A systematic comparison reveals dynamic differences in early adaptive immune responses of acute-resolving versus chronic HBV replication.

Chronic hepatitis B virus (HBV) infection has been characterized by lack of effective adaptive immune responses which are vital for the viral clearance. However, very little is known about the dynamics of adaptive immune responses during the early phase of chronic HBV infection especially in spleen and liver. Here, we used the hydrodynamic injection (HDI) mouse model to kinetically characterize differences in the features of adaptive immunity, including the frequencies, phenotypes and function of antigen-presenting cells and T cells in the spleen, peripheral blood mononuclear cells (PBMCs) and liver, of chronic versus acute-resolving HBV replication (AR). We found that mice with AR mice and mice with chronic HBV replication (CH) mice showed early splenomegaly accompanied by T cell expansion in spleen but not in liver after HDI. Interestingly, the early and continuous increase in HBV-specific CD8+ T cells in spleen of CH mice was comparable to that in the AR mice. However, the splenic T cells of CH mice showed no activation phenotype compared with those in AR mice. Besides, increases in activated effector CD8+ T cells in PBMCs and liver at later time points were only observed in AR mice but not CH mice. CH mice also showed insufficient expansion of dendritic cells (DCs) in spleen and increased programmed death-1 expression in DCs of the liver compared to AR mice. The adoptive transfer of total splenocytes or splenic CD8+ T cells of AR mice to CH mice demonstrated that their ability to break HBV tolerance varies at different stages of HBV clearance. Moreover, the adoptive transfer of splenocytes from AR mice induce functional activation of endogenous HBV-specific CD8+ T cells of CH mice. Our results suggest that early T cell priming and expansion initially happens in the periphery after HBV antigen exposure in acute-resolving and chronic replication. The paucity of T cell activation, and subsequent migration and liver infiltration is a key feature of the adaptive immune responses during the early phase of CH, which is probably caused by the dysfunction of DCs.

Prophylactic and therapeutic HBV vaccination by an HBs-expressing cytomegalovirus vector lacking an interferon antagonist in mice.

Cytomegalovirus (CMV)-based vaccines show promising effects against chronic infections in nonhuman primates. Therefore, we examined the potential of hepatitis B virus (HBV) vaccines based on mouse CMV (MCMV) vectors expressing the small HBsAg. Immunological consequences of vaccine virus attenuation were addressed by either replacing the dispensable gene m157 ("MCMV-HBsȍ) or the gene M27 ("ΔM27-HBs"), the latter encodes a potent IFN antagonist targeting the transcription factor STAT2. M27 was chosen, since human CMV encodes an analogous gene product, which also induced proteasomal STAT2 degradation by exploiting Cullin RING ubiquitin ligases. Vaccinated mice were challenged with HBV through hydrodynamic injection. MCMV-HBs and ΔM27-HBs vaccination achieved accelerated HBV clearance in serum and liver as well as robust HBV-specific CD8+ T-cell responses. When we explored the therapeutic potential of MCMV-based vaccines, especially the combination of ΔM27-HBs prime and DNA boost vaccination resulted in increased intrahepatic HBs-specific CD8+ T-cell responses and HBV clearance in persistently infected mice. Our results demonstrated that vaccines based on a replication competent MCMV attenuated through the deletion of an IFN antagonist targeting STAT2 elicit robust anti-HBV immune responses and mediate HBV clearance in mice in prophylactic and therapeutic immunization regimes.

Distinct trajectories of perinatal depression in Chinese women: application of latent growth mixture modelling.

BACKGROUND: Current research on perinatal depression rarely pays attention to the continuity and volatility of depression symptoms over time, which is very important for the early prediction and prognostic evaluation of perinatal depression. This study investigated the trajectories of perinatal depression symptoms and aimed to explore the factors related to these trajectories. METHODS: The study recruited 550 women during late pregnancy (32 ± 4 weeks of gestation) and followed them up 1 and 6 weeks postpartum. Depressive symptoms were measured using the Edinburgh Postnatal Depression Scale (EPDS). Latent growth mixture modelling (LGMM) was used to identify trajectories of depressive symptoms during pregnancy. RESULTS: Two trajectories of perinatal depressive symptoms were identified: "decreasing" (n = 524, 95.3%) and "increasing" (n = 26, 4.7%). History of smoking, alcohol use and gestational hypertension increased the chance of belonging to the increasing trajectories, and a high level of social support was a protective factor for maintaining a decreasing trajectory. CONCLUSIONS: This study identified two trajectories of perinatal depression and the factors associated with each trajectory. Paying attention to these factors and providing necessary psychological support services during pregnancy would effectively reduce the incidence of perinatal depression and improve patient prognosis.

Local Stimulation of Liver Sinusoidal Endothelial Cells with a NOD1 Agonist Activates T Cells and Suppresses Hepatitis B Virus Replication in Mice.

Functional maturation of liver sinusoidal endothelial cells (LSECs) induced by a NOD1 ligand (diaminopimelic acid [DAP]) during viral infection has not been well defined. Thus, we investigated the role of DAP-stimulated LSEC maturation during hepatitis B virus (HBV) infection and its potential mechanism in a hydrodynamic injection (HI) mouse model. Primary LSECs were isolated from wild-type C57BL/6 mice and stimulated with DAP in vitro and in vivo and assessed for the expression of surface markers as well as for their ability to promote T cell responses via flow cytometry. The effects of LSEC maturation on HBV replication and expression and the role of LSECs in the regulation of other immune cells were also investigated. Pretreatment of LSECs with DAP induced T cell activation in vitro. HI-administered DAP induced LSEC maturation and subsequently enhanced T cell responses, which was accompanied by an increased production of intrahepatic cytokines, chemokines, and T cell markers in the liver. The HI of DAP significantly reduced the HBsAg and HBV DNA levels in the mice. Importantly, the DAP-induced anti-HBV effect was impaired in the LSEC-depleted mice, which indicated that LSEC activation and T cell recruitment into the liver were essential for the antiviral function mediated by DAP application. Taken together, the results showed that the Ag-presenting ability of LSECs was enhanced by DAP application, which resulted in enhanced T cell responses and inhibited HBV replication in a mouse model.

MMP2/MMP9-mediated CD100 shedding is crucial for inducing intrahepatic anti-HBV CD8 T cell responses and HBV clearance.

BACKGROUND & AIMS: CD100 is constitutively expressed on T cells and can be cleaved from the cell surface by matrix metalloproteases (MMPs) to become soluble CD100 (sCD100). Both membrane-bound CD100 (mCD100) and sCD100 have important immune regulatory functions that promote immune cell activation and responses. This study investigated the expression and role of mCD100 and sCD100 in regulating antiviral immune responses during HBV infection. METHODS: mCD100 expression on T cells, sCD100 levels in the serum, and MMP expression in the liver and serum were analysed in patients with chronic HBV (CHB) and in HBV-replicating mice. The ability of sCD100 to mediate antigen-presenting cell maturation, HBV-specific T cell activation, and HBV clearance were analysed in HBV-replicating mice and patients with CHB. RESULTS: Patients with CHB had higher mCD100 expression on T cells and lower serum sCD100 levels compared with healthy controls. Therapeutic sCD100 treatment resulted in the activation of DCs and liver sinusoidal endothelial cells, enhanced HBV-specific CD8 T cell responses, and accelerated HBV clearance, whereas blockade of its receptor CD72 attenuated the intrahepatic anti-HBV CD8 T cell response. Together with MMP9, MMP2 mediated mCD100 shedding from the T cell surface. Patients with CHB had significantly lower serum MMP2 levels, which positively correlated with serum sCD100 levels, compared with healthy controls. Inhibition of MMP2/9 activity resulted in an attenuated anti-HBV T cell response and delayed HBV clearance in mice. CONCLUSIONS: MMP2/9-mediated sCD100 release has an important role in regulating intrahepatic anti-HBV CD8 T cell responses, thus mediating subsequent viral clearance during HBV infection. LAY SUMMARY: Chronic hepatitis B virus (HBV) infection is a major public health problem worldwide. The clearance of HBV relies largely on an effective T cell immune response, which usually becomes dysregulated in chronic HBV infection. Our study provides a new mechanism to elucidate HBV persistence and a new target for developing immunotherapy strategies in patients chronically infected with HBV.

Differential escape of HCV from CD8+ T cell selection pressure between China and Germany depends on the presenting HLA class I molecule.

Adaptation of hepatitis C virus (HCV) to CD8+ T cell selection pressure is well described; however, it is unclear if HCV differentially adapts in different populations. Here, we studied HLA class I-associated viral sequence polymorphisms in HCV 1b isolates in a Chinese population and compared viral substitution patterns between Chinese and German populations. We identified three HLA class I-restricted epitopes in HCV NS3 with statistical support for selection pressure and found evidence for differential escape pathways between isolates from China and Germany depending on the HLA class I molecule. The substitution patterns particularly differed in the epitope VTLTHPITK1635-1643 , which was presented by HLA-A*03 as well as HLA-A*11, two alleles with highly different frequencies in the two populations. In Germany, a substitution in position seven of the epitope was the most frequent substitution in the presence of HLA-A*03, functionally associated with immune escape and nearly absent in Chinese isolates. In contrast, the most frequent substitution in China was located at position two of the epitope and became the predominant consensus residue. Moreover, substitutions in position one of the epitope were significantly enriched in HLA-A*11-positive individuals in China and associated with different patterns of CD8+ T cell reactivity. Our study confirms the differential escape pathways selected by HCV that depended on different HLA class I alleles in Chinese and German populations, indicating that HCV differentially adapts to distinct HLA class I alleles in these populations. This result has important implications for vaccine design against highly variable and globally distributed pathogens, which may require matching antigen sequences to geographic regions for T cell-based vaccine strategies.

Human Glioblastoma-Derived Mesenchymal Stem Cell to Pericytes Transition and Angiogenic Capacity in Glioblastoma Microenvironment.

BACKGROUND/AIMS: Tumor vascular formation and maintenance are crucial events in glioblastoma development. Mesenchymal stem cells (MSCs) have been shown to differentiate into pericytes and contribute to neovascularization in the glioma microenvironment. Moreover, glioblastoma-derived mesenchymal stem cells (gb-MSCs), which consist of CD90-MSCs and CD90+MSCs, are a subpopulation of MSCs that are more active in glioma vascularization. However, the functions of gb-MSCs and the microRNA (miRNA) modifications in the glioblastoma microenvironment have not yet been fully elucidated. Here, we focus on the pericyte differentiation potential of gb-MSCs and miRNA modifications in gb-MSCs during new vascular formation and glioblastoma growth. METHODS: In vitro, surface markers of gb-MSCs were detected by flow cytometry; the differentiation potential was evaluated by Oil Red O staining, Alizarin Red staining and Alcian blue staining; the proliferation and migration of gb-MSCs in different conditioned media were analyzed by the cck8 test and wound-healing assay, respectively; gb-MSC to pericyte transition was detected by immunofluorescence staining and western blot assay; angiogenetic capacity was analyzed by tube formation assay; and levels of cytokines in different supernatant were determined by ELISA. Additionally, RNA was isolated from gb-MSCs, and miRNA modifications were analyzed using the RAffymetrix miRNA microarray Results: We showed that glioblastoma-conditioned medium increased gb-MSC proliferation and migration and was capable of inducing gb-MSC differentiation into pericytes. Glioblastoma secreted angiogenic factors and gb-MSCs incubated in malignant glioblastoma-conditioned medium formed more tube-like structures, and these cells also adhered to tube-like vessels formed by human umbilical vein endothelial cells (HUVECs) on Matrigel to maintain tumor vascular structure in vitro. miRNA expression were also modified in gb-MSCs cultured in malignant glioblastoma-conditioned medium in vitro. CONCLUSION: These results provide new insight into the functional effects of a subpopulation of MSCs in glioblastoma and may help in the development of novel therapies for solid tumors.

Substitutive proprioception feedback of a prosthetic wrist by electrotactile stimulation.

Objective: Sensory feedback of upper-limb prostheses is widely desired and studied. As important components of proprioception, position, and movement feedback help users to control prostheses better. Among various feedback methods, electrotactile stimulation is a potential method for coding proprioceptive information of a prosthesis. This study was motivated by the need for proprioception information for a prosthetic wrist. The flexion-extension (FE) position and movement information of the prosthetic wrist are transmitted back to the human body through multichannel electrotactile stimulation. Approach: We developed an electrotactile scheme to encode the FE position and movement of the prosthetic wrist and designed an integrated experimental platform. A preliminary experiment on the sensory threshold and discomfort threshold was performed. Then, two proprioceptive feedback experiments were performed: a position sense experiment (Exp 1) and a movement sense experiment (Exp 2). Each experiment included a learning session and a test session. The success rate (SR) and discrimination reaction time (DRT) were analyzed to evaluate the recognition effect. The acceptance of the electrotactile scheme was evaluated by a questionnaire. Main results: Our results showed that the average position SRs of five able-bodied subjects, amputee 1, and amputee 2 were 83.78, 97.78, and 84.44%, respectively. The average movement SR, and the direction and range SR of wrist movement in five able-bodied subjects were 76.25, 96.67%, respectively. Amputee 1 and amputee 2 had movement SRs of 87.78 and 90.00% and direction and range SRs of 64.58 and 77.08%, respectively. The average DRT of five able-bodied subjects was less than 1.5 s and that of amputees was less than 3.5 s. Conclusion: The results indicate that after a short period of learning, the subjects can sense the position and movement of wrist FE. The proposed substitutive scheme has the potential for amputees to sense a prosthetic wrist, thus enhancing the human-machine interaction.

Molecular characterization of the type I IFN receptor in two woodchuck species and detection of its expression in liver samples from woodchucks infected with woodchuck hepatitis virus (WHV).

Type I interferons (IFN-α/ß) serve as the first line of defense against viral infection and share the same type I IFN receptor (IFNAR) complex, which is composed of IFNAR1 and -2. The Eastern woodchuck (Marmota monax) and Chinese woodchuck (Marmota himalayana) are suitable for studying hepatitis B virus (HBV) infection. Here, the complete or partial sequences of the IFNARs of both species were obtained and analyzed. Small interference RNAs targeting wIFNAR1 and -2 specifically down-regulated the expression of wIFNAR1 and -2 and the IFN-stimulated gene MxA in a woodchuck cell line, respectively. IFNAR2 was significantly up-regulated in primary woodchuck hepatocytes stimulated with IFN-α or -γ. The expression of woodchuck IFNAR1 and -2 was decreased in woodchucks chronically infected with woodchuck hepatitis virus (WHV). These results are essential for studying type I IFN-related innate immunity and therapy in hepadnaviral infection in the woodchuck model.

Fecal Microbiota Transplantation Alters the Outcome of Hepatitis B Virus Infection in Mice.

The susceptibility of mice to hepatitis B virus (HBV) infection depends on their genetic background. The gut microbiota modulates the antiviral immune response in the liver and plays a protective role against HBV infection. However, whether HBV infection outcomes depend on the gut microbiota remains unclear. In this study, we assessed the gut microbiota composition in naïve BALB/c and C57BL/6 mice using 16S rRNA gene sequencing. The gut microbiota in BALB/c mice was depleted using broad-spectrum antibiotics (ABX) and then reconstituted with fecal microbiota from naïve BALB/c or C57BL/6 mice to evaluate the effect of fecal microbiota transplantation (FMT) on the outcomes of and immune response to HBV infection. We found that HBV infection outcomes and the gut microbiota composition differed between BALB/c and C57BL/6 mice. Commensal bacteria from the fecal microbiota selectively colonized the guts of ABX-treated BALB/c mice. Mice receiving fecal microbiota from BALB/c or C57BL/6 mice displayed different HBV infection outcomes. The fecal microbiota from C57BL/6 mice induced immune tolerance in the liver and prolonged HBV infection. In conclusion, HBV infection outcomes in mice are determined by the host genetic background and gut microbiota composition. Reconstitution of the gut microbiota by FMT can alter the susceptibility to HBV infection in mice.

[The influence of HBV and its antigens on the expressions of JAK-STAT signal transduction pathway molecules and antiviral proteins of IFN alpha].

OBJECTIVE: To investigate the possible influence of HBV and its antigens on the expressions of JAK-STAT signal transduction pathway molecules and the antiviral proteins of IFN alpha. METHODS: The HepG2 cells were transfected with pSM2, pHBS2-S and pHBc-EGFP plasmids which express HBV whole particles or S-antigen, Pre-S antigen and core antigens. The infectious supernatant from HepG2.2.15 cells and the pured HBV proteins which contained the S, Pre-S antigens were used to treat the HepG2 cells. Northern blot and RT-PCR were applied to analyse the expressions of the antiviral proteins MxA, 2' -5' OAS, 9-27 and the JAK-STAT signal transduction pathway molecules STAT1 in HepG2 cells responded to the IFN alpha treatment. RESULTS: The HepG2 cells transfected with pSM2, pHBS2-S and pHBc-EGFP plasmids could express whole HBV particles and HBsAg, Pre-S antigen and HBcAg. The quantitation of expressed HBV particles and antigens increased significantly during the course of transfection. Northern blot hybridization analysis indicated that the HepG2 cells expressed IFN alpha antiviral proteins MxA, 2' -5' OAS and 9-27. When transfected with pHBV-dimer, pHBS2-S, pHBc-EGFP plasmids, the IFN/A antiviral proteins MxA, 2' -5' OAS and 9-27 in transfected cells were reduced greatly as compared to the un-transfected HepG2 cells, and the expressed antiviral proteins decreased sharply with the development of transfection time. Furthermore, the expression of IFN alpha JAK-STAT signal transduction pathway molecule STAT1 was also inhibited with the expression of HBV particles and HBV antigens in transfected HepG2 cells. CONCLUSIONS: The HBV and its antigens influence the expressions of IFN alpha JAK-STAT signal transduction pathway molecules and antiviral proteins in the hepatocellular models in vitro. It is indicated that HBV might possess the activity to antagonise or counteract the IFN alpha antiviral action.

Depletion of Gut Microbiota Impairs Gut Barrier Function and Antiviral Immune Defense in the Liver.

Commensal gut microbiota protects the immune defense of extra-intestinal organs. Gut microbiota depletion by antibiotics can impair host antiviral immune responses and alter hepatitis B virus (HBV) infection outcomes. However, how gut microbiota modulates antiviral immune response in the liver remains unclear. Here, mice were treated with broad-spectrum antibiotics to deplete gut microbiota. Gut integrity was evaluated, and translocation of live commensal gut bacteria and their components into the liver was investigated. An HBV infection model was established to evaluate impairment of antiviral immune response in the liver after gut microbiota depletion. We found that gut microbiota depletion was associated with impairment of colon epithelial integrity, and live commensal gut microbiota could translocate to the liver. Further, T cell antiviral function in the liver was impaired, partially relying on enhanced PD-1 expression, and HBV immune clearance was hampered. In conclusion, gut microbiota depletion by antibiotics can impair gut barrier function and suppress T cell antiviral immune response in the liver.

Analysis of the Long-Term Impact on Cellular Immunity in COVID-19-Recovered Individuals Reveals a Profound NKT Cell Impairment.

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) affected over 120 million people and killed over 2.7 million individuals by March 2021. While acute and intermediate interactions between SARS-CoV-2 and the immune system have been studied extensively, long-term impacts on the cellular immune system remain to be analyzed. Here, we comprehensively characterized immunological changes in peripheral blood mononuclear cells in 49 COVID-19-convalescent individuals (CI) in comparison to 27 matched SARS-CoV-2-unexposed individuals (UI). Despite recovery from the disease for more than 2 months, CI showed significant decreases in frequencies of invariant NKT and NKT-like cells compared to UI. Concomitant with the decrease in NKT-like cells, an increase in the percentage of annexin V and 7-aminoactinomycin D (7-AAD) double-positive NKT-like cells was detected, suggesting that the reduction in NKT-like cells results from cell death months after recovery. Significant increases in regulatory T cell frequencies and TIM-3 expression on CD4 and CD8 T cells were also observed in CI, while the cytotoxic potential of T cells and NKT-like cells, defined by granzyme B (GzmB) expression, was significantly diminished. However, both CD4 and CD8 T cells of CI showed increased Ki67 expression and were fully able to proliferate and produce effector cytokines upon T cell receptor (TCR) stimulation. Collectively, we provide a comprehensive characterization of immune signatures in patients recovering from SARS-CoV-2 infection, suggesting that the cellular immune system of COVID-19 patients is still under a sustained influence even months after the recovery from disease.IMPORTANCE Wuhan was the very first city hit by SARS-CoV-2. Accordingly, the patients who experienced the longest phase of convalescence following COVID-19 reside here. This enabled us to investigate the "immunological scar" left by SARS-CoV-2 on cellular immunity after recovery from the disease. In this study, we characterized the long-term impact of SARS-CoV-2 infection on the immune system and provide a comprehensive picture of cellular immunity of a convalescent COVID-19 patient cohort with the longest recovery time. We revealed that the cellular immune system of COVID-19 patients is still under a sustained influence even months after the recovery from disease; in particular, a profound NKT cell impairment was found in the convalescent phase of COVID-19.

Corrigendum: TLR2 Expression in Peripheral CD4+ T Cells Promotes Th17 Response and Is Associated with Disease Aggravation of Hepatitis B Virus-Related Acute-On-Chronic Liver Failure.

[This corrects the article DOI: 10.3389/fimmu.2017.01609.].

Longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of SARS-CoV-2 infected patients.

BACKGROUND: The dynamic changes of lymphocyte subsets and cytokines profiles of patients with novel coronavirus disease (COVID-19) and their correlation with the disease severity remain unclear. METHODS: Peripheral blood samples were longitudinally collected from 40 confirmed COVID-19 patients and examined for lymphocyte subsets by flow cytometry and cytokine profiles by specific immunoassays. FINDINGS: Of the 40 COVID-19 patients enrolled, 13 severe cases showed significant and sustained decreases in lymphocyte counts [0·6 (0·6-0·8)] but increases in neutrophil counts [4·7 (3·6-5·8)] than 27 mild cases [1.1 (0·8-1·4); 2·0 (1·5-2·9)]. Further analysis demonstrated significant decreases in the counts of T cells, especially CD8+ T cells, as well as increases in IL-6, IL-10, IL-2 and IFN-γ levels in the peripheral blood in the severe cases compared to those in the mild cases. T cell counts and cytokine levels in severe COVID-19 patients who survived the disease gradually recovered at later time points to levels that were comparable to those of the mild cases. Moreover, the neutrophil-to-lymphocyte ratio (NLR) (AUC=0·93) and neutrophil-to-CD8+ T cell ratio (N8R) (AUC =0·94) were identified as powerful prognostic factors affecting the prognosis for severe COVID-19. INTERPRETATION: The degree of lymphopenia and a proinflammatory cytokine storm is higher in severe COVID-19 patients than in mild cases, and is associated with the disease severity. N8R and NLR may serve as a useful prognostic factor for early identification of severe COVID-19 cases. FUNDING: The National Natural Science Foundation of China, the National Science and Technology Major Project, the Health Commission of Hubei Province, Huazhong University of Science and Technology, and the Medical Faculty of the University of Duisburg-Essen and Stiftung Universitaetsmedizin, Hospital Essen, Germany.

The interferon-alpha gene family of Marmota himalayana, a Chinese marmot species with susceptibility to woodchuck hepatitis virus infection.

The interferon-alpha (IFN-alpha) gene family is an important part of the immune system. Recombinant interferon-alpha is widely used to treat viral hepatitis and malignant diseases. Marmota himalayana has been found to be susceptible to woodchuck hepatitis virus, a virus genetically related to hepatitis B virus (HBV), and is suitable as an animal model for studies on HBV infection. Here, the IFN-alpha gene family of M. himalayana (cwIFN-alpha) was characterized. Sequence data indicate that the cwIFN-alpha family consists of at least 8 functional sequences and 6 pseudogenes with high homology within the family and to IFN-alpha of Marmota monax, a related species and well-established animal model. The recombinant cwIFN-alpha subtypes were expressed and tested to be active in viral protection assay and to induce expression of MxA in a species-specific manner. This work provides essential information for future work on testing new therapeutic approaches of HBV infection based on IFN-alpha in M. himalayana.

Molecular characterization of woodchuck type I interferons and their expression by woodchuck peripheral blood lymphocytes.

Interferon (IFN)-alpha and -beta are important antiviral mediators. IFN-alpha is widely used for the treatment of chronic hepatitis B. In our previous studies, a subtype of woodchuck IFN-alpha (wIFN-alpha) was characterized and has been shown to be active in suppressing the replication of woodchuck hepatitis virus (WHV) in vitro and vivo. Here, we refined the analysis of the IFN-alpha/beta system of the woodchuck and studied the expression of wIFN-alpha/beta in peripheral blood lymphocytes (PBLs) from naïve and WHV-infected woodchucks. A number of wIFN-alpha genes were sequenced and could be classified into 10 subtypes and 3 pseudotypes. The biological activity of different subtypes of wIFN-alpha was demonstrated by their ability to protect woodchuck cells against encephalomyocarditis virus infection and to induce MxA expression in woodchuck cells. Additionally, a partial sequence of wIFN-beta was characterized. A subtyping method for wIFN-alpha based on restriction length polymorphism analysis was developed. Further, the expression of wIFN by woodchuck PBLs after stimulation with polyI/C was investigated. The maximal production of wIFN by woodchuck PBLs occurred within the first 48 h after addition poly I/C. The wIFN-alpha subtypes 1, 4, and 5 were found to be produced by poly I/C-stimulated woodchuck PBLs, indicating a selective expression of wIFN-alpha subtypes. PBLs from chronically WHV-infected woodchucks showed a reduced ability to produce wIFN when stimulated with poly I/C. The results suggest that woodchucks with chronic WHV infection have impaired immunological responses to poly I/C.

Replication of clinical hepatitis B virus isolate and its application for selecting antiviral agents for chronic hepatitis B patients.

AIM: To establish a cell model harboring replicative clinical hepatitis B virus (HBV) isolates and evaluate its application in individualized selection of anti-HBV agents for chronic hepatitis B (CHB) patients. METHODS: The full-length HBV genomic DNA from 8 CHB patients was amplified by polymerase chain reaction (PCR). All the patients were treated with lamivudine for at least seven months and finally became resistant to lamivudine. The amplified HBV DNA fragments were inserted into pHY106 vectors by Sap I digestion. The recombinant plasmids containing 1.1 copies of HBV genome were transiently transfected into Huh7 cell line, and the levels of HBsAg, HBeAg and intercellular HBV replicative intermediates were determined by ELISA and Southern blot analysis, respectively, with or without lamivudine and adefovir treatment. The antiviral treatment with adefovir was administered to the patients and analyzed in parallel. RESULTS: A total of 25 independent HBV isolates were obtained from the sera of 8 patients, each patient had at least two isolates. One isolate from each individual was selected and subcloned into pHY106 vector, including 5 isolates with YVDD mutation and 3 isolates with YIDD mutation. All recombinant plasmids harboring HBV isolates were transfected into Huh7 cells. The results indicated that HBV genome carried in HBV replicons of clinical HBV isolates could effectively replicate and express in Huh7 cells. Adefovir, but not lamivudine, inhibited HBV replication both in vitro and in vivo, and in vitro inhibition was dose-dependent. CONCLUSION: The novel method described herein enables individualized selection of anti-HBV agents in clinic and is useful in future studies of antiviral therapy for CHB.

[A study on the relationship between point mutation in pre-core region G1896A of hepatitis B virus and safety of breast feeding].

OBJECTIVE: To investigate the relationship between pre-core G1896A point mutation of hepatitis B virus (HBV) and safety of breast feeding. METHODS: Serum and breast milk samples were collected from 62 pregnant women of HBV DNA positive/HBeAg negative. PCR-solid phase hybridization was used to detect the point mutation in pre-core region G1896A of HBV from pregnant women, and HBV DNA loads in sera and breast milk were determined by fluorescence quantitative PCR (FQ-PCR). RESULTS: The prevalence of point mutation was 61.3% (38/62) in 62 pregnant women with HBsAg positive/HBeAg negative. The positive rate of HBV DNA in breast milk of group with point mutation (28.9%) was similar to that of group without mutation (29.2%, chi2=0.0003, P>0.05). However, The positive rate of HBV DNA in breast milk of group with high HBV loads (56.0%) was significantly higher than that of group with low HBV loads (10.8%, chi2=14.79, P<0.01). CONCLUSION: The point mutation in pre-core region G1896A of HBV dose not affect the positive rate of HBV DNA in breast milk and higher HBV DNA loads in serum of pregnant women might increase the risk of mother-infant transmission.