and serum HBV DNA level at the time of HBeAg loss (OR, 5.1; 95% CI, 1.3-21.6). We developed a scoring system based on these independent risk factors. No relapse was observed for patients with a risk score of 0. For patients with a risk score of 1, the relapse rates were 41%, 49% and 56% at 1, 2 and 5 years, respectively. All patients with a risk score of 2 relapsed (P = 0.01). CONCLUSION: Our new scoring system may be useful for predicting relapses in patients with lamivudine-induced HBeAg loss. Treatment strategies should be individualized according to risk score.

Sequential accumulation of the basal core promoter and the precore mutations in the progression of hepatitis B virus-related chronic liver disease.

OBJECTIVES: Despite the pathogenic role of the basal core promoter (BCP) and the precore mutations in chronic hepatitis B virus (HBV) infection, their role in the progression of liver disease is still controversial. We analyzed whether the accumulation of these mutations might enhance the progression of HBV-related chronic liver disease. METHODS: Forty consecutive patients at each clinical status were analyzed. Clinical statuses were as follows: HBeAg-positive asymptomatic carrier (HBeAg(+) ASC) (defined as HBeAg(+), anti-HBe(-), HBV-DNA(+) by hybridization, normal ALT); inactive HBsAg carrier; chronic hepatitis B; liver cirrhosis. The genotype and the BCP/precore regions were determined by PCR using genotype specific primers and direct sequencing, respectively. RESULTS: All patients except one were infected with genotype C. The A to T mutation at nucleotide 1762 and/or G to A mutation at nucleotide 1764 were found in 30% in HBeAg(+) ASC, 65.7% in inactive HBsAg carrier, 95% in chronic hepatitis B, and 90% in liver cirrhosis (p < 0.001). The prevalence of the G to A mutation at nucleotide 1896 was 5% in HBeAg(+) ASC, 22.5% in inactive HBsAg carrier, 32.5% in chronic hepatitis B, and 50% in liver cirrhosis, respectively (p < 0.001). The T to C/A mutation at nucleotide 1753 in the BCP and G to A mutation at nucleotide 1899 in the precore were more frequent in liver cirrhosis than in the other clinical statuses (p < 0.05). CONCLUSION: Sequential accumulation of mutations in the BCP/precore has an important role in the progression of HBV-related liver disease.

Chronic blockade of the angiotensin II receptor has a differential effect on adipose and vascular PAI-1 in OLETF rats.

Angiotensinogen (AGT) and plasminogen activator inhibitor-1 (PAI-1) are expressed in both vascular and adipose tissues. Angiotensin II (AG II) has an adipogenic effect and increases PAI-1 expression. To evaluate the chronic effects of AG II type 1 receptor (AT(1)R) antagonism on adipose mass and PAI-1 expression in vascular and adipose tissues, losartan (30mg/kg/day) was administered to Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of type 2 diabetes, for 20 weeks. Adipose mass and regional fat distribution in the abdomen did not change after chronic AT(1)R antagonism in OLETF rats. AGT and PAI-1 mRNA expressions in adipose tissue of OLETF rats were significantly increased compared with Long-Evans Tokushima Otsuka (LETO) rats, the normal control. Chronic losartan therapy further increased the level of adipose AGT in OLETF rats, but did not affect the level of adipose PAI-1 mRNA. In contrast, aortic PAI-1 expression in OLETF rats was attenuated by chronic losartan therapy. Our results have two implications. First, adipose tissue may be an important source of AG II in metabolic syndrome even after chronic losartan therapy. Second, chronic AT(1)R antagonism with losartan causes differential effects on vascular and adipose PAI-1 expression.

Hepatitis B virus genotypes in Korea: an endemic area of hepatitis B virus infection.

OBJECTIVES: It has been reported that distribution of hepatitis B virus (HBV) genotypes shows geographic difference and are associated with clinical outcomes of HBV infection, including response to antiviral therapy and progression of chronic liver diseases. In this study, we analyzed the distribution of HBV genotypes according to the various clinical outcomes of chronic HBV infection in Korea, which is one of the most endemic areas of HBV infection. METHODS: A total of 200 patients with chronic HBV infection were enrolled. Clinical diagnoses of the 200 patients with chronic liver diseases were as follows: hepatitis B e antigen (HBeAg)-positive healthy carrier (defined as HBeAg(+), anti-HBe(-), HBV DNA(+) by hybridization, normal transaminase; n = 40); inactive HBsAg carrier (n = 40); chronic hepatitis B (n = 40); liver cirrhosis (n = 40); hepatocellular carcinoma (n = 40). HBV genotypes were determined by nested polymerase chain reaction using genotype-specific primers. RESULTS: All patients except 2 (inactive HBsAg carriers) were positive for nested PCR and they have genotype C regardless of clinical outcomes. CONCLUSIONS: HBV genotype was genotype C regardless of various clinical outcomes of chronic HBV infection in Korea. Considering that HBV genotypes have clinical relevance, distribution of HBV genotype in each area should be monitored when management for chronic HBV infection is planned.

Serologic Markers of Hepatitis B Virus in Pregnant Women in Jeju Island / 대한간학회지

BACKGROUND/AIMS: Most cases of hepatitis B virus (HBV) are transmitted vertically in endemic areas of HBV. The positivity of serum HBeAg/HBV DNA in pregnant women is associated with vaccine failure. Recently, a national program for HBV vaccines free of charge in neonates born to HBsAg-positive pregnant women is being performed. The aim of this study was to investigate the positivity of serological markers of HBV in pregnant women in Jeju, which is an island separated from the Korean peninsula and a promising cohort to evaluate the effect of a prevention program of HBV infection. In addition, we investigated the geographic differences in the prevalence of HBV infection because it has been reported that the prevalence of HBV has been high in this area previously. METHODS: Between January 2001 and December 2002, all women who gave delivery were studied retrospectively. Women between the ages of thirty and forty, who received health screening at the Asan Medical Center health promotion center in Seoul, were analyzed as controls. RESULTS: During the study period, 1,030 pregnant women (30.8 +/- 4.3 years) and 7,270 controls (33.1 +/- 5.0 years) were enrolled. The positivity of HBsAg was high in Jeju compared with that of Seoul (6.4% vs. 4.9%) (P=0.036). The positivity of HBeAg/HBV DNA was 31.8% (21/66) in HBsAg-positive pregnant women. The positivity of anti-HBs was low in Jeju compared with that of Seoul (54.5% vs. 68.8%) (P<0.001). CONCLUSIONS: The positivity of HBsAg was found to be high in pregnant women in Jeju. Intensive supervision for HBV infection in pregnant women should be given in this area.