In vitro Production of Nuclear Polyhedrosis Virus of Helicoverpa ...

Persian Gulf Crop Protection Available online on: www.cropprotection.ir ISSN: 2251-9343 (Online)

Volume 2 Issue 3, September 2013 Pages 18-31

In vitro Production of Nuclear Polyhedrosis Virus of Helicoverpa armigera Neeta Sharma1* and Ritu Srivastava1 1-Department of Botany, Lucknow University, Lucknow, Uttar Pradesh, India (*Corresponding author e-mail: [email protected]).

Abstract: Helicoverpa armigera is currently placed on Annex I, A II of Council Directive 2000/29/EC, indicating that it is considered to be relevant for the entire Europian Union and that phytosanitary measures are required when it is found on any plants or plant products. Although, nearly 30% of total insecticides are used for controlling this pest alone on different crops, yet many of them do not prove effective because pest has been reported to have developed resistance to almost all groups of insecticides (Armes et al., 1996 and Yaqoob et al., 2006). Therefore, the demand in the present scenario is the formulation of eco-friendly means of pest control to minimize pesticide related problems and to ensure long term sustainable yield through sound ecological and sustainable management solutions. Helicoverpa armigera single nucleopolyhedrovirus (HaSNPV), a wild-type baculovirus, has the potential for use as a bio-pesticide for effective management of this pest and also incorporated successfully in integrated pest management packages in different crops. HaSNPV is specific and highly virulent to its host. The development of in-vitro production of HaSNPV is becoming increasingly important as the in-vivo production cost is expensive and labour intensive.

Key Words: Helicoverpa armigera, Nuclear polyhedrosis virus, HaNPV, In-vitro production.

Persian Gulf Crop Protection, 2(3): 18-31

18

Introduction Helicoverpa a National challenge Helicoverpa species are highly polyphagous and widely distributed in tropics and subtropics. The pest is present and widespread in Asia, Africa and Oceania (EPPO, 2006). It is established in the Bulgaria, Greece, Portugal, Romania, Spain (widespread) and Cyprus, France, Hungary and Italy (restricted distribution). In India, it’s periodic out breaks causes total crop failure in different locations, viz, Andhra Pradesh, Maharashtra, MP, UP, Rajasthan, Karnataka and Punjab. Annual loss has been estimated to US $ 500 million plus US $ 127 million spent on insecticide (Chandurkar et al., 2005). It takes a heavy toll of the crops, as happened in cotton belt in Punjab and Haryana during 1983-84; in coastal cotton belt of Andhra Pradesh during 1987-1988; in cotton growing areas of Rajasthan, Punjab and Haryana during 1990 and in different parts of Uttar Pradesh during 1995-96. The changing crop practices such as introduction of early maturing varieties of pigeonpea, summer cultivation of sunflower and ground nut, introduction of hybrids of sorghum and maize coupled with intensive and extensive agronomic practices have contributed towards greater prevalence and increased severity. Bio- management solutions The bio-control methods require detailed information about host, pathogen, effectiveness of indigenous strain and their dynamics. Residual insecticides have been employed to control insect pests of agriculture crops, but alternative control strategies are desirable because of the loss of insecticides due to pest resistance and consumer desire for pesticide free grain. The biggest impetus for the growth of biopesticides comes from the growing awareness by farmers of the value of integrated pest management as a more environmentally sound, economical, safer and a selective approach to crop protection (Fig:1). Persian Gulf Crop Protection, 2(3): 18-31

Providing safe, economical and reliable bio-management solutions for insect-pests is not only a contribution to sustainable agriculture, but also an answer to the increasing population. Bio-management solutions includes  Bio-management solutions for soil fertility includes bio-fertilizers (Rhizobium, Azoto, Azospirillum, Potash solubilizers, potash mobilizing bacteria, etc.), organic manures, compost, mulching, organic micro-nutrients solutions  Bio-chemical solution includes pheromone traps, yellow and light traps  Botanicals for crop pest and disease management  Crop protection solutions includes Trichocards, Chrysoperlla, Baculoviruses, Bacillus thuringiensis, Trichoderma spp, Verticillium spp, Beauveria spp, Pseudomonas spp and Metarhyzium spp, etc Increasing awareness of the benefits of IPM and bio-management solutions, as well as economic and environmental impacts of chemical pesticides, are the driving forces behind changes in plant protection policies. The future for entomopathogens as agent for management of insect pests in developing countries like India is promising. Viruses for control of Insect Pests Baculoviruses are target specific viruses which are capable of destroying a number of important agriculture crop pests. These are a large group of double stranded DNA viruses; the majority has been isolated from insect orders: Lepidoptera, Diptera, Hymenoptera and Coleoptera. Viral genome ranges in size from 80 to 200 kb. The most widely studied baculovirus is the Autographa californica nucleopolyhedrovirus (AcMNPV: Fig:2 and 3) (Boguslaw et al., 201). Baculoviruses are specific to their host and do not replicates in vertebrates, plants and 19

micro-organisms. Under special conditions, they enter animal cells. This unexpected property made them a valuable tool for studies of transient expression of foreign gene under vertebrate promoters introduced into baculovirus genome (Kost et al., 2005). Virions consist of one or more nucleocapsids embedded in a membranous envelop. Two morphologically distinct but genetically identical viral forms are produced during post infection: a. Budded virus particles (BV) which serve for the transmission of the virus to other tissues of the infected pest. b. Occlusion bodies (OB) which are responsible for the survival of the virus and spread of the disease. The occlusion bodies (polyhedra) of nucleopolyhedrovirus contain many occlusion-derived virions (ODV) surrounded by a matrix composed mainly of polyhedrin, a major structural protein (Braunagel et al., 2003). Polyhedrin is produced in large quantities (around 30% of total protein mass at the time of host death) but it is not needed for the transmission of the virus from cell to cell. Polyhedra are relatively stable and the protected virions in the favourable conditions can survive in the environment for more than twenty years. Under magnification of around 1000x, polyhedra resemble clear, irregular crystals of salt so they are big enough to be seen in a light microscope. Some common symptoms of the virus attack are:  Sluggishness  Discoloration of skin  Wet or extremely moist droppings  Regurgitation of fluids (a sign of stress)  Shrivelling of the hairlike antennae at either end of the caterpillar Autographa californica and Anagrapha falcifera NPVs were registered in various countries and have relatively broad host spectrum and potentially can be used on a variety of crops infested with pests Persian Gulf Crop Protection, 2(3): 18-31

including Spodoptera and Helicoverpa. Use of Spodoptera litura NPV has been tested on cabbage crops in India (Kumari et al., 2009). Granulovirus GV is the active component of a number of biopesticides used for protection of apple and pear orchards against the coddling moth, Cydia pomonella. Some of the trademarks of GVbased products are following: Granusal™ in Germany, Carpovirusine™ in France, Madex™ and Granupom™ in Switzerland, Virin-CyAP in Russia. Annually up to 250 000 hectares of orchards have been protected with MadexTM in different European countries (Vincent et al., 2007). Another granulovirus, Erinnyis ello (cassava hornworm) granulovirus, was found to be very efficient for protection of cassava plantations (Bellotti, 1999). This GV has been used for spraying cassava crops in South American countries. In China twelve baculoviruses have been authorized as commercial insecticides (Sun and Peng 2007), including H. armigera NPV (the most widely used virus in China for cotton, pepper and tobacco protection), S. litura NPV (vegetables), S. exigua NPV (vegetables), Buzura suppressaria NPV (tea), Pieris rapae GV and Plutella xylostella GV (vegetables). Use of baculoviruses in China is the greatest worldwide, regarding the number of viruses being registered for insect control. Sun and Peng (2007) also reported a Cypovirus (CPV) produced in China for control of Dendrolimus punctatus, an insect pest of pine forests. The well-known success of employing baculovirus as a biopesticide is the case of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) used to control the velvetbeen caterpillar in soybean (Moscardi, 1999). This program was implemented in Brazil in the early eighties, and came up to over 2,000,000 ha of soybean treated annually with the virus. The use of AgMNPV in Brazil brought about many economical, ecological and social benefits. The protection of soybean fields in Brazil has proven that baculoviral 20

control agents can be effectively produced on a large scale and they may be an alternative to broad-spectrum chemical insecticides. On the basis of spectacular success of a baculovirus pesticide (Table: 1), it is needless to say that the advantages of biopesticides over chemical pesticides are numerous. Safety for humans and nontarget organisms, preservation of biodiversity in the environment, reduction of toxic residues in agricultural endproducts are just the examples of potential benefits. However, the cost of biopesticide production has been usually higher than the cost of conventional pesticides. So, paradoxically, countries where the cost of human labour is low are more open towards the use of baculoviral pesticides than highly-developed countries which claim that environmental protection is one of their priorities in the development. The first viral insecticide Elcar™, introduced in 1975 (Ignoffo and Couch, 1981) was a preparation of Heliothis zea NPV which is relatively broad-range baculovirus responsible to infect species belonging to genera Helicoverpa and Heliothis. Countries with large areas of such crops like cotton, pigeonpea, tomato, pepper and maize, e.g. India and China, introduced special programs for the reduction of this pest by biological means. In Central India, H. armigera in the past was usually removed by shaking pigeonpea plants until caterpillars fell from the plants onto cotton sheets. This technique is now used to obtain caterpillars which are fed on virusinfected seeds. Baculovirus preparations obtained in this way are used by farmers to prepare a bio-insecticide spray applied on pigeonpea fields. Another baculovirus, HaSNPV is almost identical to HzSNPV, was registered in China as a pesticide in 1993 (Zhang et al., 1995). It has been used for large scale biopesticide production and has been extensively used on cotton fields. Broad spectrum biopesticide based on


HaNPV is also used in India (Srinivasa et al., 2008). The forests of temperate regions are very often attacked and defoliated by larvae of Lepidoptera (most common pest species are: Lymantria dispar, Lymantria monacha, Orgiya pseudotsugata and Panolis flammea) and some Hymenoptera species (mainly Neodiprion sertifer and Diprion pini). L. dispar MNPV formulations marketed under trade names: Gypchek, Disparivirus, Virin-ENSH, and O. pseudotsugata MNPV under trade names: TM BioControl-1 and Virtuss (Reardon et al., 1996) are sometimes used for forest protection. Forest ecosystems tend to be more stable than agricultural systems, allowing for natural or applied baculoviruses to remain in the environment for long periods of time increasing the chance of natural epizootics by these agents. Two commercial preparations based on Spodoptera NPV have been available. These are SPOD-X™ containing Spodoptera exigua NPV to control insects on vegetable crops and Spodopterin™ containing Spodoptera littolaris NPV which is used to protect cotton, corn and tomatoes (Boguslaw et al., 2011). HaNPV Production: Present Scenario In the fields, natural mortality of Helicoverpa and Spodoptera can be seen due to infestation of disease causing virus particles. Such larvae can be collected and may be utilized again for checking pest populations. The virus is specific to insect larvae causing heavy mortality but has no deleterious effect on non-target insects, animals or crops; it is therefore safe for natural enemies and the environment. Completely compatible with the all biologically based IPM approaches, field trials on chickpea in India showed that HaNPV at economic applications could control H. armigera more effectively than chemical insecticides or commercially formulated B. thuringiensis (Cherry et al, 2000). ICRISAT (Rao et al., 2007) trained several NARES scientists and farmers on 21

biopesticide production and established 96 village level NPV production units in India and Nepal to encourage their use. On-farm studies in biopesticide front indicated 20-310% increased yields in- pigeonpea and chickpea. Bio- intensive cotton IPM crops realized I-30% and vegetable farmers obtained 72% increased yields through better management of pests and augmenting natural enemies. As the selection of virulent strain of NPV is key to the development of effective bioinsecticides, local strains are always preferred for sustainability, adaptability and efficacy under a given set of agroecosystem and hold an ample scope for their wide spread multiplication and commercial use in a particular region (Gupta et al., 2007 and 2010). It is well recognized that factors such as the geographic origins of both virus and host can affect the characteristics of the doseresponse curve and the period of survival of infected hosts (Maeda et al., 1990). A study was carried out during 2001-03 under laboratory conditions in the Department of Agricultural Entomology, University of Agricultural Sciences, Dharwad, India (Kambrekar et al., 2007). The study reported that there has been a rapid growth in the production and use of NPV products in the last decade, but this has exacerbated quality problems. The causes of poor quality lay in deficiencies in production technologies and poor quality control procedures. For HaNPV production, host insect Helicoverpa armigera has to be multiplied on artificial or semi synthetic diet. Crude HaNPV is commercially produced at Dr. Punjarao Deshmukh Krishi Vidyapeeth, Krishinagar, Akola (Maharashtra) and at Agricultural Research Station Gulbarga (Karnataka). Following procedures are involved during in vivo mass production:  Host Insect Multiplication  Virus Inoculation and harvest  Extraction and purification of virus  standardization of NPV Persian Gulf Crop Protection, 2(3): 18-31

Nuclear polyhedrosis virus of Helicoverpa armigera (HaNPV) has been most extensively researched and studied with regards to its efficacy, mass production, compatibility with botanicals and other insecticides, and against several non target organisms( Hunter-Fujita et al., 19986 and Saxena and Ahmad 20057). These viruses are being producd in vivi and registered under various trade names, viz., Viron-H, Bio-control- VHZ, Elcar and Gemstar LC in various countries. The use of HaNPV is now a day is inundative and does not utilize their full potential. Large-scale production of HaNPV is currently labour intensive; it involves mass rearing and infection of insect larvae, which account for a major part in production costs. Pathogenicity of the isolates varies according to localities and therefore needs to be screened. For successful management of this specific crop pest, the viable and virulent indigenous materials should be evaluated. Economical cell culture techniques for large-scale production for this virus have not yet been developed for the agricultural use. In- Vitro production of HaNPV In the early part of the 20th century, entomologists had a dream of utilizing insect cells grown in vitro as a tool for producing entomo-pathogens. These early experiments used a simple saline solution or hemolymph as the culture medium and cultures could rarely be kept for more than a few days. A breakthrough occurred four decades ago when Grace (1962) successfully established long-term cultures of insect cells. Since then, over 500 continuous cell lines have been established from over 100 insect species (Lynn, 1999). Insect pathologists have cells capable of replicating dozens of insect-specific viruses (Granados and McKenna, 1995) while plant pathologists and vertebrate pathologists have cells capable of replicating viruses transmitted by insects (Mitsuhashi, 1989). Mass production of the virus at reasonable costs 22

is an important factor in the development of NPVs into a marketable product. Lack of appropriate formulation technologies for HaNPV limit their use. Insect cell lines Baculoviruses can be produced in vitro in infected insect cells cultivated in bioreactors. In order to develop on economically feasible process to produce baculoviruses in insect cells, low-cost culture media that satisfy the nutritional demands of both uninfected and infected cells are needed to achieve high virus yields. Fetal calf serum is the most widely used additive in insect cell culture media. However, its high cost, undefined nature and batch-to-batch variability make disadvantageous its utilization to sustain baculovirus production in large scale processes. Therefore, the replacement of fetal calf serum in insect cell culture media is the key step to develop a technically and economically feasible process to produce baculoviruses in vitro. These results demonstrate that insect’s cells are able to both proliferate permanently and replicate baculovirus in a lipid free environment. However, in order to obtain a useful medium for technological applications, it will be necessary to optimize the composition of the multiple supplements and evaluate its performance in a lipid supplemented environment. Insect cell cultures have been extensively utilized by Linda and Reid (2003) for means of production for heterologous proteins and bio-pesticides. Spodoptera frugiperda (Sf9) and Trichoplusia ni (High Five™) cell lines have been widely used for the production of recombinant proteins, thus metabolism of these cell lines have been investigated thoroughly over recent years(Table:2). The following table summarizes some general characteristics of the cell lines: A virus isolated from the alfalfa looper, Autographa californica, replicated successfully and rapidly in a suspended ovarian cell line of the cabbage looper, Trichoplusia ni (Vail and Jay, 1973). Persian Gulf Crop Protection, 2(3): 18-31

Polyhedra were observed in the nucleus of cells within 20 hr after inoculation. The cytopathological changes typical of nuclear polyhedrosis infections were observed, and an average of 64 polyhedra/cell were produced. These polyhedra were quantitatively as infectious to cabbage looper larvae as those produced in vivo. In addition, they were infective to Heliothis virescens, Spodoptera Anagrapha

Pectinophora gossypiella, exigua, A. californica, and falcifera.Bioassays have

indicated that both the H. zea and H. armigera viruses produced in vitro maintain biological activity (Suzanne, 2009 and Boguslaw et al., 2006). Chakraborty et al., (1999) studied in vitro production of virus from Helicoverpa armigera (HaSNPV) and its possible use as a specific Helicoverpa / Heliothis larvicide. Growth kinetics of Helicoverpa zea (H. zea) cells and virus occlusion body yields were compared in three SF900IIbased media, namely, SF900II (serumfree), SF900II + 1% serum, or SF900II + 10% serum. Viable cell densities were usually higher in the media supplemented with serum than in the serum-free medium; however, in the serum-free medium, cell diameters were 1.7 times greater (i.e., individual cell volumes were five times larger). Four new cell lines, designated as NTULY-1 to -4, respectively, were established from the pupal tissues of Lymantria xylina Swinhoe (Lepidoptera: Lymantriidae) (Chih and Wang, 2006). These cell lines have been cultured approximately 80 passages during 2 years in TNM–FH medium supplemented with 8% fetal bovine serum, at a constant temperature of 28 °C. Each line consists of three major morphological types: round cells, spindleshaped cells, and giant cells (Fig 4). Sundeep et al., 2005 developed two cell lines from the larval haemocyte and embryonic tissue of H. armigera and designated them as NIV-HA-1195 and NIV-HA-197, respectively. The NIV-HA197 cell lines were found highly 23

susceptible to HaSNPV, yielding a very high titre (2.88 x 107 NPV/ml) on the 10th PID83. The HaSNPV grown in the cell culture was bioassayed in different instar larvae of H. armigera. The results indicated that the OBs produced in vitro were highly virulent to 2nd and 3rd instar H. armigera larvae causing cessation of feeding on the 2nd day and mortality in 6 days. This cell lines is also growing in goat serum (GS) supplemented medium producing a comparable yield of OBs. Goat serum, being cheap and locally available will help in the large scale production of HaSNPV for use as a biopesticide in future. The cell line NIVHA-197 was found to be susceptible to the baculoviruses Autographa californica multiple nucleopolyhedrovirus, Spodoptera litura multiple nucleopolyhedrovirus, and H. armigera single nucleopolyhedrovirus (HaSNPV) (Sundeep et al., 2002). More than 90% of the cells were infected by HaSNPV on the seventh post infection day (PID), and 28.8 x 106 NPV/ml were yielded on the 10th PID. The in vitrogrown HaSNPV caused 100% mortality, when fed to the second instar H. armigera larvae, in 6 days. Cessation of feeding was observed on the second PID. Isoenzyme profile and results of 16S rRNA heteroduplex analysis clearly indicated the species specificity of the new cell line NIV-HA-1195 (Sundeep et al., 2002) and was also found susceptible to the baculoviruses, Autographa californica nucleopolyhedrovirus (AcNPV), Spodoptera litura NPV and the homologous HaNPV. Pant et al., 2002 reported that the Helicoverpa armigera cell line from the embryonic tissue was highly susceptible to HaNPV (6.3 x 106 NPV/ml). These in vitro grown HaNPV caused 100% mortality to respective 2nd instar larvae. The cultures could grow as suspension culture on shakers and may find application for in vitro production of wild type/recombinant baculoviruses as bioinsecticides. Nakat (2004) standardized the procedure for monolayer and spinner Persian Gulf Crop Protection, 2(3): 18-31

culture of Sf-9, Sf-21 and Ha-197 cells. The growth curve of different cells in spinner culture was plotted on the basis of daily viable cell count. The cell line Sf-9 was susceptible for both the baculoviruses AcMNPV and SlNPV in monolayer. The cell like Ha-197 was found susceptible for HaNPV in mobolayer. For production of HaNPV, Ha-197 cell line with HaNPV wild type MPKV strain found to be efficient, more virulent and infectious in both the cell lines. The procedure for the cell lysis was standardized to extract the PIBs from infected cells with addition of 0.1% SDS and deep freezing at -20oC followed by 15 minute sonication resulted into good separations of PIBs from 80 to 90% infected cells. The field demonstrations of in vitro and in vivo produced SlNPV and HaNPV were conducted on capsicum, gerbera, rose, soybean and chickpea. The effectiveness of in vitro produced SlNPV and HaNPV was found to be superior causing larval mortality in the range of 78-100% as compared to in vivo produced virus 70 to 88%. The insect cell line, the culture medium, the bioreactor, the virus, the infection parameters and the culture strategy are elements of the insect cell culture technology that must be optimized in order to develop in vitro production processes for insecticidal baculoviruses (Claus et al., 2012). The cell line Hz-AM1 has been used widely to examine possible factors affecting the yields and the potency of HearNPV. These factors include medium, supplemented serum, cell density at infection, multiplicity of infection, viral strain and passage effect (Chakravorty et al., 1996, Lua and Reid, 2000 and Ogembo et al., 2007). These studies confirmed that the most serious limitations on the in- vitro production of HaNPV in the cell line HzAM1 was rapid formation of polyhedral mutants of low virulence during serial passage (Chakravorty and Reid, 1999). Two cell lines from embryos of Ephestia kuehniella were developed by Lynn and Ferkovich in 2004. Strains were found 24

highly susceptible to various insect viruses, including nucleopolyhedroviruses from Autographa californica, Anagrapha falcifera, Anticarsa gemmatalis, Galleria mellonella, Heliothis armigera, and Plutella xylostella. Both strains were highly susceptible to most of the nucleopolyhedroviruses with large numbers of occlusion bodies produced in most of the inoculated cells. These results suggest that new lines can be useful in biocontrol research. Increasing occlusion body (OB) yields per cell in culture is the main challenge to enable commercialization of in vitro production of baculovirus pesticides. Isolating clones from a heterogeneous cell population may allow development of a high virus producing cell clone. An automated robotic clone picking system to establish over 250 insect clones of a Helicoverpa zea cell population to be screened for virus production has been carried out by Nguyen

et al., 2011(Fig 5 and 6). The type and degree of passage effect are dependent on the cell lines, the virus species (Krell, 1996). Homologous cell lines are desirable for the production of HaNPV. Heterologous NPV infection of cell lines decreases the productivity, and yields less virulent progeny viruses (Tompkins et al., 1988). Since, HaNPV can be produced only in living larvae, the cost of production can be quite high, as H. armigera requires isolated rearing and high labour cost. Hence, intensive search is require to yield a viral isolate with more virulence and greater resistance than the existing ones from indigenous areas and in-vitro production process may be standardized that would greatly enhance the efficacy of NPV of H. armigera as a microbial insecticide. The current technology is still insufficient to achieve economic feasibility.

Figure 1. Total Pest Management Solution

Figure 2. Baculovirus


25

Figure 3. Autographa californica nucleopolyhedrovirus

Table 1: Current use of Baculoviruses as biological insecticides Commodity Insect pest Virus Used Apple, pear, walnut and Coding moth Coding moth granulosis plum virus Cabbage, tomatoes, Cabbage army worm nuclear Cabbage moth, American bollworm, cotton, (and see pests in polyhedrosis virus diamondback moth, patato tuber moth next column) Spodoptera littoralis Spodoptera littoralis nuclear Cotton, corn, tomatoes polyhedrosis virus Helicoverpa zea nuclear Cotton and vegetables Helicoverpa zea, and Heliothis virescens polyhedrosis virus Vegetable crops, Spodoptera exigua nuclear Beet armyworm (Spodoptera exigua) greenhouse flowers polyhedrosis virus Vegetables Anagrapha falcifera nuclear Celery looper (Anagrapha falcifera) polyhedrosis virus Alfalfa and other crops Autographa californica Alfala looper (Autographa californica) nuclear polyhedrosis virus

Figure 4. Phase-contrast micrographs of new L. xylina cell strains, showing: three major cell types in each line, round cells (R), spindle-shaped cells (Sp), and giant cells (G)


26

Table:2 General characteristics of insects cell lines Cells

Sf9

Sf21

High Five™

General Characteristics Sf9 and Sf21 cell lines (Spodoptera frugiperda: ovarian tissue) are the traditional cell lines used with baculovirus and suitable for use in the Insect Select System. These two cell lines derived from the pupal ovarian tissue of the fall army worm, Spodoptera frugiperda (O'Reilly et al., 1992). Characteristics: • Grow well in monolayer and suspension culture • Adaptable to serum-free medium Origin: The High Five™ cell line was developed by the Boyce Thompson Institute for Plant Research, Ithaca, NY and originated from the ovarian cells of the cabbage looper, Trichoplusia ni (embryonic tissue). Characteristics: • Doubles in less than 24 hours • Grows well in adherent cultures, but forms irregular monolayers, thus making plaques more difficult to identify • Adaptable to suspension culture and serum-free medium • Provides 5–10 fold (for selected proteins) higher secreted expression than Sf9 cells (Davis et al., 1992)

Figure 5. SDS PAGE analysis confirming the difference in OB yield's of key clones compared to the parent cells. OB samples from 7 × 103 infected cells, collected from three infections at early (P5), mid (P15), and late (P25), passages at 7 d.p.i.

Figure 6. Cell Growth curves of the highest producing (C148, C160), and lowest producing (C150 and C160), clones as compared to the parental cell population (Parent). The low yielding clones grew faster and to a higher cell density than the control and high yielding clones.


27

References [1]

[2]

[3]

[4]

[5]

[6]

[7]

[8]

[9]

Arrnes, N.J., D.R. Jadhav and D.K.R. Souza, 1996. A survey of insecticide resistance in Helicoverpa armigera in the Indian subcontinent. Bulletin of Entomological Research, 86: 499-514. Bellotti, A. C. 1999. Recent advances in cassava pest management. Annual Review of Entomology, 44:345-370. Boguslaw Szewczyk, Liliana HoyosCarvajal, Maria Paluszek, Iwona Skrzecz and Marlinda Lobo de Souza. 2006. Baculoviruses — re-emerging biopesticides. Biotechnology Advances, 24 (2): 143–160. Boguslaw, Szewczyk, Marlinda Lobo de Souza, Maria Elita Batista de Castro, Mauricio Lara Moscardi and Moscardi, F. 2011. Baculovirus biopesticides, pesticides-formulations, effects, fate. Ed. By Margarita Stoytcheva. ISBN: 978953-307-532-7, In Tech. http://www.intechopen.com/books/pesti cides-formulations-effectsfate/baculovirus-biopesticides. Braunagel, S. C., Russell, W. K., RosasAcosta, G., Russell, D. H., and summers, M. D. 2003. Determination of protein composition of the occlusionderived virus of Autographa californica nucleopolyhedrovirus. Proceedings of the National Academy of Sciences,100: 9797-9802. Chakraborty, S., Monsour, C., Teakle, R. and Reid, S. 1999. Yield, biological activity, and field performance of a wild-type Helicoverpa nucleopolyhedrovirus produced in H. zea cell cultures. Journal of Invertebrate Pathology, 73 (2):199-205. Chakraborty, S., Green field, P. and Reid, S. 1996. In-vitro production studies with a wild type Helicoverpa baculovirus. Cytotechnology, 22: 217224. Chakravorty, S. and Reid, S. 1999. Serial passage of a Helicoverpa armigera nucleopolyhedrosis virus in Helicoverpa zea cell culture. Journal of Invertebrate Pathology, 73: 303-308. Chandurkar, P. S., Thakur, J. N. and Shukla, R. M. 2005. Indian scenario of plant protection with special reference to Helicoverpa armigera- past, present and future. Recent advances in Helicoverpa


[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

management. Proceedings of the National Symposium on Helicoverpa Management: A National Challenge. Eds: Hem Saxena, A.B. Rai, R. Ahmed and Sanjeev Gupta, IIPR, pp-1-10. Cherry, A. C., Rabindra, R. J., Gryzwacz, D., Kennedy, J. S and Satthaih, R. 2000. Field evaluation of Helicoverpa amigera NPV formulations for control of the chickpea pod-borer, H.armigera (Hubner) on chickpea in Southern India; Crop Protection, 19; 51-60. Chih-Yu Wu and Chung-Hsiung Wang. 2006. New cell lines from Lymantria xylina (Lepidoptera: Lymantriidae): Characterization and susceptibility to baculoviruses. Journal of Invertebrate Pathology, 93(3): 186–191. Claus, Juan D., Gioria, Verónica V., Micheloud, Gabriela A. and Visnovsky, G. 2012. Production of Insecticidal Baculoviruses in Insect Cell Cultures: Potential and Limitations, Insecticides Basic and Other Applications, Dr. Sonia Soloneski (Ed.), ISBN: 978-953-510007-2. In Tech, Available from: http://www.intechopen.com/books/insec ticides. Davis, T. R., Trotter, K. M., Granados, R. R. and Wood, H. A. 1992. Baculovirus Expression of Alkaline Phosphatase as a Reporter Gene for Evaluation of Production, Glycosylation, and Secretion. Bio-Technology, 10: 1148-1150. Donaldson, M. S. and Shuler, M. L. 1988. Low-cost serum-free medium for the BTI-Tn5B1-4 insect cell line. Biotechnology Progress, 14 (4):573-579. EPPO, 2006. Distribution maps of Quarantine Pests. Helicoverpa armigera. On-line available at www.eppo.org/QUARANTINE/insects/ Helicoverpa_armigera/HELIAR Grace, T. D. C. 1962. Establishment of four strains of cells from insect tissue grown in vitro. Nature, 195:788–789. Granados RR, McKenna KA. 1995. Insect Cell Culture Methods and Their Use in Virus Research. Ed by Schuler ML, Wood HA, Granados RR, Hammer DA. Baculovirus Expression Systems and Biopesticides, 13–39.New York: Wiley-Liss.

28

[18] Gupta, R. K., Raina, J. C., Arora, R. K. and Bal, K. 2010. Selection and field effectiveness of nucleopolyhedrovirus isolates against Helicoverpa armigera (Hubner). International Journal of Virology, 6 (3): 164-178. [19] Gupta, R. K., Raina, J. C. and Monobrullah, M. D. 2007. Optimization of in vivo production of nucleopolyhedrovirus in homologus host larvae of Helicoverpa armigera. Journal of Entomology, 4: 279-288. [20] Hunter-Fujita, R. F., Entwistle, P. F., Evans, F. H. and Crook, N. E. 1998. Insect virus and pest management. John Willey and Sons, Chichester, U.K. p 620. [21] Ignoffo, C. M. and Couch, T. L. 1981. The nucleopolyhedrosis virus of Heliothis species as a microbial pesticide. In Microbial Control of Pests and Plant Diseases, ed. By H. D. Burges, pp. 329-362. London: Academic Press. [22] Kambrekar, D. N. Kulkarni K. A. and Giraddi, R. S. 2007. Assessment of Quality of HaNPV Samples Produced by Private Firms. Karnataka Journal of Agricultural Sciences, 20 (2): 417-419. [23] Kost, T. A., Condreay, J. P. and Jarvis, D. L. 2005. Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nature Biotechnology, 23:567-575. [24] Krell, P. J. 1996. Passage effect of virus interaction in insect cells. In: Insect cell culture, Fundamentals and applied aspects. Ed. By J.M. Vlak, C.D. De Gooigee, J. Tramper and H. G. Miltemburger. Pp. 125-137. Kluwer Academic Publishers, A.H. Dordrecht, The NItherlands. [25] Kumari, V. and Singh, N. P. 2009. Spodoptera litura nuclear polyhedrosis virus (NPV-S) as a component in Integrated Pest Management (IPM) of Spodoptera litura (Fab.) on cabbage. Journal of Biopesticides, 2: 84-86. [26] Lammers, J. W. and MacLeod, A. 2007. Report of a Pest Risk Analysis: Helicoverpa armigera (Hübner, 1808). Plant Protection Service (NL) and Central Science Laboratory (UK) joint Pest Risk Analysis for Helicoverpa armigera.


[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

www.fera.defra.gov.uk/plants/plantHealt h/pestsDiseases/documents/Helicoverpa. Lenz, C. J., MCIntosh, A. H., Mazzacano, C. and Moodcrloh, U. 1991. Journal of Invertebrate Pathology, 57, 227-233. Linda H.L. and Lua, Steven Reid. 2003. Growth, viral production and metabolism of a Helicoverpa zea cell line in serum-free culture. Cytotechnology, 42 (3): pp 109-120. Lua, H. L. and Reid, S. 2000. Serial passage of a Helicoverpa armigera nucleopolyhedrosis virus in Helicoverpa zea serum free suspension culture. Journal of General Virology, 81: 25312543. Lynn, D. E. 1999. Development of insect cell lines: Virus susceptibility and applicability to prawn cell culture. Methods in Cell Science, 21:173–81. Lynn, D. E. and Shapiro, M. 1988. New Cell Lines from Heliothis virescens: Characterization and Susceptibility to Baculoviruses. Journal of invertebrate pathology, 72: 276–280. Maeda, S., Mukoara, Y. and Kondo, A. 1990. Characteristically distinct isolates of the nuclear polyhedrosis virus from Spodoprem litum. Journal of General Virology, 71: 2631-2639. Mitsuhashi J. 1983. A continuous cell line derived from fat bodies of the common armyworm, Leucania separata (Lepidoptera: Noctuidae). Applied Entomology and Zoology, 18: 533–539. Moscardi, F. 1999. Assessment of the application of baculoviruses for control of Lepidoptera. Annual Review of Entomology, 44: 257–289. Nakat, R.K. 2004. In vitro production of nuclear polyhedrosis virus of Helicoverpa armigeraand Spodoptera litura and its field efficacy in Western Maharashtra. Asst. Prof., Department of Entomology, Mahatma PhuleKrishiVidyapeeth, Rahuri, Ahmednagar-413722, Maharashtra (Unpublished). Nguyen, Quan, Ying Mei Qi, Yang Wu, Chan, Leslie C. L., Nielsen, Lars K. and Reid, S. 2011. In vitro production of Helicoverpa baculovirus biopesticides— Automated selection of insect cell clones

29

[37]

[38]

[39]

[40]

[41]

[42]

[43]

[44]

for manufacturing and systems biology studies. Journal of Virological Methods, 175(2):197–205 Ogemo, J. G., Chaeychomsri, S., Kamiya, K., Ishikawa, H., Katou, Y., Ikeda, M. and Kobayashi, M. 2007. Cloning and comparative characterization of nucleopolyhedrosis viruses isolated from African bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae) in different geographic regions. Journal of Insect Biotechnology and Sericology, 76: 39-49. O'Reilly, D. R., Miller, L. K. and Luckow, V. A. 1992. Baculovirus Expression Vectors: A Laboratory Manual (New York, N. Y.: W. H. Freeman and Company). Pant, U., Sudeep, A. B., Athawale, S. S. and Vipat, V. C. 2002. Baculovirus studies in new, indigenous lepidopteran cell lines. Indian Journal of Experimental Biology, 40 (1):63-8. Rao, G. V. Ranga; Rupela, O. P., Rameshwar Rao, V. and Reddy, Y. V. R. 2007. Role of Biopesticides in Crop Protection: Present status and Future Prospects. Indian Journal of Plant Protection, 35 (1): 1-9. Reardon, R., Podgwaite, J. P., and Zerillo, R. T. 1996. GYPCHECK - the gypsy moth nucleopolyhedrosis virus product. USDA Forest Service Publication FHTET-96 – 16. Saxene, Hem and Ahmad, R. 2005. NPV production, formulation and quality control. In: “Recent advances in Helicoverpa management.” Proceedings of the National Symposium on Helicoverpa Management: A National Challenge. Eds: Hem Saxena, A.B. Rai, R. Ahmed and Sanjeev Gupta, IIPR, pp1-10. Srinivasa, M., Jagadeesh Babu, C. S., Anitha, C. N., and Girish, G. 2008. Laboratory evaluation of available commercial formulations of HaNPV against Helicoverpa armigera (Hub.). Journal of Biopesticides, 1:138-139. Sudeep, A. B., Shouche, Y. S., Mourya, D. T. and Pant, U. 2002. A new cell line from the embryonic tissue of Helicoverpa armigera HBN. (Lepidoptera: Noctuidae). In Vitro


[45]

[46]

[47]

[48]

[49]

[50]

[51]

[52]

[53]

Cellular and Developmental Biology – Animal, 38 (5):262-4. Sun, X. L. and Peng, H. 2007. Recent advances in biological pest insects by using viruses in China. Virologica Sinica, 22:158-162. Sundeep, A. B., Mourya, D.T.and Mishra, A. C. 2005. Insect cell culture in research: Indian scenario. Indian Journal of Medical Research, 121, 725-738. Sundeep, A. B., Shouche, Y.S., Mourya, D.T. and Pant, U. 2002. New Helicoverpa armigera Hbn cell line from larval hemocyte for baculovirus studies. Indian Journal of Experimental Biology, 40 (1):69-73. Suzanne M. Thiem, 2009 .Baculovirus genes affecting hostfunction. In vitro Cellular and Developmental Biology Animal, 45: (3 and 4): 111-126. Thompson, C. S., Claus, J. D. and Beccaria, A. J. 2005. Evaluation of alternative low-cost supplements for the development of a serum free media for insect culture. 4th Mercosur Congress on Process Systems Engineering. Instituto Nacional de Tecnología Agropecuaria (INTA). RN 34. Km 227. (S2300) Rafaela. Santa Fe.Argentina. Tompkins, G. J., Daugherty, E. M., Adams, J. R. and Diggs, D. 1988. Changes in the virulence of nuclear polyhedrosis virus when propagated in alternate Noctuid (Lepidoptera: Noctuidae) cell lines and hosts. Journal of Economic Entomology, 81: 10271032. Vail, P. V. and Jay, D. L. 1973. Replication and infectivity of the nuclear polyhedrosis virus of the alfalfa looper, Autographa californica, produced in cells grown in vitro. Journal of Invertebrate Pathology,22, 2:231–237 Vincent, C., Andermatt, M., Valéro, J. 2007. Madex® and VirosoftCP4®, viral biopesticides for coddling moth control. In Biological Control: A Global Perspective, eds. C. Vincent, M. S. Goethel, and G. Lazarovits, pp. 336-343. Oxfordshire, UK, and Cambridge, USA: CAB International. Yaqoob, M., Arora, R. K. and Gupta, R. K. 2006. Estimation of resistance in Helicoverpa armigera (Hubner) to carbaryl and its effect on biology.

30

Resistant Pest Management. Newsletter, 16: 28-32. [54] Zhang, G. Y., Sun, X. L., Zhang, Z. X., Zhang, Z. F., and Wan, F. F. 1995.


Production and effectiveness of the new formulation of Helicoverpa virus pesticide-emulsifiable suspension. Virologica Sinica, 10:242-247.

31