Discovery of Rickettsia spp. in mosquitoes collected in Georgia by metagenomics analysis and molecular characterization.

Arthropods have a broad and expanding worldwide presence and can transmit a variety of viral, bacterial, and parasite pathogens. A number of Rickettsia and Orientia species associated with ticks, fleas, lice, and mites have been detected in, or isolated from, patients with febrile illness and/or animal reservoirs throughout the world. Mosquitoes are not currently considered vectors for Rickettsia spp. pathogens to humans or to animals. In this study, we conducted a random metagenome next-generation sequencing (NGS) of 475 pools of Aedes, Culex, and Culiseta species of mosquitoes collected in Georgia from 2018 to 2019, identifying rickettsial gene sequences in 33 pools of mosquitoes. We further confirmed the findings of the Rickettsia by genus-specific quantitative PCR (qPCR) and multi-locus sequence typing (MLST). The NGS and MLST results indicate that Rickettsia spp. are closely related to Rickettsia bellii, which is not known to be pathogenic in humans. The results, together with other reports of Rickettsia spp. in mosquitoes and the susceptibility and transmissibility experiments, suggest that mosquitoes may play a role in the transmission cycle of Rickettsia spp.

Real-world tertiary referral centre experience stopping nucleos(t)ide analogue therapy in patients with chronic hepatitis B.

BACKGROUND: Identifying strategies for stopping nucleos(t)ide analogues (NUC) in patients with chronic hepatitis B (CHB) is a major goal in CHB management. Our study describes our tertiary-centre experience stopping nucleos(t)ide analogues (NUC) in CHB. METHODS: We conducted a retrospective cohort study of all individuals with CHB seen at the Calgary Liver Unit between January 2009 and May 2020 who stopped NUC. We collected baseline demographics and HBV lab parameters before and after stopping NUC with results stratified by off-treatment durability. Clinical flare was defined as alanine aminotransferase (ALT) over twice the upper limit of normal and virological flare as HBV DNA >2000 IU/mL. RESULTS: Forty-seven (3.5%) of the 1337 individuals with CHB stopped NUC therapy. During follow-up, six patients (12.8%) restarted NUCs because of a flare. All flares occurred within six months of discontinuation. Median time to restart treatment was 90 days (Q1 65, Q3 133). Upon restarting, all showed suppression of HBV DNA and ALT normalization. Factors associated with restarting NUC therapy included hepatitis B e antigen (HBeAg) positive status at first appointment and longer NUC consolidation therapy. Age, sex, ethnicity, liver stiffness measurement, choice of NUC, and quantitative hepatitis B surface antigen (qHBsAg) level at stopping were not associated with sustained response off-treatment. Six patients had functional cure with HBsAg loss. CONCLUSIONS: Stopping long-term NUC is feasible in HBeAg negative CHB. Hepatic flares can occur despite low levels of qHBsAg. Finite NUC therapy can be considered in eligible patients who are adherent to close monitoring and follow-up, particularly in the first six months after stopping NUC therapy.

Direct-acting antiviral treatment uptake and sustained virological response outcomes are not affected by alcohol use: A CANUHC analysis.

Background: Alcohol use and hepatitis C virus (HCV) are two leading causes of liver disease. Alcohol use is prevalent among the HCV-infected population and accelerates the progression of HCV-related liver disease. Despite barriers to care faced by HCV-infected patients who use alcohol, few studies have analyzed uptake of direct-acting antiviral (DAA) treatment. Objective: We compared rates of treatment uptake and sustained virological response (SVR) between patients with and without alcohol use. Methods: Prospective data were obtained from the Canadian Network Undertaking against Hepatitis C (CANUHC) cohort. Consenting patients assessed for DAA treatment between January 2016 and December 2019 were included. Demographic and clinical characteristics were compared between patients with and without alcohol use by means of t-tests, χ2 tests, and Fisher's Exact Tests. Univariate and multivariate analyses were used to determine predictors of SVR and treatment initiation. Results: Current alcohol use was reported for 217 of 725 (30%) patients. The proportion of patients initiating DAA treatment did not vary by alcohol use status (82% versus 83%; p = 0.99). SVR rate was similar between patients with alcohol use and patients without alcohol use (92% versus 94%; p = 0.45). Univariate and multivariate analysis found no association between alcohol use and SVR or treatment initiation. Conclusion: Patients engaged in HCV treatment have highly favourable treatment uptake and outcomes regardless of alcohol use. Public health interventions should be directed toward facilitating access to care for all patients irrespective of alcohol use. Research into high-level alcohol use and DAA outcomes is needed.

Rate of brain aging and APOE ε4 are synergistic risk factors for Alzheimer's disease.

Advanced age and the APOE ε4 allele are the two biggest risk factors for Alzheimer's disease (AD) and declining cognitive function. We describe a universal gauge to measure molecular brain age using transcriptome analysis of four human postmortem cohorts (n = 673, ages 25-97) free of neurological disease. In a fifth cohort of older subjects with or without neurological disease (n = 438, ages 67-108), we show that subjects with brains deviating in the older direction from what would be expected based on chronological age show an increase in AD, Parkinson's disease, and cognitive decline. Strikingly, a younger molecular age (-5 yr than chronological age) protects against AD even in the presence of APOE ε4 An established DNA methylation gauge for age correlates well with the transcriptome gauge for determination of molecular age and assigning deviations from the expected. Our results suggest that rapid brain aging and APOE ε4 are synergistic risk factors, and interventions that slow aging may substantially reduce risk of neurological disease and decline even in the presence of APOE ε4.

Biochemical Characterization of the Active Anti-Hepatitis C Virus Metabolites of 2,6-Diaminopurine Ribonucleoside Prodrug Compared to Sofosbuvir and BMS-986094.

Ribonucleoside analog inhibitors (rNAI) target the hepatitis C virus (HCV) RNA-dependent RNA polymerase nonstructural protein 5B (NS5B) and cause RNA chain termination. Here, we expand our studies on ß-d-2'-C-methyl-2,6-diaminopurine-ribonucleotide (DAPN) phosphoramidate prodrug 1 (PD1) as a novel investigational inhibitor of HCV. DAPN-PD1 is metabolized intracellularly into two distinct bioactive nucleoside triphosphate (TP) analogs. The first metabolite, 2'-C-methyl-GTP, is a well-characterized inhibitor of NS5B polymerase, whereas the second metabolite, 2'-C-methyl-DAPN-TP, behaves as an adenosine base analog. In vitro assays suggest that both metabolites are inhibitors of NS5B-mediated RNA polymerization. Additional factors, such as rNAI-TP incorporation efficiencies, intracellular rNAI-TP levels, and competition with natural ribonucleotides, were examined in order to further characterize the potential role of each nucleotide metabolite in vivo Finally, we found that although both 2'-C-methyl-GTP and 2'-C-methyl-DAPN-TP were weak substrates for human mitochondrial RNA (mtRNA) polymerase (POLRMT) in vitro, DAPN-PD1 did not cause off-target inhibition of mtRNA transcription in Huh-7 cells. In contrast, administration of BMS-986094, which also generates 2'-C-methyl-GTP and previously has been associated with toxicity in humans, caused detectable inhibition of mtRNA transcription. Metabolism of BMS-986094 in Huh-7 cells leads to 87-fold higher levels of intracellular 2'-C-methyl-GTP than DAPN-PD1. Collectively, our data characterize DAPN-PD1 as a novel and potent antiviral agent that combines the delivery of two active metabolites.

Tenofovir disoproxil fumarate versus adefovir dipivoxil for chronic hepatitis B.

BACKGROUND: Tenofovir disoproxil fumarate (DF) is a nucleotide analogue and a potent inhibitor of human immunodeficiency virus type 1 reverse transcriptase and hepatitis B virus (HBV) polymerase. METHODS: In two double-blind, phase 3 studies, we randomly assigned patients with hepatitis B e antigen (HBeAg)-negative or HBeAg-positive chronic HBV infection to receive tenofovir DF or adefovir dipivoxil (ratio, 2:1) once daily for 48 weeks. The primary efficacy end point was a plasma HBV DNA level of less than 400 copies per milliliter (69 IU per milliliter) and histologic improvement (i.e., a reduction in the Knodell necroinflammation score of 2 or more points without worsening fibrosis) at week 48. Secondary end points included viral suppression (i.e., an HBV DNA level of <400 copies per milliliter), histologic improvement, serologic response, normalization of alanine aminotransferase levels, and development of resistance mutations. RESULTS: At week 48, in both studies, a significantly higher proportion of patients receiving tenofovir DF than of those receiving adefovir dipivoxil had reached the primary end point (P<0.001). Viral suppression occurred in more HBeAg-negative patients receiving tenofovir DF than patients receiving adefovir dipivoxil (93% vs. 63%, P<0.001) and in more HBeAg-positive patients receiving tenofovir DF than patients receiving adefovir dipivoxil (76% vs. 13%, P<0.001). Significantly more HBeAg-positive patients treated with tenofovir DF than those treated with adefovir dipivoxil had normalized alanine aminotransferase levels (68% vs. 54%, P=0.03) and loss of hepatitis B surface antigen (3% vs. 0%, P=0.02). At week 48, amino acid substitutions within HBV DNA polymerase associated with phenotypic resistance to tenofovir DF or other drugs to treat HBV infection had not developed in any of the patients. Tenofovir DF produced a similar HBV DNA response in patients who had previously received lamivudine and in those who had not. The safety profile was similar for the two treatments in both studies. CONCLUSIONS: Among patients with chronic HBV infection, tenofovir DF at a daily dose of 300 mg had superior antiviral efficacy with a similar safety profile as compared with adefovir dipivoxil at a daily dose of 10 mg through week 48. (ClinicalTrials.gov numbers, NCT00116805 and NCT00117676.)

Are chronic hepatitis C viral infections more benign in patients with hemophilia?

BACKGROUND AND AIMS: Cirrhosis is associated with thromboses of the intrahepatic vasculature. This raises the possibility that HCV infections in hemophiliacs may differ from those in non-hemophiliacs METHODS: Liver biopsy findings from 12 hemophiliacs and 20 age- and gender-matched, non-hemophiliac controls with chronic hepatitis C viral (HCV) infections were compared for inflammatory activity and fibrosis. RESULTS: The mean ages of hemophiliacs and controls were 35.0 +/- 3.0 yr and 39.6 +/- 5.6 yr, respectively (P= 0.2). Serum aspartate aminotransferase (AST) levels were lower (44 +/- 13 vs 70 +/- 43 U/L) and the duration of the partial thromboplastin (PTT) time longer (49.2 +/- 16.9 vs 31.2 +/- 1.2 s.) in hemophiliacs than in controls (P < 0.02 and <0.001, respectively). Six of the seven hemophiliac patients (86%) and 8/17 controls (46%) were infected with genotypes 1a or 1b with the remainder being infected with 2b, 3a, or 3b. Histological activity and fibrosis scores were significantly lower in hemophiliacs than in controls (1.9 +/- 0.6 vs 3.6 +/- 2.7 and 0.3 +/- 0.2 vs 1.5 +/- 1.5, P < 0.05 and P < 0.01, respectively). None of the hemophiliacs had histological evidence of advanced disease (bridging fibrosis and/or cirrhosis) as compared to 7/20 (30%) controls (P < 0.05). CONCLUSION: HCV infections in hemophiliacs may be less severe than in HCV infected patients without hemophilia.

A polyphosphate kinase 1 (ppk1) mutant of Pseudomonas aeruginosa exhibits multiple ultrastructural and functional defects.

Pseudomonas aeruginosa, of medical, environmental, and industrial importance, depends on inorganic polyphosphate (poly P) for a wide range of functions, especially survival. Mutants of PAO1 lacking poly P kinase 1, PPK1, the enzyme responsible for most poly P synthesis in Escherichia coli and other bacteria, are defective in motility, quorum sensing, biofilm formation, and virulence. We describe here multiple defects in the ppk1 mutant PAOM5, including a striking compaction of the nucleoid, distortion of the cell envelope, lack of planktonic motility and exopolymer production, and susceptibility to the beta-lactam antibiotic carbenicillin as well as desiccation. We propose that P. aeruginosa with reduced poly P levels undergoes ultrastructural changes that contribute to profound deficiencies in cellular functions.

Crystal structure of a polyphosphate kinase and its implications for polyphosphate synthesis.

Polyphosphate (polyP), a linear polymer of hundreds of orthophosphate residues, exists in all tested cells in nature, from pathogenic bacteria to mammals. In bacteria, polyP has a crucial role in stress responses and stationary-phase survival. Polyphosphate kinase (PPK) is the principal enzyme that catalyses the synthesis of polyP in bacteria. It has been shown that PPK is required for bacterial motility, biofilm formation and the production of virulence factors. PPK inhibitors may thus provide a unique therapeutic opportunity against antibiotic-resistant pathogens. Here, we report crystal structures of full-length Escherichia coli PPK and its complex with AMPPNP (beta-gamma-imidoadenosine 5-phosphate). PPK forms an interlocked dimer, with each 80 kDa monomer containing four structural domains. The PPK active site is located in a tunnel, which contains a unique ATP-binding pocket and may accommodate the translocation of synthesized polyP. The PPK structure has laid the foundation for understanding the initiation of polyP synthesis by PPK.

Crystallization and characterization of polyphosphate kinase from Escherichia coli.

Linear polyphosphate chains have been found to play a key role in bacterial responses to stresses and nutritional depletion, and are necessary for host infection of various pathogens. Polyphosphate kinase (PPK) is a critical enzyme responsible for polyphosphate synthesis in bacteria. PPK knockout mutations in several Gram-negative pathogens identify PPK as an ideal drug target for the development of a new class of antibacterial drugs. To reveal the catalytic mechanism and provide a structural basis for drug discovery, we have purified and crystallized full-length Escherichia coli PPK and its complex with AMP-PNP. The crystals diffract to a resolution of 2.5A and belong to the space group P4(2)2(1)2 with unit-cell parameters a=152.0, b=152.0, and c=150.0 A. Crystal structure of PPK is being determined by the Se-Met MAD experiment.

Origin-specific unwinding of herpes simplex virus 1 DNA by the viral UL9 and ICP8 proteins: visualization of a specific preunwinding complex.

Herpes simplex virus 1 contains three origins of replication; two copies of oriS and one of a similar sequence, oriL. Here, the combined action of multiple factors known or thought to influence the opening of oriS are examined. These include the viral origin-binding protein, UL9, and single-strand binding protein ICP8, host cell topoisomerase I, and superhelicity of the DNA template. By using electron microscopy, it was observed that when ICP8 and UL9 proteins were added together to oriS-containing supertwisted DNA, a discrete preunwinding complex was formed at oriS on 40% of the molecules, which was shown by double immunolabeling electron microscopy to contain both proteins. This complex was relatively stable to extreme dilution. Addition of ATP led to the efficient unwinding of approximately 50% of the DNA templates. Unwinding proceeded until the acquisition of a high level of positive supertwists in the remaining duplex DNA inhibited further unwinding. Addition of topoisomerase I allowed further unwinding, opening >1 kb of DNA around oriS.