Genome Homology Research Articles

Identification of a chordovirus hosted by Angelica lignescens

Summary Angelica lignescens, an endangered plant species endemic to the Azores Archipelago and member of the Apiaceae family, was subjected to Next Generation Sequencing (NGS) virome analysis. This analysis revealed the presence of contigs associated with a virus member of the Chordovirus genus. Through RT–PCR and additional Sanger sequencing, the complete genome of the virus was assembled and provisionally named angelica chordovirus (AChV). The AChV has a genome length of 8145 nt, exhibiting a similar organization to the Trivirinae subfamily (Betaflexiviridae family). Upon BLAST analysis, the highest genomic homology was uncovered with carrot chordovirus 4 (CChV4), displaying a nucleotide identity of 76%. Pairwise comparisons further emphasized this similarity, with 80% and 82% amino acid identity to the complete replicase and coat protein genes of CChV4, respectively. These values lie on the borderline of species demarcation criteria. Given that, to ensure precise classification, additional research is imperative to unravel the epidemiology and ecology of AChV. Furthermore, a more profound understanding of its impact on A. lignescens and its potential broader ecological implications is essential, especially concerning potential host crops.

Etiological characterization of multiple recombinant lineages of TJ-C6 porcine reproductive and respiratory syndrome virus in Tianjin, Northern China.

Recent outbreaks of PRRSV in live attenuated vaccine-immunized pig farms in Tianjin, China have raised questions about the etiological characteristics and pathogenicity of the PRRSV variant, which remains unknown. In this study, a multiple lineages recombinant PRRSV strain named TJ-C6, was isolated and identified. Phylogenetic trees and genome homology analyses revealed that TJ-C6 belonged to lineage 1.8 (NADC30-like) and with similar 131 discontinuous amino acid deletion pattern (111-aa + 1-a + 19-aa) in Nsp2-coding region, but it was classified in lineage 1.5 (NADC34-like) cluster based on ORF5 sequence. Furthermore, the recombination analyses revealed that TJ-C6 was a multiple recombinant virus among lineage 1.5(NADC34- like), lineage 1.8(NADC30- like), and lineage 3(GM2-Like) strains with four recombination breakpoints in Nsp9 (nt 7298/8111), ORF2 (nt 12213) and ORF6 (nt 14628), which was different from the previously prevalent PRRSV strain. Challenge experiments with 3-week-old piglets showed that TJ-C6 could cause piglets high fever, loss of appetite and severely histopathological lung lesions. Taken altogether, multiple co-circulating lineages of PRRSV strains in the swine population are accelerating the emergence of natural recombinant strains with variations in pathogenicity and highlight the importance of surveillance of newly emerging PRRSV strains in China.

Extensive homologous recombinationsafeguards oocyte genome integrity in mammals.

Meiosis in mammalian oocytes is interrupted by a prolonged arrest at the germinal vesiclestage, during which oocytes have to repair DNA lesions to ensure genome integrityor otherwise undergo apoptosis. The FIRRM/FLIP-FIGNL1 complex dissociates RAD51 from the joint DNA molecules in both homologous recombination (HR) and DNA replication. However, as a type of non-meiotic, non-replicative cells, whether this RAD51-dismantling mechanism regulates genome integrity in oocytes remains elusive. Here, we show that FIRRM/FLIP is required for disassembly of RAD51-filaments and maintenance of genome integrity in oocytes. Deletion of FIRRM in oocytes leads to formation of massive nuclear RAD51 foci in oocytes of primordial follicles and activated follicles in mice. These RAD51 foci colocalize with the sites of DNA damage repair, as indicated by RPA2 and EdU, suggesting substantial DNA damage and extensive HR in oocytes. Especially in fully-grown FIRRM-deleted oocytes, RAD51 forms a net-like structure. As a consequence, FIRRM-deleted females are infertile due to aberrant homologous chromosome segregation at metaphase I and primordial follicle insufficiency at young adulthood. Hence, our study demonstrates the physiological importance of HR in maintaining genome integrity in oocytes.

Comparative Genomic and Biological Investigation of NADC30‐ and NADC34‐Like PRRSV Strains Isolated in South Korea

Porcine reproductive and respiratory syndrome virus (PRRSV) is a globally endemic, costly swine arterivirus with wide genetic and antigenic variations, leading to the frequent appearance of novel virulent strains that hampers PRRSV control. Recently, NADC30‐like (lineage 1C, L1C) and NADC34‐like (lineage 1A, L1A) PRRSV strains were reported to be prevalent in mainland South Korea and became the main epidemic strains persistently attributed to PRRSV outbreaks nationwide, raising great concern in the domestic pork industry. Although the genotypic and pathotypic variability of NADC30‐ and NADC34‐like viruses has been explored in the United States and China, their genomic and biological characteristics have been scarcely studied in South Korea. Here, NADC34‐like GNU‐2353 and NADC30‐like GNU‐2377 strains were independently identified from vaccinated swine herds experiencing high piglet mortality. Whole‐genome sequencing and phylogenetic analysis revealed that GNU‐2353 and GNU‐2377 clustered into sublineages L1A (NADC34‐like) and L1C (NADC30‐like), respectively, sharing high genomic homology with their corresponding lineage‐representative strains and harboring the same molecular signatures of continuous 100 and discontinuous 131 amino acid deletions in the nsp2‐coding region, respectively. Recombination detection indicated that GNU‐2353 and GNU‐2377 were recombinants and evolved through natural interlineage recombination between NADC34‐like (L1A, major parent) or NADC30‐like (L1C, major parent) and RespPRRS modified live virus (MLV)–like (lineage 5, minor parent) strains, respectively. Both viruses displayed homogenous growth kinetics but replicated faster than the prototype VR‐2332 in a porcine alveolar macrophage cell line (PAM‐KNU). The transcriptional profiles of immune response genes in infected PAM‐KNU cells varied between the isolates and VR‐2332; particularly, interleukin‐10 expression was dramatically upregulated in cells infected with GNU‐2353 and GNU‐2377. Piglets with GNU‐2353 and GNU‐2377 infection had high fever; weight loss; increased viremia and nasal shedding; viral distribution in various tissues; thymic atrophy; and apparent macroscopic and microscopic lung lesions, including interstitial pneumonia and viral colonization, compared with control piglets, suggesting that both isolates were virulent to pigs. Remarkably, GNU‐2353 caused higher fever, mortality rate (40%) with cyanosis, viremia, and viral shedding within 2 weeks and significantly higher viral loads in several organs than GNU‐2377 infection. Thus, NADC34‐like GNU‐2353 was more pathogenic than NADC30‐like GNU‐2377. Our findings provide insights into the current epizootic circumstance of NADC30‐ and NADC34‐like PRRSV in South Korea and can aid in tailoring improved control strategies.

Phytocytokine genes newly discovered in Malus domestica and their regulation in response to Erwinia amylovora and acibenzolar-S-methyl.

Phytocytokines belong to a category of small secreted peptides with signaling functions that play pivotal roles in diverse plant physiological processes. However, due to low levels of sequence conservation across plant species and poorly understood biological functions, the accurate detection and annotation of corresponding genes is challenging. The availability of a high-quality apple (Malus domestica) genome has enabled the exploration of five phytocytokine gene families, selected on the basis of their altered expression profiles in response to biotic stresses. These include phytosulfokine, inflorescence deficient in abscission/-like, pathogen-associated molecular pattern induced secreted peptide, plant peptide containing sulfated tyrosine, and C-terminally encoded peptide. The genes encoding the precursors of these five families of signaling peptides were identified using a customized bioinformatics protocol combining genome mining, homology searches, and peptide motif detection. Transcriptomic analyses showed that these peptides were deregulated in response to Erwinia amylovora, the causal agent of fire blight in pome fruit trees, and in response to a chemical elicitor (acibenzolar-S-methyl). Finally, gene family evolution and the orthology relationships with Arabidopsis thaliana homologs were investigated.

Genomic Differences and Mutations in Epidemic Orf Virus and Vaccine Strains: Implications for Improving Orf Virus Vaccines.

Orf (ORF) is an acute disease caused by the Orf virus (ORFV), and poses a certain threat to animal and human health. Live attenuated vaccines play an important role in the prevention and control of ORF. The effectiveness of the live attenuated Orf virus vaccine is influenced by several factors, including the genomic match between the vaccine strain and circulating epidemic strains. Genomic differences between an ORFV epidemic strain (ORFV-2W) and a vaccine strain (ORFV-1V) were identified in this study via analysis of multiple sequence alignments, phylogenetic trees, and single nucleotide polymorphisms. Phylogenetic analysis revealed that ORFV-2W and ORFV-1V were closely related, with a whole genome homology of 99.8%. Furthermore, a deletion in the non-coding region at the end of the whole genome of ORFV-1V was detected. Such non-essential genes in the terminal regions are usually unnecessary for virus replication but may play important roles in pathogenicity, host and tissue tropism. Single nucleotide polymorphism analysis identified three missense mutations in ORF067, ORF072, and the terminal non-coding region of ORFV-1V. Moreover, a frameshift mutation in ORF102 of ORFV-1V was detected. Mutations in individual genes and deletion of terminal non-coding regions may be related to the attenuation of the vaccine strain. These results provide useful context for improving ORFV vaccines.

Using In Silico Methods to Identify Protein Tyrosine Kinase A (PtkA) Homolog in Non-Tuberculous Mycobacteria (NTM)

Non-tuberculous mycobacteria (NTM) represent a diverse group of mycobacterial species known for causing opportunistic infections, especially in individuals with underlying health conditions. Unlike Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, NTM species exhibit different pathogenic characteristics and drug resistance mechanisms, making them increasingly relevant in clinical settings. PtkA is a crucial protein tyrosine kinase that regulates bacterial growth, stress response, and virulence by phosphorylating various substrates in Mtb. Understanding whether PtkA homologs exist in NTM could provide insights into their virulence and resistance mechanisms. In silico approaches, which utilize computational tools for sequence alignment, structure prediction, and functional annotation, offer a powerful means to identify homologous proteins across different species. In this article, we have employed tools like BLAST (Basic Local Alignment Search Tool), protein structure databases, and the NTM database to identify PtkA homologs in NTM genomes, providing a foundation for further studies.

Enhanced pathogenicity and synergistic effects of co-infection with bovine viral diarrhea virus 1 and HoBi-like virus in cattle and guinea pigs.

The Bovine Viral Diarrhea Virus 1 (BVDV1) and HoBi-like virus (BVDV3), both within the same genus, share genomic homology and exhibit low antigenic cross-reactivity despite presenting similar clinical manifestations. In 2021, a bovine respiratory disease complex (BRDC) outbreak on two cattle farms in the Inner Mongolia Autonomous Region of China resulted in ten fatalities. Metagenomic and metatranscriptomic analyses were used to identify viral agents, including a co-infection case. A genetic evolution analysis assessed the relationships with related strains. Experimental infections in guinea pigs and calves evaluated the pathogenicity of the viruses. Phylogenetic analysis of the BVDV3 isolate IM2201 revealed close relatedness to Brazilian strains, with 97.06% nucleotide homology to the highly virulent strain SV478/07. Experimental co-infection in guinea pigs resulted in more severe clinical signs, including fever, cough, diarrhea, and significant pathological changes, and led to a higher mortality rate (40%) compared to no mortality from single-virus infections with BVDV1 or BVDV3. Similarly, co-infected cattle exhibited more severe clinical signs, including bloody diarrhea and rectal temperatures exceeding 40°C, along with persistent viremia and nasal viral shedding from 7 to 21 days post-infection. Blood analysis revealed significant reductions in white blood cell counts, particularly in co-infected cattle. This study highlights the enhanced pathogenicity and synergistic effects of BVDV1 and BVDV3 co-infection, exacerbating disease severity.

Isolation and identification of a novel goose-origin reovirus GD218 and its pathogenicity experiments.

Since 2020, a novel goose-derived reovirus, characterized by goose hemorrhagic hepatitis, has emerged in the goose breeding industry of Guangdong province, China, leading to significant economic losses in the poultry sector. To study the genetic variation of novel goose reovirus (NGRV) in Guangdong province, this experiment utilized goose embryonic fibroblast cells for virus isolation. RT-PCR was conducted to identify, amplify, clone, and sequence the complete genome of the NGRV isolated from Zhaoqing. The genomic sequences were compared with reference strains to construct a phylogenetic tree. Moreover, animal pathogenicity, excretion patterns, and pathological sections were examined. The results showed that liver and spleen samples from geese suspected of NGRV infection were used for isolation, resulting in the identification of a reovirus presumed to originate from geese, designated as GD218. In terms of genomic structure and sequence homology, GD218 closely resembles the novel duck reovirus, differing significantly from earlier isolated NDRV strains (J18, NP03, SD12, etc.) in genetic composition (nt: 80.6-97.9%, aa: 94.3-98.9%). However, it is similar to strains isolated after 2018, such as XT18, SY, QR, YL, LY20, etc. (nt: 95.3-98.9%, aa: 98.6-99.7%). Therefore, based on phylogenetic analysis, GD218 is hypothesized to be a novel type of goose-origin reovirus homologous to the novel duck reovirus.

Whole-genome sequencing and assessment of a novel protein- and gossypol-degrading Bacillus subtilis strain isolated from intestinal digesta of Tibetan Pigs

BackgroundWith the rapid development of animal husbandry, the demand for protein feed resources is increasing. Cottonseed meal (CSM) and soybean meal (SBM) are rich sources of protein. However, their application is limited due to the existence of anti-nutrients, which can be harmful to the digestion and absorption. A strain of Bacillus subtilis (Mafic-Y7) was isolated from digesta of intestines of Tibetan pigs. The strain showed high protease activity, which helps in degrading proteinic anti-nutritional factors in grain meal and in vitro degradation of free gossypol. In order to better understand this isolated strain, whole genome of Mafic-Y7 strain was sequenced and analyzed. Different effects on various grain meals were identified.ResultThe GC-depth Poisson distributions showed no bias suggesting high-quality genome assembly of Mafic-Y7. The whole genome sequencing showed that one chromosome with 4,248,845 base pairs(bp)and the genes total length with 3,736,524 bp was predicted in Mafic-Y7. Additionally, Mafic-Y7 possessed 4,254 protein-coding genes, and several protease genes were annotated by aligning them with databases. There are 55 protease genes, one phytase gene and one laccase gene were annotated in the gene sequence of Mafic-Y7. The average nucleotide identity between Mafic-Y7 and the GCA-000009045.1 homologous genome was 0.9938, suggesting a close genetic relationship between them at the species level. Compared with the closest four whole genomes, Mafic-Y7 was annotated the most abundant of protease genes (55 genes). The fermentation supernatant of Mafic-Y7 could increase the content of small peptides, water-soluble proteins, and acid-soluble proteins in vitro by 411%, 281% and 317% in SBM and 420%, 257% and 338% in CSM. After fermentation in grain meal by Mafic-Y7, the degradation rate of anti-nutritional factors in SBM, such as trypsin inhibitor, glycinin, and β-conglycinin was greater than 70%, and lectin was greater than 30%. The degradation rates of anti-nutritional factors in CSM, such as gossypol and phytic acid, were 82% and 26%, respectively.

Utilizing Deep Neural Networks to Fill Gaps in Small Genomes.

With the widespread adoption of next-generation sequencing technologies, the speed and convenience of genome sequencing have significantly improved, and many biological genomes have been sequenced. However, during the assembly of small genomes, we still face a series of challenges, including repetitive fragments, inverted repeats, low sequencing coverage, and the limitations of sequencing technologies. These challenges lead to unknown gaps in small genomes, hindering complete genome assembly. Although there are many existing assembly software options, they do not fully utilize the potential of artificial intelligence technologies, resulting in limited improvement in gap filling. Here, we propose a novel method, DLGapCloser, based on deep learning, aimed at assisting traditional tools in further filling gaps in small genomes. Firstly, we created four datasets based on the original genomes of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Neurospora crassa, and Micromonas pusilla. To further extract effective information from the gene sequences, we also added homologous genomes to enrich the datasets. Secondly, we proposed the DGCNet model, which effectively extracts features and learns context from sequences flanking gaps. Addressing issues with early pruning and high memory usage in the Beam Search algorithm, we developed a new prediction algorithm, Wave-Beam Search. This algorithm alternates between expansion and contraction phases, enhancing efficiency and accuracy. Experimental results showed that the Wave-Beam Search algorithm improved the gap-filling performance of assembly tools by 7.35%, 28.57%, 42.85%, and 8.33% on the original results. Finally, we established new gap-filling standards and created and implemented a novel evaluation method. Validation on the genomes of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Neurospora crassa, and Micromonas pusilla showed that DLGapCloser increased the number of filled gaps by 8.05%, 15.3%, 1.4%, and 7% compared to traditional assembly tools.

Analysis of the recombination and evolution of the new type mutant pseudorabies virus XJ5 in China

Pseudorabies have caused enormous economic losses in China’s pig industry and have recurred on many large pig farms since late 2011. The disease is caused by highly pathogenic, antigenic variant pseudorabies virus (vPRV) strains. Our laboratory isolated a pseudorabies virus in 2015 and named it XJ5. The pathogenic ability of this mutant strain was much stronger than that of the original isolate. After we sequenced its whole genome (GenBank accession number: OP512542), we found that its overall structure was not greatly changed compared with that of the previous strain Ea (KX423960.1). The whole genome alignment showed that XJ5 had a strong genetic relationship with the strains isolated in China after 2012 reported in GenBank. Based on the isolation time of XJ5 and the mutation and recombination analysis of programs, we found that the whole genome homology of XJ5 and other strains with Chinese isolates was greater than 95%, while the homology with strains outside Asia was less than 94%, which indicated that there may be some recombination and mutation patterns. We found that virulent PRV isolates emerged successively in China in 2011 and formed two different evolutionary clades from foreign isolates. At the same time, this may be due to improper immunization and the presence of wild strains in the field, and recent reports have confirmed that Bartha vaccine strains recombine with wild strains to obtain new pathogenic strains. We performed genetic evolution analysis of XJ5 isolated and sequenced in our laboratory to trace its possible mutations and recombination. We found that XJ5 may be the result of natural mutation of a virus in a branch of mutant strains widely existing in China.

Highly homologous simian T-cell leukemia virus type 1 genome in Japanese macaques: a large cohort study

BackgroundSimian T-cell leukemia virus type 1 (STLV-1) is a retrovirus closely related to human T-cell leukemia virus type 1 (HTLV-1), the causative agent of adult T-cell leukemia (ATL). It has been shown that Japanese macaques (Macaca fuscata, JMs) are one of the main hosts of STLV-1 and that a high percentage of JMs (up to 60%) are infected with STLV-1; however, the molecular epidemiology of STLV-1 in JMs has not been examined.MethodsIn this study, we analyzed full-length STLV-1 genome sequences obtained from 5 independent troops including a total of 68 JMs.ResultsThe overall nucleotide heterogeneity was 4.7%, and the heterogeneity among the troops was 2.1%, irrespective of the formation of distinct subclusters in each troop. Moreover, the heterogeneity within each troop was extremely low (>99% genome homology) compared with cases of STLV-1 in African non-human primates as well as humans. It was previously reported that frequent G-to-A single-nucleotide variants (SNVs) occur in HTLV-1 proviral genomes in both ATL patients and HTLV-1 carriers, and that a G-to-A hypermutation is associated with the cellular antiviral restriction factor, Apobec3G. Surprisingly, these SNVs were scarcely observed in the STLV-1 genomes in JMs.ConclusionsTaken together, these results indicate that STLV-1 genomes in JMs are highly homologous, at least in part due to the lack of Apobec3G-dependent G-to-A hypermutation.

High level of genomic divergence in orf-I p12 and hbz genes of HTLV-1 subtype-C in Central Australia

BackgroundHuman T cell lymphotropic virus type 1 (HTLV-1) infection remains a largely neglected public health problem, particularly in resource-poor areas with high burden of communicable and non-communicable diseases, such as some remote populations in Central Australia where an estimated 37% of adults are infected with HTLV-1. Most of our understanding of HTLV-1 infection comes from studies of the globally spread subtype-A (HTLV-1a), with few molecular studies reported with the Austral-Melanesian subtype-C (HTLV-1c) predominant in the Indo-Pacific and Oceania regions.ResultsUsing a primer walking strategy and direct sequencing, we constructed HTLV-1c genomic consensus sequences from 22 First Nations participants living with HTLV-1c in Central Australia. Phylogenetic and pairwise analysis of this subtype-C proviral gDNA showed higher levels of genomic divergence in comparison to previously published HTLV-1a genomes. While the overall genomic homology between subtypes was 92.5%, the lowest nucleotide and amino acid sequence identity occurred near the 3′ end of the proviral genome coding regulatory genes, especially overlapping hbz (85.37%, 77.46%, respectively) and orf-I product p12 (82.00%, 70.30%, respectively). Strikingly, the HTLV-1c genomic consensus sequences uniformly showed a defective translation start codon for the immune regulatory proteins p12/p8 encoded by the HTLV-1A orf-I. Deletions in the proviral genome were detected in many subjects, particularly in the structural gag, pol and env genes. Similarly, using a droplet digital PCR assay measuring the copies of gag and tax per reference host genome, we quantitatively confirmed that provirus retains the tax gene region at higher levels than gag.ConclusionsOur genomic analysis of HTLV-1c in Central Australia in conjunction with earlier Melanesian HTLV-1c sequences, elucidate substantial differences with respect to the globally spread HTLV-1a. Future studies should address the impact these genomic differences have on infection and the regionally distinctive frequency of associated pulmonary disease. Understanding the host and virus subtype factors which contribute to the differential morbidity observed, is crucial for the development of much needed therapeutics and vaccine strategies against this highly endemic infection in remote First Nations communities in Central Australia.

Identification of two archaeal GDGT lipid–modifying proteins reveals diverse microbes capable of GMGT biosynthesis and modification

Archaea produce unique membrane-spanning lipids (MSLs), termed glycerol dialkyl glycerol tetraethers (GDGTs), which aid in adaptive responses to various environmental challenges. GDGTs can be modified through cyclization, cross-linking, methylation, hydroxylation, and desaturation, resulting in structurally distinct GDGT lipids. Here, we report the identification of radical SAM proteins responsible for two of these modifications-a glycerol monoalkyl glycerol tetraether (GMGT) synthase (Gms), responsible for covalently cross-linking the two hydrocarbon tails of a GDGT to produce GMGTs, and a GMGT methylase (Gmm), capable of methylating the core hydrocarbon tail. Heterologous expression of Gms proteins from various archaea in Thermococcus kodakarensis results in the production of GMGTs in two isomeric forms. Further, coexpression of Gms and Gmm produces mono- and dimethylated GMGTs and minor amounts of trimethylated GMGTs with only trace GDGT methylation. Phylogenetic analyses reveal the presence of Gms homologs in diverse archaeal genomes spanning all four archaeal superphyla and in multiple bacterial phyla with the genetic potential to synthesize fatty acid-based MSLs, demonstrating that GMGT production may be more widespread than previously appreciated. We demonstrate GMGT production in three Gms-encoding archaea, identifying an increase in GMGTs in response to elevated temperature in two Archaeoglobus species and the production of GMGTs with up to six rings in Vulcanisaeta distributa. The occurrence of such highly cyclized GMGTs has been limited to environmental samples and their detection in culture demonstrates the utility of combining genetic, bioinformatic, and lipid analyses to identify producers of distinct archaeal membrane lipids.

Transcriptome profile of sex-related gene expression and effects of cyp19a1 siRNA-silencing on sex differentiation and determination in yellow perch

Transcriptome profile of sex-related gene expression and effects of cyp19a1 siRNA-silencing on sex differentiation and determination in yellow perch

Evidence for cross-species transmission of human coronavirus OC43 through bioinformatics and modeling infections in porcine intestinal organoids

Cross-species transmission of coronaviruses has been continuously posing a major challenge to public health. Pigs, as the major animal reservoirs for many zoonotic viruses, frequently mediate viral transmission to humans. This study comprehensively mapped the relationship between human and porcine coronaviruses through in-depth bioinformatics analysis. We found that human coronavirus OC43 and porcine coronavirus PHEV share a close phylogenetic relationship, evidenced by high genomic homology, similar codon usage patterns and comparable tertiary structure in spike proteins. Inoculation of infectious OC43 viruses in organoids derived from porcine small and large intestine demonstrated that porcine intestinal organoids (pIOs) are highly susceptible to human coronavirus OC43 infection and support infectious virus production. Using transmission electron microscopy, we visualized OC43 viral particles in both intracellular and extracellular compartments, and observed abnormalities of multiple organelles in infected organoid cells. Robust OC43 infections in pIOs result in a significant reduction of organoids viability and widespread cell death. This study bears essential implications for better understanding the evolutionary origin of human coronavirus OC43, and provides a proof-of-concept for using pIOs as a model to investigate cross-species transmission of human coronavirus.

Overlapping Streptococcus pyogenes and Streptococcus dysgalactiae subspecies equisimilis household transmission and mobile genetic element exchange

Streptococcus dysgalactiae subspecies equisimilis (SDSE) and Streptococcus pyogenes share skin and throat niches with extensive genomic homology and horizontal gene transfer (HGT) possibly underlying shared disease phenotypes. It is unknown if cross-species transmission interaction occurs. Here, we conduct a genomic analysis of a longitudinal household survey in remote Australian First Nations communities for patterns of cross-species transmission interaction and HGT. Collected from 4547 person-consultations, we analyse 294 SDSE and 315 S. pyogenes genomes. We find SDSE and S. pyogenes transmission intersects extensively among households and show that patterns of co-occurrence and transmission links are consistent with independent transmission without inter-species interference. We identify at least one of three near-identical cross-species mobile genetic elements (MGEs) carrying antimicrobial resistance or streptodornase virulence genes in 55 (19%) SDSE and 23 (7%) S. pyogenes isolates. These findings demonstrate co-circulation of both pathogens and HGT in communities with a high burden of streptococcal disease, supporting a need to integrate SDSE and S. pyogenes surveillance and control efforts.

ORBIT for E. coli: kilobase-scale oligonucleotide recombineering at high throughput and high efficiency.

Microbiology and synthetic biology depend on reverse genetic approaches to manipulate bacterial genomes; however, existing methods require molecular biology to generate genomic homology, suffer from low efficiency, and are not easily scaled to high throughput. To overcome these limitations, we developed a system for creating kilobase-scale genomic modifications that uses DNA oligonucleotides to direct the integration of a non-replicating plasmid. This method, Oligonucleotide Recombineering followed by Bxb-1 Integrase Targeting (ORBIT) was pioneered in Mycobacteria, and here we adapt and expand it for Escherichiacoli. Our redesigned plasmid toolkit for oligonucleotide recombineering achieved significantly higher efficiency than λ Red double-stranded DNA recombineering and enabled precise, stable knockouts (≤134 kb) and integrations (≤11 kb) of various sizes. Additionally, we constructed multi-mutants in a single transformation, using orthogonal attachment sites. At high throughput, we used pools of targeting oligonucleotides to knock out nearly all known transcription factor and small RNA genes, yielding accurate, genome-wide, single mutant libraries. By counting genomic barcodes, we also show ORBIT libraries can scale to thousands of unique members (>30k). This work demonstrates that ORBIT for E. coli is a flexible reverse genetic system that facilitates rapid construction of complex strains and readily scales to create sophisticated mutant libraries.

Post-genomic analysis of Monosporascus cannonballus and Macrophomina phaseolina - potential target selection

Monosporascus cannonballus Pollack & Uecker and Macrophomina phaseolina Tassi (Goid) are phytopathogenic fungi responsible for causing "root rot and vine decline" in melon (Cucumis melo L.). Currently, cultural management practices are predominantly employed to control these pathogens, as the use of pesticides not only has detrimental environmental impacts but has also proven ineffective against them. These fungi have already undergone molecular characterization, and their genomes are now available, enabling the targeted search for protein targets. Therefore, this study aimed to identify novel target proteins that can serve as a foundation for the development of fungicides for effectively managing these pathogens. The genomes of M. cannonballus (assembly ASM415492v1) and M. phaseolina (assembly ASM2087553v1) were subjected to comprehensive analysis, filtration, and comparison. The proteomes of both fungi were clustered based on functional criteria, including putative and hypothetical functions, cell localization, and function-structure relationships. The selection process for homologs in the fungal genomes included a structural search. In the case of M. cannonballus, a total of 17,518 proteins were re-annotated, and among them, 13 candidate targets were identified. As for M. phaseolina, 30,226 initial proteins were analyzed, leading to the identification of 10 potential target proteins. This study thus provides new insights into the molecular functions of these potential targets, with the further validation of inhibitors through experimental methods holding promise for expanding our knowledge in this area.