An efficient method for generating poxvirus recombinants in the absence of selection

Abstract

The use of selectable markers (ecogpt) and selection pressures to aid in detection of poxvirus (Vaccinia, VV) recombinants has been implicated in the unintended introduction of second site mutations. We have reinvestigated the use of the helper virus system described by Scheiflinger et al. and adapted by Yao and Evans which produces recombinants at a high frequency in the absence of any selection, at a rate of 6–100%. Our system uses fowlpox virus (FPV) as the infectious helper virus which in infected cells provides the enzymatic apparatus for transcription and replication of a purified, transfected VV genome and for recombination with a second transfected PCR generated DNA fragment. To optimize the system, a PCR DNA fragment was generated that contained poxvirus promoter driven gfp and lacZ genes inserted within the coding sequences of the viral thymidine kinase gene. This PCR fragment was co-transfected together with VV genomic DNA. Recombinant VV was identified by plaquing the mixture on cells non-permissive for FPV and selection of green fluorescent or LacZ positive recombinant vaccinia plaques. The system was optimized using FPV permissive cells (CEF) and non-permissive cells (A549, CV-1) for both the initial infection/transfection and the subsequent selection. Up to 70% of the progeny vaccinia virus contained the gfp/LacZ insertion. In order to test for the presence of FPV/VV intertypic recombinants or other unintended mutations, recombinant wtVV (RwtVV) was regenerated from the gfp/LacZ viruses and evaluated by RFLP analysis and pathogenesis in animals. While all RwtVVs were viable in cell culture, in many of the RwtVV isolates, RFLP differences were noted and while some recombinant viruses exhibited wild type behavior in mice, a wide range of virulence indicative of unintended changes suggests that mutants created by “rescue” systems require careful analysis particularly before use for in vivo studies employing animal models.

Keywords: fowlpox virus; helper virus; poxvirus; recombinant; vaccinia virus.

Figure 1.

Infection/transfection scheme for generation of recombinant vaccinia virus. Cells are infected with fowlpox virus (FPV) and transfected with genomic vaccinia virus (VV) DNA ± a disrupted VV PCR fragment containing the desired mutation (lacZ or gfp). The resulting virus mixture is then plaqued on mammalian cells to eliminate FPV and the desired viruses isolated.

Figure 2.

Optimization of transfection reagent for rescue of infectious vaccinia virus particles. (a) Titers of VV rescued from chicken embryo fibroblast (CEF) cells infected with FPV at two different FPV MOI and transfected with 12 μg VV genomic DNA using varying amounts of transfection reagent Lipofectamine 2000. (b) Crystal Violet staining of the initial infection/transfection mixture plaqued on CV-1 cells. Plaquing on CV-1 cells of VV rescued from CEF cells infected with FPV at an MOI of 25.

Figure 3.

Frequency of VV lacZ/gfp recombinant viruses from optimization of ratio of genomic DNA to gfp/lacZ PCR fragment in CEF cells. Cells were infected with FPV at an MOI of 25 and transfected with a total of 12 μg DNA in the ratios noted in the graph. Error bars represent SEM; n = 10.

Figure 4.

Growth of FPV on permissive CEF cells and non-permissive A549 cells. Cells were infected with FPV at an MOI of 0.001, harvested at 0, 24, 48, 72 and 96 hours post infection, and titered on CEF cells.

Figure 5.

Optimization of transfection reagent for rescue of infectious vaccinia virus particles using A549 cells. A549 cells were infected with FPV at an MOI of 1 or 25 and transfected with genomic VV DNA at varying concentrations of Lipofectamine 2000. (a) Rescue of VV in A549 cells using varying amounts of transfection reagent and 12 μg VV DNA at a low (1) and a high (25) MOI of FPV. Error bars represent SEM. (b) Plaquing stained with crystal violet on CV-1 cells of resulting virus mixture from VV rescued in A549 cells infected with FPV at an MOI of 25.

Figure 6.

Frequency of VV lacZ/gfp recombinant viruses generated from FPV infected A549 cells. A549 cells were infected with an MOI of 25 and transfected with 12 μg of total DNA in the ratios shown using 12 μL of transfection reagent. The percentages of recombinant viruses were determined by staining for the presence of LacZ and counting the number of LacZ positive plaques in relation to the total number of plaques. Error bars represent SEM. N = 4.

Figure 7.

Restriction fragment length polymorphism (RFLP) analysis of rescued wild type VV. The genomes of the viruses noted in the table underwent RFLP analysis as compared to the parental wild type virus. Any changes in banding patterns were noted as a change in that section of the genome. Changes for the five selected isolates are noted by a dark blue shadowing of the box, no shading represents no changes were detected. For comparison purposes the phenotype of the viruses in animals is also shown on the table as previously shown in Table 1.