Whole-genome based strain identification of fowlpox virus directly from cutaneous tissue and propagated virus

Fowlpox (FP) is an economically important viral disease of commercial poultry. The fowlpox virus (FPV) is primarily characterised by immunoblotting, restriction enzyme analysis in combination with PCR, and/or nucleotide sequencing of amplicons. Whole-genome sequencing (WGS) of FPV directly from clinical specimens prevents the risk of potential genome modifications associated with in vitro culturing of the virus. Only one study has sequenced FPV genomes directly from clinical samples using Nanopore sequencing, however, the study didn’t compare the sequences against Illumina sequencing or laboratory propagated sequences. Here, the suitability of WGS for strain identification of FPV directly from cutaneous tissue was evaluated, using a combination of Illumina and Nanopore sequencing technologies. Sequencing results were compared with the sequence obtained from FPV grown in chorioallantoic membranes (CAMs) of chicken embryos. Complete genome sequence of FPV was obtained directly from affected comb tissue using a map to reference approach. FPV sequence from cutaneous tissue was highly similar to that of the virus grown in CAMs with a nucleotide identity of 99.8%. Detailed polymorphism analysis revealed the presence of a highly comparable number of single nucleotide polymorphisms (SNPs) in the two sequences when compared to the reference genome, providing essentially the same strain identification information. Comparative genome analysis of the map to reference consensus sequences from the two genomes revealed that this field isolate had the highest nucleotide identity of 99.5% with an FPV strain from the USA (Fowlpox virus isolate, FWPV-MN00.2, MH709124) and 98.8% identity with the Australian FPV vaccine strain (FWPV-S, MW142017). Sequencing results showed that WGS directly from cutaneous tissues is not only rapid and cost-effective but also provides essentially the same strain identification information as in-vitro grown virus, thus circumventing in vitro culturing.

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Fungal Genomic Resources for Strain Identification and Diversity Analysis of 1900 Fungal Species

Journal of Fungi ◽

10.3390/jof7040288 ◽

2021 ◽

Vol 7 (4) ◽

pp. 288

Author(s):

Mir Asif Iquebal ◽

Sarika Jaiswal ◽

Vineet Kumar Mishra ◽

Rahul Singh Jasrotia ◽

Ulavappa B. Angadi ◽

...

Keyword(s):

Genome Sequence ◽

In Silico ◽

Sequence Data ◽

Fungal Species ◽

Genomic Research ◽

Whole Genome Sequence ◽

Strain Identification ◽

Diversity Analysis ◽

Whole Genome

Identification and diversity analysis of fungi is greatly challenging. Though internal transcribed spacer (ITS), region-based DNA fingerprinting works as a “gold standard” for most of the fungal species group, it cannot differentiate between all the groups and cryptic species. Therefore, it is of paramount importance to find an alternative approach for strain differentiation. Availability of whole genome sequence data of nearly 2000 fungal species are a promising solution to such requirement. We present whole genome sequence-based world’s largest microsatellite database, FungSatDB having >19M loci obtained from >1900 fungal species/strains using >4000 assemblies across globe. Genotyping efficacy of FungSatDB has been evaluated by both in-silico and in-vitro PCR. By in silico PCR, 66 strains of 8 countries representing four continents were successfully differentiated. Genotyping efficacy was also evaluated by in vitro PCR in four fungal species. This approach overcomes limitation of ITS in species, strain signature, and diversity analysis. It can accelerate fungal genomic research endeavors in agriculture, industrial, and environmental management.

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Assembly and Annotation of Pneumocystis jirovecii from the Human Lung Microbiome

mBio ◽

10.1128/mbio.00224-13 ◽

2013 ◽

Vol 4 (2) ◽

Cited By ~ 13

Author(s):

Melanie T. Cushion ◽

Scott P. Keely

Keyword(s):

Bioinformatic Analysis ◽

Pneumocystis Jirovecii ◽

Clinical Samples ◽

Whole Genome ◽

Content Type ◽

Aids Epidemic ◽

Lung Microbiome ◽

Immunosuppressed Patients ◽

Novel Drug

ABSTRACTPneumocystis jiroveciiis a fungus that causesPneumocystispneumonia in immunosuppressed patients and has been closely associated with AIDS since the beginning of the AIDS epidemic. Becausein vitrocultivation ofP. jiroveciiis not possible, progress has been hindered in our understanding of its life cycle, mode of transmission, metabolic function, and genome. Limited amounts ofP. jiroveciican be obtained from infected patients, but the occurrence of bacteria, other fungi, and human cells in clinical samples presents new challenges for whole-genome sequencing and downstream bioinformatic analysis. In a recent article, Cissé et al. used cell immunoprecipitation enrichment together with whole-genome amplification to generate sufficient quantities of DNA for Roche 454 and Illumina sequencing [O. H. Cissé, M. Pagni, and P. M. Hauser, mBio 4(1):e00428-12, 2012, doi:10.1128/mBio.00428-12]. In addition, a bioinformatic pipeline was devised to sort and assemble lung microbiome reads, thereby generating an 8.1-MbP. jiroveciigenome comprised of 356 contigs with an N50(median length of all contigs) of 41.6 kb. Knowledge of this genome will open new avenues of research, including the identification of nutritional requirements forin vitrocultivation as well as the identification of new and novel drug and vaccine targets.

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The Mechanism of Platelet Aggregation Induced by HLA-Related Antibodies

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650659 ◽

1996 ◽

Vol 76 (05) ◽

pp. 774-779 ◽

Cited By ~ 14

Author(s):

John T Brandt ◽

Carmen J Julius ◽

Jeanne M Osborne ◽

Clark L Anderson

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Complement Activation ◽

Thromboembolic Disease ◽

Clinical Samples ◽

Hla Antigen ◽

Aggregation Response ◽

Immune Mediated ◽

Dependent Pathway

SummaryImmune-mediated platelet activation is emerging as an important pathogenic mechanism of thrombosis. In vitro studies have suggested two distinct pathways for immune-mediated platelet activation; one involving clustering of platelet FcyRIIa, the other involving platelet-associated complement activation. HLA-related antibodies have been shown to cause platelet aggregation, but the mechanism has not been clarified. We evaluated the mechanism of platelet aggregation induced by HLA-related antibodies from nine patients. Antibody to platelet FcyRIIa failed to block platelet aggregation with 8/9 samples, indicating that engagement of platelet FcyRIIa is not necessary for the platelet aggregation induced by HLA-related antibodies. In contrast, platelet aggregation was blocked by antibodies to human C8 (5/7) or C9 (7/7). F(ab’)2 fragments of patient IgG failed to induce platelet activation although they bound to HLA antigen on platelets. Intact patient IgG failed to aggregate washed platelets unless aged serum was added. The activating IgG could be adsorbed by incubation with lymphocytes and eluted from the lymphocytes. These results indicate that complement activation is involved in the aggregation response to HLA-related antibodies. This is the first demonstration of complement-mediated platelet aggregation by clinical samples. Five of the patients developed thrombocytopenia in relationship to blood transfusion and two patients developed acute thromboembolic disease, suggesting that these antibodies and the complement-dependent pathway of platelet aggregation may be of clinical significance.

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Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01948-20 ◽

2020 ◽

Vol 65 (1) ◽

pp. e01948-20

Author(s):

Dalin Rifat ◽

Si-Yang Li ◽

Thomas Ioerger ◽

Keshav Shah ◽

Jean-Philippe Lanoix ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Clinical Evidence ◽

Clinical Samples ◽

Loss Of Function ◽

Wild Type ◽

Content Type ◽

Solid Media ◽

High Level ◽

Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

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Molecular characterization of clinical carbapenem-resistant Enterobacterales from Qatar

European Journal of Clinical Microbiology & Infectious Diseases ◽

10.1007/s10096-021-04185-7 ◽

2021 ◽

Author(s):

Fatma Ben Abid ◽

Clement K. M. Tsui ◽

Yohei Doi ◽

Anand Deshmukh ◽

Christi L. McElheny ◽

...

Keyword(s):

Common Species ◽

Clinical Samples ◽

Whole Genome ◽

E Coli ◽

Carbapenem Resistant ◽

Genes Encoding ◽

Encoding Genes ◽

Sequence Types ◽

Common Sequence

AbstractOne hundred forty-nine carbapenem-resistant Enterobacterales from clinical samples obtained between April 2014 and November 2017 were subjected to whole genome sequencing and multi-locus sequence typing. Klebsiella pneumoniae (81, 54.4%) and Escherichia coli (38, 25.5%) were the most common species. Genes encoding metallo-β-lactamases were detected in 68 (45.8%) isolates, and OXA-48-like enzymes in 60 (40.3%). blaNDM-1 (45; 30.2%) and blaOXA-48 (29; 19.5%) were the most frequent. KPC-encoding genes were identified in 5 (3.6%) isolates. Most common sequence types were E. coli ST410 (8; 21.1%) and ST38 (7; 18.4%), and K. pneumoniae ST147 (13; 16%) and ST231 (7; 8.6%).

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1280. In-Vitro Activity of Cefiderocol, Imipenem/Relebactam, & Ceftazidime/Avibactam in Ceftolozane/Tazobactam Resistant Strains of Multidrug Resistant Pseudomonas aeruginosa

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa439.1463 ◽

2020 ◽

Vol 7 (Supplement_1) ◽

pp. S655-S655

Author(s):

Daniel Navas ◽

Angela Charles ◽

Amy Carr ◽

Jose Alexander

Keyword(s):

Multidrug Resistant ◽

Resistance Mechanisms ◽

Vitro Activity ◽

Clinical Isolates ◽

Resistance Testing ◽

Clinical Samples ◽

In Vitro Activity ◽

Carbapenemase Gene ◽

Resistant Strains

Abstract Background The activity of imipenem/relebactam (I/R), ceftazidime/avibactam (CZA) and cefiderocol (FDC) were evaluated against clinical isolates of multidrug resistant (MDR) strains of P. aeruginosa which was resistant to ceftolozane/tazobactam (C/T). The recent increase of MDR P. aeruginosa strains isolated from clinical samples has prompted research and development of new antimicrobials that can withstand its multiple resistance mechanisms. C/T is an effective option for treatment of MDR P. aeruginosa in our facility with only 10% of resistance in MDR strains, but the emergence of resistance may occur due to the presence of a carbapenemase gene or an ampC mutation. Methods Antimicrobial susceptibility testing for C/T Etest® (bioMérieux, Inc.) were performed on all MDR strains initially screened by the VITEK2® (bioMérieux, Inc.). 10% (n=20) of all MDR isolates were resistant to C/T by the CLSI 2019 breakpoints. These resistant isolates were tested for presence of a carbapenemase gene using the GeneXpert CARBA-R (Cepheid®) PCR and against CZA Etest® (bioMérieux, Inc.) I/R gradient strips (Liofilchem®) and FDC broth microdilution (Thermo Scientific™ Sensititre™). Results A total of 20 clinical isolates of MDR P. aeruginosa resistant to C/T were tested following standardized CLSI protocols and techniques. All 20 isolates were screened for the presence of a carbapenemase gene (blaVIM, blaNDM, blaKPC, blaOXA-48, blaIMP). A blaVIM gene was detected in 6 (30%) out of 20 isolates. FDC demonstrated the greatest activity with 85% (n=17) of susceptible isolates (CLSI MIC <4µg/dL). CZA (CLSI MIC <8µg/dL) and I/R (FDA MIC <2µg/dL) showed 15% (n=3) and 10% (n=2) of susceptible isolates respectively. FDC was active against all 6 blaVIM isolates, where all 6 strains were resistant to CZA and I/R as expected. 3 isolates tested non-susceptible against FDC; additional characterization was not performed at this time. Conclusion Based on these results, FDC demonstrated the greatest in-vitro activity against C/T resistant strains of MDR P. aeruginosa. FDC also demonstrated activity against all 6 MDR P. aeruginosa carrying blaVIM gene. FDC is a strong option to consider on MDR P. aeruginosa strains based on a resistance testing algorithm and a cost/effective protocol. Disclosures All Authors: No reported disclosures

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Cattle connection: molecular epidemiology of BVDV outbreaks via rapid nanopore whole-genome sequencing of clinical samples

BMC Veterinary Research ◽

10.1186/s12917-021-02945-3 ◽

2021 ◽

Vol 17 (1) ◽

Author(s):

Jacqueline King ◽

Anne Pohlmann ◽

Kamila Dziadek ◽

Martin Beer ◽

Kerstin Wernike

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Economic Losses ◽

Clinical Samples ◽

Bovine Viral Diarrhea ◽

Sequence Information ◽

Whole Genome ◽

Diarrhea Virus ◽

Viral Diarrhea ◽

Viral Loads

Abstract Background As a global ruminant pathogen, bovine viral diarrhea virus (BVDV) is responsible for the disease Bovine Viral Diarrhea with a variety of clinical presentations and severe economic losses worldwide. Classified within the Pestivirus genus, the species Pestivirus A and B (syn. BVDV-1, BVDV-2) are genetically differentiated into 21 BVDV-1 and four BVDV-2 subtypes. Commonly, the 5’ untranslated region and the Npro protein are utilized for subtyping. However, the genetic variability of BVDV leads to limitations in former studies analyzing genome fragments in comparison to a full-genome evaluation. Results To enable rapid and accessible whole-genome sequencing of both BVDV-1 and BVDV-2 strains, nanopore sequencing of twelve representative BVDV samples was performed on amplicons derived through a tiling PCR procedure. Covering a multitude of subtypes (1b, 1d, 1f, 2a, 2c), sample matrices (plasma, EDTA blood and ear notch), viral loads (Cq-values 19–32) and species (cattle and sheep), ten of the twelve samples produced whole genomes, with two low titre samples presenting 96 % genome coverage. Conclusions Further phylogenetic analysis of the novel sequences emphasizes the necessity of whole-genome sequencing to identify novel strains and supplement lacking sequence information in public repositories. The proposed amplicon-based sequencing protocol allows rapid, inexpensive and accessible obtainment of complete BVDV genomes.

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Splicing factor SRSF1 promotes breast cancer progression via oncogenic splice switching of PTPMT1

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-01978-8 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Jun-Xian Du ◽

Yi-Hong Luo ◽

Si-Jia Zhang ◽

Biao Wang ◽

Cong Chen ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Progression ◽

High Risk Group ◽

Clinical Samples ◽

Binding Motif ◽

Ki 67 ◽

Tissue Samples

Abstract Background Intensive evidence has highlighted the effect of aberrant alternative splicing (AS) events on cancer progression when triggered by dysregulation of the SR protein family. Nonetheless, the underlying mechanism in breast cancer (BRCA) remains elusive. Here we sought to explore the molecular function of SRSF1 and identify the key AS events regulated by SRSF1 in BRCA. Methods We conducted a comprehensive analysis of the expression and clinical correlation of SRSF1 in BRCA based on the TCGA dataset, Metabric database and clinical tissue samples. Functional analysis of SRSF1 in BRCA was conducted in vitro and in vivo. SRSF1-mediated AS events and their binding motifs were identified by RNA-seq, RNA immunoprecipitation-PCR (RIP-PCR) and in vivo crosslinking followed by immunoprecipitation (CLIP), which was further validated by the minigene reporter assay. PTPMT1 exon 3 (E3) AS was identified to partially mediate the oncogenic role of SRSF1 by the P-AKT/C-MYC axis. Finally, the expression and clinical significance of these AS events were validated in clinical samples and using the TCGA database. Results SRSF1 expression was consistently upregulated in BRCA samples, positively associated with tumor grade and the Ki-67 index, and correlated with poor prognosis in a hormone receptor-positive (HR+) cohort, which facilitated proliferation, cell migration and inhibited apoptosis in vitro and in vivo. We identified SRSF1-mediated AS events and discovered the SRSF1 binding motif in the regulation of splice switching of PTPMT1. Furthermore, PTPMT1 splice switching was regulated by SRSF1 by binding directly to its motif in E3 which partially mediated the oncogenic role of SRSF1 by the AKT/C-MYC axis. Additionally, PTPMT1 splice switching was validated in tissue samples of BRCA patients and using the TCGA database. The high-risk group, identified by AS of PTPMT1 and expression of SRSF1, possessed poorer prognosis in the stage I/II TCGA BRCA cohort. Conclusions SRSF1 exerts oncogenic roles in BRCA partially by regulating the AS of PTPMT1, which could be a therapeutic target candidate in BRCA and a prognostic factor in HR+ BRCA patient.

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Safety Assessment of Bacillus subtilis MB40 for Use in Foods and Dietary Supplements

Nutrients ◽

10.3390/nu13030733 ◽

2021 ◽

Vol 13 (3) ◽

pp. 733

Author(s):

Jessica L. Spears ◽

Richard Kramer ◽

Andrey I. Nikiforov ◽

Marisa O. Rihner ◽

Elizabeth A. Lambert

Keyword(s):

Antibiotic Resistance ◽

Bacillus Subtilis ◽

Dietary Supplements ◽

Genome Sequencing ◽

In Silico ◽

Safety Assessment ◽

In Vivo Studies ◽

Strain Identification ◽

Consumer Safety

With the growing popularity of probiotics in dietary supplements, foods, and beverages, it is important to substantiate not only the health benefits and efficacy of unique strains but also safety. In the interest of consumer safety and product transparency, strain identification should include whole-genome sequencing and safety assessment should include genotypic and phenotypic studies. Bacillus subtilis MB40, a unique strain marketed for use in dietary supplements, and food and beverage, was assessed for safety and tolerability across in silico, in vitro, and in vivo studies. MB40 was assessed for the absence of undesirable genetic elements encoding toxins and mobile antibiotic resistance. Tolerability was assessed in both rats and healthy human volunteers. In silico and in vitro testing confirmed the absence of enterotoxin and mobile antibiotic resistance genes of safety concern to humans. In rats, the no-observed-adverse-effect level (NOAEL) for MB40 after repeated oral administration for 14 days was determined to be 2000 mg/kg bw/day (equivalent to 3.7 × 1011 CFU/kg bw/day). In a 28 day human tolerability trial, 10 × 109 CFU/day of MB40 was well tolerated. Based on genome sequencing, strain characterization, screening for undesirable attributes and evidence of safety by appropriately designed safety evaluation studies in rats and humans, Bacillus subtilis MB40 does not pose any human health concerns under the conditions tested.

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A circular RNA derived from PLXNB2 as a valuable predictor of the prognosis of patients with acute myeloid leukaemia

Journal of Translational Medicine ◽

10.1186/s12967-021-02793-7 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Leilei Lin ◽

Yu Wang ◽

Sicheng Bian ◽

Lili Sun ◽

Zhibo Guo ◽

...

Keyword(s):

Acute Myeloid Leukaemia ◽

Myeloid Leukaemia ◽

Regulatory Networks ◽

Clinical Samples ◽

Circular Rnas ◽

Qrt Pcr ◽

Rna Fish ◽

Acute Myeloid

Abstract Background As a common haematological malignancy, acute myeloid leukaemia (AML), particularly with extramedullary infiltration (EMI), often results in a high mortality rate and poor prognosis. Circular RNAs (circRNAs) regulate biological and pathogenic processes, suggesting a potential role in AML. We have previously described the overall alterations in circRNAs and their regulatory networks between patients with AML presenting with and without EMI. This study aims to find new prognostic and therapeutic targets potentially associated with AML. Methods qRT-PCR was performed on samples from 40 patients with AML and 15 healthy controls. The possibility of using circPLXNB2 (circRNA derived from PLXNB2) as a diagnostic and prognostic biomarker for AML was analysed with multiple statistical methods. In vitro, the function of circPLXNB2 was studied by lentivirus transfection, CCK-8 assays, flow cytometry, and Transwell experiments. Western blotting and qRT-PCR were performed to detect the expression of related proteins and genes. The distribution of circPLXNB2 in cells was observed using RNA fluorescence in situ hybridization (RNA-FISH). We also investigated the role of circPLXNB2 by establishing AML xenograft models in NOD/SCID mice. Results By analysing the results of qRT-PCR detection of clinical samples, the expression of the circPLXNB2 and PLXNB2 mRNAs were significantly increased in patients with AML, more specifically in patients with AML presenting with EMI. High circPLXNB2 expression was associated with an obviously shorter overall survival and leukaemia-free survival of patients with AML. The circPLXNB2 expression was positively correlated with PLXNB2 mRNA expression, as evidenced by Pearson’s correlation analysis. RNA-FISH revealed that circPLXNB2 is mainly located in the nucleus. In vitro and in vivo, circPLXNB2 promoted cell proliferation and migration and inhibited apoptosis. Notably, circPLXNB2 also increased the expression of PLXNB2, BCL2 and cyclin D1, and reduced the expression of BAX. Conclusion In summary, we validated the high expression of circPLXNB2 and PLXNB2 in patients with AML. Elevated circPLXNB2 levels were associated with poor clinical outcomes in patients with AML. Importantly, circPLXNB2 accelerated tumour growth and progression, possibly by regulating PLXNB2 expression. Our study highlights the potential of circPLXNB2 as a new prognostic predictor and therapeutic target for AML in the future.

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