Cloning Sequence Research Articles

P-724. The epidemiology, phenotype, and phylogeny of an influenza A/H3N2 virus outbreak among vaccinated U.S. Navy midshipmen

Abstract Background Characterizing influenza outbreaks among vaccinated populations may support optimization of influenza prevention. We used a surveillance study at the United States Naval Academy (USNA) to characterize the epidemiology, phenotype, and phylogeny of a recent influenza A/H3N2 outbreak among vaccinated midshipmen (undergraduate students). Figure 1 Figure 1A: Influenza A/H3N2 case counts among USNA Midshipmen and Staff Evaluated in February 2024. Day 1 refers to the collection time of the first influenza A/H3N2 outbreak clonal sequence. ‘Mitigation strategies implemented’ included case isolation, case masking, and contact chemoprophylaxis. Figure 1B: Estimated Rt (effective reproductive number) of influenza A/H3N2 virus cases in February 2024. Day 1 refers to the collection timing of first clonal influenza sequence. Methods “Acute Respiratory Infections at the Academy” (ARIA) is a study of medically attended ARIs (MAARIs) among USNA midshipmen and staff presenting to the Brigade Medical Unit for clinical care; the USNA comprises ≈ 4000 midshipmen. ARIA abstracts epidemiological, clinical and laboratory data from MAARIs. Residual clinical respiratory tract swabs also undergo multiplex PCR and sequencing of respiratory viruses, including influenza viruses. Demographic, Clinical and Viral Characteristics of Influenza A/H3N2 Positive Cases Results An outbreak of influenza A/H3N2 emerged in February 2024, with declining cases and Rt following mitigation strategies (case masking, isolation, and post-exposure oseltamivir; Fig 1). A total of 87 A/H3N2 cases were recorded, predominantly in midshipmen. HA sequence data from 83 cases confirmed local clonal transmission (3C.2a1b.2a.2a.3a.1 clade), consistent with early Rt estimates (Fig 1, Fig 2). The median age of cases was 20 years and 90% received 2023/2024 Northern Hemisphere influenza vaccine (99% egg-based inactivated influenza vaccine) by October of 2023. Over half (55%) of cases were treated with oseltamivir, and no evidence of antiviral resistance was detected. No cases were hospitalized, and the median sick-in-quarters time was 3 days (Table 1). Phylogenetic analysis did not indicate major genetic divergence from the 2023-2024 Northern Hemisphere vaccine A/H3N2 HA sequence (Fig 3). Time-scaled phylogeny of influenza A/H3N2 HA segment genes sequenced from ARIA (red branches) with context background sequences from GISAID. Phylogenetic tree inferred by IQTree and visualized in Auspice using the NextStrain environment. Inset A captures the diversity of 2a.3a.1 influenza cases involved in this outbreak investigation. Inset B shows the largest cluster of cases (n=58) with identical HA gene sequences. The 2023-2024 Northern Hemisphere vaccine strain sequences are indicated. Clade numbers are annotated by color. Conclusion Influenza A/H3N2 outbreaks can be impactful in vaccinated congregate populations; further analysis is in progress to identify strain antigenicity markers which may have contributed to this outbreak and predict other outbreaks. Non-pharmaceutical countermeasures and chemoprophylaxis appeared effective in mitigating this outbreak. Our findings highlight the importance of further research into universal influenza vaccines, alternative vaccine dosing regimens, and optimal chemoprophylaxis strategies to prevent such outbreaks. Disclosures Simon Pollett, MBBS, AstraZeneca: The IDCRP and HJF were funded to conduct an unrelated phase III COVID-19 monoclonal antibody immunoprophylaxis trial as part of US Govt COVID Response Timothy Burgess, MD, MPH, AstraZeneca: The IDCRP and HJF were funded to conduct an unrelated phase III COVID-19 monoclonal antibody immunoprophylaxis trial as part of US Govt COVID Response

Mechanisms of blaIMP-4 dissemination across diverse carbapenem resistant clinical isolates.

The IMP-4 carbapenemase is an endemic cause of carbapenem resistance in the Asia-Pacific region. Our aim was to determine the dissemination mechanism of the blaIMP-4 gene. Twelve representative Australian IMP-4 clinical isolates from The Alfred Hospital, were characterised using antimicrobial susceptibility testing and genome and plasmid assemblies analysed. Conjugation efficiencies of different plasmids were investigated using filter mating with four recipient strains across two species. Selected IMP-4 isolates included six species and four genera (Enterobacter, Klebsiella, Serratia and Acinetobacter), whereby isolates of the same species belonged to the same sequence type and were closely related. Four IMP-4 plasmid types were noted: IncHI2A type 1 and 2 (Klebsiella spp. and Enterobacter hormaechei, respectively), IncC (Serratia marcescens and Klebsiella pneumoniae), and a novel type in Acinetobacter pittii. Sequence homology was observed across all plasmids at the blaIMP-4 location, termed Region I, with IS26- on IncHI2A and IS5075-and Tn3 resistance gene cassettes present IncC plasmids, respectively. Genomic rearrangements mediated by IS26 or Tn3 and IS5075 were identified in Region I of plasmids from the same Inc type. Plasmids of each Inc type were capable of conjugative transfer with varying efficiency. IncH12A plasmids and K. pneumoniae IncC displayed higher transfer efficiencies than other plasmids examined in this study when using the recipient E. coli strain J53 (with conjugation efficiencies of 1.17×10-2 - 5.02×10-5, p<0.001). Clonal spread, Inc type, homologous region and insertion sequences are important mobility factors in the dissemination and evolution of blaIMP-4 plasmids.

Hybridization and diversity of the genus Vandenboschia in Korea insights from morphological, cytological, and genotype analyses

The genus Vandenboschia exhibits significant diversity, driven by complex hybridization events and varying ploidy levels in the natural habitats of Korea, leads to misidentifications that obscure its true distribution and classification. To address this issue, the present study employed morphological, cytological, and genotype analyses to clarify the taxonomical circumscription and distribution patterns of the Vandenboschia radicans complex in Korea. The V. radicans complex in the genus refers to V. radicans and its closely related species and exhibits significant morphological variation, making classification challenging. Previous molecular studies have revealed that it consists of several non-hybrid and hybrid species among them. Through the present study, comprehensive sampling from Jeju and Ulleung Islands identified five distinct taxa from the V. radicans complex: two non-hybrid species (Vandenboschia kalamocarpa and Vandenboschia nipponica), and three hybrids (Vandenboschia × stenosiphon, Vandenboschia × quelpaertensis, and Vandenboschia kalamocarpa × Vandenboschia nipponica × Vandenboschia striata). Notably, hybrids exhibited broader geographic distributions compared to non-hybrid species, which were confined to specific microhabitats. While overlapping morphological traits among the species complicate identification without genetic analysis, rhizome diameter and the width-to-length ratio of the involucre can serve as key morphological traits for distinguishing members of the Vandenboschia radicans complex in Korea. Additionally, cleaved amplified polymorphic sequence (CAPS) markers and cloning techniques were employed for species identification, particularly when morphological traits were inconclusive. These findings highlight the need to integrate genetic tools with traditional taxonomic methods to resolve complex species relationships within Vandenboschia in Korea and emphasize the importance of conservation efforts for geographically restricted and genetically distinct populations.

Golden Gate Cloning of Synthetic CRISPR RNA Spacer Sequences.

Prokaryotes use CRISPR-Cas systems to interfere with viruses and other mobile genetic elements. CRISPR arrays comprise repeated DNA elements and spacer sequences that can be engineered for custom target sites. These arrays are transcribed into precursor CRISPR RNAs (pre-crRNAs) that undergo maturation steps to form individual CRISPR RNAs (crRNAs). Each crRNA contains a single spacer that identifies the target cleavage site for a large variety of Cas protein effectors. Precise manipulation of spacer sequences within CRISPR arrays is crucial for advancing the functionality of CRISPR-based technologies. Here, we describe a protocol for the design and creation of a minimal, plasmid-based CRISPR array to enable the expression of specific, synthetic crRNAs. Plasmids contain entry spacer sequences with two type IIS restriction sites and Golden Gate cloning enables the efficient exchange of these spacer sequences. Factors that influence the compatibility of the CRISPR arrays with native or recombinant Cas proteins are discussed.

The mutation of ent-kaurenoic acid oxidase, a key enzyme involved in gibberellin biosynthesis, confers a dwarf phenotype to cucumber.

A dwarf mutant with short branches (csdf) was identified from EMS-induced mutagenesis. Bulked segregant analysis sequencing and map-based cloning revealed CsKAO encoding ent-kaurenoic acid oxidase as the causal gene. Plant architecture is the primary target of artificial selection during domestication and improvement based on the determinate function for fruit yield. Plant architecture is regulated by complicated genetic networks, more underlying mechanism remains to be elucidated. Here, we identified a dwarf mutant (csdf) in an EMS-induced cucumber population, and genetic analysis revealed the mutated phenotype is controlled by a single recessive gene. Optical microanalysis showed the decrease in cell length is mainly contribute to the dwarf phenotype. By strategy of BSA-seq combined with map-based cloning, CsaV3_6G006520 (CsKAO) on chromosome 6 was identified as the candidate gene for csdf. Gene cloning and sequence alignment revealed a G to A mutation in the sixth exon, which causes the premature stop codon in CsKAO of csdf. Expression analysis revealed CsKAO was expressed in various tissues with abundant transcripts, and has significant differences between WT and csdf. Gene annotation indicated CsKAO encodes a cytochrome P450 family ent-kaurenoic acid oxidase which functioned in GA biosynthesis. GA-relevant analysis showed that endogenous GA contents were significantly decreased and the dwarfism phenotype could be restored by exogenous GA3 treatment; while, some of the representative enzyme genes involved in the GA pathway were up-regulated in csdf. Besides, IAA content is decreased in the terminal bud and increased in the lateral bud in csdf as well as several IAA-related genes are differentially expressed. Overall, those findings suggest that CsKAO regulated plant height via the influence on GAs pathways, and IAA might interact with GAs on plant architecture morphogenesis in cucumber.

Single-cell analysis of a bi-clonal chronic lymphocytic leukemia reveals two clones with distinct gene expression pattern

Dual productive B-cell receptor (BCR) rearrangements have been repeatedly reported for chronic lymphocytic leukemia (CLL), but the standard population-based PCR analyses cannot distinguish whether these are bi-clonal CLL, or a monoclonal CLL with bi-allelic productive rearrangements. We investigated CLL cells by combined single-cell RNA and BCR sequencing. We identified two CLL clones using different immunoglobulin (Ig) heavy-chain V region genes (IGHV) genes and distinct Ig λ light chains. One clone is classified as Ig unmutated the other as mutated. The two CLL clones have distinct transcriptomes: Numerous genes were differentially expressed, with genes typical for unmutated or mutated CLL showing the expected representation in the two clones. Using PCR, cloning and Sanger sequencing of the IGHV rearrangements we detected both CLL clones over a period of three years without clinical progression of the CLL and thus giving insights into the disease biology of multi-clonal CLL.

Map-based cloning revealed BhAPRR2 gene regulating the black peel formation of mature fruit in wax gourd (Benincasa hispida).

Map-based cloning revealed BhAPRR2, encoding a two-component response-regulating protein that regulates the black peel formation of mature fruit in wax gourd. Wax gourd is an economically significant vegetable crop, and peel color is a crucial agronomic trait that influences its commercial value. Although genes controlling light green or white peel have been cloned in wax gourd, the genetic basis and molecular mechanism underlying black peel remain unclear. Here, we confirmed that the peel color of wax gourd is a qualitative trait governed by single gene, with black being dominant over green. Through bulked segregant analysis sequencing (BSA-seq) and map-based cloning, we identified Bh.pf3chr5g483 as the candidate gene. This gene encodes a two-component response-regulating protein and is homologous to APRR2, referred to as BhAPRR2. Compared to P170, the BhAPRR2 in YD1 exhibits multiple mutations in both its coding and promoter regions. Notably, the mutations in the coding region do not affect its nuclear localization or transcriptional activation activity. However, the mutations in the promoter region substantially increase its expression in the peel of YD1, potentially contributing to the black peel phenotype observed in this variety. Furthermore, we developed an insertion/deletion (InDel) marker based on a 93-base pair (bp) insertion/deletion mutation in the promoter region of BhAPRR2, which achieved up to 95.8% phenotypic accuracy in a natural population comprising 165 wax gourd germplasms. In summary, our findings suggest that mutations in the promoter region of BhAPRR2 may contribute to the development of black peel in wax gourd. This discovery provides new insights into the molecular and genetic mechanisms underlying peel color diversity and offers a valuable molecular marker for wax gourd breeding efforts.

Construction and Characterization of a Full-Length Infectious cDNA Clone of a Strain of Watermelon Mosaic Virus Isolated from Melon.

Watermelon mosaic virus (WMV), a member of the genus Potyvirus, causes serious economic losses in cucurbit crops. For molecular biological studies of viruses, it is necessary to construct an infectious clone that can facilitate gene functional analysis. In this study, we constructed an infectious cDNA clone of WMV genomic RNA by Gibson assembly and evaluated its virulence and symptoms on a variety of host plants. A WMV isolate (WMV-IS-me2) collected from melon in Iksan, Jeonbuk Province, Korea in 2015 caused mosaic symptoms on leaves. Four overlapping PCR fragments of the full-length genome of this isolate and the pJL89 binary vector with overhanging ends of 40 bp were amplified by reverse transcription PCR and joined in a single isothermal reaction. The complete nucleotide sequence of the infectious cDNA clone of WMV was determined and the recombinant vector was transformed into Agrobacterium tumefaciens GV3101. Following agro-infiltration, an infectious clone (pWMV-M, KACC95145P) systemically induced mosaic symptoms on watermelon (Citrullus lanatus), zucchini (Cucurbita pepo), melon (Cucumis melo), and Nicotiana benthamiana plants. This infectious clone may be useful for screening cucurbit varieties resistant to WMV, as well as for studying viral gene function.

A high-resolution two-step evolution experiment in yeast reveals a shift from pleiotropic to modular adaptation.

Evolution by natural selection is expected to be a slow and gradual process. In particular, the mutations that drive evolution are predicted to be small and modular, incrementally improving a small number of traits. However, adaptive mutations identified early in microbial evolution experiments, cancer, and other systems often provide substantial fitness gains and pleiotropically improve multiple traits at once. We asked whether such pleiotropically adaptive mutations are common throughout adaptation or are instead a rare feature of early steps in evolution that tend to target key signaling pathways. To do so, we conducted barcoded second-step evolution experiments initiated from 5 first-step mutations identified from a prior yeast evolution experiment. We then isolated hundreds of second-step mutations from these evolution experiments, measured their fitness and performance in several growth phases, and conducted whole genome sequencing of the second-step clones. Here, we found that while the vast majority of mutants isolated from the first-step of evolution in this condition show patterns of pleiotropic adaptation-improving both performance in fermentation and respiration growth phases-second-step mutations show a shift towards modular adaptation, mostly improving respiration performance and only rarely improving fermentation performance. We also identified a shift in the molecular basis of adaptation from genes in cellular signaling pathways towards genes involved in respiration and mitochondrial function. Our results suggest that the genes in cellular signaling pathways may be more likely to provide large, adaptively pleiotropic benefits to the organism due to their ability to coherently affect many phenotypes at once. As such, these genes may serve as the source of pleiotropic adaptation in the early stages of evolution, and once these become exhausted, organisms then adapt more gradually, acquiring smaller, more modular mutations.

Intra-individual polymorphisms in the mitochondrial COI gene of tick-killing Ixodiphagus wasps parasitizing Haemaphysalis flava ticks.

Ixodid ticks are significant vectors of pathogens affecting both humans and animals. Biological control with natural enemies represents a sustainable tool for managing ticks. However, there is a substantial lack of knowledge about the natural enemies of ticks. Wasps of the genus Ixodiphagus (Encyrtidae) are currently the only known tick-specific parasitoids. While these wasps have been sporadically recorded worldwide, their presence in Eastern Asia is poorly documented. In this study, we investigated the occurrence of Ixodiphagus wasps in field-collected ticks reared on rabbits under laboratory conditions. Ticks were collected from the Hokkaido, Hokuriku, Chugoku, Shikoku, and Kyushu regions in Japan. Out of 1,933 Haemaphysalis ticks attached to rabbits, adult wasps emerged from 15 engorged ticks. All the ticks from which wasps emerged were morphologically and molecularly identified as Haemaphysalis flava. Additionally, wasp DNA was detected in unfed H. flava nymphs using a newly designed Ixodiphagus-specific PCR assay. Among nine experimental sites in the Chugoku region, Ixodiphagus wasps were detected at three sites, with parasitism rates ranging from 1.8% to 8.1%. Finally, the mitochondrial COI genes of four wasp and two tick samples were characterized using shotgun sequencing, direct sequencing and in-fusion cloning approaches. Multiple intra-individual polymorphisms were observed in all the tested samples. Further studies are needed to investigate the relationship between Ixodiphagus wasps and Ixodid ticks. An increased understanding of these parasitoid wasps could contribute to future biological control measures against ticks.

Profound reduction of HIV-1 reservoir cells over 3 decades of antiretroviral therapy started in early infancy.

HIV-1 reservoir cells persist indefinitely during suppressive antiretroviral therapy (ART) in individuals who acquire infection in adulthood, but little is known about the longitudinal evolution of viral reservoir cells during long-term ART started during early infancy. We studied 2 fraternal twins who acquired HIV-1 perinatally, started ART at week 10 after birth and remained on ART for 28 years. We observed that the frequency of genome-intact proviruses, determined by single-genome near-full-length proviral sequencing, declined by approximately 4,000- to 13,000-fold during this period, indicating enhanced decay rates of intact proviruses even after adjusting for dilution effects from somatic growth. Despite analyzing more than one billion PBMC after 28 years of ART in each participant, no intact proviruses were detected in 1 participant, and 1 intact provirus was isolated in the other. The longitudinal decline of defective proviruses in the 2 participants was more similar to proviral decay kinetics reported in individuals who started ART during adulthood; moreover, clonal sequence clusters were readily detectable for defective proviruses but not for intact proviruses after 28 years of ART in the 2 twins. Together, these data suggest decreased long-term stability and increased immunological vulnerability of intact proviruses during long-term ART started in early infancy.

Bio-characteristics, tissue expression of miR-375 in hypothalamic-pituitary-ovarian axis and its regulation in reproduction-related diseases

Our study concentrated on the expression of miRNA-375 in the hypothalamic-pituitary-gonadal axis of female Hu sheep. The investigation involved cloning the precursor sequence of miR-NA-375, followed by comparison with database entries and subsequent Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. In our approach, we obtained ovaries, thalamus, cerebellum, brain, uterus, pituitary gland, hypothalamus, and pineal gland from fertile but nonpregnant Hu ewes. MiRNA extraction kit was used to extract miRNA from the above eight tissues. Real-time fluorescent quantitative polymerase chain reaction was used to evaluate the role of miR-375 in the hy-pothalamic-pituitary-gonadal axis. The results of miR-375 precursor sequence cloning were compared with those of Anopheles gambiae, Apis mellifera, Bos taurus, Drosophila melanogaster, Danio rerio, Fugu rubripes, Gallus gallus, Homo sapiens, Monodelphis domestica, Macaca mulatta, Mus musculus, Pan troglodytes, Rattus norvegicus, Tetraodon nigroviridis, Xenopus tropicalis miR-375 in miRBase database. It was found that oar-miR-375 was highly conserved. Notably, miR-375 expression in the pineal gland was significantly higher (p < 0.01) than that in the ovaries, thalamus, cerebellum, brain, uterus, pituitary gland, hypothalamus. The study also involved predicting miR-375 target genes. GO and KEGG enrichment analyses of these predicted target genes revealed that miR-375 is involved in 182 biological processes, affects 186 cellular components, and participates in 184 molecular functions. In terms of pathway enrichment, miR-375 was linked to nine pathways, including the Hippo, Wnt, and mTOR signaling pathways. This study has validated the interaction between miR-375 and its target gene FZD4, which can be recognized and bound to produce effects. These findings lead to the inference that miR-375 may play a crucial regulatory role in sheep reproduction through the Hippo pathway and Wnt pathway, laying a foundation for further exploration of miR-375’s role in this domain.

A new monopartite begomovirus infecting Melochia tomentosa in Burkina Faso.

This is the first description of the complete genome sequence of a newly characterized monopartite begomovirus isolated from an asymptomatic uncultivated plant, Melochia tomentosa, collected in Burkina Faso. The sequence was obtained through rolling-circle amplification, cloning, and Sanger sequencing. The provisional species name "Begomovirus melochiae" and common virus name "melochia associated virus" (MeAV) are proposed. The MeAV genome was found to share the most nucleotide sequence similarity with three African monopartite begomoviruses: tomato curly stunt virus (74%), pepper yellow vein Mali virus (73%), and tomato leaf curl Cameroon virus (73%). Phylogenetic analysis confirmed its relationship to Old World monopartite begomoviruses. The discovery of MeAV in an uncultivated and asymptomatic plant provides a further example of the high diversity of begomoviruses in sub-Saharan African ecosystems.

Semisynthetic Phage Display Library Construction: Generation of Filtered Libraries.

Display of antibody fragments on the surface of M13 filamentous bacteriophages is a well-established approach for the identification of antibodies binding to a target of interest. Here, we describe the second of a three-step method to construct Antibody Libraries for Therapeutic Antibody Discovery (ALTHEA) Gold Plus Libraries. The three-step method involves (1) primary library (PL) construction, (2) filtered library (FL) construction, and (3) secondary library construction. The second step, described here, involves display of the PLs as single-chain variable fragment (scFv) fusions to protein pIII of the M13 phage, as well as heat shock treatment and subsequent selection of well-folded and thermostable scFvs via protein L binding, whereas unstable and defective scFvs are removed by washing steps and centrifugation. The quality of the filtration process is assessed by sequencing clones chosen at random from the FLs. These libraries, enriched with thermostable antibodies, are then ready to be used for the third and final step of the process: generation of secondary libraries.

Semisynthetic Phage Display Library Construction: Generation of Single-Chain Variable Fragment Secondary Libraries.

Display of antibody fragments on the surface of M13 filamentous bacteriophages is a well-established approach for the identification of antibodies binding to a target of interest. Here, we describe the third and final step of a three-step method to construct Antibody Libraries for Therapeutic Antibody Discovery (ALTHEA) Libraries. The three-step method involves (1) primary library construction, (2) filtered library (FL) construction, and (3) secondary library (SL) construction. In the third step, described here, the nucleotide sequences encoding the single-chain variable fragments (scFvs) of FLs are amplified by PCR and combined with the heavy- chain CDR3 region (HCDR3) and joining fragments (H3J) obtained from a pool of donors to maximize diversity ("natural H3J fragments"). These natural H3J fragments are amplified with a set of primers designed to capture >95% of the natural H3J repertoire. The resultant fragments replace the neutral H3J fragments of the FLs, resulting in the final semisynthetic secondary libraries. The quality of these libraries is assessed by sequencing clones chosen at random from the libraries, typically 96 clones. These libraries are then ready to be used for phage selections on targets of interest, providing a robust antibody discovery platform.

AAVolve: Concatenated long-read deep sequencing enables whole capsid tracking during shuffled AAV library selection

Gene therapies using recombinant adeno-associated virus (AAV) vectors have demonstrated considerable clinical success in the treatment of genetic disorders. Improved vectors with favourable tropism profiles, decreased immunogenicity and enhanced manufacturability are poised to further improve the state of gene therapies. Such vectors can be identified through directed evolution, a process of subjecting a diverse capsid library to a selection pressure to identify individual variants with a desired trait. Currently, libraries that involve changes distributed throughout the AAV capsid coding region, such as DNA family shuffled libraries, are largely characterised using low-throughput Sanger sequencing of individual clones. However, improvements in long-read sequencing technologies have increased their applicability to capsid libraries and evaluation of the selection process. Here we explore the application of Oxford Nanopore Technologies refined by a concatemeric consensus method for initial library characterization and monitoring selection of a shuffled AAV capsid library. Furthermore, we present AAVolve, a bioinformatic pipeline for processing long-read data from AAV directed evolution experiments. Our approach allows high throughput characterisation of AAV capsids in a streamlined manner, facilitating deeper insights into library composition through multiple rounds of selection, and generalization through training of machine learning models.

Rare occurrence of cryptic 5' splice sites by downstream 3' splice site/exon boundary mutations in a heavy-ion-induced egy1-4 allele of Arabidopsis thaliana.

Pre-mRNA splicing is a fundamental process in eukaryotic gene expression, and the mechanism of intron definition, involving the recognition of the canonical GU (5'-splice site) and AG (3'-splice site) dinucleotides by splicing factors, has been postulated for most cases of splicing initiation in plants. Splice site mutations have played crucial roles in unraveling the mechanism of pre-mRNA splicing in planta. Typically, splice site mutations abolish splicing events or activate one or more cryptic splice sites surrounding the mutated region. In this report, we investigated the splicing pattern of the EGY1 gene in an Ar-ion-induced egy1-4 allele of Arabidopsis thaliana. egy1-4 has an AG-to-AC mutation in the 3'-end of intron 3, along with 4-bp substitutions and a 5-bp deletion in adjacent exon 4. RT-PCR, cDNA cloning, and amplicon sequencing analyses of EGY1 revealed that while most wild-type EGY1 mRNAs had a single splicing pattern, egy1-4 mRNAs had multiple splicing defects. Almost half of EGY1 transcripts showed 'intron retention' at intron 3, while the other half exhibited activation of 3' cryptic splice sites either upstream or downstream of the original 3'-splice site. Unexpectedly, around 8% of EGY1 transcripts in egy1-4 exhibited activation of cryptic 5'-splice sites positioned upstream of the authentic 5'-splice site of intron 3. Whole genome resequencing of egy1-4 indicated that it has no other known impactful mutations. These results may provide a rare, but real case of activation of cryptic 5'-splice sites by downstream 3'-splice site/exon mutations in planta.

Complete genome sequence of grapevine yellow speckle viroid 3, a novel apscaviroid infecting grapevine, characterized by high-throughput sequencing.

A novel grapevine viroid was discovered in an asymptomatic grapevine of Indian rootstocks. The whole genome sequence of the viroid (370 nt) was determined by high-throughput sequencing as well as RT-PCR followed by cloning and Sanger sequencing. The terminal conserved region (TCR), central conserved region (CCR) upper strand, and CCR lower strand are conserved regions found in the viroid that are unique to the members of the genus Apscaviroid. Based on our findings and the demarcation criteria for viroids, the novel viroid, which we have tentatively named "grapevine yellow speckle viroid 3" is a putative new member of the genus Apscaviroid.

Diverse Head-to-Tail Sequences in the Circular Genome of Human Bocavirus Genotype 1 among Children with Acute Respiratory Infections Implied the Switch of Template Chain in the Rolling-Circle Replication Model.

Head-to-tail sequences have been reported in human bocavirus (HBoV) 1-4. To reveal their features and functions, HBoV DNA was screened among respiratory specimens from pediatric patients with an acute respiratory infection (ARI) between April 2020 and December 2022, followed by HBoV genotyping. Head-to-tail sequences were detected using nested PCR, TA cloning, and Sanger sequencing, and these findings were confirmed by mNGS and amplicon sequencing. The secondary structure was predicted using the Mfold web server. The results indicated that head-to-tail sequences were detected in 42 specimens through TA cloning from 351 specimens positive for HBoV1 DNA, yielding 92 sequences into 32 types and 2 categories. Additionally, head-to-tail sequences were detected in 16 specimens by amplicon sequencing, yielding 60 sequences categorized into 23 types. The 374nt type, detected in 13 specimens, contains variants 374a and 374b, which differ in the unpaired loop regions of the palindrome or complementary reverse sequences, implying a switch of template chains during the replication process. The mNGS results in three specimens confirmed the presence of circular genome in copies below 1%. In conclusion, head-to-tail sequences of HBoV1 were common in children with ARI and were highly diverse in length and sequences. The variants may be generated by the switch of the template chain in the rolling-circle replication model.

Genetic and mechanistic evaluation of an individual with para-Bombay phenotype associated with a compound heterozygote comprising two novel FUT1 variants.

As is well documented, the para-Bombay phenotype is typically characterized by the reduction or absence of ABH antigens on red blood cells but the presence of corresponding antigens in saliva. Herein, the underlying molecular mechanism of an individual with para-Bombay AB phenotype combined with two novel variants of the FUT1 gene was investigated. ABH antigens and antibodies were detected in the serum of the proband using conventional serological methods. The coding region nucleotides of the ABO, FUT1, and FUT2 genes were directly sequenced by polymerase chain reaction. Moreover, the FUT1 haploid type in the proband was analyzed by TA clone sequencing. The 3D structure of wild-type and mutant fucosyltransferases were simulated and analyzed using Phyre2 and Pymol software. Lastly, the effect of missense substitution on the function of fucosyltransferase was predicted by the Polymorphism Phenotyping algorithm (PolyPhen-2) and MutationTaster. ABH antigens were noted to be absent on the surface of red blood cells of the proband. The ABO genotype was ABO*A1.02/ABO*B.01, while the FUT2 genotype was FUT2*01/FUT2*c.357T. Interestingly, two novel missense variants (c.289G>A, p.Ala97Thr and c.575G>C, p.Arg192Pro) and one synonymous SNP (c.840G>A) were identified in the FUT1 gene. Furthermore, c.289G>A was detected in one haploid type, whereas c.575G>C and c.840G>A were discovered in another haploid type. Meanwhile, in silico analysis revealed that amino acid substitution caused by missense variants altered the partial spatial structure of the α-helices where residues 97 and 298 were located using 3D homology modeling software. Finally, both missense variants were defined as probably damaging based on PolyPhen-2 prediction. Two novel FUT1 variants were identified in a Chinese individual with para-Bombay AB phenotype, which can expand our understanding of the molecular mechanism underlying the para-Bombay phenotype and contribute to improving the safety of blood transfusion.