Quality Control of Baculoviral Bioinsecticide Production

Agriculture is a discipline that has accompanied human beings since the beginning of civilization. The cultivation of different vegetables for centuries has allowed selecting varieties that far exceed the capabilities of many wild type plants originally used as a food source. That situation derived in the manipulation of natural ecosystems, transforming them into spaces where they can only grow and develop the desired species. In our world, plants are the staple diet of many organisms including invertebrates like Lepidoptera. During the larval stage, these insects can consume a large amount of leaf tissue causing serious damage to the plant. If we think that most vegetables have insect predators, agricultural crops can be transformed into an inviting habitat, allowing the development of these animals. In conclusion, all crops have pests that threaten their productivity. Given this scenario, many pest control strategies have been used by human beings to protect the health of their crops: treatment with chemical insecticides, development of transgenic plants and biological control applications (Christou et al, 2006; Gilligan, 2008). Baculovirus is a large family of insect pathogens that infect and kill different species of Lepidoptera, Hymenoptera and Diptera (Theilmann et al, 2005). In particular, many lepidopteron are pests in agriculture transforming these viruses in an important biocontrol tools for their natural hosts (Entwistle, 1998; Moscardi, 1999; Szewczyk et al, 2006). Baculoviruses have double-stranded circular DNA genomes of 80,000-180,000 bp, containing between 80 to 180 genes depending on the specie (van Oers & Vlak, 2007; Miele et al, 2011). In early stages of virus cycle, this pathogen is produced as Budded Viruses (BVs): the genome contained in a protein capsid (nucleocapsid), which is surrounded by a lipid membrane. In change, in the last phase of multiplication processes appear the Occluded Bodies (OBs): protein crystals (forming polyhedra or granules) containing nucleocapsids wrapped by a lipid membrane with a different composition (ODVs or Occluded Derived Viruses, with single or multiple nucleocapsids depending on the specie) (Rohrman, 2008). These two virus phenotypes have different biological properties; while OBs are specialists (infecting larvae by per os route with a narrow host range; responsible of primary infection in midgut cells), the BVs are generalists (infecting a wide range of different insect cells triggering their death; responsible for secondary infection). In the pest control strategies, baculoviruses (OBs) are introduced on the crops to infect and kill larvae through the production of an epizooty.


Introduction
Agriculture is a discipline that has accompanied human beings since the beginning of civilization.The cultivation of different vegetables for centuries has allowed selecting varieties that far exceed the capabilities of many wild type plants originally used as a food source.That situation derived in the manipulation of natural ecosystems, transforming them into spaces where they can only grow and develop the desired species.In our world, plants are the staple diet of many organisms including invertebrates like Lepidoptera.During the larval stage, these insects can consume a large amount of leaf tissue causing serious damage to the plant.If we think that most vegetables have insect predators, agricultural crops can be transformed into an inviting habitat, allowing the development of these animals.In conclusion, all crops have pests that threaten their productivity.Given this scenario, many pest control strategies have been used by human beings to protect the health of their crops: treatment with chemical insecticides, development of transgenic plants and biological control applications (Christou et al, 2006;Gilligan, 2008).Baculovirus is a large family of insect pathogens that infect and kill different species of Lepidoptera, Hymenoptera and Diptera (Theilmann et al, 2005).In particular, many lepidopteron are pests in agriculture transforming these viruses in an important biocontrol tools for their natural hosts (Entwistle, 1998;Moscardi, 1999;Szewczyk et al, 2006).Baculoviruses have double-stranded circular DNA genomes of 80,000-180,000 bp, containing between 80 to 180 genes depending on the specie (van Oers & Vlak, 2007;Miele et al, 2011).In early stages of virus cycle, this pathogen is produced as Budded Viruses (BVs): the genome contained in a protein capsid (nucleocapsid), which is surrounded by a lipid membrane.In change, in the last phase of multiplication processes appear the Occluded Bodies (OBs): protein crystals (forming polyhedra or granules) containing nucleocapsids wrapped by a lipid membrane with a different composition (ODVs or Occluded Derived Viruses, with single or multiple nucleocapsids depending on the specie) (Rohrman, 2008).These two virus phenotypes have different biological properties; while OBs are specialists (infecting larvae by per os route with a narrow host range; responsible of primary infection in midgut cells), the BVs are generalists (infecting a wide range of different insect cells triggering their death; responsible for secondary infection).In the pest control strategies, baculoviruses (OBs) are introduced on the crops to infect and kill larvae through the production of an epizooty.

SF2, SF9
Fall armyworm Corn and small grain crops

SLN, SL2, SLG
Oriental leafworm moth Wide range of plants, like cotton and tobacco.

Setaceous hebrew character
Huge variety of plants (tomato, tobacco, carrot, lettuce, alfalfa, potato, grape, maize, apple) Table 2. Baculovirus and pest control.The table contains some Baculoviruses with their insect hosts, revealing their possible application as bioinsecticide Actually, baculoviruses are classified in four genera according to their biological properties and gene content: Alphabaculovirus, polyhedroviruses that infect Lepidoptera (grouped into two lineages, Group I and Group II, according to their phylogenetic relationships and the identity of the fusogenic membrane protein presents in the BVs); Betabaculovirus, granuloviruses that infect Lepidoptera; Gammabaculovirus, polyhedroviruses that infect Hymenoptera; and Deltabaculovirus, polyhedroviruses that infect Diptera (Table 1) (Jehle et al, 2006a).Genomic sequence is known more than 50 different baculovirus species, being the recognized prototypes of each genus: AcMNPV, CpGV, NeleNPV and CuniNPV, respectively.Many of them have been used for biological pest control, being excellent biopesticides (Figure 1; Table 2).However, most baculoviruses cannot efficiently compete with chemical insecticides, especially in the time of death.To overcome this problem, many researchers have been focused to introduce genetic modifications in order to accelerate the lethal effects of bioinsecticide or expand their host range.One strategy that has been explored is the introduction of genes encoding insect toxins, such as different neurotoxins from eukaryotic organism or the bacterial protein Cry (Inceoglu et al, 2006;Jinn et al, 2006;De Lima et al, 2007).Thus, these genetically modified viruses (GMV) would ensure better performance in biopesticide application.
Baculoviruses are produced by infection processes in susceptible larvae or in in vitro cell cultures.First approach is appropriate and inexpensive in small-scale, but big productions prefer the use of cell bioreactors (van Beek & Davis, 2007;Micheloud et al, 2009;Mengual Gómez et al, 2010).This technology would allow the standardization of production processes and achieve bioinsecticides with reproducible quality.
The main difference among these strategies consists in the starters used, being in one case OBs (in larvae) and BVs in the other (in vitro cell cultures); but always with the goal of producing OBs (infective phenotype in nature).Although the trend is moving toward baculovirus production in cell cultures, it is important to note some problems associated with that strategy.One of them is the genome stability.Because only the BVs infect cells growing in laboratory conditions, after successive rounds of infection tend to accumulate defective viral variants with smaller genomes (Lee & Krell, 1992).These quasispecies lose genomic segments encoding late proteins important for generating OBs, because there is no selection pressure associated with oral infection in larvae.Other problems are related to the composition of culture media and the availability of susceptible insect cell lines to each baculovirus.Actually, many researchers are working on the establishment of new cell lines or modifying existing ones to improve their performance, while others have focused on developing proper and cheaper formulations of growth media for cell propagation in vitro (Agathos, 2007;Micheloud et al, 2009).

Quality control assays
The production of baculoviruses for use as bioinsecticides required quality control processes to ensure their proper formulation.In either case above (wild type viruses or GMVs) or regardless of production method applied (larvae or in vitro cell cultures), is necessary to carry out a series of phenotypic and genotypic tests against which to assess the quality of each batch produced (Figure 2).The formulation of one biological entity for some biotechnological application (e.g.baculovirus for agriculture pest control) requires its multiplication under controlled conditions and subsequent procedures for isolation and concentration.In this point, it is important to remember that all biological entities are object of evolution, natural phenomenon that can influence and alter the biological properties of the product by the accumulation of point mutation or genome rearrangements.
Fig. 2. Quality control scheme.A good quality control strategy is supported in the setting of and in the rigid adhesion to the procedures and protocols.These may include routine examinations of insect/cells stocks, microscopic examinations for infections, routine counting of ODVs, bioassays to assess bioinsecticide potency, restriction profiles of viral DNAs, and so on.First and second steps are developmental phases of the bioinsecticide production, in which the feasibility to obtain high amounts of good quality DNA is not an obstacle.In the third step, is of special importance the availability of sensitive molecular techniques to minimize the interference of formulation components Thus, quality control assays emerge as central tools for verifying the baculovirus production in each of its stages allowing generating a product that can compete with chemical insecticides, whose production is highly optimized and controlled for years.Also, quality control strategies are useful to standardize the basic studies performed in laboratory scale, necessary for the generation of improved baculovirus.

Phenotype quality controls
First of all, it is important to have good methods to quantify the number of OBs produced and isolated from larvae or in vitro cell cultures.To fulfill this purpose, it is possible to make direct eye count using hemocytometer and optical microscopes.On the other hand, there are methodologies based on immunoassays or carried out by the use of flow cytometers.In the first case, the development of ELISA kits or other similar tests based on the immune detection of OBs (through the use of polyclonal or monoclonal antibodies against polyhedrin or granulin proteins) has standardized the quantitation of baculovirus allowing a more reliable measure (Parola et al, 2003).The use of flow cytometers also provides good results, but only so far for the quantification of BVs (Shen et al, 2002;Jorio et al, 2006).
Once quantified the production of OBs, should determine their biological activity.This involves setting parameters to estimate the ability of baculovirus to kill insect pests and control their population.In view of this, parameters like median lethal time (LT 50) and median lethal dose (LD 50) work as the best indicators to characterize the baculovirus activity (Li & Bonning, 2007;Lasa et al, 2008).These tests consist of exposing susceptible larvae reared in standardized conditions of temperature, light, moisture and food to the virus under evaluation.Then, through the register of deaths and the time in which they occur can be estimated both parameters.

Genotype quality controls
The production of baculoviruses for use as bioinsecticides requires accurate determination of the number of OBs and their biological activity expressed in LT 50 and LD 50 parameters.But it is also important to apply other methodologies that allow considering genotypic evaluations.As mentioned earlier, the processes of baculovirus production in insect cell lines growing in laboratory conditions may derived in problems with the integrity of their genomes.Consequently, the productivity of OBs can be seriously affected both in quantity and activity ruining the entire production.Of course, this is particularly relevant when dealing with GMVs.The stability of putative transgenes should be considered.Most of baculoviruses applied as bioinsecticides derived from homogenous populations cloned or partially cloned by different procedures (Wang et al, 2003;Simón et al, 2004).This is a remarkable aspect since it allows establishing genotypic characteristic patterns that can be detected by different approaches.Among them, the visualization of RFLPs (Restriction Fragment Lenght Polymorphism) in agarose gel electrophoresis stained by different dyes and UV exposition is usually a good indicator of genome integrity, revealing the gain or loss of DNA (Simón et al, 2004;Eberle et al, 2009;Rowley et al, 2010).In fact, this is a classic approach to characterize genotypic variants of a viral species.The main problem that has this strategy is related to allocate part of baculovirus production to perform the isolation of viral genome, requiring high DNA masses to achieve reliable results.The complementation with hybridization assays solves part of that problem but requires the availability of suitable probes, adding experimental steps and costs of supplies and equipment.
In view of that, methodologies based on PCR (Polymerase Chain Reaction) are suitable and reproducible approaches to assess baculovirus productions because this technique can detect desired locus with high sensitivity and specificity.These characteristics transform the PCR in the best genotypic evaluation strategy due to its simple, fast and accessible properties for any laboratory production.Since the beginning of studies on the baculovirus genomes, many researchers have designed PCR tests to detect, identify and classify the different species of this virus Family.Thus, PCR assays based on polyhedrin/granulin, p74, lef8, lef9 or DNA polymerase genes, among others, were used to describe new virus isolates which are candidates to bioinsecticide applications (Faktor et al, 1996;de Moraes et al, 1999;Wang et al, 2000;Rosisnki et al, 2002;Espinel-Correal et al, 2011;Rodríguez et al, 2011).However, there are too many examples of the use of PCR as a technique for quality control in the production of a baculovirus, despite all the advantages mentioned above (Christian et al, 2001;Murillo et al, 2006).

MP-PCR to control baculovirus production
PCR amplification of several loci in the same reaction allows obtaining a profile of products that can be used for genome identification or control test in production processes.MP-PCR (Multiplex PCR) assays require the proper design of primers to amplify a set of fragments that are typical for a particular genome.This technique provides results composed of a set of enzymatic amplified fragments that are characteristic for a viral species (when primers were designed completely specific), or for a phylogenetic group (when primers derived from multiple alignments of orthologous sequences).With regard to trials designed to particular viruses, it should be noted the work developed for EpapGV (Manzán et al, 2008).Meanwhile, for the detection of groups of related viruses are not many references.Currently, the accepted practice to identify or preliminarily classify a new baculovirus is based on PCR amplification and subsequent sequencing of three genomic fragments corresponding to the polyhedrin/granulin, lef9 and lef8 genes (Jehle et al, 2006b).However, this approach is not itself an MP-PCR.In view of this, we propose an MP-PCR for alpha and betabaculovirus quality control based on universal primer designs.
Baculoviruses contain 31 core genes conserved by all known members (Miele et al, 2011).These orthologous sequences are present in each sequenced baculovirus, but their genomic distribution varies among species.From the analysis of gene distribution in genus prototypes pif2, p49, p74, lef9, 38k genes were selected to primer design targets (Figure 3).These sequences are properly distributed throughout the entire circular genome.Two genes (pif2 and p74) encode per os infectivity factors essentials to the success of primary infection in midgut cells (Song et al, 2008;Peng et al, 2010).Other two genes (p49 and 38 K) encode proteins associated to packaging, assembly, and release of virions (Wu et al, 2008;Lin et al, 2010).Meanwhile, lef9 gene encodes a polypeptide involved in virus transcription machinery (Crouch et al, 2007).Using multiple alignments derived from sequences corresponding to P74, lef9 and 38k genes from all alpha and betabaculovirus members were selected the two better regions of homology to design a set of primers (Figure 4).Thus, these three amplicons certified the presence of lepidopteron baculovirus DNA.
In change, because high divergence of pif2 and p49 sequences the primer design was conducted using multiple alignments derived from closest phylogenetic clades (Group I and Group II alphabaculovirus, and betabaculovirus).According to this, different pairs of primers were designed to generate amplicons from baculovirus genomes (Figure 5).(Brudno et al, 2004).A consensus line in the multiple alignment is a set of numbers (between 0-9) that roughly reflect the degree of local similarity among the sequences.These scores were used to generate plots.The regions with higher relative similarity were selected to design primers.These sequences are showed at the top in Sequence Logos (Crooks et al, 2004) Fig. 5. Primer design for pif-2 gene.The orthologous sequences of pif-2 gene from Group I Alphabaculovirus or Group II Alphabaculovirus or Betabaculovirus members were aligned by T-Coffee program (Notredame et al, 2000;Poirot et al, 2003).The regions with higher similarity were selected to design primers.These sequences are showed at the bottom of each multiple alignment in Sequence Logos (Crooks et al, 2004).The cladogram was made with nucleotide sequences of pif-2 Group II Alphabaculovirus using MEGA 4. It showed a significative grouping in two lineages (Group II a and Group II b), which were considered to design primers.For p49 sequence analysis a similar approach was conducted (data not shown) Sets of proposed primers for MP-PCR would allow to detect the proper integrity of genomes in a baculovirus production (Table 3).Table 3. Primer sequences to perform a MP-PCR assay.The table contains all the primer sequences designed by two different approaches and the hypothetical length of amplified fragments using the genome prototypes as reaction template.The specificity of annealing and the size of the amplicons were verified using jPCR (Kalendar et al, 2009).FW: forward primer.REV: reverse primer.Ambiguities are indicated in IUPAC code, B=C,G,T; D=A,G,T; H=A,C,T; K=G,T; M=A,C; N=A,C,G,T; R=A,G; S=C,G; V=A,C,G; W=A,T; Y=C,T

Conclusion
Integrated control management of agricultural pests requires the combination of different insecticide strategies.Among them, the use of baculovirus is an excellent solution as biological control agent.There are many known members of this viral family, with dozens of sequenced genomes.Some of the limitations that exist in their massive application are given by their time of action and modes for their production.Regarding the latter, quality control methodologies are emerging as essential to ensure proper development and formulation.In view of that, in this work are proposed a series of primers for PCR assays that would amplify a fragment profile appropriate to certify the genomic integrity and identity of batch production.Furthermore, adding other specific primers (e.g.specific of transgenes) could be confirmed genotypic stability of genetically modified viruses.Also, the methodology here proposed could be used to characterize new baculoviral isolates, which could be used as bioinsecticides and produced and controlled without the knowledge of their genome sequences.

Fig. 1 .
Fig. 1.Lepidopteron Baculovirus genome phylogeny.Cladogram based on amino acid sequence of 31 core genes.Core genes from Lepidopteron Baculoviridae family were independently aligned using MEGA 4 (GOP = 10, GEP = 1 and Dayhoff Matrix.Then, a concatemer was generated and phylogeny inferred using the same software [UPGMA; Bootstrap with 1000 replicates; gap/Missing data = complete deletion; Model = Amino (Dayhoff Matrix); patterns among sites = Same; rates among sites = Different (Gamma Distributed); gamma parameter = 2.25].Baculoviruses are identified by the acronyms given in Table1and distribution in lineages and genera are also indicated.Clades proposed for Betabaculoviruses are shown in bold letters(Miele et al, 2011)

Fig. 3 .
Fig. 3. Physical maps of ACN, LDN and CPG (Arrows shows the physical location of the 31 Core genes.The five selected Core genes for primer designs are highlighted in bold and red boxed.)

Fig. 4 .
Fig. 4. Primer design for p74, lef-9 and 38K genes.The orthologous sequences of p74, lef-9 and 38K genes from Alpha and Betabaculovirus members were aligned by CHAOS/DIALIGN program(Brudno et al, 2004).A consensus line in the multiple alignment is a set of numbers (between 0-9) that roughly reflect the degree of local similarity among the sequences.These scores were used to generate plots.The regions with higher relative similarity were selected to design primers.These sequences are showed at the top in Sequence Logos(Crooks et al, 2004) This work was supported by research funds from Agencia Nacional de Promoción Científica y Técnica (ANPCyT) and Universidad Nacional de Quilmes.PDG is member of the Research Career of CONICET (Consejo Nacional de Ciencia y Tecnología); MNB holds a postdoctoral fellowship of CONICET, SABM holds a fellowship of CONICET and MJG holds a fellowship of CICBA.