Chimeric Genomes Research Articles

Integrative omics identification, evolutionary and structural analysis of low affinity nitrate transporters in diatoms, diNPFs.

Diatoms are one of the major and most diverse groups of phytoplankton, with chimeric genomes harbouring a combination of genes of bacterial, animal and plant origin. They have developed sophisticated mechanisms to face environmental variations. In marine environments, nutrients concentration shows significant temporal and spatial variability, influencing phytoplankton growth. Among nutrients, nitrogen, present at micromolar levels, is often a limiting resource. Here, we report a comprehensive characterization of the Nitrate Transporter 1/Peptide Transporter Family (NPF) in diatoms, diNPFs. NPFs are well characterized in many organisms where they recognize a broad range of substrates, ranging from short-chained di- and tri-peptides in bacteria, fungi and mammals to a wide variety of molecules including nitrate in higher plants. Scarce information is available for diNPFs. We integrated-omics, phylogenetic, structural and expression analyses, to infer information on their role in diatoms. diNPF genes diverged to produce two distinct clades with strong sequence and structural homology with either bacterial or plant NPFs, with different predicted sub-cellular localization, suggesting that the divergence resulted in functional diversification. Moreover, transcription analysis of diNPF genes under different laboratory and environmental growth conditions suggests that diNPF diversification led to genetic adaptations that might contribute to diatoms ability to flourish in diverse environmental conditions.

Identification of the determinant of tomato yellow leaf curl Kanchanaburi virus infectivity in tomato

Geminiviruses cause devastating diseases in solanaceous crops, with the bipartite begomoviruses tomato yellow leaf curl Kanchanaburi virus (TYLCKaV) and pepper yellow leaf curl Thailand virus (PYLCThV) major threats in Southeast Asia. To determine the molecular mechanism of geminivirus infection, we constructed infectious clones of TYLCKaV and PYLCThV. Both constructs infected Nicotiana benthamiana, but only TYLCKaV could infect Solanum lycopersicum ‘A39’. A genome-swapping of TYLCKaV with PYLCThV revealed the TYLCKaV-B genome segment as the determinant of TYLCKaV infectivity in tomato. We constructed five geminivirus clones with chimeric TYLCKaV-B and PYLCThV-B genome segments to narrow down the region determining TYLCKaV infectivity in tomato. Only chimeric clones carrying the TYLCKaV intergenic region (IR) showed infectivity in S. lycopersicum ‘A39’, indicating that the IR of TYLCKaV-B is essential for TYLCKaV infectivity in tomato. Our results provide a foundation for elucidating the molecular mechanism of geminivirus infection in plants.

Hybridization and the origin of new yeast lineages.

ABSTRACTHybrids originate from the mating of two diverged organisms, resulting in novel lineages that have chimeric genomes. Hybrids may exhibit unique phenotypic traits that are not necessarily intermediate between those present in the progenitors. These unique traits may enable them to thrive in new environments. Many hybrid lineages have been discovered among yeasts in the Saccharomycotina, of which many have industrial or clinical relevance, but this might reflect a bias toward investigating species with relevance to humans. Hybridization has also been proposed to be at the root of the whole-genome duplication in the lineage leading to Saccharomyces cerevisiae. Thus, hybridization seems to have played a prominent role in the evolution of Saccharomycotina yeasts, although it is still unclear how common this evolutionary process has been during the evolution of this and other fungal clades. Similarly, the evolutionary aftermath of hybridization, including implications at the genomic, transcriptional, physiological or ecological levels, remains poorly understood. In this review, I survey recent findings from genomic analysis of yeast hybrids of industrial or clinical relevance, and discuss the evolutionary implications of genomic hybridization for the origin of new lineages, including when such hybridization results in a whole-genome duplication.

Metschnikowia pulcherrima and Related Pulcherrimin-Producing Yeasts: Fuzzy Species Boundaries and Complex Antimicrobial Antagonism.

Yeasts affiliated with the Metschnikowia pulcherrima clade (subclade) of the large ascomycetous genus Metschnikowia frequently turn out to produce the characteristic maroon-red pulcherrimin when tested for pigment production and prove to exert antagonistic effects on many types of microorganisms. The determination of the exact taxonomic position of the strains is hampered by the shortage of distinctive morphological and physiological properties of the species of the clade and the lack of rDNA barcode gaps. The rDNA repeats of the type strains of the species are not homogenized and are assumed to evolve by a birth-and-death mechanism combined with reticulation. The taxonomic division is further hampered by the incomplete biological (reproductive) isolation of the species: certain type strains can be hybridized and genome sequencing revealed chimeric genome structures in certain strains that might have evolved from interspecies hybrids (alloploid genome duplication). Various mechanisms have been proposed for the antimicrobial antagonism. One is related to pulcherrimin production. The diffusible precursor of pulcherrimin, the pulcherriminic acid is secreted by the cells into the environment where it forms the insoluble pulcherrimin with the ferric ions. The lack of free iron caused by the immobilization of ferric ions inhibits the growth of many microorganisms. Recent results of research into the complexity of the taxonomic division of the pulcherrimin-producing Metschnikowia yeasts and the mechanism(s) underlying their antimicrobial antagonism are discussed in this review.

An atypical clinicopathological manifestation of fowlpox virus associated with reticuloendotheliosis virus in commercial laying hen flocks in Brazil.

Fowlpox (FP) is a common epitheliotropic disease in chickens that is usually controlled by live attenuated vaccines. However, there have been some reports of outbreaks of FP in recent years, even in vaccinated flocks, presenting as atypical lesions and feathering abnormalities in chickens. These findings can be associated with fowlpox virus (FPV) with the reticuloendotheliosis virus (REV) integrated into its genome. In the present study, outbreaks of atypical FP were explored in vaccinated commercial laying hen flocks to determine the nature of the causative agent by histopathologic and molecular approaches. FPV and REV were detected and classified into subclade A1 of the genus Avipoxvirus and subtype 3 of REV (REV3), respectively. Additionally, heterogeneous populations of FPV with partial (containing only a remnant long terminal repeat-LTR) or total (all functional genes) integration of REV were identified by heterologous PCRs and detected considering reference integration sites. These results indicate the mechanism of chimeric genome FPV-REV associated with outbreaks and atypical clinicopathological manifestations in commercial laying hens for the first time in Brazil and in South America. In addition, this study demonstrates the emergence of REV integrated in the FPV genome in Brazilian chicken flocks.

MAT heterozygosity and the second sterility barrier in the reproductive isolation of Saccharomyces species

The genetic analysis of large numbers of Saccharomyces cerevisiae × S. uvarum (“cevarum”) and S. kudriavzevii × S. uvarum (“kudvarum”) hybrids in our previous studies revealed that these species are isolated by a postzygotic double-sterility barrier. We proposed a model in which the first barrier is due to the abruption of the meiotic process by the failure of the chromosomes of the subgenomes to pair (and recombine) in meiosis and the second barrier is assumed to be the result of the suppression of mating by allospecific MAT heterozygosity. While the former is analogous to the major mechanism of postzygotic reproductive isolation in plants and animals, the latter seems to be Saccharomyces specific. To bolster the assumed involvement of MAT in the second sterility barrier, we produced synthetic alloploid two-species cevarum and kudvarum hybrids with homo- and heterothallic backgrounds as well as three-species S. cerevisiae × S. kudvarum × S. uvarum (“cekudvarum”) hybrids by mass-mating and examined their MAT loci using species- and cassette-specific primer pairs. We found that the allospecific MAT heterozygosity repressed MAT switching and mating in the hybrids and in the viable but sterile spores produced by the cevarum hybrids that had increased (allotetraploid) genomes. The loss of heterozygosity by meiotic malsegregation of MAT-carrying chromosomes in the latter hybrids broke down the sterility barrier. The resulting spores nullisomic for the S. uvarum chromosome produced vegetative cells capable of MAT switching and conjugation, opening the way for GARMe (Genome Autoreduction in Meiosis), the process that leads to chimeric genomes.

Genetic diversity and genome recombination in Yam mild mosaic virus isolates

Yam mild mosaic virus (YMMV) is prevalent in yams (Dioscorea spp.) worldwide. To gain an insight into the genetic diversity and molecular evolution of YMMV, 89 isolates from West Africa, Asia, South Pacific, and America were analyzed phylogenetically by using sequence between the 3′-terminal of the coding region of the coat protein and the 5′-terminal of non-coding region at the 3′ end of the YMMV genome. The results revealed that there was a significant genetic diversity among isolates and a clear correlation between the coat protein gene sequence and the geographical origin of YMMV isolates. Of particular, YMMV isolates from North China and South China fell into two different groups. Furthermore, full genome comparison identified four chimeric genome patterns and six putative recombination signals representing four recombination events were detected among 12 genomes of independent isolates from China and Brazil, suggesting a high frequency of genome recombination event.

Rapid and precise genome editing in a marine diatom, Thalassiosira pseudonana by Cas9 nickase (D10A)

Diatoms are dominant phytoplankton accounting for up to 20% of annual global primary production. Diatoms are also one of the first model organisms amongst secondary endosymbionts, which have a highly chimeric genome structure accommodating genes from multiple endosymbiosis partners in their evolutionary history. Despite their ecological importance and evolutionary uniqueness, molecular-level studies have lagged behind other model organisms, hindered by difficulties in controlling their ploidy and homologous recombination system. Recently, genome editing tools, such as clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 and transcription activator-like effector nucleases (TALEN), have been established in some diatoms. However, the improvement of CRISPR/Cas9 precision technology in diatoms is still underway, and the experimental complexity of TALEN limits their application in diatom molecular genetics. Cas9 nickase, a mutated version of Cas9 with a paired-single guide RNAs (sgRNAs), was recently developed to introduce a precise double cut of target DNA, suppressing the off-target effects of native Cas9 nuclease. In this study, we developed the Cas9 nickase (D10A) system for genome editing of the marine diatom Thalassiosira pseudonana. Using this system, we successfully introduced mutations into a gene encoding a putative θ-type carbonic anhydrase, a recently discovered class of carbonic anhydrase widespread in Stramenopiles. The results showed that this system introduced precise and relatively short biallelic indels to the diatom genome with minimal off-target effects.

Nutritional geometry of mitochondrial genetic effects on male fertility.

Organismal fitness is partly determined by how well the nutritional intake matches sex-specific metabolic requirements. Metabolism itself is underpinned by complex genomic interactions involving products from both nuclear and mitochondrial genomes. Products from these two genomes must coordinate how nutrients are extracted, used and recycled, processes vital for fuelling reproduction. Given the complicated nature of metabolism, it is not well understood how the functioning of these two genomes is modulated by nutrients. Here we use nutritional geometry techniques on Drosophila lines that only differ in their mtDNA, with the aim to understand if there is nutrient-dependent mitochondrial genetic variance for male reproduction. We first find genetic variance for diet consumption, indicating that flies are consuming different amounts of food to meet new physiological requirements. We then find an interaction between mtDNA and diet for fitness, suggesting that the mtDNA plays a role in modulating diet-dependent fitness. Our results enhance our basic understanding of nutritional health and our chimeric genomes.

Chlamydia trachomatis: when the virulence-associated genome backbone imports a prevalence-associated major antigen signature.

Chlamydia trachomatis is the most prevalent sexually transmitted bacterium worldwide and the causative agent of trachoma. Its strains are classified according to their ompA genotypes, which are strongly linked to differential tissue tropism and disease outcomes [ocular disease, urogenital disease and lymphogranuloma venereum (LGV)]. While the genome-based species phylogenetic tree presents four main clades correlating with tropism/prevalence, namely ocular, LGV, urogenital T1 (more prevalent genotypes) and urogenital T2 (less prevalent genotypes), inter-clade exchange of ompA is considered a rare phenomenon probably mediating marked tropism alterations. An LGV epidemic, associated with the clonal expansion of the L2b genotype, has emerged in the last few decades, raising concerns particularly due to its atypical clinical presentation (ulcerative proctitis) and circulation among men who have sex with men (MSM). Here, we report an LGV outbreak, mostly affecting human immunodeficiency virus-positive MSM engaging in high-risk sexual practices, caused by an L2b strain with a rather unique non-LGV ompA signature that precluded the laboratory notification of this outbreak as LGV. C. trachomatis whole-genome capture and sequencing directly from clinical samples was applied to deeply characterize the genomic backbone of this novel LGV outbreak-causing clone. It revealed a chimeric genome structure due to the genetic transfer of ompA and four neighbouring genes from a serovar D/Da strain, likely possessing the genomic backbone associated with the more prevalent urogenital genotypes (T1 clade), to an LGV (L2b) strain. The hybrid L2b/D-Da strain presents the adhesin and immunodominant antigen MOMP (major outer membrane protein) (encoded by ompA) with an epitope repertoire typical of non-invasive genital strains, while keeping the genome-dispersed virulence fingerprint of a classical LGV strain. As previously reported for inter-clade ompA exchange among non-LGV clades, this novel C. trachomatis genomic mosaic involving a contemporary epidemiologically and clinically relevant LGV strain may have implications on its transmission, tissue tropism and pathogenic capabilities. The emergence of variants with epidemic and pathogenic potential highlights the need for more focused surveillance strategies to capture C. trachomatis evolution in action.

Molecular, serological and biological characterization of a novel Apple stem pitting virus strain from a local pear variety grown in China

Apple stem pitting virus (ASPV) is an important causal agent of pear diseases. Nowadays, the infection status and molecular characteristics of the virus in old pear trees have never been investigated. In this study, we provide the first complete genome sequence of an ASPV isolate LYC from an over 300-year-old tree of a local Pyrus bretschneideri cultivar ‘Chili’ specifically grown at Laiyang area in China. ASPV-LYC possesses a chimeric genome consisting of 9 273 nucleotides excluding a poly(A) tail at its 3’ end and harboring a recombination region in its open reading frame (ORF1) with Aurora-1 and KL9 identified as the major and minor parents. Western blot analysis with antisera against recombinant coat proteins (CPs) of three ASPV isolates from pear indicates that ASPV-LYC is serologically related to these ASPV isolates, but with differential activities. Further biological tests on indicator plants of Pyronia veitchii show that ASPV-LYC can induce serious leaf and stem symptoms as other ASPV isolates. The results provide an important information for understanding molecular evolution of ASPV and suggest a need to prevent dissemination of the isolate among pear trees.

Vector competence is strongly affected by a small deletion or point mutations in bluetongue virus

BackgroundTransmission of vector-borne virus by insects is a complex mechanism consisting of many different processes; viremia in the host, uptake, infection and dissemination in the vector, and delivery of virus during blood-feeding leading to infection of the susceptible host. Bluetongue virus (BTV) is the prototype vector-borne orbivirus (family Reoviridae). BTV serotypes 1–24 (typical BTVs) are transmitted by competent biting Culicoides midges and replicate in mammalian (BSR) and midge (KC) cells. Previously, we showed that genome segment 10 (S10) encoding NS3/NS3a protein is required for virus propagation in midges. BTV serotypes 25–27 (atypical BTVs) do not replicate in KC cells. Several distinct BTV26 genome segments cause this so-called ‘differential virus replication’ in vitro.MethodsVirus strains were generated using reverse genetics and their growth was examined in vitro. The midge feeding model has been developed to study infection, replication and disseminations of virus in vivo. A laboratory colony of C. sonorensis, a known competent BTV vector, was fed or injected with BTV variants and propagation in the midge was examined using PCR testing. Crossing of the midgut infection barrier was examined by separate testing of midge heads and bodies.ResultsA 100 nl blood meal containing ±105.3 TCID50/ml of BTV11 which corresponds to ±20 TCID50 infected 50% of fully engorged midges, and is named one Midge Alimentary Infective Dose (MAID50). BTV11 with a small in-frame deletion in S10 infected blood-fed midge midguts but virus release from the midgut into the haemolymph was blocked. BTV11 with S1[VP1] of BTV26 could be adapted to virus growth in KC cells, and contained mutations subdivided into ‘corrections’ of the chimeric genome constellation and mutations associated with adaptation to KC cells. In particular one amino acid mutation in outer shell protein VP2 overcomes differential virus replication in vitro and in vivo.ConclusionSmall changes in NS3/NS3a or in the outer shell protein VP2 strongly affect virus propagation in midges and thus vector competence. Therefore, spread of disease by competent Culicoides midges can strongly differ for very closely related viruses.

Yeast two- and three-species hybrids and high-sugar fermentation.

SummaryThe dominating strains of most sugar‐based natural and industrial fermentations either belong to Saccharomyces cerevisiae and Saccharomyces uvarum or are their chimeric derivatives. Osmotolerance is an essential trait of these strains for industrial applications in which typically high concentrations of sugars are used. As the ability of the cells to cope with the hyperosmotic stress is under polygenic control, significant improvement can be expected from concerted modification of the activity of multiple genes or from creating new genomes harbouring positive alleles of strains of two or more species. In this review, the application of the methods of intergeneric and interspecies hybridization to fitness improvement of strains used under high‐sugar fermentation conditions is discussed. By protoplast fusion and heterospecific mating, hybrids can be obtained that outperform the parental strains in certain technological parameters including osmotolerance. Spontaneous postzygotic genome evolution during mitotic propagation (GARMi) and meiosis after the breakdown of the sterility barrier by loss of MAT heterozygosity (GARMe) can be exploited for further improvement. Both processes result in derivatives of chimeric genomes, some of which can be superior both to the parental strains and to the hybrid. Three‐species hybridization represents further perspectives.

InFusion Cloning for the Generation of Biologically Relevant HCV Chimeric Molecular Clones.

This chapter describes how to generate chimeric molecular cassettes that are ready to receive PCR-amplified E1/E2 genes using new DNA cloning technology. The method is divided into three sections: (1) generation of a ΔCore-NS2 cassette based upon the full-length JFH-1 molecular clone; (2) insertion of a "structural gene" fragment encoding the Core, p7, and NS2 genes of a given genotype reference sequence, to generate a ΔE1/E2 cassette; and (3) insertion of patient-isolated E1/E2 genes that are genotype-matched to the structural genes. The final assembled chimeric genomes can then be analyzed in the HCV cell culture system. These cassettes allow characterization of the extensive in vivo viral diversity without the need to isolate and clone whole virus genomes. This method can be readily applied to the study of other HCV genes and other viruses.

Interspecies Hybridisation and Genome Chimerisation in Saccharomyces: Combining of Gene Pools of Species and Its Biotechnological Perspectives.

Over the last one and a half decade, interspecies hybridisation has gained continuously increasing attention as a breeding technique suitable for transferring of genetic information between Saccharomyces species and mixing of their gene pools without genetic engineering. The hybrids frequently show positive transgressive phenotypes. Segregation of the hybrid genome results in mosaic (chimeric) strains that can outperform both the parents and the hybrids or exhibit novel positive phenotypic properties. Mitotic segregation can take place during the vegetative propagation of the sterile allodiploid hybrid cells. Meiotic segregation becomes possible after genome duplication (tetraploidisation) if it is followed by break-down of sterility. The allotetraploid cells are seemingly fertile because they form viable spores. But because of the autodiploidisation of the meiosis, sterile allodiploid spores are produced and thus the hybrid genome does not segregate (the second sterility barrier). However, malsegregation of MAT-carrying chromosomes in one of the subgenomes during allotetraploid meiosis (loss of MAT heterozygosity) results in fertile alloaneuploid spores. The breakdown of (the second) sterility barrier is followed by the loss of additional chromosomes in rapid succession and recombination between the subgenomes. The process (genome autoreduction in meiosis or GARMe) chimerises the genome and generates strains with chimeric (mosaic) genomes composed of various combinations of the genes of the parental strains. Since one of the subgenomes is preferentially reduced, the outcome is usually a strain having an (almost) complete genome from one parent and only a few genes or mosaics from the genome of the other parent. The fertility of the spores produced during GARMe provides possibilities also for introgressive backcrossing with one or the other parental strain, but genome chimerisation and gene transfer through series of backcrosses always with the same parent is likely to be less efficient than through meiotic or mitotic genome autoreduction. Hybridisation and the evolution of the hybrid genome (resizing and chimerisation) have been exploited in the improvement of industrial strains and applied to the breeding of new strains for specific purposes. Lists of successful projects are shown and certain major trends are discussed.

Whole genome engineering by synthesis.

Whole genome engineering is now feasible with the aid of genome editing and synthesis tools. Synthesizing a genome from scratch allows modifications of the genomic structure and function to an extent that was hitherto not possible, which will finally lead to new insights into the basic principles of life and enable valuable applications. With several recent genome synthesis projects as examples, the technical details to synthesize a genome and applications of synthetic genome are addressed in this perspective. A series of ongoing or future synthetic genomics projects, including the different genomes to be synthesized in GP-write, synthetic minimal genome, massively recoded genome, chimeric genome and synthetic genome with expanded genetic alphabet, are also discussed here with a special focus on theoretical and technical impediments in the design and synthesis process. Synthetic genomics will become a commonplace to engineer pathways and genomes according to arbitrary sets of design principles with the development of high-efficient, low-cost genome synthesis and assembly technologies.

Genetics and Genomics of an Unusual Selfish Sex Ratio Distortion in an Insect

Diverse selfish genetic elements have evolved the ability to manipulate reproduction to increase their transmission, and this can result in highly distorted sex ratios [1]. Indeed, one of the major explanations for why sex determination systems are so dynamic is because they are shaped by ongoing coevolutionary arms races between sex-ratio-distorting elements and the rest of the genome [2]. Here, we use genetic crosses and genome analysis to describe an unusual sex ratio distortion with striking consequences on genome organization in a booklouse species, Liposcelis sp. (Insecta: Psocodea), in which two types of females coexist. Distorter females never produce sons but must mate with males (the sons of nondistorting females) to reproduce [3]. Although they are diploid and express the genes inherited from their fathers in somatic tissues, distorter females only ever transmit genes inherited from their mothers. As a result, distorter females have unusual chimeric genomes, with distorter-restricted chromosomes diverging from their nondistorting counterparts and exhibiting features of a giant non-recombining sex chromosome. The distorter-restricted genome has also acquired a gene from the bacterium Wolbachia, a well-known insect reproductive manipulator; we found that this gene has independently colonized the genomes of two other insect species with unusual reproductive systems, suggesting possible roles in sex ratio distortion in this remarkable genetic system.

It's in the mix: Reassortment of segmented viral genomes.

Segmentation of viral genomes allows exchange of intact genes between related viruses when they coinfect the same cell (Fig 1). This exchange is a type of recombination called reassortment. Classical recombination involves the joining of nucleic acid sequences derived from two different templates into one chimeric product. During reassortment, however, entire gene segments are swapped to give rise to chimeric genomes. In both cases, novel genotypes are formed, giving the potential for viruses to evolve. As with genetic change through mutation, most reassortment events yield progeny viruses that are less fit than either parent (Fig 2). Occasionally, however, reassortment gives rise to a combination of genes particularly well suited to a given set of selection pressures, and increased fitness results. Open in a separate window Fig 1 Reassortment requires viruses to meet on multiple scales. For reassortment to occur between viruses of two distinct genotypes, these viruses must infect the same host (A) and the same tissue within that host (B). Either the inoculating viruses or their progeny must come together within the same cell (C). Finally, the coinfecting viral genomes must mix within the coinfected cell, and replicated segments must be copackaged, processes which may be limited by compartmentalization of viral replication and selectivity of genome incorporation, respectively (D). When all of these criteria are met, progeny viruses of both reassortant and parental viral genotypes will emerge from the cell (E).

Bacterial Genome Containing Chimeric DNA-RNA Sequences.

Almost five decades ago Crick, Orgel, and others proposed the RNA world hypothesis. Subsequent studies have raised the possibility that RNA might be able to support both genotype and phenotype, and the function of RNA templates has been studied in terms of evolution, replication, and catalysis. Recently, we engineered strains of E. coli in which a large fraction of 2'-deoxycytidine in the genome is substituted with the modified base 5-hydroxymethyl-2'-deoxycytidine. We now report the generation of mutant strains derived from these engineered bacteria that show significant (∼40-50%) ribonucleotide content in their genome. We have begun to characterize the properties of these chimeric genomes and the corresponding strains to determine the circumstances under which E. coli can incorporate ribonucleotides into its genome and herein report our initial observations.

Vaccinia Virus Shuffling: deVV5, a Novel Chimeric Poxvirus with Improved Oncolytic Potency.

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. In this study, we used a directed evolution process, pooling different strains of VACV, including Copenhagen, Western Reserve and Wyeth strains and the attenuated modified vaccinia virus Ankara (MVA), to generate a new recombinant poxvirus with increased oncolytic properties. Through selective pressure, a chimeric VACV, deVV5, with increased cancer cell killing capacity and tumor selectivity in vitro was derived. The chimeric viral genome contains sequences of all parental strains. To further improve the tumor selectivity and anti-tumor activity of deVV5, we generated a thymidine kinase (TK)-deleted chimeric virus armed with the suicide gene FCU1. This TK-deleted virus, deVV5-fcu1 replicated efficiently in human tumor cells, and was notably attenuated in normal primary cells. These studies demonstrate the potential of directed evolution as an efficient way to generate recombinant poxviruses with increased oncolytic potency, and with high therapeutic index to improve cancer therapy.