Molecular cloning and characterization of Antheraea mylitta cytoplasmic polyhedrosis virus polyhedrin gene and its variant forms
Section snippets
Materials and methods
Silkworms, virus, and cell line. CPV-infected Indian non-mulberry silkworm A. mylitta was collected from West Bengal and Bihar state of India. The Spodoptera frugiperda cell line, Sf-9, was obtained from Invitrogen (USA) and maintained at 27 °C in TNM-FH media [Grace’s basic media supplemented with yeast and lactalbumin hydrolysate (Life Technologies) with 10% of fetal bovine serum (Hyclone)].
Purification of polyhedral bodies, isolation of total genomic RNA, and extraction of segment 10 RNA. CPV
Genetic analysis of segment 10 RNA encoding polyhedrin
Segment 10 RNA encoding polyhedrin was isolated from AmCPV, converted to cDNA and cloned into pCR2.1-TOPO vector, and analyzed by restriction enzyme digestion. Three different sizes of S10 cDNA designated as AmCPV10-1, AmCPV10-2, and AmCPV10-3 were obtained (Fig. 1). AmCPV10-1 produced 667, 452, and 383 bp fragments (lane 6), AmCPV10-2 produced 668 and 452 bp fragments (lane 4), and AmCPV10-3 produced 659, 452, and 304 bp fragments (lane 2) by EcoRI digestion. Sequence data revealed that
Discussion
Polyhedrin genes of several NPVs and CPVs including CPV of mulberry silkworm, B. mori, have been previously cloned and characterized. But the polyhedrin genes from CPVs infecting non-mulberry silkworms have not been characterized at the molecular level. The present work deals with the cloning and sequencing of segment 10 dsRNA encoding polyhedrin from CPV infecting Indian non-mulberry silkworm, A. mylitta. Three cDNA clones consisting of 1502, 1120, and 1415 nucleotides designated as AmCPV10-1,
Acknowledgments
The authors thank Prof. James I Mullins, University of Washington School of Medicine, Seattle, Washington, USA, for extending help in DNA sequencing, and Director(s) of Central Tasar Research and Training Institute, Ranchi and Jhargram for providing virus infected A. mylitta larvae. This work was supported by the Department of Biotechnology, Government of India.
References (26)
- et al.
Nucleotide sequence of the polyhedrin gene of Euxoa scandens cytoplasmic polyhedrosis virus (EsCPV)
Virology
(1989) - et al.
Sequence analysis and expression of the polyhedrin gene of Choristoneura fumiferana cytoplasmic polyhedrosis virus (CfCPV)
Gene
(1997) - et al.
Molecular characterization of segments 7–10 of Dendrolimus punctatus cytoplasmic polyhedrosis virus provides the complete genome
Virus Res.
(2003) Genome rearrangements of rotaviruses
Adv. Virus Res.
(1996)- M.H.V. van Regenmortel, C.M. Fauquet, D.H.L. Bishop, E.B Carstens, M.K. Estes, S.M. Lemon, J. Maniloff, M.A. Mayo, D.J....
- et al.
Cypoviruses
- et al.
Cytoplasmic polyhedrosis viruses
- et al.
Molecular cloning and characterization of cytoplasmic polyhedrosis virus polyhedrin and a viable deletion mutant gene
J. Virol.
(1988) - et al.
Nucleotide sequence of the polyhedrin gene of Bombyx mori cytoplasmic polyhedrosis virus A strain with nuclear localization of polyhedra
J. Gen. Virol.
(1989) - et al.
Analysis of the C polyhedrin genes from different geographical isolates of a type 5 cytoplasmic polyhedrosis virus
J. Gen. Virol.
(1994)
Nucleotide sequences of genome segments S6, S7 and S10 of Dendrolimus punctatus cypovirus 1
Arch. Virol.
Characterization of natural and induced polyhedrin gene mutants of B. mori cytoplasmic polyhedrosis viruses
Arch. Virol.
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2015, Journal of Molecular Graphics and ModellingCitation Excerpt :Our laboratory has previously characterized AmCPV by electron microscopy and its genome by electrophoresis, which revealed that it is similar to that of type-4CPV and consists of eleven segmented (S1–S11) double stranded RNA (dsRNA) molecules [6]. Molecular characterization of all these genome segments and elucidation of their functions [7–14] have shown that S2 of AmCPV codes for RNA dependent RNA polymerase (RdRp) responsible for replication/transcription [15]. But the structural basis of its enzymatic activity is still unclear.
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