Low multiplicity of infection in vivo results in purifying selection against baculovirus deletion mutants

Abstract

The in vivo fate of Autographa californica multiple nucleopolyhedrovirus deletion mutants originating from serial passage in cell culture was investigated by passaging a population enriched in these mutants in insect larvae. The infectivity of polyhedra and occlusion-derived virion content per polyhedron were restored within two passages in vivo. The frequency of occurrence of deletion mutants was determined by real-time PCR. The frequency of the non-homologous region origin (non-HR ori) of DNA replication was reduced to wild-type levels within two passages. The frequency of the polyhedrin gene did not increase and remained below wild-type levels. A low m.o.i. during the initial infection in insect larvae, causing strong purifying selection for autonomously replicating viruses, could explain these observations. The same virus population used in vivo was also passaged once at a different m.o.i. in cell culture. A similar effect (i.e. lower non-HR ori frequency) was observed at low m.o.i. only, indicating that m.o.i. was the key selective condition.The passage of baculoviruses in cultured insect cells leads to the rapid accumulation of deletion mutants, some of which have been demonstrated to have defective interfering properties (Kool et al., 1991;Pijlman et al., 2001;Wickham et al., 1991). Populations with a high frequency of these deletion mutants can completely lose infectivity for insect larvae (Heldens et al., 1996). The presence of such deletion mutants is therefore likely to be deleterious to the fitness of a virus population in vivo, although deletion mutants that can increase fitness are found in some natural baculovirus isolates (Ló pez-Ferber et al., 2003). Dai et al. (2000) found that alternate passaging of baculoviruses in insect cells and larvae resulted in sustained infectivity for insect larvae, suggesting that purifying selection (Li, 1997) occurs in these animals. In other words, there may be stabilizing selection for a particular trait, in this case the ability to replicate autonomously. Consequently, individuals that do not possess this trait are removed from the population. However, the fate and dynamics of these cell culturederived deletion mutants after reintroduction into insects have not been systematically addressed. Therefore, a clonal baculovirus was first serially passaged in insect cells to enrich for deletion mutants, and the resulting population was reintroduced into insect larvae. The effects of both in vitro and subsequent in vivo passaging were investigated by determining the virulence and occlusion-derived virion (ODV) content of polyhedra, and the frequency of occurrence of deletion mutants by quantitative real-time PCR (qPCR).In order to generate a clonal population of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), bacmid technology (Luckow et al., 1993) was employed. A bacmid with restored polyhedrin (polh) expression was generated using the pFastBac-DUAL/Polh construct (Zwart et al., 2008). This bacmid was used to transfect Spodoptera frugiperda Sf-AE-21 (Sf...

The Multiplicity of Infection of a Plant Virus Varies during Colonization of Its Eukaryotic Host

“…A large body of evidence, notably from experiments involving defective-interfering particles, suggests that the MOI in cultured mammalian cells may be very high: experiments were often performed which had virus-tocell ratios of Ն10 (e.g., 39), and the efficiency of complementation of defective-interfering particles by wild-type virus has been shown to increase up to virus-to-cell ratios above 300 (7). However, MOI levels could be much smaller in the eukaryotic host than in cell cultures, as data on complementation of defective mutants of baculoviruses in cultured cells and in insect larvae and data on HIV proviral copy numbers in infected spleen cells strongly suggest (29,63). This seems not to be the case for TMV, as MOI levels in protoplasts, derived from data in Table 4, are 2.6 to 3, within the range of values for leaf cells.…”

Mixed-genotype white spot syndrome virus infections of shrimp are inversely correlated with disease outbreaks in ponds

“…Even defective viruses, which often reduce the virulence of the virus population (e.g. Muñoz et al, 1998;Zwart et al, 2008), in some particular instances increase the virulence of the population when co-infecting with a helper virus (Lopez-Ferber et al, 2003;Lauzon et al, 2005). Here, however, we have identified a case were genetic heterogeneity is inversely correlated with disease outbreaks.…”

Experimental Evolution of Pseudogenization and Gene Loss in a Plant RNA Virus

“…As a consequence, the cellular multiplicity of infection (MOI) is also likely to increase over the course of infection (González-Jara et al 2009; Gutiérrez et al 2010; Zwart et al 2013). Different MOIs may lead to different selection pressures acting on the virus population, as exemplified by DIP viruses (e.g., Zwart et al 2008). This second model predicts conditions under which the heterologous sequence can be maintained in the virus population.…”

“…Moreover, it appears to be a very general observation that viruses expressing heterologous genes tend to be unstable (Chapman et al 1992; Dolja et al 1993; Guo et al 1998; Pijlman et al 2001; Chung et al 2007; Paar et al 2007). Furthermore, under conditions maximizing selection for competitive fitness—exemplified by undiluted serial passage in cultured cells—viruses tend to rapidly evolve defective interfering particles (DIPs): viruses with large genomic deletions are unable to replicate autonomously but with a replicative advantage at high multiplicities of infection (Huang 1973; Simon et al 2004; Zwart et al 2008; Pathak and Nagy 2009). All these observations suggest that genome size is under strong selection for viruses and that having unnecessary genomic sequences has fitness costs.…”