Persistence of foot-and-mouth disease virus in cell culture revisited: implications for contingency in evolution

Abstract

If we could rewind the tape of evolution and play it again, would it turn out to be similar to or different from what we know? Obviously, this key question can only be addressed by fragmentary experimental approaches. Twenty-two years ago, we described the establishment of BHK-21 cells persistently infected with foot-and-mouth disease virus (FMDV), a system that displayed as its major biological feature a coevolution of the cells and the resident virus in the course of persistence. Now we report the establishment of two persistently infected cell lines in parallel, starting with the same clones of FMDV and BHK-21 cells used 22 years ago. We have asked whether the evolution of the two newly established cell lines and of the earlier cell line would be similar or different. The main conclusions of the study are: (i) the basic behaviour characterized by virus-cell coevolution is similar in the three carrier cell lines, despite differences in some genetic alterations of FMDV; (ii) a strikingly parallel behaviour has been observed with the two newly established cell lines passaged in parallel, unveiling a deterministic virus behaviour during persistence; and (iii) selective RT-PCR amplifications have detected imbalances in the proportion of positive-versus negative-strand viral RNA, mediated by both viral and cellular factors. The results confirm coevolution of cells and virus as a major and reproducible feature of FMDV persistence in cell culture, and suggest that rapidly evolving viruses may constitute adequate test systems to probe the influence of historical contingency on evolutionary events.

Distance effects during polyprotein processing in the complementation between defective FMDV RNAs

“…Then, an adequate dilution in PBS (with 0.1 % powdered milk) of the primary antibody was added, and the membrane incubated for 2 h. The membrane was then washed three times for 15 min in TPBS (Tween 20 at 0.05 %,v/v,in PBS) and incubated with the corresponding secondary antibody conjugated to peroxidase (1 : 10 000 dilution in TPBS). After 1 h of incubation, three washes with PBS were carried out, and the membrane was developed by chemiluminescence (ECL; Amersham) (Herrera et al, 2008;Moreno et al, 2014;Perales et al, 2007). The amount of cell extract used for electrophoretic analysis was normalized to a constant amount of cellular actin, measured by reactivity with monoclonal antibody (anti-b-actin clone AC-15; Sigma), and corresponded to a concentration of protein in the linear region of the relationship between the Western blot signal and the protein concentration.…”

Attenuated Foot-and-Mouth Disease Virus RNA Carrying a Deletion in the 3′ Noncoding Region Can Elicit Immunity in Swine

“…PCR conditions were as follows: 95°C for 10 min (1 cycle) and then 55 cycles of 95°C for 10 s, 55°C for 5 s, and 72°C for 5 s. Primers A and B were used for amplification of positive-strand RNA and primers pair A and tag for negative-strand RNA, respectively. The RNA copy number in each sample was determined by standard curves generated in parallel using either positive-sense RNA transcribed from the pO1K clone or negative-sense RNA generated from a plasmid containing the FMDV 3D region (29), kindly provided by E. Domingo. The viral load in selected serum samples from RNA inoculated pigs was determined as described above for positive-sense RNA quantification.…”

“…The primers used (A and B), reported to amplify a 290-bp conserved region in the 3D gene by conventional RT-PCR (51), were adapted to real-time RT-qPCR. Amplification of negative-strand viral RNA was achieved by attaching to the 5Ј terminus of forward primer B a heterologous tag sequence corresponding to positions 3,663 to 3,681 of the lymphocytic choriomeningitis virus RNA, as described previously (29). RNA was reverse transcribed with Transcriptor RT (Roche) using either primer A or tag B to amplify positive-or negative-strand RNA, respectively.…”

Biological Effect of Muller's Ratchet: Distant Capsid Site Can Affect Picornavirus Protein Processing

“…To identify virus-specific proteins by Western blotting using monoclonal antibodies (37), proteins were separated electrophoretically by SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad) in transfer buffer (25 mM Tris-HCl, pH 8. PICORNAVIRUS PROTEIN PROCESSING 6749 membrane was developed by chemiluminescence (ECL kit; Amersham) (27,44). FMDV proteins were detected using mouse monoclonal antibodies that recognize capsid proteins VP1, VP3, 2C (kindly provided by E. Brocchi), monoclonal antibody anti-␤-actin clone AC-15 (Sigma), and a polyclonal antibody against the polymerase 3D (38, 44).…”

“…Procedures are described in Materials and Methods. (26,27,46,49). It is difficult to venture a molecular interpretation of the transient decrease in thermosensitivity at plaque transfer 180 that was observed in the three parallel lineages for two reasons: (i) the ts character is cumulative as a result of the addition of mutations at a constant rate in the viral genome (Fig.…”