The preparation of antigen for the generation of polyclonal antibodies against the capsid subunit, VP1, and the viral protease, 3Cpro, of Theiler's murine encephalomyelitis virus (TMEV)

The Picornaviridae is a family of viruses of economic importance that have a major impact on human and animal health. Some of the major genera found in the Picornaviridae family are Enterovirus which includes Poliovirus (PV) and Human Rhinovirus (HRV), Cardiovirus which includes Theiler’s murine encephalomyelitis virus (TMEV) and Saffold virus (SAFV), Aphthovirus of which the Foot and Mouth disease virus (FMDV) is a member and Hepatovirus which includes Hepatitis A virus (HAV). Picornaviruses have a single stranded, positive sense RNA genome which is approximately 7.5-8.4 kb pairs in size. The picornavirus genome is translated into a large polyprotein and is proteolytically cleaved by viral proteases namely 2Apro, 3Cpro and 3CDpro into mature viral structural and non-structural polypeptides encoded by the P1, P2 and P3 domains. Picornaviruses utilise host cell machinery and cellular pathways for entry and uncoating, genome replication and capsid assembly. In our laboratory, we are studying the mechanisms by which TMEV interacts with host cell components and our recent research shows that molecular chaperones are required for a production infection. To follow up on this observation, the overall aim of this study was to prepare antigen for the generation of polyclonal antibodies against the TMEV VP1 and 3Cpro proteins. To this end, the TMEV VP1 and 3Cpro amino acid sequences were analysed to identify hydrophobic, hydrophilic and antigenic regions. Homology modelling was performed in order to predict linear B cell epitopes exposed on the surface of the protein structures. The full length coding sequences of VP1 and 3Cpro were selected for amplification by the PCR and cloning into pQE-80L for expression in a bacterial system. Time course induction studies of recombinant VP1 and 3Cpro showed that the proteins were maximally expressed at 6 hrs and 4 hrs respectively. Recombinant VP1 was solubilised using the detergent, Sarcosyl and purified by Nickel affinity chromatography under native conditions. Because recombinant VP1 co-purified with an unidentified protein, the pET expression system was used. Although no protein of the estimated size was observed by SDS-PAGE analysis in the time course induction study, Western analysis using anti-His6 (2) antibodies detected a signal of ~35 kDa. Solubility studies resulted in the presence of two protein bands in the insoluble fraction resolved between 35 and 40 kDa. Recombinant 3Cpro expressed in a bacterial system was predominantly present in the insoluble fraction. Treatment with Sarcosyl had no effect on the solubility of the recombinant protein and it was therefore purified under denaturing conditions using 8M urea. Following dialysis, 3Cpro was used for immunisation of rabbits. Crude anti-TMEV 3Cpro antibodies were able to detect as little as 107 ng of bacterially expressed antigen at a dilution of 1:100 000 by Western analysis. The presence of contaminating proteins was reduced using pre-cleared anti-TMEV 3Cpro antibodies. The antibodies were unable to detect virally expressed 3Cpro in BHK-21 cell lysate supernatant. In an attempt to determine whether TMEV 3Cpro is present in the insoluble fraction, anti-TMEV 3Cpro antibodies were tested using total protein prepared from infected and mock-infected cell lysates. Once again, no protein band the size of 3Cpro was detected. The antibodies were further tested for detection of 3Cpro in TMEV-infected cells by indirect immunofluorescence and confocal microscopy. A diffuse cytoplasmic and perinuclear distribution, as well as nuclear staining, was observed in infected BHK-21 cells. This staining pattern resembled that observed for the HRV, FMDV and EMCV 3Cpro in similar experiments. Further experiments are required to confirm specificity of these antibodies for virally-expressed 3Cpro by Western analysis and indirect immunofluorescence

REASSURED diagnostics at point-of-care in sub-Saharan Africa: A scoping review.

Point-of-care (POC) diagnostics that meet the REASSURED criteria are essential in combating the rapid increase and severity of global health emergencies caused by infectious diseases. However, little is known about whether the REASSURED criteria are implemented in regions known to have a high burden of infectious diseases such as sub-Saharan Africa (SSA). This scoping review maps evidence of the use of REASSURED POC diagnostic tests in SSA. The scoping review was guided by the advanced methodological framework of Arksey and O'Malley, and Levac et al. We searched the following electronic databases for relevant literature: Scopus, Dimensions, ProQuest Central, Google Scholar, and EBSCOhost (MEDLINE, CINAHL, as well as AFRICA-WIDE). Two reviewers independently screened abstracts and full-text articles using the inclusion criteria as reference. We appraised the quality of the included studies using the mixed-method appraisal tool (MMAT) version 2018. We retrieved 138 publications, comprising 134 articles and four grey literature articles. Of these, only five articles were included following abstract and full-text screening. The five included studies were all conducted in SSA. The following themes emerged from the eligible articles: quality assurance on accuracy of REASSURED POC diagnostic tests, sustainability of REASSURED POC diagnostic tests, and local infrastructure capability for delivering REASSURED POC diagnostic tests to end users. All five articles had MMAT scores between 90% and 100%. In conclusion, our scoping review revealed limited published research on REASSURED diagnostics at POC in SSA. We recommend primary studies aimed at investigating the implementation of REASSURED POC diagnostic tests in SSA

Barriers and enablers for implementation of digital-linked diagnostics models at point-of-care in South Africa: stakeholder engagement

Abstract The integration of digital technologies holds significant promise in enhancing accessibility to disease diagnosis and treatment at point-of-care (POC) settings. Effective implementation of such interventions necessitates comprehensive stakeholder engagements. This study presents the outcomes of a workshop conducted with key stakeholders, aiming to discern barriers and enablers in implementing digital-connected POC diagnostic models in South Africa. The workshop, a component of the 2022 REASSURED Diagnostics symposium, employed the nominal group technique (NGT) and comprised two phases: Phase 1 focused on identifying barriers, while Phase 2 centered on enablers for the implementation of digital-linked POC diagnostic models. Stakeholders identified limited connectivity, restricted offline functionality, and challenges related to load shedding or rolling electricity blackouts as primary barriers. Conversely, ease of use, subsidies provided by the National Health Insurance, and 24-h assistance emerged as crucial enablers for the implementation of digital-linked POC diagnostic models. The NGT workshop proved to be an effective platform for elucidating key barriers and enablers in implementing digital-linked POC diagnostic models. Subsequent research endeavors should concentrate on identifying optimal strategies for implementing these advanced diagnostic models in underserved populations

Theiler’s murine encephalomyelitis virus infection induces a redistribution of heat shock proteins 70 and 90 in BHK-21 cells, and is inhibited by novobiocin and geldanamycin

Theiler’s murine encephalomyelitis virus (TMEV) is a positive-sense RNA virus belonging to the Cardiovirus genus in the family Picornaviridae. In addition to other host cellular factors and pathways, picornaviruses utilise heat shock proteins (Hsps) to facilitate their propagation in cells. This study investigated the localisation of Hsps 70 and 90 in TMEV-infected BHK-21 cells by indirect immunofluorescence and confocal microscopy. The effect of Hsp90 inhibitors novobiocin (Nov) and geldanamycin (GA) on the development of cytopathic effect (CPE) induced by infection was also examined. Hsp90 staining was uniformly distributed in the cytoplasm of uninfected cells but was found concentrated in the perinuclear region during late infection where it overlapped with the signal for non-structural protein 2C within the viral replication complex. Hsp70 redistributed into the vicinity of the viral replication complex during late infection, but its distribution did not overlap with that of 2C. Inhibition of Hsp90 by GA and Nov had a negative effect on virus growth over a 48-h period as indicated by no observable CPE in treated compared to untreated cells. 2C was detected by Western analysis of GA-treated infected cell lysates at doses between 0.01 and 0.125 μM, suggesting that processing of viral precursors was not affected in the presence of this drug. In contrast, 2C was absent in cell lysates of Nov-treated cells at doses above 10 μM, although CPE was evident 48 hpi. This is the first study describing the dynamic behaviour of Hsps 70 and 90 in TMEV-infected cells and to identify Hsp90 as an important host factor in the life cycle of this virus