Synergistic degradation of Azure B and sulfanilamide antibiotics by the white-rot fungus Trametes versicolor with an activated ligninolytic enzyme system.

The treatment of complex polluted wastewater has become an increasingly critical concern for the various types of hazardous organic compounds, including synthetic dyes and pharmaceuticals. Due to their efficient and eco-friendly advantages, the white-rot fungi (WRF) have been applied to degrade environmental pollutants. This study aimed to investigate the removal ability of WRF (i.e., Trametes versicolor WH21) in the co-contamination system composed of Azure B dye and sulfacetamide (SCT). Our study discovered that the decolorization of Azure B (300 mg/L) by strain WH21 was significantly improved (from 30.5% to 86.5%) by the addition of SCT (30 mg/L), while the degradation of SCT was also increased from 76.4% to 96.2% in the co-contamination system. Transcriptomic and biochemical analyses indicated that the ligninolytic enzyme system was activated by the enhanced enzymatic activities of MnPs and laccases, generating higher concentration of extracellular H2O2 and organic acids in strain WH21 in response to SCT stress. Purified MnP and laccase of strain WH21 were revealed with remarkable degradation effect on both Azure B and SCT. These findings significantly expanded the existing knowledge on the biological treatment of organic pollutants, indicating the strong promise of WRF in the treatment of complex polluted wastewater.

Characterization of algal organic matter as precursors for carbonaceous and nitrogenous disinfection byproducts formation: Comparison with natural organic matter.

Algal organic matter (AOM) and natural organic matter (NOM) from a typical eutrophic lake were comprehensively investigated in terms of their physico-chemical property, components and disinfection byproduct formation potentials (DBPFPs). The relationships between specific chemical properties of AOM and NOM with their corresponding DBPFPs were further evaluated during chlorination. Results indicated that AOM had lower specific UV absorbance (SUVA) but richer organic nitrogen contents than NOM. Fluorescence excitation emission matrix spectroscopy further demonstrated that AOM were chiefly composed of aromatic protein-like and soluble microbial byproduct-like matters, while NOM were mainly contributed from humic acid-like and soluble microbial byproduct-like substances. Although the molecular weight (MW) distribution of AOM and NOM showed no significant difference, size-exclusion chromatography with organic carbon as well as organic nitrogen detection (LC-OCD-OND) revealed that AOM were concentrated with the fraction of building blocks and NOM had higher concentrations of biopolymers and humics (HS). Moreover, AOM displayed higher DBPFPs than NOM, especially for nitrogenous DBPFP (N-DBPFP). MW < 1 kDa fractions both in AOM and NOM contributed the largest proportion to the formation of carbonaceous disinfection byproducts (C-DBPs). In addition, Pearson correlation analysis showed that bulk parameter SUVA was significantly relevant to the formation potentials of trihalomethane both in AOM and NOM, but was ineffective for carbonaceous DBPFP (C-DBPFP) prediction. Dissolved organic nitrogen contents in biopolymer and HS characterized by LC-OCD-OND had strong correlations with N-DBPFPs from AOM and NOM, indicating that LC-OCD-OND quantitative analysis could improve the prediction accuracy of the DBP formation than bulk parameters during NOM and AOM chlorination.

Development and Evaluation of a Fully Automated Molecular Assay Targeting the Mitochondrial Small Subunit rRNA Gene for the Detection of Pneumocystis jirovecii in Bronchoalveolar Lavage Fluid Specimens.

The fungal pathogen Pneumocystis jirovecii causes Pneumocystis pneumonia. Although the mitochondrial large subunit rRNA gene (mtLSU) is commonly used as a PCR target, a mitochondrial small subunit rRNA gene (mtSSU)-targeted MultiCode PCR assay was developed on the fully automated ARIES platform for detection of P. jirovecii in bronchoalveolar lavage fluid specimens in 2.5 hours. The assay showed a limit of detection of 800 copies/mL (approximately equal to 22 organisms/mL), with no cross-reactivity with other respiratory pathogens. Compared with the reference Pneumocystis-specific direct fluorescent antibody assay (DFA) and mtLSU-targeted PCR assay, the new assay demonstrated sensitivity of 96.9% (31/32) and specificity of 94.6% (139/147) in detecting P. jirovecii in 180 clinical bronchoalveolar lavage fluid specimens. This assay was concordant with all DFA-positive samples and all but one mtLSU PCR-positive sample, and detected eight positive samples that were negative by DFA and mtLSU PCR. Receiver operating characteristic curve analysis revealed an area under the curve of 0.98 and a threshold cycle (CT) cutoff of 39.1 with sensitivity of 90.9% and specificity of 99.3%. The detection of 39.1

[Restoration of River Sediment by Calcium Peroxide(CaO2) Combined with Biochar].

The internal source pollution of sediment is the main factor leading to the repetition of black-odorous river channels. In order to prevent this situation, a river channel in the Binhu District of Wuxi City was used as an experimental site. In-situ CaO2 combined with a biochar covering technology was used to repair the black odorous river sediment in this channel. The effects of this technology on the quality of mud water, sedimentary volatile sulfide (AVS) and phosphorus forms, microorganisms, and restoration of black odorous sediment were investigated. The results showed that CaO2 combined with biochar coverage could significantly increase the dissolved oxygen (DO) concentration and redox potential (ORP) of the muddy water system. The DO concentration and ORP in the overlying water were maintained above 2 mg·L-1 and 50 mV, respectively. The removal rates of interstitial water ammonia-nitrogen (NH4+-N), chemical oxygen demand (COD), and total phosphorus (TP) reached 43.40%, 41.18%, and 50.97%, respectively. The removal rate of AVS in the sediment reached 37.03%. The high-throughput sequencing showed that the relative abundance of anaerobic microorganisms in the sediment was significantly reduced, and that nitrogen and sulfur removal microorganisms appeared (e.g., Thermomonas, Dechloromonas, Proteus hauser, Desulfomicrobium, and Thiobacillus). Phosphorus in the sediment was converted into Fe/Al-P and Ca-P. Therefore, in-situ CaO2 combined with biochar coverage had a good repairing effect on black odorous sediment.

Influenza A virus directly modulates mouse eosinophil responses.

Allergic asthma and influenza are common respiratory diseases with a high probability of co-occurrence. During the 2009 influenza pandemic, hospitalized patients with influenza experienced lower morbidity if asthma was an underlying condition. We have previously demonstrated that acute allergic asthma protects mice from severe influenza and have implicated eosinophils in the airways of mice with allergic asthma as participants in the antiviral response. However, very little is known about how eosinophils respond to direct exposure to influenza A virus (IAV) or the microenvironment in which the viral burden is high. We hypothesized that eosinophils would dynamically respond to the presence of IAV through phenotypic, transcriptomic, and physiologic changes. Using our mouse model of acute fungal asthma and influenza, we showed that eosinophils in lymphoid tissues were responsive to IAV infection in the lungs and altered surface expression of various markers necessary for cell activation in a niche-specific manner. Siglec-F expression was altered in a subset of eosinophils after virus exposure, and those expressing high Siglec-F were more active (IL-5Rαhi CD62Llo ). While eosinophils exposed to IAV decreased their overall transcriptional activity and mitochondrial oxygen consumption, transcription of genes encoding viral recognition proteins, Ddx58 (RIG-I), Tlr3, and Ifih1 (MDA5), were up-regulated. CD8+ T cells from IAV-infected mice expanded in response to IAV PB1 peptide-pulsed eosinophils, and CpG methylation in the Tbx21 promoter was reduced in these T cells. These data offer insight into how eosinophils respond to IAV and help elucidate alternative mechanisms by which they regulate antiviral immune responses during IAV infection.

[Effect of Ozonation on Microorganism in the Biological Activated Carbon and Disinfection By-Products in the Effluent].

This study was carried out in the ozone (O3) and biological activated carbon (BAC) section of a drinking water plant to investigate the effects of O3 on microbial and effluent disinfection by-products (DBPs) in BAC during drinking water treatment. The water quality, dissolved organic matter (DOM) characteristics, microbial activity, and DBPs formation at different O3 concentrations were analyzed. Results showed that the effect of O3 on microorganisms is mainly that it increased the utilization efficiency of DOM. However, excessive O3 increased the amount of organic matter such as protein and microbial metabolites (SMPs) in the effluent. When the O3 concentration increased from 0 mg·L-1 to 2.0 mg·L-1, the survival rate of microorganisms in the BAC decreased from 95.10% to 62.60%. However, since O3 transforms organic matter into a biodegradable substance, we found that microbial activity increased by 62.52% and that the biofiltration of the BAC was enhanced. When the O3 concentration was further increased to 4.0 mg·L-1, the microbial survival rate decreased to 49.9% and the protein and SMPs produced by the microorganisms increased. This resulted in an increased formation of carbon-containing DBPs (C-DBPs) and nitrogen-containing DBPs (N-DBPs) by 41.93% and 7.18%, respectively. In summary, an appropriate dosage of O3 was beneficial for removing DOM by O3-BAC, but we found that an excessive O3 concentration caused the formation of new DBPs precursors.

Optimization and evaluation of a novel real-time RT-PCR test for detection of parechovirus in cerebrospinal fluid.

Human parechovirus (HPeV) infections cause a broad array of clinical manifestations ranging from gastrointestinal or respiratory illness to central nervous system (CNS) diseases. Though nucleic acid amplification tests (NAATs) for detection of HPeVs have been described, a need exists for sensitive and specific NAATs with internal control (IC). This study describes optimization and evaluation of a novel, real-time reverse transcription PCR (RT-PCR) test for detection of HPeV from CSF using EliTech HPeV Research-Use-Only detection reagent, MS2 IC and quantified HPeV control. Four RT-PCR kits were compared to select an enzyme with optimal amplification efficiency. The optimal RT-PCR enzyme volume and the best approach to add MS2 to the easyMAG extraction platform were investigated. Following assay optimization, performance characteristics were determined. SuperScript was the most efficient one-step RT-PCR kit, with 0.5 µl/reaction of enzyme being most cost-effective. Adding MS2 to samples post-lysis was better than pre-lysis. The limit of detection of the new test was 570 copies/mL. Commercially-available HPeV 1-6 were detectable, and no cross-reactivity with other CNS pathogens was observed. This test was accurate and reproducible for detection of HPeV and IC. It demonstrated good performance characteristics and is a useful addition to a suite of molecular assays for detection of viral pathogens in CSF.

The E3 ligase TRIM56 is a host restriction factor of Zika virus and depends on its RNA-binding activity but not miRNA regulation, for antiviral function.

Infection by Zika virus (ZIKV) is linked to microcephaly and other neurological disorders, posing a significant health threat. Innate immunity is the first line of defense against invading pathogens, but relatively little is understood regarding host intrinsic mechanisms that guard against ZIKV. Here, we show that host tripartite motif-containing protein 56 (TRIM56) poses a barrier to ZIKV infection in cells of neural, epithelial and fibroblast origins. Overexpression of TRIM56, but not an E3 ligase-dead mutant or one lacking a short C-terminal portion, inhibited ZIKV RNA replication. Conversely, depletion of TRIM56 increased viral RNA levels. Although the C-terminal region of TRIM56 bears sequence homology to NHL repeat of TRIM-NHL proteins that regulate miRNA activity, knockout of Dicer, which abolishes production of miRNAs, had no demonstrable effect on ZIKV restriction imposed by TRIM56. Rather, we found that TRIM56 is an RNA-binding protein that associates with ZIKV RNA in infected cells. Moreover, a recombinant TRIM56 fragment comprising the C-terminal 392 residues captured ZIKV RNA in cell-free reactions, indicative of direct interaction. Remarkably, deletion of a short C-terminal tail portion abrogated the TRIM56-ZIKV RNA interaction, concomitant with a loss in antiviral activity. Altogether, our study reveals TRIM56 is an RNA binding protein that acts as a ZIKV restriction factor and provides new insights into the antiviral mechanism by which this E3 ligase tackles flavivirus infections.

Identification of Poxvirus Genome Uncoating and DNA Replication Factors with Mutually Redundant Roles.

Genome uncoating is essential for replication of most viruses. For poxviruses, the process is divided into two stages: removal of the envelope, allowing early gene expression, and breaching of the core wall, allowing DNA release, replication, and late gene expression. Subsequent studies showed that the host proteasome and the viral D5 protein, which has an essential role in DNA replication, are required for vaccinia virus (VACV) genome uncoating. In a search for additional VACV uncoating proteins, we noted a report that described a defect in DNA replication and late expression when the gene encoding a 68-kDa ankyrin repeat/F-box protein (68k-ank), associated with the cellular SCF (Skp1, cullin1, F-box-containing complex) ubiquitin ligase complex, was deleted from the attenuated modified vaccinia virus Ankara (MVA). Here we showed that the 68k-ank deletion mutant exhibited diminished genome uncoating, formation of DNA prereplication sites, and degradation of viral cores as well as an additional, independent defect in DNA synthesis. Deletion of the 68k-ank homolog of VACV strain WR, however, was without effect, suggesting the existence of compensating genes. By inserting VACV genes into an MVA 68k-ank deletion mutant, we discovered that M2, a member of the poxvirus immune evasion (PIE) domain superfamily and a regulator of NF-κB, and C5, a member of the BTB/Kelch superfamily associated with cullin-3-based ligase complexes, independently rescued the 68k-ank deletion phenotype. Thus, poxvirus uncoating and DNA replication are intertwined processes involving at least three viral proteins with mutually redundant functions in addition to D5.IMPORTANCE Poxviruses comprise a family of large DNA viruses that infect vertebrates and invertebrates and cause diseases of medical and zoological importance. Poxviruses, unlike most other DNA viruses, replicate in the cytoplasm, and their large genomes usually encode 200 or more proteins with diverse functions. About 90 genes may be essential for chordopoxvirus replication based either on their conservation or individual gene deletion studies. However, this number may underestimate the true number of essential functions because of redundancy. Here we show that any one of three seemingly unrelated and individually nonessential proteins is required for the incompletely understood processes of genome uncoating and DNA replication, an example of synthetic lethality. Thus, poxviruses appear to have a complex genetic interaction network that has not been fully appreciated and which will require multifactor deletion screens to assess.

Novel HBV recombinants between genotypes B and C in 3'-terminal reverse transcriptase (RT) sequences are associated with enhanced viral DNA load, higher RT point mutation rates and place of birth among Chinese patients.

As one of the major global public health concerns, hepatitis B virus (HBV) can be divided into at least eight genotypes, which may be related to disease severity and treatment response. We previously demonstrated that genotypes B and C HBV, with distinct geographical distribution in China, had divergent genotype-dependent amino acid polymorphisms and variations in reverse transcriptase (RT) gene region, a target of antiviral therapy using nucleos(t)ide analogues. Recently recombination between HBV genotypes B and C was reported to occur in the RT region. However, their frequency and clinical significance is poorly understood. Here full-length HBV RT sequences from 201 Chinese chronic hepatitis B (CHB) patients were amplified and sequenced, among which 31.34% (63/201) were genotype B whereas 68.66% (138/201) genotype C. Although no intergenotypic recombination was detected among C-genotype HBV, 38.10% (24/63) of B-genotype HBV had recombination with genotype C in the 3'-terminal RT sequences. The patients with B/C intergenotypic recombinants had significantly (P<0.05) higher serum HBV DNA level than the "pure" B-genotype cohort did. Moreover, the B/C intergenotypic recombinants were prone to more substitutions at several specific residues in the RT region than genotype B or C. Besides, unlike their parental genotypes, the recombinant HBV appeared to display an altered geographic distribution feature in China. Our findings provide novel insight into the virological, clinical and epidemiological features of new HBV B/C intergenotypic recombinants at the 3' end of RT sequences among Chinese CHB patients. The highly complex genetic background of the novel recombinant HBV carrying new mutations affecting RT protein may contribute to an enhanced heterogeneity in treatment response or prognosis among CHB patients.

The Molecular Chaperone GRP78 Contributes to Toll-like Receptor 3-mediated Innate Immune Response to Hepatitis C Virus in Hepatocytes.

Toll-like receptor-3 (TLR3) senses double-stranded RNA intermediates produced during hepatitis C virus (HCV) replication, leading to activation of interferon regulatory factor-3 (IRF3) and NF-κB and subsequent antiviral and proinflammatory responses. Yet, how this TLR3-dependent pathway operates in hepatocytes is unclear. Upon fractionating cultured hepatocytes into various cellular organelles, we observed that TLR3 predominantly resides in endolysosomes of hepatocytes. To determine the critical regulators of TLR3 signaling in response to HCV infection in human hepatocytes, we isolated endolysosome fractions from mock-infected and HCV-infected hepatoma Huh7.5 cells that had been reconstituted for TLR3 expression, separated these fractions on two-dimensional gels, and identified up-regulated/down-regulated proteins by mass spectrometry. Approximately a dozen of cellular proteins were found to be differentially expressed in endolysosome fractions following HCV infection. Of these, expression of several molecular chaperone proteins was elevated. Knockdown of one of these chaperones, glucose-regulated protein 78 kDa (GRP78), compromised TLR3-dependent induction of interferon-stimulated genes and chemokines following HCV infection or poly(I:C) stimulation in cultured hepatocytes. Consistent with this finding, GRP78 depletion impaired TLR3-mediated establishment of an antiviral state. Mechanistically, although TLR3 trafficking to endolysosomes was not affected, phosphorylated IRF3 diminished faster following GRP78 knockdown. Remarkably, GRP78 transcript was significantly up-regulated in liver biopsies of chronic hepatitis C patients as compared with normal liver tissues. Moreover, the GRP78 expression level correlated with that of RANTES (regulated upon activation, normal T-cell expressed and secreted) and CXCL10, two inflammatory chemokines most frequently elevated in HCV-infected liver. Altogether, our data suggest that GRP78 contributes to TLR3-mediated, IRF3-dependent innate immune response to HCV in hepatocytes.

The C-Terminal Tail of TRIM56 Dictates Antiviral Restriction of Influenza A and B Viruses by Impeding Viral RNA Synthesis.

UNLABELLED: Accumulating data suggest that tripartite-motif-containing (TRIM) proteins participate in host responses to viral infections, either by acting as direct antiviral restriction factors or through regulating innate immune signaling of the host. Of >70 TRIMs, TRIM56 is a restriction factor of several positive-strand RNA viruses, including three members of the family Flaviviridae(yellow fever virus, dengue virus, and bovine viral diarrhea virus) and a human coronavirus (OC43), and this ability invariably depends upon the E3 ligase activity of TRIM56. However, the impact of TRIM56 on negative-strand RNA viruses remains unclear. Here, we show that TRIM56 puts a check on replication of influenza A and B viruses in cell culture but does not inhibit Sendai virus or human metapneumovirus, two paramyxoviruses. Interestingly, the anti-influenza virus activity was independent of the E3 ligase activity, B-box, or coiled-coil domain. Rather, deletion of a 63-residue-long C-terminal-tail portion of TRIM56 abrogated the antiviral function. Moreover, expression of this short C-terminal segment curtailed the replication of influenza viruses as effectively as that of full-length TRIM56. Mechanistically, TRIM56 was found to specifically impede intracellular influenza virus RNA synthesis. Together, these data reveal a novel antiviral activity of TRIM56 against influenza A and B viruses and provide insights into the mechanism by which TRIM56 restricts these medically important orthomyxoviruses. IMPORTANCE: Options to treat influenza are limited, and drug-resistant influenza virus strains can emerge through minor genetic changes. Understanding novel virus-host interactions that alter influenza virus fitness may reveal new targets/approaches for therapeutic interventions. We show here that TRIM56, a tripartite-motif protein, is an intrinsic host restriction factor of influenza A and B viruses. Unlike its antiviral actions against positive-strand RNA viruses, the anti-influenza virus activity of TRIM56 was independent of the E3 ligase activity. Rather, expression of a short segment within the very C-terminal tail of TRIM56 inhibited the replication of influenza viruses as effectively as that of full-length TRIM56 by specifically targeting viral RNA synthesis. These data reveal the remarkable multifaceted activity of TRIM56, which has developed multiple domains to inhibit multiple viral families. They also raise the possibility of developing a broad-spectrum, TRIM56-based antiviral approach for addition to influenza prophylaxis and/or control strategies.

Naturally occurring deletions/insertions in HBV core promoter tend to decrease in hepatitis B e antigen-positive chronic hepatitis B patients during antiviral therapy.

BACKGROUND: Mutations in HBV core promoter (CP) are suggested to affect viral replication and disease progression. We investigated CP deletion/insertion mutations (Del/Ins) in hepatitis B e antigen (HBeAg)-positive chronic hepatitis B (CHB) patients before and during antiviral treatment. METHODS: Direct and clone sequencings were used for detection of CP Del/Ins in 12 patients. The dynamic changes of CP Del/Ins were tracked in these cases until week 48 of treatment. The effects of Del/Ins on CP activities and hepatitis B X protein (HBx) were analysed using luciferase assay and sequence comparison, respectively. Furthermore, 292 untreated HBeAg-positive CHB cases were also analysed. RESULTS: Twelve cases with multi-peak PCR direct sequencing electropherograms at baseline were confirmed to have CP Del/Ins by clone sequencing, with detection rates varying from 14.8% to 93.3% of clones analysed. Follow-up studies showed the detection rates of CP Del/Ins in patients decreased from 100% (12/12) at baseline to 16.7% (2/12) at week 48 of treatment (P<0.001), in parallel with a decline in HBV DNA, hepatitis B surface antigen (HBsAg), alanine aminotransferase (ALT) and aspartate transaminase (AST) levels along with an increase in HBeAg loss. Luciferase assay results showed distinct promoter activities among Del/Ins-harbouring CP sequences. Importantly, 71.8% (148/206) of Del/Ins sequences potentially resulted in HBx carboxy-terminal truncations. CP Del/Ins mutations were also found in 27.4% (80/292) of untreated cases. CONCLUSIONS: Naturally occurring complex of CP Del/Ins mutants existed in untreated HBeAg-positive CHB patients. These mutations would affect HBV transcription activities and integrity of HBx, which might correlate with disease progression. Their prevalence decreases on antiviral therapy in parallel with the decline in HBV DNA, HBsAg and ALT and AST levels.

Overlapping and distinct molecular determinants dictating the antiviral activities of TRIM56 against flaviviruses and coronavirus.

UNLABELLED: The tripartite motif-containing (TRIM) proteins have emerged as a new class of host antiviral restriction factors, with several demonstrating roles in regulating innate antiviral responses. Of >70 known TRIMs, TRIM56 inhibits replication of bovine viral diarrhea virus, a ruminant pestivirus of the family Flaviviridae, but has no appreciable effect on vesicular stomatitis virus (VSV), a rhabdovirus. Yet the antiviral spectrum of TRIM56 remains undefined. In particular, how TRIM56 impacts human-pathogenic viruses is unknown. Also unclear are the molecular determinants governing the antiviral activities of TRIM56. Herein, we show that TRIM56 poses a barrier to infections by yellow fever virus (YFV), dengue virus serotype 2 (DENV2), and human coronavirus virus (HCoV) OC43 but not encephalomyocarditis virus (EMCV). Moreover, by engineering cell lines conditionally expressing various TRIM56 mutants, we demonstrated that TRIM56's antiflavivirus effects required both the E3 ligase activity that lies in the N-terminal RING domain and the integrity of its C-terminal portion, while the restriction of HCoV-OC43 relied upon the TRIM56 E3 ligase activity alone. Furthermore, TRIM56 was revealed to impair YFV and DENV2 propagation by suppressing intracellular viral RNA accumulation but to compromise HCoV-OC43 infection at a later step in the viral life cycle, suggesting that distinct TRIM56 domains accommodate differing antiviral mechanisms. Altogether, TRIM56 is a versatile antiviral host factor that confers resistance to YFV, DENV2, and HCoV-OC43 through overlapping and distinct molecular determinants. IMPORTANCE: We previously reported tripartite motif protein 56 (TRIM56) as a host restriction factor of bovine viral diarrhea virus, a ruminant pathogen. However, the impact of TRIM56 on human-pathogenic RNA viruses is unknown. Herein, we demonstrate that TRIM56 restricts two medically important flaviviruses, yellow fever virus (YFV) and dengue virus serotype 2 (DENV2), and a human coronavirus, HCoV-OC43, but not encephalomyocarditis virus, a picornavirus. Further, we show that TRIM56-mediated inhibition of HCoV-OC43 multiplication depends solely on its E3 ligase activity, whereas its restriction of YFV and DENV2 requires both the E3 ligase activity and integrity of the C-terminal portion. The differing molecular determinants appear to accommodate distinct antiviral mechanisms TRIM56 adopts to target different families of viruses; while TRIM56 curbs intracellular YFV/DENV2 RNA replication, it acts at a later step in HCoV-OC43 life cycle. These novel findings illuminate the molecular basis of the versatility and specificity of TRIM56's antiviral activities against positive-strand RNA viruses.

Amino acid similarities and divergences in the small surface proteins of genotype C hepatitis B viruses between nucleos(t)ide analogue-naïve and lamivudine-treated patients with chronic hepatitis B.

Entire C-genotype small hepatitis B surface (SHBs) sequences were isolated from 139 nucleos(t)ide analogues (NA)-naïve and 74 lamivudine (LMV)-treated chronic hepatitis B (CHB) patients. The conservation and variability of total 226 amino acids (AAs) within the sequences were determined individually, revealing significant higher mutant isolate rate and mutation frequency in LMV-treated cohort than those in the NA-naïve one (P=0.009 and 0.0001, respectively). Three absolutely conserved fragments (s16-s19, s176-s181 and s185-s188) and seven moderately conserved regions (a few AA sites acquiring increased variability after LMV-treatment) were identified. The significant mutation rate increase after LMV-treatment occurred primarily in major hydrophilic region (except 'a' determinant) and transmembrane domain 3/4, but not in other upstream functional regions of SHBs. With little influence on immune escape-associated mutation frequencies within 'a' determinant, LMV-monotherapy significantly induced classical LMVr-associated mirror changes sE164D/rtV173L, sI195M/rtM204V and sW196L/S/rtM204I, as well as non-classical ones sG44E/rtS53N, sT47K/A/rtH55R/Q and sW182stop/rtV191I outside 'a' determinant. Interestingly, another newly-identified truncation mutation sC69stop/rtS78T decreased from 7.91% (11/139) in NA-naïve cohort to 2.70% (2/74) in LMV-treated one. Altogether, the altered AA conservation and diversity in SHBs sequences after LMV-treatment in genotype-C HBV infection might shed new insights into how LMV-therapy affects the SHBs variant evolution and its antigenicity.

MCPIP1 restricts HIV infection and is rapidly degraded in activated CD4+ T cells.

HIV-1 primarily infects activated CD4+ T cells and macrophages. Quiescent CD4+ T cells, however, possess cellular factors that limit HIV-1 infection at different postentry steps of the viral life cycle. Here, we show that the previously reported immune regulator monocyte chemotactic protein-induced protein 1 (MCPIP1) restricts HIV-1 production in CD4+ T cells. While the ectopic expression of MCPIP1 in cell lines abolished the production of HIV-1, silencing of MCPIP1 enhanced HIV-1 production. Subsequent analysis indicated that MCPIP1 imposes its restriction by decreasing the steady levels of viral mRNA species through its RNase domain. Remarkably, common T-cell stimuli induced the rapid degradation of MCPIP1 in both T-cell lines and quiescent human CD4+ T cells. Lastly, blocking the proteosomal degradation of MCPIP1 by MG132 abrogated HIV-1 production in phorbol 12-myristate 13-acetate/ionomycin-stimulated human CD4+ T cells isolated from healthy donors. Overall, MCPIP1 poses a potent barrier against HIV-1 infection at a posttranscriptional stage. Although the observed HIV restriction conferred by MCPIP1 does not seem to be overcome by any viral protein, it is removed during cellular stimulation. These findings provide insights into the mechanisms of cellular activation-mediated HIV-1 production in CD4+ T cells.

TRIM56 is an essential component of the TLR3 antiviral signaling pathway.

Members of the tripartite motif (TRIM) proteins are being recognized as important regulators of host innate immunity. However, specific TRIMs that contribute to TLR3-mediated antiviral defense have not been identified. We show here that TRIM56 is a positive regulator of TLR3 signaling. Overexpression of TRIM56 substantially potentiated extracellular dsRNA-induced expression of interferon (IFN)-ß and interferon-stimulated genes (ISGs), while knockdown of TRIM56 greatly impaired activation of IRF3, induction of IFN-ß and ISGs, and establishment of an antiviral state by TLR3 ligand and severely compromised TLR3-mediated chemokine induction following infection by hepatitis C virus. The ability to promote TLR3 signaling was independent of the E3 ubiquitin ligase activity of TRIM56. Rather, it correlated with a physical interaction between TRIM56 and TRIF. Deletion of the C-terminal portion of TRIM56 abrogated the TRIM56-TRIF interaction as well as the augmentation of TLR3-mediated IFN response. Together, our data demonstrate TRIM56 is an essential component of the TLR3 antiviral signaling pathway and reveal a novel role for TRIM56 in innate antiviral immunity.

Discrepancy of potential antiviral resistance mutation profiles within the HBV reverse transcriptase between nucleos(t)ide analogue-untreated and -treated patients with chronic hepatitis B in a hospital in China.

Little is known about the discrepancy of the potential antiviral resistance mutation profiles within the hepatitis B virus (HBV) reverse transcriptase (RT) between nucleos(t)ide analogue (NA)-untreated and -treated patients with chronic hepatitis B. Full-length HBV RT sequences from 59 NA-treated and 105 NA-untreated Chinese patients were amplified and sequenced. Forty-two potential NA resistance (NAr) mutation sites were screened within these 164 RT sequences. The NAr mutation prevalence and frequency in the NA-treated group were significantly higher than those in the NA-untreated one (P < 0.001, respectively). The classical primary drug resistance and secondary/compensatory mutations were only detected at seven sites (rtL80, rtI169, rtL180, rtA181, rtT184, rtM204, and rtN236) in NA-treated patients. The non-classical putative NAr and pre-treatment mutations were observed at 22 sites (rtT38, rtN/S53, rtL82, rtL/I91, rtN/Y124, rtH126, rtT128, rtN/D134, rtN139, rtR153, rtV191, rtV207, rtS213, rtV214, rtE218, rtY/F221, rtV/I224, rtL229, rtI233, rtN/H238, rtR242, and rtS/C256) in both groups. Substitutions at seven non-classical mutation sites were of interest due to either detection only in patients with virologic breakthrough (rtL82 and rtV214), or potential ties with HBV genotypes (rtV191 and rtL229), or coexistence with rtM204I/V (rtL229), or increased mutation trends after NA-treatment (rtT128, rtV207, and rtN/H238). In conclusion, NA treatment not only constitutes a major selection factor for the primary and secondary/compensatory NAr mutations but also drives the changes of some of the putative NAr mutation sites, most of which are the genotype-independent RT sites (rtL82, rtT128, rtV191, rtV207, rtV214, rtL229, and rtN/H238). Their antiviral resistance potential calls for further investigations.

Pt and Pd in sediments from the Pearl River Estuary, South China: background levels, distribution, and source.

PURPOSE: This study assessed the concentrations of platinum (Pt) and palladium (Pd) in surface sediments and sedimentary cores collected from the Pearl River Estuary with a view of evaluating the distribution, background levels, possible sources, and contamination level of anthropogenic Pt and Pd. MATERIALS AND METHODS: Thirty-six samples of surface sediments and 12 samples from sedimentary cores were collected. Al(2)O(3) was analyzed on fused glass disks by X-ray fluorescence spectrometer. Heavy metal elements were measured by inductively coupled plasma-mass spectrometry. Pt and Pd were separated from the sample matrix by anion exchange chromatography and subsequent solvent extraction after samples had been digested in Carius tubes using aqua regia. The analysis of Pt and Pd was performed by isotopic dilution-inductively coupled plasma-mass spectrometry. RESULTS AND DISCUSSION: Pt and Pd concentrations in surface sediments were 0.28-2.11 and 0.39-38.30 ng/g, respectively, and Pt and Pd concentrations in sedimentary cores were 0.19-1.18 and 0.15-1.76 ng/g, respectively. Background values of Pt and Pd were 0.20-1.17 and 0.10-1.34 ng/g, respectively. The spatial distribution of the enrichment factor differed between Pt and Pd in surface sediments. Down-core variations in Pt, Pd, and other heavy metal elements were similar in all cases and were related to sediment type. CONCLUSIONS: Some of the Pt and Pt in surface samples were derived from anthropogenic emissions. Pt and Pd were delivered to the sediment by fluvial input. In addition to vehicle exhaust catalysts, Pt and Pd were derived from other sources (e.g., industrial process). An important post-burial remobilization process of Pt and Pd is likely to be particle mixing by billows caused by typhoon.