High affinity immobilization of proteins using the CrAsH/TC tag

Protein microarrays represent important tools for biomedical analysis. We have recently described the use of the biarsenical-tetracysteine (TC) tag for the preparation of protein microarrays. The unique feature of this tag enables the site-specific immobilization of TC-containing proteins on biarsenical-modified surfaces, resulting in a fluorescence enhancement that allows the direct quantification of the immobilized proteins. Moreover, the reversibility of the binding upon incubation with large quantities of thiols permits the detachment of the proteins from the surface, thereby enabling recovery of the substrate to extend the life time of the slide. Herein, we describe our recent results that further extend the applicability of the CrAsH/TC tag to the fabrication of biochips. With this aim, the immobilization of proteins on surfaces has been investigated using two different spacers and two TC tags, the minimal TC sequence (CCPGCC) and an optimized motif (FLNCCPGCCMEP). While the minimal peptide motif enables a rapid recycling of the slide, the optimized TC sequence reveals an increased affinity due to its greater resistance to displacement by thiols. Moreover, the developed methodology was applied to the immobilization of proteins via on-chip ligation of recombinant protein thioesters

SilkSatDb: a microsatellite database of the silkworm, Bombyx mori

The SilkSatDb (silkmoth microsatellite database) (http://www.cdfd.org.in/silksatdb) is a relational database of microsatellites extracted from the available expressed sequence tags and whole genome shotgun sequences of the silkmoth, Bombyx mori. The database has been rendered with a simple and robust web-based search facility, developed using PHP. The SilkSatDb also stores information on primers developed and validated in the laboratory. Users can retrieve information on the microsatellite and the protocols used, along with informative figures and polymorphism status of those microsatellites. In addition, the interface is coupled with Autoprimer, a primer-designing program, using which users can design primers for the loci of interest

Late Ebola virus relapse causing meningoencephalitis: a case report

Background: There are thousands of survivors of the 2014 Ebola outbreak in west Africa. Ebola virus can persist in survivors for months in immune-privileged sites; however, viral relapse causing life-threatening and potentially transmissible disease has not been described. We report a case of late relapse in a patient who had been treated for severe Ebola virus disease with high viral load (peak cycle threshold value 13·2). Methods: A 39-year-old female nurse from Scotland, who had assisted the humanitarian effort in Sierra Leone, had received intensive supportive treatment and experimental antiviral therapies, and had been discharged with undetectable Ebola virus RNA in peripheral blood. The patient was readmitted to hospital 9 months after discharge with symptoms of acute meningitis, and was found to have Ebola virus in cerebrospinal fluid (CSF). She was treated with supportive therapy and experimental antiviral drug GS-5734 (Gilead Sciences, San Francisco, Foster City, CA, USA). We monitored Ebola virus RNA in CSF and plasma, and sequenced the viral genome using an unbiased metagenomic approach. Findings: On admission, reverse transcriptase PCR identified Ebola virus RNA at a higher level in CSF (cycle threshold value 23·7) than plasma (31·3); infectious virus was only recovered from CSF. The patient developed progressive meningoencephalitis with cranial neuropathies and radiculopathy. Clinical recovery was associated with addition of high-dose corticosteroids during GS-5734 treatment. CSF Ebola virus RNA slowly declined and was undetectable following 14 days of treatment with GS-5734. Sequencing of plasma and CSF viral genome revealed only two non-coding changes compared with the original infecting virus. Interpretation: Our report shows that previously unanticipated, late, severe relapses of Ebola virus can occur, in this case in the CNS. This finding fundamentally redefines what is known about the natural history of Ebola virus infection. Vigilance should be maintained in the thousands of Ebola survivors for cases of relapsed infection. The potential for these cases to initiate new transmission chains is a serious public health concern

Real-World Outcomes of Direct-Acting Antiviral Treatment and Retreatment in United Kingdom–Based Patients Infected With Hepatitis C Virus Genotypes/Subtypes Endemic in Africa

Abstract Background Chronic hepatitis C virus (HCV) infection affects 71 million individuals, mostly residing in low- and middle-income countries (LMICs). Direct-acting antivirals (DAAs) give high rates of sustained virological response (SVR) in high-income countries where a restricted range of HCV genotypes/subtypes circulate. Methods We studied United Kingdom–resident patients born in Africa to examine DAA effectiveness in LMICs where there is far greater breadth of HCV genotypes/subtypes. Viral genome sequences were determined from 233 patients. Results Full-length viral genomic sequences for 26 known subtypes and 5 previously unidentified isolates covering 5 HCV genotypes were determined. From 149 patients who received DAA treatment/retreatment, the overall SVR was 93%. Treatment failure was associated primarily with 2 subtypes, gt1l and gt4r, using sofosbuvir/ledipasvir. These subtypes contain natural resistance-associated variants that likely contribute to poor efficacy with this drug combination. Treatment failure was also significantly associated with hepatocellular carcinoma. Conclusions DAA combinations give high SVR rates despite the high HCV diversity across the African continent except for subtypes gt1l and gt4r, which respond poorly to sofosbuvir/ledipasvir. These subtypes are widely distributed across Western, Central, and Eastern Africa. Thus, in circumstances where accurate genotyping is absent, ledipasvir and its generic compounds should not be considered as a recommended treatment option. </jats:sec

Genetic epidemiology of SARS-CoV-2 transmission in renal dialysis units - a high risk community-hospital interface

OBJECTIVES: Patients requiring haemodialysis are at increased risk of serious illness with SARS-CoV-2 infection. To improve the understanding of transmission risks in six Scottish renal dialysis units, we utilised the rapid whole-genome sequencing data generated by the COG-UK consortium. METHODS: We combined geographical, temporal and genomic sequence data from the community and hospital to estimate the probability of infection originating from within the dialysis unit, the hospital or the community using Bayesian statistical modelling and compared these results to the details of epidemiological investigations. RESULTS: Of 671 patients, 60 (8.9%) became infected with SARS-CoV-2, of whom 16 (27%) died. Within-unit and community transmission were both evident and an instance of transmission from the wider hospital setting was also demonstrated. CONCLUSIONS: Near-real-time SARS-CoV-2 sequencing data can facilitate tailored infection prevention and control measures, which can be targeted at reducing risk in these settings

Interferon lambda 4 impacts the genetic diversity of hepatitis C virus

Hepatitis C virus (HCV) is a highly variable pathogen that frequently establishes chronic infection. This genetic variability is affected by the adaptive immune response but the contribution of other host factors is unclear. Here, we examined the role played by interferon lambda-4 (IFN-λ4) on HCV diversity; IFN-λ4 plays a crucial role in spontaneous clearance or establishment of chronicity following acute infection. We performed viral genome-wide association studies using human and viral data from 485 patients of white ancestry infected with HCV genotype 3a. We demonstrate that combinations of host genetic variants, which determine IFN-λ4 protein production and activity, influence amino acid variation across the viral polyprotein - not restricted to specific viral proteins or HLA restricted epitopes - and modulate viral load. We also observed an association with viral di-nucleotide proportions. These results support a direct role for IFN-λ4 in exerting selective pressure across the viral genome, possibly by a novel mechanism

Geographical and temporal distribution of SARS-CoV-2 clades in the WHO European Region, January to June 2020

We show the distribution of SARS-CoV-2 genetic clades over time and between countries and outline potential genomic surveillance objectives. We applied three available genomic nomenclature systems for SARS-CoV-2 to all sequence data from the WHO European Region available during the COVID-19 pandemic until 10 July 2020. We highlight the importance of real-time sequencing and data dissemination in a pandemic situation. We provide a comparison of the nomenclatures and lay a foundation for future European genomic surveillance of SARS-CoV-2.Peer reviewe