Castilho Leda R., Moraes Angela Maria (Ed.) Animal Cell Technology: From Biopharmaceuticals to Gene Therapy

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An important aspect of biopesticide production using insect cell lines is

the maximization of cell growth. Culture media must satisfy the environ-

mental and nutritional requirements of the cell line. Hence, many studies

have been carried out to characterize the nutrient requirements and meta-

bolic pathways of these cells (see Chapters 4 and 5).

Polyhedra bioactivity can be compromised due to nutrient limitations,

causing abnormalities in virion development and occlusion within poly-

hedra (Rollinson et al., 1965; Slavicek et al., 1995; Belloncik et al., 1997).

The most important aspects of culture medium selection are pH, osmolar-

ity, and organic salt components. Components such as amino acids,

vitamins, and carbon source (glucose for example), are typically found in

the basal culture medium formulation. However, these simple formula-

tions cannot promote cell growth themselves, unless supplemented with

animal serum, normally 5–20% (v/v) fetal bovine serum (FBS) (Schlaeger,

1996).

Lynn (1999) demonstrated that cell maintenance for long periods in

serum-free medium can lead to a slight decline in viral production. How-

ever, Chakraborty et al. (1999) obtained lower polyhedra production

when medium with FBS was used. Therefore, medium choice is not trivial.

Even though FBS has great importance for cell growth, its usage has many

disadvantages. It is expensive, so its addition to the culture medium

increases total costs substantially. Moreover, its quality can vary from

batch to batch, with an alteration of composition, which can affect cell

growth and virus replication (Schlaeger, 1996).

Figure 19.3

Contrast micrograph of Sf9 cells.

464 Animal Cell Technology

Development of serum-free medium has great value for large-scale

biopesticide production. The latest formulations are serum-free, such as

SF900II (GIBCO

/Invitrogen) and EX-CELL

(JRH Biosciences). Cell

culture medium supplementation using yeast extract (usually from alco-

holic fermentation processes), milk, or soy protein concentrates, can also

be an alternative to decrease cell culture medium costs (more details can be

found in Chapter 5).

In vitro baculovirus production can also be affected by the cell line.

Polyhedra production yield can vary between different tissues within the

same host as well as in different cell lines. Furthermore, the cell line itself

may be a source of viral instability in the form of transposon-mediated

mutagenesis (Fraser et al., 1985). (Transposons are sequences of DNA that

can move around to different positions within the genome, a process called

‘‘transposition.’’ In the process, they can cause mutations, and change the

amount of DNA in the genome.)

Insect cell lines present different susceptibilities to virus replication. A

cell line is called permissive when it can support full virus replication

and all stages of viral cycle are completed, leading to polyhedra produc-

tion and lysis of the host cell. Semi-permissive cell lines allow partial

virus replication, possibly due to a restriction at a particular viral cycle

stage (Bilimoria et al., 1992). Abortive cell lines are those in which the

resulting virus may have a cytopathic effect, but there are no infective

particles produced (Carpenter and Bilimoria, 1983; Liu and Bilimoria,

1990).

In vitro infection can be established routinely using infected larvae

hemolymph containing viral infectious particles (BVs). These particles can

be used to inoculate susceptible cells (see secondary infection, Figure

19.2). NPV replication in insect cell lines seems to be more efﬁcient than

GV replication in vitro. While many studies have demonstrated the

susceptibility of NPVs to different cell lines, few efﬁcient replication

systems have been obtained for GVs (Miltenburger et al., 1984; Winstanley

and Crook, 1993).

In a study carried out by Castro et al. (1997), the authors evaluated the

susceptibility of four different lepidopteran cell lines to AgMNPV virus.

Anticarsia gemmatalis (UFL-AG-286) and Spodoptera frugiperda

(IPLBSF-21) infected cells were productive, whereas Choristoneura fumi-

ferana (IPRI-CF-124T) and Bombyx mori (BM-5) infected cells were

abortive.

In addition, even for permissive cells, virus yield can differ between cell

lines. In the same study (Castro et al, 1997), UFL-AG-286 cells were

shown to be more efﬁcient than IPLBSF-21 cells in producing virus. Lynn

(2003b) tested 12 insect cell lines for susceptibility to infection by three

different NPVs. The author observed that virus titer differed up to 10

fold between cell lines. Miltenburger et al. (1984) established more than

200 Cydia pomonella primary cell lines. Of these, 81 were selected for

replication of two groups of baculovirus: Choristoneura murinana NPV

and Cydia pomonella GV. Although the selected cell lines were from the

same insect species, a large difference in viral infection was noted, leading

to different polyhedra yields. Goodman et al. (2001) and Lynn (2003b)

also found substantial differences in susceptibility to baculovirus infection

Bioinsecticides 465

between cell lines of the same insect species, probably due to the kind of

tissues involved.

Other examples of in vitro baculovirus systems (virus–cell line) are:

Helicoverpa armigera SNPV and Helicoverpa zea cells, with 222 polyhe-

dra per cell (Lua et al., 2002); Trichoplusia ni NPV and Trichoplusia ni

cells, with 70 polyhedra per cell (Potter et al., 1976); Lymantria dispar

MNPV and Lymantria dispar cells, with 57 polyhedra per cell (Slavicek

et al., 1996); Spodoptera frugiperda MNPV and Spodoptera frugiperda,

with 399 polyhedra per cell (Almeida et al., 2005).

19.5 Polyhedra virulence and characteristics

Cell lines inoculated with OBs are not infectious because the virions are

not released into the neutral pH of the culture medium and thus not

available for further infection (Elam et al., 1990). In contrast, it is relatively

easy to establish continuous cultures of virus isolates in vitro with

nonoccluded virions (BVs) from infected insect hemolymph or from the

supernatants of previously infected cell lines (King and Possee, 1992). In a

comparative study, Volkman et al. (1976) estimated that BV is about

1700–1900-fold more infectious than ODV. Therefore, the use of BV is

usually the ﬁrst choice to propagate a virus in cell culture (King and

Possee, 1992).

It is possible to use ODVs as a source of infectious virions if they are

released from the OBs using alkaline solutions. But when OBs are

dissolved in alkaline solution before infection of cell lines, there is only a

low level of in vitro activity (Vail et al., 1979; Elam et al., 1990).

However, this process can be improved by using proteinases. McIntosh

et al. (1988) achieved an improvement in the insect cell culture suscept-

ibility to ODVs using a treatment with proteinase K to dissolve Helico-

verpa zea NPV (HzSNPV) polyhedra, which could infect homologous

cells. Lynn (1994) also achieved the same result when he used a treatment

of dissolving Lymantria dispar NPV (LdMNPV) polyhedra with trypsin.

How proteinase works to achieve this improvement has not yet been

elucidated. Lynn (2003a) demonstrated that, in spite of the fact that trypsin

treatment can increase infectivity of baculovirus, this enhancement does

not happen for all cell–virus systems. AcMNPV infectivity of Trichoplusia

ni (TN-368) cells increased 4750-fold using trypsin. For Anagrapha

falcifera NPV (AfMNPV), the improvement was 77 000- fold. On the

other hand, the infectivity of these two viruses for the Lymantria dispar

(LIPLB-LdE) cell line was slightly reduced.

Some studies have demonstrated that the source of polyhedra does not

affect its lethality or the time necessary to kill the caterpillars (Faulkner

and Henderson, 1972; Ignoffo et al., 1974; Wang et al., 1992; Chakraborty

et al., 1999). However, Bonning et al. (1995) found that polyhedra

produced from caterpillars kill Trichoplusia ni and Heliothis virescens

caterpillars faster than polyhedra from cell culture. This could suggest

efﬁciency differences of these polyhedra produced from different sources.

Nevertheless, there were no observed differences in lethal dose between

the polyhedra tested.

466 Animal Cell Technology

Morphological analysis showed different sizes for polyhedra produced

from cell culture and caterpillar (Bonning et al., 1995). On other hand, the

polyhedra number is inversely proportional to cell size (Federici, 1986). In

addition, the polyhedra produced in vitro is more sensitive to detergent

(sodium dodecyl sulfate, SDS) treatment than polyhedra produced in vivo

(Lua et al., 2003). All these differences may be related to biochemical

differences observed between the infected cells in vitro and in vivo

(Bonning et al., 1995). Another characteristic of polyhedra produced in

vitro is the so-called ‘‘passage effect’’, which is discussed below.

19.6 Production of viral mutants in cell culture

Viral genomes, like all genomes, evolve and adapt to changes in environ-

mental pressures. Furthermore, the rate of adaptation is speciﬁc to

individual genes exposed to a particular selective environment (Bulach and

Tribe, 1994). When conditions are radically altered, such as when baculo-

viruses are switched from in vivo to in vitro propagation, rapid changes in

the phenotype may result as the selection of optimal genotypes takes place.

An in vitro environment is radically different in many ways. This includes

the use of a speciﬁc cell type for virus propagation, the form and supply of

nutrients, and the use of BV as the sole means of genetic propagation.

Scaling up a baculovirus–insect cell process involves a large increase in

volumetric scale and requires multiple passages of the virus for completion

(Rhodes, 1996). A major difﬁculty of in vitro production of baculoviruses

is the development of virus instability or the so-called passage effect. The

passage effect can result from a variety of mutations, where a decrease of

polyhedra production and virulence are the main characteristics. These

mutations can have a signiﬁcant effect on commercial production in vitro.

The most common mutations are: defective interfering particles (DIPs)

and few polyhedra (FP) mutants.

FP mutants are characterized by reduced polyhedra and enhanced

production of BV, resulting in a selection of the FP phenotype during

serial passage in cell culture (Hink and Vail, 1973; Mackinnon et al., 1974;

Potter et al., 1976; Sohi et al., 1984; Chakraborty and Reid, 1999; Pedrini

et al., 2004). The polyhedra that are produced have practically no occluded

virions (Figure 19.4), resulting in low or no infectivity of the target pest.

Spontaneously generated FP mutants are well documented among wild-

type baculoviruses that are serially passed in cell cultures, such as:

Autographa californica NPV (AcMNPV) (Hink and Vail, 1973; Wood,

1980), Galleria mellonella NPV (GmMNPV) (Fraser and Hink, 1982),

Trichoplusia ni NPV (TnMNPV) (Potter et al., 1976), Lymantria dispar

NPV (LdMNPV) (Slavicek et al., 1995), Orgyia pseudotsugata NPV

(OpMNPV) (Russell and Rohrmann, 1993), Helicoverpa armigera NPV

(HaSNPV) (Chakraborty and Reid, 1999; Lua et al., 2002), Bombyx mori

NPV (BmMNPV) (Katsuma et al., 1999), and Spodoptera frugiperda NPV

(SfMNPV) (Pedrini et al., 2004).

The mutations have been observed predominantly within a speciﬁc

DNA region (map units 36–37) on the AcMNPV (Fraser et al., 1983;

Beames and Summers, 1988, 1989) and GmMNPV genomes (Fraser et al.,

Bioinsecticides 467

1983, 1985). This region has the 25K FP gene, which encodes a 25 kDa

protein (Harrison and Summers, 1995; Braunagel et al., 1999). This protein

is essential for polyhedron formation and virion occlusion since deletion

of this gene is sufﬁcient to eliminate these processes (Beames and

Summers, 1988, 1989, Wang et al., 1989; Harrison and Summers, 1995,

Braunagel et al., 1999). These mutations have been correlated to large

insertions of host DNA or deletions of the viral genome (Fraser and Hink,

1 mµ

Figure 19.4

Electron micrographs illustrating the phenotypic characteristics of HaSNPV

infection of H. zea cells grown in suspension cultures, comparing the use of the

wild-type MP isolate (A and B) with the plaque-puriﬁed FP8AS (C and D).

Micrographs B and D are higher magniﬁcations of micrographs A and C,

respectively, to illustrate in greater detail the ultrastructure of polyhedra and their

embedded virions within the nucleus. The typical MP phenotype is that of many

polyhedra within the nucleus and the presence of numerous virions embedded

within these polyhedra, as exempliﬁed by the wild-type MP isolate. In cont rast, the

typical FP phenotype is that of considerably fewer polyhedra within the nucleus,

with little or no virion embedment within these polyhedra, as shown by the FP8AS

variant. c, nucleus; nm, nuclear membrane; nu, nu cleus; p, polyhedra; v, virion;

vs, virogenic stroma. From Pedrini, M.R.S., et al. Properties of a Unique Mutant of

Helicoverpa armigera Single-nucleocapsid Nucleopolyhedrovirus that Exhibits a

Partial Many Polyhedra and Few Polyhedra Phenotype on Extended Serial

Passaging in Suspension Cell Cultures. In Vitro Cellular and Developmental Biology

– Animal, Vol 41, pp 289 –297. Copyright # 2005 by Society for In Vitro Biology.

Reproduced with permission of the copyright owner.

468 Animal Cell Technology

1982; Fraser et al., 1983; Beames and Summers, 1988, 1989; Wang et al.,

1989). However, for the HaSNPV (Lua et al., 2002) and LdMNPV

(Slavicek et al., 1995; Bischoff and Slavicek, 1997b), point mutations or

simple base pair insertions were observed within the 25K FP gene. The FP

mutation of HaSNPV and LdMNPV is likely to arise through a different

mechanism from AcMNPV and GmMNPV (Fraser et al., 1983; Beames

and Summers, 1988), but probably similar to both viruses (Lua et al.,

2002).

When the virus has gone through a long period of multiple infection

cycles, the ability to infect insect cells decreases due to the formation of

defective interfering particles (DIPs). DIPs are found in most animal virus

systems (Huang and Baltimore, 1977) and are well documented in many

baculovirus systems that undergo in vitro serial passaging, such as:

AcMNPV (Carstens, 1982; Kool et al., 1991; Lee and Krell, 1992; Pijlman

et al., 2001), BmMNPV (Hashimoto et al., 1993; Yanase et al., 1998),

SeMNPV (Heldens et al., 1996), H. zea baculovirus subgroup C (Burand

and Wood, 1986; Chao et al., 1990), and HaSNPV (Pedrini et al., 2005).

Some of these data are summarized in Table 19.1.

Pijlman et al. (2001) demonstrated that DIP generation and accumula-

tion are intrinsic characteristics of baculovirus propagation in insect cell

cultures, leading to a dramatic loss of BV titer. However, the origin of

DIPs is not fully understood. Pijlman et al. (2001) proposed a mechanism

involving continuous hetero- and homologous recombination of baculo-

virus DNA within insect cells, the deletion mutants of which are then

selected for, depending on their relative replicative advantages. A high

frequency of recombination between homologous baculoviruses in insect

cell cultures has been demonstrated previously (Hajo´s et al., 2000). It was

found that AcMNPV mutant virions lack around 44% of the viral genome,

including polyhedra and DNA polymerase genes (Kool et al., 1991;

Wickham et al., 1991). These particles containing only part of their

Table 19.1. Defective interfering particles (DIPs) for many baculovirus submitted to success ive

passages in insect cell cultures

Virus Cell line Native virus

size (kb)

DIP size

(kb)

%of

native virus

Reference

A. californica NPV

(AcMNPV)

S. frugiperda (Sf) 128 75 59 Carstens, 1982

A. californica NPV

(AcMNPV)

S. frugiperda

(Sf-AE-21)

130 100; 85; 70 77; 65; 54 Lee and Krell, 1992

A. californica NPV

(AcMNPV Bacmid

bGFP)

S. frugiperda

(Sf-AE-21)

Not given Not given 57 Piljman et al., 2001

B. mori NPV

(BmMNPV D1)

B. mori (BmN4) Not given Not given 74 Hashimoto et al.,

1993

S. exigua NPV

(SeMNPV)

S. exigua

(Se-UCR1)

134 109 81 Heldens et al., 1996

H. zea subgroup C

Baculovirus (Hz-1)

T. ni (Tn-368) 228 176–204 77–89 Chao et al., 1990

H. armigera NPV

(HaSNPV)

H. zea (Hz-AM1) 133.4 115.4; 92.3 87, 7 Pedrini et al. 2005

Bioinsecticides 469

genome need an intact virus as a helper (Lee and Krell, 1992), but replicate

faster than standard virus, and consequently, increase their yield with

continuous serial passage.

DIP formation in virus stocks can be avoided by infecting insect cells at

a low multiplicity of infection (MOI). According to Wickham et al.

(1991), a low MOI is used to minimize the probability of a DIP entering

in a cell along with an intact helper virion. As a result, some cells receive

only intact virus, others only defective virus, or no virus at all. Non-

infected cells or cells infected only with defective virus will not produce

any more virus, while cells infected with intact virus will produce more

intact and infectious viruses than the co-infected cells. Therefore, the DIP

fraction will decrease compared with the original inoculum. This causes an

increase of virus titer.

Other genomic mutations can also affect the viral infectivity in cater-

pillars, such as: enhancing genes (E1 and E2) (Bischoff and Slavicek, 1997a;

Popham et al., 2001), SeMNPV ORF35 (Se35) gene (Pijlman et al., 2001),

and the SeMNPV ORF36 (Se36), designated as per os infectivity factor

(pif) (Kikhno et al., 2002). An understanding of how these genes affect

viral infectivity may help to develop culture strategies that avoid these

mutations. This could improve the commercial prospects of biopesticides

produced in vitro.

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