Chimeric Genomes Research Articles

Bat Rhinacoviruses Related to Swine Acute Diarrhoea Syndrome Coronavirus Evolve under Strong Host and Geographic Constraints in China and Vietnam.

Swine acute diarrhoea syndrome coronavirus (SADS-CoV; Coronaviridae, Rhinacovirus) was detected in 2017 in Guangdong Province (China), where it caused high mortality rates in piglets. According to previous studies, SADS-CoV evolved from horseshoe bat reservoirs. Here, we report the first five Rhinacovirus genomes sequenced in horseshoe bats from Vietnam and their comparisons with data published in China. Our phylogenetic analyses provided evidence for four groups: rhinacoviruses from Rhinolphus pusillus bats, including one from Vietnam; bat rhinacoviruses from Hainan; bat rhinacoviruses from Yunnan showing a divergent synonymous nucleotide composition; and SADS-CoV and related bat viruses, including four rhinacoviruses from Vietnam sampled in Rhinolophus affinis and Rhinolophus thomasi. Our phylogeographic analyses showed that bat rhinacoviruses from Dien Bien (Vietnam) share more affinities with those from Yunnan (China) and that the ancestor of SADS-CoVs arose in Rhinolophus affinis circulating in Guangdong. We detected sequencing errors and artificial chimeric genomes in published data. The two SADS-CoV genomes previously identified as recombinant could also be problematic. The reliable data currently available, therefore, suggests that all SADS-CoV strains originate from a single bat source and that the virus has been spreading in pig farms in several provinces of China for at least seven years since the first outbreak in August 2016.

Construction of SARS-CoV-2 full - length S gene with point mutations relevant for COVID-19 vaccine strain candidate

To overcome the pandemic of COVID-19, prevent the disease or its post-conditions effectively, a vaccine with the updates of viral strains is needed. The full S gene encoding the glycoprotein spike of SARS-CoV-2 was recommended for vaccine production by the World Health Organization. To create a vaccine strain candidate for a COVID-19, we have cloned the full-length S gene of the SARS-CoV 2 containing mutations that improve thermal stability of the protein (HexaPro) and three strain-specific mutations of D614G, L452R and E484Q that are present in rapidly and widely circulating variants. The fragments of the full-length S gene were first amplified from the two commercial plasmids, in which one plasmid contains the HexaPro mutations and the other was used to amplify the 3’ fragment of the gene, these fragments were then assembled. Strain-specific mutations were generated using primers with mutations. The measles (MeV) overlaping sequences and AscI (5’- GCGCGC-3’) restriction sites were also added to the two ends of the S gene. The full length of the cloned S gene with mutations and restriction sites was confirmed by sequencing and is ready to be inserted into the measles backbone for creating a chimeric genome of MeV_SARS CoV-2.

The development of a rapid, high-throughput neutralization assay using a SARS-CoV-2 reporter

Many methods have been developed to measure the neutralizing capacity of antibodies to SARS-CoV-2. However, these methods are low throughput and can be difficult to quickly modify in response to emerging variants. Therefore, an experimental system for rapid and easy measurement of the neutralizing capacity of antibodies against various variants is needed. In this study, we developed an experimental system that can efficiently measure the neutralizing capacity of sera by using a GFP-carrying recombinant SARS-CoV-2 with spike proteins of multiple variants (B.1.1, BA.5, or XBB.1.5). For all 3 recombinant chimeric genomes generated, neutralizing antibody titers determined by measuring GFP fluorescence intensity correlated significantly with those calculated from viral RNA levels measured by RT-qPCR in the supernatant of infected cells. Furthermore, neutralizing antibody titers determined by visually assessing GFP fluorescence using microscopy were also significantly correlated with those determined by RT-qPCR. By using this high-throughput method, it is now possible to quickly and easily determine the neutralizing capacity of antibodies against SARS-CoV-2 variants.

Rhizoviticin is an alphaproteobacterial tailocin that mediates biocontrol of grapevine crown gall disease.

Tailocins are headless phage tail structures that mediate interbacterial antagonism. Although the prototypical tailocins, R- and F-pyocins, in Pseudomonas aeruginosa, and other predominantly R-type tailocins have been studied, their presence in Alphaproteobacteria remains unexplored. Here, we report the first alphaproteobacterial F-type tailocin, named rhizoviticin, as a determinant of the biocontrol activity of Allorhizobium vitis VAR03-1 against crown gall. Rhizoviticin is encoded by a chimeric prophage genome, one providing transcriptional regulators and the other contributing to tail formation and cell lysis, but lacking head formation genes. The rhizoviticin genome retains a nearly intact early phage region containing an integrase remnant and replication-related genes critical for downstream gene transcription, suggesting an ongoing transition of this locus from a prophage to a tailocin-coding region. Rhizoviticin is responsible for the most antagonistic activity in VAR03-1 culture supernatant against pathogenic A. vitis strain, and rhizoviticin deficiency resulted in a significant reduction in the antitumorigenic activity in planta. We identified the rhizoviticin-coding locus in eight additional A. vitis strains from diverse geographical locations, highlighting a unique survival strategy of certain Rhizobiales bacteria in the rhizosphere. These findings advance our understanding of the evolutionary dynamics of tailocins and provide a scientific foundation for employing rhizoviticin-producing strains in plant disease control.

Genomic insights into biased allele loss and increased gene numbers after genome duplication in autotetraploid Cyclocarya paliurus

BackgroundAutopolyploidy is a valuable model for studying whole-genome duplication (WGD) without hybridization, yet little is known about the genomic structural and functional changes that occur in autopolyploids after WGD. Cyclocarya paliurus (Juglandaceae) is a natural diploid–autotetraploid species. We generated an allele-aware autotetraploid genome, a chimeric chromosome-level diploid genome, and whole-genome resequencing data for 106 autotetraploid individuals at an average depth of 60 × per individual, along with 12 diploid individuals at an average depth of 90 × per individual.ResultsAutotetraploid C. paliurus had 64 chromosomes clustered into 16 homologous groups, and the majority of homologous chromosomes demonstrated similar chromosome length, gene numbers, and expression. The regions of synteny, structural variation and nonalignment to the diploid genome accounted for 81.3%, 8.8% and 9.9% of the autotetraploid genome, respectively. Our analyses identified 20,626 genes (69.18%) with four alleles and 9191 genes (30.82%) with one, two, or three alleles, suggesting post-polyploid allelic loss. Genes with allelic loss were found to occur more often in proximity to or within structural variations and exhibited a marked overlap with transposable elements. Additionally, such genes showed a reduced tendency to interact with other genes. We also found 102 genes with more than four copies in the autotetraploid genome, and their expression levels were significantly higher than their diploid counterparts. These genes were enriched in enzymes involved in stress response and plant defense, potentially contributing to the evolutionary success of autotetraploids. Our population genomic analyses suggested a single origin of autotetraploids and recent divergence (~ 0.57 Mya) from diploids, with minimal interploidy admixture.ConclusionsOur results indicate the potential for genomic and functional reorganization, which may contribute to evolutionary success in autotetraploid C. paliurus.

Targeted viral adaptation generates a simian-tropic hepatitis B virus that infects marmoset cells

Hepatitis B virus (HBV) only infects humans and chimpanzees, posing major challenges for modeling HBV infection and chronic viral hepatitis. The major barrier in establishing HBV infection in non-human primates lies at incompatibilities between HBV and simian orthologues of the HBV receptor, sodium taurocholate co-transporting polypeptide (NTCP). Through mutagenesis analysis and screening among NTCP orthologues from Old World monkeys, New World monkeys and prosimians, we determined key residues responsible for viral binding and internalization, respectively and identified marmosets as a suitable candidate for HBV infection. Primary marmoset hepatocytes and induced pluripotent stem cell-derived hepatocyte-like cells support HBV and more efficient woolly monkey HBV (WMHBV) infection. Adapted chimeric HBV genome harboring residues 1–48 of WMHBV preS1 generated here led to a more efficient infection than wild-type HBV in primary and stem cell derived marmoset hepatocytes. Collectively, our data demonstrate that minimal targeted simianization of HBV can break the species barrier in small NHPs, paving the path for an HBV primate model.

Novel recombinant SARS-CoV-2 lineage detected through genomic surveillance in Wales, UK.

Recombination, the process whereby a segment of genetic material from one genome is inserted into another, producing a new chimeric genome, is an important evolutionary mechanism frequently observed in coronaviruses. The risks posed by recombination include the shuffling of advantageous mutations that may increase transmissibility, severity or vaccine escape. We present a genomic and epidemiological description of a new recombinant lineage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), XR, first identified in Wales. The Pathogen Genomics Unit (Public Health Wales, UK) sequences positive SARS-CoV-2 PCR tests using the ARTIC SARS-CoV-2 sequencing protocol. Recombinants were detected using an in-house pipeline and the epidemiological data analysed in R. Nosocomial cases were defined as those with samples taken after >7 days in hospital. Between February and March 2022, we identified 78 samples with highly similar genomes, comprising a BA.1-like 5' end, a BA.2-like 3' end and a BA.2-like spike protein. This signature is consistent with recombination and was defined as XR by Pangolin (PANGO v1.8). A total of 50 % of cases had a sample collected whilst in hospital and the first three cases were immunocompromised patients. The patient median age was 58 years (range: 4-95 years) and most of the patients were fully vaccinated against SARS-CoV-2 (74 % third dose/booster). Three patients died within 28 days of their sample collection date, one of whom had COVID-19 listed amongst ICD10 (International Classification of Diseases 10) coded causes of death. Our integrated system enabled real-time monitoring of recombinant SARS-CoV-2 for early detection, in order to rapidly risk assess and respond. This work highlights the importance of setting-based surveillance of recombinant SARS-CoV-2, as well as the need to monitor immunocompromised populations through repeat testing and sequencing.

Abstract 6796: PoxSTG, a novel chimeric poxvirus with improved oncolytic potency

Abstract Oncolytic virus (OV) therapy has emerged as a promising approach for cancer treatment with the potential to be less toxic and more efficient than classic cancer therapies. Various types of OVs in clinical development, including Vaccinia virus (VACV)-derived OVs, have shown good safety profiles, but limited therapeutic efficacy as monotherapy in some cancer models. Many different methods have been employed to improve the oncolytic potency of OVs. We used a directed evolution process, pooling multiple species of orthopoxviruses to generate chimeric poxviruses with increased oncolytic properties. Through selective pressure by successive passages on human tumor cells, a new chimeric oncolytic poxvirus, named PoxSTG, was derived. The chimeric viral genome contains, in addition to sequences from several strains of Vaccinia virus, sequences of Rabbitpox virus and Cowpox virus. Compared with its parental viruses, PoxSTG has demonstrated superior oncolytic characteristics, and was notably attenuated in normal primary cells. PoxSTG has superior secretion of extracellular-enveloped virus (EEV) compared to all parental strains inducing higher dissemination of the virus into the tumors and more resistance to neutralization. PoxSTG was saved and showed potent antitumor effects in several syngeneic and xenograft mouse models of cancers. Furthermore, PoxSTG exerted low-dose antitumor effects in virus-injected and non-virus-injected distant tumors in a CRC xenograft model, demonstrating strong virus spread to distant tumors. All these data demonstrate the potential of PoxSTG as a novel therapeutic agent for cancer treatment. Citation Format: Philippe Erbs, Annie Findeli, Johann Foloppe, Juliette Kempf, Isabelle Farine, Baptiste Moreau, Julie Hortelano, Christelle Pichon, Jules Deforges, Eric Quemeneur. PoxSTG, a novel chimeric poxvirus with improved oncolytic potency. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6796.

Characterization of a Chimeric MmuPV1 Genome with HPV-16E6E7

Background/Objectives: Papilloma Viruses (PVs) are double-stranded DNA viruses that infect cutaneous and mucosal epithelium. In humans, there are over 100 types of PVs. HPV-16 is a high-risk type that causes ~50% of cervical cancers and ~70% of oropharyngeal cancers by expressing viral oncogenes E6 and E7 in replicating keratinocytes. The mouse PV, MmuPV1, displays species tropism and causes neoplastic lesions. Its discovery in 2011 introduced the opportunity to study PVs from early stages of infection to cancer development. Such an in vivo model of HPV-16 could uncover novel mechanisms for treatment intervention and disease prevention. Our study aims to characterize a small-animal infection model using a chimeric MmuPV1 genome with HPV-16 E6 and E7 in place of mouse E6 and E7 (MmuPV1-16E6E7). The goal of our study is to investigate the genome’s ability to express the HPV-16 oncogenes in vitro and to cause tumors in vivo. Methods: To increase selective pressure in vitro, we replaced the MmuPV1 L1 and L2 genes with a neomycin cassette (MmuPV1-16E6E7neo). We packaged these genomes into infectious quasiviruses using a HEK 293TTF viral packing cell line. After isolating quasiviruses, we infected two donor human foreskin keratinocytes (HFK) cell lines. Forty-eight hours post infection, we isolated mRNA for qPCR to quantify HPV-16 E6 expression. To investigate whether MmuPV1-16E6E7 can cause tumors in in vivo, we infected athymic nude (N/J) mice orally with quasiviruses to evaluate infection and viable genome persistence. Results: HPV-16 E6 cDNA was detected in three HFK isolates. Conclusions/Future Directions: The presence of HPV-16 E6 cDNA indicates that the gene can be successfully transcribed from MmuPV1-16E6E7neo in HFKs. Future studies will assess this ability in primary mouse keratinocytes. If tumors are observed, we can use this model to study the efficacy of antiviral compounds to inhibit HPV16 E6 in vivo.

Direct ITR-to-ITR Nanopore Sequencing of AAV Vector Genomes.

Recombinant adeno-associated viruses (rAAVs) are currently the most prominently investigated vector platform for human gene therapy. The rAAV capsid serves as a potent and efficient vehicle for delivering genetic payloads into the host cell, while the vector genome determines the function and effectiveness of these biotherapies. However, current production schemes yield vectors that may consist of heterogeneous populations, compromising their potencies. The development of next-generation sequencing methods within the past few years have helped investigators profile the diversity and relative abundances of heterogenous species in vector preparations. Specifically, long-read sequencing methods, like single molecule real-time (SMRT) sequencing, have been used to uncover truncations, chimeric genomes, and inverted terminal repeat (ITR) mutations in vectors. Unfortunately, these sequencing platforms may be inaccessible to investigators with limited resources, require large amounts of input material, or may require long wait times for sequencing and analyses. Recent advances with nanopore sequencing have helped to bridge the gap for quick and relatively inexpensive long-read sequencing needs. However, their limitations and sample biases are not well-defined for sequencing rAAV. In this study, we explored the capacity for nanopore sequencing to directly interrogate rAAV content to obtain full-length resolution of encapsidated genomes. We found that the nanopore platform can cover the entirety of rAAV genomes from ITR to ITR without the need for pre-fragmentation. However, the accuracy for base calling was low, resulting in a high degree of miscalled bases and false indels. These false indels led to read-length compression; thus, assessing heterogeneity based on read length is not advisable with current nanopore technologies. Nonetheless, nanopore sequencing was able to correctly identify truncation hotspots in single-strand and self-complementary vectors similar to SMRT sequencing. In summary, nanopore sequencing can serve as a rapid and low-cost alternative for proofing AAV vectors.

Naryaviridae, Nenyaviridae, and Vilyaviridae: three new families of single-stranded DNA viruses in the phylum Cressdnaviricota.

The phylum Cressdnaviricota includes viruses with circular single-stranded DNA (ssDNA) genomes and icosahedral capsids. These viruses display global environmental distribution and infect diverse eukaryotic hosts, including animals, plants, and fungi. Here, we report on the formal creation of two new orders, Rivendellvirales and Rohanvirales, and three new families, Naryaviridae, Nenyaviridae, and Vilyaviridae, of ssDNA viruses associated with protozoan parasites belonging to the genera Entamoeba and Giardia. We describe a sequence-based taxonomic framework, which was used to classify 60 ssDNA viruses into 12 genera (with 18 species) within the family Vilyaviridae; four genera (with five species) within the family Naryaviridae; and five genera (with six species) within the family Nenyaviridae. We also highlight the challenges associated with the classification of chimeric virus genomes, such as those in the families Naryaviridae and Nenyaviridae, where the replicationinitiation and capsid protein genes have undergone several independent non-orthologous replacements. The described taxonomic changes have been ratified by the International Committee on Taxonomy of Viruses (ICTV)and expand the phylum Cressdnaviricota to eight orders and 11 families.

Mapping Strengths and Weaknesses of Different Clustering Approaches to Deciphering Bacterial Chimerism

Bacterial genomes are chimeras of DNA of different ancestries. Deconstructing chimeric genomes is central to understanding the evolutionary trajectories of their disparate components and thus the organisms as a whole in the light of their evolutionary contexts. Of specific interest is to delineate and quantify native (vertically inherited) and alien (horizontally acquired) components of bacterial genomes and also specify genomic fractions that represent different donor sources. An agglomerative clustering procedure that prioritizes grouping of proximal similar genomic segments has previously been invoked for this purpose in conjunction with a recursive segmentation procedure. Surprisingly, however, the relative strengths and weaknesses of different clustering approaches to deciphering bacterial chimerism have not yet been investigated, despite the need to robustly interpret tens of thousands of completely sequenced bacterial genomes and nearly complete genome assemblies available in the public databases. To bridge this knowledge gap and develop more robust approaches, we assessed different clustering methods, including segment order based (proximal) clustering, hierarchical clustering, affinity propagation clustering, and a novel network clustering approach on chimeric genomes modeled after bacterial genomes representing a broad spectrum of compositional complexity. Although segment order-based clustering and network clustering compared favorably with the other approaches in discriminating between native and alien DNA at genome optimized settings, network clustering did consistently better than other methods at parametric settings optimized on all test genomes together. Segment order-based clustering and hierarchical clustering outperformed other methods in alien DNA identification while preserving donor identity in the genomes. Our study highlights the strengths and weaknesses of different approaches and suggests how this can be leveraged to achieve a more robust deconstruction of bacterial chimerism.

Viral Complexity.

Although traditionally viewed as streamlined and simple, discoveries over the last century have revealed that viruses can exhibit surprisingly complex physical structures, genomic organization, ecological interactions, and evolutionary histories. Viruses can have physical dimensions and genome lengths that exceed many cellular lineages, and their infection strategies can involve a remarkable level of physiological remodeling of their host cells. Virus–virus communication and widespread forms of hyperparasitism have been shown to be common in the virosphere, demonstrating that dynamic ecological interactions often shape their success. And the evolutionary histories of viruses are often fraught with complexities, with chimeric genomes including genes derived from numerous distinct sources or evolved de novo. Here we will discuss many aspects of this viral complexity, with particular emphasis on large DNA viruses, and provide an outlook for future research.

Foot-and-Mouth Disease Virus Interserotypic Recombination in Superinfected Carrier Cattle.

Viral recombination contributes to the emergence of novel strains with the potential for altered host range, transmissibility, virulence, and immune evasion. For foot-and-mouth disease virus (FMDV), cell culture experiments and phylogenetic analyses of field samples have demonstrated the occurrence of recombination. However, the frequency of recombination and associated virus–host interactions within an infected host have not been determined. We have previously reported the detection of interserotypic recombinant FMDVs in oropharyngeal fluid (OPF) samples of 42% (5/12) of heterologously superinfected FMDV carrier cattle. The present investigation consists of a detailed analysis of the virus populations in these samples including identification and characterization of additional interserotypic minority recombinants. In every animal in which recombination was detected, recombinant viruses were identified in the OPF at the earliest sampling point after superinfection. Some recombinants remained dominant until the end of the experiment, whereas others were outcompeted by parental strains. Genomic analysis of detected recombinants suggests host immune pressure as a major driver of recombinant emergence as all recombinants had capsid-coding regions derived from the superinfecting virus to which the animals did not have detectable antibodies at the time of infection. In vitro analysis of a plaque-purified recombinant virus demonstrated a growth rate comparable to its parental precursors, and measurement of its specific infectivity suggested that the recombinant virus incurred no penalty in packaging its new chimeric genome. These findings have important implications for the potential role of persistently infected carriers in FMDV ecology and the emergence of novel strains.

Factors That Influence the Choice of Markov Model Order in Discriminating DNA Sequences from Different Sources.

Markov models have frequently been used in genetic sequence analysis. The number of parameters of a Markov model increases exponentially with model order, so it is often recommended that the order be chosen based on the size of data being modeled, lower orders for small and higher orders for large dataset sizes. Approaches based on model selection criterion have also been proposed. An important problem in microbiology and evolutionary biology is to decipher chimeric genomes of microbes, particularly, identify segments of distinct ancestries in genomes and reconstruct the plausible evolutionary scenarios that might have shaped the chimeric genomes in the microbial world. In this study, we assessed a Markov model-based segmentation method for its ability to detect compositionally disparate segments in chimeric sequence constructs as a function of model order, sequence length, and phylogenetic divergence. Our results show that the choice of Markov model order depends on both sequence size and composition. Higher order Markov models were found to be more effective in delineating sequence segments arising from closely related organisms in longer constructs; on the other hand, lower order Markov models were found to be more appropriate in delineating sequence segments arising from distantly related organisms in shorter constructs. These findings are important and timely, with broad implications in fields such as epidemiology that has to deal with the emergence of novel pathogenic chimeras that arise by foreign DNA acquisition, and ecology where chimeric structures may arise in various ecosystems, necessitating more robust approaches for their deconstruction and interpretation.

Eukarya the chimera: eukaryotes, a secondary innovation of the two domains of life?

One of the most significant events in the evolution of life is the origin of the eukaryotic cell, an increase in cellular complexity that occurred approximately 2 billion years ago. Ground-breaking research has centered around unraveling the characteristics of the Last Eukaryotic Common Ancestor (LECA) and the nuanced archaeal and bacterial contributions in eukaryogenesis, resulting in fundamental changes in our understanding of the Tree of Life. The archaeal and bacterial roles are covered by theories of endosymbiogenesis wherein an ancestral host archaeon and a bacterial endosymbiont merged to create a new complex cell type - Eukarya - and its mitochondrion. Eukarya is often regarded as a unique and distinct domain due to complex innovations not found in archaea or bacteria, despite housing a chimeric genome containing genes of both archaeal and bacterial origin. However, the discovery of complex cell machineries in recently described Asgard archaeal lineages, and the growing support for diverse bacterial gene transfers prior to and during the time of LECA, is redefining our understanding of eukaryogenesis. Indeed, the uniqueness of Eukarya, as a domain, is challenged. It is likely that many microbial syntrophies, encompassing a 'microbial village', were required to 'raise' a eukaryote during the process of eukaryogenesis.

Disentangle genus microdiversity within a complex microbial community by using a multi-distance long-read binning method: example of Candidatus Accumulibacter.

SummaryComplete genomes can be recovered from metagenomes by assembling and binning DNA sequences into metagenome assembled genomes (MAGs). Yet, the presence of microdiversity can hamper the assembly and binning processes, possibly yielding chimeric, highly fragmented and incomplete genomes. Here, the metagenomes of four samples of aerobic granular sludge bioreactors containing Candidatus (Ca.) Accumulibacter, a phosphate‐accumulating organism of interest for wastewater treatment, were sequenced with both PacBio and Illumina. Different strategies of genome assembly and binning were investigated, including published protocols and a binning procedure adapted to the binning of long contigs (MuLoBiSC). Multiple criteria were considered to select the best strategy for Ca. Accumulibacter, whose multiple strains in every sample represent a challenging microdiversity. In this case, the best strategy relies on long‐read only assembly and a custom binning procedure including MuLoBiSC in metaWRAP. Several high‐quality Ca. Accumulibacter MAGs, including a novel species, were obtained independently from different samples. Comparative genomic analysis showed that MAGs retrieved in different samples harbour genomic rearrangements in addition to accumulation of point mutations. The microdiversity of Ca. Accumulibacter, likely driven by mobile genetic elements, causes major difficulties in recovering MAGs, but it is also a hallmark of the panmictic lifestyle of these bacteria.

When barcoding fails: Genome chimerization (admixing) and reticulation obscure phylogenetic and taxonomic relationships

DNA barcoding is based on the premise that the barcode sequences can distinguish individuals (strains) of different species because their sequence variation between species exceeds that within species. The primary barcodes used in fungal and yeast taxonomy are the ITS segments and the LSU (large subunit) D1/D2 domain of the homogenized multicopy rDNA repeats. The secondary barcodes are conserved segments of protein‐encoding genes, which usually have single copies in haploid genomes. This study shows that the analysis of barcode sequences fails to reconstruct accurate species trees and differentiate species when the organisms have chimeric genomes composed of admixed mosaics of different origins. It is shown that the type strains of 10 species of the pulcherrima clade of the ascomycetous yeast genus Metschnikowia cannot be differentiated with standard barcodes because their intragenomic diversity is comparable to or even higher than the interstrain diversity. The analysis of a large group of genes of the sequenced genomes of the clade and the viability and segregation of the hybrids of ex‐type strains indicate that the high intragenomic barcode differences can be attributed to admixed genome structures. Because of the mosaic structures of the genomes, the rDNA repeats do not form continuous arrays and thus cannot be homogenized. Since the highly diverse ITS and D1/D2 sequences of the type strains form a continuous pool including pseudogenes, the evolution of their rDNA appears to involve reticulation. The secondary barcode sequences and the nonbarcode genes included in the analysis show incongruent phylogenetic relationships among the type strains, which can also be attributed to differences in the phylogenetic histories of the genes.

Proteomic analysis of metabolic pathways supports chloroplast-mitochondria cross-talk in a Cu-limited diatom.

Diatoms are one of the most successful phytoplankton groups in our oceans, being responsible for over 20% of the Earth's photosynthetic productivity. Their chimeric genomes have genes derived from red algae, green algae, bacteria, and heterotrophs, resulting in multiple isoenzymes targeted to different cellular compartments with the potential for differential regulation under nutrient limitation. The resulting interactions between metabolic pathways are not yet fully understood. We previously showed how acclimation to Cu limitation enhanced susceptibility to overreduction of the photosynthetic electron transport chain and its reorganization to favor photoprotection over light harvesting in the oceanic diatom Thalassiosira oceanica (Hippmann et al., 2017, 10.1371/journal.pone.0181753). In order to gain a better understanding of the overall metabolic changes that help alleviate the stress of Cu limitation, we have further analyzed the comprehensive proteomic datasets generated in that study to identify differentially expressed proteins involved in carbon, nitrogen, and oxidative stress‐related metabolic pathways. Metabolic pathway analysis showed integrated responses to Cu limitation. The upregulation of ferredoxin (Fdx) was correlated with upregulation of plastidial Fdx‐dependent isoenzymes involved in nitrogen assimilation as well as enzymes involved in glutathione synthesis, thus suggesting an integration of nitrogen uptake and metabolism with photosynthesis and oxidative stress resistance. The differential expression of glycolytic isoenzymes located in the chloroplast and mitochondria may enable them to channel both excess electrons and/or ATP between these compartments. An additional support for chloroplast–mitochondrial cross‐talk is the increased expression of chloroplast and mitochondrial proteins involved in the proposed malate shunt under Cu limitation.

Semliki Forest Virus Chimeras with Functional Replicase Modules from Related Alphaviruses Survive by Adaptive Mutations in Functionally Important Hot Spots.

Alphaviruses (family Togaviridae) include both human pathogens such as chikungunya virus (CHIKV) and Sindbis virus (SINV) and model viruses such as Semliki Forest virus (SFV). The alphavirus positive-strand RNA genome is translated into nonstructural (ns) polyprotein(s) that are precursors for four nonstructural proteins (nsPs). The three-dimensional structures of nsP2 and the N-terminal 2/3 of nsP3 reveal that these proteins consist of several domains. Cleavage of the ns-polyprotein is performed by the strictly regulated protease activity of the nsP2 region. Processing results in the formation of a replicase complex that can be considered a network of functional modules. These modules work cooperatively and should perform the same task for each alphavirus. To investigate functional interactions between replicase components, we generated chimeras using the SFV genome as a backbone. The functional modules corresponding to different parts of nsP2 and nsP3 were swapped with their counterparts from CHIKV and SINV. Although some chimeras were nonfunctional, viruses harboring the CHIKV N-terminal domain of nsP2 or any domain of nsP3 were viable. Viruses harboring the protease part of nsP2, the full-length nsP2 of CHIKV, or the nsP3 macrodomain of SINV required adaptive mutations for functionality. Seven mutations that considerably improved the infectivity of the corresponding chimeric genomes affected functionally important hot spots recurrently highlighted in previous alphavirus studies. These data indicate that alphaviruses utilize a rather limited set of strategies to survive and adapt. Furthermore, functional analysis revealed that the disturbance of processing was the main defect resulting from chimeric alterations within the ns-polyprotein. IMPORTANCE Alphaviruses cause debilitating symptoms and have caused massive outbreaks. There are currently no approved antivirals or vaccines for treating these infections. Understanding the functions of alphavirus replicase proteins (nsPs) provides valuable information for both antiviral drug and vaccine development. The nsPs of all alphaviruses consist of similar functional modules; however, to what extent these are independent in functionality and thus interchangeable among homologous viruses is largely unknown. Homologous domain swapping was used to study the functioning of modules from nsP2 and nsP3 of other alphaviruses in the context of Semliki Forest virus. Most of the introduced substitutions resulted in defects in the processing of replicase precursors that were typically compensated by adaptive mutations that mapped to determinants of polyprotein processing. Understanding the principles of virus survival strategies and identifying hot spot mutations that permit virus adaptation highlight a route to the rapid development of attenuated viruses as potential live vaccine candidates.