Isolation, identification and molecular phylogenetic analysis of Hyblaea puera Nucleopolyhedrovirus

Department of Forest Entomology, Kerala Forest Research Institute, Thrissur, Kerala, India
DOI
10.7287/peerj.preprints.27290v1
Subject Areas
Entomology, Evolutionary Studies, Genomics, Molecular Biology, Virology
Keywords
Nucleopolyhedrovirus, Hyblaea puera, Phylogeny, HpNPV, Baculovirus, Polyhedrin
Copyright
© 2018 Vijay Krishnan et al.
Licence
This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ Preprints) and either DOI or URL of the article must be cited.
Cite this article
Vijay Krishnan S, TV S. 2018. Isolation, identification and molecular phylogenetic analysis of Hyblaea puera Nucleopolyhedrovirus. PeerJ Preprints 6:e27290v1

Abstract

Hyblaea puera (Lepidoptera: Hyblaeidae), is considered as a serious pest of teak in India and other tropical regions. It causes entire defoliation of teak trees and results in huge timber loss thereby decreasing forest productivity. Hyblaea puera Nucleopolyhedrovirus (HpNPV) is a baculovirus that has been employed in various parts of India as a bio-control agent against the pest H. puera. An unfeigned nucleopolyhedrovirus was isolated from the larvae of the moth, H. puera in Kerala, South India. Polh, lef-8, pif-2 gene sequences were amplified by PCR with degenerate primers and extracted for phylogenetic analysis. Hyblaea puera Nucleopolyhedrovirus appeared to be a distinct species of Group II NPV alphabaculovirus. Polyhedrin coding region was characterized by nucleotide sequence analysis. To date, Polyhedrin is the first isolated and characterized gene of HpNPV. It indicated the presence of ORF comprising 741 nucleotides which encode 246 amino acids with a predicted molecular mass of 28 KDa. Phylogeny based on three conserved baculovirus genes showed the highest homology of HpNPV to Helicoverpa armigera NPV. These findings were hardened by restriction endonuclease analysis, even though some differences in restriction pattern were observed. The current study will encourage future efforts to improve the efficacy of HpNPV against its natural host.

Author Comment

This is a submission to PeerJ for review.

Supplemental Information

S1: Pairwise sequence comparisons of the deduced amino acid sequences of the concatenated lef-8 & pif-2 NPV genes used for phylogenetic analysis

DOI: 10.7287/peerj.preprints.27290v1/supp-1

S2: Pairwise comparison of the deduced amino acid sequences identity(%) of 36 polyhedrin genes of NPV used for phylogenetic analysis

DOI: 10.7287/peerj.preprints.27290v1/supp-2

Nucleotide sequence of the HpNPV polyhedrin gene and its flanking regions

The deduced amino acid sequences are indicated with one-letter code designation for polyhedrin. Putative baculovirus late promoter element ATAAG is shown in pink colour. Other potential eukaryotic promoter elements, TATA box and CAAT box are also shown, in green and blue respectively.

DOI: 10.7287/peerj.preprints.27290v1/supp-3

Nucleotide sequence of the HpNPV hypotetical protein

The deduced amino acids are indicated with one-letter code for hypothetical protein.

DOI: 10.7287/peerj.preprints.27290v1/supp-4

Comparison of nucleotide sequences of HpNPV and HearNPV

Dots indicate similarity and red indicates dissimilar nucleotide(s) of HearNPV.

DOI: 10.7287/peerj.preprints.27290v1/supp-5

Comparison of polyhedrin sequences of HpNPV and HearNPV

Dots in the HearNPV sequence indicates similar amino acids to HpNPV. Red indicates dissimilar amino acid(s).

DOI: 10.7287/peerj.preprints.27290v1/supp-6

Specific PCR amplification of HpNPV pol h and lef-8 genes

DOI: 10.7287/peerj.preprints.27290v1/supp-7

Agarose gel showing PCR amplification of full-length Polyhedrin gene

DOI: 10.7287/peerj.preprints.27290v1/supp-8

Specific PCR amplification of the HpNPV pif-2 gene.

DOI: 10.7287/peerj.preprints.27290v1/supp-9

Polyhedral Occlusion Bodies(POB) under light microscope(400x)

DOI: 10.7287/peerj.preprints.27290v1/supp-10