Search

Atypical porcine pestivirus (APPV) is a recently discovered RNA virus, which mainly caused congenital tremor in piglets. Droplet digital PCR (ddPCR) is an absolute quantitative method that does not rely on the standard curve but has high sensitivity and accuracy. The present study aimed to develop a ddPCR detection assay for APPV. Furthermore, we evaluated the limit of detection, sensitivity, specificity and reproducibility of the ddPCR and real-time quantitative PCR (qPCR) and tested 135 clinical samples to calculate the detection rate of the two methods. The results showed that both methods had a strong linear relationship and quantitative correlation. The ddPCR assay had a minimum detection limit of 0.15 copies/μL for APPV, with a sensitivity 100 times that of qPCR. We tested clinical samples and found that the APPV ddPCR had a 27.4% positive detection rate, noticeably higher than that of the qPCR (14.8%). Additionally, the APPV ddPCR method had excellent repeatability and specificity. In brief, our study provided a novel, feasible and sensitive diagnostic technique to identify and monitor APPV. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-022-00728-y.

Chlamydia pecorum, an obligate intracellular bacterium, is associated with reproductive and systemic diseases in sheep, goats, pigs, cattle, and koalas. The main conditions include polyarthritis, conjunctivitis, enteritis, pneumonia, encephalomyelitis, orchitis, placentitis, and abortion. Even though there are several studies showing that C. pecorum infections are widely spread in the world, in Mexico there are no reports. During 2016, as part of a sheep restocking program in Mexico, sheep were imported from New Zealand. Briefly after their arrival in the herds in the State of Mexico, these sheep presented abortions during the last third of gestation. A total of 62 sheep vaginal swabs that had presented abortion from different municipalities of the State of Mexico were collected. Bacterial isolation was performed using L929 mouse fibroblasts, and molecular identification was achieved by 23S rRNA (Chlamydiaceae family) and ompA gene (species-specific) real-time polymerase chain reaction (PCR). In addition, the 16S rRNA subunit and ompA gene were amplified and sequenced. Seven of 62 samples were positive for C. pecorum by bacterial isolation, 23S rRNA, and ompA gene real-time PCR. The 16S rRNA subunit and ompA gene amplicons were purified and the nucleotide sequence was determined in both directions. The consensus sequences homology search was performed using BLASTn analysis and showed a 100% of homology with the C. pecorum 16S rRNA subunit and 99% with the C. pecorum ompA gene. The population structure analyses using ompA gene demonstrated 15 genetic populations or clusters of 198 sequences from GenBank and our sequences were in a particular genetic structure corresponding to genotype “O.” Herein, we describe the presence of C. pecorum in sheep imported from New Zealand into Mexico. Genetic analysis of the ompA gene showed that the isolates belong to genotype O and are related to strains isolated from sheep, cattle, and koalas. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-022-00682-9.

The influenza A virus (IAV) H1N1pdm09 induces exacerbated inflammation, contributing to disease complications. Inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), favor an inflammatory response that aids viral replication and survival. A pathway by which spontaneous TNF-α production occurs involves either the reduction of Siglec-3 (CD33) levels or the absence of its ligand, sialic acid. Influenza virus uses sialic acid to enter cells by reducing their expression; however, the role of CD33 in IAV H1N1pdm09 stimulation and its relationship with inflammation have not yet been studied. To evaluate the role of CD33 in proinflammatory cytokine production in IAV H1N1pdm09 stimulation, peripheral blood mononuclear cells from healthy subjects were incubated with IAV H1N1pdm09. We observed that the infection caused an increase in the mRNA expression of proinflammatory cytokines such as TNF-α, interleukin (IL)-1β, and IL-6 and a significant reduction in CD33 expression by monocytes at an early stage of infection. Additionally, suppressor of cytokine signaling 3 (SOCS-3) mRNA expression was upregulated at 6 h, and reactive oxygen species (ROS) production increased at 1.5 h. Moreover, a significant reduction in CD33 expression on the cell surface of monocytes from influenza patients or of IAV H1N1pdm09-stimulated monocytes incubated in vitro was observed by flow cytometry. The results suggest that the decrease in CD33 and increase of SOCS-3 expression induced by IAV H1N1pdm09 triggered TNF-α secretion and ROS production, suggesting an additional way to exacerbate inflammation during viral infection. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-021-00663-4.

In recent years, Bovine herpesvirus 4 (BoHV-4) has emerged as an attractive gene delivery viral vector, mainly for vaccination purposes in the veterinary field. In the present study, a new infectious clone of the BoHV-4 genome carrying a bacterial artificial chromosome vector (BoHV-4-BAC) was developed by homologous recombination in mammalian cell culture and bacterial systems, and exploited to express a truncated form of glycoprotein D (tgD) of Bovine herpesvirus 1 (BoHV-1) (BoHV-4-tgD∆TK) as a vaccine candidate. This construct's immunogenicity was compared to a DNA vector expressing the same antigen (pC-tgD) in a BALB/c mouse model. After the mice were immunized, total and specific antibody responses, cytokine responses, total splenocyte cells proliferation/cytotoxicity, and virus neutralization assays were conducted to analyze the immune response elicited by both constructs. Mice from both vaccine groups developed significant humoral and cellular immune responses after a booster dose regime was conducted on day 28 post-injection. In almost all immunological assays, BoHV-4-tgDΔTK induced as high an immune response as pC-tgD. In both vaccine constructs, neutralizing antibodies were a significant determining factor in protection against BoHV-1, even after the first injection. We conclude that a BoHV-4-based viral vector offers an effective immunization strategy as an alternative to DNA-based immunization platforms, at least to combat BoHV-1.

Porcine parvovirus (PPV) infection is one of the most important causes of reproductive failure in pigs impacting the piggery industry globally with huge economic losses. A cost-effective, simple, rapid, specific, and sensitive method is critical for monitoring PPV infection on pig farms. The main aim of the present study was to develop and evaluate a loop-mediated isothermal amplification (LAMP) assay for rapid visual detection of porcine parvovirus (PPV) in pigs. A set of six LAMP primers including two outer primers, two inner primers, and two loop primers were designed utilizing the conserved region of capsid protein VP2 gene sequences of PPV and was applied for detection of PPV from porcine samples. Time and temperature conditions for amplification of PPV genes were optimized to be 30 min at 63 °C. The developed assay was ten-fold more sensitive than conventional PCR with analytical sensitivity of 20 pg and 200 pg, respectively. This is the first report of detection of PPV by LAMP assay from India. The assay did not cross-react with porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), or classical swine fever virus (CSFV). The LAMP assay was assembled into a LAMP assay kit of 20 reactions and was validated in different laboratories in India. The newly developed LAMP assay was proved to be a specific, sensitive, rapid, and simple method for visual detection of PPV which does not require even costly equipments for performing the test. It complements and extends previous methods for PPV detection and provides an alternative approach for detection of PPV.

Distillation of fermented sugarcane juice produces both rum and cachaça, significant sources of revenue in Brazil and elsewhere. In this study, we provide a genomic analysis of a Saccharomyces cerevisiae strain isolated from a cachaça distillery in Brazil. We determined the complete genome sequence of a strain with high flocculation capacity, high tolerance to ethanol, osmotic and heat shock stress and high fermentation rates and compared the sequence with that of the reference S288c genome as well as those of two other cachaça strains. Single-nucleotide polymorphism analysis identified alterations in genes involved in nitrogen and organic compound metabolism, integrity of organelles and ion homeostasis. The strain exhibited fragmentation of several flocculation genes relative to the reference genome, as well as loss of a stop codon in the FLO8 gene, which encodes a transcription factor required for FLO gene expression. The strain contained no genes not present in the reference genome strain but did lack several genes, including asparaginase genes, maltose utilization loci, and several genes from the tandem array of the DUP240 family. The three cachaça strains lacked different sets of genes, but the asparaginase genes and several of the DUP240 genes were common deficiencies. This study provides new insights regarding the selective pressure of sugarcane fermentation on the genome of yeast strains and offers additional genetic resources for modern synthetic biology and genome editing tools. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-021-00444-z.

BackgroundSince the emergence of the COVID-19, health officials have struggled to devise strategies to counteract the speed of the pandemic’s spread across the globe. It became imperative to implement accurate diagnostic tests for the detection of SARS-CoV-2 RNA on respiratory samples. In many places, however, besides the limited availability of test reagents, laboratory personnel face the challenge of adapting their working routines to manipulate highly infective clinical samples. Here, we proposed the use of a virus-inactivating solution as part of a sample collection kit to decrease the infectious potential of the collected material without affecting the integrity of RNA samples used in diagnostic tests based on RT-qPCR.MethodsNasopharyngeal and oropharyngeal swab samples were collected from SARS-CoV-2-infected patients and from laboratory personnel using a commercially available viral transport solution (VTM) and the denaturing solution (DS) described here. RNA extracted from all samples was tested by RT-qPCR using probes for viral and human genes. Exposure of laboratory personnel to infective viruses was also accessed using ELISA tests.FindingsThe use of the DS did not interfere with the detection of viral genome or the endogenous human mRNA, since similar results were obtained from samples collected with VTM or DS. In addition, all tests of laboratory personnel for the presence of viral RNA and IgG antibodies against SARS-CoV-2 were negative.InterpretationThe methodology described here provides a strategy that allow high diagnostic accuracy as well as safe manipulation of clinical samples by those involved with diagnostic procedures.FundingCAPES, FAPEMIG, CNPq, MCTIC, FIOCRUZ and the UK Global Challenges Research Fund (GCRF).

Flagellar-mediated motility is a crucial virulence factor in many bacterial species. A dual flagellar system has been described in aeromonads; however, there is no flagella-related study in the emergent human pathogen Aeromonas dhakensis. Using 46 clinical A. dhakensis, phenotypic motility, genotypic characteristics (flagellar genes and sequence types), biochemical properties and their relationship were investigated in this study. All 46 strains showed swimming motility at 30 °C in 0.3% Bacto agar and carried the most prevalent 6 polar flagellar genes cheA, flgE, flgG, flgH, flgL, and flgN. On the contrary, only 18 strains (39%) demonstrated swarming motility on 0.5% Eiken agar at 30 °C and they harbored 11 lateral flagellar genes lafB, lafK, lafS, lafT, lafU, flgCL, flgGL, flgNL, fliEL, fliFL, and fliGL. No association was found between biochemical properties and motility phenotypes. Interestingly, a significant association between swarming and strains isolated from pus was observed (p = 0.0171). Three strains 187, 277, and 289 isolated from pus belonged to novel sequence types (ST522 and ST524) exhibited fast swimming and swarming profiles, and they harbored > 90% of the flagellar genes tested. Our findings provide a fundamental understanding of flagellar-mediated motility in A. dhakensis. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-021-00457-8.

We described the complete genome sequence of a novel baculovirus isolate of species Buzura suppressaria nucleopolyhedrovirus, called by isolate CNPSo-25. The occlusion bodies were found to be polyhedral in shape and to contain virions with singly embedded nucleocapsids. The size of the genome is 121,377 bp with a G+C content of 36.7%. We annotated 131 ORFs that cover 90.42% of the genome. Moreover, phylogenetic inference indicated that CNPSo-25 is a member of genus Alphabaculovirus that clustered together with two other Chinese isolates of the same species. We called the virus by Biston suppressaria nucleopolyhedrovirus isolate CNPSo-25 (BisuNPV-CNPSo-25), as Buzura was placed inside the lepidopteran genus Biston. As expected, we detected intra-population variability in the virus sample when the novel isolate was compared to the Chinese isolates: 292 single nucleotide variants were found in the genome, with 181 affecting the protein product. The closest representatives of other species to BisuNPV-CNPSo-25 was found to be Sucra jujuba nucleopolyhedrovirus and Hyposidra talaca nucleopolyhedrovirus, two other virus isolates of geometrid caterpillars. The study of baculovirus genomes is of importance for the development of tools for insect pest biological control and biotechnology. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-020-00402-1.

Foodborne viruses are becoming a global concern as they overwhelm the health system and have the potential to spread globally. Among them, some genotypes of hepatitis E virus (HEV), which is one of the main causes of acute hepatitis in humans, have a zoonotic potential and can be found in foods of animal origin. Infected farm animals are a possible source of the virus, either by direct contact with animal excreta or meat. In the present study, 240 bovine liver samples from slaughter carried out in Rio Grande do Sul (RS), Brazil, were analyzed and tested for the presence of HEV. After performing PCR, 5.4% of positive samples were observed. One of the samples could be identified by molecular phylogenetic analysis as belonging to genotype 3, for which pigs are natural reservoirs, but has not been reported in bovine meat and products so far.

Persisters are phenotypic variants of the bacterial population that survive against lethal doses of bactericidal antibiotics.These phenotypes are created in numerous bacterial species, including those of clinical significance, such as Salmonella Typhimurium. Since persister cells are associated with the failure of antibiotic treatment and infection recurrence, it is crucial to identify the mechanisms that influence the formation of these cells. The aim of this study is to investigate the persister cell formation and expression analysis as well as to predict the 3D structure of the genes involved in the production of persister cells. The presence of persisters in S. Typhimurium was determined by time dependent killing of different types of bactericidal antibiotics and expression of genes associated with persister cell formation which was assessed five hours after the addition of antibiotics by the qRT-PCR. Indeed, the 3D structural model of the proteins studied was predicted by performing several computational methods of retrieved primary protein sequences. The results of the study showed that the S. Typhimurium produced high levels of persister cells in the exposure of bactericidal antibiotics. Furthermore, qRT-PCR resulted in the fact that the expression of related genes was different depending on the type of antibiotic. Overall, this study provides information on the creation of persister cells and the role of different genes in the formation of these cells and structure of proteins involved in the production of persister cells in S. Typhimurium.

Kosmotoga olearia TBF 19.5.1 is a typical thermophile with optimal growth at 65 °C and also exhibits visible growth at an incredible minimum temperature (20 °C). It is considered an ideal model for investigating the evolutionary transition from thermophiles to mesophiles within Thermotogae. However, knowledge relevant to molecular mechanisms of K. olearia responding to cold shock is still limited. In this study, transcriptomics and proteomics were integrated to investigate the global variations at the transcript and protein level during cold shock in K. olearia. As a result, total 734 differentially expressed genes and 262 differentially expressed proteins were identified. The cold-responsive genes and proteins were associated with signaling transduction, transcription, translation and repair, cell wall/membrane reconstruction, amino acid biosynthesis, and stress response. However, most genes and proteins, involved in carbon metabolism, fatty acid biosynthesis, and energy production, were repressed. This work provides the first integrative transcriptomics and proteomics analyses of the cold shock response in K. olearia, and it offered new insights into the mechanisms of cold adaptation and post-transcriptional regulation of the distinctive thermophile within Thermotogae. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-021-00662-5.

The most common nosocomial fungal infections are caused by several species of Candida, of which Candida glabrata is the second most frequently isolated species from bloodstream infections. C. glabrata displays relatively high minimal inhibitory concentration values (MIC) to the antifungal fluconazole and is associated with high mortality rates. To decrease mortality rates, the appropriate treatment must be administered promptly. C. glabrata contains in its genome several non-identical copies of species-specific sequences. We designed three pairs of C. glabrata-specific primers for endpoint PCR amplification that align to these species-specific sequences and amplify the different copies in the genome. Using these primers, we developed a fast, sensitive, inexpensive, and highly specific PCR-based method to positively detect C. glabrata DNA in a concentration-dependent manner from mixes of purified genomic DNA of several Candida species, as well as from hemocultures and urine clinical samples. This tool can be used for positive identification of C. glabrata in the clinic. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-021-00584-2.

Medically important arboviruses such as dengue virus (DENV), Zika virus (ZIKV), and chikungunya virus (CHIKV) are primarily transmitted by the globally distributed mosquito Aedes aegypti. Increasing evidence suggests that the transmission of some viruses can be influenced by mosquito-specific and mosquito-borne viruses. Advancements in high-throughput sequencing (HTS) and bioinformatics have expanded our knowledge on the richness of viruses harbored by mosquitoes. HTS was used to characterize the presence of virus sequences in wild-caught adult Ae. aegypti from Tocantins (TO) state, Brazil. Samples of mosquitoes were collected in four cities of Tocantins state and submitted to RNA isolation, followed by sequencing at an Illumina HiSeq platform. Our results showed initially by Krona the presence of 3% of the sequenced reads belonging to the viral database. After further analysis, the virus sequences were found to have homology to two viral families found in insects Phenuiviridae and Metaviridae. Three possible viral strains including putative new viruses were detected and named Phasi Charoen-like phasivirus isolate To-1 (PCLV To-1), Aedes aegypti To virus 1 (AAToV1), and Aedes aegypti To virus 2 (AAToV2). The results presented in this work contribute to the growing knowledge about the diversity of viruses in mosquitoes and might be useful for future studies on the interaction between insect-specific viruses and arboviruses. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-021-00632-x.

NADPH oxidases are enzymes that have been reported to generate reactive oxygen species (ROS) in animals, plants and many multicellular fungi in response to environmental stresses. Six genes of the NADPH oxidase complex components, including vvnoxa, vvnoxb, vvnoxr, vvbema, vvrac1 and vvcdc24, were identified based on the complete genomic sequence of the edible fungus Volvariella volvacea. The number of vvnoxa, vvrac1, vvbema and vvcdc24 transcripts fluctuated with ageing, and the gene expression patterns of vvnoxa, vvrac1 and vvbema were significantly positively correlated. However, the expression of vvnoxb and vvnoxr showed no significant difference during ageing. In hyphae subjected to mechanical injury stress, both O(2)(−) and H(2)O(2) concentrations were increased. The expression of vvnoxa, vvrac1, vvbema and vvcdc24 was substantially upregulated, but vvnoxb and vvnoxr showed no response to mechanical injury stress at the transcriptional level. Additionally, the transcription of vvnoxa, vvrac1, vvbema and vvcdc24 could be repressed when the intracellular ROS were eliminated by diphenyleneiodonium (DPI) chloride and reduced glutathione (GSH) treatments. These results indicated a positive feedback loop involving NADPH oxidase and intracellular ROS, which might be the reason for the oxidative burst during injury stress.

Recombinant virus vectors represent a promising strategy for vaccine research. Among available viral vectors, members of the Poxviridae family—especially the modified Vaccinia virus Ankara (MVA)—stand out as immunogenic and safe vaccine platforms. Because MVA usually does not produce plaques in cell culture, visible selection markers such as the green fluorescent protein (GFP) are frequently incorporated into the constructions in order to facilitate the recognition of recombinants. However, these genetic markers have to be removed before any clinical trial. Here, we evaluated the acute responses generated in mice immunized with a MVA vector in which the GFP marker was not removed. We observed no differences in neutrophil, monocyte, or total leucocyte recruitment among animals inoculated with MVA or MVA-GFP. Likewise, there were no differences in neutrophil activation between mice groups. Hepatic functions were not altered in either MVA or MVA-GFP-inoculated mice, and we observed no histopathological alterations in different tissues from virus-inoculated animals. In conclusion, the presence of GFP is innocuous to immunized animals and do not alter acute physiopathological responses to the MVA vector. We suggest that keeping the GFP marker may be a good strategy for vaccine development, production, and evaluation.

The genus Xanthomonas comprises Gram-negative bacteria, many of which are phytopathogens. Xanthomonas fuscans subsp. fuscans is one of the most devastating pathogens affecting the bean plant, resulting in the common bacterial blight of bean (CBB). The disease is mainly foliar and affects a wide variety of bean species, thus acting as the yield-limiting factor for the bean crop. Here, we report the whole-genome sequencing of a new strain of X. fuscans subsp. fuscans, named Xff49, isolated from the infected and symptomatic beans from Capão do Leão, Southern Brazil. The genetic analysis demonstrated the presence of single-nucleotide variants (SNVs) in this strain, potentially affecting the mobilome, cell mobility, and inorganic ion metabolism. In addition, the analysis resulted in the identification of a new plasmid similar to the pAX22 derived from Achromobacter denitrificans, which was named plX, along with plA and plC, previously reported in other strains of X. fuscans subsp. fuscans. Xff49 represents the first Brazilian genome of X. fuscans subsp. fuscans and might provide useful information applicable to the studies of phylogenetics, evolution, and pathogenomics, thereby allowing a better understanding of the genomic features present in the Brazilian strains. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42770-019-00050-0) contains supplementary material, which is available to authorized users.

Cowpea (Vigna unguiculata L.) is a legume species that considerably benefits from inoculation with nitrogen fixing bacteria of the genus Bradyrhizobium. One of the strains recommended for inoculation in cowpea in Brazil is UFLA03-84 (Bradyrhizobium sp.). The aim of our study was to define the taxonomic position of the UFLA03-84 strain and of two other strains of Bradyrhizobium (UFLA03-144 and INPA237B), all belonging to the same phylogenetic group and isolated from soils of the Brazilian Amazon. Multilocus sequence analysis (MLSA) of the housekeeping genes atpD, gyrB, recA, and rpoB grouped (with similarity higher than 99%) the three strains with Bradyrhizobium viridifuturi SEMIA 690(T). The analyses of average nucleotide identity and digital DNA–DNA hybridization supported classification of the group as Bradyrhizobium viridifuturi. The three strains exhibited similar behavior in relation to the most of the phenotypic characteristics evaluated. However, some characteristics exhibited variation, indicating phenotypic diversity within the species. Phylogenetic analysis of the nodC and nifH genes showed that the three strains are members of the same symbiovar (tropici) that contains type strains of Bradyrhizobium species coming from tropical soils (SEMIA 690(T)B. viridifuturi, CNPSo 1112(T)B. tropiciagri, CNPSo 2833(T)B. embrapense, and B. brasilense UFLA03-321(T)). ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42770-019-00045-x) contains supplementary material, which is available to authorized users.

INTRODUCTION: Several arboviruses causing human disease have been reported in Brazil. In nature, arboviruses maintain a lifecycle involving vertebrates and vectors, which may contribute for periodical reemergence of those of public health concern in tropical regions, as Mato Grosso State (MT). In this study, we searched for arboviruses in mosquito body pools sampled during the rainy season of 2018 in 21 bird watching points of Cuiabá and Varzea Grande, South Central MT. METHODS: In total, 2873 (57%) males and 2167 (43%) females belonging to six urban and sylvatic mosquito genera allocated to 398 pools were subjected to RNA extraction and RT-PCR for arboviruses. Positive pools were subjected to virus isolation in C6/36 cells. RESULTS: A total of 102/398 pools, 66/233 (29.6%) of females, and 36/165 (21.8%) of males, mostly sampled in May (31/102), were positive for arboviruses. Chikungunya virus (CHIKV) was distributed in 19 points, Zika virus (ZIKV) was found in 14 points, Mayaro virus (MAYV) in 10 points, and East Equine encephalitis virus (EEEV) in three points. Culex quinquefasciatus pools (39/89 of females and 24/99 of males) were positive for CHIKV, ZIKV, and MAYV; Aedes (Stg) aegypti pools (11/46 of females and 12/33 of males) for CHIKV, ZIKV, MAYV, and EEEV; Aedes albopictus female pools (8/29) for CHIKV, ZIKV, and EEEV; and Psorophora albigenu (2/12) and Psorophora ferox female pools (4/16) for CHIKV. CONCLUSIONS: Arbovirus molecular detection in mosquito populations varies considerable between geographical regions and epidemics, influenced by genetic characteristics and microbiome interference on virus replication. Although infected females are responsible for the transmission to vertebrates during bloodfeeding, male infection by CHIKV, ZIKV, and MAYV resultant from vertical route could lead to interepidemic maintenance of these arboviruses in their natural reservoirs. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-021-00646-5.

Heterologous hosts are highly important to detect the expression of biosynthetic gene clusters that are cryptic or poorly expressed in their natural hosts. To investigate whether actinorhodin-overproducer Streptomyces coelicolor ∆ppk mutant strain could be a possible prototype as a heterologous expression host, a cosmid containing most of the elm gene cluster of Streptomyces olivaceus Tu2353 was integrated into chromosomes of both S. coelicolor A3(2) and ∆ppk strains. Interestingly, it was found that the production of tetracyclic polyketide 8-demethyl-tetracenomycin (8-DMTC) by recombinant strains caused significant changes in the morphology of cells. All the pellets and clumps were disentangled and mycelia were fragmented in the recombinant strains. Moreover, they produce neither pigmented antibiotics nor agarase and did not sporulate. By eliminating the elm biosynthesis genes from the cosmid, we showed that the morphological properties of recombinants were caused by the production of 8-DMTC. Extracellular application of 8-DMTC on S. coelicolor wild-type cells caused a similar phenotype with the 8-DMTC-producing recombinant strains. The results of this study may contribute to the understanding of the effect of 8-DMTC in Streptomyces since the morphological changes that we have observed have not been reported before. It is also valuable in that it provides useful information about the use of Streptomyces as hosts for the heterologous expression of 8-DMTC.

Four hundred endophytic fungi isolates with different colony morphologies were isolated from roots of Hordeum vulgare L. collected from un-engineered landfills (the measured cadmium was 0.9 mg kg(−1)) of Kermanshah province in West Iran. Based on morphology and phylogeny of DNA sequence data for the internal transcribed spacer (ITS) rDNA and comparing the sequences with that available in NCBI database, 11 isolates are identified as dark septate endophytes (DSE) including Alternaria alternata, Microdochium bolleyi, Bipolaris zeicola, Alternaria sp., and Pleosporales sp., and the other nine are not dark septate endophytes (non-DSE) including Fusarium redolens, Fusarium tricinctum, Fusarium monliforme, Clonostachys rosea, and Epicoccum nigrum. Tolerance of DSE and non-DSE strains for Cd were investigated in potato dextrose agar medium. Minimum inhibitory concentrations (MIC) of Cd from nitrate salt source (Cd (NO(3))(2)) and EC(50) were determined. The means of MIC and EC(50) values for DSE fungi species were 1254.5 and 209.74 mg/kg, compared to 800 and 150.3 mg/kg for non-DSEs. Among the endophytic fungi isolated, Alternaria sp. (TBR5) and Bipolaris zeicola (Tw26) showed the highest tolerance to Cd with a MIC value of 2000 mg/L and 1800 mg/L, respectively. Barley plants were inoculated with TBR5 and Tw26 in Cd-added sands (0, 10, 30, 60 mg Cd/kg sand). In terms of Cd accumulation, our results showed that TBR5 and Tw26 inoculation increased the amount of Cd in the barley roots. TBR5 and Tw26 significantly improved (p < 0.05) plant growth in the presence of Cd by enhancing plant growth attributes such as chlorophyll content, root weight, plant length, fresh weight, and dry weight of plants. This is the first study on the abundance and identification of endophytic root fungi of barley in a cadmium-contaminated soil in Iran. The results of this study showed that DSE and non-DSE have the potential to improve the efficiency of phytoremediation.

The fungus Paracoccidioides lutzii is one of the species of the Paracoccidioides genus, responsible for a neglected human mycosis, endemic in Latin America, the paracoccidioidomycosis (PCM). In order to survive in the host, the fungus overcomes a hostile environment under low levels of oxygen (hypoxia) during the infectious process. The hypoxia adaptation mechanisms are variable among human pathogenic fungi and worthy to be investigated in Paracoccidoides spp. Previous proteomic results identified that P. lutzii responds to hypoxia and it has a functional homolog of the SrbA transcription factor, a well-described hypoxic regulator. However, the direct regulation of genes by SrbA and the biological processes it governs while performing protein interactions have not been revealed yet. The goal of this study was to demonstrate the potential of SrbA targets genes in P. lutzii. In addition, to show the SrbA three-dimensional aspects as well as a protein interaction map and important regions of interaction with predicted targets. The results show that SrbA-regulated genes were involved with several biological categories, such as metabolism, energy, basal processes for cell maintenance, fungal morphogenesis, defense, virulence, and signal transduction. Moreover, in order to investigate the SrbA’s role as a protein, we performed a 3D simulation and also a protein-protein network linked to this hypoxic regulator. These in silico analyses revealed relevant aspects regarding the biology of this pathogen facing hypoxia and highlight the potential of SrbA as an antifungal target in the future. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-021-00527-x.

Candida spp. are the main causative agents of invasive fungal infections in immunocompromised patients. Candidemia has attributable mortality rates of 15 to 35% and increases hospitalisation time and costs, thus making this disease a public health concern. This study aimed to use pulsed-field gel electrophoresis (PFGE), microsatellite length polymorphism (MLP) and multilocus sequence typing (MLST) to analyse the genetic relationships among 65 Candida spp. bloodstream isolates, including 35 Candida albicans, 15 Candida glabrata and 15 Candida tropicalis isolates, all of which were obtained from patients in a Brazilian hospital. Moreover, patient clinical data were assessed. All techniques resulted in high discriminatory indexes. C. albicans and C. tropicalis isolates showed high genetic variability, while C. glabrata isolates had relatively low genetic variability. Moreover, a cluster of C. glabrata isolates was identified in a hospital unit. New MLST sequence types, diploid sequence types and alleles are described. Relationships were not observed between the molecular typing results and clinical characteristics. The molecular typing of clinical strains increases our understanding of candidemia epidemiology and promotes the development of strategies that can reduce the incidence of this disease. Moreover, this study is the first to combine these techniques to genotype these three species in Brazil. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-021-00446-x.

The aim of the present study was to evaluate the genetic diversity of Ehrlichia canis in naturally infected dogs from six mesoregions of Rio de Janeiro state. E. canis was diagnosed with a real-time polymerase chain reaction (qPCR) targeting a 93 base pair (bp) fragment of the 16S rDNA gene. To evaluate the genetic diversity of the parasite, we amplified a positive sample from each mesoregion by distinct conventional PCR assays with targets in the gp19 (414 bp), gp36 (814 bp), and p28 (843 bp) genes. A total of 267 samples were collected from dogs in Rio de Janeiro state. Among the samples analyzed, 42.3% (n = 113/267) were 16S rDNA-qPCR positive. When performing PCR for the gp19 and gp36 genes, 100% (n = 113/113) and 5.3% (n = 6/113) of the samples amplified fragments of 414 bp and 814 bp, respectively. The six PCR-positive samples for the gp36 gene also amplified the p28 gene fragment. The characterization based on the gp19 gene demonstrated that it is highly conserved. In protein analysis (TRP36), all samples showed a tandem repeat protein (TRP) that comprised 11 replicates. Seven high-entropy amino acid sites were distributed throughout the gp36 gene. Eleven high-entropy amino acid sites were found throughout the p28 gene. There is a positive selection pressure in both genes (p ≤ 0.05). Comparing and characterizing an organism are useful for providing important information about the host’s immune response and identifying new antigenic targets, as well as essential characteristics for the development of vaccines and new diagnostic tools.

Sendai virus (SeV) has been used as a model strain to reveal molecular features of paramyxovirus biology. In this study, we comprehensively analyzed the gene profiling of murine macrophages and airway epithelial cells in response to SeV using gene expression data. The significantly differentially expressed genes (DEGs) were screened by GEO2R. Gene ontology (GO) and pathway enrichment analyses were performed by DAVID. The protein-protein interaction (PPI) map of DEGs was constructed by STRING. The modules of PPI network are produced by molecular complex detection (MCODE) plug-in of Cytoscape. In total, 241 up- and 83 downregulated DEGs were identified in airway epithelial cells while 130 up- and 148 downregulated in macrophage. Particularly, Tmem119 and Colla2 are significantly downregulated in airway epithelial cells and macrophages, respectively. Functional enrichment analysis showed that upregulated DEGs are clustered in innate immunity and inflammatory response in both cell types, whereas downregulated DEGs are involved in host metabolic pathway in airway epithelial cells. PI3K-AKT signaling pathway is downregulated in macrophages. PPI network analysis indicated that some high degree of nodes exist in both cell types, such as Stat1, Tnf, and Cxcl10. In conclusion, SeV infection can induce different host cell responses in airway epithelial cells and macrophages. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42770-018-0021-6) contains supplementary material, which is available to authorized users.