İsmail DEMİR, Nurten GÜREL, Remziye NALÇACIOĞLU, İkbal A. İNCE, Zihni DEMİRBAĞ

Productive replication of Malacosoma neustria nucleopolyhedrovirus (ManeNPV) in Md203 cell line

A plaque-purified genotypic variant, derived from a field isolate of the Malacosoma neustria nucleopolyhedrovirus (ManeNPV) from Turkey was characterized based on in vitro replication properties in a cell line, Md203 derived from Malacosoma disstria. The life cycle of ManeNPV was studied based on the cytopathic effects (CPEs), polyhedral inclusion body (PIB) formation, budded virus (BV) production, viral DNA replication and polyhedrin protein expression in ManeNPV-infected Md203 cells. Infection of Md203 cells with ManeNPV resulted in cytopathic effects within 24 and 36 h post infection and most cells detachted from the bottom of the culture plate on the fourth day. Typical NPV cytopathic effects (CPE) like granulated and rounded cells, nuclear hypertrophy and impairment in cell proliferation, and lost cell shape with the extendings were observed. Budded viruses were detected at 24 h p.i. However, significant increase in BV production was observed after 48 h p.i. PIB was firstly detected at 36 h p.i. When BV’s were used for reinfection of the Md203 cells, the result showed that these cells support production of viable virus progeny. We also observed a significant level of viral DNA synthesis by 24 h p.i. The results indicate that ManeNPV can be propagated at in vitro system for further studies and thus has a potential for development into an effective biotechnological and microbial insecticidal agent.

Anahtar Kelimeler:

Key words: Baculovirus, Malacosoma neustria NPV, virus replication, Md203 cell line

Productive replication of Malacosoma neustria nucleopolyhedrovirus (ManeNPV) in Md203 cell line

Keywords:

Key words: Baculovirus, Malacosoma neustria NPV, virus replication, Md203 cell line,

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Hayakawa T, Rohrmann GF, Hashimoto Y. Patterns of genome organization and content in lepidopteran baculovirus. Virology 278: 1-12, 2000. 2.
Theilmann DA, Blissard GW, Bonning B et al. Baculoviridae.
In: Eighth report of the international committee on taxonomy
Van Oers MM. Vaccines for viral and parasitic diseases produced with baculovirus vectors. Adv Virus Res 68: 193-253, 2006.
Evans HF. Ecology and epizootiology of baculoviruses. In: Granados RR, Federici BA. eds. The Biology of Baculoviruses. Vol. 2, CRC Press, Boca Raton; 1986: pp. 89-100.
Herniou EA, Olszewski JA, Cory JS et al. The genome sequence and evolution of baculoviruses. Ann Rev Entomol 48: 211-234, 2003.
Shim H J, Roh JY, Choi JY et al. Isolation and characterization of a Lymantria dispar multinucleocapsid nucleopolyhedrovirus isolate in Korea. J Microbiol 41(4): 306-311, 2003.
Hostetter DL, Puttler B. A new broad host spectrum nuclear polyhedrosis virus isolated from a celery looper, Anagrapha falcifera (Kirby) (Lepidoptera: Noctuidae). Environ Entomol 20: 1481-1488, 1991.
Castro MEB, Souza ML, Arauja S et al. Replication of Anticarsia gemmatalis nuclear polyhedrosis virus four lepidopteran cell lines. J Invertebr Pathol 69: 40-45 1997.
Lynn DE. Comparative susceptibilities of twelve cell lines to infection by three baculoviruses. J Invertebr Pathol 82: 129-131, 2003.
Demir I, Demirbağ Z. A productive replication of Hyphantria cunea nucleopolyhedrovirus in Lymantria dispar cell line. J Microbial Biotechnol 16(10): 1485-1490, 2006.
Mitsuhashi J. A continuously growing cell line from larval hemocytes of Malacosoma neustria testacea. Jpn J Ent 64(3): 692-699, 1996.
Keddie AB, Erlandson MA, Hilchie GJ. Establishment and characterization of three Malacosoma disstria cell lines. J Invertebr Pathol 66: 136-142, 1995.
Mitsuhashi J. Cell culture. In: Hunter-Fujita FR, Entwistle PF, Evans HF et al. eds. Insect Viruses and Pest Management. Wiley, New York: 1998, pp. 485-517.
Lavina BA, Padua LE, Wu FQ et al. Biological characterization of a nucleopolyhedrovirus of Spodoptera litura (Lepidoptera: Noctuidae) isolated from the Philippines. Biol Control 20: 39- 47, 2001.
Brown M, Faulkner P. Factors affecting the yield virus in a cloned cell line of Trichoplusia ni infected with a nuclear polyhedrosis virus. J Invertebr Pathol 26: 251-257, 1975.
Invitrogen. MaxBac 2.0 Transfection and expression manual. In: Invitrogen Corporation, A Manual of Methods for Baculovirus Expression. 3985 B Sorrento Valley Blvd, San Diego; USA, 1996: pp. 35-36.
Woo SD. Rapid detection of multiple nucleopolyhedroviruses using polymerase chain reaction. Mol Cells 11: 334-340, 2001.
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970.
Sambrook Fritsch J,
EF, Maniatis T. Molecular Clonning. 2nded.
Cold Spring Habor Laboratory Press, New York; 1989.
Demirbağ Z, Beldüz AO, Ertürk Ö et al. Comparative replication of Autographa californica nuclear polyhedrosis virus in insect cell cultures. Turk J Biol 21: 63-70, 1997.
Levin DB, Huang J. Spodoptera littoralis type B nucleopolyhedrovirus infection of a grasshopper cell line. J Invertebr Pathol 74: 184-192, 1999.
Williams GV, Faulkner P. Replication patterns and cytopathology of cells infected with baculoviruses. In: Vlak JM, De Gooijer CD, Tramper J et al. eds. Insect Cell Cultures: Fundamental and Applied Aspects. Kluwer Academic Publishers, P.O. Box 17,3300, AA Dordrecht; The Netherlands, 1996: pp. 95-110. Erlandson MA, Baldwin D, Haveroen M et al. Isolation and characterization of plaque-purified strains of Malacosoma disstria nucleopolyhedrovirus. Can J Microbiol 52: 266-271, 2006.
Shirata N, Ikeda M, Kamiya K et al. Replication of nucleopolyhedrosisviruses of Autographa californica (Lepidoptera: Noctuidae), Bombyx mori (Lepidoptera: Bombycidae), Hyphantria cunea (Lepidoptera: Arctiidae) and Spodoptera exigua lepidopteran cell lines. Appl Entomol Zool 34: 507-516, 1999.
Huang J, Liu X, Levin DB. Characterization of Spodoptera littoralis type B nucleopolyhedrovirus infection in selected insect cell lines. Arch Virol 144: 935-955, 1999.
Liu HS, Bilimoria SL. Infected cell spesific protein and viral DNA synthesis in productive and abortive infections of Spodoptera frugiperda nuclear polyhedrosis virus. Arch Virol 115: 101-113, 1990.