Analysis and comparison of a spinal cord injury model with a single-axle-lever clip or a parallel-moving clip compression in rats.

STUDY DESIGN: Experimental animal study. OBJECTIVES: To assess the feasibility of a custom-designed parallel-moving (PM) clip, compared with a single-axle-lever (SAL) clip, for the development of a compressional spinal cord injury (SCI) model in rats. SETTING: Hospital laboratory in China. METHODS: We used a PM clip and a SAL clip with same compression rate, to develop a SCI model in rats, and set a sham group as a blank control. Within 3 weeks, each group of rats was evaluated for behavioral (Basso-Beattie-Bresnahan locomotor rating score, BBB), and electrophysiological changes (somatosensory evoked potential), and historical staining to observe the differences between the three groups. In particular, the mechanical results of the PM group were calculated. RESULTS: The BBB scores for the SAL and PM groups were significantly lower than those for the sham group (P < 0.05), no significant difference between the two methods (P > 0.05), but the values corresponding to the PM group had smaller standard deviations. The interpeak-latency (IPL) was significantly prolonged (P < 0.0001) and the peak-peak amplitude (PPA) was significantly reduced (P < 0.01) in SAL and PM groups than those in the sham group, but there was no statistical difference in both IPL and PPA between the two SCI groups (P > 0.05). Histological staining showed obvious pathological changes in two SCI groups, and the shape of the lesion zone in the PM group was more symmetrical than that in the SAL groups. CONCLUSIONS: The use of a compressional SCI model in rats with the PM clip we designed is an appropriate method to quantify the injury. The degree of the injury caused by this clip is more stable and uniform than those with classical methods.

Genotype Distribution and Molecular Epidemiology of Hepatitis C Virus in Guangzhou, China: Predominance of Genotype 1b and Increasing Incidence of Genotype 6a.

BACKGROUND/AIMS: Distribution of Hepatitis C virus (HCV) genotypes vary geographically and may associate with the mode of transmission. Little is known about the molecular epidemiology of HCV infection in Guangzhou, China. METHODS: A cross-sectional survey included 561 subjects with chronic HCV infection registered at Nanfang Hospital, Southern Medical University, was performed. All residents were invited for a questionnaire interview to collect information about their personal status and commercial blood donation history. RESULTS: A total of 463 chronic hepatitis C (CHC) patients were finally enrolled. Among the 463 samples, 426 were characterized by partial core-E1 sequences and classified into 7 subtypes: 1b (n=263, 61.7%), 6a (n=86, 20.2%), 2a (n=26, 6.1%), 3b (n=26, 6.1%), 3a (n=22, 5.2%), 6u (n=2, 0.5%), and 4a (n=1, 0.2%). Analysis of genotype-associated risk factors revealed that blood donation and transfusion were strongly associated with subtypes 1b and 2a, while genotype 3b and 6a were more frequent in intravenous drug users. CONCLUSIONS: Phylogeographic analyses demonstrated that the distribution of HCV genotypes in Guangzhou is complex. Interestingly, 6a has become a local endemic in Guangzhou and may be the second source region to disseminate 6a to other provinces.

A different inhibitor is required for overcoming entecavir resistance: a comparison of four rescue therapies in a retrospective study.

AIMS: Little clinical data are available regarding re-establishing the effective inhibition of entecavir (ETV)-resistant mutants. In this retrospective study, we aimed to compare the efficacies of four treatment regimens as rescue therapy for those chronic hepatitis B (CHB) patients with ETV resistance. METHODS: A total of 65 patients with ETV resistance were assigned either with tenofovir disoproxil fumarate (TDF) monotherapy (n = 21), ETV (0.5 mg) plus adefovir (ADV) combination therapy (n = 19), ETV (1.0 mg) monotherapy (n = 11) or ETV (0.5 mg) plus TDF combination therapy (n = 14). The efficacy and safety of four treatment regimens were compared. RESULTS: There were no significant differences among the four study groups in baseline characteristics, including HBV DNA levels (χ2  = 0.749, P = 0.862) and hepatitis B e antigen-positivity (χ2  = 0.099, P = 0.992). The median reduction in serum HBV DNA level from baseline at week 48 was -2.37 ± 1.07 log10 IU ml-1 , -2.16 ± 0.81 log10 IU ml-1 , -1.17 ± 1.23 log10 IU ml-1 and -2.49 ± 1.10 log10 IU ml-1 , respectively (F = 4.078, P = 0.011). The TDF group and ETV (0.5 mg) + TDF group have the highest undetectable HBV DNA rate (76.19% vs. 78.57%) compared to the ETV (0.5 mg) + ADV group and the ETV (1.0 mg) group (63.16% vs. 18.18%, respectively). Two patients in the ETV (1.0 mg) group experienced virological breakthrough at week 48 and was attributed to poor drug adherence. CONCLUSIONS: TDF monotherapy appeared to deliver the highest undetectable HBV DNA rate in patients with ETV resistance, and ADV plus ETV combination therapy could be another choice for patients with financial restraint.

Integrative functional genomics of hepatitis C virus infection identifies host dependencies in complete viral replication cycle.

Recent functional genomics studies including genome-wide small interfering RNA (siRNA) screens demonstrated that hepatitis C virus (HCV) exploits an extensive network of host factors for productive infection and propagation. How these co-opted host functions interact with various steps of HCV replication cycle and exert pro- or antiviral effects on HCV infection remains largely undefined. Here we present an unbiased and systematic strategy to functionally interrogate HCV host dependencies uncovered from our previous infectious HCV (HCVcc) siRNA screen. Applying functional genomics approaches and various in vitro HCV model systems, including HCV pseudoparticles (HCVpp), single-cycle infectious particles (HCVsc), subgenomic replicons, and HCV cell culture systems (HCVcc), we identified and characterized novel host factors or pathways required for each individual step of the HCV replication cycle. Particularly, we uncovered multiple HCV entry factors, including E-cadherin, choline kinase α, NADPH oxidase CYBA, Rho GTPase RAC1 and SMAD family member 6. We also demonstrated that guanine nucleotide binding protein GNB2L1, E2 ubiquitin-conjugating enzyme UBE2J1, and 39 other host factors are required for HCV RNA replication, while the deubiquitinating enzyme USP11 and multiple other cellular genes are specifically involved in HCV IRES-mediated translation. Families of antiviral factors that target HCV replication or translation were also identified. In addition, various virologic assays validated that 66 host factors are involved in HCV assembly or secretion. These genes included insulin-degrading enzyme (IDE), a proviral factor, and N-Myc down regulated Gene 1 (NDRG1), an antiviral factor. Bioinformatics meta-analyses of our results integrated with literature mining of previously published HCV host factors allows the construction of an extensive roadmap of cellular networks and pathways involved in the complete HCV replication cycle. This comprehensive study of HCV host dependencies yields novel insights into viral infection, pathogenesis and potential therapeutic targets.

Rethinking the pathogenesis of hepatitis B virus (HBV) infection.

Chronic hepatitis B virus (HBV) infection affects approximately 375 million people worldwide. Current antiviral treatment effectively controls, but rarely clears chronic HBV infection. In addition, a significant portion of chronic HBV infected patients are not suitable for currently available antiviral therapy, and still face higher risk for cirrhosis and hepatocellular carcinoma. The poorly understood pathogenesis of HBV infection is the main barrier for developing more effective treatment strategies. HBV has long been viewed as non-cytopathic and the central hypothesis for HBV pathogenesis lies in the belief that hepatitis B is a host specific immunity-mediated liver disease. However, this view has been challenged by the accumulating experimental and clinical data that support a model of cytopathic HBV replication. In this article we systematically review the pathogenic role of HBV replication in hepatitis B and suggest possible HBV replication related mechanisms for HBV-mediated liver injury. We propose that a full understanding of HBV pathogenesis should consider the following elements. I. Liver injury can be caused by high levels of HBV replication and accumulation of viral products in the infected hepatocytes. II. HBV infection can be either directly cytopathic, non-cytopathic, or a mix of both in an individual patient depending upon accumulation levels of viral products that are usually associated with HBV replication activity in individual infected hepatocytes.

Replication-driven HBV cccDNA loss in chimeric mice with humanized livers.

Hepatitis B virus (HBV) infection is largely noncytopathic and requires the establishment of covalently closed circular DNA (cccDNA), which is considered stable in the nuclei of infected cells. Although challenging, approaches to directly target cccDNA molecules or kill infected cells are recommended to eliminate cccDNA. Herein, cccDNA levels were investigated in HBV-infected chimeric mice with humanized livers. HBV-infected cells support robust replication, progressively retain viral products, and head for cytopathic destruction and cccDNA loss. It is difficult for infected cells to retain cccDNA and remain noncytopathic. Replication-driven cccDNA loss is observed at both phases of spread of and persistent infection. The cccDNA replenishment is required to compensate for cccDNA loss. Blocking cccDNA replenishment pathways reduces cccDNA levels by >100-fold. These results prove an unconventional cccDNA elimination strategy that does not directly target cccDNA but aims to transform spontaneous cccDNA loss into progressive cccDNA elimination by blocking cccDNA replenishment.

Epidemiological characteristics of traumatic spinal cord injuries in a multicenter retrospective study in northwest China, 2017-2020.

Background: Traumatic spinal cord injuries (TSCIs) are worldwide public health problems that are difficult to cure and impose a substantial economic burden on society. There has been a lack of extensive multicenter review of TSCI epidemiology in northwest China during the Corona Virus Disease 2019 (COVID-19) pandemic. Method: A multicenter retrospective study of 14 selected hospitals in two provinces in northwest China was conducted on patients admitted for TSCI between 2017 and 2020. Variables assessed included patient demographics, etiology, segmental distribution, treatment, waiting time for treatment, and outcomes. Results: The number of patients with TSCI showed an increasing trend from 2017 to 2019, while there were 12.8% fewer patients in 2020 than in 2019. The male-to-female ratio was 3.67:1, and the mean age was 48 ± 14.9 years. The primary cause of TSCI was high falls (38.8%), slip falls/low falls (27.7%), traffic accidents (23.9%), sports (2.6%), and other factors (7.0%). The segmental distribution showed a bimodal pattern, peak segments were C6 and L1 vertebra, L1 (14.7%), T12 (8.2%), and C6 (8.2%) were the most frequently injured segments. In terms of severity, incomplete injury (72.8%) occurred more often than complete injury (27.2%). The American Spinal Injury Association impairment scale of most patients did not convert before and after treatment in the operational group (71.6%) or the conservative group (80.6%). A total of 975 patients (37.2%) from urban and 1,646 patients (62.8%) from rural areas were included; almost all urban residents could rush to get treatment after being injured immediately (<1 h), whereas most rural patients get the treatment needed 4-7 h after injury. The rough annual incidence from 2017 to 2020 is 112.4, 143.4, 152.2, and 132.6 per million people, calculated by the coverage rate of the population of the sampling hospital. Conclusion: The incidence of TSCI in northwest China is high and on the rise. However, due to pandemic policy reasons, the incidence of urban residents decreased in 2020. The promotion of online work may be an effective primary prevention measure for traumatic diseases. Also, because of the further distance from the good conditional hospital, rural patients need to spend more time there, and the timely treatment of patients from remote areas should be paid attention to.

Necroptosis of nucleus pulposus cells involved in intervertebral disc degeneration through MyD88 signaling.

Background context: Low back pain, affecting nearly 40% of adults, mainly results from intervertebral disc degeneration (IVDD), while the pathogenesis of IVDD is still not fully elucidated. Recently, some researches have revealed that necroptosis, a programmed necrosis, participated in the progression of IVDD, nevertheless, the underlying mechanism remains unclear. Purpose: To study the mechanism of necroptosis of Nucleus Pulposus (NP) cells in IVDD, focusing on the role of MyD88 signaling. Study design: The expression and co-localization of necroptotic indicators and MyD88 were examined in vivo, and MyD88 inhibitor was applied to determine the role of MyD88 signaling in necroptosis of NP cells in vitro. Methods: Human disc specimens were collected from patients receiving diskectomy for lumbar disc herniation (LDH) or traumatic lumbar fractures after MRI scanning. According to the Pfirrmann grades, they were divided into normal (Grades 1, 2) and degenerated groups (4, 5). Tissue slides were prepared for immunofluorescence to assess the co-localization of necroptotic indicators (RIP3, MLKL, p-MLKL) and MyD88 histologically. The combination of TNFα, LPS and Z-VAD-FMK was applied to induce necroptosis of NP cells. Level of ATP, reactive oxygen species (ROS), live-cell staining and electron microscope study were employed to study the role of MyD88 signaling in necroptosis of NP cells. Results: In vivo, the increased expression and co-localization of necroptotic indicators (RIP3, MLKL, p-MLKL) and MyD88 were found in NP cells of degenerated disc, while very l low fluorescence intensity in tissue of traumatic lumbar fractures. In vitro, the MyD88 inhibitor effectively rescued the necroptosis of NP cells, accompanied by increased viability, ATP level, and decreased ROS level. The effect of MyD88 inhibition on necroptosis of NP cells was further confirmed by ultrastructure of mitochondria shown by Transmission Electron Microscope (TEM). Conclusion: Our results indicated that the involvement of MyD88 signaling in the necroptosis of NP cells in IVDD, which will replenish the pathogenesis of IVDD and provide a novel potential therapeutic target for IVDD.

Novel function of CD81 in controlling hepatitis C virus replication.

The mechanisms of hepatitis C virus (HCV) replication remain poorly understood, and the cellular factors required for HCV replication are yet to be completely defined. CD81 is known to mediate HCV entry. Our study uncovered an unexpected novel function of CD81 in the HCV life cycle that is important for HCV RNA replication. HCV replication occurred efficiently in infected cells with high levels of CD81 expression. In HCV-infected or RNA-transfected cells with low levels of CD81 expression, initial viral protein synthesis occurred normally, but efficient replication failed to proceed. The aborted replication could be restored by the transient transfection of a CD81 expression plasmid. CD81-dependent replication was demonstrated with both an HCV infectious cell culture and HCV replicon cells of genotypes 1b and 2a. We also showed that CD81 expression is positively correlated with the kinetics of HCV RNA synthesis but inversely related to the kinetics of viral protein production, suggesting that CD81 may control viral replication by directing viral RNA template function to RNA replication. Thus, CD81 may be necessary for the efficient replication of the HCV genome in addition to its role in viral entry.

Effect of Pegylated Interferon Plus Tenofovir Combination on Higher Hepatitis B Surface Antigen Loss in Treatment-naive Patients With Hepatitis B e Antigen -positive Chronic Hepatitis B: A Real-world Experience.

PURPOSE: The loss of serum hepatitis B surface antigen (HBsAg) in patients with chronic hepatitis B (CHB) is considered an ideal clinical outcome but rarely achieved with current standard of care. We evaluated the effectiveness in inducing HBsAg seroclearance in a real-world clinical cohort of Chinese patients with CHB treated with a combination of pegylated interferon (Peg-IFN) with tenofovir disoproxil fumarate (TDF) or monotherapy with each agent. METHODS: A total of 330 patients with CHB were assigned to receive Peg-IFN plus TDF for 48 weeks (Peg-IFN plus TDF group), Peg-IFN alone for 48 weeks (Peg-IFN group), or TDF alone for 144 weeks (TDF group). The primary end point was the percentages of patients who achieved HBsAg seroclearance at week 72. Differences from the baseline characteristics and treatment data were compared using the χ2 test for categorical variables or 1-way ANOVA for continuous variables. A Kaplan-Meier test was performed to compare the HBsAg loss among the 3 groups. Discrimination of responders versus nonresponders was quantified using AUC curves. Optimal cut-offs were selected based on Youden's J statistic defined as J = sensitivity + specificity-1. FINDINGS: At week 72, the Kaplan-Meier cumulative HBsAg loss was 11.5% in the Peg-IFN plus TDF group, 5.7% in the Peg-IFN group, and 0% in the TDF group. The percentage of patients with HBsAg loss was comparable in the Peg-IFN plus TDF and Peg-IFN groups (P = 0.143), but both were significantly higher than that in the TDF group (P = 0.000 and P = 0.010). In addition, a significantly higher percentage of patients in the combination group and Peg-IFN group had serum HBsAg of <100 IU/mL compared with the TDF group (32.7% vs 23.6% vs 9.2%; P < 0.001) but no significant differences in the percentages of patients with HBsAg <1000 IU/mL, the undetectable serum HBV DNA and hepatitis B e antigen seroconversion. Our model predicted serum HBsAg loss at week 72 (AUC = 0.846) if the HBsAg level was reduced by > 1.5 log10 IU/mL from baseline at treatment week 24, an optimal timepoint for prediction of HBsAg loss in this cohort. IMPLICATIONS: A 48-week course of Peg-IFN and TDF combination therapy led to profound reduction in serum HBsAg level, resulting in a significantly higher rate of HBsAg loss compared with TDF monotherapy. Patients with steep HBsAg decline >1.5 log10 IU/mL at week 24 well signaled a higher probability of achieving HBsAg loss at week 72.

Preoperative Gadoxetic Acid-Enhanced MRI Based Nomogram Improves Prediction of Early HCC Recurrence After Ablation Therapy.

PURPOSE: This study aimed to identify preoperative gadoxetic acid-enhanced MRI features and establish a nomogram for predicting early recurrence (≤ 2 years) of hepatocellular carcinoma (HCC) after ablation therapy. METHODS: A total of 160 patients who underwent gadoxetic acid-enhanced MRI and ablation HCC therapy from January 2015 to June 2018, were included retrospectively and divided into a training cohort (n = 112) and a validation cohort (n = 48). Independent clinical risk factors and gadoxetic acid-enhanced MRI features associated with early recurrence were identified by univariate and multivariate logistic regression analysis and used for construction of a nomogram. The performance of the nomogram was evaluated by discrimination, calibration, and clinical utility. RESULTS: Alpha-fetoprotein (AFP) level, tumor number, arterial peritumoral enhancement, satellite nodule and peritumoral hypointensity at hepatobiliary phases in the training cohort were identified as independent risk factors for early recurrence after ablation. A new nomogram that was constructed with these five features showed an area under the curve (AUC) of 0.843 (95%CI 0.771-0.916) and 0.835 (95%CI 0.713-0.956) in the training and validation cohort, respectively. The calibration curve and decision curve analysis (DCA) suggested that the nomogram had good consistency and clinical utility. CONCLUSIONS: A new nomogram that was constructed using four preoperative gadoxetic acid-enhanced MRI features and serum AFP level can predict the risk of early HCC recurrence after ablation therapy with AUC up to 0.843. The strong performance of this nomogram may help hepatologists to categorize patients' recurrent risk to guide selecting treatment options and improve postoperative management.

Amphipathic DNA polymers inhibit hepatitis C virus infection by blocking viral entry.

BACKGROUND & AIMS: Hepatitis C virus (HCV) gains entry into susceptible cells by interacting with cell surface receptor(s). Viral entry is an attractive target for antiviral development because of the highly conserved mechanism. METHODS: HCV culture systems were used to study the effects of phosphorothioate oligonucleotides (PS-ONs), as amphipathic DNA polymers (APs), on HCV infection. The in vivo effects of APs were tested in urokinase plasminogen activator (uPA)/severe combined immunodeficient (SCID) mice engrafted with human hepatocytes. RESULTS: We show the sequence-independent inhibitory effects of APs on HCV infection. APs were shown to potently inhibit HCV infection at submicromolar concentrations. APs exhibited a size-dependent antiviral activity and were equally active against HCV pseudoparticles of various genotypes. Control phosphodiester oligonucleotide (PO-ON) polymer without the amphipathic structure was inactive. APs had no effect on viral replication in the HCV replicon system or binding of HCV to cells but inhibited viral internalization, indicating that the target of inhibition is at the postbinding, cell entry step. In uPA/SCID mice engrafted with human hepatocytes, APs efficiently blocked de novo HCV infection. CONCLUSIONS: Our results demonstrate that APs are a novel class of antiviral compounds that hold promise as a drug to inhibit HCV entry.

AFP specificity for HCC surveillance is increased by mitigating liver injury among treated chronic hepatitis B patients with elevated AFP.

OBJECTIVE: The aim of this study was to assess AFP response in chronic hepatitis B (CHB) patients with baseline positive AFP (≥7 ng/mL) who received antiviral therapy thereafter. METHODS: A cohort study was conducted to assess AFP response in CHB patients who had baseline positive AFP and got antiviral therapy. RESULTS: This retrospective study enrolled 302 antiviral-treatment-naïve CHB patients with positive AFP. After a 12-month antiviral treatment, 144 patients normalized AFP during follow-up while the rest remained AFP-positive. There were no significant differences in baseline characteristics and virologic and ALT responses to antiviral therapy between the two groups. During a mean follow-up of 34 ± 6 months, 16 patients (5.3%) in this cohort developed HCC, and 14 (8.9%) of them emerged in the AFP positive group. There was a significant difference (P=0.004) in HCC occurrence between AFP normalized and non-normalized groups after treatment. Univariate and multivariate analyses revealed that cirrhosis (HR=9.983, 95% CI=3.609-27.617, P<0.001), and non-AFP response to antiviral treatment (HR=6.517, 95% CI=1.475-28.784, P=0.013) were two independent factors associated with HCC occurrence. CONCLUSIONS: To our knowledge, this is the first investigator-initiated cohort study to assess the performance of on-treatment AFP in CHB patients with baseline positive AFP. In contrast to the criticism that AFP is neither sensitive nor specific, the current study has provided important evidence that on-antiviral-treatment AFP normalization is a specific protective marker for HCC in patients with HBV-related chronic liver diseases who started antiviral therapy thereafter.

Serum­derived hepatitis C virus can infect human glioblastoma cell line SF268 and activate the PI3K­Akt pathway.

Extra­hepatic manifestations are frequently observed in hepatitis C virus (HCV)­infected patients; however the underlying mechanisms remain largely unknown. In the present study, the human glioblastoma SF268 cell line (the precise origin of the cell type is not clear) was infected with HCV using HCV­positive serum, and viral replication was assessed by immunofluorescence, reverse transcription­polymerase chain reaction (PCR), quantitative PCR and western blotting following infection. HCV core protein and HCV RNA were detected in HCV­positive serum­infected SF268 cells at day 4 post­infection, while no infection was observed in cells exposed to HCV­negative serum. The mean HCV RNA levels at day 4 post­infection were up to 5.00 IU/ml log10; however, HCV RNA and immunostaining for core protein were negative when cultured to day 6 or longer. The data suggest that human glioblastoma SF268 cells were transiently infected with HCV. AKT serine/threonine kinase phosphorylation was also detected in HCV­infected SF268 cells at day 4 post­infection. To the best of our knowledge, this is the first demonstration that a human glioblastoma cell line can be infected with serum­derived HCV. The results provide evidence that HCV infection can occur in cells of the central nervous system. Neurological disorder­associated phosphoinositide 3­kinase­AKT signaling pathway was activated in parallel with HCV infection, suggesting that SF268 may serve as an in vitro model for investigating HCV­nervous system cell interactions.

Sofosbuvir-Based Therapies for Patients with Hepatitis C Virus Infection: Real-World Experience in China.

Background and Aims: There is scarcity of data in literature regarding the treatment response to sofosbuvir- (SOF-) based therapies in Chinese patients with chronic Hepatitis C Virus (HCV) infection. The aim of this study was to evaluate the efficacy and safety of SOF-based regimens for chronic hepatitis C (CHC) patients without cirrhosis in a real-world setting in mainland China. Methods: A total of 226 patients receiving SOF plus daclatasvir (DCV), ledipasvir (LDV), or velpatasvir (VEL) were enrolled from December 2014 to June 2017. The primary observation point was the percentage of patients with a sustained virologic response (SVR) at posttreatment week 12 (SVR12), and all adverse events were monitored during treatment and follow-up period. Results: The overall SVR12 rate was 96% (216/226), and individual SVR12 ranged from 93% to 100% in different treatment groups. No significant differences of efficacy were detected between genotypes 1b and 6a (98% for GT 1b versus 100% for GT 6a, P=0.322). Comparing the high success rates in GT 1b and 6a patients, SVR12 was relatively low in GT 3a and 3b patients. A significant difference in efficacy was observed between GT 3 and not GT 3 patients (77% versus 98%, respectively, P<0.001). No significant differences in efficacy were detected among different regimens (93% versus 97% versus 100%, respectively, P=0.153), gender (95% for male versus 96% for female, P=0.655), or baseline HCV RNA lever (96% versus 95%, respectively, P=0.614). Similar SVR rates were also obtained in naïve and previously treated patients (98% versus 93%, respectively, P=0.100). Conclusions: NS5B polymerase inhibitor SOF plus one of the NS5A inhibitors, such as DCV, LDV, or VEL for 12 weeks was associated with high SVR12 rates and well tolerated in HCV-infected patients without cirrhosis. Moreover, patients with DAAs failure should be retreated with more effective regimens like SOF/VEL.

Duck Hepatitis B Virus cccDNA Amplification Efficiency in Natural Infection Is Regulated by Virus Secretion Efficiency.

Previous mutation based studies showed that ablating synthesis of viral envelope proteins led to elevated hepadnaviral covalently closed circular DNA (cccDNA) amplification, but it remains unknown how cccDNA amplification is regulated in natural hepadnaviral infection because of a lack of research system. In this study we report a simple procedure to prepare two identical duck hepatitis B virus inocula, but they possess 10-100-fold difference in cccDNA amplification in infected cell culture. We demonstrate that the infected cells with higher cccDNA amplification significantly reduce the virus secretion efficiency that results in higher accumulation of relaxed circular DNA (rcDNA) and DHBsAg in the cells. The infected cells with lower cccDNA amplification significantly increase the virus secretion efficiency that leads to lower intracellular rcDNA and DHBsAg accumulation. In contrast with the findings generated in the mutation based experimental system, the regulation of cccDNA amplification in natural hepadnaviral infection bypasses direct regulation of the cellular envelope proteins concentration, instead it modulates virus secretion efficiency that ultimately impacts the intracellular rcDNA concentration, an important factor determining the destination of the synthesized rcDNA in infected cells.

New perspective on the natural course of chronic HBV infection.

Chronic hepatitis B virus (HBV) infection is a significant threat to public health and an enormous burden on society. Mechanisms responsible for chronic HBV infection remain poorly understood. A better understanding of the natural course of chronic HBV infection may shed new light on the mechanisms underlying this disease and help in designing new antiviral strategies. Natural course of chronic HBV infection is conventionally viewed as an uninterrupted process that is usually marked by HBV e antigen (HBeAg) seroconversion or characterized by different phases associated with assumed host responses to HBV infection. However, none of these descriptions captures or highlights the core events that determine the natural course of chronic HBV infection. In this review, we briefly present the current knowledge on this subject and explain the significance and implication of events that occur during infection. A pre-core mutant becomes predominant in the viral population following elimination of the wild-type virus in duck hepatitis B virus-chronically infected animals. The coupled events in which first there is viral clearance that clears wild-type virus and then there is the reinfection of wild-type virus cleared livers with mutant virus are highly relevant to understanding of the natural course of chronic HBV infection under both treated and untreated conditions. In our new perspective, a general natural course of chronic HBV infection comprises cycles of viral clearance and reinfection, and such cycles prolong the chronic HBV infection course. Reviewing published data on the natural course of chronic HBV infection can reduce the possibility of missing important points in the initial data interpretation.

Age-related differences in amplification of covalently closed circular DNA at early times after duck hepatitis B virus infection of ducks.

Inoculation of 3-day-old (3D) or 3-week-old (3W) ducklings with duck hepatitis B virus results in chronic or transient infection, respectively. We previously showed that rapid production of neutralizing antibody following inoculation of 3W ducklings prevents virus from spreading in the liver and leads to a transient infection (Y.-Y. Zhang and J. Summers, J. Virol. 78:1195-1201, 2004). In this study we further investigated early events of viral infection in both 3D and 3W ducks. We present evidence that a lower level of virus replication in the hepatocytes of 3W birds is an additional factor that probably favors transient infection. We suggest that lower virus replication is due to a less rapid covalently closed circular DNA amplification, leading to lower viremias and a slower spread of infection in the liver, and that the slower spread of infection in 3W ducks makes the infection more sensitive to interruption by the host immune responses.

Rapid production of neutralizing antibody leads to transient hepadnavirus infection.

Hepatitis B virus (HBV) frequently causes transient infections in adults but chronic infections in infants. The basis of these age-related outcomes is not known. Infection of ducks with duck hepatitis B virus (DHBV) displays a similar dependence of outcome on the age of the host at the time of infection. In this study we compared the infection of ducks at 3 days and 3 weeks of age. We found that the efficiency of infection of hepatocytes by virus in the inoculum was similar between the two age groups but that spread of the infection throughout the liver was severely inhibited in the 3-week-old-old ducks, while a rapid spread of the infection was observed in 3-day-old ducklings. Inhibition of virus spread was accompanied by the appearance in the serum of virus neutralizing activity, as assayed by blocking of infection of primary hepatocyte cultures. Neutralizing activity appeared as early as 1 or 2 days postinfection and increased during the next 2 weeks. Depletion of immunoglobulins from serum eliminated the neutralizing activity. The specific depletion of IgM indicated that IgM appeared as the dominant fraction of neutralizing antibody in the first 2 days postinfection, but declined from day 3 on while IgG antibody rose. We conclude that excess neutralizing antibody arising rapidly in birds inoculated at 3 weeks of age but not in newly hatched ducks prevented secondary cycles of infection, resulting in a limited infection in the liver and contributing to the eventual transient outcome of the infection.