Abstract
Ophiusa disjungens nucleopolyhedrovirus (OpdiNPV) was newly found in Guangdong Province, China. Using BamHI, EcoRI, HindIII, PstI, XhoI, XbaI digestion, the size of the OpdiNPV genome was estimated to be 92,000 base pair. The pstI-G genomic fragment of OpdiNPV was cloned and sequenced. Searches of databases identified at least four open reading frames (ORFs) within this fragment. These ORFs encode odv-e66 (EU 623602), p87/vp80 (EU 732665), odv-ec43 (EU617337) and ac108 gene (EU 732666) respectively. The phylogenetic tree of NPVs based on the combined sequences of odv-ec43 and ac108 indicated that OpdiNPV was most closely related to Mamestra configurata NPV-A and Mamestra configurata NPV-B. The characterization of OpdiNPV in this paper would provide better understanding molecular properties of this virus and be helpful in the development of the newly isolated virus as a biopesticide or an engineered pesticide to control more species of insect pests.
Keywords: Ophiusa disjungens, Nucleopolyhedrovirus, Physical map, odc-ec43, odv-e66, p87/vp80, ac108
Anua indiscriminata is an important pest of forests and fruit trees in South China. Serious damage to the eucalyptus caused by the moth infestation has occurred these years in Guangdong and Guangxi Province of China. The nucleopolyhedrosis virus of A. indiscriminata, which was named as AiNPV by Li [1], was found when the larvae were bred in laboratory. A transmission electron micrograph revealed that the occlusion bodies (OB) contained several virions with multiple envelope packaged within a single viral envelope. We found that A. indiscriminata is a junior synonym for Ophiusa disjungens, so AiNPV is renamed as OpdiNPV by us. Bioassays on lethal concentration and lethal time were conducted by Li [1].To achieve a further understanding of this virus, the molecular properties of the virus were observed and analyzed in this study.
An OpdiNPV isolate (Guangdong strain, from Souch China Agricultural University) was obtained from the diseased larvae, O. disjungens, with nucleopolyhedrosis symptoms. The OBs were purified according to the procedure described by Peter [4] and Lin [2]. The cadavers were homogenized in approximately 10 times their own volume of 1 % SDS and left overnight at room temperature, then homogenate was filtered through four layers of muslin. The filtrate was centrifuged at 3,000×g for 15 min at 4 °C for three times and at 3,000×g for 30 min at last. In the end, pellet was suspended with ddH2O and stored at −70 °C for future use.
The OBs were dealt with dissolution buffer (0.3 M Na2CO3, 0.03 M EDTA, 0.51 M NaCl) at 37 °C for 1 h, and proteinase K at 56 °C for overnight. Then DNA was extracted by phenol/chloroform treatment. The virus DNA was dissolved in 20 μl TE buffer.
Virus DNA was digested with BamHI, EcoRI, HindIII, PstI, XhoI, XbaI (TaKaRa) at 37 °C for 2–3 h, then electrophoresed on 0.7 % agarose gels at 50 V for 15–22 h to separate the fragments. The sizes of viral DNA fragments were estimated using GelScan XL software (Pharmacia LKB).
The pst I-G fragment of OpdiNPV DNA was cloned into pUC18 (TAKARA) plasmid vector according to the methods of Sambrook [6]. The recombinant plasmids were transformed into E. coli DH 5α cells (TIANGEN), screened for lacZ complementation, recovered by using DNA Product Purification Kit (TIANGEN) from agarose gels and purified by precipitation with glass milk. The termini of the fragment was sequenced by using the universal M13 primers at first, and further sequenced by primer walking using specific primers (Sangon).
Baculovirus gene comparison was performed with sequences from GenBank and published papers. Phylogenetic trees for odv-ec43 (EU617337) and ac108 genes were constructed by the N-J method (MEGA4) with the default settings for random break tie and the distance option for mean character difference. Statistical support for each node was evaluated by bootstrap analysis with 1,000 replicates [3, 8].
Electrophoresis patterns of the OpdiNPV genome digested with restriction endonuclease are shown in Fig 1. The sizes of the BamHI, EcoRI, HindIII, PstI, XhoI and XbaI fragments of the OpdiNPV genome are shown in Table 1. The fragments were designated alphabetically starting with A for the largest fragment for each enzyme digest as proposed by Vlak and Smith [7]. The mean size estimates for the complete OpdiNPV genome ranged from 91.1 to 92.4 kbp. These estimates were determined comparing restriction endonuclease fragment mobilities with those of λDNA/HindIII, 1,000 bp Ladder and/or λ-EcoRT14 molecular mass DNA standards. The size of the entire genome was calculated to be 92 kbp, which is much smaller than certain of the better studied NPVs including the AcMNPV (133 kbp) and the Mamestra brassicae MNPV (MbMNPV) genome of 150 kbp [5].
Fig. 1.
Restriction endonuclease fragment profiles of Ophiusa disjungens NPV digested with: BamHI (lane2), EcoRI (lane3), HindIII (lane4), PstI (lane5), XhoI (lane6), XbaI (lane7), BamHI (lane9), EcoRI (lane10), HindIII (lane11), PstI (lane12), XhoI (lane13) or XbaI (lane14) and electrophoresed on a 0.7 % agarose gel at 50 V to separate the fragments. λDNA/HindIII (lane1), 1,000 bp Ladder (lane8) and λ-EcoRT14 (lane15) were used as size standards
Table 1.
Size of restriction endonuclease fragments (kbp) of Ophiusa disjungens NPV
| Fragment | Restriction enzyme | |||||
|---|---|---|---|---|---|---|
| BamHI | EcoRI | HindIII | PstI | XhoI | XbaI | |
| A | 31.5 | 18.2 | 17.8 | 9.8 | 15.8 | 35 |
| B | 26.2 | 16.0 | 14.8 | 8.1 | 14.2 | 24 |
| C | 23.2 | 9.1 | 12.0 | 8.0 | 9.5 | 13.8 |
| D | 5.6 | 8.3 | 10.2 | 7.0 | 7.2 | 11.2 |
| E | 4.6 | 7.2 | 9.5 | 7.0 | 6.8 | 8.1 |
| F | 6.8 | 7.9 | 6.5 | 5.4 | ||
| G | 6.0 | 6.5 | 5.0 | 5.0 | ||
| H | 5.1 | 5.9 | 5.0 | 4.0 | ||
| I | 4.8 | 3.0 | 4.7 | 3.5 | ||
| J | 4.5 | 2.9 | 4.5 | 3.3 | ||
| K | 2.6 | 1.9 | 3.6 | 3.3 | ||
| L | 2.2 | 3.5 | 2.5 | |||
| M | 2.0 | 3.2 | 2.2 | |||
| N | 1.5 | 3.0 | 1.9 | |||
| O | 2.6 | 1.7 | ||||
| P | 2.5 | 1.4 | ||||
| Q | 1.9 | 1.4 | ||||
| R | 1.6 | 1.3 | ||||
| S | 1.4 | 0.9 | ||||
| T | 1.2 | 0.7 | ||||
| U | 1.0 | |||||
| V | 0.8 | |||||
| Total | 91.1 | 92.0 | 92.4 | 91.9 | 92.1 | 92.1 |
pst I-G fragment of OpdiNPV genome is composed of 5,056 bp. This fragment contained four ORFs which were odv-e66 (EU 623602), p87/vp80 (EU 732665), odv-ec43 (EU617337) and ac108 gene (EU 732666) respectively. The ORFs encoding ODV-Ec43 and Ac108 are complete, and the ORFs encoding ODV-E66 and P87/VP80 are partial.
The amino acid sequences of two conserved baculovirus genes, including odv-ec43 and ac108, were used for analysis of the phylogenetic relationship between OpdiNPV and other NPVs. The phylogenetic tree (Fig. 2) of NPVs based on the combined sequences of odv-ec43 and ac108 indicated that OpdiNPV was most closely related to SeMNPV, SINPV, SfMNPV, AsNPV and MacoNPV. It also appeared that the relationships between SeMNPV, SINPV, SfMNPV, AsNPV, MacoNPV, ChChNPV and TrniNPV were closer. It is more distantly related to other NPVs, such as LdMNPV, LxMNPV, LsNPV, AcMNPV, BmNPV, EpNPV, EcobSNPV and ApCiNPV.
Fig. 2.
A phylogenetic tree for nucleopolyhedroviruses based on the amino acid sequences of odv-ec43 (EU617337) and ac108 joint genes. Statistical support (%) for each node was evaluated by bootstrap analysis with 1,000 replicates. The GenBank accession numbers for odv-43 and Ac108 protein are as follows: AdhoNPV: Adoxophyes honmai NPV (NP_818729.1, NP_818730.1); AsNPV: Agrotis segetum NPV (YP_529736.1, YP_529735.1); ApciNPV: Apocheima cinerarium NPV (ADB_84433.1, ADB_84434.1); AcNPV: Autographa californica NPV (NP_054139.1, NP_054138.1); BmNPV: Bombyx mori NPV (NP_047509.1, NP_047508.1); ChchNPV: Chrysodeixis chalcites NPV (YP_249703.1, YP_249704.1); EcobSNPV: Ecotropis obliqua NPV (YP_874278.1, YP_874279.1); EpNPV: Euproctis pseudoconspersa NPV (NP_203264.1, YP_002854704.1); HaNPV: Helicoverpa armigera NPV (NP_075163.1, YP_002332607.1); HzSNPV: Helicoverpa zea SNPV (AF334030_28, NP_542721.1); LdMNPV: Lymantria dispar MNPV (NP_047744.1, NP_047745.1); LxMNPV: Lymantria xylina MNPV (YP_003517843.1, YP_003517844.1); MacoNPV-A: Mamestra configurata NPV-A (AAQ_11099.1, NP_613162.1); MacoNPV-B: Mamestra configurata NPV-B (NP_689254.1, NP_689253.1); MvMNPV: Maruca vitrata MNPV (YP_950815.1, YP_950814.1); OlNPV: Orgyia leucostigma NPV (YP_001651003.1, YP_001651004.1); OpMNPV: Orgyia pseudotsugata MNPV (NP_046265.1, NP_046264.1); SeMNPV: Spodoptera exigua MNPV (NP_037819.1, NP_037818.1); SfMNPV: Spodoptera frugiperda MNPV (YP_001036351.1, YP_001036350.1); SlNPV: Spodoptera litura NPV (NP_258364.1, NP_258365.1); TrniNPV: Trichoplusia ni SNPV (YP_308983.1, YP_308984.1)
In conclusion, OpdiNPV was newly found nucleopolyhedrovirus in China. Restriction endonuclease analysis showed that the size of the entire genome of OpdiNPV was 92 kbp, which is smaller than most baculoviruses whose genomes have been completely sequenced so far. OpdiNPV isolated from O. disjungens is being developed as a biopesticide for the control of O. disjungens. The aforementioned characterization of the newly isolated virus would provide better understanding molecular properties of this virus and be helpful in the development of OpdiNPV as a biopesticide or an engineered pesticide to control more species of insect pests.
Acknowledgments
The authors would like to thank Pro.Chuanxi Zhang, Institute of Insect Science, Zhejiang University, for his help on the phylogenetic tree analysis. This project was supported by the National Natural Science Foundation of China (Project No. 31170612) and the Natural Science Foundation of Guangdong Province (8151064201000068).
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