Intragenic and intergenic recombination between temperature-sensitive mutants of vaccinia virus

Fathi, Zahra; Dyster, Lyn M.; Seto, Janny; Condit, Richard C.; Niles, Edward G.

doi:10.1099/0022-1317-72-11-2733

Cited by 19 publications

(11 citation statements)

References 30 publications

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“…Thus, while the mean length of a conversion track was 12 kbp, the median was only 2.6 kbp. The abundance of short conversion tracks would help favor intragenic recombination events, which can be detected between markers spaced only 54 bp apart (34). We should note that this estimate of the recombination frequency is lower than has been previously reported.…”

Section: Resultsmentioning

confidence: 65%

Genome Scale Patterns of Recombination between Coinfecting Vaccinia Viruses

Qin

Evans

2014

J Virol

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Recombination plays a critical role in virus evolution. It helps avoid genetic decline and creates novel phenotypes. This promotes survival, and genome sequencing suggests that recombination has facilitated the evolution of human pathogens, including orthopoxviruses such as variola virus. Recombination can also be used to map genes, but although recombinant poxviruses are easily produced in culture, classical attempts to map the vaccinia virus (VACV) genome this way met with little success. We have sequenced recombinants formed when VACV strains TianTan and Dryvax are crossed under different conditions. These were a single round of growth in coinfected cells, five rounds of sequential passage, or recombinants obtained using leporipoxvirusmediated DNA reactivation. Our studies showed that recombinants contain a patchwork of DNA, with the number of exchanges increasing with passage. Further passage also selected for TianTan DNA and correlated with increased plaque size. The recombinants produced through a single round of coinfection contain a disproportionate number of short conversion tracks (<1 kbp) and exhibited 1 exchange per 12 kbp, close to the ϳ1 per 8 kbp in the literature. One by-product of this study was that rare mutations were also detected; VACV replication produces ϳ1 ؋ 10 ؊8 mutation per nucleotide copied per cycle of replication and ϳ1 large (21 kbp) deletion per 70 rounds of passage. Viruses produced using DNA reactivation appeared no different from recombinants produced using ordinary methods. An attractive feature of this approach is that when it is combined with selection for a particular phenotype, it provides a way of mapping and dissecting more complex virus traits. IMPORTANCEWhen two closely related viruses coinfect the same cell, they can swap genetic information through a process called recombination. Recombination produces new viruses bearing different combinations of genes, and it plays an important role in virus evolution. Poxviruses are a family of viruses that includes variola (or smallpox) virus, and although poxviruses are known to recombine, no one has previously mapped the patterns of DNAs exchanged between viruses. We coinfected cells with two different vaccinia poxviruses, isolated the progeny, and sequenced them. We show that poxvirus recombination is a very accurate process that assembles viruses containing DNA copied from both parents. In a single round of infection, DNA is swapped back and forth ϳ18 times per genome to make recombinant viruses that are a mosaic of the two parental DNAs. This mixes many different genes in complex combinations and illustrates how recombination can produce viruses with greatly altered disease potential.

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Section: Resultsmentioning

confidence: 65%

Genome Scale Patterns of Recombination between Coinfecting Vaccinia Viruses

Qin

Evans

2014

J Virol

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“…In these cases, the distance error (blue and black horizontal error bars) was estimated from the size(s) of the restriction fragment(s) in which the mutation(s) resides (see Materials and Methods). Fathi et al reported actual standard deviations for each of the R f determinations (red-colored points with vertical error bars) (11). The error in R f is unknown for most of the individual crosses reported by Ensinger (7)(8)(9), but as a guide we show an estimated average absolute error of ϳ4% based upon what was reported for a subset of R f measurements (7).…”

mentioning

confidence: 69%

“…These data were culled from the published literature and updated (where possible) to include estimates of the marker positions on the VAC genome. These data represent many different crosses between temperature-sensitive markers and span distances up to about onethird of the VAC genome (8,9,11,16,28). Two features of these data are immediately obvious.…”

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confidence: 99%

“…Meta-analysis of VAC recombination frequencies versus distance data. These data were compiled from reports of intergenic crosses involving VAC-encoded temperature-sensitive mutations (7)(8)(9)11). All of these studies used similar experimental conditions (a 1:1 ratio of each virus at a combined multiplicity of infection of 10) and spanned distances ranging up to ϳ70 kbp.…”

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confidence: 99%

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Vaccinia Virus Particles Mix Inefficiently, and in a Way That Would Restrict Viral Recombination, in Coinfected Cells

Lin

Evans

2010

J Virol

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It is well established that poxviruses are subjected to genetic recombination, but attempts to map vaccinia virus genes using classical genetic crosses were historically confounded by high levels of experimental noise and a poor correlation between physical and genetic map distances. These virus-by-virus crosses also never produced the 50% recombinant progeny that should be seen in experiments involving distant markers. Poxviruses replicate in membrane-wrapped cytoplasmic structures called virosomes (or factories) and we have developed a method for tracking the development of these structures using live cell imaging and cells expressing phage lambda Cro protein fused to enhanced green fluorescent protein (EGFP). The EGFP-cro protein binds nonspecifically to DNA and permits live cell imaging of developing vaccinia virus factories. Using this method, we see virosomes first appearing about 4 to 5 h postinfection. The early virosomes exhibit a compact appearance and then, after a period of exponential growth lasting several hours, blur and start to dissipate in a process presumably linked to viral packaging. During the growth period, the virosomes migrate toward the nuclear periphery while colliding and fusing at a rate dependent upon the numbers of infecting particles. However, even at high multiplicities of infection (10 PFU/cell), we estimate ϳ20% of the virosomes never fuse. We have also used fluorescence in situ hybridization (FISH) methods to study virosomes formed by the fusion of viruses carrying different gene markers. FISH showed that DNA mixes rather poorly within fused virosomes and the amount of mixing is inversely dependent on the time between virosome appearance and fusion. Our studies suggest that the intracellular movement and mixing of virosomes create constraints that reduce opportunities for forming recombinants and that these phenomena create outcomes reflected in classical poxvirus genetics.Genetic recombination catalyzes the acquisition of new traits and plays a key role driving virus adaption to new hosts and ecological niches. It can also promote the reassortment of antigenic determinants and facilitate DNA repair and thus aids virus survival in response to evolving immune surveillance and other environmental hazards. Poxviruses provide several classic illustrations of the impact of recombination on viral evolution. For example, the attenuated South American form of variola virus (alastrim or variola minor) is probably a hybrid virus derived from recombination between the more virulent West African and Asian variola strains (10). Malignant rabbit virus is another example of a recombinant virus found in a facility propagating myxoma and Shope fibroma viruses (2). Genome analysis suggests that poxviruses can also slowly accrete homologs of host genes over long periods of evolutionary time. This process selects for acquisition of virulence factors, and each of the poxvirus genera carries a characteristic complement of such genes (23).Fenner provided the first experimental demonstration of vacci...

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“…Further analysis of CPXV strain Germany 1998_2 showed that genome segments clustered with either the CPXV-like 1 or the CPXV-like 2 clade, whereas for the CPXV isolate HumLit08/1 similarities to both, the VARV-like and VACV-like clade, were observed. Recombination within OPV has, so far, been described in vivo [44,45] and in vitro [46,47,48,49], and might explain the observed mosaic genomes. However, a retrospective analysis of recombination is challenging when only limited numbers of sequences are available and the underlying phylogeny is not characterized in detail.…”

Section: Discussionmentioning

confidence: 99%

Classification of Cowpox Viruses into Several Distinct Clades and Identification of a Novel Lineage

Franke

Pfaff

Jenckel

et al. 2017

Viruses

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Cowpox virus (CPXV) was considered as uniform species within the genus Orthopoxvirus (OPV). Previous phylogenetic analysis indicated that CPXV is polyphyletic and isolates may cluster into different clades with two of these clades showing genetic similarities to either variola (VARV) or vaccinia viruses (VACV). Further analyses were initiated to assess both the genetic diversity and the evolutionary background of circulating CPXVs. Here we report the full-length sequences of 20 CPXV strains isolated from different animal species and humans in Germany. A phylogenetic analysis of altogether 83 full-length OPV genomes confirmed the polyphyletic character of the species CPXV and suggested at least four different clades. The German isolates from this study mainly clustered into two CPXV-like clades, and VARV- and VACV-like strains were not observed. A single strain, isolated from a cotton-top tamarin, clustered distantly from all other CPXVs and might represent a novel and unique evolutionary lineage. The classification of CPXV strains into clades roughly followed their geographic origin, with the highest clade diversity so far observed for Germany. Furthermore, we found evidence for recombination between OPV clades without significant disruption of the observed clustering. In conclusion, this analysis markedly expands the number of available CPXV full-length sequences and confirms the co-circulation of several CPXV clades in Germany, and provides the first data about a new evolutionary CPXV lineage.

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Intragenic and intergenic recombination between temperature-sensitive mutants of vaccinia virus

Cited by 19 publications

References 30 publications

Genome Scale Patterns of Recombination between Coinfecting Vaccinia Viruses

Genome Scale Patterns of Recombination between Coinfecting Vaccinia Viruses

Vaccinia Virus Particles Mix Inefficiently, and in a Way That Would Restrict Viral Recombination, in Coinfected Cells

Classification of Cowpox Viruses into Several Distinct Clades and Identification of a Novel Lineage

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