Efficient virus extinction by combinations of a mutagen and antiviral inhibitors

Abstract

The effect of combinations of the mutagenic base analog 5-fluorouracil (FU) and the antiviral inhibitors guanidine hydrochloride (G) and heparin (H) on the infectivity of foot-and-mouth disease virus (FMDV) in cell culture has been investigated. Related FMDV clones differing up to 10(6)-fold in relative fitness in BHK-21 cells have been compared. Systematic extinction of intermediate fitness virus was attained with a combination of FU and G but not with the mutagen or the inhibitor alone. Systematic extinction of high-fitness FMDV required the combination of FU, G, and H. FMDV showing high relative fitness in BHK-21 cells but decreased replicative ability in CHO cells behaved as a low-fitness virus with regard to extinction mutagenesis in CHO cells. This confirms that relative fitness, rather than a specific genomic sequence, determines the FMDV response to enhanced mutagenesis. Mutant spectrum analysis of several genomic regions from a preextinction population showed a statistically significant increase in the number of mutations compared with virus passaged in parallel in the absence of FU and inhibitors. Also, in a preextinction population the types of mutations that can be attributed to the mutagenic action of FU were significantly more frequent than other mutation types. The results suggest that combinations of mutagenic agents and antiviral inhibitors can effectively drive high-fitness virus into extinction.

FIG. 1

BHK-21 cell viability in the presence of FU, G, and H, alone or in combination. The origin of BHK-21 cells and FMDV C-S8c1, as well as procedures for cell growth, antiviral treatment, quantification of cell viability, and infections with FMDV, are detailed in Materials and Methods. Percent cell viabilities (trypan blue exclusion) were calculated relative to those in parallel, untreated cell cultures, counting 300 to 700 cells per sample. Hours of exposure refers to the time elapsed between the addition of the mutagen or antiviral agent to cells and the determination of cell viability. Standard deviations (not included in the plots) never exceeded 20%.

FIG. 2

Effect of viral fitness on the decrease of infectivity in a single round of infection in the absence or presence of FU, G, or the mixture of both (FUG). FMDV MARLS, C-S8c1, and H clones are described in Materials and Methods. Empty columns indicate viral production in the absence of mutagen or antiviral agent; gray columns indicate viral production in the presence of 200 μg of FU/ml; striped gray columns indicate viral production in the presence of 4 mM G; and black columns show viral production in the presence of FUG. In this experiment 9 × 10⁵ BHK-21 cells were infected with 5 × 10⁴ PFU of virus. Extinction of the five H FUGp1 clones was confirmed by three additional blind passages, in the absence of mutagen and inhibitor with no evidence of infectivity, and no RT-PCR-amplifiable material in the supernatant of the third passage. Yields of H −3, H −4, and H −5 clones in the presence of FU were undetectable (<10 PFU/ml), but virus reemerged after one blind passage in the absence of the mutagen. However, these clones were extinguished upon a second passage in the presence of FU. Titrations were done in triplicate, and standard deviations are indicated. Procedures for chemical mutagenesis, antiviral treatment, and determination of viral infectivity are described in Materials and Methods.

FIG. 3

Serial passages of C-S8c1 in the absence or presence of FU, G, or FUG. In this experiment, 9 × 10⁵ BHK-21 cells were infected with 5 × 10⁴ PFU of FMDV C-S8c1, and subsequent infections were carried out with 0.1 ml of supernatant of the previous passage (the multiplicity of infection at each passage can be calculated from the titers shown in ordinate). The origin of FMDV C-S8c1, conditions for mutagenic and antiviral treatment, and those for determination of FMDV infectivity are described in Materials and Methods. The preextinction population is indicated by an arrow. Viral extinction was confirmed by three additional blind passages in the absence of mutagen and inhibitor with no evidence of infectivity, and no RT-PCR-amplifiable material was found in the supernatant of the third passage. All titrations were done in triplicate. Standard deviations (not included in the plots) never exceeded 15%.

FIG. 4

Infectivity values upon passage of FMDV MARLS in the absence or presence of FU, G, or FUG in BHK-21 and CHO cells. Conditions for mutagenic and antiviral treatments and for determination of FMDV infectivity are detailed in Materials and Methods. Left panel: FMDV MARLS was tested by infecting 9 × 10⁵ BHK-21 cells with 5 × 10⁴ PFU of virus, and the next passages were carried out by infecting cells with 0.1 ml of supernatant from the previous passage. Right panel: 9 × 10⁵ CHO cells were infected with 9 × 10⁵ PFU of FMDV MARLS, and passages were carried out as for the infections of BHK-21 cells. The multiplicity of infection at each passage can be calculated from the titers shown in ordinate. Preextinction populations are indicated by arrows. Viral extinction was ascertained by three additional blind passages in the absence of mutagen and inhibitors, with no evidence of infectivity and RT-PCR-amplifiable material in the supernatant of the third passage. All titrations were done in triplicate. Standard deviations (not included in the plots) never exceeded 15%.