Pairwise Identity Scores Research Articles

Two Novel Geminiviruses Identified in Bees (Apis mellifera and Nomia sp.).

Members of the Geminviridae family are circular single-stranded DNA plant-infecting viruses, some of which impact global food production. Geminiviruses are vectored by sap-feeding insects such as leafhoppers, treehoppers, aphids, and whiteflies. Additionally, geminivirus sequences have also been identified in other insects such as dragonflies, mosquitoes, and stingless bees. As part of a viral metagenomics study on honeybees and solitary bees (Nomia sp.), two geminivirus genomes were identified. These represent a novel citlodavirus (from honeybees collected from Westmoreland, Jamaica) and a mastrevirus-like genome (from a solitary bee collected from Tempe, Arizona, USA). The novel honeybee-derived citlodavirus genome shares ~61 to 69% genome-wide nucleotide pairwise identity with other citlodavirus genome sequences and is most closely related to the passion fruit chlorotic mottle virus identified in Brazil. Whereas the novel solitary bee-derived mastrevirus-like genome shares ~55 to 61% genome-wide nucleotide identity with other mastreviruses and is most closely related to tobacco yellow dwarf virus identified in Australia, based on pairwise identity scores of the full genome, replication-associated protein, and capsid protein sequences. Previously, two geminiviruses in the Begomovirus genus were identified in samples of stingless bee (Trigona spp.) samples. Here, we identify viruses that represent two new species of geminiviruses from a honeybee and a solitary bee, which continues to demonstrate that plant pollinators can be utilized for the identification of plant-infecting DNA viruses in ecosystems.

Identity: rapid alignment-free prediction of sequence alignment identity scores using self-supervised general linear models.

Pairwise global alignment is a fundamental step in sequence analysis. Optimal alignment algorithms are quadratic—slow especially on long sequences. In many applications that involve large sequence datasets, all what is needed is calculating the identity scores (percentage of identical nucleotides in an optimal alignment—including gaps—of two sequences); there is no need for visualizing how every two sequences are aligned. For these applications, we propose Identity, which produces global identity scores for a large number of pairs of DNA sequences using alignment-free methods and self-supervised general linear models. For the first time, the new tool can predict pairwise identity scores in linear time and space. On two large-scale sequence databases, Identity provided the best compromise between sensitivity and precision while being faster than BLAST, Mash, MUMmer4 and USEARCH by 2–80 times. Identity was the best performing tool when searching for low-identity matches. While constructing phylogenetic trees from about 6000 transcripts, the tree due to the scores reported by Identity was the closest to the reference tree (in contrast to andi, FSWM and Mash). Identity is capable of producing pairwise identity scores of millions-of-nucleotides-long bacterial genomes; this task cannot be accomplished by any global-alignment-based tool. Availability: https://github.com/BioinformaticsToolsmith/Identity.

Molecular characterization and phylogenetic analysis of tomato leaf curl Palampur virus, a bipartite begomovirus, associated with Cucumis sativus L. in Pakistan.

Leaf samples of Cucumis Sativus L. (C. sativus) (Family; Cucurbitaceae) showing vein thickening, mild leaf curling and leaf enations were collected from the farmer's field. Amplification of the full-length viral molecules was performed through rolling circle amplification (RCA). Cloning of the full-length viral molecules was done through standard cloning procedure followed by sequencing. Sequence similarity analysis and phylogenetic studies showed that the virus associated with leaf curling and enations in C. sativus was a bipartite begomovirus, where DNA-A and DNA-B showed highest nucleotide sequence homology of 98% and 97% to tomato leaf curl Palampur virus (ToLCPMV) from India. Attempts to isolate betasatellites and alphasatellites through PCR using RCA product as template, did not result in any amplification. A maximum likelihood phylogenetic tree grouped DNA-A and B components with other isolates from India. SDT was used to find the pairwise identity scores of different sequences of ToLCPMV present in the database. Phylogenetic analysis showed that sequences of ToLCPMV DNA-A and B components in this study share high degree of homology with existing viruses and are isolates of ToLCPMV-India. Infectious molecules of both components (Accessions, MG252783 and MG252784, respectively) were constructed for infectivity analysis to fulfill the Koch's postulate. Infectivity analysis revealed that ToLCPMV DNA-A is infectious to model host plant Nicotiana benthamiana and viral accumulation was confirmed through Southern blot analysis. Accumulation of DNA-B was confirmed through PCR. Infectivity analysis was also conducted using the original host, C. sativus, but plants were unable to survive the agroinoculation. To our knowledge this is the first report of ToLCPMV associated with C. sativus L. in Pakistan.

A Novel Strain of Pepper Leafroll Virus Infecting Common Bean and Soybean in Ecuador

Symptoms resembling those caused by begomoviruses (Geminiviridae: Begomovirus) have been observed in common bean and other leguminous crops in Ecuador in the last few years. The only begomovirus species found infecting legumes (common bean and cowpea) in the country to date is cabbage leaf curl virus (CabLCV) (Fiallo-Olive et al. 2018). During May to June 2017, a survey of leguminous plants was conducted in the Ecuadorian localities of Santa Elena (Santa Elena province), Milagro (Guayas province), and Pallatanga (Chimborazo province). Twenty-four plants showing leaf deformation and/or mosaic symptoms were sampled, including common bean (Phaseolus vulgaris, n = 21), soybean (Glycine max, n = 2), and pigeon pea (Cajanus cajan, n = 1). Most common bean plants were infested with whiteflies (Hemiptera: Aleyrodidae) that were also collected. Total DNA was extracted from leaf samples and used as template in polymerase chain reaction (PCR) using specific primers for CabLCV (MA2540 [5′-GATGTTGAAGCATCTGCAAACATTC-3′] and MA2541 [5′-CTAGGAACATCAGGGCTTCTCAAC-3′]) (Fiallo-Olive et al. 2018). All samples were negative for the presence of CabLCV. DNA extracts were then used as templates in rolling-circle amplification (RCA) using φ29 DNA polymerase (TempliPhi kit, GE Healthcare). Digestion of RCA products with a set of restriction enzymes yielded identical restriction patterns for six samples (five common bean and one soybean), suggesting the presence of a bipartite begomovirus. BamHI and NcoI fragments of approximately 2.6 kbp from a common bean plant were cloned in pBluescript II SK(+), and two selected clones were sequenced (Macrogen, Seoul, South Korea). BLAST analysis showed that the clones corresponded to begomoviral DNA-A (BamHI, 2,580 nt, GenBank accession no. MH481901) and DNA-B (NcoI, 2,561 nt, MH481902). Pairwise identity scores with isolates selected after BLAST analysis were calculated with SDT (Muhire et al. 2014). Cloned DNA-A and DNA-B showed the highest identity (93 and 92%, respectively) with pepper leafroll virus (PepLRV) (KC769819 and KC769820), a bipartite begomovirus identified infecting pepper, common bean, and tomato in Peru (Martinez-Ayala et al. 2014). To confirm the identity of the begomovirus present in the rest of the samples, PCR was performed with specific primers whose design was based on the cloned PepLRV DNA-A (MA2602 [5′-GTTAATGGACAATGAGGGTATGTG-3′] and MA2603 [5′-CACGAGGAGAATAATTGGCGTTAC-3′]) using Taq DNA polymerase (BIOTAQ, Bioline) and thermocycler conditions consisting of 35 cycles with 45 s annealing steps at 57°C. DNA fragments of the expected size (452 bp) were amplified from the six samples, and direct sequencing showed them to be identical to the cloned PepLRV DNA-A, supporting the presence of this virus. Based on the species/strain demarcation threshold for the viruses in the genus Begomovirus (Brown et al. 2015), the isolate characterized here belongs to a new strain of PepLRV. DNA was extracted from individual whiteflies (n = 42) using a Chelex-based method, and PCR was carried out using mitochondrial cytochrome oxidase I gene primers, C1-J-2195 (5′-TTGATTTTTTGGTCATCCAGAAGT-3′) and Btab-uni-PrimerR (5′-CTTAAATTTACTGCACTTTCTGCCAYATTAG-3′). Direct sequencing of PCR products showed that seven sequences (one deposited in GenBank with accession no. MH481903) were identical to numerous sequences of Bemisia tabaci Middle East-Asia Minor 1 (MEAM1, formerly B biotype), known to be an efficient vector of begomoviruses. To our knowledge, this is the first report of PepLRV in Ecuador and of soybean as a host for the virus, which had been identified only in Peru to date. Also, this is the first molecular evidence of the presence of B. tabaci MEAM1 in the country.

The complete genome sequence of New World jatropha mosaic virus.

Full-length sequences of a bipartite begomovirus were obtained from a plant of Jatropha multifida in Florida showing symptoms of foliar mosaic, distortion and necrosis. Sequences of four clones each of a DNA-A and DNA-B were obtained, which showed very low sequence diversity among themselves. The clones were infectious when biolistically inoculated to J. multifida, Phaseolus vulgaris and Nicotiana tabacum, but not to J. curcas. The DNA-A sequences had less than 89 % pairwise identity scores with the DNA-A of other begomoviruses. The DNA-A appeared to be a recombinant in that 18 % of the DNA-A (470 nt) had a pairwise identity score of 91.98 % with RhRGMV, indicating that this portion most likely originated from a virus closely related to RhRGMV. The remaining 82 % of the DNA-A had lower identity scores with TbMoLCV (87.84 %) and RhRGMV (87.46 %), which suggests that this part of the component originated from an undescribed virus. There was no evidence for recombination in the DNA-B. Equivalent sequences of the DNA-A had the highest identity score (94.18 %) with a 533-nt sequence obtained from J. multifida from Puerto Rico in 2001 (GenBank accession no. AF058025). Pairwise comparison, recombination and phylogenetic analysis, and biology suggest that these clones are those of jatropha mosaic virus first reported from Puerto Rico. This is the first report of the complete genome sequence of jatropha mosaic virus.

First Detection of Tomato leaf curl New Delhi virus Infecting Zucchini in Spain.

In September 2012, a novel disease syndrome was observed in zucchini (Cucurbita pepo L.) crops in Murcia Province (southeastern Spain). Symptoms included curling, vein swelling, and severe mosaic in young leaves, short internodes, and fruit skin roughness, resembling begomovirus infection. Similar symptoms were observed in May 2013 in Almería Province (southern Spain). DNA was isolated from 8 and 7 symptomatic leaf samples collected in Murcia and Almería, respectively, and analyzed by PCR with primers GemCP-V-5' and GemCP-C-3' designed to detect begomoviruses by amplifying the core of coat protein gene (CP) (3). DNA fragments of the expected size (~600 bp) were amplified supporting a begomovirus infection. The DNA sequences obtained from four samples were identical. BLAST analysis showed the highest nucleotide identity (98%) with partial CP gene sequences from isolates of Tomato leaf curl New Delhi virus (ToLCNDV) infecting cucumber in India (GenBank Accession No. KC846817). ToLCNDV, a bipartite begomovirus first reported from tomato, also infects other solanaceous and cucurbitaceous crops in India and neighboring countries (1). DNA from two samples from Murcia and three from Almería was used for rolling-circle amplification using ϕ29 DNA polymerase (TempliPhi kit, GE Healthcare, Little Chalfont, UK) and digested with a set of restriction endonucleases. All five samples yielded amplification products with identical restriction patterns. Two samples from Murcia (MU-8.1 and MU-11.1) and one from Almería (AL-661) were selected to clone the putative DNA-A and DNA-B begomovirus genome components by using single BamHI or NcoI sites. Inserts of two clones from each sample, one corresponding to DNA-A and one to DNA-B, were completely sequenced. The cloned genomes exhibited the typical organization of Old World bipartite begomoviruses (1). Sequences were aligned with begomovirus sequences available in databases using MUSCLE and pairwise identity scores were calculated with SDT (species demarcation tool [4]). DNA-A sequences obtained from Murcia (2,738 nt, KF749224 and KF749225) and Almería (2,738 nt, KF749223) shared >99% nucleotide identity, with the highest nucleotide identity (91.3 to 91.5%) with that of an Indian ToLCNDV isolate from chilli (HM007120). DNA-B sequences (2,684 nt, KF749226, KF749227, and KF749228) shared >99% nucleotide identity, and showed the highest nucleotide identity (83.1 to 83.3%) with that of a Pakistani ToLCNDV isolate from Solanum nigrum (AJ620188). Nucleotide sequence identity of DNA-A with the most closely related begomoviruses was above the 91% threshold for species demarcation (2), thus confirming that the begomoviruses found infecting zucchini in Spain are isolates of ToLCNDV. In fall 2013, the disease was widespread in zucchini both in Murcia and Almería, and ToLCNDV has also been found infecting melon and cucumber crops. To our knowledge, this is the first report of a bipartite begomovirus in Spain and Europe.

First Report of Sweet potato leaf curl virus on Blue Morning Glory in Greece.

Blue morning glory (Ipomoea indica, Convolvulaceae) plants are widespread along the Greek coast, where they grow as weeds in addition to being cultivated as ornamentals. Yellow vein symptoms are frequently observed on these plants. These symptoms are similar to those reported for isolates of Sweet potato leaf curl virus (SPLCV) infecting I. indica in Italy and Spain (1,3). SPLCV belongs to the sweepoviruses, a unique group within the genus Begomovirus in the family Geniminiviridae that infects sweet potato (I. batatas) crops around the world. In May 2013, three leaf samples of I. indica showing yellow vein symptoms were collected in Kolymbari (Crete Island), where ~50% of the observed plants were symptomatic, and five asymptomatic leaf samples were collected in Kremasti and Mandriko (Rhodes Island). Total DNA, isolated from all samples, was used as a template in rolling-circle amplification (RCA) using ϕ29 DNA polymerase (TempliPhi kit, GE Healthcare, Little Chalfont, UK) and the product was digested with a set of restriction endonucleases. The samples from Kolymbari and one sample from Kremasti yielded amplification products that were shown to contain a single BamHI site. The DNA fragments of ~2.8 kbp obtained from one sample from each island were cloned into pBluescript II SK(+) (Stratagene, La Jolla, CA). Inserts of two clones from the Kolymbari sample and one clone from the Kremasti sample were completely sequenced (Macrogen, Seoul, South Korea). Sequences were aligned with available sequences of sweepoviruses using MUSCLE and pairwise identity scores were calculated with SDT as described (4). The sequences obtained from Kolymbari (2,830 nt, GenBank Accession Nos. KF697069 and KF697070) were 98.8% similar between them and showed the highest nucleotide identity (97.7%) with a SPLCV isolate obtained from an I. indica plant in Sicily Island (Italy) (AJ586885) (1). The sequence obtained from Kremasti (2,804 nt, KF697071) showed the highest nucleotide identity (92.4%) with a SPLCV isolate (previously named as Ipomoea yellow vein virus, which is currently a synonym of SPLCV [2]) obtained from an I. indica plant from southern Spain (EU839578) (3). Nucleotide sequence identities were above the 91% threshold for begomovirus species demarcation (2), thus confirming that the begomoviruses found infecting I. indica in Greece are isolates of SPLCV. It is worth to note that the infected I. indica plant from Kremasti did not show any conspicuous symptoms, thus highlighting the importance of this species as an alternative host for SPLCV, which could thus affect the sweet potato crop that is grown in Greece in familiar plots. To our knowledge, this is the first report of SPLCV in Greece.

A genome-wide pairwise-identity-based proposal for the classification of viruses in the genus Mastrevirus (family Geminiviridae)

Recent advances in the ease with which the genomes of small circular single-stranded DNA viruses can be amplified, cloned, and sequenced have greatly accelerated the rate at which full genome sequences of mastreviruses (family Geminiviridae, genus Mastrevirus) are being deposited in public sequence databases. Although guidelines currently exist for species-level classification of newly determined, complete mastrevirus genome sequences, these are difficult to apply to large sequence datasets and are permissive enough that, effectively, a high degree of leeway exists for the proposal of new species and strains. The lack of a standardised and rigorous method for testing whether a new genome sequence deserves such a classification is resulting in increasing numbers of questionable mastrevirus species proposals. Importantly, the recommended sequence alignment and pairwise identity calculation protocols of the current guidelines could easily be modified to make the classification of newly determined mastrevirus genome sequences significantly more objective. Here, we propose modified versions of these protocols that should substantially minimise the degree of classification inconsistency that is permissible under the current system. To facilitate the objective application of these guidelines for mastrevirus species demarcation, we additionally present a user-friendly computer program, SDT (species demarcation tool), for calculating and graphically displaying pairwise genome identity scores. We apply SDT to the 939 full genome sequences of mastreviruses that were publically available in May 2012, and based on the distribution of pairwise identity scores yielded by our protocol, we propose mastrevirus species and strain demarcation thresholds of >78 % and >94 % identity, respectively.

Characterization of a New Multigene Family Encoding Isomaltases in the Yeast Saccharomyces cerevisiae, the IMA Family

It has been known for a long time that the yeast Saccharomyces cerevisiae can assimilate alpha-methylglucopyranoside and isomaltose. We here report the identification of 5 genes (YGR287c, YIL172c, YJL216c, YJL221c and YOL157c), which, similar to the SUCx, MALx, or HXTx multigene families, are located in the subtelomeric regions of different chromosomes. They share high nucleotide sequence identities between themselves (66-100%) and with the MALx2 genes (63-74%). Comparison of their amino acid sequences underlined a substitution of threonine by valine in region II, one of the four highly conserved regions of the alpha-glucosidase family. This change was previously shown to be sufficient to discriminate alpha-1,4- to alpha-1,6-glucosidase activity in YGR287c (Yamamoto, K., Nakayama, A., Yamamoto, Y., and Tabata, S. (2004) Eur. J. Biochem. 271, 3414-3420). We showed that each of these five genes encodes a protein with alpha-glucosidase activity on isomaltose, and we therefore renamed these genes IMA1 to IMA5 for IsoMAltase. Our results also illustrated that sequence polymorphisms among this family led to interesting variability of gene expression patterns and of catalytic efficiencies on different substrates, which altogether should account for the absence of functional redundancy for growth on isomaltose. Indeed, deletion studies revealed that IMA1/YGR287c encodes the major isomaltase and that growth on isomaltose required the presence of AGT1, which encodes an alpha-glucoside transporter. Expressions of IMA1 and IMA5/YJL216c were strongly induced by maltose, isomaltose, and alpha-methylglucopyranoside, in accordance with their regulation by the Malx3p-transcription system. The physiological relevance of this IMAx multigene family in S. cerevisiae is discussed.

Rapid Identification of the Coxsackievirus A24 Variant by Molecular Serotyping in an Outbreak of Acute Hemorrhagic Conjunctivitis

We evaluated the clinical applicability of a molecular serotyping method for determination of the cause of epidemic acute hemorrhagic conjunctivitis. Seventy conjunctival swab specimens from individuals involved in a nationwide acute hemorrhagic conjunctivitis outbreak were tested. Viral culture and a molecular biology-based assay were compared by directly using clinical specimens. On the one hand, virus culture was done to isolate the enteroviruses, and serotyping was done by a coxsackievirus A24 variant-specific PCR. On the other hand, the original clinical specimens were directly screened for enterovirus by reverse transcription (RT)-PCR with panenterovirus-specific primers. Enterovirus screening-positive specimens were subjected to RT-PCR for detection of the VP1 region of enterovirus, and the amplicons were sequenced. Molecular serotyping was done by calculating the pairwise identity scores for the sequences with the maximum identities to the sequences of known prototype enteroviruses. Thirty-two specimens (45.7%) were culture positive, whereas 37 specimens (52.8%) were screening PCR positive (P < 0.001). The VP1 regions were amplified from 21 of the 37 specimens (56.8%), and the products amplified from 9 specimens were appropriately sequenced. These nine sequences were homologous with the sequence of the coxsackievirus A24 variant. Molecular serotyping by direct use of clinical specimens without cell culture could be applied for the rapid identification of the causative agent of epidemic acute hemorrhagic conjunctivitis.

Organisation and structural evolution of the rice glutathione S-transferase gene family.

Glutathione S-transferases (GSTs) comprise a large family of key defence enzymes against xenobiotic toxicity. Here we describe the comprehensive characterisation of this important multigene family in the model monocot species rice [ Oryza sativa(L.)]. Furthermore, we investigate the molecular evolution of the family based on the analysis of (1) the patterns of within-genome duplication, and (2) the phylogenetic relationships and evolutionary divergence among rice, Arabidopsis, maize and soybean GSTs. By in-silico screening of the EST and genome divisions of the Genbank/EMBL/DDBJ database we have isolated 59 putative genes and two pseudogenes, making this the largest plant GST family characterised to date. Of these, 38 (62%) are represented by genomic and EST sequences and 23 (38%) are known only from their genomic sequences. A preliminary survey of EST collections shows a large degree of variability in gene expression between different tissues and environmental conditions, with a small number of genes (13) accounting for 80% of all ESTs. Rice GSTs are organised in four main phylogenetic classes, with 91% of all rice genes belonging to the two plant-specific classes Tau (40 genes) and Phi (16 genes). Pairwise identity scores range between 17 and 98% for proteins of the same class, and 7 and 21% for interclass comparisons. Rapid evolution by gene duplication is suggested by the discovery of two large clusters of 7 and 23 closely related genes on chromosomes 1 and 10, respectively. A comparison of the complete GST families in two monocot and two dicot species suggests a monophyletic origin for all Theta and Zeta GSTs, and no more than three common ancestors for all Phi and Tau genes.

Molecular evolution of the human enteroviruses: correlation of serotype with VP1 sequence and application to picornavirus classification.

Sixty-six human enterovirus serotypes have been identified by serum neutralization, but the molecular determinants of the serotypes are unknown. Since the picornavirus VP1 protein contains a number of neutralization domains, we hypothesized that the VP1 sequence should correspond with neutralization (serotype) and, hence, with phylogenetic lineage. To test this hypothesis and to analyze the phylogenetic relationships among the human enteroviruses, we determined the complete VP1 sequences of the prototype strains of 47 human enterovirus serotypes and 10 antigenic variants. Our sequences, together with those available from GenBank, comprise a database of complete VP1 sequences for all 66 human enterovirus serotypes plus additional strains of seven serotypes. Phylogenetic trees constructed from complete VP1 sequences produced the same four major clusters as published trees based on partial VP2 sequences; in contrast to the VP2 trees, however, in the VP1 trees strains of the same serotype were always monophyletic. In pairwise comparisons of complete VP1 sequences, enteroviruses of the same serotype were clearly distinguished from those of heterologous serotypes, and the limits of intraserotypic divergence appeared to be about 25% nucleotide sequence difference or 12% amino acid sequence difference. Pairwise comparisons suggested that coxsackie A11 and A15 viruses should be classified as strains of the same serotype, as should coxsackie A13 and A18 viruses. Pairwise identity scores also distinguished between enteroviruses of different clusters and enteroviruses from picornaviruses of different genera. The data suggest that VP1 sequence comparisons may be valuable in enterovirus typing and in picornavirus taxonomy by assisting in the genus assignment of unclassified picornaviruses.