Mengovirus and encephalomyocarditis virus poly(C) tract lengths can affect virus growth in murine cell culture

Abstract

Many virulent aphthoviruses and cardioviruses have long homopolymeric poly(C) tracts in the 5' untranslated regions of their RNA genomes. A panel of genetically engineered mengo-type cardioviruses has been described which contain a variety of different poly(C) tract lengths. Studies of these viruses have shown the poly(C) tract to be dispensable for growth in HeLa cells, although the relative murine virulence of the viruses correlates directly and positively with tract length. Compared with wild-type mengovirus strain M, mutants with shortened poly(C) tracts grow poorly in mice and protectively immunize rather than kill recipient animals. In the present study, several murine cell populations were tested to determine whether, unlike HeLa cells, they allowed a differential amplification of viruses with long or short poly(C) tracts. Replication and cytopathic studies with four hematopoietically derived cell lines (CH2B, RAW 264.7, A20.J, and P815) and two murine fibroblast cell lines [L929 and L(Y)] demonstrated that several of these cell types indeed allowed differential virus replication as a function of viral poly(C) tract length. Among the most discerning of these cells, RAW 264.7 macrophages supported vigorous lytic growth of a long-tract virus, vMwt (C(44)UC(10)), but supported only substantially diminished and virtually nonlytic growth of vMC(24) (C(13)UC(10)) and vMC(0) short-tract viruses. The viral growth differences evident in all cell lines were apparent early and continuously during every cycle of virus amplification. The data suggest that poly(C) tract-dependent attenuation of mengovirus may be due in part to a viral replication defect manifest in similar hematopoietic-type cells shortly after murine infection. The characterized cultures should provide excellent tools for molecular study of poly(C) tract-mediated virulence.

FIG. 1

Virus replication assays. (A) Cell monolayers (A20.J, P815, and RAW 264.7 cell lines) were infected in parallel with vMwt, vMC₂₄, and vMC₀ as described in Materials and Methods. Each point represents the amount of virus released into the supernatant since the previous point, as determined in triplicate by standard plaque assays on HeLa cells. Open symbols denote points at which >80% of the original cell monolayer was destroyed. (B) Comparison of maximum titers attained during virus replication in A20.J, P815, RAW 264.7, CHB2, L(Y), and HeLa cell lines. Each line was infected with vMwt, vMC₂₄, and vMC₀ as described in Materials and Methods. Supernatant aliquots were taken at 24, 48, 84, and 132 h p.i., and virus titers in each sample were determined by plaque assay (triplicate samples) on HeLa cells. The maximum titers (24-h time point for most cell lines; 48-h time point for A20.J) are normalized relative to those achieved by vMwt and are shown as a percentage. Typical replicate platings or repeat experiments varied in titer by <1% for any of the viruses or infectious passages.

FIG. 2

Single-step growth in RAW 267.4 cells. Cells were infected as described in Materials and Methods. The data are plotted as mean PFU/cell, with standard deviation. The inset depicts single-step growth results for the same three viruses after infection of HeLa cells.

FIG. 3

Comparison of vMwt, vMC₂₄, and vMC₀ growth in RAW 264.7 cells. (A) RAW 264.7 cells were infected with the indicated viruses as described in Materials and Methods. At 24, 48, and 72 h p.i., supernatant aliquots were removed, the virus titer was determined, and parallel aliquots were frozen until used in IFN bioassays. (B) Bioactive IFN-α/β titers were determined by the methods of Sekellick and Marcus (23) and calibrated relative to a control, IFN standard (Biosource). Data are presented as NIH units of IFN per milliliter. (C) The CPE in the infected RAW 264.7 monolayers was scored empirically on a relative scale compared to a mock-infected, control monolayer (+++++, >99% dead; +, < 10% dead). The vMwt IFN titers and CPE scores were limited to the 0- and 24-h time points, because all cells were dead after this time.

FIG. 4

Relative growth of EMCV-R, vEC₂₀, and vEC₄ in RAW 264.7 cells. Cells were infected as described in Materials and Methods. Maximum titers were reached at 24 h p.i. and are shown as a percentage normalized to the value for EMCV-R.