Pathogens

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15 pages, 1301 KiB

Open AccessEditor’s ChoiceArticle

Toxoplasma gondii Recombinant antigen AMA1: Diagnostic Utility of Protein Fragments for the Detection of IgG and IgM Antibodies

by Bartłomiej Ferra, Lucyna Holec-Gąsior, Justyna Gatkowska, Bożena Dziadek and Katarzyna Dzitko

Pathogens 2020, 9(1), 43; https://doi.org/10.3390/pathogens9010043 - 5 Jan 2020

Cited by 13 | Viewed by 4292

Abstract

Toxoplasma gondii is an important zoonotic protozoan that infects a wide variety of vertebrates as intermediate hosts. For this reason, the diagnosis of this disease is very important and requires continuous improvement. One possibility is to use recombinant antigens in serological tests. Apical [...] Read more.

Toxoplasma gondii is an important zoonotic protozoan that infects a wide variety of vertebrates as intermediate hosts. For this reason, the diagnosis of this disease is very important and requires continuous improvement. One possibility is to use recombinant antigens in serological tests. Apical membrane antigen 1 (AMA1), a protein located in specific secretory organelles (micronemes) of T. gondii, is very interesting in regard to its potential diagnostic utility. In the present study, we attempted to identify a fragment of the AMA1 protein with a high sensitivity and specificity for the serological diagnosis of human toxoplasmosis. The full-length AMA1 and two different fragments (AMA1N and AMA1C) were produced using an Escherichia coli expression system. After purification by metal affinity chromatography, recombinant proteins were tested for their utility as antigens in enzyme-linked immunosorbent assays (ELISAs) for the detection of IgG and IgM anti-T. gondii antibodies in human and mouse immune sera. Our data demonstrate that the full-length AMA1 recombinant antigen (corresponding to amino acid residues 67–569 of the native protein) has a better diagnostic potential than its N- or C-terminal fragments. This recombinant protein strongly interacts with specific anti-T. gondii IgG (99.4%) and IgM (80.0%) antibodies, and may be used for developing new tools for diagnostics of toxoplasmosis. Full article

(This article belongs to the Section Human Pathogens)

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17 pages, 1925 KiB

Open AccessEditor’s ChoiceArticle

Analysis of Porcine Pro- and Anti-Inflammatory Cytokine Induction by S. suis In Vivo and In Vitro

by Florian S. Hohnstein, Marita Meurer, Nicole de Buhr, Maren von Köckritz-Blickwede, Christoph G. Baums, Gottfried Alber and Nicole Schütze

Pathogens 2020, 9(1), 40; https://doi.org/10.3390/pathogens9010040 - 3 Jan 2020

Cited by 14 | Viewed by 4805

Abstract

Weaning piglets are susceptible to the invasive Streptococcus (S.) suis infection, which can result in septicemia. The aim of this study was to investigate the cytokine profile induced upon S. suis infection of blood, to determine the cellular sources of those cytokines, and [...] Read more.

Weaning piglets are susceptible to the invasive Streptococcus (S.) suis infection, which can result in septicemia. The aim of this study was to investigate the cytokine profile induced upon S. suis infection of blood, to determine the cellular sources of those cytokines, and to study the potential effects of the induced cytokines on bacterial killing. We measured TNF-α, IL-6, IFN-γ, IL-17A and IL-10 after an experimental intravenous infection with S. suis serotype 2 in vivo, and analyzed whole blood, peripheral blood mononuclear cells (PBMC) and separated leukocytes to identify the cytokine-producing cell type(s). In addition, we used a reconstituted whole blood assay to investigate the effect of TNF-α on bacterial killing in the presence of different S. suis-specific IgG levels. An increase in IL-6 and IL-10, but not in IFN-γ or IL-17A, was observed in two of three piglets with pronounced bacteremia 16 to 20 h after infection, but not in piglets with controlled bacteremia. Our results confirmed previous findings that S. suis induces TNF-α and IL-6 and could demonstrate that TNF-α is produced by monocytes in vitro. We further found that IL-10 induction resulted in reduced secretion of TNF-α and IL-6. Rapid induction of TNF-α was, however, not crucial for in vitro bacterial killing, not even in the absence of specific IgG. Full article

(This article belongs to the Special Issue Update on Streptococcus suis Research and Prevention in the Era of Antibiotic Restriction - 4th International Workshop on Streptococcus suis)

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13 pages, 4214 KiB

Open AccessEditor’s ChoiceArticle

Comparison of the Pathogenicity of Two Different Branches of Senecavirus a Strain in China

by Huawei Zhang, Pin Chen, Genxi Hao, Wenqiang Liu, Huanchun Chen, Ping Qian and Xiangmin Li

Pathogens 2020, 9(1), 39; https://doi.org/10.3390/pathogens9010039 - 2 Jan 2020

Cited by 17 | Viewed by 2954

Abstract

Senecavirus A (SVA), an emerging infectious disease, is associated with the porcine idiopathic vesicular disease. Here, the pathogenesis of different strains of SVA was investigated in growing-finishing pigs. We aimed to evaluate the replication characteristics, virus particle morphology, clinical signs, and vesicular lesions [...] Read more.

Senecavirus A (SVA), an emerging infectious disease, is associated with the porcine idiopathic vesicular disease. Here, the pathogenesis of different strains of SVA was investigated in growing-finishing pigs. We aimed to evaluate the replication characteristics, virus particle morphology, clinical signs, and vesicular lesions in comparison with two different strains of SVA. The animals were infected with SVA HB-CH-2016 or CH/AH-02/2017 by intranasal routes (3 mL, 10⁹TCID₅₀/mL) and monitored daily for 14 days post-inoculation (dpi) for clinical signs and vesicular lesions. Viremia or viral shedding was detected in the blood, fecal swab, and nasal swab samples. Results showed no distinct differences in plaque size, replication ability, and characteristic virions between SVA HB-CH-2016 and CH/AH-02/2017 strains. Animal experimental results showed that both SVA CH/AH-02/2017 and SVA HB-CH-2016 could infect pigs. However, an obvious difference in the pathogenicity and dynamics of infection was observed between SVA HB-CH-2016 and CH/AH-02/2017 strains. The pathogenesis of SVA CH/AH-02/2017 was similar to that of published results of USA strains, whereas the SVA HB-CH-2016 strain had low pathogenicity to pigs. Clinical signs and vesicular lesions were observed in SVA CH/AH-02/2017-infected pigs. Additionally, the different branches of SVA should be capable of inducing broad cross-reactive neutralizing antibodies, which play an important role in clearing the SVA virus. This study of animal models for SVA infection will be beneficial to develop vaccines and antivirals. Full article

(This article belongs to the Section Animal Pathogens)

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19 pages, 2112 KiB

Open AccessEditor’s ChoiceArticle

The Cell-Cycle Regulatory Protein p21^CIP1/WAF1 Is Required for Cytolethal Distending Toxin (Cdt)-Induced Apoptosis

by Bruce J. Shenker, Lisa M. Walker, Ali Zekavat, Robert H. Weiss and Kathleen Boesze-Battaglia

Pathogens 2020, 9(1), 38; https://doi.org/10.3390/pathogens9010038 - 2 Jan 2020

Cited by 13 | Viewed by 3308

Abstract

The Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) induces lymphocytes to undergo cell-cycle arrest and apoptosis; toxicity is dependent upon the active Cdt subunit, CdtB. We now demonstrate that p21^CIP1/WAF1 is critical to Cdt-induced apoptosis. Cdt induces increases in the levels of p21 [...] Read more.

The Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) induces lymphocytes to undergo cell-cycle arrest and apoptosis; toxicity is dependent upon the active Cdt subunit, CdtB. We now demonstrate that p21^CIP1/WAF1 is critical to Cdt-induced apoptosis. Cdt induces increases in the levels of p21^CIP1/WAF1 in lymphoid cell lines, Jurkat and MyLa, and in primary human lymphocytes. These increases were dependent upon CdtB’s ability to function as a phosphatidylinositol (PI) 3,4,5-triphosphate (PIP3) phosphatase. It is noteworthy that Cdt-induced increases in the levels of p21^CIP1/WAF1 were accompanied by a significant decline in the levels of phosphorylated p21^CIP1/WAF1. The significance of Cdt-induced p21^CIP1/WAF1 increase was assessed by preventing these changes with a two-pronged approach; pre-incubation with the novel p21^CIP1/WAF1 inhibitor, UC2288, and development of a p21^CIP1/WAF1-deficient cell line (Jurkat^p21−) using clustered regularly interspaced short palindromic repeats (CRISPR)/cas9 gene editing. UC2288 blocked toxin-induced increases in p21^CIP1/WAF1, and Jurkat^WT cells treated with this inhibitor exhibited reduced susceptibility to Cdt-induced apoptosis. Likewise, Jurkat^p21− cells failed to undergo toxin-induced apoptosis. The linkage between Cdt, p21^CIP1/WAF1, and apoptosis was further established by demonstrating that Cdt-induced increases in levels of the pro-apoptotic proteins Bid, Bax, and Bak were dependent upon p21^CIP1/WAF1 as these changes were not observed in Jurkat^p21− cells. Finally, we determined that the p21^CIP1/WAF1 increases were dependent upon toxin-induced increases in the level and activity of the chaperone heat shock protein (HSP) 90. We propose that p21^CIP1/WAF1 plays a key pro-apoptotic role in mediating Cdt-induced toxicity. Full article

(This article belongs to the Special Issue Aggregatibacter actinomycetemcomitans—Gram-Negative Bacterial Pathogen)

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23 pages, 4073 KiB

Open AccessEditor’s ChoiceArticle

The Xylella fastidiosa-Resistant Olive Cultivar “Leccino” Has Stable Endophytic Microbiota during the Olive Quick Decline Syndrome (OQDS)

by Marzia Vergine, Joana B. Meyer, Massimiliano Cardinale, Erika Sabella, Martin Hartmann, Paolo Cherubini, Luigi De Bellis and Andrea Luvisi

Pathogens 2020, 9(1), 35; https://doi.org/10.3390/pathogens9010035 - 31 Dec 2019

Cited by 39 | Viewed by 6747

Abstract

Xylella fastidiosa is a highly virulent pathogen that causes Olive Quick Decline Syndrome (OQDS), which is currently devastating olive plantations in the Salento region (Apulia, Southern Italy). We explored the microbiome associated with X. fastidiosa-infected (Xf-infected) and -uninfected (Xf [...] Read more.

Xylella fastidiosa is a highly virulent pathogen that causes Olive Quick Decline Syndrome (OQDS), which is currently devastating olive plantations in the Salento region (Apulia, Southern Italy). We explored the microbiome associated with X. fastidiosa-infected (Xf-infected) and -uninfected (Xf-uninfected) olive trees in Salento, to assess the level of dysbiosis and to get first insights into the potential role of microbial endophytes in protecting the host from the disease. The resistant cultivar “Leccino” was compared to the susceptible cultivar “Cellina di Nardò”, in order to identify microbial taxa and parameters potentially involved in resistance mechanisms. Metabarcoding of 16S rRNA genes and fungal ITS2 was used to characterize both total and endophytic microbiota in olive branches and leaves. “Cellina di Nardò” showed a drastic dysbiosis after X. fastidiosa infection, while “Leccino” (both infected and uninfected) maintained a similar microbiota. The genus Pseudomonas dominated all “Leccino” and Xf-uninfected “Cellina di Nardò” trees, whereas Ammoniphilus prevailed in Xf-infected “Cellina di Nardò”. Diversity of microbiota in Xf-uninfected “Leccino” was higher than in Xf-uninfected “Cellina di Nardò”. Several bacterial taxa specifically associated with “Leccino” showed potential interactions with X. fastidiosa. The maintenance of a healthy microbiota with higher diversity and the presence of cultivar-specific microbes might support the resistance of “Leccino” to X. fastidiosa. Such beneficial bacteria might be isolated in the future for biological treatment of the OQDS. Full article

(This article belongs to the Section Plant Pathogens)

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19 pages, 2039 KiB

Open AccessEditor’s ChoiceArticle

Membrane Binding, Cellular Cholesterol Content and Resealing Capacity Contribute to Epithelial Cell Damage Induced by Suilysin of Streptococcus suis

by Désirée Vötsch, Maren Willenborg, Walter M.R. Oelemann, Graham Brogden and Peter Valentin-Weigand

Pathogens 2020, 9(1), 33; https://doi.org/10.3390/pathogens9010033 - 30 Dec 2019

Cited by 7 | Viewed by 3089

Abstract

Streptococcus (S.) suis is a major cause of economic losses in the pig industry worldwide and is an emerging zoonotic pathogen. One important virulence-associated factor is suilysin (SLY), a toxin that belongs to the family of cholesterol-dependent pore-forming cytolysins (CDC). However, [...] Read more.

Streptococcus (S.) suis is a major cause of economic losses in the pig industry worldwide and is an emerging zoonotic pathogen. One important virulence-associated factor is suilysin (SLY), a toxin that belongs to the family of cholesterol-dependent pore-forming cytolysins (CDC). However, the precise role of SLY in host–pathogen interactions is still unclear. Here, we investigated the susceptibility of different respiratory epithelial cells to SLY, including immortalized cell lines (HEp-2 and NPTr cells), which are frequently used in in vitro studies on S. suis virulence mechanisms, as well as primary porcine respiratory cells, which represent the first line of barrier during S. suis infections. SLY-induced cell damage was determined by measuring the release of lactate dehydrogenase after infection with a virulent S. suis serotype 2 strain, its isogenic SLY-deficient mutant strain, or treatment with the recombinant protein. HEp-2 cells were most susceptible, whereas primary epithelial cells were hardly affected by the toxin. This prompted us to study possible explanations for these differences. We first investigated the binding capacity of SLY using flow cytometry analysis. Since binding and pore-formation of CDC is dependent on the membrane composition, we also determined the cellular cholesterol content of the different cell types using TLC and HPLC. Finally, we examined the ability of those cells to reseal SLY-induced pores using flow cytometry analysis. Our results indicated that the amount of membrane-bound SLY, the cholesterol content of the cells, as well as their resealing capacity all affect the susceptibility of the different cells regarding the effects of SLY. These findings underline the differences of in vitro pathogenicity models and may further help to dissect the biological role of SLY during S. suis infections. Full article

(This article belongs to the Special Issue Update on Streptococcus suis Research and Prevention in the Era of Antibiotic Restriction - 4th International Workshop on Streptococcus suis)

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14 pages, 5535 KiB

Open AccessEditor’s ChoiceArticle

Genome-Wide Analysis of Cyclophilin Proteins in 21 Oomycetes

by Yan Zhang, Kyle Fletcher, Rongkui Han, Richard Michelmore and Ruiwu Yang

Pathogens 2020, 9(1), 24; https://doi.org/10.3390/pathogens9010024 - 26 Dec 2019

Cited by 3 | Viewed by 3360

Abstract

Cyclophilins (CYPs), a highly-conserved family of proteins, belong to a subgroup of immunophilins. Ubiquitous in eukaryotes and prokaryotes, CYPs have peptidyl-prolyl cis–trans isomerase (PPIase) activity and have been implicated as virulence factors in plant pathogenesis by oomycetes. We identified 16 CYP orthogroups from [...] Read more.

Cyclophilins (CYPs), a highly-conserved family of proteins, belong to a subgroup of immunophilins. Ubiquitous in eukaryotes and prokaryotes, CYPs have peptidyl-prolyl cis–trans isomerase (PPIase) activity and have been implicated as virulence factors in plant pathogenesis by oomycetes. We identified 16 CYP orthogroups from 21 diverse oomycetes. Each species was found to encode 15 to 35 CYP genes. Three of these orthogroups contained proteins with signal peptides at the N-terminal end, suggesting a role in secretion. Multidomain analysis revealed five conserved motifs of the CYP domain of oomycetes shared with other eukaryotic PPIases. Expression analysis of CYP proteins in different asexual life stages of the hemibiotrophic Phytophthora infestans and the biotrophic Plasmopara halstedii demonstrated distinct expression profiles between life stages. In addition to providing detailed comparative information on the CYPs in multiple oomycetes, this study identified candidate CYP effectors that could be the foundation for future studies of virulence. Full article

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23 pages, 5544 KiB

Open AccessEditor’s ChoiceArticle

Chromosomal Conjugative and Mobilizable Elements in Streptococcus suis: Major Actors in the Spreading of Antimicrobial Resistance and Bacteriocin Synthesis Genes

by Virginie Libante, Yves Nombre, Charles Coluzzi, Johan Staub, Gérard Guédon, Marcelo Gottschalk, Sarah Teatero, Nahuel Fittipaldi, Nathalie Leblond-Bourget and Sophie Payot

Pathogens 2020, 9(1), 22; https://doi.org/10.3390/pathogens9010022 - 25 Dec 2019

Cited by 28 | Viewed by 5038

Abstract

Streptococcus suis is a zoonotic pathogen suspected to be a reservoir of antimicrobial resistance (AMR) genes. The genomes of 214 strains of 27 serotypes were screened for AMR genes and chromosomal Mobile Genetic Elements (MGEs), in particular Integrative Conjugative Elements (ICEs) and Integrative [...] Read more.

Streptococcus suis is a zoonotic pathogen suspected to be a reservoir of antimicrobial resistance (AMR) genes. The genomes of 214 strains of 27 serotypes were screened for AMR genes and chromosomal Mobile Genetic Elements (MGEs), in particular Integrative Conjugative Elements (ICEs) and Integrative Mobilizable Elements (IMEs). The functionality of two ICEs that host IMEs carrying AMR genes was investigated by excision tests and conjugation experiments. In silico search revealed 416 ICE-related and 457 IME-related elements. These MGEs exhibit an impressive diversity and plasticity with tandem accretions, integration of ICEs or IMEs inside ICEs and recombination between the elements. All of the detected 393 AMR genes are carried by MGEs. As previously described, ICEs are major vehicles of AMR genes in S. suis. Tn5252-related ICEs also appear to carry bacteriocin clusters. Furthermore, whereas the association of IME-AMR genes has never been described in S. suis, we found that most AMR genes are actually carried by IMEs. The autonomous transfer of an ICE to another bacterial species (Streptococcus thermophilus)—leading to the cis-mobilization of an IME carrying tet(O)—was obtained. These results show that besides ICEs, IMEs likely play a major role in the dissemination of AMR genes in S. suis. Full article

(This article belongs to the Special Issue Update on Streptococcus suis Research and Prevention in the Era of Antibiotic Restriction - 4th International Workshop on Streptococcus suis)

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11 pages, 1358 KiB

Open AccessEditor’s ChoiceCommunication

Human Microglia Respond to Malaria-Induced Extracellular Vesicles

by Smart Ikechukwu Mbagwu, Nils Lannes, Michael Walch, Luis Filgueira and Pierre-Yves Mantel

Pathogens 2020, 9(1), 21; https://doi.org/10.3390/pathogens9010021 - 24 Dec 2019

Cited by 23 | Viewed by 4372

Abstract

Microglia are the chief immune cells of the brain and have been reported to be activated in severe malaria. Their activation may drive towards neuroinflammation in cerebral malaria. Malaria-infected red blood cell derived-extracellular vesicles (MiREVs) are produced during the blood stage of malaria [...] Read more.

Microglia are the chief immune cells of the brain and have been reported to be activated in severe malaria. Their activation may drive towards neuroinflammation in cerebral malaria. Malaria-infected red blood cell derived-extracellular vesicles (MiREVs) are produced during the blood stage of malaria infection. They mediate intercellular communication and immune regulation, among other functions. During cerebral malaria, the breakdown of the blood–brain barrier can promote the migration of substances such as MiREVs from the periphery into the brain, targeting cells such as microglia. Microglia and extracellular vesicle interactions in different pathological conditions have been reported to induce neuroinflammation. Unlike in astrocytes, microglia–extracellular vesicle interaction has not yet been described in malaria infection. Therefore, in this study, we aimed to investigate the uptake of MiREVs by human microglia cells and their cytokine response. Human blood monocyte-derived microglia (MoMi) were generated from buffy coats of anonymous healthy donors using Ficoll-Paque density gradient centrifugation. The MiREVs were isolated from the Plasmodium falciparum cultures. They were purified by ultracentrifugation and labeled with PKH67 green fluorescent dye. The internalization of MiREVs by MoMi was observed after 4 h of co-incubation on coverslips placed in a 24-well plate at 37 °C using confocal microscopy. Cytokine-gene expression was investigated using rt-qPCR, following the stimulation of the MoMi cells with supernatants from the parasite cultures at 2, 4, and 24 h, respectively. MiREVs were internalized by the microglia and accumulated in the perinuclear region. MiREVs-treated cells increased gene expression of the inflammatory cytokine TNFα and reduced gene expression of the immune suppressive IL-10. Overall, the results indicate that MiREVs may act on microglia, which would contribute to enhanced inflammation in cerebral malaria. Full article

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15 pages, 2360 KiB

Open AccessEditor’s ChoiceArticle

Genetic Changes in Experimental Populations of a Hybrid in the Cryptococcus neoformans Species Complex

by Kelly Dong, Man You and Jianping Xu

Pathogens 2020, 9(1), 3; https://doi.org/10.3390/pathogens9010003 - 18 Dec 2019

Cited by 14 | Viewed by 2602

Abstract

Hybrids between Cryptococcus neoformans and Cryptococcus deneoformans are commonly found in patients and the environment. However, the genetic stability of these hybrids remains largely unknown. Here, we established mutation accumulation lines of a diploid C. neoformans × C. deneoformans laboratory hybrid and analyzed [...] Read more.

Hybrids between Cryptococcus neoformans and Cryptococcus deneoformans are commonly found in patients and the environment. However, the genetic stability of these hybrids remains largely unknown. Here, we established mutation accumulation lines of a diploid C. neoformans × C. deneoformans laboratory hybrid and analyzed the genotypes at 33 markers distributed across all 14 chromosomes. Our analyses found that under standard culture conditions, heterozygosity at most loci was maintained over 800 mitotic generations, with an estimated 6.44 × 10⁻⁵ loss-of-heterozygosity (LoH) event per mitotic division. However, under fluconazole stress, the observed LoH frequency increased by > 50 folds for the two markers on Chromosome 1, all due to the loss of the fluconazole susceptible allele on this chromosome. Flow cytometry analyses showed that after the 40th transfer (120 days), 19 of the 20 lines maintained the original ploidy level (2N), while one line was between 2N and 3N. The combined flow cytometry, genotyping at 33 markers, and quantitative PCR analyses showed the allelic loss was compensated for by amplification of the resistant ERG11 allele in eight of the ten fluconazole-stress lines. Our results suggest that hybrids in C. neoformans species complex are generally stable but that they can undergo rapid adaptation to environmental stresses through LoH and gene duplication. Full article

(This article belongs to the Special Issue Pathogenesis of Fungal and Bacterial Microbes)

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13 pages, 4673 KiB

Open AccessEditor’s ChoiceArticle

Endocytic Pathway of Feline Coronavirus for Cell Entry: Differences in Serotype-Dependent Viral Entry Pathway

by Tomomi Takano, Yumeho Wakayama and Tomoyoshi Doki

Pathogens 2019, 8(4), 300; https://doi.org/10.3390/pathogens8040300 - 16 Dec 2019

Cited by 14 | Viewed by 7766

Abstract

Feline coronavirus (FCoV) is a pathogen causing a lethal infectious disease in cats, feline infectious peritonitis. It has two serotypes (type I FCoV and type II FCoV). According to our previous study, type I FCoV infection is inhibited by compounds inducing intracellular cholesterol [...] Read more.

Feline coronavirus (FCoV) is a pathogen causing a lethal infectious disease in cats, feline infectious peritonitis. It has two serotypes (type I FCoV and type II FCoV). According to our previous study, type I FCoV infection is inhibited by compounds inducing intracellular cholesterol accumulation, whereas type II FCoV infection is not inhibited. Intracellular cholesterol accumulation was reported to disrupt late endosome function. Based on these findings, types I and II FCoV are considered to enter the cytosol through late and early endosomes, respectively. We investigated whether the antiviral activities of a late endosome trafficking inhibitor and cholesterol-accumulating agents are different between the FCoV serotypes. The late endosome trafficking inhibitor did not inhibit type II FCoV infection, but it inhibited type I FCoV infection. Type I FCoV infection was inhibited by cholesterol-accumulating triazoles, but not by non-cholesterol-accumulating triazoles. These phenomena were observed in both feline cell lines and feline primary macrophages. This study provides additional information on the differences in intracellular reproductive cycle between type I and type II FCoV. Full article

(This article belongs to the Special Issue Feline Infectious Peritonitis)

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13 pages, 1596 KiB

Open AccessEditor’s ChoiceArticle

Genetic Basis and Physiological Effects of Lipid A Hydroxylation in Pseudomonas aeruginosa PAO1

by Alessandra Lo Sciuto, Matteo Cervoni, Roberta Stefanelli, Maria Concetta Spinnato, Alessandra Di Giamberardino, Carmine Mancone and Francesco Imperi

Pathogens 2019, 8(4), 291; https://doi.org/10.3390/pathogens8040291 - 10 Dec 2019

Cited by 19 | Viewed by 3803

Abstract

Modifications of the lipid A moiety of lipopolysaccharide influence the physicochemical properties of the outer membrane of Gram-negative bacteria. Some bacteria produce lipid A with a single hydroxylated secondary acyl chain. This hydroxylation is catalyzed by the dioxygenase LpxO, and is important for [...] Read more.

Modifications of the lipid A moiety of lipopolysaccharide influence the physicochemical properties of the outer membrane of Gram-negative bacteria. Some bacteria produce lipid A with a single hydroxylated secondary acyl chain. This hydroxylation is catalyzed by the dioxygenase LpxO, and is important for resistance to cationic antimicrobial peptides (e.g., polymyxins), survival in human blood, and pathogenicity in animal models. The lipid A of the human pathogen Pseudomonas aeruginosa can be hydroxylated in both secondary acyl chains, but the genetic basis and physiological role of these hydroxylations are still unknown. Through the generation of single and double deletion mutants in the lpxO1 and lpxO2 homologs of P. aeruginosa PAO1 and lipid A analysis by mass spectrometry, we demonstrate that both LpxO1 and LpxO2 are responsible for lipid A hydroxylation, likely acting on different secondary acyl chains. Lipid A hydroxylation does not appear to affect in vitro growth, cell wall stability, and resistance to human blood or antibiotics in P. aeruginosa. In contrast, it is required for infectivity in the Galleria mellonella infection model, without relevantly affecting in vivo persistence. Overall, these findings suggest a role for lipid A hydroxylation in P. aeruginosa virulence that could not be directly related to outer membrane integrity. Full article

(This article belongs to the Section Human Pathogens)

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18 pages, 3669 KiB

Open AccessEditor’s ChoiceArticle

Phenotypic Characterization of Rhodococcus equi Biofilm Grown In Vitro and Inhibiting and Dissolving Activity of Azithromycin/Rifampicin Treatment

by Elisa Rampacci, Maria Luisa Marenzoni, Stefano Giovagnoli, Fabrizio Passamonti, Mauro Coletti and Donatella Pietrella

Pathogens 2019, 8(4), 284; https://doi.org/10.3390/pathogens8040284 - 4 Dec 2019

Cited by 6 | Viewed by 3484

Abstract

Microbial biofilm has been implicated in a wide range of chronic infections. In spite of the fact that Rhodococcus equi is a recognized cause of chronic disease in animals and humans, few studies have focused on the sessile phenotype of R. equi. [...] Read more.

Microbial biofilm has been implicated in a wide range of chronic infections. In spite of the fact that Rhodococcus equi is a recognized cause of chronic disease in animals and humans, few studies have focused on the sessile phenotype of R. equi. The aim of this research was to phenotypically characterize the biofilm development of R. equi and its answerability for hypo-responsiveness to macrolides and rifampicin. Biofilm formation is initiated by bacterial adhesion to the surface. In this work, the ability of R. equi to adhere to the surface of human lung epithelial cells was detected by a fluorometric adhesion test performed on 40 clinical isolates. Subsequently, the capability of R. equi to produce biofilm was investigated by colorimetric, fluorescence and scanning electron microscopy analysis, revealing a general slow growth of rhodococcal biofilm and different sessile phenotypes among field isolates, some also including filamented bacteria. Azithromycin treatment produced a higher long-term inhibition and dissolution of R. equi biofilms than rifampicin, while the two antibiotics combined boosted the anti-biofilm effect in a statistically significant manner, although this was not equally effective for all R. equi isolates. Increasing the MIC concentrations of drugs tenfold alone and in combination did not completely eradicate pre-formed R. equi biofilms, while a rifampicin-resistant isolate produced an exceptionally abundant extracellular matrix. These results have strengthened the hypothesis that biofilm production may occur as an antibiotic tolerance system in R. equi, potentially determining persistence and, eventually, chronic infection. Full article

(This article belongs to the Section Animal Pathogens)

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13 pages, 1679 KiB

Open AccessEditor’s ChoiceArticle

Transcriptomic Analysis of Aggregatibacter actinomycetemcomitans Core and Accessory Genes in Different Growth Conditions

by Natalia O. Tjokro, Weerayuth Kittichotirat, Annamari Torittu, Riikka Ihalin, Roger E. Bumgarner and Casey Chen

Pathogens 2019, 8(4), 282; https://doi.org/10.3390/pathogens8040282 - 3 Dec 2019

Cited by 5 | Viewed by 2953

Abstract

Aggregatibacter actinomycetemcomitans genome can be divided into an accessory gene pool (found in some but not all strains) and a core gene pool (found in all strains). The functions of the accessory genes (genomic islands and non-island accessory genes) are largely unknown. We [...] Read more.

Aggregatibacter actinomycetemcomitans genome can be divided into an accessory gene pool (found in some but not all strains) and a core gene pool (found in all strains). The functions of the accessory genes (genomic islands and non-island accessory genes) are largely unknown. We hypothesize that accessory genes confer critical functions for A. actinomycetemcomitans in vivo. This study examined the expression patterns of accessory and core genes of A. actinomycetemcomitans in distinct growth conditions. We found similar expression patterns of island and non-island accessory genes, which were generally lower than the core genes in all growth conditions. The median expression levels of genomic islands were 29%–37% of the core genes in enriched medium but elevated to as high as 63% of the core genes in nutrient-limited media. Several putative virulence genes, including the cytolethal distending toxin operon, were found to be activated in nutrient-limited conditions. In conclusion, genomic islands and non-island accessory genes exhibited distinct patterns of expression from the core genes and may play a role in the survival of A. actinomycetemcomitans in nutrient-limited environments. Full article

(This article belongs to the Special Issue Aggregatibacter actinomycetemcomitans—Gram-Negative Bacterial Pathogen)

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9 pages, 267 KiB

Open AccessEditor’s ChoiceArticle

The Prevalence of HSV, HHV-6, HPV and Mycoplasma genitalium in Chlamydia trachomatis positive and Chlamydia trachomatis Negative Urogenital Samples among Young Women in Finland

by Suvi Korhonen, Kati Hokynar, Tiina Eriksson, Kari Natunen, Jorma Paavonen, Matti Lehtinen and Mirja Puolakkainen

Pathogens 2019, 8(4), 276; https://doi.org/10.3390/pathogens8040276 - 1 Dec 2019

Cited by 5 | Viewed by 3944

Abstract

Chlamydia trachomatis, Mycoplasma genitalium, herpes simplex virus (HSV) and human papillomavirus (HPV) cause sexually transmitted infections. In addition, human herpesvirus 6 (HHV-6) may be a genital co-pathogen. The prevalence rates of HSV, HHV-6, HPV, M. genitalium, and the C. trachomatis [...] Read more.

Chlamydia trachomatis, Mycoplasma genitalium, herpes simplex virus (HSV) and human papillomavirus (HPV) cause sexually transmitted infections. In addition, human herpesvirus 6 (HHV-6) may be a genital co-pathogen. The prevalence rates of HSV, HHV-6, HPV, M. genitalium, and the C. trachomatis ompA genotypes were investigated by PCR in urogenital samples of the C. trachomatis nucleic acid amplification test positive (n = 157) and age-, community- and time-matched negative (n = 157) women. The prevalence of HPV DNA was significantly higher among the C. trachomatis positives than the C. trachomatis negatives (66% vs. 25%, p < 0.001). The prevalence of HSV (1.9% vs. 0%), HHV-6 (11% vs. 14%), and M. genitalium DNA (4.5% vs. 1.9%) was not significantly different between the C. trachomatis-positive and -negative women. Thirteen per cent of test-of-cure specimens tested positive for C. trachomatis. The prevalence of HSV, HHV-6, HPV, M. genitalium, and the C. trachomatis ompA genotypes did not significantly differ between those who cleared the C. trachomatis infection (n = 105) and those who did not (n = 16). The higher prevalence of HPV DNA among the C. trachomatis positives suggests greater sexual activity and increased risk for sexually transmitted pathogens. Full article

(This article belongs to the Special Issue Chlamydia trachomatis Infections)

12 pages, 6108 KiB

Open AccessEditor’s ChoiceArticle

Immunosuppressive Compounds Affect the Fungal Growth and Viability of Defined Aspergillus Species

by Stanislaw Schmidt, Michael Hogardt, Asuman Demir, Frauke Röger and Thomas Lehrnbecher

Pathogens 2019, 8(4), 273; https://doi.org/10.3390/pathogens8040273 - 29 Nov 2019

Cited by 7 | Viewed by 3076

Abstract

Immunosuppressive drugs are administered to a number of patients; e.g., to allogeneic hematopoietic stem cell transplant recipients. Immunosuppressive drugs impair the immune system and thus increase the risk of invasive fungal disease, but may exhibit antifungal activity at the same time. We investigated [...] Read more.

Immunosuppressive drugs are administered to a number of patients; e.g., to allogeneic hematopoietic stem cell transplant recipients. Immunosuppressive drugs impair the immune system and thus increase the risk of invasive fungal disease, but may exhibit antifungal activity at the same time. We investigated the impact of various concentrations of three commonly used immunosuppressive compounds—cyclosporin A (CsA), methylprednisolone (mPRED), and mycophenolic acid (MPA)—on the growth and viability of five clinically important Aspergillus species. Methods included disc diffusion, optical density of mycelium, and viability assays such as XTT. MPA and CsA had a species-specific and dose-dependent inhibitory effect on the growth of all Aspergillus spp. tested, although growth inhibition by MPA was highest in A. niger, A. flavus and A. brasiliensis. Both agents exhibited species-specific hyphal damage, which was higher when the immunosuppressants were added to growing conidia than to mycelium. In contrast, mPRED increased the growth of A. niger, but had no major impact on the growth and viability of any of the other Aspergillus species tested. Our findings may help to better understand the interaction of drugs with Aspergillus species and ultimately may have an impact on individualizing immunosuppressive therapy. Full article

(This article belongs to the Section Human Pathogens)

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12 pages, 1066 KiB

Open AccessEditor’s ChoiceArticle

Genetic Diversity among Pseudorabies Viruses Isolated from Dogs in France from 2006 to 2018

by Céline Deblanc, Aurélie Oger, Gaëlle Simon and Marie-Frédérique Le Potier

Pathogens 2019, 8(4), 266; https://doi.org/10.3390/pathogens8040266 - 26 Nov 2019

Cited by 16 | Viewed by 3120

Abstract

Pseudorabies (PR), also known as Aujeszky’s disease, is an economically important disease for the pig industry. It has been eradicated in domestic pigs in many European countries, including France, but its causative agent—Suid Herpesvirus 1—is still circulating in wild boars. The risk of [...] Read more.

Pseudorabies (PR), also known as Aujeszky’s disease, is an economically important disease for the pig industry. It has been eradicated in domestic pigs in many European countries, including France, but its causative agent—Suid Herpesvirus 1—is still circulating in wild boars. The risk of endemic PR in wild fauna lies in reintroducing the virus among domestic pigs and transmitting it to other mammals, especially hunting dogs for which the disease is rapidly fatal. As such infections are regularly reported in France, this study genetically characterized canine PR virus strains in the country to obtain information on their diversity and evolution. Partial sequencing of the glycoprotein C-encoding gene from 55 virus strains isolated from dogs between 2006 and 2018 showed that 14 strains belonged to genotype I-clade A and another 38 to genotype I-clade B, two clades usually reported in Western Europe. More surprisingly, three strains were found to belong to genotype II, suggesting an Asian origin. Genotype I-clade A strains exhibited the highest diversity as five geographically segregated genogroups were identified. Full article

(This article belongs to the Special Issue Pseudorabies Virus Infections)

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21 pages, 8574 KiB

Open AccessEditor’s ChoiceArticle

Entry of Scotophilus Bat Coronavirus-512 and Severe Acute Respiratory Syndrome Coronavirus in Human and Multiple Animal Cells

by Yi-Ning Chen, Hsiao-Chin Hsu, Sheng-Wei Wang, Hao-Chiang Lien, Hsin-Ti Lu and Sheng-Kai Peng

Pathogens 2019, 8(4), 259; https://doi.org/10.3390/pathogens8040259 - 22 Nov 2019

Cited by 7 | Viewed by 7564

Abstract

Bats are natural reservoirs of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV). Scotophilus bat CoV-512 demonstrates potential for cross-species transmission because its viral RNA and specific antibodies have been detected in three bat species of Taiwan. Understanding [...] Read more.

Bats are natural reservoirs of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV). Scotophilus bat CoV-512 demonstrates potential for cross-species transmission because its viral RNA and specific antibodies have been detected in three bat species of Taiwan. Understanding the cell tropism of Scotophilus bat CoV-512 is the first step for studying the mechanism of cross-species transmission. In this study, a lentivirus-based pseudovirus was produced using the spike (S) protein of Scotophilus bat CoV-512 or SARS-CoV as a surface protein to test the interaction between coronaviral S protein and its cell receptor on 11 different cells. Susceptible cells expressed red fluorescence protein (RFP) after the entry of RFP-bound green fluorescence protein (GFP)-fused S protein of Scotophilus bat CoV-512 (RFP-Sco-S-eGFP) or RFP-SARS-S pseudovirus, and firefly luciferase (FLuc) activity expressed by cells infected with FLuc-Sco-S-eGFP or FLuc-SARS-S pseudovirus was quantified. Scotophilus bat CoV-512 pseudovirus had significantly higher entry efficiencies in Madin Darby dog kidney epithelial cells (MDCK), black flying fox brain cells (Pabr), and rat small intestine epithelial cells (IEC-6). SARS-CoV pseudovirus had significantly higher entry efficiencies in human embryonic kidney epithelial cells (HEK-293T), pig kidney epithelial cells (PK15), and MDCK cells. These findings demonstrated that Scotophilus bat CoV-512 had a broad host range for cross-species transmission like SARS-CoV. Full article

(This article belongs to the Section Animal Pathogens)

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41 pages, 6933 KiB

Open AccessEditor’s ChoiceArticle

Genome-Wide Analyses Revealed Remarkable Heterogeneity in Pathogenicity Determinants, Antimicrobial Compounds, and CRISPR-Cas Systems of Complex Phytopathogenic Genus Pectobacterium

by Dario Arizala and Mohammad Arif

Pathogens 2019, 8(4), 247; https://doi.org/10.3390/pathogens8040247 - 20 Nov 2019

Cited by 23 | Viewed by 6018

Abstract

The Pectobacterium genus comprises pectolytic enterobacteria defined as the causal agents of soft rot, blackleg, and aerial stem rot diseases of potato and economically important crops. In this study, we undertook extensive genome-wide comparative analyses of twelve species that conform the Pectobacterium genus. [...] Read more.

The Pectobacterium genus comprises pectolytic enterobacteria defined as the causal agents of soft rot, blackleg, and aerial stem rot diseases of potato and economically important crops. In this study, we undertook extensive genome-wide comparative analyses of twelve species that conform the Pectobacterium genus. Bioinformatics approaches outlined a low nucleotide identity of P. parmentieri and P. wasabiae with other species, while P. carotovorum subsp. odoriferum was shown to harbor numerous pseudogenes, which suggests low coding capacity and genomic degradation. The genome atlases allowed for distinguishing distinct DNA structures and highlighted suspicious high transcription zones. The analyses unveiled a noteworthy heterogeneity in the pathogenicity determinants. Specifically, phytotoxins, polysaccharides, iron uptake systems, and the type secretion systems III–V were observed in just some species. Likewise, a comparison of gene clusters encoding antimicrobial compounds put in evidence for high conservation of carotovoricin, whereas a few species possessed the phenazine, carbapenem, and carocins. Moreover, three clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) systems: I-E, I-F, and III-A were identified. Surrounding some CRISPR-Cas regions, different toxin and antitoxin systems were found, which suggests bacterial suicide in the case of an immune system failure. Multiple whole-genome alignments shed light on to the presence of a novel cellobiose phosphotransferase system (PTS) exclusive to P. parmenteri, and an unreported T5SS conserved in almost all species. Several regions that were associated with virulence, microbe antagonism, and adaptive immune systems were predicted within genomic islands, which underscored the essential role that horizontal gene transfer has imparted in the dynamic evolution and speciation of Pectobacterium species. Overall, the results decipher the different strategies that each species has developed to infect their hosts, outcompete for food resources, and defend against bacteriophages. Our investigation provides novel genetic insights that will assist in understanding the pathogenic lifestyle of Pectobacterium, a genus that jeopardizes the agriculture sustainability of important crops worldwide. Full article

(This article belongs to the Section Plant Pathogens)

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14 pages, 1981 KiB

Open AccessEditor’s ChoiceArticle

LmxM.22.0250-Encoded Dual Specificity Protein/Lipid Phosphatase Impairs Leishmania mexicana Virulence In Vitro

by Natalya Kraeva, Tereza Leštinová, Aygul Ishemgulova, Karolina Majerová, Anzhelika Butenko, Slavica Vaselek, Julia Bespyatykh, Arzuv Charyyeva, Tatiana Spitzová, Alexei Yu. Kostygov, Julius Lukeš, Petr Volf, Jan Votýpka and Vyacheslav Yurchenko

Pathogens 2019, 8(4), 241; https://doi.org/10.3390/pathogens8040241 - 17 Nov 2019

Cited by 12 | Viewed by 3284

Abstract

Protein phosphorylation/dephosphorylation is an important regulatory mechanism that controls many key physiological processes. Numerous pathogens successfully use kinases and phosphatases to internalize, replicate, and survive, modifying the host′s phosphorylation profile or signal transduction pathways. Multiple phosphatases and kinases from diverse bacterial pathogens have [...] Read more.

Protein phosphorylation/dephosphorylation is an important regulatory mechanism that controls many key physiological processes. Numerous pathogens successfully use kinases and phosphatases to internalize, replicate, and survive, modifying the host′s phosphorylation profile or signal transduction pathways. Multiple phosphatases and kinases from diverse bacterial pathogens have been implicated in human infections before. In this work, we have identified and characterized the dual specificity protein/lipid phosphatase LmDUSP1 as a novel virulence factor governing Leishmania mexicana infection. The LmDUSP1-encoding gene (LmxM.22.0250 in L. mexicana) has been acquired from bacteria via horizontal gene transfer. Importantly, its orthologues have been associated with virulence in several bacterial species, such as Mycobacterium tuberculosis and Listeria monocytogenes. Leishmania mexicana with ablated LmxM.22.0250 demonstrated severely attenuated virulence in the experimental infection of primary mouse macrophages, suggesting that this gene facilitates Leishmania pathogenicity in vertebrates. Despite significant upregulation of LmxM.22.0250 expression in metacyclic promastigotes, its ablation did not affect the ability of mutant cells to differentiate into virulent stages in insects. It remains to be further investigated which specific biochemical pathways involve LmDUSP1 and how this facilitates the parasite′s survival in the host. One of the interesting possibilities is that LmDUSP1 may target host′s substrate(s), thereby affecting its signal transduction pathways. Full article

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12 pages, 1743 KiB

Open AccessEditor’s ChoiceArticle

A Single and Un-Adjuvanted Dose of a Chimpanzee Adenovirus-Vectored Vaccine against Chikungunya Virus Fully Protects Mice from Lethal Disease

by Rafael Kroon Campos, Lorena Preciado-Llanes, Sasha R. Azar, Cesar Lopez-Camacho, Arturo Reyes-Sandoval and Shannan L. Rossi

Pathogens 2019, 8(4), 231; https://doi.org/10.3390/pathogens8040231 - 12 Nov 2019

Cited by 21 | Viewed by 5314

Abstract

The mosquito-borne chikungunya virus (CHIKV) has become a major global health problem. Upon infection, chikungunya fever (CHIKF) can result in long-term joint pain and arthritis, and despite intense research, no licensed vaccine for CHIKV is available. We have developed two recombinant chimpanzee adenovirus-vectored [...] Read more.

The mosquito-borne chikungunya virus (CHIKV) has become a major global health problem. Upon infection, chikungunya fever (CHIKF) can result in long-term joint pain and arthritis, and despite intense research, no licensed vaccine for CHIKV is available. We have developed two recombinant chimpanzee adenovirus-vectored vaccines (ChAdOx1) that induce swift and robust anti-CHIKV immune responses with a single dose, without the need for adjuvants or booster vaccines. Here, we report the vaccines’ protective efficacies against CHIKV infection in a lethal A129 mouse model. Our results indicate that a single, un-adjuvanted ChAdOx1 Chik or ChAdOx1 Chik ΔCap dose provided complete protection against a lethal virus challenge and prevented CHIKV-associated severe inflammation. These candidate vaccines supported survival equal to the attenuated 181/25 CHIKV reference vaccine but without the vaccine-related side effects, such as weight loss. Vaccination with either ChAdOx1 Chik or ChAdOx1 Chik ΔCap resulted in high titers of neutralizing antibodies that are associated with protection, indicating that the presence of the capsid within the vaccine construct may not be essential to afford protection under the conditions tested. We conclude that both replication-deficient ChAdOx1 Chik vaccines are safe even when used in A129 mice and afford complete protection from a lethal challenge. Full article

(This article belongs to the Special Issue Vaccines against Alphaviruses and Flaviviruses)

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12 pages, 5962 KiB

Open AccessEditor’s ChoiceArticle

Zebrafish are Resistant to Staphylococcus aureus Endophthalmitis

by Frank Mei, Matthew Rolain, Xiao Yi Zhou, Pawan Kumar Singh, Ryan Thummel and Ashok Kumar

Pathogens 2019, 8(4), 207; https://doi.org/10.3390/pathogens8040207 - 26 Oct 2019

Cited by 5 | Viewed by 5251

Abstract

Gram-positive bacteria remain the leading cause of endophthalmitis, a blinding infectious disease of the eye. Murine models have been widely used for understanding the pathogenesis of bacterial endophthalmitis. In this study, we sought to develop an alternative zebrafish (Danio rerio) model [...] Read more.

Gram-positive bacteria remain the leading cause of endophthalmitis, a blinding infectious disease of the eye. Murine models have been widely used for understanding the pathogenesis of bacterial endophthalmitis. In this study, we sought to develop an alternative zebrafish (Danio rerio) model for Staphylococcus aureus and compare the disease pathobiology to a murine model. Endophthalmitis was induced in zebrafish and C57BL/6 mice through the intravitreal injection of S. aureus. Disease progression was monitored by assessing corneal haze, opacity, bacterial burden, and retinal histology. Our results demonstrated that, unlike the murine models, zebrafish maintained ocular integrity, corneal transparency, and retinal architecture. We found that the zebrafish was capable of clearing S. aureus from the eye via transport through retinal vessels and the optic nerve and by mounting a monocyte/macrophage response beginning at 8 hour post-infection (hpi). The bacterial burden increased up to 8 hpi and significantly decreased thereafter. An assessment of the innate retinal response revealed the induced expression of Il-1β and Il-6 transcripts. Collectively, our study shows that unlike the murine model, zebrafish do not develop endophthalmitis and rapidly clear the pathogen. Hence, a better understanding of the zebrafish protective ocular innate response may provide new insights into the pathobiology of bacterial endophthalmitis. Full article

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34 pages, 4778 KiB

Open AccessEditor’s ChoiceArticle

An Acute Stress Model in New Zealand White Rabbits Exhibits Altered Immune Response to Infection with West Nile Virus

by Willy W. Suen, Mitchell Imoda, Albert W. Thomas, Nur N.B.M. Nasir, Nawaporn Tearnsing, Wenqi Wang and Helle Bielefeldt-Ohmann

Pathogens 2019, 8(4), 195; https://doi.org/10.3390/pathogens8040195 - 18 Oct 2019

Cited by 4 | Viewed by 3215

Abstract

The immune competence of an individual is a major determinant of morbidity in West Nile virus (WNV)-infection. Previously, we showed that immunocompetent New Zealand White rabbits (NZWRs; Oryctolagus cuniculus) are phenotypically resistant to WNV-induced disease, thus presenting a suitable model for study [...] Read more.

The immune competence of an individual is a major determinant of morbidity in West Nile virus (WNV)-infection. Previously, we showed that immunocompetent New Zealand White rabbits (NZWRs; Oryctolagus cuniculus) are phenotypically resistant to WNV-induced disease, thus presenting a suitable model for study of virus-control mechanisms. The current study used corticosteroid-treated NZWRs to model acute “stress”-related immunosuppression. Maximal effects on immune parameters were observed on day 3 post dexamethasone-treatment (pdt). However, contrary to our hypothesis, intradermal WNV challenge at this time pdt produced significantly lower viremia 1 day post-infection (dpi) compared to untreated controls, suggestive of changes to antiviral control mechanisms. To examine this further, RNAseq was performed on RNA extracted from draining lymph node—the first site of virus replication and immune detection. Unaffected by dexamethasone-treatment, an early antiviral response, primarily via interferon (IFN)-I, and induction of a range of known and novel IFN-stimulated genes, was observed. However, treatment was associated with expression of a different repertoire of IFN-α-21-like and IFN-ω-1-like subtypes on 1 dpi, which may have driven the different chemokine response on 3 dpi. Ongoing expression of Toll-like receptor-3 and transmembrane protein-173/STING likely contributed to signaling of the treatment-independent IFN-I response. Two novel genes (putative HERC6 and IFIT1B genes), and the SLC16A5 gene were also highlighted as important component of the transcriptomic response. Therefore, the current study shows that rabbits are capable of restricting WNV replication and dissemination by known and novel robust antiviral mechanisms despite environmental challenges such as stress. Full article

(This article belongs to the Special Issue Pathogenesis of West Nile Virus)

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13 pages, 2346 KiB

Open AccessEditor’s ChoiceArticle

Analysis of Resistance of Ebola Virus Glycoprotein-Driven Entry Against MDL28170, An Inhibitor of Cysteine Cathepsins

by Markus Hoffmann, Svenja Victoria Kaufmann, Carina Fischer, Wiebke Maurer, Anna-Sophie Moldenhauer and Stefan Pöhlmann

Pathogens 2019, 8(4), 192; https://doi.org/10.3390/pathogens8040192 - 15 Oct 2019

Cited by 2 | Viewed by 3903

Abstract

Ebola virus (EBOV) infection can cause severe and frequently fatal disease in human patients. The EBOV glycoprotein (GP) mediates viral entry into host cells. For this, GP depends on priming by the pH-dependent endolysosomal cysteine proteases cathepsin B (CatB) and, to a lesser [...] Read more.

Ebola virus (EBOV) infection can cause severe and frequently fatal disease in human patients. The EBOV glycoprotein (GP) mediates viral entry into host cells. For this, GP depends on priming by the pH-dependent endolysosomal cysteine proteases cathepsin B (CatB) and, to a lesser degree, cathepsin L (CatL), at least in most cell culture systems. However, there is limited information on whether and how EBOV-GP can acquire resistance to CatB/L inhibitors. Here, we addressed this question using replication-competent vesicular stomatitis virus bearing EBOV-GP. Five passages of this virus in the presence of the CatB/CatL inhibitor MDL28170 were sufficient to select resistant viral variants and sequencing revealed that all GP sequences contained a V37A mutation, which, in the context of native GP, is located in the base of the GP surface unit. In addition, some GP sequences harbored mutation S195R in the receptor-binding domain. Finally, mutational analysis demonstrated that V37A but not S195R conferred resistance against MDL28170 and other CatB/CatL inhibitors. Collectively, a single amino acid substitution in GP is sufficient to confer resistance against CatB/CatL inhibitors, suggesting that usage of CatB/CatL inhibitors for antiviral therapy may rapidly select for resistant viral variants. Full article

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17 pages, 2478 KiB

Open AccessFeature PaperEditor’s ChoiceArticle

Genomic Epidemiology of Streptococcus suis Sequence Type 7 Sporadic Infections in the Guangxi Zhuang Autonomous Region of China

by Mingliu Wang, Pengcheng Du, Jianping Wang, Ruiting Lan, Jun Huang, Ming Luo, Yan Jiang, Jun Zeng, Yi Quan, Zhaohui Shi and Han Zheng

Pathogens 2019, 8(4), 187; https://doi.org/10.3390/pathogens8040187 - 12 Oct 2019

Cited by 19 | Viewed by 3913

Abstract

Streptococcus suis is an important zoonotic pathogen. Serotype 2 and sequence type (ST) 1 are the most frequently reported strains in both infected humans and pigs. ST7 is only endemic to China, and it was responsible for outbreaks in 1998 and 2005 in [...] Read more.

Streptococcus suis is an important zoonotic pathogen. Serotype 2 and sequence type (ST) 1 are the most frequently reported strains in both infected humans and pigs. ST7 is only endemic to China, and it was responsible for outbreaks in 1998 and 2005 in China. In the present study, 38 sporadic ST7 S. suis strains, which mostly caused sepsis, were collected from patients in the Guangxi Zhuang Autonomous Region (GX) between 2007 and 2018. Of 38 sporadic ST7 strains, serotype 14 was the most frequent (27 strains, 71.1%), followed by serotype 2 (11 strains, 28.9%). The phylogenetic structure of the ST7 population, including epidemic and sporadic ST7 strains, was constructed using mutational single-nucleotide polymorphisms (SNPs). High diversity within the ST7 population was revealed and divided into five lineages. Only one sporadic ST7 strain, GX14, from a Streptococcal toxic-shock-like syndrome (STSLS) patient was clustered into the same lineage as the epidemic strains. GX14 and the epidemic strains diverged in 1974. The sporadic ST7 strains of GX were mainly clustered into lineage 5, which emerged in 1980. Comparing to genome of epidemic strain, the major differences in genome of sporadic ST7 strains of GX was the absence of 89 kb pathogenicity island (PAI) specific to epidemic strain and insertion of 128 kb ICE_phage tandem MGE or ICE portion of the MGE. These mobile elements play a significant role in the horizontal transfer of antibiotic resistance genes in sporadic ST7 strains. Our results enhanced the understanding of the evolution of the ST7 strains and their ability to cause life-threatening infections in humans. Full article

(This article belongs to the Special Issue Update on Streptococcus suis Research and Prevention in the Era of Antibiotic Restriction - 4th International Workshop on Streptococcus suis)

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15 pages, 3967 KiB

Open AccessEditor’s ChoiceArticle

Whole Transcriptome Analyses Reveal Differential mRNA and microRNA Expression Profiles in Primary Human Dermal Fibroblasts Infected with Clinical or Vaccine Strains of Varicella Zoster Virus

by Soo-Jin Oh, Sooyeon Lim, Moon Jung Song, Jin Hyun Ahn, Chan Hee Lee and Ok Sarah Shin

Pathogens 2019, 8(4), 183; https://doi.org/10.3390/pathogens8040183 - 10 Oct 2019

Cited by 15 | Viewed by 3954

Abstract

Licensed live attenuated vaccines have been developed to prevent varicella zoster virus (VZV) infection, which causes chickenpox and shingles. The genomic sequences of both clinical- and vaccine-derived VZV strains have been analyzed previously. To further characterize the molecular signatures and complexity of wildtype [...] Read more.

Licensed live attenuated vaccines have been developed to prevent varicella zoster virus (VZV) infection, which causes chickenpox and shingles. The genomic sequences of both clinical- and vaccine-derived VZV strains have been analyzed previously. To further characterize the molecular signatures and complexity of wildtype (clinical) versus attenuated (vaccine-derived) VZV-mediated host cellular responses, we performed high-throughput next generation sequencing to quantify and compare the expression patterns of mRNAs and microRNAs (miRNAs) in primary human dermal fibroblasts (HDFs) infected with wildtype (YC01 low passage) and attenuated (YC01 high passage, SuduVax, and VarilRix) VZV strains. 3D-multidimensional scaling of the differentially expressed genes demonstrated the distinct grouping of wildtype and attenuated strains. In particular, we observed that HDFs infected with attenuated strains had more differentially expressed genes (DEGs) involved in the retinoic-acid inducible gene–I-like receptor and interferon-mediated signaling pathways compared with wildtype strains. Additionally, miRNA expression patterns were profiled following the infection of HDFs with VZV. Small RNA sequencing identified that several miRNAs were upregulated, including miR-146a-5p, which has been associated with other herpesvirus infections, whereas let-7a-3p was downregulated in both wildtype and attenuated VZV-infected cells. This study identified genes and miRNAs that may be essential in VZV pathogenesis. Full article

(This article belongs to the Special Issue Human Herpesviruses: Diversity and Disease)

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13 pages, 3321 KiB

Open AccessEditor’s ChoiceArticle

Atypical Dermatophytosis in 12 North American Porcupines (Erethizon dorsatum) from the Northeastern United States 2010–2017

by David B. Needle, Robert Gibson, Nicholas A. Hollingshead, Inga F. Sidor, Nicholas J. Marra, Derek Rothenheber, Anil J. Thachil, Bryce J. Stanhope, Brian A. Stevens, Julie C. Ellis, Shelley Spanswick, Maureen Murray and Laura B. Goodman

Pathogens 2019, 8(4), 171; https://doi.org/10.3390/pathogens8040171 - 30 Sep 2019

Cited by 7 | Viewed by 4619

Abstract

Twelve wild North American porcupines (Erethizon dorsatum) out of a total of 44 of this species examined in an 8-year period were diagnosed with dermatopathies while being cared for at two wildlife rehabilitation clinics. Biopsy and necropsy were performed on seven [...] Read more.

Twelve wild North American porcupines (Erethizon dorsatum) out of a total of 44 of this species examined in an 8-year period were diagnosed with dermatopathies while being cared for at two wildlife rehabilitation clinics. Biopsy and necropsy were performed on seven and five animals, respectively. Atypical dermatophytosis was diagnosed in all cases. Lesions consisted of diffuse severe epidermal hyperkeratosis and mild hyperplasia with mild lymphoplasmacytic dermatitis and no folliculitis. Dermatophytes were noted histologically as hyphae and spores in hair shafts, and follicular and epidermal keratin. Trichophyton sp. was grown in 5/6 animals where culture was performed, with a molecular diagnosis of Arthroderma benhamiae/Trichophyton mentagrophytes in these five cases. Metagenomic analysis of formalin-fixed paraffin-embedded tissue samples from three cases identified fungi from 17 orders in phyla Basidiomycota and Ascomycota. Alteration of therapy from ketaconazole, which was unsuccessful in four out of five early cases, to terbinafine or nitraconazole led to the resolution of disease and recovery to release in four subsequent animals. In all, six animals were euthanized or died due to dermatopathy, no cases resolved spontaneously, and six cases were resolved with therapy. The work we present demonstrates an atypical lesion and anatomical distribution due to dermatophytosis in a series of free-ranging wild porcupines and the successful development of novel techniques for extracting and sequencing nucleic acids from fungus in archival formalin-fixed paraffin-embedded animal tissue. Full article

(This article belongs to the Section Animal Pathogens)

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15 pages, 947 KiB

Open AccessEditor’s ChoiceArticle

Prevalence of Various Vaccine Candidate Proteins in Clinical Isolates of Streptococcus pneumoniae: Characterization of the Novel Pht Fusion Proteins PhtA/B and PhtA/D

by Mitsuyo Kawaguchiya, Noriko Urushibara, Meiji Soe Aung, Masaaki Shinagawa, Satoshi Takahashi and Nobumichi Kobayashi

Pathogens 2019, 8(4), 162; https://doi.org/10.3390/pathogens8040162 - 24 Sep 2019

Cited by 8 | Viewed by 3208

Abstract

Pneumococcal proteins unrelated to serotypes are considered to be candidates of antigens in next-generation vaccines. In the present study, the prevalence of vaccine candidate protein genes, along with serotypes and antimicrobial resistance determinants, was investigated in a total of 57 isolates obtained from [...] Read more.

Pneumococcal proteins unrelated to serotypes are considered to be candidates of antigens in next-generation vaccines. In the present study, the prevalence of vaccine candidate protein genes, along with serotypes and antimicrobial resistance determinants, was investigated in a total of 57 isolates obtained from a tertiary care hospital in Japan. All of the pediatric isolates and 76.6% of the adult isolates did not belong to PCV13 (a 13-valent pneumococcal conjugate vaccine) serotypes, and 70.2% of all isolates showed multidrug resistance. All of the isolates had ply, pavA, nanA, and nanB, and high prevalence was noted for the pspA and pspC genes (96.5% and 78.9%, respectively). Detection rates for the pneumococcal histidine triad protein (Pht) genes phtA, phtB, phtD, and phtE were 49.1%, 26.3%, 61.4%, and 100%, respectively. Two fusion-type genes, phtA/B and phtA/D, were identified, with a prevalence of 36.9% and 14.0%, respectively. These fusion types showed 78.1–90.0% nucleotide sequence identity with phtA, phtB, and phtD. The most prevalent pht profile was phtA + phtD + phtE (26.3%), followed by phtA/B + phtE (19.3%) and phtA/B + phtD + phtE (17.5%), while pht profiles including phtD and/or phtA/phtD were found in 71.9% of isolates. The present study revealed the presence of two fusion types of Pht and their unexpectedly high prevalence. These fusion types, as well as PhtA and PhtB, contained sequences similar to the B cell epitopes that have been previously reported for PhtD. Full article

(This article belongs to the Section Human Pathogens)

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12 pages, 4546 KiB

Open AccessEditor’s ChoiceArticle

New Taxon-Specific Heterobasidion PCR Primers Detect and Differentiate North American Heterobasidion spp. in Various Substrates and Led to the Discovery of Heterobasidion irregulare in British Columbia, Canada

by Simon Francis Shamoun, Craig Hammett, Grace Sumampong, Xiang Li and Matteo Garbelotto

Pathogens 2019, 8(3), 156; https://doi.org/10.3390/pathogens8030156 - 18 Sep 2019

Cited by 3 | Viewed by 4735

Abstract

Heterobasidion annosum sensu lato is a species complex of pathogenic white-rot wood decay fungi which cause root and butt rot in conifer and hardwood species across the Northern hemisphere. Annual losses to forest managers are valued in the billions of dollars, due to [...] Read more.

Heterobasidion annosum sensu lato is a species complex of pathogenic white-rot wood decay fungi which cause root and butt rot in conifer and hardwood species across the Northern hemisphere. Annual losses to forest managers are valued in the billions of dollars, due to tree mortality, reduction in timber yield, and wood decay. In North America, H. irregulare and H. occidentale have a partially overlapping host and geographic range, cause similar disease symptoms and produce similar fruiting bodies, making discrimination between the two of them often difficult. We developed two sets of primers that bind specifically to conserved, but species-specific portions of glyceraldehyde 3-phosphate dehydrogenase and elongation factor 1α alleles. The method is sensitive enough to detect either species from infected wood. Analysis of North American isolates has further clarified the distribution of both species on this continent, including the detection of H. irregulare for the first time on ponderosa pine (Pinus ponderosa) and eastern white pine (Pinus strobus) in British Columbia. This method has the potential to be a valuable tool for the detection of the pathogen in exported/imported wood products, as well as for the further identification and assessment of the distribution of North American Heterobasidion species. Full article

(This article belongs to the Section Plant Pathogens)

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14 pages, 3346 KiB

Open AccessEditor’s ChoiceArticle

A Single Dose of Modified Vaccinia Ankara Expressing Lassa Virus-like Particles Protects Mice from Lethal Intra-cerebral Virus Challenge

by Maria S. Salvato, Arban Domi, Camila Guzmán-Cardozo, Sandra Medina-Moreno, Juan Carlos Zapata, Haoting Hsu, Nathanael McCurley, Rahul Basu, Mary Hauser, Michael Hellerstein and Farshad Guirakhoo

Pathogens 2019, 8(3), 133; https://doi.org/10.3390/pathogens8030133 - 28 Aug 2019

Cited by 22 | Viewed by 8676

Abstract

Lassa fever surpasses Ebola, Marburg, and all other hemorrhagic fevers except Dengue in its public health impact. Caused by Lassa virus (LASV), the disease is a scourge on populations in endemic areas of West Africa, where reported incidence is higher. Here, we report [...] Read more.

Lassa fever surpasses Ebola, Marburg, and all other hemorrhagic fevers except Dengue in its public health impact. Caused by Lassa virus (LASV), the disease is a scourge on populations in endemic areas of West Africa, where reported incidence is higher. Here, we report construction, characterization, and preclinical efficacy of a novel recombinant vaccine candidate GEO-LM01. Constructed in the Modified Vaccinia Ankara (MVA) vector, GEO-LM01 expresses the glycoprotein precursor (GPC) and zinc-binding matrix protein (Z) from the prototype Josiah strain lineage IV. When expressed together, GP and Z form Virus-Like Particles (VLPs) in cell culture. Immunogenicity and efficacy of GEO-LM01 was tested in a mouse challenge model. A single intramuscular dose of GEO-LM01 protected 100% of CBA/J mice challenged with a lethal dose of ML29, a Mopeia/Lassa reassortant virus, delivered directly into the brain. In contrast, all control animals died within one week. The vaccine induced low levels of antibodies but Lassa-specific CD4⁺ and CD8⁺ T cell responses. This is the first report showing that a single dose of a replication-deficient MVA vector can confer full protection against a lethal challenge with ML29 virus. Full article

(This article belongs to the Special Issue Lassa Fever: Viral Replication, Disease Pathogenesis, and Host Immune Modulations)

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11 pages, 6927 KiB

Open AccessEditor’s ChoiceArticle

In Vitro Activity of Statins against Naegleria fowleri

by Aitor Rizo-Liendo, Ines Sifaoui, María Reyes-Batlle, Olfa Chiboub, Rubén L. Rodríguez-Expósito, Carlos J. Bethencourt-Estrella, Desirée San Nicolás-Hernández, Edyta B. Hendiger, Atteneri López-Arencibia, Pedro Rocha-Cabrera, José E. Piñero and Jacob Lorenzo-Morales

Pathogens 2019, 8(3), 122; https://doi.org/10.3390/pathogens8030122 - 8 Aug 2019

Cited by 26 | Viewed by 5123

Abstract

Naegleria fowleri causes a deadly disease called primary amoebic meningoencephalitis (PAM). Even though PAM is still considered a rare disease, the number of reported cases worldwide has been increasing each year. Among the factors to be considered for this, awareness about this disease, [...] Read more.

Naegleria fowleri causes a deadly disease called primary amoebic meningoencephalitis (PAM). Even though PAM is still considered a rare disease, the number of reported cases worldwide has been increasing each year. Among the factors to be considered for this, awareness about this disease, and also global warming, as these amoebae thrive in warm water bodies, seem to be the key factors. Until present, no fully effective drugs have been developed to treat PAM, and the current options are amphotericin B and miltefosine, which present side effects such as liver and kidney toxicity. Statins are able to inhibit the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which is a key enzyme for the synthesis of ergosterol of the cell membrane of these amoebae. Therefore, the in vitro activity of a group of statins was tested in this study against two types of strains of Naegleria fowleri. The obtained results showed that fluvastatin was the most effective statin tested in this study and was able to eliminate these amoebae at concentrations of 0.179 ± 0.078 to 1.682 ± 0.775 µM depending on the tested strain of N. fowleri. Therefore, fluvastatin could be a potential novel therapeutic agent against this emerging pathogen. Full article

(This article belongs to the Special Issue Emerging Parasitic Protozoa)

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11 pages, 2360 KiB

Open AccessEditor’s ChoiceArticle

Genetic Characterization of Chikungunya Virus in Field-Caught Aedes aegypti Mosquitoes Collected during the Recent Outbreaks in 2019, Thailand

by Proawpilart Intayot, Atchara Phumee, Rungfar Boonserm, Sriwatapron Sor-suwan, Rome Buathong, Supaporn Wacharapluesadee, Narisa Brownell, Yong Poovorawan and Padet Siriyasatien

Pathogens 2019, 8(3), 121; https://doi.org/10.3390/pathogens8030121 - 2 Aug 2019

Cited by 26 | Viewed by 5292

Abstract

Chikungunya virus (CHIKV) is a mosquito-borne virus belonging to the genus Alphavirus. The virus is transmitted to humans by the bite of infected female Aedes mosquitoes, primarily Aedes aegypti. CHIKV infection is spreading worldwide, and it periodically sparks new outbreaks. There [...] Read more.

Chikungunya virus (CHIKV) is a mosquito-borne virus belonging to the genus Alphavirus. The virus is transmitted to humans by the bite of infected female Aedes mosquitoes, primarily Aedes aegypti. CHIKV infection is spreading worldwide, and it periodically sparks new outbreaks. There are no specific drugs or effective vaccines against CHIKV. The interruption of pathogen transmission by mosquito control provides the only effective approach to the control of CHIKV infection. Many studies have shown that CHIKV can be transmitted among the Ae. aegypti through vertical transmission. The previous chikungunya fever outbreaks in Thailand during 2008–2009 were caused by CHIKV, the East/Central/South African (ECSA) genotype. Recently, there have been 3794 chikungunya cases in 27 provinces reported by the Bureau of Epidemiology of Health Ministry, Thailand during 1 January–16 June 2019; however, the cause of the re-emergence of CHIKV outbreaks is uncertain. Therefore, the aims of this study were to detect and analyze the genetic diversity of CHIKV infection in field-caught mosquitoes. Both female and male Ae. aegypti were collected from endemic areas of Thailand, and CHIKV detection was done by using E1-nested RT-PCR and sequencing analysis. A total of 1646 Ae. aegypti samples (900 females and 746 males) were tested. CHIKV was detected in 54 (3.28%) and 14 samples (0.85%) in female and male mosquitoes, respectively. Seventeen samples of female Ae. aegypti collected from the Ubon Ratchathani, Chiang Rai, Chiang Mai, Nakhon Sawan, and Songkhla provinces found mutation at E1: A226V. Interestingly, E1: K211E mutation was observed in 50 samples collected from Nong Khai, Bangkok, Prachuap Khiri Khan, and Krabi. In addition, the phylogenetic tree indicated that CHIKV in Ae. aegypti samples were from the Indian Ocean Clade and East/South African Clade. Both clades belong to the ECSA genotype. The information obtained from this study could be used for prediction, epidemiological study, prevention, and effective vector control of CHIKV. For instance, a novel CHIKV strain found in new areas has the potential to lead to a new outbreak. Health authorities could plan and apply control strategies more effectively given the tools provided by this research. Full article

(This article belongs to the Special Issue Chikungunya Virus Infections)

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17 pages, 9372 KiB

Open AccessEditor’s ChoiceArticle

N-acetyl Cysteine Coated Gallium Particles Demonstrate High Potency against Pseudomonas aeruginosa PAO1

by Mikaeel Young, Ali Ozcan, Briana Lee, Tyler Maxwell, Thomas Andl, Parthiban Rajasekaran, Melanie J. Beazley, Laurene Tetard and Swadeshmukul Santra

Pathogens 2019, 8(3), 120; https://doi.org/10.3390/pathogens8030120 - 1 Aug 2019

Cited by 8 | Viewed by 3802

Abstract

Nosocomial infections pose serious health concerns with over 2 million reported annually in the United States. Many of these infections are associated with bacterial resistance to antibiotics and hence, alternative treatments are critically needed. The objective of this study was to assess the [...] Read more.

Nosocomial infections pose serious health concerns with over 2 million reported annually in the United States. Many of these infections are associated with bacterial resistance to antibiotics and hence, alternative treatments are critically needed. The objective of this study was to assess the antimicrobial efficacy of a gallium (Ga)-based particle coated with N-Acetyl Cysteine (Ga-NAC) against Pseudomonas aeruginosa PAO1. Our studies showed the Minimum Inhibitory Concentration (MIC) of PAO1 treated with Ga-NAC was 1 µg/mL. Cytotoxicity of Ga-NAC against multiple cell lines was determined with no cytotoxicity observed up to concentrations of 2000 µg/mL (metal concentration), indicating a high therapeutic window. To elucidate potential antibacterial modes of action, Inductively Coupled Plasma—Mass Spectrometry (ICP-MS), infrared spectroscopy, and atomic force microscopy (AFM) were used. The results suggest improved Ga³⁺ interaction with PAO1 through Ga-NAC particles. No significant change in cell membrane chemistry or roughening was detected. As cell membrane integrity remained intact, the antimicrobial mode of action was linked to cellular internalization of Ga and subsequent iron metabolic disruption. Furthermore, Ga-NAC inhibited and disrupted biofilms seen with crystal violet assay and microscopy. Our findings suggest the Ga-NAC particle can potentially be used as an alternative to antibiotics for treatment of Pseudomonas aeruginosa infections. Full article

(This article belongs to the Section Vaccines and Therapeutic Developments)

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22 pages, 1738 KiB

Open AccessEditor’s ChoiceArticle

Multi-Lineage Evolution in Viral Populations Driven by Host Immune Systems

by Jacopo Marchi, Michael Lässig, Thierry Mora and Aleksandra M. Walczak

Pathogens 2019, 8(3), 115; https://doi.org/10.3390/pathogens8030115 - 29 Jul 2019

Cited by 10 | Viewed by 4347

Abstract

Viruses evolve in the background of host immune systems that exert selective pressure and drive viral evolutionary trajectories. This interaction leads to different evolutionary patterns in antigenic space. Examples observed in nature include the effectively one-dimensional escape characteristic of influenza A and the [...] Read more.

Viruses evolve in the background of host immune systems that exert selective pressure and drive viral evolutionary trajectories. This interaction leads to different evolutionary patterns in antigenic space. Examples observed in nature include the effectively one-dimensional escape characteristic of influenza A and the prolonged coexistence of lineages in influenza B. Here, we use an evolutionary model for viruses in the presence of immune host systems with finite memory to obtain a phase diagram of evolutionary patterns in a two-dimensional antigenic space. We find that, for small effective mutation rates and mutation jump ranges, a single lineage is the only stable solution. Large effective mutation rates combined with large mutational jumps in antigenic space lead to multiple stably co-existing lineages over prolonged evolutionary periods. These results combined with observations from data constrain the parameter regimes for the adaptation of viruses, including influenza. Full article

(This article belongs to the Special Issue Modeling Virus Dynamics and Evolution)

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10 pages, 1909 KiB

Open AccessEditor’s ChoiceArticle

Initiated Babesia ovata Sexual Stages under In Vitro Conditions Were Recognized by Anti-CCp2 Antibodies, Showing Changes in the DNA Content by Imaging Flow Cytometry

by Thu-Thuy Nguyen, Minh-Anh Dang-Trinh, Luna Higuchi, Juan Mosqueda, Hassan Hakimi, Masahito Asada, Junya Yamagishi, Rika Umemiya-Shirafuji and Shin-ichiro Kawazu

Pathogens 2019, 8(3), 104; https://doi.org/10.3390/pathogens8030104 - 17 Jul 2019

Cited by 6 | Viewed by 4915

Abstract

Sexual stage induction under in vitro conditions is useful for biological and molecular studies of Babesia parasites. Therefore, in the present study, we induced B. ovata tick stages using the chemical inducers: xanthurenic acid (XA), dithiothreitol (DTT) and tris (2-carboxyethyl) phosphine (TCEP) at [...] Read more.

Sexual stage induction under in vitro conditions is useful for biological and molecular studies of Babesia parasites. Therefore, in the present study, we induced B. ovata tick stages using the chemical inducers: xanthurenic acid (XA), dithiothreitol (DTT) and tris (2-carboxyethyl) phosphine (TCEP) at 27 °C or 37 °C conditions. Cultures at low temperature (27 °C) or treated with XA/TCEP induced a large number of extra-erythrocytic merozoites, which transformed into round shape cells at 12–24 h post-induction (pi). However, typical forms of tick stages (aggregation forms and the spiky forms/ray bodies) were only observed in the cultures treated with 40 mM or 60 mM of DTT during 3–6 h pi. The induced cells were recognized by anti-CCp2 rabbit antisera. DNA content of the cell population treated with 40 mM of DTT was analyzed by imaging flow cytometry at 0, 12 and 48 h pi. The results indicated that the parasite population with diploid-like double DNA content increased at 48 h pi. Our observations on morphological and changes in the DNA content provide useful information for understanding the life cycle of B. ovata under in vitro conditions, which will facilitate further studies on basic biology and the development of transmission blocking vaccines against bovine babesiosis. Full article

(This article belongs to the Special Issue Persistence in Babesia)

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17 pages, 3660 KiB

Open AccessEditor’s ChoiceArticle

Surface Immobilization of Nano-Silver on Polymeric Medical Devices to Prevent Bacterial Biofilm Formation

by Andri K. Riau, Thet T. Aung, Melina Setiawan, Liang Yang, Gary H. F. Yam, Roger W. Beuerman, Subbu S. Venkatraman and Jodhbir S. Mehta

Pathogens 2019, 8(3), 93; https://doi.org/10.3390/pathogens8030093 - 28 Jun 2019

Cited by 30 | Viewed by 4212

Abstract

Bacterial biofilm on medical devices is difficult to eradicate. Many have capitalized the anti-infective capability of silver ions (Ag⁺) by incorporating nano-silver (nAg) in a biodegradable coating, which is then laid on polymeric medical devices. However, such coating can be subjected [...] Read more.

Bacterial biofilm on medical devices is difficult to eradicate. Many have capitalized the anti-infective capability of silver ions (Ag⁺) by incorporating nano-silver (nAg) in a biodegradable coating, which is then laid on polymeric medical devices. However, such coating can be subjected to premature dissolution, particularly in harsh diseased tissue microenvironment, leading to rapid nAg clearance. It stands to reason that impregnating nAg directly onto the device, at the surface, is a more ideal solution. We tested this concept for a corneal prosthesis by immobilizing nAg and nano-hydroxyapatite (nHAp) on poly(methyl methacrylate), and tested its biocompatibility with human stromal cells and antimicrobial performance against biofilm-forming pathogens, Pseudomonas aeruginosa and Staphylococcus aureus. Three different dual-functionalized substrates—high Ag (referred to as 75:25 HAp:Ag); intermediate Ag (95:5 HAp:Ag); and low Ag (99:1 HAp:Ag) were studied. The 75:25 HAp:Ag was effective in inhibiting biofilm formation, but was cytotoxic. The 95:5 HAp:Ag showed the best selectivity among the three substrates; it prevented biofilm formation of both pathogens and had excellent biocompatibility. The coating was also effective in eliminating non-adherent bacteria in the culture media. However, a 28-day incubation in artificial tear fluid revealed a ~40% reduction in Ag⁺ release, compared to freshly-coated substrates. The reduction affected the inhibition of S. aureus growth, but not the P. aeruginosa. Our findings suggest that Ag⁺ released from surface-immobilized nAg diminishes over time and becomes less effective in suppressing biofilm formation of Gram-positive bacteria, such as S. aureus. This advocates the coating, more as a protection against perioperative and early postoperative infections, and less as a long-term preventive solution. Full article

(This article belongs to the Section Vaccines and Therapeutic Developments)

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22 pages, 5529 KiB

Open AccessEditor’s ChoiceArticle

Host-Pathogen Interactions of Mycoplasma mycoides in Caprine and Bovine Precision-Cut Lung Slices (PCLS) Models

by Yenehiwot Berhanu Weldearegay, Sandy Müller, Jana Hänske, Anja Schulze, Aline Kostka, Nancy Rüger, Marion Hewicker-Trautwein, Ralph Brehm, Peter Valentin-Weigand, Robert Kammerer, Joerg Jores and Jochen Meens

Pathogens 2019, 8(2), 82; https://doi.org/10.3390/pathogens8020082 - 20 Jun 2019

Cited by 16 | Viewed by 5603

Abstract

Respiratory infections caused by mycoplasma species in ruminants lead to considerable economic losses. Two important ruminant pathogens are Mycoplasma mycoides subsp. Mycoides (Mmm), the aetiological agent of contagious bovine pleuropneumonia and Mycoplasma mycoides subsp. capri (Mmc), which causes pneumonia, [...] Read more.

Respiratory infections caused by mycoplasma species in ruminants lead to considerable economic losses. Two important ruminant pathogens are Mycoplasma mycoides subsp. Mycoides (Mmm), the aetiological agent of contagious bovine pleuropneumonia and Mycoplasma mycoides subsp. capri (Mmc), which causes pneumonia, mastitis, arthritis, keratitis, and septicemia in goats. We established precision cut lung slices (PCLS) infection model for Mmm and Mmc to study host-pathogen interactions. We monitored infection over time using immunohistological analysis and electron microscopy. Moreover, infection burden was monitored by plating and quantitative real-time PCR. Results were compared with lungs from experimentally infected goats and cattle. Lungs from healthy goats and cattle were also included as controls. PCLS remained viable for up to two weeks. Both subspecies adhered to ciliated cells. However, the titer of Mmm in caprine PCLS decreased over time, indicating species specificity of Mmm. Mmc showed higher tropism to sub-bronchiolar tissue in caprine PCLS, which increased in a time-dependent manner. Moreover, Mmc was abundantly observed on pulmonary endothelial cells, indicating partially, how it causes systemic disease. Tissue destruction upon prolonged infection of slices was comparable to the in vivo samples. Therefore, PCLS represents a novel ex vivo model to study host-pathogen interaction in livestock mycoplasma. Full article

(This article belongs to the Section Animal Pathogens)

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18 pages, 1840 KiB

Open AccessEditor’s ChoiceArticle

Evolutionary Dynamics in the RNA Bacteriophage Qβ Depends on the Pattern of Change in Selective Pressures

by Pilar Somovilla, Susanna Manrubia and Ester Lázaro

Pathogens 2019, 8(2), 80; https://doi.org/10.3390/pathogens8020080 - 18 Jun 2019

Cited by 9 | Viewed by 4159

Abstract

The rate of change in selective pressures is one of the main factors that determines the likelihood that populations can adapt to stress conditions. Generally, the reduction in the population size that accompanies abrupt environmental changes makes it difficult to generate and select [...] Read more.

The rate of change in selective pressures is one of the main factors that determines the likelihood that populations can adapt to stress conditions. Generally, the reduction in the population size that accompanies abrupt environmental changes makes it difficult to generate and select adaptive mutations. However, in systems with high genetic diversity, as happens in RNA viruses, mutations with beneficial effects under new conditions can already be present in the population, facilitating adaptation. In this work, we have propagated an RNA bacteriophage (Qβ) at temperatures higher than the optimum, following different patterns of change. We have determined the fitness values and the consensus sequences of all lineages throughout the evolutionary process in order to establish correspondences between fitness variations and adaptive pathways. Our results show that populations subjected to a sudden temperature change gain fitness and fix mutations faster than those subjected to gradual changes, differing also in the particular selected mutations. The life-history of populations prior to the environmental change has great importance in the dynamics of adaptation. The conclusion is that in the bacteriophage Qβ, the standing genetic diversity together with the rate of temperature change determine both the rapidity of adaptation and the followed evolutionary pathways. Full article

(This article belongs to the Special Issue Modeling Virus Dynamics and Evolution)

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13 pages, 1404 KiB

Open AccessEditor’s ChoiceArticle

Putative Periodontal Pathogens, Filifactor alocis and Peptoanaerobacter stomatis, Induce Differential Cytokine and Chemokine Production by Human Neutrophils

by Aruna Vashishta, Emeri Jimenez-Flores, Christopher K. Klaes, Shifu Tian, Irina Miralda, Richard J. Lamont and Silvia M. Uriarte

Pathogens 2019, 8(2), 59; https://doi.org/10.3390/pathogens8020059 - 1 May 2019

Cited by 24 | Viewed by 4366

Abstract

Periodontitis is a highly prevalent infectious disease that affects ~ 50% of the adults in the USA alone. Two Gram-positive anaerobic oral bacteria, Filifactor alocis and Peptoanaerobacter stomatis, have emerged as important periodontal pathogens. Neutrophils are a major component of the innate [...] Read more.

Periodontitis is a highly prevalent infectious disease that affects ~ 50% of the adults in the USA alone. Two Gram-positive anaerobic oral bacteria, Filifactor alocis and Peptoanaerobacter stomatis, have emerged as important periodontal pathogens. Neutrophils are a major component of the innate host response in the gingival tissue, and the contribution of neutrophil-derived cytokines and chemokines plays a central role in disease progression. The pattern of cytokines and chemokines released by human neutrophils upon stimulation with newly appreciated periodontal bacteria compared to the keystone oral pathogen Porphyromonas gingivalis was investigated. Our results showed that both F. alocis and P. stomatis triggered TLR2/6 activation. F. alocis induced significant changes in gene expression of cytokines and chemokines in human neutrophils compared to unstimulated cells. However, except for IL-1ra, neutrophils released lower levels of cytokines and chemokines in response to F. alocis compared to P. stomatis. Furthermore, bacteria-free conditioned supernatant collected from neutrophils challenged with P. stomatis, but not from P. gingivalis or F. alocis, was chemotactic towards both neutrophils and monocytes. Elucidating stimuli-specific modulation of human neutrophil effector functions in the context of dysbiotic microbial community constituents provides valuable information for understanding the pathogenesis of periodontal diseases. Full article

(This article belongs to the Section Immunological Responses and Immune Defense Mechanisms)

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13 pages, 4201 KiB

Open AccessEditor’s ChoiceArticle

Inhibition of Pseudomonas aeruginosa Biofilm Formation with Surface Modified Polymeric Nanoparticles

by Tyler R. Flockton, Logan Schnorbus, Agustin Araujo, Jill Adams, Maryjane Hammel and Lark J. Perez

Pathogens 2019, 8(2), 55; https://doi.org/10.3390/pathogens8020055 - 24 Apr 2019

Cited by 30 | Viewed by 5585

Abstract

The gram-negative bacterial pathogen Pseudomonas aeruginosa represents a prominent clinical concern. Due to the observed high levels of antibiotic resistance, copious biofilm formation, and wide array of virulence factors produced by these bacteria, new treatment technologies are required. Here, we present the development [...] Read more.

The gram-negative bacterial pathogen Pseudomonas aeruginosa represents a prominent clinical concern. Due to the observed high levels of antibiotic resistance, copious biofilm formation, and wide array of virulence factors produced by these bacteria, new treatment technologies are required. Here, we present the development of a series of P. aeruginosa LecA-targeted polymeric nanoparticles and demonstrate the anti-adhesion and biofilm inhibitory properties of these constructs. Full article

(This article belongs to the Special Issue Signaling Systems in Pseudomonas aeruginosa Biofilm)

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16 pages, 2129 KiB

Open AccessEditor’s ChoiceArticle

Epidemiology of Tick-Borne Encephalitis (TBE) in Germany, 2001–2018

by Wiebke Hellenbrand, Teresa Kreusch, Merle M. Böhmer, Christiane Wagner-Wiening, Gerhard Dobler, Ole Wichmann and Doris Altmann

Pathogens 2019, 8(2), 42; https://doi.org/10.3390/pathogens8020042 - 29 Mar 2019

Cited by 58 | Viewed by 9180

Abstract

We reviewed tick-borne encephalitis (TBE) surveillance and epidemiology in Germany, as these underlie public health recommendations, foremost vaccination. We performed descriptive analyses of notification data (2001–2018, n = 6063) according to region, demographics and clinical manifestations and calculated incidence trends using negative binomial [...] Read more.

We reviewed tick-borne encephalitis (TBE) surveillance and epidemiology in Germany, as these underlie public health recommendations, foremost vaccination. We performed descriptive analyses of notification data (2001–2018, n = 6063) according to region, demographics and clinical manifestations and calculated incidence trends using negative binomial regression. Risk areas were defined based on incidence in administrative districts. Most cases (89%) occurred in the federal states of Baden-Wurttemberg and Bavaria, where annual TBE incidence fluctuated markedly between 0.7–2.0 cases/100,000 inhabitants. A slight but significantly increasing temporal trend was observed from 2001–2018 (age-adjusted incidence rate ratio (IRR) 1.02 (95% confidence interval (CI): 1.01–1.04)), primarily driven by high case numbers in 2017–2018. Mean incidence was highest in 40–69-year-olds and in males. More males (23.7%) than females (18.0%, p = 0.02) had severe disease (encephalitis or myelitis), which increased with age, as did case-fatality (0.4% overall; 2.1% among ≥70-year-olds). Risk areas increased from 129 districts in 2007 to 161 in 2019. Expansion occurred mainly within existent southern endemic areas, with slower contiguous north-eastern and patchy north-western spread. Median vaccination coverage at school entry in risk areas in 2016–2017 ranged from 20%–41% in 4 states. Increasing TBE vaccine uptake is an urgent priority, particularly in high-incidence risk areas. Full article

(This article belongs to the Special Issue Ticks and Tick Borne Diseases Surveillance)

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12 pages, 838 KiB

Open AccessEditor’s ChoiceArticle

Intra-Species and Inter-Species Differences in Cytokine Production by Porcine Antigen-Presenting Cells Stimulated by Mycoplasma hyopneumoniae, M. hyorhinis, and M. flocculare

by Sarah Fourour, Corinne Marois-Créhan, Léa Martelet, Christelle Fablet, Isabelle Kempf, Marcelo Gottschalk and Mariela Segura

Pathogens 2019, 8(1), 34; https://doi.org/10.3390/pathogens8010034 - 16 Mar 2019

Cited by 23 | Viewed by 4043

Abstract

Mycoplasma hyorhinis and M. flocculare are commonly co-isolated with M. hyopneumoniae (primary agent of swine enzootic pneumonia) in gross pneumonia-like lesions, but their involvement in the disease process remains unknown. T cells play an immuno-pathological role during mycoplasmal infections. Dendritic cells (DCs) are [...] Read more.

Mycoplasma hyorhinis and M. flocculare are commonly co-isolated with M. hyopneumoniae (primary agent of swine enzootic pneumonia) in gross pneumonia-like lesions, but their involvement in the disease process remains unknown. T cells play an immuno-pathological role during mycoplasmal infections. Dendritic cells (DCs) are major antigen-presenting cells involved in T cell activation and differentiation. In this study, we investigated cytokine (IL-6, IL-8, IL-10, IL-12, and TNF-α) production by porcine bone-marrow-derived DCs (BM-DCs) stimulated by M. hyopneumoniae, M. hyorhinis, and/or M. flocculare. Results showed that cytokine production levels were relatively homogenous for all evaluated M. hyopneumoniae strains in contrast to M. hyorhinis and M. flocculare strains. The most noteworthy inter-species differences were the overall (i) lower IL-12 production capacity of M. hyopneumoniae, and (ii) higher TNF-α production capacity of M. flocculare. Co-stimulation of BM-DCs showed that M. hyorhinis dominated the IL-12 production independently of its association with M. hyopneumoniae or M. flocculare. In addition, a decreased BM-DC production of TNF-α was generally observed in the presence of mycoplasma associations. Lastly, M. flocculare association with M. hyopneumoniae increased BM-DC ability to secrete IL-10. A higher cytotoxicity level in BM-DCs stimulated by M. hyorhinis was also observed. Overall, this study demonstrated that the combination of M. hyorhinis or M. flocculare with M. hyopneumoniae may participate to the modulation of the immune response that might affect the final disease outcome. Full article

(This article belongs to the Section Immunological Responses and Immune Defense Mechanisms)

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16 pages, 3582 KiB

Open AccessEditor’s ChoiceArticle

HIV Replication in Humanized IL-3/GM-CSF-Transgenic NOG Mice

by Federico Perdomo-Celis, Sandra Medina-Moreno, Harry Davis, Joseph Bryant and Juan C. Zapata

Pathogens 2019, 8(1), 33; https://doi.org/10.3390/pathogens8010033 - 12 Mar 2019

Cited by 15 | Viewed by 5894

Abstract

The development of mouse models that mimic the kinetics of Human Immunodeficiency Virus (HIV) infection is critical for the understanding of the pathogenesis of disease and for the design of novel therapeutic strategies. Here, we describe the dynamics of HIV infection in humanized [...] Read more.

The development of mouse models that mimic the kinetics of Human Immunodeficiency Virus (HIV) infection is critical for the understanding of the pathogenesis of disease and for the design of novel therapeutic strategies. Here, we describe the dynamics of HIV infection in humanized NOD/Shi-scid-IL2rγ^null (NOG) mice bearing the human genes for interleukin (IL)-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) (NOG-EXL mice). The kinetics of viral load, as well as the frequencies of T-cells, B-cells, Natural killer cells (NK), monocytes, and dendritic cells in blood and secondary lymphoid organs were evaluated throughout the time of infection. In comparison with a non-transgenic humanized mouse (NSG) strain, lymphoid and myeloid populations were more efficiently engrafted in humanized NOG-EXL mice, both in peripheral blood and lymphoid tissues. In addition, HIV actively replicated in humanized NOG-EXL mice, and infection induced a decrease in the percentage of CD4⁺ T-cells, inversion of the CD4:CD8 ratio, and changes in some cell populations, such as monocytes and dendritic cells, that recapitulated those found in human natural infection. Thus, the humanized IL-3/GM-CSF-transgenic NOG mouse model is suitable for the study of the dynamics of HIV infection and provides a tool for basic and preclinical studies. Full article

(This article belongs to the Section Human Pathogens)

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16 pages, 2068 KiB

Open AccessEditor’s ChoiceArticle

Characteristics of Listeria Monocytogenes Strains Persisting in a Meat Processing Facility over a 4-Year Period

by Andrea Stoller, Marc J. A. Stevens, Roger Stephan and Claudia Guldimann

Pathogens 2019, 8(1), 32; https://doi.org/10.3390/pathogens8010032 - 7 Mar 2019

Cited by 59 | Viewed by 6321

Abstract

Listeria monocytogenes can persist in food production facilities, resulting in serious threats to consumers due to the high mortality associated with listeriosis, especially in the very young, old and pregnant. We subtyped 124 strains of L. monocytogenes isolated from a meat processing facility [...] Read more.

Listeria monocytogenes can persist in food production facilities, resulting in serious threats to consumers due to the high mortality associated with listeriosis, especially in the very young, old and pregnant. We subtyped 124 strains of L. monocytogenes isolated from a meat processing facility in Switzerland by serotyping, multi locus sequence typing (MLST) typing and whole genome sequencing. We then analyzed their ability to form biofilms and their resistance to the disinfectants benzalkonium chloride (BC) and peracetic acid (PAA). The genotyping results of the strains showed that several clonal populations of L. monocytogenes belonging to CC9, CC204 and CC121 had persisted in this meat processing facility for at least four years. All of the strains showed biofilm forming capacity comparable to a known high biofilm forming strain. Known efflux pumps for BC were present in CC204, CC9 (brcABC) and CC121 (qacH) strains, while strains from other CC showed very low minimal inhibitory concentrations (MICs) for BC. For PAA, minimal bactericidal concentrations of 1.2–1.6% for 20 min and minimal inhibitory concentrations between 0.1 and 0.2% were observed. These values were close to or above the recommended concentration for use (0.5–1%), suggesting that PAA might be ineffective at controlling L. monocytogenes in this and potentially other meat processing facilities. Full article

(This article belongs to the Section Human Pathogens)

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11 pages, 1915 KiB

Open AccessEditor’s ChoiceArticle

Molecular Epidemiology and Genetic Diversity of Zika Virus from Field-Caught Mosquitoes in Various Regions of Thailand

by Atchara Phumee, Rome Buathong, Rungfar Boonserm, Proawpilart Intayot, Nucharat Aungsananta, Akanitt Jittmittraphap, Yutthana Joyjinda, Supaporn Wacharapluesadee and Padet Siriyasatien

Pathogens 2019, 8(1), 30; https://doi.org/10.3390/pathogens8010030 - 6 Mar 2019

Cited by 29 | Viewed by 8786

Abstract

Zika virus (ZIKV) infection is an emerging and re-emerging arbovirus disease that is transmitted to humans through the bite of infected mosquitoes. ZIKV infections were first described in Thailand in 1954 from the sera of indigenous residents and several travelers returning from Thailand [...] Read more.

Zika virus (ZIKV) infection is an emerging and re-emerging arbovirus disease that is transmitted to humans through the bite of infected mosquitoes. ZIKV infections were first described in Thailand in 1954 from the sera of indigenous residents and several travelers returning from Thailand in 2014. However, reported cases in Thailand have been increasing since 2015 and 2016, and epidemiological information about the vectors of ZIKV is unclear. We investigated the molecular epidemiology and genetic diversity of ZIKV from mosquitoes collected from different geographic regions experiencing ZIKV outbreaks in Thailand. Polymerase chain reaction was used to amplify the non-structural protein (NS5) gene of ZIKV, which was then sequenced. A total of 1026 mosquito samples (626 females, 367 males, and 33 larvae) were collected from active ZIKV patients’ houses. ZIKV was detected in 79 samples (7.7%), including Aedes aegypti (2.24% female, 1.27% male, and 0.19% larvae), Culex quinquefasciatus (1.85% female, 1.66% male, and 0.29% larvae), and Armigeres subalbatus (0.1% female and 0.1% male), whereas no ZIKV was detected in Aedes albopictus. Phylogenetic analysis of the 79 positive samples were classified into two clades: Those closely related to a previous report in Thailand, and those related to ZIKV found in the Americas. This is the first report of the detection of ZIKV in Ae. aegypti, Cx. quinquefasciatus, and Ar. subalbatus mosquitoes, and genetic variations of ZIKV in the mosquitoes collected from several geographic regions of Thailand were examined. Detection of ZIKV in male and larval mosquitoes suggests that vertical transmission of ZIKV occurred in these mosquito species. This study provides a more in-depth understanding of the patterns and epidemiologic data of ZIKV in Thailand; the data could be used for future development of more effective prevention and control strategies of ZIKV in Thailand. Full article

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19 pages, 1541 KiB

Open AccessEditor’s ChoiceArticle

The 3′ Untranslated Region of a Plant Viral RNA Directs Efficient Cap-Independent Translation in Plant and Mammalian Systems

by Jelena J. Kraft, Mariko S. Peterson, Sung Ki Cho, Zhaohui Wang, Alice Hui, Aurélie M. Rakotondrafara, Krzysztof Treder, Cathy L. Miller and W. Allen Miller

Pathogens 2019, 8(1), 28; https://doi.org/10.3390/pathogens8010028 - 28 Feb 2019

Cited by 11 | Viewed by 5621

Abstract

Many plant viral RNA genomes lack a 5′ cap, and instead are translated via a cap-independent translation element (CITE) in the 3′ untranslated region (UTR). The panicum mosaic virus-like CITE (PTE), found in many plant viral RNAs, binds and requires the cap-binding translation [...] Read more.

Many plant viral RNA genomes lack a 5′ cap, and instead are translated via a cap-independent translation element (CITE) in the 3′ untranslated region (UTR). The panicum mosaic virus-like CITE (PTE), found in many plant viral RNAs, binds and requires the cap-binding translation initiation factor eIF4E to facilitate translation. eIF4E is structurally conserved between plants and animals, so we tested cap-independent translation efficiency of PTEs of nine plant viruses in plant and mammalian systems. The PTE from thin paspalum asymptomatic virus (TPAV) facilitated efficient cap-independent translation in wheat germ extract, rabbit reticulocyte lysate, HeLa cell lysate, and in oat and mammalian (BHK) cells. Human eIF4E bound the TPAV PTE but not a PTE that did not stimulate cap-independent translation in mammalian extracts or cells. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) footprinting revealed that both human and wheat eIF4E protected the conserved guanosine (G)-rich domain in the TPAV PTE pseudoknot. The central G plays a key role, as it was found to be required for translation and protection from SHAPE modification by eIF4E. These results provide insight on how plant viruses gain access to the host’s translational machinery, an essential step in infection, and raise the possibility that similar PTE-like mechanisms may exist in mRNAs of mammals or their viruses. Full article

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14 pages, 2198 KiB

Open AccessEditor’s ChoiceArticle

In silico Identification of Novel Toxin Homologs and Associated Mobile Genetic Elements in Clostridium perfringens

by Jake A. Lacey, Priscilla A. Johanesen, Dena Lyras and Robert J. Moore

Pathogens 2019, 8(1), 16; https://doi.org/10.3390/pathogens8010016 - 29 Jan 2019

Cited by 14 | Viewed by 5027

Abstract

Clostridium perfringens causes a wide range of diseases in a variety of hosts, due to the production of a diverse set of toxins and extracellular enzymes. The C. perfringens toxins play an important role in pathogenesis, such that the presence and absence of [...] Read more.

Clostridium perfringens causes a wide range of diseases in a variety of hosts, due to the production of a diverse set of toxins and extracellular enzymes. The C. perfringens toxins play an important role in pathogenesis, such that the presence and absence of the toxins is used as a typing scheme for the species. In recent years, several new toxins have been discovered that have been shown to be essential or highly correlated to diseases; these include binary enterotoxin (BecAB), NetB and NetF. In the current study, genome sequence analysis of C. perfringens isolates from diverse sources revealed several putative novel toxin homologs, some of which appeared to be associated with potential mobile genetic elements, including transposons and plasmids. Four novel toxin homologs encoding proteins related to the pore-forming Leukocidin/Hemolysin family were found in type A and G isolates. Two novel toxin homologs encoding proteins related to the epsilon aerolysin-like toxin family were identified in Type A and F isolates from humans, contaminated food and turkeys. A novel set of proteins related to clostridial binary toxins was also identified. While phenotypic characterisation is required before any of these homologs can be established as functional toxins, the in silico identification of these novel homologs on mobile genetic elements suggests the potential toxin reservoir of C. perfringens may be much larger than previously thought. Full article

(This article belongs to the Section Animal Pathogens)

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21 pages, 4563 KiB

Open AccessEditor’s ChoiceArticle

Molecular Characterisation of Equine Herpesvirus 1 Isolates from Cases of Abortion, Respiratory and Neurological Disease in Ireland between 1990 and 2017

by Marie Garvey, Rachel Lyons, Ralph D Hector, Cathal Walsh, Sean Arkins and Ann Cullinane

Pathogens 2019, 8(1), 7; https://doi.org/10.3390/pathogens8010007 - 15 Jan 2019

Cited by 28 | Viewed by 4145

Abstract

Multiple locus typing based on sequencing heterologous regions in 26 open reading frames (ORFs) of equine herpesvirus 1 (EHV-1) strains Ab4 and V592 was used to characterise 272 EHV-1 isolates from 238 outbreaks of abortion, respiratory or neurological disease over a 28-year period. [...] Read more.

Multiple locus typing based on sequencing heterologous regions in 26 open reading frames (ORFs) of equine herpesvirus 1 (EHV-1) strains Ab4 and V592 was used to characterise 272 EHV-1 isolates from 238 outbreaks of abortion, respiratory or neurological disease over a 28-year period. The analysis grouped the 272 viruses into at least 10 of the 13 unique long region (U_L) clades previously recognised. Viruses from the same outbreak had identical multi-locus profiles. Sequencing of the ORF68 region of EHV-1 isolates from 222 outbreaks established a divergence into seven groups and network analysis demonstrated that Irish genotypes were not geographically restricted but clustered with viruses from all over the world. Multi-locus analysis proved a more comprehensive method of strain typing than ORF68 sequencing. It was demonstrated that when interpreted in combination with epidemiological data, this type of analysis has a potential role in tracking virus between premises and therefore in the implementation of targeted control measures. Viruses from 31 of 238 outbreaks analysed had the proposed ORF30 G2254/D752 neuropathogenic marker. There was a statistically significant association between viruses of the G2254/D752 genotype and both neurological disease and hypervirulence as defined by outbreaks involving multiple abortion or neurological cases. The association of neurological disease in those with the G2254/D752 genotype was estimated as 27 times greater than in those with the A2254/N752 genotype. Full article

(This article belongs to the Section Animal Pathogens)

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17 pages, 8731 KiB

Open AccessEditor’s ChoiceArticle

Attenuated Replication of Lassa Virus Vaccine Candidate ML29 in STAT-1^-/- Mice

by Dylan M. Johnson, Jenny D. Jokinen and Igor S. Lukashevich

Pathogens 2019, 8(1), 9; https://doi.org/10.3390/pathogens8010009 - 15 Jan 2019

Cited by 12 | Viewed by 5024

Abstract

Lassa virus (LASV), a highly prevalent mammalian arenavirus endemic in West Africa, can cause Lassa fever (LF), which is responsible for thousands of deaths annually. LASV is transmitted to humans from naturally infected rodents. At present, there is not an effective vaccine nor [...] Read more.

Lassa virus (LASV), a highly prevalent mammalian arenavirus endemic in West Africa, can cause Lassa fever (LF), which is responsible for thousands of deaths annually. LASV is transmitted to humans from naturally infected rodents. At present, there is not an effective vaccine nor treatment. The genetic diversity of LASV is the greatest challenge for vaccine development. The reassortant ML29 carrying the L segment from the nonpathogenic Mopeia virus (MOPV) and the S segment from LASV is a vaccine candidate under current development. ML29 demonstrated complete protection in validated animal models against a Nigerian strain from clade II, which was responsible for the worst outbreak on record in 2018. This study demonstrated that ML29 was more attenuated than MOPV in STAT1^-/- mice, a small animal model of human LF and its sequelae. ML29 infection of these mice resulted in more than a thousand-fold reduction in viremia and viral load in tissues and strong LASV-specific adaptive T cell responses compared to MOPV-infected mice. Persistent infection of Vero cells with ML29 resulted in generation of interfering particles (IPs), which strongly interfered with the replication of LASV, MOPV and LCMV, the prototype of the Arenaviridae. ML29 IPs induced potent cell-mediated immunity and were fully attenuated in STAT1^-/- mice. Formulation of ML29 with IPs will improve the breadth of the host’s immune responses and further contribute to development of a pan-LASV vaccine with full coverage meeting the WHO requirements. Full article

(This article belongs to the Special Issue Lassa Fever: Viral Replication, Disease Pathogenesis, and Host Immune Modulations)

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17 pages, 6695 KiB

Open AccessEditor’s ChoiceArticle

Staphylococcus aureus Superantigen-Like Protein SSL1: A Toxic Protease

by Aihua Tang, Armando R. Caballero, Michael A. Bierdeman, Mary E. Marquart, Timothy J. Foster, Ian R. Monk and Richard J. O’Callaghan

Pathogens 2019, 8(1), 2; https://doi.org/10.3390/pathogens8010002 - 1 Jan 2019

Cited by 11 | Viewed by 4979

Abstract

Staphylococcus aureus is a major cause of corneal infections that can cause reduced vision, even blindness. Secreted toxins cause tissue damage and inflammation resulting in scars that lead to vision loss. Identifying tissue damaging proteins is a prerequisite to limiting these harmful reactions. [...] Read more.

Staphylococcus aureus is a major cause of corneal infections that can cause reduced vision, even blindness. Secreted toxins cause tissue damage and inflammation resulting in scars that lead to vision loss. Identifying tissue damaging proteins is a prerequisite to limiting these harmful reactions. The present study characterized a previously unrecognized S. aureus toxin. This secreted toxin was purified from strain Newman ΔhlaΔhlg, the N-terminal sequence determined, the gene cloned, and the purified recombinant protein was tested in the rabbit cornea. The virulence of a toxin deletion mutant was compared to its parent and the mutant after gene restoration (rescue strain). The toxin (23 kDa) had an N-terminal sequence matching the Newman superantigen-like protein SSL1. An SSL1 homodimer (46 kDa) had proteolytic activity as demonstrated by zymography and cleavage of a synthetic substrate, collagens, and cytokines (IL-17A, IFN-γ, and IL-8); the protease was susceptible to serine protease inhibitors. As compared to the parent and rescue strains, the ssl1 mutant had significantly reduced virulence, but not reduced bacterial growth, in vivo. The ocular isolates tested had the ssl1 gene, with allele type 2 being the predominant type. SSL1 is a protease with corneal virulence and activity on host defense and structural proteins. Full article

(This article belongs to the Section Human Pathogens)

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