Genomic Copy Research Articles

ЦИРКОВИРУС-МИКОПЛАЗМЕННАЯ ИНФЕКЦИЯ ПОРОСЯТ В ЭКСПЕРИМЕНТЕ

The aim of the study is to investigate the manifestation of circovirus-mycoplasma infection in piglets both as a mixed infection and as a monoinfection in the experiment. Objectives: to study the interaction of circovirus type 2 with Mycoplasma hyopneumoniae in the body of weaned piglets, as well as the manifestation of monoinfection in piglets during experimental infection; to assess the pathogenicity of PCV2d/M.hyo mixed infection, PCV2d monoinfection and M. hyo monoinfection; to identify the clinical and pathomorphological manifestation of mixed and single circovirus and mycoplasma infection in the experiment. Four groups of 23-day-old piglets (5 heads each) were formed and tested for the absence of pathogens. Experimental group I – mixed circovirus-mycoplasma infection, experimental group II – PCV2d monoinfection, experimental group III – M.hyo monoinfection, group IV – control. After infection, rectal temperature was measured daily and clinical manifestations were observed, nasal swabs were taken to determine the number of copies of M. hyo nucleic acid, blood was taken from each piglet weekly to detect viral nucleic acid and antibodies. All experimental and control piglets were euthanized on the 35th day from the beginning of the experiment. Pathomorphological and pathohistological studies were carried out. The experimental study reveals the synergistic pathogenicity of PCV2d and M. hyo in mixed infection. In experimental infection of weaning piglets with both M. hyo monoinfection and M. hyo/PCV2d mixed infection, it was found that M. hyo nucleic acid was detected in nasal smears in 80 % of animals and in lung tissue in 100 %. The number of copies of the PCV2d circovirus genome in the inguinal lymph node tissue of piglets in the mixed infection group was higher than in the monoinfection group. Circovirus type 2 in piglets can proceed latently, and circovirus-mycoplasma infection can be a complex respiratory disease.

Analysis of genomic copy number variations through whole-genome scan in Yunling cattle.

Yunling cattle is a new breed of beef cattle bred in Yunnan Province, China, which has the advantages of fast growth, excellent meat quality, improved tolerance ability, and important landscape value. Copy number variation (CNV) is a significant source of gene structural variation and plays a crucial role in evolution and phenotypic diversity. Based on the latest reference genome ARS-UCD2.0, this study analyzed the genome-wide distribution of CNVs in Yunling cattle using short-read whole-genome sequencing data (n = 129) and single-molecule long-read sequencing data (n = 1), and a total of 16,507 CNVs were detected. After merging CNVs with overlapping genomic positions, 3,728 CNV regions (CNVRs) were obtained, accounting for 0.61% of the reference genome. The functional analysis indicated significant enrichment of CNVRs in 96 GO terms and 57 KEGG pathways, primarily related to cell adhesion, signal transduction, neuromodulation, and nutritional metabolism. Additionally, 111 CNVRs overlapped with 76 quantitative trait loci (QTLs), including Subcutaneous fat thickness QTL, Longissimus muscle area QTL, and Marbling score QTL. Several CNVR-overlapping genes, including BZW1, AOX1, and LOC100138449, overlap with regions associated with meat color and quality QTLs. Furthermore, Vst analysis showed that PSMB4, ERICH1, SMC2, and PPP4R3A were highly divergent between Yunling and Brahman cattle. In summary, we have constructed the genomic CNV map of Yunling cattle for the first time using whole-genome resequencing. This provides valuable genetic variation resources for the study of the Yunling cattle genome and contributes to the study of economic traits in Yunling cattle.

Impact of nebulization versus metered-dose inhaler utilization on viral particle dispersion in patients with COVID-19.

Conflicting guidance exists regarding the characterization of nebulization as an aerosol-generating procedure and subsequent risk of transmission of SARS-CoV-2 to healthcare workers. This study sought to address whether SARS-CoV-2 viral load distribution was impacted by bronchodilator delivery via nebulizer versus metered-dose inhaler (MDI). Adults infected with COVID-19 were enrolled and received a single dose of albuterol sulfate nebulized solution (2.5mg/3mL via breath-actuated nebulizer with filtered mouthpiece) or albuterol sulfate hydrofluoroalkane inhalation aerosol (90µg/actuation; two puffs via MDI with spacer) in a randomized crossover sequence. Air and surfaces were sampled at predefined locations within patients' hospital rooms to assess SARS-CoV-2 dispersion over three periods (baseline, MDI, and nebulizer). Eleven patients received crossover therapy. Six patients had detectable SARS-CoV-2 RNA during one treatment period (n = 3 each for MDI and nebulizer) and one during both treatment periods. No viral RNA was found in the rooms of four (36.4%) patients. Overall, few environmental samples (17/397; 4.3%) contained detectable viral RNA, with no meaningful differences in positivity rate across periods; RNA genome copy numbers were low in positive samples. No correlation between dispersion and patient clinical status or environmental parameters was observed. In this first prospective trial evaluating viral load distribution following use of nebulizer versus MDI in hospitalized patients with COVID-19, low environmental contamination was found regardless of administration method. Findings support the use of either device when needed to treat patients with COVID-19.

NS2 induces an influenza A RNA polymerase hexamer and acts as a transcription to replication switch

Genome transcription and replication of influenza A virus (FluA), catalyzed by viral RNA polymerase (FluAPol), are delicately controlled across the virus life cycle. A switch from transcription to replication occurring at later stage of an infection is critical for progeny virion production and viral non-structural protein NS2 has been implicated in regulating the switch. However, the underlying regulatory mechanisms and the structure of NS2 remained elusive for years. Here, we determine the cryo-EM structure of the FluAPol-NS2 complex at ~3.0 Å resolution. Surprisingly, three domain-swapped NS2 dimers arrange three symmetrical FluPol dimers into a highly ordered barrel-like hexamer. Further structural and functional analyses demonstrate that NS2 binding not only hampers the interaction between FluAPol and the Pol II CTD because of steric conflicts, but also impairs FluAPol transcriptase activity by stalling it in the replicase conformation. Moreover, this is the first visualization of the full-length NS2 structure. Our findings uncover key molecular mechanisms of the FluA transcription-replication switch and have implications for the development of antivirals.

Development of a Crystallographic Screening to Identify Sudan Virus VP40 Ligands.

The matrix protein VP40 of the highly pathogenic Sudan virus (genus Orthoebolavirus) is a multifunctional protein responsible for the recruitment of viral nucleocapsids to the plasma membrane and the budding of infectious virions. In addition to its role in assembly, VP40 also downregulates viral genome replication and transcription. VP40's existence in various homo-oligomeric states is presumed to underpin its diverse functional capabilities during the viral life cycle. Given the absence of licensed therapeutics targeting the Sudan virus, our study focused on inhibiting VP40 dimers, the structural precursors to critical higher-order oligomers, as a novel antiviral strategy. We have established a crystallographic screening pipeline for the identification of small-molecule fragments capable of binding to VP40. Dimeric VP40 of the Sudan virus was recombinantly expressed in bacteria, purified, crystallized, and soaked in a solution of 96 different preselected fragments. Salicylic acid was identified as a crystallographic hit with clear electron density in the pocket between the N- and the C-termini of the VP40 dimer. The binding interaction is predominantly coordinated by amino acid residues leucine 158 (L158) and arginine 214 (R214), which are key in stabilizing salicylic acid within the binding pocket. While salicylic acid displayed minimal impact on the functional aspects of VP40, we delved deeper into characterizing the druggability of the identified binding pocket. We analyzed the influence of residues L158 and R214 on the formation of virus-like particles and viral RNA synthesis. Site-directed mutagenesis of these residues to alanine markedly affected both VP40's budding activity and its effect on viral RNA synthesis, underscoring the potential of the salicylic acid binding pocket as a drug target. In summary, our findings lay the foundation for structure-guided drug design to provide lead compounds against Sudan virus VP40.

Amyloid-β peptide signature associated with cerebral amyloid angiopathy in familial Alzheimer’s disease with APPdup and Down syndrome

Alzheimer’s disease (AD) is characterized by extracellular amyloid plaques containing amyloid-β (Aβ) peptides, intraneuronal neurofibrillary tangles, extracellular neuropil threads, and dystrophic neurites surrounding plaques composed of hyperphosphorylated tau protein (pTau). Aβ can also deposit in blood vessel walls leading to cerebral amyloid angiopathy (CAA). While amyloid plaques in AD brains are constant, CAA varies among cases. The study focuses on differences observed between rare and poorly studied patient groups with APP duplications (APPdup) and Down syndrome (DS) reported to have higher frequencies of elevated CAA levels in comparison to sporadic AD (sAD), most of APP mutations, and controls. We compared Aβ and tau pathologies in postmortem brain tissues across cases and Aβ peptides using mass spectrometry (MS). We further characterized the spatial distribution of Aβ peptides with MS-brain imaging. While intraparenchymal Aβ deposits were numerous in sAD, DS with AD (DS-AD) and AD with APP mutations, these were less abundant in APPdup. On the contrary, Aβ deposits in the blood vessels were abundant in APPdup and DS-AD while only APPdup cases displayed high Aβ deposits in capillaries. Investigation of Aβ peptide profiles showed a specific increase in Aβx-37, Aβx-38 and Aβx-40 but not Aβx-42 in APPdup cases and to a lower extent in DS-AD cases. Interestingly, N-truncated Aβ2-x peptides were particularly increased in APPdup compared to all other groups. This result was confirmed by MS-imaging of leptomeningeal and parenchymal vessels from an APPdup case, suggesting that CAA is associated with accumulation of shorter Aβ peptides truncated both at N- and C-termini in blood vessels. Altogether, this study identified striking differences in the localization and composition of Aβ deposits between AD cases, particularly APPdup and DS-AD, both carrying three genomic copies of the APP gene. Detection of specific Aβ peptides in CSF or plasma of these patients could improve the diagnosis of CAA and their inclusion in anti-amyloid immunotherapy treatments.

Papillary tumor of the pineal region: analysis of DNA methylation profiles and clinical outcomes in 76 cases

Papillary tumor of the pineal region (PTPR) is an uncommon tumor of the pineal region with distinctive histopathologic and molecular characteristics. Experience is limited with respect to its molecular heterogeneity and clinical characteristics. Here, we describe 39 new cases and combine these with 37 previously published cases for a cohort of 76 PTPR’s, all confirmed by methylation profiling. As previously reported, two main methylation groups were identified (PTPR-A and PTPR-B). In our analysis we extended the subtyping into three subtypes: PTPR-A, PTPR-B1 and PTPR-B2 supported by DNA methylation profile and genomic copy number variations. Frequent loss of chromosome 3 or 14 was found in PTPR-B1 tumors but not in PTPR-B2. Examination of clinical outcome showed that nearly half (14/30, 47%) of examined patients experienced tumor progression with significant difference among the subtypes (p value = 0.046). Our analysis extends the understanding of this uncommon but distinct neuroepithelial tumor by describing its molecular heterogeneity and clinical outcomes, including its tendency towards tumor recurrence.

Selected Lark Mitochondrial Genomes Provide Insights into the Evolution of Second Control Region with Tandem Repeats in Alaudidae (Aves, Passeriformes).

The control region (CR) regulates the replication and transcription of the mitochondrial genome (mitogenome). Some avian mitogenomes possess two CRs, and the second control region (CR2) may enhance replication and transcription; however, the CR2 in lark mitogenome appears to be undergoing loss and is accompanied by tandem repeats. Here, we characterized six lark mitogenomes from Alaudala cheleensis, Eremophila alpestris, Alauda razae, and Calandrella cinerea and reconstructed the phylogeny of Passerida. Through further comparative analysis among larks, we traced the evolutionary process of CR2. The mitochondrial gene orders were conserved in all published lark mitogenomes, with Cytb-trnT-CR1-trnP-ND6-trnE-remnant CR2 with tandem repeat-trnF-rrnS. Phylogenetic analysis revealed Alaudidae and Panuridae are sister groups at the base of Sylvioidea, and sporadic losses of CR2 may occur in their common ancestor. CR sequence and phylogeny analysis indicated CR2 tandem repeats were generated within CR2, originating in the ancestor of all larks, rather than inherited from CR1. The secondary structure comparison of tandem repeat units within and between species suggested slipped-strand mispairing and DNA turnover as suitable models for explaining the origin and evolution of these repeats. This study reveals the evolutionary process of the CR2 containing tandem repeat in Alaudidae, providing reference for understanding the evolutionary characteristics and dynamics of tandem repeats.

Phage anti-CRISPR control by an RNA- and DNA-binding helix-turn-helix protein.

In all organisms, regulation of gene expression must be adjusted to meet cellular requirements and frequently involves helix-turn-helix (HTH) domain proteins1. For instance, in the arms race between bacteria and bacteriophages, rapid expression of phage anti-CRISPR (acr) genes upon infection enables evasion from CRISPR-Cas defence; transcription is then repressed by an HTH-domain-containing anti-CRISPR-associated (Aca) protein, probably to reduce fitness costs from excessive expression2-5. However, how a single HTH regulator adjusts anti-CRISPR production to cope with increasing phage genome copies and accumulating acr mRNA is unknown. Here we show that the HTH domain of the regulator Aca2, in addition to repressing Acr synthesis transcriptionally through DNA binding, inhibits translation of mRNAs by binding conserved RNA stem-loops and blocking ribosome access. The cryo-electron microscopy structure of the approximately 40 kDa Aca2-RNA complex demonstrates how the versatile HTH domain specifically discriminates RNA from DNA binding sites. These combined regulatory modes are widespread in the Aca2 family and facilitate CRISPR-Cas inhibition in the face of rapid phage DNA replication without toxic acr overexpression. Given the ubiquity of HTH-domain-containing proteins, it is anticipated that many more of them elicit regulatory control by dual DNA and RNA binding.

P-192 Correlation between the ratio of mitochondrial DNA to genomic DNA copy number in spent culture media and embryo quality in IVF

Abstract Abstract withdrawn by the authors

Assessment of crAssphage as a biological variable for SARS-CoV-2 data normalization in wastewater surveillance.

This study aimed to assess the use of cross-assembled phage (crAssphage) as an endogenous control employing a multivariate normalization analysis and its application as a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) data normalizer. A total of 188 twelve-hour composite raw sewage samples were obtained from eight wastewater treatment plants (WWTP) during a 1-year monitoring period. Employing the N1 and N2 target regions, SARS-CoV-2 RNA was detected in 94% (177) and 90% (170) of the samples, respectively, with a global median of 5 log10 genomic copies per liter (GC l-1). CrAssphage was detected in 100% of the samples, ranging from 8.29 to 10.43 log10 GC l-1, with a median of 9.46±0.40 log10 GC l-1, presenting both spatial and temporal variabilities. Although SARS-CoV-2 data normalization employing crAssphage revealed a correlation with clinical cases occurring during the study period, crAssphage normalization by the flow per capita per day of each WWTP increased this correlation, corroborating the importance of normalizing wastewater surveillance data in disease trend monitoring.

Accumulation of DNA G-quadruplex in mitochondrial genome hallmarks mesenchymal senescence.

Searching for biomarkers of senescence remains necessary and challenging. Reliable and detectable biomarkers can indicate the senescence condition of individuals, the need for intervention in a population, and the effectiveness of that intervention in controlling or delaying senescence progression and senescence-associated diseases. Therefore, it is of great importance to fulfill the unmet requisites of senescence biomarkers especially when faced with the growing global senescence nowadays. Here, we established that DNA G-quadruplex (G4) in mitochondrial genome was a reliable hallmark for mesenchymal senescence. Via developing a versatile and efficient mitochondrial G4 (mtG4) probe we revealed that in multiple types of senescence, including chronologically healthy senescence, progeria, and replicative senescence, mtG4 hallmarked aged mesenchymal stem cells. Furthermore, we revealed the underlying mechanisms by which accumulated mtG4, specifically within respiratory chain complex (RCC) I and IV loci, repressed mitochondrial genome transcription, finally impairing mitochondrial respiration and causing mitochondrial dysfunction. Our findings endowed researchers with the visible senescence biomarker based on mitochondrial genome and furthermore revealed the role of mtG4 in inhibiting RCC genes transcription to induce senescence-associated mitochondrial dysfunction. These findings depicted the crucial roles of mtG4 in predicting and controlling mesenchymal senescence.

Occurrence of astrovirus in young racing pigeons and genome characterization of 2 new astrovirus genomes representing 2 new species

Enteropathies are a serious concern in racing pigeons as they significantly impair performance in races and their training, and viruses are suspected to be one of the main factors. Astroviruses are well-known to be responsible for causing enteric disease in humans and various other animals including birds, although their prevalence and pathogenicity in pigeons is poorly understood. In this study, we investigated 2 groups of young racing pigeons (sick-study group and healthy-control group) to assess the correlation between the number of astrovirus genome copies in cloacal swabs and the occurrence of enteropathy. To determine this, we developed a novel TaqMan quantitative PCR (qPCR) and digital droplet PCR (ddPCR) methods for astrovirus detection and absolute quantitative analysis. We also performed high-throughput sequencing to obtain the complete genome sequences and establish the genetic similarity of the obtained strains to known astroviruses of poultry and other avian species. Two new complete genome sequences of pigeon astroviruses in the Avastrovirus genus were identified, representing 2 new species. These were found most closely related to astroviruses identified in Columbidae species and chickens. They share an average of 75.8% genome-wide pairwise identity and 57.6% and 64.6% capsid protein sequence identity with other unclassified columbid avastrovirus sequences in GenBank. Although the difference in prevalence of astrovirus in the study and control group was found statistically insignificant, there was a significant difference between the number of genome copies in positive samples from both groups. These unambiguous results leave the role of astroviruses as enteropathogenic factors in pigeons still undetermined.

A cross-species transcriptomic analysis reveals a novel 2-dimensional classification system explaining the invasiveness heterogeneity of pancreatic neuroendocrine tumor

Pancreatic neuroendocrine tumors (PanNETs), the second most common type of primary pancreatic tumors, display notable heterogeneity in invasiveness. Current knowledge regarding genomic alterations, including DAXX/ATRX, MEN1 mutations, and copy number variations (CNVs), provides some insights into tumor invasiveness. However, the underlying reasons for the significant variation in invasiveness between insulinoma and other types of PanNETs remain unclear. To construct a comprehensive model for the stratification of prognosis, we employed analysis of both the well-established Rip1-Tag2 (RT2) mouse model of PanNETs and human PanNETs with various functional types. Firstly, by applying single-cell and bulk RNA sequencing in PanNETs from different ages and strains of RT2 mice and human PanNETs, we introduced a 2-dimensional (2D) classification system. Based on the 2-D classification system, human PanNETs were mainly classified as benign insulinomas or non-insulinomas subclusters. Non-insulinomas subtypes mainly included gastrinomas, glucagonomas, VIPomas, and NF-PanNETs, which all exhibited potential invasiveness. In addition, we discovered an enrichment of specific CNV patterns and mutations in corresponding human PanNET subclusters. Then we denoted somatic DAXX/ATRX as the ‘second hit’ and confounding factors for invasiveness. Finally, by combining the 2D system, DAXX/ATRX mutation status, and tumor diameter, a group of indolent PanNETs with minimal recurrence risk was identified. In conclusion, our current work constructed a comprehensive model to elucidate the heterogeneity of invasiveness in PanNETs and improve prognostic stratification.

Safety of intravitreally delivered AAV2 vector-mediated multi-characteristic opsin genetic construct in wild type beagle dogs.

A novel adeno-associated virus 2 (AAV2)-carried multi-characteristic opsin (MCO) (MCO-010) is undergoing several clinical trials as a novel therapeutic modality for the treatment of degenerative retinal diseases including retinitis pigmentosa and Stargardt disease. The present study aimed to determine the ocular and systemic safety of MCO-010 and the AAV2 vehicle in adult Beagle dogs following intravitreal (IVT) injection. The current safety/toxicology studies spanning 13 weeks described here utilized well-documented techniques to assess the effects of IVT injection of MCO-010 up to 2.2 × 1011 genome copies (gc) per eye, or the AAV2 capsid (vehicle control) on gross behavioral and immunogenic changes, alterations in body weights, blood biochemistry, hematology, blood coagulation, gross necropsy lesions, organ weight changes and histopathology in the dogs (n = 4 per group; two males and two females per group). Immunohistochemical and functional electroretinogram studies were also conducted to determine MCO expression in the retina and determine any retinal toxicity associated with MCO-010. There were no significant deleterious effects of the MCO-010 (or the AAV2 at the tested doses) on any of the examined parameters, including the absence of any severe ocular or systemic adverse events. However, as expected, inflammation after IVT delivery of AAV2 and MCO-010 was observed in the conjunctivae of all groups of animals, although this self-resolved within 1week post-injection. Quantitative immunohistochemical analyses of MCO-010-associated mCherry revealed successful delivery of the gene therapy within the inner retina. In summary, MCO-010 demonstrated a favorable safety profile when administered to the eyes of adult Beagle dogs of both sexes at dose levels up to 2.2 × 1011 gc per eye, with no adverse effects observed. This dose was identified as the No Observed Adverse Effect Level (i.e. NOAEL) and guided selection of safe doses for human clinical trials.

Case-control study to identify the causative agents of ophthalmia and conjunctivitis in goats in Savannakhet province of Lao PDR

Pinkeye is a highly contagious disease of goats with different aetiologies. Surveys in Lao PDR have identified eye lesions typical of pinkeye as a common condition, however, this has not been confirmed diagnostically, and the responsible pathogens have not been identified. A matched case-control study was implemented in 70 goat holdings from Savannakhet province, Lao PDR, to detect agents causing pinkeye and conduct phylogenetic analysis of the identified pathogens. Fifty eye swabs from goats with infected eyes (cases) and 50 paired samples from unaffected cohorts (controls) were collected from 25 holdings. Samples were tested using quantitative PCR assays targeting known pinkeye pathogens at the genus and species levels. The prevalence of pathogens in case and control goats was as follows: Mycoplasma conjunctivae (94% and 74% respectively, P = 0.006, OR = 5.5), Chlamydia pecorum (4%, 10%), Moraxella ovis (30%, 30%), Moraxella bovis (0%, 0%) and Moraxella bovoculi (0%, 0%). M. conjunctivae was present in a high proportion of goats in both groups revealing that Lao goats are carriers of M. conjunctivae. However, the mean log10 genome copy number/µL of DNA extract was significantly higher in case goats than control goats (P < 0.05). Thus, M. conjunctivae is likely the principal causative agent of pinkeye in Lao goats with carrier status converting to clinical infection following corneal damage or other causative factors. M. conjunctivae detected in samples from different goats and districts showed low genetic diversity. Identifying the causes of pinkeye in Lao goats will assist in designing appropriate treatment and control strategies.

Field efficacy of a novel porcine reproductive and respiratory syndrome modified-live virus vaccine with an emphasis on growth performance.

This field evaluation was designed to evaluate the efficacy of a new porcine reproductive and respiratory syndrome virus-2 (PRRSV-2) modified live virus vaccine at three independent pig farms. Three farms were selected for this study based on their respiratory disease status caused by PRRSV-2 infection in post-weaning and growing pigs. Each farm housed a total of 40, 18-day-old pigs that were randomly allocated to one of two treatment groups. Pigs were administered a 1.0mL dose of the bivalent vaccine intramuscularly at 21 days of age in accordance with the manufacturer's recommendations, whereas unvaccinated pigs were administered a single dose of phosphate buffered saline at the same age. Vaccinated groups were measured and calculated significantly (p<0.05) higher in body weight and average daily weight gain on all three farms compared with unvaccinated groups. Vaccinated groups elicited PRRS antibodies and PRRSV-2-specific interferon-γ secreting cells, which reduced the amount of PRRSV-2 genomic copies in the blood and reduced macroscopic and microscopic lung lesions severity when compared with unvaccinated groups. The field evaluation data demonstrated that a new PRRSV-2 modified live virus vaccine was efficacious in swine herds suffering from respiratory diseases caused by PRRSV-2 infection.

Neonectria bordenii sp. nov., a potential symbiote of the alder bark beetle, and its detection by quantitative PCR.

A taxonomically comprehensive perspective on the fungal associates of bark beetles (Coleoptera: Curculionidae: Scolytinae), and powerful molecular tools for detection of these fungi, are imperative to understanding bark beetle impacts on forest ecosystems. The most common filamentous fungi living alongside bark beetles in infested trees are ophiostomatoids (Ascomycota: Ophiostomatales and Microascales), yet an undescribed species of Neonectria (Neonectria sp. nov.; Ascomycota: Hypocreales) was recently identified cohabitating with the alder bark beetle, Alniphagus aspericollis, in red alder, Alnus rubra. The hardwood-infesting alder bark beetle is found throughout the range of its red alder host in the Pacific Coast region of North America and is associated with Neonectria sp. nov. in southwestern British Columbia, Canada. The aim of this study was to describe and name Neonectria sp. nov. and to develop a quantitative PCR (qPCR) assay to enable rapid detection of Neonectria sp. nov. from individual adult alder bark beetles and to define the distribution of the fungus. Neonectria sp. nov. was phylogenetically and morphologically determined to represent a distinct species closely related to N. ditissima and is described herein as Neonectria bordenii sp. nov. Neonectria bordenii was reliably detected from individual whole-beetle DNA extractions using a probe-based qPCR assay targeting multi-copy internal transcribed spacers (ITS) of nuclear ribosomal DNA. The qPCR assay amplified the fungus from 87.8 % (36/41) of individual alder bark beetle samples and was highly sensitive to N. bordenii, with a lower limit of detection of 1 × 10-6 ng/μL of culture DNA (or ~262 genome copies). Application of the qPCR assay developed in this study will expedite future research evaluating N. bordenii as a potential symbiote of the alder bark beetle. Citation: Wertman DL, Tanney JB, Hamelin RC, Carroll AL (2024). Neonectria bordenii sp. nov., a potential symbiote of the alder bark beetle, and its detection by quantitative PCR. Fungal Systematics and Evolution 13: 15-28. doi: 10.3114/fuse.2024.13.02.

Heteroplasmy Is Rare in Plant Mitochondria Compared with Plastids despite Similar Mutation Rates.

Plant cells harbor two membrane-bound organelles containing their own genetic material-plastids and mitochondria. Although the two organelles coexist and coevolve within the same plant cells, they differ in genome copy number, intracellular organization, and mode of segregation. How these attributes affect the time to fixation or, conversely, loss of neutral alleles is currently unresolved. Here, we show that mitochondria and plastids share the same mutation rate, yet plastid alleles remain in a heteroplasmic state significantly longer compared with mitochondrial alleles. By analyzing genetic variants across populations of the marine flowering plant Zostera marina and simulating organelle allele dynamics, we examine the determinants of allele segregation and allele fixation. Our results suggest that the bottlenecks on the cell population, e.g. during branching or seeding, and stratification of the meristematic tissue are important determinants of mitochondrial allele dynamics. Furthermore, we suggest that the prolonged plastid allele dynamics are due to a yet unknown active plastid partition mechanism. The dissimilarity between plastid and mitochondrial novel allele fixation at different levels of organization may manifest in differences in adaptation processes. Our study uncovers fundamental principles of organelle population genetics that are essential for further investigations of long-term evolution and molecular dating of divergence events.

Landscape structure affects temporal dynamics in the bumble bee virome: Landscape heterogeneity supports colony resilience

Virus spillovers from managed honey bees, Apis mellifera, are thought to contribute to the decline of wild pollinators, including bumble bees. However, data on the impact of such viruses on wild pollinators remain scarce, and the influence of landscape structure on virus dynamics is poorly understood. In this study, we deployed bumble bee colonies in an agricultural landscape and studied changes in the bumble bee virome during field placement under varying habitat composition and configuration using a multiscale analytical framework. We estimated prevalence of viruses and viral loads (i.e. number of viral genomic equivalent copies) in bumble bees before and after placing them in the field using next generation sequencing and quantitative PCR. The results show that viral loads and number of different viruses present increased during placement in the field and that the virus composition of the colonies shifted from an initial dominance of honey bee associated viruses to a higher number (in both viral loads and number of viruses present) of bumble bee associated viruses. Especially DWV-B, typical for honey bees, drastically decreased after the time in the field. Viral loads prior to placing colonies in the field showed no effect on colony development, suggesting low impacts of these viruses in field settings. Notably, we further demonstrate that increased habitat diversity results in a lower number of different viruses present in Bombus colonies, while colonies in areas with well-connected farmland patches decreased in their total viral load after field placement. Our results emphasize the importance of landscape heterogeneity and connectivity for wild pollinator health and that these influences predominate at fine spatial scales.