Database of all currently known mobile colistin resistance genes.

We created a database of all currently known mobile colistin resistance genes and variants (n = 115). It contains accession numbers of the gene and protein sequences, mutations between the protein variants and the main proteins, and additional metadata. It is accompanied by all genetic and protein sequences as two aggregated FASTA files.

Predicting ß-lactam susceptibility from the genome of Streptococcus pneumoniae and other mitis group streptococci.

Introduction: For Streptococcus pneumoniae, ß-lactam susceptibility can be predicted from the amino acid sequence of the penicillin-binding proteins PBP1a, PBP2b, and PBP2x. The combination of PBP-subtypes provides a PBP-profile, which correlates to a phenotypic minimal inhibitory concentration (MIC). The non-S. pneumoniae Mitis-group streptococci (MGS) have similar PBPs and exchange pbp-alleles with S. pneumoniae. We studied whether a simple BLAST analysis could be used to predict phenotypic susceptibility in Danish S. pneumoniae isolates and in internationally collected MGS. Method: Isolates with available WGS and phenotypic susceptibility data were included. For each isolate, the best matching PBP-profile was identified by BLAST analysis. The corresponding MICs for penicillin and ceftriaxone was retrieved. Category agreement (CA), minor-, major-, and very major discrepancy was calculated. Genotypic-phenotypic accuracy was examined with Deming regression. Results: Among 88 S. pneumoniae isolates, 55 isolates had a recognized PBP-profile, and CA was 100% for penicillin and 98.2% for ceftriaxone. In 33 S. pneumoniae isolates with a new PBP-profile, CA was 90.9% (penicillin) and 93.8% (ceftriaxone) using the nearest recognized PBP-profile. Applying the S. pneumoniae database to non-S. pneumoniae MGS revealed that none had a recognized PBP-profile. For Streptococcus pseudopneumoniae, CA was 100% for penicillin and ceftriaxone in 19 susceptible isolates. In 33 Streptococcus mitis isolates, CA was 75.8% (penicillin) and 86.2% (ceftriaxone) and in 25 Streptococcus oralis isolates CA was 8% (penicillin) and 100% (ceftriaxone). Conclusion: Using a simple BLAST analysis, genotypic susceptibility prediction was accurate in Danish S. pneumoniae isolates, particularly in isolates with recognized PBP-profiles. Susceptibility was poorly predicted in other MGS using the current database.

One Day in Denmark: Comparison of Phenotypic and Genotypic Antimicrobial Susceptibility Testing in Bacterial Isolates From Clinical Settings.

Antimicrobial susceptibility testing (AST) should be fast and accurate, leading to proper interventions and therapeutic success. Clinical microbiology laboratories rely on phenotypic methods, but the continuous improvement and decrease in the cost of whole-genome sequencing (WGS) technologies make them an attractive alternative. Studies evaluating the performance of WGS-based prediction of antimicrobial resistance (AMR) for selected bacterial species have shown promising results. There are, however, significant gaps in the literature evaluating the applicability of WGS as a diagnostics method in real-life clinical settings against the range of bacterial pathogens experienced there. Thus, we compared standard phenotypic AST results with WGS-based predictions of AMR profiles in bacterial isolates without preselection of defined species, to evaluate the applicability of WGS as a diagnostics method in clinical settings. We collected all bacterial isolates processed by all Danish Clinical Microbiology Laboratories in 1 day. We randomly selected 500 isolates without any preselection of species. We performed AST through standard broth microdilution (BMD) for 488 isolates (n = 6,487 phenotypic AST results) and compared results with in silico antibiograms obtained through WGS (Illumina NextSeq) followed by bioinformatics analyses using ResFinder 4.0 (n = 5,229 comparisons). A higher proportion of AMR was observed for Gram-negative bacteria (10.9%) than for Gram-positive bacteria (6.1%). Comparison of BMD with WGS data yielded a concordance of 91.7%, with discordant results mainly due to phenotypically susceptible isolates harboring genetic AMR determinants. These cases correspond to 6.2% of all isolate-antimicrobial combinations analyzed and to 6.8% of all phenotypically susceptible combinations. We detected fewer cases of phenotypically resistant isolates without any known genetic resistance mechanism, particularly 2.1% of all combinations analyzed, which corresponded to 26.4% of all detected phenotypic resistances. Most discordances were observed for specific combinations of species-antimicrobial: macrolides and tetracycline in streptococci, ciprofloxacin and ß-lactams in combination with ß-lactamase inhibitors in Enterobacterales, and most antimicrobials in Pseudomonas aeruginosa. WGS has the potential to be used for surveillance and routine clinical microbiology. However, in clinical microbiology settings and especially for certain species and antimicrobial agent combinations, further developments in AMR gene databases are needed to ensure higher concordance between in silico predictions and expected phenotypic AMR profiles.

Comprehensive analysis of DNA replication timing across 184 cell lines suggests a role for MCM10 in replication timing regulation.

Cellular proliferation depends on the accurate and timely replication of the genome. Several genetic diseases are caused by mutations in key DNA replication genes; however, it remains unclear whether these genes influence the normal program of DNA replication timing. Similarly, the factors that regulate DNA replication dynamics are poorly understood. To systematically identify trans-acting modulators of replication timing, we profiled replication in 184 cell lines from three cell types, encompassing 60 different gene knockouts or genetic diseases. Through a rigorous approach that considers the background variability of replication timing, we concluded that most samples displayed normal replication timing. However, mutations in two genes showed consistently abnormal replication timing. The first gene was RIF1, a known modulator of replication timing. The second was MCM10, a highly conserved member of the pre-replication complex. Cells from a single patient carrying MCM10 mutations demonstrated replication timing variability comprising 46% of the genome and at different locations than RIF1 knockouts. Replication timing alterations in the mutated MCM10 cells were predominantly comprised of replication delays and initiation site gains and losses. Taken together, this study demonstrates the remarkable robustness of the human replication timing program and reveals MCM10 as a novel candidate modulator of DNA replication timing.

One Day in Denmark: Nationwide point-prevalence survey of human bacterial isolates and comparison of classical and whole-genome sequence-based species identification methods.

OBJECTIVES: Implementing whole-genome sequencing (WGS) technologies in clinical microbiology laboratories can increase the amount and quality of information available for healthcare practitioners. In this study, we analysed the applicability of this method and determined the distribution of bacterial species processed in clinical settings in Denmark. METHODS: We performed a point-prevalence study of all bacterial isolates (n = 2,009) processed and reported in the Clinical Microbiology Laboratories in Denmark in one day in January 2018. We compared species identification as performed by classical methods (MALDI-TOF) and by bioinformatics analysis (KmerFinder and rMLST) of WGS (Illumina NextSeq) data. We compared the national point-prevalence of bacterial isolates observed in clinical settings with the research attention given to those same genera in scientific literature. RESULTS: The most prevalent bacterium was Escherichia coli isolated from urine (n = 646), followed by Staphylococcus spp. from skin or soft tissues (n = 197). The distribution of bacterial species throughout the country was not homogeneous. We observed concordance of species identification for all methods in 95.7% (n = 1,919) of isolates, furthermore obtaining concordance for 99.7% (n = 1,999) at genus level. The number of scientific publications in the country did not correlate with the number of bacterial isolates of each genera analysed in this study. CONCLUSIONS: WGS technologies have the potential to be applied in clinical settings for routine diagnostics purposes. This study also showed that bioinformatics databases should be continuously improved and results from local point-prevalence surveys should not be applied at national levels without previously determining possible regional variations.

Danish Whole-Genome-Sequenced Candida albicans and Candida glabrata Samples Fit into Globally Prevalent Clades.

Candida albicans and Candida glabrata are opportunistic fungal pathogens with increasing incidence worldwide and higher-than-expected prevalence in Denmark. We whole-genome sequenced yeast isolates collected from Danish Clinical Microbiology Laboratories to obtain an overview of the Candida population in the country. The majority of the 30 C. albicans isolates were found to belong to three globally prevalent clades, and, with one exception, the remaining isolates were also predicted to cluster with samples from other geographical locations. Similarly, most of the eight C. glabrata isolates were predicted to be prevalent subtypes. Antifungal susceptibility testing proved all C. albicans isolates to be susceptible to both azoles and echinocandins. Two C. glabrata isolates presented azole-resistant phenotypes, yet all were susceptible to echinocandins. There is no indication of causality between population structure and resistance phenotypes for either species.

Phenotypic and genotypic characterization of mcr-1-positive multidrug-resistant Escherichia coli ST93, ST117, ST156, ST10, and ST744 isolated from poultry in Poland.

BACKGROUND: A plasmid-mediated mechanism of bacterial resistance to polymyxin is a serious threat to public health worldwide. The present study aimed to determine the occurrence of plasmid-mediated colistin resistance genes and to conduct the molecular characterization of mcr-positive Escherichia coli strains isolated from Polish poultry. METHODS: In this study, 318 E. coli strains were characterized by the prevalence of mcr1-mcr5 genes, antimicrobial susceptibility testing by minimal inhibitory concentration method, the presence of antimicrobial resistance genes was screened by PCR, and the biofilm formation ability was tested using the crystal violet staining method. Genetic relatedness of mcr-1-positive E. coli strains was evaluated by multilocus sequence typing method. RESULTS: Among the 318 E. coli isolates, 17 (5.35%) harbored the mcr-1 gene. High antimicrobial resistance rates were observed for ampicillin (100%), tetracycline (88.24%), and chloramphenicol (82.35%). All mcr-1-positive E. coli strains were multidrug-resistant, and as many as 88.24% of the isolates contained the blaTEM gene, tetracycline (tetA and tetB), and sulfonamide (sul1, sul2, and sul3) resistance genes. Additionally, 41.18% of multidrug-resistant, mcr-1-positive E. coli isolates were moderate biofilm producers, while the rest of the strains showed weak biofilm production. Nine different sequence types were identified, and the dominant ST was ST93 (29.41%), followed by ST117 (17.65%), ST156 (11.76%), ST 8979 (11.76%), ST744 (5.88%), and ST10 (5.88%). Moreover, the new ST was identified in this study. CONCLUSIONS: Our results showed a low occurrence of mcr-1-positive E. coli strains isolated from Polish poultry; however, all the isolated strains were resistant to multiple antimicrobial agents and were able to form biofilms at low or medium level.

ResFinder 4.0 for predictions of phenotypes from genotypes.

OBJECTIVES: WGS-based antimicrobial susceptibility testing (AST) is as reliable as phenotypic AST for several antimicrobial/bacterial species combinations. However, routine use of WGS-based AST is hindered by the need for bioinformatics skills and knowledge of antimicrobial resistance (AMR) determinants to operate the vast majority of tools developed to date. By leveraging on ResFinder and PointFinder, two freely accessible tools that can also assist users without bioinformatics skills, we aimed at increasing their speed and providing an easily interpretable antibiogram as output. METHODS: The ResFinder code was re-written to process raw reads and use Kmer-based alignment. The existing ResFinder and PointFinder databases were revised and expanded. Additional databases were developed including a genotype-to-phenotype key associating each AMR determinant with a phenotype at the antimicrobial compound level, and species-specific panels for in silico antibiograms. ResFinder 4.0 was validated using Escherichia coli (n = 584), Salmonella spp. (n = 1081), Campylobacter jejuni (n = 239), Enterococcus faecium (n = 106), Enterococcus faecalis (n = 50) and Staphylococcus aureus (n = 163) exhibiting different AST profiles, and from different human and animal sources and geographical origins. RESULTS: Genotype-phenotype concordance was ≥95% for 46/51 and 25/32 of the antimicrobial/species combinations evaluated for Gram-negative and Gram-positive bacteria, respectively. When genotype-phenotype concordance was <95%, discrepancies were mainly linked to criteria for interpretation of phenotypic tests and suboptimal sequence quality, and not to ResFinder 4.0 performance. CONCLUSIONS: WGS-based AST using ResFinder 4.0 provides in silico antibiograms as reliable as those obtained by phenotypic AST at least for the bacterial species/antimicrobial agents of major public health relevance considered.

The Identity of a Single Residue of the RNA-Dependent RNA Polymerase of Grapevine Fanleaf Virus Modulates Vein Clearing in Nicotiana benthamiana.

The mechanisms underlying host plant symptom development upon infection by viruses of the genus Nepovirus in the family Secoviridae, including grapevine fanleaf virus (GFLV), are poorly understood. In the systemic host Nicotiana benthamiana, GFLV strain GHu produces characteristic symptoms of vein clearing in apical leaves, unlike other GFLV strains such as F13, which cause an asymptomatic infection. In this study, we expanded on earlier findings and used reverse genetics to identify residue 802 (lysine, K) of the GFLV-GHu RNA1-encoded RNA-dependent RNA polymerase (1EPol) as a modulator of vein-clearing symptom development in N. benthamiana. Mutations to this site abolished (K to G, A, or Q) or attenuated (K to N or P) symptom expression. Noteworthy, residue 802 is necessary but not sufficient for vein clearing, as GFLV-F13 RNA1 carrying K802 remained asymptomatic in N. benthamiana. No correlation was found between symptom expression and RNA1 accumulation, as shown by reverse transcription-quantitative polymerase chain reaction. Additionally, the involvement of RNA silencing of vein clearing was ruled out by virus-induced gene silencing experiments and structure predictions for protein 1EPol suggested that residue 802 is flanked by strongly predicted stable secondary structures, including a conserved motif of unknown function (805LLKT/AHLK/RT/ALR814). Together, these results reveal the protein nature of the GFLV-GHu symptom determinant in N. benthamiana and provide a solid basis for probing and determining the virus-host proteome network for symptoms of vein clearing.

The Interaction Dynamics of Two Potato Leafroll Virus Movement Proteins Affects Their Localization to the Outer Membranes of Mitochondria and Plastids.

The Luteoviridae is an agriculturally important family of viruses whose replication and transport are restricted to plant phloem. Their genomes encode for four proteins that regulate viral movement. These include two structural proteins that make up the capsid and two non-structural proteins known as P3a and P17. Little is known about how these proteins interact with each other and the host to coordinate virus movement within and between cells. We used quantitative, affinity purification-mass spectrometry to show that the P3a protein of Potato leafroll virus complexes with virus and that this interaction is partially dependent on P17. Bimolecular complementation assays (BiFC) were used to validate that P3a and P17 self-interact as well as directly interact with each other. Co-localization with fluorescent-based organelle markers demonstrates that P3a directs P17 to the mitochondrial outer membrane while P17 regulates the localization of the P3a-P17 heterodimer to plastids. Residues in the C-terminus of P3a were shown to regulate P3a association with host mitochondria by using mutational analysis and also varying BiFC tag orientation. Collectively, our work reveals that the PLRV movement proteins play a game of intracellular hopscotch along host organelles to transport the virus to the cell periphery.

New fluorescent rosamine chelator showing promising antibacterial activity against Gram-positive bacteria.

The restricted number of antibiotics to treat infections caused by common multidrug resistant bacterial pathogens in the clinical setting demands a continuous search for new molecules with antibacterial properties. Bacterial iron deprivation represents a promising alternative, being iron chelators an attractive class for drug design in which particular compounds seem to have antibacterial effect. In this work, we report the synthesis and characterization of a new fluorescent 3-hydroxy-4-pyridinone (3,4-HPO) iron chelator functionalized with a carboxyrosamine fluorophore (MRB20). The antibacterial activity of MRB20 was assessed against representative strains from clinically relevant Gram-positive and Gram-negative bacterial species and further compared with the inhibitory effect of a set of structurally related iron chelators including Deferiprone (1,2-dimethyl-3-hydroxy-4-pyridinone). Compounds exhibiting a promising minimal inhibitory concentration (MIC < 10 mg/L) were further tested against a wider range of bacterial genera and species (Staphylococcus spp. Enterococcus spp. Listeria monocytogenes, Bacillus spp.), including multidrug resistant bacteria. With the exception of the novel compound (MRB20), all chelators inhibited the strains assayed at very high concentrations [minimum inhibitory concentrations (MIC) ranging from 70 mg/L to >180 mg/L]. MRB20 revealed a good antibacterial activity (6.7-13.2 mg/L) against Gram-positive strains from different genera and species, including clinically relevant species (Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecium, Enterococcus faecalis), which might be eventually compatible with a therapeutic application or as adjuvant.

Disruption of Chloroplast Function Through Downregulation of Phytoene Desaturase Enhances the Systemic Accumulation of an Aphid-Borne, Phloem-Restricted Virus.

Chloroplasts play a central role in pathogen defense in plants. However, most studies explaining the relationship between pathogens and chloroplasts have focused on pathogens that infect mesophyll cells. In contrast, the family Luteoviridae includes RNA viruses that replicate and traffic exclusively in the phloem. Recently, our lab has shown that Potato leafroll virus (PLRV), the type species in the genus Polerovirus, forms an extensive interaction network with chloroplast-localized proteins that is partially dependent on the PLRV capsid readthrough domain (RTD). In this study, we used virus-induced gene silencing to disrupt chloroplast function and assess the effects on PLRV accumulation in two host species. Silencing of phytoene desaturase (PDS), a key enzyme in carotenoid, chlorophyll, and gibberellic acid (GA) biosynthesis, resulted in a substantial increase in the systemic accumulation of PLRV. This increased accumulation was attenuated when plants were infected with a viral mutant that does not express the RTD. Application of GA partially suppressed the increase in virus accumulation in PDS-silenced plants, suggesting that GA signaling also plays a role in limiting PLRV infection. In addition, the fecundity of the aphid vector of PLRV was increased when fed on PDS-silenced plants relative to PLRV-infected plants.

Multiplex PCR for detection of plasmid-mediated colistin resistance determinants, mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 for surveillance purposes.

Background and aimPlasmid-mediated colistin resistance mechanisms have been identified worldwide in the past years. A multiplex polymerase chain reaction (PCR) protocol for detection of all currently known transferable colistin resistance genes (mcr-1 to mcr-5, and variants) in Enterobacteriaceae was developed for surveillance or research purposes. Methods: We designed four new primer pairs to amplify mcr-1, mcr-2, mcr-3 and mcr-4 gene products and used the originally described primers for mcr-5 to obtain a stepwise separation of ca 200 bp between amplicons. The primer pairs and amplification conditions allow for single or multiple detection of all currently described mcr genes and their variants present in Enterobacteriaceae. The protocol was validated testing 49 European Escherichia coli and Salmonella isolates of animal origin. Results: Multiplex PCR results in bovine and porcine isolates from Spain, Germany, France and Italy showed full concordance with whole genome sequence data. The method was able to detect mcr-1, mcr-3 and mcr-4 as singletons or in different combinations as they were present in the test isolates. One new mcr-4 variant, mcr-4.3, was also identified. Conclusions: This method allows rapid identification of mcr-positive bacteria and overcomes the challenges of phenotypic detection of colistin resistance. The multiplex PCR should be particularly interesting in settings or laboratories with limited resources for performing genetic analysis as it provides information on the mechanism of colistin resistance without requiring genome sequencing.

ALUMINUM RESISTANCE TRANSCRIPTION FACTOR 1 (ART1) contributes to natural variation in aluminum resistance in diverse genetic backgrounds of rice (O. sativa).

Transcription factors (TFs) regulate the expression of other genes to indirectly mediate stress resistance mechanisms. Therefore, when studying TF-mediated stress resistance, it is important to understand how TFs interact with genes in the genetic background. Here, we fine-mapped the aluminum (Al) resistance QTL Alt12.1 to a 44-kb region containing six genes. Among them is ART1, which encodes a C2H2-type zinc finger TF required for Al resistance in rice. The mapping parents, Al-resistant cv Azucena (tropical japonica) and Al-sensitive cv IR64 (indica), have extensive sequence polymorphism within the ART1 coding region, but similar ART1 expression levels. Using reciprocal near-isogenic lines (NILs) we examined how allele-swapping the Alt12.1 locus would affect plant responses to Al. Analysis of global transcriptional responses to Al stress in roots of the NILs alongside their recurrent parents demonstrated that the presence of the Alt12.1 from Al-resistant Azucena led to greater changes in gene expression in response to Al when compared to the Alt12.1 from IR64 in both genetic backgrounds. The presence of the ART1 allele from the opposite parent affected the expression of several genes not previously implicated in rice Al tolerance. We highlight examples where putatively functional variation in cis-regulatory regions of ART1-regulated genes interacts with ART1 to determine gene expression in response to Al. This ART1-promoter interaction may be associated with transgressive variation for Al resistance in the Azucena × IR64 population. These results illustrate how ART1 interacts with the genetic background to contribute to quantitative phenotypic variation in rice Al resistance.

Host Plants Indirectly Influence Plant Virus Transmission by Altering Gut Cysteine Protease Activity of Aphid Vectors.

The green peach aphid, Myzus persicae, is a vector of the Potato leafroll virus (PLRV, Luteoviridae), transmitted exclusively by aphids in a circulative manner. PLRV transmission efficiency was significantly reduced when a clonal lineage of M. persicae was reared on turnip as compared with the weed physalis, and this was a transient effect caused by a host-switch response. A trend of higher PLRV titer in physalis-reared aphids as compared with turnip-reared aphids was observed at 24 h and 72 h after virus acquisition. The major difference in the proteomes of these aphids was the up-regulation of predicted lysosomal enzymes, in particular the cysteine protease cathepsin B (cathB), in aphids reared on turnip. The aphid midgut is the site of PLRV acquisition, and cathB and PLRV localization were starkly different in midguts of the aphids reared on the two host plants. In viruliferous aphids that were reared on turnip, there was near complete colocalization of cathB and PLRV at the cell membranes, which was not observed in physalis-reared aphids. Chemical inhibition of cathB restored the ability of aphids reared on turnip to transmit PLRV in a dose-dependent manner, showing that the increased activity of cathB and other cysteine proteases at the cell membrane indirectly decreased virus transmission by aphids. Understanding how the host plant influences virus transmission by aphids is critical for growers to manage the spread of virus among field crops.

Equid herpesvirus 9 (EHV-9) isolates from zebras in Ontario, Canada, 1989 to 2007.

The objective of this study was to identify and partially characterize 3 equid herpesviruses that were isolated postmortem from zebras in Ontario, Canada in 1989, 2002, and 2007. These 3 virus isolates were characterized by plaque morphology, restriction fragment length polymorphism (RFLP) of their genomic deoxyribonucleic acid (DNA), real-time polymerase chain reaction (PCR) assay, and sequence analyses of the full length of the glycoprotein G (gG) gene (ORF70) and a portion of the DNA polymerase gene (ORF30). The isolates were also compared to 3 reference strains of equid herpesvirus 1 (EHV-1). Using rabbit kidney cells, the plaques for the isolates from the zebras were found to be much larger in size than the EHV-1 reference strains. The RFLP patterns of the zebra viruses differed among each other and from those of the EHV-1 reference strains. Real-time PCR and sequence analysis of a portion of the DNA polymerase gene determined that the herpesvirus isolates from the zebras contained a G at nucleotide 2254 and a corresponding N at amino acid position 752, which suggested that they could be neuropathogenic EHV-1 strains. However, subsequent phylogenetic analysis of the gG gene suggested that they were EHV-9 and not EHV-1.

L'objectif de la présente étude était d'identifier et de caractériser partiellement trois herpesvirus équins isolés de zèbres décédés en Ontario, Canada en 1989, 2002, et 2007. Ces trois isolats viraux furent caractérisés par morphologie des plages de lyse, par polymorphisme de taille des fragments de restriction (RFLP) de leur ADN génomique, par épreuve de réaction d'amplification en chaîne par la polymérase (PCR) en temps réel, et analyse de la séquence de la toute la longueur du gène de la glycoprotéine G (gG) (ORF70) et une portion du gène de la polymérase de l'ADN (ORF30). Les isolats furent également comparés à trois souches de référence d'herpesvirus équin de type 1 (EHV-1). L'examen de la culture des virus sur des cellules rénales de lapin a permis de constater que les plages de lyse causées par les isolats provenant des zèbres étaient beaucoup plus grandes que celles causées par les souches de référence d'EHV-1. Les patrons de RFLP des virus de zèbres différaient entre eux ainsi que des souches de référence d'EHV-1. Les analyses par PCR en temps réel et l'analyse de séquence d'une portion du gène de la polymérase de l'ADN ont permis de déterminer que les isolats d'herpesvirus provenant de zèbres avaient un G comme nucléotide à la position 2254 et un acide aminé N correspondant à la position 752, ce qui suggère qu'il pourrait s'agir de souches neuropathogènes d'EHV-1. Toutefois, des analyses phylogénétiques subséquentes du gène gG suggèrent qu'il s'agirait plutôt d'EHV-9 et non d'EHV-1.(Traduit par Docteur Serge Messier).

Use of high-resolution melting curve analysis to identify Mycoplasma species commonly isolated from ruminant, avian, and canine samples.

A real-time polymerase chain reaction assay coupled with high resolution melting curve analysis (PCR-HRM) was developed for identifying and distinguishing Mycoplasma species commonly isolated from ruminant, avian, and canine samples. The real-time PCR used 1 set of universal primers specific for the spacer region between the 16S ribosomal RNA and the 23S ribosomal RNA genes; the melting curve analysis of the PCR product used a high-resolution melt fluorescent dye. The real-time PCR-HRM assay was able to distinguish M. arginini, M. bovigenitalium, M. bovis, M. bovirhinis, M. canadense, M. cynos, M. spumans, M. iowae, M. meleagridis, and M. agalactiae reference strains. The real-time PCR-HRM assay developed was evaluated by testing field isolates of M. bovis, M. arginini, M. bovirhinis, M. bovigenitalium, M. iowae, and M. spumans with results consistent with those of the fluorescent antibody test.

Subcellular localization of the triple gene block proteins encoded by a Foveavirus infecting grapevines.

Grapevine rupestris stem pitting-associated virus (GRSPaV; Foveavirus; Flexiviridae) contains a positive-sense, ssRNA genome. GRSPaV occurs worldwide in grapes and is involved in the Rugose Wood disease complex. The GRSPaV genome contains the triple gene block (TGB), a genetic module present in several genera of plant RNA viruses. TGB encodes three proteins (TGBp1, TGBp2 and TGBp3) that are believed to work together to achieve intra- and inter-cellular transport of virions in infected plants. To reveal the subcellular localization of each TGB protein and to examine the impact that different fusion positions may have on the behavior of the native protein, we made a series of expression constructs and expressed the corresponding protein fusions in Nicotiana tabacum BY-2 cells and protoplasts. We demonstrated that TGBp1 had both a cytosolic and nuclear distribution. Two TGBp1 fusions (GFP fused at the N- or C-terminus) differ in subcellular distribution. Through the use of truncation mutants, we mapped TGBp1 regions responsible for the formation of two distinct types of aggregates. Sequence analyses predicted two and one transmembrane domains in TGBp2 and TGBp3, respectively. GFP fusions at either terminus of TGBp2 revealed identical localization to the ER network and ER-derived structures. In contrast, the two TGBp3 fusions to mRFP differed in localization. This is the first report on the subcellular localization of the viral proteins of a member of the Foveavirus genus.

Genetic diversity analyses of grapevine Rupestris stem pitting-associated virus reveal distinct population structures in scion versus rootstock varieties.

Grapevine Rupestris stem pitting-associated virus (GRSPaV) is a member of the genus Foveavirus within the family Flexiviridae. GRSPaV is closely associated with the disease Rupestris stem pitting and is frequently detected in grapevines worldwide. Previous research in several laboratories suggests that GRSPaV consists of a family of sequence variants. However, the genetic composition of GRSPaV variants in viral isolates from scion and rootstock varieties has not been studied extensively. In this report, the genetic diversity and population structure of GRSPaV isolates from scion and rootstock varieties were analysed using two pairs of primers targeting two different genomic regions encoding the helicase domain of the replicase and the capsid protein. In total, 190 cDNA clones derived from 24 isolates were sequenced and analysed. At least four major groups of GRSPaV variants were found to exist in grapevines. Interestingly, the majority of the scion varieties (9/10) that were analysed, regardless of their genetic background and geographical origin, harboured complex viral populations composed of two to four distinct viral variants. In contrast, the viral populations in isolates from rootstock varieties were homogeneous and comprised a single variant. The practice of grafting between scion and rootstock varieties commonly used in modern viticulture, coupled with the frequent regional and international exchange of propagating materials, may have played a major role in the ubiquitous distribution and mixed infections of distinct GRSPaV variants among scion varieties. The possible origin and evolution of GRSPaV are also discussed.