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MotivationThe growing number of microbial reference genomes enables the improvement of metagenomic profiling accuracy but also imposes greater requirements on the indexing efficiency, database size, and runtime of taxonomic profilers. Additionally, most profilers focus mainly on bacterial, archaeal, and fungal populations, while less attention is paid to viral communities.ResultsWe present KMCP, a novelk-mer-based metagenomic profiling tool that utilizes genome coverage information by splitting the reference genomes into chunks and then storesk-mers in a modified and optimized COBS index for fast alignment-free sequence searching. KMCP combinesk-mer similarity and genome coverage information to reduce the false positive rate ofk-mer-based taxonomic classification and profiling methods. Benchmarking results based on simulated and real data demonstrate that KMCP, despite a longer running time than all other methods, not only allows the accurate taxonomic profiling of prokaryotic and viral populations but also provides confident pathogen detection in clinical samples of low depth.Availability and ImplementationThe software is open-source under the MIT license and available athttps://github.com/shenwei356/kmcp.

Heterogeneity of antibody responses has been reported in SARS-CoV-2 vaccination recipients with underlying diseases. We investigated the impact of the presence of comorbidities on the humoral response to SARS-CoV-2 vaccination in patients with chronic disease (PWCD) and assessed the effect of the number of comorbidities on the humoral response to vaccination. In this study, neutralizing antibodies (NAbs) and IgG antibodies against the receptor-binding domain (RBD-IgG) were monitored following a full-course vaccination. In total, 1400 PWCD (82.7%, inactivated vaccines; 17.3%, subunit recombinant vaccine) and 245 healthy controls (65.7% inactivated vaccines, 34.3% subunit recombinant vaccine) vaccinated with inactivated or subunit recombinant SARS-CoV-2 vaccines, were included. The seroconversion and antibody levels of the NAbs and RBD-IgG were different in the PWCD group compared with those in the control group. Chronic hepatitis B (odds ratio [OR]: 0.65; 95% confidence interval [CI]: 0.46-0.93), cancer (OR: 0.65; 95% CI: 0.42-0.99), and diabetes (OR: 0.50; 95% CI: 0.28-0.89) were associated with lower seroconversion of NAbs. Chronic kidney disease (OR: 0.29; 95% CI: 0.11-0.76), cancer (OR: 0.38; 95% CI: 0.23-0.62), and diabetes (OR: 0.37; 95% CI: 0.20-0.69) were associated with lower seroconversion of RBD-IgG. Only the presence of autoimmune disease showed significantly lower NAbs and RBD-IgG titers. Patients with most types of chronic diseases showed similar responses to the controls, but humoral responses were still significantly associated with the presence of ≥2 coexisting diseases. Our study suggested that humoral responses following SARS-CoV-2 vaccination are impaired in patients with certain chronic diseases. This article is protected by copyright. All rights reserved.

Background and aims: Little is known regarding the antibody responses of COVID-19 vaccination in patients with autoimmune liver diseases (AILD). We aim to evaluate the antibody responses and explore the impact of immunosuppressants on SARS-CoV-2 vaccines in AILD.Methods: We conducted a prospective observational study and included participants been healthy as controls and AILD. All adverse events (AEs) were recorded. IgG antibodies against the receptor-binding domain (RBD) of spike protein (anti-RBD-IgG) and Neutralizing antibodies (NAbs) were tested after the COVID-19 vaccination. In addition, SARS-CoV-2 specific B cells were detected by flow cytometry.Results: 76 patients and 136 healthy controls (HC) were included. All AEs were mild and self-limiting, and the incidences were similar between AILD and HC groups. The seropositivity rates of anti-RBD-IgG and NAbs in AILD were 97.4% (100% in HC, p = 0.13) and 63.2% (84.6% in HC, p < 0.001), respectively. The titers of anti-RBD-IgG and NAbs were significantly lower in AILD compared with HC. After adjusting for confounders, immunosuppressive therapy was an independent risk factor for the low-level anti-RBD-IgG (adjusted odds ratio [AOR]: 4.7; 95% confidence interval [CI], 1.5-15.2; p = 0.01) and reduced probability of NAbs seropositivity (AOR, 3.0; 95% CI, 1.0-8.9; p = 0.04) in AILD patients. However, regardless of immunosuppressants, the SARS-CoV-2 specific memory B cells responses were comparable between AILD and HC groups.Conclusion: SARS-CoV-2 inactivated vaccine is safe, but its immunogenicity is compromised in patients with AILD. Moreover, immunosuppressants are significantly associated with poor antibody responses to the SARS-CoV-2 vaccine.

Inactivated COVID-19 vaccines have been widely used to vaccinate the Chinese population. However, limited literature exists to explore the effect of obesity on the humoral and cellular immune response to these vaccines. In this study, 132 high BMI (Body mass index) (obesity and overweight, BMI ≥ 24 kg/m

The safety and antibody responses of coronavirus disease 2019 (COVID-19) vaccination in patients with chronic hepatitis B (CHB) virus infection is still unclear, and exploration in safety and antibody responses of COVID-19 vaccination in CHB patients is significant in clinical practice.362 adult CHB patients and 87 healthy controls at an interval of at least 21 days after a full-course vaccination (21-105 days) were enrolled. Adverse events (AEs) were collected by questionnaire. The antibody profiles at 1, 2 and 3 months were elucidated by determination of anti-spike IgG, anti-receptor-binding domain (RBD) IgG, and RBD-angiotensin-converting enzyme 2 blocking antibody. SARS-CoV-2 specific B cells were also analysed.All AEs were mild and self-limiting, and the incidence was similar between CHB patients and controls. Seropositivity rates of three antibodies were similar between CHB patients and healthy controls at 1, 2 and 3 months, but CHB patients had lower titers of three antibodies at 1 month. Compared to healthy controls, HBeAg-positive CHB patients had higher titers of three antibodies at 3 months (all P .05) and a slower decline in antibody titers. Frequency of RBD-specific B cells was positively correlated with titers of anti-RBD IgG (OR = 1.067, P = .004), while liver cirrhosis, antiviral treatment, levels of HBV DNA, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and total bilirubin (TB) were not correlated with titers of anti-RBD IgG.Inactivated COVID-19 vaccines were well tolerated, and induced effective antibody response against SARS-CoV-2 in CHB patients.

Background and aim Globally, China has the highest chronic hepatitis C (CHC) burden, but its real-world direct-acting antiviral (DAA) data are limited. Our aim is to investigate the real-world outcome of China Food and Drug Administration-approved DAA therapies across mainland China including those with genotype (GT) 3. Methods The REAL-C is a multinational real-world interferon-free DAA-treated CHC registry of several mainland China and other Asian centers. We evaluated the sustained virological response rate 12 weeks after end of treatment (SVR12), adverse events, and treatment effect on liver function and fibrosis (fibrosis-4 index). Results We analyzed 859 DAA-treated CHC patients (6/1/2017-5/30/2019) from 12 mainland China centers (three municipalities and nine provinces): median age 52, 49.9% male, 33.1% cirrhosis, 95% treatment naive, and 2.5% HBsAg+ . The most common GT was GT1b (523, 62.2%), followed by GT2a (156, 18.5%), GT3b (74, 8.8%), GT3a (41, 4.9%), and GT6 (37, 4.4%). SVR12 rates were 98.0% overall (95% confidence interval 96.9-98.8%), 98.1% for GT1b, 96.8% GT2a, 100% GT3a, 97.3% GT3b, and 100% GT6. Baseline cirrhosis and male sex but not prior treatment history, renal dysfunction, age, and GTs were associated with SVR12. For both cirrhotic and non-cirrhotic patients, there were significant improvement in liver function tests, alpha fetoprotein, and fibrosis-4 index with SVR12. Serious adverse events were rare (1.1%) with only nine patients discontinuing therapy prematurely and anemia being the most common adverse event (13.1%, mostly with ribavirin). Conclusions In real-world Chinese patients with diverse GTs, Chinese Food and Drug Administration-approved interferon-free DAAs were well tolerated, provided high cure rates (98.0% overall) including GT3a/3b, and led to improvement of liver function.

The clinical manifestations of fascioliasis hepatica in humans are unspecific. Traditional diagnosis relies on evidence of live parasites or eggs in the bile or feces. However, due to similar imaging manifestations, they are often misdiagnosed as malignant tumors. Here, we report a case of a 43-year-old woman with fever and space-occupying liver disease. Liver biopsy, parasite-specific antibody screening, and stool testing did not find any pathogens. Therefore, metagenomic next-generation sequencing (mNGS) and routine microbiological examinations were performed. Finally, Fasciola hepatica was only identified by mNGS. The body temperature of the patient and the eosinophil count remained normal, and the space-occupying liver lesions were significantly absorbed after more than 7 months of treatment with albendazole. The details of this case highlight the timely use of mNGS to identify parasites and judge therapeutic effects after treatment, providing important help for clinical decision-making.

Long noncoding RNAs (lncRNAs) play a critical role in the regulation of many important cellular processes. However, the mechanisms by which lncRNAs regulate viral infection and host immune responses are not well understood. We sought to explore lncRNA regulation of hepatitis C virus (HCV) infection and interferon response. We performed RNA sequencing (RNAseq) in Huh7.5.1 cells with or without interferon alpha (IFNα) treatment. Clustered regularly interspaced short palindromic repeats/Cas9 guide RNA (gRNA) was used to knock out selected genes. The promoter clones were constructed, and the activity of related interferon-stimulated genes (ISGs) were detected by the secrete-pair dual luminescence assay. We constructed the full-length and four deletion mutants of an interferon-induced lncRNA RP11-288L9.4 (lncRNA-IFI6) based on predicted secondary structure. Selected gene mRNAs and their proteins, together with HCV infection, in Huh7.5.1 cells and primary human hepatocytes (PHHs) were monitored by quantitative real-time PCR (qRT-PCR) and western blot. We obtained 7,901 lncRNAs from RNAseq. A total of 1,062 host-encoded lncRNAs were significantly differentially regulated by IFNα treatment. We found that lncRNA-IFI6 gRNA significantly inhibited HCV infection compared with negative gRNA control. The expression of the antiviral ISG IFI6 was significantly increased following lncRNA-IFI6 gRNA editing compared with negative gRNA control in Japanese fulminant hepatitis 1 (JFH1)-infected Huh7.5.1 cells and PHHs. We observed that lncRNA-IFI6 regulation of HCV was independent of Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling. lncRNA-IFI6 negatively regulated IFI6 promoter function through histone modification. Overexpression of the truncated spatial domain or full-length lncRNA-IFI6 inhibited IFI6 expression and increased HCV replication. Conclusion: A lncRNA, lncRNA-IFI6, regulates antiviral innate immunity in the JFH1 HCV infection model. lncRNA-IFI6 regulates HCV infection independently of the JAK-STAT pathway. lncRNA-IFI6 exerts its regulatory function via promoter activation and histone modification of IFI6 through its spatial domain.

Background: Hemophagocytic syndrome (HPS) is a syndrome of excessive immune activation characterized by various clinical symptoms of extreme inflammation. Epstein-Barr virus (EBV) is the most frequent infection associated with HPS, often referred to as virus-associated HPS. EBV-HPS is a serious disorder in which monoclonal growth of T cells infected with EBV and activated macrophages cause pancytopenia, high fever and severe liver injury due to primary infection or reactivation of EBV. Methods: A 22-year-old patient presenting with fever, pancytopenia and splenomegaly was diagnosed with hemophagocytic syndrome (HPS) associated with chronic Epstein-Barr virus (EBV) infection. The patient had been ill for more than one year and was more sensitive to glucocorticoid therapy. However, after tapering off the hormone therapy, the patient relapsed. Results: In view of the long-term glucocorticoid therapy of the patient, glucocorticoid-related side effects may have occurred. Therefore, intermittent VP-16 chemotherapy was given, and the glucocorticoid was reduced gradually to discontinuation. The patient, his brother and his parents had mutations in the HPS related genes. There was the same HPS gene mutation in the patient and his father (the variation c.4863-4 G>A (heterozygosity) in the introns around the LYST gene), and the same HPS gene mutation in the elder brother and mother (missense mutation c.603 G>T (p.Leu 201phe heterozygosity) in the Exon8 of STXBP2 gene). Therefore, there were no suitable sibling donors for the patient, who awaited an unrelated donor. It is presumed that the secondary HPS may have a genetic background. There have been HPS related gene mutations before symptom onset, and these can induce HPS due to infection and other factors. Conclusion: The detection of hemophagocytic related gene mutations for HPS high-risk populations with chronic EBV infection, tumors and autoimmune diseases can be beneficial in predicting the risk of HPS. STXBP2 gene mutations are common in patients with HPS, while LYST gene mutations are rarely reported. This case report provides some reference for the diagnosis of other patients.

Summary Mitochondrial antiviral signaling protein (MAVS) is an adaptor of the innate immune receptor retinoic acid-inducible gene 1 (RIG-I) that links recognition of viral RNA to antiviral signaling. Upon interacting with RIG-I, MAVS undergoes lysine 63-linked poly-ubiquitination by the E3 ligase TRIM31 and subsequently aggregates to activate downstream signaling effectors. We find that the scaffold protein FAF1 forms aggregates that negatively regulate MAVS. FAF1 antagonizes the poly-ubiquitination and aggregation of MAVS by competing with TRIM31 for MAVS association. FAF1 knockout mice are more resistant to RNA virus infection, and FAF1 deficiency in myeloid cells results in enhanced innate signaling and reduced viral load and morbidity in vivo. Upon virus infection, the kinase IKKɛ directly phosphorylates FAF1 at Ser556 and triggers FAF1 de-aggregation. Moreover, Ser556 phosphorylation promotes FAF1 lysosomal degradation, consequently relieving FAF1-dependent suppression of MAVS. These findings establish FAF1 as a modulator of MAVS and uncover mechanisms that regulate FAF1 to insure timely activation of antiviral defense.

Hepatitis C virus (HCV) infection has been shown to regulate microRNA 130a (miR-130a) in patient biopsy specimens and in cultured cells. We sought to identify miR-130a target genes and to explore the mechanisms by which miR-130a regulates HCV and hepatitis B virus (HBV) replication. We used bioinformatics software, including miRanda, TargetScan, PITA, and RNAhybrid, to predict potential miR-130a target genes. miR-130a and its target genes were overexpressed or were knocked down by use of small interfering RNA (siRNA) or clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 guide RNA (gRNA). Selected gene mRNAs and their proteins, together with HCV replication in OR6 cells, HCV JFH1-infected Huh7.5.1 cells, and HCV JFH1-infected primary human hepatocytes (PHHs) and HBV replication in HepAD38 cells, HBV-infected NTCP-Huh7.5.1 cells, and HBV-infected PHHs, were measured by quantitative reverse transcription-PCR (qRT-PCR) and Western blotting, respectively. We selected 116 predicted target genes whose expression was related to viral pathogenesis or immunity for qPCR validation. Of these, the gene encoding pyruvate kinase in liver and red blood cell (PKLR) was confirmed to be regulated by miR-130a overexpression. miR-130a overexpression (via a mimic) knocked down PKLR mRNA and protein levels. A miR-130a inhibitor and gRNA increased PKLR expression, HCV replication, and HBV replication, while miR-130a gRNA and PKLR overexpression increased HCV and HBV replication. Supplemental pyruvate increased HCV and HBV replication and rescued the inhibition of HCV and HBV replication by the miR-130a mimic and PKLR knockdown. We concluded that miR-130a regulates HCV and HBV replication through its targeting of PKLR and subsequent pyruvate production. Our data provide novel insights into key metabolic enzymatic pathway steps regulated by miR-130a, including the steps involving PKLR and pyruvate, which are subverted by HCV and HBV replication. IMPORTANCE We identified that miR-130a regulates the target gene PKLR and its subsequent effect on pyruvate production. Pyruvate is a key intermediate in several metabolic pathways, and we identified that pyruvate plays a key role in regulation of HCV and HBV replication. This previously unrecognized, miRNA-regulated antiviral mechanism has implications for the development of host-directed strategies to interrupt the viral life cycle and prevent establishment of persistent infection for HCV, HBV, and potentially other viral infections.

The elongation factor Tu GTP binding domain-containing protein 2 (EFTUD2) was identified as an anti-hepatitis C virus (HCV) host factor in our recent genome-wide small interfering RNA (siRNA) screen. In this study, we sought to further determine EFTUD2's role in HCV infection and investigate the interaction between EFTUD2 and other regulators involved in HCV innate immune (RIG-I, MDA5, TBK1, and IRF3) and JAK-STAT1 pathways. We found that HCV infection decreased the expression of EFTUD2 and the viral RNA sensors RIG-I and MDA5 in HCV-infected Huh7 and Huh7.5.1 cells and in liver tissue from in HCV-infected patients, suggesting that HCV infection downregulated EFTUD2 expression to circumvent the innate immune response. EFTUD2 inhibited HCV infection by inducing expression of the interferon (IFN)-stimulated genes (ISGs) in Huh7 cells. However, its impact on HCV infection was absent in both RIG-I knockdown Huh7 cells and RIG-I-defective Huh7.5.1 cells, indicating that the antiviral effect of EFTUD2 is dependent on RIG-I. Furthermore, EFTUD2 upregulated the expression of the RIG-I-like receptors (RLRs) RIG-I and MDA5 to enhance the innate immune response by gene splicing. Functional experiments revealed that EFTUD2-induced expression of ISGs was mediated through interaction of the EFTUD2 downstream regulators RIG-I, MDA5, TBK1, and IRF3. Interestingly, the EFTUD2-induced antiviral effect was independent of the classical IFN-induced JAK-STAT pathway. Our data demonstrate that EFTUD2 restricts HCV infection mainly through an RIG-I/MDA5-mediated, JAK-STAT-independent pathway, thereby revealing the participation of EFTUD2 as a novel innate immune regulator and suggesting a potentially targetable antiviral pathway. IMPORTANCE Innate immunity is the first line defense against HCV and determines the outcome of HCV infection. Based on a recent high-throughput whole-genome siRNA library screen revealing a network of host factors mediating antiviral effects against HCV, we identified EFTUD2 as a novel innate immune regulator against HCV in the infectious HCV cell culture model and confirmed that its expression in HCV-infected liver tissue is inversely related to HCV infection. Furthermore, we determined that EFTUD2 exerts its antiviral activity mainly through governing its downstream regulators RIG-I and MDA5 by gene splicing to activate IRF3 and induce classical ISG expression independent of the JAT-STAT signaling pathway. This study broadens our understanding of the HCV innate immune response and provides a possible new antiviral strategy targeting this novel regulator of the innate response.

Objective We conducted this study to compare the efficacy and safety of entecavir and tenofovir in the treatment of treatment-naive HBV e antigen (HBeAg)-positive patients with chronic hepatitis B (CHB) for 144 weeks. Methods A total of 320 treatment-naive HBeAg-positive CHB patients who received randomly a single regimen of either entecavir capsule (ETV) (n = 160) or tenofovir disoproxil fumarate capsule (TDF) (n = 160) for 144 weeks were consecutively enrolled from 14 tertiary hospitals or university hospitals in China between January 2012 and December 2014. Results Two groups showed no difference in baseline characteristics. After 144 weeks of treatment, HBV DNA levels were similarly suppressed in both groups (ETV vs TDF; -6.6485 vs -6.692 log10IU/mL, P = .807). At the same time, both groups showed no difference in terms of the serologic and biochemical response. Of all patients, 2 dropped out due to adverse events and 5 experienced serious adverse reactions. Conclusion Both capsules (ETV or TDF) were equally effective in nucleos(t)ide-naive CHB patients with a comparable side-effect profile even in a long-term of 144 weeks.

HCV replication disrupts normal endoplasmic reticulum (ER) function and activates a signaling network called the unfolded protein response (UPR). UPR is directed by three ER transmembrane proteins including ATF6, IRE1 and PERK. HCV increases TGF-β1 and oxidative stress, which play important roles in liver fibrogenesis. HCV has been shown to induce TGF-β1 through the generation of reactive oxygen species (ROS) and p38 MAPK, JNK, ERK1/2 and NFκB-dependent pathways. However, the relationship between HCV-induced ER stress and UPR activation with TGF-β1 production has not been fully characterized. In this study, we found that ROS and JNK inhibitors block HCV up-regulation of ER stress and UPR activation. ROS, JNK and IRE1 inhibitors blocked HCV-activated NFκB and TGF-β1 expression. ROS, ER stress, NFκB and TGF-β1 signaling were blocked by JNK specific siRNA. Knockdown IRE1 inhibited JFH1-activated NFκB and TGF-β1 activity. Knockdown of JNK and IRE1 blunted JFH1 HCV up-regulation of NFκB and TGF-β1 activation. We conclude that HCV activates NFκB and TGF-β1 through ROS production and induction of JNK and the IRE1 pathway. HCV infection induces ER stress and the UPR in a JNK-dependent manner. ER stress and UPR activation partially contribute to HCV-induced NF-κB activation and enhancement of TGF-β1.

Here a new class of hydroxy- or methoxy-substituted 5-benzylidene(thio)barbiturates were designed, synthesized and their inhibitory effects on the diphenolase activity of mushroom tyrosinase were evaluated. The results showed that several compounds had more potent tyrosinase inhibitory activities than the widely used tyrosinase inhibitor kojic acid (IC50=18.25μM). In particular, 3',4'-dihydroxylated 1e was found to be the most potent inhibitor with IC50 value of 1.52μM. The inhibition mechanism analysis revealed that the potential compounds 1e and 2e exhibited such inhibitory effects on tyrosinase by acting as the irreversible inhibitors. Structure-activity relationships' (SARs) analysis also suggested that further development of such compounds might be of interest.

Background. Hepatitis B virus (HBV) infection has a tendency to be chronic. The quasi species of HBV in the pre-S/S gene and mutations in enhancer II (EnhII)/core promoter (CP)/pre-C of HBV were studied in asymptomatic carrier (AsC) mothers and their children with different virus loads, to gain a better understanding of the pathogenic mechanisms of HBV. Methods. Quasi species were analyzed using an established phylogenetic tree based on the sequences of the pre-S/S gene from 3 mother-child pairs. The mutations of EnhII/CP/pre-C were studied in 15 mother-child pairs. Results. Substitution was the main mutation model. The phylogenetic tree indicated that the pre-S/S gene in every mother-child pair had the same root. The sequences of the pre-S/S gene of each patient were somewhat different from one another, but there was limited evolutionary distance between them. The evolutionary distances between the pre-S/S gene and the base of the tree in patients with low virus loads were greater than those in patients with high virus loads. The mutations in patients with low virus loads were much more frequent than those in patients with high virus loads and were not related to age, irrespective of whether the pre-S/S gene or EnhII/CP/pre-C was considered. Conclusions. There are HBV quasi species in the sera of AsCs, and the mutations are related to virus load and hepatitis B e antigen seroconversion, irrespective of age.

To study the HBsAg transient expression in HepG2 or COS-7 cells with eukaryotic expression plasmids inserting HBsAg gene (pCI-S and pcDNA3.1-S) and the efficacy of naked DNA immunization in mice.Firstly, the recombinant plasmids of pCI-S and pcDNA3.1-S were constructed by the cloning technique and the accuracy of these constructs was confirmed by restriction enzyme digestion and DNA sequencing. Secondly, plasmids of pCI-S and pcDNA3.1-S were transferred into HepG2 and COS-7 cells, respectively by means of cationic liposome. HBsAg transient expression was assayed by ELISA in cell culture supernatants and cell lysates. Thirdly, plasmids were injected into quadriceps muscles of BALB/C mice and serum samples were obtained from individual immunized or control mice 4 weeks after injection and boost injection, respectively. Anti-HBs were assayed in mice sera by ELISA. HBsAg-specific CTL responses of spleen cells from immunized mice were tested by the LDH method.Plasmids of pCI-S and pcDNA3.1-S allowed HBsAg transient expression in cell culture supernatants and cell lysates of HepG2 or COS-7 cells. Intramuscular immunization of BALB/C mice with plasmids of pCI-S or pcDNA3.1-S elicited the antibody and cytotoxic T lymphocyte responses to HBsAg.The vectors used in this study are effective to induce prime antibody and HBsAg-specific-cytotoxic T lymphocyte responses to HBsAg in mice after intramuscular immunization.