Irradiated engineered tumor cell-derived microparticles remodel the tumor immune microenvironment and enhance antitumor immunity.

Radiotherapy (RT), administered to roughly half of all cancer patients, occupies a crucial role in the landscape of cancer treatment. However, expanding the clinical indications of RT remains challenging. Inspired by the radiation-induced bystander effect (RIBE), we used the mediators of RIBE to mimic RT. Specifically, we discovered that irradiated tumor cell-released microparticles (RT-MPs) mediated the RIBE and had immune activation effects. To further boost the immune activation effect of RT-MPs to achieve cancer remission, even in advanced stages, we engineered RT-MPs with different cytokine and chemokine combinations by modifying their production method. After comparing the therapeutic effect of the engineered RT-MPs in vitro and in vivo, we demonstrated that tIL-15/tCCL19-RT-MPs effectively activated antitumor immune responses, significantly prolonged the survival of mice with malignant pleural effusion (MPE), and even achieved complete cancer remission. When tIL-15/tCCL19-RT-MPs were combined with PD-1 monoclonal antibody (mAb), a cure rate of up to 60% was achieved. This combination therapy relied on the activation of CD8+ T cells and macrophages, resulting in the inhibition of tumor growth and the establishment of immunological memory against tumor cells. Hence, our research may provide an alternative and promising strategy for cancers that are not amenable to conventional RT.

A novel subgenotype I3 of hepatitis B virus in Guangxi, China: a 15-year follow-up study.

Genotype I of hepatitis B virus (HBV) was proposed recently following sequencing of complete HBV genomes from Vietnam and Laos. However, its long-term molecular evolution is unknown. The objectives of this study were to study the molecular evolution of this genotype from an asymptomatic HBsAg carrier from the Long An cohort over a 15-year period was studied using both NGS and clone-based sequencing. The number of complete genome sequences obtained in 2004, 2007, 2013, and 2019 are 17, 20, 19, and 10, respectively. All strains belong to subgenotype I1, except for six (five from 2007 and one from 2019) and 8 further strains from 2007 which form a cluster branching out from other subgenotype I sequences, supported by a 100% bootstrap value. Based on complete genome sequences, all of the estimated intragroup nucleotide divergence values between these strains and HBV subgenotypes I1-I2 exceed 4%. These strains are recombinants between genotype I1 and subgenotype C but the breakpoints vary. The median intrahost viral evolutionary rate in this carrier was 3.88E-4 substitutions per site per year. The Shannon entropy (Sn) ranged from 0.55 to 0.88 and the genetic diversity, D, ranged from 0.0022 to 0.0041. In conclusion, our data provide evidence of novel subgenotypes. Considering that the 8 strains disappeared after 2007, while one of the 6 strains appears again in 2019, we propose these 6 strains as a new subgenotype, provisionally designated HBV subgenotype I3 and the 8 strains as aberrant genotype.

Identification of potential plasma biomarkers in early-stage nasopharyngeal carcinoma-derived exosomes based on RNA sequencing.

BACKGROUND: Early diagnosis of nasopharyngeal carcinoma (NPC) is vital to improve the prognosis of these patients. However, early diagnosis of NPC is typically challenging. Therefore, we explored the pathogenetic roles and associated mechanisms of exosomes in plasma of patients with early-stage NPC. METHODS: Exosomes in plasma were extracted by ultra-high-speed centrifugation. Western blot and transmission electron microscopy (TEM) were used to verify the purity of exosomes. The sequencing data (6 plasma samples from healthy volunteers vs. 6 NPC plasma samples) were analyzed by principal component analysis (PCA), DESeq2, gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG), and TargetScan. The differentially expressed miRNAs (DEmiRNAs) were obtained from the dataset (GSE118720) downloaded from the Gene Expression Omnibus (GEO) repository. Additionally, the datasets downloaded from the GEO database (GSE12452, GSE13597, GSE53819, GSE64634) were used to predict the target genes and functions of hsa-miR-1301-3p. qPCR was applied to verify the differences in the expressions of hsa-miR-1301-3p between 10 normal plasma and 10 NPC plasma samples. RESULTS: Western blot, TEM, and Nanoparticle Tracking Analysis showed adequate purity of the extracted exosomes. RNA-seq analysis revealed 21 upregulated miRNAs, and 10 downregulated miRNAs in plasma exosomes of early-stage NPC patients. GO analysis showed that the target genes of DEmiRNAs were mainly enriched in DNA synthesis and transcription regulation. KEGG analysis revealed that DEmiRNAs were mainly enriched in PI3K-Akt and MAPK signaling pathways. Moreover, the expression of hsa-mir-1301-3p was verified to be significantly upregulated in enlarged samples of plasma exosomes. CONCLUSIONS: We identified several DEmiRNAs extracted from tumor-derived exosomes between normal plasma and early-stage NPC plasma. Bioinformatics analyses indicated that these DEmiRNAs may be related to NPC development. Our study may provide novel insights into underlying biomarkers and mechanisms of plasma exosomes in early-stage NPC.

Prognostic value of serum uric acid and tumor response to induction chemotherapy in locally advanced nasopharyngeal carcinoma.

BACKGROUND: To explore the combined predictive value of serum uric acid (SUA) and tumor response to induction chemotherapy (IC) in locally advanced nasopharyngeal carcinoma (LANPC) patients receiving IC followed by concurrent chemoradiation therapy (CCRT). METHODS: A total of 341 LANPC patients treated with IC + CCRT were enrolled in this retrospective study. Overall survival (OS), progression-free survival (PFS), locoregional relapse-free survival (LRFS), and distant metastasis-free survival (DMFS) were compared by the Kaplan-Meier analysis and the log-rank test, and multivariable survival analysis was carried out to investigate the independent prognostic factors. RESULTS: Univariate analysis showed that a low SUA level and unsatisfactory tumor response to two cycles of IC both were negative predictors for OS, PFS, and DMFS in patients with LANPC. multivariable analysis demonstrated that the SUA level after two cycles of IC was an independent prognostic factor for OS (P = 0.012) but of borderline significance for PFS and DMFS (P = 0.055 and P = 0.067, respectively). Furthermore, tumor response to IC was of independent significance for predicting OS, PFS, and DMFS, respectively. Finally, LANPC patients with satisfactory tumor response and a high SUA level after two cycles of IC had a better OS, PFS, and DMFS than those with unsatisfactory tumor response and a low SUA level. CONCLUSION: The SUA level and the tumor response to two cycles of IC had predictive value for LANPC patients treated with IC plus CCRT. However, more aggressive therapeutic strategies are recommended for those with a low SUA level and unsatisfactory tumor response to two cycles of IC.

Infection with Hepatitis B Virus May Increase the Serum Concentrations of Osteopontin.

BACKGROUND: Serum osteopontin (OPN) concentrations were found to be significantly increased in patients infected with hepatitis B virus (HBV) and patients with hepatocellular carcinoma (HCC). OBJECTIVE: The aim of this study was to determine the association among HCC, OPN, and HBV. METHODS: Two hundred and forty-one subjects were recruited and divided into 6 groups: healthy controls, asymptomatic HBsAg carriers, HBsAg (-) patients with other tumors, HBsAg (+) chronic liver disease patients, HBsAg (+) patients with HCC, and HBsAg (-) patients with HCC or liver cirrhosis (LC). Serum concentrations of OPN and HBsAg were measured and analyzed. RESULTS: OPN concentrations in the HBsAg (+) HCC group were significantly higher than the healthy control group and the HBsAg (-) patients with other cancers (both p = 0.0001). The OPN concentrations of the HBsAg (-) patients with HCC or LC also did not differ significantly from those of the healthy control group (p = 0.075). There is a correlation between the titer of HBsAg and concentrations of OPN in all 3 HBsAg (+) groups (all p values <0.05). CONCLUSIONS: Infection with HBV may increase the serum concentrations of OPN. The association of OPN and HCC may be not attributable to tumor development per se but, rather, to HBV infection.

Novel subgenotype D11 of hepatitis B virus in NaPo County, Guangxi, bordering Vietnam.

Hepatitis B virus has been classified into 10 genotypes and 48 subgenotypes worldwide. We found previously, through polymerase chain reaction (PCR) amplification of a sample collected in 2011, that an HBsAg carrier was infected with two genotypes (B and D) of HBV. We carried out cloning, sequencing and phylogenetic analysis of the complete genomes and, for confirmation, analysed a sample collected from the same individual in 2018. Fifteen complete sequences were obtained from each sample. The carrier was infected in 2011 by genotypes B and D and by various recombinants, but only genotype D was present in 2018. The major and minor parents of the recombinants are genotypes B and D, respectively, although the recombination breakpoints vary among them. All 23 genotype D isolates form a cluster, branching out from other subgenotype D sequences and supported by a 100 % bootstrap value. Based on complete genome sequences, almost all of the estimated intragroup nucleotide divergence values between our isolates and HBV subgenotypes D1-D10 exceed 4 %. Compared to the other subgenotypes (D1-D10), 35 unique amino acids were present in our isolates. Our data provide evidence for a novel subgenotype, provisionally designated HBV subgenotype D11.

Asymptomatic hepatitis B carriers who were vaccinated at birth.

The long-term persistence of immunity following universal infant immunization against hepatitis B virus (HBV) and the need for a subsequent booster dose in adolescence remain under debate. With data derived from Long'an County, Guangxi, China, we reported previously that the prevalence of hepatitis B surface antigen (HBsAg) among adults born from 1987 to 1993 increases with age, although these individuals had received a first dose of the vaccine within 24 hours of birth. Here, we sought the source of transmission by comparison of genotypes among their family members using phylogenetic analysis of complete HBV S gene sequences. For comparison, we screened 2199 vaccinated individuals aged 5 to 17 in Cang Wu County and 1592 vaccinated individuals aged 3 to 7 in Ling Shan County in Guangxi for HBsAg carriers and investigate their family members. In total, 50 asymptomatic HBsAg carriers who were vaccinated at birth and 152 family members were analyzed. The results showed that 25% (95% CI: 6.0-44.0) of the HBsAg-positive children had not acquired their HBV infection from their mothers. This phenomenon showed a trend that increases with age. Antibody escape mutations were detected in 22.9% (95% CI: 11.0-34.8) of the isolates. In conclusion, a booster dose may be necessary for adolescence who were vaccinated at birth in highly endemic countries.

Locus 5p13.1 may be associated with the selection of cancer-related HBV core promoter mutations.

Background: The basal core promoter (BCP) double mutations (A1762T and G1764A) of hepatitis B virus (HBV) have been reported to be an aetiological factor of hepatocellular carcinoma (HCC). What distinguishes the subset of HBV carriers in whom these mutations are selected? Methods: A genome-wide association study (GWAS) was carried out on 218 asymptomatic HBsAg carriers infected with HBV with BCP double mutations and 191 controls infected with HBV with the wild type BCP. The highest ranking nucleotide polymorphisms (SNPs) were validated with other study subjects, 203 cases and 181 controls. The expression of the gene nearest a SNP found to be significant was examined using RT-PCR. Results: Forty-five candidate SNPs were identified in the GWAS. Three SNPs were found to be associated with the selection of HBV BCP double mutations in the replication stage, including rs7717457 at 5p13.1, rs670011 at 17q21.2, rs2071611 at 6p22.2. Especially, rs7717457 (P= 4.57×10-5 combined P) reached the potential GWAS significance level. The expression of gene complement component 7 (C7), nearest to SNP rs7717457, differed significantly between the case and control groups (t=2.045, P=0.04), suggesting that SNP rs7717457 was associated with the expression of its nearest gene. Conclusions: SNP rs7717457 is associated with the selection of HBV BCP double mutations, providing an important clue to understanding the mechanisms of oncogenesis of HBV BCP double mutations.

High Prevalence of HBV Lamivudine-Resistant Mutations in HBV/HIV Co-Infected Patients on Antiretroviral Therapy in the Area with the Highest Prevalence of HIV/HBV Co-Infection in China.

OBJECTIVES: We aimed to determine the prevalence of hepatitis B virus (HBV) drug-resistant mutations in patients co- infected with HBV/human immunodeficiency virus (HIV), including both drug-naïve subjects and those who received antiretroviral therapy (ART) in Guangxi, where the prevalence of HIV/HBV co-infection is highest in China. METHODS: Two hundred and three subjects co-infected with HBV/HIV were recruited, including 123 drug-naïve patients (group 1) and 80 who received ART (group 2). The polymerase gene of HBV in the serum of all study subjects was analysed. RESULTS: The results showed that the prevalence of HBV drug-resistant mutations in group 2 (76.5%, 95% CI 56.3-96.7) was significantly higher than that in group 1 (1.4%, 95% CI -1.4 to 4.2; χ2 = 50.955, p < 0.05). The major pattern of lamivudine (3TC)-resistant mutations is L180M+M204I+L80I (35.7%). In total, 95% of subjects with resistant mutations had cross-resistance to telbivudine and entecavir. No putative tenofovir disoproxil fumarate (TDF) resistance change was found. Five subjects (6.5%) in group 2 had HBV viral loads over 10 × 106 copies/mL. Four of them had 3TC-resistant mutations. Multivariate analysis showed that ART was the only factor associated with the development of drug-resistant mutations. CONCLUSION: Treating HIV in HIV/HBV co-infection with antiretroviral agents may result in a very high prevalence of HBV 3TC-resistant mutations. TDF could not completely suppress HBV replication.

Hepatitis B Virus Core Promoter Double Mutations (A1762T, G1764A) Are Associated with Lower Levels of Serum Dihydrolipoyl Dehydrogenase.

OBJECTIVES: The aim of this study was to identify serum proteins with differential concentrations between hepatocellular carcinoma (HCC) patients and HBsAg asymptomatic carriers among individuals infected with hepatitis B virus (HBV) with basal core promoter (BCP) double mutations (A1762T, G1764A). METHODS: iTRAQ and liquid chromatography-tandem mass spectrometry were used to identify differentially expressed protein, and an ELISA test was used for the validation test. RESULTS: The total number of proteins identified was 1,125, of which 239 showed statistically significant differences in their expression. The relative concentrations of serum dihydrolipoyl dehydrogenase (DLD), which showed the most significant correlation with liver diseases and infection, were significantly lower in HCC patients than asymptomatic HBsAg carriers and individuals negative for HBsAg. However, only the difference between HCC patients with BCP double mutations and HBsAg-negative individuals could be confirmed by ELISA. Meanwhile, we found that the concentrations of serum DLD in those infected with HBV with BCP double mutations were significantly lower than in individuals with the wild-type BCP. However, the difference in the concentrations of serum DLD between individuals with wild-type BCP and those negative for HBsAg was not significant. CONCLUSIONS: HBV with BCP double mutations are associated with lower concentrations of serum DLD.

The correlation between serum HBsAg levels and viral loads depends upon wild-type and mutated HBV sequences rather than the HBeAg/anti-HBe status.

Despite several studies regarding the correlation between serum HBsAg titers and viral loads, the association remains uncertain. Eighty-nine individuals were selected randomly from a Chinese cohort of 2,258 subjects infected persistently with hepatitis B virus (HBV) for cross-sectional and longitudinal analysis. Viral loads of mutant HBV are lower than those of wild type HBV. The serum HBsAg titers correlate positively with viral loads in both HBeAg positive and negative subjects (r = 0.449, P = 0.013; r = 0.300, P = 0.018, respectively). No correlation between serum HBsAg titer and viral loads was found in any of the four phases of chronic HBV infection. The serum HBsAg titers correlate positively with viral loads in the group with wild type sequences of the PreS/S, basal core promoter (BCP), and preC regions of HBV(r = 0.502, P = 0.040). However, the correlation was not seen in the group with mutations in these regions (r = 0.165, P = 0.257). The correlation between HBsAg titers and viral loads was seen in individuals with wild type PreS/S sequences but not in the subgroup with BCP double mutations or PreC stop mutation, although their sequences in the preS/S regions were wild type. All these findings were confirmed by the longitudinal analysis. In conclusion, the correlation between serum HBsAg levels and viral loads may not differ between HBeAg positive and negative individuals but may depend on wild-type or mutated genomic sequences. Therefore, HBsAg quantitation may be used as a surrogate for viral loads in only wild-type HBV infections.

Microglia and macrophage metabolism: a regulator of cerebral gliomas.

Reciprocal interactions between the tumor microenvironment (TME) and cancer cells play important roles in tumorigenesis and progression of glioma. Glioma-associated macrophages (GAMs), either of peripheral origin or representing brain-intrinsic microglia, are the majority population of infiltrating immune cells in glioma. GAMs, usually classified into M1 and M2 phenotypes, have remarkable plasticity and regulate tumor progression through different metabolic pathways. Recently, research efforts have increasingly focused on GAMs metabolism as potential targets for glioma therapy. This review aims to delineate the metabolic characteristics of GAMs within the TME and provide a summary of current therapeutic strategies targeting GAMs metabolism in glioma. The goal is to provide novel insights and therapeutic pathways for glioma by highlighting the significance of GAMs metabolism.

Tumor cell senescence-induced macrophage CD73 expression is a critical metabolic immune checkpoint in the aging tumor microenvironment.

Background: The role of senescent cells in the tumor microenvironment (TME) is usually bilateral, and diverse therapeutic approaches, such as radiotherapy and chemotherapy, can induce cellular senescence. Cellular interactions are widespread in the TME, and tumor cells reprogram immune cells metabolically by producing metabolites. However, how senescent cells remodel the metabolism of TME remains unclear. This study aimed to explore precise targets to enhance senescent cells-induced anti-tumor immunity from a metabolic perspective. Methods: The in vivo senescence model was induced by 8 Gy×3 radiotherapy or cisplatin chemotherapy, and the in vitro model was induced by 10 Gy-irradiation or cisplatin treatment. Metabonomic analysis and ELISA assay on tumor interstitial fluid were performed for metabolites screening. Marker expression and immune cell infiltration in the TME were analyzed by flow cytometry. Cell co-culture system and senescence-conditioned medium were used for crosstalk validation in vitro. RNA sequencing and rescue experiments were conducted for mechanism excavation. Immunofluorescence staining and single-cell transcriptome profiling analysis were performed for clinical validation. Results: We innovatively reveal the metabolic landscape of the senescent TME, characterized with the elevation of adenosine. It is attributed to the senescent tumor cell-induced CD73 upregulation of tumor-associated macrophages (TAMs). CD73 expression in TAMs is evoked by SASP-related pro-inflammatory cytokines, especially IL-6, and regulated by JAK/STAT3 pathway. Consistently, a positive correlation between tumor cells senescence and TAMs CD73 expression is identified in lung cancer clinical specimens and databases. Lastly, blocking CD73 in a senescent background suppresses tumors and activates CD8+ T cell-mediated antitumor immunity. Conclusions: TAMs expressed CD73 contributes significantly to the adenosine accumulation in the senescent TME, suggesting targeting CD73 is a novel synergistic anti-tumor strategy in the aging microenvironment.

Hepatitis B virus candidate subgenotype I1 varies in distribution throughout Guangxi, China and may have originated in Long An county, Guangxi.

Sequencing of the complete hepatitis B virus (HBV) genomes from Vietnam, China and Laos led to the identification of a complex recombinant, referred to initially as an aberrant genotype and later proposed to be a new genotype, I. However, epidemiological data regarding this new genotype are lacking. A cross-sectional study was carried out to investigate the epidemiology of HBV candidate genotype I in Guangxi, China using stratified, random cluster sampling. Four thousand five hundred thirteen subjects were recruited from five counties within Guangxi. Three genotypes, B, C, and I, were identified with a prevalence of 32.6% (114/350), 64% (224/350), and 3.4% (12/350), respectively. All the genotype I isolates belong to candidate subgenotype I1 and were found in Bing Yang (15.3%, 9/59) and Na Po (5.0%, 3/60) counties only. The prevalence of this subgenotype is significantly higher in males (5.1%, 10/195) than in females (1.3%, 2/155; X(2) = 3.959, P < 0.05) but does not differ significantly with age. It was found in the Han (4.5%, 9/201) and Zhuang (3.1%, 3/97) ethnic populations only. There is no significant difference from other genotypes in the prevalence of HBV serological markers. Phylogeographic analysis revealed that genotype I1 likely arose in Long An county, then spread later to Bing Yang, Na Po counties and elsewhere in southeast Asia. In conclusion, the distribution of candidate genotype I within Guangxi is not even and it is highly endemic in some counties. Its prevalence is associated with gender and ethnicity. Subgenotype I1 likely originated in Long An county.

Combined core promoter mutations and pre-S deletion of HBV may not increase the risk of HCC: a geographical epidemiological study in Guangxi, China.

BACKGROUND: Although persistent hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC), the mechanisms of oncogenesis remain obscure. AIMS: To determine whether the findings that HBV basal core promoter (BCP) A1762T, G1764A double mutations, pre-S deletions and a combination of both are risk factors of HCC are supported by geographical epidemiology. METHODS: Study subjects were recruited from Long An county, where the incidence of HCC is the highest, and five other counties in Guangxi, where the HCC incidence is lower and varies among them. The Pre-S region and BCP of HBV from all study subjects were amplified and sequenced and the data were analysed using chi-squared tests. RESULTS: The prevalence of BCP and pre-S mutations differs significantly (χ(2) = 9.850, 5.135, respectively, all P < 0.01) between Long An and the other counties. However, the prevalence of combined BCP and pre-S mutations does not differ significantly (χ(2) = 1.510, P > 0.05). These mutations are less frequent in the young but the prevalence of pre-S deletions does not increase with age. The prevalence of these mutations does not differ significantly between men and women but is significantly higher in Zhuang than the other ethnic populations. Among the other five counties, the prevalence of BCP mutations in counties where the HCC incidence is high is significantly higher than that of counties where the HCC incidence is low. CONCLUSIONS: Combined BCP double mutations and pre-S deletion may not increase the risk of HCC, although these mutations are a risk factor of HCC when they present alone.

Low host immune pressure may be associated with the development of hepatocellular carcinoma: a longitudinal analysis of complete genomes of the HBV 1762T, 1764A mutant.

Background: It has been reported that hepatitis B virus (HBV) double mutations (A1762T, G1764A) are an aetiological factor of hepatocellular carcinoma (HCC). However, it is unclear who is prone to develop HCC, among those infected with the mutant. Exploring HBV quasispecies, which are strongly influenced by host immune pressure, may provide more information about the association of viral factors and HCC. Materials and methods: Nine HCC cases and 10 controls were selected from the Long An cohort. Serum samples were collected in 2004 and 2019 from subjects with HBV double mutations and the complete genome of HBV was amplified and sequenced using next-generation sequencing (NGS). Results: The Shannon entropy values increased from 2004 to 2019 in most cases and controls. There was no significant difference in mean intrahost quasispecies genetic distances between cases and controls. The change in the values of mean intrahost quasispecies genetic distances of the controls between 2004 and 2019 was significantly higher than that of the cases (P<0.05). The viral loads did not differ significantly between cases and controls in 2004(p=0.086) but differed at diagnosed in 2019 (p=0.009). Three mutations occurring with increasing frequency from 2004 to 2019 were identified in the HCC cases, including nt446 C→G, nt514 A→C and nt2857T→C. Their frequency differed significantly between the cases and controls (P<0.05). Conclusions: The change in the values of mean intrahost quasispecies genetic distances in HCC was smaller, suggesting that HBV in HCC cases may be subject to low host immune pressure. Increasing viral loads during long-term infection are associated with the development of HCC. The novel mutations may increase the risk for HCC.

Prevalence of hepatitis B virus infection in a highly endemic area of southern China after catch-up immunization.

The Chinese national goals for control of hepatitis B virus (HBV) infection were to achieve a prevalence of HBsAg below 7% for the entire population, and 1% for children under 5-year old, by 2010. To determine whether Guangxi, a multi-minority province with a low socio-economic status and a very high prevalence of HBV, achieved this goal, a seroepidemiological survey of HBV infection was carried out using stratified, random cluster sampling. The results show that the overall prevalence of HBsAg is 9.16% [95% confidence interval (CI) = 8.32-10%]. The prevalence in males (10.96%, 95% CI = 9.64-12.28%) is significantly higher than in females (7.71%, 95% CI = 6.64-8.78%; χ(2) = 10.5923, P < 0.05). The prevalence in children under 5-year old is 3.62% (95% CI = 0.60-6.64%) and increases with age. The prevalence of HBsAg in non-immunized individuals is significantly higher than in those immunized completely, although not within 24 hr of birth (χ(2) = 31.426, P < 0.05); a significant difference was found in those below the age of 20 years but not in older persons. Gender, age, immunization history, and familial HBsAg carriers are risk factors for infection. In conclusion, this study indicates that Guangxi has not reached the goal for the control of HBV infection. Catch-up HBV immunization may not protect adults effectively against infection in highly endemic regions.

Analysis of entire hepatitis B virus genomes reveals reversion of mutations to wild type in natural infection, a 15 year follow-up study.

It has been reported that some mutations in the genome of hepatitis B virus (HBV) may predict the outcome of the virus infection. However, evolutionary data derived from long-term longitudinal analysis of entire HBV genomes using next generation sequencing (NGS) remain rare. In this study, serum samples were collected from asymptomatic hepatitis B surface antigen (HBsAg) carriers from a long-term prospective cohort. The entire HBV genome was amplified by polymerase chain reaction (PCR) and sequenced using NGS. Twenty-eight time series serum samples from nine subjects were successfully analysed. The Shannon entropy (Sn) ranged from 0 to 0.89, with a median value of 0.76, and the genetic diversity (D) ranged from 0 to 0.013, with a median value of 0.004. Intrahost HBV viral evolutionary rates ranged from 2.39E-04 to 3.11E-03. Double mutations at nt1762(A â†’ T) and 1764(G â†’ A) and a stop mutation at nt1896(G â†’ A) were seen in all sequences from subject BO129 in 2007. However, in 2019, most sequences were wild type at these positions. Deletions between nt 2920-3040 were seen in all sequences from subject TS115 in 2007 and 2013 but these were not present in 2004 or 2019. Some sequences from subject CC246 had predicted escape substitutions (T123N, G145R) in the surface protein in 2004, 2013 and 2019 but none of the sequences from 2007 had these changes. In conclusion, HBV mutations may revert to wild type in natural infection. Clinicians should be wary of predicting long-term prognoses on the basis of the presence of mutations.

A complex hepatitis B virus (X/C) recombinant is common in Long An county, Guangxi and may have originated in southern China.

Recently, a complex (X/C) hepatitis B virus (HBV) recombinant, first reported in 2000, was proposed as a new genotype; although this was refuted immediately because the strains differ by less than 8 % in nucleotide distance from genotype C. Over 13.5 % (38/281) of HBV isolates from the Long An cohort in China were not assigned to a specific genotype, using current genotyping tools to analyse surface ORF sequences, and these have about 98 % similarity to the X/C recombinants. To determine whether this close identity extends to the full-length sequences and to investigate the evolutionary history of the Long An X/C recombinants, 17 complete genome sequences were determined. They are highly similar (96-99 %) to the Vietnamese strains and, although some reach or exceed 8 % nucleotide sequence difference from all known genotypes, they cluster together in the same clade, separating in a phylogenetic tree from the genotype C branch. Analysis of recombination reveals that all but one of the Long An isolates resembles the Vietnamese isolates in that they result from apparent recombination between genotype C and a parent of unknown genotype (X), which shows similarity in part to genotype G. The exception, isolate QL523, has a greater proportion of genotype C parent. Phylogeographic analysis reveals that these recombinants probably arose in southern China and spread later to Vietnam and Laos.

Seroconversion of hepatitis B surface antigen among those with previously successful immune response in Southern China.

Recommendations promoted worldwide have suggested a period of protection lasting more than 20 years against hepatitis B (HB) following primary immunization. Starting in 1987, universal HB vaccinations were carried out in Long An County, Guangxi Province, one of the earliest counties in which plasma-derived HB vaccine was delivered to newborns across China. Data collection targeted toward understanding the long term (26-33 years since primary immunization) immune effects of the plasma-derived HB vaccine was conducted in 2015; a second data collection was carried out in 2019 to assess seroconversion in the same cohort. This study qualitatively compared positive vs negative results and quantitatively assessed HB biomarkers - HB surface antigen (HBsAg), antibody to HBsAg (anti-HBs), HB e-antigen (HBeAg), antibody to HBeAg (anti-HBe), and antibody to HB core antigen (anti-HBc) - in serum 26-33 years after the full initial course of HB vaccination, then analyzed anti-HBs seroconversion using the two-phase sampling method in the same cohort and calculated the anti-HBs seroconversion rate from 2015 to 2019. The protective sero-conversion rate (anti-HBs ≥10mIU/mL) was 37.6% (192/511); the HBsAg-positive rate was 5.3% (27/511); the anti-HBs mean geometric titer (GMT) was 11.1 mIU/mL. Among the 143 participants involved in both 2015 and 2019 data collections, the seroconversion rate was 3.5% (5/143); two individuals had protective anti-HBs levels in 2015. These findings indicate that anti-HBs status can be seroconverted to a protective concentration level 4 years earlier in a high HBV epidemic region. The role of genomic mutations and the disappearance of immune memory and seroconversion should be investigated.