Baculovirus defective interfering particles are responsible for variations in recombinant protein production as a function of multiplicity of infection

Defective interfering particles (DIPs) are virus-like particles that arise during virus growth, fail to grow in the absence of virus, and replicate at the expense of virus during co-infections. The inhibitory effects of DIPs on virus growth are well established, but little is known about how DIPs influence their own growth. Here vesicular stomatitis virus (VSV) and its DIPs were used to coinfect BHK cells, and the effect of DIP dose on virus and DIP production was measured using a yield-reduction assay. The resulting dose-response data were used to fit and evaluate mathematical models that employed different assumptions. Our analysis supports a multiple-hit process where DIPs inhibit or promote virus and DIP production, depending on dose. Specifically, three regimes of co-infection were apparent: (i) low DIP -where both virus and DIPs are amplified, (ii) medium DIP -where amplification of both virus and DIPs is inhibited, and (iii) high DIP -with limited recovery of virus production and further inhibition of DIP growth. In addition, serial-passage infections enabled us to estimate the frequency of de novo DIP generation during virus amplification. Our combined experiments and models provide a means to understand better how DIPs quantitatively impact the growth of viruses and the spread of their infections.

Section: Introductionmentioning

confidence: 99%

Multiple-hit inhibition of infection by defective interfering particles

Thompson

Rempała

Yin

2009

“…Recently, eight cis-acting elements that may play a role in DNA replication have been identified (Pearson et al, 1992;Leisy & Rohrmann, 1993;Kool et al, 1993a, b). The nature and location of some of these cis-acting elements has firstly been inferred from the structure of defective viral genomes, which are generated in the continuous production of AcMNPV in bioreactors or upon serial, undiluted passage of virus in cell culture (Kool et al, 1991;Wickham et al, 1991; 1992). These defective viral genomes lack considerable portions of the standard viral genome, but apparently retain cis-acting elements that are essential for DNA replication and encapsidation.…”

Section: Introductionmentioning

confidence: 99%

A putative non-hr origin of DNA replication in the HindIII-K fragment of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus

Kool¹,

Goldbach

Vlak

1994

In addition to the seven known homologous regions (hrs) of Autographa ealiforniea multiple nucleocapsid polyhedrosis virus (AcMNPV) the HindlII-K fragment was also found to carry a putative ori, although this fragment does not contain an hr. Deletion analysis showed that this or~ contains several segments essential for its activity and other 'auxiliary' sequences that enhance the or~ activity. Sequence analysis identified several structures often found in other viral replication or/s, such as palindromes and other repeated motifs.

“…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).…”

mentioning

confidence: 99%

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

Zwart

Erro

Oers

et al. 2008

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...