Proposal of Helicobacter higonensis sp. nov. isolated from a human clinical specimen, and emended description of Helicobacter valdiviensis Collado, 2014.

We have previously isolated a gram-negative microaerophilic strain, PAGU2000T from a patient presenting with a fever in Kumamoto Prefecture, Japan. The present study aimed to comprehensively analyze the taxonomy of the isolated strain using a polyphasic approach. The 16S rRNA gene sequence analysis indicated that the strain was a member of enterohepatic Helicobacter. The strain PAGU2000T shared a 97.5% 16S rRNA gene nucleotide identity with Helicobacter valdiviensis, and this taxonomic position was confirmed by phylogenetic analysis of the GyrA amino acid sequences. The proposed strain PAGU2000T has a 1.482 Mbp chromosome with a DNA G + C content of 31.3 mol% and encodes 1520 predicted coding sequences. The average nucleotide identity between the strain PAGU2000T and type strain of H. valdiviensis was 70.3%, which was lower than the recommended threshold of 95% for species delineation. The strain PAGU2000T was a motile, non-spore-forming, and spiral-shaped bacterium, exhibiting catalase and oxidase activities but not urease and nitrate reduction. This study demonstrates that the isolate represents a novel species within enterohepatic Helicobacter, for which the name Helicobacter higonensis is proposed (type strain: PAGU2000T = GTC 16811T = LMG 33095T). In this study, we describe the phenotypic and morphological features of this strain and propose an emended description of some biochemical traits of H. valdiviensis.

Whole-Genome Sequencing and Analysis of Tumour-Forming Radish (Raphanus sativus L.) Line.

Spontaneous tumour formation in higher plants can occur in the absence of pathogen invasion, depending on the plant genotype. Spontaneous tumour formation on the taproots is consistently observed in certain inbred lines of radish (Raphanus sativus var. radicula Pers.). In this paper, using Oxford Nanopore and Illumina technologies, we have sequenced the genomes of two closely related radish inbred lines that differ in their ability to spontaneously form tumours. We identified a large number of single nucleotide variants (amino acid substitutions, insertions or deletions, SNVs) that are likely to be associated with the spontaneous tumour formation. Among the genes involved in the trait, we have identified those that regulate the cell cycle, meristem activity, gene expression, and metabolism and signalling of phytohormones. After identifying the SNVs, we performed Sanger sequencing of amplicons corresponding to SNV-containing regions to validate our results. We then checked for the presence of SNVs in other tumour lines of the radish genetic collection and found the ERF118 gene, which had the SNVs in the majority of tumour lines. Furthermore, we performed the identification of the CLAVATA3/ESR (CLE) and WUSCHEL (WOX) genes and, as a result, identified two unique radish CLE genes which probably encode proteins with multiple CLE domains. The results obtained provide a basis for investigating the mechanisms of plant tumour formation and also for future genetic and genomic studies of radish.

Intrahost Monkeypox Virus Genome Variation in Patient with Early Infection, Finland, 2022.

Monkeypox virus was imported into Finland during late May-early June 2022. Intrahost viral genome variation in a sample from 1 patient comprised a major variant with 3 lineage B.1.3-specific mutations and a minor variant with ancestral B.1 nucleotides. Results suggest either ongoing APOBEC3 enzyme-mediated evolution or co-infection.

Identification of the novel HLA-DPA1*01:03:43 allele resulting from an intralocus recombination involving the DPA1*04:01:01:03 and DPA1*01:03:01:27 alleles sequenced by Next Generation Sequencing (NGS).

HLA-DPA1 intralocus recombination between DPA1*04:01:01:03 and DPA1*01:03:01:27, or closely related other alleles, results in a novel allele HLA-DPA1*01:03:43.

Network analysis for estimating standardization trends in genomics using MEDLINE.

BACKGROUND: Biotechnology in genomics, such as sequencing devices and gene quantification software, has proliferated and been applied to clinical settings. However, the lack of standards applicable to it poses practical problems in interoperability and reusability of the technology across various application domains. This study aims to visualize and identify the standard trends in clinical genomics and to suggest areas on which standardization efforts must focus. METHODS: Of 16,538 articles retrieved from PubMed, published from 1975 to 2020, using search keywords "genomics and standard" and "clinical genomic sequence and standard", terms were extracted from the abstracts and titles of 15,855 articles. Our analysis includes (1) network analysis of full phases (2) period analysis with five phases; (3) statistical analysis; (4) content analysis. RESULTS: Our research trend showed an increasing trend from 2003, years marked by the completion of the human genome project (2003). The content analysis showed that keywords related to such concepts as gene types for analysis, and analysis techniques were increased in phase 3 when US-FDA first approved the next-generation sequencer. During 2017-2019, oncology-relevant terms were clustered and contributed to the increasing trend in phase 4 of the content analysis. In the statistical analysis, all the categories showed high regression values (R2 > 0.586) throughout the whole analysis period and phase-based statistical analysis showed significance only in the Genetics terminology category (P = .039*) at phase 4. CONCLUSIONS: Through comprehensive trend analysis from our study, we provided the trend shifts and high-demand items in standardization for clinical genetics.

Complete genomic sequences and comparative analysis of two Orf virus isolates from Guizhou Province and Jilin Province, China.

Orf virus (ORFV, species Orf virus) belongs to the typical species of the Parapoxvirus genus of the family Poxviridae, which infects sheep, goats, and humans with worldwide distribution. Although outbreaks of Orf have been reported sequentially in several Chinese provinces, the epidemiology of Orf and genetic diversity of ORFV strains still needs to be further characterized. To further reveal the genomic organization of the ORFV-GZ18 and ORFV-CL18 isolates, the complete genome sequences of two recently obtained ORFV isolates were sequenced using the next-generation sequencing technology and analyzed, which had been deposited in the GenBank database under accession number MN648218 and MN648219, respectively. The complete genomic sequence of ORFV-CL18 was 138,495 bp in length, including 131 potential open reading frames (ORFs) flanked by inverted terminal repeats (ITRs) of 3481 bp at both ends, which has genomic structure typical Parapoxviruses. The overall genomic organization of the fully sequenced genome of ORFV-GZ18 was consistent with ORFV-CL18 genome, with a complete genome size of 138,446 nucleotides, containing 131 ORFs flanked by ITRs of 3469 bp. Additionally, the overall G + C contents of ORFV-GZ18 and ORFV-CL18 genome sequences were about 63.9% and 63.8%, respectively. The phylogenetic analysis showed that both ORFV-GZ18 and ORFV-CL18 were genetically closely related to ORFV-SY17 derived from sheep. In summary, the complete genomic sequences of ORFV-GZ18 and ORFV-CL18 are reported, with the hope it will be useful to investigate the host range, geographic distribution, and genetic evolution of the virus in Southern West and Northern East China.

Isolation of a Bacillus cereus strain HBL-AI and its application for production of Trans-4-hydroxy-l-proline.

A new trans-4-hydroxy-l-proline (trans-Hyp) producing Bacillus cereus HBL-AI, was isolated from the air, which was screened just using l-proline as carbon and energy sources. This strain exhibited 73·4% bioconversion rate from initial l-proline (3 g l-1 ) to trans-Hyp. By sequencing the genome of this bacterium, 6244 coding sequences were obtained. Genome annotation analysis and functional expression were used to identify the proline-4-hydroxylase (BP4H) in HBL-AI. This enzyme belonged to a family of 2-oxoglutarate-related dioxygenases, which required 2-oxoglutarate and O2 as co-substrates for the reaction. Homologous modelling indicated that the enzyme had two monomers and contained conserved motifs, which included a distorted 'jelly roll' ß strand core and the residues (HXDXnH and RXS). The engineering Escherichia coli 3 Δ W3110/pTrc99a-proba-bp4h was constructed using BP4H, which transformed glucose to trans-Hyp in one step with high concentration of 46·2 g l-1 . This strategy provides a green and efficient method for synthesis of trans-Hyp and thus has a great potential in industrial application.

A DNA Sequence Based Polymer Model for Chromatin Folding.

The recent development of sequencing technology and imaging methods has provided an unprecedented understanding of the inter-phase chromatin folding in mammalian nuclei. It was found that chromatin folds into topological-associated domains (TADs) of hundreds of kilo base pairs (kbps), and is further divided into spatially segregated compartments (A and B). The compartment B tends to be located near to the periphery or the nuclear center and interacts with other domains of compartments B, while compartment A tends to be located between compartment B and interacts inside the domains. These spatial domains are found to highly correlate with the mosaic CpG island (CGI) density. High CGI density corresponds to compartments A and small TADs, and vice versa. The variation of contact probability as a function of sequential distance roughly follows a power-law decay. Different chromosomes tend to segregate to occupy different chromosome territories. A model that can integrate these properties at multiple length scales and match many aspects is highly desired. Here, we report a DNA-sequence based coarse-grained block copolymer model that considers different interactions between blocks of different CGI density, interactions of TAD formation, as well as interactions between chromatin and the nuclear envelope. This model captures the various single-chromosome properties and partially reproduces the formation of chromosome territories.

Contaminations in (meta)genome data: An open issue for the scientific community.

In recent years, the high throughput and the low cost of next-generation sequencing (NGS) technologies have led to an increase of the amount of (meta)genomic data, revolutionizing genomic research studies. However, the quality of sequencing data could be affected by experimental errors derived from defective methods and protocols. This represents a serious problem for the scientific community with a negative impact on the correctness of studies that involve genomic sequence analysis. As a countermeasure, several alignment and taxonomic classification tools have been developed to uncover and correct errors. In this critical review some of these integrated software tools and pipelines used to detect contaminations in reference genome databases and sequenced samples are reported. In particular, case studies of bacterial contaminations, contaminations of human origin, mitochondrial contaminations of ancient DNA, and cross contaminations are examined.

Reference genome and comparative genome analysis for the WHO reference strain for Mycobacterium bovis BCG Danish, the present tuberculosis vaccine.

BACKGROUND: Mycobacterium bovis bacillus Calmette-Guérin (M. bovis BCG) is the only vaccine available against tuberculosis (TB). In an effort to standardize the vaccine production, three substrains, i.e. BCG Danish 1331, Tokyo 172-1 and Russia BCG-1 were established as the WHO reference strains. Both for BCG Tokyo 172-1 as Russia BCG-1, reference genomes exist, not for BCG Danish. In this study, we set out to determine the completely assembled genome sequence for BCG Danish and to establish a workflow for genome characterization of engineering-derived vaccine candidate strains. RESULTS: By combining second (Illumina) and third (PacBio) generation sequencing in an integrated genome analysis workflow for BCG, we could construct the completely assembled genome sequence of BCG Danish 1331 (07/270) (and an engineered derivative that is studied as an improved vaccine candidate, a SapM KO), including the resolution of the analytically challenging long duplication regions. We report the presence of a DU1-like duplication in BCG Danish 1331, while this tandem duplication was previously thought to be exclusively restricted to BCG Pasteur. Furthermore, comparative genome analyses of publicly available data for BCG substrains showed the absence of a DU1 in certain BCG Pasteur substrains and the presence of a DU1-like duplication in some BCG China substrains. By integrating publicly available data, we provide an update to the genome features of the commonly used BCG strains. CONCLUSIONS: We demonstrate how this analysis workflow enables the resolution of genome duplications and of the genome of engineered derivatives of the BCG Danish vaccine strain. The BCG Danish WHO reference genome will serve as a reference for future engineered strains and the established workflow can be used to enhance BCG vaccine standardization.

Genomic characterization of two Orf virus isolates from Jilin province in China.

Orf virus (ORFV), a typical member of the Parapoxvirus genus within the family Poxviridae, which is the causative agent of Orf, a common epitheliotropic viral disease of sheep, goats, wild ruminants, and humans. In the present study, we sequenced the complete genomic sequences of two ORFV strains (ORFV-SY17, isolated from sheep, and ORFV-NA17, isolated from goat) and conducted the comparative analysis of multiple ORFVs. The complete genomic sequence of ORFV-SY17 was at length of 140,413 bp, including 131 potential open reading frames (ORFs) flanked by inverted terminal repeats (ITRs) of 4267 bp at both ends. The ORFV-NA17 strain displayed the similar genome structure with ORFV-SY17. The whole genomic sequence of ORFV-NA17 strain was 139,287 bp in length and contained 132 ORFs flanked by ITRs of 3974 bp. The overall G+C contents of ORFV-SY17 and ORFV-NA17 genome sequences were about 63.8% and 63.7%, respectively. The ITR sequences analysis showed that ORFV-SY17 and ORFV-NA17 contained the terminal BamHI sites and conserved telomere resolution sequences at both ends of their genome. In addition, comparative analysis of ORFs among ORFV-SY17, ORFV-NA17, and other ORFV strains revealed several sequence variations caused by insertions or deletions, especially in ORFs 005 and 116, which were very likely associated with host species. Phylogenetic analysis based on the complete genome sequences revealed that ORFV-SY17 was genetically closely related to NA1/11 and HN3/12 strains derived from sheep, while ORFV-NA17 was closely related to YX strain derived from goat. The multiple alignment of deduced amino acid sequences further revealed the genetic relationship between host species and genetic variations of ORFV strains. Taken together, the availability of genomic sequences of ORFV-SY17 and ORFV-NA17 strains from Jilin Province will aid in our understanding of the genetic diversity and evolution of ORFV strains in this region and can assist in distinguishing between ORFV strains that originate in sheep and goats.

Whole-genome analysis of the colonization-resistant bacterium Phytobacter sp. SCO41T isolated from Bacillus nematocida B16-fed adult Caenorhabditis elegans.

Colonization resistance is an important attribute for bacterial interactions with hosts, but the mechanism is still not completely clear. In this study, we found that Phytobacter sp. SCO41T can effectively inhibit the in vivo colonization of Bacillus nematocida B16 in Caenorhabditis elegans, and we revealed the colonization resistance mechanism. Three strains of colonization-resistant bacteria, SCO41T, BX15, and BC7, were isolated from the intestines of the free-living nematode C. elegans derived from rotten fruit and soil. The primary characteristics and genome map of one of the three isolates was investigated to explore the underlying mechanism of colonization resistance in C. elegans. In addition, we performed exogenous iron supplementation and gene cluster knockout experiments to validate the sequencing results. The results showed that relationship was close among the three strains, which was identified as belonging to the genus Phytobacter. The type strain is SCO41T (= CICC 24103T = KCTC 52362T). Whole genome analysis showed that csgA, csgB, csgC, csgE, csgF, and csgG were involved in the curli adhesive process and that fepA, fepB, fepC, fepD, and fepG played important roles in SCO41T against the colonization of B. nematocida B16 in C. elegans by competing for iron. Exogenous iron supplementation showed that exogenous iron can increase the colonization of B. nematocida B16, which was additionally confirmed by a deletion mutant strain. The csg gene family contributes to the colonization of SCO41T in C. elegans. Curli potentially contribute to the colonization of SCO41T in C. elegans, and enterobactin has a key role in SCO41T to resist the colonization of B. nematocida B16 by competing for iron.

Complete genome sequence and pathogenic analysis of a new betanodavirus isolated from shellfish.

We determined the complete genomic RNA sequence of a new type of betanodavirus Korea shellfish nervous necrosis virus (KSNNV) isolated from shellfish. Compared with other isolates representing four genotypes of betanodaviruses, the identity of the whole nucleotide sequence of the virus was in the range of 76%-83% with the presence of specific genetic motifs and formed a separate new branch in the phylogenetic analysis. In pathogenic analysis by immersion method, KSNNV-KOR1 shows 100% cumulative mortality like SFRG10/2012BGGa1 (RGNNV) in newly hatched sevenband grouper and mandarin fish, which is clearly different from those found in negative control groups. There were no significant differences in increasing rates of mortality and viral intra-tissue concentration of larval fishes infected with KSNNV-KOR1 at both 20 and 25°C water temperature. Histopathological examination of each fish species in the moribund stage revealed the presence of clear vacuoles in both brain and retinal tissues similar to typical histopathology features of RGNNV. In the present study, we first report a new betanodavirus from shellfish as the aetiological agent of viral nervous necrosis disease in fish with complete genomic nucleotide sequence and pathogenic analysis.

Characterization and comparative analysis of the genome of Puccinia sorghi Schwein, the causal agent of maize common rust.

Rust fungi are one of the most devastating pathogens of crop plants. The biotrophic fungus Puccinia sorghi Schwein (Ps) is responsible for maize common rust, an endemic disease of maize (Zea mays L.) in Argentina that causes significant yield losses in corn production. In spite of this, the Ps genomic sequence was not available. We used Illumina sequencing to rapidly produce the 99.6Mbdraft genome sequence of Ps race RO10H11247, derived from a single-uredinial isolate from infected maize leaves collected in the Argentine Corn Belt Region during 2010. High quality reads were obtained from 200bppaired-end and 5000bpmate-paired libraries and assembled in 15,722 scaffolds. A pipeline which combined an ab initio program with homology-based models and homology to in planta enriched ESTs from four cereal pathogenic fungus (the three sequenced wheat rusts and Ustilago maydis) was used to identify 21,087 putative coding sequences, of which 1599 might be part of the Ps RO10H11247 secretome. Among the 458 highly conserved protein families from the euKaryotic Orthologous Groups (KOG) that occur in a wide range of eukaryotic organisms, 97.5% have at least one member with high homology in the Ps assembly (TBlastN, E-value⩽e-10) covering more than 50% of the length of the KOG protein. Comparative studies with the three sequenced wheat rust fungus, and microsynteny analysis involving Puccinia striiformis f. sp. tritici (Pst, wheat stripe rust fungus), support the quality achieved. The results presented here show the effectiveness of the Illumina strategy for sequencing dikaryotic genomes of non-model organisms and provides reliable DNA sequence information for genomic studies, including pathogenic mechanisms of this maize fungus and molecular marker design.

Characterization of a new apple luteovirus identified by high-throughput sequencing.

BACKGROUND: 'Rapid Apple Decline' (RAD) is a newly emerging problem of young, dwarf apple trees in the Northeastern USA. The affected trees show trunk necrosis, cracking and canker before collapse in summer. In this study, we discovered and characterized a new luteovirus from apple trees in RAD-affected orchards using high-throughput sequencing (HTS) technology and subsequent Sanger sequencing. METHODS: Illumina NextSeq sequencing was applied to total RNAs prepared from three diseased apple trees. Sequence reads were de novo assembled, and contigs were annotated by BLASTx. RT-PCR and 5'/3' RACE sequencing were used to obtain the complete genome of a new virus. RT-PCR was used to detect the virus. RESULTS: Three common apple viruses and a new luteovirus were identified from the diseased trees by HTS and RT-PCR. Sequence analyses of the complete genome of the new virus show that it is a new species of the genus Luteovirus in the family Luteoviridae. The virus is graft transmissible and detected by RT-PCR in apple trees in a couple of orchards. CONCLUSIONS: A new luteovirus and/or three known viruses were found to be associated with RAD. Molecular characterization of the new luteovirus provides important information for further investigation of its distribution and etiological role.

Identification of a recombinant isolate of ungulate copiparvovirus.

To evaluate the status of parvovirus infection in free-range cows in a region of northeast China, nine serum samples were collected and analysed by sequencing and polymerase chain reaction. A new bovine parvovirus-2 (BPV2) was identified and named QQHE16. The genome of the virus is 5759 nucleotides long and retains two ORFs that are typical of the Parvovirinae family. Compared with reference BPV2 strains, BPV2 QQHE16 appeared to have a close relationship with strain BSRI isolated in the USA in 2013. A putative recombination breakpoint located at nucleotide position 2121 and in the interval between the non-structural gene and the VP gene was identified. From our analysis, we propose that strain QQHE16 originates from the natural recombination of strains ujs2665 and BSRI.

Genome characterization of sweet potato symptomless virus 1: a mastrevirus with an unusual nonanucleotide sequence.

Complete genomic sequences of nine isolates of sweet potato symptomless virus 1 (SPSMV-1), a virus of the genus Mastrevirus in the family Geminiviridae, were determined from sweet potato accessions from different countries and found to be 2,559-2,602 nucleotides in length. These isolates shared 97-100% genome sequence identity and had an unusual nonanucleotide sequence (TAAGATTCC) in a large intergenic region as well as an additional open reading frame, C3, which is conserved in dicot-infecting mastreviruses.

Characterization of a Gallid herpesvirus 2 strain with novel reticuloendotheliosis virus long terminal repeat inserts.

A bacterial artificial chromosome clone, designated LCY, was constructed from a Gallid herpesvirus 2 (GaHV-2) isolate from a GaHV-2 and reticuloendotheliosis virus co-infected clinical sample. The LCY GaHV-2 insert was sequenced and found to consist of 175,319 nucleotides. LCY GaHV-2 open reading frames (ORFs) had a high sequence identity to those of reference strains. The major difference was that two REV long terminal repeats (LTRs), in the same direction, were inserted at the internal repeat short (IRs)/unique short (Us) and Us/terminal repeat short (TRs) junctions. In addition, the a-like sequence and UL36 were different from other strains. Phylogenetic analysis revealed that LCY was closely related to pandemic strains in China. A pathogenicity study and a vaccination-challenge test were performed on LCY and the reference strain, GA. The results showed that LCY induced gross Marek's disease (MD) lesions and mortality in 71.4 and 7.1% of chickens, respectively, which are lower rates than those observed for the reference strain GA (85.7 and 35.7%). The commercially available CVI988 vaccine provided complete protection against LCY and GA (100%). These results showed that the isolate exhibited lower pathogenicity in SPF chickens. This study revealed that a novel pattern of LTR inserts was found in the strain LCY and that the strain was of low virulence. The present work expands the available genetic information for GaHV-2 and will be useful for the control of MD in China.

Molecular characterization of pathogenic and nonpathogenic fowl aviadenovirus serotype 11 isolates.

Fowl aviadenoviruses, many of which are of importance in veterinary medicine, are classified into 5 species. In this study, a pathogenic isolate and a nonpathogenic isolate of fowl aviadenovirus serotype 11 (FAdV-11) of species Fowl aviadenovirus D were characterized. Growth rates were analyzed for the 2 isolates, showing notable differences. The complete genomic sequences of the viruses were fully determined and were analyzed. The genomes of the 2 isolates showed 98.1% sequence identity and revealed 6 nonsynonymous mutations between the Ontario isolates. Two of the 6 mutations were also found in the sequences of recently published pathogenic Chinese fowl aviadenovirus 11 isolates, suggesting potential molecular markers that could be associated with pathogenesis. Deletions were found in the L5 region within the overlapping coding sequences for the 100, 22, and 33 kDa proteins, and these were found in only the nonpathogenic isolates. This molecular pattern was identified in FAdV-9, another nonpathogenic FAdV-D species virus. Furthermore, the tandem repeat regions varied dramatically; the pathogenic isolates contained a reduced number of tandem repeats compared with the nonpathogenic isolates. Lastly, a protein produced early in infection was analyzed using bioinformatics to determine its role in disease. This study highlights several candidate molecular determinants of avian adenovirus genomes related to pathogenicity.

Two novel HLA alleles, HLA-A*30:02:01:03 and HLA-C*08:113, identified in a South African bone marrow donor.

Genomic sequencing of HLA-A*30:02:01:03, an intronic variant, and HLA-C*08:113, an exonic variant.