Infection, Genetics and Evolution 1 (2001) 41–48 Transmission bottlenecks and the evolution of fitness in rapidly evolving RNA viruses Santiago F. Elena∗, Rafael Sanjuán, Antonio V. Border´ıa, Paul E. Turner1 Institut Cavanilles de Biodiversitat i Biolog´ıa Evolutiva and Departament de Genètica, Universitat de València. Apartat 2085, 46071 València, Spain Received 17 November 2000; received in revised form 10 February 2001; accepted 5 March 2001 Abstract We explored the evolutionary importance of two factors in the adaptation of RNA viruses to their cellular hosts, size of viral inoculum used to initiate a new infection, and mode of transmission (horizontal versus vertical). Transmission bottlenecks should occur in natural populations of viruses and their profound effects on viral adaptation have been previously documented. However, the role of transmission mode has not received the same attention. Here we used a factorial experimental design to test the combined effects of inoculum (bottleneck) size and mode of transmission in evolution of vesicular stomatitis virus (VSV) in tissue culture, and compared our results to the predictions of a recent theoretical model. Our data were in accord with basic genetic principles concerning the balance between mutation, selection and genetic drift. In particular, attenuation of vertically transmitted viruses was a consequence of the random accumulation of deleterious mutations, whereas horizontally transmitted viruses experiencing similar bottlenecks did not suffer the same fitness losses because effective bottleneck size was actually determined by the number of host individuals. In addition, high levels of viral fitness in horizontally transmitted populations were explained by competition among viral variants. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Beneficial mutations; Deleterious mutations; Experimental evolution; Fitness; Horizontal transmission; Vertical transmission; Vesicular stomatitis virus 1. Introduction Ewald (1987) suggests that virulence should decrease under vertical transmission because pathogen fitness is lim- Evolution of parasite fitness is governed by competition ited by host reproduction, a parasite that harms its host at two different levels, intrahost replication and interhost too much will reduce its own reproductive success (Chao transmission (Levin and Svanborg Éden, 1990; Bull, 1994; et al. (2000) for a different prediction when interference Ewald, 1994; Chao et al., 2000). Despite extensive theoreti- competition occurs between parasites within the host). cal work exploring the role of transmission mode in pathogen In contrast, vertical transmission of viral pathogens via evolution (Anderson and May, 1986; Lenski and May, 1994; plaque-to-plaque serial transfer in the laboratory is shown Lipsitch et al., 1996; Frank, 1996; Boots and Sasaki, 1999), to reduce virulence through the accumulation of deleterious relatively little empirical support exists for these models mutations (Domingo and Holland, 1997). In bottlenecked or their underlying assumptions (Bull et al., 1991; Herre, lineages harmful mutations will accumulate over time, caus- 1993; Turner et al., 1998; Messenger et al., 1999). Vertical ing the mean fitness of the population to decline; a process transmission of parasites occurs within a single host lineage analogous to Muller’s ratchet (Muller, 1964). Thus, parasite (between an infected parent and its offspring), but horizontal fitness can decrease through direct genetic consequences of transmission allows a parasite to reach any susceptible host the transmission process, rather than through a parasite’s in the population regardless of descent. Therefore, compe- effects on host fitness. tition between vertically transmitted parasites occurs only Recently, Bergstrom et al. (1999) discussed several con- at the intrahost level, while that between horizontally trans- sequences of transmission bottlenecks for the evolution of mitted parasites takes place at the interhost level as well. rapidly evolving pathogens, such as RNA viruses. (i) When inoculum size is large (absence of severe bottlenecks), changes in genotype frequency caused by sampling effects ∗ + + Corresponding author. Tel.: 34-963-983-666; fax: 34-963-983-670. become insignificant. Under vertical transmission, genotype E-mail address: [email protected] (S.F. Elena). 1 Present address: Laboratory of Clinical Investigation, National Institutes frequencies in the independently evolving lineages will of Health, Bethesda, MD 20892-1888, USA. converge on an equilibrium distribution of viral phenotypes 1567-1348/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S1567-1348(01)00006-5 42 S.F. Elena et al. / Infection, Genetics and Evolution 1 (2001) 41–48 present in the individual transmitting the infection. Thus, low Comer et al., 1990). Therefore, both modes of transmission genetic variance and similar levels of mean fitness are ex- are a natural component of VSV biology. pected among the viral populations. By a similar argument, Here we used VSV and eukaryotic cells in tissue cul- horizontally transmitted viruses that share a common gene ture to answer three questions. Under severe bottlenecks, pool will converge on an equilibrium phenotypic distribu- can horizontally transmitted viruses better combat the mu- tion (and hence the same genetic variability and fitness). But tational load and achieve greater fitness? With increased horizontal transfer allows fitter strains to export their greater inoculum size, does the fitness of vertically transmitted productivity to other host lineages, and selection should viruses approach that of their horizontally transmitted coun- maintain high fitness in horizontally transmitted viruses. terparts? Do smaller bottlenecks lead to greater genetic This effect of transmission mode on fitness is expected even variance in fitness among replicate populations regardless when the host population contains as few as five individuals. of transmission mode? (ii) When inoculum size is small (severe bottlenecks), sam- pling error causes the genotype frequency distribution in a newly infected host to deviate from that in the transmitting 2. Material and methods host and, presumably, mean fitness should decline. The evolutionary dynamics of horizontally and vertically 2.1. Viral clones, cells and culture conditions transmitted viruses should differ in two ways. First, under vertical transmission alone the viruses in each individual Viruses used in this study were originally derived from the host compose an evolutionarily separate population from Mudd-Summer strain of the VSV Indiana serotype, hereafter those in every other host. Therefore, vertically transmitted referred to as wild-type (wt). MARM C is a mutant derived → viruses from different hosts at time t = 0 will never share from wt. Both genotypes only differ on an Asp259 Ala a common host at time t>0. In contrast, horizontally substitution in the G surface protein, a mutation that allows transmitted viruses from separate hosts at t = 0 may share growth under I1-monoclonal antibody (I1-mAb) concentra- a common host at t>0. The result is that vertically trans- tions that completely neutralize wt (Lefrancois and Lyles, mitted viruses are separated into n subpopulations passing 1982; Vandepol et al., 1986). This amino acid substitution through a bottleneck of size b at each transmission event, do not have any effect on MARM C fitness and both clones whereas horizontally transmitted pathogens form a single can be considered as effectively neutrals (Holland et al., population passing through a bottleneck of size nb. Sec- 1991; Miralles et al., 1999, 2000). MARM C served as the ond, horizontally transmitted viral populations feature a common competitor in our fitness assays (see Section 2.2). greater potential for growth rate differences to counteract Baby hamster kidney (BHK) cells were grown as mono- mutational load. Under horizontal transmission a novel mu- layers under Dulbecco modified Eagle’s minimum essential tation will continue competing with its progenitor to infect medium (DMEM) supplemented with 5% newborn calf the same set of hosts until one is lost to extinction. But serum and 0.06% proteose peptone 3. Cells were grown to with vertical transmission the two mutations can compete a density of ∼ 3 × 105 cells/cm2 in 25 cm2 plastic flasks for only as long as they reside in the same individual, and infections, or in 100 cm2 plates for routine maintenance. To selection has less time to combat deleterious mutations. produce I1-mAb, hybridoma cells were grown in DMEM Thus, smaller bottleneck size and relatively weak selection containing 20% foetal bovine serum, 2 ␮g/ml thymidine, allow mutations to more severely reduce fitness in verti- 0.1 mg/ml glycine, 14 ␮g/ml hypoxanthine and 1.3 ␮l/ml of cally transmitted viruses than in horizontally transmitted Opti-mAb (GibcoBRL). All cell lines were maintained in ◦ ones. incubators at 37 C, 5% CO2 atmosphere, and 95% relative Vesicular stomatitis virus (VSV) is a typical member humidity. of the Vesiculovirus genus (family Rhabdoviridae, order Mononegavirales), and provides an excellent model to test 2.2. Experimental design predictions similar to those above. VSV and other RNA viruses feature extremely high rates of spontaneous muta- Our experimental design (Fig. 1) mimicked virus evo- tion (orders of magnitude larger than DNA genomes) that lution in a population of multicellular hosts reproduc- distinguish them from other parasites (Drake and Holland,