Nucleotide Sequence Alignment Research Articles

Whole Genome Sequencing (WGS) Analysis of Antimicrobial Resistance (AMR) Milk and Dairy-Derived Pathogens from Anand, Gujarat, India

This study aimed to evaluate the antimicrobial resistance (AMR) patterns and genomic characteristics of Enterococcus faecalis, Enterococcus faecium, and Staphylococcus aureus strains isolated from dairy products, including buttermilk, curd, ice cream, and sweets, in the Anand region of Gujarat, India. A total of 205 isolates were analyzed, with the highest contamination levels found in buttermilk and curd. The bacterial isolates were identified using biochemical tests and advanced Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) mass spectrometry. Antimicrobial susceptibility testing (AST) was performed using the Kirby-Bauer disk diffusion method, following CLSI guidelines, focusing on common antibiotics used for treating dairy-related bacterial infections. Resistance profiles were analyzed using WHONET software.s The findings revealed significant multidrug resistance (MDR), particularly among E. faecium and E. faecalis, with over 95% resistance to key antibiotics, including linezolid, ciprofloxacin, cefpodoxime, and carbapenems. Many strains were classified as MDR, XDR, and PDR. Staphylococcus aureus also exhibited substantial resistance to penicillin and enrofloxacin. whole-genome sequencing (WGS) and phylogenetic analysis to identify AMR determinants and conduct nucleotide sequence alignment. The study identified several antibiotic resistance genes, including LiaF, which regulates the expression of LiaR and LiaS genes. WGS revealed that genes such as GdpD, MprF, and PgsA encode intrinsic resistance determinants, contributing to antibiotic resistance. Additional AMR mechanisms were identified, including ABC transporter efflux pumps and the regulation of resistance genes by LiaR and LiaS. Phylogenetic analysis indicates a close relationship between Enterococcus faecium 640 1352.18624 and Enterococcus durans FB129-CNAB-4 883162.3.

First Report of Klebsiella variicola Causing Tomato Pith Necrosis in Yunnan Province, China.

Tomato (Solanum lycopersicum L.) is a key vegetable crop in China. In August 2023, an outbreak of bacterial pith necrosis in tomato occurred in Lufeng County, Yunnan Province, China, affecting over 40% of the tomato plants in a greenhouse. The stems of infected plants developed a waterlogged soft rot and the disease progressed, the lower leaves and lateral branches of infected plants gradually wilted and died. A longitudinal cut of the stem revealed hollow pith with brown vascular tissue. To isolate the pathogen, the plant surface was disinfested with 75% ethanol. Then, a piece of infected tissue from the base of the stem was excised and immersed in sterile water for 2 min. A small amount of liquid was streaked onto TTC (2,3,5-triphenyltetrazolium chloride) agar medium using an inoculation loop, and plates were incubated at 28℃ for 24 h. Colonies on the TTC plate were white, indicating that the pathogen was not Ralstonia solanacearum. Colonies grown on LB (Luria-Bertani) agar medium were randomly selected and subjected to preliminary pathogenicity tests. Based on the results, a colony named Kv4 was selected and purified through six subcultures in LB agar medium. Biochemical tests showed the strain utilized D-sorbitol, raffinose and citrate but not adonitol, and was positive for methyl red, D-glucose (acid), urea hydrolysis, lysine decarboxylase, and motility, and negative for phenylalanine deaminase, H2S production, indole production, and ornithine decarboxylase. These characteristics align with Klebsiella species (Garrity et al. 2007). To determine the species of strain Kv4, partial sequences of the 16S rDNA, phoE, leuS, and rpoB genes were amplified (Barrios-Camacho et al. 2019) and sequenced. Through BLASTn analysis, strain Kv4 sequences of 16S rDNA (OR888750) had 99.47% identity (1488/1496 bp), phoE (OR899599) had 98.69% (605/613 bp) identity, leuS (OR899598) had 99.07% identity (959/968 bp), and rpoB (OR899597) had 97.69% (633/648 bp) identity with Klebsiella variicola strain FF0907. Using the ClustalW algorithm in MEGA11 for nucleotide sequence alignment, phylogenetic trees were constructed with 16S and phoE, leuS, and rpoB via the neighbor-joining method, confirming strain Kv4 as K. variicola. To test pathogenicity, the roots of 25 'Moneymaker' tomato plants with four to five true leaves were wounded, then each plant inoculated with a 15 mL bacterial suspension (OD600=0.6) of strain Kv4, while the control plants received sterile water. Plants were incubated at 28℃ with a 16 h photoperiod. Experiments were done twice. At 15 days after inoculation (DAI), all plants inoculated with Kv4 showed yellowing, unevenly distributed small black necrotic spots on the leaf surface, and purple-brown soft rot at the stem base. By 18 DAI, there was a gradual transformation of the stem bases from green to purplish brown. At 21 DAI, 60% of the inoculated plants displayed brownish soft rot at the stem base. In contrast, the control plants remained symptom-free. The pathogen was re-isolated from the stem and identified as K. variicola via sequence analysis of 16S, phoE, leuS, and rpoB. In recent years, several new bacterial pith necrosis diseases were reported in tomato (Guo et al. 2023; Ivić et al. 2023). This is the first study documenting K. variicola causing bacterial pith necrosis in tomato. Once considered a benign plant endophyte, Sun et al. (2023) reported K. variicola causing banana sheath rot in Guangdong and Guangxi Provinces, China. Malik et al. (2023) reported that K. variicola caused leaf streak in sorghum in India. This report of bacterial pith necrosis in tomato caused by K. variicola strain Kv4 underscores the escalating threat posed by emerging pathogens to agricultural production. The emergence of K. variicola as a tomato pathogen complicates plant disease management strategies.

Development and implementation of a core genome multilocus sequence typing scheme for Haemophilus influenzae.

Haemophilus influenzae is part of the human nasopharyngeal microbiota and a pathogen causing invasive disease. The extensive genetic diversity observed in H. influenzae necessitates discriminatory analytical approaches to evaluate its population structure. This study developed a core genome multilocus sequence typing (cgMLST) scheme for H. influenzae using pangenome analysis tools and validated the cgMLST scheme using datasets consisting of complete reference genomes (N = 14) and high-quality draft H. influenzae genomes (N = 2297). The draft genome dataset was divided into a development dataset (N = 921) and a validation dataset (N = 1376). The development dataset was used to identify potential core genes, and the validation dataset was used to refine the final core gene list to ensure the reliability of the proposed cgMLST scheme. Functional classifications were made for all the resulting core genes. Phylogenetic analyses were performed using both allelic profiles and nucleotide sequence alignments of the core genome to test congruence, as assessed by Spearman's correlation and ordinary least square linear regression tests. Preliminary analyses using the development dataset identified 1067 core genes, which were refined to 1037 with the validation dataset. More than 70% of core genes were predicted to encode proteins essential for metabolism or genetic information processing. Phylogenetic and statistical analyses indicated that the core genome allelic profile accurately represented phylogenetic relatedness among the isolates (R 2 = 0.945). We used this cgMLST scheme to define a high-resolution population structure for H. influenzae, which enhances the genomic analysis of this clinically relevant human pathogen.

First report of Tomato interveinal chlorosis virus infecting Rhynchosia minima plants in Brazil.

Tomato interveinal chlorosis virus (ToICV; Begomovirus solanumintervenae, genus Begomovirus, family Geminiviridae) has been described infecting tomato (Solanum lycopersicum) and Macroptilium lathyroides in Northeastern (NE) Brazil for more than a decade (Albuquerque et al., 2012; Silva et al., 2012). During a survey in 2020, plants of the leguminous weed Rhynchosia minima exhibiting virus-like symptoms such as mosaic and interveinal chlorosis were observed in the state of Alagoas, NE Brazil. Symptomatic leaf samples of R. minima were randomly collected (n=15; supplementary figure 1). Total DNA from each sample was used as a template for PCR amplification of partial begomoviral DNA-A sequences using the degenerate primer pair PAL1v1978 and PAR1c496, universal for geminiviruses (Rojas et al., 1993). Amplicons of ~1.2 kbp were observed from 12 samples, although this should not be considered as incidence since only symptomatic plants were collected. To identify the begomovirus associated with R. minima, viral genomes were amplified from PCR-positive samples using rolling circle amplification (RCA) (Inoue-Nagata et al., 2004). The RCA products were digested with HindIII, cloned into the pBluescript II KS+ plasmid vector and bidirectionally Sanger-sequenced (Macrogen Inc., Seoul). BLASTn searches indicated that the clones (n=4) reported here corresponded to a begomovirus DNA-A component, and pairwise comparisons showed that they shared the highest identity with ToICV, at 92.4-94.7% nucleotide sequence identity. Based on the species demarcation criteria of ≥91% nucleotide identity for the genus Begomovirus (Brown et al., 2015), the begomoviruses obtained from R. minima are new isolates of ToICV. The new DNA-A sequences of 2,619-2,623 nt in length were deposited in GenBank under accession numbers PP639092 to PP639095. Multiple nucleotide sequence alignments were prepared using the MUSCLE algorithm implemented in MEGA v.11 (Kumar et al., 2018), and a maximum likelihood (ML) tree was reconstructed in RaxML-NG (Kozlov et al., 2019), assuming a general time reversible (GTR) nucleotide substitution model with a gamma (G) model of rate heterogeneity and 1,000 bootstrap replicates. The DNA-A-based tree showed that the ToICV sequences clustered into a monophyletic group, additionally supporting these isolates as members of the species Begomovirus solanumintervenae. At least two independent interspecies recombination events were predicted among the ToICV isolates, with breakpoints located in the Rep-encoding region and ToICV (GenBank Accession JF803253), tomato mottle leaf curl virus (JF803248) and soybean blistering mosaic virus (MN486865) detected as putative parents. To the best of our knowledge, this is the first report of ToICV infecting R. minima worldwide, expanding the host range of this begomovirus. Non-cultivated plants such as R. minima play a crucial role as reservoirs and sources of inoculum for begomoviruses (Paz-Carrasco et al., 2014), reinforcing their relevance to socioeconomically important crops.

Wild-type Caenorhabditis sinica, a model nematode for speciation and evolution, massively found in Vietnam

Caenorhabditis sinica is a male/female species, in which the genetic diversity is possibly high. Thus, the species is advantageous for the understanding of the mechanisms of diversity, evolution, and adaptation within the nematode genus Caenorhabditis. Previous studies reported the geographic distribution of C. sinica only in China; nonetheless, this should be more convincing for its ecology. We allegedly surveyed the Caenorhabditis species and their diversity in the forests of Vietnam. We found 59 C. sinica strains, and this therefore indicates the vast variation that is 85.88% and 100% identical within isolated wild-type strains. They unequally present in habitats and are enormous in the northern forest, and rarer in the southern forest. The comparison of 18S rDNA barcode sequences from 59 C. sinica isolates by nucleotide sequence alignment showed the consistent diversity among the strains in or off the same ecologies, and all are comparable with the ever-first isolated strain JU727.

Effect of tokenization on transformers for biological sequences.

Deep-learning models are transforming biological research, including many bioinformatics and comparative genomics algorithms, such as sequence alignments, phylogenetic tree inference, and automatic classification of protein functions. Among these deep-learning algorithms, models for processing natural languages, developed in the natural language processing (NLP) community, were recently applied to biological sequences. However, biological sequences are different from natural languages, such as English, and French, in which segmentation of the text to separate words is relatively straightforward. Moreover, biological sequences are characterized by extremely long sentences, which hamper their processing by current machine-learning models, notably the transformer architecture. In NLP, one of the first processing steps is to transform the raw text to a list of tokens. Deep-learning applications to biological sequence data mostly segment proteins and DNA to single characters. In this work, we study the effect of alternative tokenization algorithms on eight different tasks in biology, from predicting the function of proteins and their stability, through nucleotide sequence alignment, to classifying proteins to specific families. We demonstrate that applying alternative tokenization algorithms can increase accuracy and at the same time, substantially reduce the input length compared to the trivial tokenizer in which each character is a token. Furthermore, applying these tokenization algorithms allows interpreting trained models, taking into account dependencies among positions. Finally, we trained these tokenizers on a large dataset of protein sequences containing more than 400 billion amino acids, which resulted in over a 3-fold decrease in the number of tokens. We then tested these tokenizers trained on large-scale data on the above specific tasks and showed that for some tasks it is highly beneficial to train database-specific tokenizers. Our study suggests that tokenizers are likely to be a critical component in future deep-network analysis of biological sequence data. Code, data, and trained tokenizers are available on https://github.com/technion-cs-nlp/BiologicalTokenizers.

Phenol biodegradation using bio-filter tower packed column with immobilized bacterial consortium: a batch test study.

The effluents from pulp and paper manufacturing industries contain high concentrations of phenol, which when discharged directly into surface water streams, increases the biological oxygen demand (BOD) and chemical oxygen demand (COD). In this study, two dominant bacteria SP-4 and SP-8 were isolated from the effluent emanating with a pulp and paper industry. The selected phenol-degrading isolates were identified as Staphylococcus sp. and Staphylococcus sciuri respectively by using nucleotide sequence alignment and phylogenetic analysis of 16S rRNA regions of the genome. The two isolates used for the biodegradation process effectively degraded phenol concentration of pulp and paper industry effluent upto 1600 and 1800mg/L resepctively. The individual isolates and consortium were immobilized using activated carbon, wood dust, and coal ash. Additionally, the effluent was treated using a bio-filter tower packed column immobilized with bacterial cells at a constant flow rate of 5 mL/min. The present study showed that the developed immobilized microbial consortium can effectively degrade 99% of the phenol present in pulp and paper industry effluents, resulting in a significant reduction in BOD and COD of the system. This study can be well implemented on real-scale systems as the bio-filter towers packed with immobilized bacterial consortium can effectively treat phenol concentrations up to 1800mg/L. The study can be implemented for bioremediation processes in phenolic wastewater-contaminated sites.

BSAlign: A Library for Nucleotide Sequence Alignment.

Increasing the accuracy of the nucleotide sequence alignment is an essential issue in genomics research. Although classic dynamic programming (DP) algorithms (e.g., Smith-Waterman and Needleman-Wunsch) guarantee to produce the optimal result, their time complexity hinders the application of large-scale sequence alignment. Many optimization efforts that aim to accelerate the alignment process generally come from three perspectives: redesigning data structures [e.g., diagonal or striped Single Instruction Multiple Data (SIMD) implementations], increasing the number of parallelisms in SIMD operations (e.g., difference recurrence relation), or reducing search space (e.g., banded DP). However, no methods combine all these three aspects to build an ultra-fast algorithm. In this study, we developed a Banded Striped Aligner (BSAlign) library that delivers accurate alignment results at an ultra-fast speed by knitting a series of novel methods together to take advantage of all of the aforementioned three perspectives with highlights such as active F-loop in striped vectorization and striped move in banded DP. We applied our new acceleration design on both regular and edit distance pairwise alignment. BSAlign achieved 2-fold speed-up than other SIMD-based implementations for regular pairwise alignment, and 1.5-fold to 4-fold speed-up in edit distance-based implementations for long reads. BSAlign is implemented in C programing language and is available at https://github.com/ruanjue/bsalign.

Kelch 13 gene polymorphisms of Plasmodium falciparum among human population in Nasarawa-West Senatorial District, Nasarawa State, Nigeria

Kelch-13 is a gene produced by Plasmodium falciparum, one of the species of Malaria parasite. Malaria remains a major public health challenge especially in Sub-Saharan Africa, a major cause of mortality and morbidity, especially in children and pregnant women. WHO recommends Artemisinin-based combination therapies as the first-line drugs for the treatment of uncomplicated malaria but the emergence and spread of Artemisinin-resistance associated with mutations in K13 gene poses a threat to ACT efficacy. Detection of mutant K13 gene may provide the information on changes in parasite susceptibility to Artemisinin. This study was aimed at detecting K13 gene polymorphisms among human population in Nasarawa-West Senatorial District, Nigeria. A total of 385 blood samples were collected from selected hospitals and screened for malaria by microscopy (the gold standard). Species specific screening of the P. falciparum was done using RDT. Dried blood spots made from RDT positive samples were investigated for the presence of K13 genes by nested PCR. Sequencing detected mutant K13 gene. Results: 103 samples were positive for Plasmodium falciparum by RDT, PCR confirmed 48 K13 genes with a band size of 848 bp. Nucleotide sequence alignment revealed six Single Nucleotides Polymorphisms. The nucleotide sequences were converted to protein sequences and results showed a point mutation in 1(5.0%) of the 20 Pfkelch 13 gene sequenced. Conclusively, the need for continuous surveillance following the detection of mutant gene in the study population is recommended in order to have a wider picture of the parasite diversity for effective malaria control.

Evidence That Aquaporin 11 (AQP11) in the Spiny Dogfish (Squalus acanthias) May Represent a Pseudogene.

Various attempts to amplify an AQP11 cDNA from tissues of the spiny dogfish (Squalus acanthias) were made. Two pairs of deoxy-inosine-containing degenerate primers were designed based on conserved amino acid sequences from an AQP11 alignment. These primers yielded some faint bands from gill cDNA that were sequenced. Blast searches with the sequences showed they were not AQP11. An elasmobranch AQP11 nucleotide sequence alignment was produced to identify conserved regions to make further degenerate primers. One primer pair produced a short 148 bp fragment showing particularly strong amplification in gill and intestine. It was sequenced and represented a piece of the AQP11 gene. However, as the fragment may have resulted from contaminating genomic DNA (in total RNA used to make cDNA), 5' and 3' RACE were performed to amplify the two ends of the putative cDNA. Furthermore, 5' and 3' RACE amplifications depend on the presence of a 5' cap nucleotide and a poly A tail, respectively on the putative AQP11 mRNA. Hence, successful amplification was only possible from cDNA and not genomic DNA. Nested RACE amplifications were performed using gill and intestinal RACE cDNA, but none of the DNA fragments sequenced were AQP11. Consequently, the spiny dogfish AQP11 gene may represent a pseudogene.

Analysis for Mitochondrial Encoded Transfer RNA Leucine2 (MT-TL2) Gene in Breast Cancer Patients

Background: Breast cancer (BC) has been linked to a variety of nuclear DNA changes as well as mitochondrial DNA (mtDNA) alterations. The study aims to evaluate/analyze the association of mitochondrial transfer RNA (mt-tRNA) leucine 2 gene with BC patients. Methods: In the current study, 24 samples have been collected from various families in Peshawar. DNA was extracted from blood. Polymerase chain reaction was used to amplify the mt-tRNA MT-TL2 gene, and 20 samples were sequenced. Results: The sequence was compared with accession #NC-012920.1 of the Revised Cambridge Reference Sequence (rCRS). The results (chromatograph, nucleotide sequence, and rCRS alignment) show mutations in mt-tRNA MT-TL2 gene in our participants is not the cause of BC. Conclusion: Yet, a significant number of BC patients must be studied, and their full mtDNA must be analyzed. This will provide an indication of the potential DNA marker that might be used to prevent BC deaths at the earliest stages.

Identification of antimicrobial peptide genes from transcriptomes in Mandarin fish (Siniperca chuatsi) and their response to infection with Aeromonas hydrophila

Mandarin fish (Siniperca chuatsi) is a valuable freshwater fish species widely cultured in China. Its aquaculture production is challenged by bacterial septicaemia, which is one of the most common bacterial diseases. Antimicrobial peptides (AMPs) play a critical role in the innate immune system of fish, exhibiting defensive and inhibitory effects against a wide range of pathogens. This study aimed to identify the antimicrobial peptide genes in mandarin fish using transcriptomes data obtained from 17 tissue in our laboratory. Through nucleotide sequence alignment and protein structural domain analysis, 15 antimicrobial peptide genes (moronecidin, pleurocidin, lysozyme g, thymosin β12, hepcidin, leap 2, β-defensin, galectin 8, galectin 9, apoB, apoD, apoE, apoF, apoM, and nk-lysin) were identified, of which 9 antimicrobial peptide genes were identified for the first time. In addition, 15 AMPs were subjected to sequence characterization and protein structure analysis. After injection with Aeromonas hydrophila, the number of red blood cells, hemoglobin concentration, and platelet counts in mandarin fish showed a decreasing trend, indicating partial hemolysis. The expression change patterns of 15 AMP genes in the intestine after A. hydrophila infection were examined by using qRT-PCR. The results revealed, marked up-regulation (approximately 116.04) of the hepcidin gene, down-regulation of the piscidin family genes expression. Moreover, most AMP genes were responded in the early stages after A. hydrophila challenge. This study provides fundamental information for investigating the role of the different antimicrobial peptide genes in mandarin fish in defense against A. hydrophila infection.

Characterization of bovine immunodeficiency virus genes and proteins

The goal of the study was determining the effect of natural selection on the bovine immunodeficiency virus (BIV) through the identification of polymorphisms by comparing a number of genes, open reading frames and proteins and analyzing the effect of nonsynonymous substitutions in the Vif protein of BIV isolates on the Vif conformational parameters. The selection pressure on the genes was estimated by Tajima test, as well as the determination of the ratio of nonsynonymous substitutions (Ka) to synonymous (Ks) Ka/Ks. Ka and Ks coefficients, their ratio (Ka/Ks) were calculated on the basis of alignments of amino acid and nucleotide sequences of the vif gene of BIV isolates. The nonsynonymous (dN) to synonymous (dS) substitution rate ratio per nucleotide site dN/dS was calculated to estimate the effect of selection on the vif gene by SLAC method on Datamonkey server. The number of hydrogen bonds, α-helices, β-sheets, β-turns for the protein tertiary structures of proteins was determined by I-TASSER server. Genes, open reading frames (ORFs) and the U3 regulatory region of BIV genome which are under the influence of selection have been identified. Gag, pol, s, vif genes, ORF W and ORF Y are under the negative (purifying) selection pressure. Env gene and the U3 regulatory region are under positive selection. Shannon entropy analysis (peaks that are specific to the positions of amino acid residues), interpreted as polymorphisms, revealed 16, 8, and 4 nonsynonymous substitutions for Pol, Gag, and Vif BIV proteins, respectively. Significant changes in the number of hydrogen bonds, α-helices, β-layers, β-turns were determined by the analysis of the effect of four nonsynonymous substitutions in the Vif protein on the conformational parameters of Vif of two BIV isolates. The multidirectional effect of selection on BIV genes is shown

Prevalence and genotypes distribution of virus hepatitis B and hepatitis delta virus in chronic liver diseases in Kazakhstan

BackgroundThe geographical distribution of hepatitis B virus (HBV) and hepatitis D virus (HDV) genotypes is uneven and has its own clinical and organizational implications for health systems. Despite the introduction of vaccination and successful antiviral therapy the prevalence of chronic hepatitis B (with or without delta agent) increased over the past 5 years. This study aimed for the first time to investigate the molecular epidemiology of HBV and HDV in Kazakhstan.MethodsTotal 834 chronic hepatitis B (with or without delta agent) patients were included to the study from November 2017 to June 2019. The material was collected from the regional hepatological сenters from 13 cities of Kazakhstan. Genotyping of HBV/HDV isolates was carried out using phylogenetic analysis of null-binary sequences of Kazakhstani isolates, in comparison with the reference sequences. Nucleotide sequence alignment was performed using the ClustalW algorithm, the “neighbor-joining” method was used for the construction of phylogenetic trees and subsequent analysis.ResultsOverall 341 samples were PCR-positive and genotyped for HBV. Comparison and phylogenetic analysis of nucleotide sequences of HBV isolates showed that they were represented by genotypes HBV-D (95.9%), HBV-A (3.5%) and HBV-C (0.6%). At the same time, the identity of the nucleotide sequences of Kazakhstani isolates were: HBV-D (95–100%); HBV-A (97.2–100%) and HBV-C (99%). 256 samples were PCR positive and genotyped for HDV, all of them belonged to genotype 1.ConclusionThis study describes for the first time the molecular epidemiology of HBV and HDV in Kazakhstan. The data obtained expand the knowledge of the global epidemiology of viruses; have potential implications for public health policy and for further clinical research on chronic hepatitis in Kazakhstan.Trial registrationClinicalTrials.gov NCT05095181 (registered on 27/10/2021).

Molecular characterization of emerging chicken and turkey parvovirus variants and novel strains in Guangxi, China

Avian parvoviruses cause several enteric poultry diseases that have been increasingly diagnosed in Guangxi, China, since 2014. In this study, the whole-genome sequences of 32 strains of chicken parvovirus (ChPV) and 3 strains of turkey parvovirus (TuPV) were obtained by traditional PCR techniques. Phylogenetic analyses of 3 genes and full genome sequences were carried out, and 35 of the Guangxi ChPV/TuPV field strains were genetically different from 17 classic ChPV/TuPV reference strains. The nucleotide sequence alignment between ChPVs/TuPVs from Guangxi and other countries revealed 85.2–99.9% similarity, and the amino acid sequences showed 87.8–100% identity. The phylogenetic tree of these sequences could be divided into 6 distinct ChPV/TuPV groups. More importantly, 3 novel ChPV/TuPV groups were identified for the first time. Recombination analysis with RDP 5.0 revealed 15 recombinants in 35 ChPV/TuPV isolates. These recombination events were further confirmed by Simplot 3.5.1 analysis. Phylogenetic analysis based on full genomes showed that Guangxi ChPV/TuPV strains did not cluster according to their geographic origin, and the identified Guangxi ChPV/TuPV strains differed from the reference strains. Overall, whole-genome characterizations of emerging Guangxi ChPV and TuPV field strains will provide more detailed insights into ChPV/TuPV mutations and recombination and their relationships with molecular epidemiological features.

Confirmation of Groundnut ringspot orthotospovirus (GRSV) Infection in Eggplant Cultivars in Brazil.

Eggplant (Solanum melongena L.) is an economically important vegetable crop in Brazil, especially in family-based farming. Eggplant hybrids 'Ciça' and 'Napoli' (≈ 400 plants) were detected exhibiting virus-like symptoms (5-20% incidence) in field surveys (2015-2018) in Brasília-DF (Figure 1). Symptoms included chlorosis, mosaic and apical leaf deformation. Six symptomatic leaf samples were collected from fruit-bearing plants (around 100 days after planting) aiming at verifying the potential orthotospovirus infection. Double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was carried out with polyclonal antibodies (produced at Centro Nacional de Pesquisa de Hortaliças - CNPH) against the N gene coat protein of the three major orthotospoviruses: tomato spotted wilt orthotospovirus (TSWV), groundnut ringspot orthotospovirus (GRSV) and, tomato chlorotic spot orthotospovirus (TCSV). Strong serological reactions were observed only against GRSV antibodies in the extracts from symptomatic samples, but not in the controls. To confirm the causal agent of those symptoms, total RNA was extracted from infected leaf samples via the standard Trizol® (Sigma) protocol and subsequently used in a two-step reverse transcriptase polymerase chain reaction (RT-PCR) approach. Synthesis of the cDNA was carried out with the J13 primer (5'-CCC GGA TCC AGA GCA AT-3') (Cortez et al., 2001) followed by PCR assays with the primer pair BR60 (5'-AGA GCA ATC GTG TCA-3`) and BR65 (5`-ATC AAG CCT TCT GAA AGT CAT-3') (Eiras et al., 2002). This primer set amplifies a fragment of 453 bp including the 3' untranslated region at the 3' terminus of the S RNA and the protein N-coding gene of at least five species: TSWV, GRSV, TCSV, chrysanthemum stem necrosis orthotospovirus (CSNV) and zucchini lethal chlorosis orthotospovirus (ZLCV). In addition, GRSV-specific primers (LNA Reis, unpublished) were used for amplification of all three segments: L segment: LF/LR (5'-AAC AGG ATT CAG CAA TAT GG-3'/ 5'-AAT TCC TTG AAG ACA ATT GTG T -3'); M segment: MF/MR (5'-TTT GTC CAA CCA TAC CAG ACC C- 3' / 5'-GGC TTC AAT AAA GGC TTG GG-3') and, S segment: SF/SR (5'-TTC AAA CTC AGT TGT ACT CTG A-3'/5'-TTA CTT TCG ATC TGG TTG AA- 3'). Amplicons with 509 bp (MT043204), 289 bp (MT043205), and 901 bp (MT043203) were obtained for L, M and S segments of the eggplant isolate DF-687. PCR amplicons corresponding to a segment of the N-coding gene (396 bp) of a second eggplant isolate (BJL02; MK176337) were obtained with the primer pair BR60/BR65 and subjected to Sanger dideoxy sequencing at CNPH. Alignments of nucleotide sequences of both isolates revealed identity levels varying around 99% to the corresponding genomic regions of a large set of GRSV isolates from GenBank database. PCR assays using total RNA as template yielded 494 bp amplicons solely with GRSV-specific primers (Webster et al., 2011), but no products were obtained with TSWV-specific primers (Adkins and Rosskopf, 2002), confirming the former as the sole causal agent of the field symptoms. Leaves of eggplant cv. 'Ciça' and indicator hosts, including Nicotiana rustica, Capsicum chinense 'PI 159236' (with the Tsw gene), and S. lycopersicum cv. Santa Clara were rub inoculated with extracts prepared from eggplant samples naturally infected with GRSV. Mosaic, necrotic ringspots and systemic leaf deformation symptoms were observed around ten days after inoculation on newly emerged leaves of all inoculated plants. GRSV infection was confirmed by DAS-ELISA and RT-PCR ten days after inoculation. Eggplant was erroneously listed as a host of GRSV in Brazil (Kitajima, 2020). Hence, this is the first report of eggplant infection by this virus in South America. No significant yield losses have been observed in eggplant due to GRSV infection since the overall symptoms are often mild. However, this natural host of GRSV might impact disease management strategies since eggplant is quite often cultivated under family-based farming conditions as a companion crop of highly susceptible tomato, sweet-pepper, and lettuce cultivars.

Bifidobacterium breve PRL2020: Antibiotic-Resistant Profile and Genomic Detection of Antibiotic Resistance Determinants.

Antibiotics are one of the greatest scientific achievements of modern medicine, but excessive use is creating challenges for the future of medicine. Antibiotic resistance (AR) is thought to cause changes in bowel habits and an increased risk of gastroenteritis, but it may also increase the risk of overweight, obesity, autoimmune and atopic diseases, and a low response to vaccines and cancer, likely mediated by antibiotic-induced gut dysbiosis. Probiotic add-on therapy could partially prevent antibiotic-induced gut dysbiosis, but their antibiotic sensitivity features likely limits this potential. The EFSA (European Food Safety Authority) guidelines consider the use of probiotics whose antibiotic-resistant profile could be transferable an important hazard. Recently, a strain of B. breve (PRL2020) has shown to be resistant to amoxicillin and amoxicillin-clavulanate (AC) by applying the microdilution protocol according EFSA guidelines. After verifying that horizontal gene transfer is unlikely to take place, this feature suggests its concomitant use with these specific antibiotics. The results of our tests demonstrated that the strain PRL2020 is indeed endowed with amoxicillin- and AC-resistant properties and that it is also insensitive to ampicillin. In-depth analysis of the annotated genome sequence of B. breve PRL2020 was employed to query the Comprehensive Antibiotic Resistance Database (CARD) using Resistance Gene Identifier (RGI) software (version 5.2.1). The similarity among the AR determinants found was studied through nucleotide sequence alignment, and it was possible to verify not only the absence of genes explaining these features in the flanking regions but also the presence of genetic sequences (rpoB and erm(X)) putatively responsible for rifampicin and erythromycin resistance. Both features are not phenotypically expressed, and for these antibiotics, the strain is within the EFSA limits. Analysis of the flanking regions of these genes revealed possible mobile elements upstream and downstream only in the case of the erm(X) gene, but the features of the Insertion Sequences (IS) are described as not to cause horizontal transfer. Our findings on strain PRL2020 demonstrate that its AR profile is compatible with antibiotics when taken with the aim of reducing the risk of dysbiosis.

Revealing novel cytb and nad5 genes-based population diversity and benzimidazole resistance in Echinococcus granulosus of bovine origin.

Cystic echinococcosis (CE) is a neglected zoonotic disease caused by Echinococcus granulosus (sensu stricto). The parasite affects a wide range of livestock and wild animals. In this study, the population diversity of the Echinococcus species was investigated based on mitochondrial cytochrome b (cytb) and NADH dehydrogenase subunit 5 (nad5) genes. In addition to this, β-tubulin gene isoforms of Echinococcus granulosus were amplified to determine the resistance against benzimidazoles. For this purpose, 40 cyst samples from cattle (n = 20) and buffaloes (n = 20) were collected from the main abattoir of Sialkot. DNA extraction was performed using Qiagen Blood and Tissue Kits. Amplification was performed through PCR. Each amplicon was confirmed by GelRed™ stained agarose gel (2%). Samples were sequenced in a DNA analyzer and viewed for any misread nucleotide by using MEGA (v.11). Corrections in nucleotide sequence and multiple sequence alignment were made through the same software. NCBI-BLAST was used for sample specific sequences to identify them as belonging to a particular species. Diversity indices were estimated using DnaSP (v.6) while phylogenetic analysis was inferred using the Bayesian method using MrBayes (v.1.1). β-tubulin gene isoforms sequence analysis was performed to find out the candidate gene causing benzimidazole resistance. All 40 isolates were found positive for E. granulosus. BLAST-based searches of sequences of each isolate for each gene (nad5 and cytb) confirmed their maximum similarity with the G1 genotype. Overall, high haplotype diversity (Hd nad5 = 1.00; Hd cytb = 0.833) and low nucleotide diversity (π nad5 = 0.00560; π = cytb = 0.00763) was identified based on diversity indices. For both the genes, non-significant values of Tajima's D (nad5 = -0.81734; cytb = -0.80861) and Fu's Fs (nad5 = -1.012; cytb = 0.731) indicate recent population expansion. Bayesian phylogeny-based results of nad5 and cytb sequences confirmed their genotypic status as distinct from other Echinococcus species. This study shed light on the status of benzimidazole resistance in Echinococcus granulosus for the very first time from Pakistan. The findings of this study will significantly add in the information available on genetic diversity of Echinoccous granulosus based on cytb and nad5 genes sequences.

Isolation, characterization and whole genome analysis of the novel genus Lederbergvirus, phage vB_EcoP_E212 infecting enterotoxigenic Escherichia coli K88

The newly discovered phage vB_EcoP_E212 (also known as E212) was characterized and its genome was annotated in this study, which was conducted in Jilin, China. Transmission electron microscopy indicates that phage E212 belongs to the class Caudoviricetes. This phage exclusively infects enterotoxigenic E. coli K88. E212 was found to have a short latent period of 20 min, and a burst size of 125 PFU/cell. Additionally, E212 remained stable at all pH levels (3.0–12.0) and temperatures between -20 and 60 ºC. The genome of the phage E212 consists of 38,252 bp dsDNA molecule with a G + C content of 46.98%. The genome is projected to include 53 ORFs but no tRNAs. This phage lacks homologs of virulence factors or antimicrobial resistance genes, but it has lysogeny-related genes. Phage E212 was placed in the genus Lederbergvirus as a result of nucleotide sequence alignment and phylogenetic analysis.

Viral Protein 1 (VP1) Sequence-Based Genetic Diversity of SAT 2 FMDV Circulating in Ethiopia from 1990 to 2015.

Pathogen molecular epidemiology determines the origin of specific outbreaks locality of foot-and-mouth disease virus serotype South African Territories-2 sequence-based analysis of highly variable Viral Protein 1 (VP1), which helps to identify the evolution of this virus through time and space. The objective of this study was to compare the differences between SAT-2 VP1 sequences of FMDV circulated in Ethiopia from 1990 to 2015 at the genetic level. The nucleotide and amino acid sequences were analyzed using Basic Local Alignment Search Tools (BLAST), Multiple sequence alignment and sequence editing and Phylogenetic tree reconstruction. The nucleotide and amino acid sequences alignment, distance matrix, and phylogenetic tree constructions were done using the MEGA 6.0 software package. In this analysis, we found 76% nucleotide identities and amino acid similarities among the sequences. The overall group mean distance at nucleotide level was 19% with a mean intra-population diversity of 2%. The lowest sequence variation was observed among sequences obtained from the years 2007/09/10, 2014/15, and 1990/91 which was less than 5% among them. This analysis revealed that in the last 25 years, four different topotypes of the FMDV SAT-2 were circulating in Ethiopia. The Arg-Gly-Asp (RGD) amino acid (AA) motif at AA position 144-146 within the G-H loop of the VP1 protein of FMDV is conserved, but up- and downstream hyper-variable AA sequences are identified. In this study, it was observed that four topotypes (IV, XIV, XIII, and VII) were circulating in Ethiopia for 25 years. Further, compared with sequences from neighboring countries (Sudan, Kenya) confirmed the presence of these topotypes. Pertinent to this genetic diversity control strategies in Ethiopia should be based on having regular antigenic and genetic vaccine matching tests with the circulating strain within a defined period, space, transboundary nature of the disease and applying biosecurity measures.