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An extracellular alkaline protease produced byBacillus licheniformisAP-1 was purified 76-fold, yielding a single 28 kDa band on SDS-PAGE. It was optimally active at pH 11 and at 60°C (assayed over 10 min). The protease was completely inhibited by phenylmethylsulfonyl fluoride and diodopropyl fluorophosphate, with little increase upon Ca2+ and Mg2+ addition. [ABSTRACT FROM AUTHOR]

Abstract Background Single-stranded (ss) DNA viruses in the family Geminiviridae are proving to be very useful in real-time evolution studies. The high mutation rate of geminiviruses and other ssDNA viruses is somewhat mysterious in that their DNA genomes are replicated in host nuclei by high fidelity host polymerases. Although strand specific mutation biases observed in virus species from the geminivirus genus Mastrevirus indicate that the high mutation rates in viruses in this genus may be due to mutational processes that operate specifically on ssDNA, it is currently unknown whether viruses from other genera display similar strand specific mutation biases. Also, geminivirus genomes frequently recombine with one another and an alternative cause of their high mutation rates could be that the recombination process is either directly mutagenic or produces a selective environment in which the survival of mutants is favoured. To investigate whether there is an association between recombination and increased basal mutation rates or increased degrees of selection favoring the survival of mutations, we compared the mutation dynamics of the MSV-MatA and MSV-VW field isolates of Maize streak virus (MSV; Mastrevirus), with both a laboratory constructed MSV recombinant, and MSV recombinants closely resembling MSV-MatA. To determine whether strand specific mutation biases are a general characteristic of geminivirus evolution we compared mutation spectra arising during these MSV experiments with those arising during similar experiments involving the geminivirus Tomato yellow leaf curl virus (Begomovirus genus). Results Although both the genomic distribution of mutations and the occurrence of various convergent mutations at specific genomic sites indicated that either mutation hotspots or selection for adaptive mutations might elevate observed mutation rates in MSV, we found no association between recombination and mutation rates. Importantly, when comparing the mutation spectra of MSV and TYLCV we observed similar strand specific mutation biases arising predominantly from imbalances in the complementary mutations G → T: C → A. Conclusions While our results suggest that recombination does not strongly influence mutation rates in MSV, they indicate that high geminivirus mutation rates are at least partially attributable to increased susceptibility of all geminivirus genomes to oxidative damage while in a single stranded state.

Background: World Health Organization (WHO) tuberculosis (TB) screening guidelines recommend computer-aided detection (CAD) software for chest radiograph (CXR) interpretation. However, studies evaluating their diagnostic and prognostic accuracy are limited., Methods: We conducted a prospective cohort study of household contacts of rifampicin-resistant TB in South Africa. Participants underwent baseline CXR and sputum investigation (routine [single spontaneous] and enhanced [additionally 2-3 induced]) for prevalent TB and follow-up for incident TB. Three CXR-CAD software products (CAD4TBv7.0, qXRv3.0.0, and Lunit INSIGHT v3.1.4.111) were compared. We evaluated their performance to detect routine and enhanced prevalent and incident TB, comparing performance with blood tests (Xpert MTB host-response, erythrocyte sedimentation rate, C-reactive protein, QuantiFERON) in a subgroup., Results: 483 participants were followed up for 4.6 years (median). There were 23 prevalent (7 routinely diagnosed) and 38 incident TB cases. The AUC ROCs (95% CIs) to identify prevalent TB for CAD4TBv7.0, qXRv3.0.0, and Lunit INSIGHT v3.1.4.111 were .87 (.77-.96), .88 (.79-.97), and .91 (.83-.99), respectively. More than 30% with scores above recommended CAD thresholds who were bacteriologically negative on routine baseline sputum were subsequently diagnosed by enhanced sputum investigation or during follow-up. The AUC performance of baseline CAD to identify incident cases ranged between .60 and .65. Diagnostic performance of CAD for prevalent TB was superior to blood testing., Conclusions: Our findings suggest that the potential of CAD-CXR screening for TB is not maximized as a high proportion of those above current thresholds, but with a negative routine confirmatory sputum, have true TB disease that may benefit intervention., Competing Interests: Potential conflicts of interest. S. V. K. and M. R. report Free installation and use of CAD software licenses to FIND as an institution as part of research and evaluation studies. S. V. K. reports receiving consulting fees from FIND and the International Organization of Migration. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: HIV-associated tuberculosis (TB) contributes disproportionately to global tuberculosis mortality. Patients hospitalised at the time of the diagnosis of HIV-associated disseminated TB are typically severely ill and have a high mortality risk despite initiation of tuberculosis treatment. The objective of the study is to assess the safety and efficacy of both intensified TB treatment (high dose rifampicin plus levofloxacin) and immunomodulation with corticosteroids as interventions to reduce early mortality in hospitalised patients with HIV-associated disseminated TB., Methods: This is a phase III randomised controlled superiority trial, evaluating two interventions in a 2 × 2 factorial design: (1) high dose rifampicin (35 mg/kg/day) plus levofloxacin added to standard TB treatment for the first 14 days versus standard tuberculosis treatment and (2) adjunctive corticosteroids (prednisone 1.5 mg/kg/day) versus identical placebo for the first 14 days of TB treatment. The study population is HIV-positive patients diagnosed with disseminated TB (defined as being positive by at least one of the following assays: urine Alere LAM, urine Xpert MTB/RIF Ultra or blood Xpert MTB/RIF Ultra) during a hospital admission. The primary endpoint is all-cause mortality at 12 weeks comparing, first, patients receiving intensified TB treatment to standard of care and, second, patients receiving corticosteroids to those receiving placebo. Analysis of the primary endpoint will be by intention to treat. Secondary endpoints include all-cause mortality at 2 and 24 weeks. Safety and tolerability endpoints include hepatoxicity evaluations and corticosteroid-related adverse events., Discussion: Disseminated TB is characterised by a high mycobacterial load and patients are often critically ill at presentation, with features of sepsis, which carries a high mortality risk. Interventions that reduce this high mycobacterial load or modulate associated immune activation could potentially reduce mortality. If found to be safe and effective, the interventions being evaluated in this trial could be easily implemented in clinical practice., Trial Registration: ClinicalTrials.gov NCT04951986. Registered on 7 July 2021 https://clinicaltrials.gov/study/NCT04951986., (© 2024. The Author(s).)

Background: The evolution of tuberculosis (TB) disease during the clinical latency period remains incompletely understood., Methods: 250 HIV-uninfected, adult household contacts of rifampicin-resistant TB with a negative symptom screen underwent baseline 18 F-Fluorodeoxyglucose positron emission and computed tomography (PET/CT), repeated in 112 after 5-15 months. Following South African and WHO guidelines, participants did not receive preventive therapy. All participants had intensive baseline screening with spontaneous, followed by induced, sputum sampling and were then observed for an average of 4.7 years for culture-positive disease. Baseline PET/CT abnormalities were evaluated in relation to culture-positive disease., Results: At baseline, 59 (23.6%) participants had lung PET/CT findings consistent with TB of which 29 (11.6%) were defined as Subclinical TB, and 30 (12%) Subclinical TB-inactive. A further 83 (33.2%) had other lung parenchymal abnormalities and 108 (43.2%) had normal lungs. Over 1107-person years of follow-up 14 cases of culture-positive TB were diagnosed. Six cases were detected by intensive baseline screening, all would have been missed by the South African symptom-based screening strategy and only one detected by a WHO-recommended chest X-Ray screening strategy. Those with baseline Subclinical TB lesions on PET/CT were significantly more likely to be diagnosed with culture-positive TB over the study period, compared to those with normal lung parenchyma (10/29 [34.5%] vs 2/108 [1.9%], Hazard Ratio 22.37 [4.89-102.47, p<0.001])., Conclusions: These findings challenge the latent/active TB paradigm demonstrating that subclinical disease exists up to 4 years prior to microbiological detection and/or symptom onset. There are important implications for screening and management of TB.

When profiling blood samples by RNA-sequencing (RNA-seq), RNA from haemoglobin (Hgb) can account for up to 70% of the transcriptome. Due to considerations of sequencing depth and power to detect biological variation, Hgb RNA is typically depleted prior to sequencing by hybridisation-based methods; an alternative approach is to deplete reads arising from Hgb RNA bioinformatically. In the present study, we compared the impact of these two approaches on the outcome of differential gene expression analysis performed using RNA-seq data from 58 human tuberculosis (TB) patient or contact whole blood samples-29 globin kit-depleted and 29 matched non-depleted-a subset of which were taken at TB diagnosis and at six months post-TB treatment from the same patient. Bioinformatic depletion of Hgb genes from the non-depleted samples (bioinformatic-depleted) substantially reduced library sizes (median = 57.24%) and fewer long non-coding, micro, small nuclear and small nucleolar RNAs were captured in these libraries. Profiling published TB gene signatures across all samples revealed inferior correlation between kit-depleted and bioinformatic-depleted pairs when the proportion of reads mapping to Hgb genes was higher in the non-depleted sample, particularly at the TB diagnosis time point. A set of putative "globin-fingerprint" genes were identified by directly comparing kit-depleted and bioinformatic-depleted samples at each timepoint. Two TB treatment response signatures were also shown to have decreased differential performance when comparing samples at TB diagnosis to six months post-TB treatment when profiled on the bioinformatic-depleted samples compared with their kit-depleted counterparts. These results demonstrate that failure to deplete Hgb RNA prior to sequencing has a negative impact on the sensitivity to detect disease-relevant gene expression changes even when bioinformatic removal is performed., (© 2023. The Author(s).)

Although homologous recombination can potentially provide viruses with vastly more evolutionary options than are available through mutation alone, there are considerable limits on the adaptive potential of this important evolutionary process. Primary among these is the disruption of favorable coevolved genetic interactions that can occur following the transfer of foreign genetic material into a genome. Although the fitness costs of such disruptions can be severe, in some cases they can be rapidly recouped by either compensatory mutations or secondary recombination events. Here, we used a maize streak virus (MSV) experimental model to explore both the extremes of recombination-induced genetic disruption and the capacity of secondary recombination to adaptively reverse almost lethal recombination events. Starting with two naturally occurring parental viruses, we synthesized two of the most extreme conceivable MSV chimeras, each effectively carrying 182 recombination breakpoints and containing thorough reciprocal mixtures of parental polymorphisms. Although both chimeras were severely defective and apparently noninfectious, neither had individual movement-, encapsidation-, or replication-associated genome regions that were on their own "lethally recombinant." Surprisingly, mixed inoculations of the chimeras yielded symptomatic infections with viruses with secondary recombination events. These recombinants had only 2 to 6 breakpoints, had predominantly inherited the least defective of the chimeric parental genome fragments, and were obviously far more fit than their synthetic parents. It is clearly evident, therefore, that even when recombinationally disrupted virus genomes have extremely low fitness and there are no easily accessible routes to full recovery, small numbers of secondary recombination events can still yield tremendous fitness gains. Importance: Recombination between viruses can generate strains with enhanced pathological properties but also runs the risk of producing hybrid genomes with decreased fitness due to the disruption of favorable genetic interactions. Using two synthetic maize streak virus genome chimeras containing alternating genome segments derived from two natural viral strains, we examined both the fitness costs of extreme degrees of recombination (both chimeras had 182 recombination breakpoints) and the capacity of secondary recombination events to recoup these costs. After the severely defective chimeras were introduced together into a suitable host, viruses with between 1 and 3 secondary recombination events arose, which had greatly increased replication and infective capacities. This indicates that even in extreme cases where recombination-induced genetic disruptions are almost lethal, and 91 consecutive secondary recombination events would be required to reconstitute either one of the parental viruses, moderate degrees of fitness recovery can be achieved through relatively small numbers of secondary recombination events., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The plant-infecting mastreviruses (family Geminiviridae) express two distinct replication-initiator proteins, Rep and RepA. Although RepA is essential for systemic infectivity, little is known about its precise function. We therefore investigated its role in replication using 2D-gel electrophoresis to discriminate the replicative forms of Maize streak virus (MSV) mutants that either fail to express RepA (RepA(-)), or express RepA that is unable to bind the plant retinoblastoma related protein, pRBR. Whereas amounts of viral DNA were reduced in two pRBR-binding deficient RepA mutants, their repertoires of replicative forms changed only slightly. While a complete lack of RepA expression was also associated with reduced viral DNA titres, the only traces of replicative intermediates of RepA(-) viruses were those indicative of recombination-dependent replication. We conclude that in MSV, RepA, but not RepA-pRBR binding, is necessary for single-stranded DNA production and efficient rolling circle replication., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Although monocotyledonous-plant-infecting mastreviruses (in the family Geminiviridae) are known to cause economically significant crop losses in certain areas of the world, in Australia, they pose no obvious threat to agriculture. Consequently, only a few Australian monocot-infecting mastreviruses have been described, and only two have had their genomes fully sequenced. Here, we present the third full-genome sequence of an Australian monocot-infecting mastrevirus from Bromus catharticus belonging to a distinct species, which we have tentatively named Bromus catharticus striate mosaic virus (BCSMV). Although the genome of this new virus shares only 57.7% sequence similarity with that of its nearest known relative, Digitaria didactyla striate mosaic virus (DDSMV; also from Australia), it has features typical of all other known mastrevirus genomes. Phylogenetic analysis showed that both the full genome and each of its probable expressed proteins group with the two other characterised Australian monocot-infecting mastreviruses. Besides the BCSMV genome sequence revealing that Australian monocot-infecting mastrevirus diversity rivals that seen in Africa, it has enabled us, for the first, to time detect evidence of recombination amongst the Australian viruses. Specifically, it appears that DDSMV possesses a short intergenic region sequence that has been recombinationally derived from either BCSMV or a close relative that has not yet been identified.

The ongoing global spread of Tomato yellow leaf curl virus (TYLCV; Genus Begomovirus, Family Geminiviridae) represents a serious looming threat to tomato production in all temperate parts of the world. Whereas determining where and when TYLCV movements have occurred could help curtail its spread and prevent future movements of related viruses, determining the consequences of past TYLCV movements could reveal the ecological and economic risks associated with similar viral invasions. Towards this end we applied Bayesian phylogeographic inference and recombination analyses to available TYLCV sequences (including those of 15 new Iranian full TYLCV genomes) and reconstructed a plausible history of TYLCV's diversification and movements throughout the world. In agreement with historical accounts, our results suggest that the first TYLCVs most probably arose somewhere in the Middle East between the 1930s and 1950s (with 95% highest probability density intervals 1905-1972) and that the global spread of TYLCV only began in the 1980s after the evolution of the TYLCV-Mld and -IL strains. Despite the global distribution of TYLCV we found no convincing evidence anywhere other than the Middle East and the Western Mediterranean of epidemiologically relevant TYLCV variants arising through recombination. Although the region around Iran is both the center of present day TYLCV diversity and the site of the most intensive ongoing TYLCV evolution, the evidence indicates that the region is epidemiologically isolated, which suggests that novel TYLCV variants found there are probably not direct global threats. We instead identify the Mediterranean basin as the main launch-pad of global TYLCV movements.

Tomato leaf curl disease (TLCD) and and tomato yellow leaf curl (TYLCD) is caused by a number of begomovirus species that collectively threaten tomato production worldwide. We report here that an ongoing TLCD and TYLCD epidemic in Iran is caused by variants of tomato leaf curl Palampur virus (ToLCPMV), a newly proposed begomovirus species previously only detected in India. Besides infecting tomatoes, we identified ToLCPMV as the causal agent of a cucurbit disease that has devastated greenhouse cucumber and melon farms in Jiroft, southeastern Iran. We found no convincing evidence that the ToLCPMV DNA-B sequences have been derived through inter-species recombination, however, all of the currently sampled ToLCPMV DNA-A sequences are descendents of a sequence that probably arose through recombination between a ToLCNDV isolate and a currently unsampled geminivirus species that falls outside the ToLCNDV-ToLCPMV cluster. The increasing incidence of ToLCPMV in different cultivated species throughout Iran may signal the emergence of a serious new threat to agricultural production throughout the Middle East.