Nucleotide Sequence Research Articles

First report of pepper chlorosis-associated virus infecting tobacco (Nicotiana tabacum) plants in Sichuan Province in China.

Tobacco (Nicotiana tabacum) is an economically important crop in China, and more than 30 viruses have been reported to infect tobacco (Yin et al. 2022). In July 2022, we observed interveinal necrosis on tobacco leaves in fields in Sichuan Province (N 27.9172, E 105.6662) (Fig. 1). Total RNA was isolated from multiple leaves of one plant using an RNAprep Pure Polysaccharide Polyphenol Plant Total RNA Extraction Kit (TIANGEN, Beijing, China). Total RNAs were pooled, and a TruSeq Stranded Total RNA with RiboZero Gold Kit (Illumina, San Diego, CA, USA) was used to eliminate ribosomal RNA. An RNA-Seq library was constructed using VAHTS Universal V6 RNA-seq Library Prep (Nanjing Vazyme, China). High-throughput sequencing was performed on the Illumina DNBseq platform (BGI-ShenZhen, China), which yielded 20,102,087 reads with an average length of 150 nt (total size >6 Gb). Unaligned reads were assembled de novo using SPAdes (Bankevich et al. 2012). Contigs with length ≥200 nt were subjected to local BLASTn and BLASTx analyses against the GenBank nt and nr databases, respectively (Wang et al. 2022). A total of 23 contigs were identified through BLASTx (e-value cut-off = 10 -3), ranging from 631 to 1555 bp long, with 82% to 96% coverage to partial genomic sequences of pepper chlorosis-associated virus (PepCaV-Higashitsuno_2021; Accessions: LC719619 to LC719621) and one contig (6459 bp) with 99% similarity to tobacco mosaic virus (Accession: OP525281) isolate DSMZ PV-0109 from Germany. The complete genome sequence of PepCaV was obtained using primers based on the assembled contigs. The 5'- and 3'-terminal regions of the RNA genome were obtained by 5'- and 3'-rapid amplification of cDNA ends. These amplicons were cloned using the pEASY-Blunt Zero Cloning Kit (TRANSGEN, Nangjing, China) and sequenced by Sanger sequencing. Complete genome sequences of tripartite PepCaV from tobacco samples were 7697, 1808, and 1557 nucleotides long (Accession: OR451987 to OR451989) and showed genome organization typical of the genus Ophiovirus in the family Aspiviridae. The complete sequences of RNA1, RNA2 and RNA3 genome segments shared 92.36%, 90.43%, and 95.24%, nucleotide sequence identities, respectively, with the isolate PepCaV-Higashitsuno_2021 pepper isolate (Accession: LC719619 to LC719621) (Shimomoto et al. 2023), but PepCaV has not been reported to infect N. tabacum. In June 2023, 10 plants collected from each place of Macheng (N 27.9094, E 105.6740), Xiangyang (N 28.0936, E 105.6249) and Moni (N 27.8899, E 105.5936) showing interveinal necrosis symptoms were tested using RT-PCR using PepCaV-MP610-F (5'-TGTTCTCTGCTATGCGGTTG -3') and PepCaV-MP610-R (5'-AGCAATCTCGCACCTGAAGT-3') to product 610bp amplicon. Twenty-five tobacco plants were positive for PepCaV. Single sequence from each location was submitted to GenBank (Accession: PP728631 to PP728633). Sap extracts from the original field leaf samples collected from Sichuan Province were used to mechanically inoculate tobacco plants (10 plants) at the four-leaf stage. After 7 days, leaf samples were tested using RT-PCR assay specific to PepCaV and TMV while samples were positive only for TMV but failed to transmit PepCaV by mechanical inoculation. According to previous literature, ophioviruses may be transmitted though soilborne fungus (Jeong et al. 2014). Further research is needed to understand the transmission, epidemiology, and pathological properties of the PepCaV. To our knowledge, this is the first report documenting natural PepCaV infection of tobacco plants in China, providing a scientific basis for PepCaV infection control in tobacco plantations.

Molecular diagnosis and characterization of Anaplasma marginale and Ehrlichia ruminantium infecting beef cattle of Maputo Province, Mozambique

BackgroundMembers of the Anaplasmataceae family, such as the Anaplasma and Ehrlichia species, cause economic losses and public health risks. However, the exact economic impact has not been comprehensively assessed in Mozambique due to limited data available on its basic epidemiology. Therefore, we investigated the molecular occurrence and identity of Anaplasma and Ehrlichia spp. infecting beef cattle in Maputo province, Mozambique.MethodsA total of 200 whole blood samples were collected from apparently healthy beef cattle. Whole blood DNA was extracted and tested for presence of Anaplasma spp. and Ehrlichia ruminantium DNA through amplification of the 16S rRNA and map1 genes. Positive samples to Anaplasma spp. were subject to PCR assay targeting the A. marginale-msp5 gene. Amplicons obtained were purified, sequenced and subject to phylogenetic analyses.ResultsAnaplasma spp., A. marginale and E. ruminantium were detected in 153 (76.5%), 142 (71%) and 19 (9.5%) of all the samples analyzed, respectively. On this same sample group, 19 (9.5%) were co-infected with A. marginale and E. ruminantium. The 16S rRNA sequences of Anaplasma spp. obtained were phylogenetically related to A. marginale, A. centrale and A. platys. Phylogenetic analysis revealed that A. marginale-msp5 nucleotide sequences were grouped with sequences from Asia, Africa and Latin America, whereas E. ruminantium-map1 DNA nucleotide sequences were positioned in multiple clusters.ConclusionCattle in Maputo Province are reservoirs for multiple Anaplasma species. A high positivity rate of infection by A. marginale was observed, as well as high genetic diversity of E. ruminantium. Furthermore, five new genotypes of E. ruminantium-map1 were identified.

Copy number variation and elevated genetic diversity at immune trait loci in Atlantic and Pacific herring

BackgroundGenome-wide comparisons of populations are widely used to explore the patterns of nucleotide diversity and sequence divergence to provide knowledge on how natural selection and genetic drift affect the genome. In this study we have compared whole-genome sequencing data from Atlantic and Pacific herring, two sister species that diverged about 2 million years ago, to explore the pattern of genetic differentiation between the two species.ResultsThe genome comparison of the two species revealed high genome-wide differentiation but with islands of remarkably low genetic differentiation, as measured by an FST analysis. However, the low FST observed in these islands is not caused by low interspecies sequence divergence (dxy) but rather by exceptionally high estimated intraspecies nucleotide diversity (π). These regions of low differentiation and elevated nucleotide diversity, termed high-diversity regions in this study, are not enriched for repeats but are highly enriched for immune-related genes. This enrichment includes genes from both the adaptive immune system, such as immunoglobulin, T-cell receptor and major histocompatibility complex genes, as well as a substantial number of genes with a role in the innate immune system, e.g. novel immune-type receptor, tripartite motif and tumor necrosis factor receptor genes. Analysis of long-read based assemblies from two Atlantic herring individuals revealed extensive copy number variation in these genomic regions, indicating that the elevated intraspecies nucleotide diversities were partially due to the cross-mapping of short reads.ConclusionsThis study demonstrates that copy number variation is a characteristic feature of immune trait loci in herring. Another important implication is that these loci are blind spots in classical genome-wide screens for genetic differentiation using short-read data, not only in herring, likely also in other species harboring qualitatively similar variation at immune trait loci. These loci stood out in this study because of the relatively high genome-wide baseline for FST values between Atlantic and Pacific herring.

Molecular identification and characterizations of rhizobacterial isolates collected from lentil rhizosphere of Indo-gangetic plains.

Plant growth promoting rhizobacteria (PGPR) are also known to colonize in the soil rhizosphere and prevent the development of other soil borne pathogens residing in the root surface. These microorganisms play a vital role in growth and development of the plant and also enhances the soil fertility by enriching the soil with different beneficial nutrients. This study was aimed at isolation of different rhizobacteria and their molecular characterization in search of efficient bacterial strains with multiple growth regulating activities. A total 36 bacteria were isolated from lentil root nodule as well as soil from different lentil growing fields with a view to screen/evaluate their plant growth promoting potential. Morphological characterization of isolated rhizobacterial candidates were done by observing the colonies on YEMA and nutrient agar media. Determination of CFU, Congo red test and gram staining tests were done to further screen them according to their morphology. All the isolates were then undergone molecular phylogenetic analysis using the partial sequences of the 16S rDNA. Based upon the Gram staining test, all the isolates were negative in gram reaction except six Bacillus isolates, PSB2 and AB3. Results of Ribosomal Database Project (RDP) and Basic Local Alignment Search Tool for Nucleotide Sequences (BLASTn) from 16S rDNA gene sequences showed that these isolates are genetically diverse. A total of 15 isolates of Rhizobium, 6 isolates of Bacillus, 3 isolates of Pseudomonas, 2 isolates of Phosphate Solubilizing Bacteria, 4 isolates of actinomycetes were identified by molecular sequencing of their 16S rDNA region and comparing them with the other isolates enlisted in the database of NCBI for the similarity percentage, query coverage. The purpose of the present study was to select native rhizosphere bacteria from the lentil nodule and soil of Lentil field and to evaluate their plant growth promoting potential as an alternative of chemical fertilizer for sustainable, environment friendly agriculture and assessment of their phylogenetic characterization.

HIV-1 drug resistance among naïve patients in Armenia in 2017–2021

Background. The increased antiretroviral therapy (ART) coverage of patients in the absence of routine drug resistance (DR) tests highlight the importance of HIV-1 drug resistance surveillance in Armenia. Aim. The aim of this study was a determination of the prevalence of HIV-1 DR on a large-scale cohort of HIV-infected citizens of the Republic of Armenia who had no experience of taking antiretroviral drugs. Materials and methods. The study was carried out on a cohort of more than 20% of PLHIV in the Republic of Armenia. The resulting 982 nucleotide sequences of the HIV-1 pol gene fragment, encoding the protease and reverse transcriptase region, as well as 367 sequences of the integrase gene, were analyzed using the Stanford University database and the CPR tool for the presence of drug resistance mutations and determination of the resistance level to ARV drugs. The HIV-1 subtype was determined using the Stanford University database and confirmed by phylogenetic analysis. Results. The overall prevalence of HIV DR to ARV drugs in naïve patients was 13.8%. Resistance to non-nucleoside reverse transcriptase inhibitors was 11.2%, nucleotide reverse transcriptase inhibitors — 1.4%, protease inhibitors — 2.0% and integrase inhibitors — 0.5%. The predominant genetic variant among viruses containing DR mutations was subtype B. Resistance was most often recorded among men who have sex with men living in Yerevan. Conclusion. In our study, prevalence of DR was high only for the NNRTI drugs. The results show that the first-line ARV drugs recommended in current national guidelines are highly likely to be effective. The analysis was carried out on a significant proportion of HIV-infected citizens of the Republic of Armenia, which increases the reliability and accuracy of the data obtained.

XBB.1, BQ1.1 and atypical BA.4.6/XBB.1 recombinants predominate current SARS-CoV-2 Wavelets with flu-like symptoms in Cameroon: A snapshot from genomic surveillance.

As of December 2022, Cameroon had observed a slight resurgence of COVID-19, raising concerns on genomic surveillance of related-SARS-CoV-2 variants under circulation. Following a laboratory-based survey, positive SARS-CoV-2 samples detected from December-2022 through March-2023 were processed for targeted sequencing at the Chantal BIYA International Reference Centre (CIRCB) in Yaoundé-Cameroon. From all positive cases detected, 13 were successfully sequenced (mean age 34 years, 70% female); the majority of the cases were unvaccinated (70%, 9/13) and symptomatic (92%, 12/13); all with flu-like symptoms (100%, 12/12). Following RT-PCR, the median cycle threshold was 22.23 [18-24] for the N gene; and 24.09 [20-26] for the ORF gene, underscoring high viral loads. Phylogenetic analysis of nucleotide sequences identified four major sub-variants in circulation, of which BA.5 (3/13), the recombinants BQ.1.1 (4/13), XBB.1 (4/13) and novel atypical variant of BA.4.6/XBB.1 (2/13). This snapshot surveillance indicates the introduction/emergence and circulation of new Omicron sub-variants, all accompanied by minor/mild symptoms. However, these new sub-variants and recombinants call for continuous genomic surveillance to prevent further resurgence of Covid-19 epidemiological wave.

Molecular Detection and Phylogenetic Analysis of the alkB Gene in Klebsiella oxytoca Strains Isolated from the Gut of Tenebrio molitor

The challenge in polystyrene disposal has caused researchers to look for urgent innovative and ecofriendly solutions for plastic degradation. Some insects have been reported to use polystyrene as their sole carbon source, and this has been linked to the presence of microbes in their guts that aid in plastic digestion. Thus, this study focuses on the molecular detection and phylogenetic analysis of the alkane-1-monooxygenase (alkB) gene in Klebsiella oxytoca strains isolated from the gut of Tenebrio molitor. The alkB gene encodes for alkane-1-monooxygenase, an enzyme involved in the oxidation of inactivated alkanes. This gene can be used as a marker to assess bacteria’s ability to biodegrade polystyrene. Three bacterial strains were isolated from the guts of T. molitor mealworms and were confirmed using polymerase chain reaction (PCR) of the 16S ribosomal RNA gene. The primers used in the amplification of the 16S ribosomal RNA region were designed using NCBI, a bioinformatics tool. To detect the presence of the alkB gene in the isolated bacterial strains, a set of primers used in the amplification of this gene was manually designed from the conserved regions of the alkB nucleotide sequences of eleven bacterial species from GenBank. TCOFFE online tool was used to align the alkB sequences of the bacteria, while Jalview and ConSurf were used to view the alignment. The amplified alkB gene was then sequenced using the Sanger sequencing technique, blasted on NCBI to look for similar sequences, and a phylogenetic tree was constructed. Based on the 16S ribosomal RNA gene sequences, the isolated bacterial strains were confirmed to be Klebsiella oxytoca NBRC 102593, Klebsiella oxytoca JCM 1665, and Klebsiella oxytoca ATCC 13182. The alkB gene sequence identical to fourteen alkB gene sequences derived from Actinobacteria whole genome was detected in Klebsiella oxytoca for the first time to the best of our knowledge. The novel nucleotide sequence was published in the NCBI database under accession number OP959069. This gene sequence was found to be for the enzyme alkane-1-monooxygenase and may be one of the enzymes responsible for polystyrene degradation by the putative Klebsiella oxytoca ATCC 13182 in T. molitor.

ARGNet: using deep neural networks for robust identification and classification of antibiotic resistance genes from sequences

BackgroundEmergence of antibiotic resistance in bacteria is an important threat to global health. Antibiotic resistance genes (ARGs) are some of the key components to define bacterial resistance and their spread in different environments. Identification of ARGs, particularly from high-throughput sequencing data of the specimens, is the state-of-the-art method for comprehensively monitoring their spread and evolution. Current computational methods to identify ARGs mainly rely on alignment-based sequence similarities with known ARGs. Such approaches are limited by choice of reference databases and may potentially miss novel ARGs. The similarity thresholds are usually simple and could not accommodate variations across different gene families and regions. It is also difficult to scale up when sequence data are increasing.ResultsIn this study, we developed ARGNet, a deep neural network that incorporates an unsupervised learning autoencoder model to identify ARGs and a multiclass classification convolutional neural network to classify ARGs that do not depend on sequence alignment. This approach enables a more efficient discovery of both known and novel ARGs. ARGNet accepts both amino acid and nucleotide sequences of variable lengths, from partial (30–50 aa; 100–150 nt) sequences to full-length protein or genes, allowing its application in both target sequencing and metagenomic sequencing. Our performance evaluation showed that ARGNet outperformed other deep learning models including DeepARG and HMD-ARG in most of the application scenarios especially quasi-negative test and the analysis of prediction consistency with phylogenetic tree. ARGNet has a reduced inference runtime by up to 57% relative to DeepARG.ConclusionsARGNet is flexible, efficient, and accurate at predicting a broad range of ARGs from the sequencing data. ARGNet is freely available at https://github.com/id-bioinfo/ARGNet, with an online service provided at https://ARGNet.hku.hk.7nR91jX7ySRMRPZrE5tsTyVideo

Diversity of the Rysto gene conferring resistance to potato virus Y in wild relatives of potato

BackgroundPotato virus Y (PVY) is among the economically most damaging viral pathogen in production of potato (Solanum tuberosum) worldwide. The gene Rysto derived from the wild potato relative Solanum stoloniferum confers extreme resistance to PVY.ResultsThe presence and diversity of Rysto were investigated in wild relatives of potato (298 genotypes representing 29 accessions of 26 tuber-bearing Solanum species) using PacBio amplicon sequencing. A total of 55 unique Rysto-like sequences were identified in 72 genotypes representing 12 accessions of 10 Solanum species and six resistant controls (potato cultivars Alicja, Bzura, Hinga, Nimfy, White Lady and breeding line PW363). The 55 Rysto-like sequences showed 89.87 to 99.98% nucleotide identity to the Rysto reference gene, and these encoded in total 45 unique protein sequences. While Rysto-like26 identified in Alicja, Bzura, White Lady and Rysto-like16 in PW363 encode a protein identical to the Rysto reference, the remaining 44 predicted Rysto-like proteins were 65.93 to 99.92% identical to the reference. Higher levels of diversity of the Rysto-like sequences were found in the wild relatives of potato than in the resistant control cultivars. The TIR and NB-ARC domains were the most conserved within the Rysto-like proteins, while the LRR and C-JID domains were more variable. Several Solanum species, including S. antipoviczii and S. hougasii, showed resistance to PVY. This study demonstrated Hyoscyamus niger, a Solanaceae species distantly related to Solanum, as a host of PVY.ConclusionsThe new Rysto-like variants and the identified PVY resistant potato genotypes are potential resistance sources against PVY in potato breeding. Identification of H. niger as a host for PVY is important for cultivation of this plant, studies on the PVY management, its ecology, and migrations. The amplicon sequencing based on PacBio SMRT and the following data analysis pipeline described in our work may be applied to obtain the nucleotide sequences and analyze any full-length genes from any, even polyploid, organisms.

HPV prevalence and genotype distribution among Vietnamese cervical cancer patients in the northern region of Vietnam, from 2018 – 2020

Background/purposeHPV infection is the primary cause of cervical cancer, and genotype distribution varies according to geographical location and carcinogenicity. Therefore, identifying HPV genotype and its association with cervical cancer features is critical for screening, diagnosis, and therapy. MethodsData from Vietnamese women with HPV-positive cervical cancer were collected from the northern region. The HPV genotype was identified using the Cobas®®4800 HPV system, whereas the nucleotide sequences of the E6, E7, and L1 genes were used to identify lineages and sublineages using DNASTAR, Bioedit, ATGC 7.2, and MEGA 11.0.10 softwares. ResultsOf the 180 patients infected with HPV, 82.8 % revealed single infections, and 17.2 % showed multiple infections. HPV16 (64.4 %), HPV18 (28.9 %), and other HPVs (6.7 %) were the most prevalent HPV genotypes. HPV16 lineages included European (sublineage A1 (11.2 %), A2 (1.72 %), and A3 (25.86 %); Asian (sublineage A4 (53.85 %); African-American (sublineage D1 (5.17 %); and Asian-American (sublineage D3 (2.59 %). The HPV18 lineage includes Asian-Ameridian (sublineages A1 (100 %)). HPV18 revealed a higher rate of cervical cancer, cervicitis, warts, and ulcers than HPV16 and other high-risk genotypes in the 35–54–year–old group, but did not show a difference in cancer stages. ConclusionsHPV16 and HPV18 genotypes are common in cervical cancer in northern Vietnam, with European, Asian, and Asian–Amerindian lineages predominating. HPV18 causes cervical cancer at a higher rate than other genotypes in the 35–54–year–old age group; thus, early identification of the genotype is critical for more successful therapy.

Characterization of arbuscular mycorrhizal fungal species associating with Zea mays

Taxonomic identification of arbuscular mycorrhizal (AM) fungal spores extracted directly from the field is sometimes difficult because spores are often degraded or parasitized by other organisms. Single-spore inoculation of a suitable host plant allows for establishing monosporic cultures of AM fungi. This study aimed to propagate AM fungal spores isolated from maize soil using single spores for morphological characterization. First, trap cultures were established to trigger the sporulation of AM fungal species. Second, trap cultures were established with individual morphotypes by picking up only one spore under a dissecting microscope and transferring it to a small triangle of sterilized filter paper, which was then carefully inoculated below a root from germinated sorghum seeds in each pot and covered with a sterile substrate. All pots were placed in sunbags and maintained in a plant growth room for 120 days. Spores obtained from single spore trap cultures from each treatment, maize after oats (MO), maize after maize (MM), maize after peas (MP), and maize after soybean (MS), were extracted using the sieving method. Healthy spores were selected for morphological analysis. Direct PCR was conducted by crushing spores in RNAlater and applying three sets of primer pairs: ITS1 × ITS4, NS31 × AML2, and SSUmcf and LSUmBr. Nucleotide sequences obtained from Sanger sequencing were aligned on MEGA X. The phylogenetic tree showed that the closest neighbors of the propagated AM fungal species belonged to the genera Claroideoglomus, Funneliformis, Gigaspora, Paraglomus, and Rhizophagus. The morphological characteristics were compared to the descriptive features of described species posted on the INVAM website, and they included Acaulospora cavernata, Diversispora spurca, Funneliformis geosporus, Funneliformis mosseae, Gigaspora clarus, Gigaspora margarita, Glomus macrosporum, Paraglomus occultum, and Rhizophagus intraradices. These findings can provide a great contribution to crop productivity and sustainable management of the agricultural ecosystem. Also, the isolate analyzed could be grouped into efficient promoters of growth and mycorrhization of maize independent of their geographical location.

Prevalence and genetic diversity of the <i>Alongshan virus (Flaviviridae)</i> circulating in ticks in the south of Eastern Siberia

Introduction. Tick-borne infections are of great importance for many regions of Russia, including Eastern Siberia. This unfavorable epidemiological situation can be characterized not only by the circulation of well-known tick-borne infections, but also by the identification of new pathogens, the role of which remains little or generally unexplored. Multicomponent flavi-like viruses can cause infectious diseases in humans and pose a threat to public health. The purpose of the study was the identification and molecular genetic characterization of the Alongshan virus (Flaviviridae, ALSV) isolates, transmitted by ticks in the south of Eastern Siberia. Materials and methods. Total 1060 ticks were collected and analyzed from the territory of the Republics of Khakassia, Tuva, Buryatia, Irkutsk Region and Transbaikal Territory (Zabaykalsky Krai) in the spring-summer period 2023. ALSV RNA was detected by RT-PCR followed by nucleotide sequence determination and phylogenetic analysis for each segment of the genome. Results. The ALSV infection rate in Ixodes persulcatus ticks collected in the Republic of Khakassia was 3.3% (95% CI: 1.4–7.5); in Irkutsk Oblast – 1.0% (95% CI: 0.3–3.7); in the Republic of Tuva – 0.9% (95% CI: 0.3–3.4) and in Transbaikal Krai – 0.7% (95% CI: 0.2–3.6). Sequences of all four segments of ALSV genetic variants circulating in I. persulcatus ticks in the south of Eastern Siberia are grouped with sequences found in China and clustered into the Asian subgroup transmitted by taiga ticks. The level of difference in the nucleotide sequences of genome fragments among the identified genetic variants of ALSV ranged from 2 to 3%. Conclusion. The article shows the widespread distribution of ALSV in I. persulcatus ticks in the Republics of Khakassia and Tyva, Irkutsk Oblast and Transbaikal Territory. The obtained data actualize monitoring of changes in the area of distribution of potentially dangerous for humans flavi-like viruses and their vectors.

Genetic diversity and phylogenetic relationships of Clostridium perfringens strains isolated from mastitis and enteritis in Egyptian dairy farms

BackgroundClostridium perfringens, a common environmental bacterium, is responsible for a variety of serious illnesses including food poisoning, digestive disorders, and soft tissue infections. Mastitis in lactating cattle and sudden death losses in baby calves are major problems for producers raising calves on dairy farms. The pathogenicity of this bacterium is largely mediated by its production of various toxins.ResultsThe study revealed that Among the examined lactating animals with a history of mastitis, diarrheal baby calves, and acute sudden death cases in calves, C. perfringens was isolated in 23.5% (93/395) of the total tested samples. Eighteen isolates were obtained from mastitic milk, 59 from rectal swabs, and 16 from the intestinal contents of dead calves. Most of the recovered C. perfringens isolates (95.6%) were identified as type A by molecular toxinotyping, except for four isolates from sudden death cases (type C). Notably, C. perfringens was recovered in 100% of sudden death cases compared with 32.9% of rectal swabs and 9% of milk samples. This study analyzed the phylogeny of C. perfringens using the plc region and identified the plc region in five Egyptian bovine isolates (milk and fecal origins). Importantly, this finding expands the known data on C. perfringens phospholipase C beyond reference strains in GenBank from various animal and environmental sources.ConclusionPhylogenetic analyses of nucleotide sequence data differentiated between strains of different origins. The plc sequences of Egyptian C. perfringens strains acquired in the present study differed from those reported globally and constituted a distinct genetic ancestor.

Biology, phylogenetic and evolutionary relations of Tradescantia mild mosaic virus isolates from Hungary

Biology, phylogenetic and evolutionary relations of Tradescantia mild mosaic virus isolates from Hungary

211 Analysis of microbial communities from a commercial-size swine manure pit and their association with production of volatile organic compounds

Abstract Global pollution and emission concerns have been linked to concentrated animal feeding operations (CAFOs). Indeed, the U.S Environmental Protection Agency (US EPA) warns that there is a significant danger to human health, soil, and water quality from CAFOs. Volatile organic compounds (VOCs), which are amongst air contaminants that pose a serious risk, are abundantly produced in swine manure pits. While VOC emissions from swine manure have been well studied, the microbial species responsible for their production remain mostly uncharacterized. Thus, a deeper comprehension of the microorganisms responsible for VOC production in swine manure pits may help in the development of management approaches that target the microbiome in these environments. In this context, this study aimed to identify the major bacterial species populating the manure pit of a commercial-size swine wean-to-finish research facility. Manure collected from two sampling locations at different time points were analyzed using the 16S rRNA gene through PCR-amplification of the V1-V3 regions from microbial genomic DNA, followed by Illumina MiSeq 2X300 sequencing. An analysis of the most highly represented microorganisms identified 24 bacterial Operational Taxonomic Units (OTUs), with only four showing a species-level nucleotide sequence identity to currently known or valid species. As these results indicated that the majority of bacterial species from this manure pit were unknown, a metagenomics approach was used to assemble partial to full length genomes from the two most abundant OTUs: Pit_9-00239 [5.09% - 19.44%; Clostridium saccharoperbutylacetonicum (96.60%)] and Pit_ 7-00035 [5.58% - 17.30%; Bacteroides eggerthii (87.66%)]. From genome assembly analysis, 979 contigs were generated for a combined length of 12,367,510 bp, which together included 21,776 coding sequences. Predicted metabolic functions from these contig sets included pathways involved in carbohydrate utilization (glycolysis), vitamin synthesis (riboflavin), and butyrate production (volatile fatty acids). Notably, an enzyme responsible for the production of indole, a common VOC produced from swine manure pits, was also identified. Together, the results from this analysis provide insights on metabolic capabilities of abundant bacterial species from manure pits. These can in turn be used for the future development of management strategies or products that can reduce VOC emissions that result from swine CAFO manure storage.

Molecular and SEM studies on Thaparocleidus vistulensis (Siwak, 1932) (Monopisthocotyla, Ancylodiscoididae)

Presenting new molecular and scanning electron microscope (SEM) features, this study gives additional data to the better knowledge of Thaparocleidus vistulensis (Siwak, 1932) (Monopisthocotyla, Ancylodiscoididae), a parasite of the European catfish Silurus glanis Linnaeus, 1758 (Siluriformes, Siluridae) cultured in a commercial fish farm in Hungary. In addition, notes on the early development of sclerotized anchors are also provided. The main morphological difference of T. vistulensis compared to other congeneric species is associated with the male copulatory organ, which exhibits 5–7 loops in the middle of the penis length and a long open V-shaped sclerotized accessory piece, dividing terminally into two parts, securing the terminal part of the penis tube. The present study provides for the first time molecular characterization data based on the 2694 bp long nucleotide sequence of rDNA (ITS1, 5.8S, ITS2, and flanked with partial 18S and partial 28S) submitted in GenBank with the accession number OR916383. A phylogenetic tree based on ITS1 sequences supports a well-defined clade including T. vistulensis, forming a sister group with T. siluri, a species-specific monopisthocotylan parasite to S. glanis. The morphological characterization of T. vistulensis, especially for the male copulatory organ, together with the molecular data in the present study, extends knowledge about this monopisthocotylan species and provides new information for future phylogeny studies.

Frequency of HBsAg variants in occult hepatitis B virus infected patients and detection by ARCHITECT HBsAg quantitative

ObjectiveTo analyze the amino acid substitution caused by mutations in the major hydrophilic region (MHR) of the S-region genes in the serum samples of occult hepatitis B virus infection (OBI), and to explore the reasons for the missed detection of HBsAg.MethodThe full-length gene of the S-region in hepatitis B virus(HBV) in the chronic hepatitis B virus(CHB)(10 samples) and OBI groups(42 samples) was amplified using a lab-developed, two-round PCR amplification technology. The PCR amplification products were sequenced/clone sequenced, and the nucleotide sequences of the S-region gene in HBV were compared to the respective genotype consensus sequence.ResultsOnly 20 of the 42 samples in the OBI group had the S-region genes successfully amplified, with the lowest HBV DNA load of 20.1IU/ml. As S-region genes in HBV, 68 cloned strains were sequenced. In the OBI and CHB groups MHR region, with a mutation rate of 3.21% (155/4828) and 0.70% (5/710). The genetic mutation rate was significantly higher in the OBI group than in the CHB group (P<0.05). The common mutation types in the MHR region were: I126T, L162R, K122E, C124R, and C147Y.Mutations at s122, s126, and s162 were associated with subgenotypes, most of which being C genotypes. The high-frequency mutation sites L162R and K122E found in this study have not been reported in previous literature.ConclusionThe results of this study confirmed that MHR mutations can cause the missed detection of HBsAg, giving rise to OBI.

The Partial Nucleotide Sequences of the Mitochondrial Genes, COI and 16s rRNA, of Fireflies in the Genera Pygoluciola, Trisinuata, and Medeopteryx (Coleoptera: Lampyridae)

Fireflies in the genera Pygoluciola, Medeopteryx, and Trisinuata distribute throughout lower northern Thailand. Five morphospecies of these fireflies were collected and nucleotide sequences were analyzed. We sequenced the partial of two mitochondrial genes; Cytochrome c oxidase subunit I (COI) and 16s rRNA, and the phylogenetic trees were constructed and compared with the firefly nucleotide sequences in the NCBI database. The COI gene was amplified using LCO1490 and HCO2198 primers while specific primers of LR-J-13020a and LR-N-13398a were used to analyze amplicons of 16s rRNA. The results showed the average GC-contents in the nucleotide of five firefly morphospecies were found to average 30.28% and 22.15% in (COI) and 16s rRNA, respectively. The variable site in COI nucleotide sequences was 399 of the total 520 nucleotides (76.73%) whereas 91 of the total 418 nucleotides (21.77%) were found in 16s rRNA nucleotide sequences. Construction of the phylogenetic trees of those five firefly morphospecies with reference accession samples indicated five clades when analyzed by COI nucleotide sequences and two clades were revealed in 16s rRNA gene. According to these results, COI nucleotides are one of the most effective methods for differentiating between and within firefly species. The addition of the 16s rRNA gene analysis showed an increase in the precision of firefly identification. Those five firefly morphospecies were separated using both the COI and 16s rRNA nucleotide sequences.

First Report of Pseudopestalotiopsis ixorae Causing Leaf Spot of Photinia × fraseri in China.

Photinia × fraseri is a well-known ornamental shrub in southern China. In December 2021, we observed leaf spots that were circular to irregular, gray with dark red margins and violet brown with brownish violet edges on the leaves of Photinia × fraseri shrubs in the scenic area of Shenlongtan (28°46'10″N, 115°42'93″E), Jiangxi Province, China. Almost 15% of the leaves in the 1300 m2 Photinia × fraseri planting area were symptomatic. Thirty symptomatic leaves were randomly collected from different plants, and sectioned into 5-mm2 pieces, which were surface-sterilized using 1% NaOCl for 30 s. After rinsing thrice in sterile distilled water and drying, the pieces were transferred onto potato dextrose agar (PDA) and incubated at 28 ℃ for 5-7 days. A total of sixteen morphologically similar isolates were obtained. After incubation on PDA for 20 days, the fungi had irregular edges, were white to pale brown, and had spare aerial mycelium on the surface with irregularly distributed black, gregarious conidiomata. Conidia were fusoid, subcylindrical, straight to slightly curved, 4-septated, slightly constricted at the septa, and 23 to 36 × 6 to 10 μm (mean: 27.6 × 7.7 μm). The morphological characteristics were consistent with the features of Pseudopestalotiopsis species (Maharachchikumbura et al. 2014). The genomic DNA of two representative isolates (JFRL032 and JFRL033) was extracted for further identification. The internal transcribed spacer (ITS) region, translation elongation factor 1-ɑ (tef1-ɑ) and β-tubulin (tub2) genes were amplified and sequenced using primers ITS5/ITS4, EF1-526F/EF1-1567R, and Bt2A/Bt2B, respectively (Maharachchikumbura et al. 2012). The sequences of the two representative isolates were 100% identical to each other. These nucleotide sequences were deposited in GenBank with accession numbers, ON342794 and ON342795 (ITS); ON375851 and ON375852 (tef1-ɑ); ON375853 and ON375854 (tub2). BLASTn searches of the obtained sequences revealed 99%-100% to ITS (MG816316, 478/478 nucleotides), tef1-ɑ (MG816336, 924/926 nucleotides), tub2 (MG816326, 441/442 nucleotides) sequences of the ex-type strain of Pseudopestalotiopsis ixorae (NTUCC17-001.1). Phylogenetic analysis was conducted using the concatenation of multiple sequences (ITS, tef1-ɑ and tub2) with the Maximum likelihood statistics in PhyloSuite v1.2.2 (Zhang et al.2020). The phylogenetic tree showed the two isolates clustered with P. ixorae in a clade with 100% bootstrap support. The isolates were identified as P. ixorae based on morphological and molecular data. To confirm pathogenicity, eight healthy leaves of 3-year-old Photinia × fraseri were surface sterilized, scratched with a pair of sterilized tweezers, and ten µl of conidial suspension (106 conidia/ml) was sprayed on the injured leaves and the control was sprayed with sterile distilled water. Then, All plants were potted in a climate chamber at 25℃ and 85% relative humidity. After 3 days, leaf spot symptoms similar to those described above were observed on inoculated leaves, while the non-inoculated leaves remained symptomless. The pathogen was reisolated from the inoculated leaves to fulfill Koch's postulates and confirmed as P. ixorae by morphological and molecular analysis. It has been reported that P. ixorae can infect the Ixora plant (Tsai et al., 2018). To the best of our knowledge, this is the first report of P. ixorae causing leaf spot on Photinia × fraseri in China. The study provides valuable information for identifying and controlling the leaf spot on Photinia × fraseri.

Functionalizing DNA Origami by Triplex-Directed Site-Specific Photo-Cross-Linking.

Here, we present a cross-linking approach to covalently functionalize and stabilize DNA origami structures in a one-pot reaction. Our strategy involves adding nucleotide sequences to adjacent staple strands, so that, upon assembly of the origami structure, the extensions form short hairpin duplexes targetable by psoralen-labeled triplex-forming oligonucleotides bearing other functional groups (pso-TFOs). Subsequent irradiation with UVA light generates psoralen adducts with one or both hairpin staples leading to site-specific attachment of the pso-TFO (and attached group) to the origami with ca. 80% efficiency. Bis-adduct formation between strands in proximal hairpins further tethers the TFO to the structure and generates "superstaples" that improve the structural integrity of the functionalized complex. We show that directing cross-linking to regions outside of the origami core dramatically reduces sensitivity of the structures to thermal denaturation and disassembly by T7 RNA polymerase. We also show that the underlying duplex regions of the origami core are digested by DNase I and thus remain accessible to read-out by DNA-binding proteins. Our strategy is scalable and cost-effective, as it works with existing DNA origami structures, does not require scaffold redesign, and can be achieved with just one psoralen-modified oligonucleotide.