Pairwise Sequence Comparisons Research Articles

Complete genome sequence of a novel badnavirus infecting Fatsia japonica in China.

The complete genome sequence of a novel badnavirus, tentatively named "fatsia badnavirus 1" (FaBV1, OM540428), was identified in Fatsia japonica. The infected plant displayed virus-like symptoms on leaves, including yellowing and chlorosis. The genome of FaBV1 is 7313 bp in length and similar in size and organization to other members of the genus Badnavirus (family Caulimoviridae), containing four open reading frames (ORFs), three of which are found in all known badnaviruses, and the other of which is only present in some badnaviruses. The virus has the genome characteristics of badnaviruses, including a tRNAMet binding site (5'-TCTGAATTTATAGCGCTA-3') and two cysteine-rich domains (C-X-C-2X-C-4X-H-4X-C and C-2X-C-11X-C-2X-C-4X-C-2X-C). Pairwise sequence comparisons of the RT+RNase H region indicated that FaBV1 shares 61.4-71.2% nucleotide (nt) sequence identity with other known badnaviruses, which is below the threshold (80% nt sequence identity in the RT+RNase H region) used for species demarcation in the genus Badnavirus. Phylogenetic analysis revealed that FaBV1, ivy ringspot-associated virus (IRSaV, MN850490.1), and cacao mild mosaic virus (CMMV, KX276640.1) together form a separate clade within the genus Badnavirus, suggesting that FaBV1 is a new member of the genus Badnavirus in the family Caulimoviridae. To our knowledge, this is the first report of a badnavirus infecting F. japonica.

Viral metagenomic analysis reveals diverse viruses and a novel bocaparvovirus in the enteric virome of snow leopard (Panthera uncia)

The enteric virome, comprising a complex community of viruses inhabiting the gastrointestinal tract, plays a significant role in health and disease dynamics. In this study, the fecal sample of a wild snow leopard was subjected to viral metagenomic analysis using a double barcode Illumina MiSeq platform. The resulting reads were de novo assembled into contigs with SOAPdenovo2 version r240. Additional bioinformatic analysis of the assembled genome and genome annotation was done using the Geneious prime software (version 2022.0.2). Following viral metagenomic analysis and bioinformatic analysis, a total of 7 viral families and a novel specie of bocaparvovirus tentatively named Panthera uncia bocaparvovirus (PuBOV) with GenBank accession number OQ627713 were identified. The complete genome of PuBOV was predicted to contain 3 open reading frames (ORFs), contains 5433 nucleotides and has a G + C content of 47.40 %. BLASTx analysis and pairwise sequence comparison indicated the novel virus genome was a new species in the genus Bocaparvovirus based on the species demarcation criteria of the International Committee on the Taxonomy of Viruses. This study provides valuable insights into the diversity and composition of the enteric virome in wild endangered snow leopards. The identification and characterization of viruses in wildlife is crucial for developing effective strategies to manage and mitigate potential zoonotic and other viral disease threats to human and animal health.

Genomic and biological characteristics of a novel leafhopper-transmitted marafivirus infecting Triticum aestivum.

Here, we report a novel wheat-infecting marafivirus, tentatively named "Triticum aestivum marafivirus" (TaMRV). The full-length genome sequence of TaMRV comprises 6,437 nucleotides, excluding the poly(A) tail. Pairwise sequence comparisons and phylogenetic analysis revealed that TaMRV may represent a novel species within the genus Marafivirus in the family Tymoviridae. We also observed a mass of isometric particles with a diameter of about 30 nm in ultrathin sections of infected wheat leaf tissue. In addition, the leafhopper Psammotettix alienus was identified as a vector for this virus. This is the first report of the occurrence of a wheat-infecting marafivirus.

Molecular characterisation of a novel sadwavirus infecting cattleya orchids in Australia

The complete genome sequence of a novel sadwavirus infecting cattleya orchids in South East Queensland is described. Isometric virions of c. 27 nm diameter were observed in sap extracts viewed under a transmission electron microscope, and the genome sequence of this virus was determined by high-throughput sequencing. The viral genome consists of two RNA components, 5,910 and 4,435 nucleotides (nt) in length, each encoding a long polyprotein, with predicted cleavage sites at H/Y, E/G, Q/S, and Q/G for the RNA1 and T/G for the RNA2 translation products, respectively. RNA2 has an additional small ORF of 684 nt near the 3ʹ untranslated region. Phylogenetic analysis based on an amino acid sequence alignment of the Pro-Pol region suggested that this virus is most closely related to pineapple secovirus A, a member of the subgenus Cholivirus, but warrants classification as a member of a new species because it exhibited no more than 64% amino acid identity in pairwise sequence comparisons. Because of the prominent purple ringspots that were observed on the leaves of some of the plants, we propose the name “cattleya purple ringspot virus” for this virus (suggested species name: “Sadwavirus cattleyacola”).

Genomic characterization of a novel cytorhabdovirus infecting Ixeris denticulata in China.

Ixeris denticulata is a perennial herbal plant with important medical and economic value. In this study, a novel rhabdovirus from I. denticulata with leaf curling and mottle symptoms was identified through next-generation sequencing and molecular cloning approaches. Based on the host species and properties of this virus, it was tentatively named "Ixeris denticulata-associated rhabdovirus" (IdaRV). IdaRV has a negative-sense RNA genome that is 12,705 nucleotides in length and has five open reading frames (ORFs) in the order 3'-nucleoprotein -phosphoprotein -movement protein -matrix protein -large RNA-dependent RNA polymerase-5'. Pairwise sequence comparisons showed that IdaRV had 42.2-53.0% sequence identity to members of the genera Cytorhabdovirus, Varicosavirus, Betanucleorhabdovirus, Gammanucleorhabdovirus, Dichorhavirus, and Alphanucleorhabdovirus in the subfamily Betarhabdovirinae. BLASTp searches indicated that putative products of ORF1, ORF2, ORF3, ORF4, and ORF5 of IdaRV are most closely related to those of rudbeckia virus 1 (RudV1, GenBank accession number ON185810), with 32.1%, 21.3%, 52.4%, 37.6%, and 57.1% amino acid sequence identity, respectively, at the protein level. Phylogenetic analysis showed that IdaRV forms a smaller branch with RudV1, which belongs to the genus Cytorhabdovirus. These results establish IdaRV as a novel rhabdovirus in the genus Cytorhabdovirus of the family Rhabdoviridae.

Anchor Clustering for million-scale immune repertoire sequencing data

BackgroundThe clustering of immune repertoire data is challenging due to the computational cost associated with a very large number of pairwise sequence comparisons. To overcome this limitation, we developed Anchor Clustering, an unsupervised clustering method designed to identify similar sequences from millions of antigen receptor gene sequences. First, a Point Packing algorithm is used to identify a set of maximally spaced anchor sequences. Then, the genetic distance of the remaining sequences to all anchor sequences is calculated and transformed into distance vectors. Finally, distance vectors are clustered using unsupervised clustering. This process is repeated iteratively until the resulting clusters are small enough so that pairwise distance comparisons can be performed.ResultsOur results demonstrate that Anchor Clustering is faster than existing pairwise comparison clustering methods while providing similar clustering quality. With its flexible, memory-saving strategy, Anchor Clustering is capable of clustering millions of antigen receptor gene sequences in just a few minutes.ConclusionsThis method enables the meta-analysis of immune-repertoire data from different studies and could contribute to a more comprehensive understanding of the immune repertoire data space.

Complete nucleotide sequence of chrysanthemum virus D, a polero-like virus.

A putative new polerovirus, named "chrysanthemum virus D" (ChVD), was detected in a Chrysanthemum morifolium plant in South Korea. The virus was identified by high-throughput sequencing and confirmed by reverse transcription polymerase chain reaction. The entire ChVD genome is composed of 5,963 nucleotides and contains seven open reading frames (ORF0-5 and ORF3a), which are arranged similarly to those of other poleroviruses. These ORFs encode the putative proteins P0-5 and P3a, respectively. Pairwise amino acid sequence comparisons showed that the ChVD P0-5 and P3a proteins have 30.45-75% sequence identity to the corresponding proteins of other members of the genus Polerovirus. Since one of the species demarcation criteria for the genus Polerovirus is >10% difference in the amino acid sequence of any gene product, the sequence comparisons indicate that ChVD represents a new species in this genus. Phylogenetic analysis of the P1-P2 and P3 amino acid sequences further indicate that ChVD is a novel polerovirus.

Maximum likelihood estimation and natural pairwise estimating equations are identical for three sequences and a symmetric 2-state substitution model

Consider the problem of estimating the branch lengths in a symmetric 2-state substitution model with a known topology and a general, clock-like or star-shaped tree with three leaves. We show that the maximum likelihood estimates are analytically tractable and can be obtained from pairwise sequence comparisons. Furthermore, we demonstrate that this property does not generalize to larger state spaces, more complex models or larger trees. Our arguments are based on an enumeration of the free parameters of the model and the dimension of the minimal sufficient data vector. Our interest in this problem arose from discussions with our former colleague Freddy Bugge Christiansen.

Impact of Therapy Sequence on Survival Outcomes Among Patients with Relapsed or Refractory Mature T and NK Cell Neoplasms: A Global Retrospective Cohort Study

Despite evolution of therapeutic strategies, there is no universal standard of care for relapsed or refractory (RR) mature T and NK-cell neoplasms (TNKL). Most patients receive multiple lines of therapy and cycle through many available options. 1-3 There is no data to inform optimal therapy sequence, however emerging data suggest that exposure to epigenetic modifiers (EM) can sensitize tumors to other therapies. 4-6 Here we report results of comparative analyses assessing survival outcomes based on therapy sequence using a global RR TNKL patient cohort with data from 15 centers across 6 continents. We conducted a retrospective target-trial 7,8 cohort study using an updated global patient cohort. 9 Patients were excluded from analyses if they had anaplastic large cell lymphoma (as EM is infrequently used 10,11), did not receive first line cytotoxic chemotherapy (CC), or no documented second line (2L) start date. Cohort assignment was based on 2L therapy received: EM (e.g. histone deacetylase or DNA methyltransferase inhibitors), small molecule inhibitors (SI; broad or selective), or CC. Antibody-drug conjugates were included in CC. Outcomes were overall survival (OS; time from 2L start to death) and real-world progression-free-survival-2 (rwPFS2; time from 2L start to fourth line start or death 12 to assess if 2L modifies the effect of third line [3L]). Planned subgroups included histologic subtype and those who received allogeneic hematopoietic transplant (HSCT) after 2L. For sequence analyses, included patients must have received 3L. Outcomes were assessed between all prespecified 2L to 3L sequences using a single comparator, and pairwise. Kaplan Meier curves were used, and adjusted hazard ratios (aHR) were estimated using Cox regression with previously described a priori covariates. 9 Maximal, minimal, and no covariate Cox models were compared using likelihood ratio tests and Akaike's information criterion. Of the 925 RR patients in the global cohort, 472 were included who received EM (n=89), SI (n=46), or CC (n=337) at 2L. The most common SI included immunomodulatory imide drug (26.1%), PI3K inhibitor (21.7%), investigational pathway inhibitor (19.6%) and JAK inhibitor (17.4%). Within EM, SI, and CC, 42.7%, 37%, and 47.2% of patients were primary refractory to first line, respectively. The most common histologies were PTCL-NOS (51.7%, 41.3%, and 57.3%) and AITL (43.8%, 54.3%, and 26.4%) in EM, SI, and CC, respectively. Most patients had a Prognostic Index for T-cell Lymphoma (PIT) score at diagnosis of 1 (27%, 34.8%, and 27%) or 2 (28.1%, 26.1%, and 25.8%) in EM, SI, and CC, respectively. In EM, 34.8% received first-line autologous HSCT, versus 28.2% in SI, and 15.4% in CC. Following 2L, 12.4%, 17.4%, and 9.8% received allogeneic HSCT in EM, SI, and CC, respectively. The Cox model containing histology, PIT, and primary refractory status was used as the final model (vs maximal model: p=0.88). Compared to CC at 2L, EM did not affect OS (aHR 0.88, 95% CI 0.63-1.24; p=0.46) or rwPFS2 (aHR 0.84, 95% CI 0.61-1.16; p=0.28), however SI improved OS (aHR 0.61, 95% CI 0.37-0.97; p=0.038) and rwPFS2 (aHR 0.61, 95% CI 0.38-0.90; p=0.038). In the allogeneic HSCT subgroup, there was no difference in OS (EM vs CC: aHR 0.32, 95% CI 0.04-2.40; p=0.27; SI vs CC: aHR 0.51, 95% CI 0.06-4.69; p=0.55) or rwPFS2 (EM vs CC: aHR 0.30, 95% CI 0.05-1.96; p=0.21; SI vs CC: aHR 0.64, 95% CI 0.05-3.90; p=0.46). There were no differences in PTCL-NOS patients in OS (EM vs CC aHR 0.80, 95% CI 0.51-1.25; p=0.33; SI vs CC: aHR 1.00, 95% CI 0.54-1.84; p=0.99) or rwPFS2 (EM vs CC: aHR 0.91, 95% CI 0.61-1.38; p=0.66; SI vs CC: aHR 0.90, 95% CI 0.49-0.97; p=0.16). In AITL, SI improved OS (aHR 0.34, 95% CI 0.16-0.76; p=0.009) and rwPFS2 (aHR 0.36, 95% CI 0.17-0.77; p=0.008) versus CC, but EM showed no difference in OS (aHR 0.80, 95% CI 0.43-1.46; p=0.46) or rwPFS2 (aHR 0.56, 95% CI 0.31-1.02; p=0.059). There was no difference in rwPFS2 or OS across sequences overall (Figure 1) or between pairwise sequence comparisons (Figure 2). Compared to CC, SI at 2L improved OS and rwPFS2 in AITL patients. Therapy sequence did not affect OS or rwPFS2. Advanced approaches such as machine learning and dynamic treatment regimes have been initiated to fully elucidate the effect of therapy sequence. Our study highlights several equally effective therapy sequences to treat RR TNKL allowing therapy to be individualized based on patient and disease characteristics, and drug access at a given time.

Taxonomic Classification of Geminiviruses Based on Pairwise Sequence Comparisons.

Geminiviridae is the largest and one of the most diverse families of plant viruses, comprising 14 genera demarcated based on host range, type of insect vector, and phylogenetic relationships. The use of unbiased, whole-genome multiple displacement amplification techniques coupled with high-throughput sequencing has greatly expanded our knowledge of geminivirus diversity over the last two decades. As a result, a large number of new species have been described in recent years. Species demarcation criteria in the family are entirely based on sequence comparisons, but the specific cutoff values vary for each genus. The purpose of this chapter is to provide a step-by-step pipeline to classify new species in the family Geminiviridae.

Bioactivity Features of Novel Actinobacteria Isolated from Lichen and Orchid Plant

A total of 10 actinobacterial strains were isolated from a lichen and an orchid species by employing a selective isolation procedure for actinobacteria. Antimicrobial activities of the isolates were evaluated against Aspergillus niger ATCC 16404, Candida albicans ATCC 10231, Enterococcus faecalis ATCC 29212, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 700603, Pseudomonas aeroginosa ATCC 27853, Salmonella enterica ATCC 13311, Staphylococcus aureus ATCC 29213 and Listeria monocytogenes NCTC 5348. Out of the 10 isolates, eight showed antimicrobial activity against at least one pathogen. For molecular identification of the strains, 16S rRNA gene sequence analysis was performed. The pairwise comparison of the 16S rRNA gene sequences of the strains with the databases showed that the strains are members of the genus Streptomyces by sharing 98.9 – 100% gene sequence similarities. Phylogenetic relationships of the strains within the genus Streptomyces were revealed by maximum likelihood and maximum parsimony phylogenetic trees. The results revealed that unexplored environmental habitats like lichens and plant tissues may represent a potential reservoir for novel actinobacteria with promising bioactivity features.

First report of Capsicum chlorosis virus naturally infecting Ageratum conyzoides in China.

Capsicum chlorosis virus (CaCV; family Tospoviridae, genus Orthotospovirus) was first reported to infect capsicum (Capsicum annuum) and tomato (Solanum lycopersicum) in Australia in 2002 (McMichael et al., 2002). Subsequently, its infection was detected in different plants including waxflower (Hoya calycina Schlecter) in the United States (Melzer et al. 2014), peanut (Arachis hypogaea) in India (Vijayalakshmi et al. 2016), and spider lily (Hymenocallis americana) (Huang et al. 2017), Chilli pepper (Capsicum annuum) (Zheng et al. 2020), and Feiji cao (Chromolaena odorata) (Chen et al. 2022) in China. Ageratum conyzoides L. (commonly known as goat weed, family Asteraceae) is a natural weed in crop fields distributed in subtropical and tropical areas and a reservoir host of numerous plant pathogens (She et al. 2013). In April 2022, we observed that 90% of plants of A. conyzoides in maize fields in Sanya, Hainan province, China, exhibited typical virus-like symptoms of vein yellowing, leaf chlorosis, and distortion (Fig. S1 A-C). Total RNA was extracted from one symptomatic leaf of A. conyzoides. Small RNA libraries were constructed using the small RNA Sample Pre Kit (Illumina, San Diego, USA) for sequencing with an Illumina Novaseq 6000 platform (Biomarker Technologies Corporation, Beijing, China). A total 15,848,189 clean reads were obtained after removing low-quality reads. Quality-controlled qualified reads were assembled into contigs using Velvet 1.0.5 software with a k-mer value of 17. One hundred contigs shared nucleotide identity ranging from 85.7% to 100% with the CaCV using BLASTn searches online (https://blast.ncbi.nlm.nih.gov/Blast.cgi?). Numerous contigs (45, 34, and 21) obtained in this study were mapped to the L, M, and S RNA segments of the CaCV-Hainan isolate (GenBank accession no. KX078565- KX078567) from spider lily (Hymenocallis americana) in Hainan province, China, respectively. The full-length of L, M, and S RNA segments of CaCV-AC were determined to be 8,913, 4,841, and 3,629 bp, respectively (GenBank accession no. OQ597167- OQ597169). Furthermore, five symptomatic leaf samples were tested to be positive for CaCV using a CaCV enzyme-linked immunosorbent assay (ELISA) kit (MEIMIAN, Jiangsu, China) (Fig. S1-D). Total RNA from these leaves was amplified by RT-PCR with two sets of primer pairs. Primers CaCV-F (5'-ACTTTCCATCAACCTCTGT-3') and CaCV-R (5'-GTTATGGCCATATTTCCCT-3') were used for the amplification of 828 bp fragment from nucleocapsid protein (NP) on CaCV S RNA. While another, primers gL3637 (5'-CCTTTAACAGTDGAAACAT-3') and gL4435c (5'-CATDGCRCAAGARTGRTARACAGA-3') were used for the amplification of 816 bp fragment from RNA-dependent RNA polymerase (RdRP) on CaCV L RNA (Fig. S1-E and -F) (Basavaraj et al. 2020). These amplicons were cloned into the pCE2 TA/Blunt-Zero vector (Vazyme, Nanjing, China) and three independent positive colonies of Escherichia coli DH5α carrying each viral amplicon were sequenced. These sequences were deposited in the GenBank database under accession nos. OP616700-OP616709. Pairwise sequence comparison revealed that nucleotide sequences of NP and RdRP genes of the five CaCV isolates shared 99.5% (812 bp out of 828 bp) and 99.4% (799 bp out of 816 bp) nucleotide identities, respectively. They showed 86.2-99.2% and 86.5-99.1% nucleotide identities with corresponding nucleotide sequences of other CaCV isolates derived from GenBank database, respectively. The highest nucleotide sequence identity (99%) of the CaCV isolates obtained in the study was observed with the CaCV-Hainan isolate. Phylogenetic analysis based on NP amino acid demonstrated that six CaCV isolates (this study = 5 and NCBI database = 1) clustered into one distinct clade (Fig. S2). Our data confirmed for the first time the presence of CaCV naturally infecting A. conyzoides plant in China, which enriches information on the host range and will be helpful for disease management.

Corynebacterium lipophilum sp. nov., a lipophilic bacterium isolated from clinical breast specimens and emended description of the species Corynebacterium pilbarense.

Two isolates (MC-18T and MC-17D), representing the Gram-stain-positive, facultatively anaerobic, irregular rod-shaped, non-motile, and non-spore-forming actinobacteria, were isolated from clinical breast specimens in Guangzhou, China. The growth of the isolates is enhanced by supplementing 1% Tween-80 on Luria Bertani agar. Optimal growth of the isolates was observed at 37°C, pH 7-8, and with 1% (w/v) NaCl on Columbia blood agar. Pairwise comparison of the 16S rRNA gene sequences revealed that isolates MC-18T and MC-17D shared the highest sequence similarities with Corynebacterium liangguodongii 2184T (96.9%), which were lower than the threshold value for species delineation (98.65%). Phylogenetic dendrograms based on the 16S rRNA gene, rpoB gene, and core genomes indicated that two isolates formed a distinct lineage within the genus Corynebacterium. The estimated dDDH, ANIb, ANIm, and AAI values between strain MC-18T and its closely related strains were below the threshold values generally considered for recognizing a new species. The genome DNA G + C contents of both the isolates MC-18T and MC-17D are 60.6%. The two isolates have virulence-related genes of the VF classes of adhesion and antiphagocytosis, and also contain the antimicrobial resistance genes ErmX, mtrA, rpoB2, and RbpA. The major fatty acids (> 10%) of isolates MC-18T and MC-17D were C16:0, C18:1 ω9c, C18:0 and summed feature 5 (anteiso-C18:0 and/or C18:2 ω6,9c). The main respiratory quinone of strain MC-18T was MK-8(H2), and the polar lipids consisted of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, three unidentified glycolipids, an unidentified aminolipid, and four unidentified phosphoglycolipids. The two isolates lack mycolic acids in the cell envelope. Based on the above findings, the two isolates are considered to represent a novel species of the genus Corynebacterium, for which the name Corynebacterium lipophilum sp. nov. is proposed, with isolate MC-18T (= NBRC 115144T = CCTCC AB 2020201T) as the type strain. An emended description of the Corynebacterium pilbarense is also provided.

Genome-based classification of Streptomyces anatolicus sp. nov., an actinobacterium with antimicrobial and cytotoxic activities, and reclassification of Streptomyces nashvillensis as a later heterotypic synonym of Streptomyces tanashiensis.

During the course of isolating novel actinobacteria producing bioactive metabolites, strain BG9HT was obtained from an arid soil sample in Erzurum, Turkey. Pairwise sequence comparisons for 16S rRNA gene sequences showed the strain was a member of the genus Streptomyces and it shared the highest 16S rRNA gene sequence identity of 99.7% with Streptomyces huasconensis HST28T. Comparative genome analyses based on digital DNA-DNA hybridization and average nucleotide identity revealed that strain BG9HT represents a novel species within the genus Streptomyces. The polyphasic analysis also confirmed that the strain has typical characteristics of the genus Streptomyces. The strain has LL-diaminopimelic acid as diagnostic amino acid, and galactose, mannose and trace amounts of glucose and ribose as whole-cell sugars. Polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, unidentified aminolipids, phospholipids and lipids. Major isoprenoid quinones were MK-9(H6), MK-9(H4), and MK-9(H8). Its genome size is approximately 7.2Mb with 71.2% G+C content. The methanolic extract of strain BG9HT showed antimicrobial and cytotoxic activities. Further genomic analyses of strain BG9HT confirmed its high potential to produce novel secondary metabolites. On the basis of phenotypic and phylogenetic analyses, strain BG9HT represents a novel species of the genus Streptomyces, for which Streptomyces anatolicus sp. nov. is proposed, and it holds high promise for novel biosynthetic metabolites of value to the biopharmaceutical industry. We also propose Streptomyces nashvillensis as a later heterotypic synonym of Streptomyces tanashiensis as a result obtained through analysis of overall genome relatedness indices.

PhyBWT2: phylogeny reconstruction via eBWT positional clustering

BackgroundMolecular phylogenetics studies the evolutionary relationships among the individuals of a population through their biological sequences. It may provide insights about the origin and the evolution of viral diseases, or highlight complex evolutionary trajectories. A key task is inferring phylogenetic trees from any type of sequencing data, including raw short reads. Yet, several tools require pre-processed input data e.g. from complex computational pipelines based on de novo assembly or from mappings against a reference genome. As sequencing technologies keep becoming cheaper, this puts increasing pressure on designing methods that perform analysis directly on their outputs. From this viewpoint, there is a growing interest in alignment-, assembly-, and reference-free methods that could work on several data including raw reads data.ResultsWe present phyBWT2, a newly improved version of phyBWT (Guerrini et al. in 22nd International Workshop on Algorithms in Bioinformatics (WABI) 242:23–12319, 2022). Both of them directly reconstruct phylogenetic trees bypassing both the alignment against a reference genome and de novo assembly. They exploit the combinatorial properties of the extended Burrows-Wheeler Transform (eBWT) and the corresponding eBWT positional clustering framework to detect relevant blocks of the longest shared substrings of varying length (unlike the k-mer-based approaches that need to fix the length k a priori). As a result, they provide novel alignment-, assembly-, and reference-free methods that build partition trees without relying on the pairwise comparison of sequences, thus avoiding to use a distance matrix to infer phylogeny. In addition, phyBWT2 outperforms phyBWT in terms of running time, as the former reconstructs phylogenetic trees step-by-step by considering multiple partitions, instead of just one partition at a time, as previously done by the latter.ConclusionsBased on the results of the experiments on sequencing data, we conclude that our method can produce trees of quality comparable to the benchmark phylogeny by handling datasets of different types (short reads, contigs, or entire genomes). Overall, the experiments confirm the effectiveness of phyBWT2 that improves the performance of its previous version phyBWT, while preserving the accuracy of the results.

Epidemiology and genotypic diversity of canine circovirus identified in pet dogs in Harbin, China

Canine circovirus (CanineCV) is a single-stranded DNA virus that circulates in dogs and wild carnivores around the world. It has been suggested to be associated with diseases of respiratory and gastrointestinal systems, though its pathogenic potential remains unclear. Currently, CanineCV is divided into six genotypes (genotype 1−6), and genotypes 2, 3, and 4 have been described in China. In this study, 359 blood samples from pet dogs with or without clinical signs were collected in Harbin city. After PCR screening, a total of 34 samples were tested positive for CanineCV, and nine full-length genome sequences were recovered from positive samples. Pairwise sequence comparison showed that they shared 82.4–99.3% genome-wide identity with other CanineCVs available in GenBank. Additionally, recombination events were detected, all of which were determined to be associated with sequences obtained in China. The reconstructed phylogenetic tree based on the recombination-free complete genome sequences revealed that the complete genome sequences generated herein were clustered into genotypes 1 and 3. Furthermore, purifying selection was the dominant evolutionary pressure acting on the genomes of CanineCV. These results expand the knowledge about the genetic diversity of CanineCV circulating in China, and also promote us to better understand the evolution of CanineCV.

First report of Colletotrichum shisoi causing anthracnose of Perilla frutescens in the United States.

Perilla mint (Perilla frutescens (L.) Britt.) is an annual plant native to Asia and considered invasive in North America where it has escaped cultivation as an ornamental (Miller 1947; Swearingen et al. 2010). In August 2021, an anthracnose disease was observed on invasive perilla found along the disturbed margins of a forest in Frederick County, Maryland, United States. Symptoms included necrotic lesions with chlorotic halos, were concentrated in the lower canopy, and caused premature defoliation of lower leaves (Figure S1). Leaves from four plants were surface sterilized by rinsing for 30 s in 70% ethanol, 60 s in 0.8% NaClO, and 60 s in sterile water and then incubated on 2% water agar under ambient laboratory conditions to permit sporulation. After three days, spores that exuded from individual lesions were streaked onto acidified potato dextrose agar. Two single-conidium isolates were recovered from each plant. All eight isolates were identified to species using DNA sequences. A single isolate (21-067) was selected at random for morphological characterization and completion of Koch's postulates. Morphological features were recorded after seven days of growth on synthetic low-nutrient agar (SNA) and potato dextrose agar (PDA) incubated at 22°C under 12 hr UV-B and white fluorescent lighting. Measurements were based on a minimum of 20 observations per structure. Cultures on SNA were flat, hyaline to pale salmon, lacked sporodochia and grew at a rate of 1.3 mm day-1 (n = 3). Vegetative hyphal width was (minimum-maximum) 1.5-4.0 μm, (average ± standard deviation) 2.7 ± 0.9 μm. Conidiophores arose directly from hyphae, were hyaline, smooth, unbranched and measured 40.0-180.0 x 1.5-4.5 μm, 102.3 ± 33.9 x 2.7 ± 0.8 μm. Conidia were single celled, straight, hyaline, glabrous, rounded at both ends and measured 10.0-20.0 x 3.8-6.3 μm, 15.4 ± 2.5 x 4.9 ± 0.7 μm. Setae, observed only on PDA, were pale brown, 1-2 septate, straight, blunt tipped and measured 42.5-97.5 μm, 70.8 ± 13.4 μm. Appressoria formed on PDA were single-celled, pigmented, smooth, and obovoid with entire margins, measuring 5.2-7.7 x 8.6-13.8 μm, 6.8 ± 0.6 x 10.8 ± 1.4 μm. These characteristics were consistent with members of the Colletotrichum destructivum species complex. Partial DNA sequences from five loci were amplified following the procedures of Damm et al. 2014: internal transcribed spacer region of rDNA (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase 1 (CHS-1), actin (ACT), and beta-tubulin (TUB2). Pairwise sequence comparisons to references using the Blastn algorithm found 99.8% similarity between isolate 21-067 and ex-type C. shisoi isolate JCM 31818: MH660930 (ITS, 545/546 bp), MH660931 (GAPDH,192/192 bp), MH660929 (CHS-1, 278/280 bp), MH660928 (ACT, 262/262 bp), and MH660932 (TUB2, 511/511 bp) (Altschul et al. 1990; Gan et al. 2019). Subsequently, sequences from all eight isolates were aligned with Clustal Omega (Sievers and Higgins 2018), concatenated, and used in a Bayesian phylogenetic reconstruction (Huelsenbeck and Ronquist 2001) of the C. destructivum species complex (Figure S2), confirming the species identity as C. shisoi. Sequences were deposited in NCBI GenBank under accession numbers OM865277-OM865284 and OM885059-OM885090. Koch's postulates were fulfilled using perilla grown in a greenhouse until second true leaves emerged. Inoculum washed from two-week-old fungal cultures grown on potato dextrose agar was adjusted to 4 x 104 conidia mL-1 and applied to three replicate plants with an aspirator until runoff. Three plants were sprayed with sterile water as a negative control. Plants were covered with plastic bags for 72 hrs and maintained in a growth chamber at 20°C and 80% RH for 14 days. Inoculated plants displayed disease symptoms similar to those observed under field conditions, and control plants did not develop symptoms. Colletotrichum shisoi was reisolated from symptomatic tissue and reidentified based on morphology. The experiment was completed twice. To the authors' knowledge, this is the first report of C. shisoi on P. frutescens in the United States. Colletotrichum shisoi has not been reported as a pathogen on other plants in the United States and may have potential use as a biological control agent for invasive perilla.

Comparative Genomic Analysis Reveals Genetic Diversity and Pathogenic Potential of Haemophilus seminalis and Emended Description of Haemophilus seminalis.

Haemophilus seminalis is a newly proposed species that is phylogenetically related to Haemophilus haemolyticus. The distribution of H. seminalis in the human population, its genomic diversity, and its pathogenic potential are still unclear. This study reports the finding of our comparative genomic analyses of four newly isolated Haemophilus strains (SZY H8, SZY H35, SZY H36, and SZY H68) from human sputum specimens (Guangzhou, China) along with the publicly available genomes of other phylogenetically related Haemophilus species. Based on pairwise comparisons of the 16S rRNA gene sequences, the four isolates showed <98.65% sequence identity to the type strains of all known Haemophilus species but were identified as belonging to H. seminalis, based on comparable phenotypic and genotypic features. Additionally, the four isolates showed high genome-genome relatedness indices (>95% ANI values) with 17 strains that were previously identified as either "Haemophilus intermedius" or hemin (X-factor)-independent H. haemolyticus and therefore required a more detailed classification study. Phylogenetically, these isolates, along with the two previously described H. seminalis isolates (a total of 23 isolates), shared a highly homologous lineage that is distinct from the clades of the main H. haemolyticus and Haemophilus influenzae strains. These isolates present an open pangenome with multiple virulence genes. Notably, all 23 isolates have a functional heme biosynthesis pathway that is similar to that of Haemophilus parainfluenzae. The phenotype of hemin (X-factor) independence and the analysis of the ispD, pepG, and moeA genes can be used to distinguish these isolates from H. haemolyticus and H. influenzae. Based on the above findings, we propose a reclassification for all "H. intermedius" and two H. haemolyticus isolates belonging to H. seminalis with an emended description of H. seminalis. This study provides a more accurate identification of Haemophilus isolates for use in the clinical laboratory and a better understanding of the clinical significance and genetic diversity in human environments. IMPORTANCE As a versatile opportunistic pathogen, the accurate identification of Haemophilus species is a challenge in clinical practice. In this study, we characterized the phenotypic and genotypic features of four H. seminalis strains that were isolated from human sputum specimens and propose the "H. intermedius" and hemin (X-factor)-independent H. haemolyticus isolates as belonging to H. seminalis. The prediction of virulence-related genes indicates that H. seminalis isolates carry several virulence genes that are likely to play an important role in its pathogenicity. In addition, we depict that the genes ispD, pepG, and moeA can be used as biomarkers for distinguishing H. seminalis from H. haemolyticus and H. influenzae. Our findings provide some insights into the identification, epidemiology, genetic diversity, pathogenic potential, and antimicrobial resistance of the newly proposed H. seminalis.

Exploration of Multi-Gene DNA Barcode Markers to Reveal the Broad Genetic Diversity of Field Ticks (Acari: Ixodidae) in a Tropical Environment of Hainan Island, China

Ticks are hematophagous arthropods and obligate ectoparasites of humans and other animals. This study focused on the molecular discrimination of ticks in the tropical environment of Hainan according to multi-gene DNA barcode markers with the expectation of accurately distinguishing species. A total of 420 ticks, including 49 adult ticks, 203 nymphal ticks, and 168 larval ticks, were collected in the field, and the 49 adult ticks were identified as Rhipicephalus turanicus, Dermacentor marginatus, and Haemaphysalis longicornis. The mitochondrial 16S rRNA, ribosomal 28S rRNA D2, and ribosomal internal transcribed spacer 2 (ITS2) regions were used as DNA barcode markers to discriminate species. According to basic local alignment search tool analysis against the GenBank database, 16S rRNA positively identified ticks in the Rhipicephalus, Dermacentor, and Haemaphysalis genera; the 28S rRNA D2 region identified ticks in the Rhipicephalus and Dermacentor genera; and ITS2 identified ticks as D. marginatus. Pairwise sequence comparisons based on these three regions were visualized with a Sequence Demarcation Tool matrix. Substitution saturation tests using data analysis and molecular biology and evolution revealed little substitution saturation (Iss < Iss.c, p < 0.05) in the 16S rRNA region for the Haemaphysalis genus; 28S rRNA D2 region for the Rhipicephalus, Dermacentor, and Haemaphysalis genera; and ITS2 region for the Rhipicephalus and Dermacentor genera. Distinctive sequences for which it is difficult to obtain good matches with the sequences available in GenBank exist in the ticks of Hainan. Future studies should obtain complementary sequences to refine and update the database for the molecular characterization of ticks.

Henneguyosis: A novel threat to the exotic channel catfish Ictalurus punctatus cultivated in China

Channel catfish Ictalurus punctatus is a freshwater fish of economic importance and has been worldwide introduced for aquaculture purposes. China has become the main producer of channel catfish and also the primary casualty of significant economic loss caused by various contagious diseases. Hence, surveillance of the causative agents can contribute to catfish industry security. In this study, the branchial infections that emerged in the domesticated channel catfish were reported in China, and three species of Henneguya were each determined to be responsible for the infection in single locality. Among these, the species isolated from Zhijiang City exhibited multifocal infection on the gills. It showed elongated lanceolate myxospores measured 14.8 ± 0.9 (12.6–16.7) × 3.9 ± 0.3 (3.4–4.7) × 3.5 ± 0.2 (2.9–3.9) μm and was distinct from the measurement of other congeners. A blast search against all sequences available in the Genbank database showed that H. exilis of 97.35% identity is the highest similarity to the present species. Accordingly, we denominated the Zhijiang isolates as Henneguya extensa n. sp. The second species were pooled from the surface of the gill filaments of catfish specimen sampled from Anqing City, showing myxospores measured 14 ± 1.3 (11.6–16.6) × 3.6 ± 0.2 (3.1–4) × 3.1 ± 0.2 (2.8–3.6) μm. It was identified as a species novel to the science based on the characteristic morphology and morphometrics. Therefore, we denominated it Henneguya anqingensis n. sp. A heavy burden of plasmodia formed by the third species that is destructive to the gill integrity was encountered in Yancheng City. By the identical morphology and overlapping morphometrics, this species was assigned to be Henneguya postexilisMinchew, 1977 initially reported from the USA. A pairwise sequence comparison and an evolution divergence analysis indicated a conspecific nature of the American and Chinese populations of H. postexilis. Phylogenetic analysis placed H. extensa n. sp. and H. postexilis in a clade comprising species predominantly infecting Ictaluridae fish native to North America. Histologically, the aggressive propagation of both H. extensa n. sp. and H. postexilis in the gills elicited extensive lamellar deformation and conspicuous occupation in the respiratory areas, potentially imposing survival pressure by suffocating the infected fish. Overall, the emergence of the branchial Henneguyosis was determined and, as the population of Henneguya species increased by the dense anthropogenic activity, may threaten the channel catfish industry in China.