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Bombyx mori nucleopolyhedrovirus (BmNPV) is a pathogen that causes great economic losses in sericulture. Many genes play a role in viral infection of silkworms, but silkworm metabolism in response to BmNPV infection is unknown. We studied BmE cells infected with BmNPV. We performed liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)-based non-targeted metabolomics analysis of the cytosolic extract and identified 36, 76, 138, 101, 189, and 166 different molecules at 3, 6, 12, 24, 48, and 72 h post BmNPV infection (hpi) compared with 0 hpi. Compounds representing different areas of metabolism were increased in cells post BmNPV infection. These areas included purine metabolism, aminoacyl-tRNA biosynthesis, and ABC transporters. Glycerophosphocholine (GPC), 2-hydroxyadenine (2-OH-Ade), gamma-glutamylcysteine (γ-Glu-Cys), hydroxytolbutamide, and 5-pyridoxolactone glycerophosphocholine were continuously upregulated in BmE cells post BmNPV infection by heat map analysis. Only 5-pyridoxolactone was found to strongly inhibit the proliferation of BmNPV when it was used to treat BmE cells. Fewer infected cells were detected and the level of BmNPV DNA decreased with increasing 5-pyridoxolactone in a dose-dependent manner. The expression of BmNPV genes ie1, helicase, GP64, and VP39 in BmE cells treated with 5-pyridoxolactone were strongly inhibited in the BmNPV infection stage. This suggested that 5-pyridoxolactone may suppress the entry of BmNPV. The data in this study characterize the metabolism changes in BmNPV-infected cells. Further analysis of 5-pyridoxolactone, which is a robust antiviral molecule, may increase our understanding of antiviral immunity.

Canine distemper virus (CDV) is the aetiological agent that causes canine distemper (CD). Currently, no antiviral drugs have been approved for CD treatment. A77 1726 is the active metabolite of the anti-rheumatoid arthritis (RA) drug leflunomide. It inhibits the activity of Janus kinases (JAKs) and dihydroorotate dehydrogenase (DHO-DHase), a rate-limiting enzyme in de novo pyrimidine nucleotide synthesis. A77 1726 also inhibits the activity of p70 S6 kinase (S6K1), a serine/threonine kinase that phosphorylates and activates carbamoyl-phosphate synthetase (CAD), a second rate-limiting enzyme in the de novo pathway of pyrimidine nucleotide synthesis. Our present study focuses on the ability of A77 1726 to inhibit CDV replication and its underlying mechanisms. Here we report that A77 1726 decreased the levels of the N and M proteins of CDV and lowered the virus titres in the conditioned media of CDV-infected Vero cells. CDV replication was not inhibited by Ruxolitinib (Rux), a JAK-specific inhibitor, but by brequinar sodium (BQR), a DHO-DHase-specific inhibitor, and PF-4708671, an S6K1-specific inhibitor. Addition of exogenous uridine, which restores intracellular pyrimidine nucleotide levels, blocked the antiviral activity of A77 1726, BQR and PF-4708671. A77 1726 and PF-4708671 inhibited the activity of S6K1 in CDV-infected Vero cells, as evidenced by the decreased levels of CAD and S6 phosphorylation. S6K1 knockdown suppressed CDV replication and enhanced the antiviral activity of A77 1726. These observations collectively suggest that the antiviral activity of A77 1726 against CDV is mediated by targeting pyrimidine nucleotide synthesis via inhibiting DHO-DHase activity and S6K1-mediated CAD activation.

In lamivudine-refractory chronic hepatitis B (CHB) patients, discontinuation of lamivudine therapy may lead to loss of lamivudine-resistant hepatitis B virus (HBV) and reappearance of wide-type HBV as dominant strains, yet the underlying mechanism remains unclear. In this study, we cloned wide-type and lamivudine-resistant HBV genomes from the sera of a CHB patient who stopped lamivudine therapy after occurrence of resistant virus and determined the biologic properties of the two isolates in hepatoma cell lines. Sequencing reverse transcriptase region of HBV revealed that the patient developed lamivudine-resistant mutations (rtV173 L, rtL180 M, and rtM204 V) 36 months after the start of lamivudine therapy, and lamivudine-resistant mutants reversed to wild-type after the treatment was stopped for 8 months. Our data showed that the wild-type and mutant isolates had similar transcriptional and translational activity. However, comparison of intracellular and released HBV DNA levels showed that replication efficiency of the mutant virus was approximately 50% of wild-type HBV, while the infectivity of released virus was not affected by the lamivudine-resistant mutations. In conclusion, the reversion of lamivudine-resistant mutants to wild-type HBV after discontinuation of lamivudine in hepatitis B patients may be attributed to better replication fitness of wild-type HBV., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A series of hemslecin A derivatives were synthesized and evaluated for their anti-hepatitis B virus (HBV) activities, namely, inhibiting the secretion of hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and HBV DNA replication on HepG 2.2.15 cells. Most of the derivatives showed enhanced anti-HBV activities, of which compounds A1-A7, B5, C and E exhibited significant activities inhibiting HBV DNA replication with IC(50) values of 2.8-11.6 μM, comparable to that of the positive control, tenofovir. Compounds A1-A3, A5, B5, and C displayed low cytotoxicities, which resulted in high SI values of 89.7, 55.6, 77.8, >83.4, >55.8, and >150.5, respectively., (Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.)

Two new lignans, dihydrodehydrodiconiferyl alcohol 9-O-β-D-(3″-O-acetyl)-xylopyranoside (1) and threo-4,9,9'-trihydroxy-3,3'-dimethoxy-8-O-4'-neolignan 7-O-α-rhamnopyranoside (2) were isolated from Illicium henryi, together with ten known compounds, 3-12. Their structures were elucidated by extensive spectroscopic analyses. The anti-hepatitis B virus (anti-HBV) activity of compounds 1-12 inhibiting HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) secretion on Hep G2.2.15 cell line was evaluated. (-)-Dihydrodehydrodiconiferyl alcohol (4) showed moderate inhibitory activity on both HBsAg and HBeAg secretion with IC(50) values of 0.06 and 0.53 mM, respectively., (Copyright © 2011 Verlag Helvetica Chimica Acta AG, Zürich.)

A series of 4-aryl-6-chloro-quinoline derivatives were synthesized and evaluated for their anti-hepatitis B virus (HBV) activities, namely the abilities to inhibit the secretion of HBV surface antigen (HBsAg), HBV e antigen (HBeAg), and replication of HBV DNA in HepG 2.2.15 cells. Most of the compounds exhibited moderate inhibitory activity against the secretion of HBsAg and HBeAg. Nine compounds (3, 5, 6, 7, 10, 14, 17, 20, 24) showed significant inhibition against HBV DNA replication with IC(50) values in the range of 4.4-9.8 μM, which were comparative to that of positive control tenofovir. Of them, compounds 10, 17, and 20 had low cytotoxicities, resulting in high SI values, >551.2, >143.7, and >284.5, respectively., (Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.)

A series of novel 6-chloro-4-(2-chlorophenyl)-3-(2-hydroxyethyl) quinolin-2(1H)-one derivatives were synthesized and evaluated for anti-hepatitis B virus (anti-HBV) activities in vitro to explore their structure-activity relationships (SARs). Most of the synthesized compounds possessed potent anti-HBV activity, of which the promising compound 44 exhibited significantly inhibitory potency against the secretion of hepatitis surface antigen (HBsAg) (IC(50) = 0.010 mM, SI > 135), hepatitis e antigen (HBeAg) (IC(50) = 0.026 mM, SI > 51) and the replication of HBV DNA (IC(50) = 0.045 mM). Preliminary mechanism study suggested compound 44 could mainly enhance the transcript activity of HBV ENI (enhancer I), EN-II (enhancer II)., (Crown Copyright © 2010. Published by Elsevier Masson SAS. All rights reserved.)

Five new sesquiterpene lactones, henrylactones A-E ( 1- 5), together with ten known compounds: cycloparvifloralone ( 6), tashironin ( 7), tashironin A ( 8), neoanisatin ( 9), anisatin ( 10), anislactone B ( 11), 7- O-acetylanislactone B ( 12), merrillianolide ( 13), cyclomerrillianolide ( 14) and pseudomajucin ( 15), were isolated from the stems and roots of ILLICIUM HENRYI. Their structures were elucidated based on extensive spectroscopic data analyses. Among them, henrylactone A ( 1) is a novel sesquiterpene with a dilactone moiety and its structure was confirmed by X-ray diffraction. Sesquiterpene lactones 1- 15 were tested for their anti-hepatitis B virus (HBV) activities. The most active compound, tashironin ( 7), exhibited an IC (50) value of 0.48 mM (SI = 6.3) inhibiting on HBV surface antigen (HBsAg) secretion and an IC (50) value of 0.15 mM (SI = 20.1) inhibiting on HBV e antigen (HBeAg) secretion using HBV transfected Hep G2.2.15 cell line., (Georg Thieme Verlag KG Stuttgart . New York.)

Thirty-two tetra-acylated derivatives of alisol A were synthesized and evaluated for their anti-hepatitis B virus (HBV) activities and cytotoxicities in vitro. Among the series of alisol A derivatives examined, five analogues were active against HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) secretion in HepG 2.2.15 cells. These results also provide interesting structure-activity relationships of tetra-acylalisol A derivatives. Compounds tetra-acetyl alisol A (A1), tetra-methoxyacetyl alisol A (A23), and tetra-ethoxyacetyl alisol A (A24) exhibited high activities against secretion of HBsAg with IC(50) values of 0.0048, 0.0044, and 0.014 mM, respectively, HBeAg with IC(50) values of 0.011, 0.012, and 0.018 mM, respectively, and remarkable selective index values SI(HBsAg)>333, SI(HBeAg)>145; SI(HBsAg)=209, SI(HBeAg)=77; and SI(HBsAg)>200, SI(HBeAg)>156, respectively. Additional studies in rats showed that compound A1 has favorable pharmacokinetic prosperities for further development purpose, with elimination half-time (t(1/2)) of 1.63 h and oral bioavailability (F) of 40.9%.

Chemical modifications were performed on hydroxyl groups at C-11,23,24,25 positions and C-13(17) double bond of alisol A for structure-activity relationship study. Forty-one derivatives of alisol A were synthesized and assayed for their in vitro anti-hepatitis B virus (HBV) activities and cytotoxicities. Of them, 14 compounds were active against HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) secretion in HepG 2.2.15 cells, and the most promising compound 25 exhibited high activities against secretion of HBsAg (IC(50)=0.028 mM), HBeAg (IC(50)=0.027 mM) and remarkable selective indices (SI(HBsAg) >90, SI(HBeAg) >93).

Background and Aim: Although interferon-alpha (IFN-alpha) is an effective treatment for hepatitis B virus (HBV) infection, its precise mechanism of action has not been identified. In this study, we investigated the role of signal transduction pathways in the activation of anti-HBV responses mediated by IFN-alpha., Methods: Using an oligo microarray, we found that four genes in the IFN-alpha signal pathway were markedly upregulated by IFN-alpha in human hepatoma cells regardless of whether they had been transfected with a plasmid containing the HBV genome: signal transducers and activators of transcription 1 (STAT1), interferon regulatory factor-9 (IRF-9, also called ISGF3gamma or P48), IFN-alpha-inducible protein 15 (IFI-15) and IFN-alpha-inducible protein 6-16 (IFI-6-16). We also investigated the role of IFN-stimulated gene factor3 (ISGF3) complex in IFN-alpha-mediated anti-HBV responses in human hepatoma cells by measuring the mRNA of the three genes within ISGF3 (STAT1, STAT2 and IRF-9) using semiquantitative reverse-transcription PCR (RT-PCR), and expression of the three proteins by western blot, and the mRNA and protein of dsRNA-dependent protein kinase (PKR)., Results: STAT1, STAT2, IRF-9 and PKR mRNA as well as protein levels were upregulated by IFN-alpha treatment. When cells were pretreated with genistein, STAT1, STAT2 and IRF-9 mRNA levels remained unchanged after IFN-alpha stimulation, but PKR mRNA levels decreased, and the expression of the STAT1, P-STAT2, IRF-9 and PKR proteins decreased. Levels of HBV DNA decreased in the supernatants of cells treated with IFN-alpha, while ISGF3 levels increased. The quantity of HBV DNA remained unchanged by pretreating with genistein., Conclusions: These observations suggested that the Janus tyrosine kinase-STAT (JAK-STAT) pathway may play a major role in mediating the effects of IFN-alpha against HBV, and that ISGF3 might be a key factor.

A series of alisol A derivatives were synthesized and evaluated for their anti-hepatitis B virus (HBV) activities and cytotoxicities in vitro. The preliminary investigation demonstrates that simple modifications of the parent structure of alisol A can produce a number of potentially important derivatives against HBV. The most active anti-HBV compound 6a showed high activities against the secretion of HBV surface antigen (IC(50)=0.024 mM), HBV e antigen (IC(50)=0.028 mM) and remarkable selective indices (SI(HBsAg)>108, SI(HBeAg)>93), which was selected for further evaluation as a novel HBV inhibitor.

A series of 4-aryl-6-chloro-quinolin-2-ones and 5-aryl-7-chloro-1,4-benzodiazepine were synthesized and assayed for their in vitro anti-hepatitis B virus activities and cytotoxicities for the first time. Some of the tested compounds were active against HBsAg and HBeAg secretion in Hep G2.2.15 cells. Compound 5c showed IC(50) of 0.074 and 0.449 mM on HBsAg and HBeAg secretions, respectively, which were 10 times higher than that of its analog 4c and led to better selective index (SI) values (SI=23.2 and 3.4, respectively).

Two new alkaloids, hypserpanines A and B (1, 11), together with eleven known compounds, phenolbetain (2), acutumine (3), acutumidine (4), dechloroacutumine (5), dauricumine (6), dauricumidine (7), pronuciferine (8), glaziovine (9), S-reticuline (10), magnoflorine (12) and laurifoline(13), were isolated from Hypserpa nitida Miers. (Menispermaceae) and chemically elucidated through spectral analyses. All the isolated alkaloids were evaluated for their anti-HBV activities in vitro using the HBV transfected Hep G2.2.15 cell line. The most active compound, dauricumidine (7), exhibited an IC(50) value of 0.450 mM (SI=4.13) on hepatitis B virus (HBV) surface antigen (HBsAg) secretion of the Hep G2.2.15 cell line.

Objective: To study the mechanism of signal transduction in anti-HBV action of IFN-alpha., Methods: The HBV DNA in HepG 2.2.15 cell line supernatant with/without IFNalpha-2b were monitored by fluorescence real-time quantitive PCR. STAT1, STAT2, ISGF3-gamma, PKR, 2'5'-OAS mRNA levels from HepG 2 and HepG 2.2.15 cell lines that were treated with/without IFNalpha-2b at different times were detected by semi-quantitive RT-PCR. And the ISGF3-gamma protein was detected by Western blot. Then, these items were detected again after inhibiting the JAK-STAT pathway with genistein., Results: The HBV DNA in 2215 supernatant that were treated with IFNalpha-2b for 8 hours decreased 0.72 log 10 copies/ml. But the basal levels of DNA in cells pretreated with genistein? followed by IFNalpha-2b did not decrease. The STAT1, STAT2, ISGF3-gamma, 2'5'-OAS, PKR mRNA levels were upregulated by IFNalpha-2b. The same phenomena were observed with STAT1, STAT2, ISGF3-gamma mRNA when pretreated with genistein then treated with IFNalpha-2b, but the levels of 2'5'-OAS, PKR mRNA were decreased in this situation. The expression of the protein of ISGF3-gamma was also augmented by IFNalpha-2b, and was blocked by genistein., Conclusion: The JAK-STAT pathway seems to be a critical pathway in IFNalpha-2b action against HBV? and ISGF3 is most probably a key factor of the route.

Objectives: To evaluate the effectiveness and safety of N-acetylcysteine (NAC) in treating chronic hepatitis B patients., Methods: 144 patients with chronic hepatitis B (total bilirubin, TBil>170 mmol/L) from several centers were chosen for a randomized and double blind clinical trial. The patients were divided into a NAC group and a placebo group and all of them were treated with an injection containing the same standardized therapeutic drugs. A daily dose of 8 microgram NAC was added to the injection of the NAC group. The trial lasted 45 days. Hepatic function and other biochemistry parameters were checked at the experimental day 0 and days 15, 30, 45., Results: Each group consisted of 72 patients of similar demology and disease characteristics. During the trial, 28 cases of the 144 patients dropped out. In the NAC group, at day 0 and day 30, the TBil were 401.7 vs. 149.2 and 160.1+/-160.6. In the placebo group, the TBil on the corresponding days were 384.1+/-134.0 and 216.3+/-199.9. Its decrease in the NAC group was 62% and 42% in the placebo group. At day 0 and day 45 of treatment, the effective PTa increase rate was 72% in the NAC group and 54% in the placebo group. The total effective rate (TBil + PTa) was 90% in the NAC group and 69% in the placebo group. The parameters of the two groups showed a remarkable difference. The rate of side effects was 14% in the NAC and 5% in the placebo groups., Conclusion: NAC can decrease the level of serum TBil, increase the PTa and reduce the time of hospitalization. NAC showed no serious adverse effects during the period of our treatment. We find that NCA is effective and secure in treating chronic hepatitis B patients.

Background: The incorporation of human APOBEC3G (hA3G) into HIV is required for exerting its antiviral activity, therefore the mechanism underlying hA3G virion encapsidation has been investigated extensively. hA3G was shown to form low-molecular-mass (LMM) and high-molecular-mass (HMM) complexes. The function of different forms of hA3G in its viral incorporation remains unclear. Methodology/Principal Findings: In this study, we investigated the subcellular distribution and lipid raft association of hA3G using subcellular fractionation, membrane floatation assay and pulse-chase radiolabeling experiments respectively, and studied the correlation between the ability of hA3G to form the different complex and its viral incorporation. Our work herein provides evidence that the majority of newly-synthesized hA3G interacts with membrane lipid raft domains to form Lipid raft-associated hA3G (RA hA3G), which serve as the precursor of mature HMM hA3G complex, while a minority of newly-synthesized hA3G remains in the cytoplasm as a soluble LMM form. The distribution of hA3G among the soluble LMM form, the RA LMM form and the mature forms of HMM is regulated by a mechanism involving the N-terminal part of the linker region and the C-terminus of hA3G. Mutagenesis studies reveal a direct correlation between the ability of hA3G to form the RA LMM complex and its viral incorporation. Conclusions/Significance: Together these data suggest that the Lipid raft-associated LMM A3G complex functions as the cellular source of viral hA3G. [ABSTRACT FROM AUTHOR]

Abstract: Bone marrow stromal cell antigen 2 (BST-2, also known as Tetherin) inhibits HIV-1 release and thereby severely impairs viral replication. HIV-1 accessory protein Vpu induces the down-regulation of cell surface BST-2, and counteracts the antiviral function of BST-2. Blocking Vpu-mediated down-regulation of cell surface BST-2 is viewed as a new opportunity for developing anti-HIV drugs. In this study, we have developed a high-throughput cell-based ELISA to identify small molecules that antagonize HIV-1 Vpu function and consequently inhibit HIV-1 replication through rescuing the antiviral activity of host BST-2. This cell-ELISA shows an excellent correlation with results obtained by flow cytometry (FACS). Under optimal conditions, a Z′ factor of 0.605 was achieved in a 96-well format. Together, these results demonstrate that this assay can be used to quantify the cell surface level of BST-2 and be adapted to a high-throughput screening for novel anti-HIV compounds. [Copyright &y& Elsevier]

Abstract: Human APOBEC3G (hA3G), a cytidine deaminase with two cytidine deaminase domains (CDs), has been identified as an anti-HIV-1 host factor. Although the two CDs of hA3G have been extensively characterized, there is still debate on the role of the CDs in the biological function of hA3G. In this work, we constructed three hA3G mutants CD1-1, CD2-2 and CD2-1, which contain duplicate CD1 domain, duplicate CD2 domain and position switched CD domain respectively, and investigated the effect of CD domain replacement or switch upon virion encapsidation, Vif-mediated degradation, deamination and antiviral activity of hA3G. The results showed that the two CD domains were functionally equivalent in virion encapsidation and the interaction with HIV-1 Vif of hA3G, whereas CD domain switch or replacement greatly affected the sensitivity to Vif induced degradation, editing and antiviral activity of hA3G. Although the CD2 domain was shown to possess the deamination activity, CD2-2 incorporated efficiently into HIV-1 was unable to mutate viral cDNA, suggesting that CD1 also involved in the enzymatic function. Interestingly, CD2-1 retained considerable deamination activity with a different sequence preference. Taken together, our results suggest that CD domain may play a structural role in virion encapsidation and Vif-mediated degradation of hA3G, and coordination of the two CD domains is required for its editing and antiviral activity. [Copyright &y& Elsevier]

Abstract: A series of 1-aryl-6,7-dihydroxyl(methoxy)-1,2,3,4-tetrahydroisoquinolines (compounds 1–36) were synthesized via Pictet–Spengler cyclization. All the synthesized compounds were assayed for activities against HIV-1IIIB in C8166 cell cultures by MTT method for the first time. The results of the anti-HIV screening revealed that 6,7-dihydroxytetrahydroisoquinolines possessed higher selective index than 6,7-dimethoxyl analogs due to the significantly decreased cytotoxicities. Compounds 6, 24, and 36 showed potent anti-HIV activities with EC50 values of 8.2, 4.6, and 5.3μM respectively, and the cytotoxicities (CC50) of these three compounds were 784.3, 727.3, and 687.3μM, which resulted in SI values larger than 95, 159, and 130 respectively. [Copyright &y& Elsevier]