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(Kimura, 1983; Gillespie, 1991; Li, 1997), constitute the main body of population genetics to account for the dynamics of RNA viral populations.

EXPERIMENTAL VIRUS MODEL AND FITNESS ASSAYS

Vesicular stomatitis virus (VSV) is the prototype of the well-defined Rhabdoviridae family. It has a wide host range of vertebrates and arthropods. This virus is identified by its elongated bullet-shaped form of approximately 180 × 70 nm in size, with a nucleocapsid covered by a lipid-rich envelope. The RNA of the virion is complementary in its sequence to the mRNA for the viral proteins. The Rhabdoviridae are the simplest of the so-called minus-strand viruses. The VSV genome contains approximately 11.2 kb and transcribes into five mRNAs coding for five proteins (Fig. 1). All of the studies described here are in vitro experiments with VSV, in which viruses were grown in different types of cell cultures depending on the experiments, although BHK cells (from baby hamster kidney) were the usual ones.

The study of fitness effects of state variables and/or experimental regimes on viral populations is a three-step process. First, before any experimental treatment, relative fitness assays of two VSV competing clones (stock clones) were carried out as described in the third step. The first competing clone was a surrogate wild type, and the second was one of the following four different resistant to monoclonal antibodies mutant (MARM) clones. MARM C is an approximately neutral variant (with fitness 1.02 ± 0.03 relative to wild type) that contains an Asp259 → Ala substitution in the surface glycoprotein (G, Fig. 1). This amino acid substitution allows the mutant to replicate under I1 mAb concentration levels that neutralize the wild-type clone (VandePol et al., 1986). MARM R clone was isolated after repeated plaque-to-plaque transfers of MARM C and showed a lower fitness than the parental virus (0.87 ± 0.05). The I1 MAb phenotype of MARM X is conferred by an Asp257 → Val substitution in the G protein. It has a much higher fitness (2.52 ± 0.16) relative to the wild

FIGURE 1. Schematic representation of the VSV genome, with its nontranscribed leader (l), and five consecutively transcribed mRNAs (gray blocks). Each letter refers to the proteins. From left to right: N, nucleocapsid; P, phosphoprotein; M, matrix; G, glycoprotein; L, viral replicase. Numbers below each block and above lines are lengths in nts of the genes and nontranscribed sequence, respectively.



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