Biological and Molecular Characteristics of a Novel Partitivirus Infecting the Edible Fungus Lentinula edodes

A new partitivirus named Lentinula edodes partitivirus 1 (LePV1) was isolated from a diseased L. edodes strain with severe degeneration of the mycelium and imperfect browning in bag cultures. The nucleotide sequences of LePV1 dsRNA-1 and dsRNA-2 were determined; they were 2,382 bp and 2,245 bp in length, and each contained a single ORF encoding RNA-dependent RNA polymerase (RdRp) and coat protein (CP), respectively. The purified virus preparation contained isometric particles 34 nm in diameter encapsidating these dsRNAs. Phylogenetic analyses showed LePV1 to be a new member of Betapartitivirus, with the RdRp sequence most closely related to Grapevine partitivirus. RT-PCR analysis showed that 27 of the 56 Chinese L. edodes core collection strains carry LePV1, with the virus being more common in wild strains than cultivated strains. In addition, qPCR analysis suggested that coinfection with L. edodes mycovirus HKB (LeV-HKB) could increase replication of the RdRp gene of LePV1. This study may be essential for the development of more accurate disease diagnostics and the formulation of control strategies for viral diseases in L. edodes.

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An optimized protocol for stepwise optimization of real-time RT-PCR analysis

Horticulture Research ◽

10.1038/s41438-021-00616-w ◽

2021 ◽

Vol 8 (1) ◽

Author(s):

Fangzhou Zhao ◽

Nathan A. Maren ◽

Pawel Z. Kosentka ◽

Ying-Yu Liao ◽

Hongyan Lu ◽

...

Keyword(s):

Real Time ◽

Reference Genes ◽

Primer Design ◽

Plant Genome ◽

Pcr Analysis ◽

Logarithmic Scale ◽

Rt Pcr ◽

Qpcr Analysis ◽

Specificity And Sensitivity ◽

Optimized Protocol

AbstractComputational tool-assisted primer design for real-time reverse transcription (RT) PCR (qPCR) analysis largely ignores the sequence similarities between sequences of homologous genes in a plant genome. It can lead to false confidence in the quality of the designed primers, which sometimes results in skipping the optimization steps for qPCR. However, the optimization of qPCR parameters plays an essential role in the efficiency, specificity, and sensitivity of each gene’s primers. Here, we proposed an optimized approach to sequentially optimizing primer sequences, annealing temperatures, primer concentrations, and cDNA concentration range for each reference (and target) gene. Our approach started with a sequence-specific primer design that should be based on the single-nucleotide polymorphisms (SNPs) present in all the homologous sequences for each of the reference (and target) genes under study. By combining the efficiency calibrated and standard curve methods with the 2−ΔΔCt method, the standard cDNA concentration curve with a logarithmic scale was obtained for each primer pair for each gene. As a result, an R2 ≥ 0.9999 and the efficiency (E) = 100 ± 5% should be achieved for the best primer pair of each gene, which serve as the prerequisite for using the 2−ΔΔCt method for data analysis. We applied our newly developed approach to identify the best reference genes in different tissues and at various inflorescence developmental stages of Tripidium ravennae, an ornamental and biomass grass, and validated their utility under varying abiotic stress conditions. We also applied this approach to test the expression stability of six reference genes in soybean under biotic stress treatment with Xanthomonas axonopodis pv. glycines (Xag). Thus, these case studies demonstrated the effectiveness of our optimized protocol for qPCR analysis.

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Phenotypical and Functional Characteristics of Human Regulatory T Cells during Ex Vivo Maturation from CD4+ T Lymphocytes

Applied Sciences ◽

10.3390/app11135776 ◽

2021 ◽

Vol 11 (13) ◽

pp. 5776

Author(s):

Varvara G. Blinova ◽

Natalia S. Novachly ◽

Sofya N. Gippius ◽

Abdullah Hilal ◽

Yulia A. Gladilina ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Ex Vivo ◽

Pcr Analysis ◽

Mrna Levels ◽

Rt Pcr ◽

Inflammatory Reactions ◽

Effector Cells ◽

Cycle Regulation ◽

Further Development

Regulatory T cells (Tregs) participate in the negative regulation of inflammatory reactions by suppressing effector cells. In a number of autoimmune disorders, the suppressive function and/or the number of Tregs is compromised. The lack of active functioning Tregs can be restored with adoptive transfer of expanded ex vivo autologous Tregs. In our study, we traced the differentiation and maturation of Tregs CD4+CD25+FoxP3+CD127low over 7 days of cultivation from initial CD4+ T cells under ex vivo conditions. The resulting ex vivo expanded cell population (eTregs) demonstrated the immune profile of Tregs with an increased capacity to suppress the proliferation of target effector cells. The expression of the FoxP3 gene was upregulated within the time of expansion and was associated with gradual demethylation in the promotor region of the T cell-specific demethylation region. Real-time RT-PCR analysis revealed changes in the expression profile of genes involved in cell cycle regulation. In addition to FOXP3, the cells displayed elevated mRNA levels of Ikaros zinc finger transcription factors and the main telomerase catalytic subunit hTERT. Alternative splicing of FoxP3, hTERT and IKZF family members was demonstrated to be involved in eTreg maturation. Our data indicate that expanded ex vivo eTregs develop a Treg-specific phenotype and functional suppressive activity. We suggest that eTregs are not just expanded but transformed cells with enhanced capacities of immune suppression. Our findings may influence further development of cell immunosuppressive therapy based on regulatory T cells.

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Molecular Epidemiology and Whole-Genome Analysis of Bovine Foamy Virus in Japan

Viruses ◽

10.3390/v13061017 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1017

Author(s):

Hirohisa Mekata ◽

Tomohiro Okagawa ◽

Satoru Konnai ◽

Takayuki Miyazawa

Keyword(s):

Phylogenetic Analyses ◽

Foamy Virus ◽

Pcr Analysis ◽

Whole Genome ◽

Genome Sequences ◽

Whole Genome Analysis ◽

Virus Family ◽

Bovine Foamy Virus ◽

Novel Genotype

Bovine foamy virus (BFV) is a member of the foamy virus family in cattle. Information on the epidemiology, transmission routes, and whole-genome sequences of BFV is still limited. To understand the characteristics of BFV, this study included a molecular survey in Japan and the determination of the whole-genome sequences of 30 BFV isolates. A total of 30 (3.4%, 30/884) cattle were infected with BFV according to PCR analysis. Cattle less than 48 months old were scarcely infected with this virus, and older animals had a significantly higher rate of infection. To reveal the possibility of vertical transmission, we additionally surveyed 77 pairs of dams and 3-month-old calves in a farm already confirmed to have BFV. We confirmed that one of the calves born from a dam with BFV was infected. Phylogenetic analyses revealed that a novel genotype was spread in Japan. In conclusion, the prevalence of BFV in Japan is relatively low and three genotypes, including a novel genotype, are spread in Japan.

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Multiple Types of Novel Enteric Bopiviruses (Picornaviridae) with the Possibility of Interspecies Transmission Identified from Cloven-Hoofed Domestic Livestock (Ovine, Caprine and Bovine) in Hungary

Viruses ◽

10.3390/v13010066 ◽

2021 ◽

Vol 13 (1) ◽

pp. 66

Author(s):

Zoltán László ◽

Péter Pankovics ◽

Gábor Reuter ◽

Attila Cságola ◽

Ádám Bálint ◽

...

Keyword(s):

Novel Species ◽

Phylogenetic Analyses ◽

Interspecies Transmission ◽

Background Information ◽

Type Ii ◽

Rt Pcr ◽

Faecal Samples ◽

The Family ◽

Domestic Livestock

Most picornaviruses of the family Picornaviridae are relatively well known, but there are certain “neglected” genera like Bopivirus, containing a single uncharacterised sequence (bopivirus A1, KM589358) with very limited background information. In this study, three novel picornaviruses provisionally called ovipi-, gopi- and bopivirus/Hun (MW298057-MW298059) from enteric samples of asymptomatic ovine, caprine and bovine respectively, were determined using RT-PCR and dye-terminator sequencing techniques. These monophyletic viruses share the same type II-like IRES, NPGP-type 2A, similar genome layout (4-3-4) and cre-localisations. Culture attempts of the study viruses, using six different cell lines, yielded no evidence of viral growth in vitro. Genomic and phylogenetic analyses show that bopivirus/Hun of bovine belongs to the species Bopivirus A, while the closely related ovine-origin ovipi- and caprine-origin gopivirus could belong to a novel species “Bopivirus B” in the genus Bopivirus. Epidemiological investigation of N = 269 faecal samples of livestock (ovine, caprine, bovine, swine and rabbit) from different farms in Hungary showed that bopiviruses were most prevalent among <12-month-old ovine, caprine and bovine, but undetectable in swine and rabbit. VP1 capsid-based phylogenetic analyses revealed the presence of multiple lineages/genotypes, including closely related ovine/caprine strains, suggesting the possibility of ovine–caprine interspecies transmission of certain bopiviruses.

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Hepatitis E Virus Occurrence in Pigs Slaughtered in Italy

Animals ◽

10.3390/ani11020277 ◽

2021 ◽

Vol 11 (2) ◽

pp. 277

Author(s):

Eleonora Chelli ◽

Elisabetta Suffredini ◽

Paola De Santis ◽

Dario De Medici ◽

Santina Di Bella ◽

...

Keyword(s):

Hepatitis E Virus ◽

Phylogenetic Analyses ◽

Hepatitis E ◽

Rt Pcr ◽

High Seroprevalence ◽

Genotype 3 ◽

Sporadic Cases ◽

Wild Boar Meat ◽

Small Clusters ◽

Frequent Exposure

In Europe, foodborne transmission has been clearly associated to sporadic cases and small clusters of hepatitis E in humans linked to the consumption of contaminated pig liver sausages, raw venison, or undercooked wild boar meat. In Europe, zoonotic HEV-genotype 3 strains are widespread in pig farms but little information is available on the prevalence of HEV positive pigs at slaughterhouse. In the present study, the prevalence of HEV-RNA positive pigs was assessed on 585 animals from 4 abattoirs located across Italy. Twenty-one pigs (3.6%) tested positive for HEV in either feces or liver by real-time RT-PCR. In these 21 pigs, eight diaphragm muscles resulted positive for HEV-RNA. Among animals collected in one abattoir, 4 out of 91 plasma tested positive for HEV-RNA. ELISA tests for the detection of total antibodies against HEV showed a high seroprevalence (76.8%), confirming the frequent exposure of pigs to the virus. The phylogenetic analyses conducted on sequences of both ORF1 and ORF2 fragments, shows the circulation of HEV-3c and of a novel unclassified subtype. This study provides information on HEV occurrence in pigs at the slaughterhouse, confirming that muscles are rarely contaminated by HEV-RNA compared to liver, which is the most frequently positive for HEV.

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Two New Haplotypes of Bartonella sp. Isolated from Lipoptena fortisetosa (Diptera: Hippoboscidae) in SE Poland

Insects ◽

10.3390/insects12060485 ◽

2021 ◽

Vol 12 (6) ◽

pp. 485

Author(s):

Katarzyna Bartosik ◽

Weronika Maślanko ◽

Alicja Buczek ◽

Marek Asman ◽

Joanna Witecka ◽

...

Keyword(s):

Potential Role ◽

Roe Deer ◽

Phylogenetic Analyses ◽

Rpob Gene ◽

Molecular Characteristics ◽

Broad Host Range ◽

South Eastern ◽

Se Poland ◽

Pcr Method ◽

The Subject

Insects of the genus Lipoptena are parasitic arthropods with a broad host range. Due to the type of parasitism (hematophagy), their potential role as vectors of pathogens, i.e., Bartonella sp., Anaplasma phagocytophilum, Rickettsia spp., and Borrelia burgdorferi is considered. As the range of their occurrence has been changing dynamically in recent years and infestations of humans have increasingly been reported, these organisms are now the subject of numerous studies. Our research aimed to present the molecular characteristics of Bartonella sp. detected in Lipoptena fortisetosa parasitizing wild cervids in south-eastern Poland. Adults of Lipoptena spp. were collected from carcasses of roe deer and red deer between spring and autumn in 2013. The PCR method was used to detect Bartonella sp. in the insects. We report two new haplotypes of the rpoB gene of Bartonella sp. isolated from L. fortisetosa feeding on wild cervids in south-eastern Poland and the presence of this invasive ectoparasitic species in the studied area since 2013. Phylogenetic analyses of newly obtained Bartonella sp. haplotypes confirmed their unique position on the constructed tree and network topology. The rpoB gene sequences found belonging to lineage B support the view that this phylogenetic lineage represents a novel Bartonella species.

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Role of Innate Inflammation in the Regulation of Tissue Remodeling during Tooth Eruption

Dentistry Journal ◽

10.3390/dj9010007 ◽

2021 ◽

Vol 9 (1) ◽

pp. 7

Author(s):

Yusuke Makino ◽

Kaoru Fujikawa ◽

Miwako Matsuki-Fukushima ◽

Satoshi Inoue ◽

Masanori Nakamura

Keyword(s):

Bacterial Infections ◽

Tooth Movement ◽

Tooth Eruption ◽

Intercellular Adhesion Molecule ◽

Enamel Organ ◽

Pcr Analysis ◽

Maturation Stage ◽

Oral Epithelium ◽

Rt Pcr ◽

Inflammatory Reactions

Tooth eruption is characterized by a coordinated complex cascade of cellular and molecular events that promote tooth movement through the eruptive pathway. During tooth eruption, the stratum intermedium structurally changes to the papillary layer with tooth organ development. We previously reported intercellular adhesion molecule-1 (ICAM-1) expression on the papillary layer, which is the origin of the ICAM-1-positive junctional epithelium. ICAM-1 expression is induced by proinflammatory cytokines, including interleukin-1 and tumor necrosis factor. Inflammatory reactions induce tissue degradation. Therefore, this study aimed to examine whether inflammatory reactions are involved in tooth eruption. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed sequential expression of hypoxia-induced factor-1α, interleukin-1β, and chemotactic factors, including keratinocyte-derived chemokine (KC) and macrophage inflammatory protein-2 (MIP-2), during tooth eruption. Consistent with the RT-PCR results, immunohistochemical analysis revealed KC and MIP-2 expression in the papillary layer cells of the enamel organ from the ameloblast maturation stage. Moreover, there was massive macrophage and neutrophil infiltration in the connective tissue between the tooth organ and oral epithelium during tooth eruption. These findings suggest that inflammatory reactions might be involved in the degradation of tissue overlying the tooth organ. Further, these reactions might be induced by hypoxia in the tissue overlying the tooth organ, which results from decreased capillaries in the tissue. Our findings indicate that bacterial infections are not associated with the eruption process. Therefore, tooth eruption might be regulated by innate inflammatory mechanisms.

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RT-PCR analysis of Deformed wing virus in honeybees (Apis mellifera) and mites (Varroa destructor)

Journal of General Virology ◽

10.1099/vir.0.81401-0 ◽

2005 ◽

Vol 86 (12) ◽

pp. 3419-3424 ◽

Cited By ~ 210

Author(s):

Constanze Yue ◽

Elke Genersch

Keyword(s):

Varroa Destructor ◽

Pcr Analysis ◽

Body Parts ◽

Rt Pcr ◽

Larval Food ◽

Viral Pathogen ◽

Deformed Wing Virus ◽

Significant Difference ◽

Almost All ◽

Viral Sequences

Deformed wing virus (DWV) is a honeybee viral pathogen either persisting as an inapparent infection or resulting in wing deformity. The occurrence of deformity is associated with the transmission of DWV through Varroa destructor during pupal stages. Such infections with DWV add to the pathology of V. destructor and play a major role in colony collapse in the course of varroosis. Using a recently developed RT-PCR protocol for the detection of DWV, individual bees and mites originating from hives differing in Varroa infestation levels and the occurrence of crippled bees were analysed. It was found that 100 % of both crippled and asymptomatic bees were positive for DWV. However, a significant difference in the spatial distribution of DWV between asymptomatic and crippled bees could be demonstrated: when analysing head, thorax and abdomen of crippled bees, all body parts were always strongly positive for viral sequences. In contrast, for asymptomatic bees viral sequences could be detected in RNA extracted from the thorax and/or abdomen but never in RNA extracted from the head. DWV replication was demonstrated in almost all DWV-positive body parts of infected bees. Analysing individual mites for the presence of DWV revealed that the percentage of DWV-positive mites differed between mite populations. In addition, it was demonstrated that DWV was able to replicate in some but not all mites. Interestingly, virus replication in mites was correlated with wing deformity. DWV was also detected in the larval food, implicating that in addition to transmission by V. destructor DWV is also transmitted by feeding.

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Reproductive failure in mice lacking inter-alpha-trypsin inhibitor (ITI) – ITI target genes in mouse ovary identified by microarray analysis

Journal of Endocrinology ◽

10.1677/joe.1.05803 ◽

2004 ◽

Vol 183 (1) ◽

pp. 29-38 ◽

Cited By ~ 14

Author(s):

Mika Suzuki ◽

Hiroshi Kobayashi ◽

Yoshiko Tanaka ◽

Naohiro Kanayama ◽

Toshihiko Terao

Keyword(s):

Real Time ◽

Trypsin Inhibitor ◽

Target Genes ◽

Reproductive Function ◽

Pcr Analysis ◽

Rt Pcr ◽

Reproductive Process ◽

Female Mice ◽

Retinoid Metabolism ◽

Regulatory Effects

Bikunin, a Kunitz-type protease inhibitor, is found in blood and urine. It has been established by two laboratories independently that the bikunin knockout female mice display a severe reduction in fertility: the cumulus oophorus has a defect in forming the extracellular hyaluronan-rich matrix during expansion. Proteins of the inter-alpha-trypsin inhibitor (ITI) family are eliminated in mice in which the bikunin gene has been inactivated, since bikunin is essential for their biosynthesis. Proteins of the ITI family may contribute to the microenvironment in which ovulation takes place. It is not clear, however, whether a single mechanism affects the reproductive function including ovulation. For identifying the full repertoire of the ITI deficiency-related genes, a cDNA microarray hybridization screening was conducted using mRNA from ovaries of wild-type or bik−/− female mice. A number of genes were identified and their regulation was confirmed by real-time RT-PCR analysis. Our screen identified that 29 (0.7%) and 5 genes (0.1%) of the genes assayed were, respectively, up- and down-regulated twofold or more. The identified genes can be classified into distinct subsets. These include stress-related, apoptosis-related, proteases, signaling molecules, aging-related, cytokines, hyaluronan metabolism and signaling, reactive oxygen species-related, and retinoid metabolism, which have previously been implicated in enhancing follicle development and/or ovulation. Real-time RT-PCR analysis confirmed that these genes were up- and down-regulated two- to tenfold by bikunin knockout. These studies demonstrate that proteins of the ITI family may exert potent regulatory effects on a major physiological reproductive process, ovulation.

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