Recombinant Genomes Research Articles

Consecutive Affinity and Ion-Exchange Chromatography for AAV9 Vectors Purification

Background: Irrespective of the rapid development of AAV-based gene therapy, the production of clinical-grade vectors has a bottleneck resulting from product-related impurities such as empty and partially filled capsids, which lack a functional recombinant genome. Methods: In the current study, we applied the sequential affinity chromatography (AC)- and anion-exchange chromatography (AEX)-based method for purification of AAV9 vector harboring single-stranded genome encoding the fusion of firefly luciferase (fLuc)-yellow fluorescent protein (YFP) under chicken beta actin (CBA) promoter. We assessed the efficiency of two different pre-packed cross-linked sepharose and one monolithic AEX columns following AC purification to separate fully encapsulated with recombinant DNA AAV vectors from byproducts. Results: We showed the possibility to achieve approximately 20–80% recovery and over 90% calculated DNA-containing/empty capsid ratio depending on column and buffers composition. Additionally, we confirmed the infectivity of AAV by in vitro luciferase assay regardless of recovery method from different AEX columns. Conclusions: Our purification data indicate the effectiveness of dual chromatography method to obtain rAAV9 vectors with DNA-containing capsid content over 90%.

Nuclear Intron Sequence Variation of the Bulinus globosus Complex (Mollusca: Planorbidae): Implications for Molecular Systematic Analyses.

Urinary schistosomiasis is caused by the blood fluke Schistosoma haematobium, which is predominantly found in Africa. The freshwater snail Bulinus globosus is its main intermediate host. The species that make up the B. globosus group are genetically complex, and their taxonomic status remains controversial. Genetic variation, heterozygosity, and DNA recombination in B. globosus were examined using the mitochondrial cytochrome c oxidase subunit 1 (COI) and the intron 3 region of the arginine kinase gene (AkInt3). A total of 81 B. globosus snails were collected from three different localities in Kwale County, Kenya. Genomic diversity, heterozygosity, DNA recombination, and haplotype network were calculated using AkInt3 sequences. Low polymorphism in the COI sequence divided B. globosus into six haplotypes (C1-C6). However, AkInt3 sequencing studies showed high polymorphisms, classifying 81 B. globosus snails into 44 haplotypes (H1-H44). These haplotypes were separated into three haplogroups (I-III). AkInt3 sequence heterozygosity was also found. DNA recombination haplotypes between haplogroups were commonly found in heterozygous samples. AkInt3 sequence studies showed high levels of genetic polymorphism and heterozygosity, supporting its use as a genetic marker for elucidating the population genetics of B. globosus. Furthermore, our study showed that B. globosus populations in Kenya form a "species complex".

Whole-genome automated assembly pipeline for Chlamydia trachomatis strains from reference, in vitro and clinical samples using the integrated CtGAP pipeline.

Whole genome sequencing (WGS) is pivotal for the molecular characterization of Chlamydia trachomatis (Ct)-the leading bacterial cause of sexually transmitted infections and infectious blindness worldwide. Ct WGS can inform epidemiologic, public health and outbreak investigations of these human-restricted pathogens. However, challenges persist in generating high-quality genomes for downstream analyses given its obligate intracellular nature and difficulty with in vitro propagation. No single tool exists for the entirety of Ct genome assembly, necessitating the adaptation of multiple programs with varying success. Compounding this issue is the absence of reliable Ct reference strain genomes. We, therefore, developed CtGAP-Chlamydia trachomatisGenome Assembly Pipeline-as an integrated 'one-stop-shop' pipeline for assembly and characterization of Ct genome sequencing data from various sources including isolates, in vitro samples, clinical swabs and urine. CtGAP, written in Snakemake, enables read quality statistics output, adapter and quality trimming, host read removal, de novo and reference-guided assembly, contig scaffolding, selective ompA, multi-locus-sequence and plasmid typing, phylogenetic tree construction, and recombinant genome identification. Twenty Ct reference genomes were also generated. Successfully validated on a diverse collection of 363 samples containing Ct, CtGAP represents a novel pipeline requiring minimal bioinformatics expertise with easy adaptation for use with other bacterial species.

Dominance of recombinant DWV genomes with changing viral landscapes as revealed in national US honey bee and varroa mite survey

Honey bees are essential pollinators for global agriculture. The viromes of US commercial apiaries and their ectoparasitic mites are poorly characterized at a strain level and there is a need to integrate genomics into pathogen surveillance. We sequenced RNA viromes from 383 adult bees and 173 mites pooled samples from 11 major US beekeeping hubs in 2021, assembling 45 complete and 1702 partial genomes. Protein sequence similarity networks and recombinant genome identification revealed a new viral landscape. Sinaivirus (n = 312), Iflavirus sacbroodi (n = 280), and Iflavirus aladeformis (DWV, n = 135) genomes were common. Recombinant DWV genomes with high nucleotide identity were widespread, and DWV type A master variants were rare, with an indication that RT-PCR surveillance may over-detect type A due to the prevalence of recombinant DWV genomes. Future work should use genomic strategies to avoid misidentification of common honey bee virus genomes and their impact on colony health.

Metagenomic Analyses of Water Samples of Two Urban Freshwaters in Berlin, Germany, Reveal New Highly Diverse Invertebrate Viruses.

In an attempt to explore the RNA viromes of two German rivers, we searched the virus particle contents of one 50 L water sample each from the Teltow Canal and the Havel River for viruses assumed to infect invertebrates. More than 330 complete and partial virus genomes up to a length of 37 kb were identified, with noda-like and reo-like viruses being most abundant, followed by bunya-like and birna-like viruses. Viruses related to the Permutotetraviridae, Nidovirales, Flaviviridae, Rhabdoviridae and Chuviridae as well as the unclassified Jῑngmén virus and Negev virus groups were also present. The results indicate a broad extent of recombinant virus genomes, supporting the concept of the modularity of eukaryotic viruses. For example, novel combinations of genes encoding replicase and structural proteins with a jellyroll fold have been observed. Less than 35 viruses could be assigned to existing virus genera. These are (i) an avian deltacoronavirus which was represented by only one short contig, albeit with 98% similarity, (ii) a seadornavirus and a rotavirus, and (iii) some 30 nodaviruses. All remaining viruses are novel and too diverse for accommodation in existing genera. Many of the virus genomes exhibit ORFans encoding hypothetical proteins of up to 2000 amino acids without conserved protein domains.

Whole-Genome Analysis of Porcine Epidemic Diarrhea Virus from Yunnan, China.

Porcine epidemic diarrhea virus (PEDV) is a member of the genus α-coronavirus and causes severe diarrhea in piglets, leading to enormous economic losses in the pig industry. To understand the epidemic variation of PEDV strains in Yunnan province, three PEDV strains (YN2021, YNLP 2022, and YNBS 2022) and one commercially available attenuated vaccine strain (Attenuated AJ1102-R) that were previously isolated were sequenced and compared with the representative PEDV strains. NJ phylogenetic analysis showed that YN2021 strain and Attenuated CV777 strain were clustered into GI-b subtype, while YNLP 2022 and YNBS 2022 belong to GII-b subtype, accompanying ZJCZ4 and Attenuated AJ1102-R. RDP analysis revealed that YNLP 2022 was a genome recombination from both GII-b strain PEDV-7C and GII-a strain YN1, of which the recombination region is in the range nt4994-7605. YNBS 2022 strain was another recombination originated from GII-b subtype strain 17GXZC-1ORF3c and GII-a subtype strain PEDV-CHZ, of which the counterpart is in the range nt16399-22326. The Yunnan strain of PEDV was analyzed for the first time from the whole-genome perspective, and comprehensive analysis showed that the Yunnan strains have high genetic variation. This study may shed new light on the current PEDV infections in Yunnan and pave the way toward further control of PEDV infections.

Acceleration Of Recombinant Viral Sequences Search By 3SEQ Algorithm Via Adding Support Of Multi-Threaded Calculations And Considering Sample Collection Dates

The article presents an efficient multithreaded implementation of the modern 3SEQ algorithm for detecting recombinant genetic sequences, tested on viral genomes. The work was carried out within the framework of the project to create a domestic (Russian) web-platform (bioprojects.iis.nsk.su) for solving a wide range of problems related to data analysis in the field of bioinformatics, virology and epidemiology. A recombinant viral genome emerges when two different variants of virus genomes of the same species exchange their parts, which is possible in case of infection with both variants simultaneously. The emergence of recombinants is rare but important events in the context of virus evolution research. One of the most promising among the existing algorithms for searching for recombinants is 3SEQ, but the author's version works only in single-threaded mode. We implemented this algorithm with support for multithreaded computing and taking into account the dates of sample collection, which provided a significant increase in the computing speed. The developed software was used to search for recombinants in the samples of influenza A H1N1 (only PB2 segments from 2174 genomes were analyzed), Dengue fever (726 genomes), Ebola virus (865 genomes) and in two samples of SARS-CoV-2 coronavirus (776 and 2132 genomes). No recombinants were found for influenza A H1N1 (PB2 segment) and the first dataset on SARS-CoV-2 (variant from Russia), which is in agreement with the analysis of the same data by the RDP algorithm. For the second SARS-CoV-2 dataset (variants from the Siberian Federal District), the only recombinant present in the dataset was correctly found. 725 recombinants were found in Dengue fever viruses, with a recombination region length in the range from 50 to 1000 nucleotides. In Ebola viruses, the length of the recombination region was shorter – in 572 recombinants it was in the range of 50 to 100 nucleotides, and in 249 genomes – was less than 50 nucleotides.

The evolutionarily diverged single-stranded DNA-binding proteins SSB1/SSB2 differentially affect the replication, recombination and mutation of organellar genomes in Arabidopsis thaliana

Single-stranded DNA-binding proteins (SSBs) play essential roles in the replication, recombination and repair processes of organellar DNA molecules. In Arabidopsis thaliana, SSBs are encoded by a small family of two genes (SSB1 and SSB2). However, the functional divergence of these two SSB copies in plants remains largely unknown, and detailed studies regarding their roles in the replication and recombination of organellar genomes are still incomplete. In this study, phylogenetic, gene structure and protein motif analyses all suggested that SSB1 and SSB2 probably diverged during the early evolution of seed plants. Based on accurate long-read sequencing results, ssb1 and ssb2 mutants had decreased copy numbers for both mitochondrial DNA (mtDNA) and plastid DNA (ptDNA), accompanied by a slight increase in structural rearrangements mediated by intermediate-sized repeats in mt genome and small-scale variants in both genomes. Our findings provide an important foundation for further investigating the effects of DNA dosage in the regulation of mutation frequencies in plant organellar genomes.

Enhanced echovirus 11 genomic surveillance in neonatal infections in Spain following a European alert reveals new recombinant forms linked to severe cases, 2019 to 2023.

BackgroundIn 2023, a European alert was issued regarding an increase in severe enterovirus (EV) neonatal infections associated with echovirus 11 (E11) new lineage 1.AimTo analyse E11-positive cases between 2019 and 2023 to investigate whether the new lineage 1 circulated in Spain causing severe neonatal infections.MethodsEV-positive samples from hospitalised cases are sent for typing to the National Reference Enterovirus Laboratory. Available samples from 2022-23 were subjected to metagenomic next-generation sequencing.ResultsOf 1,288 samples genotyped, 103 were E11-positive (98 patients: 6 adults, 33 neonates, 89 children under 6 years; male to female ratio 1.9). E11 detection rate was similar before and after detection of the new lineage 1 in Spain in June 2022 (9.7% in 2019 vs 10.6% in 2023). The proportion of E11-infected ICU-admitted neonates in 2019-2022 (2/7) vs 2022-2023 (5/12) did not significantly differ (p=0.65). In severe neonatal infections, 4/7 E11 strains were not linked to the new lineage 1. The three novel E11 recombinant genomes were associated with severe (n=2) and non-severe (n=1) cases from 2022-2023 and clustered outside the new lineage 1. Coinfecting pathogenic viruses were present in four of 10 E11-positive samples.ConclusionThe emergence of the new lineage 1 is not linked with an increase in incidence or severity of neonatal E11 infections in Spain. The detection of two novel E11 recombinants associated with severe disease warrants enhancing genomic and clinical surveillance.

Delivering large genes using adeno-associated virus and the CRE-lox DNA recombination system.

Adeno-associated virus (AAV) is a safe and efficient gene delivery vehicle for gene therapies. However, its relatively small packaging capacity limits its use as a gene transfer vector. Here, we describe a strategy to deliver large genes that exceed the AAV's packaging capacity using up to four AAV vectors and the CRE-lox DNA recombination system. We devised novel lox sites by combining non-compatible and reaction equilibrium-modifying lox site variants. These lox sites facilitate sequence-specific and near-unidirectional recombination of AAV vector genomes, enabling efficient reconstitution of up to 16kb of therapeutic genes in a pre-determined configuration. Using this strategy, we have developed AAV gene therapy vectors to deliver IFT140, PCDH15, CEP290, and CDH23 and demonstrate efficient production of full-length proteins in cultured mammalian cells and mouse retinas. Notably, AAV-IFT140 gene therapy vectors ameliorated retinal degeneration and preserved visual functions in an IFT140-associated retinitis pigmentosa mouse model. The CRE-lox approach described here provides a simple, flexible, and effective platform for generating AAV gene therapy vectors beyond AAV's packaging capacity.

Positive selection, genetic recombination, and intra-host evolution in novel equine coronavirus genomes and other members of the Embecovirus subgenus.

There are several examples of coronaviruses in the Betacoronavirus subgenus Embecovirus that have jumped from an animal to the human host. Studying how evolutionary factors shape coronaviruses in non-human hosts may provide insight into the coronavirus host-switching potential. Equids, such as horses and donkeys, are susceptible to equine coronaviruses (ECoVs). With increased testing prevalence, several ECoV genome sequences have become available for molecular evolutionary analyses, especially those from the United States of America (USA). To date, no analyses have been performed to characterize evolution within coding regions of the ECoV genome. Here, we obtain and describe four new ECoV genome sequences from infected equines from across the USA presenting clinical symptoms of ECoV, and infer ECoV-specific and Embecovirus-wide patterns of molecular evolution. Within two of the four data sets analyzed, we find evidence of intra-host evolution within the nucleocapsid (N) gene, suggestive of quasispecies development. We also identify 12 putative genetic recombination events within the ECoV genome, 11 of which fall in ORF1ab. Finally, we infer and compare sites subject to positive selection on the ancestral branch of each major Embecovirus member clade. Specifically, for the two currently identified human coronavirus (HCoV) embecoviruses that have spilled from animals to humans (HCoV-OC43 and HCoV-HKU1), we find that there are 42 and 2 such sites, respectively, perhaps reflective of the more complex ancestral evolutionary history of HCoV-OC43, which involves several different animal hosts.IMPORTANCEThe Betacoronavirus subgenus Embecovirus contains coronaviruses that not only pose a health threat to animals and humans, but also have jumped from animal to human host. Equids, such as horses and donkeys are susceptible to equine coronavirus (ECoV) infections. No studies have systematically examined evolutionary patterns within ECoV genomes. Our study addresses this gap and provides insight into intra-host ECoV evolution from infected horses. Further, we identify and report natural selection pattern differences between two embecoviruses that have jumped from animals to humans [human coronavirus OC43 and HKU1 (HCoV-OC43 and HCoV-HKU1, respectively)], and hypothesize that the differences observed may be due to the different animal host(s) that each virus circulated in prior to its jump into humans. Finally, we contribute four novel, high-quality ECoV genomes to the scientific community.

7030 Molecular Analysis Of Congenital Adrenal Hyperplasia-X Syndrome And Development Of A Pilot Panel For Analyzing Structural Variations With Long-Read Sequencing

Abstract Disclosure: J. Kim: None. S. Park: None. J. Yoon: None. D. Kim: None. S. Hwang: None. G. Kim: None. J. Kim: None. J. Choi: None. H. Yoo: None. Purpose: Genomic recombination events between the CYP21A2 and CYP21A1P genes are frequent due to their high sequence homology. Deletions in both the CYP21A2 and TNXB genes result in a chimeric TNXA/TNXB gene, leading to congenital adrenal hyperplasia (CAH)-X syndrome, characterized by a hypermobile form of Ehlers-Danlos syndrome. The aim of study was to determine the frequency of CAH-X syndrome and to develop a pilot panel for structural variation analysis using long-read sequencing technology. Methods: Among the 165 unrelated families with confirmed genetic mutations in the CYP21A2 gene, 44 probands had large deletions in one or both alleles. Long-range PCR was performed in 12 patients out of 44 probands using a forward primer for the 5’-UTR of the CYP21A2 and CYP21A1P and a reverse primer specific for exon 32 of TNXB. The amplified PCR products were then subjected to Sanger sequencing. We constructed a custom panel of 140 kb containing the RCCX modules arranged as STK19-C4A-CYP21A1P-TNXA-STK19B-C4B-CYP21A2-TNXB. In 4 patients with deletions in both the CYP21A2 and TNXB genes, long-read sequencing was performed using the custom panel on the PacBio Sequel II platform. Results: Out of 12 probands, eight harbored TNXA/TNXB chimeras. One patient was homozygous for CAH-X CH1 with a 120 bp deletion in exon 35 and had a Beighton score of 6 for joint hypermobility and multiple diverticula in the ascending colon. Two patients were heterozygous for CAH-X CH1, while three were heterozygous for the CAH-X CH2 with an intact exon 35 and the c.P4058W in exon 40. Two patients carried heterozygous mutations p.S4175N in exon 43, which is an unclassified type. Using long-read sequencing with a custom panel, missense mutations and TNXA/TNXB chimeras were detected in four patients previously identified by conventional direct sequencing and MLPA. Conclusions: This study identified 8 patients with CAH-X syndrome carrying four different TNXA/TNXB chimeras by long-range PCR. Long-read sequencing emerged as a comprehensive and efficient method for the molecular analysis of CAH, especially for the identification of structural variations. Biallelic deletion of TNXB was associated with severe clinical manifestations. Therefore, screening for CAH-X is essential for clinical management and prevention of the long-term consequences of CAH-X syndrome. Presentation: 6/1/2024

9266 Molecular Analysis Of Congenital Adrenal Hyperplasia-X Syndrome And Development Of A Pilot Panel For Analyzing Structural Variations With Long-Read Sequencing

Abstract Disclosure: J. Kim: None. S. Park: None. J. Yoon: None. D. Kim: None. S. Hwang: None. G. Kim: None. J. Kim: None. J. Choi: None. H. Yoo: None. Purpose: Genomic recombination events between the CYP21A2 and CYP21A1P genes are frequent due to their high sequence homology. Deletions in both the CYP21A2 and TNXB genes result in a chimeric TNXA/TNXB gene, leading to congenital adrenal hyperplasia (CAH)-X syndrome, characterized by a hypermobile form of Ehlers-Danlos syndrome. The aim of study was to determine the frequency of CAH-X syndrome and to develop a pilot panel for structural variation analysis using long-read sequencing technology. Methods: Among the 165 unrelated families with confirmed genetic mutations in the CYP21A2 gene, 44 probands had large deletions in one or both alleles. Long-range PCR was performed in 12 patients out of 44 probands using a forward primer for the 5’-UTR of the CYP21A2 and CYP21A1P and a reverse primer specific for exon 32 of TNXB. The amplified PCR products were then subjected to Sanger sequencing. We constructed a custom panel of 140 kb containing the RCCX modules arranged as STK19-C4A-CYP21A1P-TNXA-STK19B-C4B-CYP21A2-TNXB. In 4 patients with deletions in both the CYP21A2 and TNXB genes, long-read sequencing was performed using the custom panel on the PacBio Sequel II platform. Results: Out of 12 probands, eight harbored TNXA/TNXB chimeras. One patient was homozygous for CAH-X CH1 with a 120 bp deletion in exon 35 and had a Beighton score of 6 for joint hypermobility and multiple diverticula in the ascending colon. Two patients were heterozygous for CAH-X CH1, while three were heterozygous for the CAH-X CH2 with an intact exon 35 and the c.P4058W in exon 40. Two patients carried heterozygous mutations p.S4175N in exon 43, which is an unclassified type. Using long-read sequencing with a custom panel, missense mutations and TNXA/TNXB chimeras were detected in four patients previously identified by conventional direct sequencing and MLPA. Conclusions: This study identified 8 patients with CAH-X syndrome carrying four different TNXA/TNXB chimeras by long-range PCR. Long-read sequencing emerged as a comprehensive and efficient method for the molecular analysis of CAH, especially for the identification of structural variations. Biallelic deletion of TNXB was associated with severe clinical manifestations. Therefore, screening for CAH-X is essential for clinical management and prevention of the long-term consequences of CAH-X syndrome. Presentation: 6/1/2024

Direct protein delivery into intact Arabidopsis cells for genome engineering

Intracellular delivery of biomolecules is a prerequisite for genetic techniques such as recombinant engineering and genome editing. Realizing the full potential of this technology requires the development of safe and effective methods for delivering protein tools into cells. In this study, we demonstrated the spontaneous internalization of exogenous proteins into intact cells and root tissue of whole plants of Arabidopsis thaliana. We termed this internalization phenomenon as protein Delivery Independent of Vehicles or Equipment (DIVE), which efficiently delivered genome engineering proteins including Cre recombinase and zinc-finger nucleases (ZFN) into plant cells. Using protein DIVE, we achieved less toxic protein delivery than electroporation with up to 94% efficiency in Arabidopsis cell culture and 19% genome modification in Arabidopsis plants that was maintained in regenerated tissue. This work illustrates the potential of protein DIVE for a wide range of applications, including genome engineering in plants.

Increased viperin expression induced by avian infectious bronchitis virus inhibits viral replication by restricting cholesterol synthesis: an in vitro study

With the emergence of new variant strains resulting from high mutation rates and genome recombination, avian infectious bronchitis virus (IBV) has caused significant economic losses to the poultry industry worldwide. Little is known about the underlying mechanisms of IBV-host interactions, particularly how IBV utilizes host metabolic pathways for efficient viral replication and transmission. In the present study, the effects of the cell membrane, viral envelope membrane, and viperin-mediated cholesterol synthesis on IBV replication were explored. Our results revealed significant increase in cholesterol levels and the expression of viperin after IBV infection. Acute cholesterol depletion in the cell membrane and viral envelope membrane by treating cells with methyl-β-cyclodextrin (MβCD) obviously inhibited IBV replication; thereafter, replenishment of the cell membrane with cholesterol successfully restored viral replication, and direct addition of exogenous cholesterol to the cell membrane significantly promoted IBV infection during the early stages of infection. In addition, overexpression of viperin effectively suppressed cholesterol synthesis, as well as IBV replication, whereas knockdown of viperin (gene silencing with siRNA targeting viperin, siViperin) significantly increased IBV replication and cholesterol levels, whereas supplementation with exogenous cholesterol to viperin-transfected cells markedly restored viral replication. In conclusion, the increase in viperin induced by IBV infection plays an important role in IBV replication by affecting cholesterol production, providing a theoretical basis for understanding the pathogenesis of IBV and discovering new potential antiviral targets.

Genotyping and phylogeographic dynamics of coxsackievirus A16

Coxsackievirus A16 (CV-A16) is one of the major pathogens of Hand, Foot and Mouth disease. Here, we analyzed 287 full-length genome sequences of CV-A16 found worldwide from 1994 to 2019 to see the genomic evolution characteristics. Full-length genome-based phylogenetic tree divided the viruses into five different genotypes, G-a to G-e. The CV-A16 strains circulating in China dominate G-a and G-c, but can also be found in other genotypes including G-b and G-e. Phylogeographic analysis showed a high diversity of CV-A16 distribution. In addition, recombination was shown to drive the genomic evolution of CV-A16 during past decades. However, the structural proteins still remain relative conserved while there is extensive genomic recombination. This study updates the phylogenetic and phylogeographic information of CV-A16 and provides insights into the genetic characteristics of CV-A16.

Frequent CHD1 deletions in prostate cancers of African American men is associated with rapid disease progression

We analyzed genomic data from the prostate cancer of African- and European American men to identify differences contributing to racial disparity of outcome. We also performed FISH-based studies of Chromodomain helicase DNA-binding protein 1 (CHD1) loss on prostate cancer tissue microarrays. We created CHD1-deficient prostate cancer cell lines for genomic, drug sensitivity and functional homologous recombination (HR) activity analysis. Subclonal deletion of CHD1 was nearly three times as frequent in prostate tumors of African American than in European American men and it associates with rapid disease progression. CHD1 deletion was not associated with HR deficiency associated mutational signatures or HR deficiency as detected by RAD51 foci formation. This was consistent with the moderate increase of olaparib and talazoparib sensitivity with several CHD1 deficient cell lines showing talazoparib sensitivity in the clinically relevant concentration range. CHD1 loss may contribute to worse disease outcome in African American men.

Alternative double strand break repair pathways shape the evolution of high recombination in the honey bee, Apis mellifera.

Social insects, particularly honey bees, have exceptionally high genomic frequencies of genetic recombination. This phenomenon and underlying mechanisms are poorly understood. To characterise the patterns of crossovers and gene conversion in the honey bee genome, a recombination map of 187 honey bee brothers was generated by whole-genome resequencing. Recombination events were heterogeneously distributed without many true hotspots. The tract lengths between phase shifts were bimodally distributed, indicating distinct crossover and gene conversion events. While crossovers predominantly occurred in G/C-rich regions and seemed to cause G/C enrichment, the gene conversions were found predominantly in A/T-rich regions. The nucleotide composition of sequences involved in gene conversions that were associated with or distant from crossovers corresponded to the differences between crossovers and gene conversions. These combined results suggest two types of DNA double-strand break repair during honey bee meiosis: non-canonical homologous recombination, leading to gene conversion and A/T enrichment of the genome, and the canonical homologous recombination based on completed double Holliday Junctions, which can result in gene conversion or crossover and is associated with G/C bias. This G/C bias may be selected for to balance the A/T-rich base composition of eusocial hymenopteran genomes. The lack of evidence for a preference of the canonical homologous recombination for double-strand break repair suggests that the high genomic recombination rate of honey bees is mainly the consequence of a high rate of double-strand breaks, which could in turn result from the life history of honey bees and their A/T-rich genome.

Modern Technologies Provide New Opportunities for Somatic Hybridization in the Breeding of Woody Plants.

Advances in cell fusion technology have propelled breeding into the realm of somatic hybridization, enabling the transfer of genetic material independent of sexual reproduction. This has facilitated genome recombination both within and between species. Despite its use in plant breeding for over fifty years, somatic hybridization has been limited by cumbersome procedures, such as protoplast isolation, hybridized-cell selection and cultivation, and regeneration, particularly in woody perennial species that are difficult to regenerate. This review summarizes the development of somatic hybridization, explores the challenges and solutions associated with cell fusion technology in woody perennials, and outlines the process of protoplast regeneration. Recent advancements in genome editing and plant cell regeneration present new opportunities for applying somatic hybridization in breeding. We offer a perspective on integrating these emerging technologies to enhance somatic hybridization in woody perennial plants.

KSHV ORF20 Promotes Coordinated Lytic Reactivation for Increased Infectious Particle Production.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a cancer-causing virus that establishes life-long infection. KSHV is implicated in the etiology of Kaposi's sarcoma, and a number of rare hematopoietic malignancies. The present study focuses on the KSHV open reading frame 20 (ORF20), a member of the conserved herpesvirus UL24 protein family containing five conserved homology domains and a conserved PD-(D/E)XK putative endonuclease motif, whose nuclease function has not been established to date. ORF20 encodes three co-linear protein isoforms, full length, intermediate, and short, though their differential functions are unknown. In an effort to determine the role of ORF20 during KSHV infection, we generated a recombinant ORF20-Null KSHV genome, which fails to express all three ORF20 isoforms. This genome was reconstituted in iSLK cells to establish a latent infection, which resulted in an accelerated transcription of viral mRNAs, an earlier accumulation of viral lytic proteins, an increase in the quantity of viral DNA copies, and a significant decrease in viral yield upon lytic reactivation. This was accompanied by early cell death of cells infected with the ORF20-Null virus. Functional complementation of the ORF20-Null mutant with the short ORF20 isoform rescued KSHV production, whereas its endonuclease mutant form failed to enhance lytic reactivation. Complementation with the short isoform further revealed a decrease in cell death as compared with ORF20-Null virus. Finally, expression of IL6 and CXCL8, previously shown to be affected by the hCMV UL24 homolog, was relatively low upon reactivation of cells infected with the ORF20-Null virus. These findings suggest that ORF20 protein, with its putative endonuclease motif, promotes coordinated lytic reactivation for increased infectious particle production.