Homologous recombination is the proposed mechanism whereby the DNA virus human herpesvirus-6 integrates into human telomeres.

When two or more viruses, each containing lethal genomic damage, infect the same host cell, the virus genomes can often pair with each other and undergo homologous recombinational repair to produce viable progeny. This process, known as multiplicity reactivation, has been studied in several bacteriophages, including phage T4. Enzymes employed in recombinational repair in phage T4 are functionally homologous to enzymes employed in bacterial and eukaryotic recombinational repair. In particular, with regard to a gene necessary for the strand exchange reaction, a key step in homologous recombinational repair, there is functional homology from viruses to humans (i. e. uvsX in phage T4; recA in E. coli and other bacteria, and rad51 and dmc1 in yeast and other eukaryotes, including humans). Multiplicity reactivation has also been demonstrated in numerous pathogenic viruses.

This is definitely time consuming but may or may not be difficult depending on the virus and what you're trying to recombine in. We may need some more information about what virus and what type of mutation you're making. You would really need to then see the latest methods that people are publishing for that virus. I don't know either if you're talking about homologous recombination inside a transfected permissive cell or BAC recombineering of the viral genome inside and E. coli host.

For recombination inside cells, if you have a highly transfectable permissive cell line and linearized plasmid with sufficient homology ends, then ease of obtaining your recombinant comes down to your positive selection method (like a drug marker) and a selection marker (like EGFP). By transfecting or electroporating in a linearized recombination plasmid with an EGFP marker and then the next day infecting the cells with the wt virus, it can be fairly straight forward to take the infectious supernatant (if applicable) from that transfection-infection to new cell monolayers--using different dilutions of sup on different dishes--and then identifying EGFP+ cells or plaques that likely contain the marker with the mutation of interest. The efficiency of recombination can be increased by co-transfecting with the plasmid and infectious, purified DNA from your virus such that the plasmid can be in a 10:1 molar ratio with the viral genome, increasing the likelihood that a cell that is transfected with viral genome also has plasmid to recombine with. By using a drug selection, too, you can go through rounds of infection and selection to enrich for your recombinants.

In either case, the next time-consuming part is to purify single clones of your virus away from the wt and other recombinants, the latter of which may have differences in the recombination region or secondary mutations. If you can infect monolayers by limiting dilution, you may be able to produce a well with a single, marker-positive plaque or infectious center. Even if there are wt or marker negative plaques, then aspirating the marker positive cells through an overlay, triturating that sample in media, and then reinfecting new monolayers can produce pure clones in about 3 rounds of isolation. If you're having to monitor the presence of wt virus by PCR of the infected cell wells, then that's a hassle but I've done that too.

You'll need to then analyze your recombinants' genomes to confirm that the recombination went as planned. If you can sequence the whole viral genome, you can look for secondary mutations that occurred as well and use the clone that has only the mutation you introduced.

The major caveats with this method is that mutations can be lethal and you wouldn't be able to isolate the recombinant to purity--it would only grow with wt helper virus co-purifying with it. I've had that happen as well as the inability to isolate a recombinant virus likely because it couldn't replicate with the transgene that I was trying to introduce since the control recombination worked. Also, some genome regions might be sensitive to introduction of any markers because nearby transcripts may be affected, so it's important to know exactly where you're going with your recombination. Hope that helps.

omologous recombination is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. It is most widely used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double-strand breaks (DSB). Homologous recombination also produces new combinations of DNA sequences during meiosis, the process by which eukaryotes make gamete cells, like sperm and egg cells in animals. These new combinations of DNA represent genetic variation in offspring, which in turn enables populations to adapt during the course of evolution. Homologous recombination is also used in horizontal gene transfer to exchange genetic material between different strains and species of bacteria and viruses.

Although homologous recombination varies widely among different organisms and cell types, most forms involve the same basic steps. After a double-strand break occurs, sections of DNA around the 5' ends of the break are cut away in a process called resection. In the strand invasion step that follows, an overhanging 3' end of the broken DNA molecule then "invades" a similar or identical DNA molecule that is not broken. After strand invasion, the further sequence of events may follow either of two main pathways discussed below (see Models); the DSBR (double-strand break repair) pathway or the SDSA (synthesis-dependent strand annealing) pathway. Homologous recombination that occurs during DNA repair tends to result in non-crossover products, in effect restoring the damaged DNA molecule as it existed before the double-strand break.

Homologous recombination is conserved across all three domains of life as well as viruses, suggesting that it is a nearly universal biological mechanism. The discovery of genes for homologous recombination in protists—a diverse group of eukaryotic microorganisms—has been interpreted as evidence that meiosis emerged early in the evolution of eukaryotes. Since their dysfunction has been strongly associated with increased susceptibility to several types of cancer, the proteins that facilitate homologous recombination are topics of active research. Homologous recombination is also used in gene targeting, a technique for introducing genetic changes into target organisms. For their development of this technique, Mario Capecchi, Martin Evans and Oliver Smithies were awarded the 2007 Nobel Prize for Physiology or Medicine; Capecchi[2] and Smithies[3] independently discovered applications to mouse embryonic stem cells, however the highly conserved mechanisms underlying the DSB repair model, including uniform homologous integration of transformed DNA (gene therapy), were first shown in plasmid experiments by Orr-Weaver, Szostack and Rothstein. Researching the plasmid-induced DSB, using γ-irradiation in the 1970s-1980s, led to later experiments using endonucleases (e.g. I-SceI) to cut chromosomes for genetic engineering of mammalian cells, where nonhomologous recombination is more frequent than in yeas

regards

Homologous recombination occurs in several groups of viruses. In DNA viruses such as herpesvirus, recombination occurs through a break-and-rejoin mechanism like in bacteria and eukaryotes. There is also evidence for recombination in some RNA viruses, specifically positive-sense ssRNA viruses like retroviruses, picornaviruses, and coronaviruses. There is controversy over whether homologous recombination occurs in negative-sense ssRNA viruses like influenza.

In RNA viruses, homologous recombination can be either precise or imprecise. In the precise type of RNA-RNA recombination, there is no difference between the two parental RNA sequences and the resulting crossover RNA region. Because of this, it is often difficult to determine the location of crossover events between two recombining RNA sequences. In imprecise RNA homologous recombination, the crossover region has some difference with the parental RNA sequences – caused by either addition, deletion, or other modification of nucleotides. The level of precision in crossover is controlled by the sequence context of the two recombining strands of RNA: sequences rich in adenine and uracil decrease crossover precision.

Homologous recombination is important in facilitating viral evolution. For example, if the genomes of two viruses with different disadvantageous mutations undergo recombination, then they may be able to regenerate a fully functional genome. Alternatively, if two similar viruses have infected the same host cell, homologous recombination can allow those two viruses to swap genes and thereby evolve more potent variations of themselves.

Homologous recombination is the proposed mechanism whereby the DNA virus human herpesvirus-6 integrates into human telomeres.

When two or more viruses, each containing lethal genomic damage, infect the same host cell, the virus genomes can often pair with each other and undergo homologous recombinational repair to produce viable progeny. This process, known as multiplicity reactivation, has been studied in several bacteriophages, including phage T4. Enzymes employed in recombinational repair in phage T4 are functionally homologous to enzymes employed in bacterial and eukaryotic recombinational repair. In particular, with regard to a gene necessary for the strand exchange reaction, a key step in homologous recombinational repair, there is functional homology from viruses to humans (i. e. uvsX in phage T4; recA in E. coli and other bacteria, and rad51 and dmc1 in yeast and other eukaryotes, including humans). Multiplicity reactivation has also been demonstrated in numerous pathogenic viruses.