Identification of infectious bursal disease virus (IBDV) receptors through the use of recombinant capsid protein, VP2.

Master of Science in Biochemistry. University of KwaZulu-Natal. Durban, 2014.Infectious bursal disease virus (IBDV) is a non-enveloped Birnavirus which infects the immature antibody producing B-cells of the bursa of Fabricius in young chickens. The virus causes infectious bursal disease (IBD) which is highly contagious and immunosuppressive. A compromised immune system in infected chickens leaves them susceptible to other opportunistic pathogens and as a result increases their mortality rate. Major economic losses in the commercial poultry industry are subsequently experienced in affected regions. Currently vaccines are used to control IBDV infection, however, their efficacy is affected by factors such as the presence of maternally derived antibodies in young chickens which reduces vaccine load, the continuous emergence of new virulent IBDV strains and bursal atrophy caused by some vaccines. It is therefore important to consider new ways of controlling the virus such as targeting specific stages in the virus life cycle. Since virus attachment to host cell receptor(s) is the most crucial step in the virus life cycle, developing novel antiviral agents which prevent viral entry represents a good alternative strategy for IBDV control. Identification of receptor binding proteins and receptors of host cell membranes is required for antiviral development. The receptor binding protein and outer capsid of IBDV is VP2, however, the receptor(s) utilised by IBDV to gain entry into host cells have not been conclusively identified. Recombinant VP2 was used to identify possible IBDV receptor(s) on bursal plasma membranes using a virus overlay protein binding assay (VOPBA) and affinity chromatography. Therefore, VP2 was heterologously expressed in an Escherichia coli and a Pichia pastoris expression system as a 64 kDa fusion protein and a 47 kDa protein respectively. In addition, both systems expressed VP2 as high molecular mass proteins which were confirmed by electro-elution and western blotting. Although purification of VP2 expressed in the E. coli system was a challenge because VP2 expressed as inclusion bodies, polyclonal chicken anti-VP2 antibodies were produced using VP2 expressed in this system. Purification of VP2 expressed in P. pastoris was easier and produced a greater yield of VP2 which was used to produce a VP2-coupled affinity matrix for the purification of chicken anti-VP2 antibodies and for the purification of VP2-binding proteins of the bursal plasma membrane. Moreover, peptides were selected from the VP2 amino acid sequence and use to raise polyclonal chicken anti-VP2 peptide antibodies for comparative identification against chicken anti-VP2 antibodies of possible IBDV receptor(s). Two IBDV VP2-binding proteins with molecular masses of 70 and 32 kDa of the bursal plasma membrane were identified in a VOPBA using recombinant VP2 or IBDV and chicken anti-VP2 antibodies. In addition to the VOPBA, four IBDV VP2-binding proteins with molecular masses of 70, 60, 45 and 32 kDa were affinity purified on a VP2-coupled affinity matrix. Analysis of the affinity purified proteins by mass spectrometry identified five proteins which share common peptides which include, the Ig-gamma chain and Ig-lambda chain of Gallus gallus, outer major protein of Serratia marcescens, the 60 kDa chaperonin of Pseudomonas fluorescens and elongation factor-Tu of Yersinia pestis. The results strongly suggest that an Ig-receptor like protein may form part of the IBDV receptor, however, much further work is required in order to establish whether the chicken homologues of the identified bacterial sequences are part of the putative bursal receptor. It is believed that the bacterial proteins contain common peptides with chicken proteins of the chicken genome which has not been fully annotated as yet. Taken together, this study successfully used VP2 to identify possible IBDV receptor(s) on bursal plasma membranes which could ultimately lead to the development of antiviral agents targeted at IBDV entry

Population-level emergence of bedaquiline and clofazimine resistance-associated variants among patients with drug-resistant tuberculosis in southern Africa: a phenotypic and phylogenetic analysis.

BACKGROUND: Bedaquiline and clofazimine are important drugs in the treatment of drug-resistant tuberculosis and are commonly used across southern Africa, although drug susceptibility testing is not routinely performed. In this study, we did a genotypic and phenotypic analysis of drug-resistant Mycobacterium tuberculosis isolates from cohort studies in hospitals in KwaZulu-Natal, South Africa, to identify resistance-associated variants (RAVs) and assess the extent of clofazimine and bedaquiline cross-resistance. We also used a comprehensive dataset of whole-genome sequences to investigate the phylogenetic and geographical distribution of bedaquiline and clofazimine RAVs in southern Africa. METHODS: In this study, we included M tuberculosis isolates reported from the PRAXIS study of patients with drug-resistant tuberculosis treated with bedaquiline (King Dinuzulu Hospital, Durban) and three other cohort studies of drug-resistant tuberculosis in other KwaZulu-Natal hospitals, and sequential isolates from six persistently culture-positive patients with extensively drug-resistant tuberculosis at the KwaZulu-Natal provincial referral laboratory. Samples were collected between 2013 and 2019. Microbiological cultures were done as part of all parent studies. We sequenced whole genomes of included isolates and measured bedaquiline and clofazimine minimum inhibitory concentrations (MICs) for isolates identified as carrying any Rv0678 variant or previously published atpE, pepQ, and Rv1979c RAVs, which were the subject of the phenotypic study. We combined all whole-genome sequences of M tuberculosis obtained in this study with publicly available sequence data from other tuberculosis studies in southern Africa (defined as the countries of the Southern African Development Community), including isolates with Rv0678 variants identified by screening public genomic databases. We used this extended dataset to reconstruct phylogenetic relationships across lineage 2 and 4 M tuberculosis isolates. FINDINGS: We sequenced the whole genome of 648 isolates from 385 patients with drug-resistant tuberculosis recruited into cohort studies in KwaZulu-Natal, and 28 isolates from six patients from the KwaZulu-Natal referral laboratory. We identified 30 isolates with Rv0678 RAVs from 16 (4%) of 391 patients. We did not identify any atpE, pepQ, or Rv1979c RAVs. MICs were measured for 21 isolates with Rv0678 RAVs. MICs were above the critical concentration for bedaquiline resistance in nine (43%) of 21 isolates, in the intermediate category in nine (43%) isolates, and within the wild-type range in three (14%) isolates. Clofazimine MICs in genetically wild-type isolates ranged from 0·12-0·5 μg/mL, and in isolates with RAVs from 0·25-4·0 μg/mL. Phylogenetic analysis of the extended dataset including M tuberculosis isolates from southern Africa resolved multiple emergences of Rv0678 variants in lineages 2 and 4, documented two likely nosocomial transmission events, and identified the spread of a possibly bedaquiline and clofazimine cross-resistant clone in eSwatini. We also identified four patients with pepQ frameshift mutations that may confer resistance. INTERPRETATION: Bedaquiline and clofazimine cross-resistance in southern Africa is emerging repeatedly, with evidence of onward transmission largely due to Rv0678 mutations in M tuberculosis. Roll-out of bedaquiline and clofazimine treatment in the setting of limited drug susceptibility testing could allow further spread of resistance. Designing strong regimens would help reduce the emergence of resistance. Drug susceptibility testing is required to identify where resistance does emerge. FUNDING: Wellcome Trust, National Institute of Allergy and Infectious Diseases and National Center for Advancing Translational Sciences of the National Institutes of Health

Whole genome sequencing Mycobacterium tuberculosis directly from sputum identifies more genetic diversity than sequencing from culture.

BACKGROUND: Repeated culture reduces within-sample Mycobacterium tuberculosis genetic diversity due to selection of clones suited to growth in culture and/or random loss of lineages, but it is not known to what extent omitting the culture step altogether alters genetic diversity. We compared M. tuberculosis whole genome sequences generated from 33 paired clinical samples using two methods. In one method DNA was extracted directly from sputum then enriched with custom-designed SureSelect (Agilent) oligonucleotide baits and in the other it was extracted from mycobacterial growth indicator tube (MGIT) culture. RESULTS: DNA directly sequenced from sputum showed significantly more within-sample diversity than that from MGIT culture (median 5.0 vs 4.5 heterozygous alleles per sample, p = 0.04). Resistance associated variants present as HAs occurred in four patients, and in two cases may provide a genotypic explanation for phenotypic resistance. CONCLUSIONS: Culture-free M. tuberculosis whole genome sequencing detects more within-sample diversity than a leading culture-based method and may allow detection of mycobacteria that are not actively replicating

Correction to: Whole genome sequencing Mycobacterium tuberculosis directly from sputum identifies more genetic diversity than sequencing from culture.

He authors reported that one of the authors' names was typeset incorrectly in the authorship list

If not now, when? Time for the European Union to define a global health strategy

Speakman, E. M., McKee, M., & Coker, R. (2017). If not now, when? Time for the European Union to define a global health strategy. Lancet Global Health, 5(4), e392-e393. https://doi.org/10.1016/S2214-109X%2817%2930085-

Dynamics of within-host <i>Mycobacterium tuberculosis</i> diversity and heteroresistance during treatment

AbstractBackgroundStudying within-host genetic diversity of Mycobacterium tuberculosis (Mtb) in patients during treatment may identify adaptations to antibiotic and immune pressure. Understanding the significance of genetic heteroresistance, and more specifically heterozygous resistance-associated variants (RAVs), is clinically important given increasing use of rapid molecular tests and whole genome sequencing (WGS).MethodsWe analyse data from six studies in KwaZulu-Natal, South Africa. Most patients (&gt;75%) had baseline rifampicin-resistance. Sputum was collected for culture at baseline and at between two and nine intervals until month six. Positive cultures underwent WGS. Mixed infections and reinfections were excluded from analysis.FindingsBaseline Mtb overall genetic diversity (at treatment initiation or major change to regimen) was associated with cavitary disease, not taking antiretroviral therapy if HIV infected, infection with lineage 2 strains and absence of second-line drug resistance on univariate analyses. Baseline genetic diversity was not associated with six-month outcome. Genetic diversity increased from baseline to weeks one and two before returning to previous levels. Baseline genetic heteroresistance was most common for bedaquiline (6/10 [60%] of isolates with RAVs) and fluoroquinolones (9/62 [13%]). Most patients with heterozygous RAVs on WGS with sequential isolates available demonstrated persistence or fixation (17/20, 85%). New RAVs emerged in 9/286 (3%) patients during treatment. We could detect low-frequency RAVs preceding emergent resistance in only one case, although validation of deep sequencing to detect rare variants is required.InterpretationIn this study of single-strain Mtb infections, baseline within-host bacterial genetic diversity did not predict outcome but may reveal adaptations to host and drug pressures. Predicting emergent resistance from low-frequency RAVs requires further work to separate transient from consequential mutations.FundingWellcome Trust, NIH/NIAID</jats:sec

Bedaquiline resistance in drug-resistant tuberculosis HIV co-infected patients

Genetic mutations linked to bedaquiline resistance were found before starting treatment and acquired during treatment in patients with drug-resistant TB and HIV in KwaZulu-Natal, South Africa. Routine bedaquiline resistance testing needs to be accelerated