Antibody-mediated spike activation promotes cell-cell transmission of SARS-CoV-2.

The COVID pandemic fueled by emerging SARS-CoV-2 new variants of concern remains a major global health concern, and the constantly emerging mutations present challenges to current therapeutics. The spike glycoprotein is not only essential for the initial viral entry, but is also responsible for the transmission of SARS-CoV-2 components via syncytia formation. Spike-mediated cell-cell transmission is strongly resistant to extracellular therapeutic and convalescent antibodies via an unknown mechanism. Here, we describe the antibody-mediated spike activation and syncytia formation on cells displaying the viral spike. We found that soluble antibodies against receptor binding motif (RBM) are capable of inducing the proteolytic processing of spike at both the S1/S2 and S2' cleavage sites, hence triggering ACE2-independent cell-cell fusion. Mechanistically, antibody-induced cell-cell fusion requires the shedding of S1 and exposure of the fusion peptide at the cell surface. By inhibiting S1/S2 proteolysis, we demonstrated that cell-cell fusion mediated by spike can be re-sensitized towards antibody neutralization in vitro. Lastly, we showed that cytopathic effect mediated by authentic SARS-CoV-2 infection remain unaffected by the addition of extracellular neutralization antibodies. Hence, these results unveil a novel mode of antibody evasion and provide insights for antibody selection and drug design strategies targeting the SARS-CoV-2 infected cells.

PTEN loss correlates with T cell exclusion across human cancers.

BACKGROUND: Recent evidences had shown that loss in phosphatase and tensin homolog deleted on chromosome 10 (PTEN) was associated with immunotherapy resistance, which may be attributed to the non-T-cell-inflamed tumor microenvironment. The impact of PTEN loss on tumor microenvironment, especially regarding T cell infiltration across tumor types is not well understood. METHODS: Utilizing The Cancer Genome Atlas (TCGA) and publicly available dataset of immunotherapy, we explored the correlation of PTEN expressing level or genomic loss with tumor immune microenvironment and response to immunotherapy. We further investigated the involvement of PI3K-AKT-mTOR pathway activation, which is known to be the subsequent effect of PTEN loss, in the immune microenvironment modulation. RESULTS: We reveal that PTEN mRNA expression is significantly positively correlated with CD4/CD8A gene expression and T cells infiltration especially T helpers cells, central memory T cell and effector memory T cells in multiples tumor types. Genomic loss of PTEN is associated with reduced CD8+ T cells, type 1 T helper cells, and increased type 2 T helper cells, immunosuppressed genes (e.g. VEGFA) expression. Furthermore, T cell exclusive phenotype is also observed in tumor with PI3K pathway activation or genomic gain in PIK3CA or PIK3CB. PTEN loss and PI3K pathway activation correlate with immunosuppressive microenvironment, especially in terms of T cell exclusion. PTEN loss predict poor therapeutic response and worse survival outcome in patients receiving immunotherapy. CONCLUSION: These data brings insight into the role of PTEN loss in T cell exclusion and immunotherapy resistance, and inspires further research on immune modulating strategy to augment immunotherapy.

Quantitative proteomic analysis of exosome protein content changes induced by hepatitis B virus in Huh-7 cells using SILAC labeling and LC-MS/MS.

Hepatitis B virus (HBV) infection could cause hepatitis, liver cirrhosis, and hepatocellular carcinoma. HBV-mediated pathogenesis is only partially understood, but X protein (HBx) reportedly possesses oncogenic potential. Exosomes are small membrane vesicles with diverse functions released by various cells including hepatocytes, and HBV harnesses cellular exosome biogenesis and export machineries for virion morphogenesis and secretion. Therefore, HBV infection might cause changes in exosome contents with functional implications for both virus and host. In this work, exosome protein content changes induced by HBV and HBx were quantitatively analyzed by SILAC/LC-MS/MS. Exosomes prepared from SILAC-labeled hepatoma cell line Huh-7 transfected with HBx, wildtype, or HBx-null HBV replicon plasmids were analyzed by LC-MS/MS. Systematic analyses of MS data and confirmatory immunoblotting showed that HBx overexpression and HBV, with or without HBx, replication in Huh-7 cells indeed caused marked and specific changes in exosome protein contents. Furthermore, specific changes in protein contents were also detected in exosomes purified from HBV-infected patients' sera compared with control sera negative for HBV markers. These results illustrate a new aspect of interactions between HBV and the host and provide the foundation for future research into roles played by exosomes in HBV infection and pathogenesis.

Novel recombinant hepatitis B virus vectors efficiently deliver protein and RNA encoding genes into primary hepatocytes.

Hepatitis B virus (HBV) has extremely restricted host and hepatocyte tropism. HBV-based vectors could form the basis of novel therapies for chronic hepatitis B and other liver diseases and would also be invaluable for the study of HBV infection. Previous attempts at developing HBV-based vectors encountered low yields of recombinant viruses and/or lack of sufficient infectivity/cargo gene expression in primary hepatocytes, which hampered follow-up applications. In this work, we constructed a novel vector based on a naturally occurring, highly replicative HBV mutant with a 207-bp deletion in the preS1/polymerase spacer region. By applying a novel insertion strategy that preserves the continuity of the polymerase open reading frame (ORF), recombinant HBV (rHBV) carrying protein or small interfering RNA (siRNA) genes were obtained that replicated and were packaged efficiently in cultured hepatocytes. We demonstrated that rHBV expressing a fluorescent reporter (DsRed) is highly infective in primary tree shrew hepatocytes, and rHBV expressing HBV-targeting siRNA successfully inhibited antigen expression from coinfected wild-type HBV. This novel HBV vector will be a powerful tool for hepatocyte-targeting gene delivery, as well as the study of HBV infection.

Characteristics and Genomic Diversity of Measles Virus From Measles Cases With Known Vaccination Status in Shanghai, China.

Although the highly effective measles vaccine has dramatically reduced the incidence of measles, measles, and outbreaks continue to occur in individuals who received the measles vaccine because of immunization failure. In this study, patients who have definite records of immunization were enrolled based on measles surveillance in Shanghai, China, from 2009 to 2017, and genomic characteristics regarding viruses retrieved from these cases provided insights into immunization failure. A total of 147 complete genomes of measles virus (MV) were obtained from the laboratory-confirmed cases through Illumina MiSeq. Epidemiological, and genetic characteristics of the MV were focused on information about age, gender, immunization record, variation, and evolution of the whole genome. Furthermore, systematic genomics using phylogeny and selection pressure approaches were analyzed. Our analysis based on the whole genome of 147 isolates revealed 4 clusters: 2 for the genotype H1 (clusters named H1-A, including 73 isolates; H1-B, including 72 isolates) and the other 2 for D8 and B3, respectively. Estimated nucleotide substitution rates of genotype H1 MV derived using genome and individual genes are lower than other genotypes. Our study contributes to global measles epidemiology and proves that whole-genome sequencing was a useful tool for more refined genomic characterization. The conclusion indicates that vaccination may have an effect on virus evolution. However, no major impact was found on the antigenicity in Shanghai isolates.

Importation and circulation of rubella virus lineages 1E-L2 and 2B-L2c between 2018 and 2021 in China: Virus evolution and spatial-temporal transmission characteristics.

To better understand the importation and circulation patterns of rubella virus lineages 1E-L2 and 2B-L2c circulating in China since 2018, 3,312 viral strains collected from 27 out of 31 provinces in China between 2018 and 2021 were sequenced and analyzed with the representative international strains of lineages 1E-L2 and 2B-L2c based on genotyping region. Time-scale phylogenetic analysis revealed that the global lineages 1E-L2 and 2B-L2c presented distinct evolutionary patterns. Lineage 1E-L2 circulated in relatively limited geographical areas (mainly Asia) and showed geographical and temporal clustering, while lineage 2B-L2c strains circulated widely throughout the world and exhibited a complicated topology with several independently evolved branches. Furthermore, both lineages showed extensive international transmission activities, and phylogeographic inference provided evidence that lineage 1E-L2 strains circulating in China possibly originated from Japan, while the source of lineage 2B-L2c isolated since 2018 is still unclear. After importation into China in 2018, the spread of lineage 1E-L2 presented a three-stage transmission pattern from southern to northern China, whereas lineage 2B-L2c spread from a single point in western China to all the other four regions. These two transmission patterns allowed both imported lineages to spread rapidly across China during the 2018-9 rubella epidemic and eventually established endemic circulations. This study provides critical scientific data for rubella control and elimination in China and worldwide.

Case Report: Area of Focus of Myocardial Infarction With Non-obstructive Coronary Arteries in Eosinophilic Granulomatosis With Polyangiitis.

Background: Eosinophilic granulomatosis with polyangitis manifested as myocardial infarction with non-obstructed coronary arteries (MINOCA) is rarely reported. Case: We report a 43-year-old male patient without any cardiovascular risk factors presenting with acute chest pain. Electrocardiogram was suggestive of acute anterior and inferior myocardial infarction. MINOCA was confirmed based on significant elevated cardiac troponin and normal coronary arteries. Cardiac magnetic resonance (CMR) imaging revealed extended late gadolinium enhancement (LGE). Further diagnosis of eosinophilic granulomatosis with polyangitis (EGPA) was based on clinical manifestations and auxiliary examination. Subsequent immunosuppressive therapy led to regression of symptoms and significant resolution of LGE on CMR. Conclusion: Our case highlights that EGPA can be a rare cause of MINOCA. CMR is useful for differentiation diagnosis and evaluation of cardiac involvement.

Virology features of a family cluster of SARS-CoV-2 infections in Shanghai, China.

The global spread of SARS-CoV-2 is currently continuing, and the World Health Organization has announced the risk assessment of the viruses as high. In this study, we analyzed virology features of SARS-CoV-2 causing a family cluster outbreak. Among the six family members, five have been laboratory-confirmed infection of SARS-CoV-2 viruses. A total of five SARS-CoV-2 viruses have been isolated from the nasopharyngeal swabs. The complete genome of the viruses exhibited 100% nucleotide identity with each other. Only two nucleotide differences have been observed between genomes of the isolated viruses and the HCoV/Wuhan/ IVDC-HB-01/2019 strain. Therefore, SARS-CoV-2 has been confirmed as the causation of the family cluster infections.

Abnormal upregulation of cardiovascular disease biomarker PLA2G7 induced by proinflammatory macrophages in COVID-19 patients.

High rate of cardiovascular disease (CVD) has been reported among patients with coronavirus disease 2019 (COVID-19). Importantly, CVD, as one of the comorbidities, could also increase the risks of the severity of COVID-19. Here we identified phospholipase A2 group VII (PLA2G7), a well-studied CVD biomarker, as a hub gene in COVID-19 though an integrated hypothesis-free genomic analysis on nasal swabs (n = 486) from patients with COVID-19. PLA2G7 was further found to be predominantly expressed by proinflammatory macrophages in lungs emerging with progression of COVID-19. In the validation stage, RNA level of PLA2G7 was identified in nasal swabs from both COVID-19 and pneumonia patients, other than health individuals. The positive rate of PLA2G7 were correlated with not only viral loads but also severity of pneumonia in non-COVID-19 patients. Serum protein levels of PLA2G7 were found to be elevated and beyond the normal limit in COVID-19 patients, especially among those re-positive patients. We identified and validated PLA2G7, a biomarker for CVD, was abnormally enhanced in COVID-19 at both nucleotide and protein aspects. These findings provided indications into the prevalence of cardiovascular involvements seen in patients with COVID-19. PLA2G7 could be a potential prognostic and therapeutic target in COVID-19.

Pan-cancer analysis of genomic properties and clinical outcome associated with tumor tertiary lymphoid structure.

How the genomic landscape of a tumor shapes the formation of tertiary lymphoid structure (TLS) and how might TLS alter the clinical outcome or response to immunotherapy had not been systematically explored. Utilizing the genomic and transcriptome data of solid tumors on TCGA, we quantified TLS based on a previous identified 12-chemokine signature and evaluated its correlation with mutation/neoantigen burden, functional mutation of oncogenes and the presence of viral infection. Clinical data was integrated to decide the prognostic significance of TLS for different cancers after surgical treatment. Publicly available data (clinical and transcriptome data) of immunotherapy clinical trials involving melanoma and lung cancer were also collected to evaluate TLS's association with therapeutic outcome. Mutation burden and predicted neoantigen counts were positively correlated with TLS scoring in multiple cancer types. Mutation in tumor suppressor genes (KEAP1, PBRM1) and genes involved in extrinsic apoptosis (CASP8), antigen-presentation (HLA-A, HLA-B), immune regulation (SMAD4) or DNA repair (BRCA1, BRCA2, TP53BP1) correlated with TLS alteration in multiple tumor types, indicating the interaction between mutation landscape and TLS formation. Epstein-Barr virus (EBV) infection in gastric cancer and human papillomavirus (HPV) infection in Head and Neck squamous cell carcinoma were associated with increased TLS scoring. High TLS scoring predicted favorable prognosis in certain cancer after surgical treatment and improved response to immunotherapy in lung cancer and melanoma. Our findings unraveled the genomic properties associated with TLS formation in different solid tumors and highlighted the prognostic and predictive significance of TLS in surgical treatment and immunotherapy.

Epidemiology and Genetic Characterization of Classical Human Astrovirus Infection in Shanghai, 2015-2016.

OBJECTIVE: Human astroviruses (HAstVs) are single-stranded RNA viruses of the Astroviridae family. Infection of classic HAstVs is one of the most common causes of acute viral gastroenteritis (infectious viral diarrhea). There is a lack of data on the prevalence and genetic characterization of classic HAstVs in acute viral gastroenteritis in the whole population. This study aimed to investigate the epidemiological trend, genotypes, viral co-infections, and viral loads of classic HAstVs in Shanghai, China, from January 2015 to December 2016. METHODS: A total of 6,051 non-redundant stool samples were collected in outpatients with acute diarrhea in Shanghai from January 2015 to December 2016. One-step real-time RT-PCR was used for screening viral diarrhea, including rotavirus A, rotavirus B, rotavirus C, norovirus genotype I and II, classic human astroviruses, and sapovirus. Real-time PCR was used for screening human enteric adenoviruses. Conventional RT-PCR was used for the amplification of viral fragments for genotyping. PCR products were sequenced and used for the construction of phylogenetic trees. RESULTS: The detection rate of classic HAstVs was 1.55% (94/6,051). The prevalence of HAstV infection displayed a typical winter/spring (December to March) seasonality and was highest in the 5-14 age group. Eighty-six samples were genotyped, which revealed HAstV-1 as the most prevalent genotype, followed by HAstV-5, HAstV-4, HAstV- 2, HAstV-8, and HAstV-3. There was a dramatic rise in the prevalence of HAstV-4 from December 2015 to March 2016, and the viral loads of HAstV-4 were significantly higher than those of other genotypes. Among the mixed infection samples, noroviruses were found to be the most frequently co-infected enteric viruses with HAstV. CONCLUSION: Multiple genotypes of classic HAstVs circulated in Shanghai from January 2015 to December 2016. For the first time, HAstV-3\4\5\8 were detected in Shanghai.

Retrospective study on the efficacy of bisphosphonates in tyrosine kinase inhibitor-treated patients with non-small cell lung cancer exhibiting bone metastasis.

Bisphosphonates (Bps) inhibit the maturation of osteoclasts and suppress the adhesion of cancer cells to the bone matrix. They are recommended as the standard treatment for tumors exhibiting bone metastasis (BM). However, whether Bps can improve the prognosis of patients with tyrosine kinase inhibitor (TKI)-treated non-small cell lung cancer (NSCLC) exhibiting BM remains unclear. A total of 129 patients with NSCLC initially diagnosed with BM at The First Affiliated Hospital of Sun Yat-Sen University (Guangzhou, China) between January 2005 and December 2017 were analyzed in the present retrospective study. Median progression free survival (mPFS) time, median bone metastasis overall survival (mBM-OS) time and bone-associated progression-free survival were analyzed. Among the 129 patients, patients treated with Bps experienced significantly prolonged PFS time [mPFS: 7.1 vs. 5.1 months; hazard ratio (HR), 0.51; confidence interval (CI), 0.30-0.87; P=0.0114] in comparison with patients not treated with Bps. Of the 49 patients treated with frontline TKIs (EGFR TKIs or ALK TKI), 32 received Bps at the same time, while 17 patients received TKIs alone. The results revealed that mPFS time was significantly greater in the TKIs plus Bps group than in the TKIs alone group (mPFS: 11.2 vs. 6.9 months; HR, 0.13; CI, 0.05-0.35; P<0.0001). Significantly prolonged BM-OS time was also observed in the combination group in comparison with the TKIs alone group (mBM-OS: 31 vs. 22 months; HR, 0.31; CI, 0.10-0.96; P=0.0413). The present study demonstrated that among the patients who received TKIs (EGFR TKIs or ALK TKIs), those who also received Bps experienced significantly longer PFS time and tended to exhibit significantly improved BM-OS time, which indicated that Bps should be added to the treatment regimen of patients with NSCLC exhibiting genetic mutations and bone metastasis who have been prescribed TKIs (EGFR TKIs or ALK TKIs).

Deciphering Microenvironment of NSCLC based on CD8+ TIL Density and PD-1/PD-L1 Expression.

Purpose: To determine whether distinct tissue immune microenvironments differentially impact on clinical outcome in non-small cell lung cancer (NSCLC), an extended analysis of PD-1/PD-L1 and Tumor Infiltrating Lymphocytes (TILs) was performed. Materials and Methods: 1016 NSCLC mRNA-sequence samples from The Genome Data Analysis Center (TCGA) and 275 NSCLC mRNA-microarray samples from Gene Expression Omnibus (GEO) were included as testing cohort and validation cohort respectively. Enrichment scores of CD8+ T cells' metagene were used for quantifying its infiltrating density. Based on the median values of CD8+ T cell density and PD-1/PD-L1 mRNA expression, the samples were classified into four Tumor Immune Microenvironment types (TIMTs). Overall survival, as well as clinicopathological features, mutational profiles, mismatch repair score etc. were compared across the four types. Results: Neither PD-1 expression nor PD-L1 expression was associated with outcome in the overall NSCLC. Classification of TIMT based on PD-1/PD-L1 and CD8+ TIL could efficiently classify patients of different survival in ADC but not SCC, with the best overall survival achieved in TIMT3 (high CD8+ TIL and low PD-1/PD-L1), whereas TIMT2 (low CD8+ TIL and high PD-1/PD-L1) manifested the worst outcome. TIMT classification based on PD-1/ CD8+ TIL could better stratify patient of different prognosis than PD-L1/ CD8+ TIL based classification. EGFR wide type and IFNγ overexpression were associated with TIMT4 (high PD-1/PD-L1 and high CD8+ TIL), whereas tumor mutational burden (TMB) manifested no significant difference across four TIMTs. Conclusion: The classification of tumors into four microenvironment subtypes based on PD-1/PD-L1 status and CD8+ TIL is an appropriate approach to stratify patients of different clinical outcome and better guide the practical use of immunotherapy.

The Detection and Characterization of Herpes Simplex Virus Type 1 in Confirmed Measles Cases.

Based on measles surveillance in Shanghai, People's Republic of China, from 2006 to 2015, we found that measles virus isolates from 40 throat swab samples exhibited atypical cytopathic effects in Vero/hSLAM cells, which was found to be a result of coinfection with measles virus (MeV) and human herpes simplex virus type 1 (HSV-1). Serological and molecular approaches were used to confirm and characterize the coinfections in these patients. Among the 40 measles cases, measles-specific IgM was detected in 37 cases, while measles-specific IgG was detected in 27 cases. HSV-1-specific IgM and IgG were detected in 7 and 34 cases, respectively, suggesting that most of the MeV infections were primary, but that HSV-1 infection was due to the reactivation of latent virus in most cases. The titers of HSV-1 IgG in patients with either measles or measles-HSV-1 coinfection were significantly higher than those in the healthy group (P = 0.0026 and P < 0.0001, respectively); however, there was no significant difference in the titers of HSV-1 IgG in the MeV and MeV-HSV-1 coinfection patients (P = 0.105). Nucleic acids from MeV and HSV-1 were detected in 40 and 39 throat swabs, respectively. Twenty five MeV RNA sequences were genotyped, and all represented genotype H1, which is the endemic genotype in China. Sequences from the glycoprotein G gene of HSV-1 were used to classify the isolates into two distinct phylogenetic groups: 34 belonged to group A and 3 belonged to group B.

Hepatocyte nuclear factor 1α downregulates HBV gene expression and replication by activating the NF-κB signaling pathway.

The role of hepatocyte nuclear factor 1α (HNF1α) in the regulation of gene expression and replication of hepatitis B virus (HBV) is not fully understood. Previous reports have documented the induction of the expression of viral large surface protein (LHBs) by HNF1α through activating viral Sp1 promoter. Large amount of LHBs can block the secretion of hepatitis B surface antigen (HBsAg). Here we found that HNF1α overexpression inhibited HBV gene expression and replication in Huh7 cells, resulting in marked decreases in HBsAg, hepatitis B e antigen (HBeAg) and virion productions. In contrast, knockdown of endogenous HNF1α expression enhanced viral gene expression and replication. This HNF1α-mediated inhibition did not depend on LHBs. Instead, HNF1α promoted the expression of NF-κB p65 and slowed p65 protein degradation, leading to nuclear accumulation of p65 and activation of the NF-κB signaling, which in turn inhibited HBV gene expression and replication. The inhibitor of the NF-κB signaling, IκBα-SR, could abrogate this HNF1α-mediated inhibition. While the dimerization domain of HNF1α was dispensable for the induction of LHBs expression, all the domains of HNF1α was required for the inhibition of HBV gene expression. Our findings identify a novel role of HNF1α in the regulation of HBV gene expression and replication.

Synergistic Cytotoxicity of Lenalidomide and Dexamethasone in Mantle Cell Lymphoma via Cereblon-dependent Targeting of the IL-6/STAT3/PI3K Axis.

At our center, relapsed mantle cell lymphoma (MCL) can be treated with maintenance therapy composed of consecutive low-dose lenalidomide and short-term, high-dose dexamethasone (LD regimen), which achieves good responses (longer overall survival and progression-free survival) and low toxicity. Cereblon is probably targeted by both lenalidomide and dexamethasone, which leads to synergistic cytotoxicity in MCL by inhibiting the interleukin-6/signal transducer and activator of transcription 3 (IL-6/STAT3), phosphatidylinositol 3-kinase (PI3K)/AKT and AKT2/Forkhead box O3 (FOXO3A)/BCL2-like 11 (BIM) pathways. The two drugs synergistically inhibit the same pathways, but through different sites. Cereblon was found expressed in most of the MCL tissues (91.3% positivity). Moreover, cereblon expression is positively correlated with LD regimen sensitivity: long-term lenalidomide exposure downregulates cereblon and induces multi-drug resistance against lenalidomide, dexamethasone, cytarabine, cisplatin, and methotrexate in vitro. Removal of lenalidomide resensitizes lenalidomide-resistant MCL cells to lenalidomide and dexamethasone. Our work suggests that rotating the LD regimen with other regimens would improve MCL maintenance therapy.

Exogenous Glycine Nitrogen Enhances Accumulation of Glycosylated Flavonoids and Antioxidant Activity in Lettuce (Lactuca sativa L.).

Glycine, the simplest amino acid in nature and one of the most abundant free amino acids in soil, is regarded as a model nutrient in organic nitrogen studies. To date, many studies have focused on the uptake, metabolism and distribution of organic nitrogen in plants, but few have investigated the nutritional performance of plants supplied with organic nitrogen. Lettuce (Lactuca sativa L.), one of the most widely consumed leafy vegetables worldwide, is a significant source of antioxidants and bioactive compounds such as polyphenols, ascorbic acid and tocopherols. In this study, two lettuce cultivars, Shenxuan 1 and Lollo Rossa, were hydroponically cultured in media containing 4.5, 9, or 18 mM glycine or 9 mM nitrate (control) for 4 weeks, and the levels of health-promoting compounds and antioxidant activity of the lettuce leaf extracts were evaluated. Glycine significantly reduced fresh weight compared to control lettuce, while 9 mM glycine significantly increased fresh weight compared to 4.5 or 18 mM glycine. Compared to controls, glycine (18 mM for Shenxuan 1; 9 mM for Lollo Rossa) significantly increased the levels of most antioxidants (including total polyphenols, α-tocopherol) and antioxidant activity, suggesting appropriate glycine supply promotes antioxidant accumulation and activity. Glycine induced most glycosylated quercetin derivatives and luteolin derivatives detected and decreased some phenolic acids compared to nitrate treatment. This study indicates exogenous glycine supplementation could be used strategically to promote the accumulation of health-promoting compounds and antioxidant activity of hydroponically grown lettuce, which could potentially improve human nutrition.

Hepatitis B virus persistence in mice reveals IL-21 and IL-33 as regulators of viral clearance.

Hepatitis B virus (HBV) generally causes self-limiting infection in immunocompetent adults, but establishes chronic infection in some adults and in most maternally infected infants. Factors determining clearance versus persistence are not fully understood. Hydrodynamic injection (HDI) of HBV replicon plasmid via tail vein generally results in quick clearance in immunocompetent adult mice. Here, we report the identification of strain-specific persistence of HBV in mice: one genotype B strain, designated BPS, persisted up to 33 weeks in ~50% of HDI mice. BPS persistence requires viral replication and multiple viral features. Compared to quickly cleared strains, BPS fails to induce robust post-exposure serum IL-21/IL-33 responses. Injection of IL-21-expressing or IL-33-expressing plasmids facilitates clearance of pre-established BPS persistence and protects cured mice from BPS re-challenge. IL-21 and IL-33 also induce clearance of pre-established HBV persistence in another mouse model. These data reveal IL-21 and IL-33 as potent regulators of HBV clearance and valid drug candidates.

Luteolin-7-O-Glucoside Present in Lettuce Extracts Inhibits Hepatitis B Surface Antigen Production and Viral Replication by Human Hepatoma Cells in Vitro.

Hepatitis B virus (HBV) infection is endemic in Asia and chronic hepatitis B (CHB) is a major public health issue worldwide. Current treatment strategies for CHB are not satisfactory as they induce a low rate of hepatitis B surface antigen (HBsAg) loss. Extracts were prepared from lettuce hydroponically cultivated in solutions containing glycine or nitrate as nitrogen sources. The lettuce extracts exerted potent anti-HBV effects in HepG2 cell lines in vitro, including significant HBsAg inhibition, HBV replication and transcription inhibition, without exerting cytotoxic effects. When used in combination interferon-alpha 2b (IFNα-2b) or lamivudine (3TC), the lettuce extracts synergistically inhibited HBsAg expression and HBV replication. By using differential metabolomics analysis, Luteolin-7-O-glucoside was identified and confirmed as a functional component of the lettuce extracts and exhibited similar anti-HBV activity as the lettuce extracts in vitro. The inhibition rate on HBsAg was up to 77.4%. Moreover, both the lettuce extracts and luteolin-7-O-glucoside functioned as organic antioxidants and, significantly attenuated HBV-induced intracellular reactive oxygen species (ROS) accumulation. Luteolin-7-O-glucoside also normalized ROS-induced mitochondrial membrane potential damage, which suggests luteolin-7-O-glucoside inhibits HBsAg and HBV replication via a mechanism involving the mitochondria. Our findings suggest luteolin-7-O-glucoside may have potential value for clinical application in CHB and may enhance HBsAg and HBV clearance when used as a combination therapy.

Engineering Hepadnaviruses as Reporter-Expressing Vectors: Recent Progress and Future Perspectives.

The Hepadnaviridae family of small, enveloped DNA viruses are characterized by a strict host range and hepatocyte tropism. The prototype hepatitis B virus (HBV) is a major human pathogen and constitutes a public health problem, especially in high-incidence areas. Reporter-expressing recombinant viruses are powerful tools in both studies of basic virology and development of antiviral therapeutics. In addition, the highly restricted tropism of HBV for human hepatocytes makes it an ideal tool for hepatocyte-targeting in vivo applications such as liver-specific gene delivery. However, compact genome organization and complex replication mechanisms of hepadnaviruses have made it difficult to engineer replication-competent recombinant viruses that express biologically-relevant cargo genes. This review analyzes difficulties associated with recombinant hepadnavirus vector development, summarizes and compares the progress made in this field both historically and recently, and discusses future perspectives regarding both vector design and application.