Factors affecting prolonged SARS-CoV-2 infection and development and validation of predictive nomograms.

Prolonged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has received much attention since it is associated with mortality and is hypothesized as the cause of long COVID-19 and the emergence of a new variant of concerns. However, a prediction model for the accurate prediction of prolonged infection is still lacking. A total of 2938 confirmed patients with COVID-19 diagnosed by positive reverse transcriptase-polymerase chain reaction tests were recruited retrospectively. This study cohort was divided into a training set (70% of study patients; n = 2058) and a validation set (30% of study patients; n = 880). Univariate and multivariate logistic regression analyses were utilized to identify predictors for prolonged infection. Model 1 included only preadmission variables, whereas Model 2 also included after-admission variables. Nomograms based on variables of Model 1 and Model 2 were built for clinical use. The efficiency of nomograms was evaluated by using the area under the curve, calibration curves, and concordance indexes (C-index). Independent predictors of prolonged infection included in Model 1 were: age ≥75 years, chronic kidney disease, chronic lung disease, partially or fully vaccinated, and booster. Additional independent predictors in Model 2 were: treated with nirmatrelvir/ritonavir more than 5 days after diagnosis and glucocorticoid. The inclusion of after-admission variables in the model slightly improved the discriminatory power (C-index in the training cohort: 0.721 for Model 1 and 0.737 for Model 2; in the validation cohort: 0.699 for Model 1 and 0.719 for Model 2). In our study, we developed and validated predictive models based on readily available variables of preadmission and after-admission for predicting prolonged SARS-CoV-2 infection of patients with COVID-19.

Lipid nanoparticle-mediated delivery of IL-21-encoding mRNA induces viral clearance in mouse models of hepatitis B virus persistence.

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), the transcription template for all viral mRNAs, is highly stable and current treatment options cannot effectively induce its clearance. Previously, we established an HBV persistence mouse model based on a clinical isolate (termed BPS) and identified interleukin-21 (IL-21) as a potent inducer of HBV clearance. Lipid nanoparticle (LNP) mediated delivery of mRNA has proven to be a highly safe and effective delivery platform. This work explored the applicability and effectiveness of the mRNA-LNP platform in IL-21-based HBV therapies. First, LNP-encapsulated murine IL-21 mRNA (LNP-IL-21) was prepared, characterized, and demonstrated to engender IL-21 expression in vitro and in vivo. Next, LNP-IL-21 was shown to induce clearance of both serum and intrahepatic HBV antigen and DNA in two HBV persistence mouse models based on BPS and recombinant cccDNA (rcccDNA), respectively, which was associated with HBV-specific humoral and cellular immune responses. Furthermore, peripheral blood mononuclear cells from BPS persistence mice treated ex vivo with LNP-IL-21 and HBV surface antigen (HBsAg) could induce similar HBV clearance upon infusion into recipient mice. These findings indicated that IL-21 combined with mRNA-LNP platform represents a valid and promising strategy for developing novel therapeutics against chronic HBV infection.

Incidence and severity of SARS-CoV-2 reinfection, a multicenter cohort study in Shanghai, China.

During March 2022 to January 2023, two Omicron waves hit Shanghai and caused a massive number of reinfections. To better understand the incidence and clinical characteristics of SARS-CoV-2 reinfection in Shanghai, China, we conducted a multicenter cohort study. COVID-19 patients first infected with BA.2 (March 1, 2022-May 23, 2022) who were quarantined in Huashan Hospital, Renji Hospital, and Shanghai Jing'an Central Hospital were followed up for reinfection from June 1, 2022 to January 31, 2023. Of 897 primary infections, 148 (16.5%) experienced reinfection. Incidence rate of reinfection was 0.66 cases per 1000 person-days. Female gender (adjusted odds ratio [aOR]= 2.19, 95% confidence interval [CI]: 1.29-3.83) was a risk factor for reinfection. The four most common symptoms of reinfections during the circulation of BA.5 sublineages were cough (62.59%), sore throat (54.42%), fatigue (48.98%), and fever (42.57%). Having received a booster vaccination was not associated with reduced severity of reinfection in comparison with not having received booster vaccination. After matched 1:1 by age and sex, we found that reinfections with BA.5 sublineages had significantly lower occurrence and severity of fever, fatigue, sore throat, and cough, as compared to primary infections with BA.5 sublineages. SARS-CoV-2 Omicron reinfections were less severe than Omicron primary infections during the circulation of the same subvariant. Protection offered by both vaccination and previous infection was poor against SARS-CoV-2 reinfection.

Lower frequency of MDSCs was significantly related to functional cure in CHB patients treated with peginterferon.

BACKGROUND AND AIMS: Myeloid-derived suppressor cells (MDSCs) and CD4+ regulatory T cells (Tregs) expand during chronic hepatitis B virus (HBV) infection and inhibit antiviral immunity. However, the relationship between antiviral effect and the frequencies of those immune suppressive cells after pegylated interferon α-2a (PegIFNα-2a) therapy is not clearly understood. This study aimed to investigate the contribution of monocytic MDSCs (mMDSCs) and CD4+ Tregs to functional cure (HBsAg seroclearance) after PegIFNα-2a therapy and evaluate the effect of PegIFNα-2a therapy on these cells. METHODS: Flow cytometry analysis was performed along with longitudinal immune monitoring of 97 hepatitis B e antigen (HBeAg) negative chronic hepatitis B (CHB) patients receiving PegIFNα-2a weekly for 48 weeks. RESULTS: The frequencies of mMDSCs and CD4+ Tregs increased in all HBV patients, and they were higher in the HBsAg persistence group than in the HBsAg seroclearance group. A significant decline in the frequency of mMDSCs was found in patients who realized functional cure after PegIFNα-2a treatment. In contrast, the frequency of CD4+ Tregs in both the HBsAg seroclearance and persistence groups significantly increased. Multivariate analyses indicated that the baseline serum HBsAg levels (p < .001) and mMDSCs frequency (p = .027) were independently associated with the HBsAg clearance, and the combined marker (HBsAg plus mMDSCs) displayed the highest specificity (93.1%) than any other markers in predicting HBsAg seroclearance. CONCLUSIONS: These results suggest that a poor response to PegIFNα-2a treatment in CHB patients may be related to the frequencies of immune suppressive cells, while the therapeutic targeting of these cells might be effective in boosting anti-HBV immunity.

Thyroid dysfunction incidence and risk factors in Chinese chronic hepatitis B patients treated with pegylated interferon alpha: A long-term follow-up study.

The long-term impact, incidence and risk factors of thyroid dysfunction in chronic hepatitis B (CHB) patients receiving pegylated interferon (IFN) alpha (PegIFN-alpha) therapy remain unclear. We aim to investigate the long-term safety of thyroid dysfunction in CHB patients receiving PegIFN-alpha. A retrospective observational study of 425 CHB patients with normal baseline thyroid function was carried out. Patients were followed up over 10 years to assess thyroid function after receiving IFN. At the end of the IFN therapy, 67 patients (15.8%) had developed thyroid dysfunction, 31 patients (46.3%) had hyperthyroidism and 64.4% presented with subclinical thyroid dysfunction. In follow-up of thyroid dysfunction patients, 37 patients (74.0%) spontaneously regained normal thyroid function. Pretreatment thyroid-stimulating hormone (TSH) level, thyroid peroxidase antibody (TPOAb) positivity and free thyroxine (FT4) were independent risk factors associated with thyroid dysfunction incidence. High TSH level (OR = 9.866, 95%CI, 3.245-29.998) was associated with a greater likelihood of hypothyroidism. High FT4 levels (OR = 0.464, 95%CI, 0.248-0.868) indicate a low likelihood of thyroid dysfunction. Thyroid dysfunction is a common but acceptable side effect of IFN therapy for CHB. Most thyroid dysfunction is reversible. Pretreatment TSH level and TPOAb positivity are risk factors for thyroid dysfunction development during IFN therapy. A high TSH level predicts an increased incidence of hypothyroidism. Moreover, FT4 may be a protective factor for thyroid dysfunction.

STAT4 genetic polymorphism significantly affected HBeAg seroconversion in HBeAg-positive chronic hepatitis B patients receiving Peginterferon-α therapy: A prospective cohort study in China.

A variant in signal transducer and activator of transcription 4 (STAT4) was reported to correlate with the response of interferon-α (IFN-α) in a retrospective study in hepatitis B e antigen (HBeAg)-positive chronic hepatitis B virus (CHB) patients. Here, we conducted a prospective study to analyze the effect of STAT4 genetic polymorphism on the response of pegylated interferon-α-2a (PegIFN-α-2a) in HBeAg-positive patients. A prospective, multicenter, open-label, parallel cohort study was performed. One hundred and fifty treatment-naïve and 156 nucleos(t)ide analog (NA)-experienced HBeAg-positive CHB patients were enrolled, respectively. All patients received PegIFN-α-2a treatment for 48 weeks and 24-week follow-up post PegIFN-α-2a treatment. Before treatment, STAT4 genetic polymorphism was determined by PCR and DNA sequencing. Serological markers, serum HBV DNA levels, and adverse events were collected at each visit. We observed a larger reduction of HBV DNA load and a significantly higher HBeAg seroconversion rate in the GT/TT group than in the GG group at week 72 (p = 0.002 and p = 0.023) in treatment-naïve patients. In NA-experienced patients, the HBeAg seroconversion rate in the GT/TT group was higher than that in the GG group at week 72 (p = 0.005). STAT4 rs7574865 gene polymorphism was the strongest independent predictor of HBeAg seroconversion in both paralleled cohorts. Also, patients in the GT/TT group had a higher hepatitis B surface antigen loss rate than in the GG group in the study. There was no significant difference in adverse events between GG and GT/TT groups. This prospective cohort study confirmed that STAT4 rs7574865 gene polymorphism is associated with HBeAg seroconversion and HBsAg loss irrespective of naïve and NA-experienced HBeAg-positive CHB patients treated with PegIFN-α-2a.

Surface Wettability Tuning of Acrylic Resin Photoresist and Its Aging Performance.

Photoresist is the key material in the fabrication of micropatterns or microstructures. Tuning the surface wettability of photoresist film is a critical consideration in its application of microfluidics. In this work, the surface wettability tuning of acrylic resin photoresist by oxygen plasma or ultra-violet/ozone, and its aging performance in different atmospheres, were systematically studied. The chemical and physical characterizations of the surfaces before and after modification show a dramatic decrease in the C-C group and increase in surface roughness for oxygen plasma treatment, while a decrease of the C-C group was found for the UV/ozone treatment. The above difference in the surface tuning mechanism may explain the stronger hydrophilic modification effect of oxygen plasma. In addition, we found an obvious fading of the wettability tuning effect with an environment-related aging speed, which can also be featured by the decrease of the C-C group. This study demonstrates the dominated chemical and physical changes during surface wettability tuning and its aging process, and provides basis for surface tuning and the applications in microfluidics.

Synaptosomal-associated protein 29 is required for the autophagic degradation of hepatitis B virus.

Hepatitis B virus (HBV) replication and envelopment is dependent on cellular autophagy. Previously, we have provided evidence for the extensive lysosomal degradation of HBV virions and the hepatitis B surface antigen (HBsAg), which is likely controlled by autophagosome-lysosome fusion. Synaptosomal-associated protein 29 (SNAP29) has been identified as a protein specifically mediating autophagosome-lysosome fusion. Thus, in the present study, we addressed the hypothesis that SNAP29 is required for the autophagic degradation of HBV virions and HBsAg. We found that silencing SNAP29 significantly increased the number of autophagosomes and concomitantly promoted HBV replication and HBsAg production. Conversely, SNAP29 overexpression decreased HBV production. Consistent with this, SNAP29 modulated HBV production by interacting with vesicle-associated membrane protein 8 (VAMP8) and synergistically regulated HBV replication with Rab7 complexes. Moreover, the production and release of the small HBsAg is strongly regulated by SNAP29 expression, suggesting that its export occurs partly through the autophagic pathway. Our findings provide new evidence, strongly suggesting that autophagic degradation critically determines the production of HBV virions and HBsAg and that this is controlled by the SNAP29-VAMP8 interaction.-Lin, Y., Wu, C., Wang, X., Liu, S., Kemper, T., Li, F., Squire, A., Zhu, Y., Zhang, J., Chen, X., Lu, M. Synaptosomal-associated protein 29 is required for the autophagic degradation of hepatitis B virus.

Differentially Expressed Intrahepatic Genes Contribute to Control of Hepatitis B Virus Replication in the Inactive Carrier Phase.

Background: The natural history of chronic hepatitis B virus (HBV) infection was divided into 4 phases. Patients in the inactive carrier (IC) status and immune tolerant (IT) phase had normal alanine aminotransferase levels but huge different viral loads. The mechanism underlying low viral replication status in IC phase is unknown. Methods: We determined the intrahepatic transcriptomes of 83 chronic hepatitis B patients by microarray analysis of liver biopsies, and screened the effect of differentially regulated genes on HBV replication using specific small interfering RNAs in vitro. Results: The gene profile distinguishing active chronic hepatitis from IT and IC was predominantly composed of immune-related genes. The liver transcriptomes between the IT and IC phase were largely similar, and 109 expressed genes were significantly different. By performing systematic screening, 5 candidate genes including EVA1A, which were expressed at a relative higher level in IC phase than IT, were identified to regulate HBV replication and gene expression in cellular models. Conclusions: The immune-related pathways were up-regulated in the active chronic hepatitis phase but not in the IT and IC phase. A number of intrahepatic genes highly expressed in the IC phase may participate in the control of HBV replication and determine the inactive status of HBV infection.

Neutrophil gelatinase-associated lipocalin accurately predicts renal tubular injury in patients with chronic hepatitis B treated with nucleos(t)ide analogs.

AIM: Little is known about the impact of long-term use of nucleos(t)ide analogs on tubular function in patients with chronic hepatitis B. Previous studies showed that neutrophil gelatinase-associated lipocalin (NGAL) elevation was associated with renal tubular injury. We evaluated renal function markers and bone mineral density in patients treated long-term with adefovir dipivoxil (ADV) or entecavir (ETV). METHODS: In this cross-sectional study, we enrolled 78 patients (ADV, 36; ETV: 42), and 21 patients matched for age, observation time, and baseline estimated glomerular filtration rate from each group. RESULTS: Patients treated with ADV showed a significant increase in serum creatinine and urine ß2 -microglobulin, and decreased estimated glomerular filtration rate and bone mineral density. Furthermore, the median levels of NGAL in patients treated with ADV were significantly higher than those of ETV (12.5 ng/mL vs. 2.5 ng/mL, P = 0.020). The proportions of patients with proteinuria and phosphate <1 mmol/L in the ADV group were higher than those in the ETV group. Additionally, age, ß2 -microglobulin, phosphate, and ADV use were associated with altered NGAL levels on multivariate analysis. Among the commonly used biomarkers, NGAL was the most useful (odds ratio = 5.72; P = 0.005) and specific (92% specificity at 18.1 ng/mL cut-off) in predicting low bone mass. CONCLUSIONS: Patients with chronic hepatitis B treated long-term with ADV showed elevated urinary NGAL levels. Neutrophil gelatinase-associated lipocalin was more specific in predicting low bone mass during therapy compared with ß2 -microglobulin, phosphate, and creatinine. In general, this analysis examined the value of NGAL as a renal tubular injury indicator, resulting from ADV use.

Role of quantitative hepatitis B core antibody levels in predicting significant liver inflammation in chronic hepatitis B patients with normal or near-normal alanine aminotransferase levels.

AIM: Chronic hepatitis B (CHB) patients with normal alanine aminotransferase (ALT) levels are not free from significant hepatic lesions. Recently, there has been an improved understanding of the clinical significance of quantitative hepatitis B core antibody levels (qAnti-HBc) during CHB management. In this cross-sectional study, we evaluated the utility of qAnti-HBc in identifying significant liver inflammation in CHB patients. METHODS: A total of 469 patients (training set, n = 363; validation set, n = 106) who underwent liver biopsy (LB) were included. The qAnti-HBc levels were quantified and the relationship between histology and serum markers was systematically analyzed. RESULTS: In the training set, qAnti-HBc levels were found to have significant diagnostic value for moderate to severe liver inflammation (≥G2) in all patients (area under the receiver operating characteristic curve [AUROC] = 0.768; 95% confidence interval [CI], 0.721-0.810; P < 0.001) and in patients with normal or near-normal ALT levels (AUROC = 0.767; 95% CI, 0.697-0.828; P < 0.001). Our novel index (AC index) for the identification of ≥G2 inflammation, which combined the qAnti-HBc and ALT levels, significantly improved diagnostic performance (AUROC = 0.813; 95% CI, 0.768-0.852) compared to the use of ALT alone (AUROC = 0.779; 95% CI, 0.732-0.821) in all patients. In the validation set, the AC index showed an improved AUROC of 0.890 (95% CI, 0.814-0.942) and 0.867 (95% CI, 0.749-0.943) in all patients and patients with normal ALT levels, respectively. CONCLUSIONS: The qAnti-HBc level predicts significant liver inflammation well, even in patients with normal or near-normal ALT levels. Compared with the conventional ALT level, the AC index is a more reliable non-invasive biomarker for significant liver inflammation in CHB patients.

Oil Motion Control by an Extra Pinning Structure in Electro-Fluidic Display.

Oil motion control is the key for the optical performance of electro-fluidic displays (EFD). In this paper, we introduced an extra pinning structure (EPS) into the EFD pixel to control the oil motion inside for the first time. The pinning structure canbe fabricated together with the pixel wall by a one-step lithography process. The effect of the relative location of the EPS in pixels on the oil motion was studied by a series of optoelectronic measurements. EPS showed good control of oil rupture position. The properly located EPS effectively guided the oil contraction direction, significantly accelerated switching on process, and suppressed oil overflow, without declining in aperture ratio. An asymmetrically designed EPS off the diagonal is recommended. This study provides a novel and facile way for oil motion control within an EFD pixel in both direction and timescale.

Partial virological response to entecavir treatment in nucleos(t)ide-naïve patients with HBeAg-positive chronic hepatitis B is not caused by reduced sensitivity.

Some patients with chronic hepatitis B (CHB) receiving entecavir (ETV) exhibit partial virological response (PVR) to ETV and the mechanism is not clear. In this study, we aim to investigate the in vitro susceptibility of residual clinical strains isolated from the sera of nucleos(t)ide-naïve hepatitis B virus e antigen (HBeAg)-positive patients with CHB and PVR to ETV, and to evaluate the clinical and virological responses to prolonged ETV monotherapy in these patients. We followed 69 nucleos(t)ide-naïve HBeAg-positive CHB patients receiving ETV treatment, with 13 partial responders to ETV. And we found that no genotypic resistance mutants were detected among the 13 PVR patients. Phenotypic analysis revealed that the residual HBV strains had normal replication capacity, and were as susceptible to ETV as wild-type HBV. All PVR patients continued to receive ETV monotherapy, and serum HBV DNA of the majority became undetectable after prolonged treatment. However, none of these patients achieved HBeAg loss. In contrast, 25.6% and 23.2% of the patients with virological response achieved HBeAg loss (P < 0.001) and HBeAg seroconversion (P < 0.001) at week 144, respectively. Thus, we conclude suboptimal response to ETV might not be due to reduced HBV susceptibility to ETV, and prolonging ETV monotherapy in patients with PVR is recommended.

Comparison of the diversity and structure of the rhizosphere microbial community between the straight and twisted trunk types of Pinus yunnanensis.

Background: Pinus yunnanensis is a major silvicultural species in Southwest China. Currently, large areas of twisted-trunk Pinus yunnanensis stands severely restrict its productivity. Different categories of rhizosphere microbes evolve alongside plants and environments and play an important role in the growth and ecological fitness of their host plant. However, the diversity and structure of the rhizosphere microbial communities between P. yunnanensis with two different trunk types-straight and twisted-remain unclear. Methods: We collected the rhizosphere soil of 5 trees with the straight and 5 trees with the twisted trunk type in each of three sites in Yunnan province. We assessed and compared the diversity and structure of the rhizosphere microbial communities between P. yunnanensis with two different trunk types by Illumina sequencing of 16S rRNA genes and internal transcribed spacer (ITS) regions. Results: The available phosphorus in soil differed significantly between P. yunnanensis with straight and twisted trunks. Available potassium had a significant effect on fungi. Chloroflexi dominated the rhizosphere soils of the straight trunk type, while Proteobacteria was predominant in the rhizosphere soils of the twisted trunk type. Trunk types significantly explained 6.79% of the variance in bacterial communities. Conclusion: This study revealed the composition and diversity of bacterial and fungal groups in the rhizosphere soil of P. yunnanensis with straight and twisted trunk types, providing proper microbial information for different plant phenotypes.

Downregulated VISTA enhances Th17 differentiation and aggravates inflammation in patients with acute-on-chronic liver failure.

BACKGROUND AND AIMS: Persistent inflammatory response and immune activation are the core mechanisms underlying acute-on-chronic liver failure (ACLF). Previous studies have shown that deficiency of V-type immunoglobulin domain-containing suppressor of T-cell activation (VISTA) exacerbates the progression of inflammatory diseases. We aimed to clarify the role of VISTA in the pathogenesis of ACLF. METHODS: Blood and liver samples were collected from healthy subjects, stable cirrhosis, and ACLF patients to characterize VISTA expression and function. An ACLF mouse model was used to ascertain potential benefits of anti-VISTA monoclonal antibody (mAb) treatment. RESULTS: VISTA expression was significantly reduced in the naïve and central memory CD4+ T cells from patients with ACLF. The expression of VISTA on CD4+ T cells was associated with disease severity and prognosis. VISTA downregulation contributed to the activation and proliferation of CD4+ T cells and enhanced the differentiation of T helper 17 cells (Th17) and secretion of inflammatory cytokines through the activated Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) signaling pathway. Moreover, agonistic anti-VISTA mAb treatment inhibited the activation and cytokine production of CD4+ T cells and reduced mortality and liver inflammation of the ACLF mice. CONCLUSIONS: The decreased expression of VISTA may facilitate development of Th17 cells and promote the progression of inflammation in ACLF patients. These findings are helpful for elucidating the pathogenesis of ACLF and for the identification of new drug targets.

Comparison of Pegylated Interferon Alfa Therapy in Combination with Tenofovir Alafenamide Fumarate or Tenofovir Disoproxil Fumarate for Treatment of Chronic Hepatitis B Patients.

Purpose: The study aims to evaluate the effectiveness of a tenofovir alafenamide fumarate (TAF) and pegylated interferon alfa (PegIFN-α) regimen compared to a tenofovir disoproxil fumarate (TDF) and PegIFN-α therapy in patients with chronic hepatitis B (CHB). Patients and Methods: Patients who were treated with PegIFN-α in combination with TAF or TDF were retrospectively enrolled. The primary outcome measured was the HBsAg loss rate. The rates of virological response, serological response for HBeAg, and normalization of alanine aminotransferase (ALT) were also calculated. The cumulative incidences of response rates were compared between the two groups using Kaplan-Meier analysis. Results: A total of 114 patients were retrospectively enrolled in the study, with 33 receiving TAF plus PegIFN-α treatment and 81 receiving TDF plus PegIFN-α treatment. The HBsAg loss rate for the TAF plus PegIFN-α group was 15.2% at 24 weeks and 21.2% at 48 weeks, while the TDF plus PegIFN-α group had rates of 7.4% at 24 weeks and 12.3% at 48 weeks (P=0.204 at 24 weeks, P=0.228 at 48 weeks). In subgroup analysis of HBeAg positive patients, the TAF group had a higher HBsAg loss rate of 25% at week 48, compared to 3.8% in the TDF group (P=0.033). According to Kaplan-Meier analysis, the TAF plus PegIFN-α group achieved virological response more quickly than the TDF plus PegIFN-α group (p=0.013). There was no statistical difference in HBeAg serological rate or ALT normalization rate. Conclusion: There was no significant difference in the HBsAg loss between the two groups. However, subgroup analysis revealed that TAF plus PegIFN-α treatment had a higher HBsAg loss rate than TDF plus PegIFN-α treatment in HBeAg-positive patients. Additionally, TAF plus PegIFN-α treatment demonstrated better virological suppression for CHB patients. Therefore, TAF plus PegIFN-α treatment regimen is recommended for CHB patients who aim to achieve functional cure.

End-of-treatment anti-HBs levels and HBeAg status identify durability of HBsAg loss after PEG-IFN discontinuation.

Background: Hepatitis B surface antigen (HBsAg) loss, namely, the functional cure, can be achieved through the pegylated interferon (PEG-IFN)-based therapy. However, it is an unignorable fact that a small proportion of patients who achieved functional cure develop HBsAg reversion (HRV) and the related factors are not well described. Methods: A total of 112 patients who achieved PEG-IFN-induced HBsAg loss were recruited. HBV biomarkers and biochemical parameters were examined dynamically. HBV RNA levels were assessed in the cross-sectional analysis. The primary endpoint was HRV, defined as the reappearance of HBsAg after PEG-IFN discontinuation. Results: HRV occurred in 17 patients during the follow-up period. Univariable analysis indicated that hepatitis B e antigen (HBeAg) status, different levels of hepatitis B surface antibody (anti-HBs), and hepatitis B core antibody (anti-HBc) at the end of PEG-IFN treatment (EOT) were significantly associated with the incidence of HRV through using the log-rank test. Additionally, time-dependent receiver operating characteristic (ROC) analysis showed that the anti-HBs was superior to anti-HBc in predictive power for the incidence of HRV during the follow-up period. Multivariable Cox proportional hazard analysis found that anti-HBs ≥1.3 log10IU/L (hazard ratio (HR), 0.148; 95% confidence interval (CI), 0.044-0.502) and HBeAg negativity (HR, 0.183; 95% CI, 0.052-0.639) at EOT were independently associated with lower incidence of HRV. Cross-sectional analysis indicated that the HBV RNA levels were significantly correlated with the HBsAg levels in patients with HRV (r=0.86, p=0.003). Conclusions: EOT HBeAg negativity and anti-HBs ≥1.3 log10IU/L identify the low risk of HRV after PEG-IFN discontinuation.

Hepatitis B virus promotes its own replication by enhancing RAB5A-mediated dual activation of endosomal and autophagic vesicle pathways.

Chronic hepatitis B virus (HBV) infection remains one of the major global public health concerns, and it develop into liver fibrosis, cirrhosis, and hepatocellular carcinoma. Recent evidence suggests that endosomal and autophagic vesicles are beneficial for HBV replication. However, it has not been well elucidated how HBV exploits such intracellular vesicle systems for its replication. RAB5A, a member of small GTPase family, plays crucial roles in early endosome biogenesis and autophagy initiation. We observed that RAB5A mRNA and protein levels were significantly increased in HBV-expressing hepatoma cell lines as well as in liver tissue samples from chronic HBV-infected patients. Moreover, RAB5A silencing inhibited HBV replication and subviral particle (SVP) expression significantly in HBV-transfected and -infected hepatoma cells, whereas RAB5A overexpression increased them. Mechanistically, RAB5A increases HBV replication through enhancement of early endosome (EE) - late endosome (LE) activation by interacting with EEA1, as well as enhancing autophagy induction by interacting with VPS34. Additionally, HBV infection enhances RAB5A-mediated dual activation of EE-LE system and autophagy. Collectively, our findings highlight that HBV utilizes RAB5A-mediated dual activation of endosomal and autophagic vesicle pathways for its own replication and persistence. Therefore, RAB5A is a potential target for chronic HBV infection treatment.

Human hepatocyte-enriched miRNA-192-3p promotes HBV replication through inhibiting Akt/mTOR signalling by targeting ZNF143 in hepatic cell lines.

Previous studies have revealed multiple tissue- or cell-specific or enriched miRNA profiles. However, miRNA profiles enriched in hepatic cell types and their effect on HBV replication have not been well elucidated. In this study, primary human hepatocytes (PHHs), Kupffer cells (KCs), liver sinusoidal endothelial cells (LSECs), and hepatic stellate cells (HSCs) were prepared from liver specimens of non-HBV-infected patients. Four hepatic cell type-enriched miRNA profiles were identified from purified liver cells miRNA microarray assay. The results revealed that 12 miRNAs, including miR-122-5p and miR-192-3p were PHH-enriched; 9 miRNAs, including miR-142-5p and miR-155-5p were KC-enriched; 6 miRNAs, including miR-126-3p and miR-222-3p were LSEC-enriched; and 14 miRNAs, including miR-214-3p and miR-199a-3p were HSC-enriched. By testing the effect of 11 PHH-enriched miRNAs on HBV production, we observed that miR-192-3p had the greatest pro-virus effect in hepatic cell lines. Moreover, we further found that miR-192-3p promoted HBV replication and gene expression through inhibiting Akt/mTOR signalling by direct targeting of ZNF143 in HepG2.2.15 cells. Additionally, the serum and hepatic miR-192-3p expression levels were significantly higher in chronic hepatitis B patients than in healthy controls and serum miR-192-3p positively correlated with the serum levels of HBV DNA and HBsAg. Collectively, we identified miRNA profiles enriched in four hepatic cell types and revealed that PHH-enriched miR-192-3p promoted HBV replication through inhibiting Akt/mTOR signalling by direct targeting of ZNF143 in hepatic cell lines. Our study provides a specific perspective for the role of hepatic cell type-enriched miRNA in interaction with viral replication and various liver pathogenesis.

CCDC88A/GIV promotes HBV replication and progeny secretion via enhancing endosomal trafficking and blocking autophagic degradation.

Hepatitis B virus (HBV) particles are thought to be secreted from hepatocytes through multivesicular bodies (MVBs); however, the cellular trafficking mechanisms prior to this process remain elusive. It has been reported that CCDC88A/GIV expression, which is involved in multiple aspects of vesicular trafficking, changes dynamically at different phases of chronic HBV infection. In this study, we focused on the role of CCDC88A/GIV in HBV replication. In the liver tissues of chronically HBV-infected patients, HBV infection significantly enhanced CCDC88A/GIV expression, and increased endoplasmic reticulum (ER) stress and autophagosome formation without changing endosome formation. Additionally, colocalization of SHBsAg with early endosomes (~30.2%) far exceeded that with autophagosomes (~3.2%). In hepatoma cells, CCDC88A/GIV and its downstream proteins, DNM2 (dynamin 2; a CCDC88A/GIV effector), CLTC and RAB5A significantly enhanced HBV replication and endosome formation but inhibited autophagosome formation. Blocking endocytosis disrupted HBsAg trafficking to endosomes and caused its accumulation in the ER lumen, which triggered ER stress to initiate the unfolded protein response (UPR). Therefore, HBsAg trafficking into autophagosomes was increased, and the lysosomal activity and maturation, which was inhibited by HBV infection, were restored. Meanwhile, core particles were prevented from entering MVBs. CCDC88A/GIV and its other effector, GNAI3, decreased autophagic flux by enhancing the insulin-induced AKT-MTOR pathway, thereby inhibiting HBV antigens autophagic degradation. In conclusion, CCDC88A/GIV enhanced HBV replication by increasing endosomal trafficking and reducing autophagic degradation of HBV antigens, suggesting that CCDC88A/GIV-mediated endosomal trafficking plays an important role in HBV replication and progeny secretion.Abbreviations: ACTB: actin beta; AO: acridine orange; ATF6: activating transcription factor 6; CCDC88A/GIV: coiled-coil domain containing 88A; CLTC: clathrin heavy chain; CQ: chloroquine; DAPI: 4',6-diamidino-2-phenylindole; DNM2: dynamin 2; ER: endoplasmic reticulum; ERN1: endoplasmic reticulum to nucleus signaling 1; EIF2A: eukaryotic translation initiation factor 2A; FBS: fetal bovine serum; GNAI3: G protein subunit alpha i3; HBV: hepatitis B virus; HBV RIs: HBV replication intermediates; HBcAg: HBV core protein; HBsAg: HBV surface antigen; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MVBs: multivesicular bodies; MTOR: mechanistic target of rapamycin kinase; PDI: protein disulfide isomerase; PHH: primary human hepatocyte; pSM2: a HBV replication-competent plasmid; HSPA5/BIP: heat shock protein family A (Hsp70) member 5; SQSTM1/p62: sequestosome 1; siRNA: small interfering RNA; SEM: standard error of the mean; UPR: unfolded protein response.